Taq activity at various temperatures?

David F. Spencer dspencer at is.dal.ca
Mon Jun 2 16:02:24 EST 1997


In article <5mfjub$1d3 at cardinal1.Stanford.EDU>, ladasky at leland.Stanford.EDU
(John Ladasky) wrote:
 
> Hi there,
> 
>         I just read a few dozen Medline abstracts trying to find what should
> be a simple piece of information: how does Taq polymerase activity vary as
> a function of temperature?  The literature says that 72 degrees is "optimal,"
> (in what way?  Yield?  Extension rate?  Error rate?) and polymerization falls
> off rapidly above this temperature.  On the cooler side, people run two-step
> PCR's at 65 to 68 degrees.  And some unconfirmed hearsay says that Taq can
> even catalyze a bit of DNA synthesis at room temperature (which is about 25
> degrees in these parts).  So has anyone actually measured various aspects of
> Taq activity at various temperatures?  If so, where is this data reported?


As a followup to my earlier post, I today stumbled on a key reference that
answers the first question above.  In the book "PCR Protocols-A Guide to Methods
and Applications" edited by Innis, Gelfand, Sninsky and White (Academic Press,
1990), in the first chapter, by Innis and Gelfand, in the section 'Primer
Extension' it says:

   "Primer extensions are traditionally performed at 72 C because this
temperature was near optimal for extending primers on an M13-based model 
template (D.Gelfand, unpublished)."


As to the second query above, touch-down PCRs do seem to use 65 or 68 C for the 
anneal-extension step.

Dave.

-- 

David F. Spencer, PhD
Dept. of Biochemistry
Dalhousie University
Halifax, Nova Scotia, Canada

dspencer at is.dal.ca
dspencer at rsu.biochem.dal.ca




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