DNA Polymerase 27
5.2. Uses ofT7 DNA Polymerase
A major use of native or modified T7 DNA polymerase is in DNA
sequencing when fragments of over 1,000 bases can be analyzed from
one reaction. The enzyme is also useful for other applications when
extension of a primer over long stretches of template are required. The
other uses of this enzyme include the rapid end-labeling of DNA probes
and the extension of primers during site-directed mutagenesis.
5.3. Experimental Procedures
A suitable reaction buffer for T7 DNA polymerase is 40 mM Tris-
HC1, pH 7.5, 10 mM MgC12, 1 mM DTT, 100 Jxg/mL BSA, 300 JaM
dNTPs (18), and reactions should be performed at 37°C. The enzyme
is stable for over 6 mo when stored at -20°C in 25 mM Tris-HC1, pH
7.5,250 mM NaC1, 5 mM DTT, and 50% glycerol.
- Taq Polymerase
Taq polymerase is a DNA polymerase isolated from the bacterium
Thermus aquaticus (19). Similar enzymes have been isolated from
other thermophilic organisms, including Thermus thermophilus, Th.
flavus, Th. litoralis, Pyrococcus furiosus, and Bacillus stearo-
thermophilus. Taq polymerase preparations have been isolated from
several different strains of Th. aquaticus itself, and each has slightly
different characteristics. The most common source is still strain YT-
1, although the enzyme is also available from recombinant clones.
6.1. Enzyme Data
Taq polymerase is a monomeric enzyme with a mol mass in the range
60,000-97,000 Da depending on the source. The enzyme exhibits a 5'-3'
polymerase activity with a high processivity and lacks a 3'-5'exonu-
clease (proofreading) activity, but does possess a 5'-3' exonuclease (strand
replacement) activity. A modified form of the enzyme is now available
from which this latter activity has been removed. DNA polymerases
from Thermus litoralis and Pyrococcusfuriosus do exhibit the 3'-5' exonu-
clease activity (20;21), which renders DNA replication less error-prone.
Taq polymerase has another activity in that it can incorporate an
additional nucleotide (usually deoxyadenosine) at the 3' terminus of
any DNA duplex in a nontemplate-dependent reaction (22). This ad-
dition often hinders subsequent manipulations, but can be turned to
advantage as a means of increasing cloning efficiency (23).
