Methylation-specific PCR: A novel PCR assay for methylation status of CpG islands
Methylation-specific PCR: A novel PCR assay for methylation status of CpG islands
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DOI：
10.1073/pnas.93.18.9821
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发表时间：
1996-09-03
影响因子：
11.1
通讯作者：
Baylin, SB
中科院分区：
文献类型：
--
作者：
Herman, JG;Graff, JR;Baylin, SB
精确定位CpG岛上的DNA甲基化模式对于了解人类癌症中印记基因的调节、X染色体失活和肿瘤抑制基因沉默等多种生物学过程至关重要。我们描述了一种新的方法，MSP(甲基化特异性PCR)，它可以独立于甲基化敏感的限制性内切酶的使用，快速评估CpG岛上几乎任何一组CpG位点的甲基化状态。这种分析需要用亚硫酸氢钠对DNA进行初始修饰，将所有未甲基化但未甲基化的胞嘧啶转化为尿嘧啶，然后用针对甲基化和未甲基化DNA的特异引物进行扩增。MSP只需要少量的DNA，对给定CpG岛基因座的0.1%甲基化等位基因敏感，可以对从石蜡包埋样本中提取的DNA进行操作。MSP消除了以前基于聚合酶链式反应的方法固有的假阳性结果，这种方法依赖差异限制性内切酶切割来区分甲基化和非甲基化的DNA。在这项研究中，我们使用MSP来确定人类癌症中与四个重要的肿瘤抑制基因(p16、p15、E-钙粘蛋白和von Hippel-Lindau)转录失活相关的启动子区域高甲基化变化。
Precise mapping of DNA methylation patterns in CpG islands has become essential for understanding diverse biological processes such as the regulation of imprinted genes, X chromosome inactivation, and tumor suppressor gene silencing in human cancer. We describe a new method, MSP (methylation-specific PCR), which can rapidly assess the methylation status of virtually any group of CpG sites within a CpG island, independent of the use of methylation-sensitive restriction enzymes. This assay entails initial modification of DNA by sodium bisulfite, converting all unmethylated, but not methylated, cytosines to uracil, and subsequent amplification with primers specific for methylated versus unmethylated DNA. MSP requires only small quantities of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples. MSP eliminates the false positive results inherent to previous PCR-based approaches which relied on differential restriction enzyme cleavage to distinguish methylated from unmethylated DNA. In this study, we demonstrate the use of MSP to identify promoter region hypermethylation changes associated with transcriptional inactivation in four important tumor suppressor genes (p16, p15, E-cadherin, and von Hippel-Lindau) in human cancer.