PCR mutagenesis and proofreading polymerase

upyers mcoon at u.washington.edu
Mon Mar 18 16:51:50 EST 1996


In article <199603181710.AA106309000 at lulu.acns.nwu.edu>, prscholl at NWU.EDU
(prscholl) wrote:

> Does anyone have any experience with using polymerases with proofreading 
> activity to do site-directed mutagenesis by PCR?
> 
> We are making mutated cDNAs by PCR using oligos to introduce single base 
> pair substitutions in a c. 1.5 kb cDNA.  To maximize fidelity in the 
> rest of the amplified sequence we were thinking of using Stratagene's 
> Pfu or similar, but will its efficiency be affected by the presence of 
> the base pair mismatch in the primer?  Any thoughts appreciated.
> 
> Paul

Paul;

Efficacy of Pfu (or whatever) enzymatic activity is probably not affected
by primer homology, but primability surely is. You may (or may not) need
to modify [Mg++] and/or annealing temperatures to get efficient annealing
of the primer. 

My point being that any problems with this strategy likely lies with the
kinetics and specificity of the annealing reaction, rather than a factor
associated with the polymerase activity itself.

Note; the presence of proofreading activity (3' - 5' exonuclease activity)
means that you will have to use a hot start.


Good luck

MEC

-- 
"The two most common things in the universe are hydrogen and stupidity" Harlan Ellison



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