Analysis of CpG island methylation using rolling Circle amplification (RCA) product microarray.
Analysis of CpG island methylation using rolling Circle amplification (RCA) product microarray.
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DOI：
10.1166/jbn.2011.1287
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发表时间：
2011-04
影响因子：
2.9
通讯作者：
Hong Zhao;Xiaodong Ma;Minli Li;Dongrui Zhou;Pengfeng Xiao;Zuhong Lu
中科院分区：
文献类型：
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作者：
Hong Zhao;Xiaodong Ma;Minli Li;Dongrui Zhou;Pengfeng Xiao;Zuhong Lu
在这里，我们报告了一种方法，甲基化分析使用滚环扩增（RCA）的产品芯片。我们用亚硫酸氢盐处理DNA样品，并为靶基因中的每个CpG区域设计一对挂锁探针，其中一个与来自甲基化CpG区域的CpG位点相匹配，另一个与来自其相应的未甲基化等位基因的TpG位点相匹配。将锁式探针与亚硫酸氢盐处理的基因组DNA的PCR产物杂交，随后连接以形成用于RCA反应的单链环状模板。将RCA产物固定于玻片上制成DNA微阵列，与一对通用双色探针杂交，检测目的基因CpG岛甲基化状态。我们用两个肿瘤相关基因P16和IGFBP7测试了RCA产物微阵列，并成功分析了两个基因中CpG岛的甲基化状态。通过甲基化特异性PCR分析进一步证实了微阵列数据。结果表明，RCA产物芯片有望用于CpG岛甲基化的高通量检测。
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.