RT-PCR problem.....HELP!

Warren Gallin wgallin at gpu.srv.ualberta.ca
Tue Jun 14 11:16:37 EST 1994


In Article <2tjuos$3hi at unicorn.ccc.nottingham.ac.uk>,
mbxspd at unicorn.nott.ac.uk (Simon Dawson) wrote:


>  Currently, I reverse transcribe a mixture of total RNA (from
>experimental samples) and in vitro control RNA. The control RNA is made
>from a deletion construct of the cDNA I'm trying to assay. The deletion
>in the control RNA is such that both the test and control RNA's can use
>the same pair of primers in the PCR step.
>  The problem is arising during analysis of the PCR products on a
>standard, ethidium bromide stained agarose gel. What seems to be the
>problem is that I'm experiencing some sort of heteroduplex formation
>between the PCR products generated from the control and sample RNA's
>(this problem dosn't occur if I leave the control RNA out of the
>procedure)! That's making things messy.
>  The PCR cycle used is:
[specifics deleted]
>  Now, unless I'm mistaken, surely all of the products should be
>homoduplexes at the end of this program?
> Anyhow, does anyone out there have any suggestions as to a reliable
>way of getting around this problem? Has anyone experienced anything
>similar? Is it possible that my primer concentrations are falling too
>low towards the end of the reaction sequence (I currently use ~20
>picomoles of each primer per reaction, 200uM dNTP's).
>  Any help/email's would be greatly appreciated!


I encountered this problem doing PCR of activin, in which there are several
closely related but non-identical mRNAs that all amplify with the same
primer pair.  What we think happens is that you can sometimes get a partial
extension on one cDNA that will then hybridize with the other form on the
next cycle and extend to completion.  The result is a chimeric molecule that
will then amplify as well as the non-chimeric templates.  The problem was
not so great for us because we sequenced a bunch of subcloned products and
were able to figure out what was going on.  My only suggestion for you is to
try a longer extension time to minimize the production of
less-than-full-length strands during amplification.
Warren Gallin,
Department of Zoology, University of Alberta
wgallin at gpu.srv.ualberta.ca



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