Does Klenow proofreading multiple-base 3' overhangs or just single base?
pnorton at lac.jci.tju.edu
Fri Mar 10 17:12:56 EST 1995
In article <MURIANAP.168.00BD2EFA at FOODSCI.PURDUE.EDU>,
MURIANAP at FOODSCI.PURDUE.EDU (Pete Muriana) wrote:
> Can anyone tell me if the Klenow proofreading activity used for trimming off
> the extra 3' nucleotide added during PCR RXNs also works for trimming back
> multi-base 3' overhangs of restriction-digested fragment ends? I've come
> across a number of papers which state in their M&M that a digested fragment
> was "blunted" with Klenow. Since you can't "fill-in" in front of the 5'
> recessed end, they must mean the use of Klenow to nibble back.
> Regards, Peter
Yes, Klenow will remove protruding 3' overhangs. In theory, one
should add all 4 dNTPs to permit re-synthesis of any segments that get
chewed in beyond the overhang. In practice, the blunt ended product appears
to be a very poor substrate for the 3' exo activity, as Klenow treatment in
the absence of dNTPs yields good blunt ends. Thus, one can usually get away
with simply adding a little Klenow right to a restriction digestion (after
checking an aliquot for complete cutting, of course).
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