Forensic DNA
Analysis
SFS3f. Compare short tandem repeat
patterns (STR) and relate to identifying
the DNA of an individual.
SFS3g. Explain the use of the DNA
database for DNA profiling.
EQ
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
What is DNA?
How is DNA evidence analyzed by
Criminalist & how has it become
useful to criminal investigations
over the past quarter century?
What is DNA?
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DNA “fingerprinting” is a common
way to ID people by their unique
genetic code.
DNA is a double stranded molecule
located in the nucleus of a cell;
Every cell in an organism contains
the same DNA.
Characteristics of DNA varies btwn
individuals w/in a species & btwn
species.
What is DNA?




DNA- deoxyribonucleic
acid.
Long-chain molecule
made up of four bases
that pair together & held
together by hydrogen
bonds.
A=T; G=C
In humans- order of
bases are 99.9% the
same.
What is DNA?


DNA is contained in
chromosomes
Chromosomes
contain DNA &
associated proteins
(which allow DNA
to
condense/wind/coil
up tightly)
What is DNA?



Genes are
sequences of
DNA nucleotides
Genes are
located on
chromosomes
Alleles: different
types of the same
gene (dominant,
recessive, codominant, etc).
Forensic DNA analysis seeks
nucleotide variation in DNA

1. Point mutations from errors in
DNA
replication


– RFLP (Restriction Fragment
Length Polymorphism) analysis
2. Variable number tandem
repeats (VNTRs)

– STR (Short tandem repeat)
analysis
Why is there nucleotide variation between
the DNA of individuals of the same
species?



All the DNA
nucleotides in a cell
must be copied
(replicated) prior to
cell division.
Random copying
errors are made
during DNA
replication.
DNA replication
copies DNA
nucleotides
 Every human cell
contains 3.4 billion
nucleotide pairs.
DNA replication
Objectives of Forensic
DNA Testing

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To link an individual to a crime
scene/criminal act.
To exonerate suspects.
To identify victims of mass
disasters.
To determine Paternity.
Specimen Samples used
for Forensic DNA
Testing
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Blood (what components?)
Semen
Saliva
Bones
Skin scrapings
Hair follicular tag
Main Procedures for
DNA Fingerprinting:
1.
2.
3.
4.
Isolation of the DNA to separate
the DNA from the cell.
Cutting w/ a restriction enzyme
to make shorter base strands.
Sorting the segments by size,
using electrophoresis
Analyzing the resulting print by
identifying specific alleles/ genes.
DNA Fingerprinting
Analysis:
1.
RFLP (Restriction Fragments
Lengths polymorphism)-use
restriction enzymes to cut the DNA in
fragments that are many diff lengths
and shapes. The length vary from
person to person.

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Restriction Enzyme-recognize a specific
sequence of bases & cut the DNA
molecule at specific points.
Electrophoresis-a procedure that sorts/
separates DNA fragments by size.
Early 1980s: Restriction Fragment
Length Polymorphism (RFLP)


Sir Alec Jeffreys

Genetic variation in the distance
between restriction enzyme sites
Template DNA digested by
enzymes, electrophoresed,
detected via Southern blotting
Power of discrimination in the
range of 106-108 for a six probe
analysis
Mechanisms
for RFLPs
The Catch:
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RFLP testing requires a relatively
large amount of HMW DNA
(~50ng = thousands of cells)
Not ideal for forensic evidence, in
which small, degraded samples
are common
2. PCR To The Rescue!


Dr. Kary Mullis
Eccentric Genius
Polymerase Chain Reaction =
molecular Xeroxing
Three temperature phases, carried
out in a Thermal Cycler, replicate/
copy or “amplify” the desired DNA
fragment(s)
PCR (cont’d)
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Works with lower quantity (1-2ng),
lower quality samples
Power of discrimination goes from
102-106...not good enough for
databasing
3. The Current Method of Choice:
Autosomal Short Tandem Repeats
(STR)
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Non-coding, tetranucleotide
sequences which vary greatly from
person to person in the number of
repeating units
Requires <1ng of DNA to type 1315 STR loci
Power of discrimination ranges
from 1014-1023. World population
is 109 so bring on the database!
Applied Biosystems 310 Genetic Analyzer
The Process In a Nutshell
Amplified DNA samples are injected
into a capillary. Fluorescent tags on
the DNA fragments are excited by a
laser as they pass a window in the
capillary, the fluorescence is recorded
by a camera, and this signal is
converted into a “peak” by the
computer software.
STR data
X, Y,
XY
STR data (cont’d)
STR data (cont’d)
STR TYPING SUMMARY SHEET
Date:
DNA Analyst / Serial #:
9/24/1999
MATTHIES
Item #
25(S)
25(E)
VICTIM
SUSPECT
AMEL
D3S1358
X, Y
17
X, Y
17
X
15, 17
X
15, 17
X
15, 17
X
15, 17
X, Y
17
X, Y
17
vWA
FGA
DR #:
V9780
D8S1179
15, 17 23, 26 14, 15
00-00-00001
D21S11
D18S51
D5S818
D13S317
26
12, 15
10
9, 13
D7S820
8, 10
D16S539
THO1
TPOX
9, 10
8, 9
9, 10
11, 12
7, 8
11
11, 12
7, 8
11
9, 10
8, 9
9, 10
8, 10
16, 18 19, 26
16, 18 19, 26
15
15
15, 17 23, 26 14, 15
28,
32.2
14, 16
28,
32.2
14, 16
26
12, 15
8, 13
12
11
11
8, 13
12
11
11
10
9, 13
8, 10
8, 10
“The DNA profile obtained from Item 25(S) matches the
DNA profile of the suspect. The combination of genetic
marker types exhibited by Item 25(S) and the suspect
occurs in approximately one in one hundred quadrillion
(1017) individuals…”
How are these astronomical figures derived?
The product rule: combined probability of a series of independent
events is determined by multiplying the probabilities of each event.
STR loci are inherited independently (unlinked)
Homozygous loci: p2 (same allele inherited from mother and father)
Heterozygous loci: 2pq (either allele could be inherited from either
parent)
p(17)2 x 2p(15)q(17) x 2p(23)q(26)….
(.223)2 x 2(.083)(.25) x 2(.14)(.02) = .000013, which is equivalent to a
probability of one in 76,000 using just 3 of the 13 loci!
STR Artifacts
-A (“minus A”): Incomplete addition of
nucleotide ‘A’ by DNA polymerase;
results in a peak that is one base pair
smaller than allele peak.
STR Artifacts
Stutter: Slippage of DNA polymerase;
results in a peak that is four base pairs
(one repeat unit) smaller than allele peak.
STR Artifacts
Pull-up: Incomplete filtration of spectral
overlap in fluorescent detection system.
Pull-up
DNA Mixtures
When more than one source of DNA is detected in a sample,
assignment of genotypes becomes more difficult.
Degraded/Trace DNA Samples
Larger alleles “drop-out” when template DNA is low in
quantity or quality, reducing certainty of genotypes.
The Combined DNA Index System
(CoDIS)
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A database of DNA profiles from
violent felons and crime scene
samples
Laws concerning who is eligible for
the database vary from state to
state
Database currently contains about
2,038,470 felons and 93,956 crime
scene profiles (19,00 hits so far)
The Mystical Power of CoDIS
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Extremely powerful investigative
tool, linking crimes, and pulling
suspects out of thin air!
Can prevent, as well as solve
crimes!
The Dark Side of CoDIS
(What the FBI doesn’t want you to know.)
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DNA mixtures and degraded DNA
profiles have lead to spurious
matches
Stringent laws explicitly permit
databasing innocent people
Adding arrestees to database
violates presumption of innocence
However, the prosecution rate on
case to offender matches is
shockingly low! (~10%)
PCR
1.
2.
3.
4.
5.
6.
7.
Isolate the DNA from crime scene evidence to
get a pure sample and cut target strands.
Mix the DNA samples, primer, DNA polymerase
enzyme & nucleotides in tube. Place in PCR
chamber.
Heat the dsDNA to separate to 95°C.
Cool to 50°C so that primers can attach to
ssDNA.
Reheat to 72 °C so that DNA polymerase can
attach to end of primer.
The DNA polymerase will now add
complementary nucleotides to the ssDNA. Two
new strands are made.
Repeat cycle until desired # of DNA is copied.
Electrophoresis
1.
2.
3.
4.
5.
6.
7.
8.
Isolate pure sample & cut the DNA into
fragments.
Make agarose gel.
Add DNA with a buffer solution to wells in gel.
Place the gel in the electrophoresis plate so
that a current passes through.
The DNA will migrate from one end to the next
based on size.
Stain the gel with Ethidium Bromide when the
electrophoresis is done.
Place under UV light to observe band.
Analyze/ compare bands for size comparison.
REview
Take this scenario:
A woman gets out of
bed, watches TV, talks
on the phone, pets her
cat, and then shoots her
husband. She hides the
gun and runs away.
Which object would be
most useful to the
forensic serologist in
finding out who the
woman was?
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A. the cat
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B. the phone
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C. the TV
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D. the TV remote
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E. none listed
What system used by the FBI
compiles DNA of known
violent offenders from all
over the nation and can be
used to match DNA with a
sample found at a crime
scene?

A. IAFIS
B. IBIN
C. SICAR
D. CODIS
E. DNAW
Review
Review: Who’s DNA is the
unknown?
Suspect A Unknown Suspect B
Review
In this gel, the base-pair
lengths are listed to
the right. Which end
of the DNA fingerprint
was plugged to the
NEGATIVE terminal
during
electrophoresis?
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A. Top
B. Bottom
C. Left
D. Right
Review
In a talk-show scandal, it
is revealed that one of
three siblings has a
different father.

GASP! Using the DNA
fingerprint of just the
mother, daughter and
two sons, which
sibling has a
DIFFERENT father.
A. Daughter
B. Son 1
C. Son 2
D. all have the same
father—go figure!
Review
Which suspect's
DNA matches
the felon's DNA
left at the crime
scene?

A. suspect 1
B. suspect 2
C. suspect 3
D. none listed
Review
Whose the daddy? This
mother is trying to
decide between two
men who desperately
want to support her
and her newborn baby.
Both want to be a part
of the baby's life,
because they love the
mother so much. Who
gets the honor and
privilege?
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A. Dad 1
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B. Dad 2
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C. neither man
D. both
REview
Compare the sperm
DNA to the two
suspects and the
boyfriend. Which
male is most
likely the
assailant?
A. suspect 1
B. suspect 2
C. boyfriend
D. none listed
Review
In this DNA fingerprint,
the blood sample
taken from a crime
scene is not the
victim's and so is
assumed to be the
perpetrator. Which
individual is the best
suspect?
A. Bob
B. Sue
C. John
D. Lisa
E. none
Review
There was a mix-up
at the hospital.
Which of these
children belong
to the parents?

A. All of the
children

B. Children 2, 3
&6
C.
Children 1 & 5
D. Children 2 & 4