Cloned genes encoding reverse transcriptase lacking RNase H activity

• US 6,589,768 B1
• Filed: 12/24/1998
• Issued: 07/08/2003
• 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:
  US09/220,329
• Time in Patent Office: 1,657 Days
• Field of Search: 536/23.2, 435/194, 435/91.1, 435/91.2, 435/69.1, 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 retroviral reverse transcriptase having DNA polymerase activity, wherein said reverse transcriptase allows an mRNA template to remain intact during cDNA synthesis by said reverse transcriptase and wherein said reverse transcriptase is modified within the RNase H domain.

2. The reverse transcriptase of claim 1, wherein said reverse transcriptase is selected from the group consisting of M-MLV, HTLV-1, BLV<

RSV and HIV reverse transcriptase.

3. The reverse transcriptase 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.

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

5. The reverse transcriptase of claim 1, wherein said reverse transcriptase may be used in the preparation of full-length cDNA.

6. The reverse transcriptase of claim 1, wherein said reverse transcriptase is a recombinantly produced reverse transcriptase.

7. The reverse transcriptase of claim 1, wherein said reverse transcriptase is purified.

8. The reverse transcriptase of claim 7, wherein said reverse transcriptase is greater than 90% pure.

9. The reverse transcriptase of claim 1, wherein said reverse transcriptase is isolated.

10. The reverse transcriptase of claim 9, wherein said reverse transcriptase is isolated using column chromatography.

11. The reverse transcriptase of claim 9, wherein said reverse transcriptase is isolated using DEAE-cellulose.

12. The reverse transcriptase of claim 9, wherein said reverse transcriptase is isolated using heparin agarose.

13. The reverse transcriptase of claim 9, wherein said reverse transcriptase is isolated using hydroxylapatite.

14.The reverse transcriptase of claim 1, wherein said reverse transcriptase has no detectable RNase H activity.

16. The reverse transcriptase of claim 1, wherein said reverse transcriptase lacks RNase H activity.

19. The reverse transcriptase of claim 1, wherein said reverse transcriptase has 0.23×

10³ units/mg or less of RNase H activity.

20. The reverse transcriptase of claim 1, wherein said reverse transcriptase has 0.1×

10³ units/mg or less of RNase H activity.

21. The reverse transcriptase of claim 1, wherein said reverse transcriptase has 0.01×

10³ units/mg or less of RNase H activity.

22. The reverse transcriptase of claim 1, wherein said DNA polymerase activity is at least 1.75×

10³ units/mg.

23. The reverse transcriptase of claim 1, wherein said DNA polymerase activity is at least 81×

10³ units/mg.

24. The reverse transcriptase of claim 1, wherein said reverse transcriptase allows an mRNA template to remain intact during cDNA synthesis as determined by gel electrophoresis.

150. A DNA molecule comprising a nucleotide sequence encoding the reverse transcriptase of any one of claims 1 to 5, 14 to 45 and 54 to 150.

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

mixing an mRNA template with the reverse transcriptase of any one of claims 1 to 150; and

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

162. A kit for the preparation of cDNA, said kit comprising a container containing the reverse transcriptase of any one of claims 1 to 150.

164. A composition comprising the reverse transcriptase of any one of claims 1-4, 26-44 and 66-124.

14. The reverse transcriptase of claim 14, wherein said no detectable RNase H activity is determined by examining the integrity of an mRNA template during DNA synthesis.

15. The reverse transcriptase of claim 14, wherein said no detectable RNase H activity is determined by gel electrophoresis.

17. The reverse transcriptase of claim 17, wherein said lack of RNase H activity is determined by examining the integrity of an mRNA template during cDNA synthesis.

18. The reverse transcriptase of claim 17, wherein said lack of RNase H activity is determined by gel electrophoresis.

25. A retroviral reverse transcriptase having DNA polymerase activity, wherein said reverse transcriptase allows an mRNA template to remain intact during a one minute cDNA synthesis reaction by said reverse transcriptase and wherein said reverse transcriptase is modified within the RNase H domain.

26. The reverse transcriptase of claim 26, 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.

27. The reverse transcriptase of claim 26, 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.

28. A retroviral reverse transcriptase having DNA polymerase activity, wherein said reverse transcriptase allows an mRNA template to remain intact during a 5 minute cDNA synthesis reaction by said reverse transcriptase and wherein said reverse transcriptase is modified within the RNase H domain.

29. A retroviral reverse transcriptase having DNA polymerase activity, wherein said reverse transcriptase allows an mRNA template to remain intact during a 10 minute cDNA synthesis reaction by said reverse transcriptase and wherein said reverse transcriptase is modified within the RNase H domain.

30. A retroviral reverse transcriptase having DNA polymerase activity, wherein said reverse transcriptase allows an mRNA template to remain intact during a 30 minute cDNA synthesis reaction by said reverse transcriptase and wherein said reverse transcriptase is modified with the RNase H domain.

31. A retroviral reverse transcriptase having DNA polymerase activity, wherein said reverse transcriptase allows an mRNA template to remain intact during a 60 minute cDNA synthesis reaction by said reverse transcriptase and wherein said reverse transcriptase is modified within the RNase H domain.

32. A retroviral reverse transcriptase having DNA polymerase activity, wherein said reverse transcriptase allows an 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.

33. The reverse transcriptase of claim 33, wherein said DNA polymerase activity is at least 17.5×

10³ units/mg.

34. The reverse transcriptase of claim 33, wherein said DNA polymerase activity is at least 81×

10³ units/mg.

35. The reverse transcriptase of claim 33, wherein said reverse transcriptase is encoded by a modified nucleotide sequence that encodes a modified amino acids sequenced modified in the region corresponding to amino acids 498-611 of M-MLV reverse transcriptase.

36. The reverse transcriptase of claim 33, 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.

37. A retroviral reverse transcriptase having DNA polymerase activity, wherein said reverse transcriptase allows an mRNA template to remain intact during a 5 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.

38. A retroviral reverse transcriptase having DNA polymerase activity, wherein said reverse transcriptase allows an mRNA template to remain intact during a 10 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.

39. A retroviral reverse transcriptase having DNA polymerase activity, wherein said reverse transcriptase allows an mRNA template to remain intact during a 30 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.

40. A retroviral reverse transcriptase having DNA polymerase activity, wherein said reverse transcriptase allows an mRNA template to remain intact during a 60 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.

41. An M-MLV reverse transcriptase having DNA polymerase activity, wherein said reverse transcriptase allows an mRNA template to remain intact during cDNA synthesis by said reverse transcriptase and wherein said reverse transcriptase is modified within the RNase H domain.

42. The reverse transcriptase of claim 42, 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.

43. The reverse transcriptase of claim 42, wherein said reverse transcriptase is encoded by a modified nucleotide sequence that encodes a modified amino acid sequence modified in the region corresponding to the amino acids 503-611 of M-MLV reverse transcriptase.

44. The reverse transcriptase of claim 42, wherein said reverse transcriptase may be used in the preparation of full-length cDNA.

45. The reverse transcriptase of claim 42, wherein said reverse transcriptase is a recombinantly produced reverse transcriptase.

46. The reverse transcriptase of claim 42, wherein said reverse transcriptase is purified.

48. The reverse transcriptase of claim 42, wherein said reverse transcriptase is isolated.

53. The reverse transcriptase of claim 42, wherein said reverse transcriptase has no detectable RNase H activity.

56. The reverse transcriptase of claim 42, wherein said reverse transcriptase lacks RNase H activity.

59. The reverse transcriptase of claim 42, wherein said reverse transcriptase has 0.23×

10³ units/mg or less of RNase H activity.

60. The reverse transcriptase of claim 42, wherein said reverse transcriptase has 0.1×

10³ units/mg or less of RNase H activity.

61. The reverse transcriptase of claim 42, wherein said reverse transcriptase has 0.01×

10³ units/mg or less of RNase H activity.

62. The reverse transcriptase of claim 42, wherein said DNA polymerase activity is at least 17.5×

10³ units/mg.

63. The reverse transcriptase of claim 42, wherein said DNA polymerase activity is at least 81×

10³ units/mg.

64. The reverse transcriptase of claim 42, wherein said reverse transcriptase allows an mRNA template to remain intact during cDNA synthesis as determined by gel electrophoresis.

47. The reverse transcriptase of claim 47, wherein said reverse transcriptase is greater than 90% pure.

49. The reverse transcriptase of claim 49, wherein said reverse transcriptase is isolated using column chromatography.

50. The reverse transcriptase of claim 49, wherein said reverse transcriptase is isolated using DEAE-cellulose.

51. The reverse transcriptase of claim 49, wherein said reverse transcriptase is isolated using heparin agarose.

52. The reverse transcriptase of claim 49, wherein said reverse transcriptase is isolated using hydroxylapatite.

54. The reverse transcriptase of claim 54, wherein said no detectable RNase H activity is determined by examining the integrity of an mRNA template during cDNA synthesis.

55. The reverse transcriptase of claim 54, wherein said no detectable RNase H activity is determined by gel electrophoresis.

57. The reverse transcriptase of claim 57, wherein said lack of RNase H activity is determined by examining the integrity of an mRNA template during cDNA synthesis.

58. The reverse transcriptase of claim 57, wherein said lack of RNase H activity is determined by gel electrophoresis.

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

66. The reverse transcriptase of claim 66, 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.

67. The reverse transcriptase of claim 66, 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.

68. An M-MLV reverse transcriptase having DNA polymerase activity, wherein said reverse transcriptase allows an mRNA template to remain intact during a 5minute cDNA synthesis reaction by said reverse transcriptase and wherein said reverse transcriptase is modified within the RNase H domain.

69. An M-MLV reverse transcriptase having DNA polymerase activity, wherein said reverse transcriptase allows an mRNA template to remain intact during a 10 minute cDNA synthesis reaction by said reverse transcriptase and wherein said reverse transcriptase is modified within the RNase H domain.

70. An M-MLV reverse transcriptase having DNA polymerase activity, wherein said reverse transcriptase allows an mRNA template to remain intact during a 30 minute cDNA synthesis reaction by said reverse transcriptase and wherein said reverse transcriptase is modified within the RNase H domain.

71. An M-MLV reverse transcriptase having DNA polymerase activity, wherein said reverse transcriptase allows an mRNA template to remain intact during a 60 minute cDNA synthesis reaction by said reverse transcriptase and wherein said reverse transcriptase is modified within the RNase H domain.

72. AN M-MLV reverse transcriptase having DNA polymerase activity, wherein said reverse transcriptase allows an 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.

73. The reverse transcriptase of claim 73, wherein said DNA polymerase activity is at least 17.5×

10³ units/mg.

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

10³ units/mg.

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 corresponding to amino acids 498-611 of M-MLV reverse transcriptase.

76. 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 corresponding to amino acids 503-611 of M-MLV reverse transcriptase.

77. An M-MLV reverse transcriptase having DNA polymerase activity, wherein said reverse transcriptase allows an mRNA template to remain intact during a 5minute cDNA synthesis reaction by said reverse transcriptase as determined by gel electrophoresis and wherein said reverse transcriptase is modified within the RNase H domain.

78. An M-MLV reverse transcriptase having DNA polymerase activity, wherein said reverse transcriptase allows an mRNA template to remain intact during a 10 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.

79. An M-MLV reverse transcriptase having DNA polymerase activity, wherein said reverse transcriptase allows an mRNA template to remain intact during a 30 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.

80. An M-MLV reverse transcriptase having DNA polymerase activity, wherein said reverse transcriptase allows an mRNA template to remain intact during a 60 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.

81. A retroviral reverse transcriptase having DNA polymerase activity and no detectable RNase H activity, wherein said reverse transcriptase is modified within the RNase H domain.

82. The reverse transcriptase of claim 82, wherein said reverse transcriptase is selected from the group consisting of M-MLV, HTLV-1, BLV, RSV, and HIV reverse transcriptase.

83. The reverse transcriptase of claim 82, wherein said no detectable RNase H activity is determined during a one minute cDNA synthesis reaction.

84. The reverse transcriptase of claim 82, wherein said no detectable RNase H activity is determined during a 5 minute cDNA synthesis reaction.

85. The reverse transcriptase of claim 82, wherein said no detectable RNase H activity is determined during a 10 minute cDNA synthesis reaction.

86. The reverse transcriptase of claim 82, wherein said no detectable RNase H activity is determined during a 30 minute cDNA synthesis reaction.

87. The reverse transcriptase of claim 82, wherein said no detectable RNase H activity is determined during a 60 minute cDNA synthesis reaction.

88. The reverse transcriptase of claim 82, wherein said no detectable RNase H activity is determined by examining the integrity of an mRNA template during a one minute cDNA synthesis reaction.

89. The reverse transcriptase of claim 82, wherein said no detectable RNase H activity is determined by examining the integrity of an mRNA template during a 5 minute cDNA synthesis reaction.

90. The reverse transcriptase of claim 82, wherein said no detectable RNase H activity is determined by examining the integrity of an mRNA template during a 10 minute cDNA synthesis reaction.

91. The reverse transcriptase of claim 82, wherein said no detectable RNase H activity is determined by examining the integrity of an mRNA template during a 30 minute cDNA synthesis reaction.

92. The reverse transcriptase of claim 82, wherein said no detectable RNase H activity is determined by examining the integrity of an mRNA template during a 60 minute cDNA synthesis reaction.

93. The reverse transcriptase of claim 82, wherein said no detectable RNase H activity is determined by examining by gel electrophoresis of the integrity of an mRNA template during cDNA synthesis.

94. The reverse transcriptase of claim 82, wherein said no detectable RNase H activity is determined by examining by gel electrophoresis the integrity of an mRNA template during a one minute cDNA synthesis reaction.

97. The reverse transcriptase of claim 82, wherein said no detectable RNase H activity is determined by examining by gel electrophoresis the integrity of an mRNA template during a 5 minute cDNA synthesis reaction.

98. The reverse transcriptase of claim 82, wherein said no detectable RNase H activity is determined by examining by gel electrophoresis the integrity of an mRNA template during a 10 minute cDNA synthesis reaction.

99. The reverse transcriptase of claim 82, wherein said no detectable RNase H activity is determined by examining by gel electrophoresis the integrity of an mRNA template during a 30 minute cDNA synthesis reaction.

100. The reverse transcriptase of claim 82, wherein said no detectable RNase H activity is determined by examining by gel electrophoresis the integrity of an mRNA template during a 60 minute cDNA synthesis reaction.

101. The reverse transcriptase of claim 82, wherein said reverse transcriptase is encoded by a modified nucleotide sequence that encodes a modified amino acids sequenced modified in the region corresponding to amino acids 498-611 of M-MLV reverse transcriptase.

102. The reverse transcriptase of claim 82, 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.

95. The reverse transcriptase of claim 95, wherein said DNA polymerase activity is at least 17.5×

10³ units/mg.

96. The reverse transcriptase of claim 95, wherein said DNA polymerase activity is at least 81×

10³ units/mg.

103. An M-MLV reverse transcriptase having DNA polymerase activity and no detectable RNase H activity, wherein said reverse transcriptase is modified within the RNase H domain.

104. The reverse transcriptase of claim 104, 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.

105. The reverse transcriptase of claim 104, 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.

106. The reverse transcriptase of claim 104, wherein said no detectable RNase H activity is determined during a one minute cDNA synthesis reaction.

107. The reverse transcriptase of claim 104, wherein said no detectable RNase H activity is determined during a 5 minute cDNA synthesis reaction.

108. The reverse transcriptase of claim 104, wherein said no detectable RNase H activity is determined during a 10 minute cDNA synthesis reaction.

109. The reverse transcriptase of claim 104, wherein said no detectable RNase H activity is determined during a 30 minute cDNA synthesis reaction.

110. The reverse transcriptase of claim 104, wherein said no detectable RNase H activity is determined during a 60 minute cDNA synthesis reaction.

111. The reverse transcriptase of claim 104, wherein said no detectable RNase H activity is determined by examining the integrity of an mRNA template during a one minute cDNA synthesis reaction.

112. The reverse transcriptase of claim 104, wherein said no detectable RNase H activity is determined by examining the integrity of an mRNA template during a 5 minute cDNA synthesis reaction.

113. The reverse transcriptase of claim 104, wherein said no detectable RNase H activity is determined by examining the integrity of an mRNA template during a 10 minute cDNA synthesis reaction.

114. The reverse transcriptase of claim 104, wherein said no detectable RNase H activity is determined by examining the integrity of an mRNA template during a 30 minute cDNA synthesis reaction.

115. The reverse transcriptase of claim 104, wherein said no detectable RNase H activity is determined by examining the integrity of an mRNA template during a 60 minute cDNA synthesis reaction.

116. The reverse transcriptase of claim 104, wherein said no detectable RNase H activity is determined by examining by gel electrophoresis the integrity of an mRNA template during cDNA synthesis.

117. The reverse transcriptase of claim 104, wherein said no detectable RNase H activity is determined by examining by gel electrophoresis the integrity of an mRNA template during a one minute cDNA synthesis reaction.

120. The reverse transcriptase of claim 104, wherein said no detectable RNase H activity is determined by examining by gel electrophoresis the integrity of an mRNA template during a 5 minute cDNA synthesis reaction.

121. The reverse transcriptase of claim 104, wherein said no detectable RNase H activity is determined by examining by gel electrophoresis the integrity of an mRNA template during a 10 minute cDNA synthesis reaction.

122. The reverse transcriptase of claim 104, wherein said no detectable RNase H activity is determined by examining by gel electrophoresis the integrity of an mRNA template during a 30 minute cDNA synthesis reaction.

123. The reverse transcriptase of claim 104, wherein said no detectable RNase H activity is determined by examining by gel electrophoresis the integrity of an mRNA template during a 60 minute cDNA synthesis reaction.

118. The reverse transcriptase of claim 118, wherein said DNA polymerase activity is at least 17.5×

10³ units/mg.

119. The reverse transcriptase of claim 118, wherein said DNA polymerase activity is at least 81×

10³ units/mg.

124. 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.

125. The reverse transcriptase of claim 125, wherein said reverse transcriptase is selected from the group consisting of M-MLV, HTLV-1, BLV, RSV and HIV reverse transcriptase.

126. The reverse transcriptase of claim 125, 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.

127. The reverse transcriptase of claim 125, 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.

128. The reverse transcriptase of claim 125, wherein said reverse transcriptase may be used in the preparation of full-length cDNA.

129. The reverse transcriptase of claim 125, wherein said reverse transcriptase is a recombinantly produced reverse transcriptase.

130. The reverse transcriptase of claim 125, wherein said reverse transcriptase has 0.1×

10³ units/mg or less of RNase H activity.

131. The reverse transcriptase of claim 125, wherein said reverse transcriptase has 0.01×

10³ units/mg or less of RNase H activity.

132. The reverse transcriptase of claim 125, wherein said DNA polymerase activity is at least 17.5×

10³ units/mg.

133. The reverse transcriptase of claim 125, wherein said DNA polymerase activity is at least 81×

10³ units/mg.

134. 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.

135. The reverse transcriptase of claim 135, 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.

136. The reverse transcriptase of claim 135, 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.

137. The reverse transcriptase of claim 135, wherein said reverse transcriptase may be used in the preparation of full-length cDNA

138. The reverse transcriptase of claim 135, wherein said reverse transcriptase is a recombinantly produced reverse transcriptase.

139. The reverse transcriptase of claim 135, wherein said reverse transcriptase has 0.1×

10³ units/mg or less of RNase H activity.

140. The reverse transcriptase of claim 135, wherein said reverse transcriptase has 0.01×

10³ units/mg or less of RNase H activity.

141. The reverse transcriptase of claim 135, wherein said DNA polymerase activity is at least 81×

10³ units/mg.

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

10³ units/mg and 0.23×

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

143. The reverse transcriptase of claim 143, 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.

144. The reverse transcriptase of claim 143, 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.

145. The reverse transcriptase of claim 143, wherein said reverse transcriptase may be used in the preparation of full-length cDNA.

146. The reverse transcriptase of claim 143, wherein said reverse transcriptase is a recombinantly produced reverse transcriptase.

147. The reverse transcriptase of claim 143, wherein said reverse transcriptase has 0.1×

10³ units/mg or less an RNase H activity.

148. The reverse transcriptase of claim 143, wherein said reverse transcriptase has 0.01×

10³ units/mg or less an RNase H activity.

149. The reverse transcriptase of claim 143, wherein said DNA polymerase activity is at least 81×

10³ units/mg.

151. A vector comprising the DNA molecule of claim 151.

152. A host cell comprising the DNA molecule of claim 151.

154. A host cell comprising the vector of claim 152.

153. The host cell of claim 153, wherein said host cell is E. coli.

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

culturing the host cell of claim 153 under conditions sufficient to produce said

reverse transcriptase; and

isolating or purifying said reverse transcriptase.

155. The host cell of claim 155, wherein said host cell is E. coli.

157. The method of claim 157, further comprising incubating said first DNA molecule under conditions sufficient to make a second DNA molecule complementary to said first DNA molecule.

160. The method of claim 157, wherein said reverse transcriptase is isolated using column chromatography.

161. The method of claim 157, wherein said reverse transcriptase is greater than 90% pure.

158. The method of claim 158, wherein said first and second DNA molecule form a double stranded DNA molecule.

163. The kit of claim 163, 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.

165. The composition of claim 165, 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.

166. A composition comprising a retroviral reverse transcriptase, wherein said composition has DNA polymerase activity and 0.23×

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

167. The composition of claim 167, wherein said reverse transcriptase is selected from the group consisting of M-MLV, HTLV-1, BLV, RSV and HIV reverse transcriptase.

168. The composition of claim 167, 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.

169. The composition of claim 167, 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.

170. The composition of claim 167, wherein said composition may be used in the preparation of full-length cDNA.

171. The composition of claim 167, wherein said reverse transcriptase is a recombinantly produced reverse transcriptase.

172. The composition of claim 167, wherein said composition has 0.1×

10³ units/mg or less of RNase H activity.

173. The composition of claim 167, wherein said composition has 0.01×

10³ units/mg or less of RNase H activity.

174. The composition of claim 167, wherein said DNA polymerase activity is at least 17.5×

10³ units/mg.

175. The composition of claim 167, wherein said DNA polymerase activity is at least 81×

10³ units/mg.

176. The composition of claim 167, 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.

177. A composition comprising an M-MLV reverse transcriptase, wherein said composition has DNA polymerase activity and 0.23×

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

178. The composition of claim 178, 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.

179. The composition of claim 178, 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.

180. The composition of claim 178, wherein said composition may be used in the preparation of full-length cDNA.

181. The composition of claim 178, wherein said reverse transcriptase is a recombinantly produced reverse transcriptase.

182. The composition of claim 178, wherein said composition has 0.1×

10³ units/mg or less of RNase H activity.

183. The composition of claim 178, wherein said composition has 0.01×

10³ units/mg or less of RNase H activity.

184. The composition of claim 178, wherein said DNA polymerase activity is at least 17.5×

10³ units/mg.

185. The composition of claim 178, wherein said DNA polymerase activity is at least 81×

10³ units/mg.

186. The composition of claim 178, 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.

187. A composition comprising an M-MLV reverse transcriptase, wherein said composition has DNA polymerase activity of at least 17.5×

10³ units/mg and 0.23×

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

188. The composition of claim 188, 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.

189. The composition of claim 188, 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.

190. The composition of claim 188, wherein said composition may be used in the preparation of full-length cDNA.

191. The composition of claim 188, wherein said reverse transcriptase is a recombinantly produced reverse transcriptase.

192. The composition of claim 188, wherein said composition has 0.1×

10³ units/mg or less of RNase H activity.

193. The composition of claim 188, wherein said composition has 0.01×

10³ units/mg or less of RNase H activity.

194. The composition of claim 188, wherein said DNA polymerase activity is at least 81×

10³ units/mg.

195. The composition of claim 188, 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.