Full text loading...
No data available.
Please log in to see this content.
You have no subscription access to this content.
No metrics data to plot.
The attempt to load metrics for this article has failed.
The attempt to plot a graph for these metrics has failed.
Backscattering from a tethered bead as a probe of DNA flexibility
1.S. B. Steven, L. Finzi, and C. Bustamante, Science 258, 1122 (1992).
2.T. T. Perkins, S. R. Quake, D. E. Smith, and S. Chu, Science 264, 822 (1994).
3.D. Bensimon, A. J. Simon, V. Croquette, and A. Bensimon, Phys. Rev. Lett. 74, 4754 (1995).
4.S. B. Steven, Y. Cui, and C. Bustamante, Science 271, 795 (1996).
5.P. Cluzel, A. Lebrun, C. Heller, R. Lavery, J. L. Viovy, D. Chatenay, and F. Caron, Science 271, 792 (1996).
6.T. R. Strick, J.-F. Allemand, D. Bensimon, A. Bensimon, and V. Croquette, Science 271, 1835 (1996).
7.M. D. Wang, H. Yin, R. Landick, J. Gelles, and S. M. Block, Biophys. J. 72, 1335 (1997).
8.B. Essevaz-Roulet, U. Bockelmann, and F. Heslot, Proc. Natl. Acad. Sci. USA 94, 11 935 (1997).
9.J. Marko and E. Siggia, Macromolecules 28, 8759 (1995).
10.G. V. Shivashankar and A. Libchaber, Appl. Phys. Lett. 71, 3727 (1997).
11.A. Ashkin, J. M. Dziedzic, J. E. Bjorkholm, and S. Chu, Opt. Lett. 11, 288 (1986).
12.R. M. Simmons, J. T. Finer, S. Chu, and J. Spudich, Biophys. J. 70, 1813 (1996).
13.To calibrate the bead displacement one chooses an immobilized bead on the coverslip. The fixed bead is aligned with respect to the laser axis and the backscattered signal is projected onto a quadrant position detector. A triangle ramp (1 μm amplitude and 1 Hz frequency) is setup to move the sample stage and the differential output signal of the quadrant detector is recorded. The minimum step size of the stage is ∼10 nm. From this data the calibration of the bead displacement is obtained.
14.Tethered beads are prepared by attaching one end of λ DNA (Promega) to a coverslip and the other end to a 3 μm latex bead (Polysciences), as described in Ref. 10. Experiments are carried out at 7.4 and in 0.1 M phosphate buffered saline. Typically the ratio of the number of beads to DNA molecules ∼1:10, for DNA attachment to the coverslip.
15.P. Howard-Flanders, S. C. West, and A. Stasiak, Nature (London) 309, 215 (1984).
16.Typical time to acquire one data set is 2 min and the polymerization reaction is carried out keeping the DNA molecule in the random coiled state (Fig. 4). The data in Fig. 4 is recorded with 100 point average, whereas in Fig. 2(a) with 10 point average. When the Rec A protein (from Promega) is reconstituted in the sample cell (volume ∼400 μl) the final concentration of Rec A ∼5 μM, ATP ∼2 mM, DTT∼2 mM, Tris-Hcl ∼20 mM, and 100 μg/400 μl acetylated BSA. The final is 7 and the experiments are done at 37 °C.
17.G. V. Shivashankar and A. Libchaber, Biophys. J. 74, A242 (1998);
17.M. Hegner and C. Bustamante, Biophys. J. 74, A150 (1998);
17.J. F. Leger, J. Robert, L. Bourdieu, D. Chatenay, and J. F. Marko (unpublished).
18.A monomer of Rec A binds to about three bases of DNA. The kinetics of polymerization depends on the state of the DNA molecule, whether it is in the random coil state or in a stretched form. Typical time scales for the polymerization of Rec A on a single DNA polymer, under the same biochemical conditions and are: 60 min for random coil and 20 min for a molecule under an applied external force of 5 pN.
Article metrics loading...