Hairpin fluorescence DNA probe for real-time monitoring of DNA methylation
Hairpin fluorescence DNA probe for real-time monitoring of DNA methylation
复制标题
用于实时监测 DNA 甲基化的发夹荧光 DNA 探针
DOI：
10.1021/ac061694i
复制
发表时间：
2007-02-01
影响因子：
7.4
通讯作者：
Zhou, Xingwang
中科院分区：
文献类型：
--
作者：
Li, Jun;Yan, Hongfei;Zhou, Xingwang
甲基化酶催化的DNA甲基化在许多生物学事件中起着重要作用。然而，传统的凝胶电泳甲基化酶活性分析方法是费力和不连续的。在本文中，我们报告了一种新的策略，研究甲基化酶活性的荧光探针结合酶联反应。制备发夹DNA探针，其中荧光团和猝灭剂连接在探针的5 ′-和3 ′-末端。DNA甲基化对茎序列的干扰会导致荧光团和猝灭剂的分离，从而导致荧光的恢复。我们使用DNA腺嘌呤甲基化（Dam）甲基转移酶（MTase）和Dpn I内切核酸酶，两者都具有5 '-G-A-T-C-3'识别序列。Dam MTase催化5 '-GATC-3'序列的甲基化，Dpn I切割5 '-G-Am-T-C-3'序列。在甲基化过程中，当发夹探针被Dpn I内切酶切割时，其荧光恢复。与传统的方法不同，该方法是在真实的时间内完成的，并且可以用于监测甲基化的动态过程。该方法简便易行，无放射性，与凝胶电泳法一样有效。这也有可能筛选合适的Dam甲基化酶抑制剂药物。
DNA methylation catalyzed by methylase plays an important role in many biological events. However, traditional methods of methylase activity analysis by gel electrophoresis were laborious and discontinuous. In this paper, we report a new strategy to study methylase activity using fluorescent probes coupled with enzyme-linkage reactions. A hairpin DNA probe is prepared with a fluorophore and a quencher linked at the 5'- and 3'-terminus of the probe. A disturbance of the stem sequence by DNA methylation would cause the separation of the fluorophore and the quencher, resulting in the restoration of the fluorescence. We used DNA adenine methylation (Dam) methyltransferase (MTase) and Dpn I endonuclease, both having a 5'-G-A-T-C-3' recognition sequence. Dam MTase catalyzed the methylation of the sequence of 5'-GATC-3', and Dpn I cut the sequence of 5'-G-Am-T-C-3'. The fluorescence of the hairpin probe was restored when it was cleaved by Dpn I endonuclease during the course of methylation. Unlike traditional methods, this assay was done in real time and could be used to monitor the dynamic process of 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 to screen suitable inhibitor drugs for Dam methylase.