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A Rapid and Inexpensive PCR-Based STR Genotyping Method for Identifying Forensic Specimens

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A Rapid and Inexpensive PCR-Based STR Genotyping Method for Identifying Forensic Specimens DOT/FAA/AM-06/14 Office of Aerospace Medicine Washington, DC 20591 A Rapid and Inexpensive PCR-Based STR Genotyping Method for Identifying Forensic Specimens Doris M. Kupfer1 Mark Huggins2 Brandt Cassidy3 Nicole Vu3 Dennis Burian1 Dennis V. Canfield1 1Civil Aerospace Medical Institute P.O. Box 25082 Oklahoma City, OK 73125 2Advancia 655 Research Parkway Oklahoma City, OK 73104 3DNA Solutions 840 Research Parkway Oklahoma City, OK 73104 June 2006 Final Report NOTICE This document is disseminated under the sponsorship of the U.S. Department of Transportation in the interest of information exchange. The United States Government assumes no liability for the contents thereof. ___________ This publication and all Office of Aerospace Medicine technical reports are available in full-text from the Civil Aerospace Medical Institute’s publications Web site: www.faa.gov/library/reports/medical/oamtechreports/index.cfm Technical Report Documentation Page 1. Report No. 2. Government Accession No. 3. Recipient's Catalog No. DOT/FAA/AM-06/14 4. Title and Subtitle 5. Report Date A Rapid and Inexpensive PCR-Based STR Genotyping Method for June 2006 6. Performing Organization Code Identifying Forensic Specimens 7. Author(s) 8. Performing Organization Report No. 1 2 3 3 Doris M. Kupfer, Mark Huggins, Brandt Cassidy, Nicole Vu, Dennis Burian,1 and Dennis V. Canfield1 9. Performing Organization Name and Address 10. Work Unit No. (TRAIS) 1FAA Civil Aerospace Medical Institute 2Advancia P.O. Box 25082 655 Research Parkway 11. Contract or Grant No. Oklahoma City, OK 73125 Oklahoma City, OK 73104 3DNA Solutions 840 Research Parkway Oklahoma City, OK 73104 12. Sponsoring Agency name and Address 13. Type of Report and Period Covered Office of Aerospace Medicine Federal Aviation Administration 800 Independence Ave., S.W. Washington, DC 20591 14. Sponsoring Agency Code 15. Supplemental Notes 16. Abstract Situations arise where forensic samples can be inadvertently misidentified during field collection. Samples identified in the field as being from the same person that return conflicting toxicological results may suggest such misidentification. Polymerase chain reaction (PCR)-based human identity testing provides a reliable and independent method to confirm sample identification. In the study presented here, the Federal Bureau of Investigation’s human Combined DNA Identity System (CODIS) loci were used on a rapid, inexpensive microfluidics chip electrophoresis platform to confirm the identity of forensic samples from an aircraft accident site. Absolute allele identification was not achieved with this method, but it was found to be suitable for comparative analysis, as demonstrated by validation of the results and conclusions from capillary electrophoresis. The nine CODIS Short Tandem Repeat loci and a gender discrimination locus used in this study have a greater than 5E+07 matching probability suitable for small sample sizes. 17. Key Words 18. Distribution Statement Forensic Science, Aerospace Medicine, DNA Typing, Short Document is available to the public through the Tandem Repeats, Microfluidics Chip Defense Technical Information Center, Ft. Belvior, VA 22060; and the National Technical Information Service, Springfield, VA 22161 19. Security Classif. (of this report) 20. Security Classif. (of this page) 21. No. of Pages 22. Price Unclassified Unclassified 18 Form DOT F 1700.7 (8-72) Reproduction of completed page authorized i ACKNOWLEDGMENTS We thank Dr. Marco Scarpetta, Orchid Cellmark, for his comments and help with power of discrimination calculations. iii A RAPID AND INEXPENSIVE PCR-BASED STR GENOTYPING METHOD FOR IDENTIFYING FORENSIC SPECIMENS The method was applied to an aviation case study where INTRODUCTION initial sample identification did not correlate with sub- sequent biochemical and toxicological evaluation. A The possibility exists that samples recovered from a fatal paternity group sample set previously used for capillary accident site can be misidentified. Results from routine electrophoresis (CE) system validation also was tested. toxicological or other biochemical testing of samples that Finally, the findings were validated on a CE system with conflict with the preliminary identification suggest this agreement in all cases. possibility but are not definitive. Genotyping, a DNA- based analysis, which is not a standard part of sample MateRIALS AND METHODS evaluation, can serve as an additional and independent test for sample identification. Capillary electrophoresis Genomic DNA extraction. Tissue or blood was stored is the method often used for analysis of the polymerase frozen at –200C. Blood was thawed and a 200 µl aliquot chain reaction (PCR) products generated in genotyping used for genomic DNA isolation with the QIAamp tests (1). This method requires expensive, dedicated in- DNA mini kit, #51304 (Qiagen; Valencia, CA) using the strumentation, software for analysis, and trained personnel protocol provided by the manufacturer. Tissue samples potentially available only in specialized facilities. There included kidney, lung, liver, muscle, and skin. A scalpel is a need for a more accessible and inexpensive human was used to cut and mince approximately 25 mg from identity testing method in instances where genotyping the frozen tissue. The minced tissue was processed as is necessary only as a confirmatory test and there are a directed by the manufacturer using the ChargeSwitch minimal number of samples. gDNA Mini Tissue kit, #CS11204 (Invitrogen; Carlsbad, Short tandem repeat (STR) loci are polymorphic CA). Yield was determined by A260 on a NanoDrop regions found in the genome that are used as genetic Spectrophotometer (Model ND-1000, NanoDrop Tech- markers for human identity testing. Typing of STR nologies; Wilmington, DE). Quality was examined on a loci by polymerase chain reaction (PCR) is becoming a Bioanalyzer DNA7500 chip (Agilent Technologies; Palo standard for nuclear DNA genotyping analysis. The FBI’s Alto, CA). Samples showing presence of material equal Combined DNA Index System (CODIS) has defined a to or greater than 10Kbp were used for PCR. core set of STR markers for human identity testing (2) Sample sets. The 15 control samples were from three which are highly discriminatory over a wide range of individuals. Samples C1-C5 were from Subject 1, Samples ethnicities. Robust primer sequences for these loci have C6-C10 from Subject 2, and Samples C11-C15 were been made publicly available (3) and are available online from Subject 3. Each control sample set contained a through the National Institute of Standards and Testing sample from blood, liver, lung, kidney and muscle. The (NIST) Website, STRbase (www.cstl.nist.gov/div831/ aviation case study contained five samples, labeled T1- strbase/) (4). The probability of samples from different T5. They were from kidney, skin, kidney, muscle, and subjects having identical alleles for all of the CODIS loci lung, respectively. The paternity set was made up of four is very small, leading to the use of these loci in forensic blood samples, 1304-1307 from the College of American evaluations (1, 5). Pathologists, AABB/CAP PARF Survey B 2005. Results will be presented here describing a qualita- PCR Primers and Amplification. Sequences for the tive method of genotyping using the Agilent 2100 ten primer pairs used in the study were as published (3) Bioanalyzer as the microfluidics platform for separation with modifications as shown at STRbase, www.cstl.nist. of PCR products amplified from STR loci selected from gov/biotech/strbase/promega_primers.htm (4). Nine tetrameric CODIS STR loci. The study demonstrates of these are from the 13 CODIS core STR loci (6) and the novel use of this instrument for rapid, inexpensive one, amelogenin, is a sex-determination locus (5). Table electrophoresis and analysis of STR PCR products to 1 lists the STR loci and primer sequences used for am- allow discrimination among samples from aviation ac- plification and the expected product sizes. Primers were cidents. Accuracy and reproducibility were demonstrated synthesized (Integrated DNA Technologies; Coralville, with control samples from a variety of tissues and blood. IA) and diluted as needed. 1 Table 1. Genetic loci and associated primer pair sequences used for PCR in the study (4 ). See Materials & Methods for PCR conditions. Product size Repeat Locus* Primer A Primer B range (bp) Structure Amel ACC TCA TCC TGG AGG CTT GAG GCC 212, 218 NA GCA CCC TGG TT AAC CAT CAG CSF1PO AAC CTG AGT CTG TTC CAC ACA CCA 291-327 [AGAT]n CCA AGG ACT AGC CTG GCC ATC TTC D3S1358 ACT GCA GTC CAA ATG AAA TCA ACA 111-143 TCTA[TCTG]n[T TCT GGG T GAG GCT TGC CTA]n D5S818 GGT GAT TTT CCT AGC CAC AGT TTA 123-155 [AGAT]n CCT CAA CAT TTG TAT CT TGG TAT CC D7S820 ATG TTG GTC AGG GAT TCC ACA TTT 211-251 [GATA]n CTG ACT ATG ATC CTC ATT GAC D13S317 ACA GAA GTC TGG GGC AGC CCA AAA 157-201 [TATC]n GAT GTG GAG GA AGA CAG A D16S539 GGG GGT CTA AGA GTT TGT GTG TGC 264-304 [GATA]n GCT TGT AAA AAG ATC TGT AAG CAT GTA TC THOI ATT CAA AGG GTA GTG GGC TGA AAA 171-215 [AATG]n TCT GGG CTC TGG GCT CCC GAT TAT TPOX ACT GGC ACA GAA GGA GGA ACT GGG 220-256 [AATG]n CAG GCA CTT AGG AAC CAC ACA GGT TA vWA† GCC CTA GTG GAT GGA CAG ATG ATA 123-181 [TCTA]n[TCTG]n[ GAT AAG AAT AAT AAT ACA TAG GAT TCTA]n CAG TAT GTG GGA TGG (TCCA[TCTA]n) *Amel (Amelogenin) is a sex-determination locus, all others listed are tetrameric STR loci †vWA repeat sequence in ( ) is not always present 2 Amplification was performed using reaction conditions gov/div831/strbase/) (4). An Agilent 2100 Bioanalyzer recommended in the Promega PowerPlex 2.1 system (7) was used for the electrophoresis and analysis of PCR except that the buffer was 1x Amplitaq gold reaction samples as a less expensive, convenient alternative to buffer, 0.1% TritonX-100, and 0.2mM each dNTP in a capillary electrophoresis.
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