Primer dimer mystery
wgallin at gpu.srv.ualberta.ca
Mon Apr 3 11:43:58 EST 2000
Susanne Rohrer wrote:
> Gregor Bucher wrote:
> > I cut out the PCR band from the
> > >first rxn and used it as a template with the Expand Pol from my DIG
> > >labeling kit. I got a product AND the primer dimers were gone (mostly)!
> > In genomic DNA you have many places where degenerate primer could prime....
> > If you use a purified PCR-template every bit of DNA is
> > template - this makes it dificult for the primers to misprime and do their
> > primer dimer stuff.
> Thanks, but I know all that. (It's kind of a theoretical question, since I
> got what I wanted)
> I thought the formation of primer dimers involves _just _ the primers -
> that's why I couldn't come up with an explanation .
I've been seeing a lot of this lately, and have a provisional explanation. If
your primers are really bad, they will make primer dimers all the time. If they
are really good, they will never make primer dimers, even if there is no
template present. However, if you have to work with less than perfect primers,
the formation of the primer dimer may be a very low probability event, only
occurring late in the cycle if there is no template present. If there is
template present, the desired PCR reaction outcompetes the primer dimer
reaction. Thus, if you got primer dimer and desired band when you were using
whole genomic template, which has very few target sites, and you then used cut
out band material, which will have a huge number of specific sites for the
primer, the reamplification will deplete the reagents before the primer dimer
reaction can get going.
Although I haven't rigorously tested this scenario, I have used serial
dilutions of gel purified PCR product, and as the amount of template dropped off
the primer dimer product increased in intensity.
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