methods to prevent digestion by REs?

Rapu Lak 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:
>Hi folks:
>	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
an 
>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
sequence 
>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
article
>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
too.
>I also thought that one can use an overlapping methylase recognition
sequence 
>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
unmethylated site. 
Hope this helps.

beilin zhang
bzhang1 at students.wisc.edu



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