DNA Replication

and the
Polymerase Chain
Reaction
Timothy G. Standish, Ph. D.

©2000 Timothy G. Standish

 History
 The Polymerase Chain Reaction (PCR) was not
a discovery, but rather an invention
 A special DNA polymerase (Taq) is used to
make many copies of a short length of DNA
(100-10,000 bp) defined by primers
 Kary Mullis, the inventor of PCR, was awarded
the 1993 Nobel Prize in Chemistry

©2000 Timothy G. Standish


What PCR Can Do
PCR can be used to make many copies of any DNA that is supplied as
a template
 Starting with one original copy an almost infinite number of copies
can be made using PCR
 “Amplified” fragments of DNA can be sequenced, cloned, probed or
sized using electrophoresis
 Defective genes can be amplified to diagnose any number of illnesses
 Genes from pathogens can be amplified to identify them (i.e., HIV)
 Amplified fragments can act as genetic fingerprints

©2000 Timothy G. Standish

 How PCR Works
 PCR is an artificial way of doing DNA replication
 Instead of replicating all the DNA present, only a
small segment is replicated, but this small
segment is replicated many times
 As in replication, PCR involves:
– Melting DNA
– Priming
– Polymerization

©2000 Timothy G. Standish

 Initiation - Forming the
Replication Eye
Origin of Replication
5’ 3’
3’ 5’

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

 Polymerizing DNA  DNA Polymerase

 Providing primer  Primase
 Joining nicks  Ligase
©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 30x
100 Melting Melting

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’

©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
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

©2000 Timothy G. Standish

Theoretical Yield Of PCR
Theoretical yield = 2n x y
Where y = the starting
number of copies and
n = the number of thermal cycles
If you start with 100 copies, how many copies are
made in 30 cycles?
2n x y
= 230 x 100
= 1,073,741,824 x 100
= 107,374,182,400
©2000 Timothy G. Standish
 How The Functions Of
Replication Are Achieved
During PCR
Function PCR
 MeltingDNA  Heat
 Polymerizing DNA  Taq DNA Polymerase

 Providing primer  Primers are added to the

reaction mix

 Joining nicks  N/A as fragments are short

©2000 Timothy G. Standish

©2000 Timothy G. Standish