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Pfu polymerase in PCR

Tom Chappell t.chappell at ucl.ac.uk
Wed Feb 19 08:29:32 EST 1997


In article <Pine.HPP.3.91.970218134513.23388B-100000 at fhs.csu.McMaster.CA>,
Martin Hughes <mhughes at fhs.csu.McMaster.CA> wrote:

>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.

Taq has VERY low processivity (that's probably spelt wrong, but) and thus
adds only a few bases at a time before hopping off the DNA strand. Another
molecule of Taq comes along and continues. If the Taq molecule dissociates
after making a mismatch mistake, it is more likely that a polymerase with
an exo activity will bind, allowing the mismatch to be taken care of
before DNA synthesis continues. The view of a primer binding to a single
stranded DNA molecule and a SINGLE Taq molecule extending that primer to
the end of the ss DNA is not how it actually works mechanistically.




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