1. Forensic Bioinformatic Services, Inc.
The prospect for more than 13 commonly used STR Tetranucleodide loci in forensic settings
Carrie Rowland
Forensic Bioinformatic Services, Inc.
Dayton, Ohio

2. An Overview: Where are we going
History of DNA testing
Generations of DNA tests and how performed
Current methodology
New prospects
Conclusions
TALKING POINTS
What I would like to accomplish

3. Six criteria for judging a DNA profiling system
Discrimination power.
Sensitivity.
Absence of, or the ability to reconcile
technical artifacts.
Speed.
Ability to deconvolve mixtures.
Amenability to database searches.

4. Brief History of Forensic DNA Typing
Ray White describes first polymorphic RFLP marker
Alec Jeffreys proposes multilocus VNTR probes be used forensically
first paper on PCR
FBI starts DNA casework
first STR paper
FSS starts UK DNA database
FBI launches CODIS database

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

6. How do they fare? RFLP DQ-alpha STR  Discriminating power
□ Sensitivity
□ Technical artifacts
□ Speed
□ Mixtures
ٱ Databasing
 Discriminating power
 Sensitivity
□ Technical artifacts
 Speed
□ Mixtures
 Databasing
□ Discriminating power
 Sensitivity
 Technical artifacts
 Speed
□ Mixtures
□ Databasing

7. like for them to look like.
RFLP technology
What we would
like for them to look like.

8. What they often
look like.
Incomplete digest

9. Available Kits for STR Analysis
Kits make it easy for labs to just add DNA samples to a pre-made mix
13 CODIS core loci
Profiler Plus and COfiler (PE Applied Biosystems)
PowerPlex 1.1 and 2.1 (Promega Corporation)
Increased power of discrimination
CTT (1994): 1 in 410
SGM Plus™ (1999): 1 in 3 trillion
PowerPlex ™ 16 (2000): 1 in 2 x 1017

10. An Example Forensic STR Multiplex Kit
AmpFlSTR® Profiler Plus™
Kit available from PE Biosystems (Foster City, CA)
9 STRs amplified along with sex-typing marker amelogenin in a single PCR
100 bp
400 bp
300 bp
200 bp
Size Separation
Color Separation D3
FGA
vWA
5-FAM (blue)
D13 D5 D7
NED (yellow) A D8
D21
D18
JOE (green)
GS500-internal lane standard
ROX (red)

11. 13 CODIS Core STR Loci with Chromosomal Positions
CSF1PO
D5S818
D21S11
TH01
TPOX
D13S317
D7S820
D16S539
D18S51
D8S1179
D3S1358
FGA
VWA
AMEL

12. STR Allele Frequencies5 10 15 20 25 30 35 40 45 6 7 8 9
9.3
TH01 Marker
Number of repeats
Frequency
Caucasians (N=427)
Blacks (N=414)
Hispanics (N=414)
*Proc. Int. Sym. Hum. ID (Promega) 1997, p. 34

13. Human Identity Testing with Multiplex STRs
Simultaneous Analysis of 10 STRs and Gender
AmpFlSTR® SGM Plus™ kit
Two different individuals
DNA Size (base pairs)
amelogenin
D19 D3 D8
TH01
VWA
D21
FGA
D16
D18 D2
Results obtained in less than 5 hours with a spot of blood the size of a pinhead
probability of a random match: ~1 in 3 trillion

14. FBI’s CODIS DNA Database
Combined DNA Index System
Launched October 1998
Used for linking serial crimes and unsolved cases with repeat offenders
Links all 50 states
Requires >4 RFLP markers and/or 13 core STR markers
Current backlog of > 600,000 samples
As of June, 2004
Total profiles = 1,857,093
Total forensic profiles = 85,477
Total convicted offender = 1,771,616

16. STR alleles with n+4 stutter products
DNA Size (bp)
D8S1179
D21S11
D18S51
Relative Fluorescence Units
TALKING P0INTS
6.3%
6.2%
5.4%

17. Schematic of Stutter Product Formation Process
Normal STR Allele Replication 1 2 3 4 5 6
Slipped Strand Mispairing Model 1 2 3 4 5
TALKING POINTS
● amplicons that differin size from primary peak by multiples of the core repeat unit (1-4 repeat units).
● the polymerization enzyme-template complex becomes destabilized and potential disassociate when it rejoins, may do so out of register
template loops out
Repeat unit bulges out when strand breathing occurs
Walsh et al (1996) Nucleic Acids Res. 24:

18. Mechanism behind n+4 stutter
DNA polymerase 1 7 2 6 3 5 4 4 5 3 2 6
TALKING POINTS
-slipped strand mispairing model
-step 1 – the DNA polymerase has extended through 4 repeat units (light gray boxes) –
Step 2 - if the polymerase becomes disassociated with the extending strand, the template and the extending strand may break apart.
Step 3 – when the two strands re-anneal, they could possibly re-anneal out of register by one repeat unit.
Step 4 – the final product contains only 6 of the 7 repeat units. 1
Template DNA
Walsh, et al. (1996) Nucleic Acids Research 24(14):2807

20. What is on the horizon ? 1. 14th …….locus 2. Pentanucleotide repeats
Mitochondrial DNA
Single nucleotide polymorphisms (SNPs)