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

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

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)