OpenAccessBelV2, France, Analysis, bibRecord, 000183

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

Identifieur interne : 000183 ( France/Analysis ); précédent : 000182; suivant : 000184

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

Auteurs : Aure Lien Crut [France] ; Be Ne Dicte Ge Ron-Landre [France] ; Isabelle Bonnet [France] ; Ste Phane Bonneau [France] ; Pierre Desbiolles [France] ; Christophe Escude [France]

Source :

Nucleic Acids Research [ 0305-1048 ] ; 2005.

RBID : ISTEX:6CDC46285C7526CA1CB86DD6590969430BEB01F4

Abstract

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.

Url:

https://api.istex.fr/document/6CDC46285C7526CA1CB86DD6590969430BEB01F4/fulltext/pdf

DOI: 10.1093/nar/gni097

Affiliations:

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ISTEX:6CDC46285C7526CA1CB86DD6590969430BEB01F4

Le document en format XML

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<front><div type="abstract" xml:lang="en">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.</div>
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Detection of single DNA molecules by multicolor quantum-dot end-labeling

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

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Abstract

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