Chemistry: electrical and wave energy – Apparatus – Electrophoretic or electro-osmotic apparatus
Reexamination Certificate
2002-03-11
2004-02-10
Noguerola, Alex (Department: 1753)
Chemistry: electrical and wave energy
Apparatus
Electrophoretic or electro-osmotic apparatus
Reexamination Certificate
active
06689267
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates to systems for the electrophoretic separation of materials present in a sample wherein the materials have a range of molecular weights. The sample is applied to a gel or similar medium, e.g., on a tray in a vessel of buffer solution, and is subjected to an electric field for a time effective to separate components thereof. In particular the invention provides a stable multi-tray system for simultaneously processing multiple samples without the use of a mechanical buffer circulation pump.
BACKGROUND OF THE INVENTION
Electrophoresis is the migration of electrically charged particles in solution or suspension under the influence of an applied electric field. Each charged particle moves toward the electrode of opposite electrical polarity to its charge. For a given set of solution conditions, the velocity with which a particle moves divided by the magnitude of the electric field is a characteristic number called the electrophoretic mobility. The electrophoretic mobility is directly proportional to the magnitude of the charge on the particle, and is inversely proportional to the size of the particle. This property has been used to determine protein molecular weights; to distinguish among molecules by virtue of their individual net electrical charge; to detect amino acid changes from charged to uncharged residues or vice versa; and to separate different molecular weight species quantitatively as well as qualitatively. In particular, electrophoresis is widely used to separate, characterize and identify large molecules such as proteins and linear molecules such as DNA and RNA, and strands and fragments thereof. More complete and detailed information regarding the basic types of electrophoresis and the many applications for which each type of electrophoresis is utilized may be found in Freifelder, D.,
Physical Biochemistry, Applications to Biochemistry and Molecular Biology
, 2nd edition, W. H. Freeman and Company, New York, 1982, pages 276-322.
The resolving power of electrophoresis can be greatly improved by the use of gel supporting media. Gel electrophoresis methods and electrophoretic apparatus which utilize gels such as starch, polyacrylamide, agarose, and agarose-acrylamide as supporting media are well established. The variety of different applications and the value of gel electrophoresis as a superior analytical and/or preparative tool is demonstrated by the many innovations in apparatus for electrophoresis. These are exemplified by U.S. Pat. Nos. 3,047,489; 4,234,400; 4,151,065; 3,980,546; 3,980,540; 3,932,265; and 3,553,097. Generally, agarose and polyacrylamide are the main types of gels used for electrophoretic analysis of nucleic acid. Typically, short to intermediate size fragments (below one thousand base pairs) are separated in polyacrylamide, while intermediate to high molecular weight fragments are separated in agarose gels. Often, the polyacrylamide gel is arranged in a vertical format or column, while the agarose gels are arranged on horizontal trays in slabs or beds. Typically, to obtain a good separation of DNA and other large molecular weight compositions (proteins and nucleic acids), electrophoresis must be performed on an extended time basis. One of the constant problems of electrophoretic analysis is the breakdown of the buffer used to control the pH of the gel medium due to the formation of acid (H
+
) at the anode and the formation of base (OH
−
) at the cathode over time. This problem is usually resolved by using a mechanical pump for circulating the buffer fluid between the anode containing buffer chamber and the cathode containing buffer chamber.
One electrophoresis device using a buffer circulation system is Applicant's previously patented gel electrophoresis apparatus shown in U.S. Pat. No. 4,702,814. That device has no mechanical circulation structure. It makes use of a pair of platinum electrodes, that are mounted in two buffer reservoirs provided at opposed ends of the device, and a tray supporting a gel bed positioned in a channel of fluid extending between the ends so that an electric field is established along the length of the channel. A hood is positioned below the surface of the buffer solution and covers the length of one of the end electrodes to capture evolved gas bubbles, and a fluid transfer passage extends from the hood at one reservoir upward and laterally to the reservoir at the other end of the apparatus. Bubbles of gas evolved from the underlying electrode enter the passage and drive buffer fluid from one reservoir to the other, creating an equalizing fluid transfer that prevents breakdown of the pH in both buffer reservoirs. The rate of transfer increases with the amount of evolved gas, and operates to transport the higher pH buffer from the cathode end and mix it with the lower pH buffer at the anode end, thus maintaining the buffer pH essentially constant throughout the whole system. The gas-driven circulation also promotes uniformity of temperature, so that the field separation characteristics remain constant and the band spacing is not subject to non-linear distortions at the central portion of the gel tray.
Other patents and marketed systems employ electromechanical pumps to effect buffer circulation and maintain substantial uniformity throughout the gel tray or trays. However, these systems are costly; the cost of a pump alone may be many hundreds of dollars. This is largely because the pump components must comply with fairly stringent standards of construction: the pump must be explosion-proof, electrically isolated, and emission-free for use in biological or laboratory situations.
While these devices have proven to be effective, recombinant DNA technologies, and the availability of polymerase chain reaction technology to amplify and produce effective sample sizes of arbitrary isolates, have expanded greatly the number of electrophoretic analyses performed, and have broadened the scope of such analyses to encompass broad applications in laboratory, industrial and clinical settings. As a result, greater throughput is needed in electrophoretic devices so that greater numbers of analyses can be run without requiring larger capital investments or taking up further space on testing benches.
Various techniques have been employed to refine the resolution or speed of the electrophoretic process for certain specific classes of molecules or conditions. For example, pulse field electrophoresis may be applied to better resolve very large DNA fragments, and extended gel lengths may be used to achieve finer resolution. Other techniques may involve the use of relatively low viscosity gels immobilized in capillary tubes for effecting the separation; the use of high field gradient, short path configurations; or the use of gradient gel concentration arrangements. In addition to having different column, capillary or bed formats, each gel material may present different effective porosity and characteristic ion mobilities. By way of example, the concentration of agarose used in the gel for DNA separations may vary from about 2% for separation of nucleic acid fragment under several thousand nucleotides in length, down to about 0.3% for separation of larger fragments having a size in the range of five thousand to sixty-thousand nucleotides. Similarly for polyacrylamide gels, the concentrations may range between about 3% for one-hundred to two-thousand nucleotide chains, up to about 20% for separating smaller molecules of lengths between about five and about one hundred nucleotides.
In electrophoretic separations, run lengths in vertical or horizontal format may range from about ten to about one-hundred centimeters, depending on the fragment size and the required degree of resolution. The voltage gradients may generally be about one to five volts per centimeter. Typically, a separation may take from one to about twenty hours depending upon resolution requirements and other parameters.
Various other techniques have been proposed to enhance speed or efficiency, or to more
Center for Blood Research
Gosz William G.
Noguerola Alex
Ropes & Gray LLP
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