Nucleic Acids Research, 2003, Vol. 31, No. 20 e125
© 2003 Oxford University Press
Sequence-specific fluorescent labeling of double-stranded DNA observed at the single molecule level
Laboratoire de Biophysique, Muséum National d’Histoire Naturelle, INSERM U565, CNRS UMR8646, 43, rue Cuvier, 75231 Paris Cedex 05, France and 1 Laboratoire Kastler Brossel, Ecole Normale Supérieure, 24, rue Lhomond, 75005 Paris, France
*To whom correspondence should be addressed. Tel: +33 1 40793774; Fax: +33 1 40793705; Email: escude{at}mnhn.fr
Present address:
Thibaut Roulon, ANOSYS Inc., 1014 Hamilton Court, Menlo Park, CA 94025, USA
This paper is dedicated to the memory of Professor Claude Hélène. We are grateful to him for initiating this project, for sharing with us his numerous ideas and for transmitting his passion for research in the field of nucleic acids.
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.
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