Pfu polymerase in PCR

Martin Hughes mhughes at fhs.csu.McMaster.CA
Tue Feb 18 13:50:25 EST 1997


On Mon, 17 Feb 1997, Dr. Duncan Clark wrote:

> Results I have seen published with mixes using Taq and a proof-reading
> enzyme all give around the same increase in fidelity ie approx 3 fold.
> Figures for Pfu on its own versus Taq vary from 10 to 12 fold
> improvement.

I'm interested to know why this should be the case.  As I 
understand it, the proofreading enzymes have their exonuclease activity 
as a subunit of the enzyme, which 'proofreads' as the strand is 
synthesised, comparing the daughter strand to the parent.  If this is the 
case (please correct me if I'm wrong), how 
(mechanistically) can a 1/50th or so addition of the proofreading enzyme 
do anything to the strands which are being synthesised by the Taq?  Does 
it 'scan' already synthesised double-stranded molecules, looking for an 
error during extension?

Thanks in advance for any light you can shed on this

Cheers

Dr. Martin Hughes
Department of Biochemistry
McMaster University
Hamilton, ONTARIO, Canada.




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