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1. WO2004061119 - AMPLIFICATION D'ARN DE TYPE CERCLE ROULANT

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CLAIMS
We claim:
1. A method of amplifying RNA sequences, the method comprising
incubating a cDNA primer and an RNA sample comprising RNA molecules under conditions that promote synthesis of first strand cDNA molecules from the RNA molecules, incubating a circularization probe and the first sfrand cDNA molecules under conditions that promote circularization ofthe first strand cDNA molecules, and
incubating the circularized first strand cDNA molecules under conditions that promote rolling circle replication ofthe circularized first sfrand cDNA molecules, thereby amplifying RNA sequences.
2. The method of claim 1 wherein the rolling circle replication results in formation of tandem sequence DNA, wherein the tandem sequence DNA is replicated to form secondary tandem sequence DNA, wherein the tandem sequence DNA and the secondary tandem sequence DNA fonn double-sfranded tandem sequence DNA.
3. The method of claim 2 wherein each double-sfranded tandem sequence DNA comprises multiple tandem repeat units ofthe same sequence, wherein each tandem repeat unit comprises one or more cleavage sites of interest, wherein the method further comprises cleaving the double-sfranded tandem sequence DNA at a plurality ofthe cleavage sites resulting in tandem sequence DNA fragments, wherein at least one ofthe tandem sequence DNA fragments does not comprise a plurality of tandem repeat units.
4. The method of claim 3 wherein each tandem repeat unit comprises a single cleavage site of interest, wherein the cDNA primer comprises the cleavage site of interest.
5. The method of claim 4 wherein the cleavage site is a cleavage site for a restriction endonuclease, wherein the restriction endonuclease has a recognition sequence comprising at least seven nucleotides.
6. The method of claim 5 wherein the restriction endonuclease has a recognition sequence comprising at least eight nucleotides.
7. The method of claim 6 wherein the restriction endonuclease is Not I, Asc I, AsiS I, Fse I, Pac I, Pme I, Sbf I, Sfi I, or Swa I.
8. The method of claim 4 wherein the cleavage site is a cleavage site for a restriction endonuclease, wherein the restriction endonuclease has a recognition sequence that occurs once every 65,536 nucleotides or more on average.

9. The method of claim 3 wherein each tandem repeat unit comprises a single cleavage site of interest, wherein the circularization probe comprises the cleavage site of interest.
10. The method of claim 3 wherein each tandem repeat unit comprises a single cleavage site of interest, wherein at least one ofthe tandem sequence DNA fragments consists of a single tandem repeat unit.
11. The method of claim 10 wherein a plurality of the tandem sequence DNA fragments consists of a single tandem repeat unit.
12. The method of claim 11 wherein a majority ofthe tandem sequence DNA fragments consists of a single tandem repeat unit.
13. The method of claim 12 wherein substantially all ofthe tandem sequence DNA fragments consists of a single tandem repeat unit.
14. The method of claim 13 further comprising cloning one or more ofthe single tandem repeat units.
15. The method of claim 14 wherein the single tandem repeat units are separated by size prior to cloning, wherein only single tandem repeat units having sizes of interest are cloned.
16. The method of claim 15 wherein only single tandem repeat units having a size the same as or greater than a size of interest are cloned.
17. The method of claim 14 wherein the cloned tandem repeat units constitute a cDNA library.
18. The method of claim 10 wherein the sequence ofthe single tandem repeat unit conesponds to the sequence of one ofthe circularized first strand cDNA molecules, wherein the sequence ofthe single tandem repeat unit is a circular permutation ofthe sequence of one ofthe circularized first sfrand cDNA molecules.
19. The method of claim 10 wherein the sequence ofthe single tandem repeat unit conesponds to the sequence of one ofthe circularized first sfrand cDNA molecules, wherein the sequence ofthe single tandem repeat is not a circular permutation ofthe sequence of one ofthe circularized first sfrand cDNA molecules.
20. The method of claim 10 wherein the single tandem repeat units constitute a cDNA library.

21. The method of claim 10 wherein rolling circle replication is primed by a rolling circle replication primer, wherein the rolling circle replication primer further comprises a promoter portion.
22. The method of claim 21 wherein the promoter portion is a promoter for a phage RNA polymerase or a bacterial RNA polymerase.
23. The method of claim 22 wherein the promoter is a promoter for T3 RNA polymerase, T7 RNA polymerase, or SP6 RNA polymerase.
24. The method of claim 21 further comprising transcribing the tandem repeat units, whereby tandem repeat transcripts are produced, wherein the tandem repeat transcripts are transcripts of single tandem repeat units.
25. The method of claim 24 wherein the tandem repeat franscripts are used, directly or indirectly, in a hybridization assay.
26. The method of claim 24 wherein the tandem repeat franscripts are associated with a solid-state subsfrate.
27. The method of claim 24 wherein the tandem repeat transcripts are translated.
28. The method of claim 10 wherein the cDNA primer further comprises a promoter portion.
29. The method of claim 28 wherein the promoter portion is a promoter for a phage RNA polymerase or a bacterial RNA polymerase.
30. The method of claim 29 wherein the promoter is a promoter for T3 RNA polymerase, T7 RNA polymerase, or SP6 RNA polymerase.
31. The method of claim 28 further comprising transcribing the tandem repeat units, whereby tandem repeat transcripts are produced, wherein the tandem repeat transcripts are transcripts of single tandem repeat units.
32. The method of claim 31 wherein the tandem repeat transcripts are used, directly or indirectly, in a hybridization assay.
33. The method of claim 31 wherein the tandem repeat transcripts are associated with a solid-state subsfrate.
34. The method of claim 31 wherein the tandem repeat franscripts are translated.
35. The method of claim 10 wherein the tandem repeat units are used, directly or indirectly, in a hybridization assay.
36. The method of claim 10 wherein the tandem repeat units are associated with a solid-state substrate.

37. The method of claim 3 further comprising inserting one or more ofthe tandem sequence DNA fragments into one or more vectors to form one or more recombinant vectors.

38. The method of claim 37 wherein the tandem sequence DNA fragments are separated by size prior to insertion into vectors, wherein only tandem sequence DNA fragments having sizes of interest are inserted into vectors.
39. The method of claim 38 wherein only tandem sequence DNA fragments having a size the same as or greater than a size of interest are cloned.
40. The method of claim 37 wherein the recombinant vectors constitute a cDNA library.
41. The method of claim 3 further comprising cloning one or more ofthe tandem sequence DNA fragments.
42. The method of claim 41 wherein the tandem sequence DNA fragments are separated by size prior to cloning, wherein only tandem sequence DNA fragments having sizes of interest are cloned.
43. The method of claim 42 wherein only tandem sequence DNA fragments having a size the same as or greater than a size of interest are cloned.
44. The method of claim 41 wherein the cloned tandem sequence DNA fragments constitute a cDNA library.
45. The method of claim 3 wherein the tandem sequence DNA fragments constitute a cDNA library.
46. The method of claim 3 wherein at least one ofthe tandem sequence DNA fragments comprises a single tandem repeat unit, wherein the method further comprises cloning one or more ofthe single tandem repeat units.
47. The method of claim 46 wherein the single tandem repeat units are separated by size prior to cloning, wherein only single tandem repeat units having sizes of interest are cloned.
48. The method of claim 47 wherein only single tandem repeat units having a size the same as or greater than a size of interest are cloned.
49. The method of claim 46 wherein the cloned tandem repeats constitute a cDNA library.
50. The method of claim 3 wherein the tandem sequence DNA fragments are used, directly or indirectly, in a hybridization assay.

51. The method of claim 3 wherein the tandem sequence DNA fragments are associated with a solid-state substrate.
52. The method of claim 1 wherein the cDNA primer comprises an RNA
complement portion, wherein the RNA complement portion hybridizes to one or more ofthe RNA molecules, wherein the cDNA primer is incoφorated into the first strand cDNA molecules during synthesis ofthe first strand cDNA molecules.
53. The method of claim 52 wherein the RNA complement portion comprises poly(dT), wherein the RNA molecules are mRNA molecules.
54. The method of claim 52 wherein the RNA complement portion is complementary to sequence in one or more RNA molecules.
55. The method of claim 54 wherein the RNA complement portion is complementary to sequence in one ofthe RNA molecules, wherein the cDNA primer is specific for the RNA molecule.
56. The method of claim 52 wherein the cDNA primer further comprises a cleavage site.
57. The method of claim 56 wherein the cleavage site is a cleavage site for a restriction endonuclease, wherein the restriction endonuclease has a recognition sequence comprising at least seven nucleotides.
58. The method of claim 57 wherein the restriction endonuclease has a recognition sequence comprising at least eight nucleotides.
59. The method of claim 58 wherein the restriction endonuclease is Not I, Asc I, AsiS I, Fse I, Pac I, Pme I, Sbf I, Sfi I, or Swa I.
60. The method of claim 56 wherein the cleavage site is a cleavage site for a restriction endonuclease, wherein the restriction endonuclease has a recognition sequence that occurs once every 65,536 nucleotides or more on average.
61. The method of claim 56 wherein the rolling circle replication results in formation of tandem sequence DNA, wherein the tandem sequence DNA is replicated to form secondary tandem sequence DNA, wherein the tandem sequence DNA and the secondary tandem sequence DNA form double-stranded tandem sequence DNA, wherein each double-sfranded tandem sequence DNA comprises multiple tandem repeats ofthe same sequence, wherein each tandem repeat comprises the cleavage site, wherein the method further comprises cleaving the double-sfranded tandem sequence DNA at a plurality ofthe cleavage sites resulting in tandem sequence DNA fragments.
62. The method of claim 52 wherein the cDNA primer further comprises a probe complement portion, wherein the probe complement portion is complementary to sequence in the circularization probe.
63. The method of claim 52 wherein the cDNA primer further comprises a primer complement portion, wherein the primer complement portion is complementary to a rolling circle replication primer.
64. The method of claim 63 wherein the cDNA primer further comprises a primer matching portion, wherein the primer matching portion matches sequence in a secondary DNA sfrand displacement primer.
65. The method of claim 64 wherein the cDNA primer further comprises a promoter portion.
66. The method of claim 52 wherein the cDNA primer further comprises a primer matching portion, wherein the primer matching portion matches sequence in a secondary DNA sfrand displacement primer.
67. The method of claim 52 wherein the cDNA primer further comprises a promoter portion.
68. The method of claim 68 wherein the promoter portion is a promoter for a phage RNA polymerase or a bacterial RNA polymerase.
69. The method of claim 68 wherein the promoter is a promoter for T3 RNA polymerase, T7 RNA polymerase, or SP6 RNA polymerase.
70. The method of claim 52 wherein the cDNA primer comprises nucleotides, wherein one or more ofthe nucleotides are ribonucleotides.
71. The method of claim 70 wherein from about 10%) to about 50% ofthe nucleotides are ribonucleotides.
72. The method of claim 70 wherein about 50% or more ofthe nucleotides are ribonucleotides.
73. The method of claim 70 wherein all ofthe nucleotides are ribonucleotides.
74. The method of claim 52 wherein the cDNA primer comprises nucleotides, wherein one or more ofthe nucleotides are 2'-O-methyl ribonucleotides.
75. The method of claim 74 wherein from about 10% to about 50% ofthe nucleotides are 2'-O-methyl ribonucleotides.

76. The method of claim 74 wherein about 50% or more ofthe nucleotides are 2'-O-methyl ribonucleotides.
77. The method of claim 74 wherein all ofthe nucleotides are 2'-O-methyl ribonucleotides.
78. The method of claim 52 wherein the cDNA primer comprises nucleotides, wherein the nucleotides are a mixture of ribonucleotides and 2'-O-methyl ribonucleotides.
79. The method of claim 52 wherein the cDNA primer comprises nucleotides, wherein the nucleotides are a mixture of deoxyribonucleotides and 2'-O-methyl
ribonucleotides.
80. The method of claim 1 wherein the cDNA primer is incoφorated into the first strand cDNA molecules during synthesis ofthe first sfrand cDNA molecules, wherein the circularization probe comprises a cDNA complement portion and a primer complement portion, wherein the cDNA complement portion hybridizes to one or more ofthe first strand cDNA molecules, wherein the primer complement portion hybridizes to the cDNA primer incoφorated into the first sfrand cDNA molecules, wherein hybridization ofthe cDNA complement portion to the first strand cDNA molecules and hybridization ofthe primer complement portion to the cDNA primer brings the ends ofthe first strand cDNA molecules into proximity thereby mediating circularization ofthe first strand cDNA molecules.
81. The method of claim 80 wherein the cDNA complement portion comprises a random or partially random sequence.
82. The method of claim 80 wherein the cDNA complement portion comprises, nucleotides, wherein one or more ofthe nucleotides comprise a universal base.
83. The method of claim 82 wherein the universal base is 5-nitroindole or 3-nitropynole.
84. The method of claim 80 wherein the cDNA complement portion comprises nucleotides, wherein one or more ofthe nucleotides can base pair with more than one type of nucleotide.
85. The method of claim 84 wherein the nucleotide that can base pair with more than one type of nucleotide is inosine or a nucleotide comprising a universal base.
86. The method of claim 85 wherein the universal base is 5-nitroindole or 3-nifropynole.
87. The method of claim 80 wherein the circularization probe further comprises a cleavage site, wherein the primer complement portion comprises the cleavage site.

88. The method of claim 87 wherein the cleavage site is a cleavage site for a restriction endonuclease, wherein the restriction endonuclease has a recognition sequence comprising at least seven nucleotides.
89. The method of claim 88 wherein the restriction endonuclease has a recognition sequence comprising at least eight nucleotides.
90. The method of claim 89 wherein the restriction endonuclease is Not I, Asc I, AsiS I, Fse I, Pac I, Pme I, Sbf I, Sfi I, or Swa I.
91. The method of claim 87 wherein the cleavage site is a cleavage site for a restriction endonuclease, wherein the restriction endonuclease has a recognition sequence that occurs once every 65,536 nucleotides or more on average.
92. The method of claim 87 wherein the rolling circle replication results in formation of tandem sequence DNA, wherein the tandem sequence DNA is replicated to form secondary tandem sequence DNA, wherein the tandem sequence DNA and the secondary tandem sequence DNA form double-sfranded tandem sequence DNA, wherein each double-sfranded tandem sequence DNA comprises multiple tandem repeats ofthe same sequence, wherein each tandem repeat comprises the cleavage site, wherein the method further comprises
cleaving the double-sfranded tandem sequence DNA at a plurality ofthe cleavage sites resulting in tandem sequence DNA fragments.
93. The method of claim 80 wherein the primer complement portion is
complementary to sequence in the cDNA primer.
94. The method of claim 80 wherein the circularization probe further comprises a second primer complement portion, wherein the second primer complement portion is complementary to a secondary DNA sfrand displacement primer.
95. The method of claim 94 wherein the circularization probe further comprises primer matching portion, wherein the primer matching portion matches sequence in a rolling circle replication primer.
96. The method of claim 95 wherein the circularization probe further comprises a promoter portion.
97. The method of claim 80 wherein the circularization probe further comprises a primer matching portion, wherein the primer matching portion matches sequence in a secondary DNA sfrand displacement primer.

98. The method of claim 80 wherein the circularization probe further comprises a promoter portion.
99. The method of claim 98 wherein the promoter portion is a promoter for a phage RNA polymerase or a bacterial RNA polymerase.
100. The method of claim 99 wherein the promoter is a promoter for T3 RNA polymerase, T7 RNA polymerase, or SP6 RNA polymerase.
101. The method of claim 80 wherein the circularization probe comprises
nucleotides, wherein one or more ofthe nucleotides are ribonucleotides.
102. The method of claim 101 wherein from about 10% to about 50% ofthe nucleotides are ribonucleotides.
103. The method of claim 101 wherein about 50% or more ofthe nucleotides are ribonucleotides.
104. The method of claim 101 wherein all ofthe nucleotides are ribonucleotides.
105. The method of claim 80 wherein the circularization probe comprises
nucleotides, wherein one or more ofthe nucleotides are 2'-O-methyl ribonucleotides.
106. The method of claim 105 wherein from about 10% to about 50% ofthe nucleotides are 2'-O-methyl ribonucleotides.
107. The method of claim 105 wherein about 50% or more ofthe nucleotides are 2'-O-methyl ribonucleotides.
108. The method of claim 105 wherein all ofthe nucleotides are 2'-O-methyl ribonucleotides.
109. The method of claim 80 wherein the circularization probe comprises
nucleotides, wherein the nucleotides are a mixture of ribonucleotides and 2'-O-methyl ribonucleotides.
110. The method of claim 80 wherein the circularization probe comprises
nucleotides, wherein the nucleotides are a mixture of deoxyribonucleotides and 2'-O-methyl ribonucleotides.
111. The method of claim 1 wherein rolling circle replication is primed by a rolling circle replication primer, wherein the rolling circle replication primer has a random sequence, wherein the rolling circle replication is primed from a plurality of locations on the
circularized first strand cDNA molecules.
112. The method of claim 111 wherein the rolling circle replication results in formation of tandem sequence DNA, wherein the tandem sequence DNA is replicated to form secondary tandem sequence DNA, wherein replication ofthe tandem sequence DNA is primed by the rolling circle replication primer.
113. The method of claim 111 wherein the circularized first sfrand cDNA molecules are amplified via exponential rolling circle amplification.
114. The method of claim 1 wherein rolling circle replication is primed by a plurality of rolling circle replication primers, wherein the rolling circle replication is primed from a plurality of locations on the circularized first sfrand cDNA molecules.
115. The method of claim 114 wherein the rolling circle replication results in formation of tandem sequence DNA, wherein the tandem sequence DNA is replicated to form secondary tandem sequence DNA, wherein replication ofthe tandem sequence DNA is primed by one or more secondary DNA strand displacement primers.
116. The method of claim 114 wherein the circularized first strand cDNA molecules are amplified via exponential rolling circle amplification.
117. The method of claim 1 wherein rolling circle replication is primed by one or more rolling circle replication primers, wherein the rolling circle replication results in formation of tandem sequence DNA, wherein the tandem sequence DNA is not replicated, wherein the tandem sequence DNA comprises sequence matching sequence in the RNA molecules.
118. The method of claim 117 wherein the tandem sequence DNA is used, directly or indirectly, in a hybridization assay.
119. The method of claim 117 wherein the tandem sequence DNA is associated with a solid-state subsfrate.
120. The method of claim 1 wherein rolling circle replication is primed by the circularization probe, wherein the rolling circle replication results in formation of tandem sequence DNA, wherein the tandem sequence DNA is not replicated, wherein the tandem sequence DNA comprises sequence matching sequence in the RNA molecules.
121. The method of claim 120 wherein the tandem sequence DNA is used, directly or indirectly, in a hybridization assay.
122. The method of claim 120 wherein the tandem sequence DNA is associated with a solid-state subsfrate.
123. The method of claim 1 wherein rolling circle replication is primed by one or more rolling circle replication primers, wherein the rolling circle replication results in formation of tandem sequence DNA, wherein the tandem sequence DNA is replicated to form secondary tandem sequence DNA, wherein replication ofthe tandem sequence DNA is primed by one or more secondary DNA strand displacement primers,
wherein the rolling circle replication primers have 5' phosphates, wherein the secondary DNA strand displacement primers have 5' hydroxyls, wherein the tandem sequence DNA has 5' phosphates, wherein the secondary tandem sequence DNA has 5' hydroxyls, wherein the method further comprises incubating the tandem sequence DNA and secondary tandem sequence DNA in the presence of a phosphate-dependent exonuclease, whereby the tandem sequence DNA is degraded and the secondary tandem sequence DNA remains.
124. The method of claim 123 wherein the phosphate-dependent exonuclease is lambda exonuclease.
125. The method of claim 123 wherein the secondary tandem sequence DNA comprises sequence complementary to sequence in the RNA molecules, wherein the secondary tandem sequence DNA is used, directly or indirectly, in a hybridization assay.
126. The method of claim 123 wherein the secondary tandem sequence DNA comprises sequence complementary to sequence in the RNA molecules, wherein the secondary tandem sequence DNA is associated with a solid-state substrate.
127. The method of claim 1 wherein rolling circle replication is primed by one or more rolling circle replication primers, wherein the rolling circle replication results in formation of tandem sequence DNA, wherein the tandem sequence DNA is replicated to form secondary tandem sequence DNA, wherein replication ofthe tandem sequence DNA is primed by one or more secondary DNA sfrand displacement primers,
wherein the rolling circle replication primers have 5' hydroxyls, wherein the secondary DNA sfrand displacement primers have 5' phosphates, wherein the tandem sequence DNA has 5' hydroxyls, wherein the secondary tandem sequence DNA has 5' phosphates,
wherein the method further comprises incubating the tandem sequence DNA and secondary tandem sequence DNA in the presence of a phosphate-dependent exonuclease, whereby the secondary tandem sequence DNA is degraded and the tandem sequence DNA remains.
128. The method of claim 127 wherein the phosphate-dependent exonuclease is lambda exonuclease.

129. The method of claim 127 wherein the tandem sequence DNA comprises sequence matching sequence in the RNA molecules, wherein the tandem sequence DNA is used, directly or indirectly, in a hybridization assay.
130. The method of claim 127 wherein the tandem sequence DNA comprises sequence matching sequence in the RNA molecules, wherein the tandem sequence DNA is associated with a solid-state substrate.
131. The method of claim 1 wherein rolling circle replication is primed by the circularization probe, wherein the rolling circle replication results in formation of tandem sequence DNA, wherein the tandem sequence DNA is replicated to form secondary tandem sequence DNA, wherein replication ofthe tandem sequence DNA is primed by one or more secondary DNA sfrand displacement primers,
wherein the circularization probe has a 5' phosphate, wherein the secondary DNA strand displacement primers have 5' hydroxyls, wherein the tandem sequence DNA has 5' phosphates, wherein the secondary tandem sequence DNA has 5' hydroxyls,
wherein the method further comprises incubating the tandem sequence DNA and secondary tandem sequence DNA in the presence of a phosphate-dependent exonuclease, whereby the tandem sequence DNA is degraded and the secondary tandem sequence DNA remains.
132. The method of claim 131 wherein the phosphate-dependent exonuclease is lambda exonuclease.
133. The method of claim 131 wherein the secondary tandem sequence DNA comprises sequence complementary to sequence in the RNA molecules, wherein the secondary tandem sequence DNA is used, directly or indirectly, in a hybridization assay.
134. The method of claim 131 wherein the secondary tandem sequence DNA comprises sequence complementary to sequence in the RNA molecules, wherein the secondary tandem sequence DNA is associated with a solid-state substrate.
135. The method of claim 1 wherein rolling circle replication is primed by the circularization probe, wherein the rolling circle replication results in formation of tandem sequence DNA, wherein the tandem sequence DNA is replicated to form secondary tandem sequence DNA, wherein replication ofthe tandem sequence DNA is primed by one or more secondary DNA strand displacement primers, wherein the circularization probe has a 5' hydroxyl, wherein the secondary DNA sfrand displacement primers have 5' phosphates, wherein the tandem sequence DNA has 5' hydroxyls, wherein the secondary tandem sequence DNA has 5' phosphates,
wherein the method further comprises incubating the tandem sequence DNA and secondary tandem sequence DNA in the presence of a phosphate-dependent exonuclease, whereby the secondary tandem sequence DNA is degraded and the tandem sequence DNA remains.
136. The method of claim 135 wherein the phosphate-dependent exonuclease is lambda exonuclease.
137. The method of claim 135 wherein the tandem sequence DNA comprises sequence matching sequence in the RNA molecules, wherein the tandem sequence DNA is used, directly or indirectly, in a hybridization assay.
138. The method of claim 135 wherein the tandem sequence DNA comprises sequence matching sequence in the RNA molecules, wherein the tandem sequence DNA is associated with a solid-state substrate.
139. The method of claim 1 wherein rolling circle replication is primed by a rolling circle replication primer, wherein the rolling circle replication primer further comprises a promoter portion, wherein the rolling circle replication results in formation of tandem sequence DNA.
140. The method of claim 139 wherein the promoter portion is a promoter for a phage RNA polymerase or a bacterial RNA polymerase.
141. The method of claim 140 wherein the promoter is a promoter for T3 RNA polymerase, T7 RNA polymerase, or SP6 RNA polymerase.
142. The method of claim 139 further comprising transcribing the tandem sequence DNA, whereby tandem sequence transcripts are produced.
143. The method of claim 142 wherein the tandem sequence transcripts are used, directly or indirectly, in a hybridization assay.
144. The method of claim 142 wherein the tandem sequence transcripts are associated with a solid-state subsfrate.
145. The method of claim 142 wherein the tandem sequence transcripts are translated.
146. The method of claim 1 wherein the cDNA primer further comprises a promoter portion, wherein the rolling circle replication results in formation of tandem sequence DNA.

147. The method of claim 146 wherein the promoter portion is a promoter for a phage RNA polymerase or a bacterial RNA polymerase.
148. The method of claim 147 wherein the promoter is a promoter for T3 RNA polymerase, T7 RNA polymerase, or SP6 RNA polymerase.
149. The method of claim 146 further comprising transcribing the tandem sequence DNA, whereby tandem sequence franscripts are produced.
150. The method of claim 149 wherein the tandem sequence transcripts are used, directly or indirectly, in a hybridization assay.
151. The method of claim 149 wherein the tandem sequence transcripts are associated with a solid-state substrate.
152. The method of claim 149 wherein the tandem sequence transcripts are translated.
153. The method of claim 1 wherein the rolling circle replication results in formation of tandem sequence DNA, wherein the method further comprises
detecting one or more target sequences in the tandem sequence DNA.
154. The method of claim 153 wherein the one or more target sequences conespond to target sequences in the RNA molecules, wherein detection ofthe one or more target sequences indicates the presence ofthe conesponding RNA molecules in the RNA sample.

155. The method of claim 153 wherein the RNA sample is derived from a subject, wherein one or more ofthe target sequences is related to a disease or condition, wherem detection ofthe one or more target sequences related to a disease or condition indicates that the subject from which the RNA sample is derived is or is not at risk for the related disease or condition.
156. The method of claim 153 wherein a plurality of first strand cDNA molecules are synthesized from a plurality of RNA molecules, wherein a plurality of tandem sequence DNAs are formed, wherein the plurality of tandem sequence DNAs conespond to the plurality of RNA molecules, wherein a plurality of target sequences are detected, wherein the plurality of target sequences detected constitute a catalog of sequences derived from the RNA sample.
157. The method of claim 156 wherein the method further comprises comparing the catalog of sequences to another catalog of sequences produced in the same way from a different RNA sample.

158. The method of claim 153 wherein the presence and absence of sequences of interest in the catalog of sequences indicates that the subject from which the RNA sample is derived is or is not at risk for a disease or condition.
159. The method of claim 1 wherein the rolling circle replication results in formation of tandem sequence DNA, wherein a plurality of first sfrand cDNA molecules are synthesized from a plurality of RNA molecules, wherein a plurality of tandem sequence DNAs are formed, wherein the plurality of tandem sequence DNAs conespond to the plurality of RNA molecules, wherein the plurality of tandem sequence DNAs constitute a catalog of sequences derived from the RNA sample.
160. The method of claim 159 wherein the catalog of sequences comprises tandem sequence DNA conesponding to a significant number ofthe RNA molecules in the RNA sample.
161. The method of claim 160 wherein the catalog of sequences comprises tandem sequence DNA conesponding to a substantial number ofthe RNA molecules in the RNA sample.
162. The method of claim 1 wherein the rolling circle replication results in formation of tandem sequence DNA, wherein the method further comprises
sequencing one or more target sequences in the tandem sequence DNA.
163. The method of claim 162 wherein the one or more target sequences conespond to target sequences in the RNA molecules, wherein the sequence ofthe one or more target sequences conesponds to sequence ofthe conesponding RNA molecules in the RNA sample.

164. The method of claim 1 wherein the rolling circle replication results in formation of tandem sequence DNA, wherein the method further comprises, following the rolling circle replication,
incubating the tandem sequence DNA under conditions that promote replication ofthe tandem sequence DNA, wherein during replication ofthe tandem sequence DNA at least one ofthe replicated sfrands is displaced from the tandem sequence DNA by strand displacement replication of another replicated strand.
165. The method of claim 1 wherein the rolling circle replication results in formation of tandem sequence DNA, wherein the tandem sequence DNA is replicated to form secondary tandem sequence DNA during the rolling circle replication, wherein the method further comprises, following the rolling circle replication, incubating the tandem sequence DNA and the secondary tandem sequence DNA under conditions that promote replication ofthe tandem sequence DNA and the secondary tandem sequence DNA, wherein during replication ofthe tandem sequence DNA and the secondary tandem sequence DNA at least one ofthe replicated sfrands is displaced from the tandem sequence DNA or the secondary tandem sequence DNA by sfrand displacement replication of another replicated sfrand.
166. The method of claim 1 wherein at least one ofthe first strand cDNA molecules is ligated to at least one other first strand cDNA molecule to form one or more first sfrand cDNA concatemers.
167. The method of claim 166 wherein at least one ofthe first sfrand cDNA concatemers is not circularized, wherein the uncircularized first stand cDNA concatemer is replicated by strand displacement replication.
168. The method of claim 166 wherein at least one ofthe first strand cDNA concatemers is circularized, wherein the circularized first strand cDNA concatemer is replicated by rolling circle replication.
169. The method of claim 166 wherein at least one ofthe first strand cDNA concatemers is not circularized, wherein the uncircularized first stand cDNA concatemer is replicated by strand displacement replication, wherein at least one ofthe first strand cDNA concatemers is circularized, wherein the circularized first sfrand cDNA concatemer is replicated by rolling circle replication.
170. The method of claim 1 wherein the conditions that promote synthesis of first strand cDNA molecules comprise incubating the cDNA primer and the RNA sample in the presence of a reverse franscriptase, wherein the conditions that promote circularization ofthe first sfrand cDNA molecules comprise incubating the circularization probe and the first strand cDNA molecules in the presence of ligase, wherein the conditions that promote rolling circle replication ofthe circularized first cDNA molecules comprise incubating the circularized first sfrand cDNA molecules in the presence of a DNA polymerase.
171. The method of claim 170 wherein the RNA molecules are mRNA molecules, wherein the cDNA primer is 5*-GTGCGGCCGCTTTTTTTTTTTTTTTTTTTT-3* (SEQ ID NO:l), wherein the circularization probe is 5'-AAAAGCGGCCGCACNNNNNN-3' (SEQ ID NO:2), wherein the reverse franscriptase is Superscript II reverse franscriptase, wherein the ligase is T4 DNA ligase, wherein the DNA polymerase is φ29 DNA polymerase.

172. The method of claim 170 wherein the method further comprises, following synthesis of first sfrand cDNA molecules and before circularization ofthe first sfrand cDNA molecules
incubating the first strand cDNA molecules in the presence of an RNAse H activity, incubating the first sfrand cDNA molecules under alkaline conditions,
neutralizing the first strand cDNA molecules, and
purifying the first strand cDNA molecules.
173. The method of claim 172 wherein the RNAse H activity is provided by an RNAse H+ reverse franscriptase, wherein the alkaline conditions are produced by adding 0.1 M NaOH, wherein the first strand cDNA molecules are neutralized by adding 0.1 M HCl.
174. A method of amplifying RNA sequences, the method comprising
incubating a cDNA primer and an RNA sample comprising RNA molecules under conditions that promote synthesis of first strand cDNA molecules from the RNA molecules, wherein the conditions that promote synthesis of first strand cDNA molecules comprise incubating the cDNA primer and the RNA sample in the presence of a reverse franscriptase, incubating the first strand cDNA molecules in the presence of an RNAse H activity, incubating the first strand cDNA molecules under alkaline conditions,
neutralizing the first sfrand cDNA molecules,
purifying the first strand cDNA molecules,
incubating a circularization probe and the first sfrand cDNA molecules under conditions that promote circularization ofthe first sfrand cDNA molecules, wherein the conditions that promote circularization ofthe first sfrand cDNA molecules comprise incubating the circularization probe and the first strand cDNA molecules in the presence of ligase,
incubating the circularized first strand cDNA molecules under conditions that promote rolling circle replication ofthe circularized first strand cDNA molecules, thereby amplifying RNA sequences, wherein the conditions that promote rolling circle replication of the circularized first cDNA molecules comprise incubating the circularized first strand cDNA molecules in the presence of a DNA polymerase.
175. The method of claim 174 wherein the RNA molecules are mRNA molecules, wherein the cDNA primer is 5'-GTGCGGCCGCTTTTTTTTTTTTTTTTTTTT-3' (SEQ ID NO:l), wherein the circularization probe is 5'-AAAAGCGGCCGCACNNNNNN-3' (SEQ ID NO:2), wherein the reverse franscriptase is Superscript II reverse franscriptase, wherein the RNAse H activity is provided by an RNAse H+ reverse franscriptase, wherein the alkaline conditions are produced by adding 0.1 M NaOH, wherein the first sfrand cDNA molecules are neutralized by adding 0.1 M HCl, wherein the ligase is T4 DNA ligase, wherein the DNA polymerase is φ29 DNA polymerase.
176. A method of amplifying RNA sequences, the method comprising
incubating a cDNA primer and an RNA sample comprising RNA molecules under conditions that promote synthesis of first strand cDNA molecules from the RNA molecules, incubating a circularization probe and the first strand cDNA molecules under conditions that promote ligation ofthe first sfrand cDNA molecules to each other to form first sfrand cDNA concatemers, and
incubating the first strand cDNA concatemers under conditions that promote strand displacement replication ofthe first sfrand cDNA concatemers, thereby amplifying RNA sequences.
177. The method of claim 176 wherein at least one ofthe first strand cDNA concatemers is circularized, wherein the circularized first sfrand cDNA concatemer is replicated by rolling circle replication.
178. The method of claim 176 wherein at least one ofthe first strand cDNA molecules is circularized, wherein the circularized first sfrand cDNA molecule is replicated by rolling circle replication.
179. The method of claim 176 wherein at least one ofthe first sfrand cDNA concatemers is circularized, wherein the circularized first sfrand cDNA concatemer is replicated by rolling circle replication, wherein at least one ofthe first sfrand cDNA molecules is circularized, wherein the circularized first sfrand cDNA molecule is replicated by rolling circle replication.