Method for isolating highly pure nucleic acid with magnetic particles

Abstract

Subject of the invention is a method for isolating nucleic acid from a sample solution, wherein the nucleic acid is separated from the sample solution by adsorption to magnetic particles, followed by separation of the magnetic particles with the nucleic acid adsorbed thereto from the sample solution by application of a magnetic field, followed by at least one washing step (w), comprising (w1) suspending the magnetic particles with the nucleic acid adsorbed thereto in a washing solution, which is an aqueous solution, which has a total salt concentration below 100 mM and does not comprise an organic solvent, (w2) incubating the magnetic particles with the nucleic acid adsorbed thereto in the washing solution, and (w3) separating the magnetic particles with the nucleic acid adsorbed thereto from the washing solution. The invention also relates to use of an aqueous solution, which has a total salt concentration below 100 mM and does not comprise an organic solvent, as a washing solution for washing magnetic particles with nucleic acid adsorbed thereto.

1 . A method for isolating nucleic acid from a sample solution, wherein the nucleic acid is separated from the sample solution by adsorption to magnetic particles, followed by separation of the magnetic particles with the nucleic acid adsorbed thereto from the sample solution by application of a magnetic field,
 followed by at least one washing step (w), comprising   (w1) suspending the magnetic particles with the nucleic acid adsorbed thereto in a washing solution, which is an aqueous solution, which has a total salt concentration below 100 mM and does not comprise an organic solvent,   (w2) incubating the magnetic particles with the nucleic acid adsorbed thereto in the washing solution, and   (w3) separating the magnetic particles with the nucleic acid adsorbed thereto from the washing solution.   
     
     
         2 . The method of  claim 1 , wherein the nucleic acid is DNA, RNA or both, DNA and RNA, preferably DNA. 
     
     
         3 . The method of at least one of the preceding claims, wherein the washing solution has a total salt concentration below 25 mM. 
     
     
         4 . The method of at least one of the preceding claims, wherein the washing solution consists of water and optionally salts. 
     
     
         5 . The method of at least one of the preceding claims, wherein washing step (w) is carried out for 5 minutes or less. 
     
     
         6 . The method of at least one of the preceding claims, wherein the nucleic acid, preferably DNA, has a chain length of at least 10 kb or kbp respectively. 
     
     
         7 . The method of at least one of  claims 1  to  5 , wherein the nucleic acid, preferably DNA, has a chain length of less than 10 kb or kbp respectively, wherein washing step (w) is carried out for 2 minutes or less. 
     
     
         8 . The method of at least one of the preceding claims, with one or more of the following characteristics being applied in washing step (w):
 the magnetic particles with the nucleic acid bound thereto are not subjected to a mechanical force   the magnetic particles with the nucleic acid bound thereto are not subjected to a mechanical force exerted oscillating and/or horizontally,   the magnetic particles with the nucleic acid bound thereto are subjected to a mechanical force exerted vertically, preferably to gravity,   the magnetic particles with the nucleic acid bound thereto are subjected to a magnetic force, preferably exerted vertically,   the magnetic particles with the nucleic acid bound thereto are suspended in the washing solution without applying a mechanical force   the magnetic particles with the nucleic acid bound thereto are suspended in the washing solution without applying a mechanical force exerted oscillating and/or horizontally   the magnetic particles with the nucleic acid bound thereto are suspended in the washing solution with applying a mechanical force exerted vertically, preferably gravity,   the magnetic particles with the nucleic acid bound thereto are suspended in the washing solution with applying a magnetic force, preferably exerted vertically.   
     
     
         9 . The method of at least one of the preceding claims, wherein before washing step (w), the magnetic particles with the nucleic acid adsorbed thereto are washed in at least one pre-washing step (p) with a washing solution, which is an aqueous solution having a total salt concentration above 500 mM and/or comprises an organic solvent. 
     
     
         10 . The method of at least one of the preceding claims, wherein after washing step (w) the nucleic acid is eluted from the magnetic particles with an elution solution. 
     
     
         11 . The method of at least one of the preceding claims, wherein the magnetic particles comprise silica. 
     
     
         12 . The method of at least one of the preceding claims, wherein the sample solution comprises a chaotropic salt and/or an alcohol. 
     
     
         13 . The method of at least one of the preceding claims, comprising the steps of
 (a) adding magnetic particles to a sample solution comprising the nucleic acid,   (b) incubating the sample solution from step (a) to allow adsorption of the nucleic acid to the magnetic particles,   (c) applying a magnetic field to the sample solution from step (b) to fixate the magnetic particles with the nucleic acid adsorbed thereto,   (d) removing the sample solution,   (e) washing the magnetic particles with a washing solution in a washing step (w) of  claim 1 , and   (f) optionally eluting the nucleic acid from said magnetic particles with an elution solution.   
     
     
         14 . The method of at least one of the preceding claims, wherein the method is an automated process. 
     
     
         15 . Use of an aqueous solution, which has a total salt concentration below 100 mM and does not comprise an organic solvent, as a washing solution for washing magnetic particles with nucleic acid adsorbed thereto in a method for isolating nucleic acid from a sample solution, wherein the magnetic particles with the nucleic acid adsorbed thereto are suspended in the washing solution.