Forensic DNA

Analysis
Summary
What is DNA?
Where is DNA found in the body?

How does DNA differ among individuals?

Forensic DNA Analysis
DNA and Statistics
What is DNA?
What is DNA?

What does DNA stand for?

Deoxyribose Nucleic Acid or Deoxyribonucleic Acid

What does DNA do?

DNA contains genetic information.
DNA codes for the proteins our
bodies make that are necessary for
survival.
What is DNA?
DNA is a code for making proteins

AGC TAG CTT ATA CTC TAT CTC TTT

Amino Amino Amino Amino Amino Amino

Acid Acid Acid Acid Acid Acid

The order of amino acids determines

what type of protein is made.
What is DNA?
Some common proteins are:
Hemoglobin - carries oxygen from lungs to cells
Insulin - regulates metabolism
Many types of enzymes - catalyze reactions in the
body, such as the breakdown of sugar for energy

DNA also determines how much of these

proteins each cell makes.
What is DNA?
What does DNA look like?

Double Helix
Like a Twisted Ladder
What is DNA?
What does DNA look like?
Sugar Phosphate
Backbone
(Sides of Ladder)

Nitrogenous
Base
(Rungs of Ladder)
What is DNA?
The DNA ladder is made up of
building blocks called nucleotides.
What is a nucleotide?
Adenine
Phosphate Group Cytosine
Base Guanine
Thymine
Deoxyribose sugar
The 4 Bases

A C
Adenine Cytosine

G T
Guanine Thymine
The 4 Bases

A C

G T
The 4 Bases
The bases pair up
to form the rungs
of the ladder.

A pairs with T

G pairs with C
What is DNA?
DNA is written as the sequence of these
bases:
AAGTCGATCGATCATCGATCATACGT

Only one side of the ladder is written.

In humans, there are three billion (3,000,000,000)
base pairs (letters) in the DNA within each cell.
What is DNA?
Among humans, most of the 3 billion bases in
the DNA sequence are exactly the same.
Our Human DNA is 99.8% similar to each
other, but the 0.2% difference is more than
enough to distinguish us from one another.
Human DNA is even 98% similar to
chimpanzees.
NO TWO PEOPLE HAVE IDENTICAL DNA*
*except identical twins
What is DNA?
Stupid Facts:
If two different people started reciting their
individual genetic code at a rate of one letter per
second, it would take almost eight and a half
minutes before they reached a difference.
If unwound and tied together, the strands of DNA
in one cell would stretch almost six feet but would
be only 50 trillionths of an inch wide.
If all the DNA in your body was put end to end, it
would reach to the sun and back over 600 times
(100 trillion times six feet divided by 92 million
miles).
Where is DNA?
Where is DNA?
DNA is found in the cells in our body.

Nucleus
(Brain of the cell)

Mitochondria
(more later)
Where is DNA?
All types of cells in our body contain a
copy of the same DNA.
Some cells important to forensic science are:

White Blood Cell Sperm Cell Cheek Cell

Where is DNA?
DNA in the nucleus is packaged into Chromosomes
Where is DNA?
Chromosomes
come in pairs

(one from Mother)

(one from Father)

There are 46
chromosomes in
each cell.
(23 pairs)
Where is DNA?
What are sources of DNA at a crime scene?
DNA can be recovered from any
substance that contains cells.
Blood Bone
Semen Teeth
Saliva Hair
Tissue Maggot Crops
Maggot Crop
How does DNA differ
among Humans?
How does DNA differ among humans?

DNA is a sequence of 4 possible letters

A G C T

Of the 3 billion letters, 99.8% of the

sequence in all humans is identical.

There are several ways the sequence can be different.

How does DNA differ among individuals?

1. One of the bases (letters) can be different.

Person 1 AGCTAGATCGTTATTCCGAG
Person 2 AGCTAGATCGTCATTCCGAG
How does DNA differ among individuals?

2. Bases (letters) can be added or removed.

Person 1 AGCTAGATCGTTATTCCGAG
Person 2 AGCTAGATCGTATTCCGAG
Person 3 AGCTAGATCGTTTATTCCGAG
Person 4 AGCTCCGAG
How does DNA differ among individuals?

2. Bases (letters) can be added or removed.

Person 1 AGCTAGATCGTTATTCCGAG
Person 2 AGCTAGATCGTATTCCGAG
Person 3 AGCTAGATCGTTTATTCCGAG
Person 4 AGCTCCGAG
 How does DNA differ among individuals?

3. Regions of DNA can be repeated a different # of times

Person 1 ..GCCAGCTAGCTAGCTAGCTAGCTAGCTTTCAT..
 How does DNA differ among individuals?

3. Regions of DNA can be repeated a different # of times

Person 1 ..GCCAGCTAGCTAGCTAGCTAGCTAGCTTTCAT..
1 2 3 4 5

Person 2 ..GCCAGCTAGCTAGCTAGCTAGCTTTCAT..
1 2 3 4 5
Person 3 ..GCCAGCTAGCTAGCTAGCTAGCTAGCTAGCTT..
1 2 3 4 5
Forensic DNA
Analysis
Forensic DNA Analysis
Collection of Evidence
Types of Unknown Samples:
Blood, Semen, Stains, Saliva
Hair, Tissue, Bones, Teeth

Types of Known Samples:

Blood or buccal swabs from suspect
or victim or other known person
 Forensic DNA Analysis
Beware of Contamination
Contamination occurs when DNA from another source gets
mixed in with the sample being collected.
An investigator touches, sneezes, bleeds on a sample.
Wear gloves and use disposable instruments
Package items separately.
Especially, do not mix known samples (from victim or
suspect) with unknown samples.
Forensic DNA Analysis
Packaging Evidence
Package each item individually.
Put evidence into paper bags, not plastic.
Moisture degrades DNA; air dry samples.
Keep samples at room temperature and out
of sun.
 Forensic DNA Analysis
Brief History of DNA- (1985)

Multilocus
RFLP

Detects VNTRs:
Variable Number of
Tandem Repeats
Forensic DNA Analysis
Brief History of DNA(Late 80s, Early 90s)

Single locus RFLP

D2S44 probe

Lanes 6 and 10 match

Lanes 8 and 11 match

Forensic DNA Analysis
Brief History of DNA(Early 90s)
PCR Strips (DQ alpha)

6 Alleles:
1.1, 1.2, 1.3
2, 3 or 4
A person can
have one or
two of these
numbers.
Forensic DNA Analysis
Two main types of analyses (90s - Present) :
Short Tandem Repeats (STRs)
Individual identification possible
Samples: Blood stains, semen

Mitochondrial DNA
Used in cases of severely degraded DNA
Individual identification not possible
Samples: Bones, hair shafts
Forensic DNA Analysis

Short Tandem Repeats (STRs)

Currently the most used of all forensic markers
Individual identification possible
Type of data used in the FBI CODIS database
People differ in length at these loci
Are located in the nuclear DNA (chromosomes)
Forensic DNA Analysis
Short Tandem Repeats (STRs)

Regions of DNA can be repeated a different # of times

Person 1 ..GCCAGCTAGCTAGCTAGCTAGCTAGCTTTCAT..
1 2 3 4 5

Person 2 ..GCCAGCTAGCTAGCTAGCTAGCTTTCAT..
1 2 3 4 5
Person 3 ..GCCAGCTAGCTAGCTAGCTAGCTAGCTAGCTT..
1 2 3 4 5
Forensic DNA Analysis
Short Tandem Repeats (STRs)

Locus or Loci:
Refers to the location on the chromosome.

Allele:
Refers to the type of DNA.
For STRs, the allele will be the number of repeats.

CCAGATAGATAGATAGATAGATAGATAGATAGATAGATCC
Forensic DNA Analysis
Example
Locus: D5S818

Alleles: 7,9

Paternal chromosome 5
CCAGATAGATAGATAGATAGATAGATAGATCC
Maternal chromosome 5
CCAGATAGATAGATAGATAGATAGATAGATAGATAGATCC
Forensic DNA Analysis
13 loci used in CODIS
STR Marker Chromosome Repeat Sequence Repeat units Other Alleles
TPOX 2 AATG 6 - 14
CSF1PO 5 AGAT 6 - 15 10 .3
D5S81 8 5 AGAT 7 -15
D7S82 0 7 GATA 6 - 14
D8S11 79 8 TATC 8 - 19
D13S3 17 13 TATC 7 - 15
D16S5 39 16 GATA 5, 8 - 15

D3S13 58 3 TCTA* 9, 11- 20 15 .2, 16.2

FGA 4 CTTT* 15 - 30 16 .2 -3 0.2
2 2.3, 34.2, 4 6.2
TH01 11 AATG* 3, 5 - 12 8.3, 9 .3, 10.3 , 13 .3
VWA 12 TCTA* 11 - 22 15 .2
D18S5 1 18 AGAA* 8 - 27 13 .2, 14.2 , 15 .2
17 .2, 19.2
D21S1 1 21 TCTA* 24 - 38 24 .2 - 35.2
Forensic DNA Analysis
Basic Steps in Analysis
Extraction:
Separates DNA from sample
Amplification or PCR:
Amplifies small portions of DNA (STR regions)
Separation:
Separates amplified fragments according to size.
PCR Hood
Forensic DNA Analysis
Basic Steps in Analysis
Extraction:
Separates DNA from sample
Amplification or PCR:
Amplifies small portions of DNA (STR regions)
Separation:
Separates amplified fragments according to size.
The Thermal Cycler

Amplifies DNA
Forensic DNA Analysis
Basic Steps in Analysis
Extraction:
Separates DNA from sample
Amplification or PCR:
Amplifies small portions of DNA (STR regions)
Separation:
Separates amplified fragments according to size.
FMBio

Separates Amplified DNA

Forensic DNA Analysis

Color image
of gel
Forensic DNA Analysis
Gel Electrophoresis

Black and white

image of STR gel.
Samples will have
one or two bands
at each loci.
ABI 310 Genetic Analyzer

Separates Amplified DNA

Forensic DNA Analysis
Capillary Electrophoresis
Sample will have one or two peaks at each loci.
Forensic DNA Analysis
Compare to a ladder that has all peaks at each loci.
Forensic DNA Analysis
Forensic DNA Analysis

TPOX CSF1PO D5S818 D8S1179

Blood stain 7,9 10,13 7,15 8,8

Suspect 1 8,9 10,10 9,10 11,12
Suspect 2 10,11 9,13 8,14 9,12
Suspect 3 7,9 10,13 7,15 8,8
Forensic DNA (mitochondria)
Mitochondria - The powerhouse of the cell.

Mitochondria have
Mitochondria their own DNA
Forensic DNA (mitochondria)
Mitochondrial DNA

Double Helix Chromosomes Ring of DNA

YES NO YES
Forensic DNA (mitochondria)
Mitochondrial DNA

Mitochondrial DNA is
only 16,569 letters long.
(compared to 3 billion
in nuclear DNA)

There is a 900 base pair

region with a 1.7%
difference (D loop).
Forensic DNA (mitochondria)
Nuclear DNA vs. Mitochondrial DNA
Double Helix Double Helix
46 Chromosomes One Ring
Multiple copies in
One copy per cell each mitochondria
Multiple mitochondria in
each cell

MtDNA used for old or degraded samples

Forensic DNA (Mitochondria)
For nuclear DNA: Length is measured
For mtDNA: Sequence is examined

Different colored peaks correspond to a different base

Forensic DNA Analysis
Basic Steps in Analysis
Extraction:
Separates DNA from sample
Amplification or PCR:
Amplifies small portion of mtDNA (D loop)
Sequencing:
Sequence is determined by another reaction
and separation of sequenced fragments
Forensic DNA (Mitochondria)

DNA Sequences are compared to each other.

Hair found on
Suspect
AGCTAGATCGTTATTCCGAG

Victim AGCTAGATCGTTATTCCGAG

Conclusion: Hair may have come from the victim.

Forensic DNA (Mitochondria)

DNA Sequences are compared to each other.

Hair found on
Suspect
AGCTAGATTGTTATTCCGAG

Victim AGCTAGATCGTTATTCCGAG

Conclusion: Hair did not come from the victim

 Forensic DNA (Mitochondria)

Cigarette butt
at crime scene
AGCTAGATTGTTATTCCGAG
Suspect #1 AGCTAGATCGTTATTCCGAG
Suspect #2 AGCTAGATTGTTATTCCGAG
Suspect #3 AGCTTGATTGTTATTCCGAG
Suspect #4 AGCTAGATTGTTATTCCGAG
Conclusion: Cigarette could be from Suspects #2, #4
or other person with the same sequence.
DNA and Statistics
The final result is presented as a statistic.
Do not say:
The DNA in the bloodstain is John Does DNA.

Do Say:
The chance that another person has this DNA
in the bloodstain is 1 in 300 billion.
DNA and Statistics
Where do the statistics come from?

First, the frequency of each allele is estimated

using data from a population data base.
Allele frequency
Locus: D5S818 from database
7 26%
Alleles: 7,9
9 11%
DNA and Statistics
Where do the statistics come from?

Next, the frequency of the genotype at

each locus is calculated.

Locus: D5S818 Genotype frequency

7,9 6%
Alleles: 7,9
DNA and Statistics
For total frequency, multiply all of the
frequencies together.
D5 = 6%
D8 = 12%
D18 = 0.5%
Total = 0.004%
 CSF1PO TPOX
7 0.00 6 0.00
8 0.00 7 0.00
9 0.03 8 0.53
10 0.25 9 0.09
11 0.31 10 0.06
12 0.33 11 0.28
13 0.06 12 0.04
14 0.01 13 0.00
15 0.00

TH01 vWA
5 0.01 13 0.00
6 0.24 14 0.13
7 0.15 15 0.08
8 0.12 16 0.21
9 0.16 17 0.27
9.3 0.33 18 0.20
10 0.01 19 0.09
11 0.00 20 0.02
21 0.00
 D16S5 39 D782 0
7 0.00 6 0.00
8 0.03 7 0.01
9 0.11 8 0.16
10 0.08 9 0.15
11 0.32 10 0.30
12 0.27 11 0.20
13 0.17 12 0.12
14 0.03 13 0.06
15 0.00 14 0.01

D13S3 17 D5S81 8
7 0.00 7 0.00
8 0.14 8 0.00
9 0.05 9 0.01
10 0.05 10 0.06
11 0.31 11 0.37
12 0.31 12 0.35
13 0.08 13 0.19
14 0.06 14 0.01
15 0.00 15 0.00
Demonstration
Calculating Frequencies