Fluorometric Identification of 5-Methylcytosine Modification in DNA: Combination of Photosensitized Oxidation and Invasive Cleavage

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• 作者：Hisatsugu Yamada ; Kazuhito Tanabe ; Sei-ichi Nishimoto
• 刊名：Bioconjugate Chemistry
• 出版年：2008
• 出版时间：January 2008
• 年：2008
• 卷：19
• 期：1
• 页码：20 - 23
• 全文大小：633K
• 年卷期：v.19,no.1(January 2008)
• ISSN：1520-4812

文摘

An efficient fluorometric detection system of DNA methylation has been developed by a combination of a photooxidative DNA cleavage reaction with 2-methyl-1,4-naphthoquinone (NQ) chromophore and an invasive cleavage reaction with human Flap endonuclease-1. Enzymatic treatment of a mixture of photochemically fragmented target oligodeoxynucleotides (ODNs) at 5-methylcytosine (^mC) and hairpin-like probe oligomer possessing a fluorophore (F) and a quencher (D) resulted in a dramatic enhancement of fluorescence. In contrast, fluorescence emission for the ODN containing cytosine but not ^mC at the target sequence was extremely weak. In addition, by monitoring the fluorescence change, this system allows for the detection of ^mC in DNA at subfemtomole amounts. This system would provide a highly sensitive protocol for determining the methylation status in DNA by fluorescence emission.