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Three generations of DNA testing
RFLP AUTORAD Allele = BAND DQ-alpha TEST STRIP Allele = BLUE DOT Automated STR ELECTROPHEROGRAM Allele = PEAK
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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
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like for them to look like.
RFLP technology What we would like for them to look like.
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What they often look like. Incomplete digest
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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
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Overview of Steps Involved in DNA Typing
AMEL D3 TH01 TPOX Penta D Penta E FGA D21 D18 CSF D16 D7 D13 D5 VWA D8 PCR Amplification with Fluorescent STR Kits and Separation with Capillary Electrophoresis Blood Stain DNA Quantitation using Slot Blot Genotyping by Comparison to Allelic Ladder
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REPEATED DNA Satellite DNA Minisatellite DNA Microsatellites
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
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SHORT TANDEM REPEATS Easy to amplify
Both heterozygote alleles amplify well Number of repeats highly variable Good for identification Many different sites
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STR NOMENCLATURE Simple Repeats Compound Repeats Complex 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
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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
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Commercial STR Kits Applied Biosystems vWA FGA TH01 TPOX
AmpFlSTR Profiler Plus 5-FAM (BLUE) Joe (GREEN) NED (YELLOW) AmpFlSTR Cofiler D3S1358 vWA FGA Amelo D8S1179 D21S11 D18S51 D5S818 D13S317 D7S820 D16S539 D3S1358 Amelo TH01 TPOX CSF1PO D7S820
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Commercial STR Kits Promega Corporation PowerPlex 1.1 PowerPlex 2.1
Fluorescein (Blue) TMR (Yellow) PowerPlex 2.1 D5S818 D13S317 D7S820 D16S539 vWA TH01 TPOX CSF1PO D3S1358 TH01 D21S11 D18S51 Penta E Amelo vWA D8S1179 TPOX FGA
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Genotyping by Comparison to Allelic Ladder
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PCR Amplification with Fluorescent STR Kits and Separation with Capillary Electrophoresis
AMEL D3 TH01 TPOX Penta D Penta E FGA D21 D18 CSF D16 D7 D13 D5 VWA D8
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Electropherogram
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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)
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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
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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
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Population database Look up how often each allele occurs at the locus in a population (the “allele” frequency)
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