Biochemistry Area, Department of Health Science, Public University of Navarre, 31008 Pamplona, Spain; IDISNA Navarra's Health Research Institute, 31008 Pamplona, Spain.
Biomarkers and Precision Medicine Unit, Health Research Institute la Fe, 46026 Valencia, Spain.
Methods. 2021 Mar;187:3-12. doi: 10.1016/j.ymeth.2020.06.021. Epub 2020 Jul 5.
Methylation of CpG dinucleotides plays a crucial role in the regulation of gene expression and therefore in the development of different pathologies. Aberrant methylation has been associated to the majority of the diseases, including cancer, neurodegenerative, cardiovascular and autoimmune disorders. Analysis of DNA methylation patterns is crucial to understand the underlying molecular mechanism of these diseases. Moreover, DNA methylation patterns could be used as biomarker for clinical management, such as diagnosis, prognosis and treatment response. Nowadays, a variety of high throughput methods for DNA methylation have been developed to analyze the methylation status of a high number of CpGs at once or even the whole genome. However, identification of specific methylation patterns at specific loci is essential for validation and also as a tool for diagnosis. In this review, we describe the most commonly used approaches to evaluate specific DNA methylation. There are three main groups of techniques that allow the identification of specific regions that are differentially methylated: bisulfite conversion-based methods, restriction enzyme-based approaches, and affinity enrichment-based assays. In the first group, specific restriction enzymes recognize and cleave unmethylated DNA, leaving methylated sequences intact. Bisulfite conversion methods are the most popular approach to distinguish methylated and unmethylated DNA. Unmethylated cytosines are deaminated to uracil by sodium bisulfite treatment, while the methyl cytosines remain unconverted. In the last group, proteins with methylation binding domains or antibodies against methyl cytosines are used to recognize methylated DNA. In this review, we provide the theoretical basis and the framework of each technique as well as the analysis of their strength and the weaknesses.
CpG 二核苷酸的甲基化在基因表达的调控中起着至关重要的作用，因此在多种病理的发生发展中起着重要作用。异常的甲基化与大多数疾病有关，包括癌症、神经退行性疾病、心血管疾病和自身免疫性疾病。分析 DNA 甲基化模式对于理解这些疾病的潜在分子机制至关重要。此外，DNA 甲基化模式可作为临床管理的生物标志物，如诊断、预后和治疗反应。如今，已经开发出多种高通量的 DNA 甲基化方法，可一次性分析大量 CpG 的甲基化状态，甚至可分析整个基因组的甲基化状态。然而，鉴定特定基因座的特定甲基化模式对于验证和作为诊断工具至关重要。在这篇综述中，我们描述了评估特定 DNA 甲基化的最常用方法。有三组主要的技术可用于鉴定差异甲基化的特定区域：基于亚硫酸氢盐转化的方法、基于限制酶的方法和基于亲和富集的测定法。在第一组中，特定的限制性内切酶识别并切割未甲基化的 DNA，使甲基化序列保持完整。亚硫酸氢盐转化方法是区分甲基化和非甲基化 DNA 的最常用方法。未甲基化的胞嘧啶在亚硫酸氢盐处理下脱氨为尿嘧啶，而甲基胞嘧啶则保持不变。在最后一组中，具有甲基化结合域的蛋白质或针对甲基胞嘧啶的抗体用于识别甲基化 DNA。在这篇综述中，我们提供了每种技术的理论基础和框架，以及对其优缺点的分析。
