Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Patent
1997-05-01
1999-03-23
Warden, Robert
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
204615, 204465, 204606, 204620, G01N 2726
Patent
active
058854312
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
This application relates to microgels for use in medical diagnosis, especially for the sequencing of nucleic acids, and to methods of making and using such gels.
DNA sequencing may be carried out using automated systems designed for laboratory application. 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 and 5,122,345, which are incorporated herein by reference.
The general methodology employed in these systems involves breaking up the sample DNA using restriction endonucleases; amplifying (for example with PCR) the restriction fragment of interest; combining the amplified DNA with a sequencing primer which may be the same as or different from the amplification primers; extending the sequencing primer in the presence of normal nucleotide (A, C, G, and T) and a chain-terminating nucleotide, such as a dideoxynucleotide, which prevents further extension of the primer once incorporated; and analyzing the product for the length of the extended fragments obtained. Analysis of fragments may be done by electrophoresis, for example on a polyacrylamide gel.
International Patent Publication No. WO93/00986 describes electrophoresis gels with a thickness of 25 to 250 microns. The gels are formed between two clamped-together plates, one of which is grooved to a depth equal to the desired gel thickness to form parallel tracks which are then filled with gel.
In performing a nucleic acid sequence analysis on a gel, the characteristics of the gel, including the size and thickness, impact the time and cost required to do the analysis. Since it is desirable to reduce the time and cost of sequencing analyses in order to improve the available of sequencing as a diagnostic tool, it would be advantageous to have a gel which permitted analysis of very small quantities of oligonucleotide fragments in a short period of time. It is an object of the present invention to provide such a gel.
It is a further object of the invention to provide single-use, disposable gel holders which are readily filled with gel to provide a gel for rapid analysis of small samples.
It is a further object of the present invention to provide a method of making gels which achieve high resolution of oligonucleotide fragments in a short period of time.
It is a further object of the invention to provide a method of evaluating a sample containing oligonucleotide fragments of various lengths.
SUMMARY OF THE INVENTION
These and other objects of the invention are achieved using an electrophoresis microgel formed in a gel holder. The gel holder comprises a top substrate, a bottom substrate and a spacer disposed between the top substrate and the bottom substrate. The spacer establishes a separation of from 250 micron or less, preferably 25 to 250 microns, between the top substrate and the bottom substrate. A gel compartment is formed by partially sealing the top substrate to the bottom substrate, while leaving an opening for the introduction of unpolymerized gel. The gel compartment is then filled with an unpolymerized or partially polymerized gel, which is polymerized in the gel compartment. Electrodes may be printed on the substrates, may be contacts to an exposed edge of gel, or may be applied through windows cut into one of the substrates.
A preferred embodiment of the invention makes use of graded beads having a diameter of 250 microns or less, preferably 25 to 250 microns, slurried in an adhesive such as an acrylic or ultraviolet-light activated adhesive or bonded to the substrate using a low-melting glass as the spacer. The slurry is printed onto the surface of one or both substrates to form a spacer of the desired shape, and then hardened using heat or light. If desired, adhesive or a low-melting glass containing solid particles can also be used to establish lanes within the gel.
The spacing between the substrates can also be established using a plurality of beads disposed on the surface of the substrates within the gel compartment. In this case, th
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Renfrew John A.
Stevens John K.
Waterhouse Paul
Zaleski Henryk
Noguerola Alex
Visible Genetics Inc.
Warden Robert
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