Department of Epigenetics, Van Andel Institute, Grand Rapids, MI, USA.
Methods Mol Biol. 2023;2660:247-262. doi: 10.1007/978-1-0716-3163-8_17.
There are many sources of endogenous and exogenous DNA damage. Damaged bases represent a threat to genome integrity and may interfere with normal cellular processes such as replication and transcription. To understand the specificity and biological consequences of DNA damage, it is essential to employ methods that are sensitive enough to detect damaged DNA bases at the level of single nucleotide resolution and genome-wide. Here we describe in detail a method we developed for this purpose, circle damage sequencing (CD-seq). This method is based on the circularization of genomic DNA that contains damaged bases and conversion of the damaged sites into double-strand breaks using specific DNA repair enzymes. Library sequencing of the opened circles yields the precise positions of the DNA lesions that are present. CD-seq can be adopted to various types of DNA damage as long as a specific cleavage scheme can be designed.
内源性和外源性 DNA 损伤有很多来源。受损的碱基代表着对基因组完整性的威胁，并可能干扰正常的细胞过程，如复制和转录。为了了解 DNA 损伤的特异性和生物学后果，必须采用足够灵敏的方法，以单核苷酸分辨率和全基因组水平检测受损的 DNA 碱基。在这里，我们详细描述了为此目的开发的一种方法，即环形损伤测序（CD-seq）。该方法基于含有受损碱基的基因组 DNA 的环化，并使用特定的 DNA 修复酶将受损部位转化为双链断裂。对打开的环进行文库测序，可获得存在的 DNA 损伤的确切位置。只要能够设计出特定的切割方案，CD-seq 就可以应用于各种类型的 DNA 损伤。
