Forensic Bioinformatic Services, Inc. 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 Current methodology New prospects Conclusions TALKING POINTS What I would like to accomplish

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.

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

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

like for them to look like. 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) 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

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

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

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

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

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

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

STR alleles with n+4 stutter products DNA Size (bp) D8S1179 D21S11 D18S51 Relative Fluorescence Units TALKING P0INTS 6.3% 6.2% 5.4%

Schematic of Stutter Product Formation Process Normal STR Allele Replication 1 2 3 4 5 6 Slipped Strand Mispairing Model 1 2 3 4 5 TALKING POINTS ● amplicons that differin size from primary peak by multiples of the core repeat unit (1-4 repeat units). ● the polymerization enzyme-template complex becomes destabilized and potential disassociate when it rejoins, may do so out of register template loops out Repeat unit bulges out when strand breathing occurs Walsh et al (1996) Nucleic Acids Res. 24: 2807-2812

Mechanism behind n+4 stutter DNA polymerase 1 7 2 6 3 5 4 4 5 3 2 6 TALKING POINTS -slipped strand mispairing model -step 1 – the DNA polymerase has extended through 4 repeat units (light gray boxes) – Step 2 - if the polymerase becomes disassociated with the extending strand, the template and the extending strand may break apart. Step 3 – when the two strands re-anneal, they could possibly re-anneal out of register by one repeat unit. Step 4 – the final product contains only 6 of the 7 repeat units. 1 Template DNA Walsh, et al. (1996) Nucleic Acids Research 24(14):2807

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