Taq - not so promiscuous???

bhjelle at carina.unm.edu bhjelle at carina.unm.edu
Wed Dec 16 15:20:26 EST 1992


In article <1992Dec15.210313.24687 at news2.cis.umn.edu> horton at molbio.cbs.umn.edu (Robert Horton) writes:
>	Your observation of a low rate of incorporation of errors by Taq
>is not unusual. We found no errors in over 6000 bases from clones amplified
>from cDNAs (J. Immunol. 145:1782-7, 1990). The reaction conditions were
>"standard" except that the [Mg++] was a little low (1mM). We had slightly
>higher error frequencies in other experiments, which happened to use slightly
>more Mg. (dNTPs were always 200uM each).
>	I think Taq often gets a bum rap as far as errors introduced into
>clones is concerned. The key may be that error _frequency_ (i.e., the
>observed fraction of errors incorporated into the product) is not directly
>correlated with error _rate_ (the fraction of wrong bases incorporated
>by Taq). This is because of "thermal proofreading" (as I believe they call
>it in California), in which DNA strands that get the wrong base added don't
>get extended efficiently by polymerase, so these errors don't show up in
>the final product.
>
What gets  *measured*  in most assays is the actual rate of misincorp-
orations into the final product, and this is what most of us
care about. Some may recall that I posted last month about
a truly spectacular misincorporation rate (11 substitutions and
3 frameshifting insertions and deletions out of 511 bp sequenced).
This was with many cycles (45) using 50ng plasmid template
and 4mM MgCl2/500mM dNTP each. Apparently high dNTP, lots
of cycles, and high MgCl2 are prone to increase error rate.
That said, on a number of previous occasions I have used 45
cycles from various templates and experience negligible
error rates.

I repeated the PCR on the plasmid that was originally used
as PCR template in the experiment with high mutation rate
using much lower Mg (1.6mM) and much lower dNTP (200 each,
I think). I ran product on a gel at 10, 15 and 20 cycles;
it was increasing only about 2x with each 5 cycles -
not a very robust PCR. However, when I pooled the 10 & 15
cycle product and cloned that, the product (so far) has
been free of misincorporations etc.

So the bottom line. If you want to decrease PCR misincorporations,
(1) lower dNTP; (2) lower Mg; and (3) lower cycle #.

Brian 



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