Methods and compositions for amplification of RNA sequences
First Claim
1. A method of generating multiple copies of the complementary sequence 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, 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 complex comprising the first primer extension product and a second primer extension product is produced;
(d) denaturing the complex of step (c); and
(e) hybridizing to the second primer extension product a propromoter polynucleotide comprising a propromoter and a region which hybridizes to the second primer extension product under conditions which allow transcription to occur by RNA polymerase, such that RNA transcripts are produced comprising sequences complementary to the target RNA;
whereby multiple copies of the complementary sequence of the RNA sequence of interest are generated.
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Abstract
The invention provides methods for linear and exponential amplification of RNA. They are particularly suitable for amplifying a plurality of RNA species in a sample. The methods are based on hybridization of polynucleotide comprising a propromoter sequence to a primer extension product to generate an intermediate polynucleotide capable of driving transcription, whereby multiple copies of RNA products comprising sequences complementary to an RNA sequence of interest are generated. 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.
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Citations
84 Claims
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1. A method of generating multiple copies of the complementary sequence of an RNA sequence of interest, said method comprising the steps of:
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(a) extending a first primer hybridized to a target RNA with an RNA-dependent DNA polymerase, 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 complex comprising the first primer extension product and a second primer extension product is produced;
(d) denaturing the complex of step (c); and
(e) hybridizing to the second primer extension product a propromoter polynucleotide comprising a propromoter and a region which hybridizes to the second primer extension product under conditions which allow transcription to occur by RNA polymerase, such that RNA transcripts are produced comprising sequences complementary to the target RNA;
whereby multiple copies of the complementary sequence of the RNA sequence of interest are generated. - View Dependent Claims (3, 4, 5, 6, 11, 12, 13, 14, 15, 16, 17, 18, 19, 28, 29, 30, 31, 32, 33, 34, 36, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 55, 56, 57, 58, 59, 60, 61, 76, 77, 78, 79, 80, 81, 82, 83, 84)
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2. A method of generating multiple copies of the complementary sequence of an RNA sequence of interest, said method comprising the steps of:
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(a) extending a first primer hybridized to a target RNA with an RNA-dependent DNA polymerase, 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 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 complex comprising the first primer extension product and a second primer extension product is produced;
(d) denaturing the complex of step (c);
(e) hybridizing to the second primer extension product a propromoter polynucleotide comprising a propromoter and a region which hybridizes to the second primer extension product under conditions which allow transcription to occur by RNA polymerase, such that RNA transcripts are produced comprising sequences complementary to the target RNA;
(f) extending a third primer hybridized to said RNA transcripts with an RNA-dependent DNA polymerase, whereby a complex comprising a third primer extension product and an RNA transcript is produced;
(g) cleaving RNA in the complex of step (f) with an enzyme that cleaves RNA from an RNA/DNA hybrid;
(h) hybridizing a propromoter polynucleotide comprising a propromoter and a region which hybridizes to a single stranded third primer extension product under conditions which allow transcription to occur by RNA polymerase, such that RNA transcripts are produced comprising sequences complementary to the target RNA;
(i) optionally repeating steps (f) to (h);
whereby multiple copies of the complementary sequence of the RNA sequence of interest are produced. - View Dependent Claims (7, 8, 9, 10, 35, 53, 54, 62, 63, 64, 65, 66, 67, 68, 70, 71, 72, 73, 74, 75)
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20. A method of generating multiple copies of the complementary sequence of an RNA sequence of interest, said method comprising incubating a reaction mixture, said reaction mixture comprising:
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(a) a target RNA;
(b) a first primer comprising a sequence that is hybridizable to the target RNA;
(c) a second primer comprising a sequence hybridizable to an extension product of the first primer;
(d) a propromoter polynucleotide comprising a propromoter and a region which is hybridizable to a single stranded second primer extension product;
(e) an RNA-dependent DNA polymerase;
(f) a DNA-dependent DNA polymerase;
(g) an RNA polymerase; and
(h) an enzyme that cleaves RNA from an RNA/DNA hybrid;
wherein the incubation is under conditions that permit primer hybridization, primer extension, RNA cleavage, propromoter polynucleotide hybridization, and RNA transcription, whereby multiple copies of the complementary sequence of the RNA sequence of interest are generated. - View Dependent Claims (22, 23, 24, 37, 69)
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21. A method of generating multiple copies of the complementary sequence of an RNA sequence of interest, said method comprising incubating a reaction mixture, said reaction mixture comprising:
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(a) a target RNA;
(b) a first primer comprising a sequence that is hybridizable to the target RNA;
(c) a second primer comprising a sequence hybridizable to an extension product of the first primer;
(d) a third primer comprising a sequence hybridizable to an RNA transcript comprising a sequence complementary to the target RNA;
(e) a propromoter polynucleotide comprising a propromoter and a region which is hybridizable to a single stranded second primer extension product;
(f) a propromoter polynucleotide comprising a propromoter and a region which is hybridizable to a single stranded third primer extension product;
(g) an RNA-dependent DNA polymerase;
(h) a DNA-dependent DNA polymerase;
(i) an RNA polymerase; and
(j) an enzyme that cleaves RNA from an RNA/DNA hybrid;
wherein the incubation is conditions that permit primer hybridization, primer extension, RNA cleavage, propromoter polynucleotide hybridization, and RNA transcription, whereby multiple copies of the complementary sequence of the RNA sequence of interest are generated. - View Dependent Claims (25, 26, 27)
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Specification