Promega Corporation

Pierce Biotechnology, Inc.

Craig E Smith

Rex M Bitner

Josephine C Grosch

Daniel J Simpson

Diana L Holmes

Jehoshua Katzenhendler

Braeden L Butler

Douglas H White

Charles K York

Jacqui Sankbeil

Steven Jerome Moore

John William Shultz

Surbhi Desai

Boguslawa Dworecki

Daniel Kephart

Steven J Ekenberg

Douglas R Storts

Susanne Selman

Paula R Brisco

Scott A Lesley

Jacqueline Sankbeil

Susan C Koller

Allan Tereba

Donald A Creswell

Judy Frederiksen

Judy Frederiksen Towne

A method for detecting a target biomolecule directly in a polyacrylamide gel in which it has been separated from other substances. Prior to binding the target to a probe and while the target biomolecule remains in the gel, the gel is immersed in a water miscible aqueous extracting medium containing a base to shrink the gel by at least about ten percent, and then the gel is washed with water to restore the gel to substantially its original size.

Direct detection of biomolecules in a gel

Methods are disclosed for using paramagnetic particles to concentrate or harvest cells. Methods are also disclosed for clearing a solution of disrupted biological material, such as a lysate of cells or a homogenate of mammalian tissue. Methods are also disclosed for using paramagnetic particles to isolate target nucleic acids, such as RNA or DNA, from a solution cleared of disrupted biological material using the same type or a different type of paramagnetic particle. Kits are also disclosed for use with the various methods of the present invention. Nucleic acids isolated according to the present methods and using the present kits are suitable for immediate use in downstream processing, without further purification.

Cell concentration and lysate clearance using paramagnetic particles

Disclosed are pH dependent ion exchange matrices, methods of making, and methods of use.

pH dependent ion exchange matrix and method of use in the isolation of nucleic acids

The present invention provides methods for isolating a defined quantity of DNA target material from other substances in a medium. The method may be carried out using a known quantity of a silica-containing solid support, such as silica magnetic particles, having a definable capacity for reversibly binding DNA target material, and DNA target material in excess of the binding capacity of the particles. The methods of the present invention involve forming a complex of the silica magnetic particles and the DNA target material in a mixture of the medium and particles, and separating the complex from the mixture using external magnetic force. The DNA target material may then be eluted from the complex. The quantity of DNA target material eluted may be determined based on a calibration model. The methods of the present invention permit isolation of DNA target material which is within a known quantity range. The methods of the invention eliminate the step of quantitating purified biological samples prior to further processing, such as amplification, Short Tandem Repeat (STR) analysis, and DNA sequencing. Samples of the DNA target materials may be obtained from liquid or solid media, such as liquid blood or paper.

Simultaneous isolation and quantitation of DNA

Mixed-bed solid phases are provided, with methods for using such solid phases to isolate target nucleic acids, such as plasmid DNA, chromosomal DNA, RNA, or nucleic acids generated by enzymatic amplification from contaminants, including proteins, lipids, cellular debris, or other nucleic acids. The mixed-bed solid phases of this invention are mixtures of at least two different solid phases, each of which has a capacity to bind to the target nucleic acid under different solution conditions, and the capacity to release the nucleic acid under similar elution conditions. By exchanging solution conditions according to the methods of this invention, one can remove contaminants from the target nucleic acid bound to the mixed-bed solid phase, then elute the target nucleic acid in an elution buffer.

Mixed-bed solid phase and its use in the isolation of nucleic acids

The present invention provides methods for isolating biological target materials, particularly nucleic acids, such as DNA or RNA or hybrid molecules of DNA and RNA, from other substances in a medium using silica magnetic particles. The methods of the present invention involve forming a complex of the silica magnetic particles and the biological target material in a mixture of the medium and particles, separating the complex from the mixture using external magnetic force, and eluting the biological target material from the complex. The preferred embodiments of magnetic silica particles used in the methods and kits of the present invention are capable of forming a complex with at least 2 &mgr;g of biological target material per milligram of particle, and of releasing at least 60% of the material from the complex in the elution step of the method. The methods of the present invention produce isolated biological target material which is substantially free of contaminants, such as metals or macromolecular substances, which can interfere with further processing or analysis, if present.

Kits for isolating biological target materials using silica magnetic particles

pH dependent ion exchange matrices are provided, with methods for making such matrices, and methods for using such matrices to isolate a target nucleic acid, such as plasmid DNA, chromosomal DNA, or RNA from contaminants, including proteins, lipids, cellular debris, or other nucleic acids. Each pH dependent ion exchange matrix of this invention comprises at least two different ion exchange functional groups, one of which is capable of acting as an anion exchanger at a first pH, and the other of which is capable of acting as a cation exchanger at a second, higher pH. The matrix has an overall neutral charge in a pH range between the first and second pH. The pH dependent ion exchange matrices of the present invention are designed to bind to the target nucleic acid at a pH wherein the overall charge of the matrix is positive, and to release the target nucleic acid as the pH of the surrounding solution is increased. The target nucleic acid can be released from the pH dependent matrix in little or no salt and at about a neutral pH. The matrices and methods of this invention enable one to isolate a target nucleic acid in very few steps, without the use of hazardous chemicals. Target nucleic acids isolated using the pH dependent ion exchange matrices according to the present invention can be used immediately without further extraction or isolation.

Kits for cell concentration and lysate clearance using paramagnetic particles

The present invention presents a novel method for removing endotoxins from nucleic acids, such as DNA, RNA, or hybrids thereof, contaminated therewith. Nucleic acid solutions which can be treated using the method of this invention include, but are not limited to, lysates of gram-negative bacteria and nucleic acid solutions contaminated with endotoxins from external sources. The present method removes endotoxins from such solutions using silica-based materials, such as silica gel particles, magnetic silica particles, or diatomaceous earth. In a preferred aspect of the method of this invention, magnetic silica particles are used to isolate plasmid DNA from a lysate of gram-negative bacteria transformed with the plasmid DNA. Application of the disclosed method produces nucleic acids which are sufficiently free of endotoxin contamination to be useful for a variety of different practical applications.

Endotoxin reduction in nucleic acid purification

The present invention provides methods for isolating biological target materials, particularly nucleic acids, such as DNA or RNA or hybrid molecules of DNA and RNA, from other substances in a medium using silica magnetic particles. The methods of the present invention involve forming a complex of the silica magnetic particles and the biological target material in a mixture of the medium and particles, separating the complex from the mixture using external magnetic force, and eluting the biological target material from the complex. The preferred embodiments of magnetic silica particles used in the methods and kits of the present invention are capable of forming a complex with at least 2 .mu.g of biological target material per milligram of particle, and of releasing at least 60% of the material from the complex in the elution step of the method. The methods of the present invention produce isolated biological target material which is substantially free of contaminants, such as metals or macromolecular substances, which can interfere with further processing or analysis, if present.

Methods of isolating biological target materials using silica magnetic

Solutions containing nucleic acids are treated with an alkaline protease to digest proteins such as nucleases that degrade the nucleic acids. In the isolation of nucleic acids, a biological sample containing nucleic acids is suspended in a solution containing water, buffer and chelating agent, the pH of the solution is adjusted to at least about 10 by adding a solution of sodium hydroxide and anionic detergent, an alkaline protease is incubated in the solution until nucleases are degraded, the pH of the solution is lowered to reduce activity of the alkaline protease by adding a solution having a pH between 3.5 and 4.5 and the alkaline protease is heat inactivated. Lowering of the pH may produce a cloudy solution which is cleared by centrifuging. Nucleic acids are isolated from the cleared solution by alcohol precipitation, or by using paramagnetic particles or a resin matrix containing silica particles. A chaotropic salt can be used to reversibly bind DNA to the resin matrix.

Growing community of inventors

Oregon, WI, United States of America

Craig E Smith