Fluorometric identification of 5-methylcytosine modification in DNA: combination of photosensitized oxidation and invasive cleavage.
Fluorometric identification of 5-methylcytosine modification in DNA: combination of photosensitized oxidation and invasive cleavage.
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DOI：
10.1021/bc7003318
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发表时间：
2008
影响因子：
4.7
通讯作者：
H. Yamada;K. Tanabe;S. Nishimoto
中科院分区：
文献类型：
--
作者：
H. Yamada;K. Tanabe;S. Nishimoto
将2-甲基-1，4-萘醌（NQ）发色团的光氧化DNA切割反应与人Flap内切酶-1的侵入性切割反应相结合，建立了一种高效的DNA甲基化荧光检测体系。酶处理光化学片段化的靶寡脱氧核苷酸（ODN）在5-甲基胞嘧啶（mC）和具有荧光团（F）和猝灭剂（D）的发夹样探针寡聚体的混合物导致荧光的显著增强。相反，在靶序列处含有胞嘧啶但不含mC的ODN的荧光发射极弱。此外，通过监测荧光变化，该系统允许检测亚飞摩尔量的DNA中的mC。该系统将提供一个高灵敏度的协议，确定在DNA中的甲基化状态的荧光发射。
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.