Method for purifying covalently closed circular DNA

US6242220A · kind A · utility

Assignee

• QIAGEN GmbH · DE

Inventors

• Stephan Wahle · Neunburg vorm Wald, DE
• Jaochim Schorr · Hilden, DE
• Martin Weber · Borgo San Lazzaro, DE

Key dates

• Filing date: Sep 9, 1999
• Grant date: Jun 5, 2001
• Expiry date: Sep 9, 2019

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12N15/1003
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

The present invention relates to a method of producing covalently closed circular (ccc) DNA from a cultured cell or unicellular organism, preferably a bacterium, essentially free of genomic DNA. The method of the invention comprises the steps of precipitating a cleared lysate with an alcohol, washing and resuspension of the precipitate, digestion of the genomic DNA with a nuclease that cleaves linear DNA or circular DNA that includes a nick or a free 3' or 5' end but not ccc DNA, and finally, separation of the purified ccc DNA from the remainder of the product of the digestion step by contacting the product with an ion exchange material. ccc DNA obtained by the method, pharmaceutical compositions comprising the ccc DNA, and a kit for carrying out the method of the invention are also contemplated.