Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-04-02
2001-05-01
Horlick, Kenneth R. (Department: 1653)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S283100, C435S286400, C435S287200, C435S287900, C435S307100, C435S309100
Reexamination Certificate
active
06225055
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to an apparatus for the parallel alignment of macromolecules on a solid support by the passage of a meniscus.
A process for the alignment of macromolecules, also called “molecular combing” has been described in the patent application in France No. 94 07444.
Controlling the conformation of macromolecules represents a major industrial challenge, for example in the manufacture of sensors or of controlled molecular assemblies, or alternatively in problems of detection and analysis. It may be useful to have an elongated molecular conformation. By way of example, in the case where polymers are grafted on a substrate, it has been proposed to extend them by the action of an electric field, a flow or with the aid of optical tweezers. In particular, in biology, the alignment of DNA—by electrophoresis (Zimmermann and Cox Nucl. Acid Res. 22, p 492, 1994), free flow (Parra and Windle, Nature Genetics, 5, p 17, 1993 and WO 93/22463) or in a gel (Schwartz et al. Science 262, p 110, 1993 and USP 33531) or with the aid of optical tweezers (Perkins et al., Science 264 p 819, 1994 and also U.S. Pat. No. 5,079,169)—opens numerous possibilities in mapping, or in the detection of pathogens.
These methods only allow in general an imperfect alignment, or alternatively a transient alignment—that is to say that relaxation of the molecule occurs once the stress disappears. In the case of optical tweezers, the method is expensive, is limited to only one molecule at a time, and is difficult to carry out by non-qualified staff.
A special technique for aligning DNA by flow after cell lysis, followed by drying, has been proposed (I. Parra and B. Windle and WO 93/22463). The alignment obtained is very imperfect and nonhomogeneous and numerous nonaligned masses are observed.
In Patent Application FR 94 07444, there has been described a novel and simple method for aligning macromolecules on the surface S of a support, characterized in that the triple line S/A/B (meniscus) resulting from the contact between a solvent A and the surface S and a medium B is caused to move on said surface S, said macromolecules having a part, especially an end, anchored on the surface S, the other part, especially the other end, being in solution in the solvent A.
It has been observed that the mere passage of a meniscus over molecules of which one part is anchored on a substrate, the remainder of the molecule existing freely in solution makes it possible to align them uniformly, perpendicularly to the moving meniscus, leaving them adsorbed on the surface behind the meniscus. This phenomenon is called “molecular combing” here.
More specifically, the stretching of the free part of the molecule is achieved by the passage of the triple line S/A/B constituting the meniscus between the surface S, the solvent A and a medium B which may be a gas (in general air) or another solvent.
In a specific embodiment, the meniscus is a water/air meniscus, that is to say that the solvent A is an aqueous solution and the medium B is air.
The movement of the meniscus can be achieved, in particular, by gradual evaporation of the solvent A or by a mechanical route by translation of the surface S.
To do this in Patent Application FR 94 07444, a drop of solvent containing the molecules to be aligned is placed between two supports of which at least one corresponds to said support of surface S and the meniscus is moved for example either by evaporation or by moving the two supports relative to each other.
By “support”, there is understood any substrate whose cohesion is sufficient to withstand the passage of the meniscus.
The support may consist, at least at the surface, of an organic or inorganic polymer, a metal especially gold, a metal oxide or sulfide, a semiconductor element or an oxide of a semiconductor element, such as a silicon oxide or a combination thereof, such as glass or a ceramic.
There may be mentioned more particularly glass, superficially oxidized silicon, graphite, mica and molybdenum sulfide.
As “support”, there may be used a single support such as a slide, beads, especially polymer beads, but also any form such as a bar, a fiber or a structured support, and also particles, whether it be powders, especially silica powders, which can moreover be made magnetic fluorescent or colored as known in the various assay technologies.
The support is advantageously in the form of cover slips.
Macromolecules, such as ordinary polymers, or biological polymers such as DNA, RNA or proteins, can be anchored by ordinary methods on a support.
The macromolecule to be aligned can be chosen from biological macromolecules such as proteins, especially antibodies, antigens, ligands or their receptors, nucleic acids, DNA, RNA or PNA, lipids, polysaccharides and derivatives thereof.
It was observed that the stretching force acts locally within the immediate vicinity of the meniscus. It is independent of the length of the molecule, of the number of molecules anchored, and within a wide range, of the speed of the meniscus. These characteristics are particularly important for aligning the molecules homogeneously and reproducibly.
It is possible to add surfactant elements into the solvent A, especially water, and/or the medium B, especially air, which modify the properties of the interfaces. The stretching can indeed be controlled by the addition of surfactants, or by an adequate surface treatment.
Too high a surface-macromolecule attraction (for example an excessively high level of adsorption) can interfere with the alignment of the molecules by the meniscus, these molecules remaining adsorbed at the surface in a state which is not necessarily stretched. Preferably, the surface exhibits a low rate of adsorption of said macromolecule, such that only the anchored molecules are aligned, the others being carried by the meniscus.
However, it is possible to vary the differences in adsorption between a part of the macromolecule, especially its ends, and its other parts (in particular for long molecules such as DNA or collagen) in order to anchor, by adsorption, the molecules by a part, especially their end(s) alone, the remainder of the molecule existing freely in solution, on a wide variety of surfaces and align them by the passage of the meniscus as described above.
The adsorption of a macromolecule onto a surface can be easily controlled by means of the pH or of the ionic medium content of the medium or of an electric voltage applied over the surface. The surface charges and the electrostatic (repulsive or attractive) interactions between the surface and the molecule are thus changed, thereby making it possible to pass from a state of complete adsorption of the molecule onto the surface to a total absence of adsorption. Between these two extreme cases, there is a range of control parameters where the adsorption occurs preferably through the end of the molecules and which will therefore be used advantageously to anchor them on the surface, and then to align them by the passage of the meniscus.
Once aligned, the molecules adhere strongly to the surface. In the case of DNA, it was possible to observe them by fluorescence several months after their alignment.
The technique therefore consists, in a first instance, in the anchoring, on the pretreated surface of a solid support in solution, of macromolecules, especially DNA, by their end(s). This specific anchoring—only the ends of the molecules adhere to the surface of the support—requires a precise control of certain physicochemical parameters of the solution: pH, temperature. This step is designated later by the name “incubation step”. The duration of incubation has an influence on the rate of anchoring per unit of surface.
In a second instance, the system passes from the configuration: surface in solution, to a configuration: surface outside the solution. This passage was first achieved by natural evaporation of a drop of solution deposited on the pretreated surface. During the evaporation, the edge of the drop, which constitutes a meniscus, that is to say a triple int
Bensimon Aaron
Bensimon David
Finnegan Henderson Farabow Garrett & Dunner LLP
Horlick Kenneth R.
Institut Pasteur
Taylor Janell E.
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