Methods for detecting target nucleic acids using coupled ligation and amplification

• US 20030190646A1
• Filed: 12/02/2002
• Published: 10/09/2003
• Est. Priority Date: 05/30/2000
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Wenz, Hans Michael, Schroth, Gary P.
• Application Number:
  US10/308,891
• Publication Number:
  US 20030190646A1
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site

Abstract

The present invention relates to the detection of nucleic acid sequences using coupled ligation and amplification. The coupling of ligation and amplification allows multiplex detection of nucleic acid sequences. The invention also relates to methods, reagents, and kits that employ addressable-support specific sequences in detecting nucleic acid sequences.

Claims

1. A method for detecting at least one target sequence in a sample comprising:

combining the sample with a probe set for each target sequence, the probe set comprising (a) at least one first probe, comprising a target-specific portion and a 5′

primer-specific portion, and (b) at least one second probe, comprising a target-specific portion and a 3′

primer-specific portion, wherein the probes in each set are suitable for ligation together when hybridized adjacent to one another on a complementary target sequence, and wherein at least one probe in each probe set further comprises an addressable support-specific portion located between the primer-specific portion and the target-specific portion;

to form a ligation reaction mixture;

subjecting the ligation reaction mixture to at least one cycle of ligation, wherein adjacently hybridizing complementary probes are ligated to one another to form a ligation product comprising the 5′

primer-specific portion, the target-specific portions, at least one addressable support-specific portion, and the 3′

primer-specific portion;

combining the ligation reaction mixture with;

(a) at least one primer set, the primer set comprising (i) at least one first primer comprising the sequence of the 5′

primer-specific portion of the ligation product, and (ii) at least one second primer comprising a sequence complementary to the 3′

primer-specific portion of the ligation product, wherein at least one primer of the primer set further comprises a reporter group, and (b) a polymerase, to form a first amplification reaction mixture;

subjecting the first amplification reaction mixture to at least one cycle of amplification to generate a first amplification product comprising at least one reporter group;

hybridizing the addressable support-specific portions of the first amplification product or a portion of the first amplification product comprising at least one reporter group to support-bound capture oligonucleotides; and

detecting the at least one reporter group.

2. The method of claim 1, wherein the first probe further comprises the addressable support-specific portion.

3. The method of claim 1, wherein the second probe further comprises the addressable support-specific portion.

4. The method of claim 1, wherein the probe set further comprises more than one pivotal complement, a pivotal complement that is not the terminal nucleotide of the target-specific portion, or both.

5. The method of claim 210, wherein the ligation agent is a ligase.

6. The method of claim 5, wherein the ligation agent is a thermostable ligase.

7. The method of claim 6, wherein the thermostable ligase is Tth ligase, Taq ligase, Pfu ligase, or an enzymatically active mutant or variant thereof.

8. The method of claim 1, wherein each probe set further comprises at least two first probes that differ in the target-specific portion by at least one nucleotide.

9. The method of claim 1, wherein each probe set further comprises at least two second probes that differ in the target-specific portion by at least one nucleotide.

10. The method of claim 1, wherein the polymerase is a thermostable polymerase.

11. The method of claim 10, wherein the thermostable polymerase is Taq, Pfu, Vent, Deep Vent, UlTma, Pwo, Tth polymerase or an enzymatically active mutant or variant thereof.

12. The method of claim 1, further comprising purifying the ligation product prior to amplification.

13. The method of claim 12, wherein the purifying comprises hybridization-based pullout.

14. The method of claim 12, wherein the purifying comprises gel filtration.

15. The method of claim 12, wherein the purifying comprises dialysis.

16. The method of claim 1, wherein the reporter group comprises a fluorescent moiety.

17. The method of claim 1, wherein the first probe of each probe set further comprises a phosphorothioate group at the 3′

-end.

18. The method of claim 1, wherein the second probe of each probe set further comprises a 5′

thymidine residue with a leaving group suitable for ligation.

19. The method of claim 18, wherein the 5′

thymidine leaving group is tosylate or iodide.

20. The method of claim 1, wherein the first amplification product comprises at least one 5′

terminal phosphate; and

further comprising;

combining the first amplification product with an exonuclease to form a digestion reaction mixture; and

incubating the digestion reaction mixture under conditions that allow the exonuclease to digest the amplification product to generate single stranded addressable support-specific portions.

21. The method of claim 1, wherein the first amplification reaction mixture comprises at least one first primer or at least one second primer for each primer set, but not both first and second primers of a primer set, and wherein the at least one first primer or the at least one second primer of a primer set, but not both, comprises a reporter group.

22. The method of claim 1, wherein the at least one first probe and the at least one second probe in the probe set further comprise an addressable support-specific portion located between the primer-specific portion and the target-specific portion, and at least two primers of the at least one primer set comprise reporter groups.

23. The method of claim 1, further comprising denaturing the first amplification product to generate single-stranded portions of the amplification product.

24. The method of claim 23, wherein denaturing comprises heating the amplification product to a temperature above the melting temperature of the amplification product to generate single stranded portions.

25. The method of claim 24, wherein denaturing comprises chemically denaturing the amplification product to generate single stranded portions.

26. The method of claim 1, wherein the molar concentration of the at least one first primer is different from the molar concentration of the at least one second primer in the at least one primer set.

210. The method of claim 1, wherein the ligation reaction mixture further comprises a ligation agent.

27. A method for detecting at least one target sequence in a sample comprising:

combining the sample with a probe set for each target sequence, the probe set comprising (a) at least one first probe, comprising a target-specific portion, and (b) at least one second probe, comprising a target-specific portion and a 3′

primer-specific portion, wherein the probes in each set are suitable for ligation together when hybridized adjacent to one another on a complementary target sequence, and wherein at least one probe in each probe set further comprises an addressable support-specific portion;

to form a ligation reaction mixture;

subjecting the ligation reaction mixture to at least one cycle of ligation, wherein adjacently hybridizing complementary probes are ligated to one another to form a ligation product comprising the target specific portions, at least one addressable support-specific portion, and the primer-specific portion;

combining the ligation reaction mixture with at least one primer comprising a sequence complementary to the primer-specific portion of the ligation product and a reporter group, and a polymerase, to form an extension reaction mixture;

subjecting the extension reaction mixture to at least one cycle of primer extension to generate a first amplification product comprising at least one reporter group;

hybridizing the addressable support-specific portions of the first amplification product or a portion of the first amplification product comprising at least one reporter group to support-bound capture oligonucleotides; and

detecting the at least one reporter group.

28. The method of claim 27, wherein the first probe further comprises the addressable support-specific portion.

29. The method of claim 27, wherein the second probe further comprises the addressable support-specific portion.

30. The method of claim 211, wherein the ligation agent is a ligase.

31. The method of claim 30, wherein the ligation agent is a thermostable ligase.

32. The method of claim 31, wherein the thermostable ligase is Pfu ligase, Tth ligase, Taq ligase, or an enzymatically active mutant or variant thereof.

33. The method of claim 27, wherein each probe set further comprises at least two first probes that differ in the target-specific portion by at least one nucleotide.

34. The method of claim 27, wherein each probe set further comprises at least two second probes that differ in the target-specific portion by at least one nucleotide.

35. The method of claim 27, wherein the polymerase is a thermostable polymerase.

36. The method of claim 35, wherein the thermostable polymerase is Taq polymerase, Pfu polymerase, Vent polymerase, Deep Vent polymerase, UlTma polymerase, Pwo polymerase, Tth polymerase or an enzymatically active mutant or variant thereof.

37. The method of claim 27, further comprising purifying the ligation product prior to amplification.

38. The method of claim 37, wherein the purifying comprises hybridization-based pullout.

39. The method of claim 37, wherein the purifying comprises gel filtration.

40. The method of claim 37, wherein the purifying comprises dialysis.

41. The method of claim 27, wherein the reporter group comprises a fluorescent moiety.

42. The method of claim 27, wherein the first probe of each probe set further comprises a phosphorothioate group at the 3′

-end.

43. The method of claim 27, wherein the second probe of each probe set further comprises a 5′

thymidine residue with a leaving group suitable for ligation.

44. The method of claim 43, wherein the 5′

thymidine leaving group is tosylate or iodide.

211. The method of claim 27, wherein the ligation reaction mixture further comprises a ligation agent.

45. A method for detecting at least one target sequence in a sample comprising:

combining the sample with a probe set for each target sequence, the probe set comprising (a) at least one first probe, comprising a target-specific portion and a 5′

primer-specific portion, and (b) at least one second probe, comprising a target-specific portion and a 3′

primer-specific portion, wherein the probes in each set are suitable for ligation together when hybridized adjacent to one another on a complementary target sequence, and wherein at least one probe in each probe set further comprises an addressable support-specific portion located between the primer-specific portion and the target-specific portion;

to form a ligation reaction mixture;

subjecting the ligation reaction mixture to at least one cycle of ligation, wherein adjacently hybridizing complementary probes are ligated to one another to form a ligation product comprising the 5′

primer-specific portion, the target specific portions, at least one addressable support-specific portion, and the 3′

primer-specific portion;

combining the ligation reaction mixture with;

(a) at least one primer set comprising;

(i) at least one first primer comprising the sequence of the 5′

primer-specific portion of the ligation product, and (ii) at least one second primer comprising a sequence complementary to the 3′

primer-specific portion of the ligation product, and (b) a polymerase, to form a first amplification reaction mixture;

subjecting the first amplification reaction mixture to at least one cycle of amplification to generate a first amplification product;

combining the first amplification product with either at least one first primer, or at least one second primer for each primer set, but not both first and second primers, wherein the at least one first primer or the at least one second primer further comprises a reporter group, to form a second amplification reaction mixture;

subjecting the second amplification reaction mixture to at least one cycle of amplification to generate a second amplification product comprising at least one reporter group;

hybridizing the addressable support-specific portions of the second amplification product or a portion of the second amplification product comprising at least one reporter group to support-bound capture oligonucleotides; and

detecting the at least one reporter group.

46. The method of claim 45, wherein the at least one first probe and the at least one second probe in the probe set further comprise an addressable support-specific portion located between the primer-specific portion and the target-specific portion, and at least two primers of the second amplification reaction mixture primer set comprise reporter groups.

212. The method of claim 45, wherein the ligation reaction mixture further comprises a ligation agent.

213. The method of claim 212, wherein the ligation agent is a ligase.

214. The method of claim 213, wherein the ligation agent is a thermostable ligase.

215. The method of claim 214, wherein the thermostable ligase is Pfu ligase, Tth ligase, Taq ligase, or an enzymatically active mutant or variant thereof.

47. A probe suitable for ligation comprising:

a 5′

-end, a 3′

end, a target-specific portion, a primer-specific portion, and an addressable support-specific portion located between the primer-specific portion and the target-specific portion.

48. The probe of claim 47, further comprising a free phosphate group at the 5′

-end.

49. The probe of claim 47, further comprising a phosphorothioate group at the 3′

-end.

50. The probe of claim 47, further comprising a thymidine residue at the 5′

-end with a leaving group suitable for ligation.

51. The probe of claim 50, wherein the 5′

thymidine leaving group is tosylate or iodide.

52. A kit for detecting at least one target sequence in a sample comprising:

at least one probe set for each target sequence to be detected, the probe set comprising (a) at least one first probe, comprising a target-specific portion and a 5′

primer-specific portion, and (b) at least one second probe, comprising a target-specific portion and a 3′

primer-specific portion, wherein the probes in each probe set are suitable for ligation together when hybridized adjacent to one another on a complementary target sequence, and wherein at least one probe in each probe set further comprises an addressable support-specific portion located between the primer-specific portion and the target-specific portion; and

optionally,

a ligation agent.

53. A kit according to claim 52, further comprising a set of nucleotide primers, the primer set comprising (i) at least one primer comprising the sequence of the 5′

primer-specific portion of the probe, and (ii) at least one primer comprising a sequence complementary to the 3′

primer-specific portion of the probe, wherein at least one primer of the primer set further comprises a reporter group; and

a polymerase.

54. A kit according to claim 52, further comprising a support, the support comprising capture oligonucleotides capable of hybridizing with addressable support-specific portions of the probes or with sequences complementary to the addressable support-specific portions of the probes.

55. A kit according to claim 54, wherein the polymerase is a thermostable polymerase.

56. A kit according to claim 55, wherein the thermostable polymerase is Taq, Pfu, Vent, Deep Vent, UlTma, Pwo, or Tth polymerase.

57. A kit according to claim 52, wherein the ligation agent is a ligase.

58. A kit according to claim 57, wherein the ligase is a thermostable ligase.

59. A kit according to claim 58, wherein the thermostable ligase is Tth or Taq ligase.

60. A kit for detecting at least one target sequence in a sample comprising:

at least one probe set for each target sequence to be detected, each probe set comprising (a) at least one first probe, comprising a target-specific portion and a 5′

primer-specific portion, and (b) at least one second probe, comprising a target-specific portion and a 3′

primer-specific portion, wherein the probes in each set are suitable for ligation together when hybridized adjacent to one another on a complementary target sequence, and wherein at least one probe in each probe set further comprises an addressable support-specific portion located between the primer-specific portion and the target-specific portion.

61. A kit according to claim 60, further comprising a support, the support comprising capture oligonucleotides capable of hybridizing with addressable support-specific portion of the at least one probe or with sequences complementary to the addressable support-specific portions of the at least one probe.

62. A kit according to claim 60, further comprising a primer set, the primer set comprising (i) at least one primer comprising the sequence of the 5′

primer-specific portion of the first probe, and (ii) at least one primer complementary to the 3′

primer-specific portion of the second probe, and wherein at least one primer of the primer set further comprises a reporter group; and

a polymerase.

63. A kit according to claim 60, wherein the reporter group comprises a fluorescent moiety.

64. A kit according to claim 60, wherein the first probe of each probe set further comprises a phosphorothioate group at the 3′

-end.

65. A kit according to claim 60, wherein the second probe of each probe set further comprises a 5′

thymidine residue with a leaving group suitable for ligation.

66. A kit according to claim 65, wherein the 5′

thymidine leaving group is tosylate or iodide.

67. A kit according to claim 60, wherein the first probe of each probe set further comprises a phosphorothioate group at the 3′

-end and wherein the second probe of each probe set further comprises a 5′

thymidine residue with. a leaving group suitable for ligation.

68. A kit according to claim 67, wherein the 5′

thymidine leaving group is tosylate or iodide.

69. A kit according to claim 60, wherein each probe set further comprises at least two first probes that differ in the target specific portion by at least one nucleotide.

70. A kit according to claim 60, wherein each probe set further comprises at least two second probes that differ in the target specific portion by at least one nucleotide.

71. A kit for detecting at least one target sequence in a sample comprising:

at least one probe set for each target sequence to be detected, the probe set comprising (a) at least one first probe, comprising a target-specific portion and (b) at least one second probe, comprising a target-specific portion and a primer-specific portion, wherein the probes in each set are suitable for ligation together when hybridized adjacent to one another on a complementary target sequence, and wherein at least one second probe in each probe set further comprises an addressable support-specific portion located between the primer-specific portion and the target-specific portion; and

optionally,

a ligation agent.

72. A kit according to claim 71, further comprising a support, the support comprising capture oligonucleotides capable of hybridizing with addressable support-specific portions of the probes or with sequences complementary to the addressable support-specific portions of the probes.

73. A kit according to claim 71, further comprising at least one primer complementary to the primer-specific portion of the second probe and a reporter group;

and a polymerase.

74. A kit according to claim 71, wherein the reporter group comprises a fluorescent moiety.

75. A kit according to claim 71, wherein the polymerase is a thermostable polymerase.

76. A kit according to claim 75, wherein the thermostable polymerase is Taq, Pfu, Vent, Deep Vent, UlTma, Pwo, or Tth polymerase or enzymatically active mutants or variants thereof.

77. A kit according to claim 71, wherein the ligation agent is a ligase.

78. A kit according to claim 77, wherein the ligase is a thermostable ligase.

79. A kit according to claim 78, wherein the thermostable ligase is Tth or Taq ligase.

80. A kit according to claim 71, wherein the first probe of each probe set further comprises a phosphorothioate group at the 3′

-end.

81. A kit according to claim 71, wherein the second probe of each probe set further comprises a 5′

thymidine residue with a leaving group suitable for ligation.

82. A kit according to claim 81, wherein the 5′

thymidine leaving group is tosylate or iodide.

83. A kit according to claim 71, wherein the first probe of each probe set further comprises a phosphorothioate group at the 3′

-end and wherein the second probe of each probe set further comprises a 5′

thymidine residue with a leaving group suitable for ligation.

84. A kit according to claim 83, wherein the 5′

thymidine leaving group is tosylate or iodide.

85. A kit according to claim 71, wherein each probe set further comprises at least two first probes that differ in the target specific portion by at least one nucleotide.

86. A kit according to claim 71, wherein each probe set further comprises at least two second probes that differ in the target specific portion by at least one nucleotide.

87. A method for detecting at least one target sequence in a sample comprising:

combining the sample with a probe set for each target sequence, the probe set comprising (a) at least one first probe, comprising a target-specific portion and a 5′

primer-specific portion, and (b) at least one second probe, comprising a target-specific portion and a 3′

primer-specific portion, wherein the probes in each set are suitable for ligation together when hybridized adjacent to one another on a complementary target sequence, and wherein at least one probe in each probe set further comprises an addressable support-specific portion located between the primer-specific portion and the target-specific portion;

to form a ligation reaction mixture;

subjecting the ligation reaction mixture to at least one cycle of ligation, wherein adjacently hybridizing complementary probes are ligated to one another to form a ligation product comprising the 5′

primer-specific portion, the target-specific portions, at least one addressable support-specific portion, and the 3′

primer-specific portion;

combining the ligation reaction mixture with;

(a) at least one primer set comprising (i) at least one first primer comprising the sequence of the 5′

primer-specific portion of the ligation product, and (ii) at least one second primer comprising a sequence complementary to the 3′

primer-specific portion of the ligation product, wherein at least one primer of the primer set further comprises a reporter group, and (b) a polymerase, to form a first amplification reaction mixture;

subjecting the first amplification reaction mixture to at least one cycle of amplification to generate a first amplification product comprising at least one reporter group;

separating the first amplification product or a portion of the first amplification product comprising at least one reporter group; and

detecting the at least one reporter group.

88. The method of claim 87, wherein separating comprises electrophoresis, gel filtration, mass spectroscopy, or HPLC.

89. The method of claim 87, wherein the first probe further comprises the addressable support-specific portion.

90. The method of claim 87, wherein the second probe further comprises the addressable support-specific portion.

91. The method of claim 216, wherein the ligation agent is a ligase.

92. The method of claim 91, wherein the ligation agent is a thermostable ligase.

93. The method of claim 92, wherein the thermostable ligase is Tth ligase, Taq ligase, Pfu ligase, or an enzymatically mutant or variant thereof.

94. The method of claim 87, wherein each probe set further comprises at least two first probes that differ in the target-specific portion by at least one nucleotide.

95. The method of claim 87, wherein each probe set further comprises at least two second probes that differ in the target-specific portion by at least one nucleotide.

96. The method of claim 87, wherein the polymerase is a thermostable polymerase.

97. The method of claim 96, wherein the thermostable polymerase is Taq, Pfu, Vent, Deep Vent, UlTma, Pwo, Tth polymerase or an enzymatically active mutant or variant thereof.

98. The method of claim 87, further comprising purifying the ligation product prior to amplification.

99. The method of claim 98, wherein the purifying comprises hybridization-based pullout.

100. The method of claim 87, wherein the reporter group comprises a fluorescent moiety.

115. The method of claim 87, wherein the addressable support-specific portion is 100 nucleotides or less long.

116. The method of claim 115, wherein the addressable support-specific portion is 40 nucleotides or less long.

117. The method of claim 116, wherein the addressable support-specific portion is 2-36 nucleotides long.

118. The method of claim 87, wherein the separating comprises at least one mobility-dependent analysis technique (MDAT).

119. The method of claim 118, wherein the MDAT comprises at least one of electrophoresis, chromatography, HPLC, mass spectroscopy, sedimentation, field-flow fractionation, or multi-stage fractionation.

120. The method of claim 119, wherein the MDAT comprises gel electrophoresis or capillary electrophoresis.

121. The method of claim 87, wherein the first probe further comprises the addressable support-specific portion.

122. The method of claim 87, wherein the second probe further comprises the addressable support-specific portion.

123. The method of claim 87, wherein the probe set further comprises more than one pivotal complement, a pivotal complement that is not the terminal nucleotide of the target-specific portion, or both.

124. The method of claim 87, wherein the ligation reaction mixture further comprises a ligase.

125. The method of claim 124, wherein the ligase is thermostable.

126. The method of claim 125, wherein the thermostable ligase is Tth ligase, Taq ligase, Pfu ligase, or an enzymatically active mutant or variant thereof.

127. The method of claim 87, wherein each probe set further comprises at least two first probes that differ in the target-specific portion by at least one nucleotide.

128. The method of claim 87, wherein each probe set further comprises at least two second probes that differ in the target-specific portion by at least one nucleotide.

129. The method of claim 87, wherein the polymerase is a thermostable polymerase.

130. The method of claim 129, wherein the thermostable polymerase is Taq polymerase, Pfu polymerase, Vent polymerase, Deep Vent polymerase, UlTma polymerase, Pwo polymerase, Tth polymerase, or an enzymatically active mutant or variant thereof.

131. The method of claim 87, further comprising purifying the ligation product prior to amplification.

132. The method of claim 131, wherein the purifying comprises hybridization-based pullout.

133. The method of claim 131, wherein the purifying comprises gel filtration.

134. The method of claim 131, wherein the purifying comprises dialysis.

135. The method of claim 87, wherein the reporter group comprises a fluorescent moiety.

136. The method of claim 87, wherein the first probe of each probe set further comprises a phosphorothioate group at the 3′

-end.

137. The method of claim 87, wherein the second probe of each probe set further comprises a 5′

thymidine residue with a leaving group suitable for ligation.

138. The method of claim 137, wherein the 5′

thymidine leaving group is tosylate or iodide.

139. The method of claim 87, wherein the first amplification product comprises at least one 5′

terminal phosphate; and

further comprising;

combining the first amplification product with an exonuclease to form a digestion reaction mixture;

incubating the digestion reaction mixture under conditions that allow the exonuclease to digest the amplification product to generate single stranded addressable support-specific portions comprising at least one reporter group;

separating the digested amplification product; and

detecting the at least one reporter group.

141. The method of claim 87, wherein the at least one first probe and the at least one second probe in the probe set further comprise an addressable support-specific portion located between the primer-specific portion and the target-specific portion, and at least two primers of the at least one primer set comprise reporter groups.

142. The method of claim 87, further comprising denaturing the first amplification product to generate single-stranded portions of the first amplification product.

143. The method of claim 142, wherein denaturing comprises heating the amplification product to a temperature above the melting temperature of the amplification product to generate single-stranded portions.

144. The method of claim 142, wherein denaturing comprises chemically denaturing the amplification product to generate single-stranded portions.

145. The method of claim 87, wherein the molar concentration of the at least one first primer is different from the molar concentration of the at least one second primer in at least one primer set.

146. The method of claim 87, wherein at least one addressable support-specific portion is complementary to a particular sequence that serves as a mobility modifier, the sequence comprising:

(i) a tag complement for selectively binding to the at least one addressable support-specific portion of the amplification product, and (ii) a tail for effecting a particular mobility in a MDAT.

216. The method of claim 87, wherein the ligation reaction mixture further comprises a ligation agent.

101. A method for detecting at least one target sequence in a sample comprising:

combining the sample with a probe set for each target sequence, the probe set comprising (a) at least one first probe, comprising a target-specific portion, and (b) at least one second probe, comprising a target-specific portion and a 3′

primer-specific portion, wherein the probes in each set are suitable for ligation together when hybridized adjacent to one another on a complementary target sequence, and wherein at least one probe in each probe set further comprises an addressable support-specific portion;

to form a ligation reaction mixture;

subjecting the ligation reaction mixture to at least one cycle of ligation, wherein adjacently hybridizing complementary probes are ligated to one another to form a ligation product comprising the target specific portions, at least one addressable support-specific portion, and the primer-specific portion;

combining the ligation reaction mixture with at least one primer comprising a sequence complementary to the primer-specific portion of the ligation product and a reporter group, and a polymerase, to form an extension reaction mixture;

subjecting the extension reaction mixture to at least one cycle of primer extension to generate a first amplification product comprising at least one reporter group;

separating the first amplification product or a portion of the first amplification product comprising at least one reporter group; and

detecting the at least one reporter group.

102. The method of claim 101, wherein separating comprises electrophoresis, gel filtration, mass spectroscopy, or HPLC.

103. The method of claim 101, wherein the first probe further comprises the addressable support-specific portion.

104. The method of claim 101, wherein the second probe further comprises the addressable support-specific portion.

105. The method of claim 217, wherein the ligation agent is a ligase.

106. The method of claim 105, wherein the ligation agent is a thermostable ligase.

107. The method of claim 106, wherein the thermostable ligase is Tth ligase, Taq ligase, Pfu ligase, or an enzymatically active mutant or variant thereof.

108. The method of claim 101, wherein each probe set further comprises at least two first probes that differ in the target-specific portion by at least one nucleotide.

109. The method of claim 101, wherein each probe set further comprises at least two second probes that differ in the target-specific portion by at least one nucleotide.

110. The method of claim 101, wherein the polymerase is a thermostable polymerase.

111. The method of claim 110, wherein the thermostable polymerase is Taq, Pfu, Vent, Deep Vent, UlTma, Pwo, Tth polymerase or an enzymatically active mutant or variant thereof.

112. The method of claim 101, further comprising purifying the ligation product prior to amplification.

113. The method of claim 112, wherein the purifying comprises hybridization-based pullout.

114. The method of claim 101, wherein the reporter group comprises a fluorescent moiety.

147. The method of claim 101, wherein the addressable support-specific portion is 0-100 nucleotides long.

148. The method of claim 147, wherein the addressable support-specific portion is 0-40 nucleotides long.

149. The method of claim 148, wherein the addressable support-specific portion is 2-36 nucleotides long.

150. The method of claim 101, wherein the separating comprises an MDAT.

151. The method of claim 101, wherein the MDAT comprises at least one of electrophoresis, chromatography, HPLC, mass spectroscopy, sedimentation, field-flow fractionation, or multi-stage fractionation.

152. The method of claim 151, wherein the MDAT comprises gel electrophoresis or capillary electrophoresis.

153. The method of claim 101, wherein the first probe further comprises the addressable support-specific portion.

154. The method of claim 101, wherein the second probe further comprises the addressable support-specific portion.

155. The method of claim 101, wherein the probe set further comprises more than one pivotal complement, a pivotal complement that is not the terminal nucleotide of the target-specific portion, or both.

156. The method of claim 101, wherein the ligation reaction mixture further comprises a ligase.

157. The method of claim 156, wherein the ligase is thermostable.

158. The method of claim 157, wherein the thermostable ligase is Tth ligase, Taq ligase, Pfu ligase, or an enzymatically active mutant or variant thereof.

159. The method of claim 101, wherein each probe set further comprises at least two first probes that differ in the target-specific portion by at least one nucleotide.

160. The method of claim 101, wherein each probe set further comprises at least two second probes that differ in the target-specific portion by at least one nucleotide.

161. The method of claim 101, wherein the polymerase is a thermostable polymerase.

162. The method of claim 161, wherein the thermostable polymerase is Taq polymerase, Pfu polymerase, Vent polymerase, Deep Vent polymerase, UlTma polymerase, Pwo polymerase, Tth polymerase, or an enzymatically active mutant or variant thereof.

163. The method of claim 101, further comprising purifying the ligation product prior to amplification.

164. The method of claim 163, wherein the purifying comprises hybridization-based pullout.

165. The method of claim 163, wherein the purifying comprises gel filtration.

166. The method of claim 163, wherein the purifying comprises dialysis.

167. The method of claim 101, wherein the reporter group comprises a fluorescent moiety.

168. The method of claim 101, wherein the first probe of each probe set further comprises a phosphorothioate group at the 3′

-end.

169. The method of claim 101, wherein the second probe of each probe set further comprises a 5′

thymidine residue with a leaving group suitable for ligation.

170. The method of claim 169, wherein the 5′

thymidine leaving group is tosylate or iodide.

171. The method of claim 101, wherein the first amplification product comprises at least one 5′

terminal phosphate; and

further comprising;

combining the first amplification product with an exonuclease to form a digestion reaction mixture; and

incubating the digestion reaction mixture under conditions that allow the exonuclease to digest the amplification product to generate single stranded addressable support-specific portions comprising at least one reporter group;

separating the digested amplification product; and

detecting the at least one reporter group.

172. The method of claim 101, further comprising denaturing the first amplification product to generate single-stranded portions of the amplification product.

173. The method of claim 172, wherein denaturing comprises heating the amplification product to a temperature above the melting temperature of the amplification product to generate single-stranded portions.

174. The method of claim 172, wherein denaturing comprises chemically denaturing the amplification product to generate single-stranded portions.

175. The method of claim 101, wherein at least one addressable support-specific portion is complementary to a particular sequence that serves as a mobility modifier, the sequence comprising:

(i) a tag complement for selectively binding to the addressable support-specific portion of the amplification product, and (ii) a tail for effecting a particular mobility in a MDAT.

217. The method of claim 101, wherein the ligation reaction mixture further comprises a ligation agent.

140. A method for detecting at least one target sequence in a sample comprising:

combining the sample with a probe set for each target sequence, the probe set comprising (a) at least one first probe, comprising a target-specific portion and a 5′

primer-specific portion, and (b) at least one second probe, comprising a target-specific portion and a 3′

primer-specific portion, wherein the probes in each set are suitable for ligation together when hybridized adjacent to one another on a complementary target sequence, and wherein at least one probe in each probe set further comprises an addressable support-specific portion located between the primer-specific portion and the target-specific portion;

to form a ligation reaction mixture;

subjecting the ligation reaction mixture to at least one cycle of ligation, wherein adjacently hybridizing complementary probes are ligated to one another to form a ligation product comprising the 5′

primer-specific portion, the target-specific portions, at least one addressable support-specific portion, and the 3′

primer-specific portion;

combining the ligation reaction mixture with;

(a) at least one primer set comprising (i) at least one first primer comprising the sequence of the 5′

primer-specific portion of the ligation product and a reporter group, or (ii) at least one second primer comprising a sequence complementary to the 3′

primer-specific portion of the ligation product and a reporter group, but not both at least one first primer and at least one second primer, and (b) a polymerase, to form a first amplification reaction mixture;

subjecting the first amplification reaction mixture to at least one cycle of amplification to generate a first amplification product comprising at least one reporter group;

separating the first amplification product or a portion of the first amplification product comprising at least one reporter group; and

detecting the reporter group.

176. A method for detecting at least one target sequence in a sample comprising:

combining the sample with a probe set for each target sequence, the probe set comprising (a) at least one first probe, comprising a target-specific portion and a 5′

primer-specific portion, and (b) at least one second probe, comprising a target-specific portion and a 3′

primer-specific portion, wherein the probes in each set are suitable for ligation together when hybridized adjacent to one another on a complementary target sequence, and wherein at least one probe in each probe set further comprises an addressable support-specific portion located between the primer-specific portion and the target-specific portion;

to form a ligation reaction mixture;

subjecting the ligation reaction mixture to at least one cycle of ligation, wherein adjacently hybridizing complementary probes are ligated to one another to form a ligation product comprising the 5′

primer-specific portion, the target specific portions, at least one addressable support-specific portion, and the 3′

primer-specific portion;

combining the ligation reaction mixture with;

(a) at least one primer set comprising;

(i) at least one first primer comprising the sequence of the 5′

primer-specific portion of the ligation product, and (ii) at least one second primer comprising a sequence complementary to the 3′

primer-specific portion of the ligation product, and (b) a polymerase, to form a first amplification reaction mixture;

subjecting the first amplification reaction mixture to at least one cycle of amplification to generate a first amplification product;

combining the first amplification product with at least one second primer set comprising at least one third primer, or at least one fourth primer for each primer set, but not both first and second primers, wherein the at least one first primer or the at least one second primer further comprises a reporter group, to form a second amplification reaction mixture;

subjecting the second amplification reaction mixture to at least one cycle of amplification to generate a second amplification product comprising at least one reporter group;

separating the second amplification product or a portion of the second amplification product comprising at least one reporter group; and

detecting the reporter group.

177. The method of claim 176, wherein the addressable support-specific portion is 0-100 nucleotides long.

178. The method of claim 177, wherein the addressable support-specific portion is 0-40 nucleotides long.

179. The method of claim 178, wherein the addressable support-specific portion is 2-36 nucleotides long.

180. The method of claim 176, wherein the separating comprises at least one MDAT.

181. The method of claim 180, wherein the MDAT comprises at least one of electrophoresis, chromatography, HPLC, mass spectroscopy, sedimentation, field-flow fractionation, or multi-stage fractionation.

182. The method of claim 181, wherein the MDAT comprises gel electrophoresis or capillary electrophoresis.

183. The method of claim 176, wherein the at least one first probe and the at least one second probe in the probe set further comprise an addressable support-specific portion located between the primer-specific portion and the target-specific portion, and wherein at least one primer of the second primer set comprises reporter groups.

184. The method of claim 176, wherein the ligation reaction mixture further comprises a ligase.

185. The method of claim 184, wherein the ligase is thermostable.

186. The method of claim 185, wherein the thermostable ligase is Tth ligase, Taq ligase, Pfu ligase, or an enzymatically active mutant or variant thereof.

187. The method of claim 176, wherein the first probe further comprises the addressable support-specific portion.

188. The method of claim 176, wherein the second probe further comprises the addressable support-specific portion.

189. The method of claim 176, wherein the probe set further comprises more than one pivotal complement, a pivotal complement that is not the terminal nucleotide of the target-specific portion, or both.

190. The method of claim 176, wherein each probe set further comprises at least two first probes that differ in the target-specific portion by at least one nucleotide.

191. The method of claim 176, wherein each probe set further comprises at least two second probes that differ in the target-specific portion by at least one nucleotide.

192. The method of claim 176, wherein the polymerase is a thermostable polymerase.

193. The method of claim 192, wherein the thermostable polymerase is Taq polymerase, Pfu polymerase, Vent polymerase, Deep Vent polymerase, UlTma polymerase, Pwo polymerase, Tth polymerase, or an enzymatically active mutant or variant thereof.

194. The method of claim 176, further comprising purifying the ligation product prior to amplification.

195. The method of claim 194, wherein the purifying comprises hybridization-based pullout.

196. The method of claim 194, wherein the purifying comprises gel filtration.

197. The method of claim 194, wherein the purifying comprises dialysis.

198. The method of claim 176, wherein the reporter group comprises a fluorescent moiety.

199. The method of claim 176, wherein the first probe of each probe set further comprises a phosphorothioate group at the 3′

-end.

200. The method of claim 176, wherein the second probe of each probe set further comprises a 5′

thymidine residue with a leaving group suitable for ligation.

201. The method of claim 200, wherein the 5′

thymidine leaving group is tosylate or iodide.

202. The method of claim 176, wherein the at least one first probe and the at least one second probe in the probe set further comprise an addressable support-specific portion located between the primer-specific portion and the target-specific portion, and at least one primer of the at least one second primer set comprises at least one reporter group.

203. The method of claim 176, wherein at least one addressable support-specific portion is complementary to a particular sequence that serves as a mobility modifier, the sequence comprising:

(i) a tag complement for selectively binding to the addressable support-specific portion of the amplification product, and (ii) a tail for effecting a particular mobility in a mobility-dependent analysis technique.

204. A probe suitable for ligation comprising:

a 5′

-end, a 3′

-end, a target-specific portion, a primer-specific portion, and a mobility modifier sequence located between the primer-specific portion and the target-specific portion.

205. The probe of claim 204, further comprising a free phosphate group at the 5′

-end.

206. The probe of claim 204, further comprising a phosphorothioate group at the 3′

-end.

207. The probe of claim 204, further comprising a thymidine residue at the 5′

-end with a leaving group suitable for ligation.

208. The probe of claim 207, wherein the 5′

thymidine leaving group is tosylate or iodide.

209. A kit comprising the probe of claim 204.

218. A method for identifying splice variants in at least one target nucleic acid sequence in a sample, comprising:

combining at least one target nucleic acid sequence with a probe set for each target nucleic acid sequence, the probe set comprising (a) at least one first probe, comprising a target-specific portion and a 5′

primer-specific portion, and (b) a plurality of second probes, each second probe comprising a 3′

primer-specific portion and one of a plurality of splice-specific portions, wherein the probes in each probe set are suitable for ligation together when hybridized adjacent to one another on the at least one target nucleic acid sequence, and wherein at least one probe in each probe set further comprises at least one addressable support-specific portion located between the primer-specific portion and the target-specific portion or between the primer-specific portion and the splice-specific portion;

to form a ligation reaction composition;

subjecting the ligation reaction composition to at least one cycle of ligation, wherein adjacently hybridized probes are ligated to one another to form a ligation product comprising the 5′

primer-specific portion, the target-specific portion, the splice-specific portion, the at least one addressable support-specific portion, and the 3′

primer-specific portion;

combining the ligation product with;

(a) at least one primer set, the primer set comprising (i) at least one first primer comprising the sequence of the 5′

primer-specific portion of the ligation product, and (ii) at least one second primer comprising a sequence complementary to the 3′

primer-specific portion of the ligation product, wherein at least one primer of the primer set further comprises at least one reporter group; and

(b) a polymerase;

to form a first amplification reaction composition;

subjecting the first amplification reaction composition to at least one cycle of amplification to generate a first amplification product comprising the at least one reporter group;

analyzing the first amplification product or a portion of the first amplification product comprising the at least one reporter group using at least a portion of the at least one addressable support-specific portion; and

identifying the splice variant in the at least one target nucleic acid sequence by detecting the at least one reporter group.

219. The method of claim 218, wherein the at least one target nucleic acid sequence comprises at least one complementary DNA (cDNA) generated from an RNA.

220. The method of claim 219, wherein the at least one cDNA is generated from a messenger RNA (mRNA).

221. The method of claim 218, wherein the at least one target nucleic acid sequence comprises at least one RNA target present in the sample.

222. The method of claim 221, wherein the ligation reaction composition further comprises a T4 DNA ligase.

223. The method of claim 218, wherein the polymerase is a DNA dependent DNA polymerase.

224. The method of claim 218, wherein the analyzing comprises hybridizing the addressable support-specific portion of the first amplification product or a portion of the first amplification product comprising at least one reporter group directly or indirectly to a support.

225. The method of claim 224, further comprising denaturing the first amplification product to generate single-stranded portions of the amplification product.

226. The method of claim 225, wherein the denaturing comprises heating the amplification product to a temperature above the melting temperature of the amplification product to generate single-stranded portions.

227. The method of claim 225, wherein the denaturing comprises chemically denaturing the amplification product to generate single-stranded portions.

228. The method of claim 218, wherein the first probe further comprises the addressable support-specific portion.

229. The method of claim 218, wherein the second probe further comprises the addressable support-specific portion.

230. The method ′

of claim 218, wherein the addressable support-specific portion comprises a mobility sequence that imparts a particular mobility on the first amplification product or a portion of the first amplification product comprising the at least one reporter group.

231. The method of claim 230, wherein the mobility sequence is less than 101 nucleotides in length.

232. The method of claim 231, wherein the mobility sequence is less than 41 nucleotides in length.

233. The method of claim 231, wherein the mobility sequence is 2-36 nucleotides in length.

234. The method of claim 230, wherein the first probe further comprises the mobility sequence.

235. The method of claim 230, wherein the second probe further comprises the mobility sequence.

236. The method of claim 230, wherein the analyzing comprises subjecting the first amplification product or a portion of the first amplification product comprising at least one reporter group to a procedure for separating nucleic acid sequences based on molecular weight or length.

237. The method of claim 236, wherein the separating comprises at least one mobility-dependent analysis technique (MDAT).

238. The method of claim 237, wherein the MDAT comprises at least one of electrophoresis, chromatography, HPLC, mass spectroscopy, sedimentation, field-flow fractionation, or multi-stage fractionation.

239. The method of claim 238, wherein the MDAT comprises gel electrophoresis or capillary electrophoresis.

240. The method of claim 236, wherein the separating comprises dialyzing the first amplification product or a portion of the first amplification product comprising at least one reporter group.

241. The method of claim 218, wherein the ligation reaction composition further comprises a ligation agent.

242. The method of claim 241, wherein the ligation agent is a ligase.

243. The method of claim 242, wherein the ligase is a thermostable ligase.

244. The method of claim 243, wherein the thermostable ligase is selected from at least one of Tth ligase, Taq ligase, Tsc ligase, and Pfu ligase.

245. The method of claim 218, wherein the polymerase is a thermostable polymerase.

246. The method of claim 245, wherein the polymerase is selected from at least one of Taq polymerase, Pfx polymerase, Pfu polymerase, Vent®

polymerase, Deep Vent™

polymerase, Pwo polymerase, and Tth polymerase.

247. The method of claim 218, wherein the reporter group comprises a fluorescent moiety.

248. The method of claim 218, wherein the molar concentration of the at least one first primer is different from the molar concentration of the at least one second primer in the at least one primer set.

249. The method of claim 218, wherein the melting temperature of the at least one first primer differs from the melting temperature of the at least one second primer by at least about 4°

C. in at least one primer set.

250. The method of claim 218, wherein the first amplification product comprises at least one 5′

terminal phosphate; and

further comprising;

combining the first amplification product with an exonuclease to form a digestion reaction composition; and

incubating the digestion reaction composition under conditions that allow the exonuclease to digest the amplification product to generate a portion of the first amplification product comprising at least one reporter group.

251. A method for identifying a splice variant in at least one target nucleic acid sequence in a sample, comprising:

combining at least one target nucleic acid sequence with a probe set for each target nucleic acid sequence, the probe set comprising (a) at least one first probe, comprising a target-specific portion and (b) a plurality of second probes, each second probe comprising a 3′

primer-specific portion and one of a plurality of splice-specific portions, wherein the probes in each probe set are suitable for ligation together when hybridized adjacent to one another on the at least one target nucleic acid sequence, and wherein at least one probe in each probe set further comprises at least one addressable support-specific portion;

subjecting the ligation reaction composition to at least one cycle of ligation, wherein adjacently hybridized probes are ligated to one another to form a ligation product comprising the target-specific portion, the splice-specific portion, the at least one addressable support-specific portion, and the 3′

primer-specific portion;

combining the ligation product with at least one primer comprising a reporter group and a sequence complementary to the 3′

primer-specific portion of the ligation product, and a polymerase or, to form an extension reaction composition;

subjecting the extension reaction composition to at least one cycle of primer extension to generate a first amplification product comprising at least one reporter group;

analyzing the first amplification product or a portion of the first amplification product comprising the at least one reporter group using at least a portion of the at least one addressable support-specific portion; and

identifying the splice variant in the at least one target nucleic acid sequence by detecting the at least one reporter group.

252. A method for identifying a splice variant in at least one target nucleic acid sequence in a sample:

combining at least one target nucleic acid sequence with a probe set for each target nucleic acid sequence, the probe set comprising (a) a first probe, comprising a first target-specific portion and a 5′

primer-specific portion, and (b) a plurality of second probes, each second probe comprising a 3′

primer-specific portion and one of a plurality of splice-specific portions, wherein the probes in each set are suitable for ligation together when hybridized adjacent to one another on the at least one target nucleic acid sequence, and wherein at least one probe in each probe set further comprises at least one addressable support-specific portion located between the primer-specific portion and the target-specific portion or between the primer-specific portion and the splice-specific portion;

to form a ligation reaction composition;

subjecting the ligation reaction composition to at least one cycle of ligation, wherein adjacently hybridized probes are ligated to one another to form a ligation product comprising;

the 5′

primer-specific portion, the target specific portion, the splice-specific portion, the at least one addressable support-specific portion, and the 3′

primer-specific portion;

combining the ligation product with;

(a) at least one primer set comprising;

(i) at least one first primer comprising the sequence of the 5′

primer-specific portion of the ligation product, and (ii) at least one second primer comprising a sequence complementary to the 3′

primer-specific portion of the ligation product; and

(b) a polymerase or;

to form a first amplification reaction composition;

subjecting the first amplification reaction composition to at least one cycle of amplification to generate a first amplification product;

combining the first amplification product with either at least one first primer, or at least one second primer for each primer set, but not both first and second primers, wherein the at least one first primer or the at least one second primer further comprises a reporter group, to form a second amplification reaction composition;

subjecting the second amplification reaction composition to at least one cycle of amplification to generate a second amplification product comprising the reporter group;

analyzing the second amplification product or a portion of the second amplification product comprising the reporter group using at least a portion of the addressable support-specific portion; and

identifying the splice variant in the at least one target nucleic acid sequence by detecting the at least one reporter group.

253. A kit for identifying a splice variant in at least one target nucleic acid sequence comprising:

at least one probe set for each target nucleic acid sequence to be detected, the probe set comprising (a) at least one first probe, comprising a target-specific portion and a 5′

primer-specific portion, and (b) a plurality of second probes, each second probe comprising a 3′

primer-specific portion and one of a plurality of splice-specific portions, wherein the probes in each probe set are suitable for ligation together when hybridized adjacent to one another on a nucleic acid sequence, and wherein at least one probe in each probe set further comprises at least addressable support-specific detection portion located between the primer-specific portion and the target-specific portion or between the primer-specific portion and the splice-specific portion.

254. A kit according to claim 253, further comprising a polymerase.

255. A kit according to claim 254, wherein the polymerase is thermostable.

256. A kit according to claim 255, wherein the thermostable polymerase is selected from at least one of Taq polymerase, Pfx polymerase, Pfu polymerase, Vent®

polymerase, Deep Vent™

polymerase, Pwo polymerase, and Tth polymerase.

257. A kit according to claim 253, further comprising a set of primers, the primer set comprising (i) at least one primer comprising the sequence of the 5′

primer-specific portion of the first probe, and (ii) at least one primer comprising a sequence complementary to the 3′

primer-specific portion of the second probe, wherein at least one primer of the primer set further comprises a reporter group.

258. A kit according to claim 257, further comprising a polymerase.

259. A kit according to claim 258, wherein the polymerase is thermostable.

260. A kit according to claim 259, wherein the thermostable polymerase is selected from at least one of Taq polymerase, Pfx polymerase, Pfu polymerase, Vent®

polymerase, Deep Vent™

polymerase, Pwo polymerase, and Tth polymerase.

261. A kit according to claim 253, wherein the addressable support-specific portion of at least one probe comprises a mobility sequence that imparts a particular mobility on the first amplification product or a portion of the first amplification product comprising the at least one reporter group.

262. A kit according to claim 253, further comprising a support, the support comprising capture oligonucleotides capable of hybridizing with addressable support-specific sequence of the at least one probe or with a sequence complementary to the addressable support-specific sequences of the at least one probe.

263. A kit according to claim 253, further comprising a ligase.

264. A kit according to claim 263, wherein the ligase is T4 DNA ligase.

265. A kit according to claim 263, wherein the ligase is a thermostable ligase.

266. A kit according to claim 265, wherein the thermostable ligase is selected from at least one of Tth ligase, Taq ligase, and Pfu ligase.

267. A kit for identifying a splice variant in at least one target nucleic acid sequence comprising:

at least one probe set for each target nucleic acid sequence to be detected, each probe set comprising (a) at least one first probe, comprising a target-specific portion and (b) a plurality of second probes, each second probe comprising a 3′

primer-specific portion and one of a plurality of splice-specific portions, wherein the probes in each set are suitable for ligation together when hybridized adjacent to one another on a nucleic acid sequence, and wherein at least one probe in each probe set further comprises at least one addressable support-specific portion.