In article <9209011818.AA04993 at genbank.bio.net> LEARN at UCRVMS.BITNET writes:
>I apologize to the network for asking a question that is almost an FAQ:
>>What methods do you all use to design primers for PCR (primarily) and
>sequencing? We have OLIGO v3.4 in our lab and many of us have tried to use it
>with mixed results. My perception of primer design is that it is a "black box."
We used conserved sequences among 4 genes, designated the primers "by hand"
looking for about 40 % of GC, calculating an approximate annealing temperature
to start and lookinf or the good one by "trials and errors" (I mean by
increasing the temperature until no more amplification...
The sequences of the primers were before use checked against the available
database for homologous sequences... then really with DNA from several plants
bacteria, fungi... They are really specific...
>>1) Has anyone done the experiment comparing the use of OLIGO to a seat-of-the-
> pants, intuitive "method?"
>>2) Our impression with the results of OLIGO is that it may be too finicky. We
> use primer-pairs that were designed before we bought software. We have
> tried to use OLIGO to analyze the primers (which work in at least 75% of a
> broad sample of different angiosperm species) and it predicts that they
> might have problems. Does anyone have the same experience?
I received Oligo some weeks ago and tested our "by hand designed" primers, the
recommended annealing temperature is a little bit too high, and according to
Oligo we should have problems (dimers formation...)
>>3) Are there reviews in the literature that specifically address the issue of
> designing primers for PCR? We are familiar with "The Simple Fool's Guide to
> PCR" v2.0 by Steve Palumbi and coworkers. Is there anything that goes
> beyond this to explain theory of primer design?
I don't know "the simple..." could you send me some precisions about this
paper?
I heard a talk from Remy Griffais (Pasteur Institute, Paris, France) about the
design of primers using non-thermodynamical approach. He developped a program
to design primers using the frequence of octamers in an organism. The program
look, in a submitted sequence, for the less 3' conserved octamers after you
indicate to the program which base (virus, mammals, Homo sapiens...) of
octamers frequences to use. Griffais tested its program (running under
Windows 3.X on IBM compatible: very nice...) for several kind of organisms
and "certifies" the corresponding fraquence bases. However he did not make
frequence databases for plants (he's working on animals).
As yet the program is not available since a company is in touch to sell the
program. I asked whether plant... frequences databases are planned or not but
he does not know exactly what to do about that: he did not test the databases
by himself. So maybe he will release "uncerticated" databases for e.g. plants,
fungi...
He tald that the designed primers were specific even at 55 degres C for the
several organisms he or other scientists of Institut Pasteur tested.
So I am waiting the commercial release of the software... and hoping that
(I tald with representatives of the company) that plant and fungi databases
will be included in the program, even not "certificated".
>>>Any help that you can provide will be greatly appreciated. I will summarize
>responses.
>>Thanks,
>>Jerry Learn
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Good luck
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Yves Bertheau
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75231 PARIS cedex 05 FRANCE Tel +33 (1) 43.31.93.97
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