Macromolécules et Microsystèmes en Biologie et en Médecine (MMBM)

Publications de l’équipe

Année de publication : 2005

Nicolas Minc, Jean-Louis Viovy, Kevin D Dorfman (2005 Aug 11)

Non-markovian transport of DNA in microfluidic post arrays.

Physical review letters : 198105 En savoir plus
Résumé

We present an analytically solvable model for the transport of long DNA through microfluidic arrays of posts. The motion is a repetitive three-part cycle: (i) collision with the post and extension of the arms; (ii) rope-over-pulley post disengagement; and (iii) a random period of uniform translation before the next collision. This cycle, inspired by geometration, is a nonseparable (Scher-Lax) continuous-time random walk on a lattice defined by the posts. Upon adopting a simple model for the transition probability density on the lattice, we analytically compute the mean DNA velocity and dispersivity in the long-time limit without any adjustable parameters. The results compare favorably with the limited amount of experimental data on separations in self-assembled arrays of magnetic beads. The Scher-Lax formalism provides a template for incorporating more sophisticated microscale models.

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Kevin D Dorfman, Max Chabert, Jean-Hugues Codarbox, Gilles Rousseau, Patricia de Cremoux, Jean-Louis Viovy (2005 Jun 1)

Contamination-free continuous flow microfluidic polymerase chain reaction for quantitative and clinical applications.

Analytical chemistry : 3700-4 En savoir plus
Résumé

We present a method for performing polymerase chain reaction (PCR) using isolated droplets flowing in an immiscible fluorinated solvent system. Thanks to an optimized control of interfacial properties, we could achieve in this capillary-based system reproducible amplification factors, without any detectable contamination between neighboring droplets. The system is readily amenable to further miniaturization and automation and serves as the first step toward a clinically viable, high-throughput, quantitative continuous flow PCR apparatus.

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Renaud Fulconis, Marie Dutreix, Jean-Louis Viovy (2005 Mar 8)

Numerical investigation of sequence dependence in homologous recognition: evidence for homology traps.

Biophysical journal : 3770-9 En savoir plus
Résumé

During the initial phase of RecA-mediated recombination, known as the search for homology, a single-stranded DNA coated by RecA protein and a homologous double-stranded DNA have to perfectly align and pair. We designed a model for the homology search between short molecules, and performed Monte Carlo Metropolis computer simulations of the process. The central features of our model are 1), the assumption that duplex DNA longitudinal thermal fluctuations are instrumental in the binding; and 2), the explicit consideration of the nucleotide sequence. According to our results, recognition undergoes a first slow nucleation step over a few basepairs, followed by a quick extension of the pairing to adjacent bases. The formation of the three-stranded complex tends to be curbed by heterologies but also by another possible obstacle: the presence of partially homologous stretches, such as mono- or polynucleotide repeats. Actually, repeated sequences are observed to trap the molecules in unproductive configurations. We investigate the dependence of the phenomenon on various energy parameters. This mechanism of homology trapping could have a strong biological relevance in the light of the genomic instability experimentally known to be triggered by repeated sequences.

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Kevin D Dorfman, Renaud Fulconis, Marie Dutreix, Jean-Louis Viovy (2005 Feb 9)

Model of RecA-mediated homologous recognition.

Physical review letters : 268102 En savoir plus
Résumé

We consider theoretically the homology search between a long double-stranded DNA and a RecA-single-stranded DNA nucleofilament, emphasizing the polymeric nature of the search and the ability of double-stranded DNA to overcome the difference in pitch between itself and the nucleofilament by thermally activated stretching from the canonical B state to the metastable, stretched S state. Our analytical first-passage-time analysis agrees well with experimental data, predicts new dependencies on the intracellular fluid viscosity and ionic strength, and strongly suggests that initial homologous recognition involves a three base-pair seed.

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Aurélien Bancaud, Gaudeline Wagner, Kevin D Dorfman, Jean-Louis Viovy (2005 Feb 1)

Measurement of the surface concentration for bioassay kinetics in microchannels.

Analytical chemistry : 833-9 En savoir plus
Résumé

We present a simple and versatile method, based on fluorescence microscopy, to reliably measure the concentration of advected molecules in the vicinity of surfaces in microchannels. This tool is relevant to many microfluidic applications such as immunoassays and single-molecule experiments, where one probes the kinetics of a reaction between an immobilized target and a reactant carried by the bulk flow. The characterization of the surface concentration highlights the dominant role of transverse diffusion, which generates an apparent diffusivity at the surface 3-4 orders of magnitude greater than molecular diffusion alone, even close to the point of injection. We directly measure the effects of the longitudinal position along the channel and of the flow rate on the concentration front and develop a simple analytical model that compares well with the data. Finally, we propose a method to properly account for concentration fronts in single-molecule measurements and use it to directly access the kinetics parameters of protamine-induced condensation of DNA.

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Zuzana Bílková, Annalisa Castagna, Gianluigi Zanusso, Alessia Farinazzo, Salvatore Monaco, Eugen Damoc, Michael Przybylski, Milan Benes, Jirí Lenfeld, Jean-Louis Viovy, Pier Giorgo Righetti (2005 Jan 26)

Immunoaffinity reactors for prion protein qualitative analysis.

Proteomics : 639-47 En savoir plus
Résumé

The cellular prion protein (PrPc) represents the substrate for generation of conformational aberrant PrP isoforms which occur in human and animal prion diseases. The published two-dimensional maps of human PrPc show a vast microheterogeneity of this glycoprotein. The main goal of this project was to develop a highly specific immunoaffinity reactor for qualitative analysis of PrP cellular isoforms isolated from brain homogenate, cerebrospinal fluid and other tissues. New techniques for affinity proteomics, carriers and immobilization chemistry were applied. The choice of matrix (chemical and magnetic properties, particle size and distribution, porosity) was the key factor that influenced the quality of the reactor and the nature of final applications. Mouse anti-prion IgGs directed to N-terminal and C-terminal epitopes (residues 23-40 and 147-165) were grafted in different manners to magnetic micro- and nanoparticles particularly developed for micro-CHIP application. High operational and storage stability of affinity reactors with minimized nonspecific absorption were achieved. The quality of the immunoreactors was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting and by two-dimensional electrophoresis.

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Nicolas Minc, Plamen Bokov, Konstantin B Zeldovich, Claus Fütterer, Jean-Louis Viovy, Kevin D Dorfman (2005 Jan 20)

Motion of single long DNA molecules through arrays of magnetic columns.

Electrophoresis : 362-75 En savoir plus
Résumé

We present a videomicroscopy study of T4 DNA (169 kbp) in microfluidic arrays of posts formed by the self-assembly of magnetic beads. We observe DNA moving through an area of 10 000 microm(2), typically containing 100-600 posts. We determine the distribution of the contact times with the posts and the distribution of passage times across the field of view for hundreds of DNA per experiment. The contact time is well approximated by a Poisson process, scaling like the inverse of the field strength, independent of the density of the array. The distribution of passage times allows us to estimate the mean velocity and dispersivity of the DNA during its motion over distances long compared to our field of view. We compare these values with those computed from a lattice Monte Carlo model and geometration theory. We find reasonable quantitative agreement between the lattice Monte Carlo model and experiment, with the error increasing with increasing post density. The deviation between theory and experiment is attributed to the high mobility of DNA after disengaging from the posts, which leads to a difference between the contact time and the total time lost by colliding. Classical geometration theory furnishes surprisingly good agreement for the dispersivity, while geometration theory with a mean free path significantly overestimates the dispersivity.

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Année de publication : 2004

Renaud Fulconis, Aurélien Bancaud, Jean-Francois Allemand, Vincent Croquette, Marie Dutreix, Jean-Louis Viovy (2004 Sep 30)

Twisting and untwisting a single DNA molecule covered by RecA protein.

Biophysical journal : 2552-63 En savoir plus
Résumé

We study dsDNA-RecA interactions by exerting forces in the pN range on single DNA molecules while the interstrand topological state is controlled owing to a magnetic tweezers setup. We show that unwinding a duplex DNA molecule induces RecA polymerization even at moderate force. Once initial polymerization has nucleated, the extent of RecA coverage still depends on the degree of supercoiling: exerting a positive or negative torsional constraint on the fiber forces partial depolymerization, with a strikingly greater stability when ATPgammaS is used as a cofactor instead of ATP. This nucleofilament’s sensitivity to topology might be a way for the bacterial cell to limit consumption of precious RecA monomers when DNA damage is addressed through homologous recombination repair.

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Jérémie Weber, Valessa Barbier, Sabine Pages-Berhouet, Virginie Caux-Moncoutier, Dominique Stoppa-Lyonnet, Jean-Louis Viovy (2004 Aug 17)

A high-throughput mutation detection method based on heteroduplex analysis using graft copolymer matrixes: application to Brca1 and Brca2 analysis.

Analytical chemistry : 4839-48 En savoir plus
Résumé

We present here a new approach to electrophoretic heteroduplex analysis (EHDA) based on improved matrixes. EHDA is an appealing technique for the detection of unknown point mutations because of its simplicity and high throughput. We present here a new matrix for electrophoretic heteroduplex analysis much more sensitive for insertions, deletions, and substitutions than reported for previous EHDA separations and also superior to DHPLC. This separation matrix is based on a copolymer with a comb architecture, poly(acrylamide-g-polydimethylacrylamide), made of a high molecular weight polyacrylamide backbone grafted with poly(dimethylacrylamide) side chains. The effect of operational parameters on electrophoretic resolution and sensitivity to single-nucleotide mismatches was studied using a collection of samples from patients bearing mutations in the breast cancer predisposition genes BRCA1 and BRCA2. Seventeen fragments (10 mutations), implying mostly substitutions on fragments with sizes ranging from 200 to 600 bp, were analyzed using a single set of separation conditions. A success rate of 94% was achieved with a qualitative analysis in terms of number of peaks, and 100% identification of mutations was obtained with a more quantitative test using peak width analysis. This strong improvement of performance with regard to previous HDA methods is attributed to a composite mechanism of separation, combining steric and chromatographic effects. It opens the route to a significant reduction of development time and operation cost for diagnostic and genomic applications.

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