Separation of nucleic acid

• US 20060024712A1
• Filed: 06/27/2005
• Published: 02/02/2006
• Est. Priority Date: 06/25/2004
• Status: Abandoned Application

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Baker, Matthew J., Stevenson, Anthony, Buckels, John
• Application Number:
  US11/167,907
• Publication Number:
  US 20060024712A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 1/06 Lysis of microorganisms
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers

Abstract

Compositions, methods and kits for separating nucleic acid from cell samples. Cells are lysed and nuclear material is flocculated/precipitated. Genomic DNA can be collected from the precipitate and purified. RNA present in the supernatant can be collected (e.g., bound to a solid phase) and purified.

Claims

1. A method for separating genomic DNA from a cell sample, comprising:

(a) lysing said cell sample under non-denaturing conditions to flocculate said genomic DNA and form a precipitate; and

(b) collecting said precipitate.

2. The method of claim 1, wherein said cells are lysed with a hypertonic monovalent cationic salt solution at a pH between about 4.0 and 10.0.

3. The method of claim 1, wherein said cell lysis occurs in the presence of a solid phase capable of binding said genomic DNA.

4. The method of claim 1, wherein said lysing does not comprise using one or more of:

(i) a chaotropic reagent;

(ii) a strong ionic detergent;

(iii) a pH that is above about 10.0 or below about 4.0;

(iv) a divalent or trivalent metal ion;

or

(v) a protein precipitant.

5. The method of claim 1, wherein said method does not involve ultracentrifugation.

6. The method of claim 2, wherein said salt is an alkali metal cationic salt or an ammonium salt.

7. The method of claim 6, wherein said salt is sodium chloride (NaCl), potassium chloride (KCl), sodium carbonate (Na₂CO₃), sodium bicarbonate (NaHCO₃), ammonium bicarbonate (NH₄HCO₃), lithium chloride (LiCl) or Cesium Chloride (CsCl).

8. The method of claim 2, wherein the concentration of said hypertonic monovalent cationic salt solution is between about 10 mM and 1.0 M.

9. The method of claim 2, wherein said hypertonic monovalent cationic salt solution has a pH of between about 6.0 and 9.0.

10. The method of claim 2, wherein said hypertonic monovalent cationic salt solution further comprises about 0.1 to 1.0% v/v of a non-ionic detergent.

11. The method of claim 1, wherein said cell sample is a mammalian cell sample or a blood cell sample.

12. The method of claim 11, wherein said cell sample is a whole blood cell sample.

13. The method of claim 3, wherein said solid phase binds at least about 50·

g of genomic DNA per mg of solid phase.

14. The method of claim 3, wherein said solid phase binds at least about 100·

g of genomic DNA per mg of solid phase.

15. The method of claim 3, further comprising:

(a) contacting said solid phase with a solution under conditions to release said precipitate of said genomic DNA and nuclear material into said solution;

(b) treating said solution to remove one or more impurities; and

(c) rebinding said genomic DNA to said solid phase.

16. The method of claim 15, further comprising contacting said solution in (b) with a protease.

17. The method of claim 15, wherein said rebinding in (c) comprises adding a precipitant, whereby said genomic DNA rebinds to said solid phase.

18. The method of claim 3, wherein said solid phase is a charge switch material.

19. The method of claim 3, wherein said solid phase is a spooling rod, a bead or particulate composition, a single bead, a mesh, a membrane, a sinter, a plastic support, a paper, a tip, a dipstick, a wall of a container, a tube, a well, a probe, a pipette, a filter, a sheet, a slide or a plug.

20. The method of claim 1, further comprising purifying said precipitate or amplifying a nucleic acid sequence within said genomic DNA.

21. A method for separating RNA from a cell sample, the method comprising:

(a) lysing said cells under non-denaturing conditions to flocculate genomic DNA and form a precipitate and a supernatant; and

(b) collecting said supernatant from said precipitate, wherein said supernatant contains said RNA.

22. The method of claim 21, wherein said cells are lysed with a hypertonic monovalent cationic salt solution at a pH between about 4.0 and 10.0.

23. The method of claim 21, wherein said cell lysis occurs in the presence of a solid phase capable of binding said genomic DNA.

24. The method of claim 21, wherein said cell lysis does not comprise using one or more of:

(i) a chaotropic reagent;

or

(ii) a strong ionic detergent;

or

(iii) a pH that is above 10.0 or below 4.0;

or

(iv) a divalent or trivalent metal ion;

or

(v) a protein precipitant.

25. The method of claim 21, wherein at least about 90% of the protein initially present in said cell sample is removed.

26. The method of claim 22, wherein said salt is an alkali metal cationic salt or an ammonium salt.

27. The method of claim 26, wherein said salt is sodium chloride (NaCl), potassium chloride (KCl), sodium carbonate (Na₂CO₃), sodium bicarbonate (NaHCO₃), ammonium bicarbonate (NH₄HCO₃), lithium chloride (LiCl) or Cesium Chloride (CsCl).

28. The method of claim 22, wherein the concentration of said hypertonic monovalent cationic salt solution is between about 10 mM and 1.0 M.

29. The method of claim 22, wherein said hypertonic monovalent cationic salt solution has a pH between about 6.0 and 9.0.

30. The method of claim 22, wherein said hypertonic monovalent cationic salt solution further comprises about 0.1 to 1.0% v/v of a non-ionic detergent.

31. The method of claim 21, wherein the cell sample is a mammalian cell sample or a blood cell sample.

32. The method of claim 31, wherein said cell sample is a whole blood cell sample.

33. The method of claim 23, wherein said solid phase binds at least 50 μ

g of genomic DNA per mg of solid phase.

34. The method of claim 23, wherein said solid phase binds at least 100 μ

g of genomic DNA per mg of solid phase.

35. The method of claim 23,further comprising:

(a) contacting said solid phase with a solution under conditions to release the precipitate of said genomic DNA and nuclear material into said solution;

(b) treating said solution to remove one or more impurities; and

(c) rebinding said genomic DNA to said solid phase.

36. The method of claim 35, further comprising contacting said solution in (b) with a protease.

37. The method of claim 35, wherein said rebinding in (c) comprises adding a precipitant, whereby said genomic DNA rebinds to said solid phase.

38. The method of claim 23, wherein said solid phase is a charge switch material.

39. The method of claim 23, wherein the solid phase is a spooling rod, a bead or particulate composition, a single bead, a mesh, a membrane, a sinter, a plastic support, a paper, a tip, a dipstick, a wall of a container, a tube, a well, a probe, a pipette, a filter, a sheet, a slide or a plug.

40. The method of claim 21, further comprising purifying said RNA.