Nucleic acid sequence amplification method, composition and kit

A method of amplifying a target ribonucleic acid sequence comprising the steps of:

• 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.

A composition for amplifying a target ribonucleic acid sequence using said target ribonucleic acid sequence as a template 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 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.

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,
• 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.

A composition for amplifying a target ribonucleic acid sequence using said target ribonucleic acid sequence as a template comprising:

• 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.