preferential amplification by proofreading Taq ???

ed ed at bio02.bio.uottawa.ca
Wed Mar 19 20:55:00 EST 1997


hi all,


just a tiny little problem...I had done some RT-PCR using regular Taq 
polymerase which yielded my intronless 1.7 Kb coding region as expected, 
so I was very happy. Since I am cloning this amplicon into an 
expression vector, I decided to repeat the RT-PCR, using the very 
same cDNA template, but using TAKARA polymerase (which has proofreading 
ability). Much to my dismay, the reaction did not yield any of the PCR 
product expected, and instead it amplified something larger (the size of 
the coding region plus the intron). Is the polymerase preferentially 
amplifying the larger fragment ? does the TAKARA Taq just not like my 
cDNA template ? 

I feel very tempted to just clone the stuff amplified by regular Taq and 
sequencing a number of clones to find one with no mutations, but for a 
1.7 Kb piece, I would probably have a very hard time finding clones with 
no point mutations.   Any ideas as to what I should do ?

thanks in advance,

Ed



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