Home

PowerPlex® Fusion 6C System

1. Create Alternatively a previously created Instrument Protocol may be used Figure 6 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 12775TA Figure 6 The Create New Instrument Protocol window The recommended settings are Application Type HID Capillary Length 36cm Polymer POP 4 Dye Set Promega J6 Run Module HID36_POP4 xl Injection Time1 15 seconds for the Applied Biosystems 3500 Genetic Analyzer 24 seconds for the Applied Biosystems 3500xL Genetic Analyzer Injection Voltage 1 2kV Run Voltage 13kV Run Time 1 500 seconds 1Injection time may be modi ed to increase or decrease peak heights Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 19 www promega com TMD045 1 15 When creating an Instrument Protocol be sure to select the same dye set that was used to perform the Promega J6 spectral calibration 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 speci c Instrument Protocols for each condition tested If a single Instrument Protocol is used follow the instructions in the Applied Biosystems 3500 3500xL Genetic Analy
2. Minor voltage changes or urea crystals passing by the laser can cause spikes or unexpected peaks Spikes sometimes appear in one color but often are easily identi ed by their presence in more than one color Re inject samples to con rm Incorrect J6 spectral was active when analyzing samples with the Applied Biosystems 3130 or 3130xl Genetic Analyzer Re run samples and con rm that the PowerPlex 6C J6 spectral is set for J6 See instructions for instrument preparation in Section 5 B Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 49 www promega com TMD045 1 15 Symptoms Causes and Comments Extra peaks visible in one Pull up or bleedthrough Pull up can occur when peak heights are or all color channels continued 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 sensitivities can vary Optimize the injection conditions See Section 5 Reboot the Applied Biosystems 3500 or 3500xL Genetic Analyzer and the instrument s computer Repeat the spectral calibration Do not allow borrowing when running the spectral calibration on the Applied Biosystems 3500 or 3500xL Genetic Analyzer Repeat sample preparation using fresh formamide Long term storage of ampli ed sample in formamide can result in artifacts The CE p
3. 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 16 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 5 A Detection of Ampli ed Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer continued Sample Preparation 1 At the rst use thaw the WEN Internal Lane Standard 500 and PowerPlex Fusion 6C Allelic Ladder Mix completely After the rst use store the reagents at 2 10 C Note Centrifuge tubes brie y to bring contents to the bottom then vortex for 15 seconds before each use Do not centrifuge after vortexing as this may cause the reagents to be concentrated at the bottom of the tube 2 Prepare a loading cocktail by combining and mixing WEN Internal Lane Standard 500 and Hi Di formamide as follows 0 5 l WEN ILS 500 samples 9 5 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 Adjust the volume added to the wells in Step 4 accordingly Do not add less than 9 5 l of formamide per well
4. erences in migration The dye label and linker also a ect 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 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 63 www promega com TMD045 1 15 Table 9 The PowerPlex Fusion 6C 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 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 11 122 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 D8S1179 14 15 13 13 12 13 D12S391 18 23 18 20 18 24 D19S433 13 14 14 15 13 14 SE33 15 16 19 29 2 23 2 26 2 D22S1045 16 16 11 14 16 18 DYS391 10 10 FGA 20 23 23 24 24 26 DYS576
5. Fax 608 277 2516 TMD045 1 15 www promega com 4 C Direct Ampli cation of DNA from Swabs in a 12 5 l Reaction Volume continued 8 For the negative ampli cation control pipet 2 0 l of Water Ampli cation Grade or TE 4 bu er instead of swab extract into a reaction well containing PCR ampli cation mix Note Additional negative controls can be included Assemble a reaction containing the swab extract prepared from a blank swab or assemble a reaction where the SwabSolution Reagent is processed as a blank without a swab 9 Seal or cap the plate or close the tubes Optional Brie y centrifuge the plate to bring contents to the bottom of the wells and remove any air bubbles Thermal Cycling Ampli cation and detection instrumentation may vary You will need to optimize protocols including the amount of template DNA cycle number 24 27 cycles injection time and loading volume for your laboratory instrumentation Testing at Promega shows that 25 cycles works well for a variety of sample types Cycle number will need to be optimized in each laboratory for each sample type 1 Place the MicroAmp plate or reaction tubes in the thermal cycler 2 Select and run the recommended protocol which is provided below and in Figure 4 The total cycling time is approximately 1 hour Notes 1 When using the Veriti 96 Well Thermal Cycler set the ramping rate to 100 2 When using the GeneAmp PCR System
6. 2003200444 and corresponding patent claims outside the US g TMR 6C CXR 6C TOM 6C and WEN 6C dyes are proprietary h This product or por ons 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 applica ons End User Terms and Condi ons Acceptance These terms and condi ons shall govern the purchase use transfer and acceptance of the products described in the purchase order quota on 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 condi onal upon End User s acceptance of these terms and condi ons Restric ons on Use End Users are speci cally not authorized to and are forbidden from reselling transferring or
7. 3 Vortex for 10 15 seconds to mix 4 Pipet 10 l of formamide internal lane standard mix into each well 5 Add 1 l of ampli ed sample or 1 l of PowerPlex Fusion 6C Allelic Ladder Mix to each well Cover wells with appropriate septa Notes 1 Instrument detection limits vary therefore injection time or the amount of sample mixed with loading cocktail may need to be increased or decreased To modify the injection time 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 ampli cation reactions or reduce the number of cycles in the ampli ca tion program to achieve the desired signal intensity If the injection time is reduced a decreased peak amplitude threshold for the orange channel may be required for proper sizing 2 Use a volume of allelic ladder that results in peak heights that are all consistently above the peak amplitude threshold determined as part of your internal validation 6 Centrifuge plate brie y to remove air bubbles from the wells 7 Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or a freezer plate block or in an ice water bath for 3 minutes Denature samples just prior to loading the instrument Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 1
8. rst then add PowerPlex Fusion 6C 5X Master Mix PowerPlex Fusion 6C 5X Primer Pair Mix and 5X AmpSolution Reagent For FTA card punches the template DNA will be added at Step 6 5 Vortex the PCR ampli cation mix for 5 10 seconds then pipet 12 5 l of PCR ampli cation mix into each reaction well Failure to vortex the PCR ampli cation mix su ciently can result in poor ampli cation or locus to locus imbalance 6 For FTA storage cards add one 1 2mm punch from a card containing buccal cells or whole blood to the appropriate wells of the reaction plate For nonFTA card punches add the PCR ampli cation mix to the well or tube containing the PunchSolution Reagent treated punch Note It also is acceptable to add the FTA card punch rst then add the PCR ampli cation mix Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 11 www promega com TMD045 1 15 7 For the positive ampli cation control vortex the tube of 2800M Control DNA then add 1 l 10ng to a reaction well containing 12 5 l of PCR ampli cation mix Notes 1 Optimization of the amount of control DNA may be required depending on cycling conditions and laboratory preferences 2 When performing more than 25 cycles with 12 5 l volume reactions you may need to dilute the 2800M Control DNA to 5ng l prior to adding 1 l 5ng
9. select Open Data in the Magic Wizard Figure 21 and click Add 12788TA Figure 21 The GeneMarker HID Magic Wizard 3 Navigate to the directory containing your raw data les and select the desired les 4 Select Open and the selected les will appear in the Data File List Note Be sure that the Auto Elevate box is not checked 38 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 7 A Creating an Analysis Method with GeneMarker HID Software Version 2 7 1 continued 5 The rst time that you use the PowerPlex Fusion 6C System with the GeneMarker HID software you will need to set the dye channels To do so click the Channels button in the bottom left of the Data Files window then select the 6 Colors tab Set the dye channels as shown in Figure 22 12789TA Figure 22 The GeneMarker HID dye channels 6 Select OK in the Open Data Files window and the data will be uploaded into the GeneMarker HID Software In the Raw Data Tree verify that the sample types allelic ladder positive control and negative control are designated If sample types are not designated designate sample types by right clicking on the le name and selecting Set sample type Note Sample types can be designated in the le name See Step 8 b Promega Corpora on 280
10. to positive control reactions 3 Do not include blank storage card punches in the positive control reactions 8 Reserve a well containing PCR ampli cation mix as a negative ampli cation 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 or cap the plate or close the tubes Brie y centrifuge reactions to bring storage card punches to the bottom of the wells and remove air bubbles Thermal Cycling Ampli cation and detection instrumentation may vary You will need to optimize protocols including the number of storage card punches cycle number 23 26 cycles injection time and loading volume for your laboratory instrumen tation Testing at Promega shows that 25 cycles works well for a variety of sample types Buccal samples may require more ampli cation cycles than blood samples NonFTA card punches may require fewer ampli cation cycles than FTA punches Cycle number should be optimized in each laboratory for each sample type 1 Place the MicroAmp plate or reaction tubes in the thermal cycler 2 Select and run the recommended protocol which is provided below and in Figure 3 The total cycling time is approximately 1 hour Notes 1 When using the Veriti 96 Well Thermal Cycler set the ramping rate to 100 2 When using the GeneAmp PCR System 9700 the program must be run with Max Mode as the ramp spee
11. 1 4 Y Marker Check Box 30 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 6 B Importing the WEN ILS 500 IDX Size Standard into GeneMapper ID X Software Version 1 4 There are two options when creating a size standard Use this protocol or the alternative protocol in Section 6 C 1 Select Tools then GeneMapper ID X Manager 2 Select the Size Standard tab 3 Select Import 4 Navigate to the location of the WEN_ILS_500_IDX xml le on your computer 5 Highlight the le 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 4 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 16 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 WEN_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 11 C Figur
12. 12 14 28 CSF1PO JOE 6C 318 362 5 16 Penta D JOE 6C 377 450 2 2 3 2 5 17 TH01 TMR 6C 72 115 3 9 9 3 10 11 13 3 vWA TMR 6C 127 183 10 24 D21S11 TMR 6C 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 6C 269 313 5 16 D5S818 TMR 6C 321 369 6 18 TPOX TMR 6C 393 441 4 16 D8S1179 CXR 6C 76 124 7 19 D12S391 CXR 6C 133 185 14 17 17 3 18 18 3 19 27 D19S433 CXR 6C 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 18 2 SE33 CXR 6C 270 408 4 2 6 3 8 20 20 2 21 21 2 22 22 2 23 2 24 2 25 2 26 2 27 2 28 2 29 2 30 2 31 2 32 2 33 2 34 2 35 37 39 D22S1045 CXR 6C 431 470 7 20 DYS391 TOM 6C 86 130 5 16 FGA TOM 6C 143 289 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 DYS576 TOM 6C 308 356 11 23 DYS570 TOM 6C 393 453 10 25 1The length of each allele in the allelic ladder has been con rmed by sequence analysis 2When using an internal lane standard such as the WEN Internal Lane Standard 500 the calculated sizes of allelic ladder components may di er from those listed This occurs because di erent sequences in allelic ladder and ILS components may cause di
13. 15 6 Assign sample names to wells 7 In the lower left portion of the screen under Assays use the Add from Library option to select the Assay created in Step 2 d or one previously created Click on the Add to Plate button and close the window 8 Under File Name Conventions use the Add from Library option to select the File Name Convention created in Step 2 e or one previously created Click on the Add to Plate button and close the window 9 Under Results Groups use the Add from Library option to select the Results Group created in Step 2 f or one previously created Click on the Add to Plate button and close the window 10 Highlight the sample wells then select the boxes in the Assays File Name Conventions and Results Groups that pertain to those samples 11 Select Link Plate for Run 12 The Load Plate window will appear Select Yes 13 In the Run Information window Figure 14 assign a Run Name Select Start Run not shown Each injection will take approximately 40 minutes 9256TA Figure 14 Assigning a run name 26 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 5 B Detection of Ampli ed Fragments Using the Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 4 0 and DC v4 6 Dye Module v1 License
14. 18 16 DYS570 17 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 2The relative peak heights for these three alleles will di er Allele 12 may not be called 64 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 11 B DNA Extraction and Quantitation Methods and Automation Support Promega o ers a wide variety of reagents and automated methods for sample preparation DNA puri cation and DNA quantitation prior to STR ampli cation For analysis of database reference and other single source samples we recommend direct ampli cation of DNA from FTA card punches or direct ampli cation of DNA from swabs and nonFTA punches following a preprocessing step with the SwabSolution Kit or PunchSolution Kit respectively The SwabSolution Kit Cat DC8271 contains reagents for rapid DNA preparation from buccal swabs prior to ampli cation The procedure lyses cells contained on the swab head and releases into solution su cient DNA for STR ampli cation A small volume of the nal swab extract is added to the PowerPlex re
15. 608 277 2516 61 www promega com TMD045 1 15 Table 7 The PowerPlex Fusion 6C System Locus Speci c Information STR Locus Label Chromosomal Location1 Repeat Sequence2 5 3 Amelogenin3 FL 6C Xp22 1 22 3 and Y NA D3S1358 FL 6C 3p21 31 45 557Mb TCTA Complex D1S1656 FL 6C 1q42 228 972Mb TAGA Complex D2S441 FL 6C 2p14 68 214Mb TCTA D10S1248 FL 6C 10q26 3 130 567Mb GGAA D13S317 FL 6C 13q31 1 81 62Mb TATC Penta E FL 6C 15q26 2 95 175Mb AAAGA D16S539 JOE 6C 16q24 1 84 944Mb GATA D18S51 JOE 6C 18q21 33 59 1Mb AGAA 19 D2S1338 JOE 6C 2q35 218 705Mb TGCC TTCC CSF1PO JOE 6C 5q33 1 149 436Mb AGAT Penta D JOE 6C 21q22 3 43 88Mb AAAGA TH01 TMR 6C 11p15 5 2 149Mb AATG 19 vWA TMR 6C 12p13 31 5 963Mb TCTA Complex 19 D21S11 TMR 6C 21q21 1 19 476Mb TCTA Complex 19 D7S820 TMR 6C 7q21 11 83 433Mb GATA D5S818 TMR 6C 5q23 2 123 139Mb AGAT TPOX TMR 6C 2p25 3 1 472Mb AATG D8S1179 CXR 6C 8q24 13 125 976Mb TCTA Complex 19 D12S391 CXR 6C 12p12 12 341Mb AGAT AGAC Complex D19S433 CXR 6C 19q12 35 109Mb AAGG Complex SE33 CXR 6C 6q14 89 043Mb AAAG Complex D22S1045 CXR 6C 22q12 3 35 779Mb ATT DYS391 TOM 6C Y TCTA FGA TOM 6C 4q28 155 866Mb TTTC Complex 19 DYS576 TOM 6C Y AAAG DYS570 TOM 6C Y TTTC 1Information about the chromosomal location of these loci can be fo
16. Materials to Be Supplied by the User 95 C dry heating block water bath or thermal cycler crushed ice ice water bath or freezer plate block centrifuge compatible with 96 well plates aerosol resistant pipette tips 3100 or 3130 capillary array 36cm plate retainer amp base set standard POP 4 polymer for the 3130 3130xl Genetic Analyzers 10X genetic analyzer bu er 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 At the rst use thaw the WEN Internal Lane Standard 500 and PowerPlex Fusion 6C Allelic Ladder Mix completely After the rst use store the reagents at 2 10 C Note Centrifuge tubes brie y to bring contents to the bottom then vortex for 15 seconds before
17. Mix is shown in Figure 28 12790TA A B C D E F Figure 27 The PowerPlex Fusion 6C System The 2800M Control DNA 1 0ng was ampli ed using the PowerPlex Fusion 6C System and 29 cycles Ampli cation products were mixed with WEN Internal Lane Standard 500 and analyzed using an Applied Biosystems 3500 Genetic Analyzer and a 1 2kV 15 second injection Results were analyzed using GeneMapper ID X software version 1 4 Panel A An electropherogram showing the peaks of the FL 6C labeled loci Amelogenin D3S1358 D1S1656 D2S441 D10S1248 D13S317 and Penta E Panel B An electropherogram showing the peaks of the JOE 6C labeled loci D16S539 D18S51 D2S1338 CSF1PO and Penta D Panel C An electropherogram showing the peaks of the TMR 6C labeled loci TH01 vWA D21S11 D7S820 D5S818 and TPOX Panel D An electropherogram showing the peaks of the CXR 6C labeled loci D8S1179 D12S391 D19S433 SE33 and D22S1045 Panel E An electropherogram showing the TOM 6C labeled loci DYS391 FGA DYS576 and DYS570 Panel F An electropherogram showing the 60bp to 500bp fragments of the WEN Internal Lane Standard 500 44 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 12791TA A B C D E Figure 28 The PowerPlex Fusion 6C Allelic Ladder Mix The PowerPlex Fusion 6C Allelic Ladde
18. This will appear as variability in peak heights between swab extracts The extraction process maximizes recovery of ampli able DNA from buccal swabs but does not normalize the amount of DNA present If variability is extreme quantitate the DNA using a uorescence based double stranded DNA quantitation method or qPCR based quantitation method The quantitation values can be used to normalize input template amounts to minimize variation in signal intensity Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 57 www promega com TMD045 1 15 9 E GeneMapper ID X Software Symptoms Causes and Comments Stutter peaks not ltered Stutter text le was not imported into the Panel Manager when the panels and bins text les were imported Be sure that the Use marker speci c stutter ratio and distance if available box is checked If the Use marker speci c stutter ratio and distance if available box is not checked stutter distance must be de ned 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 be viewed To view edits made to a
19. X Software Version 1 4 30 6 D Creating a Casework Analysis Method with GeneMapper ID X Software Version 1 4 31 6 E Creating a Databasing or Paternity Analysis Method with GeneMapper ID X Software Version 1 4 35 6 F Controls in the GeneMapper ID X Software 37 7 Data Analysis Using GeneMarker HID Software Version 2 7 1 37 7 A Creating an Analysis Method with GeneMarker HID Software Version 2 7 1 37 7 B Controls in the GeneMarker HID Software 42 8 Results 43 2 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 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 polymera
20. analyzing ampli cation products Reagents and materials used prior to ampli cation PowerPlex Fusion 6C 5X Master Mix PowerPlex Fusion 6C 5X Primer Pair Mix 2800M Control DNA and Water Ampli cation Grade are provided in a separate box and should be stored separately from those used following ampli cation PowerPlex Fusion 6C Allelic Ladder Mix and WEN 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 3 B Spectral Calibration Proper spectral calibration is critical to evaluate multicolor systems with the Applied Biosystems 3500 and 3500xL Genetic Analyzers and Applied Biosystems 3130 and 3130xl Genetic Analyzers with Data Collection Software Version 4 0 and DC v4 6 Dye Module v1 License A matrix must be generated for each individual instrument For protocols and additional information on spectral calibration on these instruments see the PowerPlex 6C Matrix Standard Technical Manual TMD046 This manual is available at www promega com
21. below and in Figure 31 The total cycling time is approximately 1 hour Notes 1 When using the Veriti 96 Well Thermal Cycler set the ramping rate to 100 2 When using the GeneAmp PCR System 9700 the program must be run with Max Mode as the ramp speed This requires a silver plated or gold plated sample block The ramp speed is set after the thermal cycling run is started When the Select Method Options screen appears select Max for the ramp speed and enter the reaction volume Thermal Cycling Protocol 96 C for 1 minute then 96 C for 5 seconds 60 C for 1 minute for 26 cycles then 60 C for 10 minutes 4 C soak 11876MB 96 C 96 C 5 seconds 1 minute 60 C 1 minute 60 C 10 minutes 4 C Optimal cycle number 1 cycle 1 cycle Hold Figure 31 Thermal cycling protocol for the GeneAmp PCR System 9700 and Veriti 96 Well Thermal Cycler 3 After completion of the thermal cycling protocol proceed with fragment analysis or store ampli ed samples at 20 C in a light protected box Note Long term storage of ampli ed samples at 4 C or higher may produce artifacts 72 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 11 E Direct Ampli cation of DNA from Swabs in a 25 l Reaction Volume continued PCR Optimization Cycle number should be optimized b
22. continued Thermal Cycling Ampli cation and detection instrumentation may vary You will need to optimize protocols including the number of storage card punches cycle number 24 27 cycles injection time and loading volume for your laboratory instrumen tation Testing at Promega shows that 26 cycles works well for a variety of sample types Buccal samples may require more ampli cation cycles than blood samples NonFTA card punches may require fewer ampli cation cycles than FTA punches Cycle number should be optimized in each laboratory for each sample type 1 Place the MicroAmp plate in the thermal cycler 2 Select and run the recommended protocol which is provided below and in Figure 30 The total cycling time is approximately 1 hour Notes 1 When using the Veriti 96 Well Thermal Cycler set the ramping rate to 100 2 When using the GeneAmp PCR System 9700 the program must be run with Max Mode as the ramp speed This requires a silver plated or gold plated sample block The ramp speed is set after the thermal cycling run is started When the Select Method Options screen appears select Max for the ramp speed and enter the reaction volume Thermal Cycling Protocol 96 C for 1 minute then 96 C for 5 seconds 60 C for 1 minute for 26 cycles then 60 C for 10 minutes 4 C soak 11876MB 96 C 96 C 5 seconds 1 minute 60 C 1 minute 60 C 10 minutes 4 C Optimal cycle numbe
23. di erent cycle number 24 27 cycles 5 Following ampli cation 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 11 E Direct Ampli cation of DNA from Swabs in a 25 l Reaction Volume Depending on the procedure used to prepare the swabs high amounts of cells may be present on each swab In these cases performing direct ampli cation in 25 l reaction volumes may provide better results Materials to Be Supplied by the User GeneAmp PCR System 9700 96 Well with a gold plated or silver plated sample block or Veriti 96 Well Thermal Cycler Applied Biosystems centrifuge compatible with a 96 well plate 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 in 25 l reaction volumes using the PowerPlex Fusion 6C System and GeneAmp PCR System 9700 or Veriti 96 Well Thermal Cycler The PowerPlex Fusion 6C System contains su cient reagents for 100 direct ampli cation reactions of 12 5 l each when performing 25 l reactions the system contains su cient reagents for 50 reactions The PowerPlex Fusion 6C System is compatible with the GeneAmp PCR System 9700 with a silver plated or gold plated sampl
24. distribu ng 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 a empt to reverse engineer parameters of any of GE Healthcare Bio Sciences Corp proprietary products or services Disclaimer of Warran es GE Healthcare Bio Sciences Corp provides no warran es to end user statutory or implied including without limita on as to product quality condi on descrip on merchantability or tness for a par cular purpose and all such warran es 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 limita on any claim of inaccurate invalid or incomplete results Exclusion of Liability GE Healthcare Bio Sciences Corp and its a liates shall have no liability to an End User including without limita on for any loss of use or pro ts business interrup on or any consequen al incidental special or other indirect damages of any kind regardless of how caused and regardless of whether an ac on in contract tort strict product liability or otherwise 2015 Promega Corpora on All Rights Reserved Plexor and PowerPlex are registered trademar
25. imported in Section 6 A 8 Ensure that the Use marker speci c stutter ratio and distance if available box is checked Doing this will assign locus speci c stutter lters and distances from the imported stutter le We recommend the settings shown in Figure 17 for proper ltering of stutter peaks when using the PowerPlex Fusion 6C System Note If you do not check the Use marker speci c stutter ratio and distance if available box you will need to optimize these settings In house validation should be performed 12781TA Figure 17 The GeneMapper ID X Software Version 1 4 Allele tab Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 33 www promega com TMD045 1 15 9 Select the Peak Detector tab Figure 18 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 rst de ned 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 Applied Biosystems 31
26. or reduced cycle number Ampli cation of excess template for a given cycle number can result in overloading of the capillary upon electrokinetic injection The presence of excess DNA in the capillary makes it di cult to maintain the DNA in a denatured single stranded state Some single stranded DNA renatures and becomes double stranded Double stranded DNA migrates faster than single stranded DNA during capillary electrophoresis and appears as shadow peaks migrating in front of the main peaks If this occurs at a heterozygous locus it is sometimes possible to see two shadow peaks that di er in size from one another by approximately the same distance as the single stranded alleles Artifacts of STR ampli cation Direct ampli cation of gt 20ng of template can result in a higher number of artifact peaks Use the recommended punch size and number of punches Optimize the cycle number See Section 8 for additional information on stutter and artifacts Artifacts of STR ampli cation Ampli cation 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 10 minute extension step at 60 C after thermal cycling Section 4 11 D or 11 E Decrease cycle number Increase the nal extension time Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608
27. 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 WEN ILS 500 peaks de ned 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 or increase the volume of WEN ILS 500 used in Section 5 If peaks are low quality rede ne the size standard for the sample to skip these peaks Signi cantly raised baseline Poor spectral calibration Perform a new spectral calibration and re run the samples Incorrect dye set was used Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 59 www promega com TMD045 1 15 10 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 Identi cation 1991 Promega Corporation 31 52 2 Edwards A et al 1991 DNA typing and genetic mapping with trimeric and tetrameric tandem repe
28. speci c stutter ratio and distance if available box is checked Doing this will assign locus speci c stutter lters and distances from the imported stutter le Ensure that the appropriate global lter is applied to this analysis method For example for a 20 lter enter 0 20 for the Global Cut o Value for Tri Tetra and Penta repeats Figure 20 Note If you do not check the Use marker speci c stutter ratio and distance if available box you will need to optimize these settings In house validation should be performed 12787TA Figure 20 The GeneMapper ID X Software Version 1 4 Allele tab with settings for using a 20 peak lter 36 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 6 E Creating a Databasing or Paternity Analysis Method with GeneMapper ID X Software Version 1 4 continued 9 Select the Peak Detector tab Figure 18 You will 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 rst de ned internal lane standard peak to help ensure proper sizing of the internal lane standard 2 The peak amplitude threshol
29. the 2800M Control DNA is stored at 2 10 C for at least 24 hours before use After the rst use store the PowerPlex Fusion 6C System components at 2 10 C where they are stable for 6 months Do not refreeze The PowerPlex Fusion 6C 5X Primer Pair Mix PowerPlex Fusion 6C Allelic Ladder Mix and WEN Internal Lane Standard 500 WEN ILS 500 are light sensitive and must be stored in the dark We strongly recommend that pre ampli cation and post ampli cation reagents be stored and used separately with di erent pipettes tube racks etc Available Separately P R O D U C T S I Z E C AT PunchSolu on Kit 100 preps DC9271 SwabSolu on Kit 100 preps DC8271 5X AmpSolu on Reagent 100 preps DM1231 The PunchSolution Kit is required to process nonFTA punches prior to direct ampli cation The SwabSolution Kit is required to process swabs prior to direct ampli cation The 5X AmpSolution Reagent is required for direct ampli ca tion of DNA from storage card punches in a 12 5 l reaction volume Both the PunchSolution Kit and SwabSolution Kit include the 5X AmpSolution Reagent The proper panels bins and stutter text les for use with GeneMapper 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 PowerPlex 6C Matrix Standard is provided se
30. to a sterile tube Table 3 PCR Ampli cation Mix for Direct Ampli cation of DNA from Swabs Using a 12 5 l Reaction Volume PCR Ampli cation Mix Component1 Volume Per Reaction Number of Reactions Final Volume Water Ampli cation Grade 5 5 l PowerPlex Fusion 6C 5X Master Mix 2 5 l PowerPlex Fusion 6C 5X Primer Pair Mix 2 5 l swab extract 2 0 l total reaction volume 12 5 l 1Add Water Ampli cation Grade to the tube rst then add PowerPlex Fusion 6C 5X Master Mix and PowerPlex Fusion 6C 5X Primer Pair Mix The swab extract will be added at Step 6 5 Vortex the PCR ampli cation mix for 5 10 seconds then pipet 10 5 l of PCR ampli cation mix into each reaction well Failure to vortex the PCR ampli cation mix su ciently can result in poor ampli cation 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 ampli cation 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 10 5 l of PCR ampli cation mix Note Optimization of the amount of 2800M Control DNA may be required depending on thermal cycling conditions and laboratory preferences 14 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330
31. 0 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 39 www promega com TMD045 1 15 7 Use the Panel Editor in the Tools menu to select a PowerPlex Fusion 6C panel Click on the plus symbol to expand the list then right click on a marker and select Edit Enter laboratory speci ed values for Min Homozygote Intensity Min Heterozygote Intensity and Min Heterozygote Imbalance Figure 23 This will set values for peaks within the marker range Values for peak amplitude thresholds are usually 50 150RFU for data generated on the 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 These settings can be applied to all markers by checking the Apply Homo Hetero Settings to All Markers checkbox Select OK close the Panel Editor window and select Save Changes Note Panels with di erent analysis values can be created by selecting Save as New Panel from the File menu 12784TA Figure 23 The GeneMarker HID Edit Marker window for the D3S1358 marker 8 In the View menu select Preferences a Navigate to the Forensic tab Select Auto Delete Alleles in V
32. 1 15 TMD045 T E C H N I C A L M A N U A L PowerPlex Fusion 6C System Instruc ons for Use of Products DC2705 and DC2720 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 1 www promega com TMD045 1 15 All technical literature is available at www promega com protocols Visit the web site to verify that you are using the most current version of this Technical Manual E mail Promega Technical Services if you have questions on use of this system genetic promega com PowerPlex Fusion 6C System 1 Description 2 2 Product Components and Storage Conditions 3 3 Before You Begin 5 3 A Precautions 5 3 B Spectral Calibration 5 4 Protocols for DNA Ampli cation Using the PowerPlex Fusion 6C System 6 4 A Ampl
33. 274 4330 Fax 608 277 2516 53 www promega com TMD045 1 15 Symptoms Causes and Comments Peak height imbalance Excessive amount of DNA Ampli cation of gt 20ng of template can result in an imbalance with smaller loci showing more product than larger loci Be sure to use the recommended number of punches Follow the manufacturer s recommendations when depositing sample onto the card Decrease cycle number The cycle number was too high Decrease the cycle number by one cycle and repeat the ampli cation AmpSolution Reagent was not included in 12 5 l reactions Be sure to include AmpSolution Reagent in all 12 5 l reactions when amplifying DNA from punches Ampli cation was inhibited when using more than one storage card punch with blood Use only one 1 2mm storage card punch with blood 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 Active PunchSolution Reagent carried over into ampli cation reactions with nonFTA card punches Larger loci are most susceptible to carryover and will drop out before the smaller loci Ensure that the heat block reached 70 C and samples were incubated for 30 minutes or until wells are dry Incubation for shorter time periods may result in incomplete inactivation of the PunchSolution R
34. 30 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 3 The normalization box can be checked regardless of whether normalization was or was not applied during data collection 12786TA Figure 18 The GeneMapper ID X Software Version 1 4 Peak Detector tab 10 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 11 Select the SQ amp GQ Settings tab You may change these settings 12 Select Save to save the new analysis method 13 Select Done to exit the GeneMapper ID X Manager 34 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 6 D Creating a Casework Analysis Method with GeneMapper ID X Software Version 1 4 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 les Highlight desired les then select Add to list followed by Add 4 In the Sample Type
35. 7 www promega com TMD045 1 15 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 bu ers and polymer pouch and perform a spatial calibration Samples may be analyzed as described in the Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 1 Open the 3500 Data Collection Software The Dashboard screen will launch Figure 5 To ensure that you are viewing the most up to date information press the Refresh button Ensure that the Consumables Information and Maintenance Noti cations are acceptable Set the oven temperature to 60 C then select Start Pre Heat When the Oven Temperature and Detection Cell Temperature turn green you may proceed with the rst injection 9247TA Figure 5 The Dashboard 18 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 5 A Detection of Ampli ed Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer continued 2 Prior to the rst analysis using the PowerPlex Fusion 6C System an Instrument Protocol Size Standard QC Protocol Assay File Name Convention and Results Group must be created a To create a new Instrument Protocol navigate to the Library select Instrument Protocols then select
36. 9700 the program must be run with Max Mode as the ramp speed This requires a silver plated or gold plated sample block The ramp speed is set after the thermal cycling run is started When the Select Method Options screen appears select Max for the ramp speed and enter the reaction volume Thermal Cycling Protocol 96 C for 1 minute then 96 C for 5 seconds 60 C for 1 minute for 25 cycles then 60 C for 10 minutes 4 C soak 11876MB 96 C 96 C 5 seconds 1 minute 60 C 1 minute 60 C 10 minutes 4 C Optimal cycle number 1 cycle 1 cycle Hold Figure 4 Thermal cycling protocol for the GeneAmp PCR System 9700 and Veriti 96 Well Thermal Cycler Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 15 www promega com TMD045 1 15 3 After completion of the thermal cycling protocol proceed with fragment analysis or store ampli ed samples at 20 C in a light protected box Note Long term storage of ampli ed 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 work ow 2
37. Analyzer Figure 8 shows one option for these settings 12777TA Figure 8 The Create New QC Protocol window Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 21 www promega com TMD045 1 15 d To create a new Assay navigate to the Library Select Assays then select Create Alternatively a previ ously created Assay may be used In the Create New Assay window Figure 9 select the Instrument Protocol created in Step 2 a and the QC Protocol created in Step 2 c Assign a descriptive assay name Select the application type HID An Assay is required for all named samples on a plate Note If autoanalysis of sample data is desired refer to the instrument user s manual for instructions 12778TA Figure 9 The Create New Assay window 22 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 5 A Detection of Ampli ed Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer continued e To create a new File Name Convention Figure 10 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 your laboratory practices and save with a des
38. Applied Biosystems 3500xL Genetic Analyzer Partial pro les were obtained with all primate species tested but these pro les can be distinguished from a human pro le because most of the alleles were called as o ladder or were outside the locus panels Table 6 Nonhuman DNA Cross Reactivity DNA Source Artifact Size Dye Label Bovine 98bp FL 6C Chicken 221bp JOE 6C 300bp TMR 6C Mouse 347bp JOE 6C Pig 259 260bp FL 6C 368 372bp JOE 6C 369 370bp CXR 6C Rat 300bp FL 6C Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 47 www promega com TMD045 1 15 9 Troubleshooting For questions not addressed here please contact your local Promega Branch O ce or Distributor Contact information available at www promega com E mail genetic promega com For questions about GeneMarker HID software contact SoftGenetics at www softgenetics com 9 A Ampli cation and Fragment Detection This section provides information about general ampli cation and detection For questions about ampli cation of extracted DNA see Section 9 B For questions about direct ampli cation see Sections 9 C and 9 D Symptoms Causes and Comments Faint or absent allele peaks The PowerPlex Fusion 6C 5X Master Mix was not vortexed well before use Vortex the 5X Master Mix for 15 seconds before dispensing in
39. EDTA from the DNA sample can negatively a ect PCR A change in pH also may a ect PCR Store DNA in TE 4 bu er 10mM Tris HCl pH 8 0 0 1mM EDTA TE 4 bu er with 20 g ml glycogen or nuclease free water Faint or absent peaks may be seen more often when using the maximum template volume or reduced ampli cation reaction volume Extra peaks visible in one Artifacts of STR ampli cation Ampli cation of excess amounts or all color channels of puri ed DNA can result in a higher number of artifact peaks Use the recommended amount of template DNA See Section 8 for additional information about stutter and artifacts The amount of template will need to be optimized if you are using reduced reaction volumes Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 51 www promega com TMD045 1 15 Symptoms Causes and Comments Peak height imbalance Excessive amount of DNA Ampli cation of gt 1 0ng of template in a 25 l reaction volume can result in an imbalance with smaller loci showing more product than larger loci Use less template or fewer cycles Degraded DNA sample DNA template was degraded and larger loci showed diminished yield Insu cient template DNA Use the recommended amount of template DNA if available Stochastic e ects can occur when amplifying low amounts of template Impure template DNA Inhibito
40. It also ensures that each reaction contains the same PCR ampli cation 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 nal volume of each reagent listed in Table 11 to a sterile tube Table 11 PCR Ampli cation Mix for Direct Ampli cation of DNA from Swabs Using a 25 l Reaction Volume PCR Ampli cation Mix Component1 Volume Per Reaction Number of Reactions Final Volume Water Ampli cation Grade 13 l PowerPlex Fusion 6C 5X Master Mix 5 0 l PowerPlex Fusion 6C 5X Primer Pair Mix 5 0 l swab extract 2 0 l total reaction volume 25 l 1Add Water Ampli cation Grade to the tube rst then add PowerPlex Fusion 6C 5X Master Mix and PowerPlex Fusion 6C 5X Primer Pair Mix The swab extract will be added at Step 6 5 Vortex the PCR ampli cation mix for 5 10 seconds then pipet 23 l of PCR ampli cation mix into each reaction well Failure to vortex the PCR ampli cation mix su ciently can result in poor ampli cation 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 ampli cation control vortex the tube of 2800M Control DNA then dilute an aliquot to 5 0ng l Add 2 l 10ng to a reaction well cont
41. Mix 200 l WEN Internal Lane Standard 500 4 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 2 Product Components and Storage Conditions continued P R O D U C T S I Z E C AT PowerPlex Fusion 6C System 200 or 400 direct amp reac ons DC2720 Not For Medical Diagnostic Use This system contains su cient reagents for 200 reactions of 25 l each or 400 direct ampli cation reactions of 12 5 l each Includes Pre ampli cation Components Box 4 250 l PowerPlex Fusion 6C 5X Master Mix 4 250 l PowerPlex Fusion 6C 5X Primer Pair Mix 25 l 2800M Control DNA 10ng l 5 1 250 l Water Ampli cation Grade Post ampli cation Components Box 4 25 l PowerPlex Fusion 6C Allelic Ladder Mix 2 200 l WEN Internal Lane Standard 500 The PowerPlex Fusion 6C Allelic Ladder Mix is provided in a separate sealed bag for shipping This component should be moved to the post ampli cation box after opening For the 200 reaction PowerPlex Fusion 6C System Cat DC2720 the Water Ampli cation Grade is provided in a separate sealed bag for shipping Store this component with the pre ampli cation components after opening Storage Conditions Upon receipt store all components at 30 C to 10 C in a nonfrost free freezer Make sure that
42. 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 di erent cycle number 24 27 cycles 4 Following ampli cation 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 Ampli ed 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 ice water bath or a freezer plate block centrifuge compatible with 96 well plates aerosol resistant pipette tips 3500 3500xL capillary array 36cm plate retainer amp base set standard POP 4 polymer for the 3500 or 3500xL anode bu er container cathode bu er 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
43. User GeneAmp PCR System 9700 96 Well with a gold plated or silver plated sample block or Veriti 96 Well Thermal Cycler Applied Biosystems centrifuge compatible with a 96 well plate 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 ampli cation of DNA from storage card punches in 25 l reaction volumes using the PowerPlex Fusion 6C System and GeneAmp PCR System 9700 or Veriti 96 Well Thermal Cycler The PowerPlex Fusion 6C System contains su cient reagents for 100 direct ampli cation reactions of 12 5 l each when performing 25 l reactions the system contains su cient reagents for 50 reactions The PowerPlex Fusion 6C System is compatible with the GeneAmp PCR System 9700 thermal cycler with a silver plated or gold plated sample block and the Veriti 96 Well Thermal Cycler This system has not been tested with the Veriti 96 Well Fast Thermal Cycler or the GeneAmp PCR System 9700 with an aluminum block For 25 l ampli cation reactions with FTA cards we recommend amplifying one or two 1 2mm punches of a storage card containing buccal cells or one 1 2mm punch of a storage card containing whole blood For nonFTA
44. We recommend 10ng of 2800M Control DNA per 12 5 l ampli cation reaction This mass of DNA should be reduced if cycle number is increased and decreased if the cycle number is increased Increase or decrease by twofold the mass of 2800M Control DNA f or every one cycle decrease or increase respectively Improper storage of the 2800M Control DNA Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 55 www promega com TMD045 1 15 Symptoms Causes and Comments Extra peaks visible in one or Swab extract was contaminated Assemble a reaction containing all color channels 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 ampli cation Ampli cation of swab extracts with high DNA concentrations can result in artifact peaks due to overampli cation resulting in saturated signal on the CE instrument We recommend 2 l of swab extract per reaction Using more than 2 l may result in overampli cation and signal saturation If signal is saturated repeat ampli cation with less swab extract or reduced cycle number Ampli cation of excess template for a given cycle number resulted in overloading of the capillary upon electrokinetic injection Excess DNA in the capillary is di cult to maintain in a denatured single st
45. X AmpSolution Reagent Cat DM1231 is required for direct ampli cation of DNA from storage card punches in a 12 5 l reaction with the PowerPlex Fusion 6C System Information about other Promega uorescent STR systems is available upon request from Promega or online at www promega com Ampli cation Setup Instrument Setup and Sample Preparation Thermal Cycling Section 4 Section 4 Section 5 GeneAmp PCR System 9700 or Veriti 96 Well Thermal Cycler Data Analysis Section 6 Applied Biosystems 3500 or 3500xL Genetic Analyzer GeneMapper ID X Software Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 4 0 with the DC v4 6 Dye Module v1 License GeneMarker HID Software Figure 1 An overview of the PowerPlex Fusion 6C System protocol 2 Product Components and Storage Conditions P R O D U C T S I Z E C AT PowerPlex Fusion 6C System 50 or 100 direct amp reac ons DC2705 Not For Medical Diagnostic Use This system contains su cient reagents for 50 reactions of 25 l each or 100 direct ampli cation reactions of 12 5 l each Includes Pre ampli cation Components Box 250 l PowerPlex Fusion 6C 5X Master Mix 250 l PowerPlex Fusion 6C 5X Primer Pair Mix 25 l 2800M Control DNA 10ng l 1 250 l Water Ampli cation Grade Post ampli cation Components Box 25 l PowerPlex Fusion 6C Allelic Ladder
46. action The PunchSolution Kit is used to process punches from nonFTA storage cards containing blood or buccal samples prior to direct ampli cation For casework or samples that require DNA puri cation we recommend the DNA IQ System Cat DC6700 which is a DNA isolation system designed speci cally for forensic samples 26 This system uses paramagnetic particles to prepare clean samples for STR analysis easily and e ciently and can be used to extract DNA from stains or liquid samples such as blood or solutions The DNA IQ System eliminates PCR inhibitors and contaminants frequently encountered in casework samples In additional DNA has been isolated from casework samples such as tissue di erentially separated sexual assault samples and stains on support materials The DNA IQ System has been tested with PowerPlex Systems to ensure a streamlined process For applications requiring human speci c DNA quanti cation the Plexor HY System Cat DC1000 was developed 27 This qPCR based method provides total human and male speci c DNA quanti cation in one reaction Additionally the Plexor HY System provides a post ampli cation melt analysis to con rm positive results and an Internal PCR Control IPC to con rm negative results Additional ordering information is available in Section 11 G For information about automation of Promega chemistries on automated workstations using Identity Automation so
47. aining 23 l of PCR ampli cation mix Note Optimization of the amount of 2800M Control DNA may be required depending on thermal cycling conditions and laboratory preferences 8 For the negative ampli cation control pipet 2 0 l of Water Ampli cation Grade or TE 4 bu er instead of swab extract into a reaction well containing PCR ampli cation mix Note Additional negative controls can be included Assemble a reaction containing the swab extract prepared from a blank swab or assemble a reaction where the SwabSolution Reagent is processed as a blank without a swab Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 71 www promega com TMD045 1 15 9 Seal or cap the plate or close the tubes Optional Brie y centrifuge the plate to bring contents to the bottom of the wells and remove any air bubbles Thermal Cycling Ampli cation 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 26 cycles works well for a variety of sample types Cycle number will need to be optimized in each laboratory for each sample type 1 Place the MicroAmp plate in the thermal cycler 2 Select and run the recommended protocol which is provided
48. arger loci ski slope e ect Use less swab extract or reduce cycle number The cycle number was too high Decrease cycle number by one cycle and repeat the ampli cation Active SwabSolution Reagent carried over from swab extracts into the ampli cation reaction Larger loci are most susceptible to reagent carryover and will drop out before the smaller loci Ensure that the heat block reached 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 to incubate tubes or plates heat transfer is ine cient and will result in poor performance Use only a heat block to maintain e cient 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 uctuate Do not re freeze as this may reduce activity DNA was not accessible on nonlytic material Small loci may amplify preferentially with large loci dropping out Pretreat swabs with SwabSolution Reagent to ensure that DNA is liberated from cellular proteins Extreme variability in sample There can be signi cant individual to individual variability in to sample peak heights cell deposition onto buccal swabs
49. ariant Bins in Allelic Ladder and enter sample name identi ers for ladder positive and negative controls b Navigate to the Display Settings tab Sample Tree section check boxes for Flag Low Quality ILS as SQ and Consider Gender for Flag In the Allele Label section uncheck Flag Variant Alleles in Ladder c Select OK 40 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 7 A Creating an Analysis Method with GeneMarker HID Software Version 2 7 1 continued 9 Click the Run Project icon green arrow in the toolbar and the Template Selection window will appear Select the PowerPlex_6C_Fusion template and the settings shown in Figure 24 Choose the panel that you created in Step 7 from the drop down menu Verify selection of Size Standard ILS_500 and Standard Color WEN Select Next 12811TA Figure 24 The Template Selection window Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 41 www promega com TMD045 1 15 10 The Data Process window will appear Figure 25 Choose settings based on your laboratory s standard operating procedures Select full range or partial range for the analysis range When using a partial range choose an appropriate analysis rang
50. ased 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 work ow 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 di erent cycle number 25 28 cycles 4 Following ampli cation use your laboratory s validated separation and detection protocols to determine the optimal cycle number for the sample type 11 F Composition of Bu ers and Solutions TE 4 bu er 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 nal volume to 1 liter with deionized water TE 4 bu er 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 nal volume to 1 liter with deionized water Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 73 www promega com TMD045 1 15 11 G Related Products STR Systems Product S
51. ats Am J Hum Genet 49 746 56 3 Edwards A et al 1992 Genetic variation at ve trimeric and tetrameric tandem repeat loci in four human population groups Genomics 12 241 53 4 Warne D et al 1991 Tetranucleotide repeat polymorphism at the human actin related pseudogene 2 actbp2 detected using the polymerase chain reaction Nucleic Acids Res 19 6980 5 Ausubel F M et al 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 ampli cation 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 Ampli cation 1989 Erlich H A ed Stockton Press New York NY 8 PCR Protocols A Guide to Methods and Applications 1990 Innis M A et al eds Academic Press San Diego CA 9 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 Identi cation 1992 Promega Corporation 245 69 11 Hartmann J M et al 1991 Guidelines for a quality assurance program for DNA analysis Cri
52. ayed in the Analysis Method Peak Quality and SQ amp GQ Settings tabs are defaults and will a ect the quality values displayed in the plot settings We recommend that you modify the values in these tabs to t your laboratory s data analysis protocols 6 F Controls in GeneMapper ID X Software 1 Observe the results for the negative control Using the protocols de ned in this manual the negative control should be devoid of ampli cation products 2 Observe the results for the 2800M Control DNA The expected 2800M DNA allele designations for each locus are listed in Table 9 Section 11 A 7 Data Analysis Using GeneMarker HID Software Version 2 7 1 The instructions in this section were written using GeneMarker HID software version 2 7 1 Due to potential di erences between individual software versions some of the instructions may not apply to other software versions 7 A Creating an Analysis Method with GeneMarker HID Software Version 2 7 1 These instructions are intended as a guide to start analyzing data in GeneMarker HID Software They are not intended as a comprehensive guide for using GeneMarker HID Software We recommend that users contact SoftGenetics at www softgenetics com for training and technical support for the software Contact SoftGenetics to obtain the current PowerPlex Fusion 6C stutter panels and bins les 1 Open the GeneMarker HID software 2 To access your data les
53. cards we recommend amplifying one 1 2mm punch of a storage card containing buccal cells or whole blood 66 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 11 D Direct Ampli cation of DNA from Storage Card Punches in 25 l Reaction Volume continued 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 swabs transferred to FTA or Indicating FTA cards Liquid blood from collection or storage Vacutainer tubes or nger 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 ampli cation mix For more information see the PunchSolution Kit Technical Manual TMD038 Failure to pretreat these samples may result in incomplete pro les Use a manual punch tool with a 1 2mm tip to manually create sample disks from a storage card Place tip near the
54. center of the sample spot and with a twisting or pressing action cut a 1 2mm sample disk Use the plunger to eject the disk into the appropriate well of a reaction plate Automated punchers also can be used to create sample disks Refer to the user s guide for your instrument for assistance with generating 1 2mm disks technical advice and troubleshooting information Note Static may be problematic when adding a punch to a well For FTA card punches adding PCR ampli cation 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 Ampli cation Setup 1 At the rst use thaw the PowerPlex Fusion 6C 5X Master Mix PowerPlex Fusion 6C 5X Primer Pair Mix and Water Ampli cation Grade completely After the rst use store the reagents at 2 10 C Note Centrifuge tubes brie y 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
55. 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 5 In the Analysis Method column select the analysis method created previously in this section 6 In the Panel column select the panels text le 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 is set to display the Analysis Requirements Summary window and Allelic Ladder Analysis Summary window if an issue is detected After analysis is complete the default setting is to show the Analysis Summary tab If these default settings are changed manual troubleshooting may be necessary 9 If all analysis requirements are met the Save Project window will open Figure 19 Figure 19 The Save Project window 10 Enter the project name 11 Choose the applicable security group from the drop down menu then select OK When the analysis is nished 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 laborator
56. criptive name 9252TA Figure 10 The Create New File Name Convention window Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 23 www promega com TMD045 1 15 f To create a new Results Group Figure 11 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 your laboratory practices Save with a descriptive name 9253TA Figure 11 The Create New Results Group window 24 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 5 A Detection of Ampli ed Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer continued 3 To create a New Plate navigate to the Library and from the Manage menu select Plates then Create 4 Assign a descriptive plate name Select the plate type HID from the drop down menu Figure 12 9254TA Figure 12 De ning plate properties 5 Select Assign Plate Contents Figure 13 9255TA Figure 13 Assigning plate contents Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 25 www promega com TMD045 1
57. ction plate or 0 2ml MicroAmp reaction tubes Applied Biosystems aerosol resistant pipette tips PunchSolution Kit Cat DC9271 for nonFTA card punches 5X AmpSolution Reagent for FTA card punches Cat DM1231 1 2mm Harris Micro Punch or equivalent manual punch and cutting mat or automated punch system This section contains a protocol for direct ampli cation of DNA from storage card punches in 12 5 l reaction volumes using the PowerPlex Fusion 6C System and GeneAmp PCR System 9700 or Veriti 96 Well Thermal Cycler A protocol for direct ampli cation of DNA from storage card punches in 25 l reaction volumes is provided in Section 11 D For 12 5 l ampli cation reactions we recommend amplifying one 1 2mm punch of an FTA or nonFTA storage card containing buccal cells or whole blood FTA based sample types include Buccal cells collected on FTA cards with Whatman EasiCollect or Fitzco Sampact devices Buccal cells collected with swabs transferred to FTA or Indicating FTA cards Liquid blood from collection or storage Vacutainer tubes or nger sticks spotted onto FTA cards NonFTA sample types include Buccal samples on Bode Buccal DNA Collector devices Blood and buccal samples on nonFTA cards e g S amp S 903 Pretreat nonFTA sample types with the PunchSolution Kit Cat DC9271 to lyse nonFTA samples before adding the PCR ampli cation mix For
58. d Sample Preparation and DNA Quanti cation Systems Product 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 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 75 www promega com TMD045 1 15 a Patents Pending b 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 c U S Pat No 6 479 235 Australian Pat No 724531 Canadian Pat No 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 d U S Pat No 6 238 863 European Pat No 1058727 Chinese Pat No ZL99802696 4 Japanese Pat No 4494630 and other patents pending e 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 Gesellscha zur F rderung der Wissenscha en e V Germany f 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
59. d This requires a silver plated or gold plated sample block The ramp speed is set after the thermal cycling run is started When the Select Method Options screen appears select Max for the ramp speed and enter the reaction volume Thermal Cycling Protocol 96 C for 1 minute then 96 C for 5 seconds 60 C for 1 minute for 25 cycles then 60 C for 10 minutes 4 C soak 12 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 4 B Direct Ampli cation of DNA from Storage Card Punches in a 12 5 l Reaction Volume continued 11876MB 96 C 96 C 5 seconds 1 minute 60 C 1 minute 60 C 10 minutes 4 C Optimal cycle number 1 cycle 1 cycle Hold Figure 3 Thermal cycling protocol for the GeneAmp PCR System 9700 and Veriti 96 Well Thermal Cycler 3 After completion of the thermal cycling protocol proceed with fragment analysis or store ampli ed samples at 20 C in a light protected box Note Long term storage of ampli ed 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
60. ds are the minimum peak heights at which the software will call a peak Values for peak amplitude thresholds are usually 50 150RFU on the 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 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 these 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 les Highlight desired les 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 contai
61. e 29 8 Select OK Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 31 www promega com TMD045 1 15 12780TA Figure 16 The GeneMapper ID X Software Version 1 4 Size Standard Editor 6 D Creating a Casework Analysis Method with GeneMapper ID X Software Version 1 4 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 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 Security Group This allows access for all users of the software Other security groups may be used 5 Enter a descriptive name for the analysis method such as PowerPlex Fusion 6C 6 Select the Allele tab Figure 17 32 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 6 D Creating a Casework Analysis Method with GeneMapper ID X Software Version 1 4 continued 7 Select the bins text le that was
62. e DNA template is stored in TE bu er that is not pH 8 0 or contains a higher EDTA concentra tion the volume of DNA added should not exceed 20 of the nal reaction volume PCR ampli cation e ciency 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 di er depending on the DNA quanti cation 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 quanti cation method 5 Vortex the PCR ampli cation mix for 5 10 seconds then pipet PCR ampli cation mix into each reaction well Failure to vortex the PCR ampli cation mix su ciently can result in poor ampli cation or locus to locus imbalance 6 Add the template DNA 1 0ng for each sample to the respective well containing PCR ampli cation mix Note The PowerPlex Fusion 6C System was optimized and balanced using 1 0ng of DNA template The amount of DNA template used in your laboratory should be based on the results of your internal validation and may be di erent 7 For the positive ampli cation control v
63. e based on your data For the allelic call range choose a start point before the rst de ned internal lane standard peak and an end point just beyond the last de ned internal lane standard peak The peak detection threshold is the minimum peak height at which the software will call a peak outside the marker range Select Next 12785TA Figure 25 The Data Process window for an analysis method 42 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 7 A Creating an Analysis Method with GeneMarker HID Software Version 2 7 1 continued 11 The Additional Settings window will appear Select the settings shown in Figure 26 The values displayed in the Allele Evaluation dialogue box are defaults and will a ect the quality values displayed in the plot settings We recommend that you modify the values in these tabs to t your laboratory s data analysis protocols Select OK Note The Auto Select Best Ladder function will analyze each sample le with the passing ladder that most closely matches If this box in not checked select an appropriate ladder in the Allelic Ladder drop down menu 12810TA Figure 26 The Additional Settings window 12 When the analysis is nished the Main Analysis window will appear We recommend that you review any yellow or red agged markers in the Rep
64. e block and the Veriti 96 Well Thermal Cycler This system has not been tested with the Veriti 96 Well Fast Thermal Cycler or the GeneAmp PCR System 9700 with an aluminum block 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 70 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 11 E Direct Ampli cation of DNA from Swabs in a 25 l Reaction Volume continued Ampli cation Setup 1 At the rst use thaw the PowerPlex Fusion 6C 5X Master Mix PowerPlex Fusion 6C 5X Primer Pair Mix and Water Ampli cation Grade completely After the rst use store the reagents at 2 10 C Note Centrifuge tubes brie y 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 ampli cation mix for all samples
65. each use Do not centrifuge after vortexing as this may cause the reagents to be concentrated at the bottom of the tube 2 Prepare a loading cocktail by combining and mixing WEN Internal Lane Standard 500 and Hi Di formamide as follows 0 5 l WEN ILS 500 samples 9 5 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 Adjust the volume added to the wells in Step 4 accordingly Do not use less than 9 5 l of formamide per well 3 Vortex for 10 15 seconds to mix 4 Pipet 10 l of formamide internal lane standard mix into each well Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 27 www promega com TMD045 1 15 5 Add 1 l of ampli ed sample or 1 l of PowerPlex Fusion 6C Allelic Ladder Mix to each well 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 o
66. eagent We recommend treating one 1 2mm nonFTA card punch with 10 l of PunchSolution Reagent and using one punch per 12 5 l ampli cation reaction Reducing the PunchSolution Reagent volume may improve results for reactions with reduced ampli cation volumes Optimization and validation are required Inactive PunchSolution Reagent was used to pretreat nonFTA punches Thaw the PunchSolution Reagent at 2 10 C Do not store reagents in the refrigerator door where the temperature can uctuate Do not refreeze as this may reduce activity Extreme variability in sample There can be signi cant individual to individual variability in the to sample peak heights number of cells on a punch resulting in peak height variability between samples The PunchSolution Kit maximizes the recovery of ampli able DNA from nonFTA punches but does not normalize the amount of DNA present 54 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 9 D Direct Ampli cation of DNA From Swabs The following information is speci c to direct ampli cation of DNA from swabs after pretreatment using the SwabSolution Kit For additional information about general ampli cation and detection see Section 9 A Symptoms Causes and Comments Faint or absent allele peaks Poor sample deposition Shedding and co
67. i cation of Extracted DNA in a 25 l Reaction Volume 6 4 B Direct Ampli cation of DNA from Storage Card Punches in a 12 5 l Reaction Volume 9 4 C Direct Ampli cation of DNA from Swabs in a 12 5 l Reaction Volume 12 5 Instrument Setup and Sample Preparation 15 5 A Detection of Ampli ed Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer 15 5 B Detection of Ampli ed Fragments Using the Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 4 0 and DC v4 6 Dye Module v1 License 26 6 Data Analysis Using GeneMapper ID X Software Version 1 4 28 6 A Importing PowerPlex Fusion 6C Panels Bins and Stutter Text Files into GeneMapper ID X Software Version 1 4 28 6 B Importing the WEN ILS 500 IDX Size Standard into GeneMapper ID X Software Version 1 4 30 6 C Creating a Size Standard with GeneMapper ID
68. ize Cat PowerPlex Fusion System 200 reactions DC2402 800 reactions DC2408 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 Fast System 100 reactions DC1611 400 reactions DC1610 PowerPlex ESX 17 Fast System 100 reactions DC1711 400 reactions DC1710 PowerPlex ESI 16 Fast System 100 reactions DC1621 400 reactions DC1620 PowerPlex ESI 17 Fast System 100 reactions DC1721 400 reactions DC1720 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 PowerPlex 6C Matrix Standard 5 preps DG4900 WEN Internal Lane Standard 500 200 l DG5001 PunchSolution Kit 100 preps DC9271 SwabSolution Kit 100 preps DC8271 5X AmpSolution Reagent 100 preps DM1231 2800M Control DNA 10ng l 25 l DD7101 2800M Control DNA 0 25ng l 500 l DD7251 Water Ampli cation Grade 6 250 l 5 1 250 l DW0991 Not for Medical Diagnostic Use 74 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 11 G Related Products continue
69. ks of Promega Corpora on AmpSolu on DNA IQ Iden ty Automa on PunchSolu on and SwabSolu on are trademarks of Promega Corpora on Applied Biosystems GeneAmp GeneMapper MicroAmp and Veri 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 GeneMarker is a registered trademark of So Gene cs Corpora on Hi Di is a trademark of Applera Corpora on POP 4 is a registered trademark of Life Technologies Corpora on POP 7 is a trademark of Life Technologies Corpora on 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 limita ons Please visit our Web site for more informa on All prices and speci ca ons are subject to change without prior no ce Product claims are subject to change Please contact Promega Technical Services or access the Promega online catalog for the most up to date informa on on Promega products
70. l Y results for Amelogenin Additionally two rapidly mutating Y STR loci are included in the system Table 9 lists the PowerPlex Fusion 6C System alleles ampli ed from commonly available standard DNA templates 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 peaks stutter or shadow peaks is due to the loss of a repeat unit during DNA ampli cation somatic variation within the DNA or both The amount of this artifact observed depends primarily on the locus and the DNA sequence being ampli ed Terminal nucleotide addition 16 17 occurs when a thermostable nonproofeading DNA polymerase adds a nucleotide generally adenine to the 3 ends of ampli ed DNA fragments in a template independent manner The e ciency with which this occurs varies with di erent primer sequences Thus an artifact peak one base shorter than expected i e missing the terminal addition is sometimes seen We have modi ed primer sequences and added a nal extension step at 60 C 18 to the ampli cation protocols to provide conditions for essentially complete terminal nucleotide addition when recommended amounts of template DNA are used Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax
71. llection of donor cells was variable Increase cycle number Inactive SwabSolution Reagent Thaw the SwabSolution Reagent completely in a 37 C water bath and mix by gentle inversion Store the SwabSolution Reagent at 2 10 C Do not store reagents in the refrigerator door where the temperature can uctuate Do not refreeze as this may reduce activity Active SwabSolution Reagent carried over into the ampli cation reaction Ensure that the heat block reached 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 to incubate tubes or plates heat transfer is ine cient and will result in poor performance Use only a heat block to maintain e cient heat transfer We have tested 60 minute incubation times and observed no di erence in performance compared to a 30 minute incubation DNA was not accessible on nonlytic material Pretreat swabs 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 make positive control reaction 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 pro le
72. lutions contact your local Promega Branch O ce or Distributor contact information available at www promega com support worldwide contacts e mail genetic promega com or visit www promega com idautomation Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 65 www promega com TMD045 1 15 11 C The WEN Internal Lane Standard 500 The WEN 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 29 Each fragment is labeled with WEN 6C dye and can be detected separately as a sixth color in the presence of PowerPlex Fusion 6C ampli ed material The WEN ILS 500 is designed for use in each CE injection to increase precision in analyses when using the PowerPlex Fusion 6C System Protocols to prepare and use this internal lane standard are provided in Section 5 12792TA Figure 29 WEN Internal Lane Standard 500 An electropherogram showing the WEN Internal Lane Standard 500 fragments 11 D Direct Ampli cation of DNA from Storage Card Punches in a 25 l Reaction Volume Depending on the procedure used to prepare storage cards high amounts of cells may be present in each punch In these cases performing direct ampli cation in 25 l reaction volumes may provide better results Materials to Be Supplied by the
73. me 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 and characterization of stutter products at the tetranucleotide repeat locus vWA Nucleic Acids Res 24 2807 12 60 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 10 References continued 19 Gri ths 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 h
74. more information see the PunchSolution Kit Technical Manual TMD038 Failure to pretreat these samples may result in incomplete pro les Use a manual punch tool with a 1 2mm tip to manually create sample disks from a storage card Place tip near the center of the sample spot and with a twisting or pressing action cut a 1 2mm sample disk Use the plunger to eject the disk into the appropriate well of a reaction plate Automated punchers also can be used to create sample disks Refer to the user s guide for your instrument for assistance with generating 1 2mm disks technical advice and troubleshooting information Note Static may be problematic when adding a punch to a well For FTA card punches adding PCR ampli cation 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 10 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 4 B Direct Ampli cation of DNA from Storage Card Punches in a 12 5 l Reaction Volume continued Ampli cation Setup 1 At the rst use thaw the PowerPlex Fusion 6C 5X Master Mix PowerPlex Fusion 6C 5X Primer Pair Mix and Water Ampli cation Grade completely After the rst use store the reagen
75. n WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 13 www promega com TMD045 1 15 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 Ampli cation Setup 1 At the rst use thaw the PowerPlex Fusion 6C 5X Master Mix PowerPlex Fusion 6C 5X Primer Pair Mix and Water Ampli cation Grade completely After the rst use store the reagents at 2 10 C Note Centrifuge tubes brie y 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 ampli cation mix for all samples It also ensures that each reaction contains the same PCR ampli cation 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 nal volume of each reagent listed in Table 3
76. n at least one allelic ladder injection that is designated as Allelic Ladder in the Sample Type column for proper genotyping 5 In the Analysis Method column select the analysis method created previously in this section 6 In the Panel column select the panels text le 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 is set to display the Analysis Requirements Summary window and Allelic Ladder Analysis Summary window if an issue is detected After analysis is complete the default setting is to show the Analysis Summary tab If these default settings are changed manual troubleshooting may be necessary 9 If all analysis requirements are met the Save Project window will open Figure 19 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 37 www promega com TMD045 1 15 10 Enter the project name 11 Choose the applicable security group from the drop down menu then select OK When the analysis is nished 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 The values displ
77. n 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 le downloaded in the Getting Started Section Select the le then Import 6 In the navigation pane highlight the PowerPlex Fusion 6C panels folder that you just imported in Step 5 7 Select File then Import Bin Set 8 Navigate to the bins text le downloaded in the Getting Started section Select the le then Import 9 In the navigation pane highlight the PowerPlex 6C 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 11 Navigate to the stutter text le imported in the Getting Started section Select the le then Import 12 In the Panel Manager check the boxes to indicate DYS391 DYS576 and DYS570 are Y markers See Figure 15 This option is not available for older versions of the GeneMapper ID X software 13 At the bottom of the Panel Manager window select OK This will save the panels bins and stutter text les and close the window 12779TA Figure 15 The GeneMapper ID X Software Version
78. nsures that you will have enough PCR ampli cation mix for all samples It also ensures that each reaction contains the same PCR ampli cation 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 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 67 www promega com TMD045 1 15 4 Add the nal volume of each reagent listed in Table 10 to a sterile tube Table 10 PCR Ampli cation Mix for Direct Ampli cation of DNA from Storage Card Punches Using a 25 l Reaction Volume PCR Ampli cation Mix Component1 Volume Per Reaction Number of Reactions Final Volume Water Ampli cation Grade 15 0 l PowerPlex Fusion 6C 5X Master Mix 5 0 l PowerPlex Fusion 6C 5X Primer Pair Mix 5 0 l total reaction volume 25 l 1Add Water Ampli cation Grade to the tube rst then add PowerPlex Fusion 6C 5X Master Mix and PowerPlex Fusion 6C 5X Primer Pair Mix For FTA card punches the template DNA will be added at Step 6 5 Vortex the PCR ampli cation mix for 5 10 seconds then pipet 25 l of PCR ampli cation mix into each reaction well Failure to vortex the PCR ampli cation mix su ciently can result in poor ampli cation or locus to locu
79. o optimize protocols including amount of template DNA cycle number and injection conditions for your laboratory instrumentation In house validation should be performed The PowerPlex Fusion 6C 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 6C 5X Master Mix This manual contains protocols for use of the PowerPlex Fusion 6C System with the GeneAmp PCR System 9 Troubleshooting 47 9 A Ampli cation and Fragment Detection 47 9 B Ampli cation of Extracted DNA 50 9 C Direct Ampli cation of DNA From Storage Card Punches 51 9 D Direct Ampli cation of DNA From Swabs 54 9 E GeneMapper ID X Software 57 10 References 59 11 Appendi
80. ogram must be run with Max Mode as the ramp speed This requires a silver plated or gold plated sample block The ramp speed is set after the thermal cycling run is started When the Select Method Options screen appears select Max for the ramp speed and enter the reaction volume Thermal Cycling Protocol 96 C for 1 minute then 96 C for 5 seconds 60 C for 1 minute for 29 cycles then 60 C for 10 minutes 4 C soak 11876MB 96 C 96 C 5 seconds 1 minute 60 C 1 minute 60 C 10 minutes 4 C Optimal cycle number 1 cycle 1 cycle Hold Figure 2 The thermal cycling protocol for the GeneAmp PCR System 9700 and Veriti 96 Well Thermal Cycler 3 After completion of the thermal cycling protocol proceed with fragment analysis or store ampli ed samples at 20 C in a light protected box Note Long term storage of ampli ed samples at 4 C or higher may produce artifacts Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 9 www promega com TMD045 1 15 4 B Direct Ampli cation of DNA from Storage Card Punches in a 12 5 l Reaction Volume Materials to Be Supplied by the User GeneAmp PCR System 9700 96 Well with a gold plated or silver plated sample block or Veriti 96 Well Thermal Cycler Applied Biosystems centrifuge compatible with a 96 well plate MicroAmp optical 96 well rea
81. olymer 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 Polymer related artifacts This system was developed using POP 4 polymer To use other polymers optimization and in house validation are required The use of POP 7 polymer can change the migration and sizing location of artifacts compared to that with POP 4 polymer 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 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 di erent injection of allelic ladder to determine sizes Poor injection of allelic ladder Include more than one ladder per instrument run Internal size standard was not assigned correctly Evaluate the sizing labels on the WEN ILS 500 and correct if necessary 50 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 9 A Ampli cation and Fragment Detection con
82. ontinued 6 In the spectral viewer select dye set J6 and con rm that the active dye set is the le generated for the PowerPlex 6C dye chemistry It is critical to select the correct J6 spectral for the PowerPlex 6C dye chemistry If the PowerPlex 6C dye chemistry is not the active dye set locate the PowerPlex 6C dye spectral in the List of Calibrations for Dye Set J6 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 ampli ed samples 9 When the plate record is linked to the plate the plate graphic changes from yellow to green and the green Run Instrument arrow will become 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 6 Data Analysis Using GeneMapper ID X Software Version 1 4 The instructions in this section were written using GeneMapper software version 1 4 Due to potential di erences between individual software versions some of the instructions may not apply to other software versions 6 A Importing PowerPlex Fusion 6C Panels Bins and St
83. ort Table window and handle them according to your laboratory s standard operating procedures 7 B Controls in the GeneMarker HID Software 1 Observe the results for the negative control Using the protocols de ned in the manual the negative controls should be devoid of ampli cation products 2 Observe the results for the 2800M Control DNA The expected 2800M allele designations for each locus are listed in Table 9 Section 11 A A le with the correct PowerPlex Fusion 6C pro le for the 2800M Control DNA should be preloaded in the GeneMarker HID software If the 2800M pro le is not preloaded in the GeneMarker HID software you can create a Positive Control Template To do so open Tools in the Main Analysis window then select Positive Control Template Editor In the Positive Control Standards dialogue box select Add Enter a descriptive name for the control such as 2800M_Fusion_6C and select OK In the Import Genotypes from Sample dialogue box select the positive control sample In the Con rm window select Yes In the Positive Control Template Editor select OK Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 43 www promega com TMD045 1 15 8 Results Representative results of the PowerPlex Fusion 6C System are shown in Figure 27 The PowerPlex Fusion 6C Allelic Ladder
84. orter time periods may result in incomplete inactivation of the PunchSolution Reagent We have not tested longer incubation times Inactive PunchSolution Reagent was used to pretreat nonFTA punches Thaw the PunchSolution Reagent at 2 10 C Do not store reagents in the refrigerator door where the temperature can uctuate Do not refreeze as this may reduce activity 52 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 9 C Direct Ampli cation of DNA From Storage Card Punches continued Symptoms Causes and Comments Faint or absent allele peaks Positive control did not amplify Do not include a blank punch in in the positive control reaction the positive control reaction Presence of blank punches may inhibit ampli cation of 2800M Control DNA Extra peaks visible in one Punch was contaminated Take punches from blank paper or all color channels between samples Ampli cation of processed punches with high amounts of DNA can result in artifact peaks due to overampli cation resulting in saturating signal on the CE instrument Be sure to use the recommended number of punches Use of a larger punch size or a smaller reaction volume may result in overampli cation and signal saturation If the signal is saturated repeat the ampli ca tion with a smaller punch a larger reaction volume
85. ortex the tube of 2800M Control DNA then dilute an aliquot to 1 0ng in the desired template DNA volume Add 1 0ng of diluted DNA to a reaction well containing PCR ampli cation mix 8 For the negative ampli cation control pipet Water Ampli cation Grade or TE 4 bu er instead of template DNA into a reaction well containing PCR ampli cation mix 9 Seal or cap the plate or close the tubes Optional Brie y centrifuge the plate to bring contents to the bottom of the wells and remove any air bubbles 8 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 4 A Ampli cation of Extracted DNA in a 25 l Reaction Volume continued Thermal Cycling Ampli cation 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 29 cycles works well for 1 0ng of puri ed DNA templates 1 Place the MicroAmp plate or reaction tubes in the thermal cycler 2 Select and run the recommended protocol which is provided below and in Figure 2 The total cycling time is approximately 1 hour Notes 1 When using the Veriti 96 Well Thermal Cycler set the ramping rate to 100 2 When using the GeneAmp PCR System 9700 the pr
86. own 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 Application drop down list and select the appropriate plate type 96 well Add entries in the owner and operator windows and select OK Note If autoanalysis of sample data is desired refer to the instrument user s manual for instructions 4 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 28 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 5 B Detection of Ampli ed Fragments Using the Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 4 and DC v4 6 Dye Module v1 License c
87. parately and is compatible with the Applied Biosystems 3500 and 3500xL Genetic Analyzers and Applied Biosystems 3130 and 3130xl Genetic Analyzers with Data Collection Software Version 4 0 and DC v4 6 Dye Module v1 License PowerPlex 6C Matrix Standard Cat DG4900 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 5 www promega com TMD045 1 15 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 puri ed DNA or direct ampli cation samples small changes in bu ers ionic strength primer concentra tions reaction volume choice of thermal cycler and thermal cycling conditions can a ect PCR success We suggest strict adherence to recommended procedures for ampli cation and uorescence detection Additional research and validation are required if any modi cations 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 ampli cation reactions and
88. project the project rst must be 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 some loci are gray When an edit is made to a locus the quality ags and marker 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 de ned An insu cient number of WEN ILS 500 fragments was de ned Be sure to de ne at least two WEN ILS 500 fragments smaller than the smallest sample peak and at least two WEN 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 WEN ILS 500 peaks de ned 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 58 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 w
89. protocols 6 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 4 Protocols for DNA Ampli cation Using the PowerPlex Fusion 6C System The PowerPlex Fusion 6C System was developed for ampli cation of extracted DNA and direct ampli cation samples Slight protocol variations are recommended for optimal performance with each template source Protocols for ampli cation 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 ampli cation sections The PowerPlex Fusion 6C System is compatible with the GeneAmp PCR System 9700 thermal cycler with a silver plated or gold plated sample block and the Veriti 96 Well Thermal Cycler This system has not been tested with the Veriti 96 Well Fast Thermal Cycler or GeneAmp PCR System 9700 with an aluminum block The use of gloves and aerosol resistant pipette tips is highly recommended to prevent cross contamination Keep all pre ampli cation and post ampli cation reagents in separate rooms Prepare ampli cation reactions in a room dedicated for reaction setup Use equipment and supplies dedicated for ampli cation setup Meticulous care must be taken to ensure successful ampli cation A guide to ampli cation troubleshooting is provided in Section 9 The concen
90. r 1 cycle 1 cycle Hold Figure 30 Thermal cycling protocol for the GeneAmp PCR System 9700 and Veriti 96 Well Thermal Cycler 3 After completion of the thermal cycling protocol proceed with fragment analysis or store ampli ed samples at 20 C in a light protected box Note Long term storage of ampli ed samples at 4 C or higher may produce artifacts Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 69 www promega com TMD045 1 15 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 you would using your normal work ow 2 Depending on your preferred protocol place one or two 1 2mm FTA storage card punches containing buccal cells one 1 2mm FTA storage card punch containing whole blood or one 1 2mm punch of a nonFTA storage card containing buccal cells or 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
91. r Mix was analyzed using an Applied Biosystems 3500xL Genetic Analyzer and a 1 2kV 24 second injection The sample le was analyzed with the GeneMapper ID X software version 1 4 and PowerPlex Fusion 6C panels and bins text les Panel A The FL 6C labeled allelic ladder components and their allele designations Panel B The JOE 6C labeled allelic ladder components and their allele designations Panel C The TMR 6C labeled allelic ladder components and their allele designations Panel D The CXR 6C labeled allelic ladder components and their allele designa tions Panel E The TOM 6C labeled allelic ladder components and their allele designations Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 45 www promega com TMD045 1 15 Artifacts and Stutter Stutter products are a common ampli cation artifact associated with STR analysis Stutter products often are observed one repeat unit smaller than the true allele peak and occasionally two repeat units smaller or one repeat unit larger than the true allele peak Frequently alleles with a greater number of repeat units will exhibit a higher percentage of 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 di er slightly between primer sets fo
92. r the same loci The mean plus three standard deviations at each locus is used for locus speci c stutter ltering for the PowerPlex Fusion 6C System In addition to stutter peaks DNA dependent artifact peaks Table 4 and DNA independent artifact peaks Table 5 can be observed at some PowerPlex Fusion 6C System loci Table 4 DNA Dependent Artifacts Observed with the PowerPlex Fusion 6C System Locus Artifact Size Amelogenin n 1 D1S1656 n 1 n 2 D13S317 n 2 D18S51 n 2 vWA n 2 elevated baseline in the locus D7S820 n 2 D5S818 n 2 D19S433 n 2 SE33 n 2 DYS391 n 1 FGA n 1 n 2 Table 5 DNA Independent Artifacts Observed with the PowerPlex Fusion 6C System Dye Label Artifact Size1 FL 6C 65 75bp 113 120bp 137 145bp JOE 6C 60 66bp TMR 6C 57 62bp 1Artifact sizes may vary depending on CE instrumentation and environmental conditions in the laboratory 46 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 8 Results continued Testing was performed with a variety of nonhuman DNA templates from bacteria yeast mammals and primates to characterize known artifacts with the PowerPlex Fusion 6C System The artifacts listed in Table 6 were noted above the 175RFU threshold with 10ng of template DNA using an
93. randed state Some single stranded DNA renatures and becomes double stranded Double stranded DNA migrates faster than single stranded DNA during capillary electrophoresis and appears as shadow peaks migrating in front of the main peaks If this occurs at a heterozygous locus it is possible to observe the presence of two shadow peaks that di er in size by approximately the same distance as the single stranded alleles Artifacts of STR ampli cation Ampli cation 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 10 minute extension step at 60 C after thermal cycling Section 4 11 D or 11 E Use 2 l of swab extract in a PowerPlex Fusion 6C 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 nal extension time 56 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 9 D Direct Ampli cation of DNA From Swabs continued Symptoms Causes and Comments Peak height imbalance Excess DNA in the ampli cation 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 l
94. range channel may be required for proper sizing 6 Centrifuge plate brie y to remove air bubbles from the wells 7 Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or a freezer plate block or in an ice water bath for 3 minutes Denature samples just prior to loading the instrument Instrument Preparation 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 Applied Biosystems 3130 or 3130xl Genetic Analyzer with the following exceptions 1 In the Module Manager select New Select Regular in the Type drop down list and select HIDFragmentAnalysis36_POP4 in the Template drop down list Con rm 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 J6 in the dye set drop d
95. 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 ampli cation mix for all samples It also ensures that each reaction contains the same PCR ampli cation 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 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 7 www promega com TMD045 1 15 4 Add the nal volume of each reagent listed in Table 1 to a sterile tube Table 1 PCR Ampli cation Mix for Ampli cation of Extracted DNA PCR Ampli cation Mix Component1 Volume Per Reaction Number of Reactions Final Volume Water Ampli cation Grade to a nal volume of 25 0 l PowerPlex Fusion 6C 5X Master Mix 5 0 l PowerPlex Fusion 6C 5X Primer Pair Mix 5 0 l template DNA 1 0ng 2 3 up to 15 l total reaction volume 25 l 1Add Water Ampli cation Grade to the tube rst then add PowerPlex Fusion 6C 5X Master Mix and PowerPlex Fusion 6C 5X Primer Pair Mix The template DNA will be added at Step 6 2Store DNA templates in TE 4 bu er 10mM Tris HCl pH 8 0 0 1mM EDTA or TE 4 bu er with 20 g ml glycogen If th
96. rs that may be present in forensic samples can lead to allele dropout or imbalance Imbalance may be seen more often when using the maximum template volume or a reduced ampli cation reaction volume 9 C Direct Ampli cation of DNA From Storage Card Punches The following information is speci c to direct ampli cation of DNA from storage card punches For additional information about general ampli cation and detection see Section 9 A Symptoms Causes and Comments Faint or absent allele peaks AmpSolution Reagent was not included in 12 5 l reactions Be sure to include 5X AmpSolution Reagent DNA was not accessible on nonlytic material Pretreat nonFTA materials with PunchSolution Reagent to ensure that DNA is liberated from cellular proteins Poor sample deposition Shedding and collection of donor cells was variable Increase cycle number Poor sample transfer to storage card or variable sampling from storage card Take punches from a di erent portion of the card Increasing cycle number can improve low peak heights Too much sample in the reaction Be sure to use the recommended number of punches Follow the manufacturer s recommendations when depositing sample onto the storage card Active PunchSolution Reagent carried over into ampli cation reactions with nonFTA card punches Ensure that the heat block reached 70 C and samples were incubated for 30 minutes or until wells are dry Incubation for sh
97. s imbalance 6 For FTA storage cards add one or two 1 2mm punches from a card containing buccal cells 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 ampli cation mix to the PunchSolution Reagent treated punch Note It also is acceptable to add the FTA card punch rst then add the PCR ampli cation mix 7 For the positive ampli cation control vortex the tube of 2800M Control DNA then add 1 l 10ng to a reaction well containing 25 l of PCR ampli cation mix Notes 1 Do not include blank storage card punches in the positive control reactions 2 Optimization of the amount of 2800M Control DNA may be required depending on cycling conditions and laboratory preferences 8 Reserve a well containing PCR ampli cation mix as a negative ampli cation 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 or cap the plate or close the tubes Brie y centrifuge the plate to bring storage card punches to the bottom of the wells and remove air bubbles 68 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 11 D Direct Ampli cation of DNA from Storage Card Punches in 25 l Reaction Volume
98. se chain reaction 5 9 Alleles of STR loci are di erentiated by the number of copies of the repeat sequence contained within the ampli ed region and are distinguished from one another using uorescence detection following electrophoretic separation The PowerPlex Fusion 6C System a h is a 27 locus multiplex for human identi cation applications including forensic analysis relationship testing and research use This six color system allows co ampli cation and uorescent detection of the 18 autosomal loci in the expanded CODIS core loci CSF1PO FGA TH01 vWA D1S1656 D2S1338 D2S441 D3S1358 D5S818 D7S820 D8S1179 D10S1248 D12S391 D13S317 D16S539 D18S51 D19S433 and D21S11 as well as Amelogenin and DYS391 for gender determination The Penta D Penta E D22S1045 TPOX and SE33 loci are also included to increase discrimination and allow searching of databases that include pro les with these loci Finally two rapidly mutating Y STR loci DYS570 and DYS576 are included in the multiplex This extended panel of STR markers is intended to satisfy both CODIS and ESS recommendations The PowerPlex Fusion 6C System is compatible with the Applied Biosystems 3500 and 3500xL Genetic Analyzers as well as Applied Biosystems 3130 and 3130xl Genetic Analyzers with Data Collection Software Version 4 0 with the DC v4 6 Dye Module v1 License Life Technologies Ampli cation and detection instrumentation may vary You may need t
99. tinued Symptoms Causes and Comments Peak height imbalance Miscellaneous balance problems At the rst use thaw the 5X Primer Pair Mix and 5X Master Mix completely Vortex the 5X Primer Pair Mix and 5X Master Mix for 15 seconds before use do not centrifuge the 5X Primer Pair Mix or 5X Master Mix after mixing Calibrate thermal cyclers and pipettes routinely PCR ampli cation mix prepared in Section 4 11 D or 11 E was not mixed well Vortex the PCR ampli cation mix for 5 10 seconds before dispensing into the reaction tubes or plate 9 B Ampli cation of Extracted DNA The following information is speci c to ampli cation of extracted DNA For information about general ampli cation and detection see Section 9 A Symptoms Causes and Comments Faint or absent allele peaks Impure template 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 Faint or absent peaks may be seen more often when using the maximum template volume or reduced ampli cation reaction volume Insu cient template Use the recommended amount of template DNA if available High salt concentration or altered pH If the DNA template is stored in TE bu er 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
100. to the PCR ampli cation mix An air bubble formed at the bottom of the reaction tube Use a pipette to remove the air bubble or centrifuge the reactions brie y before thermal cycling Thermal cycler plate or tube problems Review the thermal cycling protocol in Section 4 11 D or 11 E We have not tested other reaction tubes plates or thermal cyclers Calibrate the thermal cycler heating block if necessary Primer concentration was too low Use the recommended primer concentration Vortex the PowerPlex Fusion 6C 5X Primer Pair Mix for 15 seconds before use Poor capillary electrophoresis injection WEN ILS 500 peaks also a ected Re inject the sample Check the laser power Samples were not denatured completely Heat denature samples for the recommended time then cool on crushed ice or a freezer plate block 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 Faint or absent allele peaks Improper storage of the 2800M Control DNA for the positive control reaction 48 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 9 A Ampli cation and Fragment Detection continued Symptoms Ca
101. tration of 2800M Control DNA was determined by measuring absorbance at 260nm Quanti cation of this control DNA by other methods such as qPCR may result in a di erent value Prepare a fresh DNA dilution for each set of ampli cations Do not store diluted DNA e g 0 25ng l or less 4 A Ampli cation of Extracted DNA in a 25 l Reaction Volume Materials to Be Supplied by the User GeneAmp PCR System 9700 96 Well with a gold plated or silver plated sample block or Veriti 96 Well Thermal Cycler Applied Biosystems centrifuge compatible with a 96 well plate MicroAmp optical 96 well reaction plate or 0 2ml MicroAmp reaction tubes Applied Biosystems aerosol resistant pipette tips We routinely amplify 1 0ng of template DNA in a 25 l reaction volume using the protocol detailed below Ampli cation Setup 1 At the rst use thaw the PowerPlex Fusion 6C 5X Master Mix PowerPlex Fusion 6C 5X Primer Pair Mix and Water Ampli cation Grade completely After the rst use store the reagents at 2 10 C Note Centrifuge tubes brie y 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
102. ts at 2 10 C Note Centrifuge tubes brie y 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 ampli cation mix for all samples It also ensures that each reaction contains the same PCR ampli cation 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 nal volume of each reagent listed in Table 2 to a sterile tube Table 2 PCR Ampli cation Mix for Direct Ampli cation of DNA from Storage Card Punches Using a 12 5 l Reaction Volume PCR Ampli cation Mix Component1 Volume Per Reaction Number of Reactions Final Volume Water Ampli cation Grade 5 0 l PowerPlex Fusion 6C 5X Master Mix 2 5 l PowerPlex Fusion 6C 5X Primer Pair Mix 2 5 l 5X AmpSolution Reagent 2 5 l total reaction volume 12 5 l 1Add Water Ampli cation Grade to the tube
103. uman 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 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 Pro ling 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 26 Mandrekar P V Krenke B E and Tereba A 2001 DNA IQ The intelligent way to purify DNA Pro les in DNA 4 3 16 27 Krenke B E et al 2005 Development of a novel uorescent two primer approach to quantitative PCR Pro les in DNA 8 1 3 5 11 Appendix 11 A Advantages of Using the Loci in the PowerPlex Fusion 6C System A single PowerPlex Fusion 6C System reaction ampli es all core loci required for US expanded CODIS and European databases Tables 7 and 8 The male speci c DYS391 locus is included to identify nul
104. und 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 de ned using the rst possible 5 nucleotide of a repeat motif and 2 for STR loci not associated with a coding gene the rst database entry or original literature description shall be used 3Amelogenin is not an STR but displays an 89 base X speci c band and a 95 base Y speci c band NA Not applicable 62 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com Table 8 The PowerPlex Fusion 6C System Allelic Ladder Information STR Locus Label Size Range of Allelic Ladder Components1 2 bases Repeat Numbers of Allelic Ladder Components3 Amelogenin FL 6C 89 95 X Y D3S1358 FL 6C 103 147 9 20 D1S1656 FL 6C 161 208 9 14 14 3 15 15 3 16 16 3 17 17 3 18 18 3 19 19 3 20 3 D2S441 FL 6C 216 252 8 11 11 3 12 17 D10S1248 FL 6C 260 284 8 19 D13S317 FL 6C 308 358 5 17 Penta E FL 6C 371 471 5 25 D16S539 JOE 6C 84 132 4 16 D18S51 JOE 6C 134 214 7 10 10 2 11 13 13 2 14 27 D2S1338 JOE 6C 224 296 10
105. uses and Comments Extra peaks visible in one Contamination with another template DNA or previously or all color channels ampli ed 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 a freezer plate block 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 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 ampli cation Ampli cation 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 10 minute extension step at 60 C after thermal cycling Section 4 11 D or 11 E Decrease the amount of template DNA Using more than the recommended amount of template DNA can result in incomplete adenylation Decrease cycle number Increase the nal extension time CE related artifacts spikes
106. utter Text Files into GeneMapper ID X Software Version 1 4 To facilitate analysis of data generated with the PowerPlex Fusion 6C System we have created panels and bins text les 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 les 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 les and WEN_ILS_500_IDX xml le for the PowerPlex Fusion 6C System go to www promega com resources tools genemapper id software panels and bin sets 2 Select the PowerPlex System that you are using and select GeneMapper ID X Enter your contact information and select Submit Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 29 www promega com TMD045 1 15 3 Save the PowerPlex_Fusion_6C_Panels_IDX_vX x txt PowerPlex_Fusion_6C_Bins_IDX_vX x txt and PowerPlex_Fusion_6C_Stutter_IDX_vX x txt les where X x refers to the most recent version of the panels bins and stutter text les to a known location on your computer 4 Save the WEN_ILS_500_IDX xml le to a known location o
107. would using your normal work ow 2 Place one 1 2mm storage card punch containing buccal cells or 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 di erent cycle number 23 26 cycles 5 Following ampli cation use your laboratory s validated separation and detection protocols to determine the optimal cycle number for the sample type 4 C Direct Ampli cation of DNA from Swabs in a 12 5 l Reaction Volume Materials to Be Supplied by the User GeneAmp PCR System 9700 96 Well with a gold plated or silver plated sample block or Veriti 96 Well Thermal Cycler Applied Biosystems centrifuge compatible with a 96 well plate 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 in 12 5 l reaction volumes using the PowerPlex Fusion 6C System and GeneAmp PCR System 9700 or Veriti 96 Well Thermal Cycler A protocol for direct ampli ca tion of DNA from swabs in 25 l reaction volumes is provided in Section 11 E Promega Corpora on 2800 Woods Hollow Road Madiso
108. ww promega com 9 E GeneMapper ID X Software continued Symptoms Causes and Comments O ladder alleles An allelic ladder from a di erent 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 Panels text le selected for analysis was incorrect for the STR system used Assign correct panels text le that corresponds to the STR system used for ampli cation The allelic ladder was not identi ed as an allelic ladder in the Sample Type column The internal lane standard was not properly identi ed in the sample Manually rede ne the sizes of the size standard fragments in the sample A low quality allelic ladder was used during analysis Ensure that only high quality allelic ladders are used for analysis Incorrect polymer was used Use of a polymer other than POP 4 polymer may change migration of the fragments Alleles may migrate outside of the panel range established using POP 4 polymer Size standard not called correctly Starting data point was incorrect for the partial range chosen in Section 6 D or 6 E Adjust the starting data point in the analysis method Alternatively use a full range for the analysis Extra
109. x 60 11 A Advantages of Using the Loci in the PowerPlex Fusion 6C System 60 11 B DNA Extraction and Quantitation Methods and Automation Support 64 11 C The WEN Internal Lane Standard 500 65 11 D Direct Ampli cation of DNA from Storage Card Punches in a 25 l Reaction Volume 65 11 E Direct Ampli cation of DNA from Swabs in a 25 l Reaction Volume 69 11 F Composition of Bu ers and Solutions 72 11 G Related Products 73 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 3 www promega com TMD045 1 15 9700 thermal cycler and Veriti 96 Well Thermal Cycler in addition to protocols to separate ampli ed products and detect separated material Figure 1 A protocol to operate the uorescence detection instrument should be obtained from the instrument manufacturer The 5
110. y standard operating procedures The values displayed in the Analysis Method Peak Quality and SQ amp GQ Settings tabs are defaults and will a ect the quality values displayed in the plot settings We recommend that you modify the values in these tabs to t your laboratory s data analysis protocols Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 35 www promega com TMD045 1 15 6 E Creating a Databasing or Paternity Analysis Method with GeneMapper ID X Software Version 1 4 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 Security Group This allows access for all users of the software Other security groups may be used 5 Enter a descriptive name for the analysis method such as PowerPlex Fusion 6C 20 Filter 6 Select the Allele tab Figure 20 7 Select the bins text le that was imported in Section 6 A 8 Ensure that the Use marker
111. zers 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 b 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 a descriptive name such as WEN ILS 500 Choose Orange as the Dye Color The fragments in the size standard 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 7 12776TA Figure 7 The Create New Size Standard window 20 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD045 1 15 www promega com 5 A Detection of Ampli ed Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer continued c 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 such as WEN ILS 500 Select the size standard created in Step 2 b The settings for the QC protocol should be based on the internally validated conditions for the PowerPlex Fusion 6C System on the Applied Biosystems 3500 or 3500xL Genetic

PowerPlex® Fusion 6C System

Contents

Download Pdf Manuals

Related Search

Related Contents

PowerPlex&reg; Fusion 6C System

Contents

Download Pdf Manuals

Related Search

Related Contents

PowerPlex® Fusion 6C System