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Biophys J, August 2000, p. 1155-1167, Vol. 79, No. 2
and
Institute of Molecular Biology, University of
Oregon, Eugene, Oregon 97403, and Department of Molecular
and Cell Biology and *Department of Physics, University of California,
Berkeley, California 94720 USA
We have developed an integrated laser trap/flow control
video microscope for mechanical manipulation of single biopolymers. The
instrument is automated to maximize experimental throughput. A
single-beam optical trap capable of trapping micron-scale polystyrene beads in the middle of a 200-µm-deep microchamber is used, making it
possible to insert a micropipette inside this chamber to hold a second
bead by suction. Together, these beads function as easily exchangeable
surfaces between which macromolecules of interest can be attached. A
computer-controlled flow system is used to exchange the liquid in the
chamber and to establish a flow rate with high precision. The flow and
the optical trap can be used to exert forces on the beads, the
displacements of which can be measured either by video microscopy or by
laser deflection. To test the performance of this instrument,
individual biotinylated DNA molecules were assembled between two
streptavidin beads, and the DNA elasticity was characterized using both
laser trap and flow forces. DNA extension under varying forces was
measured by video microscopy. The combination of the flow system and
video microscopy is a versatile design that is particularly useful for the study of systems susceptible to laser-induced damage. This capability was demonstrated by following the translocation of transcribing RNA polymerase up to 650 s.
Biophys J, August 2000, p. 1155-1167, Vol. 79, No. 2
© 2000 by the Biophysical Society 0006-3495/00/08/1155/13 $2.00
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