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The prospect for more than 13 commonly used STR Tetranucleodide loci in forensic settings

The prospect for more than 13 commonly used STR Tetranucleodide loci in forensic settings. Carrie Rowland Forensic Bioinformatic Services, Inc. Dayton, Ohio www.bioforensics.com. An Overview : Where are we going. History of DNA testing Generations of DNA tests and how performed

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The prospect for more than 13 commonly used STR Tetranucleodide loci in forensic settings

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  1. The prospect for more than 13 commonly used STR Tetranucleodide loci in forensic settings Carrie Rowland Forensic Bioinformatic Services, Inc. Dayton, Ohio www.bioforensics.com

  2. An Overview: Where are we going • History of DNA testing • Generations of DNA tests and how performed • Current methodology • New prospects • Conclusions

  3. Six criteria for judging a DNA profiling system • Discrimination power. • Sensitivity. • Absence of, or the ability to reconcile • technical artifacts. • Speed. • Ability to deconvolve mixtures. • Amenability to database searches.

  4. Brief History of Forensic DNA Typing • 1980 - Ray White describes first polymorphic RFLP marker • 1985 - Alec Jeffreys proposes multilocus VNTR probes be used forensically • 1985- first paper on PCR • 1988 - FBI starts DNA casework • 1991 - first STR paper • 1995 - FSS starts UK DNA database • 1998 - FBI launches CODIS database

  5. Three generations of DNA testing RFLP AUTORAD Allele = BAND DQ-alpha TEST STRIP Allele = BLUE DOT AutomatedSTR ELECTROPHEROGRAM Allele = PEAK

  6. How do they fare? STR DQ-alpha RFLP  Discriminating power Sensitivity □ Technical artifacts  Speed □ Mixtures  Databasing  Discriminating power □ Sensitivity □ Technical artifacts □ Speed □ Mixtures ٱDatabasing □ Discriminating power  Sensitivity Technical artifacts  Speed □ Mixtures □ Databasing

  7. RFLP technology What we would like for them to look like.

  8. What they often look like. Incomplete digest

  9. Available Kits for STR Analysis • Kits make it easy for labs to just add DNA samples to a pre-made mix • 13 CODIS core loci • Profiler Plus and COfiler (PE Applied Biosystems) • PowerPlex 1.1 and 2.1 (Promega Corporation) • Increased power of discrimination • CTT (1994): 1 in 410 • SGM Plus™ (1999): 1 in 3 trillion • PowerPlex ™ 16 (2000): 1 in 2 x 1017

  10. AmpFlSTR® Profiler Plus™ Kit available from PE Biosystems (Foster City, CA) 200 bp 300 bp 400 bp 100 bp Size Separation D3 vWA FGA 5-FAM (blue) JOE (green) A D8 D21 D18 Color Separation D5 D13 D7 NED (yellow) ROX (red) GS500-internal lane standard 9 STRs amplified along with sex-typing marker amelogenin in a single PCR An Example Forensic STR Multiplex Kit

  11. 13 CODIS Core STR Loci with Chromosomal Positions TPOX D3S1358 TH01 D8S1179 D5S818 VWA FGA D7S820 CSF1PO AMEL D13S317 AMEL D16S539 D18S51 D21S11

  12. 45 40 35 30 Caucasians (N=427) 25 Frequency Blacks (N=414) 20 Hispanics (N=414) 15 10 5 0 6 7 8 9 9.3 10 Number ofrepeats STR Allele Frequencies TH01 Marker *Proc. Int. Sym. Hum. ID (Promega) 1997, p. 34

  13. Human Identity Testing with Multiplex STRs AmpFlSTR® SGM Plus™ kit DNA Size (base pairs) D3 TH01 amelogenin D8 VWA D16 D19 D21 D18 D2 FGA probability of a random match: ~1 in 3 trillion Two different individuals Results obtained in less than 5 hours with a spot of blood the size of a pinhead D3 amelogenin D19 D8 D16 VWA D21 D18 D2 FGA TH01 Simultaneous Analysis of 10 STRs and Gender

  14. FBI’s CODIS DNA Database Combined DNA Index System • Launched October 1998 • Used for linking serial crimes and unsolved cases with repeat offenders • Links all 50 states • Requires >4 RFLP markers and/or 13 core STR markers • Current backlog of > 600,000 samples As of June, 2004 • Total profiles = 1,857,093 • Total forensic profiles = 85,477 • Total convicted offender = 1,771,616

  15. http://www.fbi.gov/hq/lab/codis/aidedmap.htm

  16. 6.3% 6.2% 5.4% STR alleles with n+4 stutter products DNA Size (bp) D8S1179 D21S11 D18S51 Relative Fluorescence Units

  17. 1 2 3 4 5 6 4 5 1 2 3 Repeat unit bulges out when strand breathing occurs Schematic of Stutter Product Formation Process Normal STR Allele Replication Slipped Strand Mispairing Model Walsh et al (1996) Nucleic Acids Res. 24: 2807-2812

  18. Mechanism behind n+4 stutter DNA polymerase 7 1 2 6 3 5 4 4 3 5 2 6 1 Template DNA Walsh, et al. (1996) Nucleic Acids Research 24(14):2807

  19. What is on the horizon ? 1. 14th …….locus 2. Pentanucleotide repeats • Mitochondrial DNA • Single nucleotide polymorphisms (SNPs)

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