Profiler Plus User Bulletin.fm
Contents
1. 1 ng 600 300 o 50 0 031 ng T 7 A y 0 016 ng o j Aarin annat Aennean ai Figure 6 Effect of amplifying various amounts of DNA ranging from 16 pg to 1 ng Note that the y axis scale differs in many of these panels 8 1 2 2 Stability Species specificity sensitivity stability and mixture studies are conducted DAB 1998 As with any multi locus system the possibility exists that not every locus will amplify This is most often observed when the DNA sample contains PCR inhibitors or when the DNA sample has been severely degraded Since each locus is an independent marker whose results are not based upon information provided by the other markers results generally can still be obtained from the loci that do amplify See the AmpFLSTR Profiler Plus PCR Amplification Kit User s Manual for experiments pertaining to stability studies 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 manual to describe differences in the amplification efficiency of two alleles at a single locus Page 21 of 32 Effect of Inhibitors Degraded DNA Page 22 of 32 Preferential amplification of alleles in systems that distinguish alleles based on length polymorphisms is most likely to2. AmpF STR Profiler Plus ZD PCR Amplification Kit AmpF STR Profiler Plus D PCR Amplification Kit Components continued Kit Component Volume Description AmpF STR Profiler Plus 50 uL One tube of AmpF STR Profiler Plus Allelic Ladder Allelic Ladder See the table on pages 7 and 8 for a list of alleles included in the allelic ladder Kit Storage The table below lists the storage temperature for the kit components IMPORTANT The fluorescent dyes attached to the primers are light sensitive Protect the AmpF STR Profiler Plus D Primer Set from light when not in use Amplified DNA AmpF STR Profiler Plus Allelic Ladder and GeneScan 500 ROX Size Standard should also be protected from light Component Storage Temperature AmpF STR PCR Reaction Mix 2to8 C AmpF STR Profiler Plus D Primer Set AmpF STR Control DNA 9947A AmpF STR Profiler Plus Allelic Ladder AmpliTaq Gold DNA Polymerase 15 to 25 C AmpFLSTR_ The AmpF STR Profiler Plus D PCR Amplification Kit reagents and Profiler Plus JD protocols have been optimized to give the sensitivity and specificity PCR Amplification necessary for forensic analysis Two nanograms of the AmpF STR Kit Performance Control DNA 9947A provided in the kit will reliably type when the Characteristics Protocols described in the AmpFt STR Profiler Plus PCR Amplification Kit User s Manual P N 4303501 are followed The recommended range of input sampl
3. Figure 4 on page 17 for 1 minute hold times in the GeneAmp PCR AmpF STR Profiler Plus ZD PCR Amplification Kit System 9700 The PCR products were analyzed using the ABI PRISM 310 Genetic Analyzer ISN 120 140 160 180 200 220 240 260 280 300 320 Lahi u dil w u il 55 C Haaks vall m ua 57 C 59 C Standard Protocol 61 C lu tht id ad van oe 63 C Figure 4 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 ABI PRISM 310 Genetic Analyzer AmpF STR Profiler Plus JD PCR Amplification Kit Page 17 of 32 Species Specificity Sensitivity Stability and Mixture Studies Species Specificity 8 1 2 2 Species Specificity Species specificity sensitivity stability and mixture studies are conducted DAB 1998 The AmpF STR Profiler Plus D kit provides the required degree of specificity such that it is specific to primates Other species do not amplify for the loci tested with the exception of the Amelogenin locus Nonhuman Studies Nonhuman DNA may be present in forensic casework samples The AmpF STR Profiler Plus D 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 AmMpFZ STR
4. 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 Sensabaugh G F and von Beroldingen C 1991 The polymerase chain reaction application to the analysis of biological evidence In Forensic DNA Technology Farley M A and Harrington J J eds Chelsea MI Lewis Publishers 63 82 Sharma V and Litt M 1992 Tetranucleotide repeat polymorphism at the D21S11 locus Hum Mol Genet 1 67 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 Biotechiques 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 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 a 7 locus multiplex STR test for used in AmpF STR Profiler Plus ZD PCR Amplification Kit Page 29 of 32 Page 30 of 32 forensic casework I Mixtures ageing and degradation and species studies Int J Legal Med 109 186 194 Urquhart A Oldroy
5. with Standards 8 1 1 and 8 1 2 and its associated subsections Whereas this DNA methodology is not novel Standard 8 1 2 and its related subsections have been addressed Holt et al 2001 and Wallin et al 2001 This section will discuss many of the experiments performed by Applied Biosystems and examples of results obtained Conditions were chosen which produced maximum PCR product yield and a window in which reproducible performance characteristics were met These experiments while not exhaustive are appropriate for a manufacturer However each laboratory using the AmpF STR Profiler Plus D PCR Amplification Kit should perform their own appropriate validation studies AmpF STR Profiler Plus ZD PCR Amplification Kit Developmental Validation 8 1 1 Developmental validation that is conducted shall be appropriately Developmental documented DNA Advisory Board 1998 Validation Critical reagent concentrations and reaction conditions were tested e g Magnesium Chloride concentration annealing temperature relevant to the addition of the D8S1179 degenerate primer to produce reliable locus specific amplification and appropriate sensitivity have been determined PCR Components The concentration of the D8S1179 degenerate primer of the AmpF STR Profiler Plus D Primer Set was examined The concentration for the D8S1179 degenerate primer was established to be in the window that meets the reproducible performance characteristics
6. 13S317 13q22 31 NED 8 9 10 11 12 13 14 15 119 D78820 7q11 21 22 NED 6 7 8 9 10 11 12 10 11 13 14 15 a For CODIS purposes profile reported as 13 13 b For CODIS purposes profile reported as 30 30 c For CODIS purposes profile reported as 11 11 d For CODIS purposes profile reported as 11 11 Kit Components The AmpF STR Profiler Plus D PCR Amplification Kit P N 4330284 contains the PCR reagents necessary to co amplify the ten AmpF STR Profiler Plus loci The kit components are shown in the table below AmpF STR Profiler Plus D PCR Amplification Kit Components Kit Component Volume Description AmpF STR PCR 1 1 mL tube Two tubes of PCR Reaction Mix containing MgCl Reaction Mix dATP dGTP dCTP dTTP bovine serum albumin and 0 05 sodium azide in buffer and salt AmpFLSTR Profiler 1 1 mL One tube containing locus specific 5 FAM JOE and Plus D Primer Set NED dye labeled and unlabeled primers in buffer that amplify the STR loci D3S1358 D5S818 D7S820 D8S1179 D13S317 D18S51 D21S11 FGA vWA and the gender marker Amelogenin AmpliTaq Gold DNA 0 05 mL tube Two tubes of enzyme with an activity of 5 U uL Polymerase AmpF STR Control DNA 0 3 mL One tube containing 0 10 ng uL human cell line DNA 9947A in 0 05 sodium azide and buffer Refer to pages 7 and 8 for profile Mineral Oil 5 mL Supplied in dropper bottle Page 8 of 32
7. 32 Data Interpretation Minimum Sample Requirement PCR Cycle Number and Amplification Conditions Page 26 of 32 The AmpF STR Profiler Plus D PCR Amplification Kit has been optimized to amplify and type approximately 1 to 2 5 ng of sample DNA reliably The PCR cycle number and amplification conditions have been specified to produce low peak heights for a sample containing 35 pg human genomic DNA Thus the overall sensitivity of the assay has been adjusted to avoid or minimize stochastic effects Applied Biosystems has successfully typed samples containing less than 1 ng DNA Note Individual laboratories may find it useful to determine an appropriate minimum peak height threshold based on their own results instruments using low amounts of input DNA AmpF STR Profiler Plus ZD PCR Amplification Kit Population Data 8 1 2 3 Population Population distribution data are documented and available DAB Data 1998 8 1 2 3 1 The population distribution data would include the allele and genotype Population distributions for the locus or loci obtained from relevant populations Distribution Data Where appropriate databases should be tested for independence expectations DAB 1998 Overview 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 f
8. C et al 2001 TWGDAM validation of AmpF STR PCR Amplification Kits for Forensic DNA Casework J Forensic Sci in press Hudson T J et a 1995 An STS based map of the human genome Science 270 1945 1954 Kimpton C Walton A and Gill P 1992 A further tetranucleotide repeat polymorphism in the vWF gene Hum Mol Genet 1 287 AmpF STR Profiler Plus ZD PCR Amplification Kit Lazaruk K et al 2001 Sequence variation 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 Lerman M I Zbar B and Tory K 1993 Three tetranucleotide polymorphisms for loci D3S1352 D3S1358 D3S1359 Hum Mol Genet 2 1327 Mills K A Even D and Murray J C 1992 Tetranucleotide repeat polymorphism at the human alpha fibrinogen locus FGA Hum Mol Genet 1 779 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 Moretti T Baumstrak A Defenbaugh 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 Oldroyd N J Urquhart A J Kimpton C P Millican E S Watson
9. Profiler Plus D kit results from nonhuman DNA sources Bal 20 1 00 1 20 1 40 q 60 1 80 200 220 240 260 280 300 320 340 3000 Chimp 2000 1000 of ba didda ad nm i i 2000 Horse 2000 1000 o d 3000 Pig 2000 1000 o 4 h_A A A 3000 Dog 2000 1000 o AAA A A aoan E coli 2000 1000 o 3000 Negative 2000 Control 1000 o A Ad i A Figure 5 Representative electropherograms of a primate non primates a microorganism and a negative control are shown All samples were analyzed on an ABI PRISM 310 Genetic Analyzer The peaks depicted in red are the GeneScan 500 ROX size standard Page 18 of 32 AmpF STR Profiler Plus ZD PCR Amplification Kit The extracted DNA samples were amplified with the AmpF STR Profiler Plus D kit reactions and analyzed using the ABI PRISM 310 Genetic Analyzer Primates gorilla chimpanzee orangutan and macaque 1 0 ng each Non primates mouse dog pig cat horse chicken and cow 2 5 ng each Bacteria and yeast Escherichia and Saccharomyces 1 2 5 ng The primate DNA samples all amplified producing fragments within the 100 to 400 base pair region Wallin et a 1998 Lazaruk et al 2001 The microorganisms chicken cow cat and mouse did not yield detectable product Horse dog and pig produced a fragment near the Amelogenin locus in JOE dye see Figure 5 on page 18 Sensitivity 8 1 2 2
10. SM 377 DNA Sequencer Sequencer Protocols ABI PRISM 310 Genetic Chapter 8 ABI PRISM 310 Genetic Analyzer Analyzer Protocol Each of the 5 FAM JOE and NED labeled alleles which make up the AmpF STR Profiler Plus Allelic Ladder are combined into a single tube The AmpF STR Profiler Plus Allelic Ladder is used for both the AmpF STR Profiler Plus kit and the AmpF STR Profiler Plus D kit The AmpF STR Profiler Plus Allelic Ladder consists of one 25 uL tube containing the following alleles D3S1358 alleles 12 to 19 FGA alleles 18 to 30 and 26 2 vWA alleles 11 to 21 Amelogenin alleles X and Y D8S1179 alleles 8 to 19 D18S51 alleles 9 to 26 10 2 13 2 and 14 2 D21S11 alleles 24 to 38 24 2 28 2 29 2 30 2 31 2 32 2 33 2 34 2 and 35 2 D58818 alleles 7 to 16 D7S820 alleles 6 to 15 and D13S317 alleles 8 to 15 Please note the following changes to your AmpFt STR Profiler Plus PCR Amplification Kit User s Manual with respect to the allelic ladder preparation Page 1 7 Description of AmpF4 STR Allelic Ladders in Table 1 2 should state a single kit component named AmpF STR Profiler Plus Allelic Ladder The AmpF STR Profiler Plus Allelic Ladder is composed of the AmpF STR Blue the AmpF STR Green II and AmpF STR Yellow Allelic Ladders as currently defined This change should also be noted in the AmpF STR Profiler Plus PCR Amplification Kit Product Insert Page 7 14 Under Prepa
11. Sensitivity Species specificity sensitivity stability and mixture studies are conducted DAB 1998 The AmpF STR Profiler Plus PCR Amplification Kit has been optimized to amplify and type approximately 1 0 to 2 5 ng of sample DNA reliably The PCR cycle number and amplification conditions have been specified to produce low peak heights for a sample containing 35 pg of human genomic DNA Low peak heights should be interpreted with caution Thus the overall sensitivity of the assay has been adjusted to avoid or minimize stochastic effects Importance of The amount of input DNA added to the AmpF STR Profiler Plus D PCR Quantitation Amplification Kit should be between 1 0 and 2 5 ng The DNA sample should be quantitated prior to amplification using a system such as the QuantiBlot Human DNA Quantitation Kit P N N808 0114 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 increased amount of PCR product that is generated can result in the following AmpF STR Profiler Plus JD PCR Amplification Kit Page 19 of 32 Page 20 of 32 Fluorescence intensity that exceeds the linear dynamic range for detection by the instrument off scale data Off scale data is a problem for two rea
12. Set F module files Make a matrix file using the 5 FAM JOE NED and ROX matrix standard samples and the Filter Set F module files See Chapter 6 Multicomponent Analysis in the AmpFLSTR Profiler Plus PCR Amplification Kit User s Manual for protocols on how to make a matrix file Be sure to verify the accuracy of the matrix file Determine the quantity of DNA in samples to be amplified See Chapter 4 DNA Quantitation in the AmpFLSTR Profiler Plus PCR Amplification Kit User s Manual for more details on DNA quantitation Amplify DNA samples using the AmpF STR Profiler Plus D kit reagents See Chapter 5 Amplification of the AmpFLSTR Profiler Plus PCR Amplification Kit User s Manual The recommended range of input DNA is 1 0 2 5 ng Run AmpF STR Profiler Plus D PCR products on the ABI PRISM platforms IMPORTANT Use the GeneScan 500 ROX Internal Lane Size Standard Analyze samples using GeneScan Analysis v2 1 or higher software See Chapter 9 Results and Interpretation of the AmpFlSTR Profiler Plus PCR Amplification Kit User s Manual P N 4304501 and the GeneScan Analysis 3 1 User s Manual P N 403001 Use the Genotyper software v2 5 or higher with AmpF STR Profiler Plus kit template for automatic genotyping of samples See Chapter 9 Results and Interpretation in the AmpFtSTR Profiler Plus PCR Amplification Kit User s Manual AmpF STR Profiler Plus JD PCR Amplification Kit Pa
13. User Bulletin SUBJECT AmpF STR Profiler Plus JD PCR Amplification Kit Overview This User Bulletin describes the AmpF STR Profiler Plus D PCR Amplification Kit and experiments performed for its evaluation In This User This User Bulletin contains the following topics Bulletin Topic See Page Product Overview 3 Safety 5 About This Kit 7 How to Run Your Samples 11 Experiments Performed Using the AmpF STR Profiler Plus ID 14 PCR Amplification Kit Developmental Validation 15 Species Specificity Sensitivity Stability and Mixture Studies 18 Data Interpretation 26 Population Data 27 References 28 Applied Biosystems About This User The AmpF t STR Profiler Plus ID PCR Amplification Kit User Bulletin Bulletin provides supporting documentation for the AmpF STR Profiler Plus ID PCR Amplification Kit This user bulletin contains sections describing the following Background information on the AmpF STR Profiler Plus D kit Experiments and Results The AmpF STR Profiler Plus D PCR Amplification Kit and the AmpFLSTR Profiler Plus PCR Amplification Kit use the same protocols This User Bulletin does not contain the general protocols necessary for amplification or fragment analysis on the ABI PRISM instruments The AmpFLSTR Profiler Plus PCR Amplification Kit User s Manual P N 4303501 should be referred to for use with the AmpF STR Profiler Plus D ki
14. ation Kit Page 27 of 32 References Page 28 of 32 Akane A Matsuhara K Nakamura H Takahashi S and Kinura 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 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 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 DeFranchis R Cross N C P Foulkes N S and Cox T M 1988 A potent inhibitor of Taq DNA polymerase copurifies with human genomic DNA Nucleic Acids Res 16 10355 DNA Advisory Board Federal Bureau of Investigation U S Department of Justice 1998 Quality assurance standards for forensic DNA testing laboratories 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 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 Holt
15. be observed when the alleles differ significantly in base pair size Since most AmpF STR Profiler Plus D kit loci have small size ranges the potential for preferential amplification of alleles is low Heme compounds have been identified as PCR inhibitors in DNA samples extracted from bloodstains DeFranchis et a 1988 Akane et al 1994 It is believed that the inhibitor is co extracted and co purified with the DNA and subsequently interferes with PCR by inhibiting polymerase activity Bovine serum albumin BSA can prevent or minimize the inhibition of PCR most likely by binding to the inhibitor Comey et al 1994 Since the presence of BSA can improve the amplification of DNA from blood containing samples BSA has been included in the AmpF STR PCR Reaction Mix at 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 Comey et al 1994 See the AmpFt STR Profiler Plus PCR Amplification User s Manual for the effects of hematin on the amplification results obtained As the average size of degraded DNA approaches the size of the target sequence the amount of PCR product generated is reduced This is due to the reduced number of intact templates in the size range necessary for amplification 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 analy
16. d N J Kimpton C P and Gill P 1995 Highly discriminating heptaplex short tandem repeat PCR system for forensic identification Biotechniques 18 116 121 Wallin J Bouncristiani M Lazaruk K Fildes N Holt C and Walsh P 1998 TWGDAM validation of the AmpF STR Blue PCR Amplification Kit for forensic casework analysis J Forensic Sci 43 4 854 870 Wallin J et al 2001 Constructing universal multiplex PCR systems for comparative genotyping J Forensic Sci in press Walsh P S Erlich H A and Higuchi R 1992 Preferential PCR amplification of alleles mechanisms and solutions PCR Methods Appl 1 241 250 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 AmpF STR Profiler Plus ZD PCR Amplification Kit Copyright 2001 Applied Biosystems All rights reserved For Research Forensic or Paternity Use Only Not for use in diagnostic procedures ABI PRISM and its design is a registered trademark of Applera Corporation or its subsidiaries in the U S and certain other countries ABI and Applied Biosystems are trademarks of Applera Corporation or its subsidiaries in the U S and certain other countries All other trademarks are the sole property of their respective owners Applera Corporation is committed to providing t
17. e DNA is 1 0 2 5 ng Samples containing less than one nanogram of human DNA have successfully been typed using the AmpF STR Profiler Plus D PCR Amplification Kit PCR amplification component concentrations and thermal cycler parameters have been determined to produce specific amplification of the AmpF STR Profiler Plus D PCR Amplification loci The STR loci in the AmpF STR Profiler Plus D PCR Amplification Kit are specific for primate DNA The primers used to amplify the Amelogenin locus are known to amplify a 103 bp monomorphic band in some animals see page 12 9 of the AmpFt STR Profiler Plus PCR Amplification Kit User s Manual AmpF STR Profiler Plus ZD PCR Amplification Kit Page 9 of 32 A Certificate of Analysis is available upon request The certificate confirms that the specific combination of components that comprise a given kit lot number perform together to meet the stated performance Page 10 of 32 AmpF STR Profiler Plus ZD PCR Amplification Kit How to Run Your Samples Protocols to Use Allelic Ladder The AmpF STR Profiler Plus D PCR Amplification Kit is designed to follow the same protocols as the AmpF STR Profiler Plus PCR Amplification Kit For detailed protocols please refer to the appropriate chapters of the AmpFLSTR Profiler Plus PCR Amplification Kit User s Manual P N 4303501 Protocol Refer to Amplification Chapter 5 Amplification ABI PRISM 377 DNA Chapter 7 ABI PRI
18. ge 13 of 32 Experiments Performed Using the AmpF STR Profiler Plus 1D PCR Amplification Kit Importance of Validation of a DNA typing procedure for human identification Validation Experiments Page 14 of 32 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 proper data interpretation in casework Sparkes et al 1996a Sparkes et al 1996b Wallin et al 1998 The AmpF STR Profiler Plus PCR Amplification Kit was validated according to TWGDAM guidelines Frank et al 2001 Holt et al 2001 Moretti et al 2001 The experiments for the AmpF STR Profiler Plus ID PCR Amplification Kit were performed according to the DNA Advisory Board DAB standards which were effective October 1 1998 Experiments to evaluate the performance of AmpF STR Profiler Plus ID PCR Amplification Kit were performed relevant to the addition of the D8S1179 degenerate primer These DAB standards describe the quality assurance requirements that a laboratory should follow to ensure the quality and integrity of the data and competency of the laboratory DAB defines a laboratory as a facility in which forensic DNA testing is performed Based on these standards Applied Biosystems has conducted experiments that comply
19. he world s leading technology and information for life scientists Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses P N 4330429 Rev D Stock No 112UB02 04
20. l cycling parameters as the AmpF STR Profiler Plus kit with the addition of this second D8S1179 reverse primer The addition of the primer allows for the amplification of those D8S1179 alleles in samples containing this mutation without altering the overall performance of the AmpF STR Profiler Plus D kit See Figure 1 and Figure 2 on page 4 In Figure 2 the DNA fragments are labeled with 5 FAM dye blue JOE dye green and NED dye yellow depicted in black The GeneScan 500 Size Standard is labeled with ROX dye red AmpF STR Profiler Plus ZD PCR Amplification Kit Page 3 of 32 Page 4 of 32 2240 1960 1680 1400 1120 1400 1120 840 560 280 o Alin im 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 Profiler Plus kit primers hey 80 100 120 140 160 180 200 220 240 260 280 300 320 340 2400 1600 3 lt hi uill wow da H Figure 2 GeneScan software electropherogram showing the AmpF STR Profiler Plus D PCR Amplification Kit results for nine STR loci and the Amelogenin locus analyzed on the ABI PRISM 310 Genetic Analyzer AmpF STR Profiler Plus ZD PCR Amplification Kit Safety Documentation User Attention Words Chemical Hazard Warning Five user attention words appear in the text of all Applied Biosystems user documentation Each word implies a particular le
21. lele 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 AmpF STR Profiler Plus ZD PCR Amplification Kit Tot Panel 1 Sample A A Panel 2 A 10 1 Panel 3 A 3 1 Panel 4 k 1 1 Panel 5 n 1 3 j Panel 6 a 1 10 T Panel7 o Sample B Figure 7 Results of the two DNA samples mixed together at defined ratios and amplified with the AmpF STR Profiler Plus D PCR Amplification Kit Sample A and Sample B are a male and female sample respectively The ratios of Sample A to Sample B A B ratios shown are 10 1 3 1 1 1 1 3 and 1 10 respectively The alleles attributable to the minor component even when the major component shares an allele are highlighted in panels 2 3 5 and 6 All alleles are highlighted in panel 4 AmpF STR Profiler Plus JD PCR Amplification Kit Page 25 of
22. ll occurs follow the manufacturer s cleanup procedures as recommended on the MSDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal AmpF STR Profiler Plus ZD PCR Amplification Kit Page 5 of 32 Site Preparation A site preparation and safety guide is a separate document sent to all and Safety Guide customers who have purchased an Applied Biosystems instrument Refer to the guide written for your instrument for information on site preparation instrument safety chemical safety and waste profiles Ordering MSDSs You can order free additional copies of MSDSs for chemicals manufactured or distributed by Applied Biosystems using the contact information below To order documents by automated telephone service 1 From the U S or Canada dial 1 800 487 6809 or from outside the U S and Canada dial 1 858 712 0317 2 Follow the voice instructions to order documents for delivery by fax Note There is a limit of five documents per fax request To order documents by telephone In the U S Dial 1 800 345 5224 and press 1 To order in English dial 1 800 668 6913 and press 1 then 2 then 1 In Canada To order in French dial 1 800 668 6913 and press 2 then 2 then 1 From any other See the specific region under To Contact Technical Support by Telephone or country Fax Outside North America To view download or o
23. 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 Applied Biosystems instruments provides additional valuable data to aid in resolving mixed genotypes This quantitative value is much less subjective than comparing relative intensities of bands on a stained gel Ultimately the likelihood that any sample is a mixture must be determined by the analyst in the context of each particular case including the information provided from known reference sample s AmpF STR Profiler Plus ZD PCR Amplification Kit Page 23 of 32 Limit of Detection of the Minor Component Page 24 of 32 Mixtures of two DNA samples were examined at various ratios 1 1 to 1 10 The total amount of genomic input DNA mixed at each ratio was 1 ng The samples were amplified ina GeneAmp PCR System 9700 and were electrophoresed and detected using an ABI PRISM 310 Genetic Analyzer The results of the mixed DNA samples are shown in Figure 7 on page 25 where sample A and sample B were mixed according to the ratios provided The profiles of the samples in Figure 7 are the following Profile Al
24. of specificity and sensitivity After establishing the optimum unlabeled D8S1179 degenerate primer concentration all experiments were performed at that concentration Varying Magnesium Chloride concentrations were also tested to determine the optimum concentration see Figure 3 on page 16 AmpF STR Profiler Plus ZD PCR Amplification Kit Page 15 of 32 S 100 4500 3000 120 140 160 180 200 220 240 260 280 300 320 340 1 0 mM faal adl a i a 4500 3000 1500 4500 3000 1500 4500 3000 1500 1 15 mM 1 25 mM Standard Concentration 1 35 mM tl adlu Widi i 4500 3000 1500 1 5 mM Figure 3 A 2 ng amplification of AmpF STR Control DNA 9947A varying the Magnesium Chloride concentration analyzed on the ABI PRISM 310 Genetic Analyzer Thermal Cycler Thermal cycling parameters were established for amplification of the Parameters AmpF STR Profiler Plus D kit on the GeneAmp PCR System 9700 Page 16 of 32 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 AmpF STR Profiler Plus D kit loci were examined using AmpF STR Control DNA 9947A and two DNA samples with one mutant D8S1179 allele The annealing temperatures tested were 55 57 59 61 and 63 C see
25. rder documents through the Applied Biosystems web site Step Action 1 Go to http www appliedbiosystems com 2 Click SERVICES amp SUPPORT at the top of the page click Documents on Demand then click MSDS 3 Click MSDS Index search through the list for the chemical of interest to you then click on the MSDS document number for that chemical to open a pdf of the MSDS For chemicals not manufactured or distributed by Applied Biosystems call the chemical manufacturer Page 6 of 32 AmpF STR Profiler Plus ZD PCR Amplification Kit About This Kit AmpFtSTR Profiler Plus ZD Kit Loci The AmpF STR Profiler Plus D PCR Amplification Kit co amplifies the repeat regions of the following nine short tandem repeat loci D3S1358 Li et al 1993 D58818 Hudson et al 1995 D7S820 Green et al 1991 D8S1179 Oldroyd et al 1995 D13S317 Hudson et al 1995 D18S51 Urquhart et al 1995 and D21S11 Sharma and Litt 1992 FGA Mills et al 1992 and vWA Kimpton et al 1992 A segment of the X Y homologous gene Amelogenin is also amplified Amplifying a segment of the Amelogenin gene with a single primer pair can be used for gender identification because different length products from the X and Y chromosomes are generated Sullivan et al 1993 One primer of each locus specific primer pair is labeled with either the 5 FAM JOE or NED dyes These dyes are detected as blue green and yellow re
26. ring Samples and AmpF STR Profiler Plus Allelic Ladder steps 3 and 4 should be omitted Step 8 remains unchanged AmpF STR Profiler Plus ZD PCR Amplification Kit Page 11 of 32 Page 8 11 Under Preparing Samples and AmpF STR Profiler Plus Allelic Ladder Steps 3 and 4 should be omitted Step 6 on page 8 12 remains unchanged Page 9 8 The sub sections entitled Loading Allelic Ladder onto the ABI PRISM 310 Genetic Analyzer and Loading Allelic Ladder onto the ABI PRISM 377 DNA Sequencer and ABI PRISM 377 DNA Sequencer with XL Upgrade refer to combining the AmpF STR Blue AmpF2 STR Green II and AmpF STR Yellow Allelic Ladders into a single tube to prepare the AmpF STR Profiler Plus Allelic Ladder This reference is now irrelevant since the AmpF STR Profiler Plus Allelic Ladder is ready to use as shipped in the AmpF STR Profiler Plus and AmpF STR Profiler Plus D kits Page 12 of 32 AmpF STR Profiler Plus ZD PCR Amplification Kit Getting Started In order to get started Confirm that the Filter Set F module files are installed on the Macintosh computer connected to the ABI PRISM 377 DNA Sequencer or the ABI PRISM 310 Genetic Analyzer The Filter Set F module files must be located in the Modules folder located in the ABI PRISM 377 or ABI PRISM 310 folder See Chapter 6 Multicomponent Analysis in the AmpFLSTR Profiler Plus PCR Amplification Kit User s Manual for more information on Filter
27. rom 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 Population The AmpF STR Profiler Plus PCR Amplification Kit was used to Samples Used in generate the population data provided in the AmpFt STR Profiler Plus These Studies PCR Amplification User s Manual to include 195 African American and 200 Caucasians Homozygous samples at the D8S1179 locus were reamplified using the AmpF STR Profiler Plus D PCR Amplification Kit to confirm the homozygosity at this locus Of the 68 African American and the 60 Caucasian homozygous samples at the D8S1179 locus available for re testing all samples typed as homozygotes None of these samples were found to be heterozygous using the AmpF STR Profiler Plus ID PCR Amplification Kit Refer to Chapter 13 Population Genetics Table 13 1 in the AmpFtSTR Profiler Plus PCR Amplification Kit User s Manual for allele frequencies in the African American and Caucasian populations Holt et al 2001 AmpF STR Profiler Plus ZD PCR Amplific
28. sis If the DNA is degraded to an average of 400 base pairs in size or less adding more DNA template to the AmpF STR Profiler Plus D kit amplification reaction may help produce a typeable signal for the loci Adding more DNA to the amplification may provide more of the necessary size template for amplification AmpF STR Profiler Plus ZD PCR Amplification Kit Mixture Studies 8 1 2 2 Mixture Studies Species specificity sensitivity stability and mixture studies are conducted DAB 1998 Evidence samples may contain DNA from more than one individual The possibility of multiple contributors should be considered during data analysis and interpretation 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 Resolution of A sample containing DNA from two sources can be comprised at a Genotypes in single locus of any of the seven genotype combinations listed below Mixed Samples Heterozygote heterozygote no overlapping alleles four peaks Heterozygote heterozygote one overlapping allele three peaks Heterozygote heterozygote two overlapping alleles two peaks Heterozygote homozygote overlapping allele two peaks Heterozygote homozygote no overlapping alleles three peaks Homozygote homozygote
29. sons Quantitation peak height and area for off scale peaks is not accurate For example an allele peak that is off scale can cause the corresponding stutter peak to appear higher in relative intensity thus increasing the calculated percent stutter Multicomponent analysis of off scale data is not accurate which results in poor spectral separation pull up Incomplete A nucleotide addition The sample can be re amplified using less DNA When the total number of allele copies added to the PCR is extremely low unbalanced amplification of the two alleles of a heterozygous individual may occur Walsh et al 1992 Wallin et a 1998 due to stochastic fluctuation in the ratio of the two different alleles Sensabaugh et al 1991 The PCR cycle number and amplification conditions have been specified to produce low peak heights for a sample containing 35 pg human genomic DNA Low peak heights should be interpreted with caution see Figure 6 on page 21 Individual laboratories may find it useful to determine an appropriate minimum peak height threshold based on their own results and instruments using low amounts of input DNA AmpF STR Profiler Plus ZD PCR Amplification Kit Stability Differential and Preferential Amplification AmpF STR Profiler Plus JD PCR Amplification Kit QJ 100 120 140 160 180 200 220 240 260 tow tl i tal aaa d i te 0 5 ng 0 bidi dA 0 25 ng 0 125 ng 0 0625 ng
30. spectively on the ABI PRism instruments The loci amplified by these primers are summarized in the table below Additionally the AmpF STR Profiler Plus Allelic Ladder is used to genotype the analyzed samples The alleles contained in the allelic ladder and the genotype of the AmpFZ STR Control DNA 9947A are listed in the table as well AmpF STR Profiler Plus D Loci and Allele Information Locus Chromosome Dye AmpF STR Profiler Plus Control DNA Designation Location Label Allelic Ladder Alleles 9947A Genotype D3S1358 3p 5 FAM 12 13 14 15 16 17 14 15 18 19 vWA 12p12 pter 5 FAM 11 12 13 14 15 16 17 18 17 18 19 20 21 FGA 4q28 5 FAM 18 19 20 21 22 23 23 24 24 25 26 26 2 27 28 29 30 Amelogenin X p22 1 22 3 JOE X Y X Y p11 2 D8S1179 8 JOE 8 9 10 11 12 13 13a 14 15 16 17 18 19 D21811 21 JOE 24 2 25 26 27 28 305 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 AmpF STR Profiler Plus ZD PCR Amplification Kit Page 7 of 32 AmpF STR Profiler Plus D Loci and Allele Information continued Locus Chromosome Dye AmpF STR Profiler Plus Control DNA Designation Location Label Allelic Ladder Alleles 9947A Genotype D18S51 18q21 3 JOE 9 10 10 2 11 12 13 13 2 15 19 14 14 2 15 16 17 18 19 20 21 22 23 24 25 26 D5S818 5q21 31 NED 7 8 9 10 11 12 13 11 14 15 16 D
31. t Chapters in the AmpF STR Profiler Plus PCR Amplification Kit User s Manual that are especially useful for the Profiler Plus D kit are as follows AmpF STR Profiler Plus Kit Amplification Chapter 5 ABI PRISM 377 DNA Sequencer Protocols Chapter 7 ABI PRISM 310 Genetic Analyzer Protocol Chapter 8 Results and Interpretation Chapter 9 Automated Genotyping Chapter 10 gt gt gt gt Population Genetics Chapter 13 Page 2 of 32 AmpF STR Profiler Plus 7D PCR Amplification Kit Product Overview Purpose The AmpF STR Profiler Plus PCR Amplification Kit has proven to be extremely useful in applications for human identification The AmpF STR Profiler Plus PCR Amplification Kit co amplifies the following nine short tandem repeat loci D3S1358 D58818 D7S820 D8S1179 D13S317 D18S51 D21S11 FGA and vWA Previously Budowle Budowle et a 2000 reported an excess of homozygosity at the D8S1179 locus in a population of Chamorro and Filipinos from Guam A single G to A transition point mutation was observed in all null alleles at the D8S1179 reverse primer binding site An additional D8S1179 reverse primer with a single nucleotide difference specific for the transition was included in the primer set of the newly developed AmpF STR Identifiler PCR Amplification Kit to address this mutation The new AmpF STR Profiler Plus D kit contains the same primers and uses the same reaction conditions and therma
32. vel of observation or action as described below Note Calls attention to useful information IMPORTANT Indicates information that is necessary for proper instrument operation F RATA oN Indicates a potentially hazardous situation which if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices PANIE Indicates a potentially hazardous situation which if not avoided could result in death or serious injury eldi Indicates an imminently hazardous situation which if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations PAY Eile CHEMICAL HAZARD Some of the chemicals used with Applied Biosystems instruments and protocols are potentially hazardous and can cause injury illness or death Read and understand the material safety data sheets MSDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals e g safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation e g fume hood For additional safety guidelines consult the MSDS Check regularly for chemical leaks or spills If a leak or spi
Download Pdf Manuals
Related Search