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

spinnaker spinnaker at austin.rr.com
Wed Jul 3 22:15:50 EST 2002


Nick Theodorakis wrote:
> 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
> 

Then this begs the question- if a mispriming event occurs in the 
presence of Pfu, will the primer be edited and then the truncated primer 
allowed to continue amplification of the incorrect template?
If so, then amplification from large templates- genomic DNA for example- 
would be best performed with Taq, or other non-proof readers.

cheers,
maturin at mail.utexas.edu




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