Three generations of DNA testing RFLP AUTORAD Allele

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? DQ-alpha RFLP Discriminating power Sensitivity □ Technical artifacts Speed □ Mixtures Databasing □ Databasing Discriminating power □ □ Sensitivity □ Technical artifacts □ Speed □ Mixtures ٱ Databasing STR

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

What they often look like. Incomplete digest

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) – Power. Plex 1. 1 and 2. 1 (Promega Corporation) • Increased power of discrimination – CTT (1994): 1 in 410 – SGM Plus™ (1999): 1 in 3 trillion – Power. Plex ™ 16 (2000): 1 in 2 x 1017

Overview of Steps Involved in DNA Typing TPOX TH 01 D 3 AMEL D 5 VWA D 7 D 13 D 21 D 8 CSF D 16 D 18 FGA Penta D 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

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

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

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

THE 13 CODIS STR LOCI • Four categories • Simple repeats – TPOX, CSF 1 PO, D 5 S 818, D 13 S 317, D 16 S 539 • Simple repeats w/ non-consensus alleles – TH 01, D 18 S 51, D 7 S 820 • Compound repeats w/ non-consensus alleles – v. WA, FGA, D 3 S 1358, D 8 S 1179 • Complex repeats – D 21 S 11

Commercial STR Kits • Applied Biosystems • Amp. Fl. STR Profiler Plus – 5 -FAM (BLUE) – Joe (GREEN) – NED (YELLOW) Amelo • Amp. Fl. STR Cofiler v. WA D 3 S 1358 D 8 S 1179 D 5 S 818 FGA D 21 S 11 D 13 S 317 D 18 S 51 D 7 S 820 – 5 -FAM (BLUE) – Joe (GREEN) D 16 S 539 D 3 S 1358 – NED (YELLOW) Amelo TH 01 TPOX CSF 1 PO D 7 S 820

Commercial STR Kits • Promega Corporation • Power. Plex 1. 1 – Fluorescein (Blue) D 5 S 818 D 13 S 317 – TMR (Yellow) v. WA TH 01 D 7 S 820 TPOX D 16 S 539 CSF 1 PO • Power. Plex 2. 1 – Fluorescein (Blue)D 3 S 1358 – TMR (Yellow) Amelo TH 01 v. WA D 21 S 11 D 8 S 1179 D 18 S 51 TPOX Penta E FGA

Genotyping by Comparison to Allelic Ladder

PCR Amplification with Fluorescent STR Kits and Separation with Capillary Electrophoresis TPOX TH 01 CSF D 7 D 16 D 3 D 5 AMEL VWA D 13 D 21 D 8 D 18 FGA Penta D Penta E

Electropherogram

An Example Forensic STR Multiplex Kit Amp. Fl. STR® Profiler Plus™ Kit available from PE Biosystems (Foster City, CA) 200 bp Color Separation 100 bp Size Separation D 3 A v. WA D 8 D 5 FGA 300 bp 400 bp 5 -FAM (blue) D 21 D 18 JOE (green) D 13 D 7 NED (yellow) ROX (red) GS 500 -internal lane standard 9 STRs amplified along with sex-typing marker amelogenin in a single PCR

Human Identity Testing with Multiplex STRs Two different individuals Amp. Fl. STR® SGM Plus™ kit amelogenin D 19 D 3 DNA Size (base pairs) D 8 TH 01 VWA D 21 D 16 D 18 D 2 FGA probability of a random match: ~1 in 3 trillion amelogenin D 3 D 19 D 8 VWA TH 01 Results obtained in less than 5 hours with a spot of blood the size of a pinhead D 16 D 21 FGA D 18 Simultaneous Analysis of 10 STRs and Gender D 2

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

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