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.

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分析特定 DNA 甲基化状态的方法

CpG 二核苷酸的甲基化在基因表达的调节中起着至关重要的作用，因此在不同病理的发展中起着至关重要的作用。异常甲基化与大多数疾病有关，包括癌症、神经退行性疾病、心血管和自身免疫性疾病。DNA 甲基化模式的分析对于了解这些疾病的潜在分子机制至关重要。此外，DNA 甲基化模式可用作临床管理的生物标志物，例如诊断、预后和治疗反应。如今，已开发出多种高通量 DNA 甲基化方法来一次性分析大量 CpG 甚至整个基因组的甲基化状态。然而，在特定位点识别特定的甲基化模式对于验证至关重要，同时也是诊断的工具。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。主要有三组技术可以识别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力富集的分析。在第一组中，特定的限制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未甲基化 DNA 的最常用方法。未甲基化的胞嘧啶通过亚硫酸氢钠处理脱氨基为尿嘧啶，而甲基胞嘧啶保持未转化。在最后一组，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，并分析了它们的优缺点。