Boronic Acid-mediated Polymerase Chain Reaction for Gene- and Fragment-specific Detection of 5-Hydro

来源 :第九届全国微全分析系统学术会议、第四届全国微纳尺度生物分离分析学术会议、2014国际微流控芯片与微纳尺度生物分离分析学术 | 被引量 : 0次 | 上传用户:ZHANGXIANYU0000
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  5-hydroxymethylcytosine(5hmC)is a recently re-discovered DNA base that is converted from the well-characterized epigenetic mark 5-methylcytosine(5mC).The level of 5hmC has been observed to be altered in several diseases,including hematopoietic malignancies and a broad range of solid tumours(1,2).A lack of 5hmC was implicated as a useful biomarker for cancer diagnosis(3).Interestingly,both nutritional(e.g.,vitamin C,4)and environmental factors(e.g.,redox-active quinones,5)affect the cellular level of 5hmC.These observations may indicate that 5hmCvarying or-rich genes or sequences could be exploited as indicators or biomarkers of epigenetic states,pathogenic processes,pharmacological responses,and environmental exposures.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 qPCR assay for rapid and sensitive detection of gene-or fragment-specific 5hmC without restriction-assay-like sequence limitations.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(ES)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.
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