Consult REBASE, the restriction enzyme database, for more detailed information and specific examples upon which these guidelines are based. This table should be viewed as a guide to the behavior of the enzymes listed rather than an absolute indicator. The table below summarizes methylation sensitivity for NEB restriction enzymes, indicating whether or not cleavage is blocked or impaired by Dam, Dcm or CpG methylation if or when it overlaps each recognition site.
CpG methylation patterns are not retained once the DNA is cloned into a bacterial host. Note: The effects of CpG methylation are mainly a concern when digesting eukaryotic genomic DNA. Consequently, CpG methylation has been postulated to play a role in differentiation and gene expression (4). Patterns of CpG methylation are heritable, tissue specific and correlate with gene expression. Eukaryotic MethylationĬpG MTases, found in higher eukaryotes (e.g., Dnmt1), transfer a methyl group to the C 5 position of cytosine residues. This situation should also be considered when designing restriction enzyme digests. In this case, part of the Dam or Dcm sequence is generated by the restriction enzyme sequence, followed by the flanking sequence. Restriction sites can also be blocked if an overlapping site is present. Restriction sites that are blocked by Dam or Dcm methylation can be un-methylated by cloning your DNA into a dam –, dcm – strain of E. As a result, enzymes blocked by Dam or Dcm modification will yield partial digestion patterns with λ DNA. While pBR322 DNA is fully modified (and is therefore completely resistant to MboI digestion), only about 50% of λ DNA Dam sites are methylated, presumably because the methylase does not have the opportunity to methylate the DNA fully before it is packaged into the phage head. coli is completely resistant to cleavage by MboI, which cleaves at GATC sites. For example, plasmid DNA isolated from dam+ E. Some or all of the sites for a restriction endonuclease may be resistant to cleavage when isolated from strains expressing the Dam or Dcm methylases if the methylase recognition site overlaps the endonuclease recognition site. EcoKI methylase–methylation of adenine in the sequences AAC(N 6)GTGC and GCAC(N 6)GTT.Dcm methyltransferases–methylation at the C5 position of the second cytosine in the sequences CCAGG and CCTGG (1,3).Dam methylase–methylation at the N 6 position of the adenine in the sequence GATC (1,2).coli contain three site-specific DNA methylases. In prokaryotes, MTases have most often been identified as elements of restriction/modification systems that act to protect host DNA from cleavage by the corresponding restriction endonuclease. Methylation should be considered when digesting DNA with restriction endonucleases because cleavage can be blocked or impaired when a particular base in the recognition site is methylated. The Anza enzyme system from Thermo Fisher (Invitrogen) has been incorporated into SnapGene’s enzyme database. DNA methyltransferases (MTases) that transfer a methyl group from S-adenosylmethionine to either adenine or cytosine residues, are found in a wide variety of prokaryotes and eukaryotes.
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