Taq polymerase trivia

tom chappell tchappell at znet.com
Thu Nov 25 00:53:57 EST 1999


In article <383C3109.7FFCA949 at med.unc.edu>, Stephen Snowdy
<snowds at med.unc.edu> wrote:

> The question is *why* is it necessary to have the overhang, i.e. what
> part of the polymerazation mechanism requires that the overhang be
> added?

It isn't required or necessary and is never 100%, even with extended
incubation at 72C. With a normal Taq PCR reaction, you can clone products
that do not have the added A. All polymerases have error rates (addition
of non-base paired nucleotides), which are corrected in vivo by a variety
of mechanisms. At the end of a PCR reaction, the place where Taq's
mistakes are most visible are at the ends of the PCR products, and are
increased by extended incubation at 72 C. The mistakes it makes in the
middle of the fragment are covered up by further polymerase action.

TTTTTTATTTTT
AAAAAAAAAAAA

The A in the middle there is exactly the same type of mistake as

TTTTTA
AAAAA


If you give Taq the following substrate and only dATP,

TTTTT
AAAAAAAAAAAA

it will add an A

Pfu will also, but it will recognize it as a mistake and remove it with 3'
exonuclease activity




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