DNA-FINGERPRINTING-PCR MOLECULAR Biology

DNA fingerprinting

1. ©2000 Timothy G. Standish
DNA
Fingerprinting
Using PCR
Timothy G. Standish, Ph. D.

2. ©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

3. ©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

4. ©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

5. ©2000 Timothy G. Standish
Polymorphism In The TPA Gene
Chromosome 8
Ex11Ex10 Ex13Ex12 Ex14Ex 9Ex 1 Ex 4Ex 3 Ex 6Ex 5 Ex 8Ex 7Ex 2
The TPA Gene
13121110987654321
Exon 9Exon 8
Intron 8 Intron 9Intron 7
Exon 9Exon 8
OR
Intron 8 Intron 9Intron 7
Alu
Polymorphism

6. ©2000 Timothy G. Standish
PCR Detection of TPA
Polymorphism
Reverse
primer
Forward
primer
960 Base pairs
PCR will produce a
660 bp fragment
Exon 9Exon 8
Intron 8
Reverse
primer
Forward
primer
Alu insertion
site
Exon 9Exon 8
OR
Intron 8
Alu
300 base pairs
660 Base pairs
PCR will produce a
660 bp fragment if
these primers are used

7. ©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
TaqDNA Polymerase (or another
thermally stable DNA polymerase)

8. ©2000 Timothy G. Standish
PCR
Melting
94
o
C
Temperature
100
0
50
T i m e
5’3’
3’5’

9. ©2000 Timothy G. Standish
PCR
Melting
94
o
C
Temperature
100
0
50
T i m e
3’5’
5’3’
Heat

10. ©2000 Timothy G. Standish
PCR
Melting
94
o
C
Annealing
Primers
50
o
C
Extension
72
o
C
Temperature
100
0
50
T i m e
3’5’
5’3’
5’
5’
Melting
94
o
C

11. ©2000 Timothy G. Standish
PCR
Melting
94
o
C
Melting
94
o
C
Annealing
Primers
50
o
C
Extension
72
o
C
Temperature
100
0
50
T i m e
30x
3’5’
5’3’
Heat
Heat
5’
5’
5’

12. ©2000 Timothy G. Standish
PCR
Melting
94
o
C
Melting
94
o
C
Annealing
Primers
50
o
C
Extension
72
o
C
Temperature
100
0
50
T i m e
30x
3’5’
5’3’
5’
5’
5’
5’
5’
5’

13. ©2000 Timothy G. Standish
PCR
Melting
94
o
C
Melting
94
o
C
Annealing
Primers
50
o
C
Extension
72
o
C
Temperature
100
0
50
T i m e
30x
3’5’
5’3’
5’
5’
5’
5’
5’
5’
Heat
Heat

14. ©2000 Timothy G. Standish
PCR
Melting
94
o
C
Melting
94
o
C
Annealing
Primers
50
o
C
Extension
72
o
C
Temperature
100
0
50
T i m e
30x
3’5’
5’3’
5’
5’
5’
5’
5’
5’
5’
5’
5’
5’

15. ©2000 Timothy G. Standish
Fragments of
defined length
PCR
Melting
94
o
C
Melting
94
o
C
Annealing
Primers
50
o
C
Extension
72
o
C
Temperature
100
0
50
T i m e
30x
3’5’
5’3’
5’
5’
5’
5’
5’
5’
5’
5’
5’
5’

16. ©2000 Timothy G. Standish
DNA Between The Primers Doubles
With Each Thermal Cycle
0
Cycles
Number
1
3
8
2
4
1
2
4
16
5
32
6
64

17. ©2000 Timothy G. Standish
PCR Detection of TPA
Polymorphism
Reverse
primer
Forward
primer
960 Base pairs
PCR will produce a
660 bp fragment
Exon 9Exon 8
Intron 8
Reverse
primer
Forward
primer
Alu insertion
site
Exon 9Exon 8
OR
Intron 8
Alu
300 base pairs
660 Base pairs
PCR will produce a
660 bp fragment if
these primers are used

18. ©2000 Timothy G. Standish
Gel Electrophoresis
HeterozygousHomozygous
with Alu
Homozygous
lacking Alu

19. ©2000 Timothy G. Standish