Methods and compositions for amplification of RNA sequences using RNA-DNA composite primers

• US 6,946,251 B2
• Filed: 03/11/2002
• Issued: 09/20/2005
• Est. Priority Date: 03/09/2001
• Status: Expired due to Term

• Current Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Original Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Inventors: Kurn, Nurith
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US10/100,321
• Publication Number:
  US 20030087251A1
• Time in Patent Office: 1,289 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/24.33
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]

Abstract

The invention provides methods for isothermal amplification of RNA. The methods are particularly suitable for amplifying a plurality of RNA species in a sample. The methods employ a composite primer, a second primer and strand displacement to generate multiple copies of DNA products comprising sequences complementary to an RNA sequence of interest. In another aspect, the methods employ a single primer (which is a composite primer) and strand displacement to generate multiple copies of DNA products comprising sequences complementary to an RNA sequence of interest. In some embodiments, a transcription step is included to generate multiple copies of sense RNA of an RNA sequence of interest. The methods are useful for preparation of nucleic acid libraries and substrates for analysis of gene expression of cells in biological samples. The invention also provides compositions and kits for practicing the amplification methods, as well as methods which use the amplification products.

Claims

1. A method of generating multiple copies of a polynucleotide sequence complementary to an RNA sequence of interest, said method comprising the steps of:

(a) extending a first primer hybridized to a target RNA with at least one enzyme comprising RNA-dependent DNA polymerase activity, wherein the first primer is a composite primer comprising an RNA portion and a 3′

DNA portion, whereby a complex comprising a first primer extension product and the target RNA is produced;

(b) cleaving RNA in the complex of step (a) with at least one enzyme that cleaves RNA from an RNA/DNA hybrid;

(c) extending a second primer hybridized to the first primer extension product with at least one enzyme comprising DNA-dependent DNA polymerase activity and at least one enzyme comprising RNA-dependent DNA polymerase activity, whereby a second primer extension product is produced to form a complex of first and second primer extension products;

(d) cleaving RNA from the first primer in the complex of first and second primer extension products with at least one enzyme that cleaves RNA from an RNA/DNA hybrid such that a composite amplification primer hybridizes to the second primer extension product, wherein the composite amplification primer comprises an RNA portion and a 3′

DNA portion;

(e) extending the composite amplification primer hybridized to the second primer extension product with at least one enzyme comprising DNA-dependent DNA polymerase activity;

whereby said first primer extension product is displaced, RNA is cleaved from the composite amplification primer and another composite amplification primer hybridizes such that primer extension and strand displacement are repeated, and whereby multiple copies of a polynucleotide sequence complementary to the RNA sequence of interest are generated.

2. The method of claim 1, wherein the second primer comprises a fragment of the target RNA hybridized to the first primer extension product, said fragment generated by cleaving RNA in the complex of step (a) with an enzyme that cleaves RNA from an RNA/DNA hybrid.

3. The method of claim 1, wherein the second primer comprises DNA.

4. The method of claim 1, wherein the RNA portion of the first primer is 5′

with respect to the 3′

DNA portion.

5. The method of claim 4, wherein the 5′

RNA portion of the first primer is adjacent to the 3′

DNA portion.

6. The method of claim 1, wherein the first primer is a tailed primer that comprises a 5′

portion that is not hybridizable to the target RNA under conditions which the first primer hybridizes to the target RNA.

7. The method of claim 1, wherein the first primer comprises a poly-dT sequence.

8. The method of claim 1 or 7, wherein the target RNA is mRNA.

9. The method of claim 1, wherein first primer comprises a random primer sequence.

10. The method of claim 1, wherein the target RNA is mRNA, and the first primer comprises a poly-dT sequence, and further is a tailed primer that comprises a 5′

portion that is not hybridizable to the target mRNA under conditions which the first primer hybridizes to the target mRNA.

11. The method of claim 1, wherein plurality of different first primers are used for hybridizing to the target RNA.

12. The method of claim 1, wherein the second primer is a random primer.

13. The method of claim 1, wherein the second primer is a tailed primer that comprises a 5′

portion that is not hybridizable to the first primer extension product under conditions which the second primer hybridizes to the first primer extension product.

14. The method of claim 1, wherein the at least one enzyme that cleaves RNA from an RNA/DNA hybrid is RNase H.

15. The method of claim 1, wherein th same enzyme comprises RNA-dependent DNA polymerase activity and DNA-dependent DNA polymerase activity.

16. The method of claim 1, wherein the same enzyme comprises RNA-dependent DNA polymerase activity and cleaves RNA from RNA/DNA hybrid.

17. The method of claim 1, wherein the same enzyme comprises DNA-dependent DNA polymerase activity and cleaves RNA from an RNA/DNA hybrid.

18. The method of claim 1, wherein the same enzyme comprises DNA-dependent DNA polymerase activity, RNA-dependent DNA polymerase activity and cleaves RNA from an RNA/DNA hybrid.

19. The method of claim 1, further comprising using a labeled dNTP, whereby labeled products are generated.

20. The method of claim 1, wherein said method comprises generating multiple copies of polynucleotide sequences complementary to two or more different sequences of interest.

21. The method of claim 20, wherein said method comprises at least two different first primers.

22. The method of claim 20 or 21, wherein the first primer comprises a poly-dT sequence.

23. The method of claim 20 or 21, wherein the first primer comprises a random primer sequence.

96. A method of sequencing an RNA sequence of interest, said method comprising (a) amplifying a target RNA comprising the sequence of interest by any of the methods of claims 1, 49, 59, 82, and 89 in the presence of a mixture of dNTPs and dNTP analogs such that primer extension is terminated upon incorporation of dNTP analog;

and (b) analyzing the amplification products to determine sequence.

97. The method of claim 96, wherein the target RNA is mRNA.

100. A method of detecting a mutation in a target RNA by single stranded conformation polymorphism, comprising (a) amplifying the target RNA by any of the methods of claims 1, 24, 49, 53, 59, 82, and 89;

and (b) analyzing the amplification products for single stranded conformation, wherein a difference in conformation as compared to a reference single stranded polynucleotide indicates a mutation in the target polynucleotide.

101. The method of claim 100, wherein the target RNA is mRNA.

104. A method of producing a nucleic acid immobilized to a substrate, comprising immobilizing amplification products on a substrate, wherein the amplification products are generated by amplifying a target RNA by any of methods of claims 1, 24, 49, 59, 82, and 89.

105. The method of claim 104, wherein the target RNA is mRNA.

106. The method of claim 104, wherein the substrate is a microarray.

107. A method of characterizing an RNA sequence of interest, comprising analyzing amplification products, wherein the amplification products are generated by amplifying a target RNA comprising the sequence of interest by any of the methods of claims 1, 49, 59, 82, and 89.

108. The method of claim 107, wherein the target RNA is mRNA.

109. The method of claim 107, wherein the amplification products are labeled.

110. The method of claim 107, wherein analyzing the amplification products comprises determining amount of said products, whereby the amount of the RNA sequence of interest present in a sample is quantified.

111. The method of claim 107, wherein analyzing the amplification products comprises contacting the amplification products with a least one probe.

112. The method of claim 111, wherein the amplification products are labeled, and wherein the at least one probe is provided as a microarray.

113. The method of claim 112, wherein the microarray comprises at least one probe immobilized on a substrate fabricated from a material selected from the group consisting of paper, glass, ceramic, plastic, polypropylene, polystyrene, nylon, polyacrylamide, nitrocellulose, silicon, and optical fiber.

114. The method of claim 113, wherein the probe is immobilized on the substrate in a two-dimensional configuration or a three-dimensional configuration comprising pins, rods, fibers, tapes, threads, beads, particles, microtiter wells, capillaries, and cylinders.

123. A method of determining gene expression profile in a sample, said method comprising:

determining amount of amplification products of at least one RNA sequence of interest, wherein the amplification products are generated by amplifying at least one RNA sequence of interest in the sample using the method of any of claims 1, 49, 59, 82, and 89, whereby the gene expression profile in the sample is determined.

124. The method of claim 123, wherein the each target RNA is mRNA.

125. A method of preparing a library, said method amplifying a plurality of RNA sequences of interest using the method of any of claims 1, 24, 49, 53, 59, 82, and 89.

126. The method of claim 125, wherein the first primer comprises a random primer sequence.

127. The method of claim 125, wherein the first primer comprises a poly-dT sequence.

128. A method of preparing a subtractive hybridization probe, said method comprising generating multiple polynucleotide copies of the complement of at least one RNA sequence of interest from a first RNA population using the methods of any of claims 1, 49, 59, 82, and 96.

129. A method of performing subtractive hybridization in which at least first and second target RNA populations are compared, said method comprising:

(a) generating multiple polynucleotide copies of the complement of at least one RNA sequence of interest from a first RNA population using the method of any of claims 1, 49, 59, 82, and 89; and

(b) hybridizing the multiple copies to a second RNA population, whereby a subpopulation of the second RNA population forms a complex with a polynucleotide copy.

130. A method for differential amplification of RNA that does not hybridize to at least one RNA sequence of interest, said method comprising:

(a) hybridizing multiple polynucleotide copies of the complement of the at least one RNA sequence of interest prepared using the method of any of claims 1, 49, 59, 82, and 89 to an RNA population, whereby a subpopulation of the RNA population hybridizes to the polynucleotide copies to form complexes;

(b) cleaving RNA in the complexes of step (a) with at least one enzyme that cleaves RNA from an RNA/DNA hybrid; and

(c) amplifying an unhybridized subpopulation of the RNA population, whereby multiple copies of single stranded DNA complementary to the unhybridized subpopulation of the RNA population are generated.

131. A method for making a library, said method comprising:

preparing multiple complementary copies of the unhybridized subpopulation of a second mRNA population prepared according to claim 130.

132. The method of claim 6, wherein the RNA portion of the first primer comprises at least about 5 nucleotides and the DNA portion of the first primer comprises at least about 5 nucleotides.

134. The method of claim 5, wherein the RNA portion of the first primer comprises at least about 5 nucleotides and the DNA portion of the first primer comprises at least about 5 nucleotides.

139. The method of claim 5, wherein the RNA portion of the first primer comprises the following ribonucleotide sequence:

5′

-GACGGAUGCGGUCU-3′

(SEQ ID NO;

25).

145. The method of claim 1, wherein different enzymes comprise RNA-dependent DNA polymerase activity and DNA-dependent DNA polymerase activity.

146. The method of claim 1, wherein different enzymes comprise RNA-dependent DNA polymerase activity and cleave RNA from RNA/DNA hybrid.

147. The method of claim 1, wherein different enzymes comprise DNA-dependent DNA polymerase activity and cleave RNA from RNA/DNA hybrid.

148. The method of claim 1, wherein different enzymes comprise DNA-dependent DNA polymerase activity, RNA-dependent DNA polymerase activity and cleave RNA from an RNA/DNA hybrid.

158. The method according to claim 1, wherein the reaction mixture of step (b) comprises at least one enzyme that cleaves RNA from an RNA-DNA hybrid.

159. The method of claim 1, wherein the RNA portion of the composite amplification primer is 5 ′

with respect to the 3′

DNA portion.

160. The method of claim 159, wherein the 5′

RNA portion of the composite amplification primer is adjacent to the 3′

DNA portion.

161. The method of claim 6, wherein the RNA portion of the composite amplification primer consists of at least about 5 nucleotides an the DNA portion of the composite amplification primer consists of at least 1 nucleotide.

162. The method of claim 161, wherein the RNA portion of the composite amplification primer consists of about 5 to about 50 nucleotides and the DNA portion of the composite amplification primer consists of about 1 to about 20 nucleotides.

163. The method of claim 7, wherein the DNA portion of the first primer comprises at least 1 random nucleotide at the 3′

end.

164. The method of claim 5, wherein the RNA portion of the composite amplification primer comprises at least about 5 nucleotides an the DNA portion of the composite amplification primer comprises at least 1 nucleotide.

165. The method of claim 1, 4 or 5, wherein the first primer is a tailed primer that comprises a 5′

portion that is not hybridizable to the target RNA under conditions in which the first primer hybridizes to the target RNA.

166. The method of claim 4 or 5, wherein the target RNA is mRNA, and the first primer comprises a poly-dT sequence and further is a tailed primer that comprises a 5′

portion that is not hybridizable to the target mRNA under conditions which the first primer hybridizes to the target mRNA.

167. The method of claim 4 or 5, wherein the at least one enzyme that cleaves RNA from an RNA/DNA hybrid is RNase H.

168. The method of claim 4 or 5, further comprising using a labeled dNTP, whereby labeled products are generated.

212. The method of claim 129, wherein the second RNA population is a mRNA population.

217. The method according to claim 4 or 5, wherein the RNA portion of the composite amplification primer is 5′

with respect to the 3′

DNA portion.

218. The method of claim 217, wherein the 5′

RNA portion of the composite amplification primer is adjacent to the 3′

DNA portion.

219. The method of claim 132, wherein the RNA portion of the first primer consists of about 5 to about 50 nucleotides and the DNA portion of the first primer consists of about 5 to about 20 nucleotides.

220. The method according to claim 164, wherein the RNA portion of the composite amplification primer is 5′

with respect to the 3′

DNA portion.

221. The method of claim 220, wherein the 5′

RNA portion of the composite amplification primer is adjacent to the 3′

DNA portion.

222. The method of claim 134, wherein the RNA portion of the first primer consists of about 5 to about 50 nucleotides and the DNA portion of the first primer consists of about 5 to about 20 nucleotides.

223. The method according to claim 165, wherein the RNA portion of the composite amplification primer is 5′

with respect to the 3′

DNA portion.

224. The method of claim 223, wherein the 5′

RNA portion of the composite amplification primer is adjacent to the 3′

DNA portion.

225. The method of claim 164, wherein the RNA portion of the composite amplification primer consists of about 5 to about 50 nucleotides and the DNA portion of the composite amplification primer consists of about 1 to about 20 nucleotides.

309. A method according to claim 1, wherein the RNA portion of the first primer is 5′

with respect to the 3′

DNA portion of the first primer and the portion of the composite amplification primer is 5′

with respect to the 3′

DNA portion of the composite amplification primer.

310. A method according to claim 309, wherein the 5′

RNA portion of the first primer is adjacent to the 3′

DNA portion of the first primer and the 5′

RNA portion of the composite amplification primer is adjacent to the 3′

DNA portion of the composite amplification primer.

24. A method of generating multiple copies of an RNA sequence of interest, said method comprising the steps of:

(a) extending a first primer hybridized to a target RNA with at least one enzyme comprising RNA-dependent DNA polymerase activity, wherein the first primer is a composite primer comprising an RNA portion and a 3′

DNA portion, whereby a complex comprising a first primer extension product and the target RNA is produced;

(b) cleaving RNA in the complex of step (a) with at least one enzyme that cleaves RNA from an RNA/DNA hybrid;

(c) extending a second primer hybridized to the first primer extension product with at least one enzyme comprising DNA-dependent DNA polymerase activity and at least one enzyme comprising RNA-dependent DNA polymerase activity, whereby a second primer extension product is produced to form a complex of first and second primer extension products;

(d) cleaving RNA from the first primer in the complex of first and second primer extension products with at least one enzyme that cleaves RNA from an RNA/DNA hybrid such that a composite amplification primer hybridizes to the second primer extension product, wherein the composite amplification primer comprises an RNA portion and a 3′

DNA portion;

(e) extending said composite amplification primer hybridized to the second primer extension product with at least one enzyme comprising DNA-dependent DNA polymerase activity, whereby said first primer extension product is displaced, RNA is cleaved from the composite amplification primer and another composite amplification primer hybridizes such that primer extension and strand displacement are repeated;

(f) hybridizing the displaced first primer extension product with a polynucleotide comprising a propromoter and a region which is hybridizable to the displaced first primer extension product under conditions which allow transcription to occur by RNA polymerase, such that RNA transcripts are produced comprising sequences complementary to the displace first primer extension product,

whereby multiple copies of the RNA sequence of interest are generated.

25. The method of claim 24, wherein the second primer comprises a fragment of the target RNA hybridized to the first primer extension product, said fragment generated by cleaving RNA in the complex of step (a) with at least one enzyme that cleaves RNA from an RNA/DNA hybrid.

26. The method of claim 24, wherein the second primer comprises DNA.

27. The method of claim 24, wherein the RNA portion of the first primer is 5′

with respect to the 3′

DNA portion.

28. The method of claim 27, wherein the 5′

RNA portion of the first primer is adjacent to the 3′

DNA potion.

29. The method of claim 24, wherein the first primer is a tailed primer that comprises a 5′

portion that is not hybridizable to the target RNA under conditions which the first primer hybridizes to the target RNA.

30. The method of claim 24, wherein the first primer comprises a poly-dT sequence.

31. The method of claim 24 or 30, wherein the target RNA is mRNA.

32. The method of claim 24, wherein the first primer comprises a random primer sequence.

33. The method of claim 24, wherein the target RNA is nRNA, the first primer comprises a poly-dT sequence and further is a tailed primer that comprises a 5′

portion that is not hybridizable to the target mRNA under conditions which the first primer hybridizes to the target mRNA.

34. The method of claim 24, wherein a plurality of different first primers are used for hybridizing to the target RNA.

35. The method of claim 24, wherein the second primer is a random primer.

36. The method of claim 24, wherein the second primer is a tailed primer that comprises a 5′

portion that is not hybridizable to the first primer extension product under conditions in which the second primer hybridizes to the first primer extension product.

37. The method of claim 24, wherein the at least one enzyme that cleaves RNA from an RNA/DNA hybrid is RNase H.

38. The method of claim 24, wherein the same enzyme comprises RNA-dependent DNA polymerase activity and DNA-dependent DNA polymerase activity.

39. The method of claim 24, wherein the same enzyme comprises RNA-dependent DNA polymerase activity and cleaves RNA from RNA/DNA hybrid.

40. The method of claim 24, wherein the same enzyme comprises DNA-dependent DNA polymerase activity and cleaves RNA from RNA/DNA hybrid.

41. The method of claim 24, wherein the same enzyme comprises DNA-dependent DNA polymerase activity, RNA-dependent DNA polymerase activity and cleaves RNA from an RNA/DNA hybrid.

42. The method of claim 24, wherein the propromoter polynucleotide comprises a region at the 3′

end which hybridizes to the displaced primer extension product, whereby DNA polymerase extension of displaced primer extension product produces a double stranded promoter from which transcription occurs.

43. The method of claim 24, wherein the propromoter polynucleotide is a propromoter template oligonucleotide (PTO).

44. The method of claim 24, further comprising using a labeled rNTP, whereby labeled products are generated.

45. The method of claim 24, wherein said method comprises generating multiple copies of two or more different sequences of interest.

46. The method of claim 45, where in said method comprises at least two different first primers.

47. The method of claim 45 or 46, wherein the first primer comprises a poly-dT sequence.

48. The method of claim 45 or 46, wherein the first primer comprises a random primer sequence.

98. A method of sequencing an RNA sequence of interest, said method comprising (a) amplifying a target RNA comprising the sequence of interest by any of the methods of claims 24 and 53 in the presence of a mixture of rNTPs a and rNTP analogs such that transcription is terminated upon incorporation of a rNTP analog;

and (b) analyzing the amplification products to determine sequence.

99. The method of claim 98, wherein the target RNA is mRNA.

115. A method of characterizing an RNA sequence of interest, comprising analyzing amplification products, wherein the amplification products are generated by amplifying a target RNA comprising the sequence of interest by the methods of any of claims 24 and 53.

116. The method of claim 115, wherein the target RNA is mRNA.

117. The method of claim 115, wherein the amplification products are labeled.

118. The method of claim 115, wherein analyzing the amplification products comprises determining amount of said products, whereby the amount of the RNA sequence of interest present in a sample is quantified.

119. The method of claim 117, wherein analyzing the amplification products comprises contacting the labeled amplification product with at least one probe.

120. The method of claim 115, wherein the amplification products are labeled, and wherein the at least one probe is provided as a microarray.

121. The method of claim 120, wherein the microarray comprises at least one probe immobilized on a substrate fabricated from a material selected from the group consisting of paper, glass, ceramic, plastic, polypropylene, polystyrene, nylon, polyacrylamide, nitrocellulose, silicon, and optical fiber.

122. The method of claim 121, wherein the probe is immobilized on the substrate in a two-dimensional configuration or a three-dimensional configuration comprising pins, rods, fibers, tapes, threads, beads, particles, microtiter wells, capillaries, and cylinders.

133. The method of claim 29, wherein the RNA portion of the first primer comprises at least about 5 nucleotides and the DNA portion of the first primer comprises at least about 5 nucleotides.

135. The method of claim 28, wherein the RNA portion of the first primer comprises at least about 5 nucleotides and the DNA portion of the first primer comprises at least about 5 nucleotides.

140. The method of claim 28, wherein the RNA portion of the first primer comprises the following ribonucleotide sequence:

5′

-GACGGAUGCGGUCU-3′

(SEQ ID NO;

25).

149. The method of claim 24, wherein different enzymes comprise RNA-dependent DNA polymerase activity and DNA-dependent DNA polymerase activity.

150. The method of claim 24, wherein different enzymes comprise RNA-dependent DNA polymerase activity and cleave RNA from an RNA/DNA hybrid.

151. The method of claim 24, wherein different enzymes comprise DNA-dependent DNA polymerase activity and cleave RNA from RNA/DNA hybrid.

152. The method of claim 24, wherein different enzymes comprise DNA-dependent DNA polymerase activity, RNA-dependent DNA polymerase activity and cleave RNA from an RNA/DNA hybrid.

169. The method of claim 24, wherein the RNA portion of the composite amplification primer is 5′

with respect to the 3′

DNA portion.

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

RNA portion of the composite amplification primer is adjacent to the 3′

DNA portion.

171. The method of claim 133, wherein the RNA portion of the first primer consists of about 5 to about 50 nucleotides and the DNA portion of the first primer consists of about 5 to about 20 nucleotides.

172. The method of claim 29, wherein the RNA portion of the composite amplification primer comprises at least about 5 nucleotides and the DNA portion of the composite amplification primer comprises at least 1 nucleotide.

173. The method of claim 172, wherein the RNA portion of the composite amplification primer consists of about 5 to about 50 nucleotides and the DNA portion of the composite amplification primer consists of about 1 to about 20 nucleotides.

174. The method of claim 135, wherein the RNA portion of the first primer consists of about 5 to about 50 nucleotides and the DNA portion of the first primer consists of about 5 to about 20 nucleotides.

175. The method of claim 24, 27 or 28, wherein the first primer is a tailed primer that comprises a 5′

portion that is not hybridizable to the target RNA under conditions which the first primer hybridizes to the target RNA.

176. The method of claim 27 or 28, wherein the target RNA is mRNA, and the first primer comprises a poly-dT sequence and further is a tailed primer that comprises a 5′

portion that is not hybridizable to the target mRNA under conditions which the composite primer hybridizes to the target mRNA.

177. The method of claim 27 or 28, wherein the at least one enzyme that cleaves RNA from an RNA/DNA hybrid is RNase H.

178. The method of claim 27 or 28, further comprising using a labeled rNTP, whereby labeled products are generated.

226. The method according to claim 24, 27 or 28, wherein the RNA portion of the composite amplification primer is 5′

with respect to the 3′

DNA portion.

227. The method of claim 226, wherein the 5′

RNA portion of the composite amplification primer is adjacent to the 3′

DNA portion.

229. The method according to claim 174, wherein the RNA portion of the composite amplification primer is 5′

with respect to the 3′

DNA portion.

230. The method of claim 229, wherein the 5′

RNA portion of the composite amplification primer is adjacent to the 3′

DNA portion.

231. The method of claim 230, wherein the RNA portion of the composite amplification primer comprises at least about 5 nucleotides and the DNA portion of the composite amplification primer comprises at least 1 nucleotide.

232. The method according to claim 175, wherein the RNA portion of the composite amplification primer is 5′

with respect to the 3′

DNA portion.

233. The method of claim 232, wherein the 5′

RNA portion of the composite amplification primer is adjacent to the 3′

DNA portion.

234. The method of claim 231, wherein the RNA portion of the composite amplification primer consists of about 5 to about 50 nucleotides and the DNA portion of the composite amplification primer consists of about 1 to about 20 nucleotides.

311. A method according to claim 24, wherein the RNA portion of the first primer is 5′

with respect to the 3′

DNA portion of the first primer and the RNA portion of the composite amplification primer is 5′

with respect to the 3′

DNA portion of the composite amplification primer.

312. A method according to claim 311, wherein the 5′

RNA portion of the first primer is adjacent to the 3′

DNA portion of the first primer and the 5′

A portion of the composite amplification primer is adjacent to the 3′

DNA portion of the composite amplification primer.

49. A method of generating multiple copies of a polynucleotide sequence complementary to an RNA sequence of interest, comprising incubating a reaction mixture, said reaction mixture comprising:

(a) a complex of a first primer extension product and a second primer extension product, wherein the first primer extension product is hybridized to the second primer extension product, and wherein the second primer extension product comprises a 3′

single stranded portion, wherein the 3′

single stranded portion comprises the complement of a portion of a first primer, wherein the first primer is a composite primer comprising an RNA portion and a 3′

DNA portion and is a tailed primer that comprises a 5′

portion that is not hybridizable to the target RNA, wherein the partially double stranded complex of the first primer extension product and the second primer extension product is generated by a method comprising extension of the first primer hybridized to target RNA by at least one enzyme comprising RNA-dependent DNA polymerase activity and cleavage of RNA from the first primer from the complex of the first primer extension product and the second primer extension product by at least one enzyme that cleaves RNA from an RNA-DNA hybrid;

(b) a composite amplification primer that is hybridizable to the 3′

single stranded portion, wherein the composite amplification primer comprises an RNA portion and a 3′

DNA portion;

(c) at least one enzyme comprising DNA-dependent DNA polymerase activity; and

(d) at least one enzyme that cleaves RNA from an RNA/DNA hybrid;

wherein the incubation is under conditions that permit primer hybridization, primer extension, RNA cleavage, and displacement of the first primer extension product when another composite amplification primer hybridizes to the 3′

single stranded portion such that primer extension and strand displacement are repeated, whereby multiple copies of a polynucleotide sequence complementary to the RNA sequence of interest are generated.

50. The method of claim 49, wherein the RNA portion of the first primer is 5′

with respect to the 3′

DNA portion.

51. The method of claim 49, wherein the target RNA is mRNA.

52. The method of claim 49, wherein said reaction further comprises a labeled dNTP, wherein labeled products are generated.

179. The method of claim 49, wherein the at least one enzyme that cleaves RNA from an RNA/DNA hybrid is RNase H.

288. The method of claim 50, wherein the 5′

RNA portion of the first primer is adjacent to the 3′

DNA portion of the first primer.

313. A method according to claim 49, wherein the RNA portion of the first primer is 5′

with respect to the 3′

DNA portion of the first primer and the RNA portion of the composite amplification primer is 5′

with respect to the 3′

DNA portion of the composite amplification primer.

314. A method according to claim 313, wherein the 5′

RNA portion of the first primer is adjacent to the 3′

DNA portion of the first primer and the 5′

RNA portion of the composite amplification primer is adjacent to the 3′

DNA portion of the composite amplification primer.

53. A method of generating multiple copies of an RNA sequence of interest comprising incubating a reaction mixture, said reaction mixture comprising:

(a) a complex of a first primer extension product and a second primer extension product, wherein the first primer extension product is hybridized to the second primer extension product, and wherein the second primer extension product comprises a 3′

single stranded portion, wherein the 3′

single stranded portion comprises the complement of a portion of a first primer, wherein the first primer is a composite primer comprising an RNA portion and a 3′

DNA portion, wherein the first primer is a tailed primer that comprises a 5′

portion that is not hybridizable to target RNA and wherein the partially double stranded polynucleotide is generated by a method comprising extension of the first primer hybridized to target RNA by at least one enzyme comprising RNA-dependent DNA polymerase activity;

(b) a composite amplification primer that is hybridizable to the 3′

single stranded portion, wherein the composite amplification primer comprises an RNA portion and a 3′

DNA portion;

(c) at least one enzyme comprising DNA-dependent DNA polymerase activity; and

(d) at least one enzyme comprising RNA polymerase activity;

(e) a propromoter polynucleotide comprising a propromoter and a region which hybridizes to a first primer extension product; and

(f) at least one enzyme that cleaves RNA from an RNA/DNA hybrid;

wherein the incubation is under conditions that permit primer hybridization, primer extension, RNA cleavage, and displacement of the first primer extension product when another composite amplification primer hybridizes to the 3′

single stranded portion such that primer extension and strand displacement are repeated, hybridization of the propromoter polynucleotide to the displaced polynucleotide to form a second complex comprising the displaced polynucleotide and the propromoter polynucleotide, and RNA transcription from said second complex, whereby multiple copies of the RNA sequence of interest are generated.

54. The method of claim 53, wherein the RNA portion of the first primer is 5′

with respect to the 3′

DNA portion.

55. The method of claim 53, wherein the at least one enzyme that cleaves RNA from an RNA/DNA hybrid is RNase H.

56. The method of claim 53, wherein the propromoter polynucleotide comprises a region at the 3′

end which hybridizes to the displaced primer extension product, whereby DNA polymerase extension of displaced primer extension product produces a double stranded promoter from which transcription occurs.

57. The method of claim 53, wherein at least one type of rNTP used is a labeled rNTP, whereby labeled products are generated.

58. The method of claim 53, wherein the target RNA is mRNA.

141. The method of claim 54, wherein the RNA portion of the first primer comprises the following ribonucleotide sequence:

5′

-GACGGAUGCGGUCU-3′

(SEQ ID NO;

25).

182. The method of claim 54, wherein the 5′

RNA portion of the first primer is adjacent to the 3′

DNA portion.

207. The method of claim 53, 54 or 182, wherein the RNA portion of the composite amplification primer is 5′

with respect to the 3′

DNA portion.

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

RNA portion of the composite amplification primer is adjacent to the 3′

DNA portion.

315. A method according to claim 53, wherein the RNA portion of the first primer is 5′

with respect to the 3′

DNA portion of the first primer and the portion of the composite amplification primer is 5′

with respect to the 3′

DNA portion of the composite amplification primer.

316. A method according to claim 315, wherein the 5′

RNA portion of the first primer is adjacent to the 3′

DNA portion of the first primer and the 5′

RNA portion of the composite amplification primer is adjacent to the 3′

DNA portion of the composite amplification primer.

59. A method of generating multiple copies of a polynucleotide sequence complementary to an RNA sequence of interest comprising:

(a) incubating a reaction mixture, said reaction mixture comprising;

(i) a target RNA;

(ii) a first primer that is hybridizable to a target RNA, wherein the first primer is a composite primer comprising an RNA portion and a 3′

DNA portion; and

(iii) at least one enzyme comprising RNA-dependent DNA polymerase activity

wherein the incubation is under conditions that permit primer hybridization, formation of a complex comprising a first primer extension product and the target RNA;

(b) incubating a reaction mixture, said reaction mixture comprising;

(i) the first primer extension product;

(ii) at least one enzyme comprising DNA-dependent DNA polymerase activity;

(iii) at least one enzyme comprising RNA-dependent DNA polymerase activity; and

(iv) optionally, at least one enzyme capable of cleaving RNA from an RNA/DNA hybrid;

wherein the incubation is under conditions permitting formation of a complex comprising the first primer extension product and a second primer extension product;

(c) incubating a reaction mixture, said reaction mixture comprising;

(i) the complex comprising the first primer extension product a second primer extension product;

(ii) at least one enzyme capable of cleaving RNA from an RNA/DNA hybrid;

(iii) a composite amplification primer, wherein the composite amplification primer comprises a RNA portion and a 3′

DNA portion;

(iv) at least one enzyme comprising DNA-dependent DNA polymerase activity, wherein the incubation is under conditions that permit cleavage of RNA, composite primer hybridization, primer extension, and displacement of the first primer extension product from the complex comprising the first primer extension product and a second primer extension product, whereby another composite amplification primer hybridizes and primer extension and strand displacement are repeated;

whereby multiple copies of a polynucleotide sequence complementary to the RNA sequence of interest are generated.

60. The method of claim 59, wherein the reaction mixture of step (b) further comprises:

(v) a second primer; and

wherein the incubation is under conditions permitting hybridization of the second primer.

61. The method of claim 60, wherein the second primer comprises DNA.

62. The method of claim 59, wherein the RNA portion of the first primer is 5′

with respect to the 3′

DNA portion.

63. The method of claim 62, wherein the 5′

RNA portion of the first primer is adjacent to the 3′

DNA portion.

64. The method of claim 59, wherein the first primer is a tailed primer that comprises a 5′

portion that is not hybridizable to the target RNA under conditions which the first primer hybridizes to the target RNA.

65. The method of claim 59, wherein the first primer comprises a poly-dT sequence.

66. The method of claim 59 or 65, wherein the target RNA is mRNA.

67. The method of claim 59, wherein the first primer comprises a random primer sequence.

68. The method of claim 59, wherein the target RNA is mRNA, and the first primer comprises a poly-dT sequence and further is a tailed primer that comprises a 5′

portion that is not hybridizable to the target mRNA under conditions which the first primer hybridizes to the target mRNA.

69. The method of claim 59, wherein a plurality of different first primers are used for hybridizing to the target RNA.

70. The method of claim 60, wherein the second primer is a random primer.

71. The method of claim 60, wherein the second primer is a tailed primer that comprises a 5′

portion that is not hybridizable to the first primer extension product under conditions which the second primer hybridizes to the first primer extension product.

72. The method of claim 59, wherein the at least one enzyme that cleaves RNA from an RNA/DNA hybrid is RNase H.

73. The method of claim 59, wherein the same enzyme comprises RNA-dependent DNA polymerase activity and DNA-dependent DNA polymerase activity.

74. The method of claim 59, wherein the same enzyme comprises RNA-dependent DNA polymerase activity and cleaves RNA from an RNA/DNA hybrid.

75. The method of claim 59, wherein the same enzyme comprises DNA-dependent DNA polymerase and cleaves RNA from an RNA/DNA hybrid.

76. The method of claim 59, wherein the same enzyme comprises DNA-dependent DNA polymerase activity, RNA-dependent DNA polymerase activity and cleaves RNA from an RNA/DNA hybrid.

77. The method of claim 59, wherein the reaction mixture of step (c) further comprises a labeled dNTP, whereby labeled products are generated.

78. The method of claim 59, wherein said method comprises generating multiple copies of a polynucleotide sequences complementary to two or more different sequences of interest.

79. The method of claim 78, wherein said method comprises at least two different first primers.

80. The method of claim 78 or 79, wherein the first primer comprises a poly-dT sequence.

81. The method of claim 78 or 79, wherein the first primer comprises a random primer sequence.

136. The method of claim 63, wherein the RNA portion of the first primer comprises at least about 5 nucleotides and the DNA portion of the first primer comprises at least about 5 nucleotides.

138. The method of claim 136, wherein the RNA portion of the first primer consists of about 5 to about 50 nucleotides and the DNA portion of the first primer consists of about 5 to about 20 nucleotides.

142. The method of claim 63, wherein the RNA portion of the first primer comprises the following ribonucleotide sequence:

5′

-GACGGAUGCGGUCU-3′

(SEQ ID NO;

25).

153. The method of claim 59, wherein different enzymes comprise RNA-dependent DNA polymerase activity and DNA-dependent DNA polymerase activity.

154. The method of claim 59, wherein different enzymes comprise RNA-dependent DNA polymerase activity and cleave RNA from an RNA/DNA hybrid.

155. The method of claim 59, wherein different enzymes comprise DNA-dependent DNA polymerase activity and cleave RNA from RNA/DNA hybrid.

156. The method of claim 59, wherein different enzymes comprise DNA-dependent DNA polymerase activity, RNA-dependent DNA polymerase activity and cleave RNA from an RNA/DNA hybrid.

157. The method according to claim 59, wherein the reaction mixture of step (a) optionally comprises at least one enzyme that cleaves RNA from an RNA-DNA hybrid;

and the reaction mixture of step (b) comprises at least one enzyme that cleaves RNA from an RNA-DNA hybrid.

180. The method of claim 63, wherein the RNA portion of the composite amplification primer is 5′

with respect to the 3′

DNA portion.

181. The method of claim 180, wherein the 5′

RNA portion of the composite amplification primer is adjacent to the 3′

DNA portion.

228. The method of claim 64, wherein the RNA portion of the first primer comprises at least about 5 nucleotides and the DNA portion of the first primer comprises at least about 5 nucleotides.

238. The method according to claim 64, wherein the RNA portion of the composite amplification primer is 5′

with respect to the 3′

DNA portion.

239. The method of claim 238, wherein the 5′

RNA portion of the composite amplification primer is adjacent to the 3′

DNA portion.

240. The method of claim 228, wherein the RNA portion of the first primer consists of about 5 to about 50 nucleotides and the DNA portion of the first primer consists of about 5 to about 20 nucleotides.

243. The method of claim 64, wherein the RNA portion of the composite amplification primer comprises at least about 5 nucleotides an the DNA portion of the composite amplification primer comprises at least 1 nucleotide.

246. The method of claim 243, wherein the RNA portion of the composite amplification primer consists of about 5 to about 50 nucleotides and the DNA portion of the composite amplification primer consists of about 1 to about 20 nucleotides.

250. The method of claim 64, wherein the first primer comprises at least 1 random nucleotide at the 3′

end.

251. A method of producing a nucleic acid immobilized to a substrate, comprising immobilizing amplification products on a substrate, wherein the amplification products are generated by amplifying a target RNA by the method of claim 239.

253. A method of characterizing an RNA sequence of interest, comprising analyzing amplification products, wherein the amplification products are generated by amplifying a target RNA by the method of claim 239.

255. A method of determining gene expression profile in a sample, said method comprising:

determining amount of amplification products of at least one RNA sequence of interest, wherein the amplification products are generate by amplifying at least one RNA sequence of interest in the sample using the method of claim 239, whereby the gene expression profile in the sample is determined.

259. A method of preparing a subtractive hybridization probe, said method comprising generating multiple polynucleotide copies of the complement of at least one RNA sequence of interest from a first RNA population using the method of claim 239.

261. A method of performing subtractive hybridization in which at least first and second target RNA populations are compared, said method comprising:

(a) generating multiple polynucleotide copies of the complement of at least one RNA sequence of interest from a first RNA population using the method of claim 239; and

(b) hybridizing the multiple copies to a second RNA population, whereby a subpopulation of the second RNA population forms a complex with a polynucleotide copy.

317. A method according to claim 59, wherein the RNA portion of the first primer is 5′

with respect to the 3′

DNA portion of the first primer and the RNA portion of the composite amplification primer is 5′

with respect to the 3′

DNA portion of the composite amplification primer.

318. A method according to claim 317, wherein the 5′

RNA portion of the first primer is adjacent to the 3′

DNA portion of the first primer and the 5′

RNA portion of the composite amplification primer is adjacent to the 3′

DNA portion of the composite amplification primer.

327. The method of claim 180, wherein the RNA portion of the composite amplification primer comprises at least about 5 nucleotides and the DNA portion of the composite amplification primer comprises at least 1 nucleotide.

333. The method of claim 327, wherein the RNA portion of the composite amplification primer consists of about 5 to about 50 nucleotides and the DNA portion of the composite amplification primer consists of about 1 to about 20 nucleotides.

334. The method of claim 261, wherein the second RNA population is a mRNA population.

335. The method of claim 250, wherein the RNA portion of the first primer consists of about 5 to about 50 nucleotides and the DNA portion of the first primer consists of about 5 to about 20 nucleotides.

82. A method of generating multiple copies of a polynucleotide sequence complementary to an RNA sequence of interest, said method comprising the steps of:

(a) cleaving RNA from a complex of first and second primer extension products with at least one enzyme that cleaves KNA from an RNA/DNA hybrid wherein the first primer extension product is produced by extension of a first primer hybridized to a target RNA with at least one enzyme comprising RNA-dependent DNA polymerase activity, wherein the first primer is a composite primer comprising an RNA portion and a 3′

DNA portion, and wherein the second primer extension product is generated by extension of a second primer hybridized to the first primer extension product;

(b) hybridizing a composite amplification primer to the second primer extension product and extending the composite amplification primer with at least one enzyme comprising DNA-dependent DNA polymerase activity, wherein the composite amplification primer is a composite primer comprising an RNA portion and a 3′

DNA portion;

whereby said first primer extension product is displaced, RNA is cleaved from the composite amplification primer and another composite amplification primer hybridizes such that primer extension and strand displacement are repeated; and

whereby multiple copies of a polynucleotide sequence complementary to the RNA sequence of interest are generated.

83. The method of claim 82, wherein the second primer comprises DNA.

84. The method of claim 82, wherein the RNA portion of the first primer is 5′

with respect to the 3′

DNA portion.

85. The method of claim 84, wherein the 5′

RNA portion of the first primer is adjacent to the 3′

DNA portion.

86. The method of claim 82, wherein the first primer is a tailed primer that comprises a 5′

portion that is not hybridizable to the target RNA under conditions which the first primer hybridizes to the target RNA.

87. The method of claim 82, wherein the first primer comprises a poly-dT sequence.

88. The method of claim 82 or 87, wherein the target RNA is mRNA.

137. The method of claim 85, wherein the RNA portion of the first primer comprises at least about 5 nucleotides and the DNA portion of the first primer comprises at least about 5 nucleotides.

143. The method of claim 85, wherein the RNA portion of the first primer comprises the following ribonucleotide sequence:

5′

-GACGGAUGCGGUCU-3′

(SEQ ID NO;

25).

183. The method of claim 86, wherein the RNA portion of the first primer comprises at least about 5 nucleotides and the DNA portion of the first primer comprises at least about 5 nucleotides.

184. The method of claim 183, wherein the RNA portion of the first primer consists of about 5 to about 50 nucleotides and the DNA portion of the composite amplification primer consists of about 5 to about 20 nucleotides.

185. The method of claim 86, wherein the RNA portion of the composite amplification primer comprises at least about 5 nucleotides an the DNA portion of the composite amplification primer comprises at least 1 nucleotide.

186. The method of claim 137, wherein the RNA portion of the first primer consists of about 5 to about 50 nucleotides and the DNA portion of the first primer consists of about 5 to about 20 nucleotides.

187. The method of claim 82, 84 or 85, wherein the first primer is a tailed primer that comprises a 5′

portion that is not hybridizable to the target RNA under conditions which the first primer hybridizes to the target RNA.

188. The method of claim 84 or 85, wherein the target RNA is mRNA, and the first primer comprises a poly-dT sequence and further is a tailed primer that comprises a 5′

portion that is not hybridizable to the target mRNA under conditions which the first primer hybridizes to the target mRNA.

189. The method of claim 84 or 85, wherein the at least one enzyme that cleaves RNA from an RNA/DNA hybrid is RNase H.

190. The method of claim 84 or 85, further comprising using a labeled dNTP, whereby labeled products are generated.

235. The method according to claim 82, 84 or 85, wherein the RNA portion of the composite amplification primer is 5′

with respect to the 3′

DNA portion.

236. The method of claim 235, wherein the 5′

RNA portion of the composite amplification primer is adjacent to the 3′

DNA portion.

237. The method of claim 236, wherein the RNA portion of the composite amplification primer comprises at least about 5 nucleotides and the DNA portion of the composite amplification primer comprises at least 1 nucleotide.

241. The method according to claim 187, wherein the RNA portion of the composite amplification primer is 5′

with respect to the 3′

DNA portion.

242. The method of claim 241, wherein the 5′

RNA portion of the composite amplification primer is adjacent to the 3′

DNA portion.

249. The method of claim 185, wherein the RNA portion of the composite amplification primer consists of about 5 to about 50 nucleotides and the DNA portion of the composite amplification primer consists of about 1 to about 20 nucleotides.

252. A method of producing a nucleic acid immobilized to a substrate, comprising immobilizing amplification products on a substrate, wherein the amplification products are generated by amplifying a target RNA by the method of claim 242.

254. A method of characterizing an RNA sequence of interest, comprising analyzing amplification products, wherein the amplification products are generated by amplifying a target RNA by the method of claim 242.

256. A method of determining gene expression profile in a sample, said method comprising:

determining amount of amplification products of at least one RNA sequence of interest, wherein the amplification products are generate by amplifying at least one RNA sequence of interest in the sample using the method of claim 242, whereby the gene expression profile in the sample is determined.

257. A method of preparing a library, said method comprising:

amplifying a plurality of RNA sequences of interest using the method of claim 236.

258. A method of preparing a library, said method comprising:

amplifying a plurality of RNA sequences of interest using the method of claim 242.

260. A method of preparing a subtractive hybridization probe, said method comprising generating multiple polynucleotide copies of the complement of at least one RNA sequence of interest from a first RNA population using the method of claim 242.

262. A method of performing subtractive hybridization in which at least first and second target RNA populations are compared, said method comprising:

(a) generating multiple polynucleotide copies of the complement of at least one RNA sequence of interest from a first RNA population using the method of claim 242; and

(b) hybridizing the multiple copies to a second RNA population, whereby a subpopulation of the second RNA population forms a complex with a polynucleotide copy.

286. The method of claim 87, wherein the DNA portion of the first primer comprises at least 1 random nucleotide at the 3′

end.

287. The method of claim 82, wherein the first primer comprises a random primer sequence.

319. A method according to claim 82, wherein the RNA portion of the first primer is 5′

with respect to the 3′

DNA portion of the first primer and the RNA portion of the composite amplification primer is 5′

with respect to the 3′

DNA portion of the composite amplification primer.

320. A method according to claim 319, wherein the 5′

RNA portion of the first primer is adjacent to the 3′

DNA portion of the first primer and the 5′

RNA portion of the composite amplification primer is adjacent to the 3′

DNA portion of the composite amplification primer.

328. The method of claim 237, wherein the RNA portion of the composite amplification primer consists of about 5 to about 50 nucleotides and the DNA portion of the composite amplification primer consists of about 1 to about 20 nucleotides.

89. A method of generating multiple copies of a polynucleotide sequence complementary to an RNA sequence of interest, said method comprising the step of:

extending a composite amplification primer in a complex comprising;

(i) a complex of first and second primer extension products, wherein the first primer extension product is produced by extension of a first primer hybridized to a target RNA with at least one enzyme comprising RNA-dependent DNA polymerase activity, wherein the first primer is a composite primer comprising an RNA portion and a 3′

DNA portion, wherein the second primer extension product is generated by extension of a second primer hybridized to the first primer extension product, and wherein RNA from the complex of first and second primer extension products is cleaved with at least one enzyme that cleaves RNA from an RNA/DNA hybrid; and

(ii) a composite amplification primer, said composite amplification primer comprising an RNA portion and a 3′

DNA portion, wherein the composite amplification primer is hybridized to the second primer extension product;

whereby said first primer extension product is displaced, RNA is cleaved from the composite amplification primer and another composite amplification primer hybridizes such that primer extension and strand displacement are repeated; and

whereby multiple copies of a polynucleotide sequence complementary to the RNA sequence of interest are generated.

90. The method of claim 89, wherein the second primer comprises DNA.

91. The method of claim 89, wherein the RNA portion of the first primer is 5′

with respect to the 3′

DNA portion.

92. The method of claim 91, wherein the 5′

RNA portion of the first primer is adjacent to the 3′

DNA portion.

93. The method of claim 89, 91 or 92, wherein the first primer is a tailed primer that comprises a 5′

portion that is not hybridizable to the target RNA under conditions which the first primer hybridizes to the target RNA.

94. The method of claim 89, wherein the first primer comprises a poly-dT sequence.

95. The method of claim 89 or 94, wherein the target RNA is mRNA.

144. The method of claim 92, wherein the RNA portion of the first primer comprises the following ribonucleotide sequence:

5′

-GACGGAUGCGGUCU-3′

(SEQ ID NO;

25).

191. The method of claim 89, wherein the second primer comprises a fragment of the target RNA hybridized to the primer extension product, said fragment generated by cleaving RNA in the complex of step (a) with an enzyme that cleaves RNA from an RNA/DNA hybrid.

192. The method of claim 89, wherein the first primer comprises a random primer sequence.

193. The method of claim 89, wherein the target RNA is mRNA, and the first primer comprises a poly-dT sequence and further is a tailed primer that comprises a 5′

portion that is not hybridizable to the target mRNA under conditions which the first primer hybridizes to the target mRNA.

194. The method of claim 89, wherein a plurality of different first primers are used for hybridizing to the target RNA.

195. The method of claim 89, wherein the second primer is a random primer.

196. The method of claim 89, wherein the second primer is a tailed primer that comprises a 5′

portion that is not hybridizable to the first primer extension product under conditions in which the second primer hybridizes to the first primer extension product.

197. The method of claim 89, wherein the at least one enzyme that cleaves RNA from an RNA/DNA hybrid is RNase H.

198. The method of claim 89, wherein the same enzyme comprises RNA-dependent DNA polymerase activity and DNA-dependent DNA polymerase activity.

199. The method of claim 89, wherein the same enzyme comprises RNA-dependent DNA polymerase activity and cleaves RNA from RNA/DNA hybrid.

200. The method of claim 89, wherein the same enzyme comprises DNA-dependent DNA polymerase activity and cleaves RNA from RNA/DNA hybrid.

201. The method of claim 89, wherein the same enzyme comprises DNA-dependent DNA polymerase activity, RNA-dependent DNA polymerase activity and cleaves RNA from an RNA/DNA hybrid.

202. The method of claim 89, further comprising using a labeled dNTP, whereby labeled products are generated.

203. The method of claim 89, wherein said method comprises generating multiple copies of polynucleotide sequences complementary to two or more different sequences of interest.

204. The method of claim 203, wherein said method comprises at least two different first primers.

205. The method of claim 203 or 204, wherein the first primer a poly-dT sequence.

206. The method of claim 203 or 204, wherein the first primer comprises a random primer sequence.

209. The method of claim 91 or 92, wherein the target RNA is mRNA, and the first primer comprises a poly-dT sequence and further is a tailed primer that comprises a 5′

portion that is not hybridizable to the target mRNA under conditions which the first primer hybridizes to the target mRNA.

210. The method of claim 91 or 92, wherein the at least one enzyme that cleaves RNA from an RNA/DNA hybrid is RNase H.

211. The method of claim 91 or 92, further comprising using a labeled dNTP, whereby labeled products are generated.

213. The method of claim 89, wherein different enzymes comprise RNA-dependent DNA polymerase activity and DNA-dependent DNA polymerase activity.

214. The method of claim 89, wherein different enzymes comprise RNA-dependent DNA polymerase activity and cleave RNA from an RNA/DNA hybrid.

215. The method of claim 89, wherein different enzymes comprise DNA-dependent DNA polymerase activity and cleave RNA from RNA/DNA hybrid.

216. The method of claim 89, wherein different enzymes comprise DNA-dependent DNA polymerase activity, RNA-dependent DNA polymerase activity and cleave RNA from an RNA/DNA hybrid.

244. The method according to claim 91 or 92, wherein the RNA portion of the composite amplification primer is 5′

with respect to the 3′

DNA portion.

245. The method of claim 244, wherein the 5′

RNA portion of the composite amplification primer is adjacent to the 3′

DNA portion.

247. The method according to claim 93, wherein the RNA portion of the composite amplification primer is 5′

with respect to the 3 DNA portion.

248. The method of claim 247, wherein the 5′

RNA portion of the composite amplification primer is adjacent to the 3′

DNA portion.

321. A method according to claim 89, wherein the RNA portion of the first primer is 5′

with respect to the 3′

DNA portion of the first primer and the portion of the composite amplification primer is 5′

with respect to the 3′

DNA portion of the composite amplification primer.

322. A method according to claim 321, wherein the 5′

RNA portion of the first primer is adjacent to the 3′

DNA portion of the first primer and the 5′

RNA portion of the composite amplification primer is adjacent to the 3′

DNA portion of the composite amplification primer.

102. A method of determining presence or absence of a sequence of interest in a target RNA, said method comprising

(i) amplifying the target RNA, said amplifying comprising extending a composite amplification primer hybridized to a polynucleotide complex generated from the target RNA comprising a 3′

single stranded region corresponding to the sequence of interest, wherein the RNA portion of the composite amplification primer is known to hybridize to a reference sequence comprising the sequence of interest, and wherein the polynucleotide complex is generated by a method comprising;

(a) extending a first primer hybridized to a target RNA, wherein the first primer is a composite primer comprising an RNA portion and a 3′

DNA portion, whereby a complex comprising a first primer extension product and the target RNA is produced;

(b) extending a second primer hybridized to the first prime extension product, whereby a second primer extension product is produced to form a complex of first and second primer extension products, and

(c) cleaving RNA in the complex of first and second prime extension products, whereby the polynucleotide complex comprising the 3′

single stranded region is generated; and

(ii) comparing the amplification products if any from step (i) with the amount of amplification products from a polynucleotide complex generated from a reference template comprising a 3′

single stranded region comprising the reference sequence, wherein the 3′

single stranded region of the polynucleotide complex of the reference template corresponds to the sequence of interest,

wherein production of detectably fewer amplification products from the polynucleotide complex from the target RNA as compared to the amount of amplification products from the polynucleotide complex of the reference template indicates that the sequence of interest is absent from the target RNA and the target RNA may comprise a sequence variant with respect to the reference sequence hybridizable to the RNA portion of the composite primer.

103. The method of claim 102, wherein the target RNA is mRNA.

263. A kit for amplifying a target RNA, comprising a first composite primer and a second composite primer, wherein the first composite primer comprises an RNA portion and a 3′

DNA portion, wherein the second composite primer comprises an RNA portion and a 3′

DNA portion, and wherein the second composite primer comprises a sequence that is hybridizable to a polynucleotide comprising a complement of the first composite primer.

264. The kit of claim 263, wherein the RNA portion of the first composite primer is 5′

with respect to the 3′

DNA portion of the first composite primer and the RNA portion of the second composite primer is 5′

with respect to the 3′

DNA portion of the second composite primer.

265. The kit of claim 263, wherein the first composite primer is a tailed primer that comprises a 5′

portion that is not hybridizable to a target RNA under conditions wherein the first composite primer hybridizes to the target RNA.

266. The kit of claim 263, wherein the 3′

DNA portion of the first composite primer comprises a random primer sequence.

267. The kit of claim 263, wherein the 3′

DNA portion of the first composite primer comprises a poly-dT sequence.

268. The kit of claim 264, wherein the 5′

RNA portion of the first composite primer is adjacent to the 3′

DNA portion of the first composite primer and the 5′

RNA portion of the second composite primer is adjacent to the 3′

DNA portion of the second composite primer.

269. The kit of any of claims 263, 265, and 268, wherein the RNA portion of the second composite primer comprises at least about 5 nucleotides and the DNA portion of the second composite primer comprises of at least 1 nucleotide.

270. The kit of claim 269, wherein the RNA portion of the second composite primer consists of about 5 to about 50 nucleotides and the DNA portion of the second composite primer consists of about 1 to about 20 nucleotides.

271. The kit of claim 268, wherein the first composite primer is a tailed primer that comprises a 5′

portion that is not hybridizable to a target RNA under conditions wherein the first composite primer hybridizes to the target RNA.

272. The kit of claim 267, wherein the 3′

DNA portion of the first composite primer comprises at least 1 random nucleotide at the 3′

end.

273. The kit of claim 265, wherein the 3′

DNA portion of the first composite primer comprises a poly-dT sequence.

274. The kit of claim 273, wherein the 3′

DNA portion of the first composite primer comprises at least 1 random nucleotide at the 3′

end.

275. The kit of claim 263, further comprising an enzyme that cleaves RNA from an RNA/DNA hybrid.

276. The kit of claim 275, wherein the enzyme that cleaves RNA from an RNA/DNA hybrid is RNase H.

277. The kit of claim 263, further comprising an RNA-dependent DNA polymerase.

278. The kit of claim 263, further comprising a labeled dNTP.

279. The kit of claim 263, wherein the first composite primer consists of the RNA portion and a 3′

DNA portion.

280. The kit of claim 264, wherein the first composite primer consists of the 5′

RNA portion and a 3′

DNA portion.

281. The kit of claim 268, wherein the first composite primer consists of the 5′

RNA portion and a 3′

DNA portion.

282. The kit of claim 263, further comprising a set of instructions for carrying out a method comprising the steps of:

(a) producing a first primer extension product by extension of the first composite primer hybridized to a target RNA with at least one enzyme comprising RNA-dependent DNA polymerase activity;

(b) producing a second primer extension product by extension of a second primer hybridized to the first primer extension product;

(c) cleaving RNA from a complex of first and second primer extension products with at least one enzyme that cleaves RNA from an RNA/DNA hybrid; and

(d) hybridizing a second composite primer to the second primer extension product and extending the second composite primer with at least one enzyme comprising DNA-dependent DNA polymerase activity,

whereby said first primer extension product is displaced, RNA is cleaved from the second composite primer and another second composite primer hybridizes such that primer extension and strand displacement are repeated; and

whereby multiple copies of a polynucleotide sequence complementary to the RNA sequence of interest are generated.

283. The kit of claim 265, further comprising a set of instructions for carrying out a method comprising the steps of:

(a) producing a first primer extension product by extension of the first composite primer hybridized to a target RNA with at least one enzyme comprising RNA-dependent DNA polymerase activity;

(b) producing a second primer extension product by extension of second primer hybridized to the first primer extension product;

(c) cleaving RNA from a complex of first and second primer extension products with at least one enzyme that cleaves RNA from an RNA/DNA hybrid; and

(d) hybridizing the second composite primer to the second primer extension product and extending the second composite primer with at least one enzyme comprising DNA-dependent DNA polymerase activity,

whereby said first primer extension product is displaced, RNA is cleaved from the second composite primer and another second composite primer hybridizes such that primer extension and strand displacement are repeated; and

whereby multiple copies of a polynucleotide sequence complementary to the RNA sequence of interest are generated.

284. The kit of claim 264, wherein the first composite primer is a tailed primer that comprises a 5′

portion that is not hybridizable to the target RNA under conditions in which the first primer hybridizes to the target RNA.

285. The kit of claim 265, wherein the first composite primer comprises a random primer sequence.

325. The kit of claim 264, wherein the first composite primer is a tailed primer that comprises a 5′

portion that is not hybridizable to a target RNA under conditions wherein the first composite primer hybridizes to the target RNA.

326. The kit of claim 268, wherein the first composite primer is a tailed primer that comprises a 5′

portion that is not hybridizable to a target RNA under conditions wherein the first composite primer hybridizes to the target RNA.

330. The method of claim 263, wherein the second RNA population is a mRNA population.

337. The kit of any of claims 263, 265 and 268, wherein the RNA portion of the first composite primer comprises at least about 5 nucleotides and the DNA portion of the first composite primer comprises at least about 5 nucleotides.

338. The kit of claim 337, wherein the RNA portion of the first composite primer consists of about 5 to about 50 nucleotides and the DNA portion of the first composite primer consists of about 5 to about 20 nucleotides.

289. A method for generating multiple copies of a polynucleotide sequence complementary to an RNA sequence, said method comprising the steps of:

(a) synthesizing a polynucleotide strand by extending a first primer hybridized to a target RNA, wherein the first primer is a composite primer comprising an RNA portion and a 3′

DNA portion, and whereby a complex comprising the first polynucleotide strand and the target RNA is produced;

(b) synthesizing a second polynucleotide strand complementary to the first polynucleotide strand, whereby the second polynucleotide strand forms a complex with the first polynucleotide strand;

(c) cleaving RNA from the first composite primer in the complex of first polynucleotide strand and the second polynucleotide strand such that a composite amplification primer hybridizes to the second polynucleotide strand, wherein the composite amplification primer comprises an RNA portion and a 3′

DNA portion;

(d) extending the composite amplification primer hybridized to the second polynucleotide strand, such that said first polynucleotide strand is displaced, RNA is cleaved from the composite amplification primer and another composite amplification primer hybridizes such that primer extension and strand displacement are repeated, whereby multiple copies of a polynucleotide sequence complementary to the target RNA are generated.

290. The method of claim 289, wherein synthesis of the second polynucleotide strand is primed by a second primer comprising a fragment of the target RNA hybridized to the first polynucleotide product, said fragment generated by cleaving the target RNA in the complex of step (a) with an agent that cleaves RNA from an RNA/DNA hybrid.

291. The method of claim 290, wherein the agent is an enzyme.

292. The method of claim 291, wherein the enzyme is RNase H.

293. The method of claim 289, wherein the RNA portion of the first primer is 5′

with respect to the 3′

DNA portion.

294. The method of claim 293, wherein the 5′

RNA portion of the first primer is adjacent to the 3′

DNA portion.

295. The method of claim 292, 293 or 294, wherein the first primer is a tailed primer that comprises a 5′

portion that is not hybridizable to the target RNA under conditions in which the composite first primer hybridizes to the target RNA.

296. The method of claim 295, wherein the RNA portion of the composite amplification primer is 5′

with respect to the 3′

DNA portion.

297. The method of claim 296, wherein the RNA portion of the composite amplification primer is adjacent to the 3′

DNA portion.

298. The method of claim 289, 293 or 294, wherein the RNA portion of the composite amplification primer is 5′

with respect to the 3′

DNA portion.

299. The method of claim 298, wherein the RNA portion of the composite amplification primer is adjacent to the 3′

DNA portion.

300. The method of claim 289, wherein the first primer comprises a poly-dT sequence.

301. The method of claim 289 or 300, wherein the target RNA is an mRNA.

302. The method of claim 289, wherein the DNA portion of the first primer comprises a random primer sequence.

303. The method of claim 289, wherein the step of synthesizing the second polynucleotide strand uses at least one enzyme comprising a DNA-dependent DNA polymerase activity and a RNA-dependent DNA polymerase activity and, optionally, an activity that cleaves RNA from an RNA/DNA hybrid.

304. The method of claim 289, wherein the step of cleaving RNA from the first composite primer in the complex of first polynucleotide strand and the second polynucleotide strand uses at least one enzyme comprising an activity that cleaves RNA from an RNA/DNA hybrid.

305. The method of claim 289, wherein primer extension is carried out in the presence of at least one labeled dNTP, whereby labeled products are generated.

306. The method of claim 289, wherein said method comprises using two or more different target RNA sequences of interest and generating multiple copies of polynucleotide sequences complementary to the two or more different target RNA sequences of interest.

307. The method of claim 306, wherein said method comprises at least two different composite first primers that hybridize to target RNA wherein each primer comprises a 3′

portion comprising a random primer sequence.

308. The method of claim 306, wherein said method comprises at least two different composite first primers that hybridize to target RNA wherein each primer comprises a 3′

DNA portion comprising a poly-dT sequence.

323. A method according to claim 289, wherein the RNA portion of the first primer is 5′

with respect to the 3′

DNA portion of the first primer and the RNA portion of the composite amplification primer is 5′

with respect to the 3′

DNA portion of the composite amplification primer.

324. A method according to claim 323, wherein the 5′

RNA portion of the first primer is adjacent to the 3′

DNA portion of the first primer and the 5′

RNA portion of the composite amplification primer is adjacent to the 3′

DNA portion of the composite amplification primer.

329. The method of claim 49, 50, or 290, wherein the RNA portion of the composite amplification primer is 5′

with respect to the 3 DNA portion.

331. The method of claim 329, wherein the 5′

RNA portion of the composite amplification primer is adjacent to the 3′

DNA portion.

332. The method of claim 329, wherein the RNA portion of the composite amplification primer comprises at least about 5 nucleotides and the DNA portion of the composite amplification primer comprises at least 1 nucleotide.

336. The method of claim 332, wherein the RNA portion of the composite amplification primer consists of about 5 to about 50 nucleotides and the DNA portion of the composite amplification primer consists of about 1 to about 20 nucleotides.