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

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 fac

SUBMITTER: Forget AL 

PROVIDER: S-EPMC4329291 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature