semi-specific PCR

[bbeitzel at biomail.ucsd.edu]

In article <34F44B7B.C1957ECA at veg-physiol.med.uni-goettingen.de>,
bahn at veg-physiol.med.uni-goettingen.de wrote:

> Dear netters,
> 
> I'm working on first strand cDNA-libraries that were immobilized on
> magnetic beads. I try to amplify a "native" well known clone out of one
> of these libraries with one specific primer (universe primer) and an
> oligo dT25 (reverse primer). I tried different alternatives: 1. two
> rounds PCR, then taking the supernatant for further PCR amplification;
> 2. 5 or 10 rounds PCR and then taking the supernatant for PCR; 3. PCR
> with the library for 35 cycles; in every case a standard PCR protocol
> with an annealing of 52-55°C was used. I always get a smear!! But if I
> test the specific reverse primer I get a big specific band! I can't
> understand what went wrong. What is the problem?
> Please help asap!
> 
> Best regards
> 
> Andrew
> 


In my experience, T is a very promiscuous base that will anneal almost
equally well to a complimentary A,T,G, or C.  I think there are actually
PCR protocols that call for using a T in degenerate oligos instead of
inosine or a mix of bases.  It may work to add a defined 5' end to your
oligo(dT), do a couple of rounds of PCR using that oligo, and then switch
to a PCR using the defined 5' end as a primer (no oligo (dT)) with your
internal forward primer.  You would likely still get a smear since the
oligo(dT) can sit down in multiple places on the poly A tail, but you
might get rid of some of the non-specific priming by the oligo(dT) alone.

Hope this helps,
Brett Beitzel
bbeitzel at biomail.ucsd.edu
Infectious Disease Lab
The Salk Institute