The prospect for more than 13 commonly used STR Tetranucleodide loci in forensic

settings

Carrie Rowland

Forensic Bioinformatic Services, Inc.

Dayton, Ohio

www.bioforensics.com

An Overview: Where are we going

• History of DNA testing• Generations of DNA tests and how performed• Current methodology• New prospects• Conclusions

Six criteria for judging a DNA profiling system

1. Discrimination power.2. Sensitivity.3. Absence of, or the ability to reconcile technical artifacts.4. Speed.5. Ability to deconvolve mixtures.

6. Amenability to database searches.

Brief History of Forensic DNA Typing

• 1980 - Ray White describes first polymorphic RFLP marker

• 1985 - Alec Jeffreys proposes multilocus VNTR probes be used forensically

• 1985 - first paper on PCR• 1988 - FBI starts DNA casework• 1991 - first STR paper• 1995 - FSS starts UK DNA database• 1998 - FBI launches CODIS database

Three generations of DNA testing

DQ-alphaTEST STRIPAllele = BLUE DOT

RFLPAUTORADAllele = BAND

Automated STRELECTROPHEROGRAMAllele = PEAK

How do they fare?

□ Discriminating power

Sensitivity

Technical artifacts

Speed□ Mixtures

□ Databasing

Discriminating power □ Sensitivity□ Technical artifacts□ Speed□ MixturesDatabasing ٱ

RFLP DQ-alpha STR

Discriminating power

Sensitivity□ Technical artifacts

Speed□ Mixtures

Databasing

RFLP technology

What we would like for them to look like.

What they often look like.

Incomplete digest

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

An Example Forensic STR Multiplex Kit

D3 FGAvWA 5-FAM (blue)

D13D5 D7 NED (yellow)

A D8 D21 D18 JOE (green)

GS500-internal lane standard

ROX (red)

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 bp300 bp200 bpSize Separation

Col

or S

epar

atio

n

13 CODIS Core STR Loci with Chromosomal Positions

CSF1PO

D5S818

D21S11

TH01

TPOX

D13S317

D7S820

D16S539 D18S51

D8S1179

D3S1358

FGA

VWA

AMEL

AMEL

STR Allele Frequencies

0

5

10

15

20

25

30

35

40

45

6 7 8 9 9.3 10

Caucasians (N=427)

Blacks (N=414)

Hispanics (N=414)

TH01 Marker

*Proc. Int. Sym. Hum. ID (Promega) 1997, p. 34

Number of repeats

Fre

qu

ency

amelogenin

D19

D3

D8

TH01

VWA D21FGA

D16D18 D2

amelogeninD19

D3D8 TH01

VWA D21

FGA

D16D18 D2

Tw

o di

ffer

ent i

ndiv

idua

ls

DNA Size (base pairs)

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

Human Identity Testing with Multiplex STRs

Simultaneous Analysis of 10 STRs and Gender

AmpFlSTR® SGM Plus™ kit

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

http://www.fbi.gov/hq/lab/codis/aidedmap.htm

STR alleles with n+4 stutter products

D21S11 D18S51

D8S1179

DNA Size (bp)

6.3% 6.2% 5.4%

Rel

ativ

e F

luor

esce

nce

Uni

ts

Schematic of Stutter Product Formation Process

Walsh et al (1996) Nucleic Acids Res. 24: 2807-2812

Slipped Strand Mispairing Model

1 2 3 4 5

1 2 3 4 5 6

Normal STR Allele Replication

Repeat unit bulges out when strand breathing occurs

Mechanism behind n+4 stutter

Walsh, et al. (1996) Nucleic Acids Research 24(14):2807

DNA polymerase

Template DNA

12345

61

2

3456

7

What is on the horizon ?

1. 14th …….locus

2. Pentanucleotide repeats

3. Mitochondrial DNA

4. Single nucleotide polymorphisms (SNPs)