Methods, compositions, and kits for detecting nucleic acids in a single vessel

• US 20060223071A1
• Filed: 04/01/2005
• Published: 10/05/2006
• Est. Priority Date: 04/01/2005
• Status: Abandoned Application

• Current Assignee: Eastman Chemical Company
• Original Assignee: Eastman Chemical Company
• Inventors: Boyd, Brendan William, Wisniewski, Michele Elizabeth, Barbour, William Mark
• Application Number:
  US11/096,775
• Publication Number:
  US 20060223071A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Abstract

The invention encompasses processes, compositions, and kits for isolating and detecting nucleic acids from samples using a metal oxide coated onto a vessel. The nucleic acids can be processed and detected within this vessel.

Claims

1. A method for the detection of a microorganism comprising:

(a) lysing a microorganism to release a nucleic acid molecule;

(b) contacting the nucleic acid molecule with a vessel coated with at least one metal oxide;

(c) allowing the nucleic acid molecule to attach to the metal oxide;

(d) amplifying, in the vessel, a fragment of the nucleic acid molecule attached to the metal oxide to generate an amplified product; and

(e) detecting, in the vessel, the amplified product.

2. The method of claim 1, wherein the microorganism is lysed in step (a) with a reagent that cleaves the peptidoglycan bonds on the outer membrane, a protease, a detergent, and an alkaline solution.

3. The method of claim 2, wherein the reagent that cleaves peptidoglycan bonds is lysozyme, lysostaphin, or mutanolysin.

4. The method of claim 3, wherein the reagent that cleaves peptidoglycan bonds is lysozyme.

5. The method of claim 2, wherein the protease is proteinase K, pronase E, achromopeptidase, proteinase R, proteinase T, subtilisin DY, an alkaline serine protease from Streptomyces griseus or Bacillus lichenformis, dispase, subtilisin Calsberg, subtilopeptidase A, or thermolysin.

6. The method of claim 5, wherein the protease is proteinase K.

7. The method of claim 2, wherein the detergent is t-octylphenoxypolyethoxyethanol (Triton X-100), polyoxyethylenesorbitan monolaurate (Tween-20), polyoxyethylenesorbitan monopalmitate (Tween-40), polyoxyethylenesorbitan monostearate (Tween-60), polyoxyethylenesorbitan monooleate (Tween-80), polyoxyethylenesorbitan monotrioleate (Tween-85), (octylphenoxy)polyethoxyethanol (IGEPAL CA-630), triethyleneglycol monolauryl ether (Brij 30), or sorbitan monolaurate (Span 20).

8. The method of claim 7, wherein the nonionic detergent is polyoxyethylenesorbitan monolaurate (Tween-20).

9. The method of claim 2, wherein the alkaline solution is 50 mM-500 mM KOH or NaOH.

10. The method of claim 1, wherein the microorganism is lysed in step (a) with a chaotropic salt or a cationic surfactant.

11. The method of claim 10, wherein the chaotropic agent is a guanidinium salt.

12. The method of claim 11, wherein the guanidinium salt is guanidine hydrochloride, guanidine thiocyanate, guanidinium chloride, guanidinium hydrochloride, or guanidinium isothiocyanate.

13. The method of claim 10, wherein the cationic surfactant is cetyltrimethylammonium bromide (CTAB), cetyltrimethylammonium chloride (CTACl), tetradecetyltrimethylammonium bromide (TTAB), tetradecetyltrimethylammonium chloride (TTACl), dodecyltrimethylammonium bromide (DTAB), dodecyltrimethylammonium chloride (DTACl), dodecylethyldimethylammonium chloride (DEDTAB), decyltrimethylammonium bromide (D10TAB), or dodecyltripheylphosphonium bromide (DTPB).

14. The method of claim 1, wherein the metal oxide is an oxide of aluminum, titanium, zirconium, hafnium, scandium, yttrium, lanthanum, vanadium, tantalum, chromium, molybdenum, tungsten, boron, gallium, indium, germanium, tin, zinc, nickel, magnesium, or iron.

15. The method of claim 14, wherein the metal oxide is aluminum oxide.

16. The method of claim 1, wherein the vessel is a tube, column, plate, or well.

17. The method of claim 16, wherein the vessel is a PCR tube.

18. The method of claim 1, where the attached nucleic acid molecule is washed with a buffer that comprises t-octylphenoxypolyethoxyethanol (Triton X-100), polyoxyethylenesorbitan monolaurate (Tween-20), polyoxyethylenesorbitan monopalmitate (Tween-40), polyoxyethylenesorbitan monostearate (Tween-60), polyoxyethylenesorbitan monooleate (Tween-80), polyoxyethylenesorbitan monotrioleate (Tween-85), (octylphenoxy)polyethoxyethanol (IGEPAL CA-630), triethyleneglycol monolauryl ether (Brij 30), sorbitan monolaurate (Span 20), methanol, ethanol, or isopropanol.

19. The method of claim 18, wherein the buffer comprises at least 5% w/w ethanol and at least 0.5% w/w polyoxyethylenesorbitan monolaurate (Tween-20).

20. The method of claim 1, wherein the amplified product is detected by hybridization to a probe.

21. The method of claim 20, wherein the amplified product is detected by hybridization to a labeled DNA, PNA, RNA, o-methyl RNA, LNA, or modified nucleotide probe.

22. The method of claim 21, wherein the probe is fluorescently labeled.

23. The method of claim 1, wherein the amplified product is detected with an intercalating dye.

24. The method of claim 1, wherein the nucleic acid molecule is amplified by the polymerase chain reaction (PCR).

25. The method of claim 24, wherein amplification is detected by hybridization using a probe.

26. The method of claim 25, wherein the amplified product is detected by hybridization to a labeled DNA, PNA, RNA, o-methyl RNA, LNA, or modified nucleotide probe.

27. The method of claim 26, wherein the probe is fluorescently labeled.

28. The method of claim 24, wherein amplification is detected with an intercalating dye.

29. The method of claim 24, wherein the amplified product is detected during amplification.

30. The method of claim 24, wherein the nucleic acid molecule is amplified with a fluorescently labeled primer.

39. A kit for detecting nucleic acids in biological sample comprising:

(a) a vessel coated with at least one metal oxide and

(b) instructions for practicing the method of claim 1.

40. The kit of claim 39, further comprising primers for amplifying a nucleic acid molecule bound to the metal oxide.

41. The kit of claim 39, further comprising a fluorescent probe.

42. The kit of claim 39, further comprising a lysozyme, a protease, a nonionic detergent, and an alkaline solution.

43. The kit of claim 39, wherein the metal oxide is an oxide of aluminum, titanium, zirconium, hafnium, scandium, yttrium, lanthanum, vanadium, tantalum, chromium, molybdenum, tungsten, boron, gallium, indium, germanium, tin, zinc, nickel, magnesium, or iron.

44. The kit of claim 43, wherein the metal oxide is aluminum oxide.

45. The kit of claim 39, wherein the vessel is a tube, column, plate, or well.

46. The kit of claim 45, wherein the vessel is a PCR tube.

31. A kit for detecting nucleic acids in biological sample comprising:

(a) a vessel coated with at least one metal oxide;

(b) instructions for binding a nucleic acid molecule to the metal oxide; and

(c) instructions for amplifying and detecting, in the vessel, a nucleic acid molecule bound to the metal oxide.

32. The kit of claim 31, further comprising primers for amplifying a nucleic acid molecule bound to the metal oxide.

33. The kit of claim 31, further comprising a fluorescent probe.

34. The kit of claim 31, further comprising a lysozyme, a protease, a nonionic detergent, and an alkaline solution.

35. The kit of claim 31, wherein the metal oxide is an oxide of aluminum, titanium, zirconium, hafnium, scandium, yttrium, lanthanum, vanadium, tantalum, chromium, molybdenum, tungsten, boron, gallium, indium, germanium, tin, zinc, nickel, magnesium, or iron.

36. The kit of claim 35, wherein the metal oxide is aluminum oxide.

37. The kit of claim 31, wherein the vessel is a tube, column, plate, or well.

38. The kit of claim 36, wherein the vessel is a PCR tube.

47. A method for the detection of a nucleic acid comprising:

(a) attaching a nucleic acid molecule to a metal oxide coated onto a vessel;

(b) amplifying, in the vessel, a fragment of the nucleic acid molecule to generate an amplified product; and

(c) detecting, in the vessel, the amplified product.

48. The method of claim 47, wherein the metal oxide is an oxide of aluminum, titanium, zirconium, hafnium, scandium, yttrium, lanthanum, vanadium, tantalum, chromium, molybdenum, tungsten, boron, gallium, indium, germanium, tin, zinc, nickel, magnesium, or iron.

49. The method of claim 47, wherein the metal oxide is aluminum oxide.

50. The method of claim 49, wherein the vessel is a tube, column, plate, or well.

51. The method of claim 50, wherein the vessel is a PCR tube.

52. The method of claim 47, wherein the amplified product is detected by hybridization to a probe.

53. The method of claim 52, wherein the amplified product is detected by hybridization to a labeled DNA, PNA, RNA, o-methyl RNA, LNA, or modified nucleotide probe.

54. The method of claim 53, wherein the probe is fluorescently labeled.

55. The method of claim 47, wherein the amplified product is detected with an intercalating dye.

56. The method of claim 47, wherein the nucleic acid molecule is amplified by the polymerase chain reaction (PCR).

57. The method of claim 56, wherein amplification is detected by hybridization using a probe.

58. The method of claim 57, wherein the amplified product is detected by hybridization to a labeled DNA, PNA, RNA, o-methyl RNA, LNA, or modified nucleotide probe.

59. The method of claim 58, wherein the probe is fluorescently labeled.

60. The method of claim 56, wherein amplification is detected with an intercalating dye.

61. The method of claim 56, wherein the amplified product is detected during amplification.

62. The method of claim 56, wherein the nucleic acid molecule is amplified with a fluorescently labeled primer.