1. Generating forensic DNA profiles
Dan E. Krane, Wright State University, Dayton, OH
Forensic DNA Profiling Video Series
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. Two additional DNA tests
Mitochondrial DNA
mtDNA sequence
Sensitive but not discriminating
Y-STRs
Useful with mixtures
Paternally inherited

4. DNA content of biological samples
Trillions of cells
Roughly 100 cells
Each cell contains 6 to 7 pg of DNA
DNA profiling kits generally recommend using between 500 and 1,000 pg of template DNA
That works out to roughly 100 to 200 cells

5. What is a picogram? 1 gram = 1/4th of a packet of sugar
1 milligram = a single crystal of sugar
1 nanogram = one 1000th of a crystal of sugar
1 picogram = one billionth of a gram

6. What is a microliter? 1 liter = half of a bottle of a soft drink
1 milliliter = 1000th of a liter (about a thimble full)
100 microliters = one drop
1 microliter = one millionth of a liter

7. 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

8. 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
Electrophoresis
A technique for separating molecules according to their size

9. Automated STR Test

10. Crime Scene Samples & Reference Samples
Extract and purify DNA
Differential extraction in sex assault cases attempts to isolate DNA from sperm cells

11. Extract and Purify DNA
Add primers and other reagents

12. Setting up an amplification
Pipettors are used to transfer microliter quantities of liquids
Final reaction volumes are typically 10 or 20 microliters
There are no good visual clues that a solution contains DNA or that a reaction is proceeding correctly

13. PCR Amplification
DNA regions flanked by primers are amplified

14. PCR Amplification
Targeted regions are doubled with each round of amplification.
Instead of needle in a haystack, after 28 rounds of amplification there is a needle-stack with a piece of hay.
Amplified DNA fragments are fluorescently labeled.

15. The ABI 310 Genetic Analyzer

16. ABI 310 Genetic Analyzer: Capillary Electrophoresis
DNA pulled towards the positive electrode
DNA separated out by size:
Large DNA moves slowly
Small DNA moves faster
Detector
Window
Color of STR detected and recorded as it passes the detector

17. Profiler Plus: Raw data

18. Profiler Plus™ DNA profile

19. 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

20. Reading an electropherogram
NUMBER OF PEAKS
1 peak = homozygous
2 peaks = heterozygous
3 or more peaks = mixed sample (?)
LARGE
SMALL
POSITION OF PEAK
Smaller alleles on left
Larger alleles on right
HEIGHT OF PEAK
Proportional to amount of allele

21. Profiler Plus D3S1358 vWA FGA AMEL D8S1179 D21S11 D18S51 D5S818

22. SGM+ D3S1358 vWA D16S539 D2S1338 AMEL D8S1179 D21S11 D18S51 D19S433
THO1
FGA

23. Profiler Plus™ DNA profile

24. What weight should be given to a DNA profile match?
• Do they have the same source?
Is the match a coincidence?
Has an error occurred?

25. Generating forensic DNA profiles
Dan E. Krane, Wright State University, Dayton, OH
Forensic DNA Profiling Video Series
Forensic Bioinformatics (