Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
2001-08-02
2004-04-27
Olsen, Kaj K. (Department: 1753)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S468000, C204S469000, C204S606000
Reexamination Certificate
active
06726821
ABSTRACT:
REFERENCE TO RELATED APPLICATIONS
The present application is the national stage under 35, U.S.C. 371, of international application PCT/JP00/08465, filed Nov. 30, 2000, which designated the United States, and which international application was not published under PCT Article 21(2) in the English language.
TECHNICAL FIELD
The present invention relates to a precast polyacrylamide gel adapted for use in electrophoresis separation, production method thereof and electrophoresis using the gel.
BACKGROUND ART
Precast polyacrylamide gels for electrophoresis are extensively used as a basic investigative medium for detecting and quantitatively analyzing chemical substances: proteins, nucleic acids, carbohydrates, and lipids necessary for building all living organisms in fields as diverse as biology, medicine, fisheries, veterinary medicine, and so on. Especially, a variety of precast polyacrylamide gels different in resolving power can be made easily by varying a gel recipe because the gels are substances synthesized artificially. Accordingly, it is possible to prepare in advance the precast gels differing diversely in separation characteristic or resolution from one another. The artificial gels, since much saving the labor and effort for analytic procedure and excellent in uniformity and reproducibility, have helped the productivity and quality control in the fields stated earlier. The artificial gels which are mass-produced must have an increased shelf-life to ensure an adequate supply of these gels.
Most polyacrylamide gels for electrophoresis used extensively for analytic technique of proteins in the fields of biochemistry and medicine are based on the system developed by Ornstein [L. Ornstein, Ann. N.Y. Acad. Sci. 121, 321-349, (1964)] and Davis [B. J. Davis, Ann. N.Y. Acad. Sci. 121, 404-427(1964)] or that proposed by Laemmli [U. K. Laemmli, Nature, 227, 680 (1970)]. Gels have heretofore been produced by the users or analysts in themselves or on themselves for their private use. In particular, the Laemmli system is most extensively used, since the molecular weights of proteins can be simply presumed by adding sodium dodecyl sulfate, contracted to “SDS” hereinafter, to gels or electrophoresis buffer solutions. The Laemmli system consists of a gel buffer solution of tris(hydroxymethyl) aminomethane, contracted to “Tris” herein, which is partially neutralized with hydrochloric acid, and an electrophoresis buffer solution, or Laemmli's electrophoresis buffer solution using Tris and glycine salt. The gel buffer solution in the system stated earlier, or Laemmli's gel buffer solution contains about 10 -20 mol % Tris partially neutralized to be adjusted to pH 8.8 with hydrochloric acid. At this pH of the Laemmli's gel buffer solution, however, amide groups are subjected to hydrolysis time elapses. As the hydrolysis of gels proceeds even under low temperatures, the polyacrylamide gels come to contain partially anionic groups. As a result, the migration distances of proteins are reduced while electrophoretic patterns become vague, and therefore it is impossible to preserve the gels over a prolonged period.
The following discussions will present the electrophoresis of nucleic acids. Recently remarkable development of molecular biology has come to need the analysis and preparation of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The analysis of nucleic acids mostly depends on either the agarose electrophoresis separation process or the precast polyacrylamide gel electrophoresis separation process. As nucleic acids present strong negative charges in the neutral buffer solution while the mobility thereof depends on the molecular sieve effect of supportive matrix gels, either an about 0.3-2% agarose gel or an about 3.5-20% polyacrylamide gel is chiefly employed depending on the size of nucleic acids undergoing analysis.
The agarose gel, because of having a larger pore size, is often used for analytic separation of macromolecular nucleic acids. There are many types of agarose gels, which differ from one another in electroosmosis, gel strength, melting point, and so on. Moreover, because the agarose gels are naturally occurring materials, the differences in quality are frequently seen., even for a lot of products and even for the same gels produced in the same facility. For purifying DNA by virtue of the agarose gel electrophoresis separation process, any other purification procedures such as the phenol extract, and so on must be considered because contamination with impurities in the agarose gels would inhibit enzyme activities of restriction enzymes, DNA polymerases, DNA synthetase, and so on.
In contrast, polyacrylamide gels with relatively small pore size are useful for isolation and analysis of nucleic acids of medium and/or low molecular weight. Because the polyacrylamide gels are artificially synthetic products, they are very pure chemically, and thus involving no major problems seen in the agarose gels. Moreover, the polyacrylamide gels may be easily prepared in diverse types of gels, which are different in separation characteristics, depending on the desired gel recipe. Accordingly, it is possible to prepare in advance the precast gels differing diversely in separation characteristics from one another. The use of artificial gels mass-produced in advance diverse separation characteristics, since much saving the labor and effort for analytic procedure and excellent in uniformity and reproducibility, will help productivity and quality control in the fields stated earlier. The artificial gels obtained by mass-production methods have increased shelf-life because of preservation. The most common buffer system for using agarose and polyacrylamide as the gel material in the electrophoresis separation of DNA is a continuous buffer system of around pH 7.8 to 8.3, in which a gel buffer solution is equal in composition with an electrophoresis buffer solution: a tris-acetic acid buffer, contracted to “TAE” herein, which contains either ethylenediaminetetraacetic acid, contracted to “EDTA” or disodium ethylenediaminetetraacetate, contracted to “ETA”; a tris-boric acid buffer, abbreviated to “TBE”; and a tris-phosphoric acid, abbreviated to “TPE”.
As stated earlier, the precast gels manufactured on mass-production methods must be able to maintain the long-lasting shelf-life in preservation throughout shipping and storage. Among the prior processes for manufacturing gels which are superior in shelf-life is a process for manufacturing a polyacrylamide gel having the increased shelf-life disclosed in Published Unexamined Patent Application in Japan No. H04-184 163, which involves a gel buffer solution composed of Tris, ampholyte and acid to be storable for a prolonged period without loss of performance and also expanded remarkably in molecular weight range applicable for analytical electrophoresis. The polyacrylamide electrophoresis gel produced by the process recited just above is available for analysis using Laemmli's electrophoresis buffer solution. The polyacrylamide gel contains the ampholyte and has the pH of ranging from 4.0 to 7.5. In an example disclosed, the gel having a pH of ranging from 6.9 to 7.4 is produced, which is described to be stored under refrigeration for four months without variation in mobility of proteins.
The gel buffer solution is set high in neutralization rate of tris to depress the hydrolysis of the polyacrylamide gel, while added with the ampholyte to make gentle a variation in electric potential gradient neighboring a boundary between tris strong acid salt part and tris week acid salt part, thereby coming to exhibit the wide range in the fractionated molecular weight of species undergoing measurement, which is comparable to the gels fabricated using the Laemmli's electrophoresis buffer solution.
Nevertheless, most polyacrylamide gels containing a gel buffer solution of the pH range 6.9 to 7.4 begin to vary in electrophoretic mobility of proteins after more than five months in storage under refrigeration has g
Browdy and Neimark , P.L.L.C.
Hymo Corporation
Olsen Kaj K.
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