Fidelity of long-PCR

Brian Desany bd033054 at condor.mbcr.bcm.tmc.edu
Wed Jun 7 10:25:20 EST 1995


Hi there, I am planning to randomly mutagenize a couple of genes in the 
range of 5-9kb, and the simplicity of the long-range PCR methods I've 
been reading about recently combined with the fact that PCR is 
inherently mutagenic, makes me want to give this a try.  However, it is 
my understanding that the reason long-PCR works is because the small 
amounts of proofreading polymerase that's included in the reaction 
repairs the mismatches created by the error-prone polymerase.  So 
apparently mismatches will decrease processivity.  Yet obviously not 
every mismatch results in a terminated product, because of all the 
mutations that are found in (short) PCR products.  What I'm finally 
getting around to asking is, what is the fidelity of long PCR?  Is this 
a feasable approach to mutagenizing large ORF's?  If anyone's done this, 
I would appreciate any facts, opinions or speculations you could 
provide.  In the meantime I guess I'll just do it and see what happens.  
Thanks!

Just in case:  e-mail=  bd033054 at condor.mbcr.bcm.tmc.edu





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