Patent Application
20120004138
The present invention provides an apparatus for simultaneously loading multiple samples for conducting an electrophoresis test.
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 micro-gel 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.
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 electrophoresis apparatus for non-weighted sample deposition, including a substantially closed area, an electrophoresis gel with wells located within the electrophoresis area, and a non-liquid ion source located within the gel, eliminating the need for weighting samples before deposition into the wells.
There is provided, in accordance with another embodiment of the present invention a system for conducting electrophoresis separation including an electrical power source, a substantially closed disposable cassette for conducting an electrophoresis separation therein and having conductive elements therein, and a support for supporting the substantially closed cassette and for connecting the electrical power source to the conductive elements of the cassette, where one or more gels may be connected simultaneously. The cassette includes a body of gel for carrying therein the electrophoresis separation, a plurality of wells in the body of gel arranged in a stagger format and a plurality of apertures having loading sites leading to the plurality of wells.
There is provided, in accordance with another embodiment of the present invention a method for treating water-absorbent plastic used for electrophoresis devices, including the steps of placing the water-absorbent plastic in a humidified environment and saturating the water-absorbent plastic by leaving it in a humidified environment for a predetermined period of time.
There is provided, in accordance with another embodiment of the present invention a method for simultaneous loading of multiple samples into an electrophoresis apparatus, including the steps of providing an electrophoresis apparatus having an area with walls defining the area and a gel within the area having multiple wells arranged in stagger format, wherein the walls include apertures having loading sites and channels structurally configured to direct samples into the wells, loading the samples into the openings with a standard multiple loading mechanism, and directing the samples from the apertures to the wells.
The present invention will be understood and appreciated more fully from the following detailed description taken in conjunction with the appended drawings in which:
Reference is made to
As shown in
Bottom wall 12 and top wall 16 are preferably made of any suitable UV transparent material, such as the TPX plastic commercially available from MITSUI of Japan or the Polymethylmethacrylate (PMMA) plastic commercially available from Repsol Poliver S.P.A. of Rome, Italy. Cassette 10 may include vent holes 32 and 34 to allow for gaseous molecules that might be generated due to the electrochemical reaction (e.g., oxygen and/or hydrogen) to be released. In one embodiment, vent holes range in diameter from 0.5-2 mm. In a preferred embodiment, vent holes are 1 mm in diameter.
As seen in the cross section illustration (IV-IV) of
When the gel has set, the comb is removed to leave a row of wells 36, or holes, in the layer. In one embodiment, wells 36 are dimensions of 0.5-5 mm wide, 1-5 mm long, and 3-5 mm deep, and are used to introduce samples of the molecules to undergo molecular separation. One row or several rows may be formed. In one embodiment of the present invention, 12 rows of 8 wells per row are formed, and are arranged in a stagger format, as shown in
In addition, cassette 10 may optionally include a capture layer 37 including part of a molecular recognition pair for separating samples according to binding properties. Capture layer 37 is immobilized within gel 18, and is fabricated with resins to which the binding site of a molecule of interest will covalently bind. Some examples include avidin on acrylic beads, biotin on cross linked beaded agarose and others. The resins are mixed with agarose or other materials and poured as layers into gel 18. Alternatively, acrydite™ (available from Mosaic Technologies, Waltham, Mass., USA) may be used. Acrydite™ is a phosphoramide that is capable of copolymerization with acrylamide, and it can be used to introduce copolymerizable groups on the 5′ terminus of any oligonucleotide probe. To make the capture layer, Acrydite™ oligonucleotide capture probes may be mixed with acrylamide solutions and polymerized into gel layers.
The capture electrophoresis technique provides concentrated signals, saves time and saves material. One or multiple capture layers may be used. This technique may be performed on its own, or in combination with a standard size electrophoresis separation.
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 which emits visible light when exposed to an uttra violet (UV) light. According to one embodiment of the present invention, the samples or the gel interact with ethidium bromide or other fluorescent dyes. In this way, the results may be viewed in situ, without the need for exposing the samples to contamination by removing the gel from the enclosed area 11.
According to another embodiment of the present invention, various types of light sources may be used. In one embodiment, a light source of adjustable or non-adjustable wavelengths may be used. The light source may include visible or non-visible light.
Alternatively a colorimetric dye, such as Methylene Blue may be added to the samples, the gel, or the ion reservoir and may interact with the molecules undergoing electrophoresis separation, so as to enable visualization of the results without the need for a UV light source.
Area 11 also comprises two conductive electrodes referenced 21 and 23 which, when connected to an external direct current (DC) electrical power source, provide the electric field required to drive electrophoresis separation. In the illustrated embodiment, electrode 21 is the cathode and electrode 23 is the anode. The system may also include a support for connecting conductive elements of cassette 10 to the power source. In one embodiment, the support is configured to connect to one or more gels simultaneously. Further, the system optionally includes a camera for documentation.
In one embodiment, the gel 18 and the conductive electrodes 21 and 23 are in contact with non-liquid ion sources such as ion exchange matrices as described in U.S. Pat. Nos. 5,582,702 and 5,865,974.
It should be noted that since plastics used as cassette material are sometimes water absorbent, they may be pre-treated by placement in a humidified environment and saturation by leaving it for a predetermined period of time so as to avoid later water adsorption or uptake of liquid, thereby keeping the gel intact. In one embodiment, the period of time ranges from 1-72 hours. In another embodiment, the period of time ranges from 1-20 days. In another embodiment, the period of time is at least 10 days. In a preferred embodiment, the period of time is 10 days.
It should be noted that in conventional electrophoresis, samples must be weighted so that they sink through the buffer to the bottom of the wells. This is generally accomplished by combining a substance such as Glycerol, Sucrose, or Ficoll polymer with the sample. It will be appreciated that in one embodiment of the present invention, there is no liquid buffer present in the vicinity of the wells, and instead, a non-liquid ion source is located within said gel. Thus, the step of weighting samples before deposition into said wells may be eliminated, thereby decreasing the time necessary to perform an experiment.
Reference is now made to
Stagger format of outlet apertures 39, located on the bottom side of wall 16, corresponds to stagger format of wells 36 within a layer of gel 18, as depicted in
Thus, when electrophoresis separation takes place, the available running distance between adjacent wells 36 in the direction of electrophoresis separation is from 8-20 mm. In one embodiment, the available running distance is up to 18 mm, as shown by arrow 43. This amount is double what would be available without stagger formatting, greatly increasing the potential for larger sized molecules to be separated. If wells 36 were arranged according to a standard format, and not a stagger format, samples in each row would have a running distance of less than 1 cm, whereas in the configuration illustrated in
In the embodiment shown in
The shape of loading sites 41 may vary, but they are typically circular, so as to fit the end of a loader tip. A standard multiple loading mechanism such as a micro-titer multi-pipette loader available from, for example, Eppendorf Scientific, Inc., Westbury, N.Y., USA may be used, thus enabling simultaneous loading of as many samples as can fit in the pipette. Thus, for a 96-well configuration, loaders are available from, for example, Beckman Coulter, Inc., Fullerton, Calif., USA, that would enable loading of 96 samples all at the same time, or loading of 8 or 12 samples at a time. Similar models might be available for the other formats as well.
Loading sites 41, either located on the edges of inlet apertures 38 as in
As shown in
Reference is now made to
Reference is now made to
Reference is now made to
It will be appreciated that the embodiments described hereinabove are described by way of example only and that numerous modifications thereto, all of which fall within the scope of the present invention, exist. For example, gels may be either vertical or horizontal. In addition, apertures may be on the side wall of the apparatus, rather than directly on the top cover. In one embodiment, the entire system is in a microscale range, in which case all the dimensions described hereinabove are reduced by a factor of 10-100.
It will be appreciated by persons skilled in the art that the present invention is not limited to what has been particularly shown and described hereinabove. Rather, the scope of the present invention is defined only by the claims that follow:
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11470308 | Sep 2006 | US |
| Child | 13174400 | US | |
| Parent | 10370459 | Feb 2003 | US |
| Child | 11470308 | US | |
| Parent | 09651087 | Aug 2000 | US |
| Child | 10370459 | US |
