Tagged extendable primers and extension products

• US RE43,096 E1
• Filed: 03/13/2003
• Issued: 01/10/2012
• Est. Priority Date: 01/16/1984
• Status: Expired due to Term

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Smith, Lloyd M., Hood, Leroy E., Hunkapiller, Michael W., Hunkapiller, Timothy, Connell, Charles R.
• Primary Examiner(s): MUMMERT, STEPHANIE KANE
• Application Number:
  US10/389,663
• Time in Patent Office: 3,225 Days
• Field of Search: 435/6, 435/91.2, 536/24.33, 536/23.1, 536/24.3, 536/26.6
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: G01N 27/44721 by optical means
• CPC Class Codes: G01N 27/44726 using specific dyes, markers or binding molecules

Abstract

This invention provides a duplex comprising an oligonucleotide primer and a template, wherein the primer is coupled chemically to a chromophore or fluorophore so as to allow chain extension by a polymerase. In one embodiment, the primer is extended by a polymerase to generate the complement of the template. In a further embodiment, the extended primer is separated from the template for use in a number of methods, including sequencing reactions. Methods of generating these compositions of matter are further provided.

Claims

1. A duplex comprising an oligonucleotide primer and a template, wherein the primer is covalently coupled to a chromophore or fluorophore so as to allow chain extension by a polymerase.

2. A duplex comprising an extended oligonucleotide primer and a template, produced by providing a duplex according to claim 1 and extending the oligonucleotide primer with a polymerase.

3. A single-stranded labeled polynucleotide produced by separating the extended oligonucleotide primer from the duplex of claim 2.

4. A set of duplexes comprising two or more of the duplexes of claim 1.

5. A set of duplexes comprising two or more of the duplexes of claim 2.

6. A set of polynucleotides comprising two or more single-stranded labeled polynucleotides of claim 3.

11. The duplex of claim 1, prepared by a method comprising hybridizing an oligonucleotide primer to a template, wherein the primer is covalently coupled to a chromophore or fluorophore so as to allow chain extension by a polymerase.

12. The duplex of claim 11, wherein the chromophore or fluorophore is covalently coupled to the primer through an amine linkage.

13. The duplex of claim 11, wherein the chromophore or fluorophore is covalently coupled to the primer at its 5′

end.

14. A single-stranded labeled polynucleotide produced by the method comprising the steps of extending the oligonucleotide primer of the duplex of claim 1 by a polymerase to produce a labeled polynucleotide and separating the labeled polynucleotide from the template.

15. The polynucleotide of claim 14, wherein the chromophore or fluorophore is covalently coupled to the oligonucleotide through an amine linkage.

16. The polynucleotide of claim 14, wherein the chromophore or fluorophore is covalently coupled to the oligonucleotide at its 5′

end.

17. A chain termination DNA sequencing method comprising extending the primer of the duplex of claim 1 by a polymerase to produce a labeled polynucleotide, and separating the labeled polynucleotide from the template.

18. A chain termination DNA sequencing method comprising extending the primers of the set of duplexes of claim 4 by a polymerase to produce a set of labeled polynucleotides.

19. The chain termination DNA sequencing method of claim 18, wherein the set of duplexes comprises four DNA sequencing reactions, wherein each labeled polynucleotide is distinguishable by spectral characteristics of the chromophore or fluorophore covalently coupled thereto.

20. The oligonucleotide primer of claim 1, wherein the primer is DNA.

21. The oligonucleotide primer of claim 1 wherein the chromophore or fluorophore is detectable by exposure to a high-intensity monochromatic light source.

22. The duplex of either of claim 1 or 2, wherein the chromophore or fluorophore is detectable by exposure to a laser.

23. The set of duplexes of either of claim 4 or 5, wherein the primers are DNA.

24. The set of duplexes of either of claim 4 or 5, wherein the chromophore or fluorophore is detectable by exposure to a high-intensity monochromatic light source.

25. The set of duplexes of either of claim 4 or 5, wherein the chromophore or fluorophore is detectable by exposure to a laser.

29. The polynucleotide of any of claims 14 to 16, wherein the primer is DNA.

30. The polynucleotide of any of claims 14 to 16, wherein the chromophore or fluorophore is detectable by exposure to a high-intensity monochromatic light source.

31. The polynucleotide of any of claims 14 to 16, wherein the chromophore or fluorophore is detectable by exposure tc a laser.

32. The duplex of any of claims 11 to 13, wherein the primer is DNA.

33. The duplex of any of claims 11 to 13, wherein the chromophore or fluorophore is detectable by exposure to a high-intensity monochromatic light source.

34. The duplex of any of claims 11 to 13, wherein the chromophore or fluorophore is detectable by exposure to a laser.

35. The duplex of either of claim 1 or 2, wherein the chromophore or fluorophore is covalently coupled to the primer through an amine linkage.

36. The set of duplexes of either of claim 4 or 5, wherein the chromophore or fluorophore is covalently coupled to the primer through an amine linkage.

38. The duplex of either of claim 1 or 2, wherein the chromophore or fluorophore is covalently coupled to the primer at its 5′

end.

39. The set of duplexes of either of claim 4 or 5, wherein the chromophore or fluorophore is covalently coupled to the primer at its 5′

end.

41. The polynucleotide of claim 3, wherein the chromophore or fluorophore is covalently coupled to the primer through an amine linkage.

42. The polynucleotide of claim 3, wherein the chromophore or fluorophore is covalently coupled to the primer at its 5′

end.

43. The polynucleotide of claim 3, wherein the chromophore or fluorophore is detectable by exposure to a laser.

44. The set of polynucleotides of claim 6, wherein the primers are DNA.

45. The set of polynucleotides of claim 6, wherein the chromophore or fluorophore is detectable by exposure to a high-intensity monochromatic light source.

46. The set of polynucleotides of claim 6, wherein the chromophore or fluorophore is detectable by exposure to a laser.

47. The set of polynucleotides of claim 6, wherein the chromophore or fluorophore is covalently coupled to the primer through an amine linkage.

48. The set of polynucleotides of claim 6, wherein the chromophore or fluorophore is covalently coupled to the primer at its 5′

end.

7. A set of reagents comprising oligonucleotide primers covalently coupled to one or more chromophores or fluorophores so as to allow chain extension by a polymerase, and a polymerase.

26. The set of reagents of claim 7, wherein the primers are DNA.

27. The set of reagents of claim 7, wherein the chromophore or fluorophore is detectable by exposure to a high-intensity monochromatic light source.

28. The set of reagents of claim 7, wherein the chromophore or fluorophore is detectable by exposure to a laser.

37. The set of reagents of claim 7, wherein the chromophore or fluorophore is covalently coupled to the primer through an amine linkage.

40. The set of reagents of claim 7, wherein the chromophore or fluorophore is covalently coupled to the primer at its 5′

end.

8. A single-stranded labeled polynucleotide comprising a first portion and a second portion,

wherein the first portion comprises an oligonucleotide primer covalently coupled to a chromophore or fluorophore;

and

wherein the second portion is produced by extension of the first portion along a complementary template.

9. The polynucleotide of claim 8, wherein the chromophore or fluorophore is covalently coupled to the first portion through an amine linkage.

10. The polynucleotide of claim 8, wherein the chromophore or fluorophore is covalently coupled to the first portion at its 5′

end.

49. A duplex comprising an oligonucleotide primer and a template, wherein the primer hybridizes to a specific region of the template and wherein the primer is covalently coupled to a chromophore or fluorophore so as to allow chain extension by a polymerase.

50. A plurality of identical oligonucleotide primers of defined length and base sequences wherein each primer is covalently coupled to a fluorophore or chromophore so as to allow chain extension by a polymerase.

51. The plurality of claim 50 wherein said primers have a free 3′

hydroxyl group.

52. The plurality of claim 51 wherein the chromophore or fluorophore is covalently coupled to the primer at its 5′

end.

53. The plurality of claim 50 wherein said primers are coupled to said fluorophore or chromophore by an amine linkage.

54. A composition comprising the plurality of claim 50.

55. The composition of claim 54 further comprising a buffer.

56. A set of reagents comprising the plurality of claim 50 and a polymerase.

58. A plurality of single-stranded labeled polynucleotides produced by the method comprising the steps of hybridizing the plurality of oligonucleotide primers of claim 50 to a template thereby forming a plurality of duplexes;

extending the primers of said duplexes by a polymerase thereby forming labeled polynucleotides; and

separating said labeled polynucleotides from said duplexes.

59. A set of single stranded labeled polynucleotides comprising two or more pluralities of polynucleotides of claim 58, wherein each plurality has a different emission spectra.

60. The plurality of claim 50 wherein the chromophore or fluorophore is detectable by exposure to a high-intensity monochromatic light source.

61. The plurality of claim 50 wherein the chromophore or fluorophore is detectable by exposure to a laser.

57. A set of reagents comprising two or more pluralities of oligonucleotide primers of claim SO wherein each plurality has a different emission spectra.

62. A method of nucleic acid sequence analysis, comprising extending an oligonucleotide along a complementary strand of DNA of a duplex by a polymerase to produce a labeled extension product, wherein the duplex comprises the oligonucleotide specifically hybridized to the complementary strand of DNA, and wherein the oligonucleotide is covalently coupled to a fluorophore so as to allow chain extension by the polymerase.

63. The method of claim 62, further comprising separating said labeled extension product from said duplex.

65. The method of claim 64 or claim 62, wherein the fluorophore is covalently coupled to the oligonucleotide through an amine linkage.

70. The method of claim 62, wherein substantially all molecules of the labeled extension product individually comprise a single fluorescent nucleotide.

74. The method of claim 62, wherein substantially all molecules of the labeled extension product are individually coupled to a fluorophore by a single covalent linkage.

80. The method of claim 74, wherein substantially all molecules of the labeled extension product individually are terminally labeled with a fluorophore.

86. The method of claim 70, wherein substantially all molecules of the labeled extension product individually are terminally labeled with a fluorophore.

92. The method of claim 74, wherein substantially all molecules of the labeled extension product individually comprise a 5′

terminal fluorescent nucleotide.

98. The method of claim 86, wherein substantially all molecules of the labeled extension product individually comprise a 5′

terminal fluorescent nucleotide.

118. The method of claim 70, wherein substantially all molecules of the labeled extension product individually comprise a 3′

terminal nucleotide that is adapted to terminate polymerase extension.

121. The method of claim 74, wherein substantially all molecules of the labeled extension product individually comprise a 3′

terminal nucleotide that is adapted to terminate polymerase extension.

126. The method of claim 80, wherein substantially all molecules of the labeled extension product individually comprise a 3′

terminal nucleotide that is adapted to terminate polymerase extension.

64. A DNA sequencing method, comprising

extending oligonucleotides of a set of duplexes along hybridized complementary strands of DNA by a polymerase to produce a set of labeled extension products, wherein the set of labeled extension products comprises two or more extension products, wherein an extension product comprises an extended oligonucleotide specifically hybridized to a complementary strand of DNA,

thereby producing four sets of labeled extension products, wherein the extension products of each set are distinguishably labeled with a different type of fluorophore from the extension products of the other sets.

68. The method of claim 64, wherein the extension products comprise a terminal nucleotide having any one of four different types of terminal base components, wherein substantially all molecules of the same set of labeled extension products have the same type of terminal base component, and substantially all molecules of different sets of labeled extension products have different types of terminal base components.

71. The method of claim 64, wherein substantially all molecules of the labeled extension products individually comprise a single fluorescent nucleotide.

75. The method of claim 64, wherein substantially all molecules of the labeled extension products are individually coupled to a fluorophore by a single covalent linkage.

78. The method of claim 68, wherein substantially all molecules of the labeled extension products individually comprise a single fluorescent nucleotide.

81. The method of claim 75, wherein substantially all molecules of the labeled extension products individually are terminally labeled with a fluorophore.

84. The method of claim 68, wherein substantially all molecules of the labeled extension products individually are terminally labeled with a fluorophore.

87. The method of claim 71, wherein substantially all molecules of the labeled extension products individually are terminally labeled with a fluorophore.

90. The method of claim 78, wherein substantially all molecules of the labeled extension products individually are terminally labeled with a fluorophore.

93. The method of claim 75, wherein substantially all molecules of the labeled extension products individually comprise a 5′

terminal fluorescent nucleotide.

96. The method of claim 84, wherein substantially all molecules of the labeled extension products individually comprise a 5′

terminal fluorescent nucleotide.

99. The method of claim 87, wherein substantially all molecules of the labeled extension products individually comprise a 5′

terminal fluorescent nucleotide.

102. The method of claim 90, wherein substantially all molecules of the labeled extension products individually comprise a 5′

terminal fluorescent nucleotide.

107. The method of claim 68, wherein substantially all molecules of the labeled extension products individually comprise a 3′

terminal nucleotide that is complementary to a corresponding nucleotide on the complementary strand of DNA.

109. The method of claim 71, wherein substantially all molecules of the labeled extension products individually comprise a 3′

terminal nucleotide that is complementary to a corresponding nucleotide on the complementary strand of DNA.

111. The method of claim 75, wherein substantially all molecules of the labeled extension products individually comprise a 3′

terminal nucleotide that is complementary to a corresponding nucleotide on the complementary strand of DNA.

114. The method of claim 81, wherein substantially all molecules of the labeled extension products individually comprise a 3′

terminal nucleotide that is complementary to a corresponding nucleotide on the complementary strand of DNA.

116. The method of claim 68, wherein substantially all molecules of the labeled extension products individually comprise a 3′

terminal nucleotide that is adapted to terminate polymerase extension.

119. The method of claim 71, wherein substantially all molecules of the labeled extension products individually comprise a 3′

terminal nucleotide that is adapted to terminate polymerase extension.

122. The method of claim 75, wherein substantially all molecules of the labeled extension products individually comprise a 3′

terminal nucleotide that is adapted to terminate polymerase extension.

124. The method of claim 78, wherein substantially all molecules of the labeled extension products individually comprise a 3′

terminal nucleotide that is adapted to terminate polymerase extension.

127. The method of claim 81, wherein substantially all molecules of the labeled extension products individually comprise a 3′

terminal nucleotide that is adapted to terminate polymerase extension.

137. The method of claim 68, wherein the four different types of terminal base components are adenosine, guanosine, thymidine and cytosine.

139. The method of claim 81, wherein the oligonucleotides are fluorescently labeled before being extended.

140. The method of claim 84, wherein the oligonucleotides are fluorescently labeled before being extended.

141. The method of claim 87, wherein the oligonucleotides are fluorescently labeled before being extended.

142. The method of claim 90, wherein the oligonucleotides are fluorescently labeled before being extended.

152. The method of claim 68, wherein the oligonucleotides are fluorescently labeled before being extended.

153. The method of claim 71, wherein the oligonucleotides are fluorescently labeled before being extended.

154. The method of claim 75, wherein the oligonucleotides are fluorescently labeled before being extended.

155. The method of claim 78, wherein the oligonucleotides are fluorescently labeled before being extended.

156. The method of claim 93, wherein the oligonucleotides are fluorescently labeled before being extended.

157. The method of claim 107, wherein the oligonucleotides are fluorescently labeled before being extended.

158. The method of claim 116, wherein the oligonucleotides are fluorescently labeled before being extended.

66. A mixture comprising a polymerase and a duplex, wherein the duplex comprises an oligonucleotide specifically hybridized to a complementary strand of DNA, wherein the oligonucleotide is covalently coupled to a fluorophore so as to allow chain extension by the polymerase.

72. The mixture of claim 66, wherein substantially all oligonucleotide molecules individually comprise a single fluorescent nucleotide.

76. The mixture of claim 66, wherein substantially all oligonucleotide molecules are individually coupled to a fluorophore by a single covalent linkage.

82. The mixture of claim 76, wherein substantially all oligonucleotide molecules individually are terminally labeled with a fluorophore.

88. The mixture of claim 72, wherein substantially all oligonucleotide molecules individually are terminally labeled with a fluorophore.

94. The mixture of claim 76, wherein substantially all oligonucleotide molecules individually comprise a 5′

terminal fluorescent nucleotide.

100. The mixture of claim 88, wherein substantially all oligonucleotide molecules individually comprise a 5′

terminal fluorescent nucleotide.

67. A composition comprising four sets of oligonucleotides, wherein oligonucleotides of each of the four sets are distinguishably labeled with a different type of fluorophore from the oligonucleotides of the other three sets.

69. The composition of claim 67, wherein the oligonucleotides comprise a terminal nucleotide having any one of four different types of terminal base components, wherein substantially all oligonucleotide molecules of the same set have the same type of terminal base component, and substantially all oligonucleotide molecules of different sets have different types of terminal base components.

73. The composition of claim 67, wherein substantially all oligonucleotide molecules of each set individually comprise a single fluorescent nucleotide.

77. The composition of claim 67, wherein substantially all oligonucleotide molecules of each set are individually coupled to a fluorophore by a single covalent linkage.

79. The composition of claim 69, wherein substantially all oligonucleotide molecules of each set individually comprise a single fluorescent nucleotide.

83. The composition of claim 77, wherein substantially all oligonucleotide molecules of each set individually are terminally labeled with a fluorophore.

85. The composition of claim 69, wherein substantially all oligonucleotide molecules of each set individually are terminally labeled with a fluorophore.

89. The composition of claim 73, wherein substantially all oligonucleotide molecules of each set individually are terminally labeled with a fluorophore.

91. The composition of claim 79, wherein substantially all oligonucleotide molecules of each set individually are terminally labeled with a fluorophore.

95. The composition of claim 77, wherein substantially all oligonucleotide molecules of each set individually comprise a 5′

terminal fluorescent nucleotide.

97. The composition of claim 85, wherein substantially all oligonucleotide molecules of each set individually comprise a 5′

terminal fluorescent nucleotide.

101. The composition of claim 89, wherein substantially all oligonucleotide molecules of each set individually comprise a 5′

terminal fluorescent nucleotide.

103. The composition of claim 91, wherein substantially all oligonucleotide molecules of each set individually comprise a 5′

terminal fluorescent nucleotide.

104. The composition of claim 69, wherein substantially all oligonucleotide molecules of each set individually comprise a 3′

terminal fluorescent nucleotide.

105. The composition of claim 73, wherein substantially all oligonucleotide molecules of each set individually comprise a 3′

terminal fluorescent nucleotide.

106. The composition of claim 79, wherein substantially all oligonucleotide molecules of each set individually comprise a 3′

terminal fluorescent nucleotide.

108. The composition of claim 69, wherein substantially all oligonucleotide molecules of each set individually (i) are specifically hybridized to a complementary strand of DNA, and (ii) comprise a 3′

terminal nucleotide that is complementary to a corresponding nucleotide on the complementary strand of DNA.

110. The composition of claim 73, wherein substantially all oligonucleotide molecules of each set individually (i) are specifically hybridized to a complementary strand of DNA, and (ii) comprise a 3′

terminal nucleotide that is complementary to a corresponding nucleotide on the complementary strand of DNA.

112. The composition of claim 77, wherein substantially all oligonucleotide molecules of each set individually (i) are specifically hybridized to a complementary strand of DNA, and (ii) comprise a 3′

terminal nucleotide that is complementary to a corresponding nucleotide on the complementary strand of DNA.

113. The composition of claim 79, wherein substantially all oligonucleotide molecules of each set individually comprise a 3′

terminal nucleotide that is complementary to a corresponding nucleotide in a complementary strand of DNA.

115. The composition of claim 83, wherein substantially all oligonucleotide molecules of each set individually (i) are specifically hybridized to a complementary strand of DNA, and (ii) comprise a 3′

terminal nucleotide that is complementary to a corresponding nucleotide on the complementary strand of DNA.

117. The composition of claim 69, wherein substantially all oligonucleotide molecules of each set individually comprise a 3′

terminal nucleotide that is adapted to terminate polymerase extension.

120. The composition of claim 73, wherein substantially all oligonucleotide molecules of each set individually comprise a 3′

terminal nucleotide that is adapted to terminate polymerase extension.

123. The composition of claim 77, wherein substantially all oligonucleotide molecules of each set individually comprise a 3′

terminal nucleotide that is adapted to terminate polymerase extension.

125. The composition of claim 79, wherein substantially all oligonucleotide molecules of each set individually comprise a 3′

terminal nucleotide that is adapted to terminate polymerase extension.

128. The composition of claim 83, wherein substantially all oligonucleotide molecules of each set individually comprise a 3′

terminal nucleotide that is adapted to terminate polymerase extension.

129. The composition of claim 69, further comprising a polymerase or nucleotides adapted to terminate polymerase extension.

130. The composition of claim 73, further comprising a polymerase or nucleotides adapted to terminate polymerase extension.

131. The composition of claim 77, further comprising a polymerase or nucleotides adapted to terminate polymerase extension.

132. The composition of claim 79, further comprising a polymerase or nucleotides adapted to terminate polymerase extension.

133. The composition of claim 83, further comprising a polymerase or nucleotides adapted to terminate polymerase extension.

134. The composition of claim 85, further comprising a polymerase or nucleotides adapted to terminate polymerase extension.

135. The composition of claim 89, further comprising a polymerase or nucleotides adapted to terminate polymerase extension.

136. The composition of claim 91, further comprising a polymerase or nucleotides adapted to terminate polymerase extension.

138. The composition of claim 69, wherein the four different types of terminal base components are adenosine, guanosine, thymidine and cytosine.

143. A method of nucleic acid sequence analysis, comprising producing the composition of claim 69, and detecting the type of fluorophore on oligonucleotides of the composition.

144. A method of nucleic acid sequence analysis, comprising producing the composition of claim 73, and detecting the type of fluorophore on oligonucleotides of the composition.

145. A method of nucleic acid sequence analysis, comprising producing the composition of claim 77, and detecting the type of fluorophore on oligonucleotides of the composition.

146. A method of nucleic acid sequence analysis, comprising producing the composition of claim 83, and detecting the type of fluorophore on oligonucleotides of the composition.

147. A method of nucleic acid sequence analysis, comprising producing the composition of claim 85, and detecting the type of fluorophore on oligonucleotides of the composition.

148. A method of nucleic acid sequence analysis, comprising producing the composition of claim 104, and detecting the type of fluorophore on oligonucleotides of the composition.

149. A method of nucleic acid sequence analysis, comprising producing the composition of claim 105, and detecting the type of fluorophore on oligonucleotides of the composition.

150. A method of nucleic acid sequence analysis, comprising producing the composition of claim 108, and detecting the type of fluorophore on oligonucleotides of the composition.

151. A method of nucleic acid sequence analysis, comprising producing the composition of claim 117, and detecting the type of fluorophore on oligonucleotides of the composition.