Method for the purification covalently closed circular dna

摘要

The present invention relates to a method of producing covalently closed circular (ccc) DNA from a unicellular organism, preferably a bacterium, essentially free of genomic DNA. The method of the invention comprises the steps of precipitating a cleared lysate with isopropanol and, after washing and resuspension of the precipitate, the digestion of the genomic DNA with a nuclease that cleaves linear DNA or circular DNA that comprises a nick or comprises a free 3'- or 5'-end but not ccc DNA. Finally, the purified ccc DNA is separated from the remainder of the product of the digestion step by contacting said product with an ion exchange material. Preferably, the separation step is a chromatography using an anion exchange column. The invention also relates to purified ccc DNA obtained by the method of the invention, pharmaceutical compositions comprising said ccc DNA, as well as to a kit for carrying out the method of the invention.