Zhou Dongrui, Zhang Renmin, Fang Rujing, Cheng Lu, Xiao Pengfeng, Lu Zuhong
State Key Laboratory of Bioelectronics, Southeast University, Nanjing, P R China.
Electrophoresis. 2008 Feb;29(3):626-33. doi: 10.1002/elps.200700375.
Monitoring the methylation pattern of a single tumor cell might be important in understanding the mechanism of tumor initiation and progression. In this study, we developed a method based on molecular cloning microarray strategy for analyzing methylation patterns of a single DNA fragment from a group of tumor cells. In the method, a microarray of single monoclones of bisulfate-treated PCR products was fabricated by two-primer hyperbranched rolling circle amplification (HRCA) in polyacrylamide gel, in which a library of the bisulfate-treated PCR products with different methylated status from tumor cells were ligated to circle molecules to form HRCA templates, and one of the HRCA primers was modified with acrylamide on its 5'-end. Due to the diffusion retardation of HRCA products in a polyacrylamide matrix, the HRCA products are localized near their respective templates, and formed to a microarray of monoclones. After the nonimmobilized strands were removed, three pairs of probes were used to detect different CpG sites of each clone simultaneously by hybridization. We successfully analyzed the methylation patterns of P16 gene for three cases of stomach tumor tissues. This method could provide a significant tool in detecting the distribution of cells with different methylation patterns in one tumor tissue.
监测单个肿瘤细胞的甲基化模式对于理解肿瘤发生和进展的机制可能具有重要意义。在本研究中，我们开发了一种基于分子克隆微阵列策略的方法，用于分析一组肿瘤细胞中单个DNA片段的甲基化模式。在该方法中，通过在聚丙烯酰胺凝胶中进行双引物超分支滚环扩增（HRCA）制备经亚硫酸氢盐处理的PCR产物的单克隆微阵列，其中将来自肿瘤细胞的具有不同甲基化状态的经亚硫酸氢盐处理的PCR产物文库连接成环状分子以形成HRCA模板，并且HRCA引物之一在其5'端用丙烯酰胺修饰。由于HRCA产物在聚丙烯酰胺基质中的扩散阻滞，HRCA产物定位在其各自模板附近，并形成单克隆微阵列。去除未固定的链后，使用三对探针通过杂交同时检测每个克隆的不同CpG位点。我们成功分析了三例胃肿瘤组织中P16基因的甲基化模式。该方法可为检测一个肿瘤组织中具有不同甲基化模式的细胞分布提供重要工具。
