Promega Corporation

Rex M Bitner

Craig E Smith

Daniel J Simpson

Josephine C Grosch

Jehoshua Katzenhendler

Diana L Holmes

Braeden L Butler

Douglas H White

Paula R Brisco

Susan C Koller

Jacqui Sankbeil

Michelle N Mandrekar

Steven J Ekenberg

Daniel Kephart

Roderick G Flemming

Scott A Lesley

Jacqueline Sankbeil

Craig Smith

Allan Tereba

Michelle M Mandreker

Donald A Creswell

Jehoshua Katzhendler

The present invention relates to methods, kits, and compositions for generating purified RNA samples and purified DNA samples. In particular, the present invention provides methods for generating a purified RNA or DNA sample from a sample containing both DNA and RNA molecules using a binding matrix that preferentially binds DNA or RNA in the presence of an acidic dilution buffer, or using a binding matrix that comprises acid zeolites, as well as compositions and kits for practicing such methods.

Nucleic acid purification with a binding matrix

A formulation containing guanidine thiocyanate or guanidine hydrochloride together with acetamide, one or more acetamide derivatives, or a combination of acetamide and one or more acetamide derivatives is provided with methods to use the formulation to purify one or more nucleic acids contained in a medium.

Methods of purifying a nucleic acid and formulation and kit for use in performing such methods

A method and kit which allow the use of a discrete amount of a binding matrix to first purify nucleic acids from a medium under a first set of binding conditions wherein the amount of nucleic acid bound to the binding matrix is essentially independent of the amount of surface area of the definable amount of the binding matrix, followed by a second purification step wherein the nucleic acids are bound to a discrete amount of binding matrix under a second set of binding conditions wherein the amount of nucleic acid bound to the binding matrix is essentially dependent on the amount of surface area of the definable amount of the binding matrix, thus providing a discrete quantity of nucleic acid.

Methods of optimal purification of nucleic acids and kit for use in performing such methods

A formulation containing guanidine thiocyanate together with acetamide, one or more acetamide derivatives, or a combination of acetamide and one or more acetamide derivatives is used to purify one or more nucleic acids contained in a medium. In particular, a medium containing at least one nucleic acid is combined with a binding matrix and the formulation in order to cause the at least one nucleic acid to separate from its in vivo cellular environment and to bind to the binding matrix. The binding matrix with at least one nucleic acid bound thereto then is separated from substantially the rest of the combined medium and formulation, after which the at least one nucleic acid is eluted from the binding matrix to obtain the at least one nucleic acid in a substantially purified form. If different nucleic acids are to be selectively purified from a single medium, multiple binding matrices, each compatible with a different nucleic acid, can be used.

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

A method is provided for using a silanized silica matrix to isolate a target nucleic acids, such as plasmid DNA, fragments of DNA, chromosomal DNA, or RNA from contaminants, including proteins, lipids, cellular debris, or non-target nucleic acids. The silanized silica matrix comprises a silica based solid phase and a plurality of silane ligands covalently attached to the surface of the solid phase. Non-target material adsorbs to the silanized silica matrix in the presence of a sufficient concentration of chaotropic salt, while target nucleic acids adsorb to the matrix under other solution conditions. The method of using the silanized silica matrix of the present invention can be used to clear solutions of disrupted biological material, and to isolate nucleic acids therefrom or from other solutions containing nucleic acids and at least one contaminant.

Lysate clearance and nucleic acid isolation using silanized silica matrices

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

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.

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Cedarburg, WI, United States of America

Rex M Bitner