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PowerPlex S5 System Technical Manual, TMD021

1. 9 A Detection of Amplified Fragments Using the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 and the Applied Biosystems 3130 or 3130xl Genetic Analyzer With Data Collection Software Version 3 0 9 B Detection of Amplified Fragments Using the ABI PRISM 3100 Genetic Analyzer with Data Collection Software Version 1 0 1 or 1 1 12 C Detection of Amplified Fragments Using the ABI PRISM 310 Genetic Analyzer 14 VI Data Analysis 17 A PowerPlex Panel and Bin Sets with GeneMapper ID Version 3 2 17 B Creating an Analysis Method with GeneMapper ID Software 18 C Sample Analysis Using the GeneScan Software and PC Operating Systems 22 D Sample Analysis Using the GeneScan Software and Macintosh Operating Systems 23 E Sample Analysis Using the Genotyper Software and PowerTyper S5 Macro
2. 24 F Controls 26 G Results 27 VII Troubleshooting 29 A Amplification and Fragment Detection 29 B GeneMapper ID Analysis Software 31 C PowerTyper S5 Macro 34 VIII References 36 IX Appendix 38 A Advantages of Using the Loci in the PowerPlex S5 System 38 B DNA Extraction and Quantitation Methods 40 C The Internal Lane Standard 600 40 D Preparing the PowerPlex
3. S5 System A single DNA template 250pg was amplified using the PowerPlex S5 System The amplification products were detected using an Applied Biosystems 3130xl Genetic Analyzer and a 3kv 5 second injection The results were analyzed using GeneMapper ID software version 3 2 Panel A An electropherogram showing the peaks of the fluorescein labeled loci Amelogenin D18S51 and D8S1179 Panel B An electropherogram showing the peaks of the JOE labeled loci TH01 and FGA Panel C An electropherogram showing the 80bp to 300bp fragments of the Internal Lane Standard 600 tmd021 1008 qxp 10 13 2008 1 10 PM Page 28 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD021 Revised 10 08 Page 29 VII Troubleshooting For questions not addressed here please contact your local Promega Branch Office or Distributor Contact information available at www promega com E mail genetic promega com VII A Amplification and Fragment Detection 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 may be present in the DNA sample Insufficient template Use the recommended amount of template DNA increase injection time or voltage increa
4. For an explanation of the proper usage and effects of these settings refer to the Applied Biosystems user bulletin titled Installation Procedures and New Features for GeneMapper ID Software 3 2 and the GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide Note Some of these settings have been optimized and are different from the recommended settings in the user bulletin 10 Select the Peak Detector tab We recommend the settings shown in Figure 3 Note Select full range or partial range for the analysis range When using a partial range choose an appropriate analysis range based on the 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 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 also change these settings 13 Select OK to save your settings 7483TA Figure 3 The Peak Detector tab tmd021 1008 qxp 10 13 2008 1 10 PM Page 19 VI B Creating an Analysis Method with GeneMapper ID Software continued Creating a Size Standard 1 Select Tools then GeneMapper Manager 2 Select the Size Standard tab 3 Select New 4 Select Basic or Advanced Figure 4 The type o
5. Operating Systems 1 Analyze data using the GeneScan analysis software 2 Review the raw data for one or more sample runs Highlight the sample file name then in the Sample menu select raw data Move the cursor so that the crosshair is on the baseline to the right of the large primer peak before the first internal lane standard peak red Use the X value number shown at the bottom left of the window for the start position in the analysis parameters 3 The recommended analysis parameters are 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 TMD021 Revised 10 08 Analysis Range Start Defined in Step 2 Stop 10 000 Data Processing Baseline Checked Multicomponent Checked Smooth Options Light1 Peak Detection Peak Amplitude Thresholds2 B Y G R Min Peak Half Width 2pts Size Call Range Min 60 Max 350 Size Calling Method Local Southern Method Split Peak Correction None 1Smooth options should be determined by individual laboratories 2The peak amplitude thresholds are the minimum peak heights that the software will call as a peak Values for the peak amplitude thresholds are usually 50 200RFU and should be determined by individual laboratories tmd021 1008 qxp 10 13 2008 1 10 PM Page 23 Promega Corporation 2800 Woods Hollow Road Madison
6. Vol 2 Greene Publishing Associates Inc and 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 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 Revised Validation Guidelines Scientific Working Group on DNA Analysis Methods SWGDAM 2004 Forensic Science Communications 6 retrieved 12 03 07 from the World Wide Web www fbi gov hq lab fsc backissu july2004 standards 2004_03_standards02 htm 12 Moos M and Gallwitz D 1983 Structure of two human actin related processed genes one of which is located next to a simple repetitive sequence EMBO J 2 757 61 13 Polymeropoulos M H et al 1992 Tetranucleotide repeat polymorphism at the human
7. mix is provided in Section IX D Table 5 Note In tests performed at Promega we have found that reactions can remain at room temperature for up to 4 hours after reaction assembly and prior to thermal cycling with no adverse effect on amplification results 5 Vortex the PCR amplification mix for 5 10 seconds Note Failure to vortex the PCR amplification mix sufficiently can result in poor amplification peak height imbalance and extra peaks in the range of 50 80bp 6 Pipet the appropriate volume of PCR amplification mix into each reaction tube 7 Pipet the template DNA 0 25 0 5ng for each sample into the respective tube containing PCR amplification mix 8 For the positive amplification control dilute 9947A DNA from 10ng l to 0 5ng in the desired template DNA volume Pipet 0 5ng of diluted 9947A DNA into a reaction tube containing PCR amplification mix 9 For the negative amplification control pipet Water Amplification Grade instead of template DNA into a reaction tube containing PCR amplification mix Remove any air bubbles from the bottom of the tubes by careful pipetting or briefly centrifuging the tubes or plate Failure to remove air bubbles may result in inconsistent results Table 1 PCR Amplification Mix for the PowerPlex S5 System PCR Amplification Mix Component1 Volume Per Reaction Water Amplification Grade to a final volume of 25 0 l PowerPlex S5 5X Master Mix 5 0 l PowerPle
8. than those analyzed using the Basic or Advanced mode analysis method Advanced mode analysis methods and size standards are recommended tmd021 1008 qxp 10 13 2008 1 10 PM Page 33 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD021 Printed in USA Revised 10 08 VII B GeneMapper ID Analysis Software continued Symptoms Causes and Comments Red bar appears during analysis If none of the samples had matrices applied when run on the of samples and the following ABI PRISM 310 Genetic Analyzer no data will be displayed error message appears when data Apply a matrix file during analysis in the GeneMapper ID are displayed Some selected software and re analyze sample s do not contain analysis data Those sample s will not be shown Error message after attempting There was a conflict between different sets of panel and bin to import panel and bin files files Delete all panels and bins and re import in a different Unable to save panel data order java SQLEException ORA 00001 unique constraint IFA CKP_NNN violated Allelic ladder peaks are GeneMapper ID software was not used or microsatellite labeled off ladder analysis settings were used instead of HID analysis settings GeneMapper software does not use the same algorithms as GeneMapper ID software and cannot correct for
9. 25 0 l of prepared loading cocktail with 1 0 l of amplified sample or 1 0 l of PowerPlex S5 Allelic Ladder Mix Notes 1 Instrument detection limits vary therefore injection time or the amount of product mixed with loading cocktail may need to be increased or decreased 2 A precipitate may form during amplification This precipitate does not affect downstream analysis or capillary electrophoresis performance 4 Denature samples and ladder by heating at 95 C for 3 minutes and immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading 5 Assemble the tubes in the appropriate autosampler tray 48 or 96 tube 6 Place the autosampler tray in the instrument and close the instrument doors 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 TMD021 Revised 10 08 tmd021 1008 qxp 10 13 2008 1 09 PM Page 15 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 TMD021 Printed in USA Revised 10 08 V C Detection of Amplified Fragments Using the ABI PRISM 310 Genetic Analyzer continued Instrument Preparation Refer to the ABI PRISM 310 Genetic Analyzer User s Manual for instructions on
10. Double click on the Display JOE Data macro to display the green dye for all sample injections lanes Scroll down to observe and edit as needed tmd021 1008 qxp 10 13 2008 1 10 PM Page 25 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 TMD021 Printed in USA Revised 10 08 VI E Sample Analysis Using the Genotyper Software and PowerTyper S5 Macro continued 9 Create the appropriate table by selecting the PowerTable Make Allele Table or Make Vertical Table macro The three available table formats are shown below The PowerTable option allows up to four alleles per sample file Additional information such as low peak signal or high peak signal is also included The Allele Table and Vertical Table options include only two alleles per locus If more than two alleles are present at a locus the smallest alleles identified are included The Allele Table format displays the categories loci in columns while the Vertical table format displays the categories in rows These tables can be customized to fit needs To save data in tables go to the Table drop down menu highlight Export to File and save the file with the desired name and location The saved file can be viewed and analyzed using Microsoft Excel PowerTable Format Allele Table Format Vertical Table Format 10 Save the analyzed data go
11. Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD021 Printed in USA Revised 10 08 VI B Creating an Analysis Method with GeneMapper ID Software These instructions loosely follow the Applied Biosystems GeneMapper ID software tutorial pages 1 11 1 Select Tools then GeneMapper Manager 2 Select the Analysis Methods tab 3 Select New and a new analysis method dialog box will open 4 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 5 Enter a descriptive name for the analysis method such as PowerPlexS5 advanced 6 Select the Allele tab Figure 2 7 Select the bin set corresponding to the PowerPlex System PowerPlex_S5_Bin_ID3 2x 8 Ensure that the Use marker specific stutter ratio if available box is checked Page 18 7482TA Figure 2 The Allele tab Select the bin set PowerPlex_S5_Bins_ID3 2x tmd021 1008 qxp 10 13 2008 1 09 PM Page 18 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD021 Revised 10 08 Page 19 9 Enter the values shown in Figure 2 for proper filtering of stutter peaks when using the PowerPlex Systems
12. Notes 1 Instrument detection limits vary therefore injection time or the amount of product mixed with loading cocktail may need to be increased or decreased Use the Module Editor in the Tools menu to modify the injection time or voltage in the run module 2 A precipitate may form during amplification This precipitate does not affect downstream analysis or capillary electrophoresis performance 5 Centrifuge plate briefly to remove air bubbles from the wells if necessary 6 Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading the instrument Instrument Preparation Refer to the ABI PRISM 3100 Genetic Analyzer User s Manual for instructions on cleaning the blocks installing the capillary array performing a spatial calibration and adding polymer to the reserve syringe 1 Open the ABI PRISM 3100 data collection software 2 Change the GeneScan36_POP4DefaultModule module run time to 1 200 seconds 3 Change the injection voltage to 3kV 4 Change the injection time to 11 seconds 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 5 Save the module with a new name e g GeneScan36_POP4PowerPlexS5_3kV_11secs_1200 Use this as the initial r
13. TMD021 Revised 10 08 Page 37 20 Bever R A and Creacy S 1995 Validation and utilization of commercially available STR multiplexes for parentage analysis In Proceedings from the Fifth International Symposium on Human Identification 1994 Promega Corporation 61 8 21 Sprecher C J et al 1996 A general approach to analysis of polymorphic short tandem repeat loci BioTechniques 20 266 76 22 Lins A M et al 1996 Multiplex sets for the amplification of polymorphic short tandem repeat loci silver stain and fluorescent detection BioTechniques 20 882 9 23 Jones D A 1972 Blood samples Probability of discrimination J Forensic Sci Soc 12 355 9 24 Brenner C and Morris J W 1990 In Proceedings from the International Symposium on Human Identification 1989 Promega Corporation 21 53 25 Griffiths R et al 1998 New reference allelic ladders to improve allelic designation in a multiplex STR system Int J Legal Med 111 267 72 26 B r W et al 1997 DNA recommendations Further report of the DNA Commission of the ISFH regarding the use of short tandem repeat systems Int J Legal Med 110 175 6 27 Gill P et al 1997 Considerations from the European DNA Profiling Group EDNAP concerning STR nomenclature Forensic Sci Int 87 185 92 28 Fr geau C J et al 1995 Characterization of human lymphoid cell lines GM9947 and GM9948 as intra and interlaboratory refer
14. Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD021 Revised 10 08 Page 9 V Instrument Setup and Sample Preparation V A Detection of Amplified Fragments Using the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 and the Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 Materials to Be Supplied by the User dry heating block water bath or thermal cycler crushed ice or ice water bath centrifuge compatible with 96 well plates aerosol resistant pipette tips 3100 or 3130 capillary array 36cm performance optimized polymer 4 POP 4 for the 3100 or 3130 10X genetic analyzer buffer with EDTA MicroAmp optical 96 well plate and septa Hi Di formamide Applied Biosystems Cat 4311320 PowerPlex Matrix Standards 3100 3130 Cat DG4650 The quality of formamide is critical Use Hi Di formamide with a conductivity less than 100 S cm Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause a breakdown of the formamide Formamide with a conductivity greater than 100 S cm may contain ions that compete with DNA during injection which results in lower peak heights and reduced sensitivity A longer injection time may not increase the signal Caution Formamide is an irritant and a t
15. WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD021 Printed in USA Revised 10 08 VI D Sample Analysis Using the GeneScan Software and Macintosh Operating Systems continued 4 The analysis parameters can be saved in the Params folder 5 Apply the stored analysis parameters file to the samples 6 Assign a new size standard Select a sample file highlight the arrow next to size standard then select define new Assign the size standard peaks as shown in Figure 11 in Section IX C Store the size standard in the Size Standards folder Note With the run times recommended in this manual not all ILS 600 fragments will be detected Label all fragments present For accurate sizing the 350bp fragment must be detected If present larger fragments may be labeled also 7 Apply the size standard file to the samples then analyze the sample files See Section VI E for additional information on the use of the PowerTyper S5 Macro and Genotyper software For additional information regarding the GeneScan analysis software refer to the GeneScan Analysis Software User s Manual VI E Sample Analysis Using the Genotyper Software and PowerTyper S5 Macro To facilitate analysis of data generated with the PowerPlex S5 System we have created a file to allow automatic assignment of genotypes using the Genotyper software After samples are
16. amounts of template Miscellaneous balance problems Thaw the 10X Primer Pair Mix and 5X Master Mix completely and vortex for 5 10 seconds before use Do not centrifuge the 10X Primer Pair Mix after mixing Calibrate thermal cyclers and pipettes routinely Impure template DNA Inhibitors that may be present in forensic samples can lead to allele dropout or imbalance PCR amplification mix was not mixed thoroughly Vortex for 5 10 seconds before dispensing into the reaction tubes or plate VII B GeneMapper ID Analysis 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 9 An insufficient number of ILS 600 fragments was defined Be sure to define at least one ILS 600 fragment smaller than the smallest sample or allelic ladder peak and at least one ILS 600 fragment larger than the largest sample or allelic ladder peak 5685TA Figure 9 The error message that appears in the GeneMapper ID software when the analysis parameters and the size standard have different analysis types tmd021 1008 qxp 10 13 2008 1 10 PM Page 31 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 TMD021 Printed in USA Revis
17. appropriate matrix file Section III B 6 To analyze data automatically select the auto analyze checkbox and the appropriate analysis parameters and size standard Refer to the ABI PRISM 310 Genetic Analyzer User s Manual for specific information on these options 7 After loading the sample tray and closing the doors select Run to start the capillary electrophoresis CE system 8 Monitor electrophoresis by observing the raw data and status windows Each sample will take approximately 35 minutes for syringe pumping sample injection and sample electrophoresis Page 16 tmd021 1008 qxp 10 13 2008 1 09 PM Page 16 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 TMD021 Revised 10 08 Page 17 VI Data Analysis VI A PowerPlex Panel and Bin Sets with GeneMapper ID Version 3 2 To facilitate analysis of data generated with the PowerPlex S5 System we have created panel and bin 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 the proper operation of the software For GeneMapper ID software version 3 1 users we recommend upgrading
18. critical to evaluate multicolor systems with the ABI PRISM 310 3100 and 3100 Avant and Applied Biosystems 3130 and 3130xl Genetic Analyzers A matrix must be generated for each individual instrument The PowerPlex Matrix Standards 310 Cat DG4640 is required for matrix standardization for the ABI PRISM 310 Genetic Analyzer For best results the PowerPlex Matrix Standards 3100 3130 Cat DG4650 should not be used to generate a matrix on the ABI PRISM 310 Genetic Analyzer The PowerPlex Matrix Standards 3100 3130 Cat DG4650 is required for spectral calibration on the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xl Genetic Analyzers The PowerPlex Matrix Standards 310 Cat DG4640 cannot be used to generate a matrix on these instruments For protocols and additional information on matrix standardization see the PowerPlex Matrix Standards 310 Technical Bulletin TBD021 which is supplied with Cat DG4640 For protocols and additional information about spectral calibration see the PowerPlex Matrix Standards 3100 3130 Technical Bulletin TBD022 which is supplied with Cat DG4650 These manuals are available upon request from Promega or online at www promega com tbs tmd021 1008 qxp 10 13 2008 1 09 PM Page 5 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 ww
19. first injection on a new column for the ladder sample The base pair size of alleles was incorrect because incorrect fragment sizes were assigned to the internal lane standard Confirm that internal lane standard fragments are assigned correctly Re analyze sample using GeneScan software and redefine internal lane standard fragments tmd021 1008 qxp 10 13 2008 1 10 PM Page 35 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 TMD021 Printed in USA Revised 10 08 VIII References 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 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 actin related pseudogene 2 actbp2 detected using the polymerase chain reaction Nucleic Acids Res 19 6980 5 Ausubel F M et al 1993 Unit 15 The polymerase chain reaction In Current Protocols in Molecular Biology
20. green and red colors Page 34 tmd021 1008 qxp 10 13 2008 1 10 PM Page 34 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD021 Revised 10 08 Page 35 Symptoms Causes and Comments Error message Peak heights for one or more of alleles in the allelic ladder Could not complete the sample file were below 150RFU The allelic ladder categories Run Macro command are defined as having a minimum peak height of 150RFU If because the labeled peak peak heights of ladder alleles are below 150RFU the software could not be found will not be able to locate the allele peak Rerun the allelic ladder using more sample or longer injection time to assure peak heights above 150RFU CE spikes in the allelic ladder were identified as alleles by the macro Use a different injection of allelic ladder TH01 9 3 and 10 alleles were not separated when using heavy smoothing in the GeneScan analysis parameters Use light smoothing in the GeneScan analysis parameters The base pair size of alleles in the allelic ladder are outside of the defined category range Be sure the internal lane standard fragments are correctly sized Redefine the internal lane standard fragments and re analyze the sample using GeneScan software Compare the size of the smallest allele in the allelic ladder with the base pair si
21. other Promega fluorescent STR systems and detection of amplified STR fragments using silver staining is available upon request from Promega or online at www promega com Page 2 tmd021 1008 qxp 10 13 2008 1 09 PM Page 2 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD021 Revised 10 08 Page 3 II Product Components and Storage Conditions Product Size Cat PowerPlex S5 System 100 reactions DC6951 Not For Medical Diagnostic Use Cat DC6951 contains sufficient reagents for 100 reactions of 25 l each Includes Pre amplification Components Box Blue Label 500 l PowerPlex S5 5X Master Mix 250 l PowerPlex S5 10X Primer Pair Mix 25 l 9947A DNA 10ng l 5 1 250 l Water Amplification Grade Postamplification Components Box Beige Label 25 l PowerPlex S5 Allelic Ladder Mix 150 l Internal Lane Standard ILS 600 1 Protocol Amplification Setup Thermal Cycling Instrument Setup and Sample Preparation Data Analysis Section IV B Section V Section VI Section IV A GeneAmp PCR System 9700 GeneAmp PCR System 9600 GeneAmp PCR System 2400 Applied Biosystems 2720 Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 Section V A GeneMapper ID Software Versions 3 1 and 3 2 GeneScan S
22. patents issued to the Max Planck Gesellschaft zur F rderung der Wissenschaften e V Germany The development and use of STR loci are covered by U S Pat No 5 364 759 Australian Pat No 670231 and other pending patents assigned to Baylor College of Medicine Houston Texas Patents for the foundational PCR process European Pat Nos 201 184 and 200 362 expired on March 28 2006 In the U S the patents covering the foundational PCR process expired on March 29 2005 b This product is sold under licensing arrangements with the USB Corporation The purchase price of this product includes limited nontransferable rights under U S Patent Application Serial Number 11 171 008 owned by the USB Corporation to use only this amount of the product to practice the claims in said patent solely for activities of end users within the fields of life science research and forensic analysis of genetic material relating to or obtained as the result of criminal investigations or disaster sites conducted either by or for a governmental entity or for use in or preparation for legal proceedings as well as the compilation and indexing of the results of such analysis and also analysis for parentage determination the Forensic and Genetic Identity Applications Field The Forensic and Genetic Identity Applications Field specifically excludes tissue typing related to transplantation or other medical procedures Further licensing information may be obtained by cont
23. sets for the GeneMapper ID software version 3 2 or the PowerTyper Macro if desired Contact Promega Technical Services genetic promega com for assistance with modifications Notes 1 Peak heights outside the linear range of the instrument may generate artifact peaks due to instrument saturation i e overloading the sample Bleedthrough pull ups from one color to another may be observed Saturated signal may also appear as two peaks split peak 2 If peak heights are not within the linear range of detection of the instrument the ratio of stutter peaks to real allele peaks increases and allele designations become difficult to interpret The balance of peak heights may also appear less uniform tmd021 1008 qxp 10 13 2008 1 10 PM Page 27 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 TMD021 Printed in USA Revised 10 08 VI G Results continued Page 28 7613TA A B Figure 8 The PowerPlex S5 Allelic Ladder Mix The PowerPlex S5 Allelic Ladder Mix was analyzed using an Applied Biosystems 3130xl Genetic Analyzer and a 3kv 5 second injection The results were analyzed with the GeneMapper ID software version 3 2 Panel A The fluorescein labeled allelic ladder components Panel B The JOE labeled allelic ladder components 7612TA A B C Figure 7 The PowerPlex
24. sizing differences using the allelic ladder Promega recommends GeneMapper ID software for analysis of PowerPlex reactions If using GeneMapper ID software version 3 2 be sure that the analysis method selected is an HID method This can be verified by opening the analysis method using the GeneMapper Manager then selecting the General tab The analysis type cannot be changed If the method is not HID it should be deleted and a new analysis method created VII C PowerTyper S5 Macro Symptoms Causes and Comments File does not open Genotyper software was not installed Be certain that the on your computer Genotyper software version 2 5 Macintosh or version 3 6 or higher Windows NT is installed Incorrect version of Genotyper software The PowerTyper S5 Macro will not work with Genotyper software versions prior to version 2 5 The file was corrupted during download Download the file again Error message Allelic ladder sample files were not identified Be certain the Could not complete the sample info or color info column for each lane containing Run Macro command because PowerPlex S5 Allelic Ladder Mix contains the word ladder no dye lanes are selected The macro uses the word ladder to identify sample files containing allelic ladder All dye colors were not imported For Genotyper software versions 2 5 and 3 5 or higher set preferences in the Edit menu to import the blue
25. to the File menu and select Save as The PowerTyper Macro is a Genotyper file and can be overwritten if Save is used instead of Save as VI F Controls 1 Observe the results for the negative control The negative control should be devoid of amplification products 2 Observe the results for the 9947A DNA positive control reaction Compare the control DNA allelic repeat sizes with the locus specific allelic ladder The expected 9947A DNA allele designations for each locus are listed in Table 4 Section IX A The 9947A DNA which is cell line derived will show allelic imbalance and imbalance between STR loci Page 26 Sample Info Sample Comment Category Peak 1 Peak 2 Peak 3 Peak 4 Over flow Low Signal Satura tion Edited Label Edited Row Sample Info Category Allele 1 Category Allele 2 Category Allele 1 Category Allele 2 Category Allele 1 Category Allele 2 Category Allele 1 Category Allele 2 Sample Info Category Peak 1 Peak 2 tmd021 1008 qxp 10 13 2008 1 10 PM Page 26 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 TMD021 Revised 10 08 Page 27 VI G Results Representative results of the PowerPlex S5 System are shown in Figure 7 The PowerPlex S5 Allelic Ladder Mix is shown in Fi
26. 0 275 300 325 350 375 400 425 450 475 500 550 600 tmd021 1008 qxp 10 13 2008 1 10 PM Page 40 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 TMD021 Revised 10 08 Page 41 IX D Preparing the PowerPlex S5 System PCR Amplification Mix A worksheet to calculate the required amount of each PCR amplification mix component is provided in Table 5 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 Multiply the volume l per reaction by the total number of reactions to obtain the final PCR amplification mix volume l IX E Composition of Buffers and Solutions Table 5 PCR Amplification Mix for PowerPlex S5 System Reactions PCR Amplification Mix Component Volume Per Reaction Number of Reactions Final Volume l Water Amplification Grade1 l PowerPlex S5 5X Master Mix 5 0 l PowerPlex S5 10X Primer Pair Mix 2 5 l Per tube template DNA volume 0 25 0 50ng up to 17 5 l total reaction volume 25 l 1The total volume of PCR amplification mix volume and template DNA should be 25 l TE 4 buffer 10mM Tris HCl 0 1mM EDTA pH 8 0 2 21g Tris base 0 037g ED
27. 11 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD021 Revised 10 08 Page 25 3 In the File menu select Import and import the GeneScan project or sample files to be analyzed Import the blue green and red dye colors Note To select the dye colors to be imported select Set Preferences in the Edit menu 4 Double click on the Check ILS macro The macros are listed at the bottom left corner of the active window A plots window will be displayed to show the internal lane standard i e ILS 600 in the red dye color Scroll down to view and confirm that internal lane standard fragment sizes are correct If necessary redefine internal lane standard fragments and re analyze samples using the GeneScan software Note The software uses one ladder sample to determine allele sizes The macro uses the first ladder sample imported for allele designations 5 Double click on the POWER macro The POWER macro identifies alleles in the ladder sample and calculates offsets for all loci This process may take several minutes When completed a plots window will open to display the allelic ladders i e D8S1179 D18S51 Amelogenin etc In general allelic ladders contain fragments of the same lengths as many known alleles for the locus Allelic ladder sizes and repeat units are listed in Table 3 Section IX A Analysis using GeneScan analysis softw
28. Kit for Maxwell 16 48 preps AS1040 DNA IQ Casework Sample Kit for Maxwell 16 48 preps AS1210 Plexor HY System 200 determinations DC1001 800 determinations DC1000 Slicprep 96 Device 10 pack V1391 Not for Medical Diagnostic Use For Laboratory Use For Research Use Only Not for use in diagnostic procedures Page 42 tmd021 1008 qxp 10 13 2008 1 10 PM Page 42 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 TMD021 Revised 10 08 Page 43 ART Aerosol Resistant Tips Product Volume Size tips pack Cat ART 10 Ultramicro Pipet Tip 0 5 10 l 960 DY1051 ART 20E Ultramicro Pipet Tip 0 5 10 l 960 DY1061 ART 20P Pipet Tip 20 l 960 DY1071 ART GEL Gel Loading Pipet Tip 100 l 960 DY1081 ART 100 Pipet Tip 100 l 960 DY1101 ART 100E Pipet Tip 100 l 960 DY1111 ART 200 Pipet Tip 200 l 960 DY1121 ART 1000E Pipet Tip 1 000 l 800 DY1131 tmd021 1008 qxp 10 13 2008 1 10 PM Page 43 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 TMD021 Printed in USA Revised 10 08 Page 44 a STR loci are the subject of U S Pat No RE 37 984 German Pat No DE 38 34 636 C2 and other
29. Prepare a loading cocktail by combining and mixing Internal Lane Standard 600 and Hi Di formamide as follows 0 5 l ILS 600 injections 9 5 l Hi Di formamide injections Note The volume of internal lane standard used in the loading cocktail can be increased or decreased to adjust the intensity of 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 0 l of ILS 600 and 9 0 l of Hi Di formamide If peak heights are too high we recommend altering the loading cocktail to contain 0 25 l of ILS 600 and 9 75 l of formamide 2 Mix for 10 15 seconds using a vortex mixer 3 Pipet 10 l of formamide internal lane standard mix into each well Page 12 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD021 Printed in USA Revised 10 08 tmd021 1008 qxp 10 13 2008 1 09 PM Page 12 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD021 Revised 10 08 Page 13 4 Add 1 l of amplified sample or 1 l of allelic ladder mix Cover wells with appropriate septa
30. S5 System PCR Amplification Mix 41 E Composition of Buffers and Solutions 41 F Related Products 42 All technical literature is available on the Internet at www promega com tbs Please visit the web site to verify that you are using the most current version of this Technical Manual Please contact Promega Technical Services if you have questions on use of this system E mail genetic promega com PowerPlex S5 System tmd021 1008 qxp 10 13 2008 1 09 PM Page 1 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD021 Printed in USA Revised 10 08 I 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 radioactive silver stain or fluorescence detection following electrophoretic separation Th
31. T e c h n i c a l M a n u a l PowerPlex S5 System INSTRUCTIONS FOR USE OF PRODUCTS DC6951 AND DC6950 PRINTED IN USA REVISED 10 08 Part TMD021 tmd021 1008 qxp 10 13 2008 1 09 PM Page 1 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD021 Revised 10 08 Page 1 I Description 2 II Product Components and Storage Conditions 3 III Before You Begin 5 A Precautions 5 B Matrix Standardization or Spectral Calibration 5 IV Protocols for DNA Amplification Using the PowerPlex S5 System 6 A Amplification Setup 6 B Amplification Thermal Cycling 8 V Instrument Setup and Sample Preparation
32. TA Na2EDTA 2H2O Dissolve Tris base and EDTA in 900ml of deionized water Adjust to pH 8 0 with HCl Bring the final volume to 1 liter with deionized water tmd021 1008 qxp 10 13 2008 1 10 PM Page 41 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 TMD021 Printed in USA Revised 10 08 IX F Related Products Fluorescent STR Multiplex Systems Product Size Cat PowerPlex 16 System 100 reactions DC6531 400 reactions DC6530 PowerPlex 16 BIO System 100 reactions DC6541 400 reactions DC6540 PowerPlex Y System 50 reactions DC6761 200 reactions DC6760 PowerPlex ES System 100 reactions DC6731 400 reactions DC6730 Not for Medical Diagnostic Use Accessory Components Product Size Cat PowerPlex Matrix Standards 310 50 l each dye DG4640 PowerPlex Matrix Standards 3100 3130 25 l each dye DG4650 9947A DNA 250ng DD1001 Internal Lane Standard 600 150 l DG1071 Water Amplification Grade 6 250 l 5 1 250 l DW0991 Not for Medical Diagnostic Use For Laboratory Use Sample Preparation Systems Product Size Cat DNA IQ System 100 reactions DC6701 400 reactions DC6700 Differex System 50 samples DC6801 200 samples DC6800 Maxwell 16 Instrument each AS2000 DNA IQ Reference Sample
33. Y FGA JOE 148 270 16 18 18 2 19 19 2 20 20 2 21 21 2 22 22 2 23 23 2 24 24 2 25 25 2 26 30 31 2 43 2 44 2 45 2 46 2 TH01 JOE 93 132 4 9 9 3 10 11 13 3 1When 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 2Amelogenin is not an STR but displays a 103 base X specific band and a 109 base Y specific band tmd021 1008 qxp 10 13 2008 1 10 PM Page 38 We have carefully selected primers to avoid or minimize artifacts including those associated with Taq DNA polymerase such as repeat slippage and terminal nucleotide addition Repeat slippage 29 30 sometimes called n 4 bands stutter or shadow bands is due to the loss of a repeat unit during DNA amplification somatic variation within the DNA or both The amount of this artifact observed depends primarily on the locus and DNA sequence being amplified Terminal nucleotide addition 31 32 occurs when Taq DNA polymerase adds a nucleotide generally adenine to the 3 ends of amplified DNA fragments in a template independent manner The efficiency with which this occurs varies with different primer sequences Thus an artifact band one base shorter than expected i e mis
34. actin related pseudogene H beta Ac psi 2 ACTBP2 Nucleic Acids Res 20 1432 14 Schneider H R et al 1998 ACTBP2 nomenclature recommendations of GEDNAP Int J Leg Med 111 97 100 15 Department of Serology of the medical faculty at the Heinrich Heine University of Duesseldorf SE33 population statistics www uni duesseldorf de WWW MedFak Serology SE33b htm 16 Levedakou E et al 2001 Allele frequencies for fourteen STR loci of the PowerPlex 1 1 and 2 1 Multiplex Systems and Penta D locus in Caucasians African Americans Hispanics and other populations of the United States of America and Brazil J Forensic Sci 46 736 61 17 Lins A M et al 1998 Development and population study of an eight locus short tandem repeat STR multiplex system J Forensic Sci 43 1168 80 18 Puers C et al 1993 Identification of repeat sequence heterogeneity at the polymorphic STR locus HUMTH01 AATG n and reassignment of alleles in population analysis using a locus specific allelic ladder Am J Human Genet 53 953 8 19 Hammond H et al 1994 Evaluation of 13 short tandem repeat loci for use in personal identification applications Am J Hum Genet 55 175 89 Page 36 tmd021 1008 qxp 10 13 2008 1 10 PM Page 36 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part
35. acting the USB Corporation 26111 Miles Road Cleveland OH 44128 c This product is sold under licensing arrangements with Stratagene The purchase price of this product includes limited nontransferable rights under U S Pat Nos 5 449 603 5 605 824 5 646 019 and 5 773 257 owned by Stratagene to use only this amount of the product to practice the claims in said patent solely for activities of end users within the fields of life science research and forensic analysis of genetic material relating to or obtained as the result of criminal investigations or disaster sites conducted either by or for a governmental entity or for use in or preparation for legal proceedings as well as the compilation and indexing of the results of such analysis and also analysis for parentage determination the Forensic and Genetic Identity Applications Field The Forensic and Genetic Identity Applications Field specifically excludes tissue typing related to transplantation or other medical procedures Further licensing information may be obtained by contacting the Business Development Department Stratagene California 11011 North Torrey Pines Road La Jolla CA 92037 2008 Promega Corporation All Rights Reserved Maxwell Plexor and PowerPlex are registered trademarks of Promega Corporation Differex DNA IQ PowerTyper and Slicprep are trademarks of Promega Corporation ABI PRISM GeneMapper GeneScan Genotyper and MicroAmp are registered trademarks of A
36. amplified detected using the ABI PRISM 310 or 3100 Genetic Analyzer using data collection software version 1 0 1 or 1 1 and analyzed using the GeneScan analysis software sample files can be imported into the Genotyper program and analyzed using the PowerTyper S5 Macro The PowerTyper S5 Macro can be downloaded from the Promega web site at www promega com geneticidtools The PowerTyper S5 Macro is used in conjunction with Macintosh Genotyper software version 2 5 and Windows NT Genotyper software version 3 6 or later The Genotyper software must be installed on your computer before the PowerTyper S5 Macro can be used Be certain the sample info Macintosh computers or color info Windows NT operating systems column for each lane containing allelic ladder mix contains the word ladder The macro uses the word ladder to identify the sample file s containing allelic ladder Sample info can be added or modified after importing into the PowerTyper Macro Highlight the sample then select show dye lanes window in the Views menu 1 Download the PowerTyper S5 Macro from the Promega web site 2 Open the Genotyper software then the PowerTyper S5 Macro For questions about the Genotyper software refer to the Genotyper Analysis Software User s Manual Page 24 tmd021 1008 qxp 10 13 2008 1 10 PM Page 24 Promega Corporation 2800 Woods Hollow Road Madison WI 537
37. are and Genotyper software allows allele determination by comparing amplified sample fragments with allelic ladders and internal lane standards When using an internal lane standard the calculated lengths of allelic ladder components may differ from those listed in the table This is due to differences in migration resulting from sequence differences between allelic ladder fragments and internal size standard fragments and is not a matter of concern 6 Double click on the Allelic Ladders macro A plots window will open to display the blue fluorescein dye allelic ladders i e Amelogenin D18S51 and D8S1179 and the green JOE dye allelic ladders i e TH01 Confirm that the correct allele designations were assigned to the allelic ladders Figure 8 in Section VI G Note The software uses one ladder sample to determine allele sizes The macro uses the first ladder sample imported for allele designations If the POWER macro is run a second time the software will use the second ladder if the POWER macro is run a third time the software will use the third ladder etc until all ladders in the project are used If an allelic ladder fails to be analyzed or if many off ladder alleles are found in the samples the samples should be re analyzed using another ladder from the project 7 Double click on the Display Fluorescein Data macro to display the blue dye for all sample injections lanes Scroll down to observe and edit as needed 8
38. at 4311320 PowerPlex Matrix Standards 310 Cat DG4640 crushed ice or ice water bath Page 14 tmd021 1008 qxp 10 13 2008 1 09 PM Page 14 Page 15 The quality of formamide is critical Use Hi Di formamide with a conductivity less than 100 S cm Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause a breakdown of the formamide Formamide with a conductivity greater than 100 S cm may contain ions that compete with DNA during injection which results in lower peak heights and reduced sensitivity A longer injection time may not increase the signal Caution 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 Prepare a loading cocktail by combining Internal Lane Standard 600 and Hi Di formamide as follows 1 0 l ILS 600 injections 24 0 l Hi Di formamide injections Note The volume of internal lane standard used in the loading cocktail can be increased or decreased to adjust the intensity of size standard peaks If peak heights are too high we recommend altering the formamide internal lane standard mix to contain 0 5 l of ILS 600 and 24 5 l of Hi Di formamide 2 Mix for 10 15 seconds using a vortex mixer 3 Combine
39. cleaning the pump block installing the capillary calibrating the autosampler and adding polymer to the syringe 1 Open the ABI PRISM 310 data collection software 2 Prepare a GeneScan sample sheet as described in the ABI PRISM 310 Genetic Analyzer User s Manual Enter the appropriate sample information in the sample info column For rows containing PowerPlex S5 Allelic Ladder Mix insert the word ladder in the sample info column for the blue dye color and green dye color This information must be entered to successfully analyze your data using the PowerTyper S5 Macro 3 Create a new GeneScan injection list Select the appropriate sample sheet by using the pull down menu 4 Select the GS STR POP4 1ml A Module using the pull down menu Change the injection time to the appropriate setting and the run time to 23 minutes Keep the settings for the remaining parameters as shown below Inj Secs 2 5 Inj kV 15 0 Run kV 15 0 Run C 60 Run Time 23 You may need to optimize the injection time for individual instruments We recommend injection times of 2 5 seconds Note Migration of fragments may vary slightly over the course of a long ABI PRISM 310 Genetic Analyzer run This may be due to changes in temperature or changes in the column When analyzing many samples injections of allelic ladder at different times throughout the run can aid in accurately genotyping samples 5 Select the
40. ction of sexual assault samples using the Biomek 2000 and the DNA IQ System Profiles in DNA 5 1 3 5 39 McLaren B Bjerke M and Tereba A 2006 Automating the DNA IQ System on the Biomek 3000 Laboratory Automation Workstation Profiles in DNA 9 1 11 13 40 Cowan C 2006 The DNA IQ System on the Tecan Freedom EVO 100 Profiles in DNA 9 1 8 10 41 Bjerke M et al 2006 Forensic application of the Maxwell 16 Instrument Profiles in DNA 9 1 3 5 42 Mandrekar P et al 2007 Introduction to Maxwell 16 low elution volume configuration for forensic casework Profiles in DNA 10 2 10 12 Additional STR references can be found at www promega com geneticidentity tmd021 1008 qxp 10 13 2008 1 10 PM Page 37 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 TMD021 Printed in USA Revised 10 08 IX Appendix IX A Advantages of Using the Loci in the PowerPlex S5 System The loci included in the PowerPlex S5 System Tables 2 and 3 have been selected to include four of the current seven ENFSI loci 12 24 and four of the current CODIS loci Additionally the Amelogenin locus is included in the PowerPlex S5 System to allow gender identification of each sample Table 4 lists the PowerPlex S5 System alleles revealed in commonly available standard DNA templa
41. d in USA Part TMD021 Revised 10 08 Page 5 III Before You Begin III 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 11 The quality of purified DNA as well as 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 as well as fluorescence detection PCR based STR analysis is subject to contamination by very small amounts of nontemplate human DNA Extreme care should be taken to avoid cross contamination when preparing sample DNA handling primer pairs assembling amplification reactions and analyzing amplification products Reagents and materials used prior to amplification PowerPlex S5 5X Master Mix PowerPlex S5 10X Primer Pair Mix Water Amplification Grade and 9947A DNA are provided in a separate box and should be stored separately from those used following amplification PowerPlex S5 Allelic Ladder Mix and Internal Lane Standard 600 Always include a negative control reaction i e no template to detect reagent contamination We highly recommend the use of gloves and aerosol resistant pipette tips e g ART tips Section IX F III B Matrix Standardization or Spectral Calibration Proper generation of a matrix file is
42. e GeneScan analysis software 12 Select OK This new plate record will appear in the pending plate records table on the plate setup page of the collection software 13 Place samples in the instrument and close the instrument doors 14 Locate the pending plate record that you just created and click once on the name 15 Once the pending plate record is highlighted click on the plate graphic that corresponds to the plate on the autosampler that contains your amplified samples to link the plate to the plate record 16 When the plate record is linked to the plate the plate graphic will change from yellow to green the plate record moves from the pending plate records table to the linked plate records table and the Run Instrument button becomes enabled 17 Select the Run Instrument button on the toolbar to start the sample run 18 Monitor electrophoresis by observing the run status array and capillary views windows in the collection software Each injection will take approximately 35 minutes V C Detection of Amplified Fragments Using the ABI PRISM 310 Genetic Analyzer Materials to Be Supplied by the User 95 C dry heating block water bath or thermal cycler 310 capillaries 47cm 50 m performance optimized polymer 4 POP 4 for the 310 10X genetic analyzer buffer with EDTA sample tubes and septa aerosol resistant pipette tips Hi Di formamide Applied Biosystems C
43. e PowerPlex S5 System a c allows co amplification and detection of five loci four STR loci and Amelogenin including D8S1179 D18S51 Amelogenin FGA and TH01 One primer specific for each of Amelogenin D18S51 and D8S1179 loci is labeled with fluorescein FL and one primer specific for each of the TH01 and FGA loci is labeled with 6 carboxy 4 5 dichloro 2 7 dimethoxy fluorescein JOE All five loci are amplified simultaneously in a single tube and analyzed in a single injection The PowerPlex S5 System is compatible with the ABI PRISM 310 3100 and 3100 Avant and Applied Biosystems 3130 and 3130xl 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 cycle number and injection time or loading volume for each laboratory instrument In house validation should be performed The PowerPlex S5 System provides all of the materials necessary for amplification of STR regions of purified genomic DNA This manual contains a protocol for use of the PowerPlex S5 System with the Applied Biosystems 2720 and GeneAmp PCR system 9600 9700 and 2400 thermal cyclers in addition to protocols for separation of amplified products and detection of separated material Figure 1 Protocols for operation of the fluorescence detection instruments should be obtained from the instrument manufacturer Information on
44. e same mode either Classic or Basic or Advanced mode No alleles called but no error Panel was not selected for sample In the Panel column select message appears the appropriate panel set for the STR system that was used No size standard was selected In the size standards column be sure to select the appropriate size standard Size standard was not correctly defined or size peaks were missing Redefine size standard to include only peaks present in your sample Terminating analysis early or using short run times will cause larger ladder peaks to be missing This will cause your sizing quality to be flagged as red and no allele sizes will be called Error message The bin set assigned to the analysis method may have been Both the Bin Set used in the deleted In the GeneMapper Manager select the Analysis Analysis Method and the Panel Methods tab and open the analysis method of interest Select must belong to the same the Alleles tab and select an appropriate bin set Chemistry Kit Significantly raised baseline Poor spectral calibration for the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xl Genetic Analyzers Perform a new spectral calibration and rerun the samples Poor matrix for the ABI PRISM 310 Genetic Analyzer Rerun and optimize the matrix Use of Classic mode analysis method Use of Classic mode analysis for samples can result in baselines with more noise
45. ection Software Version 2 0 and the Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 continued 4 Add 1 l of amplified sample or 1 l of allelic ladder mix Cover wells with appropriate septa Notes 1 Instrument detection limits vary therefore the injection time voltage or amount of product mixed with loading cocktail may need to be increased or decreased Use the Module Manager in the Tools menu to modify the injection time or voltage in the run module 2 A precipitate may form during amplification This precipitate does not affect downstream analysis or capillary electrophoresis performance 5 Centrifuge plate briefly to remove air bubbles from the wells if necessary 6 Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading the instrument Instrument Preparation Refer to the instrument manual for instructions on cleaning the pump blocks installing the capillary array performing a spatial calibration and adding polymer to the reserve syringe Analyze samples as described in the user s manual for the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with data collection software version 2 0 and the Applied Biosystems 3130 or 3130xl Genetic Analyzer with the following exceptions 1 In the Module Manager select New Select Regular in the Type dr
46. ed 10 08 VII B GeneMapper ID Analysis Software continued Symptoms Causes and Comments Alleles not called Run was too short and larger peaks in ILS were not captured continued Not all ILS 600 peaks defined in the size standard were detected during the run Create a new size standard using the internal lane standard fragments present in the sample Rerun samples using a longer run time Off ladder alleles An allelic ladder from a different run than the samples was used Re analyze samples with an allelic ladder from the same run The GeneMapper ID software requires that the allelic ladder be imported from the same folder as the sample Be sure that the allelic ladder is in the same folder as the sample Create a new project and re analyze as described in Section VI B or VI C Panel file selected for analysis was incorrect for the STR system used Assign correct panel file that corresponds to the 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 sizes of the size standard fragments in the sample Size standard not called Starting data point was incorrect for the partial range chosen correctly Figure 10 in Section VI B Adjust the starting data point in the analysis me
47. ence standards for DNA typing Genomics 28 184 97 29 Levinson G and Gutman G A 1987 Slipped strand mispairing A major mechanism for DNA sequence evolution Mol Biol Evol 4 203 21 30 Schlotterer C and Tautz D 1992 Slippage synthesis of simple sequence DNA Nucleic Acids Res 20 211 5 31 Smith J R et al 1995 Approach to genotyping errors caused by nontemplated nucleotide addition by Taq DNA polymerase Genome Res 5 312 7 32 Magnuson V L et al 1996 Substrate nucleotide determined non templated addition of adenine by Taq DNA polymerase Implications for PCR based genotyping BioTechniques 21 700 9 33 Walsh P S Fildes N J and Reynolds R 1996 Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vWA Nucleic Acids Res 24 2807 12 34 Moller A Meyer E and Brinkmann B 1994 Different types of structural variation in STRs HumFES FPS HumVWA and HumD21S11 Int J Leg Med 106 319 23 35 Brinkmann B Moller A and Wiegand P 1995 Structure of new mutations in 2 STR systems Int J Leg Med 107 201 3 36 Mandrekar P V Krenke B E and Tereba A 2001 DNA IQ The intelligent way to purify DNA Profiles in DNA 4 3 16 37 Krenke B E et al 2005 Development of a novel fluorescent two primer approach to quantitative PCR Profiles in DNA 8 1 3 5 38 Greenspoon S and Ban J 2002 Robotic extra
48. eratogen 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 Prepare a loading cocktail by combining and mixing Internal Lane Standard 600 and Hi Di formamide as follows 0 5 l ILS 600 injections 9 5 l Hi Di formamide injections Note The volume of internal lane standard used in the loading cocktail can be increased or decreased to adjust the intensity of 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 0 l of ILS 600 and 9 0 l of Hi Di formamide If peak heights are too high we recommend altering the loading cocktail to contain 0 25 l of ILS 600 and 9 75 l of formamide 2 Mix for 10 15 seconds using a vortex mixer 3 Pipet 10 l of formamide internal lane standard mix into each well tmd021 1008 qxp 10 13 2008 1 09 PM Page 9 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD021 Printed in USA Revised 10 08 V A Detection of Amplified Fragments Using the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Coll
49. es for the recommended time and cool on crushed ice or in an ice water bath immediately prior to CE Poor CE injection ILS 600 peaks also affected Re inject the sample Check the syringe for leakage Check the laser power Poor quality formamide was used Use only Hi Di formamide when analyzing samples The 9947A DNA which is cell line derived will show allelic imbalance and imbalance between STR loci tmd021 1008 qxp 10 13 2008 1 10 PM Page 29 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 TMD021 Printed in USA Revised 10 08 VII A Amplification and Fragment Detection continued Symptoms Causes and Comments Extra peaks visible in one or Contamination with another template DNA or previously all of the color channels amplified DNA Cross contamination can be a problem Use aerosol resistant pipette tips and change gloves regularly Samples were not completely denatured Heat denature samples for the recommended time and cool on crushed ice or in an ice water bath immediately prior to CE Artifacts of STR amplification PCR amplification of STR systems 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 45 minute extension step at 60 C after thermal cycling Section IV B High background Decrease the in
50. f analysis method selected must match the type of analysis method created earlier Select OK 5 Enter a detailed name such as 60 350 ILS Adv in the Size Standard Editor Figure 5 6 Choose red as the color for the size standard dye 7 Enter sizes for the 60 350bp internal lane standard fragments Section IX C Figure 11 Note With the run times recommended in this manual not all ILS 600 fragments will be detected Label all fragments present For accurate sizing the 350bp fragment must be detected If present larger fragments also may be labeled 8 Select OK Page 20 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD021 Printed in USA Revised 10 08 5725TA Figure 4 The Select Dye and Analysis Method window tmd021 1008 qxp 10 13 2008 1 10 PM Page 20 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD021 Revised 10 08 Page 21 Processing Sample Data 1 Import sample files into a new project as described in the Applied Biosystems GeneMapper ID Software Human Identification Analysis Tutorial 2 In the Sample Type column use the drop down menu to select Ladder Sample Positive Control o
51. for ordering information The DNA IQ System has been fully automated on the Beckman Coulter Biomek 2000 Laboratory Automation Workstation 38 Biomek 3000 Laboratory Automation Workstation 39 and Tecan Freedom EVO Liquid Handler 40 In addition the DNA IQ Reference Sample Kit for Maxwell 16 Cat AS1040 and DNA IQ Casework Sample Kit for Maxwell 16 are available 41 42 For information on automation of laboratory processes on automated workstations contact your local Promega Branch Office or Distributor contact information available at www promega com worldwide or e mail genetic promega com IX C 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 11 For PowerPlex S5 analyses only ILS 600 fragments smaller than 350bp need to be detected Each fragment is labeled with carboxy X rhodamine CXR and may be detected separately in the presence of PowerPlex S5 amplified material The ILS 600 is designed for use in each CE injection to increase precision in analyses when using the PowerPlex S5 System Page 40 Figure 11 Internal Lane Standard 600 An electropherogram showing the fragments of the Internal Lane Standard 600 5751TA 1 200 1 000 800 600 400 200 0 60 80 100 120 140 160 180 200 225 25
52. gure 8 Artifacts and Stutter Stutter bands are a common amplification artifact associated with STR analysis Stutter products are often 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 S5 System loci Low level products can be seen in the n 2 and n 2 positions two bases below and above the true allele peak respectively with some loci such as D18S51 One or more extra peaks that are not directly related to amplification may be observed at positions 11 bases smaller than TH01 alleles 1 base smaller than FGA alleles and 1 or 8 bases smaller than Amelogenin alleles These extra peaks occur when the amplified peaks are particularly intense high signal level or template amount the formamide polymer or capillary was of poor quality or denaturation was ineffective One or more extra peaks that are not directly related to amplification may be observed at 73 bp in the fluorescein channel and at 72 76 bp in the JOE channel See Section VII for more information about how to minimize these artifacts Stutter filters can be modified in the PowerPlex panel and bin
53. imately 35 minutes tmd021 1008 qxp 10 13 2008 1 09 PM Page 11 V B Detection of Amplified Fragments Using the ABI PRISM 3100 Genetic Analyzer with Data Collection Software Version 1 0 1 or 1 1 Materials to Be Supplied by the User 95 C dry heating block water bath or thermal cycler crushed ice or ice water bath aerosol resistant pipette tips centrifuge compatible with 96 well plates 3100 capillary array 36cm performance optimized polymer 4 POP 4 for the 3100 10X genetic analyzer buffer with EDTA MicroAmp optical 96 well plate and septa for the 3100 Hi Di formamide Applied Biosystems Cat 4311320 PowerPlex Matrix Standards 3100 3130 Cat DG4650 The quality of formamide is critical Use Hi Di formamide with a conductivity less than 100 S cm Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause a breakdown of the formamide Formamide with a conductivity greater than 100 S cm may contain ions that compete with DNA during injection which results in lower peak heights and reduced sensitivity A longer injection time may not increase the signal Caution 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
54. ions 4 In the GeneMapper plate record enter sample names in the appropriate cells Scroll to the right In the results group 1 column select the desired results group In the instrument protocol 1 column select the protocol you created in Step 2 Be sure this information is present for each row that contains a sample name Select OK Note To create a new results group select New in the drop down menu in the results group column Select the General tab and enter a name Select the Analysis tab and select GeneMapper Generic in the Analysis type drop down list 5 Place samples in the instrument and close the instrument doors 6 In the spectral viewer confirm that dye set F is active and set the correct active calibration for dye set F 7 In the 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 highlighted 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 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 windows in the collection software Each injection will take approx
55. jection time See Section V CE related artifacts spikes Minor voltage changes or urea crystals passing by the laser can cause spikes or unexpected peaks Spikes sometimes appear in one color but often are easily identified by their presence in more than one color Re inject samples to confirm CE related artifacts contaminants Contaminants in the water used with the instrument or to dilute the 10X genetic analyzer buffer may generate peaks in the blue and green dye colors Use autoclaved water change vials and wash buffer reservoir Excessive amount of DNA Amplification of gt 1ng template can result in a higher number of stutter bands and other artifacts Pull up or bleedthrough Pull up can occur when peak heights are too high or if a poor or incorrect matrix has been applied to the samples For the ABI PRISM 310 Genetic Analyzer generate a new matrix and apply it to the samples For the ABI PRISM 3100 and 3100 Avant and Applied Biosystems 3130 and 3130xl Genetic Analyzers perform a new spectral calibration and rerun the samples Instrument sensitivities can vary Optimize the injection conditions See Section V 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 recom
56. lification Grade is provided in a separate sealed bag for shipping This component should be moved to the pre amplification box after opening Storage Conditions Store all components at 20 C in a nonfrost free freezer The PowerPlex S5 10X Primer Pair Mix PowerPlex S5 Allelic Ladder Mix and Internal Lane Standard 600 are light sensitive and must be stored in the dark We strongly recommend that pre amplification and postamplification reagents be stored and used separately with different pipettes tube racks etc The PowerTyper Macro S5 for use with Genotyper software can be downloaded at www promega com geneticidtools The proper panel and bin files for use with GeneMapper ID software can be obtained from the Promega web site at www promega com geneticidtools panels_bins Matrix standards are required for initial setup of the color separation matrix The matrix standards are sold separately and are available for the ABI PRISM 310 Genetic Analyzer PowerPlex Matrix Standards 310 Cat DG4640 and the ABI PRISM 3100 and 3100 Avant and Applied Biosystems 3130 and 3130xl Genetic Analyzers PowerPlex Matrix Standards 3100 3130 Cat DG4650 See Section IX F for ordering information Page 4 tmd021 1008 qxp 10 13 2008 1 09 PM Page 4 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printe
57. mended by the manufacturer PCR amplification mix was not mixed thoroughly Vortex mix for 5 10 seconds before dispensing into reaction tubes or plate An air pocket has formed at the bottom of the well Use a pipette to remove the air pocket or centrifuge briefly prior to thermal cycling Precipitate observed in A precipitate may form as a result of thermodenaturation of samples after amplification the protein associated with hot start This precipitate does not affect downstream amplification or capillary performance Page 30 tmd021 1008 qxp 10 13 2008 1 10 PM Page 30 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD021 Revised 10 08 Page 31 Symptoms Causes and Comments Allelic ladder not running the Allelic ladder and primer pair mix were not compatible the same as the sample Ensure that the allelic ladder is from the same system as the primer pair mix Poor quality formamide Use only Hi Di formamide when analyzing samples 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 Peak height imbalance Insufficient template DNA Use the recommended amount of template DNA Stochastic effects can occur when amplifying low
58. n For reactions containing 1ng of DNA the number of cycles may be reduced 25 28 cycles In house validation should be performed 1 Place the tubes or MicroAmp plate in a thermal cycler 2 Run the recommended protocol provided below for use with the GeneAmp PCR System 9600 9700 and 2400 thermal cyclers and Applied Biosystems 2720 thermal cycler For information about other thermal cyclers please contact Promega Technical Services by e mail genetic promega com 3 After completion of the thermal cycling protocol store samples at 20 C in a light protected box Notes 1 Storage of amplified samples at 4 C or higher may produce degradation products 2 A precipitate may form during amplification This precipitate does not affect downstream analysis or capillary electrophoresis performance Page 8 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD021 Printed in USA Revised 10 08 Thermal Cycling Protocol1 96 C for 2 minutes then 94 C for 30 seconds 60 C for 2 minutes 72 C for 90 seconds for 30 cycles then 60 C for 45 minutes 4 C soak 1When running the GeneAmp PCR System 9700 thermal cycler use the Method Option Ramp Speed 9600 tmd021 1008 qxp 10 13 2008 1 09 PM Page 8 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA
59. n in the analysis parameters 3 The recommended analysis parameters are shown in Figure 6 4 The analysis parameters can be saved in the Params folder in most installations this is located at C AppliedBio Shared Analysis Sizecaller Params 5 Apply the stored analysis parameters file to the samples Page 22 7329TA Figure 6 The analysis parameters window The start point of the analysis range which will vary is defined in Section VI C or VI D Step 2 tmd021 1008 qxp 10 13 2008 1 10 PM Page 22 Page 23 6 Assign a new size standard Select a sample file and highlight the arrow next to size standard Select define new Assign the size standard peaks as shown in Figure 11 in Section IX C Store the size standard in the Size Standards folder at C AppliedBio Shared Analysis Sizecaller SizeStandards Note With the run times recommended in this manual not all ILS 600 fragments will be detected Label all fragments present For accurate sizing the 350bp fragment must be detected If present larger fragments may be labeled also 7 Apply the size standard file to the samples then analyze the sample files See Section VI E for additional information on the use of the PowerTyper S5 Macro and Genotyper software For additional information regarding the GeneScan analysis software refer to the GeneScan Analysis Software User s Manual VI D Sample Analysis Using the GeneScan Software and Macintosh
60. oftware and PC Operating Systems GeneScan Software and Macintosh Operating Systems ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 Section V A ABI PRISM 3100 Genetic Analyzer with Data Collection Software Version 1 0 1 or 1 1 Section V B ABI PRISM 310 Genetic Analyzer Section V C Figure 1 An overview of the PowerPlex S5 System protocol tmd021 1008 qxp 10 13 2008 1 09 PM Page 3 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 TMD021 Printed in USA Revised 10 08 II Product Components and Storage Conditions continued Product Size Cat PowerPlex S5 System 400 reactions DC6950 Not For Medical Diagnostic Use Cat DC6950 contains sufficient reagents for 400 reactions of 25 l each Includes Pre amplification Components Box Blue Label 4 500 l PowerPlex S5 5X Master Mix 4 250 l PowerPlex S5 10X Primer Pair Mix 25 l 9947A DNA 10ng l 10 1 250 l Water Amplification Grade Postamplification Components Box Beige Label 4 25 l PowerPlex S5 Allelic Ladder Mix 4 150 l Internal Lane Standard ILS 600 1 Protocol The PowerPlex S5 Allelic Ladder Mix is provided in a separate sealed bag for shipping This component should be moved to the postamplification box after opening The Water Amp
61. op 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 Change the run time to 1 200 seconds Give a new 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 15 seconds and for the injection voltage is 1 5kV 2 In the Protocol Manager select New Type a name for your protocol Select Regular in the Type drop down list and select the run module you created in the previous step in the Run Module drop down list Lastly select F in the Dye Set drop down list Select OK Page 10 tmd021 1008 qxp 10 13 2008 1 09 PM Page 10 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD021 Revised 10 08 Page 11 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 instruct
62. pplera Corporation ART is a registered trademark of Molecular Bio Products Inc Biomek is a registered trademark of Beckman Coulter Inc FTA is a registered trademark of Flinders Technologies Pty Ltd and is licensed to Whatman Freedom EVO is a registered trademark of Tecan AG Corporation 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 Macintosh is a registered trademark of Apple Computer Inc Microsoft Windows and Windows NT are registered trademarks of Microsoft Corporation Products may be covered by pending or issued patents or may have certain limitations Please visit our Web site for more information All prices and specifications are subject to change without prior notice Product claims are subject to change Please contact Promega Technical Services or access the Promega online catalog for the most up to date information on Promega products tmd021 1008 qxp 10 13 2008 1 10 PM Page 44
63. r Negative Control Every folder in the project must contain at least one ladder that is designated as such for proper genotyping 3 In the Analysis Method column select the analysis method created earlier 4 In the Panel column select PowerPlex_S5_Panels This is the panel set that was imported in Section VI A 5 In the Size Standard column select the size standard that was created in Creating a Size Standard section 6 If analyzing data from an ABI PRISM 310 Genetic Analyzer ensure that the appropriate matrix file is selected in the Matrix column 7 Select Analyze green arrow button to start data analysis 7484TA Figure 5 The Size Standard Editor tmd021 1008 qxp 10 13 2008 1 10 PM Page 21 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 TMD021 Printed in USA Revised 10 08 VI C Sample Analysis Using the GeneScan Software and PC Operating Systems 1 Analyze data using the GeneScan analysis software 2 Review the raw data for one or more sample runs Highlight the sample file name then in the Sample menu select raw data Move the cursor so that the crosshair is on the baseline to the right of the large primer peak before the first internal lane standard peak red Use the X value number shown at the bottom left of the window for the start positio
64. se cycle number or increase the volume of amplified sample during sample preparation Insufficient enzyme activity Use the recommended amount of PowerPlex S5 5X Master Mix and vortex the 5X Master Mix prior to use Incorrect amplification program Confirm the amplification program PCR amplification mix was not mixed thoroughly Vortex mix for 5 10 seconds before dispensing into reaction tubes or plate An air pocket has formed at the bottom of the well Use a pipette to remove the air pocket or centrifuge briefly prior to thermal cycling Centrifuge samples prior to injection on the CE instrument High salt concentration or altered pH If the DNA template is stored in TE buffer that is not pH 8 0 or contains a higher EDTA concentration the DNA volume should not exceed 20 of the total reaction volume Carryover of K Na Mg2 or EDTA from the DNA sample can negatively affect PCR A change in pH may also affect PCR Store DNA in TE 4 buffer 10mM Tris HCl pH 8 0 0 1mM EDTA or nuclease free water Thermal cycler plate or tube problems Review the thermal cycling protocols in Section IV B 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 Mix the PowerPlex S5 10X Primer Pair for 15 seconds using a vortex mixer before use Samples were not completely denatured Heat denature sampl
65. sing the terminal addition is sometimes seen We have modified primer sequences and added a final extension step of 60 C for 45 minutes 33 to the amplification protocol to provide conditions for essentially full terminal nucleotide addition when recommended amounts of DNA template are used The presence of microvariant alleles alleles differing from one another by lengths other than the repeat length complicates interpretation and assignment of alleles There appears to be a correlation between a high degree of polymorphism a tendency for microvariants and increased mutation rate 34 35 FGA and D18S51 display numerous relatively common microvariants 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 TMD021 Revised 10 08 Page 39 Table 4 The PowerPlex S5 System Allele Determinations in Commonly Available Standard DNA Templates Standard DNA Templates1 STR Locus K562 9947A 9948 D8S1179 12 12 13 13 12 13 D18S51 15 16 15 19 15 18 Amelogenin X X X X X Y FGA 21 24 23 24 24 26 TH01 9 3 9 3 8 9 3 6 9 3 1Information on strains 9947A 9948 and K562 is available online at locus umdnj edu nigms Strain K562 is available from the American Type Culture Collection www atcc org Manassas VA Information about the use of 9947A and 9948 DNA as standard DNA
66. templates can be found in reference 28 tmd021 1008 qxp 10 13 2008 1 10 PM Page 39 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 TMD021 Printed in USA Revised 10 08 IX B DNA Extraction and Quantitation Methods The DNA IQ System Cat DC6700 is a DNA isolation and quantitation system designed specifically for forensic and paternity samples 36 This novel 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 larger samples the DNA IQ System delivers a consistent amount of total DNA The system has been used to isolate and quantify DNA from routine sample types including buccal swabs stains on FTA paper and liquid blood Additionally DNA has been isolated from casework samples such as tissue differentially separated sexual assault samples and stains on support materials The DNA IQ System has been tested with the PowerPlex Systems to ensure a streamlined process See Section IX F for ordering information For applications requiring human specific DNA quantification the Plexor HY System Cat DC1001 DC1000 has been developed 37 See Section IX F
67. tes Page 38 Table 2 The PowerPlex S5 System Locus Specific Information STR Locus Label Chromosomal Location GenBank Locus and Locus Definition Repeat Sequence1 5 3 D8S1179 FL 8q NA TCTA Complex 25 D18S51 FL 18q21 3 HUMUT574 AGAA 25 Amelogenin2 FL Xp22 1 22 3 and Y HUMAMEL human Y chromosomal gene for Amelogenin like protein NA FGA JOE 4q28 HUMFIBRA human fibrinogen alpha chain gene TTTC Complex 25 TH01 JOE 11p15 5 HUMTH01 human tyrosine hydroxylase gene AATG 25 1The August 1997 report 26 27 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 2Amelogenin is not an STR but displays a 103 base X specific band and a 109 base Y specific band FL fluorescein JOE 6 carboxy 4 5 dichloro 2 7 dimethoxyfluorescein Table 3 The PowerPlex S5 System Allelic Ladder Information STR Locus Label Size Range of Allelic Ladder Components1 bases Repeat Numbers of Allelic Ladder Components D8S1179 FL 208 252 7 18 D18S51 FL 123 199 8 10 10 2 11 13 13 2 14 27 Amelogenin2 FL 103 109 X
68. thod Alternatively use a full range for the analysis Page 32 5686TA Figure 10 An example showing improper assignment of size standard fragments in the GeneMapper ID software tmd021 1008 qxp 10 13 2008 1 10 PM Page 32 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD021 Revised 10 08 Page 33 Symptoms Causes and Comments Size standard not called Extra peaks in advanced mode size standard Open the size correctly Figure 10 match editor Highlight the extra peak select Edit and select continued delete size label Select auto adjust sizes Run was too short and larger peaks in ILS were not captured Not all ILS 600 peaks defined in the size standard were detected during the run Create a new size standard using the internal lane standard fragments present in the sample Rerun samples using a longer run time Peaks in size standard missing If peaks are below threshold decrease the peak threshold in the analysis method for the red channel to include peaks If peaks are low quality redefine the size standard to skip these peaks Error message The size standard and analysis method were not in the same Either panel size standard mode Classic vs Basic or Advanced Be sure both files or analysis method is invalid are set to th
69. to version 3 2 Getting Started 1 Obtain the proper panel and bin files for use with GeneMapper ID from the Promega web site at www promega com geneticidtools panels_bins 2 Enter your contact information and select GeneMapper ID version 3 2 Select Submit 3 Select the PowerPlex S5 Panels amp Bin Sets link and save the zip file to your computer 4 Open the files using the Windows WinZip program and save the unzipped files to a known location on your computer Importing Panel and Bin Files These instructions loosely follow the Applied Biosystems GeneMapper ID software tutorial pages 1 4 1 Open the GeneMapper ID software version 3 2 2 Select Tools then Panel Manager 3 Highlight the Panel Manager icon in the upper left tile navigation pane 4 Select File then Import Panels 5 Navigate to the saved panel and bin files Select PowerPlex_S5_Panels_ID3 2x txt 6 In the navigation pane highlight the PowerPlex_S5_Panels folder that you just imported 7 Select File then Import Bin Set 8 Navigate to the saved panel and bin files Select PowerPlex_S5_Bins_ID3 2x txt then Import 9 At the bottom of the panel manager window select Apply then OK The Panel Manager window will close automatically tmd021 1008 qxp 10 13 2008 1 09 PM Page 17 Promega Corporation 2800 Woods Hollow Road
70. uide to amplification troubleshooting is provided in Section VII A 1 Thaw the PowerPlex S5 5X Master Mix PowerPlex S5 10X Primer Pair Mix and 9947A DNA completely Note Mix reagents by vortexing each tube for 15 seconds before each use Do not centrifuge the 10X Primer Pair Mix as this may cause the primers to be 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 waste 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 Place one clean 0 2ml amplification tube for each reaction into a rack and label appropriately Alternatively use a MicroAmp plate and label appropriately Page 6 tmd021 1008 qxp 10 13 2008 1 09 PM Page 6 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD021 Revised 10 08 Page 7 4 Add the final volume of each reagent listed in Table 1 into a sterile 1 5ml amber colored tube Mix gently Table 1 shows the component volumes per reaction A worksheet to calculate the required amount of each component of the PCR amplification
71. un module for all runs 6 Open a new plate record Name the plate and select GeneScan Select the plate size 96 well Select Finish 7 Complete the plate record spreadsheet for the wells you have loaded Enter appropriate information into the sample name and color info columns For allelic ladder samples insert the word ladder into the color info column for the blue and green dye colors This information must be entered to successfully analyze data with the PowerTyper S5 Macro 8 In the BioLIMS Project column select 3100_Project1 from the pull down menu 9 In the Dye Set column select Z from the pull down menu tmd021 1008 qxp 10 13 2008 1 09 PM Page 13 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 TMD021 Printed in USA Revised 10 08 V B Detection of Amplified Fragments Using the ABI PRISM 3100 Genetic Analyzer with Data Collection Software Version 1 0 1 or 1 1 continued 10 When using the ABI PRISM 3100 data collection software version 1 0 1 or 1 1 select GeneScan36_POP4PowerPlexS5_3kV_11secs_1200 from the pull down menu in the Run Module 1 column 11 To collect the data without autoanalyzing select No Selection in the Analysis Module 1 column Analysis parameters can be applied after data collection and during data analysis using th
72. w promega com Part TMD021 Printed in USA Revised 10 08 IV Protocols for DNA Amplification Using the PowerPlex S5 System Materials to Be Supplied by the User Applied Biosystems 2720 or GeneAmp PCR System 9600 9700 or 2400 thermal cyclers Applied Biosystems microcentrifuge 0 2ml MicroAmp reaction tubes or MicroAmp optical 96 well reaction plate Applied Biosystems 1 5ml amber colored microcentrifuge tubes Fisher Cat 05 402 26 aerosol resistant pipette tips Section IX F We routinely amplify 0 25 0 5ng of template DNA in a 25 l reaction volume using the protocols detailed below Saturated peaks may be observed if more than the recommended amount of template is used When using high template amounts reduce the amount of template DNA or the number of cycles 25 28 cycles The PowerPlex S5 System is optimized for the GeneAmp PCR System 9700 thermal cycler Amplification protocols for the Applied Biosystems 2720 and GeneAmp PCR Systems 9600 and 2400 thermal cyclers are provided IV A Amplification Setup The use of gloves and aerosol resistant pipette tips is highly recommended to prevent cross contamination Keep all pre amplification and postamplification 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 g
73. x S5 10X Primer Pair Mix 2 5 l template DNA 0 25 0 5ng 2 up to 17 5 l total reaction volume 25 l 1Add Water Amplification Grade to the PCR amplification mix first then add PowerPlex S5 5X Master Mix and PowerPlex S5 10X Primer Pair Mix The template DNA will be added at Step 7 2Store DNA templates in Water Amplification Grade or TE 4 buffer 10mM Tris HCl pH 8 0 0 1mM EDTA If the DNA template is stored in TE buffer that is not pH 8 0 or contains a higher EDTA concentration the volume of the DNA sample 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 tmd021 1008 qxp 10 13 2008 1 09 PM Page 7 IV B Amplification Thermal Cycling Amplification and detection instrumentation may vary You may need to optimize protocols including cycle number and injection time for each laboratory instrument Testing at Promega Corporation shows that 30 cycles work well with 0 25 0 5ng of purified DNA Increased cycle number 32 34 cycles will result in increased sensitivity when using low amounts of template Decreased cycle number may be used if a higher amount of template is added to the amplificatio
74. ze and range listed in the categories for the same alleles If necessary increase the category start range in the category window to greater than 6bp and save the macro under a new name Allelic ladder peaks were too high causing stutter peaks to be called as allele peaks Use a shorter injection time decrease the amount of allelic ladder used or re analyze the allelic ladder sample using increased peak amplitude thresholds in the GeneScan analysis parameters Allelic ladder data were not compatible with the PowerTyper file used Confirm that the PowerTyper Macro file matches the allelic ladder being used The plots window or allele The macros were not run in the proper order Use the POWER table does not display all data or POWER 20 Filter macro option All three dye colors were not imported For Genotyper software versions 2 5 and 3 5 or higher set preferences in the Edit menu to import the blue green and red colors The Check ILS macro All three dye colors were not imported For Genotyper displays an empty plot software versions 2 5 and 3 5 or higher set preferences window in the Edit menu to import the blue green and red colors Off ladder peaks Migration of samples changed slightly over the course of a CE run with many samples This may be due to changes in temperature or the CE column over time Use a different injection of allelic ladder to determine sizes in the PowerTyper S5 Macro Do not use the

PowerPlex S5 System Technical Manual, TMD021

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