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

作者：Robertson Adam B; Dahl John A; Vagbo Cathrine B; Tripathi Pankaj; Krokan Hans E; Klungland Arne^*

来源：Nucleic Acids Research, 2011, 39(8): e55.

DOI：10.1093/nar/gkr051

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 beta-glucosyltransferase from T4 bacteriophage creating beta-glucosyl-5-hydroxymethylcytosine (beta-glu-5hmC). The beta-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.

• 出版日期2011-4
• 单位河北医科大学