Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-01-30
2001-08-14
Wilson, James O. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S025410, C422S091000, C203S001000, C203S002000, C203S034000, C203S035000, C203S100000, C203S100000, C203S014000, C203S014000, C203S014000
Reexamination Certificate
active
06274726
ABSTRACT:
FIELD OF THE INVENTION
The invention is in the general field of methods and devices for isolating and purifying compounds from mixtures.
BACKGROUND OF THE INVENTION
Many methods for separating biomolecules from mixtures such as cell lysates or synthetic preparations are based on a procedure in which the sample is loaded onto a column packed with a solid phase.
In the case of nucleic acids, for example, the solid phase can include an anion-exchange medium or resin. The negatively charged, anionic phosphate backbone of a nucleic acid can bind to and is thereby effectively immobilized by the resin. The resin can be washed with a low salt solution (e.g., 0.2 M sodium chloride), which flushes away the neutral, cationic, and less highly charged anionic components of the original mixture without substantially disrupting the binding of the nucleic acid molecules to the solid phase.
A high salt buffer solution (e.g., a buffer containing 1 M sodium chloride) is then used to elute the nucleic acid molecules away from the solid phase. The high salt concentration, however, can interfere with mass spectroscopy, electrophoresis, and many downstream enzymatic processes commonly employed in the laboratory or clinic, for example, for diagnostics, forensics, or genomic analysis. It is therefore necessary, in many cases, to remove at least some of the salt from the nucleic acid in an additional, frequently time-consuming step. Desalting can be accomplished by any of several procedures, including ethanol precipitation, dialysis, and purification from glass or silica beads or resin. In some cases it may also be necessary to add nuclease inhibitors to the wash and buffer solutions to prevent degradation of the nucleic acid.
SUMMARY OF THE INVENTION
The invention is based on the discovery that hyperbaric, hydrostatic pressure reversibly alters the partitioning of biomolecules between certain adsorbed and solvated phases relative to partitioning at ambient pressure. The new methods and devices disclosed herein make use of this discovery for highly selective and efficient, low salt isolation and purification of nucleic acids from a broad range of sample types, including forensic samples, blood and other body fluids, and cultured cells.
In one embodiment, the invention features a pressure-modulation apparatus. The apparatus includes an electrode array system having at least two (i.e., two, three, four, or more) electrodes; and a conduit interconnecting the electrodes. The conduit contains an electrically conductive fluid in contact with a phase positioned in a pressure chamber. The phase can be, for example, a binding medium or stationary phase. It can be a gel (e.g., a pressure-sensitive gel), a resin (e.g., an ion-exchange resin, a hydrophobic resin, a reversed phase resin, or a size exclusion resin), a plastic, a glass, hydroxyapatite, an immobilized oligonucleotide, a silica, an ion-exchange material, silicon or other metal, an alumina, a zeolite, a cellulose, a particle, a microparticle, a nanoparticle, a coating on a substrate, a soluble polymer, a micelle, a liposome, a porous solid medium, a membrane, a pressure-stable medium (e.g., DEAE-coated glass, quartz, thermoplastic polymer, gel, or a non-porous resin made up of 1 to 50 &mgr;m beads with positively charged surface), or a phase of a phase-separated liquid. The electrodes can have a protective coating (e.g., of polyacrylamide gel.
The apparatus can also include at least one (i.e., one, two, three, four, or more) reservoir in communication with the conduit to contain materials transported by the conduit. The reservoir can also be positioned in the pressure chamber. The conduit can be, for example, an electrically non-conducting tube. The apparatus can also include a pressure-transmitting apparatus (e.g., an electrically mediated pressure actuator, such as an electrostrictive apparatus, magnetostrictive apparatus, or a shape-memory alloy device) that can transmit pressure to or from the pressure chamber. If there are three electrodes or more, the electrodes can be configured in a straight line or can alternatively define two or more (i.e., two, three, four, or more) axes. The conduit can include an electrophoretic or electroosmotic capillary. The electrode array system can be configured on a microchip.
The invention also features a method for purifying nucleic acids from a sample. The method includes the steps of contacting the sample with the phase of the aforementioned apparatuses at an initial pressure (i.e., where the phase is a phase that non-specifically binds to nucleic acids with greater affinity than it does to non-nucleic acid components of the sample); transporting (e.g., electrophoretically or electroosmotically) at least some of the non-nucleic acid components (e.g., towards one electrode, or away from the nucleic acids); modifying the pressure to a level sufficient to disrupt the binding of the nucleic acids to the phase; and transporting (e.g., electrophoretically or electroosmotically) the nucleic acids (e.g., towards a second electrode, or away from the phase).
In another embodiment, the invention features another method for isolating and purifying nucleic acids from a sample. The method includes the steps of applying the sample to a phase at an initial pressure (i.e., where the phase non-specifically binds to nucleic acids with greater affinity than it does to non-nucleic acid components of the sample); spatially separating (e.g., by electrophoresis, electroosmosis, or fluid flow) at least some of the non-nucleic acid components from the phase and the nucleic acids; modifying the pressure to a level sufficient to disrupt the binding of at least some of the nucleic acids to the phase; and spatially separating the nucleic acids from the phase at the modified pressure. The “applying” and first “spatially separating” steps, at least, are carried out within a single reaction vessel (e.g., a pressure modulation apparatus, or a pressurized vessel).
The first “spatially separating” step can include transporting the non-nucleic acid components into a reservoir. The reservoir can optionally include binding materials such as ion-exchange materials, desalting (mixed ion-exchange) resin, nonspecific affinity resin, polystyrene resin, gamma-irradiated polystyrene resin, a covalent attachment resin (e.g., an aldehyde-rich surface, a carbodiimide-rich surface, an o-methylisourea-rich surface, an amidine-rich surface, a dicarbonyl-rich surface, a hydrazide-rich surface, or a thiol-rich surface), a resin or combination of resins possessing different binding functionalities, or a hydrophobic material; alternatively, an anion-exchange material can be placed at one or more electrodes of positive potential or a cation-exchange material can be placed at one or more electrodes of negative potential.
The initial pressure can be, for example, ambient pressure and the modified pressure can be an elevated pressure (e.g., 100 to 200,000 psi, 500 to 100,000 psi, 1,000 to 50,000 psi, or 2,000 to 25,000 psi).
In some instances, the sample can include cells; the method would then also include subjecting the sample to a hyperbaric pressure sufficient to lyse the cells. The cells can include both external and nuclear membranes, and the hyperbaric pressure can be sufficient to lyse both membranes, or alternatively, only to lyse the external membrane, not the nuclear membranes.
The sample can also include nucleic acid-binding proteins (e.g., nuclease enzymes); the method can thus also include subjecting the sample to a hyperbaric pressure sufficient to inactivate the nucleic acid-binding proteins.
The sample can include various sizes of nucleic acids; the modified pressure level can, for example, be sufficient only to disrupt the binding of relatively small nucleic acids to the phase. To disrupt the binding of larger nucleic acids, and the method also includes the steps of further modifying the pressure to a level sufficient to disrupt the binding of the relatively larger nucleic acids to the phase; and spatially separating the nucleic acids from the phase at
Hess Robert A.
Laugharn, Jr. James A.
Tao Feng
BBI BioSeq, Inc.
Fish & Richardson P.C.
Wilson James O.
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