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Evaluating forensic DNA evidence

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1 Evaluating forensic DNA evidence
Dan E. Krane Biological Sciences, Wright State University, Dayton OH 45435 Forensic Bioinformatics (www.bioforensics.com)

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

3 DNA in the Cell A T G C Target Region for PCR cell chromosome nucleus
Double stranded DNA molecule A T C G

4 DNA content of biological samples:
Type of sample Amount of DNA Blood 30,000 ng/mL stain 1 cm in area 2 200 ng stain 1 mm in area 2 2 ng Semen 250,000 ng/mL Postcoital vaginal swab 0 - 3,000 ng Hair plucked ng/hair shed ng/hair Saliva 5,000 ng/mL Urine ng/mL

5 Basic terminology: Genetics
DNA Polymorphism (“many forms”) Regions of DNA which differ from person to person Locus (plural = loci) Site or location on a chromosome Allele Different variants which can exist at a locus DNA Profile The combination of alleles for an individual

6 Basic terminology: Technology
Amplification or PCR (Polymerase Chain Reaction) A technique for ‘replicating’ DNA in the laboratory (‘molecular Xeroxing’) Region to be amplified defined by PRIMERS Can be ‘color coded’ Electrophoresis A technique for separating molecules according to their size

7 STR Short tandem repeat Describes a type of DNA polymorphism in which:
a DNA sequence repeats over and over again and has a short (usually 4 base pair) repeat unit A length polymorphism -- alleles differ in their length 3 repeats: AATG AATG AATG 4 repeats: AATG AATG AATG AATG 5 repeats: AATG AATG AATG AATG AATG 6 repeats: AATG AATG AATG AATG AATG AATG

8 Reading an electropherogram Peaks correspond to alleles
BLUE GREEN YELLOW RED D3 vWA FGA D8 D21 D18 D5 D13 D7 Amelogenin XX = female XY = male 75 100 139 150 160 200 245 300 bps Red = ROX size standard

9 Automated STR Test

10 Crime Scene Samples & Reference Samples
Extract and purify DNA Differential extraction in sex assault cases separates out DNA from sperm cells

11 Extract and Purify DNA Add primers and other reagents

12 PCR Amplification DNA regions flanked by primers are amplified Groups of amplified STR products are labeled with different colored dyes (blue, green, yellow)

13 The ABI 310 Genetic Analyzer: SIZE, COLOR & AMOUNT

14 ABI 310 Genetic Analyzer: Capillary Electrophoresis
Amplified STR DNA injected onto column Electric current applied DNA pulled towards the positive electrode DNA separated out by size: Large STRs travel slower Small STRs travel faster Detector Window Color of STR detected and recorded as it passes the detector

15 Profiler Plus: Raw data

16 RAW DATA PROCESSED DATA D3 vWA FGA D8 D21 D18 D5 D13 D7 Am
GENESCAN divides the raw data into a separate electropherogram for each color: Blue Green Yellow Red RAW DATA GENOTYPER identifies the different loci and makes the allele calls D3 vWA FGA D8 D21 D18 D5 D13 D7 Am The type of this sample is: D3: 16, 17 vWA: 15, 15 FGA: 21,23 Amelogenin: X, Y D8: 16, 16 D21: 28, 29 D18: 14, 19 D5: 8, 12 D13: 11, 13 D7: 10 10 PROCESSED DATA

17 Statistical estimates: the product rule
0.222 x 0.222 x 2 = 0.1

18 Statistical estimates: the product rule
1 in 10 1 in 22,200 x 1 in 111 1 in 20 = 0.1 1 in 100 1 in 14 1 in 81 1 in 113,400 x 1 in 116 1 in 17 1 in 16 1 in 31,552 x 1 in 79,531,528,960,000,000 1 in 80 quadrillion

19 Profiler Plus D3S1358 VWA FGA AMEL D8S1179 D21S11 D18S51 D5S818

20 Cofiler D3S1358 D16S539 AMEL TH01 TPOX CSF1PO D7S820

21 Identifiler D8S1179 D7S820 CSF1PO D21S11 D3S1358 TH01 D13S317 D16S539
VWA TPOX D18S51 AMEL D5S818 FGA

22 LOOKING AT A DNA REPORT

23 Components of a DNA report
The samples tested Evidence samples (crime scene) Reference samples (defendant, suspect) The lab doing the testing The test used: Profiler Plus, Cofiler, Identifiler, mtDNA The analyst who did the testing Results and conclusions: Table of alleles Narrative conclusions

24 Table of alleles Some labs include more information than others
Usually includes information about mixed samples May also include: Indication of low level results Indication of results not reported Relative amounts of different alleles (in mixed samples) No standard format

25 Narrative conclusions
Indicates which samples match Includes a statistical estimate Identifies samples as mixed May include an ‘identity statement’ i.e., samples are from the same source to a scientific degree of certainty (FBI) May allude to problems (e.g. interpretative ambiguity, contamination)

26 Looking beneath the report

27 Sources of ambiguity in STR interpretation
Degradation Allelic dropout False peaks Mixtures Accounting for relatives Threshold issues -- marginal samples

28 Degradation SMALL LARGE
When biological samples are exposed to adverse environmental conditions, they can become degraded Warm, moist, sunlight, time Degradation breaks the DNA at random Larger amplified regions are affected first Classic ‘ski-slope’ electropherogram Peaks on the right lower than peaks on the left

29 ? Allelic Dropout 1500 150 Reference sample Evidence sample
Peaks in evidence samples all very low Mostly below 150 rfu Peaks in reference sample much higher All well above 800 rfu At D13S817: Reference sample: 8, 14 Evidence sample: 8, 8 14 allele has dropped out -- or has it? Tend to see with ‘marginal samples’

30 False peaks & machine problems
Contamination Dye blob Electrical spikes Pull-up Machine problems: Noise Baseline instability Injection failures

31 Mixed DNA samples

32 How many contributors to a mixture?
How many contributors to a mixture if analysts can discard a locus? Maximum # of alleles observed in a 3 person mixture # of occurrences Percent of cases 2 0.00 3 310 4 2,498,139 5.53 5 29,938,777 66.32 6 12,702,670 28.14 8,151 1,526,550 32,078,976 11,526,219 0.02 3.38 71.07 25.53 There are 45,139,896 possible different 3-way mixtures of the 648 individuals in the MN BCI database. There are 45,139,896 possible different 3-way mixtures of the 648 individuals in the MN BCI database.

33 How many loci must have six or fewer alleles to be confident there were only three contributors?
Nine loci are commonly used. No kit tests at more than 16 loci. Approximately 144 loci must be examined for 95% confidence that there were only 3 contributors.

34 Opportunities for subjective interpretation?

35 Opportunities for subjective interpretation?
D3: 12, 17 vWA: 15, 17 FGA: 22, 26

36 Opportunities for subjective interpretation?

37 Opportunities for subjective interpretation?

38 Opportunities for subjective interpretation?

39 Opportunities for subjective interpretation?

40 Accounting for relatives

41 Likelihood ratios for allele sharing:

42 Presents all output in a web page
Automatically runs GeneScan and GenoTyper Presents all output in a web page Performs expert analysis to identify problems Generates a report detailing all testing issues

43

44 Genophiler output:

45 Cheat sheet, part 2.

46 Genophiler also flags potential problems for further review:

47 Forensic BioInformatics (bioforensics.com)
• Uses Genophiler to generate easily interpreted files • Objectively applies analysis parameters to all samples • Fast turn around times • Efficiently draws attention to problems requiring further review

48 Resources Books ‘Forensic DNA Typing’ by John M. Butler (Academic Press) Internet Applied Biosystems Website: (see human identity and forensics) Promega Website: (see Genetic Identity) STR base: (very useful) Scientists Larry Mueller (UC Irvine) Simon Ford (Lexigen, Inc. San Francisco, CA) William C. Thompson (UC Irvine) William Shields (SUNY, Syracuse, NY) Marc Taylor (Technical Associates, Ventura, CA) Carll Ladd (Connecticut State Police) Testing laboratories Technical Associates (Ventura, CA) Forensic Analytical (Haywood, CA) Other resources Forensic BioInformatics (Dayton, OH)


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