Optical Tweezers

Ineke Brouwer, Graeme A. King, Iddo Heller, Andreas S. Biebricher, Erwin J. G. Peterman, Gijs J. L. Wuite, Brouwer, Ineke, King, Graeme A., Heller, Iddo, Biebricher, Andreas S., Peterman, Erwin J. G., Wuite, Gijs J. L.

DNA metabolism and DNA compaction in vivo involve frequent interactions of remote DNA segments, mediated by proteins. In order to gain insight into such interactions, quadruple-trap optical tweezers have been developed. This technique provides an unprecedented degree of control through the ability to independently manipulate two DNA molecules in three dimensions. In this way, discrete regions of different DNA molecules can be brought into contact with one another, with a well-defined spatial configuration. At the same time, the tension and extension of the DNA molecules can be monitored. Furthermore, combining quadruple-trap optical tweezers with microfluidics makes fast buffer exchange possible, which is important for in situ generation of the dual DNA-protein constructs needed for these kinds of experiments. In this way, processes such as protein-mediated inter-DNA bridging can be studied with unprecedented control. This chapter provides a step-by-step description of how to perform a dual DNA manipulation experiment using combined quadruple-trap optical tweezers and microfluidics.