dna fingerprinting using pcr
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DNA Fingerprinting Using PCR. Timothy G. Standish, Ph. D. Polymorphism. Differences between humans can be attributed to two factors: Environmental variation impacting development Individual genes that vary between people These genes which are variable are called polymorphic - PowerPoint PPT PresentationTRANSCRIPT
©2000 Timothy G. Standish
DNA DNA Fingerprinting Fingerprinting
Using PCRUsing PCRTimothy G. Standish, Ph. D.
©2000 Timothy G. Standish
PolymorphismPolymorphism 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 FingerprintingDNA 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 GenePolymorphism 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 GenePolymorphism In The TPA GeneChromosome 8
Ex11Ex10 Ex13Ex12 Ex14Ex 9Ex 1 Ex 4Ex 3 Ex 6Ex 5 Ex 8Ex 7Ex 2
The TPA Gene13121110987654321
Exon 9Exon 8Intron 8 Intron 9Intron 7
Exon 9Exon 8
ORIntron 8 Intron 9Intron 7
Alu
Polymorphism
©2000 Timothy G. Standish
PCR Detection of TPA PCR Detection of TPA PolymorphismPolymorphism
Reverseprimer
Forwardprimer
960 Base pairsPCR will produce a 660 bp fragment
Exon 9Exon 8
Intron 8
Reverseprimer
Forwardprimer
Alu insertion site
Exon 9Exon 8
ORIntron 8
Alu300 base pairs
660 Base pairsPCR will produce a 660 bp fragment if these primers are used
©2000 Timothy G. Standish
Components of a PCR Components of a PCR ReactionReaction
Buffer (containing Mg++)Template DNA2 Primers that flank the fragment of
DNA to be amplifieddNTPsTaq DNA Polymerase (or another
thermally stable DNA polymerase)
©2000 Timothy G. Standish
PCRPCRMelting
94 oC
Tem
pera
ture
100
0
50
T i m e
5’3’
3’5’
©2000 Timothy G. Standish
PCRPCRMelting
94 oC
Tem
pera
ture
100
0
50
T i m e
3’5’
5’3’
Heat
©2000 Timothy G. Standish
PCRPCRMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
3’5’
5’3’5’
5’
Melting94 oC
©2000 Timothy G. Standish
PCRPCRMelting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’
Heat
Heat
5’
5’
5’
©2000 Timothy G. Standish
PCRPCRMelting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’5’
5’
5’
5’
5’
5’
©2000 Timothy G. Standish
PCRPCRMelting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’ 5’
5’5’
5’
5’
5’
Heat
Heat
©2000 Timothy G. Standish
PCRPCRMelting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’ 5’
5’5’
5’
5’
5’
5’
5’
5’
5’
©2000 Timothy G. Standish
Fragments of defined length
PCRPCRMelting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’ 5’
5’5’
5’
5’
5’
5’
5’
5’
5’
©2000 Timothy G. Standish
DNA Between The Primers Doubles DNA Between The Primers Doubles With Each Thermal CycleWith Each Thermal Cycle
0Cycles
Number1
3
8
2
4
1
2
4
16
5
32
6
64
©2000 Timothy G. Standish
PCR Detection of TPA PCR Detection of TPA PolymorphismPolymorphism
Reverseprimer
Forwardprimer
960 Base pairsPCR will produce a 660 bp fragment
Exon 9Exon 8
Intron 8
Reverseprimer
Forwardprimer
Alu insertion site
Exon 9Exon 8
ORIntron 8
Alu300 base pairs
660 Base pairsPCR will produce a 660 bp fragment if these primers are used
©2000 Timothy G. Standish
Gel ElectrophoresisGel ElectrophoresisHeterozygousHomozygous
with AluHomozygous lacking Alu
©2000 Timothy G. Standish