Method and device for isolating and purifying double-stranded nucleic acids

• US 9,458,452 B2
• Filed: 11/08/2011
• Issued: 10/04/2016
• Est. Priority Date: 11/09/2010
• Status: Active Grant

• Current Assignee: Qiagen GmbH (Qiagen NV)
• Original Assignee: Qiagen GmbH (Qiagen NV)
• Inventors: Singer, Thorsten, Wedler, Holger
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US13/884,416
• Publication Number:
  US 20130237699A1
• Time in Patent Office: 1,792 Days
• Field of Search: 536/25.4
• US Class Current: 1/1
• CPC Class Codes: B01D 15/34 Size selective separation, e.g. size exclusion chromatography, gel filtration, permeation
• CPC Class Codes: B01D 15/3828 Ligand exchange chromatography, e.g. complexation, chelation or metal interaction chromatography
• CPC Class Codes: C07H 1/08 from natural products
• CPC Class Codes: C12N 15/101 by chromatography, e.g. electrophoresis, ion-exchange, reverse phase

Abstract

The invention relates to a chromatographic device for isolating and/or purifying double-stranded nucleic acids, preferably double-stranded DNA, from a mixture of such nucleic acids with single-stranded nucleic acids, oligonucleotides, mononucleotides, salts and/or other such impurities. The invention also relates to a method for chromatographically isolating and/or purifying same, and to a kit for this purpose.

Claims

1. A chromatographic device for isolating and/or purifying double-stranded nucleic acids, comprising:

(1) a base body,

(2) at least one cavity within the base body, which cavity is provided with an inlet and an outlet, and

(3) a stationary phase situated in the cavity, wherein the stationary phase comprises;

(a) a size-exclusion chromatography medium for isolating and/or purifying double-stranded nucleic acids, wherein the size-exclusion chromatography medium comprises at least one porous chromatography resin having pores sufficiently small to disallow a double-stranded nucleic acid of more than 100 bp to enter, and

(b) immobilized metal ions, and

(4) a porous filter, a porous frit or a membrane that is arranged between the outlet of the cavity and the stationary phase and retains the stationary phase in the cavity.

2. The device of claim 1, wherein the double-stranded nucleic acids are double-stranded DNA.

3. The device of claim 1, further comprising at least one of the following features:

at least one closure device for closing the inlet and/or the outlet of the cavity and at least one collection vessel for collecting the mobile phase (eluate) emerging from the outlet after elution has occurred.

4. The device of claim 1, wherein the immobilized metal ions are divalent, trivalent or tetravalent metal cations or mixtures thereof.

5. The device of claim 4, wherein the immobilized metal ions are selected from the group consisting of Zn²⁺, Co²⁺, Cu²⁺, Ni²⁺, Fe²⁺, Mn²⁺, Al³⁺, Co³⁺, Ga³⁺ and Zr⁴⁺, and a combination thereof.

6. The device of claim 4, wherein the immobilized metal ions are at least one divalent metal cation selected from the group consisting of Zn²⁺, CO²⁺, Cu²⁺ and Ni²⁺, optionally in combination with Al³⁺ ions.

7. The device of claim 4, wherein the immobilized metal ions are Cu²⁺ ions, optionally in combination with Al³⁺ ions.

8. The device of claim 1, wherein the metal ions are immobilized on at least part of the stationary phase by means of at least one linker.

9. The device of claim 8, wherein the linker has the general formula A-R—

Z, where A is an anchor group for covalent bonding of the linker to at least part of the stationary phase, Z is a polydentate head group for chelating the metal ions, and R is a linear, branched or cyclic hydrocarbon chain that connects the anchor group A and the head group Z, that can be substituted or unsubstituted, and in which one or more carbon atoms can be replaced by heteroatoms.

10. The device of claim 8, wherein the linker is selected from the group consisting of iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), N-carboxymethylated aspartic acid (CM-Asp), and tris(2-ethylaminoethyl)amine (TREN).

11. The device of claim 1, wherein the porous chromatography resin is a crosslinked silicate or an organic polymer that, upon mixing with water, an aqueous solution or an aqueous buffer, forms a gel matrix having internal pores of a defined size (size-exclusion chromatography resin).

12. The device of claim 11, wherein the porous chromatography resin is selected from the group consisting of dextrans, agarose, polyacrylamides, and mixtures thereof.

13. The device of claim 1, wherein the stationary phase comprises, in addition to the at least one porous chromatography resin (size-exclusion chromatography resin), at least one additional material on which the metal ions are immobilized.

14. The device of claim 13, wherein the porous chromatography resin and the additional material in the chromatographic device

a) are arranged substantially separately from one another, or

b) are largely present as a homogeneous mixture.

15. The device of claim 14, wherein the porous chromatography resin and the additional material in the chromatographic device are arranged substantially separately from one another in the form of two successive layers in the flow direction of the mobile phase from the inlet to the outlet.

16. The device of claim 15, wherein the additional material is arranged as the upper layer, and the size-exclusion chromatography resin is arranged as the lower layer.

17. The device of claim 1, wherein the metal ions are immobilized on the porous chromatography resin.

18. The device of claim 1, wherein the chromatographic device

i) comprises exactly one stationary phase-filled cavity, or

ii) comprises, for the parallel isolation and/or purification of double-stranded nucleic acids from different samples, multiple cavities that are each filled with the stationary phase and are arranged in parallel.

19. The device of claim 18, wherein the chromatographic device is in the form of a spin column.

20. The device of claim 18, wherein the multiple cavities are arranged in the form of a filter plate.

21. The device of claim 1, wherein the base body consists of a material containing an organic polymer.

22. The device of claim 21, wherein the organic polymer is selected from the group consisting of polyethylene (PE), polypropylene (PP), polycarbonate, polystyrene, polyphenylsulfone, and polysulfone.

23. A method for chromatographically isolating and/or purifying double-stranded nucleic acids from a mixture of such nucleic acids with single-stranded nucleic acids, oligonucleotides, mononucleotides, salts and/or other contaminants, comprising:

(1) providing a sample containing at least double-stranded nucleic acids in the form of an aqueous solution,

(2) applying said solution to the inlet-facing side of the stationary phase of a chromatographic device of claim 1, and

(3) eluting the double-stranded nucleic acids and simultaneously collecting the eluate,

wherein the separation of the double-stranded nucleic acids from the further constituents present in the sample is achieved in one chromatographic step.

24. The method of claim 23, wherein the method is for isolating and/or purifying PCR amplification products from a PCR reaction mixture.

25. The method of claim 23, wherein the double-stranded nucleic acids are double-stranded deoxyribonucleic acids (DNA).

26. The method of claim 23, wherein the double-stranded nucleic acids are linear double-stranded DNA obtained as PCR amplification products.

27. The method of claim 23, wherein the double-stranded nucleic acids are eluted by application of centrifugal force to the chromatographic device.

28. The method of claim 27, additionally comprising:

preparing a gel matrix in the cavity of the chromatographic device by suspending a porous chromatography resin in water, an aqueous solution or an aqueous buffer, allowing the resin to swell in the suspension to form the gel matrix, and removing the excess water, aqueous solvent or aqueous buffer by centrifugation of the chromatographic device containing the swollen resin prior to step (2).

29. A kit for isolating and/or purifying double-stranded nucleic acids, comprising a chromatographic device of claim 1 or the starting materials thereof and optionally a buffer solution for adjusting the pH of the sample solution prior to chromatographic purification.

30. The kit of claim 29, wherein the double-stranded nucleic acids are double-stranded DNA.

31. The kit of claim 29, wherein the buffer solution is selected from the group containing Mops, Mes, Hepes and aqueous solutions of alkali metal acetates or phosphates.

32. The kit of claim 31, wherein the buffer solution is an aqueous sodium acetate solution.

33. The device of claim 1, wherein the at least one porous chromatography resin has pores sufficiently small to disallow a molecule having a molecular weight larger than 10,000 daltons to enter.

34. The device of claim 1, wherein the at least one porous chromatography resin has pores sufficiently small to disallow a double-stranded nucleic acid of more than 80 bp to enter.

35. The device of claim 1, wherein the at least one porous chromatography resin has pores that allow a molecule having a molecular weight below 10,000 daltons to enter.

36. The device of claim 1, wherein the at least one porous chromatography resin has pores that allow a molecule having a molecular weight below 1,000 daltons to enter.