Forensic DNA Typing

or
Did you kill (rape) that person?
How DNA can definitively say.
Adapted from:

National Institutes of Science & Technology

http://www.cstl.nist.gov/div831/strbase/intro.htm

Brief History of Forensic DNA Typing

1980 - Ray White describes first polymorphic RFLP marker (Restriction
Fragment Length Polymorphism [alleles]).

Different RFLP for different people

1985 - Alec Jeffreys discovers multilocus VNTR (variable number of
tandem repeats) probes.
1985 - first paper on PCR
1988 - FBI starts DNA casework
1991 - first STR paper (renaming of VNTR could be larger repeats, STR 4-6 bps.
now using mostly 4 bases )

1995 - FSS (Forensic Science Service-UK) starts UK DNA database

1998 - FBI launches CODIS (Combined DNA Information Service)
Now FBI use 13 loci: PCR identifies it: in the quadrillions except
for identical. Except for police mistakes, its done deal.

RFLPs: Sickle Cell hemoglobin

Case 1: Screening for the sickle-cell gene
Sickle cell disease is a genetic disorder in which both genes in the patient encode
the amino acid valine (Val) in the sixth position of the beta chain (beta S) of the
hemoglobin molecule. "Normal" beta chains (betaA) have glutamic acid at this
position.
The only difference between the two genes is the substitution of a T for an A in
the middle position of codon 6.
This
converts a GAG codon (for Glu) to a GTG codon for Val and
abolishes a sequence (CTGAGG, which spans codons 5, 6, and 7) recognized
and cut by one of the restriction enzymes.

Brief History of Forensic DNA Typing

1980 - Ray White describes first polymorphic RFLP (Restriction Fragment Length
Polymorphism) markerdetect to transferring to membrane. Probe w
southern blot (radiological). Diff. RFLP for dif. People. Single rflp
1985 - Alec Jeffreys discovers multilocus VNTR (variable number
of tandem repeats) probes (stat. very impressive identical 4-6 bp
that are spec. 7 and 9 repeat, one from mom and dad, on chrom. 1nowadays use pcr- but flanking sequence that is unique to
chromo1)). Jeffreys almost ident. Typing. Now use PCR.
1985 - first paper on PCR (Kerry Mullis)
1988 - FBI starts DNA casework
1991 - first STR paper ( renaming of VNTR could be larger
repeats, STR 4-6 bps. now using mostly 4 bases )
1995 - FSS (Forensic Science Service-UK) starts UK DNA
database
1998 - FBI launches CODIS (Combined DNA Information
Service) database. Now FBI use 13 loci: PCR identifies it: in the
quadrilians except for identical.

DNA Use in Forensic Cases

Most are rape cases (>2 out of 3)
Looking for match between evidence
and suspect
Must compare victims DNA profile
Challenges
Mixtures must be resolved
DNA is often degraded (stored wet- have mold, nuclease)
Inhibitors to PCR are often present

Human Identity Testing

Forensic cases -- matching suspect with evidence

Paternity testing -- identifying father
Historical investigations
Missing persons investigations
Mass disasters -- putting pieces back together
Military DNA dog tag
Convicted felon DNA databases

Steps in DNA Sample Processing

Sample Obtained from
Crime Scene or Paternity
Investigation

Biology
DNA
DNA
Quantization
Quantization

DNA
DNA
Extraction
Extraction

PCR
PCRAmplification
Amplification
of
Multiple
of MultipleSTR
STRmarkers
markers

Technology
Separation and Detection of
PCR Products
(STR Alleles)

Comparison of Sample
Comparison of Sample
Genotype to Other
Genotype to Other
Sample Results
Sample Results

Genetics

Sample Genotype
Determination

Generation of Case
Generation of Case
Report with Probability
Report with Probability
of Random Match
of Random Match

If match occurs, comparison

If match occurs, comparison
of DNA profile to population
of DNA profile to population
databases
databases

Sources of Biological Evidence

Blood
Semen
Saliva
Urine
Hair
Teeth (useful in fires).
Bone (there are cells. Decalcify it.
100,000 year old- has DNA. Has
Dinosaur!)

Tissue
All felony arrests- cheek swab.

DNA in the Cell

chromosome
cell nucleus
Double stranded
DNA molecule

Target Region for PCR

Individual
nucleotides

DNA Amplification with the

Polymerase Chain Reaction (PCR)
5

3
3

5
5

Starting DNA
Template

Separate
strands
(denature)
Forward primer

3
Make copies
Add
primers
(extend
primers)
(anneal)
5

Reverse primer

PCR (Polymerase Chain Reaction) Copies DNA

Exponentially through Multiple Thermal Cycles
Original DNA
target region

1 copy

Heat
Oligos
DNA Poly.
dUTP

Cool

Heat
Cool

2 copies

4 copies

Heat

In
In32
32cycles
cyclesat
at100%
100%efficiency,
efficiency,1.07
1.07billion
billion
copies
copiesof
oftargeted
targetedDNA
DNAregion
regionare
arecreated
created

Short Tandem Repeats (STRs)

(say chromo 3)
Identical in all people

AATG

7 repeats

Identical in all people

8 repeats
the repeat region is variable between samples while the
flanking regions where PCR primers bind are constant
Homozygote = both alleles are the same length
Heterozygote = alleles differ and can be resolved from one another

Diff. PCR primers sets, can amplify the same region. Different companies sell different kits.

195
195bp
bp

170
170bp
bp

TCAT
TCATrepeat
repeatunit
unit

Different primer sets produce different PCR product sizes for the
same STR allele

Variation Among STRs

Choosing which STRs:
Significant statistical variation but not too
many. Freq. that are measured in pop. : Loc 1
-10%. Loc 2 10%; locus 1+2 -1/100. Random
match with 13 primers 1/1013.

Multiplex PCR
Over 10 Markers Can Be
Copied at Once
Sensitivities to levels less than
1 ng of DNA
Ability to Handle Mixtures
and Degraded Samples
Different Fluorescent Dyes
Used to Distinguish STR
Alleles with Overlapping Size
Ranges
Most rxns: require 2 PCR (tubes) 7 or 8
primer pairs in one tube need total of
about 2 tubes for 13 different STRs.
$20-$25 per rxn in lab. $150 incl
labor. Cost for forensic up to $1000.

An Example Forensic STR Multiplex Kit

AmpFlSTR Profiler Plus
Kit available from PE Biosystems (Foster City, CA)
200 bp

Color Separation

100 bp

Size Separation

D3
A

vWA
D8

D5

FGA

300 bp

400 bp

5-FAM (blue)

D21

D18

JOE (green)

D13

D7

NED (yellow)
ROX (red)

GS500-internal lane standard

9 STRs amplified along with sex-typing marker amelogenin in a single PCR reaction

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

ABI Prism 310 Genetic Analyzer

5 min from inj.
to output.

capillary

Syringe with
polymer solution
Injection
electrode
Outlet
buffer

Autosampler
tray

Inlet
buffer

Close-up of ABI Prism 310 Sample Loading Area

Electrode
Capillary
Sample Vials

Autosampler Tray
See Technology section for more information on CE

D tells chromosome 21happens to be downs syndrome. 2 peaks cause heterozygotic

Human Identity Testing with Multiplex STRs

Two different individuals

Amelogenin amel protein that happens to be on sex chromosome, tooth

enamel top: 2 peaks: x and y. (universal that two diff. people.) Bottom
only 1 peak cause they have two X chromosomes.

amelogenin
D19

D3

AmpFlSTR SGM Plus kit

DNA Size (base pairs)

D8 TH01
VWA D21

D16
D18

D2

FGA

probability of a random
match: ~1 in 3 trillion
amelogenin D3
D19
D8

VWA
TH01

Results obtained in less than 5

hours with a spot of blood the
size of a pinhead
D16

D21
FGA

D18

Simultaneous Analysis of 10 STRs and Gender ID

D2

STR Allele Frequencies

45
40

TH01 Marker

Frequency

35
30

Caucasians (N=427)
Blacks (N=414)
Hispanics (N=414)

25
20
15
10
5

*Proc. Int. Sym. Hum. ID

(Promega) 1997, p. 34

9.3

Number of repeats

10

FBIs CODIS DNA Database

Combined DNA Index System --all 50 states can upload
their convicted felony and seq. of unsolved cases. In Florida to convicted felon.

Used for linking serial crimes and unsolved

cases with repeat offenders
Launched October 1998
Links all 50 states
Requires >4 RFLP markers
and/or 13 core STR markers
Current backlog of >600,000 samples

13 CODIS Core STR Loci

with Chromosomal Positions
TPOX
D3S1358
D8S1179

D5S818
FGA
CSF1PO

TH01
VWA

D7S820

AMEL
D13S317

D16S539

D18S51

D21S11

AMEL

The End