A novel method for the efficient and selective identification of 5-hydroxymethylcytosine in genomic DNA.

ABSTRACT: Recently, 5-hydroxymethylcytosine (5hmC) was identified in mammalian genomic DNA. The biological role of this modification remains unclear; however, identifying the genomic location of this modified base will assist in elucidating its function. We describe a method for the rapid and inexpensive identification of genomic regions containing 5hmC. This method involves the selective glucosylation of 5hmC residues by the ?-glucosyltransferase from T4 bacteriophage creating ?-glucosyl-5-hydroxymethylcytosine (?-glu-5hmC). The ?-glu-5hmC modification provides a target that can be efficiently and selectively pulled down by J-binding protein 1 coupled to magnetic beads. DNA that is precipitated is suitable for analysis by quantitative PCR, microarray or sequencing. Furthermore, we demonstrate that the J-binding protein 1 pull down assay identifies 5hmC at the promoters of developmentally regulated genes in human embryonic stem cells. The method described here will allow for a greater understanding of the temporal and spatial effects that 5hmC may have on epigenetic regulation at the single gene level.

SUBMITTER: Robertson AB 

PROVIDER: S-EPMC3082876 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature