Themo Scientific EpiJET 5-hmC and 5-mC Analysis Kit includes three advanced enzymes: T4 phage β-glucosyltransferase (T4 BGT), Epi MspI and Epi HpaII. The Epi MspI and Epi HpaII are isoschisomers with differing sensitivity to internal C methylation of the CCGG site. Epi MspI completely digests genomic DNA when cytosine, 5-methylcytosine (5-mC) or 5-hydroxymethylcytosine (5-hmC) is within its recognition sequence, but its activity is completely blocked when 5-hmC is glucosylated. Epi HpaII cleaves the unmodified CCGG site only.
The kit takes advantage of the T4 BGT ability to specifically modify 5-hmC residues by adding glucose moiety to 5-hmC and the sensitivity of restriction enzyme Epi MspI to glucosylated 5-hmC. The T4 BGT is formulated for highly specific, complete and fast glucosylation of 5-hmC in 15 minutes.
The analysis involves DNA sample glucosylation and subsequent restriction digestion with Epi MspI and Epi HpaII. Restriction digestion products are then analyzed by qPCR with a primer pair flanking CCGG site of interest.
Highlights
• Efficient and reliable—accurate quantification of 5-hmC and 5-mC
• Robust—broad range (from 0.2 µg to 4 µg) of input DNA
• Fast—procedure completed in 5 hours, no overnight incubation.
Applications
5-hmC and 5-mC analysis and quantification in CCGG sequences
Includes
• T4 ß – glucosyltransferase, 5 U/µL
• 10X Epi Buffer
• 10X UDP–glucose
• Epi MspI
• Epi HpaII
• 5-hmC Control DNA
• 5-mC Control DNA
• Unmodified Control DNA
• Control primer 1
• Control primer 2
• Water, nuclease-free
• Detailed Protocol
