The Combined Bisulfite Restriction Analysis (COBRA) Assay for the Analysis of Locus-Specific Changes in Methylation Patterns
The Combined Bisulfite Restriction Analysis (COBRA) Assay for the Analysis of Locus-Specific Changes in Methylation Patterns
复制标题
DOI：
10.1007/978-1-4899-7708-3_5
复制
发表时间：
2017-01-01
期刊：
影响因子：
--
通讯作者：
Kovalchuk, Igor
中科院分区：
文献类型：
--
作者：
Bilichak, Andriy;Kovalchuk, Igor
DNA甲基化是一种可遗传但可逆的控制基因表达的表观遗传机制。特定基因组区域的DNA甲基化水平与染色质凝聚、基因表达水平以及在某些情况下基因组稳定性和同源重组频率相关。在这里，我们描述了联合亚硫酸氢盐限制性分析（COBRA）测定，允许分析在特定位点的甲基化状态。该方案由以下主要步骤组成：非甲基化胞嘧啶的亚硫酸氢盐转化为尿嘧啶，转化的DNA的基因座特异性PCR扩增，限制性消化，凝胶上的限制性图谱分析，以及使用ImageJ或类似程序定量这些限制性图谱。
DNA methylation is a heritable but reversible epigenetic mechanism of control over gene expression. The level of DNA methylation of specific genomic regions correlates with chromatin condensation, the level of gene expression, and in some cases genome stability and the frequency of homologous recombination. Here, we describe the combined bisulfite restriction analysis (COBRA) assay that allows analyzing the methylation status at a specific locus. The protocol consists of the following major steps: bisulfite conversion of non-methylated cytosines to uracils, the locus-specific PCR amplification of converted DNA, restriction digestion, the analysis of restriction patterns on the gel, and the quantification of these restriction patterns using ImageJ or a similar program.