Galbiati Alessandro, d'Adda di Fagagna Fabrizio
Oncology IMED, AstraZeneca UK Ltd, Cambridge, UK.
IFOM-Foundation, The FIRC Institute of Molecular Oncology Foundation, Milan, Italy.
Methods Mol Biol. 2019;1896:11-20. doi: 10.1007/978-1-4939-8931-7_2.
Cells have evolved DNA repair mechanisms to maintain their genetic information unaltered and a DNA damage response pathway that coordinates DNA repair with several cellular events. Despite a clear role for DNA damage in the form of DNA double-strand breaks (DSBs) in several cellular processes, the most commonly used methods to detect DNA lesions are indirect, and rely on antibody-based recognition of DNA damage-associated factors, leaving several important questions unanswered. Differently, here we describe DNA damage In situ ligation followed by Proximity Ligation Assay (DI-PLA), that allows sensitive detection of physical DSBs in fixed cells, through direct labeling of the DSBs with biotinylated oligonucleotides, and subsequent signal amplification by PLA between biotin and a partner protein in the proximity of the DNA break.
细胞已经进化出DNA修复机制来维持其遗传信息不变，以及一种DNA损伤反应途径，该途径将DNA修复与多种细胞事件协调起来。尽管DNA双链断裂（DSB）形式的DNA损伤在多个细胞过程中具有明确作用，但检测DNA损伤的最常用方法是间接的，依赖于基于抗体对DNA损伤相关因子的识别，这使得几个重要问题未得到解答。不同的是，在这里我们描述了原位DNA损伤连接后进行邻近连接分析（DI-PLA），它能够通过用生物素化寡核苷酸直接标记DSB，并随后通过DNA断裂附近生物素与伴侣蛋白之间的PLA进行信号放大，在固定细胞中灵敏地检测物理性DSB。
