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Evaluating forensic DNA evidence

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Forensic BioInformatics (bioforensics.com) ... Forensic DNA Typing' by John M. Butler (Academic Press) Internet ... .com/ (see human identity and forensics) ... – PowerPoint PPT presentation

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Title: Evaluating forensic DNA evidence


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

2
Three generations of DNA testing
DQ-alpha TEST STRIP Allele BLUE DOT
RFLP AUTORAD Allele BAND
Automated STR ELECTROPHEROGRAM Allele PEAK
3
DNA in the Cell
cell
chromosome
nucleus
Double stranded DNA molecule
4
DNA content of biological samples
Type of sample
Amount of DNA
Blood
30,000 ng/mL
2
stain 1 cm in area
200 ng
2
stain 1 mm in area
2 ng
Semen
250,000 ng/mL
Postcoital vaginal swab
0 - 3,000 ng
Hair
plucked
1 - 750 ng/hair
shed
1 - 12 ng/hair
Saliva
5,000 ng/mL
Urine
1 - 20 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 electropherogramPeaks correspond to
alleles
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 AnalyzerSIZE, 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
  • Color of STR detected and recorded as it passes
    the detector

15
Profiler Plus Raw data
16
RAW DATA
  • GENESCAN divides the raw data into a separate
    electropherogram for each color
  • Blue
  • Green
  • Yellow
  • Red
  • GENOTYPER identifies the different loci and makes
    the allele calls

PROCESSED DATA
17
Statistical estimates the product rule
0.222
0.1
18
Statistical estimates the product rule
1 in 10
0.1
1 in 79,531,528,960,000,000
1 in 80 quadrillion
19
Profiler Plus
D3S1358
FGA
VWA
AMEL
D8S1179
D21S11
D18S51
D5S818
D13S317
D7S820
20
Cofiler
D3S1358
D16S539
AMEL
TH01
TPOX
CSF1PO
D7S820
21
Identifiler
D8S1179
CSF1PO
D7S820
D21S11
D2S1338
D3S1358
D16S539
TH01
D13S317
D19S433
VWA
D18S51
TPOX
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
LARGE
SMALL
  • 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
Reference sample
Evidence sample
150
?
  • 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
  • False peaks
  • 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 0.00
3 310 0.00
4 2,498,139 5.53
5 29,938,777 66.32
6 12,702,670 28.14
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
  • 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
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44
Genophiler output
 
 
45
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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 http//www.appliedbios
    ystems.com/ (see human identity and forensics)
  • Promega Website http//www.promega.com/ (see
    Genetic Identity)
  • STR base http//www.cstl.nist.gov/biotech/strbase
    / (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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