Methods for isolating nucleic acids using alkaline protease

US5981235A · kind A · utility

Assignee

• Promega Corporation · US

Inventors

• John Shultz · Verona, US
• Craig E. Smith · Oregon, US
• Douglas R. Storts · Madison, US
• Paula R. Brisco · Oregon, US
• Judy Frederiksen · Oregon, US
• Susanne Selman · Madison, US
• Josephine Grosch · Middleton, US

Key dates

• Filing date: Jul 29, 1996
• Grant date: Nov 9, 1999
• Expiry date: Jul 29, 2016

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12N15/1006
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

Solutions containing nucleic acids are treated with an alkaline protease to digest proteins such as nucleases that degrade the nucleic acids. In the isolation of nucleic acids, a biological sample containing nucleic acids is suspended in a solution containing water, buffer and chelating agent, the pH of the solution is adjusted to at least about 10 by adding a solution of sodium hydroxide and anionic detergent, an alkaline protease is incubated in the solution until nucleases are degraded, the pH of the solution is lowered to reduce activity of the alkaline protease by adding a solution having a pH between 3.5 and 4.5 and the alkaline protease is heat inactivated. Lowering of the pH may produce a cloudy solution which is cleared by centrifuging. Nucleic acids are isolated from the cleared solution by alcohol precipitation, or by using paramagnetic particles or a resin matrix containing silica particles. A chaotropic salt can be used to reversibly bind DNA to the resin matrix.