Universal PCR

[Doug Rhoads]

Dick Beeman's response to my response to his response:
>I agree with Doug, that, like PCR in general, it seems that universal PCR
>should not work.  It's a good thing people roll up their sleeves and try
>things that are theoretically impossible!  All of Doug's arguments seem
>reasonable to me.  All I can offer are some observations on the conditions
>that do work.  The universal primer (1 million-fold degenerate!) and
>specific primer 1 are used at 1 pmol per ul and 0.1 pmol per ul,
>respectively, in the first PCR reaction. In the second reaction, specific
>primer 1 and T7 are both at 1 pmol per ul.  It may be that the first 9 or
>10 bases on the 3' end of the universal primer are the only critical ones,
>and that the effective degeneracy is only about 250-500-fold (the first 4
>or so bases at the 3' end are specific).  Another point is that only a few
>copies of the target sequence may be generated in the first round, but this
>may be sufficient.  There are never any visible bands after the first
>round, and EthidBr-stained gels of round 1 product would suggest a complete
>failure of the PCR.  Remember that the 5' linker (T7 promoter sequence) is
>effectively (partly) converted to a gene-specific primer for round 2, since
>it can only anneal to three types of templates:(A) amplicons generated in
>round 1 that were primed by "forward" specific primer 1 and "reverse"
>universal primer, (B) extension products of these amplicons generated by
>specific primer 2 in cycle 1 of round 2, and (C) amplicons generated in
>round 1 that were primed on both ends by the universal primer.  Category
>(C) is apparently not a major problem, since most universal PCR clones
>turned out to be the right stuff.  
>-- 
OK.  But if your application of Universal Primer PCR  is part of your 
work with Triboleum you are working with genomes 10-100x smaller than 
mammalian and plant genomes.  Could this partly explain getting no other 
bands.  During my stay there at KSU I did degenerate PCRs on hamster DNA 
and we usually got 2-4 bands.  Now working in yeasts and fungi we only get 
one band.  Of course we were also working with genes that had 10s of 
pseudogenes around.  
Doug Rhoads                  || Dept. of Biological Sciences
drhoads at mercury.uark.edu     || 601 Science Engineering
drhoads at uafsysb.uark.edu     || University of Arkansas
501-575-3251                 || Fayetteville, AR 72701