Methylation Sensitive and Methylation Dependent Restriction Enzymes for Epigenetics

EpiMark^® validated restriction enzymes can be applied upstream of PCR and next generation sequencing for allele-specific or genome-wide methylation profiling.

Isoschizomer pair digests can be leveraged for simple detection and analysis of DNA methylation in various contexts, including CpG methylation in mammals, certain specific cytosine contexts in plants, and methyladenine (6mA) DNA which is widespread in prokaryotes and reported in eukaryotes.

Cytosine or adenine methylation status can be obtained when a methylation-sensitive restriction enzyme is used in conjunction with a methylation-insensitive isoschizomer with the same recognition site to generate DNA fragments by locus-specific cleavage.

• HpaII and MspI are isoschizomers useful for C5-methylcytosine detection within their CCGG recognition site.
  
  
  • MspI (NEB #R0106) will cleave when the internal CpG site is methylated.
  • HpaII (NEB #R0171) will cleave when the internal CpG site is unmethylated.
    
    
• DpnI and DpnII are useful for N6-methyladenine detection within their GATC recognition site.
  
  
  • DpnI (NEB #R0176) will cleave methylated adenine only.
  • DpnII (NEB #R0543) will cleave unmethylated adenine.
    
    

MspI HpaII DpnI DpnII methylation sensitive or dependent restriction enzymes for epigenetics

Hydroxymethylated DNA (5hmC) can be discriminated from methylation (5mC) using methylation-sensitive HpaII and insensitive MspI digests, after a glucosylation step that modifies 5hmC only to block cleavage by MspJI. The MspJI enzyme excises ~ 32 base pair fragments containing a centrally located 5hmC or 5mC modified residue that can be extracted and sequenced. Quantitative PCR can be used to demonstrate the relative abundance of C, 5mC, and 5hmC, at the internal CpG site of CCGG sequence.

• MspJI (NEB #R0661) is dependent on methylation or hydroxymethylation for cleavage.
• HpaII (NEB #R0171) cleavage is blocked by either 5mC or 5hmC.
• MspI (NEB #R0106) cleavage is methylation-insensitive
  
  

Fully methylated cytosine-substituted DNA controls can be prepared enzymatically with 5-methyl-dCTP (NEB #N0356). CpG Methyltransferase (M.SssI) (NEB #M0226) can be used to generate a positive control with mammalian DNA.