Primer Tm for sequencing

Wed Apr 12 09:46:11 EST 1995

Chris Silvia asks:

>   What is thought to be the optimal characteristics for
sequencing primers, specifically regarding Tm, GC clamping,
and primer self-annealing?  I use alkaline conditions to 
denature sequencing template in the standard Sequenase
way, but the availability of primer design software has
made me think that a more rational approach to sequencing
primer design is possible instead of the usual hit-and-miss
eyeball method.

The only major problem we ever have with primer design with respect
to sequencing is the inadvertant priming on secondary sites
due to chance matching.  The typical sequenase priming procedures
involve low stringency priming and hence exacerbate this problem.
Methods available to fight this off include:
1) Search against vector & other known seq. in the template 
for matching at 3' end.
2) Keep the template short.
3) Make the primer less stable on the 3' end than on the 5' end
to make weakly complexed primers unlikely to prime.
4) Use cycle sequencing or other high temperature sequencing that
allows priming at higher stringency.

Of course hairpins and other dramatic self-complementarity is obviously
bad for sequencing primers, but generally I've found that anything bad
enough to screw up sequencing is easily noticed by inspection.

If you go to high temperature priming, then you have the added constraint
that you need to be sure the primer is stable at the annealing

A final consideration is that if there's any possibility of wanting
to use the primers for PCR in the future, it pays to try to make them
all with the same Tm in the first place.

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

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