How proofreading work Re: How to avoid PCR errors?

bickle at urz.unibas.ch bickle at urz.unibas.ch
Fri Feb 18 08:37:09 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.

> 	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     *                                     *
> ********************************************************************

Sorry, not quite right. The key to the 3' to 5' exo activity is that it
is specific for single stranded DNA. A misincorporated base or a primer
mismatched at the 3' end is by definition single stranded and thus a
substrate for the exo. Blunt ended double stranded DNA is also a substrate
as the ends breath and become single stranded (very useful this for labelling
3' ends).

See the Kornberg and Baker book" DNA replication 2nd edition"
Freeman and Co 1992 ISBN 0-7167-2003-5.
-- 
Tom Bickle
Microbiology Dept, Biozentrum, Basel University
Klingelbergstrasse 70, CH-4056 Basel, Switzerland
+ 41 61 267 21 20       bickle at urz.unibas.ch



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