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AmpFlSTR ® SEfiler Plus ™ PCR Amplification Kit

1. Allele Mean Standard Deviation D18S51 7 263 07 263 14 0 025 0 05 9 271 19 271 24 0 031 0 048 10 275 24 275 3 0 024 0 039 10 2 277 24 277 3 0 023 0 047 11 279 32 279 37 0 023 0 048 12 283 36 283 41 0 03 0 048 13 287 38 287 43 0 025 0 037 13 2 289 34 291 43 0 027 0 039 14 291 37 291 43 0 027 0 039 14 2 293 35 293 4 0 033 0 045 15 295 38 295 43 0 026 0 037 16 299 37 299 43 0 032 0 043 17 303 31 303 37 0 023 0 04 18 307 29 307 34 0 027 0 044 19 311 32 311 37 0 032 0 055 20 315 46 315 5 0 024 0 036 21 319 75 319 81 0 034 0 048 22 323 93 324 0 03 0 046 23 327 97 328 04 0 03 0 049 24 332 06 332 13 0 023 0 04 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Chapter 5 Experiments and Results 5 12 Table 5 1 Precision results of five runs 16 capillaries run of the AmpF STR SEfiler Plus Allelic Ladder continued Applied Biosystems 3130x Genetic Analyzer Allele Mean Standard Deviation 25 336 12 336 18 0 027 0 036 26 340 15 340 19 0 023 0 042 27 344 27 344 29 0 022 0 038 D19S433 9 99 6 99 68 0 03 0 05 10 103 76 103 85 0 04 0 044 11 107 9 107 97 0 03 0 045 12 111 99 112 06 0 029 0 053 12 2 114 03 114 11 0 024 0 039 13 115 97 116 06 0 029 0 036 13 2 117 96 118 04 0 027 0 044 14 119 89 119 98 0 034 0 045 14 2 121 87 121 95 0 033 0 045 15 123 8 123 9 0 033 0 039 15 2 125 79 125 9 0 0
2. Figure 5 15 Amplification with AmpF STR SEfiler Plus and SEfiler kits in the presence and absence of hematin Panels 1 and 3 correspond to control samples panels 2 and 4 correspond to samples amplified in the presence of 45 uM of hematin AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Stability Comparison of SEfiler Plus and SEfiler kit performance in a simulated model of hematin inhibition Only those peaks gt 50 RFUs were counted A complete profile with control 007 DNA yields 24 peaks using the SEfiler Plus kit see Table 5 3 Table 5 3 Comparison of SEfiler Plus and SEfiler kit performance in simulated model of hematin inhibition n 3 Hematin uM SEfiler Plus kit SEfiler kit 0 24 24 24 24 24 24 24 24 24 24 24 24 30 24 24 24 24 24 24 2 24 2 24 8 24 45 24 24 21 24 24 24 0 24 0 24 0 24 55 18 24 23 24 5 24 0 24 0 24 0 24 AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 5 39 Chapter 5 Experiments and Results 5 40 Effect of Inhibitors Humic Acid Traces of humic acid may inhibit the PCR amplification of DNA evidence collected from soil In this study Applied Biosystems tested increasing amounts of humic acid in the PCR amplification of 1 ng of control DNA 007 with the SEfiler and SEfiler Plus kits for 30 cycles of amplification see Figure 5 16
3. Figure 1 2 500 pg of Control DNA 007 amplified with the AmpF STR SEfiler Plus kit and analyzed on the Applied Biosystems 3130x Genetic Analyzer 1 8 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Workflow Overview Workflow Overview Extract and Quantify DNA uA Quantifiler Total Human DNA Quantification Kit PCR Amplify DNA AmpF STR SEfiler Plus PCR Amplification Kit GeneAmp PCR System 9700 Thermal Cycler amplification should be done on silver or gold plated silver blocks a Perform Electrophoresis oe uM uk ABI PRISM ABI PRISM Applied Biosystems 310 Genetic Analyzer 3100 3100 Avant 3130 31 30x Genetic Analyzer Genetic Analyzer E Analyze Data GeneMapper D Software AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 1 9 Chapter 1 Overview Instrument and Software Overview Data Collection and Analysis Software Instrument and Software Compatibility About Multicomponent Analysis 1 10 This section provides information about the data collection and analysis software versions required to run the AmpF STR SEfiler Plus PCR Amplification Kit on specific instruments 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 records sample fluorescence on the detection s
4. 1 14 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Table 1 4 User supplied materials continued Materials and Equipment Material Source 3100 3100 Avant Analyzer materials 96 Well Plate Septa 4315933 Reservoir Septa 4315932 3130x 3100 Genetic Analyzer Capillary Array 36 cm 4315931 3130 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 600 LIZ Size Standard 4366589 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 PN 4335393 For a complete list of parts and accessories for the 3100 3100 Avant instrument refer to Appendix B of the ABI PRISM 3100 Genetic Analyzer and 3100 Avant Genetic Analyzer User Reference Guide 310 Analyzer materials 310 Genetic Analyzer Capillary 47 cm 402839 0 5 mL Sample Tray 5572 96 Well Tray Adaptor for 9700 thermal cycler trays 4305051 GeneScan 600 LIZ Size Standard 4366589 Running Buffer 10x 402824 Genetic Analyzer Septa Retainer Clips for 96 Tube Sample Tray 402866 Genetic Analysis Sample Tubes 0 5 mL 401957 AmpFSTR SEfiler Plus PCR Amplifica
5. sssseeeer eh Preparing the Reactions 000 cece eee es Performing POR uie oe RARI WDR EF ale nese gee Bas Amplification Using Bloodstained FTA Cards Les 2 Performing Electrophoresis Allelic Ladder Requirements 000 0c eee cece eens Setting Up the 3100 3100 Avant or 3130 3130xl Instrument for Electrophoresis 0 00 0c cece res Preparing Samples for Electrophoresis on the 3100 3100 Avant or 3130 3130xl Instrument 006 eee Setting Up the 310 Instrument for Electrophoresis AmpFSTR SEfiler Plus PCR Amplification Kit User Guide lii Chapter 4 Chapter 5 Appendix A Bibliography Index Preparing Samples for Electrophoresis on the 310 Instrument 3 8 Analyzing Data Overview of GeneMapper ID Software 0 0 cee eee 4 2 Setting Up GeneMapper D Software v3 2 1 for Analyzing AmpF STR SEfiler Plus Kit Data seen 4 3 Analyzing and Editing Sample Files with GeneMapper D Software 4 16 Experiments and Results OI MC 5 2 Developmental Validation llle 5 3 Accuracy Precision and Reproducibility 2 5 7 Extra Peaks in the Electropherogram 00 eee eee eee 5 21 Characterization of Loci 0 0c cee eee 5 30 Species Specificity illis 5 32 Sensitivity cusan eek ed wed RAS eae ee ur helenae 5 34 Stability sac 246 na ed geese Nee bo ee
6. Baseline Window 51 pts Size Calling Method 2nd Order Least Squares O 3rd Order Least Squares Cubic Spline Interpolation Local Southern Method Global Southern Method Peak End Factory Defaults The software uses the peak detection parameters to specify the minimum peak height to limit the number of peaks detected Although GeneMapper D software displays peaks that occur below the specified amplitude in electropherograms the software does not label or determine the genotype of these peaks IMPORTANT When analyzing data generated on an ABI PRISM 310 Genetic Analyzer running on a Windows NT platform you may need to reduce the Peak Window Size from 15 to 13 to facilitate detection of each of the 1 bp microvariant alleles 21 21 1 and 21 2 included in the SE33 allelic ladder Laboratories must perform the appropriate internal validation studies to determine the Peak Window Size value that allows for reliable interpretation of SEfiler Plus Kit data when run on an ABI PRISM 310 Genetic Analyzer running on a Windows NT platform AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Setting Up GeneMapper ID Software v3 2 1 for Analyzing AmpF4STR SEfiler Plus Kit Data Table 4 1 SEfiler Plus HID v1 Advanced Mode analysis method settings continued Tab Settings Peak Detector Note The analysis range is set by you based on continued location of the primer peaks and the
7. K AmpFLSTR SEfier Plus Panels vi IB I Bin Set ampFLSTR SEfiler Plus Bins vt v a b Sttiler Plus v 44 45 46 17 48 19 D3S1358 10 WWA c D165539 08 AMEL 08 D8s1178 SE33 07 0198433 THOI o6 FGA D21511 D18551 w 95 C3IReference Samples 0 04 4 279 287 295 303 0251338 9 Click Apply then OK to add the SEfiler Plus panel and bin set to the GeneMapper JD database IMPORTANT If you close the Panel Manager without clicking OK the panels and bins are not imported into the GeneMapper ID database 4 8 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Setting Up GeneMapper ID Software v3 2 1 for Analyzing AmpFSTR SEfiler Plus Kit Data Importing an HID Analysis Method The analysis method for the AmpF STR SEfiler Plus PCR Amplification Kit uses the HID Advanced Mode Peak Detection Algorithm This analysis method provides users with the same analysis parameters available in GeneScan Software v3 7 1 for the Windows operating system Use the following procedure to import the analysis method for the SEfiler Plus kit from the folder that you downloaded from the Applied Biosystems web site into the GeneMapper ZD software database Refer to step 1a on page 4 4 for downloading instructions To import the HID Advanced Mode analysis method into GeneMapper D software 1 Select Tools gt
8. Component Reaction uL AmpF STR SEfiler Plus Master Mix 10 0 AmpF STR SEfiler Plus Primer Set 5 0 Note Include additional reactions in your calculations to provide excess volume for the loss that occurs during reagent transfers Prepare reagents Thaw the AmpF STR SEfiler Plus Master Mix and the AmpF STR SEfiler Plus Primer Set then vortex the mix and primer set 3 seconds and centrifuge them briefly before opening the tubes IMPORTANT Thawing is required only during first use of the kit After first use reagents are stored at 2 to 8 C and therefore do not require subsequent thawing Do not refreeze the reagents Pipette the required volumes of components into an appropriately sized polypropylene tube Vortex the reaction mix for 3 seconds then centrifuge briefly Dispense 15 uL of the reaction mix into each reaction well of a MicroAmp Optical 96 Well Reaction Plate or each MicroAmp Reaction Tube AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 2 7 Chapter 2 PCR Amplification To prepare the reactions continued 6 Prepare the DNA samples DNA Sample To Prepare Negative Control Add 10 uL of low TE buffer Your Sample Dilute a portion of your DNA sample with low TE buffer so that 0 50 to 0 75 ng of total DNA is in a final volume of 10 uL Add your sample to the reaction mix Positive Control Combine 5 uL of control DN
9. Keys K Smerick J and Budowle B 2001 Validation of short tandem repeats STRs for forensic usage Performance testing of fluorescent multiplex STR systems and analysis of authentic and simulated forensic samples J Forensic Sci 46 3 647 660 Mulero J J Chang C W and Hennessy L K 2006 Characterization of N 3 stutter product in the trinucleotide repeat locus 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 Puers C Hammond H Jin L Caskey C and Schumm J 1993 Identification of repeat sequence heterogeneity at the polymorphic short tandem repeat locus HUMTHO01 AATG n and reassignment of alleles in population analysis using a locus specific allelic ladder Am J Hum Genet 53 953 958 Revised Validation Guidelines Scientific Working Group on DNA Analysis Methods SWGDAM Forensic Science Communications July 2004 Volume 6 3 Available at http www fbi gov hq lab fsc current standards 2004 03 standards02 htm Sensabaugh G F 1982 Biochemical markers of individuality In Saferstein R ed Forensic Science Handbook Prentice Hall Inc New York pp 338 415 AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide Sharma V and Litt M 1992 Tetranucleotide repeat polymorphism at the D21S11 locus Hum Mol Genet 1 67 Smith R N 1995 Accurate size comparison of sho
10. PCR Amplification Kit User Guide Worldwide Sales and Support Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches 150 countries on six continents For sales office locations and technical support please call our local office or refer to our Web site at www appliedbiosystems com Applera is committed to providing the world s leading technology and information for life scientists Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free In North America 1 800 345 5224 Fax 1 650 638 5884 03 2012 Applied Biosystems Part Number 4385739 Rev B
11. Table 4 1 SEfiler Plus HID v1 Advanced Mode analysis method settings Tab Settings General Name SEfiler Plus HID v1 Analysis Method Editor HID General Allele Peak Detector Peak Quality Quality Flags r Analysis Method Description Name SEfiler Plus HID v1 Description SEfiler Plus default Analysis Method version 1 Instrument Analysis Type HID 4 10 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Setting Up GeneMapper ID Software v3 2 1 for Analyzing AmpF4STR SEfiler Plus Kit Data Table 4 1 SEfiler Plus HID v1 Advanced Mode analysis method settings continued Tab Settings Allele Analysis Method Editor HID General A Bin Set AmpFLSTR SEfiler Plus Bins v1 Use marker specific stutter ratio if available Marker Repeat Type Tri Tetra Penta Cut off Value 0 0 oo oo MinusA Ratio 00 oo o0 Minus amp A Distance 0 0 0 0 0 0 To 00 oo o0 Minus Stutter Ratio 0 0 oo oo Minus Stutter Distance From 0 0 325 oo To 00 475 00 Plus Stutter Ratio 0 0 oo loo Plus Stutter Distance From 0 0 00 oo To 00 00 loo Amelogenin Cutoff 0 0 Range Filter Factory Defaults 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
12. 0 0000005 4 3 Analyzing and Editing Sample Files with GeneMapper ID Software 4 17 AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 4 1 Chapter 4 Analyzing Data Overview of GeneMapper D Software What Does GeneMapper D Software Do Instruments Before You Start 4 2 GeneMapper JD Software is an automated genotyping software solution for forensic paternity and database data analysis and other genotyping needs After electrophoresis the data collection software stores information for each sample in a fsa file Using GeneMapper JD v3 2 1 software you can then analyze and interpret the data Refer to Instrument and Software Overview on page 1 10 for a list of compatible instruments When using GeneMapper JD Software version 3 2 1 to perform Human Identification HID analysis with AmpF STR kits consider that HID analysis requires at least one allelic ladder sample per run folder Your laboratory can use multiple ladder samples in an analysis as long as it performs the appropriate validation studies For multiple ladder samples the GeneMapper D 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 f
13. FGA 0 034 0 044 SE33 0 019 0 02 THO1 0 103 0 119 vWA 0 067 0 077 Combined 6 47 x 10775 7 46 x 1074 The P value is the probability that two individuals selected at random will have an identical SEfiler Plus kit genotype Sensabaugh 1982 The P values for the populations described in this section are then approximately 1 1 54 x 10 4 African American and 1 1 34 x 101 U S Caucasian AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 5 49 Chapter 5 Experiments and Results Probability of Paternity Exclusion Table of Probability of Paternity of Exclusion 5 50 Table 5 8 shows the Probability of Paternity Exclusion Py values of the AmpF STR SEfiler Plus PCR Amplification Kit STR loci individually and combined Table 5 8 Probability of Paternity Exclusion values for the AmpF STR SEfiler Plus kit STR loci Locus African American U S Caucasian D2S1338 0 0745 0 621 D3S1358 0 734 0 65 D8S1179 0 477 0 763 D16S539 0 67 0 42 D18S51 0 725 0 912 D19S433 0 632 0 516 D21S11 0 745 0 734 FGA 0 784 0 676 SE33 0 745 0 792 THO1 0 578 0 734 vWA 0 613 0 705 Combined 0 999997 0 999998 The P 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 SEfiler Plus kit STR loci Chakraborty and Stivers 1996 AmpFSTR SEfiler Plus PCR Amplifica
14. GeneMapper Manager to open the GeneMapper Manager 2 Import an analysis method for HID Advanced 7 GeneMapper Manager Projects Analysis Methods Table Settings Plot Settings Matrices Size Standards a Select the Analysis Methods tab then click Import Name Last Saved SNaPshot Defaut 2005 03 16 16 00 3 gmid Owner Instrument Analysis Type Description YFiler 2005 03 28 08 29 4 gmid 3100 HID lt SNaPshot Factory ae v folder gt b Navigate to then open the GMID SEfiler Plus files the SEfiler Plus HID v1 analysis method into the GeneMapper JD database Import Analysis Method Look in c GMID SEfiler Plus files 9 cE Gs sEfiler Plus GS800 HD s sefiler_Plus_HID_v1 Desktop 3 Select SEfiler Plus HID v1 then click Import to import 2 c t File name SEfiler_Plus_HID_v1 xml My Documents Files of type ML Files xml i i AmpF 4STR SEfiler Plus PCR Amplification Kit User Guide 4 9 Chapter 4 Analyzing Data To import the HID Advanced Mode analysis method into GeneMapper D software continued 4 To view the settings in the SEfiler Plus HID vl analysis method a Select the Analysis Methods tab b Select SEfiler Plus HID v1 in the Name column then click Open Table 4 1 on page 4 10 shows the settings for each tab of the Analysis Method Editor HID
15. Table 3 1 lists data collection software and the run modules that you can use to analyze SEfiler Plus kit products For details on the procedures refer to the documents listed in the table Table 3 1 SEfiler Plus kit data collection software and reference documents for use with the 3100 3100 Avant or 3130 3130xl instruments Operating Data Collection System Software Run Module References Windows 3 0 8130 3130x e HIDFragmentAnalysis36 Applied Biosystems 3130 3130xl XP Analyzer POPA 1 Genetic Analyzers Using Data e Dye Set G5 Collection Software v3 0 Protocols for Processing AmpF4STR9 PCR Amplification Kit PCR Products User Bulletin PN 4363787 Windows 2 0 e HIDFragmentAnalysis36_POP4_1 ABI Pris M 3100 3100 Avant 2000 e Dye Set G5 Genetic Analyzers Using Data Collection Software v2 0 Protocols for Processing AmpFSTR PCR Amplification Kit PCR Products User Bulletin PN 4350218 AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 3 3 Chapter 3 Performing Electrophoresis Analysis Module GS600Analysis gsp 1 0 3100 Avant Analyzer Run Module GeneScan36Avb DyeSetG5Module Analysis Module GS600Analysis gsp Operating Data Collection System Software Run Module References Windows 1 1 3100 Run Module ABI PRISM 3100 3100 Avant NT Analyzer GeneScan36vb DyeSetG5Module Genetic Analyzers Protocols for Processing AmpFSTR PCR Amplif
16. 0 043 20 187 99 188 04 0 029 0 045 21 192 05 192 08 0 016 0 037 22 196 12 196 16 0 026 0 035 23 200 09 200 13 0 027 0 047 24 204 4 204 45 0 024 0 036 5 20 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Extra Peaks in the Electropherogram Extra Peaks in the Electropherogram Causes of Extra Peaks other than the target alleles may be detected on the Peaks electropherogram Causes for 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 that is one repeat unit smaller or less frequently one repeat unit larger than the major STR product Butler 2005 Mulero et al 2006 Sequence analysis of stutter products at tetranucleotide STR loci has revealed that the stutter product is missing a single tetranucleotide core repeat unit relative to the main allele Walsh et al 1996 The proportion of the stutter product relative to the main allele percent stutter is measured by dividing the height ofthe stutter peak by the height of the main allele peak Peak heights were measured for amplified samples n 229 at the loci used in the AmpF STR SEfiler Plus PCR Amplification Kit All data were generated on the Applied Biosystems 3130x Genetic Analyzer Some conclusions from these measurement
17. 0 052 33 2 318 75 318 82 0 033 0 049 34 2 322 93 323 0 036 0 048 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Accuracy Precision and Reproducibility Table 5 1 Precision results of five runs 16 capillaries run of the AmpF STR SEfiler Plus Allelic Ladder continued Applied Biosystems 3130x Genetic Analyzer Allele Mean Standard Deviation 35 325 01 325 07 0 038 0 054 35 2 327 05 327 09 0 033 0 044 36 329 1 329 15 0 039 0 051 37 333 21 333 22 0 042 0 05 THO1 4 159 72 159 79 0 004 0 049 5 163 79 163 85 0 028 0 048 6 167 84 167 9 0 027 0 05 7 171 88 171 96 0 023 0 039 8 175 94 176 0 029 0 039 9 179 98 180 05 0 021 0 04 9 3 183 12 183 2 0 027 0 044 10 184 09 184 14 0 022 0 036 11 188 19 188 27 0 024 0 035 13 3 199 45 199 52 0 035 0 044 vWA 11 151 27 151 32 0 025 0 034 12 155 37 155 41 0 023 0 042 13 159 52 159 56 0 006 0 042 14 163 79 163 81 0 02 0 045 15 167 7 167 74 0 018 0 045 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide 5 19 Chapter 5 Experiments and Results Table 5 1 Precision results of five runs 16 capillaries run of the AmpF STR SEfiler Plus Allelic Ladder continued Applied Biosystems 3130x Genetic Analyzer Allele Mean Standard Deviation 16 171 76 171 8 0 025 0 045 17 175 8 175 84 0 025 0 031 18 179 81 179 85 0 024 0 045 19 183 9 183 95 0 026
18. 12 figure 1 7 number per run suggested 3 2 precision results table 5 10 requirements for accurate genotyping 3 2 sample type 4 2 volume per reaction 3 6 3 9 AmpFISTR_Panels_v3 folder 4 6 analysis method for allelic ladders 4 2 analysis settings for project 4 16 annealing temperatures validation of 5 4 5 5 Applied Biosystems contacting xii customer feedback on documentation xii Information Development department xii Technical Support xii artifacts in data 5 27 B baseline noise examples 5 28 bin sets importing 4 6 viewing 4 8 Bin view displaying for a marker 4 8 biohazardous waste handling ix Bloodstained FTA Cards amplification 2 10 bold text when to use v C CAUTION description vi CEPH 5 30 characterization of loci validation 5 30 chemical safety vii chemical waste hazards viii chemical waste safety ix chromosome location locus designation 1 3 See Also allele loci contents of kit 1 12 control DNA about 1 13 contents 1 12 storage 1 12 conventions bold text v for describing menu commands v IMPORTANT v italic text v Notes v user attention words v AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide Index 1 customer feedback on Applied Biosystems documents xii cycle number validation 5 6 D D3S1358 locus designation 1 3 DANGER description vi data accuracy precision and reproducibility 5 7 artifacts 5 27 for different populations 5 47 data collection software
19. 18 19 20 20 2 21 21 1 21 2 22 2 23 2 24 2 25 2 26 2 27 2 28 2 29 2 30 2 31 2 32 2 33 2 34 2 17 25 2 THO1 11p15 5 NED 7 9 3 AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 1 5 Chapter 1 1 6 Table 1 1 AmpF STR SEfiler Plus Kit loci and alleles AmpF STR AmpF STR Locus Chromosome Dye Allelic Control Designation Location Label Ladder DNA 007 Alleles Genotype vWA 12p12 pter 6 FAM 11 12 13 14 16 14 15 16 17 18 19 20 21 22 23 24 Amelogenin X p22 1 22 3 VIC X Y X Y Y p11 2 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Product Overview Allelic Ladder Figure 1 1 shows the allelic ladder for the AmpF STR SEfiler Profile Plus kit 1358 FWA 165539 D251338 100 200 300 400 2000 1600 1200 800 400 19 433 1 GA Figure 1 1 GeneMapper D Software plot of the AmpF STR SEfiler Plus Allelic Ladder AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 1 7 Chapter 1 Overview Control DNA 007 Figure 1 2 shows amplification of Control DNA 007 using the Profile AmpF STR SEfiler Plus kit 1358 EWA 165539 51338 100 200 300 400
20. 24 24 20 24 24 24 24 24 24 0 24 0 24 0 24 40 20 24 23 24 24 24 0 24 0 24 0 24 60 9 24 8 24 12 24 0 24 0 24 0 24 AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 5 41 Chapter 5 Experiments and Results Mixture Studies SWGDAM Guideline 2 8 Mixture Studies 5 42 The ability to obtain reliable results from mixed source samples should be 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 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 locus The presence of a peak at a stutter position that is significantly greater in percentage than what is typically observed in a single source sample Significantly imbalanced alleles for a heterozygous genotype Am
21. 6 for details about these kits AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 2 5 Chapter 2 PCR Amplification Table 2 1 Methods for quantifying DNA Product Description References Quantifiler Y Human Properties Quantifiler Human Male DNA DNA Quantification Kits e Both Quantifiler kits have high specificity for human DNA The Quantifiler Y kit is highly specific for human male DNA Quantifiler Human The kit detects single stranded and DNA Quantification Kit degraded DNA PN 4343895 How it works The DNA quantification assay combines two 5 nuclease assays User s Manual PN 4344790 Quantification Kit PN 4343906 e Atarget specific human DNA or human male DNA assay which consists of two primers for amplifying human or human male DNA and one TagMan MGB probe labeled with FAM dye for detecting the amplified sequence e An 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 2 6 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Preparing the Reactions Preparing the Reactions SEfiler Plus Kit To prepare the reactions Reactions l Calculate the volume of each component needed to prepare the reactions using the table below Volume Per
22. 600 1000 800 SEfiler Plus kit 2 ng DNA 6 Units DNase 600 400 200 0 PEUT Pu A Figure 5 14 Amplification of Raji DNA samples sonicated and incubated with increasing doses of DNase I Panels 1 2 3 and 4 correspond to 0 4 5 and 6 units of DNase l AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 5 37 Chapter 5 Experiments and Results 5 38 Effect of Inhibitors Hematin Heme compounds have been identified as PCR inhibitors in DNA samples extracted from bloodstains DeFranchis et al 1988 Akane et al 1994 It is believed that the inhibitor is co extracted and co purified with the DNA and subsequently interferes with PCR by inhibiting polymerase activity To examine the effects of hematin on the amplification results obtained by the SEfiler Plus kit male DNA 007 1 ng input DNA for the SEfiler and SEfiler Plus kits was amplified with increasing concentrations of hematin for 30 cycles of amplification The concentrations of hematin used were 0 uM 30 uM 45 uM and 55 UM see Table 5 3 200 300 400 SEfiler Plus kit LA du SEfiler Plus kit 45 uM Hematin l I Mida d a m SEfiler kit 2000 L l J Untreated SEfiler kit 45 uM Hematin
23. 9700 Gold plated silver block GeneAmp PCR System 9700 AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 2 3 Chapter 2 PCR Amplification Required User Supplied Materials and Reagents Kit Contents and Storage User Supplied Reagents 2 4 Each AmpF STR SEfiler Plus PCR Amplification Kit contains materials sufficient to perform 200 reactions at a 25 uL reaction volume See Kit Contents and Storage on page 1 12 for details on SEfiler Plus kit contents 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 SEfiler Plus Allelic Ladder and GeneScan 600 LIZ Size Standard should also be protected from light Minimize freeze thaw cycles In addition to the SEfiler 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 following table or order it from Teknova Cat T0223 To prepare low TE buffer 1 Mix together JO mL of 1 M Tris HCl pH 8 0 0 2 mL of 0 5 M EDTA pH 8 0 990 mL glass distilled or deionized water Nees CHEMICAL HAZARD EDTA Exposure causes eye irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Note Adjust the volumes accordingly for specific needs 2 Aliquot and autoclave the solutions 3 Store
24. Importance of Quantitation Effect of DNA Quantity on Results When appropriate the range of DNA quantities able to produce reliable typing results should be determined SWGDAM July 2003 The optimal amount of input DNA added to the AmpF STR SEfiler Plus PCR Amplification Kit should be between 0 50 and 0 75 ng 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 05 to 0 075 ng uL so that 0 50 to 0 75 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 Figure 5 13 on page 5 35 the control DNA 007 was serially diluted from 1 ng to 0 062 ng Full profiles 24 PCR products 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 If too 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 example an allele peak that 1s off scale can cause the corresponding stutter peak to appear higher in relative intensity thus
25. Y 2 CE G5 SEfiler Plus GS600 HD v1 2 sEfiler Plus HID v1 Desktop 2 File name AmpFLSTR SEfiler Plus Panels v1 txt My Documents Files of type All Files AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 4 5 Chapter 4 Analyzing Data 6 Import AmpFLSTR SEfiler Plus Bins vl a Select the AmpFLSTR SEfiler Plus Panels v1 folder in the navigation pane F Panel Manager File Edit Bins View ir 2x m m NM WB sese amp _Panels_v Ug mpFLSTR SEfiler Plus amp jskfiler Plus v1 b Select File gt Import Bin Set to open the Import Bin Set dialog box c Navigate to then open the GMID_SEfiler_Plus_files folder d Select AmpFLSTR_SEfiler_Plus_Bins_v1 then click Import Note Importing this file associates the bin set with the panels in the AmpFLSTR_SEfiler_Plus_Panels_v1 folder Import Bin Set Look in C3 GMD sEfler Plus fles x 3 pna cC B AmpFLSTR SEfiler Plus Bins v1 e E AmpFLSTR SEfiler Plus Panels v1 e CE G5 SEfiler Plus GS600 HID v1 3 amp sEfiler Plus HID v1 Desktop File name AmpFLSTR_SEfiler _Plus_Bins_v1 tot My Documents Files of type an Fies 4 6 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Setting Up GeneMapper ID Software v3 2 1 for Analyzing AmpF4STR SEfiler Plus Kit Data G Panel Manager File Edit Bins View 7 View the
26. and Reference Documents Table 1 4 on page 1 14 lists the required materials not supplied with the AmpF STR SEfiler Plus PCR Amplification Kit IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the primer set from light when not in use Amplified DNA AmpFISTR SEfiler Plus Allelic Ladder and GeneScan 600 LIZ Size Standard should also be protected from light Minimize freeze thaw cycles Table 3 2 lists data collection software and the run modules that you can use to analyze SEfiler Plus kit products For details on the procedures refer to the documents listed in the table Table 3 2 SEfiler Plus kit data collection software and reference documents for use with the 310 instrument Operating System Data Collection Run Module References Software Windows XP 3 14 GS STR POP4 1ml G5 v2 md5 ABI PRISM 310 Genetic Analyzer User s Manual Windows PN 4317588 ABI Prisv 310 Protocols for Processing AmpFtSTR PCR Amplification Kit Products with Microsoft Windows NT Operating System User Bulletin PN 4341742 and Windows 2000 Windows NT 3 0 GS STR POP4 1ml G5 v2 md5 ABI Prisu 310 Genetic Analyzer User s Manual Windows PN 4317588 ABI Prisv 310 Protocols for Processing AmpFtSTR PCR Amplification Kit Products with Microsoft Windows NT Operating System User Bulletin PN 4341742 t Applied Biosystems conducted concord
27. at room temperature AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Quantifying DNA Quantifying DNA Importance of Quantitation Methods for Quantifying DNA Quantifying the amount of DNA in a sample before amplification allows you to 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 SEfiler Plus Kit is 0 50 to 0 75 ng in a maximum input volume of 10 uL 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 1s 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 Applied Biosystems provides several kits for quantifying DNA in samples See the reference cited in Table 2 1 on page 2
28. column 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 AmpFLSTR SEfiler Plus Panels v1 file Note For more information about allele filters refer to the GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide Chapter 3 PN 4338775 and the nstallation Procedures and New Features for GeneMapper ID Software Version 3 2 User Bulletin PN 4352543 AmpF 4STR SEfiler Plus PCR Amplification Kit User Guide 4 11 Chapter 4 Analyzing Data 4 12 Table 4 1 settings continued SEfiler_Plus_HID_v1 Advanced Mode analysis method Tab Settings Peak Detector IMPORTANT Laboratories need to perform the appropriate internal validation studies to determine the peak amplitude threshold highlighted in red below that allows for reliable interpretation of SEfiler Plus data Analysis Method Editor HID General Allele Peak Detector Peak Quality Quality Flags Peak Detection Algorithm Advanced Ranges Analysis Partial Range v Start P 2500_ Stop Pt 10000 Sizing Partial Sizes v Start Size 60 Stop Size 400 Smoothing and Baselining Smoothing O None Light O Heavy v Peak Detection Peak Amplitude Thresholds B 50 R 50 50 Min Peak Half Vidth Polynomial Degree Peak Window Size Slope Threshold Peak Start
29. commas o Aum oma rim sss se o y 33 ot 12 19 14 15 16 17 18 19 20 21 22 25 D18S51 24 25 23 27 Figure 5 8 Stutter percentages for the D21S11 and D18S51 loci 5 24 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Extra Peaks in the Electropherogram Table 5 2 Marker specific stutter percentages ratios used in GeneMapper D v3 2 1 AmpFLSTR_SEfilerPlus_panels_v1 for SEfiler Plus Kit loci Locus Stutter D21S11 12 22 D18S51 16 43 D19S433 12 64 THO1 5 77 FGA 18 23 D3S1358 13 07 vWA 11 63 D16S539 9 71 D281338 13 00 D8S1179 10 19 SE33 17 70 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 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 SEfiler Plus kit includes two design features that promote maximum A addition The primer sequences have been optimized to encourage A addition The final extension step is 60 C for 60 minutes AmpF amp TR SEfiler Plus PCR A
30. dEo kdo14 4o po dhoullo2gzd o 5 200 300 00 29 cycles a di m 30 cycles 200 300 00 ee l 31 cycles 200 300 00 E 32 cycles Figure 5 3 Representative AmpF STR SEfiler Plus kit profiles obtained from amplification of 0 50 ng DNA template using 28 29 30 31 and 32 cycles analyzed on the Applied Biosystems 3130x Genetic Analyzer Y axis scale 0 to 4 000 RFUs 5 6 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Accuracy Precision and Reproducibility Accuracy Precision and Reproducibility SWGDAM Guideline 2 9 Accuracy The extent to which a given set of measurements of the same sample agree with their mean and the extent to which these measurements match the actual values being measured should be determined SWGDAM July 2003 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 SEfiler Plus PCR Amplification Kit profiles have been determined from various sample types Figure 5 4 on page 5 8 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 5 4 on page 5 8 represents the nominal nucleotide sizes for t
31. density multiplex detection of nucleic acid sequences oligonucleotide ligation assay and sequence coded separation Nucleic Acids Res 22 4527 4534 Grubwieser P Muhlmann R Berger B Niederstatter H Palvic M Parson W 2006 A new mini STR multiplex displaying reduced amplicon lengths for the analysis of degraded DNA nt J Legal Med 120 115 120 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 4m J Hum Genet 55 175 189 Holt C Stauffer C Wallin J et al 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 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 Three tetranucleotide polymorphisms for loci D381352 D3S1358 D3S1359 Hum Mol Genet 2 1327 Magnuson V L Ally D S Nylund S J Karanjaw
32. different wavelength During data collection on the Applied Analysis Works Biosystems and ABI PRISM instruments the fluorescence signals are separated by a diffraction grating according to their wavelengths and projected onto a charge coupled device CCD camera in a predictably spaced pattern The 6 FAM dye emits at the shortest wavelength and 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 1s some overlap in the emission spectra between the dyes Figure 1 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 1 3 Emission spectra of the five dyes used in the AmpF STR SEfiler Plus PCR Amplification Kit AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 1 11 Chapter 1 Overview Materials and Equipment Kit Contents and Storage The AmpF STR SEfiler Plus PCR Amplification Kit PN 4382699 contains materials sufficient to perform 200 reactions at a 25 uL reaction volume 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 SEfiler Plus Allelic Ladder and GeneScan 600 LIZ Size Standard should also be protected from light Keep
33. freeze thaw cycles to a minimum Table 1 2 AmpF STR SEfiler Plus PCR Amplification Kit contents Reagent Contents Quantity Storage AmpF STR SEfiler Plus Forward and reverse primers 1 tube 45 to 25 C on receipt Primer Set to amplify human DNA target 0 50 mL 2 to 8 C after initial use AmpF STR SEfiler Plus Two tubes of Master Mix 2 tubes 15 to 25 C on receipt Master Mix containing enzyme salts 0 50 mL tube 2 to 8 C after initial use dNTPs carrier protein and 0 05 sodium azide AmpF STR SEfiler Plus Allelic Ladder Allelic ladder containing 1 tube 50 uL 45 to 25 C on receipt amplified alleles refer to 2 to 8 C after initial use AmpF STR SEfiler Plus Kit loci and alleles on page 1 3 for a list of alleles included in the ladder AmpF STR Control DNA 007 0 10 ng uL human male 1 tube 0 3 mL 2 to 8 C 007 DNA in 0 0296 sodium azide and buffer refer to AmpF STR SEfiler Plus Kit loci and alleles on page 1 3 for profile AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Materials and Equipment Standards for For the AmpF STR SEfiler Plus Kit the panel of standards needed Samples for PCR amplification PCR product sizing and genotyping are Control DNA 007 A positive control for evaluating the efficiency of the amplification step and STR genotyping using the AmpF STR SEfiler Plus Allelic Ladder G
34. hae tae we de vbw a 5 36 Mixture Studies 0000 ee ee 5 42 Population Data si ciii 0000 ce eee 5 47 Mutation Rate 0 000 c eee eee 5 48 Probability of Identity 00 ee eee 5 49 Probability of Paternity Exclusion 0 00 eee eee eee 5 50 Troubleshooting Troubleshooting Jie eT Genera eden E QUERN A 2 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Preface How to Use This Guide Purpose of The Applied Biosystems AmpF STR SEfiler Plus PCR This Guide Amplification Kit User Guide provides information about the Applied Biosystems instruments chemistries and software associated with the AmpF STR SEfiler Plus PCR Amplification Kit Pull Out Chapters 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 and number title Text Conventions This guide uses the following conventions Bold text indicates user action For example Type 0 then press Enter for each of the remaining fields e Italic text indicates new or important words and is also used for emphasis For example Before analyzing always prepare fresh matrix A right arrow symbol gt separates successive commands you select from a drop down or shortcut menu For example Select File Open Spot Set Right click the sample row then select View Filter gt View All Runs User Attention Two user attention words a
35. imported panels in the navigation pane a Double click AmpFLSTR SEfiler Plus Panels v1 folder to view the SEfiler Plus v1 folder B gt AmpFLSTR SEfiler Plus Panels v1 M GS Etiler Plus v1 0331358 VWA D165539 D251338 AMEL Reference Samples b Double click the SEfiler Plus v1 folder to display the panel information in the right pane and the markers below it GX DOW WO En set AmFLSTR sEfier Pus Bins i v PR CS m Marker Name Dye Color Min Size Max Size Control Alleles 1 D3S1358 blue 98 0 148 0 15 16 2 WA blue 151 0 213 5 14 16 3 D1I6S539 blue 229 0 279 0 910 4 D251338 blue 2840 350 0 2023 5 AMEL green 106 0 114 0 xv 6 D8S1179 green 118 0 183 5 1243 7 SE33 green 190 0 350 0 17252 8 D195433 yelow 101 0 148 0 14 15 9 THO1 yellow 159 0 205 0 783 10 FGA yellow 20625 360 0 2426 11 D21511 red 184 5 2475 28 31 z 12 D18S51 red 26449 3500 1215 lt AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 4 7 Chapter 4 Analyzing Data 8 View the markers and display the Bin view in the navigation pane a Select the SEfiler Plus v1 folder to display its list of markers in the right pane b Double click the SEfiler Plus v1 folder to display its list of markers below it c Select D2S1338 to display the Bin view for the marker in the right pane F Panel Manager File Edit Bins View Hx mm
36. increasing the calculated percent stutter Multicomponent analysis of off scale data is 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 due to 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 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Sensitivity vm 0 125 ng M 0 062 ng Figure 5 13 Effect of amplifying 1 ng 0 50 ng 0 25 ng 0 125 ng and 0 062 ng of control DNA 007 Note that the y axis scale is magnified for the lower amounts of DNA analyzed using the Applied Biosystems 3130x Genetic Analyzer AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 5 35 Chapter 5 Experiments and Results Stability SWGDAM Guideline 2 4 5 36 The ability to obtain results from DNA recovered from biological samples deposited 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 che
37. information d Click View Deliver Selected Documents Now Note For the MSDSs of chemicals not distributed by Applied Biosystems contact the chemical manufacturer N iue HAZARDOUS WASTE Refer to Material Safety Data Sheets and local regulations for handling and disposal NE CHEMICAL WASTE HAZARD Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury illness or death Noran 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 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Safety Chemical Waste To minimize the hazards of chemical waste Safety Guidelines t 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 requi
38. local Applied Biosystems sales representative GeneAmp PCR System 9700 with the Silver 96 Well block N8050001 GeneAmp PCR System 9700 with the Gold plated silver block 4314878 Silver 96 Well sample block N8050251 AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 1 13 Chapter 1 Overview Table 1 3 Equipment continued Equipment Source Gold plated Silver 96 Well sample block 4314443 Tabletop centrifuge with 96 well plate adapters optional Major Laboratory Supplier MLS Table 1 4 User supplied materials Material Source AmpF STR SEfiler Plus PCR Amplification Kit 4382699 3130 3100x Analyzer materials 96 Well Plate Septa 4315933 Reservoir Septa 4315932 3130x 3100 Genetic Analyzer Capillary Array 36 cm 4315931 3130 3100 Avant Genetic Analyzer Capillary Array 36 cm 4333464 POP 4 Polymer for 3130 3130x Genetic Analyzers 4352755 3100 3100 Avant Genetic Analyzer Autosampler Plate Kit 96 well 4316471 GeneScan 600 LIZ Size Standard 4366589 Running Buffer 10X 402824 DS 33 Matrix Standard Kit Dye Set G5 4345833 MicroAmp Optical 96 Well Reaction Plate N8010560 Hi Di Formamide 4311320 Guide PN 4352716 For a complete list of parts and accessories for the 3130 3130x instrument refer to Appendix A of the Applied Biosystems 3130 3130xl Genetic Analyzers Maintenance Troubleshooting and Reference
39. size standard peaks Note For information on peak detection algorithms refer to the GeneMapper ID Software v3 1 Human Identification Analysis User Guide PN 4338775 Appendix A and the nstallation Procedures and New Features for GeneMapper ID Software v3 2 User Bulletin PN 4352543 Peak Quality Analysis Method Editor HID General Allele Peak Detector Peak Quality Quality Flags Signal level Homozygous min peak height 200 0 Heterozygous min peak height 100 0 Heterozygote balance Min peak height ratio Peak morphology Max peak width basepairs yPull up peak Pull up ratio Allele number Max expected alleles Factory Defaults AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 4 13 Chapter 4 Analyzing Data Importing an HID Size Standard 4 14 Table 4 1 SEfiler Plus HID v1 Advanced Mode analysis method settings continued Tab Settings Quality Flags Analysis Method Editor HID and PQV General Allele Peak Detector Peak Quality Quality Flags Thresholds Quality weights are between 0 and 1 Quality Flag Settings Spectral Pull up os 08 os 08 Control Concordance Broad Peak Low Peak Height Off scale Peak Height Ratio Out of Bin Allele Overlap PQY Thresholds From 075 Sizing Quality 1010 From00to 025 From0 0to 0 25 Factory Defaults From 075 to1 0 Genotype Quality The size standard for
40. such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following US 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 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 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide How to Obtain More Information How to Obtain More Information Related To obtain any of the following documents go to Documentatio
41. the calling region may appear in the green 115 nt and yellow 94 nt dyes depending on the sensitivity of the instrument Figure 5 10 on page 5 28 and Figure 5 11 on page 5 29 show examples of baseline noise and artifacts in an electropherogram while using the SEfiler Plus kit Genotyping may result in the detection of these artifacts as off ladder OL alleles This occurs if the recommended amount of input DNA is exceeded and off scale data are obtained You should consider possible noise and artifacts when interpreting data from the SEfiler Plus kit on the Applied Biosystems 3130 3130x ABI PRISM 3100 3100 Avant and ABI PRISM 310 Genetic Analyzers AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 5 27 Chapter 5 Experiments and Results Dosis EWA D16s539 IDz81338 80 0 400 zy 120 130 rj 450 460 am 180 130 200 210 zo 230 240 250 260 270 200 230 300 so EJ 100 110 120 130 140 450 160 qm 180 130 200 210 20 230 22 250 260 270 200 230 300 D195433 Tao JEGA E gJ 400 10 120 130 140 150 160 m 180 190 200 240 220 230 20 250 260 270 200 230 300 Figure 5 10 Examples of baseline noise and reproducible artifacts in data produced on the Applied Biosystems 3130x Genetic Analyzer Y axis scale 0 50 RFUs 5 28 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Extra Peaks in the Electropherogram 180 160 1
42. zip file containing panels and bins from www appliedbiosystems com Support Software Downloads gt Select GeneMapper ID Software v3 2 gt Updaters amp Patches b Unzip the file Start the GeneMapper JD software then log in with the appropriate user name and password IMPORTANT If you need log on instructions refer to the GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide PN 4338775 page 2 7 Select Tools Panel Manager to open the Panel Manager Find then open the folder containing the panels and bins a Select Panel Manager in the navigation pane Panel Manager File Edit Bins View S amp H mms NE ERSS Panel Manager Highlight this b Select File gt Import Panels to open the Import Panels dialog box c Navigate to then open the GMID_SEfiler_Plus_files folder that you unzipped in step AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Setting Up GeneMapper ID Software v3 2 1 for Analyzing AmpF4STR SEfiler Plus Kit Data 5 Select AmpFLSTR SEfiler Plus Panels v1 then click Import Note Importing this file creates a new folder in the navigation pane of the Panel Manager AmpFLSTR SEfiler Plus Panels vl This folder contains the panels and associated markers Q Import Panels d com Look in le GMID SEfiler Plus files E AmpFLSTR SEfiler Plus Bins v1 AmpFLSTR_SEfiler_Plus_P
43. 0 046 44 2 321 89 321 94 0 039 0 042 45 2 326 03 326 07 0 039 0 048 46 2 330 04 330 06 0 037 0 045 47 2 334 09 334 13 0 024 0 05 48 2 338 19 338 22 0 034 0 041 50 2 346 3 346 35 0 032 0 043 51 2 350 39 350 44 0 031 0 04 SE33 4 2 201 24 201 27 0 026 0 046 6 3 210 31 210 34 0 037 0 046 8 215 36 215 4 0 03 0 042 9 219 5 219 54 0 027 0 05 11 227 65 227 7 0 032 0 051 12 231 71 231 76 0 036 0 042 13 235 79 235 83 0 031 0 041 14 239 87 239 91 0 031 0 049 15 244 04 244 08 0 033 0 048 16 248 19 248 26 0 04 0 052 17 252 26 252 31 0 029 0 047 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Chapter 5 Experiments and Results Table 5 1 Precision results of five runs 16 capillaries run of the AmpF STR SEfiler Plus Allelic Ladder continued Applied Biosystems 3130x Genetic Analyzer Allele Mean Standard Deviation 18 256 26 256 3 0 035 0 045 19 260 2 260 24 0 031 0 053 20 264 24 264 3 0 034 0 047 20 2 266 23 266 28 0 034 0 05 21 268 3 268 36 0 034 0 049 21 1 269 3 269 37 0 029 0 054 21 2 270 26 270 31 0 033 0 051 22 2 274 34 274 38 0 034 0 052 23 2 278 42 278 46 0 032 0 049 24 2 282 47 282 51 0 044 0 053 25 2 286 47 286 53 0 039 0 05 26 2 290 49 290 53 0 04 0 049 27 2 294 48 294 54 0 036 0 045 28 2 298 48 298 53 0 028 0 043 29 2 302 43 302 48 0 027 0 051 30 2 306 38 306 44 0 035 0 05 31 2 310 39 310 46 0 036 0 052 32 2 314 48 314 56 0 042
44. 001 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 produced maximum PCR product yield and that met reproducible performance standards It is the opinion of Applied Biosystems that although these experiments are not exhaustive they are appropriate for a manufacturer IMPORTANT Each laboratory using the AmpF STR SEfiler Plus PCR Amplification Kit must perform internal validation studies AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Developmental Validation Developmental Validation SWGDAM Developmental validation is the demonstration of the accuracy Guideline 1 2 1 precision and 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 Guideline 2 10 1 specificity and robustness must be determined These include thermocycling parameters the concentration of primers magnesium chloride DNA polymerase and other critical reagents SWGDAM July 2003 PCR Applied Biosystems examined the concentration of each component Components ofthe AmpF STR SEfiler Plus PCR Amplification Kit The concentration for each individual component was established to be in the window that meets t
45. 1 10 developmental validation 5 3 DNA degraded 5 36 effect of quantity on result 5 34 effect of quantity figure 5 35 methods for quantifying 2 5 mixture studies 5 42 mixture studies figure 5 44 negative control sample preparation 2 8 non human specificity 5 32 positive control sample preparation 2 8 quantitation importance of 5 34 sensitivity 5 34 stability 5 36 your sample preparation 2 8 DNA mixtures amplification figure 5 45 limit of detection 5 45 documentation related xi E electropherogram causes of extra peaks 5 10 extra peaks 5 21 species specificity 5 32 electrophoresis data collection software 3 3 3 7 preparing samples 3 5 3 8 references 3 3 3 7 runmodule 3 3 3 7 setup 3 3 3 7 emission spectra 1 11 equipment not included with kit 1 13 experiments and results 5 1 extra peaks causes 5 21 F FGA locus designation 1 3 fluorescent dyes 1 11 FTA card DNA amplification using 2 10 figure showing results 2 10 G GeneMapper ID software allele tab 4 11 analysis settings 4 16 analyzing and editing sample files 4 16 considerations 4 2 description 4 2 general tab 4 10 peak detector tab 4 12 peak quality tab 4 13 quality flags tab 4 14 size standard 4 16 viewing imported panels 4 7 GeneMapper Manager 4 9 4 14 GeneScan size standard about 1 13 dye label 1 11 fragment sizes 4 16 volume per reaction 3 5 3 8 guidelines chemical safety vii chemical waste disposal viii chemical wast
46. 20 100 80 Figure 5 11 Examples of a 2 nt reproducible artifact in the SE33 locus Data produced on the Applied Biosystems 3130x Genetic Analyzer Note that a high degree of magnification y axis is used in Figure 5 10 on page 5 28 and Figure 5 11 on page 5 29 to show the artifacts AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 5 29 Chapter 5 Experiments and Results Characterization of Loci SWGDAM Guideline 2 1 Nature of the Polymorphisms 5 30 Inheritance The basic characteristics of a genetic marker must be determined and documented SWGDAM July 2003 This section describes basic characteristics of the 11 loci and the sex determining marker amelogenin that are amplified with the AmpF STR SEfiler Plus PCR Amplification Kit These loci have been extensively characterized by other laboratories The primers for the amelogenin locus flank a six nucleotide deletion within intron 1 of the 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 SEfiler Plus kit loci except the SE33 locus 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 The SE33 locus is highly polymorphic The SE33 locus not onl
47. 3 14 15 16 15 16 17 18 19 D8S1179 8 VIC 8 9 10 11 12 13 in some 12 13 14 references 15 16 17 designated 18 19 as D6S502 D16S539 16q24 qter 6 FAM 5 8 9 10 9 10 11 12 13 14 15 D18S51 18q21 3 PET 7 9 10 12 15 10 2 11 12 13 13 2 14 14 2 15 16 17 18 19 20 21 22 23 24 25 26 27 AmpF 4STR SEfiler Plus PCR Amplification Kit User Guide 1 3 Chapter1 Overview Table 1 1 AmpF STR SEfiler Plus Kit loci and alleles AmpF STR AmpF STR Locus Chromosome Dye Allelic Control Designation Location Label Ladder DNA 007 Alleles Genotype D19S433 19q12 13 1 NED 9 10 11 14 15 12 12 2 13 13 2 14 14 2 15 15 2 16 16 2 17 17 2 D21S11 21q11 2 q21 PET 24 24 2 25 28 31 26 27 28 28 2 29 29 2 30 30 2 31 31 2 32 32 2 33 33 2 34 34 2 35 35 2 36 37 38 FGA 4q28 NED 17 18 19 24 26 20 21 22 23 24 25 26 26 2 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 1 4 AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide Table 1 1 Product Overview AmpF STR SEfiler Plus Kit loci and alleles Locus Designation Chromosome Location Dye Label AmpF STR Allelic Ladder Alleles AmpF STR Control DNA 007 Genotype SE33 ACTBP2 VIC 4 2 6 3 8 9 11 12 13 14 15 16 17
48. 3 0 04 16 127 76 127 86 0 03 0 044 16 2 129 75 129 86 0 027 0 047 17 131 72 131 83 0 035 0 057 17 2 133 72 133 84 0 034 0 044 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Accuracy Precision and Reproducibility Table 5 1 Precision results of five runs 16 capillaries run of the AmpF STR SEfiler Plus Allelic Ladder continued Applied Biosystems 3130x Genetic Analyzer Allele Mean Standard Deviation D21811 24 185 16 185 24 0 022 0 041 24 2 187 22 187 29 0 026 0 041 25 189 25 189 33 0 021 0 039 26 193 32 193 41 0 026 0 041 27 197 41 197 51 0 026 0 048 28 201 41 201 48 0 025 0 036 28 2 203 36 203 44 0 031 0 046 29 205 34 205 42 0 031 0 046 29 2 207 37 207 46 0 021 0 035 30 209 34 209 43 0 026 0 039 30 2 211 31 211 4 0 025 0 036 31 213 32 213 41 0 033 0 038 31 2 215 34 215 43 0 028 0 044 32 217 4 217 51 0 026 0 041 32 2 219 44 219 51 0 037 0 043 33 221 48 221 57 0 037 0 044 33 2 223 44 223 51 0 031 0 041 34 225 57 225 64 0 029 0 045 34 2 227 5 227 57 0 028 0 046 35 229 56 229 65 0 035 0 044 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide 5 13 Chapter 5 Experiments and Results 5 14 Table 5 1 Precision results of five runs 16 capillaries run of the AmpF STR SEfiler Plus Allelic Ladder continued Applied Biosystems 3130x Gen
49. 5 17 on page 5 45 where samples A and B were mixed according to the ratios provided The minor component allele calls at non overlapping loci are highlighted The amplification of the minor contributor at 3 1 and 7 1 0 875 0 125 ng mixture ratios was readily typeable 15 1 ratios generally resulted in partial profiles for the minor component AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 5 45 Chapter 5 Experiments and Results Table 5 6 shows the profiles of the samples in Figure 5 17 on page 5 45 Table 5 6 Genotypes of mixed DNA samples Allele oaro ONA O07 Profile Sample B D3S1358 15 16 15 18 VWA 14 16 14 D16S539 9 10 12 13 D2S1338 20 23 20 21 Amelogenin X Y X Y D8S1179 12 13 14 15 SE33 17 25 2 18 28 2 D19S433 14 15 12 2 14 2 THO1 7 93 6 9 3 FGA 24 26 21 22 D21S11 28 31 28 30 D18S51 12 15 17 19 5 46 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Population Data Population Data SWGDAM Guideline 2 7 Overview The distribution of genetic markers in populations should be determined in relevant population groups SWGDAM July 2003 To interpret the significance of a match between genetically typed samples it is necessary to know the population distribution of alleles at each locus in question If the genotype of the relevant evidence sample is different from the genotype of the suspect s reference sample then the s
50. 7 159 49 159 5 0 028 0 042 18 163 58 163 6 0 027 0 047 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Chapter 5 Experiments and Results Table 5 1 Precision results of five runs 16 capillaries run of the AmpF STR SEfiler Plus Allelic Ladder continued Applied Biosystems 3130x Genetic Analyzer Allele Mean Standard Deviation 19 167 64 167 65 0 02 0 042 FGA 17 210 09 210 1 0 032 0 038 18 214 14 214 15 0 027 0 042 19 218 28 218 3 0 026 0 032 20 222 4 222 42 0 025 0 043 21 226 47 226 5 0 033 0 046 22 230 54 230 57 0 029 0 048 23 234 63 234 65 0 033 0 041 24 238 71 238 73 0 033 0 044 25 242 86 242 89 0 029 0 037 26 247 03 247 06 0 029 0 044 26 2 249 1 249 13 0 034 0 039 27 251 1 251 14 0 03 0 037 28 255 11 255 13 0 038 0 043 29 259 08 259 11 0 027 0 035 30 263 15 263 18 0 034 0 049 30 2 264 97 265 0 036 0 049 31 2 269 03 269 06 0 035 0 048 32 2 273 09 273 11 0 031 0 045 33 2 277 17 277 2 0 036 0 047 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Accuracy Precision and Reproducibility Table 5 1 Precision results of five runs 16 capillaries run of the AmpF STR SEfiler Plus Allelic Ladder continued Applied Biosystems 3130x Genetic Analyzer Allele Mean Standard Deviation 42 2 313 41 313 48 0 039 0 053 43 2 317 67 317 72 0 028
51. A 0 1 ng uL with 5 uL of low TE buffer for a total volume of 10 uL The final sample concentration is 0 05 ng uL Add to the reaction mix Note The final reaction volume should be 25 uL 7 Centrifuge the plate at 3 000 rpm for about 20 seconds in a tabletop centrifuge with plate holders to remove any bubbles 8 Amplify the DNA in a Silver block 96 Well GeneAmp PCR System 9700 or a Gold plated silver block GeneAmp PCR System 9700 2 8 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Performing PCR Performing PCR To run PCR 1 Set the thermal cycling conditions IMPORTANT If using the Gold plated Silver or Silver 96 Well GeneAmp PCR System 9700 select the 9600 Emulation mode Initial Cycle Final Final Incubation 30 cycles Extension Hold Step De Anneal Extend nature HOLD CYCLE HOLD HOLD 95 C 94 C 59 C 72 C 60 C 4 C 11 min 20 sec 2 min 1 min 60 min eo 2 Load the plate into the thermal cycler then close the heated cover ANTITE PHYSICAL INJURY HAZARD During instrument operation the temperature of the heated cover can be as high as 108 C and the temperature of the sample block can be as high as 100 C Keep hands away from the heated cover and sample block 3 Start the run 4 Store the amplified DNA If you are storing the DNA for Store at 2 weeks 2to8 C gt 2 w
52. AmpF STR SEfiler Plus PCR Amplification Kit User Guide Applied Biosystems AmpF STR SEfiler Plus PCR Amplification Kit User Guide Copyright 2012 Life Technologies Corporation All rights reserved Printed in the U S A Applera Life Technologies Corporation AB Design ABI PRISM AmpF STR GeneAmp GeneMapper GeneScan LIZ MicroAmp PET Profiler Plus Quantifiler SGM Plus and VIC are registered trademarks and FAM Hi Di NED POP 4 SEfiler and SEfiler Plus are trademarks of Applera Corporation or its subsidiaries in the U S and or certain other countries TaqMan is a registered trademark of Roche Molecular Systems Inc Windows NT is a registered trademark of Microsoft Corporation All other trademarks are the sole property of their respective owners Part Number 4385739 Rev B 03 2012 Contents Preface Chapter 1 Chapter 2 Chapter 3 How to Use This Guide 0 0 c cc eee eens oafely ce seta SCR terere d iib tee mote daha gua BARD How to Obtain More Information llle How to Obtain Support 00 00 ee Overview Product OVetview rsss ob Sh eid S Paes Se Se Re ees Workflow Overview 0 0 eee rh Instrument and Software Overview llle 1 Materials and Equipment 000 ce eee eee 1 PCR Amplification PORANOFK Areas rU RENE RERUM cd as eus Required User Supplied Materials and Reagents Quantifying DNA
53. Consequently the CEPH family DNA sets are ideal for studying inheritance patterns Begovich et al 1992 Because the oligonucleotide sequences between the SEfiler and SEfiler Plus kits are identical no changes in the pattern of inheritance are reported Mapping The SEfiler Plus kit loci have been mapped and the chromosomal locations have been published Nakahori et al 1991 Edwards et al 1992 Kimpton ef al 1992 Mills et al 1992 Sharma and Litt 1992 Li et al 1993 Straub et al 1993 Barber and Parkin 1996 AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 5 31 Chapter 5 Experiments and Results S pecies Specificity SWGDAM For techniques designed to type human DNA the potential to detect Guideline 2 2 DNA from forensically relevant nonhuman species should be evaluated SWGDAM July 2003 The AmpF STR SEfiler 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 SEfiler Plus kit experiments on nonhuman DNA sources are shown in Figure 5 12 1800 1200 600 D 1800 1200 600 120 al NTC 100 200 300 400 I Control DNA 100 200 300 400 Chimpanzee 100 200 300 400 Microbial pool 100 200 300 400 Figure 5 12 Representative electropherograms from a species specificity stu
54. Efiler Plus GS600 HD 117 CO3 fsa 117 Sample SEfiler_Plus_HID_v1 SEfiler Plus vi CE GS SEfiler Plus GS600 HD 118 D03 fsa 118 Sample SEfiler Plus HD v1 SEfiler Plus vi CE G5 SEfiler Plus GS800 HD 119 E03 fsa 119 Sample SEfler Plus HD v1 SEfiler Plus v1 CE G5 SEfller Plus GS500 HD 120 FO03 fsa 120 Sample SEfiler Plus HID v1 SEfiler Plus v1 CE GS SEfiler Plus GS500 HD 122_A04 fsa 122 Sample SEfiler Plus HD v1 SEfiler Plus v1 CE GS SEfiler Plus GS600 HD B dk 4k dE di di d AE di Me de de di De di de di de d di d dd 123 B04 fsa 123 Sample SEfiler Plus HID v1 SEfiler Plus vi CE G5 SEfiler Plus GS amp 00 HD mHEHNENENENENENENENENENENENENENENENENENEN 3 BREHNHNENENENENENENENENENENENENENENENENEN Figure 4 1 For more information about any of these tasks refer to the Project Window before analysis GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide PN 4338775 AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 4 17 Chapter 4 Analyzing Data Examining and You can display electropherogram plots from the Samples and Editing a Project 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
55. GeneMapper ID Software v3 2 1 for the first time you need to Import panels and bins into the Panel Manager as explained in Importing Panels and Bins on page 4 4 Import an analysis method as explained in Importing an HID Analysis Method on page 4 9 Define custom views of analysis tables Refer to Chapter 1 of the GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial PN 4335523 for more information Define custom views of plots Refer to Chapter 1 of the GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial PN 4335523 for more information Note For details about GeneMapper 7D features refer to the 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 Also refer to the nstallation Procedures and New Features for GeneMapper ID Software v3 2 User Bulletin PN 4352543 Note For details about GeneMapper D X refer to GeneMapper ID X Software Version 1 0 Human Identification Analysis Getting Started Guide PN 4375574 AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 4 3 Chapter 4 Analyzing Data Importing Panels To import the SEfiler Plus kit panels and bin sets from the and Bins Applied Biosystems web site into the GeneMapper JD software database a Download the GMID SEfiler Plus files
56. J Schneider P M 2004 Preparation of degraded human DNA under controlled conditions Forensic Sci Int 139 134 140 Brinkman 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 Brinkman B Moller A and Wiegand P 1995 Structure of new mutations in 2 STR systems ntl J Legal Med 107 201 203 Butler J M 2005 Forensic DNA Typing Burlington MA Elsevier Academic Press AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide Bibliography 1 Bibliography 2 Butler J M Shen Y McCord B R 2003 The development of reduced size STR amplicons as tools for analysis of degraded DNA J Forensic Sci 48 1054 1064 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 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 Chung D T Drabek J Opel K L Butler J M and McCord B R 2004 A study of the effects of degradation and template concentration on the amplification efficiency of the Miniplex primer sets J Forensic Sci 49 733 740 Clark J M 1988 Novel non templated nucleotide addition reactions cat
57. M Holt C L Lazaruk K D Nguyen T H Walsh P S 2002 Constructing universal multiplex PCR systems for comparative genotyping J Forensic Sci 47 52 65 Walsh P S Fildes N J Reynolds R 1996 Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vWA Nucleic Acids Res 24 2807 2812 AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide Bibliography 5 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 Wiegand P and Kleiber M 2001 Less is more length reduction of STR amplicons using redesigned primers Int J Legal Med 114 285 287 Bibliography 6 AmpF4STR SEfiler Plus PCR Amplification Kit User Guide Index Symbols A nucleotide addition defined 5 25 efficiency of 5 25 lack of causes 5 27 Numerics 310 allelic ladder requirements 3 2 3100 and 3130 series allelic ladder requirements 3 2 A accuracy and reproducibility 5 7 alleles off ladder 5 8 peak height ratio table 5 42 allelic bin definitions 4 2 offsets 4 2 allelic ladder analysis method for 4 2 contents 1
58. The concentrations of humic acid tested were 0 20 40 and 60 ng uL The SEfiler Plus kit efficiently amplified the DNA at concentrations of humic acid that inhibited the amplification of DNA with the SEfiler kit see Figure 5 16 on page 5 40 200 300 400 SEfiler Plus kit 1 ng DNA 007 Untreated AL ud LL Al iL 400 SEfiler Plus kit 1 ng DNA 007 20 ng uL Humic acid SEfiler kit 1 ng DNA 007 Untreated IRE LA s SEfiler kit 1 ng DNA 007 20 ng uL Humic acid Figure 5 16 Amplification with AmpF STR SEfiler Plus and SEfiler kits in the presence and absence of humic acid Panels 1 and 3 correspond to control samples panels 2 and 4 correspond to samples amplified in the presence of 20 ng uL humic acid AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Comparison of SEfiler Plus and SEfiler kit performance ina simulated model of humic acid inhibition Stability Only those peaks gt 50 RFUs were counted A complete profile with control 007 DNA yields 24 peaks using the SEfiler Plus kit see Table 5 4 Table 5 4 Comparison of SEfiler Plus and SEfiler kit performance in simulated model of humic acid inhibition n 3 Humic Acid ng uL SEfiler Plus kit SEfiler kit 0 24 24 24 24 24 24 24 24 24 24
59. ala Z E Rayman J B Knapp J I Lowe A L Ghosh S 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 P D Budowle B McCord B R 1998 Analysis of multiplexed short tandem repeat STR systems using capillary array electrophoresis Electrophoresis 19 101 107 AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide Bibliography 3 Bibliography 4 Mills K A Even D and Murrau J C 1992 Tetranucleotide repeat polymorphism at the human alpha fibrinogen locus FGA Hum Mol Genet 1 779 Moller A and Brinkmann B 1995 PCR VNTRs PCR Variable Number of Tandem Repeats in forensic science Cell Molec Biol 41 715 724 Moller A Meyer E and Brinkmann B 1994 Different types of structural variation in STRs HumFES FPS HumVWA and HumD21 S11 Intl J Legal Med 106 319 323 Moller A Schurenkamp M and Brinkmann B 1995 Evaluation of an ACTBP2 ladder composed of 26 sequenced alleles nt J Legal Med 108 75 78 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
60. alyzed by procaryotic and eucaryotic DNA polymerases Nucleic Acids Res 16 9677 9686 Coble M D and Butler J M 2005 Characterization of new miniSTR loci to aid analysis of degraded DNA J Forensic Sci 50 43 53 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 Drabek J Chung D T Butler J M McCord B R 2004 Concordance study between Miniplex assays and a commercial STR typing kit J Forensic Sci 49 859 860 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 AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide Grossman P D Bloch W Brinson E Chang C C Eggerding F A Fung S lovannisci D M Woo S Winn Deen E S 1994 High
61. alyzer AmpFSTR SEfiler Plus PCR Amplification Kit User Guide 100 Developmental Validation Of the tested annealing temperatures 55 to 61 C produced robust profiles 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 temperature of 59 C 55 C 57 C Figure 5 2 Electropherograms obtained from amplification of 0 50 ng of control DNA 007 at annealing 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 amp TR SEfiler Plus PCR Amplification Kit User Guide 5 5 Chapter 5 Experiments and Results PCR Cycle SEfiler Plus kit reactions were amplified for 28 29 30 31 and 32 Number cycles on the Silver 96 Well GeneAmp PCR System 9700 using 0 50 ng from three DNA samples As expected the amount of PCR product increased with the number of cycles A full profile was generated at 28 cycles and off scale data were collected for several allele peaks at 32 cycles Figure 5 3 Although none of the cycle numbers tested produced nonspecific peaks 30 cycles was found to give optimal sensitivity when the amplified products were examined on Applied Biosystems 3130x Genetic Analyzers 200 300 am 28 cycles fN E E hb
62. ance studies for the SEfiler Plus kit using this configuration AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 3 7 Chapter 3 Performing Electrophoresis Preparing Samples for Electrophoresis on the 310 Instrument Preparing the Prepare the samples for electrophoresis on the 310 instrument Samples immediately before loading To prepare samples for electrophoresis l Calculate the volume of Hi Di Formamide and GeneScan 600 LIZ Internal Size Standard needed to prepare the samples using the table below Volume Reagent per reaction uL GeneScan 600 LIZ Size Standard 1 0 Hi Di Formamide 24 0 Note Include additional samples in your calculations to provide excess volume for the loss that occurs during reagent transfers IMPORTANT The volume of size standard indicated in the table is a suggested amount Determine the appropriate amount of size standard based on your results experiments Nomre CHEMICAL HAZARD Hi Di Formamide Exposure causes eye skin and respiratory tract irritation It is a possible developmental and birth defect hazard Avoid breathing vapor Use with adequate ventilation Avoid contact with eyes and skin Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Pipette the required volumes of components into an appropriately sized polypropylene tube Vortex the tube then cen
63. any of these tasks refer to Installation Procedures and New Features for GeneMapper ID Software v3 2 User Bulletin PN 4352543 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 4 18 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Part Number 4385739 Rev B 03 2012 Experiments and Results This chapter covers OVENI EW ce seemed bot A TR aha Beara RSEN Ma Poo M he He 5 2 Developmental Validation noana aaaea ees 5 3 Accuracy Precision and Reproducibility 4 5 7 Extra Peaks in the Electropherogram 00005 5 21 Characterization of Loci 0 0 ec eens 5 31 Species Specificity 0 0 ec eet 5 33 Sensitivity sees ice teche E ELE Re Se ee ew 5 36 Stabs po ok pw cette RES PM ERO NE RRR OER ee ES 5 38 Mixture St dies ee 4 ed nice adnan eh a ew ea R 5 44 Population Data 0 0 eect teens 5 49 Mutation Rate eine lorie cette t eoe REOR REE etn Ordres 5 50 Probability of Identity 2 llle 5 51 Probability of Paternity Exclusion 000000 5 52 AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 5 1 Chapter 5 Experiments and Results Overview Experiments Using AmpF STR SEfiler Plus Kit 5 2 Importance of Validation Experiments This chapter provides results of the developmental
64. ce sample s 5 44 AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 150 460 170 480 130 200 210 220 290 240 250 260 270 280 230 300 310 320 330 340 350 t Mixture Studies 30 110 130 250 260 270 280 230 300 310 320 330 340 350 a 1000 100 150 160 170 180 190 200 210 220 230 450 460 470 180 480 200 210 220 290 240 250 260 200 280 230 300 310 320 330 340 350 30 100 150 160 170 180 180 200 210 220 230 240 250 260 270 280 230 300 310 320 330 340 350 2000 30 100 110 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 230 300 310 320 330 340 350 0 1 Figure 5 17 Amplification of DNA mixtures at various ratios Limit of Detection of the Minor Component Mixtures of two DNA samples were examined at various ratios 0 1 1 1 3 1 7 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 Figure
65. dy including positive and non template controls NTC 5 32 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Species Specificity Figure 5 12 on page 5 32 shows amplification for control DNA 007 0 50 ng panel 1 chimpanzee 0 50 ng panel 2 dog 2 5 ng panel 3 cat 2 5 ng panel 4 horse 2 5 ng panel 5 microbial DNA pool equivalent to 10 copies of Candida albicans 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 SEfiler Plus kit and analyzed using the Applied Biosystems 3130x Genetic Analyzer Primates gorilla chimpanzee orangutan and macaque 0 50 ng each Non primates mouse dog sheep rabbit cat horse hamster rat chicken and cow 2 5 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 283 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 VIC dye AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 5 33 Chapter 5 Experiments and Results Sensitivity 5 34 SWGDAM Guideline 2 3
66. e results of five runs of the AmpF STR SEfiler Plus Allelic Ladder For more information on precision and genotyping see Lazaruk et al 1998 and Mansfield et al 1998 In Table 5 1 the mean size for all the alleles in each run 16 capillaries was calculated The mean range shown in the table is the lowest and highest mean size values of the 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 5 1 is the lowest and highest standard deviation values of the five runs Table 5 1 Precision results of five runs 16 capillaries run of the AmpF STR SEfiler Plus Allelic Ladder Applied Biosystems 3130x Genetic Analyzer Allele Mean Standard Deviation Amelogenin X 102 16 102 18 0 028 0 034 Y 108 14 108 15 0 015 0 038 D16S539 5 227 25 227 29 0 027 0 047 8 239 36 239 42 0 034 0 044 9 243 51 243 56 0 027 0 045 10 247 63 247 69 0 029 0 042 11 251 67 251 74 0 026 0 043 12 255 63 255 69 0 029 0 045 18 259 56 259 63 0 024 0 041 14 263 57 263 63 0 031 0 045 15 267 59 267 64 0 027 0 041 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Accuracy Precision and Reproducibility Table 5 1 Precision results of five runs 16 capillaries run of the AmpF STR SEfiler Plus Allelic Ladder continued Applied Biosystems 3130x Genetic Analyzer
67. e safety ix H hazards chemical waste viii hematin effects of 5 38 HID analysis method importing 4 9 Hi Di formamide volume per reaction 3 5 3 8 humic acid effect of 5 40 Index 2 AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide Import Panels dialog box 4 4 IMPORTANT description vi Information Development department contacting xii inheritance 5 30 italic text when to use v K kit contents 1 12 description 1 2 fluorescent dyes 1 11 instruments for use with 1 2 loci amplified 1 3 master mix 1 12 part number 1 12 primers 1 2 1 12 purpose 1 2 reagents 1 12 storage 1 12 thermal cyclers for use with 2 3 kit performance comparison DNase I figure 5 37 hematin figure 5 38 hematin table 5 39 humic acid figure 5 40 humic acid table 5 41 L LIZ size standard about 1 13 volume per reaction 3 5 3 8 loci AmpFISTR SGM Plus 1 3 amplified 1 3 characterization 5 30 mapping 5 31 low TE buffer preparation 2 4 M magnesium chloride concentration validation of 5 4 marker displaying Bin view of 4 8 master mix contents 1 12 storage 1 12 volume per reaction 2 7 materials and equipment 1 12 materials not included with kit 1 13 menu commands conventions for describing v mixed samples resolution of genotypes 5 44 mixture studies 5 42 MSDSs description vii obtaining vii multicomponent analysis 1 10 mutation rate 5 48 mutation studies 5 48 mutation STR 5 48 N navigation
68. eeks 15 to 25 C IMPORTANT Protect the amplified products from light AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 2 9 Chapter 2 PCR Amplification Amplification Using Bloodstained FTA Cards 2 10 FTA treated DNA collection cards can be useful for the collection storage and processing of biological samples You can place a small punch disk of the bloodstained card directly into an amplification tube purify it and amplify it without transferring the evidence Applied Biosystems studies indicate that a 1 2 mm bloodstained disk contains approximately 5 to 20 ng DNA An appropriate cycle number for this high quantity of DNA is 25 cycles It is recommended that each laboratory determine the cycle number based on individual validation studies In the example shown in Figure 2 1 a 1 2 mm disk of a bloodstained FTA card was purified using three washes with FTA Purification Reagent and two washes with 1X TE buffer After drying at room temperature overnight the punch was then amplified directly in the MicroAmp tube for 25 cycles 200 EJ am A Figure 2 1 AmpF STR SEfiler Plus PCR Amplification Kit results from a 1 2 mm FTA bloodstain disk 25 cycle amplification analyzed on the Applied Biosystems 3130x Genetic Analyzer AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Part Number 4385739 Rev B 03 2012 Chapter 3 Electrophoresis AmpFSTR SEfi
69. eneScan 600 LIZ Size Standard A size standard used for obtaining sizing results in the 60 to 400 nt range during use with the AmpF STR SEfiler Plus kit The GeneScan 600 LIZ Size Standard contains 36 single stranded fragments of 20 40 60 80 100 114 120 140 160 180 200 214 220 240 250 260 280 300 314 320 340 360 380 400 414 420 440 460 480 500 514 520 540 560 580 and 600 nucleotides This standard has been evaluated as an internal size standard and it yields precise sizing results for AmpF STR SEfiler Plus PCR products Order the GeneScan 600 LIZ Size Standard PN 4366589 separately AmpF STR SEfiler Plus Allelic Ladder An allelic ladder developed by Applied Biosystems for accurate characterization of the alleles amplified by the SEfiler Plus kit The AmpF STR SEfiler Plus Allelic Ladder contains most alleles reported for the 11 autosomal loci Refer to Loci Amplified by the Kit on page 1 3 for a list of the alleles included in the SEfiler Plus kit Equipment and Tables 1 3 and 1 4 list required and optional equipment and materials Materials Not not supplied with the SEfiler Plus kit Unless otherwise indicated Included many of the items are available from major laboratory suppliers MLS Table 1 3 Equipment Equipment Source Applied Biosystems 3130 3100x Genetic Analyzer ABI Prism 3100 3100 Avant Genetic Analyzer ABI PRISM 310 Genetic Analyzer Contact your
70. entages for the D3S1358 vWA D16S539 and D2S1338 loci 5 22 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Extra Peaks in the Electropherogram 19 0 18 0 17 0 16 0 s 15 0 14 0 13 0 x 12 0 a 11 0 10 0 9 0 Percent Stutter 8 0 memga e o sms 7 0 em n seasea ec so 6 0 5 0 4 0 3 0 2 0 1 0 0 0 7 8 9 10 11 12 13 44 15 16 17 18 19 20 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2122 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 D8S1179 SE33 Figure 5 6 Stutter percentages for D8S1179 and SE33 loci AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 5 23 Chapter 5 Experiments and Results 19 0 18 0 17 0 16 0 15 0 14 0 13 0 12 0 11 0 10 0 9 0 Percent Stutter 8 0 7 0 6 0 5 0 4 0 3 0 2 0 1 0 0 0 ss sennep c m ET ae t 9 9 AE Su mete ty e ee mm dib Sette zi uH cal HM ME 1 3 E 9 10 1112 13 14 15 16 17 18 19 D19S433 45678 91011121314 THO1 16 17 19 20 21 22 23 24 25 26 27 28 20 30 3132 33 3435 42 43 44 45 46 47 48 49 50 51 52 Figure 5 7 Stutter percentages for the D19S433 THO1 and FGA loci 19 0 18 0 17 0 16 0 15 0 14 0 13 0 12 0 11 0 10 0 9 0 Percent Stutter 8 0 7 0 6 0 5 0 4 0 3 0 2 0 1 0 0 0 ve eee wee anp Opes ae dak ee ee dite n ec t P
71. etic Analyzer Allele Mean Standard Deviation 35 2 231 51 231 59 0 038 0 044 36 233 5 233 58 0 029 0 046 37 237 58 237 68 0 032 0 044 38 241 59 241 67 0 03 0 04 D281338 15 288 11 288 2 0 035 0 039 16 292 09 292 18 0 039 0 041 17 296 05 296 13 0 03 0 038 18 300 300 08 0 034 0 041 19 303 91 303 98 0 031 0 038 20 307 84 307 92 0 03 0 049 21 311 86 311 93 0 044 0 05 22 315 97 316 07 0 028 0 048 23 320 22 320 28 0 033 0 041 24 324 3 324 37 0 03 0 047 25 328 36 328 44 0 031 0 046 26 332 38 332 5 0 027 0 053 27 336 44 336 53 0 029 0 038 28 340 76 340 83 0 027 0 042 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Accuracy Precision and Reproducibility Table 5 1 Precision results of five runs 16 capillaries run of the AmpF STR SEfiler Plus Allelic Ladder continued Applied Biosystems 3130x Genetic Analyzer Allele Mean Standard Deviation D3S1358 12 109 27 109 27 0 027 0 041 18 113 51 113 52 0 009 0 033 14 117 47 117 48 0 02 0 035 15 121 31 121 34 0 027 0 037 16 125 44 125 46 0 027 0 044 17 129 56 129 58 0 027 0 052 18 133 57 133 58 0 024 0 044 19 137 5 137 51 0 033 0 037 D8S1179 8 122 59 122 62 0 026 0 04 9 126 58 126 6 0 025 0 045 10 130 58 130 6 0 027 0 041 11 134 62 134 63 0 024 0 03 12 138 69 138 7 0 012 0 039 13 142 91 142 93 0 024 0 037 14 147 06 147 07 0 023 0 038 15 151 18 151 2 0 023 0 032 16 155 33 155 35 0 028 0 039 1
72. filer_Plus_HID_v1 SEfiler Plus v1 CE G5 SEfiler Plus GS500 HD 104_D01 fsa 104 Sample SEfiler Plus HID v1 SEfiler Plus v1 CE G5 SEfiler Plus GS500 HD 105 EO1 fsa 105 Sample SEfler Plus HD v1 SEfiler Plus v1 CE GS SEfiler Plus GS600 HD 106 FO01 fsa 106 Sample SEfiler Plus HID v1 SEfiler Plus vi CE G5 SEfiler Plus GS amp 00 HD 108_A02 fsa 108 Sample SEfiler Plus HID v1 SEfiler Plus v1 CE G5 SEfiler Plus GS600 HD 109 B02 fsa 108 Sample SEfiler Plus HID v1 SEfiler Plus v1 CE G5 SEfiler Plus GS600 HD 110 C02 fsa 110 Sample SEfier Plus HD v1 SEfiler Plus v1 CE GS SEfller Plus GS800 HD 111 D02 fsa 111 Sample SEfiler Plus HD v1 SEfiler Plus vi CE G5 SEfiler Plus GS600 HD 112 E02 fsa 112 Sample SEfler Plus HD v1 SEfiler Plus v1 CE G5 SEfiler Plus GS500 HD 113 F02 fsa 113 Sample SEfiler Plus HID v1 SEfiler Plus v1 CE G5 SEfiler Plus GS500 HD 114 G02 fsa 114 Sample SEfler Plus HD v1 SEfiler Plus v1 CE GS SEfiler Plus GS600 HD ladder G01 fsa ladder Allelic Ladder SEfiler_Plus_HID_v1 SEfiler Plus v1 CE G5 SEfiler Plus GSE00 HD 115 A03 fsa 115 Sample SEfiler Plus HID v1 SEfiler Plus v1 CE G5 SEfiler Plus GS500 HD 115 B03 fsa 116 Sample SEfiler Plus HID v1 SEfiler Plus v1 CE G5 S
73. h or serious injury N py elss Indicates an imminently hazardous situation that if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations rom CHEMICAL HAZARD Some of the chemicals used with Applied Biosystems instruments and protocols are potentially hazardous and can cause injury illness or death AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Safety Chemical Safety To minimize the hazards of chemicals 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 vii 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 About MSDSs Chemical manufacturers s
74. he AmpF STR SEfiler 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 allele size from the corresponding allelic ladder allele size All sample alleles are within 0 5 nt from a corresponding allele in the allelic ladder AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 5 7 Chapter 5 Experiments and Results 5 8 0 50 o N a Size Difference nt o S 100 Precision and Size Windows o a n LJ ub B a d gg 4 Sgt oR dl of BoB aiaeag H EL NUES 5 a mu o xti iy eee il o o 120 140 160 180 200 220 240 260 280 300 320 340 360 Allele Size nt Figure 5 4 Size deviation of 77 samples analyzed on the Applied Biosystems 3130x Genetic Analyzer Sizing precision allows for determining accurate and reliable genotypes Sizing 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 SEfiler 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 An off ladder allele that is an allele of a size that is not represented in the AmpF STR SEfiler Plus Allelic Ladder or An allele
75. he 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 0 50 ng of the control DNA 007 is shown in Figure 5 1 on page 5 4 Applied Biosystems observed that the performance of the multiplex is most robust within a 20 window of magnesium chloride concentration AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 5 3 Chapter 5 Experiments and Results 96 Change 400 200 300 ax 4000 m 3096 Figure 5 1 0 50 ng of control DNA 007 amplified with the AmpF STR SEfiler Plus kit in the presence of varying concentrations of magnesium chloride and analyzed on the Applied Biosystems 3130x Genetic Analyzer Thermal Cycler Thermal cycling parameters were established for amplification of the Parameters SEfiler Plus kit Thermal cycling times and temperatures of GeneAmp PCR systems were verified Varying annealing and denaturation temperature windows were tested to verify that a specific PCR product with the desired sensitivity of at least 0 50 ng of AmpF STR Control DNA 007 was produced For example annealing temperatures were tested at 55 57 59 61 and 63 C Figure 5 2 on page 5 5 for 2 minute hold times in the Silver 96 Well GeneAmp PCR System 9700 The PCR products were analyzed using the Applied Biosystems 3130x Genetic An
76. ication Kit PCR Products User Bulletin PN 4332345 t Applied Biosystems performed validation studies for the SEfiler Plus kit using this configuration 3 4 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Preparing Samples for Electrophoresis on the 3100 3100 Avant or 3130 3130xl Instrument Preparing Samples for Electrophoresis on the 3100 3100 Avant or 3130 31 30x Instrument Preparing the Prepare the samples for electrophoresis on the 3100 3 100 Avant or Samples 3 130 3130x instrument immediately before loading To prepare samples for electrophoresis l Calculate the volume of Hi Di Formamide and GeneScan 600 LIZ Internal Size Standard needed to prepare the samples using the table below Reagent Volume 9 per reaction uL GeneScan 600 LIZ9 Size Standard 0 6 Hi Di Formamide 8 4 Note Include additional samples in your calculations to provide excess volume for the loss that occurs during reagent transfers IMPORTANT The volume of size standard indicated in the table is a suggested amount Determine the appropriate amount of size standard based on your results experiments Nome CHEMICAL HAZARD Hi Di Formamide Exposure causes eye skin and respiratory tract irritation It is a possible developmental and birth defect hazard Avoid breathing vapor Use with adequate ventilation Avoid contact with eyes and skin Read the MSDS and follow the handling instructio
77. ide addition 5 26 figure 5 26 stability DNA 5 36 standards for samples 1 13 storage recommendations kit 1 12 stutter percent CSFIPO and FGA loci 5 24 D13 317 and D78820 loci 5 22 D16S 539 and D18S51 loci 5 24 D281338 and D21S111oci 5 23 foreachkitlocus 5 24 marker specific 5 24 stutter products 5 21 T Technical Support contacting xii thermal cyclers for use with kit 2 3 programming 2 9 thermal cycling parameters validation of 5 4 training information on xii troubleshooting causes and actions A 2 U user attention words described v V validation annealing temperatures 5 4 5 5 characterization of loci 5 30 developmental 5 3 effect of DNA quantity 5 34 experiments to evaluate 5 2 importance of 5 2 importance of DNA quantitation 5 34 magnesium chloride concentration 5 4 mixture studies 5 42 mutation rate 5 48 PCR components 5 3 PCR cycle number 5 6 population data 5 47 Index 4 AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide probability of identity 5 49 probability of paternity exclusion 5 50 sensitivity 5 34 size deviation sample and ladder alleles 5 8 species specificity 5 32 stability 5 36 thermal cycling parameters 5 4 vWA locus designation 1 3 W WARNING description vi waste disposal guidelines ix work area amplified DNA 2 3 PCRsetup 2 2 setup and lab design 2 2 AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide Index 5 Index 6 AmpF amp TR SEfiler Plus
78. ition See Addition of 3 A Nucleotide on page 5 25 Be sure to include the final extension step of 60 C for 45 min in the PCR Signal exceeds dynamic range of instrument off scale data Quantitate DNA and reamplify sample adding 0 5 to 0 75 ng of DNA Poor spectral separation bad matrix Follow the steps for creating a matrix file Confirm that Filter Set G5 modules are installed and used for analysis A 4 AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide Table A 1 Troubleshooting Troubleshooting causes and recommended actions continued Observation Possible Causes Recommended Actions Some but not all loci visible on electropherogram Poor peak height balance 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 Test sample contains high concentrations of a PCR inhibitor for example heme compounds certain dyes Incorrect thermal cycler parameters Quantitate DNA and add minimum necessary volume Repeat test Wash the sample in a Centricon 100 centrifugal filter unit 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 bl
79. ler Plus PCR Amplification Kit User Guide Performing Electrophoresis This chapter covers Allelic Ladder Requirements 00 0 cece eee eee Setting Up the 3100 3100 Avant or 3130 3130x Instrument for Electrophoresis 0 0 00 cece cece ence eee eee Preparing Samples for Electrophoresis on the 3100 3 100 Avant or 3130 3130x Instrument Setting Up the 310 Instrument for Electrophoresis Preparing Samples for Electrophoresis on the 310 Instrument AmpFSTR SEfiler Plus PCR Amplification Kit User Guide 3 1 Chapter 3 Performing Electrophoresis Allelic Ladder Requirements To accurately genotype samples use an Allelic Ladder sample that is run with the samples Applied Biosystems recommends that for ABI PRISM 310 Genetic Analyzer Run at least one allelic ladder for every 10 sample injections ABI PRISM 3100 or Applied Biosystems 3130 series instruments Run at least one allelic ladder for each set of 16 samples 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 IMPORTANT Variation in laboratory temperature can cause changes in fragment migration speed which can in turn cause sizing variation Applied Biosystems recommends the above freq
80. lied Biosystems instruments Applied Biosystems 3130 3130x Genetic Analyzer ABI PRISM 3100 3100 Avant Genetic Analyzer ABI PRISM 310 Genetic Analyzer Silver 96 Well GeneAmp PCR System 9700 Gold plated silver block GeneAmp PCR System 9700 The AmpF STR SEfiler Plus kit contains the same loci and primer sequences as the SEfiler kit but uses improved synthesis and purification processes to minimize the presence of dye labeled artifacts Modifications made in the production of VIC and PET dye labeled primers have greatly minimized the occurrence of the 120 VIC dye artifact as well as the PET dye labeled artifacts observed at approximately 105 to 115 bp AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Product Overview Loci Amplified by Table 1 1 shows the loci amplified by the AmpF STR SEfiler Plus the Kit kit their chromosomal locations and the corresponding fluorescent marker dyes The AmpF STR SEfiler Plus Allelic Ladder is used to genotype the analyzed samples The alleles contained in the allelic ladder and the genotype of the AmpF STR Control DNA 007 are also listed in the table Table 1 1 AmpF STR SEfiler Plus Kit loci and alleles AmpF STR AmpF STR Locus Chromosome Dye Allelic Control Designation Location Label Ladder DNA 007 Alleles Genotype D28S1338 2q35 37 1 6 FAM 15 16 17 20 23 18 19 20 21 22 23 24 25 26 27 28 D381358 3p 6 FAM 12 1
81. mical 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 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 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 SEfiler 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 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Stability 100 200 300 SEfiler Plus kit 1 ng DNA Untreated 100 200 300 1000 800 SEfiler Plus kit 2ng DNA i 4 Units DNase 400 200 300 1000 800 SEfiler Plus kit 2 ng DNA i 5 Units DNase 5 E dei al au us 300
82. mplification Kit User Guide 5 25 Chapter 5 Experiments and Results 5 26 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 ER 175 180 185 190 195 No Extension Final Extension Figure 5 9 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 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Extra Peaks in the Electropherogram Lack of complete A nucleotide addition may be observed in SEfiler Plus 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 Artifacts and anomalies are seen in all molecular biological systems Artifacts are typically reproducible Anomalies are nonreproducible intermittent occurrences that are not consistently observed in a system for example spikes and baseline noise Artifacts have been seen in data produced on genetic analyzers when using the SEfiler Plus kit Low level artifacts in
83. n tube Use Applied Biosystems MicroAmp Reaction Tubes with Caps for the GeneAmp 9700 MicroAmp Base used with tray retainer set and tubes in GeneAmp System 9700 Remove MicroAmp Base from tray retainer set and repeat test A 2 AmpF amp STR SEfiler Plus PCR Amplification Kit User Guide Troubleshooting Table A 1 Troubleshooting causes and recommended actions continued Observation Possible Causes Recommended Actions Faint or no signal from both the AmpF STR Control DNA 007 and the DNA test samples at all loci continued Insufficient PCR product electrokinetically injected For ABI Prism 3100 3100 Avant or Applied Biosystems 3130 3130x instrument runs Mix 1 0 uL of PCR product and 9 uL of Hi Di Formamide GeneScan 600 LIZ9 solution NITE CHEMICAL HAZARD Formamide causes eye skin and respiratory tract irritation It is a possible reproductive and birth defect hazard Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Degraded formamide Check the storage of formamide do not thaw and refreeze multiple times Try Hi Di Formamide N Mc CHEMICAL HAZARD Formamide causes eye skin and respiratory tract irritation It is a possible reproductive and birth defect hazard Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and glo
84. n www appliedbiosystems com then click the links for Support gt Product and Service Literature Part Document Number Applied Biosystems 3130 3100xl Genetic Analyzers Using Data Collection Software 4363787 v3 0 User Bulletin Applied Biosystems 3130 3130xl Genetic Analyzers Getting Started Guide 4352715 Applied Biosystems 3130 3130xl Genetic Analyzers Maintenance Troubleshooting and 4352716 Reference Guide Applied Biosystems 3130 3130xl Genetic Analyzers Quick Reference Card 4362825 Applied Biosystems 3130 3130xl Genetic Analyzers AB Navigator Software 4359472 Administrator Guide ABI Prisv 3100 3100 Avant Data Collection v2 0 User Guide 4347102 ABI Prisv 3100 3100 Avant Genetic Analyzers Using Data Collection Software v2 0 4350218 User Bulletin ABI Prisv 3100 Genetic Analyzer User Manual Data Collection v1 1 4315834 ABI Prism 3100 Avant Genetic Analyzer User Guide Data Collection v1 0 4333549 ABI PRISM 3100 3100 Avant Genetic Analyzers Protocols for Processing AmpF4STR9 4332345 PCR Amplification Kit PCR Products User Bulletin ABI PRISM 310 Genetic Analyzer User Guide Windows NT 4317588 New Features and Installation Procedures for GeneMapper ID Software V3 2 User 4352543 Bulletin GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial 4335523 GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide 4338775 Quantifiler Kits Q
85. ng the SEfiler 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 SEfiler 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 Also take care while handling and processing samples to prevent contamination by human DNA Wear gloves at all times and change them frequently Close sample tubes when not in use Limit aerosol dispersal by handling sample tubes and reagents carefully Note Applied Biosystems does not intend these references for laboratory design to constitute all precautions and care necessary for using PCR technology 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 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide PCR Work Areas Amplified DNA The following GeneAmp PCR Systems should be placed in the Work Area Amplified DNA Work Area Silver block 96 Well GeneAmp PCR System
86. ns Wear appropriate protective eyewear clothing and gloves Pipette the required volumes of components into an appropriately sized polypropylene tube Vortex the tube then centrifuge briefly AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 3 5 Chapter 3 Performing Electrophoresis To prepare samples for electrophoresis continued 4 Into each well of a MicroAmp Optical 96 Well Reaction Plate add 9 uL 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 mixed and collected at the bottom Heat the reaction plate in a thermal cycler for 3 minutes at 95 C ANITE PHYSICAL INJURY HAZARD During instrument operation the temperature of the heated cover can be as high as 108 C and the temperature of the sample block can be as high as 100 C Keep hands away from the heated cover and sample block Immediately place the plate on ice for 3 minutes Prepare the plate assembly on the autosampler Start the electrophoresis run AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Setting Up the 310 Instrument for Electrophoresis Setting Up the 310 Instrument for Electrophoresis Reagents and Parts Electrophoresis Setup Software
87. ocks only AmpFSTR SEfiler Plus PCR Amplification Kit User Guide A 5 Appendix A Troubleshooting A 6 AmpF amp STR SEfiler Plus PCR Amplification Kit User Guide Bibliography Akane A Matsubara K Nakamura H Takahashi S and Kimura K 1994 Identification of the heme compound copurified with deoxyribonucleic acid DNA from bloodstains a major inhibitor of polymerase chain reaction PCR amplification J Forensic Sci 39 362 372 Barber M D McKeown B J and Parkin B H 1996 Structural variation in the alleles of a short tandem repeat system at the human alpha fibrinogen locus Intl J Legal Med 108 180 185 Barber M D and Parkin B H 1996 Sequence analysis and allelic designation of the two short tandem repeat loci D18S51 and D8S1179 Intl J Legal Med 109 62 65 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 Baron H Fung S Aydin A Bahrig S Luft EC 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 VC Helmuth R C Fildes N Bugawan T L Erlich H A Klitz W 1992 Polymorphism recombination and linkage disequilibrium within the HLA class I region J Immunol 148 249 58 Bender K Farfan M
88. or calculating allelic bin offsets and subsequent genotyping Allelic ladder samples need to be identified as Allelic Ladder in the Sample Type column in a project Failure to apply this setting for ladder samples results in failed analysis Allelic bin definitions are stored in the AmpFLSTR SEfiler Plus Panels v1 in the Panel Manager 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 When using GeneMapper D X Software version 1 0 to perform Human Identification HID analysis with AmpF STR kits refer to the GeneMapper ID X Software Version 1 0 Human Identification Analysis Getting Started Guide PN 4375574 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Setting Up GeneMapper ID Software v3 2 1 for Analyzing AmpFSTR SEfiler Plus Kit Data Alleles not found in the AmpF STR Allelic Ladders do exist Off ladder 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 Ifa sample allele peak is called as an off ladder allele the sample result needs to be verified according to the laboratory s protocol Setting Up GeneMapper D Software v3 2 1 for Analyzing AmpF STR SEfiler Plus Kit Data Overview Before you can analyze sample fsa files using
89. other nine STR loci The D18S11 mutation was represented by an increase of one 4 nt repeat unit a 17 allele was inherited as an 18 single step mutation The maternal paternal source of this mutation could not be distinguished Additional Additional studies Edwards et al 1991 Edwards et al 1992 Mutation Studies Weber and Wong 1993 Hammond et al 1994 Brinkmann et al 1995 Chakraborty et al 1996 Chakraborty et al 1997 Brinkmann et al 1998 Momhinweg et al 1998 Szibor et al 1998 of direct mutation rate counts produced Larger sample sizes for some of the AmpF STR SEfiler Plus kit loci Methods for modifications of these mutation rates to infer mutation rates indirectly for those loci where these rates are not large enough to be measured directly and or to account for those events undetectable as Mendelian errors 5 48 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Probability of Identity Probability of Identity Table of Table 5 7 shows the Probability of Identity PI values of the Probability of AmpF STR SEfiler Plus PCR Amplification Kit loci individually Identity and combined Table 5 7 Probability of identity values for the AmpF STR SEfiler Plus kit STR loci Locus African American U S Caucasian D2S1338 0 025 0 038 D3S1358 0 114 0 099 D8S1179 0 079 0 072 D16S539 0 074 0 085 D18S51 0 038 0 056 D19S433 0 045 0 126 D21S11 0 051 0 057
90. ow because of measurement error is relatively rare when the standard deviation in sizing is approximately 0 15 nt or less Smith 1995 For 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 The GeneMapper JD software v3 2 1 automatically flags sample alleles that do not size within the specified window around an allelic ladder allele Although the precision within a set of capillary injections is very good the determined allele sizes vary between platforms Cross platform sizing differences arise from a number of parameters including type and concentration of polymer mixture run temperature and electrophoresis conditions Variations in sizing can occur between runs on the same instrument and between runs on different instruments because of these factors Applied Biosystems strongly recommends that the allele sizes be compared to the sizes obtained for known alleles in the AmpF STR SEfiler Plus Allelic Ladder from the same run and then be converted to genotypes as described in Before You Start on AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 5 9 Chapter 5 Experiments and Results 5 10 page 4 2 See Table 5 1 for th
91. pFSTR SEfiler Plus PCR Amplification Kit User Guide Mixture Studies 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 SEfiler Plus PCR Amplification Kit loci in unmixed population database samples are shown in Table 5 5 Table 5 5 Peak height ratios for 0 50 ng of input DNA Number of Locus ias as Mean Median Minimum Maximum Amelogenin 160 82 5 84 2 50 0 99 7 D16S539 170 84 1 86 7 50 9 99 6 D18S51 200 83 5 86 0 44 4 99 9 D19S433 185 83 3 84 1 55 9 99 9 D21S11 202 82 6 83 9 35 3 99 9 D2S1338 203 80 7 82 2 37 8 100 0 D3S1358 154 83 5 84 5 43 8 99 7 D8S1179 177 83 3 84 3 51 3 99 8 FGA 193 83 4 85 4 45 6 99 9 SE33 214 82 3 84 5 43 6 100 0 THO1 176 82 2 84 7 33 0 99 9 vWA 194 83 4 83 8 54 1 99 8 If an unusually low peak height ratio is observed for one locus and there are no other indications that the sample is a mixture the sample may be reamplified and reanalyzed to determine if the imbalance is reproducible Possible causes of imbalance at a locus are Degraded DNA Presence of inhibitors Extremely low amounts of input DNA A mutation in one of the primer binding sites AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 5 43 Chapter 5 Experimen
92. pane displaying list of panels 4 7 Panel Manager 4 4 O off ladder alleles 4 3 5 8 operating systems 1 10 3 3 3 7 P Panel Manager 4 4 panels viewing 4 7 paternity exclusion 5 50 PCR performing 2 9 setup 2 2 PCR components validation of 5 3 PCR cycle number validation 5 6 PCR inhibitor hematin 5 38 humic acid 5 40 PCR work areas 2 2 peak detection parameters 4 12 AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide Index 3 peak height ratios table of alleles 5 42 peak height minimum 4 12 percent stutter highest value for locus 5 21 off scale peaks 5 21 relation to allele length 5 21 PQV thresholds 4 14 precision and size windows 5 8 precision sizing 5 8 primers about 1 2 Amelogenin 5 30 fluorescent dyes attached 1 12 storage 1 12 volume per reaction 2 7 probability of identity 5 49 project window figure 4 17 Q quality flag settings 4 14 Quantifiler kit description 2 6 quantifying DNA methods 2 5 R radioactive waste handling ix reaction mix for PCR 2 7 reaction volume final for PCR 2 8 reactions preparing for PCR 2 7 reagents low TE buffer 2 4 not included with kit 1 13 run module electrophoresis 3 3 3 7 S safety biological hazards x chemical waste viii guidelines vii viii ix size deviation sample alleles and ladder alleles 5 8 size standard GeneMapper ID software 4 16 sizing precision 5 8 species specificity 5 32 split peaks A nucleot
93. ppear in Applied Biosystems user Words 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 ofa chemical AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide v Preface Safety Safety Alert Words Chemical Hazard vi Warning Examples of the user attention words appear below Note The Calibrate function is also available in the Control Console IMPORTANT To verify your client connection to the database you need a valid user ID and password Four safety alert words appear in Applied Biosystems user documentation at points in the 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 Definitions IMPORTANT Indicates information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical N e7 Ye Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices N Miel Indicates a potentially hazardous situation that 1f not avoided could result in deat
94. r Plus kit 60 80 100 114 120 140 160 180 200 214 220 240 250 260 280 300 314 320 340 360 380 and 400 For additional information about size standards refer to the GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide PN 4338775 Appendix D 3 Click Analyze enter a name for the project in the Save Project dialog then click OK to start analysis The status bar displays progress of analysis Asacompletion bar extending to the right with the percentage indicated With text messages on the left The table displays the row of the sample currently being analyzed in green or red if analysis failed for the sample The Genotypes tab becomes available after analysis 4 16 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Analyzing and Editing Sample Files with GeneMapper ID Software GeneMapper ID v3 2 1 Concordance Test gmid Is Logged In File Edit Analysis View Tools Help oe S Project amp 1 2007 04 amp 1 2007 04 E Ps ii LH Samples E Table Setting Concordance Status Sample File Sample Name Sample Type Analysis Method Panel Size Standard 101_A01 fsa 101 Sample SEfiler_Plus_HID_v1 SEfier Plus v1 CE GS SEfiler Plus GS800 HD 102_B01 fsa 102 Sample SEfiler_Plus_HID_v1 SEfiler Plus vi CE G5 SEfiler Plus GS800 HD 103 C01 fsa 103 Sample SE
95. rements 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 consult the MSDS Handle chemical wastes in a fume hood After emptying the 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 Waste Disposal If potentially hazardous waste is 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 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 AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide ix Preface Biological Hazard Safety ANCE BIOHAZARD Biological samples
96. rt 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 Intl J Legal Med 111 160 161 Urquhart A Kimpton C and Gill P 1993 Sequence variability of the tetranucleotide repeat of the human beta actin related pseudogene H beta Ac psi 2 ACTBP2 locus Hum Genet 92 637 638 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 70 Wallin J
97. s and observations are For each SEfiler Plus kit locus the percent stutter generally increases with allele length as shown in Figure 5 5 to Figure 5 8 on pages 5 22 through 5 24 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 The highest observed percent stutter for each locus is included as the filtering step in the GeneMapper JD software v3 2 1 These values are shown in Table 5 2 on page 5 25 Peaks in the stutter position that are above the highest observed percent stutter are not 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 5 17 on page 5 45 The measurement of percent stutter for peaks that are off scale may be unusually high AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 5 21 Chapter 5 Experiments and Results 19 0 18 0 17 0 16 0 15 0 14 0 13 0 12 0 11 0 10 0 9 0 Percent Stutter 8 0 7 0 6 0 5 0 Hi 4 0 3 0 2 0 1 0 0 0 mams o o ss r oe a a i i 11213141516 17 18 19 20 D3S1358 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 20 D2S1338 4 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 VWA 5 6 7 8 910111213141516 D16S539 Figure 5 5 Stutter perc
98. ter PCR grade MLS Tris HCL pH 8 0 MLS EDTA 0 5 M MLS Vortex MLS t 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 AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 1 17 Chapter 1 Overview 1 18 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Chapter 2 PCR Amplification AmpFSTR SEfiler Plus PCR Amplification Kit User Guide PCR Amplification This chapter covers PCR Work ATEA Sih nee aaa Re oe Re 2 2 Required User Supplied Materials and Reagents 2 4 Quantifying DNA 1 0 eee eee 2 5 Preparing the Reactions 00 0 eee eee eee eee 2 7 Performing PCR 00 ccc cee eh 2 9 Amplification Using Bloodstained FTA Cards 2 10 AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 2 1 Chapter 2 PCR Amplification PCR Work Areas Work Area Setup Many resources are available for the appropriate design of a PCR and Lab Design PCR Setup Work Area laboratory Ifyou are using the AmpF STR SEfiler 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 www nij org publications Ifyou are usi
99. that does correspond to an allelic ladder allele but whose size is just outside a window because of measurement error AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Accuracy Precision and Reproducibility The measurement error inherent in any sizing method can be defined by the degree of precision in sizing an allele multiple times Precision is measured by calculating the standard deviation in the size values obtained for an allele that is run in several injections on a capillary instrument or in several lanes of one gel Table 5 1 on page 5 10 shows typical precision results obtained from five runs 16 capillaries run of the AmpF STR SEfiler 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 600 LIZ Size Standard The results were obtained within a set of injections on a single capillary array Sample alleles may occasionally size outside 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 5 4 on page 5 8 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 ofa sample allele sizing outside of the 0 5 nt wind
100. the AmpF STR SEfiler Plus PCR Amplification Kit uses the following GS600 peaks in its sizing algorithm 60 80 100 114 120 140 160 180 200 214 220 240 250 260 280 300 314 320 340 360 380 and 400 Use the following procedure to import the size standard for the SEfiler Plus kit from the folder that you downloaded from the Applied Biosystems web site into the GeneMapper ID software database Refer to step 1a on page 4 4 for downloading instructions To import an HID Size Standard 1 Select Tools GeneMapper Manager to open the GeneMapper Manager AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Setting Up GeneMapper ID Software v3 2 1 for Analyzing AmpF4STR SEfiler Plus Kit Data To import an HID Size Standard continued Q GeneMapper Manager 2 Import a Size Standard Projects Analysis Methods Table Settings Plot Settings Matrices Size Standards a Select the Size Standards tab then click Import Name Last Saved Owner Type Description 377_G5_HID_GS500 2006 09 20 14 10 2 grid Basic Advanced Factory Provided 377_F_HID_GSS00 2006 09 20 14 10 2 amid Basic Advanced Factory Provided CE G5 HID GS500 2006 09 20 14 10 2 gmid Basic Advanced Factory Provided CE F HID GS500 2006 09 20 14 10 2 gmid Basic Advanced Factory Provided folder b Na
101. tion Kit User Guide Chapter 1 Overview Table 1 4 User supplied materials continued Material Source Septa for 0 5 mL Sample Tubes 401956 DS 33 Matrix Standard Set 6FAM VIC9 NED PETS 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 ABI PRISM 310 Genetic Analyzer User Guide PN 4317588 For a complete list of parts and accessories for the 310 instrument refer to Appendix B of the 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 Caps Strip N8010535 MicroAmp 96 Well Tray Retainer Set 403081 MicroAmp 96 Well Base N8010531 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 1 16 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Materials and Equipment Table 1 4 User supplied materials continued Material Source Tube 50 mL Falcon MLS Tube decapper autoclavable MLS Deionized wa
102. tion Kit User Guide Troubleshooting In This Appendix Follow the recommended actions for the observations described in this appendix to understand and eliminate problems you experience during analysis Troubleshooting 0 cece cette eens A 2 AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide A 1 Appendix A Troubleshooting Troubleshooting Table A 1 Troubleshooting causes and recommended actions Observation Possible Causes Recommended Actions Faint or no signal from both the AmpF STR Control DNA 007 and the DNA test samples at all loci Incorrect volume or absence of either AmpF STR SEfiler Plus Master Mix or AmpF STR SEfiler Plus Primer Set Repeat amplification No activation of enzyme Repeat amplification making sure to hold reactions initially at 95 C for 11 min Master Mix not vortexed thoroughly before aliquoting Vortex Master Mix thoroughly AmpF STR SEfiler 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 reactio
103. trifuge briefly AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Preparing Samples for Electrophoresis on the 310 Instrument To prepare samples for electrophoresis continued 4 Into each 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 appropriate septa then briefly centrifuge the tubes to ensure that the contents of each tube are mixed and collected at the bottom Heat the tubes in a thermal cycler for 3 minutes at 95 C ANTYE PHYSICAL INJURY HAZARD During instrument operation the temperature of the heated cover can be as high as 108 C and the temperature of the sample block can be as high as 100 C Keep hands away from the heated cover and sample block Immediately place the tubes on ice for 3 minutes Place the sample tray on the autosampler Start the electrophoresis run AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 3 9 Chapter 3 Performing Electrophoresis 3 10 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Part Number 4385739 Rev B 03 2012 Chapter 4 Analyzing Data AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Analyzing Data This chapter covers Overview of GeneMapper JD Software 00005 4 2 Setting Up GeneMapper ID Software v3 2 1 for Analyzing AmpF STR SEfiler Plus Kit Data
104. ts and Results Presence of an allele containing a rare sequence that does not amplify as efficiently as the other allele Resolution of Genotypes in Mixed Samples A sample containing DNA from two sources can be comprised at a single locus of any of the seven genotype combinations see below 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 allele peak heights on Applied Biosystems instruments provides additional valuable data to aid in resolving mixed genotypes This quantitative value is much less subjective than comparing relative intensities of bands on a stained gel Ultimately the likelihood that any sample 1s a mixture must be determined by the analyst in the context of each particular case including the information provided from known referen
105. uantifiler Human DNA Quantification Kit and Quantifiler Y Human 4344790 Male DNA Quantification Kit User s Manual GeneMapper ID Software v3 2 1 Patch User Bulletin 4382255 Note For additional documentation see How to Obtain Support on page xii AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide xi Preface Send Us Your Applied Biosystems welcomes your comments and suggestions for Comments improving its user documents You can e mail your comments to techpubs appliedbiosystems com IMPORTANT The e mail address above is only for submitting comments and suggestions relating to documentation To order documents download PDF files or for help with a technical question go to www appliedbiosystems com then click the link for Support See How to Obtain Support below How to Obtain Support For the latest services and support information for all locations go to www appliedbiosystems com then click the link for Support At the Support page you can Xii 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 AmpFSTR SEfiler Pl
106. uency of allelic ladder injections to account for normal variation in run speed However during internal validation studies each laboratory should verify the required allelic ladder injection frequency to ensure accurate genotyping of all samples in each laboratory environment It is critical to genotype using an allelic ladder that is run under the same conditions as the samples because Size values obtained for the same sample can differ between instrument platforms because of different polymer matrices and electrophoretic conditions Slight procedural and reagent variations between single and multiple capillaries result in greater size variation than that found between samples injected in the same capillary in a single run 3 2 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Setting Up the 3100 3100 Avant or 3130 3130xl Instrument for Electrophoresis Setting Up the 3100 3100 Avant or 3130 31 30x Instrument for Electrophoresis Reagents and Parts Electrophoresis Setup Software and Reference Documents Table 1 4 on page 1 14 lists the required materials not supplied with the AmpF STR SEfiler Plus PCR Amplification Kit 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 SEfiler Plus Allelic Ladder and GeneScan 600 LIZ Size Standard should also be protected from light Minimize freeze thaw cycles
107. upply current Material Safety Data Sheets MSDSs with shipments of hazardous chemicals to new customers They 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 MSDSs available to you free 24 hours a day To obtain MSDSs 1 Goto docs appliedbiosystems com msdssearch html 2 In the Search field of the MSDS Search page a Enter the chemical name part number or other information that you expect to appear in the MSDS of interest b Select the language of your choice c Click Search AmpF 4STR SEfiler Plus PCR Amplification Kit User Guide vii Preface Chemical Waste viii Hazards 3 To view download or print the document of interest a Right click the document title b Select Open To view the document Save Target As To download a PDF version of the document to a destination that you choose Print Target To print the document 4 To have a copy of an MSDS sent by fax or e mail in the Search Results page a Select Fax or Email below the document title b Click RETRIEVE DOCUMENTS at the end of the document list c Enter the required
108. us PCR Amplification Kit User Guide Overview This chapter covers Product Overview llle 1 2 Workflow Overview cebren ee a A e 1 8 Instrument and Software Overview 00002 ce eens 1 9 Materials and Equipment 0 0 0 cece eee esee 1 11 AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 1 1 Chapter 1 Overview Product Overview 1 2 Purpose Product Description About the Primers The AmpF STR SEfiler Plus PCR Amplification Kit is a short tandem repeat STR multiplex assay that amplifies 11 tetranucleotide repeat loci including the SE33 locus required specifically for the German DNA database The kit simultaneously coamplifies the seven loci of the European Standard Set ESSL D381358 vWA D8S1179 THO1 FGA D21S11 and D18S51 the Amelogenin locus the highly polymorphic SE33 ACTBP2 locus and the D281338 D16S539 and D19S433 loci The AmpF STR SEfiler Plus Kit has been developed to deliver improvements in performance over the original SEfiler Kit Changes to the kit include modified PCR cycling conditions for enhanced sensitivity a new buffer formulation to improve performance with inhibited samples improvements in synthesis and purification of the amplification primers and a redeveloped allelic ladder The SEfiler Plus kit contains all the necessary reagents for the amplification of human genomic DNA The reagents are designed for use with the following App
109. uspect is excluded as the donor of the biological evidence 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 the frequency of that genotype in the relevant population s The AmpF STR SEfiler Plus PCR Amplification Kit contains loci for which extensive population data are available For additional information see the population data and additional studies section of the AmpFtSTR SEfiler PCR Amplification Kit Users Manual PN 4323291 AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 5 47 Chapter 5 Experiments and Results Mutation Rate Estimating Estimation of spontaneous or induced germline mutation at genetic Germline loci may be achieved through comparison of the genotypes of Mutations offspring to those of their parents From such comparisons the number of observed mutations are counted directly In previous studies genotypes of 10 STR loci amplified by the AmpF STR SGM Plus PCR Amplification Kit were determined for a total of 146 parent offspring allelic transfers meioses at the Forensic Science Service Birmingham England One length based STR mutation was observed at the D18S11 locus mutation was not detected at any ofthe
110. validation experiments performed by Applied Biosystems using the AmpF STR SEfiler Plus PCR Amplification Kit SEfiler Plus kit Validation of a DNA typing procedure for human identification applications is an evaluation of the procedure s efficiency reliability and performance characteristics By challenging the procedure with samples commonly encountered in forensic and parentage laboratories the validation process uncovers attributes and limitations that 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 SEfiler Plus PCR Amplification Kit were performed at Applied Biosystems These 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 has performed 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 2
111. ves AmpFSTR SEfiler Plus PCR Amplification Kit User Guide A 3 Appendix A Troubleshooting Table A 1 Troubleshooting causes and recommended actions continued Observation Possible Causes Recommended Actions Positive signal from AmpF STR Control DNA 007 but partial or no signal from DNA test samples Quantity of test DNA sample is below assay sensitivity Quantitate DNA and add 0 5 to 0 75 ng of DNA Repeat test Test sample contains high concentration of PCR inhibitor for example heme compounds certain dyes Quantitate DNA and add minimum necessary volume Repeat test Wash the sample in a Centricon 100 centrifugal filter unit Repeat test Test sample DNA is severely degraded If possible evaluate the quality of DNA sample by running an agarose gel If DNA is degraded reamplify with an increased amount of DNA Dilution of test sample DNA in H O or wrong buffer for example wrong EDTA concentration Redilute DNA using 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 Too much DNA in reaction Use recommended amount of template DNA 0 5 to 0 75 ng Mixed sample Amplification of stutter product n 4 nt position See Stutter Products on page 5 21 Incomplete 3 A base addition n 1 nt pos
112. vigate to then open the GMID_SEfiler_Plus_files Import Size Standard Method Look in B GMID SEfiler Plus files 62 SEfiler Plus HID v1 3 Select CE G5 SEfiler Plus GS600 HID v1 then click Import to import the SEfiler Plus HID v1 analysis method into the GeneMapper JD database Desktop 2 File name My Documents Files of type CE GS SEfiler Plus GS600 HID v1 xml XML Files xml J o AmpF amp TR SEfiler Plus PCR Amplification Kit User Guide 4 15 Chapter 4 Analyzing Data Analyzing and Editing Sample Files with GeneMapper ID Software Analyzing 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 Advanced Analysis Method Sample Type Select the sample type Analysis Method SEfiler Plus HID v1 Panel AmpFLSTR SEfiler Plus Panels v1 Size Standard CE G5 SEfiler Plus GS600 HID Matrix Select a matrix for 310 instruments only For more information about how the Size Caller works refer to the ABI PRISM9 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 HID GS600 size standard provided with the AmpF STR SEfile
113. y possesses structural variation it also exhibits length and sequence polymorphism M ller Schurenkamp et al 1995 Among the sequence polymorphisms Type I contains the known regular four nt repeat AAAG while Type II has an additional hexanucleotide unit AAAAAG These result in additional interalleles in the SE33 locus differing by 1 to 3 nt Urquhart et al 1993 All the alleles in the AmpF STR SEfiler Plus Allelic Ladder including microvariants have been subjected to DNA sequencing at Applied Biosystems In addition other groups have sequenced alleles at some of these loci Nakahori ef al 1991 Puers et al 1993 Moller et al 1994 Barber et al 1995 Moller and Brinkmann 1995 Barber et al 1996 Barber and Parkin 1996 Brinkmann et al 1998 Momhinweg et al 1998 Watson et al 1998 Among the various sources of sequence data on the AmpF STR SEfiler Plus kit loci there is consensus on the repeat patterns and structure of the STRs 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 AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Characterization of Loci inheritance of various DNA loci Each family set contains three generations generally including four grandparents two parents and several offspring
114. ystem hardware the data collection software collects the data and stores it Information about each sample is stored in a sample file fsa which is then analyzed by the analysis software Operatin Data Instrument S ne Collection Analysis Software y Software 3130 3130x Windows XP 3 0 GeneMapper ID v3 2 1 3100 3100 Windows NT 1 1 3100 GeneMapper D v3 2 1 Avant 1 0 3100 Avant Windows 2000 2 0 GeneMapper ID v3 2 1 310 Windows XP 3 1 GeneMapper ID v3 2 1 Windows NT 3 0 GeneMapper ID v3 2 1 and Windows 2000 t Applied Biosystems performed validation studies for the SEfiler Plus kit using these configurations Applied Biosystems fluorescent multi color dye technology allows the analysis of multiple loci including loci that have alleles with overlapping size ranges Alleles for overlapping loci are distinguished by labeling locus specific primers with different colored dyes AmpFSTR SEfiler Plus PCR Amplification Kit User Guide Instrument and Software Overview 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 SEfiler Plus PCR Amplification Kit to label samples are 6 FAM VIC NED and PET dyes The fifth dye LIZ is used to label the GeneScan 600 LIZ Size Standard How Each ofthe fluorescent dyes emits its maximum fluorescence at a Multicomponent

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AmpFlSTR ® SEfiler Plus ™ PCR Amplification Kit