28 MaundersThe major distinguishing feature of Taq polymerase is its extreme
thermal stability. The enzyme can withstand temperatures in excess of
95°C for prolonged periods, and in fact, its optimum for reaction is
75°C. The rate of reaction is reduced to 50% at 60°C, and to 10% at
37°C. A range of DNApolymerases from other species ofthermostable
bacteria (including those mentioned earlier) are now becoming avail-
able, which will broaden the spectrum of possible reaction conditions.6.2. Uses ofTaq Polymerase
Taq polymerase is rapidly replacing other DNA polymerases in
many laboratory techniques, primarily because of its thermal stability
and tolerance of temperature changes. This feature allows reactions to
be performed at elevated temperatures, whereby DNA duplex melting
and annealing can be rigorously controlled. This ensures the presence
of a suitable single-stranded template for high-fidelity replication, and
also the stringency of primer association can be precisely defined.
Additional benefits are that contaminating enzyme activities are largely
eradicated, and complex but routine procedures can be automated.
The major use of Taq polymerase at present is in the polymerase
chain reaction (PCR) (24). This technique is a simple method of ampli-
fying minute quantities of DNA for a variety of subsequent proce-
dures, including cloning, sequencing, hybridization, and genome
mapping (25). The enzyme from Thermus thermophilus (when used in
a manganese-containing buffer) has an additional reverse transcriptase
activity, which extends the use of PCR directly to cDNA synthesis.
The PCR has applications beyond the reseach laboratory, including
uses in forensic science, disease diagnosis or prognosis, paternity test-
ing, and in animal and plant breeding programs.
Another use of Taq polymerase is directly in DNA sequencing,
where the high temperatures employed help reduce problems caused
by secondary structure in the template and allow an increase in the
stringency of primers used. Taq polymerase has, for the reasons men-
tioned earlier, become a very important tool for the molecular biolo-
gist and is described in detail in Chapter 4.
References- McHenry, C. S. (1988) DNA Polymerase HI holoenzyme of Escherichia coli.
Ann. Rev. Biochem. 57, 519-550.