Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
1998-08-14
2002-06-11
Tung, T. (Department: 1743)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S618000, C204S606000, C204S466000, C204S467000, C204S616000
Reexamination Certificate
active
06402914
ABSTRACT:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
Not applicable.
FIELD OF THE INVENTION
The present invention relates generally to devices for the electrophoretic separation of molecules. Specifically, the invention relates to an apparatus in which protocols can be performed and which can then load samples directly into gels for electrophoresis of molecules, and to equipment adapted to accept this apparatus.
BACKGROUND OF THE INVENTION
Electrophoresis is the process of separating molecules on the basis of the molecules' migrations in an electric field. In an electric field, a molecule will migrate towards the pole that carries a charge opposite to the charge carried by the molecule. Electrophoresis in a polymeric gel, such as an acrylamide or an agarose gel, stabilizes the electrophoretic system against convective disturbances. Further, the polymeric gel provides a porous passageway through which the molecules must travel. Since larger molecules will travel more slowly through the passageways than smaller molecules, use of a polymeric gel permits the separation by molecular size. The molecules can then be visualized by treatment with appropriate stains. If greater resolution is desired, the molecules can be radioactively labeled and the gel then exposed to X-ray film which, when developed, will reveal the migration position of the molecules.
Both vertical and horizontal assemblies are used in gel electrophoresis. In a vertical apparatus, sample wells are formed in the same plane as the gel and are loaded vertically. Horizontal gels are formed horizontally in a tray, with sample wells positioned in a vertical plane. The samples then electrophorese horizontally through the gel. Vertical gels are used particularly in analysis of proteins and in DNA sequencing, while horizontal gels are used for PCR analysis, including medical diagnostics, forensic comparisons and identifications, and preparative scale DNA purification and analytical work, for restriction enzyme digests, and for Northern blots.
Conventionally, when a vertical gel is to be formed, two glass plates are placed on top of each other, separated by a spacer on each side, and are positioned in a gel forming stand. A plastic or rubber “comb,” so called because it has a number of teeth, or prongs, extending downward from its lower edge, is placed in the space between the plates at the top of the assembly, and a liquid, typically of polyacrylamide, is carefully poured into the space between the plates up to the lower edge of the comb. In vertical gels intended for electrophoresis of proteins, the gel is poured to cover the prongs of the comb. When the comb is removed, these prongs typically leave rectangular impressions, or “wells,” in the edge of the gel. The gel is then covered with a standard “running buffer” solution, and samples to be electrophoresed, which have usually been centrifuged, and are often also subjected to heating steps, are mixed with a dense “loading solution” and then micropipetted, or drawn into and then injected from a fine gauge syringe, into the wells, a process known as “loading” the wells. The loading solution usually also contains a dye so that the technician loading the wells can confirm visually the placement of the sample into the correct well.
In DNA sequencing, the procedure is slightly different. DNA sequencing typically employs a “shark's -tooth” comb, which when seen from the front has a flat top and a bottom with prongs shaped like upside-down triangles. The comb is placed in the top of the gel forming apparatus between the glass plates, with the flat side down, and the gel poured. When the gel has set, the comb is removed, to expose the flat surface of the gel and the comb is inverted so that the teeth point down. The comb is then lowered so that the teeth just touch the surface of the gel, with a triangular space open between the teeth, the top of which extends beyond the glass plates of the apparatus. The samples are then loaded through the open spaces between the teeth and run down the interior sides of the glass plates to sit on the flat surface of the gel, separated from each other by the teeth. Gels used for DNA sequencing, in particular, are typically quite thin (between 0.2 and 0.4 mm), requiring considerable care in handling and sample loading.
Horizontal gels are typically formed of liquid agarose poured into a tray, and a comb is positioned to hang into the agarose from above. Once the gel has set, the comb is removed, leaving wells formed by the prongs of the comb. Samples to be electrophoresed are again mixed with a dense “loading solution” to reduce loss of the sample into the running buffer, and are loaded into the wells by a pipette or from a syringe. The electrophoresis of both vertical and horizontal gels is well known in the art and is set forth in, e.g., Sambrook et al.,
Molecular Cloning, A Laboratory Manual
(2nd Ed. 1989) Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; and Harlow and Lane,
Antibodies, A Laboratory Manual
(1988), Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Recently, the use of electrophoresis has undergone explosive growth. The Human Genome Project, which has undertaken to sequence the entire human genome, for example, currently consumes the efforts of a large number of laboratories, and programs are also underway to determine the complete DNA sequence of important pathogens and other organisms, including a number of plants. The use of DNA comparisons in forensic work and in medical diagnostics has also been increasing rapidly.
The high volume of DNA sequencing and other forms of electrophoresis has led to attempts to automate the process. One bottleneck in the efforts to automate electrophoresis in general, and DNA sequencing in particular, has been loading the samples in the wells. While automated equipment has been able to take over much of the repetitive work involved in preparing samples for electrophoresis, it has proven difficult to build equipment capable of loading individual wells, and especially hard to build equipment capable of loading a well in the space (only 0.2 to 0.4 mm) between the plates of a typical DNA sequencing gel. Thus, technicians load the bulk of these gels by hand.
Manual loading is generally undesirable, especially for high volume work such as sequencing. Not only does it slow the process, but it also introduces the chance for error in the order of the samples as the technician repetitively transfers sample after sample from tubes or microtiter plates to the wells. Further, manual loading increases the risk that the technician will be exposed to biohazards, or to radioactivity from radiolabeled samples. It also increases the risk of contamination of the samples due to handling errors such as a failure to change a pipette tip between samples.
Finally, the multiple transfers of samples between containers, such as centrifuge tubes and microtiter plates, and finally into the wells of a gel, are themselves undesirable. Macromolecules, which are the molecules to which electrophoresis is most often applied, are particularly susceptible to cleavage from shearing forces, which are created each time they are pipetted or transferred by syringe from one container to another. Therefore, the current multiple transfers of samples to and from microtiter plates and centrifuge tubes and finally into the wells of electrophoresis gels can alter or destroy the very molecules whose detection is sought.
Accordingly, a need exists in the art for a means of loading gels which is more amenable to automation. Further, a need exists for a means of loading gels which reduces the opportunity to alter the order of samples, reduces the exposure of lab workers to potential hazards, and reduces the number of times a particular sample has to be transferred between containers. The subject invention fills these and other needs.
SUMMARY OF THE INVENTION
The invention provides an “in situ” loader for electrophoresis gels, comprising a body which includes at least one passage. The passage contains an inlet and an outlet, whi
Noguerola Alex
The Regents of the University of California
Townsend and Townsend / and Crew LLP
Tung T.
LandOfFree
In-situ loader for electrophoretic gels does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with In-situ loader for electrophoretic gels, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and In-situ loader for electrophoretic gels will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2903835