Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
2000-08-30
2003-05-13
Tung, T. (Department: 1743)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S466000, C204S605000, C204S616000, C435S288400, C435S305200
Reexamination Certificate
active
06562213
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides an apparatus for simultaneously loading multiple samples for conducting an electrophoresis test.
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, electrophoresis 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).
In some instances, molecules are driven toward a capture layer, which has part of a molecular recognition pair e.g. antibody-antigen, DNA-DNA probe, biotin-avidin, ligand-receptor, lectin-carbohydrate or others. Only specific parts of each pair of molecules that move through the capture layer are captured (e.g., an antigen when the capture layer contains a specific antibody), while the non-specific molecules pass through the layer unimpeded.
Electrophoresis separation is carried out in a separation medium, such as a gel of agarose or acrylamide or a combination of the two. Agarose gels are cast in open trays and form a horizontal slab whereas acrylamide gels are vertically cast between two glass plates.
Prior to electrophoresis separation, wells are introduced into the gel for sample deposition by applying a comb-like structure prior to the solidification or polymerization of the gel matrix. A row of approximately 8-15 wells is formed across one end of the gel.
In order to effect the electrophoresis separation, two opposite ends of the gel are exposed to a buffered solution which is connected by electrodes, often made of platinum, to an electrical 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 molecules to move towards the cathode. 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. The running distance should be long enough to allow sufficient differentiation between molecules.
It is desirable to visualize and to document the results of the electrophoresis separation test. In electrophoresis separation of DNA molecules, this has been done by immersing the gel slab after the electrophoresis separation has been completed in a solution of a fluorescent compound, such as ethidium bromide, which intercalates within DNA molecules and emits visible light when exposed to an ultra-violet (UV) light. In order to document the results, a picture of the gel is taken through one of various photographic means.
Prior art electrophoresis systems are potential sources 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 results.
Also, conventional electrophoresis is time consuming in terms of preparation and handling. This is particularly true when a large number of samples are to be analyzed, and loading of samples is done one by one.
Several inventions have been directed towards eliminating contamination, such as U.S. Pat. No. 5,972,188, which describes the use of a membrane loader for gel electrophoresis; and an electrophoresis apparatus with a cover, in U.S. Pat. Nos. 5,582,702, and 5,865,974 incorporated herein by reference. The apparatus is directed towards the running of electrophoresis separation, as well as detecting and analyzing the results, within a self-contained, disposable unit.
Attempts have been made to reduce the time it takes to run an electrophoresis separation as well by loading many samples at once. Further, simultaneous loading of samples could reduce contamination and human error. Standards in cell culture, ELISA and PCR analysis provide different sized plates, with corresponding pipettes for ease in sample loading and analysis. For example, 96-well plates are typically used. Correspondingly, pipettes that fit this configuration are available and are widely used. Use of standard microtiter pipettes would greatly reduce the loading time for electrophoresis.
Saito et al., in U.S. Pat. No. 5,785,835, address this issue by providing an apparatus for loading of samples into wells within an exposed gel with standard pipettes. However, the testing apparatus has limited resolution capacity since a running distance of only 0.8 cm is available. In U.S. Pat. No. 6,071,396 a gel-matrix layer is described with wells arranged for loading of samples with standard pipettes. In this patent, the running distance is increased by diagonally offsetting the entire array of wells. U.S. Pat. No. 6,013,166 describes a method for reducing the linear dimension necessary for electrophoresis separation in a microgel format.
In addition, several needle guide designs have been developed to aid in loading samples directly into wells in a way that would save time and prevent inaccuracies. For example, U.S. Pat. No. 5,656,145 provides a needle guide for loading samples into a vertical slab gel. Similarly, U.S. Pat. No. 5,843,295 is directed towards a combination comb/loading guide unit. In both of these designs, the loading sites are positioned directly on top of the wells so as to allow for simple, direct loading of samples.
SUMMARY OF THE INVENTION
This invention provides, in accordance with an embodiment of the present invention, an apparatus for simultaneous loading of multiple samples for molecular separation, including a separation area with walls wherein at least one of the walls has multiple apertures with loading sites, a gel located within the separation area, and a plurality of wells within the gel. The apertures are connected to the plurality of wells by channels structurally configured to convey samples from the apertures to the wells. In one embodiment, the loading sites are spaced at predetermined intervals so as to conform with intervals between tips on a loader.
In one embodiment, the plurality of wells is arranged in rows, and the rows are arranged in stagger format, providing a running distance for molecular separation which is longer than the distance between two adjacent rows.
There is provided, in accordance with another embodiment of the present invention an apparatus for electrophoresis separation having a substantially closed electrophoresis area, an electrophoresis gel located within the electrophoresis area, and multiple rows of wells within the electrophoresis gel, wherein the rows are arranged in a stagger format.
There is provided, in accordance with another embodiment of the present invention, a gel layer for molecular separation having a plurality of wells within the gel layer. The wells are arranged in a plurality of rows, and wells of one row are horizontally shifted from wells of a neighboring row by a predetermined distance. The horizontal shift is alternated from left to right, so as to form a staggered format of wells within the gel layer.
There is provided, in accordance with another embodiment of the present invention a device for delivering samples into wells for molecular separation, having a flat surface with a top side and a bottom side, multiple loading sites on the top side arranged in standard format, multiple apertures on the bottom side arranged in stagger format and leading to the wells, and a channel through the flat surface connecting the loading sites to the apertures.
There is provided, in accordance with another embodiment of the present invention an
Cabilly Shmuel
Margalit Ilana
Yogev Uri
Eitan, Pearl, Latzer & Cohen Zedek LLP.
Ethrog Biotechnology Ltd.
Noguerola Alex
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