Nucleic acid sequence amplification method, composition and kit
First Claim
1. A method of amplifying a target ribonucleic acid sequence comprising the following steps:
- a) incubating a mixture comprising;
a target nucleic acid comprising said target ribonucleic acid sequence,one or more promoter-primers comprising a single nucleic acid sequence comprising a promoter recognizable by an RNA polymerase and a primer located 3'"'"' relative to said promoter, said primer being sufficiently complementary to said target nucleic acid to form a promoter-primer;
target nucleic acid complex at or near the 3'"'"'-end of said target ribonucleic acid sequence, and able to be extended to form a complement of said target ribonucleic acid sequence by a DNA polymerase,said DNA polymerase, and said RNA polymerase,at a temperature and in a solution effective to allow amplification of said target ribonucleic acid sequence, said mixture lacking a primer which forms a hybrid with said complement of said target ribonucleic acid sequence; and
b) producing multiple copies of an RNA sequence complementary to said target ribonucleic acid sequence using said target ribonucleic acid sequence as a template.
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Accused Products
Abstract
A method, composition and kit for amplifying a target nucleic acid sequence under conditions of substantially constant temperature, ionic strength, and pH and using only a single promoter-primer. To effect the amplification, a supply of a single promoter-primer having a promoter and a primer complementary to the 3'"'"'-end of the target sequence, and a reverse transcriptase and an RNA polymerase are provided to a mixture including the target sequence; the amplification proceeds accordingly. The invention is useful for generating copies of a nucleic acid target sequence for purposes that include assays to quantitate specific nucleic acid sequences in clinical, environmental, forensic and similar samples, cloning and generating probes.
379 Citations
41 Claims
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1. A method of amplifying a target ribonucleic acid sequence comprising the following steps:
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a) incubating a mixture comprising; a target nucleic acid comprising said target ribonucleic acid sequence, one or more promoter-primers comprising a single nucleic acid sequence comprising a promoter recognizable by an RNA polymerase and a primer located 3'"'"' relative to said promoter, said primer being sufficiently complementary to said target nucleic acid to form a promoter-primer;
target nucleic acid complex at or near the 3'"'"'-end of said target ribonucleic acid sequence, and able to be extended to form a complement of said target ribonucleic acid sequence by a DNA polymerase,said DNA polymerase, and said RNA polymerase, at a temperature and in a solution effective to allow amplification of said target ribonucleic acid sequence, said mixture lacking a primer which forms a hybrid with said complement of said target ribonucleic acid sequence; and b) producing multiple copies of an RNA sequence complementary to said target ribonucleic acid sequence using said target ribonucleic acid sequence as a template. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 29, 30)
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15. A method of amplifying a target ribonucleic acid sequence comprising the steps of:
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a) incubating a mixture consisting essentially of;
a target nucleic acid comprising said target ribonucleic acid sequence,a supply of promoter-primers comprising a single nucleic acid sequence comprising a promoter recognizable by an RNA polymerase and a primer located 3'"'"' relative to said promoter, said primer being sufficiently complementary to said target ribonucleic acid to form a promoter-primer;
target nucleic acid complex at or near the 3'"'"'-end of said target ribonucleic acid sequence, said supply comprising one or more modified promoter-primers and one or more unmodified promoter-primers, wherein the ratio of said one or more modified promoter-primers to said one or more unmodified promoter-primers is effective to produce greater amplification compared to said one or more modified promoter-primers or said one or more unmodified promoter primers alone,a reverse transcriptase, and said RNA polymerase, at a temperature and in a solution effective to allow amplification of said target ribonucleic acid sequence, said incubating comprising essentially constant temperature during said amplification; and b) producing multiple copies of an RNA sequence complementary to said target ribonucleic sequence using said target ribonucleic acid sequence as a template. - View Dependent Claims (16, 17, 18, 19, 20)
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21. A method for amplifying a target ribonucleic acid sequence, comprising the steps of:
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a) contacting said target ribonucleic acid sequence with a plurality of promoter-primers comprising a single nucleic acid sequence comprising a promoter recognizable by an RNA polymerase and a primer located 3'"'"' relative to said promoter, said primer being able to complex at or near a 3'"'"'-end of said target ribonucleic acid sequence, and wherein one or more of said plurality of promoter-primers is an unmodified promoter-primer and one or more of said plurality of promoter-primers is a modified promoter-primer comprising a modified nucleotide at its 3'"'"'-end to prevent or decrease a nucleic acid extension reaction from proceeding therefrom, under conditions effective to allow said amplifying; and b) producing multiple copies of an RNA sequence complementary to said target ribonucleic acid sequence.
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22. A composition for amplifying a target ribonucleic acid sequence using said target ribonucleic acid sequence as a template comprising:
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said target ribonucleic acid sequence, one or more promoter-primers comprising a single nucleic acid sequence comprising a promoter recognizable by an RNA polymerase and a primer located 3'"'"' relative to said promoter, said primer being sufficiently complementary to said target ribonucleic acid sequence to form a complex at or near the 3'"'"'-end of said target ribonucleic acid sequence, a reverse transcriptase, said RNA polymerase, and a solution of reagents able to allow amplification of said target ribonucleic acid sequence at essentially constant temperature;
wherein a primer able to hybridize to a nucleic acid sequence complementary to said target sequence is not present. - View Dependent Claims (23, 25, 26, 27)
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24. The composition of 22 wherein said target ribonucleic acid sequence is present on RNA which comprises nucleotides located 3'"'"' of said complex.
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28. Nucleic acid consisting of a sequence chosen from the group consisting of:
- SEQ ID No 6, SEQ ID No 8, and SEQ ID No 9.
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31. A method of amplifying a target ribonucleic acid sequence comprising the following steps:
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a) incubating a mixture comprising; a target nucleic acid comprising said target ribonucleic acid sequence, one or more promoter-primers comprising a single nucleic acid sequence comprising a promoter recognizable by an RNA polymerase and a primer located 3'"'"' relative to said promoter, said primer being sufficiently complementary to said target nucleic acid to form a promoter-primer;
target nucleic acid complex at or near the 3'"'"'-end of said target ribonucleic acid sequence,a DNA polymerase, and said RNA polymerase, at a temperature and in a solution effective to allow amplification of said target ribonucleic acid sequence, wherein said mixture lacks a primer which forms a hybrid with said complement of said target ribonucleic acid sequence; and b) producing multiple copies of an RNA sequence complementary to said target ribonucleic acid sequence using said target ribonucleic acid sequence as a template; wherein at least one of said one or more promoter-primers is a modified promoter-primer comprising a modification at its 3'"'"'-end to prevent or decrease a nucleic acid extension reaction from proceeding therefrom. - View Dependent Claims (32, 33, 34, 35, 36, 37)
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38. A composition for amplifying a target ribonucleic acid sequence using said target ribonucleic acid sequence as a template comprising:
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said target ribonucleic acid sequence, and one or more promoter-primers comprising a single nucleic acid sequence comprising a promoter recognizable by an RNA polymerase and a primer located 3'"'"' relative to said promoter, said primer being sufficiently complementary to said target ribonucleic acid sequence to form a complex at or near the 3'"'"'-end of said target ribonucleic acid sequence, a reverse transcriptase, said RNA, and a solution of reagents able to allow amplification of said target ribonucleic acid sequence at essentially constant temperature;
wherein a primer able to hybridize to a nucleic acid sequence complementary to said target ribonucleic acid sequence is not present;wherein said one or more promoter-primers comprises one or more modified promoter-primers and one or more unmodified promoter-primers, said one or more modified promoter-primers and said one or more unmodified promoter primers being present in a ratio effective to produce amplification. - View Dependent Claims (39, 40, 41)
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Specification