Real-time single-molecule observation of rolling-circle DNA replication

作者：Nathan A. Tanner, Joseph J. Loparo, Samir M. Hamdan, Slobodan Jergic, Nicholas E. Dixon, Antoine M. van Oijen · 发表于：Nucleic Acids Research · 年份：2009 · DOI：10.1093/nar/gkp006 · 被引用次数：1283 · 研究领域：DNA and Nucleic Acid Chemistry、DNA Repair Mechanisms、Bacterial Genetics and Biotechnology

We present a simple technique for visualizing replication of individual DNA molecules in real time. By attaching a rolling-circle substrate to a TIRF microscope-mounted flow chamber, we are able to monitor the progression of single-DNA synthesis events and accurately measure rates and processivities of single T7 and Escherichia coli replisomes as they replicate DNA. This method allows for rapid and precise characterization of the kinetics of DNA synthesis and the effects of replication inhibitors.