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Non-cognate methylation of specific nucleotides of the recognition sequence could inhibit the methylase but not the endonudease of the same restriction-modification system. This permits to increase the cleavage specificity of the restriction enzyme by two-step methylation of the DNA (1), as shown here for the three-component M-Mspl-MFo/d-Fo/d combination, changing Fo/d specificity from 5 to 7 bp. The cleavage specificity of Fo/d is 5'-GGATG(N)9/13 (2). MFokl methylates only one strand of the recognition site, resulting in GGmATG sequence (3). Msp\ and Fold sites can overlap in the 7-bp sequence CCGGATG or in the 8-bp sequence CCGGGATG. Methylation of such sites by MMsp\ (methylation specificity: mCCGG)produces non-cognate methylation of the overlapping Fo/d sites, resulting in (a) STST' IB o r () m8SrIB sequences, respectively. We show here that in case (a), MFo/d is inhibited and Fok\ is unaffected (Fig. 2, lanes 3 and 2, respectively), while in case (b), MFo/cl is unaffected and Fo/d is partially inhibited (Fig. 2, lanes 6 and 5, respectively, where MHpati was used to produce the (b) type non-cognate methylation of Fo/d sites on the same 7-bp sequences). As the result, when the DNA is premethylated by M-Msp\, MFo/d cannot methylate those 7-bp CCGGATG sequences (which thus remain susceptible to Fo/d), whereas all other Fo/d sites would be methylated and thus protected from Fold cleavage. However, cleavage by Fo/d at the CCGGATG sites may not be complete (max. 90%), because MFo/d has some residual affinity to the sites premethylated by M-Mspl, especially when excess M-Fo/d is used (data not shown). Nevertheless, in many cases this does not affect the applicability of the system.

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