Methods and compositions for transcription-based nucleic acid amplification
First Claim
1. A method of generating multiple copies of a nucleic acid sequence of interest, said method comprising the steps of:
- (a) hybridizing a single stranded target polynucleotide comprising the sequence of interest with a first primer;
(b) hybridizing a propromoter template switch oligonucleotide (TSO) comprising a propromoter sequence and a region that is hybridizable to a region of the target polynucleotide which is 5′
with respect to hybridization of the first primer to the target polynucleotide;
(c) extending the first primer with DNA polymerase such that a first primer extension product comprising a sequence complementary to the propromoter sequence of the propromoter TSO is produced, whereby a complex of first primer extension product, target polynucleotide and propromoter TSO is generated, wherein said complex comprises a double stranded promoter region;
(d) transcribing from the double stranded promoter region with a DNA-dependent RNA polymerase to generate a sense RNA transcript;
(e) hybridizing a second primer to the sense RNA transcript of step (d);
(f) extending the second primer with RNA-dependent DNA polymerase to generate a complex comprising a second primer extension product and an RNA transcript;
(g) cleaving RNA in the complex of step (f) with an enzyme that cleaves RNA in an RNA/DNA hybrid;
(h) hybridizing a single stranded second primer extension product with a propromoter polynucleotide, wherein the propromoter polynucleotide comprises a propromoter and a region which hybridizes to the single stranded second primer extension product under conditions which allow transcription to occur by RNA polymerase, such that sense RNA transcripts comprising the sequence of interest are produced;
whereby multiple copies of the nucleic acid sequence of interest are produced.
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Abstract
Methods for isothermal exponential amplification of a target polynucleotide are disclosed. The methods employ two transcription modules, the first module providing linear amplification resulting in RNA transcripts, and a second module providing for further (generally cyclical) amplification resulting in more RNA transcripts. In one aspect, the amplification of the first module is composite primer based. In a second aspect, the amplification of the first module is based on target switching to generate a primer extension product comprising a promoter sequence. In all aspects, the RNA transcripts of the first transcription module are subjected to further amplification by creating an intermediate product comprising a double stranded promoter region from which transcription can occur. The invention further provides compositions and kits for practicing said methods, as well as methods which use the amplification results.
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Citations
32 Claims
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1. A method of generating multiple copies of a nucleic acid sequence of interest, said method comprising the steps of:
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(a) hybridizing a single stranded target polynucleotide comprising the sequence of interest with a first primer; (b) hybridizing a propromoter template switch oligonucleotide (TSO) comprising a propromoter sequence and a region that is hybridizable to a region of the target polynucleotide which is 5′
with respect to hybridization of the first primer to the target polynucleotide;(c) extending the first primer with DNA polymerase such that a first primer extension product comprising a sequence complementary to the propromoter sequence of the propromoter TSO is produced, whereby a complex of first primer extension product, target polynucleotide and propromoter TSO is generated, wherein said complex comprises a double stranded promoter region; (d) transcribing from the double stranded promoter region with a DNA-dependent RNA polymerase to generate a sense RNA transcript; (e) hybridizing a second primer to the sense RNA transcript of step (d); (f) extending the second primer with RNA-dependent DNA polymerase to generate a complex comprising a second primer extension product and an RNA transcript; (g) cleaving RNA in the complex of step (f) with an enzyme that cleaves RNA in an RNA/DNA hybrid; (h) hybridizing a single stranded second primer extension product with a propromoter polynucleotide, wherein the propromoter polynucleotide comprises a propromoter and a region which hybridizes to the single stranded second primer extension product under conditions which allow transcription to occur by RNA polymerase, such that sense RNA transcripts comprising the sequence of interest are produced; whereby multiple copies of the nucleic acid sequence of interest are produced. - View Dependent Claims (2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)
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4. A method of generating multiple copies of a nucleic acid sequence of interest, said method comprising the steps of:
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(a) combining; a target polynucleotide; a first primer which is hybridizable to the target polynucleotide; a propromoter template switch oligonucleotide comprising a propromoter sequence and a region that is hybridizable to a region of the target polynucleotide which is 5′
with respect to hybridization of the first primer to the target polynucleotide;optionally a DNA-dependent DNA polymerase; an RNA polymerase; a second primer that is hybridizable to a sense RNA transcript comprising the sequence of interest; an RNA-dependent DNA polymerase; an enzyme that cleaves RNA from an RNA/DNA hybrid; and a propromoter polynucleotide comprising a propromoter and a region which hybridizes to a second primer extension product; and (b) incubating the mixture of step (a) under conditions that permit primer hybridization and extension, RNA cleavage, hybridization of a propromoter polynucleotide to a primer extension product to form a complex comprising a primer extension product and a propromoter polynucleotide, and RNA transcription, whereby multiple copies of the nucleic acid sequence of interest are generated.
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Specification