Lyophilized beads containing mannitol

• US 20050069898A1
• Filed: 09/25/2003
• Published: 03/31/2005
• Est. Priority Date: 09/25/2003
• Status: Abandoned Application

• Current Assignee: Cepheid (Danaher Corporation)
• Original Assignee: Cepheid (Danaher Corporation)
• Inventors: Valdez, Johnny, Jones, Martin, Moon, Byung Sook
• Application Number:
  US10/672,266
• Publication Number:
  US 20050069898A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12N 9/98 Preparation of granular or free-flowing enzyme compositions C12N9/96 takes precedence
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2545/101 with an internal standard/control

Abstract

Mannitol in certain weight percentages can be used to produce lyophilized beads of consistent size, consistent morphology, and reduced moisture content. These mannitol-containing lyophilized beads are useful in a variety of biological applications where precise reagent amounts are required or moisture-sensitive components are utilized. PCR technologies represent one biological application where mannitol-containing lyophilized beads can be used.

Claims

1. A lyophilized bead suitable for use in the amplification of a nucleic acid sequence, said lyophilized bead comprising:

a thermally stable enzyme; and

mannitol;

wherein said lyophilized bead has a weight percentage of said mannitol of between about 53% and about 75% (w/w).

2. The lyophilized bead of claim 1, wherein said amplification occurs in a reaction mixture comprising a volume of between about 5 μ

L and about 200 μ

L.

3. The lyophilized bead of claim 1, further comprising a nucleoside triphosphate or a derivative thereof.

4. The lyophilized bead of claim 1, wherein said lyophilized bead has an average cross-section of between about 1 millimeter and about 4.5 millimeters.

5. The lyophilized bead of claim 1, wherein said weight percentage is between about 62% and about 75% (w/w).

6. The lyophilized bead of claim 5, wherein said weight percentage is between about 68% and about 75% (w/w).

7. The lyophilized bead of claim 1, wherein said thermally stable enzyme is selected from the group consisting of polymerase, ligase, and combinations thereof.

8. The lyophilized bead of claim 1, further comprising a hot start methodology.

9. The lyophilized bead of claim 1, further comprising HEPES.

10. The lyophilized bead of claim 1, further comprising a probe.

11. The lyophilized bead of claim 1, further comprising a reverse transcriptase.

12. The lyophilized bead of claim 1, further comprising an internal control.

13. A lyophilized bead suitable for use in the amplification of a nucleic acid sequence, said lyophilized bead comprising:

a forward polynucleotide primer;

a reverse polynucleotide primer; and

mannitol;

wherein said lyophilized bead has a weight percentage of said mannitol of between about 53% and about 75% (w/w).

14. The lyophilized bead of claim 13, wherein said amplification occurs in a reaction mixture comprising a volume of between about 5 μ

L and about 200 μ

L.

15. The lyophilized bead of claim 13, wherein said lyophilized bead has an average cross-section of between about 1 millimeter and about 4.5 millimeters.

16. The lyophilized bead of claim 13, wherein said weight percentage is between about 62% and about 75% (w/w).

17. The lyophilized bead of claim 16, wherein said weight percentage is between about 68% and about 75% (w/w).

18. The lyophilized bead of claim 13, further comprising HEPES.

19. The lyophilized bead of claim 13, further comprising a probe.

20. The lyophilized bead of claim 13, further comprising an internal control.

21. The lyophilized bead of claim 13, wherein said nucleic acid sequence is selected from the group consisting of bacterial, fungal, and viral nucleic acid sequences.

22. The lyophilized bead of claim 21, wherein said bacterial nucleic acid sequence is derived from a member selected from the group consisting of Bacillus Anthracis, Yersinia pestis, Clostridium botulinum, Francisella tularensis, Group B Streptococcus, Neisseria gonorrhoeae, Chlamydia trachomatis, and Xylella fastidiosa.

23. The lyophilized bead of claim 21, wherein said viral nucleic acid sequence is derived from a member selected from the group consisting of Vaccinia, West Nile Fever virus, Equine Encephalitis virus, and Foot and Mouth Disease virus.

24. A method for the amplification of a nucleic acid sequence, said method comprising:

(a) dissolving a lyophilized bead in a liquid, wherein said lyophilized bead comprises;

a thermally stable enzyme; and

mannitol;

wherein said lyophilized bead has a weight percentage of said mannitol of between about 53% and about 75% (w/w), thus forming a reaction mixture; and

(b) subjecting said reaction mixture to an amplification reaction.

25. The method of claim 24, wherein said reaction mixture further comprises a volume of between about 5 μ

L and about 200 μ

L.

26. The method of claim 24, wherein said reaction mixture further comprises a nucleoside triphosphate or a derivative thereof.

27. The method of claim 24, wherein said thermally stable enzyme is selected from the group consisting of polymerase, ligase, and combinations thereof.

28. The method of claim 24, wherein said reaction mixture further comprises a forward polynucleotide primer.

29. The method of claim 24, wherein said reaction mixture further comprises a reverse polynucleotide primer.

30. The method of claim 24, wherein said reaction mixture further comprises a probe.

31. The method of claim 24, wherein said reaction mixture further comprises a nucleic acid comprising said nucleic acid sequence.

32. The method of claim 24, wherein said reaction mixture further comprises HEPES.

33. The method of claim 24, wherein said reaction mixture further comprises an internal control.

34. The method of claim 24, wherein said reaction mixture further comprises a hot start methodology.

35. The method of claim 24, wherein said lyophilized bead has an average cross-section of between about 1 millimeter and about 4.5 millimeters.

36. A method for the amplification of a nucleic acid sequence, said method comprising:

(a) dissolving a lyophilized bead in a liquid, wherein said lyophilized bead comprises;

a forward polynucleotide primer;

a reverse polynucleotide primer; and

mannitol; and

wherein said lyophilized bead has a weight percentage of said mannitol of between about 53% and about 75% (w/w), thus forming a reaction mixture; and

(b) subjecting said reaction mixture to an amplification reaction.

37. The method of claim 36, wherein said reaction mixture further comprises a volume of between about 5 μ

L and about 200 μ

L.

38. The method of claim 36, wherein said reaction mixture further comprises a nucleoside triphosphate or a derivative thereof.

39. The method of claim 36, wherein said reaction mixture further comprises a probe.

40. The method of claim 36, wherein said reaction mixture further comprises a nucleic acid comprising said nucleic acid sequence.

41. The method of claim 36, wherein said reaction mixture further comprises HEPES.

42. The method of claim 36, wherein said reaction mixture further comprises a thermally stable enzyme.

43. The method of claim 36, wherein said reaction mixture further comprises an internal control.

44. The method of claim 36, wherein said lyophilized bead has an average cross-section of between about 1 millimeter and about 4.5 millimeters.

45. A lyophilized bead suitable for use in the amplification of a nucleic acid sequence, prepared by a process comprising:

(a) creating an aqueous solution, said aqueous solution comprising;

a thermally stable enzyme; and

mannitol;

wherein said solution has a concentration of said mannitol between about 0.38 M (moles of mannitol/liter of solution) and about 0.99 M (moles of mannitol/liter of solution);

(b) quick-freezing the product of (a); and

(c) freeze-drying the product of (b).

46. The lyophilized bead of claim 45, wherein the product of (c) has an average cross-section of between about 1 millimeter and about 4.5 millimeters.

47. The lyophilized bead of claim 45, wherein the product of (c) further comprises a nucleoside triphosphate or a derivative thereof.

48. The lyophilized bead of claim 45, wherein said thermally stable enzyme is selected from the group consisting of polymerase, ligase, and combinations thereof.

49. The lyophilized bead of claim 45, wherein the product of (c) further comprises a reverse transcriptase.

50. The lyophilized bead of claim 45, wherein the product of (c) further comprises a hot start methodology.

51. The lyophilized bead of claim 45, wherein the product of (c) further comprises HEPES.

52. The lyophilized bead of claim 45, wherein the product of (c) further comprises a probe.

53. The lyophilized bead of claim 45, wherein the product of (c) further comprises an internal control.

54. A lyophilized bead suitable for use in the amplification of a nucleic acid sequence, prepared by a process comprising:

(a) creating an aqueous solution, said aqueous solution comprising;

a forward polynucleotide primer;

a reverse polynucleotide primer; and

mannitol;

wherein said solution has a concentration of said mannitol between about 0.38 M (moles of mannitol/liter of solution) and about 0.99 M (moles of mannitol/liter of solution);

(b) quick-freezing the product of (a); and

(c) freeze-drying the product of (b).

55. The lyophilized bead of claim 54, wherein the product of (c) has an average cross-section of between about 1 millimeter and about 4.5 millimeters.

56. The lyophilized bead of claim 54, wherein the product of (c) further comprises a nucleoside triphosphate or a derivative thereof.

57. The lyophilized bead of claim 54, wherein the product of (c) further comprises HEPES.

58. The lyophilized bead of claim 54, wherein the product of (c) further comprises a probe.

59. The lyophilized bead of claim 54, wherein the product of (c) further comprises an internal control.

60. A lyophilized bead suitable for use in microanalytic systems comprising:

a moisture-sensitive reactant; and

mannitol;

wherein said lyophilized bead has a weight percentage of said mannitol of between about 53% and about 75% (w/w); and

wherein said lyophilized bead has an average cross-section of between about 1 millimeter and about 4.5 millimeters.

61. The lyophilized bead of claim 60, wherein said weight percentage is between about 62% and about 75% (w/w).

62. The lyophilized bead of claim 60, wherein said weight percentage is between about 68% and about 75% (w/w).