Detection of average methylation level of specific genes by binary-probe hybridization
Detection of average methylation level of specific genes by binary-probe hybridization
复制标题
通过双探针杂交检测特定基因的平均甲基化水平
DOI：
10.1016/j.talanta.2021.122630
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发表时间：
2021
期刊：
影响因子：
6.1
通讯作者：
Zhang Xin-Xiang
中科院分区：
文献类型：
--
作者：
Zhong Xin-Ying;Zhou Qian-Yu;Dong Jia-Hui;Yu Yue;Zhou Ying-Lin;Zhang Xin-Xiang
我们建立了一种基于二元探针DNA杂交的5-甲基胞嘧啶检测特定基因序列的简单、高选择性方法。与靶标互补的序列被设计成两个探针，每个探针片段与靶标的一个相对较短的序列结合，这使得它对亚硫酸氢盐处理引入的碱基错配敏感。二探针杂交的成功设计证明了二探针杂交具有甲基化丰度0.1%的低检测限和10 pM的高灵敏度等优点。二元探针的成功设计使得量化五个CpG位点的平均甲基化水平成为可能。用同一对二元探针成功分析了含有5、4、3、2、1和0个CpG位点的32条DNA链。靶基因的平均甲基化水平越高，杂交反应的程度越高。基于二元探针杂交结构的简单性，所研制的生物传感器所显示的信号与基因的平均甲基化水平成正比，可用于评价人工混合物的平均甲基化水平。此外，该方法已被用于在基因组背景下检测甲基化，具有良好的特异性，这表明它在甲基化相关疾病的预诊断中具有潜力。
We developed a simple and highly-selective method for 5-methylcytosine detection of specific gene sequence based on binary-probe DNA hybridization. The sequence complementary to the target was designed into two probes, and each fragment of binary probes bound to a relatively short sequence of the target, which made it sensitive to the base mismatches introduced by bisulfite treatment. The advantages of a low detection limit of methylation abundance of 0.1% for the fully methylated target and high sensitivity of 10 pM have been proved by the successful design of binary-probe hybridization. The successful design of the binary probes makes it possible to quantify the average methylation levels of five CpG sites. Thirty-two DNA strands containing 5, 4, 3, 2, 1 and 0 CpG sites were successfully analyzed with the same pair of binary probes. The higher the average methylation level of the target was, the higher the degree of the hybridization reaction. Based on the simple construction of the binary-probe hybridization, the developed biosensor exhibited signals proportional to the average methylation level of thevimentingene and could evaluate the average methylation level of artificial mixtures. Furthermore, the method has been used to detectvimentinmethylation in a genomic context with good specificity, which indicated its potential in the pre-diagnosis of methylation related disease.