I would like to open a discussion on DNA melting temperature - in particular
with regard to oligonucleotides. I would like to design primers for PCR, and
I am currently using the Breslauer nearest neighbour values of entropy and
enthalpy to calculate the Tm based on the nucleotide sequence.
HOWEVER, what if there is a mismatch? (obviously this is not desirable for
designing primers but it is useful to know if it binds anywhere else...) In
this case, how is the Tm calculated? I have seen, in Manatius, the suggestion
that 1 degree C should be subtracted for each mismatch (?) but this is based
on the Suggs equation ( Tm = 4 * (GC) + 2 * (AT) etc.) !
I have also recently read a paper that calculated delta G for a sequence that
DID have a mismatch in it and there is NO WAY I can duplicate the math (in
fact, if I ignore the mismatch completely, I still am off by a factor of 2).
The paper is:
"Use of an RNA Folding Algorithm to Choose Regions for Amplification by the
Polymerase Chain Reaction". Analytical Biochemistry 185: 57 - 62 (1990).
Luke Pallansch, Howard Beswick, John Talian, and Peggy Zelenka.
Does anyone have any thoughts on this subject and/or e-mail addresses of any
of the authors? Any help would be appreciated!
Dorothy
--
Dorothy Lowry
Dept. Medical Biochemistry
University of Geneva, Switzerland dlowry at cmu.unige.ch
Graduate student and PC/GENE programmer phone: +41 22 70.25.490
