Properties of Thermostable
DNA Polymerase
Yufei TU
Discovery--history of Taq DNA polymerase
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The original report of this enzyme,
purified from the hot springs
bacterium Thermus aquaticus, was
published in 1976.
Roughly 10 years later, the
polymerase chain reaction was
developed and shortly thereafter
"Taq" became a household word in
molecular biology circles.
*THE DARNDEST PLACES: Scientists isolated the
thermostable DNA polymerase Taq, an enzyme that drives
PCR, from Thermus aquaticus Yellowstone type-1, a
resident of geysers like this one at Yellowstone National
Park.
Properties
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The thermophilic DNA polymerases,
like other DNA polymerases, catalyze
template-directed synthesis of DNA
from nucleotide triphosphates.
A primer having a free 3‘ hydroxyl is
required to initiate synthesis
Magnesium ion is necessary.
In general, they have maximal catalytic
activity at 75 to 80℃, and substantially
reduced activites at lower temperatures.
At 37℃, Taq polymerase has only about
10% of its maximal activity.
Taq DNA Polymerase
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Recombinant Taq DNA Polymerase is the
enzyme of choice for most PCR
applications.
The half-life of enzyme is >40 minutes at
95°C.
The error rate of Taq DNA Polymerase in
PCR is 2.2x10-5 errors per nt per cycle;
Other thermostable Polymerases
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In addition to Taq DNA polymerase, several other thermostable DNA
polymerases have been isolated and expressed from cloned genes. Three of the
most-used polymerases are described in the following table:
Polymerase
3'->5'
Exonuclease
Source and Properties
Taq
No
From Thermus aquaticus.
Halflife at 95℃is 1.6 hours.
Pfu
Yes
From Pyrococcus furiosus.
Appears to have the lowest error rate of
known thermophilic DNA polymerases.
Vent
Yes
From Thermococcus litoralis; also
known as Tli polymerase.
Halflife at 95 C is approximately 7
hours.
The Error Rate
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One of the most discussed characteristics of
thermostable polymerases is their error rate.
Error rates are measured using several different assays,
and as a result, estimates of error rate vary, particularly
when the assays are performed by different labs.
The total error rate
Taq
Pfu
1 x 10-4 to 2 x 10-5 errors per base pair
Vent
between Taq and Pfu
appears to have the lowest error rate at roughly 1.5 x 10-6
error per base pair
Reliability/Fidelity
Average error rates(mutation
frequency/bp/duplication)
increased as follows:
Pfu (1.3 x 10-6)
Deep Vent (2.7 x 10-6)
Vent (2.8 x 10-6)
Taq (8.0 x 10-6)
exo- Pfu and UlTma
(approximately 50 x 10-6).
Reference
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Voet,D, Voet,J. Biochemistry Vol.1 3rd ed.
Alberts, Johnson, Lewis. Molecular Biology of The
Cell 4th ed.
http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/enzymes/hotpolys.htm
l
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=146123&rendertyp
e=abstract
http://www.fermentas.com/techinfo/pcr/dnaamplprotocol.htm
http://www.fermentas.com/techinfo/pcr/pcrprotocolpfu.htm