胞嘧啶在CpG二核苷酸上甲基化成5-甲基胞嘧啶，是与基因表达调控密切相关的表观遗传学修饰中最常研究的一种。正常组织中，组织特异性的CpG甲基化模式是在发育期间建立的。相反，异常细胞（如癌细胞）中观察到甲基化模式的改变。已经鉴定了癌症特定的CpG甲基化模式，并被用作癌症诊断的生物标志物。在本研究中，我们开发了一种基于杂交的CpG甲基化水平传感系统，使用一个甲基化CpG结合结构域（MBD）融合的荧光蛋白质。在这个系统中，目标DNA通过互补的甲基化探针DNA捕获。当目标DNA被甲基化时，在双链DNA中形成对称甲基化的CpG。MBD特异性识别双链DNA上对称甲基化的CpG，因此通过测量绑定MBD融合荧光蛋白质的荧光强度来量化甲基化水平。我们制备了MBD-AcGFP1融合蛋白质，并使用MBD-AcGFP1量化了针对SEPT9、BRCA1和长间隔核元素-1（LINE-1）的目标DNA的CpG甲基化水平。这一检测原理可以应用于同时并全基因组修饰碱基检测系统，使用融合于荧光蛋白质的修饰碱基结合蛋白质的微阵列。

Methylation of cytosine to 5-methylcytosine on CpG dinucleotides is the most frequently studied epigenetic modification involved in the regulation of gene expression. In normal tissues, tissue-specific CpG methylation patterns are established during development. In contrast, alterations in methylation patterns have been observed in abnormal cells, such as cancer cells. Cancer type-specific CpG methylation patterns have been identified and used as biomarkers for cancer diagnosis. In this study, we developed a hybridization-based CpG methylation level sensing system using a methyl-CpG-binding domain (MBD)-fused fluorescent protein. In this system, the target DNA is captured by a complementary methylated probe DNA. When the target DNA is methylated, a symmetrically methylated CpG is formed in the double-stranded DNA. MBD specifically recognizes symmetrical methyl-CpG on double-stranded DNA; therefore, the methylation level is quantified by measuring the fluorescence intensity of the bound MBD-fused fluorescent protein. We prepared MBD-fused AcGFP1 and quantified the CpG methylation levels of the target DNA against SEPT9, BRCA1, and long interspersed nuclear element-1 (LINE-1) using MBD-AcGFP1. This detection principle can be applied to the simultaneous and genome-wide modified base detection systems using microarrays coupled with modified base binding proteins fused to fluorescent proteins.