Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
2000-04-26
2004-01-27
Warden, Jill (Department: 1743)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S465000, C204S606000, C204S615000, C204S616000, C204S618000, C204S619000, C204S620000, C425S179000, C425S234000, C425S543000
Reexamination Certificate
active
06682641
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an apparatus for casting electrophoresis gels and performing electrophoresis for separation and analysis of DNA molecules, proteins and other charged molecules. The present invention also relates to a method of casting electrophoresis gels.
BACKGROUND OF THE INVENTION
Prior art electrophoresis assemblies include vertical and horizontal arrangements constructed of impermeable, non-conducting plates of glass or plastic between which electrophoresis gels are molded and contained during electrophoresis. Horizontal assemblies are less commonly used due to a number of inherent disadvantages and difficulties in casting, loading and electrophorescing horizontal gels. For instance, sample wells of horizontal assemblies that serve as sample loading sites are typically constructed of the same materials used to form gels. Sample wells are defined by molding well walls into a thickness of a gel. This molding technique is convenient and enables samples wells to be formed simultaneously with or immediately after the casting of gels. However, well walls formed of gel matrix materials contain the same electrolytic buffer ions as gels and are capable of conducting electrical current, which often results in problems with the introduction of samples into gels. When electrical current is applied to assemblies for sample injection, electrolytic buffer ions in well walls compete with similar ions in samples, causing the introduction of sample ions into gels to occur slowly. This effect produces broader sample peaks and limits the ability of the electrophoresis assemblies to resolve molecules of different sizes. In addition, well walls formed of gel materials are not mechanically reliable and susceptible to breakage and tearing during gel casting and sample loading.
Vertical electrophoresis assemblies well known in the art avoid problems associated with samples wells formed of gel materials by using non-conducting well dividers, often referred to as “shark's tooth combs”, constructed of durable materials. A non-conducting comb, such as that disclosed in U.S. Pat. Nos. 4,883,577, 4,909,918 and 5,164,065, is inserted between the assembly plates and placed in a plane of a gel to provide a non-conducting, “hard well” sample loading site. Such non-conducting combs are difficult and inconvenient to insert between the plates of horizontal electrophoresis assemblies. When used in horizontal assemblies, such combs do not provide sample wells that are cable of containing samples by gravity. Therefore, sample wells of horizontal assemblies are invariably formed from gel materials and are electrically conductive.
Another disadvantage of horizontal assemblies is that they require external biasing mechanisms to maintain the assembly plates and buffer reservoirs in precise relationship during the casting and electrophorescing of gels. These biasing mechanisms are typically built with very high tolerances and require continual and difficult manual adjustment. Often external biasing systems work in conjunction with a base or other substrate, wherein the assembly plates and reservoirs are maintained in precise relationship by connection of the assembly components to the base. As disclosed in U.S. Pat. Nos. 5,137,613 and 5,228,971, a horizontal gel assembly is connected to a base by means of adjustable clamps. Uniform and effective biasing requires accurate and frequent manual adjustment of the clamps. In addition, since external pressure is required to cast gels in such a horizontal assembly, the assembly cannot be removed from its casting position in the base without the risk of the assembly coming apart.
The base disclosed in U.S. Pat. Nos. 5,137,613 and 5,228,971 also serves as a water jacket for temperature control of gel during electrophoresis. Such a water jacket cannot be constructed of a sufficiently transparent material to permit the use of photoinitiators, such as riboflavin or benzoin methyl ether, to initiate gel polymerization, and is limited to the use of chemical initiators. Chemically-initiated gel polymerization takes several hours, while photo-initiated gels polymerize in only a few minutes. In addition, the bases and water jackets as disclosed in U.S. Pat. Nos. 5,137,613 and 5,228,971 are expensive and difficult to manufacture.
Prior art horizontal assemblies are also prone to leak fluid from buffer reservoirs. In addition, horizontal assemblies are often not of sufficient size to accommodate various high performance electrophoretic techniques, such as DNA sequencing and DNA fragment size analysis. The technique of DNA sequencing and fragment analysis require longer electrophoresis assemblies on the order of 50 cm or longer. Longer horizontal assemblies require a greater number of amp-hours of electrophoretic current and, hence, a greater supply of buffer ions for a single electrophoretic run. Therefore, buffer reservoirs must be larger in horizontal assemblies used in these techniques to provide a sufficient supply of buffer ions to maintain a consistent electric current for the duration of an electrophoretic run. Buffer reservoirs of prior art horizontal assemblies as those disclosed in U.S. Pat. Nos. 5,137,613, 5,228,971 and 5,242,568, cannot be significantly enlarged without such designs becoming difficult to handle. In addition, such prior art buffer reservoirs are sealed only by means of gaskets, and an increase in reservoir size would render such buffer reservoirs more prone to leak fluid.
Therefore, it is desirable to provide a horizontal electrophoresis assembly for use in high performance electrophoretic techniques, such as DNA sequencing and DNA fragment analysis, that overcomes the limitations and disadvantages of prior art assemblies. It is desirable that the horizontal electrophoresis assembly include sample wells with well walls constructed of a durable and electrically non-conductive material to help ensure uniform and consistent sample injection into a gel. It is also desirable that the horizontal assembly is structured and constructed such that gels are optically accessible to permit photopolymerization and optical detection of separated molecules. The horizontal assembly that provides flexibility to increase the size of buffer reservoirs is also desirable. In addition, it is desirable that the structure and construction of the horizontal assembly facilitate formation of leak-proof seals between assembly components.
SUMMARY OF THE INVENTION
The invention provides an electrophoresis cassette to cast electrophoresis gels and to separate and analyze molecular components by electrophoresis. The invention also provides a method of casting electrophoresis gels.
A first embodiment of the invention includes an electrophoresis cassette comprising a top plate assembly seated on a bottom plate with a continuous spacer therebetween to define a thickness of the electrophoresis cassette and a molding space. The spacer seals an outer perimeter of the electrophoresis cassette.
The top plate assembly includes a central plate and a bottom plate, the bottom plate longer in length than the central plate. The central plate and the bottom plate are similarly rectangular in shape. The central plate has a first terminal edge or a cathode edge that is connected to a cathode buffer reservoir, and a second terminal edge or an anode edge that is connected to an debuffer reservoir. Side edges of the central plate are substantially covered by side rails that protect the side edges of the central plate during use. The side rails extend beyond the terminal ends of the central plate and connect with the side walls of the cathode and the anode buffer reservoirs to provide mechanical support to the top plate assembly.
The cathode buffer reservoir is a substantially rectangular receptacle of a sufficient depth to provide an adequate supply of buffer solution to maintain a consistent electrical current for the duration of an electrophoretic run. The cathode buffer reservoir includes a planar base connected to a body extending from the base to define the rec
Andre Charles P.
Bowers William D.
Finney Michael J.
Linton John R.
Nislow Corey
Brown Jennine
Lahive & Cockfield LLP
MJ Research, Inc.
Warden Jill
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