Method of plasmid DNA production and purification
First Claim
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1. A method for isolation and purification of plasmid DNA, comprising the steps of:
- a) determining the irreversible alkaline denaturation value of said plasmid DNA;
b) growing a culture of bacterial host cells to a cell density within the range of about 2 g to about 200 g per liter dry weight units, wherein said host cells contain plasmid DNA having an irreversible alkaline denaturation value;
c) lysing said bacterial cells by contacting said culture during exponential growth with an amount of alkali sufficient to reach a pH value that is about 0.1 pH unit to about 0.2 pH unit below the irreversible alkaline denaturation value of said plasmid DNA; and
d) isolating a said plasmid DNA preparation.
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Abstract
A scalable method for the production of highly purified plasmid DNA in Escherichia coli is described, which method includes growing plasmid-containing cells to a high biomass in exponential growth and lysing the cells by raising the pH of the culture to a carefully controlled pH value in which chromosomal DNA is denatured but plasmid DNA is reversibly renatured. The method has been developed for the production of pharmaceutical grade DNA for use in in vivo and ex vivo gene therapy.
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36 Claims
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1. A method for isolation and purification of plasmid DNA, comprising the steps of:
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a) determining the irreversible alkaline denaturation value of said plasmid DNA; b) growing a culture of bacterial host cells to a cell density within the range of about 2 g to about 200 g per liter dry weight units, wherein said host cells contain plasmid DNA having an irreversible alkaline denaturation value; c) lysing said bacterial cells by contacting said culture during exponential growth with an amount of alkali sufficient to reach a pH value that is about 0.1 pH unit to about 0.2 pH unit below the irreversible alkaline denaturation value of said plasmid DNA; and d) isolating a said plasmid DNA preparation. - View Dependent Claims (2, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)
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3. A method for determining the optimum lysis conditions for lysing host cells containing plasmid DNA, comprising the steps of:
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a) growing a culture of bacterial host cells to a cell density within the range of about 2 g to about 200 g per liter dry weight units; b) lysing said bacterial cells during exponential growth at a pH of said culture sufficient to cause cell lysis and to cause denaturation of no greater than 50% of plasmid DNA contained in said cells; and c) selecting a pH value for optimum lysis conditions which is about 0.1 pH units below said pH of step b). - View Dependent Claims (4)
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Specification