oligo dA analogues as PCR primer

Bernard Murray, PhD spam at 127.0.0.1
Tue Jul 11 15:58:26 EST 2000


In article <8ker1v$8tl$1 at nnrp1.deja.com>, tweissen at my-deja.com wrote:

> I have had a fair amount of trouble with genomic DNA contamination
> in RT-PCR: I want to know whether a transgenic transcript is
> expressed in certain tissues. Even very, very little could matter,
> so I need maximum sensitivity.
> Unfortunately the transgene construct has no introns which could be
> used to distinguish PCR products generated from cDNA and
> contaminating genomic DNA.

I sympathise as I've been in exactly the same situation.  My guess is that
your transgene has at least some heterologous sequence present (5' UTR
or 3' UTR) from construction.  Rather than playing with something
targeting the polyA tail I'd try a primer binding to one of these flanks.
The target doesn't have to be 100% heterologous, just make sure the 3'
end of the primer is in that region.  I had very good success with that
(a primer in the region between the promoter and the start of the cDNA).
I used a second genomic-selective pair (upstream primer within the
promoter itself, same downstream primer) to check the level of
DNA contamination.

Other things I tried successfully are RNase protection using probes
across the 5' or 3' ends and primer extension.  Of course you need
much more RNA for these but the RNase protection is easier to
quantify than RT-PCR.

Note that if you are using intron-spanning primers to assess genomic
contamination you'll almost always see a positive (large band)
result if you push the sensitivity too hard as you'll start to pick
up hnRNA (which is actually nice as it gives a rough assessment of
transcriptional activity of the endogenous gene).

   Bernard

-- 
Bernard P. Murray, PhD
bpmurray at cgl . ucsf . edu
Department of Cellular & Molecular Pharmacology, UCSF






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