Chromatographic device and method for isolating and purifying nucleic acids

Abstract

The present invention relates to a chromatographic device for isolating and purifying nucleic acids, preferably genomic DNA, by gel filtration chromatography, a method for isolating and purifying nucleic acids, preferably genomic DNA, using this device and a kit comprising this device.

1 . A chromatographic device for isolating and purifying nucleic acids, preferably comprising DNA, in particular genomic DNA, from contaminants by gel filtration chromatography, comprising at least one chromatographic unit comprising:
 (a) a hollow body ( 1 ) having an inlet ( 2 ) and an outlet ( 3 ), the hollow body ( 1 ) comprising a solid matrix ( 4 ), preferably forming a gel bed,   (b) a porous frit, filter, fleece or membrane ( 5 ), placed between the outlet ( 3 ) and the solid matrix ( 4 ), to retain the solid matrix ( 4 ) within the chromatographic unit, preferably allowing nucleic acids of any size to pass,   (c) preferably a non-porous ring ( 6 ), placed between the porous frit, filter, fleece or membrane ( 5 ) and the matrix ( 4 ), sealing the outer area of the frit, filter, fleece or membrane ( 5 ), to prevent the mobile phase from entering the frit ( 5 ) without passing the matrix ( 4 ),   (d) optionally at least one removable closing device ( 7 ) to seal the inlet ( 2 ) and/or outlet ( 3 ) of the chromatographic unit, and   (e) optionally at least one collection tube to collect the mobile phase (eluate) after having passed the matrix ( 4 ),   wherein the solid matrix is a gel-forming polymer having a size exclusion limit of 150 to 500 bp, preferably 200 to 400 bp, and most preferably 250 to 300 bp.   
     
     
         2 . The device according to  claim 1 , wherein the gel-forming polymer is selected from the group consisting of dextrans, agarose, polyacrylamide, and mixtures thereof, preferably a mixture of a dextran and a polyacrylamide. 
     
     
         3 . The device according  claim 1 , wherein the removable closing device is a disposable closing device selected from the group consisting of lid foils, seals and break-away ends, and a re-closable closing device selected from screw caps and snap-on caps. 
     
     
         4 . The device according  claim 1 , wherein the inlet and the outlet of the chromatographic unit are sealed with a removable closing device, and the solid matrix is supplied in form of a gel, pre-swollen in a solvent selected from the group consisting of water and homogenous mixtures of organic solvents with water or aqueous buffers, wherein the solvent is purged from the chromatographic unit and the matrix (bed) is established by centrifugation (pre-spinning) immediately prior to use. 
     
     
         5 . The device according  claim 1 , comprising a plurality of chromatographic units in a parallel fashion, preferably in form of a multiwell plate, wherein each well of the multiwell plate contains one separate chromatographic unit. 
     
     
         6 . A method for purifying nucleic acids, preferably comprising DNA, in particular genomic DNA, by gel filtration chromatography using a device according to  claim 1 , comprising the steps of:
 (a) providing a sample comprising the nucleic acids to be purified, wherein the sample is in the form of a solution or a suspension in a liquid, preferably aqueous eluent,   (b) establishing a matrix in the chromatographic unit, preferably by centrifugation (pre-spinning),   (c) applying the sample to the matrix upper surface, and   (d) eluting the nucleic acids from the chromatographic unit preferably by centrifugation and simultaneously collecting the eluate.   
     
     
         7 . The method according to  claim 6 , wherein the sample is a lysate obtained from a biological sample, preferably from a cell-containing biological sample selected from the group consisting of fresh and frozen tissue, blood, other body liquids and Gram-negative Bacteria. 
     
     
         8 . The method according to  claim 6 , wherein the step of pre-spinning is carried out by centrifuging the device at 500 to 900×g for 1 to 7 min, preferably at 700×g for 2 to 5 min, and most preferably at 700×g for 3 min. 
     
     
         9 . The method according to  claim 6 , wherein the volume of matrix bed per chromatographic unit is in the range of 100 μL to 2 mL, preferably in the range of 500 μL to 1 mL, and most preferably is 800 μL. 
     
     
         10 . The method according to  claim 6 , wherein the sample volume per 600 μL to 800 μL matrix bed is in the range of 10 to 100 μL. 
     
     
         11 . The method according to  claim 6 , wherein the step of eluting he nucleic acids from the chromatographic unit is carried out by centrifuging the device at 500 to 900×g for 1 to 7 min, preferably at 700×g for 2 to 5 min, and most preferably at 700×g for 3 min. 
     
     
         12 . The method according to  claim 6 , wherein the sample is a lysate of a cell-containing biological sample obtained by a preceding lysis procedure comprising the steps of:
 (a) Mixing the cell-containing sample with a lysis buffer,   (b) incubating the mixture to obtain a lysate comprising at least DNA, RNA and proteins,   (c) optionally disintegrating the RNA contained in the lysate, and   (d) optionally selectively precipitating solutes other than DNA.   
     
     
         13 . Kit for the isolation and purification of nucleic acids, preferably genomic DNA, comprising:
 (a) a device according to  claim 1 , and   (b) one or more components selected from the group consisting of
 (i) a lysis buffer, 
 (ii) a source of monovalent ions of alkali earth metals and/or divalent ions of alkaline earth metals, and 
 (iii) primers for the direct amplification of one or more target nucleic acid. 
   
     
     
         14 . (canceled)