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?
What does DNA do?• DNA contains genetic information.• DNA codes for the proteins our bodies
make that are necessary for survival.
Deoxyribose Nucleic Acid or Deoxyribonucleic Acid
What is DNA?
DNA is a code for making proteins
AGC TAG CTT ATA CTC TAT CTC TTT
AminoAcid
AminoAcid
AminoAcid
AminoAcid
AminoAcid
AminoAcid
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)
NitrogenousBase
(Rungs of Ladder)
What is DNA?The DNA ladder is made up of building blocks called nucleotides.
What is a nucleotide?
Phosphate Group
Deoxyribose sugar
Base
AdenineCytosineGuanineThymine
The 4 Bases
AAdenine
GGuanine
CCytosine
TThymine
The 4 Bases
G
C
T
A
The 4 Bases
A pairs with T
G pairs with C
The bases pair up to form the rungs of the ladder.
What is DNA?
DNA is written as the sequence of these bases:
AAGTCGATCGATCATCGATCATACGT
• In humans, there are three billion (3,000,000,000) base pairs (letters) in the DNA within each cell.
• Only one side of the ladder is written.
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?
(one from Mother)
(one from Father)
Chromosomes
come in pairs
There are 46 chromosomes in
each cell. (23 pairs)
Where is DNA?
What are sources of DNA at a crime scene?
• Blood• Semen• Saliva• Tissue
• Bone• Teeth• Hair• Maggot Crops
DNA can be recovered from any substance that contains cells.
Maggot Crop
How does DNA differ among Humans?
How does DNA differ among humans?
DNA is a sequence of 4 possible letters
GA 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 2 AGCTAGATCGTCATTCCGAGPerson 1 AGCTAGATCGTTATTCCGAG
How does DNA differ among individuals?
2. Bases (letters) can be added or removed.
Person 1 AGCTAGATCGTTATTCCGAGPerson 2 AGCTAGATCGTATTCCGAGPerson 3 AGCTAGATCGTTTATTCCGAGPerson 4 AGCTCCGAG
How does DNA differ among individuals?
2. Bases (letters) can be added or removed.
Person 1 AGCTAGATCGTTATTCCGAGPerson 2 AGCTAGATCGTATTCCGAGPerson 3 AGCTAGATCGTTTATTCCGAGPerson 4 AGCTCCGAG
How does DNA differ among individuals?
Person 1 ..GCCAGCTAGCTAGCTAGCTAGCTAGCTTTCAT..
3. Regions of DNA can be repeated a different # of times
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 6
Person 2 ..GCCAGCTAGCTAGCTAGCTAGCTTTCAT..
Person 3 ..GCCAGCTAGCTAGCTAGCTAGCTAGCTAGCTT..
1 2 3 4 5
1 2 3 4 5 6 7
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, 1.32, 3 or 4
A person canhave one ortwo of these
numbers.
Forensic DNA Analysis
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
Two main types of analyses (90s - Present) :
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..
Person 2 ..GCCAGCTAGCTAGCTAGCTAGCTTTCAT..
Person 3 ..GCCAGCTAGCTAGCTAGCTAGCTAGCTAGCTT..
1 2 3 4 5 6
1 2 3 4 5
1 2 3 4 5 6 7
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
Locus: D5S818
Alleles: 7,9
CCAGATAGATAGATAGATAGATAGATAGATCC
Paternal chromosome 5
Maternal chromosome 5
CCAGATAGATAGATAGATAGATAGATAGATAGATAGATCC
Example
Forensic DNA Analysis
STR Marker Chromosome Repeat Sequence Repeat units Other AllelesTPOX 2 AATG 6 - 14
CSF1PO 5 AGAT 6 - 15 10.3D5S818 5 AGAT 7 -15D7S820 7 GATA 6 - 14
D8S1179 8 TATC 8 - 19D13S317 13 TATC 7 - 15D16S539 16 GATA 5, 8 - 15
D3S1358 3 TCTA* 9, 11- 20 15.2, 16.2FGA 4 CTTT* 15 - 30 16.2 -30.2
22.3, 34.2, 46.2TH01 11 AATG* 3, 5 - 12 8.3, 9.3, 10.3, 13.3VWA 12 TCTA* 11 - 22 15.2
D18S51 18 AGAA* 8 - 27 13.2, 14.2, 15.217.2, 19.2
D21S11 21 TCTA* 24 - 38 24.2 - 35.2
13 loci used in CODIS
Forensic DNA Analysis
Basic Steps in Analysis
Extraction:• Separates DNA from sample
Separation:• Separates amplified fragments according to size.
Amplification or PCR:• Amplifies small portions of DNA (STR regions)
PCR Hood
Forensic DNA Analysis
Basic Steps in Analysis
Extraction:• Separates DNA from sample
Separation:• Separates amplified fragments according to size.
Amplification or PCR:• Amplifies small portions of DNA (STR regions)
The Thermal Cycler
Amplifies DNA
Forensic DNA Analysis
Basic Steps in Analysis
Extraction:• Separates DNA from sample
Separation:• Separates amplified fragments according to size.
Amplification or PCR:• Amplifies small portions of DNA (STR regions)
FMBio
Separates Amplified DNA
Forensic DNA Analysis
Color image of gel
Forensic DNA Analysis
Black and white image of STR
gel.Samples will have one or two bands
at each loci.
Gel Electrophoresis
ABI 310 Genetic Analyzer
Separates Amplified DNA
Forensic DNA Analysis
Sample will have one or two peaks at each loci.
Capillary Electrophoresis
Forensic DNA Analysis
Compare to a ladder that has all peaks at each loci.
Forensic DNA Analysis
Forensic DNA Analysis
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
TPOX CSF1PO D5S818 D8S1179
Forensic DNA (mitochondria)
Mitochondria - The powerhouse of the cell.
Mitochondria
Mitochondria have their own DNA
Forensic DNA (mitochondria)
Mitochondrial DNA
Double HelixYES
ChromosomesNO
Ring of DNAYES
Forensic DNA (mitochondria)
Mitochondrial DNA
Mitochondrial DNA is only 16,569 letters long.
There is a 900 base pair region with a 1.7% difference (D loop).
(compared to 3 billion in nuclear DNA)
Forensic DNA (mitochondria)
Nuclear DNA vs. Mitochondrial DNA
Double Helix Double Helix
One copy per cell Multiple copies in
each mitochondriaMultiple mitochondria in
each cell
One Ring 46 Chromosomes
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
Sequencing:• Sequence is determined by another reaction
and separation of sequenced fragments
Amplification or PCR:• Amplifies small portion of mtDNA (D loop)
Forensic DNA (Mitochondria)
DNA Sequences are compared to each other.
AGCTAGATCGTTATTCCGAG
AGCTAGATCGTTATTCCGAG
Hair found onSuspect
Victim
Conclusion: Hair may have come from the victim.
Forensic DNA (Mitochondria)
DNA Sequences are compared to each other.
AGCTAGATTGTTATTCCGAG
AGCTAGATCGTTATTCCGAG
Hair found onSuspect
Victim
Conclusion: Hair did not come from the victim
Forensic DNA (Mitochondria)
AGCTAGATTGTTATTCCGAG
AGCTAGATCGTTATTCCGAG
Cigarette butt at crime scene
Suspect #1
Conclusion: Cigarette could be from Suspects #2, #4 or other person with the same sequence.
AGCTAGATTGTTATTCCGAGSuspect #2
AGCTTGATTGTTATTCCGAGSuspect #3
AGCTAGATTGTTATTCCGAGSuspect #4
DNA and Statistics
The final result is presented as a statistic.
Do Say:“The chance that another person has this DNA
in the bloodstain is 1 in 300 billion.”
Do not say:“The DNA in the bloodstain is John Doe’s DNA.”
DNA and Statistics
Where do the statistics come from?
First, the frequency of each allele is estimated using data from a population data base.
Locus: D5S818
Alleles: 7,9
Allele frequencyfrom database
7 26%
9 11%
DNA and Statistics
Where do the statistics come from?
Next, the frequency of the genotype at each locus is calculated.
Locus: D5S818
Alleles: 7,9 7,9 6%
Genotype frequency
DNA and Statistics
For total frequency, multiply all of the frequencies together.
D5 = 6%
D8 = 12%
D18 = 0.5%
Total = 0.004%
7 0.00 6 0.008 0.00 7 0.009 0.03 8 0.53
10 0.25 9 0.0911 0.31 10 0.0612 0.33 11 0.2813 0.06 12 0.0414 0.01 13 0.0015 0.00
CSF1PO TPOX
5 0.01 13 0.006 0.24 14 0.137 0.15 15 0.088 0.12 16 0.219 0.16 17 0.27
9.3 0.33 18 0.2010 0.01 19 0.0911 0.00 20 0.02
21 0.00
TH01 vWA
7 0.00 6 0.008 0.03 7 0.019 0.11 8 0.16
10 0.08 9 0.1511 0.32 10 0.3012 0.27 11 0.2013 0.17 12 0.1214 0.03 13 0.0615 0.00 14 0.01
D16S539 D7820
7 0.00 7 0.008 0.14 8 0.009 0.05 9 0.01
10 0.05 10 0.0611 0.31 11 0.3712 0.31 12 0.3513 0.08 13 0.1914 0.06 14 0.0115 0.00 15 0.00
D13S317 D5S818
Demonstration
Calculating Frequencies
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