oligo dA analogues as PCR primer

tweissen at my-deja.com tweissen at my-deja.com
Tue Jul 11 04:57:23 EST 2000


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 have tried various kits for mRNA isolation, but so far with little
success (not much difference in product yield from cDNA versus RT
control).

I have thought of trying using a 3' primer complementary to the
polyT tail (as in 3' RACE) to selectively amplify the cDNA, then
probe with an internal oligo, or use the Taq man technology.
PolyA primer will have relatively low Tms and may give very low
yields.

Has anyone got PCR experience with dA analogues with increased
DNA duplex stability?
Could sensitivity could be improved by adding a few dAs to the
3' end?

Thank you for your help

Thomas



Thomas Weissensteiner
Edward Jenner Institute for Vaccine Research
Compton RG20 7NN, U.K.
T.:   0044 1653 577900 x3931
Fax: 0044 1635 577901

PCR Jump Station: http://www.highveld.com/pcr.html


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