Filling in without chewing back

Dom Spinella dspinella at chugaibio.com
Thu Jul 2 19:14:11 EST 1998


Hi All.  I have been forced to develop an esoteric cloning strategy
which, at one step, requires that a a small DNA fragment with a 3' and
5' overhang be treated such that the 5' overhang is filled in, but the
3' overhang is left unaffected.  That is, I need to convert this:

5'-NNNNNNNNNNNNNNNNNNNNNN
        NNNNNNNNNNNN-5'

into this:

5'-NNNNNNNNNNNNNNNNNNNNN
   NNNNNNNNNNNNNNNN-5'

(I sure hope this gets posted like I drew it)

Anybody have any ideas on how best to accomplish this?  It seems to me
that I need a polymerase with no 3'->5' exo activity, but reasonable
polymerase activity. Is there such an enzyme?  Modified T7 polymerase
(Sequenase) is the closest thing I can think of, but I don't think its
3'->5' exo activity is completely absent -- just markedly reduced. BTW,
just to make things more difficult, the fragment is rather small and
will melt apart at high temperature which would probably rule out
thermostable polymerases. Any thoughts on the subject will be
appreciated. Thanks.
-Dom Spinella



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