How proofreading work Re: How to avoid PCR errors?

chai_z at wehi.edu.au chai_z at wehi.edu.au
Thu Feb 17 00:59:21 EST 1994


In article <2jej4o$s8o at scunix2.harvard.edu>, roth at husc7.harvard.edu (Frederick Roth) writes:
> In article <oliver.4.2D5A3C2F at molgen.biologie.uni-marburg.de> oliver at molgen.biologie.uni-marburg.de writes:
>>I want to amplify 2 kb genomic DNA and then to subclone the DNA (actually I 
>>have to amplify it two times, because I perform a site directed mutagensis). 
>>It is essential, that the sequence of the subcloned DNA is the SAME as the 
>>original sequence. Until now I did not manage to get a clone without PCR 
>>errors.
>>Has anyone experience with such a problem and knows how to circumvent this? 
>>I think there are some polymerases wich have a proof reading function. Has 
>>anyone ever used such an enzyme and can tell me wich is the best to use for 
>>this purpos 
> Check out Pfu....  Lundberg et al., Gene 108:1-6 (1991)
> 
> Pfu (from Pyrococcus furiosus-- 'rushing fireball') is a proofreading 
> polymerase w/ 1 or 2x10^-6 error rate.

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?

Zhonglin Chai
Melbourne






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