Promega Corporation

Diana L Holmes

Daniel J Simpson

Rex M Bitner

Craig E Smith

Josephine C Grosch

Jehoshua Katzenhendler

Craig Smith

Jehoshua Katzhendler

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

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.

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Crystal Lake, IL, United States of America

Diana L Holmes