Genentech, Inc.

Darien L Cohen

Marjorie E Winkler

David W Kahn

Michelle D Butler

A process is described for purifying plasmid DNA from prokaryotic cells comprised thereof. This process comprises the steps of: (a) digesting the cells; (b) incubating the cells for about 4 to 24 hours to effect lysis and solubilization thereof, without effecting enzymatic digestion of RNA; (c) removing lysate contaminants from the cells to provide a plasmid DNA solution; (d) filtering the solution through a tangential flow filtration device to obtain a retentate containing the plasmid DNA; and (e) collecting the retentate.

Purification of plasmid DNA

A process is described for preparing transfectacons, or particles, of calcium phosphate and a desired nucleic acid comprising admixing calcium divalent cation, phosphate multivalent anion, and the desired nucleic acid to form a precipitation mixture, wherein the precipitation mixture comprises an initial phosphate anion concentration of about 0.2 to 0.5 mM; and incubating the precipitation mixture for about 10 to 60 minutes to form transfectacons comprising calcium phosphate and the desired nucleic acid. A process is also provided for delivering desired nucleic acid to eukaryotic tissue or cells comprising introducing to the tissue or cells the transfectacons so prepared. Additionally, a process is disclosed for introducing a desired nucleic acid into a eukaryotic host cell comprising the above two steps, followed by diluting the precipitation mixture and admixing it with a eukaryotic host cell lacking a cell wall to form a transfection mixture; and incubating the transfection mixture to allow the eukaryotic host cell to take up the transfectacons to form a transfected cell.

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Oakland, CA, United States of America

Darien L Cohen