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PowerPlex® CS7 System

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1. ID Software Version 3 2 Ha pa p gt a Select Tools then GeneMapper Manager Select the Size Standard tab Select New Select Basic or Advanced Figure 3 The type of analysis method selected must match the type of analysis method created earlier Select OK 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 TMD042 Page 20 Printed in USA Revised 6 14 Select Dye and Analysis Method C Ss z va EP eS 5725TA Figure 3 The Select Dye and Analysis Method window 5 Enter a detailed name such as ILS 600 advanced in the Size Standard Editor Figure 4 Size Standard Editor go Size in Basepais T A ai m ee ee ta i _ ee j 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 Note Definition and detection of the 600bp fragment is optional 8 Select OK 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 TMD042 Revised 6 14 Page 21 6 C Creating a Databasing or
2. Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD042 Page 30 Printed in USA Revised 6 14 Symptoms Causes and Comments C Allelic ladder not running Allelic ladder and primer pair mix were not compatible Ensure o the same as samples that the allelic ladder is from the same kit as the primer pair i he ee Promega 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 is 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
3. C for 2 minutes 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 20 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 For the GneAmp PCR System 9700 thermal cycler with a 96 well block the program must be run in the 9600 ramp mode The 9600 ramp mode on the GeneAmp PCR System 9700 thermal cycler with the 384 well dual block ramps does not exist and is not required to program ramp rates 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 appropriate ramp rates for the GeneAmp PCR System 9700 thermal cycler The ramp mode is set after start is selected for the thermal cycling run A Select Method Options screen appears Select 9600 ramp mode and enter the reaction volume Using 10 20 cycles works well for routine testing For maximum sensitivity cycle number can be
4. You will need to optimize protocols including the number of storage card punches cycle number injection conditions and loading volume for each laboratory instrument Testing at Promega Corporation shows that 10 17 cycling works well for a variety of sample types Buccal samples may require more amplification cycles than blood samples Cycle number will need to be optimized in each laboratory for each sample type that is amplified 1 Place the MicroAmp plate in the 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 TMD042 Page 12 Printed in USA Revised 6 14 2 Select and run the recommended protocol The preferred protocol for use C with the GeneAmp PCR System 9700 thermal cycler is provided below eI Thermal Cycling Protocol 96 C for 2 minutes 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 For the GeneAmp PCR System 9700 thermal cycler with a 96 well block the program must be run in the 9600 ramp mode The 9600 ramp mode o
5. 2516 www promega com Printed in USA Part TMD042 Revised 6 14 Page 5 C 4 A Amplification of Extracted DNA o We routinely amplify 0 5ng of template DNA in a 25y reaction volume using the protocols detailed below Expect to see high peak heights at the smaller loci and relatively 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 Materials to Be Supplied by the User GeneAmp PCR System 9600 and 9700 thermal cycler Applied Biosystems microcentrifuge e MicroAmp optical 96 well reaction plate or 0 2ml MicroAmp reaction tubes Applied Biosystems e aerosol resistant pipette tips see Section 9 E Amplification Setup 1 Thaw the PowerPlex HS 5X Master Mix PowerPlex CS7 10X Primer Pair Mix and Water Amplification Grade completely Notes 1 PowerPlex HS 5X Master Mix and PowerPlex CS7 10X Primer Pair Mix are manufactured as a matched set for optimal performance Do not combine components from kits with different lot numbers printed on the boxes and Certificates of Analyses If lots are mixed locus to locus imbalance and variation in signal intensity may occur 2 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 or 5X Master Mix after vortexing as this may cause the reagents to be
6. 60 C after thermal cycling Section 4 Peak height imbalance Excessive amount of DNA Amplification of gt 20ng of template can result in an imbalance with smaller loci showing more product than larger loci Use one or two 1 2mm punches from a storage card containing a buccal sample or one 1 2mm punch from a storage card containing whole blood per 251 amplification reaction Follow the manufacturer s recommendations when depositing sample onto the storage card Decrease number of cycles The reaction volume was too low This system is optimized for a final reaction volume of 2511 to overcome inhibitors present in FTA cards and PunchSolution Reagent Decreasing the reaction volume can result in suboptimal performance Amplification was inhibited when using more than one storage card punch with blood Use only one 1 2mm storage card punch with blood 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 TMD042 Revised 6 14 Page 33 7 B Direct Amplification of DNA from Storage Card Punches continued Symptoms Causes and Comments Peak height imbalance continued Active PunchSolution Reagent carried over into the amplification reaction Larger loci are most suspectible to carryover and will drop out before the smaller loci Ensure that the hea
7. CS7 System are shown in Figure 9 The PowerPlex CS7 Allelic Ladder Mix is shown in Figure 10 ws ne new m a i _ 2 is a m _ ar n gt re n 1 J NE on ENS em oe Sew oe ee ee es ee ee ee ee ees ee ss ee ee ee ee es et 3 Figure 9 The PowerPlex CS7 System A single source template DNA 0 5ng was amplified using the PowerPlex CS7 System 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 and PowerPlex CS7 panels and bins text files Panel A An electropherogram showing the peaks of the fluorescein labeled loci LPL F13B FESFPS F13A01 and Penta D Panel B An electropherogram showing the peaks of the JOE labeled locus Penta C Panel C An electropherogram showing the peaks of the TMR labeled locus Penta E 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 Printed in USA Part TMD042 Revised 6 14 Page 27 O Promega 6 F Results continued r iili Hl im finn mn ll mat Ei Sele shin REEE Pee ca SENPASIE 7 OS mn ae
8. Dat Atma ysis aie cccssasssssccecssaiusisssscsnevans ssn benevoyssesenSovendsssassasscussaaisuastebiiadiaiyssnsnessgassssssnssssyansn 19 A Importing PowerPlex CS7 Panels and Bins Text Files with GeneMapper ID Versin 3 2 ccsscccccsvescccsssssecresvevscseisasetsevevectevsassssnecvsivensssssteessisoneuasss 19 B Creating a Size Standard with GeneMapper ID Software Version 3 2 20 C Creating a Databasing or Paternity Analysis Method Using a Global Filter with GeneMapper ID Software Version 3 2 ssssssssesssssseeeeeen 22 D Creating an Analysis Method Without a General Filter in GeneMapper 1D Software Version 9 2 ccscc asiisasiesassiaibiiitesigscttesvssseesecesssssesoreeszen 24 E Controls dl F ROSUILES ss scssssssorsssssoesssssesossssssssanesssssssssssannnssssasesscasssssssanssssssssssesasass abesbesonsnssssonensnsorbesoisore 27 7 TtOubleSMOOtitngescsscassasecsccessadiasasesseansvssveeabsnnvvsesenbsspcestsaaisastentvaaisanttesiteassnssssreassunasississaiinasen A Amplification and Fragment Detection i B Direct Amplification of DNA from Storage Card Punches 32 C Direct Amplification of DNA from Swab3 ccssccsssssseecsssssssseeeesssssssseeeessssssneeees 34 D GeneMapper ID Software sscssssssissssssssssssssssessscsssassssscccsssssssescssiavesssestnsssisnestnesssennestn 36 B Referentes sisia ERN T 39 9 Appendia a n e E EORR tiers 40 A Ady nt ges of STR TYYPIN nirien rasiassa rsna ras sa 40 B DNA Extr
9. ID Version 3 2 continued 6 B Getting Started 1 To obtain the panels and bins text files for the PowerPlex CS7 System go to www promega com resources tools genemapper id software panels and bin sets Enter your contact information and select GeneMapper ID and the control DNA that you use Select Submit Save the PowerPlex_CS7_Panels_vX x txt and PowerPlex_CS7_Bins_vX x txt files where X x refers to the most recent version of the panels 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 oe SS NMP 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 Navigate to the panels text file that was obtained in the Getting Started Section above Select the file then Import In the navigation pane highlight the Promega 16 HS CS7 panels folder that you just imported in Step 5 Select File then Import Bin Set Navigate to the bins text file that was obtained in the Getting Started Section above Select the file then Import At the bottom of the Panel Manager window select OK The Panel Manager window will close automatically Creating a Size Standard with GeneMapper
10. New York 7 PCR Technology Principles and Applications for DNA Amplification 1989 Erlich H A ed Stockton Press New York NY 8 PCR Protocols A Guide to Methods and Applications 1990 Innis M A et al eds Academic Press San Diego CA 9 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 10 Hartmann J M et al 1991 Guidelines for a quality assurance program for DNA analysis Crime Laboratory Digest 18 44 75 11 Internal Validation of STR Systems Reference Manual GE053 Promega Corporation 12 Kline M C et al 2005 Results from the NIST 2004 DNA quantitation study J Forensic Sci 50 571 8 13 Levinson G and Gutman G A 1987 Slipped strand mispairing A major mechanism for DNA sequence evolution Mol Biol Evol 4 203 21 14 Schl tterer C and Tautz D 1992 Slippage synthesis of simple sequence DNA Nucleic Acids Res 20 211 5 15 Bar W et al 1997 DNA recommendations Further report of the DNA Commission of the ISFH regarding the use of short tandem repeat systems Int J Legal Med 110 175 6 16 Gill P et al 1997 Considerations from the European DNA Profiling Group EDNAP concerning STR nomenclature Forensic Sci Int 87 185 92 17 Fr geau CJ et al 1995 Characterization of human lymphoid c
11. 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 Prepare three identical reaction plates with aliquots of the same swab extracts 3 Amplify samples using the thermal cycling protocol provided above but subject each plate to a different cycle number 10 17 10 18 and 10 19 cycling Note This recommendation is for 2 1 of swab extract Additional cycle number testing may be required 4 Following amplification use your laboratory s validated separation and detection protocols to determine the optimal cycle number for the sample type 5 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 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 e 3100 or 3130 capillary array 36cm e performance optimized polymer 4 POP 4 for the 3100 or 3130 e 10X genetic analyzer buffer with EDTA e MicroAmp optical 96 well plate and septa e Hi Di formamide Applied Biosystems Cat 4311320 The quality of formamide is critical Use Hi Di formamide Freeze formami
12. Symptoms Causes and Comments Faint or absent allele peaks The reaction volume was too low This system is optimized for a final reaction volume of 25 11 to overcome inhibitors present in FTA cards and PunchSolution Reagent Decreasing the reaction volume may result in suboptimal performance 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 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 or two 1 2mm storage card punches see Section 4 B Follow the manufacturer s recommendations when depositing sample onto the storage card With storage cards reducing the reaction volumes below 25pl may result in amplification failure Amplification was inhibited when using more than one storage card punch with blood Use only one 1 2mm storage card punch with blood Make sure that the PCR amplification mix also contained AmpSolution Reagent Omission of AmpSolution Reagent from amplification reactions will result in amplification failure Active PunchSolution Reagent carried over into the amplification reaction 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 W
13. cycler with a 96 well block the program must be run in the 9600 ramp mode The 9600 ramp mode on the GeneAmp PCR System 9700 thermal cycler with the 384 well dual block ramps does not exist and is not required to program ramp rates 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 appropriate ramp rates for the GeneAmp PCR System 9700 thermal cycler The ramp mode is set after start is 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 TMD042 Page 16 Printed in USA Revised 6 14 PCR Optimization C o Cycle number should be optimized based on the results of an initial experiment to determine the sensitivity with your collection method sample types and
14. 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 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 TMD042 Page 2 Printed in USA Revised 6 14 Amplification Setup C Section 4 o t Promega Thermal Cycling Section 4 GeneAmp PCR System 9700 GeneAmp PCR System 9600 y Instrument Setup and Sample Preparation Section 5 Applied Biosystems 3130 or ABI PRISM 3100 or 3130x Genetic Analyzer with 3100 Avant Genetic Analyzer Data Collection Software with Data Collection Software Version 3 0 Version 2 0 y Data Analysis Section 6 GeneMapper ID Software Version 3 2 Figure 1 An overview of the PowerPlex CS7 System protocol 2 Product Components and Storage Conditions Product Size Cat PowerPlex CS7 System 100 reactions DC6613 Not For Medical Diagnostic Use This system contains sufficient reagents for 100 reactions of 25l each Includes Pre amplification Components Box 500u1 PowerPlex HS 5X Master Mix 250ul PowerPlex CS7 10X Primer Pair Mix 25ul 2800M Control DNA 10ng ul 2x1 25ml Water Amplification Grade Post amplification Components Box 501 PowerPlex CS7 Allelic Ladder Mix 150p1 Inter
15. for sample preparation DNA purification and DNA quantitation prior to STR amplification For analysis of database reference and other single source samples we recommend direct amplification from FTA punches or preprocessing of swabs and nonFTA punches with the SwabSolution Kit or PunchSolution Kit The SwabSolution Kit Cat DC8271 contains reagents for rapid DNA preparation from buccal swab samples prior to 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 For casework or samples that require DNA purification we recommend the DNA IQ System Cat DC6700 which is a DNA isolation system designed specifically for forensic samples 18 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 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 an
16. one or two 1 2mm punches from a storage card containing a buccal sample or one 1 2mm punch from a storage card containing whole blood per 25 1 amplification 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 the 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 i e smaller in size 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 F for additional information on 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
17. peaks are particularly intense one or more extra peaks can be seen occasionally in the fluorescein channel at 254bp 273bp 301bp 357bp 379bp 429bp or 479bp 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 TMD042 Page 28 Revised 6 14 8831TA 7 Troubleshooting w 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 Promega 7 A Amplification and Fragment Detection This section provides information about general amplification and detection For questions about direct amplification see Sections 7 B and 7 C Symptoms 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 Incorrect amplification program Confirm the amplification program The PowerPlex HS 5X Master Mix was not vortexed well before use Vortex the 5X Master Mix for 15 seconds before dispensing into reaction tubes or plates An air bubble formed at the bottom of the reaction tube Use a pipette to remove the air bubble or centrifuge th
18. 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 Printed in USA Part TMD042 Revised 6 14 Page 35 7 C Direct Amplification of DNA from Swabs continued Symptoms Causes and Comments Extreme variability in sample There can be significant individual to individual variability to sample peak heights in cell deposition onto buccal swabs This will appear as variability in peak heights between swab extracts The 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 Software 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 m
19. the tube of 2800M Control DNA then add 1pl 10ng of the 2800M Control DNA to a reaction well containing 25pl of PCR amplification mix Notes 1 Do not include blank storage card punches in the positive control reactions 2 Optimization of the amount of 2800M Control DNA may be required based 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 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 Seal the plate and briefly centrifuge the plate to bring storage card punches to the bottom of the wells and remove any air bubbles Note Place the 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 Thermal Cycling Amplification and detection instrumentation may vary
20. 24 1The length of each allele in the allelic ladder has been confirmed by sequence analyses 2When 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 3For 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 Table 4 The PowerPlex CS7 System Allele Determinations in Commonly Available Standard DNA Templates Standard DNA Template STR Locus 9947A1 2800M LPL 11 12 11 13 F13B 8 10 6 9 FESFPS 10 12 11 11 F13A01 6 16 By Penta D 12 12 1213 Penta C iL 2 JOPI Penta E 12 13 7 14 Information on strain 9947A is available online at http ccr coriell org Sections Search Sample_Detail aspx Ref GM09947 Information about the use of 9947A DNA as a standard DNA template can be found in reference 17 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 TMD042 Revised 6 14 Page 41 9 B DNA Extraction and Quantitation Methods and Automation Support Promega offers a wide variety of reagents and automated methods
21. 25 0u1 x a PowerPlex HS 5X Master Mix 5 0ul x PowerPlex CS7 10X Primer Pair Mix 2 5ul x template DNA 0 5ng 4 up to 17 5ul total reaction volume 25ul 1Add Water Amplification Grade to the tube first then add PowerPlex HS 5X Master Mix and PowerPlex CS7 10X Primer Pair Mix The template DNA will be added at Step 6 Store DNA templates in TE buffer 10mM Tris HCl 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 PCR amplification 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 3Apparent DNA concentrations can differ depending on the DNA quantification method used 12 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 particular DNA quantification method 5 Vortex the PCR amplification mix for 5 10 seconds then pipet PCR amplification mix into each reaction well Failure to vortex the PCR amplification mix sufficiently can resul
22. 3B FL 1q31 q32 1 HUMBFXII Human factor AAAT XIII b subunit gene FESFPS FL 15q25 qter HUMFESFPS Human c AAAT fes fps proto oncogene F13A01 FL 6p24 p25 HUMF13A01 Human AAAG coagulation factor XIII a subunit gene Penta D FL 21q NA AAAGA Penta C JOE 9p13 NA AAAAC Penta E TMR 15q NA AAAGA FL fluorescein NA not applicable TMR carboxy tetramethylrhodamine JOE 6 carboxy 4 5 dichloro 2 7 dimethoxyfluorescein 1The August 1997 report 15 16 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 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 TMD042 Page 40 Printed in USA Revised 6 14 Table 3 The PowerPlex CS7 System Allelic Ladder Information 4 Size Range of Allelic Ladder Repeat Numbers of Allelic Ladder STR Locus Label Components bases Components Promega LPL FL 105 133 7 14 F13B EE 169 193 6 12 FESFPS FL 222 250 7 14 F13A01 Fl 279 331 3 16 Penta D FL 373 446 2 2 3 2 5 17 Penta C JOE 104 169 4 15 17 Penta E TMR 376 471 5
23. Not for use in diagnostic procedures ART Aerosol Resistant Tips Product Volume Size tips pack Cat ART 10 Ultramicro Pipet Tip 0 5 101 960 DY1051 ART 20E Ultramicro Pipet Tip 0 5 10p1 960 DY1061 ART 20P Pipet Tip 20ul 960 DY1071 ART GEL Gel Loading Pipet Tip 100u1 960 DY1081 ART 100 Pipet Tip 10041 960 DY1101 ART 100E Pipet Tip 100u1 960 DY1111 ART 200 Pipet Tip 200p1 960 DY1121 ART 1000E Pipet Tip 1 000p1 800 DY1131 9 F Summary of Changes The following change was made to the 6 14 revision of this document Legal disclaimers were updated 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 TMD042 Revised 6 14 Page 45 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 No 6 221 598 and Canadian Pat No 2 118 048 U S Pat No 6 242 235 European Pat No 1088060 Japanese Pat No 3673175 and other patents pending 2012 2014 Promega Corporation All Rights Reserved Maxwell Plexor and PowerPlex are registered trademarks of Promega Corporation AmpSoluti
24. Paternity Analysis Method Using a Global Filter 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 Pm GG fe 5 Enter a descriptive name for the analysis method such as PowerPlexCS7_20 filter 6 Select the Allele tab Figure 5 7 Select the bins text file that was obtained in Section 6 A 8 Ensure that the Use marker specific stutter ratio if available box is checked 9 Enter the values shown in Figure 5 for proper filtering of peaks when using the PowerPlex CS7 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 Dabo Pumega IEL CS7 Pa 220 e ima mamerqpertis cute cate t maistre Modei Repast fore Tas Perto Hwa Cason vone 60 o2 2 Abe et Bee 60 oo oo Mausa Datons o tem Ye O90 oo 90 Meas Enso Agte 49 T Mosas Into atoes o fem G0 238 a o 479 575 Pia itno tns eo oo Pim Divine ataa Ton eo d amare gene Catra 0 Range fener posty potosi a gw 8836TA Figure 5 The Allele tab with settings for using a 20 pe
25. TECHNICAL MANUAL PowerPlex CS7 System Instructions for use of Product DC6613 NOTE PowerPlex CS7 System Cat DC6613 and this Technical Manual TMD042 are replacements for PowerPlex CS7 System Custom Cat X6613 and Technical Manual TMDO030 Q Revised 6 14 TMD042 PowerPlex CS7 System 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 Veo DLE e o e OORE TEE 2 2 Product Components and Storage Conditions cccccccssssseessesssssssssseeeeesssssseeseesssssseesees 3 Be Before You Begifinusisenimenssnnensnnnasinn iiit 4 A Precautions B Spectral Calibration sinini aE EE EE Esa 5 4 Protocols for DNA Amplification Using the PowerPlex CS7 System 5 A Amplification of Extracted DNA B Direct Amplification of DNA from Storage Card Punches 10 C Direct Amplification of DNA from Swab3 ssccssssssssssssssssssssessssssssenessssssssneeees 14 5 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 cssssssseesesssssseessssssneeeessen 17 6
26. 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 TMD042 Revised 6 14 Page 29 7 A Amplification and Fragment Detection continued Symptoms Causes and Comments Extra peaks visible in one or all color channels Contamination with another template DNA or previously amplified DNA Cross contamination can be a problem Use aerosol resistant pipette tips and change gloves regularly Samples were not denatured completely Heat denature samples for the recommended time and cool on crushed ice or in an ice water bath immediately prior to capillary 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 F for additional information on 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 High background Load less amplification product or decrease injection time See Section 5 CE related artifacts spikes Minor voltage chan
27. action and Quantitation Methods and Automation Support 42 C The Internal Lane Standard 600 43 D Composition of Buffers and Solutions 43 E Related Prodtacts scscscscsvssssssssssssssssssssssssssssssssteccscsssassscssccssassscscccssiavvssassennssvsssnestnecassnseren 44 Bi Summary OF Changes ynan n 45 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 TMD042 Revised 6 14 Page 1 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 CS7 System is used for human identification applications and research use The system allows co amplification and three color detection of seven STR loci including LPL F13B FESFPS F13A01 Penta D Penta C and Penta E One primer for each of the LPL F13B PESFPS F13A01 and Penta D loci is labeled with fluorescein FL one primer for the Penta E locus is labe
28. age Card Punches PCR Amplification Mix Volume x Number of _ Final Component Per Reaction Reactions Volume Water Amplification Grade 12 5u1 x PowerPlex HS 5X Master Mix 5 0ul x PowerPlex CS7 10X Primer Pair Mix 2 51 x 5X AmpSolution Reagent 5 0u x total reaction volume 25ul 1Add Water Amplification Grade to the tube first then add PowerPlex HS 5X Master Mix and PowerPlex CS7 10X Primer Pair Mix For FTA card punches the template DNA will be added at Step 6 5 Vortex the PCR amplification mix for 5 10 seconds then pipet 2511 of PCR amplification mix into each reaction well Failure to vortex the PCR amplification mix sufficiently can result in poor amplification or locus to locus imbalance 6 For FTA storage cards add one or two 1 2mm punches from a card containing a buccal sample or one 1 2mm punch from a card containing whole blood to the appropriate wells of the reaction plate For nonFTA card punches add PCR amplification mix to the pretreated punches Note It also is acceptable to add the FTA card punch first then add the PCR amplification mix 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 TMD042 Revised 6 14 Page 11 4 B Direct Amplification of DNA from Storage Card Punches continued Ta For the positive amplification control vortex
29. ak filter 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 TMD042 Printed in USA Page 22 Revised 6 14 10 Select the Peak Detector tab We recommend the settings shown in Figure 6 A Notes 1 Select full range or partial range for the analysis range When using a Promega 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 Ba Carers amate Fusk Dalasi Paak Quality Quality Flaget Paak Detection Algotthty Advanced Ranges Peak Detection Analysis ie Sieg i Pea Ampitede Thresholds Full Range 2 J Pata siz yj B 100 R 10 Start Size 00 pez 1 Stop Size 600 G 100 Or 00 Y 100 Smoothing and Basslining Min Peak Halt Widin 2 pe Smoothing O Hons Potynamial Degtee 3 O Heaw Peak Window Size pts y Baseline 61 vb Slope Threstedd Weak Start 00 Hee C afine Pesk End 00 2nd Order Leart tigaser 31d Order Least Sqeares Catic Spline tntempotation Lov
30. al Sostens Method O Global Southam Method Figure 6 The Peak Detector tab 8835TA 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 Printed in USA Part TMD042 Revised 6 14 Page 23 C 6 C Creating a Databasing or Paternity Analysis Method Using a Global Filter o with GeneMapper ID Software Version 3 2 continued Promega 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 injection that is designated as Ladder in the Sample Type column for proper genotyping 5 In the Analysis Method column select the analysis method created previously in this section 6 In the Panel colum
31. concentrated at the bottom of the tube 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 2m1 reaction tubes required and label appropriately 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 TMD042 Printed in USA Page 6 Revised 6 14 4 Add the final volume of each reagent listed in Table 1 to a sterile tube C o Amplification of gt 1 0ng of DNA template results in an imbalance in peak heights from locus to locus The smaller loci show greater amplification Promega 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 x Numberof _ Final PCR Amplification Mix Component Per Reaction Reactions Volume to a final Water Amplification Grade volume of
32. ction In addition to signal saturation excess DNA in the capillary is difficult to maintain 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 i e smaller in size 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 cycle number Active protease carried over from swab extracts into the amplification reaction Larger loci are most suspectible to protease carryover and will drop out before the 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 incomplete protease 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 Inactive SwabSolution Reagent Thaw the SwabSolution Reagent completely in a 37 C waterbath and mix by gentle inversion Store the SwabSolution Reagent at 2 10 C Do not store reagents in the
33. cts Reagents and materials used prior to amplification PowerPlex HS 5X Master Mix 2800M Control DNA and PowerPlex CS7 10X Primer Pair Mix are provided in a separate box and should be stored separately from those used following amplification PowerPlex CS7 Allelic Ladder Mix and 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 TMD042 Page 4 Printed in USA Revised 6 14 Always include a negative control reaction i e no template to detect reagent C contamination We highly recommend the use of gloves and aerosol resistant A pipette tips e g ART tips Section 9 E Promega Some reagents used in the analysis of STR products are potentially hazardous and should be handled accordingly 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 3 B Spectral Calibration Proper spectral calibration is critical to evaluate multicolor systems with the ABI PRISM 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 3100 3130 Cat DG4650 is required for spectral calib
34. d 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 PowerPlex Systems to ensure a streamlined process For applications requiring human specific DNA quantification the Plexor HY System Cat DC1000 was developed 19 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 Part TMD042 Page 42 Printed in USA Revised 6 14 9 C 9 D The Internal Lane Standard 600 The Internal Lane Standard ILS 600 contains 22 DNA fragments of 60 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 500 550 and 600 bases in length Figure 13 Each fragment is labeled with carboxy X rhodamine CXR and can be detected separately as a fourth color in the presence of PowerPlex CS7 amplified material The ILS 600 is designed for use in each CE injection to increase precision in analyses when using the PowerPlex CS7 S
35. de in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause a 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 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 TMD042 Revised 6 14 Page 17 5 Detection of Amplified Fragments Using the ABI PRISM 3100 or 3100 Avant v Genetic Analyzer with Data Collection Software Version 2 0 or the Applied Pro Biosystems 3130 or 3130x Genetic Analyzer with Data Collection Software m G Version 3 0 continued 2 Prepare a loading cocktail by combining and mixing Internal Lane Standard 600 and Hi Di formamide as follows 0 5u1 ILS 600 x sampl
36. e 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 avoid multiple freeze thaw cycles as this may reduce activity Faint or absent peaks for the positive control reaction If the positive control reaction failed to amplify check to make sure that the correct amount of 2800M Control DNA was added to the reaction We recommend 10ng of 2800M Control DNA per 25pl amplification reaction Do not include a blank punch in the positive control reaction Presence of a blank punch may inhibit amplification of 2800M Control DNA e Optimize the amount of 2800M Control DNA for your thermal cycling conditions and laboratory preferences Improper storage of the 2800M Control DNA 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 TMD042 Page 32 Printed in USA Revised 6 14 Symptoms Causes and Comments C Extra peaks visible in one or Punch was contaminated Clean the punch by taking blank o all color channels punches between samples Amplification of processed punches with high amounts of Promega DNA can result in artifact peaks due to overamplification resulting in saturating signal on the CE instrument We recommend
37. e 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 Nat 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 1mM EDTA or TE buffer with 20ug ml glycogen 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 Calibrate the thermal cycler heating block if necessary Primer concentration was too low Use the recommended primer concentration Vortex the PowerPlex CS7 10X Primer Pair Mix for 15 seconds before use Poor capillary electrophoresis injection ILS 600 peaks also affected Re inject the sample Check the syringe or pump system for leakage Check the laser power Samples were not denatured completely Heat denature samples for the recommended time then cool on crushed ice or in an ice water bath immediately prior to capillary electrophoresis Do not cool samples in a thermal cycler set at 4 C as this may lead to artifacts due to DNA re annealing Poor quality formamide was used Use only Hi Di formamide when analyzing samples Promega Corporation 2800 Woods Hollow Road Madison
38. ell lines GM9947 and GM9948 as intra and interlaboratory reference standards for DNA typing Genomics 28 184 97 18 Mandrekar P V Krenke B E and Tereba A 2001 DNA IQ The intelligent way to purify DNA Profiles in DNA 4 3 16 19 Krenke B E et al 2005 Development of a novel fluorescent two primer approach to quantitative PCR Profiles in DNA 8 1 3 5 Additional STR references can be found at www promega com geneticidentity 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 TMD042 Revised 6 14 Page 39 C 9 Appendix I 9 A Advantages of STR Typing The loci included in the PowerPlex CS7 System are listed in Tables 2 and 3 Table 4 lists the PowerPlex CS7 System alleles revealed in commonly available standard DNA templates 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 CS7 System Table 2 The PowerPlex CS7 System Locus Specific Information Chromosomal GenBank Locus and Repeat Sequence STR Locus Label Location Locus Definition 5a 3 LPL FL 8p22 HUMLIPOL Human AAAT lipoprotein lipase gene F1
39. es 9 5u1 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 01 of ILS 600 and 9 0u1 of Hi Di formamide If peak heights are too high we recommend altering the loading cocktail to contain 0 25ul of ILS 600 and 9 75y1 of formamide 3 Vortex for 10 15 seconds to mix 4 Pipet 10p1 of formamide internal lane standard mix into each well 5 Add 1pl of amplified sample or 1ul of PowerPlex CS7 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 use less DNA template in the amplification reaction 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 min
40. ges 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 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 Pull up or bleedthrough Pull up can occur when peak heights are too high or if a poor or incorrect matrix was applied to the samples Perform a new spectral calibration and re run the samples Instrument sensitivities can vary Optimize the injection conditions See Section 5 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 blue and green dye colors Use autoclaved deionized water change vials and wash buffer reservoir Long term storage of amplified sample in formamide can result in degradation Repeat sample preparation using fresh formamide 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 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526
41. id software panels and bin sets Product Size Cat PowerPlex Matrix Standards 3100 3130 25ul each dye DG4650 Not For Medical Diagnostic Use 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 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130x1 Genetic Analyzers PowerPlex Matrix Standards 3100 3130 See Section 9 E for ordering information 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 9 10 Guidelines for the validation process are published in the Internal Validation of STR Systems Reference Manual 11 The quality of purified DNA 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 are made to the recommended protocols 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 sample DNA handling primer pairs assembling amplification reactions and analyzing amplification produ
42. ighted click the plate graphic that corresponds 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 6 Data Analysis 6 A Importing PowerPlex CS7 Panels and Bins Text Files with GeneMapper ID Version 3 2 To facilitate analysis of data generated with the PowerPlex CS7 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 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 TMD042 Revised 6 14 Page 19 6 A Importing PowerPlex CS7 Panels and Bins Text Files with GeneMapper
43. in USA Revised 6 14 Symptoms Causes and Comments C Off ladder alleles An allelic ladder from a different run than the samples was o used Re analyze samples with an allelic ladder from the same i Promega 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 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 A low quality allelic ladder was used during analysis Ensure that only high quality allelic ladders are used for analysis Size standard not called Starting data point was incorrect for the partial range chosen correctly Figure 12 in Section 6 D Adjust the starting data point in the analysis method Alternatively use a full range for the analysis Extra peaks in advanced mode size standard Open the Size Match Editor Highlight the extra peak select Edit and select delete
44. increased to 10 22 3 tmp 10 cycles 94 0 C 3 tmp 22 cycles 29 1 1 1 i L 100 100 1 70 0 C 1 70 0 C i 23 23 1 60 0 C 60 0 C 1 1 1 1 1 74g6MA 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 Printed in USA Revised 6 14 Part TMD042 Page 9 4 B Direct Amplification of DNA from Storage Card Punches Materials to Be Supplied by the User GeneAmp PCR System 9700 thermal cycler Applied Biosystems microcentrifuge e MicroAmp optical 96 well reaction plate Applied Biosystems e aerosol resistant pipette tips see Section 9 E PunchSolution Kit Cat DC9271 for nonFTA card punches this kit includes the 5X AmpSolution Reagent 1 2mm Harris Micro Punch or equivalent manual punch and cutting mat or automated punch system This section contains a protocol for direct amplification of DNA from storage card punches using the PowerPlex CS7 System and GeneAmp PCR System 9700 thermal cycler When using the protocol detailed below add the number of 1 2mm storage card punches indicated below to each 25pl amplification reaction Note You will need to optimize and validate the number of storage card punches per reaction in your laboratory FTA based sample types incl
45. ining whole blood into each well of a reaction plate Be sure to pretreat nonFTA samples with the PunchSolution Kit Cat DC9271 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 TMD042 Revised 6 14 Page 13 4 B Direct Amplification of DNA from Storage Card Punches continued Prepare three identical reaction plates with punches from the same samples Amplify samples using the thermal cycling protocol provided above but subject each plate to a different cycle number 10 16 10 17 and 10 18 cycling Following amplification use your laboratory s validated separation and detection protocols to determine the optimal cycle number for the sample type and number of storage card punches 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 E SwabSolution Kit Cat DC8271 This section contains a protocol for amplifying swab extracts using the PowerPlex CS7 System and GeneAmp PCR System 9700 thermal cycler Pretreat cotton or OmniSwabs GE Healthcare swabs with the SwabSolution Kit Cat DC8271 as described in the SwabSolution Kit Technical Manual TMD037 t
46. iw f mE lt ey ATAT TREA IIIT Lisan Su z biti Ce tE TIT Figure 10 The PowerPlex CS7 Allelic Ladder Mix The PowerPlex CS7 Allelic Ladder 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 CS7 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 Panel D The Internal Lane Standard 600 showing fragments of 80bp to 500bp Artifacts and Stutter Stutter products are a common amplification artifact associated with STR analysis 13 14 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 In addition to stutter peaks other artifact peaks can be observed at some of the PowerPlex CS7 loci Low level products can be seen at n 1 at LPL and Penta C at n 9 and n 1 at F13B and at n 12 to n 13 at FESPS and F13A01 When the amplified
47. led with carboxy tetramethylrhodamine TMR and one primer for the Penta C locus is labeled with 6 carboxy 4 5 dichloro 2 7 dimethoxy fluorescein JOE All seven loci are amplified simultaneously in a single tube and analyzed in a single injection The PowerPlex CS7 System contains two loci that overlap with loci included in the PowerPlex 16 HS System Penta D and Penta E This feature allows the PowerPlex CS7 System to be used as a confirmatory kit in paternity applications using the five unshared STR loci to supplement the genotype and increase the available information The PowerPlex CS7 System is compatible with the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130x1 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 amount of template DNA cycle number injection conditions and loading volume for your laboratory instrumentation In house validation should be performed The PowerPlex CS7 System provides all materials necessary to amplify STR regions of purified human genomic DNA This manual contains separate protocols for use of the PowerPlex CS7 System with GeneAmp PCR System 9600 and 9700 thermal cyclers in addition to protocols to separate amplified products and detect separated material Figure 1 Protocols to operate the fluorescence
48. loading volume for your laboratory instrumentation Testing at Promega Corporation shows that 10 20 cycles work well for 0 5ng of purified DNA templates The cycle number can be increased to 10 22 to maximize sensitivity For higher template amounts or to decrease sensitivity fewer cycles such as 10 18 should be evaluated In house validation should be performed 1 Place MicroAmp plate or reaction tubes in the 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 TMD042 Printed in USA Page 8 Revised 6 14 2 Select and run the recommended protocol The preferred protocols for use with the GeneAmp PCR System 9600 and 9700 thermal cyclers 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 Protocol for the GeneAmp PCR System 9700 Thermal Cycler Protocol for the GeneAmp PCR System 9600 Thermal Cycler 96 C for 2 minutes 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 20 cycles then 60 C for 30 minutes 4 C soak 96
49. lution Reagent completely in a 37 C waterbath and mix by gentle inversion Store 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 Active protease carried over into the amplification reaction from the SwabSolution Reagent 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 protease 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 incubation Make sure that the PCR amplification mix also contained AmpSolution Reagent Omission of AmpSolution Reagent from amplification reactions will result in amplification failure 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 TMD042 Page 34 Printed in USA Revised 6 14 Symptoms Causes and Comments C Faint or absent peaks for the If the positive control reaction failed to amplify check
50. mation 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 TMD042 Page 10 Printed in USA Revised 6 14 Note Static may be problematic when adding a punch to a well For FTA card C punches adding PCR amplification mix to the well before adding the punch eI may help alleviate static problems Amplification Setup 1 Thaw the PowerPlex HS 5X Master Mix PowerPlex CS7 10X Primer Pair Mix and Water Amplification Grade completely Note 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 or 5X Master Mix after vortexing as this may cause the reagents to be concentrated at the bottom of the tube 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 2 to a sterile tube Table 2 PCR Amplification Mix for Direct Amplification of DNA from Stor
51. ms user Pro bulletin titled Installation Procedures and New Features for GeneMapper ID m g 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 B Uonorsi Allele Paak Detertor Peake Quality Quality I lage Bie Sek Promega_16 5_C amp 7_ ins_320 x Z Use makarspeotio stutter rate available Mater Nepeat Type Th Teta Vents Hexa Cubott Vados oo 00 00 00 Minuta Ratio oo 0a on oo Minsa Oistance fom 00 99 90 oo Te oo on 00 oo Minut titeter Hatia oo 00 oo 00 Minus Gestas Distance fom 00 3 26 ais oc Te oo 476 676 oo Pius Gtutter Ratio oo 00 oo oo Plut Stutter Ditaace Isa oo 00 oo 00 Te eo 0a oo oo Ameiegema Cutan oo Mange fumed Factory Oetauits lt 18 Figure 7 The Allele tab 10 Select the Peak Detector tab We recommend the settings shown in Figure 8 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 Promega Corporati
52. n select the panels text file that was imported in Section 6 A 7 In the Size Standard column select the size standard that was created in Section 6 B 8 Select Analyze green arrow button to start the data analysis me mor 6 D Creating an Analysis Method Without a General Filter in GeneMapper ID Software Version 3 2 These instructions loosely follow the Applied Biosystems GeneMapper ID software tutorial pages 1 11 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 Enter a descriptive name for the analysis method such as PowerPlexCS7 advanced Select the Allele tab Figure 7 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 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 TMD042 Page 24 Printed in USA Revised 6 14 9 Enter the values shown in Figure 7 for proper filtering of stutter peaks C when using the PowerPlex CS7 System For an explanation of the proper o usage and effects of these settings refer to the Applied Biosyste
53. n the GeneAmp PCR System 9700 thermal cycler with the 384 well dual block ramps does not exist and is not required to program ramp rates 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 appropriate ramp rates for the GeneAmp PCR System 9700 thermal cycler The ramp mode is set after start is 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 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 Depending on your preferred protocol place one or two 1 2mm storage card punches containing a buccal sample or one 1 2mm punch of a storage card conta
54. nal Lane Standard 600 The PowerPlex CS7 Allelic Ladder Mix is provided in a separate sealed bag for shipping This component should be moved to the post amplification box after opening PowerPlex HS 5X Master Mix and PowerPlex CS7 10X Primer Pair Mix are manufactured as a matched set for optimal performance Do not combine components from kits with different lot numbers printed on the boxes and Certificates of Analysis If lots are mixed locus to locus imbalance and variation in signal intensity may occur 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 TMD042 Revised 6 14 Page 3 Product Components and Storage Conditions continued 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 CS7 10X Primer Pair Mix PowerPlex CS7 Allelic Ladder Mix and Internal Lane Standard 600 ILS 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 Available Separately The proper panels and bins text files for use with GeneMapper ID software can be obtained from the Promega web site at www promega com resources tools genemapper
55. o generate a swab extract Amplification Setup T Thaw the PowerPlex HS 5X Master Mix PowerPlex CS7 10X Primer Pair Mix and Water Amplification Grade completely Note 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 or 5X Master Mix after vortexing as this may cause the reagents to be concentrated at the bottom of the tube 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 MicroAmp plate for reaction assembly and label appropriately 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 TMD042 Page 14 Printed in USA Revised 6 14 4 Add the final volume of each reagent listed in Table 3 to a sterile tube C o Table 3 PCR Amplification Mix for Direct Amplification of DNA From Swabs PCR Amplification Mix Volume Per Number of Final Component Reaction x Reactions Volume Water Amplification Grade 10 5 x PowerPlex HS 5X Master Mix 5 0
56. odule drop down list Lastly select F in the Dye Set drop down list Select OK Pro mega 3 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 4 Inthe 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 5 Place samples in the instrument and close the instrument doors 6 Inthe spectral viewer confirm that dye set F is active and set the correct active calibration for dye set F 7 Inthe run scheduler locate the plate record that you just created in Steps 3 and 4 and click once on the name to highlight it 8 Once the plate record is highl
57. on Differex DNA IQ PunchSolution Slicprep and SwabSolution are trademarks of Promega Corporation ABI PRISM and MicroAmp are registered trademarks of Applera Corporation Applied Biosystems and GeneMapper are registered trademarks of Applied Biosystems ART is a registered trademark of Molecular Bio Products Inc Bode Buccal DNA Collector is a trademark of the Bode Technology Group Inc EasiCollect and OmniSwab are trademarks of Whatman 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 GeneAmp is a registered trademark of Roche Molecular Systems Inc Hi Di and POP 4 are trademarks of Applera Corporation Sampact is a trademark of Fitzco Vacutainer is a registered trademark of Becton Dickinson and Company 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 Part TMD042 Page 46 Printed in USA Revised 6 14
58. on 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 TMD042 Revised 6 14 Page 25 6 D Creating an Analysis Method Without a General Filter in GeneMapper ID Software Version 3 2 continued Analysis Method Editor HID Genel Ansie Peak Datuei Peake Quality Quality Flaget Paak Uatectan Algonthmt Advanced co Ranger Peak Detection Analysis z Sieg Peat Amgitete Thresholds Full Range xj Parisi Siz yj B 100 R co Start Size 00 1 Stop Size 600 G 100 100 Y 10 Smoothing and Baselining Min Peak Hait Width 2 pe Smoothing O None Potynamial Degtee 3 O Heaw Peak Window Size 1 pts Bassline 61 bb Slope Threstebd Pesk Stat 00 FEM pode tale Peak End 00 2nd Order Leart gases 31d Onder Least Squares Catic Spline tntampotation Local Sevthem Methed oO Global Southam Methyd Figure 8 The Peak Detector tab 11 12 13 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 Select the Quality Flags tab You may change these settings Select OK to save your settings Processing Data for Samples Without a General Filter 1 2 3 Select File then New Project Select Edit then Add Samples to Project Browse to the location of
59. ottom of the wells and remove any air bubbles 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 TMD042 Revised 6 14 Page 15 4 C Direct Amplification of DNA from Swabs continued Thermal Cycling Amplification and detection instrumentation may vary You will need to optimize protocols including amount of template DNA cycle number injection conditions and loading volume for your laboratory instrumentation Testing at Promega Corporation shows that 10 18 cycling 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 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 96 C for 2 minutes 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 1When using the GeneAmp PCR System 9700 thermal cycler the ramp rates indicated in the cycling program must be set For the GeneAmp PCR System 9700 thermal
60. problems Thaw the 10X Primer Pair Mix and 5X Master Mix completely and vortex for 15 seconds before use Do not centrifuge the 10X Primer Pair Mix after mixing Calibrate thermal cyclers and pipettes routinely Using a 59 C annealing temperature instead of 60 C has been shown to improve balance in some instances Impure template DNA Inhibitors that may be present in forensic samples can lead to allele dropout or imbalance Impure template DNA Include a proteinase K digestion prior to DNA purification PCR amplification mix prepared in Section 4 was not mixed well Vortex the PCR amplification mix for seconds before dispensing into the reaction tubes or plate Tubes of 5X Master Mix and 10X Primer Pair Mix from different lots were used The PowerPlex HS 5X Master Mix and PowerPlex CS7 10X Primer Pair Mix are manufactured as a matched set for optimal performance If lots are mixed locus to locus imbalance and variation in signal intensity may occur 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 TMD042 Revised 6 14 Page 31 7 B Direct Amplification of DNA from Storage Card Punches The following information is specific to direct amplification of DNA from storage card punches For information about general amplification and detection see Section 7 A
61. rPlex ESX 16 System 100 reactions DC6711 400 reactions DC6710 PowerPlex ESX 17 System 100 reactions DC6721 400 reactions DC6720 PowerPlex ESI 16 System 100 reactions DC6771 400 reactions DC6770 PowerPlex ESI 17 Pro System 100 reactions DC7781 400 reactions DC7780 Not for Medical Diagnostic Use Accessory Components Product Size Cat PowerPlex Matrix Standards 3100 3130 25ul each dye DG4650 Internal Lane Standard 600 150ul DG1071 Water Amplification Grade 6 250ul 5 x 1 25041 DW0991 2800M Control DNA 10ng ul 25pl DD7101 2800M Control DNA 0 25ng ul 500pl DD7251 PunchSolution Kit 100 preparations DC9271 SwabSolution Kit 100 preparations DC8271 5X AmpSolution Reagent 500 ul DM1231 Not for Medical Diagnostic 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 TMD042 Page 44 Printed in USA Revised 6 14 Sample Preparation Systems 4 Product Size Cat DNA IQ System 100 reactions DC6701 Promega 400 reactions DC6700 Differex System 50 samples DC6801 200 samples DC6800 Maxwell 16 Forensic Instrument leach AS3060 DNA IQ Reference Sample Kit for Maxwell 16 48 preps AS1040 DNA IQ Casework Pro Kit for Maxwell 16 48 preps AS1240 Slicprep 96 Device 10 pack V1391 Not for Medical Diagnostic Use For Research Use Only
62. ration on the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xl Genetic Analyzers For protocols and additional information on spectral calibration see the PowerPlex Matrix Standards 3100 3130 Technical Bulletin TBD022 available online at www promega com protocols 4 Protocols for DNA Amplification Using the PowerPlex CS7 System The PowerPlex CS7 System is optimized for the GeneAmp PCR System 9700 thermal cycler An amplification protocol for the GeneAmp PCR System 9600 thermal cycler also is 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 qPCR may result in a different value Prepare a fresh DNA dilution for each set of amplifications Do not store dilute DNA e g 0 25ng l or less 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
63. redefine the size standard for the sample to skip these peaks Error message Either panel size standard or analysis method is invalid The size standard and analysis method were not in the same mode Classic vs Basic or Advanced Be sure both files are set to the same mode either Classic or Basic or Advanced mode No alleles called but no error message appears Panels text file was not selected for sample In the Panel column select the appropriate panels text file for the STR system 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 Both the Bin Set used in the Analysis Method and the Panel must belong to the same Chemistry Kit The bins text file assigned to the analysis method was deleted In the GeneMapper Manager select the Analysis Methods tab and open the analysis method of interest Select the Alleles tab and select an appropriate bins text file The wrong bins text file was chosen in the analysis method Allele tab Be sure to choose the appropriate bins tex
64. 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 Definition and detection of the 600bp fragment is optional 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 mept more Preetoria LI ed JIOA We clauon ON Mure Fart ADT oo m joe Size m File a View Tumis rao a x oo sso Standard u Han manear swe Catas Come i co Quality 5s no tang Cray 08 Ovome s9 r mno A j w sano z r aano E G vano xa 8 n ne uano A a a re sano A 1 G A i 2 nm A a ponam m ne A a z re m 5 G um a m n A E re A E a A E 5 5686TA 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 Printed in USA Part TMD042 Revised 6 14 Page 37 7 D GeneMapper ID Software continued Symptoms Causes and Comments Peaks in size standard missing If peaks are below threshold decrease the peak amplitude threshold in the analysis method for the red channel to include peaks If peaks are low quality
65. t be changed If the method is not HID it should be deleted and a new analysis method created 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 TMD042 Page 38 Printed in USA Revised 6 14 8 References C o 1 Edwards A et al 1991 DNA typing with trimeric and tetrameric tandem repeats Polymorphic loci detection systems and population genetics In The Second International Symposium on Human Promega Identification 1991 Promega Corporation 31 52 2 Edwards A et al 1991 DNA typing and genetic mapping with trimeric and tetrameric tandem repeats Am J Hum Genet 49 746 56 3 Edwards A et al 1992 Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups Genomics 12 241 53 4 Warne D et al 1991 Tetranucleotide repeat polymorphism at the human b actin related pseudogene 2 actbp2 detected using the polymerase chain reaction Nucleic Acids Res 19 6980 5 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 6 Sambrook J Fritsch E F and Maniatis T 1989 Chapter 14 In vitro amplification of DNA by the polymerase chain reaction In Molecular Cloning A Laboratory Manual Second Edition Cold Spring Harbor Laboratory Press Cold Spring Harbor
66. t 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 e Using a smaller amplification reaction volume may compromise performance when using 10 1 of PunchSolution Reagent Reducing the PunchSolution Reagent volume may improve results for reactions with reduced amplification volumes Optimization and validation are required Inactive PunchSolution Reagent Thaw PunchSolution 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 Extreme variability in sample to sample peak heights There can be significant individual to individual variability in 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 Amplification 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 SwabSo
67. t file as shown in Figure 5 Significantly raised baseline Poor spectral calibration Perform a new spectral calibration and re run the samples 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 Error message after attempting to import panels and bins text files Unable to save panel data java SQLEException ORA 00001 unique constraint IFA CKP_NNN violated There was a conflict between different sets of panels and bins text files Check to be sure that the bins are installed properly If not delete all panels and bins text files and re import files in a different order Allelic ladder peaks are labeled off ladder GeneMapper ID software was not used or microsatellite 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 canno
68. t in poor amplification peak height imbalance and extra peaks in the range of 50 80bp 6 Add the template DNA 0 5ng for each sample to the respective well containing PCR amplification mix 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 TMD042 Revised 6 14 Page 7 C 4 A Amplification of Extracted DNA continued o 7 For the positive amplification control vortex the tube of 2800M Control Pr omega DNA then dilute an aliquot to 0 5ng in the desired template DNA volume Add 0 5ng of the diluted DNA to a reaction well containing PCR amplification mix 8 For the negative amplification control pipet Water Amplification Grade or TE buffer instead of template DNA into a reaction tube containing PCR amplification mix 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 manual contains protocols for use of the PowerPlex CS7 System with the GeneAmp PCR System 9600 and 9700 thermal cyclers For information on other thermal cyclers contact Promega Technical Services by e mail at genetic promega com Amplification and detection instrumentation may vary You may need to optimize protocols including the amount of template DNA cycle number injection conditions and
69. the run files Highlight desired files then select Add to list followed by Add 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 Ladder in the Sample Type column for proper genotyping In the Analysis Method column select the analysis method previously created in this section 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 TMD042 Page 26 Printed in USA Revised 6 14 6 Inthe Panel column select the panels text file that was imported in C Section 6 A o 7 Inthe Size Standard column select the size standard that was created in Promega Section 6 B 8 Select Analyze green arrow button to start the data analysis 6 E Controls 1 Observe the results for the negative control Using the protocols defined in 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 4 Section 9 A 6 F Results Representative results of the PowerPlex
70. to o ositive control reaction make sure that the correct amount of 2800M Control DNA P was added to the reaction Due to the reduced cycle numbers Promega 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 Improper storage of the 2800M Control DNA Extra peaks visible in one Swab extract was contaminated Assemble a reaction or all color channels 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 Artifacts of STR amplification Amplification of swab extracts with high DNA concentrations can result in artifact peaks due to overamplification resulting in saturated signal on the CE instrument We recommend 2 of swab extract per 25y1 reaction Using more than 2u in a 25u reaction or using 2u1 with a smaller reaction volume may result in overamplification and signal saturation If signal is saturated repeat amplification with less swab extract or reduced cycle number Amplification of excess template for a given cycle number resulted in overloading of the capillary upon electrokinetic inje
71. ude Buccal cells collected on FTA cards with Whatman EasiCollect or Fitzco Sampact devices one or two punches per 25ul amplification reaction Buccal cells collected with sterile swabs transferred to FTA or Indicating FTA cards one or two punches per 25y amplification reaction Liquid blood from collection or storage Vacutainer tubes or finger sticks spotted onto FTA cards one punch per 25ul amplification reaction NonFTA sample types include Buccal samples on Bode Buccal DNA Collector devices one punch per 25ul amplification reaction e Blood and buccal samples on nonFTA card punches e g S amp S 903 one punch per 25y1 amplification reaction Pretreat these sample types with the PunchSolution Reagent 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 infor
72. ul x PowerPlex CS7 10X Primer Pair Mix 2 5ul x 5X AmpSolution Reagent 5 0ul x swab extract 2 0ul total reaction volume 25ul 1Add Water Amplification Grade to the tube first then add PowerPlex HS 5X Master Mix and PowerPlex CS7 10X Primer Pair Mix The swab extract 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 Failure to vortex the PCR amplification mix sufficiently can result in poor amplification or locus to locus imbalance D 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 DNA then dilute an aliquot to 2 5ng pl and add 2pl 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 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 9 Seal the plate Optional Briefly centrifuge the plate to bring contents to the b
73. ust be defined An insufficient number of ILS 600 fragments was defined Be sure to define at least two ILS 600 fragments smaller than the smallest sample peak and at least two ILS 600 fragments larger than the largest sample 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 Re run samples using a longer run time A low quality allelic ladder was used during analysis Ensure that only high quality allelic ladders are used for analysis LL OS Aae toto namwona EPT Data Sample tuformation Saaple File CRE _172h_ H0S 2004 06 17 fea Sample Origin Parh Gt Privare Teennical Servier GT date genetice CRE_172h_HOS_2004 06 17 ts0 Status Message 1 Changed size standard from 1is8050Gndv co TLSS00 Classic File Source Disk aedis Frio Message fatal genetica CRE 172n_ HOO 2004 06 17 fsa Either Panel Sire Standard ort alysis Method vas invalid 5685TA Figure 11 The error message that appears in the GeneMapper ID software when the analysis parameters 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 Part TMD042 Page 36 Printed
74. utes Denature samples just prior to loading the instrument 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 3130x Genetic Analyzer with the following exceptions 1 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 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 TMD042 Printed in USA Page 18 Revised 6 14 2 Inthe Protocol Manager select New Type a name for your protocol Select C Regular in the Type drop down list and select the run module you created o in the previous step in the Run M
75. ystem Protocols to prepare and use this internal lane standard are provided in Section 5 a e 10349TA Figure 13 Internal Lane Standard 600 An electropherogram showing the Internal Lane Standard 600 fragments Composition of Buffers and Solutions TE buffer 10mM Tris HCl TE buffer with 20u ml glycogen 0 1mM EDTA pH 8 0 iig Tris base 1 21g Tris base 0 037 EDTA 0 037g EDTA Na EDTA 2H O Na EDTA 2H 0 ug ml glycogen Dissolve Tris base and EDTA in 900ml of deionized water Adjust to Pea i pe ca i nee i tt pH 8 0 with HCI Bring the final ee pH 8 0 with HCI Add glycogen Bring the final volume to 1 liter with deionized water volume to 1 liter with deionized water 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 14 Part TMD042 Page 43 9 E Related Products STR Systems Product Size Cat PowerPlex Fusion System 200 reactions DC2402 800 reactions DC2408 PowerPlex 21 System 200 reactions DC8902 PowerPlex 18D System 200 reactions DC1802 800 reactions DC1808 PowerPlex 16 HS System 100 reactions DC2101 400 reactions DC2100 PowerPlex 16 Monoplex System Penta E Fluorescein 100 reactions DC6591 PowerPlex 16 Monoplex System Penta D JOE 100 reactions DC6651 Powe

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