CA2416963A1 - Methods and compositions for amplification of rna sequences - Google Patents

Methods and compositions for amplification of rna sequences Download PDF

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Publication number
CA2416963A1
CA2416963A1 CA002416963A CA2416963A CA2416963A1 CA 2416963 A1 CA2416963 A1 CA 2416963A1 CA 002416963 A CA002416963 A CA 002416963A CA 2416963 A CA2416963 A CA 2416963A CA 2416963 A1 CA2416963 A1 CA 2416963A1
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rna
primer
dna
extension product
composite
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CA2416963C (en
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Nurith Kurn
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Nugen Technologies Inc
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Nugen Technologies, Inc.
Nurith Kurn
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates

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 (161)

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 an RNA-dependent DNA polymerase, 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 (b) with an enzyme that cleaves RNA
from an RNA/DNA hybrid;
(c) extending a second primer hybridized to the first primer extension product with a DNA-dependent DNA polymerase, whereby a second primer extension product is produced to form a complex of first and second primer extension products;
(d) cleaving RNA from the composite primer in the complex of first and second primer extension products with an enzyme that cleaves RNA from an RNA/DNA
hybrid such that a composite primer hybridizes to the second primer extension product, wherein the composite primer comprises an RNA portion and a 3' DNA portion;
(e) extending the composite primer hybridized to the second primer extension product with a DNA-dependent DNA polymerase;
whereby said first primer extension product is displaced, 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 primer extension product, said fragment generated by~

cleaving RNA in the complex of step (b) 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 composite primer that hybridizes to target RNA is 5' with respect to the 3' DNA portion.
5. The method of claim 4, wherein the 5' RNA portion is adjacent to the 3' DNA portion.
6. The method of claim 1, wherein the composite primer that hybridizes to target RNA comprises a 5' portion that is not hybridizable to the target RNA
under conditions which the composite primer hybridizes to the target RNA.
7. The method of claim 1, wherein the composite primer that hybridizes to target RNA comprises a poly-dT sequence.
8. The method of claim 7, wherein the target RNA is mRNA.
9. The method of claim 1, wherein the composite primer that hybridizes to target RNA comprises a random sequence.
10. The method of claim 1, wherein the target RNA is mRNA, and the composite primer that hybridizes to target RNA comprises a poly-dT sequence, and further comprises a 5' portion that is not hybridizable to the target mRNA under conditions which the composite primer hybridizes to the target mRNA.
11. The method of claim 1, wherein a plurality of different composite 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 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 enzyme that cleaves RNA from an RNA/DNA hybrid is RNase H.
15. The method of claim 1, wherein the RNA -dependent DNA polymerase and DNA-dependent DNA polymerase are the same enzyme.
16. The method of claim 1, wherein the RNA-dependent DNA polymerase and the enzyme that cleaves RNA from an RNA/DNA hybrid are the same enzyme.
17. The method of claim 1, wherein the DNA-dependent DNA polymerase and the enzyme that cleaves RNA from an RNA/DNA hybrid are the same enzyme.
18. The method of claim 1, wherein the DNA-dependent DNA polymerase, the RNA-dependent DNA polymerase and the enzyme that cleaves RNA from an RNA/DNA
hybrid are the same enzyme.
19. The method of claim 1, wherein at least one type of dNTP used is a labeled dNTP, whereby labeled products are generated.
20. The method of claim 1, wherein the composite primer that hybridizes to target RNA and the composite primer that hybridizes to second primer extension product are the same.
21. The method of claim 1, wherein the composite primer that hybridizes to target RNA and the composite primer that hybridizes to second primer extension product are different.
22. The method of claim 1, wherein said method comprises generating multiple copies of a polynucleotide sequence complementary to two or more different sequences of interest.
23. The method of claim 22, wherein said method comprises at least two different composite primers that hybridize to target RNA.
24. The method of claim 22 or 23, wherein the composite primer that hybridizes to target RNA comprises a poly-dT portion.
25. The method of claim 22 or 23, wherein the composite primer that hybridizes to target RNA is a random primer.
26. 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 an RNA-dependent DNA polymerase, 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 (b) with an enzyme that cleaves RNA
from an RNA/DNA hybrid;
(c) extending a second primer hybridized to the first primer extension product with a DNA-dependent DNA polymerase, whereby a second primer extension product is produced to form a complex of first and second primer extension products;
(d) cleaving RNA from the composite primer in the complex of first and second primer extension products with an enzyme that cleaves RNA from an RNA/DNA
hybrid such that a composite primer hybridizes to the second primer extension product, wherein the composite primer comprises an RNA portion and a 3' DNA portion;

(e) extending said composite primer hybridized to the second primer extension product with a DNA-dependent DNA polymerase, whereby said first primer extension product is displaced;
(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 displaced first primer extension product, whereby multiple copies of the RNA sequence of interest are generated.
27. The method of claim 26, 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 (b) with an enzyme that cleaves RNA from an RNA/DNA hybrid.
28. The method of claim 26, wherein the second primer comprises DNA.
29. The method of claim 26, wherein the RNA portion of the composite primer that hybridizes to target RNA is 5' with respect to the 3' DNA portion.
30. The method of claim 29, wherein the 5' RNA portion is adjacent to the 3' DNA portion.
31. The method of claim 26, wherein the composite primer that hybridizes to target RNA comprises a 5' portion that is not hybridizable to the target RNA
under conditions which the composite primer hybridizes to the target RNA.
32. The method of claim 26, wherein the composite primer that hybridizes to target RNA comprises a poly-dT sequence.
33. The method of claim 26, wherein the target RNA is mRNA.

gccttcgacg aagccggtcc ggacgcagcg
34. The method of claim 26, wherein the composite primer that hybridizes to target RNA comprises a random sequence.
35. The method of claim 26, wherein the target RNA is mRNA, and the composite primer that hybridizes to target RNA comprises a poly-dT sequence, and further comprises a 5' portion that is not hybridizable to the target mRNA under conditions which the composite primer hybridizes to the target mRNA.
36. The method of claim 26, wherein a plurality of different composite primers are used for hybridizing to the target RNA.
37. The method of claim 26, wherein the second primer is a random primer.
38. The method of claim 26, wherein the second primer comprises a 5' portion that is not hybridizable to the first primer extension product under conditions which the second primer hybridizes to the fist primer extension product.
39. The method of claim 26, wherein the enzyme that cleaves RNA from an RNA/DNA hybrid is RNase H.
40. The method of claim 26, wherein the RNA-dependent DNA polymerase and DNA-dependent DNA polymerase are the same enzyme.
41. The method of claim 26, wherein the RNA-dependent DNA polymerase and the enzyme that cleaves RNA from an RNA/DNA hybrid are the same enzyme.
42. The method of claim 26, wherein the DNA-dependent DNA polymerase and the enzyme that cleaves RNA from an RNA/DNA hybrid are the same enzyme.
43. The method of claim 26, wherein the DNA-dependent DNA polymerase, the RNA-dependent DNA polymerase and the enzyme that cleaves RNA from an RNA/DNA
hybrid are the same enzyme.
44. The method of claim 26, wherein at least one type of dNTP used is a labeled dNTP, whereby labeled products are generated.
45. The method of claim 26, wherein the composite primer that hybridizes to target RNA and the composite primer that hybridizes to second primer extension product are the same.
46. The method of claim 26, wherein the composite primer that hybridizes to target RNA and the composite primer that hybridizes to second primer extension product are different.
47. The method of claim 26, 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.
48. The method of claim 26, wherein the propromoter polynucleotide is a propromoter template oligonucleotide (PTO).
49. The method of claim 26, wherein at least one type of rNTP used is a labeled rNTP, whereby labeled products are generated.
50. The method of claim 26, wherein said method comprises generating multiple copies of two or more different sequences of interest.
51. The method of claim 50, wherein said method comprises at least two different composite primers that hybridize to target RNA.
52. The method of claim 50 or 51, wherein the composite primer that hybridizes to target RNA comprises a poly-dT portion.
53. The method of claim 50 or 51, wherein the composite primer that hybridizes to target RNA is a random primer.
54. A method of amplifying an RNA sequence of interest, comprising incubating a reaction mixture, said reaction mixture comprising:
(a) the complex of first and second primer extension products of step (c) of claim 1;
(b) a composite primer that is hybridizable to the second primer extension product, wherein the composite primer comprises an RNA portion and a 3' DNA portion;
(c) a DNA-dependent DNA polymerase; and (d) an enzyme that cleaves RNA from an RNA/DNA hybrid;
wherein the incubation is under conditions that permit primer hybridization, RNA
cleavage, and displacement of the first primer extension product from the complex of step (c) of claim 1 when its RNA is cleaved and a composite primer binds to the second primer extension product in the complex and is extended, whereby multiple copies of a polynucleotide sequence complementary to the RNA sequence of interest are generated.
55. The method of claim 54, wherein the RNA portion of the composite primer that hybridizes to target RNA is 5' with respect to the 3' DNA portion, and the 5' RNA
portion is adjacent to the 3' DNA portion.
56. The method of claim 54, wherein the target RNA is mRNA.
57. The method of claim 54, wherein at least one type of dNTP used is a labeled dNTP, wherein labeled products are generated.
58. A method of amplifying an RNA sequence of interest comprising incubating a reaction mixture, said reaction mixture comprising:

(a) a first complex, wherein the first complex is the complex of first and second primer extension products of step (c) of claim 1;
(b) a composite primer that is hybridizable to the second primer extension product, wherein the composite primer comprises an RNA portion and a 3' DNA portion;
(c) a DNA-dependent DNA polymerase;
(d) an RNA polymerase;
(e) a propromoter polynucleotide comprising a propromoter and a region which hybridizes to a second primer extension product; and (f) an enzyme that cleaves RNA from an RNA/DNA hybrid;
wherein the incubation is under conditions that permit primer hybridization, RNA
cleavage, displacement of the first primer extension product from the first complex when its RNA is cleaved and a composite primer binds to the second primer extension product in the first complex, hybridization of the propromoter polynucleotide to the displaced first primer extension product to form a second complex comprising the displaced primer extension product and the propromoter polynucleotide, and RNA transcription from said second complex, whereby multiple copies of the RNA sequence of interest are generated.
59. The method of claim 58, wherein the RNA portion of the composite primer that hybridizes to target RNA is 5' with respect to the 3' DNA portion, and the 5' RNA
portion is adjacent to the 3' DNA portion.
60. The method of claim 58, wherein the enzyme that cleaves RNA from an RNA/DNA hybrid is RNase H.
61. The method of claim 58, 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.
62. The method of claim 58, wherein at least one type of rNTP used is a labeled rNTP, whereby labeled products are generated.
63. The method of claim 58, wherein the target RNA is mRNA.
64. A method of generating multiple copies of (amplifying) 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;
(iii) a RNA-dependent DNA polymerase; and (iv) an enzyme capable of cleaving RNA from an RNA/DNA hybrid;
wherein the incubation is under conditions that permit primer hybridization, formation of a complex comprising a first primer extension product and the target RNA
and cleavage of RNA in the 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) a DNA-dependent DNA polymerase; and (iii) optionally, an 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, and optionally, cleavage of RNA in the complex comprising a first primer extension product and the target RNA;
(c) incubating a reaction mixture, said reaction mixture comprising:
(i) the complex comprising the first primer extension product and a second primer extension product;
(ii) an enzyme capable of cleaving RNA from an RNA/DNA hybrid;

(iii) a composite primer, wherein the composite primer comprises a RNA
portion and a 3' DNA portion;
(iv) the cleaved complex comprising the first primer extension product and a second primer extension product; and (v) DNA-dependent DNA polymerase, wherein the incubation is under conditions that permit cleavage of RNA in the complex comprising the first primer extension product and a second primer extension product, composite primer hybridization, and displacement of the first primer extension product from the complex comprising the first primer extension product and a second primer extension product;
whereby multiple copies of a polynucleotide sequence complementary to the RNA
sequence of interest are generated.
65. The method of claim 64, wherein step (b) includes incubating a reaction mixture further comprising: (iv) a second primer; and wherein the incubation is under conditions permitting hybridization of the second primer.
66. The method of claim 65, wherein the second primer comprises DNA.
67. The method of claim 64, wherein the RNA portion of the composite primer that hybridizes to target RNA is 5' with respect to the 3' DNA portion.
68. The method of claim 67, wherein the 5' RNA portion is adjacent to the 3' DNA portion.
69. The method of claim 64, wherein the composite primer that hybridizes to target RNA comprises a 5' portion that is not hybridizable to the target RNA
under conditions which the composite primer hybridizes to the target RNA.
70. The method of claim 64, wherein the composite primer that hybridizes to target RNA comprises a poly-dT sequence.
71. The method of claim 64, wherein the target RNA is mRNA.
72. The method of claim 64, wherein the composite primer that hybridizes to target RNA comprises a random sequence.
73. The method of claim 64, wherein the target RNA is mRNA, and the composite primer that hybridizes to target RNA comprises a poly-dT sequence, and further comprises a 5' portion that is not hybridizable to the target mRNA under conditions which the composite primer hybridizes to the target mRNA.
74. The method of claim 64, wherein a plurality of different composite primers are used for hybridizing to the target RNA.
75. The method of claim 64, wherein the second primer is a random primer.
76. The method of claim 64, wherein the second primer 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.
77. The method of claim 64, wherein the enzyme that cleaves RNA from an RNA/DNA hybrid is RNase H.
78. The method of claim 64, wherein the RNA -dependent DNA polymerase and DNA-dependent DNA polymerase are the same enzyme.
79. The method of claim 64, wherein the RNA-dependent DNA polymerase and the enzyme that cleaves RNA from an RNA/DNA hybrid are the same enzyme.
80. The method of claim 64, wherein the DNA-dependent DNA polymerase and the enzyme that cleaves RNA from an RNA/DNA hybrid are the same enzyme.
81. The method of claim 64, wherein the DNA-dependent DNA polymerase, the RNA-dependent DNA polymerase and the enzyme that cleaves RNA from an RNA/DNA
hybrid are the same enzyme.
82. The method of claim 64, wherein at least one type of dNTP used is a labeled dNTP, whereby labeled products are generated.
83. The method of claim 64, wherein the composite primer that hybridizes to target RNA and the composite primer that hybridizes to second primer extension product are the same.
84. The method of claim 64, wherein the composite primer that hybridizes to target RNA and the composite primer that hybridizes to second primer extension product are different.
85. The method of claim 64, wherein said method comprises generating multiple copies of a polynucleotide sequence complementary to two or more different sequences of interest.
86. The method of claim 85, wherein said method comprises at least two different composite primers that hybridize to target RNA.
87. The method of claim 85 or 86, wherein the composite primer that hybridizes to target RNA comprises a poly-dT portion.
88. The method of claim 85 or 86, wherein the composite primer that hybridizes to target RNA is a random primer.
89. 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 an enzyme that cleaves RNA from an RNA/DNA hybrid such that a composite primer hybridizes to the second primer extension product, wherein the composite primer comprises an RNA portion and a 3' DNA portion, wherein the first primer extension product is produced by extension of a first primer hybridized to a target RNA
with a RNA-dependent DNA polymerase, wherein the first primer is a composite primer comprising an RNA portion and a 3' DNA portion;

(b) hybridizing a composite primer to the second primer extension product and extending the composite primer with a DNA-dependent DNA polymerase;
whereby said first primer extension product is displaced, 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 composite primer that hybridizes to target RNA is 5' with respect to the 3' DNA portion.
92. The method of claim 91, wherein the 5' RNA portion is adjacent to the 3' DNA portion.
93. The method of claim 89, wherein the composite primer that hybridizes to target RNA comprises a 5' portion that is not hybridizable to the target RNA
under conditions which the composite primer hybridizes to the target RNA.
94. The method of claim 89, wherein the composite primer that hybridizes to target RNA comprises a poly-dT sequence.
95. The method of claim 94, wherein the target RNA is mRNA.
96. 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 primer in a complex comprising:
(i) a complex of a 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 a RNA-dependent DNA polymerase, 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 an enzyme that cleaves RNA from an RNA/DNA hybrid; and (ii) a composite primer, said composite primer comprising an RNA portion and a 3' DNA portion, wherein the composite primer is hybridized to the second primer extension product, and wherein the composite primer may be the same or different from the first primer;
whereby said first primer extension product is displaced, and whereby multiple copies of a polynucleotide sequence complementary to the RNA sequence of interest are generated.
97. The method of claim 96, wherein the second primer comprises DNA.
98. The method of claim 96, wherein the RNA portion of the composite primer that hybridizes to target RNA is 5' with respect to the 3' DNA portion.
99. The method of claim 98, wherein the 5' RNA portion is adjacent to the 3' DNA portion.
100. The method of claim 96, wherein the composite primer that hybridizes to target RNA comprises a 5' portion that is not hybridizable to the target RNA
under conditions which the composite primer hybridizes to the target RNA.
101. The method of claim 96, wherein the composite primer that hybridizes to target RNA comprises a poly-dT sequence.
102. The method of claim 101, wherein the target RNA is mRNA.
103. A method of sequencing an RNA sequence of interest, said method comprising (a) amplifying a target RNA containing the sequence of interest by any of the methods of claims 1, 54, 64, 89, and 96 in the presence of a mixture of dNTPs and dNTP
analogs such that primer extension is terminated upon incorporation of a dNTP
analog; and (b) analyzing the amplification products to determine sequence.
104. The method of claim 103, wherein the target RNA is mRNA.
105. A method of sequencing an RNA sequence of interest, said method comprising (a) amplifying a target RNA containing the sequence of interest by any of the methods of claims 26 and 58 in the presence of a mixture of rNTPs and rNTP
analogs such that transcription is terminated upon incorporation of a rNTP analog; and (b) analyzing the amplification products to determine sequence.
106. The method of claim 105, wherein the target RNA is mRNA.
107. 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, 26, 54, 58, 64, 89, and 96; 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.
108. The method of claim 107, wherein the target RNA is mRNA.
109. A method of determining presence or absence of a sequence of interest, said method comprising (i) amplifying a target RNA containing the sequence of interest, said amplifying comprising extending a composite primer hybridized to the complex of step (d) of claim 1, wherein the sequence of the RNA portion of the composite primer is known, and (ii) comparing the amplification products if any from step (i) with the amount of amplification products from a reference template wherein (1) production of detestably fewer amplification products from the template as compared to the amount of amplification products from the reference template which comprises a region hybridizable to the RNA portion of the composite primer indicates that the second primer extension product does not comprise a sequence hybridizable to the RNA portion of the composite primer and is a sequence variant with respect to the sequence hybridizable to the RNA portion of the composite primer; or (2) production of detectably more amplification products from the template as compared to the amount of amplification products from the reference template which does not comprise a region which is hybridizable to the RNA portion of the composite primer indicates that the second primer extension product comprises a sequence hybridizable to the RNA portion of the composite primer and is not a sequence variant with respect to the sequence hybridizable to the RNA portion of the composite primer.
110. The method of claim 109, wherein the target RNA is mRNA.
111. A method of producing a nucleic acid immobilized to a substrate, comprising (a) amplifying a target RNA by any of the methods of claims 1, 26, 54, 58, 64, 89, and 96; and (b) immobilizing the amplification products on a substrate.
112. The method of claim 111, wherein the target RNA is mRNA.
113. The method of claim 111, wherein the substrate is a microarray.
114. A method of characterizing an RNA sequence of interest, comprising (a) amplifying a target RNA by any of the methods of claims 1, 54, 64, 89, and 96;
and (b) analyzing the DNA products.
115. The method of claim 114, wherein the target RNA is mRNA.
116. The method of claim 114, wherein the DNA products are labeled.
117. The method of claim 114, wherein step (b) of analyzing the DNA products comprises determining amount of said products, whereby the amount of the RNA
sequence of interest present in a sample is quantified.
118. The method of claim 114, wherein step (b) comprises contacting the DNA
products with at least one probe.
119. The method of claim 118, wherein the DNA products are labeled, and wherein the at least one probe is provided as a microarray.
120. The method of claim 119, 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.
121. The method of claim 120, 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.
122. A method of characterizing an RNA sequence of interest, comprising (a) amplifying a target RNA by the methods of any of claims 26 and 58; and (b) analyzing the RNA products.
123. The method of claim 122, wherein the target RNA is mRNA.
124. The method of claim 122, wherein the RNA products are labeled.
125. The method of claim 122, wherein step (b) of analyzing the RNA products comprises determining amount of said products, whereby the amount of the RNA
sequence of interest present in a sample is quantified.
126. The method of claim 122, wherein step (b) comprises contacting the labeled RNA products with at least one probe.
127. The method of claim 122, wherein the RNA products are labeled, and wherein the at least one probe is provided as a microarray.
128. The method of claim 127, 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.
129. The method of claim 128, 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.
130. A method of determining gene expression profile in a sample, said method comprising: (a) amplifying at least one RNA sequence of interest in the sample using the method of any of claims 1, 54, 64, 89, and 96; and (b) determining amount of amplification products of each RNA sequence of interest, wherein each said amount is indicative of amount of each RNA sequence of interest in the sample, whereby the gene expression profile in the sample is determined.
131. The method of claim 130, wherein each target RNA is mRNA.
132. A method of preparing a library, said method comprising: amplifying at least one RNA sequences of interest using the method of any of claims 1, 26, 54, 58, 64, 89, and 96.
133. The method of claim 132, wherein the first primer that hybridizes to target RNA is a random primer
134. The method of claim 132, wherein the first primer that hybridizes to target RNA comprises a poly-dT portion.
135. A method of preparing a subtractive hybridization probe, said method comprising generating multiple DNA 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, 54, 64, 89, and 96.
136. A method of performing subtractive hybridization, said method comprising:
(a) generating multiple DNA 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, 54, 64, 89, and 96; and (b) hybridizing the multiple copies to a second mRNA population, whereby a subpopulation of the second mRNA population forms a complex with a DNA copy.
137. The method of claim 136, said method further comprising: (c) cleaving RNA in the complex of step (b) with an enzyme that cleaves RNA from an RNA/DNA
hybrid; and (d) amplifying an unhybridized subpopulation of the second mRNA
population, whereby multiple copies of single stranded DNA complementary to the unhybridized subpopulation of the second mRNA population are generated.
138. A method for differential amplification of one or more RNA sequence of interest, said method comprising:
(a) hybridizing multiple polynucleotide copies of the complement of one or more RNA sequence of interest from a first RNA population using the methods of any of claims 1, 54, 64, 89, and 96 to a second mRNA population, whereby a subpopulation of the second mRNA population hybridizes to the polynucleotide copies to form complexes;
(b) cleaving RNA in the complexes of step (b) with an enzyme that cleaves RNA
from an RNA/DNA hybrid; and (c) amplifying an unhybridized subpopulation of the second mRNA population, whereby multiple copies of single stranded DNA complementary to the unhybridized subpopulation of the second mRNA population are generated.
139. A method for making a cDNA library, said method comprising: preparing a subtractive hybridization probe according to claim 84.
140. A composition comprising a composite primer, wherein the composite primer comprises an RNA portion and a 3' DNA portion, and a second primer.
141. The composition of claim 140, wherein the second primer comprises DNA.
142. The composition of claim 141, wherein the second primer is a random primer.
143. The composition of claim 140, wherein the second primer comprises a fragment of the target RNA hybridized to the primer extension product.
144. The composition of claim 140, further comprising an RNA-dependent DNA
polymerase.
145. The composition of claim 140, wherein the composite primer further comprises a 5' region that is not hybridizable to the RNA sequence of interest under conditions which the composite primer hybridizes to the target RNA.
146. A composition comprising a composite primer and a second primer that comprises a sequence that is not hybridizable to a first primer extension product under conditions which the composite primer hybridizes to the target RNA.
147. A composition comprising: (a) a composite primer; (b) a second primer;
and (c) a propromoter polynucleotide.
148. The composition of claim 147, wherein the second primer is a random primer.
149. A composition comprising a complex of (a) a first primer extension product, wherein the first primer is a composite primer comprising an RNA portion and a 3' DNA
portion; and (b) a propromoter polynucleotide.
150. A composition comprising a complex of (a) a first primer extension product, wherein the first primer is a composite primer comprising an RNA portion and a 3' DNA
portion; and (b) a second primer extension product, wherein the second primer comprises DNA.
151. A composition comprising a complex of (a) a first primer extension product, wherein the first primer is a composite primer comprising an RNA portion and a 3' DNA

portion; and (b) a second primer extension product, wherein the second primer comprises a fragment of the RNA target.
152. A composition comprising a complex of (a) a cleaved primer extension product, wherein the primer is a composite primer comprising an RNA portion and a 3' DNA portion; (b) a second primer extension product; and (c) a composite primer that hybridizes to second primer extension product.
153. The method of claim 152, wherein the composite primer that hybridizes to target RNA and the composite primer that hybridizes to second primer extension product are the same.
154. The method of claim 152, wherein the composite primer that hybridizes to target RNA and the composite primer that hybridizes to second primer extension product are different.
155. A reaction mixture comprising (a) a target RNA; (b) a composite primer comprising a 3' DNA portion and an RNA portion; (c) a second primer; and (d) a DNA
polymerase.
156. The reaction mixture of claim 155, further comprising: (e) an enzyme which cleaves RNA from an RNA/DNA hybrid.
157. The reaction mixture of claim 155, further comprising: (e) a propromoter polynucleotide.
158. A kit for amplifying a target RNA, comprising: (a) a composite primer comprising a 3' DNA portion and an RNA portion; (b) a second primer; and (c) instructions for amplifying RNA according to any of methods 1, 26, 54, 58, 64, 89, and 96.
159. The kit of claim 158, further comprising: (d) a propromoter polynucleotide.
160. The kit of claim 157 or 158, further comprising an enzyme which cleaves RNA from an RNA/DNA hybrid.
161. A method of making a complex of first and second primer extension products comprising a 3' single stranded portion comprising:
(a) extending a first primer hybridized to a target RNA with an RNA-dependent DNA polymerase, 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 (b) with an enzyme that cleaves RNA
from an RNA/DNA hybrid;
(c) extending a second primer hybridized to the first primer extension product with a DNA-dependent DNA polymerase, whereby a second primer extension product is produced to form a complex of first and second primer extension products;
(d) cleaving RNA from the composite primer in the complex of first and second primer extension products with an enzyme that cleaves RNA from an RNA/DNA
hybrid;
whereby a complex of first and second primer extension products comprising a 3' single stranded portion is generated;
whereby the 3' single stranded portion is complementary to the RNA portion of the composite primer.
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