Download DNA Polymerases

DNA polymerases
Taq polymerase
Pfu polymerase
Pwo polymerase
Approximate error rate
10-5
10-6
10-6
Extension rate
0.5–1.0 min/kb
2.0 min/kb
0.75 min/kb
<10 kb
1 U/100l
2.5-5.0 U/100l
>10 kb
unfit
2.5-10.0 U/100l
dNTP requirement
200 μM each
100-250 M each
150-250 M each
Exonuclease Activity
5’  3’
3’  5’ proofreading
3’  5’ proofreading
Termination
Extendase activity
Blunt end
Blunt end
Half life
60 min at 94°C
95% still active after
1h at 95°C
2 h at 100°C
Origin:
Thermus aquaticus
Pyrococcus furiosus
Pyrococcus woesei
Concentrations
1.0-10.0 U/100l
Extendase activity
Taq, T7, Vent and Klenow polymerase exhibit 3’ extendase activity, thus adding single bases
to the 3’-end of the PCR products. This can be a problem for blunt-end cloning.
3’-end nucleotide
A
C
G
T
3’-extension/replacement
+A at low efficiency
+A > +C
+G > +A > +C
-T , +A
Amplifying long PCR products
Taq makes many errors and transcription stops, thus, no long PCR products can be made with
Taq. Adding 1/10 of a proof-reading polymerase, like Pfu polymerase, to Taq allows the
amplification of long PCR products. TaqPlus is a commercial such mixture for long PCR. A
pure proofreading polymerase like Pfu can also be used directly for long PCR, but these
enzymes are slower, so more time has to be allowed for amplification.
Caution
1. A-T TOPO cloning and some other methods depends on A-overhangs, so Taq has to be
used!
2. Proofreading polymerases degrade the primers if the reactions are not set up at 0 °C!