Methylation-sensitive restriction enzymes

An alternative, perhaps more advanced, form (called ExoMeth) of Exo Ill-based sequencing strategies is to incorporate 5-methyl-dCTP in place of dCTP during the repair synthesis. Digestion of the (5-Me-dCMP)-containing DNA with suitable frequent cutter restriction enzymes sensitive to the m C gives constant 5 -end points that allow one to obtain far more sequence information (up to 10 kb) from a nested set of Exo Ill-digested templates (29). 

Restriction enzymes also display different sensitivities to noncanonically hemimethylated DNAs which are created, for example, by incorporating m C during the second-strand DNA synthesis catalyzed by DNA polymerases (40). At high enzyme-to-substrate ratios, some enzymes (e.g., Accl, BawiHl, Pstl, Sail, Smal, Sstl, and Xhol) are unable to cleave their recognition sites, whereas other enzymes (e.g., EcoRl, Hindlll, Dpnl, Pvull, and Xbal) are only partially sensitive to hemi-methylation. 

In the methylation of DNA, specific methylases catalyse transfer of methyl groups from SAM to the 6-amino groups of adenine residues and C5 of cytosine. A specific pattern of methylation serves to protect DNA from the cell s own Restriction endonucleases (see) these enzymes destroy the DNA of invading viruses. The DNA of viruses that are able to replicate within a particular host are protected from the endonucleases by methylation of bases at the endonuclease-sensitive sites. 

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