Methylation sites

This post was written by admin on April 9, 2009
Posted Under: Molecular biology

Are you wondering why this enzime you are using doesn’t cut your plasmid? It happened to me! I was doing some easy cloning and there was no way I could get my plasmid cut. I tried several times, I bought a new restriction enzime… until I realized that my enzime was methylation sensitive.

What’s that? There is a very good explanation in the New England Biolabs website:

DNA methyltransferases (MTases), which transfer a methyl group from S-adenosylmethionine to either adenine or cytosine residues, are found in a wide variety of prokaryotes and eukaryotes. Methylation needs to 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.

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. Most laboratory strains of E. coli contain three site-specific DNA methylases. The methylase encoded by the dam gene (Dam methylase) transfers a methyl group to the N6 position of the adenine residues in the sequence GATC (1,2). The Dcm methylase, encoded by the dcm gene, methylates the internal cytosine residues in the sequences CCAGG and CCTGG (1,3) at the C5 position. The EcoKI methylase, M. EcoKI, modifies adenine residues in the sequences AAC(N6)GTGC and GCAC(N6)GTT. 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. For example, plasmid DNA isolated from Dam+ E. coli is completely resistant to cleavage by MboI, which cleaves at GATC sites.

Not all DNA isolated from E. coli is methylated to the same extent. While pBR322 DNA is fully modified (and is therefore completely resistant to Mbo I 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. As a result, enzymes blocked by Dam or Dcm modification will yield partial digestion patterns with l DNA.

CpG MTases, found in higher eukaryotes (e.g., Dnmt1), transfer a methyl group to the C5 position of cytosine residues. Patterns of CpG methylation are heritable, tissue specific, and correlate with gene expression. Consequently CpG methylation has been postulated to play a role in differentiation and gene expression (4). The effects of CpG methylation are mainly of concern when digesting eukaryotic genomic DNA. It should be noted that CpG methylation patterns are not retained once the DNA is cloned into a bacterial host.

Also, if you go to the NEB web site, there’s a list of all the methylation sensitive enzimes.

What do you have to do to solve a methylation problem? In my opinion, the fastest way is to transform your plasmid in a dam and dcm negative bacteria strain, so it won’t get methylated. There are several strains of this characteristics: SCS110 or JM110 (Stratagene) for example.

One of the most used plasmids, pEGFP, has a methylation sensitive site. You will not be able to cut this plasmid with XbaI unless you transform it first in bacteria that generates unmethylated dna.  Hope this helps!

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