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Exploring protein-DNA interactions in 3D using in situ construction, manipulation and visualization of individual DNA dumbbells with optical traps, microfluidics and fluorescence microscopy.

Authors
Type
Published Article
Journal
Nature Protocols
Publisher
Springer Nature
Volume
8
Issue
3
Pages
525–538
Identifiers
DOI: 10.1038/nprot.2013.016
Source
Kowalczykowski Lab
License
Unknown

Abstract

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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