Enhancement of PCR? (by boiling primers)

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Tue Mar 7 11:45:16 EST 1995


Richard Hastings (mbxrh at unicorn.ccc.nottingham.ac.uk) wrote:
: I've recently been having some problems with contamination of PCR, i've
: had some advice as to how to minimise contamination and smearing.  One
: such piece of information was to boil the PCR prmers for 5 minutes then
: snap freeze in liquid nitrogen and thaw on ice before using in the PCR
: reaction.  This apparently reduces smearing of products, i found that
: this treatment also enhanced the PCR reaction somewhat, producing
: stronger bands in both my main reaction as well (unfortunately) as my
: no template negative control.
: I generally store aliquoted stocks of primers at -20C then take an
: aliquot and store at 4C and use that rather than refreezing each time
: (which is supposed to have a detrimental effect on the primers).
: Does anybody know why this enhancement is occuring?


The contamination problem is an unrelated issue.  

There has been a recurrent thread in this newsgroup that PCR primers
go bad on storage and can be rescued by boiling.  Hairpinning wouldn't
behave this way; it would have to be an intermolecular interaction,
maybe even an  aggregation.  As such it might be true that you'd have
to boil the stock; ie. if you take out a sample and boil it, you might
have left the primer behind in the stock tube.  I'm guessing that this
is the case because otherwise the denaturing step might be expected to 
accomplish the same dissociation.  In any case, the end result of this
problem  would be that you simply don't deliver the correct amount of
usable primer to the rxn tube and hence starve the rxn.  Smeared bands
or no bands would be two reasonable outcomes.  What you're calling
enhancement would simply be restoring the rxn to its nominal
condition.   

I presume the freeze thaw was intended as a suggestion for how to
store the stock so that reboiling wouldn't be necessary.  Snap
freezing inhibits all manner of  intermolecular interactions (but not
intramolecular rxns. like  hairpinning).  I also presume that the
freeze/thaw would be irrelevant if you intended to use the primer
right after boiling it; and that storing at 4 C after freeze thawing
would be counterproductive.

All of the above is guesswork.  I'd be interested in hearing from 
anyone who has a better grasp on this problem. 

Steve Hardies, Assoc. Prof. of Biochem., Univ. of Texas HSC at San
Antonio
Hardies at uthscsa.edu


 



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