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