1 research outputs found
Jump-seq: Genome-Wide Capture and Amplification of 5-Hydroxymethylcytosine Sites
5-Hydroxymethylcytosine
(5hmC) arises from the oxidation of 5-methylcytosine
(5mC) by Fe2+ and 2-oxoglutarate-dependent 10–11
translocation (TET) family proteins. Substantial levels of 5hmC accumulate
in many mammalian tissues, especially in neurons and embryonic stem
cells, suggesting a potential active role for 5hmC in epigenetic regulation
beyond being simply an intermediate of active DNA demethylation. 5mC
and 5hmC undergo dynamic changes during embryogenesis, neurogenesis,
hematopoietic development, and oncogenesis. While methods have been
developed to map 5hmC, more efficient approaches to detect 5hmC at
base resolution are still highly desirable. Herein, we present a new
method, Jump-seq, to capture and amplify 5hmC in genomic DNA. The
principle of this method is to label 5hmC by the 6-N3-glucose moiety and connect a hairpin DNA oligonucleotide carrying
an alkyne group to the azide-modified 5hmC via Huisgen cycloaddition
(click) chemistry. Primer extension starts from the hairpin motif
to the modified 5hmC site and then continues to “land”
on genomic DNA. 5hmC sites are inferred from genomic DNA sequences
immediately spanning the 5-prime junction. This technology was validated,
and its utility in 5hmC identification was confirmed