title: 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 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.
authors: Department of Microbiology and Molecular Genetics, University of California, Davis, California, USA.
authors: Anthony L
authors: AL
authors: Forget
authors: Christopher C
authors: CC
authors: Dombrowski
authors: Ichiro
authors: I
authors: Amitani
authors: Stephen C
authors: SC
authors: Kowalczykowski
mesh_terms: D001665:Binding Sites; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D044085:Microfluidics / Q000295:instrumentation / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D009690:Nucleic Acid Conformation; D052898:Optical Tweezers; D017434:Protein Structure, Tertiary; D011938:Rec A Recombinases / Q000737:chemistry*
chemical_list: D004268:DNA-Binding Proteins; D004247:DNA; D011938:Rec A Recombinases