nash at nrcbsa.bio.nrc.ca
Mon Mar 14 15:58:33 EST 1994
In article <wgallin.1114058593A at news.srv.ualberta.ca>,
Warren Gallin <wgallin at gpu.srv.ualberta.ca> wrote:
>In Article <2m24e5$ps7 at news.u.washington.edu>, findley at u.washington.edu
>(Seth Findley) wrote:
>>Does anybody out there have experience with doing PCR with highly
>>degenerate oligo pairs?
>>I was thinking degeneracy on the order of 128 or 256 complexity.
>>(I have a number of "A or C or G or T" positions when I reverse
>>translate a segment of a protein I am looking for homologs of)
>>Is this commonly problematic?
>>Does one have to add much more of the primer to make up for the complexity,
>>or do you just go a few more cycles?
>>Any tips greatly appreciated from people who have had success with this.
> We've been doing this, and have been successful in pulling out the clones
>we want, in particular ion channels from jellyfish. So have a number of
>other labs. The short answer is, it works, but with problems. The good
>news is that you don't have to use any more primer than a normal PCR, nor do
>you have to run more cycles; because you are using degenerate primers, once
>you start priming off PCR product rather than template, there are always
>several primers that only differe by one base from the PCR product template.
>You do need to optimize the buffer and the annealing temperature. Both of
>these factors haved turned out to be critical in our hands, and totally
I couldn't agree more. I would optimize the Mg++ and the anneal time
and temperature. In my hands, I found that lowering the anneal temp
too much VASTLY increased the number of spurious bands. I think that
you're better off trying to keep the anneal temp high-ish, and
lengthen the anneal time. With the last set of degenerates I did, the
"theoretical" anneal temp was 40 deg (assuming worse case), a
quick-and-dirty "anneal and release" experiment looking at
temperature-related release of radiolabelled primer after annealing
gave a peak at 58 deg, so I used 55 deg in my reactions. I got the
best yield with a two min anneal instead of my usual 1 min.
> The bad news is that you will obtain lots of bands that are not what you
>want; this isn't too surprising, since the degeneracy you are introducing is
>decreasing the specificity. You may have to sequence a lot of PCR products
>to get the one that you are looking for, and that is probably the fastest
>and most rigorous way to check your products. There are a number of
>chapters in handbooks about this approach. We found that once we had basic
>PCR down, it was just a matter of trying a lot of conditions and grinding
>through the sequencing.
Again, well said - good point. In my case, the band with the
brightest intensity was not the desired product (so I wasted several
months cloning and sequencing crap!). Now, I don't do anything until
after I have sequenced the band and verified it's what I want!!! I
had variable results sequencing PCR bands using degenerate primers
(but NO problems sequencing PCR bands using non-degenerate primers) -
even with cycle-sequencing. Since it only took an extra 2-3 days to
clone the products, I just cloned a few bands and sequenced them.
That way, I had heaps of probe for the colony lifts.
It's a pain, but it works if you avoid shortcuts, and check your tail!
John Nash (nash at nrcbsa.bio.nrc.ca)
Institute for Biological Sciences, National Research Council of Canada,
Yet another Aussie-in-exile ;-)
*** Disclaimer: All opinions are mine, not NRC's! ***
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