University of Pittsburgh Laboratory of Physical Molecular Biology; Sanford Leuba, Ph.D.
researchpeoplegallerypublicationsLeuba lab home

Download theplayer if you don't have it on your computer.


How to Catch A DNA Molecule
Video of single lambda-DNA molecule attaching to polystyrene beads

This video (duration three minutes) shows how a single lambda-DNA molecule (16.4 microns long) is attached to two polystyrene beads, as seen through the microscope with built-in optical tweezers. The exact position of the optical trap is indicated with four red lines. The arrow at the bottom left indicates the presence and direction of the flow. In the beginning, strepavidin-coated beads are flowed into the cell, and the first bead is captured in the optical trap. This bead is then transferred to a glass micropipette and held there by suction. A second bead is captured and maintained in the optical trap. Next, biotin-end-labeled lambda-DNA molecules are flowed in. At the moment a single DNA molecule attaches to the trapped bead, the force (indicated by the top bar) increases sharply. Following attachment to the trapped bead, the second bead is moved around in the vicinity of the position where the free end of the DNA is expected to be. After the connection to the second bead is made, the micropipette bead is moved to the left of the screen. Moving the micropipette bead away from the optical trapped bead and back, we demonstrate that the trapped bead moves out and in in the optical trap. This demonstrates that the two beads are tethered together by a DNA molecule.


Chromatin Real Time Assembly Video

This video (duration 38 seconds) shows the movement of the two beads to which a single lambda-DNA molecule is attached, after the X. laevis egg extract is introduced in the flow cell. A reduction in the distance between the beads is observed indicating nucleosome formation. This video is speeded up (the real time is ~10 min). The optical trap is turned off during this portion of the experiment.
Animation Assembly And Stretching
Animation of the assembly and then disassembly of a single chromatin fiber

This animation (duration 50 seconds) illustrates our experimental setup and how we envision our dynamic experiments occur. We have a single lambda-DNA molecule suspended, via-end-labeled-biotins, between two strepavidin-coated, two-micron-polystyrene beads. One bead is held by suction to a micropipette, the other one is held by an optical trap. In the first half of this animation, a X. laevis assembly extract is flowed into the liquid cell, and a corresponding reduction of distance between the two beads is observed, signifying the assembly of a chromatin fiber by DNA wrapping around each histone octamer. The optical trap is turned off during this portion of the experiment to avoid trapping of cell debris present in the egg extract. (For simplicity in this animation we only show nine nucleosomes out of the ~240 assembled on the lambda-DNA). In the second half of this animation, the free bead is placed in a force-measuring optical trap (signified by the four red bars) following the assembly process, and the micropipette bead is pulled away from the trap. Deflections of the bead in the optical trap are indicated as the disruptions in the force signal in force-extension curve (presented in the upper left-hand corner graph).

University of Pittsburgh Home University of Pittsburgh School of Medicine Pitt SOM Department of Cell Biology and Physiology Read a bio of Sanford Leuba, Ph.D.