Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-07-29
2001-07-31
Brusca, John S. (Department: 1631)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S026430
Reexamination Certificate
active
06268492
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to biotechnology, and more specifically, to a method for purifying non-chromosomal nucleic acid molecules from a cell lysate.
BACKGROUND OF THE INVENTION
Since the discovery of the structure of nucleic acid molecules (DNA and RNA) in the 1950's, research into the structure, function, and use of these molecules has increased dramatically. In particular, a tremendous amount of research has been undertaken in order to utilize these molecules to diagnose and treat disease. In order however to obtain sufficient quantities of nucleic acids for use in experiments, and for diagnosis and treatment of disease, it is first necessary to purify and or isolate substantial quantities of nucleic acid molecules. For example, in the field of gene therapy, patients can be vaccinated or treated with nucleic acid molecules in order to protect against, or, remedy a disease (see, e.g., PCT publication WO 95/07994). However, preparation of large-quantities of purified, pharmaceutical-grade nucleic acid molecules is presently time-consuming and costly.
More specifically, while many methods exist for the purification of nucleic acid molecules, such methods are often limited when the nucleic acid molecules are to be produced for therapeutic purposes, since they must be prepared free of any contaminants such as toxic compounds and antigenic molecules. Prior art methods of DNA purification often utilized highly toxic chemicals such as ethidium bromide, phenol, chloroform, and cesium chloride (Sambrook, et al., 1992, Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring Harbor, N.Y.). Additionally, many methods involving both physical or chemical lysis often relied upon centrifugation to remove cellular debris, a step which is undesirable because of the difficulty in maintaining sterility. Furthermore, procedures using alkali lysis (Bimboim and Doly,
Nucleic Acids Res.
7;1513, 1979) may lead to the loss of a significant amount of DNA through degradation caused by the increase in pH.
The present invention provides methods for producing purified nucleic acid molecules that are suitable for use in pharmaceutical applications, and on a large scale. Such methods do not require traditional techniques such as toxic extractants, mutagenic reagents, or steps such as centifugation. The present invention also provides other, related advantages.
SUMMARY OF THE INVENTION
Briefly stated, the present invention provides methods for purifying non-chromosomal nucleic acid molecules from cells, comprising the general steps of (a) lysing cells to form a nucleic acid-molecule containing lysate, (b) precipitating chromosomal DNA and cell wall components from the lysate, in order to form a flocculent solution, and (c) applying the flocculent solution to a depth filter, such that non-chromosomal nucleic acid molecules are purified.
Within further embodiments, the non-chromosomal nucleic acid molecules can be further isolated by chromatographically separating non-chromosomal nucleic acid molecules (e.g., by ion-exchange chromatography or preparative HPLC) from proteins and other compounds which are in the lysate. Within other embodiments, the clarified solution may be concentrated (e.g., by centrifugation or tangential flow). Within yet other embodiments the purification and/or isolation methods described herein can be performed in a sterile environment and/or in a continuous manner.
The methods provided herein may be utilized to purify and/or isolate a variety of non-chromosomal nucleic acid molecules, including for example plasmids and RNA.
Within certain embodiments of the invention, the depth filter has a nominal pore size of between about 0.2 and 20 microns. Within other embodiments, the non-chromosomal nucleic acid molecules are precipitated utilizing a neutralizing agent such as sodium acetate or potassium acetate.
The methods provided herein may be utilized for purifying and/or isolating non-chromosomal nucleic acid molecules from a wide variety of cells, including for example, bacterial cells, yeast cells, and mammalian cells (i.e., both eukaryotic and prokaryotic cells).
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth herein which describe in more detail certain procedures or compositions (e.g., plasmids, etc.), and are therefore incorporated by reference in their entirety.
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Lacoste-Bourgeacq et al., “A New Procedure Using Membrane Chromatography for the Valorization of Fraction IV from Kistler and Nitschmann's Fractionation of Blood Plasma”, Chromatographia, 1991, vol. 32, No. 1-2, pp. 27-32.*
Van Reis et al., “Industrial Scale Harvest of Proteins from Mammalian Cell Culture by Tangential Flow Filtration; Protein Purification ...”, Biotechnology and Bioengineering, 1991, vol. 38, No. 4, pp. 413-422.*
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Brasch et al, “Isolation and Analysis of Nuclear Bodies from Estrogen-Stimulated Chick Liver”, Experimental Cell Research, 1989, vol. 182, No. 2, pp. 425-435.
Hsu David Chi-Tang
Mittelstaedt Denice M.
Blackburn Robert P.
Brusca John S.
Chiron Corporation
Dollard Anne S.
Kim Young
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