Isolation of DNA molecules
View Patent ↗The present invention relates to a process for separating nucleic acid molecules, preferably open circular and supercoiled plasmid DNA and RNA molecules from each other, comprising the steps of providing a solution comprising the molecules; adsorbing the molecules to adsorbing groups on a carrier; and optionally washing the column with a suitable solution. The present process is especially suitable for large-scale isolation of supercoiled ccc DNA to be used in gene therapy.
1. A process for separating nucleic acid molecules form a solution, comprising the steps of:
(a) subjecting a mixture of nucleic acid molecules to a matrix of a carrier surface with an S-aryl ligand containing a pyridyl moiety attached thereto, such that the nucleic acids are adsorbed onto said S-aryl ligand containing a pyridyl moiety;
(b) subjecting the nucleic acid molecules to an elution step;
(c) isolating one or more different fractions containing the different nucleic acid molecules; and
(d) optionally washing the carrier with a washing solution wherein the nucleic acid molecules include open circle plasmid DNA, closed circle complex plasmid DNA, genomic DNA and RNA.
2. The process of claim 1 , further comprising an initial step of lysing cells which express the nucleic acid molecules to provide the solution comprising nucleic acid molecules.
3. The process of claim 1 , which further comprises a step of eluting the nucleic acid molecules by contacting with an eluent.
4. The process of claim 1 , wherein the carrier is an inorganic matrix.
5. The process of claim 4 , wherein the carrier is glass.
6. The process of claim 4 , wherein the carrier is a zeolite.
7. The process of claim 4 , wherein the carrier is a silica.
8. The process of claim 1 , wherein the carrier is an organic matrix.
9. The process of claim 8 , wherein the carrier is a resin or polymer.
10. The process of claim 9 , wherein the carrier is selected from the group consisting of agarose, cross-linked agarose, dextran, polystyrene/divinylbenzene, coated polystyrene, acrylic polymer, dextran acrylic polymer, vinylic grafted polymer and vinylic polymer.
11. The process of claim 9 , wherein the carrier includes one or more silica based resins in the form of silica-dextran, silica-acrylic polymer or silica-polyethyleneimine.
12. The process of claim 1 , wherein the carrier is present as a membrane which comes in contact with the nucleic acid molecule mixture.
13. The process of claim 1 , wherein the carrier is present as a chromatography matrix in a column through which the nucleic acid molecule mixture is passed.
14. The process of claim 1 , wherein the S-aryl ligand is a pyridyl-S ligand having the S-moiety bound in 2 (ortho) or 3 (meta) position to the pyridyl nitrogen atom.
15. The process of claim 14 , wherein the S-aryl ligand is separated from the pyridyl moiety via an alkylene chain having 1 to 7 carbon atoms.