Download BASIC DNA

Evaluating forensic DNA
evidence
Dan E. Krane
Biological Sciences, Wright State University,
Dayton OH 45435
Forensic Bioinformatics
(www.bioforensics.com)
[email protected]
Three generations of DNA testing
RFLP
AUTORAD
Allele = BAND
DQ-alpha
TEST STRIP
Allele = BLUE DOT
Automated STR
ELECTROPHEROGRAM
Allele = PEAK
DNA in the Cell
cell
chromosome
nucleus
Target Region for PCR
Double
stranded DNA
molecule
A
T
T G
A T
C
A
DNA content of biological samples:
Type of sample
Blood
stain 1 cm2 in area
stain 1 mm2 in area
Semen
Postcoital vaginal swab
Amount of DNA
30,000 ng/mL
200 ng
2 ng
250,000 ng/mL
0 - 3,000 ng
Hair
plucked
shed
Saliva
Urine
1 - 750 ng/hair
1 - 12 ng/hair
5,000 ng/mL
1 - 20 ng/mL
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
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
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
4
5
6
repeats:
repeats:
repeats:
repeats:
AATG
AATG
AATG
AATG
AATG
AATG
AATG
AATG
AATG
AATG AATG
AATG AATG AATG
AATG AATG AATG AATG
Reading an electropherogram
Peaks correspond to alleles
BLUEFGA
D3 vWA
D8
D21
GREEN
Amelogenin
XX = female
XY = male
Red = ROX size standard
Electropherogram
D18
Amelogenin
D5
D13 D7
YELLOW
75 100 139
200
RED
150
160
245
300 bps
Automated STR Test
Crime Scene Samples &
Reference Samples
• Extract and purify DNA
Differential extraction in sex
assault cases separates out
DNA from sperm cells
Extract and Purify DNA
• Add primers and other reagents
PCR Amplification
• DNA regions flanked by
primers are amplified
Groups of amplified STR products are
labeled with different colored dyes
(blue, green, yellow)
The ABI 310 Genetic Analyzer:
SIZE, COLOR & AMOUNT
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
Detector
Window
Profiler Plus: 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
•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
RAW DATA
D3 vWA
Am D8
D5
FGA
D21
D18
D13 D7
PROCESSED DATA
Statistical estimates: the product rule
0.222 x 0.222 x 2
= 0.1
Statistical estimates: the product rule
1 in 10 x 1 in 111 x 1 in 20
= 0.1
1 in 22,200
1 in 100
x 1 in 14 x 1 in 81
1 in 113,400
1 in 116 x 1 in 17
x 1 in 16
1 in 31,552
1 in 79,531,528,960,000,000
1 in 80 quadrillion
Profiler Plus
D3S1358
AMEL
VWA
D8S1179
D5S818
FGA
D21S11
D13S317
D18S51
D7S820
Cofiler
D3S1358
AMEL
D16S539
TH01
TPOX
CSF1PO
D7S820
Identifiler
D8S1179
D7S820
CSF1PO
D21S11
D3S1358
TH01
D13S317
D16S539
D2S1338
D19S433
VWA
AMEL
D5S818
TPOX
D18S51
FGA
LOOKING AT A DNA REPORT
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
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
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)
Looking beneath the report
Sources of ambiguity in STR
interpretation
• Degradation
• Allelic dropout
• False peaks
• Mixtures
• Accounting for relatives
• Threshold issues -- marginal
samples
Degradation
S
M
A
L
L
L
A
R
G
E
• 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
Allelic Dropout
Reference sample
1500
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’
150
False peaks & machine problems
• False peaks:
– Contamination
– Dye blob
– Electrical spikes
– Pull-up
• Machine problems:
– Noise
– Baseline instability
– Injection failures
Mixed DNA samples
QuickTime™ and a
Photo - JPEG decompressor
are needed to see this picture.
How many contributors to a mixture?
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
8,151
310
0.02
0.00
4
11,526,219
2,498,139
25.53
5.53
5
32,078,976
29,938,777
6
1,526,550
12,702,670
71.07
66.32
3.38
28.14
There are 45,139,896 possible different 3-way mixtures of the 648
individuals in the MN BCI database.
How many loci must have six or
fewer alleles to be confident there
were only three contributors?
4-Way Mixtures, MN Data, Average
80.00%
70.00%
% Misclassified
60.00%
50.00%
40.00%
30.00%
20.00%
10.00%
0.00%
12
24
36
48
60
72
84
96
108
120
132
144
Loci
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.
Opportunities for subjective
interpretation?
Opportunities for subjective
interpretation?
D3: 12, 17
vWA: 15, 17
FGA: 22, 26
Opportunities for subjective
interpretation?
Opportunities for subjective
interpretation?
Opportunities for subjective
interpretation?
Opportunities for subjective
interpretation?
Accounting for relatives
20.00%
18.00%
16.00%
Percent of Total
14.00%
12.00%
Synthetic
10.00%
Cousins
Siblings
8.00%
6.00%
4.00%
2.00%
0.00%
1
3
5
7
9
11
13
15
Num ber of Shared Alleles
17
19
21
23
Likelihood ratios for allele
sharing:
100.00%
90.00%
80.00%
Likelihood
70.00%
60.00%
Synthetic
50.00%
Cousins
Siblings
40.00%
30.00%
20.00%
10.00%
0.00%
1
3
5
7
9
11 13 15 17 19 21 23
Number of Shared Alleles
• 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
Genophiler output:
Genophiler also flags potential problems for further review:
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
Resources
•
•
•
•
•
Books
– ‘Forensic DNA Typing’ by John M. Butler (Academic Press)
Internet
– Applied Biosystems Website: http://www.appliedbiosystems.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)