SELECTRe: RE:Fill-in enzymes

ryan at mbcf.stjude.org ryan at mbcf.stjude.org
Wed Jul 28 11:35:00 EST 1993


> Subject: Fill-in enzymes
> ------------------------------------------------------------------------------
> 
> We are searching for an enzyme that can reliably fill-in
> 5' overhangs such that they become blunt ends ... but
> without ever losing even a base off the end.  Our luck so
> far, even with 3'-5'exo lacking enzymes, has not been
> great.  Would anyone have recommendations concerning either
> the enzyme of choice and/or optimal reaction conditions ?
> 
> Many thanks,
> 
> Greg Lennon
> 

Just an additional point to keep in mind about using Klenow to generate blunt
ends:
the traditional method of preparing Klenow fragment from DNA pol I is to cleave
the enzyme with subtilisin and purify the large fragment. This digestive
treatment is occasionally less than 100% complete for a given batch, and a very
minor trace of 5'->3' exo activity can co-purify with the desired product. In
normal use this minor contaminating activity doesn't matter much, but in a case
where the last nucleotide of a fill-in is added inefficiently (eg if any needed
dNTP is limiting) then the small amount of 5'-3' exo activity may be evidenced
by the removal of a terminal base. Usually this won't matter, as the fragment
end is still made blunt. However, if you want to precisely know the bases left
at the ligation junction, it is a good idea to use Klenow enzyme expressed from
a cloned gene that lacks coding sequences for the pol I "small fragment." No
proteolytic digestion is used to purify this enzyme, so no contaminating 5'-3'
exo activity is possible. The "source" comments in the supplier's catalog will 
describe which kind of Klenow they are selling.

Best wishes,
 Kevin.
-- 
==========================================
Kevin W. Ryan
Department of Virology & Molecular Biology
St. Jude Children's Research Hospital
Memphis, Tennessee, U.S.A.
  phone (901) 522-0411, fax (901) 523-2622
    Internet: ryan at mbcf.stjude.org
==========================================



More information about the Methods mailing list