Tanner Nathan A, Loparo Joseph J, Hamdan Samir M, Jergic Slobodan, Dixon Nicholas E, van Oijen Antoine M
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Nucleic Acids Res. 2009 Mar;37(4):e27. doi: 10.1093/nar/gkp006. Epub 2009 Jan 20.
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.
我们展示了一种实时可视化单个DNA分子复制的简单技术。通过将滚环底物连接到安装在全内反射荧光（TIRF）显微镜上的流动腔室，我们能够监测单链DNA合成事件的进程，并准确测量单个T7和大肠杆菌复制体在复制DNA时的速率和持续合成能力。该方法能够快速、精确地表征DNA合成动力学以及复制抑制剂的作用。
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