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Brief History of Forensic DNA Typing

Brief History of Forensic DNA Typing. 1980 - Ray White describes first polymorphic RFLP marker 1985 - Alec Jeffreys discovers multilocus VNTR probes 1985 - first paper on PCR 1988 - FBI starts DNA casework 1991 - first STR paper 1995 - FSS starts UK DNA database

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Brief History of Forensic DNA Typing

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  1. Brief History of Forensic DNA Typing • 1980 - Ray White describes first polymorphic RFLP marker • 1985 - Alec Jeffreys discovers multilocus VNTR probes • 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

  2. DNA Use in Forensic Cases • Most are rape cases (>2 out of 3) • Looking for match between evidence and suspect • Must compare victim’s DNA profile Challenges • Mixtures must be resolved • DNA is often degraded • Inhibitors to PCR are often present

  3. Human Identity Testing • Forensic cases -- matching suspect with evidence • Paternity testing -- identifying father • Historical investigations • Missing persons investigations • Mass disasters -- putting pieces back together • Military DNA “dog tag” • Convicted felon DNA databases

  4. DNA Quantitation PCR Amplification of Multiple STR markers DNA Extraction Separation and Detection of PCR Products (STR Alleles) Sample Genotype Determination Comparison of Sample Genotype to Other Sample Results Generation of Case Report with Probability of Random Match If match occurs, comparison of DNA profile to population databases Steps in DNA Sample Processing Sample Obtained from Crime Scene or Paternity Investigation Biology Technology Genetics

  5. Sources of Biological Evidence • Blood • Semen • Saliva • Urine • Hair • Teeth • Bone • Tissue

  6. chromosome cell nucleus Double stranded DNA molecule Target Region for PCR Individual nucleotides DNA in the Cell

  7. 5’ 3’ 5’ 3’ Starting DNA Template 3’ 5’ 3’ 5’ Separate strands (denature) Forward primer 5’ 3’ 3’ 5’ 5’ 3’ 5’ 3’ Make copies (extend primers) Reverse primer Add primers (anneal) DNA Amplification with the Polymerase Chain Reaction (PCR)

  8. Original DNA target region Thermal cycle Thermal cycle Thermal cycle PCR Copies DNA Exponentially through Multiple Thermal Cycles In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created

  9. AATG Short Tandem Repeats (STRs) 7 repeats 8 repeats the repeat region is variable between samples while the flanking regions where PCR primers bind are constant Homozygote = both alleles are the same length Heterozygote = alleles differ and can be resolved from one another

  10. 170 bp 195 bp TCAT repeat unit Different primer sets produce different PCR product sizes for the same STR allele

  11. Over 10 Markers Can Be Copied at Once Sensitivities to levels less than 1 ng of DNA Ability to Handle Mixtures and Degraded Samples Different Fluorescent Dyes Used to Distinguish STR Alleles with Overlapping Size Ranges Multiplex PCR

  12. 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 reaction An Example Forensic STR Multiplex Kit

  13. 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

  14. capillary Syringe with polymer solution Injection electrode Autosampler tray Outlet buffer Inlet buffer ABI Prism 310 Genetic Analyzer

  15. Electrode Capillary Sample Vials Autosampler Tray Close-up of ABI Prism 310 Sample Loading Area See Technology section for more information on CE

  16. 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 ID

  17. Microvariant allele STR genotyping is performed by comparison of sample data to allelic ladders

  18. 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 of repeats STR Allele Frequencies TH01 Marker *Proc. Int. Sym. Hum. ID (Promega) 1997, p. 34

  19. FBI’s CODIS DNA Database Combined DNA Index System • Used for linking serial crimes and unsolved cases with repeat offenders • Launched October 1998 • Links all 50 states • Requires >4 RFLP markers and/or 13 core STR markers • Current backlog of >600,000 samples

  20. 13 CODIS Core STR Loci with Chromosomal Positions TPOX D3S1358 TH01 D8S1179 D5S818 VWA FGA D7S820 CSF1PO AMEL D13S317 AMEL D16S539 D18S51 D21S11

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