[ please ignore header contents, tin seems to be out to lunch ...]
micha at myhost.subdomain.domain wrote:
:ajb at seqnet.dl.ac.uk wrote:
: : Does anyone have a citation for the basis of the melting point calculation
: : used in the PRIMER program?
: The Q&A document, however, says it is _not_ using GC/AT ratios but thermo-
: dynamic data ...
: We will check this out, but we are running PRIMER 0.5, so YMMV :-)
We checked it :
1) Primer 0.5 ( the up-to-date version ) does not have
any quote in the source code,
2) comparison with the energy rule tables we keep here (see quote
below :-) ... ) revealed that Primer is using the Breslauer
parameter set ( Breslauer et al. (1986) PNAS 83, 3746-3750)
One short comment on these parameters:
The Breslauer set is 'exaggerating' the stability of C/A stacks (compared
with other data sets and compared with experimental results obtained by
us),
and one PCR user in our lab definitely got the stability of an A/T rich
primer wrong by this flaw.
Maybe the old rule-of-thumb (2 deg per AT, 4 deg per GC) would sometimes
be nearer the mark ... :-)
Now that I got a fresh copy of the Primer sources, I will try to modify
the energy parameters to either the Klump or the Gotoh parameter set.
A table of these parameters plus the appropriate references is found
on p. 2761 in the recent NAR issue.(see below)
: Nucleic Acids Research, 1994, Vol. 22 No. 14 pp 2760-2768
: G. Steger :
: Thermal denaturation of double-stranded nucleic acids:
: prediction of temperatures critical for gradiemt gel electrophoresis
: and polymerase chain reaction
: : Thanks in Advance
: : Alan Bleasby
: : daresbury
Michael Schmitz
Biophysics Dept.
University of Duesseldorf
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e-mail: michael at alpha.biophys.uni-duesseldorf.de
