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AmpFlSTR® Identifiler® PCR Amplification Kit User Guide

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1. Allele Mean Standard Deviation 8 313 30 0 10 9 317 55 0 11 10 321 97 0 12 11 325 86 0 11 D 12 329 97 0 13 13 334 00 0 10 3 14 338 04 0 11 z 15 341 84 0 08 amp D251338 15 307 30 0 11 gt 16 311 65 0 11 17 315 91 0 12 18 320 16 0 12 19 324 35 0 12 20 328 45 0 08 21 332 58 0 11 22 336 62 0 09 23 340 57 0 11 25 344 18 0 07 25 347 78 0 07 26 351 39 0 07 27 355 08 0 07 28 358 77 0 05 0351358 12 111 96 0 06 13 116 04 0 04 14 119 99 0 04 15 123 89 0 02 16 128 06 0 05 17 132 24 0 05 18 136 30 0 06 19 140 43 0 03 055818 7 134 14 0 05 8 138 21 0 04 9 142 56 0 04 AmpFtSTR Identifiler PCR Amplification Kit User Guide 71 Chapter 5 Experiments and Results Accuracy precision and reproducibility 310 Genetic Analyzer Allele Mean Standard Deviation 10 147 02 0 06 11 151 31 0 01 12 155 63 0 05 13 159 81 0 06 14 164 04 0 07 15 167 95 0 05 16 172 09 0 05 D7S820 6 259 15 0 08 7 259 21 0 07 8 263 24 0 07 9 267 26 0 09 10 271 32 0 08 11 275 35 0 06 12 279 42 0 07 13 283 42 0 06 14 287 48 0 10 15 291 58 0 06 D8S1179 8 123 29 0 07 9 127 32 0 05 10 131 41 0 05 11 135 49 0 04 12 139 73 0 04 13 144 25 0 03 14 148 71 0 06 15 153 16 0 07 16 157 51 0 07 17 161 72 0 05 18 165 84 0 07 19 169 92 0 05 013531
2. Plus green ji 8 SGM_Plus_v2 THO1 1590 2050 893 p D13S317 green 20585 25046 11 0851179 0165539 green 2553 301 81 11 12 D75820 9 0251338 green 3048 37031 19 23 m 10 yellow r CSFAPO D19S433 li 101 0 148 0 1415 r on 358 11 WA yellow 1 510 2135 1718 0135317 12 TPOX yellow 216 99 260 99 8 0165539 13 1018551 yellow 264 49 3500 15 19 0251338 AMEL red 106 0 1140 x D1 95433 ar incco40o0 vaal anon n 44 7 View the markers and display the Bin view in the navigation pane a Select the AmpFLSTR Panels v2 folder to display its list of kits in the right pane b Double click the Identifiler v2 folder to display its list of markers below it AmpFtSTR Identifiler PCR Amplification Kit User Guide 37 Chapter 4 Analyze Data Set up GeneMapper ID Software for data analysis c Select D8S1179 to display the Bin view for the marker in the right pane F Panel Manager File Edit Bins View Bi X TET m e sec oerte v2 Me m m i li HES Panel Manager HES AmpFLSTR_Panels_v2 Cf jBIue_v2 Profiler_Plus_v2 cotiler_v2 CSGM_Plus_v2 ES ldentifiler v2 021511 075820 CSF1PO 0351358 THO1 0135317 0165539 0251338 0195433 018551 AMEL D55818 L FGA 91 5
3. 125 PGR setupwork area HHHH HHHH PD T PM BUE NB AD Sep 125 Amplified DNA work area 126 APPENDIX D Safety 127 Gh mical Safety Sb c gees dete eed shies Heat ei ale ede 128 Specific chemical handling a an UE cee LA Ak A AE A eee e 128 Biologicalliazard safety 43 5 u bw ea eee ie 129 Documentation and Support 131 Related documentation Si ss ek ka wn W a a ka eee tence rn 131 Obtain SDSS eri ce beret icine ce edes 132 Obtain SUpDOLE see ie d ILI OG St Maes EK E AE Sea deh eta a or ees 132 Limited Product Warranty 132 Bibliography kac x YO er ew ausis o Ed deo Ead E 133 Index kk rek ya En aetna b reso erp ala Liebe b dead 137 AmpFtSTR Identifiler PCR Amplification Kit User Guide 7 Contents 8 AmpFtSTR Identifiler PCR Amplification Kit User Guide About This Guide IMPORTANT Before using this product read and understand the information the Safety appendix in this document Revision history Revision Date Description A May 2001 New document B May 2001 Update GeneAmp System 9700 part numbers C May 2005 Change to limited licensing information D August 2006 Change to li
4. 29 Set up the 3500 3500xL instrument for electrophoresis 29 Prepare samples for electrophoresis on the 3500 3500xL instrument 29 Section 3 3 310 Instrument 0 y Yu waqa ua Naa een eee 31 Set up the 310 instrument for electrophoresis 31 Prepare samples for electrophoresis on the 310 instrument 31 Allelic ladder requirements To accurately genotype samples you must run an allelic ladder sample along with the unknown samples Number of allelic One Number of samples per Instrument injection ladders allelic ladder s equals 3100 Avant or 3130 1 per 4 injections 4 samples 15 samples 1 allelic ladder 3100 or 3130xl 1 per injection 16 samples 15 samples 1 allelic ladder 3500 1 per 3 injections 8 samples 23 samples 1 allelic ladder 3500xL 1 per injection 24 samples 23 samples 1 allelic ladder 310 1 per 10 injections 1 sample 9 samples 1 allelic ladder IMPORTANT Variation in laboratory temperature can cause changes in fragment migration speed and sizing variation between both single and multiple capillary runs with larger size variations seen between samples injected in multiple capillary runs We recommend the above frequency of allelic ladder injections which should account for normal variation in run speed However during internal validation studies verify the r
5. lt 27 Prepare samples for electrophoresis on the 3100 3100 Avant or 3130 3130xl instrument 28 Section 3 2 3500 3500xL Series instruments 29 Set up the 3500 3500xL instrument for electrophoresis 29 Reagents and parts zi socer ror ya Hey e Ue tte coe EAM ERR sees 29 Electrophoresis software setup and reference lt 29 Prepare samples for electrophoresis on the 3500 3500xL instrument 29 Section 3 3 310 Instrument gt sox 2 3 cee teen saus 31 Set up the 310 instrument for electrophoresis 31 Reagents and parts DR RE l al ke Seth ie See AK Ka ROMs 31 Electrophoresis software setup and reference lt 31 Prepare samples for electrophoresis on the 310 instrument 31 CHAPTER 4 Analyze Data 33 Section 4 1 GeneMapper ID Software 33 Overview of GeneMapper ID Software 33 nStFup enter un sa conan EO Re ae up eel NS gu oe Bee eee 33 Before youistarE ruay uma satu 34 Set up GeneMapper ID Software for data analysis 34 Filena
6. 17 Materials and equipment 18 Kittcontents andStOra ge u usursa wade e DERI eH 18 Standards for samples 18 E CHAPTER 2 Perform PCR sinan kak EL aer Wn NR Qusan siasa 19 Required user supplied reagents 19 DNAGd antifi ca tO zu Aa aA tee tese AE LAS S da dan Men he uha BR Trece te 19 Importance of quantification 19 Methods of quantifying DNA 20 Prepare the amplification kit reactions 21 Perform PORA Su ERR rRNA A KOYA e ENA betula cen tee can Sek A 22 Amplification using bloodstained FTA cards 22 AmpFtSTR Identifiler PCR Amplification Kit User Guide 3 Contents m CHAPTER Perform Electrophoresis 25 Allelic ladder requirements 25 Section 3 1 3100 3100 Avant and 3130 3130xl instruments 27 Set up the 3100 3100 Avant or 3130 3130xl instrument for electrophoresis 27 Reagents andiparts icr nM boo sa hamana a EUER NIE ceded cee dec DERE Med 27 Electrophoresis software setup and reference
7. 3 Chapter 3 Perform Electrophoresis Prepare samples for electrophoresis on the 3100 3100 Avant or 3130 3130xl instrument Prepare samples for electrophoresis on the 3100 3100 Avant or 3130 3130xl instrument Prepare the samples for electrophoresis immediately before loading 1 28 O RO SOR ety SON Calculate the volume of Hi Di Formamide and size standard needed to prepare the samples Reagent Volume per Reagent volume per reaction reaction GeneScan 500 0 3 uL OR GeneScan 600 0 5 pL LIZ Size Standard LIZ Size Standard v2 0 Hi Di Formamide 87 Hi Di Formamide 8 5 pL Note Include additional samples in your calculations to provide excess volume for the loss that occurs during reagent transfers IMPORTANT The volume of size standard indicated in the table is a suggested amount Determine the appropriate amount of size standard based on your experiments and results Pipette the required volumes of components into an appropriately sized polypropylene tube Vortex the tube then centrifuge briefly Into each well of a MicroAmp Optical 96 Well Reaction Plate add 9uL of the formamide size standard mixture 1 uL of PCR product or allelic ladder Note For blank wells add 10 uL of Hi Di Formamide Seal the reaction plate with appropriate septa then centrifuge the plate to ensure that the contents of each well are collected at the bottom Heat the r
8. SU k la hh 91 Data interpretation ra M HRH HHHH HHHHU UH REH HHHH n 94 Minimum sample requirement 94 Population data er EXER UR Rb ee hid tad RE ODER wea 94 DAB 8 1 2 3 Population Data 94 DAB 8 1 2 3 1 Population Distribution Data 94 Dn D N DD a DD cc mae wane Magan Mave 95 Population samples used in these studies 95 Allele frequencies ig sm a ERI Re UR ives was abe ce began k rk 95 Analyzing the four databases 104 E owesfrequency alleles umo IRR EET k e UP 104 Miata tonite tess e eoim ttn oh Aa see his s Al uL ortae d ies kir 105 Estimating germline mutations 105 Additional mutation studies 105 22 2 sie EXER ER a an as i 105 Probability of paternity exclusion 106 Section 5 2 Performance Verification After Primer Manufacturing Process Improvements 4 a eise one Du bI DUE 107 Section 5 3 Performance Validation After Buffer and Enzyme Component R place MN bm nn EOD CEN tis 108
9. 9 5 90 gt Highlight this E Panel Manager b Select File Import Panels to open the Import Panels dialog box c Navigate to then open the x V Applied Biosystems GeneMapper Panels folder where x is the drive on which the GeneMapper ID Software is installed 4 Select AmpFLSTR Panels v2 txt then click Import Note Importing this file creates a new folder in the navigation pane of the Panel Manager AmpFLSTR Panels v2 This folder contains the panels and associated markers Import Panels Look fa Panels j j cle qi 2 AmpFLSTR_Bins_v2 txt ampFLSTR Panels v2t Recent AmpFISTR Yfiler Bin v2 bd AmpFISTR_Yfiler_Panel_v2 txt Desktop Wao My Network File name amp mpFLSTR Panels v2 txt Import Files of type Im Files v Cancel 5 Import AmpFLSTR_Bins_v2 txt a Select the AmpFLSTR_Panels_v2 folder in the navigation pane AmpFtSTR Identifiler PCR Amplification Kit User Guide 35 4 Chapter 4 Analyze Data Set up GeneMapper ID Software for data analysis Panel Manager File Edit Bins View jit X m BI IBI er EHE Panel Manager F AmpFLSTR_Panels_v2 lue b Select File Import Bin Set to open the Import Bin Set dialog box c Navigate to then open the x V Applied Biosystems GeneMapper Panels folder d Select AmpFLSTR Bins v2 txt then click Import Note Importing th
10. Equipment Source 3100 3100 Avant Genetic Analyzer Applied Biosystems 3130 3130xl Genetic Analyzer Applied Biosystems 3500 3500xL Genetic Analyzer for Human Identification Applied Biosystems 310 Genetic Analyzer Contact your local Life Technologies sales representative GeneAmp PCR System 9700 with the Silver 96 Well Block N8050001 GeneAmp PCR System 9700 with the gold plated silver 96 well block 4314878 Veriti 96 Well Thermal Cycler 4375786 Silver 96 well sample block N8050251 Gold plated silver 96 well sample block 4314443 Tabletop centrifuge with 96 well plate adapters optional MLS Table 12 User supplied materials Itemt Source AmpF amp STR Identifiler PCR Amplification Kit 4322288 3100 Analyzer materials 96 well plate septa 4315933 Reservoir septa 4315932 3100 3130xl Genetic Analyzer capillary array 36 cm 4315931 POP 4 polymer for 3100 3100 Avant Genetic Analyzers 4316355 3100 3100 Avant Genetic Analyzer Autosampler Plate Kit 96 well 4316471 GeneScan 500 LIZ Size Standard 4322682 OR OR GeneScan 600 LIZ Size Standard v2 0 4408399 Running Buffer 10X 402824 Hi Di Formamide 4311320 AmpFtSTR Identifiler PCR Amplification Kit User Guide 121 Appendix Ordering Information Equipment and materials not included Itemt Source DS 33 Matrix Standard Kit Dye Set G5 4345833 MicroAmp Optical 96 w
11. reference documents Genetic pata Operatin Collection p g Run modules and conditions References Analyzer System Software Applied 3 0 Windows HIDFragmentAnalysis36_POP4_1 Applied Biosystems 3130 3130xl Biosystems XP Injection conditions Genetic Analyzers Using Data 3130 3130xl 3130 3 kV 5 sec Collection Software v3 0 Protocols for Processing AmpFtSTR PCR 3130x 3 kV 10 sec Amplification Kit PCR Products User Dye Set G5 Bulletin Pub no 4363787 3100 2 0 Windows e HlIDFragmentAnalysis36 POPA 1 3100 3100 Avant Genetic Analyzers 2000 Injection condition 3kV 10 sec Using Data Collection Software v2 0 Protocols for Processing AmpFtSTR Dye Set G5 00 PCR Amplification Kit PCR Products User Bulletin Pub no 4350218 1 1 Windows e GeneScan3 vb_DyeSetG5Module 3100 3100 Avant Genetic Analyzers NT Injection condition 3kV 10 sec Protocols for Processing AmpFtSTR PCR Amplification Kit PCR Products e GS600v2 0Analysis JU User Bulletin Pub no 4332345 3100 Avant 1 0 Windows e GeneScan36Avb DyeSetG5Module 3100 3100 Avant Genetic Analyzers NT Injection condition 3 kV 5sec e GS600v2 0Analysis gsp Protocols for Processing AmpFtSTR PCR Amplification Kit PCR Products User Bulletin Pub no 4332345 AmpFtSTR Identifiler PCR Amplification Kit User Guide 27 9 ES T gt S 5 m a x lt Q 8 ER 5 Q ra E 5 E
12. DNA sample To prepare Negative control Add 10 pL of low TE buffer 10mM Tris 0 1mM EDTA pH 8 0 Test sample Dilute a portion of the test DNA sample with low TE buffer so that 1 0 ng of total DNA is in a final volume of 10 uL Add 10 uL of the diluted sample to the reaction mix Positive control Add 10 uL of 9947A control DNA 0 1 ng uL The final reaction volume sample or control plus master mix is 25 uL 7 Sealthe plate with MicroAmp Clear Adhesive Film or MicroAmp Optical Adhesive Film or cap the tubes 8 Centrifuge the tubes at 3000 rpm for about 20 seconds in a tabletop centrifuge with plate holders if using 96 well plates 9 Amplify the samples in a GeneAmp PCR System 9700 with the silver or gold plated silver 96 well blocks or a Veriti 96 Well Thermal Cycler Note The Identifiler Kit is not validated for use with the GeneAmp PCR System 9700 with the aluminium 96 well block Use of this thermal cycling platform may adversely affect performance of the Identifiler Kit AmpFtsTR Identifiler9 PCR Amplification Kit User Guide 21 Perform PCR Perform PCR Chapter 2 Perform PCR 1 Program the thermal cycling conditions When using the GeneAmp PCR System 9700 with either 96 well silver or gold plated silver block select the 9600 Emulation Mode When using the Veriti 96 Well Thermal Cycler refer to the following document for instructions on how to configure the Verit
13. BW 14uM BW 14uM BW 14uM BW 14uM 78 YS BW 16uM BW 16uM BW 16uM BW 16uM BW 16uM BW 16uM BW 16uM BW 16uM 8B LIN ev A11 BW 18uM BW 18uM A11 BW 18uM BW 18uM BW 18uM BW 18uM BW 18uM BW 18uM 9B B2 BW 20uM BW 20uM 82 Bw 20uM BW 200 Degraded DNA 88 9G B2 BW 20uM BW 20uM SR B2 BW 20uM BW 20uM As the average size of degraded DNA approaches the size of the target sequence the amount of PCR product generated is reduced This is due to the reduced number of intact templates in the size range necessary for amplification Degraded DNA was prepared to examine the potential for differential amplification of loci High molecular weight DNA was incubated with the enzyme DNase I for varying amounts of time The DNA was examined by agarose gel analysis to determine the average size of the DNA fragments at each time point Four nanograms of degraded DNA or 1 ng undegraded DNA was amplified using the Identifiler Kit all 16 primer pairs together As the DNA became increasingly degraded the loci became undetectable according to size Preferential amplification was not observed The loci failed to robustly amplify in the order of decreasing size as the extent of degradation progressed CSF1PO and D251338 were the first loci to exhibit decreased amplification followed 0165539
14. Panel Name Ofiler v1 1X SGM Plus v1 1X null NGM SElect v2 1X null Identifiler Plus v1 1x null i fy Panel Manager C mpFLSTR NGMsSElect v2X AmpFLSTR NGM v3X AmpFLSTR_Panels_v1X esa eet AMpFLSTR_Pane b Select File Import Marker Stutter to open the Import Marker Stutter dialog box c Navigate to then open the AmpFLSTR Analysis Files GMIDX folder AmpFtSTR Identifiler PCR Amplification Kit User Guide 53 4 Chapter 4 GeneMapper ID X Software Set up GeneMapper ID X Software for data analysis d Select AmpFLSTR_Stutter_v2X then click Import Note Importing this file associates the marker stutter ratio with the bin set in the AmpFLSTR v2X folder Import Marker Stutter Lookin G AmpFLSTR Analysis Files GMIDX ampFLsTR_Bins_v2x 4 AmpFLSTR_Panels_v2 My Recent B AmpFLSTR Stutter v2X Documents El ReadMe AmpFLSTR v2X My Documents AmpFLSTR_Stutter_v2X txt My Computer Files of type All Files 10 View the imported marker stutters in the navigation pane a Double click the AmpFLSTR Panels v2X folder to display its list of kits in the right pane b Double click the Identifiler v1 1X folder to display its list of markers below it c Double click 0165539 to display the Stutter Ratio amp Distance view for the marker in the right pane Panel Manager File Edit B
15. b Select File Import Bin Set to open the Import Bin Set dialog box c Navigate to then open the AmpFLSTR Analysis Files GMIDX folder AmpFtSTR Identifiler PCR Amplification Kit User Guide 51 Chapter 4 GeneMapper ID X Software Set up GeneMapper ID X Software for data analysis d Select AmpFLSTR_Bins_V2X txt then click Import Note Importing this file associates the bin set with the panels in the AmpFLSTR_Panels_v2X folder Import Bin Set Lookin J AmpFLSTR Analysis Files GMIDX 2 po My Recent B AmpFLSTR_Stutter_v2X Documents EJ ReadMe AmpFLSTR v2X My Documents 48 File name AmpFLSTR Bins v2X Ext My Computer Files of type all Files 7 View the imported panels in the navigation pane a Double click the AmpFLSTR_Panels_v2X folder b Double click the Identifiler_v1 1X folder to display the panel information in the right pane Panel Manager File Edit Bins View Help a x N N NN o S Bin Set AmpFLSTR Bins li m m m EI EI Panel Name Comment Ofiler v1 1X null SGM Plus v1 1X null NGM SElect v2 1X null Identifiler Plus v1 1x null NGM v3 1X null Identifiler Direct v1 1X null S gf Panel Manager C3AmpFLSTR NGMsElect v2X D AmpFLSTR_NGM_v3X AmpFLSTR_Panels_v1X mp K COfiler v1 1x CD SGM_Plus_v1 1x CQNGM_SElect_v2 1 dentifiler_Plus_v1 1 NGM _v3 1x amp C3Identifiler Direct v1 1x 3 Prof
16. D18551 and so forth A similar result at each time point was obtained whether the DNA samples were amplified for each locus alone or co amplified with the Identifiler Kit Figure 18 on page 89 AmpFtSTR Identifiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation 5 Stability Figure 18 Multiplex amplifications of a DNA sample in the absence of DNase and the sample incubated for 30 sec 1 min 4 min and 8 min with DNase analyzed using the 310 Genetic Analyzer 150 225 250 275 300 ges Er mE zt a 1 no DNase BIJ 16 A1 no DNase 1 no DNase IR cC Al no DNase M b a A2 30 seconds A2 80 seconds 2 30 seconds 2 30 seconds g lt e 3 o 2 e E e 2 1 amp 4 1 minute 4 1 minute 4 1 minute 4 1 minute LA A A A BB 4 minutes 4 minutes BY A6 4 minutes 4 minutes 8B AS 8 minutes 8 8 minutes DIB 8Y AS 8 minutes AB 8 minutes Multiplex DNA samples were amplified in 16 separate reactions containing primers for only one amplifications Identifiler Kit locus singleplex and a reaction containing all primers for the Identifiler Kit loci multiplex DNA used as PCR template consisted of a sample that had been degraded for 1 min with DNase I Amplified samples were analyzed using the 310 Gen
17. Probability of paternity exclusion Table 8 shows the Probability of Paternity Exclusion Pg values of the Identifiler Kit STR loci individually and combined Table 8 Probability of Paternity Exclusion for the Identifiler Kit STR loci Locus African American U S Caucasian U S Hispanic Native American CSF1PO 0 545 0 496 0 450 0 409 D2S1338 0 748 0 725 0 671 0 399 D3S1358 0 591 0 630 0 495 0 510 D5S818 0 506 0 440 0 525 0 601 D7S820 0 591 0 582 0 574 0 492 D8S1179 0 580 0 680 0 599 0 601 D13S317 0 383 0 487 0 638 0 370 D16S539 0 649 0 566 0 567 0 428 D18S51 0 760 0 731 0 767 0 329 D19S433 0 601 0 531 0 678 0 360 D21S11 0 737 0 708 0 586 0 399 FGA 0 760 0 766 0 739 0 309 THO1 0 492 0 566 0 618 0 646 TPOX 0 521 0 329 0 392 0 687 vWA 0 709 0 625 0 555 0 528 Combined 0 9999996 0 9999992 0 9999990 0 9999527 106 AmpFtSTR dentifiler9 PCR Amplification Kit User Guide Chapter 5 Performance Verification After Primer Manufacturing Process Improvements Section 5 2 Performance Verification After Primer Manufacturing Process Improvements As part of our continual efforts to improve the quality of our products several improvements and updates have been made to the manufacturing process of the Identifiler Kit Part no 4322288 since its introduction in 2001 Effective from kit lot number 0310018 modifications were made to the manufacturing process of the Identifiler Kit
18. Set up GeneMapper D X Software for data analysis 49 Panel bin and stutter file version 49 Before using the software for the first time 49 For more informatl n 2 cce n WALE eee an KA di ka Ea K EE ERA 49 Check panel bin and stutter file version 50 Import panels bins and marker stutter 50 Create an analysis method 55 General tab settings 56 Allel tab setlings eerie dixe Gacy 57 Peak Detector tab settings 58 Peak Quality tab senings gt 22111555 bead Seana eee h dr 59 SQ 6 GOitab DD u r AA Bees esas 60 Analyze and edit sample files with GeneMapper D X Software 61 Examine and edit a project 62 For more information Reheat I E A RE Ta 62 CHAPTER 5 Experiments and Results 65 Section 5 1 Developmental Validation 66 OVERVIEWS kat Pei tne Ae re AN AA E 66
19. 36 cm for 3500 Genetic Analyzers 4404683 24 Capillary array 36 cm for 3500xL Genetic Analyzers 4404687 96 well retainer amp base set Standard 3500 3500xL Genetic Analyzers 4410228 8 Tube retainer amp base set Standard for 3500 3500xL Genetic Analyzers 4410231 8 Strip Septa for 3500 3500xL Genetic Analyzers 4410701 96 Well Septa for 3500 3500xL Genetic Analyzers 4412614 Septa Cathode Buffer Container 3500 series 4410715 GeneScan 600 LIZ9 Size Standard v2 0 4408399 DS 33 Matrix Standard Kit Dye Set G5 4345833 For a complete list of parts and accessories for the 3500 3500xL instrument refer to the Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Pub no 4401661 122 AmpFtSTR dentifiler9 PCR Amplification Kit User Guide Appendix B Ordering Information Equipment and materials not included Itemt Source 310 Analyzer materials 310 DNA Analyzer capillary array 47 cm 402839 0 5 mL sample tray 5972 96 well tray adaptor for 9700 thermal cycler trays 4305051 GeneScan 500 LIZ Size Standard 4322682 OR OR GeneScan 600 LIZ Size Standard v2 0 4408399 Running Buffer 10x 4335643 Genetic analyzer septa retainer clips for 96 tube sample tray 402866 Genetic analysis sample tubes 0 5 mL 401957 Septa for 0 5 mL sample tubes 401956 05 33 Matrix Standard Set 6 FAM VIC9 NED PET and LIZ9 dyes for 310 377 systems 4318159 MicroAmp 8 tube strip 0 2
20. 57 59 and 61 C produced robust profiles At 63 C the yield of the majority of loci was significantly reduced This should pose no problem with routine thermal cycler calibration and when following the recommended amplification protocol Preferential amplification was not observed at any of the tested annealing temperatures Figure 6 An amplification of 1 ng of genomic DNA amplified while varying the annealing temperature analyzed on the 310 Genetic Analyzer 55 C 57 C 59 C standard protocol 61 C 63 C Identifiler Kit reactions were amplified for 27 28 29 30 and 31 cycles on the GeneAmp PCR System 9700 using 1 0 ng of three DNA samples As expected PCR product increased with the number of cycles A full profile was generated at 27 cycles off scale data were collected for several allele peaks at 31 cycles While none of the cycle numbers tested produced nonspecific peaks 28 cycles was found to give optimal sensitivity when the amplified products were examined on 310 Genetic Analyzers Additionally the cycle number was set to avoid detection of low quantities of DNA 20 pg or less At 28 cycles 1 0 ng of AmpF STR Control DNA 9947A amplifies reliably and specifically following the conditions outlined in this guide AmpFtSTR Identifiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation Accuracy precision and reproducibility Accuracy precision and reproducibi
21. Importance of validation lt i ad Seki nu bI SIC SN Reg 66 Experiment conditions salya a silka nala k ha Seley tegen A pdt en DEUS ee ee 66 Developmental validation 67 DAB 8 1 1 Developmental Validation 67 PGR COMPONENTS Ar A sys hrs ese An h n vete FS pu ue 67 Thermal cycler parameters 67 AmpliTaq Gold DNA Polymerase activation 68 Accuracy precision and reproducibility 69 DABIS MEZA CCUPAGY es ir tue Socr teo Ser op ME D Eus LIA A LO AR 69 Precision and size windows 69 Extra Peaks in the electropherogram 77 Causes of extra peaks 2 k68 sika Aw eed ER ad gens ELO CUL E Gale ee sl 77 Stutter products 4 2 4 0 esses MN NnNHnE EEHnE EENNREEEHEREEEnEnEnEEnE EEE EnHnEE H HH H 77 Additional An acleolide uu mr i ERATIS e eileen eed aya 81 yA ER je ol ARA SEBE QIR HEE N Ka M Me ray E Sek a fee ee UE on ale rn ET 81 Characterization of loci usis ana enced Yan a rie EE RE EE RE Das WS u s s sas 82 DAB 8 1 2 1 Documentation J u ke k ka kwa kk aa dil a kk a eee REM SEE 82 OVERVIEW sisse
22. Test A amp Test B gt Test C M EN e e r 3 2 U o ER e 3 o 5 a 2 e 3 Ww 2 m N lt 3 o o E o Figure 31 Inhibition study minimum referenced peak height Inhibitors HA Humic Acid HE Hematin PRI Pristine or Uninhibited DNA LOT Contro H Control B 9 Test A Test 8 gt Test to s e m EN 1 2 Minimum Referenced Peak Height o m AmpFtSTR Identifiler PCR Amplification Kit User Guide 115 5 Chapter 5 Performance Validation After Buffer and Enzyme Component Replacement Inhibition study Figure 32 Inhibition study intracolor balance Y axis intracolor balance percentage versus X axis dye color Inhibitors HA Humic Acid HE Hematin PRI Pristine or Uninhibited DNA LOT Control Control B 4 Test 9 Test 8 Test C Intracolor Balance Representative electropherograms from the inhibition study are shown in Figure 33 34 and 35 Figure 33 Inhibition study representative electropherograms using uninhibited Control DNA 007 Y scale 4000 RFU Control A Muri ampi Nor Cureta x Control Mart Campi for Catton Test A Met Lamia hor Cuterce nad as Mart Sampe hor Ctr Test C 116 AmpFtSTR dentifiler9
23. ep Seat ee 108 2 a co Mu S ee t Sa Y ku IU ELEM oer 108 Izeprogucibility Study e Ro p MIS aea 109 Intracolorf balance c kula l ka We Mar kaka KWA ped etd e Sb dg 109 Stutter percentages 110 grum kanaa 110 Sens StUY T suan asas IER 111 Mean referenced peak height 111 DNA concentration and peak height 112 AmpFtSTR Identifiler PCR Amplification Kit User Guide Contents Allelic drop lU sae teen Mot eas cae Re MEME urd ka an fe NAN 113 Genotype concordance 5o den eo eee PER n hal e iet edes 114 Inhibition study seater RN ed cote dee e Oed tet Een ae s did 114 Mean referenced peak height minimum referenced peak height and intracolor balance 114 lele N rusya HERR OEC tun ua el TE 117 CONCLUSIONS ac Alte ayd AA S cba tatus pie cd A res 118 APPENDIX A Troubleshooting 119 APPENDIX B Ordering Information 121 Equipment and materials not included 00 cece kk KK eh 121 APPENDIX PCR Work 125 Work area setup and lab design
24. no GeneMapper ID X Software Version 1 1 Mixture Analysis Quick Reference Guide 4402094 GeneMapper ID X Software Version 1 2 Reference Guide 4426481 GeneMapper ID X Software Version 1 2 Quick Reference Guide 4426482 Obtain SDSs Obtain support Portable document format PDF versions of this guide and the documents listed above are available at www lifetechnologies com Note To open the user documentation available from the Applied Biosystems web site use the Adobe Acrobat Reader software available from www adobe com Safety Data Sheets SDSs are available from www lifetechnologies com support Note For the SDSs of chemicals not distributed by Life Technologies contact the chemical manufacturer For HID support In North America Send an email to HIDTechSupport lifetech com or call 888 821 4443 option 1 Outside North America Contact your local support office For the latest services and support information for all locations go to www lifetechnologies com At the website you can Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities Search through frequently asked questions FAQs Submit a question directly to Technical Support Search for user documents SDSs vector maps and sequences application notes formulations handbooks certificates of analysis citations and other product support documents Obtain information about customer train
25. no 4426481A 3 Click Analyze enter a name for the project in the Save Project dialog box then click OK to start analysis The status bar displays the progress of analysis as a completion bar extending to the right with the percentage indicated The table displays the row of the sample currently being analyzed in green or red if analysis failed for the sample The Analysis Summary tab is displayed and the Genotypes tab becomes available upon completion of the analysis AmpFtSTR Identifiler PCR Amplification Kit User Guide 61 O D 3 0 lt i e 0 e v gt p 2 0 Chapter 4 GeneMapper ID X Software Examine and edit a project Analysis summary window after analysis GeneMapper ID X Identifiler Example gmidx Is Logged In Database GBOLDROYNJO9E File Edit Analysis View Tools Admin Help GO F m E E ill S ah Project m JC Ider tifiler Example de Table Setting 31XX Data Ar alysis Samples Analysis Summary Genotypes Analysis Summary Select run folder to display Sample Status Total of Samples Unanalyzed 0 Analyzed 65 Analysis Setting Changed 0 Click a link below to display a filtered Samples Table containing only the samples selected Allelic Ladder Quality per run folder based on SQ and CGQ only Identifiler Example Run Folder __ total of Analyzed adders
26. 310 Genetic Analyzer Allele Mean Standard Deviation THO1 4 163 29 0 04 5 167 36 0 03 6 171 40 0 05 7 175 40 0 03 8 179 38 0 04 9 183 36 0 05 9 3 186 93 0 02 10 187 29 0 04 11 191 23 0 03 13 3 201 94 0 05 TPOX 6 222 07 0 04 7 226 02 0 06 8 229 91 0 03 9 233 86 0 06 10 237 88 0 07 11 241 83 0 06 12 245 77 0 07 13 249 78 0 08 vWA 11 154 59 0 08 12 158 87 0 07 13 163 00 0 05 14 167 27 0 05 15 171 15 0 05 16 175 15 0 04 17 179 15 0 04 18 183 08 0 04 19 187 00 0 04 20 190 93 0 05 21 194 80 0 05 22 198 62 0 06 23 202 44 0 05 24 206 69 0 08 76 AmpFtSTR Identifiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation Extra Peaks in the electropherogram Extra Peaks in the electropherogram Causes of extra peaks Stutter products To further demonstrate reproducibility 1187 population database DNA samples have been typed using the Identifiler Kit These samples have been previously genotyped with concordant results of the same loci using other AmpF STR kits Peaks other than the target alleles may be detected on the electropherogram displays Several causes for the appearance of extra peaks including the stutter product at the n 4 position incomplete 3 A nucleotide addition at the n 1 position artifacts and mixed DNA samples see DAB 8 1 2 2 Mixture Studies on page
27. 91 detecting 91 92 detection limit 92 94 resolving genotypes in mixed samples 92 software instrument compatibility 16 STRBase 104 stutter check version 49 50 import 50 stutter peak or product 77 support obtaining 132 T technical support 132 thermal cyclers for use with kit 126 programming conditions 22 training information on 132 troubleshooting 119 U user supplied reagents 19 V validation accuracy precision reproducibility 69 characterization of loci 82 developmental 66 experiments to evaluate 66 importance of 66 minimum sample requirement 94 mixture studies 91 mutation rate 105 population data 94 probability of identity 105 probability of paternity exclusion 106 sensitivity 85 139 Index species specificity 83 stability 86 thermal cycler parameters 67 warranty 132 work area amplified DNA 126 PCR setup 125 setup and lab design 125 workflow overview 15 140 AmpF amp TR Identifiler PCR Amplification Kit User Guide C 1628528 Headquarters a 5791 Van Allen Way Carlsbad CA 92008 USA Phone 1 760 603 7200 Toll Free in USA 800 955 6288 Bn For support visit www lifetechnologies com support technologies www lifetechnologies com
28. Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Data Collection v1 0 4401661 Applied Biosystems 3500 3500xL Genetic Analyzer User Bulletin Solutions to issues related to software data 4445098 hardware and consumables Note Additional user bulletins may be available at www lifetechnologies com Applied Biosystems 3730 3730xl Genetic Analyzer Getting Started Guide 4359476 GeneAmp PCR System 9700 Base Module Users Manual N805 0200 Veriti 96 Well Thermal Cycler AmpFtSTR Kit Validation User Bulletin 4440754 Quantifiler Kits Quantifiler Human DNA Quantification Kit and Quantifiler Y Human Male DNA 4344790 Quantification Kit Users Manual PrepFiler9 Forensic DNA Extraction Kit User Guide 4390932 GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide 4338775 GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial 4335523 Installation Procedures and New Features for GeneMapper ID Software v3 2 User Bulletin 4352543 GeneMapper ID X Software Version 1 0 Getting Started Guide 4375574 GeneMapper ID X Software Version 1 0 Quick Reference Guide 4375670 GeneMapper ID X Software Version 1 0 Reference Guide 4375671 GeneMapper ID X Software Version 1 1 Mixture Analysis Getting Started Guide 4396773 AmpFtSTR Identifiler PCR Amplification Kit User Guide 131 Documentation and Support Obtain SDSs Document title Pub
29. D07 0 25ng Sample Identifiler AnalysisMethod v1 Identifiler v2 CE G5 HID GS500 75 45t 22 Ident O07 Q 5ng CO1 fsa Ident 007 Q 5ng Sample Identifiler amp nalysisMethod v1 Identifiler v2 CE G5 HID GSS00 75 45t 23 Ildent O07 0 5ng CO2 fsa Ident O07 Q 5ng Sample Identifiler amp nalysisMethod v1 Identifiler v2 CE G5 HID GSS00 75 45t 24 Ident O07 Q 5ng CO3 fsa Ident 007 Q 5ng Sample Identifiler amp nalysisMethod v1 Identifier v2 CE G5 HID GSS00 75 45t 25 Ident O07 0 5ng CO4 fsa Ident O07 0 5ng Sample Identifiler amp nalysisMethod v1 Identifiler v2 CE G5 HID GSS00 75 45t _ For more information about any of these tasks refer to the GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide Pub no 4338775 Examine and edit a project You can display electropherogram plots from the Samples and Genotypes tabs of the Project window to examine the data These procedures start with the Samples tab of the Project window assuming the analysis is complete For more information For details about GeneMapper ID Software features allele filters peak detection algorithms and project editing refer to ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial Pub no 4335523 GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide Pub no 4338775 Installation Procedures and New Features for GeneMapper ID Software S
30. J J 5 s o Control Quality per project based on sample PQVs SOS SSPK MIX OMR SQ CGQ Control Type Total of Samples All thresholds met One or more thresholds not met Positive Control 0 0 0 Custom Control 0 0 0 Negative Control 0 0 0 Total 0 0 0 Sample Quality per project based on sample PQVs SOS SSPK MIX OMR SQ CGQ Samples Total of Samples All thresholds met more thresholds not met 60 Analysis Completed Examine and edit a project You can display electropherogram plots from the Samples and Genotypes tabs of the Project window to examine the data These procedures start with the Analysis Summary tab of the Project window assuming the analysis is complete For more information 62 For more information refer to GeneMapper ID X Software Version 1 0 Getting Started Guide Pub no 4375574 GeneMapper ID X Software Version 1 0 Quick Reference Guide Pub no 4375670 GeneMapper ID X Software Version 1 0 Reference Guide Pub no 4375671 GeneMapper ID X Software Version 1 1 Mixture Analysis Getting Started Guide Pub no 4396773 AmpFtSTR Identifiler PCR Amplification Kit User Guide Section 4 2 GeneMapper ID X Software For more information GeneMapper ID X Software Version 1 2 Reference Guide Pub no 4426481 GeneMapper ID X Software Version 1 2 Quick
31. N N 225 Allele Size bp Sizing precision allows for determining accurate and reliable genotypes Sizing precision was measured on the 310 Genetic Analyzer The recommended method for genotyping is to use a 0 5 bp window around the size obtained for each allele in the AmpF STR Identifiler Allelic Ladder A 0 5 bp window allows for the detection and correct assignment of alleles An allele that sizes only one base pair different from an allele in the allelic ladder will not be incorrectly typed and will be identified as off ladder Any sample allele that sizes outside a window could be either of the following e off ladder allele for example an allele of a size that is not represented in the AmpF STR Identifiler Allelic Ladder An allele that does correspond to an allelic ladder allele but whose size is just outside a window because of measurement error AmpFtSTR Identifiler PCR Amplification Kit User Guide 69 g lt e 3 D e E e 2 70 Chapter 5 Experiments and Results Accuracy precision and reproducibility The measurement error inherent in any sizing method can be defined by the degree of precision in sizing an allele multiple times Precision is measured by calculating the standard deviation in the size values obtained for an allele that is run in several injections in one capillary run Table 3 on page 70 indicates typical precision resu
32. Oldroyd N Clayton T Barnett L Arnold J Thompson C Hale R Chapman J Urquhart A and Gill P 1996a The validation of a 7 locus multiplex STR test for use in forensic casework I Mixtures ageing degradation and species studies Int J Legal Med 109 186 194 Sparkes R Kimpton C Gilbard S Carne P Andersen J Oldroyd N Thomas D Urquhart A and Gill P 1996b The validation of a 7 locus multiplex STR test for use in forensic casework II Artifacts casework studies and success rates Int J Legal Med 109 195 204 Straub R E Speer M C Luo Y Rojas K Overhauser J Ott J and Gilliam T C 1993 A microsatellite genetic linkage map of human chromosome 18 Genomics 15 48 56 AmpFtSTR Identifiler PCR Amplification Kit User Guide 135 Bibliography 136 Szibor R Lautsch S Plate I Bender K and Krause D 1998 Population genetic data of the STR HumD351358 in two regions of Germany Int J Legal Med 111 160 161 Wallin J M Buoncristiani M R Lazaruk K D Fildes N Holt C L Walsh P S 1998 SWGDAM validation of the AmpFISTR blue PCR amplification kit for forensic casework analysis J Forensic Sci 43 854 870 Wallin J M Holt C L Lazaruk K D Nguyen T H and Walsh P S 2002 Constructing universal multiplex PCR systems for comparative genotyping J Forensic Sci 47 52 65 Walsh P S Fildes N J and Reynolds R 1996 Sequence ana
33. Performing the exact test of Hardy Weinberg proportion for multiple alleles Biometrics 48 361 372 Hammond Jin L Zhong Y Caskey C and Chakraborty 1994 Evaluation of 13 short tandem repeat loci for use in personal identification applications Am J Hum Genet 55 175 189 Holt C Stauffer C Wallin J Lazaruk L Nguyen T Budowle B and Walsh P 2000 Practical applications of genotypic Surveys for forensic STR testing Forensic Sci Int 112 91 109 Kimpton C Walton A and Gill P 1992 A further tetranucleotide repeat polymorphism in the vWF gene Hum Mol Genet 1 287 Kong X Murphy K Raj T He C White P S and Matise T C 2004 A combined linkage physical map of the human genome Am J Hum Genet 75 1143 1148 Kwok S and Higuchi R 1989 Avoiding false positives with PCR Nature 339 237 238 Lazaruk K Walsh P S Oaks F Gilbert D Rosenblum B B Menchen S Scheibler D Wenz H M Holt C Wallin J 1998 Genotyping of forensic short tandem repeat STR systems based on sizing precision in a capillary electrophoresis instrument Electrophoresis 19 86 93 Li H Schmidt L Wei M H Hustad T Leman M I Zbar B and Tory 1993 Three tetranucleotide polymorphisms for loci D351352 0351358 0351359 Hum Mol Genet 2 1327 Magnuson V L Ally D S Nylund S J Karanjawala Z E Rayman J B Knapp J I Lowe A L Ghosh S and Collins F
34. S 1996 Substrate nucleotide determined non templated addition of adenine by Taq DNA polymerase implications for PCR based genotyping and cloning Biotechniques 21 700 709 Mansfield E S Robertson J M Vainer M Isenberg A R Frazier R R Ferguson K Chow S Harris D W Barker D L Gill P D Budowle B and McCord B R 1998 Analysis of multiplexed short tandem repeat STR systems using capillary array electrophoresis Electrophoresis 19 101 107 AmpFtSTR Identifiler PCR Amplification Kit User Guide Bibliography Mills K A Even D and Murrau J C 1992 Tetranucleotide repeat polymorphism at the human alpha fibrinogen locus FGA Hum Mol Genet 1 779 Moller A Meyer E and Brinkmann B 1994 Different types of structural variation in STRs HumFES FPS HumVWA and HumD215S11 Intl J Legal Med 106 319 323 Momhinweg E Luckenbach C Fimmers R and Ritter H 1998 D351358 sequence analysis and gene frequency in a German population Forensic Sci Int 95 173 178 Moretti T Baumstark A Defenbaugh D Keys K Smerick J and Budowle B 2001 Validation of short tandem repeats STRs for forensic usage Performance testing of fluorescent multiplex STR systems and analysis of authentic and simulated forensic samples J Forensic Sci 46 3 647 660 Mulero J J Chang C W and Hennessy L K 2006 Characterization of N 3 stutter product in the trinucleotide repeat locus DYS3
35. SEfiler_v2 E Reference Samples 38 1 0 03 08 0 7 0 6 0 5 04 0 3 0 2 0 1 0 0 113 117 121 125 128 133 137 141 145 149 153 0851179 157 161 165 169 173 177 181 185 X 118 33 Y 0 49 Marker 0851179 118 00 183 50 OK Cancel Apply 8 Click Apply then OK to add the AmpF STR panel and bin set to the GeneMapper ID Software database IMPORTANT If you close the Panel Manager without clicking OK the panels and bins are not imported into the GeneMapper ID Software database AmpFtSTR Identifiler PCR Amplification Kit User Guide Section 4 1 GeneMapper ID Software Set up GeneMapper ID Software for data analysis Create an analysis The HID Advanced analysis method for the Identifiler Kit uses the method AmpFLSTR_Bins_v2 file described in step 5 on page 35 Use the following procedure to create a HID analysis method for the Identifiler Kit 1 Select Tools GeneMapper Manager to open the GeneMapper Manager 2 Manager Projects Analysis Methods Table Settings Plot Settings Matrices Size Standards Name Last Saved Owner Instrument Analysis Type Descrip HID_Advanced 2009 06 18 16 22 2 gmid HID I HID Classic 2007 08 06 10 03 0 grid HID m Microsatellite Default 2004 05 28 11 34 3 gmid Microsatellite Factor ji Open Save As Import Export New 2 Select the Analy
36. The AmpF STR loci have been validated by family studies to demonstrate their mode s of inheritance The Centre d Etude du Polymorphisme Humain CEPH has collected DNA from 39 families of Utah Mormon French Venezuelan and Amish descent These DNA sets have been extensively studied all over the world and are routinely used to characterize the mode of inheritance of various DNA loci Each family set contains three generations generally including four grandparents two parents and several offspring Consequently the CEPH family DNA sets are ideal for studying inheritance patterns Begovich et al 1992 g lt e 3 e E s 2 Four family DNA sets were examined and a half nanograms of DNA from each sample were amplified using the AmpF STR SGM Plus kit followed by analysis using an 377 DNA Sequencer The families examined included 1331 11 offspring 13291 9 offspring 13292 9 offspring and 13294 8 offspring representing 37 meiotic divisions The results confirmed that the loci are inherited according to Mendelian rules as has been reported in the literature Nakahori et al 1991 Edwards et al 1992 Kimpton et al 1992 Mills et al 1992 Sharma and Litt 1992 Li et al 1993 Straub et al 1993 Mapping The Identifiler Kit loci Amelogenin CSF1PO 0251338 0351358 055818 075820 0851179 0135317 0165539 018551 0195433 021511 FGA TH01 and v
37. X Software or with GeneMapper ID Software version 3 2 1 1 Select Tools GeneMapper ID X Manager to open the GeneMapper ID X Manager GeneMapper ID X Manager Find Name Containing O 0 2 0 lt i o 0 p e v gt O p 2 0 mM Projects Analysis Methods Table Settings Plot Settings Matrices Size Standards Report Settings 2 Select the Analysis Methods tab then click New to open the Analysis Method Editor with the General tab selected 3 The figures below show the settings for each tab of the Analysis Method Editor Configure the Analysis Method Editor tab settings as shown in the figures below unless the instructions state otherwise Note The Analysis Method Editor closes when you save your settings To complete this step quickly do not save the analysis method until you finish entering settings in all of the tabs 4 After you enter settings in all tabs click Save AmpFtSTR Identifiler PCR Amplification Kit User Guide 55 4 Chapter 4 GeneMapper ID X Software Set up GeneMapper ID X Software for data analysis General tab settings Analysis Method Editor General allele Peak Detector Peak Quality SQ amp GQ Settings r nalysis Method Description Name Identifiler_AnalysisMethod_v2x Security Group GeneMapper ID X Security Group Description Instrument Analysis Type
38. and the safety information provided in this document Before using an instrument or device read and understand the safety information provided in the user documentation provided by the manufacturer of the instrument or device Before handling chemicals read and understand all applicable Safety Data Sheets SDSs and use appropriate personal protective equipment gloves gowns eye protection etc To obtain SDSs see the Documentation and Support section in this document AmpFtSTR Identifiler PCR Amplification Kit User Guide 127 Appendix D Safety Chemical safety Chemical safety AN WARNING GENERAL CHEMICAL HANDLING To minimize hazards ensure laboratory personnel read and practice the general safety guidelines for chemical usage storage and waste provided below and consult the relevant SDS for specific precautions and instructions Read and understand the Safety Data Sheets SDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials To obtain SDSs see the Documentation and Support section in this document Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood Check regularly for chemical leaks
39. appropriately sized polypropylene tube 3 Vortex the tube then centrifuge briefly 4 Into each 0 2 mL sample tube add e 25 uL of the formamide size standard mixture 1 5 uL of PCR product or allelic ladder Note For blank wells add 25 uL of Hi Di Formamide 5 Sealthe tubes with the appropriate septa then briefly centrifuge to ensure that the contents of each tube are mixed and collected at the bottom Heat the tubes in a thermal cycler for 3 minutes at 95 C Immediately place the tubes on ice for 3 minutes Place the sample tray on the autosampler Ensure that an injection list is prepared Start the electrophoresis 32 AmpFtSTR Identifiler PCR Amplification Kit User Guide Analyze Data Section 4 1 GeneMapper ID Software 33 Overview of GeneMapper ID Software 33 Set up GeneMapper ID Software for data 34 Analyze and edit sample files with GeneMapper ID Software 46 Examine and edita project W K KK KK KK KK KK KK KK KK KK KK KK KK KK KK KK 47 For more information Wk kk kk kk kk K k k k l l le l le l le lel ee a 47 Section 4 2 GeneMapper ID X Software 48 Overview of GeneMapper ID X Software 48 Set up GeneMapper ID X Software for data
40. care necessary for using PCR technology PCR setup work area IMPORTANT These items should never leave the PCR Setup Work Area Calculator Gloves disposable Marker pen permanent Microcentrifuge Microcentrifuge tubes 1 5 mL or 2 0 mL or other appropriate clean tube for Master Mix preparation Microcentrifuge tube rack Pipette tips sterile disposable hydrophobic filter plugged Pipettors AmpFtSTR Identifiler PCR Amplification Kit User Guide 125 Appendix C PCR Work Areas Amplified DNA work area Tube decapper autoclavable e Vortex Amplified DNA work area IMPORTANT Place the thermal cyclers in the Amplified DNA Work Area You can use the following systems GeneAmp PCR System 9700 with the Silver 96 Well Block e GeneAmp PCR System 9700 with Gold plated Silver 96 Well Block IMPORTANT The Identifiler Kit is not validated for use with the GeneAmp PCR System 9700 with the Aluminium 96 Well Block Use of this thermal cycling platform may adversely affect performance of the Identifiler Kit Veriti 96 Well Thermal Cycler 126 AmpFtSTR Identifiler PCR Amplification Kit User Guide Safety WARNING GENERAL SAFETY Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device Ensure that anyone using this product has received instructions in general safety practices for laboratories
41. ldentifiler AnalysisMethod v1 Identifiler v2 CE G5 HID GS500 75 45 e 10 Ident 007 0 0625ng F01 fsa Ident 007 0 0625ng Sample Identifiler_AnalysisMethod_v1 Identifiler v2 CE G5 HID GS500 75 45t b 11 Ident 007 0 0625ng F02 fsa Ident 007 0 0625ng Sample Identifiler_AnalysisMethod_v1 Identifiler_v2 CE G5 HID GS500 75 45t 12 Ident 007 0 0625ng F03 fsa Ident 007 0 0625ng Sample Identifiler_AnalysisMethod_v1 Identifiler v2 CE G5 HID GS500 75 45 13 Ident 007 0 0625ng F04 fsa Ident 007 0 0625ng Sample Identifiler_AnalysisMethod_v1 Identifiler v2 CE G5 HID GS500 75 45 14 Ident 007 0 125ng E01 fsa Ident 007 0 125ng Sample Identifiler_AnalysisMethod_v1 Identifiler_v2 CE G5 HID GS500 75 45 15 Ident 007 0 125ng E02 fsa Ident 007 0 125ng Sample Identifiler_AnalysisMethod_v1 Identifiler_v2 CE G5 HID GS500 75 45t 16 Ident_007_0 125ng_E03 fsa Ident 007 0 125ng Sample Identifiler AnalysisMethod v1 Identifiler v2 CE G5 HID GS500 75 45t 17 Ident_007_0 125ng_E04 fsa Ident_007_0 125ng Sample Identifiler amp nalysisMethod v1 Identifiler v2 CE G5 HID GS500 75 45t 18 Ident_007_0 25ng_D01 fsa Ident_007_0 25ng Sample Identifiler amp nalysisMethod v1 Identifiler v2 CE G5 HID GS500 75 45t 19 Ident Q07 0 25ng D02 fsa Ident 007 0 25ng Sample Identifiler amp nalysisMethod v1 Identifiler v2 CE G5 HID GSS00 75 45t 20 Ident 007 0 25ng D03 fsa Ident 007 0 25ng Sample Identifiler amp nalysisMethod v1 Identifiler v2 CE G5 HID GSS00 75 45t 2 Ident_007_0 25ng_D04 fsa Ident
42. mL N8010580 MicroAmp 96 well base holds 0 2 mL reaction tubes N8010531 MicroAmp 96 well full plate cover N8010550 MicroAmp 96 well tray retainer set 403081 POP 4 polymer for the 310 Genetic Analyzer 402838 For a complete list of parts and accessories for the 310 instrument refer to Appendix B of the 370 Genetic Analyzer User Guide Pub no 4317588 PCR Amplification MicroAmp 96 well tray N8010541 MicroAmp reaction tube with cap 0 2 mL N8010540 MicroAmp 8 tube strip 0 2 mL N8010580 MicroAmp 8 cap strip N8010535 MicroAmp 96 well tray retainer set 403081 MicroAmp 96 well base N8010531 MicroAmp clear adhesive film 4306311 MicroAmp optical adhesive film 4311971 MicroAmp optical 96 well reaction plate N8010560 Other user supplied materials Hi Di Formamide 25 mL 4311320 Aerosol resistant pipette tips MLS Microcentrifuge tubes MLS Pipettors MLS Tape labeling MLS Tube 50 mL Falcon MLS Tube decapper autoclavable MLS Deionized water PCR grade MLS AmpFtSTR Identifiler PCR Amplification Kit User Guide 123 El Appendix B Ordering Information Equipment and materials not included Item Source Tris HCL pH 8 0 MLS EDTA 0 5 M MLS Vortex MLS t For the Safety Data Sheet SDS of any chemical not distributed by Life Technologies contact the chemical manufacturer Before handling any chemicals refer to the SDS
43. retainer set and tubes in GeneAmp 9700 Insufficient PCR product electrokinetically injected Remove MicroAmp Base from tray retainer set and repeat test Prepare PCR product as described in Chapter 3 Perform Electrophoresis on page 25 Degraded formamide Check the storage of formamide do not thaw and refreeze multiple times Try Hi Di Formamide AmpFtSTR Identifiler PCR Amplification Kit User Guide 119 Appendix A Troubleshooting Observation Possible causes Recommended actions Positive signal from AmpF STR Control DNA 9947A but partial or no signal from DNA test samples Quantity of test DNA sample is below assay sensitivity Quantitate DNA and add 1 0 ng of DNA Repeat test Test sample contains high concentration of PCR inhibitor for example heme compounds certain dyes Quantitate DNA and add minimum necessary volume Repeat test Wash the sample in a Centricon 100 centrifugal filter unit Repeat test Test sample DNA is severely degraded If possible evaluate the quality of DNA sample by running an agarose gel If DNA is degraded reamplify with an increased amount of DNA or use the AmpF STR MiniFiler Kit Dilution of test sample DNA in water or wrong buffer for example TE formula with incorrect EDTA concentration Redilute DNA using low TE Buffer with 0 1 mM EDTA More than two allele present at a locus Some but not
44. showing equivalence with the degenerate primer has been published AmpFtSTR9 Identifiler9 PCR Amplification Kit User Guide 11 Loci amplified by the kit Chapter 1 Overview Product overview Non nucleotide linkers are used in primer synthesis for the following loci CSF1PO D135317 D165539 D251338 and TPOX For these primers non nucleotide linkers are placed between the primers and the fluorescent dye during oligonucleotide synthesis Butler 2005 Grossman et al 1994 and Baron et al 1996 Non nucleotide linkers enable reproducible positioning of the alleles to facilitate interlocus spacing The combination of a five dye fluorescent system and the inclusion of non nucleotide linkers allows for simultaneous amplification and efficient separation of the 15 STR loci and Amelogenin during automated DNA fragment analysis The following table shows the loci amplified their chromosomal locations and the corresponding fluorescent marker dyes The AmpF STR Identifiler Allelic Ladder is used to genotype the analyzed samples The alleles contained in the allelic ladder and the genotype of the AmpF STR Control DNA 9947A are also listed in the table Table 1 Identifiler9 Kit loci and alleles Lacus desia ation Chromosome Alleles included in AmpF STR Dye Control DNA g location Identifiler Allelic Ladder label 9947A D8S1179 8 8 9 10 11 12 13 14 15 16 17 18 19 6 FAM 131 D
45. study known artifacts Y scale 100 RFU VIC9 dye labeled artifact at 70 bp NED dye labeled artifact at 88 bp viet Sewe Te oss Control A Control B 110 AmpFtSTR dentifiler9 PCR Amplification Kit User Guide Section 5 3 Performance Validation After Buffer and Enzyme Component Replacement 5 Sensitivity study Sensitivity study For the sensitivity study dilution series of three genomic DNA samples were amplified 1 ng three replicates each 0 5 ng 0 25 ng and 0 125 ng four replicates each The results were evaluated for mean referenced peak height degree of linearity between input DNA concentration and peak height level of allelic dropout at 125 pg and genotype concordance Mean referenced Mean referenced peak height observations were consistent between all Test and peak height Control mixes Figure 25 demonstrating equivalent performance Figure 26 Figure 25 Sensitivity study mean referenced peak heights three genomic DNA samples 125 250 500 1000 Reagent Lot Contro B Control Test A Test 8 b Test C U ES e 3 D o 5 Q S e gt Ww E 5 m 5 N lt 3 D e e o E o ke 3 i 125 250 500 1000 125 250 500 1000 Input DNA Concentration pg AmpFtSTR Identifiler PCR Amplification Kit User Guide 111 5 Chapter 5 Performance Validation After Buffer and En
46. to obtain and compare quantitative values for the different allele peak heights on Applied Biosystems instruments provides additional valuable data to aid in resolving mixed genotypes This quantitative value is much less subjective than comparing relative intensities of bands on a stained gel Ultimately the likelihood that any sample is a mixture must be determined by the analyst in the context of each particular case including the information provided from known reference sample s Limit of detection of the minor component Mixtures of two DNA samples were examined at various ratios 1 1 to 1 10 The total amount of genomic input DNA mixed at each ratio was 1 ng The samples were amplified in a GeneAmp PCR System 9700 with a silver or gold plated silver block and were electrophoresed and detected using a 310 Genetic Analyzer AmpFtSTR Identifiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation Mixture studies The results of the mixed DNA samples are shown in Figure 20 where sample A and sample B were mixed according to the ratios provided The profiles of the samples in Figure 20 are listed in Table 5 Table 5 Mixture profiles Profile Allele Sample A Sample B J Amelogenin X X Y CSF1P0 10 12 11 12 3 D2S1338 17 25 20 23 0351358 15 18 15 16 D5S818 11 13 11 2 D7S820 9 10 7 12 D8S1179 13 12 13 0135317 11 11 016
47. to reduce the occurrence of artifacts in the PET dye and VIC dye channels that may interfere with the interpretation of casework samples We amplified negative control samples using lot number 0301011 and lot number 0310018 and generated data using the 310 Genetic Analyzer with the Windows NT OS using the G5 module Results show that the VIC and PET labeled artifacts are greatly reduced in the after the manufacturing process improvements Figure 21 Figure 21 Comparison of the observed VIC dye and PET dye labeled artifacts for negative control amplifications with Identifiler9 Kit lot numbers before and after kit lot number 0310018 The artifacts have been highlighted for illustrative purposes VIC dye labelled artifact at 120 bp PET dye labeled artifacts between Amelogenin and 055818 loci U e 3 hy 2 e zi e 2 10 3 5 e ER 2 20 9 e p 7 7 3 9 lt 3 o 2 Lot no 0301011 Lot no 0301011 representative of lots representative of lots 0103001 0308017 0103001 0308017 Before process improvements a Lot 0310018 Lot no 0310018 representative of lots representative of lots After 0310018 and later 0310018 and later process improvements For more information refer to AmpFtSTR Identifiler PCR Amplification Kit Human Identification Application Note Pub no 040302 A
48. 0 o 1080 540 l L 15 5 D18 D18 A4 CSF CSF 110 1 D2 D2 90 AmpFtSTR dentifiler9 PCR Amplification Kit User Guide Section 5 1 Developmental Validation Mixture studies Mixture studies DAB 8 1 2 2 Mixture Studies Mixed specimen studies Species specificity sensitivity stability and mixture studies are conducted DAB 1998 Evidence samples may contain DNA from more than one individual The possibility of multiple contributors should be considered when interpreting the results We recommend that individual laboratories assign a minimum peak height threshold based on validation experiments performed in each laboratory to avoid typing when stochastic effects are likely to interfere with accurate interpretation of mixtures Evidence samples that contain body fluids and or tissues originating from more than one individual are an integral component of forensic casework Therefore it is essential to ensure that the DNA typing system is able to detect DNA mixtures In the case of STRs stutter peaks may be informative in the interpretation of mixed samples Furthermore alleles amplified with the Identifiler Kit have similar peak height values for a heterozygous genotype within a locus This balance can be used as an aid in detecting and interpreting mixtures Detection of mixed samples Each of the following can aid in determining whether a sample i
49. 0 791 15 t 0 291 0 341 t D2S1338 15 0 14t t t t 16 5 32 4 73 2 41 2 62 AmpFtSTR Identifiler PCR Amplification Kit User Guide 95 Chapter 5 Experiments and Results Population data Allele African American U S Caucasian U S Hispanic Native American n 357 n 349 290 n 191 17 10 78 17 34 21 21 9 95 18 5 60 6 30 4 14 7 07 19 14 15 13 75 22 76 29 58 20 6 02 14 61 13 79 9 69 21 14 01 2 58 2 59 2 36 22 13 17 4 01 7 41 15 18 23 10 78 11 46 11 38 11 78 24 9 80 11 75 8 45 7 85 25 8 12 10 60 5 17 3 14 26 1 96 2 72 0 69t 0 791 27 0 14t 0 14t t t 28 96 AmpFtSTR Identifiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation Population data Allele African American U S Caucasian U S Hispanic Native American 357 349 n 290 191 D3S1358 lt 11 0 421 0 141 1 b 11 t t t 0 261 12 0 561 t 0 171 t 13 0 701 0 291 0 171 t 14 12 04 15 76 7 41 6 81 15 30 53 25 36 39 14 40 84 15 2 0 141 t t t 16 28 57 22 78 26 72 32 98 17 19 47 18 19 16 03 9 95 18 6 72 16 48 8 97 8 38 19 0 84 1 00 1 03 0 791 20 t t 0 341 t D5S818 7 0 141 t 6 72 15 71 8 5 46 t 0 691 t 9 1 68 4 15 5 17 6 02 10 6 72 5 44 5 17 4 19 11 25 49 39 26 39 14 41 10 12 36 41 35 24 29 31 23 30 13 21 57 15 47 12 59 9 42 14 2 38 0 141 0 691 0 261 15 t 0 291 0 18
50. 1 49 Analyze and edit sample files with GeneMapper ID X Software 61 Examine and edita project 6 eee een 62 Section 4 1 GeneMapper ID Software Overview of GeneMapper D Software GeneMapper ID Software is an automated genotyping software for forensic casework databasing and paternity data analysis After electrophoresis the data collection software stores information for each sample in a fsa file Using GeneMapper ID Software v3 2 1 software you can then analyze and interpret the data from the fsa files Instruments Refer to Instrument and software overview on page 16 for a list of compatible instruments AmpFtSTR Identifiler PCR Amplification Kit User Guide 33 4 Chapter 4 Analyze Data Set up GeneMapper ID Software for data analysis Before you start When using GeneMapper ID Software v3 2 1 to perform human identification HID analysis with AmpF STR kits be aware that HID analysis requires at least one allelic ladder sample per run folder Your laboratory can use multiple ladder samples in an analysis provided individual laboratories conduct the appropriate validation studies For multiple ladder samples the GeneMapper ID Software calculates allelic bin offsets by using an average of all ladders that use the same panel within a run folder Allelic ladder samples in an individual run folder are considered to be from a single run When the softwar
51. 1 t 16 t t 0 17t t 17 0 141 t 0 17t t AmpFtSTR Identifiler PCR Amplification Kit User Guide 97 g lt 3 2 EI 2 Chapter 5 Experiments and Results Population data Allele African American U S Caucasian U S Hispanic Native American n 357 n 349 n 290 n 191 D7S820 6 T 0 14 0 17 t 7 0 421 1 29 1 72 0 521 8 18 77 16 48 11 72 13 09 9 13 73 17 62 6 21 8 12 10 34 45 27 22 27 41 21 99 11 19 89 18 05 28 79 28 80 12 10 78 14 76 20 17 24 08 13 1 54 3 72 3 45 3 40 14 0 421 0 72 0 341 Li 15 D8S1179 8 0 421 2 29 0 341 0 521 9 0 421 1 15 0 341 0 261 10 2 38 9 74 8 45 4 71 11 3 92 6 02 5 86 3 40 12 13 31 14 04 12 07 11 52 13 23 25 32 52 32 93 37 43 14 30 11 21 35 26 21 30 63 15 20 17 9 89 10 86 9 42 16 4 62 2 72 2 41 1 57 17 1 12 0 291 0 521 0 521 18 0 281 t t t 19 t t T D13S317 8 3 08 12 18 9 66 4 97 9 2 52 7 74 21 72 17 80 10 3 78 4 44 9 14 13 61 11 24 51 29 80 23 10 24 35 12 46 22 30 80 20 86 23 04 13 15 41 11 17 10 17 7 85 14 4 34 3 72 5 34 8 12 15 0 141 0 141 t 0 261 98 AmpFtSTR Identifiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation Population data Allele African American U S Caucasia
52. 21S11 21q11 2 q21 24 24 2 25 26 27 28 28 2 29 29 2 30 30 30 2 31 31 2 32 32 2 33 33 2 34 34 2 35 35 2 36 37 38 D7S820 7411 21 22 6 7 8 9 10 11 12 13 14 15 10 11 CSF1PO 5433 3 34 6 7 8 9 10 11 12 13 14 15 10 12 0351358 12 13 14 15 16 17 18 19 vic 14 15 THO1 11p15 5 4 5 6 7 8 9 9 3 10 11 13 3 8 9 3 D13S317 13q22 31 8 9 10 11 12 13 14 15 118 D16S539 16q24 qter 5 8 9 10 11 12 13 14 15 11 12 D2S1338 2435 37 1 15 16 17 18 19 20 21 22 23 24 25 26 19 23 27 28 0195433 19912 13 1 9 10 11 12 12 2 13 13 2 14 14 2 15 NED 14 15 15 2 16 16 2 17 17 2 wA 12p12 pter 11 12 13 14 15 16 17 18 19 20 21 22 17 18 23 24 TPOX 2p23 2per 6 7 8 9 10 11 12 13 gtt D18S51 18q21 3 7 9 10 10 2 11 12 13 13 2 14 14 2 15 15 19 16 17 18 19 20 21 22 23 24 25 26 27 Amelogenin X p22 1 22 3 X Y PET X Y p11 2 D5S818 5g21 31 7 8 9 10 11 12 13 14 15 16 1144 FGA 4q28 17 18 19 20 21 22 23 24 25 26 26 2 27 23 24 28 29 30 30 2 31 2 32 2 33 2 42 2 43 2 44 2 45 2 46 2 47 2 48 2 50 2 51 2 t For CODIS purposes profi For CODIS purposes profi 8 For CODIS purposes profi tt For CODIS purposes profi ttFor CODIS purposes profi 12 e reported as 13 13 e reported as 30 30 e reported as 11 11 e reported as 8 8 e reported as 11 11 AmpFtSTR Identifiler PCR Amplific
53. 33 i e E dt Zt IF UC 5 UU 3 4 H 3 0 e 38 2 1 0 9 10 11 12 13 14 15 16 17 11 12 13 14 15 16 17 18 19 20 21 67 8 9 1011 12 11 12 13 14 15 16 17 18 19 20 21 22 23 D19S433 vWA TPOX D18S51 Allele AmpFtSTR Identifiler PCR Amplification Kit User Guide 79 Chapter 5 Experiments and Results Extra Peaks in the electropherogram Figure 11 Stutter percentages for the D5S818 and FGA loci are e 9494994 MM 5 5 z 2 i 2 o a 7 8 9 10 11 12 13 14 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 D5S818 FGA Allele Figure 12 Sample 1 in panel A and panel B has a profile of 15 16 for 0351358 The amount of stutter can not be accurately measured due to the VIC dye artifact Note the degree of magnification y axis used in panels B and to illustrate the artifact Data was produced on the 310 Genetic Analyzer Negative Control 80 AmpFtSTR dentifiler9 PCR Amplification Kit User Guide Addition of 3 A nucleotide Artifacts Section 5 1 Developmental Validation Extra Peaks in the electropherogram AmpliTaq Gold enzyme like many other DNA polymerases can catalyze the addition of a single nucleotide predominately adenosine to the 3 ends of double stranded PCR products Clark 1988 Magnuson et al 1996 This non template addition results in a PCR produ
54. 4 4 30 4 66 3 40 11 21 43 25 93 27 24 39 27 12 3 08 4 73 10 52 14 92 13 1 1 1 AmpFtSTR Identifiler PCR Amplification Kit User Guide 103 g lt e 3 2 EX S Chapter 5 Experiments and Results Population data Allele African American U S Caucasian U S Hispanic Native American n 357 n 349 290 191 11 0 281 t 0 171 t 12 t t t 0 261 13 1 26 0 431 t 0 261 14 7 14 8 31 6 90 4 45 15 20 03 11 32 10 00 7 07 16 26 75 23 35 34 31 32 98 17 20 59 24 50 21 55 33 51 18 14 71 22 49 18 45 15 45 19 6 72 8 31 7 07 4 71 20 1 96 1 15 1 38 1 051 21 0 281 t 0 171 0 261 22 0 281 t t f 23 t t t t 24 t 0 141 t t Aminimum allele frequency 0 7 for the African American database 0 7 for the U S Caucasian database 0 9 for the U S Hispanic database and 1 3 for the Native American database is suggested by the National Research Council in forensic calculations Analyzing the four databases Low frequency alleles 104 Analysis across the four databases of 2274 total chromosomes per locus revealed the following number of different alleles 10 CSF1PO alleles 13 D2S1338 alleles at least 12 0351358 alleles 11 055818 alleles 9 075820 alleles 11 0851179 alleles 8 0135317 alleles 8 0165539 alleles 20 018551 alleles 17 0195433 alleles 26 021511 alleles 31 FGA alleles 9 1 alle
55. 5 0 05 15 2 127 43 0 05 16 129 44 0 05 16 2 131 46 0 05 17 133 42 0 03 17 2 135 44 0 06 D21S11 24 184 86 0 04 24 2 186 82 0 02 25 188 77 0 03 26 192 69 0 05 27 196 56 0 04 28 200 41 0 05 28 2 202 36 0 05 29 204 32 0 03 29 2 206 31 0 02 30 208 29 0 07 30 2 210 24 0 05 31 212 23 0 05 31 2 214 14 0 06 32 216 14 0 04 32 2 218 10 0 04 33 220 14 0 05 33 2 222 07 0 04 74 AmpFtSTR Identifiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation Accuracy precision and reproducibility 310 Genetic Analyzer Allele Mean Standard Deviation 34 224 10 0 07 34 2 226 02 0 06 35 228 07 0 06 35 2 230 01 0 07 D 36 232 04 0 07 37 236 00 0 03 3 38 239 94 0 08 z FGA 5 17 214 81 0 07 18 218 80 0 06 gt 19 222 79 0 07 20 226 81 0 06 21 230 76 0 08 22 234 78 0 07 23 238 81 0 05 24 242 83 0 07 25 246 88 0 06 26 250 96 0 06 26 2 253 00 0 09 27 254 97 0 08 28 259 02 0 10 29 263 12 0 08 30 267 26 0 09 30 2 269 07 0 10 31 2 273 17 0 09 32 2 277 25 0 08 33 2 281 33 0 09 42 2 319 83 0 14 43 2 324 04 0 14 44 2 328 26 0 13 45 2 332 42 0 16 46 2 336 43 0 14 47 2 340 42 0 14 48 2 344 15 0 10 50 2 351 45 0 05 51 2 355 13 0 05 AmpFtSTR Identifiler PCR Amplification Kit User Guide 75 Chapter 5 Experiments and Results Accuracy precision and reproducibility
56. 5 to1 0 Genotype Quality From zs to1 0 FromO 0to 0 25 From to 025 Factory Defaults OK Cancel IMPORTANT The values shown the software defaults the values we used during developmental validation Perform the appropriate internal validation studies to determine the appropriate values for interpretation of Identifiler Kit data Create size The size standards for the Identifiler Kit use the following size standard peaks in standard their definitions GeneScan 500 LIZ Size Standard GeneScan 600 LIZ Size Standard v2 0 75 100 139 150 160 200 300 340 350 400 and 450 80 100 114 120 140 160 180 200 214 220 240 250 260 280 300 314 320 340 360 380 400 414 420 440 and 460 Note The 250 nt peak in the GeneScan 500 LIZ Size Standard is not included in the size standard definition This peak can be used as an indicator of precision within a run Use the following procedure to create the appropriate size standard 1 Select Tools GeneMapper Manager to open the GeneMapper Manager 44 AmpFtSTR dentifiler9 PCR Amplification Kit User Guide Section 4 1 GeneMapper ID Software Set up GeneMapper ID Software for data analysis 2 Select the Size Standards tab click New select the Basic or Advanced radio button then click OK Manager x Projects Analysis Methods Table Setting
57. 5539 11 12 9 10 018551 17 19 12 15 0195433 13 14 15 021511 30 30 2 28 31 FGA 23 2 24 24 26 THO1 7 9 7 9 3 TPOX 8 9 8 wA 17 19 14 16 For these 1 ng total DNA mixture studies the limit of detection is when the minor component is present at approximately one tenth of the concentration of the major component and a threshold of 50 RFU The limit of detection for the minor component is influenced by the combination of genotypes in the mixture AmpFtSTR Identifiler PCR Amplification Kit User Guide 93 Chapter 5 Experiments and Results Data interpretation Figure 20 Results of the two DNA samples mixed together at defined ratios and amplified with the Identifiler Kit Sample A and Sample are a female and male sample respectively The ratios of Sample A to Sample B A B ratios shown are 10 1 3 1 1 1 1 3 and 1 10 respectively The alleles attributable to the minor component even when the major component shares an allele are highlighted in panels 2 3 5 and 6 All alleles are highlighted in panel 4 Panel 1 Sample A Sample B Data interpretation Minimum sample The Identifiler Kit has been optimized to reliably amplify and type approximately The PCR cycle number and amplification conditions have been specified to produce low peak heights fo
58. 7 8 216 87 0 05 9 220 83 0 05 10 224 77 0 07 11 228 88 0 07 72 AmpFtSTR Identifiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation Accuracy precision and reproducibility 310 Genetic Analyzer Allele Mean Standard Deviation 12 232 81 0 05 13 236 68 0 07 14 240 69 0 06 15 244 68 0 09 g 0165539 5 252 37 0 08 3 8 264 30 0 07 z 9 268 32 0 08 5 10 272 32 0 06 11 276 37 0 07 2 12 280 37 0 09 13 284 34 0 07 14 288 44 0 09 15 292 51 0 07 018551 7 262 07 0 08 9 270 22 0 06 10 274 34 0 09 10 2 276 36 0 06 11 278 41 0 08 12 282 49 0 05 13 286 57 0 06 13 2 288 63 0 05 14 290 77 0 04 14 2 292 78 0 05 15 294 91 0 07 16 299 07 0 06 17 303 50 0 07 18 307 94 0 09 19 312 40 0 11 20 316 71 0 09 21 320 99 0 14 22 325 24 0 11 23 329 40 0 11 24 333 54 0 15 25 337 67 0 11 AmpFtSTR Identifiler PCR Amplification Kit User Guide 73 Chapter 5 Experiments and Results Accuracy precision and reproducibility 310 Genetic Analyzer Allele Mean Standard Deviation 26 341 56 0 09 27 345 24 0 08 D19S433 9 101 99 0 05 10 105 88 0 05 11 109 78 0 04 12 113 64 0 02 12 2 115 61 0 02 13 117 56 0 03 13 2 119 55 0 02 14 121 46 0 03 14 2 123 47 0 02 15 125 4
59. 72 8 79 10 73 16 2 38 4 15 4 31 3 93 16 2 2 38 1 72 2 93 1 83 17 t 0 291 0 171 0 791 17 2 0 281 0 291 t 2 88 18 2 0 141 0 291 t 1 051 100 AmpFtSTR Identifiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation Population data Allele African American U S Caucasian U S Hispanic Native American 357 349 n 290 191 D21S11 24 Y t t t 24 2 0 14t 0 431 0 171 t 24 3 0 281 t t t 25 1 y 25 2 0 141 0 171 t 8 26 0 14t 0 14t 0 171 t 3 27 5 04 4 58 1 21 0 521 s 28 22 97 16 76 9 14 6 28 o 282 t t t 9 29 19 33 20 49 21 21 16 75 29 2 0 14 t 0 52t 0 26t 29 3 0 141 t t t 30 17 23 25 21 29 31 34 29 30 2 1 40 3 30 2 93 1 83 31 7 98 7 16 6 72 5 76 31 2 7 98 9 46 8 62 18 85 32 1 12 1 43 1 55 0 791 32 2 5 88 7 16 12 93 9 69 33 0 561 t t 0 521 33 2 3 78 3 30 4 14 3 66 34 1 26 t t t 34 1 0 141 t t t 34 2 0 141 0 291 0 861 0 791 35 2 94 t 0 341 t 35 1 0 141 t t i 352 t 0 141 36 0 84 t T 37 0 28 T t t 38 0 141 T t t AmpFtSTR Identifiler PCR Amplification Kit User Guide 101 Chapter 5 Experiments and Results Population data Allele African American U S Caucasian U S Hispanic Native American n 357 n 349 n 290
60. 89 AmpF amp TR Identifiler PCR Amplification Kit User Guide Index developmental validation 66 differential amplification of loci 88 DNA control about 18 effect of DNA quantity on results 85 86 how degraded DNA affects which loci amplify 88 89 mixed samples causing extra peaks 91 negative control reaction 21 positive control reaction 21 quantification 19 quantification methods 20 sample preparation 21 test sample 21 using agarose gel analysis to examine the DNA 88 documentation related 131 E effect of inhibitors 87 electropherogram addition of a 3 A nucleotide 81 causes for extra peaks 92 93 DNA from more than one individual 91 stutter peak 77 electrophoresis Data Collection Software 27 29 31 preparing samples on the 310 instrument 31 preparing samples on the 3100 3100 A vant or 3130 3130xl instrument 28 preparing samples on the 3500 3500xL instrument 29 reagents and parts 27 29 31 references 27 29 31 run module 27 29 31 setup 27 29 31 emission spectra 17 enzyme new 108 equipment not included with kit 121 evidence exclusion of suspects 95 137 Index F fluorescent dyes 16 FTA cards amplification 22 bloodstained 22 G gels 88 GeneMapper ID Software analyze project 46 create analysis method 39 create size standard 44 examine and edit project 47 import panels and bins 35 overview 16 33 setup 34 GeneMapper ID X Software analyze project 61 check version of panels bins and st
61. 91 The PCR amplification of tetranucleotide STR loci typically produces a minor product peak four bases shorter n 4 than the corresponding main allele peak This is referred to as the stutter peak or product Sequence analysis of stutter products at tetranucleotide STR loci has revealed that the stutter product is missing a single tetranucleotide core repeat unit relative to the main allele Walsh et al 1996 The proportion of the stutter product relative to the main allele percent stutter is measured by dividing the height of the stutter peak by the height of the main allele peak Such measurements have been made for amplified samples at the loci used in the Identifiler Kit All data were generated on the 310 Genetic Analyzer Some of the general conclusions from these measurements and observations are as follows For each Identifiler Kit locus the percent stutter generally increases with allele length as shown in Figure 8 through Figure 12 on the following pages Smaller alleles display a lower level of stutter relative to the longer alleles within each locus This is reflected in Figure 8 through Figure 11 where minimal data points are plotted for some smaller alleles as stutter could not be detected for many of these samples Forthe alleles within a particular locus the percent stutter is generally greater for the longer allele in a heterozygous sample this is related to the first point above Each allele within a loc
62. 92 J Forensic Sci 51 826 830 Nakahori Y Takenaka O and Nakagome Y 1991 A human X Y homologous region encodes amelogenin Genomics 9 264 269 National Research Council 1996 The evaluation of forensic DNA evidence National Academy Press Washington D C Nei M 1978 Estimation of average heterozygosity and genetic distance from a small number of individuals Genetics 89 583 590 Nei M 1973 Analysis of gene diversity in subdivided populations Proc Natl Acad Sci USA 70 3321 3323 Revised Validation Guidelines Scientific Working Group on DNA Analysis Methods SWGDAM Forensic Science Communications July 2004 Volume 6 3 Available at www fbi gov hq lab fsc current standards 2004_03_standards02 htm Puers C Hammond H Jin L Caskey C and Schumm J 1993 Identification of repeat sequence heterogeneity at the polymorphic short tandem repeat locus HUMTH01 AATG n and reassignment of alleles in population analysis using a locus specific allelic ladder Am J Hum Genet 53 953 958 Sensabaugh G F 1982 Biochemical markers of individuality In Saferstein R ed Forensic Science Handbook Prentice Hall Inc New York pp 338 415 Sharma V and Litt M 1992 Tetranucleotide repeat polymorphism at the 021511 locus Hum Mol Genet 1 67 Smith R N 1995 Accurate size comparison of short tandem repeat alleles amplified by PCR Biotechniques 18 122 128 Sparkes R Kimpton C Watson S
63. A 94 1041 1046 Clark J M 1988 Novel non templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases Nucleic Acids Res 16 9677 9686 AmpFtSTR Identifiler PCR Amplification Kit User Guide 133 Bibliography 134 DeFranchis R Cross N C P Foulkes N S and Cox T M 1988 A potent inhibitor of Taq DNA polymerase copurifies with human genomic DNA Nucleic Acids Res 16 10355 DNA Advisory Board Federal Bureau of Investigation U S Department of Justice 1998 Quality assurance standards for forensic DNA testing laboratories Edwards A Civitello A Hammond H and Caskey C 1991 DNA typing and genetic mapping with trimeric and tetrameric tandem repeats Am J Hum Genet 49 746 756 Edwards Hammond Lin J Caskey and Chakraborty 1992 Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups Genomics 12 241 253 Frank W Llewellyn B Fish P et al 2001 Validation of the AmpF STR Profiler Plus PCR Amplification Kit for use in forensic casework J Forensic Sci 46 642 646 Grossman P D Bloch W Brinson E Chang C C Eggerding F A Fung S Iovannisci D M Woo S and Winn Deen E S 1994 High density multiplex detection of nucleic acid sequences oligonucleotide ligation assay and sequence coded separation Nucleic Acids Res 22 4527 4534 Guo S W and Thompson E A 1992
64. Analyze Data Set up GeneMapper ID Software for data analysis Peak Detector tab settings Analysis Method Editor HID General Allele Peak Detector Peak Quality Quality Flags Peak Detection Algorithm Advanced M Ranges Peak Detection Perform Analysis Sizing internal Ful Range All Sizes validation studies to determine settings Smoothing and Baselining Smoothing O None Light Polynomial Degree O Heavy Peak Window Size Slope Threshold Peak Start Min Peak Half Width Baseline Window 51 pts r Size Calling Method 2nd Order Least Squares 3rd Order Least Squares Cubic Spline Interpolation Local Southern Method Global Southern Method Peak End om Factory Defaults IMPORTANT Perform the appropriate internal validation studies to determine the peak amplitude thresholds for interpretation of Identifiler Kit data Fields include Peak amplitude thresholds The software uses these parameters to specify the minimum peak height in order to limit the number of detected peaks Although GeneMapper ID Software displays peaks that fall below the specified amplitude in electropherograms the software does not label or determine the genotype of these peaks Size calling method The Identifiler Kit has been validated using the Local Southern sizing method Before using other sizing methods perform internal
65. Fluorescence intensity that exceeds the linear dynamic range for detection by the instrument off scale data Off scale data are problematic because Quantitation peak height and area for off scale peaks is not accurate For example an allele peak that is off scale can cause the corresponding stutter peak to appear higher in relative intensity thus increasing the calculated percent stutter Multicomponent analysis of off scale data is not accurate and it results in poor spectral separation pull up Incomplete A nucleotide addition When the total number of allele copies added to the PCR is extremely low allelic dropout can occur resulting in a partial profile Methods of quantifying DNA Life Technologies provides several kits for quantifying DNA in samples See the references cited in the following table for details about these kits Product Description Quantifiler Human DNA Quantification Kit Part no 4343895 and Quantifiler9 Y Human Male DNA Quantification Kit Part no 4343906 For more information see Quantifiler Human DNA Quantification Kits Users Manual Pub no 4344790 Properties The Quantifiler Human and Quantifiler Y Human Male Kits are highly specific for human DNA and they individually detect total human or male DNA respectively The kits detect single stranded and degraded DNA How they work The Quantifiler DNA Quantification Kits consist of target specif
66. GeneMapper ID X Software 4 Set up GeneMapper ID X Software for data analysis Peak Quality tab settings Analysis Method Editor General Allele Peak Detector Peak Quality SQ amp GQ Settings Min Max Peak Height LPHIMPH Homozygous min peak height Perform Heterozygous min peak height internal validation Max Peak Height MPH studies determine settings Peak Height Ratio PHR Min peak height ratio Broad Peak BD Max peak width basepairs Allele Number AN Max expected alleles Allelic Ladder Spike Cut off value Factory Defaults Save Cancel IMPORTANT Perform the appropriate internal validation studies to determine the minimum heterozygous and homozygous minimum peak height thresholds maximum peak height threshold and the minimum peak height ratio threshold for interpretation of Identifiler Kit data AmpFtSTR Identifiler PCR Amplification Kit User Guide 59 O 0 2 0 lt o 0 e v gt n e e 2 0 4 Chapter 4 GeneMapper ID X Software Set up GeneMapper ID X Software for data analysis SQ amp GQ tab settings Analysis Method Editor Quality weights are between and 1 Sample and Control GQ Weighting Broad Peak BD 0 8 Allele Number Out of Bin Allele BIN 8 Low Peak Height LPH Overlap OVL Max Peak Height Marke
67. HID Save Cancel In the Name field either type the name as shown or enter a name of your choosing In the Security Group field select the Security Group appropriate to your software configuration from the dropdown list The Description and Instrument fields are optional 56 AmpFtSTR dentifiler9 PCR Amplification Kit User Guide Section 4 2 GeneMapper ID X Software 4 Set up GeneMapper ID X Software for data analysis Allele tab settings Analysis Method Editor General Allele Peak Detector Peak Quality SQ amp GQ Settings Bin Set AmpFLSTR_Bins Use marker specific stutter ratio and distance if available Marker Repeat Type Tri Tetra Penta Global Cut off Value 0 0 0 0 0 0 Ratio 0 0 0 0 0 0 MinusA Distance 0 0 0 0 0 0 0 0 0 0 0 0 Global Minus Stutter Ratio 0 0 0 0 0 0 O 0 2 0 lt o 0 e v gt O e p 2 0 Global Minus Stutter Distance From 0 0 3 25 0 0 To 0 0 4 75 0 0 Global Plus Stutter Ratio 0 0 0 0 0 0 Global Plus Stutter Distance From 00 0 0 0 0 To 0 0 0 0 0 0 Amelogenin Cutoff 0 0 Range Filter Factory Defaults Save Cancel Inthe Bin Set field select the AmpFLSTR Bins v2X bin set and configure the stutter distance parameters as shown GeneMapper ID X So
68. It is important to note that while the precision within a set of capillary injections is very good the determined allele sizes vary between platforms Cross platform sizing differences arise from a number of parameters including type and concentration of polymer mixture run temperature and electrophoresis conditions Variations in sizing can also be found between runs on the same instrument and between runs on different instruments because of these parameters We strongly recommend that the allele sizes obtained be compared to the sizes obtained for known alleles in the AmpF STR Identifiler Allelic Ladder from the same run and then converted to genotypes For more information on precision and genotyping see Lazaruk et al 1998 and Mansfield et al 1998 1187 population database DNA samples have been typed using the Identifiler Kit see About the primers on page 11 These samples have been previously genotyped with concordant results of the same loci using other AmpF STR kits Table 3 Example of precision results of seven injections of the AmpF STR Identifiler Allelic Ladder 310 Genetic Analyzer Allele Mean Standard Deviation Amelogenin X 107 02 0 04 Y 112 61 0 02 CSF1PO 6 304 69 0 08 7 309 01 0 10 AmpFtSTR Identifiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation Accuracy precision and reproducibility 310 Genetic Analyzer
69. Laboratory Biosafety Manual third edition found at www who int csr resources publications biosafety WHO CDS CSR LYO 2004 11 AmpFtSTR Identifiler PCR Amplification Kit User Guide 129 Appendix D Safety Biological hazard safety 130 AmpFtSTR dentifiler9 PCR Amplification Kit User Guide Documentation and Support Related documentation Document title Pub no AmpFtSTR Identifiler PCR Amplification Kit Human Identification Application Note 040302 3100 3100 Avant Data Collection v2 0 User Guide 4347102 3100 3100 Avant Genetic Analyzers Using Data Collection Software v2 0 User Bulletin 4350218 3100 Genetic Analyzer User Manual Data Collection v1 1 4315834 3100 3100 Avant Genetic Analyzers Protocols for Processing AmpFISTR9 PCR Amplification Kit PCR Products 4332345 User Bulletin Applied Biosystems 3130 3100xl Genetic Analyzers Using Data Collection Software v3 0 User Bulletin 4363787 Applied Biosystems 3130 3130xl Genetic Analyzers Getting Started Guide 4352715 Applied Biosystems 3130 3130xl Genetic Analyzers Maintenance Troubleshooting and Reference Guide 4352716 Applied Biosystems 3130 3130xl Genetic Analyzers Quick Reference Card 4362825 Applied Biosystems 3130 3130xl Genetic Analyzers AB Navigator Software Administrator Guide 4359472 Applied Biosystems 3130 3100xl DNA Analyzers User Guide 4331468 Applied Biosystems 3500 3500xL Genetic Analyzer Quick Reference Card 4401662
70. M 10 uM 12 uM 14 uM 16 uM 18 uM and 20 uM When the amount of hematin was increased to a concentration that started to inhibit the PCR CSF1PO and D2S1338 were the first loci to exhibit decreased amplification followed by 0165539 and D18S51 Differential amplification was observed in the presence of increasing amounts of hematin Moreover as the concentration of hematin was increased the overall yield of products was reduced particularly for the larger loci AmpFtSTR Identifiler PCR Amplification Kit User Guide 87 g lt o e 3 e E s Chapter 5 Experiments and Results Stability Figure 17 DNA amplified with the Identifiler Kit in the presence of varying concentrations of hematin 0 10 uM 12 uM 14 uM 16 uM 18 uM and 20 uM analyzed on the 310 Genetic Analyzer 120 1 150 1 240 270 48003 32003 16003 of 111 di illas 4800 32003 LIN 3Y 8B A1 BW No hematin BW No hematin A1 BW No hematin BW No hematin iau Ea A1 BW No hematin BW No hematin 1 BW No hematin BW No hematin __ BW 100 BW 10uM BW 10uM BW 10uM 11 BW 100 BW 10uM BW 10uM BW 10uM A 4 A5 BW 12uM BW 12uM S BW 12uM BW 12uM BW 12uM BW 12uM BW 12uM BW 12uM aa ss BW 14uM BW 14uM A BW 14uM BW 14uM
71. OS is a registered trademark of Apple Inc 2012 Life Technologies Corporation All rights reserved Contents About This Guide USE CE 9 REVISION san bakan kulk ka l kk ee kt ka hi PRIA Tee E CAR et 2 9 Purpose oe a wi DN e n D MN A a Aine Das ae 9 B CHAPTER 1 11 Produet ove vieWrt x cess it one Re dt te dale tte qur e suse yukaywan gg le2z9 11 Purpose iud nus eg EE Em 11 Product description peka INC aes etn 11 Aboutthe primers la Reb be a nE HEnNREE HE Ea EEEEE suya eee 11 Loci amplifled by the lt Ayan k al S ER eei IN Ie EISE 12 Allelic ladder profile 13 Control DNA 2947A profile of siman eet s ua eh eb ER dvi be Hob 14 Workflow 0verview gace saz y kawe Seat Mad a Ka WL da ka DOR ka 3 uyu d l ka ERE Ka 15 Instrument and software overview 16 Data Collection and GeneMapper ID or D X Software 16 Instrument and software compatibility 16 About multicomponent analysis 16 How multicomponent analysis works
72. PCR Amplification Kit User Guide Section 5 3 Performance Validation After Buffer and Enzyme Component Replacement 5 Inhibition study Figure 34 Inhibition study representative electropherograms using Control DNA 007 inhibited with 34 uM Hematin Y scale 4000 RFU Mark sangka for li Control A Sangin Ror Dite Control B Mark Sampie for bosan Si e 3 5 2 e 3 Ww m N lt 3 D o o E o Figure 35 Inhibition study representative electropherograms using Control DNA 007 inhibited with15 25 ng uL Humic Acid Y scale 4000 RFU T Mek ampla hor Control A De Sample for Dino Control ws Semele for Allelic dropout No allelic dropout events were seen for any Test or Control mixes tested on uninhibited Control DNA 007 and Control DNA 007 inhibited with Hematin or Humic Acid AmpFtSTR Identifiler PCR Amplification Kit User Guide 117 5 Chapter 5 Performance Validation After Buffer and Enzyme Component Replacement Conclusions Conclusions Laboratories can expect to obtain equivalent quality profiles across a wide range of forensic samples when using the Identifiler Kit containing the AmpliTaq Gold enzyme and 10X PCR Buffer II manufactured by Life Technologies as compared to the original Identifiler Kit conta
73. PCR Amplification Kit User Guide 85 g lt e 3 e ei E s 2 Chapter 5 Experiments and Results Stability Figure 16 Effect of amplifying various amounts of DNA ranging from 16 pg to 1 ng Note that the y axis scale is magnified for the lower amounts of DNA Data analyzed using the 310 Genetic Analyzer 0 03 1ng Negative Control Stability DAB 8 1 2 2 Stability Lack of amplification of some loci 86 Species specificity sensitivity stability and mixture studies are conducted DAB 1998 As with any multi locus system the possibility exists that not every locus will amplify This is most often observed when the DNA sample contains PCR inhibitors or when the DNA sample has been severely degraded Because each locus is an independent marker whose results are not based upon information provided by the other markers results generally can still be obtained from the loci that do amplify AmpFtSTR Identifiler PCR Amplification Kit User Guide Differential and preferential amplification Effect of inhibitors Section 5 1 Developmental Validation 5 Stability Differential amplification can be defined as the difference in the degree of amplification of each locus within a co amplified system such that one or more loci may amplify to a greater extent compared to the other loci Preferential amplification is used in th
74. Reference Guide Pub no 4426482 GeneScan Analysis Software for the Windows NT Operating System Overview of the Analysis Parameters and Size Caller User Bulletin Pub no 4335617 O 2 D lt me 0 e i gt O D AmpFtSTR Identifiler PCR Amplification Kit User Guide 63 For more information Chapter 4 GeneMapper ID X Software 64 AmpFtSTR Identifiler PCR Amplification Kit User Guide Experiments and Results Section 5 1 Developmental Validation 66 Overview soe et ea ere eem A pU Ed d 66 Developmental validation LL KAK KK KK KK KK KK KK KK KK KK KK KK eee 67 Accuracy precision and reproducibility 0 0 KK KK KK KK 69 Extra Peaks in the electropherogram 06 6 e cece KK KK KK KK KK KK KK 77 Characterization Of 106 cesa bowed es ne kasu Eee aa es dace gach tana 82 Speciesispecificity sj oi Mek Pee Pde oe ad qe AK e kn 83 SOTI SI VIE at ya e AH LU NE Oi Rc Lect yek ed too e re Ao t 85 Stability 4 2 4535454405 de Sate eh eed wins bn l n 86 Mixture Studie Siei 2 0 E bed bol ER RU E OPER eR 91 Data mterpretation ie este a ee RN tag oh ale Lees 94 Population data sina s de eg e e ese ee eee Saves 94 Mutation l auqa aoe els EV EVER tea a 105 Probability of Identity sek oie ehe le ate aha NEN
75. USER GUIDE applied biosystems by Life technologies AmpF STR Identifiler PCR Amplification Kit for use with 200 reaction kit Part no 4322288 Publication Number 4323291 Rev J Revision Date August 2012 technologies For Forensic or Paternity Use Only Information in this document is subject to change without notice LIFE TECHNOLOGIES CORPORATION AND OR ITS AFFILIATE S DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT TO THE EXTENT ALLOWED BY LAW IN NO EVENT SHALL LIFE TECHNOLOGIES AND OR ITS AFFILIATE S BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF TRADEMARKS The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners TaqMan and AmpliTaq Gold are registered trademarks of Roche Molecular Systems Inc Windows and Windows Vista are registered trademarks of Microsoft Corporation EasiCollect is a registered trademark of Whatman Limited FTA is a registered trademark of Whatman International Limited Whatman is a registered trademark of GE Healthcare Companies Minitab is a registered trademark of Minitab Inc Mac
76. WA have been mapped and the chromosomal locations have been published Nakahori et al 1991 Edwards et al 1992 Kimpton et al 1992 Mills et al 1992 Sharma and Litt 1992 Li et al 1993 Straub et al 1993 Barber and Parkin 1996 Species specificity DAB 8 1 2 2 Species specificity sensitivity stability and mixture studies are conducted DAB 1998 Species Specificity The Identifiler Kit provides the required degree of specificity such that it is specific to primates Other species do not amplify for the loci tested with the exception of the Amelogenin locus Nonhuman Studies Nonhuman DNA may be present in forensic casework samples The Identifiler Kit provides the required degree of specificity such that it is specific to primates for the species tested with the exception of the Amelogenin locus Figure 15 AmpFtSTR Identifiler PCR Amplification Kit User Guide 83 Chapter 5 Experiments and Results Species specificity Figure 15 Representative electropherograms of a primate non primates a microorganism and a negative control are shown All samples were analyzed on an 310 Genetic Analyzer The peaks shown in orange are the GeneScan 500 LIZ Size Standard Negative Control The following experiments were conducted to investigate interpretation of Identifiler Kit results from nonhuman DNA sources The extracted DNA samples were amplified in Identifiler Kit reactions an
77. able reference documents Data Operatin Collection g Run modules and conditions References System Software 3 11 Windows GS STR P0P4 1mL G5 v2 md5 310 Genetic Analyzer User s Manual Windows or or Injection condition Pub no 4317588 3 01 Windows 15 kV 5 sec 310 Protocols for Processing AmpFtSTR PCR NT and Amplification Kit Products with Microsoft Windows Windows NT Operating System User Bulletin 2000 Pub no 4341742 t We conducted concordance studies for the Identifiler Kit using this configuration Prepare samples for electrophoresis on the 310 instrument Prepare the samples for electrophoresis immediately before loading 1 Calculate the volume of Hi Di Formamide and size standard needed to prepare the samples Reagent Volume per reaction GeneScan 500 LIZ9 Size Standard or 0 75 uL GeneScan 600 LIZ Size Standard v2 0 Hi Di Formamide 24 5 uL Note Include additional samples in your calculations to provide excess volume for the loss that occurs during reagent transfers AmpFtSTR Identifiler PCR Amplification Kit User Guide 31 Chapter 3 Perform Electrophoresis Prepare samples for electrophoresis on the 310 instrument IMPORTANT The volume of size standard indicated in the table is a suggested amount Determine the appropriate amount of size standard based on your results and experiments 2 Pipette the required volumes of components into an
78. all loci visible on electropherogram of DNA test samples Presence of exogenous DNA Use appropriate techniques to avoid introducing foreign DNA during laboratory handling Amplification of stutter product See Stutter products on page 77 Mixed sample Test sample DNA is severely degraded If possible evaluate the quality of DNA sample by running an agarose gel If DNA is degraded reamplify with an increased amount of DNA or use the AmpF STR MiniFiler Kit Test sample contains high concentrations of a PCR inhibitor for example heme compounds certain dyes Quantitate DNA and add minimum necessary volume Repeat test Wash the sample in a Centricon 9 100 centrifugal filter unit Repeat test Poor peak height Incorrect thermal cycler parameters Check the protocol for correct thermal cycler balance parameters GeneAmp PCR System 9700 with Use Applied Biosystems GeneAmp PCR System Aluminum 96 Well block or third 9700 with silver or gold plated silver blocks only party thermal cyclers or the Veriti 96 Well Thermal Cycler 120 AmpFtSTR dentifiler9 PCR Amplification Kit User Guide Equipment and materials not included Ordering Information Table 11 and Table 12 list required and optional equipment and materials not supplied with the Identifiler Kit Unless otherwise noted many of the items are available from major laboratory suppliers MLS Table 11 Equipment
79. and sub E EB Panel Manager folders to identify the analysis file version already installed for your kit choice 4 Check the version of files available for import into the Panel Manager a Select Panel Manager then select File Import Panels to open the Import Panels dialog box b Navigate to then open the Panels folder and check the version of panel bin and stutter files installed 5 If newer versions are available on the website download and import as described below Import panels To import the Identifiler Kit panel bin set and marker stutter from our web site into bins and marker the GeneMapper ID X Software database stutter 1 Download and open the file containing panels bins and marker stutter a Goto www lifetechnologies com support Software Patches amp Updates gt GeneMapper ID X Software Download the file AmpFLSTR Analysis Files GMIDX b Unzip the file 2 Start the GeneMapper ID X Software then log in with the appropriate user name and password IMPORTANT For logon instructions refer to the GeneMapper ID X Software Version 1 0 Getting Started Guide Pub no 4375574 3 Select Tools Panel Manager 4 Find then open the folder containing the panels bins and marker stutter a Select Panel Manager in the navigation pane s Panel Manager b Select File Import Panels to open the Import File Edit Bins View Hel Panels dialog box eee e
80. and analyzed on the Applied Biosystems 3130xl Genetic Analyzer Mark Sample For Deletion 370 Mark Sample for Deletion Mark Sample For Deletion 370 Mark Sample for Deleti 370 14 AmpFtSTR dentifiler9 PCR Amplification Kit User Guide Chapter 1 Overview 1 Workflow overview Workflow overview Perform PCR 25 xa ui E S Quantifiler Duo DNA Quantification Kit ee a5 AmpF STR Identifiler PCR Amplification Kit e E Ay f 6 Q a j GeneAmp PCR System 9700 Cycler Veriti 96 Well Thermal Cycler Perform U 1 nz ar 3 M as phoresis 3100 3100 Avant 3130 3130xl 3500 3500xL P E Genetic Analyzer Genetic Analyzer Genetic Analyzer nalyzer Analyze d data 8 q GeneMapper D X or GeneMapper D Software AmpFtSTR Identifiler PCR Amplification Kit User Guide 15 1 Chapter 1 Overview Instrument and software overview Instrument and software overview Data Collection and GeneMapper D or D X Software Instrument and software compatibility About multicomponent analysis 16 This section provides information about the Data Collection Software versions required to run the AmpF STR Identifiler PCR Amplification Kit on specific instruments The Data Collection Software provides instructions to firmware running on the in
81. atio ha foo foo foo ES MinusA Distance From o fo lo lo E To foo oo Minus Stutter Ratio fo lo lo foo gt Minus Stutter Distance From 525 2 To joo ja 75 Plus Stutter Ratio foo Plus Stutter Distance From foo ro foo ho bo po Amelogenin Cutoff po Range Filter Inthe Bin Set field select the AmpFLSTR Bins v2 bin set imported previously and configure the stutter distance parameters as shown GeneMapper ID Software v3 2 1 allows you to specify four types of marker repeat motifs tri tetra penta and hexa You can enter parameter values for each type of repeat in the appropriate column Specify the stutter ratio apply the stutter ratios listed in the Allele tab for single source data deselect the Use marker specific stutter ratio if available check box selected by default Perform appropriate internal validation studies to determine the appropriate filter setting to use Note Applying global stutter ratios may reduce the editing required for single source sample data To apply the stutter ratios contained in the AmpFLSTR Panels v2 txt file select the Use marker specific stutter ratio if available check box selected by default Perform appropriate internal validation studies to determine the appropriate filter setting to use AmpFtSTR9 Identifiler PCR Amplification Kit User Guide 41 4 Chapter 4
82. ation Kit User Guide Chapter 1 Overview 1 Product overview Allelic ladder Figure 1 shows the allelic ladder for the Identifiler Kit See Allelic ladder profile requirements on page 25 for information on ensuring accurate genotyping Figure 1 GeneMapper D X Software plot of the AmpF STR Identifiler Allelic Ladder Mark Sample for Deletion 370 bl Go ba 3 ba s ks bes kz lel B Is E ll bl lel ba lal bs bs bs E El F bo bx lal bs bs bs Mark Sample for Deletion 210 250 290 330 430 170 370 s 4105 Be 2 281 l s 5 E bba k E Do rx aded hs Bs E ko ba fs be 05 he Bz he Bo Bo B ea Baled es ies e Ba fio Mark Sample for Deletion 370 ja ls D bs be z Ba Be Eo b B Es E E E E bol a sz he l Ba Esp ps pe 2 Bs 5 Eo EJ Ba Esl Es Es Be Ez 10 2 13 2 14 2 Mark Sample For Deletion 170 210 250 230 330 370 e Go k kaka 640505 Bz Be 29 Bo Ex 2 23 B 25 280627 Bs be Bo 1 2 5 2 AmpFtSTR Identifiler PCR Amplification Kit User Guide 13 Chapter 1 Overview Product overview Control DNA 9947A Figure 2 shows amplification of Control DNA 9947A using the Identifiler Kit profile Figure 2 1 ng of Control DNA 9947A amplified with the Identifiler Kit
83. be beneficial If too much DNA is added to the PCR reaction then the increased amount of PCR product that is generated can result in the following Fluorescence intensity that exceeds the linear dynamic range for detection by the instrument off scale data Off scale data is a problem for two reasons Quantitation peak height and area for off scale peaks is not accurate For example an allele peak that is off scale can cause the corresponding stutter peak to appear higher in relative intensity thus increasing the calculated percent stutter Multicomponent analysis of off scale data is not accurate which results in poor spectral separation pull up Incomplete A nucleotide addition The sample can be re amplified using less DNA When the total number of allele copies added to the PCR is extremely low unbalanced amplification of the two alleles of a heterozygous individual may occur Walsh et al 1992 Wallin et al 1998 due to stochastic fluctuation in the ratio of the two different alleles Sensabaugh et al 1991 The PCR cycle number and amplification conditions have been specified to produce low peak heights for a sample containing 20 pg human genomic DNA Low peak heights should be interpreted with caution Individual laboratories may find it useful to determine an appropriate minimum peak height threshold based on their own results and instruments using low amounts of input DNA AmpFtSTR Identifiler
84. ct that is one base pair longer than the actual target sequence and the PCR product with the extra nucleotide is referred to as the A form Figure 13 The efficiency of A addition is related to the particular sequence of the DNA at the 3 end of the PCR product The Identifiler Kit includes two main design features that promote maximum A addition The primer sequences have been optimized to encourage A addition The final extension step is 60 C for 60 minutes This final extension step gives the AmpliTaq Gold DNA Polymerase extra time to complete A addition to all double stranded PCR product STR systems that have not been optimized for maximum A addition may have split peaks where each allele is represented by two peaks one base pair apart Figure 13 Split peaks resulting from incomplete A nucleotide addition due to omission of the 60 minute extension step No Extension A Final Extension The AmpliTaq Gold DNA Polymerase generally requires extra time to complete the A nucleotide addition at the 3 end of the PCR products Lack of full A nucleotide addition may be observed in Identifiler Kit results when the amount of input DNA is greater than recommended protocols This is because more time is needed for AmpliTaq Gold DNA Polymerase to add the A nucleotide to all molecules as more PCR product is generated Amplification of too much input DNA will also resu
85. d as a possible source of the evidence sample The probability that another unrelated individual would also match the evidence sample is estimated by the frequency of that genotype in the relevant population s The Identifiler Kit prior to the addition of the D8S1179 degenerate primer was used to generate the population data provided in this section Samples were collected from individuals throughout the United States with no geographical preference Population samples used in these studies g lt e 3 e E e 2 Number Population of samples Samples provided by African American 357 U S Caucasian 349 U S Hispanic 290 Native American 191 Kentucky State Police and the Federal Bureau of Investigation Minnesota Bureau of Criminal Apprehension Memorial Blood Center of Minneapolis Table 6 shows the Identifiler Kit allele frequencies in four populations listed as percentages Allele frequencies Table 6 Identifiler Kit allele frequencies Allele African American U S Caucasian U S Hispanic Native American n 357 n 349 n 290 n 191 CSF1P0 1 t t t 7 4 62 0 14 0 34 t 8 7 56 0 29 0 17 0 52 9 3 78 1 72 0 86 8 38 10 27 87 24 21 23 10 30 89 11 20 59 31 81 28 28 21 99 11 3 0 14 t t t 12 29 13 32 81 39 6 32 72 13 5 32 7 31 6 38 4 71 14 0 98 1 43 0 861
86. d analyzed using the 310 Genetic Analyzer Primates Gorilla chimpanzee orangutan and macaque 1 0 ng each Non primates Mouse dog pig cat horse chicken and cow 2 5 ng each Bacteria and yeast Brochothrix Escherichia Neisseria Pseudomonas Bacillus Staphylococcus approximately 5 ng each and Saccharomyces 1 ng The primate DNA samples all amplified producing fragments within the 100 400 base pair region Lazaruk et al 2001 Wallin et al 1998 The microorganisms chicken cow cat and mouse did not yield detectable product Horse dog and pig produced a 103 bp fragment near the Amelogenin locus in PET dye 84 AmpFtSTR dentifiler9 PCR Amplification Kit User Guide Sensitivity DAB 8 1 2 2 Sensitivity Effect of DNA quantity on results and importance of quantitation Section 5 1 Developmental Validation Sensitivity Species specificity sensitivity stability and mixture studies are conducted DAB 1998 The amount of input DNA added to the Identifiler Kit should be between 0 5 and 1 25 ng Figure 16 on page 86 The DNA sample should be quantitated prior to amplification using a system such as the Quantifiler Human DNA Quantitation Kit Part no 4343895 The final DNA concentration should be in the range of 0 05 0 125 ng uL so that 0 5 1 25 ng of DNA will be added to the PCR reaction in a volume of 10 uL If the sample contains degraded DNA amplification of additional DNA may
87. dn 105 Probability of paternity exclusion W K KK KK KK KK KK KK KK KK KK KK 106 Section 5 2 Performance Verification After Primer Manufacturing Process Improvements beet g2gg293g g 107 Section 5 3 Performance Validation After Buffer and Enzyme Component Replacement Man ee ete aoe hey ey ee ee br 108 wa Dor r 108 u la ul t b tm D dek D y r d toate eee n OR 108 Reproducibility study si syur b y lel e 109 Sensitivity Study Kume y niet eerta eR Mae Eyn suce quu N n 111 Inhibition study Qusa See eben did ao 114 GConclt slonS uy ua k n L tuwa xata er eux be mie but 118 AmpFtSTR Identifiler9 PCR Amplification Kit User Guide 65 5 Chapter 5 Experiments and Results Overview Section 5 1 Developmental Validation Overview Importance of validation Experiment conditions 66 This chapter provides results of the developmental validation experiments we performed using the AmpF STR Identifiler PCR Amplification Kit The data contained in this section was generated during the original developmental validation of the Identifiler Kit before its release in 2001 Since that time we have made a series of improvements to the Identifiler Kit For information on changes to the Identifiler Kit since 2001 and associated data see Performance Verification After Primer Manufacturing Process Improvements on pag
88. e 107 Performance Validation After Buffer and Enzyme Component Replacement on page 108 Validation of a DNA typing procedure for human identification applications is an evaluation of the procedure s efficiency reliability and performance characteristics By challenging the procedure with samples commonly encountered in forensic and parentage laboratories the validation process uncovers attributes and limitations which are critical for sound data interpretation in casework Sparkes Kimpton Watson et al 1996 Sparkes Kimpton Gilbard et al 1996 Wallin et al 1998 We performed experiments to evaluate the performance of the Identifiler Kit according to the DNA Advisory Board DAB Quality Assurance Standards effective October 1 1998 DNA Advisory Board 1998 The DAB standards describe the quality assurance requirements that a laboratory should follow to ensure the quality and integrity of the data and competency of the laboratory These DAB standards describe the quality assurance requirements that a laboratory should follow to ensure the quality and integrity of the data and competency of the laboratory DAB defines a laboratory as a facility in which forensic DNA testing is performed Based on these standards we conducted experiments which comply with Standards 8 1 1 and 8 12 and its associated subsections Whereas this DNA methodology is not novel Standard 8 1 2 and its related subsections have been addressed Holt et a
89. e Amelogenin gender determining marker in a single PCR amplification All thirteen of the required loci for the Combined DNA Index System CODIS Budowle et al 1998 Two additional loci D251338 and D195433 Product The Identifiler Kit contains all the necessary reagents for the amplification of human description genomic DNA The reagents are designed for use with the following Applied Biosystems instruments 3100 3100 Avant Genetic Analyzer Applied Biosystems 3130 3130x Genetic Analyzer Applied Biosystems 3500 3500xL Genetic Analyzer 310 Genetic Analyzer GeneAmp PCR System 9700 with the Silver 96 Well Block GeneAmp PCR System 9700 with the Gold plated Silver 96 Well Block Veriti 96 Well Thermal Cycler About the primers The Identifiler Kit employs the same primer sequences for all loci common to other AmpF STR kits except the MiniFiler kit A degenerate unlabeled primer for the D8S1179 locus was added to the AmpF STR Identifiler Primer Set in order to address a mutation observed in a population of Chamorros and Filipinos from Guam Budowle et al 1998b and Budowle et al 2000 The addition of the degenerate primer allows for the amplification of those alleles in samples containing this mutation without altering the overall performance of the Identifiler Kit The original validation data in this guide Section 5 1 on page 66 were generated prior to the addition of the degenerate primer Data
90. e and buffer manufactured by Life Technologies n Control A mix Control B mix Test A mix Test B mix Test C mix Material Buffer Control Buffer Control Buffer Test Buffer Test Buffer Test Buffer Lot 1 Lot 2 Lot 1 Lot 2 Lot 3 Enzyme Control Control TestEnzyme Control Test Enzyme Enzyme Enzyme Lot 1 Enzyme Lot 2 Lot 1 Lot 2 Lot 2 Each of the five mixes listed above were used to conduct reproducibility sensitivity and inhibition studies All amplifications were performed using a GeneAmp PCR System 9700 with either silver or gold plated silver block using the recommended amplification conditions and cycle number for the Identifiler Kit All data was run on an Applied Biosystems 3130 Genetic Analyzer running Data Collection Software v3 0 and analyzed using GeneMapper ID X Software Subsequent data analysis was performed using Minitab Statistical Software To minimize the effect of injection to injection variation on result interpretation peaks heights for all studies were normalized using an in house multicolor reference standard AmpFtSTR Identifiler PCR Amplification Kit User Guide Section 5 3 Performance Validation After Buffer and Enzyme Component Replacement 5 Reproducibility study Reproducibility study For the reproducibility study 12 replicates of control DNA 007 at 1 ng input and three negative control replicates were amplified The results were evaluated for intracolor balance stutte
91. e eee c Navigate to then open the AmpFLSTR Analysis Files GMIDX folder that you unzipped in step 1 on page 50 50 AmpFtSTR dentifiler9 PCR Amplification Kit User Guide Section 4 2 GeneMapper ID X Software 4 Set up GeneMapper ID X Software for data analysis 5 Select AmpFLSTR_Panels_v2X or the version you installed then click Import Note Importing this file creates a new folder in the navigation pane of the Panel Manager Identifiler_v1 1X This folder contains the panel and associated markers Import Panels Lookin G AmpFLSTR Analysis Files GMIDX c E AmpFLSTR_Bins_v2X My Recent EJ AmpFLSTR Stutter v2X Documents E ReadMe_AmpFLSTR_v2x My Documents ys File name AmpFLSTR Panels v2X bxt My Computer of type All Files O 0 2 lt o 0 p e v gt O e p 2 0 6 Import AmpFLSTR Bins V2X txt a Select the AmpFLSTR Panels v2X folder in the navigation pane Panel Manager File Edit Bins Yiew Help gt lt ke Bin Set i sf Panel Manager AmpFLSTR Bins v jlji amp I E i BI EI EI E Comment Panel Name AmpFLSTR_NGMSElect_v2x Ofiler v1 1X null tig SGM Plus vi 1X C AmpFLSTR_P mpFLSTR_Panels_v1X NGM_SElect_v2 1 Inul AmpFLSTR_Panels_v2 Identifiler_Plus_v1 1X nul MAM es ut
92. e imports multiple run folders into a project only the ladder s within their respective run folders are used for calculating allelic bin offsets and subsequent genotyping Allelic ladder samples must be labeled as Allelic Ladder in the Sample Type column in a project Failure to apply this setting for ladder samples results in failed analysis Injections containing the allelic ladder must be analyzed with the same analysis method and parameter values that are used for samples to ensure proper allele calling Alleles that are not in the AmpF STR Allelic Ladders do exist Off ladder OL alleles may contain full and or partial repeat units An off ladder allele is an allele that occurs outside the 0 5 nt bin window of any known allelic ladder allele or virtual bin Note If a sample allele peak is called as an off ladder allele the sample result needs to be verified according to the laboratory s protocol Set up GeneMapper D Software for data analysis File names Before using the software for the first time 34 The file names shown in this section may differ from the file names you see when you download or import files If you need help determining the correct files to use contact your local Life Technologies Human Identification representative or go to www lifetechnologies com support gt Software Patches amp Updates GeneMapper ID Software Before you can analyze sample fsa files using GeneMapper ID Sof
93. e number for this high quantity of DNA is 25 cycles as determined by our validation studies However it is recommended that each laboratory determine the optimum cycle number based on internal validation studies In the example shown in Figure 4 a 1 2 mm disc of a bloodstained FTA card was purified using three washes with FTA Purification Reagent and two washes with 1X low TE buffer The purified punch disc was then amplified in the MicroAmp tube for 25 cycles AmpFtSTR Identifiler PCR Amplification Kit User Guide Chapter 2 Perform PCR 2 Amplification using bloodstained FTA cards Figure 4 AmpF STR Identifiler9 Kit PCR Amplification Kit results from a 1 2 mm FTA bloodstain disc 25 cycle amplification analyzed on the Applied Biosystems 3130xl Genetic Analyzer AmpFtSTR Identifiler PCR Amplification Kit User Guide 23 2 Chapter 2 Perform PCR Amplification using bloodstained FTA cards 24 AmpFtSTR Identifiler PCR Amplification Kit User Guide Perform Electrophoresis Allelic ladder requirements 0 KK KK KK KK KK KK KK KK KK KK KK KK KK KK 25 Section 3 1 3100 3100 Avant and 3130 3130xl instruments 27 Set up the 3100 3100 Avant or 3130 3130xl instrument for electrophoresis 27 Prepare samples for electrophoresis on the 3100 3100 Avant or 3130 3130xl aa oho api upyakuqku ii up ye tee denote soit eric ees 28 Section 3 2 3500 3500xL Series
94. eaction plate in a thermal cycler for 3 minutes at 95 C Immediately place the plate on ice for 3 minutes Prepare the plate assembly then place on the autosampler Ensure that a plate record is completed and link the plate record to the plate Start the electrophoresis run AmpFtSTR Identifiler PCR Amplification Kit User Guide Chapter 3 Perform Electrophoresis 3 Set up the 3500 3500xL instrument for electrophoresis Section 3 2 3500 3500xL Series instruments Set up the 3500 3500xL instrument for electrophoresis Reagents and parts Ordering Information on page 121 lists the required materials not supplied with the AmpF STR Identifiler PCR Amplification Kit IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the primer set amplified DNA allelic ladder and size standard from light when not in use Keep freeze thaw cycles to a minimum Electrophoresis The following table lists Data Collection Software and the run modules that can be software setup and used to analyze Identifiler Kit PCR products For details on the procedures refer to the documents listed in the table e a 2 E 5 a o x a 0 5 reference documents Genetic vat Operatin Collection 3 Run modules and conditions References Analyzer System Software Applied 3500 Data Windows HID36_POP4 Applied Biosystems 3500 3500xL Biosystems Collection Injection cond
95. echnologies com support Software Patches amp Updates GeneMapper ID X Software The instructions and examples in this section refer to the latest version of panel bin and stutter file available at the time of publication Before using the Before you use GeneMapper ID X Software v1 0 1 or higher for fsa files v1 2 or software for the first time 1 higher for hid files to analyze data for the first time you must do the following Check the version of panel bin and stutter files installed with the GeneMapper ID X Software as explained in Check panel bin and stutter file version below Check www lifetechnologies com support gt Software Patches amp Updates GeneMapper ID X Software to determine if newer files are available If updated files are available download and import the files into the GeneMapper ID X Software as explained in Import panels bins and marker stutter on page 50 Note When downloading new versions of analysis files refer to the associated Read Me file for details of changes between software file versions If you have validated previous file versions for data analysis conduct the appropriate internal verification studies before using new file versions for operational analysis Create an analysis method as explained in Create an analysis method on page 55 Define custom views of analysis tables Refer to Chapter 1 of the GeneMapper ID X Software Ve
96. eile anu oe ws 82 Nature of the polymorphisms 82 cem carmi Rz mA MI UE una AERE E 83 Mappinga chica cei E 83 AmpFtSTR Identifiler PCR Amplification Kit User Guide 5 Contents lele lS S93 ellils li AR Lean EU PEEL 83 DAB 8 1 2 2 Species Specificily 83 Sensitivity corem niacin eu exe eee Ex E UP ERE ee RU eR AL abn DU CR Ca dome 85 DAB38 1 22 Sensitivity k uerbo e Re review e PCIE TM bure la E MEE E PER 85 Effect of DNA quantity on results and importance of quantitation 85 Stability yA kn s ere eren due AN A MR A EE rs e 86 DAB38 1 22 Stability 1 csi D Hele ali eee eee e REIP E E 86 Lack of amplification of some loci 86 Differential and preferential amplification 87 Effectofanhibitots xua Peet cat be n ete Herb Ma a CA 87 Degraded DNA cse i Dem SUDORE RIDERE Le ER 88 Multiplex amplifications uu s devl da n bal 89 Mixture studies xas kunika nea kina kab E ka Mak lara ke a de ed KAWE a ERR e le sa 91 DAB 8 1 2 2 Mixture Studies WWW kk kk kk kk kk kk kk kK kK kK kk kk kk kk kk kk kk kk kk kk kk kk 91 Mixed specimen studieS J kil lk n kila b ki y na k k
97. ell reaction plate N8010560 250 uL glass syringe array fill syringe 4304470 5 0 mL glass syringe polymer reserve syringe 628 3731 For a complete list of parts and accessories for the 3100 3100 Avant instrument refer to Appendix B of the 3100 Genetic Analyzer and 3100 Avant Genetic Analyzer User Reference Guide Pub no 4335393 3130xl Analyzer materials 96 well plate septa 4315933 Reservoir septa 4315932 3100 3130xl Genetic Analyzer capillary array 36 cm 4315931 POP 49 polymer for 3130 3130xl Genetic Analyzers 4352755 3100 3100 Avant Genetic Analyzer Autosampler Plate Kit 96 well 4316471 GeneScan 500 LIZ9 Size Standard 4322682 OR OR GeneScan 600 LIZ9 Size Standard v2 0 4408399 Running Buffer 10x 402824 DS 33 Matrix Standard Kit Dye Set G5 4345833 MicroAmp Optical 96 well reaction plate N8010560 Hi Di Formamide 4311320 For a complete list of parts and accessories for the 3130 3130xl instrument refer to Appendix A of the Applied Biosystems 3130 3130xl Genetic Analyzers Maintenance Troubleshooting and Reference Guide Pub no 4352716 3500 3500xL Analyzer materials Anode buffer container ABC 4393927 Cathode buffer container CBC 4408256 POP 4 polymer 960 samples for 3500 3500xL Genetic Analyzers 4393710 POP 49 polymer 384 samples for 3500 3500xL Genetic Analyzers 4393715 Conditioning reagent 4393718 8 Capillary array
98. entifiler Example gmid Is Logged In File Edit Analysis View Tools Help HH LL EI Hl El EE y B S eS Project Samples Genotypes identifier Example Status Sample File Sample Name Sample Type Analysis Method Panel Size Standard 1 Allelic Ladder H02 fsa Allelic Ladder Allelic Ladder Identifiler amp nalysisMethod v1 Identifiler v2 CE G5 HID GS500 75 45t 2 Allelic Ladder H04 fsa Allelic Ladder Allelic Ladder Identifiler amp nalysisMethod v1 Identifiler v2 CE G5 HID GS500 75 45t Q 3 Allelic_Ladder_HO6 fsa Allelic_Ladder Allelic Ladder Identifiler amp nalysisMethod v1 Identifiler v2 CE G5 HID GS500 75 45t 4 Allelic_Ladder_HO8 tsa Allelic_Ladder Allelic Ladder Identifiler amp nalysisMethod v1 Identifiler v2 CE G5 HID GS500 75 45t lt 5 Allelic Ladder H10 fsa Allelic Ladder Allelic Ladder Identifiler amp nalysisMethod v1 Identifiler v2 CE G5 HID GS500 75 45t z 5 Ident 007 0 0312ng GO01 fsa Ident O07 0 0312ng Sample Identifiler amp nalysisMethod v1 Identifiler v2 CE G5 HID GS500 75 45t 5 7 Ident_007_0 031 2ng_G02 tsa Ident 007 0 0312ng Sample Identifiler amp nalysisMethod v1 Identifiler v2 CE G5 HID GS500 75 45 EJ 8 Ident 007 0 0312ng G03 fsa Ident 007 0 0312ng Sample ldentifiler AnalysisMethod v1 Identifiler v2 CE G5 HID GS500 75 45 9 Ident_007_0 0312ng_G04 fsa Ident 007 0 0312ng Sample
99. equired allelic ladder injection frequency to ensure accurate genotyping of all samples in your laboratory environment AmpFtsTR Identifiler9 PCR Amplification Kit User Guide 25 26 Chapter 3 Perform Electrophoresis Allelic ladder requirements It is critical to genotype using an allelic ladder run under the same conditions as the samples because size values obtained for the same sample can differ between instrument platforms because of different polymer matrices and electrophoretic conditions AmpFtSTR Identifiler PCR Amplification Kit User Guide Section 3 1 3100 3100 Avant and 3130 3130xl instruments Set up the 3100 3100 Avant or 3130 3130xl instrument for electrophoresis Section 3 1 3100 3100 Avant and 3130 3130xl instruments Set up the 3100 3100 Avant or 3130 3130xl instrument for electrophoresis Reagents and parts Electrophoresis software setup and Ordering Information on page 121 lists the required materials not supplied with the AmpF STR Identifiler PCR Amplification Kit IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the primer set amplified DNA allelic ladder and size standard from light when not in use Keep freeze thaw cycles to a minimum The following table lists Data Collection Software and the run modules that can be used to analyze Identifiler Kit PCR products For details on the procedures refer to the documents listed in the table
100. erate a profile of expected genotype The AmpF STR Control DNA 9947A is not designed to be used as a DNA quantitation control and you may see variation from the labelled concentration when quantitating aliquots of the AmpF STR Control DNA 9947A Standards for For the Identifiler Kit the panel of standards needed for PCR amplification PCR samples product sizing and genotyping are AmpFE STR Control DNA 9947A A positive control for evaluating the efficiency of the amplification step and STR genotyping using the AmpF STR Identifiler Allelic Ladder GeneScan 500 LIZ Size Standard or GeneScan 600 LIZ Size Standard v2 0 Used for obtaining sizing results These standards which have been evaluated as internal size standards yield precise sizing results for Identifiler Kit PCR products Order the GeneScan 500 LIZ Size Standard Part no 4322682 or the GeneScan 600 LIZ Size Standard v2 0 Part no 4408399 separately AmpE STR Identifiler Allelic Ladder Allelic ladder developed by Life Technologies for accurate characterization of the alleles amplified by the Identifiler Kit The AmpF STR Identifiler Allelic Ladder contains most of the alleles reported for the 15 autosomal loci Refer to Table 1 on page 12 for a list of the alleles included in the AmpFSTR Identifiler Allelic Ladder 18 AmpFtSTR dentifiler9 PCR Amplification Kit User Guide Perform PCR m Required user supplied
101. erolemia Nat Biotechnol 14 1279 1282 Begovich A B McClure G R Suraj V C Helmuth R C Fildes N Bugawan T L Erlich H A and Klitz W 1992 Polymorphism recombination and linkage disequilibrium within the HLA class II region J Immunol 148 249 58 Bender K Farfan M J and Schneider P M 2004 Preparation of degraded human DNA under controlled conditions Forensic Sci Int 139 134 140 Brinkmann B Moller A and Wiegand P 1995 Structure of new mutations in 2 STR systems Intl J Legal Med 107 201 203 Brinkmann B Klintschar M Neuhuber F Huhne J and Rolf B 1998 Mutation rate in human microsatellites Influence of the structure and length of the tandem repeat Am J Hum Genet 62 1408 1415 Budowle B et al 1998 CODIS and PCR Based Short Tandem Repeat Loci Law Enforcement Tools Second European Symposium on Human Identification 73 88 Butler J M 2005 Forensic DNA Typing Burlington MA Elsevier Academic Press Chakraborty R Stivers D and Zhong Y 1996 Estimation of mutation rates from parentage exclusion data applications to STR and VNTR loci Mutat Res 354 41 48 Chakraborty R and Stivers D N 1996 Paternity exclusion by DNA markers effects of paternal mutations J Forensic Sci 41 671 677 Chakraborty R Kimmel M Stivers D Davison L and Deka R 1997 Relative mutation rates at di tri and tetranucleotide microsatellite loci Proc Natl Acad Sci US
102. etic Analyzer Similar results were obtained genotype and peak height whether the DNA samples were amplified for each locus alone or co amplified in the Identifiler Kit reaction Figure 19 on page 90 When degraded DNA is suspected to have compromised amplification of one or more loci the molecular weight of the DNA can be assessed by agarose gel analysis If the DNA is degraded to an average of 400 base pairs in size or less adding more DNA template to the Identifiler Kit amplification reaction may help produce a typeable signal for the loci Adding more DNA to the amplification may provide more of the necessary size template for amplification AmpFtSTR Identifiler PCR Amplification Kit User Guide 89 5 Chapter 5 Experiments and Results Stability Figure 19 Multiplex and singleplex amplifications of a DNA sample incubated for 1 min with DNase 1 analyzed on the 310 Genetic Analyzer BA 125 150 J hala AK 00 58 41 minute HEJ 56 44 1 minute sv A41 minute SR c44 1 minute L 165 6 AMEL AMEL 12Y B2 D19 D19 J ASDS DS A1D8 D8 17R B7 D5 DS 1 Y B3 VWA A2 D21 D21 BM ier B8 FGA FGA sec Az pia pia 14 4 4 A3 D7 D7 106 A8 D16 D16 1080 540 1080 540 o 1080 54
103. ftware v1 0 1 or higher allows you to specify 4 types of marker repeat motifs tri tetra penta and hexa You can enter parameter values for each type of repeat in the appropriate column Specify the stutter ratio apply the stutter ratios listed in the Allele tab for single source data deselect the Use marker specific stutter ratio if available check box selected by default Perform appropriate internal validation studies to determine the appropriate filter setting to use Note Applying global stutter ratios may reduce the editing required for single source sample data To apply the stutter ratios contained in the AmpFLSTR Panels v2 txt file select the Use marker specific stutter ratio if available check box selected by default Perform appropriate internal validation studies to determine the appropriate filter setting to use AmpFtSTR Identifiler PCR Amplification Kit User Guide 57 4 Chapter 4 GeneMapper ID X Software Set up GeneMapper ID X Software for data analysis Peak Detector tab settings Analysis Method Editor General Allele 1 Peak Quality SQ amp GQ Settings Peak Detection Algorithm Advanced Ranges Peak Detection Analysis Sizing Peak Amplitude Thresholds Perform Full Range all Sizes internal R validation o studies to determine settings Smoothing and Baselining Min Peak Half Width 2 pts Smoothing L J Li
104. ght Polynomial Degree O Heavy Peak Window Size Baseline Window 51 pts ee erna Peak Start r Size Calling Method 2nd Order Least Squares 3rd Order Least Squares Cubic Spline Interpolation Local Southern Method Global Southern Method Peak End Factory Defaults IMPORTANT Perform the appropriate internal validation studies to determine the appropriate peak amplitude thresholds for interpretation of Identifiler Kit data Fields include Peak amplitude thresholds The software uses these parameters to specify the minimum peak height in order to limit the number of detected peaks Although GeneMapper ID X Software displays peaks that fall below the specified amplitude in electropherograms the software does not label or determine the genotype of these peaks Size calling method The Identifiler Kit has been validated using the Local Southern sizing method Select alternative sizing methods only after you perform the appropriate internal validation studies e Normalization A Normalization checkbox is available on this tab in GeneMapper ID X Software v1 2 for use in conjunction with data run on the Applied Biosystems 3500 Series Genetic Analyzers Users of this version of software should perform laboratory evaluations to determine whether to use the Normalization feature for analysis of Identifiler Kit data 58 AmpFtSTR dentifiler9 PCR Amplification Kit User Guide Section 4 2
105. i instrument to run in the 9600 Emulation Mode User Bulletin Veriti 96 Well Thermal Cycler AmpF STR Kit Validation Pub no 4440754 Initial Denature Anneal Extend Final Final hold incubation step extension HOLD CYCLE 28 HOLD HOLD 95 C 94 C 59 C 72 C 60 C 4 25 C 11 min 1 min 1min 1min 60 min co 2 Load the plate into the thermal cycler and close the heated cover IMPORTANT If using the 9700 thermal cycler with silver or gold plated silver block and adhesive clear film instead of caps to seal the plate wells be sure to place a MicroAmp compression pad Part no 4312639 on top of the plate to prevent evaporation during thermal cycling The Veriti Thermal Cycler does not require a compression pad 3 Start the run 4 On completion of the run store the amplified DNA and protect from light If you are storing the DNA Then place at 2 to 8 C 15 to 25 C 2 weeks 2 weeks IMPORTANT Store the amplified products so that they are protected from light Amplification using bloodstained FTA cards 22 FTA cards can be useful for collecting storing and processing biological samples A small punch disc of the card containing the sample can be placed directly into an amplification tube purified and amplified without transferring the disc Our studies indicate that a 1 2 mm bloodstained disc contains approximately 5 20 ng DNA An appropriate cycl
106. ic and internal control 5 nuclease assays The Quantifiler Human and Quantifiler Y Human Male Kits contain different target specific assays human DNA or human male DNA respectively that each consist of two locus specific PCR primers and one TaqMan MGB probe labeled with FAM dye for detecting the amplified sequence The kits each contain a separate internal PCR control IPC assay which consists of an IPC template DNA a synthetic sequence not found in nature two primers for amplifying the IPC template and one TaqMan MGB probe labeled with VIC dye for detecting the amplified IPC Quantifiler Duo DNA Quantification Kit Part no 4387746 For more information see Quantifiler Duo DNA Quantification Kit User s Manual Pub no 4391294 Properties The Quantifiler Duo Kit is highly specific for human DNA This kit combines the detection of both total human and male DNA in one PCR reaction The kit detects single stranded and degraded DNA How it works The Quantifiler Duo DNA Quantification Kit consists of target specific and internal control 5 nuclease assays The Quantifiler Duo kit combines two human specific assays in one PCR reaction for total human DNA and human male DNAJ The two human DNA specific assays each consist of two PCR primers and a TagMan probe The TagMan probes for the human DNA and human male DNA assays are labeled with VIC and FAM dyes respectively In addition the kit contain
107. iler_Plus_v1 1X H E Profiler v1 1 H SEfiler_Plus_v1 1X amp C3Identifiler v1 1x c amp C3MiniFiler v1 1X il AProfiler Plus CODIS v1 1X Profiler_Plus_v1 1 null Profiler v1 1X null SEFiler Plus v1 1X null Identifiler v1 1X null MiniFiler v1 1X null Profiler Plus CODIS v1 1 null Vfiler v1 1X null AmpFtSTR Identifiler PCR Amplification Kit User Guide Section 4 2 GeneMapper ID X Software 4 Set up GeneMapper ID X Software for data analysis 8 Select and expand Identifiler_v1 1X in the navigation pane then select D8S1179 to display the Bin view for the marker in the right pane Panel Manager File Edit Bins View Help x d uu Bin Set AmpFLSTR_Bins_v2x S Identifiler_v1 1X 9 D21511 D75820 CSF1PO D351358 THOL 0135317 0165539 0251338 0195433 VWA 018551 AMEL D55818 E FGA m ZO MiniFiler_v1 1x m l Profiler_Plus_CODIS_v1 1X 8C filer v1 1x I 420 H O COfiler_CODIS_v1 1X fe Aldentifiler CODIS v1 1 asy Reference Samples lt Ti X 178 57 Y 0 40 9 Import AmpFLSTR Stutter v2X a Select the AmpFLSTR Panels v2X folder in the navigation panel O gt lt o 19 o e e gt gt O E g Panel Manager File Edit Bins Yiew Help i x BF BI HHB Bin Set v W Be BBE Comment null
108. ing Download software updates and patches Limited Product Warranty 132 Applied Biosystems and or its affiliate s warrant their products as set forth in the Life Technologies General Terms and Conditions of Sale found on Life Technologies website at www lifetechnologies com termsandconditions If you have any questions please contact Life Technologies at www lifetechnologies com support AmpFtSTR Identifiler PCR Amplification Kit User Guide Bibliography Akane A Matsubara K Nakamura H Takahashi S and Kimura K 1994 Identification of the heme compound copurified with deoxyribonucleic acid DNA from bloodstains a major inhibitor of polymerase chain reaction PCR amplification J Forensic Sci 39 362 372 Barber M D Piercy R C Andersen J F and Parkin B H 1995 Structural variation of novel alleles at the Hum vWA and Hum FES FPS short tandem repeat loci Intl J Legal Med 108 31 35 Barber M D McKeown and Parkin 1996 Structural variation in the alleles of a short tandem repeat system at the human alpha fibrinogen locus Intl J Legal Med 108 180 185 Barber M D and Parkin B H 1996 Sequence analysis and allelic designation of the two short tandem repeat 018551 and 0851179 Intl J Legal Med 109 62 65 Baron H Fung S Aydin A Bahrig S Luft F C and Schuster H 1996 Oligonucleotide ligation assay OLA for the diagnosis of familial hypercholest
109. ining AmpliTaq Gold enzyme and 10X PCR Buffer manufactured by Roche Molecular Systems 118 AmpFtSTR Identifiler PCR Amplification Kit User Guide Troubleshooting Follow the actions recommended in this appendix to troubleshoot problems that occur during analysis Table 10 Troubleshooting Observation Possible causes Recommended actions Faint or no signal from both the AmpF amp STR Control DNA 9947A and the DNA test samples at all loci Incorrect volume or absence of PCR Master Mix or Identifiler Primer Set Repeat amplification No activation of AmpliTaq Gold DNA Polymerase Repeat amplification making sure to hold reactions initially at 95 C for 11 minutes Master Mix not vortexed thoroughly before aliquoting Vortex the Master Mix thoroughly Identifiler Primer Set exposed to too much light Store the Primer Set protected from light GeneAmp PCR System malfunction Refer to the thermal cycler user s manual and check instrument calibration Use of incorrect thermal cycling parameters Check the protocol for correct thermal cycling parameters Tubes not seated tightly in the thermal cycler during amplification Push reaction tubes firmly into contact with block after first cycle Repeat test Wrong PCR reaction tube Use Applied Biosystems MicroAmp Reaction Tubes with Caps for the GeneAmp PCR System 9700 MicroAmp Base used with tray
110. ins View Help BE NEN NW Bin Set AmpFLSTR_Bins_v2x v H amp S O Identifiler_v1 1x 090851179 Please enter the stutter filter s for D16S539 marker here If left blank the global stutt 8 D21511 be applied p D75820 5 1 D351358 Minus Stutter Plus Stutter THO1 Ratio From Distance To Distance Ratio From Distance 0135317 100 3 25 4 75 0165539 m H E E E m Stutter Ratio amp Distance D251338 D195433 VWA 4 gt lt TPOX macra EI New Edi Delete 11 Click Apply then OK to add the Identifiler Kit panel bin set and marker stutter to the GeneMapper ID X Software database o N gt o S 148 8 IMPORTANT If you close the Panel Manager without clicking Apply the panels bin sets and marker stutter will not be imported into the GeneMapper ID X Software database 54 AmpFtSTR dentifiler9 PCR Amplification Kit User Guide Section 4 2 GeneMapper ID X Software 4 Set up GeneMapper ID X Software for data analysis Create an analysis Use the following procedure to create an analysis method for the Identifiler Kit method IMPORTANT Analysis methods are version specific so you must create an analysis method for each version of the software For example an analysis method created for GeneMapper ID X version 1 2 is not compatible with earlier versions of GeneMapper ID
111. ion describes basic characteristics of the 16 loci that are amplified with the Identifiler Kit These loci have been previously characterized The primers for the Amelogenin locus flank a six base pair deletion within intron 1 of the X homologue Amplification results in 107 bp and 113 bp products from the X and Y chromosomes respectively Sizes are the actual base pair size according to sequencing results including 3 A nucleotide addition The remaining Identifiler Kit loci are all tetranucleotide short tandem repeat STR loci The length differences among alleles of a particular locus result from differences in the number of 4 bp repeat units AmpFtSTR Identifiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation Species specificity We have subjected to DNA sequencing some alleles in the AmpF STR Identifiler Allelic Ladder containing partial repeat units in population database and nonhuman primate DNA samples Lazaruk et al 2001 In addition other groups in the forensic community have sequenced alleles at some of these loci Nakahori et al 1991 Puers et al 1993 Moller et al 1994 Barber et al 1995 M ller and Brinkmann 1995 Barber et al 1996 Barber and Parkin 1996 Brinkmann et al 1998 Momhinweg et al 1998 Watson et al 1998 Among the various sources of sequence data on the Identifiler Kit loci there is consensus on the repeat patterns and structure of the STRs Inheritance
112. is file associates the bin set with the panels in the AmpFLSTR Panels v2 folder Xj Import Bin Set Look in a Panels ei EB AmpFLSTR_Bins_v2 txt AmpFLSTR_Panels_v2 txt AmpFISTR_Yfiler_Bin_v2 txt ArmpFISTR_Yfiler Panel v2txt My Network File name amp mpFLSTR Bins v2bd Import Files of type All Files v Cancel 6 View the imported panels in the navigation pane a Double click the AmpFLSTR_Panels_v2 folder 36 AmpFtSTR Identifiler PCR Amplification Kit User Guide Section 4 1 GeneMapper ID Software 4 Set up GeneMapper ID Software for data analysis b Double click the Identifiler_v2 folder to display the panel information in the right pane and the markers below it Panel Manager File Edit Bins View m m m mmm Bin Set AmpFLSTR_Bins_v2 lili Bek EHE Panel Manager Marker Name Dye Color Min Size Size Control Alleles EH amp S AmpFLSTR_Panels_v2 D851178 pue 1180 hess fs Blue_v2 021511 blue 1845 2475 30 Green v2 DTS820 Dae laaa E 075820 bli 251 0 2985 10 1 _ 2 r LL ET Profiler Pius v2 CSF1PO blue 30212 348 63 1012 0351358 green 98 0 1480 11415 EI Cofiler_v2 co j MD p Wi NM G o lt D Ke Ke e dS 2 o
113. is guide to describe differences in the amplification efficiency of two alleles at a single locus Preferential amplification of alleles in systems that distinguish alleles based on length polymorphisms is most likely to be observed when the alleles differ significantly in base pair size Since most Identifiler Kit loci have small size ranges the potential for preferential amplification of alleles is low Heme compounds have been identified as PCR inhibitors in DNA samples extracted from bloodstains DeFranchis et al 1988 Akane et al 1994 It is believed that the inhibitor is co extracted and co purified with the DNA and subsequently interferes with PCR by inhibiting polymerase activity Bovine serum albumin BSA can prevent or minimize the inhibition of PCR most likely by binding to the inhibitor Comey et al 1994 Since the presence of BSA can improve the amplification of DNA from blood containing samples BSA has been included in the AmpFSTR PCR Reaction Mix at 4 ug per 25 uL amplification BSA has also been identified as an aid in overcoming inhibition from samples containing dyes such as in denim Comey et al 1994 To examine the effects of hematin on the amplification results obtained by the Identifiler Kit DNA samples were amplified using the Identifiler Kit reagents including the BSA containing PCR reaction mix in the presence of varying concentrations of purified hematin The concentrations of hematin used were 0 u
114. ited control Humic Acid at a final concentration of 15 25 ng uL and Hematin at a final concentration of 34 uM in replicates of five was amplified using each of Test and Control mixes The amount of each inhibitor tested was titrated to cause an approximate 50 reduction in overall peak height of the samples Results were evaluated for mean referenced peak height minimum referenced peak height intracolor balance and levels of allelic dropout Mean referenced Uninhibited Control DNA 007 Test and Control mixes displayed no significant peak height difference in mean referenced peak height minimum referenced peak height and mini intracolor balance For the Humic Acid inhibited and Hematin inhibited DNA n mum f d Heal however the Test mixes showed slightly improved performance compared to the re renced Control mixes for mean referenced peak height intracolor balance minimum height and referenced peak height Figure 30 31 and 32 All results obtained for all Test and intracolor balance Control mixes fall within the expected range of performance for the Identifiler Kit 114 AmpFtSTR Identifiler PCR Amplification Kit User Guide Section 5 3 Performance Validation After Buffer and Enzyme Component Replacement 5 Inhibition study Figure 30 Inhibition study mean referenced peak height Inhibitors HA Humic Acid HE Hematin PRI Pristine or Uninhibited DNA LOT 9 Control A W Control E 4
115. itions 1 2kV 15 sec Genetic Analyzer User Guide 3500 Software Pub no 4401661 10 or Dye Set G5 Applied Windows e HID36 POP4 Applied Biosystems 3500 and 3500xL Bi DT Mn Genetic Analyzers Quick Reference losystems Vista Injection conditions 1 2kV 24 sec Card Pub no 4401662 3500xL Dye Set G5 Prepare samples for electrophoresis on the 3500 3500xL instrument Prepare the samples for electrophoresis immediately before loading 1 Calculate the volume of Hi Di Formamide and GeneScan 600 LIZ Size Standard v2 0 needed to prepare the samples Reagent Volume per reaction GeneScan 600 LIZ9 Size Standard v2 0 0 5 uL Hi Di Formamide 8 5 uL Note Include additional samples in your calculations to provide excess volume for the loss that occurs during reagent transfers IMPORTANT The volume of size standard indicated in the table is a suggested amount Determine the appropriate amount of size standard based on your results and experiments AmpFtSTR Identifiler PCR Amplification Kit User Guide 29 30 Chapter 3 Perform Electrophoresis Prepare samples for electrophoresis on the 3500 3500xL instrument 9 Pipette the required volumes of components into an appropriately sized polypropylene tube Vortex the tube then centrifuge briefly Into each well of a MicroAmp Optical 96 Well Reaction Plate or each MicroAmp optical strip tube add 9uL of the formamide s
116. ity study electropherogram of 125 pg Sample 1 amplified with Test A mix One allele at the FGA locus in the PET dye channel is below the analysis threshold of 50 RFU Y scale 300 RFU U o ER e 3 D 5 a 2 e 3 Ww 2 m N lt 3 o o E o Figure 29 Sensitivity study electropherogram of 125 pg Sample 3 amplified with Control B mix One allele at the FGA locus in the PET Red dye channel is below the analysis threshold of 50 RFU Y scale 300 RFU 12 do xe w EJ x ALI AmpFtSTR Identifiler PCR Amplification Kit User Guide 113 5 Chapter 5 Performance Validation After Buffer and Enzyme Component Replacement Inhibition study Genotype Genotypes for Test and Control mixes were 100 concordant Table 9 concordance Table 9 Sensitivity study genotype concordance DNA Input Amount Reagent Mix Genotype Concordance 125 pg Test A 100 Test B 100 Test C 100 Control A 100 Control 100 250 pg Test A 100 Test B 100 Test C 100 Control A 100 Control B 100 500 pg Test A 100 Test B 100 Test C 100 Control A 100 Control B 100 1 ng Test A 100 Test B 100 Test C 100 Control A 100 Control B 100 Inhibition study An inhibition series of 1 ng control DNA 007 consisting of uninhib
117. ize standard mixture 1uL of PCR product or allelic ladder Note For blank wells add 10 uL of Hi Di Formamide Seal the reaction plate or strip tubes with the appropriate septa then centrifuge to ensure that the contents of each well are collected at the bottom Heat the reaction plate or strip tubes in a thermal cycler for 3 minutes at 95 C Immediately put the plate or strip tubes on ice for 3 minutes Prepare the plate assembly then place on the autosampler Ensure that a plate record is completed and link the plate record to the plate Start the electrophoresis run AmpFtSTR Identifiler PCR Amplification Kit User Guide Chapter 3 Perform Electrophoresis Set up the 310 instrument for electrophoresis Section 3 3 310 Instrument Set up the 310 instrument for electrophoresis Reagents and parts Ordering Information on page 121 lists the required materials not supplied with the AmpF STR Identifiler PCR Amplification Kit IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the primer set amplified DNA allelic ladder and size standard from light when not in use Keep freeze thaw cycles to a minimum e zi o 3 Z Electrophoresis The following table lists Data Collection Software and the run modules that can be software setup and used to analyze Identifiler Kit PCR products For details on the procedures refer to the documents listed in the t
118. l 2001 and Wallin et al 2001 This chapter will discuss many of the experiments we performed and examples of the results we obtained We used conditions that produced maximum PCR product yield and a window in which reproducible performance characteristics were met These experiments while not exhaustive are appropriate for a manufacturer in our opinion Each laboratory using the Identifiler Kit should perform appropriate validation studies AmpFtSTR Identifiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation Developmental validation Developmental validation DAB 8 1 1 Developmental validation that is conducted shall be appropriately documented DNA Validation Critical reagent concentrations and reaction conditions such as thermal cycling parameters AmpliTaq Gold DNA polymerase activation cycle number to produce reliable locus specific amplification and appropriate sensitivity have been determined PCR components The concentration of each component of the Identifiler Kit was examined The PCR components are Tris HCl pH 8 3 KCl dNTPs primers AmpliTaq Gold DNA Polymerase MgCl bovine serum albumin and sodium azide The concentration for a particular component was established to be in the window that meets the reproducible performance characteristics of specificity and sensitivity Figure 5 g lt e 3 D e E e 2 Figure 5 A 1 ng amplificatio
119. le run When the software imports multiple run folders into a project only the ladder s within their respective run folders are used for calculating allelic bin offsets and subsequent genotyping Allelic ladder samples must be labeled as Allelic Ladder in the Sample Type column in a project Failure to apply this setting for ladder samples results in failed analysis Injections containing the allelic ladder must be analyzed with the same analysis method and parameter values that are used for samples to ensure proper allele calling Alleles that are not in the AmpF STR Allelic Ladders do exist Off ladder OL alleles may contain full and or partial repeat units An off ladder allele is an allele that occurs outside the 0 5 nt bin window of any known allelic ladder allele or virtual bin Note If a sample allele peak is called as an off ladder allele the sample result needs to be verified according to the laboratory s protocol AmpFtSTR Identifiler PCR Amplification Kit User Guide Section 4 2 GeneMapper ID X Software 4 Set up GeneMapper ID X Software for data analysis Set up GeneMapper D X Software for data analysis Panel bin and The file names shown in this section may differ from the file names you see when you stutter file version download or import files If you need help determining the correct files to use contact your local Life Technologies Human Identification representative or go to www lifet
120. les 7 alleles and 13 vWA alleles In addition to the alleles that were observed recorded the our databases other known alleles have been published or reported to us by other laboratories see STRBase www cstl nist gov div831 strbase Some alleles of the Identifiler Kit loci occur at a low frequency For these alleles a minimum frequency 5 21 where the number of individuals in the database was assigned for the Identifiler Kit African American U S Caucasian U S Hispanic and Native American databases as suggested in the 1996 report of the Committee on DNA Forensic Science National Research Council 1996 These databases are summarized in Table 6 on page 95 through page 104 The minimum reportable genotype frequency at each locus is as follows 1 19 x 10 4 for the African American database 1 19 x 10 4 for the U S Caucasian database 1 70 x 10 4 for the U S Hispanic database and 2 97 x 10 4 for the Native American database p2 p 1 p where 0 01 Hence the minimum combined multilocus genotype frequency at 15 loci is as follows 1 36 x 10 99 for the African American database 1 36 x 10 5 for the U S Caucasian database 2 86 x 10 5 for the U S Hispanic database and 1 23 x 10 93 for the Native American database AmpFtSTR Identifiler PCR Amplification Kit User Guide Mutation rate Estimating germline mutations Additional mutation studies Mutation rate Section 5 1 Developmen
121. lity DAB 8 1 2 Accuracy Precision and size windows Novel forensic DNA methodologies shall undergo developmental validation to ensure the accuracy precision and reproducibility of the procedure DAB 1998 Laser induced fluorescence detection systems of length polymorphism at short tandem repeat loci is not a novel methodology Holt et al 2001 and Wallin et al 2001 However accuracy and reproducibility of Identifiler Kit profiles have been determined from various sample types Figure 7 illustrates the size differences that are typically observed between sample alleles and allelic ladder alleles on the 310 Genetic Analyzer with POP 4 polymer The x axis in Figure 7 represents the nominal base pair sizes for the AmpF STR Identifiler Allelic Ladder and the dashed lines parallel to the x axis represent the 0 5 bp windows The y axis is the deviation of each sample allele size from the corresponding allelic ladder allele size The data include a total of 2269 alleles from 70 population database samples All sample alleles are within 0 5 bp of a corresponding allele in an allelic ladder Figure 7 Size deviation of 70 samples and two allelic ladders from one injection of allelic ladder on a single 310 Genetic Analyzer run 00851179 021511 X 075820 0351358 ATHO1 00135317 00165539 X 0251338 0195433 OvWA ADIBSSI X AMEL ADSSB18 FGA a A B 5 2 8 gt a o
122. logical samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following In the U S U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories found at www cdc gov biosafety Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 found at www access gpo gov nara cfr waisidx 01 29cfr1910a 01 html e Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials Additional information about biohazard guidelines is available at www cdc gov In the EU Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best practices published in the World Health Organization WHO
123. lt in off scale data Artifacts or anomalies have been seen in data produced on the 310 Genetic Analyzer when using the Identifiler Kit The shape of these artifacts is not consistent with the shape of labeled DNA fragments as seen on the 310 Genetic Analyzer Artifacts may or not be reproducible AmpFtSTR Identifiler PCR Amplification Kit User Guide 81 g lt e 3 e ei E s 2 Chapter 5 Experiments and Results Characterization of loci Artifacts can be intermittent and are not always reproducible In our experience non reproducible artifacts can be correlated to sources other than the kit that is spikes An intermittent artifact is not observed in the same position upon re injection Figure 14 demonstrates reproducible artifacts while using the Identifiler Kit Consider these artifacts when interpreting data Figure 14 Reproducible anomalies in the blue green yellow and red dye electropherograms when using the Identifiler Kit Genotyping may result in the detection of these artifacts as off ladder alleles or OL Alleles Note the degree of magnification y axis used in this figure to illustrate these artifacts Data produced on the 310 Genetic Analyzer Characterization of loci DAB 8 1 2 1 Documentation Overview Nature of the polymorphisms 82 Documentation exists and is available which defines and characterizes the locus DAB 1998 This sect
124. lts obtained from the seven injections of the AmpF STR Identifiler Allelic Ladder analyzed on the 310 Genetic Analyzer 47 cm capillary and POP 4 polymer The internal size standard used was GeneScan 500 LIZ Size Standard These results were obtained within a set of injections on a single capillary As indicated above sample alleles may occasionally size outside of the 0 5 bp window for a respective allelic ladder allele because of measurement error The frequency of such an occurrence is lowest in detection systems having the smallest standard deviations in sizing Figure 7 on page 69 illustrates the tight clustering of allele sizes obtained on the 310 Genetic Analyzer where the standard deviation in sizing is typically less than 0 15 bp The instance of a sample allele sizing outside of the 0 5 bp window because of measurement error is relatively rare when the standard deviation in sizing is approximately 0 15 bp or less Smith 1995 For sample alleles that do not size within a 0 5 bp window the PCR product must be rerun to distinguish between a true off ladder allele vs measurement error of a sample allele that corresponds with an allele in the allelic ladder Repeat analysis when necessary provides an added level of confidence to the final allele assignment GeneMapper ID Software and GeneMapper ID X Software automatically flags sample alleles that do not size within the prescribed window around an allelic ladder allele
125. lysis and characterization of stutter products at the tetranucleotide repeat locus Nucleic Acids Res 24 2807 2812 Watson S Kelsey Z Webb R Evans J and Gill P 1998 The development of a third generation STR multiplex system TGM In Olaisen B Brinkmann B and Lincoln P J eds Progress in Forensic Genetics 7 Proceedings of the 17th International ISFH Congress Oslo 2 6 September 1997 Elsevier Amsterdam pp 192 194 Weber J and Wong C 1993 Mutation of human short tandem repeats Hum Mol Genet 2 1123 1128 Weir B S 1996 Genetic data analysis II Sunderland MA Sinauer Associates Inc AmpFtSTR Identifiler PCR Amplification Kit User Guide Numerics 310 instrument 31 3130 3130xl instrument 27 3500 3500xL instrument 29 A A nucleotide addition by AmpliTaq Gold to 3 end of amplicon 81 agarose gel using to examine DNA 88 allele frequencies in the population databases 95 104 allelic ladder about 18 precision 69 profile 13 requirements for accurate genotyping 25 volume per reaction 28 30 32 amplification differential amplification of loci 88 loci 12 using bloodstained FTA cards 22 AmpliTaq Gold DNA Polymerase catalyzing the ad dition of a3 A nucleotide 81 B bins check version 50 import 35 50 biohazard safety 129 buffer new 108 C chemical safety 128 contents of kit 18 control DNA 9947A 14 18 D Data Collection Software overview 16 degraded DNA 88
126. mes i sie doque I pe Re Rp Mee ge EP gU FR ep le wees 34 Before using the software for the first time 34 Import panels and bins 35 Create an analysis method 39 General t b Settings xis kk Awak tang keeles ete aie anus tee 40 All le tab SettingS vole ree ule lx he ha tese ul aia tic wed E YR 41 Peak Detector tab settings 42 Peak Quality tab settings po esse ernaria rnahi k kalak Sk Ak ak ky kak iya KUR ea 43 Quality Flags Iab Sell l0S A six l u bbe Vibe hive da yan ad 44 Create size standard al Ana ELAN AL d h k e 44 Analyze and edit sample files with GeneMapper ID Software 46 Examine and edit a project Jx c hin k 47 For moreintormatioM usa uc a n MA eo 47 AmpFtSTR Identifiler PCR Amplification Kit User Guide Contents Section 4 2 GeneMapper D X Software 48 Overview of GeneMapper D X Software 48 IristrurmmentS a setae eec o eoa HOC ER adeb be Palas wud Dacian et eee Moe ds 48 Before you Stafti eec e Ue up Reed abiur Yum qr ue 48
127. meter Settings Sample Type Select the sample type Analysis Method Identifiler AnalysisMethod v1 or the name of the analysis method you created Panel Identifiler v2 Size Standard CE G5 Identifiler GS500 or the name of the size standard you created t The Identifiler Kit was originally validated using the GeneScan 500 LIZ Size Standard If you use the GeneScan 600 LIZ Size Standard v2 0 as an alternative perform the appropriate internal validation studies to support the use of this size standard with the Identifiler Kit Note For more information about how the Size Caller works refer to the GeneScan Analysis Software for the Windows NT Operating System Overview of the Analysis Parameters and Size Caller User Bulletin Pub no 4335617 3 Click P Analyze enter a name for the project in the Save Project dialog box then click OK to start analysis The status bar displays the progress of analysis Asacompletion bar extending to the right with the percentage indicated With text messages on the left e The table displays the row of the sample currently being analyzed in green or red if analysis failed for the sample The Genotypes tab becomes available after analysis see the figure on the next page 46 AmpFtSTR dentifiler9 PCR Amplification Kit User Guide Section 4 1 GeneMapper ID Software Examine and edit a project Project window after analysis Q GeneMapper ID v3 2 1 Id
128. mited licensing information E September 2010 Change to limited licensing information F April 2011 Change to limited licensing information G March 2012 Change to limited licensing information H May 2012 e Remove Mac OS procedures Add 3100 3100 Avant 3130 3130xl 3500 3500xL Genetic Analyzer information Added GeneMapper D Software and GeneMapper ID X Software information J August 2012 Add validation experiments and results for primer manufacturing process improvements and buffer and enzyme kit component changes Purpose The Applied Biosystems AmpF STR Identifile PCR Amplification Kit User Guide provides information about the Applied Biosystems instruments chemistries and software associated with the AmpF STR Identifiler PCR Amplification Kit AmpFtSTR Identifiler PCR Amplification Kit User Guide About This Guide Purpose 10 AmpFtSTR Identifiler PCR Amplification Kit User Guide Overview B Product 11 Workflow overview 15 B Instrument and software overview 16 m Materials and equipment l kal kk l l e kl k l k 18 Product overview Purpose The AmpF STR Identifiler PCR Amplification Kit is a short tandem repeat STR multiplex assay that amplifies 15 tetranucleotide repeat loci and th
129. mpFtSTR Identifiler PCR Amplification Kit User Guide 107 Chapter 5 Performance Validation After Buffer and Enzyme Component Replacement Overview Section 5 3 Performance Validation After Buffer and Enzyme Component Replacement Overview Experiments 108 As part of an ongoing program to exercise greater control over raw materials used in the AmpF amp STR PCR Amplification Kits manufacturing of the AmpliTaq Gold enzyme and 10X PCR Buffer II Tris KCl buffer components is transitioning from Roche Molecular Systems to Life Technologies Manufacturing of both components by Life Technologies will be conducted according to the same specifications used previously by Roche The in house components are established raw materials in our next generation kits for example the NGM NGM SElect and Identifiler Plus Kits We performed studies to compare the performance of the Identifiler Kit containing the in house components updated kit with the performance of the original kit focusing on studies most relevant to forensic DNA testing see SWGDAM Guidelines effective January 1 2011 These studies while not exhaustive are in our opinion appropriate for a manufacturer Our studies compared the performance of two Roche manufactured enzyme and buffer lots Control mixes with three new lots of buffer and two new lots of enzyme manufactured by Life Technologies Test mixes Studies were performed using Test mixes containing both the enzym
130. n 191 FGA 16 0 141 t t 16 1 0 141 t t t 17 t 0 291 0 171 t 17 2 0 141 t t t 18 0 701 2 72 0 521 1 31 18 2 1 40 t t t 19 6 72 6 16 7 07 10 21 19 2 0 281 t t t 20 7 00 13 90 7 41 12 30 20 2 ib 0 14t t t 21 12 89 16 91 14 66 12 83 21 2 t 0 291 0 171 t 22 21 57 16 91 17 24 10 47 22 2 0 28t 1 29 0 34 0 261 22 3 0 141 t t F 23 14 99 15 19 11 90 15 97 23 2 0 141 t 0 861 0 261 24 17 51 13 75 15 34 15 71 24 2 t 0 141 0 171 t 25 7 98 8 60 14 14 14 14 26 3 50 2 72 6 90 4 45 26 2 t t t 0 52 27 1 82 0 72 2 41 0 791 28 1 40 0 141 0 691 0 521 29 0 561 t t 30 30 2 0 141 t t 31 2 t t f 32 2 t t t 33 2 t t t 34 2 0 141 t t 42 2 t t t 43 2 t t 44 2 0 281 t t 45 2 t t 0 261 46 2 0 141 t t t 102 AmpFtSTR Identifiler PCR Amplification Kit User Guide Section 5 1 Developmental Validation Population data Allele African American U S Caucasian U S Hispanic Native American 357 n 349 290 191 FGA continued 47 2 t t t 48 2 0 141 t t 50 2 t t t 51 2 t t THO1 4 t 5 0 281 0 431 0 171 t 6 11 06 20 49 22 76 20 68 7 42 86 21 78 33 62 43 98 8 20 73 11 46 8 45 5 24 8 3 0 141 t t 9 12 32 16 19 14 14 6 28 9 3 11 62 29 08 20 34 23 56 10 0 98 0 431 0 521 0 261 11 1 13 3 0 141 t t t TP0X 6 6 72 0 141 0 341 t 7 2 24 t 0 341 0 261 8 36 13 53 30 49 66 37 96 9 21 15 11 60 7 24 4 19 10 9 2
131. n U S Hispanic Native American 357 349 n 290 191 D16S539 5 t t 1 8 3 22 1 72 1 72 0 791 9 19 05 10 46 9 31 12 30 10 10 92 5 59 15 69 15 45 11 31 51 31 95 30 17 30 89 12 18 77 30 23 29 48 27 75 13 14 85 16 76 11 55 10 73 14 1 54 3 01 2 07 2 09 15 0 141 0 291 t t D18S51 7 9 0 141 t t t 10 0 281 0 86 0 521 0 791 10 2 0 141 t t t 11 0 281 1 15 1 21 t 12 7 00 13 90 10 34 14 92 13 4 34 12 18 14 48 9 16 13 2 0 421 t t t 14 6 86 16 76 15 52 26 96 14 2 0 281 t t i 15 19 47 13 61 16 55 12 04 16 16 53 13 61 11 72 10 73 17 18 21 12 32 14 14 14 66 18 11 90 7 74 6 72 2 62 19 6 02 4 44 4 14 3 93 20 4 90 1 72 2 24 1 83 21 2 10 1 00 1 03 1 31 22 0 70 0 43 0 52 0 79 23 0 421 0 141 0 521 0 261 24 U 0 141 0 171 E 25 t t 0 171 t 26 t t t t 27 t t 1 AmpFtSTR Identifiler PCR Amplification Kit User Guide 99 g lt 3 2 EI S 2 Chapter 5 Experiments and Results Population data Allele African American U S Caucasian U S Hispanic Native American n 357 n 349 n 290 n 191 D19S433 9 t 0 141 0 171 t 10 1 54 t t t 11 7 14 0 72 0 521 0 521 11 2 0 141 t 0 171 t 12 10 78 7 74 6 21 3 14 12 2 6 30 0 571 1 90 t 13 29 83 28 94 16 03 17 80 13 2 5 74 1 72 8 62 15 45 14 21 01 34 10 31 72 24 87 14 2 4 20 0 86 5 00 3 66 15 4 76 15 76 13 45 13 35 15 2 3 36 2
132. n of genomic DNA varying the MgCl concentration analyzed on the 310 Genetic Analyzer 20 8 Optimal 896 dl d 20 Thermal cycler Thermal cycling parameters were established for amplification of the Identifiler Kit parameters in the GeneAmp PCR Systems 9600 and 9700 Thermal cycling times and temperatures of GeneAmp PCR systems were verified Annealing and denaturation temperature windows were tested around each stipend to verify that a 1 5 C window produced a specific PCR product with the desired sensitivity of at least 1 ng of AmpF STR Control DNA 9947A The effects of denaturation and annealing temperatures on the amplification of Identifiler Kit loci were examined using AmpFISTR Control DNA 9947A and two DNA samples AmpFtSTR Identifiler PCR Amplification Kit User Guide 67 Chapter 5 Experiments and Results Developmental validation AmpliTaq Gold DNA Polymerase activation 68 The denaturation temperatures tested were 92 5 94 and 95 5 C all for 1 minute hold times on the GeneAmp PCR System 9700 The annealing temperatures tested were 55 57 59 61 and 63 C Figure 6 also for 1 minute hold times in the GeneAmp PCR System 9700 The PCR products were analyzed using the 310 Genetic Analyzer Neither preferential nor differential amplification was observed in the denaturation temperature experiments Of the tested annealing temperatures 55
133. nd equipment Kit contents and The AmpF STR Identifiler PCR Amplification Kit Part no 4322288 contains storage materials sufficient to perform 200 amplifications at 25 uL amplification IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the primer set amplified DNA allelic ladder and size standard from light when not in use Keep freeze thaw cycles to a minimum Component Description 200X Volume Storage AmpF STR9 PCR Contains MgCl deoxynucleotide 2 tubes 1 1 mL 15 to 25 C on receipt Reaction Mix triphosphates and bovine serum albumin in each 2 to 8 C after initial use buffer with 0 0596 sodium azide AmpF STR Identifiler9 Contains fluorescently labeled primers and 1 tube 1 1 mL Primer Set non labeled primers AmpF STR Identifiler9 Contains amplified alleles 1 tube 0 05 mL Allelic Ladder See Table 1 on page 12 for a list of alleles included in the allelic ladder AmpliTaq Gold DNA Contains enzyme with an activity of 5 U uL 2 tubes 15 to 25 C Polymerase 0 05 mL tube AmpF STR Control Contains 0 10 ng uL human female 9947A 1 tube 0 3 2to 8 C DNA 9947A DNA in 0 05 sodium azide and buffert See Table 1 on page 12 for profile t The AmpF STR Control DNA 9947A is included at a concentration appropriate to its intended use as an amplification control i e to provide confirmation of the capability of the kit reagents to gen
134. oftware Version v3 2 User Bulletin Pub no 4352543 AmpFtSTR9 Identifiler PCR Amplification Kit User Guide 47 Chapter 4 GeneMapper ID X Software Overview of GeneMapper ID X Software Section 4 2 GeneMapper D X Software Overview of GeneMapper D X Software GeneMapper ID X Software is an automated genotyping software for forensic casework databasing and paternity data analysis After electrophoresis the data collection software stores information for each sample in a fsa file or a hid file Using GeneMapper ID X Software you can then analyze and interpret the data from fsa files GeneMapper ID X Software v1 0 1 or higher or hid files GeneMapper ID X Software v1 2 or higher Instruments Refer to Instrument and software overview on page 16 for a list of compatible instruments Before you start When using GeneMapper ID X Software v1 0 1 or higher to perform human identification HID analysis with AmpF STR kits be aware that 48 HID analysis requires at least one allelic ladder sample per run folder Your laboratory can use multiple ladder samples in an analysis provided individual laboratories conduct the appropriate validation studies For multiple ladder samples the GeneMapper ID X Software calculates allelic bin offsets by using an average of all ladders that use the same panel within a run folder Allelic ladder samples in an individual run folder are considered to be from a sing
135. or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the SDS Handle chemical wastes in a fume hood Ensure use of primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage After emptying a waste container seal it with the cap provided Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure that the waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply pennig chemical CAS Chemical Phrase handling 26628 22 8 Sodium Azide Sodium azide may react with lead and copper plumbing to form highly explosive metal azides 128 AmpFtSTR dentifiler9 PCR Amplification Kit User Guide Appendix D Safety Biological hazard safety Biological hazard safety Q WARNING Potential Biohazard Depending on the samples used on this instrument the surface may be considered a biohazard Use appropriate decontamination methods when working with biohazards A WARNING BIOHAZARD Bio
136. provided by the manufacturer and observe all relevant precautions 124 AmpFtSTR Identifiler PCR Amplification Kit User Guide PCR Work Areas Work area setup and lab design W K kK KK KK KK KK KK KK KK KK KK KK KK KK 125 PCRsetup workarea k n m k y Wale E ha ne 125 Amplified DNA work area 6 W K KK KK KK KK KK KK KK KK KK KK KK KK KK KK 126 Work area setup and lab design Many resources are available for the appropriate design of a PCR laboratory If you are using the AmpF STR Identifiler PCR Amplification Kit for Forensic DNA testing refer to Forensic Laboratories Handbook for Facility Planning Design Construction and Moving National Institute of Justice 1998 Parentage DNA testing refer to the Guidance for Standards for Parentage Relationship Testing Laboratories American Association of Blood Banks 7th edition 2004 The sensitivity of the Identifiler Kit and other PCR based tests enables amplification of minute quantities of DNA necessitating precautions to avoid contamination of samples yet to be amplified Kwok and Higuchi 1989 Also take care while handling and processing samples to prevent contamination by human DNA Wear gloves at all times and change them frequently Close sample tubes when not in use Limit aerosol dispersal by handling sample tubes and reagents carefully Note We do not intend these references for laboratory design to constitute all precautions and
137. r Spike 5 Off scale OS Peak Height Ratio Control Concordance CC Weight 1 0 Only applicable to controls 5Q Weighting Broad Peak BD Allelic Ladder GQ Weighting Spike SSPK SPK Off scale OS 5Q amp GQ Ranges Sizing Quality From 0 75 to 1 0 From to 0 25 Genotype Quality From 0 75 to 1 0 From to 0 25 Reset Defaults IMPORTANT The values shown are the software defaults and are the values we used during developmental validation Perform appropriate internal validation studies to determine the appropriate values to use 60 AmpFtSTR dentifiler9 PCR Amplification Kit User Guide Section 4 2 GeneMapper ID X Software 4 Analyze and edit sample files with GeneMapper ID X Software Analyze and edit sample files with GeneMapper ID X Software 1 In the Project window select File Add Samples to Project then navigate to the disk or directory containing the sample files 2 Apply analysis settings to the samples in the project Parameter Settings Sample Type Select the sample type Analysis Method Identifiler_AnalysisMethod_v2X or the name of the analysis method you created Panel Identifiler_v1 1X Size Standard CE_G5_GS500 75 450 For more information about how the Size Caller works or about size standards refer to the GeneMapper ID X Software v1 2 Reference Guide Pub
138. r a sample containing 20 pg human genomic DNA Thus the overall sensitivity of the assay has been adjusted to avoid or minimize stochastic effects We have successfully typed samples containing less than 0 5 ng DNA Note Individual laboratories may find it useful to determine an appropriate minimum peak height threshold based on their own results instruments using low amounts of input DNA Population data DAB 8 1 2 3 Population distribution data are documented and available D AB 1998 Population Data DAB 8 1 2 3 1 The population distribution data would include the allele and genotype distributions for the Population locus or loci obtained from relevant populations Where appropriate databases should be tested Distribution Data for independence expectations DAB 1998 94 AmpFtSTR dentifiler9 PCR Amplification Kit User Guide Section 5 1 Developmental Validation Population data To interpret the significance of a match between genetically typed samples it is necessary to know the population distribution of alleles at each locus in question If the genotype of the relevant evidence sample is different from the genotype of the suspect s reference sample then the suspect is excluded as the donor of the biological evidence tested An exclusion is independent of the frequency of the two genotypes in the population Overview If the suspect and evidence samples have the same genotype then the suspect is include
139. r percentage and the presence signal intensity and location of artifacts Intracolor balance No significant difference lt 10 increase or decrease in the level of intracolor balance was observed between the Test and Control mixes Figure 22 Figure 22 Reproducibility study intracolor balance Reagent Lot e Control E Control B 4 Test A Test gt Test C U ES e 3 D o 5 Q S e gt Ww E 5 a m 5 N lt 3 D e D e D 3 o ke 3 9 S t g o 9 5 5 o x t AmpFtSTR Identifiler PCR Amplification Kit User Guide 109 5 Chapter 5 Performance Validation After Buffer and Enzyme Component Replacement Reproducibility study Stutter Stutter percentage results for each marker were comparable across all Test and Control percentages mixes Figure 23 Figure 23 Reproducibility study mean stutter percentage be Reagent Lot Control B Control B Test A Test B Test C 9 o he g N O N O e G_D135317 G D165539 9 G 51338 Y 0195433 1 Artifacts Known artifacts observed showed the same morphology signal intensity and location in all Test and Control mixes and did not exceed 50 RFU Figure 24 No new artifacts were observed in the Test mixes Figure 24 Reproducibility
140. reagents een 19 a DNA quantification cioe K N K R Ha ae bs W EUH ESSET 19 m Prepare the amplification kit 21 Perform PCR Darrere 24 1285443 e obe entes 22 m Amplification using bloodstained FTA cards 22 Required user supplied reagents In addition to the Identifiler Kit reagents the use of low TE buffer 10 mM Tris 0 1 mM EDTA pH 8 0 is recommended You can prepare the buffer as described in the procedure below or order it from Teknova Cat T0223 To prepare low TE buffer 1 Mix together e 10mLof 1 M Tris HCI pH 8 0 02 mL of 0 5 M EDTA pH 8 0 990 mL glass distilled or deionized water Note Adjust the volumes accordingly for specific needs 2 Aliquot and autoclave the solutions 3 Store at room temperature DNA quantification Importance of Quantifying the amount of DNA in a sample before amplification allows you to quantification determine whether or not sufficient DNA is present to permit amplification and to calculate the optimum amount of DNA to add to the reaction The optimum amount of DNA for the Identifiler Kit is 1 0 ng in a maximum input volume of 10 uL for 28 PCR cycles AmpFtsTR Identifiler9 PCR Amplification Kit User Guide 19 Chapter 2 Perform PCR DNA quantification If too much DNA is added to the PCR reaction then the increased amount of PCR product that is generated can result in e
141. rsion 1 0 Getting Started Guide Pub no 4375574 for more information Define custom views of plots Refer to Chapter 1 of the GeneMapper ID X Software Version 1 0 Getting Started Guide Pub no 4375574 for more information For more For quick set up instructions refer to the GeneMapper ID X Software Version 1 0 Getting information Started Guide Pub no 4375574 For details about GeneMapper ID X Software features refer to GeneMapper ID X Software Version 1 0 Getting Started Guide Pub no 4375574 GeneMapper ID X Software Version 1 0 Quick Reference Guide Pub no 4375670 GeneMapper ID X Software Version 1 0 Reference Guide Pub no 4375671 AmpFtSTR Identifiler PCR Amplification Kit User Guide 49 O 0 2 0 lt i o 0 e v gt O p E 0 4 Chapter 4 GeneMapper ID X Software Set up GeneMapper ID X Software for data analysis Check panel bin 1 Start the GeneMapper ID X Software then log in with the appropriate user and stutter file name and password version IMPORTANT For logon instructions refer to the GeneMapper ID X Software Version 1 0 Getting Started Guide Pub no 4375574 2 Select Tools Panel Manager 3 Check the version of files imported into the Panel Manager s Panel Manager File Edit Bins View Help a Select Panel Manager in the navigation pane lt Ly b Expand the Panel Manager folder
142. s Plot Settings Matrices Name Last Saved Owner Type Description 377 F HID GS500 2004 05 28 11 34 3 gmid Basic amp dvanced Factory Provided CE G5 HID GS500 2004 05 28 11 34 3 gmid Basic amp dvanced Factory Provided CE F HID 65500 2004 05 28 11 34 3 gmid Basic Advanced Factory Provided 3 Enter a name for example CE G5 Identifiler GS500 as shown below In the Size Standard Dye field select Orange In the Size Standard Table enter the sizes specified in on page 44 The example below is for the GeneScan 500 LIZ Size D lt 5 5 90 gt Standard 7 Size Standard Editor Edit r Size Standard Description Name CE G5 Identifiler GS500 Description Size Standard Dye orange r Size Standard Table Size in Basepairs pr 100 0 138 0 150 0 160 0 300 0 340 0 1 2 3 4 5 6 2000 7 8 9 350 0 10 4000 11 4500 AmpFtSTR Identifiler PCR Amplification Kit User Guide 45 4 Chapter 4 Analyze Data Analyze and edit sample files with GeneMapper ID Software Analyze and edit sample files with GeneMapper ID Software 1 In the Project window select File Add Samples to Project then navigate to the disk or directory containing the sample files 2 Apply analysis settings to the samples in the project Para
143. s a mixture The presence of more than two alleles at a locus The presence of a peak at a stutter position that is significantly greater in percentage than what is typically observed in a single source sample Significantly imbalanced alleles for a heterozygous genotype The peak height ratio is defined as the height of the lower peak in RFU divided by the height of the higher peak in RFU expressed as a percentage Mean median and minimum and maximum peak height ratios observed for alleles in the Identifiler Kit loci in unmixed population database samples are listed in Table 4 Table 4 Peak height ratios Allele Meant Median Minimumt Maximumt CSF1PO 84 86 88 63 6 99 8 D2S1338 93 84 86 42 8 99 7 D3S1358 91 88 90 64 3 99 7 D5S818 82 89 91 64 9 99 7 D7S820 96 89 90 66 2 99 5 D8S1179 89 90 93 57 5 99 8 0135317 96 87 87 63 3 100 0 0165539 92 88 91 61 5 99 9 018551 99 82 83 56 3 99 9 0195433 98 88 92 48 8 100 0 021511 92 88 89 66 4 99 6 FGA 94 85 87 60 9 99 5 AmpFtSTR Identifiler PCR Amplification Kit User Guide 91 g lt e 3 e E s 2 92 Chapter 5 Experiments and Results Mixture studies Allele HE Lan Meant Mediant Minimum Maximumt THO1 99 86 88 48 8 99 9 TPOX 87 87 92 55 9 99 8 wA 101 86 88 62 8 99 1 t Peak height ratios were determined for those heterozygous
144. s an internal PCR control IPC assay similar in principle to that used in the other Quantifiler kits but labeled with NED dye 20 AmpFtSTR Identifiler PCR Amplification Kit User Guide Chapter 2 Perform PCR 2 Prepare the amplification kit reactions Prepare the amplification kit reactions 1 Calculate the volume of each component needed to prepare the reactions using the table below DNA sample Volume per reaction AmpF STR PCR Reaction Mix 10 5 uL AmpliTaq Gold DNA Polymerase 0 5 uL AmpF STR Identifiler Primer Set 5 5 uL Note The volumes indicated above include a slight overfill to account for the loss that occurs during reagent transfers 2 Prepare reagents Thaw the PCR Reaction Mix and the Identifiler Primer Set then vortex all reagent tubes including the enzyme for 3 seconds and centrifuge briefly before opening the tubes IMPORTANT Thawing is required only during first use of the Primer Set and PCR Reaction Mix After first use these reagents are stored at 2 to 8 C and do not require subsequent thawing Do not refreeze these reagents 3 Prepare the master mix Pipette the required volumes of components into an appropriately sized polypropylene tube 4 Vortex the master mix for 3 seconds then centrifuge briefly 5 Dispense 15 uL of the reaction mix into each reaction well of a MicroAmp Optical 96 Well Reaction Plate or each MicroAmp tube 6 Prepare the DNA samples
145. samples with peak heights gt 200 RFU For all 15 loci the mean peak height ratios indicate that the two alleles of a heterozygous individual are generally very well balanced If an unusually low peak height ratio is observed for one locus and there are no other indications that the sample is a mixture the sample may be reamplified and reanalyzed to determine if the imbalance is reproducible Possible causes of imbalance at a locus are degraded DNA presence of inhibitors extremely low amounts of input DNA or the presence of an allele containing a rare sequence that does not amplify as efficiently as the other allele Resolution of genotypes in mixed samples A sample containing DNA from two sources can be comprised at a single locus of any of the seven genotype combinations listed below Heterozygote heterozygote no overlapping alleles four peaks Heterozygote heterozygote one overlapping allele three peaks Heterozygote heterozygote two overlapping alleles two peaks e Heterozygote homozygote no overlapping alleles three peaks e Heterozygote homozygote overlapping allele two peaks Homozygote homozygote no overlapping alleles two peaks Homozygote homozygote overlapping allele one peak Specific genotype combinations and input DNA ratios of the samples contained in a mixture determine whether it is possible to resolve the genotypes of the major and minor component s at a single locus The ability
146. sis Methods tab then click New to open the New Analysis Method dialog box 3 Select HID and click OK to open the Analysis Method Editor with the General Tab selected 4 Enter the settings in each tab of the Analysis Method Editor as shown in the figures below unless the instructions state otherwise Note The Analysis Method Editor closes when you save your settings To complete this step quickly do not save the analysis method until you finish entering settings in all of the tabs 5 After you enter settings in all tabs click Save AmpFtSTR Identifiler PCR Amplification Kit User Guide 39 G lt 97 9 5 90 gt 4 Chapter 4 Analyze Data Set up GeneMapper ID Software for data analysis General tab settings Analysis Method Editor HID ldentifiler _AnalysisMethod_v1 2 In the Name field either type the name as shown or enter a name of your choosing The Description and Instrument fields are optional AmpFtSTR Identifiler PCR Amplification Kit User Guide 40 Section 4 1 GeneMapper ID Software 4 Set up GeneMapper ID Software for data analysis Allele tab settings Analysis Method Editor HID General Allele Peak Detector Peak Quality Quality Flags Bin Set ges IV Use marker specific stutter ratio if available Marker Repeat Tri Tetra Penta Hexa Cut off Value oo joo foo oo e MinusA R
147. spectral components The four dyes used in the Identifiler Kit to label samples are 6 FAM VIC NED and PET dyes The fifth dye LIZ is used to label the GeneScan 500 LIZ Size Standard or the GeneScan 600 LIZ9 Size Standard v2 0 AmpFtSTR Identifiler PCR Amplification Kit User Guide How multicomponent analysis works Chapter 1 Overview 1 Instrument and software overview Each of these fluorescent dyes emits its maximum fluorescence at a different wavelength During data collection on the Applied Biosystems instruments the fluorescence signals are separated by diffraction grating according to their wavelengths and projected onto a charge coupled device CCD camera in a predictably spaced pattern The 6 FAM dye emits at the shortest wavelength and it is displayed as blue followed by the VIC dye green NED dye yellow PET dye red and LIZ dye orange Although each of these dyes emits its maximum fluorescence at a different wavelength there is some overlap in the emission spectra between the dyes Figure 3 The goal of multicomponent analysis is to correct for spectral overlap Figure 3 Emission spectra of the five dyes used in the AmpF STR Identifiler Kit Dyes VIC NED PET c o 2 N E 9 2 Wavelength nm AmpFtSTR Identifiler PCR Amplification Kit User Guide 17 1 Chapter 1 Overview Materials and equipment Materials a
148. strument and displays instrument status and raw data in real time As the instrument measures sample fluorescence with its detection system the Data Collection Software collects the data and stores it The Data Collection Software stores information about each sample in a sample file fsa which is then analyzed by the GeneMapper ID or ID X Software Table 2 Software specific to each instrument Instrument Operating Data Collection Analysis software system Software 3500 3500xL Windows XP 3500 Series GeneMapper D X Software Windows Data Collection v1 2 or higher Vista Software v1 0 3130 3130xl Windows XP 3 0 GeneMapper ID Software v3 2 1 3100 3100 Avant Windows NT 1 1 3100 1 0 3100 Avant Windows 2000 2 0 310 Windows XP 3 1 Windows NT 3 0 e Windows 2000 and GeneMapper D X Software v1 0 1 or higher Note We conducted validation studies for the AmpF STR Identifiler PCR Amplification Kit using the 310 Genetic Analyzer running Mac OS This configuration is now obsolete Applied Biosystems fluorescent multi color dye technology allows the analysis of multiple loci including loci that have alleles with overlapping size ranges Alleles for overlapping loci are distinguished by labeling locus specific primers with different colored dyes Multicomponent analysis is the process that separates the five different fluorescent dye colors into distinct
149. tal Validation Estimation of spontaneous or induced germline mutation at genetic loci may be achieved through comparison of the genotypes of offspring to those of their parents From such comparisons the number of observed mutations are counted directly In previous studies genotypes of ten STR loci amplified by the AmpF STR SGM Plus PCR Amplification Kit were determined for a total of 146 parent offspring allelic transfers meioses at the Forensic Science Service Birmingham England One length based STR mutation was observed at the D18511 locus mutation was not detected at any of the other nine STR loci The D18511 mutation was represented by an increase of one 4 bp repeat unit a 17 allele was inherited as an 18 single step mutation The maternal paternal source of this mutation could not be distinguished Additional studies Edwards et al 1991 Edwards et al 1992 Weber and Wong 1993 Hammond et al 1994 Brinkmann et al 1995 Chakraborty et al 1996 Chakraborty et al 1997 Brinkmann et al 1998 Momhinweg et al 1998 Szibor et al 1998 of direct mutation rate counts produced Larger sample sizes for some of the Identifiler Kit loci Methods for modifications of these mutation rates to infer mutation rates indirectly for those loci where these rates are not large enough to be measured directly and or to account for those events undetectable as Mendelian errors Probability of identity Table 7 shows the Probabilit
150. tion that this identified artifact may add to the observed peak height or area The highest percent stutter for D351358 is not inconclusive of allele 15 Figure 8 Stutter percentages for the 0851179 021511 075820 and CSF1PO loci oe re 2 o o 9 me ame 9 00 9 WO 99949 OO 9990 4000 9 0000 999 vo mam 99999 8 9 10 11 12 13 14 15 16 17 25 26 27 28 29 30 31 32 33 34 35 36 8 9 10 11 12 13 14 8 9 10 11 12 13 14 D8S1179 D21S11 D7S820 CSF1PO Allele 78 AmpFtSTR dentifiler9 PCR Amplification Kit User Guide Section 5 1 Developmental Validation Extra Peaks in the electropherogram Figure 9 Stutter percentages for the 0351358 TH01 0135317 0165539 and 0251338 loci See the comment on page 78 regarding stutter at allele 15 of D3S1358 g lt e 3 D m S E e 2 z o c o o 12 13 14 15 16 17 18 19 6 7 8 9 1011 8 9 1011121314 8 9 1011121314 16 17 18 19 20 21 22 23 24 25 26 D3S1358 TH01 D13S317 D16S539 D2S1338 Allele Figure 10 Stutter percentages for the 0195433 vWA 018551 loci 16 T 15 14 ha 13 at 12 t 33 11 gt Pas 30 10 ef e 2 e PAg 0 5 6 9 iit i56 a te e oo te zt ei t 1 7 SAU Sete is
151. tware v3 2 1 for the first time you need to Import panels and bins into the Panel Manager as explained in Import panels and bins on page 35 Create an analysis method as explained in Create an analysis method on page 39 Create a size standard as explained in Create size standard on page 44 Define custom views of analysis tables Refer to Chapter 1 of the GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial Pub no 4335523 for more information Define custom views of plots Refer to Chapter 1 of the GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial Pub no 4335523 for more information AmpFtSTR Identifiler PCR Amplification Kit User Guide Section 4 1 GeneMapper ID Software 4 Set up GeneMapper ID Software for data analysis Import panels and To import the Identifiler panel and bin set into the GeneMapper ID Software v3 2 1 bins database 1 Start the GeneMapper ID Software then log in with the appropriate user name and password IMPORTANT If you need logon instructions refer to page 2 7 of the GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide Pub no 4338775 2 Select Tools Panel Manager 3 Find then open the folder containing the panels and bins a Select Panel Manager in the navigation pane Panel Manager File Edit Bins View emimmm mi D lt 97
152. us displays percent stutter that is reproducible The highest percent stutter observed for each allele is as follows CSF1PO 9 2 0251338 11 1 0351358 10 7 055818 6 8 075820 8 2 0851179 8 2 0135317 8 0 0165539 10 4 018551 17 0 0195433 13 3 021511 9 4 14 7 1 5 1 4 8 vWA 12 6 The highest observed percent stutter for each locus is included as the filter in the GeneMapper ID Software and the GeneMapper ID X Software Peaks in the stutter position that are above the highest observed percent stutter will not be filtered Peaks in the stutter position that have not been filtered and remain labeled can be further evaluated For evaluation of mixed samples see Mixture studies on page 91 AmpFtSTR Identifiler PCR Amplification Kit User Guide 77 g lt e 3 e E s 2 Chapter 5 Experiments and Results Extra Peaks in the electropherogram The percent stutter does not change significantly with the recommended quantity of input DNA for on scale data The measurement of percent stutter may be unusually high for main peaks that are off scale The percent stutter for allele 15 in D351358 Figure 9 is artificially increased due to a reproducible artifact Figure 4 8 observed in the green dye lanes at this position When analyzing samples which contain a 0351358 allele 15 we recommend careful examination due to the contribu
153. utter 49 50 create analysis method 55 examine and edit project 62 overview 16 48 setup 49 GeneScan size standard about 18 dye label 16 volume per reaction 28 29 31 genetics 94 104 allele frequencies 95 104 populations and samples used in studies 95 probability of identity 105 probability of paternity exclusion 106 genotype determining 69 exclusion of suspects 95 resolving in mixed samples 92 H hematin effecton DNA samples 87 Hi Di formamide volume per reaction 28 29 31 import HID size standard 44 panels and bins 35 panels bins and stutter 50 138 instrumentation 310 genetic analyzer 16 31 3100 3100 Avant genetic analyzer 16 27 3130 3130xl genetic analyzer 16 27 3500 3500xL genetic analyzer 16 27 29 software compatibility 16 kit allelic ladder 18 amplification 11 contents 18 control DNA 18 description 11 DNA polymerase 18 21 fluorescent dyes 16 loci amplification 12 PCR reaction mix 18 21 primers 11 18 20 reagents 18 supported instruments 11 thermal cyclers for use with 126 L limited product warranty 132 LIZ size standard about 18 volume per reaction 28 29 31 loci allele frequencies in the population databases 95 104 chromosomal location 12 differential amplification 88 dye label 12 lack of amplification effect of DNA quantity on results 85 86 population data allele frequencies 95 104 population data probability of identity 105 population data probability of paternit
154. validation studies 42 AmpFtSTR dentifiler9 PCR Amplification Kit User Guide Section 4 1 GeneMapper ID Software 4 Set up GeneMapper ID Software for data analysis Peak Quality tab setti ngs Analysis Method Editor HID gx General Allele Peak Detector Peak Quality Quality Flags Signal level Perform internal Homozygous min peak height validation Heterozygous min peak height studies to determine Heterozygote balance settings Q Min peak height ratio 3 Peak morphology Max peak width basepairs j 5 Pull up peak gt i e Pull up ratio 0 05 number 5 Max expected alleles Factory Defaults IMPORTANT Perform the appropriate internal validation studies to determine the minimum heterozygous and homozygous minimum peak height thresholds and the minimum peak height ratio threshold for interpretation of Identifiler Kit data AmpFtSTR9 Identifiler PCR Amplification Kit User Guide 43 4 Chapter 4 Analyze Data Set up GeneMapper ID Software for data analysis Quality Flags tab settings x General Allele Peak Detector Peak Quality i Quality weights are between and 1 r Quality Flag Settings Spectral Pull up bs Control Concordance j 0 Broad Peak bs Low Peak Height bs Out of Bin Allele bs Off scale os Overlap jos Peak Height Ratio jos r PQV Thresholds Sizing Quality From 07
155. y exclusion 106 population data samples used in studies 95 locus See loci low TE buffer 19 M materials and equipment included in kit 18 AmpF amp TR Identifiler PCR Amplification Kit User Guide not included with kit 121 mixed samples 91 multicomponent analysis 16 17 N negative control sample preparation 21 0 operating systems 16 27 29 31 P panels check version 49 50 import 35 50 PCR amplification of tetranucleotide STR loci stutter peak 77 inhibitor causing lack of amplification 87 performing 22 setup 125 thermal cycling conditions programming 22 PCR work areas 121 125 population genetics 94 104 allele frequencies 95 104 populations and samples used in the studies 95 Probability of Exclusion Pg from Paternity 106 Probability of Identity P 105 See Also allele positive control sample preparation 21 precision results 71 75 primer manufacturing process improvements 107 primers volume per reaction 21 Q quantification DNA 19 R reaction mix volume per reaction 21 reactions preparing for PCR 21 reagents not included with kit 121 user supplied 19 references 133 run module electrophoresis 27 29 31 AmpF amp TR Identifiler PCR Amplification Kit User Guide Index S safety biohazard 129 chemical 128 Safety Data Sheets SDSs obtaining 132 sample preparation 21 DNA negative control 21 DNA positive control 21 standards 18 samples DNA from more than one individual
156. y of Identity Pr values of the Identifiler Kit loci individually and combined Table 7 Probability of Identity values for the Identifiler Kit STR loci Locus African American U S Caucasian U S Hispanic Native American CSF1PO 0 079 0 132 0 141 0 123 0251338 0 023 0 027 0 038 0 043 0351358 0 097 0 076 0 112 0 158 055818 0 104 0 147 0 115 0 110 075820 0 085 0 063 0 083 0 081 0851179 0 074 0 064 0 089 0 104 0135317 0 132 0 079 0 056 0 056 0165539 0 077 0 097 0 090 0 082 018551 0 033 0 031 0 031 0 046 0195433 0 042 0 087 0 049 0 044 021511 0 037 0 044 0 047 0 074 FGA 0 034 0 035 0 032 0 031 THO1 0 109 0 079 0 097 0 134 TPOX 0 089 0 188 0 168 0 159 AmpFtSTR Identifiler PCR Amplification Kit User Guide 105 g lt e 3 e E s 2 Chapter 5 Experiments and Results Probability of paternity exclusion Locus African American U S Caucasian U S Hispanic Native American vWA 0 066 0 066 0 080 0 103 Combined 1 31 x 10 18 5 01 x 10 18 7 65 x 10 18 3 62 x 10 17 The P value is the probability that two individuals selected at random will have an identical Identifiler Kit genotype Sensabaugh 1982 The P values for the populations described in this section are then approximately 1 7 64 x 1017 African American 1 2 00 x 1017 U S Caucasian 1 1 31 x 1017 U S Hispanic and 1 2 76 x 1016 Native American
157. zyme Component Replacement Sensitivity study Figure 26 Sensitivity study representative electropherograms for Sample 2 amplified using 125 pg input DNA Y scale 500 RFU Control A Mat Sampie He Ctt i Li Control B dek Sampie for Caton Test B Mek Lampe fon Ctr Test C DNA concentration The calculated slope and R values for each of the plotted curves were equivalent and peak height showing comparable relationships between peak height and DNA input amount for the Test and Control mixes Figure 27 In general the Test mixes showed a slight increase in peak height compared to the Control mixes Figure 27 Sensitivity study linear regression plot of combined mean referenced peak height for three genomic DNA samples Reagent Lct Control A Control B Test A TestB Test C Referenced Peak Height 400 600 800 DNA Input Amount pg 112 AmpFtSTR dentifiler9 PCR Amplification Kit User Guide Section 5 3 Performance Validation After Buffer and Enzyme Component Replacement 5 Sensitivity study Allelic dropout Allelic dropout was observed only for amplifications of 125 pg where dropout of a single allele was observed for Test A Sample 1 Figure 28 and Control B Sample 3 Figure 29 These results can be explained by stochastic variation and sampling from dilute DNA solutions Allelic dropout results can therefore be considered equivalent between Test and Control mixes Figure 28 Sensitiv

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