Detection and discrimination of maintenance and de novo CpG methylation events using MethylBreak

William Hsu, Augustus T. Mercado, George Hsiao, Jui Ming Yeh, Chung Yung Chen

研究成果: 雜誌貢獻文章同行評審


Understanding the principles governing the establishment and maintenance activities of DNA methyltransferases (DNMTs) can help in the development of predictive biomarkers associated with genetic disorders and diseases. A detection system was developed that distinguishes and quantifies methylation events using methylation-sensitive endonucleases and molecular beacon technology. MethylBreak (MB) is a 22-mer oligonucleotide with one hemimethylated and two unmethylated CpG sites, which are also recognition sites for Sau96I and SacII, and is attached to a fluorophore and a quencher. Maintenance methylation was quantified by fluorescence emission due to the digestion of SacII when the hemimethylated CpG site is methylated, which inhibits Sau96I cleavage. The signal difference between SacII digestion of both MB substrate and maintenance methylated MB corresponds to de novo methylation event. Our technology successfully discriminated and measured both methylation activities at different concentrations of MB and achieved a high correlation coefficient of R2=0.997. Additionally, MB was effectively applied to normal and cancer cell lines and in the analysis of enzymatic kinetics and RNA inhibition of recombinant human DNMT1.
頁(從 - 到)658-663
期刊Biosensors and Bioelectronics
出版狀態已發佈 - 5月 15 2017

ASJC Scopus subject areas

  • 生物技術
  • 生物物理學
  • 生物醫學工程
  • 電化學


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