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AmpFlSTR® Profiler Plus® and Profiler Plus® ID PCR Amplification

1. S panel Manager a Meke Name Dye Color Mim Size Diax Size Control Alleles Marker E Marker Comments Ladder Alleles e Eb AmpFLSTR Panels v2 4 pssi358 bue 980 fso fisas 041 none 1243141516171819 o Bue v2 2 vwa bue 1510 2030 17418 4 011 none 1142 3 4 15 15 7 18 19 20 21 z G v2 S erenn l 3 FGA bue 206 25 3600 23 24 4 041 none 18 19 20 21 22 23 24 25 26 26 2 27 26 29 30 gt Profiler v2 gel amp Profiler Pus v3 4 AMEL green 1060 1140 x 9 00 none XY xe D381358 s D8s1179 green 1180 fess 13 4 012 none 8 9 10 11 12 13 14 15 16 17 18 19 S vA 6 D21S11 green 1845 2475 30 4 043 none 24 2 25 26 27 28 28 2 29 29 2 30 30 2 31 31 2 32 32 2 33 33 2 34 34 2 35 35 2 36 38 ue m 7 D18551 green 26449 3500 15 19 4 048 none 9 10 10 2 1 12 13 13 2 4 14 2 5 16 17 16 19 20 21 22 23 24 25 26 Oo DBS1178 8 D55818 yellow 1280 180 0 11 4 01 none 788 0 1 12 3 14 15 6 del D24511 8 D135317 yelow 1920 2420 t 4 04 none 89101112131415 e D18551 10 D75820 yelow 2510 2985 10 11 4 0 09 none 6 7 8 9 10 11 12 13 14 15 E D55818 S D135317 m D75820 l e nli Y lt E 7 View the markers and display the Bin view in the navigation pane a Double click the AmpFLSTR_Panels_v2 folder to display its list of kits in the right pane b Double click the Profiler_Plus_v2 folder to display its list of markers below it c Select D8S1179 to display the Bin view for the marker in the right pane C Panel Manager F
2. 98 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Section 5 2 Developmental Validation of the Profiler Plus ID Kit 5 Developmental validation Thermal cycler Thermal cycling parameters were established for amplification of the Profiler Plus ID parameters Kit on the GeneAmp PCR Systems 9700 run in 9600 emulation mode Varying annealing temperature windows were tested to verify that a 2 0 C window produced a specific PCR product with the desired sensitivity of at least 2 ng of AmpF STR Control DNA 9947A The effects of annealing temperatures on the amplification of Profiler Plus ID Kit loci were examined using AmpF STR Control DNA 9947A and two DNA samples with one mutant D851179 allele The annealing temperatures tested were 55 57 59 61 and 63 C see Figure 19 for 1 minute hold times in the GeneAmp PCR System 9700 The PCR products were analyzed using the 377 Genetic Analyzer Figure 19 An amplification of 2 ng AmpF STR Control DNA 9947A amplified with the Profiler Plus D Kit while varying the annealing temperature analyzed on the Applied Biosystems 310 Genetic Analyzer 55 C g 0 lt o e me 3 o 2 a 5 for 9 1 p 9 Ex oy I 7 e iw a 57 C 620 59 C standard ah wall w a a 77 2470 61 C 63 C AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 99
3. Allelic dropout No allelic dropout was observed for any of the Test or Control mixes Genotype Genotypes for Test and Control mixes were 100 concordant Table 7 concordance Table 7 Sensitivity study genotype concordance DNA Input Amount Reagent Mix Genotype Concordance 3 250 pg Test A 100 E Test B 100 Si Test C 10096 Control A 10096 T Control B 100 zi 500 pg Test A 10096 S Test B 100 3 Test C 100 p Control A 100 5 Control B 100 3 1 ng Test A 10096 Test B 100 Test C 100 Control A 100 Control B 100 2ng Test A 100 Test B 100 Test C 100 Control A 100 Control B 100 Conclusions Laboratories can expect to obtain equivalent quality profiles across a wide range of forensic samples when using the Profiler Plus and Profiler Plus ID Kits containing the AmpliTaq Gold enzyme and 10X PCR Buffer II manufactured by Life Technologies as compared to the original Profiler Plus and Profiler Plus ID Kits containing AmpliTaq Gold enzyme and 10X PCR Buffer II manufactured by Roche Molecular Systems AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 107 5 Chapter 5 Performance Validation After Buffer and Enzyme Component Replacement Conclusions 108 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Troubleshooting Follow the actions recommended in this appendix to troubleshoot problems that occur during analysis
4. Im o r7 I I Gl Sample Quality per project based on sample PQVs SOS SSPK MIX OMR SQ CGQ Total of Samples All thresholds met One or more thresholds not met Samples 2 i 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 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 63 Chapter 4 GeneMapper ID X Software For more information For more information For more information refer to e GeneMapper ID X Software Version 1 0 Getting Started Guide Pub no 4375574 e GeneMapper ID X Software Version 1 0 Quick Reference Guide Pub no 4375670 e GeneMapper ID X Software Version 1 0 Reference Guide Pub no 4375671 s GeneMapper ID X Software Version 1 1 Mixture Analysis Getting Started Guide Pub no 4396773 s GeneMapper ID X Software Version 1 2 Reference Guide Pub no 4426481 e GeneMapper ID X Software Version 1 2 Quick Reference Guide Pub no 4426482 64 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Experiments and Results E Section 5 1 Developmental Validation of the Profiler Plus Kit 66 Overview io Noel See ee ie Aen iin asthe dep xs 66 Dev
5. Chapter 5 Developmental Validation of the Profiler Plus ID Kit Species specificity Species specificity DAB 8 1 2 2 Species Specificity Nonhuman studies 100 Species specificity sensitivity stability and mixture studies are conducted DAB 1998 Nonhuman DNA may be present in forensic casework samples The Profiler Plus ID Kit provides the required degree of specificity such that it is specific to primates for the species tested with the exception of the amelogenin locus The following experiments were conducted to investigate interpretation of Profiler Plus ID Kit results from nonhuman DNA sources The extracted DNA samples were amplified in Profiler Plus ID Kit reactions and analyzed using the Applied Biosystems 310 DNA Sequencer Primates Gorilla chimpanzee and orangutan and macaque 1 0 ng each e Non primates Mouse cat dog pig chicken cow and horse 2 5 ng each e Bacteria and yeast Escherichia and Saccharomyces 1 2 5 ng each The primate DNA samples all amplified producing fragments within the 100 400 base pair region Wallin et al 1998 Lazaruk et al 2001 The bacteria yeast mouse chicken cow and cat samples did not yield detectable product The dog pig and horse samples produced a a fragment near the Amelogenin locus in JOE dye see Figure 20 on page 100 Figure 20 Representative electropherograms of a primate non primates a microorganism and a negative c
6. Note We conducted validation studies for the Profiler Plus and Profiler Plus ID Kits using the Applied Biosystems 310 Genetic Analyzer running Mac OS This configuration is now obsolete Applied Biosystems fluorescent multi color dye technology allows the analysis of multiple loci including loci that have alleles with overlapping size ranges Alleles for overlapping loci are distinguished by labeling locus specific primers with different colored dyes Multicomponent analysis is the process that separates the four different fluorescent dye colors into distinct spectral components The three dyes used in the Profiler Plus and Profiler Plus ID Kits to label samples are 5 FAM JOE and NED dyes The fourth dye ROX is used to label the GeneScan 500 ROX Size Standard AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Chapter 1 Overview 1 Instrument and software overview How Each of these fluorescent dyes emits its maximum fluorescence at a different multicomponent wavelength During data collection on the Applied Biosystems and Applied Biosystems instruments the fluorescence signals are separated by diffraction grating according to their wavelengths and projected onto a charge coupled device CCD camera in a predictably spaced pattern The 5 FAM dye emits at the shortest wavelength and it is displayed as blue followed by the JOE dye green NED dye yellow and ROX dye
7. R reaction mix for PCR 21 reactions preparing for PCR 21 reagents not included with kit 113 user supplied 19 references 121 run module electrophoresis 27 29 31 Index S safety biohazard 118 chemical 118 Safety Data Sheets SDSs obtaining 128 sample preparation 21 DNA negative control 21 DNA positive control 21 standards 18 samples DNA from more than one individual 77 78 89 102 sexual assault DNA mixtures 89 software instrument compatibility 16 stutter check version 49 import 50 stutter peak or product 73 support obtaining 128 T technical support 128 thermal cyclers for use with kit 112 programming conditions 22 training information on 128 troubleshooting 109 U user supplied reagents 19 V validation Profiler Plus ID Kit developmental 98 mixture studies 102 optimizing PCR components 98 population data 103 sensitivity 101 species specificity 100 thermal cycler parameters 99 validation Profiler Plus Kit accuracy 70 characterization of loci 80 developmental 66 importance of 66 mixture studies 89 optimizing PCR components 66 AmpF amp STR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 131 Index performance after buffer and enzyme replacement 104 population studies 81 precision 70 probability of identity 96 probability of paternity exclusion 97 reproducibility 67 sensitivity 82 sexual assault DNA mixtures 89 species specificity 81
8. Walsh P S Metzger D A and Higuchi R 1991 Chelex 100 as a Medium for Simple Extraction of DNA for PCR Based Typing From Forensic Material Biotechniques 10 506 518 Walsh P S Varlaro J and Reynolds R 1992 A rapid chemiluminescent method for quantitation of human DNA Nucleic Acids Res 20 5061 5065 Weir B S 1996 Genetic data analysis II Sunderland MA Sinauer Associates Inc Zhou H G Sato K Nishimaki Y Fang L and Hasekura H 1997 The HumD21S11 system of short tandem repeat DNA polymorphisms in Japanese and Chinese Forensic Sci Intl 86 109 118 Ziegle J S Su Y Corcoran K P Nie L Mayrand P E Hoff L B McBride L J Kronick M N and Diehl S R 1992 Application of automated DNA sizing technology for genotyping microsatellite loci Genomics 14 1026 1031 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 125 Bibliography 126 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Documentation and Support Related documentation Document title Pub no AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits Human Identification Application Note 040302 3100 3100 Avant Data Collection v2 0 User Guide 4347102 3100 3100 Avant Genetic Analyzers Using Data Collection Software v2 0 User Bulletin 4350218 3100 Genetic Analyzer User Manual Data Collection v1 1 4315834 3100 3100 A
9. 1 1 Windows GeneScan36_POP4DyeSetF 3100 3100 Avant Genetic Analyzers NT Injection condition 3kV 10 sec Protocols for Processing AmpFI STRO PCR Amplification Kit PCR Products e GS500Analysis e User Bulletin Pub no 4332345 Applied 1 0 Windows GeneScan36A_POP4DyeSetFModule 3100 3100 Avant Genetic Analyzers Biosystems NT Injection condition 3 kV 5sec Protocols for Processing AmpFI STRO 3100 Avant e GS500Analysis os PCR Amplification Kit PCR Products Ties User Bulletin Pub no 4332345 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 27 wo En e e 69 zx e ur g 23 gt g fex wo i ies e I wo i S x 5 e x 3 bU 3 gt 7 3 Chapter 3 Electrophoresis Prepare samples for electrophoresis on the 3100 3100 Avant or 3130 3130xl instrument Prepare samples for electrophoresis on the 3100 3100 Avant or 3130 3130xl instrument Prepare the samples for electrophoresis immediately before loading 1 Calculate the volume of Hi Di Formamide and size standard needed to prepare the samples Reagent Volume per g reaction GeneScan 500 ROX Size Standard OSL Hi Di Formamide 85yL 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 Det
10. AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 15 1 Chapter 1 Overview Instrument and software overview Instrument and software overview Data Collection and GeneMapper D or D X Software Instrument and software compatibility About multicomponent analysis 16 This section provides information about the Data Collection Software versions required to run the Profiler Plus and Profiler Plus ID Kits on specific instruments The Data Collection Software provides instructions to firmware running on the instrument and displays instrument status and raw data in real time As the instrument measures sample fluorescence with its detection system the Data Collection Software collects the data and stores it The Data Collection Software stores information about each sample in a sample file fsa which is then analyzed by the GeneMapper ID or ID X Software Table 2 Software specific to each instrument Instrument Operating Data Colection Analysis software system Software 3500 3500xL e Windows XP 3500 Series GeneMapper D X Software e Windows Data Collection v1 2 or higher Vista Software v1 0 3130 3130xl Windows XP 3 0 GeneMapper D Software v3 2 1 3100 3100 Avant Windows NT 1 1 3100 and e GeneMapper D X Software v1 0 1 or higher 1 0 3100 Avant Windows 2000 2 0 310 Windows XP 3 1 e Windows NT 3 0 e Windows 2000
11. Amplified DNA work area IMPORTANT Place the thermal cyclers in the Amplified DNA Work Area You can use the following systems s GeneAmp PCR System 9700 with the Silver 96 Well Block s GeneAmp PCR System 9700 with the Gold plated Silver 96 Well Block IMPORTANT The Profiler Plus and Profiler Plus ID Kits are 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 kits Veriti 96 Well Thermal Cycler 112 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide C Ordering Information Equipment and materials not included Table 9 and Table 10 list required and optional equipment and materials not supplied with the Profiler Plus Kit Unless otherwise noted many of the items are available from major laboratory suppliers MLS Table 9 Equipment Equipment Source Applied Biosystems 3100 3100 Avant Genetic Analyzer Applied Biosystems 3130 3130xl Genetic Analyzer Applied Biosystems 3500 3500xL Genetic Analyzer for Human Identification Applied Biosystems 310 Genetic Analyzer Contact your local Life Technologies sales representative GeneAmp PCR System 9700 with the Silver 96 Well Block N8050001 GeneAmp PCR System 9700 with the gold plated silver 96 well block 4314878 Veriti 96 Well Ther
12. File names The file names shown in this section may differ from the file names you see when you download or import files If you need help determining the correct files to use contact your local Life Technologies Human Identification representative or go to www lifetechnologies com support gt Software Patches amp Updates gt GeneMapper ID Software Use the same analysis files for analysis of data from Profiler Plus and Profiler Plus ID Kits Before using the Before you can analyze sample fsa files using GeneMapper ID Software v3 2 1 for software for the the first time you need to first time Import panels and bins into the Panel Manager as explained in Import panels and bins on page 35 Createananalysis method as explained in Create an analysis method on page 38 Create a size standard as explained in Create size standard on page 43 Define custom views of analysis tables 34 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Section 4 1 GeneMapper ID Software 4 Set up GeneMapper ID Software for data analysis Refer to Chapter 1 of the GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial Pub no 4335523 for more information Define custom views of plots Refer to Chapter 1 of the GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial Pub no 4335523 for more information Import panels an
13. Kloosterman A D Kratzer A Lareu M V Meldegaard M Phillips C Pfitzinger H Rand S Sabatier M Scheithauer R Schmitter H Schneider P and Vide M C 1997 Report of the European DNA Profiling Group EDNAP an investigation of the complex STR loci D21511 and HUMFIBRA FGA Green E D et al 1991 Systematic generation of sequence tagged sites for physical mapping of human chromosome 7 using yeast artificial chromosomes Genomics 11 548 564 Hartl D L and Clark A G 1989 Principles of population genetics 2nd edition Sunderland MA Sinauer Associates Inc Holt C et al 2001 TWGDAM validation of AmpF STR PCR Amplification Kits for Forensic DNA Casework J Forensic Sci 47 1 Hudson TJ et al 1995 An STS based map of the human genome Science 270 1945 1954 Karlin S Cameron E C and Williams P T 1981 Sibling and parent offspring correlation estimation with variable family size Proc Natl Acad Sci USA 78 2664 2668 Kimpton C Walton A and Gill P 1992 A further tetranucleotide repeat polymorphism in the vWF gene Hum Mol Genet 1 287 Kimpton C P Gill P Walton A Urquhart A Millican E S and Adams M 1993 Automated DNA profiling employing multiplex amplification of short tandem repeat loci PCR Methods Appl 3 13 22 Kwok S and Higuchi R 1989 Avoiding false positives with PCR Nature 339 237 238 Lazaruk K et al 2001 Sequence variati
14. and Profiler Plus ID Kits Ordering Information on page 113 lists the required materials not supplied with the IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the primer set amplified DNA allelic ladder and size standard from light when not in use Keep freeze thaw cycles to a minimum The following table lists Data Collection Software and the run modules that can be used to analyze Profiler Plus and Profiler Plus ID Kits PCR products For details on the procedures refer to the documents listed in the table reference documents Data Operatin Run modules and Collection p g m References System conditions Software 3t Windows XP e GS STR POP4 1mL 310 Genetic Analyzer User s Manual Windows or or Injection condition Pub no 4317588 3 0t Windows9 NT 15 kV 5 sec 310 Protocols for Processing AmpF amp STR PCR Amplification and Windows 2000 User Bulletin Pub no 4341742 Kit Products with Microsoft Windows NT Operating System t We conducted concordance studies for the Profiler Plus and Profiler Plus D Kits using this configuration Prepare samples for electrophoresis on the 310 instrument Prepare the samples for electrophoresis immediately before loading 1 Calculate the volume of Hi Di Formamide and size standard needed to prepare the samples Reagent Volume per reaction GeneScan 500 ROX Size Standard 0 75 uL Hi
15. s GeneScan 500 ROX Size Standard Used for obtaining sizing results This standard which has been evaluated as an internal size standard yields precise sizing results for PCR products generated by the kit Order the GeneScan 500 ROX Size Standard Part no 4322682 separately s AmpF STR Profiler Plus Allelic Ladder or AmpF STR Profiler Plus ID Allelic Ladder Allelic ladders developed by Life Technologies for accurate characterization of the alleles amplified by the Profiler Plus and Profiler Plus ID Kits The allelic ladders contain most of the alleles reported for the 9 autosomal loci Refer to Table 1 on page 12 for a list of the alleles included in the allelic ladders AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Perform PCR S Required user supplied reagents ne 19 a DNA quantification emo iresi nE eR reri pleas Ye pee eis 19 B Prepare the amplification kit reactions 000 e ccc ee eee 21 S Select the correct PCR cycle number 1 0 6 eee 22 a Pertorm PCR euet ala ae Sher Poaceae oa eee bee ewes ale 22 m Amplification using bloodstained FTA cards 0 00 cece eee eee 23 Required user supplied reagents In addition to the Profiler Plus and Profiler Plus ID Kits 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
16. 50 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Section 4 2 GeneMapper ID X Software 4 Set up GeneMapper ID X Software for data analysis 5 Select AmpFLSTR_Panels_v2X or the version you installed then click Import Note Importing this file creates a new folder in the navigation pane of the Panel Manager AmpFLSTR_Panels_v2X This folder contains panels for multiple AmpF STR kits and associated markers Import Panels Look in G AmpFLSTR Analysis Files GMIDX i cu E AmpFLsTR Bins v2x My Recent gl AmpFLSTR Stutter v2X Documents E ReadMe_AmpFLSTR_v2x My Documents ys File name AmpFLSTR_Panels_v2x txt My Computer Files of type All Files C 0 2 0 D oc o 0 e v Xx GD e p 2 0 6 Import AmpFLSTR Bins v2X txt a Select the AmpFLSTR Panels v2X folder in the navigation pane Panel Manager File Edit Bins View Help iX ON UN R RS jonset ampristrons_ v i amp N S NU 8888888 Panel Name Comment Ofiler v1 1X null S sy Panel Manager C AmpFLSTR NGMSElect v2X 1 f Lar 2 SGM_Plus_v1 1X null da 3 NGM SElect v2 1X null 4 Identifiler Plus v1 1X null 5 NGM v3 1X null 1 6 Identifier Direct v1 1X null 7 b Select File Import Bin Set to open the Import Bin Set dialog box c Navigate to then open the AmpFLSTR Analysis Files GMIDX folder AmpFtSTR Prof
17. Cat T0223 To prepare low TE buffer 1 Mix together e 10mLof1M Tris HCl pH 8 0 e 0 2 mL of 0 5 M EDTA pH 8 0 e 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 Profiler Plus and Profiler Plus ID Kits is 1 0 2 5 ng in a maximum input volume of 20 uL for 28 PCR cycles AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 19 Chapter 2 Perform PCR DNA quantification If too much DNA is added to the PCR reaction then the increased amount of PCR product that is generated can result in e Fluorescence intensity that exceeds the linear dynamic range for detection by the instrument off scale data Off scale data are problematic because Quantitation peak height and area for off scale peaks is not accurate For example an allele peak that is off scale can cause the corresponding stutter peak to appear higher in relative intensity thus increasing the calculated percent stutter Multicomponent analysis of off scale data is not accurate and it results in poor spectral se
18. Check the version of files available for import into the Panel Manager a Select Panel Manager then select File Import Panels to open the Import Panels dialog box b Navigate to then open the Panels folder and check the version of panel bin and stutter files installed 5 If newer versions are available on the website download and import as described below Import panels To import the Profiler Plus Kit panel bin set and marker stutter from our web site bins and marker into the GeneMapper ID X Software database stutter 1 Download and open the file containing panels bins and marker stutter a Go to www lifetechnologies com support gt Software Patches amp Updates GeneMapper ID X Software Download the file AmpFLSTR Analysis Files GMIDX b Unzip the file 2 Start the GeneMapper ID X Software then log in with the appropriate user name and password IMPORTANT For logon instructions refer to the GeneMapper ID X Software Version 1 0 Getting Started Guide Pub no 4375574 3 Select Tools Panel Manager 4 Find then open the folder containing the panels bins and marker stutter a Select Panel Manager in the navigation pane Panel Manager File Edit Bins View SE Bae Be Panel Manager b Select File Import Panels to open the Import Panels dialog box c Navigate to then open the AmpFLSTR Analysis Files GMIDX folder that you unzipped in step 1 on page 50
19. Determine the appropriate amount of size standard based on your results and experiments AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 29 20 Chapter 3 Electrophoresis Prepare samples for electrophoresis on the 3500 3500xL instrument ener DES NS Pipette the required volumes of components into an appropriately sized polypropylene tube Vortex the tube then centrifuge briefly Into each well of a MicroAmp Optical 96 Well Reaction Plate or each MicroAmp optical strip tube add s 9uL of the formamide size standard mixture e luL of PCR product or allelic ladder Note For blank wells add 10 uL of Hi Di Formamide Seal the reaction plate or strip tubes with the appropriate septa then centrifuge to ensure that the contents of each well are collected at the bottom Heat the reaction plate or strip tubes in a thermal cycler for 3 minutes at 95 C Immediately put the plate or strip tubes on ice for 3 minutes Prepare the plate assembly then place on the autosampler Ensure that a plate record is completed and link the plate record to the plate Start the electrophoresis run AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Chapter 3 Electrophoresis 3 Set up the 310 instrument for electrophoresis Section 3 3 310 Instrument Set up the 310 instrument for electrophoresis Reagents and parts Electrophoresis software setup and Profiler Plus
20. equivalent to 50 ng human DNA and Rhodotorula The primate DNA samples all amplified producing fragments within the 75 350 base pair region Wallin et al 1998 The primate samples were subsequently sequenced by Life Technologies scientists The data revealed significant sequence homology between the primate and human DNA for the Profiler Plus Kit loci The bacteria yeast mouse chicken and horse samples did not yield detectable product The dog pig and cow samples produced a 103 bp fragment This 103 bp fragment was also amplified using the amelogenin primers alone This confirms amplification of the product obtained by Buel et al 1995 The 103 bp fragment is 4 bp shorter than the primate 107 bp X specific product including A addition Species specificity sensitivity stability and mixture studies are conducted DAB 1998 The amount of input DNA added to the PCR reaction should be 1 0 2 5 ng The DNA sample should be quantitated prior to amplification using a system such as the Quantifiler Human DNA Quantitation Kit Part no 4343895 Figure 13 on page 83 shows the effect of different amounts of AmpF STR Control DNA 9947A The final DNA concentration should be in the range of 0 05 0 125 ng uL so that 1 0 2 5 ng of DNA will be added to the PCR reaction in a volume of 20 uL If the sample contains degraded DNA amplification of additional DNA may be beneficial If too much DNA is added to the PCR reaction then the in
21. 11B 12 12 Non degraded DNA Profiler Plus BE 116 12 12 Non degraded DNA Profiler Plus 2 OW 11 12012 Non degraded DNA Profiler Plus oO 600 e 400 E 200 D 0 E 128 13 13 DNase 30 seconds Profiler Plus EH 126 13 13 DNase 30 seconds Profiler Plus 2 OW 12v 13013 DNase 30 seconds Profiler Plus Cm o 600 400 Q e 200 o 0 LA LL d T an AA POTETE E EHE 138 14514 DNase 1 minute Profiler Plus BDE 136 14 14 DNase 1 minute Profiler Plus l LIE 12v 14014 DNase 1 minute Profiler Plus U 300 S 200 ka 100 EX 2 Z DE 148 15 15 DNase 4 minutes Profiler Plus EHE 146 15 15 DNase 4 minutes Profiler Plus 7 LIO 14v 15 15 DNase 4 minutes Profiler Plus 5 A 300 E 200 100 0 DE 156 16 16 DNase 8 minutes Profiler Plus EH 156 1616 DNase 8 minutes Profiler Plus LIE 15v 16916 DNase 8 minutes Profiler Plus The loci failed to amplify in the order of decreasing size as the extent of degradation progressed D18551 was the first locus to drop out followed by FGA and so forth A similar result at each timepoint was obtained whether the DNA samples were amplified for each locus alone or co amplified with the Profiler Plus Kit Figure 17 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 87 Chapter 5 Experiments and Results Stability Matrix studies 88 Figure 17 Multiplex and single locus amplifications of the DNA sample incubated for 1 min
22. 1X null Identifiler Plus v1 1x null i gf Panel Manager O AmpFLSTR_NGMSElect_v2X C AmpFLSTR_NGM_v3x i D AmpFLSTR_Panels_v1X figi AmpFLSTR_Panel X b Select File Import Marker Stutter to open the Import Marker Stutter dialog box c Navigate to then open the AmpFLSTR Analysis Files GMIDX folder AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 53 n Chapter 4 GeneMapper ID X Software Set up GeneMapper ID X Software for data analysis d Select AmpFLSTR_Stutter_v2X then click Import Note Importing this file associates the marker stutter ratio with the bin set in the AmpFLSTR Panels v2X folder Import Marker Stutter Look in l AmpFLSTR Analysis Files GMIDX EJ ampFLSTR_Bins_v2x 4 E AmpFLSTR_Panels_v2 My Recent B AmpFLSTR Stutter v2X Documents 2 ReadMe AmpFLSTR v2X E Desktop My Documents 448 Filename AmpFLSTR_Stutter_v2 txt My Computer Ples of type All Files 10 View the imported marker stutters in the navigation pane a Double click the AmpFLSTR Panels v2X folder to display its list of kits in the right pane b Double click the Profiler Plus v1 1X folder to display its list of markers below it c Double click D21S11 to display the Stutter Ratio amp Distance view for the marker in the right pane File Edit Bins View Help Bx EEE HN S Binset AmpFLSTR
23. At 0 25 ng the FGA alleles of this sample both amplified in equal proportions and at 0 03 ng both alleles were no longer detectable These data underscore the importance of interpreting single peaks of low fluorescence signal with caution and on a case by case basis Effect of inhibitors Heme compounds have been identified as PCR inhibitors in DNA samples extracted from bloodstains Akane et al 1994 DeFranchis et al 1988 It is believed that the inhibitor is co extracted and co purified with the DNA and subsequently interferes with PCR by inhibiting polymerase activity AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Section 5 1 Developmental Validation of the Profiler Plus Kit 5 Stability Bovine serum albumin BSA can prevent or minimize the inhibition of PCR most likely by binding to the inhibitor Comey et al 1994 Since the presence of BSA can improve the amplification of DNA from blood containing samples BSA has been included in the AmpF STR PCR Reaction Mix at a concentration of 8 ug per 50 uL amplification BSA has also been identified as an aid in overcoming inhibition from samples containing dyes such as in denim To examine the effects of hematin on the Profiler Plus Kit amplification results DNA samples were amplified using the Profiler Plus Kit reagents including the BSA containing PCR reaction mix in the presence of varying concentrations of purified hematin The conce
24. Be Bs Be Ez Es Es Bo bs 2 C Mark Sample fce Deletion B RHI fesse 210 m 130 170 290 330 4006 n o mene c di Dci S raj 7 Mark Sample for Deletion 130 170 210 250 290 330 n 800 00 an 200 o a LA A d ae Dwell L ene A eda E EJ E bs ky ba by Bd k3 bs E E bol GGG bs sd os E E EJ E Bol ud Bod sd d os AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 13 1 Chapter 1 Overview Product overview Control DNA 9947A Figure 3 shows amplification of Control DNA 9947A using the Profiler Plus Kit profile Figure3 1 ng of Control DNA 9947A amplified with the Profiler Plus9 Kit and analyzed on the Applied Biosystems 3130xl Genetic Analyzer 14 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Chapter 1 Overview 1 Workflow overview Workflow overview Perform g PCR es ka ui Ea s e Quantifiler Duo DNA Quantification Kit 9 S Profiler Plus PCR Amplification Kit or a Profiler Plus D PCR Amplification Kit m ii F E y a GeneAmp PCR System 9700 Cycler Veriti 96 Well Thermal Cycler Perform le 3 e sec E 5 STG phoresis 3100 3100 Avant 3130 3130xl 3500 3500xL RY S Genetic Analyzer Genetic Analyzer Genetic Analyzer natyzer Analyze data 3 GeneMapper ID X or GeneMapper D Software
25. Bins v2X ciH LRR BEBNHEBNH o F Please enter the stutter filter s for D21511 marker here If left blank the global stutter filter will be applied G vWA i FGA Minus Stutter Plus Stutter GH AMEL D851179 Ratio From Distance To Distance Ratio lt D21511 p ee 4 75 1 Stutter Ratio amp Distance E 2 D18551 D55818 3 D135317 a D75820 H E COfiler_v1 1X Me Edt L Delete H E SGM Plus v1 1X amp CS Identifiler v1 1X amp C SEFiler Plus v1 1X amp C Profiler Plus CODIS v1 1X amp C3 COFiler CODIS v1 1X Ii A Idankifilae CODTS si 1v ai Reference Samples From Distance To Distance 11 Click Apply then OK to add the panel bin set and marker stutter to the GeneMapper ID 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 54 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Section 4 2 GeneMapper ID X Software 4 Set up GeneMapper ID X Software for data analysis Create an analysis Use the following procedure to create an analysis method for the Profiler Plus and method Profiler Plus ID Kits IMPORTANT Analysis methods are version specific so you must create an analysis method for each version of the software For examp
26. D8S1179 8 T 1 75 9 0 51t 1 00 10 3 08 8 00 11 4 36 6 25 12 10 51 14 25 13 19 74 34 75 14 33 33 18 75 15 21 03 13 00 16 6 41 2 00 17 1 03 0 25t 18 T T 19 T t D21S11 24 2 0 50 25 t di AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Section 5 1 Developmental Validation of the Profiler Plus Kit Population data Allele African American U S Caucasian n 195 n 200 26 0 261 t 27 5 90 3 75 28 21 80 16 25 28 2 t 1 29 20 00 20 75 29 2 t 0 50 29 3 0 26 t 30 16 15 26 25 30 2 2 56 250 31 8 97 5 50 31 2 5 90 10 50 32 0 77t 125 32 2 6 92 7 25 33 0 51t 0 25t 33 1 0 26t T 33 2 4 10 4 00 34 0 26t t 34 2 t 0 75t 35 3 59 t 35 2 t F 36 1 54 t 38 0 26t T D18S51 9 t 7 10 0 51f 0 50f 10 2 0 51f t 11 1 54 2 00 12 5 64 14 25 13 4 10 15 00 13 2 0 51t t 14 6 67 16 75 14 2 0 51t t 15 17 69 14 25 16 17 44 14 00 17 16 41 10 50 18 11 03 6 00 19 10 26 3 75 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 93 g t lt o e E 3 D EE 2 S Q E Q 2 1 U cA GQ b oy ni RU 7 e 2 Chapter B Experiments and Results Population data Allele African American
27. Di Formamide 24 5 uL Note Include additional samples in your calculations to provide excess volume for the loss that occurs during reagent transfers IMPORTANT The volume of size standard indicated in the table is a suggested amount Determine the appropriate amount of size standard based on your results and experiments AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 31 ta S zi 7 o E s Sj D 3 7 3 Chapter 3 Electrophoresis Prepare samples for electrophoresis on the 310 instrument 2 Pipette the required volumes of components into an appropriately sized polypropylene tube 3 Vortex the tube then centrifuge briefly 4 Into each 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 Note For blank wells add 25 uL of Hi Di Formamide 5 Sealthe tubes with the appropriate septa then briefly centrifuge to ensure that the contents of each tube are mixed and collected at the bottom Heat the tubes in a thermal cycler for 3 minutes at 95 C Immediately place the tubes on ice for 3 minutes Place the sample tray on the autosampler Ensure that an injection list is prepared St Ber eS Start the electrophoresis run 32 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Data Analysis m GeneMapper ID Software 33 Overview of GeneMapper
28. Group GeneMapper ID X Security Group Description Instrument Analysis Type HID In the Name field either type the name as shown or enter a name of your choosing In the Security Group field select the Security Group appropriate to your software configuration from the dropdown list The Description and Instrument fields are optional 56 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Section 4 2 GeneMapper ID X Software 4 Set up GeneMapper ID X Software for data analysis Allele tab settings Analysis Method Editor General Allele Peak Detector Peak Quality 5Q amp GQ Settings Bin Set AmpFLSTR_Bins Use marker specific stutter ratio and distance if available Marker Repeat Type Tri Tetra Penta Global Cut off Value 0 0 0 0 0 0 Minus Ratio 0 0 0 0 0 0 MinusA Distance 0 0 0 0 0 0 0 0 0 0 0 0 Global Minus Stutter Ratio 0 0 0 0 0 0 Global Minus Stutter Distance From 0 0 3 25 0 0 To 10 0 4 75 0 0 C 0 2 0 ict oc o 0 p e v Xx GD 2 p 2 0 Global Plus Stutter Ratio 0 0 0 0 0 0 Global Plus Stutter Distance From 0 0 0 0 0 0 To 0 0 0 0 0 0 Amelogenin Cutoff 0 0 Range Filter Factory Defaults Save Cancel e In the Bin Set field select the AmpFLSTR Bins
29. ID Software 0 0 2 6 00sec e cence eee 33 Set up GeneMapper ID Software for data analysis 34 Analyze and edit sample files with GeneMapper ID Software 45 Examine and edit a project 6 eens 46 For more information 2 005006 0canesawn e Re hear ne ERR Ren ea 46 B GeneMapper ID X Software 48 Overview of GeneMapper ID X Software sisse 48 Set up GeneMapper ID X Software for data analysis 49 Analyze and edit sample files with GeneMapper ID X Software 62 Examine and edita project een 63 Examine and edita project es seres REE anidar N aE T TE ee 63 Section 4 1 GeneMapper ID Software Overview of GeneMapper D Software GeneMapper ID Software is an automated genotyping software for forensic casework databasing and paternity data analysis After electrophoresis the data collection software stores information for each sample in a fsa file Using GeneMapper ID Software v3 2 1 software you can then analyze and interpret the data from the fsa files Instruments Refer to Instrument and software overview on page 16 for a list of compatible instruments AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 33 n Chapter 4 Data Analysis Set up GeneMapper ID Software for data analysis Before you start When using GeneMapper ID Software v3 2 1 to perform human identification HID analysis with AmpF STR kits be aware that HID analysi
30. OG SURES 2 Sa eco eodd ton R manana ect eddie hoch R ALT 102 DAB 8 1 2 2 Mixture Studies xes Pi ee Se dnd ee ed ne 102 Limit of detection of the minor component cece e cece rrn eee eae 102 Population data oe ee bei ee pa Re pe eee e eae e au e 103 DAB 841 2 3 Population Data gx gos di a Phish Sek ie AG Pe ee ee a tee 103 Population samples used in these studies cece eee eee eee 103 Section 5 3 Performance Validation After Buffer and Enzyme Component Replacement 00sec cece n 104 dl MR 104 Experiments Leon oc wA gl RB Gale weir de TIE ue bi oat Tone hop RETO d 104 Sensitivity Study cz tinet vb eei cep eR To beer em tende theatre 105 Mean peak height 105 DNA concentration and peak height 0000 cece eee eee eee eens 106 Allelic dropout 5 en nc yes viene A REL nA ade the E e nay dee Tae eee ceed 107 Genotype concordance 00 cece nnr eee tee eect eee en 107 CON CUUSIONS a HEE see EE Une DID bL i eL Lib ratu s ade 107 APPENDIX A Troubleshooting e e e e e cece eee 109 APPENDIX B PCR Work Areas xxe e e 00 ce eee ee eens 111 Work area setup and lab design n n 0c eee eee eee nent eee e eens 111 PCR setup work area n s 2 0 acere 111 Amplified DNA work area 0 600 eee eee n 112 APPENDIX C Ordering Information Lsslussse 113 Equipment and materials not included ccc cece eee eens 113 APPENDIX D Safety 0 0c cece cece eee 117 Chemica
31. Table 8 Troubleshooting Observation Possible causes Recommended actions Faint or no signal from both the AmpF amp STR Control DNA 9947A and the DNA test samples at all loci Incorrect volume or absence of AmpF STR PCR Reaction Mix Profiler Plus Kit or Profiler Plus D Kit Primer Set or AmpliTaq Gold DNA Polymerase Repeat amplification No activation of AmpliTaq Gold DNA Polymerase Repeat amplification making sure to hold reactions initially at 95 C for 11 minutes Master Mix not vortexed thoroughly before aliquoting Profiler Plus Kit or Profiler Plus D Kit Primer Set exposed to too much light Vortex the Master Mix thoroughly Store the Primer Set protected from light GeneAmp PCR System malfunction Refer to the thermal cycler user s manual and check instrument calibration Use of incorrect thermal cycling parameters Check the protocol for correct thermal cycling parameters Tubes not seated tightly in the thermal cycler during amplification Push reaction tubes firmly into contact with block after first cycle Repeat test Wrong PCR reaction tube Use Applied Biosystems MicroAmp Reaction Tubes with Caps or a MicroAmp optical 96 well plate MicroAmp Base used with tray retainer set and tubes in GeneAmp 9700 Remove MicroAmp Base from tray retainer set and repeat test Insufficient PCR product electrokinetically injected
32. and PCR products were analyzed using an Applied Biosystems 310 Genetic Analyzer and GeneScan Analysis 2 1 Software DNA isolated from each of the different tissues fluids from each individual yielded the same genotype Figure 6 illustrates the size differences that are typically observed between sample alleles and AmpF STR Profiler Plus Allelic Ladder alleles on the Applied Biosystems 310 Genetic Analyzer with POP 4 polymer The x axis in Figure 6 represents the nominal base pair sizes for a single injection of AmpF STR Profiler Plus Allelic Ladder and the dashed lines parallel to the x axis represent the 0 5 bp windows The y axis is the deviation of each sample allele size from the corresponding allelic ladder allele size The data include a total of 542 alleles from 31 population database samples In this representative example all sample alleles are within 0 5 bp of a corresponding allelic ladder allele Figure 6 Size deviation of 31 samples and two allelic ladders from one injection of allelic ladder on a single Applied Biosystems 310 instrument run Size Deviation bp 1 050 L 0 75 E MM 4 75 125 175 225 275 325 375 Allele Size bp AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Section 5 1 Developmental Validation of the Profiler Plus Kit 5
33. and reproducibility of the procedure DAB 1998 Laser induced fluorescence detection systems of length polymorphism at short tandem repeat loci is not a novel methodology Holt et al 2001 and Wallin et al 2001 However accuracy and reproducibility of Profiler Plus Kit profiles have been determined from various sample types AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 67 g o lt o 9 me 3 o 2 e S G S Q D w G I e 7 2 Chapter B Experiments and Results Accuracy reproducibility and precision Accuracy 68 In the following study body fluids and tissues were collected and DNA extracts were prepared by the Santa Clara County Crime Laboratory DNA Unit San Jose CA Blood saliva hair and either a semen sample or a vaginal swab were collected from four individuals and DNA was extracted following a phenol chloroform procedure and stored at the crime laboratory for approximately 1 year at 15 to 25 C DNA was also extracted from the brain kidney liver muscle and skin of a human cadaver using the phenol chloroform procedure Additionally four individuals contributed blood and saliva and two individuals contributed blood saliva and hair that were processed using a Chelex DNA extraction protocol and stored at the crime laboratory for approximately 1 week at 4 C These thirty samples were amplified using Profiler Plus Kit reagents
34. binding site This primer amplifies D851179 alleles in samples containing this mutation and does not alter the overall performance of the kit Figure 1 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 11 Product overview Loci amplified by the kits Chapter 1 Overview Figure 1 Profile of a DNA sample heterozygous with one mutant allele amplified without A and with B the D8S1179 unlabeled primer along with the standard primers aal 140 100 22402 ETE 680 TTE EE 40 Keu 200 D A4 Ada ves 4002 B 1202 210 sco sani ni m i The following table shows the loci amplified their chromosomal locations and the corresponding fluorescent marker dyes The Allelic Ladder in each kit is used to genotype the analyzed samples from both the Profiler Plus and Profiler Plus ID Kits The alleles contained in the allelic ladders and the genotype of the AmpFSTR Control DNA 9947A are also listed in the table Table 1 Profiler Plus Kit and Profiler Plus D Kit loci and alleles both kits use the same allelic ladder configuration Alleles included in AmpF STR Profiler Locus designation eee Plus Allelic Ladder and AmpF STR9 uu cb Profiler Plus D Allelic Ladder D3S1358 3p 12 13 14 15 16 17 5 FAM 14 15 18 19 vWA 12p12 pter 11 12 13 14 15 16 17 18 17 18 19 20 21 FGA 4q28 18 19 20 21 22 23 23 24 24
35. cycles to a minimum Electrophoresis The following table lists Data Collection Software and the run modules that can be software setup and used to analyze Profiler Plus and Profiler Plus ID Kits PCR products For details on a a 5 E S a o e x e 3 a l e s D gt 7 reference the procedures refer to the documents listed in the table documents Genetic ata Operatin Collection p g Run modules and conditions References Analyzer System Software Applied 3500 Data Windows e HID36_POP4 Applied Biosystems 3500 3500xL Biosystems Collection XP Injection conditions 1 2kV 15 sec Genetic Analyzer User Guide 3500 Software Pub no 4401661 V1 0 or Dye Set F Applied Windows e HID36 POP4 Applied Biosystems 3500 and 3500xL Biosystems Vista Injection conditions 1 2kV 24 sec ee ne 3500xL J Rs Card Pub no 4401662 Dye Set F Prepare samples for electrophoresis on the 3500 3500xL instrument Prepare the samples for electrophoresis immediately before loading 1 Calculate the volume of Hi Di Formamide and size standard needed to prepare the samples Reagent Volume per reaction GeneScan 500 ROX Size Standard 0 5 uL Hi Di Formamide 8 5 uL Note Include additional samples in your calculations to provide excess volume for the loss that occurs during reagent transfers IMPORTANT The volume of size standard indicated in the table is a suggested amount
36. deposited on wool cotton nylon leather blue denim acetate vinyl upholstery facial tissue a condom with spermicide 5 nonoxynol 9 a condom with water soluble lubricant and a latex glove AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Section 5 1 Developmental Validation of the Profiler Plus Kit 5 Mixture studies Specimens were stored at room temperature and at specified time points a sampling of each stain was removed for extraction The blood and semen stains were extracted using the organic extraction procedure Additionally a portion of each blood specimen was extracted using the Chelex method followed by Centricon 100 ultrafiltration Extracted samples were then stored at 15 to 25 C for 3 4 years The 1 week and 1 year time points were analyzed by Life Technologies scientists The samples were amplified using Profiler Plus Kit reagents and analyzed using the Applied Biosystems 310 Genetic Analyzer A complete 10 locus genotype was obtained for all 36 of the blood and semen samples exposed to the tested matrices for 1 week or 1 year Mixture studies DAB 8 1 2 2 Mixture Studies Analysis of sexual assault DNA mixture evidence Species specificity sensitivity stability and mixture studies are conducted DAB 1998 Evidence samples may contain DNA from more than one individual The possibility of multiple contributors should be considered when interpreting the resu
37. determine settings Smoothing and Baselining Min Peak Half Width Smoothing None Light Polynomial Degree O Heavy Peak Window Size Baseline Window 51 Slope Threshold Peak Start Size Calling Method 2nd Order Least Squares 3rd Order Least Squares Cubic Spline Interpolation Local Southern Method Global Southern Method Peak End Factory Defaults IMPORTANT Perform the appropriate internal validation studies to determine the appropriate peak amplitude thresholds for interpretation of data Fields include e Peak amplitude thresholds The software uses these parameters to specify the minimum peak height in order to limit the number of detected peaks Although GeneMapper ID X Software displays peaks that fall below the specified amplitude in electropherograms the software does not label or determine the genotype of these peaks e Size calling method The Profiler Plus and Profiler Plus ID Kits has been validated using the Local Southern sizing method Before using other sizing methods perform internal validation studies e Normalization A Normalization checkbox is available on this tab in GeneMapper ID X Software v1 2 for use in conjunction with data run on the Applied Biosystems 3500 Series Genetic Analyzers Normalization cannot be applied to 4 dye data so this feature is not for use with data from Profiler Plus and Profiler Plus ID Kits 58 AmpFtSTR Pr
38. number was set to avoid detection of low quantities of DNA 35 pg or less At 28 cycles 2 0 ng ofAmpF STR Control DNA 9947A amplifies reliably and specifically following the conditions outlined in this manual The effects of denaturation and annealing temperatures on the amplification of Profiler Plus Kit loci were examined using 1 2 ng of the AmpF STR Control DNA 9947A The denaturation temperatures tested were 92 94 and 96 C all for 1 minute hold times The annealing temperatures tested were 57 59 61 and 63 C also for 1 minute hold times The majority of these were tested on both the DNA Thermal Cycler 480 and the GeneAmp PCR System 9600 and 2400 The PCR products were analyzed using the Applied Biosystems 377 DNA Sequencer and GeneScan Analysis 2 1 Software Neither preferential nor differential amplification was observed in any of these denaturation temperature experiments Of the tested annealing temperatures 57 59 and 61 C did not induce any differential amplification At 63 C the yield of the majority of loci was significantly reduced This should pose no problem if the thermal cyclers are calibrated routinely and the recommended amplification protocol is followed Preferential amplification was not observed at any of the tested annealing temperatures Accuracy reproducibility and precision DAB 8 1 2 Accuracy Novel forensic DNA methodologies shall undergo developmental validation to ensure the accuracy precision
39. occurrence is lowest in detection systems with the smallest standard deviations in sizing Figure 6 on page 68 illustrates the tight clustering of allele sizes obtained on the Applied Biosystems 310 Genetic Analyzer where the standard deviation in sizing is typically less than 0 15 bp The instance of a sample allele sizing outside of the 0 5 bp window because of measurement error is relatively rare when the standard deviation in sizing is approximately 0 15 bp or less Smith 1995 For sample alleles that do not size within a 0 5 bp window the PCR product must be rerun to distinguish between a true off ladder allele versus measurement error of a sample allele that corresponds with an allele in the allelic ladder AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 69 70 Chapter 5 Experiments and Results Accuracy reproducibility and precision It is important to note that while the precision within a set of capillary injections is very good the determined allele sizes vary between platforms Cross platform sizing differences arise from a number of conditions including type and concentration of polymer mixture run temperature and electrophoresis conditions Variations in sizing can also be found between runs on the same instrument and between runs on different instruments because of these conditions We strongly recommend that the allele sizes obtained be compared to the sizes obtained for known alleles i
40. or 3130 3130xl instrument for electrophoresis Reagents and parts Electrophoresis software setup and Ordering Information on page 113 lists the required materials not supplied with the Profiler Plus and Profiler Plus ID Kits IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the primer set amplified DNA allelic ladder and size standard from light when not in use Keep freeze thaw cycles to a minimum The following table lists Data Collection Software and the run modules that can be used to analyze Profiler Plus and Profiler Plus ID Kits PCR products For details on the procedures refer to the documents listed in the table reference documents Genetic 22 Operatin Collection p Run modules and conditions References Analyzer g System Software Applied 3 0 Windows HIDFragmentAnalysis36 POPA 1 Applied Biosystems 3130 3130xl Biosystems XP Injection conditions Genetic Analyzers Using Data Collection 3130 3130xl Software v3 0 Protocols for Processing 3130 3 kV 5 dins AmpFISTR PCR Amplification Kit PCR 3130x 3 kV 10 sec Products User Bulletin Dye Set F Pub no 4363787 Applied 2 0 Windows e HIDFragmentAnalysis36 POPA 1 3100 3100 Avant Genetic Analyzers Biosystems 2000 Injection condition 3kV 10 sec Using Data Collection Software v2 0 3100 e Dye Set F Protocols for Processing AmpFI STRO y PCR Amplification Kit PCR Products User Bulletin Pub no 4350218
41. or ethnic groups These loci were amplified and typed for 200 U S Caucasian and 195 African American individuals For more information regarding analysis of these samples see Population data on page 90 Species specificity DAB 8 1 2 2 Species specificity sensitivity stability and mixture studies are conducted DAB 1998 Species Specificity Nonhuman studies Nonhuman DNA may be present in forensic casework samples The Profiler Plus Kit provides the required degree of specificity such that it is specific to primates for the species tested with the exception of the amelogenin locus The following experiments were conducted to investigate interpretation of Profiler Plus Kit results from nonhuman DNA sources AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 81 g o lt o 9 me 3 o 2 e S G S Q D w G I 7 2 Chapter 5 Experiments and Results Sensitivity Sensitivity DAB 8 1 2 2 Sensitivity Effect of DNA quantity on results 82 The extracted DNA samples were amplified in Profiler Plus Kit reactions and analyzed using the Applied Biosystems 377 DNA Sequencer Primates Gorilla chimpanzee and orangutan 2 5 ng each e Non primates Mouse cat dog pig chicken cow and horse 50 ng each e Bacteria and yeast Legionella Escherichia Listeria Neisseria Vibrio Citrobacter Salmonella Candida Saccharomyces
42. red analysis works 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 4 The goal of multicomponent analysis is to correct for spectral overlap Figure 4 Emission spectra of the four dyes used in the Profiler Plus9 and Profiler Plus D Kits SS KUE e SS RS SSX gt E m E z tu o z ul o o I c o 2 u a m N z c o z UE 560 600 620 WAVELENGTH nm 5 FAM JOE NED RUAL Filter 1 Filter 2 Filter 3 Filter 4 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 17 Chapter 1 Overview Materials and equipment Materials and equipment Kit contents and storage The Profiler Plus Kit Part no 4303326 and the Profiler Plus ID Kit Part no 4330284 contain materials sufficient to perform 100 amplifications at 50 uL amplification IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the primer set amplified DNA allelic ladder and size standard from light when not in use Keep freeze thaw cycles to a minimum Component Description Volume Storage AmpF STR9 PCR Reaction Mix Contains MgCl deoxynucleotide triphosphates and bovine serum albumin in buffer with 0 05 sodium azide 2 tubes 1 1 mL each AmpF STR Profiler Plus or AmpF STR Profiler Plus 9 D Prime
43. standard deviations from the mean For example if the percent stutter for a particular allele averages 5 for multiple replicates and if the average standard deviation at the allele is 0 5 then the expected range in percent stutter for this allele is 5 1 5 3 5 6 5 This range also provides an estimate of the maximum expected stutter percent for each allele The highest percent stutter observed for any D5S818 D13S317 or D75820 allele was lt 8 for any D851179 allele lt 9 for any D351358 vWA FGA or D21S11 allele lt 10 and for any D18551 allele lt 13 An upper limit stutter percent interpretational threshold can be estimated for each locus as 3 standard deviations above the highest percent stutter observed at the locus see above two observations Peaks at the stutter position that are above this threshold are not expected to be observed in single source samples and therefore can be noted for closer examination The upper limit threshold values for each locus are as follows 9 D75820 10 D55818 and D13S317 11 D3S1358 vWA and FGA 12 D8S1179 13 D21511 and 16 D18551 For evaluation of mixed samples see Mixed samples on page 77 The measurement of percent stutter may be unnaturally high for main peaks that are off scale Loading or injecting less of the PCR product will yield accurate quantitation See DNA quantification on page 19 for information on off scale data AmpFtSTR Profiler Plus
44. stutter product relative to the main allele percent stutter is measured by dividing the height of the stutter peak by the height of the main allele peak Such measurements have been made for hundreds of samples at the loci used in the Profiler Plus Kit AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 73 74 Chapter 5 Experiments and Results Extra peaks in the electropherogram Some of the general conclusions from these measurements and observations are as follows For each Profiler Plus Kit locus the percent stutter generally increases with allele length as shown in Figure 7 through Figure 9 on page 75 through page 76 Smaller alleles display a lower level of stutter relative to the longer alleles within each locus This is reflected in Figure 7 through Figure 9 where minimal data points are plotted for some smaller alleles because stutter was not detected for many of these samples For the alleles within a particular locus the percent stutter is generally greater for the longer allele in a heterozygous sample this is related to the first point above Each allele within a locus displays a percent stutter that is quite reproducible the average of the standard deviation values measured for each allele at each locus is 0 6 for D3S1358 vWA FGA D5S818 D135317 and D7S820 0 8 for D8S1179 D21511 and D18551 The expected range of percent stutter for any particular allele can be estimated as 3
45. tab settingS esse errara e 0 02 T T r Z Ra T R N aa N R Ra LR ATK 42 Quality Flags tab Setungs tee raai i aa Ree ET ETERNI ULP RIT ME 43 Create size standard ren 43 Analyze and edit sample files with GeneMapper ID Software 000 c eee e nee ene 45 Examine and edit a project n dse enea esses 46 FOr moredpnrorrmiatiop se zs ced Eti oar Bhs he fu niece dra fcn i re at 46 AmpF amp TR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Contents Section 4 2 GeneMapper D X Software 2 0 cece cece ee 48 Overview of GeneMapper D X Software 2 nuunuu ccc cece en 48 Ie el aa a setae eec o eoa HOC ER adeb be Palas wud Dacian et eee Moe ds 48 Before you start me eec e Ue up Reed abiur Yum qr ue 48 Set up GeneMapper D X Software for data analysis 0 c ccc cece cece een eee ees 49 Panel bin and stutter file version 0 cece cee RR un 49 Before using the software for the first time cece eee ee eee eee 49 Check panel bin and stutter file version cece cece eee 49 Import panels bins and marker stutter 0 cece eee eee cece eee ees 50 Create an analysis method ccc cece eect tte eee ee eee 55 General tab settingS m eee eee 56 Allele tab settings sn 9 ee enc K eR ev eee ee bees eee beeen ieee egies 57 Peak Detector tab settings unnur rnnr rnnr cee eee eee e enna 58 Peak Quality tab settings scs ercer nsere KR KC NK gR R eee Ea a VE R R 59 SUS 60
46. the IPC template and one TaqMan MGB probe labeled with VIC dye for detecting the amplified IPC Quantifiler Duo DNA Quantification Kit Part no 4387746 For more information see Quantifiler9 Duo DNA Quantification Kit User s Manual Pub no 4391294 Properties The Quantifiler Duo Kit is highly specific for human DNA This kit combines the detection of both total human and male DNA in one PCR reaction The kit detects single stranded and degraded DNA How it works The Quantifiler Duo DNA Quantification Kit consists of target specific and internal control 5 nuclease assays The Quantifiler Duo kit combines two human specific assays in one PCR reaction for total human DNA and human male DNAJ The two human DNA specific assays each consist of two PCR primers and a TagMan probe The TagMan probes for the human DNA and human male DNA assays are labeled with VIC and FAM dyes respectively In addition the kit contains an internal PCR control IPC assay similar in principle to that used in the other Quantifiler kits but labeled with NED dye 20 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Chapter 2 Perform PCR Prepare the amplification kit reactions Prepare the amplification kit reactions 1 Calculate the volume of each component needed to prepare the reactions using the table below DNA sample Volume per reaction AmpF STR PCR Rea
47. the two DNA samples used for this study Figure 11 Reference samples for mixture study shown in next figure 1B Sample A only EHE 16 Sample A only LIN iY Sample only 3B Sample B only E 36 5ample B only LI 2Y Sample B only AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 79 Chapter 5 Experiments and Results Characterization of loci Figure 12 Results of the two DNA samples from previous figure mixed together at defined ratios and amplified with the Profiler Plus Kit The A B ratios shown are 1 1 1 3 1 5 1 10 and 1 20 top to bottom Alleles attributable only to the minor component are highlighted 8B 1 1 Profiler Plus LI SY 1 1 Profiler Plus 7B 3 1 Profiler Plus LIE 7 3 Profiler Plus BB 5 1 Profiler Plus OW 6v 5 1 Profiler Plus 5B 10 1 Profiler Plus LIO SY 10 1 Profiler Plus 4B 20 1 Profiler Plus E 46 20 Profiler Plus OW 4Y 20 1 Profiler Plus Characterization of loci DAB 8 1 2 1 Documentation Nature of the polymorphisms 80 Documentation exists and is available which defines and characterizes the locus DAB 1998 The primers for the amelogenin locus flank a six base pair deletion within intron 1 of the X homologue Amplification results in 107 bp and 113 bp products from the X and Y chromosomes respectively Sizes are the actual base pair size according to sequencing results including 3
48. with MicroAmp Clear Adhesive Film or MicroAmp Optical Adhesive Film or cap the tubes 8 Centrifuge the tubes or plate at 3000 rpm for 20 seconds in a tabletop centrifuge with plate holders if using 96 well plates 9 Amplify the samples in a GeneAmp PCR System 9700 with the silver or gold plated silver 96 well block or a Veriti 96 Well Thermal Cycler Note The Profiler Plus and Profiler Plus ID Kits are 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 kits AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 21 2 Chapter 2 Perform PCR Select the correct PCR cycle number Select the correct PCR cycle number Perform PCR 22 All AmpF STR kits are optimized for a specific number of amplification cycles to deliver well balanced and high quality results However increases in the number of low level DNA samples being submitted for analysis have prompted many laboratories to evaluate increasing the number of amplification cycles to increase the sensitivity of the assay Before increasing the cycle number perform a comprehensive validation study to establish new performance criteria for the higher cycle number Higher cycle numbers can cause the following to occur e Exaggerated stochastic effects resulting from low DNA input amounts Greater difference between the presence
49. 0xL instrument refer to the Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Pub no 4401661 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 114 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Appendix C Ordering Information Equipment and materials not included Itemt Source GeneScan 500 ROX Size Standard 401734 Running Buffer 10X 4335643 Genetic analyzer septa retainer clips for 96 tube sample tray 402866 Genetic analysis sample tubes 0 5 mL 401957 Septa for 0 5 mL sample tubes 401956 DS 32 Matrix Standard Kit Dye Set F for the 310 Genetic Analyzer 4312131 MicroAmp 8 tube strip 0 2 mL N8010580 MicroAmp 96 well base holds 0 2 mL reaction tubes N8010531 MicroAmp 96 well full plate cover N8010550 MicroAmp 96 well tray retainer set 403081 POP 4 polymer for the 310 Genetic Analyzer 402838 For a complete list of parts and accessories for the 310 instrument refer to Appendix B of the 370 Genetic Analyzer User Guide Pub no 4317588 PCR Amplification MicroAmp 96 well tray N8010541 MicroAmp reaction tube with cap 0 2 mL N8010540 MicroAmp 8 tube strip 0 2 mL N8010580 MicroAmp 8 cap strip N8010535 MicroAmp 96 well tray r
50. 25 26 26 2 27 28 29 30 Amelogenin X p22 1 22 3 X Y JOE X Y p11 2 D8S1179 8 8 9 10 11 12 13 13t 14 15 16 17 18 19 D21S11 21 24 2 25 26 27 28 30T 28 2 29 29 2 30 30 2 31 31 2 32 32 2 33 33 2 34 34 2 35 35 2 36 38 D18S51 18q21 3 9 10 10 2 11 12 13 13 2 14 14 2 15 16 15 19 17 18 19 20 21 22 23 24 25 26 D5S818 5q21 31 7 8 9 10 11 12 13 NED 118 14 15 16 D13S317 13q22 31 8 9 10 11 12 13 14 15 qitt D7S820 7q11 21 22 6 7 8 9 10 11 12 10 11 13 14 15 t For CODIS purposes profi T For CODIS purposes profi 8 For CODIS purposes profi tt For CODIS purposes profi 12 e reported as 13 13 e reported as 30 30 e reported as 11 11 e reported as 11 11 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Chapter 1 Overview 1 Product overview Allelic ladder Figure 2 shows the allelic ladder profile for the Profiler Plus and Profiler Plus ID profile Kits See Allelic ladder requirements on page 25 for information on ensuring accurate genotyping Figure 2 GeneMapper D X Software plot of the AmpF STR Profiler Plus Allelic Ladder and the AmpF STR Profiler Plus D Allelic Ladder ST 210 250 290 330 e 4000 B lt 800 600 400 lt G es end A 22 23 ie ks e o be os D b3 23 Bd les B 22 e ed Ee Bal p bs Es x Ea E
51. 3130 3130xl Genetic Analyzers 4352755 3100 3100 Avant Genetic Analyzer Autosampler Plate Kit 96 well 4316471 GeneScan 500 ROX Size Standard 401734 Running Buffer 10X 402824 DS 32 Matrix Standard Kit Dye Set F 4345831 MicroAmp Optical 96 well reaction plate N8010560 Hi Di Formamide 4311320 For a complete list of parts and accessories for the 3130 3130xl instrument refer to Appendix A of the Applied Biosystems 3130 3130xl Genetic Analyzers Maintenance Troubleshooting and Reference Guide Pub no 4352716 3500 3500xL Analyzer materials Anode buffer container ABC 4393927 Cathode buffer container CBC 4408256 POP 4 polymer 960 samples for 3500 3500xL Genetic Analyzers 4393710 POP 49 polymer 384 samples for 3500 3500xL Genetic Analyzers 4393715 Conditioning reagent 4393718 8 Capillary array 36 cm for 3500 Genetic Analyzers 4404683 24 Capillary array 36 cm for 3500xL Genetic Analyzers 4404687 96 well retainer amp base set Standard 3500 3500xL Genetic Analyzers 4410228 8 Tube retainer amp base set Standard for 3500 3500xL Genetic Analyzers 4410231 8 Strip Septa for 3500 3500xL Genetic Analyzers 4410701 96 Well Septa for 3500 3500xL Genetic Analyzers 4412614 Septa Cathode Buffer Container 3500 series 4410715 GeneScan 500 ROX Size Standard 401734 DS 33 Matrix Standard Kit Dye Set G5 4345833 For a complete list of parts and accessories for the 3500 350
52. 5 2 Developmental Validation of the Profiler Plus D Kit Developmental validation DAB 8 1 1 Developmental validation that is conducted shall be appropriately documented DNA Validation Critical reagent concentrations and reaction conditions such as magnesium chloride concentration thermal cycling parameters AmpliTaq Gold DNA polymerase activation cycle number to produce reliable locus specific amplification and appropriate sensitivity have been determined PCR components The concentration of D8S1179 degenerate primer of the AmpF STR Profiler Plus 9 ID Primer Set was examined The concentration for the D851179 degenerate primer was established to be in the window that meets the reproducible performance characteristics of specificity and sensitivity After establishing the optimum unlabeled D851179 degenerate primer concentration all experiments were performed at that concentration Varying magnesium chloride concentrations were also tested to determine the optimum concentration Figure 18 Figure 18 A 2 ng amplification of AmpF STR9 Control DNA 9947A varying the magnesium chloride concentration analyzed on the Applied Biosystems 310 Genetic Analyzer HEN 100 120 140 160 180 200 220 240 260 280 300 320 340 4500 3000 1 15 mM 1500 o 4500 3000 1 25 mM o 4500 Standard z D dal id i Ai W hd a 4500 3000 1 35 mM 1500 3L il 1 Ai Al W nu hd a 4500 3000 1 5 mM 1500 o
53. 7 29 31 setup 27 29 31 emission spectra 17 enzyme new 104 equipment not included with kit 113 AmpF amp STR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 129 Index evidence exclusion of suspects 90 F fluorescent dyes 16 FTA cards amplification 23 bloodstained 23 G gels 85 GeneMapper ID Software analyze project 45 create analysis method 38 create size standard 43 examine and edit project 46 import panels and bins 35 overview 16 33 setup 34 GeneMapper ID X Software analyze project 62 check version of panels bins and stutter 49 create analysis method 55 create size standard 60 examine and edit project 63 import panels bins and stutter 50 overview 16 48 setup 49 GeneScan size standard about 18 dye label 16 volume per reaction 28 29 31 genetics 90 allele frequencies 91 populations and samples used in studies 91 103 genotype exclusion of suspects 90 resolving in mixed samples 78 H hematin effecton DNA samples 84 Hi Di formamide volume per reaction 28 29 31 l import HID size standard 43 60 panels and bins 35 panels bins and stutter 50 instrumentation 310 genetic analyzer 16 31 3100 3100 Avant genetic analyzer 16 27 3130 3130xl genetic analyzer 16 27 3500 3500xL genetic analyzer 16 27 29 software compatibility 16 kit allelic ladders 18 amplification 11 contents 18 control DNA 18 description 11 DNA polymerase 18 21 fluorescent dyes 16 l
54. 982 Biochemical markers of individuality In Saferstein R ed Forensic Science Handbook Prentice Hall Inc New York pp 338 415 Sensabaugh G F von Beroldingen C The polymerase chain reaction application to the analysis of biological evidence In Farley MA Harrington JJ editors Forensic DNA Technology Michigan Lewis 1991 63 82 Sharma V and Litt M 1992 Tetranucleotide repeat polymorphism at the D21511 locus Hum Mol Genet 1 67 Singer Sam J and Tanguay R 1989 Use of Chelex to Improve the PCR Signal From a Small Number of Cells Amplifications 3 Smith R N 1995 Accurate size comparison of short tandem repeat alleles amplified by PCR Biotechniques 18 122 128 Straub R E Speer M C Luo Y Rojas K Overhauser J Ott J and Gilliam T C 1993 A microsatellite genetic linkage map of human chromosome 18 Genomics 15 48 56 Sullivan K M Mannucci A Kimpton C P and Gill P 1993 A rapid and quantitative DNA sex test fluorescence based PCR analysis of X Y homologous gene amelogenin Biotechniques 15 636 641 Sparkes R Kimpton C Gilbard S Carne P Anderson J Oldroyd N Thomas D Urquhart A and Gill P 1996a 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 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Bibliogr
55. A nucleotide addition The remaining Profiler 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 bp repeat units see Table 1 on page 12 Alleles in the AmpF STR Profiler Plus Allelic Ladder alleles containing partial repeat units population database and nonhuman primate DNA samples have been subjected to DNA sequencing at Life Technologies In addition other groups in the forensic community have sequenced alleles at some of these loci Nakahori et al 1991 Puers et al 1993 M ller et al 1994 Barber et al 1995 M ller and Brinkmann 1995 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Section 5 1 Developmental Validation of the Profiler Plus Kit 5 Species specificity 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 Profiler Plus Kit loci there is consensus on the repeat patterns and structure of the STRs see Table 1 on page 12 Inheritance The Profiler Plus Kit loci have been validated by family studies in order to demonstrate their mode s of inheritance The Centre d Etude du Polymorphisme Humain CEPH has collected DNA from 39 families of Utah Mormon French Venezuelan and Amish descent These DNA sets have been extensively studied all
56. Accuracy reproducibility and precision The AmpF STR Profiler Plus Allelic Ladder contains the majority of alleles for the D3S1358 vWA FGA amelogenin D8S1179 D21S11 D18S51 D55818 D13S317 and D7S820 loci However alleles not found in the AmpF STR Profiler Plus Allelic Ladder do exist These off ladder alleles may contain full and or partial repeat units An off ladder allele should flag itself by not falling inside the 0 5 bp window of any known allelic ladder allele Note If a sample allele peak is found to be gt 0 5 bp from the corresponding allelic ladder peak then the sample must be rerun to verify the result Reproducibility The reproducible nature of purified DNA samples from various individuals gt 10 used routinely at Life Technologies as well as validation samples processed during characterization of AmpF STR PCR Amplification Kits for example from body fluid mixture studies environmental studies matrix studies nonprobative studies CEPH family DNA sets was without exception All samples yielded the correct genotype Precision and size As indicated in the previous section the recommended method for genotyping is to windows employ a 0 5 bp window around the size obtained for each allele in the AmpF STR Profiler Plus Allelic Ladder A 0 5 bp window allows for the detection and correct assignment of potential off ladder sample alleles whose true size is only one base different from an al
57. Control B est A Tete K Tes C e e Mean Peak Height 700 600 500 400 3 200 100 j 1000 2000 1000 2000 DNA Concentration on AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 105 Chapter 5 Performance Validation After Buffer and Enzyme Component Replacement Sensitivity study DNA concentration and peak height 106 Figure 24 Sensitivity study representative electropherograms for Sample 2 amplified using 250 pg input DNA Y scale 500 RFU Control A O Mark Samia for Dalston Control B m Test A l Test B i l Li ll LLI Test C The calculated slope and R values for each of the plotted curves are equivalent showing comparable relationships between peak height and DNA input amount for the Test and Control mixes Figure 25 Figure 25 Sensitivity study linear regression plot of combined mean peak height for three genomic DNA samples 700 Control A E Control B Te amp A 600 A Texts Test C 500 2 Z 400 x z 300 200 100 0 0 500 1000 1500 2000 DNA Concentration pg AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Section 5 3 Performance Validation After Buffer and Enzyme Component Replacement 5 Conclusions
58. DNA EH 8G 08 8 16 pg 33474 Control DNA OM sY 08 8 16 pg 99474 Control DNA Species specificity sensitivity stability and mixture studies are conducted DAB 1998 As with any multi locus system the possibility exists that not every locus will amplify This is most often observed when the DNA substrate has been severely degraded or when the DNA sample contains PCR inhibitors Because each locus is an independent marker results generally can still be obtained from the loci that do amplify AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 83 g g lt o 9 E 3 o 2 e S GL S Q D w 9 Em E RS e 7 2 Chapter B Experiments and Results Stability Differential and preferential amplification 84 Differential amplification can be defined as the difference in the degree of amplification of each locus within a co amplified system such that one or more loci may amplify to a greater extent compared to the other loci Preferential amplification is used in this guide to describe differences in the amplification efficiency of two alleles at a single locus and is observed when the peak height ratio between the two alleles is lt 70 see Mixed samples on page 77 Preferential amplification of alleles in systems that distinguish alleles based on length polymorphisms is most likely to be observed when the alleles differ significantly in bas
59. Kits User Guide 61 n Chapter 4 GeneMapper ID X Software Analyze and edit sample files with GeneMapper ID X Software Analyze and edit sample files with GeneMapper D X Software 1 In the Project window select File Add Samples to Project then navigate to the disk or directory containing the sample files 2 Apply analysis settings to the samples in the project Parameter Settings Sample Type Select the sample type Analysis Method Profiler Plus AnalysisMethod v1X or the name of the analysis method you created Panel Profiler Plus v1 1X Size Standard CE E 6GS500 75 450 or the name of the size standard you created t The Profiler Plus and Profiler Plus D Kits were originally validated using the GeneScan 500 ROX Size Standard If you use the GeneScan 400 HD Size Standard as an alternative perform the appropriate internal validation studies to support the use of this size standard with the Profiler Plus and Profiler Plus D Kits Note For more information about how the Size Caller works refer to the GeneScan Analysis Software for the Windows NT Operating System Overview of the Analysis Parameters and Size Caller User Bulletin Pub no 4335617 62 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Section 4 2 GeneMapper ID X Software Examine and edit a project 3 Click Analyze enter a name for the project in the Save Project dialog bo
60. PCR Amplification Kits User Guide Section 5 3 Performance Validation After Buffer and Enzyme Component Replacement 5 Sensitivity study Each of the five mixes listed above were used to conduct reproducibility and sensitivity studies All amplifications were performed using a GeneAmp PCR System 9700 with either silver or gold plated silver block using the recommended amplification conditions and cycle number for the Profiler Plus Kit All data was run on an Applied Biosystems 3130x Genetic Analyzer running Data Collection Software v3 0 and analyzed using GeneMapper ID X Software Subsequent data analysis was performed using Minitab Statistical Software Sensitivity study For the sensitivity study dilution series of three genomic DNA samples were amplified 2 ng three replicates 1 ng 0 5 ng and 0 25 ng four replicates each The results were evaluated for mean peak height degree of linearity between input DNA concentration and peak height level of allelic dropout at 250 pg and genotype concordance Mean peak height Mean peak height observations were consistent between all Test and Control mixes Figure 23 demonstrating equivalent performance Figure 24 U 0 ES e 3 D 5j O o Q S e gt Ww GC E D m o 5 a m 5 N lt 3 D pa 0 o D O o E o Figure 23 Sensitivity study mean peak heights three genomic DNA samples 1000 2000 IBB286 IBB404 1BB676 Lot Control A B
61. Plus and Profiler Plus ID PCR Amplification Kits User Guide Section 5 1 Developmental Validation of the Profiler Plus Kit 5 Extra peaks in the electropherogram Allele n Mean S D 16 3 169 53 0 06 D13S317 8 8 205 04 0 02 p 9 4 209 05 0 06 S 10 3 213 03 0 07 3 11 13 217 06 0 09 S 12 25 221 02 0 05 S 13 10 225 01 0 05 3 14 7 229 00 0 05 E 15 3 233 02 0 03 2 D7S820 z c 6 4 256 05 0 05 5 7 3 260 09 0 08 z 8 12 264 18 0 04 9 8 268 15 0 06 10 22 27224 0 06 11 14 276 29 0 06 12 12 280 31 0 07 13 6 284 38 0 06 14 3 288 49 0 05 15 3 292 43 0 04 Extra peaks in the electropherogram Overview Peaks other than the target alleles may be detected on the electropherogram displays Described below are several causes for the appearance of extra peaks including the stutter product found at the n 1 repeat unit position incomplete 3 A nucleotide addition found at the n 1 position and mixed DNA samples Stutter products The PCR amplification of tetranucleotide STR loci typically produces a minor product peak one repeat unit shorter than the corresponding main allele peak This is referred to as the stutter peak or product Sequence analysis of stutter products at tetranucleotide STR loci has revealed that the stutter product is missing a single tetranucleotide core repeat unit relative to the main allele Walsh et al 1996 The proportion of the
62. Prepare PCR product as described in Chapter 3 Electrophoresis on page 25 Degraded formamide Check the storage of formamide do not thaw and refreeze multiple times Try Hi Di Formamide AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 109 Appendix A Troubleshooting Observation Possible causes Recommended actions Positive signal from AmpF STR Control DNA 9947A but partial or no signal from DNA test samples Quantity of test DNA sample is below assay sensitivity Quantitate DNA and add 1 0 2 5 ng of DNA Repeat test Test sample contains high concentration of PCR inhibitor for example heme compounds certain dyes Quantitate DNA and add minimum necessary volume Repeat test Wash the sample in a Centricon 9 100 centrifugal filter unit Repeat test Test sample DNA is severely degraded If possible evaluate the quality of DNA sample by running an agarose gel If DNA is degraded reamplify with an increased amount of DNA or use the AmpF STR MiniFiler Kit Dilution of test sample DNA in water or wrong buffer for example TE formula with incorrect EDTA concentration Redilute DNA using low TE Buffer with 0 1 mM EDTA More than two alleles present at a locus Some but not all loci visible on electropherogram of DNA test samples Presence of exogenous DNA Use appropriate techniques to avoid introducing forei
63. R Profiler Plus and Profiler Plus ID PCR Amplification Kits are short tandem repeat STR multiplex assays that amplify 9 tetranucleotide repeat loci D3S1358 vWA FGA D8S1179 D21S11 D18S51 D55818 D135317 D7S820 and the Amelogenin gender determining marker in a single PCR amplification Both kits use the same reaction conditions and thermal cycling parameters The Profiler Plus and Profiler Plus ID Kits contain 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 e Applied Biosystems 3130 3130x Genetic Analyzer Applied Biosystems 3500 3500xL Genetic Analyzer Applied Biosystems 310 Genetic Analyzer s GeneAmp PCR System 9700 with the Silver 96 Well Block s GeneAmp PCR System 9700 with the Gold plated Silver 96 Well Block s Veriti 96 Well Thermal Cycler The Profiler Plus and Profiler Plus ID Kits employ the same primer sequences for all loci common to other AmpF STR kits except the MiniFiler kit The Profiler Plus ID Kit includes the degenerate unlabeled primer for the D8S1179 locus that was also included in later AmpF STR kits This primer was added to address a mutation observed in a population of Chamorros and Filipinos from Guam Budowle et al 2000 A single G to A transition point mutation was observed in all null alleles at the D8S1179 reverse primer
64. R Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 119 Appendix D Safety Biological hazard safety 120 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Bibliography Advanced DNA Technologies Workshop 1997 49th Annual Meeting of the American Academy of Forensic Sciences Akane A Matsubara K Nakamura H Takahashi S and Kimura K 1994 Identification of the heme compound copurified with deoxyribonucleic acid DNA from bloodstains a major inhibitor of polymerase chain reaction PCR amplification J Forensic Sci 39 362 372 Bar W Brinkmann B Budowle B Carracedo A Gill P Lincoln P Mayr W and Olaisen B 1997 DNA recommendations Further report of the DNA Commission of the ISFH regarding the use of short tandem repeat systems Intl J Legal Med 110 175 176 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 D18551 and D851179 Intl J Legal Med 109 62 65 Begovich A B McClure G R Suraj V C He
65. Software 4 Set up GeneMapper ID X Software for data analysis 2 Select the Size Standards tab then click New GeneMapper ID X Manager Find Name Containing n Projects Analysis Methods Table Settings Plot Settings Matrices Size Standards Report Settings Name Last Saved Owner Type Description CE F HID GS500 75 400 2007 08 09 13 23 gmidx Advanced CE P HID G5500 75 450 2007 08 09 13 24 gmidx Advanced CE G5 HID G5500 2006 10 11 13 12 z gmidx Advanced GS600 LIZ 2007 06 26 10 43 1 gmidx Advanced GS600 LIZ Normalization 80 400 2007 06 27 01 43 1 gmidx Advanced GS600 LIZ 80 400 2007 06 27 01 43 1 gmidx Advanced Open ort Delete C D o 9 o e e P x lt GD S E g o 3 Enter a name In the Size Standard Dye field select Red In the Size Standard Table enter the sizes specified in on page 60 The example below is for the GeneScan 500 ROX Size Standard Size Standard Editor Edit rSize Standard Description Name CE_F_HID_GS500 75 450 Security Group GeneMapper ID X Security Group Description Size Standard Dye Size Standard Table Size in Basepairs p 100 0 139 0 150 0 160 0 200 0 300 0 340 0 350 0 400 0 450 0 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification
66. U S Caucasian n 195 n 200 20 4 87 1 50 21 1 03f 1 00f 22 1 03 0 25t 23 0 26t 0 25 24 t 25 t F 26 t x D5S818 7 0 26 0 25 8 5 13 0 50 2 05 2 25 10 7 44 6 75 11 25 39 39 25 12 32 56 33 25 13 24 87 16 50 14 2 05 1 00f 15 0 261 t i i 0 25 D13S317 2 T 0 25 8 3 59 11 50 2 31 7 75 10 2 31 6 75 11 27 18 31 25 12 44 36 28 25 13 14 10 9 75 14 6 15 4 25 G d 0 25 D7S820 6 0 51 t gs 0 25 j 2 50 8 17 95 17 50 7 11 80 13 00 10 33 59 24 00 11 22 82 23 00 12 9 49 16 00 94 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Section 5 1 Developmental Validation of the Profiler Plus Kit 5 Population data Allele African American U S Caucasian n 195 n 200 13 3 59 275 14 0 26t 0 75t 2 0 25 t A minimum allele frequency of 1 396 is suggested by the National Research Council in forensic calculations using either of these African American or U S Caucasian databases analyzed using the Profiler Plus Kit Analyzing the two databases Analysis across both databases of 790 total chromosomes revealed a total of 11 different D3S1358 alleles 11 different vWA alleles 18 different FGA alleles 10 different D8S1179 alleles 20 different D21S11 alleles 17 different D18S51 alleles 10 different D5S818 alleles 9 different D13S317 alleles and 11 different D7S820 alleles In addition to the alleles that were observed and recorded in the App
67. USER GUIDE applied biosystems by Life technologies AmpF STR Profiler Plus and Profiler Plus D PCR Amplification Kits for use with Profiler Plus PCR Amplification Kit 100 reaction kit Part no 43033246 Profiler Plus D PCR Amplification Kit 100 reaction kit Part no 4330284 Publication Part Number 4476488 Rev B Revision Date August 2012 6 technologies For Forensic or Paternity Use Only Information in this document is subject to change without notice LIFE TECHNOLOGIES CORPORATION AND OR ITS AFFILIATE S DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT TO THE EXTENT ALLOWED BY LAW IN NO EVENT SHALL LIFE TECHNOLOGIES AND OR ITS AFFILIATE S BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF TRADEMARKS The trademarks mentioned herein are the property of Life Technologies and or its affiliate s or their respective owners TaqMan and AmpliTaq Gold are registered trademarks of Roche Molecular Systems Inc Windows and Windows Vista are registered trademarks of Microsoft Corporation EasiCollect is a registered trademark of Whatman Limited FTA is a registered trademar
68. Z 12 11 140 22 0 11 13 17 144 77 0 05 14 16 149 24 0 04 15 14 153 64 0 07 16 4 157 98 0 06 17 3 162 13 0 08 18 3 166 25 0 04 19 3 170 33 0 06 D21511 24 2 3 187 10 0 07 25 3 189 08 0 02 26 3 192 97 0 04 27 8 196 93 0 07 28 14 200 77 0 06 28 2 3 202 76 0 02 29 10 204 73 0 06 29 2 3 206 73 0 04 30 12 208 68 0 05 30 2 5 210 63 0 03 31 7 212 67 0 06 31 2 7 214 57 0 07 32 4 216 61 0 05 32 2 6 218 54 0 06 33 3 220 55 0 05 332 5 222 48 0 06 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 71 Chapter B Experiments and Results Accuracy reproducibility and precision Allele n Mean S D 34 3 224 52 0 02 34 2 3 226 49 0 02 35 6 228 60 0 08 35 2 3 230 45 0 04 36 6 232 46 0 06 38 4 240 42 0 06 D18S51 9 3 270 49 0 06 10 4 274 62 0 02 10 2 5 276 61 0 06 11 4 278 70 0 07 12 5 282 83 0 05 13 5 286 93 0 05 13 2 4 288 97 0 04 14 9 291 03 0 06 14 2 3 293 01 0 06 15 10 295 06 0 02 16 16 299 16 0 06 17 10 303 54 0 05 18 9 307 94 0 05 19 10 312 31 0 04 20 5 316 61 0 04 21 4 320 93 0 04 22 4 325 11 0 02 23 3 329 28 0 09 24 3 333 45 0 04 25 3 337 55 0 01 26 3 341 56 0 06 D5S818 7 3 131 32 0 06 8 8 135 40 0 06 9 5 139 61 0 06 10 10 144 04 0 05 11 14 148 47 0 05 12 21 152 85 0 06 13 12 157 13 0 04 14 4 161 39 0 08 15 3 165 41 0 03 72 AmpFtSTR Profiler
69. action DNA and included the suspect as a possible semen donor e Case 3 contained a victim reference blood sample and a victim vaginal swab The Profiler Plus Kit genotype of the epithelial cell fraction was the same as that of the victim reference and did not contain alleles foreign to the victim In accordance with the victim s account Profiler Plus Kit genotypes of the sperm fraction revealed DNA from multiple semen donors No suspect s were developed in this case e Case 4 contained victim and suspect reference samples and a victim vaginal swab The Profiler Plus Kit genotype of the epithelial cell fraction was the same as that of the victim reference and did not contain alleles foreign to the victim The major sperm fraction genotype included the suspect A minor genotype attributable to carryover from the epithelial cell fraction was present in the sperm fraction AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 89 g 0 lt Us 9 E 2 0 ES E S e S O e 1 RU 9 ety Oy vss RU 7 e 2 Chapter B Experiments and Results Population data Limit of detection of the minor component Simulation of forensic casework scenarios was achieved by combining various body fluids blood blood semen blood saliva blood and semen saliva from two donors in defined ratios by volume from 1 1 1 50 Mixed stains were prepared and DNA was extracted at the Santa Clara Cou
70. alidated previous file versions for data analysis conduct the appropriate internal verification studies before using new file versions for operational analysis 4 Create an analysis method as explained in Create an analysis method on page 55 5 Define custom views of analysis tables Refer to Chapter 1 of the GeneMapper ID X Software Version 1 0 Getting Started Guide Pub no 4375574 for more information 6 Define custom views of plots Refer to Chapter 1 of the GeneMapper ID X Software Version 1 0 Getting Started Guide Pub no 4375574 for more information 1 Start the GeneMapper ID X Software then log in with the appropriate user name and password IMPORTANT For logon instructions refer to the GeneMapper ID X Software Version 1 0 Getting Started Guide Pub no 4375574 2 Select Tools Panel Manager AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 49 fo 0 2 0 i oc o o p e v X GD Q p 2 0 n Chapter 4 GeneMapper ID X Software Set up GeneMapper ID X Software for data analysis 3 Check the version of files imported into the Panel Manager Panel Manager File Edit Bins View Sain D BD B Panel Manager a Select Panel Manager in the navigation pane b Expand the Panel Manager folder and any sub folders to identify the analysis file version already installed for your kit choice mi Lea 4
71. and New Features for GeneMapper ID Software Software Version v3 2 User Bulletin Pub no 4352543 46 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Section 4 1 GeneMapper ID Software For more information O D 5 D zZ 5 o o D 5 GD fe E B o AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 47 n Chapter 4 GeneMapper ID X Software Overview of GeneMapper ID X Software Section 4 2 GeneMapper D X Software Overview of GeneMapper D X Software GeneMapper ID X Software is an automated genotyping software for forensic casework databasing and paternity data analysis After electrophoresis the data collection software stores information for each sample in a fsa or hid file Using GeneMapper ID X Software v1 0 1 or higher you can then analyze and interpret the data from the fsa or hid files Instruments Refer to Instrument and software overview on page 16 for a list of compatible instruments Before you start When using GeneMapper ID X Software v1 0 1 or higher to perform human identification HID analysis with AmpF STR kits be aware that 48 HID analysis requires at least one allelic ladder sample per run folder Your laboratory can use multiple ladder samples in an analysis provided individual laboratories conduct the appropriate validation studies For multiple ladder samples the GeneMapper ID X Software calc
72. and Profiler Plus ID PCR Amplification Kits User Guide Section 5 1 Developmental Validation of the Profiler Plus Kit Extra peaks in the electropherogram Figure 7 Stutter percentages for the D3S1358 vWA and FGA loci e o eo eo N o o o e o B o P o z 3 2 c o o P4 o n w o 12 13 14 15 16 17 18 D3S1358 14 15 16 17 18 19 20 vWA 19 20 21 22 23 24 25 26 27 28 FGA Figure 8 Stutter percentages for the D8S1179 D21S11 and D18S51 loci 13 0 12 0 11 0 10 0 T 3 9 0 3 t 8 0 oe KEL t 9947 5 E Y i DOE T i ee Bro T o geo H l t L 8 33 t gg U i 1 3333 i il D ii f Fee 6 0 i T T H 33 3 be Soe 53 s g Fe 33 Sot c 5 0 KE Ad H j 3 12 g i 3 3 6 4 0 L UH L ka 3 1 3 0 t M t E 20 10 0 0 8 9 10 1112131415 16 17 D8S1179 242 27 26 29 30 31 32 28 292 302 31 2 32 33 33 2 342 36 D21S11 2 33 1 34 35 38 14 10 11 13 10 2 12 132 142 15 16 17 18 19 20 21 2223 D18S51 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 75 g g lt Us 9 EK 3 0 ES E S GL O 1 RU 9 b Oy ES RU 7 e a Chapter B Experiments and Results Extra peaks in the electropherogram Addition of 3 A nucleotide 76 Figure 9 Stutter percenta
73. 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 Profiler Plus and Profiler Plus ID Kits are optimized for 28 cycles of amplification only The results of developmental validation studies are shown in Developmental Validation of the Profiler Plus Kit on page 66 1 Program the thermal cycling conditions Whenusing 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 AmpF STR Kit Validation Pub no 4440754 Initial Melt Anneal Extend Final Final incubation step extension hold HOLD CYCLE 28 HOLD HOLD 95 C 94 C 59 C 7296 60 C 25 C 11 min 1 min 1 min 1 min 45 min co 2 Load the plate into the thermal cycler and close the heated cover IMPORTANT If using the 9700 thermal cycler with silver or gold plated silver block and adhesive clear film instead of caps to seal the plate wells be sure to place a MicroAmp compression pad Part no 4312639 on top of the plate to prevent evaporation during thermal cycling The Veri
74. aphy 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 1996b The validation of a7 locus multiplex STR test for used in forensic casework I Mixtures ageing and degradation and species studies Int J Legal Med 109 186 194 Technical Working Group on DNA Analysis Methods 1995 Guidelines for a quality assurance program for DNA analysis Crime Lab Digest 22 21 43 Urquhart A Oldroyd N J Kimpton C P and Gill P 1995 Highly discriminating heptaplex short tandem repeat PCR system for forensic identification Biotechniques 18 116 121 U S Department of Health and Human Services 1993 Biosafety in Microbiological and Biomedical Laboratories 3rd edition U S Government Printing Office U S Department of Health and Human Services OSHA Bloodborne Pathogen Standard 29 CFR part 1910 1030 Wallin J M Buoncristiani M R Lazaruk K D Fildes N Holt C L Walsh P S 1998 TWGDAM validation of the AmpFISTR blue PCR amplification kit for forensic casework analysis J Forensic Sci 43 854 870 Walsh P S Erlich H A and Higuchi R 1992 Preferential PCR amplification of alleles mechanisms and solutions PCR Methods Appl 1 241 250 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
75. ation 1000 300 120 160 200 240 280 320 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 23 7 Chapter 2 Perform PCR Amplification using bloodstained FTA cards 24 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Electrophoresis Allelic ladder requirements 0 e 3100 3100 Avant and 3130 3130xl instruments Set up the 3100 3100 Avant or 3130 3130xl instrument for electrophoresis Prepare samples for electrophoresis on the 3100 3100 Avant or 3130 3130xl AISLE UCM 5 eene betur iere tse dte ie rtv e Ete LA et bu cda Es dentine m 3500 3500xL Series instruments RI an Set up the 3500 3500xL instrument for electrophoresis isses Prepare samples for electrophoresis on the 3500 3500xL instrument BW 310 Instrum nt 2 e d Eu A aaa duae dau aA Set up the 310 instrument for electrophoresis lleeeeeeeee Prepare samples for electrophoresis on the 310 instrument Allelic ladder requirements To accurately genotype samples you must run an allelic ladder sample along with the unknown samples Number of allelic One Number of samples per Instrument injection ladders to run allelic ladder s equals 3100 Avant or 3130 1 per 4 injections 4 samples 15 samples 1 allelic ladder 3100 or 3130xl 1 per injection 16 samples 15 samples 1 allelic lad
76. baugh D Keys K Smerick J and Budowle B 2001 Validation of short tandem repeats STR s 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 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 and Roychoudhury A K 1974 Sampling variances of heterozygosity and genetic distance Genetics 76 379 390 Nei M 1978 Estimation of average heterozygosity and genetic distance from a small number of individuals Genetics 89 583 590 Oldroyd N J Urquhart A J Kimpton C P Millican E S Watson S K Downes T and Gill P D 1995 A highly discriminating octoplex short tandem repeat polymerase chain reaction system suitable for human individual identification Electrophoresis 16 334 337 Oppitz E 1969 Arkhiv Fur Kriminologie 1969 144 145 Perkin Elmer Corporation 1993 AmpliType User s Guide Version 2 Foster City CA Prince A M and Andrus L 1992 PCR How to kill unwanted DNA Biotechniques 12 358 Sambrook J Fritsch E F and Maniatis T eds 1989 Molecular Cloning A Laboratory Manual 2nd Edition Volume 2 Cold Spring Harbor Laboratory Press New York pp E10 E14 Sensabaugh G F 1
77. by the height of the higher peak in RFU expressed as a percentage Mean peak height ratios and standard deviations observed for alleles in the Profiler Plus Kit loci in unmixed population database samples are as follows Allele Mean Peak Height Ratio Number of Observations n D3S1358 93 x 4 68 vWA 93 x 5 74 FGA 93 x 5 80 Amelogenin 90 696 46 D8S1179 92 x 696 93 D21S11 91 x 796 95 D18S51 91 x 695 100 D5S818 92 x 5 65 D13S317 93 5 73 D7S820 93 6 79 For all 10 loci the mean peak height ratios indicate that the two alleles of a heterozygous individual are generally very well balanced Ratios lt 70 are rare in normal unmixed samples If the peak height ratio is lt 70 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 Reproducible imbalance at only one locus may indicate a mixture of significantly overlapping genotypes Other AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 77 g o lt o 9 E 3 o 2 e S G S Q D w G I 7 2 78 Chapter 5 Experiments and Results Extra peaks in the electropherogram possible causes of imbalance at a locus are degraded DNA presence of inhibitors extremely low amounts of input DNA or the presence of an allele containing a rare seq
78. creased amount of PCR product that is generated can result in the following Fluorescence intensity that exceeds the linear dynamic range for detection by the instrument off scale data Off scale data is a problem for two reasons Quantitation peak height and area for off scale peaks is not accurate For example an allele peak that is off scale can cause the corresponding stutter peak to appear higher in relative intensity thus increasing the calculated percent stutter Multicomponent analysis of off scale data is not accurate which results in poor spectral separation pull up Identification of off scale peaks and multicomponent analysis are discussed in DNA quantification on page 19 and About multicomponent analysis on page 16 Incomplete A nucleotide addition To avoid these issues the sample can be re amplified using less DNA AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Stability DAB 8 1 2 2 Stability Lack of amplification of some loci Section 5 1 Developmental Validation of the Profiler Plus Kit 5 Stability When the total number of allele copies added to the PCR is extremely low unbalanced amplification of the two alleles of a heterozygous individual may occur Wallin et al 1998 Walsh et al 1992 This is due to stochastic fluctuation in the ratio of the two different alleles Sensabaugh et al 1991 The PCR cycle number and amplification conditio
79. ction Mix 21 0 uL AmpF STR Profiler Plus or AmpF STR Profiler 11 0 uL Plus 9 D Primer Set AmpliTaq Gold DNA Polymerase 1 0 uL Note The volumes above include a slight overfill to provide excess volume for the loss that occurs during reagent transfers 2 Prepare reagents Thaw the PCR Reaction Mix and the Primer Set then vortex all reagent tubes including the enzyme for 3 seconds and centrifuge briefly before opening the tubes IMPORTANT Thawing is required only during first use of the kit After first use reagents are stored at 2 to 8 C and therefore they do not require subsequent thawing Do not refreeze these reagents 3 Pipette the required volumes of components into an appropriately sized polypropylene tube to create a master mix 4 Vortex the master mix for 3 seconds then centrifuge briefly 5 Dispense 30 uL of the reaction mix into each reaction well of a MicroAmp Optical 96 Well Reaction Plate or each MicroAmp tube 6 Prepare the DNA samples DNA sample To prepare Negative control Add 20 pL of low TE buffer 10mM Tris 0 1mM EDTA pH 8 0 Test sample Dilute a portion of the test DNA sample with low TE buffer so that 1 0 2 5 ng of total DNA is in a final volume of 20 uL Add 20 uL of the diluted sample to the reaction mix Positive control Add 20 uL of 9947A control DNA 0 1 ng uL The final reaction volume sample or control plus master mix is 50 uL 7 Sealthe plate
80. d To import the Profiler Plus Kit panel and bin set into the GeneMapper ID Software bins v3 2 1 database 1 Start the GeneMapper ID Software then log in with the appropriate user name and password IMPORTANT If you need logon instructions refer to page 2 7 of the GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide Pub no 4338775 e D o z D o 9 o 5 GD fe E E 2 Select Tools Panel Manager 3 Find then open the folder containing the panels and bins a Select Panel Manager in the navigation pane F Panel Manager File Edit Bins View E m m m m ese ERSS Panel Manager b Select File Import Panels to open the Import Panels dialog box c Navigate to then open the x V Applied Biosystems GeneMapper Panels folder where x is the drive on which the GeneMapper ID Software is installed AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 35 n Chapter 4 Data Analysis Set up GeneMapper ID Software for data analysis 4 Select AmpFLSTR Panels v2 txt then click Import Note Importing this file creates a new folder in the navigation pane of the Panel Manager AmpFLSTR Panels v2 This folder contains the panels and associated markers 2 Import Panels Look in e Panels 2 E AmpFLSTR_Bins_v1 txt Z AmpFLSTR_Bins_v2 txt My Recent E AmpFLSTR_Panels_ v1 txt Documents T T ESL NT eat
81. der 3500 1 per 3 injections 8 samples 23 samples 1 allelic ladder 3500xL 1 per injection 24 samples 23 samples 1 allelic ladder 310 1 per 10 injections 1 sample 9 samples 1 allelic ladder IMPORTANT Variation in laboratory temperature can cause changes in fragment migration speed and sizing variation between both single and multiple capillary runs with larger size variations seen between samples injected in multiple capillary runs We recommend the above frequency of allelic ladder injections which should account for normal variation in run speed However during internal validation studies verify the required allelic ladder injection frequency to ensure accurate genotyping of all samples in your laboratory environment AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 25 26 Chapter 3 Electrophoresis Allelic ladder requirements It is critical to genotype using an allelic ladder run under the same conditions as the samples because size values obtained for the same sample can differ between instrument platforms because of different polymer matrices and electrophoretic conditions AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Section 3 1 3100 3100 Avant and 3130 3130xl instruments T Set up the 3100 3100 Avant or 3130 3130xl instrument for electrophoresis Section 3 1 3100 3100 Avant and 3130 3130xl instruments Set up the 3100 3100 Avant
82. ducibility and precision After the optimal concentration was determined for a single component the others were tested sequentially until it was determined that each component was at the optimal concentration relative to the concentrations of the other components in the master mix The optimized Profiler Plus Kit provides the required degree of specificity such that it is specific to primates for the species tested with the exception of the amelogenin locus see Nonhuman studies on page 81 and does not produce nonspecific mispriming artifacts Thermal cycling parameters were established for amplification of the Profiler Plus Kit in the DNA Thermal Cycler 480 and GeneAmp PCR Systems 2400 9600 and 9700 Thermal cycling times and temperatures met GeneAmp PCR Instrument specifications Annealing and denaturation temperature windows were tested around each setpoint to verify that a 2 C window DNA Thermal Cycler 480 or 1 5 C window GeneAmp PCR System 2400 9600 and 9700 yielded specific PCR product with the desired sensitivity of at least 1 ng of AmpF STR Control DNA 9947A Profiler Plus Kit reactions were amplified for 27 28 29 and 30 cycles on both the DNA Thermal Cycler 480 and the GeneAmp PCR System 9600 While none of the cycle numbers tested produced nonspecific peaks 28 cycles was found to give optimal sensitivity when the amplified products were examined on Applied Biosystems instruments Additionally the cycle
83. e Check panel bin and stutter file version The file names shown in this section may differ from the file names you see when you download or import files If you need help determining the correct files to use contact your local Life Technologies Human Identification representative or go to www lifetechnologies com support gt Software Patches amp Updates GeneMapper ID X Software Use the same analysis files for analysis of data from Profiler Plus and Profiler Plus ID Kits The instructions and examples in this section refer to the latest version of panel bin and stutter file available at the time of publication Before you use GeneMapper ID X Software v1 0 1 or higher for fsa files v1 2 or higher for hid files to analyze data for the first time you must do the following 1 Check the version of panel bin and stutter files installed with the GeneMapper ID X Software as explained in Check panel bin and stutter file version below 2 Check www lifetechnologies com support gt Software Patches amp Updates GeneMapper ID X Software to determine if newer files are available 3 If updated files are available download and import the files into the GeneMapper ID X Software as explained in Import panels bins and marker stutter on page 50 Note When downloading new versions of analysis files refer to the associated Read Me file for details of changes between software file versions If you have v
84. e pair size Because most Profiler Plus Kit loci have small size ranges the potential for preferential amplification of alleles is low DNA samples with FGA alleles in the 300 350 bp range have been observed One such DNA sample containing FGA alleles separated by 90 bp 240 and 330 bp was analyzed in some of our studies to determine the potential for preferential amplification In assessing potential for differential and preferential amplification the following four variables were examined Low template copy number Effectof PCR inhibitor in a DNA sample Degraded DNA Amplification denaturation and annealing temperatures Low template copy number To determine if the amount of input DNA affected either differential or preferential amplification varying quantities of two DNA samples were amplified One nanogram 0 5 ng 0 25 ng 0 125 ng 0 06 ng 0 03 ng and 0 015 ng of AmpF STR Control DNA 9947Aand the sample containing the widespread FGA alleles were amplified and then analyzed using the Applied Biosystems 377 DNA Sequencer No loci in any of the samples tested differentially amplified at any of the ten loci The sample with the widespread FGA alleles preferentially amplified the shorter allele at 0 06 ng input DNA such that the shorter allele 240 bp was detected and the longer allele 330 bp was not However the shorter allele peak height 30 RFU was significantly below the recommended minimum threshold of 150 RFU
85. e reamplified using the Profiler Plus ID Kit to confirm the homozygosity at this locus Of the 68 African American and the 6076 Caucasian homozygous samples at the D851179 locus available for re testing all samples typed as homozygotes None of these samples were found to be heterozygous using the Profiler Plus ID Kit For allele frequencies in the African American and Caucasian populations Holt et al 2001 see Table 4 on page 91 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 103 g 0 lt o e me 3 0 2 E E 5 os 9 1 p 9 Ex I 7 e iw a Chapter 5 Performance Validation After Buffer and Enzyme Component Replacement Overview Section 5 3 Performance Validation After Buffer and Enzyme Component Replacement Overview Experiments 104 As part of an ongoing program to exercise greater control over raw materials used in the AmpF STRO PCR Amplification Kits manufacturing of the AmpliTaq Gold enzyme and 10X PCR Buffer II Tris KCl buffer components is transitioning from Roche Molecular Systems to Life Technologies Manufacturing of both components by Life Technologies will be conducted according to the same specifications used previously by Roche The in house components are established raw materials in our next generation kits for example the NGM NGM SElect and Identifiler Plus Kits We performed studies to compare the performance
86. eak BD Allele Number AN Out of Bin Allele BIN Low Peak Height LPH Overlap OVL Max Peak Height MPH Marker Spike SP 0 3 Off scale OS Peak Height Ratio PHR Control Concordance CC Weight 1 0 Only applicable to controls r5Q Weighting Broad Peak BD os Allelic Ladder GQ Weighting Spike SSPK SPK 1 ov Off scale 05 SQ amp GQ Ranges Sizing Quality From 0 75 to 1 0 From 0to 0 25 Genotype Quality From 0 75 to1 0 From 0to 0 25 IMPORTANT The values shown are the software defaults and are the values we used during developmental validation Perform appropriate internal validation studies to determine the appropriate values to use The CE F HID GS500 75 450 size standard definition is installed with the software for use with the Profiler Plus and Profiler Plus D Kits and contains the following size standard peaks GeneScan 500 ROX Size Standard 75 100 139 150 160 200 300 340 350 400 and 450 Note The 250 nt peak in the GeneScan 500 ROX Size Standard is not included in the size standard definition This peak can be used as an indicator of precision within a run Use the following procedure if you want to create your own size standard 1 Select Tools r GeneMapper Manager to open the GeneMapper Manager AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Section 4 2 GeneMapper ID X
87. elopmental validation 00 eee eee eee ee 66 Accuracy reproducibility and precision 0 eee eee eee eee ee 67 Extra peaks in the electropherogram 0 cece cece eee eee ee 73 Characterization Of loci scere ge phan Ea ee pane e Da ele gale 80 Species specificitys Jeu e peo ed ree Fete qe ep e 81 SensiDvity oos Seat Seis tot eet Soha Lec onto ub ubt s doo rptu hor 82 Stability M 83 Mixture studies 4e aU Sth ER PERRA VERE Eu EGRE ERES 89 Population data eese ete ee e eR 90 Probability ofidentity esa Z e Raa r imeem nee eee e PR xe a 96 Probability of paternity exclusion 00 6c ccc eee ee 97 Section 5 2 Developmental Validation of the Profiler Plus ID Kit 98 Developmental validation 00 98 Species specificity es ins Moding aaa ake mamie od wea NAY wind oak edad 100 SEHSID VID eta need Rah bee Ud os Sedet i p tiu vane ote ride aa hat 101 Stability oon et IMs eun cma sro MS MO gu eee itur 101 M xt re st dies osea dee Ru eee RR SU eate RU PR I E qus 102 Population datas veste M Ree TER ELA XIV ET iA RI E eat 103 Section 5 3 Performance Validation After Buffer and Enzyme Component Replacement ziehe RR DARREN EFE UNE EM PE ER Mr v 104 Overview tise woobp Ne cre Up boe te eto eve 104 EXDetriments 23 34 aude erbe pnt d eed dose oboe be E 104 Sensitivity study eicere eR ERU EO tee re Su a Fhe a a eg 105 Conclusions 5 eene UU eee Pee ena Ea ette eg
88. emical manufacturer Obtain support For HID support In North America Send an email to HIDTechSupport lifetech com or call 888 821 4443 option 1 e 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 e Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities e 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 Life Technologies Corporation 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 128 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Numerics 310 instrument 31 31xx instrument 28 3500 3500 xL instrument 29 A A nucleotide addition by AmpliTaq Gold to 3 end of amplicon 76 agarose gel using to examine DNA 85 allele f
89. en ort Desktop D Filename AmpFLSTR_Panels_v2 txt Lamp My Documents Files oftype all pies v Can 5 Import AmpFLSTR_Bins_v2 txt a Select the AmpFLSTR_Panels_v2 folder in the navigation pane Panel Manager File Edit Bins View Ex mmm mH Panel Manager t KA AmpFLSTR Panels v2 b Select File r Import Bin Set to open the Import Bin Set dialog box c Navigate to then open the x V Applied Biosystems GeneMapper Panels folder d Select AmpFLSTR Bins v2 txt then click Import Note Importing this file associates the bin set with the panels in the AmpFLSTR Panels v2 folder Import Bin Set Look in la Panels x a E Ez E E AmpFLSTR_Bins_v1 txt i My Recent E AmpFLSTR Panels v1 txt Documents E AmpFLSTR_Panels_v2 txt E Desktop G File name AmpFLSTR Bins v2 txt My Documents Files of type All Files 36 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Section 4 1 GeneMapper ID Software 4 Set up GeneMapper ID Software for data analysis 6 View the imported panels in the navigation pane a Double click the AmpFLSTR_Panels_v2 folder b Double click the Profiler_Plus_v2 folder to display the panel information in the right pane and the markers below it F Panel Manager File Edit Bins View HEX OU SB Bn Set ampristr_pins_v2
90. ence sample s Limit of detection of the minor component Mixtures of two DNA samples were examined at various ratios 1 1 to 1 20 The total amount of genomic input DNA mixed at each ratio was 1 ng The samples were amplified in a GeneAmp PCR System 9600 and were electrophoresed and detected using an Applied Biosystems 377 DNA Sequencer The results of the mixed DNA samples shown separately in Figure 11 on page 79 are displayed in Figure 12 on page 80 where sample A was the minor component and sample B was the major component AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Section 5 1 Developmental Validation of the Profiler Plus Kit 5 Extra peaks in the electropherogram The genotypes of the samples in Figure 11 are the following Genotype Allele Sample A Male Sample B Femalje C Amelogenin X Y X X E D3S1358 15 16 15 18 El WA 14 16 17 19 E FGA 24 26 23 24 EN D8S1179 12 13 13 13 a D21S11 28 31 30 33 ej D18551 12 15 17 19 y D55818 1 11 11 13 D13S317 11 11 11 11 D D7S820 7 12 9 10 E ES For these 1 ng total DNA mixture studies the limit of detection is when the minor component is present at approximately 1 20 of the concentration of the major component The limit of detection for the minor component is influenced by the combination of genotypes in the mixture The following figures show the reference samples and
91. ermine the appropriate amount of size standard based on your experiments and results 2 Pipette the required volumes of components into an appropriately sized polypropylene tube 3 Vortex the tube then centrifuge briefly 4 Into each well of a MicroAmp Optical 96 Well Reaction Plate add e 9uL of the formamide size standard mixture e luL of PCR product or allelic ladder Note For blank wells add 10 uL of Hi Di Formamide 5 Seal the reaction plate with appropriate septa then centrifuge the plate to ensure that the contents of each well are collected at the bottom Heat the 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 then place on the autosampler Ensure that a plate record is completed and link the plate record to the plate S sO SOR Pay BOs Start the electrophoresis run 28 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Set up the 3500 3500xL rai a Section 3 2 3500 3500xL Series instruments Set up the 3500 3500xL instrument for electrophoresis Reagents and parts Ordering Information on page 113 lists the required materials not supplied with the Profiler Plus and Profiler Plus ID Kits IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the primer set amplified DNA allelic ladder and size standard from light when not in use Keep freeze thaw
92. es 107 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 65 Chapter B Experiments and Results Overview Section 5 1 Developmental Validation of the Profiler Plus Kit Overview Importance of validation Experiment conditions This section provides results of the developmental validation experiments we performed using the Profiler Plus Kit These studies meet or exceed those recommended in the Technical Working Group on DNA Analysis Methods TWGDAM guidelines as well as the DNA Advisory Board DAB Quality Assurance Standards effective October 1 1998 Technical Working Group on DNA Analysis Methods 1995 DNA Advisory Board Federal Bureau of Investigation U S Department of Justice 1998 These studies also address the guidelines outlined in the ENFSI DNA Working Group Quality Assurance Programme for DNA Laboratories Validation of a DNA typing procedure for human identification applications is an evaluation of the procedure s efficiency reliability and performance characteristics By challenging the procedure with samples commonly encountered in forensic and parentage laboratories the validation process uncovers attributes and limitations which are critical for sound data interpretation in casework Sparkes Kimpton Watson et al 1996 Sparkes Kimpton Gilbard et al 1996 Wallin et al 1998 This chapter discusses many of the experiments we performed and provides examples of
93. etainer set 403081 MicroAmp 96 well base N8010531 MicroAmp clear adhesive film 4306311 MicroAmp optical adhesive film 4311971 MicroAmp optical 96 well reaction plate N8010560 Other user supplied materials Hi Di Formamide 25 mL 4311320 Aerosol resistant pipette tips MLS Microcentrifuge tubes MLS Pipettors MLS Tape labeling MLS Tube 50 mL Falcon MLS Tube decapper autoclavable MLS Deionized water PCR grade MLS Tris HCL pH 8 0 MLS EDTA 0 5 M MLS Vortex MLS For the Safety Data Sheet SDS of any chemical not distributed by Life Technologies contact the chemical manufacturer Before handling any chemicals refer to the SDS provided by the manufacturer and observe all relevant precautions AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 115 C Appendix C Ordering Information Equipment and materials not included 116 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Safety WARNING GENERAL SAFETY Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device Ensure that anyone using this product has received instructions in general safety practices for laboratories and the safety information provided in this document Before using an instrument or device read and understand the safety information provided in the user documentation provided by the manufactu
94. filer_v1 1 nul ES SEfiler Plus v1 1X amp C3Identifiler v1 1x lisa id null B IC3MiniFiler v1 AX Profiler Plus CODIS v1 ijnull il AProfiler Plus CODIS v1 1X VFiler v1 1X null AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Section 4 2 GeneMapper ID X Software 4 Set up GeneMapper ID X Software for data analysis 8 Select and expand Profiler_Plus_v1 1X in the navigation pane then select D8S1179 to display the Bin view for the marker in the right pane Panel Manager Bin Set AmpFLSTR Bins v2X amp C9Profiler Plus v1 t E D351358 E G vWA FGA D21511 D18551 D55818 sj D135317 D75820 COfiler_v1 1X SGM Plus_v1 1X Identifiler v1 1X amp C SEfiler_Plus_v1 1x O D z o Ke gal D p S z X GD 9 Profiler Plus COD 3 cofiler_CODIS_v1 n ia conc Jill sd Reference Samples 0 0 113 115 117 119 121 123 125 127 129 131 133 135 137 139 141 143 145 147 149 151 153 155 157 153 161 163 155 167 163 171 173 175 177 179 12 183 185 187 Dpss1179 lt Marker D891179 118 00 183 50 WW Fe es US 9 Import AmpFLSTR Stutter v2X a Select the AmpFLSTR Panels v2X folder in the navigation panel Panel Manager File Edit Bins Yiew Help a x a N N JN S inset ampristr Bins v W NE S D BEBE Panel Name Ofiler v1 1X null a SGM Plus v1 1X null NGM SElect v2
95. ges for the D5S818 D13S317 and D7S820 loci x o z 23 z o c o o S o n 9 10 11 12 13 14 15 8 9 10 11 12 13 14 9 10 11 12 13 14 D5S818 D13S317 D7S820 AmpliTaq Gold enzyme like many other DNA polymerases can catalyze the addition of a single nucleotide predominately adenosine to the 3 ends of double stranded PCR products Clark 1988 Magnuson et al 1996 This non template addition results in a PCR product that is one base pair longer than the actual target sequence and the PCR product with the extra nucleotide is referred to as the A form The efficiency of A addition is related to the particular sequence of the DNA at the 3 end of the PCR product The Profiler Plus Kit includes two main design features that promote maximum A addition The primer sequences have been optimized to promote A addition e The last thermal cycling step is 60 C for 45 minutes This final extension step gives the AmpliTaq Gold DNA polymerase extra time to complete A addition to all double stranded PCR product STR systems that have not been optimized for maximum A addition may have split peaks where each allele is represented by two peaks one base pair apart Figure 10 Figure 10 Split peaks resulting from incomplete A nucleotide addition due to omission of the 45 minute extension step AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Section 5 1 Deve
96. ghts for a sample containing 35 pg human genomic DNA Low peak heights should be interpreted with caution Individual laboratories may find it useful to determine an appropriate minimum peak height interpretational threshold based on their own results using low amounts of input DNA Figure 21 Effect of amplifying various amounts of AmpF STR Control DNA 9947A ranging from 16 pg to 1 ng Note that the y axis scale differs in many of these panels See Stability on page 83 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 101 g 0 lt o e me 3 o 2 a 5 for 9 1 p 9 Ex I 7 e iw a 5 Chapter5 Developmental Validation of the Profiler Plus ID Kit Mixture studies Mixture studies DAB 8 1 2 2 Species specificity sensitivity stability and mixture studies are conducted DAB 1998 Mixture Studies Limit of detection Mixtures of two DNA samples Sample A male Sample B female were amplified of the minor at various ratios 1 1 to 1 10 with the Profiler Plus ID Kit The total amount of component genomic input DNA mixed at each ratio was 1 ng The samples were amplified in a GeneAmp PCR System 9700 and were electrophoresed and detected using an Applied Biosystems 310 Genetic Analyzer The results of the mixed DNA samples are shown in Figure 22 where sample A and sample B were mixed according to the ratios listed Fig
97. gn DNA during laboratory handling Amplification of stutter product Mixed sample Test sample DNA is severely degraded See Stutter products on page 73 If possible evaluate the quality of DNA sample by running an agarose gel If DNA is degraded reamplify with an increased amount of DNA or use the AmpF STR MiniFiler Kit Test sample contains high concentrations of a PCR inhibitor for example heme compounds certain dyes Quantitate DNA and add minimum necessary volume Repeat test Wash the sample in a Centricon 9 100 centrifugal filter unit Repeat test Poor peak height Incorrect thermal cycler parameters Check the protocol for correct thermal cycler balance parameters GeneAmp PCR System 9700 with Use Applied Biosystems GeneAmp PCR System Aluminum 96 Well block or third 9700 with silver or gold plated silver blocks only party thermal cyclers or the Veriti 96 Well Thermal Cycler 110 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide PCR Work Areas Work area setup and lab design cece eens 111 PCR setup work area Ee eens 111 Amplified DNA work area eee eee 112 Work area setup and lab design Many resources are available for the appropriate design of a PCR laboratory If you are using the AmpF STR Profiler Plus and Profiler Plus ID PCR Amplification Kits for Forensic DNA testing refer to Forensic Labora
98. gs Analysis Method Editor HID General Allele Peak Detector Peak Quality Quality Flags BinSet AmpFLSTR Bins v2 Use marker specific stutter ratio if available Marker Repeat Type Tri Tetra Penta Hexa Cut off Value 00 00 oo 00 Minus Ratio oo 0 0 loo oo MinusA Distance 0 0 0 0 i 10 0 To 00 0 0 o oo Minus Stutter Ratio 0 0 oo oo Minus Stutter Distance From 00 325 oo oo 475 loo loo Plus Stutter Ratio oo 00 oo 00 Plus Stutter Distance 0 0 00 o0 0 0 f 00 Amelogenin Cutoff Range Filter Factory Defaults In the Bin Set field select the AmpFLSTR Bins v2 bin set imported previously and configure the stutter distance parameters as shown e GeneMapper ID Software v3 2 1 allows you to specify four types of marker repeat motifs tri tetra penta and hexa You can enter parameter values for each type of repeat in the appropriate column Specify the stutter ratio To apply the stutter ratios listed in the Allele tab for single source data deselect the Use marker specific stutter ratio if available check box selected by default Perform appropriate internal validation studies to determine the appropriate filter setting to use Note Applying global stutter ratios may reduce the editing required for single source sample data To apply the stutter ratios contained in the AmpFLSTR Panels
99. ian databases as suggested in the 1996 report of the Committee on DNA Forensic Science National Research Council 1996 These databases are summarized in Table 4 on page 91 The minimum reportable genotype frequency at each locus is then 1 69 X 10 4 giving a minimum combined multilocus genotype frequency of 1 12 X 10 4 for both the African American and U S Caucasian databases Probability of identity 96 Table 5 shows the Probability of Identity Pr values of the Profiler Plus Kit loci individually and combined Table 5 Probability of Identity values for the Profiler Plus Kit STR loci Locus African American U S Caucasian D3S1358 0 102 0 078 vWA 0 058 0 065 FGA 0 035 0 036 D8S1179 0 075 0 067 D21S11 0 033 0 045 D18S51 0 028 0 030 D5S818 0 097 0 140 D13S317 0 131 0 074 D7S820 0 081 0 061 Combined 1 48 x 10711 1 04 x 10711 The Pr value is the probability that two individuals selected at random will have an identical Profiler Plus Kit genotype Sensabaugh 1982 The P values for the populations described in this section are then approximately 1 6 8 X 101 African American and 1 96 X 101 U S Caucasian Of 18 915 and 19 900 pairs of Profiler Plus Kit profiles represented by the African American and U S Caucasian databases respectively no 9 locus matches were observed AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Section 5 1 Deve
100. ic DNA Extraction Kit User Guide 4390932 GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide 4338775 GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial 4335523 Installation Procedures and New Features for GeneMapper ID Software v3 2 User Bulletin 4352543 GeneMapper ID X Software Version 1 0 Getting Started Guide 4375574 GeneMapper ID X Software Version 1 0 Quick Reference Guide 4375670 GeneMapper ID X Software Version 1 0 Reference Guide 4375671 GeneMapper ID X Software Version 1 1 Mixture Analysis Getting Started Guide 4396773 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 127 Documentation and Support Obtain SDSs Document title Pub no GeneMapper ID X Software Version 1 1 Mixture Analysis Quick Reference Guide 4402094 GeneMapper ID X Software Version 1 2 Reference Guide 4426481 GeneMapper ID X Software Version 1 2 Quick Reference Guide 4426482 Portable document format PDF versions of this guide and the documents listed above are available at www lifetechnologies com Note To open the user documentation available from the Applied Biosystems web site use the Adobe Acrobat Reader software available from www adobe com Obtain SDSs Safety Data Sheets SDSs are available from www lifetechnologies com support Note For the SDSs of chemicals not distributed by Life Technologies contact the ch
101. ification Degraded DNA was prepared to examine the potential for differential amplification of loci High molecular weight DNA was incubated with the enzyme DNase I for varying amounts of time The DNA was examined by agarose gel analysis to determine the average size of the DNA fragments at each timepoint Gel analysis results of the degraded DNA are shown in Figure 15 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 85 5 Chapter 5 Experiments and Results Stability Figure 15 Agarose gel of degraded genomic DNA c S Q 3 o E o bd Q Zz a o c pGEM size marker 30 sec 1 min 4 min 8 min 12 min 20 min 30 min 1 hr 2 hrs 4 hrs 8 hrs 24 hrs 222 bp Four 4 ng of degraded DNA or 2 ng undegraded DNA was amplified using the Profiler Plus Kit all 10 primer pairs together and also in reactions containing each locus specific primer pair individually The electropherograms in Figure 16 show the Profiler Plus Kit amplification results of a DNA sample with no DNase I treatment 1 5 ng amplified and those of the 30 second 1 4 and 8 minute incubations approximately 4 ng amplified 86 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Section 5 1 Developmental Validation of the Profiler Plus Kit 5 Stability Figure 16 Amplifications of DNA incubated for various times with DNase g
102. ile Edit Bins View X BM IB DH gust amprustR_sins_v2 X TTT mimmimmm GjPanel Manager E S AmpFLSTR Panels v2 amp Blue v2 Green 1 v2 LProtiler v2 a ES Profiler Plus v2 D381358 D21511 D18551 D55818 D13S317 D75820 1 cotiler v2 m ac ETRAS Reference Samples 0 0 i tt a aoa 113 117 121 125 129 133 137 141 145 149 153 157 161 165 169 173 177 181 185 D8s1179 lt EZ X 115 98 Y 0 61 OK Cancel Apply AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 37 Chapter 4 Data Analysis Set up GeneMapper ID Software for data analysis Create an analysis method 38 8 Click Apply then OK to add the Profiler_Plus_v2 panel and bin set to the GeneMapper ID Software database IMPORTANT If you close the Panel Manager without clicking OK the panels and bins are not imported into the GeneMapper ID Software database The HID Advanced analysis method for the Profiler Plus and Profiler Plus ID Kits uses the AmpFLSTR_Bins_v2 file described in step 5 on page 36 Use the following procedure to create a HID analysis method for the Profiler Plus and Profiler Plus ID Kits 1 Select Tools GeneMapper Manager to open the GeneMapper Manager GeneMapper Manager Name Last Saved Owner Instrument Analysis 1 s L i IdentifilerDirect_HID_v1 2010 05 05 10 24 1 mg HID Identifiler Plus amp na
103. iler Plus and Profiler Plus ID PCR Amplification Kits User Guide 51 Chapter 4 GeneMapper ID X Software Set up GeneMapper ID X Software for data analysis d Select AmpFLSTR_Bins_v2X txt then click Import Note Importing this file associates the bin set with the panels in the AmpFLSTR_Panels_v2X folder Import Bin Set Lookin 2 AmpFLSTR Analysis Files GMIDX v amp paa My Recent B ampFistR_Stutter_v2x Documents El ReadMe AmpFLSTR v2X My Documents 48 File name AmpFLSTR Bins v2X Ext My Computer Files of type all Files 7 View the imported panels in the navigation pane a Double click the AmpFLSTR_Panels_v2X folder b Double click the Profiler_Plus_v1 1X folder to display the panel information in the right pane Panel Manager File Edit Bins View Help i x E NN JE S ein Set lampristroins L E S D Saal i Panel Manager Panel Name Comment AmpFLSTR_NGMSElect_v2x Ofller v1 1X null CQAmpFLSTR_NGM_v3x SGM_Plus_v1 1 null B AmpFLSTR_Panels_v1X EDIR Panels vix NGM SElect v2 1X null Iz um AmpFLSTR Pan X E COfiler_v1 1 Identifiler_Plus_v1 1 null 9 SGM_Plus_v1 1X NGM v3 1X null Ez rco n Identifler Direct v1 1x null ES Identifiler Plus v1 1X zie Fil AX MGN va IX Pro jer Pius vi 1 null amp amp CIdentifiler Direct v1 1x Profiler_v1 1 null 3 Profiler_Plus_v1 1X SEfiler Plus v1 1X null E Identi
104. ixed sampl S 255 stessa bp iat est be eter utis RN pn Nas 77 AmpF ZSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 5 Contents Characterization Ol lOc 6 eee ta sen ate oid Ha ae at pneu eA ee LC dE 80 DAB 8 1 2 1 Documentation oen tesan hina Steph inp n tb RM hee hed 80 Nature of the polvnmaorphisanms cece eee eee s rn 80 Inheritance oes K Re eke R e PE 9 bet nner eae bot cena ease Ra ene doves Woke ened Ovals RR a Aa 81 MET e 81 Populati ri stidie5 Za erbe tosis an beh stem Plura Eos Ad Manca ets d dts 81 Species specifIGily eL ee Lb ee ee eee ee wem 81 DAB 8 1 2 2 Species Specificity 2 0 20 0 00 see 81 Nonhuman studies iesin errei et ene suc m dine e nm Ree ed Nove EEES 81 SIM CIE 82 DAB8 1 22 Sensitivity Z Tec 095 eluaca C Xa RAT E EXUERIT ET Ee iade 82 Effect of DNA quantity on results 2 2 0 sse 82 Stability iiscecebx E RUD si envio ee a eee edie Pome ERIS onde ae hes 83 DAB 8 1 2 2 Stability ori p Re Reis e d eI ue Avo eee PITE HE 83 Lack of amplification of some loci 0 000s 83 Differential and preferential amplification 0c cece eee ee eee ee eae 84 Matrik SNES sni aie OH eel ad UB ceat tenute erede eie 88 Mixt re studies mee three erbe DU Puke dane EH candied ea x eR RR ea 89 DAB 8 1 2 2 Mixture Studies 20 0 c cece eee n 89 Analysis of sexual assault DNA mixture evidence 0000 cece ee eee nents 89 Limit of detection of the minor compone
105. k of Whatman International Limited Whatman is a registered trademark of GE Healthcare Companies Mac OS is a registered trademark of Apple Inc Minitab is a registered trademark of Minitab Inc 2012 Life Technologies Corporation All rights reserved Contents About THIS 6191 LT 9 Revision HISTORY nes eser seek ed RE E ecd RR bog weed Bau e ie e ec tod d 9 PUPDOSE oi fee L pt E enr rint i SERES A IA EA MEC 9 User attention WOndS oett ne EE EIN e Eee CE eR tete Elle wets 9 M CHAPTER Overview seeeseeeenn I I 11 Productoverview e 9 9 ee viu enar Sue KR dim RRR R 3T Ra Pen d des 11 Purpose usted EET Ft pee du aie eae tee e ob 11 Producbdescplptlobss Soto fett eA E eiie E tu eU Ae Bat 11 About the primers sce ene e RIS ebbe E ewe etes ish 11 Loci amplified by the kte 12 Allelic ladder profile II III Il 13 Control DNA 99474 profile i ca ose R x T X R UC b EU eA SER 14 WorktloW OVervig We x exce tec e D Gand en LEE eU EIU OU ULT QURE DERE 15 Instrument and software overview 6 cece eee een ent eee t teen eee 16 Data Collection and GeneMapper ID or ID X Software 2 0 c cece cece cece nee 16 Instrument and software compatibility 20000 000 e eee eee eee eae 16 About multicomponent analysis 000 c eee eee eee eee e eens 16 How multicomponent analysis works 0000 cece ence eee cence eee ees 17 Materials and equipment annn cece eee eee eee rns 18 Kit content
106. l safety vs ga fe S de newt a eed Bat eda ce R RE de eee Red eb ee da 118 Biological hazard Safe x K ce cellier Ra Rn ee 119 Bibliography cisci adita eire bosco Borm dg ade EE ne cae ete las 121 Documentation and Support ee e e eee eere 127 Related doctimentationc i san 95 IR de gah EEEa Sa eee Gah ee a eee ee a 127 ObtainiSDSsucis do etos astitit tae Leake A TT 128 Obtain Support een oat oe Sale eme eee ee dct ee Ed 128 Limited Product Warranty 0c eee mn 129 WEE KB seat aan nlc 137 AmpF amp TR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 7 Contents 8 AmpF amp TR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide About This Guide IMPORTANT Before using this product read and understand the information the Safety appendix in this document Revision history Revision Date Description A May 2012 New document Combines information for AmpF STR Profiler Plus and Profiler Plus D PCR Amplification Kits into one user guide Add validation experiments and results for buffer and enzyme kit component changes B August 2012 Update language in validation experiments and results for buffer and enzyme kit component changes Purpose The Applied Biosystems AmpFlSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide provides information about the Applied Biosystems instrument
107. le an analysis method created for GeneMapper ID X version 1 2 is not compatible with earlier versions of GeneMapper ID X Software or with GeneMapper ID Software version 3 2 1 1 Select Tools GeneMapper ID X Manager to open the GeneMapper ID X Manager EE GeneMapper ID X Manager Find Name Containing C 0 2 0 i oc o 0 p e v X GD Q p 2 0 Projects Analysis Methods Table Settings Plot Settings Matrices Size Standards Report Settings 2 Select the Analysis Methods tab then click New to open the Analysis Method Editor with the General tab selected The figures below show the settings for each tab of the Analysis Method Editor Configure the Analysis Method Editor tab settings as shown in the figures below unless the instructions state otherwise Note The Analysis Method Editor closes when you save your settings see step 3 on page 55 To complete this step quickly do not save the analysis method until you finish entering settings in all of the tabs 3 Click Save AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 55 n Chapter 4 GeneMapper ID X Software Set up GeneMapper ID X Software for data analysis General tab settings Analysis Method Editor General Allele Peak Detector Peak Quality SQ amp GQ Settings r Analysis Method Description Name ProfilerPlus AnalysisMethod v1X Security
108. le frequencies observed in the Life Technologies African American and U S Caucasian databases Random association Existence of random association linkage equilibrium between all 10 STR loci was established through two separate statistical tests Results of the first test which considers the observed variance of the number of heterozygous loci Brown et al 1980 Budowle et al 1995 indicate that in both population samples all Profiler Plus Kit loci are inherited independently AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 95 Chapter 5 Experiments and Results Probability of identity Pairwise interclass correlation tests were performed between every possible two locus combination across the African American and U S Caucasian databases Karlin et al 1981 Mendelian behavior between the nine STR loci was observed Profiler Plus Kit multilocus genotype frequency estimates may be derived through direct multiplication of each single locus genotype frequency the product rule estimated from the Applied Biosystems African American and U S Caucasian databases Low frequency alleles Some alleles of the Profiler Plus Kit loci occur at a low frequency less than five times in either database For these alleles a minimum frequency of 0 013 five divided by 2n where n equals the number of individuals in the database was assigned for the Profiler Plus Kit African American and U S Caucas
109. lelic ladder allele Alleles of all possible sizes within the range of 75 400 bp should be readily identifiable Any sample allele that sizes outside a window could be either of the following g o lt o 9 me 3 o 2 e S G S Q D w G I 7 2 An off ladder allele i e an allele of a size that is not represented in the AmpF STR Profiler Plus Allelic Ladder go to http www cstl nist gov strbase for examples of known off ladder alleles 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 in one capillary run Table 3 on page 70 indicates typical precision results obtained from 31 database samples and three AmpF STR Profiler Plus Allelic Ladder samples analyzed on the Applied Biosystems 310 Genetic Analyzer 47 cm capillary POP 49 polymer GeneScan 500 ROX Size Standard These results were obtained within a set of injections on a single capillary As indicated above sample alleles may occasionally size outside of the 0 5 bp window for a respective allelic ladder allele because of measurement error The frequency of such an
110. les provided by African American 195 Laboratory Corporation of America U S Caucasian 200 Allele frequencies Table 4 shows the Profiler Plus Kit allele frequencies in two populations listed as percentages Table 4 Profiler Plus Kit allele frequencies g g lt o 9 E 3 o 2 e S GL S Q D w 9 Em I 7 2 Allele African American U S Caucasian n 195 n 200 D3S1358 7 0 26 t 10 t 3 11 0 26t 0 25t 12 0 51f 0 25t G t 0 50 14 11 80 11 25 15 27 95 28 25 15 2 0 26t t 16 32 31 22 25 17 21 80 22 25 18 4 62 14 50 id i 0 50t vWA 11 0 26t t 12 t a 13 1 54 t 14 7 70 8 50 15 22 05 8 25 16 26 92 19 75 17 16 92 25 00 18 13 85 25 75 19 8 46 11 00 20 2 05 1 50 A li 0 25 22 0 261 t AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 91 92 Chapter 5 Experiments and Results Population data Allele African American U S Caucasian n 195 n 200 FGA 16 2 0 26 T 17 0 26 T 18 0 51t 1 50 19 4 62 6 25 19 2 0 26 T 20 4 36 16 25 20 2 T 0 75 21 13 33 17 75 21 2 0 26 t 22 18 97 16 50 22 2 0 26t 0 50t 23 18 97 14 00 24 16 41 13 25 25 12 31 11 25 26 4 10 1 50 26 2 f T 27 4 10 0 50t 28 0 771 T 29 0 26t T 30 t T
111. lied Biosystems databases other known alleles listed in Table 1 on page 12 have either been published or reported to us by other laboratories g o lt o 9 me 3 o 2 e S G S Q D w 9 Em E RS e 7 2 Independent allele frequencies Independence of allelic frequencies within a locus can be expressed by the Hardy Weinberg HW relationship Approximation of HW expectations in a sample population allows estimation of genotypic frequencies HW proportions from observed allelic frequencies using the HW equation expanded binomial square law Hartl and Clark 1989 Weir 1996 Several biostatistical tests were used to survey HW relationships at the Profiler Plus Kit STR loci in each sample population Independence was found between alleles within each locus as p values gt 0 05 were obtained from the homozygosity test Chakraborty et al 1988 Nei and Roychoudhury 1974 Nei 1978 likelihood ratio test Edwards et al 1992 Weir 1992 and Guo Thompson exact test Guo and Thompson 1992 Additionally allele frequency data were analyzed for independence based on the total number of observed distinct homozygous and heterozygous genotype classes Nei 1978 Chakraborty et al 199 Observed values were within two standard errors of expected values for each locus These sets of data demonstrate that appropriate estimations of Profiler Plus Kit genotype frequencies are generated from alle
112. lmuth R C Fildes N Bugawan T L Erlich H A and Klitz W 1992 Polymorphism recombination and linkage disequilibrium within the HLA class II region J Immunol 148 249 58 Brinkmann B Junge A Meyer E and Wiegand P 1998 Population genetic diversity in relation to microsatellite heterogeneity Hum Mutat 11 135 144 Brinkmann B Klintschar M Neuhuber F Huhne J and Rolf B 1998 Mutation rate in human microsatellites Influence of the structure and length of the tandem repeat Am J Hum Genet 62 1408 1415 Brown A H D Feldman M W and Nevo E 1980 Multilocus structure of natural populations of Hordeum spontaneum Genetics 96 523 536 Budowle B et al 1995 D1S80 population data in African Americans Caucasians Southeastern Hispanics Southwestern Hispanics and Orientals J Forensic Sci 40 38 44 Budowle B DeFenbaugh D A Keys K M 2000 Genetic variations at nine short tandem repeat loci in Chamorros and Filipinos Legal Med 2 1 26 30 Buel E Wang G and Schwartz M 1995 PCR amplification of animal DNA with human X Y amelogenin primers used in gender determination J Forensic Sci 40 641 644 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 121 Bibliography 122 Chakraborty R Smouse P E and Neel J V 1988 Population amalgamation and genetic variation observations on artificially agglomerated tribal populations of Central and S
113. lopmental Validation of the Profiler Plus Kit 5 Extra peaks in the electropherogram Lack of full A nucleotide addition may be observed in Profiler Plus Kit results when the amount of input DNA is greater than approximately 5 0 ng The reason for this is that more time is needed for AmpliTaq Gold DNA Polymerase to add the A nucleotide to all molecules as more PCR product is generated Amplification of too much input DNA will also result in off scale data see DNA quantification on page 19 for more information on off scale data Mixed samples Evidence samples may contain DNA from more than one individual The possibility of multiple contributors should be considered when interpreting the results In the discussion below a peak is defined as any peak that is greater than 150 RFU We recommend a minimum peak height threshold to avoid typing less than 35 pg of input DNA see Effect of DNA quantity on results on page 82 Detection of mixed samples Each of the following can aid in determining whether a sample is a mixture The presence of more than two alleles at a locus The presence of a peak at a stutter position that is significantly greater in percentage than what is typically observed in a single source sample see Stutter products on page 73 and Figure 7 through Figure 9 Significantly imbalanced alleles for a heterozygous genotype The peak height ratio is defined as the height of the lower peak in RFU divided
114. lopmental Validation of the Profiler Plus Kit 5 Probability of paternity exclusion Linkage disequilibrium between the Profiler Plus Kit loci and the AmpF STR Green I loci TH01 TPOX and CSF1PO was not detected The combination of these 12 AmpF STR loci offers an average probability of identity of approximately 1 1 18 x 10 4 African American and 1 2 52 x 10 4 U S Caucasian Probability of paternity exclusion Table 6 shows the Probability of Paternity Exclusion Pg values of the Profiler Plus Kit STR loci individually and combined Table 6 Probability of paternity exclusion values for the Profiler Plus Kit loci g g lt o 9 me 3 o 2 e S GL S Q D w GQ I 7 2 Locus African American U S Caucasian D3S1358 0 5260 0 5797 vWA 0 6394 0 6170 FGA 0 7202 0 7173 D8S1179 0 5930 0 6128 D21S11 0 7281 0 6835 D18S51 0 7518 0 7414 D5S818 0 5375 0 4554 D13S317 0 4725 0 5948 D7S820 0 5742 0 6307 Combined 0 999989 0 999982 The Pg value is the probability averaged over all possible mother child pairs that a random alleged father will be excluded from paternity after DNA typing of the Profiler Plus Kit STR loci Chakraborty and Stivers 1996 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 97 Chapter 5 Developmental Validation of the Profiler Plus ID Kit Developmental validation Section
115. lts 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 Profiler Plus Kit reactions with DNA extracted from adjudicated and non probative sexual assault evidence were examined to assess the performance of the Profiler Plus Kit on typical casework samples comprised of mixed body fluids These samples were extracted amplified and analyzed in collaboration with the Santa Clara County Crime Laboratory DNA extracts from four adjudicated sexual assault cases were prepared by DNA analysts at the Santa Clara County Crime Laboratory Sexual assault evidence materials were processed using the differential lysis and organic extraction procedure while victim suspect reference blood samples were processed using the Chelex extraction procedure Following amplification with the Profiler Plus Kit reagents the PCR products were analyzed using the Applied Biosystems 377 DNA Sequencer Case 1 and Case 2 contained a victim reference blood sample a suspect reference blood sample and a victim vaginal swab The Profiler Plus Kit genotype of the epithelial cell fraction was the same as that of the victim reference and did not contain alleles foreign to the victim The Profiler Plus Kit genotype of the sperm cell fraction did not contain detectable epithelial cell fr
116. lus ID PCR Amplification Kits User Guide 43 e D o z D o 9 5 GD fe E n Chapter 4 Data Analysis Set up GeneMapper ID Software for data analysis 2 Select the Size Standards tab click New select the Basic or Advanced radio button then click OK Q GeneMapper Manager l eC Projects Analysis Methods Table Settings Plot Settings Matrices Size Standards Last Saved Owner Type Description CE G5 HD GS500 201 0 09 08 15 04 3 gmid BasiciAdvanced 3 Enter a name for example CE_F_HID_GS500 75 450 In the Size Standard Dye field select Red In the Size Standard Table enter the sizes specified in on page 43 The example below is for the GeneScan 500 ROX Size Standard 77 Size Standard Editor Edit r Size Standard Description Name CE_F_HID_GS500 75 450 Description Size Standard Dye SIze Standard Table Size in Basepairs 75 0 100 0 139 0 150 0 160 0 200 0 300 0 340 0 350 0 400 0 450 0 44 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Section 4 1 GeneMapper ID Software 4 Analyze and edit sample files with GeneMapper ID Software Analyze and edit sample files with GeneMapper ID Software 1 In the Project window select File Add Samples to Project
117. lysisMet 2011 05 19 14 41 1 mg HID Microsatellite Default 2010 01 27 14 58 0 amd Microsetel Select the Analysis Methods tab then click New to open the New Analysis Method dialog box Select HID and click OK to open the Analysis Method Editor with the General Tab selected The figures below show the settings for each tab of the Analysis Method Editor Configure settings as shown unless the instructions state otherwise Note The Analysis Method Editor closes when you save your settings see step 5 on page 38 To complete this step quickly do not save the analysis method until you finish entering settings in all of the tabs Click Save AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Section 4 1 GeneMapper ID Software 4 Set up GeneMapper ID Software for data analysis General tab settings Analysis Method Editor HID General Allele Peak Detector Peak Quality Quality Flags r Analysis Method Description Name ProfilerPlus amp nalysisMethod v1 Description Instrument Analysis Type HID BEMYOS G oddeu In the Name field either type the name as shown or enter a name of your choosing The Description and Instrument fields are optional AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 39 n Chapter 4 Data Analysis Set up GeneMapper ID Software for data analysis Allele tab settin
118. mal 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 10 User supplied materials Itemt Source AmpF STR Profiler Plus and Profiler Plus D PCR Amplification Kits 4303326 3100 Analyzer materials 96 well plate septa 4315933 Reservoir septa 4315932 3100 3130x Genetic Analyzer capillary array 36 cm 4315931 POP 4 polymer for 3100 3100 Avant Genetic Analyzers 4316355 3100 3100 Avant Genetic Analyzer Autosampler Plate Kit 96 well 4316471 GeneScan 500 ROX Size Standard 401734 Running Buffer 10X 402824 Hi Di Formamide 4311320 DS 32 Matrix Standard Kit Dye Set F 4345831 MicroAmp Optical 96 well reaction plate N8010560 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 113 C Appendix C Ordering Information Equipment and materials not included 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 3100 Avant instrument refer to Appendix B of the 3100 Genetic Analyzer and 3100 Avant Genetic Analyzer User Reference Guide Pub no 4335393 3130xl Analyzer materials 96 well plate septa 4315933 Reservoir septa 4315932 3100 3130xl Genetic Analyzer capillary array 36 cm 4315931 POP 4 polymer for
119. n the AmpF STR Profiler Plus Allelic Ladder from the same run and then converted to genotypes see Allelic ladder requirements on page 25 For more information on precision and genotyping see Lazaruk et al 1998 Table 2 Example of precision results on a 310 Genetic Analyzer Allele n Mean S D D3S1358 12 3 111 34 0 01 13 3 115 51 0 04 14 11 119 43 0 07 15 14 123 42 0 14 15 22 127 48 0 10 17 16 131 62 0 15 18 6 135 75 0 11 19 4 139 96 0 08 vWA 11 4 154 59 0 05 12 3 158 84 0 07 13 3 163 01 0 02 14 6 167 18 0 12 15 13 171 09 0 07 16 13 175 09 0 05 17 11 179 11 0 06 18 12 183 04 0 05 19 13 187 00 0 09 20 4 190 93 0 08 21 3 194 86 0 04 FGA 18 3 216 23 0 05 19 5 220 27 0 07 20 6 224 28 0 02 21 16 228 30 0 05 24 9 240 41 0 07 25 12 244 50 0 06 26 3 248 53 0 05 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Section 5 1 Developmental Validation of the Profiler Plus Kit 5 Accuracy reproducibility and precision Allele n Mean S D 26 2 3 250 56 0 05 27 5 252 61 0 04 28 3 256 67 0 06 g 29 4 260 74 0 07 a 30 3 264 85 0 08 E Amelogenin S X 34 103 44 0 05 S Y 22 109 12 0 05 3 D8S1179 F y 8 3 123 68 0 05 9 9 4 127 69 0 02 z 10 6 131 82 0 04 A 11 7 135 95 0 06
120. 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 After emptying a waste container seal it with the cap provided Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure that the waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply pennig chemical CAS Chemical Phrase handling 26628 22 8 Sodium Azide Sodium azide may react with lead and copper plumbing to form highly explosive metal azides 118 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Appendix D Safety Biological hazard safety Biological hazard safety Q WARNING Potential Biohazard Depending on the samples used on this instrument the surface may be considered a biohazard Use appropriate decontamination methods when working with biohazards A WARNING BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit infectious di
121. ns have been specified to produce peak heights of lt 150 RFU for a sample containing 35 pg human genomic DNA corresponding to ten total allele copies Peak heights lt 150 RFU should be interpreted with caution Individual laboratories may find it useful to determine an appropriate minimum peak height interpretational threshold based on their own results using low amounts of input DNA Figure 13 Effect of amplifying various amounts of AmpF STR Control DNA 9947A ranging from 16 pg to 1 ng Note that the y axis scale differs in many of these panels E 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 C A Jh n o AN MA Mo A 2B 02 2 1 0 ng 99474 Control DNA GM 26 02 2 1 0 ng 99474 Control DNA OW 2Y 02 92 71 0 ng 99474 Control DNA AMM A oa uA A 3B 03 3 0 5 ng 99474 Control DNA E 36 03 3 0 5 ng 88474 Control DNA LIO 3y 08 3 0 5 ng 99474 Control DNA 4B 04 4 250 pg 99474 Control DNA GM 46 0494 250 pg 39474 Control DNA LIO 4v 04 4 250 pg 99474 Control DNA 5B 05 5 125 pg 3947 Control DNA HE 56 05 5 125 pg 99474 Control DNA OW 5Y 05 5 125 pg 99474 Control DNA HE 6B 06 5 62 5 pg 99474 Control DNA EE 66 0696 62 5 pg 39474 Control DNA OW eY 08 6 62 5 pg 99474 Control DNA 7B 07 7 81 pg 99474 Control DNA GM 76 07 7 431 pg 39474 Control DNA LIO 7 07 7 31 pg 99474 Control DNA 8B 08 8 16 pg 99474 Control
122. nt cece cece cece eee e eens 90 Population data s eee e RR a ee ede beter ede be d N wie po rue ule yc AR RR ees 90 DAB 8 1 2 3 Population Data s lt e aga RN KK aR eee eee eens 90 DAB 8 1 2 3 1 Population Distribution Data 0000s 90 OVERVIEW todd tid emit adeeb BE vie baled REETA glee e E Rd 90 Population samples used in these studies cece eee eee eee eee 91 Allele frequencies i vec ccr eter en eite Lahde d Te bases 91 Probability of identity esa snc euer R H a Eog e EUER ede es ud MED ead 96 Probability of paternity exclusion 2 00 ccc eee eee e 97 Section 5 2 Developmental Validation of the Profiler Plus D Kit 98 Developmental validations esisi n r a eee RE n 98 DAB 8 1 1 Developmental Validation nannan annann nennen neee 98 PGR components Lore pL CRI ue EOS ee eee Fed EROS 98 Thermal cycler parameters sssssussssselslllllll ss 99 Species Specificity u ts ges kcu bac due Ti e tee mE cupo uM ite A ate A 100 DAB 8 1 2 2 Species Specificity 000 cece sse 100 Nenlfiuman studies ice cod RR xem ex et Yd e aae 100 Sensitivity cues er ck vw ue dr x ons N e RO ee Se eee ee eee IY R e 101 DAB 8 1 2 Z SensitlVily a eit ese bei PRU Hb ERN Re libe 101 Effect of DNA quantity on results sss 101 ec 101 DAB 8 1 22 Stability cese gale t e EUM REA K eb uie enki 101 AmpF amp TR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Contents
123. ntrations of hematin used were 0 uM 20 uM 22 uM 28 uM and 30 uM When the amount of hematin was increased to a concentration that started to inhibit the PCR D75820 and D18551 were the first loci to drop out in each experiment followed by FGA Figure 14 Figure 14 DNA amplified with the Profiler Plus Kit in the presence of varying concentrations of hematin 20 uM 22 uM 26 uM and 30 uM g g lt o 9 E 3 o 2 e S GL S Q D w GQ I 7 2 120 150 180 210 240 270 300 n i n BB 05 5 no hematin Profiler Plus HE 56 05 5 no hematin Profiler Plus SY 05 S no hematin Profiler Plus it A Ah AN Ad AA AA A A 7B 07 7 20 uM hematin Profiler Plus EH 76 077 20 4M hematin Profiler Plus 7Y 07 7 20 jM hematin Profiler Plus 8B 08 8 22 uM hematin Profiler Plus EH 8G 08 8 22 M hematin Profiler Plus 8Y 08 8 22 JM hematin Profiler Plus 10B 10 10 26 uM hematin Profiler Plus DE 106 10 10 4 26 4M hematin Profiler Plus 10Y 10510 26 4M hematin Profiler Plus 12B 12 12 30 uM hematin Profiler Plus IE 126 12012 30 4M hematin Profiler Plus 12Y 12512 30 uM hematin Profiler Plus Degraded DNA As the average size of degraded DNA approaches the size of the target sequence the amount of PCR product generated is reduced This is due to the reduced number of intact templates in the size range necessary for ampl
124. nty Crime Laboratory San Jose CA following the phenol chloroform procedure differential lysis was performed on stains containing semen At Applied Biosystems approximately 3 ng of DNA was amplified from each dried fluid mixture in a GeneAmp PCR System 9600 and detected using the Applied Biosystems 310 Genetic Analyzer The limit of detection of the minor genotype component of each body fluid mixture ratio was determined The limit of detection is defined here as the ratio below which a mixture is not recognized but rather appears to contain DNA from a single source the major contributor of the mixture The limit of detection occurred in blood blood mixtures when the minor component was present at one tenth the volume of the major genotype In epithelial cell fractions of differential extractions of semen blood or semen saliva stains blood and saliva were detectable at one tenth the volume of semen Sperm DNA carryover into the epithelial cell fraction of these stains was detectable at one tenth the volume of blood or saliva In sperm fractions the male genotype was detectable from every semen blood or semen saliva mixture with no trace of the female DNA Note that the limit of detection for the minor component is influenced by the specific combination of genotypes present in mixtures Population data DAB 8 1 2 3 Population Data DAB 8 1 2 3 1 Population Distribution Data Overview 90 Population distribution data are doc
125. oci amplification 12 PCR reaction mix 18 primers 11 18 20 reagents 18 supported instruments 11 thermal cyclers for use with 112 L limited product warranty 129 LIZ size standard about 18 volume per reaction 28 29 31 loci allele frequencies in the population databases 91 amplified by kit 12 chromosomal location 12 differential amplification 85 dye label 12 effect of DNA quantity on results 82 inhibitors 84 lack of amplification 82 83 population data allele frequencies 91 population data samples used in studies 91 103 low TE buffer 19 M master mix volume per reaction 21 materials and equipment included in kit 18 not included with kit 113 130 AmpF amp TR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide mixed samples 77 multicomponent analysis 16 17 N negative control sample preparation 21 nonprobative evidence reference samples 89 0 operating systems 16 27 29 31 ordering information 113 P panel check version 49 import 35 50 PCR amplification of tetranucleotide STR loci stutter peak 73 inhibitor 84 performing 22 setup 111 thermal cycling conditions programming 22 PCR work areas 111 113 population genetics 90 allele frequencies 91 populations and samples used in the studies 91 103 population studies 81 positive control sample preparation 21 primers volume per reaction 21 probability of identity definition 96 values 96 Q quantification DNA 19
126. of the Profiler Plus Kit containing the in house components updated kit with the performance of the original kit focusing on studies most relevant to forensic DNA testing see SWGDAM Guidelines effective January 1 2011 Because of the similarity between the Profiler Plus and Profiler Plus ID Kits the results generated with the Profiler Plus Kit can be considered representative of expected performance of the Profiler Plus ID Kit These studies while not exhaustive are in our opinion appropriate for a manufacturer Additional studies were performed with inhibited samples using the AmpF STR SGM Plus Kit and represent the expected performance for 4 dye chemistries Refer to the AmpFtSTR SGM Plus Kit User Guide Pub no 4309589 for further details Our studies compared the performance of two Roche manufactured enzyme and buffer lots Control mixes with three new lots of buffer and two new lots of enzyme manufactured by Life Technologies Test mixes Studies were performed using Test mixes containing both the enzyme and buffer manufactured by Life Technologies Test Control A mix Control B mix Test A mix Test B mix Test C mix Material Buffer Control Buffer Control Buffer Test Buffer Test Buffer Test Buffer Lot 1 Lot 2 Lot 1 Lot 2 Lot 3 Enzyme Control Control TestEnzyme Control Test Enzyme Enzyme Enzyme Lot 1 Enzyme Lot 2 Lot 1 Lot 2 Lot 2 AmpFtSTR Profiler Plus and Profiler Plus ID
127. ofiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Section 4 2 GeneMapper ID X Software 4 Set up GeneMapper ID X Software for data analysis Peak Quality tab Analysis Method Editor settings General Allele Peak Detector Peak Quality so amp GQ Settings Min Max Peak Height LPH MPH Homozygous min peak height Perform Heterozygous min peak height internal a validation Max Peak Height MPH studies to determine settings Peak Height Ratio PHR Min peak height ratio Broad Peak BD Max peak width basepairs C 0 2 0 ict oc o 0 p e v Xx GD 2 p 2 0 Allele Number AN Max expected alleles Allelic Ladder Spike Cut off Value Factory Defaults Save Cancel IMPORTANT Perform the appropriate internal validation studies to determine the minimum heterozygous and homozygous minimum peak height thresholds maximum peak height threshold and the minimum peak height ratio threshold for interpretation of data AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 59 n Chapter 4 GeneMapper ID X Software Set up GeneMapper ID X Software for data analysis SQ amp GQ tab settings Create size standard optional 60 Analysis Method Editor Quality weights are between 0 and 1 r Sample and Control GQ Weighting Broad P
128. on in humans and other primates at six short tandem repeat loci used in forensic identity testing Forensic Sci Intl 119 1 1 12 Li H Schmidt L Wei M H Hustad T Leman M I Zbar B and Tory K 1993 Three tetranucleotide polymorphisms for loci D351352 D351358 D351359 Hum Mol Genet 2 1327 Luna L G ed Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology McGraw Hill Book Co New York 1968 Mancuso D J Tuley E A Westfield L A Worrall N K Shelton Inloes B B Sorace J M Alevy Y G and Sadler J E 1989 Structure of the gene for human von Willebrand factor J Biol Chem 264 19514 19527 Mills K A Even D and Murrau J C 1992 Tetranucleotide repeat polymorphism at the human alpha fibrinogen locus FGA Hum Mol Genet 1 779 M ller A Meyer E and Brinkmann B 1994 Different types of structural variation in STRs HumFES FPS HumVWA and HumD215S11 Intl J Legal Med 106 319 323 Moller A and Brinkmann B 1995 PCR VNTRs PCR Variable Number of Tandem Repeats in forensic science Cell Molec Biol 41 715 724 Moretti T R Baumstark A L Defenbaugh D A Keys K M Brown A L Budowle B 2001 Validation of STR typing by capillary electrophoresis J Forensic Sci May 46 3 661 76 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 123 Bibliography 124 Moretti T Baumstrak A Defen
129. ontrol are shown All samples were analyzed on an Applied Biosystems 310 Genetic Analyzer The peaks depicted in red are the GeneScan 500 ROX size standard Be LONE i g20 240 2000 4 ETTTE Chimp 1000 7 03 20003 20003 Horse 1000 3 oJ nid 2000 1000 4 0 5000 3 2000 4 1000 4 T mas E coli of 14 Add l u ek Negative n i i i T i control AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Sensitivity DAB 8 1 2 2 Sensitivity Effect of DNA quantity on results Stability DAB 8 1 2 2 Stability Section 5 2 Developmental Validation of the Profiler Plus ID Kit 5 Sensitivity Species specificity sensitivity stability and mixture studies are conducted DAB 1998 The amount of input DNA added to the PCR reaction should be 1 0 2 5 ng The DNA sample should be quantitated prior to amplification using a system such as the Quantifiler Human DNA Quantitation Kit Part no 4343895 Figure 21 on page 101 shows the effect of different amounts of AmpF STR Control DNA 9947A The final DNA concentration should be in the range of 0 05 0 125 ng uL so that 1 0 2 5 ng of DNA will be added to the PCR reaction in a volume of 20 uL If the sample contains degraded DNA amplification of additional DNA may be beneficial The PCR cycle number and amplification conditions have been specified to produce low peak hei
130. oresis 000 ccc cece cece cece eens 29 Reagents and parts zi socer ror ev ema Hey ede Ue tte coe EAM ERR sees 29 Electrophoresis software setup and reference documents ee eeee 29 Prepare samples for electrophoresis on the 3500 3500xL instrument 0 0 0005 29 Section 3 3 310 Instrument o cerie ietie Z cee e N eees 31 Set up the 310 instrument for electrophoresis 000000 c cece eee ee eee eee ee eees 31 Reagents and parts esgaret DR s Bie weg eee Seth ie IERI ER ROMs 31 Electrophoresis software setup and reference documents ee eeee 31 Prepare samples for electrophoresis on the 310 Instrument cece eee ees 31 CHAPTER 4 Data Analysis 0c cece cece eee eee eee 33 Section 4 1 GeneMapper ID Software 6 cece ee 33 Overview of GeneMapper ID Software 0 cc ccc ccc nnn e rne 33 STRUM GIES aces core d sir Sd eds E Ct Eos 33 Before youistarE n tero Lu Vere Eee e vert 34 Set up GeneMapper D Software for data analysis esee 34 File names sei iei e eg du RR Sova ee ees ee eee Se Ae eee PIT EU 34 Before using the software for the first nme cece cece eee ee eee 34 Import panels and bins annann annann anaana eee eee n 35 Create an analysis method n n 0 0 eect eee eee eens 38 General tab Settings nunnan unner rnrn adedihe ohh ei naan ewan ca 39 Allele tab settinigS sees la eve rule lv ee ha dec tese ieu tiec rue eres 40 Peak Detector tab settings ss eens 41 Peak Quality
131. outh America Am J Hum Genet 43 709 725 Chakraborty R Fornage M Guegue R and Boerwinkle E 1991 Population genetics of hypervariable loci analysis of PCR based VNTR polymorphism within a population In Burke T Doif G Jeffreys A J and Wolff R eds DNA Fingerprinting Approaches and Applications Birkhauser Verlag Berlin pp 127 143 Chakraborty R and Stivers D N 1996 Paternity exclusion by DNA markers effects of paternal mutations J Forensic Sci 41 671 677 Clark J M 1988 Novel non templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases Nucleic Acids Res 16 9677 9686 Comey C T Koons B W Presley K W Smerick J B Sobieralski C A Stanley D M and Baechtel F S 1994 DNA extraction strategies for amplified fragment length polymorphism analysis J Forensic Sci 39 1254 1269 Cone R W and Fairfax M R 1993 Protocol for ultraviolet irradiation of surfaces to reduce PCR contamination PCR Methods Appl 3 515 S17 D135317 Cooperative Human Linkage Center CHLC accession number 512 GenBank accession number G09017 D55818 Cooperative Human Linkage Center CHLC accession number 415 GenBank accession number G08446 D75820 Cooperative Human Linkage Center CHLC accession number 511 GenBank accession number G08616 DeFranchis R Cross N C P Foulkes N S and Cox T M 1988 A potent inhibitor of Taq DNA polymerase copurifies
132. 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 Four CEPH family DNA sets were examined One and a half nanograms of DNA from each sample was amplified using the Profiler Plus Kit followed by analysis using an Applied Biosystems Genetic Analyzer The families examined included 884 14 offspring 1340 eight offspring 1341 ten offspring and 1345 eight offspring representing eighty meiotic divisions The results confirmed that the loci are inherited according to Mendelian rules as has also been reported in the literature Nakahori et al 1991 Kimpton et al 1992 Mills et al 1992 Sharma and Litt 1992 Li et al 1993 Straub et al 1993 Begovich et al 1992 Mapping The Profiler Plus Kit loci D3S1358 vWA FGA amelogenin D8S1179 D21S11 D18551 D55818 D135317 and D7S820 have been mapped and the chromosomal locations have been published Nakahori et al 1991 Mills et al 1992 Sharma and Litt 1992 Straub et al 1993 Barber and Parkin 1996 Hudson et al 1995 Green et al 1991 They are listed in Table 1 on page 12 Population studies Population distribution data of the 9 Profiler Plus Kit STR loci have been established in different racial and
133. paration pull up Incomplete A nucleotide addition When the total number of allele copies added to the PCR is extremely low allelic dropout can occur resulting in a partial profile Methods of quantifying DNA Life Technologies provides several kits for quantifying DNA in samples See the reference cited in the following table for details about these kits Product Description Quantifiler Human DNA Quantification Kit Part no 4343895 and Quantifiler9 Y Human Male DNA Quantification Kit Part no 4343906 For more information see Quantifiler Human DNA Quantification Kits User s Manual Pub no 4344790 Properties The Quantifiler Human and Quantifiler Y Human Male Kits are highly specific for human DNA and they individually detect total human or male DNA respectively The kits detect single stranded and degraded DNA How they work The Quantifiler DNA Quantification Kits consist of target specific and internal control 5 nuclease assays The Quantifiler Human and Quantifiler Y Human Male Kits contain different target specific assays human DNA or human male DNA respectively that each consist of two locus specific PCR primers and one TaqMan MGB probe labeled with FAM dye for detecting the amplified sequence The kits each contain a separate internal PCR control IPC assay which consists of an IPC template DNA a synthetic sequence not found in nature two primers for amplifying
134. r ID Software 4 Set up GeneMapper ID Software for data analysis Quality Flags tab 3 Analysis Method Editor HID settings General Allele Peak Detector Peak Quality Quality Flags Quality weights are between 0 and 1 rQualty Flag Settings Spectral Pull up Control Concordance Broad Peak Low Peak Height Out of Bin Allele Off scale Peak Height Ratio Overlap rPGV Thresholds Sizing Quality From 0 75 to1 0 FromO 0to 0 25 Genotype Quality From 0 75 to1 0 FromO 0to 0 25 Factory Defaults IMPORTANT The values shown are the software defaults and are the values we used during developmental validation Perform the appropriate internal validation studies to determine the appropriate values for interpretation of data Create size The GeneScan 500 ROX Size Standard for the Profiler Plus and Profiler Plus ID standard Kits uses the following size standard peaks in its definitions 75 100 139 150 160 200 300 340 350 400 and 450 Note The 250 nt peak in the GeneScan 500 ROX Size Standard is not included in the size standard definition This peak can be used as an indicator of precision within a run Use the following procedure to create the appropriate size standard 1 Select Tools GeneMapper Manager to open the GeneMapper Manager AmpFtSTR Profiler Plus and Profiler P
135. r Set Contains fluorescently labeled primers and non labeled primers 1 tube 1 1 mL AmpF STR Profiler Plus Allelic Ladder or AmpF STR Profiler Plus D Allelic Ladder Contains amplified alleles See Table 1 on page 12 for a list of alleles included in the allelic ladders 1 tube 0 05 mL 15 to 25 C on receipt 2 to 8 C after initial use AmpFSTR Control Contains 0 10 ng L human female 9947A 1 tube 0 3 mL DNA 9947A DNA in 0 05 sodium azide and buffert See Table 1 on page 12 for profile AmpliTaq Gold DNA Contains enzyme with an activity of 5 U uL 2 tubes 15 to 25 C Polymerase 0 05 mL tube t The AmpF STR Control DNA 9947A is included at a concentration appropriate to its intended use as an amplification control i e to provide confirmation of the capability of the kit reagents to generate a profile of expected genotype The AmpF STR Control DNA 9947A is not designed to be used as a DNA quantitation control and you may see variation from the labelled concentration when quantitating aliquots of the AmpF STR Control DNA 9947A Standards for samples 18 For the Profiler Plus and Profiler Plus ID Kits the panel of standards needed for PCR amplification PCR product sizing and genotyping are s AmpFE STR Control DNA 9947A A positive control for evaluating the efficiency of the amplification step and STR genotyping using the allelic ladder in the kit
136. requencies in the population databases 91 allelic dropout 107 allelic ladder about 18 precision 70 profile 13 requirements for accurate genotyping 25 using to determine genotypes 69 volume per reaction 28 30 32 amplification differential amplification of loci 85 loci 12 using bloodstained FTA cards 23 AmpliTaq Gold DNA Polymerase catalyzing the ad dition of a3 A nucleotide 76 B bins check version 49 import 35 50 biohazard safety 119 buffer new 104 C chemical safety 118 contents of kit 18 control DNA 007 14 18 Index D Data Collection Software overview 16 degraded DNA 85 developmental validation 66 differential amplification 85 DNA control about 18 effect of DNA quantity on results 82 how degraded DNA affects which loci amplify 85 mixed samples 77 89 102 negative control reaction 21 positive control reaction 21 quantification 19 quantification methods 20 sample preparation 21 test sample 21 using agarose gel analysis to examine the DNA 85 DNA from more than one individual 77 89 102 documentation related 127 E electropherogram addition of a3 A nucleotide 76 causes for extra peaks 73 77 89 102 stutter peak 73 electrophoresis Data Collection Software 27 29 31 preparing samples on the 310 instrument 31 preparing samples on the 3100 3100 Avant or 3130 3130xl instrument 28 preparing samples on the 3500 3500xL instrument 29 reagents and parts 27 29 31 references 27 29 31 run module 2
137. rer of the instrument or device Before handling chemicals read and understand all applicable Safety Data Sheets SDSs and use appropriate personal protective equipment gloves gowns eye protection etc To obtain SDSs see the Documentation and Support section in this document AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 117 Appendix D Safety Chemical safety Chemical safety AN WARNING GENERAL CHEMICAL HANDLING To minimize hazards ensure laboratory personnel read and practice the general safety guidelines for chemical usage storage and waste provided below and consult the relevant SDS for specific precautions and instructions Read and understand the Safety Data Sheets SDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials To obtain SDSs see the Documentation and Support section in this document Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the SDS Handle chemical wastes in a fume hood Ensure use of primary a
138. results obtained We chose conditions that produced optimum PCR product yield and that met reproducible performance standards It is our opinion that while these experiments are not exhaustive they are appropriate for a manufacturer of STR kits intended for forensic and or parentage testing use Each laboratory using the Profiler Plus Kit should perform their own internal validation studies Developmental validation DAB 8 1 1 Developmental Validation PCR components 66 Developmental validation that is conducted shall be appropriately documented DNA Advisory Board 1998 Critical reagent concentrations and reaction conditions such as thermal cycling parameters AmpliTaq Gold DNA polymerase activation cycle number to produce reliable locus specific amplification and appropriate sensitivity have been determined The concentration of each component of the Profiler Plus Kit Tris HCl pH 8 3 KCL dNTPs primers AmpliTaq Gold DNA Polymerase MgCL bovine serum albumin and sodium azide was optimized to give the most reliable performance The optimal concentration for a particular component was established to be in the middle of a window that meets the reproducible performance characteristics of specificity and sensitivity AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Thermal cycler parameters Section 5 1 Developmental Validation of the Profiler Plus Kit 5 Accuracy repro
139. s chemistries and software associated with the AmpF STR Profiler Plus and Profiler Plus ID PCR Amplification Kits User attention words Five user attention words may appear in this document Each word implies a particular level of observation or action as described below Note Provides information that may be of interest or help but is not critical to the use of the product IMPORTANT Provides information that is necessary for proper instrument operation or accurate chemistry kit use AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 9 About This Guide User attention words CAUTION Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices WARNING Indicates a potentially hazardous situation that if not avoided could result in death or serious injury DANGER Indicates an imminently hazardous situation that if not avoided will result in death or serious injury 10 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Overview B Product OvervieW lssseseeseeeeeeee hm 11 M Workflow overview 6 000 ce e hr 15 B Instrument and software overview 000 cece cette eens 16 R Materials and equipment sese e s e e s e c e e e eee nes 18 Product overview Purpose Product description About the primers The AmpF ST
140. s and storage 0c essen 18 Standards for samples n 0000 c cece eee eee nent le 18 B CHAPTER 2 Perform PCR eese 19 Required user supplied reagents esse 19 DNA quantificatlon erue bebe pec Lp RIS bi bebe tbe he MS 19 Importance of quantification seen 19 Methods of quantifying DNA 2 naaraan nenene eee ee ee ee enna 20 Prepare the amplification kit reactions cece cence eee aee 21 Select the correct PCR cycle number 00 00 cee cece eect eee eee ee 22 Perform PCR eren id ei dee X rude eder dede vite ade er We bathe vee hab nyo s 22 Amplification using bloodstained FTA cards 02 cece cece n enn nen eens 23 AmpF ZSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 3 Contents M CHAPTER 2 Electrophoresis 0 cece cece eee eens 25 Allelic ladder requirements n cee eee eee eee eect cette e eee eeeees 25 Section 3 1 3100 3100 Avant and 3130 3130xl instruments issues 27 Set up the 3100 3100 Avant or 3130 3130xl instrument for electrophoresis 27 Reagents andiparts sinises tangina e EER RR EUER NIE ceded cee dec DERE Med 27 Electrophoresis software setup and reference documents ee eeee 27 Prepare samples for electrophoresis on the 3100 3100 Avant or 3130 3130xl instrument 28 Section 3 2 3500 3500xL Series instruments 0 0 cece eee eee eens 29 Set up the 3500 3500xL instrument for electroph
141. s requires at least one allelic ladder sample per run folder Your laboratory can use multiple ladder samples in an analysis provided individual laboratories conduct the appropriate validation studies For multiple ladder samples the GeneMapper ID Software calculates allelic bin offsets by using an average of all ladders that use the same panel within a run folder Allelic ladder samples in an individual run folder are considered to be from a single run When the 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 e 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 e Alleles that are not in the AmpF STR Allelic Ladders do exist Off ladder OL alleles may contain full and or partial repeat units An off ladder allele is an allele that occurs outside the 0 5 nt bin window of any known allelic ladder allele or virtual bin Note If a sample allele peak is called as an off ladder allele the sample result needs to be verified according to the laboratory s protocol Set up GeneMapper D Software for data analysis
142. seases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following In the U S U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories found at www cdc gov biosafety Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 found at www access gpo gov nara cfr waisidx 01 29cfr1910a 01 html e Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials Additional information about biohazard guidelines is available at www cdc gov In the EU Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best practices published in the World Health Organization WHO Laboratory Biosafety Manual third edition found at www who int csr resources publications biosafety WHO CDS CSR LYO 2004 11 en AmpFtST
143. software uses these parameters to specify the minimum peak height in order to limit the number of detected peaks Although GeneMapper ID Software displays peaks that fall below the specified amplitude in electropherograms the software does not label or determine the genotype of these peaks e Size calling method The Profiler Plus and Profiler Plus ID Kits have been validated using the Local Southern sizing method Before using other sizing methods perform internal validation studies AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 41 n Chapter 4 Data Analysis Set up GeneMapper ID Software for data analysis Peak Quality tab Analysis Method Editor HID settings E General Allele Peak Detector Peak Quality Quality Flags Perform Signal level internal Homozygous min peak height validation f studies to Heterozygous min peak height x E g 7 determine Heterozygote balance settings Min peak height ratio Peak morphology Max peak width basepairs Pull up peak Pull up ratio Allele number Max expected alleles Factory Defaults IMPORTANT Perform the appropriate internal validation studies to determine the minimum heterozygous and homozygous minimum peak height thresholds and the minimum peak height ratio threshold for interpretation of data 42 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Section 4 1 GeneMappe
144. stability 83 thermal cycler parameters 67 W warranty 129 work area amplified DNA 112 PCR setup 111 setup and lab design 111 workflow overview 15 132 AmpF amp TR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Headquarters a 5791 Van Allen Way Carlsbad CA 92008 USA Phone 1 760 603 7200 Toll Free in USA 800 955 6288 0 For support visit www appliedbiosystems com support technologies www lifetechnologies com
145. tab settings 4 beoe nce Ri eel eene Rial aE r dh dats 60 Create size standard optional ccc ccc nnn nen ene ne rnn 60 Analyze and edit sample files with GeneMapper D X Software 2 000 ccc cece ee ene 62 Examine and edit a project 0 0 nn 63 For more information asedio oree aiaa ZE Kea Ta 22 EA a e n 64 CHAPTER 5 Experiments and Results 0 ccc cence eee 65 Section 5 1 Developmental Validation of the Profiler Plus Kit 66 OVERVIEW kas Lom Fes So es BS ba oe pua e tn ELA IE cue ht ee T acts d A els a 66 Itriportance of validation cc bI A aE TRT a Lae 66 Experiment conditionS 5 00 4 pk eR Seley tegen r LOS eee eee ee 66 Developmental validation 000 ccc een eee e eee e eens 66 DAB 8 1 1 Developmental Validation 0c eee eee eee eens 66 PCR COMPONENTS esu s ences s ecd Ev H cope eap ue Area eet ee 66 Thermal cycler parameters sss 67 Accuracy reproducibility and precision e 67 DAB 8 1 2 Accuracy i ie Lese E i It RE UL E ROITUDE PERITUS 67 7 cle ci ds v Am cue ne NI SS ceri t E ty A ENDE 68 Reproducibility asare deco or orale akt dos fA soa T nao pe tidie 69 Precision and size windows 0c eee eee e eee eee 69 Extra peaks in the electropherogram 2000 cece eee eee nr nrnna 73 OVErVIEW su ener he T d EE E g sete E RR g 0 de hot eee RAS 73 Stutter products ree eh ot e aote s pi epo Sas RR e tps 73 Addition of 3 A nucleotide 0 0 ieee see 76 M
146. 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 Profiler Plus AnalysisMethod v1 or the name of the analysis method you created Panel Profiler Plus v2 Size Standard CE E HID GS500 75 450 or the name of the size standard you created t The Profiler Plus and Profiler Plus D Kits were originally validated using the GeneScan 500 ROX Size Standard If you use the GeneScan 400 HD Size Standard as an alternative perform the appropriate internal validation studies to support the use of this size standard with the Profiler Plus and Profiler Plus D Kits Note For more information about how the Size Caller works refer to the GeneScan Analysis Software for the Windows NT Operating System Overview of the Analysis Parameters and Size Caller User Bulletin Pub no 4335617 3 Click B Analyze enter a name for the project in the Save Project dialog box then click OK to start analysis The status bar displays the progress of analysis As a completion bar extending to the right with the percentage indicated With text messages on the left The table displays the row of the sample currently being analyzed in green or red if analysis failed for the sample The Genotypes tab becomes available after analysis Project window after anal
147. ti Thermal Cycler does not require a compression pad 3 Start the run AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Chapter 2 Perform PCR 7 Amplification using bloodstained FTA cards 4 On completion of the run store the amplified DNA and protect from light If you are storing the DNA Then place at lt 2 weeks 2 to 8 C gt 2 weeks 15 to 25 C IMPORTANT Store the amplified products so that they are protected from light Amplification using bloodstained FTA cards FTA cards can be useful for collecting storing and processing biological samples A small punch disc of the card containing the sample can be placed directly into an amplification tube purified and amplified without transferring the disc Our studies indicate that a 1 2 mm bloodstained disc contains approximately 5 20 ng DNA An appropriate cycle number for this high quantity of DNA is 25 cycles as determined by our validation studies However it is recommended that each laboratory determine the optimum cycle number based on internal validation studies In the example shown in Figure 5 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 a MicroAmp tube for 25 cycles Figure 5 Profiler Plus Kit results from a 1 2 mm FTA bloodstain disc 25 cycle amplific
148. tories Handbook for Facility Planning Design Construction and Moving National Institute of Justice 1998 e Parentage DNA testing refer to the Guidance for Standards for Parentage Relationship Testing Laboratories American Association of Blood Banks 7th edition 2004 The sensitivity of the AmpF STR kits and other PCR based tests enables amplification of minute quantities of DNA necessitating precautions to avoid contamination of samples yet to be amplified Kwok and Higuchi 1989 Also take care while handling and processing samples to prevent contamination by human DNA Wear gloves at all times and change them frequently Close sample tubes when not in use Limit aerosol dispersal by handling sample tubes and reagents carefully Note We do not intend these references for laboratory design to constitute all precautions and care necessary for using PCR technology PCR setup work area IMPORTANT These items should never leave the PCR Setup Work Area Calculator Gloves disposable Marker pen permanent Microcentrifuge e Microcentrifuge tubes 1 5 mL or 2 0 mL or other appropriate clean tube for Master Mix preparation Microcentrifuge tube rack Pipette tips sterile disposable hydrophobic filter plugged Pipettors AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 111 Appendix B PCR Work Areas Amplified DNA work area Tube decapper autoclavable e Vortex
149. uence that does not amplify as efficiently as the other allele Amplification and analysis of additional loci may assist in the interpretation of the sample Resolution of genotypes in mixed samples A sample containing DNA from two sources can be comprised at a single locus of any of the seven genotype combinations listed below Heterozygote heterozygote no overlapping alleles four peaks Heterozygote heterozygote one overlapping allele three peaks Heterozygote heterozygote two overlapping alleles two peaks 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 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 Life Technologies instruments provides additional valuable data to aid in resolving mixed genotypes This quantitative value is much less subjective than comparing relative intensities of bands on a stained gel Ultimately the likelihood that any sample is a mixture must be determined by the analyst in the context of each particular case including the information provided from known refer
150. ulates 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 Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Section 4 2 GeneMapper ID X Software 4 Set up GeneMapper ID X Software for data analysis Set up GeneMapper D X Software for data analysis Panel bin and stutter file version Before using the software for the first tim
151. umented and available DAB 1998 The population distribution data would include the allele and genotype distributions for the locus or loci obtained from relevant populations Where appropriate databases should be tested for independence expectations DAB 1998 To interpret the significance of a match between genetically typed samples it is necessary to know the population distribution of alleles at each locus in question If the genotype of the relevant evidence sample is different from the genotype of the suspect s reference sample then the suspect is excluded as the donor of the biological evidence tested An exclusion is independent of the frequency of the two genotypes in the population 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 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Section 5 1 Developmental Validation of the Profiler Plus Kit 5 Population data Popu lation The Profiler Plus Kit was used to generate the population data provided in this samples used in section Samples were collected from individuals throughout the United States with no these studies geographical preference Population Number of samples Samp
152. ure 22 Results of Sample A to Sample B mixtures The mixture profiles below highlight the alleles attributable to the minor component even when the major component shares an allele Sample A Ine ND UCT ACU CN 1 1 all alleles 1 3 IRH RR d COC LLL e 102 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Section 5 2 Developmental Validation of the Profiler Plus ID Kit 5 Population data The profiles of the samples in Figure 22 are listed below Profile Allele Sample A Sample B Amelogenin X Y X D3S1358 15 16 15 18 D5S818 11 11 13 D7S820 7 12 9 10 D8S1179 12 13 13 D13S317 11 11 D18S51 12 15 17 19 D21S11 28 31 30 32 2 FGA 24 26 23 2 24 vWA 14 16 17 19 For these 1 ng total DNA mixture studies the limit of detection is when the minor component is present at approximately one tenth of the concentration of the major component and a threshold of 50 RFU The limit of detection for the minor component is influenced by the combination of genotypes in the mixture Population data DAB 8 1 2 3 Population Data Population samples used in these studies Population distribution data are documented and available DAB 1998 The Profiler Plus Kit was used to generate the population data provided in Population data on page 90 for 195 African Americans and 200 Caucasians Homozygous samples at the D851179 locus wer
153. ute with DNase E 120 150 180 210 240 270 300 N 30 0 GM 15 011 7 Profiler 111 min DNase BE 16 01 1 Profiler 111 min DNase LI 1 01 1 Profiler Il 1 min DNase 180 IH 26 02 2 amelogenin 1 min DNase GIN sy 03 3 58818 1 min DNase Seti te laa 7B 07 7 VWA 1 min DNase o das BE 46 044 D21511 1 min DNase LI 10v 10 10 40138317 1 min DNase 180 30 0 99 88 08 8 FGA min DNase 180 30 o O 11v 1111 7075820 1 min DNase 180 30 0 T Ned aucto a A ad IH 56 05 5 D18S51 1 min DNase meeec When degraded DNA is suspected to have compromised amplification of one or more loci the molecular weight of the DNA can be assessed by agarose gel analysis If the DNA is degraded to an average of 400 bp in size or less adding more DNA template to the Profiler Plus Kit amplification reaction can help produce a typeable signal for all loci Adding more DNA to the amplification provides more of the necessary size template for amplification Analysts at the Santa Clara Crime Laboratory prepared a panel of blood and semen specimens deposited on a variety of commonly encountered substrates Blood samples from two donors were deposited individually on wool cotton nylon metal glass leather one donor and blue denim Semen samples from two donors were
154. v2 txt file select the Use marker specific stutter ratio if available check box selected by default Perform appropriate internal validation studies to determine the appropriate filter setting to use 40 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Section 4 1 GeneMapper ID Software 4 Set up GeneMapper ID Software for data analysis Peak Detector tab Analysis Method Editor HID settings General Allele Peak Detector Peak Quality Quality Flags Peak Detection Algorithm Advanced v Ranges Peak Detection Analysis Sizing Peak Amplitude Thresholds Perfo G internal All Sizes k R S 3 validation 0 studies to determine settings rSmoothing and Baselining Smoothing O None Light O Heavy Peak Window Size Slope Threshold Peak Start r Size Calling Method Peak End 2nd Order Least Squares 3rd Order Least Squares Cubic Spline Interpolation Local Southern Method Global Southern Method Min Peak Half Vadi Polynomial Degree Baseline Window 51 e D o z D o 9 5 GD fe E E Factory Defaults IMPORTANT Perform the appropriate internal validation studies to determine the peak amplitude thresholds for interpretation of data Fields include e Peak amplitude thresholds The
155. v2X bin set and configure the stutter distance parameters as shown s GeneMapper ID X Software v1 0 1 or higher allows you to specify 4 types of marker repeat motifs tri tetra penta and hexa You can enter parameter values for each type of repeat in the appropriate column Specify the stutter ratio To apply the stutter ratios listed in the Allele tab for single source data deselect the Use marker specific stutter ratio if available check box selected by default Perform appropriate internal validation studies to determine the appropriate filter setting to use Note Applying global stutter ratios may reduce the editing required for single source sample data To apply the stutter ratios contained in the AmpFLSTR Stutter v2X file select the Use marker specific stutter ratio if available check box selected by default Perform appropriate internal validation studies to determine the appropriate filter setting to use AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 57 n Chapter 4 GeneMapper ID X Software Set up GeneMapper ID X Software for data analysis Peak Detector tab Analysis Method Editor settings General Allele Pe Peak Quality SQ amp GQ Settings Peak Detection Algorithm Advanced Ranges Peak Detection Analysis Sizing Peak Amplitude Thresholds Perform Ful Range AllSizes amp internal S d validation studies to
156. vant Genetic Analyzers Protocols for Processing AmpFISTR9 PCR Amplification Kit PCR Products 4332345 User Bulletin Applied Biosystems 3130 3100xl Genetic Analyzers Using Data Collection Software v3 0 User Bulletin 4363787 Applied Biosystems 3130 3130xl Genetic Analyzers Getting Started Guide 4352715 Applied Biosystems 3130 3130xl Genetic Analyzers Maintenance Troubleshooting and Reference Guide 4352716 Applied Biosystems 3130 3130xl Genetic Analyzers Quick Reference Card 4362825 Applied Biosystems 3130 3130xl Genetic Analyzers AB Navigator Software Administrator Guide 4359472 Applied Biosystems 3130 3100xl DNA Analyzers User Guide 4331468 Applied Biosystems 3500 3500xL Genetic Analyzer Quick Reference Card 4401662 Applied Biosystems 3500 3500xL Genetic Analyzer User Guide Data Collection v1 0 4401661 Applied Biosystems 3500 3500xL Genetic Analyzer User Bulletin Solutions to issues related to software data 4445098 hardware and consumables Note Additional user bulletins may be available at www lifetechnologies com Applied Biosystems 3730 3730xl Genetic Analyzer Getting Started Guide 4359476 GeneAmp PCR System 9700 Base Module User s Manual N805 0200 Veriti 96 Well Thermal Cycler AmpFeSTR Kit Validation User Bulletin 4440754 Quantifiler Kits Quantifiler Human DNA Quantification Kit and Quantifiler Y Human Male DNA 4344790 Quantification Kit Users Manual PrepFiler Forens
157. 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 DNA Recommendations 1994 Report concerning further recommendations of the DNA Commission of the ISFH regarding PCR based polymorphisms in STR short tandem repeat systems Intl J Legal Med 107 159 160 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 Liewellyn B Fish P Riech A Marcacci T Gandor D Parker D Carter R and Thibault S 2001 Validation of the AmpF STR Profiler Plus PCR Amplification Kit for use in forensic casework J Forensic Sci 46 3 642 646 Fr geau C J and Fourney R M 1993 DNA typing with fluorescently tagged short tandem repeats a sensitive and accurate approach to human identification Biotechniques 15 100 119 Gill P Urquart A Millican E Oldroyd N Watson S Sparkes R and Kimpton C P 1996 A new method of STR interpretation using inferential logic development of a criminal intelligence database Int J Leg Med 109 14 22 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide Bibliography Gill P d Aloja E Andersen J Dupuy B Jangblad M Johnsson V
158. x then click OK to start analysis The status bar displays the progress of analysis as a completion bar extending to the right with the percentage indicated The table displays the row of the sample currently being analyzed in green or red if analysis failed for the sample The Analysis Summary tab is displayed and the Genotypes tab becomes available upon completion of the analysis Analysis summary window after analysis GeneMapper ID X Profiler Plus Kit Example gmidx Is Logged In Database GBOLDROYNJOSE File Edit Analysis View Tools Admin Help G G H E Ey amp Ll re gt amp Table Setting 31XX Data Analysis Gai Project Samples Analysis Summary Genotypes Profiler Plus Kit Example Analysis Summary C 0 2 0 ict oc o 0 p e v Xx GD 2 p 2 0 Select run Folder to display Sample Status It Total of Samples Unanalyzed 0 Analyzed 6 Analysis Setting Changed 0 Click a link below to display a filtered Samples Table containing only the samples selected Allelic Ladder Quality per run folder based on SQ and CGQ only riis T 8 5 R 9 L IIS T RER UNI Profiler Plus Kit Example Control Quality per project based on sample PQVs SOS SSPK MIX OMR SQ CGQ Control Type Total of Samples B All thresholds met One or more thresholds not met Positive Control 0 Custom Control Negative Control Total
159. ysis Fle E Andy e Took Help tad B WM DNE bd ree versie hd rote be Ferret type 500 ate 5a Lo x Saure Fite Sarr ira Sarpi Type Act est etad P4 umma Surg Prote Phir Antya ma HO gt OHIO Serge ProterPhus rv etit FW M23 1 3 Saree Proser Act ya Le Lois IU ProtlerPlus Anales MTC DO DOA fam NIC Negative Cartroi Prag yw i g I DU Prat tA O06 C 4 19 PostweCo oU POMN Corto ProtierPlus evil etie hod For more information about any of these tasks refer to the GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide Pub no 4338775 AmpFtSTR Profiler Plus and Profiler Plus ID PCR Amplification Kits User Guide 45 e D o z D o 9 5 GD fe E E n Chapter 4 Data Analysis Examine and edit a project Examine and edit a project You can display electropherogram plots from the Samples and Genotypes tabs of the Project window to examine the data These procedures start with the Samples tab of the Project window assuming the analysis is complete For more information For details about GeneMapper ID Software features allele filters peak detection algorithms and project editing refer to e GeneMapper ID Software Versions 3 1 and 3 2 Human Identification Analysis Tutorial Pub no 4335523 e GeneMapper ID Software Version 3 1 Human Identification Analysis User Guide Pub no 4338775 e Installation Procedures

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AmpFlSTR® Profiler Plus® and Profiler Plus® ID PCR Amplification