Robert K. Pope
POPE at SCI.WFBR.EDU
Sun Mar 2 14:36:58 EST 1997
Dr Koen A.L. De Smet wrote:
> I have the sequence of a gene of two related bacteria. I would now
> like to set up PCRs to amplify this gene from other species in the
> genus, and then sequence the PCR product. Can anybody give any tips on
> degenerate PCR. Advise on the following questions are much
> 1. How much redundancy is still acceptable in the primers?
> 2. Should I use all possible codons, or allow for mismatches, to
> minimise redundancy?
> 3. Should I increase primer concentration in the PCR?
> 4. Any tips on annealing conditions?
> 5. I intend to use these degenerate oligos as sequencing primers as
> well (which works fine for specific oligos), should I use more primer
> than in standard reacions?
> Any help, tips or advise muc happreciated!
Answers to your questions based on our limited, but successful,
experience with cloning using degenerate primers:
1. Minimize redundancy as much as possible by using inosines in
positions of 4-fold degeneracy. Elsewhere, use mixes of the possible
nts. The exception to this is the 3' five or six bp, which should
contain all possible nts.
2. If your template is not present in high abundance, you are asking
for trouble by allowing for mismatches. Use all possible codons and
inosine, as above.
3. We used 200 nM of each degenerate primer.
4. Annealing conditions will depend upon your primers. We had luck
with touchdown PCR. To estimate the starting temperature range, we
determined the Tms predicted if all unknown positions were GC and then
again assuming that all unknown positions were AT. The starting
annealing temperature was a few degrees above the higher temperature and
the final cycles were performed 4-5 degrees below the lower value.
Then, we adjusted the range up or down depending upon whether we had a
lot or no bands. If there were only 1 or 2 bands, we cloned and
sequenced the suckers. If they were not the right thing, we both tried
another temperature range AND ordered new primers. It wasn't elegant,
but it eventually worked.
5. My best advice here is: DON'T! You'll probably be much happier if
you end-sequence your candidate products using known, tested primers
after cloning into a bacterial plasmid. Then, you can use known
sequences inside the primer sites for primer walking or for re-PCR.
Elizabeth J. Luna
Worcester Foundation for Biomedical Research
222 Maple Avenue
Shrewsbury, MA 01545
Tel: (508)-842-8921, x275
LUNA at sci.wfbr.edu
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