DNA Polymerases and mutants thereof

• US 20070020622A1
• Filed: 09/16/2002
• Published: 01/25/2007
• Est. Priority Date: 09/14/2001
• Status: Abandoned Application

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Shandilya, Harini, Gerard, Gary, Gibbs, Moreland, Bergquist, Peter, Lee, Jun, Potter, Robert, Griffiths, Katherine
• Application Number:
  US10/244,081
• Publication Number:
  US 20070020622A1
• 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 9/1276 RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
• CPC Class Codes: C12N 9/14 Hydrolases (3)
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Y 306/01 in phosphorus-containing anhydrides (3.6.1)

Abstract

The present invention provides polypeptides having a nucleotide polymerase activity and method of enhancing polymerase activity. The polypeptides of the present invention may posses both a DNA-dependent DNA polymerase activity and an RNA-dependent DNA polymerase activity, i.e., a reverse transcriptase activity. The polypeptides of the present invention may be used in any application including, but not limited to, DNA sequencing reactions, amplification reactions, cDNA synthesis reactions, and combined cDNA synthesis and amplification reactions, e.g., RT-PCR.

Claims

1. An isolated nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 80% identical to forty contiguous amino acids disclosed in any one of Tables 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24, wherein the polypeptide has a nucleotide polymerase activity.

2. A nucleic acid according to claim 1, wherein the polypeptide encoded by the nucleic acid has both a DNA-dependent and an RNA-dependent nucleotide polymerase activity.

3. An isolated nucleic acid comprising a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid comprising a sequence complementary to a sequence of any one of Tables 1, 3 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23, and encodes a polypeptide having a nucleotide polymerase activity.

4. A nucleic acid according to claim 3, wherein the hybridization is under following conditions:

42°

C. in 50% formamide, a first wash at 65°

C. in 2×

SSC and 1% SDS, and a second wash at 65°

C. in 0.1×

SSC.

5. A nucleic acid according to claim 4, wherein the polypeptide encoded by the nucleic acid has both a DNA-dependent and an RNA-dependent nucleotide polymerase activity.

6. A polypeptide comprising an amino acid sequence that is at least 80% identical to forty contiguous amino acids disclosed in any one of Tables 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24, and mutants, fragments and fragments of mutants thereof wherein the polypeptide, mutant, fragment or fragment of mutant has a nucleotide polymerase activity.

7. A polypeptide according to claim 6, wherein the polypeptide has both a DNA-dependent and an RNA-dependent nucleotide polymerase activity.

8. A composition comprising a polypeptide according to claim 6.

9. A composition according to claim 8, further comprising a DNA polymerase.

10. A composition according to claim 9, wherein the DNA polymerase is a thermostable DNA polymerase.

11. A composition according to claim 9, wherein the DNA polymerase is selected from a group consisting of Taq DNA polymerase, Tne DNA polymerase, Tma DNA polymerase, Pfu DNA polymerase, Tfl DNA polymerase, Tth DNA polymerase, Thr DNA polymerase, Pwo DNA polymerase, Bst DNA polymerase, Bca DNA polymerase, VENT DNA polymerase, T7 DNA polymerase, T5 DNA polymerase, DNA polymerase III, Klenow fragment DNA polymerase, Stoffel fragment DNA polymerase, and mutants, fragments or derivatives thereof having DNA polymerase activity.

12. A composition according to claim 9, further comprising a nucleic acid molecule.

13. A composition according to claim 12, wherein the nucleic acid molecule is an mRNA.

14. A composition according to claim 13, further comprising an oligonucleotide primer.

15. A composition according to claim 14, wherein the primer has a sequence comprising at least 10 contiguous thymidine residues.

16. A method of sequencing a DNA molecule, comprising:

(a) hybridizing a primer to a first DNA molecule to form a complex;

(b) contacting the complex with deoxyribonucleoside triphosphates, a polypeptide according to claim 6, and a terminator molecule to form a mixture;

(c) incubating the mixture under conditions sufficient to synthesize a random population of DNA molecules complementary to the first DNA molecule and wherein the synthesized DNA molecules comprise a terminator nucleotide at their 3′

termini; and

(d) separating the synthesized DNA molecules by size so that at least a portion of the nucleotide sequence of the first DNA molecule can be determined.

17. A method according to claim 16, wherein the polypeptide is selected from the group consisting of polypeptides having a sequence in Tables 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24, mutants, fragments and fragments of mutants thereof, wherein the mutant, fragment, or fragment of a mutant has DNA polymerase activity.

18. A method according to claim 17, wherein the polypeptide has at least one mutation selected from the group consisting of (1) a mutation that reduces, substantially reduces or eliminates 5′

-3′

exonuclease activity of the DNA polymerase, (2) a mutation that results in the DNA polymerase becoming non-discriminating against dideoxynucleotides, and (3) a mutation that increases thermostability of an activity of the polypeptide.

19. A method according to claim 17, wherein the polypeptide has an O-helix comprising a mutation that results in the polypeptide becoming non-discriminating against dideoxynucleotides.

20. A method according to claim 19, wherein the mutation in the O-helix is a substitution of Phe at a position corresponding to position 754 of the polypeptide of Table 2 with an amino acid selected from the group consisting of Lys, Arg, His, Asp, Glu, Ala, Val, Ile, Leu, Pro, Met, Trp, Gly, Ser, Tyr, Cys, Thr, Asn, and Gln.

21. A method according to claim 19, wherein the mutation in the O-helix is a Phe to Tyr substitution at a position corresponding to position 754 of the polypeptide of Table 2.

22. A method according to claim 19, wherein the polypeptide further comprises an additional mutation that reduces, substantially reduces or eliminates 5′

-3′

exonuclease activity of the polypeptide.

23. A method according to claim 21, wherein the polypeptide further comprises an additional mutation that reduces, substantially reduces or eliminates 5′

-3′

exonuclease activity of the polypeptide.

24. A method according to claim 22, wherein the additional mutation is deletion of all or a portion of the amino acids corresponding to amino acids 1-304 of the polypeptide of Table 6.

25. A method according to claim 23, wherein the additional mutation is deletion of all or a portion of the amino acids corresponding to amino acids 1-304 of the polypeptide of Table 6.

26. A method according to 16, wherein the deoxyribonucleoside triphosphates are selected from the group consisting of:

dATP, dCTP, dGTP, dTTP, dITP, 7-deaza-dGTP, dUTP, [α

-S]dATP, [α

-S]dTTP, [α

-S]dGTP, and [α

-S]dCTP.

27. A method according to claim 16, wherein the terminator nucleotide is selected from the group consisting of:

ddTTP, ddATP, ddGTP, ddITP, and ddCTP.

28. A method of amplifying a double-stranded DNA molecule comprising:

(a) providing a first and second primer, wherein the first primer is complementary to a sequence of the first strand of the DNA molecule and the second primer is complementary to a sequence of the second strand of the DNA molecule;

(b) hybridizing the first primer to the first strand and the second primer to the second strand in the presence of a polypeptide according to claim 6, under conditions such that a third DNA molecule complementary to the first strand and a fourth DNA molecule complementary to the second strand are synthesized.

29. A method according to claim 28, further comprising:

(c) denaturing the first and third strand, the second and fourth strands; and

(d) repeating steps (a) to (c) one or more times.

30. A method according to claim 29, wherein the polypeptide is selected from the group consisting of polypeptides having a sequence in Tables 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24, mutants, fragments and fragments of mutants thereof, wherein the mutant, fragment, or fragment of a mutant has DNA polymerase activity.

31. A method according to claim 30, wherein the polypeptide has at least one mutation selected from the group consisting of (1) a mutation that reduces, substantially reduces or eliminates 5′

-3′

exonuclease activity of the DNA polymerase, (2) a mutation that results in the DNA polymerase becoming non-discriminating against dideoxynucleotides, and (3) a mutation that increases thermostability of an activity of the polypeptide.

32. A method according to claim 30, wherein the polypeptide has an O-helix comprising a mutation that results in the polypeptide becoming non-discriminating against dideoxynucleotides.

33. A method according to claim 32, wherein the mutation in the O-helix is a substitution of Phe at a position corresponding to position 754 of the polypeptide of Table 2 with an amino acid selected from the group consisting of Lys, Arg, His, Asp, Glu, Ala, Val, Ile, Leu, Pro, Met, Trp, Gly, Ser, Tyr, Cys, Thr, Asn, and Gln.

34. A method according to claim 32, wherein the mutation in the O-helix is a Phe to Tyr substitution at a position corresponding to position 754 of the polypeptide of Table 2.

35. A method according to claim 32, wherein the polypeptide further comprises an additional mutation that reduces, substantially reduces or eliminates 5′

-3′

exonuclease activity of the polypeptide.

36. A method according to claim 34, wherein the polypeptide further comprises an additional mutation that reduces, substantially reduces or eliminates 5′

-3′

exonuclease activity of the polypeptide.

37. A method according to claim 35, wherein the additional mutation is deletion of all or a portion of the amino acids corresponding to amino acids 1-304 of the polypeptide of Table 6.

38. A method according to claim 36, wherein the additional mutation is deletion of all or a portion of the amino acids corresponding to amino acids 1-304 of the polypeptide of Table 6.

39. A kit for sequencing a DNA molecule comprising a first container containing a polypeptide according to claim 6.

40. A kit according to claim 39, further comprising one or more containers selected from the group consisting of a second container containing one or more terminator nucleotides and a third container containing one or more deoxyribonucleoside triphosphates.

41. A kit according to claim 39, wherein the polypeptide is selected from the group consisting of polypeptides having a sequence in Tables 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24, mutants, fragments and fragments of mutants thereof, wherein the mutant, fragment, or fragment of a mutant has DNA polymerase activity.

42. A kit according to claim 41, wherein the polypeptide has at least one mutation selected from the group consisting of (1) a mutation that reduces, substantially reduces or eliminates 5′

-3′

exonuclease activity of the DNA polymerase, (2) a mutation that results in the DNA polymerase becoming non-discriminating against dideoxynucleotides, and (3) a mutation that increases thermostability of an activity of the polypeptide.

43. A kit according to claim 41, wherein the polypeptide has an O-helix comprising a mutation that results in the polypeptide becoming non-discriminating against dideoxynucleotides.

44. A kit according to claim 43, wherein the mutation in the O-helix is a substitution of Phe at a position corresponding to position 754 of the polypeptide of Table 2 with an amino acid selected from the group consisting of Lys, Arg, His, Asp, Glu, Ala, Val, Ile, Leu, Pro, Met, Trp, Gly, Ser, Tyr, Cys, Thr, Asn, and Gln.

45. A kit according to claim 43, wherein the mutation in the O-helix is a Phe to Tyr substitution at a position corresponding to position 754 of the polypeptide of Table 2.

46. A kit according to claim 43, wherein the polypeptide further comprises an additional mutation that reduces, substantially reduces or eliminates 5′

-3′

exonuclease activity of the polypeptide.

47. A kit according to claim 45, wherein the polypeptide further comprises an additional mutation that reduces, substantially reduces or eliminates 5′

-3′

exonuclease activity of the polypeptide.

48. A kit according to claim 46, wherein the additional mutation is deletion of all or a portion of the amino acids corresponding to amino acids 1-304 of the polypeptide of Table 6.

49. A kit according to claim 47, wherein the additional mutation is deletion of all or a portion of the amino acids corresponding to amino acids 1-304 of the polypeptide of Table 6.

50. A kit according to claim 40, wherein the deoxyribonucleoside triphosphates are selected from the group consisting of:

dATP, dCTP, dGTP, dTTP, dITP, 7-deaza-dGTP, dUTP, [α

-S]dATP, [α

-S]dTTP, [α

-S]dGTP, and [α

-S]dCTP.

51. A kit according to claim 40, wherein the terminator nucleotide is selected from the group consisting of:

ddTTP, ddATP, ddGTP, ddITP, and ddCTP.

52. A kit according to claim 39, wherein the kit further comprises a container containing a pyrophosphatase.

53. A kit for amplifying a DNA molecule, comprising a first container containing a polypeptide according to claim 6.

54. A kit according to claim 53, further comprising a second container containing one or more deoxyribonucleoside triphosphates.

55. A kit according to claim 53, wherein the polypeptide is selected from the group consisting of polypeptides having a sequence in Tables 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24, mutants, fragments and fragments of mutants thereof, wherein the mutant, fragment, or fragment of a mutant has DNA polymerase activity.

56. A kit according to claim 55, wherein the polypeptide has at least one mutation selected from the group consisting of (1) a mutation that reduces, substantially reduces or eliminates 5′

-3′

exonuclease activity of the DNA polymerase, (2) a mutation that results in the DNA polymerase becoming non-discriminating against dideoxynucleotides, and (3) a mutation that increases thermostability of an activity of the polypeptide.

57. A kit according to claim 55, wherein the polypeptide has an O-helix comprising a mutation that results in the polypeptide becoming non-discriminating against dideoxynucleotides.

58. A kit according to claim 57, wherein the mutation in the O-helix is a substitution of Phe at a position corresponding to position 754 of the polypeptide of Table 2 with an amino acid selected from the group consisting of Lys, Arg, His, Asp, Glu, Ala, Val, Ile, Leu, Pro, Met, Trp, Gly, Ser, Tyr, Cys, Thr, Asn, and Gln.

59. A kit according to claim 57, wherein the mutation in the O-helix is a Phe to Tyr substitution at a position corresponding to position 754 of the polypeptide of Table 2.

60. A kit according to claim 57, wherein the polypeptide further comprises an additional mutation that reduces, substantially reduces or eliminates 5′

-3′

exonuclease activity of the polypeptide.

61. A kit according to claim 59, wherein the polypeptide further comprises an additional mutation that reduces, substantially reduces or eliminates 5′

-3′

exonuclease activity of the polypeptide.

62. A kit according to claim 60, wherein the additional mutation is deletion of all or a portion of the amino acids corresponding to amino acids 1-304 of the polypeptide of Table 6.

63. A kit according to claim 61, wherein the additional mutation is deletion of all or a portion of the amino acids corresponding to amino acids 1-304 of the polypeptide of Table 6.

64. A kit according to claim 53, wherein the deoxyribonucleoside triphosphates are selected from the group consisting of:

DATP, dCTP, dGTP, dTTP, dITP, 7-deaza-dGTP, dUTP, [α

-S]dATP, [α

-S]dTTP, [α

-S]dGTP, and [α

-S]dCTP.

65. A kit according to claim 53, wherein the kit further comprises a container containing a pyrophosphatase.

66. A method for synthesizing a DNA molecule comprising:

(a) hybridizing a primer to a first nucleic acid molecule to form a complex; and

(b) incubating the complex in the presence of a polypeptide according to claim 6, and one or more deoxyribonucleoside triphosphates under conditions sufficient to synthesize a second DNA molecule complementary to all or a portion of the first nucleic acid molecule.

67. A method according to claim 66, wherein the primer and/or one or more of the deoxyribonucleoside triphosphates are fluorescently labeled.

68. A method according to claim 66, wherein the polypeptide is selected from the group consisting of polypeptides having a sequence in Tables 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24, mutants, fragments and fragments of mutants thereof, wherein the mutant, fragment, or fragment of a mutant has DNA polymerase activity.

69. A method according to claim 68, wherein the polypeptide has at least one mutation selected from the group consisting of (1) a mutation that reduces, substantially reduces or eliminates 5′

-3′

exonuclease activity of the DNA polymerase, (2) a mutation that results in the DNA polymerase becoming non-discriminating against dideoxynucleotides, and (3) a mutation that increases thermostability of an activity of the polypeptide.

70. A method according to claim 68, wherein the polypeptide has an O-helix comprising a mutation that results in the polypeptide becoming non-discriminating against dideoxynucleotides.

71. A method according to claim 70, wherein the mutation in the O-helix is a substitution of Phe at a position corresponding to position 754 of the polypeptide of Table 2 with an amino acid selected from the group consisting of Lys, Arg, His, Asp, Glu, Ala, Val, Ile, Leu, Pro, Met, Trp, Gly, Ser, Tyr, Cys, Thr, Asn, and Gln.

72. A method according to claim 70, wherein the mutation in the O-helix is a Phe to Tyr substitution at a position corresponding to position 754 of the polypeptide of Table 2.

73. A method according to claim 70, wherein the polypeptide further comprises an additional mutation that reduces, substantially reduces or eliminates 5′

-3′

exonuclease activity of the polypeptide.

74. A method according to claim 72, wherein the polypeptide further comprises an additional mutation that reduces, substantially reduces or eliminates 5′

-3′

exonuclease activity of the polypeptide.

75. A method according to claim 73, wherein the additional mutation is deletion of all or a portion of the amino acids corresponding to amino acids 1-304 of the polypeptide of Table 6.

76. A method according to claim 74, wherein the additional mutation is deletion of all or a portion of the amino acids corresponding to amino acids 1-304 of the polypeptide of Table 6.

77. A method according to 66, wherein the deoxyribonucleoside triphosphates are selected from the group consisting of:

DATP, dCTP, dGTP, dTTP, dITP, 7-deaza-dGTP, dUTP, [α

-S]dATP, [α

-S]dTTP, [α

-S]dGTP, and [α

-S]dCTP.

89. A method of amplifying a double-stranded DNA molecule, comprising:

(a) providing a first and a second primer, wherein the first primer is complementary to a sequence of the first strand of the DNA molecule and the second primer is complementary to a sequence of the second strand of the DNA molecule;

(b) hybridizing the first primer to the first strand and the second primer to the second strand in the presence of a polypeptide according to claim 6, under conditions such that a third DNA molecule complementary to the first strand and a fourth DNA molecule complementary to the second strand are synthesized;

(c) denaturing the first and third strands, and the second and fourth strands; and

optionally

(d) repeating steps (a) to (c) one or more times.

90. A kit for sequencing a DNA molecule comprising a container containing a polypeptide according to claim 6 and one or more containers containing one or more of the following:

(a) one or more dideoxyribonucleoside triphosphates; and

(b) one or more deoxyribonucleoside triphosphates;

wherein the Clostridium DNA polymerase is selected from the group consisting of a mutant Clostridium DNA polymerase according to claim 78, and a fragment of the mutant Clostridium DNA polymerase, wherein the fragment has DNA polymerase activity.

91. A kit for sequencing according to claim 90, wherein the kit further comprises a container containing a pyrophosphatase.

92. A kit for amplifying a DNA molecule, comprising a container containing a polypeptide according to claim 6 and one or more containers containing one or more of the following:

(a) one or more buffers or buffer salts; and

(b) one or more deoxyribonucleoside triphosphates.

93. A kit for amplifying according to claim 92, wherein the kit further comprises a container containing a pyrophosphatase.

94. A method for synthesizing a DNA molecule comprising:

(a) hybridizing a primer to a first nucleic acid molecule to form a complex; and

(b) incubating the complex in the presence of a polypeptide according to claim 6 and one or more deoxyribonucleoside triphosphates under conditions sufficient to synthesize a second DNA molecule complementary to all or a portion of the first DNA molecule.

95. A method of synthesizing a DNA molecule according to claim 94, wherein the primer and/or one or more of the deoxyribonucleoside triphosphates are fluorescently labeled.

96. A method for reverse transcribing RNA into complementary DNA (cDNA) and amplifying the cDNA, comprising

(a) hybridizing a first primer to the RNA molecule in the presence of a polypeptide according to claim 6 to form a reaction mixture;

(b) incubating the reaction mixture under conditions such that a cDNA molecule complementary to the target RNA is synthesized;

(c) treating the reaction mixture to provide single stranded cDNA;

(d) hybridizing a second primer to the cDNA molecule in the presence of the DNA polymerase of the invention, under conditions such that an extension product is synthesized to provide a double-stranded cDNA molecule; and

(e) amplifying the double-stranded cDNA molecule of (d) by a polymerase chain reaction.

97. A kit for RT/PCR, comprising a first container containing a polypeptide according to claim 6;

and one or more containers selected from the group consisting of a second container containing one or more deoxyribonucleoside triphosphates and a third container containing a thermostable DNA polymerase.

78. A polypeptide having a sequence in Table 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24, and fragments thereof, wherein the polypeptide has a substitution of an amino acid corresponding to Arg at position 724 of the polymerase in Table 2 with an amino acid selected from the group consisting of Asp, Glu, Ala, Val, Leu, Ile, Pro, Met, Phe, Trp, Gly, Ser, Thr, Cys, Tyr, Gln, Asn, Lys, and His.

79. A polypeptide according to claim 78, wherein the mutation is a substitution of an amino acid corresponding to Arg at position 724 of the polymerase in Table 2 with an amino acid selected from the group consisting of His, Lys, Tyr, and Ala.

80. A polypeptide according to claim 78, wherein the DNA polymerase further comprises a mutation that reduces, substantially reduces or eliminates 5′

-3′

exonuclease activity of the polypeptide, wherein the mutation is in the 5′

-3′

exonuclease domain of the polypeptide.

81. A recombinant nucleic acid molecule encoding the polypeptide of claim 78.

82. A host cell comprising a recombinant nucleic acid molecule encoding the polypeptide of claim 78.

83. A method of producing a polypeptide, comprising:

(a) culturing a host cell as claimed in claim 82;

(b) expressing the polypeptide; and

(c) isolating the polypeptide.

84. A method of sequencing a DNA molecule comprising:

(a) hybridizing a primer to a first DNA molecule to form a complex;

(b) contacting the complex with deoxyribonucleoside triphosphates, a polypeptide according to claim 78, and a terminator molecule to form a mixture;

(c) incubating the mixture under conditions sufficient to synthesize a random population of DNA molecules complementary to the first DNA molecule and wherein the synthesized DNA molecules comprise a terminator at their 3′

termini; and

(d) separating the synthesized DNA molecules by size so that at least a portion of the nucleotide sequence can be determined.

85. A method of sequencing according to claim 84, wherein the deoxyribonucleoside triphosphates are selected from the group consisting of:

dATP, dCTP, dGTP, dTTP, dITP, 7-deaza-dGTP, dUTP, [α

-S]dATP, [α

-S]dTTP, [α

-S]dGTP, and [α

-S]dCTP.

86. A method of sequencing according to claim 84, wherein the terminator nucleotide is selected from the group consisting of:

ddTTP, ddATP, ddGTP, ddITP, or ddCTP.

87. A method of sequencing according to claim 84, wherein the primer comprises a detectable moiety.

88. A method of sequencing according to claim 87, wherein the detectable moiety is a fluorescent moiety.

98. A nucleic acid polymerase having an RNA-dependent DNA polymerase activity, wherein the activity occurs in the presence of manganese and/or magnesium.

107. A nucleic acid polymerase according to claim 98, wherein the activity occurs in the presence of manganese at a concentration of from about 0.1 to 5.0 mM.

108. A nucleic acid polymerase according to claim 98, wherein the activity occurs in the presence of manganese at a concentration of from about 0.5 to 3 mM.

109. A nucleic acid polymerase according to claim 98, wherein the activity occurs in the presence of manganese at a concentration of from about 1 to 2.5 mM.

99. A nucleic acid polymerase having an RNA-dependent DNA polymerase activity, wherein the activity occurs in the presence of a manganese/magnesium ratio>

1.

100. A nucleic acid polymerase according to claim 99, wherein the activity occurs in the presence of a manganese/magnesium ratio of at least 2.

101. A nucleic acid polymerase according to claim 99, wherein the activity occurs in the presence of a manganese/magnesium ratio of at least 4.

102. A nucleic acid polymerase according to any one of claims 98-101, wherein the polymerase further has a DNA-dependent DNA polymerase, the DNA-dependent DNA polymerase activity occurring under the same manganese/magnesium ratios as the RNA-dependent DNA polymerase activity.

110. A nucleic acid polymerase according to any one of claims 98-101 and 103-109, wherein the activity occurs in the presence of magnesium at a concentration of from about 0 to about 2 mM.

111. A nucleic acid polymerase according to claim 102, wherein the activities occur in the presence of magnesium at a concentration of from about 0 to about 2 mM.

112. A nucleic acid polymerase according to claim 110, wherein the activity occurs in the absence of magnesium.

113. A nucleic acid polymerase according to claim 111, wherein the activity occurs in the absence of magnesium.

103. A nucleic acid polymerase having a DNA-dependent DNA polymerase activity, wherein the activity occurs in the presence of manganese and/or magnesium.

104. A nucleic acid polymerase having a DNA-dependent DNA polymerase activity, wherein the activity occurs in the presence of a manganese/magnesium ratio>

1.

105. A nucleic acid polymerase according to claim 104, wherein the activity occurs in the presence of a manganese/magnesium ratio of at least about 2.

106. A nucleic acid polymerase according to claim 105, wherein the first activity occurs in the presence of a manganese/magnesium ratio of at least about 4.

114. A polypeptide having an RNA-dependent DNA polymerase specific activity and a DNA-dependent DNA polymerase specific activity, wherein the ratio of RNA-dependent DNA polymerase specific activity to DNA-dependent DNA polymerase specific activity is greater than about 0.05.

115. A polypeptide according to claim 114, wherein the ration is greater than about 0.01.

116. A polypeptide according to claim 114, wherein the ratio is greater than about 0.2.

117. A polypeptide having an RNA-dependent DNA polymerase specific activity and a DNA-dependent DNA polymerase specific activity, wherein the RNA-dependent specific activity is greater than about 500 units/mg polypeptide.

118. A polypeptide according to claim 117, wherein the DNA-dependent DNA polymerase specific activity is greater than about 10,000 units/mg polypeptide.

119. A polypeptide according to claim 118, wherein the RNA-dependent DNA polymerase activity is greater than about 1,000 units/mg polypeptide.

120. A polypeptide according to claim 118, wherein the RNA-dependent DNA polymerase activity is greater than about 2,000 units/mg polypeptide.

121. A polypeptide according to claim 118, wherein the RNA-dependent DNA polymerase activity is greater than about 3,000 units/mg polypeptide.

122. A polypeptide according to claim 118, wherein the RNA-dependent DNA polymerase activity is greater than about 4,000 units/mg polypeptide.