Hydroxymethylcytosine analysis: T4 Beta-glucosyltransferase
5-hydroxymethylcytosine (5-hmC) is an epigenetic modification (methylated cytosine) that is thought to be a derivative of 5-methylcytosine (5-mC). T4 β-glucosyltransferase catalyzes the transfer of glucose from uridine diphosphoglucose (UDP-Glucose) to the hydroxymethyl group 5-hmC, but does not modify 5-mC. This modifying enzyme can be used to differentiate these types of cytosine methylation. Specifically, after T4 β-glucosyltransferase treatment, restriction enzyme (e.g., MspI/HpaII) digestion or immunodetection can be used identify β-glucosyl-5-hydroxymethylcytosine (β-glu-5hmC) at particular loci, or genome-wide analyses can be accomplished by selective pull-down of β-glu-5hmC.
- Addition of glucose to 5-hydroxymethylcytosine (to create β-glucosyl-5-hydroxymethylcytosine) in mammalian or plant genomic DNA
- Detection of 5-hydroxymethylcytosine when coupled with digestion with a restriction enzyme that is sensitive to β-glucosyl-5-hydroxymethylcytosine
|10X T4-BGT Buffer||120 µl|
|50X UDP-Glucose||25 µl|
Escherichia coli carrying a plasmid that enables expression of β-glucosyltransferase from bacteriophage T4
Nuclease activity is not detected after incubation of 1 µg of DNA (pBR322 and λ-HindIII digest) with 50 units of this enzyme for 4 hours at 37°C as assessed by agarose gel electrophoresis
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