Laboratory of Bioinformatics and Systems Biology, Centre of New Technologies, University of Warsaw, Warsaw, Poland.
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch at Galveston, Galveston, TX, USA.
Nat Protoc. 2021 Feb;16(2):1034-1061. doi: 10.1038/s41596-020-00448-3. Epub 2020 Dec 21.
DNA double-strand breaks (DSBs) are implicated in various physiological processes, such as class-switch recombination or crossing-over during meiosis, but also present a threat to genome stability. Extensive evidence shows that DSBs are a primary source of chromosome translocations or deletions, making them a major cause of genomic instability, a driving force of many diseases of civilization, such as cancer. Therefore, there is a great need for a precise, sensitive, and universal method for DSB detection, to enable both the study of their mechanisms of formation and repair as well as to explore their therapeutic potential. We provide a detailed protocol for our recently developed ultrasensitive and genome-wide DSB detection method: immobilized direct in situ breaks labeling, enrichment on streptavidin and next-generation sequencing (i-BLESS), which relies on the encapsulation of cells in agarose beads and labeling breaks directly and specifically with biotinylated linkers. i-BLESS labels DSBs with single-nucleotide resolution, allows detection of ultrarare breaks, takes 5 d to complete, and can be applied to samples from any organism, as long as a sufficient amount of starting material can be obtained. We also describe how to combine i-BLESS with our qDSB-Seq approach to enable the measurement of absolute DSB frequencies per cell and their precise genomic coordinates at the same time. Such normalization using qDSB-Seq is especially useful for the evaluation of spontaneous DSB levels and the estimation of DNA damage induced rather uniformly in the genome (e.g., by irradiation or radiomimetic chemotherapeutics).
DNA 双链断裂 (DSBs) 参与多种生理过程，如类别转换重组或减数分裂中的交叉，同时也对基因组稳定性构成威胁。大量证据表明，DSBs 是染色体易位或缺失的主要来源，使它们成为基因组不稳定性的主要原因，是许多文明病（如癌症）的驱动力。因此，非常需要一种精确、敏感和通用的 DSB 检测方法，以便既能研究其形成和修复机制，又能探索其治疗潜力。我们提供了一种详细的方案，用于我们最近开发的超灵敏和全基因组 DSB 检测方法：固定直接原位断裂标记、链亲和素富集和下一代测序（i-BLESS），该方法依赖于细胞在琼脂糖珠中的包封和用生物素化接头直接和特异性标记断裂。i-BLESS 以单核苷酸分辨率标记 DSBs，允许检测超稀有断裂，需要 5 天完成，并且可以应用于任何生物体的样本，只要能够获得足够的起始材料。我们还描述了如何将 i-BLESS 与我们的 qDSB-Seq 方法结合使用，以便同时测量每个细胞的绝对 DSB 频率及其精确的基因组坐标。使用 qDSB-Seq 进行这种归一化对于评估自发 DSB 水平和估计基因组中均匀诱导的 DNA 损伤（例如，通过辐照或类放射化疗药物）特别有用。
