Many restriction enzymes are sensitive to the DNA methylation states. Cleavage can be blocked or impaired when a particular base in the recognition site is modified. Scientists at NEB recently identified the MspJI (NEB #R0661) family of restriction enzymes, which are dependent on methylation and hydroxymethylation for cleavage to occur. These enzymes excise ~ 32 base pair fragments containing a centrally located 5-hmC or 5-mC modified residue that can be extracted and sequenced. Due to the known position of this epigenetic modification, bisulfite conversion is not required prior to downstream analysis. These EpiMark® validated, methylation-dependent restriction enzymes expand the potential for mapping epigenetic modifications and simplify the study of DNA methylation. Additionally, they provide an opportunity to better understand the role of 5-hydroxymethylcytosine in the genome. Several of our existing restriction enzymes can also be used to study epigenetic modifications of DNA, such as DpnI (NEB #R0176) and DpnII (NEB #R0543) that recognize the same sequence, but different methylation patterns. McrBC also only cleaves DNA that is methylated cytosine on one or both strands. MspI (NEB #R0106) and HpaII (NEB #R0171) recognize the same sequence (CCGG) but are sensitive to different methylation statuses. HpaII cleaves only a completely unmodified site: any modification (5-mC, 5-hmCo or 5-ghmC) at either cytosine blocks cleavage. MspI will recognize and cleave 5-hmC and 5-hmC, but not 5-ghmc. These enzymes are included in the EpiMark® 5-hmC and 5-mC Analysis Kit (NEB #E3317). Simplify DNA Methylation Analysis with MspJI MspJI (NEB #R0661) recognizes methylated and hydroxymethylated DNA and cleaves out ~ 32 bp fragments for downstream sequencing analysis. Overnight digestion of 1 µg of genomic DNA from various sources with or without MspJI is shown. Note: Yeast DNA does not contain methylated DNA and thus, no 32-mer is detected. EpiMark® is a registered trademark of New England Biolabs, Inc.
