Gene Cloning and DNA Analysis in Forensic Science

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Gene Cloning and DNA Analysis in Forensic Science

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

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We already know

• The human genome contains coding and non-coding
  regions of the genome.
• Among individual humans, the genome is 99.8
  similar, but differences exist in coding regions
  (very few) and non-coding regions (not quite as
  few).
• No two humans have identical DNA.
• (except identical twins)

3
We already know

• Also
• Genetic markers are inherited from parents can
  be used to test kinship.

YES
NO
YES?
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Summary

• Identifying suspect
• History
• STRs
• Kinship Analysis
• Sex Identification

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Genetic Testing

• The basis of genetic fingerprinting is that no
  two humans DNA is alike (except identical twins).

Polymorphisms are positions where the nucleotide
sequence is not the same in every member of the
population.
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DNA Markers

• Restriction fragment length polymorphisms
  (RFLPs) digest PCR product, gives unique pattern.

• Short tandem repeats (STRs) different repeats at
  a locus.

• Single Nucleotide Polymorphisms base change
  possible in genome.

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History of DNA Testing

• RFLP Multilocus (fingerprint)
• RFLP single locus
• PCR Strips
• PCR STR
• Mitochondrial DNA

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Restriction Fragment Length Polymorphisms (RFLP)
History of DNA Testing

• Restriction Enzymes
• Individuals differ in number of cleavage sites
  and distance separating cleavage sites

• Length variation of fragments occurs two ways
• Base Change at restriction site
• Variable Number of Tandem Repeats (VNTR)

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History of DNA Testing
RFLP

• Length variation of fragments occurs two ways
• Base Change at restriction site
• Variable Number of Tandem Repeats (VNTR)

• Individuals differ in number of cleavage sites
  and distance separating sites

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Base Change at Restriction Site
History of DNA Testing gt RFLP
Person A
5'
3'
GGCC
GGCC
GGCC
GGCC
Person B
5'
3'
GGCC
GGCC
GGTC
GGCC
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Base Change at Restriction Site
History of DNA Testing gt RFLP
Hae III cuts at
5 GGCC 3
5'
3'
GGCC
GGCC
GGCC
GGCC
CC
GG
CC
GG
CC
GG
5'
3'
GGCC
GGCC
GGTC
GGCC
CC
GG
CC
GG
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VNTR
History of DNA Testing gt RFLP
VNTR - repeat unit 2-30 b.p. long repeated 5-50
times Example D1S7
GGTGGAGAG ( 9 b.p.)
29 repeat units
GGCC
GGCC
21 repeat units
GGCC
GGCC
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History of DNA Testing gt RFLP

• Sir Alec Jeffreys

Forensic Applications of DNA Fingerprinting
Jeffreys AJ, Wilson V, Thein SL. Hypervariable
minisatellite regions in human DNA. Nature
1985 31467-73.
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RFLP - Multilocus
History of DNA Testing gt RFLP
No amplification - Requires high quality DNA
Procedure

• Extract DNA
• Digest with restriction enzyme
• Separate on agarose gel
• Southern Blot
• Label with sequence specific probe

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History of DNA Testing gt RFLP
RFLP - Multilocus
Multilocus - Probe binds to multiple sites with
similar sequences, but different lengths
Locus 1
Probe
Locus 2
Binds to approximately 25 loci producing complex
pattern of 30-50 bands
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History of DNA Testing gt RFLP gt Multilocus
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History of DNA Testing gt RFLP
RFLP - Multilocus

• Probe binds to multiple sites
• Produces complex pattern of 30-50 bands

• Disadvantages
• Requires high quality DNA
• Overlap of Loci
• Cannot differentiate mixed samples

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History of DNA Testing gt RFLP
RFLP Single Locus
Probes bind to only one VNTR locus Produces
one band (homozygous) or two bands
(heterozygous)

• Advantages
• Examine one locus at a time

• Disadvantages
• Limited resolution
• Requires high quality DNA

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History of DNA Testing gt RFLP gt Single Locus
D2S44 probe Hae III digested Lanes 6 and 10
match Lanes 8 and 11 match
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History of DNA Testing gt RFLP gt Single Locus
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History of DNA Testing gt RFLP
RFLP Problems

• With each method, the quantity and quality of DNA
  had to be high.
• Different intensities made fingerprint difficult
  to interpret. Sizes could not be exactly
  measured (binning)

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Chris Mullin

• Position Forward
  Born 7/30/63
  
  Height 6-7 / 2m 1cm
  
  Weight 215 lbs. / 97.5kg
  
  College - St. John's (N.Y.) '85

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Kary Mullis

• 1993 Nobel Prize Winner

Inventor of the polymerase chain reaction (PCR)
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History of DNA Testing gt PCR
PCR Based Methods

• Sequence Specific Oligonucleotide (SSO) probe
• Amplified fragment-length polymorphism (AMP-FLP)
• Large VNTR regions (10-30 b.p. repeat)
• Short Tandem Repeats (STR) (2-7 b.p. repeat)

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History of DNA Testing gt PCR gt SSO Probe
Sequence variation among individuals at certain
loci

• Procedure
• Amplify region of DNA containing sequence
  variation using labeled primers
• Add labeled PCR product to sequence specific
  probe strips
• Labeled DNA attaches to homologous sequences

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History of DNA Testing gt PCR gt SSO Probe

• Example DQA
• 6 possible alleles based on sequence

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History of DNA Testing gt PCR gt SSO Probe

• Example DQA

1.2 1.3 4
All but 1.3
1
2
3
4
C
1.1
1.3
DQ-Alpha
1.2 1.3 4
All but 1.3
1
2
3
4
C
1.1
1.3
DQ-Alpha
Sample 1 2, 4
1.2 1.3 4
All but 1.3
1
2
3
4
C
1.1
1.3
DQ-Alpha
Sample 2 1.1, 1.2
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History of DNA Testing gt PCR gt STR
Short Tandem Repeats

• Repeat unit 2-7 b.p. in length
• Repeated 5-30 times
• Length is appropriate for PCR amplification

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History of DNA Testing gt PCR gt STR
Cells
XX XX XX XX
1 2 3 4
XX XX XX XX
X
5 6 7 8
X
X
X
XX XX XX XX
X
X
X
X
X
9 10 11 12
X
XX XX XX XX
13 14 15 16
XX XX XX XX
17 18 19 20
XX XX XY
21 22 23
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History of DNA Testing gt PCR gt STR
Short Tandem Repeats
X X
10 repeats
5
8 repeats
D5S818
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History of DNA Testing gt PCR gt STR
Short Tandem Repeats

• Units 4-7 bp in length repeated 5-30 times

Example D5S818 (7 allele) 4 b.p. AGAT
repeated 7 times
5'---AGAT AGAT AGAT AGAT AGAT AGAT AGAT---3'
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History of DNA Testing gt PCR gt STR
13 CODIS Loci
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History of DNA Testing gt PCR gt STR
DNA Monoplex

• Monoplex means only one locus is amplified
• For this example, we will look at D5S818.

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History of DNA Testing gt PCR gt STR
DNA Monoplex
11 repeats
12 repeats
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History of DNA Testing gt PCR gt STR
DNA Monoplex
11 repeats
12 repeats
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History of DNA Testing gt PCR gt STR
DNA Monoplex
11 repeats
12 repeats
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History of DNA Testing gt PCR gt STR
D5S818
7 8 9 10 11 12 13 14 15
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History of DNA Testing gt PCR gt STR
D5S818
11 12
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History of DNA Testing gt PCR gt STR
Multiplex

• In a multiplex run, more than one locus is
  amplified at one time.

• In order to avoid overlap
• The primer sites are moved
• The dye is changed

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History of DNA Testing gt PCR gt STR
DNA Multiplex
11 repeats
12 repeats
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History of DNA Testing gt PCR gt STR
DNA Multiplex
11 repeats
12 repeats
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History of DNA Testing gt PCR gt STR
DNA Multiplex
11 repeats
12 repeats
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History of DNA Testing gt PCR gt STR
DNA Multiplex
11 repeats
12 repeats
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History of DNA Testing gt PCR gt STR
DNA Multiplex
11 repeats
12 repeats
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D5S818
D13S317
D7S820
D16S539
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vWA
TH01
TPOX
CSF1PO
Am
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Ladders
History of DNA Testing gt PCR gt STR gt Multiplex
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Samples
History of DNA Testing gt PCR gt STR gt Multiplex
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History of DNA Testing gt PCR gt STR gt Multiplex
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History of DNA Testing gt PCR gt STR gt Multiplex
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Kinship by DNA profiling

• STR Analysis
• Using STR analysis with a known mother, potential
  fathers can be eliminated.

• Mitochondrial Analysis
• mtDNA is maternally inherited (cant use for
  paternity)
• Good for tracing ancestors

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Sex Identification
Y chromosome-specific loci

• Some genetic markers are found only on the Y
  chromosome.
• Presence of male only amplified band identifies
  the subject as male.
• Some of these Y markers can be used to ID the
  individual male in mixed samples.

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Sex Identification
Amelogenin gene

• Y chromosome testing cannot differentiate between
  female DNA and a failed sample.
• Amelogenin gene is found on each sex chromosome,
  but is longer on Y.
• Male gives 2 bands, female gives just one band.