Accurate and Long PCR

Anthony V. Furano avf at helix.nih.gov
Fri Jul 7 15:18:27 EST 1995


Hello all,

We've been fooling around with mixtures of Taq (or the Stoffel fragment or
KlenTaq) and Pfu (to provide proof reading function, e.g.,  see Barnes
1994 PNAS vol. 91, pp. 2216-2220) to accurately amplify a somewhat long
(~3.5 kb) open reading frame (ORF) that we want to clone and express.  We
have yet to compare the fidelity achieved by our enzyme mixtures with that
of Taq (or its derivatives) alone.

Recently several commercial products have come on the market claiming to
do the same:  Elongase, from Gibco/BRL; Expand, from Boerhinger-Mannheim. 
In addition Perkin Elmer sells a proof reading DNA polymerase enzyme from
Thermotoga maritima that they claim is both efficient and accurate in the
PCR.

Does anyone have any experience with the above. Any comments will be
appreciated and I'll post a summary of anything sent to me directly.

Thanks in advance,

Anthony Furano
avf at helix.nih.gov



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