期刊
NATURE PROTOCOLS
卷 8, 期 3, 页码 525-538出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2013.016
关键词
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资金
- American Cancer Society [PF-08-046-01-GMC]
- US National Institutes of Health (NIH) [T32 CA108459]
- NIH [GM-62653, GM-64745]
In this protocol, we describe a procedure to generate 'DNA dumbbells'-single molecules of DNA with a microscopic bead attached at each end-and techniques for manipulating individual DNA dumbbells. We also detail the design and fabrication of a microfluidic device (flow cell) used in conjunction with dual optical trapping to manipulate DNA dumbbells and to visualize individual protein-DNA complexes by single-molecule epifluorescence microscopy. Our design of the flow cell enables the rapid movement of trapped molecules between laminar flow channels and a flow-free reservoir. The reservoir provides the means to examine the formation of protein-DNA complexes in solution in the absence of external flow forces while maintaining a predetermined end-to-end extension of the DNA. These features facilitate the examination of the role of 3D DNA conformation and dynamics in protein-DNA interactions. Preparation of flow cells and reagents requires 2 days each; in situ DNA dumbbell assembly and imaging of single protein-DNA complexes require another day.
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