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BCH5425 Molecular Biology and Biotechnology

BCH5425 Molecular Biology and Biotechnology
Spring 1998
Dr. Michael Blaber
blaber@sb.fsu.edu


Lecture 23

Polymerase Chain Reaction (PCR) cont.


Choice of Polymerases for PCR

Thermostable DNA polymerases and their sources

DNA Polymerase

Natural or recombinant

Source

Taq

Natural

Thermus aquaticus

Amplitaq®

Recombinant

T. aquaticus

Amplitaq (Stoffel fragment)®

Recombinant

T. aquaticus

Hot Tub™

Natural

Thermus flavis

Pyrostase™

Natural

T. flavis

Vent™

Recombinant

Thermococcus litoralis

Deep Vent™

Recombinant

Pyrococcus GB-D

Tth

Recombinant

Thermus thermophilus

Pfu

Natural

Pyrococcus furiosus

ULTma™

Recombinant

Thermotoga maritima

Taq/Amplitaq®

Stoffel fragment

Vent™

Deep Vent™

Pfu

Tth

ULTma™

95 C half-life

40 min

80 min

400 min

1380 min

>120 min

20 min

>50 min

5'3' exo

+

       

+

 

3'5' exo

   

+

+

+

 

+

Extension rate (nt/sec)

75

>50

>80

?

60

>33

?

RT activity

Weak

Weak

?

?

?

Yes

?

Resulting ends

3' A

3' A

>95% blunt

>95% blunt

blunt

3' A

blunt

Strand displacement

   

+

+

     

M.W. (kDa)

94

61

?

?

92

94

70

 

Buffers and MgCl2 in PCR reactions

A typical reaction buffer for PCR would something like:

Generally,

Primers

Primer design

Melting temperature (Tm) of primers

(1) Tm = [(number of A+T residues) x 2 C] + [(number of G+C residues) x 4 C]

(2) Tms = 81.5 + 16.6(log10[J+]) + 0.41(%G+C) - (600/l)

(3) Tmp = 22 + 1.46([2 x (G+C)] + (A+T))

G

A

T

C

Tm

Tms

Tmp

15 mer

3

5

2

5

46

42

56

20 mer

6

5

4

5

62

52

67

30 mer

8

6

8

8

92

62

89

Calculating primer concentrations


1998 Dr. Michael Blaber