Detection of single DNA molecules by multicolor quantum-dot end-labeling.
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.
Crut
Aurélien
A
Laboratoire 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.
Géron-Landre
Bénédicte
B
Bonnet
Isabelle
I
Bonneau
Stéphane
S
Desbiolles
Pierre
P
Escudé
Christophe
C
D019921:Biotinylation; D003116:Color; D004247:DNA / Q000032:analysis*; D004076:Digoxigenin / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods*; D045663:Quantum Dots
D004247:DNA; D004076:Digoxigenin