Error prone PCR with nucleotide analogs and high fidelity polymerase

David Liu via methods%40net.bio.net (by amanosz from yahoo.com)
Thu Apr 5 16:57:14 EST 2012



I am trying to do error prone PCR using nucleotide analogs (dPTP and 8-oxo-2'-dGTP).  However for the particular construct I would like to do error prone PCR, I have had trouble amplifying the target by normal PCR unless I use specific enzymes (eg. LongAmp and OneTaq amplify fine only with a small amount of Phusion Hot Start spiked in; KOD, Phusion High Fidelity only, LongAmp and OneTaq only do not).  It is about 3kb, and ~63% GC.  I have used Taq in the past for error prone PCR by nucleotide analogs but Taq does not amplify the construct well at all.  

So my question is, would error prone PCR by nucleotide analogs be incompatible with the exonuclease and error correction activity of high fidelity polymerases (in general or specifically with Phusion Hot Start)?  


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