Cloned genes encoding reverse transcriptase lacking RNase H activity

• US 6,610,522 B1
• Filed: 12/24/1998
• Issued: 08/26/2003
• Est. Priority Date: 01/13/1988
• Status: Expired due to Fees

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Kotewicz, Michael Leslie, Gerard, Gary Floyd
• Primary Examiner(s): Achutamurthy, Ponnathapu
• Assistant Examiner(s): Moore, William W.
• Application Number:
  US09/220,330
• Time in Patent Office: 1,706 Days
• Field of Search: 536/23.2, 435/194, 435/69.1, 435/252.3, 435/252.33, 435/320.1, 435/91.1, 435/91.2, 435/475, 435/975, 435/471
• US Class Current: 435/194
• CPC Class Codes: C12N 9/1276 RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Y 207/07049 RNA-directed DNA polymerase (2.7.7.49), i.e. telomerase or reverse-transcriptase
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 435/975 Kit

Abstract

The invention relates to a gene which encodes reverse transcriptase having DNA polymerase activity and substantially no RNase H activity. The invention also relates to vectors containing the gene and hosts transformed with the vectors of the invention. The invention also relates to a method of producing reverse transcriptase having DNA polymerase activity and substantially no RNase H activity by expressing the reverse transcriptase genes of the present invention in a host. The invention also relates to a method of producing cDNA from mRNA using the reverse transcriptase of the invention. The invention also relates to a kit for the preparation of cDNA from mRNA comprising the reverse transcriptase of the invention.

Claims

1. A composition, comprising

(a) an mRNA template;

and

(b) a retroviral reverse transcriptase having DNA polymerase activity, wherein said reverse transcriptase allows the mRNA template to remain intact during a one minute cDNA synthesis reaction by said reverse transcriptase as determined by gel electrophoresis and wherein said reverse transcriptase is modified within the RNase H domain.

2. The composition of claim 1, wherein said reverse transcriptase is encoded by a modified nucleotide sequence that encodes a modified amino acid sequence modified in the region corresponding to amino acids 498-611 of M-MLV reverse transcriptase.

3. The composition of claim 1, wherein said reverse transcriptase is encoded by a modified nucleotide sequence that encodes a modified amino acid sequence modified in the region corresponding to amino acids 503-611 of M-MLV reverse transcriptase.

4. The composition of claim 1, wherein said reverse transcriptase allows the mRNA template to remain intact during a 5 minute cDNA synthesis reaction as determined by gel electrophoresis.

5. The composition of claim 1, wherein said reverse transcriptase allows the mRNA template to remain intact during a 10 minute cDNA synthesis reaction as determined by gel electrophoresis.

6. The composition of claim 1, wherein said reverse transcriptase allows the mRNA template to remain intact during a 30 minute cDNA synthesis reaction as determined by gel electrophoresis.

7. The composition of claim 1, wherein said reverse transcriptase allows the mRNA template to remain intact during a 60 minute cDNA synthesis reaction as determined by gel electrophoresis.

8. A composition, comprising

(a) an mRNA template;

and

(b) an M-MLV reverse transcriptase having DNA polymerase activity, wherein said reverse transcriptase allows the mRNA template to remain intact during a one minute cDNA synthesis reaction by said reverse transcriptase as determined by gel electrophoresis and wherein said reverse transcriptase is modified within the RNase H domain.

9. The composition of claim 8, wherein said reverse transcriptase is encoded by a modified nucleotide sequence that encodes a modified amino acid sequence modified in the region of amino acids 498-611.

10. The composition of claim 8, wherein said reverse transcriptase is encoded by a modified nucleotide sequence that encodes a modified amino acid sequence modified in the region of amino acids 503-611.

11. The composition of claim 8, wherein said reverse transcriptase allows the mRNA template to remain intact during a 5 minute cDNA synthesis reaction as determined by gel electrophoresis.

12. The composition of claim 8, wherein said reverse transcriptase allows the mRNA template to remain intact during a 10 minute cDNA synthesis reaction as determined by gel electrophoresis.

13. The composition of claim 8, wherein said reverse transcriptase allows the mRNA template to remain intact during a 30 minute cDNA synthesis reaction as determined by gel electrophoresis.

14. The composition of claim 8, wherein said reverse transcriptase allows the mRNA template to remain intact during a 60 minute cDNA synthesis reaction as determined by gel electrophoresis.

15. A composition, comprising

(a) an mRNA template;

and

(b) a retroviral reverse transcriptase having DNA polymerase activity, wherein said reverse transcriptase has no detectable RNase H activity as determined by examining by gel electrophoresis the integrity of an mRNA template during a one minute cDNA synthesis reaction by said reverse transcriptase and wherein said reverse transcriptase is modified within the RNase H domain.

16. The composition of claim 15, wherein said reverse transcriptase is encoded by a modified nucleotide sequence that encodes a modified amino acid sequence modified in the region corresponding to amino acids 498-611 of M-MLV reverse transcriptase.

17. The composition of claim 15, wherein said reverse transcriptase is encoded by a modified nucleotide sequence that encodes a modified amino acid sequence modified in the region corresponding to amino acids 503-611 of M-MLV reverse transcriptase.

18. The composition of claim 15, wherein said no detectable RNase H activity is determined by examining by gel electrophoresis the integrity of the mRNA template during a 5 minute cDNA synthesis reaction.

19. The composition of claim 15, wherein said no detectable RNase H activity is determined by examining by gel electrophoresis the integrity of the mRNA template during a 10 minute cDNA synthesis reaction.

20. The composition of claim 15, wherein said no detectable RNase H activity is determined by examining by gel electrophoresis the integrity of the mRNA template during a 30 minute cDNA synthesis reaction.

21. The composition of claim 15, wherein said no detectable RNase H activity is determined by examining by gel electrophoresis the integrity of the mRNA template during a 60 minute cDNA synthesis reaction.

22. A composition, comprising

(a) an mRNA template;

and

(b) an M-MLV reverse transcriptase having DNA polymerase activity, wherein said reverse transcriptase has no detectable RNase H activity as determined by examining by gel electrophoresis the integrity of an mRNA template during a one minute cDNA synthesis reaction by said reverse transcriptase and wherein said reverse transcriptase is modified within the RNase H domain.

23. The composition of claim 22, wherein said reverse transcriptase is encoded by a modified nucleotide sequence that encodes a modified amino acid sequence modified in the region of amino acids 498-611.

24. The composition of claim 22, wherein said reverse transcriptase is encoded by a modified nucleotide sequence that encodes a modified amino acid sequence modified in the region of amino acids 503-611.

25. The composition of claim 22, wherein said no detectable RNase H activity is determined by examining by gel electrophoresis the integrity of the mRNA template during a 5 minute cDNA synthesis reaction.

26. The composition of claim 22, wherein said no detectable RNase H activity is determined by examining by gel electrophoresis the integrity of the mRNA template during a 10 minute cDNA synthesis reaction.

27. The composition of claim 22, wherein said no detectable RNase H activity is determined by examining by gel electrophoresis the integrity of the mRNA template during a 30 minute cDNA synthesis reaction.

28. The composition of claim 22, wherein said no detectable RNase H activity is determined by examining by gel electrophoresis the integrity of the mRNA template during a 60 minute cDNA synthesis reaction.

29. A composition, comprising

(a) an mRNA template;

and

(b) a retroviral reverse transcriptase having DNA polymerase activity and 0.23×

10³ Units/mg or less of RNase H activity wherein said reverse transcriptase is modified within the RNase H domain.

30. The composition of claim 29, wherein said reverse transcriptase is encoded by a modified nucleotide sequence that encodes a modified amino acid sequence modified in the region corresponding to 498-611 of M-MLV reverse transcriptase.

31. The composition of claim 29, wherein said reverse transcriptase is encoded by a modified nucleotide sequence that encodes a modified amino acid sequence modified in the region corresponding to amino acids 503-611 of M-MLV reverse transcriptase.

32. The composition of claim 29, wherein said reverse transcriptase has 0.1×

10³ Units/mg or less of RNase H activity.

33. The composition of claim 29, wherein said reverse transcriptase has 0.01×

10³ Units/mg or less of RNase H activity.

34. The composition of claim 29, wherein said DNA polymerase activity is at least 17.5×

10³ Units/mg.

35. The composition of claim 29, wherein said DNA polymerase activity is at least 81×

10³ Units/mg.

36. A composition, comprising

(a) an mRNA template;

and

(b) an M-MLV reverse transcriptase having DNA polymerase activity and 0.23×

10³ Units/mg or less of RNase H activity wherein said reverse transcriptase is modified within the RNase H domain.

37. The composition of claim 36, wherein said reverse transcriptase is encoded by a modified nucleotide sequence that encodes a modified amino acid sequence modified in the region of amino acids 498-611.

38. The composition of claim 36, wherein said reverse transcriptase is encoded by a modified nucleotide sequence that encodes a modified amino acid sequence modified in the region of amino acids 503-611.

39. The composition of claim 36, wherein said reverse transcriptase has 0.1×

10³ Units/mg or less of RNase H activity.

40. The composition of claim 36, wherein said reverse transcriptase has 0.01×

10³ Units/mg or less of RNase H activity.

41. The composition of claim 36, wherein said DNA polymerase activity is at least 17.5×

10³ Units/mg.

42. The composition of claim 36, wherein said DNA polymerase activity is at least 81×

10³ Units/mg.

85. A DNA molecule comprising a nucleotide sequence encoding the reverse transcriptase of any one of claims 42-49, 53-59, 63-69, and 73-79.

86. A vector comprising the DNA molecule of claim 85.

87. A host cell comprising the DNA molecule of claim 85.

88. The host cell of claim 87, wherein said host cell is E. coli.

89. A host cell comprising the vector of claim 86.

90. The host cell of claim 89, wherein said host cell is E. coli.

91. A method for preparing a DNA molecule, said method comprising:

mixing an mRNA template with the reverse transcriptase of any one of claims 42-81; and

incubating said mixture under conditions sufficient to make a first DNA molecule complementary to said mRNA template.

92. The method of claim 91, further comprising incubating said first DNA molecule under conditions sufficient to make a second DNA molecule complementary to said first DNA molecule.

93. The method of claim 91, wherein said first and second DNA molecule form a double stranded DNA molecule.

94. A method for producing a reverse transcriptase, said method comprising:

culturing the host cell of claim 87 under conditions sufficient to produce said reverse transcriptase; and

isolating or purifying said reverse transcriptase.

95. The method of claim 94, wherein said reverse transcriptase is isolated using column chromatography.

96. The method of claim 94, wherein said reverse transcriptase is greater than 90% pure.

97. A kit for the preparation of cDNA, said kit comprising a container containing the reverse transcriptase of any one of claims 42-81.

98. The kit of claim 97, said kit further comprising one or more additional containers selected from the group consisting of:

a container containing one or more nucleoside triphosphates;

a container containing an oligo(dT) primer; and

a container containing a buffer suitable for use in making cDNA.

43. A retroviral reverse transcriptase having DNA polymerase activity and less than 1,100 Units/mg RNase H activity wherein said reverse transcriptase is modified within the RNase H domain.

44. The reverse transcriptase of claim 43, wherein said reverse transcriptase is encoded by a modified nucleotide sequence that encodes a modified amino acid sequence modified in the region corresponding to amino acids 498-611 of M-MLV reverse transcriptase.

45. The reverse transcriptase of claim 43, wherein said reverse transcriptase is encoded by a modified nucleotide sequence that encodes a modified amino acid sequence modified in the region corresponding to amino acids 503-611 of M-MLV reverse transcriptase.

46. The reverse transcriptase of claim 43, wherein said reverse transcriptase may be used in the preparation of full-length cDNA.

47. The reverse transcriptase of claim 43, wherein said reverse transcriptase is a recombinantly produced reverse transcriptase.

48. The reverse transcriptase of claim 43, wherein said DNA polymerase activity is at least 81×

10³ Units/mg.

49. The reverse transcriptase of claim 43, wherein said DNA polymerase activity is at least 230×

10³ Units/mg.

50. The reverse transcriptase of claim 43, wherein said reverse transcriptase is isolated.

51. The reverse transcriptase of claim 43, wherein said reverse transcriptase is purified.

52. The reverse transcriptase of claim 43, wherein said reverse transcriptase is greater than 90% pure.

83. A composition, comprising the reverse transcriptase of any one of claims 43-82.

84. The composition of claim 83, wherein said composition further comprises at least one component selected from the group consisting of one or more nucleoside triphosphates, an oligo(dT) primer, a buffer and an mRNA template.

53. An M-MLV reverse transcriptase having DNA polymerase activity and less than 1,100 Units/mg RNase H activity wherein said reverse transcriptase is modified within the RNase H domain.

54. The reverse transcriptase of claim 53, wherein said reverse transcriptase is encoded by a modified nucleotide sequence that encodes a modified amino acid sequence modified in the region of amino acids 498-611.

55. The reverse transcriptase of claim 53, wherein said reverse transcriptase is encoded by a modified nucleotide sequence that encodes a modified amino acid sequence modified in the region of amino acids 503-611.

56. The reverse transcriptase of claim 53, wherein said reverse transcriptase may be used in the preparation of full-length cDNA.

57. The reverse transcriptase of claim 53, wherein said reverse transcriptase is a recombinantly produced reverse transcriptase.

58. The reverse transcriptase of claim 53, wherein said DNA polymerase activity is at least 81×

10³ Units/mg.

59. The reverse transcriptase of claim 53, wherein said DNA polymerase activity is at least 230×

10³ Units/mg.

60. The reverse transcriptase of claim 53, wherein said reverse transcriptase is isolated.

61. The reverse transcriptase of claim 53, wherein said reverse transcriptase is purified.

62. The reverse transcriptase of claim 53, wherein said reverse transcriptase is greater than 90% pure.

63. A retroviral reverse transcriptase having DNA polymerase activity of at least 17.5×

10³ Units/mg and less than 1,100 Units/mg RNase H activity wherein said reverse transcriptase is modified within the RNase H domain.

64. The reverse transcriptase of claim 63, wherein said reverse transcriptase is encoded by a modified nucleotide sequence that encodes a modified amino acid sequence modified in the region corresponding to amino acids 498-611 of M-MLV reverse transcriptase.

65. The reverse transcriptase of claim 63, wherein said reverse transcriptase is encoded by a modified nucleotide sequence that encodes a modified amino acid sequence modified in the region corresponding to amino acids 503-611 of M-MLV reverse transcriptase.

66. The reverse transcriptase of claim 63, wherein said reverse transcriptase may be used in the preparation of full-length cDNA.

67. The reverse transcriptase of claim 63, wherein said reverse transcriptase is a recombinantly produced reverse transcriptase.

68. The reverse transcriptase of claim 63, wherein said DNA polymerase activity is at least 81×

10³ Units/mg.

69. The reverse transcriptase of claim 63, wherein said DNA polymerase activity is at least 230×

10³ Units/mg.

70. The reverse transcriptase of claim 63, wherein said reverse transcriptase is isolated.

71. The reverse transcriptase of claim 63, wherein said reverse transcriptase is purified.

72. The reverse transcriptase of claim 63, wherein said reverse transcriptase is greater than 90% pure.

73. An M-MLV reverse transcriptase having DNA polymerase activity of at least 17.5×

10³ Units/mg and less than 1,100 Units/mg RNase H activity wherein said reverse transcriptase is modified within the RNase H domain.

74. The reverse transcriptase of claim 73, wherein said reverse transcriptase is encoded by a modified nucleotide sequence that encodes a modified amino acid sequence modified in the region of amino acids 498-611.

75. The reverse transcriptase of claim 73, wherein said reverse transcriptase is encoded by a modified nucleotide sequence that encodes a modified amino acid sequence modified in the region of amino acids 503-611.

76. The reverse transcriptase of claim 73, wherein said reverse transcriptase may be used in the preparation of full-length cDNA.

77. The reverse transcriptase of claim 73, wherein said reverse transcriptase is a recombinantly produced reverse transcriptase.

78. The reverse transcriptase of claim 73, wherein said DNA polymerase activity is at least 81×

10³ Units/mg.

79. The reverse transcriptase of claim 73, wherein said DNA polymerase activity is at least 230×

10³ Units/mg.

80. The reverse transcriptase of claim 73, wherein said reverse transcriptase is isolated.

81. The reverse transcriptase of claim 73, wherein said reverse transcriptase is purified.

82. The reverse transcriptase of claim 73, wherein said reverse transcriptase is greater than 90% pure.