Multicolor detection of combed DNA molecules using quantum dots.

DNA combing is a useful strategy for manipulating single DNA molecules and has a wide range of applications in genetics, single molecule studies, and nanobiotechnology. Visualization of combed DNA molecules is usually performed by using DNA binding organic dyes. Such dyes are not suitable in all circumstances, especially because of their photoreactivity. We have developed a method for the detection of combed DNA molecules by fluorescence microscopy that avoids the use of DNA-staining agents and does not perturb the structure of the DNA molecule. Biotin- and/or digoxigenin-modified DNA fragments are covalently linked at both ends of a DNA molecule via sequence-specific hybridization and subsequent ligation. After the modified DNA molecules have been combed on a polystyrene-coated surface, their ends are visualized by multicolor fluorescence microscopy using conjugated quantum dots.

Stem-loop oligonucleotides as tools for labelling double-stranded DNA.

We report on a sequence-specific double-stranded DNA labelling strategy in which a stem-loop triplex forming oligonucleotide (TFO) is able to encircle its DNA target. Ligation of this TFO to either a short hairpin oligonucleotide or a long double-stranded DNA fragment leads to the formation of a topological complex. This process requires the hybridization of both extremities of the TFO to each other on a few base pairs. The effects of different factors on the formation of these complexes have been investigated. Efficient complex formation was observed using both GT or TC TFOs. The stem-loop structure enhances the specificity of the complex. The topologically linked TFO remains associated with its target even under conditions that do not favour triple-helix formation. This approach is sufficiently sensitive for detection of a 20-bp target sequence at the subfemtomolar level. This study provides new insights into the mechanics and properties of stem-loop TFOs and their complexes with double-stranded DNA targets. It emphasizes the interest of such molecules in the development of new tools for the specific labelling of short DNA sequences.

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.

Sequence-specific fluorescent labeling of double-stranded DNA observed at the single molecule level.

Fluorescent labeling of a short sequence of double-stranded DNA (dsDNA) was achieved by ligating a labeled dsDNA fragment to a stem-loop triplex forming oligonucleotide (TFO). After the TFO has wound around the target sequence by ligand-induced triple helix formation, its extremities hybridize to each other, leaving a dangling single-stranded sequence, which is then ligated to a fluorescent dsDNA fragment using T4 DNA ligase. A non-repeated 15 bp sequence present on lambda DNA was labeled and visualized by fluorescence microscopy after DNA combing. The label was found to be attached at a specific position located at 4.2 +/- 0.5 kb from one end of the molecule, in agreement with the location of the target sequence for triple helix formation (4.4 kb from one end). In addition, an alternative combing process was noticed in which a DNA molecule becomes attached to the combing slide from the label rather than from one of its ends. The method described herein provides a new tool for the detection of very short sequences of dsDNA and offers various perspectives in the micromanipulation of single DNA molecules.