methods to prevent digestion by REs?
rapulak at macc.wisc.edu
Mon Apr 25 21:37:34 EST 1994
In article <9404221408.AA29527 at venus.med.pitt.edu> Basavaraju
Shankarappa, bsh at MED.PITT.EDU writes:
> We have a situation with one of our clones where we thought the
>restriction site was an unique one, but now it turns out that there is
>extra one in the vector. Now we need a method that will prevent
>digestion by restriction enzyme at one site while allowing it to cut at
>the other site.
> I remember seeing an article or a note somewhere
>which said that it is possible to use an oligo as a band-aid on the
>where you want to avoid digestions by the RE. More like forming a triple
>helix at the position. Does anyone know of the full reference of this
>or opinions concerning how efficiently does the digestion get blocked.
>If you have any new ways, that this can be done, I would appreciate that
>I also thought that one can use an overlapping methylase recognition
>to block some of the sites. Please correct me or guide me.
>I can use any help.
I once learned a way from my friend, who is working on recA. The basic
idea is to make a oligo complementary to the site you want to cut and
put the DNA, the oligo and some recA together. recA will anneal the
oligo to the site and protect it. Then you add some methylase to
methylate the other site. After removing the oligo and recA, you can cut
your DNA with the restriction enzyme and it will only cut the
Hope this helps.
bzhang1 at students.wisc.edu
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