Methylation-Specific PCR
Methylation-Specific PCR
复制标题
DOI：
10.1007/978-1-62703-547-7_7
复制
发表时间：
2013-01-01
期刊：
影响因子：
--
通讯作者：
Murphy, Susan K.
中科院分区：
文献类型：
--
作者：
Huang, Zhiqing;Bassil, Christopher F.;Murphy, Susan K.
定义基因组中的DNA甲基化模式对于理解不同的生物学过程，包括基因表达的调节，印记基因和X染色体失活以及这些模式在人类疾病中是如何失调的至关重要。甲基化特异性（MS）-PCR是一种用于定性DNA甲基化分析的有用工具，具有多种优点，包括易于设计和执行，检测少量甲基化DNA的能力灵敏度，以及快速筛选大量样品而无需购买昂贵的实验室设备的能力。该测定需要通过亚硫酸氢钠修饰基因组DNA和用于PCR扩增的两个独立引物组，一对设计用于识别亚硫酸氢钠修饰序列的甲基化版本，另一对设计用于识别亚硫酸氢盐修饰序列的未甲基化版本。扩增子在琼脂糖凝胶电泳后使用溴化乙锭染色可视化。由任一引物对产生的预期大小的扩增子指示原始样品中存在具有相应甲基化状态的DNA。
Defining DNA methylation patterns in the genome has become essential for understanding diverse biological processes including the regulation of gene expression, imprinted genes, and X chromosome inactivation and how these patterns are deregulated in human diseases. Methylation-specific (MS)-PCR is a useful tool for qualitative DNA methylation analysis with multiple advantages, including ease of design and execution, sensitivity in the ability to detect small quantities of methylated DNA, and the ability to rapidly screen a large number of samples without the need for purchase of expensive laboratory equipment. This assay requires modification of the genomic DNA by sodium bisulfite and two independent primer sets for PCR amplification, one pair designed to recognize the methylated and the other pair the unmethylated versions of the bisulfite-modified sequence. The amplicons are visualized using ethidium bromide staining following agarose gel electrophoresis. Amplicons of the expected size produced from either primer pair are indicative of the presence of DNA in the original sample with the respective methylation status.