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AmpFlSTR® Identifiler® Plus PCR Amplification Kit User`s Guide

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1. Figure 30 Amplification of DNA mixtures at various ratios using the 28 PCR cycle protocol Minor allele peaks that do not overlap with the major contributor peaks are highlighted 99 AmpFtSTR Identifiler Plus User Guide Mixture studies 330 130 170 210 210 250 290 290 330 E 6000 5000 4000 3000 2000 1 1000 Figure 31 peaks are highlighted 3 1 10 1 0 1 Amplification of DNA mixtures at various ratios using the 29 PCR cycle protocol Minor allele peaks that do not overlap with the major contributor Table 9 Genotypes of mixed DNA samples Locus Profile Sample A Profile Sample B D8S1179 1
2. Allele Mean Standard Deviation 24 242 37 242 52 0 044 0 067 25 246 42 246 57 0 044 0 056 26 250 48 250 62 0 038 0 069 26 2 252 49 252 64 0 046 0 066 27 254 5 254 65 0 047 0 057 28 258 55 258 71 0 045 0 064 29 262 63 262 78 0 049 0 062 30 266 72 266 88 0 052 0 069 30 2 268 53 268 7 0 049 0 065 31 2 272 62 272 78 0 036 0 062 32 2 276 71 276 86 0 05 0 068 33 2 280 77 280 94 0 043 0 069 42 2 317 89 318 06 0 045 0 062 43 2 322 01 322 16 0 038 0 055 44 2 326 14 326 27 0 034 0 05 45 2 330 28 330 39 0 039 0 048 46 2 334 28 334 4 0 044 0 05 47 2 338 3 338 49 0 039 0 055 48 2 342 51 342 66 0 034 0 055 50 2 350 59 350 76 0 041 0 061 51 2 354 54 354 7 0 039 0 063 THO1 4 162 72 162 77 0 025 0 04 5 166 78 166 84 0 027 0 035 6 170 82 170 87 0 03 0 046 7 174 83 174 9 0 029 0 045 8 178 84 178 9 0 02 0 046 9 182 82 182 89 0 027 0 034 9 3 185 84 185 9 0 022 0 042 10 186 77 186 83 0 026 0 036 11 190 71 190 77 0 027 0 034 12 201 48 201 55 0 026 0 037 TPOX 6 221 82 221 91 0 029 0 05 7 225 8 225 88 0 029 0 053 8 229 79 229 86 0 034 0 048 AmpFtSTR Identifiler Plus User Guide 80 Chapter 5 Experiments and Results Table 4 Precision results of five runs 16 capillaries run of the AmpF STR Identifiler Plus Allelic Ladder continued Applied Biosystems 3130x Genetic Analyzer
3. NM zan p Quantifiler Duo DNA Quantification Kit Perform PCR AmpF STR Identifiler Plus PCR Amplification Kit A o a GeneAmp PCR System Veriti 96 Well Thermal 9700 Thermal Cycler Cycler Perform Electrophoresis gas the 32 ABI PRISM Applied Biosystems ABI PRISM 310 3100 3100 Avant 3130 3130x Genetic Genetic Analyzer Genetic Analyzer Analyzer Analyze Data GeneMapper ID or GeneMapper D X Software 6 AmpFtSTR Identifiler Plus User Guide Instrument and software overview Instrument and software overview This section provides information about the Data Collection Software versions required to run the AmpF STR Identifiler Plus PCR Amplification Kit on specific instruments Data Collection and GeneMapper D or ID X Software The Data Collection Software provides instructions to firmware running on the instrument 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 Instrument and by the GeneMapper JD or ID X Software Operating Data Collection A software Instrument system Software Analysis software compatibility 3130 3130xI Window
4. Figure 4 AmpF STR Identifiler Plus PCR Amplification Kit results from a 1 2 mm FTA bloodstain disc 24 cycle amplification analyzed on the Applied Biosystems 3130x Genetic Analyzer AmpFtSTR Identifiler Plus User Guide Part Number 4440211 Rev D 03 2012 Chapter 3 Electrophoresis AmpFtSTR Identifiler Plus User Guide AmpFtSTR Identifiler Plus User Guide Electrophoresis This chapter covers amp Allelic ladder requirements uuaeaaaaaa aaa KK KK KK KK KK iii 27 Section 3 1 3100 3100 Avant and 3130 3130xlinstruments 28 m Set up the 3100 3100 Avant or 3130 3130xl instrument for electrophoresis 28 n Prepare samples for electrophoresis on the 3100 3100 Avant or 3130 3130xl Instr merit ND ZPO ho PKO ska c DNI AA ova tc verbe WR 20 Section 3 2 310 instrument e k kk kk KK kk k kk k 30 n Set up the 310 instrument for electrophoreSis 4 2221111 30 n Prepare samples for electrophoresis on the 310 instrument 31 AmpFtSTR Identifiler Plus User Guide 26 Chapter 3 Electrophoresis Allelic ladder requirements To accurately genotype samples you must run an allelic ladder sample along with the unknown samples For samples run on the ABI PRISM 310 Genetic Analyzer Run at least one allelic ladder for every 10 sample injections ABI PRISM 3100 and Applied Biosystems 3130 series Genetic Analyzers Run at least one
5. 1200 0 062 ng 115 155 195 235 275 315 355 395 1000 moj 0 031 ng 600 400 200 i i Figure 26 Effect of amplifying 0 5 ng 0 25 ng 0 125 ng 0 062 ng and 0 031 ng of Control DNA 9947A using the 29 PCR cycle protocol Note that the y axis scale is magnified for the lower amounts of DNA analyzed using the Applied Biosystems 3130x Genetic Analyzer AmpFtSTR Identifiler Plus User Guide 92 Chapter 5 Experiments and Results Stability SWGDAM The ability to obtain results from DNA recovered from biological samples deposited guideline 2 4 on various substrates and subjected to various environmental and chemical insults has been extensively documented In most instances assessment of the effects of these factors on new forensic DNA procedures is not required However if substrates and or environmental and or chemical insults could potentially affect the analytical process then the process should be evaluated using known samples to determine the effects of such factors SWGDAM July 2003 Degraded DNA As the average size of degraded DNA approaches the size of the target sequence the amount of PCR product generated is reduced This reduction 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 Raji DNA was sonicated and
6. Master Mix not vortexed thoroughly before aliquoting Vortex Master Mix thoroughly AmpF STR Identifiler Plus Primer Set exposed to too much light Store Primer Set protected from light GeneAmp PCR System malfunction Refer to the thermal cycler user s manual and check instrument calibration Incorrect thermal cycler parameters Check the protocol for correct thermal cycler 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 retainer set and tubes in GeneAmp PCR System 9700 Remove MicroAmp Base from tray retainer set and repeat test Insufficient PCR product electrokinetically injected For ABI PR sM 3100 Avant or Applied Biosystems 3100 3130x runs Mix 1 0 uL of PCR product and 9 uL of Hi DiTM Formamide GeneScan 500 LIZ solution For ABI PRISM 310 instrument runs Mix 1 5 uL of PCR product and 25 uL of Hi Di Formamide GeneScan 500 LIZ solution Degraded formamide Check the storage of formamide do not thaw and refreeze multiple times Try Hi DiTM Formamide AmpFtSTR Identifiler Plus User Guide 118 Appendix A Troubleshooting Table 14 Troubleshooting continued Observation P
7. Allele Mean Standard Deviation 9 233 77 233 86 0 039 0 048 10 237 76 237 83 0 025 0 053 11 241 75 241 83 0 028 0 04 12 245 78 245 85 0 04 0 049 13 249 76 249 85 0 036 0 048 vWA 11 154 07 154 14 0 028 0 042 12 158 26 158 34 0 028 0 04 18 162 42 162 49 0 031 0 043 14 166 66 166 73 0 031 0 05 15 170 59 170 66 0 029 0 044 16 174 62 174 68 0 03 0 04 17 178 61 178 67 0 028 0 051 18 182 54 182 61 0 021 0 037 19 186 5 186 56 0 024 0 043 20 190 43 190 49 0 028 0 04 21 194 29 194 36 0 024 0 044 22 198 17 198 24 0 023 0 036 23 202 01 202 09 0 028 0 041 24 206 36 206 42 0 034 0 041 Extra peaks in the electropherogram 81 Causes of extra peaks Peaks other than the target alleles may be detected on the electropherogram Causes for the appearance of extra peaks include stutter products incomplete 3 A nucleotide addition at the n 1 position dye artifacts and mixed DNA samples see DAB Standard 8 1 2 2 Stutter products A stutter is a well characterized PCR artifact that refers to the appearance of a minor peak one repeat unit smaller or less frequently one repeat larger than the major STR product Butler 2005 and Mulero et a 2006 Sequence analysis of stutter products at tetranucleotide STR loci has revealed that the stutter product 1s missing a single tetranucleotide core repeat unit relative to the main allele Walsh et al 1996 AmpFtSTR Identifiler Plus User Guide Extra peaks in the electropherogram
8. 19 20 21 22 17 18 23 24 TPOX 2p23 2per 6 7 8 9 10 11 12 13 gt 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 D58818 5q21 31 7 8 9 10 11 12 13 14 15 16 1188 FGA 49028 17 18 19 20 21 22 23 24 25 26 26 2 23 24 27 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 For CODIS purposes profile reported as 13 13 For CODIS purposes profile reported as 30 30 For CODIS purposes profile reported as 11 11 For CODIS purposes profile reported as 8 8 For CODIS purposes profile reported as 11 11 AmpFtSTR Identifiler Plus User Guide 3 Chapter 1 Overview Allelic ladder Figure 1 shows the allelic ladder for the AmpF STR Identifiler Plus Kit See Allelic profile ladder requirements on page 27 for information on ensuring accurate genotyping ladder m mg m jm IT Mark Sample for Deletion 130 170 210 250 290 330 370 1200 800 400 o 3432222422424 BAAD S aa l a a AA AAAAAAAAAA ARAARARAAA E 2 bo ba ba 3 fs bs ha lz ls l ps ESEo E7 Eo E E2 E EsE5 Es ETE I E E 2 Belo dp ds k E E Akdede 4 2 s E22 42 9 2 12 3 2 B52 ladder jm ja Mark Sample for Deletion 130 170 210 250 290 330 370 1200 j 800 400 D A 2 A hkkk fis 17 re 15
9. Files of type xw Files xml z Cancel To view the settings for Identifiler Plus_AnalysisMethod_v1 select the Analysis Methods tab then select Identifiler_Plus_AnalysisMethod in the Name column and click Open 43 AmpFtSTR Identifiler Plus User Guide Set up GeneMapper ID Software for data analysis jJ GeneMapper Manager 2 Projects Analysis Methods Table Settings Plot Settings Matrices Size Standards Mame Last Saved Owner Instrument Analysis Type t Default 2009 08 31 11 27 45 695 gmid Hio x Identifiler Plus AnalysisMethod v1 2009 09 02 11 13 05 191 gmid Hio New Open Save As Import Export Delete Done Figure 5 Analysis Method Editor HID General tab settings Figures 6 through 9 below show the settings for each tab of the Analysis Method Editor HID C D 2 o lt ko go D e n e Eh 0 Analysis Method Editor HID General Allele Peak Detector Peak Quality Quality Flags Bin Set REM OIC TA v Use marker specific stutter ratio if available Marker Repeat Type Tri Tetra Penta Hexa Cut off Value bo bo po ho MinusA Ratio ao foo bo fo Minus Distance From bo foo loo loo To foo ao bo fo Minus Stutter Ratio o o 0 loo bo Minus Stutter Distance From o B 25 bo foo To ho fus foo ho Plus Stutter Ratio ao foo loo fo Plus Stutter Distance From o 0 o 0 fo fo
10. Hispanic Pania CSF1PO 0 545 0 496 0 450 0 409 D281338 0 748 0 725 0 671 0 399 D381358 0 591 0 630 0 495 0 510 D58818 0 506 0 440 0 525 0 601 D78820 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 The Pg value is the probability averaged over all possible mother child pairs that a random alleged father will be excluded from paternity after DNA typing of the AmpF STR Identifiler Plus Kit STR loci Chakraborty and Stivers 1996 AmpFtSTR Identifiler Plus User Guide 116 Chapter 5 Experiments and Results 117 AmpFtSTR Identifiler Plus User Guide Troubleshooting Table 14 Troubleshooting Follow the actions recommended in Table 14 to troubleshoot problems that occur during analysis Observation Possible causes Recommended actions Faint or no signal from both the 9947A and the DNA test samples at all loci Incorrect volume or absence of either AmpF STR Identifiler Plus Master Mix or AmpF STR Identifiler Plus Primer Set Repeat amplification No activation of enzyme Repeat amplification making sure to hold reactions initially at 95 C for 11 min
11. Neither the 250 nt nor the 340 nt peak are included in the size standard definition These peaks can be used as an indicator of precision within a run 3 Click PP 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 completed 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 see figure on next page is displayed upon completion of the analysis C o D lt ko o 0 8 w gt n 9 zh 2 0 AmpFtSTR Identifiler Plus User Guide 64 Section 4 2 GeneMapper ID X Software s GeneMapperf ID X 09 02 09 Dickens Analysis Method Example gmidx Is Logged In Database FOSWANGD1L03 File Edit Analysis View Tools Admin Help H he DE ul gt g Table Setting sex Data Analysis Mm 2 amp E e Samples Analysis Summary Genotypes I aa Project Analysis Summary n a Identifiler Plus Analysis Examples Select run folder to display Identifiler Plus Analysis Examples Unanalyzed 0 Analyzed 39 Ur Analysis Setting Changed Click a link below to display a filtered Samples Table containing only the samples selected Allelic Ladder Quality per run folder base
12. Polymer for 3130 3130x Genetic Analyzers 4352755 3130 3130x Genetic Analyzer Autosampler Plate Kit 96 well 4316471 GeneScan 500 LIZ Size Standard 4322682 Running Buffer 10X 402824 DS 33 Matrix Standard Kit Dye Set G5 4345833 MicroAmp Optical 96 Well Reaction Plate N8010560 For a complete list of parts and accessories for the 3130x instrument refer to Appendix A of the Applied Biosystems 3130 3130xl Genetic Analyzers Maintenance Troubleshooting and Reference Guide PN 4352716 310 Analyzer materials 310 DNA Analyzer Capillary Array 47 cm 402839 0 5 mL Sample Tray 5572 96 Well Tray Adaptor for 9700 thermal cycler trays 4305051 GeneScan 500 LIZ Size Standard 4322682 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 DS 33 Matrix Standard Set 6 FAM VIC NED PET and LIZ dyes for 4318159 ABI PRISM 310 377 systems MicroAmp 8 Tube Strip 0 2 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 Analyzer User Guide PN 4317588 For a complete list of parts and accessories for the 310 instrument refer to Appendix B of the AB PRISM 310 Genetic
13. consult the MSDS Handle chemical wastes in a fume hood After emptying a waste container seal it with the cap provided Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or national environmental and health regulations If potentially hazardous waste 1s generated when you operate the instrument you must Characterize by analysis 1f necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure the health and safety of all personnel in your laboratory AmpFtSTR Identifiler Plus User Guide Biological hazard safety Ensure that the instrument 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 Biological hazard safety General WARNING BIOHAZARD Biological samples such as tissues body fluids biohazard 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 t
14. k E 5 El E beth 133 El bJ belles ps le e hro u 12 13 14 15 FE F3 Fd GI FC ES 22 ERI FECI FEES ELE fader mj pa m Mark Sample for Deletion 130 470 210 250 290 330 370 1200 800 400 AIZ WA EMI a L lele jJl sl sl J Bela eas fas kl E ll bl hejl JleI s e hejl ill p hs he hs e s eo l ka o Ja5 es o 12 2 14 2 16 2 10 2 32 fis 72 ladder m jam T Mark Sample for Deletion Figure 1 GeneMapper D X Software plot of the AmpF STR Identifiler Plus Kit Allelic Ladder 4 AmpFtSTR Identifiler Plus User Guide Product overview Control DNA Figure 2 shows amplification of Control DNA 9947A using the AmpF STR 9947A profile _ Identifiler Plus Kit iP pvsenzsc ig ss47A m m m m S B T Mark Sample for Deletion 130 170 210 250 290 330 370 IDP DYSen 28C ng 947A E g jm r B Q 7 Mark Sample for Deletion HEHE TORO Z I o WRZ LILO 130 170 210 250 290 330 370 IDP_DVSen 28C_1ng_9947A gj r jm r B r Mark Sample for Deletion 130 170 210 250 290 330 370 Figure 2 1 ng of Control DNA 9947A amplified with the AmpF STR Identifiler Plus Kit and analyzed on the Applied Biosystems 3130x Genetic Analyzer AmpFtSTR Identifiler Plus User Guide 5 Chapter 1 Overview Workflow overview Extract DNA PrepFiler Forensic DNA Extraction Kit Quantify DNA
15. 1 for the first time Import panels and bins into the Panel Manager as explained in Import panels and bins on page 39 Import an analysis method as explained in Import an HID analysis method on page 42 C D 2 lt ko zo 0 iw 2 e Eh E 0 Import a size standard as explained in Import an HID size standard on page 47 Define custom views of analysis tables Define custom views of plots For more info For details about GeneMapper ID Software workflow and features refer to e GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide PN 4338775 GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial PN 4335523 Installation Procedures and New Features for GeneMapper ID Software v3 2 User Bulletin PN 4352543 AmpFtSTR Identifiler Plus User Guide 38 Section 4 1 GeneMapper ID Software Import panels To import the AmpF STR Identifiler Plus Kit panel and bin set from the and bins Applied Biosystems web site into the GeneMapper ID Software v3 2 1 database 1 Download and open the file containing panels and bins a From the Support menu of www appliedbiosystems com select Software Downloads Patches amp Updates Select GeneMapper ID Software v3 2 from the drop down menu Select Updaters amp Patches and download the file Identifiler Plus Analysis Files GMID b Unzip the file 2 Start the Gene
16. 2 07 2 09 15 0 14 0 29 t t D18S51 7 t 9 0 14t t t 10 0 28 0 86 0 524 0 79 10 2 0 14t t 2 11 0 28 1 15 1 21 12 7 00 13 90 10 34 14 92 13 4 34 12 18 14 48 9 16 13 2 0 42 t 2 14 6 86 16 76 15 52 26 96 14 2 0 28 t t t 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 66 105 AmpFtSTR Identifiler Plus User Guide Population Data Table 10 AmpF STR Identifiler Plus Kit allele frequencies continued Allele ARS Gale siak n sj Ani n 357 n 349 n 191 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 42 0 14 0 52 0 26 24 t 0 14 0 17 t 25 t t 0 17 t 26 t t t t 27 t t t t D19S433 9 t 0 14 0 17 t 10 1 54 t t t 11 7 14 0 72 0 52 0 52 11 2 0 147 t 0 17 t 12 10 78 7 74 6 21 3 14 12 2 6 30 0 57 1 90 t 13 29 83 28 94 16 03 17 80 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 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 29 0 174 0 79 17 2 0 28 0 29 t 2 88 18 2 0 14 0 29 t 1 05 D21S11 24 t 24 2 0 14 0 43 0 17 t 24 3 1 i t 25 t t t 5 25 2 t 0 14 0 17 t 26 0 14 0 14 0 17 t 27 5 04 4 58 1 21 0 52 28 22 97 16 76 9 14 6 28 28 2 t AmpFt
17. 3 w Cerebus 3130XL IB_0331 Sample Identifiler Plus amp nalysisMethod v1 Identifiler Plus Panels v1 CE GS ldentifiler Plus GS500 amp BI3130 4 In Cerebus 3130XL IB 0332 Sample Identifiler Plus amp nalysisMethod v1 Identifiler Plus Panels v1 CE GS ldentifiler Plus GS500 amp BI3130 5 In Cerebus 3130XL IB 0333 Sample Identifiler Plus amp nalysisMethod v1 Identifiler Plus Panels v1 CE G5 ldentifiler Plus GS500 amp BI3130 B m Cerebus_3130XL IB_0334 Sample Identifiler_Plus_AnalysisMethod_v1 Identifiler Plus Panels v1 CE G5 ldentifiler Plus GS500 amp BI3130 7 ie Cerebus_3130XL IB_0335 Sample Idertifiler_Plus_AnalysisMethod_v1 Identifiler Plus Panels v1 CE G5 ldentifiler Plus GS500 amp BI3130 8 i Cerebus_3130XL IB_0336 Sample Identifiler Plus amp nalysisMethod v1 Identifiler Plus Panels v1 CE GS ldentifiler Plus GS500 amp BI3130 9 iy Cerebus 3130XL IB 0337 Sample Identifiler Plus amp nalysisMethod v1 Identifiler Plus Panels v1 CE GS Identifiler Plus GS500 amp BI3130 10 L3 Cerebus 3130XL IB 0338 Sample Identifiler Plus amp nalysisMethod v1 Identifiler Plus Panels v1 CE G5 ldentifiler Plas GS500 amp BI3130 11 w Cerebus_3130XL IB_0339 Sample Identifiler Plus amp nalysisMethod v1 Identifiler Plus Panels v1 CE GS Identifiler Plus GS500 amp BI3130 12 L3 Cerebus 3130XL IB 0340 Sample Identifiler Plus amp nalysisMethod v1 Identifiler Plus Panels v1 CE G3 Identifiler Plus GS500 amp BI3130 13 i Cere
18. 315 355 395 29 cycles 75 115 155 195 235 275 315 355 395 j 30 cycles Figure 17 Representative AmpF STR Identifiler Plus Kit profiles obtained from amplification of 1 0 ng DNA template using 26 27 28 29 and 30 cycles analyzed on the Applied Biosystems 3130x Genetic Analyzer Y axis scale 0 to 4 000 RFUs AmpFtSTR Identifiler Plus User Guide 72 Chapter 5 Experiments and Results Accuracy precision and reproducibility SWGDAM The extent to which a given set of measurements of the same sample agree with guideline 2 9 their mean and the extent to which these measurements match the actual values being measured should be determined SWGDAM July 2003 Accuracy Laser induced fluorescence detection of length polymorphism at short tandem repeat loci is not a novel methodology Holt et al 2000 and Wallin et al 2002 However accuracy and reproducibility of AmpF STR Identifiler Plus profiles have been determined from various sample types Figure 18 shows the size differences that are typically observed between sample alleles and allelic ladder alleles on the Applied Biosystems 3130x Genetic Analyzer with POP 4 polymer The x axis in Figure 18 represents the nominal nucleotide sizes for the AmpF STR Identifiler Plus Allelic Ladder The dashed lines parallel to the x axis represent the 0 25 nt windows The y axis represents the deviation of each sample a
19. Biosystems 3130x Genetic Analyzer 79 Allele Mean Standard Deviation 13 159 55 159 61 0 027 0 051 14 163 63 163 7 0 032 0 038 15 167 68 167 74 0 026 0 051 16 171 7 171 76 0 035 0 04 D7S820 6 255 08 255 19 0 029 0 058 7 259 13 259 22 0 04 0 056 8 263 16 263 25 0 037 0 053 9 267 19 267 29 0 046 0 053 10 271 25 271 34 0 039 0 051 11 275 28 275 4 0 037 0 06 12 279 34 279 45 0 034 0 05 13 283 38 283 49 0 039 0 049 14 287 44 287 54 0 039 0 051 15 291 51 291 62 0 043 0 052 D8S1179 8 122 49 122 61 0 03 0 044 9 126 56 126 68 0 037 0 045 10 130 66 130 76 0 026 0 044 11 134 8 134 89 0 031 0 041 12 138 98 139 09 0 019 0 043 13 143 58 143 68 0 028 0 042 14 148 03 148 14 0 03 0 046 15 152 43 152 54 0 025 0 043 16 156 73 156 83 0 026 0 039 17 160 93 161 04 0 031 0 042 18 165 03 165 12 0 024 0 046 19 169 1 169 2 0 035 0 044 FGA 17 214 11 214 23 0 041 0 05 18 218 14 218 26 0 043 0 052 19 222 17 222 3 0 039 0 054 20 226 21 226 35 0 044 0 057 21 230 26 230 38 0 045 0 055 22 234 29 234 42 0 05 0 058 23 238 33 238 47 0 038 0 057 AmpFtSTR Identifiler Plus User Guide Accuracy precision and reproducibility Table 4 Precision results of five runs 16 capillaries run of the AmpFfSTR Identifiler Plus Allelic Ladder continued Applied Biosystems 3130x Genetic Analyzer
20. D21S11 locus Hum Mol Genet 1 67 AmpFtSTR Identifiler Plus User Guide 130 Bibliography 131 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 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 nt 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 Szibor R Lautsch S Plate I Bender K and Krause D 1998 Population genetic data of the STR HumD3S1358 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 PS 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 Wal
21. EX 0 a Select the AmpFLSTR JIdentifiler Plus v1 folder in the navigation pane File Edit Bins View zim m m Comment ur X m m IBi BI en set Panel Name 1 fdertifiler_Plus_v1 nut i amp iPanel Manager AmpFLSTR_Panels_v2_ LL ArmpFLSTR_Identifiler_Plus_ 1 b Select File Import Bin Set to open the Import Bin Set dialog box c Navigate to then open the Identifiler Plus Analysis Files GMID folder d Select Identifiler_Plus_Bins_v1 then click Import Note Importing this file associates the bin set with the panels in the Identifiler Plus Panels v1 folder Look in e Identifiler Plus Analysis Files GMID m i2 Im Fe CE_G5_ldertifiler_Plus_GS500 xml Idertifiler_Plus_AnalysisMethod_v1 xml My Recent D B der tifiler _Plus_Bins_v1 txt E Identifiler_Plus_Panels_v1 txt Desktop AI My Documents File name identifier Plus_Bins_v1 txt Import JE Files of type a Files v Cancel AmpFtSTR Identifiler Plus User Guide 40 Section 4 1 GeneMapper ID Software 7 View the imported panels in the navigation pane a Double click the AmpFLSTR_Identifiler_Plus_v1 folder to view the Panel Manager Identifiler Plus Panels v1 folder b Double click the Identifiler Plus Panels v1 folder to display the panel information in the right pane File Edit Bins View uf x m M mmm l E CIT IL ICI uj gr Manager H 7
22. Gx wmmi m Highlight this fF b Select File gt Import Panels to open the Import Panels dialog box c Navigate to then open the Identifiler Plus Analysis Files GMIDX folder that you unzipped in step 1 on page 52 5 Select Identifiler_Plus_Panels_v1X then click Import Note Importing this file creates a new folder in the navigation pane of the Panel Manager AmpFLSTR_Identifiler_Plus_v1X This folder contains the panel and associated markers z Import Panels X Look in je Identifiler Plus Analysis Files GMIDX Y i2 m HEES CE G5 Identifiler Plus GS500 xml Identifier Plus Bins vix txt B Identifier Plus Panels v1X txt Identifier Plus Stutter vix txt Identifiler Plus AnalysisMethod v1x xml My Recent Documents Desktop Cem Filename Identifier Plus Panels vix bxt Import Files of type fan Files Cancel 6 Import Identifiler Plus Bins v1X a Select the AmpFLSTR JIdentifiler Plus v1X folder in the navigation pane g Panel Manager File Edit Bins View Help u x W m m enset az Panel Manager AmpFLSTR_Panels_v1x dentifiler_Plus_Panels_v1X null RJ fa AmpFLSTR _Identifiler _Plus_v1X b Select File Import Bin Set to open the Import Bin Set dialog box c Navigate to then open the AmpFLSTR_Identifiler_Plus_v1X folder 53 AmpFtSTR Identifiler Plus User Guide Set up GeneMapper ID X Software for dat
23. Native Americans D21811 p 0 0118 D5S818 p 0 0205 These are no more than would be expected by chance No more alleles were observed to be in linkage disequilibrium than would be expected by chance alone The average observed heterozygosity across the 15 STR loci was 0 804 in the African American population 0 792 in the U S Caucasian sample population 0 793 in the Hispanic sample population and 0 757 in the Native Americans The most heterozygous locus was FGA mean observed heterozygosity across all populations of 0 875 and the least heterozygous STR locus was TPOX mean observed heterozygosity across all populations of 0 677 Table 11 Heterozygosity and p values for Hardy Weinberg tests of the 15 Identifiler Plus Kit STR loci in four U S populations felisi Aye n r b paw n 357 n 349 n 191 CSF1PO HW X p 0 13649 0 926431 0 951476 0 839278 HW G p 0 08902 0 894972 0 918038 0 728023 HW Exact p 0 0762 0 2688 0 5456 0 6148 HExp 0 7829 0 7267 0 7051 0 7398 Ho 0 7703 0 7421 0 7138 0 7958 AmpFtSTR Identifiler Plus User Guide 110 Chapter 5 Experiments and Results 111 Table 11 Heterozygosity and p values for Hardy Weinberg tests of the 15 Identifiler Plus Kit STR loci in four U S populations continued A nerk di GE as io Ari aj aa n 357 n 349 n 191 D2S1338 HW
24. Plus Bins v1X folder 55 AmpFtSTR Identifiler Plus User Guide Set up GeneMapper ID X Software for data analysis g Import Marker Stutter xl Look ir je Identifiler Plus Analysis Files GMIDX bud D RE My Recent Documents CE_G5_Identifiler_Plus_G5500 xml Identifier Plus Bins vix txt Identifier Plus Panels v1x txt B Identifier Plus Stutter v1x txt Identifiler Plus AnalysisMethod v1x xml Desktop My sore Filename Identifier _Plus_Stutter_v1x txt Import JE Files of type fan Files Cancel 10 View the imported marker stutters in the navigation pane a Select the Identifiler Plus Panel v1X folder to display its list of markers in the right pane b Double click the Identifiler Plus Panel v1X folder to display its list of markers below it c Double click D5S818 to display the Stutter Ratio amp Distance view for the marker in the right pane g Panel Manager File Edit Bins Yiew Help aP x lf i og sos Identifiler_Plus_Bins_v1X z Ii amp amp B u u i m Ho p inii T Please enter the stutter filter s for D5S818 marker here If left blank the global stutter filter will be applied E E AmpFLSTR Identifiler Plus vix Cj Identifier Plus Panels v1x Minus Stutter Plus Stutter D251338 Ratio From Distance To Distance Ratio From Distanc
25. See kod A 132 Related documentation lt s awa au xl k Db ay ee hs 132 Send us your comments x reei Xa ale Ku al EWAN eee In 133 Bel gt C NET EEEE IEEE 134 AmpFtSTR Identifiler Plus User Guide V Contents vi AmpFtSTR Identifiler Plus User Guide Preface Safety information Note For general safety information see this Preface and Appendix B Safety on page 122 When a hazard symbol and hazard type appear by a chemical name or instrument hazard see the Safety Appendix for the complete alert on the chemical or instrument Safety alert Four safety alert words appear in Applied Biosystems user documentation at points words inthe document where you need to be aware of relevant hazards Each alert word IMPORTANT CAUTION WARNING DANGER implies a particular level of observation or action as defined below IMPORTANT Indicates information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical CAUTION Indicates a potentially hazardous situation that 1f not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices WARNING Indicates a potentially hazardous situation that 1f not avoided could result in death or serious injury DANGER Indicates an imminently hazardous situation that 1f not avoided will result in death or serious injury This signal word is t
26. 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 Peak heights were measured for samples n 500 amplified using the 28 cycle protocol DNA input 1ng at the loci used in the Identifiler Plus Kit All data were generated on the Applied Biosystems 3130xl Genetic Analyzer Some conclusions from these measurements and observations are For each AmpF STR Identifiler Plus Kit locus the percent stutter generally increases with allele length as shown in Figure 19 to Figure 22 on pages 83 through 85 Smaller alleles display a lower level of stutter relative to the longer alleles within each locus Each allele within a locus displays a percent stutter that is consistent with other alleles in the locus The stutter value for each locus shown in Table 5 on page 85 was determined by taking the mean plus three times the standard deviation These values are the stutter filter percentages in the Identifiler Plus stutter file they will be used during the filtering step in the GeneMapper ID Software v3 2 1 or GeneMapper ID X Software v1 0 1 v1 1 or v1 1 1 Peaks in the stutter position that are above the stutter filter percentage 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 Figure 30 on page 99 The p
27. View All Runs Two user attention words appear in Applied Biosystems user documentation Each word implies a particular level of observation or action as described below Note Provides information that may be of interest or help but is not critical to the use of the product IMPORTANT Provides information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical AmpFtSTR Identifiler Plus User Guide How to obtain support How to obtain support For the latest services and support information for all locations go to www appliedbiosystems com At the Applied Biosystems web site you can Access worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities Search through frequently asked questions FAQs Submit a question directly to Technical Support Order Applied Biosystems user documents MSDSs certificates of analysis and other related documents Download PDF documents Obtain information about customer training Download software updates and patches AmpFtSTR Identifiler Plus User Guide ix Preface x AmpFtSTR Identifiler Plus User Guide Overview This chapter covers Bl Prod ct overview i 224 eg Se ebe e diate Me RT A aa EE M Workflow overview i gwa ob WE kK KK KK KK KK KK KK KK KK KK k kk k i E Instrument and software overview nsns nonoa KK KK RR RR RR RR KK KK n M
28. X2 p 0 409878 0 537758 0 975972 0 722543 HW G p 0 962501 0 407932 0 973054 0 760953 HWExactp 0 7838 0 3488 0 9794 0 5825 HExp 0 8936 0 8823 0 8529 0 8428 Ho 0 8768 0 8653 0 8379 0 801 D3S1358 HW X2 p 0 947371 0 670787 0 681659 0 087223 HW G p 0 907905 0 654776 0 852278 0 175807 HWExactp 0 2967 0 2814 0 4684 0 0614 HExp 0 7681 0 7986 0 7361 0 7028 Ho 0 7955 0 8166 0 7414 0 7382 D5S818 HW X2 p 0 993751 0 859805 0 944725 0 073002 HW G2 p 0 989776 0 520417 0 979044 0 08025 HW Exact p 0 958 0 462 0 4662 0 0205 HExp 0 7476 0 6931 0 7351 0 7378 Ho 0 7479 0 7077 0 7586 0 6806 D7S820 HW X2 p 0 987668 0 571989 0 336834 0 324754 HW G2 p 0 969887 0 44694 0 687948 0 289733 HW Exact p 0 9818 0 2286 0 4028 0 1276 HExp 0 7758 0 8117 0 7822 0 7858 Ho 0 7955 0 7908 0 7862 0 7487 D8S1179 HW X2 p 0 067164 0 545414 0 047783 0 446248 HW G2 p 0 568837 0 275218 0 302937 0 760077 HW Exactp 0 2176 0 3264 0 0304 0 1656 HExp 0 7925 0 8047 0 7853 0 7403 Ho 0 7899 0 8424 0 8 0 6806 D13S317 HW X2 p 0 014379 0 711127 0 353995 0 813948 HW G p 0 609389 0 871173 0 190736 0 814681 HW Exactp 0 3818 0 667 0 2415 0 6851 HExp 0 6977 0 7797 0 8251 0 8222 Ho 0 6695 0 7364 0 8207 0 8168 AmpFtSTR Identifiler Plus User Guide Population Data Table 11 Heterozygosity and p values for Hardy Weinberg tests of the 15 Identifiler Plus Kit STR loci in fo
29. able to produce reliable typing results should be determined SWGDAM July 2003 The optimal amount of input DNA added to the AmpF STR Identifiler Plus PCR Amplification Kit should be between 0 75 and 1 0 ng for 28 cycle amplification The DNA sample should be quantitated before amplification using a system such as the Quantifiler Human DNA Quantification Kit PN 4343895 The final DNA concentration should be 0 075 to 0 1 ng uL so that 0 75 to 1 0 ng of DNA is added to the PCR reaction in a volume of 10 uL If the sample contains degraded or inhibited DNA amplification of additional DNA may be beneficial In Figures 25 and 26 the control DNA 9947A was serially diluted from 1 ng to 0 031 ng With the 28 PCR cycle protocol full profiles 26 alleles were consistently obtained at 0 125 ng but occasional partial profiles that are missing anywhere from 1 to 3 alleles were observed at 0 062 ng With the 29 PCR cycle protocol full profiles 26 alleles were consistently obtained at 0 062 ng but occasional partial profiles that are missing anywhere from 1 to 5 alleles were observed at 0 031 ng Iftoo much DNA is added to the PCR reaction the increased amount of PCR product that is generated can result in Fluorescence intensity that exceeds the linear dynamic range for detection by the instrument off scale data Off scale data is a problem because Quantitation peak height and area for off scale peaks is not accurate For ex
30. allelic ladder per every set of 16 samples IMPORTANT Variation in laboratory temperature can affect fragment migration speed and result in sizing variation Applied Biosystems recommends the following frequency of allelic ladder injections which should account for normal variation in run speed However during internal validation studies verify the required allelic ladder injection frequency to ensure accurate genotyping of all samples in your laboratory environment Applied Biosystems 3130x or ABI PRISM 3100 systems One ladder per injection one injection 16 samples 15 samples 1 allelic ladder Applied Biosystems 3130 or ABI PRISM 3100 Avant One ladder for every 4 injections one injection 4 samples When genotyping it is critical to use an allelic ladder run under the same conditions as the samples because 27 Size values obtained for the same sample can differ between instrument platforms because of different polymer matrices and electrophoretic conditions Variation in laboratory temperature can affect migration speed see IMPORTANT above AmpFtSTR Identifiler Plus User Guide Section 3 1 3100 3100 Avant and 3130 3130xl instruments Section 3 1 3100 3100 Avant and 3130 3130x instruments Set up the 3100 3100 Avant or 3130 3130x instrument for electrophoresis Reagents and parts 3100 3100 Avant Table 3 on page 10 lists the required materials not supplied with the AmpF STR Ide
31. also provide MSDSs with the first shipment of a hazardous chemical to a customer after an MSDS has been updated MSDSs provide the safety information you need to store handle transport and dispose of the chemicals safely Each time you receive a new MSDS packaged with a hazardous chemical be sure to replace the appropriate MSDS in your files Obtaining The MSDS for any chemical supplied by Applied Biosystems is available to you free MSDSs 24 hours a day To obtain MSDSs 1 Goto www appliedbiosystems com click Support then select MSDS 2 In the Keyword Search field enter the chemical name product name MSDS part number or other information that appears in the MSDS of interest Select the language of your choice then click Search 3 Find the document of interest right click the document title then select any of the following Open To view the document Print Target To print the document Save Target As To download a PDF version of the document to a destination that you choose Note For the MSDSs of chemicals not distributed by Applied Biosystems contact the chemical manufacturer AmpFtSTR Identifiler Plus User Guide 124 Appendix B Safety Chemical waste safety Chemical waste WARNING HAZARDOUS WASTE Refer to Material Safety Data Sheets hazards and local regulations for handling and disposal WARNING CHEMICAL WASTE HAZARD Wastes produced by Applied Biosystems instruments are poten
32. be gk Panel Manager n E AmpFLSTR_Panels_v1X AmpFLSTR_Identifiler_Plus_v1X w 7 8 9 10 11 12 13 14 15 16 1 CjIdentifiler Plus Panels vix ER 03 ja 0 8 CSF1PO D351358 THO1 0 7 D135317 D165539 D251338 us D195433 vWA 0 5 TPOX D18551 AMEL 0 4 D55818 FGA 03 02 ss Reference Samples 04 0 0 113145117 419121423125 127 129131 133135 137 139141 143145 147149 151 153155 157159161 163165 157159 171173175 177179 181183185 187 D851178 4 gt Marker D851173 118 00 183 50 _ cmm ay nen 9 Import Identifiler_Plus_Stutter_v1X a Select the AmpFLSTR_Identifiler_Plus_v1X folder in the navigation panel g5 Panel Manager File Edit Bins View Help i X u n gw Bin Set tdertfiler Plus Bins vix dj sB Panel Manager Panel Name Comment AmpFLSTR_Panels_v1X Identifiler_Plus_Panels_v1x null GI f AmpFLSTR Identifiler _Plus_ b Select File Import Marker Stutter to open the Import Marker Stutter dialog box c Navigate to then open the Identifiler Plus Analysis Files GMIDX folder d Select Identifiler Plus Stutter v1X then click Import Note Importing this file associates the marker stutter ratio with the bin set in the Identifiler
33. causes 81 F fluorescent dyes 7 FTA cards amplification 22 bloodstained 22 G GeneMapper ID Software data analysis 38 overview 7 37 GeneMapper ID X Software data analysis 52 overview 7 GeneScan size standard about 9 dye label 7 volume per reaction 29 31 guidelines chemical safety 123 chemical waste disposal 125 chemical waste safety 125 H hazards See safety hematin 94 hematin effects of 94 Hi Di formamide volume per reaction 29 31 humic acid effects of 95 IMPORTANT description vii 135 Information Development department contacting 133 inheritance 88 instrumentation 310 genetic analyzer 7 27 30 3100 3100 Avant genetic analyzer 7 27 28 3130 3130x genetic analyzer 7 27 28 software compatibility 7 italic text when to use viii K kit allelic ladder 9 amplification 2 contents 9 control DNA 9 description 2 fluorescent dyes 7 loci amplification 3 master mix 9 primers 2 9 18 purpose 2 reagents 9 supported instruments 2 kit performance comparisons DNase I 93 hematin 94 humic acid 95 96 L LIZ size standard about 9 volume per reaction 29 31 loci characterization 88 chromosomal location 3 dye label 3 genotype frequency in population 109 mapping 89 low TE buffer 17 M magnesium chloride concentration validation of 70 master mix volume per reaction 19 materials and equipment included in kit 9 not included in kit 10 menu commands conventions for describing viii mi
34. details about these kits Product Description References Quantifiler Human DNA Quantification Kit PN 4343895 and Quantifiler Y Human Male DNA Quantification Kit PN 4343906 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 specific 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 DNA and one TaqMan MGB probe labeled with VIC dye for detecting the amplified IPC DNA Quantifiler Human DNA Quantification Kits User s Manual PN 4344790 Quantifiler Duo DNA Quantification Kit PN 4387746 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 deg
35. 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 USA 94 1041 1046 AmpFtSTR Identifiler Plus User Guide 128 Bibliography 129 Clark J M 1988 Novel non templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases Nucleic Acids Res 16 9677 9686 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 A Hammond H A Lin J Caskey C T and Chakraborty R 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 a
36. of the Control DNA 99474 for 28 cycles is shown in Figure 15 Applied Biosystems observed that the performance of the multiplex is robust within a 30 window of the optimum magnesium chloride concentration E E as as ass 96 Change am 3096 x 209 T 2096 i 1096 i gt 0 E 1096 E 2096 i so 3096 Figure 15 1 0 ng of Control DNA 9947A amplified with the AmpF STR Identifiler Plus Kit for 28 cycles in the presence of varying concentrations of magnesium chloride and analyzed on the Applied Biosystems 3130x Genetic Analyzer AmpFtSTR Identifiler Plus User Guide 70 Chapter 5 Experiments and Results Thermal cycler parameters Thermal cycling parameters were established for amplification of the AmpF STR Identifiler Plus PCR Amplification Kit Thermal cycling times and temperatures of GeneAmp PCR systems were verified Varying annealing extension and denaturation temperature windows were tested to verify that a specific PCR product with the desired sensitivity of at least 1 0 ng of AmpF STR Control DNA 9947A was produced For example annealing extension temperatures were tested at 55 57 59 61 and 63 C for 3 minute hold times in the Silver 96 Well GeneAmp PCR System 9700 Figure 16 The PCR products were analyzed using the Applied Biosystems 3130x Genetic Analyzer Of the tested annealing extension temperatures 55 to 61 C produced robust p
37. run of the AmpF STR Identifiler Plus Allelic Ladder continued Applied Biosystems 3130x Genetic Analyzer Allele Mean Standard Deviation D19S433 9 101 25 101 34 0 022 0 034 10 105 16 105 25 0 028 0 037 11 109 09 109 17 0 021 0 033 12 113 04 113 12 0 024 0 036 12 2 115 06 115 13 0 027 0 035 13 117 02 117 09 0 026 0 036 13 2 119 03 119 1 0 027 0 038 14 121 02 121 07 0 025 0 038 14 2 123 05 123 1 0 028 0 037 15 125 03 125 09 0 03 0 041 15 2 127 08 127 13 0 027 0 04 16 129 08 129 13 0 031 0 039 16 2 131 13 131 19 0 023 0 042 17 133 16 133 21 0 034 0 046 17 2 135 23 135 28 0 034 0 041 D21S11 24 184 41 184 46 0 024 0 042 24 2 186 39 186 45 0 025 0 04 25 188 35 188 4 0 024 0 038 26 192 27 192 34 0 027 0 035 27 196 21 196 28 0 024 0 039 28 200 06 200 13 0 026 0 039 28 2 202 03 202 1 0 026 0 036 29 204 02 204 09 0 025 0 044 29 2 206 08 206 14 0 027 0 041 30 208 06 208 11 0 028 0 04 30 2 210 03 210 09 0 031 0 037 31 212 04 212 12 0 031 0 037 31 2 214 03 214 1 0 023 0 04 32 216 04 216 11 0 028 0 042 32 2 218 03 218 09 0 023 0 0351 33 220 05 220 1 0 031 0 043 33 2 221 98 222 05 0 033 0 038 34 224 12 224 18 0 024 0 033 34 2 226 03 226 09 0 029 0 041 77 AmpFtSTR Identifiler Plus User Guide Table 4 Precision results of five runs 16 capillaries run of the Accuracy precision and reproducibility AmpFfST
38. the African American database 0 7 for the U S Caucasian database 0 996 for the U S Hispanic database and 1 396 for the Native American database is suggested by the National Research Council in forensic calculations Low frequency alleles Some alleles of the AmpF STR Identifiler Plus Kit loci occur at a low frequency For these alleles a minimum frequency 5 divided by 2n where n equals the number of individuals in the database was assigned for the AmpF STR Identifiler Plus Kit African American Native American U S Caucasian and U S Hispanic databases as suggested in the 1996 report of the Committee on DNA Forensic Science National Research Council 1996 These databases are summarized in Table 13 on page 116 The minimum reportable genotype frequency at each locus is 1 19 X 104 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 0 where 0 0 01 AmpFtSTR Identifiler Plus User Guide Population Data Evaluation of Estimates of expected heterozygosity HExp were computed as described by Nei Hardy Weinberg M 1973 using the program PopGene 1 32 Possible divergence from equilibrium Hardy Weinberg expectations HWE was tested using various methods Bycalculating the unbiased estimate of the expected homozygote heterozygote frequencies Nei M 1978 Using chi square HW X p an
39. 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 Plus User Guide Set up GeneMapper ID Software for data analysis If you are using GeneMapper w Software to perform Human Identification HID analysis with AmpF STR kits go to Set up GeneMapper ID X Software for data analysis on page 52 or refer to the GeneMapper ID X Software Version 1 0 Human Identification Analysis Getting Started Guide PN 4375574 Set up GeneMapper D Software for data analysis Workflow Before you can analyze sample fsa files using GeneMapper ZD Software v3 2
40. use of the kit After first use reagents are stored at 2 to 8 C and therefore they do not require subsequent thawing Do not refreeze the reagents 3 Pipette the required volumes of components into an appropriately sized polypropylene tube 4 Vortex the reaction 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 DNA sample To prepare Negative control Add 10 uL 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 reaction mix is 25 uL 7 Seal the MicroAmp Optical 96 Well Reaction Plate with MicroAmp Clear Adhesive Film or MicroAmp Optical Adhesive Film or cap the tubes 8 Vortex the reaction mix for 3 seconds then centrifuge the tubes at 3000 rpm for about 20 seconds in a tabletop centrifuge with plate holders 1f using 96 well plates to remove bubbles AmpFtSTR Identifiler Plus User Guide 19 Chapter 2 PCR Amplification 9 Amplify the samples in a GeneAmp PCR System 9700 with the silver 96 well block a GeneAmp PCR System 9700 with the gol
41. 0 3430xX INStTFUME E s coe GRES PR n FE a wayan ov eee ate Sie athe eed a8 29 Section 3 2 310instrument naaaaaaa na kk KK KK KK RR R KK KK KK KK KK kk 30 Set up the 310 instrument for electrophoresis kk kk kK KK RR KK KK KK RR KK eee 30 Prepare samples for electrophoresis on the 310 instrument 31 AmpFtSTR Identifiler Plus User Guide iii Contents Chapter 4 Chapter 5 Appendix A Appendix B Bala ANAVSIS sica Shara n EN MEETS eae r ka D RES 36 Section 4 1 GeneMapper ID Software JX kk cece eee eee eee 37 Overview of GeneMapper ID Software kk kK KK KK KK KK KK KK KK KK KK KK KK 37 Set up GeneMapper ID Software for data analysis aaaaaaaaaaa111 38 Analyze and edit sample files with GeneMapper ID Software 48 Examine and edita project kk kk kk kk kk kK kK kK KK aaa aaa kk kK kK kk kk kk lk kk kk 49 Section 4 2 GeneMapper D X Software JW KEKE KRE KRE KK 51 Overview of GeneMapper D X Software WW kk kK KK KK KK KK eee eens 51 Set up GeneMapper D X Software for data analysis 44411411111 52 Analyze and edit sample files with GeneMapper D X Software 64 Examine and edit a project kk kk kk kk kk KK kK kK aaa ee 65 Experiments and Results XWA KEK KK KK KN 68 OVerVIGW A i ya aya tuyo DONA a aise A Ce tuere UM od t Rer a Rr de A zer ve aka 69 Developmental validation kk kk kk kK kk KK KK KK KK KK KK KK KK KK KK KK KK
42. 0 st H 50 M 4 0 Percent Stutter eee oe ome ve we e occo os oe on ace o o 0 o e oe 3 0 N o oe ee dawano oo RR RR i e HK 9 10111213 14 15 16 17 18 11121314 15 16 17 18 19 20 212223 678 0 1112 10 1112 13 14 15 16 17 18 1920 21222324 2526 D19S433 VWA TPOX D18S51 Figure 21 Stutter percentages for the D19S443 vWA TPOX and D18S51 loci AmpFtSTR Identifiler Plus User Guide 84 Chapter 5 Experiments and Results Percent Si o b hezim emp cem m o be m o o e A so cema o om e mp c coe oo w o em w te 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 3132333435 42 43 44 45 46 47 48 49 50 51 52 53 D5S818 FGA Figure 22 Stutter percentages for the D5S818 and FGA loci Table 5 Marker specific stutter filter percentages for AmpF STR Identifiler Plus PCR Amplification Kit loci Locus Stutter CSF1PO 9 2041 D13S317 9 9348 D16S539 10 3945 D18S51 13 6799 D19S433 11 2096 D21S11 10 6714 D2S1338 12 4409 D3S1358 12 2719 D5S818 10 0599 D7S820 9 6926 D8S1179 10 3155 FGA 13 028 THO1 4 0813 85 AmpFtSTR Identifiler Plus User Guide Extra peaks in the electropherogram Table 5 Marker specific stutter filter perce
43. 0 to a25 Genotype Quality From zs ta 1 0 From 0 0 to azs Factory Defaults Figure 9 Analysis Method Editor HID Quality Flags tab settings The size standard for the Identifiler Plus Kit uses the following GS500 peaks in its sizing algorithm 75 100 139 150 160 200 300 350 400 and 450 Use the following procedure to import the size standard for the AmpF STR Identifiler Plus PCR Amplification Kit from the folder that you downloaded from the Applied Biosystems web site into the GeneMapper ID Software database Refer to step 1 on page 39 for downloading instructions 1 Select Tools GeneMapper Manager to open the GeneMapper Manager 2 Import a size standard a Select the Size Standards tab then click Import G GeneMapper Manager xl Projects Analysis Methods Table Settings Plot Settings matrice Name Last Saved Owner Type Description 377_F_HD_GS500 2004 05 28 11 34 3 amid Basic Advanced CE G5 HID GS500 2004 05 28 11 34 3 amid Basiciadvanced CE F HID GS500 2004 05 28 11 34 3 amid Basic Advanced Factory Provided Factory Provided Factory Provided Import Export Delete b Navigate to then open the Identifiler Plus Analysis Files GMID folder AmpFtSTR Identifiler Plus User Guide Analyze and edit sample files with GeneMapper ID Software c Select CE G5
44. 1 11 27 45 695 gmidx HID 4 Identifiler Plus AnalysisMethod v1X 2009 09 02 11 13 05 191 gmidx HID a New Open Save 4s Import Export Figure 10 Analysis Method Editor General tab settings Figures 11 through 14 below show the settings for each tab of the Analysis Method Editor Q D el D lt 0 o go 0 8 o Xx 2 e EB lt B 0 AmpFtSTR Identifiler Plus User Guide 58 Section 4 2 GeneMapper ID X Software Analysis Method Editor X General Allele Peak Detector Peak Quality 5Q amp GQ Settings Bin Set Identifiler Plus Bins v1X v Use marker specific stutter ratio and distance if available Marker Repeat Type Tri Tetra Penta Global Cut off value 0 0 MinusA Ratio MinusA Distance From To Global Minus Stutter Ratio Global Minus Stutter Distance From To Global Plus Stutter Ratio Global Plus Stutter Distance From PPEPPPPEISE MINE N Hd li TEEEREEER lt PEEEEFEL To Amelogenin Cutoff f o Range Filter Factory Defaults Save Car cel Help Figure 11 Analysis Method Editor Allele tab settings e GeneMapper D X Software 1 0 1 1 1 or 1 1 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 The Use marker specific stutter ratio if available check box is selected by
45. 1 14 10 11 D21S11 29 35 31 2 32 2 AmpFtSTR Identifiler Plus User Guide 100 Chapter 5 Experiments and Results Table 9 Genotypes of mixed DNA samples continued Locus Profile Sample A Profile Sample B D7S820 8 10 11 CSF1PO 8 10 12 D3S1358 14 17 15 16 THO1 7 8 6 9 3 D138317 12 13 9 11 D16S539 10 11 9 12 D281338 17 23 17 20 D19S433 11 17 2 13 VWA 14 17 17 19 TPOX 9 10 8 11 D18S51 15 16 13 14 AMEL X Y X D5S818 11 13 8 10 FGA 19 25 22 23 101 AmpFtSTR Identifiler Plus User Guide Population Data Population Data SWGDAM The distribution of genetic markers in populations should be determined in relevant guideline 2 7 population groups SWGDAM July 2003 Overview To interpret the significance of a match between genetically typed samples you must 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 a suspects reference sample then the suspect is excluded as the donor of the biological evidence that was tested An exclusion is independent of the frequency of the two genotypes in the population If the suspect and evidence samples have the same genotype then the suspect is included as a possible source of the evidence sample The probability that another unrelated individual would also match the evidence sample is estimated by t
46. 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 14t 0 69 0 26 15 i 0 29 0 18 i 16 i i 0 172 17 0 144 i 0 172 D7S820 6 t 0 14 0 172 7 0 421 1 29 1 72 0 522 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 34 15 t t t t D8S1179 8 0 421 2 29 0 34 0 52 9 0 421 1 15 0 34 0 26 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 42 104 AmpFtSTR Identifiler Plus User Guide Chapter 5 Experiments and Results Table 10 AmpF STR Identifiler Plus Kit allele frequencies continued Allele kin r G le cd T5 oar Amedeo n 357 n 349 n 191 16 4 62 2 72 2 41 1 57 17 1 12 0 29 0 52 0 522 18 0 28 t t t 19 t D138317 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 14 0 14 t 0 26 D16S539 5 8 3 22 1 72 1 72 0 79 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
47. 539 D18S51 D19S433 D21S11 FGA THO1 TPOX and vWA have been mapped and the chromosomal locations have been published Nakahori et al 1991 Edwards et al 1992 Kimpton et al 1992 Kong et al 2004 Mills et al 1992 Sharma and Litt 1992 Li et al 1993 Straub et al 1993 Barber and Parkin 1996 Species specificity 89 SWGDAM Guideline 2 2 For techniques designed to type human DNA the potential to detect DNA from forensically relevant nonhuman species should be evaluated SWGDAM July 2003 The AmpF STR Identifiler Plus PCR Amplification Kit provides the required specificity for detecting human alleles Nonhuman studies Nonhuman DNA may be present in forensic casework samples The data from AmpF STR Identifiler Plus PCR Amplification Kit experiments on nonhuman DNA sources are shown in Figure 24 on page 90 Figure 24 shows amplification for Control DNA 9947A 1 0 ng panel 1 chimpanzee 1 0 ng panel 2 dog 10 ng panel 3 cat 10 ng panel 4 horse 10 ng panel 5 microbial DNA pool equivalent to 105 copies of Candida albicans Staphylococcus aureus Neisseria gonorrhoeae E coli 0157 H7 Bacillus subtilis and Lactobacillus rhamnosus panel 6 and the negative control panel 7 The extracted DNA samples were amplified with the AmpF STR Identifiler Plus PCR Amplification Kit and analyzed using the Applied Biosystems 3130x Genetic Analyzer Primates gorilla chimpanzee orangutan and
48. 75 180 185 190 195 No Extension A Final Extension Figure 23 Omitting the final extension step results in split peaks due to incomplete A nucleotide addition Data are from an ABI PRISM 310 Genetic Analyzer using another AmpF STR kit Lack of complete A nucleotide addition may be observed in AmpF STR Identifiler Plus PCR Amplification Kit results when the amount of input DNA is greater than the recommended protocols because more time is needed for the enzyme to add the A nucleotide to all molecules as more PCR product is generated Amplification of too much input DNA may also result in off scale data Artifacts and anomalies are seen in all molecular biological systems Artifacts are typically reproducible while anomalies are non reproducible intermittent occurrences that are not observed consistently in a system for example spikes and baseline noise Reproducible artifacts have not been seen in data produced with either 28 or 29 cycles of amplification on the genetic analyzers used during developmental validation of the Identifiler Plus Kit However you should consider possible noise and artifacts when interpreting data from the AmpF STR Identifiler Plus PCR Amplification Kit on the Applied Biosystems 3130 3130x ABI PRISM 3100 3100 Avant and ABI PRISM 310 Genetic Analyzers AmpFtSTR Identifiler Plus User Guide Characterization of loci Characterization of loci SWGDAM The basic ch
49. 820 Blue 2510 2985 10 11 4 none 6 7 8 9 10 11 12 13 14 amp D21511 4 CSFIPO Blue 302 12 348 63 10 12 4 none 6 7 8 9 10 11 12 13 14 D75820 5 D351358 Green 98 0 148 0 14 15 4 none 12 13 14 15 16 17 18 1 i CSFIPO 6 THOI Green 159 0 205 0 8 9 4 none 45 6 7 8 9 9 3 10 11 1 Bi a 7 D135317 Green 205 65 250 16 11 4 none 8 9 10 11 12 13 14 15 amp D135317 8 D165539 Green 255 3 301 81 11 12 4 none 5 8 9 10 11 12 13 14 1E fH D165539 9 D251338 Green 304 8 370 31 19 23 4 none 15 16 17 18 19 20 21 2 H D251338 10 D195433 Yelow 101 0 148 0 14 15 4 none 9 10 11 12 12 2 13 13 2 fH D195433 l Boa 11 VWA Yelow 151 0 213 5 17 18 4 none 11 12 13 14 15 16 17 14 l TPOX 12 TPOX Yelow 216 99 260 99 8 4 none 6 7 8 9 10 11 12 13 D18551 13 D18551 Yelow 264 49 350 0 15 19 4 mone 7 9 10 10 2 11 12 13 12 E AMEL 14 AMEL Red 106 0 114 0 x 9 noe XY D55818 SL FCA 15 D55818 Red 128 0 180 0 11 4 none 7 8 9 10 11 12 13 14 1E 16 FGA Red 206 25 360 0 23 24 4 none 17 18 19 20 21 22 23 2 8 Select D2S1388 to display the Bin view for the marker in the right pane e o e lt 5 15 9 eo 5 o x de ej E 0 AmpFtSTR Identifiler Plus User Guide 54 Section 4 2 GeneMapper ID X Software g5 Panel Manager 4 File Edit Bins View Help sf x um m diss Bin set entier Pus Bins vx ji ETET u u u j m m e 17 18
50. 9 n 191 24 17 51 13 75 15 34 15 71 24 2 t 0 14 0 17 t 25 7 98 8 60 14 14 14 14 26 3 50 2 72 6 90 4 45 26 2 t t 0 52 29 0 56 t t t 30 t t t t 30 2 0 14 t t t 31 2 t t t t 32 2 t t t t 31 2 t t t t 33 2 t t t t 34 2 0 14 t t f 42 2 t t t t 43 2 t t t t 44 2 0 28 t t 45 2 0 26 46 2 0 147 t 47 2 t t t t 48 2 0 14 t t t 50 2 t t t t 51 2 t t t t THO1 4 t 5 0 28 0 437 0 17 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 t 0 14 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 43 0 52 0 26 11 t t t t 13 3 0 147 t t t TPOX 6 72 0 14 0 34 t 2 24 t 0 34 0 26 108 AmpFtSTR Identifiler Plus User Guide Chapter 5 Experiments and Results 109 Table 10 AmpF STR Identifiler Plus Kit allele frequencies continued Allele ao Gu a n Aman ar n 357 n 349 n 191 8 36 13 53 30 49 66 37 96 9 21 15 11 60 7 24 4 19 10 9 24 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 t t t t vWA 11 0 28 t 0 17 t 12 t t t 0 26 13 1 26 0 43 t 0 26 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 05 21 0 28 t 0 17 0 26 22 0 28 t t t 23 t t t t 24 t 0 14 t t t A minimum allele frequency 0 7 for
51. AmpF STR Identifiler Plus PCR Amplification Kit User s Guide Applied Bibsystems 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 and or their affiliates or their respective owners TaqMan is a registered trademark of Roche Molecular Systems Inc used under permission and license Windows NT is a trademark of Microsoft Corporation FTA is a trademark of GE Healthcare companies All other trademarks are the sole property of their respective owners 2012 Life Technologies Corporation All rights reserved Part Number 4440211 Rev D 03 2012 Contents Bm N DR NAA ACZ EZR vii Safety information 2 gx Reden RO Ae e ORC eRe he n Ro ena ee ae vii FH w to usethis guide ks gw a oh be alay akon close Sie ye Sete ate eed nad viii How
52. AmpF STR Identifiler Plus Kit contains all the necessary reagents for the amplification of human genomic DNA The reagents are designed for use with the following Applied Biosystems instruments e ABI PRISM 3100 3100 Avant Genetic Analyzer Applied Biosystems 3130 3130x Genetic Analyzer Applied Biosystems 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 The AmpF STR Identifiler Plus Kit employs the latest improvements in primer synthesis and purification techniques to minimize the presence of dye labeled artifacts These improvements result in a much cleaner electropherogram background that enhances the assay s signal to noise ratio and simplifies the interpretation of results Non nucleotide linkers are used in primer synthesis for the following loci CSF1PO D138317 D168539 D2S1338 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 AmpFtSTR Identifiler Plus User Guide Product overview interlocus spacing The combination of a five dye fluorescent system and the inclusion of non nucleotide linkers allows for simultaneous amplification and efficient separation
53. AmpFLSTR Panels v2 CHES AmpFLSTR Idertifiler Plus v1 S D881178 D21811 D75820 CSF1PO D381358 THO1 D138317 D16S539 D281338 D183433 WA TPOX D18551 j AMEL D55818 FGA Maker Name Dye Color Min Size Max Sire Control Alleles er E Marker Ladder Alleles 1 pesuzs Jewe nso 1835 fis 4 0 1032 none 8 9 10 11 12 13 1 2 D21511 bue M845 2475 30 4 0 1067 none 24 24 2 25 26 27 3 D75820 bue 2510 298 5 10 11 4 0 0969 none 6 7 8 9 10 11 12 4 CSF1PO blue 302112 348 63 10 12 4 0 082 none 6 7 8 9 10 11 12 s D351358 green 98 0 148 0 14 15 4 0 1227 none 12 13 14 15 16 1 8 THO green 1590 2050 8 3 4 0 0408 none 4567899310 7 D135317 green 205 65 250 16 m 4 0 0993 none 8 9 10 11 12 13 1 8 D165539 green 255 3 301 8 11 12 4 0 1039 none 8 9 10 11 12 1 9 D251338 green 3048 370 31 19 23 4 0 1244 none 15 18 17 18 19 2 10 D195433 Jyelow Moto 480 fisas 4 0 1121 none 9 10 11 12 12 2 1 11 WA yellow 151 0 2135 17 18 4 0 1245 none 11121314151 12 TPOX yelow 21699 260 99 8 4 0 0638 none 6 7 8 9 10 11 12 13 D18551 yellow 26449 3500 f1519 4 0 1368 none 79101021117 14 AMEL red 1060 1140 x 9 00 hone XY 15 D55818 red 128 0 180 0 m 4 0 1006 none 789104124 18 FGA red 20625 3600 23 24 4 0 1303 none 17 18 19 20 21 2 8 Select D8S1179 to display
54. AmpFtSTR Identifiler Plus User Guide 11 Chapter 1 Overview Table 3 User supplied materials continued Item Source 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 430631 1 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 Tris HCl pH 8 0 MLS EDTA 0 5 M MLS Vortex MLS For the Material Safety Data Sheet MSDS of any chemical not distributed by Applied Biosystems contact the chemical manufacturer Before handling any chemicals refer to the MSDS provided by the manufacturer and observe all relevant precautions 12 AmpFtSTR Identifiler Plus User Guide Chapter 2 PCR Amplification AmpFtSTR Identifiler Plus User Guide AmpFtSTR Identifiler Plus User Guide PCR Amplification This chapter covers m PCR work areas DNA quantification Perform PCR AmpFtSTR Identifiler Plus User Guide Required user su
55. Heavy Peak Window Size I pts Baseline Window B pts Slope Threshold Peak Start b 0 Size Calling Method Peak End bo 2nd Order Least Squares C 3rd Order Least Squares C Cubic Spline Interpolation Local Southern Method Global Southern Method Factory Defaults oo Cancel Figure 7 Analysis Method Editor HID Peak Detector tab settings IMPORTANT Laboratories need to perform the appropriate internal validation studies to determine the peak amplitude thresholds that allow for reliable interpretation of AmpF STR Identifiler Plus PCR Amplification Kit data 45 AmpF STR Identifiler Plus User Guide Set up GeneMapper ID Software for data analysis The software uses the peak amplitude threshold parameters to specify the minimum peak height to limit the number of detected peaks Although GeneMapper D Software displays peaks that fall below the specified amplitude in electropherograms the software does not label or determine the genotype of these peaks Note The analysis range is set by the user based on the locations of the primer peaks and size standard peaks For more information about peak detection algorithms refer to e GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide PN 4338775 GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial Appendix A PN 4335523 Installation Procedures and New Features for GeneM
56. Identifiler Plus User Guide Accuracy precision and reproducibility Table 4 Precision results of five runs 16 capillaries run of the AmpFfSTR Identifiler Plus Allelic Ladder continued Applied Biosystems 3130x Genetic Analyzer Allele Mean Standard Deviation D16S539 5 252 01 252 15 0 05 0 06 8 264 264 15 0 05 0 061 9 268 268 14 0 05 0 063 10 272 272 15 0 045 0 059 11 276 02 276 17 0 04 0 064 12 280 03 280 18 0 039 0 067 13 284 05 284 22 0 045 0 06 14 288 08 288 23 0 044 0 054 15 292 12 292 26 0 038 0 059 D18S51 7 261 8 261 9 0 037 0 049 9 269 94 270 03 0 037 0 051 10 274 02 274 12 0 043 0 051 10 2 276 03 276 13 0 037 0 048 11 278 11 278 22 0 042 0 059 12 282 2 282 29 0 037 0 046 13 286 29 286 39 0 039 0 051 13 2 288 29 288 4 0 034 0 052 14 290 38 290 49 0 032 0 051 14 2 292 39 292 5 0 043 0 057 15 294 48 294 59 0 029 0 049 16 298 57 298 69 0 035 0 053 17 302 69 302 81 0 039 0 056 18 306 83 306 95 0 039 0 056 19 310 96 311 07 0 034 0 049 20 315 08 315 18 0 036 0 045 21 319 2 319 31 0 036 0 045 22 323 39 323 5 0 03 0 044 23 327 46 327 53 0 025 0 052 24 331 59 331 65 0 026 0 043 25 335 69 335 76 0 032 0 043 26 339 8 339 88 0 036 0 047 27 343 87 343 99 0 031 0 048 AmpFtSTR Identifiler Plus User Guide 76 Chapter 5 Experiments and Results Table 4 Precision results of five runs 16 capillaries
57. Identifiler Plus GS500 then click Import to import the CE G5 Identifiler Plus GS500 size standard into the GeneMapper D Software database Import Size Standard Method xl Lookin ED entfier Plus Analysis Files GMD z m re kej CE G5 ldentifiler Plus GS500 xml 4 Identifiler_Plus_AnalysisMethod_v1 xml My Recent D 9 z B oO Desktop zo zo o My Documents cM er U File name CE_G5_ldentifiler_Plus_GS500 xmi Import Ej 3 Files of type m Files xml v Cancel oO Analyze and edit sample files with GeneMapper D Software Analyze a project 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 Plus AnalysisMethod v1 Panel Identifiler Plus Panels v1 Size Standard CE G5 Identifiler Plus GS5005 t For more information about how the Size Caller works refer to the ABI PRISM GeneScan Analysis Software for the Windows NT Operating System Overview of the Analysis Parameters and Size Caller User Bulletin PN 4335617 S The following fragments are defined for the CE G5 Identifiler Plus GS500 size standard provided with the AmpF STR Identifiler Plus Kit 75 100 139 150 160 200 300 350 400 and 450 For additional information about size standar
58. KK kk k 70 Accuracy precision and reproducibility kk kk kk kk KK KK KK ee 73 Extra peaks in the electropherogram kk kk kk KK KK KK KK KK K KK KK KI KK KK KK KIR 81 Characterization of loci kk kk kk nk kk kK kK KK KK KK KEK KK KI KK KK KK KK KK KK kK KK kk kk 88 SPECIES Specificity 2 ou xn k A Waki a ia ls tee J ASA RAE aate WZW WRAY ai 89 Sensitivity uox soy re xd ee s baa ao eee i CARNE EE Ri eas 91 Stability wozie we cuu EDO be ee AW Akai W WA bee ERIT ER MERE PE 93 Mixt re studies s onu e a daa bid ce dete PODA ck debe Wad pady 96 Population Data 2 00 00 be aw RE pre ree REG Ge ster Rum d us 102 Mutation Rate ss de an mate t ebbe dS pred da belt mb E cetus PAGE 114 Probability of Identity is lt k gas ad Oss peed A eG SER URN PALA CR 115 Probability of Paternity Exclusion Jk kk kk kk kK KK KK K KK KK KIRI K KOK Ih 116 Troubleshooting 000 kK KK KIR K KK KIR aa aa KK IK KG 118 Sdlely asso As AAY wizaz K A ken ier ME E 122 Chemical safety cile ashe ein ee ek la esos Room Gea a dae aa aes 123 Chemical waste safety iesu Sette px bd e cred Rid x e ko ux se A Ba a ala 125 Biological hazard safety yee nn la wa D cee QR KA dik SA AZ Ki ne 126 Chemical alerts 22d a dk Ra balk dak a Aa dya WY Odo lad 127 Bibliography Ga Sk kk kk kk KK KK K KK K KK E DIM KI KK ee 128 AmpFtSTR Identifiler Plus User Guide Contents Documentation uo 4 kk kk ou es eee
59. Kit STR loci in four U S populations continued Amica GS W e er W Ai n 357 n 349 n 191 TPOX HW X p 0 765163 0 801518 0 875348 0 333914 HW G p 0 611014 0 757735 0 913091 0 229017 HW Exact p 0 7247 0 5775 0 8356 0 0647 HExp 0 7643 0 6311 0 6607 0 6765 Ho 0 7563 0 6304 0 6759 0 6178 vWA HW X p 0 925176 0 005048 0 641684 0 994248 HW G p 0 964308 0 218817 0 934427 0 997184 HW Exact p 0 7033 0 0564 0 7066 0 8845 HExp 0 8141 0 8081 0 7818 0 7457 Ho 0 8571 0 8138 0 7759 0 7277 HW X p probability value of X test for Hardy Weinberg equilibrium HW G p probability value of the G statistic of the Likelihood Ratio test for multinomial proportions HW Exact p A Markov chain unbiased exact test to estimate the P value of the Hardy Weinberg test with multiple alleles Hexp Expected heterozygosity Ho observed heterozygosity Applied Biosystems analyzed 7500 samples by comparing allele calls between the AmpF STR Identifiler and Identifiler Plus Kits The genotype data from all the analyzed samples showed 100 concordance between the Identifiler and Identifiler Plus Kits AmpFtSTR Identifiler Plus User Guide Mutation Rate Mutation Rate Estimation of spontaneous or induced germline mutation at genetic loci can be achieved by comparing 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
60. Mapper JD 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 PN 4338775 3 Select Tools gt Panel Manager 4 Find then open the folder containing the panels and bins a Select Panel Manager in the navigation pane File Edit Bins View a MEE E Hi ES Panel Manager Highlight this b Select File gt Import Panels to open the Import Panels dialog box c Navigate to then open the Identifiler Plus Analysis Files GMID folder that you unzipped in step 1 5 Select Identifiler Plus Panels vl then click Import Note Importing this file creates a new folder in the navigation pane of the Panel Manager AmpFLSTR JIdentifiler Plus v1 This folder contains the panel and associated markers 39 AmpFtSTR Identifiler Plus User Guide Set up GeneMapper ID Software for data analysis Look in E Idertifiler Plus Analysis Files GMID S GB E CE_G5_ldentifiler _Plus_GS500 xml Identifiler_Plus_AnalysisMethod_v1 xml E Identifiler_Plus_Bins_v1 txt identifler _Plus_Panels_v1 tt r My Recent D Desktop My Documents File name dentitier Plus Panels v1 txt Import E Files of type Im Files Y Cancel 6 Import Identifiler Plus Bins vl CD D D lt ied zo go D iw e e z e
61. Ps carrier protein and 0 04 sodium azide 2 tubes 1 0 mL each 15 to 25 C on receipt 2 to 8 C after initial use AmpF STR Identifiler Plus Allelic Ladder Contains amplified alleles See Table 1 on page 3 for a list of alleles included in the allelic ladder 1 tube 50 0 uL 15 to 25 C on receipt 2 to 8 C after initial use AmpF STR Control DNA 9947A Contains 0 10 ng uL human female 9947A DNA in 0 05 sodium azide and buffer 1 tube 0 3 mL 2to8 C See Table 1 on page 3 for profile 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 generate a profile of expected genotype The AmpFZSTR Control DNA 9947A is not designed to be used as a DNA quantitation control and laboratories may expect to see variation from the labelled concentration when quantitating aliquots of the AmpF STR Control DNA 9947A Standards for For the AmpF STR Identifiler Plus Kit the panel of standards needed for PCR samples amplification PCR product sizing and genotyping are Control DNA 9947A A positive control for evaluating the efficiency of the amplification step and STR genotyping using the AmpF STR Identifiler Plus Allelic Ladder GeneScan 500 LIZ Size Standard Standard used for obtaining sizing results It contains 16 single stran
62. R Identifiler Plus Allelic Ladder continued Applied Biosystems 3130x Genetic Analyzer Allele Mean Standard Deviation 35 228 1 228 17 0 03 0 048 35 2 230 03 230 09 0 03 0 037 36 232 02 232 09 0 03 0 047 37 236 08 236 17 0 026 0 041 38 240 04 240 1 0 033 0 045 D2S1338 15 306 27 306 39 0 033 0 058 16 310 35 310 47 0 031 0 055 17 314 39 314 53 0 029 0 042 18 318 45 318 58 0 029 0 046 19 322 52 322 63 0 025 0 046 20 326 58 326 67 0 029 0 039 21 330 66 330 74 0 034 0 045 22 334 71 334 8 0 031 0 043 23 338 74 338 85 0 026 0 045 24 342 75 342 89 0 026 0 05 25 346 78 346 92 0 026 0 051 26 350 77 350 89 0 028 0 049 27 354 69 354 81 0 026 0 045 28 358 87 359 01 0 028 0 045 D3S1358 12 111 12 111 22 0 024 0 047 13 115 23 115 32 0 03 0 046 14 119 2 119 31 0 03 0 044 15 123 14 123 22 0 031 0 045 16 127 32 127 41 0 032 0 042 17 131 54 131 62 0 027 0 039 18 135 64 135 71 0 021 0 042 19 139 72 139 81 0 024 0 045 D5S818 7 133 69 133 75 0 029 0 039 8 137 8 137 86 0 031 0 037 9 142 17 142 24 0 022 0 035 10 146 64 146 71 0 025 0 039 11 151 05 151 12 0 031 0 043 12 155 32 155 39 0 028 0 041 AmpFtSTR Identifiler Plus User Guide 78 Chapter 5 Experiments and Results Table 4 Precision results of five runs 16 capillaries run of the AmpF STR Identifiler Plus Allelic Ladder continued Applied
63. STR Identifiler Plus User Guide 106 Chapter 5 Experiments and Results Table 10 AmpF STR Identifiler Plus Kit allele frequencies continued Allele ele G le cd T n Amen n 357 n 349 n 191 29 19 33 20 49 21 21 16 75 29 2 0 14 t 0 522 0 26 29 3 0 14 t t i 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 79 32 2 5 88 7 16 12 93 9 69 33 0 56 t t 0 522 33 2 3 78 3 30 4 14 3 66 34 1 26 t t t 34 1 0 14t t t 34 2 0 14 0 29 0 86 0 79 35 2 94 t 0 34 i 35 1 0 14 t t 35 2 t 0 14 t t 36 0 84 t t i 37 0 28 t t 38 0 14 t t FGA 16 t 0 14 t t 16 1 0 14 t t 17 t 0 29 0 17 17 2 0 14 t t 18 0 70 2 72 0 522 1 31 18 2 1 40 t t t 19 6 72 6 16 7 07 10 21 19 2 0 28t t t t 20 7 00 13 90 7 41 12 30 20 2 i 0 14t t t 21 12 89 16 91 14 66 12 83 22 21 57 16 91 17 24 10 47 222 0 28 1 29 0 34 0 26 22 3 0 14 0 14 t t 23 14 99 15 19 11 90 15 97 23 2 0 14 0 14 0 86 0 26 107 AmpFtSTR Identifiler Plus User Guide Population Data Table 10 AmpF STR Identifiler Plus Kit allele frequencies continued Allele Am Ga a ogy And i n 357 n 34
64. Setup GeneMapper ID X Software for data analysis 52 m Analyze and edit sample files with GeneMapper ID X Software 64 m Examine and edit a project nauuna KK KK KK KK KK KK KK KK KK eh 65 AmpFtSTR Identifiler Plus User Guide 36 Section 4 1 GeneMapper ID Software Section 4 1 GeneMapper D Software Overview of GeneMapper D Software 37 Instruments Before you start GeneMapper JD 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 Refer to Instrument and software overview on page 7 for a list of compatible instruments 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 ZD 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
65. Software Overview of GeneMapper D X Software 51 Instruments Before you start 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 Using GeneMapper ID X Software v1 0 1 v1 1 or v1 1 1 you can then analyze and interpret the data from the fsa files Refer to Instrument and software overview on page 7 for a list of compatible instruments When using GeneMapper D X Software v1 0 1 v1 1 or v1 1 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 7D 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 single 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 ap
66. a analysis d Select Identifiler_Plus_Bins_v1X then click Import Note Importing this file associates the bin set with the panels in the Identifiler_Plus_Panels_v1X folder Look in je Identifiler Plus Analysis Files GMIDX Y 1d CE re My Recent Documents CE G5 Identifiler Plus GS500 xml B Identifier Plus Bins vix txt Identifier Plus Panels v1x txt Identifier Plus Stutter vix txt Identifiler Plus AnalysisMethod vix xml Desktop 7 View the imported panels in the navigation pane a Double click the AmpFLSTR_Identifiler_Plus_v1X folder to view the Identifiler_Plus_Panel_v1X folder b Double click the Identifiler_Plus_Panel_v1X folder to display the panel information in the right pane and the markers below it File Edit Bins View Help w s mo uja Marker Comments a X MMM NS Bn set fra lentifiler_Plus_Bins_v1X Marker Name Dye Color Mim Size Max Size sfa Panel Manager Control Alleles c Ladder Alleles C AmpFLSTR_Panels_v1X 1 pss Blue 11 amp 0 183 5 13 4 nom 8 9 10 11 12 13 14 15 1 QQ AmpFLSTR Identifiler Plus vix 2 D21511 Blue 1845 247 5 30 4 none 24 24 2 25 26 27 28 28 zB Identifiler Plus Panels v1 RATE d 3 D75
67. allele frequencies Allele NA GG a z 5 asa n 357 n 349 n 191 CSF1PO 6 t t t t 7 4 62 0 14 0 34 i 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 91 28 28 21 99 11 3 0 14 t t t 12 29 13 32 81 39 66 32 72 13 5 32 7 31 6 38 4 71 14 0 98 1 43 0 86 0 79 15 t 0 29 t t D2S1338 15 0 14 t t t 16 5 32 4 73 2 41 2 62 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 38 22 13 17 4 01 7 41 15 18 23 10 78 11 46 11 36 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 69 0 79 27 0 14 0 14 t t 28 t t t t D3S1358 11 0 42 0 14 t t 11 t t t 0 26 12 0 56 t 0 17 t 13 0 70 0 29 0 17 t 14 12 04 15 76 7 41 6 81 15 30 53 25 36 39 14 40 84 15 2 0 14 t t t AmpFtSTR Identifiler Plus User Guide Population Data Table 10 AmpF STR Identifiler Plus Kit allele frequencies continued Allele mon RE n sj Amari n 357 n 349 n 191 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 08 0 79 20 i 0 34 i D5S818 7 0 14t i 6 72 15 71 8 5 46 t 0 69 t 9 1 68 4 15 5 17 6 02 10 6 72
68. ample 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 1s not accurate This inaccuracy results in poor spectral separation pull up Incomplete A nucleotide addition The sample can be reamplified using less DNA When the total number of allele copies added to the PCR is extremely low unbalanced amplification of the alleles may occur because of stochastic fluctuation 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 For information on selecting the appropriate cycle number for your DNA input amount see Select the appropriate PCR cycle number on page 20 AmpFtSTR Identifiler Plus User Guide Sensitivity 73 115 155 195 235 275 315 355 395 40001 3000 1 ng 2000 1000 o 75 115 155 185 235 275 315 355 395 d 3000 0 50 ng 2000 4000 D 75 115 155 185 235 275 315 355 395 2000 4600 1200 0 25 ng cy 0 125 ng aa 0 062 ng Figure 25 Effect of amplifying 1 ng 0 50 ng 0 25 ng 0 125 ng and 0 062 ng of Control DNA 9947A using the 28 PCR cycle protocol avo 0 50 ng 2000 f 0 25 ng 75 115 455 195 235 275 315 355 395 2000 al 0 125 ng 75 115 455 195 235 275 315 355 395
69. apper D X Software workflow and features refer to e GeneMapper D X Software Version 1 0 Getting Started Guide PN 4375574 GeneMapper JD X Software Version 1 0 Quick Reference Guide PN 4375670 GeneMapper ID X Software Version 1 0 Reference Guide PN 4375671 Import To import the AmpF STR Identifiler Plus Kit panels bin sets and marker stutter panels bins and from the Applied Biosystems web site into the GeneMapper D X Software v1 0 1 marker stutter v1 1 or v1 1 1 database 1 Download and open the file containing panels bins and marker stutter a From the Support menu of www appliedbiosystems com select Software Downloads Patches amp Updates Select GeneMapper ID X Software from the drop down menu Select Updaters amp Patches and download the file Identifiler Plus Analysis Files GMIDX b Unzip the file 2 Start the GeneMapper D X Software then log in with the appropriate user name and password IMPORTANT If you need logon instructions refer to the GeneMapper ID X Software Version 1 0 Getting Started Guide PN 4375574 C o mi D ko go 0 8 w gt n 9 zh 2 0 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 AmpFtSTR Identifiler Plus User Guide 52 Section 4 2 GeneMapper ID X Software g5 Panel Manager File Edit Bins View Help
70. apper ID Software database Refer to step 1 on page 39 for downloading instructions Note The Identifiler Plus AnalysisMethod vl has been provided to assist you in getting started with Identifiler Plus kit data analysis Analysis parameters should be established by each individual laboratory based on the laboratory s internal validation studies 1 Select Tools GeneMapper Manager to open the GeneMapper Manager AmpFtSTR Identifiler Plus User Guide 42 Section 4 1 GeneMapper ID Software 2 Import an analysis method for HID_Advanced a Select the Analysis Methods tab then click Import 7 GeneMapper Manager x 3 i Table Settings Plot Settings Matrices Size Standards Name Last Saved Owner Instrument Analysis Type Description HID Advanced 2009 06 18 16 22 2 gmid HID Classic 2007 08 06 10 03 0 gmid li Microsstelite Default 2004 05 28 11 34 3 grid Factory Provided b Navigate to then open the Identifiler Plus Analysis Files GMID folder c Select Identifiler Plus AnalysisMethod v1 then click Import to import the Identifiler Plus AnalysisMethod vl into the GeneMapper ID Software database Import Analysis Method Look in e Idertifiler Plus Analysis Files GMID i2 oma mn Fe Ej CE G5 Identifiler Plus GS500 xml o zj Idertifiler_Plus_AnalysisMethod_v1 My Recent D xml File name identifier Plus AnalysisMethod v1 xml Import
71. apper ID Software v3 2 User Bulletin PN 4352543 Analysis Method Editor HID i ji xi General Allele Peak Detector Peak Quality Quality Flags C D 2 lt ko zo 0 iw 2 e Eh E 0 r Signal level Homozygous min peak height j 00 0 Heterozygous min peak height so 0 rHeterozygote balance Min peak height ratio ja r Peak morphology Max peak width basepairs j 5 Pull up peak Pull up ratio 0 05 Allele number Max expected alleles B Factory Defaults _ok Cancel Figure 8 Analysis Method Editor HID Peak Quality tab settings IMPORTANT Laboratories need to perform the appropriate internal validation studies to determine the minimum heterozygous and homozygous minimum peak height thresholds and the minimum peak height ratio threshold that allow for reliable interpretation of AmpF STR Identifiler Plus PCR Amplification Kit data AmpFtSTR Identifiler Plus User Guide 46 Section 4 1 GeneMapper ID Software Import an HID size standard 47 Analysis Method Editor HID General Allele Peak Detector Peak Quality Quality Flags Quality weights are between 0 and 1 r Quality Flag Settings Spectral Pull up os Control Concordance j 0 Broad Peak os Low Peak Height os Out of Bin Allele os Off scale os Overlap os Peak Height Ratio fos r PQY Thresholds Sizing Quality From ozs to 1 0 From 0
72. aracteristics of a genetic marker must be determined and documented guideline 2 1 SWGDAM July 2003 This section describes basic characteristics of the 15 loci and the sex determining marker Amelogenin which are amplified with the AmpF STR Identifiler Plus Kit These loci have been extensively characterized by other laboratories Nature of the The primers for the Amelogenin locus flank a 6 nucleotide deletion within intron 1 polymorphisms ofthe X homologue Amplification results in 107 nt and 113 nt products from the X and Y chromosomes respectively Sizes are the actual nucleotide size according to sequencing results including 3 A nucleotide addition The remaining AmpF STR Identifiler Plus 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 nt repeat units All the alleles in the AmpF STR Identifiler Plus Allelic Ladder including microvariants have been subjected to sequencing at Applied Biosystems In addition other groups in the scientific community have sequenced alleles at some of these loci Nakahori et al 1991 Puers et al 1993 M ller et al 1994 Barber et al 1995 Brinkmann and M ller 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 AmpF STR Identifiler Plus Kit l
73. ased on your results and experiments 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 briefly centrifuge the plate to ensure that the contents of each well are collected at the bottom Heat the reaction plate in a thermal cycler for 3 minutes at 95 C Immediately place the plate on ice for 3 minutes Prepare the plate assembly on the autosampler Start the electrophoresis run AmpFtSTR Identifiler Plus User Guide Section 3 2 310 instrument Section 3 2 310 instrument Set up the 310 instrument for electrophoresis Reagents and Table 3 on page 10 lists the required materials not supplied with the AmpF STR parts Identifiler Plus Kit IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the AmpF STR Identifiler Plus Primer Set from light when not in use Amplified DNA AmpF STR Identifiler Plus Allelic Ladder and GeneScan 500 LIZ Size Standard should also be protected from light Keep freeze thaw cycles to a minimum 310 instrument The following table lists Data Collection Software and the run modules that ca
74. aterials and equipment aeaeaaaa aaa aaa aaa KK KK KK K AmpFtSTR Identifiler Plus User Guide Chapter 1 Overview Product overview Purpose The AmpF STR Identifiler Plus PCR Amplification Kit is a short tandem repeat Product description About the primers STR multiplex assay that amplifies 15 tetranucleotide repeat loci and the Amelogenin gender determining marker in a single PCR amplification All thirteen of the required loci for the Combined DNA Index System CODIS loci are included in this kit for known offender databasing in the United States Budowle et al 1998 Two additional loci D281338 and D19S433 are included These loci are consistent with the AmpF STR SGM Plus PCR Amplification Kit The combination of the 15 loci are consistent with several worldwide database recommendations The AmpF STR Identifiler Plus Kit delivers a 16 locus multiplex with the same power of discrimination as better sensitivity than and better robustness than the earlier generation of the AmpF STR Identifiler Kit The kit uses modified PCR cycling conditions for enhanced sensitivity a new buffer formulation to improve performance with inhibited samples and an improved process for DNA synthesis and purification of the amplification primers to deliver a much cleaner electrophoretic background The AmpF STR Identifiler Plus Kit uses the same primer sequences as the earlier generation AmpF STR Identifiler Kit The
75. bus_3130XL IB_0341 Sample Identifiler Plus amp nalysisMethod v1 Identifiler Plus Panels v1 CE G5 Identifiler Plus GS500 amp BI3130 14 L3 Cerebus 3130XL IB 0342 Sample Identifiler Plus AnalysisMethod v1 Identifiler Plus Panels v1 CE G3 Identifiler Plus GS500 amp BI3130 15 ie Cerebus 3130XL ladder3 Allelic Ladder Identifiler Plus AnalysisMethod v1 Identifiler Plus Panels v1 CE G3 ldentifiler Plus GS500 amp BI3130 For more information about any of these tasks refer to the GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide PN 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 about any of these tasks refer to GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial PN 4335523 GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide PN 4338775 Installation Procedures and New Features for GeneMapper ID Software Version v3 2 User Bulletin PN 4352543 49 AmpF STR Identifiler Plus User Guide 2 D lt ied xe 5 2 AE Xx ie e 2 0 0 AmpFtSTR Identifiler Plus User Guide 50 Section 4 2 GeneMapper ID X Software Section 4 2 GeneMapper D X
76. d likelihood ratio HW G p tests as implemented in the program PopGene 1 32 With an exact test HW Exact p which is a Markov chain method based on 1000 shuffling experiments to estimate without bias the exact P value of the Hardy Weinberg test with multiple alleles Guo and Thompson 1992 as implemented in the program GenePop 3 4 Aninter class correlation test analysis Burrows composite measure of linkage disequilibria between pairs of loci and X tests for significance Weir 1996 was performed separately in each population to detect any correlations between alleles at any of the pair wise comparisons of the 15 loci using the program PopGene 1 32 Observed heterozygosity Ho expected heterozygosity information content and tests for detecting departures from Hardy Weinberg equilibrium are shown for each population in Table 11 While a number of the chi square tests gave seemingly significant p values putatively indicating departures from Hardy Weinberg equilibrium chi squared tests are very sensitive to small expected values as in the case of multiple rare alleles where the expected number of certain genotypes is 1 or fewer such as with some of these markers and can greatly inflate the test statistic in this situation Weir 1990 With the exact test the number of tests with p value 0 05 were 0 in the African American and U S Caucasian populations 1 in the U S Hispanic population D8S1179 p 0 0304 and 2 in the
77. d on SQ and CGQ only Run Folder Total of Analyzed Ladders m a 3 3 0 0 Identifiler Plus Analysis Examples Control Quality per project based on sample PQVs SOS SSPK MIX OMR SQ CGQ Control Type Total of Samples EJ All thresholds met One or more thresholds not met Positive Control Custom Control Negative Control Total Pale Oo io ojojo Sample Quality per project based on sample PQVs SOS SSPK MIX OMR SQ CGQ Ly jT Total of Samples EJ All thresholds met One or more thresholds not met Samples 29 1 12 Analysis Completed J rm For more information about any of these tasks refer to GeneMapper JD X Software Version 1 0 Getting Started Guide PN 4375574 GeneMapper ID X Software Version 1 0 Quick Reference Guide PN 4375670 GeneMapper ID X Software Version 1 0 Reference Guide PN 4375671 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 about any of these tasks refer to GeneMapper ID X Software Version 1 0 Getting Started Guide PN 4375574 GeneMapper ID X Software Version 1 0 Quick Reference Guide PN 4375670 GeneMapper ID X Software Version 1 0 Reference Guide PN 4375671 65 AmpFtSTR Identifi
78. d plated silver 96 well block or a Veriti 96 Well Thermal Cycler Note The AmpF STR Identifiler Plus 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 AmpF STR Identifiler Plus Kit Select the appropriate PCR cycle number All AmpF STR kits are optimized for a specific number of amplification cycles to deliver well balanced and high quality results However increases in the number of low level DNA samples being submitted for analysis have prompted many laboratories to evaluate increasing the number of amplification cycles to increase the sensitivity of the assay Before increasing the cycle number perform a comprehensive validation study to establish new performance criteria for the higher cycle number Higher cycle numbers can cause the following to occur Exaggerated stochastic effects resulting from low DNA input amounts Greater difference between the presence and absence of an allele Greater heterozygote peak imbalance Possible differences in expected stutter position and percentage Possible increase in artifacts and or background in the profile to accompany the increase in sample allele signal The Identifiler Plus Kit offers two PCR cycle number options Standard 28 PCR cycle protocol Provides high sensitivity to consistently generate full STR profiles with 125 pg of DNA input Use with t
79. ded labeled fragments of 35 50 75 100 139 150 160 200 250 300 340 350 400 450 490 and 500 nucleotides This standard which has been evaluated as an internal lane size standard yields precise sizing results for AmpF STR Identifiler Plus Kit PCR products Order the GeneScan 500 LIZ Size Standard PN 4322682 separately AmpF STR Identifiler amp Plus Allelic Ladder Allelic ladder developed by Applied Biosystems for accurate characterization of the alleles amplified by the AmpF STR Identifiler Plus Kit The AmpF STR Identifiler Plus Allelic Ladder contains most of the alleles reported for the 15 autosomal loci Refer to Table 1 on page 3 for a list of the alleles included in the AmpF STR Identifiler Plus Allelic Ladder AmpFtSTR Identifiler Plus User Guide 9 Chapter 1 Overview Equipment and Tables 2 and 3 list required and optional equipment and materials not supplied with materials not the AmpF STR Identifiler Plus Kit Unless otherwise noted many of the items are included available from major laboratory suppliers MLS Table 2 Equipment Equipment Source ABI Prism 3100 3100 Avant Genetic Analyzer Applied Biosystems 3130 3130x Genetic Analyzer Applied Biosystems 310 Genetic Analyzer Contact your local Applied Biosystems sales representative GeneAmp PCR System 9700 with the Silver 96 Well Block N8050001 G
80. default Consequently the software applies the stutter ratio filters supplied in the Identifiler Plus Stutter v1X file For more information about allele filters refer to GeneMapper ID X Software Version 1 0 Getting Started Guide PN 4375574 GeneMapper D X Software Version 1 0 Quick Reference Guide PN 4375670 GeneMapper JD X Software Version 1 0 Reference Guide PN 4375671 59 AmpFtSTR Identifiler Plus User Guide Set up GeneMapper ID X Software for data analysis Analysis Method Editor lE xi D F Peak Quality 5Q amp GQ Settings Peak Detection Algorithm Advanced Ranges Peak Detection Analysis Sizing Peak Amplitude Thresholds Fut Range Al Sizes B Go R go Start Pt tooo Start AN PT Bo 50 50 Stop Pt i000 Stap Size i00 G 0 Y po Smoothing and Baselining j Min Peak Half width j pts Smoothing C None i b Light Polynomial Degree C Heavy Peak Window Size 15 pts Baseline Window g pts Slope Threshold Peak Start o o Size Calling Method Peak End o 2nd Order Least Squares 3rd Order Least Squares Cubic Spline Interpolation Local Southern Method Global Southern Method Factory Defaults Save As Save Cancel Help Figure 12 Analysis Method Editor Peak Detector tab settings The software uses the peak amplitude threshold parameters to specify the minimum peak height to limit the numbe
81. ds refer to the GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide PN 4338775 Appendix D Neither the 250 nt nor the 340 nt peak are included in the size standard definition These peaks can be used as an indicator of precision within a run 3 Click PP 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 completed indicated With text messages on the left AmpFtSTR Identifiler Plus User Guide 48 Section 4 1 GeneMapper ID Software The Samples table figure below 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 2 1 Untitled gmid Is Logged In ew Tools Help 5 MM IL Bi gt 6 Tavie setting HD_LsdderTable_FileName Hi d m Samples Genotypes Status Sample File Sample Name Sample Type Analysis Method Panel Size Standard Instrument Type 1 w Cerebus 3130XL IB_0329 Sample Identifiler Plus amp nalysisMethod v1 Identifiler Plus Panels v1 CE G5 Identifiler Plus GS500 4513130 2 w Cerebus 3130XL IB_0330 Sample Identifiler Plus amp nalysisMethod v1 Identifiler Plus Panels v1 CE G5 Identifiler Plus GS500 4513130
82. e To open the user documentation available from the Applied Biosystems web site use the Adobe Acrobat Reader software available from www adobe com Send us your comments 133 Applied Biosystems welcomes your comments and suggestions for improving its user documents You can e mail your comments to techpubs appliedbiosystems com IMPORTANT The e mail address above is for submitting comments and suggestions relating only to documentation To order documents download PDF files or for help with a technical question see How to obtain support on page ix AmpFtSTR Identifiler Plus User Guide Index Symbols fsa sample files 38 52 A nucleotide addition defined 86 efficiency of 86 lack of causes 87 A accuracy and reproducibility 70 73 alleles low frequency 109 off ladder 74 peak height ratio table 96 allelic ladder about 9 figure 4 number per run suggested 27 precision results table 75 requirements for accurate genotyping 27 volume per reaction 29 31 amplification amplified DNA 16 loci 3 using bloodstained FTA cards 22 work area tools 16 annealing temperatures validation of 71 Applied Biosystems customer feedback on documentation 133 Information Development department 133 artifacts in data 87 B biohazardous waste handling 126 bold text when to use viii C CAUTION description vii CEPH 88 characterization of loci validation 88 chemical safety 123 chemical waste safety 125 c
83. e To Distance T 21 1 1006 3 25 4 75 1 v TPOX 2 2 D18551 3 3 AMEL 4 4 D55818 gag Stutter Ratio amp Distance __New e __pelete FGA D851179 HB 11 Click Apply then OK to add the AmpF STR Identifiler Plus Kit panels bin sets and marker stutter to the GeneMapper ZD X Software database 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 Q D el D lt 5 o go 0 8 o Xx 2 e EB B 0 AmpFtSTR Identifiler Plus User Guide 56 Section 4 2 GeneMapper ID X Software Import an Use the following procedure to import the analysis method for the AmpF STR analysis method Identifier Plus PCR Amplification Kit from the folder that you downloaded from the Applied Biosystems web site into the GeneMapper D X Software database Refer to step 1 on page 52 for downloading instructions Note The Identifiler Plus AnalysisMethod vl1x has been provided to assist you in getting started with Identifiler Plus kit data analysis Analysis parameters should be established by each individual laboratory based on the laboratory s internal validation studies 1 Select Tools GeneMapper ID X Manager to open the GeneMapper D X Manager 2 Import an analysis method a Select the Analysis Methods tab then click Import s Ge
84. ed optimum PCR product yield and that met reproducible performance standards While these experiments are not exhaustive they are appropriate for a manufacturer of STR kits intended for forensic and or parentage testing use IMPORTANT Each laboratory using the AmpF STR Identifiler Plus PCR Amplification Kit must perform internal validation studies AmpFtSTR Identifiler Plus User Guide Developmental validation Developmental validation SWGDAM Developmental validation is the demonstration of the accuracy precision and guideline 1 2 1 reproducibility of a procedure by the manufacturer technical organization academic institution government laboratory or other party SWGDAM July 2003 SWGDAM The reaction conditions needed to provide the required degree of specificity and guideline 2 10 1 robustness must be determined These include thermal cycling parameters the concentration of primers magnesium chloride DNA polymerase and other critical reagents SWGDAM July 2003 PCR components Applied Biosystems examined the concentration of each component of the AmpF STR Identifiler Plus PCR Amplification Kit The concentration for each individual component was established to be in the window that meets the reproducible performance characteristics of specificity and sensitivity For example various magnesium chloride concentrations were tested on the Applied Biosystems 3130x Genetic Analyzer The amplification of 1 0 ng
85. eight 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 A mutation in one of the primer binding sites 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 comprise at a single locus any of the seven genotype combinations see below 97 AmpFtSTR Identifiler Plus User Guide Mixture studies Heterozygote heterozygote no overlapping alleles four peaks Heterozygote heterozygote one overlapping allele three peaks Heterozygote heterozygote two overlapping alleles two peaks Heterozygote homozygote no overlapping alleles three peaks 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 or not it is possible to resolve the genotypes of the major and minor component s at a single locus The ability to obtain and compare quantitative values for the different alle
86. eneAmp 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 3 User supplied materials Item Source AmpF STR Identifiler Plus PCR Amplification Kit 4427368 3100 3100 Avant Genetic Analyzer materials 96 Well Plate Septa 4315933 Reservoir Septa 4315932 3100 3100 Avant Genetic Analyzer Capillary Array 36 cm 4333464 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 Running Buffer 10X 402824 DS 33 Matrix Standard Kit Dye Set G5 4345833 MicroAmp Optical 96 Well 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 instrument refer to Appendix B of the AB PR sM 3100 Genetic Analyzer and 3100 Avant Genetic Analyzer User Reference Guide PN 4335393 10 AmpFtSTR Identifiler Plus User Guide Table 3 User supplied materials continued Materials and equipment Item Source 3130 3130x Analyzer materials 96 Well Plate Septa 4315933 Reservoir Septa 4315932 3100 3130x Genetic Analyzer Capillary Array 36 cm 4315931 POP 4
87. er Plus Kit Hematin uM Alleles detected Total alleles 200 26 26 26 26 26 26 300 26 26 26 26 26 26 t Only those peaks gt 50 RFUs were counted A complete profile with Control 9947A DNA yields 26 peaks using the AmpF4STR Identifiler Plus Kit Effect of Traces of humic acid may inhibit the PCR amplification of DNA evidence collected inhibitors from soil In this study Applied Biosystems tested increasing amounts of humic acid humic acid in the PCR amplification of 1 ng of Control DNA 9947A with the Identifiler Plus Kit for 28 cycles of amplification see Figure 29 The concentrations of humic acid tested were 0 50 100 and 150 ng uL see Table 7 9947A b L m 7 Mark Sample for Deletior 125 165 205 245 285 325 4000 Control samples 3000 20001 1000 HASO b DJ Je Tm Tm m I 7 Mark Sample For Deletior 425 465 205 245 285 325 4000 3000 50 n g Li L 20004 1000 0 HA100 b m ja m C Mark Sample for Deletior 125 165 205 245 285 325 4000 100 ng uL 3000 2000 1000 0 HA150 b Mark Sample for Deletior 125 165 205 245 285 325 4000 150 ng uL 3000 20004 1000 Figure 29 Amplification with the AmpF STR Identifiler Plus PCR Amplification Kit in the presence and absence of humic acid Panel 1 corresponds to control samples panels 2 4 correspond to sa
88. ercent stutter cannot be accurately measured for allele peaks that are off scale and may appear unusually high relative to the main peak AmpFtSTR Identifiler Plus User Guide 82 Chapter 5 Experiments and Results 83 Percent Stutter 20 0 19 0 18 0 17 0 16 0 15 0 14 0 13 0 12 0 11 0 10 0 9 0 8 0 7 0 6 0 5 0 4 0 3 0 2 0 1 0 0 0 EER EJ o EO IE annee ne meo ce m DE e e twe e comme mw oe o ee c e m ve 3 1 boc i 8 9 10 1112 13 14 15 16 17 24 25 26 27 28 29 30 31 32 33 34 35 36 6 7 8 9 10 1112 13 14 15 7 8 9 10 1112 13 14 15 D8S1179 D21S11 D7S820 CSF1PO Figure 19 Stutter percentages for the D8S1179 D21S11 D7S820 and CSF1PO loci AmpFtSTR Identifiler Plus User Guide Extra peaks in the electropherogram 19 0 18 0 17 0 16 0 15 0 14 0 13 0 12 0 11 0 10 0 9 0 8 0 7 0 HE Percent Stutter 6 0 5 0 B 40 30 x a 1 0 i 0 0 H i pot a O 11121314 15 16 17 18 1920 567891011 8 9 101112131415 8 9 1011121314 15 16 17 18 1920 2122232425262728 D3S1358 THO1 D13S317 D16S539 D2S1338 Figure 20 Stutter percentages for the D3S1358 THO1 D13S317 D16S539 and D2S1338 loci 19 0 18 0 17 0 16 0 15 0 14 0 13 0 td 12 0 z 11 0 10 0 e 9 0 sjit 8 0 ilil a 3 7 0 1 6
89. feres with PCR by inhibiting polymerase activity To examine the effects of hematin on the amplification results obtained by the Identifiler Plus Kit Control DNA 9947A 1 ng input DNA was amplified with increasing concentrations of hematin for 28 cycles of amplification Figure 28 on page 94 The concentrations of hematin used were 0 uM 100 uM 200 uM and 300 uM see Table 6 LL Mark Sample for Deletio 170 210 250 290 330 m m m Control samples IT Mark Sample for Deletio 10004 100 uM HE200 b 4000 3000 2000 1000 0 HE300 b 4000 3000 20004 1000 AmpFtSTR Identifiler Plus User Guide m m s s s Mark Sample for Deletio 170 210 250 230 330 m m m 200 uM IT Mark Sample for Deletio 170 210 250 230 330 300 uM Figure 28 Amplification with the AmpF STR Identifiler Plus Kit in the presence and absence of hematin Panel 1 corresponds to control samples panels 2 4 correspond to samples amplified in the presence of 100 200 and 300 uM of hematin Table6 Performance in simulated model of hematin inhibition n 3 Identifiler Plus Kit Hematin uM Alleles detected Total alleles 0 26 26 26 26 26 26 100 26 26 26 26 26 26 94 Chapter 5 Experiments and Results Table 6 Performance in simulated model of hematin inhibition n 3 continued Identifil
90. g 121 AmpFtSTR Identifiler Plus User Guide Safety This appendix covers Bi Chemical safety sese sese vH rs Re oe EA e We Po k 123 M Chemical waste safety kk kk kK KK KK KK KK KK KK KK ees 125 m Biological hazard safety 0 kK KK KK KK KK KK KK KK KK K KK KK KK KK 126 m Chemical alerts od o ee Raa hes RY Rees Ee EA CEA 127 AmpFtSTR Identifiler Plus User Guide 122 Appendix B Safety Chemical safety Chemical hazard WARNING CHEMICAL HAZARD Before handling any chemicals refer warning to the Material Safety Data Sheet MSDS provided by the manufacturer and observe all relevant precautions WARNING CHEMICAL HAZARD All chemicals in the instrument including liquid in the lines are potentially hazardous Always determine what chemicals have been used in the instrument before changing reagents or instrument components Wear appropriate eyewear protective clothing and gloves when working on the instrument n WARNING CHEMICAL HAZARD Four liter reagent and waste bottles can crack and leak Each 4 liter bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles m WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shatter
91. he frequency of that genotype in the relevant population s Population The AmpF STR Identifiler PCR Amplification Kit prior to the addition of the samples used in D851179 degenerate primer was used to generate the population data provided in these studies this section Samples were collected from individuals throughout the United States with no geographical preference African American 357 samples were provided by the Kentucky State Police and the Federal Bureau of Investigation U S Caucasian 349 samples were provided by the Kentucky State Police and the Federal Bureau of Investigation U S Hispanic 290 samples were provided by the Minnesota Bureau of Criminal Apprehension Memorial Blood Center of Minneapolis and the Federal Bureau of Investigation Native American 191 samples were provided by the Minnesota Bureau of Criminal Apprehension Memorial Blood Center of Minneapolis In addition to the alleles that were observed and recorded in the Applied Biosystems databases other alleles have been published or reported to Applied Biosystems by other laboratories see the STRBase at www cstl nist gov div831 strbase AmpFtSTR Identifiler Plus User Guide 102 Chapter 5 Experiments and Results AmpF STR Identifiler Plus 103 Kit allele frequencies Table 10 shows the AmpF STR Identifiler Plus Kit allele frequencies in four populations listed as percentages Table 10 AmpF STR Identifiler Plus Kit
92. he AmpF STR Identifiler Plus Kit STR loci Locus edes GRE U S Hispanic moli CSF1PO 0 079 0 132 0 141 0 123 D281338 0 023 0 027 0 038 0 043 D381358 0 097 0 076 0 112 0 158 D58818 0 104 0 147 0 115 0 110 D78820 0 085 0 063 0 083 0 081 D8S1179 0 074 0 064 0 089 0 104 D13S317 0 132 0 079 0 056 0 056 D16S539 0 077 0 097 0 090 0 082 D18S51 0 033 0 031 0 031 0 046 D19S433 0 042 0 087 0 049 0 044 D21S11 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 vWA 0 066 0 066 0 080 0 103 Combined 1 31 x 10 18 5 01 X 10 18 7 65 X 10718 3 62 X 10777 The P value is the probability that two individuals selected at random will have an identical AmpF STR Identifiler Plus 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 10 U S Caucasian 1 1 31 X 10 U S Hispanic and 1 2 76 X 10 6 Native American 115 AmpFtSTR Identifiler Plus User Guide Probability of Paternity Exclusion Probability of Paternity Exclusion Table 13 shows the Probability of Paternity Exclusion Py values of the AmpF STR Identifiler Plus Kit STR loci individually and combined Table 13 Probability of Paternity Exclusion values for the AmpF STR Identifiler Plus Kit loci LOCUS AA Gie a U S
93. he 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 U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories stock no 017 040 00547 4 bmbl od nih gov Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 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 AmpFtSTR Identifiler Plus User Guide 126 Appendix B Safety Chemical alerts For the definitions of the alert words IMPORTANT CAUTION WARNING and DANGER see Safety alert words on page vii General alerts for Avoid contact with skin eyes and or clothing Read the MSDS and follow the all chemicals handling instructions Wear appropriate protective eyewear clothing and gloves Specific ZN CAUTION CHEMICAL HAZARD AmpF STR Identifiler Plus PCR chemical alerts Amplification Kit may cause eye skin and respiratory tract irritation Sodium azide may react with lead and copper plumbing t
94. he optimum 1 0 ng DNA input amount in a maximum input volume of 10 uL 29 PCR cycle protocol Adds the extra sensitivity when amplifying 125 pg DNA inputs Recommended for use when the total DNA input amount is 0 5 ng The results of the developmental validation at both PCR cycle numbers is presented in Chapter 5 on page 68 20 AmpFtSTR Identifiler Plus User Guide Perform PCR Perform PCR ZN WARNING PHYSICAL INJURY HAZARD Thermal cycler 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 Veriti instrument to run in the 9600 Emulation Mode User Bulletin Veriti 96 Well Thermal Cycler AmpFtSTR Kit Validation PN 4440754 Initial incubation Cycle 28 or 29 cycles Final Final hold Siep Denature Anneal Extend extension HOLD CYCLE HOLD HOLD 95 C 94 C 59 C 60 C 4 C 11 min 20 sec 3 min 10 min 00 Refer to the previous section for selecting the appropriate PCR cycle number 2 Load the plate into the thermal cycler and close the heated cover IMPORTANT If using adhesive clear film instead of caps to seal the plate wells be sure to place a MicroAmp compression pad PN 4312639 on top of the plate to prevent evaporation during ther
95. icity 89 split peaks A nucleotide addition 86 STRBase 102 stutter products 81 T text conventions vili thermal cycling parameters validation of 71 programming conditions 21 training information on ix U user attention words described viii user supplied reagents 17 V validation annealing temperatures 71 characterization of loci 88 developmental 69 effect of DNA quantity 91 experiments to evaluate 69 importance of 69 magnesium chloride concentration 70 mixture studies 96 mutation rate 114 PCR cycle number 71 population data 102 probability of identity 115 probability of paternity exclusion 116 sensitivity 91 size deviation sample and ladder alleles 73 species specificity 89 thermal cycling parameters 71 136 Index W WARNING description vii waste disposal guidelines 125 waste profiles description 125 work area amplified DNA tools 16 PCR tools 16 setup 16 workflow overview 6 137 AmpF STR Identifiler Plus User Guide DI PS 5 reg Part Number 4440211 Rev D 03 2012 A ie d Headquarters International Sales Bi t 850 Lincoln Centre Drive Foster City CA 94404 USA For our office locations please call the division IOSyS ems Phone 650 638 5800 Toll Free 800 345 5224 headquarters or refer to our Web site at www appliedbiosystems com www appliedbiosystems com about offices cfm
96. incubated with increasing doses of DNase I 0 to 6 Units for 20 minutes Bender et al 2004 The DNA was examined by agarose gel analysis to determine the average size of the DNA fragments at each time point One nanogram of degraded DNA was amplified using the AmpF STR Identifiler Plus Kit 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 30 130 330 2000 Pa ll Untreated i Li ALL Ali A db i J Ad Ll m sr z 3 Units DNase I 30 130 470 210 250 280 330 400 z 4 Units DNase 30 130 470 210 250 290 330 300 1 ool 5 Units DNase 30 130 170 210 250 290 330 6 Units DNase I Figure 27 28 PCR cycle amplification of Raji DNA samples sonicated and incubated with increasing doses of DNase I Panels 1 2 3 4 and 5 correspond to 0 3 4 5 and 6 units of DNase I 93 AmpFtSTR Identifiler Plus User Guide 9947A b 3000 2000 0 Effect of inhibitors hematin m ja a Stability Heme compounds have been identified as PCR inhibitors in DNA samples extracted from bloodstains DeFranchis er al 1988 Akane et al 1994 It is believed that the inhibitor is co extracted and co purified with the DNA and that it subsequently inter
97. ing Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles Chemical safety To minimize the hazards of chemicals 123 guidelines Read and understand the Material Safety Data Sheets MSDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials See About MSDSs on page 124 Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the MSDS Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the MSDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal AmpFtSTR Identifiler Plus User Guide Chemical safety About MSDSs Chemical manufacturers supply current Material Safety Data Sheets MSDSs with shipments of hazardous chemicals to new customers They
98. laboratory s internal policies and procedures for additional information and references 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 Tube decapper autoclavable Vortex Amplified DNA The following PCR systems should be placed in the amplified DNA work area work area tools 16 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 AmpFtSTR Identifiler Plus User Guide Required user supplied reagents Required user supplied reagents In addition to the AmpF STR Identifiler Plus 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 10mL of 1 M Tris HCl pH 8 0 02mL of0 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 a
99. le 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 Limit of detection of the minor component Mixtures of two genomic DNA samples were examined at various ratios 0 1 1 1 3 1 7 1 10 1 15 1 1 0 The total amount of genomic input DNA mixed at each ratio was 1 ng The samples were amplified in a GeneAmp PCR System 9700 then electrophoresed and detected using an Applied Biosystems 3130x Genetic Analyzer The results of the mixed DNA samples are shown in Figures 30 28 PCR cycles and 31 29 PCR cycles on page 99 where samples A and B were mixed according to the ratios provided Using either the 28 or 29 PCR cycle protocol the minor component allele calls at non overlapping loci are highlighted The amplification of the minor contributor at 3 1 7 1 0 875 0 125 ng and 10 1 0 9 0 09 ng mixture ratios was readily typeable 15 1 0 9375 0 0625 ng mixture ratios resulted in full or partial profiles for the minor component The profiles of these samples are described in Table 9 AmpFtSTR Identifiler Plus User Guide 98 Chapter 5 Experiments and Results 30 130 170 210 250 290 330
100. ler Plus User Guide Part Number 4440211 Rev D 03 2012 Experiments and Results This chapter covers M OVER VIS Wie gt Aye la bete und epe Qd e ho data PE done Re e1 s aet te 69 E Developmental validation 0 0 ccc KK KK KK KK KK KK KK KK KO 70 n Accuracy precision and reproducibility essel 73 m Extra peaks in the electropherogram kk KK KK RR RR KK KI KK 81 n Characterization of loci 1 2 kk KK KK KK KK KK KK KK KK KK KK KK KK KK KK 88 B Species specificity iep neea EEG LO HHHH O 89 B Sensitivity 00 Sicha ela es BO OO LA HHH a E a 91 B Stability y yz w H l o ATE PARZE OE Se AO bte o Leeds 93 m Mixture Studies s tese a KA AO ers ets 96 M Population Data xe eren inoia a K mmm 102 B Mutation Rate eisses gt a l xe dalal dhe aha hh An Al 114 n Probability of Identity 0 0 0 KK KK KK KK KK KK KK KK KK K KK KK KK KK 115 m Probability of Paternity Exclusion KK KK KK KK KK KK eee eee 116 AmpFtSTR Identifiler Plus User Guide 68 Chapter 5 Experiments and Results Overview Experiments using the AmpF STR Identifiler Plus Kit Importance of validation Experiment conditions 69 This chapter provides results of the developmental validation experiments performed by Applied Biosystems using the AmpF STR Identifiler Plus PCR Amplification Kit Validation of a DNA typing procedure for human identification applications is a
101. llele but whose size is just outside a window because of measurement error 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 on a capillary instrument or in several lanes of one gel Table 4 on page 75 shows typical precision results obtained from five runs 16 capillaries run of the AmpF STR Identifiler Plus Allelic Ladder on the Applied Biosystems 3130x Genetic Analyzer 36 cm capillary and POP 4 polymer The internal size standard that was used was GeneScan 500 LIZ Size Standard The results were obtained within a set of injections on a single capillary array Sample alleles may occasionally size outside of the 0 5 nt 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 18 on page 73 illustrates the tight clustering of allele sizes obtained on the Applied Biosystems 3130x Genetic Analyzer where the standard deviation in sizing is typically less than 0 15 nt The instance of a sample allele sizing outside the 0 5 nt window because of measurement error is relatively rare when the standard deviation in sizing is approximately 0 15 nt or less Smith 1995 For
102. llele size from the corresponding allelic ladder allele size All sample alleles are within 0 5 nt from a corresponding allele in the allelic ladder 0 50 CSF1PO D2S1338 D3S1358 D5S818 D7S820 D8S1179 D13S317 D16S539 D18S51 D19S433 D21S11 FGA THO1 TPOX vWA AMEL OtuoobOobotutbo ono Size Difference nt 0 50 TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT 100 120 140 160 180 200 220 240 260 280 300 320 340 360 Allele Size nt Figure 18 Size deviation of 200 samples analyzed on the Applied Biosystems 3130x Genetic Analyzer For each sample 1 0 ng of DNA was amplified for 28 PCR cycles 73 AmpFtSTR Identifiler Plus User Guide Accuracy precision and reproducibility Precision and Sizing precision allows for determining accurate and reliable genotypes Sizing size windows precision was measured on the Applied Biosystems 3130x Genetic Analyzer The recommended method for genotyping is to employ a 0 5 nt window around the size obtained for each allele in the AmpF STR Identifiler Plus Allelic Ladder A 0 5 nt window allows for the detection and correct assignment of alleles Any sample allele that sizes outside the specified window could be e An off ladder allele that is an allele of a size that is not represented in the AmpF STR Identifiler Plus Allelic Ladder or e An allele that does correspond to an allelic ladder a
103. macaque 1 0 ng each Non primates mouse dog sheep rabbit cat horse hamster rat chicken and cow 10 ng each Microorganisms Candida albicans Staphylococcus aureus Escherichia coli Neisseria gonorrhoeae Bacillus subtilis and Lactobacillus rhamnosus equivalent to 10 copies The chimpanzee and gorilla DNA samples produced partial profiles within the 70 to 350 nucleotide region The microorganisms chicken hamster mouse rabbit and rat did not yield detectable products Dog horse sheep and cow produced a 98 bp fragment near the amelogenin locus in the PET dye AmpFtSTR Identifiler Plus User Guide Species specificity 75 115 155 195 235 275 315 355 395 Control DNA 9947A 75 115 155 195 235 275 315 355 395 2000 Chimpanzee 115 155 195 235 275 315 355 385 115 155 195 235 275 315 355 395 75 115 155 195 235 275 315 355 385 wl Microbial pool 20d MS A85 49s 225 zs i 315 i 355 395 m NTC 420 4l Figure 24 Representative electropherograms from a species specificity study including positive and non template controls NTC run for 28 PCR cycles AmpFtSTR Identifiler Plus User Guide 90 Chapter 5 Experiments and Results Sensitivity SWGDAM guideline 2 3 Importance of quantitation Effect of DNA quantity on results 91 When appropriate the range of DNA quantities
104. mal cycling 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 lt 2 weeks 2to8 C gt 2 weeks 15 to 25 C IMPORTANT Store the amplified products so that they are protected from light AmpFtSTR Identifiler Plus User Guide 21 Chapter 2 PCR Amplification Amplification using bloodstained FTA cards 2 cycle 3 22 2800 2000 1600 1200 400 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 Applied Biosystems studies indicate that a 1 2 mm bloodstained disc contains approximately 5 to 20 ng DNA An appropriate cycle number for this high quantity of DNA is 24 cycles as determined by Applied Biosystems 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 24 cycles Mark Sample for Deletion 155 195 235 275 315 355 395 A L An MANI
105. mount 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 AmpF STR Identifiler Plus Kit is 1 0 ng in a maximum input volume of 10 uL for 28 PCR cycles and 0 5 ng in a maximum input volume of 10 uL for 29 PCR cycles If too much DNA is added to the PCR reaction then the increased amount of PCR product that is generated can result in 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 1s 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 AmpFtSTR Identifiler Plus User Guide 17 Chapter 2 PCR Amplification Methods of Applied Biosystems provides several kits for quantifying DNA in samples See the quantifying DNA reference cited in the following table for
106. mples amplified in the presence of 50 100 and 150 ng uL humic acid 95 AmpFtSTR Identifiler Plus User Guide Mixture studies Table 7 Performance in simulated model of humic acid inhibition n 3 Humic Acid ng uL Identifiler Plus Kit 0 26 26 26 26 26 26 50 26 26 26 26 26 26 100 26 26 26 26 26 26 150 26 26 26 26 26 26 t Only those peaks gt 50 RFUs were counted A complete profile with Control 9947A DNA yields 26 peaks using the AmpFZSTR Identifiler Plus Kit Mixture studies SWGDAM The ability to obtain reliable results from mixed source samples should be guideline 2 8 determined SWGDAM July 2003 Evidence samples may contain DNA from more than one individual The possibility of multiple contributors should be considered when interpreting the results Applied Biosystems recommends 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 Mixture Studies 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 Mixed samples can be distinguished from single source samples by The presence of more than two alleles at a locu
107. mulated 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 DYS392 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 HUMTHO1 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
108. n 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 and Wallin et al 1998 Experiments to evaluate the performance of the AmpF STR Identifiler Plus PCR Amplification Kit were performed at Applied Biosystems The experiments were performed 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 Additional validation was performed according to the revised guidelines from the Scientific Working Group on DNA Analysis Methods SWGDAM July 10 2003 Based on these guidelines Applied Biosystems conducted experiments that comply with guidelines 1 0 and 2 0 and its associated subsections This DNA methodology is not novel Moretti et al 2001 Frank et al 2001 Wallin et al 2002 and Holt et al 2000 This chapter discusses many of the experiments performed by Applied Biosystems and provides examples of results obtained Applied Biosystems chose conditions that produc
109. n be requirements used to analyze Identifiler Plus PCR products For details on the procedures refer to the documents listed in the table Operating Data system Collection Run modules and conditions References Software Windows 3 17 GS STR POP4 1mL G5 v2 md5 ABI Prism 310 Genetic Analyzer User s Manual XE or Injection condition 15 kV 5 sec Windows PN 4311988 or ABI PRISM 310 Protocols for Processing 3 0 AmpFtSTR PCR Amplification Kit Products with Windows Microsoft Windows NT Operating System User NT and Bulletin PN 4341742 Windows 2000 t Applied Biosystems conducted concordance studies for the Identifiler Plus kit using this configuration 2 u U D 7 3 o re o 3 e ct o gt 5 D N e AmpFtSTR Identifiler Plus User Guide 30 Section 3 2 310 instrument Prepare samples for electrophoresis on the 310 instrument Prepare the Prepare the samples for capillary electrophoresis on the 310 instrument immediately samples before loading l 31 Calculate the volume of Hi Di Formamide and GeneScan 500 LIZ Internal Size Standard needed to prepare the samples using the table below Reagent Volume per reaction uL GeneScan 500 LIZ9 Size Standard 0 5 Hi Di Formamide 24 5 Note Include additional samples in your calculations to provide excess volume for the loss that occurs during reagent transfers IMPORTANT The volume
110. nd 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 Performing the exact test of Hardy Weinberg proportion for multiple alleles Biometrics 48 361 372 Hammond H Jin L Zhong Y Caskey C and Chakraborty R 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 K 1993 Th
111. neMapper ID X Manager xi Find Name Containing Projects Analysis Methods Table Settings Plot Settings Matrices Size Standards Report Settings Name Last Saved Owner Instrument Analysis Type Description AmpFLSTR Analysis Method 2007 12 18 16 18 2 gmidx HID New Open Save 5 Import Export Delete Help Done b Navigate to then open the Identifiler Plus Analysis Files GMIDX folder c Select Identifiler_Plus_AnalysisMethod_v1X then click Import to import Identifiler Plus AnalysisMethod v1X into the GeneMapper ID X Software database g8 Import Analysis Method x Look in Identifiler Plus Analysis Files GMIDX M i2 HE ES My Recent Documents Desktop WPESTUZUEJ File name Identifier Plus AnalysisMethod v1x xml Import T Files of type xv Files xml Cancel CE G5 Identifiler Plus GS500 xml le Identifiler Plus AnalysisMethod v1X xml 3 To view the settings for Identifiler Plus AnalysisMethod v1X select the Analysis Methods tab then select Identifiler Plus AnalysisMethod v1X in the Name column and click Open 57 AmpFtSTR Identifiler Plus User Guide Set up GeneMapper ID X Software for data analysis Find Name Containing Projects Analysis Methods Table Settings Plot Settings Matrices Size Standards Report Settings Name Last Saved Owner pere Analysis Type t Default j2009 08 3
112. ntages for AmpF STR9 Identifiler Plus PCR Amplification Kit loci continued Locus 9o Stutter TPOX 6 3832 VWA 12 446 t These percentages are used as stutter filters in used in GeneMapper ID v3 2 1 Identifiler Plus Panels v1 and GeneMapper D X software v1 0 1 v1 1 or v1 1 1 Identifiler Plus Panels v1x Addition of 3 A nucleotide Many DNA polymerases can catalyze the addition of a single nucleotide predominately adenosine to the 3 ends of double stranded PCR products Clark 1988 and Magnuson et al 1996 This nontemplate addition results in a PCR product that is one nucleotide longer than the actual target sequence The PCR product with the extra nucleotide is referred to as the A form The efficiency of A addition is related to the particular sequence of the DNA at the 3 end of the PCR product The Identifiler Plus 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 10 min This final extension step gives the DNA polymerase additional time to complete A addition to all double stranded PCR products STR systems where each allele is represented by two peaks that are one nucleotide apart that have not been optimized for A addition may have split peaks AmpFtSTR Identifiler Plus User Guide 86 Chapter 5 Experiments and Results 87 Artifacts ER 1
113. ntifiler Plus Kit IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the AmpF STR Identifiler Plus Primer Set from light when not in use Amplified DNA AmpF STR Identifiler Plus Allelic Ladder and GeneScan 500 LIZ Size Standard should also be protected from light Keep freeze thaw cycles to a minimum The following table lists Data Collection Software and the run modules that can be or 3130 3130x used to analyze Identifiler Plus PCR products For details on the procedures refer instrument to the documents listed in the table requirements Operatin Data r stem Collection Run modules and conditions References y Software Windows 3 0 e HIDFragmentAnalysis36 POPA 1 Applied Biosystems 3130 3130xl Genetic XP 3130 3130x Injection conditions Analyzers Using Data Collection Software v3 0 Analyzer Protocols for Processing AmpF STR PCR 3130 3 kV S sec Amplification Kit PCR Products User Bulletin 3130x 3 kV 10 sec PN 4363787 e Dye Set G5 Windows 2 0 e HIDFragmentAnalysis36_POP4_1 ABI Prism 3100 3100 Avant Genetic 2000 3100 Injection condition 3 kV 10 sec Analyzers Using Data Collection Software v2 0 Analyzer D G Protocols for Processing AmpF STR PCR Dye Set G5 Ambplification Kit PCR Products User Bulletin PN 4350218 Windows 1 1 GeneScan36vb_DyeSetG5Module ABI Prism 3100 3100 Avant Genetic NT 3100 Injection condition 3 kV 10 sec Analyzers Protocols f
114. o To foo oo bo fo Amelogenin Cutoff po Range Filter Figure 6 Analysis Method Editor HID Allele tab settings 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 AmpFtSTR Identifiler Plus User Guide 44 Section 4 1 GeneMapper ID Software The Use marker specific stutter ratio if available check box is selected by default Consequently the software applies the stutter ratio filters supplied in the Identifiler Plus Panels v1 file For more information about allele filters refer to GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide Chapter 3 PN 4338775 GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial PN 4335523 Installation Procedures and New Features for GeneMapper ID Software v3 2 User Bulletin PN 4352543 Analysis Method Editor HID x General Allele Peak Detector Peak Quality Quality Flags Peak Detection Algorithm Advanced Ranges Peak Detection Analysis Sizing Peak Amplitude Thresholds Fun Range All Sizes m p B so R 150 Start PE fo Start SIZen 5 G so O 150 LE so ii Stop PE 0000 Stop Size iso Smoothing and Baselining Min Peak Half Width j pts Smoothing None Light Polynomial Degree j C
115. o be limited to the most extreme situations MSDSs The MSDSs for any chemicals supplied by Applied Biosystems or Ambion are available to you free 24 hours a day For instructions on obtaining MSDSs see Obtaining MSDSs on page 124 IMPORTANT For the MSDSs of chemicals not distributed by Applied Biosystems or Ambion contact the chemical manufacturer AmpFtSTR Identifiler Plus User Guide vii Preface How to use this guide Purpose of this guide Pull out chapters Text conventions viii User attention words The Applied Biosystems AmpF STR Identifiler Plus PCR Amplification Kit User Guide provides information about the Applied Biosystems instruments chemistries and software associated with the AmpF STR Identifiler Plus PCR Amplification Kit This guide is designed to allow users to pull out chapters 2 3 and 4 The pull out chapters have title and back pages which indicate the chapter number and title This guide uses the following conventions Bold text indicates user action For example Type 0 then press Enter for each of the remaining fields Italic text indicates new or important words and is also used for emphasis For example Before analyzing always prepare fresh matrix Aright arrow symbol separates successive commands you select from a drop down or shortcut menu For example Select File Open gt Spot Set Right click the sample row then select View Filter
116. o form highly explosive metal azides WARNING CHEMICAL HAZARD POP 4 Polymer for 3130 3130x Genetic Analyzers causes skin eye and respiratory tract irritation WARNING CHEMICAL HAZARD Running Buffer 10X causes skin eye and respiratory tract irritation WARNING CHEMICAL HAZARD Hi Di Formamide is harmful if swallowed inhaled or absorbed through skin and causes irritation to skin eyes and respiratory tract It affects the central nervous system and may affect the reproductive system WARNING CHEMICAL HAZARD POP 4 Polymer for 3100 3100 Avant Genetic Analyzers is irritating to eyes respiratory system and skin It causes adverse cardiovascular effects It contains a known or suspected reproductive toxin and a known or suspected mutagen 127 AmpFtSTR Identifiler Plus 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 B J and Parkin B H 1996 Structural variation in the alleles of a short tandem repeat system at the human alpha fib
117. oci there is consensus on the repeat patterns and structure of the STRs Inheritance The Centre d Etude du Polymorphisme Humain CEPH has collected DNA from 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 Three CEPH family DNA sets were examined One nanogram of DNA from each sample was amplified using the Identifiler Plus Kit followed by analysis using an Applied Biosystems 3130x Genetic Analyzer The families examined included 1333 9 offspring 1340 7 offspring and 1345 7 offspring representing 23 meiotic divisions In family 1340 we observed two parent offspring pairs with mutations at locus D8S1179 The genotypes differed by one repeat unit between the two generations Calculation of a mutation rate based on these data would be inaccurate due to the small sample size The other parent offspring allele transfers were in accordance with Mendelian rules AmpFtSTR Identifiler Plus User Guide 88 Chapter 5 Experiments and Results Mapping The Identifiler Plus Kit loci Amelogenin CSF1PO D2S1338 D3S1358 D5S818 D7S1179 D13S317 D16S
118. of size standard indicated in the table is a suggested amount Determine the appropriate amount of size standard based on your results and experiments Pipette the required volumes of components into an appropriately sized polypropylene tube Vortex the tube then centrifuge briefly Into a 0 2 mL or 0 5 mL sample tube add 25 uL of the formamide size standard mixture 1 5 uL of PCR product or allelic ladder Seal the tubes with the appropriate septa then briefly vortex and centrifuge the tubes to ensure that the contents of each tubes 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 Start the electrophoresis run AmpFtSTR Identifiler Plus User Guide Part Number 4440211 Rev D 03 2012 Chapter 4 Data Analysis AmpFtSTR Identifiler Plus User Guide AmpFtSTR Identifiler Plus User Guide Data Analysis This chapter covers Section 4 1 GeneMapper ID Software esee 37 m Overview of GeneMapper ID Software 37 m Setup GeneMapper ID Software for data analysis 38 m Analyze and edit sample files with GeneMapper ID Software 48 m Examine and edit a project 0 KK KK KK KK KK KK KK KK eh 49 Section 4 2 GeneMapper ID X Software sese 51 m Overview of GeneMapper ID X Software 4444111111 51 m
119. of the 15 STR loci and Amelogenin during automated DNA fragment analysis Loci amplified by the kit The following table shows the loci amplified their chromosomal locations and the corresponding fluorescent marker dyes The AmpF STR Identifiler Plus Allelic Ladder is used to genotype the analyzed samples The alleles contained in the allelic ladder and the genotype of the AmpF STR Identifiler Plus Control DNA 9947A are also listed in the table Table 1 AmpF STR Identifiler Plus Kit loci and alleles gt 3 Chromosome Alleles included in Identifiler Plus Dye Control DNA Locus designation location Allelic Ladder label 9947A D8S1179 8 8 9 10 11 12 13 14 15 16 17 18 19 6 FAM 137 D21511 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 7q11 21 22 6 7 8 9 10 11 12 13 14 15 10 11 CSF1PO 5q33 3 34 6 7 8 9 10 11 12 13 14 15 10 12 D381358 3p 12 13 14 15 16 17 18 19 VIC9 14 15 THO1 11p15 5 4 5 6 7 8 9 9 3 10 11 13 3 8 9 D13S317 13q22 31 8 9 10 11 12 13 14 15 117 D165S539 16q24 qter 5 8 9 10 11 12 13 14 15 11 12 D281338 2035 37 1 15 16 17 18 19 20 21 22 23 24 25 19 23 26 27 28 D195433 19q12 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 vWA 12p12 pter 11 12 13 14 15 16 17 18
120. on page 21 Repeat PCR amplification using fewer PCR cycles or use your laboratory s SOP to analyze off scale data Poor spectral separation bad matrix Follow the steps for creating a spectral file Confirm that Filter Set G5 modules are installed and used for analysis Too much DNA in reaction Use recommended amount of template DNA 1 0 ng Some but not all loci visible on electropherogram 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 100 centrifugal filter unit Repeat test 119 AmpFtSTR Identifiler Plus User Guide Table 14 Troubleshooting continued Observation Possible causes Recommended actions ance Poor peak height bal Incorrect thermal cycler parameters Check the protocol for correct thermal cycler parameters GeneAmp PCR System 9700 with Aluminum 96 Well block or third party thermal cyclers Use Applied Biosystems GeneAmp PCR System 9700 with silver or gold plated silver blocks only AmpFtSTR Identifiler Plus User Guide 120 Appendix A Troubleshootin
121. oncordance studies 113 AmpF amp TR Identifiler Plus User Guide contents of kit 9 18 control DNA 9947A 5 9 conventions bold text viii for describing menu commands viii IMPORTANTS viii in this guide viii italic text viii Notes viii user attention words viii customer feedback on Applied Biosystems documents 133 cycle number validation 71 D DANGER description vii data accuracy precision and reproducibility 73 Data Collection Software 7 Data Collection Software overview 7 data accuracy precision and reproducibility of 70 data artifacts 87 data for different populations 102 developmental validation 69 DNA amplified 16 control about 9 degraded 93 effect of quantity figure 92 mixture studies 96 mixture studies figure 98 negative control reaction 19 positive control reaction 19 quantification 17 quantification methods 18 sample preparation 19 sensitivity 91 test sample 19 tools 16 DNA mixtures amplification figure 100 limit of detection 98 documentation related 132 134 Index E electropherogram causes of extra peaks 75 81 extra peaks 81 species specificity 90 93 electrophoresis Data Collection Software 28 30 preparing samples on the 310 instrument 31 preparing samples on the 3100 3100 Avant or 3130 3130x instrument 29 reagents and parts 28 30 references 28 30 run module 28 30 setup 28 30 emission spectra 8 equipment not included in kit 10 experiments and results 68 extra peaks
122. or Processing AmpFtSTR Analyzer OAnalvsi PCR Amplification Kit PCR Products User GS500Analysis gsp Bulletin PN 4332345 1 0 e GeneScan36Avb_DyeSetG5Module ABI PRISM 3100 3100 Avant Genetic 3100 Avant Injection condition 3 KV 5 sec Analyzers Protocols for Processing AmpF STR Analyzer PCR Amplification Kit PCR Products User GS500Analysis gsp Bulletin PN 4332345 Applied Biosystems conducted validation studies for the Identifiler Plus Kit using this configuration AmpFtSTR Identifiler Plus User Guide 28 e Q e EX o ES 3 szej w 2 a o G S EX n e e 2 o 2 0 Section 3 1 3100 3100 Avant and 3130 3130xl instruments Prepare samples for electrophoresis on the 3100 3100 Avant or 3130 3130x instrument 29 Prepare the Prepare the samples for electrophoresis on the 3100 3100 Avant or 3130 3130x instrument immediately before loading Calculate the volume of Hi Di Formamide and GeneScan 500 LIZ Size Standard needed to prepare the samples using the table below Reagent Volume per reaction uL GeneScan 500 LIZ Size Standard 0 3 Hi Di Formamide 8 7 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 b
123. ossible 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 vol ume Repeat test Wash the sample in a Centricon 100 centrifugal filter unit Repeat test Test sample DNA is severely degrad ed If possible evaluate the quality of DNA sample by running an agarose gel If DNA is degraded ream plify 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 one allele present at a locus Presence of exogenous DNA Use appropriate techniques to avoid introducing foreign DNA during laboratory handling Amplification of stutter product Mixed sample See Stutter products on page 81 Incomplete 3 A base addition n 1 nt position See Addition of 3 A nucleotide on page 86 Be sure to include the final extension step of 60 C for 10 min in the PCR Signal exceeds dynamic range of instrument off scale data Ensure cycle number is optimized according to instructions
124. ply 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 Plus User Guide Set up GeneMapper ID X Software for data analysis Set up GeneMapper D X Software for data analysis Workflow Before you can analyze sample fsa files using GeneMapper ID X Software v1 0 1 v1 1 or v1 1 1 for the first time Import panels bins and marker stutter into the Panel Manager as explained in Import panels bins and marker stutter on page 52 Import an analysis method as explained in Import an analysis method on page 57 Importa size standard as explained in Import a HID size standard on page 62 Define custom views of analysis tables Define custom views of plots For more info For quick set up instructions refer to the GeneMapper D X Software Version 1 0 Getting Started Guide PN 4375574 For details about the GeneM
125. pplied reagents u aaaaaaaaa KK KK KK KK KK KK KK Prepare the amplification kit reactions e aeaaaaaa aaa KK KK Chapter 2 PCR Amplification PCR work areas Work area setup and lab design PCR setup tools Many resources are available for the appropriate design of a PCR laboratory e Ifyou are using the AmpF STR Identifiler Plus PCR Amplification Kit for forensic DNA testing refer to Forensic Laboratories Handbook for Facility Planning Design Construction and Moving National Institute of Justice 1998 http nij ncjrs gov publications pubs db asp Ifyou are using the AmpF STR Identifiler Plus Kit for 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 AmpF STR Identifiler Plus 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 To prevent contamination by human DNA be careful while handling and processing samples 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 These laboratory design resources and guidances constitute only a sample of the precautions that need to be observed when using PCR technology Refer to your
126. r Plus Allelic Ladder For more information on precision and genotyping see Lazaruk et al 1998 and Mansfield et al 1998 In Table 4 the mean sizes for all the alleles in each run 16 capillaries were calculated The mean range shown in the table represents the lowest and highest mean size values obtained across all five runs Similarly the standard deviation for the allele sizing was calculated for all the alleles in each run The standard deviation range shown in Table 4 represents the lowest and highest standard deviation values obtained across all five runs Table 4 Precision results of five runs 16 capillaries run of the AmpF STF Identifiler Plus Allelic Ladder Applied Biosystems 3130x Genetic Analyzer 75 Allele Mean Standard Deviation Amelogenin X 106 03 106 13 0 033 0 045 Y 111 69 111 8 0 03 0 042 CSF1PO 6 303 99 304 12 0 041 0 063 7 308 04 308 17 0 037 0 058 8 312 1 312 2 0 039 0 065 9 316 13 316 25 0 035 0 045 10 320 18 320 3 0 034 0 055 11 324 24 324 34 0 03 0 046 12 328 3 328 39 0 025 0 047 13 332 36 332 44 0 032 0 037 14 336 39 336 49 0 024 0 039 15 340 42 340 53 0 038 0 05 D13S317 8 216 36 216 48 0 031 0 064 9 220 34 220 48 0 035 0 051 10 224 32 224 45 0 034 0 059 11 228 31 228 45 0 031 0 065 12 282 42 32 55 0 031 0 063 13 236 3 236 43 0 038 0 066 14 240 24 240 37 0 043 0 058 15 244 23 244 37 0 037 0 066 AmpFtSTR
127. r of detected peaks Although GeneMapper D X Software displays peaks that fall below the specified amplitude in electropherograms the software does not label or determine the genotype of these peaks IMPORTANT Laboratories need to perform the appropriate internal validation studies to determine the peak amplitude thresholds that allow for reliable interpretation of AmpF STR Identifiler Plus PCR Amplification Kit data For more information about peak detection algorithms refer to GeneMapper ID X Software Version 1 0 Getting Started Guide PN 4375574 GeneMapper ID X Software Version 1 0 Quick Reference Guide PN 4375670 GeneMapper D X Software Version 1 0 Reference Guide PN 4375671 C o mi D lt ko o 0 8 w gt n 9 zh 2 0 AmpFtSTR Identifiler Plus User Guide 60 Section 4 2 GeneMapper ID X Software Analysis Method Editor xl General Allele Peak Detector Peak Quality sq amp GQ Settings r MinfMax Peak Height LPH MPH Homozygous min peak height 100 0 Heterozygous min peak height m Max Peak Height MPH 8000 0 r Peak Height Ratio PHR Min peak height ratio o 7l Broad Peak BD Max peak width basepairs Allele Number AN Max expected alleles Allelic Ladder Spike Cut off value Factory Defaults Save As Save Car cel Help Figure 13 Analysis Method Editor Peak Quality tab set
128. raded 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 DNA The two human DNA specific assays each consist of two PCR primers and a TaqMan probe The TaqMan probes for the human DNA and human male DNA assays are labeled with VIC and FAM dyes respectively In addition the kit contains an internal PCR control IPC assay similar in principle to that used in the other Quantifiler kits but labeled with NED dye Quantifiler Duo DNA Quantification Kit User s Manual PN 4391294 18 AmpFtSTR Identifiler Plus User Guide 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 uL AmpF STR Identifiler Plus Master Mix 10 0 AmpF STR Identifiler Plus Primer Set 5 0 Note Include additional reactions in your calculations to provide excess volume for the loss that occurs during reagent transfers 2 Prepare reagents Thaw the AmpF STR Identifiler Plus Kit Master Mix and the AmpF STR Identifiler Plus Kit Primer Set then vortex 3 seconds and centrifuge briefly before opening the tubes IMPORTANT Thawing is required only during first
129. ree tetranucleotide polymorphisms for loci D3S1352 D3S1358 D3S1359 Hum Mol Genet 2 1327 AmpFtSTR Identifiler Plus User Guide 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 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 PD Budowle B and McCord B R 1998 Analysis of multiplexed short tandem repeat STR systems using capillary array electrophoresis Electrophoresis 19 101 107 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 M ller A Meyer E and Brinkmann B 1994 Different types of structural variation in STRs HumFES FPS HumVWA and HumD21S11 Intl J Legal Med 106 319 323 Momhinweg E Luckenbach C Fimmers R and Ritter H 1998 D3S1358 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 si
130. rinogen locus ntl 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 loci D18S51 and D8S1179 Intl J Legal Med 109 62 65 Baron H Fung S Aydin A Bahrig S Luft FC and Schuster H 1996 Oligonucleotide ligation assay OLA for the diagnosis of familial hypercholesterolemia 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 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
131. rofiles At 63 C the yield of the majority of loci was significantly reduced Routine thermal cycler calibration is recommended when you follow the amplification protocol No preferential amplification was observed at the standard annealing extension temperature of 59 C PCR cycle number 71 Figure 16 Electropherograms obtained from amplification of 1 0 ng of Control DNA 9947A at annealing extension temperatures of 55 C 57 C 59 C 61 C and 63 C analyzed on the Applied Biosystems 3130x Genetic Analyzer Y axis scale 0 to 4 000 RFUs AmpF STR Identifiler Plus PCR Amplification Kit reactions were amplified for 26 27 28 29 and 30 cycles on the Silver 96 Well GeneAmp PCR System 9700 using 1 0 ng from three DNA samples As expected the amount of PCR product increased with the number of cycles A full profile was generated at 26 cycles and off scale data were collected for several allele peaks at 30 cycles Figure 17 AmpFtSTR Identifiler Plus User Guide Developmental validation Although none of the cycle numbers tested produced nonspecific peaks 28 cycles was found to give optimal peak heights with 1 ng of DNA input when the amplified products were examined on Applied Biosystems 3130x Genetic Analyzers 75 115 155 195 235 275 315 355 395 3o00 26 cycles 115 155 195 235 275 315 355 395 wo 27 cycles 28 ada 2000 1000 0 75 115 155 195 235 275
132. s 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 minimum and maximum peak height ratios observed for alleles in the AmpF STR Identifiler Plus PCR Amplification Kit loci in unmixed population database samples are shown in Table 8 AmpFtSTR Identifiler Plus User Guide 96 Chapter 5 Experiments and Results Table 8 Peak height ratios for 1 0 ng of input DNA amplified for 28 PCR cycles Number of Locus bak Mean Median Minimum Maximum Amel 320 90 23 91 29 46 21 99 89 CSF1PO 378 90 37 91 56 67 19 99 96 D13S317 375 90 61 92 09 68 28 99 94 D16S539 389 90 03 91 15 68 11 99 87 D18S51 432 90 06 91 24 62 26 99 94 D19S433 399 90 2 91 2 57 29 99 96 D21S11 428 90 3 91 12 69 05 100 D281388 436 90 36 91 56 63 86 100 D381358 356 91 39 92 66 62 82 99 94 D5S818 357 91 15 92 21 66 07 100 D7S820 395 90 49 92 07 46 61 99 94 D881179 396 91 22 92 7 67 42 100 FGA 429 89 83 91 07 60 38 99 87 THO1 362 91 62 93 04 70 09 100 TPOX 333 91 17 92 18 70 65 100 VWA 414 91 16 92 33 65 22 100 Actual DNA input amounts 0 7 ng to 1 3 ng If an unusually low peak h
133. s XP 3 0 GeneMapper D Software v3 2 1 3100 3100 Windows NT 1 1 3100 and Avant 1 0 3100 Avant e GeneMapper D X Software v1 0 1 or higher Windows 2000 2 0 310 Windows XP 3 1 Window NT and 3 0 Windows 2000 t Applied Biosystems conducted validation studies for the AmpF STR9 Identifiler Plus Kit using this configuration About Applied Biosystems fluorescent multi color dye technology allows the analysis of multicomponent multiple loci including loci that have alleles with overlapping size ranges Alleles for analysis 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 spectral components The four dyes used in the AmpF STR Identifiler Plus PCR Amplification Kit to label samples are 6 FAM VIC NED and PET dyes The fifth dye LIZ dye is used to label the GeneScan 500 LIZ Size Standard AmpFtSTR Identifiler Plus User Guide 7 Chapter 1 Overview How Each of these fluorescent dyes emits its maximum fluorescence at a different multicomponent wavelength During data collection on the Applied Biosystems and ABI PRISM analysis works 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 shorte
134. s Analysis Files GMIDX v S m HEES e My Recent Documents Desktop 4 Le File name cE_65_Identifler_Plus_G5500 xml Import w T Files of type xw Files xml Cancel w CE G5 Identifiler Plus GSS500 xml Identifiler Plus AnalysisMethod v1x xml 63 AmpFtSTR Identifiler Plus User Guide Analyze and edit sample files with GeneMapper ID X Software Analyze and edit sample files with GeneMapper D X Software Analyze a project 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 Plus AnalysisMethod v1X Panel Identifiler Plus Panel v1X Size Standard CE G5 Identifiler Plus GS500 t For more information about how the Size Caller works refer to the ABI PRISM GeneScan Analysis Software for the Windows NT9 Operating System Overview of the Analysis Parameters and Size Caller User Bulletin PN 4335617 The following fragments are defined for the CE G5 Identifiler Plus GS500 size standard provided with the AmpF STR Identifier Plus Kit 75 100 139 150 160 200 300 350 400 and 450 For additional information about size standards refer to the GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide PN 4338775 Appendix D
135. sample alleles that do not size within a 0 5 nt window the PCR product must be rerun to distinguish between a true off ladder allele versus 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 JD Software and GeneMapper ID X Software automatically flag sample alleles that do not size within the prescribed window around an allelic ladder allele by labelling the allele as OL off ladder Maximum precision is obtained with a set of capillary injections on each of the supported platforms however the determined allele sizes will vary between the different platforms Cross platform sizing differences occur from a number of factors including type and concentration of polymer run temperature and electrophoresis conditions Variations in sizing can also occur between runs on the same instrument and between runs on different instruments of the same platform type because of these factors AmpFtSTR Identifiler Plus User Guide 74 Chapter 5 Experiments and Results Applied Biosystems strongly recommends that the allele sizes be compared to the sizes obtained for known alleles in the AmpF STR Identifiler Plus Allelic Ladder from the same run and then be converted to genotypes as described in Before you start on pages 37 and 51 See Table 4 for the results of five runs of the AmpF STR Identifile
136. sh 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 analysis and characterization of stutter products at the tetranucleotide repeat locus vWA 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 Plus User Guide Documentation Related documentation For additional documentation see How to obtain support on page ix Document title Part number ABI PRISM 3100 3100 Avant Data Collection v2 0 User Guide 4347102 ABI PRisM 3100 3100 Avant Genetic Analyzers Using Data Collection Software v2 0 User Bulletin 4350218 ABI PRISM 3100 Genetic Analyzer User Manual Data Collection v1 1 4315834 ABI PRISM 3100 3100 Avant Genetic Analyzers Protocols for Processing AmpF STR PCR Amplification 4332345 Kit PCR Products User Bulletin Applied Bios
137. sizing algorithm 75 100 139 150 160 200 300 350 400 and 450 Use the following procedure to import the size standard for the AmpF STR Identifiler Plus PCR Amplification Kit from the folder that you downloaded from the Applied Biosystems web site into the GeneMapper D X Software database Refer to step 1 on page 52 for downloading instructions C o mi D ko go 0 8 w gt n 9 zh 2 0 1 Select Tools GeneMapper ID X Manager to open the GeneMapper D X Manager AmpFtSTR Identifiler Plus User Guide 62 Section 4 2 GeneMapper ID X Software 2 Import a size standard a Select the Size Standards tab then click Import s GeneMapper ID X Manager xl Find Name Containing Projects Analysis Methods Table Settings Plot Settings Matrices Size Standards Report Settings Name Last Saved Owner Type Description cE_F_HID_G5500 75 400 2007 08 09 13 23 gmidx Advanced CE_F_HID_GS500 75 450 2007 08 09 13 24 gmidx Advanced cE_G5_HID_G5500 2006 10 11 13 12 gmidx Advanced New Open Save As Import Export b Navigate to then open the Identifiler Plus Analysis Files GMIDX folder c Select CE G5 Identifiler Plus GS500 then click Import to import the CE G5 Identifiler Plus GS500 analysis method into the GeneMapper ID X Software database Import Size Standard Method xl Look in je Identifiler Plu
138. st 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 Dyes 6 FAM VIC NED PET LIZ Normalized Emission 500 550 600 650 700 Wavelength nm Figure 3 Emission spectra of the five dyes used in the AmpF STR Identifiler Plus Kit 8 AmpFtSTR Identifiler Plus User Guide Materials and equipment Kit contents and storage Materials and equipment The AmpF STR Identifiler Plus PCR Amplification Kit PN 4427368 contains materials sufficient to perform 200 amplifications at 25 uL reaction volumes IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the primer set from light when not in use Amplified DNA AmpF STR Identifiler Plus Allelic Ladder and GeneScan 500 LIZ Size Standard should also be protected from light Keep freeze thaw cycles to a minimum Component Description 200X Volume Storage AmpF STR Identifiler Plus Primer Set Contains forward and reverse primers to amplify human DNA targets 1 tube 1 0 mL 15 to 25 C on receipt 2 to 8 C after initial use AmpF STR Identifiler Plus Master Mix Contains enzyme salts dNT
139. ten STR loci that were 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 D18S11 locus mutations were not detected at any of the other nine STR loci The D18S11 mutation was represented by an increase of one 4 nt repeat unit allele 17 was inherited as allele 18 single step mutation The maternal paternal source of this mutation could not be distinguished Additional Additional studies Edwards er al 1991 Edwards er al 1992 Weber and Wong mutation studies 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 AmpF STR Identifiler Plus Kit loci Methods for modifications of these mutation rates to infer mutation rates indirectly for those loci where the rates are not large enough to be measured directly and or to account for those events undetectable as Mendelian errors AmpFtSTR Identifiler Plus User Guide 114 Chapter 5 Experiments and Results Probability of Identity Table 12 shows the Probability of Identity PI values of the AmpF STR Identifiler Plus Kit loci individually and combined Table 12 Probability of Identity values for t
140. the Bin view for the marker in the right pane 41 AmpFtSTR Identifiler Plus User Guide Set up GeneMapper ID Software for data analysis OZ Manager File Edit Bins View BX m JE H Ba set entier Pus es lli E mms i a e jw mim L amp E Panel Manager n E E AmpFLSTR Panels v2 7 8 la ho m 2 hal ha A45 16 47 18 hol 20 H AmpFLSTR Identifier Plus v1 1 0 T 2 lder tifiler Plus Panels v1 D851178 D21811 03 D7S820 CSF1PO 08 D381358 Q THO1 B D13S317 07 D D165539 Z 0251338 0195433 06 D Tox R k D18551 s U I AMEL e D55818 04 2 L FGA 1 e o 02 amp iReference Samples 04 T 0 0 113 119 125 131 137 143 149 155 151 167 173 178 185 D851179 4 gt 9 Click Apply then OK to add the AmpF STR Identifiler Plus Kit 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 ZD Software database Import an HID The HID Advanced analysis method for the AmpF STR Identifiler Plus PCR analysis method Amplification Kit uses the Identifiler Plus Bins vl file described in step 6 on page 40 Use the following procedure to import the analysis method from the folder that you downloaded from the Applied Biosystems web site into the GeneM
141. tially hazardous and can cause injury illness or death WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles Chemical waste To minimize the hazards of chemical waste safety guidelines Waste disposal 125 Read and understand the Material Safety Data Sheets MSDSs provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste Provide 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 Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines
142. tings IMPORTANT Laboratories need to perform the appropriate internal validation studies to determine the minimum heterozygous and homozygous minimum peak height thresholds and the minimum peak height ratio threshold that allow for reliable interpretation of AmpF STR Identifiler Plus PCR Amplification Kit data 61 AmpFtSTR Identifiler Plus User Guide Set up GeneMapper ID X Software for data analysis Analysis Method Editor xl General Allele Peak Detector Peak Quality SQ amp GQ Settings Quality weights are between and 1 Sample and Control GQ Weighting Broad Peak BD jos Allele Number AN o Out of Bin Allele BIN os Low Peak Height LPH fos Overlap OYL fos Max Peak Height MPH 3 Marker Spike SPK o3 Off scale OS fos Peak Height Ratio PHR o5 Control Concordance CC Weight 1 0 Only applicable to controls 5Q Weighting Broad Peak BD fos Allelic Ladder GQ Weighting Spike SSPK SPK h gt Off scale 05 fi SQ amp GQ Ranges PassRange BE Sizing Quality From 75 to 1 0 From to 0 25 Genotype Quality From 75 to 1 0 From 0 0 to p zs Reset Defaults Save As Save Cancel Help Figure 14 Analysis Method Editor SQ and GQ tab settings Import a HID size The size standard for the AmpF STR Identifiler Plus PCR Amplification Kit uses standard the following GeneScan 500 LIZ size standard peaks in its
143. to obtain support kk kk kk kk kK kK aaa K KK KK KK aaa KK KK kk kk aaa lk kk ix Chapter 1 OVBEVIGW oh ars aod o ww RES b EA alaya b 2 y RAE Ald x 1 Product OVerview x xd baw Gael nor REDE Ee UE adr M RE Neu 2 Workflow overview Sk kk kk kk kk kk kk kk kk kK aaa aaa aaa hh nn 6 Instrument and software overview kk kk kk kk kk kk KK KK KK kK kK KK kK KK KK KK KK kK kk 7 Materials and equipment kk kk kK KK KK KK KOK KK KK KK kk kk kk kk kk kk lk lk lk kk 9 Chapter 2 PCR Amplification llle 15 PCOR w rkareaS xcu ihe one Sam bdo breed Ox ed wad Eth Ian 16 Required user supplied reagents 4 kk kk kk kk kk KK KK KK KK KK KK KK KK kK KK kk k 17 DNA quantification ze ryk oe WA ara l WPRAWY ae UR ar ER EE sal 17 Prepare the amplification kit reactions kk kk KK KK KK KK KK KIR KK KK KK 19 Select the appropriate PCR cycle number kk kk kk kK KK KK KK KK KK KK RR KK KK 20 Perform POR css k2 new E SEMA DA eee pae nea MS Eee ET 21 Amplification using bloodstained FTA cards aaaaaaaaaaa KK KK KK YY 22 Chapter 3 Electrophoresis A d Wl sik utens ete imd Pune tees 26 Allelic ladder requirements 000 kk kK KK KK KK KIR KK KK KK KK KK KK KK KK KK KK kk 27 Section 3 1 3100 3100 Avant and 3130 3130x instruments 28 Set up the 3100 3100 Avant or 3130 3130x instrument for electrophoresis 28 Prepare samples for electrophoresis on the 3100 3100 Avant or 313
144. ur U S populations continued Amin TBE T S s Amede n 357 n 349 n 191 D16S539 HW X p 0 433216 0 67702 0 058631 0 996396 HW G p 0 482435 0 594871 0 37601 0 981384 HW Exact p 0 3753 0 4328 0 3068 0 9986 HExp 0 7939 0 7632 0 7747 0 7766 Ho 0 8263 0 7822 0 7828 0 7853 D18S51 HW X p 0 999844 0 628334 0 999203 0 343027 HW G p 1 0 872113 0 999492 0 798859 HW Exact p 0 978 0 0982 0 9152 0 2265 HExp 0 8694 0 8769 0 8761 0 8463 Ho 0 8824 0 8682 0 8862 0 8377 D19S433 HW X p 0 91703 0 806717 0 731222 0 810711 HW G p 0 83419 0 999765 0 975476 0 898389 HW Exact p 0 4517 0 69 0 3475 0 4301 HExp 0 8364 0 7659 0 8310 0 8430 Ho 0 8011 0 7622 0 8414 0 822 D21S11 HW X p 0 985687 0 936146 0 0 HW G p 1 0 999757 0 999794 0 712937 HW Exact p 0 7627 0 7861 0 6476 0 0118 HExp 0 8585 0 8427 0 8290 0 8003 Ho 0 8711 0 8567 0 7931 0 801 FGA HW X p 0 0 904953 0 263223 0 999686 HW G p 1 0 999812 0 960137 0 999946 HW Exact p 0 9761 0 4459 0 0891 0 9161 HExp 0 8659 0 8686 0 8751 0 8746 Ho 0 8824 0 8854 0 8724 0 8482 THO1 HW X p 0 961911 0 997905 0 649467 0 329461 HW G p 0 940414 0 99169 0 617212 0 318591 HW Exact p 0 8286 0 9716 0 4495 0 1377 HExp 0 7323 0 7866 0 7666 0 7016 Ho 0 7395 0 7822 0 8103 0 6492 112 AmpFtSTR Identifiler Plus User Guide Chapter 5 Experiments and Results 113 Concordance studies Table 11 Heterozygosity and p values for Hardy Weinberg tests of the 15 Identifiler Plus
145. xed samples resolution of genotypes 97 mixture studies 96 MSDSs about vii AmpF amp TR Identifiler Plus User Guide description 124 obtaining ix l24 multicomponent analysis 7 8 mutation studies 114 mutation STR 114 N negative control sample preparation 19 O off ladder alleles 74 operating systems 7 28 30 P PCR hematin inhibitor 94 humic acid inhibitor 95 performing 21 setup tools 16 thermal cycling conditions programming 21 work area setup 16 PCR components validation of 70 PCR cycle number validation 71 peak height ratios table ofalleles 96 percent stutter highest value for locus 82 off scale peaks 82 relation to allele length 82 positive control sample preparation 19 precision and size windows 74 precision sizing 74 primers Amelogenin 88 volume per reaction 19 probability of identity definition 115 values 115 Q quantification DNA 17 R radioactive waste handling 126 reaction mix for PCR 19 reactions preparing for PCR 19 reagents user supplied 17 run module electrophoresis 28 30 AmpF amp TR Identifiler Plus User Guide Index S safety biological hazards 126 chemical waste 125 guidelines 123 125 sample files fsa 38 52 sample preparation 19 DNA negative control 19 DNA positive control 19 standards 9 setup tools PCR 16 size deviation sample alleles and ladder alleles 73 sizing precision 74 software instrument compatibility 7 species specif
146. ystems 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 Quantifiler Kits Quantifiler Human DNA Quantification Kit and Quantifiler Y Human Male DNA 4344790 Quantification Kit User s Manual AmpF STR Identifiler PCR Amplification Kit User s Manual 4323291 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 Veriti 96 Well Thermal Cycler AmpFtSTR Kit Validation User Bulletin 4440754 AmpFtSTR Identifiler Plus User Guide 132 Documentation Portable document format PDF versions of this guide and the documents listed above are available at www appliedbiosystems com Not

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