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

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

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

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 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

STR NOMENCLATURE Microvariants Alleles that contain incomplete units aatg aatg aatg aatg aatg aatg aatg aatg aatg aatg - 10 aatg aatg aatg aatg aatg aatg atg aatg aatg aatg - 9.3

STRs Used In Forensic Science Need lots of variation - polymorphic Overall short segments - 100-400 bp Can use degraded DNA samples Segment size usually limits preferential amplification of smaller alleles Single base resolution TH01 9.3

STRs Used In Forensic Science TETRANUCLEOTIDE REPEATS Narrow allele size range - multiplexing Reduces allelic dropout (stochastic effects) Use with degraded DNA possible Reduced stutter rates - easier to interpret mixtures

STR NOMENCLATURE Use the 5’ to 3’ (Top) strand Start with the first 5’ nucleotide in the repeat Microvariants designated by decimal places Number of complete repeats Number of bases in incomplete repeat (9.3) Allelic ladders used as reference Contain all common alleles

ALLELIC LADDERS Artificial mixture of common alleles Reference standards Enable forensic scientists to compare results Different instruments Different detection methods Allele quantities balanced Produced with same primers as test samples Commercially available in kits

Profiler Plus Allelic Ladders D3S1358 VWA FGA AMEL D8S1179 D21S11 D18S51 D5S818 D13S317 D7S820

ALLELIC LADDERS

THE 13 CODIS STR LOCI November 1997 Average random match probability is greater than 1in 1 trillion US slower than UK in selection of loci for database

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

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

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

Power of Discrimination Same DNA Sample Run with Each of the ABI STR Kits TH01 Amel D16S539 D7S820 CSF1PO TPOX D3S1358 D18S51 D21S11 D8S1179 D13S317 D5S818 D19S433 D2S1338 FGA vWA PCR Product Size (bp) Power of Discrimination 1:5000 1:410 1:3.6 x 109 1:9.6 x 1010 1:8.4 x 105 1:3.3 x 1012 Profiler Plus COfiler SGM Plus Green I Profiler Blue

AmpFlSTR® Identifiler™ D8S1179 D21S11 D7S820 CSF1PO D3S1358 TH01 D13S317 D16S539 D2S1338 D19S433 D18S51 TPOX VWA AMEL D5S818 FGA GS500 LIZ size standard 6FAM (blue) VIC (green) NED (yellow) PET (red) LIZ (orange)

Genotyping by Comparison to Allelic Ladder

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

Electropherogram

STR LOCI ALLELES CSF1PO FGA c-fms proto-oncogene Chromosome 5 AGAT repeat 6 to 15 repeats FGA alpha fibrinogen locus Chromosome 4 CTTT repeat 15 to 51.2 repeats

STR LOCI ALLELES TH01 TPOX TYROSINE HYDROXYLASE Chromosome 11 TCTA repeat (Bottom strand) 4 to 11 repeats Common microvariant 9.3 TPOX THYROID PEROXIDASE Chromosome 2 AATG repeat 6 to 13 repeats

STR LOCI ALLELES vWA D3S1358 von Willebrand Factor Chromosome 12 TCTA with TCTG repeat 10 to 22 repeats D3S1358 Chromosome 3 AGAT with AGAC repeat 12 to 20 repeats

STR LOCI ALLELES D5S818 D7S820 Chromosome 5 AGAT repeat 7 to 16 repeats D7S820 Chromosome 7 GATA repeat Some Microvariants 6 to 15 repeats

STR LOCI ALLELES D8S1179 D13S317 Chromosome 8 TCTA repeat with TCTG in alleles >13 7 to 19 repeats D13S317 Chromosome 13 GATA repeat 7 to 15 repeats

STR LOCI ALLELES D16S539 D18S15 Chromosome 16 AGAT repeat with TCTG in alleles >13 8 to 15 repeats D18S15 Chromosome 18 AGAA repeat Some x.2 Microvariants 8 to 27 repeats

STR LOCI ALLELES D21S11 Chromosome 21 TCTA repeat with TCTG Some x.2 Microvariants 24 to 38 repeats Over 70 reported alleles Fine differences must be sequenced 4 alleles are designated as 30 repeats

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

Position of Forensic STR Markers on Human Chromosomes CSF1PO D5S818 D21S11 TH01 TPOX D13S317 D7S820 D16S539 D18S51 D8S1179 D3S1358 FGA VWA D2S1338 D19S433 13 CODIS Core STR Loci AMEL Sex-typing Penta E Penta D

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)

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

Exclusions don’t require numbers Matches do require statistics

Hardy - Weinberg Equilibrium A1A1 A1A2 A2A2 A1 A2 A1A2 A2A2 A1A1 p12 2p1p2 p22 p1p2 p12 freq(A1) = p1 p22 freq(A2) = p2 (p1 + p2 )2 = p12 + 2p1p2 + p22

A Hardy-Weinberg Population LARGE POPULATION NO NATURAL SELECTION NO MUTATION NO IMMIGRATION / EMIGRATION RANDOM MATING

Estimate genotype frequency: 1. Frequency at each locus 2. Frequency across all loci Product Rule

Criteria for Use of Product Rule The frequency of a multi-locus STR profile is the product of the genotype frequencies at the individual loci ƒ locus1 x ƒ locus2 x ƒ locusn = ƒcombined Criteria for Use of Product Rule Inheritance of alleles at one locus have no effect on alleles inherited at other loci

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

Item D3S1358 D16S539 TH01 TPOX CSF1P0 D7S820 Q1 16,16 10,12 8,9.3 9,10 12,12 8,11 Item D3S1358 vWA FGA D8S1179 D21S11 D18S51 D5S818 D13S317 D7S820 Q1 16,16 15,17 21,22 13,13 29,30 16,20 8,12 12,12 8,11 CoFIler ProfIler Plus

D3S1358 = 16, 16 (homozygote) Frequency of 16 allele = ??

Frequency = genotype frequency (p2) D3S1358 = 16, 16 (homozygote) Frequency of 16 allele = 0.3071 When same allele: Frequency = genotype frequency (p2) (for now!) Genotype freq = 0.3071 x 0.3071 = 0.0943

Item D3S1358 D16S539 TH01 TPOX CSF1P0 D7S820 Q1 16,16 10,12 8,9.3 9,10 12,12 8,11 Item D3S1358 vWA FGA D8S1179 D21S11 D18S51 D5S818 D13S317 D7S820 Q1 16,16 15,17 21,22 13,13 29,30 16,20 8,12 12,12 8,11 CoFIler ProfIler Plus

VWA = 15, 17 (heterozygote) Frequency of 15 allele = ?? Frequency of 17 allele = ??

VWA = 15, 17 (heterozygote) Frequency of 15 allele = ?? Frequency of 17 allele = ??

Frequency = 2 X allele 1 freq X allele 2 freq VWA = 15, 17 (heterozygote) Frequency of 15 allele = 0.2361 Frequency of 17 allele = 0.1833 When heterozygous: Frequency = 2 X allele 1 freq X allele 2 freq (2pq) Genotype freq = 2 x 0.2361 x 0.18331 = 0.0866 Overall profile frequency = Frequency D3S1358 X Frequency vWA 0.0943 x 0.0866 = 0.00817

What if… We encounter alleles not represented in the population database… …or alleles that are extremely rare in the database???

Where the random match probability is the sum of the squares of the observed phenotype/genotype frequencies in a database, The Power of Exclusion of a genetic locus is based on the 1 – the sum of squares of all the expected phenotypes/genotypes!

These measures tell us two things about our markers and databases: Power of Discrimination – how powerful our loci are at individualizing Power of Exclusion – how powerful our marker panel is at excluding particular genotypes

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