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AmpFlSTR Identifiler Plus PCR Amplification Kit User Guide (Pub

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1. amp u j j j m ii so WSE wen Please enter the stutter filter s for D55818 marker here If left blank the global stutter filter will be applied E E AmpFLSTR Identifller Plus vix S Identifiler Plus Panels v1x Minus Stutter Plus Stutter fel D251338 Ratio From Distance To Distance Ratio From Distance To Distance T R 1 1006 3 25 4 75 1 v TPOX 2 2 3 3 4 4 __New eoe delete D851179 11 Click Apply then OK to add the AmpF STR Identifiler Plus Kit panels bin sets and marker stutter to the GeneMapper ZD X Software database IMPORTANT If you close the Panel Manager without clicking Apply the panels bin sets and marker stutter will not be imported into the GeneMapper ID X Software database Q D i D lt 5 o go 8 o Xx e EB B 0 AmpFtSTR Identifiler Plus User Guide 65 Section 4 2 GeneMapper ID X Software Import an Use the following procedure to import the analysis method for the AmpF STR analysis method _ Identifiler Plus PCR Amplification Kit from the folder that you downloaded from our web site into the GeneMapper ZD X Software database Refer to step 1 on page 61 for downloading instructions Note The Identifiler_Plus_AnalysisMethod_v1x has been provided to assist you in get
2. AmpFLSTR_Panels_v1X IIdentitiler Plus Panels v1X null E fis AmpFLSTR Identifiler Plus v1X b Select File Import Marker Stutter to open the Import Marker Stutter dialog box c Navigate to then open the Identifiler Plus Analysis Files GMIDX folder d Select Identifiler Plus Stutter v1X then click Import Note Importing this file associates the marker stutter ratio with the bin set in the Identifiler Plus Bins v1X folder 64 AmpFtSTR Identifiler Plus User Guide Set up GeneMapper ID X Software for data analysis g5 Import Marker Stutter xl Look in je Identifiler Plus Analysis Files GMIDX v id CE My Recent Documents E Desktop KA AI WID Filename identfier_Plus_Stutter_v1x bt Import r CZA Fesottwe sine TT TTS _ Grad CE G5 Identifiler Plus GS500 xml Identifier Plus Bins vix txt Identifier Plus Panels v1x txt Identifier Plus Stutter v1X txt Identifiler Plus AnalysisMethod v1x xml 10 View the imported marker stutters in the navigation pane a Selectthe Identifiler Plus Panel v1X folder to display its list of markers in the right pane b Double click the Identifiler Plus Panel v1X folder to display its list of markers below it c Double click D5S818 to display the Stutter Ratio amp Distance view for the marker in the right pane Panel Manager E File Edit Bins View Help uw x FMM Bin Set identfier_Pius_Bins_v1x z i amp
3. Perform PCR Pek WARNING PHYSICAL INJURY HAZARD Thermal cycler 1 Program the thermal cycling conditions When using the GeneAmp PCR System 9700 with either 96 well silver or gold plated silver block select the 9600 Emulation Mode When using the Veriti 96 Well Thermal Cycler refer to the following document for instructions on how to configure the Veriti instrument to run in the 9600 Emulation Mode User Bulletin Veriti 96 Well Thermal Cycler AmpFtSTR Kit Validation PN 4440754 When using the ProFlex PCR System refer to the ProFlex PCR System Kit Validation User Bulletin Pub no 100031595 for more information Initial incubation Cycle 28 or 29 cycles Final Final hold step Denature Anneal Extend AH HOLD CYCLE HOLD HOLD 95 C 94 C 59 C 60 C 4 C 11 min 20 sec 3 min 10 min Up to 24 hours t Referto the previous section for selecting the appropriate PCR cycle number The infinity setting allows an unlimited hold time 2 Load the plate into the thermal cycler and close the heated cover IMPORTANT If using adhesive clear film instead of caps to seal the plate wells be sure to place a MicroAmp compression pad PN 4312639 on top of the plate to prevent evaporation during thermal cycling 3 Start the run 4 On completion of the run store the amplified DNA and protect from light If you are storing the DNA Then place at 2 weeks 2to
4. Save Cancel Help Figure 11 Analysis Method Editor Allele tab settings e GeneMapper D X Software 1 0 1 1 1 or 1 1 1 allows you to specify four types of marker repeat motifs tri tetra penta and hexa You can enter parameter values for each type of repeat in the appropriate column The Use marker specific stutter ratio if available check box is selected by default Consequently the software applies the stutter ratio filters supplied in the Identifiler_Plus_Stutter_v1X file For more information about allele filters refer to GeneMapper ID X Software Version 1 0 Getting Started Guide PN 4375574 e GeneMapper D X Software Version 1 0 Quick Reference Guide PN 4375670 GeneMapper ID X Software Version 1 0 Reference Guide PN 4375671 68 AmpFtSTR Identifiler Plus User Guide Set up GeneMapper ID X Software for data analysis Analysis Method Editor x Peak Quality SQ amp GQ Settings Peak Detection Algorithm Advanced r Rar ges Peak Detectior Analysis Sizing Full Range Al Sizes Start Pt tooo Start EAN Stop Pt fioooo 5 ize Peak Amplitude Thresholds B fsa R o a fe of u o r Smoothing and Baselining E Min Peak Half width E pts Smoothing None i Light Polynomial Degree C Heavy Peak Window Size fis pts Baseline Window g pts Slope Threshold Peak Start o o r Size Calling Method Rek Ed po C 2nd Order Least Squ
5. 6 Prepare the DNA samples DNA sample To prepare Negative control Add 10 uL of low TE buffer 10mM Tris 0 1mM EDTA pH 8 0 Test sample Dilute a portion of the test DNA sample with low TE buffer so that 1 0 ng of total DNA is in a final volume of 10 uL Add 10 uL of the diluted sample to the reaction mix Positive control Add 10 uL of 9947A control DNA 0 1 ng uL The final reaction volume sample or control plus reaction mix is 25 UL 7 Seal the MicroAmp Optical 96 Well Reaction Plate with MicroAmp Clear Adhesive Film or MicroAmp Optical Adhesive Film or cap the tubes 8 Vortex the reaction mix for 3 seconds then centrifuge the tubes at 3000 rpm for about 20 seconds in a tabletop centrifuge with plate holders 1f using 96 well plates to remove bubbles 28 AmpFtSTR Identifiler Plus User Guide Select the appropriate PCR cycle number 9 Amplify the samples in a GeneAmp PCR System 9700 with the silver 96 well block a GeneAmp PCR System 9700 with the gold plated silver 96 well block a Veriti 96 Well Thermal Cycler or a ProFlex PCR System Note The AmpF STR Identifiler Plus Kit is not validated for use with the GeneAmp PCR System 9700 with the aluminium 96 well block Use of this thermal cycling platform may adversely affect performance of the AmpF STR Identifiler Plus Kit Select the appropriate PCR cycle number All AmpF STR kits are optimized for a specific number o
6. Plus Kit STR loci in four U S populations en nuc iion N SU AREA n 357 n 349 n 191 CSF1PO HW X p 0 13649 0 926431 0 951476 0 839278 HW G p 0 08902 0 894972 0 918038 0 728023 HW Exact p 0 0762 0 2688 0 5456 0 6148 HExp 0 7829 0 7267 0 7051 0 7398 Ho 0 7703 0 7421 0 7138 0 7958 AmpFtSTR Identifiler Plus User Guide 119 Chapter 5 Experiments and Results 120 Table 11 Heterozygosity and p values for Hardy Weinberg tests of the 15 Identifiler Plus Kit STR loci in four U S populations continued aaa E 5 ds AO ug ARCE n 357 n 349 n 191 D2S1338 HW X2 p 0 409878 0 537758 0 975972 0 722543 HW G p 0 962501 0 407932 0 973054 0 760953 HWExactp 0 7838 0 3488 0 9794 0 5825 HExp 0 8936 0 8823 0 8529 0 8428 Ho 0 8768 0 8653 0 8379 0 801 D3S1358 HW X2 p 0 947371 0 670787 0 681659 0 087223 HW G p 0 907905 0 654776 0 852278 0 175807 HW Exactp 0 2967 0 2814 0 4684 0 0614 HExp 0 7681 0 7986 0 7361 0 7028 Ho 0 7955 0 8166 0 7414 0 7382 D5S818 HW X2 p 0 993751 0 859805 0 944725 0 073002 HW G2 p 0 989776 0 520417 0 979044 0 08025 HW Exactp 0 958 0 462 0 4662 0 0205 HExp 0 7476 0 6931 0 7351 0 7378 Ho 0 7479 0 7077 0 7586 0 6806 D7S820 HW X2 p 0 987668 0 571989 0 336834 0 324754 HW G p 0 969887 0 44694 0 687948 0 289733 HWExactp 0 9818 0 2286 0 4028 0 1276 HExp 0 7758 0 8117 0 7822 0 7858 Ho 0 7955
7. Related documentation Document title Part number Applied Biosystems 3100 3100 Avant Data Collection v2 0 User Guide 4347102 Applied Biosystems 3100 3100 Avant Genetic Analyzers Using Data Collection Software v2 0 User 4350218 Bulletin Applied Biosystems 3100 Genetic Analyzer User Manual Data Collection v1 1 4315834 Applied Biosystems 3100 3100 Avant Genetic Analyzers Protocols for Processing AmpF STR PCR 4332345 Amplification Kit PCR Products User Bulletin Applied Biosystems 3130 3100xl Genetic Analyzers Using Data Collection Software v3 0 User Bulletin 4363787 Applied Biosystems 3130 3130xl Genetic Analyzers Getting Started Guide 4352715 ae Biosystems 3130 3130xl Genetic Analyzers Maintenance Troubleshooting and Reference 4352716 uide Applied Biosystems 3130 3130xl Genetic Analyzers Quick Reference Card 4362825 Applied Biosystems 3130 3130xl Genetic Analyzers AB Navigator Software Administrator Guide 4359472 Applied Biosystems 3130 3100xl DNA Analyzers User Guide 4331468 Quantifiler Kits Quantifiler Human DNA Quantification Kit and Quantifiler Y Human Male DNA 4344790 Quantification Kit User s Manual Quantifiler Duo DNA Quantification Kit User Guide 4391294 Quantifiler HP and Trio DNA Quantification Kits User Guide 4485354 AmpFtSTR Igentifiler PCR Amplification Kit User s Manual 4323291 Veriti 96 Well Thermal Cycler AmpFLSTR Kit Validation User Bulle
8. cycles Number of Locus o a Mean Median Minimum Maximum Amel 320 90 23 91 29 46 21 99 89 CSF1PO 378 90 37 91 56 67 19 99 96 D13S317 375 90 61 92 09 68 28 99 94 D16S539 389 90 03 91 15 68 11 99 87 D18S51 432 90 06 91 24 62 26 99 94 D19S433 399 90 2 91 2 57 29 99 96 D21S11 428 90 3 91 12 69 05 100 D2S1388 436 90 36 91 56 63 86 100 D381358 356 91 39 92 66 62 82 99 94 D5S818 357 91 15 92 21 66 07 100 D7S820 395 90 49 92 07 46 61 99 94 D8S1179 396 91 22 92 7 67 42 100 FGA 429 89 83 91 07 60 38 99 87 THO1 362 91 62 93 04 70 09 100 TPOX 333 91 17 92 18 70 65 100 VWA 414 91 16 92 33 65 22 100 Actual DNA input amounts 0 7 ng to 1 3 ng If an unusually low peak 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 Presence of an allele containing a rare sequence that does not amplify as efficiently as the other allele Resolution of genotypes in mixed samples A sample containing DNA from two sources can comprise at a single locus any of the seven genotype combinations see below 106 AmpFtSTR Identifiler Plus User Guide Mixture studies Heterozygote heterozygote no overlapping alleles
9. ii X tee uw Bin Set identifier Plus_Bins_v1X a d a 0 n ju n j ad Panel Manager i Ladder Alleles a ED AmpFLSTR_Panels_v1X D6si179 Blue 118 0 183 5 13 4 none EN AET E C AmpFLSTR_Identifiler Plus D21511 Blue 1845 247 5 30 4 none 24 24 2 25 26 27 28 28 CETT D75820 Blue 251 0 2985 10 11 4 none 6 7 8 9 10 11 12 13 14 D21511 CSFIPO Blue 302 12 348 63 10 12 4 none 6 7 8 9 10 11 12 13 14 F D75820 D351358 Green 98 0 148 0 14 15 4 none 12 13 14 15 16 17 18 14 F CSFIPO THO1 Green 159 0 205 0 8 9 3 4 none 4 5 6 7 8 9 9 3 10 11 1 a D135317 Green 205 65 250 16 11 4 none 8 9 10 11 12 13 14 15 j D135317 D165539 Green 255 3 301 81 11 12 4 none 5 8 9 10 11 12 13 14 1 J D165539 D251338 Green 304 8 370 31 19 23 4 none 15 16 17 18 19 20 21 2 D251338 D195433 Yellow 101 0 148 0 14 15 4 none 9 10 11 12 12 2 13 13 2 r So YWA Yelow 151 0 213 5 17 18 4 none 11 12 13 14 15 16 17 1t Li TPOX TPOX Yelow 216 99 260 99 8 4 mone 6 7 8 9 10 11 12 13 D18551 D18551 Yelow 264 49 350 0 15 19 4 none 7 9 10 10 2 11 12 13 12 AMEL AMEL Red 106 0 1140 x 9 moe XY D55818 D55818 Red 128 0 180 0 11 4 none 7 8 9 10 11 12 13 14
10. 11 0 42 0 14 t t 11 t t t 0 26 12 0 56 t 0 17 t 13 0 70 0 29 0 17 t 14 12 04 15 76 7 41 6 81 15 30 53 25 36 39 14 40 84 15 2 0 14 t t t AmpFtSTR Identifiler Plus User Guide Population Data Table 10 AmpF STR Identifiler Plus Kit allele frequencies continued Allele een AE eco ra Amari n 357 n 349 n 191 16 28 57 22 78 26 72 32 98 17 19 47 18 19 16 03 9 95 18 6 72 16 48 8 97 8 38 19 0 84 1 00 1 03 0 79 20 i i 0 34 D5S818 7 0 14t i 6 72 15 71 8 5 46 t 0 69 t 9 1 68 4 15 5 17 6 02 10 6 72 5 44 5 17 4 19 11 25 49 39 26 39 14 41 10 12 36 41 35 24 29 31 23 30 18 21 57 15 47 12 59 9 42 14 2 38 0 14t 0 69 0 26 15 i 0 29 0 18 i 16 i i 0 172 17 0 144 0 172 D7S820 6 t 0 14t 0 172 7 0 421 1 29 1 72 0 522 8 18 77 16 48 11 72 13 09 9 13 73 17 62 6 21 8 12 10 34 45 27 22 27 41 21 99 11 19 89 18 05 28 79 28 80 12 10 78 14 76 20 17 24 08 18 1 54 3 72 3 45 3 40 14 0 421 0 72 0 34 15 t t t t D8S1179 8 0 421 2 29 0 34 0 52 9 0 421 1 15 0 34 0 26 10 2 38 9 74 8 45 471 11 3 92 6 02 5 86 3 40 12 13 31 14 04 12 07 11 52 13 23 25 32 52 32 93 37 43 14 30 11 21 35 26 21 30 63 15 20 17 9 89 10 86 9 42 113 AmpFtSTR Identifiler Plus User Guide Chapter 5 Experiments and Results Table 10 AmpF STR Identifiler Plus Kit allele frequencies
11. 16 1 0 14 t t t 17 t 0 29 0 17 17 2 0 14 t t t 18 0 70 2 72 0 522 1 31 18 2 1 40 t t t 19 6 72 6 16 7 07 10 21 19 2 0 28t t t t 20 7 00 13 90 7 41 12 30 20 2 i 0 14t t t 21 12 89 16 91 14 66 12 83 22 21 57 16 91 17 24 10 47 222 0 28 1 29 0 34 0 26 22 3 0 14 0 14 t t 23 14 99 15 19 11 90 15 97 23 2 0 14 0 14 0 86 0 26 116 AmpFtSTR Identifiler Plus User Guide Population Data Table 10 AmpF STR Identifiler Plus Kit allele frequencies continued Allele Aa Se A in ac ra And i n 357 n 349 n 191 24 17 51 13 75 15 34 15 71 24 2 t 0 14 0 17 t 25 7 98 8 60 14 14 14 14 26 3 50 2 72 6 90 4 45 26 2 t t 0 52 29 0 56 t t t 30 t t t t 30 2 0 14 t t t 31 2 t t t t 32 2 t t t t 31 2 t t t t 33 2 t t t t 34 2 0 14 t t f 42 2 t t t t 43 2 t t t t 44 2 0 28 t t 45 2 0 26 46 2 0 147 t 47 2 t t t t 48 2 0 14 t t t 50 2 t t t t 51 2 t t t t THO1 4 t t t t 5 0 28 0 437 0 17 t 6 11 06 20 49 22 76 20 68 7 42 86 21 78 33 62 43 98 8 20 73 11 46 8 45 5 24 8 3 0 147 t 9 12 32 16 19 14 14 6 28 9 3 11 62 29 08 20 34 23 56 10 0 98 0 43 0 52 0 26 11 t t t t 13 3 0 147 t t t TPOX 6 72 0 14 0 34 t 2 24 t 0 34 0 26 117 AmpFtSTR Identifiler Plus User Guide Chapter 5 Experiments and Re
12. 3 Find the document of interest right click the document title then select any of the following Open To view the document Print Target To print the document Save Target As To download a PDF version of the document to a destination that you choose Note For the MSDSs of chemicals not distributed by us contact the chemical manufacturer AmpFtSTR Identifiler Plus User Guide 133 Appendix B Safety Chemical waste safety Chemical waste WARNING HAZARDOUS WASTE Refer to Material Safety Data Sheets hazards and local regulations for handling and disposal WARNING CHEMICAL WASTE HAZARD Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury illness or death WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles Chemical waste To minimize the hazards of chemical waste safety guidelines 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 a
13. STR Control DNA 9947A Standards for samples For the AmpF STR Identifiler Plus Kit the panel of standards needed for PCR amplification PCR product sizing and genotyping are Control DNA 9947A A positive control for evaluating the efficiency of the amplification step and STR genotyping using the AmpF STR Identifiler Plus Allelic Ladder GeneScan 500 LIZ Size Standard Standard used for obtaining sizing results It contains 16 single stranded labeled fragments of 35 50 75 100 139 150 160 200 250 300 340 350 400 450 490 and 500 nucleotides This standard which has been evaluated as an internal lane size standard yields precise sizing results for AmpF STR Identifiler Plus Kit PCR products Order the GeneScan 500 LIZ Size Standard PN 4322682 separately AmpF STR Identifiler Plus Allelic Ladder Allelic ladder for accurate characterization of the alleles amplified by the AmpF STR Identifiler Plus Kit The AmpF STR Identifiler Plus Allelic Ladder contains most of the alleles reported for the 15 autosomal loci Refer to Table 1 on page 11 for a list of the alleles included in the AmpF STR Identifiler Plus Allelic Ladder AmpFtSTR Identifiler Plus User Guide 17 Chapter 1 Overview Equipment and Tables 2 and 3 list required and optional equipment and materials not supplied with materials not the AmpF STR Identifiler Plus Kit Unless otherwise noted many of the items are in
14. laboratories conduct the appropriate validation studies For multiple ladder samples the GeneMapper 7D X Software calculates allelic bin offsets by using an average of all ladders that use the same panel within a run folder Allelic ladder samples in an individual run folder are considered to be from a single run When the software imports multiple run folders into a project only the ladder s within their respective run folders are used for calculating allelic bin offsets and subsequent genotyping Allelic ladder samples must be labeled as Allelic Ladder in the Sample Type column in a project Failure to apply this setting for ladder samples results in failed analysis Injections containing the allelic ladder must be analyzed with the same analysis method and parameter values that are used for samples to ensure proper allele calling Alleles that are not in the AmpF STR Allelic Ladders do exist Off ladder OL alleles may contain full and or partial repeat units An off ladder allele is an allele that occurs outside the 0 5 nt bin window of any known allelic ladder allele or virtual bin Note If a sample allele peak is called as an off ladder allele the sample result needs to be verified according to the laboratory s protocol AmpFtSTR Identifiler Plus User Guide Set up GeneMapper ID X Software for data analysis Set up GeneMapper D X Software for data analysis Workflow Before you can analyze sample fsa f
15. 0 044 15 123 14 123 22 0 031 0 045 16 127 32 127 41 0 032 0 042 17 131 54 131 62 0 027 0 039 18 135 64 135 71 0 021 0 042 19 139 72 139 81 0 024 0 045 D5S818 7 133 69 133 75 0 029 0 039 8 137 8 137 86 0 031 0 037 9 142 17 142 24 0 022 0 035 10 146 64 146 71 0 025 0 039 11 151 05 151 12 0 031 0 043 12 155 32 155 39 0 028 0 041 AmpFtSTR Identifiler Plus User Guide 87 Chapter 5 Experiments and Results 88 Table 4 Precision results of five runs 16 capillaries run of the AmpF STR Identifiler Plus Allelic Ladder continued Applied Biosystems 3130x Genetic Analyzer Allele Mean Standard Deviation 13 159 55 159 61 0 027 0 051 14 163 63 163 7 0 032 0 038 15 167 68 167 74 0 026 0 051 16 171 7 171 76 0 035 0 04 D7S820 6 255 08 255 19 0 029 0 058 7 259 13 259 22 0 04 0 056 8 263 16 263 25 0 037 0 053 9 267 19 267 29 0 046 0 053 10 271 25 271 34 0 039 0 051 11 275 28 275 4 0 037 0 06 12 279 34 279 45 0 034 0 05 13 283 38 283 49 0 039 0 049 14 287 44 287 54 0 039 0 051 15 291 51 291 62 0 043 0 052 D8S1179 8 122 49 122 61 0 03 0 044 9 126 56 126 68 0 037 0 045 10 130 66 130 76 0 026 0 044 11 134 8 134 89 0 031 0 041 12 138 98 139 09 0 019 0 043 13 143 58 143 68 0 028 0 042 14 148 03 148 14 0 03 0 046 15 152 43 152 54 0 025 0 043 16 156 73 156 83 0 026 0 039 17 160 93 161 04 0 031 0 042 18 165 03
16. 0 046 9 182 82 182 89 0 027 0 034 9 8 185 84 185 9 0 022 0 042 10 186 77 186 83 0 026 0 036 11 190 71 190 77 0 027 0 034 12 201 48 201 55 0 026 0 037 TPOX 6 221 82 221 91 0 029 0 05 7 225 8 225 88 0 029 0 053 8 229 79 229 86 0 034 0 048 AmpFtSTR Identifiler Plus User Guide 89 Chapter 5 Experiments and Results Table 4 Precision results of five runs 16 capillaries run of the AmpF STR Identifiler Plus Allelic Ladder continued Applied Biosystems 3130x Genetic Analyzer Allele Mean Standard Deviation 9 233 77 233 86 0 039 0 048 10 237 76 237 83 0 025 0 053 11 241 75 241 83 0 028 0 04 12 245 78 245 85 0 04 0 049 13 249 76 249 85 0 036 0 048 vWA 11 154 07 154 14 0 028 0 042 12 158 26 158 34 0 028 0 04 18 162 42 162 49 0 031 0 043 14 166 66 166 73 0 031 0 05 15 170 59 170 66 0 029 0 044 16 174 62 174 68 0 03 0 04 17 178 61 178 67 0 028 0 051 18 182 54 182 61 0 021 0 037 19 186 5 186 56 0 024 0 043 20 190 43 190 49 0 028 0 04 21 194 29 194 36 0 024 0 044 22 198 17 198 24 0 023 0 036 23 202 01 202 09 0 028 0 041 24 206 36 206 42 0 034 0 041 Extra peaks in the electropherogram 90 Causes of extra peaks Peaks other than the target alleles may be detected on the electropherogram Causes for the appearance of extra peaks include stutter products incomplete 3 A nucleotide addition at the n 1 position dye artifacts an
17. 0 70 0 43 0 52 0 79 23 0 42 0 14 0 52 0 26 24 t 0 14 0 17 t 25 t t 0 17 t 26 t t t t 27 t t t t D19S433 9 t 0 14 0 17 t 10 1 54 t t t 11 7 14 0 72 0 52 0 52 11 2 0 147 t 0 17 t 12 10 78 7 74 6 21 3 14 12 2 6 30 0 57 1 90 t 13 29 83 28 94 16 03 17 80 14 21 01 34 10 31 72 24 87 14 2 4 20 0 86 5 00 3 66 15 4 76 15 76 13 45 13 35 15 2 3 36 2 72 8 79 10 73 16 2 38 4 15 4 31 3 93 16 2 2 38 1 72 2 93 1 83 17 t 0 29 0 174 0 79 17 2 0 28 0 29 t 2 88 18 2 0 14 0 29 t 1 05 D21S11 24 t 24 2 0 14 0 43 0 17 t 24 3 1 i 25 t 25 2 t 0 14 0 17 t 26 0 14 0 14 0 17 t 27 5 04 4 58 1 21 0 52 28 22 97 16 76 9 14 6 28 28 2 t AmpFtSTR Identifiler Plus User Guide 115 Chapter 5 Experiments and Results Table 10 AmpF STR Identifiler Plus Kit allele frequencies continued Allele kalen a G le lk in Bou Arenas n 357 n 349 n 191 29 19 33 20 49 21 21 16 75 29 2 0 14 t 0 522 0 26 29 3 0 14 t t t 30 17 28 25 21 29 31 34 29 30 2 1 40 3 30 2 93 1 83 81 7 98 7 16 6 72 5 76 31 2 7 98 9 46 8 62 18 85 32 1 12 1 43 1 55 0 79 32 2 5 88 7 16 12 93 9 69 33 0 56 t t 0 522 33 2 3 78 3 30 4 14 3 66 34 1 26 t t t 34 1 0 14t t t t 34 2 0 14 0 29 0 86 0 79 35 2 94 t 0 34 35 1 0 14 t t t 35 2 t 0 14 i t 36 0 84 t t i 37 0 28 t t t 38 0 14t t t t FGA 16 t 0 14 t t
18. 1 FGA Red 206 25 360 0 23 24 4 none 17 18 19 20 21 22 23 2 8 Select D2S1388 to display the Bin view for the marker in the right pane e o e lt 5 15 9 eo 5 o x de e p 0 AmpFtSTR Identifiler Plus User Guide 63 Section 4 2 GeneMapper ID X Software File Edit Bins View Help EE x G MH kl QU Bin Set identifier _Plus_Bins_v1x i EGS u o Eh aig Panel Manager 7 ls le hol 1 faz laj ha las lie 17 z0 El ED AmpFLSTR_Panels_v1X 10 ampFLSTR Identifiler Plus v1x 1 C3gIdentifiler Plus Panels vix a T 03 H D21511 D75820 08 CSF1PO D351358 THO1 0 7 D135317 D165539 D251338 gt D195433 E YWA 0 5 TPOX D18551 AMEL 0 4 D55818 FGA 03 02 as Reference Samples 04 0 0 113115 117 119121 123125 127 123131 133135 137 133141 143145 147149 151 153155 157159 161 163165 167163 171173175 177173 181183185 187 D851179 Marker D851179 118 00 183 50 L9 cm mh ep 9 Import Identifiler Plus Stutter v1X a Select the AmpFLSTR JIdentifiler Plus v1X folder in the navigation panel g Panel Manager File Edit Bins View Help ir x E li E a Bin Set identifier Pus Bins 1x a EJ sh Panel Manager Panel Name Comment i
19. 10X 4335643 Genetic Analyzer Septa Retainer Clips for 96 Tube Sample Tray 402866 Genetic Analysis Sample Tubes 0 5 mL 401957 Septa for 0 5 mL Sample Tubes 401956 DS 33 Matrix Standard Set 6 FAM VIC NED PET and LIZ dyes for 4318159 ABI PRISM 310 377 systems MicroAmp 8 Tube Strip 0 2 mL N8010580 MicroAmp 96 Well Base holds 0 2 mL reaction tubes N8010531 MicroAmp 96 Well Full Plate Cover N8010550 AmpFtSTR Identifiler Plus User Guide 19 Chapter 1 Overview Table 3 User supplied materials continued Item Source MicroAmp 96 Well Tray Retainer Set 403081 POP 4 Polymer for the 310 Genetic Analyzer 402838 Analyzer User Guide PN 4317588 For a complete list of parts and accessories for the 310 instrument refer to Appendix B of the ABI PRISM 310 Genetic PCR Amplification MicroAmp 96 Well Tray N8010541 MicroAmp Reaction Tube with Cap 0 2 mL N8010540 MicroAmp 8 Tube Strip 0 2 mL N8010580 MicroAmp 8 Cap Strip N8010535 MicroAmp 96 Well Tray Retainer Set 403081 MicroAmp 96 Well Base N8010531 MicroAmp Clear Adhesive Film 4306311 MicroAmp Optical Adhesive Film 4311971 MicroAmp Optical 96 Well Reaction Plate N8010560 Other user supplied materials Hi Di Formamide 25 mL 4311320 Aerosol resistant pipette tips MLS Microcentrifuge tubes MLS Pipettors MLS Tape labeling MLS Tube 50 mL F
20. 119 125 131 137 143 149 155 161 167 173 178 185 D851179 4 gt 9 Click Apply then OK to add the AmpF STR Identifiler Plus Kit panel and bin set to the GeneMapper ID Software database IMPORTANT If you close the Panel Manager without clicking OK the panels and bins are not imported into the GeneMapper ZD Software database Import an HID The HID Advanced analysis method for the AmpF STR Identifiler Plus PCR analysis method Amplification Kit uses the Identifiler Plus Bins vl file described in step 6 on page 49 Use the following procedure to import the analysis method from the folder that you downloaded from our web site into the GeneMapper ID Software database Refer to step 1 on page 48 for downloading instructions Note The Identifiler Plus AnalysisMethod vl has been provided to assist you in getting started with Identifiler Plus kit data analysis Analysis parameters should be established by each individual laboratory based on the laboratory s internal validation studies 1 Select Tools GeneMapper Manager to open the GeneMapper Manager AmpFtSTR Identifiler Plus User Guide 51 Section 4 1 GeneMapper ID Software 2 Import an analysis method for HID_Advanced a Select the Analysis Methods tab then click Import GeneMapper Manager Name Last Saved Owner HID Advanced 2009 06 18 16 22 2 omic HID_Classic 2007 08 06 10 03 0 gmid Microsatellite Default 2004 05 28
21. 165 12 0 024 0 046 19 169 1 169 2 0 035 0 044 FGA 17 214 11 214 23 0 041 0 05 18 218 14 218 26 0 043 0 052 19 222 17 222 3 0 039 0 054 20 226 21 226 35 0 044 0 057 21 230 26 230 38 0 045 0 055 22 234 29 234 42 0 05 0 058 23 238 33 238 47 0 038 0 057 AmpFtSTR Identifiler Plus User Guide Table 4 Precision results of five runs 16 capillaries run of the Accuracy precision and reproducibility AmpFfSTR Identifiler Plus Allelic Ladder continued Applied Biosystems 3130x Genetic Analyzer Allele Mean Standard Deviation 24 242 37 242 52 0 044 0 067 25 246 42 246 57 0 044 0 056 26 250 48 250 62 0 038 0 069 26 2 252 49 252 64 0 046 0 066 27 254 5 254 65 0 047 0 057 28 258 55 258 71 0 045 0 064 29 262 63 262 78 0 049 0 062 30 266 72 266 88 0 052 0 069 30 2 268 53 268 7 0 049 0 065 31 2 272 62 272 78 0 036 0 062 32 2 276 71 276 86 0 05 0 068 33 2 280 77 280 94 0 043 0 069 42 2 317 89 318 06 0 045 0 062 43 2 322 01 322 16 0 038 0 055 44 2 326 14 326 27 0 034 0 05 45 2 330 28 330 39 0 039 0 048 46 2 334 28 334 4 0 044 0 05 47 2 338 3 338 49 0 039 0 055 48 2 342 51 342 66 0 034 0 055 50 2 350 59 350 76 0 041 0 061 51 2 354 54 354 7 0 039 0 063 THO1 4 162 72 162 77 0 025 0 04 5 166 78 166 84 0 027 0 035 6 170 82 170 87 0 03 0 046 7 174 83 174 9 0 029 0 045 8 178 84 178 9 0 02
22. 2 Start the GeneMapper D X Software then log in with the appropriate user name and password IMPORTANT If you need logon instructions refer to the GeneMapper ID X Software Version 1 0 Getting Started Guide PN 4375574 0 0 5 e lt D T zo 0 2 P x e b g o 3 Select Tools Panel Manager 4 Find then open the folder containing the panels bins and marker stutter a Select Panel Manager in the navigation pane AmpFtSTR Identifiler Plus User Guide 61 Section 4 2 GeneMapper ID X Software Panel Manager File Edit Bins View Help Cix tee a Highlight this E b Select File gt Import Panels to open the Import Panels dialog box c Navigate to then open the Identifiler Plus Analysis Files GMIDX folder that you unzipped in step 1 on page 61 5 Select Identifiler_Plus_Panels_v1X then click Import Note Importing this file creates a new folder in the navigation pane of the Panel Manager AmpFLSTR_Identifiler_Plus_v1X This folder contains the panel and associated markers Import Panels xl Look in je Identifiler Plus Analysis Files GMIDX w I de FERES CE G5 Identifier Plus GS500 xml Identifier Plus Bins vix txt B Identifier Plus Panels v1xX txt E Identifier_Plus_Stutter_v1X txt Identifiler_Plus_AnalysisMethod_v1X xml My Recent Documents Desktop Cee ee File name fidentifier_Plus_Panels_v1x txt Im
23. AK kK KK KK KK KK KK KK KK KK KK KK KK KK 74 AmpFtSTR Identifiler Plus User Guide 45 Section 4 1 GeneMapper ID Software Section 4 1 GeneMapper D Software Overview of GeneMapper D Software GeneMapper JD Software is an automated genotyping software for forensic casework databasing and paternity data analysis After electrophoresis the Data Collection Software stores information for each sample in a fsa file Using GeneMapper ID Software v3 2 1 software you can then analyze and interpret the data from the fsa files Instruments Refer to Instrument and software overview on page 15 for a list of compatible instruments Before you start When using GeneMapper ID Software v3 2 1 to perform human identification HID analysis with AmpF STR kits be aware that 46 HID analysis requires at least one allelic ladder sample per run folder Your laboratory can use multiple ladder samples in an analysis provided individual laboratories conduct the appropriate validation studies For multiple ladder samples the GeneMapper JD Software calculates allelic bin offsets by using an average of all ladders that use the same panel within a run folder Allelic ladder samples in an individual run folder are considered to be from a single run When the software imports multiple run folders into a project only the ladder s within their respective run folders are used for calculating allelic bin offsets and subsequent g
24. GeneAmp PCR System 9700 Remove MicroAmp Base from tray retainer set and repeat test Insufficient PCR product electrokinetically injected For ABI PRISM 3100 Avant or Applied Biosystems 3100 3130x runs Mix 1 0 uL of PCR product and 9 uL of Hi Di Formamide GeneScan 500 LIZ solution For ABI PRISM 310 instrument runs Mix 1 5 uL of PCR product and 25 uL of Hi Di Formamide GeneScan 500 LIZ solution Degraded formamide Check the storage of formamide do not thaw and refreeze multiple times Try Hi Di Formamide AmpFtSTR Identifiler Plus User Guide 127 Appendix A Troubleshooting Table 14 Troubleshooting continued Observation Possible causes Recommended actions Positive signal from AmpFZSTR Control DNA 9947A but partial or no signal from DNA test samples Quantity of test DNA sample is below assay sensitivity Quantitate DNA and add 1 0 ng of DNA Repeat test Test sample contains high concentration of PCR inhibitor for example heme compounds certain dyes Quantitate DNA and add minimum necessary vol ume Repeat test Wash the sample in a Centricon 100 centrifugal filter unit Repeat test Test sample DNA is severely degrad ed If possible evaluate the quality of DNA sample by running an agarose gel If DNA is degraded ream plify with an increased amount of DNA or use the AmpF STR MiniFiler Kit Dilution of test sample D
25. H Takahashi S and Kimura K 1994 Identification of the heme compound copurified with deoxyribonucleic acid DNA from bloodstains a major inhibitor of polymerase chain reaction PCR amplification J Forensic Sci 39 362 372 Barber M D Piercy R C Andersen J F and Parkin B H 1995 Structural variation of novel alleles at the Hum vWA and Hum FES FPS short tandem repeat loci Intl J Legal Med 108 31 35 Barber M D McKeown B J and Parkin B H 1996 Structural variation in the alleles of a short tandem repeat system at the human alpha 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 Baron H Fung S Aydin A Bahrig S Luft FC and Schuster H 1996 Oligonucleotide ligation assay OLA for the diagnosis of familial hypercholesterolemia Nat Biotechnol 14 1279 1282 Begovich A B McClure G R Suraj V C Helmuth R C Fildes N Bugawan T L Erlich H A and Klitz W 1992 Polymorphism recombination and linkage disequilibrium within the HLA class II region J mmunol 148 249 58 Brinkmann B Moller A and Wiegand P 1995 Structure of new mutations in 2 STR systems Intl J Legal Med 107 201 203 Brinkmann B Klintschar M Neuhuber F Huhne J and Rolf B 1998 Mutation rate in human microsatellites Influ
26. Human Identification Analysis User Guide PN 4338775 3 Select Tools gt Panel Manager 4 Find then open the folder containing the panels and bins a Select Panel Manager in the navigation pane File Edit Bins View on m mmm zar Panel Manager Highlight this b Select File Import Panels to open the Import Panels dialog box c Navigate to then open the Identifiler Plus Analysis Files GMID folder that you unzipped in step 1 5 Select Identifiler Plus Panels v1 then click Import Note Importing this file creates a new folder in the navigation pane of the Panel Manager AmpFLSTR Identifiler Plus v1 This folder contains the panel and associated markers 48 AmpFtSTR Identifiler Plus User Guide Set up GeneMapper ID Software for data analysis Look in le Identifiler Plus Analysis Files GMID c CE GS ldentifiler Plus GS500 xml Identifiler Plus amp nalysisMethod 1 xml E Idertifiler_Plus_Bins_v1 txt B identifiler_Plus_Panels_v1 txt r My Recent D Desktop 2 My Documents File name Jdertifier_Plus_Panels_v1 txt Import JE Files of type aj Files m cnca 6 Import Identifiler Plus Bins vl CD D D lt ied zo go D o e e z o E o a Select the AmpFLSTR_Identifiler_Plus_v1 folder in the navigation pane File Edit Bins View zim Bi m Comment lik x mm M BEJI om se Panel Name 1 fdertifiler_Plus_v1
27. K Overhauser J Ott J and Gilliam T C 1993 A microsatellite genetic linkage map of human chromosome 18 Genomics 15 48 56 Szibor R Lautsch S Plate I Bender K and Krause D 1998 Population genetic data of the STR HumD3S1358 in two regions of Germany Int J Legal Med 111 160 161 Wallin J M Buoncristiani M R Lazaruk K D Fildes N Holt C L Walsh PS 1998 SWGDAM validation of the AmpFISTR blue PCR amplification kit for forensic casework analysis J Forensic Sci 43 854 870 Wallin J M Holt C L Lazaruk K D Nguyen T H and Walsh P S 2002 Constructing universal multiplex PCR systems for comparative genotyping J Forensic Sci 47 52 65 Walsh P S Fildes N J and Reynolds R 1996 Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vWA Nucleic Acids Res 24 2807 2812 Watson S Kelsey Z Webb R Evans J and Gill P 1998 The development of a third generation STR multiplex system TGM In Olaisen B Brinkmann B and Lincoln P J eds Progress in Forensic Genetics 7 Proceedings of the 17th International ISFH Congress Oslo 2 6 September 1997 Elsevier Amsterdam pp 192 194 Weber J and Wong C 1993 Mutation of human short tandem repeats Hum Mol Genet 2 1123 1128 Weir B S 1996 Genetic data analysis II Sunderland MA Sinauer Associates Inc AmpFtSTR Identifiler Plus User Guide Documentation
28. P S and Matise T C 2004 A combined linkage physical map of the human genome Am J Hum Genet 75 1143 1148 Kwok S and Higuchi R 1989 Avoiding false positives with PCR Nature 339 237 238 Lazaruk K Walsh P S Oaks F Gilbert D Rosenblum B B Menchen S Scheibler D Wenz H M Holt C Wallin J 1998 Genotyping of forensic short tandem repeat STR systems based on sizing precision in a capillary electrophoresis instrument Electrophoresis 19 86 93 Li H Schmidt L Wei M H Hustad T Leman M I Zbar B and Tory K 1993 Three tetranucleotide polymorphisms for loci D3S1352 D3S1358 D3S1359 Hum Mol Genet 2 1327 AmpFtSTR Identifiler Plus User Guide Magnuson V L Ally D S Nylund S J Karanjawala Z E Rayman J B Knapp J I Lowe A L Ghosh S and Collins F S 1996 Substrate nucleotide determined non templated addition of adenine by Taq DNA polymerase implications for PCR based genotyping and cloning Biotechniques 21 700 709 Mansfield E S Robertson J M Vainer M Isenberg A R Frazier R R Ferguson K Chow S Harris D W Barker D L Gill P D Budowle B and McCord B R 1998 Analysis of multiplexed short tandem repeat STR systems using capillary array electrophoresis Electrophoresis 19 101 107 Mills K A Even D and Murrau J C 1992 Tetranucleotide repeat polymorphism at the human alpha fibrinogen locus FGA Hum Mol Genet 1 77
29. Reference Guide PN 4375671 Examine and edit a project You can display electropherogram plots from the Samples and Genotypes tabs of the Project window to examine the data These procedures start with the Analysis Summary tab of the Project window assuming the analysis is complete For more information about any of these tasks refer to GeneMapper ID X Software Version 1 0 Getting Started Guide PN 4375574 GeneMapper ID X Software Version 1 0 Quick Reference Guide PN 4375670 GeneMapper ID X Software Version 1 0 Reference Guide PN 4375671 74 AmpFtSTR Identifiler Plus User Guide Part Number 4440211 Rev F 2 2015 Experiments and Results This chapter covers Qay o A GO a Pk PE A dtu 78 Developmental validation 00 aaa aaa KK KK KK KK KK KK KK KK 79 Accuracy precision and reproducibility sssaaa eese 82 Extra peaks in the electropherogram uaaaaa aaa aaa aaa aaa ia 90 Characterization of loci W K kk KK KK KK KK KK aaa KK KK KK KK KK iii 97 See leS S o Zil SJ M _DE gt MDERDMDNDMDNMNMNnEMMIAMNDMDIEnENERaREnEnENn EaNna En En T HD DJ HHR 98 Sensitivity 3 24 35 T 100 Stability en tie done O YE eS LR REEL EEE e ed OR AE 102 Mil XtUr Studies sca iit tutae ee eel et aE A ARR A 105 Population Datas vs xe wuna indii Ear WOW EFG di vig Atha 111 Mutation Rate e ool eme ua buk Wd me ud RM SA EDS 123 Probability of Identity o kk kk kk KK KK KK KK KK KK KK KK KK KK K
30. Xe jM msp e CSF1PO D351358 THO1 D135317 D165539 3 stel 0 1039 none 8 9 10 11 12 12 D251338 370 31 j 9 23 0 1244 none 15 18 17 18 19 2 10 D195433 yellow 101 148 0 14 15 0 1121 none 9 10 11 12 12 2 1 d ex bd rd Sf on M yellow 151 0 213 5 17 18 0 1245 none yellow 215 99 260 99 Je Ja 0 0638 none 13 D18551 yelow 264 49 350 0 15 19 4 0 1368 14 AMEL red 106 0 114 0 15 D55818 1280 haoo f1 0 1006 none 78910111244 20625 3600 23 24 0 1303 none 17 18 19 20 21 2 11 12 13 14 15 1 8 Select D8S1179 to display the Bin view for the marker in the right pane 50 AmpFtSTR Identifiler Plus User Guide Set up GeneMapper ID Software for data analysis Panel Manager xl File Edit Bins View Bi XE MI M HT fein set fuer Pus 5e Bi Ti Wi i m m JB Bij amp iPanel Manager n AmpFLSTR_Panels_y2 z bl hol m 12 hal ha hs 46 17 18 hal 20 EE AmpFLSTR_Identitiler_Plus_v1 1 0 1 4S Identifier Plus Panels v1 l D851179 I D21811 09r D75820 CSF1PO 081 D381358 O THO1 T o I D138317 071 a 0165539 Z D251338 0195433 0 51 O zo WA 4 o TPOX D18551 OST U I AMEL 05s818 oat E FGA 031 o 021 GjReference Samples i 041 00 113
31. a MicroAmp Optical 96 Well Reaction Plate add 9 uL of the formamide size standard mixture I UL of PCR product or allelic ladder Note For blank wells add 10 uL of Hi Di Formamide Sealthe reaction plate with appropriate septa then briefly centrifuge the plate to ensure that the contents of each well are collected at the bottom Heat the reaction plate in a thermal cycler for 3 minutes at 95 C Immediately place the plate on ice for 3 minutes Prepare the plate assembly on the autosampler Start the electrophoresis run AmpFtSTR Identifiler Plus User Guide Section 3 2 310 instrument Section 3 2 310 instrument Set up the 310 instrument for electrophoresis Reagents and Table 3 on page 18 lists the required materials not supplied with the AmpF STR parts Identifiler Plus Kit IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the AmpF STR Identifiler Plus Primer Set from light when not in use Amplified DNA AmpF STR Identifiler Plus Allelic Ladder and GeneScan 500 LIZ Size Standard should also be protected from light Keep freeze thaw cycles to a minimum 310 instrument The following table lists Data Collection Software and the run modules that can be requirements used to analyze Identifiler Plus PCR products For details on the procedures refer to the documents listed in the table Operating Data E system Collection Run modules and condi
32. and 3 2 Human Identification Analysis Tutorial PN 4335523 Installation Procedures and New Features for GeneMapper ID Software v3 2 User Bulletin PN 4352543 Analysis Method Editor HID FulRange Z start Pt jo Stop Pt Figure 7 Analysis Method Editor HID Peak Detector tab settings IMPORTANT Laboratories need to perform the appropriate internal validation studies to determine the peak amplitude thresholds that allow for reliable interpretation of AmpF STR Identifiler Plus PCR Amplification Kit data 54 AmpFtSTR Identifiler Plus User Guide Set up GeneMapper ID Software for data analysis The software uses the peak amplitude threshold parameters to specify the minimum peak height to limit the number of detected peaks Although GeneMapper D Software displays peaks that fall below the specified amplitude in electropherograms the software does not label or determine the genotype of these peaks Note The analysis range is set by the user based on the locations ofthe primer peaks and size standard peaks For more information about peak detection algorithms refer to GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide PN 4338775 GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial Appendix A PN 4335523 Installation Procedures and New Features for GeneMapper ID Software v3 2 User Bulletin PN 4352543 Analysis Me
33. et al 1998 Momhinweg et al 1998 Szibor et al 1998 of direct mutation rate counts produced Larger sample sizes for some of the AmpF STR Identifiler Plus Kit loci Methods for modifications of these mutation rates to infer mutation rates indirectly for those loci where the rates are not large enough to be measured directly and or to account for those events undetectable as Mendelian errors AmpFtSTR Identifiler Plus User Guide 123 Chapter 5 Experiments and Results Probability of Identity Table 12 shows the Probability of Identity PI values of the AmpF STR Identifiler Plus Kit loci individually and combined Table 12 Probability of Identity values for the AmpF STR Identifiler Plus Kit STR loci Locus AEG GE m U S Hispanic Amati CSF1PO 0 079 0 132 0 141 0 123 D281338 0 023 0 027 0 038 0 043 D381358 0 097 0 076 0 112 0 158 D5S818 0 104 0 147 0 115 0 110 D7S820 0 085 0 063 0 083 0 081 D8S1179 0 074 0 064 0 089 0 104 D13S317 0 132 0 079 0 056 0 056 D168539 0 077 0 097 0 090 0 082 D18S51 0 033 0 031 0 031 0 046 D19S433 0 042 0 087 0 049 0 044 D21S11 0 037 0 044 0 047 0 074 FGA 0 034 0 035 0 032 0 031 THO1 0 109 0 079 0 097 0 134 TPOX 0 089 0 188 0 168 0 159 vWA 0 066 0 066 0 080 0 103 Combined 1 31 X 10718 5 01 X 10718 7 65 X 10718 3 62 X 10717 The P value is the probability that two individuals selected at random will have an identical Am
34. 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 is a mixture must be determined by the analyst in the context of each particular case Limit of detection of the minor component Mixtures of two genomic DNA samples were examined at various ratios 0 1 1 1 3 1 7 1 10 1 15 1 1 0 The total amount of genomic input DNA mixed at each ratio was 1 ng The samples were amplified in a GeneAmp PCR System 9700 then electrophoresed and detected using an Applied Biosystems 3130x Genetic Analyzer The results of the mixed DNA samples are sho
35. multiple contributors should be considered when interpreting the results We recommend that individual laboratories assign a minimum peak height threshold based on validation experiments performed in each laboratory to avoid typing when stochastic effects are likely to interfere with accurate interpretation of mixtures Mixture Studies Evidence samples that contain body fluids and or tissues originating from more than one individual are an integral component of forensic casework Therefore it is essential to ensure that the DNA typing system is able to detect DNA mixtures Mixed samples can be distinguished from single source samples by The presence of more than two alleles at a locus The presence of a peak at a stutter position that is significantly greater in percentage than what is typically observed in a single source sample Significantly imbalanced alleles for a heterozygous genotype The peak height ratio is defined as the height of the lower peak in RFU divided by the height of the higher peak in RFU expressed as a percentage Mean median minimum and maximum peak height ratios observed for alleles in the AmpF STR Identifiler Plus PCR Amplification Kit loci in unmixed population database samples are shown in Table 8 AmpFtSTR Identifiler Plus User Guide 105 Chapter 5 Experiments and Results Table 8 Peak height ratios for 1 0 ng of input DNA amplified for 28 PCR
36. nui i amp iPanel Manager E AmpFLSTR Panels v2 Bill 4mpFLSTR Identifiler Plus v1 b Select File gt Import Bin Set to open the Import Bin Set dialog box c Navigate to then open the Identifiler Plus Analysis Files GMID folder d Select Identifiler Plus Bins v1 then click Import Note Importing this file associates the bin set with the panels in the Identifiler Plus Panels v1 folder Look in le Identifiler Plus Analysis Files GMID m i2 e A CE_G5_Identifiler_Plus_G5500 xml Idertifiler_Plus_AnalysisMethod_v1 xml My Recent D B dentifiler _Plus_Bins_v1 ixt E E Identifiler Plus Panels v1 txt Desktop File name Jdentifier_Plus_Bins_vt bt Import Files of type m Files Cancel AmpFtSTR Identifiler Plus User Guide 49 Section 4 1 GeneMapper ID Software 7 View the imported panels in the navigation pane a Double click the AmpFLSTR_Identifiler_Plus_v1 folder to view the Identifiler_Plus_Panels_v1 folder b Double click the Identifiler_Plus_Panels_v1 folder to display the panel information in the right pane Panel Manager File Edit Bins View E EPanel Manager AmpFLSTR_Panels_v2 CHES AmpFLSTR_Idertifiler_Plus_v1 EHE Identifiler Plus Panels v1 D851178 I D21811 D75820 I CSF1PO gt D3S1358 TH01 0135317 0165539 D281338 D195433 vWA TPOX D18551 AMEL D55818 FGA
37. p 0 7627 0 7861 0 6476 0 0118 HExp 0 8585 0 8427 0 8290 0 8003 Ho 0 8711 0 8567 0 7931 0 801 FGA HW X p 0 0 904953 0 263223 0 999686 HW G p 1 0 999812 0 960137 0 999946 HW Exact p 0 9761 0 4459 0 0891 0 9161 HExp 0 8659 0 8686 0 8751 0 8746 Ho 0 8824 0 8854 0 8724 0 8482 THO1 HW X p 0 961911 0 997905 0 649467 0 329461 HW G p 0 940414 0 99169 0 617212 0 318591 HW Exact p 0 8286 0 9716 0 4495 0 1377 HExp 0 7323 0 7866 0 7666 0 7016 Ho 0 7395 0 7822 0 8103 0 6492 121 AmpFtSTR Identifiler Plus User Guide Chapter 5 Experiments and Results 122 Concordance studies Table 11 Heterozygosity and p values for Hardy Weinberg tests of the 15 Identifiler Plus Kit STR loci in four U S populations continued Fai NSM us AO E Aii n 357 n 349 n 191 TPOX HW X p 0 765163 0 801518 0 875348 0 333914 HW G p 0 611014 0 757735 0 913091 0 229017 HW Exact p 0 7247 0 5775 0 8356 0 0647 HExp 0 7643 0 6311 0 6607 0 6765 Ho 0 7563 0 6304 0 6759 0 6178 vWA HW X p 0 925176 0 005048 0 641684 0 994248 HW G p 0 964308 0 218817 0 934427 0 997184 HW Exact p 0 7033 0 0564 0 7066 0 8845 HExp 0 8141 0 8081 0 7818 0 7457 Ho 0 8571 0 8138 0 7759 0 7277 HW X p probability value of X test for Hardy Weinberg equilibrium HW G p probability value of the G statistic of the Likelihood Ratio test for multinomial proportions HW Exact p A Markov chain unbiased exact test to estimate the P va
38. panel 3 cat 10 ng panel 4 horse 10 ng panel 5 microbial DNA pool equivalent to 105 copies of Candida albicans Staphylococcus aureus Neisseria gonorrhoeae E coli 0157 H7 Bacillus subtilis and Lactobacillus rhamnosus panel 6 and the negative control panel 7 The extracted DNA samples were amplified with the AmpF STR Identifiler Plus PCR Amplification Kit and analyzed using the Applied Biosystems 3130x Genetic Analyzer Primates gorilla chimpanzee orangutan and macaque 1 0 ng each Non primates mouse dog sheep rabbit cat horse hamster rat chicken and cow 10 ng each Microorganisms Candida albicans Staphylococcus aureus Escherichia coli Neisseria gonorrhoeae Bacillus subtilis and Lactobacillus rhamnosus equivalent to 10 copies The chimpanzee and gorilla DNA samples produced partial profiles within the 70 to 350 nucleotide region The microorganisms chicken hamster mouse rabbit and rat did not yield detectable products Dog horse sheep and cow produced a 98 bp fragment near the amelogenin locus in the PET dye AmpFtSTR Identifiler Plus User Guide Species specificity 75 115 155 195 235 275 315 355 395 Control DNA 9947A 75 115 155 195 235 275 315 355 395 2000 Chimpanzee 115 155 195 235 275 315 355 395 115 155 195 235 275 315 355 395 sof Horse 30004 2000 100
39. s E E EILo E2 3 lt 2 24 2 5 2 50 2 22 2 B2 29 2 E12 53 2 5 2 ladder m jm jm jm Mark Sample for Deletion 130 170 210 250 230 330 370 1200 800 400 o a_ R 5 BABE an co E E E a EE A AAAA AE RR S AE RSEN mM balises s zll sll s lJ 5 e El ll eles 1 afojn mim mmm e Thef hs be fas 15 16 17 fas 19 2o 21 22 23 24 25 26 27 28 10 ladder mm m j m I Mark Sample for Deletion 130 170 210 250 230 330 370 1200 800 400 spes des Ipsos pepe bob EE bs E E e belg pos gt bel lsd espe ps es pps b Le pe eod bl EIE Es es bs 7 10 2 13 2 4 2 ladder ma Mark Sample for Deletion 12 Figure 1 GeneMapper D X Software plot of the AmpF STR Identifiler Plus Kit Allelic Ladder AmpFtSTR Identifiler Plus User Guide Product overview Control DNA Figure 2 shows amplification of Control DNA 9947A using the AmpF STR 9947A profile Identifiler Plus Kit iP pvsenzsc ia ss7A gg m m m S B Mark Sample for Deletion HETTA DUN ME HT 130 170 210 250 290 330 370 IDP DVSen 28C ng 947A jjj jm jm jm ma r Mark Sample for Deletion mess 130 170 210 250 290 330 370 IDP_DVSen 28C_1ng_9347A jj jm jm jm jm jm IT Mark Sample for Deletion 130 170 210 250 290 330 370 Figure 2 1 ng of Control DNA 9947A amplified with the AmpF STR Identifil
40. 0 75 115 155 195 235 275 315 355 395 200 wl Microbial pool 1204 200 As ss 195 225 zs A 315 355 395 m NTC 1204 al al Figure 24 Representative electropherograms from a species specificity study including positive and non template controls NTC run for 28 PCR cycles AmpFtSTR Identifiler Plus User Guide 99 Chapter 5 Experiments and Results Sensitivity 100 SWGDAM guideline 2 3 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 Identifiler Plus PCR Amplification Kit should be between 0 75 and 1 0 ng for 28 cycle amplification The DNA sample should be quantitated before amplification using a system such as the Quantifiler Human DNA Quantification Kit PN 4343895 or the Quantifiler HP Human Plus DNA Quantification Kit PN 4482911 The final DNA concentration should be 0 075 to 0 1 ng uL so that 0 75 to 1 0 ng of DNA is added to the PCR reaction in a volume of 10 UL If the sample contains degraded or inhibited DNA amplification of additional DNA may be beneficial In Figures 25 and 26 the control DNA 9947A was serially diluted from 1 ng to 0 031 ng With the 28 PCR cycle protocol full profiles 26 alleles were consistently obtained at 0 125 ng but occasional partial profiles that are miss
41. 0 7908 0 7862 0 7487 D8S1179 HW X2 p 0 067164 0 545414 0 047783 0 446248 HW G2 p 0 568837 0 275218 0 302937 0 760077 HW Exactp 0 2176 0 3264 0 0304 0 1656 HExp 0 7925 0 8047 0 7853 0 7403 Ho 0 7899 0 8424 0 8 0 6806 D13S317 HW X2 p 0 014379 0 711127 0 353995 0 813948 HW G2 p 0 609389 0 871173 0 190736 0 814681 HW Exact p 0 3818 0 667 0 2415 0 6851 HExp 0 6977 0 7797 0 8251 0 8222 Ho 0 6695 0 7364 0 8207 0 8168 AmpFtSTR Identifiler Plus User Guide Population Data Table 11 Heterozygosity and p values for Hardy Weinberg tests of the 15 Identifiler Plus Kit STR loci in four U S populations continued alt PBE TE e AG Amede n 357 n 349 n 191 D16S539 HW X p 0 433216 0 67702 0 058631 0 996396 HW G p 0 482435 0 594871 0 37601 0 981384 HW Exact p 0 3753 0 4328 0 3068 0 9986 HExp 0 7939 0 7632 0 7747 0 7766 Ho 0 8263 0 7822 0 7828 0 7853 D18S51 HW X p 0 999844 0 628334 0 999203 0 343027 HW G p 1 0 872113 0 999492 0 798859 HW Exact p 0 978 0 0982 0 9152 0 2265 HExp 0 8694 0 8769 0 8761 0 8463 Ho 0 8824 0 8682 0 8862 0 8377 D19S433 HW X p 0 91703 0 806717 0 731222 0 810711 HW G p 0 83419 0 999765 0 975476 0 898389 HW Exact p 0 4517 0 69 0 3475 0 4301 HExp 0 8364 0 7659 0 8310 0 8430 Ho 0 8011 0 7622 0 8414 0 822 D21S11 HW X p 0 985687 0 936146 0 0 HW G p 1 0 999757 0 999794 0 712937 HW Exact
42. 11 34 3 grid Microsatellite ma KI b Navigate to then open the Identifiler Plus Analysis Files GMID folder c Select Identifiler_Plus_AnalysisMethod_v1 then click Import to import the Identifiler_Plus_AnalysisMethod_v1 into the GeneMapper ID Software database Import Analysis Method Look in la Identifiler Plus Analysis Files GMID i2 SO 3 EJ E CE_G5_Identifiler_Plus_GS500 xml zj Identifiler Plus _AnalysisMethod_v1 xml re My Recent D Desktop S My Documents File name identifier Plus AnalysisMethod v1 xml Import er Files of type mt Files xml v Cancel To view the settings for Identifiler Plus_AnalysisMethod_v1 select the Analysis Methods tab then select Identifiler_Plus_AnalysisMethod in the Name column and click Open 52 AmpFtSTR Identifiler Plus User Guide Set up GeneMapper ID Software for data analysis 7 GeneMapper Manager E Projects Analysis Methods Table Settings Plot Settings Matrices Size Standards Mame Default Last Saved Owner 2009 08 31 11 27 45 695 gmid Instrument Analysis Type HID Identifiler Plus AnalysisMethod v1 2009 09 02 11 13 05 191 gmid HID New Open Save f45 Import Export s Delete Done Figure 5 Analysis Method Editor HID General tab settings Figures 6 through 9 below show the settings for each tab of the Ana
43. 14 2 292 39 292 5 0 043 0 057 15 294 48 294 59 0 029 0 049 16 298 57 298 69 0 035 0 053 17 302 69 302 81 0 039 0 056 18 306 83 306 95 0 039 0 056 19 310 96 311 07 0 034 0 049 20 315 08 315 18 0 036 0 045 21 319 2 319 31 0 036 0 045 22 323 39 323 5 0 03 0 044 23 327 46 327 53 0 025 0 052 24 331 59 331 65 0 026 0 043 25 335 69 335 76 0 032 0 043 26 339 8 339 88 0 036 0 047 27 343 87 343 99 0 031 0 048 AmpFtSTR Identifiler Plus User Guide 85 Chapter 5 Experiments and Results Table 4 Precision results of five runs 16 capillaries run of the AmpF STR Identifiler Plus Allelic Ladder continued Applied Biosystems 3130x Genetic Analyzer Allele Mean Standard Deviation D19S433 9 101 25 101 34 0 022 0 034 10 105 16 105 25 0 028 0 037 11 109 09 109 17 0 021 0 033 12 113 04 113 12 0 024 0 036 12 2 115 06 115 13 0 027 0 035 18 117 02 117 09 0 026 0 036 13 2 119 03 119 1 0 027 0 038 14 121 02 121 07 0 025 0 038 14 2 123 05 123 1 0 028 0 037 15 125 03 125 09 0 03 0 041 15 2 127 08 127 13 0 027 0 04 16 129 08 129 13 0 031 0 039 16 2 131 13 131 19 0 023 0 042 17 133 16 133 21 0 034 0 046 17 2 135 23 135 28 0 034 0 041 D21S11 24 184 41 184 46 0 024 0 042 24 2 186 39 186 45 0 025 0 04 25 188 35 188 4 0 024 0 038 26 192 27 192 34 0 027 0 035 27 196 21 196 28 0 024 0 039 28 200 06 200 13 0 026 0 039 28 2 20
44. 17 0 16 0 15 0 14 0 13 0 12 0 11 0 10 0 9 0 8 0 7 0 eye Percent Stutter 6 0 5 0 e 4 0 J 30 x 1 0 see c comm o so e ne o comu o ox 9 co o mean e 4 compe LE sss c SE e 00 a9 00 e m ce meoo 00 00 1 RR oe ien ee ZAR 11121314 1516 17 18 1920 567891011 8 9 101112131415 8 9 1011121314 15 16 17 18 1920 21222324 25262728 D3S1358 THO1 D13S317 D16S539 D2S1338 Figure 20 Stutter percentages for the D3S1358 TH01 D13S317 D16S539 and D2S1338 loci 19 0 18 0 17 0 16 0 15 0 14 0 13 0 12 0 11 0 LA 10 0 a 9 0 Percent Stutter 8 0 7 0 6 0 e 50 4 0 3 0 1 0 1 A A RR 9 10111213 1415 16 17 18 111213 14 15 16 17 18 19 20 212223 6 7 8 9 101112 10 11121314 15 16 17 18 1920 2122 2324 2526 D19S433 vWA TPOX D18S51 Figure 21 Stutter percentages for the D19S443 vWA TPOX and D18S51 loci AmpFtSTR Identifiler Plus User Guide 93 Chapter 5 Experiments and Results 94 ciii 5 as maam RE a E H Li t cob e H e HE SR Li MEM 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 3132 33 34 35 42 43 44 45 46 47 48 49 50 51 52 53 D5S818 FGA Figure 22 Stutter percentages for the D5S818 and FGA loci Table 5
45. 1PO 6 303 99 304 12 0 041 0 063 7 308 04 308 17 0 037 0 058 8 312 1 312 2 0 039 0 065 9 316 13 316 25 0 035 0 045 10 320 18 320 3 0 034 0 055 11 324 24 324 34 0 03 0 046 12 328 3 328 39 0 025 0 047 13 332 36 332 44 0 032 0 037 14 336 39 336 49 0 024 0 039 15 340 42 340 53 0 038 0 05 D13S317 8 216 36 216 48 0 031 0 064 9 220 34 220 48 0 035 0 051 10 224 32 224 45 0 034 0 059 11 228 31 228 45 0 031 0 065 12 282 42 32 55 0 031 0 063 13 236 3 236 43 0 038 0 066 14 240 24 240 37 0 043 0 058 15 244 23 244 37 0 037 0 066 AmpFtSTR Identifiler Plus User Guide Table 4 Precision results of five runs 16 capillaries run of the Accuracy precision and reproducibility AmpF STR Identifiler Plus Allelic Ladder continued Applied Biosystems 3130x Genetic Analyzer Allele Mean Standard Deviation D16S539 5 252 01 252 15 0 05 0 06 8 264 264 15 0 05 0 061 9 268 268 14 0 05 0 063 10 272 272 15 0 045 0 059 11 276 02 276 17 0 04 0 064 12 280 03 280 18 0 039 0 067 18 284 05 284 22 0 045 0 06 14 288 08 288 23 0 044 0 054 15 292 12 292 26 0 038 0 059 D18S51 7 261 8 261 9 0 037 0 049 9 269 94 270 03 0 037 0 051 10 274 02 274 12 0 043 0 051 10 2 276 03 276 13 0 037 0 048 11 278 11 278 22 0 042 0 059 12 282 2 282 29 0 037 0 046 13 286 29 286 39 0 039 0 051 13 2 288 29 288 4 0 034 0 052 14 290 38 290 49 0 032 0 051
46. 2 03 202 1 0 026 0 036 29 204 02 204 09 0 025 0 044 29 2 206 08 206 14 0 027 0 041 30 208 06 208 11 0 028 0 04 30 2 210 03 210 09 0 031 0 037 31 212 04 212 12 0 031 0 037 31 2 214 03 214 1 0 023 0 04 32 216 04 216 11 0 028 0 042 32 2 218 03 218 09 0 023 0 0351 33 220 05 220 1 0 031 0 043 33 2 221 98 222 05 0 033 0 038 34 224 12 224 18 0 024 0 033 34 2 226 03 226 09 0 029 0 041 86 AmpFtSTR Identifiler Plus User Guide Table 4 Precision results of five runs 16 capillaries run of the Accuracy precision and reproducibility AmpFfSTR Identifiler Plus Allelic Ladder continued Applied Biosystems 3130x Genetic Analyzer Allele Mean Standard Deviation 35 228 1 228 17 0 03 0 048 35 2 230 03 230 09 0 03 0 037 36 232 02 232 09 0 03 0 047 37 236 08 236 17 0 026 0 041 38 240 04 240 1 0 033 0 045 D2S1338 15 306 27 306 39 0 033 0 058 16 310 35 310 47 0 031 0 055 17 314 39 314 53 0 029 0 042 18 318 45 318 58 0 029 0 046 19 322 52 322 63 0 025 0 046 20 326 58 326 67 0 029 0 039 21 330 66 330 74 0 034 0 045 22 334 71 334 8 0 031 0 043 23 338 74 338 85 0 026 0 045 24 342 75 342 89 0 026 0 05 25 346 78 346 92 0 026 0 051 26 350 77 350 89 0 028 0 049 27 354 69 354 81 0 026 0 045 28 358 87 359 01 0 028 0 045 D3S1358 12 111 12 111 22 0 024 0 047 13 115 23 115 32 0 03 0 046 14 119 2 119 31 0 03
47. 30 3130x instruments 37 Set up the 3100 3100 Avant or 3130 3130x instrument for electrophoresis 37 Prepare samples for electrophoresis on the 3100 3100 Avant or 3130 3130xLinstrument s w SY a Ak a d hene e Wa OE adage BATE a ER DE sh 38 Section 3 2 310 instrument kk kk kk KK KK KK RR RR KK KK KK KK IK kk 39 Set up the 310 instrument for electrophoresis kk kk KK KK KK RR KK RR KK KK 39 Prepare samples for electrophoresis on the 310 instrument LL 40 AmpFtSTR Identifiler Plus User Guide 3 Contents Chapter 4 Chapter 5 Appendix A Appendix B Data Analysis c i ataca c a RACER n RAO BUE Ea A ged EIN 45 Section 4 1 GeneMapper ID Software 00 0c ccc eee RR KK KK KS 46 Overview of GeneMapper ID Software uuuaauaaaaaaaaaaaaaaaa KK KK KK 46 Set up GeneMapper ID Software for data analysis n on cece eee eens 47 Analyze and edit sample files with GeneMapper ID Software 57 Examine and edita project lk kk kk kk kk kk KK KK KK KK KK eee 58 Section 4 2 GeneMapper D X Software llle 60 Overview of GeneMapper D X Software WW kk kK KK KK KK KK KK K K KK eens 60 Set up GeneMapper D X Software for data analysis aa4444111111 61 Analyze and edit sample files with GeneMapper D X Software 73 Examine and edit a project kk kk kk kk kk kK kk KK KK ee 74 Experiments and Results aaaaaaa
48. 33 MicroAmp Optical 96 Well Reaction Plate N8010560 18 AmpFtSTR Identifiler Plus User Guide Table 3 User supplied materials continued Materials and equipment Item Source 250 UL Glass Syringe array fill syringe 4304470 5 0 mL Glass Syringe polymer reserve syringe 628 3731 For a complete list of parts and accessories for the 3100 instrument refer to Appendix B of the ABI PR sM 3100 Genetic Analyzer and 3100 Avant Genetic Analyzer User Reference Guide PN 4335393 3130 3130x Analyzer materials 96 Well Plate Septa 4315933 Reservoir Septa 4315932 3100 3130x Genetic Analyzer Capillary Array 36 cm 4315931 POP 4 Polymer for 3130 3130x Genetic Analyzers 4352755 3130 3130x Genetic Analyzer Autosampler Plate Kit 96 well 4316471 GeneScan 500 LIZ Size Standard 4322682 Running Buffer 10X 402824 DS 33 Matrix Standard Kit Dye Set G5 4345833 MicroAmp Optical 96 Well Reaction Plate N8010560 For a complete list of parts and accessories for the 3130x instrument refer to Appendix A of the Applied Biosystems 3130 3130xl Genetic Analyzers Maintenance Troubleshooting and Reference Guide PN 4352716 310 Analyzer materials 310 DNA Analyzer Capillary Array 47 cm 402839 0 5 mL Sample Tray 5572 96 Well Tray Adaptor for 9700 thermal cycler trays 4305051 GeneScan 500 LIZ Size Standard 4322682 Running Buffer
49. 4 25 19 23 26 27 28 D19S433 19q12 13 1 9 10 11 12 12 2 13 13 2 14 14 2 15 NED 14 15 15 2 16 16 2 17 17 2 vWA 12p12 pter 11 12 13 14 15 16 17 18 19 20 21 22 17 18 23 24 TPOX 2p23 2per 6 7 8 9 10 11 12 13 gt D18S51 18q21 3 7 9 10 10 2 11 12 13 13 2 14 14 2 15 15 19 16 17 18 19 20 21 22 23 24 25 26 27 Amelogenin X p22 1 22 3 X Y PET X Y p11 2 D58818 5q21 31 7 8 9 10 11 12 13 14 15 16 115 FGA 4q28 17 18 19 20 21 22 23 24 25 26 26 2 23 24 27 28 29 30 30 2 31 2 32 2 33 2 42 2 43 2 44 2 45 2 46 2 47 2 48 2 50 2 51 2 For CODIS purposes profile reported as 13 13 For CODIS purposes profile reported as 30 30 For CODIS purposes profile reported as 11 11 For CODIS purposes profile reported as 8 8 For CODIS purposes profile reported as 11 11 AmpFtSTR Identifiler Plus User Guide 11 Chapter 1 Overview Allelic ladder Figure 1 shows the allelic ladder for the AmpF STR Identifiler Plus Kit See Allelic profile ladder requirements on page 36 for information on ensuring accurate genotyping EB e m j m L Mark Sample for Deletion 130 170 210 250 290 330 370 1200 800 400 0 J_ 2 3 2 2 2 2 2 2 2 2 SA PAR EBR BAB RA SBASASAAAAS ABARARBRARAA Es bJ bo fa 2 l 16 17 18 15 EALE Ee Eo aps E s s Es Is E E ed hejl ia l z l l
50. 7001 800 5004 400 300 f 200 400 o 30 600 10 1 500 400 300 200 100 oks 30 6000 u 15 1 ARAM LH ALLJ j EN SN M hha A Lala Nak 130 170 210 250 290 330 5000 0 1 4000 3000 2000 1000 0 Figure 31 Amplification of DNA mixtures at various ratios using the 29 PCR cycle protocol Minor allele peaks that do not overlap with the major contributor peaks are highlighted Table 9 Genotypes of mixed DNA samples Locus Profile Sample A Profile Sample B D881179 11 14 10 11 D21S11 29 35 31 2 32 2 AmpFtSTR Identifiler Plus User Guide 109 Chapter 5 Experiments and Results Table 9 Genotypes of mixed DNA samples continued Locus Profile Sample A Profile Sample B D7S820 8 10 11 CSF1PO 8 10 12 D3S1358 14 17 15 16 THO1 7 8 6 9 3 D138317 12 13 9 11 D16S539 10 11 9 12 D281338 17 23 17 20 D198433 11 17 2 13 VWA 14 17 17 19 TPOX 9 10 8 11 D18S51 15 16 13 14 AMEL X Y X D5S818 11 13 8 10 FGA 19 25 22 23 110 AmpFtSTR Identifiler Plus User Guide Population Data Population Data SWGDAM The distribution of genetic markers in populations should be determined in relevant guideline 2 7 p
51. 8 C 2 weeks 15 to 25 C IMPORTANT Store the amplified products so that they are protected from light 30 AmpFtSTR Identifiler Plus User Guide Amplification using bloodstained FTA cards Amplification using bloodstained FTA cards 2 cycle 3 2800 2000 4600 1200 800 400 FTA cards can be useful for collecting storing and processing biological samples A small punch disc of the card containing the sample can be placed directly into an amplification tube purified and amplified without transferring the disc Our studies indicate that a 1 2 mm bloodstained disc contains approximately 5 to 20 ng DNA An appropriate cycle number for this high quantity of DNA is 24 cycles as determined by our validation studies However it is recommended that each laboratory determine the optimum cycle number based on internal validation studies In the example shown in Figure 4 a 1 2 mm disc of a bloodstained FTA card was purified using three washes with FTA Purification Reagent and two washes with 1X low TE buffer The purified punch disc was then amplified in the MicroAmp tube for 24 cycles m B T Mark Sample for Deletion 155 195 235 275 315 355 395 A L I An M Figure 4 AmpF STR Identifiler Plus PCR Amplification Kit results from a 1 2 mm FTA bloodstain disc 24 cycle amplification analyzed on the Applied Biosystems 3130x Genetic Analyzer AmpFtSTR Iden
52. 9 Moller A Meyer E and Brinkmann B 1994 Different types of structural variation in STRs HumFES FPS HumV WA and HumD21S11 Intl J Legal Med 106 319 323 Momhinweg E Luckenbach C Fimmers R and Ritter H 1998 D3S1358 sequence analysis and gene frequency in a German population Forensic Sci Int 95 173 178 Moretti T Baumstark A Defenbaugh D Keys K Smerick J and Budowle B 2001 Validation of short tandem repeats STRS for forensic usage Performance testing of fluorescent multiplex STR systems and analysis of authentic and 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 National Research Council 1996 The evaluation of forensic DNA evidence National Academy Press Washington D C Nei M 1978 Estimation of average heterozygosity and genetic distance from a small number of individuals Genetics 89 583 590 Nei M 1973 Analysis of gene diversity in subdivided populations Proc Natl Acad Sci USA 70 3321 3323 Revised Validation Guidelines Scientific Working Group on DNA Analysis Methods SWGDAM Forensic Science Communications July 2004 Volume 6 3 Available at www fbi g
53. Applied Biosystems 3130x Genetic Analyzer The recommended method for genotyping is to employ a 0 5 nt window around the size obtained for each allele in the AmpF STR Identifiler Plus Allelic Ladder A 0 5 nt window allows for the detection and correct assignment of alleles Any sample allele that sizes outside the specified window could be e An off ladder allele that is an allele of a size that is not represented in the AmpF STR Identifiler Plus Allelic Ladder or e An allele that does correspond to an allelic ladder allele but whose size is just outside a window because of measurement error The measurement error inherent in any sizing method can be defined by the degree of precision in sizing an allele multiple times Precision is measured by calculating the standard deviation in the size values obtained for an allele that is run in several injections on a capillary instrument or in several lanes of one gel Table 4 on page 84 shows typical precision results obtained from five runs 16 capillaries run of the AmpF STR Identifiler Plus Allelic Ladder on the Applied Biosystems 3130x Genetic Analyzer 36 cm capillary and POP 4 polymer The internal size standard that was used was GeneScan 500 LIZ Size Standard The results were obtained within a set of injections on a single capillary array Sample alleles may occasionally size outside of the 0 5 nt window for a respective allelic ladder allele be
54. E a eR d d Ae 10 B Workflow overview L lle 14 B Instrument and software overview uaeaeaea aaa aaa aaa aaa 15 EM Materials and equipment aaaaeaaaaaa aaa aaa eh 17 AmpFtSTR Identifiler Plus User Guide 9 Chapter 1 Overview Product overview Purpose Product description About the primers 10 The AmpF STR Identifiler Plus PCR Amplification Kit is a short tandem repeat STR multiplex assay that amplifies 15 tetranucleotide repeat loci and the Amelogenin gender determining marker in a single PCR amplification All thirteen of the required loci for the Combined DNA Index System CODIS loci are included in this kit for known offender databasing in the United States Budowle et al 1998 Two additional loci D281338 and D19S433 are included These loci are consistent with the AmpF STR SGM Plus PCR Amplification Kit The combination of the 15 loci are consistent with several worldwide database recommendations The AmpF STR Identifiler Plus Kit delivers a 16 locus multiplex with the same power of discrimination as better sensitivity than and better robustness than the earlier generation of the AmpF STR Identifiler Kit The kit uses modified PCR cycling conditions for enhanced sensitivity a new buffer formulation to improve performance with inhibited samples and an improved process for DNA synthesis and purification of the amplification primers to deliver a much cleaner electrophoretic backgroun
55. JD Software database Refer to step 1 on page 48 for downloading instructions 1 Select Tools GeneMapper Manager to open the GeneMapper Manager 2 Import a size standard a Select the Size Standards tab then click Import GeneMapper Manager Name Last Save owner frype _ pescrpton HOSS Qmoeceo iius _ emseamcei tory ona e Jesse masa stajni pese Factory one Jego ss O aA o pase Factory rowed b Navigate to then open the Identifiler Plus Analysis Files GMID folder 56 AmpFtSTR Identifiler Plus User Guide Analyze and edit sample files with GeneMapper ID Software c Select CE_G5_Identifiler_Plus_GS500 then click Import to import the CE_G5_Identifiler_Plus_GS500 size standard into the GeneMapper D Software database Import Size Standard Method xi Look in le Identifiler Plus Analysis Files GMID z 2 PIE Fe ej CE GS Identifiler Plus GS500 xml Idertifiler_Plus_AnalysisMethod_v1 xml My Recent D O oO Desktop D go o My Documents cM jg U e icp e File name CE_G5_ldentifiler_Plus_GS500 xmi Import Ej 3 e Files of type jew Files xml v Cancel d Analyze and edit sample files with GeneMapper D Software Analyze a project 1 In the Project window select File Add Samples to Project then navigate to the disk or directory containing the sample files 2 Apply analysis settings to the samples in the project Para
56. K KK KK 124 Probability of Paternity Exclusion 00002 cee KK KK KK KK 125 AmpFtSTR Identifiler Plus User Guide 77 Chapter 5 Experiments and Results Overview Experiments using the AmpF STRS Identifiler Plus Kit Importance of validation Experiment conditions 78 This chapter provides results of the developmental validation experiments performed using the AmpF STR Identifiler Plus PCR Amplification 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 which are critical for sound data interpretation in casework Sparkes Kimpton Watson et al 1996 Sparkes Kimpton Gilbard et al 1996 and Wallin et al 1998 Experiments to evaluate the performance of the AmpF STR Identifiler Plus PCR Amplification Kit were performed The experiments were performed according to the DNA Advisory Board DAB Quality Assurance Standards effective October 1 1998 DNA Advisory Board 1998 The DAB standards describe the quality assurance requirements that a laboratory should follow to ensure the quality and integrity of the data and competency of the laboratory Additional validation was performed according to the revised guidelines from th
57. Marker specific stutter filter percentages for AmpF STR Identifiler Plus PCR Amplification Kit loci Locus Stutter CSF1PO 9 2041 D13S317 9 9348 D16S539 10 3945 D18S51 13 6799 D19S433 11 2096 D21S11 10 6714 D281338 12 4409 D381358 12 2719 D5S818 10 0599 D7S820 9 6926 D8S1179 10 3155 FGA 13 028 THO1 4 0813 AmpFtSTR Identifiler Plus User Guide Extra peaks in the electropherogram Table 5 Marker specific stutter filter percentages for AmpF STR9 Identifiler Plus PCR Amplification Kit loci continued Locus 9o Stutter TPOX 6 3832 VWA 12 446 t These percentages are used as stutter filters in used in GeneMapper ID v3 2 1 Identifiler_Plus_Panels_v1 and GeneMapper D X software v1 0 1 v1 1 or v1 1 1 Identifiler_Plus_Panels_v1x Addition of 3 A nucleotide Many DNA polymerases can catalyze the addition of a single nucleotide predominately adenosine to the 3 ends of double stranded PCR products Clark 1988 and Magnuson et al 1996 This nontemplate addition results in a PCR product that is one nucleotide longer than the actual target sequence The PCR product with the extra nucleotide is referred to as the A form The efficiency of A addition is related to the particular sequence of the DNA at the 3 end of the PCR product The Identifiler Plus Kit includes two main design features that promote maximum A addition The prim
58. NA in water or wrong buffer for example TE formula with incorrect EDTA concentration Redilute DNA using low TE Buffer with 0 1 mM EDTA More than one allele present at a locus Some but not all loci visible on electropherogram Presence of exogenous DNA Use appropriate techniques to avoid introducing foreign DNA during laboratory handling Amplification of stutter product Mixed sample See Stutter products on page 90 Incomplete 3 A base addition n 1 nt position See Addition of 3 A nucleotide on page 95 Be sure to include the final extension step of 60 C for 10 min in the PCR Signal exceeds dynamic range of instrument off scale data Poor spectral separation bad matrix Ensure cycle number is optimized according to instructions on page 30 Repeat PCR amplification using fewer PCR cycles or use your laboratory s SOP to analyze off scale data Follow the steps for creating a spectral file Confirm that Filter Set G5 modules are installed and used for analysis Too much DNA in reaction Test sample DNA is severely degraded Use recommended amount of template DNA 1 0 ng If possible evaluate the quality of DNA sample by running an agarose gel If DNA is degraded reamplify with an increased amount of DNA or use the AmpF STR MiniFiler Kit Test sample contains high concentrations of a PCR inhibitor for example heme compounds certain dyes Quantit
59. Peak Height MPH 8000 0 Peak Height Ratio PHR Min peak height ratio fo 7 r Broad Peak BD Max peak width basepairs Allele Number AN Max expected alleles Allelic Ladder Spike Cut off value Factory Defaults i Save As Save Cancel Help Figure 13 Analysis Method Editor Peak Quality tab settings IMPORTANT Laboratories need to perform the appropriate internal validation studies to determine the minimum heterozygous and homozygous minimum peak height thresholds and the minimum peak height ratio threshold that allow for reliable interpretation of AmpF STR Identifiler Plus PCR Amplification Kit data 70 AmpFtSTR Identifiler Plus User Guide Set up GeneMapper ID X Software for data analysis Analysis Method Editor xl General Allele Peak Detector Peak Quality SQ amp GQ Settings Quality weights are between and 1 r Sample and Control GQ Weighting Broad Peak BD as Allele Number AN Jo Out of Bin Allele BIN fos Low Peak Height LPH ps Overlap OYL fs Max Peak Height MPH fos Marker Spike SPK NEN Off scale 05 os Peak Height Ratio PHR os Control Concordance CC Weight 1 0 Only applicable to controls r SQ Weighting Broad Peak BD os Allelic Ladder GQ Weighting Spike SSPK SPK h gt Off scale 05 fi SQ amp GQ Ranges Sizing Quality From 7s to 1 0 Fro
60. Plus Allelic Ladder including microvariants have been subjected to sequencing In addition other groups in the scientific community have sequenced alleles at some of these loci Nakahori ef al 1991 Puers et al 1993 Moller et al 1994 Barber et al 1995 Brinkmann and Moller 1995 Barber et al 1996 Barber and Parkin 1996 Brinkmann et al 1998 Momhinweg et al 1998 Watson et al 1998 Among the various sources of sequence data on the AmpF STR Identifiler Plus Kit loci there is consensus on the repeat patterns and structure of the STRs Inheritance The Centre d Etude du Polymorphisme Humain CEPH has collected DNA from families of Utah Mormon French Venezuelan and Amish descent These DNA sets have been extensively studied all over the world and are routinely used to characterize the mode of inheritance of various DNA loci Each family set contains three generations generally including four grandparents two parents and several offspring Consequently the CEPH family DNA sets are ideal for studying inheritance patterns Begovich et al 1992 Three CEPH family DNA sets were examined One nanogram of DNA from each sample was amplified using the Identifiler Plus Kit followed by analysis using an Applied Biosystems 3130x Genetic Analyzer The families examined included 1333 9 offspring 1340 7 offspring and 1345 7 offspring representing 23 meiotic divisions In family 1340 we observed two parent offsprin
61. Plus User Guide DNA quantification Product Description References Quantifiler HP DNA Quantification Kit Cat no 4482911 and Quantifiler Trio DNA Quantification Kit Cat no 4482910 Properties The Quantifiler HP Kit is designed to quantify the total amount of amplifiable human DNA in a sample The Quantifiler Trio Kit is designed to simultaneously quantify the total amount of amplifiable human DNA and human male DNA in a sample How it works The Quantifiler HP and Trio DNA Quantification Kits use multiple copy target loci for improved detection sensitivity The human specific target loci Small Autosomal Large Autosomal and Y chromosome targets each consist of multiple copies dispersed on various autosomal chromosomes Small Autosomal and Large Autosomal or multiple copies on the Y chromosome Quantifiler Trio Kit only To maximize the consistency of quantification results genomic targets were selected with conserved primer and probe binding sites within individual genomes and also with minimal copy number variability between different individuals and population groups As a result the detection sensitivity of the Quantifiler HP and Trio assays is improved over Quantifiler Duo Human and Y Human Male DNA Quantification Kit assays The primary quantification targets Small Autosomal and Y consist of relatively short amplicons 75 to 80 bases to improve the detection of degraded DNA s
62. R Identifiler Plus Allelic Ladder and GeneScan 500 LIZ Size Standard should also be protected from light Keep freeze thaw cycles to a minimum The following table lists Data Collection Software and the run modules that can be or 3130 3130x used to analyze Identifiler Plus PCR products For details on the procedures refer instrument to the documents listed in the table requirements Operatin pan l stem Collection Run modules and conditions References y Software Windows 3 0 e HiDFragmentAnalysis36 POPA 1 Applied Biosystems9 3130 3130xl Genetic XP 8130 3130xl Injection conditions Analyzers Using Data Collection Software v3 0 Analyzer Protocols for Processing AmpF STR PCR 3130 3 kV 5 sec Amplification Kit PCR Products User Bulletin 3130x 3 kV 10 sec PN 4363787 e Dye Set G5 Windows 2 0 e HIDFragmentAnalysis36_POP4_1 ABI PRisM 3100 3100 Avant Genetic 2000 3100 Injection condition 3 kV 10 sec Analyzers Using Data Collection Software v2 0 Analyzer e D G Protocols for Processing AmpFISTR PCR ye Set G5 Amplification Kit PCR Products User Bulletin PN 4350218 Windows 1 1 e GeneScan36vb DyeSetG5Module ABI PRISM 3100 3100 Avant Genetic NT 3100 Injection condition 3 kV 10 sec Analyzers Protocols for Processing AmpF STR Analyzer e GS500Analvsi PCR Amplification Kit PCR Products User S00Analysis gsp Bulletin PN 4332345 1 0 e GeneScan36Avb DyeSetG5Module ABI PRISM 3100 3100 Avant Gene
63. R Identifiler Plus User Guide Contents Documentation 2 3 ru ae Sonos ee Ak yay Dk Ems 141 Related documentation messaer atakehin end Wl lal b d A A Wn EN an iii 141 How to obtain support LLL a ha Wa a ak kk lay kk aaa eee 142 Limited product warranty sucres ak ka reniei a Kala k la kk kb Wa kk Wl e WE di 142 lel SEE MR TER 143 AmpFtSTR Identifiler Plus User Guide 5 Contents 6 AmpFtSTR Identifiler Plus User Guide Preface Revision history Revision Date Description A September 2008 New document B September 2010 Remove patent numbers C April 2011 Remove patent numbers D March 2012 Update legal information E August 2014 Add information about 50 100 and 1000 reaction kit sizes F February 2015 Add information for the ProFlex PCR System Purpose The Applied Biosystems AmpFtSTR Identifiler Plus PCR Amplification Kit User Guide provides information about the Applied Biosystems instruments chemistries and software associated with the AmpF STR Identifiler Plus PCR Amplification Kit 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 number and title AmpFtSTR Identifiler Plus User Guide Preface 8 AmpFtSTR Identifiler Plus User Guide Overview This chapter covers M PrOdUC OVETVIEW sacs Ua A oO Dee RO Reed O
64. TTTTTTTTTTTTTTTTTTTTT loci is not a novel methodology Holt et al 2000 and Wallin et al 2002 However accuracy and reproducibility of AmpF STR Identifiler Plus profiles have been determined from various sample types Figure 18 shows the size differences that are typically observed between sample alleles and allelic ladder alleles on the Applied Biosystems 3130x Genetic Analyzer with POP 4 polymer The x axis in Figure 18 represents the nominal nucleotide sizes for the AmpF STR Identifiler Plus Allelic Ladder The dashed lines parallel to the x axis represent the 0 25 nt windows The y axis represents the deviation of each sample 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 0 50 CSF1PO D2S1338 D3S1358 D5S818 D7S820 D8S1179 D13S317 D168539 D18S51 D19S433 D21S11 FGA THO1 TPOX vWA AMEL 0 u O O 0 t gt gt O O 100 120 140 160 180 200 220 240 260 280 300 320 340 360 Allele Size nt Figure 18 Size deviation of 200 samples analyzed on the Applied Biosystems 3130x Genetic Analyzer For each sample 1 0 ng of DNA was amplified for 28 PCR cycles AmpFtSTR Identifiler Plus User Guide Accuracy precision and reproducibility Precision and Sizing precision allows for determining accurate and reliable genotypes Sizing size windows precision was measured on the
65. USER GUIDE applied biosystems kije technologies AmpF STR Identifiler Plus PCR Amplification Kit User Guide Publication Number 4440211 Revision F o For Forensic or Paternity Use Only technologies Information in this document is subject to change without notice DISCLAIMER LIFE TECHNOLOGIES CORPORATION AND OR ITS AFFILIATE S DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT TO THE EXTENT ALLOWED BY LAW IN NO EVENT SHALL LIFE TECHNOLOGIES AND OR ITS AFFILIATE S BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF Important Licensing Information These products may be covered by one or more Limited Use Label Licenses By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses TRADEMARKS All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified 2015 Thermo Fisher Scientific Inc All rights reserved TaqMan is a registered trademark of Roche Molecular Systems Inc used under permission and license Microsoft and Excel are registered trademarks of Microsoft Corporation Adobe Acroba
66. aaa aaa iii 77 OVerVIGW 4 u GN DR a eee Te eae SETA RU Rt Ae ian zer EE At elle 78 Developmental validation kk kk kk kk kk kK kK KK KK KK ee 79 Accuracy precision and reproducibility kk kk kk kK kK KK KK ee 82 Extra peaks in the electropherogram 00 kk kk KK KK KI KIR K K KK KK KI KK KK KK IK 90 Characterization of loci kk kk kk kk kk kK KK KK eee 97 Species Specificity yata xn eon tg rcl ye her AE eer e av ae ye e e des 98 Sensitivity sx Mo Naty a Eye xd cae thes eat peu ae la we IRA ka a s Baraye a 100 Stability ww ET 102 Mixt re studies oe bawia uA was a a HH HE HHHH Heg 105 Population Data sta kn Aya wa WA ER Lente es Gov b l exstet EV aus 111 Mutatio iRate sek Ab dn swaps EE RE baat DSi ees Sto 123 Probability of Identity Sk osad Oss eek dn ale adw dop C An DO A 124 Probability of Paternity Exclusion LX kk kk kk kK kK kK KK KI K K KOK K KIRI KI K KOK K KK KI KK KK kk 125 Troubleshooting aaa aaa KK KK aaa KIR KIR KK KIR k 127 Sdloty caos pu eis AA quet fue ee hae E EPI I eio adi 131 Chemical safety osuere eben RUE wets kS L dei Baw aes 132 Chemical Wast safety isses neve pem S eee eee bee aaa e na E nal 134 Biological hazard safety yes nn kla AW DE KEKE V WE QA KA dik i Sk E A D k z 135 Chemical alent tiii 550 Kan An ee daka a ak ke ERR SEA AA O AE i lad 136 Bibliography Ga kk kk kk kK kK KK KK KK EE DIM SE 137 AmpFtST
67. able 5 on page 94 was determined by taking the mean plus three times the standard deviation These values are the stutter filter percentages in the Identifiler Plus stutter file they will be used during the filtering step in the GeneMapper ID Software v3 2 1 or GeneMapper ID X Software v1 0 1 v1 1 or v1 1 1 Peaks in the stutter position that are above the stutter filter percentage will not be filtered Peaks in the stutter position that have not been filtered and remain labeled can be further evaluated For evaluation of mixed samples see Figure 30 on page 108 The percent stutter cannot be accurately measured for allele peaks that are off scale and may appear unusually high relative to the main peak AmpFtSTR Identifiler Plus User Guide 91 Chapter 5 Experiments and Results 92 er 5 St Percent 20 0 19 0 18 0 17 0 16 0 15 0 14 0 13 0 12 0 11 0 10 0 9 0 8 0 70 6 0 5 0 4 0 3 0 2 0 1 0 0 0 e e twe e comme wm o o ee e c ee s ce t t E a 1 I 8 9 10 1112 13 14 15 16 17 24 25 26 27 28 29 30 31 32 33 34 35 36 6 7 8 9 10 1112 13 14 15 7 8 9 10 1112 13 14 15 D8S1179 D21S11 D7S820 CSF1PO Figure 19 Stutter percentages for the D8S1179 D21S11 D7S820 and CSF1PO loci AmpFtSTR Identifiler Plus User Guide Extra peaks in the electropherogram 19 0 18 0
68. aboratories American Association of Blood Banks 7th edition 2004 The sensitivity of the AmpF STR Identifiler Plus Kit and other PCR based tests enables amplification of minute quantities of DNA necessitating precautions to avoid contamination of samples yet to be amplified Kwok and Higuchi 1989 To prevent contamination by human DNA be careful while handling and processing samples Wear gloves at all times and change them frequently Close sample tubes when not in use Limit aerosol dispersal by handling sample tubes and reagents carefully Note These laboratory design resources and guidances constitute only a sample of the precautions that need to be observed when using PCR technology Refer to your laboratory s internal policies and procedures for additional information and references IMPORTANT These items should never leave the PCR setup work area Calculator Gloves disposable Marker pen permanent Microcentrifuge Microcentrifuge tubes 1 5 mL or 2 0 mL or other appropriate clean tube for Master Mix preparation Microcentrifuge tube rack Pipette tips sterile disposable hydrophobic filter plugged Pipettors Tube decapper autoclavable Vortex Amplified DNA The following PCR systems should be placed in the amplified DNA work area work area tools 24 GeneAmp PCR System 9700 with the Silver 96 Well Block GeneAmp PCR System 9700 with the Gold plated Silver 96 Well Blo
69. acid amplification of 1 ng of Control DNA 9947A with the Identifiler Plus Kit for 28 cycles of amplification see Figure 29 The concentrations of humic acid tested were 0 50 100 and 150 ng uL see Table 7 9947A b m L m C m 7 Mark Sample for Deletior 2000 Control samples HASO b j w m m I SI Mark Sample for Deletior sooo 50 ng uL HA100 5 z jm B B jm J Mark Sample for Deletior 2000 100 ng uL HA160 b ja m ma In jm IT Mark Sample for Deletior 4000 3000 2000 1000 104 Figure 29 Amplification with the AmpF STR Identifiler Plus PCR Amplification Kit in the presence and absence of humic acid Panel 1 corresponds to control samples panels 2 4 correspond to samples amplified in the presence of 50 100 and 150 ng L humic acid AmpFtSTR Identifiler Plus User Guide Mixture studies Table 7 Performance in simulated model of humic acid inhibition n 3 Humic Acid ng uL Identifiler Plus Kit 0 26 26 26 26 26 26 50 26 26 26 26 26 26 100 26 26 26 26 26 26 150 26 26 26 26 26 26 t Only those peaks gt 50 RFUs were counted A complete profile with Control 9947A DNA yields 26 peaks using the AmpFZSTR Identifiler Plus Kit Mixture studies SWGDAM The ability to obtain reliable results from mixed source samples should be guideline 2 8 determined SWGDAM July 2003 Evidence samples may contain DNA from more than one individual The possibility of
70. alcon MLS Tube decapper autoclavable MLS Deionized water 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 20 AmpFtSTR Identifiler Plus User Guide Chapter 2 PCR Amplification AmpFtSTR Identifiler Plus User Guide AmpFtSTR Identifiler Plus User Guide PCR Amplification This chapter covers m PCR work areas DNA quantification Perform PCR AmpFtSTR Identifiler Plus User Guide Required user supplied reagents uaaaaaaaaaaaa aaa KK KK KK KK Prepare the amplification kit reactions 00 02 cece eee eee 23 Chapter 2 PCR Amplification PCR work areas Work area setup and lab design PCR setup tools Many resources are available for the appropriate design of a PCR laboratory Ifyou are using the AmpF STR Identifiler Plus PCR Amplification Kit for forensic DNA testing refer to Forensic Laboratories Handbook for Facility Planning Design Construction and Moving National Institute of Justice 1998 http nij ncjrs gov publications pubs db asp Ifyou are using the AmpF STR Identifiler Plus Kit for parentage DNA testing refer to the Guidance for Standards for Parentage Relationship Testing L
71. amples In addition the Quantifiler HP and Trio Kits each contain a Large Autosomal target with a longer amplicon 2200 bases to aid in determining if a DNA sample is degraded Quantifiler HP and Trio DNA Quantification Kits User Guide Pub no 4485354 AmpFtSTR Identifiler Plus User Guide 27 Chapter 2 PCR Amplification Prepare the amplification kit reactions 1 Calculate the volume of each component needed to prepare the reactions using the table below DNA sample Volume per reaction uL AmpF STR Identifiler Plus Master Mix 10 0 AmpF STR Identifiler Plus Primer Set 5 0 Note Include additional reactions in your calculations to provide excess volume for the loss that occurs during reagent transfers 2 Prepare reagents Thaw the AmpF STR Identifiler Plus Kit Master Mix and the AmpF STR Identifiler Plus Kit Primer Set then vortex 3 seconds and centrifuge briefly before opening the tubes IMPORTANT Thawing is required only during first use of the kit After first use reagents are stored at 2 to 8 C and therefore they do not require subsequent thawing Do not refreeze the reagents 3 Pipette the required volumes of components into an appropriately sized polypropylene tube 4 Vortex the reaction mix for 3 seconds then centrifuge briefly 5 Dispense 15 UL of the reaction mix into each reaction well of a MicroAmp Optical 96 Well Reaction Plate or each MicroAmp tube
72. ares 3rd Order Least Squares Cubic Spline Interpolation Local Southern Method C Global Southern Method Factory Defaults Save As Save Cancel Help Figure 12 Analysis Method Editor Peak Detector tab settings The software uses the peak amplitude threshold parameters to specify the minimum peak height to limit the number of detected peaks Although GeneMapper D X Software displays peaks that fall below the specified amplitude in electropherograms the software does not label or determine the genotype of these peaks IMPORTANT Laboratories need to perform the appropriate internal validation studies to determine the peak amplitude thresholds that allow for reliable interpretation of AmpF STR Identifiler Plus PCR Amplification Kit data For more information about peak detection algorithms refer to GeneMapper ID X Software Version 1 0 Getting Started Guide PN 4375574 GeneMapper D X Software Version 1 0 Quick Reference Guide PN 4375670 GeneMapper JD X Software Version 1 0 Reference Guide PN 4375671 AmpFtSTR Identifiler Plus User Guide 69 C D mi D lt ko o 0 8 w gt n 9 zh 2 0 Section 4 2 GeneMapper ID X Software Analysis Method Editor xj General Allele Peak Detector Peak Quality sq amp GQ Settings r Min Max Peak Height LPH MPH Homozygous min peak height 100 0 Heterozygous min peak height Jk Max
73. ate DNA and add minimum necessary volume Repeat test Wash the sample in a Centricon 100 centrifugal filter unit Repeat test 128 AmpFtSTR Identifiler Plus User Guide Table 14 Troubleshooting continued Observation Possible causes Recommended actions ance Poor peak height bal Incorrect thermal cycler parameters Check the protocol for correct thermal cycler parameters GeneAmp PCR System 9700 with Aluminum 96 Well block or third party thermal cyclers Use Applied Biosystems GeneAmp PCR System 9700 with silver or gold plated silver blocks only AmpFtSTR Identifiler Plus User Guide 129 Appendix A Troubleshooting 130 AmpFtSTR Identifiler Plus User Guide Safety This appendix covers m Chemical safety aaa yen beer vH ee AA ed eie doa he 132 m Chemical waste safety W K kk kk KK KK KK KK KK KK KK KK KK KK KK KK KK 134 m Biological hazard safety kk kk kK kK KK KK KK KK KK KK KK KK KK K KK KI KK 135 M Chemical alerts i ss ks k lk k ccc III 136 AmpFtSTR Identifiler Plus User Guide 131 Appendix B Safety Chemical safety Chemical hazard WARNING CHEMICAL HAZARD Before handling any chemicals refer warning to the Material Safety Data Sheet MSDS provided by the manufacturer and observe all relevant precautions T WARNING CHEMICAL HAZARD All chemicals in the instrument including liquid in the l
74. c mapping with trimeric and tetrameric tandem repeats Am J Hum Genet 49 746 756 Edwards A Hammond H A Lin J Caskey C T and Chakraborty R 1992 Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups Genomics 12 241 253 Frank W Llewellyn B Fish P et al 2001 Validation of the AmpF STR Profiler Plus PCR Amplification Kit for use in forensic casework J Forensic Sci 46 642 646 Grossman P D Bloch W Brinson E Chang C C Eggerding F A Fung S Iovannisci D M Woo S and Winn Deen E S 1994 High density multiplex detection of nucleic acid sequences oligonucleotide ligation assay and sequence coded separation Nucleic Acids Res 22 4527 4534 Guo S W and Thompson E A 1992 Performing the exact test of Hardy Weinberg proportion for multiple alleles Biometrics 48 361 372 Hammond H Jin L Zhong Y Caskey C and Chakraborty R 1994 Evaluation of 13 short tandem repeat loci for use in personal identification applications Am J Hum Genet 55 175 189 Holt C Stauffer C Wallin J Lazaruk L Nguyen T Budowle B and Walsh P 2000 Practical applications of genotypic Surveys for forensic STR testing Forensic Sci Int 112 91 109 Kimpton C Walton A and Gill P 1992 A further tetranucleotide repeat polymorphism in the vWF gene Hum Mol Genet 1 287 Kong X Murphy K Raj T He C White
75. cause of measurement error The frequency of such an occurrence is lowest in detection systems having the smallest standard deviations in sizing Figure 18 on page 82 illustrates the tight clustering of allele sizes obtained on the Applied Biosystems 3130x Genetic Analyzer where the standard deviation in sizing is typically less than 0 15 nt The instance of a sample allele sizing outside the 0 5 nt window because of measurement error is relatively rare when the standard deviation in sizing is approximately 0 15 nt or less Smith 1995 For sample alleles that do not size within a 0 5 nt window the PCR product must be rerun to distinguish between a true off ladder allele versus measurement error of a sample allele that corresponds with an allele in the allelic ladder Repeat analysis when necessary provides an added level of confidence to the final allele assignment GeneMapper JD Software and GeneMapper ID X Software automatically flag sample alleles that do not size within the prescribed window around an allelic ladder allele by labelling the allele as OL off ladder Maximum precision is obtained with a set of capillary injections on each of the supported platforms however the determined allele sizes will vary between the different platforms Cross platform sizing differences occur from a number of factors including type and concentration of polymer run temperature and electrophoresis conditions Variations in sizing can also
76. ck Veriti 96 Well Thermal Cycler ProFlex PCR System AmpFtSTR Identifiler Plus User Guide Required user supplied reagents Required user supplied reagents In addition to the AmpF STR Identifiler Plus Kit reagents the use of low TE buffer 10 mM Tris 0 1 mM EDTA pH 8 0 is recommended You can prepare the buffer as described in the procedure below or order it from Teknova Cat T0223 To prepare low TE buffer 1 Mix together 10mL of 1 M Tris HCl pH 8 0 0 2 mL of 0 5 M EDTA pH 8 0 990 mL glass distilled or deionized water Note Adjust the volumes accordingly for specific needs 2 Aliquot and autoclave the solutions 3 Store at room temperature DNA quantification Importance of Quantifying the amount of DNA in a sample before amplification allows you to quantification determine whether or not sufficient DNA is present to permit amplification and to calculate the optimum amount of DNA to add to the reaction The optimum amount of DNA for the AmpF STR Identifiler Plus Kit is 1 0 ng in a maximum input volume of 10 uL for 28 PCR cycles and 0 5 ng in a maximum input volume of 10 uL for 29 PCR cycles If too much DNA is added to the PCR reaction then the increased amount of PCR product that is generated can result in Fluorescence intensity that exceeds the linear dynamic range for detection by the instrument off scale data Off scale data are problematic because Quantitation pea
77. cluded available from major laboratory suppliers MLS Table 2 Equipment Equipment Source ABI Prism 3100 3100 Avant Genetic Analyzer Applied Biosystems 3130 3130x Genetic Analyzer Applied Biosystems 310 Genetic Analyzer Contact your local sales representative GeneAmp PCR System 9700 with the Silver 96 Well Block N8050001 GeneAmp PCR System 9700 with the Gold plated Silver 96 Well Block 4314878 ProFlex PCR System 4484075 Veriti 96 Well Thermal Cycler 4375786 Silver 96 Well Sample Block N8050251 Gold plated Silver 96 Well Sample Block 4314443 Tabletop centrifuge with 96 Well Plate Adapters optional MLS Table 3 User supplied materials Item Source 50 reaction AmpF STR Identifiler Plus PCR Amplification Kit A26182 100 reaction AmpFLSTR Identifiler Plus PCR Amplification Kit A26364 200 reaction AmpFLSTR Identifiler Plus PCR Amplification Kit 4427368 1000 reaction AmpF STR Identifiler Plus PCR Amplification Kit 4486467 3100 3100 Avant Genetic Analyzer materials 96 Well Plate Septa 4315933 Reservoir Septa 4315932 3100 3100 Avant Genetic Analyzer Capillary Array 36 cm 4333464 POP 4 Polymer for 3100 3100 Avant Genetic Analyzers 4316355 3100 3100 Avant Genetic Analyzer Autosampler Plate Kit 96 well 4316471 GeneScan 500 LIZ Size Standard 4322682 Running Buffer 10x 402824 DS 33 Matrix Standard Kit Dye Set G5 43458
78. continued Allele ed G te cd Sa Amoi n 357 n 349 n 191 16 4 62 2 72 2 41 1 57 17 1 12 0 29 0 52 0 522 18 0 28 t t t 19 t t t t D138317 8 3 08 12 18 9 66 4 97 9 2 52 7 74 21 72 17 80 10 3 78 4 44 9 14 13 61 11 24 51 29 80 23 10 24 35 12 46 22 30 80 20 86 23 04 13 15 41 11 17 10 17 7 85 14 4 34 3 72 5 34 8 12 15 0 14 0 14 t 0 26 D16S539 5 t 8 3 22 1 72 1 72 0 79 9 19 05 10 46 9 31 12 30 10 10 92 5 59 15 69 15 45 11 31 51 31 95 30 17 30 89 12 18 77 30 23 29 48 27 75 13 14 85 16 76 11 55 10 73 14 1 54 3 01 2 07 2 09 15 0 14 0 29 t t D18S51 7 t 9 0 14t t t t 10 0 28 0 86 0 524 0 79 10 2 0 14t t t t 11 0 28 1 15 1 21 12 7 00 13 90 10 34 14 92 13 4 34 12 18 14 48 9 16 13 2 0 42 t t t 14 6 86 16 76 15 52 26 96 14 2 0 28 t t t 15 19 47 13 61 16 55 12 04 16 16 53 13 61 11 72 10 73 17 18 21 12 32 14 14 14 66 114 AmpFtSTR Identifiler Plus User Guide Population Data Table 10 AmpF STR Identifiler Plus Kit allele frequencies continued Allele Aa Ge n WW Brod Am de i n 357 n 349 n 191 18 11 90 7 74 6 72 2 62 19 6 02 4 44 4 14 3 93 20 4 90 1 72 2 24 1 83 21 2 10 1 00 1 03 1 31 22
79. d The AmpF STR Identifiler Plus Kit uses the same primer sequences as the earlier generation AmpF STR Identifiler Kit The AmpF STR Identifiler Plus Kit contains all the necessary reagents for the amplification of human genomic DNA The reagents are designed for use with the following Applied Biosystems instruments Applied Biosystems 3100 3100 Avant Genetic Analyzer Applied Biosystems 3130 3130x Genetic Analyzer Applied Biosystems 310 Genetic Analyzer GeneAmp PCR System 9700 with the Silver 96 Well Block GeneAmp PCR System 9700 with the Gold plated Silver 96 Well Block Veriti 96 Well Thermal Cycler ProFlex PCR System The AmpF STR Identifiler Plus Kit employs the latest improvements in primer synthesis and purification techniques to minimize the presence of dye labeled artifacts These improvements result in a much cleaner electropherogram background that enhances the assay s signal to noise ratio and simplifies the interpretation of results Non nucleotide linkers are used in primer synthesis for the following loci CSF1PO D138317 D16S539 D2S1338 and TPOX For these primers non nucleotide linkers are placed between the primers and the fluorescent dye during oligonucleotide synthesis Butler 2005 Grossman et al 1994 and Baron et al 1996 Non nucleotide linkers enable reproducible positioning of the alleles to facilitate AmpFtSTR Identifiler Plus User Guide Product overview interl
80. d mixed DNA samples see DAB Standard 8 1 2 2 Stutter products A stutter is a well characterized PCR artifact that refers to the appearance of a minor peak one repeat unit smaller or less frequently one repeat larger than the major STR product Butler 2005 and Mulero et 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 AmpFtSTR Identifiler Plus User Guide Extra peaks in the electropherogram The proportion of the stutter product relative to the main allele percent stutter is measured by dividing the height of the stutter peak by the height of the main allele peak Peak heights were measured for samples n 500 amplified using the 28 cycle protocol DNA input 1ng at the loci used in the Identifiler Plus Kit All data were generated on the Applied Biosystems 3130xl Genetic Analyzer Some conclusions from these measurements and observations are For each AmpF STR Identifiler Plus Kit locus the percent stutter generally increases with allele length as shown in Figure 19 to Figure 22 on pages 92 through 94 Smaller alleles display a lower level of stutter relative to the longer alleles within each locus Each allele within a locus displays a percent stutter that is consistent with other alleles in the locus The stutter value for each locus shown in T
81. d the Federal Bureau of Investigation Native American 191 samples were provided by the Minnesota Bureau of Criminal Apprehension Memorial Blood Center of Minneapolis In addition to the alleles that were observed and recorded in our databases other alleles have been published or reported by other laboratories see the STRBase at www cstl nist gov div831 strbase AmpFtSTR Identifiler Plus User Guide 111 Chapter 5 Experiments and Results AmpFiSTR Identifiler Plus 112 Kit allele frequencies Table 10 shows the AmpF STR Identifiler Plus Kit allele frequencies in four populations listed as percentages Table 10 AmpF STR Identifiler Plus Kit allele frequencies Allele Paa Gie x E s Fama n 357 n 349 n 191 CSF1PO 6 t t t t 7 4 62 0 14 0 34 8 7 56 0 29 0 17 0 52 9 3 78 1 72 0 86 8 38 10 27 87 24 21 23 10 30 89 11 20 59 31 91 28 28 21 99 11 3 0 14 t i t 12 29 13 32 81 39 66 32 72 13 5 32 7 31 6 38 4 71 14 0 98 1 43 0 86 0 79 15 t 0 29 t t D2S1338 15 0 14 t t t 16 5 32 4 73 2 41 2 62 17 10 78 17 34 21 21 9 95 18 5 60 6 30 4 14 7 07 19 14 15 13 75 22 76 29 58 20 6 02 14 61 13 79 9 69 21 14 01 2 58 2 59 2 38 22 13 17 4 01 7 41 15 18 23 10 78 11 46 11 36 11 78 24 9 80 11 75 8 45 7 85 25 8 12 10 60 5 17 3 14 26 1 96 2 72 0 69 0 79 27 0 14 0 14 t t 28 t t t t D3S1358
82. ditional information about biohazard guidelines is available at www cdc gov AmpFtSTR Identifiler Plus User Guide 135 Appendix B Safety Chemical alerts General alerts for all chemicals handling instructions Wear appropriate protective eyewear clothing and gloves 136 Specific chemical alerts Avoid contact with skin eyes and or clothing Read the MSDS and follow the CAUTION CHEMICAL HAZARD AmpF STR Identifiler Plus PCR Amplification Kit may cause eye skin and respiratory tract irritation Sodium azide may react with lead and copper plumbing to form highly explosive metal azides WARNING CHEMICAL HAZARD POP 4 Polymer for 3130 3130x Genetic Analyzers causes skin eye and respiratory tract irritation WARNING CHEMICAL HAZARD Running Buffer 10X causes skin eye and respiratory tract irritation WARNING CHEMICAL HAZARD Hi Di Formamide is harmful if swallowed inhaled or absorbed through skin and causes irritation to skin eyes and respiratory tract It affects the central nervous system and may affect the reproductive system WARNING CHEMICAL HAZARD POP 4 Polymer for 3100 3100 Avant Genetic Analyzers is irritating to eyes respiratory system and skin It causes adverse cardiovascular effects It contains a known or suspected reproductive toxin and a known or suspected mutagen AmpFtSTR Identifiler Plus User Guide Bibliography Akane A Matsubara K Nakamura
83. e Scientific Working Group on DNA Analysis Methods SWGDAM July 10 2003 Based on these guidelines we conducted experiments that comply with guidelines 1 0 and 2 0 and its associated subsections This DNA methodology is not novel Moretti et al 2001 Frank et al 2001 Wallin et al 2002 and Holt et al 2000 This chapter discusses many of the experiments performed by us and provides examples of results obtained We chose conditions that produced optimum PCR product yield and that met reproducible performance standards While these experiments are not exhaustive they are appropriate for a manufacturer of STR kits intended for forensic and or parentage testing use IMPORTANT Each laboratory using the AmpF STR Identifiler Plus PCR Amplification Kit must perform internal validation studies AmpFtSTR Identifiler Plus User Guide Developmental validation Developmental validation SWGDAM Developmental validation is the demonstration of the accuracy precision and guideline 1 2 1 reproducibility of a procedure by the manufacturer technical organization academic institution government laboratory or other party SWGDAM July 2003 SWGDAM The reaction conditions needed to provide the required degree of specificity and guideline 2 10 1 robustness must be determined These include thermal cycling parameters the concentration of primers magnesium chloride DNA polymerase and other critical reagents SWGDAM J
84. e compatibility 15 K kit allelic ladder 17 amplification 10 contents 17 control DNA 17 description 10 fluorescent dyes 15 loci amplification 11 144 master mix 17 primers 10 17 26 purpose 10 reagents 17 supported instruments 10 kit performance comparisons DNase I 102 hematin 103 humic acid 104 105 L limited product warranty 142 LIZ size standard about 17 volume per reaction 38 40 loci characterization 97 chromosomal location 11 dye label 11 genotype frequency in population 118 mapping 98 low TE buffer 25 M magnesium chloride concentration validation of 79 master mix volume per reaction 28 materials and equipment included in kit 17 not included in kit 18 mixed samples resolution of genotypes 106 mixture studies 105 MSDSs description 133 obtaining 133 multicomponent analysis 15 16 mutation studies 123 mutation STR 123 N negative control sample preparation 28 O off ladder alleles 83 operating systems 37 39 P PCR hematin inhibitor 103 humic acid inhibitor 104 performing 30 AmpF amp TR Identifiler Plus User Guide setup tools 24 thermal cycling conditions programming 30 work area setup 24 PCR components validation of 79 PCR cycle number validation 80 peak height ratios table ofalleles 105 percent stutter highest value for locus 91 off scale peaks 91 relation to allele length 91 positive control sample preparation 28 precision and size windows 83
85. e peaks can be used as an indicator of precision within a run 3 Click PP Analyze enter a name for the project in the Save Project dialog box then click OK to start analysis The status bar displays the progress of analysis as a completion bar extending to the right with the percentage completed indicated The table displays the row of the sample currently being analyzed in green or red if analysis failed for the sample The Analysis Summary tab see figure on next page is displayed upon completion of the analysis C o gt D ko go 0 8 w gt n o zh 2 0 AmpFtSTR Identifiler Plus User Guide 73 Section 4 2 GeneMapper ID X Software GeneMapper ID X 09 02 09 Dickens Analysis Method Example gmidx Is Logged In Database FOSWANGD1L03 TRAFE BE gt 6 reese nooo E Project H C3 Identifiler Plus Analysis Examples Bersabee gt gt Sample Status Total tof Samples A Analyzed 88 EZS mel MARO a E identifier Plus Analyss Examples ao o o 3 0 0 Control Type _ Total tof Samples _ i llthresholis met Euston Control 00 Negative Control E S S Hd lid For more information about any of these tasks refer to GeneMapper JD X Software Version 1 0 Getting Started Guide PN 4375574 GeneMapper D X Software Version 1 0 Quick Reference Guide PN 4375670 GeneMapper ID X Software Version 1 0
86. ence of the structure and length of the tandem repeat Am J Hum Genet 62 1408 1415 Budowle B et al 1998 CODIS and PCR Based Short Tandem Repeat Loci Law Enforcement Tools Second European Symposium on Human Identification 73 88 Butler J M 2005 Forensic DNA Typing Burlington MA Elsevier Academic Press Chakraborty R Stivers D and Zhong Y 1996 Estimation of mutation rates from parentage exclusion data applications to STR and VNTR loci Mutat Res 354 41 48 Chakraborty R and Stivers D N 1996 Paternity exclusion by DNA markers effects of paternal mutations J Forensic Sci 41 671 677 Chakraborty R Kimmel M Stivers D Davison L and Deka R 1997 Relative mutation rates at di tri and tetranucleotide microsatellite loci Proc Natl Acad Sci USA 94 1041 1046 AmpFtSTR Identifiler Plus User Guide 137 Bibliography 138 Clark J M 1988 Novel non templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases Nucleic Acids Res 16 9677 9686 DeFranchis R Cross N C P Foulkes N S and Cox T M 1988 A potent inhibitor of Taq DNA polymerase copurifies with human genomic DNA Nucleic Acids Res 16 10355 DNA Advisory Board Federal Bureau of Investigation U S Department of Justice 1998 Quality assurance standards for forensic DNA testing laboratories Edwards A Civitello A Hammond H and Caskey C 1991 DNA typing and geneti
87. enotyping Allelic ladder samples must be labeled as Allelic Ladder in the Sample Type column in a project Failure to apply this setting for ladder samples results in failed analysis Injections containing the allelic ladder must be analyzed with the same analysis method and parameter values that are used for samples to ensure proper allele calling Alleles that are not in the AmpF STR Allelic Ladders do exist Off ladder OL alleles may contain full and or partial repeat units An off ladder allele is an allele that occurs outside the 0 5 nt bin window of any known allelic ladder allele or virtual bin Note If a sample allele peak is called as an off ladder allele the sample result needs to be verified according to the laboratory s protocol AmpFtSTR Identifiler Plus User Guide Set up GeneMapper ID Software for data analysis If you are using GeneMapper D X Software to perform Human Identification HID analysis with AmpF STR kits go to Set up GeneMapper ID X Software for data analysis on page 61 or refer to the GeneMapper ID X Software Version 1 0 Human Identification Analysis Getting Started Guide PN 4375574 Set up GeneMapper D Software for data analysis Workflow Before you can analyze sample fsa files using GeneMapper ID Software v3 2 1 for the first time Import panels and bins into the Panel Manager as explained in Import panels and bins on page 48 Import an analysis method as e
88. er Plus Kit and analyzed on the Applied Biosystems 3130x Genetic Analyzer AmpFtSTR Identifiler Plus User Guide 13 Chapter 1 Overview Workflow overview Perform a U PCR Bg 3 x O Li lt S e Quantifiler Duo DNA Quantification Kit Quantifiler HP and Trio DNA Quantification Kits n 5 AmpF STR Identifiler Plus PCR Amplification Kit EE ag SK 7 g T 0 GeneAmp PCR System 9700 Cycler ProFlex PCR System Veriti 96 Well Thermal Cycler j jer Perform i electro 31 J gt phoresis Ed 2 ae 3100 3100 Avant 3130 3130xl 05 Genetic Analyzer Genetic Analyzer nayzer Analyze EM data gt LG GeneMapper D X or GeneMapper D Software 14 AmpFtSTR Identifiler Plus User Guide Instrument and software overview Instrument and software overview This section provides information about the Data Collection Software versions required to run the AmpF STR Identifiler Plus PCR Amplification Kit on specific instruments Data Collection The Data Collection Software provides instructions to firmware running on the and instrument and displays instrument status and raw data in real time As the GeneMapper D instrument measures sample fluorescence with its detection system the Data or ID X Software Collection Software collects the data and stores it The Data Collection Software stores information about each sample in a sample file fsa which i
89. er on page 29 AmpFtSTR Identifiler Plus User Guide Sensitivity 73 115 155 195 235 275 315 355 395 ano 3000 1 ng 2000 1000 D 75 115 155 195 235 275 315 355 385 ao ome 0 50 ng 2000 4000 0 75 115 155 195 235 275 315 355 395 2000 4600 4200 0 25 ng 800 400 0 75 115 155 195 235 275 315 355 395 sw 0 125 ng 800 400 0 75 115 455 495 235 275 315 355 395 000 800 600 0 062 ng 400 200 0 Figure 25 Effect of amplifying 1 ng 0 50 ng 0 25 ng 0 125 ng and 0 062 ng of Control DNA 9947A using the 28 PCR cycle protocol 75 115 155 195 235 275 315 355 395 5000 I avo 0 50 ng 3000 2000 4000 0 75 115 155 195 235 275 315 355 395 4000 2000 f 0 25 ng 75 115 455 195 235 275 315 355 395 2000 xl 0 125 ng 75 115 455 195 235 275 315 355 395 m 0 062 ng 75 115 155 195 235 275 315 355 395 1000 oj 0 031 ng 600 400 200 j 0 Figure 26 Effect of amplifying 0 5 ng 0 25 ng 0 125 ng 0 062 ng and 0 031 ng of Control DNA 9947A using the 29 PCR cycle protocol Note that the y axis scale is magnified for the lower amounts of DNA analyzed using the Applied Biosystems 3130x Genetic Analyzer AmpFtSTR Identifiler Plus User Guide 101 Chapter 5 Experiments and Results Stability SWGDAM guideline 2 4 Deg
90. er sequences have been optimized to encourage A addition The final extension step is 60 C for 10 min This final extension step gives the DNA polymerase additional time to complete A addition to all double stranded PCR products STR systems where each allele is represented by two peaks that are one nucleotide apart that have not been optimized for A addition may have split peaks AmpFtSTR Identifiler Plus User Guide 95 Chapter 5 Experiments and Results 96 Artifacts ER 175 180 185 130 195 No Extension Final Extension Nokia Figure 23 Omitting the final extension step results in split peaks due to incomplete A nucleotide addition Data are from an ABI PRISM 310 Genetic Analyzer using another AmpF STR kit Lack of complete A nucleotide addition may be observed in AmpF STR Identifiler Plus PCR Amplification Kit results when the amount of input DNA is greater than the recommended protocols because more time is needed for the enzyme to add the A nucleotide to all molecules as more PCR product is generated Amplification of too much input DNA may also result in off scale data Artifacts and anomalies are seen in all molecular biological systems Artifacts are typically reproducible while anomalies are non reproducible intermittent occurrences that are not observed consistently in a system for example spikes and baseline noise Reproducible artifacts have not been seen in da
91. f amplification cycles to deliver well balanced and high quality results However increases in the number of low level DNA samples being submitted for analysis have prompted many laboratories to evaluate increasing the number of amplification cycles to increase the sensitivity of the assay Before increasing the cycle number perform a comprehensive validation study to establish new performance criteria for the higher cycle number Higher cycle numbers can cause the following to occur Exaggerated stochastic effects resulting from low DNA input amounts Greater difference between the presence and absence of an allele Greater heterozygote peak imbalance Possible differences in expected stutter position and percentage Possible increase in artifacts and or background in the profile to accompany the increase in sample allele signal The Identifiler Plus Kit offers two PCR cycle number options Standard 28 PCR cycle protocol Provides high sensitivity to consistently generate full STR profiles with 125 pg of DNA input Use with the optimum 1 0 ng DNA input amount in a maximum input volume of 10 uL 29 PCR cycle protocol Adds the extra sensitivity when amplifying 125 pg DNA inputs Recommended for use when the total DNA input amount is 0 5 ng The results of the developmental validation at both PCR cycle numbers is presented in Chapter 5 on page 77 AmpFtSTR Identifiler Plus User Guide 29 Chapter 2 PCR Amplification
92. g pairs with mutations at locus D8S1179 The genotypes differed by one repeat unit between the two generations Calculation of a mutation rate based on these data would be inaccurate due to the small sample size The other parent offspring allele transfers were in accordance with Mendelian rules AmpFtSTR Identifiler Plus User Guide 97 Chapter 5 Experiments and Results Mapping The Identifiler Plus Kit loci Amelogenin CSF1PO D2S1338 D3S1358 D5S818 D7S1179 D13S317 D16S539 D18S51 D19S433 D21S11 FGA THO1 TPOX and vWA have been mapped and the chromosomal locations have been published Nakahori et al 1991 Edwards et al 1992 Kimpton et al 1992 Kong et al 2004 Mills et al 1992 Sharma and Litt 1992 Li et al 1993 Straub et al 1993 Barber and Parkin 1996 Species specificity 98 SWGDAM Guideline 2 2 For techniques designed to type human DNA the potential to detect DNA from forensically relevant nonhuman species should be evaluated SWGDAM July 2003 The AmpF STR Identifiler Plus PCR Amplification Kit provides the required specificity for detecting human alleles Nonhuman studies Nonhuman DNA may be present in forensic casework samples The data from AmpF STR Identifiler Plus PCR Amplification Kit experiments on nonhuman DNA sources are shown in Figure 24 on page 99 Figure 24 shows amplification for Control DNA 9947A 1 0 ng panel 1 chimpanzee 1 0 ng panel 2 dog 10 ng
93. hod v1X in the Name column and click Open 66 AmpFtSTR Identifiler Plus User Guide Set up GeneMapper ID X Software for data analysis z GeneMapper ID X Manager X Find Name Containing Projects Analysis Methods Table Settings Plot Settings Matrices Size Standards Report Settings Name Last Saved Owner Ea Analysis Type t Default 2009 08 31 11 27 45 695 jamidx HID r Identifiler Plus AnalysisMethod v1X 2009 09 02 11 13 05 191 gmidx HID E New Open Save 4s Import Export Figure 10 Analysis Method Editor General tab settings Figures 11 through 14 below show the settings for each tab of the Analysis Method Editor CD o el 0 lt 0 3 o e 2 Xx e E 2 0 AmpFtSTR Identifiler Plus User Guide 67 Section 4 2 GeneMapper ID X Software Analysis Method Editor xl General Allele Peak Detector Peak Quality 5Q amp GQ Settings Bin Set Identifiler Plus Bins v1X v Use marker specific stutter ratio and distance if available Marker Repeat Type Tri Tetra Penta Global Cut off value 0 0 MinusA Ratio MinusA Distance From To Global Minus Stutter Ratio Global Minus Stutter Distance From To Global Plus Stutter Ratio Global Plus Stutter Distance From PPPEPEPEPI NEHE N HUN li ba il il ed aa ik TTTTTT TT T To Amelogenin Cutoff fo Range Filter Factory Defaults
94. ied 24 control about 17 degraded 102 effect of quantity figure 101 mixture studies 105 mixture studies figure 107 negative control reaction 28 positive control reaction 28 quantification 25 quantification methods 26 sample preparation 28 sensitivity 100 test sample 28 tools 24 DNA mixtures amplification figure 109 limit of detection 107 E electropherogram causes of extra peaks 84 90 extra peaks 90 species specificity 99 102 electrophoresis Data Collection Software 37 39 preparing samples on the 310 instrument 40 preparing samples on the 3100 3100 Avant or 3130 3130x instrument 38 reagents and parts 37 39 references 37 39 run module 37 39 setup 37 39 emission spectra 16 143 Index equipment not included in kit 18 experiments and results 77 extra peaks causes 90 F fluorescent dyes 15 FTA cards amplification 31 bloodstained 31 G GeneMapper ID Software data analysis 47 overview 15 46 GeneMapper ID X Software data analysis 61 overview 15 GeneScan size standard about 17 dye label 15 volume per reaction 38 40 guidelines chemical safety 132 chemical waste disposal 134 chemical waste safety 134 H hazards See safety hematin 103 hematin effects of 103 Hi Di formamide volume per reaction 38 40 humic acid effects of 104 inheritance 97 instrumentation 310 genetic analyzer 15 36 39 3100 3100 Avant genetic analyzer 15 36 37 3130 3130x genetic analyzer 15 36 37 softwar
95. iles using GeneMapper ID X Software v1 0 1 v1 1 or v1 1 1 for the first time Import panels bins and marker stutter into the Panel Manager as explained in Import panels bins and marker stutter on page 61 Import an analysis method as explained in Import an analysis method on page 66 Importa size standard as explained in Import a HID size standard on page 71 Define custom views of analysis tables Define custom views of plots For more info For quick set up instructions refer to the GeneMapper D X Software Version 1 0 Getting Started Guide PN 4375574 For details about the GeneMapper D X Software workflow and features refer to GeneMapper ID X Software Version 1 0 Getting Started Guide PN 4375574 GeneMapper D X Software Version 1 0 Quick Reference Guide PN 4375670 GeneMapper ID X Software Version 1 0 Reference Guide PN 4375671 Import To import the AmpF STR Identifiler Plus Kit panels bin sets and marker stutter panels bins and from our web site into the GeneMapper ID X Software v1 0 1 v1 1 or v1 1 1 marker stutter database 1 Download and open the file containing panels bins and marker stutter a From the Support menu of www appliedbiosystems com select Software Downloads Patches amp Updates Select GeneMapper ID X Software from the drop down menu Select Updaters amp Patches and download the file Identifiler Plus Analysis Files GMIDX b Unzip the file
96. ines are potentially hazardous Always determine what chemicals have been used in the instrument before changing reagents or instrument components Wear appropriate eyewear protective clothing and gloves when working on the instrument WARNING CHEMICAL HAZARD Four liter reagent and waste bottles can crack and leak Each 4 liter bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles Chemical safety To minimize the hazards of chemicals 132 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 133 Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For add
97. ing anywhere from 1 to 3 alleles were observed at 0 062 ng With the 29 PCR cycle protocol full profiles 26 alleles were consistently obtained at 0 062 ng but occasional partial profiles that are missing anywhere from to 5 alleles were observed at 0 031 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 is off scale can cause the corresponding stutter peak to appear higher in relative intensity thus increasing the calculated percent stutter Multicomponent analysis of off scale data is not accurate 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 1s extremely low unbalanced amplification of the alleles may occur because of stochastic fluctuation Individual laboratories may find it useful to determine an appropriate minimum peak height threshold based on their own results and instruments using low amounts of input DNA For information on selecting the appropriate cycle number for your DNA input amount see Select the appropriate PCR cycle numb
98. ion 134 work area amplified DNA tools 24 PCR tools 24 setup 24 workflow overview 14 145 Index 146 AmpFSTR Identifiler Plus User Guide Headquarters 5791 Van Allen Way Carlsbad CA 92008 USA Phone 1 760 603 7200 Toll Free in USA 800 955 6288 For support visit www lifetechnologies com support technologies www lifetechnologies com A Thermo Fisher Scientific Brand 5 February 2015
99. itional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the MSDS Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the MSDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal AmpFtSTR Identifiler Plus User Guide Chemical safety About MSDSs Chemical manufacturers supply current Material Safety Data Sheets MSDSs with shipments of hazardous chemicals to new customers They also provide MSDSs with the first shipment of a hazardous chemical to a customer after an MSDS has been updated MSDSs provide the safety information you need to store handle transport and dispose of the chemicals safely Each time you receive a new MSDS packaged with a hazardous chemical be sure to replace the appropriate MSDS in your files Obtaining The MSDS for any chemical supplied by Applied Biosystems is available to you free MSDSs 24 hours a day To obtain MSDSs 1 Goto www appliedbiosystems com click Support then select MSDS 2 In the Keyword Search field enter the chemical name product name MSDS part number or other information that appears in the MSDS of interest Select the language of your choice then click Search
100. k height and area for off scale peaks is not accurate For example an allele peak that is off scale can cause the corresponding stutter peak to appear higher in relative intensity thus increasing the calculated percent stutter Multicomponent analysis of off scale data 1s not accurate and it results in poor spectral separation pull up Incomplete A nucleotide addition When the total number of allele copies added to the PCR is extremely low allelic dropout can occur resulting in a partial profile AmpFtSTR Identifiler Plus User Guide 25 Chapter 2 PCR Amplification Methods of quantifying DNA We provide several kits for quantifying DNA in samples See the reference cited in the following table for details about these kits Product Description References Quantifiler Human DNA Quantification Kit PN 4343895 and Quantifiler Y Human Male DNA Quantification Kit PN 4343906 Properties The Quantifiler Human and Quantifiler Y Human Male Kits are highly specific for human DNA and they individually detect total human or male DNA respectively The kits detect single stranded and degraded DNA How they work The Quantifiler DNA Quantification Kits consist of target specific and internal control 5 nuclease assays The Quantifiler Human and Quantifiler Y Human Male Kits contain different target specific assays human DNA or human male DNA respectively that each consist of two l
101. lue of the Hardy Weinberg test with multiple alleles Hexp Expected heterozygosity Ho observed heterozygosity We analyzed gt 500 samples by comparing allele calls between the AmpF STR Identifiler and Identifiler Plus Kits The genotype data from all the analyzed samples showed 100 concordance between the Identifiler and Identifiler Plus Kits AmpFtSTR Identifiler Plus User Guide Mutation Rate Mutation Rate Estimation of spontaneous or induced germline mutation at genetic loci can be achieved by comparing the genotypes of offspring to those of their parents From such comparisons the number of observed mutations are counted directly In previous studies genotypes of ten STR loci that were amplified by the AmpF STR SGM Plus PCR Amplification Kit were determined for a total of 146 parent offspring allelic transfers meioses at the Forensic Science Service Birmingham England One length based STR mutation was observed at the D18S11 locus mutations were not detected at any of the other nine STR loci The D18S11 mutation was represented by an increase of one 4 nt repeat unit allele 17 was inherited as allele 18 single step mutation The maternal paternal source of this mutation could not be distinguished Additional Additional studies Edwards et al 1991 Edwards et al 1992 Weber and Wong mutation studies 1993 Hammond et al 1994 Brinkmann et al 1995 Chakraborty et al 1996 Chakraborty et al 1997 Brinkmann
102. lysis Method Editor HID Analysis Method Editor HID B xi General Allele Peak Detector Peak Quality Quality Flags Bin Set REMI l A IV Use marker specific stutter ratio if available Marker Repeat Type Cut off value Minus amp Ratio Minus Distance Minus Stutter Ratio Minus Stutter Distance Plus Stutter Ratio Plus Stutter Distance Tri bo TEM From bo To foo TEN From bo To bo TE From bo To bo Tetra TEE Penta Hexa POCECE POCECE Amelogenin Cutoff Ta Range Filter n P 2 a s lt e m 2 r I2 oo Cancel Figure 6 Analysis Method Editor HID Allele tab settings GeneMapper ID Software v3 2 1 allows you to specify four types of marker repeat motifs tri tetra penta and hexa You can enter parameter values for each type of repeat in the appropriate column AmpFtSTR Identifiler Plus User Guide 53 C D 2 D lt ko zo D iw n e Eh 0 Section 4 1 GeneMapper ID Software The Use marker specific stutter ratio if available check box is selected by default Consequently the software applies the stutter ratio filters supplied in the Identifiler Plus Panels v1 file For more information about allele filters refer to GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide Chapter 3 PN 4338775 GeneMapper ID Software Versions 3 1
103. m 0 0 to p zs Genotype Quality From 75 to 1 0 From 0 0 to 2s Reset Defaults Save As Save Cancel Help Figure 14 Analysis Method Editor SQ and GQ tab settings Import a HID size The size standard for the AmpF STR Identifiler Plus PCR Amplification Kit uses standard the following GeneScan 500 LIZ size standard peaks in its sizing algorithm 75 100 139 150 160 200 300 350 400 and 450 Use the following procedure to import the size standard for the AmpF STR Identifiler Plus PCR Amplification Kit from the folder that you downloaded from our web site into the GeneMapper JD X Software database Refer to step 1 on page 61 for downloading instructions C D mi D lt ko o 0 8 w gt n 9 zh 2 0 1 Select Tools GeneMapper ID X Manager to open the GeneMapper D X Manager AmpFtSTR Identifiler Plus User Guide 71 Section 4 2 GeneMapper ID X Software 2 Import a size standard a Select the Size Standards tab then click Import GeneMapper ID X Manager Mme Jlastseved ome ie Description _ areas wzm lm BI oF ss 0 zwani ae LED a awana qn Save As b Navigate to then open the Identifiler Plus Analysis Files GMIDX folder c Select CE G5 Identifiler Plus GS500 then click Import to import the CE G5 Identifiler Plus GS500 analysis method into the GeneMapper ID X Software database Import Size Standard Method C Iden
104. meter Settings Sample Type Select the sample type Analysis Method Identifiler Plus AnalysisMethod v1 Panel Identifiler Plus Panels v1 Size Standard CE G5 Identifiler Plus GS5005 t For more information about how the Size Caller works refer to the ABI PRISM GeneScan Analysis Software for the Windows NT Operating System Overview of the Analysis Parameters and Size Caller User Bulletin PN 4335617 S The following fragments are defined for the CE G5 Identifiler Plus GS500 size standard provided with the AmpF STR Identifiler Plus Kit 75 100 139 150 160 200 300 350 400 and 450 For additional information about size standards refer to the GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide PN 4338775 Appendix D Neither the 250 nt nor the 340 nt peak are included in the size standard definition These peaks can be used as an indicator of precision within a run 3 Click PP Analyze enter a name for the project in the Save Project dialog box then click OK to start analysis The status bar displays the progress of analysis Asacompletion bar extending to the right with the percentage completed indicated With text messages on the left AmpFtSTR Identifiler Plus User Guide 57 Section 4 1 GeneMapper ID Software The Samples table figure below displays the row of the sample currently being analyzed in green or red if analysis failed for the sample The Ge
105. ming the analysis is complete For more information about any of these tasks refer to GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial PN 4335523 GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide PN 4338775 Installation Procedures and New Features for GeneMapper ID Software Version v3 2 User Bulletin PN 4352543 AmpFtSTR Identifiler Plus User Guide 2 D lt ied xe 5 2 AE Xx e 2 0 0 AmpFtSTR Identifiler Plus User Guide 59 Section 4 2 GeneMapper ID X Software Section 4 2 GeneMapper D X Software Overview of GeneMapper D X Software 60 Instruments Before you start GeneMapper ID X Software is an automated genotyping software for forensic casework databasing and paternity data analysis After electrophoresis the Data Collection Software stores information for each sample in a fsa file Using GeneMapper ID X Software v1 0 1 v1 1 or v1 1 1 you can then analyze and interpret the data from the fsa files Refer to Instrument and software overview on page 15 for a list of compatible instruments When using GeneMapper D X Software v1 0 1 v1 1 or v1 1 1 to perform human identification HID analysis with AmpF STR kits be aware that HID analysis requires at least one allelic ladder sample per run folder Your laboratory can use multiple ladder samples in an analysis provided individual
106. nd secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the MSDS Handle chemical wastes in a fume hood After emptying a waste container seal it with the cap provided Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or national environmental and health regulations Waste disposal Ifpotentially hazardous waste is generated when you operate the instrument you must 134 Characterize by analysis 1f necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure the health and safety of all personnel in your laboratory AmpFtSTR Identifiler Plus User Guide Biological hazard safety Ensure that the instrument waste is stored transferred transported and disposed of acco
107. notypes tab becomes available after analysis 2 1 Untitled gmid Is Logged In ew Tools Help 5 jl m Mie gu gt Table Setting HD_LsdderTable_FilName 8 amp BB Samples Genotypes Status Sample File Sample Name Sample Type Analysis Method Panel Size Standard Instrument Type 1 m Cerebus_3130XL IB_0329 Sample Identifiler Plus amp nalysisMethod v1 Identifiler Plus Panels v1 CE G5 ldentifiler Plus GS500 amp BI3130 2 m Cerebus 3130XL I 0330 Sample Identifiler Plus AnalysisMethod v1 ldertifiler Plus Panels v1 CE GS ldentifiler Plus GS500 ABI3130 3 i Cerebus 3130XL IB_0331 Sample Identifiler Plus amp nalysisMethod v1 Identifiler Plus Panels v1 CE GS Identifiler Plus GS500 amp BI3130 4 i Cerebus 3130XL IB_0332 Sample Identifiler_Plus_AnalysisMethod_v1 Identifiler Plus Panels v1 CE G5 Identifiler Plus GS500 amp BI3130 5 i Cerebus_3130XL IB_0333 Sample Identifiler_Plus_AnalysisMethod_v1 Identifiler Plus Panels v1 CE_G5_ldentifiler_Plus_GS500 amp BI3130 6 ih Cerebus_3130XL IB_0334 Sample Identifiler_Plus_AnalysisMethod_v1 Identifiler Plus Panels v1 CE_G5_ldentifiler_Plus_GS500 amp BI3130 7 lx Cerebus 3130XL IB 0335 Sample Identifiler Plus amp nalysisMethod v1 Identifiler Plus Panels v1 CE G5 Identifiler Plus GS500 amp BI3130 8 i Cerebus 3130XL IB 0336 Sample Identifiler Plus amp nalysisMethod v1 Identifiler Plus Panel
108. nt and tests for detecting departures from Hardy Weinberg equilibrium are shown for each population in Table 11 While a number of the chi square tests gave seemingly significant p values putatively indicating departures from Hardy Weinberg equilibrium chi squared tests are very sensitive to small expected values as in the case of multiple rare alleles where the expected number of certain genotypes is 1 or fewer such as with some of these markers and can greatly inflate the test statistic in this situation Weir 1990 With the exact test the number of tests with p value lt 0 05 were 0 in the African American and U S Caucasian populations 1 in the U S Hispanic population D8S1179 p 0 0304 and 2 in the Native Americans D21S11 p 0 0118 D5S818 p 0 0205 These are no more than would be expected by chance No more alleles were observed to be in linkage disequilibrium than would be expected by chance alone The average observed heterozygosity across the 15 STR loci was 0 804 in the African American population 0 792 in the U S Caucasian sample population 0 793 in the Hispanic sample population and 0 757 in the Native Americans The most heterozygous locus was FGA mean observed heterozygosity across all populations of 0 875 and the least heterozygous STR locus was TPOX mean observed heterozygosity across all populations of 0 677 Table 11 Heterozygosity and p values for Hardy Weinberg tests of the 15 Identifiler
109. occur between runs on the same instrument and between runs on different instruments of the same platform type because of these factors AmpFtSTR Identifiler Plus User Guide 83 Chapter 5 Experiments and Results We strongly recommend that the allele sizes be compared to the sizes obtained for known alleles in the AmpF STR Identifiler Plus Allelic Ladder from the same run and then be converted to genotypes as described in Before you start on pages 46 and 60 See Table 4 for the results of five runs of the AmpF STR Identifiler Plus Allelic Ladder For more information on precision and genotyping see Lazaruk et al 1998 and Mansfield er al 1998 In Table 4 the mean sizes for all the alleles in each run 16 capillaries were calculated The mean range shown in the table represents the lowest and highest mean size values obtained across all five runs Similarly the standard deviation for the allele sizing was calculated for all the alleles in each run The standard deviation range shown in Table 4 represents the lowest and highest standard deviation values obtained across all five runs Table 4 Precision results of five runs 16 capillaries run of the AmpF STR Identifiler Plus Allelic Ladder Applied Biosystems 3130x Genetic Analyzer 84 Allele Mean Standard Deviation Amelogenin X 106 03 106 13 0 033 0 045 Y 111 69 111 8 0 03 0 042 CSF
110. ocus spacing The combination of a five dye fluorescent system and the inclusion of non nucleotide linkers allows for simultaneous amplification and efficient separation of the 15 STR loci and Amelogenin during automated DNA fragment analysis Loci amplified by the kit The following table shows the loci amplified their chromosomal locations and the corresponding fluorescent marker dyes The AmpF STR Identifiler Plus Allelic Ladder is used to genotype the analyzed samples The alleles contained in the allelic ladder and the genotype of the AmpF STR Identifiler Plus Control DNA 9947A are also listed in the table Table 1 AmpF STR Identifiler Plus Kit loci and alleles s Chromosome Alleles included in Identifiler Plus Dye Control DNA Locus designation location Allelic Ladder label 9947A D8S1179 8 8 9 10 11 12 13 14 15 16 17 18 19 6 FAM 137 D21S11 21q11 2 q21 24 24 2 25 26 27 28 28 2 29 29 2 30 30 30 2 31 31 2 32 32 2 33 33 2 34 34 2 35 35 2 36 37 38 D7S820 7q11 21 22 6 7 8 9 10 11 12 13 14 15 10 11 CSF1PO 5q33 3 34 6 7 8 9 10 11 12 13 14 15 10 12 D381358 3p 12 13 14 15 16 17 18 19 VIC9 14 15 THO1 11p15 5 4 5 6 7 8 9 9 3 10 11 13 3 8 9 3 D13S317 13q22 31 8 9 10 11 12 13 14 15 11 D165S539 16q24 qter 5 8 9 10 11 12 13 14 15 11 12 D281338 2035 37 1 15 16 17 18 19 20 21 22 23 2
111. ocus specific PCR primers and one TaqMan MGB probe labeled with FAM dye for detecting the amplified sequence The kits each contain a separate internal PCR control IPC assay which consists of an IPC template DNA a synthetic sequence not found in nature two primers for amplifying the IPC template DNA and one TaqMan MGB probe labeled with VIC dye for detecting the amplified IPC DNA Quantifiler Human DNA Quantification Kits User s Manual PN 4344790 Quantifiler Duo DNA Quantification Kit PN 4387746 Properties The Quantifiler Duo Kit is highly specific for human DNA This kit combines the detection of both total human and male DNA in one PCR reaction The kit detects single stranded and degraded DNA How it works The Quantifiler Duo DNA Quantification Kit consists of target specific and internal control 5 nuclease assays The Quantifiler Duo kit combines two human specific assays in one PCR reaction for total human DNA and human male DNA The two human DNA specific assays each consist of two PCR primers and a TagMan probe The TaqMan probes for the human DNA and human male DNA assays are labeled with VIC and FAM dyes respectively In addition the kit contains an internal PCR control IPC assay similar in principle to that used in the other Quantifiler kits but labeled with NED dye Quantifiler Duo DNA Quantification Kit User s Manual PN 4391294 26 AmpFtSTR Identifiler
112. ollected for several allele peaks at 30 cycles Figure 17 AmpFtSTR Identifiler Plus User Guide Developmental validation Although none of the cycle numbers tested produced nonspecific peaks 28 cycles was found to give optimal peak heights with 1 ng of DNA input when the amplified products were examined on Applied Biosystems 3130x Genetic Analyzers 355 395 3000 26 cycles 75 115 455 195 235 275 315 355 395 27 cycles j 3000 28 cycles 2000 1000 o 395 75 115 155 195 235 275 n 355 29 cycles 75 115 155 195 235 275 315 355 395 30 cycles Figure 17 Representative AmpF STR Identifiler Plus Kit profiles obtained from amplification of 1 0 ng DNA template using 26 27 28 29 and 30 cycles analyzed on the Applied Biosystems 3130x Genetic Analyzer Y axis scale 0 to 4 000 RFUs AmpFtSTR Identifiler Plus User Guide 81 Chapter 5 Experiments and Results Accuracy precision and reproducibility 82 SWGDAM The extent to which a given set of measurements of the same sample agree with guideline 2 9 their mean and the extent to which these measurements match the actual values being measured should be determined SWGDAM July 2003 Accuracy Laser induced fluorescence detection of length polymorphism at short tandem repeat Size Difference nt 0 50 rrrFETTTTTTTTTTTTTTTTTTTTETTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
113. olume Volume Storage A26182 A26364 4427368 4486467 AmpF STR Contains enzyme salts 1 tube 2 tubes 2 tubes 10 tubes 15 to 25 C on Identifiler Plus dNTPs carrier protein and 0 5 mL 0 5 mL tube 1 0 mL tube 1 0 mL tube receipt 2 to 8 C Master Mix 0 04 sodium azide after initial use AmpF STR Contains 0 10 ng uL human 1 tube 1 tube 1 tube 5 tubes Control DNA female 9947A DNA in 0 05 0 3 mL 0 3 mL 0 3 mL 0 3 mL tube 9947A sodium azide and buffer See Table 1 on page 11 for profile AmpF STR Contains forward and 1 tube 2 tubes 1 tube 5 tubes 15 to 25 C on Identifiler Plus reverse primers to amplify 0 25mL 0 25 mL tub 1 0 mL 1 0 mL tube receipt 2 to 8 C Primer Set human DNA targets e after initial use AmpF STR Contains amplified alleles 1tube 1 tube 1 tube Stlbes Jen n n Identifiler Plus See Table 1 on page 11 for a 0 025 mL 0 025 mL 0 05 mL 0 05 mL tube oe Allelic Ladder pag list of alleles included in the allelic ladder The AmpF STR Control DNA 9947A is included at a concentration appropriate to its intended use as an amplification control i e to provide confirmation of the capability of the kit reagents to generate a profile of expected genotype The AmpFZSTR Control DNA 9947A is not designed to be used as a DNA quantitation control and laboratories may expect to see variation from the labelled concentration when quantitating aliquots of the AmpF
114. opulation groups SWGDAM July 2003 Overview To interpret the significance of a match between genetically typed samples you must know the population distribution of alleles at each locus in question If the genotype of the relevant evidence sample is different from the genotype of a suspects reference sample then the suspect is excluded as the donor of the biological evidence that was tested An exclusion is independent of the frequency of the two genotypes in the population If the suspect and evidence samples have the same genotype then the suspect is included as a possible source of the evidence sample The probability that another unrelated individual would also match the evidence sample is estimated by the frequency of that genotype in the relevant population s Population The AmpF STR Identifiler PCR Amplification Kit prior to the addition of the samples used in D8S1179 degenerate primer was used to generate the population data provided in these studies this section Samples were collected from individuals throughout the United States with no geographical preference African American 357 samples were provided by the Kentucky State Police and the Federal Bureau of Investigation U S Caucasian 349 samples were provided by the Kentucky State Police and the Federal Bureau of Investigation U S Hispanic 290 samples were provided by the Minnesota Bureau of Criminal Apprehension Memorial Blood Center of Minneapolis an
115. or Deletio 30004 2000 0 10004 100 uM HE200 b 4000 130 IT Mark Sample for Deletio 170 210 250 290 330 3000 2000 1000 0 200 uM HE300 b IT Mark Sample for Deletio 470 210 250 290 330 4000 3000 2000 1000 AmpFtSTR Identifiler Plus User Guide 300 uM Figure 28 Amplification with the AmpF STR Identifiler Plus Kit in the presence and absence of hematin Panel 1 corresponds to control samples panels 2 4 correspond to samples amplified in the presence of 100 200 and 300 uM of hematin Table 6 Performance in simulated model of hematin inhibition n 3 Identifiler Plus Kit Hematin uM Alleles detected Total alleles 0 26 26 26 26 26 26 100 26 26 26 26 26 26 103 Chapter 5 Experiments and Results Table 6 Performance in simulated model of hematin inhibition n 3 continued Hematin uM Alleles detected Total alleles Identifiler Plus Kit 200 26 26 26 26 26 26 300 26 26 26 26 26 26 t Only those peaks gt 50 RFUs were counted A complete profile with Control 9947A DNA yields 26 peaks using the AmpF STR Identifiler Plus Kit Effect of Traces of humic acid may inhibit the PCR amplification of DNA evidence collected inhibitors from soil In this study We tested increasing amounts of humic acid in the PCR humic
116. oration and or its affiliate s warrant their products as set forth in the Life Technologies General Terms and Conditions of Sale found on Life Technologies website at www lifetechnologies com termsandconditions If you have any questions please contact Life Technologies at www lifetechnologies com support AmpFtSTR Identifiler Plus User Guide Index Symbols fsa sample files 47 61 A nucleotide addition defined 95 efficiency of 95 lack of causes 96 A accuracy and reproducibility 79 82 alleles low frequency 118 off ladder 83 peak height ratio table 105 allelic ladder about 17 figure 12 number per run suggested 36 precision results table 84 requirements for accurate genotyping 36 volume per reaction 38 40 amplification amplified DNA 24 loci 11 using bloodstained FTA cards 31 work area tools 24 annealing temperatures validation of 80 artifacts in data 96 B biohazardous waste handling 135 C CEPH 97 characterization of loci validation 97 chemical safety 132 chemical waste safety 134 concordance studies 122 contents of kit 17 26 control DNA 9947A 13 17 cycle number validation 80 AmpFSTR Identifiler Plus User Guide D data accuracy precision and reproducibility 82 Data Collection Software 15 Data Collection Software overview 15 data accuracy precision and reproducibility of 79 data artifacts 96 data for different populations 111 developmental validation 78 DNA amplif
117. ortable genotype frequency at each locus is 1 19 X 10 4 for the African American database 1 19 X 10 4 for the U S Caucasian database 1 70 X 10 4 for the U S Hispanic database and 2 97 X 10 4 for the Native American database p2 p 1 p 9 where 0 0 01 AmpFtSTR Identifiler Plus User Guide Population Data Evaluation of Estimates of expected heterozygosity HExp were computed as described by Nei Hardy Weinberg M 1973 using the program PopGene 1 32 Possible divergence from equilibrium Hardy Weinberg expectations HWE was tested using various methods By calculating the unbiased estimate of the expected homozygote heterozygote frequencies Nei M 1978 Using chi square HW X p and likelihood ratio HW G p tests as implemented in the program PopGene 1 32 With an exact test HW Exact p which is a Markov chain method based on 1000 shuffling experiments to estimate without bias the exact P value of the Hardy Weinberg test with multiple alleles Guo and Thompson 1992 as implemented in the program GenePop 3 4 e An inter class correlation test analysis Burrows composite measure of linkage disequilibria between pairs of loci and X tests for significance Weir 1996 was performed separately in each population to detect any correlations between alleles at any of the pair wise comparisons of the 15 loci using the program PopGene 1 32 Observed heterozygosity Ho expected heterozygosity information conte
118. orty and Stivers 1996 AmpFtSTR Identifiler Plus User Guide 125 Chapter 5 Experiments and Results 126 AmpFtSTR Identifiler Plus User Guide Troubleshooting Follow the actions recommended in Table 14 to troubleshoot problems that occur during analysis Table 14 Troubleshooting Observation Possible causes Recommended actions Faint or no signal from both the 9947A and the DNA test samples at all loci Incorrect volume or absence of either AmpF STR Identifiler Plus Master Mix or AmpFZSTR Identifiler 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 ZSTR Identifiler Plus Primer Set exposed to too much light Store Primer Set protected from light GeneAmp PCR System malfunction Refer to the thermal cycler user s manual and check instrument calibration Incorrect thermal cycler parameters Check the protocol for correct thermal cycler parameters Tubes not seated tightly in the thermal cycler during amplification Push reaction tubes firmly into contact with block after first cycle Repeat test Wrong PCR reaction tube Use Applied Biosystems MicroAmp Reaction Tubes with Caps for the GeneAmp PCR System 9700 MicroAmp Base used with tray retainer set and tubes in
119. ov hq lab fsc current standards 2004_03_standards02 htm Puers C Hammond H Jin L Caskey C and Schumm J 1993 Identification of repeat sequence heterogeneity at the polymorphic short tandem repeat locus HUMTHO1 AATG n and reassignment of alleles in population analysis using a locus specific allelic ladder Am J Hum Genet 53 953 958 Sensabaugh G F 1982 Biochemical markers of individuality In Saferstein R ed Forensic Science Handbook Prentice Hall Inc New York pp 338 415 Sharma V and Litt M 1992 Tetranucleotide repeat polymorphism at the D21S11 locus Hum Mol Genet 1 67 AmpFtSTR Identifiler Plus User Guide 139 Bibliography 140 Smith R N 1995 Accurate size comparison of short tandem repeat alleles amplified by PCR Biotechniques 18 122 128 Sparkes R Kimpton C Watson S Oldroyd N Clayton T Barnett L Arnold J Thompson C Hale R Chapman J Urquhart A and Gill P 1996a The validation of a 7 locus multiplex STR test for use in forensic casework I Mixtures ageing degradation and species studies Int J Legal Med 109 186 194 Sparkes R Kimpton C Gilbard S Carne P Andersen J Oldroyd N Thomas D Urquhart A and Gill P 1996b The validation of a 7 locus multiplex STR test for use in forensic casework II Artifacts casework studies and success rates Int J Legal Med 109 195 204 Straub R E Speer M C Luo Y Rojas
120. pF STR Identifiler Plus Kit genotype Sensabaugh 1982 The P values for the populations described in this section are then approximately 1 7 64 X 10 African American 1 2 00 X 10 U S Caucasian 1 1 31 X 10 U S Hispanic and 1 2 76 X 10 6 Native American 124 AmpFtSTR Identifiler Plus User Guide Probability of Paternity Exclusion Probability of Paternity Exclusion Table 13 shows the Probability of Paternity Exclusion Pp values of the AmpF STR Identifiler Plus Kit STR loci individually and combined Table 13 Probability of Paternity Exclusion values for the AmpF STR Identifiler Plus Kit loci Locus nineteen Sieja U S Hispanic Pyar aa CSF1PO 0 545 0 496 0 450 0 409 D2S1338 0 748 0 725 0 671 0 399 D381358 0 591 0 630 0 495 0 510 D5S818 0 506 0 440 0 525 0 601 D7S820 0 591 0 582 0 574 0 492 D881179 0 580 0 680 0 599 0 601 D13S317 0 383 0 487 0 638 0 370 D16S539 0 649 0 566 0 567 0 428 D18S51 0 760 0 731 0 767 0 329 D19S433 0 601 0 531 0 678 0 360 D21S11 0 737 0 708 0 586 0 399 FGA 0 760 0 766 0 739 0 309 THO1 0 492 0 566 0 618 0 646 TPOX 0 521 0 329 0 392 0 687 VWA 0 709 0 625 0 555 0 528 Combined 0 9999996 0 9999992 0 9999990 0 9999527 The 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 AmpF STR Identifiler Plus Kit STR loci Chakrab
121. port Fa Files of type At Files Cancel 6 Import Identifiler Plus Bins v1X a Select the AmpFLSTR JIdentifiler Plus v1X folder in the navigation pane Panel Manager File Edit Bins View Help u xa N ELICEI x Panel Name Comment E am Panel Manager i ED AmpFLSTR_Panels_v1X 1 fidentifiler_Plus_Panels_vix null fimi AmpFLSTR Identifiler Plus v1X b Select File Import Bin Set to open the Import Bin Set dialog box c Navigate to then open the AmpFLSTR Identifiler Plus v1X folder 62 AmpFtSTR Identifiler Plus User Guide Set up GeneMapper ID X Software for data analysis d Select Identifiler_Plus_Bins_v1X then click Import Note Importing this file associates the bin set with the panels in the Identifiler_Plus_Panels_v1X folder Look in je Identifiler Plus Analysis Files GMIDX v CIE r My Recent Documents Ef CE_G5_Identifiler_Plus_G5500 xml Identifier_Plus_Bins_v1X txt E Identifier Plus Panels _v1X txt E Identifier_Plus_Stutter_v1X txt Identifiler Plus AnalysisMethod v1x xml Desktop 7 View the imported panels in the navigation pane a Double click the AnpFLSTR_Identifiler_Plus_v1X folder to view the Identifiler_Plus_Panel_v1X folder b Double click the Identifiler_Plus_Panel_v1X folder to display the panel information in the right pane and the markers below it Panel Manager File Edit Bins View Help
122. precision sizing 83 primers Amelogenin 97 volume per reaction 28 probability of identity definition 124 values 124 Q quantification DNA 25 R radioactive waste handling 135 reaction mix for PCR 28 reactions preparing for PCR 28 reagents user supplied 25 run module electrophoresis 37 39 S safety biological hazards 135 chemical waste 134 guidelines 132 134 sample files fsa 47 61 sample preparation 28 DNA negative control 28 DNA positive control 28 standards 17 setup tools PCR 24 size deviation sample alleles and ladder alleles 82 sizing precision 83 software instrument compatibility 15 species specificity 98 split peaks A nucleotide addition 95 STRBase 111 stutter products 90 AmpF amp TR Identifiler Plus User Guide Index T thermal cycling parameters validation of 80 programming conditions 30 training information on 142 U user supplied reagents 25 V validation annealing temperatures 80 characterization of loci 97 developmental 78 effect of DNA quantity 100 experiments to evaluate 78 importance of 78 magnesium chloride concentration 79 mixture studies 105 mutation rate 123 PCR cycle number 80 population data 111 probability of identity 124 probability of paternity exclusion 125 sensitivity 100 size deviation sample and ladder alleles 82 species specificity 98 thermal cycling parameters 80 W warranty 142 waste disposal guidelines 134 waste profiles descript
123. raded DNA 30 130 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 chemical insults could potentially affect the analytical process then the process should be evaluated using known samples to determine the effects of such factors SWGDAM July 2003 As the average size of degraded DNA approaches the size of the target sequence the amount of PCR product generated is reduced This reduction is due to the reduced number of intact templates in the size range necessary for amplification Degraded DNA was prepared to examine the potential for differential amplification of loci High molecular weight Raji DNA was sonicated and incubated with increasing doses of DNase I 0 to 6 Units for 20 minutes Bender et al 2004 The DNA was examined by agarose gel analysis to determine the average size of the DNA fragments at each time point One nanogram of degraded DNA was amplified using the AmpF STR Identifiler Plus Kit As the DNA became increasingly degraded the loci became undetectable according to size Preferential amplification was not observed The loci failed to robustly amplify in the order of decreasing size as the extent of degrada
124. rding to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply Biological hazard safety General AN WARNING BIOHAZARD Biological samples such as tissues body fluids biohazard infectious agents and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using 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 U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories stock no 017 040 00547 4 bmbl od nih gov Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 www access gpo gov nara cfr waisidx_01 29cfr1910a_01 html Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials Ad
125. s then analyzed by the GeneMapper ID or ID X Software Instrument and software Instrument Pata Collection Analysis software DA Software compatibility 3500 3500xL 3500 Series GeneMapper ID X Software Data Collection v1 2 or later Software v1 0 or later 3130 3130xI 3 0 or later e GeneMapper ID Software v3 2 1 3100 3100 1 1 3100 and Avant 1 0 31 00 Avant GeneMapper ID X Software v1 0 1 or later 2 0 310 3 1 3 0 t We conducted validation studies for the AmpF STR9 Identifiler Plus Kit using this configuration About Applied Biosystems fluorescent multi color dye technology allows the analysis of multicomponent multiple loci including loci that have alleles with overlapping size ranges Alleles for analysis overlapping loci are distinguished by labeling locus specific primers with different colored dyes Multicomponent analysis is the process that separates the five different fluorescent dye colors into distinct spectral components The four dyes used in the AmpF STR Identifiler Plus PCR Amplification Kit to label samples are 6 FAM VIC NED and PET dyes The fifth dye LIZ dye is used to label the GeneScan 500 LIZ Size Standard AmpFtSTR Identifiler Plus User Guide 15 Chapter 1 Overview How Each of these fluorescent dyes emits its maximum fluorescence at a different multicomponent wavelength During data collection on the Applied Biosystems and ABI PRISM anal
126. s v1 CE G5 Identifiler Plus GS500 amp BI3130 9 I Cerebus_3130XL IB_0337 Sample Identifiler Plus amp nalysisMethod v1 Identifiler Plus Panels v1 CE GS ldentifiler Plus GS500 amp BI3130 10 lm Cerebus_3130XL IB_0338 Sample Identifiler Plus amp nalysisMethod v1 Identifiler Plus Panels v1 CE GS ldentifiler Plus GS500 amp BI3130 11 L3 Cerebus 3130XL IB 0339 Sample Identifiler Plus amp nalysisMethod v1 Identifiler Plus Panels v1 CE G5 ldentifiler Plus GS500 amp BI3130 12 i Cerebus_3130XL IB_0340 Sample Identifiler Plus amp nalysisMethod v1 Identifiler Plus Panels v1 CE G5 ldentifiler Plus GS500 amp BI3130 13 i Cerebus_3130XL IB_0341 Sample Identifiler Plus amp nalysisMethod v1 Identifiler Plus Panels v1 CE GS ldentifiler Plus GS500 amp BI3130 14 i Cerebus_3130XL IB_0342 Sample Identifiler Plus amp nalysisMethod v1 Identifiler Plus Panels v1 CE G5 ldentifiler Plus GS500 amp BI3130 15 ie Cerebus 3130XL ladder3 Allelic Ladder Identifiler_Plus_AnalysisMethod_v1 Idertifiler_Plus_Panels_v1 CE G5 ldentifiler Plus GS500 amp BI3130 For more information about any of these tasks refer to the GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide PN 4338775 Examine and edit a project 58 You can display electropherogram plots from the Samples and Genotypes tabs of the Project window to examine the data These procedures start with the Samples tab of the Project window assu
127. sized polypropylene tube Vortex the tube then centrifuge briefly Into a 0 2 mL or 0 5 mL sample tube add e 25 uL of the formamide size standard mixture 1 5 UL of PCR product or allelic ladder Seal the tubes with the appropriate septa then briefly vortex and centrifuge the tubes to ensure that the contents of each tubes are mixed and collected at the bottom Heat the tubes in a thermal cycler for 3 minutes at 95 C Immediately place the tubes on ice for 3 minutes Place the sample tray on the autosampler Start the electrophoresis run AmpFtSTR Identifiler Plus User Guide Part Number 4440211 Rev F 2 2015 Chapter 4 Data Analysis AmpFtSTR Identifiler Plus User Guide AmpFtSTR Identifiler Plus User Guide Data Analysis This chapter covers Section 4 1 GeneMapper ID Software eese 46 m Overview of GeneMapper ID Software 4444444111111 46 m Set up GeneMapper ID Software for data analysis 47 m Analyze and edit sample files with GeneMapper ID Software 57 m Examine and edit a project WA kK KK KK KK KK KK KK KK KK KK KK KK KK KK 58 Section 4 2 GeneMapper ID X Software cese 60 m Overview of GeneMapper ID X Software 424444111112 60 m Set up GeneMapper ID X Software for data analysis 61 m Analyze and edit sample files with GeneMapper ID X Software 73 B Examine and edit a project
128. sults 118 Table 10 AmpF STR Identifiler Plus Kit allele frequencies continued Allele Fiir Gi le ai ic dede n 2 357 n 349 n 191 8 36 13 53 30 49 66 37 96 9 21 15 11 60 7 24 4 19 10 9 24 4 30 4 66 3 40 11 21 43 25 93 27 24 39 27 12 3 08 4 73 10 52 14 92 13 t t t t vWA 11 0 28 t 0 17 t 12 t t t 0 26 13 1 26 0 43 t 0 26 14 7 14 8 31 6 90 4 45 15 20 03 11 32 10 00 7 07 16 26 75 23 35 34 31 32 98 17 20 59 24 50 21 55 33 51 18 14 71 22 49 18 45 15 45 19 6 72 8 31 7 07 4 71 20 1 96 1 15 1 38 1 05 21 0 28 t 0 17 0 26 22 0 28 t t t 23 t t t t 24 t 0 14 t t t Aminimum allele frequency 0 796 for the African American database 0 796 for the U S Caucasian database 0 996 for the U S Hispanic database and 1 396 for the Native American database is suggested by the National Research Council in forensic calculations Low frequency alleles Some alleles of the AmpF STR Identifiler Plus Kit loci occur at a low frequency For these alleles a minimum frequency 5 divided by 2n where n equals the number of individuals in the database was assigned for the AmpF STR Identifiler Plus Kit African American Native American U S Caucasian and U S Hispanic databases as suggested in the 1996 report of the Committee on DNA Forensic Science National Research Council 1996 These databases are summarized in Table 10 on page 112 The minimum rep
129. t and Reader are registered trademarks of Adobe Systems Inc FTA is a registered trademark of Whatman Limited Contents Preface San en kid hoe n na A S RA EIN Naa hus 7 Revislof MISTORY A xx rhe ay Ran ee QU e RUPEE ERR ue CREE TE 7 PUrDOSE Mens aa Helle oe EP 7 Pullout chapters sites E a Dn a abay RA COR ce S PRA Stee 7 Chapter 1 OVEIVIOWS th a desde d cree ee b E AO e d Eb ZEE 00 w 9 Product OVervIew e Rota rec mete ete DO NR ok OWE qa dep dw at der sl 10 Workflow overview 4S kk kk kk kk kk kk kk kk RR hr nn 14 Instrument and software overview ssl 15 Materials and equipment uuuauaeaaaaaa aaa eee 17 Chapter 2 PGR AmplifiCationm s 2 cet bis ee pote lk kt EG ae 23 PCR Wwork areas ecu eR waski UR US ob te vee wa ER dev GG a 24 Required user supplied reagents 4 kk kk kk kk kK KK KK KK KK KK KK KK KK KK KK KK kk 25 DNA quantifiCation m sets Re i a RARE aa ta ep ca Ske hans 25 Prepare the amplification kit reactions uuaaaaaaaaaaaaa aaa KK KK ee 28 Select the appropriate PCR cycle number kk kk kk kK KK KK KK KK KK RR K KK KK 29 Perform POR za Qisa S ka es hee Dv Sed hay te wis Bie P edet soie E 30 Amplification using bloodstained FTA cards 0 0 ccc KK KRE RR RY KK 31 Chapter 3 Electrophoresis 2 353 9 ede utes a a gute l Wl k Ara prx 35 Allelic ladder requirements lk kk kk kk kk KK kk KK KK KK KK KK KK KK KK KK KK KK KK kk 36 Section 3 1 3100 3100 Avant and 31
130. ta produced with either 28 or 29 cycles of amplification on the genetic analyzers used during developmental validation of the Identifiler Plus Kit However you should consider possible noise and artifacts when interpreting data from the AmpF STR Identifiler Plus PCR Amplification Kit on the Applied Biosystems 3130 3130x ABI PRISM 3100 3100 Avant and ABI PRISM 310 Genetic Analyzers AmpFtSTR Identifiler Plus User Guide Characterization of loci Characterization of loci SWGDAM The basic characteristics of a genetic marker must be determined and documented guideline 2 1 SWGDAM July 2003 This section describes basic characteristics of the 15 loci and the sex determining marker Amelogenin which are amplified with the AmpF STR Identifiler Plus Kit These loci have been extensively characterized by other laboratories Nature of the The primers for the Amelogenin locus flank a 6 nucleotide deletion within intron 1 polymorphisms ofthe X homologue Amplification results in 107 nt and 113 nt products from the X and Y chromosomes respectively Sizes are the actual nucleotide size according to sequencing results including 3 A nucleotide addition The remaining AmpF STR Identifiler Plus Kit loci are all tetranucleotide short tandem repeat STR loci The length differences among alleles of a particular locus result from differences in the number of 4 nt repeat units All the alleles in the AmpF STR Identifiler
131. th the desired sensitivity of at least 1 0 ng of AmpF STR Control DNA 9947A was produced For example annealing extension temperatures were tested at 55 57 59 61 and 63 C for 3 minute hold times in the Silver 96 Well GeneAmp PCR System 9700 Figure 16 The PCR products were analyzed using the Applied Biosystems 3130x Genetic Analyzer Of the tested annealing extension temperatures 55 to 61 C produced robust 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 extension temperature of 59 C 155 195 235 275 315 355 395 4000 30004 55 i C 2000 10001 o 115 155 195 235 275 315 355 395 80 PCR cycle number Figure 16 Electropherograms obtained from amplification of 1 0 ng of Control DNA 9947A at annealing extension temperatures of 55 C 57 C 59 C 61 C and 63 C analyzed on the Applied Biosystems 3130x Genetic Analyzer Y axis scale 0 to 4 000 RFUs AmpF STR Identifiler Plus PCR Amplification Kit reactions were amplified for 26 27 28 29 and 30 cycles on the Silver 96 Well GeneAmp PCR System 9700 using 1 0 ng from three DNA samples As expected the amount of PCR product increased with the number of cycles A full profile was generated at 26 cycles and off scale data were c
132. thod Editor HID xj General Allele Peak Detector Peak Quality Quality Flags C D 2 lt ko zo 0 iw e e Eh E 0 r Signal level Homozygous min peak height j 00 0 Heterozygous min peak height 0 0 rHeterozygote balance Min peak height ratio oz r Peak morphology Max peak width basepairs j 5 r Pull up peak Pull up ratio 0 05 Allele number Max expected alleles B Factory Defaults X Cancel Figure 8 Analysis Method Editor HID Peak Quality tab settings IMPORTANT Laboratories need to perform the appropriate internal validation studies to determine the minimum heterozygous and homozygous minimum peak height thresholds and the minimum peak height ratio threshold that allow for reliable interpretation of AmpF STR Identifiler Plus PCR Amplification Kit data AmpFtSTR Identifiler Plus User Guide 55 Section 4 1 GeneMapper ID Software Analysis Method Editor HID Figure 9 Analysis Method Editor HID Quality Flags tab settings Import an HID The size standard for the Identifiler Plus Kit uses the following GS500 peaks in its size standard sizing algorithm 75 100 139 150 160 200 300 350 400 and 450 Use the following procedure to import the size standard for the AmpF STR Identifiler Plus PCR Amplification Kit from the folder that you downloaded from our web site into the GeneMapper
133. tic 3100 Avant Injection condition 3 KV 5 sec Analyzers Protocols for Processing AmpF STR Analyzer PCR Amplification Kit PCR Products User GS500Analysis gsp Bulletin PN 4332345 t We conducted validation studies for the Identifiler Plus Kit using this configuration AmpFtSTR Identifiler Plus User Guide 37 Q e o T ES 5 mej w EJ a o S ES e e e z o 5 2 7 Section 3 1 3100 3100 Avant and 3130 3130xl instruments Prepare samples for electrophoresis on the 3100 3100 Avant or 3130 3130x instrument 38 Prepare the Prepare the samples for electrophoresis on the 3100 3100 Avant or 3130 3130x instrument immediately before loading Calculate the volume of Hi Di Formamide and GeneScan 500 LIZ Size Standard needed to prepare the samples using the table below Reagent Volume per reaction uL GeneScan 500 LIZ Size Standard 0 3 Hi Di Formamide 8 7 Note Include additional samples in your calculations to provide excess volume for the loss that occurs during reagent transfers IMPORTANT The volume of size standard indicated in the table is a suggested amount Determine the appropriate amount of size standard based on your results and experiments Pipette the required volumes of components into an appropriately sized polypropylene tube Vortex the tube then centrifuge briefly Into each well of
134. tifiler Plus User Guide 31 Part Number 4440211 Rev F 2 2015 Chapter 3 Electrophoresis AmpFtSTR Identifiler Plus User Guide AmpFtSTR Identifiler Plus User Guide Electrophoresis This chapter covers amp Allelic ladder requirements uuaeaaaaaa aaa KK KK KK KK KK KK KK 36 Section 3 1 3100 3100 Avant and 3130 3130xlinstruments 37 m Set up the 3100 3100 Avant or 3130 3130x1 instrument for electrophoresis 37 Em Prepare samples for electrophoresis on the 3100 3100 Avant or 3130 3130xl Instr mert SAND Dy Mc eene aa bun eras ete tat on acne Don eect 38 Section 3 2 310 instrument 0 cee ce cee kk kk kk kk kk 39 m Set up the 310 instrument for electrophoreSis 2 42221111 39 n Prepare samples for electrophoresis on the 310 instrument 40 AmpFtSTR Identifiler Plus User Guide 35 Chapter 3 Electrophoresis Allelic ladder requirements To accurately genotype samples you must run an allelic ladder sample along with the unknown samples For samples run on the ABI PRISM 310 Genetic Analyzer Run at least one allelic ladder for every 10 sample injections ABI PRISM 3100 and Applied Biosystems 3130 series Genetic Analyzers Run at least one allelic ladder per every set of 16 samples IMPORTANT Variation in laboratory temperature can affect fragment migration speed and result in sizing variation We recommend the following freq
135. tifiler Plus Analysis Files GMIDX e CE G5 Identifiler Plus GS500 xml Identifiler Plus AnalysisMethod v1X xml ICE G5 Identifiler Plus G9500 xml ED punecom mo 72 AmpFtSTR Identifiler Plus User Guide Analyze and edit sample files with GeneMapper ID X Software Analyze and edit sample files with GeneMapper D X Software Analyze a project 1 In the Project window select File Add Samples to Project then navigate to the disk or directory containing the sample files 2 Apply analysis settings to the samples in the project Parameter Settings Sample Type Select the sample type Analysis Method Identifiler_Plus_AnalysisMethod_v1X Panel Identifiler_Plus_Panel_v1X Size Standard CE G5 Identifiler Plus GS500 t For more information about how the Size Caller works refer to the ABI PRISM GeneScan Analysis Software for the Windows NT9 Operating System Overview of the Analysis Parameters and Size Caller User Bulletin PN 4335617 S The following fragments are defined for the CE G5 Identifiler Plus GS500 size standard provided with the AmpF STR Identifiler Plus Kit 75 100 139 150 160 200 300 350 400 and 450 For additional information about size standards refer to the GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide PN 4338775 Appendix D Neither the 250 nt nor the 340 nt peak are included in the size standard definition Thes
136. tin 4440754 ProFlex PCR System Kit Validation User Bulletin 100031595 GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide 4338775 GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial 4335523 Installation Procedures and New Features for GeneMapper ID Software v3 2 User Bulletin 4352543 GeneMapper ID X Software Version 1 0 Getting Started Guide 4375574 GeneMapper ID X Software Version 1 0 Quick Reference Guide 4375670 GeneMapper ID X Software Version 1 0 Reference Guide 4375671 AmpFtSTR Identifiler Plus User Guide 141 Documentation How to obtain support For HID support In North America Send an email to HIDTechSupport lifetech com or call 888 821 4443 option 1 Outside North America Contact your local support office For the latest services and support information for all locations go to www lifetechnologies com At the website you can Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities Search through frequently asked questions FAQs Submit a question directly to Technical Support Search for user documents SDSs vector maps and sequences application notes formulations handbooks certificates of analysis citations and other product support documents Obtain information about customer training Download software updates and patches Limited product warranty 142 Life Technologies Corp
137. ting started with Identifiler Plus kit data analysis Analysis parameters should be established by each individual laboratory based on the laboratory s internal validation studies 1 Select Tools GeneMapper ID X Manager to open the GeneMapper D X Manager 2 Import an analysis method a Select the Analysis Methods tab then click Import z GeneMapper ID X Manager x Find Name Containing Projects Analysis Methods Table Settings Plot Settings Matrices Size Standards Report Settings Name Last Saved Owner Instrument Analysis Type Description AmpFLSTR Analysis Method 2007 12 18 16 18 2 gmidx HID New Open Save As Import Export Delete Help Done b Navigate to then open the Identifiler Plus Analysis Files GMIDX folder c Select Identifiler_Plus_AnalysisMethod_v1X then click Import to import Identifiler_Plus_AnalysisMethod_v1X into the GeneMapper ID X Software database Import Analysis Method x Lookin e Identifiler Plus Analysis Files GMIDX v i2 E353 CE G5 Identifiler Plus GS500 xml fe Identifiler Plus AnalysisMethod v1X xml My Recent Documents 6 Desktop A WAPEETUZUEJ File name fidentifiler_Plus_AnalysisMethod_v1x xml Import j f Files of type m Files xml 7 Cancel 3 To view the settings for Identifiler Plus AnalysisMethod v1X select the Analysis Methods tab then select Identifiler Plus AnalysisMet
138. tion 470 2000 30 800 1 udi ali l progressed Untreated Ld ull 4 30 130 3 Units DNase 170 210 250 230 330 400 30 130 300 w 4 Units DNase I 170 210 250 290 330 s0 430 200 1 5 Units DNase I 170 210 250 230 330 160 420 80 40 0 102 6 Units DNase I Figure 27 28 PCR cycle amplification of Raji DNA samples sonicated and incubated with increasing doses of DNase I Panels 1 2 3 4 and 5 correspond to 0 3 4 5 and 6 units of DNase I AmpFtSTR Identifiler Plus User Guide 9947A b Effect of inhibitors hematin Stability 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 that it subsequently interferes with PCR by inhibiting polymerase activity To examine the effects of hematin on the amplification results obtained by the Identifiler Plus Kit Control DNA 9947A 1 ng input DNA was amplified with increasing concentrations of hematin for 28 cycles of amplification Figure 28 on page 103 The concentrations of hematin used were 0 UM 100 uM 200 uM and 300 UM see Table 6 Mark Sample for Deletio 4000 3000 20004 1000 ns Ta Ta 4000 470 210 250 290 330 Control samples IT Mark Sample f
139. tions References Software Windows 3 1 GS STR POP4 1mL G5 v2 md5 ABI PRISM 310 Genetic Analyzer User s Manual w dr Injection condition 15 kV 5 sec ARROWS EN 4317588 ABI PRISM 310 Protocols for Processing 3 0 AmpFtSTR PCR Amplification Kit Products with Windows Microsoft Windows NT Operating System User NT and Bulletin PN 4341742 Windows 2000 t We conducted concordance studies for the Identifiler Plus kit using this configuration gt w U J 7 E 3 e je o c o 2 5 m lt N re AmpFtSTR Identifiler Plus User Guide 39 Section 3 2 310 instrument Prepare samples for electrophoresis on the 310 instrument Prepare the Prepare the samples for capillary electrophoresis on the 310 instrument immediately samples before loading l 40 Calculate the volume of Hi Di Formamide and GeneScan 500 LIZ Internal Size Standard needed to prepare the samples using the table below Reagent Volume per reaction uL GeneScan 500 LIZ9 Size Standard 0 5 Hi Di Formamide 24 5 Note Include additional samples in your calculations to provide excess volume for the loss that occurs during reagent transfers IMPORTANT The volume of size standard indicated in the table is a suggested amount Determine the appropriate amount of size standard based on your results and experiments Pipette the required volumes of components into an appropriately
140. uency of allelic ladder injections which should account for normal variation in run speed However during internal validation studies verify the required allelic ladder injection frequency to ensure accurate genotyping of all samples in your laboratory environment Applied Biosystems 3130x or ABI PRISM 3100 systems One ladder per injection one injection 16 samples 15 samples 1 allelic ladder Applied Biosystems 3130 or ABI PRISM 3100 Avant One ladder for every 4 injections one injection 4 samples When genotyping it is critical to use an allelic ladder run under the same conditions as the samples because 36 Size values obtained for the same sample can differ between instrument platforms because of different polymer matrices and electrophoretic conditions Variation in laboratory temperature can affect migration speed see IMPORTANT above AmpFtSTR Identifiler Plus User Guide Section 3 1 3100 3100 Avant and 3130 3130xl instruments Section 3 1 3100 3100 Avant and 3130 3130xl instruments Set up the 3100 3100 Avant or 3130 3130x instrument for electrophoresis Reagents and parts 3100 3100 Avant Table 3 on page 18 lists the required materials not supplied with the AmpF STR Identifiler Plus Kit IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the AmpF STR Identifiler Plus Primer Set from light when not in use Amplified DNA AmpF ST
141. uly 2003 PCR components We examined the concentration of each component of the AmpF STR Identifiler Plus PCR Amplification Kit The concentration for each individual component was established to be in the window that meets the reproducible performance characteristics of specificity and sensitivity For example various magnesium chloride concentrations were tested on the Applied Biosystems 3130x Genetic Analyzer The amplification of 1 0 ng of the Control DNA 9947A for 28 cycles is shown in Figure 15 We observed that the performance of the multiplex is robust within a 30 window of the optimum magnesium chloride concentration a z zir si B 96 Change m 30 on 20 10 f a 0 zm 10 m 20 m 30 Figure 15 1 0 ng of Control DNA 9947A amplified with the AmpF STR Identifiler Plus Kit for 28 cycles in the presence of varying concentrations of magnesium chloride and analyzed on the Applied Biosystems 3130x Genetic Analyzer AmpFtSTR Identifiler Plus User Guide 79 Chapter 5 Experiments and Results Thermal cycler 75 parameters 115 Thermal cycling parameters were established for amplification of the AmpF STR Identifiler Plus PCR Amplification Kit Thermal cycling times and temperatures of GeneAmp PCR systems were verified Varying annealing extension and denaturation temperature windows were tested to verify that a specific PCR product wi
142. wn in Figures 30 28 PCR cycles and 31 29 PCR cycles on page 108 where samples A and B were mixed according to the ratios provided Using either the 28 or 29 PCR cycle protocol the minor component allele calls at non overlapping loci are highlighted The amplification of the minor contributor at 3 1 7 1 0 875 0 125 ng and 10 1 0 9 0 09 ng mixture ratios was readily typeable 15 1 0 9375 0 0625 ng mixture ratios resulted in full or partial profiles for the minor component The profiles of these samples are described in Table 9 AmpFtSTR Identifiler Plus User Guide 107 Chapter 5 Experiments and Results 15 1 Y 8 9 16007 1200 soot 400 A Figure 30 Amplification of DNA mixtures at various ratios using the 28 PCR cycle protocol Minor allele peaks that do not overlap with the major contributor peaks are highlighted 108 AmpFtSTR Identifiler Plus User Guide Mixture studies 30 130 170 210 250 290 330 3000 2000 1 0 1000 0 90 130 3001 100 800 130 170 210 250 290 330
143. xplained in Import an HID analysis method on page 51 C D 2 0 lt ko go 0 we n 9 EB E 0 Importa size standard as explained in Import an HID size standard on page 56 Define custom views of analysis tables Define custom views of plots For more info For details about GeneMapper ID Software workflow and features refer to e GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide PN 4338775 GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial PN 4335523 Installation Procedures and New Features for GeneMapper ID Software v3 2 User Bulletin PN 4352543 AmpFtSTR Identifiler Plus User Guide 47 Section 4 1 GeneMapper ID Software Import panels To import the AmpF STR Identifiler Plus Kit panel and bin set from our web site and bins into the GeneMapper D Software v3 2 1 database 1 Download and open the file containing panels and bins a From the Support menu of www appliedbiosystems com select Software Downloads Patches amp Updates Select GeneMapper ID Software v3 2 from the drop down menu Select Updaters amp Patches and download the file Identifiler Plus Analysis Files GMID b Unzip the file 2 Start the GeneMapper JD Software then log in with the appropriate user name and password IMPORTANT If you need logon instructions refer to page 2 7 of the GeneMapper ID Software Version 3 1
144. ysis works instruments the fluorescence signals are separated by diffraction grating according to their wavelengths and projected onto a charge coupled device CCD camera in a predictably spaced pattern The 6 FAM dye emits at the shortest wavelength and it is displayed as blue followed by the VIC dye green NED dye yellow PET dye red and LIZ dye orange Although each of these dyes emits its maximum fluorescence at a different wavelength there is some overlap in the emission spectra between the dyes Figure 3 The goal of multicomponent analysis is to correct for spectral overlap Dyes 6 FAM VIC NED PET LIZ Normalized Emission 500 550 600 650 700 Wavelength nm Figure 3 Emission spectra of the five dyes used in the AmpF STR Identifiler Plus Kit 16 AmpFtSTR Identifiler Plus User Guide Materials and equipment Kit contents and Materials and equipment The AmpF STR Identifiler Plus PCR Amplification Kit contains materials storage sufficient to perform 50 100 200 or 1000 amplifications at 25 UL reaction volumes IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the primer set from light when not in use Amplified DNA AmpF STR Identifiler Plus Allelic Ladder and GeneScan 500 LIZ Size Standard should also be protected from light Keep freeze thaw cycles to a minimum 50X 100X 200X 1000X Component Description Volume Volume V

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