Professional Documents
Culture Documents
and the
Polymerase Chain
Reaction
Timothy G. Standish, Ph. D.
3’ 5’
5’ 3’
3’ 5’
5’ 3’
3’ 5’
5’ 3’
3’ 5’
5’ 3’
©2000 Timothy G. Standish
Extension - The Replication
3’
Fork
5’
5’ 3’
3’ 5’ 3’ 5’
Primase
Single-strand
Lagging Strand binding
5’ proteins
Okazaki 5’
fragment 3’
5’
RNA
Primers
DNA
Polymerase
5’
3’
Helicase
Leading Strand
5’
3’ ©2000 Timothy G. Standish
Functions And Their
Associated Enzymes
Function Enzyme
Melting DNA
Helicase
SSB Proteins
Topisomerase
Temperature
94 oC Extension 94 oC
Annealing 72 oC
Primers
50 50 oC
T i m e 3’ 5’
3’ 5’
3’ 5’
5’ 5’
3’ 5’
5’
3’ 5’
5’ 3’ 5’
5’ 3’
5’ 5’
5’ 3’
5’ 3’
5’ ©2000 Timothy G. Standish 3’
PCR
Temperature
100
Melting
94 oC
50
0
T i m e
3’ 5’
5’ 3’
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
Number
Cycle
1 2 4 8 16 32 64
0 1 2 3 4 5 6
Cycles
©2000 Timothy G. Standish
More Cycles = More DNA
Size Number of cycles
Marker 0 10 15 20 25 30