degenerate PCR

Andre Hamel hamel at ccu.umanitoba.ca
Tue Apr 13 15:23:48 EST 1993


Some questions regarding your choice of degnerate primers for PCR:

 Do the primers have any degeneracies at either end, esp. the 3'
end?
What is the starting/target source .. DNA or RNA?
Were the primers desalted and either gel or RP-HPLC purified?
What thermostable DNA pol are you using?
Taq is NOT particularly stable after 30 cycles, especially when large temp
fluctuations (35oC to 95oC) occur for many cycles (greater than 30 cycles).
The Vent pol. from NEW England Biolabs, and Pfu pol from Stratagene are
VERY stable and can withstand up to 3 min per cycle at 95oC for at least
up to 50 cycles! (I tried this with serial dilutions of a control DNA
template, down to 10 E-10 dilution, for 30, 40, 50 cycles at 95oC for 3
min, 45oC for 30 sec, 70oC for 30 sec up to 5 min by the end ... product
intensity DID increase noticeably by 50 cycles, versus 40 and 30 cycles,
and ONLY observed product from most dilute samples when 50 cycles was
used. This involved a 16 fold deg. pair of primers for a 400 bp product
from a cDNA contained in a cloning vector.
 
The couple of times I'd used primers with inosines were fine, just takes some
"playing" with the conditions in order to get specific product
(95oC for 30 sec, then anneal/elong. temp/times to adjust.) .. once again,
avoid placing inosines at the ends.
Minimum primer length would usually be dictated by annealing temp ... want
to keep Tm as high as possible in order to reduce non specific background
product formation.
Higher degeneracy primers tend to need more mass of primer per reaction ..
for example if non degenerate, 50 ng of a pair of 15 mer primers should
work, whereas if 32 fold degenerate, perhaps 1 ug of each primer would do.

Have you tried 40 to 50 cycles, starting with your human cDNA (say a few ng of
target DNA) ... 95oC for 3 min, then 40 to 50 cycles of: 95oC for 30 sec, 35oC
for 30 sec, 70oC for 30 sec, with the 70oC step increased by about 3 
seconds per cycle or by 15 seconds after every 5 cycles, then hold for 5
to 10 min at 70oC at end. 

good luck

----------------------------------------------------------------------------
Andre Hamel                                 email: hamel at ccu.umanitoba.ca
Manitoba Veterinary Services                lab tel.: (204) 945-7630
545 University Crescent                    home tel.: (204) 275-1204
Winnipeg, Manitoba                               FAX: (204) 945-8062
CANADA
R3T 5S6
------------------------------------------------------------------------------

 >
>
>Dear Netters,
>
>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. Al
>
>
>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. 
> 
>
>The problem: 
>
>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, the
re 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 suffic
>
>
>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;
>
>Questions:
>
>
>- 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 p
lace?
>- 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, 
>Olli.



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