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Nucleic Acids Research 2005 33(11):e98; doi:10.1093/nar/gni097
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Published online 20 June 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
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Methods Online

Detection of single DNA molecules by multicolor quantum-dot end-labeling

Aurélien Crut1, Bénédicte Géron-Landre2, Isabelle Bonnet1, Stéphane Bonneau1,3, Pierre Desbiolles1 and Christophe Escudé2,*

1Laboratoire Kastler Brossel, Unité de Recherche de l'Ecole Normale Supérieure et de l'Université Pierre et Marie Curie, associée au CNRS, Département de Physique 24 rue Lhomond, F-75005 Paris, France 2Laboratoire ‘Régulation et Dynamique des Génomes’, Département ‘Régulations, Développement et Diversité Moléculaire’, USM 0503 Muséum National d'Histoire Naturelle, CNRS UMR5153, INSERM U565 Case Postale 26, 43 rue Cuvier, F-75231 Paris Cedex 05, France 3CEREMADE, Université Paris IX -Dauphine F-75775 Paris, France

*To whom correspondence should be addressed. Tel: +33 1 40793774; Fax: +33 1 40793705; Email: escude{at}mnhn.fr

Received April 13, 2005. Revised May 31, 2005. Accepted May 31, 2005.

Observation of DNA–protein interactions by single molecule fluorescence microscopy is usually performed by using fluorescent DNA binding agents. However, such dyes have been shown to induce cleavage of the DNA molecule and perturb its interactions with proteins. A new method for the detection of surface-attached DNA molecules by fluorescence microscopy is introduced in this paper. Biotin- and/or digoxigenin-modified DNA fragments are covalently linked at both extremities of a DNA molecule via sequence-specific hybridization and ligation. After the modified DNA molecules have been stretched on a glass surface, their ends are visualized by multicolor fluorescence microscopy using conjugated quantum dots (QD). We demonstrate that under carefully selected conditions, the position and orientation of individual DNA molecules can be inferred with good efficiency from the QD fluorescence signals alone. This is achieved by selecting QD pairs that have the distance and direction expected for the combed DNA molecules. Direct observation of single DNA molecules in the absence of DNA staining agent opens new possibilities in the fundamental study of DNA–protein interactions. This work also documents new possibilities regarding the use of QD for nucleic acid detection and analysis.

Correspondence may also be addressed to Pierre Desbiolles. Tel: +33 1 44323380; Fax: +33 1 44323434; Email: pierre.desbiolles{at}lkb.ens.fr

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors


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