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Methods. 2015 Jan 15;72:9-15. doi: 10.1016/j.ymeth.2014.08.009. Epub 2014 Aug 28.

oxBS-450K: a method for analysing hydroxymethylation using 450K BeadChips.

Author information

1
Department of Cancer Biology, UCL Cancer Institute, University College London, London WC1E 6BT, UK.
2
Scottish Centre for Regenerative Medicine, Edinburgh EH16 4UU, UK.
3
Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, UK.
4
Department of Cancer Biology, UCL Cancer Institute, University College London, London WC1E 6BT, UK. Electronic address: s.beck@ucl.ac.uk.

Abstract

DNA methylation analysis has become an integral part of biomedical research. For high-throughput applications such as epigenome-wide association studies, the Infinium HumanMethylation450 (450K) BeadChip is currently the platform of choice. However, BeadChip processing relies on traditional bisulfite (BS) based protocols which cannot discriminate between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Here, we report the adaptation of the recently developed oxidative bisulfite (oxBS) chemistry to specifically detect both 5mC and 5hmC in a single workflow using 450K BeadChips, termed oxBS-450K. Supported by validation using mass spectrometry and pyrosequencing, we demonstrate reproducible (R(2)>0.99) detection of 5hmC in human brain tissue using the optimised oxBS-450K protocol described here.

KEYWORDS:

450K BeadChip; Bisulfite conversion; DNA methylation; Epigenetics; Hydroxymethylation; Oxidation

PMID:
25175075
PMCID:
PMC4304834
DOI:
10.1016/j.ymeth.2014.08.009
[Indexed for MEDLINE]
Free PMC Article

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