Help: high mutation rate during RT-PCR

Ian A. York iayork at panix.com
Tue Aug 15 08:35:45 EST 2000


In article <B7868D2D2540D311BED900105AD9DBA957E41B at n4s03451.grunenthal.com>,
Gillen, Dr. Clemens <Clemens.Gillen at grunenthal.de> wrote:
>Hi everybody,
>we are using the Advantage polymerase to amplify genes via RT-PCR and we
>face the problems of various mutations. Do you know of certain experimental
>conditions or better polymerases to overcome this problem?

Have you tried Pfx Platinum (Life Tech; no affiliation)?  I think you get
significantly higher fidelity than with the various proofreading/Taq mixes
I've tried, and it seems to be just about as robust and high-yield as Taq
alone.  

It's still not error free, though.  For example, I recently made a series
of PCR fragments off a plasmid (which should give better fidelity than
RT-PCR, though the primers were far from optimal) and cloned and sequenced
them.  I ended up with reliable sequence (say, 500 bp each) on six
fragments; one had a point mutation and an insert.  So, depending on how
you count, I saw 5/6 error-free fragments, or 2/3000 error-free bases.  

They hype a much better rate than that, but in my experience (probably
because I don't try to optimize much) that's typical.

Ian 
-- 
    Ian York   (iayork at panix.com)  <http://www.panix.com/~iayork/>
    "-but as he was a York, I am rather inclined to suppose him a
     very respectable Man." -Jane Austen, The History of England






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