Chemistry: electrical and wave energy – Apparatus – Electrophoretic or electro-osmotic apparatus
Reexamination Certificate
1999-09-01
2002-04-30
Warden, Jill (Department: 1743)
Chemistry: electrical and wave energy
Apparatus
Electrophoretic or electro-osmotic apparatus
C264S267000
Reexamination Certificate
active
06379519
ABSTRACT:
FIELD OF THE INVENTION
The present invention is concerned with a disposable electrophoresis cassette particularly suitable for pre-cast electrophoretic media for protein and nucleic acid electrophoresis. The invention also comprises a support plate for the said cassette to ensure proper rigidity thereof into the electrophoresis apparatus, and acting as a heat sink, thus providing a more uniform migration front in operation because the temperature of the medium is substantially even anywhere inside the cassette during electrophoresis operation. The invention also comprises a comb for injection of electrophoresis medium into the cassette, as well as the corresponding method of injection.
BACKGROUND OF THE INVENTION
Electrophoresis is a well known separation technique that requires the application of electrical current at both poles of a cassette or plate to force samples through an electrophoretic medium that acts as a molecular sieve. The application of a difference of potential between the upper section and the lower section of the cassette assumes the creation of two areas sealed from each other. Because current is transmitted via two separate buffer reservoirs, it is necessary to apply a pressure or force on the cassette so that the seals properly operate. It is therefore imperative that the whole system, including the cassette, possess some rigidity.
Conventional electrophoresis cassettes are made of two glass plates spaced apart with plastic spacers or tongues to create a space therebetween while ensuring that the sides of the assembly are properly sealed. Importantly, the spacers must not conduct electrical current. The assembly is generally maintained together with clamps, and it is often necessary to reinforce the seals with hot agar. When the gel is cast into the cassette, a comb element is introduced at one end of the assembly to create one or more reservoirs or wells thereafter wherein the sample(s) will be received later. The shape of the comb may comprise various numbers and sizes of reservoirs, depending on the application required and the size of the cassette. For example, a preparation gel necessitate less reservoirs, while an analytical gel will require more reservoirs and the width thereof will depend on the resolution desired.
However, such assemblies have several drawbacks and limitations. The assembling operation requires dexterity and is a time-consuming operation, because it is done manually. The plates are conventionally made of glass, and thus must be handled with care. Further, they must be carefully cleaned to obtain good results. Finally, manipulation of acrylamide gel, a commonly used electrophoretic medium, represents a long-term danger for the health of operators since such gel is highly toxic.
More recently, to simplify the assembling work of operators and reduce poisoning and manipulation hazards, pre-cast cassettes already containing the gel have been made available commercially. The cassettes comprise an acrylamide gel, and a comb is provided at one extremity thereof However, the cost of these cassettes is prohibitive, and demolding thereof, for visualization of the results, is a delicate and complicated procedure. In addition, the comb is produced by injection molding, and is used to form the wells or reservoirs in the gel. They generally represent an important part of the total cost of the cassette.
To be economically feasible and capable of supporting, without substantial bending, the mechanical forces applied thereon, cassettes containing pre-cast electrophoresis medium, must be rigid enough and made of a material economically sound and preferably recyclable, such as for example thermoplastic materials like polymethylmethacrylate (PMMA). However, conventionally, in order to be sufficiently rigid, the plates must be relatively thick. Two obvious problems therefore become apparent: a) the amount of thermoplastic material required is significant, thus increasing the cost, which is not suitable for a disposable device; and b) maintaining the gel at an appropriate operating temperature is complicated, because the thick walls of the thermoplastic material act as a dielectric material. Thicker plastic walls also affect the diffusion of the heat generated during the electrophoretic process, creating temperature gradients within the electrophoresis medium, and non-uniform migration of the samples analyzed.
Conventional processes for filling the cassettes are generally standard, irrespective of the electrophoretic medium. Typically, a gel comprising a mixture of acrylamide and bis-acrylamide, a buffer like tris-borate ethylenediamine (EDTA), tris-acetate-EDTA, tris-glycine, tricine, and a polymerization initiator are injected or cast into the cassette. Some of these products are neurotoxic and/or irritant, and must therefore be handled with extreme care. A laboratory pipette or a pump can be used to fill the cassette from the top with the liquid medium. Once the cassette is filled, a comb closes the top of the cassette. The comb has a design such that it contains one or more teeth forming reservoirs in the gel wherein the sample will be placed later. After polymerization of the medium, the comb is removed, as well as a separator present in the lower portion of the cassette. The cassette is then placed in an electrophoresis apparatus wherein the lower and upper portions of the gel will be in contact with two independent buffer solutions. The samples are then introduced in the reservoirs, and current is applied to separate the various components of each sample. After completion of the separation, the medium is removed from the cassette for further processing, i.e., coloration, photograph and analysis.
Again, such system and procedure have various major drawbacks and limitations. As stated above, manual filling of the cassette requires great care and dexterity, not to mention exposure of the operator to toxic chemicals. Further, undesirable bubbles often form during filling, and installation of the comb after filling may also create bubbles at the bottom of the teeth. Such air bubbles must be avoided at all times, since they interfere significantly with the samples migrating in the polymerized gel during the electrophoresis procedure.
Pre-cast gels have been marketed recently, but have not been able to overcome other problems mentioned above for cassettes containing the same, such as prohibitive costs. One of the main reason is that the cassettes are obtained by injection molding, which is a costly and relatively slow process because of the significant amount of plastic required for injection, the cost of the plastic material itself, and the time necessary to allow complete cooling of the cassette thus obtained. In addition, because the cassettes are made of a thermoplastic material, gel polymerization is greatly affected and slowed down because the polymer absorbs free radicals generated by the chain reaction of the polymerization. As a result, the polymerized electrophoretic medium does not “stick” do the cassette inner surfaces. An expensive coating layer or overlay must therefore be applied on the thermoplastic material surfaces to minimize this problem and ensure proper polymerization quality and speed.
The electrophoresis operation necessitates the application of a voltage across the gel that generates heat that must be somehow dissipated. During the heat dissipation process, if the temperature of the gel is not uniform, it causes distortion in the separated protein or polynucleic acid bands. Such heat is therefore a critical problem because it limits the rate at which gels can be run. Increasing temperatures reduces the resistance and increases current at a given voltage. Although the net effect is a shorter run, excessive temperature can lead to undesirable band broadening. It is therefore preferable to run at a higher voltage and a constant lower temperature.
SUMMARY OF THE INVENTION
The present invention is concerned with an electrophoresis cassette comprising:
first and second thermoformed surfaces comprising their edges in hermetic contact
Roy Dominique
Sevigny Pierre
Côté France
Mirador DNA Design Inc.
Noguerola Alex
Swabey Ogilvy Renault
Warden Jill
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