Universal reagents for rolling circle amplification and methods of use

• US 20040191784A1
• Filed: 03/31/2003
• Published: 09/30/2004
• Est. Priority Date: 03/31/2003
• Status: Active Grant

• Current Assignee: Qiagen GmbH (Qiagen NV), Agilix Corp. (Revvity, Inc.)
• Original Assignee: Qiagen GmbH (Qiagen NV)
• Inventors: Sparkowski, Jason, Smelkova, Natalia, Abarzua, Patricio
• Granted Patent: US 8,043,834 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2525/125 incorporating agents resulting in resistance to degradation
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

Disclosed are compositions and methods useful for labeling and detection of analytes. The compositions generally are associations of three components: reporter binding agents, amplification target circles, and DNA polymerase. The compositions are assembled prior to their use in a rolling circle amplification reaction and can be stored and transported prior to use without substantial loss of activity. The reporter binding agents generally are composed of a specific binding molecule and a rolling circle replication primer. The specific binding molecule can be specific for a target molecule. The rolling circle replication primer has sequence complementary to the amplification target circle. The DNA polymerase can interact with the rolling circle replication primer and amplification target circle. For use as a general reagent, the specific binding molecule is not bound to the target molecule until the composition is used in an assay.

Claims

1. A composition comprising a reporter binding agent, an amplification target circle, and DNA polymerase,

wherein the reporter binding agent comprises a specific binding molecule and a rolling circle replication primer,

wherein the specific binding molecule is specific for a target molecule, wherein the specific binding molecule is not bound to the target molecule, wherein the composition does not comprise tandem sequence DNA.

2. The composition of claim 1 wherein the reporter binding agent, the amplification target circle, and the DNA polymerase are associated with each other.

3. The composition of claim 2 wherein the reporter binding agent, the amplification target circle, and the DNA polymerase are associated via non-covalent interactions.

4. The composition of claim 1 wherein the specific binding molecule comprises an antibody.

5. The composition of claim 4 wherein the antibody comprises a whole antibody or an antibody fragment.

6. The composition of claim 1 wherein the amplification target circle is associated with the rolling circle replication primer.

7. The composition of claim 6 wherein the DNA polymerase is associated with the amplification target circle and the rolling circle replication primer.

8. The composition of claim 1 wherein the reporter binding agent comprises a plurality of rolling circle replication primers.

9. The composition of claim 8 wherein the reporter binding agent does not comprise a plurality of specific binding molecules.

10. The composition of claim 8 wherein the amplification target circles are associated with a plurality of the rolling circle replication primers.

11. The composition of claim 10 wherein the rolling circle replication primers are the same.

12. The composition of claim 8 wherein the rolling circle replication primers are the same.

13. The composition of claim 8 wherein the rolling circle replication primers are different.

14. The composition of claim 13 wherein the amplification target circles are associated with a plurality of the rolling circle replication primers, wherein the amplification target circles are different.

15. The composition of claim 13 wherein an amplification target circle is associated with each of the rolling circle replication primers, wherein a different amplification target circle is associated with each different rolling circle replication primer.

16. The composition of claim 8 wherein the reporter binding agent comprises three or more rolling circle replication primers.

17. The composition of claim 8 wherein the reporter binding agent comprises two or three rolling circle replication primers.

18. The composition of claim 1 wherein the composition is stored at or below about 25°

C.

19. The composition of claim 18 wherein the composition is stored at or below about 4°

C.

20. The composition of claim 19 wherein the composition is stored at or below about −

20°

C.

21. The composition of claim 20 wherein the composition is stored at or below about −

70°

C.

22. The composition of claim 1 wherein the composition is stored at or below room temperature.

23. The composition of claim 1 wherein the DNA polymerase is φ

29 DNA polymerase.

24. The composition of claim 1 further comprising glycerol.

25. The composition of claim 1 further comprising from about 20% glycerol to about 50% glycerol.

26. The composition of claim 1 wherein the amplification target circle comprises a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to the rolling circle replication primer.

27. A method of making a composition for rolling circle amplification, the method comprising

annealing an amplification target circle to a rolling circle replication primer, wherein a reporter binding agent comprises the rolling circle replication primer and a specific binding molecule, and

incubating the reporter binding agent and amplification target circle with DNA polymerase,

wherein the specific binding molecule is specific for a target molecule, wherein the specific binding molecule is not bound to the target molecule, wherein the composition does not comprise tandem sequence DNA.

28. The method of claim 27 wherein the reporter binding agent, the amplification target circle, and the DNA polymerase are associated with each other.

29. The composition of claim 28 wherein the reporter binding agent, the amplification target circle, and the DNA polymerase are associated via non-covalent interactions.

30. The method of claim 27 wherein the specific binding molecule comprises an antibody.

31. The method of claim 30 wherein the antibody comprises a whole antibody or an antibody fragment.

32. The method of claim 27 wherein the amplification target circle is associated with the rolling circle replication primer.

33. The method of claim 32 wherein the DNA polymerase is associated with the amplification target circle and the rolling circle replication primer.

34. The method of claim 27 wherein the reporter binding agent comprises a plurality of rolling circle replication primers.

35. The method of claim 34 wherein the reporter binding agent does not comprise a plurality of specific binding molecules.

36. The method of claim 34 wherein the amplification target circles are associated with a plurality of the rolling circle replication primers.

37. The method of claim 36 wherein the rolling circle replication primers are the same.

38. The method of claim 34 wherein the rolling circle replication primers are the same.

39. The method of claim 34 wherein the rolling circle replication primers are different.

40. The method of claim 39 wherein the amplification target circles are associated with a plurality of the rolling circle replication primers, wherein the amplification target circles are different.

41. The method of claim 39 wherein an amplification target circle is associated with each of the rolling circle replication primers, wherein a different amplification target circle is associated with each different rolling circle replication primer.

42. The method of claim 34 wherein the reporter binding agent comprises three or more rolling circle replication primers.

43. The composition of claim 34 wherein the reporter binding agent comprises two or three rolling circle replication primers.

44. The method of claim 27 further comprising storing the composition at or below about 25°

C.

45. The method of claim 44 further comprising storing the composition at or below about 4°

C.

46. The method of claim 45 wherein the composition is stored at or below about −

20°

C.

47. The method of claim 46 further comprising storing the composition at or below about −

70°

C.

48. The method of claim 27 wherein the composition is stored at or below room temperature.

49. The method of claim 27 wherein the amplification target circle is annealed to the rolling circle replication primer in the presence of from about 50 mM to about 150 mM salt.

50. The method of claim 27 wherein the concentration of reporter binding agent during annealing of the amplification target circle and the rolling circle replication primer is about 50 nM or higher.

51. The method of claim 27 wherein the concentration of the amplification target circle during annealing is from about 1 to about 3 times the concentration the reporter binding agent.

52. The method of claim 27 wherein the amplification target circle is annealed to the rolling circle replication primer for from about 20 minutes to about 60 minutes at from about 35°

C. to about 40°

C.

53. The method of claim 52 wherein the amplification target circle is annealed to the rolling circle replication primer for from about 30 minutes to about 45 minutes at about 37°

C.

54. The method of claim 27 wherein the reporter binding agent and amplification target circle are incubated with the DNA polymerase substantially in the absence of Mg²⁺.

55. The method of claim 27 wherein the concentration of the DNA polymerase during incubation is from about 2 to about 6 times the concentration the amplification target circle.

56. The method of claim 55 wherein the concentration of the DNA polymerase during incubation is from about 3 to about 5 times the concentration the rolling circle replication primer.

57. The method of claim 27 wherein the reporter binding agent and amplification target circle are incubated with the DNA polymerase for from about 10 minutes to about 20 minutes at from about 30°

C. to about 34°

C.

58. The method of claim 57 wherein the reporter binding agent and amplification target circle are incubated with the DNA polymerase for about 15 minutes at about 31°

C.

59. The method of claim 27 wherein the reporter binding agent and amplification target circle are incubated with the DNA polymerase at from about 25°

C. to about 33°

C.

60. The method of claim 59 wherein the reporter binding agent and amplification target circle are incubated with the DNA polymerase at about 31°

C.

61. The method of claim 27 wherein the DNA polymerase is 100 29 DNA polymerase.

62. The method of claim 27 wherein the composition further comprises glycerol.

63. The composition of claim 27 wherein the composition further comprises from about 20% glycerol to about 50% glycerol.

64. A method of making a composition for rolling circle amplification, the method comprising

incubating an amplification target circle and a rolling circle replication primer under conditions that promote association of the amplification target circle with the rolling circle replication primer, wherein a reporter binding agent comprises the rolling circle replication primer and a specific binding molecule, and

incubating the reporter binding agent and amplification target circle with DNA polymerase under conditions that promote association of the DNA polymerase with the reporter binding agent,

wherein the specific binding molecule is specific for a target molecule, wherein the specific binding molecule is not bound to the target molecule, wherein the composition does not comprise tandem sequence DNA.

65. A method comprising

labeling a molecule of interest, wherein the molecule of interest is labeled by associating a target molecule with the molecule of interest, associating a composition with the target molecule, and generating a signal by performing rolling circle amplification of the composition,

wherein the composition comprises a reporter binding agent, an amplification target circle, and DNA polymerase,

wherein the reporter binding agent comprises a specific binding molecule and a rolling circle replication primer,

wherein the specific binding molecule is specific for the target molecule, wherein the specific binding molecule is not bound to the target molecule prior to association of the composition with the target molecule, wherein the composition does not comprise tandem sequence DNA prior to association of the composition with the target molecule.

66. The method of claim 65 wherein, following associating the target molecule with the molecule of interest, the molecule of interest is separated from other molecules.

67. The method of claim 65 wherein, following associating the composition with the target molecule, the target molecule is separated from other molecules.

68. The method of claim 65 wherein a sample comprises the molecule of interest, wherein, following associating the target molecule with the molecule of interest, the molecule of interest is separated from other molecules in the sample.

69. The method of claim 65 wherein the target molecule comprises biotin, wherein the specific binding molecule comprises an antibody, wherein the antibody is an antibody specific for biotin.

70. The method of claim 65 wherein the specific binding molecule comprises an antibody, wherein the target molecule comprises a second antibody, wherein the second antibody is an animal immunoglobulin, wherein the antibody of the reporter binding agent is specific for immunoglobulins from the same type of animal of which the second antibody is an immunoglobulin.

71. The method of claim 70 wherein the antibody of the reporter binding agent is specific for the allotype corresponding to the second antibody.

72. The method of claim 70 wherein the antibody of the reporter binding agent is specific for the isotype corresponding to the second antibody.

73. The method of claim 65 wherein the target molecule is coupled to a second specific binding molecule.

74. The method of claim 73 wherein the second specific binding molecule is specific for the molecule of interest.

75. The method of claim 73 wherein the second specific binding molecule is specific for a second target molecule, wherein the second target molecule is associated with the molecule of interest.

76. The method of claim 65 wherein the molecule of interest comprises a peptide, protein, lipid, carbohydrate, complex carbohydrate, proteolipid, antibody, membrane fragment, nucleic acid, cofactor, metabolite, enzyme substrate, metal ion, or metal chelate.

77. The method of claim 65 wherein the molecule of interest is associated with a solid support.

78. The method of claim 77 wherein the solid support comprises acrylamide, agarose, 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, polyamino acid, or a combination.

79. The method of claim 77 wherein the solid support comprises a thin film, membrane, bead, bottle, dish, fiber, optical fiber, woven fiber, chip, compact disk, shaped polymer, particle, microparticle, or a combination.

80. The method of claim 77 the solid support comprises a plurality of molecules of interest.

81. The method of claim 80 wherein each molecule of interest is immobilized on the solid support in a different predefined region of the solid support.

82. The method of claim 81 wherein the distance between the different predefined regions of the solid support is fixed.

83. The method of claim 82 wherein the solid support comprises thin film, membrane, bead, bottle, dish, fiber, optical fiber, woven fiber, chip, compact disk, shaped polymer, particle, microparticle, or a combination.

84. The method of claim 81 wherein the distance between at least two of the different predefined regions of the solid support is variable.

85. The method of claim 81 wherein the molecules of interest are immobilized to the solid support at a density exceeding 400 different molecules of interest per cubic centimeter.

86. The method of claim 81 wherein the solid support comprises at least 1,000 different molecules of interest immobilized on the solid support.

87. The method of claim 81 wherein the solid support comprises at least 10,000 different molecules of interest immobilized on the solid support.

88. The method of claim 81 wherein the solid support comprises at least 100,000 different molecules of interest immobilized on the solid support.

89. The method of claim 81 wherein the solid support comprises at least 1,000,000 different molecules of interest immobilized on the solid support.

90. The method of claim 81 wherein each of the different predefined regions is physically separated from each other of the different regions.

91. The method of claim 90 wherein the solid support comprises a plurality of beads, microparticles, or a combination.

92. The method of claim 81 wherein the solid support comprises thin film, membrane, bead, bottle, dish, fiber, optical fiber, woven fiber, chip, compact disk, shaped polymer, particle, microparticle, or a combination.

93. The method of claim 81 wherein the solid support comprises acrylamide, agarose, 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, polyamino acid, or a combination.

94. The method of claim 81 wherein the solid support is porous.

95. The method of claim 81 wherein the molecules of interest in the different predefined regions are at least 20% pure.

96. The method of claim 81 wherein the molecules of interest in the different predefined regions are at least 50% pure.

97. The method of claim 81 wherein the molecules of interest in the different predefined regions are at least 80% pure.

98. The method of claim 81 wherein the molecules of interest in the different predefined regions are at least 90% pure.

99. The method of claim 77 wherein, following associating the molecule of interest with the solid support, the molecule of interest is separated from other molecules.

100. The method of claim 77 wherein a sample comprises the molecule of interest, wherein, following associating the molecule of interest with the solid support, the molecule of interest is separated from other molecules in the sample.

101. The method of claim 65 wherein the molecule of interest comprises a protein of interest.

102. The method of claim 101 wherein the protein of interest is immobilized on a solid support.

103. The method of claim 102 wherein the protein of interest is immobilized on a solid support prior to associating the composition with the target molecule.

104. The method of claim 103 wherein the target molecule is associated with the molecule of interest prior to immobilizing the protein of interest on the solid support.

105. The method of claim 103 wherein the target molecule is associated with the molecule of interest after the protein of interest is immobilized on the solid support.

106. The method of claim 102 wherein the protein of interest is immobilized on a solid support prior to associating the target molecule with the molecule of interest.

107. The method of claim 102 wherein a plurality of other proteins are immobilized on the solid support.

108. The method of claim 107 wherein one or more other compositions are associated with target molecules associated with one or more of the other proteins, wherein one or more other signals are generated by performing rolling circle amplification of one or more of the other compositions,

wherein the other compositions each comprise a reporter binding agent, an amplification target circle, and DNA polymerase,

wherein the reporter binding agent comprises a specific binding molecule and a rolling circle replication primer,

wherein the specific binding molecule is specific for the target molecule, wherein the specific binding molecule is not bound to the target molecule prior to association of the composition with the target molecule, wherein the composition does not comprise tandem sequence DNA prior to association of the composition with the target molecule.

109. The method of claim 108 wherein the target molecules are all the same.

110. The method of claim 108 wherein the target molecules are all different.

111. The method of claim 108 wherein at least two of the target molecules are all different.

112. The method of claim 101 wherein the protein of interest comprises a cell surface marker.

113. The method of claim 112 wherein the cell surface marker is on a cell.

114. The method of claim 113 wherein the cell is immobilized on a solid support.

115. The method of claim 113 wherein the cell is separated from other cells by fluorescent activated cell sorting, wherein rolling circle amplification results in an amplification product comprising a fluorescent label, wherein the fluorescent activated cell sorting is accomplished using the fluorescent label of the amplification product.

116. The method of claim 113 wherein the cell is separated from other cells, counted, or both, by flow cytometry.

117. The method of claim 65 wherein rolling circle amplification results in an amplification product comprising a fluorescent label.

118. The method of claim 117 wherein a bead comprises the fluorescent label, wherein the molecule of interest is separated from other molecules by fluorescent activated cell sorting, wherein the fluorescent activated cell sorting is accomplished using the fluorescent label of the bead.

119. The method of claim 117 wherein the molecule of interest is separated from other molecules, counted, or both, by flow cytometry.

120. The method of claim 119 wherein a bead comprises the fluorescent label.

121. The method of claim 65 wherein the association of the composition with the target molecule is performed as part of or in conjunction with a cytometric bead assay, immuno-histochemistry, flow cytometry, immuno-fluorescence microscopy, in situ hybridization, fluorescence in situ hybridization, multiparametric fluorescence in situ hybridization, or a combination.

122. A method comprising

labeling a target molecule, wherein the target molecule is labeled by associating a composition with the target molecule, and generating a signal by performing rolling circle amplification of the composition,

wherein the composition comprises a reporter binding agent, an amplification target circle, and DNA polymerase,

wherein the reporter binding agent comprises a specific binding molecule and a rolling circle replication primer,

wherein the specific binding molecule is specific for the target molecule, wherein the specific binding molecule is not bound to the target molecule prior to association of the composition with the target molecule, wherein the composition does not comprise tandem sequence DNA prior to association of the composition with the target molecule.

123. The method of claim 122 wherein, following associating the composition with the target molecule, the target molecule is separated from other molecules.

124. The method of claim 122 wherein a sample comprises the target molecule, wherein, following associating the composition with the target molecule, the target molecule is separated from other molecules in the sample.

125. The method of claim 122 wherein the target molecule comprises a peptide, protein, lipid, carbohydrate, complex carbohydrate, proteolipid, antibody, membrane fragment, nucleic acid, cofactor, metabolite, enzyme substrate, metal ion, or metal chelate.

126. The method of claim 122 wherein the target molecule is associated with a solid support.

127. The method of claim 126 wherein the solid support comprises acrylamide, agarose, 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, polyamino acid, or a combination.

128. The method of claim 126 the solid support comprises a plurality of target molecules.

129. The method of claim 128 wherein each target molecule is immobilized on the solid support in a different predefined region of the solid support.

130. The method of claim 129 wherein the distance between the different predefined regions of the solid support is fixed.

131. The method of claim 130 wherein the solid support comprises thin film, membrane, bead, bottle, dish, fiber, optical fiber, woven fiber, chip, compact disk, shaped polymer, particle, microparticle, or a combination.

132. The method of claim 129 wherein the distance between at least two of the different predefined regions of the solid support is variable.

133. The method of claim 129 wherein the target molecules are immobilized to the solid support at a density exceeding 400 different target molecules per cubic centimeter.

134. The method of claim 129 wherein the solid support comprises at least 1,000 different target molecules immobilized on the solid support.

135. The method of claim 129 wherein the solid support comprises at least 10,000 different target molecules immobilized on the solid support.

136. The method of claim 129 wherein the solid support comprises at least 100,000 different target molecules immobilized on the solid support.

137. The method of claim 129 wherein the solid support comprises at least 1,000,000 different target molecules immobilized on the solid support.

138. The method of claim 129 wherein each of the different predefined regions is physically separated from each other of the different regions.

139. The method of claim 138 wherein the solid support comprises a plurality of beads, microparticles, or a combination.

140. The method of claim 129 wherein the solid support comprises thin film, membrane, bead, bottle, dish, fiber, optical fiber, woven fiber, chip, compact disk, shaped polymer, particle, microparticle, or a combination.

141. The method of claim 129 wherein the solid support comprises acrylamide, agarose, 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, polyamino acid, or a combination.

142. The method of claim 129 wherein the solid support is porous.

143. The method of claim 129 wherein the target molecules in the different predefined regions are at least 20% pure.

144. The method of claim 129 wherein the target molecules in the different predefined regions are at least 50% pure.

145. The method of claim 129 wherein the target molecules in the different predefined regions are at least 80% pure.

146. The method of claim 129 wherein the target molecules in the different predefined regions are at least 90% pure.

147. The method of claim 126 wherein, following associating the target molecule with the solid support, the target molecule is separated from other molecules.

148. The method of claim 126 wherein a sample comprises the target molecule, wherein, following associating the target molecule with the solid support, the target molecule is separated from other molecules in the sample.

149. The method of claim 122 wherein the target molecule comprises a protein of interest.

150. The method of claim 149 wherein the protein of interest is immobilized on a solid support.

151. The method of claim 150 wherein the protein of interest is immobilized on a solid support prior to associating the composition with the target molecule.

152. The method of claim 150 wherein a plurality of other proteins are immobilized on the solid support.

153. The method of claim 152 wherein one or more other compositions are associated with one or more of the other proteins, wherein one or more other signals are generated by performing rolling circle amplification of one or more of the other compositions,

wherein the other compositions each comprise a reporter binding agent, an amplification target circle, and DNA polymerase,

wherein the reporter binding agent comprises a specific binding molecule and a rolling circle replication primer,

wherein the specific binding molecule is specific for the target molecule, wherein the specific binding molecule is not bound to the target molecule prior to association of the composition with the target molecule, wherein the composition does not comprise tandem sequence DNA prior to association of the composition with the target molecule.

154. The method of claim 149 wherein the protein of interest comprises a cell surface marker.

155. The method of claim 154 wherein the cell surface marker is on a cell.

156. The method of claim 155 wherein the cell is immobilized on a solid support.

157. The method of claim 155 wherein the cell is separated from other cells by fluorescent activated cell sorting, wherein rolling circle amplification results in an amplification product comprising a fluorescent label, wherein the fluorescent activated cell sorting is accomplished using the fluorescent label of the amplification product.

158. The method of claim 155 wherein the cell is separated from other cells, counted, or both, by flow cytometry.

159. The method of claim 122 wherein rolling circle amplification results in an amplification product comprising a fluorescent label.

160. The method of claim 159 wherein a bead comprises the fluorescent label, wherein the target molecule is separated from other molecules by fluorescent activated cell sorting, wherein the fluorescent activated cell sorting is accomplished using the fluorescent label of the bead.

161. The method of claim 159 wherein the target molecule is separated from other molecules, counted, or both, by flow cytometry.

162. The method of claim 161 wherein a bead comprises the fluorescent label.

163. The method of claim 122 wherein the association of the composition with the target molecule is performed as part of or in conjunction with a cytometric bead assay, immuno-histochemistry, flow cytometry, immuno-fluorescence microscopy, in situ hybridization, fluorescence in situ hybridization, multiparametric fluorescence in situ hybridization, or a combination.

164. A method for detecting one or more analytes, the method comprising

(a) bringing into contact one or more analyte samples and one or more compositions, wherein each composition comprises a reporter binding agent, an amplification target circle, and DNA polymerase, wherein the reporter binding agent comprises a specific binding molecule and a rolling circle replication primer,

wherein the specific binding molecule is specific for a target molecule, wherein the specific binding molecule is not bound to the target molecule prior to association of the composition with the target molecule, wherein the composition does not comprise tandem sequence DNA prior to association of the composition with the target molecule,

wherein each target molecule either comprises an analyte or is associated with an analyte, wherein each specific binding molecule interacts with an analyte directly or indirectly, and

incubating the analyte samples and the compositions under conditions that promote interaction of the specific binding molecules and analytes,

(b) incubating the compositions under conditions that promote replication of the amplification target circles,

wherein replication of the amplification target circles results in the formation of tandem sequence DNA, wherein detection of tandem sequence DNA indicates the presence of the corresponding analytes.

165. The method of claim 164 wherein the analyte samples include one or more first analyte samples and one or more second analyte samples, wherein the compositions include one or more first compositions and one or more second compositions,

the method further comprising, following step (a) and prior to step (b),

mixing one or more of the first analyte samples and one or more of the second analyte samples,

wherein for each first composition there is a matching second composition, wherein the specific binding molecules of the first composition interact with the same analyte as the specific binding molecules of the matching second composition,

wherein the rolling circle replication primer of each different composition is different, wherein each different rolling circle replication primer primes replication of a different one of the amplification target circles, wherein each different amplification target circle produces a different tandem sequence DNA,

wherein the presence or absence of the same analyte in different analyte samples is indicated by the presence or absence of corresponding tandem sequence DNA.

166. The method of claim 165 wherein the analytes are immobilized on a solid support, wherein the tandem sequence DNA corresponding to one of the analytes and produced in association with a first composition is in the same location on the solid support as tandem sequence DNA corresponding to the same analyte and produced in association with the matching second composition,

wherein the presence or absence of the same analyte in different analyte samples is indicated by the presence or absence of corresponding tandem sequence DNA.

167. A kit comprising a composition and a buffer for rolling circle amplification,

wherein the composition comprises a reporter binding agent, an amplification target circle, and DNA polymerase,

wherein the reporter binding agent comprises a specific binding molecule and a rolling circle replication primer,

wherein the specific binding molecule is specific for a target molecule, wherein the specific binding molecule is not bound to the target molecule, wherein the composition does not comprise tandem sequence DNA.

168. The kit of claim 167 further comprising nucleotide triphosphates.

169. The kit of claim 167 further comprising a solid support, wherein the solid support comprises one or more second specific binding molecules, wherein each second specific binding molecule is specific for a molecule of interest.

170. The kit of claim 169 further comprising one or more third specific binding molecules, wherein each third specific binding molecule is specific for one of the molecules of interest, wherein the third specific binding molecule is the target molecule, wherein the third specific binding molecule is an animal immunoglobulin, wherein the specific binding molecule of the reporter binding agent comprises an antibody specific for immunoglobulins from the same type of animal of which the third specific binding molecule is an immunoglobulin.

171. The kit of claim 169 wherein the target molecule is one of the molecules of interest.

172. The kit of claim 167 further comprising a solid support, wherein the solid support comprises a second specific binding molecule, wherein the second specific binding molecule is specific for a molecule of interest.

173. The kit of claim 172 further comprising a third specific binding molecule, wherein the third specific binding molecule is specific for the molecule of interest, wherein the third specific binding molecule comprises the target molecule, wherein the third specific binding molecule is an animal immunoglobulin, wherein the specific binding molecule of the reporter binding agent comprises an antibody specific for immunoglobulins from the same type of animal of which the third specific binding molecule is an immunoglobulin.

174. The kit of claim 172 wherein the target molecule comprises the molecule of interest.