Zusammenfassung
We demonstrate that the DNA polymerase isolated from Thermococcus
litoralis (VentTM DNA polymerase) is the first thermostable DNA
polymerase reported having a 3'----5' proofreading exonuclease activity.
This facilitates a highly accurate DNA synthesis in vitro by the
polymerase. Mutational frequencies observed in the base substitution
fidelity assays were in the range of 30 x 10(-6). These values were
5-10 times lower compared to other thermostable DNA polymerases
lacking the proofreading activity. All classes of DNA polymerase
errors (transitions, transversions, frameshift mutations) were assayed
using the forward mutational assay (1). The mutation frequencies
of Thermococcus litoralis DNA polymerase varied between 15-35 x
10(-4) being 2-4 times lower than the respective values obtained
using enzymes without proofreading activity. We also noticed that
the fidelity of the DNA polymerase from Thermococcus litoralis responds
to changes in dNTP concentration, units of enzyme used per one reaction
and the concentration of MgSO4 relative to the total concentration
of dNTPs present in the reaction. The high fidelity DNA synthesis
in vitro by Thermococcus litoralis DNA polymerase provides good
possibilities for maintaining the genetic information of original
target DNA sequences intact in the DNA amplification applications.
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