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Three generations of DNA testing

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Presentation on theme: "Three generations of DNA testing"— Presentation transcript:

1 Three generations of DNA testing
RFLP AUTORAD Allele = BAND DQ-alpha TEST STRIP Allele = BLUE DOT Automated STR ELECTROPHEROGRAM Allele = PEAK

2 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

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

4 What they often look like. Incomplete digest

5 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

6 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

7 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

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

9 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

10 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

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

12 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

13 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

14 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

15

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

17 ALLELIC LADDERS

18 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

19 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

20 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

21 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

22 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

23 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)

24 Genotyping by Comparison to Allelic Ladder

25 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

26 Electropherogram

27 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

28 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

29 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

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

31 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

32 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

33 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

34 13 CODIS Core STR Loci with Chromosomal Positions
CSF1PO D5S818 D21S11 TH01 TPOX D13S317 D7S820 D16S539 D18S51 D8S1179 D3S1358 FGA VWA AMEL

35 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

36 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)

37 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

38 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

39 Exclusions don’t require numbers Matches do require statistics

40 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

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

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

43 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

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

45 Item D3S1358 D16S539 TH01 TPOX CSF1P0 D7S820
Q ,16 10, , , , ,11 Item D3S vWA FGA D8S D21S11 D18S51 D5S818 D13S317 D7S820 Q , , , , , , , , ,11 CoFIler ProfIler Plus

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

47 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 = x =

48 Item D3S1358 D16S539 TH01 TPOX CSF1P0 D7S820
Q ,16 10, , , , ,11 Item D3S vWA FGA D8S D21S11 D18S51 D5S818 D13S317 D7S820 Q , , , , , , , , ,11 CoFIler ProfIler Plus

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

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

51 Frequency = 2 X allele 1 freq X allele 2 freq
VWA = 15, 17 (heterozygote) Frequency of 15 allele = Frequency of 17 allele = When heterozygous: Frequency = 2 X allele 1 freq X allele 2 freq (2pq) Genotype freq = 2 x x = Overall profile frequency = Frequency D3S1358 X Frequency vWA x =

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

53

54 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!

55 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

56

57 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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