Quantitative PCR (qPCR) and "tripple bands"
gal-1 at bones
Thu Jun 15 09:11:55 EST 1995
Yeah, we had it happen once, doing PCR on activins. The problem appears
to arise when synthesis doesn't go completely to the end of every strand
in one round. Then in the next round, the incomplete strands will act as
a primer. Becasue there are two closely related but non-identical
templates, some of this partially elongated pseudo primer will anneal to
the inappropriate template, finish elongating, and thus yield a chimeric
molecule that can then be amplified. It happened rarely enough that it
wasn't a problem for our purposes, but we did all our subsequent work
with cloned and sequenced PCR products.
On 15 Jun 1995, Holger Bohnemeier wrote:
> In order to quantitate mRNA from several tissues I've established an
> RT-qPCR. I'm dealing with a 450 bp wildtype amplicon and a 410 bp dele-
> tion mutante. If either one of the above is shown on an EtBr-stained
> agarose gel all seems rather fine. If both are put in a single PCR
> together (as it is usual for qPCR) sometimes I can see a so called
> "tripple band" instead of the expected double band (the signal of the
> wildtype and the deletion-mutante). This extra band is just between
> the to expected one. I guess it is a double stranded hybrid synthesized
> out of a wildtype and a deletion-strand.
> The question to the community now: Has anyone notified such an artefact
> Any suggestions or ideas are welcome!
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