DNA Analysis: STR Multiplex

1
DNA Analysis STR Multiplex

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

2
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)

3
Summary

• We know how to amplify DNA and what area of DNA
  to amplify (Short Tandem Repeats)

• We know how to analyze (measure) the DNA (sort of)

• We do not know how to measure many STRs (16
  pairs) at the same time.

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Forensic DNA Analysis
One Region of DNA 10 million copies
Sample Buccal swab Blood stain Semen stain
PCR
Extraction
All DNA Hundreds of copies
PCR
16 Regions of DNA 10 million copies
This is what we want to do this week.
Run on Genetic Analyzer
5
Short Tandem Repeats gt Capillary Electrophoresis
9
12
Last week, we raced 2 alleles down the tube and
measured them.
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DNA Detection gt Fluorescent Label
We also mentioned the DNA fragments are labeled
with a fluorescent dye.
Fluorescent Light
Laser
DNA
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Short Tandem Repeats gt Capillary Electrophoresis
9
12
Well add the dyes to our drawing.
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Short Tandem Repeats gt Capillary Electrophoresis
TPOX
We can determine the size of the fragments (when
compared to the size standard).
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Short Tandem Repeats gt Capillary Electrophoresis
TPOX
TPOX
The sizes are compared to the allelic ladder to
determine the alleles.
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Short Tandem Repeats gt Capillary Electrophoresis
From TPOX
9
12
From vWA
10
11
What if we wanted to look at 2 loci at the same
time?
11
Short Tandem Repeats gt Capillary Electrophoresis
From TPOX
9
12
From vWA
10
11
What if we wanted to look at 2 loci at the same
time?
12
Short Tandem Repeats gt Capillary Electrophoresis
TPOX
TPOX
vWA
vWA
Who knows what size goes with which loci?
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Short Tandem Repeats gt Capillary Electrophoresis
TPOX
TPOX
vWA
vWA
Who knows what size goes with which loci?
14
Short Tandem Repeats gt Capillary Electrophoresis
TPOX
TPOX
vWA
vWA
Who knows what size goes with which loci?
15
Short Tandem Repeats gt Capillary Electrophoresis
From TPOX
9
12
From vWA
10
11
There are 2 ways to differentiate loci.
16
Short Tandem Repeats gt Capillary Electrophoresis
From TPOX
9
12
From vWA
10
11
1. Use a different fluorescent dye.
17
Short Tandem Repeats gt Capillary Electrophoresis
From TPOX
9
12
From vWA
10
11
When TPOX fragments cross, the camera sees red
light.
18
Short Tandem Repeats gt Capillary Electrophoresis
9
12
From vWA
10
11
When vWA fragments cross, the camera sees blue
light.
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Short Tandem Repeats gt Capillary Electrophoresis
TPOX
TPOX
vWA
vWA
Now the alleles can be determined.
20
Short Tandem Repeats gt Capillary Electrophoresis
From TPOX
9
12
From vWA
10
11
2. The location of primers can be moved so all
alleles for vWA come out after TPOX.
21
Short Tandem Repeats gt Capillary Electrophoresis
From TPOX
9
12
From vWA
10
11
2. The location of primers can be moved so all
alleles for vWA come out after TPOX.
22
Short Tandem Repeats gt Capillary Electrophoresis
TPOX
TPOX
vWA
vWA
Moving primers changes the sizes of alleles on
the vWA allelic ladder.
23
Short Tandem Repeats gt Capillary Electrophoresis
TPOX
TPOX
vWA
vWA
Moving primers changes the sizes of alleles on
the vWA allelic ladder.
24
Short Tandem Repeats gt Capillary Electrophoresis
A multiplex reaction analyzes gt1 loci at one time.
25
Forensic DNA Analysis
One Region of DNA 10 million copies
Sample Buccal swab Blood stain Semen stain
PCR
Extraction
All DNA Hundreds of copies
This is the reason we can analyze 16 loci at one
time.
PCR
16 Regions of DNA 10 million copies
Run on Genetic Analyzer