RT-PCR Using Oligo-dT Primer

bhjelle at carina.unm.edu bhjelle at carina.unm.edu
Wed Mar 3 12:13:09 EST 1993


A friend of mine wants to use RT-PCR and oligo-dT primer to
capture an elusive 3' end. He believes they are no more than
a few 100 bp, max, to the end of their gene's cDNA sequence,
but have not so far been able to get the polyA tail.

He insists that if he uses an oligo dT primer, coupled with
a primer from known sequence (as far 3' as they have been
able to get), the reiterative PCR cycles will eventually
select for the polyA sequence *closest* to the 3' end
of the transcript. I say that it might be necessary
to place a single base at the (C, G, A) to "anchor" the
end to the next base in the RNA, and that otherwise you
will get a smear.

Has anyone had experience with this? Do you get a smear, or
a single species of PCR product?

Brian



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