用于检测DNA甲基化的高通量方法。
High-throughput method for detecting DNA methylation.
作者信息
Hou Peng, Ji Meiju, Li Song, He Nongyue, Lu Zuhong
机构信息
Chien-Shiung Wu Laboratory, Department of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China.
出版信息
J Biochem Biophys Methods. 2004 Aug 31;60(2):139-50. doi: 10.1016/j.jbbm.2004.05.001.
Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in cancer. Detection of promoter hypermethylation of cancer-related gene may be useful for cancer diagnosis or the detection of recurrence. However, most of the studies have focused on a single gene only and gave little information about the concurrent methylation status of multiple genes. In this study, we attempted to develop a microarray method coupled with linker-PCR for detecting methylation status of multiple genes in the tumor tissue. A series of synthesized oligonucleotides were synthesised and purified to completely match with 16 investigated targets. Then they were immobilized on the aldehyde-coated glass slide to fabricate a DNA microarray for detecting methylation status of these genes. The results indicated that these genes were all methylated in the positive control. However, no methylated was found in these genes for the negative control. Only p16 and p15 genes were methylated in investigated genes for the gastric tumor tissue, whereas others were not methylated. The above results were validated by bisulfite DNA sequencing. Our experiments successfully demonstrated that the DNA microarray could be applied as a high-throughput tool to determine methylation status of the investigated genes.
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。然而，大多数研究仅关注单个基因，关于多个基因同时甲基化状态的信息很少。在本研究中，我们尝试开发一种结合接头 PCR 的微阵列方法来检测肿瘤组织中多个基因的甲基化状态。合成并纯化了一系列寡核苷酸，使其与 16 个研究靶点完全匹配。然后将它们固定在醛包被的载玻片上，制备用于检测这些基因甲基化状态的 DNA 微阵列。结果表明，这些基因在阳性对照中均被甲基化。然而，在阴性对照的这些基因中未发现甲基化。在胃癌组织的研究基因中，只有 p16 和 p15 基因被甲基化，而其他基因未被甲基化。上述结果通过亚硫酸氢盐 DNA 测序得到验证。我们的实验成功证明，DNA 微阵列可作为一种高通量工具来确定所研究基因的甲基化状态。
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