A variety of methods are available for isolating and purifying plasmid DNA. In general, these methods take advantage of the physical differences between chromosomal DNA and plasmid DNA. In terms of size, chromosomal DNA is larger than plasmid DNA. When cells are lysed, the larger chromosomal DNA becomes linearized and entangled in the cellular debris and may be separated from the cell lysate.
Prior art methods of isolating and purifying plasmid DNA include lysis by boiling (Holmes and Quigley, Anal. Biochem. 114, 193 (1981)), lysis with alkali (Birnboim and Doly, Nuc. Acids Res. 7, 1513 (1979)), and lysis with detergent (Godson and Vapnek, Biochem. Biophys. Acta 299, 516 (1973)). PCT publication no. 95/21250 discloses a method of isolating plasmid DNA using detergent in combination with alkali treatment. Prior art methods also use highly toxic chemicals to extract and isolate the plasmid DNA; such as ethidium bromide, cesium chloride, phenol, and chloroform. Moreover, these methods are most effective with smaller plasmids; e.g. plasmids less than approximately 8-10 kb. As plasmid size increases, plasmid DNA isolation becomes more difficult using the existing prior art methods.
In general, methods that employ alkali quickly add a standard amount of sodium hydroxide to the cellular suspension (Birnboim and Doly, supra). The pH of the resulting solution rises rapidly, in some cases, to over 13, which results in much degradation of the plasmid DNA. In addition, the concentration of the cells in suspension is dilute (i.e., of the order of an optical density (OD) of 1-3 units at a wavelength 600 nm for a 1 cm light path) to maximize recovery of plasmid DNA.
An object of the invention is to provide a method of preparing pharmaceutical grade DNA.
Another object of the invention is to provide a plasmid DNA preparation which is substantially free of bacterial host RNA.
Another object of the invention is to provide a plasmid DNA preparation which is substantially free of bacterial host protein.
Another object of the invention is to provide a plasmid DNA preparation which is substantially free of bacterial host chromosomal DNA.
Another object of the invention is to provide a plasmid DNA preparation which is substantially free of bacterial host endotoxins.
Another object of the invention is to provide a method of isolating and purifying relatively large plasmid DNAs.
Another object of the invention is to provide a scalable method of isolating large amounts of plasmid DNA in sufficiently pure form for use in gene therapy.
Another object of the invention is to maximize the yield of plasmid DNA from a host cell/plasmid DNA combination.
USPTO Backgrounds
USPTO Backgrounds