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Publications de l’équipe
Macromolécules et Microsystèmes en Biologie et en Médecine (MMBM)
Année de publication : 2006
Max Chabert, Kevin D Dorfman, Patricia de Cremoux, Johan Roeraade, Jean-Louis Viovy (2006
Nov 16)
Automated microdroplet platform for sample manipulation and polymerase
chain reaction.
Analytical chemistry : 7722-8
Résumé
We present a fully automated system performing continuous sampling, reagent mixing, and
polymerase chain reaction (PCR) in microdroplets transported in immiscible oil. Sample
preparation and analysis are totally automated, using an original injection method from a
modified 96-well plate layered with three superimposed liquid layers and in-capillary laserinduced fluorescence endpoint detection. The process is continuous, allowing sample
droplets to be carried uninterruptedly into the reaction zone while new drops are aspirated
from the sample plate. Reproducible amplification, negligible cross-contamination, and
detection of low sample concentrations were demonstrated on numerous consecutive
sample drops. The system, which opens the route to strong reagents and labor savings in
high-throughput applications, was validated on the clinically relevant quantification of
progesterone receptor gene expression in human breast cancer cell lines.
Renaud Fulconis, Judith Mine, Aurélien Bancaud, Marie Dutreix, Jean-Louis Viovy (2006 Sep 2)
Mechanism of RecA-mediated homologous recombination revisited by single
molecule nanomanipulation.
The EMBO journal : 4293-304
Résumé
The mechanisms of RecA-mediated three-strand homologous recombination are investigated
at the single-molecule level, using magnetic tweezers. Probing the mechanical response of
DNA molecules and nucleoprotein filaments in tension and in torsion allows a monitoring of
the progression of the exchange in real time, both from the point of view of the RecA-bound
single-stranded DNA and from that of the naked double-stranded DNA (dsDNA). We show
that strand exchange is able to generate torsion even along a molecule with freely rotating
ends. RecA readily depolymerizes during the reaction, a process presenting numerous
advantages for the cell’s ‘protein economy’ and for the management of topological
constraints. Invasion of an untwisted dsDNA by a nucleoprotein filament leads to an
exchanged duplex that remains topologically linked to the exchanged single strand,
suggesting multiple initiations of strand exchange on the same molecule. Overall, our results
seem to support several important assumptions of the monomer redistribution model.
Zuzana Bílková, Marcela Slováková, Nicolas Minc, Claus Fütterer, Roxana Cecal, Daniel Horák,
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 1
Publications de l’équipe
Macromolécules et Microsystèmes en Biologie et en Médecine (MMBM)
Milan Benes, Isabelle le Potier, Jana Krenková, Michael Przybylski, Jean-Louis Viovy (2006 Apr 29)
Functionalized magnetic micro- and nanoparticles: optimization and application
to micro-chip tryptic digestion.
Electrophoresis : 1811-24
Résumé
The preparation of an easily replaceable protease microreactor for micro-chip application is
described. Magnetic particles coated with poly(N-isopropylacrylamide), polystyrene, poly(2hydroxyethyl methacrylate-co-ethylene dimethacrylate), poly(glycidyl methacrylate), [(2amino-ethyl)hydroxymethylen]biphosphonic acid, or alginic acid with immobilized trypsin
were utilized for heterogeneous digestion. The properties were optimized, with the constraint
of allowing immobilization in a microchannel by a magnetic field gradient. To obtain the
highest digestion efficiency, sub-micrometer spheres were organized by an inhomogeneous
external magnetic field perpendicularly to the direction of the channel. Kinetic parameters of
the enzyme reactor immobilized in micro-chip capillary (micro-chip immobilized magnetic
enzyme reactor (IMER)) were determined. The capability of the proteolytic reactor was
demonstrated by five model (glyco)proteins ranging in molecular mass from 4.3 to 150 kDa.
Digestion efficiency of proteins in various conformations was investigated using SDS-PAGE,
HPCE, RP-HPLC, and MS. The compatibility of the micro-chip IMER system with total and
limited proteolysis of high-molecular-weight (glyco)proteins was confirmed. It opens the
route to automated, high-throughput proteomic micro-chip devices.
Aurélien Bancaud, Natalia Conde e Silva, Maria Barbi, Gaudeline Wagner, Jean-François
Allemand, Julien Mozziconacci, Christophe Lavelle, Vincent Croquette, Jean-Marc Victor, Ariel
Prunell, Jean-Louis Viovy (2006 Apr 20)
Structural plasticity of single chromatin fibers revealed by torsional
manipulation.
Nature structural & molecular biology : 444-50
Résumé
Magnetic tweezers were used to study the mechanical response under torsion of single
nucleosome arrays reconstituted on tandem repeats of 5S positioning sequences. Regular
arrays are extremely resilient and can reversibly accommodate a large amount of
supercoiling without much change in length. This behavior is quantitatively described by a
molecular model of the chromatin three-dimensional architecture. In this model, we assume
the existence of a dynamic equilibrium between three conformations of the nucleosome,
corresponding to different crossing statuses of the entry/exit DNAs (positive, null or negative,
respectively). Torsional strain displaces that equilibrium, leading to an extensive
reorganization of the fiber’s architecture. The model explains a number of long-standing
topological questions regarding DNA in chromatin and may provide the basis to better
understand the dynamic binding of chromatin-associated proteins.Note: In the
supplementary information initially published online to accompany this article,
Supplementary Figure 2 was mistakenly replaced by Supplementary Equation 2. The error
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 2
Publications de l’équipe
Macromolécules et Microsystèmes en Biologie et en Médecine (MMBM)
has been corrected online.
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 3