Does Klenow proofreading multiple-base 3' overhangs or just single base?

Tracy Aquilla aquilla at salus.med.uvm.edu
Tue Mar 14 09:14:17 EST 1995


In Article <pnorton-100395170507 at norton1.jmc.tju.edu>,
pnorton at lac.jci.tju.edu (Pamela Norton) wrote:
>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 
>
>
>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).
>
>             Good luck,
>
>                                                                               
Pam Norton

True. I do this frequently for 3' overhangs and I just add Klenow, no NTPs.
It works.
    tracy



More information about the Methods mailing list