Northeastern University

Hybridon, Inc.

Massachusetts Institute of Technology

Aharon S Cohen

Barry L Karger

Maria Vilenchik

Alexei Belenky

Andre Bourque

Roger W Giese

William G Thilly

Frantisek Foret

Robert J Nelson

Katalin Ganzler

Christopher M Ott

Aran Paulus

Phouthone Koehavong

David N Heiger

Konstaintin Khrapko

A capillary column for high performance electrophoretic separation and detection of SDS proteins and system for using the same is disclosed. A preferred column includes a capillary, a static or dynamic layer of coating material on the inner surface of the capillary wall, and a hydrophilic polymer network which is UV-transparent in the tube, filling it. The capillary column is particularly useful in any electrophoresis system requiring UV-transparent materials.

Capillary column for high performance electrophoretic separation and

Disclosed is a substrate useful for separating unmodified and modified mononucleotides and oligonucleotides. The substrate includes at least 12% polymer in at least 50% (volume:volume) organic solvent, the organic solvent being a denaturing agent. This substrate is easily removable from the capillary using low pressure. Also provided is a method of separating unmodified and modified mononucleotides and oligonucleotides utilizing this substrate.

Substrate useful for separating modified oligonucleotides

Disclosed is a method of detecting a single-stranded target oligonucleotide in a biological fluid. In this method, a biological fluid sample to-be-tested is contacted with helper and primer oligonucleotides, thereby forming a labelled, double-stranded molecule if the sample contains an oligonucleotide which is complementary to the nucleotide sequences of the primer and the helper oligonucleotides. The primer so annealed to the target oligonucleotide is then ligated to the helper annealed to the target oligonucleotide. The presence of the ligation product is being indicative of the presence of the target oligonucleotide in the biological fluid.

Method of detecting sub-ppb levels of oligonucleotides in biological

The disclosure relates to a method for resolving double-stranded DNA species differing by at least one base pair. Each of the species is characterized by an iso-melting domain with a unique melting temperature contiguous with a melting domain of higher thermal stability.

Electrophoretic detection and separation of mutant DNA using replaceable

Disclosed is a substrate useful for separating unmodified and modified mononucleotides and oligonucleotides. The substrate includes at least 12% (weight:volume) polymer in at least 5M urea and at least 32% (volume:volume) organic solvent, the organic solvent being a chemically stable liquid at room temperature and having a dielectric constant of at least 20. Also provided is a method of separating unmodified and modified mononucleotides and/or oligonucleotides utilizing this substrate.

Method for analyzing oligonucleotide analogs

Disclosed is a method for determining the nucleotide sequence of a target oligonucleotide. In this method a single-stranded ligation product is prepared which contains a target oligonucleotide-to-be sequenced and an auxiliary oligonucleotide. A primer complementary to a portion of the auxiliary oligonucleotide and having a label covalently attached thereto is annealed to the auxiliary oligonucleotide portion of the ligation product. The primer is extended with chain-extending nucleoside triphosphates and chain-terminating nucleoside triphosphates in the presence of a polymerase to yield a plurality of primer extension products. These extension products are then separated on the basis of their base length; and the nucleoside sequence of the target oligonucleotide is determined from the mobilities of the primer extension products obtained during their separation.

Method of sequencing [short] oligonucleotides

Disclosed is a method for detecting and quantitating oligonucleotides with charged internucleotide linkages in biological fluids. In this method, a biological fluid sample is contacted with an anion exchange resin at from 40.degree. C. to 65.degree. C. for a time sufficient to enable oligonucleotides in the sample to adsorb to the resin. The absorbed oligonucleotides are then desorbed with a buffer having a salt concentration of about 1 M to 2.5 M and a pH in the range of about 6.5 to 7.5, the desorption being performed at about 40.degree.-65.degree. C. The oligonucleotides so released are then detected and quantitated.

Method for detecting charged oligonucleotides in biological fluids

A method for separating a mixture of 5'-phosphorothioate mononucleotides, a mixture of 5'-phosphorothioate oligonucleotides, or a mixture of both 5'-phosphorothioate mononucleotides and 5'-phosphorothioate oligonucleotides comprising electro- phoresis through a gel of at least 12% polymerized linear acrylamide containing at least 50% formamide or other denaturing organic solvent. This method can resolve phosphorothioate oligonucleotides differing by only a single base. Samples of less than 1 nanogram in microliter volumes can be conveniently handled.

Separation of phosphorothioate oligonucleotides by capillary gel

Apparatus and method for conducting electrophoresis in a capillary tube under the influence of a pulsed electric field. Apparatus for separating and detecting molecular species of a sample in a conductive medium includes a capillary tube for containing conductive medium, means for introducing a sample into a conductive medium-filled capillary tube, means for applying a pulsed electric field across the conductive medium-filled capillary tube between its ends, and means for detecting constituent molecular species of a sample in a conductive medium-filled capillary tube as they traverse the tube. A method for separating and detecting molecular species of a sample in a conductive medium includes the steps of introducing the sample into a capillary tube filled with conductive medium, applying a pulsed electric field of up to 500 volts/centimeter between the ends of the capillary, and detecting the constituent molecular species of the sample as they traverse the capillary.

Pulsed field capillary electrophoresis

A microcapillary column for high performance electrophoresis. A preferred column includes a microcapillary, a thin layer of coating material covalently bonded to the inner surface of the microcapillary wall, and a gel comprising polyacrylamide polymerized in the tube, filling it. The gel-containing microcapillary is prepared by covalently bonding a layer of a suitable coating material to the inner surface of the microcapillary wall, and then causing a mixture of monomer with or without crosslinking agent, initiator, and polymerization catalyst to react in the bore of the microcapillary to form a polymeric matrix. In electrophoresis, the gel-containing microcapillary provides peak efficiencies in excess of 100,000 theoretical plates and in some instances over 1,000,000 theoretical plates within separation times of less than thirty minutes, permits trace level determinations of molecular weights, and permits electrophoretic operation at fields of 300 V/cm or higher, resulting in extremely high resolution separations.

High performance microcapillary gel electrophoresis

Selective high performance electrokinetic separations employing the

A microcapillary column for high performance electrophoresis. A preferred column includes a microcapillary, a thin layer of coating material covalently bonded to the inner surface of the microcapillary wall, a thin layer of a hydrophilic polymer absorbed on the layer of coating material, and a gel comprising polyacrylamide polymerized in the tube, filling it. The gel-containing microcapillary is prepared by covalently bonding a layer of a suitable coating material to the inner surface of the microcapillary wall, applying a layer of hydrophilic polymer, and then causing a mixture of monomer with or without crosslinking agent, initiator, and polymerization catalyst to react in the bore of the microcapillary to form a polymeric matrix. In electrophoresis, the gel-containing microcapillary provides peak efficiencies in excess of 100,000 theoretical plates and in some instances over 1,000,000 theoretical plates within separation times of less than thirty minutes, permits trace level determinations of molecular weights, and permits electrophoretic operation at fields of 300 V/cm or higher, resulting in extremely high resolution separations.

An integrated temperature control/alignment system for use with a high performance capillary electrophoretic apparatus is disclosed. In one exemplary embodiment the integrated temperature control/alignment system comprises a pair of capillary column mounting plates for mounting a capillary column as a constituent of the electrophoretic apparatus, a pair of secondary support plates, a pair of thermoelectric plates and external heat sink plates. Detection openings are formed in each of the plates as required so that a continuous detection path exists through the integrated temperature control/alignment system. The capillary column is seated and locked in grooves formed in the capillary column mounting plates so that the detection windows of the capillary column are coordinated with the aligned detection openings formed in the applicable plates of the integrated temperature control/alignment system. The temperature of substantially the entire capillary column, including the detection zone, is controlled or regulated by the temperatures of the thermoelectric plates which comprise one or more thermoelectric circuits utilizing the Peltier effect. Regulation of current flow in the thermoelectric plates controls the temperature effect exerted by the thermoelectric plates, thereby controlling both the direction and rate of heat transfer with the capillary column.

Integrated temperature control/alignment system for high performance

An improved microcapillary column for high performance electrophoresis includes a microcapillary, a hydrophilic polymer within a gel of crosslinked polyacrylamide polymerized in the tube, and preferably, a thin layer of connecting material covalently bonded to the inner surface of the microcapillary wall and to the polymeric gel. The microcapillary is prepared by first covalently bonding a suitable bifunctional reagent to the inner surface of the microcapillary wall, and then causing a mixture of the hydrophilic polymer, monomer, crosslinking agent, and polymerization catalyst to react in the bore of the microcapillary to form a hydrophilic polymer-containing gel matrix which is covalently bonded to the microcapillary wall via the bifunctional reagent. In electrophoresis, this improved gel-containing microcapillary can provide peak efficiencies in excess of 100,000 theoretical plates within separation times of less than thirty minutes, permits trace level determinations of molecular weights, and permits electrophoretic operation at fields of 1000 V/cm or higher, resulting in extremely high resolution separations.

Capillary gel electrophoresis columns

A microcapillary column for high performance electrophoresis includes a fused silica microcapillary, a gel of crosslinked polyacrylamide polymerized in the tube, and a thin layer of connecting material covalently bonded to the inner surface of the microcapillary wall and to the polymeric gel. The gel-containing microcapillary is prepared by first covalently bonding a suitable bifunctional reagent to the inner surface of the microcapillary wall, and then causing a mixture of monomer, crosslinking agent, and polymerization catalyst to react in the bore of the microcapillary to form a polymeric matrix which is covalently bonded to the microcapillary wall via the bifunctional reagent. In electrophoresis, the gel-containing microcapillary provides peak efficiencies in excess of 100,000 theoretical plates within separation times of less than thirty minutes, and permits trace level determinations of molecular weights.

Growing community of inventors

Brookline, MA, United States of America

Aharon S Cohen