Will I get amplification if my last 3' nucleotide is not paired?

Nick Theodorakis nicholas_theodorakis at urmc.rochester.edu
Thu Jun 27 16:44:22 EST 2002


On Thu, 27 Jun 2002 11:27:38 -0400, Sylvain Foisy
<foisys at medcn.umontreal.ca> wrote:

>Hi,
>
>I am trying to isolate clones from transfection experiments with PCR and 
>I have lots of background with a faint band that is at the right 
>molecular weight. Is it possible to get amplification if the last 
>nucleotide of one of my primers (at the 3' end) does not hybridize with 
>the template?

In theory this should not work for a non-proofreading polymerase (like
Taq). A proofreading polymerase (with 3'exo activity) should be able
to delete the upaired base and move on.

Nick

-- 
Nick Theodorakis
nicholas_theodorakis at urmc.rochester.edu



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