Method for nucleic acid preparation
Concentration of oligonucleotides in salt solutions is accomplished by loading a solution of the oligonucleotide dissolved in aqueous sodium chloride or other salt solution onto a reverse-phase poly(styrene-divinylbene) liquid chromatography (LC) column. The column bearing oligonucleotide is then washed with water to low conductivity and eluted with an organic eluent such as ethanol, thus effecting a combination desalting concentration procedure in one step, thus this procedure has utility in the desalting and concentration of oligonucleotides that have been purified and/or treated by anion exchange chromatography. In situ cationic exchange of the associated cation of the oligonucleotide can also be incorporated into the procedure of the new invention.
1 . A method of desalting and concentrating a nucleic acid within a sample, said method comprising the steps of:
contacting the sample with a binding medium comprising a strongly hydrophobic base matrix; and
eluting the nucleic acid with an aqueous organic solvent.
2 . The method of claim 1 , wherein the binding medium is comprised of poly(styrene-divinylbenzene).
3 . The method of claim 1 , wherein the binding medium is a column comprised of particles having a diameter of about 1 micron to about 250 microns.
4 . The method of claim 3 , wherein the binding medium is a column comprised of particles having a diameter of about 50 to about 75 microns.
5 . The method of claim 1 , further comprising the step of:
rinsing the binding medium with an unbuffered aqueous solution prior to elution.
6 . The method of claim 5 , wherein the unbuffered aqueous solution is water.
7 . The method of claim 5 , wherein an effluent conductivity following rinsing is at or below 100 microSiemens/cm.
8 . The method of claim 7 , wherein the effluent conductivity following rinsing is at or below 25 microSiemens/cm.
9 . The method of claim 1 , wherein the nucleic acid has been modified with a compound selected from the group consisting of: biotin, fluorescein and related dyes, spacers, thiol modifiers, amino modifiers, carboxylate modifiers, or any combination of these.
10 . The method of claim 1 , wherein the nucleic acid is selected from the group consisting of: a DNA phosphodiester, RNA phosphodiester, phosphorothioate, methylphosphonate, 2′-methyl RNA, 2′-O-alkyl RNA, 2′-O-methyl DNA, 2′-O-alkyl DNA and chimeras containing such structures.
11 . The method of claim 1 , wherein the nucleic acid comprises nucleotide bases selected from the group consisting of: 5-methylcytidine, inosine, halogenated uridines, etheno-bases, dideoxynucleosides, and inverted bases.
12 . The method of claim 1 , wherein the nucleic acid is comprised of inverted 3′-5′ linkages.
13 . The method of claim 1 , wherein the nucleic acid is comprised of 5′-2′ linkages.
14 . The method of claim 1 , wherein the nucleic acid is an oligonucleotide comprised of about 1 to about 100 nucleotides.
15 . The method of claim 1 , wherein the sample is the product of strong anion exchange chromatography.
16 . The method of claim 1 , wherein the sample is the product of weak anion exchange chromatography.
17 . The method of claim 1 , wherein the sample is derived from a biological source material.
18 . The method of claim 1 , wherein the aqueous organic solvent is selected from the group consisting of acetonitrile, n-propanol, isopropanol, or methanol.
19 . The method of claim 1 wherein the aqueous organic solvent is aqueous ethanol.
20 . A method of exchanging a cation associated with a nucleic acid in a sample, comprising the steps of:
contacting a nucleic acid associated with a first cation with a binding medium comprising a strongly hydrophobic base matrix;
rinsing the nucleic acid bound to the binding medium with an unbuffered aqueous solution prior to elution;
contacting the bound nucleic acid with a solution comprised of a second cation; and
eluting the nucleic acid associated with the second cation from the binding medium;
wherein the second cation effectively displaces the first cation in the effluent sample.