Methylation of cytosine at the 5-position is a common epigenetic modification in mammalian DNA and plays an important role in regulating gene expression. Oxidized derivatives of 5-methylcytosine were discovered recently, and some of these oxidized derivatives, in addition to being intermediates in an active demethylation pathway, might also function as epigenetic modifications. Oxidized derivatives of thymine are known to be products of DNA damage, although evidence exists that the oxidized thymine derivative 5-hydroxymethyluracil might have an epigenetic role. There is a pressing need to learn more about these modifications, as epigenetic modifications have roles in development and in diseases, including cancer. This emerging area of research requires highly accurate and sequence-specific methods for the detection of cytosine and thymine modifications in DNA. In this Review, we introduce the biochemistry of cytosine and thymine modifications and discuss established detection methods, such as bisulfite sequencing and its modifications, as well as newer methods, which have been developed to overcome the substantial obstacles associated with studying these modifications in genomic DNA.

5位胞嘧啶的甲基化是哺乳动物DNA中常见的表观遗传修饰，在调节基因表达中起重要作用。最近发现了5-甲基胞嘧啶的氧化衍生物，这些氧化衍生物中的一些，除了作为活性脱甲基途径中的中间体外，还可能作为表观遗传修饰。胸腺嘧啶的氧化衍生物是DNA损伤的产物，尽管有证据表明氧化胸腺嘧啶的衍生物5-羟甲基尿嘧啶可能具有表观遗传作用。迫切需要更多地了解这些修饰，因为表观遗传修饰在发育和包括癌症在内的疾病中具有重要作用。这个新兴的研究领域需要用于检测DNA中胞嘧啶和胸腺嘧啶修饰的高度准确和特定于序列的方法。在本综述中，我们介绍了胞嘧啶和胸腺嘧啶修饰的生物化学，并讨论了已建立的检测方法，例如亚硫酸氢盐测序及其修饰，以及开发出的新方法，以克服与研究基因组DNA中的这些修饰相关的重大障碍。