Compositions, methods, and kits for isolating and analyzing nucleic acids using an anion exchange material
The present disclosure relates to methods for isolating, amplifying, and/or analyzing nucleic acids in the presence of an anion exchange material by performing the isolation, amplification and/or analysis step in the presence of at least one anionic compound.
1. A method of eluting a nucleic acid from an anion exchange material to form an eluate, said method comprising:
a) providing a nucleic acid-anion exchange complex; and
b) eluting the nucleic acid from the nucleic acid-anion exchange complex, wherein said eluting comprises addition of an elution buffer comprising an anionic compound to the nucleic acid-anion exchange complex, wherein said anionic compound comprises at least two anionic groups,
wherein said anionic compound disrupts the nucleic acid-anion exchange complex, and
wherein said nucleic acid-anion exchange complex is provided by a method comprising:
i) providing a sample comprising a nucleic acid; and
ii) adding the sample to an anion exchange material under pH and salt conditions at which the anion exchange material reversibly complexes with the nucleic acid,
wherein the elution buffer has a pH and salt concentration that does not exceed the pH and salt conditions at which the nucleic acid-anion exchange complex is formed,
wherein said nucleic acid-anion exchange complex is formed at a pH of 7 or greater and
wherein said nucleic acid-anion exchange complex is not exposed to a pH of less than 7 prior to said eluting of step (b).
2. The method of claim 1 , wherein the anionic compound is present in the elution buffer at a concentration sufficient to disrupt the nucleic acid-anion exchange complex, and wherein the elution buffer possesses a pH such that, in the absence of the anionic compound, the nucleic acid-anion exchange complex would not be disrupted.
3. The method of claim 1 wherein the anion exchange material comprises a positively ionizable capture moiety selected from the group consisting of:
a) An amine of the formula R 3 N;
b) An amine of the formula R 2 NH;
c) An amine of the formula RNH 2 ; and
d) An amine of the formula X—(CH 2 ) n —Y,
wherein:
X is R 2 N, RNH or NH 2 ,
Y is R 2 N, RNH or NH 2 ,
R is independently of each other a linear, branched or cyclic alkyl, alkenyl, alkynyl or aryl substituent which may comprise one or more heteroatoms, preferably selected from O, N, S and P, and
n is an integer in the range of from 0 to 20.
4. The method of claim 1 wherein the anion exchange material comprises a polyethylenimine (PEI)-modified magnetic silica bead.
5. The method of claim 1 wherein the anionic compound is selected from the group consisting of:
a) a non-polymeric compound selected from the group consisting of: a carboxylic acid, an oligomer of polymerizable or condensable acidic monomers, an organosulfonic acid, an organophosphonic acid, an organophosphate, a carbonate, and a diacetylacetone; and
b) a polyanionic compound selected from the group consisting of: a polymerized unsaturated carboxylic acid; a copolymer of an unsaturated carboxylic acid and at least one other monomer; a polypeptide of an acidic amino acid; a copolymer of an acidic amino acid with at least one other amino acid; a polycarbohydrate bearing a covalently attached ionizable group selected from the group consisting of carboxylic acid, sulfonic acid, phosphonic acid, phosphate, and carbonate; a polystyrene bearing a covalently attached ionizable group selected from the group consisting of carboxylic acid, sulfonic acid, phosphonic acid, phosphate, and carbonate; a second nucleic acid; and a nucleic acid analog.
6. The method of claim 1 wherein the anionic compound comprises at least three anionic groups.
7. The method of claim 1 wherein the anionic compound is a non-polymeric compound comprising from 2 to 20 carbon atoms.
8. The method of claim 1 wherein the anionic compound is selected from the group consisting of: oxalic acid, mellitic acid, pyromellitic acid, citric acid, polyacrylic acid (PAA), polymethacrylic acid (PMA), polyglutamic acid (PGA), and dextran sulfate (DS).
9. The method of claim 1 wherein the elution buffer has a pH such that, in the absence of the anionic compound, the nucleic acid-anion exchange complex would not be disrupted.
10. The method of claim 1 wherein the elution buffer has a pH that does not exceed the pKa of the positively ionizable groups of the capture moiety.
11. The method of claim 1 wherein the elution buffer has a pH in a range from 7 to 13.
12. The method of claim 1 wherein the elution buffer has a pH in a range from 7 to 8.5.
13. The method of claim 1 wherein the elution buffer has a total salt concentration of approximately 1M or less.
14. The method of claim 1 wherein the elution buffer has a total salt concentration of approximately 0.5M or less.
15. The method of claim 1 wherein the elution buffer has a pH in a range from 8.2 to 9.
16. The method of claim 1 wherein the elution buffer further comprises an enzyme having a polymerase activity and, optionally, a source of Mg 2+ .
17. The method of claim 2 , wherein the elution buffer possesses a pH and salt concentration suitable for performing a nucleic acid amplification reaction.
18. The method of claim 2 , wherein the elution buffer further comprises an enzyme having a polymerase activity and optionally comprising Mg 2+ or a source thereof.