PRIMER: melting points

micha at myhost.subdomain.domain micha at myhost.subdomain.domain
Thu Aug 11 04:35:31 EST 1994


[ 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

--------------------------------------------------------------------------------

	e-mail: michael at alpha.biophys.uni-duesseldorf.de






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