Universal PCR

Dick Beeman beeman at crunch.usgmrl.ksu.edu
Mon Aug 1 11:07:09 EST 1994

In article <4060220020 at uamercury.uark.edu>, DRHOADS at MERCURY.UARK.EDU ("Doug
Rhoads") wrote, in response to my post about universal PCR:
> When does one decide to add extra of a degenerate primer.  The `universal' 
> primer Dr. Beeman describes would have an approximate million-fold   
> (4^10=3D1,048,576)=degeneracy.  We have worked on amplifying homologous gene 
>  regions based on 
> degenerate primers developed from reverse-translation of conserved 
> protein domains.  Usually when we have 30 or 40,000 fold degeneracies we 
> up the primer 2-3x (from around 0.8 uM in the reaction).  In other tests 
> with specific, non-degenerate primers we have obtained much more 
> specificity in PCRs when we drop the primer concentrations to around 0.1 
> uM.  But with degenerate primers such as the `universal' primer, after the
> first cycle, don't you generate a million-fold excess of potential 
> competitive inhibitors for the priming site, plus reduce the concentration  
> of effective primers a few thousand fold??...(stuff deleted)

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.  
Dick Beeman
beeman at crunch.usgmrl.ksu.edu
"One of the advantages of being disorderly is that one is constantly making
exciting discoveries".  A. A. Milne

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