DNA Analysis: Short Tandem Repeats STRs

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DNA Analysis Short Tandem Repeats (STRs)

• Dr. Jason Linville
• University of Alabama at Birmingham
• jglinvil_at_uab.edu

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Summary

• We know what DNA is and where it is found in the
  body.

• We know how to identify substances that may
  contain DNA (blood, semen, saliva)

• We know how to extract DNA
• (evidence gt DNA in a tube)

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Summary

• We know how to amplify DNA.

• We do not yet know what area of DNA to amplify,
  or how to analyze the DNA.

First, lets decide what area to amplify.
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Forensic DNA Analysis
Two main areas (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

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Forensic DNA Analysis gt STR

• 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)

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Forensic DNA Analysis gt STR

• Short Tandem Repeats (STRs)

Person 1 ..GCCAGCTAGCTAGCTAGCTAGCTAGCTTTCAT..
1 2 3 4 5 6
Person 2 ..GCCAGCTAGCTAGCTAGCTAGCTTTCAT..
1 2 3 4 5
Person 3 ..GCCAGCTAGCTAGCTAGCTAGCTAGCTAGCTT..
1 2 3 4 5 6 7
This is not a true representation since each
person has two copies of DNA (mother, father).
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Person 1..GCCAGCTAGCTAGCTAGCTTTCAT..
1 2 3 4
..GCCAGCTAGCTAGCTAGCTAGCTAGCTTTCAT..
1 2 3 4 5 6
Person 2..GCCAGCTAGCTAGCTAGCTAGCTAGCTTTCAT..
1 2 3 4 5 6
..GCCAGCTAGCTAGCTAGCTAGCTAGCTAGCTTTCAT..
1 2 3 4 5 6 7
Person 3..GCCAGCTAGCTAGCTAGCTAGCTTTCAT..
1 2 3 4 5
..GCCAGCTAGCTAGCTAGCTAGCTTTCAT..
1 2 3 4 5
This is a better representation since each person
has two copies of DNA (mother, father).
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Person 1..GCCAGCTAGCTAGCTAGCTTTCAT..
..CGGTCGATCGATCGATCGAAAGTA..
1 2 3 4
..GCCAGCTAGCTAGCTAGCTAGCTAGCTTTCAT..
..CGGTCGATCGATCGATCGATCGATCGAAAGTA..
1 2 3 4 5 6
Person 2..GCCAGCTAGCTAGCTAGCTAGCTAGCTTTCAT..
..CGGTCGATCGATCGATCGATCGATCGAAAGTA..
1 2 3 4 5 6
..GCCAGCTAGCTAGCTAGCTAGCTAGCTAGCTTTCAT..
..CGGTCGATCGATCGATCGATCGATCGATCGAAAGTA..
1 2 3 4 5 6 7
This is an even better representation.
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Forensic DNA Analysis gt STR
Locus or Loci Refers to the location on the
chromosome.
Allele Refers to the type of DNA. For STRs, the
allele is the number of repeats.
Each person has 2 copies of each chromosome, so
each person has 2 alleles at each locus.
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Forensic DNA Analysis gt STR
Example
Locus D5S818
Alleles 7,9
Paternal chromosome 5
CCAGATAGATAGATAGATAGATAGATAGATCC
Maternal chromosome 5
CCAGATAGATAGATAGATAGATAGATAGATAGATAGATCC
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Forensic DNA Analysis gt STR
DNA profile can be different for each person.
Wes and Rena have the same genotype. Is this
possible?
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Forensic DNA Analysis gt STR
13 loci used in CODIS
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Forensic DNA Analysis gt STR
Rare for people to match at many loci.
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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.
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Forensic DNA Analysis gt Statistics
Where do the statistics come from?
First, the frequency of each allele is estimated
using data from a population data base.
Allele frequency from database
Locus D5S818
11 0.37
Alleles 11,13
13 0.19
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Forensic DNA Analysis gt Statistics
Next, the frequency of the genotype at each locus
is calculated.
If alleles are the same, use p x p
If alleles are different, use (p x q) x 2
Locus D5S818
(0.37 x 0.19) x 2
Alleles 11,13
0.14
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Forensic DNA Analysis gt Statistics
Next, the frequency of the genotype at each locus
is calculated.
If alleles are the same, use p x p
If alleles are different, use (p x q) x 2
Locus D5S818
Genotype frequency
11,13 0.14
Alleles 11,13
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Forensic DNA Analysis gt Statistics

• For total frequency, multiply all of the
  frequencies together.

D5 0.14 D8 0.12 D18 0.005 Total
0.000084
See worksheet and homework.
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Summary

• We know we want to amplify 13 regions of DNA (all
  will be STR loci).

How do we amplify 13 regions of DNA at the same
time?
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Forensic DNA Analysis
One Segment of DNA 10 million copies
Sample Buccal swab Blood stain Semen stain
Agarose Gel
Cycle Sequencing
PCR
One Segment of Sequenced DNA Millions of copies
Extraction
All DNA Hundreds of copies
Run on Genetic Analyzer
PCR
16 Segments of DNA 10 million copies
Run on Genetic Analyzer
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Forensic DNA Analysis
One Region of DNA 10 million copies
Sample Buccal swab Blood stain Semen stain
This is what we did last week.
PCR
Extraction
All DNA Hundreds of copies
This is what we want to do this week.
PCR
16 Regions of DNA 10 million copies
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Forensic DNA Analysis

• Basic Steps in Analysis

• Extraction
• Separates DNA from sample

• Amplification or PCR
• Amplifies small portion(s) of DNA

• Analysis
• Measure DNA length or Sequence DNA.

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PCR
Setting up a PCR reaction for one locus or 16
loci is basically the same procedure.
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PCR
Start 500 copies 1 cycle 1000 copies 2 cycles
2000 copies 3 cycles 4000 copies 4 cycles 8000
copies 5 cycles 16000 copies 10 cycles 512,000
copies 15 cycles 16,384,000 copies 20 cycles
524,288,000 copies
Both will result in millions of copies of the
regions to be amplified.
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The Thermal Cycler
Both will use a thermal cycler
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PCR
Both PCR reactions take place in a tube.
What goes into the tube?
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PCR

• PCR components for one region

• DNA
• Taq polymerase
• Primers (1 set Primer 1, Primer 2)
• dNTPs
• KCl, Tris-HCl, MgCl2, BSA

Its in there!
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PCR

• PCR components for 16 regions

• DNA
• Taq polymerase
• Primers (16 sets 32 primers)
• Primers are labeled
• dNTPs
• KCl, Tris-HCl, MgCl2, BSA

Its in there!
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PCR

• Negative Control

• Instead of DNA, water is added.

• PCR product indicates a reagent is contaminated
  with DNA.

Positive Control

• Put a known amount of DNA in the tube.
• Ensures reaction components and thermal cycling
  parameters are working.

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PCR gt Issues
Contamination

• Since 20 copies of DNA can be used in PCR,
  inadvertent contamination is possible.

• From scientist setting up reaction.
• From police or crime scene investigator.
• From other samples