Function of dUTP in real-time PCR?

Dag Rune Gjellesvik dagrgREMOVE at NOSPAMonline.no
Mon Nov 1 15:58:32 EST 2004


"Paul Wary" <paul_wary at yahoo.com> skrev i melding
news:c4879cbb.0410290643.b887be1 at posting.google.com...
> Tom Knight wrote:
> > paul_wary at yahoo.com (Paul Wary) writes:
> > > I read in a description of mixes used for real-time PCR that they
> > > contain dUTP "to prevent re-amplification of carryover PCR products".
> > > Can anyone tell me how this works or give me a reference?
> >
> > The mix also contains an enzyme which selectively degrades dUTP
> > containing DNA.  This enzyme is degraded early in the PCR process.
> > Carryover PCR product will have dUTP and will be degraded.  Real
> > template will have dTTP, and will not be degraded.  The final
> > amplification product will contain dUTP, and will be a poor template
> > for the next PCR reaction.
>
> Ummm, where do I actually get any carryover PCR product from? I use
> real-time PCR for quantification of gene expression levels, so I
> reverse transcribe mRNA to cDNA and use that cDNA for the real-time
> PCR. I can't see where I get that carryover PCR product from.
>
> Paul

Hello,
Well, if you use the same amplification primers, you still will amplify any
PCR products along with your cDNA. And you won't know if your amplicon came
from cDNA or PCR product. Maybe one or two extra template molecules doesn't
matter too much in gene expression studies - it's worse if you want to know
the presence of HIV virions, though.

Dag Rune Gjellesvik
Biotec Pharmacon ASA





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