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.
Original language | English (US) |
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Pages (from-to) | 525-538 |
Number of pages | 14 |
Journal | Nature Protocols |
Volume | 8 |
Issue number | 3 |
DOIs | |
State | Published - Feb 2013 |
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)