Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Patent
1997-05-01
1999-12-28
Beisner, William H.
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
204620, 204457, 204608, 425174, G01N 2726
Patent
active
060076895
DESCRIPTION:
BRIEF SUMMARY
I. BACKGROUND OF THE INVENTION
This application relates to an apparatus for preparing gels particularly polyaciylamide gels, for use in electrophoretic separation of biomolecules and similar applications.
Polyacrylamide gel electrophoresis (PAGE) separation of biomolecules is now routinely performed. Current Protocols in Molecular Biology, Chap. 10, John Wiley & Sons, (1994). A polyacrylamide gel provides a suitably insoluble sieve that separates biomolecules in solution by size and conformation as they are drawn through the sieve under electromotive force. Such separation of biomolecules provides valuable insights into their structures and functions. For example, PAGE separation can separate two polypeptides of the same size but of different isoforms or polypeptides only 100 daltons difference in size (Current Protocols, 1994). Another use for PAGE is in separation of nucleic acids based on size of fragments, such as in the extremely important application of DNA sequence determination. Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Chap. 13 (1987).
The prior art on PAGE is extensive. Many patents and scientific papers disclose uses for PAGE in research applications. DNA sequencing may be carried out using automated systems designed for laboratory application. These techniques have historically been important for sequencing long stretches of unknown DNA, such as is the focus of the Human Genome Project. Methods and apparatus for sequencing of DNA are described in U.S. Pat. Nos. 4,811,218; 4,823,007; 5,062,942; 5,091,652; 5,119,316; 5,122,345; 5,228,971, and 5,338,426 which are incorporated herein by reference.
Unfortunately, the traditional techniques for preparation of gels for use in electrophoresis are inadequate for use in clinical diagnostic services, such as the emerging field of clinical diagnostic DNA sequencing. For clinical diagnostic DNA sequencing purposes, it is desirable to sequence hundreds of DNA sequences per day. Existing methods do not provide for such capacity. For example, typical methods of DNA sequencing require that a skilled technician spend up to four hours constructing a gel holder, filling the gel holder with actively polymerizing acrylamide solution, inserting a well-forming comb before substantial polymerization has occurred, and then waiting for the gel to polymerize. (U.S. Pat. Nos. 5,338,426; 5,069,773; Maniatis, 1987). Using the gel is equally cumbersome. Loading sample to be electrophoresed requires painstaking care to ensure the integrity of loading wells and to prevent samples from running together. Thus, in order to make maximum clinical use of the opportunities presented by our ever increasing, knowledge of the human genetics and the genetic causes of many disease, it would be advantageous to have a method of rapidly making polyacrylamide gels which are convenient for more efficient sample loading and running, particularly for use in clinical diagnostic applications.
It is an object of the invention to provide an apparatus for filling a gel holder with a polymerizable solution, and catalyzing the polymerization of the solution using ultraviolet light to form a gel usable for electrophoretic separation of biomolecules, particularly nucleic acids.
It is a further object of the invention to provide a cartridge for use as a reservoir in the gel filling and polymerizing apparatus of the invention.
It is a further object of the invention to provide an apparatus for polymerizing an already filled gel holder.
It is a further object of the invention to provide a method for preparing an electrophoresis gel which uses the apparatus of the invention.
It is a further object of the invention to provide an apparatus for the rapid and convenient formation of gels which are more easily loaded with sample.
II. SUMMARY OF THE INVENTION
These and other objects are achieved using an apparatus specifically designed for the rapid filling and polymerization of electrophoresis gels. This apparatus comprises: receive a gel holder having an internal gel compartment; polym
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Renfrew John A.
Steinbach Eric
Stevens John K.
Zaleski Henryk
Beisner William H.
Visible Genetics Inc.
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