Developmental Therapeutics Branch, Center for Cancer Research, NCI, NIH, Bethesda, MD, USA.
STAR Protoc. 2022 Apr 12;3(2):101290. doi: 10.1016/j.xpro.2022.101290. eCollection 2022 Jun 17.
In this protocol, the progression of DNA synthesis is profiled at a single-molecule resolution. DNA fibers are uniformly stretched on silanized coverslips, and replicating DNA can be traced with thymidine analogs using specific antibodies against distinct analogs. Single DNA fibers are visualized by an anti-single stranded DNA antibody. The protocol can be used to study DNA replication dynamics, the cellular response to replication stress, and replication fork progression at specific chromosomal regions when combined with fluorescent hybridization. For complete details on the use and execution of this protocol, please refer to Conti et al. (2007), Fu et al. (2021), Kaykov et al. (2016), Redmond et al. (2018), and Schwob et al. (2009).
在本方案中，以单分子分辨率描绘 DNA 合成的进展。DNA 纤维在硅烷化盖玻片上均匀拉伸，使用针对不同类似物的特异性抗体可以追踪胸腺嘧啶类似物掺入的复制 DNA。通过抗单链 DNA 抗体可视化单 DNA 纤维。当与荧光杂交结合使用时，该方案可用于研究 DNA 复制动力学、细胞对复制应激的反应以及特定染色体区域的复制叉进展。有关该方案使用和执行的完整详细信息，请参考 Conti 等人（2007 年）、Fu 等人（2021 年）、Kaykov 等人（2016 年）、Redmond 等人（2018 年）和 Schwob 等人（2009 年）。
