Hou Peng, Ji Meiju, Ge Cunwang, Shen Jiayao, Li Song, He Nongyue, Lu Zuhong
Chien-Shiung Wu Laboratory, Department of Biological Science and Medical Engineering, Southeast University, Nanjing, 210096, China.
Nucleic Acids Res. 2003 Aug 15;31(16):e92. doi: 10.1093/nar/gng092.
Aberrant DNA methylation of the CpG site is among the earliest and most frequent alterations in cancer. Detection of promoter hypermethylation of cancer-related genes may be useful for cancer diagnosis or the detection of recurrence. p16, an inhibitor of the cyclin D-dependent protein kinases, is a classical tumor suppressor gene, and its inactivation is closely associated with carcinogenesis. p16 hypermethylation could be detected in each stage, which is consistent with the finding that aberrant methylation of p16 is a very early event in carcinogenesis. We have developed an electrochemical procedure for detecting DNA methylation of the human p16(Ink4a) gene. The procedure is based on the coupling of DNA electrochemical sensors with linker-PCR- amplified DNA from human gastric tumor tissue and whole blood cells of healthy human. The synthesized oligonucleotide was immobilized on the modified gold electrode to fabricate a DNA biosensor. The hybridization reaction on the electrode surface was monitored by cyclic voltammogram (CV) and square wave voltammogram (SWV), using Co(phen)(3)(3) as a redox indicator. Methylation status of human p16(Ink4a) gene was detected and the results were validated by bisulfite DNA sequencing. A good reproducibility was observed in several parallel experiments. The coupling of DNA electrochemical sensors with PCR allowed quick detection and have the potential of the quantitative evaluation of the methylation status of the human p16(Ink4a) gene.
CpG 位点的异常 DNA 甲基化是癌症中最早且最常见的改变之一。检测癌症相关基因的启动子高甲基化可能有助于癌症诊断或复发检测。p16 是细胞周期蛋白 D 依赖性蛋白激酶的抑制剂，是一种经典的肿瘤抑制基因，其失活与致癌作用密切相关。在各个阶段都能检测到 p16 高甲基化，这与 p16 异常甲基化是致癌过程中非常早期的事件这一发现一致。我们开发了一种用于检测人 p16(Ink4a)基因 DNA 甲基化的电化学方法。该方法基于将 DNA 电化学传感器与来自人胃肿瘤组织和健康人全血细胞的接头 - PCR 扩增 DNA 相结合。将合成的寡核苷酸固定在修饰的金电极上以制备 DNA 生物传感器。以Co(phen)(3)(3)作为氧化还原指示剂，通过循环伏安法（CV）和方波伏安法（SWV）监测电极表面的杂交反应。检测了人 p16(Ink4a)基因的甲基化状态，并通过亚硫酸氢盐 DNA 测序验证了结果。在几个平行实验中观察到了良好的重现性。DNA 电化学传感器与 PCR 的结合实现了快速检测，并具有对人 p16(Ink4a)基因甲基化状态进行定量评估的潜力。
