DNA methylation is the modification of DNA bases by adding a methyl group by a DNA methyltransferase. It is important to consider methylation when planning a restriction digest, as methylated DNA may block or impair the binding of restriction enzymes to DNA. The 3 most common types of methylation that impact restriction digestions are dam and dcm methylation, which are found in prokaryotes such as E. coli, and CpG methylation, which is found in eukaryotes such as plants and mammals.
If present within the recognition site, a methyl group can prevent the restriction enzyme from binding and cleaving. However, dam and dcm methylation will only impact restriction digestion of a plasmid if the restriction and methylation sites overlap.
To illustrate this, let’s look at two XbaI restriction sites to see if they may be impaired by dam methylation. Keep in mind that dam methyltransferase recognizes the sequence GATC and methylates the adenine. In this first example, XbaI is able to digest the sequence because it does not overlap with a dam methylation site. However, in this second example, XbaI is unable to digest the DNA because a dam methylation site overlaps with the restriction site. dcm methyltransferases behave the same way and recognize the sequence CCAGG and CCTGG. In these cases, they will methylate the cytosine. If you find that your restriction site is blocked by dam or dcm methylation, you can use NEB’s dam and dcm negative Competent E. coli to propagate your plasmid.
Alternatively, you can amplify your vector using a high-fidelity polymerase, such as Q5, as PCR amplified DNA will not be methylated. CpG methylation is only an issue when digesting eukaryotic genomic DNA. When working with DNA from a eukaryotic source, you may want to choose an enzyme that is not impacted by CpG methylation, or, amplify a region of the DNA.
You can also use NEB’s interactive tool, Enzyme Finder, to search for alternative restriction enzymes. NEB provides dam, dcm and CpG methylation sensitivity data for all of our restriction enzymes. If you are working with a methylation sensitive enzyme and digesting a plasmid, we recommend using our interactive tool, NEBcutter, to determine if your plasmid or sequence of interest will be impacted by overlapping methylation. Methylation information is also available on REBASE.
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