Need Help: "Blocking Primer" in PCR

Edwin Fink fink at clinbio.med.uni-muenchen.de
Tue Sep 12 11:24:48 EST 1995


From:       Edwin Fink 
To:           methods at daresbury.ac.uk
Subject:         "Blocking Primer" in PCR

In a PCR-experiment, our forward primer binds at 2 different sites:

For-primer>       For-primer>
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<
                                               <Rev-primer

Therfore, if we use a polymerase without 5'-3' exonuclease activity 
(e.g. the Stoffel fragment) we amplify only the short fragment;  if we 
use a polymerase with 5'-3' exonuclease (or 3'-5' exonuclease!!?) 
activity we obtain a mixture of both the short and the long fragment.

We need to optimize the yield of the long fragment!  Is there a 
polymerase with high 5'-3' exonuclease activity, a way to enhance this 
activity, or another method?






Edwin Fink
Department of Clinical Biochemistry
University of Munich
E-Mail: fink at clinbio.med.uni-muenchen.de
Tel.: +49 89  5160 2601
Fax:  +49 89  5160 4735



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