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Published online 20 May 2004

Nucleic Acids Research, 2004, Vol. 32, No. 9 e73
© 2004 Oxford University Press

Single-particle tracking for DNA tether length monitoring

Noëlle Pouget1,2, Cynthia Dennis3, Catherine Turlan2, Mikhail Grigoriev3, Michaël Chandler2 and Laurence Salomé*,1

1 Institut de Pharmacologie et Biologie Structurale (UMR CNRS 5089), 205 route de Narbonne, 31077 Toulouse Cedex, France, 2 Laboratoire de Microbiologie et Génétique Moléculaire (UMR CNRS 5100), 118 route de Narbonne, 31062 Toulouse Cedex, France and 3 Laboratoire de Biologie Moléculaire Eucaryote (UMR CNRS 5099), 118 route de Narbonne, 31062 Toulouse Cedex, France

*To whom correspondence should be addressed. Tel: +33 5 61 17 59 39; Fax: +33 5 61 17 59 94; Email: laurence.salome{at}ipbs.fr
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors

Received February 9, 2004; Revised and Accepted April 28, 2004

We describe a simple single-particle tracking approach for monitoring the length of DNA molecules in tethered particle motion experiments. In this method, the trajectory of a submicroscopic bead tethered by a DNA molecule to a glass surface is determined by videomicroscopy coupled to image analysis. The amplitude of motion of the bead is measured by the standard deviation of the distribution of successive positions of the bead in a given time interval. We were able to describe theoretically the variation of the equilibrium value of the amplitude of the bead motion with the DNA tether length for the entire applicable DNA length range (up to ~3500 bp). The sensitivity of the approach was illustrated by the evidence obtained for conformational changes introduced into a Holliday junction by the binding of the Escherichia coli RuvA protein. An advantage of this method is that the trajectory of the tethered bead, rather than its averaged motion, is measured, allowing analysis of the conformational dynamics of DNA chains at the single-molecule level.


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