How proofreading work Re: How to avoid PCR errors?

Brian Foley brianf at med.uvm.edu
Thu Feb 17 14:03:42 EST 1994


: Does proofreading activity correct the mutant primers used in the 
: mutagenesis
: PCR reaction? I meant, that the bases of mutant primers which don't 
: match the 
: template might be cut off and replaced by matching nucleotide bases 
: when Vent
: or Pfu being used. I was told that they wouldn't correct the primers, 
: but only
: the sequences being synthesized in between the primers. If so, could any 
: one tell me how the proofreading works?

	The "proofreading" is a 3' to 5' exonuclease activity.  I believe 
that the current theory is that this activity is always there, but the 5' to 
3' polymerization of nucleotides proceeds far faster than the exonuclease 
activity EXCEPT when a base is misincorporated.  Following 
misincorporation, the polymerase is a bit slow to add the next base, and 
thus there is time for the exonuclease to chew back the misincorporated base.
	This is quite different from mismatch repair enzymes that excise 
mismatched bases from the middle of a long strand of duplex DNA.  I 
suspect that if you added your primers and proofreading polymerase in the 
absence of nucleotides, a few of your primers would get destroyed (only 
those that were annealed to template) but this would happen even if there 
was no mismatch.  In the absence of nucleotides the polymerase will run 
in reverse.
	Thus the exonuclease activity is not truly "proofreading" because 
it will remove correctly matched bases as well as misincorporated bases.  
It is only the fact that the polymerase activity pauses after a mismatch 
that allows the differential removal of mismatched bases, vs correctly 
matched bases.

	I do not have a publication of the above theory.  I could be 
wrong on some points.  Has anyone seen publications that would clearly 
show what I have described?


--
********************************************************************
*  Brian Foley               *     If we knew what we were doing   *
*  Molecular Genetics Dept.  *     it wouldn't be called research  *
*  University of Vermont     *                                     *
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