Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
1996-10-22
2002-04-30
Warden, Jill (Department: 1743)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S450000, C204S466000, C204S600000, C204S606000, C204S616000
Reexamination Certificate
active
06379516
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to electrophoresis generally and more particularly to apparatus for conducting an electrophoresis test therein.
BACKGROUND OF THE INVENTION
A great deal of diagnostic procedures and laboratory research are carried out wherein DNA, RNA or proteins are separated according to their physical and chemical properties via electrophoresis. This process is widely used and has many applications. For example, it is used to analyze DNA molecules according to their resultant size after being digested by restriction enzymes. It is also used to analyze the products of a polymerase chain reaction (PCR).
Typically, electrophoresis separation is carried out in a separation medium, such as a gel of agarose or acylamide or a combination of the two. Usually, agarose gels are cast in open trays and form a slab whereas acrylamide gels are cast between two glass plates.
In order to effect the electrophoretic separation, two opposite ends of the gels are exposed to an electrically conducting buffer which is connected by electrodes, typically carbon or platinum, to an electric power source. Once the electrical power source is switched on, the electric field forces negatively charged molecules to move towards the anode and positively charged molecule to move towards the cathode. One characteristic of conventional electrophoresis is the use of large volumes of buffer having a relatively low salt concentration to maintain the required electric field.
DNA is negatively charged and therefore, in the agarose or acrylamide gels which provide sieving action, DNA molecules move towards the anode at a rate which depends on their size, wherein the smaller the molecules the faster they move.
In the electrophoretic separation of proteins, the proteins are often treated with an ionic detergent, such as sodium dodecylsulphate (SDS). The negatively charged dodecylsulphate anions interact with hydrophobic domains on the protein molecules, thus creating negatively charged protein/SDS complexes that undergoing electrophoresis separation similarly to DNA molecules.
Typically, it is desirable to visualize and to document the results of the electrophoretic separation test. In electrophoretic separation of DNA molecules, this has been done by immersing the gel slab after the electrophoretic separation has been completed in a solution of a fluorescent compound which emits visible light when exposed to a ultra violet (UV) light. A widely used compound in ethidium bromide.
Conventional electrophoretic separation systems are deficient in many respects, a few of which are listed below.
Prior art electrophoresis separation systems are a potential source of contamination to the working environment in which the tests are performed. The two major sources of contamination are ethidium bromide and PCR products. Ethidium bromide is a hazardous chemical due to its mutagenic activity and therefore, exposure to ethidium bromide may induce malignant tumors. PCR is an extremely sensitive method to the extent that a single molecule of DNA product from one PCR (out of the trillions of molecules being produced) may interfere with the subsequent PCR such that it will produce incorrect result.
Conventional electrophoresis is also deficient in other respects, one being that it is time consuming.
Various attempts have been made to solve the deficiencies of conventional electrophoresis. Most attempts have been addressed to overcome the deficiency of conventional electrophoresis systems with respect to the use of buffers therein.
U.S. Pat. No. 4,874,491 to Stalberg describes an electrophoresis system having a high concentration buffer containing gel.
U.S. Pat. No. 4,892,639 to Sarrine et al. describes an electrophoresis plate with improved buffer circulation.
U.S. Pat. No. 5,045,164 to Tansamrit et al. describes an electrophoresis plate having thickened ends as buffer reservoirs.
U.S. Pat. No. 5,209,831 to MacConnel describes a bufferless disposable cassette having open ends and conductive film electrodes.
U.S. Pat. Nos. 5,407,552 to Lebacq and 5,411,657 to Leka describe open electrophoresis devices requiring a buffer tank for operation.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide an improved apparatus for electrophoresis.
A major object of the present invention is to provide a closed cassette for electrophoresis which is substantially closed before, during and after an electrophoresis test conducted therein.
According to an aspect of the present invention the cassette is a disposable cassette.
The cassette of the present invention overcomes drawbacks associated with prior art electrophoresis cassettes, plates or slabs. Since the cassette is a closed one, its outer environment is not susceptible to contamination. Moreover, since it is ready to use, the preparation time required for preparing prior art cassettes is saved.
Another object of the present invention is to provide an electrophoresis system in which both the electrophoretic separation and the visualization of the results thereof are done while the cassette is in situ.
According to one aspect of the present invention, there is provided a substantially closed disposable cassette with openings for introducing a sample of molecules thereinto, the openings being preferably opened only just before the electrophoresis test.
According to another aspect of the present invention, the cassette includes all the chemical compounds required to drive the electrophoretic separation.
In accordance with yet another aspect of the present invention, when DNA, RNA and protein molecules are separated, the cassette includes the compounds required to stain the separated DNA, RNA and proteins, respectively.
According to yet another aspect of the invention, the volume of the ion source utilized for providing the ions required for the electrophoresis separation is smaller than the volume of the gel utilized as the electrophoresis separation matrix and preferably smaller than the volume of gel utilized for actual separation during an electrophoresis test.
According to a preferred embodiment of the present invention, the ions (cations and anions) required to drive the electrophoretic separation are provided by a cation exchange matrix and an anion exchange matrix, respectively.
According to another preferred embodiment of the present invention, the ions exchange matrix also provides the ions required to stain the separated molecules in order to enable visualization thereof when the cassette is illuminated with a UV light source in the case of DNA molecules separation and with a visible light source when protein molecules are being electorphoretically separated.
According to an alternative embodiment of the present invention the ions required to drive the electrophoresis separation are provided by a reservoir, preferably a breakable ampoule containing a buffer characterized by relatively high concentration of these ions.
One advantage of the cassette of the present invention is that it is disposable.
Another advantage of the cassette of the present invention is that the user is not exposed to any hazardous chemical constituent, such as ethidium bromide, as in prior art cassettes.
Yet another advantage of the cassette of the present invention is that PCR-DNA products are contained within the cassette and are disposed therewith so as to substantially reduce the contamination of the working environment in which the tests are performed.
There is thus provided, in accordance with a preferred embodiment of the present invention, an apparatus for conducting electrophoresis separation therein which includes a housing having at least bottom and side walls defining a chamber, wherein the chamber includes in contact therebetween a body of gel for carrying therein the electrophoresis separation, at least one ion source for providing ions for driving the electrophoresis, the at least one ion source having a volume smaller than the volume of the body of gel, and electrodes for connecting the chamber to an external electrical power source,
Cabilly Shmuel
Margalit Ilana
Yogev Uri
Eitan Pearl Latzer & Cohen-Zedek
Ethrog Biotechnology Ltd.
Starsiak Jr. John S.
LandOfFree
Apparatus and method for electrophoresis does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Apparatus and method for electrophoresis, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Apparatus and method for electrophoresis will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2839574