Plastic and nonmetallic article shaping or treating: processes – Direct application of electrical or wave energy to work – Polymerizing – cross-linking – or curing
Reexamination Certificate
1998-08-19
2001-02-13
Vargot, Mathieu D. (Department: 1732)
Plastic and nonmetallic article shaping or treating: processes
Direct application of electrical or wave energy to work
Polymerizing, cross-linking, or curing
C204S470000, C204S620000, C264S040600, C264S145000, C264S299000, C425S144000, C425S145000, C425S174400, C425S308000, C425S371000, C425S812000
Reexamination Certificate
active
06187250
ABSTRACT:
TECHNICAL FIELD AND INDUSTRIAL APPLICATION OF INVENTION
The invention relates to the production of acrylamide or other gels for use in separations of proteins, nucleic acids or other biological materials. The invention further relates to a continuous gel casting method and apparatus. In particular, the invention is intended to provide a method and apparatus for casting gels, and particularly, gradient gels, in a continuous manner as compared to the batch methods presently used.
BACKGROUND OF THE INVENTION
The separation of macromolecules through the use of gel electrophoresis has been common practice in biological laboratories for over twenty years. The technique is in even greater use in the last decade due to the tremendous explosion in protein analysis and the sequencing of genes. Since the late 1980's, rapid protein analysis and gene sequencing has become not only ubiquitous, but absolutely necessary for researchers. In particular, gels are often the starting points for new drug targets and also for sequencing as it relates to overall scientific endeavors such as the human genome project.
In general, electrophoresis gels can be either in a slab gel or tube gel form. For slab gels, the apparatus used to prepare them usually consists of two glass or plastic plates with a space disposed between them by means of a spacer or gasket material and the apparatus is held together by a clamping means so that the space created is closed on three sides and open at the top. A solution of unpolymerized gel, such as polyacrylamide, is poured into the space while in its liquid state. A means of creating wells or depressions in the top of the gel (such as a comb) in which to place samples is then placed in the space. The gel is then polymerized and becomes solid. After polymerization is complete, the comb device is removed and the gel, while still held within the plates, is then ready for use. Examples of such apparatus are well known and are described in U.S. Pat. No. 4,337,131 (Vesterberg), U.S. Pat. No. 4,339,327 (Tyler), U.S. Pat. No. 3,980,540 (Hoefer et al.), U.S. Pat. No. 4,142,960 (Hahn et al.), U.S. Pat. No. 4,560,459 (Hoefer), and U.S. Pat. No. 4,574,040 (Delony et al.). Tube gels are produced in a similar manner, however, instead of glass or plastic plates, glass capillary tubing is used to contain the liquid gel. It is important to note that each slab gel must be produced one at a time in this manner.
In addition to ordinary acrylamide gels, methods of making the composition of the gel vary along one or two directions, so called gradient gels have also been devised. Gradient gels are gels that have characteristics such as pH or pore size or percentage of crosslinked acrylamide which vary as a sample travels through the gel in one direction. A good general overview of the common method of preparing gradient gels is provided in Westermeier,
Electrophoresis in Practice,
VCH: New York, 1993, pp. 25-27, 174-177, and 197-214. In particular,
Westermeier
describes the pouring of a single linear pH gradient gel. It is even possible to construct gels that vary in two directions, such as pH from left to right across the slab while increasing in pore size from the top to the bottom of the slab. U.S. Pat. No. 5,071,531 (Soane) describes an apparatus for making such a gradient slab gel. In addition, U.S. Pat. No. 5,597,480 (Zhou) describes an apparatus which can be adapted to create a gradient slab gel.
Because of the tremendous need for faster and faster analysis of proteins and sequencing of genes due to their value as drug and disease targets in all aspects of biology, there exists a critical need for users to have access to hundreds or even thousands of gels for use in separations in a short period of time. Users, such as genomics companies, must be able to rely on gels that are produced quickly and inexpensively yet are absolutely consistent in their performance from batch to batch. To satisfy this need, a method of producing multiple gels in a batch manner on a large scale was needed.
A number of patents disclose methods for batch production of gels. U.S. Pat. No. 5,047,135 (Nieman) describes a modified slab gel apparatus where the gels are actually very thin wafers kept separate through the use of thin divider sheets. The multiple gels are all sandwiched together and run as one slab, but then may be peeled off and separately processed. Another method of producing a plurality of slab gels is disclosed in U.S. Pat. No. 5,520,790 (Chopas et al.), wherein a casting stand is described which can accommodate a number of separate gel molds held vertically. The individual gel molds can then be filled simultaneously from the bottom and can be ordinary gels or gradient gels via the use of a gradient former in combination with the apparatus. The article by P. G. Righetti, “Modern Aspects of Isoelectric Focusing: Two-Dimensional Maps and Immobilized pH Gradients,”
Journal of Biochemical and Biophysical Methods,
8:89-108 (1983), describes a gel casting method in which 20 gels are made concurrently for a single isoelectric focusing run. The gel casting method comprises pouring the gelling mixture into a loading trough, which is lowered into a tank partly filled with water. As the trough sinks to the bottom, the gelling mixture is displaced into tubes at the desired level. The gels are then allowed to polymerize in the tubes.
What is needed presently, is an apparatus which could allow one skilled in the art to make either gradient or non-gradient slab gels continuously so that the gels produced are uniformly formed, polymerized and cut to a specific size as needed, in a mass-produced, assembly line manner. The present invention accomplishes this goal and is described in specific detail below.
SUMMARY OF THE INVENTION
The present invention discloses an apparatus for the continuous casting of a gel by introducing the gel reaction mixture into a molding space and a casting manifold which encloses the molding space and provides a continuously moving, sealed molding space for holding the reaction mixture during gelation.
Specifically, the present invention describes a means of introducing a reaction (monomer) solution into a molding space or manifold in a controlled manner. The invention also describes a means of introducing the reaction solution in either a temporally or spatially varying manner so the final gel contains a concentration gradient.
Additionally, the present invention describes a casting manifold and within it a molding space to hold the reaction solution (gel) and initiators of polymerization without leaking at a maximum pressure of less than one atmosphere during the time it takes for the polymer to gel, but not necessarily complete polymerization. The invention also describes allowances for shrinkage of the gel of a few percent and a venting mechanism to displace entrained air in the mold space because air or other fluids can be entrained in the molding space in some applications where the gel is molded in a discontinuous fashion into a support cassette or backing or mask containing holes or spaces to be filled with gel. This air or other fluid is inadvertently carried into the molding space with the support cassettes or backing or masks as they are introduced into the casting manifold.
The invention further describes a controlled means of initiating the free radical polymerization process as the reaction mixture enters the mold space. In addition, it discloses methods to prevent premature polymerization when using chemical free radical initiators and catalysts requiring thorough mixing into the reaction mixture just prior to entering the mold space. The present invention allows ultraviolet free radical generation to occur through the use of a UV transparent window in the side of the mold cavity.
The present invention additionally discloses a means of controlling the reaction temperatures in the molding space during the polymerization, removing excess reaction heat and a means of removing the gels from the mold cavity.
REFERENCES:
patent: 3422178 (1969-01-01), Junker et al.
Champagne James
Shanks & Herbert
Vargot Mathieu D.
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