GC melt

Tom Knight tk at pasteur.ai.mit.edu
Fri Nov 5 18:50:54 EST 1999


Another useful reference with a view consistent with Swanson's is
Wojciech Rychlik's chapter "Selection of Primers for Polymerase Chain
Reaction" in White, BA, PCR Protocols, Current Methods and
Applications, Methods in Molecular Biology Vol. 15, Humana.

He asserts that what is important about good primers is their ability
to bind at the middle and 5' end before binding at the 3' end, if one
is concerned about specificity.  Otherwise, the 5' end binds and
primes nonspecifically.  Makes sense to me.  I have noticed that the
good (i.e. they work for me) primer generating programs, such as
Primer3 produce primers of this kind (see

http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi

)

> ssands at my-deja.com wrote:
> > In the article from The Scientist the author says
> > that one should not put a string of G or C bases at
> > the 3' end of a PCR primer because they could cause
> > nonspecific amplification products.  I had heard
> > that I _should_ do this to get a GC-clamp.

"R. John Lye" <rjl6n at Virginia.edu> writes:
> Well, Debra Swanson suggests avoiding *three* G's or
> C's at the 3' end of the primer.  I aim for *two* G's
> or C's to get a GC clamp, so there's no conflict




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