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PowerPlex ESX 17 System Technical Manual, TMD024

1. 10 0 1 Hi Di formamide x samples Note The volume of internal lane standard used in the loading cocktail can be increased or decreased to adjust the intensity of the size standard peaks Use a volume of CC5 ILS 500 that results in peak heights that are all consistently above the peak amplitude threshold of the orange dye channel determined as part of your internal validation Keep the volume of formamide at 10 0ul and adjust the volume added to the wells in Step 4 accordingly 3 Vortex for 10 15 seconds to mix 4 Pipet 111 of formamide internal lane standard mix into each well 5 Add 1 l of amplified sample or 111 of PowerPlex ESX 17 Allelic Ladder Mix Cover wells with appropriate septa Notes 1 Instrument detection limits vary therefore injection time injection voltage or the amount of product mixed with loading cocktail may need to be adjusted Use the Module Manager in the data collection software to modify the injection time or voltage in the run module see Instrument Preparation below If the injection time or voltage is reduced a decreased peak amplitude threshold for the orange channel may be required for proper sizing 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 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Pho
2. 3 Determine the number of reactions to be set up This should include positive and negative control reactions Add 1 or 2 reactions to this number to compensate for pipetting error While this approach does consume a small amount of each reagent it ensures that you will have enough PCR amplification mix for all samples It also ensures that each reaction contains the same PCR amplification mix 4 Usea clean MicroAmp plate for reaction assembly and label appropriately 5 Add the final volume of each reagent listed in Table 2 to a sterile tube Table 2 PCR Amplification Mix for Direct Amplification of DNA from Storage Card Punches PCR Amplification Mix Volume x Numberof _ Final Component Per Reaction Reactions Volume Water Amplification Grade 12 5ul x PowerPlex ESX 5X Master Mix 5 0ul x PowerPlex ESX 17 10X Primer Pair Mix 2 5ul x 5X AmpSolution Reagent 5 0p1 x total reaction volume 25ul 1Add Water Amplification Grade to the tube first then add PowerPlex ESX 5X Master Mix PowerPlex ESX 17 10X Primer Pair Mix and 5X AmpSolution Reagent For FTA card punches the template DNA will be added at Step 6 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 11 C 4 B Direct Amplification of DNA from Storage Card Punches conti
3. At the bottom of the Panel Manager window select OK This will save the panels bins and stutter text files and close the window Importing the CC5 ILS 500 Size Standard into GeneMapper ID X Software Version 1 2 There are two options when creating a size standard Use this protocol or the alternative protocol in Section 6 C The CC5_ILS_500_IDX xml file is available for download at www promega com resources tools genemapper id software panels and bin sets Save the CC5_ILS_500_IDX xml file to a known location on your computer Ha Go a Select Tools then GeneMapper ID X Manager Select the Size Standard tab Select Import Navigate to the location of the CC5_ILS_500_IDX xml file on your computer Highlight the file then select Import Select Done to save changes and close the GeneMapper ID X Manager Creating a Size Standard with GeneMapper ID X Software Version 1 2 He 192 hs e Select Tools then GeneMapper ID X Manager Select the Size Standard tab Select New In the Size Standard Editor window Figure 12 select GeneMapper ID X Security Group as the Security Group This allows access for all users of the software Other security groups may be used Enter a detailed name such as CC5_ILS_500_IDX Choose Orange for the Size Standard Dye Enter the sizes of the internal lane standard fragments 60 65 80 100 120 140 160 1
4. Note Centrifuge tubes briefly to bring contents to the bottom then vortex reagents for 15 seconds before each use Do not centrifuge the 10X Primer Pair Mix or 5X Master Mix after vortexing as this may cause the reagents to be concentrated at the bottom of the tube Vortex the 5X AmpSolution Reagent for 10 15 seconds Note The 5X AmpSolution Reagent should be thawed completely mixed by vortexing and stored at 2 10 C The reagent may be turbid after thawing or storage at 4 C If this occurs warm the buffer briefly at 37 C then vortex until clear Do not store reagents in the refrigerator door where the temperature can fluctuate Storing reagents in the refrigerator door can compromise stability Determine the number of reactions to be set up This should include positive and negative control reactions Add 1 or 2 reactions to this number to compensate for pipetting error While this approach does consume a small amount of each reagent it ensures that you will have enough PCR amplification mix for all samples It also ensures that each reaction contains the same PCR amplification mix Use a clean MicroAmp plate for reaction assembly and label appropriately Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Page 14 Printed in USA Revised 6 14 5 Add the final volume of each reagent listed
5. threshold of 175RFU when using the default injection conditions C However individual laboratories should determine their peak o amplitude thresholds from internal validation studies 3 Peak heights for the CC5 ILS 500 are generally lower than those for the other dyes Therefore the threshold for the orange dye may be lower than that for the other dyes Be sure that all CC5 ILS 500 peaks are consistently above the peak amplitude threshold for the orange dye channel determined as part of your internal validation 4 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 files Highlight desired files then select Add to list followed by Add 4 In the Sample Type column use the drop down menu to select Allelic Ladder Sample Positive Control or Negative Control as appropriate for the sample Every f
6. Instrument Protocol then select Create Alternatively a previously created Instrument Protocol may be used Figure 3 shows the settings used at Promega for the Applied Biosystems 3500xL Genetic Analyzer for the application type dye set capillary length polymer run module and appropriate protocol information The only setting that was changed from the default settings is dye set boiei EM POPI Pemmegettt jtm 2a behed pe een A Rite tep Gems it btn aep nuts a Cae tee ne ue bba mee oo oe ee Pate bate oe Am ee pee Itema ees tet te emnat ts bee Maman map tape et wapeane a weap hp beessi A Pea Reed tne teeme 7e wsanihami ihe Teme eee te a eT et eee aae Pen een Peete Montane tte 14 Lae 9393TA Figure 3 Create New Instrument Protocol window When creating an Instrument Protocol be sure to select the same dye set that was used to perform the Promega 5 dye spectral calibration We recommend using a run time of 1 210 seconds and the default injection conditions Run time and other instrument settings should be optimized and validated in your laboratory Assign a descriptive protocol name Note For more detailed information refer to the Applied Biosystems 3500 3500xL Genetic Analyzers User Guide Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Printed i
7. A low quality allelic ladder was used during analysis Ensure that only high quality allelic ladders are used for analysis Incorrect polymer 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 E Adjust the starting data point in the analysis method Alternatively use a full range for the analysis Extra peaks in size standard Open the Size Match Editor Highlight the extra peak select Edit and select delete size label Select auto adjust sizes Run was too short and larger peaks in ILS were not captured Not all CC5 ILS 500 peaks defined in the size standard were detected during the run e Create a new size standard using the internal lane standard fragments present in the sample Re run samples using a longer run time 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 CC5 ILS 500 used in Section 5 If peaks are low quality redefine the size standard for the sample to skip these peaks Significantly raised baseline e Poor spectral calibration for the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosy
8. FTA based sample types include Buccal cells collected on FTA cards with Whatman EasiCollect or Fitzco Sampact devices one or two punches per 25ul amplification reaction e Buccal cells collected with sterile swabs transferred to FTA or Indicating FTA cards one or two punches per 25l amplification reaction Liquid blood from collection or storage Vacutainer tubes or finger sticks spotted onto FTA cards one punch per 25ul amplification reaction NonFTA sample types include Buccal samples on Bode Buccal DNA Collector devices one punch per 25ul amplification reaction e Blood and buccal samples on nonFTA card punches e g S amp S 903 one punch per 25y amplification reaction Pretreat nonFTA sample types with the PunchSolution Kit Cat DC9271 to lyse nonFTA samples before adding the amplification mix For more information see the PunchSolution Kit Technical Manual TMD038 Failure to pretreat these samples may result in incomplete profiles Use a manual punch tool with a 1 2mm tip to manually create sample disks from a storage card Place tip near the center of the sample spot and with a twisting or pressing action cut a 1 2mm sample disk Use the plunger to eject the disk into the appropriate well of a reaction plate Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com P
9. type 5 Instrument Setup and Sample Preparation 5 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer Materials to Be Supplied by the User 95 C dry heating block water bath or thermal cycler crushed ice or ice water bath centrifuge compatible with 96 well plates aerosol resistant pipette tips 3500 3500xL capillary array 36cm 96 well retainer amp base set standard Applied Biosystems Cat 4410228 POP 4 polymer in a pouch for the Applied Biosystems 3500or 3500xL Genetic Analyzer anode buffer container cathode buffer container MicroAmp optical 96 well plate or equivalent and septa 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 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phon
10. 2 assistens 43 H Creating a Size Standard with GeneMapper ID Software Version 3 2 44 I Creating a Casework Analysis Method with GeneMapper ID Software Version 32siri 45 J Creating a Databasing or Paternity Analysis Method with GeneMapper ID Software Version 3 2 sssssssssecssssssescsssssueeessssssseseesssn 48 K Sample Analysis Using the GeneScan Software and Windows Operating Systems ssssseecsessssssseeeessssssseeesssssssnseeesssssseeeeessssy 50 L Sample Analysis Using the Genotyper Software and PowerTyper ESX 17 Magro niina 52 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 1 Mi Cmts aninion E iiiad 54 N RESUS sssssscssscsessissssssssssssssssssssesssssssasssacccsssaasssczescssaaasacssscisasssastapssnnsbassensnessbosasnesccccssaisen 55 Trot bles hooting sco ciascssssisznsvsssissssensssnscnssnacsubsvsauvssaccussaasaaistutssssssisssssnsvsssansssesvsssainsteangeccvaaen A Amplification and Fragment Detection Pe B Direct Amplification of DNA from Storage Card Punches 62 C Amplification of DNA from Swabs sssri ssassn sienas 64 D GeneMapper ID X Software a E GeneMapper ID SoftWare sisipin s EEr israr sr ra isisisi F PowerTyper ESX 17 Macro References eceania GE RRN UARAN 72 APPOMAn a scccissiiscssscocsssssssssszs
11. 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Printed in USA Page 36 Revised 6 14 Analysis Method Editor f C 4 Gormal Abele Fes Detector Peak Quality SQ GQ Settings Hanger Peak Detection Andysa Sig Pex Amplitude Thresholde Ful Range Y Puta Soe v e so re 0 art Soe 60 pog Stop Sere 500 2 P00 Y so so Mn Peak Malf With Polynomial Degree Pook Window Soe Sope Theeshok 1 Peak Start Peak End Smocthing and Besig Smoothing Ore Normainwter Z Use Normatnston applicable Eactory Defmas 8259TA Figure 14 The GeneMapper ID X Peak Detector tab Notes 1 Select full range or partial range for the analysis range When using a partial range choose an appropriate analysis range based on your data Choose a start point after the primer peak and just before the first defined internal lane standard peak to help ensure proper sizing of the internal lane standard 2 The peak amplitude thresholds are the minimum peak heights at which the software will call a peak Values for peak amplitude thresholds are usually 50 150RFU for data generated on the ABI PRISM 310 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130x Genetic Analyzers For the Applied Biosystems 3500 and 3500xL Genetic Analyzers Life Technologies suggests an analysis threshold of 175RFU when using the de
12. PowerPlex ESX panels folder that you just imported in Step 5 7 Select File then Import Bin Set 8 Navigate to the bins text file downloaded in the Getting Started section above Select the file then Import 9 At the bottom of the Panel Manager window select OK The Panel Manager window will close automatically 6 G Importing the CC5 ILS 500 Size Standard into GeneMapper ID Software Version 3 2 There are two options when creating a size standard Use this protocol or the alternative protocol in Section 6 H The CC5_ILS_500 xml file is available for download at www promega com resources tools genemapper id software panels and bin sets Save the CC5_ILS_500 xml file to a known location on your computer Select Tools then GeneMapper Manager Select the Size Standard tab Select Import Browse to the location of the CC5_ILS_500 xml file Highlight the file then select Import Sy Pi ae Se B me Select Done to save changes and exit the GeneMapper Manager Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 43 O 6 H Creating a Size Standard with GeneMapper ID Software Version 3 2 3 1 Select Tools then GeneMapper Manager 2 Select the Size Standard tab 3 Select New 4
13. The PowerTable option allows up to four alleles per sample file Additional information such as low peak signal or high peak signal is also included The Allele Table and CODIS Table options include only two alleles per locus If more than two alleles are present at a locus the smallest alleles identified are included The Allele Table format displays the categories loci in columns while the CODIS table format displays the categories in rows These tables can be customized to fit needs To save data in tables go to the Table drop down menu highlight Export to File and save the file with the desired name and location The saved file can be viewed and analyzed using Microsoft Excel Save the analyzed data Go to the File menu and select Save as The PowerTyper ESX 17 Macro is a Genotyper file and can be overwritten if Save is used instead of Save as PowerTable Format Sample Sample Info Comment Category 1 2 3 4 flow Signal tion Label Row Peak Peak Peak Peak Over Low Satura Edited Edited Allele Table Format Sample Category Category Category Category Category Category Category Category Info Allele1 Allele2 Allele1 Allele2 Allele1 Allele2 Allele1 Allele 2 CODIS Table Format Sample Info Category Peak 1 Peak 2 1 6 M Controls Observe the results for the negative co
14. 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 2 Ensure that the Consumables Information and Maintenance Notifications are acceptable Set the oven temperature to 60 C then select Start Pre Heat at least 30 minutes prior to the first injection to preheat the oven dewey Mus T iee e Mee Lang tee oam oh ee can POPE Petpmee ABE node EC atadni Wam Ji cap Anay acest ee gt gt umna neno bonr fhe oa _ ett ye tnes de ow Dabe She atapas Tues airo in da a fow Ce Derestan et Temperate CO a a rn dt tet ete tee Q Nene j gt a Pe mbono iqomba anness Pet thenter tae Kes sap t remun me umo x ene ee a De ey Ae he LLY paie sonnet ene ee OK thans hem hiet ea me Oqir eee hse bg temeeg th enn OT taae tte ce v Peete Nett ee Das os 9247TA Figure 2 The Dashboard Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 19 C 5 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or o 3500xL Genetic Analyzer continued Promega 2 To create a new Instrument Protocol navigate to the Library select
15. method such as PowerPlexESX 17 6 Select the Allele tab Figure 19 7 Select the bins text file that was imported in Section 6 F Analysis Method Editor HID Generali Aldie Peak Octector Peat Quality Guilty Pipe BnS PowerPiex_ES Bins v2 0 F Use marber specific stutter ratio if avaiable Marker Repeat Type Tero O4 otf Vake 09 00 Mrut Kato 0o oo Mousa Distance 00 00 oo oo Mrus Studer Fino 00 00 Mirus Ser Distance 23 3 25 375 an Pius Stutter Ratio 02 00 Pass Shutter Distance 225 00 375 00 Co Cee 8123TA Figure 19 The GeneMapper ID Allele tab Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 45 C 6I Creating a Casework Analysis Method with GeneMapper ID Software A Version 3 2 continued Promega 8 Ensure that the Use marker specific stutter ratio if available box is checked 9 Enter the values shown in Figure 19 for proper filtering of stutter peaks when using the PowerPlex ESX 17 System For an explanation of the proper usage and effects of these settings refer to the Applied Biosystems user bulletin titled Installation Procedures and New Features for GeneMapper ID Software 3 2 Note Some of these settings have been optimized and are different from the recommended settings in the user bulletin 1
16. of the panels bins and stutter text files to a known location on your computer Importing Panels Bins and Stutter Text Files 1 Open the GeneMapper ID X software Select Tools then Panel Manager Highlight the Panel Manager icon in the upper left navigation pane Select File then Import Panels pr W GS hf Navigate to the panels file downloaded in the Getting Started Section Select the file then Import 6 In the navigation pane highlight the PowerPlex ESX panels folder that you just imported in Step 5 7 Select File then Import Bin Set 8 Navigate to the bins text file downloaded in the Getting Started Section Select the file then Import 9 In the navigation pane highlight the PowerPlex ESX 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 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 33 6 A Importing PowerPlex ESX Panels Bins and Stutter Text Files into GeneMapper ID X Software Version 1 2 continued 6 B 6 C 11 12 Navigate to the stutter file downloaded in the Getting Started Section Select the file then Import
17. s technology or intellectual property other than expressly provided herein End Users may not use sequence s in an attempt to reverse engineer parameters of any of GE Healthcare Bio Sciences Corp proprietary products or services Disclaimer of Warranties GE Healthcare Bio Sciences Corp provides no warranties to end user statutory or implied including without limitation as to product quality condition description merchantability or fitness for a particular purpose and all such warranties are hereby expressly disclaimed GE Healthcare Bio Sciences Corp hereby expressly disclaims any warranty regarding results obtained through the use of the products including without limitation any claim of inaccurate invalid or incomplete results Exclusion of Liability GE Healthcare Bio Sciences Corp and its affiliates shall have no liability to an End User including without limitation for any loss of use or profits business interruption or any consequential incidental special or other indirect damages of any kind regardless of how caused and regardless of whether an action in contract tort strict product liability or otherwise 2009 2014 Promega Corporation All Rights Reserved Maxwell Plexor and PowerPlex are registered trademarks of Promega Corporation AmpSolution Differex DNA IQ PowerTyper PunchSolution Slicprep and SwabSolution are trademarks of Promega Corporation ABI PRISM Applied Biosystems GeneAmp GeneMapper and M
18. the desired template volume Add 0 5ng of diluted DNA to a reaction well containing PCR amplification mix Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Page 8 Printed in USA Revised 6 14 8 For the negative amplification control pipet Water Amplification Grade C or TE buffer instead of template DNA into a reaction well containing o PCR amplification mix Promega 9 Seal the plate Optional Briefly centrifuge the plate to bring contents to the bottom and remove any air bubbles Thermal Cycling This section contains a protocol for use of the PowerPlex ESX 17 System with the GeneAmp PCR System 9700 and 2720 thermal cyclers 1 Place the MicroAmp plate or reaction tubes in the thermal cycler 2 Select and run the recommended protocol The preferred protocol for use with the GeneAmp PCR System 9700 and 2720 thermal cyclers is provided below Thermal Cycling Protocol 96 C for 2 minutes then 94 C for 30 seconds 59 C for 2 minutes 72 C for 90 seconds for 30 cycles then 60 C for 45 minutes 4 C soak 1When using the GeneAmp PCR System 9700 thermal cycler the program must be run with 9600 as the ramp speed The ramp speed is set after the thermal cycling run is started The Select Method Options screen appears Select 9600 for the ramp speed and enter the react
19. to genotyping errors caused by nontemplated nucleotide addition by Taq DNA polymerase Genome Res 5 312 7 18 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 19 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 20 Griffiths R et al 1998 New reference allelic ladders to improve allelic designation in a multiplex STR system Int J Legal Med 111 267 72 21 Butler J M 2006 Genetics and genomics of core STR loci used in human identity testing J Forensic Sci 51 253 65 22 Hill C R et al 2008 Characterization of 26 miniSTR loci for improved analysis of degraded DNA samples J Forensic Sci 53 73 80 23 B r W et al 1997 DNA recommendations Further report of the DNA Commission of the ISFH regarding the use of short tandem repeat systems Int J Legal Med 110 175 6 24 Gill P et al 1997 Considerations from the European DNA Profiling Group EDNAP concerning STR nomenclature Forensic Sci Int 87 185 92 25 Fr geau CJ 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 20
20. 0 Select the Peak Detector tab We recommend the settings shown in Figure 20 Analysis Method Editor HID Ceneul Abele Peak Oatu Pea Quality Quality flage Peak Datection Algatith Advanced v Ranges Peak Detection Analysis Sines Peak Amgitete Thresholds Full Range Y Patai Si Y B so R 2 Start Size 00 Stop Size 500 G 0 0 Y 40 Smoothing and Baselining Min Peak Halt Width Smoothing C None Uses O Heavy Peak Window Size Blope Threshetd Ves Stat Potynamial Degree Basenne Win sow 61 Saw Canoga Menot C 2nd Ortar Leart gosier 31d Order Least Squares Catic Spline Intemotation Loval Sosten Methed O Global Southern Method Peak End 8187TA Figure 20 The GeneMapper ID Peak Detector tab Notes 1 Select full range or partial range for the analysis range When using a partial range choose an appropriate analysis range based on your data Choose a start point after the primer peak and just before the first defined internal lane standard peak to help ensure proper sizing of the internal lane standard 2 The peak amplitude thresholds are the minimum peak heights at which the software will call a peak Values for peak amplitude thresholds are usually 50 150RFU and should be determined by individual laboratories Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega
21. 0 5ng of template can result in an imbalance with smaller loci showing more product than larger loci Use less template Degraded DNA sample DNA template was degraded and larger loci showed diminished yield Repurify template DNA Insufficient template DNA Use the recommended amount of template DNA if available Stochastic effects can occur when amplifying low amounts of template Miscellaneous balance problems Thaw the 10X Primer Pair Mix and 5X Master Mix completely and vortex for 15 seconds before use Do not centrifuge the 10X Primer Pair Mix after mixing Calibrate thermal cyclers and pipettes routinely PCR amplification mix prepared in Section 4 was not mixed well Vortex the PCR amplification mix for 5 10 seconds before dispensing into the reaction tubes or plate Impure template DNA Inhibitors that may be present in forensic samples can lead to allele dropout or imbalance Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 61 7 B Direct Amplification of DNA from Storage Card Punches The following information is specific to direct amplification For information about general amplification and detection see Section 7 A Symptoms Causes and Comments Faint or absent allele peaks The reaction volume was too low This sys
22. 01 DNA IQ The intelligent way to purify DNA Profiles in DNA 4 3 16 27 Krenke B E et al 2005 Development of a novel fluorescent two primer approach to quantitative PCR Profiles in DNA 8 1 3 5 9 Appendix 9 A Advantages of Using the Loci in the PowerPlex ESX 17 System The loci included in the PowerPlex ESX 17 System Tables 4 and 5 were selected because they meet the recommendations of the European Network of Forensic Science Institutes ENFSI The PowerPlex ESX 17 System amplifies all ENFSI core loci plus SE33 in a single reaction Table 6 lists the PowerPlex ESX 17 System alleles revealed in commonly available standard DNA templates Terminal nucleotide addition 17 18 occurs when Taq DNA polymerase adds a nucleotide generally adenine to the 3 ends of amplified DNA fragments in a template independent manner The efficiency with which this occurs varies with different primer sequences Thus an artifact band one base shorter than expected Le missing the terminal addition is sometimes seen We have modified primer sequences and added a final extension step of 60 C for 45 minutes 19 to the amplification protocols to provide conditions for essentially complete terminal nucleotide addition when recommended amounts of template DNA are used Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Pr
23. 2 2 23 2 24 2 D2 ROM DN RM DIM SOB MY YL 382 34 2 35 37 39 42 D198433 CXR ET 193 245 5 2 6 2 8 12 12 2 13 13 2 14 14 2 15 15 2 16 16 2 17 17 2 18 18 2 D125391 CXR ET 130 182 14 17 17 3 18 18 3 19 27 D25441 CXR ET 88 124 8 11 11 3 12 17 1The length of each allele in the allelic ladder has been confirmed by sequence analyses 2When using an internal lane standard such as the CC5 Internal Lane Standard 500 the calculated sizes of allelic ladder components may differ from those listed This occurs because different sequences in allelic ladder and ILS components may cause differences in migration The dye label and linker also affect migration of alleles 3For a current list of microvariants see the Variant Allele Report published at the U S National Institute of Standards and Technology NIST web site at www cstl nist gov div831 strbase 4Amelogenin is not an STR but displays an 87 base X specific band and a 93 base Y specific band Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 75 9 A Advantages of Using the Loci in the PowerPlex ESX 17 System continued a Table 6 The PowerPlex ESX 17 System Allele Determinations in Commonly Available Standard Promega DNA Templates Standard DNA Templates STR Locus 994
24. 2S1045 SE33 D19S433 D125391 and D2S441 The PowerPlex ESX 17 System is designed with the new loci recommended by the European Network of Forensic Science Institutes ENFSI and European DNA Profiling Group EDNAP loci as mini STRs lt 125bp D25441 D10S1248 and D2251045 or midi STRs 125 185bp D1S1656 and D125391 To complement this design and allow maximal recovery of allelic information from degraded samples the PowerPlex ESI 17 Pro System which amplifies the same seventeen loci present in the PowerPlex ESX 17 System is designed with six of the original seven European Standard Set ESS loci D3S1358 D18551 TH01 vWA D8S1179 and the more common FGA alleles along with D165539 and D195433 as smaller amplicons lt 250bp while the new ENFSI EDNAP loci are present as larger amplicons Therefore these two STR systems can be used to complement each other when analyzing degraded or challenging samples to maximize recovery of allelic information from as many loci as possible and also allow confirmation of results obtained with the other system Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Page 2 Printed in USA Revised 6 14 The PowerPlex ESX 17 System is compatible with the ABI PRISM 310 3100 and C 3100 Avant Genetic Analyzers and Applied Biosystems 3130 3130x1 3500 and 3500xL o Geneti
25. 3 2 and PowerPlex ESX panels and bins text files Panel C The TMR ET labeled allelic ladder components and their allele designations Panel D The CXR ET labeled allelic ladder components and their allele designations Note Panels A and B are shown on the previous page Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 57 6 N Results continued Artifacts and Stutter Stutter products are a common amplification artifact associated with STR analysis 15 16 Stutter products are often observed one repeat unit below the true allele peak and occasionally two repeat units smaller or one repeat unit larger than the true allele peak Frequently alleles with a greater number of repeat units will exhibit a higher percent stutter The pattern and intensity of stutter may differ slightly between primer sets for the same loci Increased stutter often is associated with D2251045 as it is a trinucleotide repeat marker The highest stutter observed at each locus is used in the PowerPlex ESX panels text files for locus specific filtering in the GeneMapper ID software version 3 2 GeneMapper ID X software and PowerTyper ESX 17 Macro for Genotyper software In addition to stutter peaks the following low level artifact peaks may be observed with the PowerPlex ESX 17 System loci Locu
26. 43987 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 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 81 0851867 Switzerland Pat Nos 0743987 and 0851867 United Kingdom Pat Nos 0743987 and 0851867 U S Pat Nos 5 654 419 5 688 648 5 869 255 6 177 247 5 707 804 6 028 190 6 544 744 7 015 000 and 5 728 528 and other pending and foreign patent applications End User Terms and Conditions Acceptance These terms and conditions shall govern the purchase use transfer and acceptance of the products described in the purchase order quotation or invoice which products are sold and distributed by Promega to the buyer transferee of such products the End User The transfer sale of products to the End User is expressly conditional upon End User s acceptance of these terms and conditions Restrictions on Use End Users are specifically not authorized to and are forbidden from reselling transferring or distributing any products either as a stand alone product or as a component of another product The right to use the products does not in and of itself include or carry any right of the End User to any GE Healthcare Bio Sciences Corp
27. 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 51 6 L Sample Analysis Using the Genotyper Software and PowerTyper ESX 17 Macro To facilitate analysis of data generated with the PowerPlex ESX 17 System we have created a file to allow automatic assignment of genotypes using the Genotyper software After samples are amplified detected using the ABI PRISM 310 or 3100 Genetic Analyzer and analyzed using the GeneScan software sample files can be imported into the Genotyper program and analyzed using the PowerTyper ESX 17 Macro The PowerTyper ESX Macros are available for download at www promega com resources tools powertyper macros The PowerTyper ESX 17 Macro is used in conjunction with Macintosh Genotyper software version 2 5 and Windows NT Genotyper software version 3 6 or later The Genotyper software must be installed on your computer before the PowerTyper ESX 17 Macro can be used Be certain the Sample Info Macintosh computers or Color Info Windows NT operating systems column for each lane containing allelic ladder mix contains the word ladder The macro uses the word ladder to identify the sample file s containing allelic ladder Sample info can be added or modified after importing into the PowerTyper ESX 17 Macro Highlight the sample then select show dye lanes wi
28. 74 4330 Fax 608 277 2516 www promega com Part TMD024 Page 52 Printed in USA Revised 6 14 5 For casework double click on the POWER macro The POWER macro C identifies alleles in the ladder sample and calculates offsets for all loci A This process may take several minutes When completed a plots window Pro will open to display the allelic ladders i e D18551 D21S11 TH01 m G D3S1358 and Amelogenin Alternatively for databasing or paternity double click on the POWER 20 Filter macro This macro has a higher level of filtering than the standard POWER macro to reduce the need for manual editing of peak labels The POWER 20 Filter should not be used if mixtures may exist 6 Double click on the Allelic Ladders macro A plots window will open to display the fluorescein allelic ladders i e D18551 D21S11 TH01 D3S1358 and Amelogenin JOE allelic ladders i e D16S539 D2S1338 D1S1656 and D10S1248 TMR ET allelic ladders i e FGA D8S1179 vWA and D22S1045 and CXR ET allelic ladders i e SE33 D19S433 D12S391 and D25441 Confirm that the correct allele designations were assigned to the allelic ladders Figure 24 in Section 6 N and Table 5 in Section 9 A The software uses one ladder sample to determine allele sizes The macro uses the first ladder sample imported for allele designations If the POWER macro is run a second time the software will use the second ladder if the POWER macro is run a third time the
29. 7A 9948 2800M D18551 15 19 15 18 16 18 D21S11 30 30 29 30 29 31 2 TH01 8 9 3 6 9 3 6 9 3 D3S1358 14 15 15 iy 17 18 Amelogenin X X X Y X Y D16539 ii 12 iil UL 9 13 D2S1338 19 23 23 23 22 25 D1S1656 18 3 18 3 14 17 12 113 D10S1248 13 15 12 15 13 15 FGA 23 24 24 26 20 23 D8S1179 13 13 12 13 14 15 vWA 17 18 7 iy 16 19 D22S1045 11 14 16 18 16 16 SE33 19 29 2 23 2 26 2 15 16 D19S433 14 15 13 14 13 14 D128391 18 20 18 24 18 23 D2S441 10 14 11 12 10 14 1Information about strains 9947A and 9948 is available online at http ccr coriell org Sections Collections NIGMS SsId 8 Information about the use of 9947A and 9948 DNA as standard DNA templates can be found in reference 25 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Printed in USA Page 76 Revised 6 14 9 B DNA Extraction and Quantitation Methods and Automation Support C o Promega offers a wide variety of reagents and automated methods for sample preparation DNA purification and DNA quantitation prior to STR Promega amplification For analysis of database reference and other single source samples we recommend direct amplification from FTA punches or preprocessing of swabs and nonFTA punches with the SwabSolution Kit or PunchSolution Kit respectively The SwabSolution Kit Cat DC8271 conta
30. 80 200 225 250 275 300 325 350 375 400 425 450 475 and 500 bases See Section 9 C Figure 25 Select OK Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Page 34 Printed in USA Revised 6 14 Size Standard Editor C Ede va See Layi 0esopton ccs asooo Promega 8257TA Figure 12 The GeneMapper ID X Size Standard Editor 6 D Creating a Casework Analysis Method with GeneMapper ID X Software Version 1 2 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 Select Tools then GeneMapper ID X Manager Select the Analysis Methods tab Select New and a new analysis method dialog box will open PrP eo rN a 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 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 35 C 6 D Creating a Casework Analysis Method
31. 9 MnusA Distance from 00 oo 0 0 0 0 To 08 0 0 0 0 00 Goba Minus stutter Rabo 0 0 oo 0 0 00 Goba Mus Rutte Oetarce From 2 25 3 25 0 0 0 0 I to 3 75 78 0 0 0 0 Geba Plus Quete Ratio 0 1 0 0 0 0 0 0 Gebel Pas Sutter Ontarce From 2 35 T 0 0 00 l To 3 75 0 0 0 0 amalsganin Otor 02 i Figure 16 The GeneMapper ID X Allele tab 9 Select the Peak Detector tab Figure 14 shows an example of settings used at Promega You may need to optimize these settings In house validation should be performed Notes 1 Select full range or partial range for the analysis range When using a partial range choose an appropriate analysis range based on your data Choose a start point after the primer peak and just before the first defined internal lane standard peak to help ensure proper sizing of the internal lane standard 2 The peak amplitude thresholds are the minimum peak heights at which the software will call a peak Values for peak amplitude thresholds are usually 50 150RFU on the ABI PRISM 310 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130x Genetic Analyzers For the Applied Biosystems 3500 and 3500xL Genetic Analyzers Life Technologies suggests an analysis Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Printed in USA Page 40 Revised 6 14
32. BI PRISM 310 Genetic Analyzer Materials to Be Supplied by the User e 95 C dry heating block water bath or thermal cycler e crushed ice or ice water bath e 310 capillaries 47cm x 501m e performance optimized polymer 4 POP 4 polymer e 10X genetic analyzer buffer with EDTA e sample tubes and septa e aerosol resistant pipette tips e Hi Di formamide Applied Biosystems Cat 4311320 The quality of formamide is critical Use Hi Di formamide Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause breakdown of formamide Poor quality formamide may contain ions that compete with DNA during injection which results in lower peak heights and reduced sensitivity A 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 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Page 30 Printed in USA Revised 6 14 Sample Preparation C o 1 Thaw the CC5 Internal Lane Standard 500 Note Centrifuge tube briefly to bring contents to the bottom then vortex Promega for 15 seconds before each use Do not centrifuge after vor
33. DNA templates Developmental validation of the kit showed routine generation of full profiles using 30 cycles of amplification with lower amounts of DNA template down to 62 5pg Partial profiles were typically observed for DNA template of 32pg and below 13 In house optimization and validation should be performed to establish the performance of the kit in your laboratory 12 Materials to Be Supplied by the User GeneAmp PCR System 9700 or 2720 thermal cycler Applied Biosystems microcentrifuge e MicroAmp optical 96 well reaction plates or 0 2ml MicroAmp reaction tubes Applied Biosystems e aerosol resistant pipette tips see Section 9 E Amplification Setup 1 Thaw the PowerPlex ESX 5X Master Mix PowerPlex ESX 17 10X Primer Pair Mix and Water Amplification Grade completely Note Centrifuge tubes briefly to bring contents to the bottom then vortex reagents for 15 seconds before each use Do not centrifuge the 10X Primer Pair Mix or 5X Master Mix after vortexing as this may cause the primers to be concentrated at the bottom of the tube 2 Determine the number of reactions to be set up This should include positive and negative control reactions Add 1 or 2 reactions to this number to compensate for pipetting error While this approach does consume a small amount of each reagent it ensures that you will have enough PCR amplification mix for all samples It also ensures that each reaction contains the same PCR ampl
34. Mix 4 x 300ul CC5 Internal Lane Standard 500 The PowerPlex ESX 17 Allelic Ladder Mix is provided in a separate sealed bag for shipping This component should be moved to the post amplification box after opening The Water Amplification Grade is provided in a separate sealed bag for shipping This component should be moved to the pre amplification box after opening Storage Conditions Store all components except the 2800M Control DNA at 30 C to 10 C in a nonfrost free freezer Store the 2800M Control DNA at 2 10 C The Water Amplification Grade can be stored at 2 10 C long term The PowerPlex ESX 17 10X Primer Pair Mix PowerPlex ESX 17 Allelic Ladder Mix and CC5 Internal Lane Standard 500 CC5 ILS 500 are light sensitive and must be stored in the dark We strongly recommend that pre amplification and post amplification reagents be stored and used separately with different pipettes tube racks etc For daily use the PowerPlex ESX 17 10X Primer Pair Mix and PowerPlex ESX 5X Master Mix can be stored at 2 10 C for up to 1 month without loss of activity Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Page 4 Printed in USA Revised 6 14 Available Separately C o The proper panels bins and stutter files for use with GeneMapper ID and ID X software are available for download at Promega w
35. Select Basic or Advanced Figure 17 The type of analysis method selected must match the type of analysis method created earlier Select OK Select Dye and Analysis Method 5725TA Figure 17 The Select Dye and Analysis Method window 5 Enter a detailed name such as CC5 ILS 60 to 500 in the Size Standard Editor Figure 18 8199TA Figure 18 The Size Standard Editor Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Printed in USA Page 44 Revised 6 14 6 Choose Orange for the Size Standard Dye C o 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 Promega 500 bases See Section 9 C Figure 25 8 Select OK 6 1 Creating a Casework Analysis Method with GeneMapper ID Software Version 3 2 These instructions loosely follow the Applied Biosystems GeneMapper ID software tutorial pages 5 11 Select Tools then GeneMapper Manager Select the Analysis Methods tab Select New and a new analysis method dialog box will open Select HID and select OK Note If you do not see the HID option you do not have the GeneMapper ID software Contact Applied Biosystems pe NY E 5 Enter a descriptive name for the analysis
36. Select Regular in the Type drop down list and select the run module you created in the previous step in the Run Module drop down list Lastly select G5 in the dye set drop down list Select OK 3 In the Plate Manager create a new plate record as described in the instrument user s manual In the dialog box that appears select GeneMapper Generic in the 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 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com P
37. TECHNICAL MANUAL PowerPlex ESX 17 System Instructions for use of Products DC6720 and DC6721 Pro Revised 6 14 TMDO24 PowerPlex ESX 17 System O All technical literature is available on the Internet at www promega com protocols Please visit the web site to verify that you are using the most current version of this Technical Manual Please contact Promega Technical Services if you have questions on use of this system E mail genetic promega com 1 Description ssccciesccecssscsccusstzecsaadzcansssssssttssazsictsssainessazotecddsasccecsussctcavasececsuiscessbeovissnesovessbe ovine 2 2 Product Components and Storage Conditions ss sssssssssssssssrsstrssssssrssnrsssersnnernnrnnnes 4 3e Before You Began ssisssssssscssssssssssscscccssesssscanssnsssvesonosstesinareesecesssssisissssesessiassssssssssisssissstssssssaannsnin 5 Pc eee CO a E E 5 B Matrix Standardization or Spectral Calibration ssccssscsssssseesessssesssssseesseesssssnenees 6 4 Protocols for DNA Amplification Using the PowerPlex ESX 17 System 6 A Amplification of Extracted DNA sssssssssssssssssssessssssasasesssesssssassesccvessassccscssesvesseuseteanevssiiens Z B Direct Amplification of DNA from Storage Card Punches 10 C Direct Amplification of DNA from Swab sesscsscssssssesssesssssseeseesssssssssnesesssssssneesees 14 5 Instrument Setup and Sample Preparation 0 cccssssssesesssssssseeseessssssss
38. ard were detected during the run e Create a new size standard using the internal lane standard fragments present in the sample e Re run samples using a longer run time A low quality allelic ladder was used during analysis Ensure that only high quality allelic ladders are used for analysis Off ladder alleles An allelic ladder from a different run than the samples was used Re analyze samples with an allelic ladder from the same run The GeneMapper ID X software requires that the allelic ladder be imported from the same folder as the sample Be sure that the allelic ladder is in the same folder as the sample Create a new project and re analyze as described in Section 6 D or 6 E Panels text file selected for analysis was incorrect for the STR system used Assign correct panels text file that corresponds to the STR system used for amplification The allelic ladder was not identified as an allelic ladder in the Sample Type column The internal lane standard was not properly identified in the sample Manually redefine the sizes of the size standard fragments in the sample Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 67 7 D GeneMapper ID X Software continued Symptoms Causes and Comments Off ladder alleles continued
39. art TMD024 Page 10 Printed in USA Revised 6 14 Automated punchers also can be used to create sample disks Refer to the user s C guide for your instrument for assistance with generating 1 2mm disks technical A advice and troubleshooting information Note Static may be problematic when adding a punch to a well For FTA card punches adding PCR amplification mix to the well before adding the punch may help alleviate static problems For nonFTA card punches adding PunchSolution Reagent to the well before adding the punch during pretreatment may help alleviate static problems Amplification Setup 1 Thaw the PowerPlex ESX 5X Master Mix PowerPlex ESX 17 10X Primer Pair Mix and Water Amplification Grade completely Note Centrifuge tubes briefly to bring contents to the bottom then vortex reagents for 15 seconds before each use Do not centrifuge the 10X Primer Pair Mix or 5X Master Mix after vortexing as this may cause the reagents to be concentrated at the bottom of the tube 2 Vortex the 5X AmpSolution Reagent for 10 15 seconds Note The 5X AmpSolution Reagent should be thawed completely mixed by vortexing and stored at 2 10 C The reagent may be turbid after thawing or storage at 4 C If this occurs warm the buffer briefly at 37 C then vortex until clear Do not store reagents in the refrigerator door where the temperature can fluctuate Storing reagents in the refrigerator door can compromise stability
40. ates heat transfer is inefficient and will result in poor performance Only use a heat block to maintain efficient heat transfer We have tested 60 minute incubation times and observed no difference in performance compared to a 30 minute incubation Make sure that the PCR amplification mix also contained AmpSolution Reagent Omission of AmpSolution Reagent from amplification reactions will result in amplification failure Faint or absent peaks for the If the positive control reaction failed to amplify check to positive control reaction make sure that the correct amount of 2800M Control DNA was added to the reaction Due to the reduced cycle numbers used with swab extracts it is necessary to increase the mass of 2800M Control DNA to obtain a profile We recommend 5ng of 2800M Control DNA per 25pl amplification reaction This mass of DNA should be reduced if the cycle number is increased and decreased if the cycle number is increased Increase or decrease by twofold the mass of 2800M Control DNA for every one cycle decrease or increase respectively Extra peaks visible in one Swab extract was contaminated Assemble a reaction or all color channels containing the swab extract prepared from a blank swab or assemble a reaction where the SwabSolution Reagent is processed and incubated as a blank without a swab Artifacts of STR amplification Amplification of swab extracts with high DNA concentrations can result in artifact peaks due to o
41. ay be labeled by the software This peak is not used during sizing of the peaks present in the sample t o we we we wee mo me zno z sme wns ae tee ane emo asse eme ame 8248TA s Figure 25 CC5 Internal Lane Standard 500 An electropherogram showing the CC5 Internal Lane Standard 500 fragments Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Printed in USA Page 78 Revised 6 14 9 D Composition of Buffers and Solutions w TE buffer 10mM Tris HCl TE buffer with 20ug ml glycogen Pro mega 0 1mM EDTA pH 8 0 121g Tris base 1 21g Tris base 0 037g EDTA 0 037g EDTA Na EDTA 2H 0 Na EDTA 2H 0 Dissolve Tris base and EDTA in 900ml of deionized water Adjust to pH 8 0 with HCI Bring the final volume to 1 liter with deionized water 20ug ml glycogen Dissolve Tris base and EDTA in 900ml of deionized water Adjust to pH 8 0 with HCI Add glycogen Bring the final volume to 1 liter with deionized water 9 E Related Products Fluorescent STR Systems Product Size Cat PowerPlex ESX 17 Fast System 100 reactions DC1711 400 reactions DC1710 PowerPlex ESI 17 Fast System 100 reactions DC1721 400 reactions DC1720 PowerPlex ESX 16 System 100 reactions DC6711 400 reactions DC6710 PowerPlex ESI 16 S
42. box is checked Stutter distance was not defined in the analysis method Allele tab Samples in the project not analyzed Analysis Requirement Summary window was not active and there was an analysis requirement that was not met Turn on Analysis Requirement Summary in the Options menu and correct the necessary analysis requirements to continue analysis Edits in label edit viewer cannot To view edits made to a project the project must first be be viewed saved Close the plot view window go back 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 When an edit is made to a locus the quality flags and marker are gray header bar automatically change to gray To change the GQ and marker header bar for a locus to green override the GQ in the plot window Alleles not called To analyze samples with GeneMapper ID X software at least one allelic ladder must be defined An insufficient number of CC5 ILS 500 fragments was defined Be sure to define at least two CC5 ILS 500 fragments smaller than the smallest sample peak or allelic ladder peak and at least two CC5 ILS 500 fragments larger than the largest sample peak or allelic ladder peak In this instance the allelic ladder would have failed the allelic ladder quality check Run was too short and larger peaks in ILS were not captured Not all CC5 ILS 500 peaks defined in the size stand
43. c Analyzers Amplification and detection instrumentation may vary You may Pro need to optimize protocols including the amount of template DNA cycle number m g injection conditions and loading volume for your laboratory instrumentation In house validation should be performed The PowerPlex ESX 17 System provides all materials necessary to amplify STR regions of human genomic DNA including hot start Tag DNA polymerase which is a component of the PowerPlex ESX 5X Master Mix This manual contains a protocol for use of the PowerPlex ESX 17 System with the GeneAmp PCR system 9700 and 2720 thermal cyclers in addition to protocols to separate amplified products and detect separated material Figure 1 Protocols to operate the fluorescence detection instruments should be obtained from the instrument manufacturer Information about other Promega fluorescent STR systems is available upon request from Promega or online at www promega com Amplification Setup Vid 4 Thermal Cycling Section 4 GeneAmp PCR System 9700 y GeneAmp PCR System 2720 Instrument Setup and Sample Preparation Section 5 Applied Biosystems 3500 or ABI PRISM 3100 or 3500xL Genetic Analyzer 3100 Avant Genetic Analyzer Section 5 A with Data Collection Software Version 2 0 Applied Biosystems 3130 or SENON E 3130x Genetic Analyzer with Data Collection Software ABI PRISM 310 Genetic Version 3 0 Analyzer y Section 5 B Sectio
44. claved deionized water change vials and wash buffer reservoir Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Page 60 Printed in USA Revised 6 14 Symptoms Causes and Comments C Extra peaks visible in one Repeat sample preparation using fresh formamide Long term o or all color channels continued storage of amplified samples in formamide can result in aimee aaa Promega The CE polymer was beyond its expiration date or polymer was stored at room temperature for more than one week Maintain instrumentation on a daily or weekly basis as recommended by the manufacturer Allelic ladder not running Allelic ladder and primer pair mix were not compatible Ensure the same as samples that the allelic ladder is 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 different injection of allelic ladder to determine sizes Poor injection of allelic ladder Include more than one ladder per instrument run Peak height imbalance Excessive amount of DNA Amplification of gt
45. com Part TMD024 Printed in USA Page 46 Revised 6 14 3 Peak heights for the CC5 ILS 500 are generally lower than those for C the other dyes Therefore the threshold for the orange dye may be o lower than that for the other dyes Be sure that all CC5 ILS 500 peaks are consistently above the peak amplitude threshold for the orange dye channel determined as part of your internal validation 11 Select the Peak Quality tab You may change the settings for peak quality Note For Steps 11 and 12 see the GeneMapper ID user s manual for more information 12 Select the Quality Flags tab You may change these settings 13 Select OK to save your settings Processing Data for Casework Samples 1 Select File then New Project 2 Select Edit then Add Samples to Project 3 Browse to the location of the run files Highlight desired files then select Add to list followed by Add 4 In the Sample Type column use the drop down menu to select Ladder Sample Positive Control or Negative Control as appropriate for the sample Every folder in the project must contain at least one allelic ladder injection that is designated as Ladder in the Sample Type column for proper genotyping 5 In the Analysis Method column select the analysis method created previously in this section 6 In the Panel column select the panels file that was imported in Section 6 F 7 Inthe S
46. concentration the DNA volume should not exceed 20 of the total reaction volume Carryover of K Nat Mg or EDTA from the DNA sample can negatively affect PCR A change in pH also may affect PCR Store DNA in TE buffer 10mM Tris HCl pH 8 0 0 1mM EDTA TE buffer with 20ug ml glycogen or nuclease free water The reaction volume was too low This system is optimized for a final reaction volume of 25p1 Decreasing the reaction volume may result in suboptimal performance Thermal cycler plate or tube problems Review the thermal cycling protocols in Section 4 We have not tested other reaction tubes plates or thermal cyclers Calibrate the thermal cycler heating block if necessary Primer concentration was too low Use the recommended primer concentration Vortex the PowerPlex ESX 17 10X Primer Pair Mix for 15 seconds before use Samples were not denatured completely Heat denature samples for the recommended time then cool on crushed ice or in an ice water bath immediately prior to capillary electrophoresis Do not cool the samples in a thermal cycler set at 4 C as this may lead to artifacts due to DNA re annealing Poor capillary electrophoresis injection CC5 ILS 500 Pro peaks also affected Re inject the sample Check the 310 instrument syringe pump system for leakage Poor capillary electrophoresis injection CC5 ILS 500 Pro peaks also affected Check the laser power Poor quality formamide was used Use only Hi Di formamide
47. d a new analysis method created Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Page 70 Printed in USA Revised 6 14 7 F PowerTyper ESX 17 Macro Symptoms Causes and Comments File does not open on your computer Genotyper software was not installed Be certain that the Genotyper software version 2 5 Macintosh or version 3 6 or higher Windows NT is installed Incorrect version of Genotyper software The PowerTyper ESX 17 Macro will not work with Genotyper software versions prior to version 2 5 Macintosh or 3 6 Windows NT Error message Could not complete the Run Macro command because no dye lanes are selected Allelic ladder sample files were not identified Be certain the Sample Info or Color Info column for each lane containing PowerPlex ESX 17 Allelic Ladder Mix contains the word ladder The macro uses the word ladder to identify sample files containing allelic ladder All five dye colors were not imported For Genotyper software versions 2 5 and 3 6 or higher set preferences in the Edit menu to import fluorescein JOE TMR ET CXR ET and CC5 data Error message Could not complete the Run Macro command because the labeled peak could not be found Peak heights for one or more alleles in t
48. e 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 17 5 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer continued Sample Preparation 1 Thaw the CC5 Internal Lane Standard 500 Note Centrifuge tube briefly to bring contents to the bottom then vortex for 15 seconds before each use Do not centrifuge after vortexing as this may cause the size standard to be concentrated at the bottom of the tube Prepare a loading cocktail by combining and mixing CC5 Internal Lane Standard 500 and Hi Di formamide as follows 1 0p1 CC5 ILS 500 x samples 10 0p1 Hi Di formamide x samples Note The volume of internal lane standard used in the loading cocktail can be increased or decreased to adjust the intensity of the size standard peaks Use a volume of CC5 ILS 500 that results in peak heights that are all consistently above the peak amplitude threshold of the orange dye channel determined as part of your internal validation Keep the volume of formamide at 10 0p1 and adjust the volume added to the wells in Step 4 accordingly Vortex for 10 15 seconds to mix Pipet 11 1 of formamide internal lane standard mix into each well Add 11 of amplified sample or 1p of PowerPlex ESX 17 Allelic Ladder Mix Cover wells with appropriate septa Notes 1 Instrument detection limits vary therefore injection time injecti
49. e taken to avoid cross contamination when preparing template DNA handling primer pairs assembling amplification reactions and analyzing amplification products Reagents and materials used prior to amplification PowerPlex ESX 5X Master Mix PowerPlex ESX 17 10X Primer Pair Mix 2800M Control DNA and Water Amplification Grade are provided in a separate box and should be stored separately from those used following amplification PowerPlex ESX 17 Allelic Ladder Mix and CCS 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 e g ART tips Section 9 E Some reagents used in the analysis of STR products are potentially hazardous and should be handled accordingly Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning label and take appropriate precautions when handling this substance Always wear gloves and safety glasses when working with formamide Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 5 3 B Matrix Standardization or Spectral Calibration Proper generation of a matrix file is critical to evaluate multicolor systems with the ABI PRISM 310 3100 and 3100 Avant Genet
50. ed protocol for use with the GeneAmp PCR System 9700 thermal cycler is provided below Promega Thermal Cycling Protocol 96 C for 2 minutes then 94 C for 30 seconds 59 C for 2 minutes 72 C for 90 seconds for 24 cycles then 60 C for 45 minutes 4 C soak When using the GeneAmp PCR System 9700 thermal cycler the program must be run with 9600 as the ramp speed The ramp speed is set after the thermal cycling run is started The Select Method Options screen appears Select 9600 for the ramp speed and enter the reaction volume 3 After completion of the thermal cycling protocol store amplified samples at 20 C in a light protected box Note Long term storage of amplified samples at 4 C or higher may produce artifacts PCR Optimization Cycle number should be optimized based on the results of an initial experiment to determine the sensitivity with your collection method sample types 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 workflow 2 Depending on your preferred protocol place one or two 1 2mm storage card punches containing a buccal sample or one 1 2mm punch of a storage card containing whole blood in each well of a reaction plate Be sure to pretreat nonFTA samples with the PunchSolution Kit Cat DC9271 3 Prepare three identical reaction plates wi
51. egative 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 10 Seal the plate Optional Briefly centrifuge the plate to bring contents to the bottom of the wells and remove any air bubbles Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 15 4 C Direct Amplification of DNA from Swabs continued Thermal Cycling Amplification and detection instrumentation may vary You will need to optimize protocols including the amount of template DNA cycle number injection conditions 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 that is amplified see below 1 Place the MicroAmp plate in the thermal cycler 2 Select and run the recommended protocol The preferred protocol for use with the GeneAmp PCR System 9700 thermal cycler is provided below Thermal Cycling Protocol 96 C for 2 minutes then 94 C for 30 seconds 59 C for 2 minutes 72 C for 90 seconds for 26 cycles then 60 C for 45 minutes 4 C soak 1When using the GeneAmp PCR S
52. eotide of a repeat motif and 2 for STR loci not associated with a coding gene the first database entry or original literature description shall be used 3Amelogenin is not an STR but displays an 87 base X specific band and a 93 base Y specific band Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Page 74 Printed in USA Revised 6 14 Table 5 The PowerPlex ESX 17 System Allelic Ladder Information 4 Size Range of Allelic Ladder Components Repeat Numbers of Allelic Ladder Promega STR Locus Label bases Components D18551 Fluorescein 286 366 7 10 10 2 11 13 13 2 14 27 D21511 Fluorescein 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 THO1 Fluorescein 152 195 3 9 9 3 10 11 13 3 D3S1358 Fluorescein 103 147 9 20 Amelogenint Fluorescein 87 93 X Y D165539 JOE 273 321 4 16 D251338 JOE 197 269 10 12 14 28 D1S1656 JOE 137 184 9 14 14 3 15 15 3 16 16 3 17 17 3 18 18 3 19 19 3 20 3 D10S1248 JOE 83 127 8 19 FGA TMR ET 264 410 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 D8S1179 TMR ET 203 251 7 19 vWA TMR ET 124 180 10 24 D22S1045 TMR ET 79 118 7 20 SE33 CXR ET 267 417 4 2 6 3 8 20 20 2 21 21 2 22 2
53. ernal validation studies 3 Peak heights for the CC5 ILS 500 are generally lower than those for the other dyes Therefore the threshold for the orange dye may be lower than that for the other dyes Be sure that all CC5 ILS 500 peaks are consistently above the peak amplitude threshold for the orange dye channel determined as part of your internal validation 11 Select the Peak Quality tab You may change the settings for peak quality Note For Steps 11 and 12 see the GeneMapper ID user s manual for more information 12 Select the Quality Flags tab You may change these settings 13 Select OK to save your settings Processing Data for Databasing or Paternity Samples 1 Select File then New Project 2 Select Edit then Add Samples to Project 3 Browse to the location of the run files Highlight desired files then select Add to list followed by Add 4 Inthe Sample Type column use the drop down menu to select Ladder Sample Positive Control or Negative Control as appropriate for the sample Every folder in the project must contain at least one allelic ladder injection that is designated as Ladder in the Sample Type column for proper genotyping 5 In the Analysis Method column select the analysis method created previously in this section 6 In the Panel column select the panels text file that was imported in Section 6F 7 Inthe Size Standard colum
54. es were assigned to the internal lane standard Confirm that internal lane standard fragment sizes are assigned correctly Re analyze the sample using GeneScan software and redefine the internal lane standard fragments ds 10 11 12 13 8 References Edwards A et al 1991 DNA typing with trimeric and tetrameric tandem repeats Polymorphic loci detection systems and population genetics In The Second International Symposium on Human Identification 1991 Promega Corporation 31 52 Edwards A et al 1991 DNA typing and genetic mapping with trimeric and tetrameric tandem repeats Am J Hum Genet 49 746 56 Edwards A et al 1992 Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups Genomics 12 241 53 Warne D et al 1991 Tetranucleotide repeat polymorphism at the human b actin related pseudogene 2 actbp2 detected using the polymerase chain reaction Nucleic Acids Res 19 6980 Ausubel F M et al 1996 Unit 15 The polymerase chain reaction In Current Protocols in Molecular Biology Vol 2 John Wiley and Sons NY Sambrook J Fritsch E F and Maniatis T 1989 Chapter 14 In vitro amplification of DNA by the polymerase chain reaction In Molecular Cloning A Laboratory Manual Second Edition Cold Spring Harbor Laboratory Press Cold Spring Harbor New York PCR Technology Principles and Applications for DNA Amplification 1989 Erlic
55. fault injection conditions However individual laboratories should determine their peak amplitude thresholds from internal validation studies 3 Peak heights for the CC5 ILS 500 are generally lower than those for the other dyes Therefore the threshold for the orange dye may be lower than that for the other dyes Be sure that all CC5 ILS 500 peaks are consistently above the peak amplitude threshold for the orange dye channel determined as part of your internal validation 4 The normalization box can be checked regardless of whether normalization was or was not applied during data collection Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 37 C 6 D Creating a Casework Analysis Method with GeneMapper ID X Software o Promega 11 1 2 3 12 13 14 Version 1 2 continued 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 Select the SQ amp GQ Settings tab You may change these settings Select Save to save the new analysis method Select Done to exit the GeneMapper ID X Manager Processing Data for Casework Samples Select File then New Project Select Edit then Add Samples to Project Browse to
56. from samples but does not normalize the amount of DNA present 7 C Amplification of DNA from Swabs The following information is specific to amplification of DNA from swabs after pretreatment using the SwabSolution Kit For information about general amplification and detection see Section 7 A Symptoms Causes and Comments Faint or absent allele peaks Poor sample deposition Shedding and collection of donor cells was variable Increase cycle number Inactive SwabSolution Reagent Thaw the SwabSolution Reagent completely in a 37 C water bath and mix by gentle inversion Store the SwabSolution Reagent at 2 10 C Do not store reagents in the refrigerator door where the temperature can fluctuate Do not refreeze as this may reduce activity Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Page 64 Printed in USA Revised 6 14 Symptoms Causes and Comments C Faint or absent allele peaks Active SwabSolution Reagent carried over into the o continued amplification reaction Ensure that the heat block is heating to 70 C 90 C if using a 2 2ml Square Well Deep Well Plate mi Promega samples were incubated for the full 30 minutes Incubation for shorter time periods may result in incomplete reagent inactivation Do not use an incubator set at 70 C to incubate tubes or pl
57. ghts that are all consistently above the peak amplitude threshold determined as part of your internal validation 5 Centrifuge tubes briefly to remove air bubbles from the wells 6 Denature samples and ladder by heating at 95 C for 3 minutes and immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading 7 Place the tubes in the appropriate autosampler tray 8 Place the autosampler tray in the instrument and close the instrument doors Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 31 5 C Detection of Amplified Fragments Using the ABI PRISM 310 Genetic Analyzer continued Instrument Preparation Refer to the instrument user s manual for instructions on cleaning the pump block installing the capillary calibrating the autosampler and adding polymer to the syringe 1 2 Open the ABI PRISM 310 Data Collection Software Prepare a GeneScan sample sheet as described in the ABI PRISM 310 Genetic Analyzer User s Manual Enter the appropriate sample information in the Sample Info column For rows containing PowerPlex ESX 17 Allelic Ladder Mix insert the word ladder in the Sample Info column for the fluorescein JOE TMR ET CXR ET and CC5 dyes when using the GeneScan and Genotype
58. h H A ed Stockton Press New York NY PCR Protocols A Guide to Methods and Applications 1990 Innis M A et al eds Academic Press San Diego CA Butler J M 2005 Forensic DNA Typing 2nd ed Elsevier Academic Press London Presley L A et al 1992 The implementation of the polymerase chain reaction PCR HLA DQ alpha typing by the FBI laboratory In The Third International Symposium on Human Identification 1992 Promega Corporation 245 69 Hartmann J M et al 1991 Guidelines for a quality assurance program for DNA analysis Crime Laboratory Digest 18 44 75 Internal Validation of STR Systems Reference Manual GE053 Promega Corporation Tucker V C et al 2012 Developmental validation of the PowerPlex ESX 16 and PowerPlex ESX 17 Systems Forensic Sci Int Genet 6 124 31 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Page 72 Printed in USA Revised 6 14 14 Kline M C et al 2005 Results from the NIST 2004 DNA quantitation study J Forensic Sci 50 570 8 oe 15 Levinson G and Gutman G A 1987 Slipped strand mispairing A major mechanism for DNA va sequence evolution Mol Biol Evol 4 203 21 Promega 16 Schlotterer C and Tautz D 1992 Slippage synthesis of simple sequence DNA Nucleic Acids Res 20 211 5 17 Smith J R et al 1995 Approach
59. he Create New Assay window Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 23 5 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer continued 6 To create a new File Name Convention Figure 7 navigate to the Library Select File Name Conventions then select Create Alternatively a previously created File Name Convention may be used Select the File Name Attributes according to laboratory practices and save with a descriptive name t idy Metmsrce Tosh Manage Arh Setup n File Name Convestnn Pier 5s repered Natt Premade rege eater Nae aiet Fos lisma Amtn Derren si Fle Iome Sempre Home gt Areir aetate Ampi so hieme Aadrnn Pata d Nase Aivey Maree pars Phew Coston Tet Catam Trt Cotan Tot Ade AMA b torn vibee ta p phsbie Wm ar ipaesi Can Tot Coaten Tet Select P r louan O Deina idr imee D hpphed Borrinm DOF De 9252TA Figure 7 The Create New File Name Convention window Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Page 24 Printed in USA Revised 6 14 7 To create a new Results Group Figure 8 navigate to the Librar
60. he allelic ladder sample file were below 150RFU The allelic ladder categories are defined as having a minimum peak height of 150RFU If peak heights of ladder alleles are below 150RFU the software will not be able to locate the allele peak Re run the allelic ladder using more sample or longer injection time to assure peak heights above 150RFU CE spikes in the allelic ladder sample were identified as alleles by the macro Use a different injection of allelic ladder Allelic ladder data were not compatible with the PowerTyper file used Confirm that the PowerTyper Macro file matches the allelic ladder being used The base pair size of alleles in the allelic ladder are outside of the defined category range Be sure internal lane standard fragments are correctly sized Redefine internal lane standard fragments and re analyze the sample using GeneScan software Compare the size of the smallest allele in the allelic ladder with the base pair size and range listed in the categories for the same alleles If necessary increase the category start range in the category window and save the macro under a new name Allelic ladder peaks were too high causing stutter peaks to be called as allele peaks Use a shorter injection time decrease the amount of allelic ladder used or re analyze the allelic ladder sample using increased peak amplitude thresholds in the GeneScan analysis parameters Allelic ladder data were not compatible with the Po
61. his may reduce activity DNA was not accessible on nonlytic material Pretreat swabs with SwabSolution Reagent to ensure that DNA is liberated from cellular proteins Extreme variability in sample to sample peak heights There can be significant individual to individual variability in cell deposition onto buccal swabs This will appear as variability in peak heights between swab extracts The extraction process maximizes recovery of amplifiable DNA from buccal swabs but does not normalize the amount of DNA present If variability is extreme quantitate the DNA using a fluorescence based double stranded DNA quantitation method or qPCR based quantitation method The quantitation values can be used to normalize input template amounts to minimize variation in signal intensity DNA was not accessible on nonlytic material Pretreat swabs with SwabSolution Reagent to ensure that DNA is liberated from cellular proteins Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Page 66 Printed in USA Revised 6 14 7 D GeneMapper ID X Software C o Symptoms Causes and Comments Stutter peaks not filtered Stutter file was not imported into the Panel Manager when Promega the panels and bin text files were imported Be sure that the Use marker specific stutter ratio and distance if available
62. ic Analyzers and Applied Biosystems 3130 3130x1 3500 and 3500xL Genetic Analyzers A matrix must be generated for each individual instrument The PowerPlex 5 Dye Matrix Standards 310 Cat DG4600 is required for matrix standardization for the ABI PRISM 310 Genetic Analyzer The PowerPlex 5 Dye Matrix Standards 3100 3130 Cat DG4700 cannot be used to generate a matrix on the ABI PRISM 310 Genetic Analyzer The PowerPlex 5 Dye Matrix Standards 3100 3130 Cat DG4700 is required for spectral calibration on the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 3130x1 3500 and 3500xL Genetic Analyzers The PowerPlex 5 Dye Matrix Standards 310 cannot be used to generate a matrix on these instruments For protocols and additional information about matrix standardization see the PowerPlex 5 Dye Matrix Standards 310 Technical Bulletin 1BD023 For protocols and additional information about spectral calibration see the PowerPlex 5 Dye Matrix Standards 3100 3130 Technical Bulletin TBD024 These manuals are available online at www promega com protocols Protocols for DNA Amplification Using the PowerPlex ESX 17 System The PowerPlex ESX 17 System is optimized for the GeneAmp PCR System 9700 thermal cycler An amplification protocol for the GeneAmp PCR Systems 2720 thermal cycler also is provided for extracted DNA The use of gloves and aerosol resistant pipette tips is highl
63. icroAmp are registered trademarks of Applied Biosystems ART is a registered trademark of Molecular Bio Products Inc Bode Buccal DNA Collector is a trademark of the Bode Technology Group Inc EasiCollect and OmniSwab are trademarks of Whatman Excel Microsoft Windows and Windows NT are registered trademarks of Microsoft Corporation FTA is a registered trademark of Flinders Technologies Pty Ltd and is licensed to Whatman GeneScan and Genotyper are registered trademarks of Applera Corporation Hi Di is a trademark of Applera Corporation Macintosh is a registered trademark of Apple Computer Inc POP 4 is a registered trademark of Life Technologies Corporation Sampact is a trademark of Fitzco Vacutainer is a registered trademark of Becton Dickinson and Company Products may be covered by pending or issued patents or may have certain limitations Please visit our Web site for more information All prices and specifications are subject to change without prior notice Product claims are subject to change Please contact Promega Technical Services or access the Promega online catalog for the most up to date information on Promega products Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Page 82 Printed in USA Revised 6 14
64. ification 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 Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 7 4 A Amplification of Extracted DNA continued 4 Add the final volume of each reagent listed in Table 1 to a sterile tube Table 1 PCR Amplification Mix for the PowerPlex ESX 17 System Volume Number of _ Final PCR Amplification Mix Component Per Reaction Reactions Volume to a final Water Amplification Grade volume of 25 0ul x PowerPlex ESX 5X Master Mix 5 0ul x PowerPlex ESX 17 10X Primer Pair Mix 2 5ul x template DNA 0 5ng 54 up to 17 5p1 total reaction volume 25ul 1Add Water Amplification Grade to the tube first then add PowerPlex ESX 5X Master Mix and PowerPlex ESX 17 10X Primer Pair Mix The template DNA will be added at Step 6 Store DNA templates in nuclease free water or TE buffer 10mM Tris HCI pH 8 0 0 1mM EDTA If the DNA template is stored in TE buffer that is not pH 8 0 or contains a higher EDTA concentration the volume of DNA added should not exceed 20 of the final reaction volume PCR amplification efficiency and quality can be greatly altered by changes in
65. in Table 3 to a sterile tube 4 Table 3 PCR Amplification Mix for Direct Amplification of DNA From Swabs PCR Amplification Mix Volume Per Number of Final Component Reaction x Reactions Volume Water Amplification Grade 10 5 x S PowerPlex ESX 5X Master Mix 5 0ul x PowerPlex ESX 17 10X Primer Pair Mix 2 5ul x 5X AmpSolution Reagent 5 0p1 x swab extract 2 0ul total reaction volume 25ul 1Add Water Amplification Grade to the tube first then add PowerPlex ESX 5X Master Mix and PowerPlex ESX 17 10X Primer Pair Mix The swab extract will be added at Step 6 6 Vortex the PCR amplification mix for 5 10 seconds then pipet 231 of PCR amplification mix into each reaction well Failure to vortex the PCR amplification mix sufficiently can result in poor amplification or locus to locus imbalance N Pipet 2 0ul of swab extract for each sample into the appropriate well of the reaction plate 8 For the positive amplification control vortex the tube of 2800M Control DNA then dilute to 2 5ng pl Add 2pl 5ng to a reaction well containing 23pl of PCR amplification mix Note Optimization of the amount of 2800M Control DNA may be required depending on thermal cycling conditions and laboratory preferences 9 For the negative amplification control pipet 21 of Water Amplification Grade or TE buffer instead of swab extract into a reaction well containing PCR amplification mix Note Additional n
66. ins reagents for rapid DNA preparation from buccal swabs prior to amplification The procedure lyses cells contained on the swab head and releases into solution sufficient DNA for STR amplification A small volume of the final swab extract is added to the PowerPlex reaction The PunchSolution Kit is used to process punches from nonFTA storage cards containing buccal samples prior to direct amplification For casework or samples that require DNA purification we recommend the DNA IQ System Cat DC6700 which is a DNA isolation and quantitation system designed specifically for forensic and paternity samples 26 This system uses paramagnetic particles to prepare clean samples for STR analysis easily and efficiently and can be used to extract DNA from stains or liquid samples such as blood or solutions The DNA IQ Resin eliminates PCR inhibitors and contaminants frequently encountered in casework samples With DNA rich samples the DNA IQ System delivers a consistent amount of total DNA The system has been used to isolate DNA from routine sample types including buccal swabs stains on FTA paper and liquid blood Additionally DNA has been isolated from casework samples such as tissue differentially separated sexual assault samples and stains on support materials The DNA IQ System has been tested with the PowerPlex Systems to ensure a streamlined process See Section 9 E for ordering information For applications requiring hu
67. inted in USA Part TMD024 Revised 6 14 Page 73 9 A Advantages of Using the Loci in the PowerPlex ESX 17 System continued a Table 4 The PowerPlex ESX 17 System Locus Specific Information Promega Repeat Sequence STR Locus Label Chromosomal Location 5 fi y D18551 Fluorescein 18q21 33 AGAA 20 59 1Mb D21S11 Fluorescein 21q21 1 TCTA Complex 20 19 476Mb THO1 Fluorescein 11p15 5 AATG 20 2 149Mb D3S1358 Fluorescein 3p21 31 TCTA Complex 45 557Mb Amelogenin Fluorescein Xp22 1 22 3 and Y NA D16S539 JOE 16q24 1 GATA 84 944Mb D2S1338 JOE 2q35 TGCC TTCC 218 705Mb D1S1656 JOE 1942 TAGA Complex 228 972Mb D10S1248 JOE 10q26 3 GGAA 130 567Mb FGA TMR ET 4q28 THE 155 866Mb Complex 20 D8S1179 TMR ET 8q24 13 TCTA Complex 20 125 976Mb vWA TMR ET 12p13 31 TCTA 5 963Mb Complex 20 D22S1045 TMR ET 22q12 3 ATT 35 779Mb SE33 CXR ET 6ql4 AAAG Complex 89 043Mb D19S433 CXR ET 19q12 AAGG Complex 35 109Mb D128391 CXR ET 12p12 AGAT AGAC Complex 12 341Mb D25441 CXR ET 2p14 TCTA 68 214Mb 1Information about chromosomal location of these loci can be found in references 21 and 22 and at www cstl nist gov biotech strbase chrom htm The August 1997 report 23 24 of the DNA Commission of the International Society for Forensic Haemogenetics ISFH states 1 for STR loci within coding genes the coding strand shall be used and the repeat sequence motif defined using the first possible 5 nucl
68. ion volume 3 After completion of the thermal cycling protocol store amplified samples at 20 C in a light protected box Note Long term storage of amplified samples at 4 C or higher may produce artifacts Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 9 4 B Direct Amplification of DNA from Storage Card Punches Materials to Be Supplied by the User GeneAmp PCR System 9700 thermal cycler Applied Biosystems microcentrifuge e MicroAmp optical 96 well reaction plate Applied Biosystems e aerosol resistant pipette tips see Section 9 E PunchSolution Kit Cat DC9271 5X AmpSolution Reagent Cat DM1231 also supplied with the PunchSolution Kit 1 2mm Harris Micro Punch or equivalent manual punch and cutting mat or automated punch system This section contains a protocol for direct amplification of DNA from storage card punches using the PowerPlex ESX 17 System and GeneAmp PCR System 9700 thermal cycler When using the protocol detailed below add the number of 1 2mm storage card punches indicated below to each 25pl amplification reaction Note You will need to optimize and validate the number of storage card punches per reaction in your laboratory See the PCR Optimization recommendations at the end of this section
69. ize Standard column select the size standard that was imported in Section 6 G or created in Section 6 H 8 If analyzing data from an ABI PRISM 310 Genetic Analyzer ensure that the appropriate matrix file is selected in the Matrix column 9 Select Analyze green arrow button to start data analysis Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 47 6J Creating a Databasing or Paternity Analysis Method with GeneMapper ID Software Version 3 2 w p e Select Tools then GeneMapper Manager Select the Analysis Methods tab Select New and a new analysis method dialog box will open Select HID and select OK Note If you do not see the HID option you do not have the GeneMapper ID software Contact Applied Biosystems Enter a descriptive name for the analysis method such as PowerPlexESX 17_20 filter Select the Allele tab Figure 21 Select the bins text file that was imported in Section 6 F Ensure that the Use marker specific stutter ratio if available box is checked Enter the values shown in Figure 21 for proper filtering of peaks when using the PowerPlex ESX 17 System For an explanation of the proper usage and effect of these settings refer to the Applied Biosystems user bulletin titled In
70. ll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 27 5 B Detection of Amplified Fragments Using the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 or the Applied Biosystems 3130 or 3130x Genetic Analyzer with Data Collection Software Version 3 0 continued The quality of formamide is critical Use Hi Di formamide Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause breakdown of formamide Poor quality formamide may contain ions that compete with DNA during injection which results in lower peak heights and reduced sensitivity A longer injection time may not increase the signal Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning label and take appropriate precautions when handling this substance Always wear gloves and safety glasses when working with formamide Sample Preparation 1 Thaw the CC5 Internal Lane Standard 500 Note Centrifuge tube briefly to bring contents to the bottom then vortex for 15 seconds before each use Do not centrifuge after vortexing as this may cause the size standard to be concentrated at the bottom of the tube 2 Prepare a loading cocktail by combining and mixing CC5 Internal Lane Standard 500 and Hi Di formamide as follows 1 0p1 CC5 ILS 500 x samples
71. llowing error message appears when data are displayed Some selected sample s do not contain analysis data Those sample s will not be shown Error message after attempting Unable to save panel data java SQLEException ORA 00001 unique constraint IFA CKP_NNN violated to import panels and bins text files If none of the samples had matrices applied when run on the ABI PRISM 310 Genetic Analyzer no data will be displayed Apply a matrix file during analysis in the GeneMapper ID software and re analyze There was a conflict between different sets of panels and bins text files Check to be sure that the bins are installed properly If not delete all panels and bins text files and re import files in a different order Allelic ladder peaks labeled off ladder GeneMapper ID software was not used or microsatellite analysis settings were used instead of HID analysis settings GeneMapper software does not use the same algorithms as GeneMapper ID software and cannot correct for sizing differences using the allelic ladder Promega recommends using GeneMapper ID software to analyze PowerPlex reactions If using GeneMapper ID software version 3 2 be sure that the analysis method selected is an HID method This can be verified by opening the analysis method using the GeneMapper Manager then selecting the General tab The analysis type cannot be changed If the method is not HID it should be deleted an
72. llowing change was made to the 6 14 revision of this document Legal disclaimers were updated 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 U S Pat Nos 5 843 660 6 479 235 6 221 598 and 7 008 771 Australian Pat No 724531 Canadian Pat Nos 2 118 048 and 2 251 793 Korean Pat No 290332 Singapore Pat No 57050 Japanese Pat Nos 3602142 and 4034293 Chinese Pat Nos ZL99813729 4 and ZL97194967 0 European Pat No 0960207 and other patents pending STR loci are the subject of U S Pat No RE 37 984 German Pat No DE 38 34 636 C2 and other patents issued to the Max Planck Gesellschaft zur F rderung der Wissenschaften e V Germany Allele sequences for one or more of the loci WA FGA D8S1179 D21S11 and D18551 in allelic ladder mixtures is licensed under U S Pat Nos 7 087 380 7 645 580 Australia Pat No 2003200444 and corresponding patent claims outside the US TMR ET CXR ET and CC5 dyes are proprietary This product or portions thereof is manufactured and sold under license from GE Healthcare under Australia Pat No 692230 Austria Pat No E236994 Belgium Pat No 0743987 Canada Pat No 2231475 EP Pat Nos 0743987 and 0851867 France Pat Nos 0743987 and 0851867 Germany Pat Nos 19581489 69530286 8 and 0851867 Italy Pat Nos 07
73. lysis using GeneMapper ID software version 3 2 you will need the proper panels and bins text files PowerPlex_ESX_Panels_vX x txt and PowerPlex_ESX_Bins_vX x txt files where X x refers to the most recent version of the panels and bins text files Getting Started 1 To obtain the panels and bins text files for the PowerPlex ESX 17 System go to www promega com resources tools genemapper id softwarepanels and bin sets 2 Select the PowerPlex System that you are using and select GeneMapper ID Enter your contact information and select Submit 3 Save the PowerPlex_ESX_Panels_vX x txt and PowerPlex_ESX_Bins_vX x txt files to a known location on your computer Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Page 42 Printed in USA Revised 6 14 Importing Panels and Bins Text Files C o These instructions loosely follow the Applied Biosystems GeneMapper ID software tutorial pages 1 4 Promega Open the GeneMapper ID software version 3 2 Select Tools then Panel Manager Highlight the Panel Manager icon in the upper left navigation pane Select File then Import Panels or oe oe oN oe Navigate to the panels text file downloaded in the Getting Started section above Select the file then Import 6 In the navigation pane highlight the
74. man specific DNA quantification the Plexor HY System Cat DC1000 was developed 27 See Section 9 E for ordering information For information about automation of Promega chemistries on automated workstations using Identity Automation solutions contact your local Promega Branch Office or Distributor contact information available at www promega com support worldwide contacts e mail genetic promega com or visit www promega com idautomation Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 77 C 9 C The CC5 Internal Lane Standard 500 o The CC5 Internal Lane Standard 500 contains 21 DNA fragments of 60 65 80 Promega 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 and 500 bases in length Figure 25 Each fragment is labeled with CC5 dye and can be detected separately as a fifth color in the presence of PowerPlex ESX 17 amplified material The CC5 ILS 500 is designed for use in each CE injection to increase precision in analyses when using the PowerPlex ESX 17 System Protocols to prepare and use this internal lane standard are provided in Section 5 A low level artifact peak at approximately 172 bases may be observed with the CC5 ILS 500 in the orange channel The peak height of this artifact may vary from lot to lot and m
75. n Figure 15 10 Enter the project name 11 Choose the applicable security group from the drop down menu then select OK When the analysis is finished the Analysis Summary screen will appear We recommend that you review any yellow or red marker header bars in the plots view and handle them according to laboratory standard operating procedures Navigate to the Genotype tab or Samples tab To assist the review of any low quality samples use the default Data Interpretation plot settings and review the contents in the Quality Value Details table The values displayed in the Analysis Method Peak Quality and SQ amp GQ Settings tabs are defaults and will affect the quality values displayed in the plot settings We recommend that you modify the values in these tabs to fit your laboratory s data analysis protocols Importing PowerPlex ESX Panels and Bins Text Files into GeneMapper ID Software Version 3 2 To facilitate analysis of data generated with the PowerPlex ESX 17 System we have created panels and bins text files to allow automatic assignment of genotypes using GeneMapper ID software version 3 2 We recommend that users of GeneMapper ID software version 3 2 complete the Applied Biosystems GeneMapper ID Software Human Identification Analysis Tutorial to familiarize themselves with proper operation of the software For GeneMapper ID software version 3 1 users we recommend upgrading to version 3 2 For ana
76. n select the size standard that was imported in Section 6 G or created in Section 6 H 8 If analyzing data from an ABI PRISM 310 Genetic Analyzer ensure that the appropriate matrix file is selected in the Matrix column 9 Select Analyze green arrow button to start the data analysis Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 49 C 6 K Sample Analysis Using the GeneScan Software and Windows Operating o Systems Promega 1 Analyze data using the GeneScan software 2 Review the raw data for one or more sample runs Highlight the sample file name then in the Sample menu select raw data Move the cursor so that the crosshair is on the baseline to the right of the large primer peak before the first internal lane standard peak orange Use the X value number shown at the bottom left of the window for the start position in the analysis parameters 3 The recommended analysis parameters are shown in Figure 22 A PowerPlex_ESX gsp R Analysis Range Size Call Range C Full Range C Full Range This Range Data Points This Range Base Pairs Stat E600 Min 50 Stop fioooo Max foo Data Processing Size Calling Method 2nd Order Least Squares Smooth Optio ts C 31d Order Least Squares C None c A y Lish Cubic Spline Interpola
77. n 5 C Data Analysis Section 6 GeneMapper D X Software GeneScan Software Version 1 2 and Windows Operating Systems GeneMapper D Software Version 3 2 Figure 1 An overview of the PowerPlex ESX 17 System protocol Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 3 Product Components and Storage Conditions Product Size Cat PowerPlex ESX 17 System 100 reactions DC6721 Not For Medical Diagnostic Use This system contains sufficient reagents for 100 reactions of 25 ul each Includes Pre amplification Components Box 500ul PowerPlex ESX 5X Master Mix 250ul PowerPlex ESX 17 10X Primer Pair Mix 25ul 2800M Control DNA 10ng ul 5x 1 250u1 Water Amplification Grade Post amplification Components Box 50ul PowerPlex ESX 17 Allelic Ladder Mix 300ul CC5 Internal Lane Standard 500 Product Size Cat PowerPlex ESX 17 System 400 reactions DC6720 Not For Medical Diagnostic Use This system contains sufficient reagents for 400 reactions of 25 ul each Includes Pre amplification Components Box 4 x 500u1 PowerPlex ESX 5X Master Mix 4 x 250ul PowerPlex ESX 17 10X Primer Pair Mix 25ul 2800M Control DNA 10ng ul 10 x 1 250ul Water Amplification Grade Post amplification Components Box 4 x 50ul PowerPlex ESX 17 Allelic Ladder
78. n USA Page 20 Revised 6 14 3 To create a new Size Standard for the QC protocol navigate to the Library CG Select Size Standards then select Create Alternatively a previously eo created Size Standard may be used Promega Assign the Size Standard the name ILS500 or another appropriate name 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 4 a ra Oeteced ia gt Die Me T o Ma I a o o a imanan f Po Jie Gip Aaa wie Pip A re Anth Hiter we Santed DES Dugase stye 30 voe Aiia G eee are Ben Samid detesta eg ALA A TH 9227TA Figure 4 The Create New Size Standard window Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 21 C 5 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or o 3500xL Genetic Analyzer continued Promega 4 Tocreate a new QC Protocol navigate to the Library Select QC Protocols then select Create Alternatively a previously created QC Protocol may be used Assign a descriptive protocol name Select the size standard created in Step 3 The settings for the QC protocol should be based on the internally validated condition
79. nd from the Manage menu select Plates then Create 9 Assign a descriptive plate name Select the plate type HID from the drop down menu Figure 9 ete pee eee elem 9254TA Figure 9 Defining plate properties 10 Select Assign Plate Contents Figure 10 11 Assign sample names to wells amp nm amme Deha Gta e i ee ee a ae Ebre Hinna f asa whore i Caw i8 ome ine Gm Be O g B 9255TA Figure 10 Assigning plate contents Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Printed in USA Page 26 Revised 6 14 12 In the lower left portion of the screen under Assays use the Add from C Library option to select the Assay created in Step 5 or one previously o created Click on the Add to Plate button and close the window Promega 13 Under File Name Convention use the Add from Library option to select the File Name Convention created in Step 6 or one previously created Click on the Add to Plate button and close the window 14 Under Results Groups use the Add from Library option to select the Results Group created in Step 7 or one previously created Click on the Add to Plate button and close the window 15 Highlight the sample wells then select the boxes in the Assays File Name Conve
80. ndow in the Views menu 1 Transfer the PowerTyper_ESX_17 Macro file to a designated location on your computer hard drive 2 Open the Genotyper software then the PowerTyper_ESX_17 Macro file For questions about the Genotyper software refer to the Genotyper Analysis Software User s Manual 3 Inthe File menu select Import and import the GeneScan project or sample files to be analyzed Import the fluorescein JOE TMR ET CXR ET and CC5 dyes Note To select the dye colors to be imported select Set Preferences in the Edit menu 4 Double click on the Check ILS macro The macros are listed at the bottom left corner of the active window A plots window will be displayed to show the internal lane standard i e CC5 ILS 500 in the CC5 orange channel Scroll down to view and confirm that the internal lane standard fragment sizes are correct If necessary re analyze samples using the GeneScan software and redefine internal lane standard fragments Notes 1 The software uses one ladder sample to determine allele sizes The macro uses the first ladder sample imported for allele designations 2 The Macintosh version of the Genotyper software displays the fragment sizes for the internal lane standard However it displays the raw data for the electropherogram instead of the analyzed data Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 2
81. ne 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Page 28 Printed in USA Revised 6 14 6 Centrifuge plate briefly to remove air bubbles from the wells C o 7 Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to Promega 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 ABI PRISM 3100 or 3100 A vant Genetic Analyzer with Data Collection Software Version 2 0 and the Applied Biosystems 3130 or 3130x1 Genetic Analyzer with Data Collection Software Version 3 0 with the following exceptions 1 Inthe Module Manager select New Select Regular in the Type drop down list and select HIDFragmentAnalysis36_POP4 in the Template drop down list Confirm that the injection time is 5 seconds the injection voltage is 3kV and the run time is 1 500 seconds Give a descriptive name to your run module and select OK Note Instrument sensitivities can vary The injection time and voltage may be adjusted in the Module Manager A suggested range for the injection time is 3 22 seconds and for the injection voltage is 1 3kV 2 Inthe Protocol Manager select New Type a name for your protocol
82. ning PCR amplification mix as a negative amplification control Note An additional negative control with a blank punch may be performed to detect contamination from the storage card or punch device 10 Seal the plate and briefly centrifuge the plate to bring storage card punches to the bottom of the wells and remove any air bubbles Note Place the amplification plate in the thermal cycler and start the thermal cycling program as soon as the PCR amplification mix is added to all wells Prolonged storage of assembled reactions prior to cycling may result in poor performance i e lower peak heights for large amplicons Thermal Cycling Amplification and detection instrumentation may vary You will need to optimize protocols including the number of storage card punches cycle number injection conditions and loading volume for your laboratory instrumentation Testing at Promega shows that 24 cycles works well for a variety of sample types Buccal samples may require more amplification cycles than blood samples Cycle number will need to be optimized in each laboratory for each sample type that is amplified Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Printed in USA Page 12 Revised 6 14 1 Place the MicroAmp plate in the thermal cycler A 2 Select and run the recommended protocol The preferr
83. ntions and Results Groups that pertain to those samples 16 Select Link Plate for Run 17 The Load Plate window will appear Select Yes 18 Inthe Run Information window Figure 11 assign a Run Name Select Start Run not shown Onbbend faa gt dewey Mertens Toi Mew tt hae ha bti ernea Pes san edt the Pun Hiara ber memes ted Utes e emer tee Bare Commeited iha iots Adea Cwta Hane Poems senp ra conn Petes mm oe E me romen Pee A me woth i Pete i bt Pare OC gt e neesan Tes iE reene are Aa Bowe Sents ven enaere Ree a amp vem hatt Rome iii eL a Figure 11 Assigning a run name 5 B Detection of Amplified Fragments Using the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 or the Applied Biosystems 3130 or 3130x Genetic Analyzer with Data Collection Software Version 3 0 Materials to Be Supplied by the User 95 C dry heating block water bath or thermal cycler e crushed ice or ice water bath centrifuge compatible with 96 well plates e aerosol resistant pipette tips e 3100 or 3130 capillary array 36cm performance optimized polymer 4 POP 4 polymer for the 3100 or 3130 e 10X genetic analyzer buffer with EDTA e MicroAmp optical 96 well plate or equivalent and septa e Hi Di formamide Applied Biosystems Cat 4311320 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA To
84. ntrol Using the protocols defined in this manual the negative control should be devoid of amplification products Observe the results for the 2800M Control DNA Compare the 2800M DNA allelic repeat sizes with the locus specific allelic ladder The expected 2800M DNA allele designations for each locus are listed in Table 6 Section 9 A Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Page 54 Printed in USA Revised 6 14 6 N Results C o Representative results of the PowerPlex ESX 17 System are shown in Figure 23 P The PowerPlex FSX 17 Allelic Ladder Mix is shown in Figure 24 mega Figure 23 The PowerPlex ESX 17 System A single source template DNA 0 5ng was amplified using the PowerPlex ESX 17 System Amplification products were mixed with CC5 Internal Lane Standard 500 and analyzed with an Applied Biosystems 3130 Genetic Analyzer using a 3kV 5 second injection Results were analyzed using GeneMapper ID software version 3 2 Panel A An electropherogram showing the peaks of the fluorescein labeled loci Amelogenin D3S1358 TH01 D21S11 and D18551 Panel B An electropherogram showing the peaks of the JOE labeled loci D10S1248 D1S1656 D2S1338 and D165539 Panel C An electropherogram showing the peaks of the TMR ET labeled loci D2251045 vWA D8S1179 and FGA Panel D An electropher
85. nued o 6 Vortex the PCR amplification mix for 5 10 seconds then pipet 25ul of PCR Promega amplification mix into each reaction well Failure to vortex the PCR amplification mix sufficiently can result in poor amplification or locus to locus imbalance N For FTA storage cards add one or two 1 2mm punches from a storage card containing a buccal sample or one 1 2mm punch from a storage card containing whole blood to the appropriate wells of the reaction plate For nonFTA card punches add PCR amplification mix to the PunchSolution Reagent treated punches Note It also is acceptable to add the FTA card punch first then add the PCR amplification mix 8 For the positive amplification control add 1p 10ng of the 2800M Control DNA to a reaction well containing 25pl of PCR amplification mix Notes 1 Do not include blank storage card punches in the positive control reactions 2 Optimization of the amount of 2800M Control DNA may be required depending on thermal cycling conditions and laboratory preferences Typically 10ng of 2800M Control DNA is sufficient to provide a robust profile using the cycle numbers recommended here A one cycle reduction in cycle number will require a twofold increase in mass of DNA template to generate similar signal intensity Similarly a one cycle increase in cycle number will require a twofold reduction in the amount of 2800M Control DNA to avoid signal saturation 9 Reserve a well contai
86. 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 e Be sure to perform the 20 minute extension step at 60 C after thermal cycling Section 4 Decrease cycle number e Increase the final extension time Peak height imbalance Excess DNA in the amplification reaction can result in locus to locus imbalance within a dye channel such that the peak heights at the smaller loci are greater than those at the larger loci ski slope effect Use less swab extract or reduce the cycle number Active SwabSolution Reagent carried over into the amplification reaction Larger loci are most susceptible to reagent carryover and will drop out before the smaller loci Ensure that the heat block is heating to 70 C 90 C if using 2 2ml Square Well Deep Well Plates and samples were incubated for the full 30 minutes Incubation for shorter time periods may result in incomplete reagent inactivation Do not use an incubator set at 70 C to incubate tubes or plates heat transfer is inefficient and will result in poor performance Only use a heat block to maintain efficient heat transfer Inactive SwabSolution Reagent Thaw the SwabSolution Reagent completely in a 37 C water bath and mix by gentle inversion Store the SwabSolution Reagent at 2 10 C Do not store reagents in the refrigerator door where the temperature can fluctuate Do not re freeze as t
87. ogram showing the peaks of the CXR ET labeled loci D2S441 D12S391 D19S433 and SE33 Panel E An electropherogram showing the 60bp to 500bp fragments of the CC5 Internal Lane Standard 500 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 55 8247TA 8468TA Figure 24 The PowerPlex ESX 17 Allelic Ladder Mix The PowerPlex ESX 17 Allelic Ladder Mix was analyzed with an Applied Biosystems 3130 Genetic Analyzer using a 3kV 5 second injection The sample file was analyzed with the GeneMapper ID software version 3 2 and PowerPlex ESX panels and bins text files Panel A The fluorescein labeled allelic ladder components and their allele designations Panel B The JOE labeled allelic ladder components and their allele designations Note Panels C and D are shown on the next page Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Printed in USA Page 56 Revised 6 14 8468TB Figure 24 The PowerPlex ESX 17 Allelic Ladder Mix continued The PowerPlex ESX 17 Allelic Ladder Mix was analyzed with an Applied Biosystems 3130 Genetic Analyzer using a 3kV 5 second injection The sample file was analyzed with the GeneMapper ID software version
88. older 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 In the Analysis Method column select the analysis method created previously in this section 5 In the Panel column select the panels file that was imported in Section 6 A 6 In the Size Standard column select the size standard that was imported in Section 6 B or created in Section 6 C 7 If analyzing data from an ABI PRISM 310 Genetic Analyzer ensure that the appropriate matrix file is selected in the Matrix column 8 Select Analyze green arrow button to start data analysis Note By default the software displays the Analysis Requirement Summary Allelic Ladder Analysis Summary and Analysis Summary windows after quality review by the software Ensure that all requirements are met as each window appears If you do not have the Analysis Requirement Summary window activated you may need to do additional manual troubleshooting Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 41 Oo Promega 6 E 6 F Creating a Databasing or Paternity Analysis Method with GeneMapper ID X Software Version 1 2 continued 9 If all analysis requirements are met the Save Project window will ope
89. on voltage or the amount of product mixed with loading cocktail may need to be increased or decreased To modify the injection time or injection voltage in the run module select Instrument Protocol from the Library menu in the data collection software If peak heights are higher than desired use less DNA template in the amplification reactions or reduce the number of cycles in the amplification program by 2 4 cycles to achieve the desired signal intensity If the injection time or voltage is reduced a decreased peak amplitude threshold for the orange channel may be required for proper sizing 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 Centrifuge plate briefly to remove air bubbles from the wells Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading the instrument Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Page 18 Printed in USA Revised 6 14 Instrument Preparation 4 Refer to the Applied Biosystems 3500 3500xL Genetic Analyzer User Guide for the instrument maintenance schedule and instructions to install the capillary array Promega buffers and polymer pouch
90. on re annealing Artifacts of STR amplification Amplification of STRs can result in artifacts that appear as peaks one base smaller than the allele due to incomplete addition of the 3 A residue Be sure to perform the 45 minute extension step at 60 C after thermal cycling Section 4 CE related artifacts spikes Minor voltage changes or urea crystals passing by the laser can cause spikes or unexpected peaks Spikes sometimes appear in one color but often are easily identified by their presence in more than one color Re inject samples to confirm Incorrect G5 spectral was active Re run samples and confirm that the PowerPlex 5 dye G5 spectral is set for G5 See instructions on instrument preparation in Section 5 Pull up or bleedthrough Pull up can occur when peak heights are too high or if a poor or incorrect matrix is applied to the samples For the ABI PRISM 310 Genetic Analyzer generate a new matrix and apply it to the samples For the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 3130x1 3500 and 3500xL Genetic Analyzers perform a new spectral calibration and re run the samples e Instrument sensitivities can vary Optimize the injection conditions See Section 5 CE related artifacts contaminants Contaminants in the water used with the instrument or to dilute the 10X genetic analyzer buffer may generate peaks in the fluorescein and JOE channels Use auto
91. pH due to added Tris HCI available magnesium concentration due to chelation by EDTA or other PCR inhibitors which may be present at low concentrations depending on the source of the template DNA and the extraction procedure used 3Apparent DNA concentrations can differ depending on the DNA quantification method used 14 The amount of DNA template recommended here is based on DNA concentrations determined by measuring absorbance at 260nm We strongly recommend that you perform experiments to determine the optimal DNA amount based on your DNA quantification method The PowerPlex ESX 17 System is optimized and balanced for 0 5ng of DNA template The amount of DNA template used in your laboratory should be based on the results of your internal validation and may be different 5 Vortex the PCR amplification mix for 5 10 seconds then pipet PCR amplification mix into each reaction well Failure to vortex the PCR amplification mix sufficiently can result in poor amplification or locus to locus imbalance os Add the template DNA for each sample to the respective well containing PCR amplification mix Note The PowerPlex ESX 17 System is optimized and balanced for 0 5ng of DNA template The amount of DNA template used in your laboratory should be based on the results of your internal validation and may be different 7 For the positive amplification control vortex the tube of 2800M Control DNA then dilute an aliquot to 0 5ng in
92. ples were incubated for 30 minutes until dry Incubation for shorter time periods may result in incomplete inactivation of the PunchSolution Reagent Using a smaller amplification reaction volume may compromise performance when using 10 1 of PunchSolution Reagent Reducing the PunchSolution Reagent volume may improve results for reactions with reduced amplification volumes Optimization and validation are required Inactive PunchSolution Reagent Thaw the PunchSolution Reagent at 2 10 C Do not store reagents in the refrigerator door where the temperature can fluctuate Do not refreeze avoid multiple freeze thaw cycles as this may reduce activity Carryover of excess PunchSolution Reagent into amplification reaction We recommend treating one 1 2mm nonFTA card punch with 101 of PunchSolution Reagent and using one punch per 25ul amplification reaction Use of a smaller amplification reaction volume may compromise performance if using 10u of PunchSolution Reagent Reducing the PunchSolution Reagent volume may improve results when using a reduced amplification reaction volume Laboratory optimization and validation is required Extreme variability in sample to sample peak heights There can be significant individual to individual variability in the deposition of cells onto a punch resulting in peak height variability between samples The PunchSolution Kit increases the recovery of amplifiable DNA
93. plification Grade 6 250ul 5 x 1250p DW0991 5X AmpSolution Reagent 500ul DM1231 Not for Medical Diagnostic Use Sample Preparation Systems Product Size Cat DNA IQ System 100 reactions DC6701 400 reactions DC6700 Differex System 50 samples DC6801 200 samples DC6800 Tissue and Hair Extraction Kit for use with DNA IQ 100 reactions DC6740 Maxwell 16 Forensic Instrument each AS3060 DNA IQ Reference Sample Kit for Maxwell 16 48 preps AS1040 DNA IQ Casework Pro Kit for Maxwell 16 48 preps AS1240 Plexor HY System 200 reactions DC1001 800 reactions DC1000 Slicprep 96 Device 10 pack V1391 Not for Medical Diagnostic Use For Research Use Only Not for use in diagnostic procedures ART Aerosol Resistant Tips Product Volume Size tips pack Cat ART 10 Ultramicro Pipet Tip 0 5 10ul 960 DY1051 ART 20E Ultramicro Pipet Tip 0 5 10ul 960 DY1061 ART 20P Pipet Tip 20ul 960 DY1071 ART GEL Gel Loading Pipet Tip 100p1 960 DY1081 ART 100 Pipet Tip 10041 960 DY1101 ART 100E Pipet Tip 10041 960 DY1111 ART 200 Pipet Tip 20041 960 DY1121 ART 1000E Pipet Tip 1 000u1 800 DY1131 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Page 80 Printed in USA Revised 6 14 9 F Summary of Changes C o The fo
94. promega com Part TMD024 Page 38 Printed in USA Revised 6 14 10 If all analysis requirements are met the Save Project window will open C Figure 15 o Save Project Name Security Group GeneMapper ID Securty Group M Lice JL cne J e Figure 15 The Save Project window 11 Enter the project name 12 Choose the applicable security group from the drop down menu then select OK When the analysis is finished the Analysis Summary screen will appear We recommend that you review any yellow or red marker header bars in the plots view and handle them according to laboratory standard operating procedures Navigate to the Genotype tab or Samples tab To assist the review of any low quality samples use the default Data Interpretation plot settings and review the contents in the Quality Value Details table The values displayed in the Analysis Method Peak Quality and SQ amp GQ Settings tabs are defaults and will affect the quality values displayed in the plot settings We recommend that you modify the values in these tabs to fit your laboratory s data analysis protocols 6 E Creating a Databasing or Paternity Analysis Method with GeneMapper ID X Software Version 1 2 These instructions are intended as a guide to start analyzing data in GeneMapper ID X software They are not intended as a comprehensive guide for using the GeneMapper ID X software We recommend that users contact Applied Biosystem
95. proximately 35 minutes for syringe pumping sample injection and sample electrophoresis Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Page 32 Printed in USA Revised 6 14 6 Data Analysis C eI 6 A Importing PowerPlex ESX Panels Bins and Stutter Text Files into P GeneMapper ID X Software Version 1 2 mega To facilitate analysis of data generated with the PowerPlex ESX 17 System we have created panels and bins text files to allow automatic assignment of genotypes using GeneMapper ID X software We recommend that users receive training from Applied Biosystems on the GeneMapper ID X software to familiarize themselves with proper operation of the software Note The panels bins and stutter text files mentioned here are compatible with earlier versions of the GeneMapper ID X software Getting Started 1 To obtain the proper panels bins and stutter text files for the PowerPlex ESX 17 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 3 Save the PowerPlex_ESX_Panels_IDX_vX x txt PowerPlex_ESX_Bins_IDX_vX x txt and PowerPlex_ESX_Stutter_IDX_vX x txt files where X x refers to the most recent version
96. r software This information must be entered to successfully analyze your data using the PowerTyper ESX 17 Macro Create a new GeneScan injection list Select the appropriate sample sheet by using the drop down menu Select the GS STR POP4 1ml G5 Module using the drop down menu Change the injection time to 3 seconds and the run time to 28 minutes Keep the settings for the remaining parameters as shown below Inj Secs 3 Inj kV 15 0 Run kV 15 0 Run C 60 Run Time 28 You may need to optimize the injection time for individual instruments Injection times of 2 5 seconds are suggested for samples that contain 0 5ng of template DNA Note Migration of fragments may vary slightly over the course of a long ABI PRISM 310 Genetic Analyzer run This may be due to changes in temperature or changes in the column When analyzing many samples injections of allelic ladder at different times throughout the run can aid in accurately genotyping samples Select the appropriate matrix file To analyze data automatically select the auto analyze checkbox and the appropriate analysis parameters and size standard Refer to the ABI PRISM 310 Genetic Analyzer User s Manual for specific information about these options After loading the sample tray and closing the doors select Run to start the capillary electrophoresis system Monitor electrophoresis by observing the raw data and status windows Each sample will take ap
97. rinted in USA Part TMD024 Revised 6 14 Page 29 5 B 5 C Detection of Amplified Fragments Using the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 or the Applied Biosystems 3130 or 3130x Genetic Analyzer with Data Collection Software Version 3 0 continued 6 Inthe spectral viewer select dye set G5 and confirm that the active dye set is the file generated for the PowerPlex 5 dye chemistry It is critical to select the correct G5 spectral for the PowerPlex 5 dye chemistry If the PowerPlex 5 dye chemistry is not the active dye set locate the PowerPlex 5 dye spectral in the List of Calibrations for Dye Set G5 and select Set 7 Inthe run scheduler locate the plate record that you just created in Steps 3 and 4 and click once on the name to highlight it 8 Once the plate record is highlighted click the plate graphic that corresponds to the plate on the autosampler that contains your amplified samples 9 When the plate record is linked to the plate the plate graphic will change from yellow to green and the green Run Instrument arrow becomes enabled 10 Click on the green Run Instrument arrow on the toolbar to start the sample run 11 Monitor electrophoresis by observing the run view array or capillaries viewer window in the data collection software Each injection will take approximately 40 minutes Detection of Amplified Fragments Using the A
98. s Instrument Artifact Sizes D21S11 D25441 ABI PRISM 310 and Applied Biosystems n 2 n 2 D1S1656 and SE33 3130 Genetic Analyzers Amelogenin ABI PRISM 310 and Applied Biosystems n 15 3130 Genetic Analyzers 65 71 bases Artifact migrates before Amelogenin ABI PRISM 310 Genetic 72 73 bases Analyzer only male and female samples 77 78 bases male samples only Applied Biosystems 3130 76 bases Genetic Analyzer only male and female samples 85 bases male samples only D10S1248 ABI PRISM 310 and Applied Biosystems 60 64 bases 3130 Genetic Analyzers Artifact migrates before D10S1248 The artifacts listed here are DNA dependent Two bases below and above the true allele peak respectively 5The n 1 artifact is more noticeable with high template amounts and allele peak heights These variably sized peaks on the ABI PRISM 310 and Applied Biosystems 3130 Genetic Analyzers may represent double stranded DNA derived from the Amelogenin amplicon double stranded DNA is known to migrate faster than single stranded DNA on capillary electrophoresis instruments This artifact is only seen with high peak heights for the X and Y alleles 5Low level DNA dependent artifact is noticeable only with high template amounts and allele peak heights The artifact migrates approximately 13 17 bases in front of the smallest allele allele 8 in D10S1248 This peak may be above or below analysis threshold depending on the
99. s for the PowerPlex ESX 17 System on the Applied Biosystems 3500 or 3500xL Genetic Analyzer Figure 5 shows one option for these settings Note Peak heights for the CC5 ILS 500 are generally lower than those for the other dyes Therefore the threshold for the orange dye may be lower than that for the other dyes TERE Dettowd t iney Metes Tosi Manage Pit O trey Hermer Sine sie B ya A iyt Fiye vam eect Heat wey Tom Pesi Lergttnate Thewvhett wm ates Catton eterna dta o f Une Bossimurng Mesrine Word P P t Meore Pess Hat Hih i Frsl Menten ee n Prirmammsi Degg se Shope Wer tuned Proh tat depo Thewstnatd Pooh Heed 9228TA Figure 5 The Create New QC Protocol window Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Printed in USA Page 22 Revised 6 14 5 To create a new Assay navigate to the Library Select Assays then select C Create Alternatively a previously created Assay may be used A In the Create New Assay window Figure 6 select the Instrument Promega Protocol created in Step 2 and the QC Protocol created in Step 4 Assign a descriptive assay name Select the application type HID An Assay is required for all named samples on a plate Pitoded Colar Bach butranent Protecet QC Protocol 9229TA Figure 6 T
100. s for training on the software Select Tools then GeneMapper ID X Manager Select the Analysis Methods tab Select New and a new analysis method dialog box will open we Ge Pa ya 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 PowerPlexESX 17 20 Filter 6 Select the Allele tab Figure 16 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 39 C 6 E Creating a Databasing or Paternity Analysis Method with GeneMapper ID X A Software Version 1 2 continued Promega 7 Select the bins text file that was imported in Section 6 A 8 We recommend the values shown in Figure 16 for proper filtering of stutter peaks when using the PowerPlex ESX 17 System You may need to optimize these settings In house validation should be performed Analysis Method Editor P General Mitlt Peak Detector Peak Quality SQ amp GQ Settings Dn Set PowePiax CSX Dns JOK v2 0 X J Use marker spectic stutter ratio and dstarce avaliable tanya Marker Repeat Type Tei Tere Porta Hexa Goba C t off Value 0 2 02 0 0 6 0 Mirus Ratio 0 0 T 0 0 0
101. se cycle number e Increase the final extension time Peak height imbalance Excessive amount of DNA Amplification of gt 20ng of template can result in an imbalance with smaller loci showing more product than larger loci e Use one or two 1 2mm punches from a storage card containing a buccal sample or one 1 2mm punch from a storage card containing whole blood Follow the manufacturer s recommendations when depositing sample onto the storage card e Decrease cycle number The reaction volume was too low This system is optimized for a final reaction volume of 25411 to overcome inhibitors present in FTA cards and PunchSolution Reagent Decreasing the reaction volume can result in suboptimal performance Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 63 7 B Direct Amplification of DNA from Storage Card Punches continued Symptoms Causes and Comments Peak height imbalance continued Amplification was inhibited when using more than one storage card punch with blood Use only one 1 2mm storage card punch with blood Active PunchSolution Reagent carried over into the amplification reaction Larger loci are most susceptible to carryover and will drop out before the smaller loci Ensure that the heat block was set at 70 C and sam
102. sensitivity of the capillary electrophoresis instrument Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Page 58 Printed in USA Revised 6 14 7 Troubleshooting w For questions not addressed here please contact your local Promega Branch Office or Distributor Contact information available at www promega com E mail genetic promega com Promega 7 A Amplification and Fragment Detection This section provides information about general amplification and detection For questions about direct amplification see Sections 7 B and 7 C Symptoms Causes and Comments Faint or absent allele peaks Impure template DNA Because of the small amount of template used this is rarely a problem Depending on the DNA extraction procedure used and sample source inhibitors might be present in the DNA sample Insufficient template Use the recommended amount of template DNA if available The PowerPlex ESX 5X Master Mix was not vortexed well before use Vortex the 5X Master Mix for 15 seconds before dispensing into the PCR amplification mix An air bubble formed at the bottom of the reaction tube Use a pipette to remove the air bubble or centrifuge the reactions briefly before thermal cycling High salt concentration or altered pH If the DNA template is stored in TE buffer that is not pH 8 0 or contains a higher EDTA
103. software will use the third ladder etc until all ladders in the project are used If an allelic ladder fails to be analyzed or if many off ladder alleles are found in the samples samples should be re analyzed using another ladder from the project Note FGA alleles 50 2 and 48 2 exhibit high stutter The PowerTyper ESX 17 Macro will label these stutter peaks as alleles 49 2 and 47 2 7 Double click on the Display Blue Data macro to display fluorescein data for all sample injections Scroll down to observe and edit as needed 8 Double click on the Display Green Data macro to display JOE data for all sample injections Scroll down to observe and edit as needed 9 Double click on the Display Yellow Data macro to display TMR ET data for all sample injections Scroll down to observe and edit as needed 10 Double click on the Display Red Data macro to display CXR ET data for all sample injections Scroll down to observe and edit as needed Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 53 6 L Sample Analysis Using the Genotyper Software and PowerTyper ESX 17 A Promega 1L 12 Macro continued Create the appropriate table by selecting the PowerTable Make Allele Table or Make CODIS Table macro The three available table formats are shown below
104. sseeeesssssneees 17 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or 3500xL Genetic AmalyZet sssssssssssecessssssnsee 17 B Detection of Amplified Fragments Using the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 or the Applied Biosystems 3130 or 3130x1 Genetic Analyzer with Data Collection Software Version 3 0 veces 27 C Detection of Amplified Fragments Using the ABI PRISM 310 Genetic ANALY Zetec das stsctecssedsssisnsittssssazibibecssssasianstcdecsaisecdecccsiavesc scdsaatvansseensnersuaseeereedl 30 6 Data Analysis usinnan E E AORE 33 A Importing PowerPlex ESX Panels Bins and Stutter Text Files into GeneMapper ID X Software Version 1 2 33 B Importing the CC5 ILS 500 Size Standard into GeneMapper ID X Software Version 1 2 csssssssssssesssssssscsssssssieesesssssseesesssey 34 C Creating a Size Standard with GeneMapper ID X Software Version 1 2 34 D Creating a Casework Analysis Method with GeneMapper ID X Software Version 1 2 cssssssssssssssssseeesssssssssseesssssseeeesessss 35 E Creating a Databasing or Paternity Analysis Method with GeneMapper ID X Software Version 1 2 39 F Importing PowerPlex ESX Panels and Bins Text Files into GeneMapper ID Software Version 3 2 sssssssssssssssssssssssssssemessesssesssesessee 42 G Importing the CC5 ILS 500 Size Standard into GeneMapper ID Software Version 3
105. ssssssasssznsssssssatscecccssaassscescssaaasscsdeconstassotaptnensborangsbensvussnbocsecdesstasies 73 A Advantages of Using the Loci in the PowerPlex ESX 17 System ald B DNA Extraction and Quantitation Methods and Automation Support lT C The CC5 Internal Lane Standard 500 scccssssssessesssssssssseeeeessssssseeeesssssssees 78 D Composition of Buffers and Solutions il E Related Products ald F Summary of Cham pesssssssssssssssssssssssssssscsssassscscccssaassssscccssinssasasnstessvesonsntsesdvesasosdeccbsostseess 81 Description STR short tandem repeat loci consist of short repetitive sequence elements 3 7 base pairs in length 1 4 These repeats are well distributed throughout the human genome and are a rich source of highly polymorphic markers which may be detected using the polymerase chain reaction 5 9 Alleles of STR loci are differentiated by the number of copies of the repeat sequence contained within the amplified region and are distinguished from one another using fluorescence detection following electrophoretic separation The PowerPlex ESX 17 System is used for human identification applications including forensic analysis relationship testing and research use This system allows co amplification and four color fluorescent detection of seventeen loci sixteen STR loci and Amelogenin including D18551 D21S11 TH01 D3S1358 Amelogenin D16S539 D2S1338 D1S1656 D10S1248 FGA D8S1179 vWA D2
106. stallation Procedures and New Features for GeneMapper ID Software 3 2 Analysis Method Editor HID Generali Alio Pask Detector Peat Quality Guilty Plage Ben Set PowerPex_ ESX Bins v2 0 F Use marker specitic stutter ratio it ava atte Marker Repeat Type Th Tetra O4 otf Vake 02 Q2 MirnutA Rado 0o oo MnusA Distance oo 00 Mirus Shior Rado oo Minus Saar Distance 2 3 25 Pius Stutter Rato 2 oo Pais Shutter Distance 25 oo oo Factory Deimas Lox _swrea_ 8188TA Figure 21 The GeneMapper ID Allele tab with settings for using a 20 peak filter Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Page 48 Printed in USA Revised 6 14 10 Select the Peak Detector tab We recommend the settings shown in Figure 20 Notes 1 Select full range or partial range for the analysis range When using a Promega partial range choose an appropriate analysis range based on your data Choose a start point after the primer peak and just before the first defined internal lane standard peak to help ensure proper sizing of the internal lane standard 2 The peak amplitude thresholds are the minimum peak heights at which the software will call a peak Values for peak amplitude thresholds are usually 50 150RFU Individual laboratories should determine their peak amplitude thresholds from int
107. stems 3130 3130x1 3500 and 3500xL Genetic Analyzers Perform a new spectral calibration and re run the samples e Poor matrix for the ABI PRISM 310 Genetic Analyzer Re run and optimize the matrix Make sure that the matrix applied was generated on the same instrument Incorrect G5 spectral was active Re run samples and confirm that the PowerPlex 5 dye G5 spectral is set for G5 See instructions for instrument preparation in Section 5 7 E GeneMapper ID Software Symptoms Causes and Comments Alleles not called To analyze samples with GeneMapper ID software the analysis parameters and size standard must both have Basic or Advanced as the analysis type If they are different an error is obtained To analyze samples with GeneMapper ID software at least one allelic ladder must be defined An insufficient number of CC5 ILS 500 fragments was defined Be sure to define at least two CC5 ILS 500 fragments smaller than the smallest sample peak or allelic ladder peak and at least two CC5 ILS 500 fragments larger than the largest sample peak or allelic ladder peak Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Page 68 Printed in USA Revised 6 14 Symptoms Causes and Comments C Alleles not called continued Run was too short and larger peaks in ILS were not cap
108. sult in overamplification and signal saturation If the signal is saturated repeat the amplification with a smaller punch a larger reaction volume or reduced cycle number Amplification of excess template for a given cycle number can result in overloading of the capillary upon electrokinetic injection The presence of excess DNA in the capillary makes it difficult to maintain the DNA in a denatured single stranded state Some single stranded DNA renatures and becomes double stranded Double stranded DNA migrates faster than single stranded DNA during capillary electrophoresis and appears as shadow peaks migrating in front of the main peaks If this occurs at a heterozygous locus it is sometimes possible to see two shadow peaks that differ in size from one another by approximately the same distance as the single stranded alleles Artifacts of STR amplification Direct amplification of gt 20ng of template can result in a higher number of artifact peaks Use the recommended punch size and number of punches Optimize the cycle number Do not reduce the reaction volume below 25ul See Section 6 N for additional information on stutter and artifacts Artifacts of STR amplification Amplification of STRs can result in artifacts that appear as peaks one base smaller than the allele due to incomplete addition of the 3 A residue e Be sure to perform a 45 minute extension step at 60 C after thermal cycling Section 4 e Decrea
109. t in incomplete inactivation of the PunchSolution Reagent We have not tested longer incubation times Inactive PunchSolution Reagent Thaw the PunchSolution Reagent at 2 10 C Do not store reagents in the refrigerator door where the temperature can fluctuate Do not refreeze as this may reduce activity Faint or absent peaks for the positive control reaction If the positive control reaction failed to amplify check to make sure that the correct amount of 2800M Control DNA was added to the reaction Do not include a blank punch in the positive control reaction Presence of a blank punch may inhibit amplification of 2800M Control DNA Optimize the amount of 2800M Control DNA for your thermal cycling conditions and laboratory preferences Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Page 62 Printed in USA Revised 6 14 Symptoms Causes and Comments C Extra peaks visible in one or Punch was contaminated Take punches from blank paper o or all color channels between samples Amplification of processed punches with high amounts of Promega DNA can result in artifact peaks due to overamplification resulting in saturating signal on the CE instrument We recommend one 1 2mm punch per 251 reaction Use of a larger punch size or a smaller reaction volume may re
110. tem is optimized for a final reaction volume of 25ul to overcome inhibitors present in FTA cards and PunchSolution Reagent Decreasing the reaction volume may result in suboptimal performance 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 different portion of the card Increasing cycle number can improve low peak heights Too much sample in the reaction Use one or two 1 2mm storage card punches Follow the manufacturer s recommendations when depositing sample onto the storage card With storage cards reducing the reaction volumes below 25p may result in amplification failure Amplification was inhibited when using more than one storage card punch with blood Use only one 1 2mm storage card punch with blood Make sure that the PCR amplification mix also contained AmpSolution Reagent Omission of AmpSolution Reagent from amplification reactions will result in amplification failure Active PunchSolution Reagent carried over into the amplification reaction when using nonFTA card punches Ensure that the heat block was set at 70 C and samples were incubated for 30 minutes until dry Incubation for shorter time periods may resul
111. texing as this may cause the size standard to be concentrated at the bottom of the tube 2 Prepare a loading cocktail by combining the CC5 Internal Lane Standard 500 and Hi Di formamide as follows 1 01 CCS ILS 500 x samples 24 0u1 Hi Di formamide x samples Note The volume of internal lane standard used in the loading cocktail can be increased or decreased to adjust the intensity of the size standard peaks Use a volume of CC5 ILS 500 that results in peak heights that are all consistently above the peak amplitude threshold of the orange dye channel determined as part of your internal validation If peak heights are too high we recommend altering the loading cocktail to contain 0 51 of CC5 ILS 500 and 24 5ul of Hi Di formamide 3 Vortex for 10 15 seconds to mix 4 Combine 25 0u1 of prepared loading cocktail and 1 0u1 of amplified sample or 1ul of PowerPlex ESX 17 Allelic Ladder Mix Notes 1 Instrument detection limits vary therefore injection time injection voltage or the amount of product mixed with loading cocktail may need to be increased or decreased Use the Module Manager in the data collection software to modify the injection time or voltage in the run module see Instrument Preparation below If the injection time or voltage is reduced a decreased peak amplitude threshold for the orange channel may be required for proper sizing 2 Use a volume of allelic ladder that results in peak hei
112. th punches from the same samples 4 Amplify samples using the thermal cycling protocol provided above but subject each plate to a different cycle number 5 Following amplification use your laboratory s validated separation and detection protocols to determine the optimal cycle number for the sample type and number of storage card punches Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 13 4 C Direct Amplification of DNA from Swabs Materials to Be Supplied by the User GeneAmp PCR System 9700 thermal cycler Applied Biosystems microcentrifuge MicroAmp optical 96 well reaction plate Applied Biosystems aerosol resistant pipette tips see Section 9 E SwabSolution Kit Cat DC8271 5X AmpSolution Reagent Cat DM1231 also supplied with the SwabSolution Kit This section contains a protocol for amplifying DNA from swab extracts using the PowerPlex ESX 17 System and GeneAmp PCR System 9700 thermal cycler Pretreat OmniSwab GE Healthcare or cotton swabs using the SwabSolution Kit Cat DC8271 as described in the SwabSolution Kit Technical Manual TMD037 to generate a swab extract Amplification Setup 1 Thaw the PowerPlex ESX 5X Master Mix PowerPlex ESX 17 10X Primer Pair Mix and Water Amplification Grade completely
113. the fragments Alleles may migrate outside of the panel range established using POP 4 polymer Size standard not called Starting data point was incorrect for the partial range chosen correctly in Section 6 K Adjust the starting data point in the analysis method Alternatively use a full range for the analysis Extra peaks in advanced mode size standard Open the Size Match Editor Highlight the extra peak select Edit and select delete size label Select auto adjust sizes Run was too short and larger peaks in ILS were not captured Not all CC5 ILS 500 peaks defined in the size standard were detected during the run e 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 were below threshold decrease the peak amplitude threshold in the analysis method for the orange channel to include peaks or increase the volume of CC5 ILS used in Section 5 If peaks were low quality redefine the size standard for the sample to skip these peaks Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 69 Symptoms 7 E GeneMapper ID Software continued Causes and Comments Error message Ei
114. the location of the run files Highlight the desired files then select Add to list followed by Add In the Sample Type column use the drop down menu to select Allelic Ladder Sample Positive Control or Negative Control as appropriate for the sample Every folder in the project must contain at least one allelic ladder injection that is designated as Allelic Ladder in the Sample Type column for proper genotyping In the Analysis Method column select the analysis method created previously in this section In the Panel column select the panels text file that was imported in Section 6 A In the Size Standard column select the size standard that was imported in Section 6 B or created in Section 6 C If analyzing data from an ABI PRISM 310 Genetic Analyzer ensure that the appropriate matrix file is selected in the Matrix column Select Analyze green arrow button to start data analysis Note By default the software displays the Analysis Requirement Summary Allelic Ladder Analysis Summary and Analysis Summary windows after quality review by the software Ensure that all requirements are met as each window appears If you do not have the Analysis Requirement Summary window activated you may need to do additional manual troubleshooting Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www
115. ther panel size standard or analysis method is invalid No alleles called but no error message appears The size standard and analysis method were not in the same mode Classic vs Basic or Advanced Be sure both files are set to the same mode either Classic or Basic or Advanced mode Panels text file was not selected for sample In the Panel column select the appropriate panels text file for the STR system that was used No size standard was selected In the Size Standard column be sure to select the appropriate size standard Size standard was not correctly defined or size peaks were missing Redefine size standard to include only peaks present in your sample Terminating analysis early or using short run times will cause larger ladder peaks to be missing This will cause your sizing quality to be flagged as red and no allele sizes will be called Error message Both the Bin Set used in the Analysis Method and the Panel must belong to the same Chemistry Kit The bins text file assigned to the analysis method was deleted In the GeneMapper Manager select the Analysis Methods tab and open the analysis method of interest Select the Allele tab and select an appropriate bins text file The wrong bins text file was chosen in the analysis method Allele tab Be sure to choose the appropriate bins text file as shown in Figure 19 Red bar appears during analysis of samples and the fo
116. tion eaw Local Southem Method Global Southem Method Peak Detection Baselning Peak Ampli Thr l BaseLine Window Size Auto Analysis Only Size Standard Min Peak Half Width B Pts lt iona gt Polynomial Degree 3 Peak Window Size fs Pts Slope Threshold for bo Peak Start Slope Threshold for bo Peak End 8200TA Figure 22 The Analysis Parameters window The start point of the analysis range which will vary is defined in Section 6 K Step 2 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Printed in USA Page 50 Revised 6 14 Notes C oj 1 The peak amplitude thresholds are the minimum peak heights at which the software will call a peak Values for peak amplitude Promega thresholds are usually 50 150RFU and should be determined by individual laboratories 2 Peak heights for the CC5 ILS 500 are generally lower than those for the other dyes Therefore the threshold for the orange dye may be lower than that for the other dyes Be sure that all CC5 ILS 500 peaks are consistently above the peak amplitude threshold for the orange dye channel determined as part of your internal validation 4 The analysis parameters can be saved in the Params folder in most installations this is located at C AppliedBio Shared Analysis Sizecaller Params 5 Apply the stored analysis parameters file
117. to the samples 6 Assign a new size standard Select a sample file and highlight the arrow next to size standard Select define new Assign the size standard peaks of 60 65 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 and 500 bases Store the size standard in the Size Standards folder at C Applied Bio Shared Analysis Sizecaller SizeStandards 7 Apply the size standard file to the samples then analyze the sample files See Section 6 L for additional information about the use of the PowerTyper ESX 17 Macro and Genotyper software Notes 1 Peak heights outside the linear range of the instrument may generate artifact peaks due to instrument saturation i e overloading the sample Bleedthrough pull ups from one color to another may be observed Saturated signal also may appear as two peaks split peak 2 If peak heights are not within the linear range of detection of the instrument the ratio of stutter peaks to real allele peaks increases and allele designations become difficult to interpret The balance of peak heights also may appear less uniform 3 There can be variation between instruments regarding the relative fluorescence levels detected using the same sample Furthermore different instruments vary in the relative efficiency of color detection affecting the dye color to dye color balance Promega Corporation 2800 Woods Hollow Road Madison WI 53711
118. tured o Not all CC5 ILS 500 peaks defined in the size standard were detected during the on Promega e Create a new size standard using the internal lane standard fragments present in the sample Re run samples using a longer run time A low quality allelic ladder was used during analysis Ensure that only high quality allelic ladders are used for analysis Off ladder alleles An allelic ladder from a different run than the samples was used Re analyze samples using an allelic ladder from the same run The GeneMapper ID software requires that the allelic ladder be imported from the same folder as the sample Be sure that the allelic ladder is in the same folder as the sample Create a new project and re analyze as described in Section 6 1 or 6 Panels text file selected for analysis was incorrect for the STR system used Assign correct panels text file that corresponds to the STR system used for amplification The allelic ladder was not identified as an allelic ladder in the Sample Type column The wrong analysis type was chosen for the analysis method Be sure to use the HID analysis type The internal lane standard was not properly identified in the sample Manually redefine the sizes of the size standard fragments in the sample A low quality allelic ladder was used during analysis Ensure that only high quality allelic ladders are used for analysis Incorrect polymer used Use of a polymer other than POP 4 polymer may change migration of
119. veramplification resulting in saturated signal on the CE instrument We recommend 2 of swab extract per 25y1 reaction Using more than 2u in a 25yl reaction or using 2u1 with a smaller reaction volume may result in overamplification and signal saturation If signal is saturated repeat the amplification with less swab extract or a reduced cycle number Amplification of excess template for a given cycle number resulted in overloading of the capillary upon electrokinetic injection In addition to signal saturation excess DNA in the capillary is difficult to maintain in a denatured single stranded state Some single stranded DNA renatures and becomes double stranded Double stranded DNA migrates faster than single stranded DNA during capillary electrophoresis and appears as shadow peaks migrating in front of the main peaks If this occurs at a heterozygous locus it is possible to observe the presence of two shadow peaks that differ in size by approximately the same distance as the single stranded alleles Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 65 7 C Amplification of DNA from Swabs continued Symptoms Causes and Comments Extra peaks visible in one or all color channels continued Artifacts of STR amplification Amplification
120. werTyper_ESX_17 Macro file used Confirm that the PowerTyper_ESX_17 Macro file matches the allelic ladder being used The plots window or allele table does not display all data The macros were not run in the proper order Use the POWER or POWER 20 Filter macro option All five dye colors were not imported For Genotyper software versions 2 5 and 3 6 or higher set preferences in the Edit menu to import fluorescein JOE TMR ET CXR ET and CC5 data Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Revised 6 14 Part TMD024 Page 71 C 7 F PowerTyper ESX 17 Macro continued o Symptoms Causes and Comments Promega The Check ILS macro All five dye colors were not imported For Genotyper displays an empty plot software versions 2 5 and 3 6 or higher set preferences in the window Edit menu to import fluorescein JOE TMR ET CXR ET and CC5 data Off ladder peaks Migration of samples changed slightly over the course of a CE run with many samples This may be due to changes in temperature or the CE column over time Use a different injection of allelic ladder to determine sizes in the PowerTyper ESX 17 Macro Do not use the first injection on a new column for the allelic ladder sample The base pair size of alleles was incorrect because incorrect fragment siz
121. when analyzing samples Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 59 7 A Amplification and Fragment Detection continued Symptoms Causes and Comments Extra peaks visible in one or all color channels Contamination with another template DNA or previously amplified DNA Cross contamination can be a problem Use aerosol resistant pipette tips and change gloves regularly Artifacts of STR amplification Amplification of gt 0 5ng template can result in a higher number of artifact peaks Use less template DNA See Section 6 N for additional information on stutter and artifacts Samples were not denatured completely Heat denature samples for the recommended time and cool on crushed ice or in an ice water bath immediately prior to capillary electrophoresis Do not cool the 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 distant as the main peaks in a heterozygote can indicate the presence of double stranded DNA due to incomplete denaturation or post injecti
122. with GeneMapper ID X Software A Version 1 2 continued Promega 5 Enter a descriptive name for the analysis method such as PowerPlexESX 17 6 Select the Allele tab Figure 13 7 Select the bins text file that was imported in Section 6 A 8 Ensure that the Use marker specific stutter ratio and distance if available box is checked 9 We recommend the values shown in Figure 13 for proper filtering of stutter peaks when using the PowerPlex ESX 17 System You may need to optimize these settings In house validation should be performed Analysis Method Editor mp ij me General Niele Peak Detector Peak Quality SQ amp GQ Settings Dn Set PowerPlex_ Sx firs IDK w20 T Use marker spectic stutter rane and datance awatable Marker Repeat Type Tri Tetra Perta Hexa Goba Cut off Value 0 0 00 0 0 0 0 Mrana Ratio 0 0 30 o o 0 0 MinusA Distance From 0 0 00 0 0 0 0 to 0 0 00 6 0 0 0 Goba Minus Sutter Ratio 0 0 00 0 0 0 0 Gobel Mrax Sutter Ontarxe From 2 25 3 25 0 0 0 to 3 75 4 75 0 0 0 0 Godal Pin Rutter Ratio 0 1 00 9 0 0 0 Goba Plus Sutter Distance From 2 25 6 0 0 0 0 0 Yo 3 5 02 6 0 0 0 Amdoi OSAI 0 0 tanya 8258TA Figure 13 The GeneMapper ID X Allele tab 10 Select the Peak Detector tab Figure 14 shows an example of settings used at Promega You may need to optimize these settings In house validation should be performed Promega Corporation
123. ww promega com resources tools genemapper id software panels and bin sets The PowerTyper ESX Macros are available for download at www promega com resources tools powertyper macros Matrix standards are required for initial setup of the color separation matrix The matrix standards are provided separately and are available for the ABI PRISM 310 Genetic Analyzer PowerPlex 5 Dye Matrix Standards 310 Cat DG4600 and ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 3130xl 3500 and 3500xL Genetic Analyzers PowerPlex 5 Dye Matrix Standards 3100 3130 Cat DG4700 3 Before You Begin 3 A Precautions The application of PCR based typing for forensic or paternity casework requires validation studies and quality control measures that are not contained in this manual 10 11 Guidelines for the validation process are published in the Internal Validation of STR Systems Reference Manual 12 The quality of purified DNA or direct amplification samples small changes in buffers ionic strength primer concentrations choice of thermal cycler and thermal cycling conditions can affect PCR success We suggest strict adherence to recommended procedures for amplification and fluorescence detection Additional research and validation are required if any modifications to the recommended protocols are made PCR based STR analysis is subject to contamination by very small amounts of human DNA Extreme care should b
124. y Select C Results Group then select Create Alternatively a previously created eo Results Groups may be used Promega Select the Results Group Attributes according to laboratory practices and save with a descriptive name Sow j Bowe a Setup a Meets Grow Q Nee bread eld Poete p iompo ramet werkt Gng imien Pres of Brats emp hone sRontii Goap Mimes Be erais beers baam liere an baat maay hiara ge tems Ies Dodate O Aa betonem menar botes p cantassi satas m ahas Ge Pontes oe Tale etal Pese tect Eeee Dee Fates Oven are eege tows empie Airi m o separ ate D ompa tone Abhor i amet bowel ox bye Des Viethen OO Wan eegee empi Avi eed agaa orga Bie iame werat brist f ob r Opte Oet hebu D Append Brim DAP Dair P iobin Arret Omp timoa Heide h etter Cunan tie tec tte imide oe estnareeas Pue Pamese fehber CA be bote s Bradt oanp Hemme tabon E bedde on Doe thors Nother Co 9253TA Figure 8 The Create New Results Group window Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 25 C 5 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or o 3500xL Genetic Analyzer continued Promega 8 To create a New Plate navigate to the Library a
125. y recommended to prevent cross contamination Keep all pre amplification and post amplification reagents in separate rooms Prepare amplification reactions in a room dedicated for reaction setup Use equipment and supplies dedicated for amplification setup Meticulous care must be taken to ensure successful amplification A guide to amplification troubleshooting is provided in Section 7 A The concentration of 2800M Control DNA was determined by measuring absorbance at 260nm Quantification of this control DNA by other methods such as qPCR may result in a different value Prepare a fresh DNA dilution for each set of amplifications Do not store diluted DNA e g 0 25ng tl or less Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Page 6 Printed in USA Revised 6 14 4 A Amplification of Extracted DNA C o Amplification and detection instrumentation may vary You may need to optimize protocols including the amount of template DNA cycle number Promega injection conditions and loading volume for your laboratory instrumentation The PowerPlex ESX 17 System is optimized and balanced for 0 5ng of DNA template The amount of DNA template used in your laboratory should be based on the results of your internal validation and may be different Testing at Promega shows that 30 cycles work well for 0 5ng of purified
126. ystem 100 reactions DC6771 400 reactions DC6770 PowerPlex ESI 17 Pro System 100 reactions DC7781 400 reactions DC7780 PowerPlex 16 Monoplex System Penta E Fluorescein 100 reactions DC6591 PowerPlex 16 Monoplex System Penta D JOE 100 reactions DC6651 PowerPlex ES Monoplex System SE33 JOE 100 reactions DC6751 PowerPlex Fusion System 200 reactions DC2402 800 reactions DC2408 PowerPlex Y23 System 50 reactions DC2305 200 reactions DC2320 PowerPlex 21 System 200 reactions DC8902 PowerPlex 18D System 200 reactions DC1802 800 reactions DC1808 PowerPlex 16 HS System 100 reactions DC2101 400 reactions DC2100 PowerPlex S5 System 100 reactions DC6951 400 reactions DC6950 PowerPlex CS7 System 100 reactions DC6613 Not for Medical Diagnostic Use Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TMD024 Revised 6 14 Page 79 9 E Related Products continued Accessory Components Product Size Cat PowerPlex 5 Dye Matrix Standards 310 50ul each dye DG4600 PowerPlex 5 Dye Matrix Standards 3100 3130 25ul each dye DG4700 CC5 Internal Lane Standard 500 300ul DG1521 PunchSolution Kit 100 preparations DC9271 SwabSolution Kit 100 preparations DC8271 2800M Control DNA 10ng l 25ul DD7101 2800M Control DNA 0 25ng 1 500 DD7251 Water Am
127. ystem 9700 thermal cycler the program must be run with 9600 as the ramp speed The ramp speed is set after the thermal cycling run is started The Select Method Options screen appears Select 9600 for the ramp speed and enter the reaction volume 3 After completion of the thermal cycling protocol store amplified samples at 20 C in a light protected box Note Long term storage of amplified samples at 4 C or higher may produce artifacts PCR Optimization Cycle number should be optimized based on the results of an initial experiment to determine the sensitivity with your collection method sample types and instrumentation 1 Choose several samples that represent typical sample types you encounter in the laboratory Prepare them as you would using your normal workflow 2 Prepare three identical reaction plates with aliquots of the same swab extracts Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD024 Page 16 Printed in USA Revised 6 14 3 Amplify samples using the thermal cycling protocol provided above but C subject each plate to a different cycle number 25 26 and 27 cycles o Note This recommendation is for 2l of swab extract Promega 4 Following amplification use your laboratory s validated separation and detection protocols to determine the optimal cycle number for the sample

PowerPlex ESX 17 System Technical Manual, TMD024

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