Mechanics and Single-Molecule Interrogation of DNA Recombination: Supplemental Video 9

A supplemental video from the 2016 review by Jason C. Bell and Stephen C. Kowalczykowski, "Mechanics and Single-Molecule Interrogation of DNA Recombination," from the Annual Review of Biochemistry. 

Supplemental Video 9 Composite video depicting the experimental procedure used to visualize DNA pairing on single λ DNA-dumbbell molecules by optical trapping.

A DNA pairing reaction (2 min) was performed with the 430 nt substrate at a 2 μm bead distance. Text and illustrations were inserted at appropriate places to facilitate description. A four-channel flow cell with a flow-free reservoir was used. Solution flow is top to bottom. First, two 1-μm streptavidin-coated polystyrene beads are captured in dual optical traps. Next, a single DNA molecule is captured on one bead. The DNA-dumbbell is made by sliding the DNA along the other bead, until the biotinylated end attaches. The DNA end-to-end distance is set, and the YOYO-1 dye is removed. The DNA-dumbbell is incubated with fluorescent RecA nucleoprotein filaments in the flow-free reservoir for 2 min. Afterward, the DNA-dumbbell is moved to the observation channel and is extended to near contour length to image the pairing products. Published with permission from Reference 152.