Nucleic acid purification method

• US 9,663,779 B2
• Filed: 12/23/2009
• Issued: 05/30/2017
• Est. Priority Date: 12/23/2008
• Status: Expired due to Fees

• Current Assignee: Qiagen GmbH (Qiagen NV)
• Original Assignee: Qiagen GmbH (Qiagen NV)
• Inventors: Fabis, Roland, Petzel, Jan, Scheltinga, Sabine
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US13/141,885
• Publication Number:
  US 20120245337A1
• Time in Patent Office: 2,715 Days
• Field of Search: 536 254, 536 2541, 536 2542
• CPC Class Codes: C12N 15/101 by chromatography, e.g. electrophoresis, ion-exchange, reverse phase

Abstract

The present invention relates to a method for purifying nucleic acids from a sample containing nucleic acids, the method comprising at least the following steps: a. bringing the sample containing nucleic acids into contact with a nucleic acid binding phase comprising protonatable groups, wherein the protonatable groups have a pKs value of 9 to 12; b. binding the nucleic acids to the nucleic acid phase at a pH (binding pH) that is at least one pH unit less than the pKs value of at least one of the protonatable groups; c. eluting the nucleic acids at a pH greater than the binding pH but at least one pH unit less than the pKs value of at least one of the protonatable groups. Also disclosed are corresponding kits and nucleic acid binding phases that can be used for purifying nucleic acids.

Claims

1. A method of purifying nucleic acids from a nucleic acid-containing sample, the method comprising:

(a) contacting the nucleic acid-containing sample with a nucleic acid-binding phase comprising nucleic acid-binding groups covalently bound to a support, the nucleic acid-binding groups having one or more protonatable groups having a pKa from 9 to 12;

(b) binding the nucleic acids to the nucleic acid-binding phase at a binding pH which is more than one pH unit below the pKa of at least one of the one or more protonatable groups; and

(c) eluting the nucleic acids at an eluting pH which is above the binding pH, but one, or more than one, pH unit below the pKa of at least one of the one or more protonatable groups,

wherein the nucleic acid-binding phase further comprises;

(i) diluting groups, wherein the proportion of nucleic acid-binding groups relative to the diluting groups is <

50%; and

(ii) functional groups which are bound to the support and/or the nucleic acid-binding groups, and which functional groups have a negative charge during the eluting and promote the release of the nucleic acids at the eluting pH.

2. The method according to claim 1 wherein the functional groups are cation exchangers.

3. The method according to claim 1, wherein the binding comprises one or more of the following:

(a) binding at a pH from 3 to 8, from 4 to 7.5, from 4.5 to 7, from 5.5 to 7, or from 6.5 to 7; and

/or

(b) binding at a salt concentration of less than 1 M.

4. The method according to claim 1, wherein the eluting comprises one or more of the following:

(a) eluting at a pH of from 7.5 to 10, 8 to 9, or 8.2 to 8.8; and

/or

(b) eluting at a salt concentration of less than 1 M; and

/or

(c) eluting in a solution selected from the group consisting of water, biological buffers and organic buffers.

5. The method according to claim 1, further comprising washing the nucleic acid binding phase with a washing solution after the binding and before the elution of the nucleic acids.

6. The method according to claim 5 wherein the washing solution is water or an aqueous solution, the aqueous solution having a salt concentration of less than 400 mM.

7. The method according to claim 1, wherein the one or more protonatable groups comprise amino groups.

8. The method of claim 2, wherein the functional groups are acidic groups.

9. The method of claim 8, wherein the acidic groups are carboxyl groups.

10. The method of claim 7, wherein the one or more protonatable groups comprise from one to ten, two to eight, or two to six amino groups per protonatable group.

11. The method of claim 7, wherein the amino groups have a pKa of from 10 to 12.

12. The method of claim 7, wherein the amino groups are not conjugated to one or more electron density-reducing groups.

13. The method of claim 1, wherein the support is an organic polymer, hydrogel, or inorganic material.

14. The method of claim 13, wherein the support is an organic polymer selected from the group consisting of polystyrenes, polyacrylates, polymethacrylates, polyurethanes, nylon, polyethylene, polypropylene, polysaccharides, polybutylene and copolymers thereof.

15. The method of claim 13, wherein the support is a hydrogel selected from the group consisting of agarose, cellulose, dextran, cross-linked dextran gel, cross-linked allyl dextran N,N′

-methylene bisacrylamide, and chitosan.

16. The method of claim 13, wherein the support is an inorganic material selected from the group consisting of silica gels, silica particles, glass, metal oxides, semi-metal oxides, boron oxide, magnetic particles and substrates with a metal surface.

17. The method of claim 16, wherein the inorganic material is a substrate with a gold surface.

18. The method of claim 1, wherein the nucleic acid-binding groups comprise primary and secondary amines.

19. The method of claim 18, wherein the nucleic acid-binding groups are spermine and/or spermidine.

20. The method of claim 1, wherein the one or more protonatable groups have, or are, ion exchangers.

21. The method according to claim 3, wherein the binding occurs at a salt concentration of 0.5 M or less, 0.25 M or less, or 0.1 M or less.

22. The method according to claim 4, wherein the eluting occurs at a salt concentration of 0.5 M or less, 0.25 M or less, 0.1 M or less, 50 mM or less, 0.25 mM or less, 0.15 mM or less, or 10 mM or less.

23. The method according to claim 5 wherein the washing solution is water or an aqueous solution, the aqueous solution having a salt concentration of 200 mM or less, 100 mM or less, 50 mM or less, or 25 mM or less.

24. The method according to claim 1, wherein the proportion of nucleic acid-binding groups relative to the diluting groups is ≦

25%.

25. The method according to claim 1, wherein the proportion of nucleic acid-binding groups relative to the diluting groups is ≦

15%.