DNA methylation: Bisulphite modification and analysis
DNA methylation: Bisulphite modification and analysis
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DOI：
10.1038/nprot.2006.324
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Frommer, Marianne
中科院分区：
文献类型：
--
作者：
Clark, Susan J.;Statham, Aaron;Frommer, Marianne
DNA甲基化是哺乳动物基因组中一种重要的表观遗传修饰。DNA甲基化模式在发育早期建立，在组织特异性分化期间受到调节，并在许多疾病状态（包括癌症）中被破坏。为了进一步了解这些变化的生物学功能，需要准确和可重复的方法来全面分析DNA甲基化序列。在这里，我们描述了“金标准”亚硫酸氢盐转换协议，可用于重新测序DNA从哺乳动物细胞，以确定和量化的甲基化状态的基因或基因组区域在单核苷酸分辨率。亚硫酸氢盐处理过程利用了胞嘧啶和5-甲基胞嘧啶（5-MeC）在酸性条件下对亚硫酸氢盐脱氨基的不同敏感性-其中胞嘧啶转化为尿嘧啶，而5-MeC保持不反应。在单管或96孔格式中，DNA的亚硫酸氢盐转化可以在最短8小时和最长18小时内进行，这取决于起始DNA的量和质量。
DNA methylation is an important epigenetic modification of DNA in mammalian genomes. DNA methylation patterns are established early in development, modulated during tissue-specific differentiation and disrupted in many disease states, including cancer. To understand further the biological functions of these changes, accurate and reproducible methods are required to fully analyze the DNA methylation sequence. Here, we describe the 'gold-standard' bisulphite conversion protocol that can be used to re-sequence DNA from mammalian cells in order to determine and quantify the methylation state of a gene or genomic region at single-nucleotide resolution. The process of bisulphite treatment exploits the different sensitivities of cytosine and 5-methylcytosine (5-MeC) to deamination by bisulphite under acidic conditions - in which cytosine undergoes conversion to uracil, whereas 5-MeC remains unreactive. Bisulphite conversion of DNA, in either single tubes or in a 96-well format, can be performed in a minimum of 8 h and a maximum of 18 h, depending on the amount and quality of starting DNA.