RT-PCR Using Oligo-dT Primer

Ed Rybicki ED at micro.uct.ac.za
Thu Mar 4 02:37:54 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?

You do most definitely get a smear if you use a simple oligo-dT: this
detracts from detectability, and looks a mess, and complicates cloning,
etc.  Yes, it most definitely does help to use a 3'-degenerate oligo-dT,
with or without restriction site at 5'-end: a useful reference, which also
has a good 1-tube protocol for cDNA PCR [sound of horn blowing, slightly
muted] is :

Pappu, Brand, Pappu, Rybicki, Gough, Frenkel and Niblett, 1993.  A
polymerase chain reaction method adapted for selective amplification and
cloning of 3' sequences of potyviral genomes: application to dasheen
mosaic virus.  Journal of Virological Methods 41:9-20.

The paper describes use of a selection of unique degenerate second primers
with a 3'-degenerate 22-mer oligo-dT to amplify up to 700 bases to the
5' side of the beginning of the poly(A) tail of several potyvirus
genomes.

Good luck!

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