Nuclease Inhibitor Cocktail

• US 20070148174A1
• Filed: 12/06/2006
• Published: 06/28/2007
• Est. Priority Date: 09/24/1999
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Ambion Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Pasloske, Brittan, Winkler, Matthew, Kudlicki, W.
• Granted Patent: US 7,445,901 B2
• Time in Patent Office: Days
• US Class Current: 424/146.100
• CPC Class Codes: C07K 16/40 against enzymes
• 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 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 1/34 involving hydrolase
• CPC Class Codes: G01N 2333/922 Ribonucleases (RNAses); Deoxyribonucleases (DNAses)
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes

Abstract

Methods and compositions for inhibiting and/or inactivating nucleases by employing nuclease inhibitors are provided. The nuclease inhibitors comprise anti-nuclease antibodies and non-antibody nuclease inhibitors. The anti-nuclease antibodies of the present invention may be a polyclonal or monoclonal antibodies, and may be anti-ribonuclease antibodies, anti-deoxyribonuclease antibodies, or antibodies to non-specific nucleases. A preferred embodiment comprises at least two nuclease inhibitors, and is referred to as a nuclease inhibitor cocktail. In some specific embodiments, the invention concerns methods of performing in vitro translation comprising obtaining a first nuclease inhibitor, which inhibitor is further defined as an anti-nuclease antibody, and placing the anti-nuclease antibody in an in vitro translation reaction. In many cases, the in vitro translation reaction comprises at least one nuclease, which may be a ribonuclease, a deoxyribonuclease, or a nonspecific nuclease. The invention also relates to kits for the performance of various microbiological procedures, which kits comprise the nuclease inhibitors described herein.

Claims

1-50. -50. (canceled)

51. A method comprising:

a) obtaining at least a first soluble anti-nuclease antibody;

b) obtaining at least a second soluble anti-nuclease antibody wherein said first and said at least second soluble anti-nuclease antibodies bind to different nucleases;

c) obtaining a composition; and

d) admixing the anti-nuclease antibodies and the composition to form an admixture;

wherein nucleases that may be present in the admixture are inhibited and

wherein at least one of the first and second soluble anti-nuclease antibodies is a monoclonal antibody.

52. The method of claim 51, wherein the first and the second soluble anti-nuclease antibodies are monoclonal antibodies.

53. The method of claim 51, wherein the first soluble anti-nuclease antibody is an anti-ribonuclease monoclonal antibody.

54. The method of claim 51, wherein the first soluble anti-nuclease antibody is an anti-RNase A monoclonal antibody and the second soluble anti-nuclease antibody is an anti-RNase 1 monoclonal antibody.

55. The method of claim 51, wherein the first soluble anti-nuclease antibody is an anti-RNase 1 monoclonal antibody and the second soluble anti-nuclease antibody is an anti-RNase T1 monoclonal antibody.

56. The method of claim 51, wherein the first soluble anti-nuclease antibody is an anti-RNase T1 monoclonal antibody and the second soluble anti-nuclease antibody is an anti-RNase A monoclonal antibody.

57. The method of claim 51, wherein the first soluble anti-nuclease antibody is an anti-deoxyribonuclease monoclonal antibody.

58. The method of claim 51, comprising obtaining at least an anti-RNase A antibody, an anti-RNase 1 antibody, and an anti-RNase T1 antibody and admixing them with the composition, wherein at least one of the anti-RNase A antibody, the anti-RNase 1 antibody, and the anti-RNase T1 antibody is a monoclonal antibody.

59. The method of claim 51, comprising obtaining at least an anti-RNase A monoclonal antibody, an anti-RNase 1 monoclonal antibody, and an anti-RNase T1 monoclonal antibody and admixing them with the composition.

60. A method comprising:

a) obtaining at least a first nuclease inhibitor further defined as a soluble anti-nuclease monoclonal antibody;

b) obtaining at least a second nuclease inhibitor further defined as a non-antibody nuclease inhibitor;

c) obtaining a composition; and

d) admixing the first nuclease inhibitor, the second nuclease inhibitor, and the composition to form an admixture;

wherein nucleases that may be present in the admixture are inhibited.

61. The method of claim 60, wherein the soluble anti-nuclease monoclonal antibody is an anti-ribonuclease monoclonal antibody.

62. The method of claim 61, wherein the soluble anti-ribonuclease monoclonal antibody is an anti-RNase 1 monoclonal antibody.

63. The method of claim 61, wherein the soluble anti-ribonuclease monoclonal antibody is an anti-RNase T1 monoclonal antibody.

64. The method of claim 61, wherein the soluble anti-ribonuclease monoclonal antibody is an anti-RNase A monoclonal antibody.

65. The method of claim 60, wherein the second nuclease inhibitor is human placental ribonuclease inhibitor.

66. A kit comprising:

a) a first soluble anti-ribonuclease monoclonal antibody that binds to at least a first ribonuclease;

b) at least one of;

a second soluble anti-nuclease antibody that binds to a nuclease that is different from the first nuclease, or

a non-antibody ribonuclease inhibitor; and

c) a reagent used in molecular biology.

67. The kit of claim 66 comprising:

a) a first soluble anti-ribonuclease monoclonal antibody that binds to at least a first ribonuclease;

b) a second soluble anti-nuclease antibody that binds to a nuclease that is different from the first nuclease; and

c) a reagent used in molecular biology.

68. The kit of claim 66 comprising:

a) a first soluble anti-ribonuclease monoclonal antibody that binds to at least a first ribonuclease;

b) a non-antibody ribonuclease inhibitor; and

c) a reagent used in molecular biology.

69. The kit of claim 66 wherein the first soluble anti-ribonuclease monoclonal antibody is an anti-RNase A monoclonal antibody, an anti-RNase T1 monoclonal antibody, or an anti-RNase 1 monoclonal antibody.

70. The kit of claim 68 wherein the non-antibody ribonuclease inhibitor is placental ribonuclease inhibitor.