Analysis of DNA Replication by Optical Mapping in Nanochannels.
DNA replication is essential to maintain genome integrity in S phase of the cell division cycle. Accumulation of stalled replication forks is a major source of genetic instability, and likely constitutes a key driver of tumorigenesis. The mechanisms of regulation of replication fork progression have therefore been extensively investigated, in particular with DNA combing, an optical mapping technique that allows the stretching of single molecules and the mapping of active region for DNA synthesis by fluorescence microscopy. DNA linearization in nanochannels has been successfully used to probe genomic information patterns along single chromosomes, and has been proposed to be a competitive alternative to DNA combing. Yet this conjecture remains to be confirmed experimentally. Here, two complementary techniques are established to detect the genomic distribution of tracks of newly synthesized DNA in human cells by optical mapping in nanochannels. Their respective advantages and limitations are compared, and applied them to detect deregulations of the replication program induced by the antitumor drug hydroxyurea. The developments here thus broaden the field of applications accessible to nanofluidic technologies, and can be used in the future as part for molecular diagnostics in the context of high throughput cancer drug screening.
CNRS, LAAS, 7 avenue du colonel Roche, F-31400, Toulouse, France.|Univ de Toulouse, LAAS, F-31400, Toulouse, France.
Joris
J
Lacroix
CNRS, LAAS, 7 avenue du colonel Roche, F-31400, Toulouse, France.|Univ de Toulouse, LAAS, F-31400, Toulouse, France.
Sandrine
S
Pélofy
CNRS, LAAS, 7 avenue du colonel Roche, F-31400, Toulouse, France.|Univ de Toulouse, LAAS, F-31400, Toulouse, France.
Charline
C
Blatché
Univ de Toulouse, LAAS, F-31400, Toulouse, France.|Equipe "Labellisée LA LIGUE CONTRE LE CANCER 2013" - Laboratoire d'Excellence Toulouse Cancer LABEX TOUCAN - Cancer Research Center of Toulouse, Inserm U1037, CNRS ERL5294, 2 Avenue Hubert Curien, CS 53717, 31037, Toulouse, France.
Marie-Jeanne
MJ
Pillaire
CNRS, UMR 6061, Institut Génétique et Développement de Rennes, F-35043, Rennes, France.|Université Rennes 1, UEB, UMR 6290, Faculté de Médecine, F-35043, Rennes, France.
Sébastien
S
Huet
CNRS, UMR 6061, Institut Génétique et Développement de Rennes, F-35043, Rennes, France.|Université Rennes 1, UEB, UMR 6290, Faculté de Médecine, F-35043, Rennes, France.
Catherine
C
Chapuis
Univ de Toulouse, LAAS, F-31400, Toulouse, France.|Equipe "Labellisée LA LIGUE CONTRE LE CANCER 2013" - Laboratoire d'Excellence Toulouse Cancer LABEX TOUCAN - Cancer Research Center of Toulouse, Inserm U1037, CNRS ERL5294, 2 Avenue Hubert Curien, CS 53717, 31037, Toulouse, France.
Jean-Sébastien
JS
Hoffmann
CNRS, LAAS, 7 avenue du colonel Roche, F-31400, Toulouse, France.|Univ de Toulouse, LAAS, F-31400, Toulouse, France.
Aurélien
A
Bancaud
D004261:DNA Replication; D004058:Diffusion; D006801:Humans; D018625:Microscopy, Atomic Force; D053758:Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D061848:Optical Imaging / Q000379:methods*; D013052:Spectrometry, X-Ray Emission; D017968:Vanadium Compounds / Q000737:chemistry
D017968:Vanadium Compounds; C066075:vanadium pentoxide
DNA replication; chromosome manipulation; nanochannels; optical mapping; single DNA molecule microscopy