Error rate in Taq pol. 1:270?

Richard P Grant rgrant at worf.molbiol.ox.ac.uk.remove_this_bit
Wed Feb 12 10:10:36 EST 1997


Tom Chappell (t.chappell at ucl.ac.uk) wrote:

} This is just the mathematics. I would assume that the error rate of Taq
} actually increases during a PCR reaction as you differentially use up and
} heat destroy the nucleotides. In addition, I would also assume that very

I don't think that this assumption holds.  I can't recall a reference, but
lower [dNTP] _decreases_ the error rate - at least to a point.  In my
(limited) experience with Taq, a starting [dNTP] of 50 uM gave zero errors
in target products of 1.3 and 1.9 kb, sequencing the equivalent of 3 or 4
different clones for each reaction.

<handwaving>
The reason for this is that the Km for dNTPs with Taq is around 20 uM -
and as you limit the [dNTP] the enzyme slows down and therefore the
occurence of errors is reduced.  With the [dNTP] waay above the Km the
enzyme zips along and can easily force in an incorrect nucleotide before
it is 'noticed' - and too late! The pubs are all shut.
</handwaving>

Then again, perhaps I was lucky :-)

} From my limited experience in sequencing cloned PCR products, it's not as
} bad as this. 

Just my tuppence.

R

--
Richard P. Grant  MA  DPhil        University of Oxford
Nuffield Department Obstetrics and Gynaecology      FFPGP
http://users.ox.ac.uk/~lady0266    http://www.molbiol.ox.ac.uk/~rgrant
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