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1. 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 TMD012 Page 50 Printed in USA Revised 6 13 Symptoms Causes and Comments C Error message The base pair size of alleles in the allelic ladder are outside of A Could not complete the the defined category range Be sure internal lane standard Run Macro command fragments are correctly sized Redefine internal lane standard Promega because the labeled peak fragments and re analyze the sample using GeneScan could not be found continued software Compare the size of the smallest allele in the allelic ladder with the base pair size and range listed in the categories for the same alleles If necessary increase the category start range in the category window to greater than 6bp and save the macro under a new name Allelic ladder peaks were too high causing stutter peaks to be called as allele peaks Use a shorter injection time decrease the amount of allelic ladder used or re analyze the allelic ladder sample using increased peak amplitude thresholds in the GeneScan analysis parameters Allelic ladder data were not compatible with the PowerTyper Macro file used Confirm that the PowerTyper Macro file matches the allelic ladder being used The plots window or allele The macros were not run in the proper order Use the POWER table does not display al
2. shadow peaks that differ in size from one another by approximately the same distance as the single stranded alleles Artifacts of STR amplification Direct amplification of gt 20ng of template can result in a higher number of artifact peaks Use the recommended punch size and number of punches Optimize the cycle number Do not reduce the reaction volume below 25ul See Section 6 H for additional information about stutter and artifacts Excessive amount of DNA Amplification of gt 20ng of template can result in an imbalance with smaller loci showing more product than larger loci e Use one 1 2mm punch from a nonFTA card containing a buccal sample Follow the manufacturer s recommendations when depositing sample onto the storage card e Decrease cycle number cycle Amplification was inhibited when using more than one storage card punch Use only one 1 2mm storage card punch Active PunchSolution Reagent carried over into the amplification reaction Larger loci are most susceptible to carryover and will drop out before smaller loci e Ensure that the heat block was set at 70 C and samples were incubated for 30 minutes Incubation for shorter time periods may result in incomplete inactivation of the PunchSolution Reagent Using a smaller amplification reaction volume may compromise performance when using 10 of PunchSolution Reagent Reducing the PunchSolution Reagent volume may improve results for reactions with re
3. 1998 Development and population study of an eight locus short tandem repeat STR multiplex system J Forensic Sci 43 1168 80 29 Puers C et al 1993 Identification of repeat sequence heterogeneity at the polymorphic STR locus HUMTH01 AATG n and reassignment of alleles in population analysis using a locus specific allelic ladder Am J Hum Genet 53 953 8 30 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 31 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 32 Sprecher C J et al 1996 General approach to analysis of polymorphic short tandem repeat loci BioTechniques 20 266 76 33 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 34 Jones D A 1972 Blood samples Probability of discrimination J Forensic Sct Soc 12 355 9 35 Brenner C and Morris J W 1990 In Proceedings from the International Symposium on Human Identification 1989 Promega Corporation 21 53 36 Mandrekar P V Krenke B E and Tereba A 2001 DNA IQ The intelligent way to purify DNA Profiles in DNA 4 3 16 37 Krenke
4. Meyer E and Brinkmann B 1994 Different types of structural variation in STRs HumFES FPS HumVWA and HumD21S11 Int J Leg Med 106 319 23 Brinkmann B Moller A and Wiegand P 1995 Structure of new mutations in 2 STR systems Int J Leg Med 107 201 3 Griffiths R et al 1998 New reference allelic ladders to improve allelic designation in a multiplex STR system Int J Legal Med 111 267 72 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 TMD012 Page 52 Printed in USA Revised 6 13 24 Bar W et al 1997 DNA recommendations Further report of the DNA Commission of the ISFH ZA regarding the use of short tandem repeat systems Int J Legal Med 110 175 6 o 25 Gill P et al 1997 Considerations from the European DNA Profiling Group EDNAP concerning STR nomenclature Forensic Sci Int 87 185 92 Pro mega 26 Fr geau C J et al 1995 Characterization of human lymphoid cell lines GM9947 and GM9948 as intra and interlaboratory reference standards for DNA typing Genomics 28 184 97 27 Levadokou E N et al 2001 Allele frequencies for fourteen STR loci of the PowerPlex 1 1 and 2 1 multiplex systems and Penta D locus in Caucasians African Americans Hispanics and other populations of the United States of America and Brazil J Forensic Sci 46 736 61 28 Lins A M et al
5. 32 2 30 30 2 3434 239 35 2 36 38 TH01 FL 156 195 4 9 9 3 10 11 13 3 D3S1358 FL 115 147 12 20 FGA TMR 322 444 16 18 18 2 19 19 2 20 20 2 DA DAN 2 DIDS PG DE DD aN 24 2 25 25 2 26 30 31 2 43 2 44 2 45 2 46 2 TPOX TMR 262 290 6 13 D8S1179 TMR 203 247 7 18 vWA TMR 123 171 10 22 Amelogenin TMR 106 112 AY Penta D JOE 376 449 2 2 3 2 5 7 17 CSF1PO JOE 321 357 6 15 D16S539 JOE 264 304 5 8 15 D7S820 JOE 215 247 6 14 D13S317 JOE 176 208 7 15 D5S818 JOE 119 155 7 16 The length of each allele in the allelic ladder has been confirmed by sequence analyses When using an internal lane standard such as the Internal Lane Standard 600 the calculated sizes of allelic ladder components may differ from those listed This occurs because different sequences in allelic ladder and ILS components may cause differences in migration The dye label also affects migration of alleles The alleles listed are those with a frequency of gt 1 1000 For a current list of microvariants see the Variant Allele Report published at the U S National Institute of Standards and Technology NIST web site at www cstl nist gov div831 strbase Amelogenin is not an STR but displays a 106 base X specific band and a 112 base Y specific band 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 TMD012 Printed
6. Occasionally an off ladder artifact can be seen in the 270 271bp position in the JOE dye channel One or more extra peaks that are not directly related to amplification may be observed at positions 8 26 bases smaller than TPOX alleles and 6 21 bases smaller than vWA alleles These extra peaks occur when the amplified peaks are particularly intense high signal intensity or template amount formamide polymer or capillary was of poor quality or denaturation was ineffective See Section 7 for more information about how to minimize these artifacts 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 TMD012 Revised 6 13 Page 39 CQ 6 H Results continued A pP A low level artifact in the D55818 region of the JOE channel may be observed mega between 114 120bp In addition low level artifacts in the TMR channel may be observed at 142 144 and 400 405bp These artifacts are not template derived and may appear in the negative control and in low product yield analyses The peak heights of these artifacts may increase with longer injection time or higher injection voltage 7 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 7 A Amplification and Fragment Detection Sympto
7. Part TMD012 Page 16 Printed in USA Revised 6 13 9 Seal the plate Optional Briefly centrifuge the plate to bring contents to CQ the bottom of the wells and remove any air bubbles va Thermal Cycling Promega Amplification and detection instrumentation may vary You will need to optimize protocols including the amount of template DNA cycle number injection conditions and loading volume for your laboratory instrumentation Testing at Promega shows that 28 cycles works well for a variety of sample types Cycle number will need to be optimized in each laboratory for each sample type that is amplified see below 1 Place the MicroAmp plate in the thermal cycler 2 Select and run the recommended protocol The preferred protocol for use with the GeneAmp PCR System 9700 thermal cycler is provided below Thermal Cycling Protocol 95 C for 11 minutes then 96 C for 1 minute then ramp 100 to 94 C for 30 seconds ramp 29 to 60 C for 30 seconds ramp 23 to 70 C for 45 seconds for 10 cycles then ramp 100 to 90 C for 30 seconds ramp 29 to 60 C for 30 seconds ramp 23 to 70 C for 45 seconds for 18 cycles then 60 C for 30 minutes 4 C soak When using the GeneAmp PCR System 9700 thermal cycler the ramp rates indicated in the cycling program must be set and the program must be run in 9600 ramp mode The ramp rates are set in the Ramp Rate Modification screen While viewing the cycling program navigate
8. Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD012 Revised 6 13 Page 1 72 0 C E ER ane eet one eRe MICE eee RCT ere re ener meer EAE N EA eee re 54 Advantages of Using the Loci in the PowerPlex 16 System 54 B Poweror Dica minoon esea E E 58 C DNA Extraction and Quantitation Methods and Automation Suppott 59 D dhelnernal Lane nrandard 0U meee neem ntere mene tees meee tee teetennene re rere 60 E Composition of Buffers and Solutions ssssssssssssessssssrrsessssssrrreesssssssreeeesssssrreessssses 60 E E OCCU E E A A rae aoe eteis enemies 61 Description STR short tandem repeat loci consist of short repetitive sequence elements 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 which may be detected using the polymerase chain reaction 5 8 Alleles of STR loci are differentiated by the number of copies of the repeat sequence contained within the amplified region and are distinguished from one another using fluorescence detection following electrophoretic separation The PowerPlex 16 Systeme 9 10 is used for human identification applications including forensic analysis relationship testing and research use The system allows co amplification and three color detection of sixteen loci fifteen STR loci and Amelogenin including Penta E D18551 D21S11 TH01 D3S1358 FGA T
9. Category 1 2 3 4 flow Signal tion Label Row Allele Table Format Sample Category Category Category Category Category Category Category Category Info Allele 1 Allele2 Allele1 Allele2 jAllele1 Allele2 jAllele1 Allele 2 CODIS Table Format C 11 Save the analyzed data Go to the File menu and select Save as The PowerTyper Macro is a Genotyper file and can be overwritten if Save is used instead of Save as 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 TMD012 Revised 6 13 Page 37 C 6 G Controls o pP 1 Observe the results for the negative control Using the protocols defined in mega this manual the negative control should be devoid of amplification products 2 Observe the results for the 2800M Control DNA Compare the 2800M Control DNA allelic repeat sizes with the locus specific allelic ladder The expected 2800M Control DNA allele designations for each locus are listed in Table 6 Section 9 A 6 H Results Representative results of the PowerPlex 16 System are shown in Figure 9 The PowerPlex 16 Allelic Ladder Mix is shown in Figure 10 A DIST m o B D55818 _ pusr Imsa D135317_ DTS820 pisses __ CSFIPO Penta Dp _____ z 400 C WA 100 200 300 400 500 Figure 9 The PowerPlex 16 System A single source templat
10. DNA typing with trimeric and tetrameric tandem repeats Polymorphic loci detection systems and population genetics In The Second International Symposium on Human Identification 1991 Promega Corporation 31 52 Edwards A et al 1991 DNA typing and genetic mapping with trimeric and tetrameric tandem repeats Am J Hum Genet 49 746 56 Edwards A et al 1992 Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups Genomics 12 241 53 Warne D et al 1991 Tetranucleotide repeat polymorphism at the human actin related pseudogene 2 actbp2 detected using the polymerase chain reaction Nucleic Acids Res 19 6980 Ausubel F M et al 1996 Unit 15 The polymerase chain reaction In Current Protocols in Molecular Biology Vol 2 John Wiley and Sons NY 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 Press Cold Spring Harbor New York PCR Technology Principles and Applications for DNA Amplification 1989 Erlich H A ed Stockton Press New York NY PCR Protocols A Guide to Methods and Applications 1990 Innis M A et al eds Academic Press San Diego CA Krenke B et al 2002 Validation of a 16 locus fluorescent multiplex system J Forensic Sci 47 773 85 Budowle B et al 2001 STR primer
11. Fax 608 277 2516 www promega com Part TMD012 Page 6 Printed in USA Revised 6 13 4 A Amplification of Extracted DNA C A Materials to Be Supplied by the User e model 480 or GeneAmp PCR System 9600 9700 or 2400 thermal cycler Promega Applied Biosystems e microcentrifuge e MicroAmp optical 96 well reaction plate or 0 5ml GeneAmp or 0 2ml MicroAmp reaction tubes Applied Biosystems aerosol resistant pipette tips see Section 9 F e AmpliTaq Gold DNA polymerase Applied Biosystems e Nuclease Free Water Cat P1193 Mineral Oil Cat DY1151 for use with the model 480 thermal cycler We routinely amplify 0 5 1ng of template DNA in a 25 l reaction volume using the protocols detailed below Developmental validation of the kit showed routine generation of full profiles with lower amounts of DNA template down to 125pg 14 Partial profiles were typically observed for DNA template of 62p and below Expect to see higher peak heights at the smaller loci and lower peak heights at the larger loci if more than the recommended amount of template is used Reduce the amount of template DNA or number of cycles to correct this We recommend that you perform optimization and validation of the kit to establish its performance in your laboratory Store DNA to be used for sensitivity studies at 4 C overnight before use Amplification Setup 1 Thaw the Gold ST R 10X Buffer and PowerPlex 16 10X Primer Pair Mix completely
12. Fragments Using the ABI PRISM 310 Genetic Analyzer continued 7 After loading the sample tray and closing the doors select Run to start the capillary electrophoresis system 8 Monitor electrophoresis by observing the raw data and status windows Each sample will take approximately 40 minutes for syringe pumping sample injection and sample electrophoresis Data Analysis 6 A Importing PowerPlex Panels and Bins Text Files into GeneMapper ID Version 3 2 To facilitate analysis of data generated with the PowerPlex 16 System we have created panels and bins text files to allow automatic assignment of genotypes using GeneMapper ID software version 3 2 We recommend that users of GeneMapper ID software version 3 2 complete the Applied Biosystems GeneMapper ID Software Human Identification Analysis Tutorial to familiarize themselves with proper operation of the software For GeneMapper ID software version 3 1 users we recommend upgrading to version 3 2 Getting Started 1 To obtain the panels and bins text files for use with the PowerPlex 16 System go to www promega com resources tools genemapper id software panels and bin sets 2 Select the PowerPlex System that you are using and select GeneMapper ID Enter your contact information and select Submit 3 Save the Promega_Panels_ID3 2 X txt and Promega_Bins_ID3 2 X txt files where X refers to the most recent version of the panels
13. GATA D75820 JOE 7qi1 21 22 NA GATA D135317 JOE 13q22 q31 NA TATC D5S818 JOE 9q23 3 32 NA AGAT The August 1997 report 24 25 of the DNA Commission of the International Society for Forensic Haemogenetics ISFH states 1 for STR loci within coding genes the coding strand shall be used and the repeat sequence motif defined using the first possible 5 nucleotide of a repeat motif and 2 for STR loci not associated with a coding gene the first database entry or original literature description shall be used Amelogenin is not an STR but displays a 106 base X specific band and a 112 base Y specific band TMR carboxy tetramethylrhodamine FL fluorescein JOE 6 carboxy 47 5 dichloro 27 7 dimethoxyfluorescein NA not applicable 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 TMD012 Revised 6 13 Page 55 Promega 9 A Advantages of Using the Loci in the PowerPlex 16 System continued Table 5 The PowerPlex 16 System Allelic Ladder Information Size Range of Allelic Ladder Components Repeat Numbers of Repeat Numbers of Allelic Alleles Not Present STR Locus Label bases Ladder Components in Allelic Ladder Penta E FL 379 474 5 24 20 3 D18S51 FL 290 366 8 10 10 2 11 13 13 2 14 27 D215 FL 203 259 24 24 2 25 25 2 26 28 28 2 29 29 2 30 90 2 Oly 2 L202
14. 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 TMD012 Printed in USA Page 62 Revised 6 13 ART Aerosol Resistant Tips CQ A Product Volume Size tips pack Cat ART 10 Ultramicro Pipet Tip 0 5 104 960 DY1051 Promega ART 20E Ultramicro Pipet Tip 0 5 10ul 960 DY1061 ART 20P Pipet Tip 20ul 960 DY1071 ART GEL Gel Loading Pipet Tip 100ul 960 DY1081 ART 100 Pipet Tip 100u1 960 DY1101 ART 100E Pipet Tip 100u1 960 DY1111 ART 200 Pipet Tip 200u1 960 DY1121 ART 1000E Pipet Tip 1 000p1 800 DY1131 STR loci are the subject of U S Pat No RE 37 984 German Pat No DE 38 34 636 C2 and other patents issued to the Max Planck Gesellschaft zur Forderung der Wissenschaften e V Germany U S Pat No 6 238 863 Chinese Pat No ZL99802696 4 European Pat No 1058727 Japanese Pat No 4494630 and other patents pending U S Pat Nos 5 843 660 6 479 235 6 221 598 and 7 008 771 Australian Pat No 724531 Canadian Pat Nos 2 118 048 and 2 251 793 Korean Pat No 290332 Singapore Pat No 57050 Japanese Pat Nos 3602142 and 4034293 Chinese Pat Nos ZL99813729 4 and ZL97194967 0 European Pat No 0960207 and other patents pending The purchase of this product does not convey a license to use AmpliTaq Gold DNA polymerase You should purchase AmpliTag Gold DNA polymerase licensed for the forensic and human identity field directly from
15. Ladder Mix and Internal Lane Standard 600 Always include a negative control reaction i e no template to detect reagent contamination We highly recommend the use of gloves and aerosol resistant pipette tips e g ART tips Section 9 F 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 TMD012 Revised 6 13 Page 5 Promega 3 B Matrix Standardization or Spectral Calibration Proper generation of a matrix file is critical to evaluate multicolor systems with the ABI PRISM 310 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130x Genetic Analyzers A matrix must be generated for each individual instrument The PowerPlex Matrix Standards 310 Cat DG4640 is required for matrix standardization for the ABI PRISM 310 Genetic Analyzer The PowerPlex Matrix Standards 3100 3130 Cat DG4650 cannot be used to generate a matrix on the ABI PRISM 310 Genetic Analyzer The PowerPlex Matrix Standards 3100 3130 Cat DG4650 is required for spectral calibration on the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130x Genetic Analyzers The PowerPlex Matrix Standards 310 Cat DG4640 cannot be used to generate a matrix on these instruments For protocols and additional information about matrix generation and spectral
16. PRISM 310 Genetic Analyzer Analyzer with Data Collection Section 5 C Software Version 1 0 1 or 1 1 Section 5 B Data Analysis Section 6 GeneMapper D Software GeneScan Software and Version 3 2 Windows Operating Systems Figure 1 An overview of the PowerPlex 16 System protocol 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 TMD012 Revised 6 13 Page 3 CQ 2 Product Components and Storage Conditions A pP m Product Size Cat ega PowerPlex 16 System 100 reactions DC6531 Not For Medical Diagnostic Use Cat DC6531 contains sufficient reagents for 100 reactions of 25ul each Includes Pre amplification Components Box 1 x 300ul Gold ST R 10X Buffer 1 x 250ul PowerPlex 16 10X Primer Pair Mix 25ul 2800M Control DNA 10ng ul Post amplification Components Box 1 x 50ul PowerPlex 16 Allelic Ladder Mix 1x 1504 Internal Lane Standard ILS 600 Product Size Cat PowerPlex 16 System 400 reactions DC6530 Not For Medical Diagnostic Use Cat DC6530 contains sufficient reagents for 400 reactions of 25ul each Includes Pre amplification Components Box 4 x 300ul Gold ST R 10X Buffer 4 x 250ul PowerPlex 16 10X Primer Pair Mix 25ul 2800M Control DNA 10ng tl Post amplification Components Box 4 x 50ul PowerPlex 16 Allelic Ladder Mix 4x150ul Internal Lane Standard ILS 600 Th
17. Revised 6 13 Page 57 Promega 9 B Power of Discrimination The fifteen STR loci amplified with the PowerPlex 16 System provide powerful discrimination Population statistics for these loci and their various multiplex combinations are displayed in Table 7 These data were generated as part of a collaboration 27 with The Bode Technology Group Springfield VA North Carolina Bureau of Investigation Raleigh NC Palm Beach County Sheriff s Office West Palm Beach FL Virginia Division of Forensic Science Richmond VA and Charlotte Mecklenburg Police Department Laboratory NC Data generation included analysis of over 200 individuals from African American Caucasian American and Hispanic American populations Data for Asian Americans include analysis of more than 150 individuals For additional population data for STR loci see references 28 33 and the Short Tandem Repeat DNA Internet DataBase at www cstl nist gov div831 strbase Table 7 shows the matching probability 34 for the PowerPlex 16 System in various populations The matching probability ranges from 1 in 1 83 x 1017 for Caucasian Americans to 1 in 1 41 x 10 8 for African Americans A measure of discrimination often used in paternity analyses is the paternity index PI a means for presenting the genetic odds in favor of paternity given the genotypes for the mother child and alleged father 35 The typical paternity indices for the PowerPlex 16 System are shown
18. Take punches from a different portion of the card Increasing cycle number also can improve low peak heights Too much sample in the reaction Use one 1 2mm nonFTA storage card punch Follow the manufacturer s recommendations when depositing sample onto the storage card Blood card punches used We do not recommend analysis of blood card punches Make sure that the PCR amplification mix contained AmpSolution Reagent Omission of AmpSolution Reagent from amplification reactions will result in amplification failure Active PunchSolution Reagent carried over into the amplification reaction when using nonFTA card punches Ensure that the heat block was set at 70 C and samples were incubated for 30 minutes until dry Incubation for shorter time periods may result in incomplete inactivation of the PunchSolution Reagent We have not tested longer incubation times Inactive PunchSolution Reagent Thaw the PunchSolution Reagent at 2 10 C Do not store reagents in the refrigerator door where the temperature can fluctuate Do not refreeze as this may reduce activity Faint or absent peaks for the If the positive control reaction failed to amplify check to positive control reaction make sure that the correct amount of 2800M Control DNA was added to the reaction Optimize the amount of 2800M Control DNA for your thermal cycling conditions and laboratory preferences Promega Corporation 2800 Woods Hollow Road Madison WI 53711 53
19. USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD012 Revised 6 13 Page 61 CQ 9 F Related Products continued A Accessory Components Product Size Cat PowerPlex Matrix Standards 310 50ul each dye DG4640 PowerPlex Matrix Standards 3100 3130 25ul each dye DG4650 PunchSolution Kit 100 preparations DC9271 SwabSolution Kit 100 preparations DC8271 Internal Lane Standard 600 150ul DG1071 2800M Control DNA 10ng ul 25pl DD7101 2800M Control DNA 0 25ng ul 500ul DD7251 Water Amplification Grade 5 x 1 250u1 DW0991 Gold ST R 10X Buffer 1 2ml DM2411 Mineral Oil 12ml DY1151 Not for Medical Diagnostic Use Sample Preparation and DNA Quantitation Systems Product Size Cat DNA IQ System 100 reactions DC6701 400 reactions DC6700 Differex System 50 samples DC6801 200 samples DC6800 Maxwell 16 Forensic Instrument each AS3060 DNA IQ Reference Sample Kit for Maxwell 16 48 preps AS1040 DNA IQ Casework Pro Kit for Maxwell 16 48 preps AS1240 Plexor HY System 800 reactions DC1000 200 reactions DC1001 Slicprep 96 Device 10 pack V1391 Not for Medical Diagnostic Use For Research Use Only Not for use in diagnostic procedures Polyacrylamide Gel Electrophoresis Reagents Product Size Cat Ammonium Persulfate 25 V3131 TBE Buffer 10X 1L V4251 Urea 1kg V3171 Blue Dextran Loading Solution 3ml DV4351 Promega Corporation 2800 Woods
20. When using an internal lane standard the calculated lengths of allelic ladder components might differ from those listed in the table This is due to differences in migration resulting from sequence differences between the allelic ladder fragments and internal size standard and is not a matter of concern Double click on the Allelic Ladders macro A plots window will open to display the blue fluorescein dye allelic ladders i e Penta E D18551 D21S11 TH01 and D351358 green JOE dye allelic ladders i e Penta E CSF1PO D165539 D75820 D13S317 and D55818 and yellow TMR dye allelic ladders i e FGA TPOX D851179 vWA and Amelogenin Confirm that the correct allele designations were assigned to the allelic ladders Figure 10 in Section 6 H The software uses one ladder sample to determine allele sizes The macro uses the first ladder sample imported for allele designations If the POWER macro is run a second time the software will use the second ladder if the POWER macro is run a third time the software will use the third ladder etc until all ladders in the project are used If an allelic ladder fails to be analyzed or if many off ladder alleles are found in the samples samples should be re analyzed using another ladder from the project 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 TMD012 Page 36 Pr
21. and bins text files to a known location on your computer Importing Panels and Bins Text Files These instructions loosely follow the Applied Biosystems GeneMapper ID software tutorial pages 1 4 Open the GeneMapper ID software version 3 2 Select Tools then Panel Manager Highlight the Panel Manager icon in the upper left navigation pane Select File then Import Panels a pe ae Pa m Navigate to the panels text file downloaded in the Getting Started section above Select the file then Import 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 TMD012 Page 26 Printed in USA Revised 6 13 6 Inthe navigation pane highlight the Promega Panels ID3 2 X folder that C you just imported in Step 5 I 7 Select File then Import Bin Set Promega 8 Navigate to the bins text file downloaded in the Getting Started section above Select the file then Import 9 At the bottom of the Panel Manager window select OK The Panel Manager window will close automatically 6 B Creating a Size Standard with GeneMapper ID Software Version 3 2 Select Tools then GeneMapper Manager Select the Size Standard tab Select New amp iS Select Basic or Advanced Figure 3 The type of analysis method selected must match the type of
22. calibration see the PowerPlex Matrix Standards 310 Technical Bulletin TBD021 For protocols and additional information about spectral calibration see the PowerPlex Matrix Standards 3100 3130 Technical Bulletin TBD022 These manuals are available online at www promega com protocols Protocols for DNA Amplification Using the PowerPlex 16 System The PowerPlex 16 System is optimized for the GeneAmp PCR System 9700 thermal cycler Amplification protocols for the GeneAmp PCR Systems 9600 and 2400 thermal cyclers and Perkin Elmer model 480 thermal cycler are provided The use of gloves and aerosol resistant pipette tips is highly recommended to prevent cross contamination Keep all pre amplification and post amplification reagents in separate rooms Prepare amplification reactions in a room dedicated for reaction setup Use equipment and supplies dedicated for amplification setup Meticulous care must be taken to ensure successful amplification A guide to amplification troubleshooting is provided in Section 7 The concentration of 2800M Control DNA was determined by measuring absorbance at 260nm Quantification of this control DNA by other methods such as gPCR may result in a different value Prepare a fresh DNA dilution for each set of amplifications Do not store diluted DNA e g 0 25ng tl or less Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330
23. concordance study Forensic Sct Int 124 47 54 Presley L A et al 1992 The implementation of the polymerase chain reaction PCR HLA DQ alpha typing by the FBI laboratory In The Third International Symposium on Human Identification 1992 Promega Corporation 245 69 Hartmann J M et al 1991 Guidelines for a quality assurance program for DNA analysis Crime Laboratory Digest 18 44 75 Internal Validation of STR Systems Reference Manual GE053 Promega Corporation Krenke B et al 2002 Validation of a 16 locus fluorescent multiplex system J Forensic Sci 47 773 85 Kline M C et al 2005 Results from the NIST 2004 DNA quantitation study J Forensic Sci 50 570 8 Levinson G and Gutman G A 1987 Slipped strand mispairing A major mechanism for DNA sequence evolution Mol Biol Evol 4 203 21 Schlotterer C and Tautz D 1992 Slippage synthesis of simple sequence DNA Nucleic Acids Res 20 211 5 Smith J R et al 1995 Approach to genotyping errors caused by nontemplated nucleotide addition by Tag DNA polymerase Genome Res 5 312 7 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 Walsh P S Fildes N J and Reynolds R 1996 Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vWA Nucleic Acids Res 24 2807 12 Moller A
24. handling this substance Always wear gloves and safety glasses when working with formamide Sample Preparation 1 Thaw the Internal Lane Standard 600 Note Centrifuge tube briefly to bring contents to the bottom then vortex for 15 seconds before each use Do not centrifuge after vortexing as this may cause the size standard to be concentrated at the bottom of the tube 2 Prepare a loading cocktail by combining Internal Lane Standard 600 ILS 600 and Hi Di formamide as follows 1 Ou1 ILS 600 x samples 24 0u1 Hi Di formamide x samples Note The volume of internal lane standard used in the loading cocktail can be increased or decreased to adjust the intensity of the size standard peaks The optimal peak height for the 100 base fragment of the internal lane standard is 500 1 000RFU If peak heights are too high we recommend altering the loading cocktail to contain 0 5ul of ILS 600 and 24 5ul of Hi Di formamide 3 Vortex for 10 15 seconds to mix 4 Combine 25 0ul of prepared loading cocktail and 1 0u1 of amplified sample or 1ul of PowerPlex 16 Allelic Ladder Mix Note Instrument detection limits vary therefore injection time injection voltage or the amount of product mixed with loading cocktail may need to be increased or decreased If peak heights are higher than desired samples can be diluted in Gold ST R 1X Buffer before mixing with loading cocktail This may result in uneven allele peak heig
25. in USA Page 56 Revised 6 13 Table 6 The PowerPlex 16 System Allele Determinations in Commonly Available Standard C DNA Templates Standard DNA Templates Promega STR Locus K5622 9947A 99483 2800M Penta E 5 14 12 15 1 7 14 D18551 lay NE 15 19 l5 the 16 18 D21511 29 30 31 30 30 29 30 29 Ohad TH01 9395 8I 099 Gn D3S1358 16 16 14 15 15 17 17 18 FGA 21 24 23 24 24 26 2023 TPOX 8 9 8 8 8 9 11 11 D8S1179 2 Di is 14 15 vWA 16 16 17 18 17 47 16 19 Amelogenin DIS X X X Y X Penta D 9 13 22 8 12 I CSF1PO 90 10 12 10 11 12 o D16S539 11 12 11 12 dd Ad 9 10 D7S820 I 10 11 Dh al 8 11 D138317 8 8 ila 11 11 ae il D5S818 O ah m PiL Information on strains 9947A and 9948 is available online at http ccr coriell org Sections Search Sample_Detail aspx Ref GM09947 and http ccr coriell org Sections Search Sample_Detail aspx Ref GM09948 Strain K562 is available from the American Type Culture Collection www atcc org Manassas VA Information about the use of 9947A and 9948 DNA as standard DNA templates can be found in reference 26 Strain K562 displays three alleles at the D21S11 locus Strain 9948 displays three alleles at the CSF1PO locus The peak height for allele 12 is much lower than those for alleles 10 and 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 TMD012
26. program must be run in 9600 ramp mode The ramp rates are set in the Ramp Rate Modification screen While viewing the cycling program navigate to the Ramp Rate Modification screen by selecting More then Modify On the Ramp Rate Modification screen the default rates for each step are 100 The rate under each hold step is the rate at which the temperature will change to that hold temperature Figure 2 shows the ramp rates for the GeneAmp PCR System 9700 thermal cycler The ramp mode is set after start has been selected for the thermal cycling run A Select Method Options screen appears Select 9600 ramp mode and enter the reaction volume l l l l l i WEG l l l l l l l 7486MA Figure 2 The ramp rates for the GeneAmp PCR System 9700 thermal cycler 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 TMD012 Page 10 Printed in USA Revised 6 13 4 B Direct Amplification of DNA from nonFTA Storage Card Punches C A Materials to Be Supplied by the User e GeneAmp PCR System 9700 thermal cycler Applied Biosystems Pi omega e microcentrifuge e MicroAmp optical 96 well reaction plate Applied Biosystems aerosol resistant pipette tips see Section 9 F e AmpliTag Gold DNA polymerase Applied Biosystems e Nuclease Free Water Cat P1193 e PunchSolution Kit Cat DC9271
27. to instrument saturation i e overloading the sample Bleedthrough pull ups from one color to another may be observed Saturated signal also may appear as two peaks split peak 2 If peak heights are not within the linear range of detection of the instrument the ratio of stutter peaks to real allele peaks increases and allele designations become difficult to interpret The balance of peak heights also may appear less uniform 3 There can be variation between instruments regarding the relative fluorescence levels detected using the same sample Furthermore different instruments vary in the relative efficiency of color detection affecting the dye color to dye color balance 6 F Sample Analysis Using the Genotyper Software and PowerTyper 16 Macro To facilitate analysis of data generated with the PowerPlex 16 System we have created a file to allow automatic assignment of genotypes using the Genotyper software After samples are amplified detected using the ABI PRISM 310 or 3100 Genetic Analyzer using Data Collection Software Version 1 0 1 or 1 1 and analyzed using the GeneScan software sample files can be imported into the Genotyper program and analyzed using the PowerTyper 16 Macro Release 2 0 The PowerTyper 16 Macro Release 2 0 can be downloaded from the Promega web site at www promega com resources tools powertyper macros The PowerTyper 16 Macro Release 2 0 is used in conjunction with Windo
28. to the Ramp Rate Modification screen by selecting More then Modify On the Ramp Rate Modification screen the default rates for each step are 100 The rate under each hold step is the rate at which the temperature will change to that hold temperature Figure 2 shows the ramp rates for the GeneAmp PCR System 9700 thermal cycler The ramp mode is set after start has been selected for the thermal cycling run A Select Method Options screen appears Select 9600 ramp mode and enter the reaction volume 3 After completion of the thermal cycling protocol store amplified samples at 20 C in a light protected box Note Long term storage of amplified samples at 4 C or higher may produce artifacts 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 TMD012 Revised 6 13 Page 17 Promega 5 4 C Direct Amplification of DNA from Swabs continued PCR Optimization Cycle number should be optimized based on the results of an initial experiment to determine the sensitivity with your collection method sample types and instrumentation 1 Choose several samples that represent typical sample types you encounter in the laboratory Prepare them as you would using your normal workflow 2 Prepare three identical reaction plates with aliquots of the same swab extracts 3 Amplify sampl
29. your authorized enzyme supplier Allele sequences for one or more of the loci vWA FGA D8S1179 D21S11 and D18551 in allelic ladder mixtures is licensed under U S Pat Nos 7 087 380 7 645 580 Australia Pat No 2003200444 and corresponding patent claims outside the US 2000 2013 Promega Corporation All Rights Reserved Maxwell Plexor and PowerPlex are registered trademarks of Promega Corporation AmpSolution Differex DNA IQ PowerTyper PunchSolution Slicprep and SwabSolution are trademarks of Promega Corporation ABI PRISM Applied Biosystems GeneAmp GeneMapper and MicroAmp are registered trademarks of Applied Biosystems AmpliTag Gold is a registered trademark of Roche Molecular Systems Inc ART is a registered trademark of Molecular Bio Products Inc Bode Buccal DNA Collector is a trademark of the Bode Technology Group Inc Excel Microsoft Windows and Windows NT are registered trademarks of Microsoft Corporation 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 GeneScan and Genotyper are registered trademarks of Applera Corporation Hi Di is a trademark of Applera Corporation Liqui Nox is a registered trademark of Alconox Inc Long Ranger and Long Ranger Singel are registered trademarks of Cambrex Corporation Nalgene is a registered trademark of Nalge Nunc International POP 4 is a registered tr
30. 0 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 or the Applied Biosystems 3130 or 3130x Genetic Analyzer with Data Collection Software Version 3 0 continued Instrument Preparation Refer to the instrument users manual for instructions on cleaning installing the capillary array performing a spatial calibration and adding polymer Analyze samples as described in the user s manual for the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 and the Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 with the following exceptions k In the Module Manager select New Select Regular in the Type drop down list and select HIDFragmentAnalysis36_POP4 in the Template drop down list Confirm that the injection time is 5 seconds and the injection voltage is 3kV Lengthen the run time to 2 000 seconds Give a descriptive name to your run module and select OK Note Instrument sensitivities can vary The injection time and voltage may be adjusted in the Module Manager A suggested range for the injection time is 3 22 seconds and for the injection voltage is 1 3kV In the Protocol Manager select New Type a name for your protocol Select Regular in the Type drop down list and select the run module you created in the previous step in the Run Module drop down list Lastly select F in the
31. 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD012 Printed in USA Page 60 Revised 6 13 TE buffer 10mM Tris HCl TE buffer with 20ug ml glycogen C A 0 1mM EDTA pH 8 0 1 21g Tris base 1 21g Tris base 0 037g EDTA Promega 0 037g EDTA Na EDTA 2H 0 Na EDTA 2H 0 20ug ml glycogen Dissolve Tris base and EDTA in Dissolve Tris base and EDTA in 900ml of deionized water Adjust to 900ml of deionized water Adjust to pH 8 0 with HCI Bring the final pH 8 0 with HCI Add glycogen volume to 1 liter with deionized Bring the final volume to 1 liter with water deionized water 9 F Related Products STR Systems Product Size Cat PowerPlex 16 Monoplex System Penta E Fluorescein 100 reactions DC6591 PowerPlex 16 Monoplex System Penta D JOE 100 reactions DC6651 PowerPlex Fusion System 200 reactions DC2402 800 reactions DC2408 PowerPlex 21 System 200 reactions DC8902 PowerPlex 16 HS System 100 reactions DC2101 400 reactions DC2100 PowerPlex ESX 17 Fast System 100 reactions DC1711 400 reactions DC1710 PowerPlex ESI 17 Fast System 100 reactions DC1721 400 reactions DC1720 PowerPlex ESX 16 System 100 reactions DC6711 400 reactions DC6710 PowerPlex ESI 16 System 100 reactions DC6771 400 reactions DC6770 PowerPlex Y23 System 50 reactions DC2305 200 reactions DC2320 Not for Medical Diagnostic Use Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in
32. 99 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD012 Revised 6 13 Page 43 7 B Direct Amplification of DNA from nonFTA Storage Card Punches continued Symptoms Extra peaks visible in one or or all color channels Peak height imbalance Causes and Comments Punch was contaminated Take punches from blank paper samples and include a reaction with one blank punch as a negative control Amplification of processed punches with high amounts of DNA can result in artifact peaks due to overamplification resulting in saturating signal on the CE instrument We recommend one 1 2mm punch per 25 reaction Use of a larger punch size or a smaller reaction volume may result in overamplification and signal saturation If the signal is saturated repeat the amplification with a smaller punch a larger reaction volume or reduced cycle number Amplification of excess template for a given cycle number can result in overloading of the capillary upon electrokinetic injection The presence of excess DNA in the capillary makes it difficult to maintain DNA in a denatured single stranded state Some single stranded DNA renatures and becomes double stranded Double stranded DNA migrates faster than single stranded DNA during capillary electrophoresis and appears as shadow peaks migrating in front of the main peaks If this occurs at a heterozygous locus it is sometimes possible to see two
33. ABI PRISM 310 Genetic Analyzer ensure that the appropriate matrix file is selected in the Matrix column 9 Select Analyze green arrow button to start the data analysis 6 E Sample Analysis Using the GeneScan Software and Windows Operating Systems 1 Analyze data using the GeneScan software 2 Review the raw data for one or more sample runs Highlight the sample file name then in the Sample menu select raw data Move the cursor so that 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 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 TMD012 Revised 6 13 Page 33 Promega 6 E Sample Analysis Using the GeneScan Software and Windows Operating Systems continued 3 The recommended analysis parameters are shown in Figure 8 di PowerPlex 16 Analysis gsp x Analysis Range C Full Range This Range Data Points Start 200 Stop fi ggg Data Processing Smooth Options C None f Light C Heavy Feak Detection Feak Amplitude Thresholds Min Feak Half vwidth Polynomial Degree Peak Window Size Slope Threshold tor Peak St
34. Amp plate for reaction assembly and label appropriately Add the final volume of each reagent listed in Table 2 to a sterile tube Table 2 PCR Amplification Mix for Direct Amplification of DNA from Storage Card Punches Volume Number of _ Final PCR Amplification Mix Component Per Reaction Reactions Volume nuclease free water 14 2ul x Gold ST R 10X Buffer 2 5ul x PowerPlex 16 10X Primer Pair Mix 2 5 x AmpliTaq Gold DNA polymerase 0 8ul 4u x 5X AmpSolution Reagent 5 0ul x total reaction volume 251 1A dd nuclease free water to the tube first then add Gold ST R 10X Buffer PowerPlex 16 10X Primer Pair Mix AmpliTag Gold DNA polymerase and 5X AmpSolution Reagent The template DNA will be added at Step 6 3 Vortex the PCR amplification mix for 5 10 seconds Failure to vortex the PCR amplification mix sufficiently can result in poor amplification or locus to locus imbalance 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 TMD012 Page 12 Printed in USA Revised 6 13 6 Pipet 25ul of PCR amplification mix into each reaction well with one C pretreated 1 2mm punch from a nonFTA storage card containing a buccal I sample Pipet 25ul of PCR amplification mix into each reaction well for the Promega control reactions 7 For the positive amplification control vortex the tube of 2800
35. B E et al 2005 Development of a novel fluorescent two primer approach to quantitative PCR Profiles in DNA 8 1 3 5 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 TMD012 Revised 6 13 Page 53 Promega 9 Appendix 9 A Advantages of Using the Loci in the PowerPlex 16 System The loci included in the PowerPlex 16 System Tables 4 and 5 were selected because they satisfy the needs of several major standardization bodies throughout the world For example the United States Federal Bureau of Investigation FBI has selected 13 STR core loci for typing prior to searching or including submitting samples in CODIS Combined DNA Index System the U S national database of convicted offender profiles The PowerPlex 16 System amplifies all CODIS core loci in a single reaction The PowerPlex 16 System also contains two low stutter highly polymorphic pentanucleotide repeat loci Penta E and Penta D These additional loci add significantly to the discrimination power of the system making the PowerPlex 16 System a single amplification system with a power of exclusion sufficient to resolve paternity disputes definitively In addition the extremely low level of stutter seen with Penta E and Penta D makes them ideal loci to evaluate DNA mixtures often encountered in forensic casework Finally t
36. Calibrate the thermal cycler heating block if necessary Primer concentration was too low Use the recommended primer concentration Vortex the 10X PowerPlex 16 Primer Pair for 15 seconds before 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 Part TMD012 Printed in USA Page 40 Revised 6 13 Symptoms Causes and Comments C Faint or absent allele peaks Samples were not denatured completely Heat denature A continued samples for the recommended time then cool on crushed ice or in an ice water bath immediately prior to electrophoresis hi omega Do not cool samples in a thermal cycler set at 4 C as this may lead to artifacts due to DNA re annealing Poor capillary electrophoresis injection ILS 600 peaks also affected Re inject the sample Check the instrument syringe pump system for leakage Poor capillary electrophoresis injection ILS 600 peaks also affected Check the laser power Poor quality formamide was used Use only Hi Di formamide when analyzing samples Extra peaks visible in one Contamination with another template DNA or previously or all color channels amplified DNA Cross contamination can be a problem Use aerosol resistant pipette tips and change gloves regularly Samples were not completely denatured Heat denature samples for the recommended time and cool on crushed ice or in an ice water ba
37. Dye Set drop down list Select OK In the Plate Manager create a new plate record as described in the instrument user s manual In the dialog box that appears select GeneMapper Generic in the Application drop down list and select the appropriate plate type 96 well Add entries in the owner and operator windows and select OK Note If autoanalysis of sample data is desired refer to the instrument user s manual for instructions In the GeneMapper plate record enter sample names in the appropriate cells Scroll to the right In the Results Group 1 column select the desired results group In the Instrument Protocol 1 column select the protocol you created in Step 2 Be sure this information is present for each row that contains a sample name Select OK Note To create a new results group select New in the drop down menu in the Results Group column Select the General tab and enter a name Select the Analysis tab and select GeneMapper Generic in the Analysis type drop down list Place samples in the instrument and close the instrument doors In the spectral viewer confirm that dye set F is active and set the correct active calibration for dye set F 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 TMD012 Page 20 Printed in USA Revised 6 13 7 Inthe run schedul
38. He awyr Baseline Wiin dor f1 pts Size Calling Method Polynomial Degree Peak Window Size Slope Threshold Peak Start Peak End 2nd Order Least Squares 3rd Order Least Squares Cubic Spline Interpolation f Local Southern Method f Global Southern Method 5723TA Figure 6 The Peak Detector tab 11 Select the Peak Quality tab You may change the settings for peak quality Note For Steps 11 and 12 see the GeneMapper ID user s manual for more information 12 Select the Quality Flags tab You may change these settings 13 Select OK to save your settings 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 TMD012 Printed in USA Page 30 Revised 6 13 Processing Data for Casework Samples C A 1 Select File then New Project 2 Select Edit then Add Samples to Project 3 Browse to the location of the run files Highlight desired files then select Add to list followed by Add 4 In the Sample Type column use the drop down menu to select Ladder Sample Positive Control or Negative Control as appropriate for the sample Every folder in the project must contain at least one allelic ladder that is designated as such for proper genotyping 5 Inthe Analysis Method column select the analysis method created previou
39. M 3100 Genetic Analyzer with Data Collection Software Version 1 0 1 or 1 1 21 C Detection of Amplified Fragments Using the ABI PRISM 310 Oe E A ce eect T sete ere eee 24 o Da aS ee E me ete 26 A Importing PowerPlex Panels and Bins Text Files into Gene Mapper I VIO eronneen T ttc eee 26 B Creating a Size Standard with GeneMapper ID Software Version 3 2 a C Creating a Casework Analysis Method with Geneliapper ID SOmwWare Versio 0 2 sesvesasessccastace sce aroseussstecunstsadeinsetdeserseemnsneescees 28 D Creating a Databasing or Paternity Analysis Method with GeneMapper ID Software Version 9 2 sicssiese scisscsererisdcsseriieewccietecsntntennn 31 E Sample Analysis Using the GeneScan Software and Windows Operating Dy SCI essesi err tiro Cinania 33 F Sample Analysis Using the Genotyper Software aT dl wey iy ed per E MA Oien a meee cert renee eee 35 e CONTO ennenen AE EA E 38 CE o E ee E E A A ere 38 Te Tro bl eshool nt sasn 40 A Amphicanon and Fragment Delectoiersssann 40 B Direct Amplification of DNA from nonFTA Storage Card Punches 43 C Direct Amplification of DNA from SwabS sssssssressssssssscsersssssrsecssssosssrirssssssssscerssssss 45 B Cne ppe Barolo haus E E eee ent per Terre erence Tee erent tern rer 47 E MOE yet NG IANO aaiesisterae cence terse A 50 D SC 0s 0 are E eee ee ee A D2 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526
40. M Control DNA then add 1pl 10ng to a reaction well containing 25p1 of PCR amplification mix Notes 1 Do not include a blank storage card punch in the positive control reactions 2 Optimization of the amount of 2800M Control DNA may be required depending on thermal cycling conditions and laboratory preferences Typically 10ng of 2800M Control DNA is sufficient to provide a robust profile using the cycle numbers recommended here A one cycle reduction in cycle number will require a twofold increase in mass of DNA template to generate similar signal intensity Similarly a one cycle increase in cycle number will require a twofold reduction in the amount of 2800M Control DNA to avoid signal saturation 8 Reserve a well containing PCR amplification mix as a negative amplification control Note An additional negative control with a blank punch may be performed to detect contamination from the storage card or punch device 9 Seal the plate and briefly centrifuge the plate to bring the storage card punch to the bottom of the wells and remove any air bubbles Note Place the amplification plate in the thermal cycler and start the thermal cycling program as soon as the PCR amplification mix is added to all wells Prolonged storage of assembled reactions prior to cycling may result in poor performance i e lower peak heights for large amplicons Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in U
41. Notes 1 Centrifuge tubes briefly to bring contents to the bottom then vortex reagents for 15 seconds before each use Do not centrifuge the 10X Primer Pair Mix after vortexing as this may cause the reagents to be concentrated at the bottom of the tube 2 A precipitate may form in the Gold ST R 10X Buffer If this occurs warm the solution briefly at 37 C then vortex until the precipitate is in solution 2 Determine the number of reactions to be set up This should include positive and negative control reactions Add 1 or 2 reactions to this number to compensate for pipetting error While this approach does consume a small amount of each reagent it ensures that you will have enough PCR amplification mix for all samples It also ensures that each reaction contains the same PCR amplification mix 3 Use a clean MicroAmp plate for reaction assembly and label appropriately Alternatively determine the number of clean 0 2ml reaction tubes required and label appropriately Note If using the GeneAmp PCR System 9600 9700 or 2400 thermal cyclers use a MicroAmp plate or 0 2ml MicroAmp 8 strip reaction tubes For the model 480 thermal cycler we recommend 0 5m GeneAmp thin walled reaction tubes 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 TMD012 Revised 6 13 Page 7 4 A Amplificati
42. POX D8S1179 vWA Amelogenin Penta D CSF1PO D165539 D7S820 D135317 and D55818 One primer for each of the Penta E D18551 D21S11 TH01 and D351358 loci is labeled with fluorescein FL one primer for each of the FGA TPOX D8S1179 vWA and Amelogenin loci is labeled with carboxy tetramethylrhodamine TMR and one primer for each of the Penta D CSF1PO D165539 D75820 D135317 and D55818 loci is labeled with 6 carboxy 4 5 dichloro 2 7 dimethoxy fluorescein JOE All sixteen loci are amplified simultaneously in a single tube and analyzed in a single injection or gel lane The PowerPlex 16 Monoplex System Penta E Fluorescein Cat DC6591 and PowerPlex 16 Monoplex System Penta D JOE Cat DC6651 are available to amplify the Penta E and Penta D loci respectively Each monoplex system allows amplification of a single locus to confirm results obtained with the PowerPlex 16 System The monoplex systems also can be used to re amplify DNA samples when one or more of the loci do not amplify initially due to nonoptimal amplification conditions or poor DNA template quality The PowerPlex 16 System is compatible with the ABI PRISM 310 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130x Genetic Analyzers The protocols presented in this manual were tested at Promega Corporation Amplification and detection instrumentation may vary You may need to optimize protocols including the amount of templ
43. SA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD012 Revised 6 13 Page 13 Promega 4 B Direct Amplification of DNA from nonFTA Storage Card Punches continued Thermal Cycling Amplification and detection instrumentation may vary You will need to optimize protocols including cycle number injection conditions and loading volume for each laboratory instrument Testing at Promega shows that 27 cycles works well for a variety of nonFTA sample types Cycle number will need to be optimized in each laboratory for each sample type that is amplified Place the MicroAmp plate in the thermal cycler Select and run the recommended protocol The preferred protocol for use with the GeneAmp PCR System 9700 thermal cycler is provided below Thermal Cycling Protocol 95 C for 11 minutes then 96 C for 1 minute then ramp 100 to 94 C for 30 seconds ramp 29 to 60 C for 30 seconds ramp 23 to 70 C for 45 seconds for 10 cycles then ramp 100 to 90 C for 30 seconds ramp 29 to 60 C for 30 seconds ramp 23 to 70 C for 45 seconds for 17 cycles then 60 C for 30 minutes 4 C soak 1When using the GeneAmp PCR System 9700 thermal cycler the ramp rates indicated in the cycling program must be set and the program must be run in 9600 ramp mode The ramp rates are set in the Ramp Rate Modification screen While viewing the cycling program navigate to the Ramp Rate Modi
44. TECHNICAL MANUAL PowerPlex 16 System Instructions for use of Products DC6530 and DC6531 Pg TMD012 PowerPlex 16 System O All technical literature is available on the Internet at www promega com protocols 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 1 TDS PUOI a EEE E RERE T S AER 2 2 Product Components and Storage Conditions 0 ce eeeseseesessesseeseseeeesenteseeneeneeneeneees 4 w Perie Yon Deo eere EE ER 5 e NOC AON eer a E A sat caves E tae eae cuereteention 9 B Matrix Standardization or Spectral Calibration 0 essesesesesseceensessesseneesennes 6 4 Protocols for DNA Amplification Using the PowerPlex 16 System cee 6 A Ampihiationof FA DNA geese I B Direct Amplification of DNA from nonFTA Storage Card Punches 11 C Direct Amplincatonot DNA from Swabs cigecscesiesscoscassssravenizeicenees teastecteusedantduceades 15 5 Instrument Setup and Sample Preparation ceecssesseseeseseceeseeseeceeneeneeneeeeeenenees 18 A Detection of Amplified Fragments Using the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 or the Applied Biosystems 3130 or 3130 1 Genetic Analyzer with Data Collection Software Version 3 0 esses 18 B Detection of Amplified Fragments Using the ABI PRIS
45. ach fragment is labeled with carboxy X rhodamine CXR and can be detected separately as a fourth color in the presence of PowerPlex 16 amplified material The ILS 600 is designed for use in each gel lane or CE injection to increase precision in analyses when using the PowerPlex 16 System Protocols for preparation and use of this internal lane standard are provided in Section 5 300 400 10349TA Figure 13 Internal Lane Standard 600 An electropherogram showing the Internal Lane Standard 600 fragments 9 E Composition of Buffers and Solutions 10 ammonium persulfate TAE 50X buffer pH 7 2 Add 0 05g of ammonium persulfate 242g Tris base to 500ul of deionized water 57 1ml glacial acetic acid 100ml 0 5M EDTA stock Add Tris base and EDTA stock to 500ml of deionized water Add glacial acetic acid Bring the volume to 1 liter with deionized water Blue Dextran Loading Solution 88 25 formamide 15mg ml blue dextran 41mM EDTA pH 8 0 Gold ST R 10X Buffer TBE 10X buffer 500mM KCI 100mM Tris HCI 107 8g Tris 7 44g EDT H 8 3 at 25 C 15mM o _ NaEDTA 2H O 1 Triton X 100 ae Wonca 2mM each dNTP Dissolve Tris base and EDTA in 1 6mg ml BSA 800ml of deionized water Slowly add the boric acid and monitor the pH until the desired pH of 8 3 is obtained Bring the final volume to 1 liter with deionized water Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356
46. action volume 25ul 1A dd nuclease free water to the tube first then add Gold ST R 10X Buffer PowerPlex 16 10X Primer Pair Mix AmpliTag Gold DNA polymerase and 5X AmpSolution Reagent The template DNA will be added at Step 6 5 Vortex the PCR amplification mix for 5 10 seconds then pipet 23ul of PCR amplification mix into each reaction well O Failure to vortex the PCR amplification mix sufficiently can result in poor amplification or locus to locus imbalance z Pipet 2 0ul of swab extract for each sample into the appropriate well of the reaction plate 7 For the positive amplification control vortex the tube of 2800M Control DNA then dilute to 2 5ng pl Add 2ul Sng to a reaction well containing 23pl of PCR amplification mix Note Optimization of the amount of 2800M Control DNA may be required depending on thermal cycling conditions and laboratory preferences 8 For the negative amplification control pipet 2ul of Water Amplification Grade or TE buffer instead of swab extract into a reaction well containing PCR amplification mix Note Additional negative controls can be included Assemble a reaction containing the swab extract prepared from a blank swab or assemble a reaction where the SwabSolution Reagent is processed as a blank without a swab 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
47. ademark of Life Technologies Corporation 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 information 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 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 TMD012 Revised 6 13 Page 63
48. al cyclers and pipettes routinely Using a 59 C annealing temperature instead of 60 C has been shown to improve balance in some instances 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 TMD012 Printed in USA Page 42 Revised 6 13 Symptoms Causes and Comments C Peak height imbalance continued PCR amplification mix prepared in Section 4 was not mixed A well Vortex the PCR amplification mix for 5 10 seconds before dispensing into ae tubes or plate Promega The reaction volume was too low This system is optimized for a final reaction volume of 25ypl Decreasing the reaction volume can result in suboptimal performance Impure template DNA Inhibitors that may be present in forensic samples can lead to allele dropout or imbalance 7 B Direct Amplification of DNA from NonFTA Storage Card Punches The following information is specific to direct amplification For information about general amplification and detection see Section 7 A Symptoms Causes and Comments Faint or absent allele peaks DNA was not accessible on nonlytic material Pretreat nonFTA materials with PunchSolution Reagent to ensure that DNA is liberated from cellular proteins Poor sample deposition Shedding and collection of donor cells was variable Increase cycle number Poor sample transfer to storage card or variable sampling from the storage card
49. analysis method created earlier Select OK Select Dye and Analysis Method O Classic D ye Analysis Method Default Select Sample 5725TA Figure 3 The Select Dye and Analysis Method window 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 TMD012 Revised 6 13 Page 27 Promega 6 B Creating a Size Standard with GeneMapper ID Software Version 3 2 continued 5 Enter a detailed name such as ILS 600 Advanced in the Size Standard Editor Figure 4 Size Standard Editor ILS GOO Advanced 20 0 5726TA Figure 4 The Size Standard Editor 6 Choose Red for the Size Standard Dye 7 Enter the sizes of the internal lane standard fragments 60 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 500 550 and 600 bases See Section 9 D Figure 13 8 Select OK 6 C Creating a Casework Analysis Method with GeneMapper ID Software Version 3 2 These instructions loosely follow the Applied Biosystems GeneMapper ID software tutorial pages 5 11 1 Select Tools then GeneMapper Manager 2 Select the Analysis Methods tab 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 Par
50. art Slope Threshold for Peak End Size Call Range Full Range C This Range Base Pairs hlin T hil az f ood Size Calling Method 2nd Order Least Squares C 3rd Order Least Squares C Cubic Spline Interpolation f Local Southern Method Global Southern Method Baselining BaseLine Window Size 51 Pts Auto Analysis Only Size Standard 5684TA Figure 8 The Analysis Parameters window The start point of the analysis range which will vary is defined in Step 2 4 The analysis parameters can be saved in the Params folder in most installations this is located at C AppliedBio Shared Analysis Sizecaller Params Apply the stored analysis parameters file to the samples Assign a new size standard Select a sample file and highlight the arrow next to size standard Select define new Assign the size standard peaks as shown in Figure 13 in Section 9 D Store the size standard in the Size Standards folder at C AppliedBio Shared Analysis Sizecaller SizeStandards 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 TMD012 Page 34 Printed in USA Revised 6 13 7 Apply the size standard file to the samples then analyze the sample files C A Notes 1 Peak heights outside the linear range of the instrument may generate Promega artifact peaks due
51. ate DNA cycle number injection conditions and loading volume for your laboratory instrumentation In house validation should be performed 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 TMD012 Page 2 Printed in USA Revised 6 13 The PowerPlex 16 System provides all materials necessary to amplify STR regions C of human genomic DNA except for AmpliTaq Gold DNA polymerase This manual SA contains protocols for use of the PowerPlex 16 System with the Perkin Elmer model 480 and GeneAmp PCR System 9600 9700 and 2400 thermal cyclers in addition to protocols to separate amplified products and detect separated material Figure 1 Protocols to operate the fluorescence detection instruments should be obtained from the instrument manufacturer Information about other Promega fluorescent STR systems is available upon request from Promega or online at www promega com Amplification Setup Section 4 Thermal Cycling Section 4 GeneAmp PCR System 9700 GeneAmp PCR System 9600 GeneAmp PCR System 2400 Model 480 Thermal Cycler Instrument Setup and Sample Preparation Section 5 Applied Biosystems 3130 or ABI PRISM 3100 or 3100 Avant 3130x Genetic Analyzer with Data Genetic Analyzer with Data Collection Software Version 3 0 Collection Software Version 2 0 Section 5 A Section 5 A ABI PRISM 3100 Genetic ABI
52. ater bath for 3 minutes Denature samples just prior to loading the instrument Instrument Preparation Refer to the ABI PRISM 3100 Genetic Analyzer User s Manual for instructions on cleaning the blocks installing the capillary array performing a spatial calibration and adding polymer to the reserve syringe 1 2 Open the ABI PRISM 3100 Data Collection Software Change the GeneScan36_POP4DefaultModule module run time to 2 000 seconds Change the injection voltage to 3kV 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 TMD012 Page 22 Printed in USA Revised 6 13 4 Change the injection time to 11 seconds C A Note Instrument sensitivities can vary Injection time and voltage may be adjusted in the Module Manager A suggested range for the injection time Promega is 3 22 seconds and for the injection voltage is 1 3kV 5 Save the module with a new name e g GeneScan36_POP4PowerPlex16_3kV_11secs_2000 Use this as the initial run module for all runs 6 Opena new plate record Name the plate and select GeneScan Select the plate size 96 well Select Finish 7 Complete the plate record spreadsheet for the wells you have loaded Enter appropriate information into the Sample Name and Color Info columns For allelic ladder samples insert the word ladder into the Color Inf
53. blue and green dye colors Use autoclaved deionized water change vials and wash buffer reservoir 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 TMD012 Revised 6 13 Page 41 CQ 7 A Amplification and Fragment Detection continued A Symptoms Causes and Comments Promega Extra peaks visible in one Pull up or bleedthrough Pull up can occur when peak heights or all color channels continued are too high or if a poor or incorrect matrix has been applied to the samples e For the ABI PRISM 310 Genetic Analyzer generate a new matrix and apply it to the samples For the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130x1 Genetic Analyzers perform a new spectral calibration and re run the samples Instrument sensitivities can vary Optimize the injection or gel loading conditions See Section 5 Repeat sample preparation using fresh formamide Long term storage of amplified sample in formamide can result in artifacts The CE polymer was beyond its expiration date or polymer was stored at room temperature for more than one week Maintain instrumentation on a daily or weekly basis as recommended by the manufacturer Allelic ladder not running Allelic ladder and primer pair mix were not compatible Ensure the same as samples that the allelic ladder is fro
54. de thresholds from internal validation studies 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 TMD012 Printed in USA Page 32 Revised 6 13 11 Select the Peak Quality tab You may change the settings for peak quality CQ Note For Steps 11 and 12 see the GeneMapper ID user s manual for more information Promega 12 Select the Quality Flags tab You may change these settings 13 Select OK to save your settings Processing Data for Databasing or Paternity Samples 1 Select File then New Project 2 Select Edit then Add Samples to Project 3 Browse to the location of the run files Highlight desired files then select Add to list followed by Add 4 Inthe Sample Type column use the drop down menu to select Ladder Sample Positive Control or Negative Control as appropriate for the sample Every folder in the project must contain at least one allelic ladder that is designated as Ladder in the Sample Type column for proper genotyping 5 Inthe Analysis Method column select the analysis method created previously in this section 6 Inthe Panel column select the panels text file that was imported in Section 6 A 7 Inthe Size Standard column select the size standard that was created in Section 6 B 8 If analyzing data from an
55. duced amplification volumes Optimization and validation are required 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 TMD012 Page 44 Printed in USA Revised 6 13 Symptoms Causes and Comments C Peak height imbalance continued Inactive PunchSolution Reagent Thaw the PunchSolution So Reagent at 2 10 C Do not store reagents in the refrigerator door where the temperature can fluctuate Do not refreeze Promega avoid multiple freeze thaw cycles as this may reduce activity Carryover of excess PunchSolution Reagent into amplification reaction We recommend treating one 1 2mm nonFTA card punch with 10u1 of PunchSolution Reagent and using one punch per 25ul amplification reaction Use of a smaller amplification reaction volume may compromise performance if using 10ul of PunchSolution Reagent Reducing the PunchSolution Reagent volume may improve results when using a reduced amplification reaction volume Laboratory optimization and validation are required Extreme variability in sample There can be significant individual to individual variability in to sample peak heights the deposition of cells onto a punch resulting in peak height variability between samples The PunchSolution Kit increases the recovery of amplifiable DNA from samples but does not normalize the amount of DNA present 7 C Direct Amp
56. e 5X AmpSolution Reagent Cat DM1231 also supplied with the PunchSolution Kit e 1 2mm Harris Micro Punch or equivalent manual punch and cutting mat This section contains a protocol for direct amplification of DNA from nonFTA storage card punches using the PowerPlex 16 System and GeneAmp PCR System 9700 thermal cycler When using the protocol detailed below add one 1 2mm storage card punch to each 25ul amplification reaction NonFTA sample types include e Buccal samples on Bode Buccal DNA Collector devices e Buccal samples on nonFTA card punches e g S amp S 903 We do not recommend amplification of DNA from blood samples on nonFTA cards using the PowerPlex 16 System Pretreat nonFTA sample types with the PunchSolution Kit Cat DC9271 to lyse nonFTA samples before adding the amplification mix For more information see the PunchSolution Kit Technical Manual TMD038 Failure to pretreat these samples may result in incomplete profiles Use a manual punch tool with a 1 2mm tip to manually create sample disks from a storage card Place tip near the center of the sample spot and with a twisting or pressing action cut a 1 2mm sample disk Use the plunger to eject the disk into the appropriate well of a reaction plate Automated punchers also can be used to create sample disks Refer to the user s guide for your instrument for assistance with generating 1 2mm disks technical advice and troubleshooting
57. e DNA 1 0ng was amplified using the PowerPlex 16 10X Primer Pair Mix Amplification products were mixed with Internal Lane Standard 600 and analyzed with an Applied Biosystems 3130 Genetic Analyzer using a 3kV 5 second injection Results were analyzed using GeneMapper ID software version 3 2 Panel A An electropherogram showing the peaks of the fluorescein labeled loci D3S1358 TH01 D21S11 D18551 and Penta E Panel B An electropherogram showing the peaks of the JOE labeled loci D5S818 D135317 D75820 D16S539 CSF1PO and Penta D Panel C An electropherogram showing the peaks of the TMR labeled loci Amelogenin vWA D8S1179 TPOX and FGA Panel D An electropherogram showing the 60bp to 500bp fragments of the Internal Lane Standard 600 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 TMD012 Printed in USA Page 38 Revised 6 13 5683TA 800 600 Promega 200 i Wes meee R ie eit mmaa yg223033 0 apale Palle all Jae podelia Beheko 04 ledled ba 13 15 7 bs aleman oA aidhe bal bal m i ERICE iii 13 2 B Sate ae ee A R C POX SSF 200 300 400 1200 T nee e O oe AAAA G ENN ete I nag ebb Hee Jaare 3 2 46 2 iA bs by bA bs ed bal bA pid paal 45 2 5682TA Bi Er Figure 10 The PowerPlex 16 Allelic Ladder Mix The PowerPlex 16 Allelic Ladd
58. e PowerPlex 16 Allelic Ladder Mix is provided in a separate sealed bag for shipping This component should be moved to the post amplification box after opening Storage Conditions Store all components except the 2800M Control DNA at 30 C to 10 C in a nonfrost free freezer Store the 2800M Control DNA at 2 10 C The PowerPlex 16 10X Primer Pair Mix PowerPlex 16 Allelic Ladder Mix and Internal Lane Standard 600 are light sensitive and must be stored in the dark We strongly recommend that pre amplification and post amplification reagents be stored and used separately with different pipettes tube racks etc 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 TMD012 Printed in USA Page 4 Revised 6 13 Available Separately C v Product Size Cat Blue Dextran Loading Solution 3ml DV4351 Promega The PowerTyper Macros Release 2 0 for use with Genotyper software can be downloaded at www promega com resources tools powertyper macros The proper panels and bins text files for use with GeneMapper ID software are available for download at www promega com resources tools genemapper id software panels and bin sets Matrix standards are required for initial setup of the color separation matrix The matrix standards are sold separately and are available for the ABI PRISM 310 Genetic Analyzer PowerPlex Mat
59. e analysis Extra peaks in advanced mode size standard Open the Size Match Editor Highlight the extra peak select Edit and select hi delete size label Select auto adjust sizes Run was too short and larger peaks in ILS were not captured Not all ILS 600 peaks defined in the size standard were detected during the run e Create a new size standard using the internal lane standard fragments present in the sample e Re run samples using a longer run time le no epon ppa mi Aate Powerex 6v4 IL5600 a IDA Re ct 3oe sm 03 Eun Fodera aaao 0 Ho o no Size Match Editor ZA o ajp ann Size Mi na File Edt Yew Tools Ae g 1 1 es Ee PF EET tandari Size Pelatohes Size Calling Cura Qu ality Sizing Quality 0 0 Overide S0 Fy la E m xja m A B A e E Cle a E ia i E A E 28 E 28 E 30 A E i l E 8 Figure 12 An example showing improper assignment of size standard fragments in the GeneMapper ID software 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 TMD012 Printed in USA Page 48 Revised 6 13 Symptoms Causes and Comments C Peaks in size standard missing If peaks are below threshold decrease the peak amplitude o threshold in the analysis method
60. er locate the plate record that you just created in Steps 3 C and 4 and click once on the name to highlight it I 8 Once the plate record is highlighted click the plate graphic that corresponds Promega to the plate on the autosampler that contains your amplified samples 9 When the plate record is linked to the plate the plate graphic will change from yellow to green and the green Run Instrument arrow becomes enabled 10 Click on the green Run Instrument arrow on the toolbar to start the sample run 11 Monitor electrophoresis by observing the run view array or capillaries viewer window in the data collection software Each injection will take approximately 45 minutes 5 B Detection of Amplified Fragments Using the ABI PRISM 3100 Genetic Analyzer with Data Collection Software Version 1 0 1 or 1 1 Materials to Be Supplied by the User e 95 C dry heating block water bath or thermal cycler e crushed ice or ice water bath e centrifuge compatible with 96 well plates aerosol resistant pipette tips see Section 9 F e 3100 capillary array 36cm e performance optimized polymer 4 POP 4 polymer for the 3100 e 10X genetic analyzer buffer with EDTA e MicroAmp optical 96 well plate or equivalent and septa for the 3100 e Hi Di formamide Applied Biosystems Cat 4311320 The quality of formamide is critical Use Hi Di formamide Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long te
61. er Mix was analyzed with an Applied Biosystems 3130 Genetic Analyzer using a 3kV 5 second injection The sample file was analyzed with the GeneMapper ID software version 3 2 and PowerPlex 16 panels and bins text files Panel A The fluorescein labeled allelic ladder components and their allele designations Panel B The JOE labeled allelic ladder components and their allele designations Panel C The TMR labeled allelic ladder components and their allele designations Artifacts and Stutter Stutter products are a common amplification artifact associated with STR analysis Stutter products often are observed one repeat unit below the true allele peak and occasionally two repeat units smaller or one repeat unit larger than the true allele peak Frequently alleles with a greater number of repeat units will exhibit a higher percent stutter The pattern and intensity of stutter may differ slightly between primer sets for the same loci The level of stutter was determined and published as part of the PowerPlex 16 System validation 9 In addition to stutter peaks other artifact peaks can be observed at some of the PowerPlex 16 System loci Low level products can be seen in the n 2 and n 2 positions two bases below and above the true allele peak respectively with some loci such as D21511 Samples may show low level artifacts in the noncalling regions between the D75820 and D135317 allele ranges and between the D3S1358 and TH01 allele ranges
62. es using the thermal cycling protocol provided above but subject each plate to a different cycle number 27 28 and 29 cycles Note This recommendation is for 2ul of swab extract 4 Following amplification use your laboratory s validated separation and detection protocols to determine the optimal cycle number for the sample type Instrument Setup and Sample Preparation 5 A Detection of Amplified Fragments Using the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 or the Applied Biosystems 3130 or 3130x Genetic Analyzer with Data Collection Software Version 3 0 Materials to Be Supplied by the User e 95 C dry heating block water bath or thermal cycler e crushed ice or ice water bath e centrifuge compatible with 96 well plates e aerosol resistant pipette tips see Section 9 F e 3100 or 3130 capillary array 36cm e performance optimized polymer 4 POP 4 polymer for the 3100 or 3130 e 10X genetic analyzer buffer with EDTA e MicroAmp optical 96 well plate or equivalent and septa e Hi Di formamide Applied Biosystems Cat 4311320 The quality of formamide is critical Use Hi Di formamide Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause breakdown of formamide Poor quality formamide may contain ions that compete with DNA during injection which results in lower peak heights and reduced sensitivity A lon
63. fication screen by selecting More then Modify On the Ramp Rate Modification screen the default rates for each step are 100 The rate under each hold step is the rate at which the temperature will change to that hold temperature Figure 2 shows the ramp rates for the GeneAmp PCR System 9700 thermal cycler The ramp mode is set after start has been selected for the thermal cycling run A Select Method Options screen appears Select 9600 ramp mode and enter the reaction volume After completion of the thermal cycling protocol store amplified samples at 20 C in a light protected box Note Long term storage of amplified samples at 4 C or higher may produce artifacts 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 TMD012 Page 14 Printed in USA Revised 6 13 PCR Optimization CQ A Cycle number should be optimized based on the results of an initial experiment to determine the sensitivity with your collection method sample types and Promega instrumentation 1 Choose several samples that represent typical sample types you encounter in the laboratory Prepare them as you would using your normal workflow 2 Place one 1 2mm nonFTA storage card punch in each well of a reaction plate Be sure to pretreat nonFTA samples with the PunchSolution Kit Cat DC9271 3 Prepare three identical
64. for the red channel to include peaks Promega If peaks are low quality redefine the size standard for the sample to skip these peaks Error message The size standard and analysis method were not in the same Either panel size standard mode Classic vs Basic or Advanced Be sure both files or analysis method is invalid are set to the same mode either Classic or Basic or Advanced mode No alleles called but no error Panels text file was not selected for sample In the Panel message appears column select the appropriate panels text file for the STR system that was used No size standard was selected In the Size Standards column be sure to select the appropriate size standard Size standard was not correctly defined or size peaks were missing Redefine size standard to include only peaks present in your sample Terminating analysis early or using short run times will cause larger ladder peaks to be missing This will cause your sizing quality to be flagged as red and no allele sizes will be called Error message The bins text file assigned to the analysis method was deleted Both the Bin Set used in the In the GeneMapper Manager select the Analysis Methods Analysis Method and the Panel tab and open the analysis method of interest Select the Alleles must belong to the same tab and select the appropriate bins text file Chemistry Kit The wrong bins text file was chosen in the analysis method Al
65. ger injection time may not increase the signal Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning label and take appropriate precautions when handling this substance Always wear gloves and safety glasses when working with formamide 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 TMD012 Page 18 Printed in USA Revised 6 13 Sample Preparation C v 1 Thaw the Internal Lane Standard 600 Note Centrifuge tube briefly to bring contents to the bottom then vortex Promega for 15 seconds before each use Do not centrifuge after vortexing as this may cause the size standard to be concentrated at the bottom of the tube 2 Prepare a loading cocktail by combining and mixing Internal Lane Standard 600 and Hi Di formamide as follows 0 5pl ILS 600 x samples 9 5ul Hi Di formamide x samples Note The volume of internal lane standard used in the loading cocktail can be increased or decreased to adjust the intensity of the size standard peaks The optimal peak height for the 100 base fragment of the internal lane standard is 500 1 000RFU If peak heights are too low we recommend altering the formamide internal lane standard mix to contain 1 0ul of ILS 600 and 9 0ul of Hi Di formamide If peak heights are too high we recommend altering the loading c
66. he Amelogenin locus is included in the PowerPlex 16 System to allow gender identification of each sample Table 6 lists the PowerPlex 16 System alleles revealed in commonly available standard DNA templates We have carefully selected STR loci and primers to avoid or minimize artifacts including those associated with Tag DNA polymerase such as repeat slippage and terminal nucleotide addition Repeat slippage 16 17 sometimes called n 4 peaks stutter or shadow bands is due to the loss of a repeat unit during DNA amplification somatic variation within the DNA or both The amount of this artifact observed depends primarily on the locus and the DNA sequence being amplified Terminal nucleotide addition 18 19 occurs when Tag DNA polymerase adds a nucleotide generally adenine to the 3 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 We have modified primer sequences and added a final extension step of 60 C for 30 minutes 20 to the amplification protocols to provide conditions for essentially complete terminal nucleotide addition when recommended amounts of template DNA are used The presence of microvariant alleles alleles differing from one another by lengths other than the repeat length complicates interpretat
67. hts across loci For best results use less template DNA in the amplification reactions or reduce the number of cycles in the amplification program by 2 4 cycles i e 10 18 or 10 20 cycling 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 TMD012 Page 24 Printed in USA Revised 6 13 5 Centrifuge tubes briefly to remove air bubbles from the wells CQ A 6 Denature samples by heating at 95 C for 3 minutes and immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just Promega prior to loading 7 Assemble tubes in the appropriate autosampler tray 8 Place the autosampler tray in the instrument and close the instrument doors Instrument Preparation Refer to the instrument users manual for instructions on cleaning the pump block installing the capillary calibrating the autosampler and adding polymer to the syringe 1 Open the ABI PRISM 310 Data Collection Software 2 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 For rows containing PowerPlex 16 Allelic Ladder Mix insert the word ladder in the Sample Info column for the blue dye color yellow dye color and green dye color This information must be entered to successfully analyze you
68. ied Be certain the Could not complete the Sample Info or Color Info column for each lane containing Run Macro command because PowerPlex 16 Allelic Ladder Mix contains the word ladder no dye lanes are selected The macro uses the word ladder to identify sample files containing allelic ladder All four dye colors were not imported For Genotyper software versions 2 5 and 3 5 or higher set preferences in the Edit menu to import the blue green yellow and red colors Error message Peak heights for one or more alleles in the allelic ladder Could not complete the sample file were below 150RFU The allelic ladder categories Run Macro command are defined as having a minimum peak height of 150RFU If because the labeled peak peak heights of ladder alleles are below 150RFU the software could not be found will not be able to locate the allele peak Re run the allelic ladder using more sample or longer injection time to ensure that peak heights are above 150RFU CE spikes in the allelic ladder sample were identified as alleles by the macro Use a different injection of allelic ladder TH01 9 3 and 10 alleles were not separated when using heavy smoothing in the GeneScan analysis parameters Use light smoothing in the GeneScan analysis parameters Allelic ladder data were not compatible with the PowerTyper file used Confirm that the PowerTyper Macro file matches the allelic ladder being used
69. imized and balanced for 0 5 1 0ng Promega of DNA template The amount of DNA template used in your laboratory should be based on the results of your internal validation and may be different 7 For the positive amplification control vortex the tube of 2800M Control DNA then dilute an aliquot to 0 5ng in the desired template DNA volume Add 0 5ng of diluted DNA to a reaction well or tube containing PCR amplification mix 8 For the negative amplification control pipet nuclease free water or TE buffer instead of template DNA into a reaction well containing PCR amplification mix 9 If using the model 480 thermal cycler and GeneAmp reaction tubes add one drop of mineral oil to each tube before closing If using the GeneAmp PCR System 9600 9700 or 2400 thermal cycler and MicroAmp reaction tubes or plates no addition of mineral oil to the reaction wells or tubes is required 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 9 Seal the plate or close the tubes Optional Briefly centrifuge the plate to bring contents to the bottom of the wells and remove any air bubbles Thermal Cycling This section contains protocols for use of the PowerPlex 16 System with the model 480 and GeneAmp PCR system 9600 9700 and 2400 thermal cyclers For information about other thermal cyclers contact Promega Technical Services by e mail genetic pr
70. in Table 7 The PowerPlex 16 System provides typical paternity indices exceeding 500 000 in each population eroup An alternative calculation used in paternity analyses is the power of exclusion 35 This value calculated for the PowerPlex 16 System exceeds 0 999998 in all populations tested Table 7 Matching Probabilities Paternity Indices and Power of Exclusion of the PowerPlex 16 System in Various Populations African American Caucasian American MHispanic American Asian American Matching Probability 1 in 1 41 x 1038 1 in 1 83 x 10 1 in 2 93 x 101 1 in 3 74 x 10 Paternity Index 2 510 000 1 520 000 522 000 4 110 000 Power of Exclusion 0 9999996 0 9999994 0 9999983 0 9999998 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 TMD012 Page 58 Printed in USA Revised 6 13 9 C DNA Extraction and Quantitation Methods and Automation Support C A Promega offers a wide variety of reagents and automated methods for sample pP preparation DNA purification and DNA quantitation prior to STR amplification mega For analysis of database reference and other single source samples we recommend preprocessing of swabs and nonFTA punches with the SwabSolution Kit or PunchSolution Kit respectively The SwabSolution Kit Cat DC8271 contains reagents for rapid DNA preparation from buccal swabs prior t
71. in overamplification and signal saturation If signal is saturated repeat the amplification with less swab extract or a reduced cycle number Amplification of excess template for a given cycle number resulted in overloading of the capillary upon electrokinetic injection The presence of excess DNA in the capillary makes it difficult to maintain DNA in a denatured single stranded state Some single stranded DNA renatures and becomes double stranded Double stranded DNA migrates faster than single stranded DNA during capillary electrophoresis and appears as shadow peaks migrating in front of the main peaks If this occurs at a heterozygous locus it is possible to observe the presence of two shadow peaks that differ in size by approximately the same distance as the single stranded alleles Peak height imbalance Excess DNA in the amplification reaction can result in locus to locus imbalance within a dye channel such that the peak heights at the smaller loci are greater than those at the larger loci ski slope effect Use less swab extract or reduce the cycle number Active SwabSolution Reagent carried over into the amplification reaction Larger loci are most susceptible to reagent carryover and will drop out before smaller loci Ensure that the heat block is heating to 70 C 90 C if using 2 2ml Square Well Deep Well Plates and samples were incubated for the full 30 minutes Incubation for shorter time periods may result in inco
72. information Note Static may be problematic when adding a punch to a well Adding PunchSolution Reagent to the well before adding the punch during pretreatment may help alleviate static problems 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 TMD012 Revised 6 13 Page 11 C 4 B Direct Amplification of DNA from nonFTA Storage Card Punches continued Pro mega Amplification Setup 1 lt 4 Thaw the Gold ST R 10X Buffer and PowerPlex 16 10X Primer Pair Mix completely Notes 1 Centrifuge tubes briefly to bring contents to the bottom then vortex reagents for 15 seconds before each use Do not centrifuge the 10X Primer Pair Mix after vortexing as this may cause the reagents to be concentrated at the bottom of the tube 2 A precipitate may form in the Gold ST R 10X Buffer If this occurs warm the solution briefly at 37 C then vortex until the precipitate is in solution Determine the number of reactions to be set up This should include positive and negative control reactions Add 1 or 2 reactions to this number to compensate for pipetting error While this approach does consume a small amount of each reagent it ensures that you will have enough PCR amplification mix for all samples It also ensures that each reaction contains the same PCR amplification mix Use a clean Micro
73. inted in USA Revised 6 13 7 Double click on the Display Fluorescein Data macro to display the blue C dye for all sample injections or lanes Scroll down to observe and edit as So E Promega 8 Double click on the Display TMR Data macro to display the yellow dye for all sample injections or lanes Scroll down to observe and edit as needed 9 Double click on the Display JOE Data macro to display the green dye for all sample injections or lanes Scroll down to observe and edit as needed 10 Create the appropriate table by selecting the PowerTable Make Allele Table or Make CODIS Table macro The three available table formats are shown below The PowerTable option allows up to four alleles per sample file Additional information such as low peak signal or high peak signal also is included The Allele Table and CODIS Table options include only two alleles per locus If more than two alleles are present at a locus the smallest alleles identified are included The Allele Table format displays the categories loci in columns while the CODIS table format displays the categories in rows These tables can be customized to fit needs To save data in tables go to the Table drop down menu highlight Export to File and save the file with the desired name and location The saved file can be viewed and analyzed using Microsoft Excel PowerTable Format Sample Sample Peak Peak Peak Peak Over Low Satura Edited Edited Info Comment
74. ion and assignment of alleles There appears to be a correlation between a high degree of polymorphism a tendency for microvariants and increased mutation rate 21 22 Thus FGA and D21S11 display numerous relatively common microvariants For reasons yet unknown the highly polymorphic Penta E locus does not display frequent microvariants Table 5 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 TMD012 Page 54 Printed in USA Revised 6 13 Table 4 The PowerPlex 16 System Locus Specific Information C A GenBank Locus and Repeat Sequence STR Locus Label Chromosomal Location Locus Definition 54 gt 3 Promega Penta E FL 15q NA AAAGA D18551 FL 18q21 3 HUMUT574 AGAA 22 D21S11 FL 21q11 21q21 HUMD21LOC TCTA Complex 22 THO1 lll 11p15 5 HUMTHO01 human tyrosine AATG 22 hydroxylase gene D3S1358 FL 3p NA TCTA Complex FGA TMR 4q28 HUMFIBRA human TOKE fibrinogen alpha chain gene Complex 22 TPOX TMR 2p24 2pter HUMTPOx human thyroid AATG peroxidase gene D8S1179 TMR 8q24 13 NA TCTA Complex 22 vWA TMR 12p13 31 HUMVWFA31 human von TCTA Willebrand factor gene Complex 22 Amelogenin TMR Xp22 1 22 3 and Y HUMAMEL human Y NA chromosomal gene for Amelogenin like protein Penta D JOE 21q NA AAAGA CSFIPO JOE 5q33 3 34 HUMCSF1PO human c fms AGAT proto oncogene for CSF 1 receptor gene D165539 JOE 16q24 1 NA
75. l data or POWER 20 Filter macro option All four dye colors were not imported For Genotyper software versions 2 5 and 3 5 or higher set preferences in the Edit menu to import the blue green yellow and red colors The Check ILS macro displays All four dye colors were not imported For Genotyper an empty plot window software versions 2 5 and 3 5 or higher set preferences in the Edit menu to import the blue green yellow and red colors Off ladder peaks Migration of samples changed slightly over the course of a CE run with many samples This may be due to changes in temperature or the CE column over time Use a different injection of allelic ladder to determine sizes in the PowerTyper 16 Macro Release 2 0 Do not use the first injection on a new column for the ladder sample The base pair size of alleles was incorrect because incorrect fragment sizes were assigned to the internal lane standard Confirm that internal lane standard fragment sizes are assigned correctly Re analyze the sample using GeneScan software and redefine the internal lane standard fragments 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 TMD012 Revised 6 13 Page 51 C 8 References o Promega 10 11 12 13 14 15 16 7 18 19 20 21 22 23 Edwards A et al 1991
76. lele tab Be sure to choose the appropriate bins text file as shown in Figure 5 Significantly raised baseline e Poor spectral calibration for the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130x Genetic Analyzers Perform a new spectral calibration and re run the samples e Poor matrix for the ABI PRISM 310 Genetic Analyzer Re run and optimize the matrix Use of Classic mode analysis method Use of Classic mode analysis on samples can result in baselines with more noise than those analyzed using the Basic or Advanced mode analysis method Advanced mode analysis methods and size standards are recommended Red bar appears during analysis If none of the samples had matrices applied when run on the of samples and the following ABI PRISM 310 Genetic Analyzer no data will be displayed error message appears when data Apply a matrix file during analysis in the GeneMapper ID are displayed Some selected software and re analyze sample s do not contain analysis data Those sample s will not be shown 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 TMD012 Revised 6 13 Page 49 7 D GeneMapper ID Software continued Symptoms Causes and Comments Error message after attempting to There was a conflict between different sets of panels and bins impor
77. lification of DNA from Swabs The following information is specific to amplification of DNA from swabs For information about general amplification and detection see Section 7 A Symptoms Causes and Comments Faint or absent allele peaks Poor sample deposition Shedding and collection of donor cells was variable Increase cycle number Inactive SwabSolution Reagent Thaw the SwabSolution Reagent completely in a 37 C water bath and mix by gentle inversion Store the SwabSolution Reagent at 2 10 C Do not store reagents in the refrigerator door where the temperature can fluctuate Do not refreeze avoid multiple freeze thaw cycles as this may reduce activity Make sure that the PCR amplification mix contained AmpSolution Reagent Omission of AmpSolution Reagent from amplification reactions will result in amplification failure Active SwabSolution Reagent carried over into the amplification reaction Ensure that the heat block is heating to 70 C 90 C if using a 2 2ml Square Well Deep Well Plate and samples were incubated for the full 30 minutes Incubation for shorter time periods may result in incomplete reagent inactivation Do not use an incubator set at 70 C to incubate tubes or plates Heat transfer is inefficient and will result in poor performance Only use a heat block to maintain efficient heat transfer We have tested 60 minute incubation times and observed no difference in performance compared to a 30 minute i
78. m the same kit as the primer pair mix Buffer incompatibility Samples were diluted in the wrong buffer Use Gold STXR 1X Buffer to dilute samples Poor quality formamide Use only Hi Di formamide when analyzing samples Be sure the allelic ladder and samples are from the same instrument run Migration of samples changed slightly over the course of a CE run with many samples This may be due to changes in temperature or the CE column over time Use a different injection of allelic ladder to determine sizes Poor injection of allelic ladder Include more than one ladder per instrument run Peak height imbalance Excessive amount of DNA Amplification of gt 1ng of template can result in an imbalance with smaller loci showing more product than larger loci Use less template or reduce the number of cycles in the amplification program by 2 4 cycles 10 20 or 10 18 cycling to improve locus to locus balance Note Dilution of overamplified samples can result in dropout of larger loci Degraded DNA sample DNA template was degraded and larger loci show diminished yield Repurify template DNA Insufficient template DNA Use the recommended amount of template DNA Stochastic effects can occur when amplifying low amounts of template Miscellaneous balance problems Thaw the 10X Primer Pair Mix and Gold ST R 10X Buffer completely and vortex for 15 seconds before using Do not centrifuge the 10X Primer Pair Mix after mixing Calibrate therm
79. mplete reagent inactivation Do not use an incubator set at 70 C to incubate tubes or plates Heat transfer is inefficient and will result in poor performance Use only a heat block to maintain efficient heat transfer Inactive SwabSolution Reagent Thaw the SwabSolution Reagent completely in a 37 C water bath and mix by gentle inversion Store the SwabSolution Reagent at 2 10 C Do not store reagents in the refrigerator door where the temperature can fluctuate Do not re freeze avoid multiple freeze thaw cycles as this may reduce activity 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 TMD012 Printed in USA Page 46 Revised 6 13 Symptoms Causes and Comments C Extreme variability in sample There can be significant individual to individual variability in A to sample peak heights cell deposition onto buccal swabs This will appear as variability in peak heights between swab extracts The Promega extraction process maximizes recovery of amplifiable DNA from buccal swabs but does not normalize the amount of DNA present If variability is extreme quantitate the DNA using a fluorescence based double stranded DNA quantitation method or qPCR based quantitation method The quantitation values can be used to normalize input template amounts to minimize variation in signal intensity 7 D GeneMapper ID Sof
80. ms Causes and Comments Faint or absent allele peaks Impure template DNA Because of the small amount of template used this is rarely a problem Depending on the DNA extraction procedure used and sample source inhibitors might be present in the DNA sample Insufficient template Use the recommended amount of template DNA if available Insufficient enzyme activity Use the recommended amount of AmpliTag Gold DNA polymerase Check the expiration date on the tube label Incorrect amplification program Confirm the amplification program An air bubble formed at the bottom of the reaction well Use a pipette to remove the air bubble or centrifuge the reactions briefly before thermal cycling High salt concentration or 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 Mg or EDTA from the DNA sample can negatively affect PCR A change in pH also may affect PCR Store DNA in TE buffer 10mM Tris HCl pH 8 0 0 lmM EDTA TE buffer with 20p ml glycogen or nuclease free water The reaction volume was too low This system is optimized for a final reaction volume of 25 11 Decreasing the reaction volume may result in suboptimal performance Thermal cycler plate or tube problems Review the thermal cycling protocols in Section 4 We have not tested other reaction tubes plates or thermal cyclers
81. nabled 17 Select Run Instrument on the toolbar to start the sample run 18 Monitor electrophoresis by observing the run status array and capillary views windows in the collection software Each injection will take approximately 45 minutes 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 TMD012 Revised 6 13 Page 23 Promega 5 C Detection of Amplified Fragments Using the ABI PRISM 310 Genetic Analyzer Materials to Be Supplied by the User 95 C dry heating block water bath or thermal cycler 310 capillaries 47cm x 50um performance optimized polymer 4 POP 4 polymer 10X genetic analyzer buffer with EDTA sample tubes and septa aerosol resistant pipette tips see Section 9 F Hi Di formamide Applied Biosystems Cat 4311320 crushed ice or ice water bath The quality of formamide is critical Use Hi Di formamide Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause breakdown of formamide Poor quality formamide may contain ions that compete with DNA during injection which results in lower peak heights and reduced sensitivity A longer injection time may not increase the signal Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning label and take appropriate precautions when
82. ncubation 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 TMD012 Revised 6 13 Page 45 CQ 7 C Direct Amplification of DNA from Swabs continued A Symptoms Causes and Comments Promega Faint or absent peaks for the If the positive control reaction failed to amplify check to positive control reaction make sure that the correct amount of 2800M Control DNA was added to the reaction Due to the reduced cycle numbers used with swab extracts it is necessary to increase the mass of 2800M Control DNA to obtain a profile We recommend 5ng of 2800M Control DNA per 25pl amplification reaction This mass of DNA should be reduced if the cycle number used is increased and decreased if the cycle number is increased Increase or decrease by twofold the mass of 2800M Control DNA for every one cycle decrease or increase respectively Extra peaks visible in one Swab extract was contaminated Include a blank swab as a or all color channels negative control when processing samples Artifacts of STR amplification Amplification of swab extracts with high concentrations of DNA can result in artifact peaks due to overamplification resulting in saturated signal on the CE instrument We recommend 2 of swab extract per 25 11 reaction Using more than 2ul in a 251 reaction or using 2 with a smaller reaction volume may result
83. nsity of the size standard peaks The optimal peak height for the 100 base fragment of the internal lane standard is 500 1 000RFU If peak heights are too low we recommend altering the formamide internal lane standard mix to contain 1 0ul of ILS 600 and 9 0ul of Hi Di formamide If peak heights are too high we recommend altering the loading cocktail to contain 0 25u1 of ILS 600 and 9 75 ul of formamide Vortex for 10 15 seconds to mix Pipet 10ul of formamide internal lane standard mix into each well Add 1ul of amplified sample or 1ul of PowerPlex 16 Allelic Ladder Mix Cover wells with appropriate septa Note Instrument detection limits vary therefore injection time injection voltage or the amount of product mixed with loading cocktail may need to be increased or decreased Use the Module Editor in the data collection software to modify injection time or voltage in the run module If peak heights are higher than desired samples can be diluted in Gold ST amp R 1X Buffer before mixing with loading cocktail The use of too much template DNA 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 to achieve the desired signal intensity Centrifuge plate briefly to remove air bubbles from the wells Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or in an ice w
84. o amplification The procedure lyses cells contained on the swab head and releases into solution sufficient DNA for STR amplification A small volume of the final swab extract is added to the PowerPlex reaction The PunchSolution Kit is used to process punches from nonFTA storage cards containing blood or buccal samples prior to direct amplification When performing direct amplification with the PowerPlex 16 System make sure that the PCR amplification mix contains AmpSolution Reagent Omission of AmpSolution Reagent from amplification reactions will result in amplification failure The SwabSolution Kit Cat DC8271 contains reagents for rapid DNA preparation from single source buccal swab samples prior to PowerPlex System analysis The procedure lyses cells contained on the swab head and releases into solution sufficient DNA for STR amplification A small volume of the final swab extract is added to the PowerPlex reaction For casework or samples that require DNA purification we recommend the the DNA IQ System Cat DC6700 which is a DNA isolation system designed specifically for forensic and paternity samples 36 This system 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 eliminates PCR inhibitors and contaminants frequently encountered in casework samples With DNA
85. o column for the blue yellow and green dye colors This information must be entered to successfully analyze data with the PowerTyper 16 Macro Release 2 0 8 In the BioLIMS Project column select 3100_Project1 from the drop down menu 9 Inthe Dye Set column select Z from the drop down menu 10 When using the ABI PRISM 3100 Data Collection Software Version 1 0 1 or 1 1 select GeneScan36_POP4PowerPlex16_3kV_11secs_2000 from the drop down menu in the Run Module 1 column 11 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 software 12 Select OK This new plate record will appear in the pending plate records table on the plate setup page of the collection software 13 Place samples in the instrument and close the instrument doors 14 Locate the pending plate record that you just created and click once on the name 15 Once the pending plate record is highlighted click on the plate graphic that corresponds to the plate on the autosampler that contains your amplified samples to link the plate to the plate record 16 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 e
86. ocktail to contain 0 251 of ILS 600 and 9 75 ul of formamide 3 Vortex for 10 15 seconds to mix 4 Pipet 10ul of formamide internal lane standard mix into each well 5 Add 1pl of amplified sample or 1ul of PowerPlex 16 Allelic Ladder Mix Cover wells with appropriate septa Note Instrument detection limits vary therefore injection time injection voltage or the amount of product mixed with loading cocktail may need to be adjusted Use the Module Manager in the data collection software to modify the injection time or voltage in the run module If peak heights are higher than desired samples can be diluted in Gold STXR 1X Buffer before mixing 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 to achieve the desired signal intensity 6 Centrifuge plate briefly to remove air bubbles from the wells 7 Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading the instrument 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 TMD012 Revised 6 13 Page 19 Promega 5 A Detection of Amplified Fragments Using the ABI PRISM 310
87. omega com Amplification and detection instrumentation may vary You may need to optimize protocols including the amount of template DNA cycle number injection conditions and loading volume for your laboratory instrumentation Testing at Promega shows that 10 22 cycles work well for 0 5 1ng of purified DNA templates For higher template amounts or to decrease sensitivity fewer cycles such as 10 16 10 18 or 10 20 should be evaluated In house validation should be performed 1 Place the MicroAmp plate or reaction tubes in the thermal cycler 2 Select and run the recommended protocol The preferred protocols for use with the GeneAmp PCR System 9600 9700 and 2400 thermal cyclers and model 480 thermal cycler are provided below 3 After completion of the thermal cycling protocol store amplified samples at 20 C in a light protected box Note Long term storage of amplified samples at 4 C or higher may produce artifacts 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 TMD012 Revised 6 13 Page 9 Protocol for the GeneAmp PCR System 9700 Thermal Cycler 95 C for 11 minutes then 96 C for 1 minute then ramp 100 to 94 C for 30 seconds ramp 29 to 60 C for 30 seconds ramp 23 to 70 C for 45 seconds for 10 cycles then ramp 100 to 90 C for 30 seconds ramp 29 to 60 C for 30
88. on of Extracted DNA continued 4 Add the final volume of each reagent listed in Table 1 to a sterile tube Amplification of gt 1ng of DNA template results in an imbalance in peak heights from locus to locus The smaller loci show greater amplification yield than the larger loci Reducing the number of cycles in the amplification program by 2 to 4 cycles i e 10 20 or 10 18 cycling can improve locus to locus balance Table 1 PCR Amplification Mix for Amplification of Extracted DNA Volume Numberof _ Final PCR Amplification Mix Component Per Reaction Reactions Volume to a final nuclease free water volume of 25 0ul x Gold ST R 10X Buffer 2 5ul x PowerPlex 16 10X Primer Pair Mix 2 5 x AmpliTag Gold DNA polymerase 0 8ul 4u x template DNA 0 5 1 0ng 34 up to 19 2ul total reaction volume 25ul 1Add nuclease free water to the tube first then add Gold ST R 10X Buffer PowerPlex 16 10X Primer Pair Mix and AmpliTag Gold DNA polymerase The template DNA will be added at Step 6 Assumes the AmpliTaq Gold DNA polymerase is at 5u yl If the enzyme concentration is different the volume of enzyme must be adjusted accordingly Store DNA templates in TE buffer 10mM Tris HCI pH 8 0 0 1mM EDTA or TE buffer with 20ug ml glycogen If the DNA template is stored in TE buffer that is not pH 8 0 or contains a higher EDTA concentration the volume of DNA added should not exceed 20 of the final reaction volume Am
89. on the Check ILS macro The macros are listed at the bottom left corner of the active window A plots window will be displayed to show the internal lane standard i e ILS 600 in the red dye color Scroll down to view and confirm that the internal lane standard fragment sizes are correct If necessary re analyze samples using the GeneScan software and redefine internal lane standard fragments Note The software uses one ladder sample to determine allele sizes The macro uses the first ladder sample imported for allele designations For casework double click on the POWER macro The POWER macro identifies alleles in the ladder sample and calculates offsets for all loci This process may take several minutes When completed a plots window will open to display the allelic ladders i e Penta E D18551 D21S11 TH01 and D3S1358 Alternatively for databasing or paternity double click on the POWER 20 Filter macro This macro has a higher level of filtering than the standard POWER macro to reduce the need for manual editing of peak labels The POWER 20 Filter should not be used if mixtures may exist In general allelic ladders contain fragments of the same lengths as many known alleles for the locus Allelic ladder sizes and repeat units are listed in Table 5 Section 9 A Analysis using GeneScan and Genotyper software allows allele determination by comparing amplified sample fragments with allelic ladders and internal lane standards
90. plification efficiency and quality can be greatly altered by changes in pH due to added Tris HCl available magnesium concentration due to chelation by EDTA or other PCR inhibitors which may be present at low concentrations depending on the source of the template DNA and the extraction procedure used 4Apparent DNA concentrations can differ depending on the DNA quantification method used 15 The amount of DNA template recommended here is based on DNA concentrations determined by measuring absorbance at 260nm We strongly recommend that you perform experiments to determine the optimal DNA amount based on your DNA quantification method The PowerPlex 16 System is optimized and balanced for 0 5 1 0ng of DNA template The amount of DNA template used in your laboratory should be based on the results of your internal validation and may be different 5 Vortex the PCR amplification mix for 5 10 seconds then pipet PCR amplification mix into each reaction well or tube Failure to vortex the PCR amplification mix sufficiently can result in poor amplification or locus to locus imbalance 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 TMD012 Page 8 Printed in USA Revised 6 13 6 Add template DNA for each sample to the respective well or tube C containing PCR amplification mix I Note The PowerPlex 16 System is opt
91. r data using the PowerTyper 16 Macro Release 2 0 3 Create a new GeneScan injection list Select the appropriate sample sheet from the drop down menu 4 Select the GS STR POP4 1ml F Module using the drop down menu Change the injection time to 3 seconds and the run time to 30 minutes Keep the settings for the remaining parameters as shown below Inj Secs 3 Inj kV 15 0 Run kV 15 0 Run C 60 Run Time 30 O You may need to optimize the injection time for individual instruments Injection times of 2 5 seconds are suggested for samples that contain 1ng of template DNA Note Migration of fragments may vary slightly over the course of a long ABI PRISM 310 Genetic Analyzer run This may be due to changes in temperature or changes in the column When analyzing many samples injections of allelic ladder at different times throughout the run can aid in accurately genotyping samples 5 Select the appropriate matrix file Section 3 B 6 To analyze data automatically 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 TMD012 Revised 6 13 Page 25 Promega 5 C Detection of Amplified
92. reaction plates with punches from the same samples 4 Amplify samples using the thermal cycling protocol provided above but subject each plate to a different cycle number 5 Following amplification use your laboratory s validated separation and detection protocols to determine the optimal cycle number for the sample type 4 C Direct Amplification of DNA from Swabs Materials to Be Supplied by the User GeneAmp PCR System 9700 thermal cycler Applied Biosystems microcentrifuge MicroAmp optical 96 well reaction plate Applied Biosystems aerosol resistant pipette tips see Section 9 F AmpliTaq Gold DNA polymerase Applied Biosystems Nuclease Free Water Cat P1193 SwabSolution Kit Cat DC8271 5X AmpSolution Reagent Cat DM1231 also supplied with the SwabSolution Kit This section contains a protocol for amplifying DNA from swab extracts using the PowerPlex 16 System and GeneAmp PCR System 9700 thermal cycler Pretreat OmniSwab GE Healthcare or cotton swabs with the SwabSolution Kit Cat DC8271 as described in the SwabSolution Kit Technical Manual TMD037 to generate a swab extract Amplification Setup 1 Thaw the Gold ST R 10X Buffer and PowerPlex 16 10X Primer Pair Mix completely Notes 1 Centrifuge tubes briefly to bring contents to the bottom then vortex reagents for 15 seconds before each use Do not centrifuge the 10X Primer Pair Mix after vortexing as this may cause the
93. reagents to be concentrated at the bottom of the tube 2 A precipitate may form in the Gold ST R 10X Buffer If this occurs warm the solution briefly at 37 C then vortex until the precipitate is in solution 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 TMD012 Revised 6 13 Page 15 Promega 4 C Direct Amplification of DNA from Swabs continued 2 Determine the number of reactions to be set up This should include positive and negative control reactions Add 1 or 2 reactions to this number to compensate for pipetting error While this approach does consume a small amount of each reagent it ensures that you will have enough PCR amplification mix for all samples It also ensures that each reaction contains the same PCR amplification mix 3 Use a clean MicroAmp plate for reaction assembly and label appropriately 4 Add the final volume of each reagent listed in Table 3 to a sterile tube Table 3 PCR Amplification Mix for Direct Amplification of DNA from Storage Card Punches Volume Number of _ Final PCR Amplification Mix Component Per Reaction Reactions Volume nuclease free water 12 2ul x Gold ST R 10X Buffer 2 5ul x PowerPlex 16 10X Primer Pair Mix Zoi x AmpliTagq Gold DNA polymerase 0 8ul 4u x 5X AmpSolution Reagent 5 0ul x swab extract 2ul total re
94. rich samples the DNA IQ System delivers a consistent amount of total DNA The system has been used to isolate DNA from routine sample types including buccal swabs stains on FTA paper and liquid blood Additionally DNA has been isolated from casework samples such as tissue differentially separated sexual assault samples and stains on support materials The DNA IQ System has been tested with the PowerPlex Systems to ensure a streamlined process See Section 9 F for ordering information For applications requiring human specific DNA quantification the Plexor HY System Cat DC1000 was developed 37 See Section 9 F for ordering information For information about automation of Promega chemistries on automated workstations using Identity Automation solutions contact your local Promega Branch Office or Distributor contact information available at www promega com support worldwide contacts e mail genetic promega com or visit www promega com idautomation 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 TMD012 Revised 6 13 Page 59 CO 9 D The Internal Lane Standard 600 gt The Internal Lane Standard ILS 600 contains 22 DNA fragments of 60 80 100 Promega 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 500 550 and 600 bases in length Figure 13 E
95. rix Standards 310 and ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130x Genetic Analyzers PowerPlex Matrix Standards 3100 3130 See Section 9 F for ordering information 3 Before You Begin 3 A Precautions 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 11 12 Guidelines for the validation process are published in the Internal Validation of STR Systems Reference Manual 13 The quality of purified DNA or direct amplification samples small changes in buffers ionic strength primer concentrations choice of thermal cycler and thermal cycling conditions can affect PCR success We suggest strict adherence to recommended procedures for amplification and fluorescence detection Additional research and validation are required if any modifications to the recommended protocols are made PCR based STR analysis is subject to contamination by very small amounts of human DNA Extreme care should be taken to avoid cross contamination when preparing template DNA handling primer pairs assembling amplification reactions and analyzing amplification products Reagents and materials used prior to amplification Gold ST R 10X Buffer and PowerPlex 16 10X Primer Pair Mix are provided in a separate box and should be stored separately from those used following amplification PowerPlex 16 Allelic
96. rm storage at 4 C may cause breakdown of formamide Poor quality formamide may contain ions that compete with DNA during injection which results in lower peak heights and reduced sensitivity A longer injection time may not increase the signal O Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning label and take appropriate precautions when handling this substance Always wear gloves and safety glasses when working with formamide Sample Preparation 1 Thaw the Internal Lane Standard 600 Note Centrifuge tube briefly to bring contents to the bottom then vortex for 15 seconds before each use Do not centrifuge after vortexing as this may cause the size standard to be concentrated at the bottom of the tube 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 TMD012 Revised 6 13 Page 21 Promega 5 B Detection of Amplified Fragments Using the ABI PRISM 3100 Genetic Analyzer with Data Collection Software Version 1 0 1 or 1 1 continued 2 Prepare a loading cocktail by combining and mixing Internal Lane Standard 600 and Hi Di formamide as follows 0 5ul ILS 600 x samples 9 5ul Hi Di formamide x samples Note The volume of internal lane standard used in the loading cocktail can be increased or decreased to adjust the inte
97. rs and size standard have different analysis types 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 TMD012 Revised 6 13 Page 47 CQ 7 D GeneMapper ID Software continued A Symptoms Causes and Comments I omega Off ladder alleles An allelic ladder from a different run than the samples was used Re analyze samples with an allelic ladder from the same run The GeneMapper ID software requires that the allelic ladder be imported from the same folder as the sample Be sure that the allelic ladder is in the same folder as the sample Create a new project and re analyze as described in Section 6 C or 6 D Panels text file selected for analysis was incorrect for the STR system used Assign correct panels text file that corresponds to the STR system used for amplification The allelic ladder was not identified as an allelic ladder in the Sample Type column The wrong analysis type was chosen for the analysis method Be sure to use the HID analysis type The internal lane standard was not properly identified in the sample Manually redefine the sizes of the size standard fragments in the sample Size standard not called Starting data point was incorrect for the partial range chosen correctly Figure 12 in Section 6 C Adjust the starting data point in the analysis method Alternatively use a full range for th
98. seconds ramp 23 to 70 C for 45 seconds for 22 cycles then 60 C for 30 minutes 4 C soak Protocol for the GeneAmp PCR System 9600 Thermal Cycler 95 C for 11 minutes then 96 C for 1 minute then 94 C for 30 seconds ramp 68 seconds to 60 C hold for 30 seconds ramp 50 seconds to 70 C hold for 45 seconds for 10 cycles then 90 C for 30 seconds ramp 60 seconds to 60 C hold for 30 seconds ramp 50 seconds to 70 C hold for 45 seconds for 22 cycles then 60 C for 30 minutes 4 C soak 3 tmp 10 cycles l i 29 i l l 3 tmp 22 cycles 94 0 C 190 0 C 100 29 Protocol for the GeneAmp PCR System 2400 Thermal Cycler 95 C for 11 minutes then 96 C for 1 minute then ramp 100 to 94 C for 30 seconds ramp 100 to 60 C for 30 seconds ramp 23 to 70 C for 45 seconds for 10 cycles then ramp 100 to 90 C for 30 seconds ramp 100 to 60 C for 30 seconds ramp 23 to 70 C for 45 seconds for 22 cycles then 60 C for 30 minutes 4 C soak Protocol for the Model 480 Thermal Cycler 95 C for 11 minutes then 96 C for 2 minutes then 94 C for 1 minute 60 C for 1 minute 70 C for 1 5 minutes for 10 cycles then 90 C for 1 minute 60 C for 1 minute 70 C for 1 5 minutes for 22 cycles then 60 C for 30 minutes 4 C soak When using the GeneAmp PCR System 9700 thermal cycler the ramp rates indicated in the cycling program must be set and the
99. sly in this section 6 Inthe Panel column select the panels text file that was imported in Section 6 A 7 Inthe Size Standard column select the size standard that was created in Section 6 B 8 If analyzing data from an ABI PRISM 310 Genetic Analyzer ensure that the appropriate matrix file is selected in the Matrix column 9 Select Analyze green arrow button to start data analysis 6 D Creating a Databasing or Paternity Analysis Method with GeneMapper ID Software Version 3 2 Select Tools then GeneMapper Manager Select the Analysis Methods tab Select New and a new analysis method dialog box will open Select HID and select OK Note If you do not see the HID option you do not have the GeneMapper ID software Contact Applied Biosystems 2S p a 5 Enter a descriptive name for the analysis method such as PowerPlex16_20 filter 6 Select the Allele tab Figure 7 7 Select the bins text file that was imported in Section 6 A 8 Ensure that the Use marker specific stutter ratio if available box is checked 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 TMD012 Revised 6 13 Page 31 C 6 D Creating a Databasing or Paternity Analysis Method with GeneMapper ID Software Version 3 2 continued Promega 9 Enter the
100. t TMD012 Page 28 Printed in USA Revised 6 13 Select New and a new analysis method dialog box will open CO amp Select HID and select OK Note If you do not see the HID option you do not have the GeneMapper ID software Contact Applied Biosystems Enter a descriptive name for the analysis method such as PowerPlex16 advanced Select the Allele tab Figure 5 Select the bins text file that was imported in Section 6 A Ensure that the Use marker specific stutter ratio if available box is checked Enter the values shown in Figure 5 for proper filtering of stutter peaks when using the PowerPlex 16 System For an explanation of the proper usage and effects of these settings refer to the Applied Biosystems user bulletin titled Installation Procedures and New Features for GeneMapper ID Software 3 2 Note Some of these settings have been optimized and are different from the recommended settings in the user bulletin Analysis Method Editor HID x General Allele Peak Detector Feak Quality Quality Flags Bin Set Promega_Bins_ID3 2 x mj Use makerspecific stutter ratio if available Tri TE TE TEE T TE TEE TE TE TE TE Maker Repeat Type Cut off Value Minus Ratio MinusA Distance hdinus Stutter Ratio TECE TEET Minus Stutter Distance From J ih Flus Stutter Ratio Flus Stutter Distance TUCC TU Amelogenin Cutoff Range Fil
101. t panels and bins text files text files Check to be sure that the bins are installed properly Unable to save panel data If not delete all panels and bins text files and re import files java SQLEException in a different order ORA 00001 unique constraint IFA CKP_NNN violated Allelic ladder peaks are GeneMapper ID software was not used or microsatellite labeled off ladder analysis settings were used instead of HID analysis settings GeneMapper software does not use the same algorithms as GeneMapper ID software and cannot correct for sizing differences using the allelic ladder Promega recommends using GeneMapper ID software to analyze PowerPlex reactions If using GeneMapper ID software version 3 2 be sure that the analysis method selected is an HID method This can be verified by opening the analysis method using the GeneMapper Manager then selecting the General tab The analysis type cannot be changed If the method is not HID it should be deleted and a new analysis method created 7 E PowerTyper 16 Macro Symptoms Causes and Comments File does not open Genotyper software was not installed Be certain that the on your computer Genotyper software version 3 6 or higher Windows NT is installed Incorrect version of Genotyper software The PowerTyper 16 Macro will not work with Genotyper software versions prior to version 2 5 Error message Allelic ladder sample files were not identif
102. ter Factory Defaults Ook Cancel 5724TA Figure 5 The Allele tab 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 Revised 6 13 Part TMD012 Page 29 C 6 C Creating a Casework Analysis Method with GeneMapper ID Software Version 3 2 continued Promega 10 Select the Peak Detector tab We recommend the settings shown in Figure 6 Notes 1 Select full range or partial range for the analysis range When using a partial range choose an appropriate analysis range based on your data Choose a start point after the primer peak and just before the first defined internal lane standard peak to help ensure proper sizing of the internal lane standard 2 The peak amplitude thresholds are the minimum peak heights at which the software will call a peak Values for peak amplitude thresholds are usually 50 150RFU and should be determined by individual laboratories Analysis Method Editor HID B General Allele Peak Detector Peak Quality Quality Flags Peak Detection Algorithm Advanced Ranges Feak Detection Analysis Sizing Feak Amplitude Thresholds B 100 R Full Range ba Partial Sizes Star Peo Start size fon Stop Pt 10000 Stop Size 500 Smoothing and Baselining G 100 QO ye 100 Mlin Peak Half Width Smoothing None f Light B
103. th immediately prior to electrophoresis Do not cool samples in a thermal cycler set at 4 C as this may lead to artifacts due to DNA re annealing Artifacts of STR amplification Amplification of STRs can result in artifacts that appear as faint peaks one repeat unit smaller than the allele Stutter product peak heights can be high if samples are overloaded See Section 6 H for additional information about stutter and artifacts Artifacts of STR amplification Amplification of STRs can result in artifacts that appear as peaks one base smaller than the allele due to incomplete addition of the 3 A residue Be sure to perform the 30 minute extension step at 60 C after thermal cycling Section 4 Excessive amount of DNA Amplification of gt 2ng template can result in a higher number of artifact peaks Use less template DNA or reduce the number of cycles in the amplification program by 2 4 cycles 10 20 or 10 18 cycling High background Load less amplification product or decrease injection time See Section 5 CE related artifacts spikes Minor voltage changes or urea crystals passing by the laser can cause spikes or unexpected peaks Spikes sometimes appear in one color but often are easily identified by their presence in more than one color Re inject samples to confirm CE related artifacts contaminants Contaminants in the water used with the instrument or to dilute the 10X genetic analyzer buffer may generate peaks in the
104. tware Symptoms Causes and Comments Alleles not called To analyze samples with GeneMapper ID software the analysis parameters and size standard must both have Basic or Advanced as the analysis type If they are different an error is obtained Figure 11 To analyze samples with GeneMapper ID software at least one allelic ladder must be defined An insufficient number of ILS 600 fragments was defined Be sure to define at least one ILS 600 fragment smaller than the smallest sample peak or allelic ladder peak and at least one ILS 600 fragment larger than the largest sample peak or allelic ladder peak Run was too short and larger peaks in ILS were not captured Not all ILS 600 peaks defined in the size standard were detected during the run e Create a new size standard using the internal lane standard fragments present in the sample e Re run samples using a longer run time hh LU A jl E E Table Setting Eric default Info Raw Data EPT Data Sample Information Sample File CRE_1 2h_H0G_2004 06 17 fsa Sample Origin Fath G Frivate Technical Service GI datai genetica CRE 172h HOS 2004 06 17 fsa Status Message Changed size standard from 1ls80500adv to LS600_Classic File Source Disk media Error Message Analysis Method was invalid Current Settings Sample Type Sample 5685TA Figure 11 The error message that appears in the GeneMapper ID software when the analysis paramete
105. values shown in Figure 7 for proper filtering of peaks when using the PowerPlex 16 System For an explanation of the proper usage and effect of these settings refer to the Applied Biosystems user bulletin titled Installation Procedures and New Features for GeneMapper ID Software 3 2 Analysis Method Editor HID xi General Allele Peak Detector Feak Quality Quality Flags Bin Set Promega_Bins_1D3 2 x T m Use makerspecific stutter ratio if available 4 TECE Maker Repeat Type Cut off Value Minus Ratio MinusA Distance hdinus Stutter Ratio Winus Stutter Distance From J TTT TE Ta Flus Stutter Ratio Plus Stutter Distance TTT TE TUCE Amelogenin Cutoff jo2 Range Filter Factory Defaults 5785TA Figure 7 The Allele tab with settings for using a 20 peak filter 10 Select the Peak Detector tab We recommend the settings shown in Figure 6 Notes 1 Select full range or partial range for the analysis range When using a partial range choose an appropriate analysis range based on your data Choose a start point after the primer peak and just before the first defined internal lane standard peak to help ensure proper sizing of the internal lane standard 2 The peak amplitude thresholds are the minimum peak heights at which the software will call a peak Values for peak amplitude thresholds are usually 50 150RFU Individual laboratories should determine their peak amplitu
106. ws NT Genotyper software version 3 6 or later The Genotyper software must be installed on your computer before the PowerTyper 16 Macro Release 2 0 can be used Be certain the Color Info Windows NT operating systems column for each lane containing allelic ladder mix contains the word ladder The macro uses the word ladder to identify the sample file s containing allelic ladder Sample info can be added or modified after importing into the PowerTyper Macro Highlight the sample then select show dye lanes window in the Views menu 1 Transfer the PowerTyper 16 Macro Release 2 0 to a designated location on your computer hard drive 2 Open the Genotyper software then the PowerTyper 16 Macro Release 2 0 For questions about the Genotyper software refer to the Genotyper Analysis Software User s Manual 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 TMD012 Revised 6 13 Page 35 Promega 6 F Sample Analysis Using the Genotyper Software and PowerTyper 16 Macro continued 3 In the File menu select Import and import the GeneScan project or sample files to be analyzed Import the blue yellow green and red dye colors Note To select the dye colors to be imported select Set Preferences in the Edit menu Double click

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