TA cloning nightmare
horto005 at maroon.tc.umn.edu
Fri Mar 8 06:54:18 EST 1996
Catherine I. DiOrio wrote:
> The polymerase you used to amplify your PCR product, KlenTaq, has
> 3'to 5' exonuclease activity...
KlenTaq is a truncated form of Taq, with the 5'-3' exonuclease domain removed. Taq
doesn't have 3'-5' exo activity to start with, and none was added. It does have a lower
mutation rate than regular Taq, but it is not due to any added exonuclease activity.
Here's the ref:
Barnes WM. The fidelity of Taq polymerase catalyzing PCR is improved by an N-terminal
deletion. Gene. 112(1):29-35, 1992 Mar 1.
KlenTaq DNA polymerase is an N-terminally truncated Thermus aquaticus
(Taq) DNA polymerase I. As expressed from a gene construct in Escherichia
coli, translation initiates at Met236, bypassing the 5'----3' exonuclease
domain of the DNA polymerase-encoding gene. A sensitive forward mutation
assay was used to measure the relative number of mutations introduced into
the entire lacZ gene by the polymerase chain reaction (PCR) under various
conditions which allow the amplification of such a large DNA span. Two
selectable markers, one at each end of the test lacZ fragment, were
employed to avoid the plating and scoring of PCR artefacts such as primer
initiation in the midst of the lacZ gene, and cloning artefacts such as
empty vector plasmid. The measured relative mutation rate was twofold
lower for KlenTaq as compared to the full-length Taq DNA polymerase.
Robert M. Horton
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