EipMark 5-hmC and 5-mC Analysis Kit
To further the study of 5-mC and 5-hmC in DNA, New England Biolabs (NEB) has developed a robust, simple method that utilizes beta-glucosyltransferase (beta-GT), an enzyme that glucosylates 5-hmC in the presence of UDP-Glucose (UDP-Glc), and two methylation state sensitive restriction enzymes (MspI and HpaII) that differ in their ability to cut methylated DNAs containing a CpG dinucleotide.
To definitively determine whether a specific locus is 5-hmC modified, DNAs are treated with beta-GT, modifying all 5-hmC to glucosylated 5-hmC (5-ghmC) (Figure, step 1a). A control reaction is also set up without beta-GT, leaving all 5-hmC modifications intact (Figure, step 1b).
Digestion of the two reactions with MspI can be used to differentiate between 5-ghmC and 5-mC modified cytosines, as MspI will cleave 5-mC and 5-hmC, but not 5-ghmC (Figure, steps 2a and 2d). Digestion of the beta-GT treated sample with HpaII further enables the determination of the percentage of total methylation (5-mC, 5-hmC, and 5-ghmC), as all three modifications block HpaII cleavage (Figure, step 2b).
Finally, a control of beta-GT treated uncut DNA should be analyzed to determine the total amount of the DNA of interest (Figure, step 2c). Following digestion, qPCR with primers flanking the DNA sequence of interest can be used to quantify levels of 5-hmC and 5-mC at the given locus (Figure, step 3).
Amplification is dependent on the levels of MspI and HpaII cleavage and, therefore, is a direct correlation of the quantities of 5-hmC and both 5-mC and 5-hmC, respectively. Validation of this method is shown in this article using synthetic DNAs with different ratios of the various states of cytosine methylation.