Taq activity at various temperatures?

Darren Natale dnatale at box-d.nih.gov
Fri May 30 15:56:15 EST 1997


Bryan L. Ford wrote:
> 
> Jerry Kropp wrote:
> 
> > Lemme respectfully suggest that there's something missing here,
> > viz.(whatever that means):
> > There's a pretty good reason for melting the ds template in the first step
> > of the PCR--so's the primers (hint hint) can get access to the ss target

> Jerry:
> 
> Quite clever, and sorta sounds reasonable... but for the "offspring" of
> the "impossible" matings at low temperatures that are often seen to
> disappear when "hot start" (that is adding a crucial ingredient of the
> PCR after it has reached a denaturing temperature) is used.
> 
> Looking for mechanisms to explain the hot start empirical data, one
> might reasonably suspect that typical genomic template may have enough
> damage (e.g. from the isolation prep) to expose some single-stranded DNA
> irrespective of the low temperature

<snip>

> The inevitable presence of damage and perhaps a few genomes from cells
> caught in the S phase at death/prep time can provide a lot of
> primer annealing possibilities at 10-20 degrees (not to mention
> primer/primer and even some untoward template/template hybridizations).

<another snip>

I too thought that the beneficial component of hot start was to reduce 
background extensions (as you propose above). But if this were true,
then 
leaving out the template from the bottom phase would be expected to kill 
any untoward annealings at low temperature.  This seems not to be
the case: If hot start is done by leaving the enzyme or the primer out
of the lower phase, it works fine.  However, if the template is the 
sequestered component, hot start does not seem to have any benefit.  The
true
benefit of hot start is preventing the elongation of primer-dimers
(based
on the above information).

Darren Natale
dnatale at box-d.nih.gov



More information about the Methods mailing list