primer selection

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Mon Dec 12 16:28:25 EST 1994


Michael Gorry writes:

> Can anyone indicate a source that can be used to obtain the information
> needed when selecting primers?
> What I mean, is there any written explanation of what selection parameters
> (Tm, primer-dimer formation, hairpin
> loops, and the energy values associated with these, salt concentrations,
> primer concentrations, etc) to use as cutoffs
> when selecting a possible primer.  

These are my personal recommendations:

Tm:  Match primers within 1 degree if possible.  As a matter of
convenience I try to make all primer pairs so they work with an
annealing temp of 55 C.   For NCBI's Oligo program (no affiliation),
this would be a predicted Tm of 52.  For the Whitehead PRIMERS program
this would be a predicted Tm of 60.  If you find youself having to
titrate the temperature downward a lot (or adding extra Mg) and then
raising spurious bands, then your prediction algorithm is inaccurate
and you're subjecting yourself to imbalanced primers.  You can
arbitrarily pick a higher or lower target Tm to balance on.  Tm's just
under the annealing temperature can clean up difficult specificity
problems, but put a lot of stress on the accuracy of your Tm
prediction.  Tm's below 45-50 may subject you to problems with template
hairpinning.  Any template of over 60% G+C may require special
handling and special primer design.

I try hard to find oligos where the Tm of the 3' 5 bp in isolation is
lower than other 5bp windows at the 5' end or the middle of the oligo.
This seems to cut down on spurious priming.  

Salt conditions:  I insist that the primers work at 1.3 mM Mg, 50 mM
Na.  Again, if you find you have to titrate Mg a lot, then your
prediction algorithm is unsatisfactory and you're subjecting yourself
to a lot of side effect problems.

Primer dimer:  I arbitrarily chose not to accept primers with 3/3
matches involving the 3' end, or 6/7 matches involving any 3' end. 
This is probably conservative, but I haven't seen a primer dimer since
adopting this criterion.

Hairpins:  If they involve a 3' end, then the above applies;
otherwise, they only are a problem if the stem holds together at the
annealing temperature.  Unfortunately, most software makes the
stability prediction for irrelevant conditions, like 25C or 1 M Na, 0
Mg.  However, stable hairpins aren't that common, so simply avoiding
anything with a negative delta G however it's calculated will keep you
out of trouble.

Primer concentration:  I've never seen this to be an issue, but we
never stray from the standard concentration.  Sometimes people cut
primer way back because they are trying to label with it, and end up
starving the reaction.  If you want super high sepcific activity, you
amplify with cold primer first, purify the fragment, and then
polymerize with the labelled primer, not expecting much amplification.

Steve Hardies, Dept. of Biochem., Univ. of Texas HSC at San Antonio
Hardies at thorin.uthscsa.edu




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