State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China.
J Biomed Nanotechnol. 2011 Apr;7(2):292-9. doi: 10.1166/jbn.2011.1287.
Here we report a method for methylation analysis using rolling circle amplification (RCA) product microarray. We treated DNA samples with bisulfite and designed, for each CpG region in the target gene, a pair of padlock probes with one matching to the CpG sites derived from methylated CpG region, and the other matching to the TpG sites derived from their corresponding unmethylated allele. The padlock probes were hybridized to the PCR products of the bisulfite-treated genomic DNA, and were subsequently ligated to form single-strand, circular template for RCA reaction. The RCA products were immobilized on the slide to fabricate DNA microarray which hybridized a pair of universal dual-color probes to detect the methylation status of the CpG islands in the target gene. We tested the RCA product microarray with two tumor-related genes, P16 and IGFBP7, and successfully analyzed the methylation status of the CpG islands in the two genes. The microarray data were further confirmed by methylation-specific PCR analysis. Our results demonstrated that the RCA product microarray was hopeful for high-throughput detection of CpG island methylation.
在这里，我们报告了一种使用滚环扩增（RCA）产物微阵列进行甲基化分析的方法。我们用亚硫酸氢盐处理 DNA 样本，并为目标基因中的每个 CpG 区域设计了一对锁式探针，其中一个与来自甲基化 CpG 区域的 CpG 位点匹配，另一个与来自相应未甲基化等位基因的 TpG 位点匹配。锁式探针与经亚硫酸氢盐处理的基因组 DNA 的 PCR 产物杂交，随后连接形成单链、环状模板，用于 RCA 反应。RCA 产物固定在载玻片上以制备 DNA 微阵列，该微阵列杂交一对通用双色探针以检测目标基因中 CpG 岛的甲基化状态。我们用两个与肿瘤相关的基因 P16 和 IGFBP7 测试了 RCA 产物微阵列，并成功分析了这两个基因中 CpG 岛的甲基化状态。微阵列数据进一步通过甲基化特异性 PCR 分析得到证实。我们的结果表明，RCA 产物微阵列有望用于高通量检测 CpG 岛甲基化。
