saunders at bio.vu.nl
Thu Nov 19 08:04:39 EST 1998
Shiao Wang wrote:
> If contaminating dsDNA was tailed at the 3'end with C, both strands
> would serve as template when (GGG)n is used as primer. However, the
> newly synthesized strand (containing GGG) would not be a useful template
> for further PCR unless it contains an annealing site for the gene
> specific primer. Thus, am I correct in thinking that if the the
> contaminating DNA lacked annealing sites for the gene specific primer,
> the DNA would not be a problem?
> I know several students who have had success with 3'RACE but are
> struggling with 5'RACE. Other than the fact that longer cDNAs are more
> difficult to obtain than short cDNAs, is there a good reason for this?
> Recommendations of references on 5'RACE that you think were particularly
> helpful would be appreciated also. Thank you for your attention.
> Shiao Y. Wang
> Department of Biological Sciences
> University of Southern Mississippi
I guess the answer is that DNA lacking the gene-specific primer site
wouldn't be a problem, but if there were contaminating DNA there might
always be the possibility that tailed DNA plus the site was present, hence
an RNase-free DNase treatment of the RNA is essential. And there's always
the possibility that the gene specific primer is not so gene specific! I've
found that primers often differ in their usefulness when used for different
things; e.g. a good sequencing or PCR primer may not be a good cDNA
synthesis primer, and so on.
Two good references on 5'-RACE:
Frohman, M.A. Methods in Enzymology 218: 340-356
This contains detailed protocols by the originator of the method. I've
always used these virtually as published, with good results.
Schaefer, B.C. Analytical Biochemistry 227: 255-273
This is a very detailed discussion of all the RACE variations, with lots of
refs. and a protocol for ligation-mediated RACE.
Department of Molecular Cell Physiology,
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email: saunders at bio.vu.nl
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