methylation/restriction enzymes

Mikhail Alexeyev malexe at biost1.thi.tmc.edu
Wed Sep 4 14:30:38 EST 1996


In article <50kcao$3s3 at lyra.csx.cam.ac.uk>, rw200 at cus.cam.ac.uk (R.
Woodward) wrote:

> A quick question for someone who has always been puzzled by methylation
> inhibiting restriction enzyme digestion. I believe that the ClaI site
> can be methylated and inhibit cleavage by this enzyme, is this always
the case?
> In a host which does have dam activity are all ClaI sites uncleavable with
> ClaI or only a percentage of the sites? and finally does XL1Blue strain 
> methylate DNA. If the answer to all of these questions is YES how can
> I ClaI digest plasmid DNA which was grown up in XL1Blue cells, or is the
> answer obvious?  Thanks for your help
> 
> Robert
> 
> Email  rw200 at cus.cam.ac.uk
  

Pam Norton made a good clarification on this subject awhile ago. Cla I is
inhibited by overlapping dam methylation. dam methylase recognizes GATC
sequence. ClaI recognizes ATCGAT. Therefore, only if  ClaI site is
preceded by g nucleotide (sequence gATCGAT), it will be methylated and
therefore resistant to cleavage. Statistically, it will happen to only 1/4
of ClaI sites.

Plasmids are to be expected to be fully methylated, lambda DNA is
partially methylated in methylase-proficient strains. Methylation usually
blocks enzyme completely. To my recollection XL1 blue is dam-positive.

M.A.



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