Rapid Amplification of cDNA Ends (RACE)

Phillip SanMiguel pmiguel at bilbo.bio.purdue.edu
Mon Sep 23 10:23:13 EST 1996


3' RACE works okay with an oligo-T primed reverse transcription --
but I never had any luck using just the oligo-T as a PCR primer.  Instead
I used an oligo-T+adaptor primer where the adaptor was 20 bases of some
reasonable G+C content containing a couple of restriction site for subsequent
cloning of the band. The PCR I did with just the adaptor (no oligo-T; i.e.
another primer) and the gene-specific primer.
  Even so I had to do 2 sets of 40 cycle PCR rxns -- the second set with
a nested gene-specific primer.  I never could trust the bands I got as
being the real thing -- I always used a Southern to check out which bands
were a specific product.  I think this may be an organism-specific problem.  
I did my amplification from maize endosperm mRNA.  Most, if not all, maize
RNA contains lots of LTR-retrotransposon RNA in it.  I doubt this is true
of many organisms.
  Also, products larger than 500 bases were very weak.  I don't think I ever
saw a product larger than 700 bases without hybridization.  PCR techniques
are more sophisticated now.  So this may not be a great problem.


In article <51vdfa$c8i at pulp.ucs.ualberta.ca>, plagali at gpu2.srv.ualberta.ca
(Pamela Lagali) wrote:

> Hi,
> 
> I have been attempting to look for full-length cDNAs using both 5' and 3'
> RACE, however have had no success with the 3' RACE procedures I have
> tried.  I think the problem may lie in the steps subsequent to
> first-strand cDNA synthesis with a gene specific primer, namely PCR using
> an oligo(dT)-based reverse primer.  Control PCR reactions using two
> gene-specific primers have worked, but I have been unable to obtain
> further downstream sequences closer to the 3' poly(A)+ tail.  Does anyone
> have any experience with RACE?  Has anyone experienced success with PCR
> using an oligo(dt)-based primer to amplify 3'-end cDNA sequences?
> Any assistance would be greatly appreciated!
> Thanks,
> Pamela Lagali



More information about the Methods mailing list