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Promega`s GammaSTR® Fluorescent STR System

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1. 23 E Reuse of Glass Plates 24 VIII Sample Preparation Gel Electrophoresis and Detection on the ABI PRISM 377 DNA Sequencers and the ABI PRISM 310 Genetic Analyzer 24 A Matrix Standardization 24 B Instrument Preparation 25 C Sample Preparation and Loading 26 D Electrophoresis and Detection 27 E Data Analysis 28 F Reuse of Glass Plates 28 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD006 Revised 7 06 GenePrint Fl
2. 6 A Choice of Thermal Cycling Protocol 7 B Amplification Setup 12 C Amplification Thermal Cycling 14 V Polyacrylamide Gel Preparation 15 A Gel Preparation for the Hitachi FMBIO and FMBIO II Fluorescence Imaging Systems 15 B Gel Preparation for the ABI PRISM 377 DNA Sequencer 18 VI Polyacrylamide Gel Electrophoresis and Detection 19 VII Sample Preparation Gel Electrophoresis and Detection on the Hitachi FMBIO and FMBIO II Fluorescence Imaging Systems 20 A Gel Pre Run 20 B Sample Preparation Loading and Electrophoresis 21 C Detection 22 D Data Analysis
3. 32 XI Troubleshooting 35 XII References 36 A Cited References 36 B Additional STR References 38 XIII Appendix 39 A Advantages of STR Typing 39 B Advantages of Using the Loci in the GenePrint Fluorescent STR Systems 40 C Power of Discrimination 43 D DNA Extraction and Quantitation Methods 44 E Agarose Gel Electrophoresis of Amplification Products 45 F Composition of Buffers and Solutions 46 G Organizational Sheets
4. 48 H Related Products 51 I Description STR a short tandem repeat loci consist of short repetitive sequence elements of 3 7 base pairs in length 1 4 These repeats are well distributed throughout the human genome and are a rich source of highly polymorphic markers that may be detected using PCR 5 8 Alleles of these loci are differentiated by the number of copies of the repeat sequence contained within the amplified region and are distinguished from one another using radioactive silver stain or fluorescence detection following electrophoretic separation The GenePrint Fluorescent STR Systems contain all of the materials except for Taq DNA polymerase and sample DNA required to perform 100 or 400 amplification reactions Accessory components are available to simplify many of the procedures related to STR analysis Section XIII H All of the GenePrint Fluorescent STR Systems contain 10X primer pairs In each pair one of the primers is labeled with fluorescein FL and the matched primer is unlabeled In the GenePrint Fluorescent Identification System TMR the Amelogenin specific primer is labeled with tetramethylrhodamine TMR STR 10X Buffer loading solution the appropriate allelic ladder and K562 DNA positive control template are also provided Page 3 The GenePrint Fluorescent STR Sys
5. Page 47 XIII F Composition of Buffers and Solutions continued Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD006 Revised 7 06 5X loading solution 5 Ficoll 400 0 1 bromophenol blue 0 1 xylene cyanol 100mM EDTA Na2EDTA 2H2O 10mM Tris HCl pH 7 5 STR 2X Loading Solution 10mM NaOH 95 formamide 0 05 bromophenol blue 0 05 xylene cyanol FF STR 10X Buffer 500mM KCl 100mM Tris HCl pH 9 0 15mM MgCl2 1 Triton X 100 2mM each dNTP 50X TAE buffer pH 7 2 242g Tris base 57 1ml glacial acetic acid 100ml 0 5M EDTA pH 8 0 Add Tris base and EDTA to 500ml of deionized water Add the glacial acetic acid Bring the volume to 1 liter with deionized water 1X TAE buffer pH 7 2 Add 20ml of 50X TAE to 980ml of deionized water 0 5X TBE buffer Add 50ml of 10X TBE to 950ml of deionized water 10X TBE buffer 107 8g Tris base 7 44g EDTA Na2EDTA 2H2O 55 0g boric acid Dissolve Tris base and EDTA in 800ml of deionized water Slowly add the boric acid and monitor the pH until the desired pH of 8 3 is obtained Bring the volume to 1 liter with deionized water XIII G Organizational Sheets Sample Preparation Page 48 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in
6. X Representative STR Data Representative data are shown in Figures 1 2 and 3 Page 33 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD006 Revised 7 06 5857TA CTTv Lane Trace CSF1PO TPOX TH01 vWA FFFL Lane Trace F13A01 FESFPS F13B LPL GammaSTR Lane Trace D16S539 D7S820 D13S317 D5S818 CSF1PO TPOX TH01 vWA 51 53 NA NA Locus Allele Height Shorter Peak Higher Peak F13A01 FESFPS F13B LPL 38 45 NA 50 Locus Allele Height Shorter Peak Higher Peak D16S539 D7S820 D13S817 D5S818 42 83 NA 59 Locus Allele Height Shorter Peak Higher Peak Figure 1 K562 DNA amplified using the GenePrint Fluorescent STR Systems Five nanograms of K562 DNA were amplified using the GenePrint CTTv and FFFL Multiplex systems and one nanogram of K562 DNA was amplified using the GammaSTR Multiplex The amplified DNA was separated on a 43cm 4 denaturing polyacrylamide gel for 1 hour at 65 watts then scanned using the Hitachi FMBIO Fluorescence Imaging System Lane traces for K562 DNA amplified using each of the three GenePrint Systems are shown The table below each lane trace indicates the percent differences in allele height i e peak heights minus typical int
7. 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD006 Printed in USA Revised 7 06 XIII B Advantages of Using the Loci in the GenePrint Fluorescent STR Systems The STR loci and primers contained in the GenePrint Fluorescent STR Systems Tables 11 and 12 have been carefully selected to minimize artifacts including those associated with Taq DNA polymerase such as repeat slippage and terminal nucleotide addition as well as genetic artifacts called microvariant alleles Repeat slippage 21 22 sometimes called n 4 bands stutter or shadow bands is due to the loss of a repeat unit during DNA amplification The amount of this artifact observed is dependent primarily on the locus and the DNA sequence being replicated We have chosen loci that exhibit little or no repeat slippage The vWA locus is an exception revealing as much as 10 stutter This locus has been included primarily for its popularity in the forensics community Terminal nucleotide addition 23 24 occurs when Taq DNA polymerase adds a nucleotide generally adenine to the ends of amplified DNA fragments in a template independent manner The efficiency with which this occurs varies with different primer sequences Thus an artifact band one base shorter than expected i e missing the terminal addition is sometimes seen Redefinition of the primer seq
8. CTTv 10X Primer Pair Mix 2 50 Amelogenin Fluorescein 10X Primer Pair2 1 0 Taq DNA polymerase at 5u l 1 0 25 1 25u total volume 22 50 PCR Master Mix Component Volume Per Sample l Number of Reactions Final Volume l sterile water 17 45 STR 10X Buffer 2 50 locus specific 10X primer pair 2 50 Taq DNA polymerase at 5u l 1 0 05 0 25u total volume 22 50 Page 14 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD006 Printed in USA Revised 7 06 IV B Amplification Setup continued 6 Add 22 5 l of PCR master mix to each tube and place on ice Failure to keep the reagents and samples on ice can produce imbalanced amplification of multiplexed loci If using AmpliTaq Gold DNA polymerase it is not necessary to keep the reactions on ice 7 Hitachi FMBIO Users Pipet 2 5 l 1 25ng template DNA of each sample into the respective tube containing 22 5 l of PCR master mix ABI PRISM 377 DNA Sequencer ABI PRISM 310 Genetic Analyzer and ABI PRISM 3100 Genetic Analyzer Users Use only 1 2ng template DNA Protocol 10 Users See Table 4 for the amount of template DNA to use with each instrument and GenePrint system Note If the template DNA is stored in TE buffer 10mM Tris HCl 1mM EDTA pH 7 5 the volume of the DNA sample a
9. Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD006 Printed in USA Revised 7 06 VIII C Sample Preparation and Loading The Fluorescent Ladder CXR 60 400 Bases is available as the internal size marker for two color detection and analysis of amplified samples With this approach only 2 3 lanes of allelic ladder are required per gel 1 Prepare samples according to the instructions given in Table 10 2 Briefly centrifuge the samples to bring the contents to the bottom of the tubes 3 Denature the samples by heating at 95 C for 2 minutes and immediately chill on crushed ice or in an ice water bath Denature the samples just prior to loading the gel Sample DNA will partially reanneal if denatured hours before loading Table 10 Instrument Specific Instructions for Sample Preparation Using the ABI PRISM 377 DNA Sequencer and ABI PRISM 310 Genetic Analyzer Notes 1 Samples amplified with the GammaSTR Multiplex 1 2ng of template will need to be diluted 1 5 i e 1 part sample 4 parts 1X STR Buffer before mixing with the loading solution 2 To analyze the CTTv Multiplex with Amelogenin reactions mix the corresponding ladders 1 1 before mixing with loading solution The number of ladder lanes depends on personal preference and the number of samples analyzed 3 To use the TH01 allele 9 3 alone mix 0 5 l of allele 9 3 with 1 5 l of 1X STR Buffer before mixing with the loading so
10. www promega com geneticidtools powerstats PowerStats is a Microsoft Excel workbook spreadsheet that allows genotype data from STaRCall or Genotyper software to be pasted directly into the workbook to obtain standard population statistics on the distribution of alleles within particular population subsets If you cannot access the web site please contact your local Promega branch office or distributor 1 The Short Tandem Repeat DNA Database Web Site Butler J M and Reeder D J 1997 STRBase A short tandem repeat DNA database Profiles in DNA 1 2 10 2 PowerStats Analysis of Population Data Tereba A 1999 Tools for analysis of population statistics Profiles in DNA 2 3 14 6 XIII Appendix XIII A Advantages of STR Typing The GenePrint Fluorescent STR Systems provide a rapid non radioactive method that can be used to evaluate small amounts e g 1ng of human DNA The protocols in this manual describe the use of a fluorescein label to detect the presence of amplified STR products following their separation by denaturing polyacrylamide gel electrophoresis For information on multicolor fluorescent STR systems refer to the PowerPlex 16 System Technical Manual TMD012 PowerPlex 16 BIO System Technical Manual TMD016 PowerPlex ES System Technical Manual TMD017 PowerPlex 1 1 System Technical Manual TMD008 PowerPlex 1 2 System Technical Manual TMD009 and PowerPlex 2 1 System Technical Manua
11. 4 Prepare 1 liter of TAE 1X buffer for the electrophoresis running buffer 5 Place the gel and tray in the electrophoresis gel box Pour enough running buffer into the tank to cover the gel to a depth of at least 0 65cm Gently remove the comb 6 Load each sample mixed with 5X loading solution prepared in Step 3 7 Set the voltage at 5 volts cm measured as the distance between the two electrodes Allow the gel to run for 2 hours 8 After electrophoresis stain the gel in TAE 1X buffer containing 0 5 g ml ethidium bromide Gently rock for 20 minutes at room temperature Remove the ethidium bromide solution and replace with deionized water Allow the gel to destain for 20 minutes 9 Using a UV transilluminator 302nm photograph the gel e g with Polaroid 667 film Note When analyzing the data do not be alarmed by extra bands in addition to the alleles DNA heteroduplexes can be expected when performing nondenaturing agarose gel electrophoresis The sole purpose of the agarose gel is to confirm the success of the PCR reaction Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD006 Revised 7 06 40 acrylamide bis 19 1 Dissolve 380g of acrylamide and 20g of bisacrylamide in 500ml of deionized water Bring volume to 1 liter with deionized water 10 ammonium
12. 6 9600 96 C for 50 seconds to 94 C 1 minute 45 seconds to 90 C 1 minute None Refer to 2 minutes 30 seconds to 64 C 1 minute 26 seconds to 64 C 1 minute 4 C Note 3 15 seconds to 70 C 1 5 minutes 15 seconds to 70 C 1 5 minutes 7 480 96 C for None 94 C 1 minute None 90 C 1 minute 60 C for Refer to 2 minutes 60 C 1 minute 60 C 1 minute 30 minutes 4 C Note 1 70 C 1 5 minutes 70 C 1 5 minutes 8 9600 96 C for None 94 C 1minute None 90 C 1 minute 60 C for Refer to 2 minutes 60 C 1 minute 60 C 1 minute 30 minutes 4 C Note 2 70 C 1 5 minutes 70 C 1 5 minutes Table 5 Amplification Protocols Page 10 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD006 Printed in USA Revised 7 06 Protocol Thermal Initial Programmed Cycling for Programmed Cycling for Extension Hold Number Cycler1 Incubation2 Ramp Times First 10 Cycles Ramp Times Last 20 Cycles Step Step 9 9600 96 C for 50 seconds to 94 C 30 seconds 45 seconds to 90 C 1 minute 60 C for Refer to 2 minutes 34 seconds to 60 C 1 minute 30 seconds to 60 C 1 minute 30 minutes 4 C Note 3 25 seconds to 70 C 1 5 minutes 25 seconds to 70 C 1 5 minutes 10 9600 96 C for Default ramp to 94 C 30 seconds Default ramp to 90 C 30 seconds 60 C fo
13. C Multiple freeze thaw cycles or long term storage at 4 C may cause a breakdown of the formamide Formamide with a conductivity greater than 100 S cm may contain ions that compete with DNA during injection This results in lower peak heights and reduced sensitivity A longer injection time may not increase the signal IX A Spectral Calibration The PowerPlex Matrix Standards 3100 3130 Cat DG4650 is required for spectral calibration on the ABI PRISM 3100 Genetic Analyzer For protocols and additional information on spectral calibration see the PowerPlex Matrix Standards 3100 3130 Technical Bulletin TBD022 supplied with Cat DG4650 available upon request from Promega or online at www promega com tbs Proper spectral calibration is critical to evaluate multicolor systems with the ABI PRISM 3100 Genetic Analyzer Spectral calibration must be performed for each ABI PRISM 3100 Genetic Analyzer IX B Sample Preparation The Fluorescent DNA Ladder CXR 60 400 Bases Cat DG6221 is available as the internal lane standard for four color detection and analysis of amplified samples 1 Prepare a loading cocktail by combining and mixing the Fluorescent DNA Ladder CXR 60 400 Bases and deionized formamide as follows 1 l Fluorescent DNA Ladder injections 9 l deionized formamide injections 2 Pipet 10 l of formamide fluorescent ladder loading cocktail into each well 3 Add 1 l of a
14. Corporation 161 72 27 Puers C et al 1994 Allelic ladder characterization of the short tandem repeat polymorphism located in the 5 flanking region to the human coagulation factor XIII A subunit gene Genomics 23 260 4 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD006 Revised 7 06 Page 38 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD006 Printed in USA Revised 7 06 28 B r W et al 1997 DNA Recommendations Further report of the DNA Commission of the ISFH regarding the use of short tandem repeat systems Int J Leg Med 110 175 6 29 Mandrekar P V Krenke B E and Tereba A 2001 DNA IQ The intelligent way to purify DNA Profiles in DNA 4 3 16 30 Mandrekar M N et al 2001 Development of a human DNA quantitation system Profiles in DNA 4 3 9 12 31 Greenspoon S and Ban J 2002 Robotic extraction of sexual assault samples using the Biomek 2000 and the DNA IQ System Profiles in DNA 5 1 3 5 32 Procedures for the Detection of Restriction Fragment Length Polymorphisms in Human DNA 1990 FBI Laboratory Quantico VA 33 Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloni
15. Part TMD006 Revised 7 06 Analysis Range Start Defined in Step 2 Stop 10 000 Data Processing Baseline Checked Multicomponent Checked Smooth Options Light1 Peak Detection Peak Amplitude Thresholds2 B Y G R Min Peak Half Width 2pts Size Call Range Min 60 Max 600 Size Calling Method Local Southern Method Split Peak Correction None Page 32 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD006 Printed in USA Revised 7 06 IX D Sample Detection continued 5 Assign a new size standard Select a sample file and highlight the arrow next to size standard then select define new Assign the size standard peaks as shown in Figure 1 Store the size standard in the SizeStandards folder 6 Apply the size standard file to the samples then analyze the sample files 7 See Section IX E for further data analysis For additional information regarding the GeneScan analysis software refer to the GeneScan analysis software user s manual Notes 1 Peak heights outside the linear range of the instrument may generate artifact peaks due to instrument saturation i e overloading the sample 2 If the sample peak heights are not within the linear detection of the instrument the ratio of stutter peaks to real allele peaks increases and allele designations
16. System Note Protocol 12 see Table 5 has been developed optimal performance of the GenePrint Fluorescent STR System F13A01 FESFPS F13B LPL Fluorescein with the GeneAmp PCR system 9600 thermal cycler 16 This protocol uses Gold ST R 10X Buffer and AmpliTaq Gold DNA polymerase Materials to Be Supplied by the User Solution compositions are provided in Section XIII F thermal cycler Model 480 or GeneAmp System 9600 Perkin Elmer microcentrifuge Taq DNA polymerase Nuclease Free Water Cat P1193 or equivalent Mineral Oil Cat DY1151 or equivalent 0 5ml microcentrifuge tubes 1 5ml microcentrifuge tubes aerosol resistant pipet tips IV A Choice of Thermal Cycling Protocol The CTTv and FFFL Multiplexes their corresponding monoplexes the GenePrint Fluorescent Sex Identification System Amelogenin Fluorescein and the GenePrint Fluorescent STR System HPRTB are optimized for use with Perkin Elmer GeneAmp reaction tubes and the Perkin Elmer Model 480 thermal cycler The GammaSTR Multiplex its corresponding monoplex systems and the GenePrint Fluorescent Sex Identification System Amelogenin TMR are optimized for use on the GeneAmp PCR system 9600 thermal cycler However each system may be used with either thermal cycler Please refer to Tables 2 and 3 for recommended and alternative protocols for each system and thermal cycler Table 4 describes the special
17. allelic ladder is from the same kit as the 10X Primer Pair Mix Poor quality formamide used Be sure that high quality formamide is used when running samples on the ABI PRISM 310 or 3100 Genetic Analyzer Uneven peak heights Thermal cycler problems Review the thermal cycling protocols between loci in Section IV We have not tested other thermal cyclers Excessive amount of DNA Use the recommended amount of template See Table 4 Section IV A for recommendations Use of FTA paper Results may be similar to use of excess amounts of DNA template Reduce the number of cycles in the amplification program by 2 4 cycles 10 18 or 10 16 cycling to improve the locus to locus balance Degraded DNA sample DNA template is degraded into smaller fragments with the larger loci showing diminished yield High background with low Part of the spacers were scanned Re scan the gel being careful signal not to scan any portion of the spacers Plates were improperly washed Improper washing of the plates can cause a soap residue to build up on the plates This can cause background fluorescence XII References XII A Cited References 1 Edwards A et al 1991 DNA typing with trimeric and tetrameric tandem repeats Polymorphic loci detection systems and population genetics In Proceedings from The Second International Symposium on Human Identification 1991 Promega Corporation 31 52 2 Edwards A et al 1991 DNA typing and geneti
18. from lane to lane when the allele size is compared to the Fluorescent Ladder contained within each lane When using the Hitachi FMBIO and FMBIO II Fluorescence Imaging Systems inclusion of the Fluorescent Ladder CXR is optional If alleles are called visually by comparing the sample alleles directly against the allelic ladders the Fluorescent Ladder CXR is not needed However if the Hitachi Software STaRCall is used for identifying alleles we recommend including the Fluorescent Ladder CXR Note The Internal Lane Standard 600 Cat DG2611 contains the same DNA fragments as the Fluorescent Ladder CXR 60 400 Bases with additional DNA fragments of 425 450 475 500 550 and 600 bases VII Sample Preparation Gel Electrophoresis and Detection on the Hitachi FMBIO and FMBIO II Fluorescence Imaging Systems VII A Gel Pre Run 1 Remove the clamps from the polymerized acrylamide gel and clean the glass plates with paper towels saturated with deionized water 2 Shave any excess polyacrylamide away from the comb and remove the comb 3 Add 0 5X TBE Buffer to the bottom chamber of the electrophoresis apparatus 4 Gently lower the gel glass plates into the bottom chamber with the longer plate facing out and the well side on top 5 Secure the glass plates to the sequencing gel apparatus 6 Add 0 5X TBE Buffer to the top chamber of the electrophoresis apparatus 7 Use a 50 100cc syringe filled wi
19. persulfate Add 0 5g of ammonium persulfate to 5ml of deionized water Use 200 l for one acrylamide gel solution 30ml Store the remaining volume in 200 l aliquots at 20 C Blue Dextran Loading Solution 4 1mM EDTA 88 25 formamide ACS grade 15mg ml blue dextran Bromophenol Blue Loading Solution 10mM NaOH 95 formamide 0 05 bromophenol blue 0 5M EDTA pH 8 0 186 1g Na2EDTA 2H2O Add EDTA to 800ml of deionized water with vigorous stirring Adjust the pH to 8 0 with NaOH about 20g of NaOH pellets Adjust final volume to 1 liter Dispense into aliquots and sterilize by autoclaving ethidium bromide solution 10mg ml 1 0g ethidium bromide Dissolve in 100ml of deionized water Wrap in aluminum foil or transfer to a dark bottle and store at room temperature Caution Ethidium bromide is a powerful mutagen Wear gloves at all times and use a mask when weighing out ethidium bromide powder Gel Tracking Dye 10mM NaOH 95 formamide 0 05 bromophenol blue 0 05 xylene cyanol FF Gold ST R 10X Buffer 500mM KCl 100mM Tris HCl pH 8 3 at 25 C 15mM MgCl2 1 Triton X 100 2mM each dNTP 1 6mg ml BSA Page 46 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD006 Printed in USA Revised 7 06 XIII F Composition of Buffers and Solutions
20. plates The following protocol is for the preparation of a 36cm denaturing polyacrylamide gel for use with the ABI PRISM 377 DNA Sequencer Low fluorescence glass plates are recommended and may be obtained from the instrument manufacturer 1 Thoroughly clean the glass plates with hot water and a 1 Liqui Nox solution Rinse extremely well using deionized water Allow the glass plates to air dry 2 Assemble the glass plates by placing 0 2mm side gel spacers between the front and rear glass plates Hold the plates together using binder clamps 4 clamps on each side Place the assembly horizontally on a test tube rack or similar support 3 Prepare a 5 Long Ranger acrylamide gel total of 50ml by combining the ingredients listed in Table 8 Stir the solution until the urea has dissolved Table 8 Preparation of a 5 Long Ranger Polyacrylamide Gel Component 5 Gel Final Concentration urea 18g 6M deionized water 26ml 10X TBE buffer 5 0ml 0 5X 50 Long Ranger gel solution 5 0ml 5 total volume 50 0ml Page 19 4 Filter the acrylamide solution through a 0 2 micron filter e g Nalgene tissue culture filter and de gas for an additional 5 minutes 5 Add 35 l of TEMED and 250 l of 10 ammonium persulfate to 50ml of acrylamide solution and mix gently 6 Using a disposable 30cc syringe pour the gel by starting at the well end of the plates and carefully injecting the acrylamide so
21. used according to the precedent of Edwards et al 2 The published article DNA Guidelines Further Report of the DNA Commission of the ISFH Regarding the use of Short Tandem Repeat Systems 28 describes different rules for STR allele nomenclature Allele designations for all listed loci are identical using both methods except for the locus F13B In this case alleles are one repeat unit larger when using the method described by the ISFH For this locus the community will have to decide whether to follow the new nomenclature or maintain the Edwards nomenclature to avoid confusion The DNA Commission of the ISFH states If a repeat designation of a commonly used STR system does not follow these guidelines the established nomenclature for the sequence can continue to be used to avoid new confusion Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD006 Revised 7 06 STR Locus Chromosomal Location GenBank Locus and Locus Definition Repeat Sequence 5 3 Amelogenin1 Xp22 1 22 3 and Y HUMAMEL Human Y chromosomal gene for amelogenin like protein NA CSF1PO 5q33 3 34 HUMCSF1PO Human c fms proto oncogene for CSF 1 receptor gene AGAT2 D16S539 16q24 qter NA AGAT2 D7S820 7q11 21 q22 NA AGAT2 D13S317 13q22 q31 NA AGAT2 D5S818 5q23 3 32 NA
22. yield We recommend Sigma BSA Cat A2153 Performance may vary depending on the source of this component 5 Use MicroAmp reaction tubes in the MicroAmp 9600 tray This allows a maximum of 96 simultaneous reactions No mineral oil is needed Cover reactions with the system 9600 thermal cycler lid Optional Add BSA Fraction V final concentration 60 g ml to all reactions This may result in a slight increase in yield We recommend Sigma BSA Cat A2153 Performance may vary depending on the source of this component Page 11 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD006 Revised 7 06 IV B Amplification Setup The use of gloves and aerosol resistant pipet tips see Section XIII H is highly recommended to prevent cross contamination Helpful organizational sheets are provided in Section XIII G Alternative steps are included in the following procedure for laboratories using the Hitachi FMBIO and FMBIO II Fluorescence Imaging Systems and ABI PRISM 377 DNA Sequencers and ABI PRISM 310 and 3100 Genetic Analyzers 1 Thaw the STR 10X Buffer and 10X Primer Pair s and place on ice Note Mix the STR 10X Buffer and 10X Primer Pair by vortexing each tube for 15 seconds before each use Do not centrifuge the 10X Primer Pair Mix as this may cause the primers to be c
23. 0 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD006 Printed in USA Revised 7 06 Internal Lane Standards Product Size Cat Internal Lane Standard 600 150 l DG2611 Fluorescent Ladder CXR 60 400 Bases 65 l DG6221 For Laboratory Use Sample Preparation Systems Product Size Cat DNA IQ System 100 reactions DC6701 400 reactions DC6700 Differex System 50 samples DC6801 200 samples DC6800 AluQuant Human DNA Quantitation System 80 determinations DC1010 400 determinations DC1011 Slicprep 96 Device 10 pack V1391 Not for Medical Diagnostic Use For Laboratory Use ART Aerosol Resistant Tips Product Volume Size tips pack Cat ART 10 Ultramicro Pipet Tip 0 5 10 l 960 DY1051 ART 20E Ultramicro Pipet Tip 0 5 20 l 960 DY1061 ART 20P Pipet Tip 20 l 960 DY1071 ART GEL Gel Loading Pipet Tip 100 l 960 DY1081 ART 100 Pipet Tip 100 l 960 DY1101 ART 100E Pipet Tip 100 l 960 DY1111 ART 200 Pipet Tip 200 l 960 DY1121 ART 1000E Pipet Tip 1 000 l 800 DY1131 Page 53 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD006 Revised 7 06 a STR loci are the subject of U S Pat No RE 37 984 German Pat No DE 38 34 636 C2 and other p
24. 00 reactions DC6171 400 reactions DC6170 Not for Medical Diagnostic Use The GenePrint Fluorescent Sex Identification System Amelogenin Fluorescein can be amplified independently or simultaneously with the CTTv Multiplex X specific and Y specific chromosome bands will fall between the TPOX and TH01 loci if amplified along with the CTTv Multiplex In the GenePrint Fluorescent Sex Identification System Amelogenin TMR the Amelogenin specific primer is labeled with tetramethylrhodamine TMR and can be co amplified with the PowerPlex 1 1 System compatible with the Hitachi FMBIO Fluorescence Imaging Systems The CTTv component of the PowerPlex System is labeled with TMR so the TMR labeled Amelogenin is required II D GenePrint Fluorescent Monoplex Systems Each GenePrint Fluorescent Monoplex System contains the specific primer and allelic ladder plus other components sufficient to perform 100 reactions These systems are available by custom order Please contact your local Promega Branch Office or Distributor for ordering information Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD006 Revised 7 06 Page 6 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www prom
25. 16 www promega com Part TMD006 Printed in USA Revised 7 06 Page 13 Table 6 PCR Amplification Reaction Setup Multiplex Reactions Containing Four Loci Combined CTTv Multiplex and Amelogenin Reactions Monoplex or Amelogenin Only Reactions 1The volume given assumes a Taq DNA polymerase concentration of 5u l For different enzyme concentrations the volume of enzyme added must be adjusted accordingly If using AmpliTaq Gold DNA polymerase use the Gold ST R 10X Buffer instead of the STR 10X Buffer 2Use of more Amelogenin primer has produced extra bands below the expected 212 and 218 base fragments with some samples using protocol 7 especially when 25ng or more of template are used Amelogenin TMR is only for use with the PowerPlex Systems and should not be used with the CTTv Multiplex system Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD006 Revised 7 06 PCR Master Mix Component Volume Per Sample l Number of Reactions Final Volume l sterile water 17 30 STR 10X Buffer 2 50 Multiplex 10X Primer Pair Mix 2 50 Taq DNA polymerase at 5u l 1 0 2 1 0u total volume 22 50 PCR Master Mix Component Volume Per Sample l Number of Reactions Final Volume l sterile water 16 25 STR 10X Buffer1 2 50
26. AGAT2 F13A01 6p24 3 p25 1 HUMF13A01 Human coagulation factor XIII a subunit gene AAAG2 F13B 1q31 q32 1 HUMBFXIII Human factor XIII b subunit gene AAAT2 FESFPS 15q25 qter HUMFESFPS Human c fes fps proto oncogene AAAT2 HPRTB Xq26 HUMHPRTB Human hypoxanthine phosphoribosyl transferase gene AGAT2 LPL 8p22 HUMLIPOL Human lipoprotein lipase gene AAAT2 TH01 11p15 5 HUMTH01 Human tyrosine hydroxylase gene AATG2 TPOX 2p25 1 pter HUMTPOX Human thyroid peroxidase gene AATG2 vWA 12p12 pter HUMVWFA31 Human von Willebrand factor gene AGAT2 Table 12 Additional Locus Specific Information NA not applicable 1Lengths of each allele in the allelic ladders have been confirmed by sequence analyses 2Alleles that represent lt 0 2 of the population may not be listed in this table 3Amelogenin is not an STR but displays a 212 base X specific band and a 218 base Y specific band K562 DNA female displays only the 212 base X specific band 4Allele 10 307 bases is not included because it is rare and its exclusion creates a gap that simplifies interpretation of the allelic ladder 27 28 5F13A01 allele 5 appears more intense than allele 4 in the K562 control sample The K562 strain contains an unusual number of chromosomes and some are represented more than twice per cell It is hypothesized that in this strain the allele 5 version of chromosome 6 is present twice while the all
27. DNA Sequencers and ABI PRISM 310 and 3100 Genetic Analyzers 1ng 1ng 1ng Page 9 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD006 Revised 7 06 Protocol Thermal Initial Programmed Cycling for Programmed Cycling for Extension Hold Number Cycler1 Incubation2 Ramp Times First 10 Cycles Ramp Times Last 20 Cycles Step Step 1 480 96 C for None 94 C 1 minute None 90 C 1 minute None Refer to 2 minutes 60 C 1 minute 60 C 1 minute 4 C Note 1 70 C 1 5 minutes 70 C 1 5 minutes 2 480 96 C for None 94 C 1 minute None 90 C 1 minute None Refer to 2 minutes 64 C 1 minute 64 C 1 minute 4 C Note 1 70 C 1 5 minutes 70 C 1 5 minutes 3 9600 96 C for None 94 C 1 minute None 90 C 1 minute None Refer to 2 minutes 60 C 1 minute 60 C 1 minute 4 C Note 2 70 C 1 5 minutes 70 C 1 5 minutes 4 9600 96 C for 50 seconds to 94 C 1 minute 45 seconds to 90 C 1 minute None Refer to 2 minutes 34 seconds to 60 C 1 minute 30 seconds to 60 C 1 minute 4 C Note 3 25 seconds to 70 C 1 5 minutes 25 seconds to 70 C 1 5 minutes 5 9600 96 C for None 94 C 1 minute None 90 C 1 minute None Refer to 2 minutes 64 C 1 minute 64 C 1 minute 4 C Note 2 70 C 1 5 minutes 70 C 1 5 minutes
28. T e c h n i c a l M a n u a l GenePrint Fluorescent STR Systems For use with the Hitachi FMBIO and ABI PRISM 377 DNA Sequencers the ABI PRISM 310 and ABI PRISM 3100 Genetic Analyzers INSTRUCTIONS FOR USE OF PRODUCTS DC5081 DC5091 DC5101 DC5111 DC5121 DC5131 DC5141 DC5151 DC5161 DC5170 DC5171 DC6300 DC6301 DC6310 DC6311 DC6070 DC6071 DC6131 DC6141 DC6151 DC6161 DC6170 DC6171 DG2121 DG2131 AND DG3291 PRINTED IN USA Revised 7 06 Part TMD006 Page 1 I Description 2 II Product Components and Storage Conditions 4 A GenePrint Fluorescent STR Multiplex Systems 4 B Allelic Ladders and Size Markers 5 C GenePrint Fluorescent Sex Identification Systems 5 D GenePrint Fluorescent Monoplex Systems 5 III Before You Begin 6 IV Amplification
29. USA Revised 7 06 Notes continued with loading cocktail This may result in uneven allele peak heights across loci For best results use less DNA template in the amplification reactions or reduce the number of cycles in the amplification program by 2 4 cycles 4 For allelic ladders dilute the ladder 1 5 i e 1 part sample 4 parts 1X STR Buffer then combine 1 l of diluted ladder with 9 0 l of deionized formamide and 1 0 l of Fluorescent Ladder CXR Cover wells with appropriate septa 5 Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or in an ice water bath for 3 minutes Denature the samples just prior to loading into the ABI PRISM 3100 Genetic Analyzer Note Brief centrifugation of prepared samples will remove bubbles that may affect analysis IX C Instrument Preparation 1 Refer to the ABI PRISM 3100 Genetic Analyzer user s manual for instructions on cleaning the pump blocks installing the capillary array performing a spatial calibration and adding polymer to the reserve syringe 2 Open the ABI PRISM 3100 data collection software 3 Open a new plate record Name the plate and select GeneScan Select the plate size 96 well or 384 well Select Finish 4 Complete the plate record spreadsheet for the wells you have loaded 5 In the BioLIMS Project column select 3100_Project1 from the pull down menu 6 In the Dye Set colum
30. USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD006 Printed in USA Revised 7 06 Tube Number Sample ID negative control Sample Conc ng l Sample l reaction 0 Sterile Water l 2 5 Tube Number Sample ID Sample Conc ng l Sample l reaction Sterile Water l Tube Number Sample ID Sample Conc ng l Sample l reaction Sterile Water l Tube Number Sample ID Sample Conc ng l Sample l reaction Sterile Water l Tube Number Sample ID Sample Conc ng l Sample l reaction Sterile Water l Tube Number Sample ID Sample Conc ng l Sample l reaction Sterile Water l Page 49 Master Mix Preparation Date Name GenePrint STR Systems Locus Reaction volume sample master mix 25 l Number of reactions Add 2 5 l of DNA to each tube containing 22 5 l of master mix Thermal Cycling Profile Perkin Elmer Thermal Cycler Model Number Annealing Temperature File Number Full Program Description ______cycles _______ C _______ minutes _______ C _______ minutes _______ C _______ minutes ______cycles _______ C _______ minutes _______ C _______ minutes _______ C _______ minutes Hold 4 C indefinitely Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phon
31. When loading is complete run the gel using the same conditions as in Section VII A gel pre run Note In 6 gels bromophenol blue migrates at approximately 25 bases and xylene cyanol migrates at approximately 105 bases In 4 gels bromophenol blue migrates at approximately 40 bases and xylene cyanol migrates at approximately 170 bases 8 Use the size ranges for each locus see Table 12 Section XIII B and the migration characteristics of the dyes see Step 7 to stop electrophoresis any time after the locus of interest has passed the midpoint of the gel If running more than one locus or a multiplex be careful not to run the smallest locus off the bottom of the gel VII C Detection 1 After electrophoresis remove the gel glass plate unit from the apparatus Remove the comb and side spacers but do not separate the glass plates 2 The plates must be very clean for scanning Clean both sides of the gel glass plate unit with deionized water and paper towels Do not use ethanol to clean the plates The ethanol fluoresces and is detected by the FMBIO instruments 3 Scan the gel according to the parameters listed in Table 9 Use the 505nm filter to detect fluorescein labeled fragments and the 650nm filter to detect the Fluorescent Ladder CXR 60 400 Bases Different laboratories may wish to modify these parameters according to their specific preferences Note If the signal is too intense dilute the samples in 1X STR Buff
32. alleles in population analysis using a locus specific allelic ladder Am J Hum Genet 53 953 8 10 Hammond H et al 1994 Evaluation of 13 short tandem repeat loci for use in personal identification applications Am J Hum Genet 55 175 89 11 Bever R A and Creacy S 1995 Validation and utilization of commercially available STR multiplexes for parentage analysis In Proceedings from the Fifth International Symposium on Human Identification 1994 Promega Corporation 61 8 12 Sprecher C J et al 1996 General approach to analysis of polymorphic short tandem repeat loci BioTechniques 20 266 76 13 Lins A M et al 1996 Multiplex sets for the amplification of polymorphic short tandem repeat loci silver stain and fluorescent detection BioTechniques 20 882 9 14 Presley L A et al 1992 The implementation of the polymerase chain reaction PCR HLA DQ alpha typing by the FBI laboratory In Proceedings from the Third International Symposium on Human Identification 1992 Promega Corporation 245 69 15 Hartmann J M et al 1991 Guidelines for a quality assurance program for DNA analysis Crime Laboratory Digest 18 44 75 16 Micka K A et al 1999 TWGDAM validation of a nine locus and a four locus fluorescent STR multiplex system J Forensic Sci 44 1243 57 17 Bassam B J Caetano Anolles G and Gresshoff P M 1991 Fast and sensitive silver staining of DNA in polyacrylami
33. altered pH If the DNA template is stored in TE buffer that is not pH 8 0 or contains a higher EDTA concentration the DNA volume should not exceed 20 of the total reaction volume Carryover of K Na Mg2 or EDTA from the DNA sample can negatively affect PCR A change in pH may also affect PCR Store DNA in TE 4 buffer 10mM Tris HCl pH 8 0 0 1mM EDTA or nuclease free water Thermal cycler or tube problems Review the thermal cycling protocols in Section IV We have not tested other reaction tubes or thermal cyclers Calibration of the thermal cycler heating block may be required Primer concentration too low Use the recommended primer concentration Mix well before use Ice not used to set up reactions Set up the reactions on ice Very light allele intensity is obtained with some loci if ice is not used when setting up the reactions The use of AmpliTaq Gold DNA polymerase will also remedy this problem Samples not denatured before loading in the gel Be sure the samples are heated at 95 C for 2 minutes immediately prior to loading Poor CE injection Re inject the sample Poor quality formamide Be sure that high quality formamide is used when running samples on the ABI PRISM 310 or 3100 Genetic Analyzer Extra bands visible in Contamination with another template DNA or previously one or all of the lanes amplified DNA Cross contamination can be a problem Use aerosol resistant pipet tips and change gloves regularly Artifacts
34. at 338 254 and 161 bases 4Special template requirements for use of protocol 10 with certain multiplex system and detection instrument combinations are described in Table 4 Table 4 Recommended Amounts of Template For Various Instruments Using Protocol 10 Page 8 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD006 Printed in USA Revised 7 06 GenePrint STR System Recommended Protocols1 Alternative Protocols2 CTTv Multiplex 7 1 CTTv Multiplex with Amelogenin3 7 1 FFFL Multiplex 7 1 GammaSTR III Multiplex 7 NA Amelogenin CSF1PO F13A01 TH01 or TPOX 2 1 D16S539 D7S820 D13S317 or D5S818 7 NA F13B FESFPS or HPRTB 1 NA LPL or vWA 7 1 GenePrint STR System Recommended Protocols1 Alternative Protocols2 CTTv Multiplex 8 9 or 104 3 4 11 CTTv Multiplex with Amelogenin3 8 9 or 104 3 4 11 FFFL Multiplex using AmpliTaq DNA polymerase 8 9 or 104 3 4 11 FFFL Multiplex using AmpliTaq Gold DNA polymerase 12 NA GammaSTR III Multiplex 104 9 Amelogenin CSF1PO F13A01 F13B FESFPS HPRTB TH01 or TPOX 3 4 NA D16S539 D7S820 D13S317 or D5S818 9 or 104 NA LPL or vWA 8 9 3 4 Fluorescent STR System CTTv FFFL GammaSTR Hitachi FMBIO and FMBIO II Fluorescence Imaging Systems 5ng 2 5ng 1ng ABI PRISM 377
35. atents issued to the Max Planck Gesellschaft zur F rderung der Wissenschaften e V Germany The development and use of STR loci are covered by U S Pat No 5 364 759 Australian Pat No 670231 and other pending patents assigned to Baylor College of Medicine Houston Texas Patents for the foundational PCR process European Pat Nos 201 184 and 200 362 expired on March 28 2006 In the U S the patents covering the foundational PCR process expired on March 29 2005 b U S Pat Nos 5 843 660 and 6 221 598 Australian Pat No 724531 Canadian Pat No 2 118 048 Korean Pat No 290332 Singapore Pat No 57050 and Japanese Pat No 3602142 have been issued to Promega Corporation for multiplex amplification of STR loci Other patents are pending c U S Pat No 5 843 660 Australian Pat No 724531 Korean Pat No 290332 Singapore Pat No 57050 and Japanese Pat No 3602142 have been issued to Promega Corporation for multiplex amplification of STR loci Other patents are pending d U S Pat Nos 5 843 660 and 6 221 598 Australian Pat No 724531 Canadian Pat No 2 118 048 and Korean Pat No 290332 have been issued to Promega Corporation for multiplex amplification of STR loci Other patents are pending 1993 2006 Promega Corporation All Rights Reserved AluQuant GammaSTR GenePrint pGEM PowerPlex and SilverSTR are registered trademarks of Promega Corporation Differex DNA IQ and Slicprep are trademarks of Promega Cor
36. become difficult to interpret The balance of the peak heights may also appear less uniform 3 There may be variation between instruments regarding their relative fluorescent units detected using the same sample IX E Data Analysis Controls Observe the lanes containing the negative controls They should be devoid of amplification products Observe the lanes containing the K562 DNA positive control reactions Compare the K562 alleles with the locus specific allelic ladder The expected K562 alleles for each locus are listed in Table 12 Section XIII B The K562 DNA contains imbalanced alleles at several loci This result is due to the unusual chromosome content of the K562 cell line and is not a function of the GenePrint Fluorescent STR Systems performance Allelic Ladders In general the allelic ladders contain fragments of the same lengths as either several or all known alleles for the locus The allelic ladder sizes and repeat units are listed in Table 12 Section XIII B Analysis using GeneScan analysis software allows allele determination by comparison of amplified sample fragments with either allelic ladders internal size standards or both When using an internal size standard the calculated lengths of the allelic ladder components will differ from those listed in Table 12 This is due to differences in migration resulting from sequence differences between the allelic ladder fragments and those of the internal size standard
37. c mapping with trimeric and tetrameric tandem repeats Am J Hum Genet 49 74 56 3 Edwards A et al 1992 Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups Genomics 12 241 53 4 Warne D et al 1991 Tetranucleotide repeat polymorphism at the human beta actin related pseudogene 2 ACTBP2 detected using the polymerase chain reaction Nucl Acids Res 19 6980 5 Ausubel F M et al 1993 Unit 15 The polymerase chain reaction In Current Protocols in Molecular Biology Greene Publishing Associates and Wiley Interscience NY Page 36 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD006 Printed in USA Revised 7 06 Page 37 6 Sambrook J Fritsch E F and Maniatis T 1989 Chapter 14 In vitro amplification of DNA by the polymerase chain reaction In Molecular Cloning A Laboratory Manual Second Edition Cold Spring Harbor Laboratory Cold Spring Harbor NY 7 PCR Technology Principles and Applications for DNA Amplification 1989 ed Erlich H A Stockton Press NY 8 PCR Protocols A Guide to Methods and Applications 1990 eds Innis M A et al Academic Press San Diego CA 9 Puers C et al 1993 Identification of repeat sequence heterogeneity at the polymorphic STR locus HUMTH01 AATG n and reassignment of
38. ce or in an ice water bath Denature the samples just prior to loading the gel Sample DNA will re anneal if denatured hours before loading This may produce fragments of indeterminate migration 5 If using a sharkstooth comb flush the urea from the well area with a 50 100cc syringe filled with buffer Carefully insert the comb teeth into the gel approximately 1 2mm Leave the comb inserted in the gel during gel loading and electrophoresis If a square tooth comb is used clean the individual wells with buffer using a 50 100cc syringe and do not reinsert the comb The samples will be loaded directly into the wells Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD006 Revised 7 06 Page 22 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD006 Printed in USA Revised 7 06 VII B Sample Preparation Loading and Electrophoresis continued 6 Load 3 l of each sample into the respective wells We recommend using gel loading tips to load the wells formed by the square tooth combs The loading process should take no longer than 20 minutes to prevent the gel from cooling Note An organizational sheet for gel loading is provided in Section XIII G 7
39. dded should not exceed 20 of the final reaction volume PCR amplification efficiency and quality can be greatly altered by changes in pH due to added Tris HCl or available magnesium concentration due to chelation by EDTA DNA samples stored or diluted in sterile deionized water are not subject to this caution but may contain other PCR inhibitors at low concentrations 8 Hitachi FMBIO Users Pipet 2 5 l 25ng of K562 DNA into a 0 5ml microcentrifuge tube containing 22 5 l of PCR master mix as a positive amplification control ABI PRISM 377 DNA Sequencer ABI PRISM 310 Genetic Analyzer and ABI PRISM 3100 Genetic Analyzer Users Use only 1 2ng of K562 template DNA as a positive amplification control 9 Pipet 2 5 l of sterile water instead of template DNA into a 0 5ml microcentrifuge tube containing 22 5 l of PCR master mix as a negative amplification control 10 If recommended by the cycling protocol add 1 drop of mineral oil to each tube Close the tubes Note Allow the mineral oil to flow down the side of the tube and form an overlay to limit sample loss or cross contamination due to splattering 11 Centrifuge the samples briefly to bring the aqueous contents to the bottom of the tube IV C Amplification Thermal Cycling 1 Assemble the tubes in a thermal cycler 2 Select and run a recommended protocol from Table 2 or 3 Section IV A 3 After completion of the thermal cycling protocol store
40. de gels Anal Biochem 196 80 3 18 Budowle B et al 1991 Analysis of the VNTR locus D1S80 by the PCR followed by high resolution PAGE Am J Hum Genet 48 137 44 19 Nakamura Y et al 1987 Variable number of tandem repeat VNTR markers for human gene mapping Science 235 1616 22 20 Budowle B and Monson K L 1989 In Proceedings of an International Symposium on the Forensic Aspects of DNA Analysis Government Printing Office Washington D C 21 Levinson G and Gutman G A 1987 Slipped strand mispairing A major mechanism for DNA sequence evolution Mol Biol Evol 4 203 21 22 Schlotterer C and Tautz D 1992 Slippage synthesis of simple sequence DNA Nucl Acids Res 20 211 5 23 Smith J R et al 1995 Approach to genotyping errors caused by nontemplated nucleotide addition by Taq DNA polymerase Genome Res 5 312 7 24 Magnuson V L et al 1996 Substrate nucleotide determined non templated addition of adenine by Taq DNA polymerase Implications for PCR based genotyping BioTechniques 21 700 9 25 Walsh P S Fildes N J and Reynolds R 1996 Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vWA Nucl Acids Res 24 2807 12 26 Puers C et al 1994 Analysis of polymorphic STR loci using well characterized allelic ladders In Proceedings from the Fourth International Symposium on Human Identification 1993 Promega
41. de solution into a squeeze bottle 6 Add the following amounts of TEMED and 10 ammonium persulfate and mix gently 7 Pour the gel by starting at the well end of the plates Carefully pour the acrylamide between the horizontal glass plates Allow the solution to fill the top width of the plates Slightly tilt the plates to assist the movement of the solution to the bottom of the plates while maintaining a constant flow of the solution When the solution begins to flow out from the bottom position the plates horizontally 8 Insert the straight side of a 14cm doublefine 49 point sharkstooth comb 6mm of the comb should be between the two glass plates If using a square tooth comb insert the comb between the glass plates until the teeth are almost completely inserted into the gel 9 Secure the comb with 3 evenly spaced clamps 10 Pour the remaining acrylamide solution into a disposable conical tube as a polymerization control Rinse the squeeze bottle including the spout with water 11 Allow polymerization to proceed for at least 1 hour Check the polymerization control to be sure that polymerization has occurred Note The gel may be stored overnight if a paper towel saturated with deionized water and plastic wrap are placed around the top and bottom to prevent the gel from drying out crystallization of the urea will destroy the gel Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll F
42. dled accordingly Table 1 describes the potential hazards associated with such reagents Table 1 Hazardous Reagents IV Amplification The GenePrint Fluorescent STR Systems have been developed for amplification without artifacts using standard Taq DNA polymerase Special enzymes such as AmpliTaq Gold DNA polymerase are not required for peak performance However if using AmpliTaq Gold DNA polymerase we recommend using the Gold ST R 10X Buffer Cat DM2411 instead of the STR 10X Buffer Currently the STR 10X Buffer pH 9 0 is not compatible with AmpliTaq Gold DNA polymerase because the optimal pH for the modified Taq DNA polymerase is pH 8 3 Also when using AmpliTaq Gold DNA polymerase an additional incubation at 95 C for 11 minutes must be incorporated prior to initiating the thermal cycling program Reagent Hazard acrylamide suspected carcinogen toxic ammonium persulfate oxidizer corrosive bisacrylamide toxic irritant formamide STR 2X Loading Solution irritant teratogen bind silane methacryloxypropyltrimethoxysilane toxic moisture sensitive TEMED corrosive flammable urea irritant xylene cyanol FF STR 2X Loading Solution irritant Page 7 The following section gives detailed amplification protocols for using the GenePrint Fluorescent STR Systems Thermal cycling protocols for the Perkin Elmer Model 480 and the GeneAmp System 9600 thermal cyclers are given for each GenePrint
43. e 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD006 Revised 7 06 Master Mix Component Lot Number Volume l Per Sample Number of Reactions Final Volume l sterile water 17 45 for monoplex 17 35 for quadriplex STR 10X Buffer 2 50 10X Primer Pair 2 50 Taq DNA polymerase 5u l 0 05 for monoplex 0 20 for quadriplex total volume Page 50 Experiment Date Name Electrophoresis Pre run minutes Starting time Stopping time Watts Watts Milliamps Milliamps Voltage Voltage Notes Gel Number Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD006 Printed in USA Revised 7 06 Lane Sample Description 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Lane Sample Description 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Page 51 XIII H Related Products Fluorescent STR Multiplex Systems Product Size Cat PowerPlex 1 1 System 100 reactions DC6091 400 reactions DC6090 PowerPlex 2 1 System 100 reactions DC6471 400 reactions DC6470 PowerPlex 1 2 System 100
44. e assignment of alleles Analysis using specific instrumentation also allows allele determination by Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD006 Revised 7 06 Parameter Hitachi FMBIO Fluorescence Imaging System Hitachi FMBIO II Fluorescence Imaging System Material Type Acrylamide gel Acrylamide gel Resolution Horizontal Vertical 150dpi 150dpi 150dpi 150dpi Rate 0 1024s line NA Repeat 1 time 256 times Gray Level Correction Type Range Range Cutoff Threshold Low Background High signal 50 1 50 1 Reading Sensitivity 80 100 505nm channel 100 650nm channel Focusing Point NA 0mm Page 24 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD006 Printed in USA Revised 7 06 comparison of amplified sample fragments with either allelic ladders internal size standards or both see software documentation from instrument manufacturer When using an internal size standard the calculated lengths of the allelic ladder components will differ from those listed in Table 12 This is due to differences in migration resulting from sequence differences between the allelic ladder fragments and those of the int
45. e sample run 15 Monitor electrophoresis by observing the run status array and capillary views windows in the collection software Each run 16 samples capillaries will take approximately 45 minutes IX D Sample Detection 1 Analyze the data using the GeneScan analysis software 2 Review the raw data for one or more sample runs Highlight the sample file name then under the sample menu select raw data Move the cursor so the crosshair is on the baseline to the right of the large primer peak before the first internal lane standard peak red Use the X value number shown at the bottom left of the window for the start position in the analysis parameters The recommended analysis parameters are 1Smoothing options should be determined by individual laboratories Occasionally the separation control alleles and the TH01 alleles 9 3 and 10 will not be distinguished using heavy smoothing 2The peak amplitude thresholds are the minimum peak height that the software will call as a peak Values for the peak amplitude thresholds are usually 50 200RFU and should be determined by individual laboratories 3 The analysis parameters can be saved in the Params folder 4 Apply the stored analysis parameters file to the samples Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA
46. ega com Part TMD006 Printed in USA Revised 7 06 III Before You Begin The application of PCR based typing for forensic or paternity casework requires validation studies and quality control measures that are not contained in this manual 14 15 The quality of the purified DNA sample as well as small changes in buffers ionic strength primer concentrations choice of thermal cycler and thermal cycling conditions can affect the success of PCR amplification We suggest strict adherence to recommended procedures for amplification as well as for denaturing gel electrophoresis and fluorescence detection STR analysis is subject to contamination by very small amounts of nontemplate human DNA Extreme care should be taken to avoid cross contamination in preparing sample DNA handling primer pairs setting up amplification reactions and analyzing amplification products Reagents and materials used prior to amplification e g STR 10X Buffer K562 control DNA and fluorescein labeled 10X primer pairs should be stored separately from those used following amplification e g fluorescein labeled allelic ladders loading solutions and Gel Tracking Dye Always include a negative control reaction i e no template to ensure reagent purity We highly recommend the use of gloves and aerosol resistant pipet tips e g ART tips Section XIII H Some of the reagents used in the analysis of STR products are potentially hazardous and should be han
47. ele 4 version of chromosome 6 is present only once 6Alleles in bold are present in greater amounts than other alleles This simplifies interpretation Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD006 Printed in USA Revised 7 06 Page 42 STR Locus Allelic Ladder Size Range1 bases STR Ladder Alleles of repeats Other Known Alleles2 of repeats K562 DNA Allele Sizes of repeats Comments Amelogenin3 212 218 NA None 212 212 1 2 CSF1PO 295 327 7 8 9 10 11 12 13 14 15 6 10 9 1 D16S539 264 304 5 8 9 10 11 12 13 14 15 None 12 11 1 D7S820 215 247 6 7 8 9 10 11 12 13 14 None 11 9 1 D13S317 165 197 7 8 9 10 11 12 13 14 15 None 8 8 1 D5S818 119 151 7 8 9 10 11 12 13 14 15 16 12 11 1 F13A01 283 331 4 5 6 7 8 9 11 12 13 14 15 16 3 2 104 5 45 1 3 F13B 169 189 6 7 8 9 10 11 12 10 10 1 FESFPS 222 250 7 8 9 10 11 12 13 14 None 12 10 1 HPRTB 259 303 6 7 86 9 10 11 12 13 14 15 16 17 None 13 13 1 LPL 105 133 7 9 10 11 12 13 14 8 12 10 1 TH01 179 203 5 6 7 8 9 10 11 9 3 9 3 9 3 1 4 TPOX 224 252 6 7 8 9 10 11 12 13 None 9 8 1 vWA 139 167 13 14 15 16 17 18 19 20 10 11 21 22 16 16 1 Page 43 Promega Corporation 2800 Woods Hollow Road Mad
48. er before mixing with loading solution or use less DNA template in the amplification reactions Page 23 Table 9 Instrument Parameters for the Hitachi FMBIO and FMBIO II Fluorescence Imaging Systems NA Not applicable VII D Data Analysis Controls Observe the lanes containing the negative controls They should be devoid of amplification products Observe the lanes containing the K562 DNA positive controls Compare the K562 alleles with the locus specific ladder The expected K562 alleles for each locus are listed in Table 12 Figure 1 Section X B shows an example of results obtained after amplification of the positive control K562 DNA using the GenePrint Multiplex CTTv FFFL and GammaSTR Systems The K562 DNA contains imbalanced alleles at several loci This result is due to the unusual chromosome content of this cell line and is not a function of the GenePrint Fluorescent STR System performance Representative STR Data Representative STR data obtained using the Hitachi FMBIO II Fluorescent Scanner and the GenePrint Fluorescent STR Multiplex GammaSTR are shown in Figure 2 Section X Allelic and Fluorescent Ladders In general the allelic ladders contain fragments of the same lengths as either several or all known alleles for the locus The allelic ladder sizes and repeat units are listed in Table 12 Visual comparison between the allelic ladder and amplified samples of the same locus allows for precis
49. erlocus background value occurring as a result of the unusual chromosome content of the K562 cell line These variations are not a consequence of primer imbalance in amplification Page 34 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD006 Printed in USA Revised 7 06 L 1 2 L 3 4 L 5 6 L CSF1P0 CTTv Multiplex TPOX TH01 vWA 15 7 13 6 11 5 20 13 F13A01 FESFPS F13B LPL 16 4 14 7 11 6 14 7 L 1 2 L 3 4 L 5 6 L FFFL Multiplex D16S539 D7S820 D13S317 D5S818 15 5 14 6 15 7 15 7 L 1 2 L 3 4 L 5 6 L GammaSTR Multiplex 5858TA Figure 2 STR analyses performed using the fluorescein labeled GenePrint STR Multiplex systems and the Hitachi FMBIO Fluorescence Imaging System DNA samples amplified using the CTTv FFFL and GammaSTR Multiplex systems are shown For each system six DNA samples were amplified lanes 1 6 and are shown with allelic ladders for the corresponding loci lanes L Each allelic ladder is labeled to its right with the number of copies of the repeated sequence contained within the corresponding largest and smallest alleles of each locus All materials were separated using 4 denaturing
50. ernal size standard VII E Reuse of Glass Plates Separate the glass plates and discard the gel Clean the glass plates with deionized water and a 1 solution of Liqui Nox detergent The use of Liqui Nox detergent is extremely important as other kinds of soap can build up on the glass plates Buildup will result in low signal and high background on the gels If the glass plates have a soap residue buildup on them we recommend soaking in 10 sodium hydroxide for 1 hour and rinsing well in deionized water If bind silane is used to fix the gel to the smaller glass plate soak the plate in 10 sodium hydroxide for 1 hour or until the gel comes off the plate and clean as described VIII Sample Preparation Gel Electrophoresis and Detection on the ABI PRISM 377 DNA Sequencers and the ABI PRISM 310 Genetic Analyzer Instructions for use with the ABI PRISM 377 DNA Sequencer and ABI PRISM 310 Genetic Analyzer are given below Please refer to the user s manuals provided with these instruments for specific operating instructions VIII A Matrix Standardization Proper generation of a matrix file is critical to evaluate multi color systems with the ABI PRISM 377 DNA Sequencers or the ABI PRISM 310 Genetic Analyzer A new matrix must be generated for use with the GenePrint Fluorescent STR Systems and the Fluorescent Ladder CXR 60 400 Bases because the dyes used in the GenePrint Systems may differ from those obtained fr
51. ga Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD006 Printed in USA Revised 7 06 Materials to Be Supplied by the User continued Liqui Nox detergent Use of Liqui Nox detergent is extremely important as other kinds of detergent can build up on the glass plates clamps e g large office binder clips diamond pencil for marking glass plates Unpolymerized acrylamide is a neurotoxin and suspected carcinogen avoid inhalation and contact with skin Read the warning label and take the necessary precautions when handling this substance Always wear gloves and safety glasses when working with acrylamide powder or solutions Hitachi FMBIO Fluorescence Imaging Systems 1 Thoroughly clean the shorter and longer glass plates twice with 95 ethanol and Kimwipes tissues Note The plates require bind silane treatment if using a square tooth comb see below The plates do not require a special bind silane treatment when using a sharkstooth comb Bind Silane Treatment of Glass Plate Prepare fresh binding solution in a chemical fume hood Add 1 5 l of bind silane to a 1 5ml microcentrifuge tube containing 0 5ml of 0 5 acetic acid in 95 ethanol Wipe the etched side of the shorter glass plate in the comb region using a Kimwipes tissue saturated with the freshly prepared binding solution Wait 5
52. ic Analyzer 1 Refer to the ABI PRISM 310 Genetic Analyzer user s manual for instructions on cleaning the pump block installing the capillary calibrating the autosampler and adding polymer to the syringe 2 Open the ABI PRISM 310 data collection software 3 Prepare a GeneScan sample sheet as described in the ABI PRISM 310 Genetic Analyzer user s manual Enter the appropriate sample information in the sample info column 4 Create a new GeneScan injection list Select the appropriate sample sheet by using the pull down menu 5 Select the GS STR POP4 1ml A Module using the pull down menu Use the settings shown below Inj Secs 5 Inj kV 15 0 Run kV 15 0 Run C 60 Run Time min 24 You may need to optimize the injection time for individual instruments 6 Select the appropriate gel matrix file created in Section VIII A 7 To automatically analyze the data select the Auto Analyze checkbox and the appropriate analysis parameters and size standard Refer to the ABI PRISM 310 Genetic Analyzer user s manual for specific information on these options Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD006 Revised 7 06 Page 26 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526
53. ified sample fragments with either allelic ladders internal size standards or both When using an internal size standard the calculated lengths of the allelic ladder components will differ from those listed in Table 12 This is due to differences in migration resulting from sequence differences between the allelic ladder fragments and those of the internal size standard VIII F Reuse of Glass Plates For the ABI PRISM 377 DNA Sequencers separate the glass plates and discard the gel Clean the plates in the following manner rinse with hot water wash with 1 Liqui Nox solution rinse well with hot water wash with 1N NaOH rinse extremely well with deionized water and allow the plates to air dry IX Detection of Amplified Fragments Using the ABI PRISM 3100 Genetic Analyzer and Data Collection Software Version 1 1 Materials to Be Supplied by the User dry heating block water bath or thermal cycler crushed ice or ice water bath aerosol resistant pipet tips 3100 capillary array 36cm performance optimized polymer 4 POP 4 for the 3100 10X genetic analyzer buffer with EDTA sample tubes and septa for the 3100 Hi Di formamide Applied Biosystems Cat 4311320 PowerPlex Matrix Standards 3100 3130 Cat DG4650 Page 29 The quality of the formamide is critical Use deionized formamide with a conductivity lt 100 S cm Formamide can be dispensed into aliquots and frozen at 20
54. ison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD006 Revised 7 06 XIII C Power of Discrimination The GenePrint Fluorescent STR Systems provide extremely powerful discrimination The combined matching probability of the CTTv FFFL and GammaSTR quadriplexes range from 1 in 303 000 000 000 in Caucasian Americans to 1 in 4 610 000 000 000 in African Americans see Table 13 Typical paternity indices for the GenePrint Fluorescent STR Systems are shown in Table 14 An alternative calculation in paternity analyses is the power of exclusion Table 15 gives typical values for the power of exclusion for the GenePrint Fluorescent STR Systems in various populations Table 13 Matching Probability of Various Populations Table 14 Typical Paternity Indices of the Multiplex GenePrint STR Systems in Various Populations Table 15 Power of Exclusion of the GenePrint STR Systems in Various Populations Matching Probability STR System African American Caucasian American Hispanic American CTTv quadriplex CSF1PO TPOX TH01 vWA 1 in 25 236 1 in 6 796 1 in 7 219 FFFL quadriplex F13A01 FESFPS F13B LPL 1 in 16 802 1 in 2 658 1 in 3 276 GammaSTR III quadriplex D16S539 D7S820 D13S317 D5S818 1 in 10 872 1 in 16 790 1 in 20 106 All 3 quadriplexes 12 loci 1 in 4 61 1012 1 in 3 03 1011 1 in 4 75 1011 Ty
55. l TMD011 Refer to the GenePrint STR Systems Technical Manual TMD004 for information about detecting STR products using silver staining 17 These Technical Manuals are available at www promega com tbs STR typing is more tolerant of the use of degraded DNA templates than other methods of individual identification because the amplification products are less than 400bp long much smaller than the material detected with AMP FLP 18 or VNTR 19 analysis This format is also amenable to a variety of rapid DNA purification techniques In addition to these advantages the STR loci chosen for inclusion in the GenePrint systems contain alleles of discrete and separable lengths This allows the construction of allelic ladders which contain fragments of the same lengths as several or all known alleles for the locus Visual comparison between the allelic ladder and amplified samples of the same locus allows rapid and precise assignment of alleles Results obtained using the GenePrint Fluorescent STR Systems can be recorded in a digitized format allowing direct comparison with stored databases Population analyses do not require the use of arbitrarily defined fixed bins for population data 20 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD006 Revised 7 06 Page 39 Page 40 Promega Corporation
56. lanes If allelic ladders are used for making allele determinations visually use either a sharkstooth or a square tooth comb Materials to Be Supplied by the User Solution compositions are provided in Section XIII F 40 acrylamide bis 19 1 and TEMED 10X TBE Buffer Cat V4251 10 Ammonium Persulfate Cat V3131 Urea Cat V3171 bind silane methacryloxypropyltrimethoxysilane if square tooth combs are to be used 0 5 acetic acid in 95 ethanol Nalgene tissue culture filter 0 2 micron 32cm 19cm 0 4mm h w thickness low fluorescence glass plates MiraiBio spacers for SA 32 low fluorescence glass plates 43cm 19cm 0 4mm h w thickness low fluorescence glass plates Whatman Biometra SA 43 Spacer Set Whatman Biometra SA 43 Extension Whatman Biometra for use with SA 43 glass plates power supply polyacrylamide gel electrophoresis apparatus for gels 30cm glass plates and side spacers for polyacrylamide gel 30cm 14cm vinyl doublefine sharkstooth comb s 49 point 0 4mm thick or square tooth comb 35cm 60 wells cut in half for 30 wells gel 0 4mm thick Owl Scientific Cat S2S 60A Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD006 Revised 7 06 Page 16 Prome
57. lood Additionally DNA has been isolated from casework samples such as tissue differentially separated sexual assault samples and stains on support materials See Section XIII B for additional information For applications requiring human specific DNA quantitation the AluQuant Human DNA Quantitation System Cat DC1011 and DC1010 has been developed to work with the PowerPlex Systems 30 See Section XIII B for additional information Both the DNA IQ System and AluQuant Human DNA Quantitation System have been fully automated on the Beckman Coulter Biomek 2000 Laboratory Automation Workstation 31 For information on automation of laboratory processes on Beckman Coulter or other workstations contact your local Promega Branch Office or Distributor contact information available at www promega com or e mail genetic promega com Traditional DNA extraction methods such as phenol chloroform extraction methods 32 33 or inorganic methods 34 35 can be used for STR analysis For stains from blood and saliva scientists at the FBI Academy have suggested an alternative method for DNA extraction see reference 36 DNA isolation methods such as Chelex 100 see reference 37 and cell lysis followed by proteinase K digestion see reference 38 are capable of extracting DNA from bloodstains or from as little as 3 l of whole blood Because both of these methods produce single stranded DNA they should not be used for VNTR variable number
58. lution To use in combination with the TH01 monoplex or the CTTv Allelic Ladder mix 0 5 l of allele 9 3 with 2 0 l of the allelic ladder before mixing with the appropriate loading dye ABI PRISM 377 DNA Sequencer For PCR amplified samples combine 1 l of sample with 1 5 l of Blue Dextran Loading Solution and 0 5 l of Fluorescent Ladder CXR For allelic ladders dilute the ladder 1 10 in 1X STR Buffer then combine 1 l of diluted ladder with 1 5 l of Blue Dextran Loading Solution and 0 5 l of Fluorescent Ladder CXR ABI PRISM 310 Genetic Analyzer For PCR amplified samples combine 1 l of sample with 24 5 l of formamide deionized and 0 5 l of Fluorescent Ladder CXR For allelic ladders dilute the ladder 1 10 in 1X STR Buffer then combine 1 l of diluted ladder with 24 5 l of formamide deionized and 0 5 l of Fluorescent Ladder CXR Page 27 ABI PRISM 377 DNA Sequencer 1 Pre run the gel to achieve a surface temperature of approximately 50 C After the 15 20 minute pre run pause the instrument by clicking on Pause 2 Use a 30cc syringe filled with buffer to flush the urea from the well area 3 Load 1 5 l of each denatured sample into the respective wells 4 Place the lid on the upper buffer chamber and close the instrument door ABI PRISM 310 Genetic Analyzer 1 Assemble the tubes in the appropriate autosampler tray 48 tube or 96 tube 2 Place the autosampler tray in the instr
59. lution between the horizontal glass plates Allow the solution to fill the top width of the plates While maintaining a constant flow of solution gently tap the glass plates to assist the movement of solution to the bottom of the plates 7 Insert the straight edge of one 36 well sharkstooth comb or insert a 34 well square tooth comb between the glass plates 8 Secure the comb with 3 evenly spaced clamps 9 Pour the remaining acrylamide solution into a disposable conical tube as a polymerization control 10 Allow polymerization to proceed for at least 2 hours Check the polymerization control Step 9 to ensure that polymerization has occurred Note The gel may be stored overnight if a paper towel saturated with 1X TBE and plastic wrap are placed around the top and bottom of the gel to prevent the gel from drying out crystallization of the urea will destroy the gel VI Polyacrylamide Gel Electrophoresis and Detection Electrophoresis protocols are dependent on the type of instrument used for fluorescence detection In the following sections procedures for loading and running gels on the Hitachi FMBIO and FMBIO II Fluorescence Imaging Systems Section VII and the ABI PRISM 310 Genetic Analyzer Section VIII and the ABI PRISM 3100 Genetic Analyzer Section IX are provided If a different instrument is used for detection please refer to the manufacturer s recommendations for that particular instrument The Fluore
60. minutes for the binding solution to dry Wipe the shorter glass plate 3 4 times with 95 ethanol and Kimwipes tissues in the comb area to remove the excess binding solution 2 Assemble the glass plates by placing 0 4mm side spacers between the plates and using clamps to hold them in place 3 4 clamps on each side A bottom spacer is neither required nor recommended Place the assembly horizontally on a test tube rack or similar support 3 Prepare a 4 or 6 acrylamide solution total of 30ml for a 32cm plate or 45ml for a 43cm plate by combining the ingredients listed in Table 7 Table 7 Preparation of 4 and 6 Polyacrylamide Gels Note If preparing multiple gels on a daily basis a larger 4 or 6 stock solution may be prepared filtered as in Step 4 below and stored at 4 C in the dark for up to one month To prepare a single gel remove either 30ml or 45ml of the stock solution and continue with Step 6 Component 4 Gel 32cm 4 Gel 43cm 6 Gel 32cm 6 Gel 43cm Final Concentration urea 12 6g 18 9g 12 6g 18 9g 7M deionized water 16 0ml 24 0ml 14 5ml 21 75ml 10X TBE buffer 1 5ml 2 25ml 1 5ml 2 25ml 0 5X 40 acrylamide bis 19 1 3 0ml 4 5ml 4 5ml 6 75ml 4 or 6 total volume 30 0ml 45 0ml 30 0ml 45 0ml Page 17 4 Filter the acrylamide solution through a 0 2 micron filter e g Nalgene tissue culture filter 5 Slowly pour the filtered acrylami
61. mplified sample Notes 1 Samples 1 2ng of template amplified with the FFFL CTTv and GammaSTR Systems will need to be diluted 1 5 i e 1 part sample 4 parts 1X STR Buffer before mixing with the loading solution 2 To analyze the CTTv Multiplex with Amelogenin reactions mix the corresponding ladders 1 1 before mixing with loading solution The number of ladder lanes depends on personal preference and the number of samples analyzed 3 To use the TH01 allele 9 3 alone mix 0 5 l of allele 9 3 with 1 5 l of 1X STR Buffer before mixing with the loading solution To use in combination with the TH01 monoplex or the CTTv Allelic Ladder mix 0 5 l of allele 9 3 with 2 0 l of the allelic ladder before mixing with the appropriate loading dye 4 Instrument detection limits vary therefore injection time or the amount of product mixed with loading cocktail may need to be increased or decreased If the peak heights are too high gt 4 000RFU the samples can be diluted in Gold ST R 1X Buffer before mixing Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD006 Revised 7 06 Page 30 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD006 Printed in
62. n select Z from the pull down menu 7 In the Run Module 1 column select GeneScan36_POP4DefaultModule from the pull down menu 8 To collect the data without autoanalyzing select No Selection in the Analysis Module 1 column Analysis parameters can be applied after data collection and during data analysis using the GeneScan analysis software To analyze the data during data collection an appropriate analysis module must be selected in the Analysis Module 1 column Refer to the ABI PRISM 3100 Genetic Analyzer user s manual for specific instructions on creating analysis modules 9 Select OK This new plate record will appear in the pending plate records table on the plate setup page of the data collection software 10 Place samples in instrument and close the instrument doors 11 In the pending plate records table click once on the name of the plate record you just created Page 31 12 Once the plate record is highlighted click the plate graphic that corresponds to the plate on the autosampler that contains your amplified samples to link the plate to the plate record 13 When the plate record is linked to the plate the plate graphic will change from yellow to green the plate record moves from the pending plate records table to the linked plate records table and the Run Instrument button becomes enabled 14 Select Run Instrument on the toolbar to start th
63. ng A Laboratory Manual Second Edition Cold Spring Harbor Laboratory Cold Spring Harbor NY 9 16 34 Grimberg J et al 1989 A simple and efficient non organic procedure for the isolation of genomic DNA from blood Nucl Acids Res 17 8390 35 Miller S Dykes D and Polesky H 1988 A simple salting out procedure for extracting DNA from human nucleated cells Nucl Acids Res 16 1215 36 Comey C et al 1994 DNA extraction strategies for amplified fragment length polymorphism analysis J Forensic Sci 39 1254 69 37 Walsh P S Metzger D A and Higuchi R 1991 Chelex 100 as a medium for simple extraction of DNA for PCR based typing from forensic material BioTechniques 10 506 13 38 Higuchi R 1989 Rapid efficient DNA extraction for PCR from cells or blood In Amplifications A Forum for PCR Users May 1989 Perkin Elmer Norwalk CT Issue 2 XII B Additional STR References A substantial reference list of publications describing STRs and much related information can be found at a web site created by the National Institutes of Science and Technology NIST Biotechnology Division This web site www cstl nist gov div831 strbase is occasionally updated and has numerous links to many other useful sites The references below provide an overview of the listed topics and may be found on the Promega web site at www promega com profiles The spreadsheet for the PowerStats application is available at
64. of STR amplification PCR amplification sometimes generates artifacts that appear as faint bands one or four bases below an allele Refer to Section XIII B for locus specific information regarding this event Samples not completely denatured Heat denature the samples at 95 C for 2 minutes immediately prior to loading the gel Insufficient pre run of gel Pre run gels until a temperature of 50 C is reached before loading samples Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD006 Revised 7 06 XI Troubleshooting continued Symptoms Causes and Comments Bands are fuzzy Poor quality polyacrylamide gel Prepare acrylamide and throughout the lanes buffer solutions using high quality reagents Store acrylamide solutions in the dark Electrophoresis temperature is too high Run gel at a lower temperature 40 60 C Extra peaks visible in one CE related artifacts Minor voltage changes or urea crystals or all of the color channels passing by the laser may cause spikes or unexpected peaks ABI PRISM 310 or 3100 Spikes sometimes appear in one color but often are easily Genetic Analyzer identified by their presence in more than one color Re inject the samples to confirm Allelic ladder not running the Wrong allelic ladder or primer pair mix used Be sure that the same as the sample
65. om other commercial sources The PowerPlex Matrix Standards 310 Cat DG4640 is required for spectral calibration on the ABI PRISM 377 DNA Sequencer and ABI PRISM 310 Genetic Analyzer Please refer to the ABI PRISM 377 DNA Sequencer or ABI PRISM 310 Genetic Analyzer user s manual for instructions on how to generate a matrix file using virtual filter set A To prepare a matrix a set of four standards is run using the same conditions as those used for samples and allelic ladders The matrix can be generated using the PowerPlex Matrix Standards 310 For protocols and additional information on the use of the PowerPlex Matrix Standards 310 see the PowerPlex Matrix Standards 310 Technical Bulletin TBD021 supplied with Cat DG4640 which is available upon request from Promega or online at www promega com tbs Page 25 VIII B Instrument Preparation ABI PRISM 377 DNA Sequencer 1 Open the GeneScan data collection software 2 Prepare a GeneScan sample sheet as described in the GeneScan analysis software user s manual 3 Create a new GeneScan run using the following settings Plate Check Module Plate Check A PreRun Module PR GS 36A 2400 Run Module GS 36A 2400 Collect Time 2 25 hours Well to Read Distance 36cm 4 Select the appropriate sample sheet and comb selection by using the pull down menus 5 Select the appropriate gel matrix file created in Section VIII A ABI PRISM 310 Genet
66. omophenol Blue Loading Solution and 2 l of sample or allelic ladder we recommend running two lanes of allelic ladder per gel when using the Fluorescent Ladder CXR Note The Bromophenol Blue Loading Solution does not contain xylene cyanol because it fluoresces and is detected by the FMBIO instruments 2 After the samples are prepared centrifuge the tubes briefly to bring the contents to the bottom of the tube 3 Optional Place 6 l of Gel Tracking Dye in one tube The Gel Tracking Dye contains both bromophenol blue and xylene cyanol This dye may be loaded in the outermost lane of the gel and used as a visual indicator of migration We recommend leaving two empty lanes between the gel tracking dye and the sample lanes so the xylene cyanol fluorescence does not interfere with sample interpretation Notes 1 To analyze the CTTv Multiplex with Amelogenin reactions mix the corresponding ladders 1 1 before mixing with loading solution The number of ladder lanes depends on personal preference and the number of samples analyzed 2 To use the TH01 allele 9 3 alone mix 0 5 l of allele 9 3 with 1 5 l of 1X STR Buffer before mixing with the loading solution To use in combination with the TH01 monoplex or the CTTv Allelic Ladder mix 0 5 l of allele 9 3 with 2 0 l of the allelic ladder before mixing with the appropriate loading dye 4 Denature the samples by heating at 95 C for 2 minutes and immediately chill on crushed i
67. oncentrated at the bottom of the tube 2 Place one clean autoclaved 0 5ml reaction tube for each reaction into a rack and label appropriately Note If using the GeneAmp PCR system 9600 thermal cycler refer to the notes for Table 5 for tube selection 3 Determine the number of reactions to be set up This should include a positive and negative control reaction Add 1 or 2 reactions to this number to compensate for pipetting error While this approach does waste a small amount of each reagent it ensures that you will have enough PCR master mix for all samples 4 Calculate the required amount of each component of the PCR master mix Table 6 Multiply the volume l per sample by the total number of reactions from Step 3 to obtain the final volume l Note The CTTv Multiplex and Amelogenin locus can be amplified simultaneously 5 In the order listed in Table 6 add the final volume of each reagent to a sterile tube Mix well and place on ice Note If the final volume of Taq DNA polymerase added to the master mix is less than 0 5 l you may wish to dilute the enzyme with STR 1X Buffer first and add a larger volume The amount of sterile water should be adjusted accordingly so that the final volume per reaction is 25 l Do not store diluted Taq DNA polymerase Page 12 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 25
68. ormation All prices and specifications are subject to change without prior notice Product claims are subject to change Please contact Promega Technical Services or access the Promega online catalog for the most up to date information on Promega products
69. pical Paternity Index STR System African American Caucasian American Hispanic American CTTv quadriplex CSF1PO TPOX TH01 vWA 29 4 19 26 10 51 FFFL quadriplex F13A01 FESFPS F13B LPL 16 83 15 28 8 23 GammaSTR III quadriplex D16S539 D7S820 D13S317 D5S818 16 93 13 51 30 40 All 3 quadriplexes 12 loci 8 373 3 976 2 627 Power of Exclusion STR System African American Caucasian American Hispanic American CTTv quadriplex CSF1PO TPOX TH01 vWA 0 967 0 953 0 918 FFFL quadriplex F13A01 FESFPS F13B LPL 0 946 0 941 0 902 GammaSTR III quadriplex D16S539 D7S820 D13S317 D5S818 0 946 0 934 0 967 All 3 quadriplexes 12 loci 0 99990 0 99981 0 99974 Page 44 XIII D DNA Extraction and Quantitation Methods The DNA IQ System Cat DC6700 is a DNA isolation and quantitation system designed specifically for forensic and paternity samples 29 This novel technology uses paramagnetic particles to prepare clean samples for STR analysis easily and efficiently and can be used to extract DNA from stains or liquid samples such as blood or solutions The DNA IQ Resin is designed to eliminate PCR inhibitors and contaminants frequently encountered in casework samples For larger samples the DNA IQ System delivers a consistent amount of total DNA The system has been used isolate and quantify DNA from routine sample types including buccal swabs stains on FTA paper and liquid b
70. polyacrylamide gels The CTTv FFFL and GammaSTR Multiplex was detected using the Hitachi FMBIO II Fluorescence Imaging System Figure 3 STR analyses performed using the GenePrint Fluorescent STR System GammaSTR the Fluorescent Ladder CXR 60 400 Bases and the ABI PRISM 310 Genetic Analyzer The upper panel shows the alleles of loci D5S818 D13S317 D7S820 and D16S539 amplified using the GammaSTR System and 1ng DNA template The sample was diluted 1 5 1 part sample 4 parts STR 1X Buffer prior to loading The bottom panel shows the relevant portion of the Fluorescent Ladder CXR 60 400 Bases The numbers above the peaks indicate the sizes of the fragments in the ladder 5860TA 100 120 140 160 180 200 225 250 275 300 325 350 375 400 D5S818 D13S317 D7S820 D16S539 Page 35 XI Troubleshooting For questions not addressed here please contact your local Promega Branch Office or Distributor Contact information available at www promega com E mail genetic promega com Symptoms Causes and Comments Faint or no bands allele peaks Impure template DNA Inhibitors may exist in the DNA sample Insufficient template DNA Use the recommended amount of template DNA Insufficient enzyme activity Use the recommended amount of Taq DNA polymerase Check the expiration date on the tube label Wrong amplification program Choose the correct amplification program for each locus High salt concentration or
71. poration ABI PRISM GeneScan Genotyper and MicroAmp are registered trademarks of Applera Corporation AmpliTaq AmpliTaq Gold and GeneAmp are registered trademarks of Roche Molecular Systems Inc ART is a registered trademark of Molecular BioProducts Inc Biomek is a registered trademark of Beckman Coulter Inc Biometra is a registered trademark of Biometra Biomedizinische Analytik GmbH Chelex is a registered trademark of Bio Rad Laboratories Inc Ficoll is a registered trademark of GE Healthcare Bio sciences FMBIO is a registered trademark of Hitachi Software Engineering Company Ltd FTA is a registered trademark of Flinders Technologies Pty Ltd and is licensed to Whatman GenBank is a registered trademark of the U S Dept of Health and Human Services Kimwipes is a registered trademark of Kimberly Clark Liqui Nox is a registered trademark of Alconox Inc Long Ranger is a registered trademark of Cambrex Corporation Microsoft is a registered trademark of Microsoft Corporation Nalgene is a registered trademark of Nalge Nunc International Polaroid is a registered trademark of Polaroid Corporation POP 4 is a trademark of Applera Corporation STaRCall is a registered trademark of Hitachi Software Engineering Company Ltd Triton is a registered trademark of Union Carbide Chemicals and Plastics Technology Corporation Products may be covered by pending or issued patents or may have certain limitations Please visit our Web site for more inf
72. r Refer to 1 minute 68 seconds to 60 C 30 seconds 60 seconds to 60 C 30 seconds 30 minutes 4 C Note 4 50 seconds to 70 C 45 seconds 50 seconds to 70 C 45 seconds 11 9600 96 C for 50 seconds to 94 C 1 minute 45 seconds to 90 C 1 minute 60 C for Refer to 2 minutes 34 seconds to 60 C 1 minute 30 seconds to 60 C 1 minute 30 minutes 4 C Note 5 25 seconds to 70 C 1 5 minutes 25 seconds to 70 C 1 5 minutes Protocol Thermal Initial Programmed Cycling for Programmed Cycling for Extension Hold Number Cycler1 Incubation2 Ramp Times First 10 Cycles Ramp Times Last 22 Cycles3 Step Step 12 9600 96 C for 50 seconds to 94 C 1 minute 45 seconds to 90 C 1 minute None Refer to 2 minutes 30 seconds to 64 C 1 minute 26 seconds to 64 C 1 minute 4 C Note 4 15 seconds to 70 C 1 5 minutes 15 seconds to 70 C 1 5 minutes Table 5 Amplification Protocols continued 1480 refers to the Perkin Elmer model 480 thermal cycler 9600 refers to the Perkin Elmer GeneAmp PCR system 9600 thermal cycler 2Initial incubation performed using AmpliTaq DNA polymerase When using AmpliTaq Gold DNA polymerase include an additional incubation at 95 C for 11 minutes prior to initiation of the thermal cycling program with any protocol Also when using AmpliTaq DNA polymerase be sure to use the Gold ST R 10X Buffer 3Please note that Protocol 12 incorporates 2 additional cycles in the last po
73. reactions DC6101 PowerPlex 16 System 100 reactions DC6531 400 reactions DC6530 PowerPlex 16 BIO System 100 reactions DC6541 400 reactions DC6540 PowerPlex ES System 100 reactions DC6731 400 reactions DC6730 Not for Medical Diagnostic Use The PowerPlex 1 1 2 1 and 16 BIO Systems are compatible with the Hitachi FMBIO Fluorescence Imaging Systems Accessory Components Product Size Cat Acrylamide 100g V3111 Ammonium Persulfate 25g V3131 Bisacrylamide 25g V3141 Blue Dextran Loading Solution 3ml DV4351 Bromophenol Blue Loading Solution 3ml DV4371 Gel Tracking Dye 1ml DV4361 Gold ST R 10X Buffer 1 2ml DM2411 K562 DNA High Molecular Weight 30 g DD2011 Mineral Oil 12ml DY1151 Nuclease Free Water 50ml P1193 150ml P1195 PowerPlex Matrix Standards 310 50 l each dye DG4640 PowerPlex Matrix Standards 3100 3130 25 l each dye DG4650 STR 10X Buffer 1 2ml DM2211 STR 2X Loading Solution 3ml DV4331 TBE Buffer 10X 1L V4251 Urea 1kg V3171 Not for Medical Diagnostic Use For Laboratory Use Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD006 Revised 7 06 Page 52 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 80
74. ree in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD006 Revised 7 06 Component 32cm gel 30ml 43cm gel 45ml TEMED 20 l 30 l 10 ammonium persulfate 200 l 300 l Page 18 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD006 Printed in USA Revised 7 06 V B Gel Preparation for the ABI PRISM 377 DNA Sequencer When working with the glass plates for the ABI PRISM 377 DNA Sequencer it is extremely important to avoid contact between the gel side of the plates and paper towels Rinse the plates extremely well with deionized water and allow to air dry in a dust free environment before use Materials to Be Supplied by the User Solution compositions are provided in Section XIII F Long Ranger gel solution Cambrex Cat 50611 10X TBE Buffer Cat V4251 10 Ammonium Persulfate Cat V3131 TEMED Urea Cat V3171 Nalgene tissue culture filter 0 2 micron 36cm front and rear glass plates refer to the instrument manual for recommendations 36cm gel spacers 0 2mm thick 36 well sharkstooth comb or 34 well square tooth comb 0 2mm thick clamps Liqui Nox detergent Use of Liqui Nox detergent is extremely important as other kinds of detergent can build up on the glass
75. rtion of the cycling protocol When using AmpliTaq Gold with protocols 1 11 use the Gold ST R 10X Buffer instead of the STR 10X Buffer An additional incubation at 95 C for 11 minutes must also be incorporated prior to the initiation of the thermal cycling program Notes for Table 5 1 Use GeneAmp reaction tubes and overlay all reactions with mineral oil 2 Use GeneAmp reaction tubes in combination with the GeneAmp thin walled tray This reduces the maximum number of simultaneous reactions to 48 due to the spacing of holes in the tray Add mineral oil to all reactions 3 Use MicroAmp reaction tubes in the MicroAmp 9600 tray This allows a maximum of 96 simultaneous reactions Add mineral oil to all reactions Do not cover the reactions with the system 9600 thermal cycler lid Cover the reaction tubes loosely with aluminum foil Optional Add BSA Fraction V final concentration 60 g ml to all reactions This may result in a slight increase in yield We recommend Sigma BSA Cat A2153 Performance may vary depending on the source of this component 4 See Table 4 for recommended amounts of template Use MicroAmp reaction tubes in the MicroAmp 9600 tray This allows a maximum of 96 simultaneous reactions No mineral oil is needed Cover reactions with the System 9600 thermal cycler lid Optional Add BSA Fraction V final concentration 60 g ml to all reactions This may result in a slight increase in
76. s allelic ladder loading solutions and Gel Tracking Dye are sealed in separate packages to prevent cross contamination We strongly recommend that pre amplification and post amplification reagents be stored and used separately with different pipettes tube racks etc Store amplified material at 20 C Page 5 II B Allelic Ladders and Size Markers Product Size Cat Fluorescent Ladder CXR 60 400 Bases 65 l DG6221 For Laboratory Use Allelic Ladders Product Size Cat CTTv Allelic Ladder Mix Fluorescein a 150 l DG2121 FFFL Allelic Ladder Mix Fluorescein a 150 l DG2131 GammaSTR Allelic Ladder Mix Fluorescein a 150 l DG3291 For Laboratory Use The Fluorescent Ladder CXR 60 400 Bases is a size marker composed of 16 evenly spaced DNA fragments labeled with carboxy X rhodamine When this marker is included in each gel lane the instruments recommended for fluorescence detection are capable of monitoring and correcting lane to lane sample migration differences The Internal Lane Standard 600 contains the same DNA fragments found in the Fluorescent Ladder CXR with additional fragments in the range of 425 600 bases II C GenePrint Fluorescent Sex Identification Systems Product Size Cat GenePrint Fluorescent Sex Identification System Amelogenin Fluorescein 100 reactions DC5171 400 reactions DC5170 GenePrint Fluorescent Sex Identification System Amelogenin TMR 1
77. scent Ladder The Fluorescent Ladder CXR 60 400 Bases contains 16 evenly spaced DNA fragments of 60 80 100 120 140 160 180 200 225 250 275 300 325 350 375 and 400 bases in length This ladder may be used as an internal size standard in each lane to increase precision in analyses Inclusion of the Fluorescent Ladder CXR in each lane reduces the number of allelic ladder lanes needed per gel and therefore increases the number of lanes available for samples Allelic ladders still need to be run on one or two lanes on every gel as a control to verify that the gel ran correctly and that the allele sizes are correct Be aware that the sizes determined by the software may not exactly correspond to the sequenced sizes given in Table 12 because samples migrate on a gel according to length sequence and dye label Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD006 Revised 7 06 The Fluorescent Ladder CXR 60 400 Bases is required when using the ABI PRISM 377 DNA Sequencer ABI PRISM 310 Genetic Analyzer or ABI PRISM 3100 Genetic Analyzer The Genescan software provided with these instruments requires use of a sizing ladder The Fluorescent Ladder CXR must be included in all lanes to account for lane to lane or run to run variability Adjustments in the allele sizes will be made
78. son WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD006 Revised 7 06 Page 28 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD006 Printed in USA Revised 7 06 VIII E Data Analysis Representative STR data obtained using the ABI PRISM 377 DNA Sequencer the GenePrint Fluorescent STR Multiplex GammaSTR and the Fluorescent Ladder CXR 60 400 Bases is shown in Figure 3 Section X Controls Observe the lanes containing the negative controls They should be devoid of amplification products Observe the lanes containing the K562 DNA positive control reactions Compare the K562 alleles with the locus specific allelic ladder The expected K562 alleles for each locus are listed in Table 12 Section XIII B The K562 DNA contains imbalanced alleles at several loci This result is due to the unusual chromosome content of the K562 cell line and is not a function of the GenePrint Fluorescent STR Systems Allelic Ladders In general the allelic ladders contain fragments of the same lengths as either several or all known alleles for the locus The allelic ladder sizes and repeat units are listed in Table 12 Section XIII B Analysis using GeneScan analysis software allows allele determination by comparing ampl
79. t be observed if the system was not optimized on that particular thermal cycler This manual provides a number of cycling protocol options so that excellent results can be obtained regardless of the thermal cycler used Allele frequencies for African Americans Caucasian Americans and Hispanic Americans for all currently available STR systems can be found at www promega com techserv apps hmnid referenceinformation popstat custstat_Allelefreq htm Additional population data for STR loci can be found in references 3 and 9 13 Additional STR references are listed in Section XIII B Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD006 Revised 7 06 Page 4 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD006 Printed in USA Revised 7 06 II Product Components and Storage Conditions II A GenePrint Fluorescent STR Multiplex Systems Below is a description of the components of the GenePrint Fluorescent STR Multiplex Systems All of the GenePrint STR Multiplex Systems include the required fluorescein labeled 10X primer pairs as a mixture for simultaneous amplification of more than one locus and a mixture of the fluorescein labeled allelic ladders for the same se
80. t of loci is also provided Additional components include STR 10X Buffer K562 DNA loading solutions and Gel Tracking Dye Product Size Cat GenePrint Fluorescent STR Multiplex CSF1PO TPOX TH01 vWA Fluorescein a b 100 reactions DC6301 400 reactions DC6300 GenePrint Fluorescent STR Multiplex GammaSTR Fluorescein a c D16S539 D7S820 D13S317 D5S818 100 reactions DC6071 400 reactions DC6070 GenePrint Fluorescent STR Multiplex F13A01 FESFPS F13B LPL Fluorescein a d 100 reactions DC6311 400 reactions DC6310 Not for Medical Diagnostic Use Cat DC6300 DC6070 and DC6310 contain sufficient reagents for 400 reactions of 25 l each Each system includes 4 250 l FFFL CTTv or GammaSTR 10X Primer Pair Mix Fluorescein 4 150 l FFFL CTTv or GammaSTR Allelic Ladder Mix Fluorescein 4 300 l STR 10X Buffer 3 g K562 DNA High Molecular Weight 10ng l 2 1ml Bromophenol Blue Loading Solution 2 1ml Blue Dextran Loading Solution 250 l Gel Tracking Dye 1 100 l TH01 Allele 9 3 Fluorescein 200 lanes CTTv system only 1 1 5ml Gold ST R 10X Buffer FFFL system only 1 Protocol Storage Conditions Store all components at 20 C The fluorescent 10X Primer Pair Mix and fluorescent Allelic Ladder Mix are light sensitive therefore minimize light exposure and store in the dark The post amplification component
81. tandem repeat polymorphism analysis Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD006 Printed in USA Revised 7 06 Page 45 XIII E Agarose Gel Electrophoresis of Amplification Products Optional This procedure is optional if PCR is routinely performed in your laboratory Agarose gel electrophoresis can be used to rapidly confirm the success of the amplification reaction prior to performing polyacrylamide gel Materials to Be Supplied by the User Solution compositions are provided in Section XIII F TAE 1X buffer agarose 5X loading solution ethidium bromide solution 0 5 g ml Ethidium bromide is a powerful mutagen Wear gloves at all times and use a mask when weighing out ethidium bromide powder 1 Prepare a 2 agarose gel approximately 150cm2 by adding 2 0g of agarose to 100ml of TAE 1X buffer Mark the liquid level on the container then boil or heat in a microwave oven to dissolve the agarose Add preheated 60 C deionized water to make up for any volume lost due to evaporation 2 Cool the agarose to 55 C before pouring into the gel tray Be sure that the gel tray is level Pour the agarose into the tray insert the gel comb and allow to set for 20 30 minutes 3 Prepare the samples by mixing 10 l of each amplified sample with 2 5 l of 5X loading solution
82. template requirement of each multiplex system for use in combination with various detection instruments when using protocol 10 Many customers prefer protocol 10 because it uses the GeneAmp PCR system 9600 thermal cycler with the thermal cycler lid MicroAmp reaction tubes and no mineral oil Specific details for each protocol including number of cycles incubation temperatures and times and ramp times are provided in Table 5 When using a thermal cycler for which a system was not optimized there may be a small loss in product yield or sensitivity and the balance between loci may change slightly in the multiplex systems Meticulous care must be taken to ensure successful amplification A guide to amplification troubleshooting is provided in Section XI Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD006 Revised 7 06 Table 2 Protocol Options for the Model 480 Thermal Cycler Table 3 Protocol Options for the GeneAmp PCR System 9600 Thermal Cycler NA Not applicable 1Recommended protocols offer similar performance characteristics 2Alternative protocols also work but may trade off performance characteristics such as greater speed or convenience for less sensitivity 3The amplification of 25ng or more of K562 DNA using the CTTv system with Amelogenin may result in extra bands
83. tems can be detected using any of the following instruments the Hitachi FMBIO and Hitachi FMBIO II Fluorescence Imaging Systems the ABI PRISM 377 DNA Sequencers the ABI PRISM 310 and 3100 Genetic Analyzers This manual describes methods that we have evaluated and recommend for preparation of sample amplification of sample separation of amplified products and detection of separated material Instructions to operate fluorescence detecting instrumentation should be obtained from the instrument manufacturer The GenePrint Fluorescent STR Multiplex Systems CSF1PO TPOX TH01 vWA CTTv F13A01 FESFPS F13B and LPL FFFL and D16S539 D7S820 D13S317 and D5S818 GammaSTR and all of the GenePrint Fluorescent STR Monoplex Systems are currently quality certified on the Hitachi FMBIO II Fluorescent Imaging System The CTTv and FFFL Multiplex Systems their corresponding monoplex systems HPRTB and the GenePrint Fluorescent Sex Identification System Amelogenin are quality certified for amplification using the Perkin Elmer Model 480 thermal cycler while the GammaSTR Multiplex System and its corresponding monoplex systems are quality certified for amplification using the Perkin Elmer GeneAmp PCR system 9600 thermal cycler All of the GenePrint Fluorescent Systems can be amplified on either the Perkin Elmer Model 480 or GeneAmp PCR system 9600 System thermal cyclers but slight differences in yield or balance between loci migh
84. th buffer to remove any air bubbles on top of the gel Be certain the well area is devoid of air bubbles and small pieces of polyacrylamide Use a syringe with a bent 19 gauge needle to remove air bubbles from the bottom of the gel 8 Pre run the gel to achieve a surface temperature of approximately 50 C Consult the manufacturer s instruction manual for recommended electrophoresis conditions Note As a reference we generally use 40 45 watts for 30 minutes for a 32cm gel or 60 65 watts for 30 minutes for a 43cm gel The gel running conditions may have to be adjusted in order to reach a temperature of 50 C Page 20 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD006 Printed in USA Revised 7 06 Page 21 VII B Sample Preparation Loading and Electrophoresis The Fluorescent Ladder CXR is optional with the Hitachi instruments If the alleles are called visually we recommend running an allelic ladder in every third lane so that each sample is next to a ladder 1 If the Fluorescent Ladder CXR is not used prepare the PCR samples or allelic ladders by combining 2 5 l of Bromophenol Blue Loading Solution with 2 5 l of PCR sample or allelic ladder If the Fluorescent Ladder CXR ladder is used prepare PCR samples and allelic ladders by combining 1 l of Fluorescent Ladder CXR 3 l of Br
85. the samples at 20 C Note Storage of amplified samples at 4 C or above may produce degradation products Page 15 V Polyacrylamide Gel Preparation Gel preparation is dependent on the type of instrument used for fluorescent detection Outlined below are procedures for preparing gels for the Hitachi FMBIO and FMBIO II Fluorescence Imaging Systems and the ABI PRISM 377 DNA Sequencer If you are using a different instrument please refer to the manufacturer s recommendations New glass plates should be soaked in 10 NaOH for 1 hour then rinsed thoroughly with deionized water before use New plates should also be etched with a diamond pencil in the corner of one side to distinguish the sides of the plates in contact with the gel V A Gel Preparation for the Hitachi FMBIO and FMBIO II Fluorescence Imaging Systems There are two size options for gels on the Hitachi FMBIO and FMBIO II Fluorescence Imaging Systems either 32cm 19cm 0 4mm h w thickness or 43cm 19cm 0 4mm The 43cm 19cm 0 4mm low fluorescence glass plates are strongly recommended for better separation The use of the longer glass plates enables the instrument to distinguish one base pair differences and differences in alleles over 300bp more easily If the Hitachi Software STaRCall is used for identifying alleles use square tooth combs to maximize software performance Square tooth combs provide better separation between the
86. uences and or the addition of a final extension step of 60 C for 30 minutes to the amplification protocol can lead to essentially full terminal nucleotide addition 25 Notes for Table 12 1 PCR amplification sometimes generates artifacts that appear as faint bands below the alleles These products probably result from a process known as slippage commonly observed in PCR amplification of regions containing tandem repeats of short sequences 21 22 This characteristic is most pronounced with the vWA locus 2 A strong extra band may be observed below the 212bp Amelogenin allele when more than 25ng of template DNA is amplified 3 Locus F13A01 has a common allele 3 2 It contains 4 copies of the repeat but has a 2 base deletion in the region flanking the repeat 26 27 4 Locus TH01 has a common 9 3 allele 9 A one base deletion is present in the allele that contains 10 repeats Note that reference 9 refers to this allele as 10 1 rather than 9 3 This allele was renamed 9 3 at the ISFH Conference in Venice in October 1993 Page 41 Table 11 Locus Specific Information NA not applicable 1Amelogenin is not an STR but displays a 212 base X specific band and a 218 base Y specific band K562 DNA female displays only the 212 base X specific band 2Repeat sequences represent all four possible permutations e g AGAT is used for AGAT GATA ATAG or TAGA The first alphabetic representation of the repeat e g AGAT is
87. ument and close the instrument doors VIII D Electrophoresis and Detection ABI PRISM 377 DNA Sequencer 1 After loading select Cancel to stop the pre run Select Run to begin electrophoresis 2 Monitor the electrophoresis by observing the gel image and status windows 3 Allow electrophoresis to proceed for 2 25 hours At this point the 400 base fragment will have migrated past the laser 4 Analyze the gel according to the GeneScan analysis software user s manual Note If the signal is too intense a peak height greater than 3 000RFU dilute the samples in 1X STR Buffer before mixing with loading solution or use less DNA template in the amplification reactions ABI PRISM 310 Genetic Analyzer 1 After loading the sample tray and closing the doors select Run to start the capillary electrophoresis system 2 Monitor the electrophoresis by observing the raw data and status windows 3 Each sample will take approximately 30 minutes for syringe pumping sample injection and sample electrophoresis 4 Analyze the data according to the GeneScan analysis software user s manual Note Peak heights outside the linear range of the instrument may generate artifact peaks due to instrument saturation i e overloading the sample Bleedthrough pull ups from one color to another may be observed Saturated signal may also appear as two peaks split peak Promega Corporation 2800 Woods Hollow Road Madi
88. uorescent STR Systems All technical literature is available on the Internet at www promega com tbs Please visit the web site to verify that you are using the most current version of this Technical Manual Please contact Promega Technical Services if you have questions on use of this system E mail genetic promega com Page 2 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD006 Printed in USA Revised 7 06 IX Detection of Amplified Fragments Using the ABI PRISM 3100 Genetic Analyzer and Data Collection Software Version 1 1 28 A Spectral Calibration 29 B Sample Preparation 29 C Instrument Preparation 30 D Sample Detection 31 E Data Analysis 32 X Representative STR Data

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