Restriction enzyme digestion of hemimethylated DNA

Abstract

Hemimethylated duplex DNA of the bacteriophage ØX 174 was synthesized using primed repair synthesis in vitro with E. coli DNA polymerase I followed by ligation to produce the covalently closed circular duplex (RFI). Single-stranded ØX DNA was used as template, a synthetic oligonucleotide as primer and 5-methyldeoxycytidine-5′-triphosphate (5mdCTP) was used in place of dCTP. The hemimethylated product was used as substrate for cleavage by various restriction enzymes. Out of the 17 enzymes tested, only 5 (BstN I, Taq I, Hinc II, Hinf I and Hpa I) cleaved the hemimethylated DNA. Two enzymes (Msp I and Hae III) were able to produce nicks on the unmethylated strand of the cleavage site. Msp I, which is known to cleave at CCGG when the internal cytosine residue is methylated, does not cleave when both cytosines are methylated. Another enzyme, Apy I, cleaves at the sequence CCGG when the internal cytosine is methylated, but is inactive on hemimethylated DNA in which both cytosines are methylated. Hemimethylated molecules should be useful for studying DNA methylation both in vivo and in vitro. © 1981 Oxford University Press.