title: Boronic acid-mediated polymerase chain reaction for gene- and fragment-specific detection of 5-hydroxymethylcytosine.
abstract: The gene- or fragment-specific detection of newly recognized deoxyribonucleic acid (DNA) base 5-hydroxymethylcytosine (5hmC) will provide insights into its critical functions in development and diseases, and is also important for screening 5hmC-rich genes as an indicator of epigenetic states, pathogenic processes and pharmacological responses. Current analytical technologies for gene-specific detection of 5hmC are heavily dependent on glucosylated 5hmC-resistant restriction endonuclease cleavage. Here, we find that boronic acid (BA) can inhibit the amplification activity of Taq DNA polymerase for replicating glucosylated 5hmC bases in template DNA by interacting with their glucose moiety. On the basis of this finding, we propose for the first time a BA-mediated polymerase chain reaction (PCR) assay for rapid and sensitive detection of gene- or fragment-specific 5hmC without restriction-assay-like sequence limitations. To optimize the BA-mediated PCR assay, we further tested BA derivatives and show that one BA derivative, 2-(2'-chlorobenzyloxy) phenylboronic acid, displays the highest inhibitory efficiency. Using the optimized assay, we demonstrate the enrichment of 5hmC in an intron region of Pax5 gene (a member of the paired box family of transcription factors) in mouse embryonic stem cells. Our work potentially opens a new way for the screening and identification of 5hmC-rich genes and for high throughput analysis of 5hmC in mammalian cells.
authors.affiliation: State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China hlwang@rcees.ac.cn.
authors.forename: Hailin
authors.initials: H
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China.
authors.forename: Bailin
authors.initials: B
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China.
authors.forename: Cuiping
authors.initials: C
authors.lastname: Li
authors.affiliation: State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China.
authors.forename: Ruichuan
authors.initials: R
authors.lastname: Yin
authors.affiliation: State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China.
authors.forename: Maoyong
authors.initials: M
authors.lastname: Song
authors.affiliation: State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China.
authors.forename: Baodong
authors.initials: B
authors.lastname: Liu
authors.affiliation: Department of Chemistry, Nanjing University, Nanjing 210093, China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China.
authors.forename: Guibin
authors.initials: G
authors.lastname: Jiang
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives; D000818:Animals; D001483:Base Sequence; D001897:Boronic Acids / Q000737:chemistry*; D002478:Cells, Cultured; D003596:Cytosine / Q000031:analogs & derivatives* / Q000737:chemistry; D053595:Embryonic Stem Cells / Q000378:metabolism; D007438:Introns; D051379:Mice; D051757:PAX5 Transcription Factor / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA
chemical_list: D001897:Boronic Acids; D051757:PAX5 Transcription Factor; C497282:Pax5 protein, mouse; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine