Calculating Tm - Help Needed

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Wed Jul 19 16:21:57 EST 1995


Shiao Wang writes [concerning the equation:
Tm=81.5-16.6(log[Na+]) + 0.41 (%G+C)-600/N to calculate Tms of
oligos]:

> By the way, someone suggested I made a mistake where
> 0.41(fraction G+C) perhaps should be 0.41(% G+C) or 0.41(50) instead of
> 0.41(0.5). Sambrook et al. does say fraction and not %.

Lest we sow unnecessary confusion, it is %G+C, Sambrook quotes it
correctly on pg. 11.46 and again on pg. 11.52 {I won't be surprised
if there is a pg. with an error in there somewhere}, and Sambrook
gives the original refs.  

As Sambrook states, it does work for oligos 14-70.  Oligo reports Tm's
by this methods, a nearest neighbor methods, and the 4xG+2xA method. 
These seldom vary by more than a few degrees from each other for the oligos
I have designed; and so any of them are adequate for designing PCR
primers.  The only cases I have seen where there is significant
variation of this method from nearest neighbor methods is when the
sequence is runny, which is a bad idea anyway.

> There does not appear to be a simple solution to the Tm of
> oligos question. The best method seems to be the nearest neighbor method
> which is not easily done with a calculator. The software Oligo uses it but
> the last time I checked, it was $640. Anyone knows of a less expensive
> software that uses the nearest neighbor method? Primer is free but I'm not
> sure about the method used.

Getting the Tm alone is a trivial component of the services offered by
the various packages out there.  Hang in for protection against
hairpin, primer dimer and false priming problems. Primer reports Tm's
about 8 degrees  higher than the true Tm, which curiously just about
covers the Mg  effect although they make no mention of intentionally
doing this. They do use a nearest neighbor method. We gave up on
primer because the version we tried was full of bugs in the user
interface, doing crazy stuff like choosing both primers on the same
strand, etc., and often simply refusing to run altogether.
Hopefully, it's been improved since then; but at the time it was
clearly only debugged for the specific job that interested the author
[choosing primers around CA repeats]. It does have the very nice
feature that it automatically finds matched primers even if they have
to be of different lengths.  Oligo (at least the version I have)
doesn't do that, so I almost never get what I want using its
automatic search functions.  Oligo impresses me as very powerful,
but very user-unfriendly.  

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



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