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PowerPlex® Fusion System

1. 67 F Summary of Changes 68 1 Description STR short tandem repeat loci consist of short repetitive sequence elements 3 7 base pairs in length 1 4 These repeats are well distributed throughout the human genome and are a rich source of highly polymorphic markers which may be detected using the polymerase chain reaction 5 9 Alleles of STR loci are differentiated by the number of copies of the repeat sequence contained within the amplified region and are distinguished from one another using fluorescence detection following electrophoretic separation The PowerPlex Fusion System a g is a 24 locus multiplex for human identification applications including forensic analysis relationship testing and research use This five color system allows co amplification and fluorescent detection of the 13 core CODIS US loci CSF1PO FGA TH01 TPOX vWA D3S1358 D5S818 D7S820 D8S1179 D13S317 D16S539 D18S51 and D21S11 the 12 core European Standard Set loci TH01 vWA FGA D21S11 D3S1358 D8S1179 D18S51 D10S1248 D22S1045 D2S441 D1S1656 and D12S391 and Amelogenin for gender determination In addition the male specific DYS391 locus is included to identify null Y allele results for Amelogenin The Penta D and Penta E loci are included to increase discrimination and allow searching of databases that include profiles with these Pent
2. 73 and 75 77 bases in TH01 214 bases in D18S51 and 247 bases in D2S1338 These artifacts are typically below common minimum thresholds The PowerPlex Fusion System is optimized for use with POP 4 polymer This system was not developed for use with POP 7 polymer if using POP 7 polymer optimization and in house validation are required Some DNA independent artifacts specific to the PowerPlex Fusion System with POP 7 polymer have been noted The global filters used for database analyses will generally filter these artifact peaks Artifact peaks may be seen in the fluorescein channel at 74 76 bases 85 87 bases and 99 101 bases In the JOE channel artifacts may be seen at 88 90 bases The signal strength of the JOE channel artifact increases with storage of the amplification plate at 4 C most commonly when plates are left at 4 C for a few days We recommend storing amplification products at 20 C Page 47 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 TMD039 Revised 10 12 Page 48 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 TMD039 Printed in USA Revised 10 12 Figure 22 The PowerPlex Fusion System A single source male template DNA
3. navigate to the Library Select Results Group then select Create Alternatively a previously created Results Group may be used Select the Results Group Attributes according to laboratory practices Save with a descriptive name 8 To create a New Plate navigate to the Library and from the Manage menu select Plates then Create Figure 8 The Create New Results Group window 9253TA Page 23 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 TMD039 Revised 10 12 5 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer continued 9 Assign a descriptive plate name Select the plate type HID from the drop down menu Figure 9 10 Select Assign Plate Contents Figure 10 11 Assign sample names to wells 12 In the lower left portion of the screen under Assays use the Add from Library option to select the Assay created in Step 5 or one previously created Click on the Add to Plate button and close the window Figure 10 Assigning plate contents 9255TA Figure 9 Defining plate properties Page 24 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD039
4. 0 5ng was amplified using the PowerPlex Fusion System and 30 cycles Amplification products were mixed with CC5 Internal Lane Standard 500 and analyzed with an Applied Biosystems 3130 Genetic Analyzer using a 3kV 5 second injection Results were analyzed using GeneMapper ID software version 3 2 Panel A An electropherogram showing the peaks of the fluorescein labeled loci Amelogenin D3S1358 D1S1656 D2S441 D10S1248 D13S317 and Penta E Panel B An electropherogram showing the peaks of the JOE labeled loci D16S539 D18S51 D2S1338 CSF1PO and Penta D Panel C An electropherogram showing the peaks of the TMR ET labeled loci TH01 vWA D21S11 D7S820 D5S818 TPOX and DYS391 Panel D An electropherogram showing the peaks of the CXR ET labeled loci D8S1179 D12S391 D19S433 FGA and D22S1045 Panel E An electropherogram showing the 60bp to 500bp fragments of the CC5 Internal Lane Standard 500 11080TA A B C D E Page 49 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 TMD039 Revised 10 12 11048TA Figure 23 The PowerPlex Fusion Allelic Ladder Mix The PowerPlex Fusion Allelic Ladder Mix was analyzed with an Applied Biosystems 3130 Genetic Analyzer using a 3kV 3 second injection The sample file was analyzed with the GeneMapper ID software version 3 2 and Powe
5. Do not re freeze avoid multiple freeze thaw cycles as this may reduce activity DNA was not accessible on nonlytic material Small loci may amplify preferentially with large loci dropping out Pretreat nonFTA materials with PunchSolution Reagent to ensure that DNA is liberated from cellular proteins 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 TMD039 Revised 10 12 Page 55 7 E GeneMapper ID X Software Symptoms Causes and Comments Stutter peaks not filtered Stutter text file was not imported into the Panel Manager when the panels and bins text files were imported Be sure that the Use marker specific stutter ratio and distance if available box is checked Stutter distance was not defined in the Analysis Method Allele tab Samples in the project not analyzed The Analysis Requirement Summary window was not active and there was an analysis requirement that was not met Turn on Analysis Requirement Summary in the Options menu and correct the necessary analysis requirements to continue analysis Edits in label edit viewer cannot To view edits made to a project the project first must be be viewed saved Close the plot view window return to the main GeneMapper ID X page and save the project Display the plot window again then view the label edit table Marker header bar for s
6. Fusion Allelic Ladder Mix 2 300 l CC5 Internal Lane Standard 500 Product Size Cat PowerPlex Fusion System 800 reactions DC2408 Not For Medical Diagnostic Use This system contains sufficient reagents for 800 reactions of 25 l each Includes Pre amplification Components Box 4 1ml PowerPlex Fusion 5X Master Mix 4 1ml PowerPlex Fusion 5X Primer Pair Mix 25 l 2800M Control DNA 10ng l 10 1 250 l Water Amplification Grade Post amplification Components Box 4 100 l PowerPlex Fusion Allelic Ladder Mix 8 300 l CC5 Internal Lane Standard 500 The PowerPlex Fusion Allelic Ladder Mix is provided in a separate sealed bag for shipping This component should be moved to the post amplification box after opening The Water Amplification Grade is provided in a separate sealed bag for shipping This component should be moved to the pre amplification box after opening Storage Conditions For long term storage store all components except the 2800M Control DNA at 30 C to 10 C in a nonfrost free freezer Store the 2800M Control DNA at 2 10 C For daily use the PowerPlex Fusion System components can be stored for up to 1 week at 2 10 C The PowerPlex Fusion 5X Primer Pair Mix PowerPlex Fusion Allelic Ladder Mix and CC5 Internal Lane Standard 500 CC5 ILS 500 are light sensitive and must be stored in the dark We strongly recommend that pre amplification and post ampli
7. It also ensures that each reaction contains the same PCR amplification mix 3 Use a clean MicroAmp plate for reaction assembly and label appropriately 4 Add the final volume of each reagent listed in Table 3 to a sterile tube 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 Printed in USA Part TMD039 Revised 10 12 Table 3 PCR Amplification Mix for Direct Amplification of DNA from Swabs PCR Amplification Mix Component1 Volume Per Reaction Number of Reactions Final Volume Water Amplification Grade 13 l PowerPlex Fusion 5X Master Mix 5 0 l PowerPlex Fusion 5X Primer Pair Mix 5 0 l swab extract 2 0 l total reaction volume 25 l 1Add Water Amplification Grade to the tube first then add PowerPlex Fusion 5X Master Mix and PowerPlex Fusion 5X Primer Pair Mix The swab extract will be added at Step 6 4 C Direct Amplification of DNA from Swabs continued 5 Vortex the PCR amplification mix for 5 10 seconds then pipet 23 l of PCR amplification mix into each reaction well Failure to vortex the PCR amplification mix sufficiently can result in poor amplification or locus to locus imbalance 6 Pipet 2 0 l of swab extract for each sample into the appropriate well of the reaction plate 7 For the positive amplification
8. Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD039 Revised 10 12 6 J Creating a Databasing or Paternity Analysis Method with GeneMapper ID Software Version 3 2 continued 11 Select the Peak Quality tab You may change the settings for peak quality Note For Steps 11 and 12 see the GeneMapper ID user s manual for more information 12 Select the Quality Flags tab You may change these settings 13 Select OK to save your settings Processing Data for Databasing or Paternity Samples 1 Select File then New Project 2 Select Edit then Add Samples to Project 3 Browse to the location of the run files Highlight desired files then select Add to list followed by Add 4 In the Sample Type column use the drop down menu to select Ladder Sample Positive Control or Negative Control as appropriate for the sample Every folder in the project must contain at least one allelic ladder injection that is designated as Ladder in the Sample Type column for proper genotyping Note The positive control DNA defined in the GeneMapper ID panel file is the 2800M Control DNA Redefine the genotype in the panel file if using a different positive control DNA 5 In the Analysis Method column select the analysis method created previously in this section 6 In the Panel column select the panels text file that
9. User Guide 1 Open the 3500 Data Collection Software The Dashboard screen will launch Figure 2 Ensure that the Consumables Information and Maintenance Notifications are acceptable Set the oven temperature to 60 C then select Start Pre Heat at least 30 minutes prior to the first injection to preheat the oven 2 To create a new Instrument Protocol navigate to the Library select Instrument Protocol then select Create Alternatively a previously created Instrument Protocol may be used 9247TA Figure 2 The Dashboard Page 17 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD039 Revised 10 12 5 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer continued Figure 3 shows the settings used at Promega for the Applied Biosystems 3500xL Genetic Analyzer for the application type dye set capillary length polymer run module and appropriate protocol information The only setting that was changed from the default settings is dye set The recommended settings are Figure 3 The Create New Instrument Protocol window 9393TA Application Type HID Capillary Length 36cm Polymer POP 4 Dye Set G5 Promega G5 spectral Run Module HID36_POP4 xl Injection Time1 24 seconds Injection Voltage
10. WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD039 Printed in USA Revised 10 12 Page 14 Thermal Cycling Protocol1 96 C for 1 minute then 94 C for 10 seconds 59 C for 1 minute 72 C for 30 seconds for 27 cycles then 60 C for 20 minutes 4 C soak 1When using the GeneAmp PCR System 9700 thermal cycler the program must be run with Max mode as the ramp speed This requires a silver or gold plated silver sample block The ramp speed is set after the thermal cycling run is started The Select Method Options screen appears Select Max for the ramp speed and enter the reaction volume 3 After completion of the thermal cycling protocol store amplified samples at 20 C in a light protected box Note Long term storage of amplified samples at 4 C or higher may produce artifacts PCR Optimization Cycle number should be optimized based on the results of an initial experiment to determine the sensitivity with your collection method sample types and instrumentation 1 Choose several samples that represent typical sample types you encounter in the laboratory Prepare them as you would using your normal workflow 2 Prepare four identical reaction plates with aliquots of the same swab extracts 3 Amplify samples using the thermal cycling protocol provided above but subject each plate to a different cycle number 25 28 cy
11. select the analysis method created previously in this section 6 In the Panel column select the panels text file that was imported in Section 6 F 7 In the Size Standard column select the size standard that was imported in Section 6 G or created in Section 6 H 8 Select Analyze green arrow button to start data analysis Note Sizing of Penta E and DYS391 alleles 475 bases will not use Local Southern Method For Penta E alleles gt 24 will be labeled as OL 6 J Creating a Databasing or Paternity Analysis Method with GeneMapper ID Software Version 3 2 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 PowerPlex_Fusion_20 filter 6 Select the Allele tab Figure 21 7 Select the bins text file that was imported in Section 6 F 8 Ensure that the Use marker specific stutter ratio if available box is checked Page 44 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 TMD039 Printed in USA Revised 10 12 9 Enter the values
12. when using the PowerPlex Fusion System For an explanation of the proper usage and effects of these settings refer to the Applied Biosystems user bulletin titled Installation Procedures and New Features for GeneMapper ID Software 3 2 Note Some of these settings have been optimized and are different from the recommended settings in the user bulletin 11052TA Figure 19 The GeneMapper ID Allele tab 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 Part TMD039 Printed in USA Revised 10 12 10 Select the Peak Detector tab We recommend the settings shown in Figure 20 Notes 1 Select full range or partial range for the analysis range When using a partial range choose an appropriate analysis range based on your data Choose a start point after the primer peak and just before the first defined internal lane standard peak to help ensure proper sizing of the internal lane standard 2 The peak amplitude thresholds are the minimum peak heights at which the software will call a peak Values for peak amplitude thresholds are usually 50 150RFU and should be determined by individual laboratories Peak heights for the CC5 ILS 500 are generally lower than those for the other dyes Therefore the threshold for the orange dye may be lower than that for the other dyes 11 Select the Peak Quali
13. 1 2kV Run Time 1 210 1 500 seconds 1Injection time may be modified 2 24 seconds to increase or decrease peak heights 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 TMD039 Printed in USA Revised 10 12 Page 18 When creating an Instrument Protocol be sure to select the same dye set that was used to perform the Promega 5 dye spectral calibration We recommend using a run time of 1 210 1 500 seconds and the default injection conditions Run time and other instrument settings should be optimized and validated in your laboratory When optimizing injection conditions in your laboratory you may choose to create specific Instrument Protocols for each condition tested If a single Instrument Protocol is used follow the instructions in the Applied Biosystems 3500 3500xL Genetic Analyzers User Guide to edit a library entry Assign a descriptive protocol name Note For more detailed information refer to the Applied Biosystems 3500 3500xL Genetic Analyzers User Guide 3 To create a new Size Standard for the QC protocol navigate to the Library Select Size Standards then select Create Alternatively a previously created Size Standard may be used Assign the size standard the name ILS500 or another appropriate name Choose Orange as the Dye Color The fragments in the size s
14. 2 40 H Creating a Size Standard with GeneMapper ID Software Version 3 2 40 I Creating a Casework Analysis Method with GeneMapper ID Software Version 3 2 41 J Creating a Databasing or Paternity Analysis Method with GeneMapper ID Software Version 3 2 44 K Controls 46 L Results 47 PowerPlex Fusion System All technical literature is available on the Internet at www promega com protocols Please visit the web site to verify that you are using the most current version of this Technical Manual Please contact Promega Technical Services if you have questions on use of this system E mail genetic promega com 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 TMD039 Revised 10 12 Page 1 7 Troubleshooting 50 A Amplification and
15. 26 Mandrekar P V Krenke B E and Tereba A 2001 DNA IQ The intelligent way to purify DNA Profiles in DNA 4 3 16 27 Krenke B E et al 2005 Development of a novel fluorescent two primer approach to quantitative PCR Profiles in DNA 8 1 3 5 9 Appendix 9 A Advantages of Using the Loci in the PowerPlex Fusion System A single PowerPlex Fusion System reaction amplifies all core loci required for US CODIS and European databases Tables 4 and 5 Table 6 lists the PowerPlex Fusion System alleles revealed in commonly available standard DNA templates Additionally the male specific DYS391 locus is included to identify null Y results for Amelogenin We have carefully selected primers to avoid or minimize artifacts including those associated with DNA polymerases such as repeat slippage and terminal nucleotide addition 14 15 Repeat slippage 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 the DNA sequence being amplified Terminal nucleotide addition 16 17 occurs when a thermostable nonproofeading 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 a
16. Application drop down list and select the appropriate plate type 96 well Add entries in the owner and operator windows and select OK Note If autoanalysis of sample data is desired refer to the instrument user s manual for instructions 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 TMD039 Revised 10 12 Page 27 5 B Detection of Amplified Fragments Using the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 or the Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 continued 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 select dye set G5 and confirm that the activ
17. Applied Biosystems 3500 or 3500xL Genetic Analyzer 15 B Detection of Amplified Fragments Using the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 or the Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 26 6 Data Analysis 29 A Importing PowerPlex Fusion Panels Bins and Stutter Text Files with GeneMapper ID X Software Version 1 2 29 B Importing the CC5 ILS 500 IDX Size Standard into GeneMapper ID X Software Version 1 2 30 C Creating a Size Standard with GeneMapper ID X Software Version 1 2 30 D Creating a Casework Analysis Method with GeneMapper ID X Software Version 1 2 31 E Creating a Databasing or Paternity Analysis Method with GeneMapper ID X Software Version 1 2 35 F Importing PowerPlex Fusion Panels and Bins Text Files with GeneMapper ID Software Version 3 2 38 G Importing the CC5 ILS 500 Size Standard into GeneMapper ID Software Version 3
18. Instrument detection limits vary therefore injection time injection voltage or the amount of sample mixed with loading cocktail may need to be increased or decreased To modify the injection time or injection voltage in the run module select Instrument Protocol from the Library menu in the data collection software If peak heights are higher than desired use less DNA template in the amplification reactions or reduce the number of cycles in the amplification program to achieve the desired signal intensity If the injection time or voltage is reduced a decreased peak amplitude threshold for the orange channel may be required for proper sizing 6 Centrifuge plate briefly to remove air bubbles from the wells 7 Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading the instrument 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 Part TMD039 Printed in USA Revised 10 12 Instrument Preparation Refer to the Applied Biosystems 3500 3500xL Genetic Analyzer User Guide for the instrument maintenance schedule and instructions to install the capillary array buffers and polymer pouch and perform a spatial calibration Samples may be analyzed as described in the Applied Biosystems 3500 3500xL Genetic Analyzer
19. Printed in USA Revised 10 12 13 Under File Name Convention use the Add from Library option to select the File Name Convention created in Step 6 or one previously created Click on the Add to Plate button and close the window 14 Under Results Groups use the Add from Library option to select the Results Group created in Step 7 or one previously created Click on the Add to Plate button and close the window 15 Highlight the sample wells then select the boxes in the Assays File Name Conventions and Results Groups that pertain to those samples 16 Select Link Plate for Run 17 The Load Plate window will appear Select Yes 18 In the Run Information window Figure 11 assign a Run Name Select Start Run not shown Figure 11 Assigning a run name 9256TA 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 Printed in USA Part TMD039 Revised 10 12 5 B Detection of Amplified Fragments Using the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 or the Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 Materials to Be Supplied by the User 95 C dry heating block water bath or thermal cycler crushed ice or ice water bath centrifuge compatible with 96 wel
20. Size Cat DNA IQ System 100 reactions DC6701 400 reactions DC6700 Plexor HY System 200 reactions DC1001 800 reactions DC1000 Not for Medical Diagnostic Use 9 F Summary of Changes The following change was made to the 5 14 revision of this document Legal disclaimers were updated a U S Pat No 6 242 235 Australian Pat No 761757 Canadian Pat No 2 335 153 Chinese Pat No ZL99808861 7 Hong Kong Pat No HK 1040262 Japanese Pat No 3673175 European Pat No 1088060 and other patents pending b U S Pat Nos 5 843 660 6 479 235 6 221 598 and 7 008 771 Australian Pat No 724531 Canadian Pat No 2 118 048 and 2 251 793 Korean Pat No 290332 Singapore Pat No 57050 Japanese Pat Nos 3602142 and 4034293 Chinese Pat Nos ZL99813729 4 and ZL97194967 0 European Pat No 0960207 and other patents pending c U S Pat No 6 238 863 European Pat No 1058727 Chinese Pat No ZL99802696 4 Japanese Pat No 4494630 and other patents pending d STR loci are the subject of U S Pat No RE 37 984 German Pat No DE 38 34 636 C2 and other patents issued to the Max Planck Gesellschaft zur F rderung der Wissenschaften e V Germany e Allele sequences for one or more of the loci vWA FGA D8S1179 D21S11 and D18S51 in allelic ladder mixtures is licensed under U S Pat Nos 7 087 380 7 645 580 Australia Pat No 2003200444 and corresponding patent claims outside the US f TMR ET CXR ET a
21. blood in a 25 l reaction volume using the protocols detailed below The PowerPlex Fusion System is optimized for the GeneAmp PCR System 9700 thermal cycler Note You will need to optimize and validate the number of storage card punches per reaction in your laboratory See the PCR optimization recommendations at the end of the section FTA based sample types include Buccal cells collected on FTA cards with Whatman EasiCollect or Fitzco Sampact devices Buccal cells collected with sterile swabs transferred to FTA or Indicating FTA cards Liquid blood from collection or storage Vacutainer tubes or finger sticks spotted onto FTA cards NonFTA sample types include Buccal samples on Bode Buccal DNA Collector devices Blood and buccal samples on nonFTA card punches e g S amp S 903 Pretreat nonFTA sample types with the PunchSolution Kit Cat DC9271 to lyse nonFTA samples before adding the PCR amplification mix For more information see the PunchSolution Kit Technical Manual TMD038 Failure to pretreat these samples may result in incomplete profiles Use a manual punch tool with a 1 2mm tip to manually create sample disks from a storage card Place tip near the center of the sample spot and with a twisting or pressing action cut a 1 2mm sample disk Use the plunger to eject the disk into the appropriate well of a reaction plate Page 9 Promega Corporation 2800 Wood
22. control vortex the tube of 2800M Control DNA then dilute an aliquot to 5 0ng l Add 2 l 10ng to a reaction well containing 23 l of PCR amplification mix 8 For the negative amplification control pipet 2 0 l of Water Amplification Grade or TE 4 buffer instead of swab extract into a reaction well containing PCR amplification mix 9 Seal the plate Optional Briefly centrifuge the plate to bring contents to the bottom of the wells and remove any air bubbles Thermal Cycling Amplification and detection instrumentation may vary You will need to optimize protocols including the amount of template DNA cycle number 25 28 cycles injection time and loading volume for your laboratory instrumentation Testing at Promega shows that 27 cycles works well for a variety of sample types Cycle number will need to be optimized in each laboratory for each sample type that is amplified 1 Place the MicroAmp plate in the thermal cycler 2 Select and run the recommended protocol Be sure that Max mode is selected as the ramp speed The preferred protocol for use with the GeneAmp PCR System 9700 thermal cycler is provided below The estimated total cycle time is 1 5 hours Note The final extension for direct amplification was extended to 20 minutes compared to 10 minutes for the extracted DNA protocol to allow sufficient time for adenylation of large amounts of amplicon Promega Corporation 2800 Woods Hollow Road Madison
23. shown in Figure 21 for proper filtering of peaks when using the PowerPlex Fusion System For an explanation of the proper usage and effect of these settings refer to the Applied Biosystems user bulletin titled Installation Procedures and New Features for GeneMapper ID Software 3 2 Note Ensure that the appropriate 20 filter is applied to this analysis method by entering 0 20 for the Global Cut off Value for Tri Tetra and Penta repeats 10 Select the Peak Detector tab We recommend the settings shown in Figure 20 Notes 1 Select full range or partial range for the analysis range When using a partial range choose an appropriate analysis range based on your data Choose a start point after the primer peak and just before the first defined internal lane standard peak to help ensure proper sizing of the internal lane standard 2 The peak amplitude thresholds are the minimum peak heights that the software will call as a peak Values for peak amplitude thresholds are usually 50 150RFU and should be determined by individual laboratories Peak heights for the CC5 ILS 500 are generally lower than those for the other dyes Therefore the threshold for the orange dye may be lower than that for the other dyes 11053TA Figure 21 The GeneMapper ID Allele tab with settings for using a 20 peak filter Page 45 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526
24. the orange dye may be lower than that for the other dyes 3 The normalization box can be checked regardless of whether normalization was or was not applied during data collection 11046TA Figure 14 The GeneMapper ID X Peak Detector tab Page 33 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD039 Revised 10 12 6 D Creating a Casework Analysis Method with GeneMapper ID X Software Version 1 2 continued 11 Select the Peak Quality tab You may change the settings for peak quality Note For Steps 11 and 12 see the GeneMapper ID X user s manual for more information 12 Select the SQ amp GQ Settings tab You may change these settings 13 Select Save to save the new analysis method 14 Select Done to exit the GeneMapper ID X Manager Processing Data for Casework Samples 1 Select File then New Project 2 Select Edit then Add Samples to Project 3 Browse to the location of the run files Highlight desired files then select Add to list followed by Add 4 In the Sample Type column use the drop down menu to select Allelic Ladder Sample Positive Control or Negative Control as appropriate for the sample Every folder in the project must contain at least one allelic ladder i
25. used during analysis Ensure that only high quality allelic ladders are used for analysis Size standard not called Starting data point was incorrect for the partial range chosen correctly in Section 6 I Adjust the starting data point in the analysis method Alternatively use a full range for the analysis Extra peaks in advanced mode size standard Open the Size Match Editor Highlight the extra peak select Edit and select delete size label Select auto adjust sizes Run was too short and larger peaks in ILS were not captured Not all CC5 ILS 500 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 Re run samples using a longer run time Peaks in size standard missing If peaks are below threshold decrease the peak amplitude threshold in the analysis method for the orange channel to include peaks If peaks are low quality redefine the size standard for the sample to skip these peaks Error message The size standard and analysis method were not in the same Either panel size standard mode Classic vs Basic or Advanced Be sure both files or analysis method is invalid are set to the same mode either Classic or Basic or Advanced mode No alleles called but no error Panels text file was not selected for sample In the Panel message appears column select the appropriate panels text fil
26. used is increased and decreased if the cycle number is increased Increase or decrease by twofold the mass of 2800M Control DNA for every one cycle decrease or increase respectively Improper storage of the 2800M Control DNA 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 TMD039 Printed in USA Revised 10 12 Page 54 Symptoms Causes and Comments Extra peaks visible in one or Swab extract was contaminated Assemble a reaction all color channels containing the swab extract prepared from a blank swab or assemble a reaction where the SwabSolution Reagent is processed and incubated as a blank without a swab Artifacts of STR amplification Amplification of swab extracts with high DNA concentrations can result in artifact peaks due to overamplification resulting in saturated signal on the CE instrument We recommend 2 l of swab extract per 25 l reaction Using more than 2 l in a 25 l reaction or using 2 l with a smaller reaction volume may result in overamplification and signal saturation If signal is saturated repeat amplification with less swab extract or reduced cycle number Artifacts of STR amplification Amplification of STRs can result in artifacts that appear as peaks one base smaller than the allele due to incomplete addition of the 3 A residue Be sure to perform the 20 minute extension step
27. 0 12 Page 20 5 To create a new Assay navigate to the Library Select Assays then select Create Alternatively a previously created Assay may be used In the Create New Assay window Figure 6 select the Instrument Protocol created in Step 2 and the QC Protocol created in Step 4 Assign a descriptive assay name Select the application type HID An Assay is required for all named samples on a plate Figure 6 The Create New Assay window 9229TA 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 TMD039 Revised 10 12 Page 21 5 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer continued 6 To create a new File Name Convention Figure 7 navigate to the Library Select File Name Conventions then select Create Alternatively a previously created File Name Convention may be used Select the File Name Attributes according to laboratory practices and save with a descriptive name Figure 7 The Create New File Name Convention window 9252TA Page 22 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD039 Printed in USA Revised 10 12 7 To create a new Results Group Figure 8
28. 0 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 TMD039 Revised 10 12 Page 59 8 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 b actin related pseudogene 2 actbp2 detected using the polymerase chain reaction Nucleic Acids Res 19 6980 5 Ausubel F M et al 1996 Unit 15 The polymerase chain reaction In Current Protocols in Molecular Biology Vol 2 John Wiley and Sons NY 6 Sambrook J Fritsch E F and Maniatis T 1989 Chapter 14 In vitro amplification of DNA by the polymerase chain reaction In Molecular Cloning A Laboratory Manual Second Edition Cold Spring Harbor Laboratory Press Cold Spring Harbor New York 7 PCR Technology Principles and Applications for DNA Amplification 1989 Erlich H A ed Stockton Press
29. 050TA Figure 13 The GeneMapper ID X Allele tab Page 32 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD039 Printed in USA Revised 10 12 10 Select the Peak Detector tab Figure 14 shows an example of settings used at Promega You may need to optimize these settings In house validation should be performed Notes 1 Select full range or partial range for the analysis range When using a partial range choose an appropriate analysis range based on your data Choose a start point after the primer peak and just before the first defined internal lane standard peak to help ensure proper sizing of the internal lane standard 2 The peak amplitude thresholds are the minimum peak heights at which the software will call a peak Values for peak amplitude thresholds are usually 50 150RFU for data generated on the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xl Genetic Analyzers For the Applied Biosystems 3500 and 3500xL Genetic Analyzers Life Technologies suggests an analysis threshold of 175RFU under their default injection conditions However individual laboratories should determine their peak amplitude thresholds from internal validation studies Peak heights for the CC5 ILS 500 are generally lower than those for the other dyes Therefore the threshold for
30. 15 15 17 D1S1656 12 13 18 3 18 3 14 17 D2S441 10 14 10 14 11 12 D10S1248 13 15 13 15 12 15 D13S317 9 11 11 11 11 11 Penta E 7 14 12 13 11 11 D16S539 9 13 11 12 11 11 D18S51 16 18 15 19 15 18 D2S1338 22 25 19 23 23 23 CSF1PO 12 12 10 12 10 11 Penta D 12 13 12 12 8 12 TH01 6 9 3 8 9 3 6 9 3 vWA 16 19 17 18 17 17 D21S11 29 31 2 30 30 29 30 D7S820 8 11 10 11 11 11 D5S818 12 12 11 11 11 13 TPOX 11 11 8 8 8 9 DYS391 10 10 D8S1179 14 15 13 13 12 13 D12S391 18 23 18 20 18 24 D19S433 13 14 14 15 13 14 FGA 20 23 23 24 24 26 D22S1045 16 16 11 14 16 18 1Information on strains 9947A and 9948 is available online at http ccr coriell org Sections Search Sample_Detail aspx Ref GM09947 and http ccr coriell org Sections Search Sample_Detail aspx Ref GM09948 Information about the use of 9947A and 9948 DNA as standard DNA templates can be found in reference 25 9 B DNA Extraction and Quantitation Methods and Automation Support Promega offers a wide variety of reagents and automated methods for sample preparation DNA purification and DNA quantitation prior to STR amplification For analysis of database reference and other single source samples we recommend direct amplification from FTA punches or preprocessing of swabs and nonFTA punches with the SwabSolution Kit or P
31. 16 www promega com Printed in USA Part TMD039 Revised 10 12 3 B Spectral Calibration Proper spectral calibration is critical to evaluate multicolor systems with the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 3130xl 3500 and 3500xL Genetic Analyzers A matrix must be generated for each individual instrument For protocols and additional information on spectral calibration on these instruments see the PowerPlex 5 Dye Matrix Standards 3100 3130 Technical Bulletin TBD024 This manual is available online at www promega com protocols 4 Protocols for DNA Amplification Using the PowerPlex Fusion System The PowerPlex Fusion System was developed for amplification of extracted DNA and direct amplification samples Slight protocol variations are recommended for optimal performance for each template source Protocols for amplification using extracted DNA Section 4 A FTA and nonFTA storage card punches Section 4 B and swabs Section 4 C are included in the following amplification sections The PowerPlex Fusion System is optimized for the GeneAmp PCR System 9700 thermal cycler The use of gloves and aerosol resistant pipette tips is highly recommended to prevent cross contamination Keep all pre amplification and post amplification reagents in separate rooms Prepare amplification reactions in a room dedicated for reaction setup Use equipment and supplies dedicated for amplifica
32. 18 2 FGA CXR ET 265 411 14 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 32 2 33 2 42 2 43 2 44 2 45 2 46 2 48 2 50 2 D22S1045 CXR ET 425 464 7 20 1The length of each allele in the allelic ladder has been confirmed by sequence analysis 2When using an internal lane standard such as the CC5 Internal Lane Standard 500 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 and linker also affect migration of alleles 3For a current list of microvariants see the Variant Allele Report published at the U S National Institute of Standards and Technology NIST web site at www cstl nist gov div831 strbase 4Amelogenin is not an STR but displays an 89 base X specific band and a 95 base Y specific band 9 A Advantages of Using the Loci in the PowerPlex Fusion System continued Page 64 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 TMD039 Printed in USA Revised 10 12 Table 6 The PowerPlex Fusion System Allele Determinations in Commonly Available Standard DNA Templates STR Locus Standard DNA Templates1 2800M 9947A 9948 Amelogenin X Y X X X Y D3S1358 17 18 14
33. 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD039 Revised 10 12 Table 5 The PowerPlex Fusion System Allelic Ladder Information STR Locus Label Size Range of Allelic Ladder Components1 2 bases Repeat Numbers of Allelic Ladder Components3 Amelogenin4 Fluorescein 89 95 X Y D3S1358 Fluorescein 103 147 9 20 D1S1656 Fluorescein 161 208 9 14 14 3 15 15 3 16 16 3 17 17 3 18 18 3 19 19 3 20 3 D2S441 Fluorescein 214 250 8 11 11 3 12 17 D10S1248 Fluorescein 256 280 8 19 D13S317 Fluorescein 302 350 5 17 Penta E Fluorescein 371 466 5 24 D16S539 JOE 84 132 4 16 D18S51 JOE 134 214 7 10 10 2 11 13 13 2 14 27 D2S1338 JOE 224 296 10 12 14 28 CSF1PO JOE 318 362 5 16 Penta D JOE 377 450 2 2 3 2 5 17 TH01 TMR ET 72 115 3 9 9 3 10 11 13 3 vWA TMR ET 127 183 10 24 D21S11 TMR ET 203 259 24 24 2 25 25 2 26 28 28 2 29 29 2 30 30 2 31 31 2 32 32 2 33 33 2 34 34 2 35 35 2 36 38 D7S820 TMR ET 269 313 5 16 D5S818 TMR ET 321 369 6 18 TPOX TMR ET 393 441 4 16 DYS391 TMR ET 442 486 5 16 D8S1179 CXR ET 76 124 7 19 D12S391 CXR ET 133 185 14 17 17 3 18 18 3 19 27 D19S433 CXR ET 193 245 5 2 6 2 8 12 12 2 13 13 2 14 14 2 15 15 2 16 16 2 17 17 2 18
34. DNA Use the recommended amount of template DNA if available Stochastic effects can occur when amplifying low amounts of template Impure template DNA Inhibitors that may be present in forensic samples can lead to allele dropout or imbalance Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD039 Printed in USA Revised 10 12 Page 52 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 TMD039 Revised 10 12 7 C Direct Amplification of DNA From Storage Card Punches The following information is specific to direct amplification of DNA from storage card punches For additional information about general amplification and detection see Section 7 A Symptoms Causes and Comments Faint or absent allele peaks The reaction volume was too low This system is optimized for a final reaction volume of 25 l to overcome inhibitors present in FTA cards and DNA samples Decreasing the reaction volume may result in suboptimal performance Poor sample transfer to storage card or variable sampling from storage card Take punches from a different portion of the card Increasing cycle number can improve low peak heights DNA was not accessible on nonlytic material Pretreat nonFTA material
35. Fragment Detection 50 B Amplification of Extracted DNA 52 C Direct Amplification of DNA From Storage Card Punches 53 D Direct Amplification of DNA From Swabs 54 E GeneMapper ID X Software 56 F GeneMapper ID Software 57 8 References 60 9 Appendix 61 A Advantages of Using the Loci in the PowerPlex Fusion System 61 B DNA Extraction and Quantitation Methods and Automation Support 65 C The CC5 Internal Lane Standard 500 66 D Composition of Buffers and Solutions 67 E Related Products
36. JOE 2q35 218 705Mb TGCC TTCC CSF1PO JOE 5q33 1 149 436Mb AGAT Penta D JOE 21q22 3 43 88Mb AAAGA TH01 TMR ET 11p15 5 2 149Mb AATG 19 vWA TMR ET 12p13 31 5 963Mb TCTA Complex 19 D21S11 TMR ET 21q21 1 19 476Mb TCTA Complex 19 D7S820 TMR ET 7q21 11 83 433Mb GATA D5S818 TMR ET 5q23 2 123 139Mb AGAT TPOX TMR ET 2p25 3 1 472Mb AATG DYS391 TMR ET Y TCTA D8S1179 CXR ET 8q24 13 125 976Mb TCTA Complex 19 D12S391 CXR ET 12p12 12 341Mb AGAT AGAC Complex D19S433 CXR ET 19q12 35 109Mb AAGG Complex FGA CXR ET 4q28 155 866Mb TTTC Complex 19 D22S1045 CXR ET 22q12 3 35 779Mb ATT 1Information about the chromosomal location of these loci can be found in references 20 21 and 22 and at www cstl nist gov biotech strbase chrom htm 2The August 1997 report 23 24 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 3Amelogenin is not an STR but displays an 89 base X specific band and a 95 base Y specific band Page 63 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356
37. Mapper ID Software Version 3 2 There are two options when creating a size standard Use this protocol or the alternative protocol in Section 6 H The CC5_ILS_500 xml file is available for download at www promega com resources tools genemapper id software panels and bin sets Save the CC5_ILS_500 xml file to a known location on your computer 1 Select Tools then GeneMapper Manager 2 Select the Size Standard tab 3 Select Import 4 Browse to the location of the CC5_ILS_500 xml file 5 Highlight the file then select Import 6 Select Done to save changes and exit the GeneMapper Manager 6 H Creating a Size Standard with GeneMapper ID Software Version 3 2 1 Select Tools then GeneMapper Manager 2 Select the Size Standard tab 3 Select New 4 Select Basic or Advanced Figure 17 The type of analysis method selected must match the type of analysis method created earlier Select OK Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD039 Printed in USA Revised 10 12 Page 40 5725TA Figure 17 The Select Dye and Analysis Method window 5 Enter a detailed name such as CC5 ILS 60 to 500 in the Size Standard Editor Figure 18 6 Choose Orange for the Size Standard Dye 7 Enter th
38. 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 Butler J M 2005 Forensic DNA Typing 2nd ed Elsevier Academic Press London 10 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 11 Hartmann J M et al 1991 Guidelines for a quality assurance program for DNA analysis Crime Laboratory Digest 18 44 75 12 Internal Validation of STR Systems Reference Manual GE053 Promega Corporation 13 Kline M C et al 2005 Results from the NIST 2004 DNA quantitation study J Forensic Sci 50 570 8 14 Levinson G and Gutman G A 1987 Slipped strand mispairing A major mechanism for DNA sequence evolution Mol Biol Evol 4 203 21 15 Schl tterer C and Tautz D 1992 Slippage synthesis of simple sequence DNA Nucleic Acids Res 20 211 5 16 Smith J R et al 1995 Approach to genotyping errors caused by nontemplated nucleotide addition by Taq DNA polymerase Genome Res 5 312 7 17 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 18 Walsh P S Fildes N J and Reynolds R 1996 Sequence analysis a
39. Revised 5 14 TMD039 PowerPlex Fusion System Instruc ons for use of Products DC2402 AND DC2408 T E C H N I C A L M A N U A L 1 Description 2 2 Product Components and Storage Conditions 4 3 Before You Begin 5 A Precautions 5 B Spectral Calibration 6 4 Protocols for DNA Amplification Using the PowerPlex Fusion System 6 A Amplification of Extracted DNA 6 B Direct Amplification of DNA from Storage Card Punches 9 C Direct Amplification of DNA from Swabs 13 5 Instrument Setup and Sample Preparation 15 A Detection of Amplified Fragments Using the
40. a silver or gold plated silver sample block The ramp speed is set after the thermal cycling run is started The Select Method Options screen appears Select Max for the ramp speed and enter the reaction volume 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 TMD039 Printed in USA Revised 10 12 3 After completion of the thermal cycling protocol store amplified samples at 20 C in a light protected box Note Long term storage of amplified samples at 4 C or higher may produce artifacts 4 B Direct Amplification of DNA from Storage Card Punches Materials to Be Supplied by the User GeneAmp PCR System 9700 thermal cycler Applied Biosystems microcentrifuge MicroAmp optical 96 well reaction plate or 0 2ml MicroAmp reaction tubes Applied Biosystems aerosol resistant pipette tips PunchSolution Kit Cat DC9271 for nonFTA card punches 1 2mm Harris Micro Punch or equivalent manual punch and cutting mat or automated punch system This section contains a protocol for direct amplification of DNA from storage card punches using the PowerPlex Fusion System and GeneAmp PCR System 9700 thermal cycler We recommend amplifying one or two 1 2mm punches of a storage card containing a buccal sample or one 1 2mm punch of a storage card containing whole
41. a in GeneMapper ID X software They are not intended as a comprehensive guide for using GeneMapper ID X software We recommend that users contact Applied Biosystems for training on the software 1 Select Tools then GeneMapper ID X Manager 2 Select the Analysis Methods tab 3 Select New and a new analysis method dialog box will open 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 TMD039 Revised 10 12 Page 31 8257TA Figure 12 The GeneMapper ID X Size Standard Editor 6 D Creating a Casework Analysis Method with GeneMapper ID X Software Version 1 2 continued 4 In the Analysis Method Editor window select GeneMapper ID X Security Group as the Security Group This allows access to all users of the software Other security groups may be used 5 Enter a descriptive name for the analysis method such as PowerPlex Fusion 6 Select the Allele tab Figure 13 7 Select the bins text file that was imported in Section 6 A 8 Ensure that the Use marker specific stutter ratio and distance if available box is checked 9 We recommend the values shown in Figure 13 for proper filtering of stutter peaks when using the PowerPlex Fusion System You may need to optimize these settings In house validation should be performed 11
42. a loci Finally the D2S1338 and D19S433 loci which are popular loci included in a number of databases were incorporated to further increase the power of discrimination This extended panel of STR markers is intended to satisfy both CODIS and ESS recommendations The PowerPlex Fusion System is compatible with the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 3130xl 3500 and 3500xL Genetic Analyzers Amplification and detection instrumentation may vary You may need to optimize protocols including amount of template DNA cycle number injection conditions and loading volume for your laboratory instrumentation In house validation should be performed 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 TMD039 Printed in USA Revised 10 12 Page 2 The PowerPlex Fusion System provides all materials necessary to amplify STR regions of human genomic DNA including a hot start thermostable DNA polymerase which is a component of the PowerPlex Fusion 5X Master Mix This manual contains protocols for use of the PowerPlex Fusion System with the GeneAmp PCR System 9700 thermal cycler in addition to protocols to separate amplified products and detect separated material Figure 1 Protocols to operate the fluorescence detection instruments should be obtained from the instrument manufacture
43. amplification cycles than blood samples Cycle number should be optimized in each laboratory for each sample type that is amplified 1 Place the MicroAmp plate in the thermal cycler Page 11 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD039 Revised 10 12 4 B Direct Amplification of DNA from Storage Card Punches continued 2 Select and run the recommended protocol Be sure that Max mode is selected as the ramp speed The preferred protocol for use with the GeneAmp PCR System 9700 thermal cycler is provided below The estimated total cycle time is 1 5 hours Note The final extension for direct amplification was extended to 20 minutes compared to 10 minutes for the extracted DNA protocol to allow sufficient time for adenylation of large amounts of amplicon 3 After completion of the thermal cycling protocol store amplified samples at 20 C in a light protected box Note Long term storage of amplified samples at 4 C or higher may produce artifacts PCR Optimization Cycle number should be optimized based on the results of an initial experiment to determine the sensitivity with your collection method sample types number of punches and instrumentation 1 Choose several samples that represent typical sample types you encounter in the laboratory Prepare them as
44. anels text file selected for analysis was incorrect for the STR system used Assign correct panels text file that corresponds to the STR system used for amplification The allelic ladder was not identified as an allelic ladder in the Sample Type column The internal lane standard was not properly identified in the sample Manually redefine the sizes of the size standard fragments in the sample 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 TMD039 Printed in USA Revised 10 12 Page 56 Symptoms Causes and Comments Off ladder alleles continued A low quality allelic ladder was used during analysis Ensure that only high quality allelic ladders are used for analysis An allelic ladder from a different run than the samples was used Re analyze samples with an allelic ladder from the same run Size standard not called Starting data point was incorrect for the partial range chosen correctly in Section 6 E Adjust the starting data point in the analysis method Alternatively use a full range for the analysis Extra peaks in size standard Open the Size Match Editor Highlight the extra peak select Edit and select delete size label Select auto adjust sizes Run was too short and larger peaks in ILS were not captured Not all CC5 ILS 500 peaks defined in the size standard were detected during the ru
45. at 60 C after thermal cycling Section 4 C Use 2 l of swab extract in a 25 l PowerPlex Fusion reaction A larger volume of swab extract may contain more than the recommended amount of DNA template resulting in incomplete adenylation Decrease cycle number Increase the final extension time Peak height imbalance Excess DNA in the amplification reaction can result in locus to locus imbalance within a dye channel such that the peak heights at the smaller loci are greater than those at the larger loci ski slope effect Use less swab extract or reduce cycle number Active SwabSolution Reagent carried over from swab extracts into the amplification reaction Larger loci are most susceptible to reagent carryover and will drop out before the smaller loci Ensure that the heat block is heating to 70 C 90 C if using 2 2ml Square Well Deep Well Plates and samples were incubated for the full 30 minutes Incubation for shorter time periods may result in incomplete reagent inactivation Do not use an incubator set at 70 C to incubate tubes or plates heat transfer is inefficient and will result in poor performance Use only a heat block to maintain efficient heat transfer Inactive SwabSolution Reagent Thaw the SwabSolution Reagent completely in a 37 C water bath and mix by gentle inversion Store the SwabSolution Reagent at 2 10 C Do not store reagents in the refrigerator door where the temperature can fluctuate
46. ce Always wear gloves and safety glasses when working with formamide Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD039 Revised 10 12 Page 15 5 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer continued Sample Preparation 1 Thaw the CC5 Internal Lane Standard 500 Note Centrifuge tube briefly to bring contents to the bottom then vortex for 15 seconds before each use Do not centrifuge after vortexing as this may cause the size standard to be concentrated at the bottom of the tube 2 Prepare a loading cocktail by combining and mixing CC5 Internal Lane Standard 500 and Hi Di formamide as follows 1 0 l CC5 ILS 500 samples 10 0 l Hi Di formamide samples Note The volume of internal lane standard used in the loading cocktail can be increased or decreased to adjust the intensity of the size standard peaks based on laboratory preferences Keep the volume of formamide at 10 0 l per well and adjust the volume added to the wells in Step 4 accordingly 3 Vortex for 10 15 seconds to mix 4 Pipet 11 l of formamide internal lane standard mix into each well 5 Add 1 l of amplified sample or 1 l of PowerPlex Fusion Allelic Ladder Mix Cover wells with appropriate septa Note
47. cles 4 Following amplification use your laboratory s validated separation and detection protocols to determine the optimal cycle number for the sample type 5 Instrument Setup and Sample Preparation 5 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer Materials to Be Supplied by the User 95 C dry heating block water bath or thermal cycler crushed ice or ice water bath centrifuge compatible with 96 well plates aerosol resistant pipette tips 3500 3500xL capillary array 36cm 96 well retainer amp base set standard Applied Biosystems Cat 4410228 POP 4 polymer for the Applied Biosystems 3500 or 3500xL Genetic Analyzer anode buffer container cathode buffer container MicroAmp optical 96 well plate and septa or equivalent Hi Di formamide Applied Biosystems Cat 4311320 The quality of formamide is critical Use Hi Di formamide Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause breakdown of formamide Poor quality formamide may contain ions that compete with DNA during injection which results in lower peak heights and reduced sensitivity A longer injection time may not increase the signal Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning label and take appropriate precautions when handling this substan
48. contact information and select GeneMapper ID Select Submit 3 Save the PowerPlex_Fusion_Panels_IDX_vX x txt and PowerPlex_Fusion_Bins_IDX_vX x txt files where X x refers to the most recent version of the panels and bins text files to a known location on your computer Importing Panels and Bins Text Files These instructions loosely follow the Applied Biosystems GeneMapper ID software tutorial pages 1 4 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 navigation pane 4 Select File then Import Panels 5 Navigate to the panels text file imported in the Getting Started section above Select the file then Import 6 In the navigation pane highlight the PowerPlex Fusion panels folder that you just imported in Step 5 7 Select File then Import Bin Set 8 Navigate to the bins text file imported in the Getting Started section above Select the file then Import 9 At the bottom of the Panel Manager window select OK The Panel Manager window will close automatically 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 TMD039 Revised 10 12 Page 39 6 G Importing the CC5 ILS 500 Size Standard into Gene
49. d in Step 5 7 Select File then Import Bin Set 8 Navigate to the bins text file imported in the Getting Started Section Select the file then Import 9 In the navigation pane highlight the PowerPlex Fusion panels folder that you just imported in Step 5 10 Select File then Import Marker Stutter A warning box will appear asking if you want to overwrite current values Select Yes 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 Printed in USA Part TMD039 Revised 10 12 6 A Importing PowerPlex Fusion Panels Bins and Stutter Text Files with GeneMapper ID X Software Version 1 2 continued 11 Navigate to the stutter text file imported in the Getting Started Section Select the file then Import 12 At the bottom of the Panel Manager window select OK This will save the panels bins and stutter text files and close the window 6 B Importing the CC5 ILS 500 IDX Size Standard into GeneMapper ID X Software Version 1 2 There are two options when creating a size standard Use this protocol or the alternative protocol in Section 6 C The CC5_ILS_500_IDX xml file is available for download at www promega com resources tools genemapper id software panels and bin sets Save the CC5_ILS_500_IDX xml file to a known location on your c
50. e dye set is the file generated for the PowerPlex 5 dye chemistry It is critical to select the correct G5 spectral for the PowerPlex 5 dye chemistry If the PowerPlex 5 dye chemistry is not the active dye set locate the PowerPlex 5 dye spectral in the List of Calibrations for Dye Set G5 and select Set 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 on the green Run Instrument arrow on the toolbar to start the sample run 11 Monitor electrophoresis by observing the run view array or capillaries viewer window in the data collection software Each injection will take approximately 40 minutes Page 28 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD039 Printed in USA Revised 10 12 6 Data Analysis 6 A Importing PowerPlex Fusion Panels Bins and Stutter Text Files with GeneMapper ID X Software Version 1 2 To facilitate analysis of data generated with the PowerPlex Fus
51. e for the STR system that was used No size standard was selected In the Size Standard 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 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 TMD039 Printed in USA Revised 10 12 Page 58 Symptoms Causes and Comments Error message The bins text file assigned to the analysis method was deleted Both the Bin Set used in the In the GeneMapper Manager select the Analysis Methods tab Analysis Method and the Panel and open the analysis method of interest Select the Allele tab must belong to the same and select an appropriate bins text file Chemistry Kit The wrong bins text file was chosen in the analysis method Allele tab Be sure to choose the appropriate bins text file as shown in Figure 19 Significantly raised baseline Poor spectral calibration Perform a new spectral calibration and re run the samples Use of Classic mode analysis method Use of Classic mode analysis on samples can result in baselines with more noise
52. e sizes of the internal lane standard fragments 60 65 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 and 500 bases See Section 9 C Figure 24 8 Select OK 6 I Creating a Casework Analysis Method with GeneMapper ID Software Version 3 2 These instructions loosely follow the Applied Biosystems GeneMapper ID software tutorial pages 5 11 1 Select Tools then GeneMapper Manager 2 Select the Analysis Methods tab 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 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 TMD039 Revised 10 12 Page 41 8199TA Figure 18 The Size Standard Editor 6 I Creating a Casework Analysis Method with GeneMapper ID Software Version 3 2 continued 5 Enter a descriptive name for the analysis method such as PowerPlex Fusion 6 Select the Allele tab Figure 19 7 Select the bins text file that was imported in Section 6 F 8 Ensure that the Use marker specific stutter ratio if available box is checked 9 Enter the values shown in Figure 19 for proper filtering of stutter peaks
53. eles with a greater number of repeat units will exhibit a higher percent stutter A trinucleotide repeat locus like D22S1045 will have more pronounced stutter in both n 3 and n 3 positions than a typical tetranucleotide repeat locus The pattern and intensity of stutter may differ slightly between primer sets for the same loci The mean plus three standard deviations at each locus is used in the PowerPlex Fusion panels text file for locus specific filtering in the GeneMapper ID software version 3 2 and in the PowerPlex Fusion stutter text file for locus specific filtering in GeneMapper ID X software In addition to stutter peaks other artifact peaks can be observed at some of the PowerPlex Fusion System loci Low level products can be seen in the n 2 and n 2 positions with some loci such as D1S1656 D13S317 D18S51 D21S11 D7S820 D5S818 D12S391 and D19S433 N 1 peaks are sometimes present at amelogenin and D2S441 N 3 peaks are sometimes present at D12S391 Amplification independent artifacts may be observed in template and no template samples in the fluorescein channel at 64 65 69 71 and 88 90 bases and in the JOE channel at 74 76 bases Artifact peaks may be seen outside the locus panels in the fluorescein channel at 70 74 bases in the TMR ET channel at 66 68 bases and in the CXR ET channel at 58 65 bases Artifacts that may be seen within the locus panels include allele 5 84 bases in D16S539 and peaks at 71
54. ence to recommended procedures for amplification and fluorescence detection Additional research and validation are required if any modifications to the recommended protocols are made PCR based STR analysis is subject to contamination by very small amounts of human DNA Extreme care should be taken to avoid cross contamination when preparing template DNA handling primer pairs assembling amplification reactions and analyzing amplification products Reagents and materials used prior to amplification PowerPlex Fusion 5X Master Mix PowerPlex Fusion 5X Primer Pair Mix 2800M Control DNA and Water Amplification Grade are provided in a separate box and should be stored separately from those used following amplification PowerPlex Fusion Allelic Ladder Mix and CC5 Internal Lane Standard 500 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 Some reagents used in the analysis of STR products are potentially hazardous and should be handled accordingly Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning label and take appropriate precautions when handling this substance Always wear gloves and safety glasses when working with formamide Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 25
55. enter the reaction volume 4 C Direct Amplification of DNA from Swabs Materials to Be Supplied by the User GeneAmp PCR System 9700 thermal cycler Applied Biosystems microcentrifuge MicroAmp optical 96 well reaction plate or 0 2ml MicroAmp reaction tubes Applied Biosystems aerosol resistant pipette tips SwabSolution Kit Cat DC8271 This section contains a protocol for amplifying DNA from swab extracts using the PowerPlex Fusion System and GeneAmp PCR System 9700 thermal cycler Pretreat OmniSwab GE Healthcare or cotton swabs with the SwabSolution Kit Cat DC8271 as described in the SwabSolution Kit Technical Manual TMD037 to generate a swab extract Amplification Setup 1 Thaw the PowerPlex Fusion 5X Master Mix PowerPlex Fusion 5X Primer Pair Mix and Water Amplification Grade completely Note Centrifuge tubes briefly to bring contents to the bottom then vortex reagents for 15 seconds before each use Do not centrifuge the 5X Primer Pair Mix or 5X Master Mix after vortexing as this may cause the reagents to be concentrated at the bottom of the tube 2 Determine the number of reactions to be set up This should include positive and negative control reactions Add 1 or 2 reactions to this number to compensate for pipetting error While this approach does consume a small amount of each reagent it ensures that you will have enough PCR amplification mix for all samples
56. erPlex Fusion 5X Primer Pair Mix The template DNA will be added at Step 6 2Store DNA templates in TE 4 buffer 10mM Tris HCl pH 8 0 0 1mM EDTA or TE 4 buffer with 20 g ml glycogen If the DNA template is stored in TE buffer that is not pH 8 0 or contains a higher EDTA concentration the volume of DNA added should not exceed 20 of the final reaction volume PCR amplification efficiency and quality can be greatly altered by changes in pH due to added Tris HCl available magnesium concentration due to chelation by EDTA or other PCR inhibitors which may be present at low concentrations depending on the source of the template DNA and the extraction procedure used 3Apparent DNA concentrations can differ depending on the DNA quantification method used 13 The amount of DNA template recommended here is based on DNA concentrations determined by measuring absorbance at 260nm We strongly recommend that you perform experiments to determine the optimal DNA amount based on your DNA quantification method 4 A Amplification of Extracted DNA continued 5 Vortex the PCR amplification mix for 5 10 seconds then add the PCR amplification mix to each reaction well Failure to vortex the PCR amplification mix sufficiently can result in poor amplification or locus to locus imbalance 6 Add the template DNA 0 25 0 5ng for each sample to the respective well containing PCR amplification mix 7 For the positive amplification contro
57. ese settings In house validation should be performed Notes 1 Select full range or partial range for the analysis range When using a partial range choose an appropriate analysis range based on your data Choose a start point after the primer peak and just before the first defined internal lane standard peak to help ensure proper sizing of the internal lane standard 2 The peak amplitude thresholds are the minimum peak heights at which the software will call a peak Values for peak amplitude thresholds are usually 50 150RFU on the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xl Genetic Analyzers For the Applied Biosystems 3500 and 3500xL Genetic Analyzers Life Technologies suggests an analysis threshold of 175RFU under their default injection conditions However individual laboratories should determine their peak amplitude thresholds from internal validation studies Peak heights for the CC5 ILS 500 are generally lower than those for the other dyes Therefore the threshold for the orange dye may be lower than that for the other dyes 3 The normalization box can be checked regardless of whether normalization was or was not applied during data collection 10 Select the Peak Quality tab You may change the settings for peak quality Note For Steps 10 and 11 see the GeneMapper ID X user s manual for more information 11 Select the SQ amp GQ Settings tab You may change t
58. fication reagents be stored and used separately with different pipettes tube racks etc Page 4 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD039 Printed in USA Revised 10 12 Page 5 Available Separately The proper panels bins and stutter text files for use with GeneMapper ID and ID X software are available for download at www promega com resources tools genemapper id software panels and bin sets Matrix standards are required for initial setup of the color separation matrix The matrix standards are provided separately and are available for ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 3130xl 3500 and 3500xL Genetic Analyzers PowerPlex 5 Dye Matrix Standards 3100 3130 Cat DG4700 3 Before You Begin 3 A Precautions The application of PCR based typing for forensic or paternity casework requires validation studies and quality control measures that are not contained in this manual 10 11 Guidelines for the validation process are published in the Internal Validation of STR Systems Reference Manual 12 The quality of purified DNA or direct amplification samples small changes in buffers ionic strength primer concentrations reaction volume choice of thermal cycler and thermal cycling conditions can affect PCR success We suggest strict adher
59. h CC5 dye and can be detected separately as a fifth color in the presence of PowerPlex Fusion amplified material The CC5 ILS 500 is designed for use in each CE injection to increase precision in analyses when using the PowerPlex Fusion System Protocols to prepare and use this internal lane standard are provided in Section 5 Note Sizing of Penta E and DYS391 alleles 475 bases will not use Local Southern Method For Penta E alleles gt 24 will be labeled as OL Figure 24 CC5 Internal Lane Standard 500 An electropherogram showing the CC5 Internal Lane Standard 500 fragments 8248TA Page 67 9 D Composition of Buffers and Solutions 9 E Related Products STR Systems Product Size Cat PowerPlex Y23 System 50 reactions DC2305 200 reactions DC2320 PowerPlex 21 System 200 reactions DC8902 4 200 reactions DC8942 PowerPlex 18D System 200 reactions DC1802 800 reactions DC1808 PowerPlex ESX 16 System 100 reactions DC6711 400 reactions DC6710 PowerPlex ESX 17 System 100 reactions DC6721 400 reactions DC6720 PowerPlex ESI 16 System 100 reactions DC6771 400 reactions DC6770 PowerPlex ESI 17 Pro System 100 reactions DC7781 400 reactions DC7780 PowerPlex 16 HS System 100 reactions DC2101 400 reactions DC2100 PowerPlex CS7 System 100 reactions DC6613 Not for Medical Diagnostic Use Accessory Components Product Size Cat PowerPle
60. he Size Standard column select the size standard that was imported in Section 6 B or created in Section 6 C 7 Select Analyze green arrow button to start data analysis Note By default the software displays the Analysis Requirement Summary Allelic Ladder Analysis Summary and Analysis Summary windows after quality review by the software Ensure that all requirements are met as each window appears If you do not have the Analysis Requirement Summary window activated you may need to do additional manual troubleshooting 8 If all analysis requirements are met the Save Project window will open Figure 15 9 Enter the project name 10 Choose the applicable security group from the drop down menu then select OK Note Sizing of Penta E and DYS391 alleles 475 bases will not use Local Southern Method For Penta E alleles gt 24 will be labeled as OL When the analysis is finished the Analysis Summary screen will appear We recommend that you review any yellow or red marker header bars in the plots view and handle them according to laboratory standard operating procedures Navigate to the Genotype tab or Samples tab To assist the review of any low quality samples use the default Data Interpretation plot settings and review the contents in the Quality Value Details table The values displayed in the Analysis Method Peak Quality and SQ amp GQ Settings tabs are defaults and will affect the quality values d
61. hese settings 12 Select Save to save the new analysis method 13 Select Done to exit the GeneMapper ID X Manager Processing Data for Databasing or Paternity Samples 1 Select File then New Project 2 Select Edit then Add Samples to Project 3 Browse to the location of run files Highlight desired files then select Add to list followed by Add 4 In the Sample Type column use the drop down menu to select Allelic Ladder Sample Positive Control or Negative Control as appropriate for the sample Every folder in the project must contain at least one allelic ladder injection that is designated as Allelic Ladder in the Sample Type column for proper genotyping Note The positive control DNA defined in the GeneMapper ID X panel file is the 2800M Control DNA Redefine the genotype in the panel file if using a different positive control DNA In the Analysis Method column select the analysis method created above 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 TMD039 Revised 10 12 Page 37 6 E Creating a Databasing or Paternity Analysis Method with GeneMapper ID X Software Version 1 2 continued 5 In the Panel column select the panels text file that was imported in Section 6 A 6 In t
62. ion System we have created panels and bins text files to allow automatic assignment of genotypes using GeneMapper ID X software We recommend that users receive training from Applied Biosystems on the GeneMapper ID X software to familiarize themselves with proper operation of the software Note The panels bins and stutter text files mentioned here are compatible with earlier versions of the GeneMapper ID X software Getting Started 1 To obtain the proper panels bins and stutter text files for the PowerPlex Fusion System go to www promega com resources tools genemapper id software panels and bin sets 2 Enter your contact information and select GeneMapper ID X Select Submit 3 Save the PowerPlex_Fusion_Panels_IDX_vX x txt PowerPlex_Fusion_Bins_IDX_vX x txt and PowerPlex_Fusion_Stutter_IDX_vX x txt files where X x refers to the most recent version of the panels bins and stutter text files to a known location on your computer Importing Panels Bins and Stutter Text Files 1 Open the GeneMapper ID X software 2 Select Tools then Panel Manager 3 Highlight the Panel Manager icon in the upper left navigation pane 4 Select File then Import Panels 5 Navigate to the panels text file imported in the Getting Started Section Select the file then Import 6 In the navigation pane highlight the PowerPlex Fusion panels folder that you just importe
63. isplayed in the plot settings We recommend that you modify the values in these tabs to fit your laboratory s data analysis protocols 6 F Importing PowerPlex Fusion Panels and Bins Text Files with GeneMapper ID Software Version 3 2 To facilitate analysis of data generated with the PowerPlex Fusion System we have created panels and bins text files to allow automatic assignment of genotypes using GeneMapper ID software version 3 2 We recommend that users of GeneMapper ID software version 3 2 complete the Applied Biosystems GeneMapper ID Software Human Identification Analysis Tutorial to familiarize themselves with proper operation of the software For GeneMapper ID software version 3 1 users we recommend upgrading to version 3 2 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD039 Printed in USA Revised 10 12 Page 38 For analysis using GeneMapper ID software version 3 2 you will need the proper panels and bins text files PowerPlex_Fusion_Panels_vX x txt and PowerPlex_Fusion_Bins_vX x txt files where X x refers to the most recent version of the panels and bins text files Getting Started 1 To obtain the proper panels and bins text files for the PowerPlex Fusion System go to www promega com resources tools genemapper id software panels and bin sets 2 Enter your
64. l vortex the tube of 2800M Control DNA then dilute an aliquot to 0 5ng in the desired template DNA volume Add 0 5ng of diluted DNA to a reaction well containing PCR amplification mix 8 For the negative amplification control pipet Water Amplification Grade or TE 4 buffer instead of template DNA into a reaction well containing PCR amplification mix 9 Seal the plate or close the tubes Optional Briefly centrifuge the plate to bring contents to the bottom of the wells and remove any air bubbles Thermal Cycling Amplification and detection instrumentation may vary You may need to optimize protocols including the amount of template DNA cycle number injection conditions and loading volume for your laboratory instrumentation Testing at Promega shows that 30 cycles works well for 0 5ng of purified DNA templates 1 Place the MicroAmp plate or reaction tubes in the thermal cycler 2 Select and run the recommended protocol Be sure that Max mode is selected as the ramp speed The preferred protocol for use with the GeneAmp PCR System 9700 thermal cycler is provided below The estimated total cycling time is 1 5 hours Thermal Cycling Protocol1 96 C for 1 minute then 94 C for 10 seconds 59 C for 1 minute 72 C for 30 seconds for 30 cycles then 60 C for 10 minutes 4 C soak 1When using the GeneAmp PCR System 9700 thermal cycler the program must be run with Max mode as the ramp speed This requires
65. l 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 or equivalent Hi Di formamide Applied Biosystems Cat 4311320 The quality of formamide is critical Use Hi Di formamide Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause breakdown of formamide Poor quality formamide may contain ions that compete with DNA during injection which results in lower peak heights and reduced sensitivity A longer injection time may not increase the signal Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning label and take appropriate precautions when handling this substance Always wear gloves and safety glasses when working with formamide Sample Preparation 1 Thaw the CC5 Internal Lane Standard 500 Note Centrifuge tube briefly to bring contents to the bottom then vortex for 15 seconds before each use Do not centrifuge after vortexing as this may cause the size standard to be concentrated at the bottom of the tube 2 Prepare a loading cocktail by combining and mixing CC5 Internal Lane Standard 500 and Hi Di formamide as follows 1 0 l CC5 ILS 500 samples 10 0 l Hi Di formamide samples Note The volume of i
66. late DNA Because a small amount of template is used this is rarely a problem Depending on the DNA extraction procedure used and sample source inhibitors might be present in the DNA sample Insufficient template Use the recommended amount of template DNA if available 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 also may affect PCR Store DNA in TE 4 buffer 10mM Tris HCl pH 8 0 0 1mM EDTA or TE 4 buffer with 20 g ml glycogen The reaction volume was too low This system is optimized for a final reaction volume of 25 l Decreasing the reaction volume may result in suboptimal performance Extra peaks visible in one Artifacts of STR amplification Amplification of excess or all color channels amounts of purified DNA can result in a higher number of artifact peaks Use the recommended amount of template DNA See Section 6 L for additional information on stutter and artifacts Peak height imbalance Excessive amount of DNA Amplification of gt 0 5ng of template can result in an imbalance with smaller loci showing more product than larger loci Decrease number of cycles Degraded DNA sample DNA template was degraded and larger loci showed diminished yield Insufficient template
67. lification of STRs can result in artifacts that appear as peaks one base smaller than the allele due to incomplete addition of the 3 A residue Be sure to perform an extension step 10 minutes for purified DNA samples at 60 C after thermal cycling Section 4 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD039 Printed in USA Revised 10 12 Page 50 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 TMD039 Revised 10 12 Symptoms Causes and Comments Extra peaks visible in one CE related artifacts spikes Minor voltage changes or urea or all color channels continued 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 Incorrect G5 spectral was active Re run samples and confirm that the PowerPlex 5 dye G5 spectral is set for G5 See instructions on instrument preparation in Section 5 Pull up or bleedthrough Pull up can occur when peak heights are too high or if a poor or incorrect matrix is applied to the samples Perform a new spectral calibration and re run the samples Instrument
68. man specific DNA quantification the Plexor HY System Cat DC1000 was developed 27 This qPCR based method provides total human and male specific DNA quantification in one reaction Additionally the Plexor HY System provides a post amplification melt analysis to confirm positive results and and Internal PCR Control IPC to confirm negative results Additional ordering information is available in Section 9 E For information about automation of Promega chemistries on automated workstations using Identity Automation solutions contact your local Promega Branch Office or Distributor contact information available at www promega com support worldwide contacts e mail genetic promega com or visit www promega com idautomation Page 65 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 TMD039 Revised 10 12 Page 66 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 TMD039 Printed in USA Revised 10 12 9 C The CC5 Internal Lane Standard 500 The CC5 Internal Lane Standard 500 contains 21 DNA fragments of 60 65 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 and 500 bases in length Figure 24 Each fragment is labeled wit
69. n Create a new size standard using the internal lane standard fragments present in the sample Re run samples using a longer run time Peaks in size standard missing If peaks are below threshold decrease the peak amplitude threshold in the analysis method for the orange channel to include peaks If peaks are low quality redefine the size standard for the sample to skip these peaks Significantly raised baseline Poor spectral calibration Perform a new spectral calibration and re run the samples Incorrect G5 spectral was active Re run samples and confirm that the PowerPlex 5 dye G5 spectral is set for G5 See instructions for instrument preparation in Section 5 7 F GeneMapper ID Software Symptoms Causes and Comments Alleles not called To analyze samples with GeneMapper ID software the analysis parameters and size standard must both have Basic or Advanced as the analysis type If they are different an error is obtained To analyze samples with GeneMapper ID software at least one allelic ladder must be defined An insufficient number of CC5 ILS 500 fragments was defined Be sure to define at least two CC5 ILS 500 fragments smaller than the smallest sample peak and at least two CC5 ILS 500 fragments larger than the largest sample peak Run was too short and larger peaks in ILS were not captured Not all CC5 ILS 500 peaks defined in the size standard were detected during the run Create a new size sta
70. n Refer to the instrument user s manual for instructions on cleaning installing the capillary array performing a spatial calibration and adding polymer Analyze samples as described in the user s manual for the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 and the Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 with the following exceptions 1 In the Module Manager select New Select Regular in the Type drop down list and select HIDFragmentAnalysis36_POP4 in the Template drop down list Confirm that the injection time is 5 seconds the injection voltage is 3kV and the run time is 1 500 seconds Give a descriptive name to your run module and select OK Note Instrument sensitivities can vary The injection time and voltage may be adjusted in the Module Manager A suggested range for the injection time is 3 22 seconds and for the injection voltage is 1 3kV 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 G5 in the dye set drop down list Select OK 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
71. n contract tort strict product liability or otherwise 2012 2014 Promega Corporation All Rights Reserved Plexor and PowerPlex are registered trademarks of Promega Corporation DNA IQ Identity Automation PunchSolution and SwabSolution are trademarks of Promega Corporation ABI PRISM Applied Biosystems GeneMapper and MicroAmp are registered trademarks of Applied Biosystems Bode Buccal DNA Collector is a trademark of the Bode Technology Group Inc EasiCollect and OmniSwab are trademarks of Whatman FTA is a registered trademark of Flinders Technologies Pty Ltd and is licensed to Whatman GeneAmp is a registered trademark of Roche Molecular Systems Inc Hi Di and POP 7 are trademarks of Applera Corporation POP 4 is a registered trademark of Life Technologies Corporation Sampact is a trademark of Fitzco Vacutainer is a registered trademark of Becton Dickinson and Company Products may be covered by pending or issued patents or may have certain limitations Please visit our Web site for more information All prices and specifications are subject to change without prior notice Product claims are subject to change Please contact Promega Technical Services or access the Promega online catalog for the most up to date information on Promega products
72. nd CC5 dyes are proprietary Page 69 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 TMD039 Revised 10 12 g This product or portions thereof is manufactured and sold under license from GE Healthcare under Australia Pat No 692230 Austria Pat No E236994 Belgium Pat No 0743987 Canada Pat No 2231475 EP Pat Nos 0743987 and 0851867 France Pat Nos 0743987 and 0851867 Germany Pat Nos 19581489 69530286 8 and 0851867 Italy Pat Nos 0743987 and 0851867 Japan Pat No 3066984 Liechtenstein Pat Nos 0743987 and 0851867 Netherlands Pat Nos 0743987 and 0851867 Spain Pat Nos 2197193 and 2173310 Sweden Pat Nos 0743987 and 0851867 Switzerland Pat Nos 0743987 and 0851867 United Kingdom Pat Nos 0743987 and 0851867 U S Pat Nos 5 654 419 5 688 648 5 869 255 6 177 247 5 707 804 6 028 190 6 544 744 7 015 000 and 5 728 528 and other pending and foreign patent applications End User Terms and Conditions Acceptance These terms and conditions shall govern the purchase use transfer and acceptance of the products described in the purchase order quotation or invoice which products are sold and distributed by Promega to the buyer transferee of such products the End User The transfer sale of products to the End User is expressly conditional upon End User s acceptance of the
73. nd DYS391 alleles 475 bases will not use Local Southern Method For Penta E alleles gt 24 will be labeled as OL When the analysis is finished the Analysis Summary screen will appear We recommend that you review any yellow or red marker header bars in the plots view and handle them according to laboratory standard operating procedures Navigate to the Genotype tab or Samples tab To assist the review of any low quality samples use the default Data Interpretation plot settings and review the contents in the Quality Value Details table The values displayed in the Analysis Method Peak Quality and SQ amp GQ Settings tabs are defaults and will affect the quality values displayed in the plot settings We recommend that you modify the values in these tabs to fit your laboratory s data analysis protocols 6 E Creating a Databasing or Paternity Analysis Method with GeneMapper ID X Software Version 1 2 These instructions are intended as a guide to start analyzing data in GeneMapper ID X software They are not intended as a comprehensive guide for using the GeneMapper ID X software We recommend that users contact Applied Biosystems for training on the software 1 Select Tools then GeneMapper ID X Manager 2 Select the Analysis Methods tab 3 Select New and a new analysis method dialog box will open 4 In the Analysis Method Editor window select GeneMapper ID X Security Group as the Secu
74. nd characterization of stutter products at the tetranucleotide repeat locus vWA Nucleic Acids Res 24 2807 12 19 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 20 Butler J M 2006 Genetics and genomics of core STR loci used in human identity testing J Forensic Sci 51 253 65 21 Hill C R et al 2008 Characterization of 26 miniSTR loci for improved analysis of degraded DNA samples J Forensic Sci 53 73 80 22 Lu D J Liu Q L and Zhao H 2011 Genetic data of nine non CODIS STRs in Chinese Han population from Guangdong Province Southern China Int J Legal Med 125 133 7 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 TMD039 Printed in USA Revised 10 12 Page 60 23 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 24 Gill P et al 1997 Considerations from the European DNA Profiling Group EDNAP concerning STR nomenclature Forensic Sci Int 87 185 92 25 Fr geau C J et al 1995 Characterization of human lymphoid cell lines GM9947 and GM9948 as intra and interlaboratory reference standards for DNA typing Genomics 28 184 97
75. ndard using the internal lane standard fragments present in the sample Re run samples using a longer run time A low quality allelic ladder was used during analysis Ensure that only high quality allelic ladders are used for analysis 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 TMD039 Revised 10 12 Page 57 7 F GeneMapper ID Software continued Symptoms Causes and Comments Off ladder alleles An allelic ladder from a different run than the samples was used Re analyze samples with an allelic ladder from the same run The GeneMapper ID software requires that the allelic ladder be imported from the same folder as the sample Be sure that the allelic ladder is in the same folder as the sample Create a new project and re analyze as described in Section 6 I or 6 J Panels text file selected for analysis was incorrect for the STR system used Assign correct panels text file that corresponds to the STR system used for amplification The allelic ladder was not identified as an allelic ladder in the Sample Type column The wrong analysis type was chosen for the analysis method Be sure to use the HID analysis type The internal lane standard was not properly identified in the sample Manually redefine the sizes of the size standard fragments in the sample A low quality allelic ladder was
76. njection that is designated as Allelic Ladder in the Sample Type column for proper genotyping Note The positive control DNA defined in the GeneMapper ID X panel file is the 2800M Control DNA Redefine the genotype in the panel file if using a different positive control DNA 5 In the Analysis Method column select the analysis method created above 6 In the Panel column select the panels text file that was imported in Section 6 A 7 In the Size Standard column select the size standard that was imported in Section 6 B or created in Section 6 C 8 Select Analyze green arrow button to start data analysis Note By default the software displays the Analysis Requirement Summary Allelic Ladder Analysis Summary and Analysis Summary windows after quality review by the software Ensure that all requirements are met as each window appears If you do not have the Analysis Requirement Summary window activated you may need to do additional manual troubleshooting Page 34 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD039 Printed in USA Revised 10 12 9 If all analysis requirements are met the Save Project window will open Figure 15 10 Enter the project name 11 Choose the applicable security group from the drop down menu then select OK Note Sizing of Penta E a
77. nsures that you will have enough PCR amplification mix for all samples It also ensures that each reaction contains the same PCR amplification mix 3 Use a clean MicroAmp plate for reaction assembly and label appropriately Alternatively determine the number of clean 0 2ml reaction tubes required and label appropriately 4 Add the final volume of each reagent listed in Table 2 to a sterile tube Page 10 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD039 Printed in USA Revised 10 12 Table 2 PCR Amplification Mix for Direct Amplification of DNA from Storage Card Punches PCR Amplification Mix Component1 Volume Per Reaction Number of Reactions Final Volume Water Amplification Grade 15 l PowerPlex Fusion 5X Master Mix 5 0 l PowerPlex Fusion 5X Primer Pair Mix 5 0 l total reaction volume 25 l 1Add Water Amplification Grade to the tube first then add PowerPlex Fusion 5X Master Mix and PowerPlex Fusion 5X Primer Pair Mix For FTA card punches the template DNA will be aded at Step 6 5 Vortex the PCR amplification mix for 5 10 seconds then pipet 25 l of PCR amplification mix into each reaction well Failure to vortex the PCR amplification mix sufficiently can result in poor amplification or locus to locus imbalance 6 F
78. nternal lane standard used in the loading cocktail can be increased or decreased to adjust the intensity of the size standard peaks based on laboratory preferences Keep the volume of formamide at 10 0 l per well and adjust the volume added to the wells in Step 4 accordingly 3 Vortex for 10 15 seconds to mix 4 Pipet 11 l of formamide internal lane standard mix into each well 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 TMD039 Printed in USA Revised 10 12 Page 26 5 Add 1 l of amplified sample or 1 l of PowerPlex Fusion Allelic Ladder Mix Cover wells with appropriate septa Note Instrument detection limits vary therefore injection time injection voltage or the amount of sample mixed with loading cocktail may need to be adjusted Use the Module Manager in the data collection software to modify the injection time or voltage in the run module see Instrument Preparation below If the injection time or voltage is reduced a decreased peak amplitude threshold for the orange channel may be required for proper sizing 6 Centrifuge plate briefly to remove air bubbles from the wells 7 Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading the instrument Instrument Preparatio
79. ntle inversion Store the SwabSolution Reagent at 2 10 C Do not store reagents in the refrigerator door where the temperature can fluctuate Do not refreeze avoid multiple freeze thaw cycles as this may reduce activity Active SwabSolution Reagent carried over into the amplification reaction Ensure that the heat block is heating to 70 C 90 C if using a 2 2ml Square Well Deep Well Plate and samples were incubated for the full 30 minutes Incubation for shorter time periods may result in incomplete reagent inactivation Do not use an incubator set at 70 C to incubate tubes or plates heat transfer is inefficient and will result in poor performance Use only a heat block to maintain efficient heat transfer We have tested 60 minute incubation times and observed no difference in performance compared to a 30 minute incubation DNA was not accessible on nonlytic material Pretreat nonFTA materials with SwabSolution Reagent to ensure that DNA is liberated from cellular proteins Faint or absent peaks for the If the positive control reaction failed to amplify check to positive control reaction make sure that the correct amount of 2800M Control DNA was added to the reaction Due to the reduced cycle numbers used with swab extracts it is necessary to increase the mass of 2800M Control DNA to obtain a profile We recommend 10ng of 2800M Control DNA per 25 l amplification reaction This mass of DNA should be reduced if the cycle number
80. ome loci When an edit is made to a locus the quality flags and marker are gray header bar automatically change to gray To change the GQ and marker header bar for a locus to green override the GQ in the plot window Alleles not called To analyze samples with GeneMapper ID X software at least one allelic ladder must be defined An insufficient number of CC5 ILS 500 fragments was defined Be sure to define at least two CC5 ILS 500 fragments smaller than the smallest sample peak and at least two CC5 ILS 500 fragments larger than the largest sample peak In this instance the allelic ladder would have failed the allelic ladder quality check Run was too short and larger peaks in ILS were not captured Not all CC5 ILS 500 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 Re run samples using a longer run time A low quality allelic ladder was used during analysis Ensure that only high quality allelic ladders are used for analysis 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 X software requires that the allelic ladder be imported from the same folder as the sample Be sure that the allelic ladder is in the same folder as the sample Create a new project and re analyze as described in Section 6 D or 6 E P
81. omputer 1 Select Tools then GeneMapper ID X Manager 2 Select the Size Standard tab 3 Select Import 4 Navigate to the location of the CC5_ILS_500_IDX xml file on your computer 5 Highlight the file then select Import 6 Select Done to save changes and close the GeneMapper ID X Manager 6 C Creating a Size Standard with GeneMapper ID X Software Version 1 2 1 Select Tools then GeneMapper ID X Manager 2 Select the Size Standard tab 3 Select New 4 In the Size Standard Editor window Figure 12 select GeneMapper ID X Security Group as the Security Group This allows access for all users of the software Other security groups may be used 5 Enter a detailed name such as CC5_ILS_500_IDX 6 Choose Orange for the Size Standard Dye 7 Enter the sizes of the internal lane standard fragments 60 65 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 and 500 bases See Section 9 C Figure 24 8 Select OK Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD039 Printed in USA Revised 10 12 Page 30 6 D Creating a Casework Analysis Method with GeneMapper ID X Software Version 1 2 These instructions are intended as a guide to start analyzing dat
82. or FTA storage cards add one or two 1 2mm punches from a card containing a buccal sample or one 1 2mm punch from a card containing whole blood to the appropriate wells of the reaction plate For nonFTA card punches add the PCR amplification mix to the PunchSolution Reagent treated punches Note It also is acceptable to add the FTA card punch first then add the PCR amplification mix 7 For the positive amplification control add 1 l of 2800M Control DNA 10ng to a reaction well containing 25 l of PCR amplification mix Notes 1 Do not include blank storage card punches in the positive control reactions 2 Optimization of the amount of control DNA may be required depending on cycling conditions and laboratory preferences 8 Reserve a well containing PCR amplification mix as a negative amplification control Note An additional negative control with a blank punch may be performed to detect contamination from the storage card or punch device 9 Seal the plate and briefly centrifuge the plate to bring storage card punches to the bottom of the wells Thermal Cycling Amplification and detection instrumentation may vary You will need to optimize protocols including the number of storage card punches cycle number 25 28 cycles injection time and loading volume for your laboratory instrumentation Testing at Promega shows that 27 cycles works well for a variety of sample types Buccal samples may require more
83. r Information about other Promega fluorescent STR systems is available upon request from Promega or online at www promega com Amplification Setup Thermal Cycling Instrument Setup and Sample Preparation Data Analysis Section 4 Section 5 Section 6 Section 4 GeneAmp PCR System 9700 Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 Section 5 B ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 Section 5 B Figure 1 An overview of the PowerPlex Fusion System protocol Applied Biosystems 3500 or 3500xL Genetic Analyzer Section 5 A GeneMapper ID Software Version 3 2 GeneMapper ID X Software Version 1 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 TMD039 Revised 10 12 Page 3 2 Product Components and Storage Conditions Product Size Cat PowerPlex Fusion System 200 reactions DC2402 Not For Medical Diagnostic Use This system contains sufficient reagents for 200 reactions of 25 l each Includes Pre amplification Components Box 1ml PowerPlex Fusion 5X Master Mix 1ml PowerPlex Fusion 5X Primer Pair Mix 25 l 2800M Control DNA 10ng l 5 1 250 l Water Amplification Grade Post amplification Components Box 100 l PowerPlex
84. rPlex Fusion panels and bins text files Panel A The fluorescein labeled allelic ladder components and their allele designations Panel B The JOE labeled allelic ladder components and their allele designations Panel C The TMR ET labeled allelic ladder components and their allele designations Panel D The CXR ET labeled allelic ladder components and their allele designations 7 Troubleshooting For questions not addressed here please contact your local Promega Branch Office or Distributor Contact information available at www promega com E mail genetic promega com 7 A Amplification and Fragment Detection This section provides information about general amplification and detection For questions about amplification of extracted DNA see Section 7 B For questions about direct amplification see Sections 7 C and 7 D Symptoms Causes and Comments Faint or absent allele peaks The PowerPlex Fusion 5X Master Mix was not vortexed well before use Vortex the 5X Master Mix for 15 seconds before dispensing into the PCR amplification mix An air bubble formed at the bottom of the reaction tube Use a pipette to remove the air bubble or centrifuge the reactions briefly before thermal cycling Thermal cycler plate or tube problems Review the thermal cycling protocol in Section 4 We have not tested other reaction tubes plates or thermal cyclers Calibrate the thermal cycler heating block if necessary Primer concentration was too low Use
85. rity Group This allows access to all users of the software Other security groups may be used 5 Enter a descriptive name for the analysis method such as PowerPlex Fusion 20 Filter 9430TA Figure 15 The Save Project window 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 Printed in USA Part TMD039 Revised 10 12 6 E Creating a Databasing or Paternity Analysis Method with GeneMapper ID X Software Version 1 2 continued 6 Select the Allele tab Figure 16 7 Select the bins text file that was imported in Section 6 A 8 We recommend the values shown in Figure 16 for proper filtering of stutter peaks when using the PowerPlex Fusion System You may need to optimize these settings In house validation should be performed Note Ensure that the appropriate 20 filter is applied to this analysis method by entering 0 20 for the Global Cut off Value for Tri Tetra and Penta repeats 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 TMD039 Printed in USA Revised 10 12 Page 36 11051TA Figure 16 The GeneMapper ID X Allele tab 9 Select the Peak Detector tab Figure 14 shows an example of settings used at Promega You may need to optimize th
86. rtifact peak one base shorter than expected i e missing the terminal addition is sometimes seen We have modified primer sequences and added a final extension step at 60 C 18 to the amplification protocols to provide conditions for essentially complete terminal nucleotide addition when recommended amounts of template DNA are used Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD039 Revised 10 12 Page 61 9 A Advantages of Using the Loci in the PowerPlex Fusion System continued Page 62 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 TMD039 Printed in USA Revised 10 12 Table 4 The PowerPlex Fusion System Locus Specific Information STR Locus Label Chromosomal Location1 Repeat Sequence2 5 3 Amelogenin3 Fluorescein Xp22 1 22 3 and Y NA D3S1358 Fluorescein 3p21 31 45 557Mb TCTA Complex D1S1656 Fluorescein 1q42 228 972Mb TAGA Complex D2S441 Fluorescein 2p14 68 214Mb TCTA D10S1248 Fluorescein 10q26 3 130 567Mb GGAA D13S317 Fluorescein 13q31 1 81 62Mb TATC Penta E Fluorescein 15q26 2 95 175Mb AAAGA D16S539 JOE 16q24 1 84 944Mb GATA D18S51 JOE 18q21 33 59 1Mb AGAA 19 D2S1338
87. s to be concentrated at the bottom of the tube 2 Determine the number of reactions to be set up This should include positive and negative control reactions Add 1 or 2 reactions to this number to compensate for pipetting error While this approach does consume a small amount of each reagent it ensures that you will have enough PCR amplification mix for all samples It also ensures that each reaction contains the same PCR amplification mix 3 Use a clean MicroAmp plate for reaction assembly and label appropriately Alternatively determine the number of clean 0 2ml reaction tubes required and label appropriately 4 Add the final volume of each reagent listed in Table 1 to a sterile tube Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD039 Revised 10 12 Page 7 Table 1 PCR Amplification Mix for Amplification of Extracted DNA PCR Amplification Mix Component1 Volume Per Reaction Number of Reactions Final Volume l Water Amplification Grade1 to a final volume of 25 0 l PowerPlex Fusion 5X Master Mix 5 0 l PowerPlex Fusion 5X Primer Pair Mix 5 0 l template DNA 0 25 0 5ng 2 3 up to 15 l total reaction volume 25 l 1Add Water Amplification Grade to the tube first then add PowerPlex Fusion 5X Master Mix and Pow
88. s 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 TMD039 Revised 10 12 4 B Direct Amplification of DNA from Storage Card Punches continued Automated punchers also can be used to create sample disks Refer to the user s guide for your instrument for assistance with generating 1 2mm disks technical advice and troubleshooting information Note Static may be problematic when adding a punch to a well For FTA card punches adding PCR amplification mix to the well before adding the punch may help alleviate static problems For nonFTA card punches adding PunchSolution Reagent to the well before adding the punch during pretreatment may help alleviate static problems Amplification Setup 1 Thaw the PowerPlex Fusion 5X Master Mix PowerPlex Fusion 5X Primer Pair Mix and Water Amplification Grade completely Note Centrifuge tubes briefly to bring contents to the bottom then vortex reagents for 15 seconds before each use Do not centrifuge the 5X Primer Pair Mix or 5X Master Mix after vortexing as this may cause the reagents to be concentrated at the bottom of the tube 2 Determine the number of reactions to be set up This should include positive and negative control reactions Add 1 or 2 reactions to this number to compensate for pipetting error While this approach does consume a small amount of each reagent it e
89. s with PunchSolution Reagent to ensure that DNA is liberated from cellular proteins Too much sample in the reaction Use one or two 1 2mm storage card punches Follow the manufacturer s recommendations when depositing sample onto the storage card With FTA cards reducing the reaction volumes below 25 l may result in amplification failure Positive control did not amplify Do not include a blank punch in the positive control reaction Presence of blank punches may inhibit amplification of 2800M Control DNA Extra peaks visible in one Punch may be contaminated Take punches from blank paper or all color channels between samples Artifacts of STR amplification Direct amplification of gt 20ng of template can result in a higher number of artifact peaks Use the recommended punch size and number See Section 6 L for additional information on stutter and artifacts Artifacts of STR amplification Amplification of STRs can result in artifacts that appear as peaks one base smaller than the allele due to incomplete addition of the 3 A residue Be sure to perform a 20 minute extension step at 60 C after thermal cycling Section 4 B Decrease cycle number Increase the final extension time Peak height imbalance Excessive amount of DNA Amplification of gt 20ng of template can result in an imbalance with smaller loci showing more product than larger loci Use one or two 1 2mm punches from a storage card containing a buccal
90. sample or one 1 2mm punch from a storage card containing whole blood Follow the manufacturer s recommendations when depositing sample onto the card Decrease cycle number The reaction volume was too low This system is optimized for a final reaction volume of 25 l to overcome inhibitors present in FTA cards and PunchSolution Reagent Decreasing the reaction volume can result in suboptimal performance Amplification was inhibited when using more than one storage card punch with blood Use only one 1 2mm storage card punch with blood Page 53 7 C Direct Amplification of DNA From Storage Card Punches continued Symptoms Causes and Comments Peak height imbalance continued DNA was not accessible on nonlytic material Small loci may amplify preferentially with large loci dropping out Pretreat nonFTA materials with PunchSolution Reagent to ensure that DNA is liberated from cellular proteins 7 D Direct Amplification of DNA From Swabs The following information is specific to direct amplification of DNA from swabs after pretreatment using the SwabSolution Kit For additional information about general amplification and detection see Section 7 A Symptoms Causes and Comments Faint or absent allele peaks Poor sample deposition Shedding and collection of donor cells was variable Increase cycle number Inactive SwabSolution Reagent Thaw the SwabSolution Reagent completely in a 37 C water bath and mix by ge
91. se terms and conditions Restrictions on Use End Users are specifically not authorized to and are forbidden from reselling transferring or distributing any products either as a stand alone product or as a component of another product The right to use the products does not in and of itself include or carry any right of the End User to any GE Healthcare Bio Sciences Corp s technology or intellectual property other than expressly provided herein End Users may not use sequence s in an attempt to reverse engineer parameters of any of GE Healthcare Bio Sciences Corp proprietary products or services Disclaimer of Warranties GE Healthcare Bio Sciences Corp provides no warranties to end user statutory or implied including without limitation as to product quality condition description merchantability or fitness for a particular purpose and all such warranties are hereby expressly disclaimed GE Healthcare Bio Sciences Corp hereby expressly disclaims any warranty regarding results obtained through the use of the products including without limitation any claim of inaccurate invalid or incomplete results Exclusion of Liability GE Healthcare Bio Sciences Corp and its affiliates shall have no liability to an End User including without limitation for any loss of use or profits business interruption or any consequential incidental special or other indirect damages of any kind regardless of how caused and regardless of whether an action i
92. sensitivities can vary Optimize the injection conditions See Section 5 CE related artifacts contaminants Contaminants in the water used with the instrument or to dilute the 10X genetic analyzer buffer may generate peaks in the fluorescein and JOE channels Use autoclaved deionized water change vials and wash buffer reservoir Repeat sample preparation using fresh formamide Long term storage of amplified sample in formamide can result in artifacts The CE polymer was beyond its expiration date or polymer was stored at room temperature for more than one week Maintain instrumentation on a daily or weekly basis as recommended by the manufacturer POP 7 related artifacts This system was not developed for use with POP 7 polymer if using POP 7 polymer optimization and in house validation are required The use of POP 7 CE polymer can change the migration and sizing location of artifacts compared to the POP 4 locations An artifact can be seen with the POP 7 polymer at 88 90bp in the JOE channel and at 74 76 85 87 or 99 101bp in the fluorescein channel Allelic ladder not running Allelic ladder and primer pair mix were not compatible Ensure the same as samples that the allelic ladder is from the same kit as the primer pair mix Poor quality formamide Use only Hi Di formamide when analyzing samples Be sure the allelic ladder and samples are from the same instrument run Migration of samples changed slightly over
93. tandard are 60 65 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 and 500 bases See Figure 4 Figure 4 The Create New Size Standard window 9227TA 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 TMD039 Revised 10 12 Page 19 5 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer continued 4 To create a new QC Protocol navigate to the Library Select QC Protocols then select Create Alternatively a previously created QC Protocol may be used Assign a descriptive protocol name Select the size standard created in Step 3 The settings for the QC protocol should be based on the internally validated conditions for the PowerPlex Fusion System on the Applied Biosystems 3500 or 3500xL Genetic Analyzer Figure 5 shows one option for these settings Note Peak heights for the CC5 ILS 500 are generally lower than those for the other dyes Therefore the threshold for the orange dye may be lower than that for the other dyes Figure 5 The Create New QC Protocol window 9228TA 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 TMD039 Printed in USA Revised 1
94. than those analyzed using the Basic or Advanced mode analysis method Advanced mode analysis methods and size standards are recommended Incorrect G5 spectral was active Re run samples and confirm that the PowerPlex 5 dye G5 spectral is set for G5 See instructions for instrument preparation in Section 5 Error message after attempting There was a conflict between different sets of panels and bins to import panels and bins text files text files Check to be sure that the bins are installed properly Unable to save panel data If not delete all panels and bins text files and re import files java SQLEException in a different order ORA 00001 unique constraint IFA CKP_NNN violated Allelic ladder peaks GeneMapper ID software was not used or microsatellite labeled off ladder analysis settings were used instead of HID analysis settings GeneMapper software does not use the same algorithms as GeneMapper ID software and cannot correct for sizing differences using the allelic ladder Promega recommends using GeneMapper ID software to analyze PowerPlex reactions If using GeneMapper ID software version 3 2 be sure that the analysis method selected is an HID method This can be verified by opening the analysis method using the GeneMapper Manager then selecting the General tab The analysis type cannot be changed If the method is not HID it should be deleted and a new analysis method created Promega Corporation 280
95. the course of a CE run with many samples This may be due to changes in temperature or the CE column over time Use a different injection of allelic ladder to determine sizes Poor injection of allelic ladder Include more than one ladder per instrument run Internal size standard not assigned correctly Evaluate the sizing labels on the CC5 ILS 500 and correct if necessary Page 51 7 A Amplification and Fragment Detection continued Symptoms Causes and Comments Peak height imbalance The reaction volume was too low This system is optimized for a final reaction volume of 25 l to overcome inhibitors present in DNA samples Decreasing the reaction volume can result in suboptimal performance Miscellaneous balance problems Thaw the 5X Primer Pair Mix and 5X Master Mix completely and vortex for 15 seconds before use Note that the 5X Master Mix will take longer to thaw than the 5X Primer Pair Mix Do not centrifuge the 5X Primer Pair Mix or 5X Master Mix after mixing Calibrate thermal cyclers and pipettes routinely PCR amplification mix prepared in Section 4 was not mixed well Vortex the PCR amplification mix for 5 10 seconds before dispensing into the reaction tubes or plate 7 B Amplification of Extracted DNA The following information is specific to amplification of extracted DNA For information about general amplification and detection see Section 7 A Symptoms Causes and Comments Faint or absent allele peaks Impure temp
96. the recommended primer concentration Vortex the PowerPlex Fusion 5X Primer Pair Mix for 15 seconds before use Poor capillary electrophoresis injection CC5 ILS 500 peaks also affected Re inject the sample Check the syringe pump system for leakage Check the laser power Samples were not denatured completely Heat denature samples for the recommended time then cool on crushed ice or in an ice water bath immediately prior to capillary electrophoresis Do not cool samples in a thermal cycler set at 4 C as this may lead to artifacts due to DNA re annealing Poor quality formamide was used Use only Hi Di formamide when analyzing samples Extra peaks visible in one Contamination with another template DNA or previously or all color channels amplified DNA Cross contamination can be a problem Use aerosol resistant pipette tips and change gloves regularly Samples were not denatured completely Heat denature samples for the recommended time and cool on crushed ice or in an ice water bath immediately prior to loading the capillary Double stranded DNA migrates faster than single stranded DNA during capillary electrophoresis Appearance of shadow peaks migrating in front of the main peaks especially if the shadow peaks are separated by the same distance as the main peaks in a heterozygote can indicate the presence of double stranded DNA due to incomplete denaturation or post injection re annealing Artifacts of STR amplification Amp
97. tion setup Meticulous care must be taken to ensure successful amplification A guide to amplification troubleshooting is provided in Section 7 The concentration of 2800M Control DNA was determined by measuring absorbance at 260nm Quantification of this control DNA by other methods such as qPCR may result in a different value Prepare a fresh DNA dilution for each set of amplifications Do not store diluted DNA e g 0 25ng l or less 4 A Amplification of Extracted DNA Materials to Be Supplied by the User GeneAmp PCR System 9700 thermal cycler Applied Biosystems microcentrifuge MicroAmp optical 96 well reaction plate or 0 2ml MicroAmp reaction tubes Applied Biosystems aerosol resistant pipette tips We routinely amplify 0 25 0 5ng of template DNA in a 25 l reaction volume using the protocol detailed below 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 TMD039 Printed in USA Revised 10 12 Page 6 Amplification Setup 1 Thaw the PowerPlex Fusion 5X Master Mix PowerPlex Fusion 5X Primer Pair Mix and Water Amplification Grade completely Note Centrifuge tubes briefly to bring contents to the bottom then vortex reagents for 15 seconds before each use Do not centrifuge the 5X Primer Pair Mix or 5X Master Mix after vortexing as this may cause the reagent
98. ty tab You may change the settings for peak quality Note For Steps 11 and 12 see the GeneMapper ID user s manual for more information 12 Select the Quality Flags tab You may change these settings 13 Select OK to save your settings 11047TA Figure 20 The GeneMapper ID Peak Detector tab Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD039 Revised 10 12 Page 43 6 I Creating a Casework Analysis Method with GeneMapper ID Software Version 3 2 continued Processing Data for Casework Samples 1 Select File then New Project 2 Select Edit then Add Samples to Project 3 Browse to the location of the run files Highlight desired files then select Add to list followed by Add 4 In the Sample Type column use the drop down menu to select Ladder Sample Positive Control or Negative Control as appropriate for the sample Every folder in the project must contain at least one allelic ladder injection that is designated as Ladder in the Sample Type column for proper genotyping Note The positive control DNA defined in the GeneMapper ID panel file is the 2800M Control DNA Redefine the genotype in the panel file if using a different positive control DNA 5 In the Analysis Method column
99. unchSolution Kit The SwabSolution Kit Cat DC8271 contains reagents for rapid DNA preparation from buccal swabs prior to amplification The procedure lyses cells contained on the swab head and releases into solution sufficient DNA for STR amplification A small volume of the final swab extract is added to the PowerPlex reaction The PunchSolution Kit is used to process punches from nonFTA storage cards containing blood or buccal samples prior to direct amplification For casework or samples that require DNA purification we recommend the DNA IQ System Cat DC6700 which is a DNA isolation system designed specifically for forensic samples 26 This system uses paramagnetic particles to prepare clean samples for STR analysis easily and efficiently and can be used to extract DNA from stains or liquid samples such as blood or solutions The DNA IQ Resin eliminates PCR inhibitors and contaminants frequently encountered in casework samples With DNA rich samples the DNA IQ System delivers a consistent amount of total DNA The system has been used to isolate DNA from routine sample types including buccal swabs stains on FTA paper 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 PowerPlex Systems to ensure a streamlined process For applications requiring hu
100. was imported in Section 6 F 7 In the Size Standard column select the size standard that was imported in Section 6 G or created in Section 6 H 8 Select Analyze green arrow button to start the data analysis Note Sizing of Penta E and DYS391 alleles 475 bases will not use Local Southern Method For Penta E alleles gt 24 will be labeled as OL 6 K Controls 1 Observe the results for the negative control Using the protocols defined in this manual the negative control should be devoid of amplification products 2 Observe the results for the 2800M Control DNA Compare the 2800M DNA allelic repeat sizes with the locus specific allelic ladder The expected 2800M DNA allele designations for each locus are listed in Table 6 Section 9 A Page 46 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD039 Printed in USA Revised 10 12 6 L Results Representative results of the PowerPlex Fusion System are shown in Figure 22 The PowerPlex Fusion Allelic Ladder Mix is shown in Figure 23 Artifacts and Stutter Stutter products are a common amplification artifact associated with STR analysis Stutter products often are observed one repeat unit below the true allele peak and occasionally two repeat units smaller or one repeat unit larger than the true allele peak Frequently all
101. x 5 Dye Matrix Standards 3100 3130 25 l each dye DG4700 PunchSolution Kit 100 preparations DC9271 SwabSolution Kit 100 preparations DC8271 CC5 Internal Lane Standard 500 300 l DG1521 2800M Control DNA 10ng l 25 l DD7101 2800M Control DNA 0 25ng l 500 l DD7251 Water Amplification Grade 6 250 l 5 1 250 l DW0991 Not for Medical Diagnostic Use Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD039 Revised 10 12 TE 4 Buffer 10mM Tris HCl 0 1mM EDTA pH 8 0 1 21g Tris base 0 037g EDTA 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 TE 4 Buffer with 20 g ml Glycogen 1 21g Tris base 0 037g EDTA Na2EDTA 2H2O 20 g ml glycogen Dissolve Tris base and EDTA in 900ml of deionized water Adjust to pH 8 0 with HCl Add glycogen Bring the final volume to 1 liter with deionized water Page 68 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 TMD039 Printed in USA Revised 10 12 9 E Related Products continued Sample Preparation and DNA Quantification Systems Product
102. you would using your normal workflow 2 Depending on your preferred protocol place one or two 1 2mm storage card punches containing a buccal sample or one 1 2mm punch of a storage card containing whole blood in each well of a reaction plate Be sure to pretreat nonFTA samples with the PunchSolution Kit Cat DC9271 3 Prepare four identical reaction plates with punches from the same samples 4 Amplify samples using the thermal cycling protocol provided above but subject each plate to a different cycle number 25 28 cycles 5 Following amplification use your laboratory s validated separation and detection protocols to determine the optimal cycle number for the sample type and number of storage card punches 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 TMD039 Printed in USA Revised 10 12 Thermal Cycling Protocol1 96 C for 1 minute then 94 C for 10 seconds 59 C for 1 minute 72 C for 30 seconds for 27 cycles then 60 C for 20 minutes 4 C soak 1When using the GeneAmp PCR System 9700 thermal cycler the program must be run with Max mode as the ramp speed This requires a silver or gold plated silver sample block The ramp speed is set after the thermal cycling run is started The Select Method Options screen appears Select Max for the ramp speed and

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