Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
2001-03-05
2003-08-05
Nguyen, Nam (Department: 1753)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S455000, C204S605000, C204S613000, C204S451000, C204S601000
Reexamination Certificate
active
06602391
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates generally to devices and methods for performing separation of biological macromolecules (electrophoresis or gel chromatography) and transfer (electro-blot, capillary blot or other means of blotting). The present invention particularly relates to a device, which can perform separation and blotting of protein and nucleic acid samples within one capillary element.
Separation of macromolecules such as proteins and nucleic acids is a necessary step in numerous applications of protein and DNA-RNA analyses in multiple biological, scientific, medical, and forensic applications. The most prominent and widely used techniques for separating macromolecules are chromatography and gel electrophoresis. Following separation by said principles, proteins and nucleic acids are then generally collected in separate volumes or fixed (blotted) to special chemical compounds, mostly nitrocellulose or nylon (F. Ausubel et al (Ed.),
Current Protocols in Molecular Biology
, Ed. Current Protocols, Wiley, N.Y., 1994), for further processing and analyses. Separation and blotting must be performed independently in time and space, otherwise highly chemically reactive blotting membrane may be contaminated with separated molecules. For this reason after separation is performed, the gel with separated molecules is placed in contact with blotting membrane by mechanical transfer.
Electrophoresis, used for separation, utilizes a physical phenomenon of charged particles' ability to migrate toward the pole possessing the charge opposite to that of a particle, when it is suspended between opposite poles in an electric field. Conventional gel electrophoresis utilizes a gel slab assembly. Pore matrix of a gel slab is usually made of agarose or polyacrialamide. Pores, which retain macromolecules depending on their physical properties (size, hydrodynamic radius, weight, composition, electrical charge, etc.), form an interacting or sieving matrix. Wells are formed in the upper part of the gel slab during casting procedure. A gel slab assembly is placed into a device for electrophoresis. Samples are then introduced in a band within wells; and an electric field is applied across the slab. The upper and the lower portions of the gel are submerged into separate buffer solution reservoirs. The electric field forces macromolecules of the samples to migrate through the gel. During migration, the macromolecules are “sieved” by their properties, most often their molecular weight. Specific species of macromolecules will be in bands arranged from top to bottom of the gel (Andrews, A. T.
Electrophoresis: Theory, Techniques, and Biochemical and Clinical Applications
. Clarendon Press-Oxford (1986), 2
nd
Ed).
In the process of DNA separation, the DNA fragments (DNA ladders) can be labeled before or after separation with either radioactive or florescence labels (F. Ausubel et al (Ed.),
Current Protocols in Molecular Biology
. Current Protocols, Wiley, N.Y., 1994, Chapter 2). In DNA sequencing procedures, each of the four types of a nucleotide can be labeled with specific probe, which will appear at termination of DNA fragment. A mixture of different reactions can be electrophorized and separated according to the label from one reservoir in the gel.
After separation is complete, deposition membrane with blotting material is placed in contact with gel. A common transfer process is called “electro-blot” transfer. In the “electro-blot” transfer process, the macromolecules in the gel slab move under an electric field to a blotting membrane. In designing an electro-blot transfer system it is essential that the blotting membrane be in close contact with the gel slab. Presence of gas bubbles between the gel slab and blotting membrane will prevent the band images from being transferred properly. It is also important to maintain a uniform electric field across the electro-blot sandwich. Transfer of the gel slab onto the nitrocellulose membrane must be carefully performed so that the macromolecules on the gel membrane are not removed or contaminated. After transfer, a labeling procedure must be employed, and a detection technique must be utilized so that the samples can be analyzed. A commonly used detection method involves staining and de-staining of the gel slab. This technique imposes staining of the entire gel with a dye that only adheres to the macromolecules. Then a de-staining process is performed, wherein dye not adhered to the macromolecules is washed away; bands of macromolecules thus become detectable. Another common detection method is the use of antibodies. Bands of proteins or samples are blotted or transferred to a binding membrane, which binds macromolecules. Then, a known antibody is introduced. The antibody combines with a specific protein if it is present in a sample. In order to detect the antibody-protein combination, the antibodies are labeled with fluorescent or radioactive tags or have enzyme activity, which is further detected by separate methods (F. Ausubel et al (Ed.),
Current Protocols in Molecular Biology
, Ed. Current Protocols, Wiley, N.Y., 1994, Chapter 10).
Capillary electrophoresis offers some advantages not available with other separation methods, such as slab gel separation, HPLC, or column chromatography (Krylov, S. N., and Dovichi, N. J. Capillary electrophoresis for the analyses of biopolymers.
Analytical Chemistry,
2000, Vol. 72, No. 12:111R-128R). The major advantage of the capillary electrophoresis is the speed of the analysis (few minutes, compared to few hours by other methods). Highly efficient dissipation of electro-resistive generated heat in the capillary is provided by a large surface to volume ratio. Decoupling of gel temperature from electro-resistive generated energy provides greater effective field strength. Migration rate varies directly with the field strength over a linear range, which can be extended using gel-filled capillaries. Thus, separations at higher fields can be performed with reduced running time. The advantage of a single capillary can be further extended by use of an array of coupled capillaries with equal characteristics. A possibility to process them together under identical conditions allows technology for protein separation and DNA sequencing (Dolnik V. DNA sequencing by capillary electrophoresis.
J Biochem. and Biophys. Methods.
1999, V.41, No. 2-3:103-119).
Collection of samples in capillary electrophoresis poses, however, substantial technical problems (Altria, K. D. Overview of capillary electrophoresis and capillary chromatography.
J. Chromatography,
1999, Vol. 286, No 1-2:443-63; Swinney, K., Bornhop, D. J. Detection in capillary electrophoresis.
Electrophoresis,
2000, Vol. 21, no. 7:1239-50). Samples are eluted from the capillary at a certain point by either pressurized flow or electroelution. Method for sample collection by electroelution employs standard capillary electrophoretic equipment. Several parameters of the system must be precisely controlled (velocity of migration of a sample, distance between the detection point and the end of the capillary, etc). To collect fractions in appropriate vials, capillaries, etc, one must know the exact time, when a zone appears at the exit end of the capillary, as the variation of migration rates in capillary electrophoresis can be more than 2%. The time necessary for the zone of interest to move through the distance between the detection point and the capillary exit is calculated after detection of the zone of interest is done. Electric current is then turned off, and the capillary is removed from the apparatus and placed into a collection vial. Current is applied for a predetermined time so that the zone migrates into the collection reservoir with buffer from the capillary. Pressure can also be applied to remove the sample from the capillary. After collection of the zone is accomplished, the capillary is placed back into the electrophoretic device, and separation continues. A set of collection reservoirs, or capillaries, containing a collection buffer
Nguyen Nam
Noguerola Alex
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