DNA

Fingerprinting
Using PCR
Timothy G. Standish, Ph. D.

©2000 Timothy G. Standish

 Polymorphism
● Differences between humans can be attributed to
two factors:
1. Environmental variation impacting development
2. Individual genes that vary between people
● These genes which are variable are called
polymorphic
● Each person is genetically unique because of
their unique set of polymorphic genes
● All people are related in that the vast majority of
their genes do not vary, but are identical from
person to person
©2000 Timothy G. Standish
 DNA Fingerprinting
● DNA fingerprinting involves identification of
DNA segments which vary between individuals
● A set of DNA fragments polymorphic enough to
provide a unique set of fragments for all
individuals, can be used to identify any specific
individual in a population
● No single fragment will uniquely identify an
individual, just as no single polymorphic genetic
trait will uniquely identify a person, but a unique
set of polymorphic DNA traits/fragments can serve
as a reliable means of identification
©2000 Timothy G. Standish
 Polymorphism In The TPA Gene
● Tissue Plasminogen Activator (TPA) is a
protein that functions in the cascade of
reactions which break down blood clots
● The gene contains 14 exons and 13 introns
● Scattered within the introns are 28 Alu
transposon sequences
● Within intron 8 a single Alu sequence may be
present, or it may be missing
● Thus, as intron 8 varies with the presence or
absence of Alu, it is polymorphic
©2000 Timothy G. Standish
 Polymorphism In The TPA Gene
Chromosome 8

The TPA Gene Polymorphism

1 2 3 4 5 6 7 8 9 10 11 12 13
Ex 1 Ex 2 Ex 3 Ex 4 Ex 5 Ex 6 Ex 7 Ex 8 Ex 9 Ex10 Ex11 Ex12 Ex13 Ex14

Intron 7 Intron 8 Intron 9

Exon 8 Exon 9
OR
Intron 7 Intron 8 Intron 9
Exon 8 Alu Exon 9
©2000 Timothy G. Standish
 PCR Detection of TPA
Polymorphism
Intron 8
Forward Alu insertion
primer
site
Exon 8 Reverse
Exon 9
primer

PCR will produce a

660 Base pairs 660 bp fragment if
these primers are used
OR
Forward
Intron 8
primer

Exon 8 Alu Reverse

Exon 9
300 base pairs primer

PCR will produce a

660 bp fragment 960 Base pairs ©2000 Timothy G. Standish
 Components of a PCR
Reaction
● Buffer(containing Mg++)
● Template DNA
● 2 Primers that flank the fragment of
DNA to be amplified
● dNTPs
● Taq DNA Polymerase (or another
thermally stable DNA polymerase)
©2000 Timothy G. Standish
 PCR
Temperature
100
Melting
94 oC

50

0
T i m e

3’ 5’
5’ 3’

©2000 Timothy G. Standish

 PCR
Temperature
100
Melting
94 oC

50

0
T i m e
3’ 5’

Heat

5’ 3’
©2000 Timothy G. Standish
 PCR
Temperature
100
Melting Melting
94 oC Extension 94 oC
Annealing
Primers 72 oC
50 50 oC

0
T i m e
3’ 5’
5’

5’
5’ 3’
©2000 Timothy G. Standish
 PCR
Temperature
100
Melting Melting 30x
94 oC Extension 94 oC
Annealing
Primers 72 oC
50 50 oC

0
T i m e
3’ 5’

Heat
5’

5’

Heat
5’
5’ 3’ ©2000 Timothy G. Standish
 PCR
Temperature
100
Melting Melting 30x
94 oC Extension 94 oC
Annealing
Primers 72 oC
50 50 oC

0
T i m e
3’ 5’
5’

5’
5’

5’
5’

5’
5’ 3’ ©2000 Timothy G. Standish
 PCR
Temperature
100
Melting Melting 30x
94 oC Extension 94 oC
Annealing
Primers 72 oC
50 50 oC

0
3’ 5’ 5’ T i m e
5’
5’
5’ 3’

Heat
5’

5’

Heat
5’
©2000 Timothy G. Standish
 PCR
Temperature
100
Melting Melting 30x
94 oC Extension 94 oC
Annealing
Primers 72 oC
50 50 oC

0
3’ 5’ 5’ T i m e
5’
5’ 5’
5’ 3’

5’
5’

5’
5’

5’
5’
©2000 Timothy G. Standish
 PCR
Temperature
100
Melting Melting 30x
94 oC Extension 94 oC
Annealing
Primers 72 oC
50 50 oC

0
3’ 5’ 5’ T i m e
5’
5’ 5’
5’ 3’

5’
5’
Fragments of
defined length 5’
5’

5’
5’
©2000 Timothy G. Standish
DNA Between The Primers Doubles
With Each Thermal Cycle
Number
1 2 4 8 16 32 64

0 1 2 3 4 5 6
Cycles
©2000 Timothy G. Standish
 PCR Detection of TPA
Polymorphism
Intron 8
Forward Alu insertion
primer
site
Exon 8 Reverse
Exon 9
primer

PCR will produce a

660 Base pairs 660 bp fragment if
these primers are used
OR
Forward
Intron 8
primer

Exon 8 Alu Reverse

Exon 9
300 base pairs primer

PCR will produce a

660 bp fragment 960 Base pairs ©2000 Timothy G. Standish
Gel Electrophoresis
Homozygous Homozygous Heterozygous
lacking Alu with Alu

©2000 Timothy G. Standish

©2000 Timothy G. Standish