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Three generations of DNA testing

Three generations of DNA testing. RFLP AUTORAD Allele = BAND. DQ-alpha TEST STRIP Allele = BLUE DOT. Automated STR ELECTROPHEROGRAM Allele = PEAK. How do they fare?. STR. DQ-alpha. RFLP.  Discriminating power  Sensitivity □ Technical artifacts  Speed □ Mixtures

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Three generations of DNA testing

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

  2. 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

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

  4. What they often look like. Incomplete digest

  5. 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

  6. TPOX TH01 CSF D7 D16 D3 D5 D13 Penta D D8 D18 FGA VWA D21 AMEL Penta E Blood Stain PCR Amplification with Fluorescent STR Kits and Separation with Capillary Electrophoresis DNA Quantitation using Slot Blot Genotyping by Comparison to Allelic Ladder Overview of Steps Involved in DNA Typing

  7. REPEATED DNA • Satellite DNA • Around chromosomal centromere • Long repeats can be 100s to 1000s bp long • Minisatellite DNA • VNTR • Medium repeats 10 to 100 bp long • Microsatellites • STR • Short repeats 2 to 6 bp long

  8. SHORT TANDEM REPEATS • Easy to amplify • Both heterozygote alleles amplify well • Number of repeats highly variable • Good for identification • Many different sites

  9. STR NOMENCLATURE • Simple Repeats • Identical length and sequence • agat agat agat agat agat • Compound Repeats • Two or more adjacent simple repeats • agat agat agat ttaa ttaa ttaa • Complex Repeats • Variable unit length & possible intervening seq • agat agat aggat agat agat ttaacggccat agat agat

  10. THE 13 CODIS STR LOCI • Four categories • Simple repeats • TPOX, CSF1PO, D5S818, D13S317, D16S539 • Simple repeats w/ non-consensus alleles • TH01, D18S51, D7S820 • Compound repeats w/ non-consensus alleles • vWA, FGA, D3S1358, D8S1179 • Complex repeats • D21S11

  11. Commercial STR Kits • Applied Biosystems • AmpFlSTR Profiler Plus • 5-FAM (BLUE) • Joe (GREEN) • NED (YELLOW) • AmpFlSTR Cofiler • 5-FAM (BLUE) • Joe (GREEN) • NED (YELLOW) D3S1358 vWA FGA Amelo D8S1179 D21S11 D18S51 D5S818 D13S317 D7S820 D16S539 D3S1358 Amelo TH01 TPOX CSF1PO D7S820

  12. Commercial STR Kits • Promega Corporation • PowerPlex 1.1 • Fluorescein (Blue) • TMR (Yellow) • PowerPlex 2.1 • Fluorescein (Blue) • TMR (Yellow) D5S818 D13S317 D7S820 D16S539 vWA TH01 TPOX CSF1PO D3S1358 TH01 D21S11 D18S51 Penta E Amelo vWA D8S1179 TPOX FGA

  13. Genotyping by Comparison to Allelic Ladder

  14. TPOX TH01 CSF D7 D16 D3 D5 D13 Penta D D8 D18 FGA VWA D21 AMEL Penta E PCR Amplification with Fluorescent STR Kits and Separation with Capillary Electrophoresis

  15. Electropherogram

  16. 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

  17. 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

  18. 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

  19. Population database • Look up how often each allele occurs at the locus in a population (the “allele” frequency)

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