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GeneScan® Reference Guide

1. Step Action 1 From the Project containing your matrix standard Sample files open the Analysis Control window In the Analysis Control window select the colors for each sample HS P2 1 copy Analysis Control O Print Results Print Setup Sample File Size Standard PP Parameters 02eP27 2 1 HS P27 Standard GS 350 k lt Analysis Parameters gt 03eP27 3 1 HS P27 Standard GS 350 gt lt Analysis Parameters gt de as ad econ Y 19 osep27 s 1 HS P27 Standard GS 350 lt Analysis Parameters gt a Created Thu Jul 18 1996 2 34 PM Select the four matrix standard Sample files Choose Assign New Matrix in the Project menu Select the matrix file Select numbers 1 2 3 and 4 on the left side of the window to highlight the colors for each row 5 Use the Set Analysis Parameters dialog box in the Settings menu to set the Analysis Range Click Analyze Choose Results from the Windows menu and check each electropherogram by a Clicking 4 in the of Panels menu b Clicking 1 under Dye Samples c Clicking 1 on the Sample Files side of the Results window 8 If Then each peak is one color with the other the matrix is good colors flat under it the other colors are not flat under the the matrix is poor peaks as shown below 9 If Then the matrix is good Save the matrix file to the ABI folder the matrix is poor Redo the matrix by using different start and
2. Step Action 1 Highlight the sample files of interest From the Sample window select Sample Info 38 The Sample File Information window appears 2 Each dye is listed in the Analysis records Select the Red dye as shown below Sample File Information gt Run Information gt Data Collection Settings gt Gel Information gt Sample Information Y Analysis Records gt Analyzed 1 00 51 PM Wed Sep 3 gt Analyzed 1 00 51 PM Wed Sep 3 gt Analyzed 1 00 51 PM Wed Sep 3 Y Analyzed 1 00 51 PM Wed Sep 3 Parameters lt Analysis Parameters gt Standard 310 400 HD Peak Totals Analysis Range 3100 6100 pts Dye Std R Found In Baselined Yes Multi Componented Yes Size Method Local Southern Method Sample 21 Data Smoothing None Size Range All Sizes Dye Std 21 Peak Detection Threshold 200 Std Peak Det Threshold 200 Std Defined 21 Peak Detection Min Half width 3 Split Pk Corr None Std Matched 21 Note The sample must already be analyzed 3 In the column marked Peak Totals confirm that the Dye Std and the Sample contain the same numbers of peaks General Analysis and Evaluation Techniques 3 7 Evaluating Data Quality Good Data Looks Electropherogram peaks should be sharp well defined and scaled between 150 and Like This 4000 relative fluorescent units RFU as shown in Figure 3 1 The peaks corresponding to different color dyes need not be of equal intensity but the data for the less intense
3. Step Action 1 Verify data peaks are present in all four samples Peak data should be on scale and the dye of interest should have a value of at least 200 2 Check for any data anomalies such as an unstable baseline Rerun samples that have an unstable baseline 3 Select a starting point for the matrix data as shown below The starting point for matrix data should be slightly beyond the point where the primer peak falls back to the baseline approximately 2950 scans in this example EN Ajened EE 2700 3000 3300 3600 3900 4200 4500 start point 4 Choose a stop point such that at least three matrix standard peaks will be within the range analyzed To generate the matrix files Step Action 1 Choose New from the File menu Create New QO Project S 1 nalysi Si 3 Matri ee Theet Parameters Standard 2 Click the Matrix icon This will open the Make New Matrix dialog box Make New Matrix Select the Matrix Standard Sample Files No File Selected for B Data Start At C6 No File Selected for G Data Start At m No File Selected for Y Data Start At No File Selected for R Data Start At Points 100000 To generate the matrix files continued Step Action 3 Click the B G Y and R buttons to choose the standard sample files Choose the sample file representing blue dye for B green dye for G etc
4. Sizing and Size Standards 5 1 Introduction to Sizing Introduction Process Overview Size standards and sample fragments loaded on the same gel undergo the same electrophoretic forces Therefore the relative electrophoretic mobility of any sample fragment is a good indicator of its molecular weight because lane to lane variation in electrophoretic forces no longer contributes to measurement error The three steps in the size calling process are the following Fitting the Internal Lane Size Standard to the Size Standard Definition During this first step the GeneScan Analysis Software tries to match the peaks of the internal lane standard with the peaks of the size standard definition so that the overall fit Maximizes the number of matched peaks To be considered a match an internal lane standard peak must lie within 400 scans of its expected position as defined by the corresponding size standard definition peak Y Minimizes the total squared error of the matched peaks Once a size standard peak is matched the error is defined as the difference in scan number between its expected position and its actual position in the internal lane standard To complete Step 1 successfully the GeneScan Analysis Software must match at least three peaks Generating a Sizing Curve From the fragment migration times of the internal lane standard the GeneScan Analysis Software generates a sizing curve giving size in base
5. Data Field Setting Plate Check Module Plate Check PreRun Module PreRun Run Module GS Run Collect Time 4 hours for a 12 cm gel 8 hours for a 24 cm gel or 12 hours for a 34 cm gel Note The default collect time is 12 hours Sample Sheet The name of the sample sheet that you created for this run Well to Read distance 12 24 or 34 cm Filter Set A Lanes As appropriate for the comb used Run Mode XL only Full scan 24 34 or 36 lanes XL scan 50 or 66 lanes Operator Your name Gel s Matrix File The name of the matrix file that you created for this set of run conditions or None if you have not yet created a matrix AutoAnalyze On or off as desired Note You must create a matrix file before analyzing the data continued on next page Microsatellite Analysis 8 9 Preparing and Preparing and Loading Experimental Samples Loading Samples 8 10 Microsatellite Analysis Note Often you will need to dilute the PCR product from 1 5 1 20 in distilled deionized HzO before loading 1 uL into the sample tube Step Action 1 Prepare Loading Mix sufficient for loading 100 samples 250 uL deionized formamide 50 uL GeneScan size standard GeneScan 350 GeneScan 400 HD or GeneScan 500 recommended 25 uL Loading buffer 25 mM EDTA 50 mg mL blue dextran WARNING CHEMICAL HAZARD Formamide is a known teratogen It can caus
6. 401736 GeneScan 350 TAMRA 402985 GeneScan 400HD ROX 401734 GeneScan 500 ROX 401733 GeneScan 500 TAMRA 403040 GeneScan 500XL TAMRA 403039 GeneScan 500XL ROX 401098 GeneScan 1000 ROX 401100 GeneScan 2500 ROX 401545 GeneScan 2500 TAMRA 401144 Loading Buffer Fluorescent dNTPs For fluorescent labeling of DNA during PCR amplification 401894 FIdUTP Set R110 R6G and TAMRA 3 3 and 12 nmol 3 x 30 pL 401896 R110 dUTP 6 nmol 2 x 30 pL 401897 R6G dUTP 6 nmol 2 x 30 pL 401895 TAMRA dUTP 24 nmol 2 x 30 uL 402793 F JdCTP Set R110 R6G and TAMRA 3 3 and 12 nmol 3 x 30 pL 402795 R110 dCTP 6 nmol 2 x 30 pL 402796 R6G dCTP 6 nmol 2 x 30 pL 402794 TAMRA dUTP 24 nmol 2 x 30 uL Note TAMRAJdNTP is supplied at a concentration four times higher than R110 dNTP and R6G dNTP because it produces approximately four times less signal continued on next page Part Numbers F 1 Fluorescent dNTP Each kit listed below includes a GeneAmp kit as specified 100 reactions along with PCR Kits an FJdNTP set that contains 30 uL each of R110 JdNTP 3 nmol REG ANTP 3 nmol and TAMRA dNTP 12 nmol N808 0220 GeneAmp PCR Reagent Kit with AmpliTag DNA Polymerase with F dUTP Set N808 0221 GeneAmp PCR Core Reagents with F JdUTP Set N808 0222 G
7. C C 100 15 100 20 100 25 100 30 100 35 90 14 90 18 90 23 90 29 90 33 80 12 80 16 80 21 80 26 80 31 70 10 70 14 70 19 70 24 70 29 60 12 60 17 60 21 60 26 50 10 50 14 50 19 50 23 40 10 40 15 40 19 30 10 30 15 20 9 SSCP Analysis 7 9 PCR Amplification Labeling and Controls for SSCP Primer Choice Labeling Rules 7 10 SSCP Analysis Choose primers so that the resulting PCR product is no longer than 400 preferably 250 base pairs in length Note Empirical observation indicates that the efficiency of mutation detection is optimal for fragments between 130 and 250 base pairs in length To use the ABI PRism multicolor fluorescent dye technology for SSCP analysis follow these general rules Use 5 End Labeled Primers The success of SSCP analysis depends upon the ability to detect slight mobility shifts The reproducible sizing and sharp peaks obtained when using the 5 end labeling method are crucial to the success of this application Note Post PCR end labeling with FJdNTPs is an alternative lwahana et al 1995 Inazuka et al 1996 Inazuka et al 1997 Use a Different Dye for Each Strand When you first perform SSCP analysis on a region of DNA you should label the forward and reverse strands with different dyes Using different colors for the forward and reverse strands will permit detection of residual double stranded molecules remaining after denaturation as indicated by overlapping
8. 0 ccc een e nee n ene nee 5 14 OVER VICW a a A A A ii Sash 6 1 Choosing Reaction Volumes and Tube Types 0 0 00 cece eee eee ee eee 6 2 Designing Custom Primers 00 aE cee ec ee eee eee nen eens 6 3 Determining Reagent Concentrations 0 0 0 0 cece ee eee ene 6 6 Choosing the Right Enzyme 0 ec eee e ence eee 6 8 Multiplexing PER e pats gin ia Sd Ce he Ag aig ae meena ea eek didas 6 10 Using RNA Template ida a Mada date Ye ode Sa 6 12 Preventing Competing Side Reactions Hot Start PCR 0 0 eee eee eee 6 13 Modifying Thermal Cycling Parameters 0 0 0 cece eee eee 6 15 Avoiding Contamination oo 6 16 3 A Nucleotide Additions eissart 38 ea ee ee lela oe pail ee Meee risa 6 18 Stutter Products as o ce eine a Dal hea was Veet Oita dem ake 6 21 Preparing PCR Products for Analysis 0 0 0 eee eee eee eee eens 6 23 7 SSCP Analysis A tee tite keel cowie et Fal OVA a UA aes tite ayia We AD day a ria ta do 7 1 Introduction to SSCP Analysis 2 0 0 0 cece cee een ene n ene n en eas 7 2 Before You Begin didactica 7 3 Running at Subambient Temperatures on the ABI PRISM 377 DNA Sequencer 7 4 PCR Amplification Labeling and Controls for SSCP 0 0 00 0 02 7 10 To Save Time Prerun Checklist 0 0 0 ee cece eee nee 7 13 Preparing for a Run ABI PRISM 377 ABI PRISM 377 with XL Upgrade and ABI 373 with XL Update od Sica has oie aie ud eine eae Ria las er
9. AFLP Mapping 10 13 10 14 AFLP Mapping Table 10 8 Primer Combinations for Maize Species EcoRI Primers CAA CAC Msel Primers CAG CAT CTA CTC CTG AAC O AAG ACA O O ACC ACG ACT AGC DDO AGG Table 10 9 Primer Combinations for Sugar Beet Species EcoRI Primers CAA CAC Msel Primers CAG CAT CTA CTC CTG CTT AAC AAG ACA ACC ACG ACT AGC AGG Table 10 10 Primer Combinations for Tomato Species Msel Primers CAA CAC CAG CAT CTA CTC CTG CTT AAC AAG O 9 o ACA ACC lo M ACG ACT AGC AGG Table 10 11 Primer Combinations for Lettuce Species Msel Primers CAA CAC CAG CAT CTA CTC CTG CTT AAC AAG O O Y o ACA ACC lo M aca O O ACT AGC AGG O AFLP Mapping 10 15 Table 10 12 Primer Combinations for Arabidopsis Species Small Plant Genome Msel Primers CAA CAC CAG CAT CTA CTC CTG CTT AA AC y O 5 AG Sar O iz AT M TA O Table 10 13 Primer Combinations for Cucumber Species Small Plan
10. Enter the starting point for each file The Start At point should be after the primer peak Define the Points value This is the number of points after the start point to be analyzed Click OK A successful matrix opens an untitled Matrix Values window with a 4x4 matrix of numerical values OD 377 D Matrix File E Reactions G Y R 0 0028 0 4001 0 0088 0 2193 0 0427 0 0888 Use the Save As command to name and save the matrix file Choose a name that reflects the chemistry the virtual filter set and the run conditions How to Check Check the quality of the matrix by Matrix Quality 4 reviewing the values in the Matrix Values window reviewing the analyzed data of the matrix run Review the matrix values in the Matrix Values window as follows Step Action 1 View the Matrix Values window CS 377 D Matrix File E Reactions B G Y R 1 0000 0 0028 0 0088 00427 10000 The numbers on the diagonal Blue against Blue Green against Green etc must all be 1 00 The numbers off the diagonal should be less than 1 00 Note In Virtual Filter C Green under Blue the second box from the top in the first column on the left is sometimes slightly above 1 00 This is acceptable Creating Matrix Files C 3 C 4 Creating Matrix Files Check matrix quality as follows
11. LOH Screening What is LOH Advantages Limitations The body has many tumor suppressing genes These genes are functional unless one or both of the alleles is lost or inactive the remaining allele contains recessive mutations or both alleles contain recessive mutations This allele loss is called loss of heterozygosity LOH Analysis of DNA for LOH is a useful tool for the detection of cancer The microsatellite markers used in LOH screening map either to known oncogenes or to tumor suppressor genes Therefore the loss of the DNA region containing the linked oncogene or tumor supressor gene often correlates both with the loss of LOH markers and with the onset or susceptibility to certain types of cancer LOH is the second hit in the two hit model One needs to inherit a mutant oncogene or tumor suppressor gene for LOH to cause cancer Mulligan et a 1990 LOH screening has demonstrated reliability in the early detection of nonpolyposis colon cancer as well as for prognosis in confirmed cases Aaltonen et al 1993 de la Chapelle and Peltomaki 1995 Canzian et al 1996 The application of this technique to other tumor types only awaits further characterization of the relevant genes and linked polymorphic markers The original LOH studies Dryja et al 1984 Mannens et al 1988 employed DNA probes that recognize RFLPs throughout the genome Southern analysis PCR based detection of LOH has the following advantages ove
12. Note A note calls attention to useful and or interesting information IMPORTANT This information is emphasized because it is critical to the success of your experiments CAUTION A caution explains damage that could occur to the instrument WARNING Indicates that physical injury to yourself or others could result if you do not follow the recommended precautions For information on the safe operation of the ABI 373 or ABI PRISM 377 DNA Sequencer refer to the 373A DNA Sequencing System Preinstallation Manual P N 901173 or ABI PRISM 377 DNA Sequencer Safety Summary P N 903526 respectively Visit our World Wide Web site www appliedbiosystems com techsupport for updates to this manual This manual is designed to be used in conjunction with the following manuals Y ABI PRISM 377 DNA Sequencer User s Manual P N 903433 or 373 DNA Sequencing System User s Manual P N 902376 Y GeneScan Analysis Software User s Manual P N 904435 or GeneScan 672 Software User s Manual P N 902842 Technical Support To Reach Us on the Web Hours for Telephone Technical Support To Reach Us by Telephone or Fax in North America Applied Biosystems web site address is http www appliedbiosystems com techsupport We strongly encourage you to visit our web site for answers to frequently asked questions and to learn more about our products You can also order technical documents and or an index of available documents and have the
13. run modules set of dyes gel parameters gel polymer type buffer denaturing or non denaturing conditions etc After running the matrix standards use their sample files to generate a matrix file using ABI PRISM GeneScan Analysis Software How to Verify the Before creating the matrix file verify that the raw data from the standards is good Raw Data To view the raw data in GeneScan Analysis Software Step Action 1 Create a new project if you did not select Autoanalyze in the GeneScan Run Defaults preferences in the data collection software a Choose New from the File menu b Select the Project icon An untitled Analysis Control window opens c Choose Add Sample Files from the Project menu d Find and open the Run Folder for the matrix standards run e Select the four Sample files representing the blue green yellow and red dye labeled runs and then click Add f Click Done after the Sample files are transferred 2 In the Analysis Control window select the four matrix standard Sample files by clicking on the first Sample file holding down the mouse button and releasing on the last Sample file 3 Choose Raw Data from the Project menu Electropherograms displaying raw data from the four matrix standard Sample files appear Creating Matrix Files C 1 How to Generate The Matrix File C 2 Creating Matrix Files To verify the raw data
14. 104 68 y 0 50 continued on next page Microsatellite Analysis 8 25 Binning Alleles Using the Make from Labels Feature 8 26 Microsatellite Analysis You can use the Make from Labels feature in Genotyper 2 0 software to generate category members allele bins automatically This method is ideal for the following types of linkage mapping genotyping projects A single family pedigree typed with a number of markers Small studies in which all markers for all individuals fit on a single gel Unlike the other binning methods presented in this manual using this method requires Working with one marker at a time to make categories from labels Y Clearing all labels between markers categories To bin alleles using the Make from Labels feature Step Action 1 From the Views menu choose Show Categories Window 38 K and set up the main categories groups for your markers as follows e 012883 e Unknown All peaks from 98 00 to 113 00 bp in blue e 075517 e Unknown All peaks from 235 00 to 261 00 bp in blue e 025391 Unknown All peaks from 139 00 to 153 00 bp in green e 0139171 Unknown All peaks from 171 00 to 197 00 bp in green From the Edit menu choose Select All 8 A to select all categories From the Edit menu choose Unmark 8 U to unmark the categories Select the first category in the list From the Edit menu choose Mark 38 M e D125
15. 403107 403136 18 12 13 403108 403137 19 12 13 403109 403138 20 14 403110 403139 21 15 16 403111 403140 22 15 16 403112 403141 23 17 18 403113 403142 24 17 18 403114 403143 25 19 20 21 22 403115 403144 26 19 20 21 22 403116 403145 27 19 20 21 22 403117 403146 28 Xx 450096 Individual Primer Pairs from the ABI PRISM Linkage Mapping 3000 pmol Set Version 2 Must be ordered through Applied Biosystems Custom Oligonucleotide Synthesis Service specify locus name 403061 True Allele PCR Premix 18mL enough for 2000 rxns 403062 Control DNA CEPH 1347 02 180 uL enough for 150 rxns ABI PRISM 377 DNA Sequencer 401878 48 cm Glass Plates Spacers Kit Includes two sets of 48 cm Well to Read Glass Plates and Gel Spacers 401876 36 cm Glass Plates Spacers Kit Includes two sets of 36 cm Well to Read Glass Plates and Gel Spacers 401877 12 cm Glass Plates Spacers Kit Includes two sets of 12 cm Well to Read Glass Plates and Gel Spacers 401835 48 cm Rear Glass Plate 401838 48 cm Front Glass Plate 401837 Two 48 cm Gel Spacers 0 2 mm thick 401839 36 cm Rear Glass Plate 401840 36 cm Front Glass Plate 401836 Two 36 cm Gel Spacers 0 2 mm thick 401833 12 cm Rear Glass Plate 401834 12 cm Front Glass Plate 401904 24 well Squaretooth Comb 0 2 mm thick 401907 32 well Squaretooth Comb 0 2 mm thick 401910 36 well Squaretooth C
16. Amplification primers B Run Selective Amplification G A CAG TGT GTC ACA Figure 10 10 Selective amplification with fluorescent dye labeled primers Use the AFLP Selective Amplification Start Up Modules Regular Plant Genomes P N 4303050 Small Plant Genomes P N 4303051 or individual AFLP Selective Primers see Table 10 3 on page 10 11 continued on next page Testing New Genomes Primer Selection Guidelines Microbial Fingerprinting When testing novel genomes you must be sure that the DNA restriction digest with EcoRI and Msel generates enough fragments for comparison of samples There is a large variability in the number of restriction sites within microbial genomes No assurances of kit performance are made for organisms not listed Empirical guidelines suggest that if the G C content of the genome is gt 65 Msel will not give a significant number of fragments Optimal results are obtained with Msel when the G C content is lt 50 EcoRI also tends to produce more fragments in G C poor genomes In cases where an organism s G C content is unknown the effectiveness of the restriction enzymes must be determined empirically For genomes that restrict well with the EcoRI and Msel restriction endonuclease combination some general recommendations can be made in terms of the genome size and the selective nucleotides to choose for subsequent amplification Table 10 1 Table
17. Be aware that preferential amplification can make LOH measurements less accurate Figure 9 2 shows an electropherogram example of preferential amplification of the D5S346 marker In both the normal top panel and tumor bottom panel samples the peak height of the larger 124 bp fragment is much less than that of the smaller 110 bp fragment 36 101 106 111 116 121 126 131 1 1800 1200 600 0 MI Lane 1 Normal 1800 1200 600 0 MN Lane 1 Tumor Figure 9 2 Example of preferential amplification of the shorter PCR product at D58346 9 12 Microsatellite Analysis Applications RER Screening What is RER Advantages Limitations Replication error RER also called microsatellite instability describes the reduced fidelity during the replication of repetitive DNA often occurring in tumor cells RER leads to the appearance of multiple alleles at microsatellite loci It is thought to be caused by strand slippage during DNA replication due to mutations in DNA mismatch repair genes The technique for detecting RER involves comparing microsatellite alleles after PCR amplification in normal and tumor samples from the same host You calculate a raw RER score using an algebraic formula that quantifies the relative strength of the stutter bands in the two samples after normalizing for differences in PCR efficiency RER is a simple inexpensive and reliable tool for the analysis of tumors The RER phenotype can be variable
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19. FIANTP Blue A TET Phosphoramidite Green C JOE Labeled primer in reagent kits Green A F R6G FIANTP Green A HEX Phosphoramidite Green A D Yellow C NEDb NHS ester phosphoramidite Yellow D F TAMRA NHS ester F dNTP or GeneScan Internal Yellow A Red C Lane Size Standard ROX NHS ester or GeneScan Internal Lane Size Red A D F Standard a 5 FAM and JOE are only available as labeled primers in certain reagent kits see Table 4 4 on page 4 8 b NED labeled primers are available only in kits or through the Applied Biosystems Custom Oligo Service Call Applied Biosystems or visit the Applied Biosystems WorldWideWeb site at www appliedbiosystems com techsupport for information on how to order custom labeled oligos ABI PRISM Dyes 4 7 Table 4 3 Recommended Dye Virtual Filter Set Combinations Chemical Forms Filter Set Virtual Filter Set Dye Combination Combined ABI 373 ABI PRISM 377 6 FAM HEX TAMRA ROX std Phosphoramidites A A NHS esters R110 R6G TAMRA ROX std FIdNTPs 5 FAM JOE TAMRA ROX std Reagent kit primers NHS esters 6 FAM TET HEX TAMRA std Phosphoramidites B C 6 FAM HEX NED ROX std Phosphoramidites A D 5 FAM JOE NED ROX std Reagent kit primers F NHS esters a This combination although recommended can sometimes give poor quality data due to spectral overlap among the dye signals If you experience matrix p
20. Figure 5 9 Electropherogram of the GeneScan 2500 size standard run under denaturing conditions on the ABI PRISM 377 DNA Sequencer Fragments were run on a 36 cm 5 Long Ranger 6M Urea gel at 51 C Note Under denaturing conditions the two strands of the doubly labeled GeneScan 2500 size standard fragments migrate at different rates appearing as split peaks To ensure size calling precision and a reliable size standard definition you must explicitly define one peak from each split peak pair in the size standard definition To improve matching of size standard peaks choose either LeftMost Peak or RightMost Peak in the Split Peak Correction section of the Analysis Parameters window Figure 5 10 shows the peak patterns of GeneScan 2500 fragments run under non denaturing conditions 7200 6400 5600 4800 4000 3200 2400 1600 800 Figure 5 10 Electropherogram of the GeneScan 2500 size standard run under non denaturing conditions on the ABI Prism 377 DNA Sequencer Fragments were run on a 36 cm 5 Long Ranger Native gel at 30 C Sizing and Size Standards 5 17 Optimizing PCR Overview In This Chapter The success or failure of most GeneScan fragment analysis experiments depends upon the success or failure of the PCR amplification step This chapter presents strategies that you can employ to optimize polymerase chain reaction PCR product yield in your experiments Because no single set of PCR conditions is optimal for
21. ranging from a simple increase in the strength of the stutter bands to the presence of extra bands on top of variable strength stutter bands Although the formula for determining the raw RER score partially corrects for differences in the amplification efficiency of normal and tumor samples extreme discrepancies in amplification efficiency can lead to false positive or false negative results For example if the amplification of the normal sample is unusually poor Genotyper might only recognize the relatively strong peaks corresponding to the main alleles In an efficiently amplified tumor sample both the main allele peaks and the stutter peaks would be recognized In this hypothetical case you would obtain a false positive determination of RER Because RER often appears in the same types of tumors as LOH RER screening should be performed in conjunction with LOH screening A false negative RER diagnosis can be obtained in LOH positive samples continued on next page Microsatellite Analysis Applications 9 13 Advantages of Using Increasing Reliability of RER Results ABI PRISM Traditional approaches using radioactivity are labor intensive time consuming and Technology difficult to automate For example multiple exposures of films are often required and if normal and tumor DNA do not amplify with equal efficiency interpretation becomes difficult Moreover automation is highly desirable to reduce the arbitrariness in analysis encour
22. 0 4 mm thick 402182 64 well Sharktooth Comb 0 4 mm thick 402214 Casting Comb for Sharktooth 0 4 mm thick 402048 Uni length Glass Plate Spacer 0 4 mm thick 402054 50 well Squaretooth Comb 0 3 mm thick 402184 66 well Squaretooth Comb 0 3 mm thick 401582 24 well Sharktooth Comb 0 3 mm thick 401581 32 well Sharktooth Comb 0 3 mm thick 401583 36 well Sharktooth Comb 0 3 mm thick 402178 48 well Sharktooth Comb 0 3 mm thick 402181 64 well Sharktooth Comb 0 3 mm thick 402215 Casting Comb for Sharktooth 0 3 mm thick 402047 Uni length Glass Plate Spacer 0 3 mm thick 401617 48 cm Notched Glass Plate Stretch Configuration 401618 48 cm Plain Glass Plate 401069 24 cm non Stretch Configuration or 34 cm Stretch Configuration Notched Glass Plate 401498 24 cm non Stretch Configuration or 34 cm Stretch Configuration Plain Glass Plate 401620 24 cm Notched Glass Plate Stretch Configuration 401621 24 cm Plain Glass Plate Stretch Configuration 401479 6 cm non Stretch Configuration or 12 cm Stretch Configuration Notched Glass Plate 401480 6 cm non Stretch Configuration or 12 cm Stretch Configuration Plain Glass Plate 401477 12 cm Notched Glass Plate non Stretch configuration 401478 12 cm Plain Glass Plate non Stretch configuration 401623 6 cm Notched
23. 09 2000 Part Number 4303188B
24. 162 08 186 58 horse 4 Results 7 Green std dev taq 1500 1000 500 HTG6 AHTS HMS6 ASB2 83 15 132 03 158 98 254 83 139 63 168 87 horse 4 Results 7 Yellow std dev taq 2000 1500 1000 500 HTG10 111 82 HTG HMS3 HMS2 93 79 128 93 159 75 223 88 165 73 226 17 Figure 9 5 Genotyper plot of the equine control DNA For More Refer to the StockMarks for Cattle Bovine Paternity PCR Typing Kit Protocol Information P N 401917 and the StockMarks for Horses Equine Paternity PCR Typing Kit Protocol P N 402828 for more information You can also find information about animal paternity testing on the Agriculture page of the Applied Biosystems Web site www appliedbiosystems com techsupport 9 18 Microsatellite Analysis Applications Human Identification Human Identification with STRs Advantages of STR Analysis Short tandem repeat STR markers also referred to as microsatellites are polymorphic DNA loci that contain a repeated nucleotide sequence The STR repeat unit can be from two to seven nucleotides in length The number of nucleotides per repeat unit is the same for a majority of repeats within an STR locus The number of repeat units at an STR locus may differ so alleles of many different lengths are possible Polymorphic STR loci are therefore very useful for human identification purposes Edwards et al 1992 STR loci can be amplified using the polymerase chain reaction PCR process and the PCR products are then a
25. 17 genotyping 9 18 improved breeding 9 17 kits 9 17 primer dye set storing dyes D 9 STR short tandem repeat markers See microsatellite marker analysis stutter products 6 21 to 6 22 definition 6 21 evaluating data with stutter preparing PCR products for analysis 6 23 stutter facts 6 21 4 8 6 22 T TBE buffer preparing A 7 technical support 1 3 to 1 8 e mail address 1 3 Documents on Demand 1 4 regional offices 1 5 to 1 8 temperature control parameters modifying 6 15 template concentration determining reagent concentration 6 7 RNA using to optimize PCR 6 12 troubleshooting Linkage Mapping Set LMS V2 9 4 LOH and RER screening 9 16 microsatellite analysis common problems 8 33 stutter aiding in allele calling 8 36 stutter example 8 33 to 8 35 PCR amplification 11 1 to 11 6 problems with extra peaks 11 5to 11 6 problems with missing peaks 11 6 problems with poor amplification 11 4 PCR product detection 11 13 problems with automatic data analysis 11 8 problems with current 11 9 problems with gel or gel image 11 7 to 11 8 problems with peak number and position 11 11 to 11 12 problems with peak quality and resolution 11 13 problems with signal strength 11 1 to 11 7 to and quality 11 9 to 11 10 single strand conformation polymorphism SSCP 7 29 size standard problems 5 5 to 5 6 possible problems 5 5 to 5 6 preventing sizing problems 5 5 Tth DNA Polymerase recombinant rTth
26. 2504 2514 Li L Li X Francke U and Cohen S N 1997 The TSG101 tumor susceptibility gene Is located in chromosome 11 band p15 and is mutated in human breast cancer Cell 88 143 154 Lindqvist A Magnusson P Balciuneiene J Wadelius C Lindholm E Alarcon Riquelme M and Gyllensten U 1996 Chromosome specific panels of tri and tetranucleotide microsatellite markers for multiplex fluorescent detection and automated genotyping evaluation of their utility in pathology and forensics Genome Res 6 1170 1176 References E 5 E 6 References Litt M and Luty J A 1989 A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene Am J Hum Genet 44 397 401 Magnuson V Ally D Nylund S Karanjawala Z Rayman J Knapp J Lowe A Ghosh S and Collins F 1996 Substrate nucleotide determined non templated addition of adenine by Taq DNA polymerase Implications for PCR based genotyping and cloning Bio Techniques 21 700 709 Mayrand P Corcoran K Ziegle J Robertson J Hoff L and Kronick M 1992 The use of fluorescence detection and internal lane standards to size PCR products automatically Applied and Theoretical Electrophoresis 3 1 11 Mayrand P Robertson J Ziegle J Hoff L McBride L Chamberlain J and Kronick M 1990 Automated genetic analysis Annales de Biologie Clinique 91 224 230 Middl
27. 3 5 Analyzed data from sample in Figure 3 4 General Analysis and Evaluation Techniques 3 9 Quantitating Nucleic Acids Introduction How to Proceed Ensuring Precise Relative Quantitation You can determine the relative quantities of two 5 end labeled fragments run on your ABI PRISM instrument by comparing the corresponding peak areas or peak heights on a GeneScan electropherogram IMPORTANT Do not use internally labeled F dNTP labeled fragments in your quantitative experiments Variations in the per fragment number of labeled nucleotides and the increased peak spreading with this method make relative quantitation unreliable To determine the relative number of molecules of two different sized fragments you calculate the ratio of respective peak areas or heights Always compare peak area to peak area and peak height to peak height If two fragments are similar in size it is often better to compare peak heights especially if the peaks overlap slightly If the peaks are well defined peak area and peak height will give similar results If the peaks are irregularly shaped e g have shoulders peak heights will often give better results than peak areas If two fragments are far apart in size it is often better to compare peak areas because large peaks tend to spread considerably more than small peaks Choosing the Minimum Peak Half Width and the Smoothing Option Depending upon the value you set for the Minimum Peak H
28. 650 570 6667 800 345 5224 Fax 650 572 2743 Latin America Del A Obregon Mexico Tel 305 670 4350 Fax 305 670 4349 Europe Austria Wien Hungary Budapest Tel 43 0 1 867 35 750 Tel 36 0 1 270 8398 Fax 43 0 1 867 35 75 11 Fax 36 0 1 270 8288 Belgium Tel 32 0 2 712 5555 Fax 32 0 2 7125516 Italy Milano Tel 39 0 39 83891 Fax 39 0 39 838 9492 Czech Republic and Slovakia Praha Tel 420 2 61 222 164 Fax 420 2 61 222 168 The Netherlands Nieuwerkerk a d IJssel Tel 31 0 180 331400 Fax 31 0 180 331409 Denmark Naerum Tel 45 45 58 60 00 Fax 45 45 58 60 01 Norway Oslo Tel 47 23 12 06 05 Fax 47 23 12 05 75 Introduction 1 5 1 6 Introduction Europe Finland Espoo Tel 358 0 9 251 24 250 Fax 358 0 9 251 24 243 Poland Lithuania Latvia and Estonia Warszawa Tel 48 22 866 40 10 Fax 48 22 866 40 20 France Paris Tel 33 0 1 69 59 85 85 Fax 33 0 1 69 59 85 00 Portugal Lisboa Tel 351 0 22 605 33 14 Fax 351 0 22 605 33 15 Germany Weiterstadt Tel 49 0 6150 1010 Fax 49 0 6150 101 101 Russia Moskva Tel 7 095 935 8888 Fax 7 095 564 8787 Spain Tres Cantos Tel 34 0 91806 1210 Fax 34 0 91 806 1206 South Africa Johannesburg Tel 27 11 478 0411 Fax 27 11 478 0349 Sweden Stockholm Tel 46 0 8 619 4400 Fax 46 0 8 619 4401 Un
29. 8 29 which will become part of the name of the alleles Figure 8 2 on page 8 28 and Figure 8 4 on page 8 29 In the First number box enter the number of the first allele the smallest allele expected in the data for example 101 for the marker or the starting number for example 1 if using a prefix In the Number increment box enter a numeric value This is the value by which software automatically increases each successive allele number For example enter 2 for dinucleotide markers if alleles are expected every 2 bp Enter 1 to number alleles sequentially for example A1 A2 A3 and so on c Select the With checkbox and the group name button d Enter the group marker name in the field to the right of the group name parameter This indicates that the category members created belong to the group marker that you are currently working with e The appropriate dye color box should have been selected automatically by Genotyper software If not check the appropriate box f Select the Exclusive checkbox if not automatically selected g Click OK 10 Return to step 3 to define the remaining categories 11 When all the categories markers have been defined choose Select All from the Edit menu 8 A to select all categories From the Edit menu choose Mark 8 M to mark all the categories 12 From the Analysis menu choose Clear All Labels You can now label the alleles with the newly defined bin names Mic
30. A However if you are experiencing problems with signal strength using TAMRA or you want to load less sample you should use this combination Relative signal strength is not indicated in these normalized spectra Virtual Filter Set F is used in the AmpF STR Profiler and AmpF STR Profiler Plus PCR Amplification Kits and in the AFLP Plant Mapping and AFLP Microbial Fingerprinting Kits 100 80 60 40 alicia O 7 AN 20 WSS NORMALIZED FLUORESCENCE INTENSITY ZO 560 580 600 620 640 WAVELENGTH nm 5 FAM JOE NED ROX Filter 1 Filter 2 Filter 3 Filter 4 Sizing and Size Standards Overview In This Chapter This chapter describes the size calling process in detail including the following 4 The distinction between absolute accuracy and precision in size calling How cross platform sizing differences arise How to avoid or recognize the most common sizing problems This chapter also describes the available GeneScan Internal Lane Size Standards including each standard s useful range This chapter contains the following topics Topic See Page Introduction to Sizing 5 2 Preventing Troubleshooting Sizing Problems 5 5 GeneScan Internal Lane Size Standards 5 7 GeneScan 350 Size Standard 5 8 GeneScan 400HD Size Standard 5 10 GeneScan 500 500XL Size Standard 5 12 GeneScan 1000 Size Standard 5 14 GeneScan 2500 Size Standard 5 16
31. Chooser window Note If you are using GeneScan Analysis Software version 2 0 2 or higher follow the instructions for the ABI PRISM 377 DNA Sequencer on pages 7 14 through 7 18 To set the Preprocess Parameters Step Action 1 Restart the Macintosh computer to defragment the RAM memory 2 Launch the GeneScan 672 Fragment Analysis Software 3 Select Preprocess Parameters from the Analysis menu 4 Specify the settings as shown below Select your own matrix file in the Multi Component area of the dialog box Preprocess Info EJ Multi Component _ Process Amount Matrix File Start at Scan 373 dye amidite matrix Switch Matrix File End at Scan 10000 x Use Sample Sheet Process Functions Sheet Name EJ Auto Lane Track 12 11 98 Sample Sheet Switch Sample Sheet Gel Scaling Estimated Peak Heights 4096 J Baseline Data Note Refer to the GeneScan 672 Software User s Manual for more information on creating and using sample sheets SSCP Analysis 7 19 7 20 SSCP Analysis To set the Preprocess Parameters continued Step Action 5 Click OK when done Note The matrix file must be created previously and saved to the ABI folder which is located in the System folder Refer to Appendix B in the 373 DNA Sequencing System User s Manual for instructions on making a matrix Be sure to use the Dye Primer Matrix Standard Kit P N 401
32. DNA added because of inaccurate quantitation Evaluate the quality and concentration of the DNA sample by Using the QuantiBlot Human DNA Quantitation Kit for human DNA Running an agarose yield gel If DNA is degraded or inaccurately quantitated reamplify with an increased amount of DNA Incorrect pH Verify buffer pH and concentration If correct quantitate sample DNA Too little or too much DNA can alter the pH Primer choice not optimal for example primers may be annealing to sites of template secondary structure or may have internal secondary structure Use different primers See Designing Custom Primers on page 6 3 for more information Tm Of primers is lower than expected Decrease the annealing temperature by 2 C increments Troubleshooting 11 3 Table 11 1 Problems with Poor Amplification continued Observation Possible Causes Recommended Actions Poor yield for multiplex PCR Non optimal thermal cycling parameters Between the denaturation and annealing stages add a 2 minute down ramp time to thermal cycling profile Note For multiplex PCR a short down ramp time is not necessarily optimal Competition from mispriming and other competing side reactions Use AmpliTag Gold DNA Polymerase See Designing Custom Primers on page 6 3 Multiplexing PCR on page 6 10 and Preventing Competing Side Reactions Hot Start PCR o
33. Fragment Analysis Kits and Reagents 0 00 00 00 0000 F 1 ABI PRISM 377 DNA Sequencer 0 0 0 cece eee cnet n ene e en ens F 5 ABI373 DNA Sequencer mim Geer hese A Wai we Godse Wa as AONE AR F 6 User s Manuals di a AA ered eds F 7 Updates oia A AA AAA A tae A da F 7 Index Introduction Using This Manual How This Manual Is This manual contains four major topic divisions Organized 4 General knowledge Chapter 2 through Chapter 6 Designing experiments Analyzing and evaluating data Fluorescent dyes and recommended dye sets Choosing size standards and size calling methods and troubleshooting sizing problems Optimizing PCR Applications Chapter 7 through Chapter 10 SSCP protocols and optimization suggestions Generic microsatellite analysis protocols and optimization suggestions Specialized applications of microsatellite analysis AFLP microbial fingerprinting and plant mapping applications Troubleshooting Chapter 11 PCR amplification PCR product detection Appendices A through F Reagent preparation Loading volumes for samples Creating matrix files Preparing 5 end labeled primers Literature references Part numbers continued on next page Introduction 1 1 Conventions Used in This Manual Safety Information Updates Other Manuals 1 2 Introduction The following words and styles draw your attention to specific details of the information presented in this manual
34. HAZARD Long Ranger gel solution contains acrylamide Acrylamide is a neurotoxin Avoid skin contact with Long Ranger gel solution because acrylamide can be absorbed through the skin Always work in a fume hood Obtain a copy of the MSDS from the manufacturer Wear appropriate protective eyewear clothing and gloves 4 Slowly add distilled deionized H O to bring the liquid level to approximately 45 mL Note Gently tap the cylinder while adding the water to release any air bubbles trapped by the urea 5 Stopper and then invert the cylinder to dissolve the urea Note Although the cylinder and its contents become very cold the urea dissolves rapidly 6 Allow the solution to warm to room temperature 7 Add distilled deionized H5O to a final volume of 50 mL 8 Stopper the cylinder and then mix the contents thoroughly 9 Filter the mixture through 0 2 um cellulose nitrate filter 10 Degas the filtrate by one of the following methods Purging with argon or helium Applying a vacuum for at least five minutes 11 Add fresh 10 APS to the gel solution Swirl gently to mix Be careful to avoid introducing air bubbles 12 Add 25 uL of TEMED tetramethylethylenediamine to the solution Swirl gently to mix Be careful to avoid introducing air bubbles WARNING TEMED Tetramethylethylenediamine is extremely flammable and can be very destructive to the skin eyes nose and respiratory system Keep TEMED in a tightly closed container Avoid i
35. Intensity Multiplexing PCR You can multiplex the PCR by combining more than one pair of primers in the same PCR reaction tube Do not multiplex primers with similar product lengths labeled with similar dyes Note For microsatellite applications do not multiplex primers labeled with the same fluorescent dye for loci with overlapping allele size ranges If you do not know the full extent of the allele size ranges leave at least 15 20 base pairs between the known size ranges Before performing the PCR perform a preliminary check for primer compatibility Avoid excessive regions of complementarity among the primers Also choose primer pairs with similar melting temperatures For more information on primer pair compatibility see Designing Custom Primers on page 6 3 After identifying compatible primer pairs test the pairs for successful coamplification You will often need to optimize reaction conditions and occasionally you will need to redesign the primers For more information on optimizing reaction conditions see Multiplexing PCR on page 6 10 Accounting for Differences in Dye Signal Strengths The intensity of emitted fluorescence is different for each dye For example to generate signals of equal intensity you need to load approximately eight times as much ROX as you do 6 FAM or TET The following lists ABI PRISM dyes in order of increasing emission intensity when excited by the dual mode argon laser ROX lowest s
36. LOH Screening isis oa Solus ee lada deed 9 7 RER Screenings sctereirt city Gat wv eae Gate PR ae eae See A eee Gee 9 13 Troubleshooting LOH and RER Screening 00 0 0 cece ee eee eee 9 16 Animal Paternity iii a eae de estate add a A AA aaa dt 9 17 Human Identification ee eseri 2 nea mi reiii atie e e cece E Ea een aa 9 19 AREPMAappite ii ADA Troubleshooting Lita A a ta A Troubleshooting PCR Amplification 0 0 eee ee eee ence eens 11 1 Troubleshooting PCR Product Detection 0 0 0 cece eee eee eee 11 7 Reagent Preparation 0 ccc ccc ccc cece e eee e ee And Preparing Gels sy ii e ites aa anh Riedie WG Se ted ith a a Mada a A 1 Preparing TBE Buffer and Deionized Formamide 0 0 ee eee eee ee A 7 Sample Loading Volumes 0 0 c cece cece eee e eee Bal Creating Matrix Files 0 ccc ccc cee cece eee Cn Creating the GeneScan Matrix File 2 ee cee eens C 1 Preparing 5 End Labeled Primers o D I OVEDVIEW 004 A AA Ad dia ci D 1 Introduction to 5 end Labeling 0 0 cece eee nen rare D 2 Instriiment Set pisses cessive o Bae cae area Me Bia ae eee eae de ee a Sees A D 4 Synthesis and Purification of 5 end Labeled Primers 0 000 000 eee D 6 Calculating Absorbance for DNA Samples 0 00 c eee eee ee eens D 10 iii E REOTOFONTCES 0 ANDAR A AAA yet a E l F POTENUMDERS 030 A sl ABI PRISM DNA
37. M C 1992 Closing in on a breast cancer gene on chromosome 17q Am J Hum Genet 50 1235 1242 Hearne C M Ghosh S and Todd J A 1992 Microsatellites for linkage analysis of genetic traits Trends in Genetics 8 288 294 Houwen R Baharloo S Blankenshipp K Raeymaekers P Juyn J Sandkuijl L and Freimer N 1994 Genome screening by searching for shared segments mapping a gene for benign recurrent intrahepatic cholestasis Nature Genetics 8 380 386 Huang T H M Hejtmancik J R Edwards A Pettigrew A L Herrera C A Hammond H A Caskey C T Zoghbi H Y and Ledbetter D H 1991 Linkage of the gene for an X linked mental retardation disorder to a hypervariable AGAT repeat motif within the human hypoxanthine phosphoribosyltransferase HPRT locus Xq26 Am J Hum Genet 49 1312 1319 Kimpton C P Gill P Walton A Urquhart A Millican E S and Adams M 1993 Automated DNA profiling employing multiplex amplification of short tandem repeat loci PCR Methods Appl 3 13 22 Kwiatkowski T J Beaudet A L Trask B J and Zoghbi H Y 1991 Linkage mapping and fluorescence in situ hybridization of TCETE1 on human chromosome 6p Analysis of dinucleotide polymorphisms on native gels Genomics 10 921 926 Lawler M Humphries P and McCann S R 1991 Evaluation of mixed chimerism by in vitro amplification of dinucleotide repeat sequences using the polymerase chain reaction Blood 77
38. Na EDTA Distilled deionized HO to 50 mL IMPORTANT Be sure to use disodium EDTA to make 10X TBE stock Some major laboratory suppliers provide monosodium EDTA or tetrasodium EDTA Mix ingredients thoroughly by vortexing Verify that the pH is between 8 2 and 8 3 Note 10X TBE lasts indefinitely at 2 6 C and for 6 months at room temperature If a precipitate forms in the TBE buffer discard it and prepare fresh buffer Deionized IMPORTANT Always use deionized formamide Over time formamide hydrolyzes to formic Formamide acid and formate Deionized formamide stock lasts for 3 months at 15 to 25 C Step Action 1 Mix 50 mL of formamide and 5 g of AG501 X8 ion exchange resin WARNING CHEMICAL HAZARD Formamide is a known teratogen It can cause birth defects Wash thoroughly after handling formamide Obtain a copy of the MSDS from the manufacturer Wear appropriate protective eyewear clothing and gloves Wash thoroughly after handling formamide Stir for 30 minutes at room temperature Check that the pH is greater than 7 0 using pH paper If the pH is not greater than 7 0 decant the formamide into a beaker containing another 5 g of ion exchange resin and repeat 30 minute stirring at room temperature When the pH is greater than 7 0 allow the beads to settle to the bottom of the beaker Remove the supernatant formamide taking care not to disturb the beads Dispense th
39. PRISM 377 DNA Sequencer Fragments were run on a 36 cm 5 Long Ranger Native gel at 30 C Sizing and Size Standards 5 13 GeneScan 1000 Size Standard Useful Range You can use the GeneScan 1000 size standard to determine fragment lengths Special Uses Fragment Lengths 5 14 Preparation between 100 and 900 base pairs Under non denaturing conditions you can use the GeneScan 1000 size standard to determine fragment lengths between 100 and 900 base pairs Under denaturing conditions you can determine fragment lengths between 100 and 539 base pairs the linear range with respect to length in bp versus scan number of the separation The GeneScan 1000 size standard fragments are labeled on both strands and thus are most suited for non denaturing applications If run under denaturing conditions some or all of the peaks will appear split making interpretation difficult Under denaturing conditions all fragments will run 18 nucleotides smaller that the sizes shown in Table 5 5 The following table lists the lengths of the 17 fragments comprising the GeneScan 1000 size standard Table 5 5 GeneScan 1000 Non denatured Fragment Lengths bp 47 93 292 695 51 99 317 946 55 126 439 82 136 557 85 262 692 Note Non denatured fragments are 18 nucleotides longer than denatured fragments The GeneScan 1000 size standard is prepared by digesting pBR322 with the restriction enzyme Alu I followed by ligating a ROX labeled 22 mer
40. accurately for the assay to be successful Estimate DNA yields by comparing the isolated DNA to DNA standards of known molecular weight and concentration A 5 um section of 1 cm paraffin embedded tissue generally yields 100 500 ng of DNA The size of the DNA typically ranges from 200 base pairs bp to one kilobase pair kb or more depending on how the tissue was processed continued on next page 9 8 Microsatellite Analysis Applications PCR Amplification The protocols described here use the Microsatellite RER LOH Assay which is optimized for LOH RER assays and includes appropriate controls This kit is a convenient way to become familiar with these assays even if your loci of interest are not in the kit Preparing PCR Samples This procedure describes how to prepare DNA samples for amplifying specific loci within the human genome Refer to the Microsatellite RER LOH Assay User s Manual for more information Step Action 1 Label ten one for each marker 0 2 mL MicroAmpe tubes for each control sample and ten for each tumor sample Thaw and gently vortex the PCR Mix In an area free of PCR products aliquot 15 uL of the PCR Mix into each MicroAmp tube Dilute the DNA to a final concentration of 10 ng uL using sterile distilled water or TE buffer 10 mM Tris 0 1 mM EDTA pH 8 0 Note If 1X TE buffer is used to dilute genomic DNA the EDTA in the buffer will alter the magnesium concentration in the P
41. amplifications are run to reduce the complexity of the mixture further Amplification so that the fragments can be resolved on a polyacrylamide gel These amplifications Microbial Use primers chosen from the 18 available AFLP Microbial Fingerprinting Kit Selective Fingerprinting Primers nine EcoRI fluorescent dye labeled primers and nine unlabeled Msel primers After PCR amplification with these primers a portion of the samples is analyzed on a Applied Biosystems DNA Sequencer Selective amplification with an EcoRI and an Msel primer amplifies primarily EcoRI Msel ended fragments The EcoRI EcoRI fragments do not amplify well The Msel Msel fragments are not visualized because they do not contain fluorescent dye labels Only the EcoRI containing strands are detected Figure 10 7 A Choose selective AFLP primers Y MMM 0 X AX one of nine different fluorescent dye labeled AFLP EcoRI selective amplification primers 0 X CX one of nine different AFLP Msel selective amplification primers B Run selective amplification Primers l Thermal Core Mix Cycling m Figure 10 7 Selective amplification with fluorescent dye labeled primers continued on next page 10 6 AFLP Mapping Simplifying Complex Figure 10 8 shows examples of AFLP fingerprint patterns that were prepared using Patterns different selective primers Note that the EcoRI selective primers with one nucleotide Presel
42. and assemble the glass plates as described in your instrument user s manual Combine the following 10 0 mL 2X MDE Mutation Detection Gel Solution 4 0 mL 10X TBE see page A 7 2 0 g glycerol WARNING Mutation Detection Gel Solution MDE contains acrylamide Acrylamide is a neurotoxin Avoid skin contact with MDE because acrylamide can be absorbed through the skin Always work in a fume hood Obtain a copy of the MSDS from the manufacturer Wear appropriate protective eyewear clothing and gloves Add sufficient distilled deionized H3O to bring the total volume to 40 0 mL Mix well to obtain a homogenous solution Note This step is critical because glycerol is viscous and it is difficult to obtain a homogeneous solution Use a 0 2 um filter unit to vacuum filter the MDE solution containing glycerol and 1X TBE for 5 minutes With the filtered MDE solution at room temperature add 200 uL of 10 APS Swirl gently to mix Be careful to avoid air bubbles Add 25 uL of TEMED to the solution Swirl gently to mix Be careful to avoid air bubbles Immediately cast the gel as described in your instrument user s manual Allow the gel to polymerize for at least 2 hours before electrophoresis Preparing TBE Buffer and Deionized Formamide 10X TBE To make 50 mL of 10X TBE Step Action 1 To a 50 mL screw cap tube add the following Y 5 4g Tris 2 8 g Boric acid 0 4g
43. both strands Thus peaks tend to be wider with FJdNTPs and can appear to be split Consistent quantitation Every fragment in a peak contributes a single fluorophore to the total signal Thus peak area is directly proportional to the number of molecules A population of fragments labeled with FIANTPs has a variable number of attached fluorophores The average number of attached fluorophores depends upon the fragment s base composition and length and upon the ratio of FJdNTPs to dNTPs added to the reaction mixture Thus it is inadvisable to compare peak areas between fragments labeled with FIANTPs Distinguishing between the forward and reverse strands By attaching a different fluorophore to the forward and reverse primers you can distinguish the peaks corresponding to the forward strand the reverse strand and residual double stranded product Advantages of Using F dNTPs 4 High sensitivity Because most fragments contain multiple fluorophores a given number of FIANTP labeled fragments will produce a greater signal than the same number of 5 end labeled fragments The increased signal strength allows you to use smaller reaction volumes and fewer amplification cycles during PCR Low cost You can add FIANTPs to any PCR reaction You do not need to order or synthesize fluorescently labeled primers before each PCR and you can use FIANTPs with your existing primer sets Experimental Design Considerations 2 5 Y Post P
44. collected by the ABI 373 and In This Chapter ABI PRISM 377 DNA Sequencers to size and quantitate DNA fragments GeneScan analysis of sample files includes establishing a baseline adjusting for spectral overlap of the dyes peak detection and size calling The GeneScan Analysis Software sizes and quantitates DNA fragments automatically allowing faster and more accurate analysis than traditional methods such as radiolabeling Depending on your run conditions you can achieve resolution sufficient to differentiate between fragments that have apparent sizes up to 5000 base pairs When you use the GeneScan system you can label different DNA fragments with up to three different color fluorescent dyes A fourth color is reserved for the GeneScan Internal Lane Size Standard The size standard is used for precise size calling without the problems often encountered using other techniques such as band shift artifacts and run to run variation You can display the results of an experiment as electropherograms as tabular data or as both Electropherograms show fluorescence intensity as a function of fragment size or migration time Each electropherogram represents a single gel lane The tabular data provides precise sizing and quantitative information The data can be exported to downstream applications such as Genotyper software You can also display the results from the ABI 373 and ABI PRISM 377 DNA Sequencers as a reconstructed gel image The g
45. denaturing conditions you can use the GeneScan 2500 size standard to determine fragment lengths between 100 and 5000 base pairs Under denaturing conditions you can determine fragment lengths between 100 and 536 base pairs the linear range with respect to length in bp versus scan number of the separation Special Uses The GeneScan 2500 size standard fragments are labeled on both strands and thus are most suited for non denaturing applications If run under denaturing conditions some or all of the peaks will appear split making interpretation difficult Under denaturing conditions all fragments will run 18 nucleotides smaller than the sizes shown in Table 5 6 Fragment Lengths The following table lists the lengths of the 27 fragments comprising the GeneScan 2500 size standard Table 5 6 GeneScan 2500 Non denatured Fragment Lengths bp 55 240 488 1740 4547 112 251 508 2026 4789 127 256 554 2180 5117 134 287 845 2483 14097 190 304 1133 2499 204 379 1199 2878 Note Non denatured fragments are 18 nucleotides longer than denatured fragments Preparation The GeneScan 2500 size standard is prepared by digesting phage DNA with Pst I followed by ligating a TAMRA or ROX labeled 22 mer oligodeoxynucleotide continued on next page 5 16 Sizing and Size Standards Denaturing Figure 5 9 shows the peak patterns of GeneScan 2500 fragments run under Electropherogram denaturing conditions Non denaturing Electropherogram
46. designated as D6S502 c R can represent either an A or G nucleotide continued on next page Microsatellite Analysis Applications 9 21 AmpF STR Kits The following AmpF STR kits are currently available from Applied Biosystems AmpF STR Blue PCR Amplification Kit P N 402800 AmpF STR Green PCR Amplification Kit P N 402902 Y AmpF STR Profiler PCR Amplification Kit P N 403038 AmpF STR Profiler Plus PCR Amplification Kit P N 4303326 Each AmpF STR kit contains preformulated AmpF STR PCR Reaction Mix blended primer set AmpliTaq Gold DNA Polymerase control DNA of known genotype mineral oil and AmpF STR Allelic Ladders The PCR license rights for forensic testing and research use are also included in the kit The AmpF STR loci contained in the AmpF STR kits are shown in Table 9 5 Table 9 5 Loci in the AmpF STR Kits Locus AmpF STR AmpF STR Blue AmpF STR Green AmpF STR Profiler Profiler Plus D3S1358 X X X vWA X FGA X Amelogenin X X X THO1 TPOX CSF1PO X X X X X Xx lt x X Xx D8S1179 D21S11 D18S51 D5S818 X D13S317 X D7S820 X X X X Xx Xx X Note AmpF STR Profiler and AmpF STR Profiler Plus detection are not supported on the ABI 373 DNA Sequencer or ABI 373 DNA Sequencer with XL Upgrade platforms as the appropriate band pass glass filter for the NED dy
47. eye irritation Wear gloves and safety glasses when handling and always use in a well ventilated area 3 Check that all tubing is tightly connected 4 Turn on power to the external water bath start the circulating pump and add water as needed to maintain the proper level IMPORTANT If the water level drops below the level recommended by the manufacturer the circulation pump shuts off and the required temperature is not maintained 5 Set the external cold water bath temperature about 3 4 C below the desired gel plate temperature and monitor the temperature in the Status screen under the Window menu Place a lid on the bath to minimize evaporation Add water and antifreeze during operation as needed to maintain the manufacturer s recommended water level 8 Monitor the temperature and humidity in the lab see Avoiding Condensation below continued on next page Avoiding Condensation CAUTION Condensation can cause decreased performance and severe damage to the instrument Monitor the room temperature and humidity consult the following condensation tables and maintain the water bath temperature setting above the corresponding dew point Table 7 1 Dew Points 15 C 20 C 25 C 30 C 35 C Approx Approx Approx Approx Approx Relative Dew Relative Dew Relative Dew Relative Dew Relative Dew Humidity Point Humidity Point Humidity Point Humidity Point Humidity Point C C C
48. gapped duplex structures is more important than actual percent G C Effects of Base Order Two primer template complexes with identical G C content will have different melting temperatures because base order influences the overall stability You can determine the exact effect of base order on complex stability using Table 6 1 adapted from Salser 1978 Table 6 1 Base Pairing Energies kcal dinucleotide pair 3 Nucleotide 5 Nucleotide A Cc G T A 1 2 2 1 2 1 1 8 Cc 2 1 4 8 3 0 2 1 G 2 1 4 3 4 8 2 1 T 1 8 2 1 2 1 1 2 Note In the table larger negative values represent more stable interactions Optimizing PCR 6 3 Maximizing Specificity 6 4 Optimizing PCR To illustrate the use of the table consider the two sequences 3 GAC 5 and 3 CGA 5 The sequence 3 GAC 5 contained within a primer would contribute 4 2 kcal to the binding energy 2 1 kcal 3 GA 57 2 1 kcal 3 AC 57 4 2 kcal However if the G and C are next to each other as in 3 CGA 5 the contribution increases to 6 4 kcal 4 3 kcal 3 CG 5 2 1 kcal 3 GA 5 6 4 kcal Note Although a G C dinucleotide at the 3 end of the primer can stabilize the binding complex when using thermostable enzymes such as AmpliTaq DNA Polymerase a 3 G C can also lead to false priming if you do not optimize PCR conditions See Minimizing Binding to Secondary Sites on page 6 4 Effects
49. in a fume hood Obtain a copy of the MSDS from the manufacturer Wear appropriate protective eyewear clothing and gloves Denature the samples for 5 minutes at 95 C Chill the samples by placing directly on ice 4 Run an appropriate volume of these four samples in the first gel lanes of an experimental run See Appendix B for more information about loading volumes Note To ensure the best possible quality matrix load the matrix samples into every other lane and leave at least one empty lane between the matrix samples and the experimental samples continued on next page SSCP Analysis 7 17 Creating the The ABI PRISM 377 run modules are either voltage limiting or wattage limiting To run Run Module ata constant voltage select a voltage limiting module such as GS Run 2140V CHILLER A C or D To run at a constant wattage select a wattage limiting module such as GS Run 60W CHILLER A C or D As there is no dedicated SSCP module currently you will need to edit the module that you choose 7 18 SSCP Analysis IMPORTANT Always run MDE gels at a constant wattage Otherwise a loss of current could result if the gel resistance increases during the run ABI PRISM 377 Collection Software Version 1 1 Step Action 1 Open the Modules folder located in the ABI PRISM folder 2 Duplicate one of the run modules and rename the new module GS SSCP V A or C or D or GS SSCP W A or C or D d
50. intensity than the allele peak itself The larger 98 bp allele peak is of lower intensity than the smaller 90 bp allele In heterozygotes the higher molecular weight allele often produces a fluorescent signal of lower intensity than the lower molecular weight peak suggesting a less efficient amplification of the larger fragment 33 ss 93 23 103 Figure 8 6 Typical pattern for dinucleotide repeat heterozygote Alleles differ by 8 bp Example 3 The GeneScan electropherogram from a dinucleotide repeat marker of a heterozygous individual 86 bp 90 bp is shown in Figure 8 7 on page 8 34 Allele sizes differ by 4 bp When the difference between the allele sizes is 4 bp or less a shift occurs in the height ratio between the two allele peaks compare with Figure 8 6 The fluorescent signal from the 4 bp stutter of the 90 bp allele is added to the signal from the 86 bp allele 73 33 33 93 2400 1600 300 0 Figure 8 7 Typical pattern for dinucleotide repeat heterozygote Alleles differ by 4 bp Example 4 The GeneScan electropherogram from a dinucleotide repeat marker of a heterozygous individual 92 bp 94 bp is shown in Figure 8 8 Allele sizes differ by 2 bp The fluorescent signal from the 2 bp stutter of the 94 bp allele is added to the signal of the 92 bp allele The signal from the 4 bp stutter band of the 94 bp allele is added to the signal from the 2 bp stutter band of the 92 bp allele A dinucleotide
51. mix will last for 1 week at 2 6 C Aliquot 4 5 uL of the mix into each sample tube before adding 1 uL of PCR product Place the samples in a heat block or thermal cycler for 5 minutes at 95 C Remove the tubes from the heat source and place immediately in an ice water bath Load 4 uL of sample into each gel lane SSCP Analysis 7 21 7 22 SSCP Analysis Preparing the Matrix Samples Step Action 1 To each of four sample tubes add the following 3 0 uL deionized formamide 0 5pL 0 1 N NaOH 0 5 uL EDTA blue dextran 50 mM EDTA 50 mg mL blue dextran 1L of one of the matrix standards Dye Primer or Fluorescent Amidite WARNING CHEMICAL HAZARD Formamide is a known teratogen It can cause birth defects Wash thoroughly after handling formamide Obtain a copy of the MSDS from the manufacturer Wear appropriate protective eyewear clothing and gloves Wash thoroughly after handling formamide WARNING CHEMICAL HAZARD Sodium hydroxide NaOH can cause severe burns to the skin eyes and respiratory tract Always work in a fume hood Obtain a copy of the MSDS from the manufacturer Wear appropriate protective eyewear clothing and gloves Denature the samples for 5 minutes at 95 C Chill the samples by placing directly on ice Run 4 uL of each of these four samples in the first gel lanes of an experimental run Note To ensure the best possible quality matri
52. of 6 8 LD description of 6 8 Stoeffel Fragment description of 6 8 AmpliTaq Gold DNA Polymerase 3 A addition 6 18 to 6 20 enzymatic treatment 6 20 modifying Mg 6 19 modifying thermal cycling conditions 6 19 reverse primer tailing 6 19 why incomplete 3 A addition causes problems 6 18 description of 6 8 enzyme concentration 6 7 AmpliWax PCR Gem mediated Hot Start performing 6 14 analyzing data evaluating data quality 3 8 to 3 9 bad data example 3 9 good dataexample 3 8 matrix evaluating quality 3 11 to 3 14 purpose of matrix 3 11 recognizing problems 3 11 to 3 14 solving matrix problems 3 14 when to remake matrix 3 11 nucleic acids quantitating 3 10 sample files analyzing 3 2 size standard peak assignments verifying 3 5 to 3 7 animal paternity 9 17 to 9 18 allele frequency 9 18 DNA based tests advantages 9 17 genotyping 9 18 improved breeding 9 17 StockMarks Kits 9 17 10 9 9 25 6 19 to Index 2 applications AFLP bacterial and fungal genomes analyzed 10 10 for more information 10 4 literature references E 9 microbial fingerprinting 10 1 preselective amplification primer selection selective amplification plant genomes analyzed 10 17 plant mapping preselective amplification primer selection selective amplification simplifying complex patterns 10 7 template preparation and adaptor ligation 10 5 testing new genomes 10 9 whatis AFLP 10 1 animal paternity 9 17 to 9 18 allele frequency 9 1
53. of Primer 2 Structure Strings of Gs and Cs can form internal non Watson Crick basepairs Sarocchi et al 1970 that disrupt stable primer binding Although this anomalous behavior is difficult to predict a good general rule is to avoid runs of more than three consecutive Gs in primers See the following Note for exceptions to this rule Note A short run of G s at or near the 5 end of a primer will not disrupt stable primer binding because 5 positioning does not lead to involvement in disruptive secondary structures for example primer dimer or duplex loops Similarly self complementarity can lead to the formation of hairpin structures that disrupt stable primer binding A stable hairpin can form with just four G C basepairs in the stem and three bases in the loop Summer et al 1985 Effects of Template 2 Structure Primers do not bind effectively to target sequences with known secondary structure For example RNA sequences often have regions of looped secondary structure Maximizing binding specificity requires minimizing primer binding to secondary sites in the DNA and to other primers Minimizing Binding to Secondary Sites Note This section is most applicable if your starting template is genomic DNA The probability of binding to secondary sites is greatly diminished for low complexity templates such as plasmid DNA Ideally the binding of the primer to the desired template is Y Strongest at the 5 end More
54. on a mini gel If after ethidium bromide staining the product signal is visible but not oversaturated try a 1 10 dilution on your ABI PRISM instrument After determining the optimal dilution ratio you can use the same dilutions for subsequent analyses as PCR yields should be fairly consistent Purification If the expected product length is within 50 nucleotides of the primer length remove unincorporated primers before performing electrophoresis lt is often a good idea to remove unincorporated primers anyway unless the PCR is run to completion Optimizing PCR 6 23 SSCP Analysis Overview In This Chapter This chapter provides detailed instructions for performing Single Strand Conformation Polymorphism SSCP mutation analysis on the ABI PRISM 377 and ABI 373 DNA Sequencers SSCP analysis is highly sensitive to electrophoresis conditions The protocol contained in this section is a good starting point for beginning your own optimization trials and includes suggestions for improving performance in your system This chapter contains the following topics Topic See Page Introduction to SSCP Analysis 7 2 Before You Begin 7 3 Running at Subambient Temperatures on the ABI PRISM 377 DNA Sequencer 7 4 PCR Amplification Labeling and Controls for SSCP 7 10 To Save Time Prerun Checklist 7 13 Preparing for a Run ABI PRISM 377 ABI PRISM 377 with XL Upgrade and 7 14 ABI 373 with XL Upgrade Prepar
55. overlap among the dyes the GeneScan Analysis Software color codes the intensity displays from the four light collection regions These appear as the blue green yellow and red peaks in the Raw Data window The blue display represents the total light intensity from the shortest wavelength range monitored and the red display represents the total light intensity from the longest wavelength range monitored It is important to realize that the same four colors are used to color code fluorescence data from all dye virtual filter set combinations Thus the display colors represent the relative not the actual detection wavelengths The process is similar to using a physical filter to separate light of different wavelengths However the ABI PRISM 377 filter sets are called virtual filters because the instruments use no physical filtering hardware to perform the separation 2 ABI 373 and ABI 373 with XL Upgrade The ABI 373 and ABI 373 with XL Upgrade DNA Sequencers use physical filtering hardware on a filter wheel to separate light of different wavelengths The instruments record the light intensity in four regions centered at the following wavelengths Y Filter Set A 531 nm 560 nm 580 nm and 610 nm Y Filter Set B 531 nm 545 nm 560nm and 580 nm continued on next page 1 Each GeneScan run module contains instructions for a specific virtual filter set Once you choose a module the choice of virtual filter set is automatic 2
56. pairs or nucleotides as a function of scan number i e migration time using one of the following operator chosen sizing methods Global sizing methods 2nd Order Least Squares 8rd Order Least Squares Global Southern Local sizing methods Local Southern Cubic Spline Global methods which generate the best fit curve from all matched fragments in the size standard are less affected than local methods by anomalies in the run times of single size standard fragments Local methods which generate the best fit curve from nearby internal lane standard data points are less affected by changes in the electrophoresis conditions or in the analysis range A change in the analysis range will change the subset of size standard fragments that is available for generating the sizing curve IMPORTANT For the Local Southern Method to work you must have at least two size standard fragments larger than your largest unknown fragment 5 2 Sizing and Size Standards Accuracy Versus Precision Comparing Sizes Obtained Within and Across Instrument Platforms For detailed information on the different sizing methods refer to the GeneScan Analysis Software User s Manual Converting Fragment Migration Times to Sizes This step is a straightforward mapping of any given fragment s migration time onto the sizing curve Accuracy in size calling is a measure of the instrument s ability to generate fragment sizes that are close t
57. peaks from 98 00 to 113 00 bp in blue Al 00 Highest peak at 100 82 0 50 bp in blue A2 X Highest peak at 102 80 0 50 bp in blue AS X Highest peak at 104 75 0 50 bp in blue A4 00 Highest peak at 108 61 0 50 bp in blue AS X gt Highest peak at 110 60 0 50 bp in blue Figure 8 4 Example of allelic bin names generated from the Make from Labels dialog box configured as shown in Figure 8 3 You can use the Add Multiple Categories feature to create a defined set of category members allele bins that are equally spaced with a fixed tolerance Once Genotyper software create the categories you can Label and filter peaks Use the Histogram window to fine tune the category definitions To define a set of equally spaced allelic bins with a fixed tolerance Step Action 1 Set up the main categories groups for your markers as follows All peaks from 98 00 to 113 00 bp in blue Unknown All peaks from 235 00 to 261 00 bp in blue e 025391 Unknown All peaks from 139 00 to 153 00 bp in green e 0139171 Unknown All peaks from 171 00 to 197 00 bp in green Microsatellite Analysis 8 29 8 30 Microsatellite Analysis To define a set of equally spaced allelic bins with a fixed tolerance continued Step Action 2 From the Category menu choose Add Multiple Categories Choose the appropriate settings for the first marker as follows Add Multiple Categories Sta
58. positive than 9 8 kcal mole at the 3 end This is equivalent to saying that the binding at the 3 end is weaker than 9 8 kcal mole Polymerases only require the binding of the nucleotides at the 3 end to begin elongation If the 3 nucleotides bind strongly perhaps because of a 3 G C any template sequences that are complementary to the 3 end are amplified In this case because the entire primer is not used to discriminate among target sequences specificity is lost Conversely if binding is strongest at the 5 end the typical binding event on the template DNA begins at the 5 end Polymerases however cannot begin elongation until the 3 end binds Therefore the entire primer is used to distinguish among target sequences Post Amplification Manipulation Self complementarity can lead to the formation of hairpin structures that decrease binding specificity as well as disrupt binding stability as discussed earlier Nucleotides in the hairpin structure are not available for recognition of the target sequence The available nucleotides can be thought of as forming a smaller and therefore less specific primer When performing a computer assisted search to evaluate binding to secondary sites in the target DNA consider the potential for gapped duplex formation 1 Note Binding to secondary sites can also involve the formation of stable non Watson Crick base pairs Topal and Fresco 1976 Stable base pairing
59. protective eyewear clothing and gloves Note For convenience you can combine the formamide NaOH GeneScan size standard and EDTA blue dextran into a master mix 300 pL deionized formamide 50 uL 0 1 N NaOH 50 uL GeneScan size standard 50 yL EDTA blue dextran This mix will last for 1 week at 2 6 C Aliquot 4 5 uL of the mix into each sample tube before adding 1 uL of PCR product Place the samples in a heat block or thermal cycler for 5 minutes at 95 C Remove the tubes from the heat source and place immediately in an ice water bath Load an appropriate volume of sample into each lane depending upon the instrument and type of comb used See Appendix B for more information about loading volumes Preparing the Matrix Samples Step Action 1 To each of four sample tubes add the following 3 0 uL deionized formamide 0 5 uL0 1 N NaOH 0 5 uL EDTA blue dextran 50 mM EDTA 50 mg mL blue dextran 4 1 uL of one of the matrix standards Dye Primer or Fluorescent Amidite see page 7 24 WARNING CHEMICAL HAZARD Formamide is a known teratogen It can cause birth defects Wash thoroughly after handling formamide Obtain a copy of the MSDS from the manufacturer Wear appropriate protective eyewear clothing and gloves Wash thoroughly after handling formamide WARNING CHEMICAL HAZARD Sodium hydroxide NaOH can cause severe burns to the skin eyes and respiratory tract Always work
60. work contaminated surface using a cloth soaked with 50 bleach IMPORTANT Before cleaning the sample block of a thermal cycler refer to the instrument manual for the proper procedure Close sample tubes when not using them Always run a no DNA negative control A negative control contains no template DNA only primers and the DNA diluent usually water or buffer Aliquot reaction reagents so as to minimize the number of times you use a particular stock solution Definition PCR product carryover is the contamination of an unamplified sample with previously amplified DNA Why Carryover Is a Particular Concern PCR product carryover is a particular concern because amplified PCR product serves as an ideal template for subsequent amplifications of that same target A single PCR amplification produces a large number of copies as many as 1013 The inadvertent transfer of even a minute volume or aerosol of amplified product can mean significant contamination This can result in false positives and the detection and amplification of the contaminating sequence at the expense of the target sequence Precautionary Measures Adopting these precautionary measures can help you minimize the likelihood of PCR product carryover Use positive displacement pipettes or filter plugged pipette tips Y Physically separate reactions prior to and following amplification Handle pre and post PCR solutions with separate sets of the followin
61. 0 1 on page 10 2 The first 24 lanes show six samples each of four different Escherichia coli strains each of the six samples represents a different growth phase of the organism The final 11 AFLP Mapping 10 1 10 2 AFLP Mapping lanes show different growth phases of a single strain of Legionella pneumophila Note that the E coli fingerprints are similar to each other and different from the Legionella fingerprint Within a strain all of the bands are reproducible Large population studies provide data for the linkage of a band with a given phenotype such as pathogenicity E coli strains Legionella strain Figure 10 1 AFLP fingerprints of four E coli strains and one Legionella strain Applications for AFLP in plant mapping include establishing linkage groups in crosses Y saturating regions of introgression with markers for gene landing efforts assessing the degree of relatedness or variability among cultivars Examples of AFLP plant mapping are shown in Figure 10 2 and Figure 10 3 on page 10 3 and Figure 10 4 on page 10 4 Figure 10 2 AFLP plant mapping gel Note that the pattern is much more complicated for plants than for bacteria You can build a genetic map of markers showing Mendelian inheritance from AFLP data such as that shown in Figure 10 3 The four electropherogram panels in Figure 10 3 contain data from tomato DNA samples prepared using the AFLP technique Samples were run on an ABI 373 DNA Sequencer a
62. 0 92n7 where nx number of residues of base x in the oligonucleotide Two useful facts follow from this One Azs unit of single stranded DNA contains 33 pg mL One Azs unit of double stranded DNA contains 50 pg mL Preparing 5 End Labeled Primers D 11 References Optimizing PCR Post PCR Labeling with F JdNTPs 5 End Labeling Ginot F Bordelais l Nguyen S and Gyapay G 1996 Correction of some genotyping errors in automated fluorescent microsatellite analysis by enzymatic removal of one base overhangs Nucleic Acids Res 21 540 541 Hunter W N Brown T and Kennard O 1986 Structural features and hydration of d C G C G A A T T A G C G a double helix containing two G A mispairs J Biomolec Struct Dynam 4 173 191 Summers M F Byrd R A Gallo K A Samsom C J Zon G and Egan W 1985 Nuclear magnetic resonance and circular dichroism studies of a duplex single stranded hairpin loop equilibrium for the oligodeoxyribonucleotide sequence d CGCGATTCGCG Nucleic Acids Res 13 6375 6386 Topal M D and Fresco J R 1976 Complementary base pairing and the origin of substitution mutations Nature 263 285 289 Walsh P S Fildes N J and Reynolds R 1996 Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vWA Nucleic Acids Res 24 2807 2812 Inazuka M Tahira T and Hayashi K 1996 One tube post PCR fluorescent labeling of DNA
63. 000 RFU will ensure that data remains within this range even if there is slight sample to sample and run to run variation Preliminary Data Analysis Follow the protocols for creating a matrix setting Analysis Parameters and analyzing sample files provided in Chapter 8 Microsatellite Analysis For detailed instructions on using Genotyper software to assess LOH see Using Analyze and Calculate in Table Commands An LOH Example on page 8 26 of the Genotyper User s Manual or Chapter 6 of the Microsatellite RER LOH Assay User s Manual Average Across Independent Injections Once GeneScan software determines the peak height and area for all alleles of all relevant microsatellite loci use Genotyper software to pool the data from all the independent injections of each N or T sample to obtain an average peak height and area for every allele in every sample IMPORTANT Do not combine N data with T data to obtain this average IMPORTANT In almost any microdissection there are contaminating normal cells so normal DNA is present Your results will depend upon the amount of contaminating normal DNA in your sample Calculate the LOH Value LOH can be defined mathematically as follows height of normal allele two height of normal allele one height of tumor allele two height of tumor allele one LOH Eq 1 An LOH value lt 0 5 indicates that the tumor sample shows significant loss of the longer allele whereas an LO
64. 10 1 Guide to Choosing Selective Primers for Microbial Fingerprinting Nucleotide Application Addition EcoRI Primers Msel Primers Cosmids BACs P1 0 0 EcoRI 0 FAM Msel 0 mapping YACs some larger 0 1 EcoRI 0 FAM Msel A BACs Msel C Msel G Msel T 1 0 EcoRI A FAM Msel 0 EcoRI C NED EcoRI G JOE EcoRI T JOE Bacteria 0 2 EcoRI 0 FAM Msel CA Msel CC Msel CG Msel CT 1 1 EcoRI A FAM Msel A EcoRI C NED Msel C EcoRI G JOE Msel G EcoRI T JOE Msel T 2 0 EcoRI AA JOE Msel 0 EcoRI AC FAM EcoRI AG JOE EcoRI AT NED Yeast small fungi 2 2 EcoRI AA JOE Msel CA genomes EcoRI AC FAM Msel CC EcoRI AG JOE Msel CG EcoRI AT NED Msel CT Large fungi genomes 2 3 Use the primers from the AFLP Regular and Small 3 2 Plant Genome Mapping Kits See Table 10 3 on page 10 11 for the primers available continued on next page AFLP Mapping 10 9 Genome Analysis Some bacterial and fungal genomes that have been analyzed successfully using Guide EcoRI Msel and the primers in the AFLP Microbial Fingerprinting Kit are shown in Table 10 2 Table 10 2 Genomes analyzed with EcoRI and Msel Primer Pairs Primer Pairs Used Organism Successfully Primer Pairs to Avoid gt Acinetobacter sp Aeromonas sp Aspergillus sp Bacillus sp Candida utilis Clostridium sp Vancomycin resistant Enterobacter Escherichia coli Eutypa sp Legionella pneumophila Nensenula anomola Paenibacillus larvae Pichia membrefacie
65. 114 and Filter Set A To set the Analysis Parameters Step Action 1 Choose Analysis Parameters from the Analysis menu 2 Set the parameters as shown below Analysis Parameters Smooth Options Minimum Peak Heights Per Dye None Blue Dye yellow Dye so Light Channel Averaging Size Range __ Channels B Allow all Sizes Min Peak Half Width Minimur size Half Width Is 3 Mania Size xeaae Split Peak Correction Size Calling Method None 2nd Order Least Squares O GENESCAN 2500 3rd Order Least Squares O LeftMost Peak O Cubic Spline Interpolation O RightMost Peak Local Southern Method Carr Limir Seans Global Southern Method Save Settings 0k 3 Click the Save Settings button Click OK when done Quit the GeneScan 672 Fragment Analysis Software continued on next page Plate Check Refer to the 373 DNA Sequencing System User s Manual for instructions on loading and Pre run plates verifying instrument settings and performing the plate check No pre run is required for SSCP analysis Step Action 1 On the ABI 373 keyboard press CALIBRATION 2 Press CONFIGURE 3 Configure the instrument settings as follows SCANNER WILL NOT STOP ON DATACOM ERRORS LASER POWER IS SET TO 40 MILLIWATTS Y FILTER SEQUENCE 531 560 580 610 set A Set the PMT voltage such that the blue l
66. 13 to 9 15 materials required 8 3 PCR amplification labeling and controls 8 5 to 8 6 preparing for a run 8 8 to 8 11 creating a new matrix file 8 11 gel preparation 8 8 instrument setup 8 8 to 8 9 preparing and loading samples 8 10 prerun checklist 8 7 troubleshooting 8 33 to 8 36 common problems 8 33 LOH and RER screening 9 16 stutter aiding in allele calling 8 36 stutter example 8 33 to 8 35 multiplexing PCR increasing throughput 2 2 to 2 3 optimizing 6 10 to 6 11 PCR howto 2 3 N NHS esters dye chemical form 4 3 Note user attention word 1 2 O off scale data how effects size calling 5 6 oligonucleotide sequence analysis of D 7 Custom Oligonucleotide Synthesis 2 7 deprotection D 6 purification of D 7 typical synthesis yields D 8 P part numbers F 1 to F 7 PCR and microsatellite marker analysis 8 5 to 8 6 and single strand conformation polymorphism SSCP analysis 7 10 to 7 12 co electrophoresis guidelines 2 2 multiplexing howto 2 3 optimizing 3 A addition 6 18 to 6 20 contamination avoiding 6 16 to 6 17 designing custom primers 6 3 to 6 5 enzyme choosing the right enzyme 6 8 to 6 9 Hot Start technique 6 13 to 6 14 literature references E 1 multiplexing PCR 6 10 to 6 11 reaction volumes and tube types choosing 6 2 reagent concentrations determining 6 6 to 6 7 RNA templates using 6 12 stutter products 6 21 to 6 22 temperature control parameters modifying 6 15 products guidelines for w
67. 2 Thermal Cycling Parameters for DNA Samples Isolated from Paraffin embedded Tissue Step AmpliTaq PCR Final Preserve Gold extension Sample Activation Hold 45 Cycles Hold Hold Denature Anneal Extend Time 10 10 30 3 30 Forever minutes seconds seconds minutes minutes Temperature 95 C 96 C 55 C 70 C 70 C 4 C Pooling the Markers For specific information on pooling markers refer to Chapter 2 of the ABI PRISM Linkage Mapping Set Version 2 User s Manual P N 904999 Why Pool the Markers It is necessary to pool markers in different volumes because 4 Primers amplifying markers are labeled with fluorescent dyes that vary in intensity 4 The yields of the PCR products from the few markers are different Y DNA varies in amplification efficiency especially DNA extracted from paraffin embedded tissue Optimizing Pooling Ratios Occasionally one marker may amplify either more or less than expected If the fluorescent intensity is too high or too low Load and run that one marker sample individually or Adjust the pooling ratios Note Loading the gel with a volume of sample that gives a fluorescent signal of approximately 500 1000 relative fluorescent units RFU will produce the most consistently interpretable data Although the dynamic range of the DNA sequencing instruments is 9 10 Microsatellite Analysis Applications Analyzing LOH Data 50 6000 RFU a target of approximately 1
68. 4 3 5 Flush to 5 10 10 6 Interrupt 104 0 7 Flush to 5 10 5 8 Phos Prep 101 15 9 5 to Waste 54 1 10 18 to Waste 64 7 11 Block Flush 1 5 12 End 107 0 continued on next page D 4 Preparing 5 End Labeled Primers Creating a Begin Reduce to 1 second the delivery to waste from the dye phosphoramidite bottle position The following procedure is for the ABI 392 instrument with the dye phosphoramidite at bottle position 5 Other instruments or bottle positions are similarly Procedure programmed To create a Begin procedure 392 Begin Procedure Step Function Number Time 1 Begin 106 0 2 Phos Prep 101 15 3 A to Waste 50 2 4 G to Waste 51 2 5 C to Waste 52 2 6 T to Waste 53 2 7 5 to Waste 54 1 8 Tet to Waste 58 2 9 18 to Waste 64 10 10 Block Flush 1 10 11 End 107 0 Preparing 5 End Labeled Primers D 5 Synthesis and Purification of 5 end Labeled Primers Synthesizing the Primer Step Action 1 Enter the oligonucleotide sequence with the dye phosphoramidite denoted by its bottle position at the 5 end 2 Create a user defined cycle with an additional 120 second wait after the coupling wait Note You can use dye phosphoramidites with standard cycles and coupling times however an additional 120 second coupling wait significantly improves coupling efficiency 3 Using the base specifier field set the dye phosphoramidite bottle position to Yes for the 120 second wait step All other bottle pos
69. 4 Enters the allele member set name to the rounded size in bp Y Enters the Member of group name i e the marker name Y Selects the Highest peak button 4 Enters the allele size range from x to y bp Y Selects the dye color Y Selects the Exclusive checkbox Add Category Nome ESMAS Qu gt J Member of group D12 83 Comment All peaks Highest peak O Highest 2 O Left peak O Right peak Size 104 40 tov 105 00 with dye color s X blue green yelow red O with scaled height of at least 1 O with scaled height of at most 9999 E Exclusive clears previous labels at same peak Cx Note Check the information entered automatically for accuracy Note Genotyper software will not allow the addition of a new group if a group or category with the same name already exists 9 Click OK to create a category member allele bin such as the one shown here e 012583 Unknown All peaks from 98 00 to 113 00 bp in blue e 105 OO Highest peak from 104 40 to 105 00 bp in blue e 075517 e 10 Optional The bin shown in Step 9 was created with the size as a range You can also create a bin centered around the median size of the range with a set tolerance for example 104 68 0 5 bp as follows a Hold down the Shift key while choosing Add Category from the Category menu b Edit the bin tolerance as desired The size is displayed in the dialog box as shown here Size 104 68 0 50 Note The category member gene
70. 6 9 tube types choosing 6 2 when using small amounts of template 6 2 U UlTma DNA Polymerase description of 6 9 updates using Internet to access 1 2 user attention words 1 2 V Virtual Filter Set A representative emission spectra 4 10 Virtual Filter Set C making matrix files for 3 11 matrix problems resulting from using 4 11 Virtual Filter Set D representative emission spectra 4 11 Virtual Filter Set F representative emission spectra 4 12 virtual filter set available sets 4 7 Ww Warning user attention word 1 2 wavelengths table of emission and excitation 4 5 Index 7 Worldwide Sales Offices Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches into 150 countries on six continents For international office locations please call our local office or refer to our web site at www appliedbiosystems com Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free 1 800 345 5224 Fax 1 650 638 5884 Technical Support For technical support Toll Free 1 800 831 6844 www appliedbiosystems com techsupport www appliedbiosystems com qv AR Applied D Biosystems PE Corporation is committed to providing the world s leading technology and information for life scientists PE Corporation consists of the Applied Biosystems and Celera Genomics businesses Printed in the USA
71. 8 DNA based tests advantages 9 17 genotyping 9 18 improved breeding 9 17 StockMarks Kits 9 17 human identification 9 19 to 9 25 advantages of STRs 9 19 AmpFISTR kits 9 22 AmpFISTR loci table of 9 21 automated sizing and genotyping 9 20 for more information 9 25 high throughput about 9 20 using allelic ladders 9 23 to 9 24 using STRs 9 19 Linkage Mapping Set 9 2 to 9 4 loss of heterozygosity screening for 9 5 to 9 12 advantages 9 5 analyzing LOH data 9 11 to 9 12 example 9 12 limitations 9 5 what is LOH 9 5 replication error screening for 9 13 to 9 15 advantages 9 13 examples 9 14 limitations 9 13 10 5 10 9 10 6 10 12 to 10 2 10 7 10 11 10 8 whatis RER 9 13 single strand conformation polymorphism SSCP to 7 29 about SSCP 7 2 analyzing the data 7 24 to 7 25 creating matrix file 7 24 setting analysis parameters 7 25 materials required 7 3 optimizing run conditions 7 26 to 7 28 PCR amplification labeling and controls 7 10 to 7 12 preparing for a run 7 14 to 7 18 creating the run module 7 18 gel preparation 7 14 instrument setup 7 14 preparing and loading samples 7 16 preparing for a run using the ABI 373 7 19 to 7 23 gel preparation 7 19 instrument setup 7 19 plate check and pre run 7 21 preparing and loading samples 7 21 to 7 22 setting preprocess and analysis parameters 7 19 starting the run 7 23 prerun checklist 7 13 running at subambient temperatures 7 4 to 7 9 troubleshoot
72. 8 Characteristic appearance of an elevated baseline caused by a bad or incorrect matrix In this example the green baseline is elevated in the region between two large black peaks representing the yellow dye signal because too much green signal is subtracted from the yellow signal see Figure 3 9 on page 3 14 The GeneScan software uses these low data points to calculate the baseline for the green signal Therefore the original baseline is elevated Note If the baseline is sufficiently elevated random fluctuations in the baseline can lie above the Peak Amplitude Threshold and might be falsely interpreted as product peaks If you suspect that an elevated interpeak baseline is caused by matrix problems inspect the data before baselining This can be done by reanalyzing the data with baselining deselected in the Analysis Parameters dialog box As shown in Figure 3 9 low data points are apparent as troughs in one color beneath peaks in another General Analysis and Evaluation Techniques 3 13 O E BEERA Peak PAANAN Figure 3 9 The data of Figure 3 8 before baselining What To Do If You If problems related to bleedthrough peaks or to an elevated interpeak baseline appear Have Matrix with any regularity you should remake the matrix Apply the new matrix to the old Problems sample data and reanalyze the data For directions on how to create a matrix file see Appendix C 3 14 General Analysis and Eval
73. 83 e Unknown All peaks from 98 00 to 113 00 bp in blue 075517 Unknown All peaks from 235 00 to 261 00 bp in blue D25391 Unknown All peaks from 139 00 to 153 00 bp in green 0138171 Unknown All peaks from 171 00 to 197 00 bp in green 018220 Unknown All peaks from 226 00 to 260 00 bp in yellow 03S 1266 Unknown All peaks from 281 00 to 303 00 bp in yellow 5 If the first category Then is currently being defined proceed directly to step 6 is defined already open the Analysis menu and select Clear All Labels Select the appropriate dye lanes by clicking on the appropriate color button From the Analysis menu choose Label Peaks Label peaks with Size in bp only From the Analysis menu choose Filter Labels Filter labels using the default settings best for dinucleotide repeat markers To bin alleles using the Make from Labels feature continued Step Action 9 From the Category menu choose Make from Labels to display the Make Categories from Labels dialog box Set the parameters as follows a Select Unmark overlapping categories and deselect Skip overlapping categories To enable you to correct for overlaps Genotyper software automatically unmarks two or more category members that overlap in size based on the tolerance b Inthe Name box Either leave the Prefix field blank Figure 8 1 on page 8 28 or enter a name for the allele in the Prefix field Figure 8 3 on page
74. C or D modules with fluorescent amidites see Table 4 3 on page 4 8 and Creating a Matrix File on page 7 24 6 Close the Run Window and open a new one to be able to select the modified module from the Run Module pop up menu Preparing for a Run ABI 373 Gel Preparation Try both non denaturing 6 5 polyacrylamide gels and 0 5X MDE Mutation Instrument Setup Setting the Preprocess and Analysis Parameters Detection gels to see which gel type gives the highest mutation detection rate in your system See Preparing Polyacrylamide Gels for SSCP on page A 4 and MDE Gels for SSCP on page A 6 for detailed instructions Note In place of polyacrylamide you can substitute Long Ranger gel solution FMC Corporation P N 50610 for standard polyacrylamide stock See Preparing a Long Ranger Gel for SSCP on page A 3 for detailed instructions Use 24 cm well to read plates standard 0 4 mm spacers and a square tooth comb Refer to the 373 DNA Sequencing System User s Manual for the general procedure The specific equipment and reagents needed for an SSCP analysis run are listed in Before You Begin on page 7 3 Note If your Macintosh computer is connected to a network Applied Biosystems recommends that you disconnect it from the network during the gel run To do so select Chooser under the Apple menu In the bottom right area of the Chooser window click the AppleTalk Inactive button then close the
75. CR Mix and potentially influence the amplification results Combine the following to prepare 60 uL of 12X Master Mix TE buffer 27 6 uL AmpliTaq Gold DNA Polymerase 5 U uL 2 4 uL Normal or tumor genomic DNA 10 ng uL 30 uL Aliquot 5 uL of the Master Mix into each MicroAmp tube containing the PCR Mix Close the MicroAmp tubes Place the MicroAmp tray with samples into a centrifuge with a 96 well adapter Spin the tubes for 20 seconds at 150 x g Place the MicroAmp tray with samples into the thermal cycler Program the GeneAmp PCR System 9600 as described in Thermal Cycling below Note Upon completion of thermal cycling you may store the samples at 2 6 C Microsatellite Analysis Applications 9 9 Thermal Cycling The thermal cycling conditions in Table 9 1 and Table 9 2 are optimized for the GeneAmp 9600 PCR System Other thermal cyclers may require reoptimization of cycling conditions IMPORTANT DNA from paraffin embedded tissue requires more amplification cycles than DNA from fresh or frozen tissue Table 9 1 Thermal Cycling Parameters for Samples Isolated from Fresh or Frozen Tissue Step AmpliTaq PCR Final Preserve Gold Extension Sample Activation Hold 30 Cycles Hold Hold Denature Anneal Extend 10 10 30 3 30 Forever Time minutes seconds seconds minutes minutes Temperature 95 C 96 C 55 C 70 C 70 C 4 C Table 9
76. CR end labeling Post PCR end labeling with FJdNTPs using Klenow is an alternative to labeling during PCR See Iwahana et al 1995 and Inazuka et al 1996 for details You can also label with F JdNTPs using traditional techniques such as random priming or nick translation Figure 2 1 and Figure 2 2 compare the results obtained using 5 end labeled primers and F JdNTPs 5 end labeled primers give better resolution but F JdNTPs give better sensitivity Note also the unincorporated fluorescently labeled nucleotides in the FIdNTP labeled sample bottom panel of Figure 2 1 E 30 60 30 120 150 180 210 240 270 300 330 360 390 420 450 480 510 lt 3600 3200 2800 2400 2000 1600 1200 800 400 o Veni reta fte t ati TV TE TT i A A Pa 1B DYE PRIMER 276 SAMPLE 341 7200 6400 5600 4800 4000 3200 2400 1600 800 o 2B 2 2 5PM EACH 276 Figure 2 1 Results from 5 end labeling top and F ANTPs bottom N 210 240 3 3600 3200 2800 2400 2000 1600 1200 800 _ 400 _ 0 1B DYE PRIMER 276 SAMPLE 341 2B 2 4 2 5PM EACH 276 7 Figure 2 2 Expanded view of the electropherogram from Figure 2 1 showing the differences in resolution and peak height between the two labeling methods continued on next page 2 6 Experimental Design Considerations For More You can obtain custom 5 end labeled primers from the Applied Bi
77. Cc hannels B Min Peak Half Width Half Width Is 3 Size Range Allow all Sizes Split Peak Correction None GENESCAN 2500 O LeftMost Peak RightMost Peak Carr Limit seans Size Calling Method 2nd Order Least Squares 3rd Order Least Squares Cubic Spline Interpolation Local Southern Method Global Southern Method Save Settings ok IMPORTANT The settings listed here are intended as suggestions to get you started with microsatellite analysis Alter them as necessary for your system However if you choose to use different settings be sure to define the Analysis Range to exclude the primer peak 3 Click the Save Settings button Click OK when done 5 Quit the GeneScan Analysis Software Refer to the 373 DNA Sequencing System User s Manual for instructions on loading plates verifying instrument settings and performing the plate check Refer to the following table for run parameters 12cm 24 cm 34 cm Timer 4 hr a 8 hr a 12 hr a Power W 15 30 30 a The listed run times will enable you to see the 350 bp peak of the size standard Adjust the run time accordingly if the expected product is significantly longer or shorter than 350 base pairs in length Prerun the instrument for 10 minutes to 1 hour continued on next page Preparing and Preparing and Loading Experimental Samples Loadin
78. D 1 Dye phosphoramidites use 6 carboxyfluorescein and its chlorinated analogs Preparing 5 End Labeled Primers D 3 Instrument Setup Installing Dye on To install dye labeled phosphoramidites on the DNA RNA synthesizer Synthesizer Step Action 1 Open the dye labeled phosphoramidites and desiccant at room temperature 2 Dilute with dry acetonitrile lt 50 ppm water by the manual method with a dry syringe For 0 2 umol 1 umol and 10 umol scale syntheses add 1 mL of dry acetonitrile to prepare a 0 1 M solution For 40 nmol scale syntheses add 2 mL to prepare a 0 05 M solution Note Do not perform autodilution on the ABI 392 and ABI 394 instruments Significant acetonitrile is lost during argon bubbling Place the diluted dye phosphoramidite at any monomer position on your DNA RNA synthesizer typically position 5 or greater Refer to the Functions Cycles and Procedures section in your synthesizer user s manual for more detailed directions Creating a Bottle This program reduces the time for the delivery to waste from the dye phosphoramidite Change Procedure bottle to one second The program is for the ABI 392 instrument with the dye phosphoramidite at bottle position 5 Program other instruments or bottle positions similarly To create a Bottle Change procedure 392 Bottle Change Procedure Step Function Number Time 1 Begin 106 0 2 Block Flush 1 5 3 18 to Waste 64 7 4 18to5 7
79. DNA and decreasing the signal strength Adjusting the Signal Intensity For more information on adjusting the signal intensity see Determining Loading Concentrations for Samples on page 2 8 and Optimizing Electrophoresis Conditions on page 2 9 2 4 Experimental Design Considerations Choosing Fluorescent Labeling Methods Recommended Most GeneScan applications analyze PCR products You can use either of two Methods fluorescent labeling methods during PCR amplification Comparing Labeling Methods 4 4 Incorporating a 5 end labeled primer during the primer annealing step Incorporating fluorescent dye labeled dUTPs or dCTPs FIANTPs during the primer extension step IMPORTANT With any labeling technique you should use only ABI PRISM dyes Other dyes or mixed isomers of dyes have variable spectral shifts that will interfere with making a multicomponent matrix The matrix compensates for the spectral overlap between the dyes Advantages of Using 5 end Labeled Primers 4 High precision All fluorophores affect DNA mobility to some degree Different fluorophores have different mobilities Because fragments labeled with 5 end labeled primers have a single fluorophore at the 5 end the mobility of all detectable fragments is comparably affected Therefore fragment peaks tend to be narrow By contrast fragments labeled with F JdNTPs have a variable number of fluorophores attached in variable positions on
80. GeneScan Reference Guide Chemistry Reference for the ABI PRISM 377 and ABI 373 DNA Sequencers Applied BS Biosystems Copyright 2000 Applied Biosystems For Research Use Only Not for use in diagnostic procedures ABI PRISM GeneScan Genotyper MicroAmp OPC StockMarks and StockMarks for Cattle are registered trademarks of PE Corporation or its subsidiaries in the U S and certain other countries ABI AmpF STR AmpF STR Blue AmpF STR Green Applied Biosystems BigDye MicroGel POP POP 4 Primer Express Profiler Profiler Plus and True Allele are trademarks of PE Corporation or its subsidiaries in the U S and certain other countries AmpErase AmpliTaq AmpliType AmpliWax GeneAmp QuantiBlot TaqMan and UlTma are registered trademarks of Roche Molecular Systems Inc AmpliTaq Gold is a trademark of Roche Molecular Systems Inc Apple AppleTalk Macintosh and Power Macintosh are registered trademarks of Apple Computer Inc AFLP is a trademark of Keygene N V P N 4303188B Contents Introduction we bo A eee BEE eR Reese Del Using This Mandala orcas Gaeta 8 a Wa OR Sees RR a Re Re ee da 1 1 Technical Supports siiig ei a sacks ek Pec es at hb ae aoa ae ode saat nade es 1 3 Experimental Design Considerati0ns oooooooo o 2 1 Using GeneScan Analysis Software to Analyze DNA Fragments ooo oooooooooo o 2 1 Working with Multiple Colors tirer nai aana ne ia i a a aea ene nee 2 2 Choo
81. GeneScan Analysis software version 2 0 2 or higher Genotyper software version 2 0 Microsatellite Analysis 8 3 8 4 Microsatellite Analysis For the ABI 373 You will need the following software to perform and analyze a microsatellite analysis run GeneScan 672 software version 1 1 or higher Note If you use the GeneScan 672 Fragment Analysis Software on the ABI 373 instrument and want to analyze the data using GeneScan Analysis Software version 2 0 2 or higher you must transfer your data to a Power Macintosh computer and perform additional steps Refer to the ABI Prism GeneScan Analysis 2 1 User s Manual P N 904435 for details Genotyper software version 2 0 PCR Amplification Labeling and Controls PCR Amplification Reactant Concentrations and Volumes The following table provides guidelines for beginning multiplex PCR in your system In many systems the 7 5 uL reaction volume produces sufficient product without extensive optimization Table 8 1 Preparing PCR Reaction Mixtures Volume pL Volume pL Reaction Component 15 pL rxn 7 5 pL rxn DNA 1 20 1 20 50 ng uL stock 25 ng uL stock PCR Primer Mix 5 uM each primer 1 00 0 50 10X GeneAmp PCR Buffer II 1 50 0 75 GeneAmp dNTP Mix 250 uM each dNTP 1 50 0 75 AmpliTaq Gold DNA Polymerase 0 12 0 06 5 U uL MgCl 25 mM 1 50 0 75 Distilled deionized HO 8 18 3 49 a Applied Biosystems recommends adding the s
82. Glass Plate non Stretch configuration 401624 6 cm Plain Glass Plate non Stretch configuration F 6 Part Numbers 603750 Small Upper Buffer Assembly 503751 Large Upper Buffer Assembly 603410 Lower Buffer Assembly 200576 Foam Cord for Upper Buffer Chamber Seal 201410 Gasket Adhesive use with P N 200576 User s Manuals 903433 ABI PRISM 377 DNA Sequencer User s Manual 902376 373 DNA Sequencing System User s Manual 904435 GeneScan Analysis Software User s Manual 902842 GeneScan 672 Software User s Manual Updates Part numbers are subject to change Consult the Applied Biosystems World Wide Web Site www appliedbiosystems com techsupport for updated information Part Numbers F 7 Index Symbols FIANTPs advantages of using 2 5 dye chemical form 4 3 literature references E 1 Numerics 3 A addition 6 18 to 6 20 enzymatic treatment modifying Mg 6 19 modifying thermal cycling conditions 6 19 reverse primer tailing 6 19 why incomplete 3 A addition causes problems 6 18 5 end labeled primers advantages of using 2 5 determining relative quantities 3 10 literature references E 1 preparing abbreviations and definitions D 1 calculating absorbance for DNA samples D 10to D 11 instrument setup D 4 to D 5 creating a Begin procedure D 5 creating a bottle change procedure D 4 installing dye on the synthesizer D 4 introduction to 5 e
83. H value gt 1 5 indicates that the tumor sample shows significant loss of the shorter allele Note If a particular locus in an N sample is homozygous you cannot use that locus to diagnose LOH for the corresponding N T pair Peak Height vs Peak Area Although a number of examples in the literature e g Canzian et al 1996 Cawkwell et al 1993 calculate LOH using peak area we find peak height to be a more reliable metric continued on next page Microsatellite Analysis Applications 9 11 Sample Calculation Here is an example of a LOH calculation for the TP53 Penta marker which is located near the p53 gene Using Equation 1 1343 1723 _ 0 2073 0 162 LOR 2315 1 283 i 480 An LOH value of 0 16 clearly indicates loss of heterozygosity in the tumor sample In Figure 9 1 the first electropherogram corresponds to the normal tissue sample and the second electropherogram to the tumor tissue sample Peak heights are as shown in Figure 9 1 36 141 146 151 156 161 166 171 176 iaio 1723 1343 1200 600 i 0 IN MI Lane 1 Normal 1800 2315 1200 480 600 0 WB Lane 1 Tumor Figure 9 1 Example of LOH at TP53 Penta Preferential When alleles differ in size by ten or more base pairs you will likely observe preferential Amplification amplification of shorter PCR products over longer ones Walsh et al 1992 This will also occur when amplifying low copy number DNA or DNA isolated from paraffin embedded tissues
84. M 16 17 ee Ho pg D138317 y D7S820 o NN BE R 17 Figure 9 7 GeneScan electropherogram of AmpF STR Profiler Plus alleles in AmpF STR Control DNA 9947A continued on next page 9 24 Microsatellite Analysis Applications Discrimination The Probability of Identity P and Probability of Paternity Exclusion P values have Power For More Information been determined for each AmpF STR kit and are shown in Table 9 6 Allele frequencies for each population database are provided in the respective user s manuals Existence of random association linkage equilibrium among all 12 loci was established Table 9 6 Discrimination Power of the AmpF STR Kits African American U S Caucasian AmpF STR Kit P PE P Pe AmpF STR Blue 0 00021 N A 0 00020 N A AmpF STR Green 0 00058 N A 0 0024 N A AmpF STR Profiler 1 23 x 10 10 0 9996 2 79 x 10 10 0 9994 AmprF STR Profiler Plus 1 48 x 10 11 0 999989 1 04 x 10 11 0 999982 Refer to the following manuals 4 4 4 4 AmpFtSTR Blue PCR Amplification Kit User s Manual P N 402827 AmpFtSTR Green PCR Amplification Kit Users Manual P N 402944 AmpFtSTR Profiler PCR Amplification Kit Users Manual P N 402945 AmpFtSTR Profiler Plus PCR Amplification Kit User s Manual P N 4303501 These manuals contain sections describing the following o gt 1 1 oo 1 oo oo o o background information on the respective AmpF STR ki
85. NA Polymerase is a recombinant form of Taq DNA Polymerase obtained by expressing a modified Taq DNA Polymerase gene in an E coli host Like native Taq DNA Polymerase it lacks endonuclease and 3 5 exonuclease AmpliTaq Gold DNA Polymerase is a chemically modified form of AmpliTaq DNA Polymerase It provides the benefits of Hot Start PCR that is higher specific product yield increased sensitivity and success with multiplex PCR without the extra steps and modifications of experimental conditions that make Hot Start impractical for high throughput applications AmpliTaq Gold DNA Polymerase is delivered in an inactive state A prePCR heating step of 10 12 minutes at 95 C which can be programmed into the thermal cycling profile activates the enzyme For low template copy number amplifications step wise activation of AmpliTaq Gold DNA Polymerase or Time Release PCR can prove useful 2 AmpliTaq DNA Polymerase LD Low DNA is the same enzyme as AmpliTaq DNA Polymerase However the LD formulation has undergone a further purification process The purification step insures that false positive PCR products will be effectively minimized when amplifying bacterial sequences AmpliTaq DNA Polymerase LD is especially useful for low copy number amplifications AmpliTaq DNA Polymerase Stoeffel Fragment is a modified form of AmpliTaq DNA Polymerase from which the N terminal 289 amino acids have been deleted It is approximately twofold more thermos
86. Nature Gen 13 98 100 Skolnick M H and Wallace R B 1988 Simultaneous analysis of multiple polymorphic loci using amplified sequence polymorphisms ASPs Genomics 2 273 279 Smith J Carpten J Brownstein M Ghosh S Magnuson V Gilbert D Trent J and Collins F 1995 Approach to genotyping errors caused by nontemplated nucleotide addition by Taq DNA polymerase Genome Res 5 312 317 Smith J Freije D Carpten J Gronberg H Xu J Isaacs S Brownstein M Bova G Guo H Bujnovszky P et al 1996 Major susceptibility locus for prostate cancer on chromosome 1 suggested by a genome wide search Science 274 1371 1374 LOH RER Sullivan K M Mannucci A Kimpton C P and Gill P 1993 A rapid and quantitative DNA sex test fluorescence based PCR analysis of X Y homologous gene amelogenin Bio Techniques 15 636 641 Sutherland G and Richards R 1994 DNA repeats a treasury of human variation New Engl J Med 331 191 193 Urquhart A Oldroyd N J Kimpton C P and Gill P 1995 Highly discriminating heptaplex short tandem repeat PCR system for forensic identification Bio Techniques 18 116 121 Walsh P S Fildes N J and Reynolds R 1996 Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vWA Nucleic Acids Res 24 2807 2812 Weber J L 1990 Human DNA polymorphisms based on length variations in simple sequence tandem rep
87. Problems with Poor Amplification Observation Possible Causes Recommended Actions Faint or no signal from sample DNA and from positive control Insufficient enzyme in reactions Use the recommended amount of enzyme Incomplete activation of AmpliTag Gold DNA Polymerase Repeat amplification making sure to Hold reactions initially at 95 C for 10 15 minutes Y Use the recommended buffer Note Both buffer pH and buffer composition affect enzyme activation Note At temperatures gt 95 C the enzyme is susceptible to irreversible denaturation If you suspect insufficient activation increase the incubation time not the incubation temperature Too little sample DNA added to reaction Note This is especially critical in human identification experiments because sample quality is often poor Quantitate DNA and use the amount recommended in the protocol Note For accurate quantitation of human DNA samples use the QuantiBlot Human DNA Quantitation Kit P N N808 0114 Troubleshooting 11 1 Table 11 1 Problems with Poor Amplification continued Observation Possible Causes Recommended Actions Faint or no signal from sample DNA and from positive control Incorrect or suboptimal thermal cycler parameters Check protocol for correct thermal cycler parameters If the correct parameters were used they may need to be optimized for your specific applicat
88. R components under the melted wax The wax also serves as a vapor barrier during cycling b Program temperature control parameters for conventional thermal cycling c Cool samples to 2 35 C at the end of the run Run the PCR Note After thermal cycling ends the wax will form a solid shield preventing spillage and evaporation For post PCR analysis you can penetrate the wax layer with a pipet tip and withdraw the PCR product Reheat the reaction tube to seal for long term storage Modifying Thermal Cycling Parameters Guidelines The following table summarizes the effects of modifying temperature control Temperature Optimization parameters on PCR performance Specific Change in Thermal Cycling Parameter Effect on PCR Performance Raising denaturation temperatures up to 96 C Can be necessary to allow denaturation especially with GC rich templates Can also cause template degradation by depurination Lowering annealing temperatures Can increase yield but can reduce specificity Raising annealing temperatures Increases specificity but can reduce yield Setting the denaturation annealing and extension step to at least 15 seconds preferably 30 seconds with the GeneAmp PCR System 9600 9700 or 2400 Y 45 seconds using thin walled tubes with the DNA Thermal Cycler 480 Y 1 minute using thick walled tubes with the DNA Thermal Cycler 480 or the DNA Thermal C
89. Remove comb slowly Wet comb or well former Ensure that comb is clean and dry before inserting into gel Note To ease insertion of comb you can wet the comb with acrylamide or the appropriate gel polymer Air bubbles trapped when comb inserted Ensure that no air is trapped by comb Severely bowed gel image Clamping bottom of gel plates Do not clamp the bottom of the gel Note Use clamps of equal tension along the side of the gel The clamps must be placed over the spacers Gel extruded between plates into upper buffer reservoir After cleaning plates soak for 10 minutes in 3M HCI then rinse with distilled deionized HO Remake gel Note This will remove any residual charge on the plates that might cause the gel to become oppositely charged Red or green smearing on gel Gel dried out before running Before storing a poured gel wrap the gel ends with damp Kimwipes and plastic wrap IMPORTANT Use gels within 24 hours of pouring Appearance of donut shaped band with black central hole Too much sample loaded Load less sample or remake sample with a higher dilution Large migrating region showing no fluorescence Plates not rinsed free of Alconox Rinse plates thoroughly with distilled deionized H O Troubleshooting 11 7 Table 11 4 Problems with Gel or Gel Image continued Observation Possible Causes Recommended Actions Lan
90. Runs with External Bath Attached 7 6 SSCP Analysis Water Bypasses the Gel Temperature Control Unit With the external cold water bath attached water passes directly into the front and back gel heat transfer plates in the instrument bypassing the gel temperature control unit Figure 7 2 External Valve arrow down Flow sensor a NS I I 1 l pr uml H Front plate i I External Back plate water i I i gt H Gel A i temperature control unit is O a E by passed P eat oe LJ exchange Heater GRO969 JU Figure 7 2 ABI PRISM 377 interior view with arrows showing water flow Dotted line shows ABI PRISM 377 components used with the external water bath If the external water bath you use has heating capacity to 60 C you can run the instrument under standard configuration conditions ambient temperature plus 10 C to 60 C with the external bath attached Frequent monitoring of the water level is required when the external bath is used during standard runs For long periods of use disconnect the external water bath reinstall the water bottle and tubes and use the standard run modules not the chiller modules in the Modules folder Refer to the AB Prism 377 DNA Sequencer Data Collection Software 2 1 Enhancements User Bulletin for the names of all the ABI PRISM 377 Collection Softwa
91. Settings for the ABI PRISM 377 and ABI PRISM 377XL 7 14 SSCP Analysis Data Field Setting Plate Check Module Plate Check A C or D PreRun Module PR GS 36A 2400 PR GS 36C 2400 or PR GS 36D 2400 Run Module GS Run 2140V A C or D CHILLER or GS Run 60W A C or D CHILLER Note Use one of the wattage limiting modules for MDE gels Collect Time 6 to 10 hours depending on the length and conformation of the PCR product Sample Sheet The name of the sample sheet created for this run Well to read distance 36 cm Lanes As appropriate for the comb used Run Mode XL only Full scan 24 34 or 36 lanes XL scan 50 or 66 lanes Operator Your name Gel Matrix The name of the matrix file created for this set of run conditions or None if you have not yet created a matrix Matrix Same as Gel Matrix AutoAnalyze On or off as desired Note You must create a matrix file before analyzing the data Table 7 3 Run Window Settings for the ABI 373 with XL Upgrade Data Field Setting Plate Check Module Plate Check PreRun Module PreRun Run Module GS Run Collect Time 4 hours for a 12 cm gel 8 hours for a 24 cm gel or 12 hours for a 34 cm gel Note The default collect time is 12 hours Sample Sheet The name of the sample sheet that you created for this run Well to Read distance 12 24 or 34 cm Filter Set A Lanes As appropriate fo
92. Store the tubes and water bottle for later use 3 Step to the rear of the instrument and turn the external valve so that the arrow points down for the external position 4 Cut two pieces of polyurethane tubing approximately three feet one meter long to connect the external water bath to the intake and outlet openings on the instrument Insert one end of a piece of tubing into the intake port on the instrument 6 Repeat step 5 with the second piece of tubing in the outlet port SSCP Analysis 7 7 Attaching the External Cold Water Bath Step Action 1 Wrap the threads of the intake and outlet ports on the external water bath with Teflon tape Attach a metal tubing connector to each port Connect the free end of the intake tube to the outlet fitting on the back of the external water bath Connect the free end of the outlet tube to the intake fitting on the back of the external water bath Performing a Run at After your ABI PRISM 377 DNA Sequencer is modified for subambient temperature runs perform the following steps each time you use it Subambient Temperatures 7 8 SSCP Analysis Step Action 1 Follow all instructions in the external water bath user s manual 2 Fill the external water bath with cold deionized distilled water and add antifreeze as recommended by the manufacturer WARNING CHEMICAL HAZARD Antifreeze can cause respiratory tract skin and
93. The ABI Prism 377 DNA Sequencer has a long pass filter to prevent light from the instrument s argon ion laser from interfering with the detection of the dye signals Available Filter Sets Rules for Dye Choice ABI PRISM 377 and ABI PRISM 377 with XL Upgrade The ABI PRISM 377 and ABI PRISM 377 with XL Upgrade DNA Sequencers use six virtual filter sets A F Virtual Filter Sets A C D and F are used for GeneScan applications while A B and E are used for DNA sequencing applications ABI 373 and ABI 373 with XL Upgrade The ABI 373 and ABI 373 with XL Upgrade DNA Sequencers use filter sets A and B Both filter sets are used for GeneScan applications and for DNA sequencing applications Each filter set should be used with a combination of four or fewer dyes including the dye reserved for the internal lane size standard that Display in four different colors Are available in compatible chemical forms You can combine phosphoramidite labels with NHS ester labels You should not combine FIdANTP labeling with any other labeling method Table 4 2 through Table 4 4 on pages 4 7 and 4 8 will help you chose the appropriate dye and filter set combination for your particular application Table 4 2 Chemical Forms and Color Displays of ABI Prism Dyes Color Display Dye Available As in Virtual Filter Set 5 FAMa Labeled primer in reagent kits Blue A F 6 FAM Phosphoramidite Blue A C D R110
94. We recommend GeneScan 350 or GeneScan 500 Y Sodium hydroxide NaOH 0 1 N stored in a plastic container For the ABI PRISM 377 ABI PRISM 377 with XL Upgrade and ABI 373 with XL Upgrade You will need the following software to perform and analyze an SSCP run Y ABI PRISM Module GS Run 2140V CHILLER voltage limiting or GS Run 60W CHILLER wattage limiting Use as a template for creating a dedicated SSCP module Be sure to use a version that is compatible with the chosen dye set ABI PRISM 377 Collection Software version 1 1 or higher IMPORTANT ABI PRISM 377 Collection Software version 2 1 is required if you want to run experiments at subambient temperatures See Running at Subambient Temperatures on the ABI Prism 377 DNA Sequencer on page 7 4 for more information Y GeneScan Analysis Software version 2 0 2 or higher Genotyper software version 2 0 is also highly recommended Using Genotyper software you can obtain numerical sizing data and use the generated numbers to flag potential mutations automatically For the ABI 373 You will need the following software to perform and analyze an SSCP run GeneScan 672 Collection Software version 1 1 or higher GeneScan 672 Fragment Analysis Software version 1 1 or higher Note If you use the GeneScan 672 Fragment Analysis Software on the ABI 373 instrument and want to analyze the data using GeneScan Analysis Software version 2 0 2 or higher you must transfer your dat
95. a to a Power Macintosh computer and perform additional steps Refer to the ABI Prism GeneScan Analysis 2 1 User s Manual P N 904435 for details We also recommend Genotyper software version 2 0 Using Genotyper software you can obtain numerical sizing data and use the generated numbers to flag potential mutations automatically SSCP Analysis 7 3 Running at Subambient Temperatures on the ABI PRISM 377 DNA Sequencer Purpose of an External Cooling System Hardware Requirements 7 4 SSCP Analysis The ABI Prism 377 DNA Sequencer provides gel temperature control from 10 C above the ambient temperature to a maximum of 60 C To perform PCR Single stranded Conformation Polymorphism SSCP protocols at lower temperatures below 10 C above ambient you must attach a cold water bath to the instrument To provide gel temperature control for operations at reduced temperatures you must have all of the following Y External cold water bath connected to the ABI PRISM 377 instrument 30 k ohm thermistors installed on the instrument ABI PRISM 377 Collection Software version 2 1 The water bath polyurethane tubing and tubing connectors needed for the instrument modification must meet the following minimum requirements Hardware Minimum Requirements Water Bath Cooling capacity 250 watts at 20 C Heating capacity 500 watts up to 60 C Pump flow rate 1 gal min or 4 L min at pressure head of 3m Automatic Shutdow
96. achieve acceptable size standard peak heights when loading the small amounts required by 50 lane and 66 lane gels The following table lists the lengths of the 16 fragments comprising the GeneScan 500 size standard Table 5 4 GeneScan 500 Fragment Lengths nt 35 139 250 400 50 150 300 450 75 160 340 490 100 200 350 500 The GeneScan 500 size standard is prepared by digesting a proprietary DNA plasmid with Pst I followed by ligating a TAMRA or ROX labeled 22 mer oligodeoxynucleotide to the cut ends A subsequent enzymatic digestion with BstU yields DNA fragments containing a single TAMRA or ROX dye Although the GeneScan 500 size standard is made of double stranded DNA fragments only one of the strands is labeled Consequently even if the two strands migrate at different rates under denaturing conditions you will not need to worry about peak splitting Figure 5 5 shows the peak patterns of GeneScan 500 fragments run under denaturing conditions Figure 5 5 Electropherogram of the GeneScan 500 size standard run under denaturing conditions on the ABI PRISM 377 DNA Sequencer Fragments were run on a 36 cm 5 Long Ranger 6M Urea gel at 51 C continued on next page 5 12 Sizing and Size Standards Non denaturing Figure 5 6 shows the peak patterns of GeneScan 500 fragments run under Electropherogram non denaturing conditions Figure 5 6 Electropherogram of GeneScan 500 run under non denaturing conditions on the ABI
97. age Microsatellite Analysis Applications 9 19 Applied Biosystems Applied Biosystems fluorescent multicolor dye technology allows multiple loci Fluorescent Dye including loci that have alleles with overlapping size ranges to be analyzed in a single Technology gel lane or capillary injection Alleles for overlapping loci are distinguished by labeling locus specific primers with different color dyes Because only one primer of each pair is labeled the ABI PRISMO instruments detect only one strand for each amplified DNA fragment The detection of only one strand eliminates doublets arising from the different mobilities of complementary strands that are often observed when using gel staining detection methods Automated Sizing Amplified samples can be analyzed in a slab gel format or can be injected into a and Genotyping capillary An internal lane size standard is loaded with each sample to allow for automatic sizing of the PCR products and to normalize differences in electrophoretic mobility between gel lanes or injections Ziegle et al 1992 GeneScan software automatically analyzes the collected data which can then be imported into Genotyper software for automatic genotyping of alleles High Throughput Laboratories can analyze hundreds of loci in a single day using four dye fluorescent labeling This is a dramatic increase in productivity compared with gel staining techniques which visualize all PCR products in the same color or with other s
98. aged by the variability of the RER phenotype Co electrophoresis To Increase Throughput One of the primary advantages of using multiple dyes in RER screening is valid for any microsatellite application you can increase throughput by co loading multiple different reactions covering many relevant microsatellite loci for a single individual in one capillary injection Co loading allows you to screen hundreds of individuals in a single day Rapid Screening The ABI PRISM 310 Genetic Analyzer allows extremely rapid separations Fragments that are 300 bp or less in length can be separated in under 30 minutes This translates to a throughput of at least 48 samples in a 24 hour period Protocol RER screening uses the same protocols as LOH screening see page 9 7 Examples of RER This section presents two classic examples of RER Refer to them when interpreting your own data Figure 9 3 shows the electropherogram of the dinucleotide repeat marker D18S35 from a homozygous individual The appearance of numerous extra alleles at lower molecular weights in the tumor sample bottom panel indicates significant genomic instability 73 83 23 103 113 123 133 143 400 300 200 0 MA Lane N 400 300 200 100 0 MA Lane T Figure 9 3 An example of RER for a homozygote allele at D18835 Figure 9 4 on page 9 15 shows the electropherogram of two dinucleotide repeat markers NM23 and D5S346 from one individual heterozygous in both alleles The ap
99. airs in length 6 Select the Collect button on the controller panel to start data collection IMPORTANT If you omit this step no data will be collected If chosen as a Preference upon completion of electrophoresis the computer will automatically launch the GeneScan 672 Software to generate a gel image on the screen using the most recently saved gel processing parameters You can view data collection at any time during the run by selecting the Scan Map or Gel buttons However you should not leave the gel window open for long periods of time during the run because it uses a large block of memory You must create a matrix file before analyzing microsatellite data for the first time For more information on creating matrices see Appendix C For directions on preparing matrix samples for microsatellite analysis see Preparing the Matrix Samples on page 8 15 To create the matrix file use one of the following Dye Primer Matrix Standards Kit 5 FAM JOE TAMRA and ROX P N 401114 and Filter Set A Dye Primer Matrix Standards Kit 5 FAM JOE and ROX P N 401114 the NED Matrix Standard P N 402996 and Filter Set A Fluorescent Amidite Matrix Standards Kit 6 FAM TET HEX and TAMRA P N 401546 and Filter Set B Fluorescent Amidite Matrix Standards Kit 6 FAM HEX and ROX P N 401546 the NED Matrix Standard P N 402996 and Filter Set A Note Usually you create and save a single matrix file for ea
100. alf Width and the smoothing option that you choose GeneScan may interpret a single noisy peak as multiple peaks Thus these parameters can influence the measurement of peak area and peak height Overestimating the number of peaks can be a particular problem with the jagged peaks characteristic of noisy data Either increase the Minimum Peak Half Width or use a stronger smoothing option when analyzing noisy data Use Consistent Channel Averaging Numbers Before comparing peak areas or heights analyze all samples using the same channel averaging numbers Verify the Position of the Tracker Lane Before comparing peaks areas or heights verify that the tracker lane follows the center of the band 3 10 General Analysis and Evaluation Techniques Evaluating Matrix Quality Purpose of a Matrix Why the Matrix Must Be Remade How to Recognize Matrix Problems While the most intense fluorescence emitted by an ABI PRiSM dye will fall within a small wavelength detection range some fluorescence emission in the detection ranges of the other dyes will always occur The multicomponent matrix compensates for this overlap by subtracting out in each dye s detection range the portion of the signal due to fluorescence from other dyes For directions on how to create a matrix file see Appendix C When you create a matrix you must run each relevant dye matrix standard separately to determine the proportional amount of fluorescence that is emitt
101. all applications consider the optimization strategies discussed in this section as hints or tips for improving PCR product yield This chapter contains the following topics Topic See Page Choosing Reaction Volumes and Tube Types 6 2 Designing Custom Primers 6 3 Determining Reagent Concentrations 6 6 Choosing the Right Enzyme 6 8 Multiplexing PCR 6 10 Using RNA Templates 6 12 Preventing Competing Side Reactions Hot Start PCR 6 13 Modifying Thermal Cycling Parameters 6 15 Avoiding Contamination 6 16 3 A Nucleotide Addition 6 18 Stutter Products 6 21 Preparing PCR Products for Analysis 6 23 Optimizing PCR 6 1 Reaction Volumes Tube Types Using Small Amounts of Template 6 2 Optimizing PCR Choosing Reaction Volumes and Tube Types With Applied Biosystems PCR Instrument Systems reaction volumes in the range of 25 100 uL are generally used However reactions with less than 25 uL have been successful The following table lists the type of reaction tube to use with each thermal cycler Thermal Cycler Reaction Tube Type Uses DNA Thermal Cycler GeneAmp PCR Reaction Tubes Optimized for PCR amplification of reaction volumes between 25 uL and 100 uL Mineral oil required DNA Thermal Cycler 480 0 5 mL GeneAmp Thin Walled Reaction Tubes with domed or flat capsa Allows you to progra
102. alysis Applications 9 1 Microsatellite Analysis Using the LMS V2 What is the The ABI Prism Linkage Mapping Set Version 2 LMS V2 contains 400 fluorescently LMS V2 labeled PCR primers which amplify a highly informative subset of the microsatellite loci from the G n thon human linkage map Weissenbach et al 1992 Gyapay et al 1994 The LMS V2 consists of 28 panels each containing 10 18 primer pairs It expands the capabilities of the original ABI PRISM Linkage Mapping Set with New markers for improved resolution Tailed reverse primers for improved automatic allele calling Anew dye set containing NED for improved spectral resolution of allele peaks 4 True Allele PCR Premix P N 403061 with AmpliTaq Gold DNA Polymerase This premix contains an optimized solution of AmpliTaq Gold DNA Polymerase dNTPs and magnesium in a buffer for simpler more reliable PCR amplification Advantages Product Performance and Quality Control The primer pair combinations in all panels have been optimized for maximum reliability All have been tested on CEPH family 1347 and various sample DNAs to confirm PCR conditions and to verify allele size ranges In addition Applied Biosystems performs a final use test on all manufactured lots of primers using the CEPH 1347 02 control DNA P N 403062 New patented reverse primer tailing pig tailing chemistry improves allele calling efficiency by eliminating problems associated with non templated n
103. ame volume of template DNA to the 15 uL and 7 5 uL reactions because manual pipetting of volumes smaller than 1 uL is inaccurate IMPORTANT To avoid the inaccuracies associated with pipetting small volumes you should combine all reaction components except sample DNA in a PCR Master Mix Using the ratios in the preceding table prepare sufficient mix for at least one extra reaction volume Aliquot 13 8 uL of the mix into each 15 uL reaction or 6 3 uL of the mix into each 7 5 uL reaction PCR Master Mix lasts for 1 2 weeks at 2 6 C Thermal Cycling Profile The following table lists recommended thermal cycling times and temperatures on a GeneAmp PCR System 2400 9600 or 9700 for both the 15 and 7 5 uL reactions Initial Each of 10 Cycles Each of 20 Cycles Incubation Melt Anneal Extend Melt Anneal Extend Final Final Step Extension Step HOLD CYCLE CYCLE HOLD HOLD 95 C 94 C 55 C 72 C 89 C 55 C 72 C 72 C 4 C 12 min 15sec 15sec 15sec 15sec 15sec 15 sec 10 min forever If some loci amplify poorly see Multiplexing PCR on page 6 10 and Troubleshooting PCR Amplification on page 11 1 for suggestions on improving PCR performance continued on next page Microsatellite Analysis 8 5 Labeling Rules Size Standard Control DNA 8 6 Microsatellite Analysis Use 5 end labeled primers The success of microsatellite analysis depends upon the ability to det
104. amide on page A 7 for directions Note Formamide pH should be between 7 and 9 Small bubble between load and read region Cast gel as described in protocol Note Make sure plates are lint free before pouring gel Incomplete strand separation due to Make sure the samples are heated at insufficient heat denaturation 95 C for 5 minutes prior to loading in gel Misshapen wells To prevent suction when removing the comb Note If only a few wells are misshapen you can still load the gel using only those 1 Lay gel flat lanes with flat well formed wells 2 Pour 1X TBE over comb 3 Remove comb slowly Ensure that no air is trapped by comb during insertion Wrong TBE buffer formulation Remake buffer carefully following protocol Troubleshooting 11 13 Reagent Preparation Preparing Gels 10 Ammonium IMPORTANT Prepare the 10 ammonium persulfate solution APS no more than 2 hours Persulfate before pouring the gel Step Action 1 Weigh out 0 50 0 005 g of ammonium persulfate into a 15 mL polypropylene tube WARNING CHEMICAL HAZARD Ammonium persulfate APS is harmful is swallowed inhaled or absorbed through the skin It is extremely destructive to mucous membranes eyes and skin Inhalation can be fatal Always work in a fume hood Obtain a copy of the MSDS from the manufacturer Wear appropriate protective eyewear clothing and gloves Using a P 5000 Pipetman or
105. amination problems Note The manual Hot Start protocol requires the use of mineral oil to prevent evaporation Thus you cannot perform a manual Hot Start in the GeneAmp PCR Systems 2400 9600 and 9700 Step Action 1 Mix all reagents except one key component choose from dNTPs MgCl or primers below a mineral oil cap 2 Load all tubes into the PCR instrument system Define temperature control parameters so that the temperature rises to 70 80 C Add the missing component to each tube changing pipet tips after each sample continued on next page Optimizing PCR 6 13 Performing an AmpliWax PCR Gem mediated Hot Start 6 14 Optimizing PCR Step Action At room temperature add primers MgCl dNTPs and buffer to reaction tube Add a single AmpliWax PCR Gem to each reaction tube Note The mass of the PCR Gem necessary for proper performance depends upon the reaction volume and reaction tube geometry Define temperature control parameters so that the temperature rises to 70 80 C for 5 10 minutes then cools to 2 35 C Note This creates a wax barrier over the aqueous layer Add the polymerase and sample buffer above the solid wax layer Create a PCR thermal profile as follows a Rapidly heat samples to the first denaturation temperature This melts the wax layer denatures the DNA template and creates enough thermal convection to assure complete mixing of all PC
106. amount of target DNA Incomplete restriction and or ligation if performing AFLP Repeat restriction and or ligation If performing AFLP too much DNA in reaction so that insufficient adaptor is present Use the recommended amount of template DNA Mixed sample Verify quality and integrity of sample Troubleshooting 11 5 Table 11 2 Problems with Extra Peaks continued Observation Possible Causes Recommended Actions Presence of split peaks differing in size by one base pair Extra peak of size n 1 Partial nontemplate addition of an extra nucleotide usually adenosine to the blunt end of the PCR product Add the correct amount of Mg2 to the reaction mix Note Increasing Mg concentrations can increase the frequency of nontemplated nucleotide addition and vice versa Increasing the extension time at 72 C will increase the frequency of nontemplate nucleotide addition For more suggestions see 3 A Nucleotide Addition on page 6 18 Presence of peaks differing in size by two three or four base pairs Extra peak of size n 2 n 3 or n 4 Stutter product formed during amplification of di tri or tetranucleotide STR loci See Stutter Products on page 6 21 for suggestions Table 11 3 Problems with Missing Peaks Observation Possible Causes Recommended Actions Some but not all loci visible on electropherogram S
107. ample DNA is degraded indicated if shorter amplicons are favored Quantitate DNA and add more template Repeat amplification Wash the sample in an Amicon Centricon 100 column and repeat amplification Note For fragments smaller than 130 bp the Amicon Centricon 30 column is preferable Sample contains PCR inhibitor e g heme compounds EDTA or certain dyes Quantitate DNA and add minimum necessary volume of PCR product Repeat amplification Individual alleles are data is examined missing when inheritance Mutation in primer annealing site of one allele Change the primer 11 6 Troubleshooting Troubleshooting PCR Product Detection Topics This section offers troubleshooting suggestions for the following problem areas 4 gt Problems with gel or gel image page 11 7 Problems with automatic data analysis page 11 8 Problems with current page 11 9 Problems with signal strength and quality page 11 9 Problems with peak number and position page 11 11 Problems with peak quality and resolution page 11 13 Table 11 4 Problems with Gel or Gel Image Observation Possible Causes Recommended Actions Misshapen wells Note If only a few wells are misshapen you can still load the gel using only those lanes with flat well formed wells Suction when comb removed When removing the comb 1 Lay gel flat 2 Pour 1X TBE over comb 3
108. an Analysis Software User s Manual If the Analysis Control window is not visible choose Analysis Control from the Windows pull down menu Assign a size standard to all sample files as described in Define and Select the Size Standard on page 3 4 Analyze Sample Files Step 1 2 3 4 5 Highlight the appropriate dye colors for each sample General Analysis and Evaluation Techniques To analyze sample files continued Step Action 6 Define analysis parameters a From the File menu select New b Click Analysis Parameters The Analysis Parameters dialog box appears Analysis Parameters Analysis Range Size Call Range Full Range All Sizes This Range Data Points This Range Base Pairs Start min 8 Stop 6000 Max Data Processing Size Calling Method e Basel ne 2nd Order Least Squares Y PUES PED O 3rd Order Least Squares Pe options O Cubic Spline Interpolation one O Light Local Southern Method O Heavy O Global Southern Method Peak Detection Split Peak Correction Peak Amplitude Thresholds None B Y O GENESCAN 2500 G R LeftMost Peak 1 RightMost Peak Min Peak Half Width Pts Correction Limit 30__ Data Pts ox c Set the appropriate analysis parameters for your application as described in Chapter 4 of the GeneScan Analysis Software User s Manual Be sure to exclude the primer peak from the analysis range 7 Click Analyze 8 After the
109. analysis is complete verify the peak assignments for the size standard in all sample files using one of the methods described below continued on next page General Analysis and Evaluation Techniques 3 3 Define and Select the Size Standard Step Action 1 Select one of the sample files in the Analysis Control window 2 If a size standard file already exists proceed to step 8 Otherwise continue to step 3 and create a size standard file now 3 Open the Size Standard pop up menu for the highlighted sample and select Define New 4 Define the size standard See Chapter 5 for the sizes of the fragments in the size standards For detailed information on defining size standards refer to Chapter 4 of the GeneScan Analysis Software User s Manual 5 Close the window 6 Click Save 7 Name the size standard file and click Save 8 Open the Size Standard pop up menu at the top of the Size Standard column and select the appropriate size standard A diamond in this column indicates the size standard will be applied to the corresponding sample file Size standard pop up menu GeneScan Project 5 22 97 Analysis Control CJ Print Results rint Setup Sample File Y lt None gt Parameters lt Collection Setting gt Analysis Parameters gt 4102500 1000 2 Analysis Parameters gt GS 400HD ROH Analysis Parameters gt A
110. andards 5 9 GeneScan 400HD Size Standard Useful Range You can use the GeneScan 400HD High Density size standard to determine fragment lengths between 50 and 400 base pairs Special Uses The high density of marker bands in this standard makes it particularly useful for microsatellite analysis All fragments have been checked for migration that is true to size under a wide variety of run conditions on all ABI PRISM instruments There are no anomalous fragments e g the 250 bp fragment in GeneScan 350 and GeneScan 500 on the ABI PRISM 310 Genetic Analyzer Note GeneScan 400HD is the recommended size standard for use with the ABI PRISM Linkage Mapping Set Version 2 Fragment Lengths The following table lists the lengths of the 21 fragments comprising the GeneScan 400HD size standard Table 5 3 GeneScan 400HD Fragment Lengths nt 50 160 260 360 60 180 280 380 90 190 290 400 100 200 300 120 220 320 150 240 340 Preparation All aspects of the preparation of the GeneScan 400HD size standard are proprietary Each fragment contains a single TAMRA or ROX fluorophore continued on next page 5 10 Sizing and Size Standards Denaturing Although the GeneScan 400HD size standard is made of double stranded DNA Electropherogram fragments only one of the strands is labeled Consequently even if the two strands Non denaturing Electropherogram migrate at different rates under denaturing conditions you will not need to worry about pea
111. anually Figure 6 1 Split peaks resulting from incomplete 3 A nucleotide addition How to Avoid Problems Caused by Incomplete 3 A Nucleotide Addition Modify the thermal cycling conditions either to promote or to inhibit 3 A nucleotide addition Modify Mg concentration either to promote or to inhibit 3 A nucleotide addition Modify or tail the 5 end of the reverse primer either to promote or to inhibit 3 A nucleotide addition to the forward labeled strand Y Treat PCR products enzymatically to remove the 3 A overhangs In general the most reliable strategy is to maximize 3 A nucleotide addition by modifying thermal cycling conditions and Mg2 concentration and if necessary by tailing the reverse primer continued on next page Modifying Thermal Cycling Conditions Modifying Mg Concentration Reverse Primer Tailing Enzymatic Treatment Increasing the time spent between 60 and 72 C promotes 3 A nucleotide addition Decreasing the time spent between 60 and 72 C inhibits 3 A nucleotide addition To use this method effectively you need to determine the optimal thermal cycling conditions for each marker in each set of reaction conditions Promoting 3 A nucleotide addition has proven to be the more successful strategy Residual polymerase activity at room temperature or even at 4 C is often sufficient to catalyze enough 3 A nucleotide addition to create genot
112. balance in intensity between loci labeled with the same color dye Figure 9 7 Component concentrations of the reagents combined for PCR amplification including PCR reaction mix primer set and enzyme are set in the middle of a range of concentrations that give acceptable performance To increase the success of obtaining a result in the presence of enzyme inhibitors bovine serum albumin BSA has been included in the AmpF STR PCR Reaction Mix GeneAmp PCR Instrument times and temperatures have been developed to produce specific amplification while providing the necessary degree of sensitivity The recommended range of input DNA is 1 0 2 5 ng Furthermore instrument detection protocols have been optimized to provide reproducible results with excellent resolution and precision in sizing alleles Each AmpF STR Kit has been validated according to the Technical Working Group on DNA Analysis Methods TWGDAM guidelines Validation was performed by scientists at Applied Biosystems in conjunction with forensic laboratories These validation studies demonstrate that the AmpF STR kits can be used reliably with Applied Biosystems instruments and software to analyze samples commonly encountered in forensic casework 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 EESTE ee O II A ee O AA dudo uo ll a ndi i BE 18 14 GM 6 17 OM iv 17 BE 1 17 1200 D21511 Sog D18S51 400 0 4 G
113. baseline when viewing raw data Y Make matrix using same gel formulation buffer and run conditions as samples Contamination of buffer with fluorescent species for example from laboratory pen used to label lanes gel plates or comb Do not label gel plates or comb Lower buffer chamber not clean i e contaminated with fluorescent species Clean and replenish buffer in lower buffer chamber Signal gets progressively weaker for successive gels run over time Degraded primers Store primers in the dark at 15 to 25 C when not in use Avoid unnecessary freeze thaw cycles Keep primers on ice during run setup Expired or mishandled reagents Check expiration dates on all reagents If not expired verify that reagents are being stored and used according to manufacturer s instructions Compare with signal strength of the same samples prepared and run using fresh reagents Inconsistent signal from lane to lane Some samples not thoroughly mixed with formamide size standard mixture Mix samples into formamide size standard mixture by pipetting up and down several times 11 10 Troubleshooting continued on next page Table 11 8 Problems with Peak Number and Position Observation Possible Causes Recommended Actions Extra peaks in additional colors displayed underneath the position of one strong peak Too much sample loaded causing off scale data Indicated
114. bed ion exchange resin Y 25 mL distilled deionized H 0 3 Stir well until all the urea crystals have dissolved Filter the polyacrylamide solution through a 0 2 um cellulose nitrate filter for 5 minutes Note During this step you both filter and degas the solution 5 Add 4 0 mL of filtered 10X TBE and sufficient distilled deionized H O to bring the total volume to 40 0 mL 6 With the filtered polyacrylamide solution at room temperature add 200 uL of 10 APS ammonium persulfate Swirl gently to mix Be careful to avoid air bubbles 7 Add 25 uL of TEMED tetramethylethylenediamine to the solution Swirl gently to mix Be careful to avoid air bubbles WARNING TEMED Tetramethylethylenediamine is extremely flammable and can be very destructive to the skin eyes nose and respiratory system Keep TEMED in a tightly closed container Avoid inhalation and contact with the skin eyes and clothing Always work in a fume hood Obtain a copy of the MSDS from the manufacturer Wear appropriate protective eyewear clothing and gloves 8 Immediately cast the gel as described in your instrument user s manual 9 Allow the gel to polymerize for at least 2 hours before performing electrophoresis continued on next page Reagent Preparation A 5 MDE Gels for SSCP A 6 Reagent Preparation Mutation Detection Gel Solution MDE can be obtained from FMC P N 50621 Step Action 1 Wash
115. can 672 Fragment Analysis Software to generate a gel image on the screen if chosen as a Preference The gel processing parameters last saved in this application are used to generate the gel image SSCP Analysis 7 23 Analyzing the Data Creating a Matrix You must create a matrix file before analyzing SSCP data for the first time For more 7 24 SSCP Analysis File information on creating matrix files see Appendix C For directions on preparing matrix samples for SSCP analysis see Preparing the Matrix Samples on page 7 17 Note Usually you create and save a single matrix file for each set of run conditions However if you experience persistent problems such as spectral overlap in the analyzed data you should re create the matrix file For the ABI PRISM 377 ABI PRISM 377 with XL Upgrade and ABI 373 with XL Upgrade To create the matrix file use one of the following Dye Primer Matrix Standards Kit 5 FAM JOE TAMRA and ROX P N 401114 and module GS SSCP A Fluorescent Amidite Matrix Standards Kit 6 FAM TET HEX and TAMRA P N 401546 and module GS SSCP C Y Fluorescent Amidite Matrix Standards Kit 6 FAM HEX and ROX P N 401546 the NED Matrix Standard P N 402996 and module GS SSCP D Name the new matrix SSCP Matrix A SSCP Matrix C or SSCP Matrix D as appropriate For the ABI 373 Use the Dye Primer Matrix Standards Kit 5 FAM JOE TAMRA and ROX P N 401114 and Filter Set A to create the matrix
116. cation Primers for Plant Mapping EcoRI Primers Regular Plant Genomes Part Number Part Number Primer 250 reactions 500 reactions EcoRI ACT FAM 402045 402037 EcoRI ACA FAM 402038 402030 EcoRI AAC NED 4303053 4303054 EcoRI ACC NED 4303055 4303056 EcoRI AGC NED 4303057 4303058 EcoRI AAG JOE 402042 402034 EcoRI AGG JOE 402043 402035 EcoRI ACG JOE 402044 402036 EcoRI Primers Small Plant Genomes Part Number Primer 250 reactions EcoRI TG FAM 402264 EcoRI TC FAM 402265 EcoRI AC FAM 402269 EcoRI TT NED 4304352 EcoRI AT NED 402955 500 reactions EcoRI TA JOE 402267 EcoRI AG JOE 402268 EcoRI AA JOE 402271 Msel Primers Regular and Small Plant Genomes Part Number Part Number Primer 250 reactions 500 reactions Msel CAA 402021 402029 Msel CAC 402020 402028 Msel CAG 402019 402027 Msel CAT 402018 402026 Msel CTA 402017 402025 Msel CTC 402016 402024 Msel CTG 402015 402023 Msel CTT 402014 402022 continued on next page AFLP Mapping 10 11 Plant Genomes Ten different crop species genomes were analyzed using the AFLP technique For Mapped Using each crop species primer combinations that produce the best DNA fingerprints were AFLP determined 10 12 AFLP Mapping The names of each crop species tested and corresponding primer combination tables are given in Table 10 4 Those combinations of EcoRI and Msel Selective Amplification primers that are best suited for amplification screening of th
117. cation of amplified restriction fragment polymorphism AFLP markers tightly linked to the tomato Cf 9 gene for resistance to Cladosporum fulvum Plant J 8 785 794 Valsangiacomo C Baggi F Gaia V Balmelli T Peduzzi R and Piffaretti J C 1995 Use of amplified fragment length polymorphism in molecular typing of Legionella pneumophila and application to epidemiological studies Journal of Clinical Microbiology 33 1716 1719 Van Eck H J Rouppe van der Voort J Draaistra J van Zandwoort P van Enckevort E Segers B Peleman J Jacobsen E Helder J and Bakker J 1995 The inheritance and chromosomal location of AFLP markers in a non inbred potato offspring Molecular Breeding 1 397 410 Vos P Hogers R Bleeker M Reijans M van de Lee T Hornes M Fritjers A Pot J Peleman J Kuiper M and Zabeau M 1995 AFLP a new concept for DNA fingerprinting Nucl Acids Res 23 4407 4414 Zabeau M and Vos P 1993 Selective restriction fragment amplification a general method for DNA fingerprinting European Patent Application EP 0534858 Part Numbers ABI PRISM DNA Fragment Analysis Kits and Reagents Internal Lane Size Standards GeneScan 350 500 and 400HD contain enough material for 800 lanes GeneScan 1000 and 2500 contain enough material for 400 lanes GeneScan 500XL contains enough material for 1600 lanes Loading buffer is included 401735 GeneScan 350 ROX
118. ch set of run conditions However if you experience persistent problems such as spectral overlap in the analyzed data you should remake the matrix file even if you have not altered the run conditions Analyzing the Data Part I Using GeneScan After A Run The following diagram summarizes the data analysis process using GeneScan Process Overview Analysis Software Setting the analysis parameters is covered in more detail on page 8 18 For brief directions on analyzing sample files see page 3 2 Data analysis using GeneScan 672 Fragment Analysis Software is covered in more detail on page 8 13 Check automatic gel tracking Manually retrack misaligned lanes Re extract retracked lanes Install a new matrix if necessary Set the Analysis Parameters Define the size standard Analyze the data When using the ABI 373 DNA Sequencer with GeneScan 672 Fragment Analysis Software i e without the XL Upgrade if a new matrix is installed the gel lanes must be re extracted again continued on next page Microsatellite Analysis 8 17 Setting the Analysis For the ABI PRISM 377 ABI PRISM 377 with XL Upgrade and the ABI 373 with XL Parameters Upgrade Step Action 1 Open the GeneScan Analysis application version 2 0 2 or higher 2 Choose New from the File menu and click on the Analysis Parameters icon 3 Specify the settings as shown below SS Analysis Parameters Analysis Range Full Ra
119. color lying directly under a large peak of another color even though there is no PCR product corresponding to the smaller peak The large peak signal is pulling up peaks in other colors An example is shown in Figure 3 6 on page 3 12 General Analysis and Evaluation Techniques 3 11 EL KDM01 82297 Beach n Display 2 150 5400 4800 4200 3600 3000 A 2400 1800 1200 pull up peaks wi ta IN GM se 0ses GM s5 05 MN sr 05 5 WM sr 0s s Figure 3 6 Characteristic appearance of bleedthrough peaks Bleedthrough can occur for two reasons Y The matrix was made with the wrong dyes or filter set For a list of recommended dye filter set combinations see Table 4 3 on page 4 8 The signal from the large peak is off scale because of sample overloading In the example shown in Figure 3 6 the peak showing bleedthrough is actually off scale Figure 3 7 Figure 3 7 Raw data from the bleedthrough example shown in Figure 3 6 Keep peak heights between approximately 150 and 4000 RFU If sample data is off scale do one of the following Rerun the samples loading a smaller volume Dilute the samples and rerun them 3 12 General Analysis and Evaluation Techniques Elevated Interpeak Baseline Figure 3 8 shows a typical example of an elevated interpeak baseline Figure 3
120. colors should be clearly resolvable at higher magnification Figure 3 2 O3eCEPH 1347 02 Figure 3 1 Example of data from the Fluorescent Genotyping Demonstration Kit B Two dinucleotide repeat loci are labeled with each dye except for red which is used for the size standard Ela O3eCEPH 1347 02 Figure 3 2 Expanded view of the D12S83 locus from the same sample as Figure 3 1 continued on next page 3 8 General Analysis and Evaluation Techniques Bad Data Looks Like This An example of data where the sample was greatly overloaded is shown in Figure 3 3 Note that even the size standard peak is truncated KDM01 8229 Beach w Display 1 3B 03 3 36 03 3 3Y 0343 3R 03 3 Figure 3 3 Ridiculously overloaded sample A more subtle example of overloading is shown in Figure 3 4 and Figure 3 5 The off scale peaks in the raw data in Figure 3 4 note that the peaks are truncated at 8100 RFU appear smaller in the analyzed data in Figure 3 5 The peaks are broadened and blue pull up peaks see page 3 11 appear under the green peaks Ela A1 1325 1 2 Figure 3 4 Raw data from an overloaded sample GeneScan Project 8 20 97 Display 4 pull up peaks As GH 2c Ge 2 Figure
121. ctrophoresis voltage but this can decrease the resolution continued on next page Experimental Design Considerations 2 9 Modifying Perform native applications and non denaturing applications such as SSCP at lower Electrophoresis temperatures 27 42 C Temperature Protocols for most denaturing applications specify a 51 C run temperature For More For information on setting electrophoresis parameters refer to the ABI PRISM 377 Information DNA Sequencer User s Manual or the 373 DNA Sequencing System User s Manual 2 10 Experimental Design Considerations Using Control DNA Purpose of Control DNA Control DNA Guidelines for Use CEPH 1347 02 Control DNA 4 Serves as a positive control for troubleshooting PCR amplification Knowing whether the control DNA amplifies will allow you to distinguish between problems with the sample DNA the control DNA amplifies but samples do not and problems with reagents thermal cyclers or protocols the control DNA does not amplify Allows you to monitor sizing precision Because the control DNA is not used to calculate the sizing curve you can use the sizes obtained in different gel lanes to verify that sizing precision reproducibility is within acceptable limits Allows you to correlate the fragment sizes that you obtain with the fragment sizes obtained by others e g for the CEPH families in the Linkage Mapping Set Amplify at least one control DNA sample for every
122. d around the average size with a tolerance limit A typical allele definition would look like this 101 5 0 5 bp Allele binning has several benefits Y As the sample size increases for a particular marker or set of markers new or previously undefined alleles can appear in the sample Allele binning helps you to accommodate undefined alleles Allele sizes tend to vary among electrophoresis runs due to subtle differences in gels or capillaries and electrophoresis conditions Allele binning allows you to define appropriate tolerances for this variance You can define alleles more precisely by binning alleles based on sample size If using the same control DNA e g CEPH 1347 02 on every run you can adjust allele definitions against the reference alleles automatically by binning alleles You can perform allele binning using any of the following Genotyper 2 0 features Histogram window Plot window Make from Labels o gt Add Multiple Categories Offset Calculate Offset In general we recommend using the Histogram window for binning alleles This method works best when the full data set from a study is available for each marker before the allele bins are determined Note For users of the ABI Prism Fluorescent Genotyping Demonstration Kit To familiarize yourself with the allele binning methods described in this section you can use the sample files from the Fluorescent Genotyping Demonstration Kit The Tutor
123. d conformation polymorphism SSCP 7 29 about SSCP 7 2 analyzing the data 7 24 to 7 25 creating matrix file 7 24 setting analysis parameters 7 25 literature references E 2 to E 3 materials required 7 3 optimizing run conditions 7 26 to 7 28 PCR amplification labeling and controls 7 10 to 7 12 preparing fora run 7 14 to 7 18 creating the run module 7 18 gel preparation 7 14 instrument setup 7 14 preparing and loading samples 7 16 preparing for a run using the ABI 373 7 19 to 7 23 gel preparation 7 19 instrument setup 7 19 plate check and pre run 7 21 preparing and loading samples 7 21 to 7 22 setting preprocess and analysis parameters 7 19 starting the run 7 23 prerun checklist 7 13 running at subambient temperatures 7 4 to 7 9 troubleshooting 7 29 size calling See size standard size standard accuracy versus precision 5 3 comparing sizes from across platforms 5 3 to 5 4 precision results table of defining 3 4 dye chemical form 4 3 7 2 to 5 4 GeneScan Internal Lane Size Standards 5 7 to 5 17 GeneScan 1000 5 14 to 5 15 GeneScan 2500 5 16 to 5 17 GeneScan 350 5 8 to 5 9 GeneScan 400HD 5 10 to 5 11 GeneScan 500 5 12 to 5 13 peak assignments verifying 3 5 to 3 7 process overview 5 2 to 5 3 troubleshooting 5 5 to 5 6 possible problems 5 5 to 5 6 preventing sizing problems 5 5 SSCP See single strand conformation polymorphism SSCP StockMarks 9 17 to 9 18 allele frequency 9 18 DNA based tests advantages 9
124. d on next page Fluorescent Genotyping 402246 Kit A PCR Reagents Demonstration Kits Contains six fluorescent labeled PCR primer pairs labeled with HEX TET amp A and B FAM two control DNAs CEPH 1347 02 and 1347 10 and a ready made mix of PCR reagents containing AmpliTaq Gold DNA Polymerase GeneAmp PCR Buffer Il dNTPs and magnesium chloride Also includes GeneScan 350 Internal Lane Size Standard and loading buffer 402247 Kit B Amplified PCR Products Contains four tubes of pooled combined PCR products To generate the products each DNA sample CEPH 1347 01 1347 02 1347 10 1347 15 has been amplified with the same six fluorescent labeled PCR primer pairs in kit A All of the PCR products from one tube can be detected in one gel lane continued on next page Part Numbers F 3 ABI PRISM Linkage Mapping Set Version 2 F 4 Part Numbers 50 Rxn Kits 300 Rxn Kits Panel Chromosome 403089 403118 Complete Set 1 22 X 403090 403119 1 1 403091 403120 2 1 403092 403121 3 2 403093 403122 4 2 403094 403123 5 3 4 403095 403124 6 3 4 403096 403125 7 3 4 403097 403126 8 5 6 403998 403127 9 5 6 403099 403128 10 5 6 403100 403129 11 7 8 403101 403130 12 7 8 403102 403131 13 9 10 11 403103 403132 14 9 10 11 403104 403133 15 9 10 11 403105 403134 16 9 10 11 403106 403135 17 12 13
125. determining reagent concentration 6 6 matrix file creating C 1 to C 4 checking matrix quality C 3 to C 4 generating the matrix file C 2 to C 3 how to verify raw data C 1 to C 2 evaluating quality of 3 11 to 3 14 purpose of matrix 3 11 recognizing problems 3 11 to 3 14 solving matrix problems 3 14 when to remake matrix 3 11 matrix problems how effects size calling 5 5 microsatellite marker analysis creating new matrix file 8 11 maximum emission wavelength table of 4 5 MDE gels preparing A 6 microsatellite marker analysis ABI 373 preparing for a run 8 12 to 8 16 creating a new matrix file 8 16 data collection viewing 8 16 gel preparation 8 12 instrument setup and prerun 8 14 preparing and loading samples 8 15 preprocess analysis parameters setting 8 13 to 8 14 starting the run 8 16 about 8 2 analyzing the data using GeneScan 8 17 to 8 18 setting analysis parameters 8 18 analyzing the data using Genotyper 8 19 to 8 32 allele binning methods 8 19 allele binning definition 8 19 benefits of allele binning 8 19 creating defined set of category members 8 29 to 8 32 generating category members automatically 8 26 to 8 29 using the Histogram window 8 20 to 8 24 using the Plot window 8 24 to 8 25 applications animal paternity 9 17 to 9 18 applications loss of heterozygosity screening for 9 5to 9 12 human identification 9 19 to 9 25 Linkage Mapping Set 9 2 to 9 4 replication error screening for 9
126. duction traits and maintenance of ancestral records by the breeding and racing associations Recently researchers have turned to microsatellite markers to identify genetically desirable animals much more quickly reliably and inexpensively The StockMarks kits have been used to perform the following 4 Parental identification for accurate pedigree analysis Quantitative trait loci QTL research to find markers linked to desirable traits The amplification capability of PCR combined with the information content of microsatellites provides the following advantages PCR based tests are easy to standardize and automate ensuring reproducible results PCR based tests can be run on a variety of samples including blood semen and hair Obtaining samples from hair eliminates the expense of having a veterinarian draw blood In addition hair is easier to transport than blood Very little sample is required for a positive result unlike traditional serological blood assays Quicker more accurate parentage identification can be obtained from DNA analysis than from serological testing DNA analysis allows inclusion as well as exclusion An animal can be identified positively as the parent rather than merely being eliminated as a possibility One of the primary advantages of using multiple dyes in StockMarks analysis is valid for any mapping or identification application you can increase throughput by co loading multiple differen
127. e pipetting small volumes for example make master mixes Note You may also want to consider using a 2 uL or other high precision pipette 11 4 Troubleshooting continued on next page Table 11 2 Problems with Extra Peaks Observation Possible Causes Recommended Actions Extra peaks appear with no discernible pattern Presence of exogenous DNA Use appropriate techniques to avoid introducing foreign DNA during laboratory handling Nonspecific priming e primer template mismatch Check for good primer design See Designing Custom Primers on page 6 3 for more information Add less template DNA Note High DNA concentrations promote nonspecific annealing Optimize Mg2 concentration Add less primer DNA Note High primer concentrations promote nonspecific annealing If you are not using AmpliTaq Gold DNA Polymerase consider using a Hot Start Technique Increase annealing temperature in 2 5 C increments Decrease annealing and or extension times Increase primer length Perform a second amplification with nested primers Perform Touchdown PCR Primer dimer and primer oligomer artifacts likelihood increases with multiplex PCR Check primers for 3 complementarity Design longer primers Reduce primer concentration Reduce number of cycles Raise the annealing temperature in 2 5 C increments Increase
128. e Successful Amplification Melting Temperature Maximizing Stability A PCR primer pair consists of two oligonucleotides typically 15 30 nucleotides long that hybridize to complementary strands of the DNA template and flank the region of interest Choose primers with similar melting temperatures T Choose primers that maximize the stability and specificity of binding to the desired template Note For best results evaluate potential primers with the aid of primer design software e g Primer Express Choosing primers with similar T s makes it possible to find thermal cycling parameters that are optimal for all members of a primer pair or pairs Be aware that the calculated T is only a guideline based on base composition The actual Tm is also influenced by the concentration of Mg2 K and cosolvents Binding stability is influenced by Primer template base composition 4 Primer template base order Primer or template secondary structure Effects of Base Composition G C bonds contribute more to the stability increased melting temperature of primer template binding than do A T bonds To ensure stable binding of primer and template while avoiding problems with the internal secondary structure of primers or long stretches of any one base choose primers with a 40 60 G C content However do not let this rule interfere with primer choice based on T and primer length considerations Avoiding primer dimers and
129. e birth defects Wash thoroughly after handling formamide Obtain a copy of the MSDS from the manufacturer Wear appropriate protective eyewear clothing and gloves Wash thoroughly after handling formamide Note Vortex gently for 3 5 seconds Loading Mix lasts for 2 weeks at 2 6 C To each sample tube add the following 3 0 uL Loading Mix from step 1 1 pL PCR product Place the samples in a heat block or thermal cycler for 5 minutes at 95 C Remove the tubes from the heat source and place immediately in an ice water bath Load an appropriate volume of sample into each lane depending upon the instrument and type of comb used Note See Appendix B for more information about loading volumes Preparing the Matrix Samples Step Action 1 To each of four sample tubes add the following 2 5 uL deionized formamide 2 5 uL of one of the matrix standards Dye Primer Fluorescent Amidite or NED matrix standard WARNING CHEMICAL HAZARD Formamide is a known teratogen It can cause birth defects Wash thoroughly after handling formamide Obtain a copy of the MSDS from the manufacturer Wear appropriate protective eyewear clothing and gloves Wash thoroughly after handling formamide Denature the samples for 5 minutes at 95 C Cool the samples by placing directly on ice Run an appropriate volume of these four samples either alone on a gel or in the first lanes of an expe
130. e deionized formamide into aliquots of 500 uL and store for up to 3 months at 15 to 25 C Use one aliquot per set of samples Discard any unused deionized formamide Reagent Preparation A 7 Sample Loading Volumes Loading Volumes The following table summarizes total loading volumes for various instrument platforms and comb configurations Number Loading Volume Instrument Platform of Wells uL ABI 373 24 or 34 4 ABI PRISM 373 XL 50 2 3 66 1 ABI PRISM 377 24 or 34 1 5 ABI PRISM 377 XL 50 1 15 66 0 5 1 Loading the small volumes required by the ABI PRiSM 377 DNA Sequencer with XL Upgrade is easier if you use a gel loading syringe such as a multiplexed loader with four or eight syringes arrayed to match the ABI PRISM 377 with XL Upgrade s well spacing Alternatively you can use an adjustable volume pipet with 0 17 mm gel loading tips Loading volumes smaller than 1 uL with a pipet can be difficult Sample Loading Volumes B 1 Creating Matrix Files Creating the GeneScan Matrix File Overview The matrix file contains the information necessary for software to correct the spectral overlap of the dyes in the filter and virtual filter sets see Chapter 4 for more information on the dyes available Once a matrix file has been created it can be used for subsequent runs performed With the same kit or chemistry Onthe same instrument Using the same
131. e designated crop genomes are shown in Table 10 5 through Table 10 14 Table 10 4 Primer Combination Tables for Crop Species Crop Species Primer Combination Table Regular Plant Genomes Sunflower Table 10 5 on page 10 12 Pepper Table 10 6 on page 10 13 Barley Table 10 7 on page 10 13 Maize Table 10 8 on page 10 14 Sugar beet Table 10 9 on page 10 14 Tomato Table 10 10 on page 10 15 Lettuce Table 10 11 on page 10 15 Small Plant Genomes Arabidopsis Table 10 12 on page 10 16 Cucumber Table 10 13 on page 10 16 Rice Table 10 14 on page 10 17 The following symbol indicates unacceptable primer combinations for amplification screening of designated species Table 10 5 Primer Combinations for Sunflower Species Msel Primers CAA CAC CAG CAT CTA CTC CTG CTT AAC O sac O O ACA O O O ACC ACG O O ACT EcoRI Primers AGC VO AGG Table 10 6 Primer Combinations for Pepper Species Msel Primers CAA CAC CAG CAT CTA CTC CTG CTT AAC O O S AAG O O E ACA ACC le Oo i ACG act O AGC AGG S Table 10 7 Primer Combinations for Barley Species Msel Primers CAA CAC CAG CAT CTA CTC CTG CTT AAC AAG O O Y E ACA ACC e M ACG ACT O acc O AGG
132. e entire chromosomes at a 20 30 cM resolution for 12 tumor normal pairs in a single experiment For any given sample you can detect LOH in 1 day Microsatellite Any microsatellite markers can be used in LOH and replication error RER screening RER LOH Assay Applied Biosystems offers two options 4 The Applied Biosystems Microsatellite RER LOH Assay P N K0015 is a fluorescent microsatellite assay that detects RER and loss of heterozygosity LOH in genomic DNA isolated from microdissected normal and tumor tissue pairs This assay is for research purposes only It can be used to help determine if tumor cells are positive for the RER or the LOH phenotype The Microsatellite RER LOH Assay includes reagents used for the polymerase chain reaction PCR amplification of ten microsatellite markers located on several chromosomes near known and suspected cancer genes The assay also includes control DNA Refer to the Microsatellite RER LOH Assay User s Manual P N L0089 for more information The ABI Prism Linkage Mapping Set Version 2 see page 9 2 a 10 cM microsatellite map has 400 markers that can also be used for LOH and RER screening However this kit may require optimization for your application because of the quantitative nature of LOH RER screening 9 6 Microsatellite Analysis Applications Performing LOH Screening Overview Microsatellites are amplified using one fluorescently labeled and one unlabeled primer Run Setup Handl
133. e is not available continued on next page 9 22 Microsatellite Analysis Applications Genotyping Using The AmpF STR Allelic Ladders contain the most common alleles of the loci and are the AmpF STR used to genotype the analyzed samples The AmpF STR Allelic Ladders in the Allelic Ladders AmprF STR Profiler Plus PCR Amplification Kit are shown in Figure 9 6 val ta NIN m y a te ve E eal a Pal 351358 YWA ra E ad et ech a e al eee a eae lalele les le 10 12 13 18 fis 25 27 28 2 E0 2 62 E3242 10 12 23 29 2 14 2 Amelogenin D8S1179 D21811 D18551 ae ee e Ben pa amec e ma D55818 D135317 D75820 Figure 9 6 Genotyper plot of the AmpF STR Allelic Ladders contained in the AmpF STR Profiler Plus PCR Amplification Kit When interpreting AmpF STR kit results genotypes are assigned to sample alleles by comparison of their sizes to those obtained for the known alleles in the AmpF STR Allelic Ladders Genotypes not sizes are used for comparison of data between runs instruments and laboratories We strongly recommend that laboratories use the AmpF STR Allelic Ladders on each gel or set of capillary injections to convert the allele sizes to genotypes The main reasons for this approach are outlined below 4 The size values obtained for the same
134. e ne Us 7 14 Preparing for a Run ABI 373 0 rr 7 19 Analyzing the Data ic estes g eee A a we ews 7 24 Optimizing SSCP Run Conditions 0 0 0 2 eee ec cece ene 7 26 Troubleshooting eiii ce cea ee ee ee a Gearon a ated g Deke ee res 7 29 8 Microsatellite Analysis 0 ccc ccc cece eee cee cee Ol IA O Gets fet hai da Oe ee a dhe a ted pease 8 1 Introduction to Microsatellite AnalySiS o ooo oooooooorcrroccror nee 8 2 Before YOU BM a tanta Seger etl Gane ate Aten de ean Rega Cie 8 3 PCR Amplification Labeling and Controls 0 0 00 cece cece eee eee 8 5 To Save Time Prerun Checklist 0 2 0 eee cece nets 8 7 Preparing for a Run ABI PRISM 377 ABI PRISM 377 with XL Upgrade and ABI 373 with XL Upgraden sie antec te ee teenie x Sat 2a ee Re Ba EEE RS Te ICAA 8 8 Preparing for a Run ABI 373 1 0 cc teen nen ence enn 8 12 Analyzing the Data Part I Using GeneScan 2 0 0 0 cece eee eee 8 17 10 Il Analyzing the Data Part II Allele Binning Using Genotyper 2 0 8 19 Troubleshooting Microsatellite Analysis 0 2 0 eee eee eee eee 8 33 Microsatellite Analysis Applications oooooooo 91 VEL VIEW arinean 8 we Ba A A Dye ying die e A AA 9 1 Microsatellite Analysis Using the LMS V2 1 0 0 ee cee eee 9 2 Troubleshooting the LMS V2 o oooooocoococoocroro eee erener 9 4 LOH Screenin ati ai Paik wee eed Gas COND e Dade Gallen 9 5 Performing
135. e target away from sequences that amplify only linearly i e those with one modified end Prepared template genomic DNA fragment modified with adaptors a Preselective primers MI EcoRI adaptor recognition site Adaptors Thermal Core Mix Cycling E Figure 10 6 Preselective amplification of the prepared template L Meel adaptor recognition site In the microbial genomes targeted by the AFLP Microbial Fingerprinting Kit P N 402948 the core primer sequence is used In larger genomes such as plants AFLP Mapping 10 5 and some fungi this amplification would create too many fragments In those cases the preselective amplification is performed with additional nucleotides on the end of each primer see page 10 7 Each added nucleotide reduces the number of sequences by a factor of four The thermal cycling conditions of the preselective amplification step have been optimized to generate a constant final mass of fragments Band intensity in subsequent reactions can therefore be correlated with relative differences in representation of the fragments within the genome and not to the overall amount of genomic DNA that went into the initial restriction ligation mix It is not necessary to perform this step if 4 relative peak height information is not desired methods are available to normalize the final signal Y very accurate quantitation of the input DNA is performed routinely Selective Additional PCR
136. e wide region s of fluorescence Gel wide region of migrating fluorescence can be of any color Contaminated loading buffer or sample Contamination of buffer with fluorescent species e g laboratory pen used to label lanes gel plates or comb Remake solutions co loaded with sample Do not mark gel plates or comb Lower buffer chamber not clean i e contaminated with fluorescent species Clean and replenish buffer in lower buffer chamber Tilted or deformed bands Small bubble in lane Cast gel as described in protocol Make sure plates are lint free before pouring gel Gel not clamped properly Use clamps of equal tension along the side of the gel The clamps must be placed over the spacers only IMPORTANT of the gel Do not clamp the bottom Dirty plates Clean plates with 3 M HCI Table 11 5 Problems with Automatic Data Analysis Observation Possible Causes Recommended Actions Data was not automatically analyzed Sample Sheet not completed or completed incorrectly Complete Sample Sheet as described GeneScan Analysis Software Run Defaults set incorrectly in data collection program in the Preferences under the Window menu Make sure that the Autoanalyze box is checked and that the GeneScan Run Default appears with the correct pathname Insufficient free RAM Restart the computer before collecting data Note You should always restart
137. eaks associated with a single marker 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 c From the Views menu choose Zoom In Selected Range or type R to display the plots for the individual alleles se b ob aa 96 98 100 102 104 106 108 110 112 114 116 tie 120 122 5 Draw a box around the first tall peak from the left 98 100 102 104 106 108 110 11 To bin alleles directly using the individual allele plots continued Step Action 6 From the Category menu choose Add Category Genotyper software automatically enters the size information for the category definition Enter the following information Name of the allele member set to the rounded size in bp Member of group name marker name Highest peak button Size range of the allele from x to y bp Color of dye gt gt gt Exclusive checkbox Note You cannot add a new group if a group or category with the same name already exists 7 Click OK to add the category bin 8 Continuing to move from left to right repeat step 4 through step 6 for the remaining peaks 9 Optional To create a bin centered around the median size of the range with a set tolerance for example 104 68 0 5 bp follow these steps a Hold down the Shift key while choosing Add Category from the Category menu b Edit the bin tolerance as desired The size is displayed in the dialog box as shown here Size
138. eats Genome Analysis Volume 1 Genetic and Physical Mapping Cold Spring Harbor Laboratory Press Weber J L and May P E 1989 Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction Proc Natl Acad Sci USA 44 388 396 Weber J L 1989 Length polymorphisms in dC dA and dG dT sequences detected using the polymerase chain reaction Current Communications in Molecular Biology Cold Spring Harbor Laboratory Press Weissenbach J et al 1992 A second generation linkage map of the human genome Nature 359 794 801 Yuille M A R Goudie D R Affara N A and Ferguson Smith M A 1991 Rapid determination of sequences flanking microsatellites Nucleic Acids Res 19 1950 Ziegle J S Su Y Corcoran K P Nie L Mayrand P E Hoff L B McBride L J Kronick M N and Diehl S R 1992 Application of automated DNA sizing technology for genotyping microsatellite loci Genomics 14 1026 1031 Aaltonen L A Peltomaki P Leach F S ef a 1993 Clues to the pathogenesis of familial colorectal cancer Science 260 812 816 Bookstein R Levy A MacGrogan D Lewis T B et al 1994 Yeast artificial chromosome and radiation hybrid map of loci in chromosome band 8p22 a common region of allelic loss in multiple human cancers Genomics 24 317 323 Bottaro D P Rubin J S Faletto D L et al 1991 Identification of the hepatocyte growth factors the c met proto onco
139. ect the mobility of only one strand Size Standard Conversely if a mutation causes only a slight mobility shift in one of the strands the likelihood of a false positive result diminishes if this shift is correlated with a mobility shift however slight in the other strand A size standard must be defined for each run condition Many point mutations cause only slight mobility shifts Internal lane size standards in a dedicated color greatly enhance the sensitivity of mutation detection We also recommend adding wild type DNA labeled with the same dye as the size standard to the size standard However the wild type DNA has to have a similar peak height to the size standard fragment peak heights to facilitate peak recognition by the GeneScan Analysis Software Inazuka et al 1997 Figure 7 3 shows the GeneScan 500 TAMRA Internal Lane Size Standard run under non denaturing conditions at 25 C El 65 500 0 54 MDE w 5 glyc 25 C Figure 7 3 GeneScan 500 TAMRA run in 0 5X MDE with 5 glycerol at 25 C Note The migration rates of the fragments in the size standard do not necessarily conform to their sizes The standards are denatured and the resulting single stranded DNA molecules like the samples for analysis adopt unpredictable three dimensional conformations The goal is to use the size standards to align samples and compare migration rates not to determine s
140. ect small mobility differences The reproducible sizing and sharp peaks obtained when using the 5 end labeling method are crucial to the success of this application Always use a GeneScan Internal Lane Size Standard In this context control DNA satisfies the following criteria The DNA comes from a single individual with known genotype The DNA sample is in sufficiently good condition to serve as a positive control for PCR amplification Applied Biosystems recommends using control DNA such as the CEPH 1347 02 standard used to generate the G n thon map of the human genome to monitor several stages of the experimental process Control DNA Serves as a positive control for troubleshooting PCR amplification For example if the sample DNA amplifies poorly knowing whether the control DNA amplifies will allow you to distinguish between problems with the sample DNA control DNA amplifies and problems with reagents instruments or protocols control DNA does not amplify Serves as a sizing reference for monitoring gel to gel variation Because the control DNA is not used to calculate the sizing curve the size obtained for the control DNA across gels will alert you to potential problems with sizing precision Facilitates allele binning Allele binning is a simple statistical method for converting peak sizes to alleles Briefly the method involves generating a frequency histogram of called sizes Refer to the Genotyper User
141. ective Amplification Plant Mapping extensions EcoRI A EcoRI T and EcoRI G give simpler patterns than that obtained using the primer with no extra nucleotide EcoRI 0 E 60 30 120 150 180 210 240 270 300 330 360 390 420 450 480 Ss es A a a on Oe UMMM aL o 12B W3110 CA O i Mlk I 13B W3110 CAZA 7 DE 146 W3110 casts 166 W3110 CA G Figure 10 8 AFLP fingerprints of E coliW3110 Reference DNA The Msel CA and fluorescent dye labeled EcoRI 0 EcoRI A EcoRI T and EcoRI G selective primers shown here top to bottom respectively were used If the complexity of the AFLP pattern is still too high at the 2 2 level we recommend reamplifying the preselective amplification sample with the preselective primers from the AFLP Ligation and Preselective Amplification Modules of the AFLP Regular and Small Plant Genome Mapping Kits P N 402004 and 402273 respectively The sequences of the adaptors and the restriction site serve as primer binding sites for a subsequent low level selection or preselective amplification of the restriction fragments The Msel complementary primer contains a 3 C The EcoRI complementary primer contains a 3 A Regular Plant Genome Kit modules or no base addition Small Plant Genome Kit modules Only those genomic fragments that have an adaptor on each end amplify exponentially during PCR amplification Figure 10 9 on page 10 8 This step effect
142. ed Use spacers and combs that are of equal thickness Peak positions off throughout size range Inadvertent change in Analysis Parameters Check settings for GeneScan Analysis Parameters Incorrect Sample Sheet selected Check Sample Sheet selection in data collection program Change in size calling method Use consistent size calling method Incorrect internal size standard Use correct GeneScan Internal Lane Size Standard Incorrectly defined size standard Define size standard peak sizes within each lane 11 12 Troubleshooting continued on next page Table 11 9 Problems with Peak Quality and Resolution Observation Possible Causes Recommended Actions Fuzzy or smeared bands Dirty gel plates Clean plates with Alconox cleaner and a in electropherogram soft cloth then rinse thoroughly with distilled deionized water Excess urea in wells before loading Flush wells immediately before loading Incorrect TBE buffer formulation Remake buffer carefully following protocol Worn or damaged spacers causing variable Replace damaged spacers gel thickness Poor resolution Poor quality or old reagents Remake 10X TBE and gel solution stock throughout size range using fresh reagents from a reliable source IMPORTANT Urea must be ultrapure Formamide degraded to formic acid and Use freshly deionized formamide See formate ions denaturing applications only Deionized Form
143. ed Capillaries However the dye phosphoramidite imparts greater hydrophobicity to the oligonucleotide The fluorescently labeled oligonucleotide will migrate approximately one base slower than on PAGE and will be visible under long wavelength UV light Also by HPLC and MicroGel CE the fluorescently labeled oligonucleotide will elute later than it does for unlabeled sequences Figure D 2 on page D 8 shows a typical fluorescently labeled oligonucleotide analyzed by MicroGel capillary electrophoresis on the Applied Biosystems Model 270A HT Preparing 5 End Labeled Primers D 7 mo mo AD alo io oo Ab mo Figure D 2 MicroGel electropherogram of an OPC purified 18 mer with the sequence 5 HEX TGT AAA ACG ACG GCC AGT 3 Evaluating Yield The following are typical yields of a fluorescently labeled 20 mer Base composition purine pyrimidine ratio and length may affect yield Scale Expected Yield Purification 40 nmol 1 2 O D 33 66 ug OPC 0 2 mol 5 10 O D 165 330 ug OPC 1 umol 20 40 O D 0 66 1 32 mg HPLC 10 umol 100 200 O D 3 3 6 6 mg HPLC Molecular Weight and Emission Emission Specifications Dye Linker MW Amax nm 6 FAM 491 5 494 TET 629 3 521 HEX 698 2 535 continued on next page D 8 Preparing 5 End Labeled Primers Storing the Dyes 4 Store dry dye phosphoramidite powder over desiccant in a freezer at 15 to 25 C Note These products are stable for at least one year under these condit
144. ed in all four detection regions Because the emission spectra of the dyes vary with the physical environment such as the pH or polymer type and concentration the matrix must be remade if run conditions change Factors Affecting Matrix Quality Aging reagents Buffer type and concentration Gel polymer type Denaturing vs non denaturing conditions Run temperature Virtual Filter Set C The emission maximum of 6 FAM the recommended blue displaying dye for this filter set is very close to the laser wavelength of 514 5 nm Thus the window for collected blue light intensity data is offset to longer wavelengths and does not contain the emission maximum of 6 FAM It is also very close to the detection region for the green displaying dye TET see Virtual Filter Set C on page 4 11 Matrix files made for Virtual Filter Set C are especially susceptible to minor changes in run conditions If you are using Virtual Filter Set C for GeneScan applications watch for evidence of matrix problems and remake the matrix as soon as problems appear A poor or incorrect matrix results in too much or too little subtraction of dye spectral overlap during data analysis Each causes a recognizable electropherogram anomaly Bleedthrough peaks also called pull ups caused by too little subtraction 4 Elevated interpeak baseline caused by too much subtraction Bleedthrough Peaks or Pull ups Bleedthrough peaks are small peaks of one
145. ee page 6 18 for a discussion of the plus A phenomenon and for suggestions Y Stutter See page 6 21 for a discussion of the stutter phenomenon and for suggestions See below for examples of stutter patterns in dinucleotide repeat loci Examples of Successful amplification of dinucleotide repeat markers yields allele peaks and Stutter associated PCR stutter bands within a maximum range of eight base pairs from the Dinucleotide Repeats allele peak The number of allele peaks depends on whether the individual tested is a heterozygote or homozygote Dinucleotide repeats give specific stutter patterns that are illustrated in Figure 8 5 through Figure 8 9 on pages 8 33 through 8 35 Example 1 The GeneScan electropherogram of a dinucleotide repeat marker from a homozygous individual 118 6 bp 118 6 bp is shown in Figure 8 5 The peaks at 116 6 bp 114 6 bp and 112 6 bp are the typical 2 bp stutter pattern seen with dinucleotide repeats They represent the 2 bp 4 bp and 6 bp stutters from the true 118 6 bp allele 111 116 121 2400 2000 1600 1200 Figure 8 5 Typical pattern for dinucleotide repeat homozygote Microsatellite Analysis 8 33 8 34 Microsatellite Analysis Example 2 The GeneScan electropherogram of a dinucleotide repeat marker from a heterozygous individual 90 bp 98 bp is shown in Figure 8 6 Allele sizes differ by 8 bp The 2 bp stutter peak to the left of each allele peak is always of lower
146. el image provides a qualitative picture of the run This chapter summarizes the basic design factors that you should consider before beginning any GeneScan fragment analysis experiment The chapter contains the following topics Topic See Page Working with Multiple Colors 2 2 Choosing Fluorescent Labeling Methods 2 5 Determining Loading Concentrations for Samples 2 8 Optimizing Electrophoresis Conditions 2 9 Using Control DNA 2 11 Experimental Design Considerations 2 1 Working with Multiple Colors Introduction Guidelines Multiplexing to Increase Throughput Fluorescent labeling enables you to analyze many independent loci in the same lane using color in addition to size to distinguish between fragments To take advantage of the increased discrimination power you need to consider more factors than you would with traditional techniques For Performing Any Experiment You should always Use the same GeneScan size standard labeled with the same dye for all samples in a single study Compare peak areas heights and sizes in nucleotide bases only for fragments that are labeled with the same dye For example compare FAM labeled fragments only to other FAM labeled fragments Note Itis possible to compare samples labeled with different dyes when comparing relative sizes and peak height and area ratios For Working with Similarly Sized DNA Fragments If the sizes of different fragments overla
147. en it is in crystalline form Avoid skin contact when it is in solution because acrylamide can be absorbed through the skin Always work in a fume hood Obtain a copy of the MSDS from the manufacturer Wear appropriate protective eyewear clothing and gloves 2 Dissolve the crystalline acrylamide and bisacrylamide in a sufficient distilled deionized H O to bring the total volume to 135 mL 3 Add 15 g of mixed bed ion exchange resin Stir at room temperature until all crystals dissolve Continue stirring for 5 10 minutes Filter the mixture through a 0 2 um cellulose nitrate filter Transfer the filtrate to a graduated cylinder and bring the total volume to 150 mL with distilled deionized H O 7 Store at 2 6 C Note 40 polyacrylamide stock lasts for 1 month at 2 6 C Reagent Preparation A 3 A 4 Reagent Preparation Preparing Polyacrylamide Gels for SSCP To prepare a standard 6 5 nondenaturing polyacrylamide gel for SSCP Step Action 1 Wash and assemble the glass plates as described in your instrument user s manual Combine the following Y 6 5 mL 40 polyacrylamide stock 37 5 1 1 0 g mixed bed ion exchange resin Y 1 25 g glycerol 25 mL distilled deionized H 0 Mix well to obtain a homogenous solution Note This step is critical because glycerol is viscous and it is difficult to obtain a homogeneous solution Filter the mixture through a 0 2 u
148. eneAmp Thermostable rTth Reverse Transcriptase RNA PCR Kit with FIdUTP Set N808 0223 GeneAmp PCR Reagent Kit with AmpliTaq DNA Polymerase with F JdCTP Set N808 0224 GeneAmp PCR Core Reagents with F JdCTP Set N808 0225 GeneAmp Thermostable r7th Reverse Transcriptase RNA PCR Kit with FJACTP Set Fluorescent For direct 5 end labeling on an automated DNA synthesizer Phosphoramidites Fluorescent NHS Esters Matrix Standard Sets F 2 Part Numbers 401527 6 FAM Phosphoramidite 85 mg 401533 TET Phosphoramidite 100 mg 401526 HEX Phosphoramidite 105 mg For post synthesis labeling of primers containing a 5 Aminolink 2 400981 TAMRA NHS Ester 5 mg 60 uL in DMSO 400980 ROX NHS Ester 5 mg 60 uL in DMSO 400808 Aminolink 2 0 259 401114 Dye Primer Matrix Standards Kit Filter Set A for NHS ester labeling Contains one tube each of 5 FAM JOE TAMRA and ROX labeled DNA 402792 FJANTP Matrix Standards Contains one tube each of R110 R6G TAMRA and ROX labeled DNA 401546 Fluorescent Amidite Matrix Standards Kit Filter Set C for fluorescent phosphoramidite labeling Contains one tube each of 6 FAM TET HEX TAMRA and ROX labeled DNA 402996 NED Matrix Standard Used in combination with the 5 FAM JOE and ROX dyes in the Dye Primer Matrix Standards Kit or the 6 FAM HEX and ROX dyes in the Fluorescent Amidite Matrix Standards Kit continue
149. epending on the dye set you are using and whether the module is voltage or wattage limiting respectively IMPORTANT Use virtual filter set A modules with dye primers and virtual filter set C or D modules with fluorescent amidites see Table 4 3 on page 4 8 and Creating a Matrix File on page 7 24 Click the filename once to select it and choose Get Info from the file menu Deselect the checkbox labeled Locked and close the Get Info dialog box Double click the ABI PRISM 377 Collection icon if the program is not currently open O OJ a Open the Run window and click the small document icon to the right of the Module pop up menu Enter the revised numbers in the appropriate data fields Click on Save as Default ABI PRISM 377 Collection Software Version 2 0 ABI 373XL and ABI PRISM 377XL and ABI PRISM 377 Collection Software Version 2 1 Step Action 1 Open a GeneScan Run window 2 Select the appropriate module in the Run Module pop up menu 3 Click the small document icon next to the Run Module pop up menu 4 In the Settings dialog box enter the revised numbers in the appropriate data fields 5 Click Save Copy in and rename the new module GS SSCP W A or C or D or GS SSCP V A or C or D depending on the dye set you are using and whether the module is voltage or wattage limiting IMPORTANT Use virtual filter set A modules with dye primers and virtual filter set
150. equivalent add 5 mL of deionized water to the tube Vortex until all crystals dissolve Long Ranger Gels IMPORTANT The 5 Long Ranger FMC Corporation P N 50610 gel solution is hazardous waste Dispose of the unused working solution in accordance with all applicable federal sate and local health and safety regulations After you finish pouring the gel thoroughly clean all work surfaces with water Note A variation of the following protocol suitable for SSCP appears after the protocol To prepare a 36 cm 5 Long Ranger 6 M urea gel Step Action 1 Wash and assemble the glass plates as described in your instrument user s manual Weigh out 18 0 g of urea and transfer it carefully to a graduated cylinder WARNING CHEMICAL HAZARD Urea is a potential mutagen Dangers cited in toxicity studies show reproductive and tumorigenic effects Urea can cause irritation to the skin eyes and respiratory tract Avoid inhalation and contact with skin eyes and clothing Always work in a fume hood Obtain a copy of the MSDS from the manufacturer Wear appropriate protective eyewear clothing and gloves Reagent Preparation A 1 A 2 Reagent Preparation To prepare a 36 cm 5 Long Ranger 6 M urea gel continued Step Action 3 Using a pipette add the following 5 0 mL 50 Long Ranger gel solution concentrate 5 0 mL 10X TBE WARNING CHEMICAL
151. er Y Perform a DNA titration with 1 5 less DNA than the original concentration 1 2 less DNA than the original concentration Twice as much DNA as the original concentration Five times as much DNA as the original concentration Increase the number of PCR cycles from 30 to 33 35 by increasing the second set of melt anneal extend cycles If amplification occurs using samples containing less DNA inhibitors might be present Washing the samples in a Centricon 100 may help remove inhibitors If amplification occurs using samples containing more DNA the original concentration of DNA in the sample may not have been high enough or the sample may be degraded Increasing Signal Strength Y Increase the amount of a particular marker in your sample by adjusting the pooling ratios for that marker Increase the number of PCR cycles from 30 to 33 35 by increasing the second set of melt anneal extend cycles Increase the magnesium chloride concentration by performing a titration as described on page 6 6 Background may increase as well Decrease the annealing temperature 2 3 C at a time Background may increase Decreasing Background Non specific Amplification Y Decrease the amount of the marker used by adjusting the pooling ratios if background is interfering with allele calls of other markers Increase the annealing temperature 2 3 C at a time Overall signal may decrease 9 4 Microsatellite Analysis Applications
152. er Bath Functions ABI PRISM 377 Collection Software version 2 1 contains all required features to accommodate the external cold water bath including new chiller modules Refer to the AB Prism 377 DNA Sequencer Data Collection Software 2 1 Enhancements User Bulletin P N 904945 for software installation instructions descriptions of features and instructions for using the new modules Software Thermistor External Run Conditions Version ohms Bath gt 10 C above ambient to 60 C 1 1 or 2 1 100 k not required SSCP from 22 C to ambient 2 1 100 k required 10 C SSCP at lt 22 C 2 1 30 k required Disengages Internal System The ABI PRISM 377 configuration with the external cold water bath attached is similar to Figure 7 1 when viewed from the back New modules in the ABI PRISM 377 Collection Software version 2 1 disengage the instrument s internal pump system and switch to the external cold water bath When you turn the external valve so the arrow points down access to the internal gel temperature control unit is closed off External valve arrow down o o a o E GR0686 Figure 7 1 Rear view of the ABI PRISM 377 DNA Sequencer with external water bath attached SSCP Analysis 7 5 Standard Configuration
153. ers all of the peaks associated with a single marker c From the Views menu choose Show Histogram Window SS Histogram Microsat template 1 Category Member Locate in Table T T T T T T T T T T 102 103 104 105 106 107 108 109 110 d Make sure the correct marker name is displayed in the Category field Leave the Member field blank Note All labeled peaks in the selected range for a given marker display as vertical bars in the Histogram window Each bar represents a particular size x axis value The height represents the number of labeled peaks found for that size y axis counts If you place the cursor on a particular peak bar Genotyper software displays the corresponding value and counts Draw a box around a bar or group of bars that represent one allele The area inside the box is the allelic bin Genotyper software displays the size range for the bin and the number of peaks found in that range in the status box at the bottom of the window BE Histogram Microsat template 1 Category Member Lyd Locate in Table T T T T T T T T T T 107 108 109 Count 8 Value 104 40 105 00 Category D12583 Unknown To bin alleles using the Histogram window continued Step Action 8 From the Category menu choose Add Category Genotyper software automatically
154. ervation Possible Cause Recommended Action No bands on GeneScan gel PCR amplification failed due to presence of PCR inhibitors Run DNA sample on an agarose gel If DNA is present repurify the sample using the QlAamp Tissue Kit or other commercial kit to remove PCR inhibitors DNA degraded Incorrect DNA loading or mispipetting If no DNA is observed on an agarose gel prepare new DNA from the tissue sample Preferential amplification of shorter PCR products over longer ones Alleles are separated by ten base pairs or more Make sure the DNA is quantitated accurately for both the normal N and tumor T samples 9 16 Microsatellite Analysis Applications Animal Paternity StockMarks Kits Improved Breeding Advantages of DNA based Tests Advantages of Using ABI PRISM Technology The StockMarks for Cattle Bovine Paternity PCR Typing Kit uses 11 microsatellite loci to automate the genotyping of cattle for breeding purposes The StockMarks for Horses Equine Paternity PCR Typing Kit uses 12 microsatellite loci to automate the genotyping of horses for breeding purposes Humans have been breeding cattle and horses selectively for centuries Animals that exhibit superior production traits such as high milk production lean carcasses speed or strength are used as breeding stock for subsequent generations This classical method requires the measurement of quantitative pro
155. erwise Formula O D Aggg X Stock Volume mL x Dilution Factor Dilution Factor Dilution Volume mL Aliquot Volume mL Example 1 Question How many O D units are present in a 1 5 mL PD 10 column fraction A0 1 mL aliquot is brought to 1 0 mL The absorbance at 260 nm is 0 16 in a 1 mL cuvette with a 1 cm path length Answer O D 0 16x1 5mLx1 0mL 2 40 D units 0 1 mL Example 2 Question How many O D units are present in 0 5 mL of a purified dye primer 0 3 mL of this solution is loaded into a 0 3 mL cuvette 1 cm path length and the absorbance at 260 nm is 0 6 Y Since no dilution of the stock occurred the dilution factor is 1 The sample can be recovered from the cuvette and pooled with the rest of the stock dye primer Answer O D 0 6x0 5mLx0 3mL 0 3 O D unit 0 3 mL continued on next page D 10 Preparing 5 End Labeled Primers Converting A to Azgq values can be converted into pg mL using Beer s Law Concentration Absorbance 260 nm sum of extinction coefficient contributions x cuvette pathlength x oligonucleotide concentration The following formulas which are derived from Beer s Law convert Apso readings into pmol uL concentrations Single stranded DNA C pmol uL A250 10 x S where S size of DNA in kilobases Double stranded DNA C pmol L Aggo 13 2 x S where S size of DNA in kilobases Y Oligonucleotides C pmol UL Aso x 100 1 54n 0 75ng 1 17Ng
156. eton Price H R Harding A D Monteiro C Berciano J and Malcolm S 1990 Linkage of hereditary motor and sensory neuropathy type 1 to the pericentromeric region of chromosome 17 Am J Hum Genet 46 92 94 Murray J M Davies K E Harper P S Meredith L Mueller C R and Wiliamson R 1982 Linkage relationship of a cloned DNA sequence on the short arm of the X chromosome to Duchenne muscular dystrophy Nature 300 69 71 Ostrander E A Jong P M Rine J and Duyk G 1991 Construction of small insert genomic DNA libraries highly enriched for microsatellite repeat sequences Proc Nail Acad Sci USA 89 3419 3423 Pritchard L Kawaguchi Y Reed P Copeman J Davies J Barnett A Bain S and Todd J 1995 Analysis of the CD3 gene region and type 1 diabetes application of fluorescence based technology to linkage disequilibrium mapping Hum Mol Genet 4 197 202 Reed P Davies J Copeman J Bennett S Palmer S Pritchard L Gough S Kawaguchi Y Cordell H Balfour K et al 1994 Chromosome specific microsatellite sets for fluorescence based semi automated genome mapping Nature Gen 7 390 395 Schuster H Wienker T E Bahring S Bilginturan N Toka H R Neitzel H Jeschke E Toka O Gilbert D Lowe A Ott J Haller H and Luft F C 1996 Severe autosomal dominant hypertension and brachydactyly in a unique Turkish kindred maps to human chromosome 12
157. example faint bands in a position one repeat unit smaller than the main allele can be interpreted either as a stutter band or as an allele in a minor component of a mixed sample The possibility of stutter makes precise quantitation especially important to enable the Genotyper software s filtering algorithm to interpret the peak pattern accurately Fortunately the percent stutter for a given allele is reproducible In particular the percent stutter does not depend on the quantity of input DNA or the number of loci amplified during multiplex PCR The relative invariance of percent stutter is important for a few reasons In many cases you can adjust the Minimum Peak Height threshold in the Analysis Parameters pop up window of the GeneScan software so that the analysis program ignores stutter peaks while recognizing true allele peaks Results showing an abnormally high percent stutter can indicate mixed samples See page 8 33 for examples of stutter patterns in dinucleotide repeat loci Preparing PCR Products for Analysis Dilution You will often need to dilute PCR amplification products Typically the required dilution lies in the range from 1 3 1 80 PCR product distilled deionized H O or loading buffer A good way to proceed is to begin by optimizing PCR run conditions for your specific application Then run a dilution series on your ABI PRiSM instrument to determine the optimal dilution Alternatively run 1 uL of PCR product
158. ey C T 1991 DNA typing and genetic mapping with trimeric and tetrameric tandem repeats Am J Hum Genet 49 746 756 Fr geau C J and Fourney R M 1993 DNA typing with fluorescently tagged short tandem repeats a sensitive and accurate approach to human identification BioTechniques 15 100 119 Georges M Lathrop M Hilbert P Marcotte A Schwers A Swillens S Vassart G and Hanset R 1990 On the use of DNA fingerprints for linkage studies in cattle Genomics 6 461 474 Ghosh S Karanjawala Z E Hauser E R Ally D Knapp J Rayman J B Musick A Tannenbaum J Te C Shapiro S et al 1997 Methods for precise sizing automated binning of alleles and reduction of error rates in large scale genotyping using fluorescently labeled dinucleotide markers Genome Methods 7 165 178 Gill K S Lubbers E L Gill G S Raupp W J and Cox T S 1991 A genetic linkage map of Triticum tauschii DD and its relationship to the D genome of bread wheat AABBDD Genome 32 362 372 Gyapay G Morissette J Vignal A Dib C Fizames C Millasseau P Marc S Berrnardi G Lathrop M and Weissenbach J 1994 The 1993 94 G n thon Human Genetic Linkage Map Nature Gen 7 246 249 Hall J LeDuc C Watson A and Roter A 1996 An Approach to High throughput Genotyping Genome Res 6 781 790 Hall J M Friedman L Guenther C Lee M K Weber J L Black D M and King
159. f the internal lane size standards that are often seen in SSCP runs it can be difficult for the GeneScan Analysis Software s peak detection algorithm to recognize peaks reproducibly Examine your data for each injection to ensure that the correct peaks are being called Adjust the Peak Height Threshold in the Analysis Parameters window to get the most consistent data Define the size standard so that each unknown peak is flanked by at least two size standard peaks on either side Adjust the values in the Analysis Range section of the Analysis Parameters window to include only the peaks of interest Sodium Hydroxide Using poor quality or old sodium hydroxide solutions can lead to decreased capillary lifetimes IMPORTANT Do not store sodium hydroxide in glass bottles NaOH etches glass SSCP Analysis 7 29 Microsatellite Analysis Overview In This Chapter This chapter provides detailed instructions for performing microsatellite analysis on the ABI PRISM 377 and ABI 373 DNA Sequencers This chapter contains the following topics Topic See Page Introduction to Microsatellite Analysis 8 2 Before You Begin 8 3 PCR Amplification Labeling and Controls 8 5 To Save Time Prerun Checklist 8 7 Preparing for a Run ABI PRISM 377 ABI PRISM 377 with XL Upgrade and 8 8 ABI 373 with XL Upgrade Preparing for a Run ABI 373 8 12 Analyzing the Data Part I Using GeneScan 8 17 A
160. file continued on next page Setting the Analysis For the ABI PRISM 377 ABI PRISM 377 with XL Upgrade and ABI 373 with XL Parameters Upgrade IMPORTANT If you choose to use analysis parameters different from those recommended here be sure to define the Analysis Range to exclude the primer peak Step Action 1 Start the GeneScan Analysis Software version 2 0 2 or higher 2 Choose New from the File menu and click on the Analysis Parameters icon 3 Specify the settings as shown below e untitled Analysis Range Size Call Range O Full Range All Sizes This Range Data Points O This Range Base Pairs Start o Mir Data Processing EJ Baseline EX MultiComponent Smooth Options None Light Heavy Size Calling Method 2nd Order Least Squares O 3rd Order Least Squares O Cubic Spline Interpolation Local Southern Method O Global Southern Method Peak Detection Split Peak Correction Peak Amplitude Thresholds None B Y GENESCAN 2500 G R O LeftMost Peak RightMost Peak Min Peak Half Width Pts Correction Limit 30 _ Data Choose Save As from the File menu Save the settings as SSCP Analysis Parameters 6 Click OK when done Note In SSCP analysis all the peaks including the standard are typically broader than in other types of analysis Therefore we reco
161. fragments Genome Res 6 551 557 Inazuka M Wenz H M Sakabe M Tahira T and Hayashi K 1997 A streamlined mutation detection system multicolor post PCR fluorescence labeling and single strand conformational polymorphism analysis by capillary electrophoresis Genome Res 7 1094 1103 Iwahana H Adzuma K Takahashi Y Katashima R Yoshimoto K and Itakura M 1995 Multiple fluoresce based PCR SSCP analysis with postlabeling PCR Methods Appl 4 275 282 Applied Biosystems 1994 Synthesis and purification of fluorescently labeled oligonucleotides using dye phosphoramidites In User Bulletin Number 78 for ABI 38x 39x DNA Synthesizers Foster City CA Applied Biosystems Applied Biosystems 1991 New applications for the oligonucleotide purification cartridge In User Bulletin Number 59 for ABI 38x 39x DNA Synthesizers Foster City CA Applied Biosystems References E 1 E 2 References SSCP Applied Biosystems 1991 40 nanomole polystyrene New highly efficient DNA synthesis columns In User Bulletin Number 61 for ABI 38x 39x DNA Synthesizers Foster City CA Applied Biosystems Applied Biosystems 1992 4FAM amidite fluorescent dye labeling of oligonucleotides on the DNA synthesizer In User Bulletin Number 67 for ABI 38x 39x DNA Synthesizers Foster City CA Applied Biosystems Eadie J S McBride L J Efcavitch J W Hoff L B and Cathcart R 1987 High performance liquid chromatographic anal
162. g Pipettes Pipette tips Microcentrifuge tubes Gloves Use AmpErase UNG in reaction mixtures to prevent the subsequent reamplification of dU containing PCR products For More Applied Biosystems supplies the GeneAmp PCR Carryover Prevention Kit Information P N N808 0068 and AmpErase UNG P N N808 0096 to ensure that PCR products cannot be reamplified in subsequent PCR amplifications Optimizing PCR 6 17 3 A Nucleotide Addition Introduction The AmpliTaq and AmpliTaq Gold DNA Polymerases like many other DNA 6 18 Optimizing PCR polymerases catalyze the addition of a single nucleotide usually an adenosine to the 3 ends of the two strands of a double stranded DNA fragment This non template complementary addition results in a denatured PCR product that is one nucleotide longer than the target sequence A PCR product containing the extra nucleotide is referred to as the plus A form Why Incomplete 3 A Nucleotide Addition Can Cause Problems Because 3 A nucleotide addition rarely goes to completion without a long extension step at the end of thermal cycling e only a fraction of the fragments receive the extra nucleotide single base ladders often form Figure 6 1 creating peak patterns that Genotyper software might not interpret correctly The resulting allele calls can be inconsistent incorrect or missing entirely forcing you to inspect all allele calls and to correct erroneous calls m
163. g Samples Note Often you will need to dilute the PCR product from 1 5 1 20 in distilled deionized H O before loading 1 uL into the sample tube Step Action 1 Prepare Loading Mix sufficient for loading 100 samples 250 uL deionized formamide 50 uL GeneScan size standard GeneScan 350 GeneScan 400 HD or GeneScan 500 recommended 25 uL Loading Buffer 25 mM EDTA 50 mg mL blue dextran WARNING CHEMICAL HAZARD Formamide is a known teratogen It can cause birth defects Wash thoroughly after handling formamide Obtain a copy of the MSDS from the manufacturer Wear appropriate protective eyewear clothing and gloves Wash thoroughly after handling formamide Note Vortex gently for 3 5 seconds Loading Mix lasts for 2 weeks at 2 6 C 2 To each sample tube add the following 3 0 uL Loading Mix 1 pL PCR product 3 Place the samples in a heat block or thermal cycler for 5 minutes at 95 C Remove the tubes from the heat source and place immediately in an ice water bath 5 Load 4 uL of sample into each gel lane Preparing the Matrix Samples Step Action 1 To each of four sample tubes add the following 2 5 uL deionized formamide 2 5 uL of one of the matrix standards Dye Primer Fluorescent Amidite or NED matrix standard WARNING CHEMICAL HAZARD Formamide is a known teratogen It can cause birth defects Wash thoroughly after handling formamide Obtain a copy
164. gene product Science 251 802 804 Bronner C E Baker S M Morrison P T et al 1994 Mutation in the DNA mismatch repair gene homolog hMLH1 is associated with hereditary non polyposis colon cancer Nature 368 258 261 Canzian F Salovaara R Hemminki A Kristo P Chadwick R B Aaltonen L A and de la Chapelle A 1996 Semiautomated assessment of loss of heterozygosity and replication error in tumors Cancer Res 56 3331 3337 Cawkwell L Lewis F A and Quirke P 1994 Frequency of allele loss of DCC p53 WT1 NF1 NM23 and APC MCC in colorectal cancer assayed by fluorescent multiplex polymerase chain reaction Br J Cancer 70 813 818 References E 7 E 8 References Cawkwell L Bell S M Lewis F A Dixon M F Taylor G R and Quirke P 1993 Rapid detection of allele loss in colorectal tumors using microsatellites and fluorescent DNA technology Br J Cancer 67 1262 1267 Chuaqui R F Sanz Ortega J Vocke C ef al 1995 Loss of heterozygosity on the short arm of chromosome 8 in male breast carcinomas Cancer Res 55 4995 4998 Cher M L Bova G S Moore D H et al 1996 Genetic alterations in untreated metastases and androgen independent prostate cancer detected by comparative genomic hybridization and allelotyping Cancer Res 56 3091 3102 Cooper C S 1992 The met oncogene from detection by transfection to transmembrane receptor for hepatocyte growth factor Oncogene 7 3 7 Co
165. h the dyes in the Dye Primer Matrix Standards Kit 5 FAM JOE TAMRA and ROX 100 80 60 SY N ad 40 S a EE EA A J SS 20 NORMALIZED FLUORESCENCE INTENSITY ZA WAVELENGTH nm Filter 1 Filter 2 Filter 3 Filter 4 TAMRA ROX continued on next page Virtual Filter Set C Virtual Filter Set D The following figure demonstrates why matrix problems can occur using this virtual filter set The light collection windows for 6 FAM and TET are close to one another and the spectral overlap between the two dyes is significant me 00 MDBW a aon NORMALIZED FLUORESCENCE INTENSITY ER WSS 80 60 40 20 SS 4 520 540 560 580 600 620 640 WAVELENGTH nm Filter 1 Filter 2 Filter 3 Filter 4 HEX TAMRA The spectral resolution of this dye virtual filter set combination is much greater than the spectral resolution of 6 FAM TET HEX and TAMRA with Virtual Filter Set C Switching to this combination reduces the potential for matrix problems and may yield cleaner data Xx K NORMALIZED FLUORESCENCE INTENSITY y WAVELENGTH nm Filter 1 Filter 2 Filter 3 Filter 4 continued on next page ABI PRISM Dyes 4 11 Virtual Filter Set F 4 12 ABI PRISM Dyes The spectral resolution of this dye virtual filter set combination is similar to the spectral resolution of 5 FAM JOE TAMRA and ROX with Virtual Filter Set
166. he ABI Prism BigDye Filter Wheel was developed Its filter sets are as follows Filter Set A 540 nm 570 nm 595 nm and 625 nm Y Filter Set B 540 nm 555 nm 570 and 595 nm From this it follows that the only adequate 4 dye combination available for GeneScan applications would be TET HEX NED and ROX on Filter Set B see Table 4 1 on page 4 5 Therefore we do not recommend performing GeneScan experiments on ABI 373 instruments with the ABI PRISM BigDye Filter Wheel installed Refer to the ABI PRISM BigDye Filter Wheel User Bulletin P N 4304367 for more information ABI PRISM Dyes 4 9 Emission Spectra for Representative Dye Virtual Filter Set Combinations About the Spectra Virtual Filter Set A 4 10 ABI PRISM Dyes In the following four figures the collection windows of the four GeneScan virtual filter sets are shown overlaid with the normalized emission spectra of a recommended dye set The wavelength of maximum emission of the dyes shown in these figures differs from the value given in Table 4 1 on page 4 5 for the following reasons The long pass blocking filter see footnote on page 4 6 blocks much of the light emitted by 6 FAM and 5 FAM the dyes with emission maxima closest to the laser wavelength The environment of the gel polymer shifts the emission spectra of ABI PRISM dyes See Factors That Affect Spectra on page 4 4 for an explanation of the observed shift Virtual Filter Set A is used wit
167. he probability that two individuals are related Their even distribution in the genome makes them very good markers for constructing genetic maps Edwards et al 1992 PCR based microsatellite analysis has the following advantages over conventional genotyping methods e g Restriction Fragment Length Polymorphism RFLP The small size of microsatellite loci improves the chance of obtaining a result particularly for samples containing minute amounts of DNA and or degraded DNA The small size range of microsatellite loci makes them ideal candidates for co amplification while keeping all amplified alleles smaller than 350 base pairs Many microsatellite loci can therefore be typed from a single PCR Y Microsatellite alleles have discrete sizes allowing for simplified interpretation of results PCR based tests are rapid giving results in 24 hours or less PCR based tests are easy to standardize and automate ensuring reproducible results To exploit the potential for increased throughput using ABI PRISM multicolor fluorescent dye technology you will want to multiplex electrophoresis by co loading the products of multiple PCR reactions in the same lane The ABI PRISM 377 and ABI 373 instruments allow extremely rapid separations Fragments that are 300 bp or less in length can be separated in 2 hours 4 hours on the ABI 373 Before You Begin Materials Required Software Required You will need the following materials to
168. he size standard peaks did not lie on the sizing curve for any samples define a new size standard for those samples as described in the GeneScan Analysis Software User s Manual and reanalyze Second Method Step Action 1 Select Results Control from the Window menu 38 R 2 From the View menu select Align By Size Note If fragments are aligned by size this option will not be available 3 Examine the GeneScan size standard peaks in overlapping groups of 16 samples Quick Tile Off Verify that the size standard peaks are superimposed as shown below General Analysis and Evaluation Techniques 3 5 Second Method continued Step Action EE 210 240 270 E 2700 tt i 4 BR A692 BE 7R 47 3 BR A894 MM sr ases 10R A1096 2400 2100 Size Peak Height Peak Area Data Point 4571 4738 4894 5054 a i 5214 29 644 6244 5294 4 In the Results display table peaks in the size standard definition are marked with a bullet as shown below Minutes Scroll through the tables to verify the size standard peak assignments and note which samples if any have incorrect assignments 5 Define a new size standard for those samples with incorrect peak assignments as described in the GeneScan Analysis Software User s Manual 3 6 General Analysis and Evaluation Techniques Third Method
169. hemical forms table of 4 3 dye spectra understanding 4 4 to 4 5 wavelengths table of 4 5 dye filter sets choosing 4 6 to 4 8 chemical forms table of 4 7 reagents and primer dye sets table of 4 8 recommended combinations table of 4 8 dye filter sets emission spectra 4 10 to 4 12 Virtual Filter Set A 4 10 Virtual Filter SetC 4 11 Virtual Filter Set D 4 11 Virtual Filter Set F 4 12 E electrophoresis conditions optimizing 2 9 to 2 10 electrophoresis changes and how effects size calling 5 6 e mail address technical support emission spectrum definition of enzyme choosing 6 8 to 6 9 enzyme choice table 6 9 PCR enzyme overview 6 8 to 6 9 concentration determining reagent concentration 6 7 evaluating data quality 3 8 to 3 9 bad data example 3 9 good data example 3 8 excitation efficiency definition of excitation spectrum definition of excitation wavelength table of experiment design factors See GeneScan fragment analysis design factors 1 3 4 4 4 4 4 4 4 5 F filter sets available sets 4 7 See Also individual filter set filter wheel 4 2 4 6 ABI PRISM BigDye Filter Wheel 4 9 fluorescent dNTPs See FIANTPs fluorescent labeling 2 2 to 2 4 available dyes 4 2 control DNA using 2 11 dye spectra understanding 4 4 to 4 5 wavelengths table of 4 5 dye filter sets choosing 4 6 to 4 8 chemical forms table of 4 7 reagents and primer dye set table of 4 8 recommended combinations table of 4 8 dye fi
170. hermal Cycler 480 Push reaction tubes firmly into contact with block after first cycle Repeat amplification GeneAmp PCR System 9600 heated cover misaligned Align the heated cover so that white stripes align after twisting the top portion clockwise 11 2 Troubleshooting Table 11 1 Problems with Poor Amplification continued Observation Possible Causes Recommended Actions Faint or no signal from sample DNA and from positive control Poor thermal cycler performance Check instrument calibration Use a Applied Biosystems thermal cycler Good signal from positive control but faint or no signal from sample DNA Sample contains PCR inhibitor for example heme compounds EDTA or certain dyes Quantitate DNA Dilute if possible in order to add minimum necessary volume Repeat amplification Wash the sample in an Amicon Centricon 100 column and repeat amplification Note For fragments smaller than 130 bp use the Amicon Centricon 30 column instead Add bovine serum albumin BSA to the PCR reaction mixture Use 8 16 ug BSA for every 50 uL PCR reaction volume Sample DNA is degraded Evaluate the quality and concentration of the DNA sample by Using the QuantiBlot Human DNA Quantitation Kit for human DNA Running an agarose yield gel If DNA is degraded or inaccurately quantitated reamplify with an increased amount of DNA Insufficient sample
171. ial disk supplied with the installation disks for Genotyper 2 0 software contains the data files continued on next page Microsatellite Analysis 8 19 Using the Histogram Window 8 20 Microsatellite Analysis To bin alleles using the Histogram window Step Action 1 Define the bin size as follows a From the Analysis menu choose Set Statistics Options b Select the following buttons as shown below Plot selection Size in bp Starting bin determined automatically c Enter 0 10 in the Bin size field Set Statistics Options source ___ _Jalue Plot selection 8 Size in bp O Range of first selected category O Scan number O Fined range 0 00 to 100 00 O Peak height O Table selection O divided by scale factor O Table column s Ly O Peak area to Ly O divided by scale factor Label text Value in table column when name of first selected category group Cell value is in table column Ly O Cell text Bin size Starting bin 8 Determined automatically Note A bin size of 0 1 bp gives the most precise allele binning If insufficient data is available however the Genotyper software displays an error message stating that the bin size is too small If this occurs increase the bin size From the View menu choose Show Categories Window or ty
172. if any peak is greater than 4000 RFU on the ABI 373 greater than approximately 5000 RFU in the raw data on the ABI PRISM 377 Dilute PCR sample before adding to formamide size standard mix Reamplify using less DNA Incorrect matrix chosen or poor matrix Check matrix selection If correct you may want to create a new matrix Extra peaks when sample is known to contain DNA from a single source If extra peaks are 1 4 nt larger or smaller than the expected peak it may be a PCR artifact See Problems with Extra Peaks on page 11 5 Samples not fully denatured Make sure the samples are heated at 95 C for 5 minutes prior to loading Unoptimized PCR Check efficiency of the PCR See Chapter 6 Optimizing PCR for detailed suggestions Renaturation of denatured samples Load samples immediately following denaturation or store on ice until you are ready to load IMPORTANT Do not store samples on ice for more than 2 hours before loading Note Too much DNA also promotes renaturation but before you add less DNA you will need to assess the signal strength and quality Well spillover Follow the recommended sample loading protocol Gel image not showing upper or lower bands Gel ran slower or faster than expected Adjust scan numbers for Display Gel Image under Gel Processing Parameters and choose Regenerate Gel Image Gel polymer concentration is inco
173. ignal strength TAMRA HEX JOE NED 5 FAM 6 FAM TET highest signal strength o Refer to Chapter 4 for more information about ABI PRISM dyes dye spectra and the argon ion laser Decreasing the Salt Concentration If the salt concentration in loaded samples is too high the samples will run more slowly than expected You may also notice exaggerated bowing or smiling along the length of the gel For best results you should desalt and potentially concentrate the samples To desalt Use an Amicon Centricon 100 Microconcentrator or Centricon 30 for fragments smaller than 130 base pairs in length Precipitate the pooled PCR product and resuspend in TE 4 10 mM Tris 0 1 mM EDTA buffer or distilled deionized water Experimental Design Considerations 2 3 Y Dialyze the sample on a filter membrane to remove salt from the solution Step Action 1 Float a Millipore VS filter Millipore P N VSWP 02500 shiny side up on top of 50 mL of deionized autoclaved water in a 50 mL conical plastic tube 2 Carefully spot approximately 15 uL of sample on top of the filter using an appropriate pipette Dialyze the sample for 20 minutes 3 Using a pipette very carefully remove the sample and dilute Note Do not increase sample concentration by evaporating the samples without performing a desalting step This increases the salt concentration making it more difficult to denature the
174. in a mixed population of cells A comparison of SSCP and direct sequencing GATA 9 143 145 Suzuki Y Orita M Shiraishi M Hayashi K and Sekiya T 1990 Detection of ras gene mutations in human lung cancers by single strand conformation polymorphism analysis of polymerase chain reaction products Oncogene 5 1037 1043 Takahashi Fuji A Ishino Y Shimada A and Kato I 1993 Practical application of fluorescence based image analyzer for PCR single stranded conformation polymorphism analysis used in detection of multiple point mutations PCR Methods Appl 2 323 327 continued on next page References E 3 E 4 References STR Beckman J S and Weber J L 1992 Survey of human and rat microsatellites Genomics 12 627 631 Boerwinkle E Xiong W Fourest E and Chan L 1989 Rapid typing of tandemly repeated hypervariable loci by the polymerase chain reaction Application to the apolipoprotein B 3 hypervariable region Proc Natl Acad Sci USA 86 212 216 Bonyadi M Rusholme S Cousins F Su H Biron C Farrall M and Akhurst R 1997 Mapping of a major genetic modifier of embryonic lethality in TGF1 knockout mice Nature Gen 15 207 211 Brennan M and Hochgeschwender U 1995 Commentary So many needles so much hay Hum Mol Genet 4 153 156 Brownstein M Carpten J and Smith J 1996 Modulation of non templated nucleotide addition by Tag DNA polymerase Primer modification
175. ine position in the plate check window is from 800 1000 on the y axis Press MAIN MENU when done Note The instrument settings should be as follows for an SSCP run Preparing and Preparing and Loading Experimental Samples Loading Samples Note Sometimes it is necessary to dilute the PCR product from 1 5 to 1 20 in distilled deionized H O before adding 1 L to the sample tube Step Action 1 To each sample tube add the following 3 0 uL deionized formamide 0 5 uL GeneScan Internal Lane Size Standard GeneScan 350 or GeneScan 500 recommended 1 pL PCR product 0 5 uL of 0 1 N NaOH 0 5 uL EDTA blue dextran 50 mM EDTA 50 mg mL blue dextran WARNING CHEMICAL HAZARD Formamide is a known teratogen It can cause birth defects Wash thoroughly after handling formamide Obtain a copy of the MSDS from the manufacturer Wear appropriate protective eyewear clothing and gloves Wash thoroughly after handling formamide WARNING CHEMICAL HAZARD Sodium hydroxide NaOH can cause severe burns to the skin eyes and respiratory tract Always work in a fume hood Obtain a copy of the MSDS from the manufacturer Wear appropriate protective eyewear clothing and gloves Note For convenience you can combine the formamide NaOH GeneScan size standard and EDTA blue dextran into a master mix 300 uL deionized formamide 50 uL 0 1 N NaOH 50 uL GeneScan size standard 50 yL EDTA blue dextran This
176. ing 7 29 artifact peaks result of too much signal 2 8 7 2 B Beer s Law converting Azgo to concentration D 11 buffers preparing TBE A 7 C cattle StockMarks 9 18 CEPH 1347 02 part number See Also DNA co electrophoresis guidelines 2 2 contamination avoiding 6 16 to 6 17 conventions used 1 2 2 11 cross platform size comparing sizes 5 3 to 5 4 precision results table of 5 4 Custom Oligonucleotide Synthesis Service help 2 7 custom primers storage of D 9 See Also primers designing custom primers D data analyzing 3 2 to 3 7 sample files 3 2 size standard peak assignments verifying 3 5 to 3 7 evaluating data quality 3 8 to 3 9 bad dataexample 3 9 good dataexample 3 8 data collection how it works 4 6 deionized formamide preparing A 7 design factors 2 2 to 2 11 control DNA using 2 11 electrophoresis conditions optimizing 2 9 to 2 10 fluorescent labeling methods choosing 2 5 to 2 7 samples determining loading concentrations 2 8 working with multiple colors 2 2 to 2 4 ensuring signal intensity 2 3 to 2 4 guidelines 2 2 increasing throughput multiplexing 2 2 to 2 3 diluting PCR amplification products 6 23 Documents on Demand telephone number 1 4 DNA control DNA using 2 11 control for microsatellite marker analysis 8 6 salt concentration decreasing 2 3 dNTP concentration when determining reagent concentration 6 6 dyes accounting for differences in signal strengths 2 3 available dyes 4 2 c
177. ing Samples for each locus Two amplifications are run for each microsatellite one using sample genomic DNA isolated from normal cells and one using sample DNA isolated from tumor cells from the same individual An GeneScan Internal Lane Size Standard is added to each sample before denaturation and loading The amplification products are separated and detected using the ABI PRISM 310 Genetic Analyzer and the data is analyzed using GeneScan Analysis Software Further analysis can be done using Genotyper software LOH run setup and operation in preparation for data analysis is the same as for the basic microsatellite protocol given in Chapter 8 with the following minor modifications Run two DNA samples from each individual One from normal tissue N One from tumor tissue T Note Some normal tissue contaminating the tumor tissue sample is typical 4 Run 3 4 independent lanes for each sample N and T This allows you to obtain sufficiently accurate quantitative estimates for subsequent data analysis In addition to the controls suggested for basic microsatellite analysis see Control DNA on page 8 6 obtain 8 10 confirmed N T sample pairs to test the ability to detect LOH Preventing Contamination When using the PCR process certain laboratory practices are necessary in order to avoid false positive amplifications Kwok and Higuchi 1989 This is because the PCR process is capable of amplifying single DNA mo
178. ing for a Run ABI 373 7 19 Analyzing the Data 7 24 Optimizing SSCP Run Conditions 7 26 Troubleshooting 7 29 SSCP Analysis 7 1 Introduction to SSCP Analysis What is SSCP Advantages Limitations Advantages of Using ABI PRISM Technology For More Information 7 2 SSCP Analysis Single strand conformation polymorphism SSCP analysis is an application that detects mutations based upon the ability of a single or multiple nucleotide change to alter the electrophoretic mobility of a single stranded DNA molecule under non denaturing conditions Under non denaturing conditions most single stranded DNA molecules will assume one or more stable three dimensional conformations that depend on the nucleotide sequence In many cases the change of a single nucleotide will cause a conformational change that can be detected as a change in the electrophoretic mobility as compared to the wild type sequence You can analyze a large number of samples using SSCP technique because the technique is simple and fast The only step necessary after PCR amplification is a heat denaturation in formamide and NaOH Moreover as with any PCR based technique you can analyze mutations in a specified DNA region by choosing PCR primers that span that region SSCP analysis indicates only that a mutation exists You must perform subsequent DNA sequencing to determine the nature of the mutation that caused an electrophoretic mobility shift in a gi
179. ion For example allow a linear increase in extension time with increasing cycle number or increase time at the denaturation plateau PCR Master Mix not well mixed before aliquoting Vortex PCR Master Mix thoroughly Primer concentration too low Use the recommended primer concentration Primers degraded Use new primers Note _Preincubation at 95 C for 5 10 minutes should inactivate proteases or nucleases Note To prevent primer degradation during storage store primers at 15 to 25 C either lyophilized or in TE Avoid excessive more than 3 4 freeze thaw cycles Too little free Mg2 in reaction Check that you added sufficient total Mg given concentration of the dNTPs and EDTA Note Free Mg Total Mg2 Total dNTP 2 EDTA Incorrect pH Verify buffer pH and buffer concentration Wrong PCR tube Use Applied Biosystems GeneAmp Thin Walled Reaction Tubes for the DNA Thermal Cycler 480 Y MicroAmp Reaction Tubes with Caps for the GeneAmp PCR Systems 9600 and 2400 MicroAmp Base used with tray retainer set and tubes in GeneAmp PCR System 9600 or 2400 Remove MicroAmp Base from tray retainer set and repeat amplification Verify GeneAmp PCR System protocols and programmed parameters Refer to the thermal cycler user s manual and check instrument calibration Tubes not seated tightly in the thermal cycler during amplification DNA T
180. ion concentration 6 6 template concentration 6 7 preparation A 1 to A 7 10 ammonium persulfate solution A 1 5 Long Ranger gels A 1 to A 3 MDE gels A 6 polyacrylamide gels A 3 to A 5 references 5 end labeled primers E 1 loss of heterozygosity screening for E 7 optimizing PCR E 1 post labeling with FIdNTPs_ E 1 replication error screening for E 7 shorttandem repeat markers E 4 to E 7 single strand conformation polymorphism SSCP E 2 to E 3 replication error screening for 9 15 advantages 9 13 examples 9 14 limitations 9 13 literature references E 7 troubleshooting 9 16 whatis RER 9 13 RER See replication error screening for resolution definition of 2 9 resolution modifying run voltage effect of 2 9 to 2 10 RNA templates using to optimize PCR 6 12 run time modifying 2 9 9 13 to S salt concentration decreasing for adequate signal intensity 2 3 samples loading concentrations determining 2 8 loading volumes B 1 short tandem repeat markers human identification 9 19 to 9 25 advantages of STRs 9 19 AmpFISTR kits 9 22 AmpFISTR loci table Index 6 of 9 21 automated sizing and genotyping 9 20 for more information 9 25 high throughput about 9 20 using allelic ladders 9 23 to 9 24 using STRs 9 19 literature references E 4 to E 7 See Also microsatellite marker analysis signal intensity adjusting See samples loading concentrations determining signals problems how to proceed 2 8 single stran
181. ion to 5 end Labeling Ease of Preparation Designing Fluorescently labeled Primers You can prepare 5 end labeled primers on any Applied Biosystems DNA synthesizer using one of three dye phosphoramidites 6 FAM HEX or TET Fluorescently labeled primers are as easy to make as unlabeled oligonucleotides The principles of custom primer design are the same for fluorescently labeled primers as for unlabeled primers used in traditional procedures Choose a sequence for the custom primer that binds to the desired template location with maximum stability and specificity and with minimum destabilization of internal structures For guidelines on choosing sequences for custom primers see page 6 3 continued on next page D 2 Preparing 5 End Labeled Primers Dye Preparation For spectral homogeneity all three dye phosphoramidites are prepared from single isomer fluorescein dyes 6 carboxyfluorescein and its tetra and hexachlorinated analogs The phosphoramidites are synthesized purified and formulated specifically for use on all Applied Biosystems nucleic acid synthesizers The dyes and their linkers are stable under standard detritylation coupling capping and oxidation conditions Figure D 1 shows the structures of the dye phosphoramidites C CHs 3 gt 6 FAM OCH2CH2ON HN P O NCH CHs s HgC 3C C CHs 3 HEX OCCH2CH2ON HN P O NCH CHs s H3C 3C C ChHa3 3 TET OCH2CH2ON A ae OT NCH CHa o Figure
182. ions Use diluted dye phosphoramidites as soon as possible Coupling efficiency may fall below 90 after four days on the instrument Although OPC purification will remain effective less fluorescently labeled oligonucleotide may be produced IMPORTANT As with nucleoside phosphoramidites dye phosphoramidites in solution undergo significant loss of coupling efficiency if removed from the DNA synthesizer regardless of care or technique Preparing 5 End Labeled Primers D 9 Calculating Absorbance for DNA Samples Introduction Optical Density Spectrophotometric absorbance readings are used to determine quantitative information about DNA samples The method of expressing this information differs between research specialties however creating the potential for confusion and error For example the O D unit is the standard of DNA quantification in the field of DNA synthesis In molecular biology concentration values of pmol uL or ug L are far more relevant The information in this section is designed to help you correctly convert absorbance information into several different units Not all of these conversions are for the procedures outlined in this appendix they are provided for your convenience Definition One Optical Density O D unit is the amount of a substance dissolved in 1 0 mL which gives an absorbance reading of 1 00 in a spectrophotometer with a 1 cm path length The wavelength is assumed to be 260 nm unless stated oth
183. ions All changes in electrophoresis parameters buffers and gel composition affect the fragment migration rate and can therefore affect sizing 5 6 Sizing and Size Standards GeneScan Internal Lane Size Standards Definition Internal size standards are fluorescently labeled DNA ladders that you load in the same lane as your experimental samples The size standard fragments are subject to the same electrophoretic forces as the experimental samples and compensate for lane to lane variation in these forces The uniform spacing of size standard fragments ensures precise size calling throughout the size calling range Available Standards Applied Biosystems provides five different size standards labeled with either TAMRA or ROX GeneScan 350 GeneScan 400HD GeneScan 500 or 500XL GeneScan 1000 only available labeled with ROX GeneScan 2500 IMPORTANT Choose a size standard such that there at least two size standard fragments larger than your largest unknown fragment Sizing and Size Standards 5 7 GeneScan 350 Size Standard Useful Range Fragment Lengths Preparation Denaturing Electropherogram You can use the GeneScan 350 size standard to determine fragment lengths between 35 and 350 base pairs The following table lists the lengths of the 12 fragments comprising the GeneScan 350 size standard Table 5 2 GeneScan 350 Fragment Lengths nt 35 139 250 50 150 300 75 160 340 100 200 350 The GeneSca
184. is most likely to occur between G and T but A C and G A pairs can also be stable Hunter 1986 All software programs have difficulty modeling these sorts of interactions Minimizing Binding to Other Primers Complementarity between two primers especially at the 3 ends can lead to the formation of product artifacts arising from amplified primer dimers and primer oligomers Avoid primers with regions of complementarity between members of a primer pair or pairs Adding 5 primer extensions that are not complementary to the template can facilitate a variety of useful post amplification manipulations of the PCR product without adversely affecting yield Examples include 5 extensions that contain restriction sites universal primer binding sites or promoter sequences 1 A gapped duplex can form when the primer and target are completely complementary except for a single base Miller Kirchoff et al 1987 Miller Wlodawer et al 1987 Optimizing PCR 6 5 Determining Reagent Concentrations Factors to Consider dNTP Concentration Magnesium Ion Concentration 6 6 Optimizing PCR When preparing reaction mixtures consider the following factors that can affect overall yield of specific DNA target sequences dNTP concentration Magnesium ion concentration Primer concentration Template concentration gt Enzyme concentration In the standard GeneAmp PCR protocol the concentration of each deoxynucleoside t
185. is recommended when a high degree of fidelity is required Derivatives of Tth DNA Polymerase Applied Biosystems supplies two modified forms of Thermus thermophilus Tth DNA Polymerase Y rTthDNA Polymerase rTth DNA Polymerase is obtained by expression of a modified form of the Tth gene in an E coli host Y rTih DNA Polymerase XL rTth DNA Polymerase XL Extra Long provides the same features as r7th DNA Polymerase for target sequences from 5 40 kb in length An inherent 3 5 exonuclease activity allows for the correction of nucleotide misincorporations that might otherwise terminate synthesis prematurely If your application has special requirements the table below will help you choose the best enzyme Table 6 2 Application Requirements and Recommended Enzymes If your application requires Use High specificity AmpliTaq Gold DNA Polymerase High sensitivity AmpliTaq Gold DNA Polymerase High fidelity UlTma DNA Polymerase High temperatures AmpliTag DNA Polymerase Stoeffel Fragment UlTma DNA Polymerase Multiplex PCR AmpliTaq Gold DNA Polymerase Amplification of low copy number template AmpliTaq Gold DNA Polymerase AmpliTaq DNA Polymerase LD for bacterial sequences High specificity at high ionic strength AmpliTaq Gold DNA Polymerase AmpliTaq DNA Polymerase Stoeffel Fragment Amplification of extra long fragments gt 5 kb rTth DNA Polyme
186. itch Matrix File and Switch Sample Sheet buttons Preprocess Info E Multi Component _ Process Amount Matrix File E Start at Scan 100 373 dye amidite matrix rood _ Switch Matrix File End at Scan 10000 EJ Use Sample Sheet Process Functions Sheet Name A Auto Lane Track 12 11 98 Sample Sheet A Baseline Data Switch Sample Sheet Gel Scaling Estimated Peak Heights 4096 IMPORTANT If you have not yet created a matrix file for microsatellite analysis do not select the Multi Component checkbox Continue with normal run setup and electrophoresis and then see Creating a New Matrix File on page 8 16 for further instructions You will need to reprocess the gel file after running the matrix samples and creating the matrix file Note Refer to the GeneScan 672 Software User s Manual for more information on creating and using sample sheets Click OK when done Microsatellite Analysis 8 13 Instrument Setup and Prerun 8 14 Microsatellite Analysis To set the Analysis Parameters Step Action 1 Select Analysis Parameters from the Analysis menu 2 Set the parameters as shown below Analysis Parameters Smooth Options None Light Heavy Minimum Peak Heights Per Dye Blue Dye 50 Yellow Dye so Green Dye so Red Dye 50 erame Averaging
187. ited Kingdom Warrington Cheshire Tel 44 0 1925 825650 Fax 44 0 1925 282502 Switzerland Rotkreuz Tel 41 0 41 799 7777 Fax 41 0 41 790 0676 South East Europe Zagreb Croatia Tel 385 1 34 91 927 Fax 385 1 34 91 840 Middle Eastern Countries and North Africa Monza Italia Tel 39 0 39 8389 481 Fax 39 0 39 8389 493 Africa English Speaking and West Asia Fairlands South Africa Tel 27 11 478 0411 Fax 27 11 478 0349 All Other Countries Not Listed Warrington UK Tel 44 0 1925 282481 Fax 44 0 1925 282509 Japan Japan Hatchobori Chuo Ku Tokyo Tel 81 3 5566 6100 Fax 81 3 5566 6501 Eastern Asia China Oceania Australia Scoresby Victoria Malaysia Petaling Jaya Tel 61 3 9730 8600 Tel 60 3 758 8268 Fax 61 3 9730 8799 Fax 60 3 754 9043 China Beijing Singapore Tel 86 106238 1156 Tel 65 896 2168 Fax 86 10 6238 1162 Fax 65 896 2147 Eastern Asia China Oceania Hong Kong Tel 852 2756 6928 Fax 852 2756 6968 Taiwan Taipei Hsien Tel 886 2 2698 3505 Fax 886 2 2698 3405 Korea Seoul Tel 82 2 593 6470 6471 Fax 82 2 593 6472 Thailand Bangkok Tel 66 2 719 6405 Fax 66 2 319 9788 Introduction 1 7 Experimental Design Considerations Using GeneScan Analysis Software to Analyze DNA Fragments Introduction The GeneScan Analysis Software analyzes the data
188. itions should be set to No For 0 2 umol 1 umol and 10 umol scale syntheses use 0 1 M phosphoramidites For 40 nmol scale syntheses use 0 05 M phosphoramidite and the 0 2 umol scale cycle 4 Choose Trityl on Consumption Dye phosphoramidites yield the following approximate number of couplings Couplings Scale M Cycle 12 40 nmol 0 05 0 2 umol 6 0 2 umol 0 1 0 2 mol 4 1 mol 0 1 1 0 umol 1 10 umol 0 1 10 umol Deprotecting Perform ammonia deprotection for 4 hours at 55 C IMPORTANT Monitor the deprotection time and temperature for HEX labeled oligonucleotides with special care Extended treatment in concentrated aqueous ammonia will cause degradation of the dye and subsequent formation of side products Although these impurities can be removed by OPC purification corresponding product yields will be lower continued on next page D 6 Preparing 5 End Labeled Primers Purifying Purify with the Oligonucleotide Purification Cartridge OPC following the protocol Analyzing the Oligos below The selective OPC media binds only the fluorescently labeled product Unlabeled impurities are washed away For more details regarding OPC purification see DNA User Bulletin 59 New Applications for the Oligonucleotide Purification Cartridge March 1991 Step Action 1 Dry the crude fluorescently labeled oligonucleotide and dissolve it in 1 mL of 0 1 M TEAA P N 400613 2 Pass 5 mL of dry acetoni
189. ity of mutation detection in a slab gel format enabling effective analysis of fragments as large as 800 bp in length SSCP has been performed successfully at temperatures ranging from 4 40 C If your lab setup permits try run temperatures below 30 C first Sometimes however raising the temperature above 30 C improves results 40 C appears to be an empirical upper limit to improving performance Figure 7 4 shows mobility differences between p53 mutant and wild type SSCP samples at 25 C 30 C and 35 C for both forward blue and reverse strands green The Namalwa sample left gives the best differentiation between wild type and mutant at 25 C the H596 sample middle gives the best differentiation at 30 C and the Colo320 sample right gives the best differentiation at 35 C Namalwa H596 Colo 320 10 60 0 50 4 8 10 E g 0 20 ES g 20 3 10 See 3 3 0 F 10 20 50 30 2556 30 C 35 C 25 C 30 C 35 C 25 C 30 C 35 C Temperature Temperature Temperature Figure 7 4 Temperature dependence of p53 mutant and wild type SSCP mobility profiles Mutant sample mobilities are given as differences in scan number from the wild type sample mobilities defined as zero Forward strands are shown in blue and reverse strands are shown in green For the ABI PRISM 377 Note The ABI PRISM 377 DNA Sequencer is equi
190. ive this method has serious limitations because it Requires a post PCR enzymatic treatment step Y Requires titrating each lot of T4 DNA polymerase to determine optimal enzyme concentrations and treatment times IMPORTANT Excess T4 treatment can cause PCR product degradation whereas insufficient treatment will fail to correct the problem and can even make some alleles more difficult to call Optimizing PCR 6 19 For More Information 6 20 Optimizing PCR Specific Suggestion To begin optimization trials use 0 5 1 unit of T4 DNA polymerase in 10 uL of pooled PCR product Incubate at 37 C for 30 minutes Refer to the AmpF STR Profiler Plus PCR Amplification Kit User s Manual P N 4303501 for more information on maximizing 3 A nucleotide addition Tailed primers are available through the Applied Biosystems Custom Oligonucleotide Synthesis Service Call 800 345 5224 for price and availability Stutter Products What is Stutter During the PCR amplification of di tri and tetranucleotide microsatellite loci minor products that are 1 4 repeat units shorter than the main allele are produced The minor product peaks are referred to as stutter peaks Stutter peaks might be caused by polymerase slippage during elongation Stutter Facts You can estimate the percent stutter by calculating the ratio of the combined heights of the stutter peaks with the height of the main allele peak Some general conclusions about perce
191. ively purifies the target away from sequences that amplify only linearly i e those with one modified end AFLP Mapping 10 7 Selective Amplification Plant Mapping 10 8 AFLP Mapping Prepared Template Genomic DNA Fragment Modified with Adaptors m Preselective Primers MI A EcoRI adaptor recognition site A or EcoRI adaptor recognition site Adaptors Thermal Core Mix Cycling C Msel adaptor recognition site C Mc TEN IG Al Figure 10 9 Preselective amplification of the prepared template Additional PCR amplifications are run to further reduce the complexity of the mixture so that it can be resolved on a polyacrylamide gel These amplifications use primers chosen from the 24 available AFLP Selective Primers eight Msel and sixteen EcoRI primers After PCR amplification with these primers a portion of each sample is analyzed on a Applied Biosystems DNA Sequencer Selective amplification with an EcoRI and an Msel primer amplifies primarily EcoRI Msel ended fragments The EcoRI EcoRI fragments do not amplify well The Msel Msel fragments are not visualized because they do not contain fluorescent dye labels Only the EcoRI containing strands are detected Figure 10 10 A Choose Selective AFLP Primers X HEN Axx E Cxx K Fluorescent dye Y ME Axx one of sixteen different fluorescent dye labeled AFLP EcoRI Selective Amplification primers Cxx one of eight different AFLP Msel Selective
192. izes continued on next page SSCP Analysis 7 11 Controls Post PCR Purification Step 7 12 SSCP Analysis Determining Run to run Variation in the Wild Type Sample Because the mobility shifts caused by many mutations are slight you must always run a wild type control to obtain an estimate of run to run variation in the wild type sample Run 3 5 lanes of the wild type sample Example Control Setup If you are running a 24 or 36 lane gel run at least three wild type controls one in either end lane and one in a middle lane Note Running five control lanes will allow you to obtain a satisfactory estimate for the reproducibility under the chosen experimental conditions Use the values of the five injections to determine the standard deviation of the wild type sample Any sample that deviates from the wild type mean by more than three standard deviations is 99 7 likely to be caused by a mutation and not by run to run variation in the wild type strand Estimate Percent Detectable Mutations If possible examine known mutant samples to obtain a rough estimate of percent detectable mutations before mounting a large scale analysis Using the initial set of electrophoresis conditions described in the following sections amplify DNA from the wild type and confirmed mutant samples Tabulate the percent detectable mutations for several repeat experiments If the detection rate is unacceptably low see the suggestions for optimizing SSCP r
193. k splitting Figure 5 3 shows the peak patterns of GeneScan 400HD fragments run under denaturing conditions 3 S 3 a 3 3 Parenezal Wi hess na a fh s a il de A Seeds amal Ne ena al nafis tiss fe tte ste TET Figure 5 3 Electropherogram of the GeneScan 400HD size standard run under denaturing conditions on the ABI PRISM 377 DNA Sequencer Fragments were run on a 36 cm 5 Long Ranger 6M Urea gel at 51 C 50 90 100 20 160 190 220 240 260 280 320 360 150 200 Figure 5 4 shows the peak patterns of GeneScan 400HD fragments run under non denaturing conditions Figure 5 4 Electropherogram of the GeneScan 400HD size standard run under non denaturing conditions on the ABI PRISM 377 DNA Sequencer Fragments were run on a 36 cm 5 Long Ranger Native gel at 30 C Sizing and Size Standards 5 11 GeneScan 500 500XL Size Standard Useful Range Special Uses Fragment Lengths Preparation Denaturing Electropherogram You can use the GeneScan 500 or 500XL size standard to determine fragment lengths between 35 and 500 base pairs This size standard is recommended for analysis of tri and tetranucleotide microsatellite loci which can often exceed 400 base pairs in length The GeneScan 500XL size standard is a concentrated form of the GeneScan 500 size standard for use on the ABI PRISM 377 DNA Sequencer with XL Upgrade and ABI 373 DNA Sequencer with XL Upgrade instruments The higher concentration is needed to
194. larvae with emended descriptions of P larvae as P larvae subsp larvae and P larvae subsp pulvifaciens International Journal of Systematic Bacteriology 46 270 279 Huys G Coopman R Janssen P and Kersters K 1996 High resolution genotypic analysis of the genus Aeromonas by AFLP fingerprinting International Journal of Systematic Bacteriology 46 572 580 Janssen P Coopman R Huys G Swings J Bleeker M Vos P Zabeau M and Kersters K 1996 Evaluation of the DNA fingerprinting method AFLP as a new tool in bacterial taxonomy Microbiology 142 1881 1893 Lin J J Kuo J Saunders J A Beard H S MacDonald M H Kenworthy W Ude G N and Matthews B F 1996 Identification of molecular markers in soybean comparing RFLP RAPD and AFLP DNA mapping techniques Plant Molecular Biology Reporter 14 156 169 Meksem K Leister D Peleman J Zabeau M Salamini F and Gebhardt C 1995 A high resolution map of the R1 locus on chromosome V of potato based on RFLP and AFLP markers Mol Gen Genet 249 74 81 Money T Reader S Qu L J Dunford R P and Moore G 1996 AFLP based mRNA fingerprinting Nucleic Acids Res 24 2616 2617 Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Cold Spring Harbor Press NY Thomas C M Vos P Zabeau M Jones D A Norcott K A Chadwick B and Jones J D G 1995 Identifi
195. lecules Saiki et al 1985 Mullis and Faloona 1987 Y Wear a clean lab coat one never worn while handling amplified PCR products or doing sample preparation and clean gloves when preparing samples for PCR amplification Change gloves whenever contamination is possible Maintain separate areas and dedicated equipment and supplies for Sample preparation PCR setup PCR amplification and analysis of PCR products Use aerosol resistant filter plugged PCR pipet tips Never bring amplified PCR products into the PCR setup area Open and close all sample tubes carefully Try not to splash or spray PCR samples Keep reactions and components capped as much as possible Clean lab benches and equipment regularly with 10 bleach solution Microsatellite Analysis Applications 9 7 Precautions for Working with Human Tissue WARNING BIOHAZARD Tissue samples have the potential to transmit infectious diseases Follow the latest guidelines published by the Centers for Disease Control CDC and National Institutes of Health NIH concerning the principles of risk assessment biological containment and safe laboratory practices for activities involving clinical specimens These principles can be found in the U S Department of Health and Human Services HHS publication Biosafety in Microbiological and Biomedical Laboratories publication number 93 8395 stock number 017 040 523 7 The biosafety Level 2 containment elements are c
196. lification Microbial Fingerprinting The first step of the AFLP technique is to generate restriction fragments by using two restriction endonucleases EcoRI and Msel in the AFLP Microbial Fingerprinting Kit and AFLP Plant Mapping Kits Double stranded adaptors supplied with each kit are ligated to the ends of the DNA fragments generating template DNA for subsequent polymerase chain reaction PCR amplification Restriction and ligation may take place in a single reaction if the buffers are compatible Figure 10 5 Adaptor sequences have been designed such that ligation of the adaptor oligonucleotide to the restricted DNA does not regenerate the recognition site If the buffers are not compatible the reactions must be run sequentially A Cut genomic DNA into fragments with the restriction enzymes Msel and EcoRI aS ja B Ligate adaptors EcoRI E es Msel C Modify genomic DNA fragments Msel Msel EcoRI EcoRI Figure 10 5 Example of template preparation and AFLP adaptor ligation The sequences of the adaptors and the restriction site serve as primer binding sites for a subsequent low level selection or preselective amplification of the restriction fragments Only those genomic fragments that have an adaptor on each end amplify exponentially during PCR amplification Figure 10 6 This step effectively purifies th
197. listed values due to the influence of the physical environment upon the dye Table 4 1 Maximum Emission and Excitation Wavelengths Emission Amax Excitation Amax Dye nm nm 6 FAM 517 494 5 FAM 522 493 R110 525 501 TET 538 522 R6G 549 529 HEX 553 535 JOE 554 528 TAMRA F JANTP 572 555 NED 575 553 TAMRA 583 560 ROX 607 587 a All dye labeled oligonucleotides were run at 10 7 Min 1X TE buffer pH 8 0 room temperature on a TaqMan LS 50B PCR Detection System ABI PRISM Dyes 4 5 Choosing Dye Filter Set Combinations How Data Collection ABI PRISM 377 and ABI PRISM 377 with XL Upgrade Works The ABI Prism 377 DNA Sequencer determines the light intensity in four 4 6 ABI PRISM Dyes non overlapping regions on a CCD camera Each region corresponds to a wavelength range that contains or is close to the emission maximum of an ABI PRISM dye The exact positions of the regions and the dye combinations appropriate to these positions depend upon the virtual filter set used For example with Virtual Filter Set A the instrument records the light intensity in four regions or windows centered at 535 5 nm 559 nm 586 nm and 615 nm The window positions in each virtual filter set have been optimized to provide the maximum possible separation among the centers of detection for the different dyes while maintaining an excellent signal to noise ratio After correcting for the spectral
198. ll lanes To verify check the Analysis Record in the Sample Information window Define the same size standard peaks in the size standard definition for all lanes if using more than one size standard definition To verify overlay the size standard peaks from each lane or display the sizing curve for each sample file Verify that all defined size standard peaks are called within all sample files In the Analysis Record in the Sample Information window make sure that Standard Defined and Standard Matched are the same Use the same Analysis Range in the size standard definition and in the sample files to be analyzed To verify check defined data points in the Analysis Parameters window If the position of a size standard peak differs by more than 400 scans from the Wrong definition peak the GeneScan Analysis Software will not recognize the peak The most common causes of failure to recognize a size standard peak are the following Inconsistent prerun times Bleedthrough peaks in the in lane size standard color caused by Matrix problems Off scale data e g too much sample is loaded or the primer peak is not removed from the analysis range Changes in electrophoresis conditions Inconsistent Prerun Times Staggered loading of even and odd lanes to simplify sample loading is one of the most common causes of inconsistency in the scan position of size standard peaks If you
199. lowing the individuals to be genotyped or differentiated based on the alleles they carry AFLP technology combines the power of restriction fragment length polymorphism RFLP with the flexibility of PCR based technology by ligating primer recognition sequences adaptors to the restricted DNA Some of the advantages of the AFLP technique are the following Only small amounts of DNA are needed Unlike randomly amplified polymorphic DNAs RAPDs that use multiple arbitrary primers and lead to unreliable results the AFLP technique uses only two primers and gives reproducible results Many restriction fragment subsets can be amplified by changing the nucleotide extensions on the adaptor sequences Hundreds of markers can be generated reliably High resolution is obtained because of the stringent PCR conditions The AFLP technique works on a variety of genomic DNA samples No prior knowledge of the genomic sequence is required Applications for AFLP in microbial fingerprinting include differentiation and tracking of highly related microbes at the species or strain level high resolution genotyping for taxonomic applications detection of DNA polymorphisms in genome evolution studies determining the relatedness of pathogenic organisms in epidemiological studies o gt gt mapping of cloned fragments in bacterial and yeast artificial chromosomes BACs and YACs An example of AFLP microbial fingerprints is shown in Figure 1
200. lter sets emission spectra 4 10 to 4 12 Virtual Filter Set A 4 10 Virtual Filter SetC 4 11 Virtual Filter SetD 4 11 Virtual Filter Set F 4 12 electrophoresis conditions optimizing 2 9 to 2 10 ensuring signal intensity 2 3 to 2 4 guidelines 2 2 increasing throughput multiplexing 2 2 to 2 3 methods choosing 2 5 to 2 7 samples determining loading concentrations 2 8 G gel preparation recipes A 1 to A 6 SSCP ABI 373 7 19 SSCP all instruments but ABI 373 7 14 GeneScan analyzing microsatellite markers 8 17 to 8 18 setting analysis parameters 8 18 control DNA using 2 11 design factors 2 2 to 2 11 electrophoresis conditions optimizing 2 9 to 2 10 ensuring signal intensity 2 3 to 2 4 fluorescent labeling methods choosing 2 5 to 2 7 increasing throughput multiplexing 2 2 to 2 3 samples determining loading concentrations 2 8 working with multiple colors 2 2 to 2 4 guidelines 2 2 matrix file creating C 1 to C 4 checking matrix quality C 3 to C 4 generating the matrix file C 2 to C 3 how to verify raw data C 1 to C 2 size standards 5 2 to 5 17 accuracy versus precision 5 3 comparing sizes across platforms 5 3 to 5 4 GeneScan Internal Lane Size Standards 5 7 to 5 17 GeneScan 1000 5 14to 5 15 GeneScan 2500 5 16to 5 17 GeneScan 350 5 8 to Index 3 5 9 GeneScan 400HD 5 10 to 5 11 GeneScan 500 5 12 to 5 13 process overview 5 2 to 5 3 troubleshooting 5 5 to 5 6 possible problems 5 5 to 5 6 preventing sizi
201. m cellulose nitrate filter for 5 minutes Note During this step you both filter and degas the solution Add 4 0 mL of filtered 10X TBE and sufficient distilled deionized HO to bring the total volume to 40 0 mL With the filtered polyacrylamide solution at room temperature add 200 uL of 10 APS ammonium persulfate Swirl gently to mix Be careful to avoid air bubbles Add 25 ul of TEMED tetramethylethylenediamine to the solution Swirl gently to mix Be careful to avoid air bubbles WARNING TEMED Tetramethylethylenediamine is extremely flammable and can be very destructive to the skin eyes nose and respiratory system Keep TEMED in a tightly closed container Avoid inhalation and contact with the skin eyes and clothing Always work in a fume hood Obtain a copy of the MSDS from the manufacturer Wear appropriate protective eyewear clothing and gloves Immediately cast the gel as described in your instrument user s manual Use standard 0 2 mm spacers and a square tooth comb Allow the gel to polymerize for at least 2 hours before performing electrophoresis Preparing Polyacrylamide Denaturing Gels To prepare 40 mL of an N polyacrylamide 6 M urea gel Step Action 1 Wash and assemble the glass plates as described in your instrument user s manual 2 Combine the following NmL 40 polyacrylamide stock 19 1 Note N isthe desired gel percentage Y 144g urea 1 0 g mixed
202. m faxed or e mailed to you through our site see the Documents on Demand section below In the United States and Canada technical support is available at the following times Product Hours Chemiluminescence 9 00 a m to 5 00 p m Eastern Time LC MS 9 00 a m to 5 00 p m Pacific Time All Other Products 5 30 a m to 5 00 p m Pacific Time See the Regional Offices Sales and Service section below for how to contact local service representatives outside of the United States and Canada Call Technical Support at 1 800 831 6844 and select the appropriate option below for support on the product of your choice at any time during the call To open a service call for other support needs or in case of an emergency press 1 after dialing 1 800 831 6844 For Support On This Product Dial 1 800 831 6844 and ABI Prism 3700 DNA Analyzer Press FAX 8 650 638 5891 a gt ABI PRISM 3100 Genetic Analyzer Press FAX 26 650 638 5891 Biolnformatics includes BioLIMS BioMerge and SQL GT applications icai 5 505 982 7690 DNA Synthesis Press FAX 21 650 638 5981 Fluorescent DNA Sequencing Press FAX 22 650 638 5891 Fuoresceni Fragment Analysis includes Press FAX GeneScan applications 23 650 638 5891 Integrated Thermal Cyclers Press FAX 24 650 638 5891 Introduction 1 3 For Supp
203. m shorter hold times 45 seconds or more at each temperature in the PCR cycle Mineral oil required GeneAmp PCR System 9600 0 2 mL MicroAmp Reaction Tubes 0 5 mL GeneAmp Thin walled Reaction Tubes with domed caps Optimized for fast PCR amplification of reaction volumes between 25 uL and 100 uL Use without mineral oil Optimized for fast PCR amplification of 100 uL reaction volumes 48 well adapter required Mineral oil overlay or AmpliWax PCR Gems required GeneAmp PCR System 9700 0 2 mL MicroAmp Reaction Tubes Optimized for fast PCR amplification of reaction volumes between 25 uL and 100 uL Use without mineral oil GeneAmp PCR System 2400 0 2 mL MicroAmp Reaction Tubes Optimized for fast PCR amplification of reaction volumes between 25 uL and 100 uL Use without mineral oil a Can also be used with the DNA Thermal Cycler TC1 using hold times of at least 60 seconds Although reaction tubes usually do not need to be sterilized or siliconized autoclave tubes when dealing with small quantities approximately 150 500 pg of starting DNA template Autoclaved PCR tubes can also be ordered from Applied Biosystems MicroAmp Autoclaved Reaction Tubes with Caps P N N801 0612 for the GeneAmp PCR Systems 9600 9700 and 2400 GeneAmp Autoclaved Thin walled Reaction Tubes P N N801 0611 for the DNA Thermal Cycler 480 Designing Custom Primers Definition To Ensur
204. mmend setting the Minimum Peak Half Width to 10 You can increase or decrease this setting based on the average peaks widths that you obtain Analyzing For brief directions on analyzing sample files see page 3 2 S le Fil A i aa Detailed protocols can be found in the GeneScan Analysis Software User s Manual SSCP Analysis 7 25 Optimizing SSCP Run Conditions Sensitivity to Run Conditions Fragment Size Mutation Position Polymer and Cross Linker Concentration 7 26 SSCP Analysis SSCP analysis is more sensitive to electrophoresis conditions than many ABI PRISM applications You should optimize run conditions for your particular system The following factors have an unpredictable effect on electrophoretic mobility under non denaturing conditions Fragment size Fragment position within the DNA region of interest Polymer and cross linker concentration Percentage of glycerol or other cosolvents in polymer Electrophoresis voltage o gt gt 4 gt Run temperature If your results are poor and the fragment length is well outside the optimal size range of 130 250 base pairs consider choosing new primers so that the fragment length is within the optimal size range Hayashi et al 1992 found that at least 90 of single base pair substitutions can be detected if the PCR products are kept under 200 bp in length and that 80 of single base pair substitutions can be detected if the PCR products are kept under 400 bp i
205. n At high temperature and low liquid level Polyurethane Tubing 6 ft 1 4 in i d 3 8 inch o d Tubing Connectors 1 4 in female NPT 1 4 in male barbed fittings NESLAB P N 126000000007 Any external water bath that meets these requirements should work properly The NESLAB Model RTE111 meets these requirements and has been tested with the instrument Refer to the user manual for the water bath you select follow proper procedures and ensure that all product safety and regulatory requirements applicable to your location are met Changing Thermistors for SSCP ABI PRISM 377 instruments with serial numbers beginning with 9 have 100 k ohm thermistors installed To perform SSCP at temperatures less than 22 C on these machines you must replace the 100 k ohm thermistors with 30 k ohm thermistors Note Instruments with serial number 100000001 and higher have 30 k ohm thermistors To determine which thermistors are installed on your instrument upgrade to the Data Collection 2 1 software and check the Log file from a recent run The configuration information in the Log makes reference to the type of thermistors installed Schedule a service call with your Applied Biosystems service engineer for replacement of the 100 k ohm thermistors if you wish to run SSCP at the lower temperatures continued on next page Software Requirements Requirements for Specific Run Conditions How the External Cold Wat
206. n length A given point mutation will interact differently with the various regions in the surrounding DNA Changing the primer positions to amplify different regions of the surrounding DNA can change the relative mobility difference between the mutant and the wild type fragments Although increasing polymer percentage increases your ability to detect a fixed mobility difference between two fragments polymer concentration has an unpredictable effect on three dimensional conformation and can change the mobility difference between the mutant and the wild type fragments Moreover increasing polymer or cross linker concentration will increase run times If you are altering polymer concentration try polymer concentrations in the 5 8 range Polyacrylamide concentrations from 4 20 have been used effectively in manual systems Low cross linker concentration appears to give the best results 37 5 1 for acrylamide gels continued on next page Glycerol Percentage Buffer pH Run Temperature Glycerol at concentrations of 5 10 usually stabilizes three dimensional DNA conformations and thus enhances mutation detection especially at higher temperatures If you decide to add glycerol as a cosolvent try concentrations in the range of 5 10 Other cosolvents you might want to consider include the following Y Urea Formamide Sucrose Glucose DMSO e Kukita et al 1997 suggest that low pH greatly increases the sensitiv
207. n page 6 13 for additional suggestions Problems with primer choice concentration or degradation See Designing Custom Primers on page 6 3 Determining Reagent Concentrations on page 6 6 and Multiplexing PCR on page 6 10 for additional suggestions Yield gets progressively poorer for successive PCR amplifications performed over time Expired or mishandled reagents Check expiration dates on all reagents If not expired verify that reagents are being stored and used according to manufacturer s instructions Compare with PCR performance using fresh reagents Inconsistent yields with control DNA Combined reagents not spun to bottom of PCR sample tube Place all reagents in apex of tube and spin briefly after combining Combined reagents left at room temperature or on ice for extended periods of time encouraging mispriming and other primer artifacts Put tubes in block immediately after combining reagents Combined reagents not thoroughly mixed Primers not uniformly suspended before adding to reaction mixture Primers can aggregate and settle to the bottom of the tube Vortex all primers reagents and reaction mixes minus enzyme thoroughly to ensure uniform concentration Pipetting errors Follow all these precautionary measures Y Calibrate pipettes Attach tips firmly Check all phases of pipetting technique 4 Whenever possible minimiz
208. n 350 size standard is prepared by digesting a proprietary DNA plasmid with Pst I followed by ligating a TAMRA or ROX labeled 22 mer oligodeoxynucleotide to the cut ends A subsequent enzymatic digestion with BstU yields DNA fragments containing a single TAMRA or ROX dye Although the GeneScan 350 size standard is made of double stranded DNA fragments only one of the strands is labeled Consequently even if the two strands migrate at different rates under denaturing conditions you will not need to worry about peak splitting Figure 5 1 shows the peak patterns of GeneScan 350 fragments run under denaturing conditions Figure 5 1 Electropherogram of the GeneScan 350 size standard run under denaturing conditions on the ABI PRISM 377 DNA Sequencer Fragments were run on a 36 cm 5 Long Ranger 6M Urea gel at 51 C continued on next page 5 8 Sizing and Size Standards Non denaturing Figure 5 2 shows the peak patterns of GeneScan 350 fragments run under Electropherogram non denaturing conditions 2700 2400 o o So o LO o 2100 o Q a e AN A O Lo o 150 oo NR Y 0 1800 o oO m LO 1500 ite M 1200 300 600 200 o at ha Ps ST pd AL Figure 5 2 Electropherogram of the GeneScan 350 size standard run under non denaturing conditions on the ABI PRISM 377 DNA Sequencer Fragments were run on a 36 cm 5 Long Ranger Native gel at 30 C Sizing and Size St
209. nalysis Parameters gt LA lt anatysis Parameters gt azezson iooa 2 2 ol lt Analysis Parameters 7 l 4202500 1000 3 None M lt Anatusis Parameters gt 9 The size standard must be selected for all the samples except matrix standard samples The size standard is selected if a diamond appears in the R red column for a particular sample To select or deselect the size standard hold down the command key and click in the appropriate square continued on next page 3 4 General Analysis and Evaluation Techniques Verifying First Method Size Standard Peak Assignments Step Action 1 Highlight each sample file of interest and display its sizing curve by selecting Size Curve from the Sample menu Sample File 07 Size Curve Best Fit 2nd Order Curve AO 2 923352E 01 Al 9 240988E 02 A2 1 290388E 07 R 2 1 000 lign By Size Curve Best Fit 2nd Order Curve Size Calling Curve Local Southern Method y g 1 I I 2800 3500 4200 4900 5600 Data Point The R 2 value and the coefficients of the curve are provided The R 2 value is a measure of the accuracy of fit of the best fit second order curve Note You can only display the sizing curve for a sample if a valid sizing curve exists for that sample 2 Check whether all the defined size standard peaks fall on the red sizing curve and note peaks that lie off the curve 3 If all of t
210. nalyzed by electrophoresis to separate the alleles according to size PCR amplified STR alleles can be detected using various methods such as fluorescent dye labeling silver staining or fluorescent dye staining The analysis of short tandem repeat loci is an important complement to the length and sequence based DNA typing systems already in use for human identification A majority of the STRs that have been evaluated by the forensic community are composed of four nucleotide repeat units Fr geau and Fourney 1993 Kimpton et al 1993 Urquhart et al 1995 PCR based STR analysis has the following advantages over conventional methods of DNA analysis such as Restriction Fragment Length Polymorphism RFLP PCR based tests are rapid giving results in 24 hours or less The small size of STR loci improves the chance of obtaining a result particularly for samples containing minute amounts of DNA and or degraded DNA The small size range of STR loci makes them ideal candidates for co amplification while keeping all amplified alleles smaller than 350 base pairs Many STR loci can therefore be typed from a single PCR STR alleles have discrete sizes allowing for simplified interpretation of results The STR alleles can be combined to form an allelic ladder which is used to genotype individuals PCR based STR tests can be automated increasing laboratory throughput while decreasing analysts hands on time continued on next p
211. nalyzing the Data Part II Allele Binning Using Genotyper 2 0 8 19 Troubleshooting Microsatellite Analysis 8 33 Microsatellite Analysis 8 1 Introduction to Microsatellite Analysis Definition What is Microsatellite Analysis Background Advantages of PCR Based Microsatellite Analysis Advantages of Using ABI PRISM Technology 8 2 Microsatellite Analysis Microsatellite markers also called short tandem repeat STR markers are polymorphic DNA loci that contain a repeated nucleotide sequence The repeat unit can be from 2 7 nucleotides in length The number of nucleotides per repeat unit is the same for a majority of repeats within a microsatellite locus Microsatellite loci are PCR amplified and the PCR products are then analyzed by electrophoresis to separate the alleles according to size PCR amplified microsatellite alleles can be detected using various methods such as fluorescent dye labeling silver staining or fluorescent dye staining The number of repeat units at a microsatellite locus may differ so alleles of many different lengths are possible Microsatellite loci occur throughout the genome of most organisms and therefore have been used as markers to establish linkage groups in crosses and to map genetically identified mutations to chromosomal positions If allele frequencies are known highly polymorphic microsatellite loci are very useful for identifying individuals in a population and for determining t
212. nd labeled primers D 2 to D 3 synthesis and purification D 6 to D 9 analyzing the oligos D 7 to D 8 consumption D 6 deprotecting D 6 evaluating yield D 8 molecular weight and emission specifications D 8 purifying D 7 storing the dyes D 9 synthesizing the primer D 6 6 19 to 6 20 A ABI 373 analyzing data 3 2 to 3 7 size standard peak assignments verifying 3 5 to 3 7 data collection how it works 4 6 evaluating data quality 3 8 to 3 9 bad data example 3 9 good data example 3 8 matrix evaluating quality 3 11 to 3 14 purpose of matrix 3 11 recognizing problems 3 11 to 3 14 solving matrix problems 3 14 when to remake matrix 3 11 nucleic acids quantitating 3 10 preparing for a run microsatellite marker analysis 8 12 to 8 16 creating a new matrix file 8 16 data collection viewing 8 16 gel preparation 8 12 instrument setup and prerun 8 14 preparing and loading samples 8 15 preprocess analysis parameters setting 8 13 to 8 14 starting the run 8 16 sample loading volumes B 1 ABI 373 with XL Upgrade data collection how it works 4 6 sample loading volumes B 1 See Also ABI 373 ABI Prism 310 analyzing data sample files analyzing 3 2 ABI PRISM 377 analyzing data 3 2 to 3 7 size standard peak assignments verifying 3 5 to 3 7 data collection how it works 4 6 evaluating data quality 3 8 to 3 9 bad dataexample 3 9 good data example 3 8 matrix evaluating quality 3 11 to 3 14 purpose of matrix 3 11 recognizing p
213. nd the resulting data analyzed using GeneScan Analysis Software P1 P2 F1 328 Gl sie F2 1 JN EE P F2 2 F2 3 x 330__ Y 380 Figure 10 3 Tomato AFLP samples showing Mendelian segregation The overlapping electropherograms in the top panel are AFLP results of sample DNA from three individuals parent one P1 parent two P2 and F1 from a cross A and B are the two significant peaks on this panel and appear only in P2 and F1 The lower three electropherogram panels are AFLP results of sample DNA from three F2 generations Peak A appears in F2 3 but does not appear in either F2 1 or F2 2 Peak B is inherited in all three F2 individuals The remaining non polymorphic AFLP Mapping 10 3 For More Information 10 4 AFLP Mapping peaks appear in all three F2 electropherograms and show that the overall AFLP patterns are reproducible T 7 80 30 120 150 180 210 240 270 300 330 360 390 420 450 sem 0 3500 3000 2500 2000 1500 1000 500 Any l il j NU LU A J LL VIAL AIA A MUL h A 138 3500 3000 2500 2000 100 1000 MIU ul i o N pag UII A A Jua 168 x 364 v 132 0 E Dye Sample Minutes Size Peak Height Peak Area Data P
214. nematode resistance gene Gro1 Molecular and General Genetics 249 82 90 Bates S R E Knorr D A Weller J N and Ziegle J S 1996 Instrumentation for automated molecular marker acquisition and data analysis In Sobral B W S ed The Impact of Plant Molecular Genetics Birkha ser Boston MA pp 239 255 Becker J Vos P Kuiper M Salamini F and Heun M 1995 Combined mapping of RFLP and AFLP markers in barley Mol Gen Genet 249 65 73 Dijkshoorn L Aucken H Gerner Smidt P Janssen P Kaufmann M E Garaizar J Ursing J and Pitt T L 1996 Comparison of outbreak and nonoutbreak Acinetobacter baumanii strains by genotypic and phenotypic methods Journal of Clinical Microbiology 34 1519 1525 Doyle J and Doyle J 1990 Isolation of plant DNA from fresh tissue Focus 12 13 15 References E 9 E 10 References Folkertsma R T Rouppe van der Voort J N A de Groot K E van Zandvoort P M Schots A Gommers F J Helder J and Bakker J 1996 Gene pool similarities of potato cyst nematode populations assessed by AFLP analysis Molecular Plant Microbe Interactions 9 47 54 Heyndrickx M Vandemeulebroecke K Hoste B Janssen P Kersters K Vos P Logan N A Ali N and Berkeley R C W 1996 Reclassification of Paenibacillus formerly Bacillus pulvifaciens a later subjective synonym of Paenibacillus formerly Bacillus larvae as a subspecies of P
215. ng problems 5 5 GeneScan 1000 size standard denaturing electropherogram 5 15 fragment lengths table of 5 14 non denaturing electropherogram 5 15 preparation of 5 14 special uses 5 14 what to use it for 5 14 GeneScan 2500 size standard denaturing electropherogram 5 17 fragment lengths table of 5 16 non denaturing electropherogram 5 17 preparation of 5 16 special uses 5 16 what to use it for 5 16 GeneScan 350 size standard denaturing electropherogram 5 8 fragment lengths table of 5 8 non denaturing electropherogram 5 9 preparation of 5 8 what to use it for 5 8 GeneScan 400HD size standard denaturing electropherogram 5 11 fragment lengths table of 5 10 non denaturing electropherogram 5 11 preparation of 5 10 special uses 5 10 what to use it for 5 10 GeneScan 500 size standard denaturing electropherogram 5 12 fragment lengths table of 5 12 non denaturing electropherogram 5 13 preparation of 5 12 special uses 5 12 what to use itfor 5 12 GeneScan 500XL size standard See GeneScan 500 size standard Genotyper analyzing microsatellite markers 8 19 to 8 32 allele binning methods 8 19 Index 4 allele binning definition 8 19 benefits of allele binning 8 19 creating defined set of category members 8 29 to 8 32 generating category member automatically 8 26 to 8 29 using the Histogram window 8 20 to 8 24 using the Plot window 8 24 to 8 25 StockMarks example 9 18 global sizing methods 5 2 guidelines co electrophore
216. ng buttons as shown below Plot selection Size in bp Starting bin determined automatically c Enter 0 10 in the Bin size field Set Statistics Options Source ___ _Jalue Plot selection a Size in bp Range of first selected category Scan number O Fixed range 0 00 to 100 00 O Peak height O Table selection O divided by scale factor O Table column s O Peak area to Ly O divided by scale factor O Value in table column O Label text when name of first selected category group O Cell value is in table column Lv O Cell text Bin size Starting bin Determined automatically Note A bin size of 0 1 bp gives the most precise allele binning If insufficient data is available however Genotyper software displays an error message stating that the bin size is too small If this occurs increase the bin size Click B to choose all blue dye lanes In the Plot window draw a box around all the peaks associated with a single marker 80 4 From the Views menu choose Show Histogram Window Microsatellite Analysis 8 31 8 32 Microsatellite Analysis To fine tune the category definitions using the Histogram window continued Step Action 5 Check the marker name displayed by the Category
217. nge This Range Data Points Start Stop Data Processing x Baseline EJ MultiComponent Smooth Options None Light Heavy Size Call Range All Sizes O This Range Base Pairs Size Calling Method 2nd Order Least Squares O 3rd Order Least Squares O Cubic Spline Interpolation Local Southern Method O Global Southern Method Peak Detection Peak Amplitude Thresholds Split Peak Correction None O GENESCAN 2500 LeftMost Peak RightMost Peak Correction Limit 50 Data Pts Min Peak Half Width Pts IMPORTANT The settings listed here are intended as suggestions to get you started with microsatellite analysis Alter them as necessary for your system However if you choose to use different settings be sure to define the Analysis Range to exclude the primer peak Choose Save As from the File menu Save the settings as Microsatellite Analysis Parameters Click OK 8 18 Microsatellite Analysis Analyzing the Data Part II Allele Binning Using Genotyper 2 0 What is Allele Binning Benefits of Allele Binning Methods Used to Bin Alleles Allele definitions for microsatellite markers are based on the fragment length size of the PCR products as estimated by gel or capillary electrophoresis Experimental variation in sizing has led to the practice of binning alleles grouping allele fragments belonging to a particular size into a range bin centere
218. nhalation and contact with the skin eyes and clothing Always work in a fume hood Obtain a copy of the MSDS from the manufacturer Wear appropriate protective eyewear clothing and gloves 13 Immediately cast the gel as described in your instrument user s manual Use standard 0 2 mm spacers and a square tooth comb 14 Allow the gel to polymerize for at least 2 hours before performing electrophoresis Polyacrylamide Gels Preparing a Long Ranger Gel for SSCP The protocol for making a non denaturing 8 1 Long Ranger gel for SSCP differs from the preceding protocol in two places Delete step 2 e do not add urea Step 3 Add the following 8 1 mL 50 Long Ranger gel solution concentrate 1 25 g glycerol 5 0 mL 10X TBE Preparing 40 Polyacrylamide Stock To make 150 mL of 40 polyacrylamide stock solution use Application Acrylamide g Bisacrylamide g SSCP 58 5 1 56 All others 57 3 Note For SSCP always use a low cross linker concentration 237 5 1 acrylamide bisacrylamide in the polyacrylamide stock solution For other applications typically use a 19 1 stock solution To make 40 polyacrylamide stock solution Step Action 1 In a well ventilated area combine the acrylamide and bisacrylamide in a glass beaker See the preceding table for the amounts WARNING CHEMICAL HAZARD Acrylamide and bis acrylamide are neurotoxins Avoid inhalation of acrylamide wh
219. ns Saccharomyces sp Schizosaccharomyces pombe Xanthomonas sp EcoRI C Msel T EcoRI A Msel T EcoRI A Msel G EcoRI A Msel CA EcoRI C Msel CA EcoRI T Msel A EcoRI 0 Msel A EcoRI G Msel A EcoRI C Msel C EcoRI A Msel T EcoRI G Msel A EcoRI T Msel C EcoRI 0 Msel C EcoRI A Msel C EcoRI G Msel A EcoRI T Msel C EcoRI A Msel CA EcoRI AC Msel C EcoRI A Msel G EcoRI AC Msel C EcoRI A Msel T EcoRI G Msel A EcoRI C Msel A EcoRI AC Msel C EcoRI A Msel CA EcoRI AC Msel C EcoRI AC Msel C EcoRI 0 Msel C EcoRI 0 Msel A EcoRI 0 Msel G EcoRI 0 Msel A a Producing 25 130 bands evenly dispersed from 50 500 bases with intensities of 100 2000 relative fluorescent units b Too few or too many bands or uneven size distribution Note The list in Table 10 2 is not exhaustive Refer to the publications listed in Appendix E for in depth discussion of primer choices continued on next page 10 10 AFLP Mapping Primers Available Plant Mapping If you want to use a specific primer combination for the AFLP Selective Amplification reactions you can order primer pairs in any combination of one EcoRI primer and one Msel primer This gives you 128 possible primer pair combinations from which you can choose for either regular or small plant genomes Order the AFLP Amplification Core Mix Module P N 402005 and the desired AFLP Selective Amplification Primers shown in Table 10 3 Table 10 3 AFLP Selective Amplifi
220. nt stutter follow 4 The longer the length of the repeat unit the less stutter product made Figure 6 2 In other words among microsatellite loci with the same number of repeat units the percent stutter is greater for dinucleotide microsatellite loci than it is for trinucleotide microsatellite loci and so on Walsh et al 1996 zi 233 235 Figure 6 2 Comparison of the amounts of stutter in dinucleotide left and tetranucleotide right repeat loci Each locus is homozygous with the largest peak in each picture representing the true allele The percent stutter increases with increasing allele length i e with increasing number of consecutive repeat units as shown in Figure 6 3 on page 6 22 This rule is likely to be violated if the core repeat units are not consecutive e g if some of the repeats are partial repeats or if the core repeat structure is interrupted by other repeat units Optimizing PCR 6 21 Evaluating Data with Stutter 6 22 Optimizing PCR 6 0 q 5 0 te ao Es 5 t t 3 30 3 E t EN 3 20 ree a E 3 eo e i 1 0 f t e 3 3 o 0 0 AA AAA AAA 567899310 6 7 8 9 10 11 12 6 7 8 9 10111213 14 THO1 TPOX CSF1PO Figure 6 3 Percent stutter observed for the STR loci in the AmpF STR Green PCR Amplification Kit The multiband stutter pattern can complicate analysis particularly for dinucleotide repeat loci For
221. ntaining a 5 Aminolink 2 Phosphoramidite Preparing custom 5 end labeled 6 FAM HEX TET NED reagents primers directly on any Applied Biosystems DNA synthesizerb F JdNTPs Simple internal fluorescent labeling of R6G R110 TAMRA Labeled primers in reagent kits Microsatellite and human identification applications 5 FAMC JOES 6 FAM HEX TET NEDa Labeled size standard Generating the sizing curve TAMRA ROX to size unknown sample fragments a NED labeled primers are available only in kits or through the Applied Biosystems Custom Oligo Service Call Applied Biosystems or visit the Applied Biosystems WorldWideWeb page at www appliedbiosystems com techsupport for information on how to order custom labeled oligos b For directions on synthesizing labeled oligonucleotides see Appendix C c 5 FAM and JOE are only available as labeled primers in select reagent kits See Choosing Fluorescent Labeling Methods on page 2 5 for a detailed comparison of fluorescent labeling with 5 end labeled primers and F JdNTPs ABI PRISM Dyes 4 3 Understanding Dye Spectra Background Definitions Factors That Affect Spectra Experimental Considerations 4 4 ABI PRISM Dyes An electron in a molecule or atom that has been transferred to an excited high energy state e g after absorption of a photon will eventually return to the more stable lower energy ground state The return to the gr
222. o the actual size of the fragment as determined by sequencing Precision or reproducibility in size calling is a measure of the instrument s ability to generate the same size consistently for a given fragment independent of whether the called size is close to the actual size for a given set of run conditions If care is taken to control for variations in run conditions ABI Prism instruments are highly precise within a single set of injections or a single gel However the called size for the same fragment can differ between run conditions on a single instrument In other words the generated sizes are not necessarily accurate Between run sizing differences arise from a number of factors including Differences in the type and concentration of gel polymer Well to read or time to read differences Differences in run temperature 4 Differences in electrophoresis conditions e g the denaturing ability of the separation matrix Changes in the sizing method or specific GeneScan size standard used to generate the sizing curve When comparing across injections it is important to use the same sizing method and the same size standard definition Table 5 1 on page 5 4 compares precision within and between the three instrument platforms for a typical data set from the AmpF STR Blue PCR Amplification Kit The three instrument platforms represented are the following ABI PRISM 377 DNA Sequencer 36 cm wtr plates Y ABI 373 DNA Se
223. of the MSDS from the manufacturer Wear appropriate protective eyewear clothing and gloves Wash thoroughly after handling formamide Denature the samples for 5 minutes at 95 C Cool the samples by placing directly on ice 4 Run 4 uL of each of these four samples either alone on a gel or in the first lanes of an experimental run Note To ensure the best possible quality matrix load the matrix samples into every other lane and leave at least one empty lane between the matrix samples and the experimental samples continued on next page Microsatellite Analysis 8 15 Starting the Run Viewing Data Collection Creating a New Matrix File 8 16 Microsatellite Analysis Step Action 1 On the ABI 373 DNA Sequencer press the MAIN MENU soft key pad 2 Select the CHOOSE RUN option and then select GENESCAN RUN Note This starts the electrophoresis run not data collection 3 Immediately go to the computer 4 Make sure that the GeneScan Analysis application is closed and that the GeneScan Collection application is still open 5 Choose Settings from the Edit menu Set the Timer for 4 hours 12 cm plates 8 hours 24 cm plates or 12 hours 34 cm plates and name the run file Note The suggested data collection times will enable you to detect 350 bp peak of the GeneScan size standard Adjust the time accordingly if the expected product is significantly longer or shorter than 350 base p
224. oftware User s Manual or the GeneScan 672 Software User s Manual for details Create a microsatellite analysis matrix file if necessary For directions on preparing matrix samples for microsatellite analysis see page 8 10 ABI PRISM 377 and ABI PRISM 377 with XL Upgrade or page 8 15 ABI 373 and ABI 373 with XL Upgrade See Appendix C for instructions on creating a matrix file Usually you create and then reuse a single matrix file for each set of run conditions Note You must create a matrix file before analyzing your data Microsatellite Analysis 8 7 Preparing for a Run ABI PRISM 377 ABI PRISM 377 with XL Upgrade and ABI 373 with XL Upgrade Gel Preparation Instrument Setup 8 8 Microsatellite Analysis The specific equipment and reagents needed for a microsatellite analysis run are listed in Before You Begin on page 8 3 Note Appendix A contains protocols for preparing both Long Ranger and polyacrylamide denaturing gels For the ABI PRISM 377 and ABI PRISM 377 with XL Upgrade Applied Biosystems recommends using a 5 Long Ranger 6 M urea gel Alternatively you can use a 4 25 polyacrylamide 6 M urea gel Use 36 cm well to read plates 0 2 mm spacers Y Square or shark s tooth comb For the ABI 373 with XL Upgrade Applied Biosystems recommends using a 5 Long Ranger 6 M urea gel Alternatively you can use a 6 polyacrylamide 8 3 M urea gel Use Y 12 24 or 34 cm well to read plates Fo
225. oint E Peak 165 69 218 10 303 43 1461 12443 2181 16B 70 223 70 313 05 394 2759 2237 16B 71 225 70 316 50 1482 11577 2257 16B 72 227 90 320 31 71 559 2279 16B 73 229 70 323 43 133 1433 2297 16B 74 242 80 346 11 130 763 2428 16B 75 244 30 348 65 394 1692 2443 A 361 25 16B 77 260 80 376 12 157 1249 2608 po 168 78 278 50 406 44 50 564 2785 E E HE 16B 79 299 90 445 60 942 7602 2999 Figure 10 4 Rice AFLP samples showing near isogenic regions The two electropherogram panels shown in Figure 10 4 contain data from rice DNA samples prepared using the AFLP technique Samples were run on an ABI 373 DNA Sequencer and the resulting data analyzed using GeneScan Analysis Software The rice DNA was isolated from near isogenic lines almost identical genetic material lt was selected for an introgressed region carrying a disease resistance gene By comparing peak patterns in the two electropherograms you will find that the rice lines differ by only 1 2 One of the peaks distinguishing the two lines has been highlighted in both the electropherogram display and the related tabular data beneath the electropherogram panels For examples of other applications refer to the literature cited in Appendix E Refer to the AFLP Microbial Fingerprinting Protocol P N 402977 and the AFLP Plant Mapping Protocol P N 4303146 for more information The AFLP Technique Template Preparation and Adaptor Ligation Preselective Amp
226. oligodeoxynucleotide to the cut ends continued on next page Sizing and Size Standards Denaturing Electropherogram Non denaturing Electropherogram Figure 5 7 shows the peak patterns of GeneScan 1000 fragments run under denaturing conditions Figure 5 7 Electropherogram of the GeneScan 1000 size standard run under denaturing conditions on the ABI PRISM 377 DNA Sequencer Fragments were run on a 36 cm 5 Long Ranger 6M Urea gel at 51 C Note Under denaturing conditions the two strands of the doubly labeled GeneScan 1000 size standard fragments migrate at different rates appearing as split peaks To ensure size calling precision and a reliable size standard definition you must explicitly define one peak from each split peak pair in the size standard definition To improve matching of size standard peaks choose either LeftMost Peak or RightMost Peak in the Split Peak Correction section of the Analysis Parameters window Figure 5 8 shows the peak patterns of GeneScan 1000 fragments run under non denaturing conditions Figure 5 8 Electropherogram of the GeneScan 1000 size standard run under non denaturing conditions on the ABI PRISM 377 DNA Sequencer Fragments were run on a 36 cm 5 Long Ranger Native gel at 30 C Sizing and Size Standards 5 15 GeneScan 2500 Size Standard Useful Range You can use the GeneScan 2500 size standard to determine fragment lengths between 100 and 5000 base pairs Under non
227. omb 0 2 mm thick 402053 50 well Squaretooth Comb 0 2 mm thick 402183 66 well Squaretooth Comb 0 2 mm thick 401827 24 well Sharktooth Comb 0 2 mm thick 401922 32 well Sharktooth Comb 0 2 mm thick 401828 36 well Sharktooth Comb 0 2 mm thick 402177 48 well Sharktooth Comb 0 2 mm thick 402180 64 well Sharktooth Comb 0 2 mm thick 402186 50 well Plastic Overlay 402187 66 well Plastic Overlay 603627 Gel Cassette 603947 Top and Bottom Gel Pouring Fixtures 401969 Top Pouring Fixture 604014 Bottom Pouring Fixture 603873 Upper Buffer Chamber 603875 Lower Buffer Chamber 603822 Upper Buffer Electrode Assembly 603823 Lower Buffer Electrode Assembly 603833 Front 36 cm Well to Read Heat Plate Part Numbers F 5 ABI 373 DNA Sequencer 401444 24 well Squaretooth Comb 0 4 mm thick 401778 34 well Squaretooth Comb 0 4 mm thick 401497 36 well Squaretooth Comb 0 4 mm thick 402185 50 well Squaretooth Comb 0 4 mm thick 402253 66 well Squaretooth Comb 0 4 mm thick 402186 50 well Squaretooth Plastic Overlay 402187 66 well Squaretooth Plastic Overlay 401472 24 well Sharktooth Comb 0 4 mm thick 401580 32 well Sharktooth Comb 0 4 mm thick 401473 36 well Sharktooth Comb 0 4 mm thick 402179 48 well Sharktooth Comb
228. on polymorphism SSCP technique for detection of point mutations Human Mutation 2 404 414 Hayashi K 1992 PCR SSCP A method for detection of mutations GATA 9 73 79 Inazuka M Tahira T and Hayashi K 1996 One tube post PCR fluorescent labeling of DNA fragments Genome Res 6 551 557 Inazuka M Wenz H M Sakabe M Tahira T and Hayashi K 1997 A streamlined mutation detection system multicolor post PCR fluorescence labeling and single strand conformational polymorphism analysis by capillary electrophoresis Genome Res 7 1094 1103 Iwahana H Adzuma K Takahashi Y Katashima R Yoshimoto K and ltakura M 1995 Multiple fluorescence based PCR SSCP analysis with postlabeling PCR Methods Appl 4 275 282 Iwahana H Fujimura M Takahashi Y Iwabuchi T Yoshimoto K and Itakura M 1996 Multiple fluorescence based PCR SSCP analysis using internal fluorescent labeling of PCR products Bio Techniques 21 510 519 Iwahana H Yoshimoto K and ltakura J 1994 Multiple fluorescence based PCR SSCP analysis Bio Techniques 16 296 304 Kukita Y Tahira T Sommer S S and Hayashi K 1997 Human Mutation 10 400 407 Makino R Yazyu H Kishimoto Y Sekiya T and Hayashi K 1992 F SSCP Fluorescence based polymerase chain reaction single strand conformation polymorphism PCR SSCP analysis PCR Methods Appl 2 10 13 Mannens M Slater R M Heyting C Bliek J de Krake
229. onsistent with the Occupational Health and Safety Administration OSHA requirements contained in the HHS OSHA Bloodborne Pathogen Standard 29 CFR part 1910 1030 Preparing DNA Extract DNA Genomic DNA for the RER LOH Assay can be extracted from fresh frozen or paraffin embedded tissue For successful results particularly when extracting DNA from paraffin embedded tissue you can use the following Nucleon Genomic DNA Kit Scotlab P N SL 8501 PureGene DNA Isolation Kit Gentra Systems Inc P N D 5500A QlAamp Tissue Kit Qiagen Inc P N 29304 Good yields of DNA have been obtained in Applied Biosystems laboratories using any of these kits and have also found that they work well to eliminate PCR inhibitors from the DNA Extract the DNA as described in the protocol provided with these kits Note The quality of genomic DNA isolated from paraffin embedded tissue will vary with the type of fixative used the time in formalin the age of the sample and the method of DNA isolation Evaluate DNA Quality and Quantity After DNA extraction it is critical to evaluate DNA quantity This can be done using the QuantiBlot Human DNA Quantitation Kit P N N808 0114 or spectrophotometrically using Picogreen Molecular Probes P N P 7581 DNA quality and quantity can also be evaluated by electrophoresing 5 uL of genomic DNA through a 1 Seakem GTG agarose gel with 0 8 ug mL ethidium bromide Note The genomic DNA must be quantitated
230. oper C S Park M Blair D G et al 1984 Molecular cloning of a new transforming gene from a chemically transformed human cell line Nature 311 29 33 Dean M Park M LeBeau M Robins T S ef al 1985 The human met oncogene is related to the tyrosine kinase oncogenes Nature 318 385 388 Fishel R Lescoe M K Rao M R S ef al 1993 The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer Cell 75 1027 1038 Kwok S and Higuchi R 1989 Avoiding false positives with PCR Nature 339 237 238 Leone A McBride O W Weston A et al 1991 Somatic allelic deletion of nm23 in human cancer Cancer Res 51 2490 2493 Levine A J Momand J and Finlay C A 1991 The p53 tumor suppressor gene Nature 351 453 456 Lindblom A Tannergard P Werelius B and Nordenskjold M 1993 Genetic mapping of a second locus predisposing to hereditary non polyposis colon cancer Nature Gen 5 270 282 Liu B Nicolaides N C Markowitz S et al 1995 Mismatch repair gene defects in sporadic colorectal cancers with microsatellite instability Nature Gen 9 48 55 Magnusson P Wilander E and Gyllensten U 1996 Analysis of loss of heterozygosity in microdissected tumor cells from cervical carcinoma using fluorescent dUTP labeling of PCR products Bio Techniques 21 844 847 Mullis K B and Faloona F A 1987 Specific synthesis of DNA in vitro via a polymerase cataly
231. oresis parameters in the application modules are set to ensure the following 4 Detection of all fragments in the typical size range permitted by the application For example microsatellite loci are rarely over 400 base pairs in length Acceptable run times Acceptable resolution The resolution R of two peaks in an electropherogram is defined as follows R gt ot 05x W W where the P are the peak positions measured below the peak apex and the W are the peak widths measured at half peak maximum Determining Required Run Time To determine the minimal acceptable run time for a given run voltage you will need to perform a trial run To ensure that you collect sufficient data to perform analysis set the electrophoresis run time approximately 10 higher than the migration time of the largest fragment of interest In general the migration time of the 400 bp fragment in the GeneScan 400HD Internal Lane Size Standard is 2 hours on a 36 cm well to read 5 Long Ranger gel Note The largest fragment of interest will most probably be a size standard peak that is needed for sizing the largest sample fragments of interest The set of size standard peaks that GeneScan uses to generate the sizing curve can vary with the size calling method In general be sure to include the two size standard peaks immediately larger than the largest sample fragment of interest Decreasing Run Time For faster run times you can increase the ele
232. orking with 2 2 troubleshooting amplification 11 1 to 11 6 product detection 11 7 to 11 13 PCR product carryover avoiding contamination from 6 16 Applied Biosystems Custom Oligonucleotide Synthesis Service help 2 7 phosphoramidite reagents dye chemical form 4 3 polyacrylamide gels preparing A 3 to A 5 post PCR end labeling advantages of using 2 6 precision in size calling 5 3 primer dye kits table of 4 8 primers 5 end labeled primers preparing abbreviations and definitions D 1 calculating absorbance for DNA samples D 10to D 11 instrument setup D 4 to D 5 introduction to 5 end labeled primers D 2 to D 3 synthesis and purification D 6 to D 9 designing custom primers 6 3 to 6 5 definition 6 3 ensuring successful amplification 6 3 maximizing specificity 6 4 to 6 5 maximizing stability 6 3 to 6 4 melting temperature 6 3 post amplification manipulation 6 5 Q QuantiBlot kit quantitating human DNA 9 8 11 1 11 3 quantitation 3 10 choosing fluorescent labeling methods 2 5 converting Azgp to concentration D 11 importance in interpreting stutter patterns 6 22 importance in LOH RER assays 9 8 troubleshooting weak signals 11 3 quantum yield definition of 4 4 R reaction volumes choosing 6 2 when using small amounts of template 6 2 Index 5 reagents determining concentrations 6 6 to 6 7 dNTP concentration 6 6 enzyme concentration 6 7 magnesium
233. ort On This Product Dial 1 800 831 6844 and PCR and Sequence Detection Press FAX 5 or call 1 800 762 4001 and press 1 for PCR or 2 for Sequence Detection 240 453 4613 FMAT Telephone FAX 1 800 899 5858 and press 1 then press 6 508 383 7855 Peptide and Organic Synthesis Press FAX 31 650 638 5981 Protein Sequencing Press FAX 32 650 638 5981 Chemiluminescence Telephone FAX 1 800 542 2369 781 275 8581 U S only or Tropix 1 781 271 0045 9 00 a m to Tropix 5 00 p m ET LC MS Telephone FAX 1 800 952 4716 650 638 6223 9 00 a m to 5 00 p m PT Documents on Free 24 hour access to Applied Biosystems technical documents including MSDSs is Demand available by fax or e mail 1 4 Introduction You can access Documents on Demand through the internet or by telephone If you want to order Then through the Use http www appliedbiosystems com techsupport interner You can search for documents to order using keywords Up to five documents can be faxed or e mailed to you by title by phone fromthe a Call 1 800 487 6809 from a touch tone phone Have your fax United States or Canada number ready b Press 1 to order an index of available documents and have it faxed to you Each document in the index has an ID number Use this as your order number in step d below c
234. osystems Custom Information Oligonucleotide Synthesis Service either by phone 800 345 5224 by e mail Support appliedbiosystems com or online www appliedbiosystems com techsupport For information on synthesizing 5 end labeled primers see Appendix D For information on labeling with F JdNTPs consult the FJANTP Reagents Protocol P N 402774 Experimental Design Considerations 2 7 Determining Loading Concentrations for Samples Why Problems Arise How to Proceed Using too little or too much sample can cause problems Your ABI Prism instrument can convert a limited range of fluorescent signal into digital values For optimal results you should keep the fluorescent signal between approximately 150 and 4000 relative fluorescent units RFU Too Little Signal Below this range the signal to noise ratio is too low to discriminate between sample peaks and background fluctuations Too Much Signal The Most Common Problem Above this range the instrument cannot measure the true value of the signal and consequently cannot compensate for the spectral overlap among the dyes As a result artifact peaks called bleedthrough or pull up peaks see page 3 11 can appear in other colors Artifact peaks can corrupt both automated size calling extra peaks in the size standard color and the analysis of co loaded samples 4 Typically load 0 5 2 0 uL of each PCR product and 0 5 uL of the GeneScan size standard in the a
235. ot Start Limitations and Alternatives How Hot Start Works Hot Start Options Performing a Manual Hot Start Consider using the Hot Start technique whenever you need to improve the specificity and sensitivity of your PCR amplifications Loss of specificity and sensitivity are often caused by competing side reactions which usually occur during the prePCR setup period while all reactants sit together at permissive temperatures A common competing side reaction involves the amplification of nontarget sequences in background DNA either due to mispriming or to primer oligomerization The technique is cumbersome If you have high throughput needs switching to AmpliTaq Gold DNA Polymerase will give the same benefits as performing the Hot Start technique without the need for using wax barriers or opening reaction tubes In particular if you are already using AmpliTaq Gold DNA Polymerase performing the Hot Start technique will not improve the specificity and sensitivity of PCR amplification In the Hot Start technique components necessary for amplification are separated so that critical reactants do not mix until reaching a temperature sufficiently high to suppress primer self annealing or annealing to nontarget sequences You can perform either a manual Hot Start or an AmpliWax PCR Gem mediated Hot Start Note Although manual Hot Start can increase specificity and yield it is inconvenient and you can encounter reproducibility and cont
236. ound state can occur either through the evolution of heat radiationless decay or through the emission of a photon fluorescence or phosphorescence The emission spectrum of a fluorescent dye is the intensity of emitted light fluorescence as a function of the wavelength of the emitted light The excitation spectrum of a dye is the intensity of emitted light as a function of the wavelength of the exciting light The excitation efficiency of a dye is a measure of the probability that it will absorb light of a certain wavelength as a percentage of the probability of absorption at the wavelength of maximum absorption The quantum yield of a dye is the probability that its excited state will emit a photon as it decays back to the ground state The emission and excitation spectra of a dye attached to DNA depend upon the dye s Chemical structure Physical environment Relevant parameters include the buffer pH and concentration the gel polymer composition and whether the DNA is single or double stranded The effect of the physical environment on dye spectra explains why you need to remake the matrix every time you change the run conditions Although altered by the physical environment the wavelengths of maximum emission and excitation for each ABI PRISM dye always lie within a small wavelength range This relative invariance of the emission spectra is crucial to experimental success Detecting a Dye Signal The ability of the inst
237. p then you can do one of the following to differentiate between them Label overlapping products with different dye colors Choose new primer sites to alter the PCR product fragment lengths Load overlapping products in different lanes To exploit the potential for increased throughput using ABI PRISM multicolor fluorescence technology you will want to multiplex electrophoresis by co loading the products of multiple PCR reactions in the same lane Depending upon your application you may also want to multiplex the PCR Co electrophoresis Before performing PCR for co electrophoresis be sure that you follow these guidelines Use different dye labels for PCR reactions with overlapping product sizes Use a combination of dyes that display in different colors and can be detected by the same filter set or virtual filter set Table 4 2 on page 4 7 lists recommended filter dye set combinations Use greater dye concentrations in PCR reactions with dyes of low emission intensity than for dyes of high emission intensity See Accounting for Differences in Dye Signal Strengths later in this section After pooling PCR products you may need to perform a desalting step prior to loading samples Pooling products from multiple PCR reactions often increases the salt concentration in the loaded samples See Decreasing the Salt Concentration on page 2 3 2 2 Experimental Design Considerations Ensuring Adequate Signal
238. pe K To bin alleles using the Histogram window continued Step Action 3 Follow these steps to set up a Category Group for each marker a b C From the Analysis menu choose Clear Category List From the Category menu choose Add Category Enter the marker name size range and dye color for the first marker as shown below Click OK Repeat these steps for the remaining markers From the Category menu choose Add Category Enter the marker name size range and dye color Click OK Add Category Name Unknown m y X Member of group D12 83 comment All peaks O Highest peak Highest 2 peaks Left peak O Right peak Size ms with dye color s XK blue green yellow red O with scaled height of at least M O with scaled height of at most 9999 O Exclusive clears previous labels at same peak From the Analysis menu choose Label Peaks Label peaks with Size in bp only From the Analysis menu choose Filter Labels Filter labels using the default settings best for dinucleotide repeat markers Microsatellite Analysis 8 21 8 22 Microsatellite Analysis To bin alleles using the Histogram window continued Step Action 6 Working with one dye color at a time in the Main window a Click B to choose all blue dye lanes b Draw a box in the plot window that cov
239. peaks in the two colors If you choose not to label both strands you could mistake a band produced by residual double stranded product for a band produced by mutant single stranded product Note Under certain circumstances both strands can have identical mobilities Using a different color for each strand will also allow you to detect mutations that cause the forward and reverse strands to switch positions without significantly affecting the relative mobilities of the wild type and mutant samples Once data interpretation is well established differential labeling of the two strands will not always be necessary You can switch to using a single dye to increase throughput by running multiple differently labeled samples in a single gel lane Dedicate a Color to Each Sample Because single stranded DNA molecules can adopt multiple stable conformations extra peaks will often be present in an electropherogram Dedicating a color to each sample e g labeling the wild type one color and the mutant another allows you to confirm the origin of extra peaks Label Both Strands Even if you decide to use a single dye for each sample it is important that you label both strands Labeling both strands increases detection sensitivity and can indicate potential false positive results Often mutations will cause an observable mobility shift in only one of the two strands By labeling both strands you increase your chances of detecting mutations that aff
240. pearance of numerous extra alleles at both higher and lower molecular weight in the tumor bottom panel sample indicates significant genomic instability 9 14 Microsatellite Analysis Applications 73 83 i pa 103 113 123 f 133 r E 800 600 400 200 o A A SM MI Lane N 800 600 400 200 i i 0 MI Lane T Figure 9 4 An example of RER for heterozygote alleles at markers NM23 and D58346 RER is often more subtle than is shown in these examples Virtually all difference between normal and tumor samples that are not LOH are interpreted as replication error In general you should see RER in more than one marker to be sure that it is really genomic instability that you are observing and not merely an artifact Note While both microsatellite instability and loss of heterozygosity are indicative of cancerous tissue if an electropherogram shows RER at a given marker location an LOH calculation for that allele region is complicated or even invalid Canzian et al 1996 We do not recommend LOH calculations in regions that show clear signs of RER Microsatellite Analysis Applications 9 15 Troubleshooting LOH and RER Screening Common LOH and RER Problems Most problems in LOH and RER screening are common to all microsatellite applications See Chapter 11 for more information A few problems that are more specific to LOH and RER screening are described in Table 9 3 Table 9 3 Troubleshooting LOH and RER Screening Obs
241. perform a microsatellite analysis run The items marked with an asterisk are consumable items that should be discarded after a single use Reagents for making denaturing Long Ranger or polyacrylamide gels Square or shark s tooth comb GeneScan Internal Lane Size Standard GeneScan 350 GeneScan 400 HD or GeneScan 500 recommended Deionized formamide Loading buffer 25 mM EDTA 50 mg mL blue dextran Y 1X TBE buffer For the ABI PRISM 377 and ABI PRISM 377 with XL Upgrade 36 cm well to read plates Y 0 2 mm spacers For the ABI 373 and ABI 373 with XL Upgrade Y 12 24 or 34 cm well to read plates Y 0 4 mm spacers For the ABI PRISM 377 and ABI PRISM 377 with XL Upgrade You will need the following software to perform and analyze a microsatellite run ABI PRISM 377 or 377XL Collection Software version 2 0 or higher ABI Prism Run Modules Plate Check PreRun Module PR GS36 2400 for 36 cm well to read plates Run Module GS36 2400 for 36 cm well to read plates Be sure to use versions of these modules A C D or F that are compatible with the chosen dye set GeneScan Analysis software version 2 0 2 or higher Genotyper software version 2 0 For the ABI 373 with XL Upgrade ABI PRISM 373 XL Collection Software version 2 0 or higher ABI Prism Run Modules Plate Check PreRun GS Run Be sure to use the filter set A or B that is compatible with the chosen dye set
242. pop up menu If incorrect select the correct marker from the Category pop up menu EBE Histogram Microsat template 1 Category 7 Unknown 249 M 250 251 252 253 254 25 Note Leave the Member pop up field blank 6 Open the Member pop up menu to see a list of all the defined allele bins category members The boundaries for each bin are displayed as vertical dotted lines in the histogram All the peaks alleles belonging to a particular bin should fall between the two outer boundaries of that bin If they do not proceed to the next step to adjust the bin size 7 Open the Member pop up menu and choose the name of the bin you want to adjust Two handles are displayed on the corresponding bin boundaries EE Histogram Microsat template 1 E Category g Member Ros y T T T T T T T T T E I 246 247 248 pS 249 250 251 252 253 254 2590 8 Move the cross hair cursor onto one of the handles until the cursor becomes a cross hairs in a circle Click and drag the handle in or out as appropriate to redefine the bin Genotyper software automatically updates the bin size category definition as you move the handles Troubleshooting Microsatellite Analysis Common Problems The most commonly encountered problems during microsatellite analysis are Poor or non specific amplification See Chapter 6 Optimizing PCR and Troubleshooting PCR Amplification on page 11 1 for suggestions Incomplete 3 A nucleotide addition S
243. pped with a double sided temperature control for running 36 cm gels This minimizes differential heating effects both from front to back within a lane and between the edge and center lanes minimizing the effect known as smiling You can control temperature from 10 C above the ambient temperature to 60 C To perform electrophoresis at temperatures lower than 10 C above ambient connect a cooling water bath to the ABI PRISM 377 instrument Refer to the Modifications for Subambient Temperature Operations User Bulletin P N 904056 for detailed information about using an external water bath SSCP Analysis 7 27 7 28 SSCP Analysis Note Thermal control systems have limited capacity for maintaining a gel at a given temperature setting Electrophoresis at high power may produce more heat than the thermal control system can regulate The actual gel temperature is therefore higher than the temperature setting CAUTION Condensation from heat plates at low temperatures can lead to electrical shorts resulting in damage to the instrument Troubleshooting Preventing Problems As with any high resolution application it is advisable to display the migration time of at least one size standard peak for every lane Verify that the migration times of the size standard peaks are similar from lane to lane before flagging a potential mutant This will decrease the number of false positive results Size Standards Because of the complex peak patterns o
244. ppropriate volume of formamide blue dextran EDTA loading mix Refer to the application specific protocols and Appendix B Sample Loading Volumes for more information WARNING CHEMICAL HAZARD Formamide is a known teratogen It can cause birth defects Wash thoroughly after handling formamide Obtain a copy of the MSDS from the manufacturer Wear appropriate protective eyewear clothing and gloves Wash thoroughly after handling formamide 4 If you anticipate an extremely high sample concentration run dilutions of the sample as a precautionary measure Ifthe signal is too weak try concentrating the sample see Decreasing the Salt Concentration on page 2 3 4 Ifthe signal intensity is still too low after concentration consult Problems with Signal Strength and Quality on page 11 9 or Problems with Poor Amplification on page 11 1 2 8 Experimental Design Considerations Optimizing Electrophoresis Conditions Introduction Definition of Resolution Modifying Run Time Optimizing electrophoresis conditions run time run voltage and run temperature can greatly improve data quality run to run precision and or throughput When selecting values for these parameters consider the following factors Range of fragment lengths Required degree of resolution Type of genetic analysis you will be performing For example does the application require native or denaturing conditions The preset electroph
245. prerun the even or odd lanes before loading the rest of the lanes it is important not to prerun for more than 2 3 minutes If you are unavoidably delayed during the prerun period define separate size standards for the even and the odd lanes Differences in prerun times between gels can also cause sizing problems You can easily avoid this problem by defining the size standard every time you run a gel Matrix Problems Ifthe multicomponent matrix is not correct sample peaks in other colors will often bleed through to the size standard color creating false peaks and affecting sizing Sizing and Size Standards 5 5 Off Scale Data Off scale data can also be the source of peaks that bleed through to the size standard color even when the matrix is good See Determining Loading Concentrations for Samples on page 2 8 for suggestions on evaluating and modifying signal strength Even if you load the correct amount of sample sometimes the 35 bp fragment of the GeneScan 350 and GeneScan 500 size standards co migrates with one of the primer peaks In most cases the unused primer runs as a number of clustered off scale peaks Because matrix files cannot correct for off scale data bleedthrough peaks inevitably appear in other colors If the GeneScan Analysis Software mistakes one of the bleedthrough peaks for the 35 bp size standard peak the size calling curve will be inaccurate over part or all of its range Changes in Electrophoresis Condit
246. quencer 24 cm wtr plates ABI Prism 310 Genetic Analyzer POP 4 polymer Sizing and Size Standards 5 3 All results were obtained within a gel or within a set of injections from a single capillary Table 5 1 Cross platform precision results obtained from the AmpF STR Blue PCR Amplification Kit ABI Prism 377 ABI 373 ABI Prism 310 Allele n Actual Size Mean S D Mean S D Mean S D D3S1358 12 3 114 114 23 0 05 11453 0 15 111 89 0 07 19 3 142 143 36 0 08 143 06 0 04 140 55 0 01 vWA 11 3 157 157 25 0 06 157 62 0 03 155 20 0 01 21 3 197 196 98 0 03 197 16 0 05 195 50 0 06 FGA 18 4 219 220 25 0 05 217 73 0 11 217 15 0 05 30 18 267 268 87 0 05 265 20 0 13 265 68 0 10 For example consider D3S1358 allele 12 On all three platforms three times the standard deviation is less than 0 5 bp By contrast the mean called size for this allele differs by more than 2 bp between the ABI PRISM 310 and ABI PRISM 377 instruments IMPORTANT Because the called size for a fragment can differ from its actual size you should convert fragment sizes to alleles before comparing microsatellite data generated on different instruments 5 4 Sizing and Size Standards Preventing Troubleshooting Sizing Problems Preventing Sizing The following guidelines will help you avoid some of the more common sources of inconsistent sizing within a single experiment Problems What Can Go Guidelines Comments Use the same sizing method for a
247. r J Coad N de Pagter Holthuizen P and Pearson P L 1988 Molecular nature of genetic changes resulting in loss of heterozygosity of chromosome 11 in Wilms tumours Hum Genet 81 41 48 Mok S C H Lo K W and Tsao S W 1993 Direct cycle sequencing of mutated alleles detected by PCR single strand conformation polymorphism analysis BioTechniques 14 790 794 Mulligan L M Matlashewski G J Scrable H J and Cavenee W K 1990 Mechanisms of p53 loss in human sarcomas Proc Natl Acad Sci USA 87 5863 5867 Orita M lwahana H Kanazawa H Hayashi K and Sekiya T 1989 Detection of polymorphisms of human DNA by gel electrophoresis as single strand conformation polymorphisms SSCP Proc Natl Acad Sci USA 86 2766 2770 Orita M Suzuki Y Sekiya T and Hayashi K 1989 Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction Genomics 5 874 879 Seto D and Sukumar S 1992 Improved detection of mutations in the p53 gene in human tumors as single stranded conformation polymorphs and double stranded heteroduplex DNA PCR Methods Appl 2 96 98 Sheffield V C Beck J S Kwitek A E Sandstrom D W and Stone E W 1993 The sensitivity of single stand conformation polymorphism analysis for the Detection of single base substitutions Genomics 13 441 443 Smith T A Whelan J and Parry PJ 1992 Detection of single base mutations
248. r Southern based LOH Itis faster than Southern based detection of LOH Y It requires only minute nanogram amounts of tumor DNA 4 Itis suitable for formalin fixed and paraffin embedded archival tissue The third feature of PCR based LOH will prove extremely useful in developing tests to diagnose and predict the course of new types of cancer Existing archival tissue contains a wealth of genetic material along with complete patient histories Because LOH often appears in the same types of tumors as RER replication error see page 9 13 in some instances you will need to perform LOH screening in conjunction with RER screening The undetected presence of RER can mask the presence of LOH leading to a false negative LOH diagnosis Extraction of high quality DNA from formalin fixed and paraffin embedded tissue can be difficult If you cannot perform accurate DNA quantitation before PCR amplification interpretation of results can be difficult continued on next page Microsatellite Analysis Applications 9 5 Advantages of Using In one study of cervical cancer fluorescent detection had several key advantages over ABI PRISM radioactive detection Technology 4 Quantitative results were repeatable for a given locus Y Results from contiguous loci were entirely consistent When two contiguous loci indicated LOH the calculated LOH ratios were similar Rapid Screening Analysis is rapid Under ideal circumstances you can analyz
249. r details Create an SSCP analysis matrix file For directions on preparing matrix samples for SSCP analysis see Preparing the Matrix Samples on page 7 17 See Appendix C for instructions on creating a matrix file Usually you create and then reuse a single matrix file for each set of run conditions SSCP Analysis 7 13 Preparing for a Run ABI PRISM 377 ABI PRISM 377 with XL Upgrade and ABI 373 with XL Upgrade Gel Preparation Try both non denaturing 6 5 polyacrylamide gels and 0 5X MDE Mutation Detection gels to see which gel type gives the highest mutation detection rate in your system See Preparing Polyacrylamide Gels for SSCP on page A 4 and MDE Gels for SSCP on page A 6 for detailed instructions Note You can substitute Long Ranger gel solution FMC Corporation P N 50610 for standard polyacrylamide stock See Preparing a Long Ranger Gel for SSCP on page A 3 for detailed instructions Use 36 cm well to read plates standard 0 2 mm spacers and a square tooth comb Refer to the ABI Prism 377 DNA Sequencer User s Manual the ABI PRISM 377 DNA Sequencer User s Manual XL Upgrade or the ABI 373 DNA Sequencing System XL Upgrade User s Manual for the general procedure Instrument Setup The specific equipment and reagents needed for an SSCP analysis run are listed in Before You Begin on page 7 3 Run window settings are listed in Table 7 2 below and Table 7 3 on page 7 15 Table 7 2 Run Window
250. r most applications you can obtain acceptable results using a 12 cm gel If time is not limiting longer plates will give better resolution and greater precision 0 4 mm spacers Square or shark s tooth comb Refer to the ABI Prism 377 DNA Sequencer User s Manual the 373 DNA Sequencing System Users Manual or the 373 DNA Sequencer with XL Upgrade User s Manual for the general procedure Table 8 2 Run Window Settings for the ABI PRISM 377 and 377XL Data Field Setting Plate Check Module Plate Check A C D or F PreRun Module PR GS 36A 2400 or 36C D or F Run Module GS 36A 2400 or 36C D or F Collect Time 2 hours Note To see all product fragments you might need to increase the run time to 2 5 hours Sample Sheet The name of the sample sheet that you created for this run Well to Read distance 36 cm Lanes As appropriate for the comb used Table 8 2 Run Window Settings for the ABI PRISM 377 and 377XL continued Data Field Seiting Run Mode XL only Full scan 24 34 or 36 lanes XL scan 50 or 66 lanes Operator Your name Gel Matrix The name of the matrix file that you created for this set of run conditions or None if you have not yet created a matrix Matrix Same as Gel Matrix AutoAnalyze On or off as desired Note You must create a matrix file before analyzing the data Table 8 3 Run Window Settings for the ABI 373 with XL Upgrade
251. r pairs the increased cost per reaction and the decreased reproducibility over time do not justify this short term solution When buying or making primers make sure that they are length purified and that they are free of contaminants Typically start out with equal concentrations for all primer pairs It will often be necessary to adjust the concentration of primer pairs in the multiplex reaction until the peak heights are relatively even Increase the primer pair concentration for fragments showing weak amplification Decrease the primer pair concentration for fragments showing significantly greater than average amplification continued on next page Troubleshooting Consider amplifying separately any primer pair that fails to amplify after its Multiplex PCR concentration is increased To get rid of interfering background peaks try Swapping primer pairs between different multiplex reactions Removing primer pairs from the multiplex reaction Optimizing PCR 6 11 Using RNA Templates Suitable Templates Two Step RNA PCR Single Step RNA PCR For More Information 6 12 Optimizing PCR RNA templates can be single or double stranded If your starting sample is RNA then your template can be Total cellular RNA Poly A RNA Viral RNA tRNA rRNA gt To synthesize first strand cDNA prior to performing conventional PCR amplification you can use a reverse transcriptase such as MuLV or you can use
252. r the comb used Run Mode XL only Full scan 24 34 or 36 lanes XL scan 50 or 66 lanes Operator Your name Gel s Matrix File The name of the matrix file that you created for this set of run conditions or None if you have not yet created a matrix AutoAnalyze On or off as desired Note You must create a matrix file before analyzing the data continued on next page SSCP Analysis 7 15 Preparing and Preparing and Loading Experimental Samples Loading Samples Note deionized H O before loading 1 uL into the sample tube 7 16 SSCP Analysis Sometimes it is necessary to dilute the PCR product from 1 5 1 20 in distilled Step Action 1 To each sample tube add the following 3 0 uL deionized formamide Y 0 5 uL GeneScan Internal Lane Size Standard GeneScan 350 or GeneScan 500 recommended Y 1 uL PCR product Y 0 5 uL of 0 1 M NaOH 0 5 uL EDTA blue dextran 50 mM EDTA 50 mg mL blue dextran WARNING CHEMICAL HAZARD Formamide is a known teratogen It can cause birth defects Wash thoroughly after handling formamide Obtain a copy of the MSDS from the manufacturer Wear appropriate protective eyewear clothing and gloves Wash thoroughly after handling formamide WARNING CHEMICAL HAZARD Sodium hydroxide NaOH can cause severe burns to the skin eyes and respiratory tract Always work in a fume hood Obtain a copy of the MSDS from the manufacturer Wear appropriate
253. rTih DNA Polymerase In the presence of MnClL r Tth DNA Polymerase will efficiently reverse transcribe RNA to cDNA After chelating the manganese ions with EGTA and adding MgCl r Tth DNA Polymerase can act as a thermostable DNA polymerase in a subsequent reaction in the same tube Note For RNA templates with a high GC content or complex secondary structure Applied Biosystems recommends using r7th DNA polymerase because of its high temperature reverse transcriptase activity and thermostable DNA polymerase activity Using the GeneAmp EZ rTth RNA PCR Kit P N N808 0178 you can perform reverse transcription and PCR amplification in successive reactions in the same tube without buffer changes or reagent additions By combining programmed changes in temperature with the action of Bicine which is capable of buffering both metal and hydrogen ions in the EZ Buffer the GeneAmp EZ r7th RNA PCR Kit enables you to control a complex interaction of factors metal ion concentration pH and ionic strength affecting enzyme activity Note When amplifying multiple samples making buffer changes is time consuming and increases the likelihood of contamination The EZ Buffer affords full dUTP compatibility for AmpErase UNG mediated carryover prevention Refer to the TaqMan Gold RT PCR Kit Protocol P N 402876 and the TaqMan EZ RT PCR Kit Protocol P N 402877 for more information Preventing Competing Side Reactions Hot Start PCR When to Use H
254. rase XL PrePCR conversion to cDNA rTth DNA Polymerase Extra cycles AmpliTaq DNA Polymerase Stoeffel Fragment UlTma DNA Polymerase High Mg2 concentration AmpliTaq Gold DNA Polymerase AmpliTaq DNA Polymerase Stoeffel Fragment Optimizing PCR 6 9 Multiplexing PCR Definition Advantages Limitations Enzyme Choice Primer Quality Primer Pair Concentrations 6 10 Optimizing PCR Multiplex PCR is a technique for simultaneously amplifying multiple DNA targets using multiple primer pairs in the same PCR reaction Multiplex PCR can Simplify PCR setup Y Increase throughput Decrease cost per amplification Potential limitations to multiplex PCR include Y Primer oligomer formation Loss of specificity Decreased yield of specific products Overcoming these limitations can require a significant amount of optimization The high specificity of AmpliTaq Gold DNA Polymerase typically permits amplifying with elevated Mg concentrations for increased yield Because reactants such as dNTPs are often limiting during multiplex PCR using high quality primers is particularly important For example the decreased specificity and thus the increased reagent consumption of one pair of degraded PCR primers can prevent the success of the entire multiplex reaction Although you can compensate for a degraded pair of primers to some extent by increasing the concentration of the other prime
255. rated appears as follows e D12583 e Unknown All peaks from 98 00 to 113 00 bp in blue 105 lt X gt Highest peak at 104 68 0 50 bp in blue Microsatellite Analysis 8 23 Using the Plot Window 8 24 Microsatellite Analysis To bin alleles using the Histogram window continued Step Action 11 Repeat these steps to continue adding categories for each allele Remember to Y Select the markers by color Verify that the Histogram window displays the correct marker name To bin alleles directly using the individual allele plots Step Action 1 From the Views menu choose Show Categories Window or type 8 K and set up the main categories Groups for each marker as shown here e D12583 e Unknown All peaks from 98 00 to 113 00 bp in blue e D75517 Unknown All peaks from 235 00 to 261 00 bp in blue e 025391 Unknown All peaks from 139 00 to 153 00 bp in green D135171 Unknown All peaks from 171 00 to 197 00 bp in green From the Analysis menu choose Label Peaks Label peaks with Size in bp only From the Analysis menu choose Filter Labels Filter labels using the default settings best for dinucleotide repeat markers 4 In the Main window working with one dye color at a time a Choose all blue dye lanes by clicking on the Blue color button to the left of the dye lanes window b Draw a box in the plot window that covers all of the p
256. re listed in Before You Begin on page 8 3 Applied Biosystems recommends using a 5 Long Ranger 6 M urea gel to begin microsatellite analysis Alternatively you can use a 6 polyacrylamide 8 3 M urea gel Applied Biosystems recommends using a 5 Long Ranger 6 M urea gel Alternatively you can use a 6 polyacrylamide 8 3 M urea gel Use Y 12 24 or 34 cm well to read plates For most applications you can obtain acceptable results using a 12 cm gel If time is not limiting longer plates will give better resolution and greater precision Y 0 4 mm spacers Square or shark s tooth comb Note Appendix A Reagent Preparation contains protocols for preparing both Long Ranger and polyacrylamide gels continued on next page Setting the Preprocess and Analysis Parameters Note If the computer is connected to a network Applied Biosystems recommends that you disconnect it from the network during the gel run To do so select Chooser under the Apple menu In the bottom right area of the Chooser window click the AppleTalk Inactive button then close the Chooser window To set the Preprocess Parameters Step Action 1 Restart the Macintosh computer to defragment the RAM memory Open the GeneScan Analysis Software Select Preprocess Parameters from the Analysis menu 2 3 4 Specify the settings as shown below Select the appropriate matrix file and Sample Sheet by clicking on the Sw
257. re version 2 1 modules and for setting preferences to switch between the standard run modules and the chiller modules continued on next page Installing the Positioning the Machines External Cold Water Bath Step Action 1 Place the ABI PRISM 377 DNA Sequencer on a table or counter that provides easy rear access 2 Facing the DNA sequencer position the water bath on the right side of the DNA sequencer and at the same height 3 Plug both machines into an external power supply Determining Intake and Outlet Step Action 1 Turn on the ABI Prism 377 DNA Sequencer Select the Pump On command from the Manual Control window then click Execute 2 Open the right side panel where the water bottle for the internal pump system is mounted 3 Unscrew the water bottle and slowly pull it down to expose the ends of the tubes but do not remove the bottle 4 Observe the direction of water flow from the tubes into and out of the bottle to identify the intake and outlet tubes Note Water coming into the bottle is from the instrument outlet and water being drawn out of the bottle is pumped into the instrument intake Some instruments have a small cutout at the bottom of the water intake tube 5 Turn off the ABI PRISM 377 instrument Disconnecting the Internal Pump Step Action 1 Remove the water bottle and pull down the intake and outlet tubes to remove them 2
258. repeat marker for a heterozygous individual shows this typical triangle pattern when the alleles differ by 2 bp Figure 8 8 Typical pattern for dinucleotide repeat heterozygote Alleles differ by 2 bp Example 5 A GeneScan electropherogram for a dinucleotide repeat marker displaying peaks at 1 bp intervals is shown in Figure 8 9 AmpliTag Gold and other DNA polymerases can add a non templated A to the end of a PCR product during amplification When both the true allele and allele plus A products show 2 bp stutter bands a ladder of peaks differing by 1 bp may be seen for PCR products This phenomenon tends to be locus specific Figure 8 9 Typical pattern for dinucleotide repeat heterozygote showing 1 bp stutter See Table 8 4 for explanation of peak labels One allele in Figure 8 9 is labeled to indicate the origin of the peaks The pattern produced is a combination of both the 2 bp stutter peaks from the true allele and the allele plus the non templated A The resulting peaks differ by 1 bp Table 8 4 Table 8 4 Origin of 1 bp peak pattern in dinucleotide repeat marker Peak Origin 1 A product of allele peak 2 True allele peak based on DNA sequence 3 2 bp stutter of A peak 4 2 bp stutter to true allele peak 5 4 bp stutter of A peak continued on next page Microsatellite Analysis 8 35 Is Stutter a Real Stutter once understood does not pose a real problem for microsatellite analysi
259. rimental run Note See Appendix B for more information about loading volumes Note To ensure the best possible quality matrix load the matrix samples into every other lane and leave at least one empty lane between the matrix samples and the experimental samples continued on next page Creating a New You must create a matrix file before analyzing microsatellite data for the first time For Matrix File more information on creating matrices see Appendix C To create the matrix file use one of the following Dye Primer Matrix Standards Kit 5 FAM JOE TAMRA and ROX P N 401114 and module GS 36A 2400 Fluorescent Amidite Matrix Standards Kit 6 FAM TET HEX and TAMRA P N 401546 and module GS 36C 2400 Fluorescent Amidite Matrix Standards Kit 6 FAM HEX and ROX P N 401546 the NED Matrix Standard P N 402996 and module GS 36D 2400 Dye Primer Matrix Standards Kit 5 FAM JOE and ROX P N 401114 the NED Matrix Standard P N 402996 and module GS 36F 2400 Note Usually you create and save a single matrix file for each set of run conditions However if you experience persistent problems such as spectral overlap in the analyzed data you should remake the matrix file even if you have not altered the run conditions Microsatellite Analysis 8 11 Preparing for a Run ABI 373 Gel Preparation 8 12 Microsatellite Analysis The specific equipment and reagents needed for a microsatellite analysis run a
260. riphosphate dNTP is 200 uM In most cases lower dNTP concentrations do not significantly affect the yield of PCR amplification product and will increase the fidelity However for efficient base incorporation keep the four dNTP concentrations balanced and above the estimated Km of each dNTP 10 15 uM Some applications might require higher dNTP concentration especially when dNTP analogues are used However excess dNTPs decrease enzyme fidelity DNA polymerases require free magnesium ion in solution for activity For most PCR amplifications you can relate product yield and specificity and enzyme fidelity to the free magnesium ion Mg2 concentration free Mg total Mg total dNTP 2 EDTA In general increasing the free magnesium concentration increases yield and decreases specificity and fidelity To identify the magnesium concentration that gives the best compromise between yield and specificity or fidelity for your particular application perform the following experiment In the presence of 800 uM total dNTP concentration run a MgCl reaction series in 0 5 mM increments over the range from 1 4 mM MgCl to identify the optimal concentration continued on next page Template Concentration Enzyme Concentration The concentration of template in your sample can affect the success of PCR amplification in a variety of ways Too much template promotes nonspecific binding of primers to secondary sites or changes
261. roblems 3 11 to 3 14 solving matrix problems 3 14 when to remake matrix 3 11 nucleic acids quantitating 3 10 running at subambient temperatures 7 4 to 7 9 sample loading volumes B 1 ABI PRISM 377 with XL Upgrade data collection how it works 4 6 sample loading volumes B 1 See Also ABI PRISM 377 ABI PRISM dyes available dyes 4 2 dye spectra understanding 4 4 to 4 5 dye filter sets choosing 4 6 to 4 8 chemical forms table of 4 7 reagents and primer dye sets table of 4 8 recommended combinations table of 4 8 ABI PRISM Linkage Mapping Set primer dye set 4 8 ABI PRISM multicolor fluorescence technology increasing throughput multiplexing 2 2 to 2 3 AFLP bacterial and fungal genomes analyzed 10 10 for more information 10 4 literature references E 9 microbial fingerprinting 10 1 preselective amplification 10 5 primer selection 10 9 selective amplification 10 6 plant genomes analyzed 10 12 to 10 17 plant mapping 10 2 preselective amplification 10 7 primer selection 10 11 selective amplification 10 8 primer dye sets 4 8 Index 1 simplifying complex patterns 10 7 template preparation and adaptor ligation 10 5 testing new genomes what is AFLP 10 1 allele binning See Genotyper Amicon Centricon 100 Microconcentrator using to desalt DNA 2 3 ammonium persulfate solution 10 A 1 AmpFISTR kits 9 22 discrimination power primer dye sets 4 8 reliability 9 24 table of loci 9 21 AmpliTaq DNA Polymerase description
262. roblems with this combination you may see an improvement in data quality by switching to 6 FAM HEX NED and ROX and using Virtual Filter Set D Table 4 4 Reagent Kit and Custom labeled Primer Dye Sets Filter Set Virtual Filter Set Application Dye Set ABI 373 ABI PRISM 377 AmpF STR Blue PCR Amplification Kit 5 FAM Blue ROX Red std A A F AmpF STR Green PCR Amplification JOE Green ROX Red std A F Kit StockMarks for Horses Equine Paternity 5 FAM Blue JOE Green A PCR Typing Kit TAMRA Yellow ROX Red std ABI Prism Linkage Mapping Set 6 FAM Blue TET Green B C StockMarks for Cattle Bovine Paternity HEX Yellow TAMRA Red PCR Typing Kit std Custom labeled primers for fragment 6 FAM Blue HEX Green A D analysis NED Yellow ROX Red std ABI PRISM Linkage Mapping Set Version 2 AFLP Microbial Fingerprinting Kit 5 FAM Blue JOE Green F AFLP Plant Mapping Kits AmpF STR Profiler PCR Amplification Kit AmpF STR Profiler Plus PCR Amplification Kit NED Yellow ROX Red std not supported for use with the ABI 373 and ABI 373 with XL Upgrade DNA Sequencers 4 8 ABI PRISM Dyes continued on next page BigDye Filter Wheel To use the new dRhodamine terminator BigDye primer and BigDye terminator sequencing chemistries on the ABI 373 and ABI 373 with XL Upgrade DNA Sequencers t
263. rosatellite Analysis 8 27 Make Categories from Labels Category tolerance EJ Unmark overlapping categories O Skip overlapping categories Name Prefix First number Number increment comment group name un O member name Biante for dye color s blue green Jyellow Jred EX Exclusive clears previous labels at same peak O with scaled height of at least 1 O with scaled height of at most 9999 D12583 Unknown All peaks from 98 00 to 113 00 bp in blue 101 lt X Highest peak at 100 82 4 0 50 bp in blue 103 X Highest peak at 102 80 0 50 bp in blue 105 X Highest peak at 104 75 0 50 bp in blue 107 X gt Highest peak at 108 61 0 50 bp in blue 109 X Highest peak at 110 60 0 50 bp in blue Figure 8 2 Example of allelic bin names generated from the Make from Labels dialog box configured as shown in Figure 8 1 8 28 Microsatellite Analysis Using the Add Multiple Categories Feature Make Categories from Labels Category tolerance EJ Unmark overlapping categories O Skip overlapping categories Name First number fi Number increment comment mwin member name for dye color s blue green yellow red E Exclusive clears previous labels at same peak O with scaled height of atleast 1 O with scaled height of at most 9999 Figure 8 3 The Make from Labels dialog box configured to use a prefix for the allele name D12583 Unknown All
264. round of PCR amplification Run at least one lane of amplified control DNA on every gel In addition to the GeneScan Internal Lane Size Standards Applied Biosystems sells the CEPH 1347 02 standard used to generate the G n thon map of the human genome P N 403062 Experimental Design Considerations 2 11 General Analysis and Evaluation Techniques Overview In This Chapter This chapter will help you evaluate and analyze data obtained on the ABI PRISM 377 and ABI 373 DNA Sequencers In particular it should help you avoid many common causes of poor quality data This chapter contains the following topics Topic See Page Analyzing the Data 3 2 Evaluating Data Quality 3 8 Quantitating Nucleic Acids 3 10 Evaluating Matrix Quality 3 11 General Analysis and Evaluation Techniques 3 1 Analyzing the Data 3 2 Process Overview The following diagram summarizes the data analysis process using GeneScan Analysis Software Data analysis using GeneScan 672 Fragment Analysis Software is covered in more detail on page 8 13 Set the Analysis Parameters Define the size standard Analyze the data To analyze sample files Action Launch the GeneScan Analysis Software Assign the matrix file to the sample files if you have not already done so in the ABI PRISM 310 Collection Software Note For detailed instructions see Assigning a New Matrix to a Sample File in Chapter 4 of the GeneSc
265. rrect Check gel composition and rerun samples Size standard peaks not recognized when defining size standard Height of a size standard peak less than the Peak Amplitude Threshold for the size standard color in Analysis Parameters Note 50 RFU is the default threshold Gel image does not display all size standard peaks Minimum Peak Half Width is set too high in Analysis Parameters Rerun sample adding recommended amount of size standard Adjust scan numbers for Display Gel Image under Gel Processing Parameters and choose Regenerate Gel Image Lower the value for the Minimum Peak Half Width Troubleshooting 11 11 Table 11 8 Problems with Peak Number and Position continued Observation Possible Causes Recommended Actions Inconsistent mobilities from gel to gel Total polymer percentage wrong Remake gel carefully following protocol Wrong TBE buffer concentration Remake buffer carefully following protocol Poor quality or old reagents Remake 10X TBE and gel solution stock using fresh reagents from a reliable source IMPORTANT ultrapure Urea must be Too much dissolved O remaining in gel Make sure that you Y Keep vacuum strength at 20 Hg and leave under vacuum for 5 minutes when degassing acrylamide Y Stir and pour gel solutions gently Filter and pour gels at 20 23 C Variations in spacers that is some may be worn or damag
266. rting size Category tolerance Category spacing Number of categories EJ Group name D S517 Name First number fi Number increment fi with dye color s blue green yellow red O with scaled height of atleast 1 O with scaled height of at most 9999 EX Exclusive clears previous labels at same peak 3 Click OK to generate a set of categories for the marker as follows e D75517 e Unknown All peaks from 235 00 to 261 00 bp in blue Al X Highest peak at 235 00 0 50 bp in blue A2 X Highest peak at 237 00 0 50 bp in blue AS 00 Highest peak at 239 00 0 50 bp in blue A4 X Highest peak at 241 00 0 50 bp in blue e AS 00 Highest peak at 243 00 0 50 bp in blue AG X Highest peak at 245 00 0 50 bp in blue A X Highest peak at 247 00 0 50 bp in blue AS Xx Highest peak at 249 00 0 50 bp in blue 4 Repeat step 2 and step 3 for the rest of the markers making sure to enter the appropriate starting size dye color and marker name in the Add Multiple Categories dialog box 5 From the Analysis menu choose Label Peaks Label peaks with Size in bp only 6 From the Analysis menu choose Filter Labels Filter labels using the default settings best for dinucleotide repeat markers To fine tune the category definitions using the Histogram window Step Action 1 Define the bin size as follows a From the Analysis menu choose Set Statistics Options b Select the followi
267. rument to detect a dye signal depends upon the dye s Excitation efficiency at the wavelengths of light emitted by the laser The argon ion laser in the ABI PRISM 377 and the ABI 373 instruments emits the greatest intensity of light at 488 and 514 5 nm Quantum yield Concentration Distinguishing Between Two Dye Signals The instrument identifies a dye by determining the ratio of the dye s emission intensities in a series of filters Each dye s maximum emission intensity lies in a unique filter The ability of any instrument to distinguish between two dye signals is determined by the relative differences in the measurements of the two dyes in each of two filters The ABI PRISM Dye Maximum Emission and Excitation Wavelengths larger the difference in the intensity of the signal in the two filters the easier it is to identify each dye The closeness of the ratios of the measurements in each of the filters will be determined by the amount of overlap in the two emission spectra In general the accuracy with which two dye signals can be discerned is enhanced by A larger separation between the emission maxima of the two dyes Y A narrower emission spectrum of one or both dyes The following table lists the maximum fluorescence emission and excitation wavelengths for oligonucleotides labeled with ABI PRISM dye NHS esters dye phosphoramidites and F JdNTPs The actual maximum emission and excitation wavelengths may differ from the
268. s In Problem fact stutter can actually aid in allele calling in two cases Distinguishing true allele peaks from non specific PCR products Non specific PCR products are not associated with stutter bands 4 Identifying alleles that fall far outside the reported allele range The percent stutter is often specific to a particular locus You can sometimes identify alleles that fall far outside the previously reported range on the basis of percent stutter 8 36 Microsatellite Analysis Microsatellite Analysis Applications Overview In This Chapter This chapter provides instructions for performing specialized applications of microsatellite analysis on the ABI PRISM 377 and ABI 373 DNA Sequencers 4 Performing microsatellite analysis using the ABI PRIsM Linkage Mapping Set Version 2 LMS V2 Screening for the loss of heterozygosity LOH of microsatellite markers linked to known oncogenes or tumor supressor genes Screening for evidence of replication error RER using microsatellite markers Determining animal paternity with the StockMarks kits Determining human identity with the AmpF STR kits This chapter contains the following topics Topic See Page Microsatellite Analysis Using the LMS V2 9 2 Troubleshooting the LMS V2 9 4 LOH Screening 9 5 RER Screening 9 13 Troubleshooting LOH and RER Screening 9 16 Animal Paternity 9 17 Human Identification 9 19 Microsatellite An
269. s Manual for a detailed discussion of allele binning IMPORTANT To convert a fragment s called size to an allele you cannot simply round the called size to the nearest allele size If you choose a commonly used standard such as CEPH 1347 02 P N 403062 you can correlate the allele sizes that you obtain with the allele sizes obtained by others such as the CEPH Genotype Database http www cephdb fr cephdb For more information on the CEPH Genotype Database refer to page 7 33 of the ABI PRISM Linkage Mapping Set Version 2 User s Manual You can also use an allelic ladder to genotype analyzed samples see page 9 23 Guidelines for Using Control DNA Amplify at least one control DNA sample for every round of PCR amplification Run at least one lane of amplified control DNA for every set of microsatellite markers used Run at least one lane of amplified control DNA whenever you change the electrophoresis conditions To Save Time Prerun Checklist Stock Solutions Having stocks of the following reagents buffers saves time during run setup Y Deionized formamide Lasts for 3 months at 15 to 25 C See Deionized Formamide on page A 7 for details Loading Mix 10 2 1 mixture of formamide size standard loading buffer Lasts for 2 weeks at 2 6 C See step 1 on page 8 10 for details At Any Time Perform the following at any time before running Beforea Run Complete the Sample Sheet Refer to the GeneScan Analysis S
270. s that facilitate genotyping Bio Techniques 20 1004 1010 Clemens P R Fenwick R G Chamberlain J S Gibbs R A de Andrade M Chakraborty R and Caskey C T 1991 Carrier detection and prenatal diagnosis in Duchenne and Becker muscular dystrophy families using dinucleotide repeat polymorphisms Am J Hum Genet 49 951 960 Collins F 1992 Positional cloning Let s not call it reverse anymore Nature Gen 1 3 6 Comuzzie A Hixson J Almasy L Mitchell B Mahaney M Dyer T Stern M MacCluer J and Blangero J 1997 A major quantitative trait locus determining serum leptin levels and fat mass is located on human chromosome 2 Nature Gen 15 273 276 Cook E H Lindgren V Leventhal B L Courchesne R Lincoln A Shulman C Lord C and Courchesne E 1997 Autism or atypical autism in maternally but not paternally derived proximal 15q duplication Am J Hum Genetics 60 928 934 Copeman J Cuccan F Hearne C Cornall R Reed P Ronningen K Undlien D Nistico L Buzzetti R Tosi R et al 1995 Linkage disequilibrium mapping of a type 1 diabetes susceptibility gene IDDM7 to chromosome 2q31 q33 Nature Gen 9 80 85 Edwards A Hammond H A Jin L Caskey C T and Chakraborty R 1992 Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups Genomics 12 241 253 Edwards A Civitello A Hammond H A and Cask
271. sample can differ between instrument platforms ABI PRISM 310 Genetic Analyzer versus ABI PRISM 377 DNA Sequencer because of differences in the type and concentration of the gel polymer matrices and in electrophoresis conditions Y Sizes may differ between protocols for the same instrument platform because of differences in gel or polymer concentration run temperature gel or capillary thickness and well to read length Y Slight procedural and reagent variations between gels or between capillaries result in greater size variation than that found between samples on the same gel or between samples injected in the same capillary The internal lane size standard is run with every sample and is used to normalize lane to lane or injection to injection migration differences thereby providing excellent sizing precision within a gel or within a set of capillary injections Size windows based on the allelic ladder are used to assign allele designations and genotypes to the samples The genotyping of samples by comparison to the AmpF STR Allelic Ladder Microsatellite Analysis Applications 9 23 can be automated using Genotyper 2 0 software and the custom AmpF STR template files Reliable Results The AmpF STR kit reagents and protocols have been optimized to produce the quality of results necessary for human identification applications Primers have been designed to give a high degree of target specificity maximum 3 A nucleotide addition and
272. sh with back heat transfer plate and alignment pins ABI PRISM 377 DNA Sequencer only Place cassette flush against back heat transfer plate IMPORTANT The spacers must touch the alignment pins You should be able to see the alignment pins pressing against the spacers Difficulty in loading sample because wells not flushed Flush the wells prior to loading the gel Troubleshooting 11 9 Table 11 7 Problems with Signal Strength and Quality continued Observation Possible Causes Recommended Actions Signal too low e peak heights are lower than usual Bad formamide Use freshly deionized formamide See Deionized Formamide on page A 7 for directions Note Formamide pH should be between 7 and 9 Insufficient FJdNTPs added to PCR reaction Optics detector misaligned Reamplify using more FIANTPs or examine the efficiency of the PCR Call service representative Signal too high streaks appearing or peaks heights are greater than 4000 RFU Too much sample added to some or all lanes Load less sample Reamplify using less F JdNTPs Unincorporated F dNTPs Purify the PCR product High noisy baseline Matrix made incorrectly resulting in too much correction also indicated by troughs under peaks Remake matrix Be sure to Remove primer peak or aberrant off scale peaks from scan range Y Pick start and stop points on flat parts of the
273. sing Fluorescent Labeling Methods 0 0 eee eee eee 2 5 Determining Loading Concentrations for Samples o ooooo oocoooocoocrcoroooo oo 2 8 Optimizing Electrophoresis Conditions 00 0 cece eee eee eee eens 2 9 Using Control DNA 3 deca ed he eee Ge eed See le dae ae oes 2 11 General Analysis and Evaluation Techniques 3 1 OVA aio fied 3 1 Analyzing the D ta osie ar Dd e a aide E N E 3 2 Evaluating Data Quality oooooooocoorrr cece cence ences 3 8 Quantitating Nucleic Acids 2 0 cece eee een S 3 10 Evaluating Matrix Quality ABI PRISM Dyes cis a bees ses ee Oe eel OVINA A A bi 4 1 Av llable DY ES coi Ai A EA Eee A IA 4 2 Understanding Dye Spectra oo oooocooooororrr nee e eens 4 4 Choosing Dye Filter Set Combinations 00 0 0 eee cee eee 4 6 Emission Spectra for Representative Dye Virtual Filter Set Combinations 4 10 Sizing and Size Standards ccc ccc cee cence ween es Sel OVERVIEW a A A ORO we ee ae had Aes 5 1 Introduction to S121 RA AA A ae aa NS 5 2 Preventing Troubleshooting Sizing Problems 0 0 c eee eee eee eee nee 5 5 GeneScan Internal Lane Size Standards 0 cece eee cece nen eee 5 7 GeneScan 350 Size Standard 0 0 cece ence een ences 5 8 GeneScan 400HD Size Standard 0 0 ee cc eee eee eens 5 10 GeneScan 500 SOOXL Size Standard 0 0 cee een eens 5 12 GeneScan 1000 Size Standard
274. sis 2 2 control DNA using 2 11 for performing experiments 2 2 temperature control parameters modifying 6 15 H help Custom Oligonucleotide Synthesis Service 2 7 e mail address 1 3 Documents on Demand 1 4 Internet address 1 3 regional offices 1 7 to 1 9 support 1 3 to 1 9 horse StockMarks 9 18 Hot Start technique 6 13 to 6 14 AmpliWax PCR Gem mediated Hot Start performing 6 14 Hot Start procedure 6 13 human identification 9 19 to 9 25 advantages of STRs 9 19 AmpFISTR kits 9 22 discrimination power 9 25 reliability 9 24 AmpFISTR loci table of 9 21 automated sizing and genotyping 9 20 for more information 9 25 high throughput about 9 20 using allelic ladders 9 23 to 9 24 using STRs 9 19 I Important user attention word 1 2 inconsistent prerun times how effects size calling 5 5 Internet address Custom Oligonucleotide Synthesis Service 2 7 manual updates 1 2 technical support 1 3 L Linkage Mapping Set advantages 9 2 collecting and analyzing LMS V2 microsatellite data 9 3 for more information 9 3 primer dye set 4 8 troubleshooting 9 4 whatis LMS 9 2 local sizing methods 5 2 LOH See loss of heterozygosity screening for Long Ranger gels preparing A 1 to A 3 loss of heterozygosity screening for 9 5 to 9 12 advantages 9 5 analyzing LOH data 9 11 to 9 12 example 9 12 limitations 9 5 literature references E 7 troubleshooting 9 16 whatis LOH 9 5 9 2 to 9 4 M magnesium ion concentration
275. stop points If this does not improve the matrix data run new matrix standards Preparing 5 End Labeled Primers Overview In This Appendix This appendix provides detailed instructions for preparing 5 end labeled primers directly on any Applied Biosystems DNA synthesizer using any of the Applied Biosystems dye phosphoramidites This appendix contains the following topics Topic See Page Abbreviations and Definitions D 1 Introduction to 5 end Labeling D 2 Instrument Setup D 4 Synthesis and Purification of 5 end Labeled Primers D 6 Calculating Absorbance for DNA Samples D 10 Abbreviations and Definitions Abbreviation Definition O D Optical Density unit The amount of a substance dissolved in 1 0 mL that will give an absorbance reading of 1 00 in a spectrophotometer with a 1 cm path length The wavelength is assumed to be 260 nm unless stated otherwise 3 3 prime hydroxyl end of an oligonucleotide 5 5 prime hydroxyl end of an oligonucleotide A Absorbance of a solution at wavelength x nm in a spectrophotometer with a 1 cm path length kcal kilocalories OPCO Oligonucleotide Purification Cartridge q s Quantity sufficient to bring solution to desired volume TBE buffer Tris borate EDTA buffer TEAA Triethylammonium acetate TEMED N N N N tetramethylethylenediamine U Enzyme unit viv Volume per volume Preparing 5 End Labeled Primers D 1 Introduct
276. t New Engl J Med 319 525 532 Walsh P S Erlich H A and Higuchi R 1992 Preferential PCR amplification of alleles Mechanisms and solutions PCR Methods Appl 1 241 Yaremko M L Recant W M and Westbrook C A 1995 Loss of heterozygosity from the short arm of chromosome 8 is an early event in breast cancers Genes Chromosomes Cancer 13 186 191 Yaremko M L Kutsa C Lyzak J Mick R Recant W M and Westbrook C A 1996 Loss of heterozygosity from the short arm of chromosome 8 is associated with invasive behavior in breast cancer Genes Chromosomes Cancer 16 189 191 Ausubel F M Brent R Kingstin R E Moore D D Seidman J G Smith J A and Struhl K eds 1987 Current Protocols in Molecular Biology Greene Publishing Associates and Wiley Interscience John Wiley and Sons New York Bachem C W B van der Hoeven R S de Bruijn S M Vreugdenhil D Zabeau M and Visser R G F 1996 Visualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP analysis of gene expression during potato tuber development Plant J 9 745 753 Baker S B Rugh C L and Kamalay J C 1990 RNA and DNA isolation from recalcitrant plant tissue Bio Techniques 9 268 272 Ballvora A Hesselbach J Niewohner J Leister D Salamini F and Gebhardt C 1995 Marker enrichment and high resolution map of the segment of potato chromosome VII harbouring the
277. t Genome Msel Primers CAA CAC CAG CAT CTA CTC CTG CTT AA O ac O EcoRI Primers NOT DETERMINED 10 16 AFLP Mapping Table 10 14 Primer Combinations for Rice Species Small Plant Genome Msel Primers CAA CAC CAG CAT CTA CTC CTG CTT EcoRI Primers Fluorescent Applied Biosystems has adapted the AFLP technique for use with our ABI PRISM Dye labeling and fluorescent dye labeling and detection technology PCR products are dye labeled Marker Detection during amplification using a 5 dye labeled primer For high throughput you can co load up to three different reactions labeled with different colored dyes in a single lane on the ABI 373 or ABI PRISM 377 DNA Sequencer Load a GeneScan Internal Lane Size Standard in a fourth color in every lane or injection to size all amplification fragments accurately You can automate the scoring of the large numbers of markers that are typically generated by analyzing your results with GeneScan Analysis and Genotyper software AFLP Mapping 10 17 Troubleshooting Troubleshooting PCR Amplification Topics This section offers troubleshooting suggestions for the following problem areas Y Problems with poor amplification page 11 1 Problems with extra peaks page 11 5 Problems with missing peaks page 11 6 Table 11 1
278. t reactions covering all relevant microsatellite loci for a single individual in one gel lane Co loading allows you to genotype hundreds of animals in a single day You can also automate genotyping by analyzing your results with GeneScan Analysis and Genotyper software Figure 9 5 on page 9 18 continued on next page Microsatellite Analysis Applications 9 17 Allele Frequencies StockMarks for Cattle Allele frequency information is currently available for the Holstein dairy cattle breed Similar frequency information is being collected for Aberdeen Angus Red Angus Simmental Gelbvieh Salers and South Devon To test non Holstein breeds with the StockMarks for Cattle kit you will first need to genotype approximately 20 unrelated animals to determine if the Holstein frequencies apply to the breed of interest StockMarks for Horses Allele frequency information is currently available for a number of horse breeds Warmblood 1 and 2 Standardbred Fjord Friesian Throroughbred and Tennessee Walkers Genotyping A Genotyper plot of GeneScan results from the equine control DNA is shown in Example Figure 9 5 All 12 alleles amplified by the StockMarks for Horses Equine Paternity PCR Typing Kit are plotted T T T TT T T T T T T Li T T T T T T T T T T T T T T T T T T T T T T T T T T 30 100 120 140 160 180 200 220 240 260 horse 4 Results 7 Blue std dev taq 1500 1000 500 VHL20 HTG4 AHT4 HMS 96 71 129 49 148 31 176 50 99 27 131 47
279. table than AmpliTaq DNA Polymerase allowing higher denaturation temperatures for GC rich templates or templates with complex secondary structure It lacks 5 3 exonuclease activity making it useful in multiplex PCR Finally it is active and specific over a wide range of magnesium A prePCR incubation heating step of 10 minutes at 95 C activates approximately 40 of the enzyme molecules This is sufficient to perform efficient amplification during the early cycles when target copy number is low Because more enzyme is activated during each denaturation step enzyme activity increases as the number of target molecules increases providing optimal PCR performance AmpliTaqg DNA Polymerase proofreading activities but has a 5 3 exonuclease activity AmpliTaq Gold DNA Polymerase AmpliTaq DNA Polymerase LD AmpliTag DNA Polymerase Stoeffel Fragment ion concentrations 2 10 mM 1 2 In Time Release PCR the prePCR heating step is omitted and the total number of cycles is increased Because the activation step is omitted very little active enzyme is present during the first few PCR cycles and many additional cycles are necessary for good results Enzyme Choice Table UlTma DNA Polymerase UlTma DNA Polymerase is obtained by expressing a modified form of the Thermotoga maritima Tma DNA polymerase gene in an E coli host It lacks a 5 3 exonuclease activity but retains a 3 5 exonuclease proofreading activity It
280. the computer before collecting data Conflicting extensions Choose Extensions Manager from the Control Panels Turn off any extensions that were not part of the original installation and restart computer 11 8 Troubleshooting continued on next page Table 11 6 Problems with Current Observation Possible Causes Recommended Actions No current Incorrect TBE buffer formulation Use correct TBE buffer concentrations 10X TBE in the gel for a final concentration of 1X 1X TBE running buffer Instrument set up incorrectly Verify that The electrode leads are secure and plugged in The bottom and top of the gel are immersed in running buffer Broken instrument parts Check for breaks in Y Electrode leads Y Buffer chambers Y Platinum wire in buffer chamber Corrupted firmware Resend firmware by performing a total reset Table 11 7 Problems with Signal Strength and Quality Observation Possible Causes Recommended Actions Signal too low e peak heights are lower than usual Insufficient sample added to some or all lanes Did you add a full 1 5 uL of PCR product to formamide size standard mix If not add 1 5 uL PCR product to formamide size standard mix If so concentrate your PCR product before adding to formamide size standard mix or examine the efficiency of the PCR Check pipette calibration Cassette not flu
281. the pH of the reaction mix Too little template can result in poor yields especially if the template is degraded Even very low template concentrations ten copies are often sufficient for successful PCR amplification If your starting sample is DNA you can use up to 20 000 copies of the target to start optimization trials In general this translates to 1 5 ng of cloned template 200 ng to 1 ug of genomic DNA Start optimization trials with less genomic DNA if starting material is limited With clean good quality genomic DNA 1 2 ng of starting material almost always works well Use fewer PCR cycles for very large amounts of genomic DNA For most PCR applications 2 0 2 5 units of AmpliTaq Gold DNA Polymerase is recommended for each 100 uL reaction volume Note You can avoid the tedium and inaccuracies involved in pipetting 0 5 uL amounts of enzyme by adding the enzyme to a fresh Master Mix prepared for a number of reactions Optimizing PCR 6 7 Choosing the Right Enzyme Introduction For most applications AmpliTaq Gold DNA Polymerase is the enzyme of choice However Applied Biosystems supplies a number of PCR enzymes that have been optimized for specific needs For quick reference an enzyme choice table is included at the end of this section PCR Enzyme Overview 6 8 Optimizing PCR A brief summary of the seven PCR enzymes supplied by Applied Biosystems follows Derivatives of Native Taq DNA Polymerase AmpliTaq D
282. tion the now resolved fluorescence intensities are color coded and displayed as peaks in the electropherogram ABI 373 DNA Sequencer During an electrophoresis run the ABI 373 DNA Sequencer records the fluorescence intensity as a function of time using a moving stage that contains the optical equipment filter wheel and photomultiplier tube Each time the stage traverses across the gel a different bandpass filter is positioned in front of the photomultiplier tube A single scan of the gel with one filter takes 1 5 seconds A complete scan with four filters takes 6 seconds A complete scan equals one data point A multicomponent matrix is applied to the fluorescence intensity data to correct for spectral overlap among the dyes After correction the now resolved fluorescence intensities are color coded and displayed as peaks in the electropherogram continued on next page Dye Chemical Forms ABI PRISM dyes are available in multiple chemical forms Some forms are supplied coupled to primers and others you can use to label your own custom primers Each form has distinct advantages and disadvantages depending upon the intended application and your laboratory setup The following table summarizes the uses of and dyes available in each chemical form Chemical Form Used For Available Dyes multiple nucleotides during PCR amplification NHS esters Post synthesis 5 end labeling of NEDa TAMRA ROX oligonucleotides co
283. trile followed by 5 mL of 2 M TEAA through the OPC to waste 3 Pass the fluorescently labeled oligonucleotide solution through the OPC at a rate of about one drop per second Collect the eluate and pass it through a second time 4 Pass 5 mL of 8 acetonitrile in 0 1 M TEAA v v followed by 5 mL of water through the OPC to waste 5 Elute drop by drop the purified dye labeled oligonucleotide with 1 mL of 20 acetonitrile in water v v P N 400314 6 Store the purified fluorescently labeled oligonucleotide in the freezer either dry or as a neutral aqueous solution Keep it in the dark The capacity of an OPC cartridge allows for maximum recovery of purified product from 8 10 O D of crude fluorescently labeled oligonucleotide All of the product from a 40 nmol synthesis can be purified using a single OPC cartridge However a 0 2 umol synthesis requires three or four cartridges for total product purification depending on crude yield You can also purify fluorescently labeled oligonucleotides by reverse phase HPLC employing the same column and mobile phase conditions as for unlabeled oligonucleotides The greater hydrophobicity of fluorescently labeled oligonucleotides results in significantly longer elution times than those of unlabeled species You can analyze crude or OPC purified fluorescently labeled oligonucleotides by reverse phase HPLC PAGE and capillary electrophoresis using MicroGel Gel Fill
284. ts guidelines for setting up a laboratory for PCR DNA analysis recommended protocols for DNA extraction the importance of DNA quantitation prior to STR analysis protocols for PCR amplification of the kit loci information on the multicomponent analysis of fluorescence data protocols for detection and analysis of PCR products guidelines for interpretation of results the use of Genotyper software for automated genotyping of alleles guidelines for troubleshooting results summaries of the validation work for the respective kits according to the guidelines established by the Technical Working Group on DNA Analysis Methods TWGDAM population genetics data for the kit loci The sections detailing how to set up a laboratory for PCR DNA analysis and how to perform the recommended protocols for DNA extraction can be used by all laboratories that perform PCR analysis These sections are updated from the Applied Biosystems AmpliType User s Guide Versions 1 and 2 Microsatellite Analysis Applications 9 25 AFLP Mapping Introduction What is AFLP Advantages of AFLP Applications of AFLP The AFLP amplified fragment polymorphism technique is used to visualize hundreds of amplified DNA restriction fragments simultaneously The AFLP band patterns or fingerprints can be used for many purposes such as monitoring the identity of an isolate or the degree of similarity among isolates Polymorphisms in band patterns map to specific loci al
285. uation Techniques ABI PRISM Dyes Overview In This Chapter This chapter describes the dyes available for fluorescent labeling of oligonucleotides used in GeneScan fragment analysis applications It also provides the background information necessary to understand the following issues relevant to experimental design and data evaluation Y How to choose dye filter set combinations Y What factors determine the relative signal intensities of ABI PRISM dyes Y Why matrix files depend on the run conditions This chapter contains the following topics Topic See Page Available Dyes 4 2 Understanding Dye Spectra 4 4 Choosing Dye Filter Set Combinations 4 6 Emission Spectra for Representative Dye Virtual Filter Set Combinations 4 10 ABI PRISM Dyes 4 1 Available Dyes Introduction Applied Biosystems supplies ten fluorescent dyes to use on ABI PRISM instruments 4 2 ABI PRISM Dyes 5 FAM 6 FAM R110 TET JOE R6G HEX NED TAMRA and ROX Each emits a continuous spectrum of light upon laser excitation ABI PRISM 377 DNA Sequencer During an electrophoresis run the ABI PRISM 377 DNA Sequencer records the fluorescence intensity as a function of time and wavelength from regions on a CCD camera that correspond to different detection wavelength ranges A multicomponent matrix is applied to the fluorescence intensity data to correct for spectral overlap among the dyes After correc
286. ucleotide addition ABI PRISM Technology The ABI PRISM 377 and ABI 373 instruments allow extremely rapid separations Fragments that are 300 bp or less in length can be separated in 2 hours 4 hours on the ABI 373 The PCR conditions and optimized protocols described in this section were developed using the following GeneAmp PCR System 9600 4 True Allele PCR Premix with AmpliTag Gold DNA Polymerase CEPH family 1347 DNA When using other instruments reagents or DNA some optimization might be required continued on next page 9 2 Microsatellite Analysis Applications Data Collection and Data collection and analysis are performed as for any microsatellite application For Analysis more information refer to the ABI PRISM Linkage Mapping Set Version 2 User s Manual P N 904999 Microsatellite Analysis Applications 9 3 Troubleshooting the LMS V2 If No Amplification Occurs Optimizing Marker Performance For PCR failures repeat PCR on the CEPH 1347 02 control DNA using the recommended protocol in the AB Prism Linkage Mapping Set Version 2 User s Manual Applied Biosystems reagents consumables and thermal cyclers Make sure that pipettes are calibrated and that reagents have been stored properly If the control DNA is amplified the problem may lie with the sample DNA We suggest you try the following Use the PureGene DNA Isolation Kit Gentra Systems Inc P N D 5500A Y Increase the pooling ratio of that mark
287. un conditions in Troubleshooting on page 7 29 To remove excess primer purify the amplified PCR product using either a Centricon 100 column for fragments greater than 130 base pairs in length or a Centricon 30 column for fragments less than 130 base pairs in length Note Performing this step will simplify the analysis If you will not be sequencing putative mutants this step is not absolutely necessary However if you intend to sequence putative mutants isolated during SSCP analysis you must perform this purification step To Save Time Prerun Checklist Stock Solutions At the Beginning of a Run At Any Time Before a Run Having stocks of the following reagents buffers saves time during run setup 4 Deionized formamide Lasts for 3 months at 15 to 20 C See Deionized Formamide on page A 7 for details Sodium hydroxide NaOH 0 1 N stored in a plastic container at room temperature Formamide NaOH Blue Dextran EDTA Size Standard Master Mix Lasts for 1 week at 2 6 C See step 1 on page 7 16 for details For the ABI PRISM 377 Only Set the instrument run temperature to the desired temperature 30 C by default immediately before run setup Refer to the ABI Prism 377 DNA Sequencer User s Manual for details Perform the following at any time before running 4 4 Complete the Sample Sheet Refer to the GeneScan Analysis Software User s Manual or the GeneScan 672 Software User s Manual fo
288. ven sample Moreover not all point mutations in a given sequence will cause a detectable change in electrophoretic mobility However by optimizing PCR reactions and run conditions before attempting a large scale analysis you can increase the sensitivity See Optimizing SSCP Run Conditions on page 7 26 for more details Changes in relative mobility due to minor variations in electrophoresis conditions limit the ability to compare results obtained on different instrument platforms or even in different laboratories Ability to Vary Electrophoresis Temperature The mobility difference between the wild type and a mutant strand is very sensitive to temperature Within the temperature range from 25 C if your laboratory permits to 40 C the temperature for the best differentiation of the two strands will depend upon the particular mutant wild type strand combination Whatever temperature regime you choose strict temperature control is crucial to ensure consistency because three dimensional conformation is highly sensitive to changes in temperature Rapid Analysis The ABI PRISM 377 and ABI 373 instruments allow extremely rapid separations Fragments that are 300 bp or less in length can be separated in 2 hours 4 hours on the ABI 373 Refer to PCR SSCP Analysis P N 904413 Stock No 237825 001 for more information Before You Begin Materials Required Software Required Optional Y GeneScan Internal Lane Size Standard
289. x load the matrix samples into every other lane and leave at least one empty lane between the matrix samples and the experimental samples continued on next page Starting the Run Step Action 1 On the ABI 373 DNA Sequencer select MAIN MENU button 2 Select SETUP RUN from the Main menu Verify the length of time voltage and wattage of the run Enter the appropriate values Note The ABI 373 is limited by its lack of temperature control To best maintain consistent run temperatures we recommend that you run at 10 W constant power Depending on the run type glycerol percentage and fragment size of interest run times can vary from 12 24 hours 3 Select CHOOSE RUN option Then select GENESCAN RUN 4 Immediately go to the computer 5 Make sure that the GeneScan 672 Fragment Analysis Software is closed 6 Confirm that the GeneScan 672 Collection Software is still open then choose Settings from the Edit menu Set the Timer for the appropriate run time in hours 8 Name the run file 9 Select the Collect button on the controller panel Data collection will begin Note The data collection can be viewed at any time during the run by selecting either the Scan Map or Gel buttons However the gel window should not be left open for long periods of time during the run as it occupies a large block of memory After the electrophoresis is complete the computer automatically launches the GeneS
290. ycler TC1 Allows samples to reach thermal equilibrium at each stage Using the autoextension or AutoX function of a thermal cycler to allow longer extension times in later cycles Increases yield by allowing complete extension of PCR product in later cycles a For most applications an extension temperature of 72 C is effective and rarely requires optimization In the two temperature PCR process the combined annealing extension step temperature should range from 60 70 C To find the optimal thermal cycling parameters perform a series of runs varying the annealing or denaturation temperatures in 2 C increments Note Do not vary more than one parameter at a time Optimizing PCR 6 15 Avoiding Contamination Introduction Avoiding Contamination from the Environment Avoiding PCR Product Carryover 6 16 Optimizing PCR PCR protocols are extremely sensitive to contaminants in the DNA Although many protocols that describe simple or fast extraction or purification methods have been published recently you should carefully evaluate any changes or improvements in extraction or purification methods Also be sure that the physical and chemical condition of the sample itself are adequate for the intended labeling and assay methods To avoid general contamination take the following precautionary measures Change pipet tips between samples Use filter plugged pipet tips Clean any
291. yping problems Many protocols increase the final extension step to 30 45 minutes to promote 3 A nucleotide addition Increasing the Mg2 concentration promotes 3 A nucleotide addition Decreasing the Mg concentration inhibits plus A addition In general optimizing the Mg2 concentration is best employed in conjunction with other strategies If you choose to maximize 3 A nucleotide addition consider using AmpliTaq Gold DNA Polymerase at 2 5 mM MgCl What It Is Brownstein et al 1996 found that adding additional nucleotides a tail to the 5 end of the reverse PCR primer either promoted or inhibited 3 A nucleotide addition to the forward labeled strand depending upon the sequence of the added nucleotides General Rule Magnuson et al 1996 noticed a correlation between tail sequence and the amount of 3 A nucleotide addition In particular they found that adding a single G to the 5 end of the reverse PCR primer generally resulted in almost complete 3 A nucleotide addition Therefore using a tail to promote 3 A nucleotide addition can yield a consistently callable pattern Things to Consider Reverse primer tailing has advantages compared to other methods because it Y Works well under diverse reaction conditions Does not require additional experimental steps Ginot et al 1996 used T4 DNA polymerase to remove the 3 A overhangs from pooled PCR products Things to Consider Although effect
292. ysis of oligodeoxyribonucleotide base composition Anal Biochem 165 442 447 McBride L J McCollum C Davidson S Efcavitch J W Andrus A and Lombardi S J 1988 A new reliable cartridge for the rapid purification of synthetic DNA BioTechniques 6 362 367 McBride L J and O Neill M D 1991 Automated analysis of mutations responsible for genetic diseases in humans Amer Laboratory 52 59 Theisen P McCollum C Upadhya K Jacobson K Vu H and Andrus A 1992 Fluorescent dye labeling of oligonucleotides via phosphoramidite chemistry Tetrahedron Lett 33 5033 5036 Vu H McCollum C Jacobson K Theisen P Vinayak R Spiess E and Andrus A 1990 Fast oligonucleotide deprotection phosphoramidite chemistry for DNA synthesis Tetrahedron Lett 31 7269 7272 Boutin P Hani E H Vasseur F Roche C Bailleul B Hager J and Froguel P 1997 Automated fluorescence based screening for mutation by SSCP use of universal M13 dye primers for labeling and detection Bio Techniques 23 358 362 Dryja T P Cavenee W White R Rapaport J M Petersen R Albert D M and Bruns G A 1984 Homozygosity of chromosome 13 in retinoblastoma N Engl J Med 310 550 553 Ellison J Dean M and Goldman D 1993 Efficacy of fluorescence based PCR SSCP for detection of point mutations Bio Techniques 15 684 691 Glavac D and Dean M 1993 Optimization of the single strand conformati
293. ystems that are limited to one or two colors continued on next page 9 20 Microsatellite Analysis Applications AmpFSTR Loci The AmpF STR PCR Amplification Kits co amplify the repeat regions of various short tandem repeat loci Table 9 4 In some kits a segment of the X Y homologous gene amelogenin is also amplified Amplifying a segment of the amelogenin gene with a single primer pair can be used for gender identification because different length products from the X and Y chromosomes are generated Sullivan et a 1993 Table 9 4 AmpF STR loci Locus Chromosome Size Range Designation Location Common Sequence Motif bp a Dye Label D3S1358 3p TCTA TCTG 3 TCTA 114 142 5 FAM vWA 12p12 pter TCTA TCTG s3 4 TCTA 157 197 5 FAM FGA 4q28 TTTC 3 TTTT TTCT CTTT CTCC TTCO gt 219 267 5 FAM Amelogenin X p22 1 22 3 107 JOE Y p11 2 113 D8S1179b 8 TCTR 128 168 JOE THO1 11p15 5 AATG 169 189 JOE D21S11 21 TCTA TCTG TCTA 3 TA TCTA 3 TCA TCTA gt 189 243 JOE TCCA TA TCTA TPOX 2p23 2per AATG 218 242 JOE D18S51 18q21 3 AGAA 273 341 JOE CSF1PO 5q33 3 34 AGAN 281 317 JOE D5S818 5q21 31 AGAT 135 171 NED D138317 13q22 31 GATA 206 234 NED D7S820 7q GATA 258 294 NED a The size range is the actual base pair size of sequenced alleles contained in the AmpF STR Allelic Ladders The sizes in the table include the 3 A nucleotide addition b In some literature references this locus is
294. zed chain reaction In Wu R ed Methods in Enzymology Vol 155 Academic Press Inc San Diego CA pp 335 350 Nishisho l Nakamura Y Miyoshi Y et al 1991 Mutations of chromosome 5q21 genes in FAP and colorectal cancer patients Science 253 665 669 Papadopoulos N Nicolaides N C Wei Y F et al 1994 Mutation of a mutL homolog in hereditary colon cancer Science 263 1625 1629 Parsons R Li G M Longley M J Fang W H et al 1993 Hypermutability and mismatch repair deficiency in RER tumor cells Cell 75 1227 1236 Peltomaki P Aaltonen L A Sistonen P et al 1993 Genetic mapping of a locus predisposing to human colorectal cancer Science 260 810 812 AFLP Saiki R K Scharf S J Faloona F A Mullis K B Horn G T Erlich H A and Amheim N 1985 Enzymatic amplification of B globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia Science 230 1350 1354 Smith J R Freije D Carpten J D et al 1996 Major susceptibility locus for prostate cancer on chromosome 1 suggested by a genome wide search Science 274 1371 1374 Vocke C D Pozzatti R O Bostwick D G et al 1996 Analysis of 99 microdissected prostate carcinomas reveals a high frequency of allelic loss on chromosome 8p12 21 Cancer Res 15 2411 2416 Vogelstein B Fearon E R Hamilton S R Kern S E et a 1988 Genetic alterations during colorectal tumor developmen

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