DNA Analysis: Detection of Short Tandem Repeats

1
DNA Analysis Detection of Short Tandem
Repeats

• 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.

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

• We do not know how to analyze (measure) the DNA.

4
Forensic DNA Analysis

• Short Tandem Repeats (STRs)
• Individual identification possible
• Samples Blood stains, semen

STRs are measured by separating them (racing
them) through a small tube.
STRs are separated by electrophoresis.
5
Short Tandem Repeats

• Electrophoresis of DNA

• Phosphate groups on the DNA backbone gives it a
  negative charge.
• In an electric field, will migrate toward the
  anode ().

Can migrate through slab-gel or capillary.
6
Short Tandem Repeats

• Electrophoresis of DNA

• Phosphate groups on DNA backbone gives it a
  negative charge.
• In an electric field, will migrate toward the
  anode ()

Can migrate through slab-gel or capillary.
7
Short Tandem Repeats

• Capillary Electrophoresis

• Samples separated through a viscous polymer
  filled capillary
• Detection measures time span from sample
  injection to detection

8
Short Tandem Repeats gt Capillary Electrophoresis
7
9
10
12
9
Short Tandem Repeats gt Capillary Electrophoresis
7
9
10
12
10
Short Tandem Repeats gt Capillary Electrophoresis
7
9
10
12
11
Short Tandem Repeats gt Capillary Electrophoresis
7
9
10
12
12
Short Tandem Repeats gt Capillary Electrophoresis
7
9
10
12
13
Short Tandem Repeats gt Capillary Electrophoresis
7
9
10
12
14
Short Tandem Repeats gt Capillary Electrophoresis
Time
7
47 seconds
9
52 seconds
10
55 seconds
12
59 seconds
15
Short Tandem Repeats gt Capillary Electrophoresis
Horse
47
52
55
59
Time (seconds)
7
9
10
12
16
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
We are separating DNA fragments.
17
Short Tandem Repeats gt Capillary Electrophoresis
7
9
10
12

• Instead of horses, we are separating amplified
  fragments of DNA.

18
Short Tandem Repeats gt Capillary Electrophoresis
7
9
10
12

• Remember, there isnt just one fragment, there
  are millions of identical fragments.

19
Short Tandem Repeats gt Capillary Electrophoresis
9
12

• For each person at each loci, there are only 2
  types of fragments (2 alleles).

20
Short Tandem Repeats gt Capillary Electrophoresis
9
12

• David Caruso is not the detector.
• A CCD camera is the detector.

21
Short Tandem Repeats gt Capillary Electrophoresis
9
12
The fragments race down the tube. The smaller
fragments are always faster.
22
Short Tandem Repeats gt Capillary Electrophoresis
9
12
The fragments race down the tube. The smaller
fragments are always faster.
23
Short Tandem Repeats gt Capillary Electrophoresis
19.5 minutes
9
23.6 minutes
12
24
Short Tandem Repeats gt Capillary Electrophoresis
DNA Signal
19.5
23.6
Time (minutes)
9
12
25
Short Tandem Repeats gt Capillary Electrophoresis
?
?
DNA Signal
19.5
23.6
Time (minutes)
Problem The fragments will not always finish
at the same time.
26
DNA Detection gt STR Genotyping
Problem The fragments will not always finish
at the same time.

• Solution
• First, use a Size Standard to estimate size (in
  base pairs).
• Use an Allelic Ladder to determine the allele (
  of repeats).

27
DNA Detection gt STR Genotyping
Use Size Standard to Estimate Size

• Size Standard DNA fragments of known sizes that
  are added to each sample before separation.

• Times of sample peaks are compared to times of
  size standard peaks.

28
Short Tandem Repeats gt Capillary Electrophoresis
?
?
DNA Signal
190
200
210
Size (base pairs)
Instead of time, the size (in base pairs) is
estimated.
29
Short Tandem Repeats gt Capillary Electrophoresis
Size 194 Allele ?
Size 206 Allele ?
DNA Signal
190
200
210
Size (base pairs)
Instead of time, the size (in base pairs) is
estimated.
30
DNA Detection gt STR Genotyping
31
DNA Detection gt STR Genotyping
Use Allelic Ladder to Call Allele

• Allelic Ladder sample that contains millions of
  copies of all possible alleles.

• The size of the sample peaks are compared to the
  sizes of the allelic ladder peaks (run
  separately).

32
Short Tandem Repeats gt Capillary Electrophoresis
DNA Signal
7 8 9 10 11 12
13
Alleles
190
200
210
Size (base pairs)
Since the alleles in the ladder are known, you
can assign a size to each allele.
33
Short Tandem Repeats gt Capillary Electrophoresis
Sizes
186 190 194 198 202
206 210
DNA Signal
7 8 9 10 11 12
13
Alleles
190
200
210
Size (base pairs)
Since the alleles in the ladder are known, you
can assign a size to each allele.
34
Short Tandem Repeats gt Capillary Electrophoresis
Since the alleles in the ladder are known, you
can assign a size to each allele.
35
Short Tandem Repeats gt Capillary Electrophoresis
Remember, for different instruments in different
labs, this chart will be different.
36
Short Tandem Repeats gt Capillary Electrophoresis
Size 194 Allele ?
Size 206 Allele ?
DNA Signal
190
200
210
Size (base pairs)
For our sample, we know the size, but not what
the alleles are.
37
Short Tandem Repeats gt Capillary Electrophoresis
We can compare the sizes to this chart to figure
out what alleles they are.
38
DNA Detection gt Fluorescent Label

• How does a CCD camera see DNA?

• DNA fragment is labeled with a fluorescent dye.
• Fluorescent dye is attached to the primer.
• Fluorescent dye releases light as it relaxes.

39
DNA Detection gt Fluorescent Label
How does a CCD camera see DNA?

• At the detection window, the fluorescent dye is
  excited by a laser
• Different dyes will fluoresce in different
  colors.

No DNA
40
DNA Detection gt Fluorescent Label
How does a CCD camera see DNA?

• At the detection window, the fluorescent dye is
  excited by a laser
• Different dyes will fluoresce in different
  colors.

DNA
41
DNA Detection gt Detectors
Applied Biosystems 310

• Capillary Electrophoresis sample run and
  detected during run (30 min/sample)
• Fluorescence emissions detected by splitting
  light with prism and collecting data on CCD
  camera
• CCD camera converts light into electric signal.

42
DNA Detection gt Detectors
Applied Biosystems 310
43
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
44
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
45
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.

46
Forensic DNA Analysis

• Summary

• Extraction
• Many weeks ago we extracted DNA from students
  (buccal swabs), a blood stain, and a saliva stain.

• Amplification or PCR
• Before spring break, we combined this DNA with
  Taq, dNTPs, primers, and buffers. We put it into
  a thermal cycler.

47
Forensic DNA Analysis

• Analysis
• Tonight we begin with millions of copies of
  amplified DNA in a tube. We need to prepare it
  for electrophoresis.

What goes into the tube?
48
Forensic DNA Analysis
What goes into the tube?

• PCR Product (amplified DNA)
• Formamide keeps DNA in single stranded form for
  good separation
• Size standard Used to determine the size of our
  PCR product (base paris)

Also include tube of formamide size standard
allelic ladder