Increasing the Foki cleavage specificity from 5 to 7 base pairs by two-step methylation

Non-cognate methylation of specific nucleotides of the recognition sequence could inhibit the methylase but not the endonuclease of the same restriction-modification system. This permits to increase the cleavage specificity of the restriction enzyme by two-step methylation of the DNA, as shown here for the three-component M multiplied by MspI -M multiplied by FokI-FokI combination, changing FokI specificity from 5 to 7 bp. The cleavage specificity of FokI is 5'-GGATG(N)9/13. M multiplied by FokI methylates only one strand of the recognition site, resulting in GGmATG sequence.