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US 5,981,179 AGranted
Continuous amplification reaction
AssigneeDigene Diagnostics, Incorporated
InventorsAttila T. Lorinez, Abel DeLaRosa
Life of this patent
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Abstract
Continuous amplification reaction provide a method of amplifying a specific nucleic acid without the need to cycle a reaction. The method produces RNA transcripts which can be detected by a variety of methods. Amplification and detection kits are also provided.
Claims26
A method of amplifying a target nucleic acid comprising the steps of:
- providing a single-stranded nucleic acid containing a target region;
- hybridizing the target region of said nucleic acid to a promoter-primer having a central promoter portion and two regions homologous to non-contiguous portions of the target region forming a circular hybrid;
- trimming back single-stranded sequence 3'to the target region generating a flushed 3' end of said hybrid;
- extending 3' ends of the target region and the promoter-primer forming a double-stranded intermediate;
- and
- transcribing the double-stranded intermediate producing many RNA transcripts from each target region.
3 more elements
A method of detecting a nucleic acid containing a target region comprising the steps of:
- providing a single-stranded nucleic acid comprising said target region;
- hybridizing said nucleic acid to a single stranded promoter-primer having a 5' primer portion and a 3' promoter portion, said 3' primer portion comprising a sequence complementary to a 3' portion of said target region, said 3' primer portion further comprising at least one modified nucleotide or modified phosphodiester linkage to resist exonuclease activity, to form a hybrid;
- trimming back single-stranded sequence 3' to the target with an exonuclease activity, thereby generating a flushed 3' end of said hybrid;
- extending 3' ends of said target region and the promoter-primer forming a double-stranded intermediate;
- transcribing the double-stranded intermediate producing many RNA transcripts from each target region;
- and
- detecting RNA transcripts.
4 more elements
A kit for amplification of nucleic acid in a biological sample comprising:
- a promoter-primer having a central promoter portion and two regions homologous to non-contiguous sequences in the target region;
- a trimming back agent;
- a nucleic acid polymerase;
- an RNA polymerase;
- and
- a sample transport medium for stabilization of the biological sample.
3 more elements
A kit for amplification of nucleic acid in a biological sample comprising:
- a promoter-primer having a 5' promoter portion and a 3' primer portion, said primer portion comprising a sequence complementary to a 3' portion of said target region, said promoter-primer further comprising at least one modified nucleotide or phosphodiester linkage to resist exonuclease activity;
- a trimming back agent comprising an exonuclease activity;
- a nucleic acid polymerase;
- a RNA polymerase;
- and
- a sample transport medium for stabilization of the biological sample.
3 more elements
Dependencies are inferred from the claim text.