Why use Klenow on a PCR product to clone?

[Sheryl White]

Brain Foley (brianf at med.uvm.edu) wrote:

: Dear BioNetters:

: 	I was recently asked why so many of the protocols people have 
: supplied to this list for cloning a PCR product include treating
: the PCR product with Klenow fragment to "clean up" the PCR
: product ends prior to blunt-end ligation.

: 	The PCR product has a 3' overhang, usually an A, added by the
: polymerase.  Klenow lacks 3'-5' exonuclease activity, and is only
: a 5'-3' polymerase (unless I'm mistaken).  Thus it does not seem
: to us that Klenow should actaully do anything to "clean up" the
: 3' overhanging A.

: 	Are we missing something???

: --
: ********************************************************************
: *  Brian Foley               *     If we knew what we were doing   *
: *  Molecular Genetics Dept.  *     it wouldn't be called research  *
: *  University of Vermont     *                                     *

  Hi Brian, nice to see a fellow-UVMer on the bionet.  In answer to your
question, I believe Klenow possesses a 3'to 5' exonuclease activity, but
lacks the 5' to 3' exonuclease (just checked a couple of polymerase stats
to be sure).  Some klenow that is now sold is specifically altered to be
exo-.  Personally, I would probably go for T4 DNA polymerase, it has a
very active exonuclease activity and I use it frequently to remove
3'-overhangs for cloning purposes.  Just thought I'd add my 2 cents worth.

-- . ----------------------- Sheryl L White  -------------------------
    Dept of Mol Physiology, Given Med, UVM, Burlington, VT 05405
       Voice: (802) 656-4433  E-Mail: slw at salus.med.uvm.edu
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