Thermodynamic equilibrium extension of primers

• US 20040115643A1
• Filed: 12/12/2002
• Published: 06/17/2004
• Est. Priority Date: 12/12/2002
• Status: Abandoned Application

• Current Assignee: Yale University
• Original Assignee: Yale University
• Inventors: Gribanov, Oleg G., Lizardi, Paul M.
• Application Number:
  US10/318,416
• Publication Number:
  US 20040115643A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis

Abstract

Disclosed is a method and materials for amplifying nucleic acid sequences by limited primer extension. The disclosed method involves association of a primer with a template, extension of the primer for a short distance, termination of extension, and dissociation of the primer from the template, whereupon the events repeat with a new primer. The repeated association, extension, and dissociation of primers from a single template sequence results in amplification of the extended sequences. The termination of extension can be effected by a feature of the template sequence. The reaction can be carried under a single set of conditions, such as isothermal conditions, based on the thermodynamics of dissociation of the extended primers. The disclosed method is particularly suited to detection of nucleic acid sequences. Multiple sequences can be amplified and detected in the same reaction by targeting multiple sequences with extension primers.

Claims

1. A method of amplifying nucleic acid sequences, the method comprising

bringing into contact one or more extension primers and one or more target templates and incubating under conditions that promote interaction of the extension primers and the target templates, extension of the extension primers using the interacting target templates as template, and dissociation of the extended extension primers from the target templates, whereby multiple extended extension primers are produced from at least one target template,

wherein the target templates each comprise a replication terminating feature.

2. The method of claim 1 wherein the extension primers and target templates are incubated under isothermal conditions.

3. The method of claim 1 wherein the extension primers and target templates are incubated under a single set of conditions.

4. The method of claim 1 wherein the target templates are nucleic acid sequences of interest.

5. The method of claim 1 wherein the extension primers each comprise a target complement portion.

6. The method of claim 5 wherein the extension primers each consist of a target complement portion.

7. The method of claim 5 wherein the extension primers each comprise nucleotides, wherein the nucleotides consist of the target complement portion.

8. The method of claim 5 wherein the extension primers each further comprise a non-target complement portion.

9. The method of claim 5 wherein the target complement portion of at least one of the extension primers has a melting temperature of about 5°

C. to about 16°

C. lower than the temperature at which the extension primers and target templates are incubated.

10. The method of claim 9 wherein the target complement portion of at least one of the extension primers has a melting temperature of about 10°

C. to about 16°

C. lower than the temperature at which the extension primers and target templates are incubated.

11. The method of claim 10 wherein the target complement portion of at least one of the extension primers has a melting temperature of about 11°

C. to about 13°

C. lower than the temperature at which the extension primers and target templates are incubated.

12. The method of claim 5 wherein the target complement portion of each extension primer has a melting temperature of about 10°

C. to about 16°

C. lower than the temperature at which the extension primers and target templates are incubated.

13. The method of claim 5 wherein the target complement portion of each extension primer has a calculated melting temperature of about 10°

C. to about 16°

C. lower than the temperature at which the extension primers and target templates are incubated.

14. The method of claim 5 wherein the target complement portion of at least one of the extension primers has a melting temperature of about 10°

C. to about 16°

C. lower than the temperature at which the extension primers and target templates are incubated, and wherein the extended extension primers produced by extension of the at least one extension primer has a melting temperature of about 0°

C. to about 6°

C. higher than the temperature at which the extension primers and target templates are incubated.

15. The method of claim 5 wherein the target complement portion of each of the extension primers has a melting temperature of about 10°

C. to about 16°

C. lower than the temperature at which the extension primers and target templates are incubated, and wherein each of the extended extension primers has a melting temperature of about 0°

C. to about 6°

C. higher than the temperature at which the extension primers and target templates are incubated.

16. The method of claim 5 wherein the melting temperature of the target complement portion of each of the extension primers is about 10°

C. to about 22°

C. lower than the melting temperature of the extended extension primer.

17. The method of claim 5 wherein the extension primers and target templates are incubated at about 68°

C. to about 74°

C., wherein the target complement portion of each extension primer has a melting temperature of about 55°

C. to about 61°

C.

18. The method of claim 17 wherein the extension primers and target templates are incubated at about 70°

C. to about 72°

C., wherein the target complement portion of each extension primer has a melting temperature of about 55°

C. to about 61°

C.

19. The method of claim 5 wherein the extension primers and target templates are incubated at about 68°

C. to about 74°

C., wherein the target complement portion of each extension primer has a melting temperature of about 55°

C. to about 61°

C., wherein the melting temperature of each extended extension primer is about 72°

C. to about 77°

C.

20. The method of claim 19 wherein the extension primers and target templates are incubated at about 70°

C. to about 72°

C., wherein the target complement portion of each extension primer has a melting temperature of about 55°

C. to about 61°

C., wherein the melting temperature of each extended extension primer is about 74°

C. to about 76°

C.

21. The method of claim 1 wherein at least one of the extended extension primers has a melting temperature of about 2°

C. lower than to about 7°

C. higher than the temperature at which the extension primers and target templates are incubated.

22. The method of claim 21 wherein at least one of the extended extension primers has a melting temperature of about 0°

C. to about 6°

C. higher than the temperature at which the extension primers and target templates are incubated.

23. The method of claim 22 wherein at least one of the extended extension primers has a melting temperature of about 2°

C. to about 5°

C. higher than the temperature at which the extension primers and target templates are incubated.

24. The method of claim 1 wherein the extended extension primers have melting temperatures of about 0°

C. to about 6°

C. higher than the temperature at which the extension primers and target templates are incubated.

25. The method of claim 1 wherein the extended extension primers have calculated melting temperatures of about 0°

C. to about 6°

C. higher than the temperature at which the extension primers and target templates are incubated.

26. The method of claim 1 wherein the extension primers and target templates are incubated at about 68°

C. to about 74°

C., wherein the melting temperature of each extended extension primer is about 72°

C. to about 77°

C.

27. The method of claim 26 wherein the extension primers and target templates are incubated at about 70°

C. to about 72°

C., wherein the melting temperature of each extended extension primer is about 74°

C. to about 76°

C.

28. The method of claim 1 wherein the extension primers are extended 5 to 11 nucleotides.

29. The method of claim 28 wherein the extension primers are extended 5 to 8 nucleotides.

30. The method of claim 28 wherein the extended extension primers have melting temperatures of about 0°

C. to about 6°

C. higher than the temperature at which the extension primers and target templates are incubated.

31. The method of claim 1 wherein the extended extension primers each comprise an extension portion, wherein the extension portion comprises nucleotides added to the extension primer by extension, wherein the extended extension primers each comprise an extended target complement portion, wherein the extended target complement portion comprises the target complement portion and the extension portion.

32. The method of claim 31 wherein the extended extension primers each further comprise a non-target complement portion.

33. The method of claim 32 wherein the extension primers each further comprise a non-target complement portion, wherein the non-target complement portion of an extended extension primer is the non-target complement portion of the extension primer from which the extended extension primer is produced.

34. The method of claim 31 wherein the extended extension primers each consist of an extended target complement portion.

35. The method of claim 31 wherein the extended extension primers each comprise nucleotides, wherein the nucleotides consist of the extended target complement portion.

36. The method of claim 31 wherein the target complement portion of each of the extension primers has a melting temperature of about 10°

C. to about 16°

C. lower than the temperature at which the extension primers and target templates are incubated, and wherein the extended target complement portion of each of the extended extension primers has a melting temperature of about 0°

C. to about 6°

C. higher than the temperature at which the extension primers and target templates are incubated.

37. The method of claim 31 wherein the melting temperature of the target complement portion of each of the extension primers is about 10°

C. to about 22°

C. lower than the melting temperature of the extended target complement portion of the extended extension primer.

38. The method of claim 31 wherein the extension primers and target templates are incubated at about 68°

C. to about 74°

C., wherein the target complement portion of each extension primer has a melting temperature of about 55°

C. to about 61°

C., wherein the melting temperature of the extended target complement portion of each extended extension primer is about 72°

C. to about 77°

C.

39. The method of claim 31 wherein the extended target complement portions of the extended extension primers have melting temperatures of about 0°

C. to about 6°

C. higher than the temperature at which the extension primers and target templates are incubated.

40. The method of claim 1 wherein at least 50 extended extension primers are produced from at least one of the target templates.

41. The method of claim 40 wherein at least 100 extended extension primers are produced from at least one of the target templates.

42. The method of claim 41 wherein at least 200 extended extension primers are produced from at least one of the target templates.

43. The method of claim 42 wherein at least 400 extended extension primers are produced from at least one of the target templates.

44. The method of claim 43 wherein at least 800 extended extension primers are produced from at least one of the target templates.

45. The method of claim 44 wherein at least 1000 extended extension primers are produced from at least one of the target templates.

46. The method of claim 45 wherein at least 2000 extended extension primers are produced from at least one of the,target templates.

47. The method of claim 1 wherein an average of at least 50 extended extension primers are produced from each of the target templates.

48. The method of claim 47 wherein an average of at least 100 extended extension primers are produced from each of the target templates.

49. The method of claim 48 wherein an average of at least 200 extended extension primers are produced from each of the target templates.

50. The method of claim 49 wherein an average of at least 400 extended extension primers are produced from each of the target templates.

51. The method of claim 50 wherein an average of at least 800 extended extension primers are produced from each of the target templates.

52. The method of claim 51 wherein an average of at least 1000 extended extension primers are produced from each of the target templates.

53. The method of claim 52 wherein an average of at least 2000 extended extension primers are produced from each of the target templates.

54. The method of claim 1 further comprising detecting one or more of the extended extension primers.

55. The method of claim 54 wherein each extended extension primer corresponds to one or more of the target templates, wherein the target templates correspond to nucleic acid sequences of interest, wherein detection of an extended extension primer indicates detection of the nucleic acid sequence of interest to which a target template corresponding to the detected extended extension primer corresponds.

56. The method of claim 54 wherein detection of the extended extension primers is performed simultaneously with incubation of the extension primers and target templates.

57. The method of claim 54 wherein the extension primers comprise a detection label, wherein the extended extension primers are detected via the detection label.

58. The method of claim 54 wherein the extended extension primers comprise a detection label, wherein the extended extension primers are detected via the detection label.

59. The method of claim 54 wherein the extended extension primers are detected via interaction of the extended extension primers with detection probes.

60. The method of claim 59 wherein the interaction is a base pairing interaction.

61. The method of claim 59 wherein the interaction is a hybridization interaction.

62. The method of claim 59 wherein the extended extension primers are covalently coupled to the detection probes.

63. The method of claim 62 wherein the extended extension primers are covalently coupled to the detection probes by ligation.

64. The method of claim 59 wherein the detection probes are stem-loop probes.

65. The method of claim 59 wherein the extended extension primers are covalently coupled to anchor probes, wherein covalent coupling of the extended extension primers is facilitated by the interaction of the extended extension primers with the detection probes.

66. The method of claim 65 wherein the anchor probes are associated with a substrate.

67. The method of claim 65 wherein the extended extension primers are covalently coupled to the anchor probes by ligation.

68. The method of claim 59 wherein the detection probes each comprise an oligonucleotide comprising modified nucleotides or an oligonucleotide analog, wherein the detection probes have a higher melting temperature than an oligonucleotide consisting of unmodified nucleotides having the same nucleotide base composition as the oligonucleotide comprising modified nucleotides or the oligonucleotide analog.

69. The method of claim 68 wherein the detection probes comprise peptide nucleic acid.

70. The method of claim 69 wherein the melting temperature of the extended extension primers interacting with the detection probes is at least 5°

C. higher than the melting temperature of the extended extension primers interacting with the target templates.

71. The method of claim 69 wherein the melting temperature of the extended extension primers interacting with the detection probes is at least 10°

C. higher than the melting temperature of the extended extension primers interacting with the target templates.

72. The method of claim 69 wherein the melting temperature of the extended extension primers interacting with the detection probes is at least 6°

C. higher than the temperature at which the extension primers and target templates are incubated.

73. The method of claim 69 wherein the melting temperature of the extended extension primers interacting with the detection probes is at least 11°

C. higher than the temperature at which the extension primers and target templates are incubated.

74. The method of claim 69 wherein the melting temperature of the extended extension primers interacting with the detection probes is at least 20°

C. higher than the temperature at which the extension primers and target templates are incubated.

75. The method of claim 59 wherein detection probes each comprise a primer complement portion.

76. The method of claim 75 wherein the primer complement portion of the detection probes each comprise an oligonucleotide comprising modified nucleotides or an oligonucleotide analog, wherein the primer complement portions of the detection probes have a higher melting temperature than an oligonucleotide consisting of unmodified nucleotides having the same nucleotide base composition as the oligonucleotide comprising modified nucleotides or the oligonucleotide analog.

77. The method of claim 75 wherein the primer complement portion comprises peptide nucleic acid.

78. The method of claim 59 wherein the extended extension primers each comprise an extension portion, wherein the extension portion comprises nucleotides added to the extension primer by extension, wherein the extended extension primers each comprise an extended target complement portion and a non-target complement portion, wherein the extended target complement portion comprises the target complement portion and the extension portion.

79. The method of claim 78 wherein the detection probes interact with both the extended target complement portion and the non-target complement portion of the extended extension primers.

80. The method of claim 79 wherein the melting temperature of the extended extension primers interacting with the detection probes is at least 5°

C. higher than the melting temperature of the extended extension primers interacting with the target templates.

81. The method of claim 79 wherein the melting temperature of the extended extension primers interacting with the detection probes is at least 10°

C. higher than the melting temperature of the extended extension primers interacting with the target templates.

82. The method of claim 79 wherein the melting temperature of the extended extension primers interacting with the detection probes is at least 6°

C. higher than the temperature at which the extension primers and target templates are incubated.

83. The method of claim 79 wherein the melting temperature of the extended extension primers interacting with the detection probes is at least 11 °

C. higher than the temperature at which the extension primers and target templates are incubated.

84. The method of claim 79 wherein the melting temperature of the extended extension primers interacting with the detection probes is at least 20°

C. higher than the temperature at which the extension primers and target templates are incubated.

85. The method of claim 59 wherein the detection probes are associated with a substrate.

86. The method of claim 85 wherein the detection probes are attached to the substrate.

87. The method of claim 86 wherein the detection probes are covalently coupled to the substrate.

88. The method of claim 85 wherein the detection probes are indirectly associated with the substrate.

89. The method of claim 85 wherein the detection probes are directly associated with the substrate.

90. The method of claim 85 wherein the substrate is a single substrate structure, wherein all of the detection probes are associated with a single substrate.

91. The method of claim 90 wherein the substrate is a thin film, membranes, bead, microbead, bottle, dish, slide, fiber, optical fiber, woven fiber, chip, compact disk, shaped polymer, particles, or microparticle.

92. The method of claim 85 wherein the substrate is comprised of a plurality of substrate structures.

93. The method of claim 92 wherein the substrate comprises a plurality of beads.

94. The method of claim 93 wherein the beads are microbeads.

95. The method of claim 93 wherein the beads are paramagnetic beads.

96. The method of claim 85 wherein the substrate is acrylamide, cellulose, nitrocellulose, glass, gold, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, polysilicate, polycarbonate, teflon, fluorocarbon, nylon, silicon rubber, polyanhydride, polyglycolic acid, polylactic acid, polyorthoester, functionalized silane, polypropylfumerate, collagen, glycosaminoglycan, or polyamino acid.

97. The method of claim 1 wherein a plurality of extension primers are brought into contact with a plurality of target templates, wherein multiple extended extension primers are produced from each of a plurality of the target templates.

98. The method of claim 97 wherein the extension primers and target templates are incubated in the same reaction.

99. The method of claim 97 wherein the extension primers and target templates are incubated in a plurality of different reactions.

100. The method of claim 97 wherein each extension primer is incubated with the target templates in a different reaction.

101. The method of claim 97 wherein at least two extension primers are incubated with the target templates in a different reaction.

102. The method of claim 97 wherein the target templates each comprise a primer complement region, wherein the extension primers each comprise a target complement portion, wherein the target complement portion is complementary to a primer complement region of one or more of the target templates, wherein the extension primers comprise a set of extension primers, wherein each target complement portion of each extension primer in the set has similar hybrid stability.

103. The method of claim 102 wherein the extended extension primers comprise a set of extended extension primers, wherein each extended extension primer in the set has similar hybrid stability.

104. The method of claim 102 wherein the melting temperatures of each target complement portion of each extension primer in the set is within 5°

C.

105. The method of claim 104 wherein the melting temperatures of each target complement portion of each extension primer in the set is within 3°

C.

106. The method of claim 105 wherein the melting temperatures of each target complement portion of each extension primer in the set is within 2°

C.

107. The method of claim 97 wherein the extended extension primers comprise a set of extended extension primers, wherein each extended extension primer in the set has similar hybrid stability.

108. The method of claim 107 wherein the melting temperatures of each extended extension primer in the set is within 7°

C.

109. The method of claim 108 wherein the melting temperatures of each extended extension primer in the set is within 5°

C.

110. The method of claim 109 wherein the melting temperatures of each extended extension primer in the set is within 3°

C.

111. The method of claim 97 wherein the extended extension primers each comprise an extension portion, wherein the extension portion comprises nucleotides added to the extension primer by extension, wherein the extended extension primers each comprise an extended target complement portion, wherein the extended target complement portion comprises the target complement portion and the extension portion,

wherein the extended extension primers comprise a set of extended extension primers, wherein the extended target complement portion of each extended extension primer in the set has similar hybrid stability.

112. The method of claim 111 wherein the melting temperature of the extended target complement region of each extended extension primer in the set is within 7°

C.

113. The method of claim 112 wherein the melting temperature of the extended target complement region of each extended extension primer in the set is within 5°

C.

114. The method of claim 113 wherein the melting temperature of the extended target complement region of each extended extension primer in the set is within 3°

C.

115. The method of claim 111 wherein the extended extension primers each further comprise a non-target complement region,

wherein each extended extension primer in the set has similar hybrid stability.

116. The method of claim 115 wherein the melting temperature of the extended extension primers is at least 5°

C. higher than the melting temperature of the extended target complement portion of the extended extension primers.

117. The method of claim 115 wherein the melting temperature of the extended extension primers is at least 10°

C. higher than the melting temperature of the extended target complement portion of the extended extension primers.

118. The method of claim 115 wherein the melting temperature of the extended extension primers is at least 6°

C. higher than the temperature at which the extension primers and target templates are incubated.

119. The method of claim 115 wherein the melting temperature of the extended extension primers is at least 11°

C. higher than the temperature at which the extension primers and target templates are incubated.

120. The method of claim 115 wherein the melting temperature of the extended extension primers is at least 20°

C. higher than the temperature at which the extension primers and target templates are incubated.

121. The method of claim 97 further comprising detecting one or more of the extended extension primers.

122. The method of claim 121 wherein each extended extension primer corresponds to a different target template, wherein each target template corresponds to a different nucleic acid sequence of interest, wherein detection of an extended extension primer indicates detection of the nucleic acid sequence of interest to which the target template corresponding to the detected extended extension primer corresponds.

123. The method of claim 122 wherein detection of a plurality of extended extension primers indicates detection of the nucleic acid sequences of interest to which the target templates corresponding to the detected extended extension primers correspond.

124. The method of claim 121 wherein detection of the extended extension primers is performed simultaneously with incubation of the extension primers and target templates.

125. The method of claim 121 wherein the extension primers comprise a detection label, wherein the detection label of each extension primer is the same, wherein the extended extension primers are detected via the detection label.

126. The method of claim 121 wherein the extended extension primers comprise a detection label, wherein the detection label of each extended extension primer is the same, wherein the extended extension primers are detected via the detection label.

127. The method of claim 121 wherein the extension primers comprise a detection label, wherein the detection label of each extension primer is different, wherein the extended extension primers are detected via the detection label.

128. The method of claim 121 wherein the extended extension primers comprise a detection label, wherein the detection label of each extended extension primer is different, wherein the extended extension primers are detected via the detection label.

129. The method of claim 121 wherein a signal quotient is calculated for one or more of the detected extended extension primers.

130. The method of claim 121 wherein the extended extension primers are detected via detection labels, wherein a signal quotient is calculated for one or more of the detected extended extension primers using the detection labels.

131. The method of claim 121 wherein the extended extension primers are detected via detection labels, wherein the detection labels generate signals, wherein a signal quotient is calculated for one or more of the detected extended extension primers using the signals.

132. The method of claim 121 wherein the extended extension primers are detected via detection labels, wherein the detection labels generate signals, wherein the intensity of the signal generated for each detected extended extension primer is measured, wherein a signal quotient is calculated for one or more of the detected extended extension primers using the intensity of the measured signals.

133. The method of claim 132 wherein the signal quotient is a median signal quotient, wherein the median signal quotient of a given extended extension primer is calculated by dividing the signal intensity generated for that extended extension primer by the median signal intensity generated for the detected extended extension primers.

134. The method of claim 133 wherein the median signal quotient of a given extended extension primer is calculated by dividing the signal intensity generated for that extended extension primer by the median signal intensity generated for all of the detected extended extension primers.

135. The method of claim 133 wherein the median signal quotient of a given extended extension primer is calculated by dividing the signal intensity generated for that extended extension primer by the median signal intensity generated for a subset of all of the detected extended extension primers.

136. The method of claim 132 wherein the signal quotient is a ratio of signal quotients, wherein the ratio of signal quotients of a given extended extension primer is calculated by dividing the median signal quotient for that extended extension primer by the average signal quotient for that extended extension primer.

137. The method of claim 136 wherein each of the plurality of amplifications is carried out in the same way.

138. The method of claim 136 wherein the average signal quotient of a given extended extension primer is calculated by dividing the average signal intensity generated for that extended extension primer in 10 or more amplifications by the average signal intensity generated for the detected extended extension primers in the 10 or more amplifications.

139. The method of claim 136 wherein the average signal quotient of a given extended extension primer is calculated by dividing the average signal intensity generated for that extended extension primer in 20 or more amplifications by the average signal intensity generated for the detected extended extension primers in the 20 or more amplifications.

140. The method of claim 136 wherein the average signal quotient of a given extended extension primer is calculated by dividing the average signal intensity generated for that extended extension primer in 50 or more amplifications by the average signal intensity generated for the detected extended extension primers in the 50 or more amplifications.

141. The method of claim 136 wherein the average signal quotient of a given extended extension primer is calculated by dividing the average signal intensity generated for that extended extension primer in all of the amplifications by the average signal intensity generated for the detected extended extension primers in all of the amplifications.

142. The method of claim 136 wherein the average signal quotient of a given extended extension primer is calculated by dividing the average signal intensity generated for that extended extension primer in a subset of all of the amplifications by the average signal intensity generated for the detected extended extension primers in a subset of all of the amplifications.

143. The method of claim 121 wherein the extended extension primers are detected via interaction of the extended extension primers with detection probes, wherein each extended extension primer corresponds to a different detection probe, wherein each extended extension primer can interact with the detection probe to which the extended extension primer corresponds.

144. The method of claim 143 wherein the interaction is a base pairing interaction.

145. The method of claim 143 wherein the interaction is a hybridization interaction.

146. The method of claim 143 wherein the extended extension primers are covalently coupled to the detection probes.

147. The method of claim 146 wherein the extended extension primers are covalently coupled to the detection probes by ligation.

148. The method of claim 143 wherein the detection probes are stem-loop probes.

149. The method of claim 143 wherein the extended extension primers are covalently coupled to anchor probes, wherein covalent coupling of the extended extension primers is facilitated by the interaction of the extended extension primers with the detection probes, wherein each extended extension primer corresponds to a different anchor probe.

150. The method of claim 149 wherein the anchor probes are associated with a substrate.

151. The method of claim 149 wherein the extended extension primers are covalently coupled to the anchor probes by ligation.

152. The method of claim 143 wherein the detection probes comprise peptide nucleic acid.

153. The method of claim 152 wherein the melting temperature of the extended extension primers interacting with the detection probes to which the extended extension primers correspond is at least 5°

C. higher than the melting temperature of the extended extension primers interacting with the target templates to which the extended extension primers correspond.

154. The method of claim 152 wherein the melting temperature of the extended extension primers interacting with the detection probes to which the extended extension primers correspond is at least 10°

C. higher than the melting temperature of the extended extension primers interacting with the target templates to which the extended extension primers correspond.

155. The method of claim 152 wherein the melting temperature of the extended extension primers interacting with the detection probes to which the extended extension primers correspond is at least 6°

C. higher than the temperature at which the extension primers and target templates are incubated.

156. The method of claim 152 wherein the melting temperature of the extended extension primers interacting with the detection probes to which the extended extension primers correspond is at least 11°

C. higher than the temperature at which the extension primers and target templates are incubated.

157. The method of claim 152 wherein the melting temperature of the extended extension primers interacting with the detection probes to which the extended extension primers correspond is at least 20°

C. higher than the temperature at which the extension primers and target templates are incubated.

158. The method of claim 143 wherein detection probes each comprise a primer complement region.

159. The method of claim 158 wherein the primer complement region comprises peptide nucleic acid.

160. The method of claim 143 wherein the detection probes are associated with a substrate.

161. The method of claim 160 wherein the detection probes are attached to the substrate.

162. The method of claim 161 wherein the detection probes are covalently coupled to the substrate.

163. The method of claim 160 wherein the detection probes are indirectly associated with the substrate.

164. The method of claim 160 wherein the detection probes are directly associated with the substrate.

165. The method of claim 160 wherein the substrate is a single substrate structure, wherein all of the detection probes are associated with a single substrate.

166. The method of claim 165 wherein the detection probes are each associated with a different region of the substrate.

167. The method of claim 166 wherein the region of the substrate where an extended extension primer is detected is indicative of the identity of the detected extended extension primer.

168. The method of claim 165 wherein the substrate is a thin film, membranes, bead, microbead, bottle, dish, slide, fiber, optical fiber, woven fiber, chip, compact disk, shaped polymer, particles, or microparticle.

169. The method of claim 160 wherein the substrate is comprised of a plurality of substrate structures.

170. The method of claim 169 wherein the detection probes are each associated with a different substrate structure.

171. The method of claim 170 wherein the substrate structure on which an extended extension primer is detected is indicative of the identity of the detected extended extension primer.

172. The method of claim 169 wherein the detection probes are each associated with a different region of a substrate structure, a different substrate structure, or a different region of a different substrate structure.

173. The method of claim 172 wherein the substrate structure and region of the substrate structure where an extended extension primer is detected is indicative of the identity of the detected extended extension primer.

174. The method of claim 169 wherein the substrate comprises a plurality of beads.

175. The method of claim 174 wherein the beads are microbeads.

176. The method of claim 174 wherein the beads are paramagnetic beads.

177. The method of claim 160 wherein the substrate is acrylamide, cellulose, nitrocellulose, glass, gold, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, polysilicate, polycarbonate, teflon, fluorocarbon, nylon, silicon rubber, polyanhydride, polyglycolic acid, polylactic acid, polyorthoester, functionalized silane, polypropylfumerate, collagen, glycosaminoglycan, or polyamino acid.

178. The method of claim 1 wherein the target templates each comprise a primer complement region, an extension region, and replication terminating feature.

179. The method of claim 178 wherein the extension primers each comprise a target complement portion, wherein the target complement portion is complementary to a primer complement region of one or more of the target templates.

180. The method of claim 178 wherein the replication terminating feature is a 5′

end, one or more abasic nucleotides, or one or more derivatized nucleotides.

181. The method of claim 180 wherein the replication terminating feature is a 5′

end.

182. The method of claim 181 wherein the target templates correspond to nucleic acid molecules comprising nucleic acid sequences of interest, wherein the replication terminating feature of at least one of the target templates is produced by cleavage of the nucleic acid molecule to which the target template corresponds.

183. The method of claim 192 wherein the cleavage is accomplished using one or more restriction endonucleases, cleavase I, T4 endonuclease, endonuclease IV, one or more resolvases, or a combination.

184. The method of claim 182 wherein the nucleic acid molecule is DNA containing deoxyuridine, wherein the cleavage is accomplished by excising uracil residues in the nucleic acid molecule using uracil DNA glycosylase and cleaving the backbone of the nucleic acid molecule with endonuclease.

185. The method of claim 184 wherein the uracil residues are excised in the presence of single stranded binding protein, wherein excision of uracil in loops of the nucleic acid molecule is favored over excision of uracil in single-stranded regions of the nucleic acid molecule.

186. The method of claim 184 wherein the DNA containing deoxyuridine is produced by replicating a nucleic acid molecule using one or more DNA primers containing one or more deoxyuridines.

187. The method of claim 182 wherein one or more mismatch probes are hybridized to the nucleic acid molecule, wherein there is at least one mismatch between each mismatch probe and the nucleic acid molecule, wherein the cleavage is accomplished by using cleavase I, wherein cleavase I cleaves at the mismatches.

188. A method of detecting nucleic acid sequences, the method comprising bringing into contact one or more extension primers and one or more target templates and incubating under conditions that promote interaction of the extension primers and the target templates, extension of the extension primers using the interacting target templates as template, and dissociation of the extended extension primers from the target templates, whereby multiple extended extension primers are produced from at least one target template, and

detecting one or more of the extended extension primers.

189. The method of claim 188 wherein each extension primer corresponds to one or more of the target templates, wherein the target templates correspond to nucleic acid sequences of interest, wherein detection of an extended extension primer indicates detection of the nucleic acid sequence of interest to which a target template corresponding to the detected extended extension primer corresponds.

190. A method of detecting nucleic acid sequences, the method comprising

bringing into contact one or more extension primers and one or more target templates and incubating under conditions that promote interaction of the extension primers and the target templates, extension of the extension primers using the interacting target templates as template, and dissociation of the extended extension primers from the target templates, whereby multiple extended extension primers are produced from at least one target template, and

detecting one or more of the extended extension primers.

191. The method of claim 190 wherein each extension primer corresponds to one or more of the target templates, wherein the target templates correspond to nucleic acid sequences of interest, wherein detection of an extended extension primer indicates detection of the nucleic acid sequence of interest to which a target template corresponding to the detected extended extension primer corresponds.

192. A method of amplifying nucleic acid sequences, the method comprising

bringing into contact one or more extension primers and one or more target templates and incubating under conditions that promote interaction of the extension primers and the target templates, extension of the extension primers using the interacting target templates as template, and dissociation of the extended extension primers from the target templates, whereby multiple extended extension primers are produced from at least one target template.