3' overlaps in PCR

Brain Foley brianf at dna.uvm.edu
Wed Dec 16 11:28:17 EST 1992


In my experience a three-base comlimentarity 5'-GAC-3' on one and
5'-GTC-3' on the other lead to complete failure to get the desired PCR
product.  I got all primer-dimer.  With a two-base complimentarity I
was able to get some product if the initial template concentration was
high enough.

Although 3 bases of anealing does not seem like much, consider the
high concentraion of primer relative to template DNA in the initial
rounds. And consider that once a single primer-dimer is formed it acts
as a template with 100% complimentarity to BOTH primers.

I say avoid 3' complimentarity at all costs!
--
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*  Brian Foley               *     If we knew what we were doing   *
*  Molecular Genetics Dept.  *     it wouldn't be called research  *
*  University of Vermont     *                                     *
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