preferential amplification by proofreading Taq ???
ed at bio02.bio.uottawa.ca
Wed Mar 19 20:55:00 EST 1997
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,
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