Chemistry: electrical and wave energy – Apparatus – Electrophoretic or electro-osmotic apparatus
Reexamination Certificate
2000-02-07
2003-09-02
Warden, Jill (Department: 1743)
Chemistry: electrical and wave energy
Apparatus
Electrophoretic or electro-osmotic apparatus
C204S600000, C204S601000, C204S452000, C436S043000, C436S063000, C356S344000, C250S458100, C422S050000, C422S068100, C422S082050, C422S082080
Reexamination Certificate
active
06613212
ABSTRACT:
The present invention relates to multicapillary electrophoresis systems.
It is known that conventional gel electrophoresis techniques, in which various samples are injected along a plurality of tracks defined in a gel placed between two plates, are not satisfactory, given that, on the one hand, they require a number of manual operations and, on the other hand, they do not allow very high migration velocities and therefore very high treatment throughputs.
However, the major sequencing and genotyping programs require a very high rate of separation and identification of DNA molecules.
Moreover, electrophoresis techniques are known which use, for the migration, a capillary filled with gel or with another separating matrix having the advantage of being particularly handleable, easy to load and which allow substantially automatic operation, with higher separation rates than in electrophoresis using gel slabs by virtue of a high electric field that can be applied.
However, the use of a single capillary does not make it possible to achieve the same rates as those allowed by electrophoresis techniques using slabs which possess many parallel tracks, even if nevertheless the electric fields that can be applied to a capillary, and therefore the migration velocities obtained, are high.
This is why systems called multicapillary systems comprising a linear array of several juxtaposed capillaries have also been proposed.
In particular, multicapillary electrophoresis systems are known in which the laser beam for exciting the molecules is sent into the capillaries through their walls, along an axis in the plane of the linear array, perpendicular to the direction along which the capillaries extend, the fluorescence of the molecules being observed by receiving means having an optical axis perpendicular to the plane of the linear array of capillaries.
In this regard, reference may be made, for example, to the publication: “A Capillary Array Gel Electrophoresis System Using Multiple Laser Focusing For DNA Sequencing”—T. Anazawa, S. Takahashi and H. Kambara, Anal. Chem., Vol. 68, No. 15, Aug. 1, 1996, pp. 2699-2704.
However, such a technique is not very satisfactory on account of the detection noise resulting from the interaction of the excitation light and the fluorescence from the walls of the capillary. Furthermore, the laser beam loses intensity as it passes through the capillaries, so that the molecules which are located in the capillaries furthest from the laser source are less excited than those moving in the first capillaries.
Because of these major drawbacks, the systems of the type of those described in the publication: “Analysis of Nucleic Acids by Capillary Electrophoresis” by C. Heller, pp. 236 to 254, Editions Vieweg, 1997, or else in the patents and patent applications U.S. Pat. No. 5,567,294 or EP-723,149, are generally preferred to multicapillary electrophoresis systems in which the laser beam for exciting the molecules is sent into the latter through the walls of the capillaries.
In the systems described in that publication or those patents, the capillaries are held one with respect to the other in a glass cuvette along which said capillaries extend. The molecules which travel along the capillaries are excited after having exited said capillaries by a beam of laser radiation which is sent, just at the exit of the linear array, in the plane of said linear array and perpendicular to the direction along which the capillaries extend.
The fluorescence of the molecules excited by this radiation is detected, for example, by means of a CCD camera which is oriented with an axis perpendicular to the plane of the linear array of capillaries or else with an axis parallel to the capillaries.
However, such a system requires means to be provided, such as laminar flow means or guiding elements, which prevent the molecules from diverging too significantly at the exit of the various capillaries. To do this, the cuvette requires a high-precision mechanical construction in glass. In particular, the device will have to provide a very uniform flow and avoid any gas bubbles or dust disturbing the flow.
Furthermore, this technique requires the use of different materials—at least as regards the viscosity—for the capillaries and the cuvette, which have different functions, one serving for separating the molecules and the other for channeling the flows. It therefore becomes necessary to use large volumes of solutions to produce the flow.
As will have been understood, such a technique has the drawback of being particularly expensive.
One aim of the invention is therefore to propose a multicapillary electrophoresis system which, for chemical and pharmaceutical applications, is robust, inexpensive, reliable and easy to use and whose performance allows high-throughput sequencing and genotyping.
For this purpose, the invention proposes a multicapillary electrophoresis system comprising a plurality of juxtaposed capillaries, at least one source for the emission of a beam intended to excite molecules lying in its path and inside the capillaries and means for detecting the fluorescence of the molecules excited by said beam.
In order to alleviate the drawbacks which, in the known systems in the prior art, caused those skilled in the art to move away from this type of system, the invention proposes to arrange the detection means so as to detect the light which emerges at the exit of said capillaries and which propagates along the direction in which said capillaries extend, as well as to use detection means having a high enough resolution to distinguish the light which emerges at the exit of the capillaries from that coming from the walls of the latter and/or from the medium which surrounds them.
Such a structure makes it possible to detect molecules inside the capillaries while considerably reducing the detection noise.
This system is advantageously completed by the following various advantageous characteristics taken by themselves or in any of their technically possible combinations:
it includes a matrix of capillaries;
it includes means, such as microlenses, for producing multiple focusing on a linear array of capillaries;
one linear array of capillaries produces multiple focusing at the entry of the following linear array;
the excitation beam is of elongate cross section and strikes several superposed capillaries simultaneously;
the space between the capillaries is filled, at least along the path of the excitation beam, by a material whose refractive index is chosen so that the excitation beam does not diverge after having traveled along a capillary;
said material is transparent and non-fluorescent;
it includes means for applying pressure in the detection cuvette, which pressure allows the capillaries to be filled with the separating matrix;
it includes dispersion means for spatially separating the various fluorescence wavelengths;
the detection means provide a complete image of the light exiting the capillaries;
the detection means comprise detection means of the charge-coupled device (CCD) type, as well as focusing means;
the detection means comprise detection means of the charge-coupled device (CCD) type, as well as a fiber bundle interposed between the exits of the capillaries and the detection means of the charge-coupled device type.
REFERENCES:
patent: 5324401 (1994-06-01), Yeung et al.
patent: 5567294 (1996-10-01), Dovichi et al.
patent: 5616228 (1997-04-01), Nasu et al.
patent: 5667656 (1997-09-01), Kambara
patent: 5833827 (1998-11-01), Anazawa et al.
patent: 5938908 (1999-08-01), Anazawa et al.
patent: 6063251 (2000-05-01), Kane et al.
patent: 6103083 (2000-08-01), Merenkova et al.
patent: 6191425 (2001-02-01), Imai
patent: 6224733 (2001-05-01), Takahashi et al.
patent: 19616824 (1997-05-01), None
patent: 0723149 (1996-07-01), None
patent: 2312505 (1997-10-01), None
patent: 2314622 (1998-01-01), None
patent: WO94/29712 (1994-12-01), None
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Dovichi N J: “Laser-based Microchemical analysis” review of scientific instruments, vol. 61, N
Bottani Samuele
Rebscher Hans
Siebert Rainer
Valentin Luc
Blakely & Sokoloff, Taylor & Zafman
Centre National de la Recherche Scientifique
Sines Brian
Warden Jill
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