Presentation on theme: "Forensic Bioinformatic Services, Inc."— Presentation transcript:
1 Forensic Bioinformatic Services, Inc. The prospect for more than 13 commonly used STR Tetranucleodide loci in forensic settingsCarrie RowlandForensic Bioinformatic Services, Inc.Dayton, Ohio
2 An Overview: Where are we going History of DNA testingGenerations of DNA tests and how performedCurrent methodologyNew prospectsConclusionsTALKING POINTSWhat I would like to accomplish
3 Six criteria for judging a DNA profiling system Discrimination power.Sensitivity.Absence of, or the ability to reconciletechnical artifacts.Speed.Ability to deconvolve mixtures.Amenability to database searches.
4 Brief History of Forensic DNA Typing Ray White describes first polymorphic RFLP markerAlec Jeffreys proposes multilocus VNTR probes be used forensicallyfirst paper on PCRFBI starts DNA caseworkfirst STR paperFSS starts UK DNA databaseFBI launches CODIS database
5 Three generations of DNA testing RFLPAUTORADAllele = BANDDQ-alphaTEST STRIPAllele = BLUE DOTAutomated STRELECTROPHEROGRAMAllele = PEAK
6 How do they fare? RFLP DQ-alpha STR Discriminating power □ Sensitivity□ Technical artifacts□ Speed□ Mixturesٱ Databasing Discriminating power Sensitivity□ Technical artifacts Speed□ Mixtures Databasing□ Discriminating power Sensitivity Technical artifacts Speed□ Mixtures□ Databasing
7 like for them to look like. RFLP technologyWhat we wouldlike for them to look like.
9 Available Kits for STR Analysis Kits make it easy for labs to just add DNA samples to a pre-made mix13 CODIS core lociProfiler Plus and COfiler (PE Applied Biosystems)PowerPlex 1.1 and 2.1 (Promega Corporation)Increased power of discriminationCTT (1994): 1 in 410SGM Plus™ (1999): 1 in 3 trillionPowerPlex ™ 16 (2000): 1 in 2 x 1017
10 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 PCR100 bp400 bp300 bp200 bpSize SeparationColor SeparationD3FGAvWA5-FAM (blue)D13D5D7NED (yellow)AD8D21D18JOE (green)GS500-internal lane standardROX (red)
11 13 CODIS Core STR Loci with Chromosomal Positions CSF1POD5S818D21S11TH01TPOXD13S317D7S820D16S539D18S51D8S1179D3S1358FGAVWAAMEL
12 STR Allele Frequencies 5101520253035404567899.3TH01 MarkerNumber of repeatsFrequencyCaucasians (N=427)Blacks (N=414)Hispanics (N=414)*Proc. Int. Sym. Hum. ID (Promega) 1997, p. 34
13 Human Identity Testing with Multiplex STRs Simultaneous Analysis of 10 STRs and GenderAmpFlSTR® SGM Plus™ kitTwo different individualsDNA Size (base pairs)amelogeninD19D3D8TH01VWAD21FGAD16D18D2Results obtained in less than 5 hours with a spot of blood the size of a pinheadprobability of a random match: ~1 in 3 trillion
14 FBI’s CODIS DNA Database Combined DNA Index SystemLaunched October 1998Used for linking serial crimes and unsolved cases with repeat offendersLinks all 50 statesRequires >4 RFLP markers and/or 13 core STR markersCurrent backlog of > 600,000 samplesAs of June, 2004Total profiles = 1,857,093Total forensic profiles = 85,477Total convicted offender = 1,771,616
16 STR alleles with n+4 stutter products DNA Size (bp)D8S1179D21S11D18S51Relative Fluorescence UnitsTALKING P0INTS6.3%6.2%5.4%
17 Schematic of Stutter Product Formation Process Normal STR Allele Replication123456Slipped Strand Mispairing Model12345TALKING 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 registertemplate loops outRepeat unit bulges out when strand breathing occursWalsh et al (1996) Nucleic Acids Res. 24:
18 Mechanism behind n+4 stutter DNA polymerase172635445326TALKING 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.1Template DNAWalsh, et al. (1996) Nucleic Acids Research 24(14):2807
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