Dr. A. Rosenthal
arosenth at crc.ac.uk
Tue Apr 13 11:27:57 EST 1993
I am currently experiencing problems with a set of degenerate primers, that I am using in an attempt to PCR-amplify cross species. The primers I generated were chosen with two criteria in mind. Firstly I looked at conserved exonic regions of the gene in human, chick, rat, and drosphila and then at the degeneracy of the amino acid codons. Since there is relatively little conservation, with drosophila being far off most of the time, there are only to possible areas that I could design primers from. Also I c
hose to make a set of nested primers covering that conserved region, so as to allow specific amplification from cDNA by nested PCR. My primers are 17 mers and I can not easily extend them, without increasing the degeneracy dramatically. The present primers have 4 to 32 fold redundancy.
I did not manage to get any PCR product what so ever, not even with the human cDNA control, but that may have all sorts of reasons to do with the primer quality or something like that. I did check them on a gel and although they did not look too bad, there must still be a general problem. The annealing temperature for the two primers in a set was about 41C and 55 C respectively and I did the annealing at 35 C for 1 min (I also tried 40, and 30 C). Extension time was 1 min at 72C which shoul be sufficient f
or a 1.5kb expected product. I ran 38 cycles. The PCR itself worked fine, since a control experiment with another specific primer set on the cDNA works nicely. However, here are some general questions I have;
- Does anyone of you have experience with degenerated primers in PCR?
- What is the minimum length of primers to use for this kind of stuff?
- If you have also used nucleotide analogues (eg inosine) to reduce redundancy,perhaps you could let me know with what results and what difference they made when compared to the degenerated primers or did you never use the degenerated ones in the first place?
- Is it a good idea to use inosine only to replace purines or do you use it for all four bases?
- What is the maximal primer redundancy that you have used successfully?
- What PCR conditions do you suggest, any variations from the ordinary?
Thanks v. much for your comments,
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