COMPOSITIONS AND METHODS FOR RECOVERY OF NUCLEIC ACIDS OR PROTEINS FROM TISSUE SAMPLES FIXED IN CYTOLOGY MEDIA

• US 20110065906A1
• Filed: 09/14/2010
• Published: 03/17/2011
• Est. Priority Date: 09/14/2009
• Status: Active Grant

• Current Assignee: QIAGEN Gaithersburg, Inc. (Qiagen NV)
• Original Assignee: QIAGEN Gaithersburg, Inc. (Qiagen NV)
• Inventors: LIU, Shubing, Ramachandran, Suganthi
• Granted Patent: US 9,410,146 B2
• Time in Patent Office: Days
• US Class Current: 536/23.1
• 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 15/1013 by using magnetic beads
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/708 for papilloma
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: C12Q 2565/531 characterised by the capture moiety being a protein for target oligonucleotides

Abstract

The present invention provides compositions and methods for improving nucleic acid or protein recovery from fixed biological samples.

Claims

1. A composition for lysing a fixed biological sample, the composition comprising at least two amines.

2. The composition of claim 1, wherein at least one of the amines is selected from the group consisting of methylamine, dimethylamine, diethylamine, trimethylamine, triethylamine, monoethanolamine, diethanolamine, triethanolamine, ethylenediamine, diethylenetriamine, tris(hydroxymethyl)aminomethane, hexamethylenetetramine, aniline, and an amino acid.

3. The composition of claim 1, wherein at least one of the amines is a buffering agent.

4. The composition of claim 3, wherein the buffering agent is selected from the group consisting of tris(hydroxymethyl)aminomethane (“

TRIS”

), N-tris-(hydroxymethyl)methyl-3-aminopropanesulfonic acid (“

TAPS”

), 3-[N-tris-(hydroxymethyl)-methyl-amino]-2-hydroxypropanesulfonic acid (“

TAPSO”

);

N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (“

TES”

);

N-[tris(hydroxymethyl)methyl]-glycine (“

TRICINE”

);

bis(2-hydroxyethyl)iminotris-(hydroxymethyl)methane (“

bis-TRIS”

); and

1,3-bis[tris(hydroxymethyl)methylamino]propane (“

bis-TRIS PROPANE”

).

5. The composition of claim 3, wherein the buffering agent is TRIS.

6. The composition of claim 1, wherein at least one of the amines is diazoamine.

7. The composition of claim 1 further comprising at least one detergent.

8. The composition of claim 6 wherein the detergent is selected from the group consisting of polyoxyethyleneglycol dodecyl ether, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, Nonidet P-40, Igepal CA-630, deoxycholate, Triton X-100, sodium dodecyl sulfate, and polysorbate surfactants.

9. The composition of claim 1 wherein the pH is 7 or greater.

10. The composition of claim 1, further comprising at least one DNA-binding magnetic bead.

11. The composition of claim 1 comprising:

150 mM Tris-HCl;

300 mM diethanolamine;

3% Brij-58;

0.09% sodium azide;

wherein the composition has a pH of approximately 9.4.

25. A method of lysing a fixed biological sample, the method comprising treating the fixed biological sample with a composition of claim 1.

26. The method of claim 25, wherein the fixed biological sample is fixed with a cross-linking fixative agent.

27. The method of claim 26, wherein the cross-linking fixative agent comprises an aldehyde.

28. The method of claim 27, wherein the aldehyde is selected from the group consisting of formaldehyde and glutaraldehyde.

29. The method of claim 25, wherein the fixed biological sample is fixed with a fixative agent comprising an alcohol.

30. The method of claim 29, wherein the alcohol is selected from the group consisting of methanol and ethanol.

31. The method of claim 25, wherein the fixative agent is selected from the group consisting of SUREPATH and PRESERVECYT.

32. The method of claim 25, wherein the fixed biological sample is a cervical sample.

65. Use of a composition according to claim 1 for lysing a fixed biological sample.

12. A composition for lysing a fixed biological sample, the composition comprising a detergent, an amine, and a buffering agent.

13. The composition of claim 12 wherein the detergent is selected from the group consisting of polyoxyethyleneglycol dodecyl ether, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, Nonidet P-40, Igepal CA-630, deoxycholate, Triton X-100, sodium dodecyl sulfate, and polysorbate surfactants.

14. The composition of claim 12, wherein the detergent is Triton-X100.

15. The composition of claim 12, wherein the amine is selected from the group consisting of methylamine, dimethylamine, diethylamine, trimethylamine, triethylamine, monoethanolamine, diethanolamine, triethanolamine, ethylenediamine, diethylenetriamine, tris(hydroxymethyl)aminomethane, hexamethylenetetramine, aniline, and an amino acid.

16. The composition of claim 12, wherein the amine is diethanolamine.

17. The composition of claim 12, wherein the buffering agent is selected from the group consisting of tris(hydroxymethyl)aminomethane (“

TRIS”

), N-tris-(hydroxymethyl)methyl-3-aminopropanesulfonic acid (“

TAPS”

), 3-[N-tris-(hydroxymethyl)-methyl-amino]-2-hydroxypropanesulfonic acid (“

TAPSO”

);

N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (“

TES”

);

N-[tris(hydroxymethyl)methyl]-glycine (“

TRICINE”

);

bis(2-hydroxyethyl)iminotris-(hydroxymethyl)methane (“

bis-TRIS”

);

1,3-bis[tris(hydroxymethyl)methylamino]propane (“

bis-TRIS PROPANE”

);

carbonate-bicarbonate;

glycine;

phosphate;

4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (“

HEPES”

);

N,N-bis(2-hydroxyethyl)glycine (“

Bicine”

); and

3-(N-morpholino)propanesulfonic acid (“

MOPS”

).

18. The composition of claim 12, wherein the buffering agent is TRIS.

19. The composition of claim 12, further comprising a preservative.

20. The composition of claim 12, wherein the preservative is selected from the group consisting of sodium azide, gentomycin, 2-Methyl-4-isothiazolin-3-one, 5-Chloro-2-methyl-4-isothiazolin-3-one, and 1,2-Benzisothiazolin-3-one.

21. The composition of claim 12, wherein the preservative is sodium azide.

22. The composition of claim 12 wherein the pH is 7 or greater.

23. The composition of claim 12, further comprising at least one DNA-binding magnetic bead.

24. The composition of claim 12 comprising:

150 mM Tris-HCl;

300 mM diethanolamine;

3% Brij-58;

0.09% sodium azide;

wherein the composition has a pH of approximately pH 9.4.

33. A method of isolating a component of a fixed biological sample, the method comprising:

a. lysing the fixed biological sample according to the method of claim 24; and

b. isolating the component from the lysate.

34. The method of claim 33, wherein the component is a nucleic acid molecule.

35. The method of claim 34 wherein the nucleic acid molecule comprises a specific sequence.

36. The method of claim 35, wherein the nucleic acid is isolated according to a method comprising hybridizing a nucleic acid probe to the specific sequence of the nucleic acid molecule.

37. The method of claim 36, wherein the nucleic acid probe is adapted to be bound to a solid phase or is bound to a solid phase.

38. The method of claim 37, wherein the solid phase is a magnetic bead.

39. The method of claim 36, wherein the hybridizing of the nucleic acid probe to the specific sequence of the nucleic acid molecule results in the formation of a DNA:

RNA hybrid between the nucleic acid molecule and the nucleic acid probe.

40. The method of claim 39, wherein the DNA:

RNA hybrid is bound by a DNA;

RNA hybrid-binding antibody.

41. The method of claim 40, wherein the DNA:

RNA hybrid-binding antibody is bound to a solid phase or adapted to be bound to a solid phase.

42. The method of claim 41, wherein the solid phase is a magnetic bead.

43. The method of claim 34, wherein the nucleic acid molecule is a viral nucleic acid molecule.

44. The method of claim 33, wherein the fixed biological sample is a cervical sample.

45. A method of lysing a fixed biological sample, the method comprising treating the fixed biological sample with a composition of claim 12.

46. The method of claim 45, wherein the fixed biological sample is fixed with a cross-linking fixative agent.

47. The method of claim 46, wherein the cross-linking fixative agent comprises an aldehyde.

48. The method of claim 47, wherein the aldehyde is selected from the group consisting of formaldehyde and glutaraldehyde.

49. The method of claim 45, wherein the fixed biological sample is fixed with a fixative agent comprising an alcohol.

50. The method of claim 49, wherein the alcohol is selected from the group consisting of methanol and ethanol.

51. The method of claim 45, wherein the fixative agent is selected from the group consisting of SUREPATH and PRESERVECYT.

52. The method of claim 45, wherein the fixed biological sample is a cervical sample.

53. A method of isolating a component of a fixed biological sample, the method comprising:

a. lysing the fixed biological sample according to the method of claim 44; and

b. isolating the component from the lysate.

54. The method of claim 53, wherein the component is a nucleic acid molecule.

55. The method of claim 54 wherein the nucleic acid molecule comprises a specific sequence.

56. The method of claim 55, wherein the nucleic acid is isolated according to a method comprising hybridizing a nucleic acid probe to the specific sequence of the nucleic acid molecule.

57. The method of claim 56, wherein the nucleic acid probe is adapted to be bound to a solid phase or is bound to a solid phase.

58. The method of claim 57, wherein the solid phase is a magnetic bead.

59. The method of claim 56, wherein the hybridizing of the nucleic acid probe to the specific sequence of the nucleic acid molecule results in the formation of a DNA:

RNA hybrid between the nucleic acid molecule and the nucleic acid probe.

60. The method of claim 59, wherein the DNA:

RNA hybrid is bound by a DNA;

RNA hybrid-binding antibody.

61. The method of claim 60, wherein the DNA:

RNA hybrid-binding antibody is bound to a solid phase or adapted to be bound to a solid phase.

62. The method of claim 61, wherein the solid phase is a magnetic bead.

63. The method of claim 54, wherein the nucleic acid molecule is a viral nucleic acid molecule.

64. The method of claim 53, wherein the fixed biological sample is a cervical sample.

66. Use of a composition according to claim 12 for lysing a fixed biological sample.