Hairpin Fluorescence DNA Probe for Real-Time Monitoring of DNA Methylation

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• 作者：Jun Li ; Hongfei Yan ; Kemin Wang ; Weihong Tan ; Xingwang Zhou
• 刊名：Analytical Chemistry
• 出版年：2007
• 出版时间：February 1, 2007
• 年：2007
• 卷：79
• 期：3
• 页码：1050 - 1056
• 全文大小：327K
• 年卷期：v.79,no.3(February 1, 2007)
• ISSN：1520-6882

文摘

DNA methylation catalyzed by methylase plays an important role in many biological events. However, traditionalmethods of methylase activity analysis by gel electrophoresis were laborious and discontinuous. In this paper,we report a new strategy to study methylase activity usingfluorescent probes coupled with enzyme-linkage reactions.A hairpin DNA probe is prepared with a fluorophore anda quencher linked at the 5'- and 3'-terminus of the probe.A disturbance of the stem sequence by DNA methylationwould cause the separation of the fluorophore and thequencher, resulting in the restoration of the fluorescence.We used DNA adenine methylation (Dam) methyltransferase (MTase) and Dpn I endonuclease, both having a5'-G-A-T-C-3' recognition sequence. Dam MTase catalyzedthe methylation of the sequence of 5'-GATC-3', and DpnI cut the sequence of 5'-G-Am-T-C-3'. The fluorescenceof the hairpin probe was restored when it was cleaved byDpn I endonuclease during the course of methylation.Unlike traditional methods, this assay was done in realtime and could be used to monitor the dynamic processof methylation. Our method is easy, simple, and nonradioactive, yet as efficient as gel electrophoresis in detecting the activity of methylase. It also had the potential toscreen suitable inhibitor drugs for Dam methylase.