Proofreading x engineering terminal sites

Brian Foley brianf at med.uvm.edu
Mon Mar 14 09:39:55 EST 1994


Petr GRUZ (gruz at nihs.nihs.go.jp) wrote:
:         I am trying to introduce terminal restriction enzyme sites into my 
: PCR product by primers which have about 15 bp homology with the template at 
: their 3'-end and some mismatches with the template at their 5'-end (the 
: region of hopefully engineered restriction site).  I am using the 
: ...[deletion to save space]

: I have a suspicion that the other side of the 
: high fidelity of Pfu is that the proofreading activity of Pfu causes it to 
: chew back into the primer and reach the mismatches between the 5'-end and 
: the template.  That may correct the sequence of restriction site according 
: to the sequence of template and that may be why the PCR product does not cut 
: and does not ligate.

	Only the ORIGINAL template has a mismatch with your PCR primer.  All
PCR product of the correct length is made using your primer (now 
elongated by the polymerase) as template for the other strand.  I suppose 
it might be possible fot the Pfu to correct a few of your primers, but I 
doubt it could "repair" more than a few of them.

--
********************************************************************
*  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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