EP0361983A2
Expired / Lapsed
EP
⏱️ 0.0 years remaining
RNA template end-linked probe constructs and methods for use
Assignee: BP Corp North America Inc
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Filing: 1989-10-02
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Granted: —
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Expires: 2009-10-02
Abstract
Abstract New probes are provided for the detection of target RNA comprising RNA templates replicated by Q-beta replicase having a target specific probe attached through specific sequences at both the template's 3′ and 5′ ends whereby hybridization of the probe with target results in the formation of a cleavage inducible ribozyme. Following cleavage, the RNA template may be advantageously amplified by Q-beta replicase resulting in the generation of a large signal correlatable to the presence of RNA target in the sample. Suitable methods employing such probes are also provided.
Inventors
- 👤 James E. Stefano
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-30 13:26
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/682; C12N; C12N15/00; C12N15/09; C12N15/11; C12N15/113; C12Q1/6823; C12Q1/6844; C12Q1/6867; C12Q1/6876; C12Q1/6888; C12Q1/689; C12Q1/70; C12Q1/701; C12Q1/702; C12Q1/703
Claims Analysis
Total Claims: 28
Claim 1
Independent Claim
218 words
A method of detecting a target nucleic acid having a first and second nucleic acid sequence in a sample suspected of comprising the target nucleic acid, the method comprising: where necessary obtaining the target nucleic acid in single stranded form; further providing a probe comprising: an RNA template having a 3′ end and a 5′ end and replicable by an RNA directed RNA polymerase; a first nucleic acid probe sequence at the 5′ end of the RNA template and substantially complementary to the first nucleic acid sequence of the target nucleic acid (first target sequence); and a second nucleic acid probe sequence at the 3′ end of the RNA template and substantially complementary to the second nucleic acid sequence of the target nucleic acid (second target sequence); contacting the sample with the probe under conditions to allow hybridization to occur between the first and second probe sequences and the first and second target sequences, respectively, if present, to form a ribozyme comprising sequences from the target nucleic acid and the probe; inducing cleavage of the ribozyme to release the RNA template from the target nucleic acid; allowing the released RNA template to be replicated for a predetermined period of time; and detecting replicated RNA template as an indication of the presence or absence of the target nucleic acid.
Claim 2
Independent Claim
218 words
A method of detecting a target nucleic acid having a first and second nucleic acid sequence in a sample suspected of comprising the target nucleic acid, the method comprising: where necessary obtaining the target nucleic acid in single stranded form; further providing a probe comprising: an RNA template having a 3′ end and a 5′ end and replicable by an RNA directed RNA polymerase; a first nucleic acid probe sequence at the 5′ end of the RNA template and substantially complementary to the first nucleic acid sequence of the target nucleic acid (first target sequence); and a second nucleic acid probe sequence at the 3′ end of the RNA template and substantially complementary to the second nucleic acid sequence of the target nucleic acid (second target sequence); contacting the sample with the probe under conditions to allow hybridization to occur between the first and second probe sequences and the first and second target sequences, respectively, if present, to form a ribozyme comprising sequences from the target nucleic acid and the probe; inducing cleavage of the ribozyme to release the RNA template from the target nucleic acid; allowing the released RNA template to be replicated for a predetermined period of time; and detecting replicated RNA template as an indication of the presence or absence of the target nucleic acid.
Claim 3
Independent Claim
261 words
A method of detecting a target nucleic acid having a first and second nucleic acid sequence, in a sample suspected of comprising the target nucleic acid, the method comprising: where necessary, obtaining the target nucleic acid in single stranded form; further providing a first probe comprising: an RNA template having a 3′ end and a 5′ end and replicable by an RNA directed RNA polymerase; and a first nucleic acid probe sequence at one end, such as the 5′end, of the RNA template and substantially complementary to the first nucleic acid sequence of the target nucleic acid (first target sequence); additionally providing a second probe comprising: a second nucleic acid probe sequence substantially complementary to the second nucleic acid sequence of the target nucleic acid (second target sequence) and having a third sequence at one end, such as the 5′ probe end substantially complementary to a portion of the first probe; contacting the sample with the first and second probes simultaneously or separately under conditions to allow hybridization to occur between the first and second probe sequences and the first and second target sequences respectively, if present, and between the first and second probes to form a ribozyme comprising sequences from the target nucleic acid, first nucleic acid probe and second nucleic acid probe; inducing cleavage of the ribozyme to release the RNA template from the target nucleic acid; allowing the released RNA template to be replicated for a predetermined period of time; and detecting replicated RNA template as an indication of the presence or absence of the target nucleic acid.
Claim 4
Independent Claim
261 words
A method of detecting a target nucleic acid having a first and second nucleic acid sequence, in a sample suspected of comprising the target nucleic acid, the method comprising: where necessary, obtaining the target nucleic acid in single stranded form; further providing a first probe comprising: an RNA template having a 3′ end and a 5′ end and replicable by an RNA directed RNA polymerase; and a first nucleic acid probe sequence at one end, such as the 5′end, of the RNA template and substantially complementary to the first nucleic acid sequence of the target nucleic acid (first target sequence); additionally providing a second probe comprising: a second nucleic acid probe sequence substantially complementary to the second nucleic acid sequence of the target nucleic acid (second target sequence) and having a third sequence at one end, such as the 5′ probe end substantially complementary to a portion of the first probe; contacting the sample with the first and second probes simultaneously or separately under conditions to allow hybridization to occur between the first and second probe sequences and the first and second target sequences respectively, if present, and between the first and second probes to form a ribozyme comprising sequences from the target nucleic acid, first nucleic acid probe and second nucleic acid probe; inducing cleavage of the ribozyme to release the RNA template from the target nucleic acid; allowing the released RNA template to be replicated for a predetermined period of time; and detecting replicated RNA template as an indication of the presence or absence of the target nucleic acid.
Claim 5
Dependent Claim
27 words
A method as claimed in claim 1 or 2 wherein cleavage is induced by contacting the ribozyme with a divalent cation which is Mn++, Mg++ or Ca++.
Claim 6
Independent Claim
48 words
A method as claimed in any of claims 1 to 3 wherein the first and second target sequences are separated by a sequence comprising 5′ -GAAA-3′, the second nucleic acid probe sequence has a length of at least about 4 bases and the RNA template is a midivariant.
Claim 7
Independent Claim
32 words
A method as claimed in any of claims 1 to 4 further comprising separating unhybridized probe from hybridized probe following contact of the probe and target nucleic acid and before inducing cleavage.
Claim 8
Independent Claim
26 words
A method as claimed in any of claims 1 to 5 further comprising correlating the detected replicated RNA templates with the amount of target nucleic acid.
Claim 9
Independent Claim
28 words
A method as claimed in any of claims 1 to 6 wherein the first nucleic acid probe sequence remains hybridized to the first target sequence while inducing cleavage.
Claim 10
Independent Claim
98 words
A method of detecting a target nucleic acid sequence in a sample suspected of comprising the target nucleic acid , the method comprising contacting the sample with a probe comprising an RNA template and a sequence substantially complementary with a portion of the target nucleic acid, allowing hybridization of the probe with the target nucleic acid sequence to form a ribozyme comprising sequences from the target nucleic acid and the probe; inducing cleavage at the ribozyme to release the RNA template; replicating said RNA template with an RNA directed RNA polymerase and detecting the resultant replicated RNA templates.
Claim 11
Independent Claim
98 words
A method of detecting a target nucleic acid sequence in a sample suspected of comprising the target nucleic acid , the method comprising contacting the sample with a probe comprising an RNA template and a sequence substantially complementary with a portion of the target nucleic acid, allowing hybridization of the probe with the target nucleic acid sequence to form a ribozyme comprising sequences from the target nucleic acid and the probe; inducing cleavage at the ribozyme to release the RNA template; replicating said RNA template with an RNA directed RNA polymerase and detecting the resultant replicated RNA templates.
Claim 12
Dependent Claim
53 words
A process for the preparation of a probe comprising an RNA template, having a 3′ end and a 5′ end and susceptible of replication by an RNA-directed RNA polymerase, and a nucleic acid attached to either or both said 3′ and 5′ ends, the process comprising coupling successive nucleotides together or ligating oligonucleotides.
Claim 13
Dependent Claim
53 words
A process for the preparation of a probe comprising an RNA template, having a 3′ end and a 5′ end and susceptible of replication by an RNA-directed RNA polymerase, and a nucleic acid attached to either or both said 3′ and 5′ ends, the process comprising coupling successive nucleotides together or ligating oligonucleotides.
Claim 14
Dependent Claim
24 words
A process as claimed in claim 9 wherein the nucleic acid is specific for, and hybridizable under suitable conditions with, a target nucleic acid.
Claim 15
Dependent Claim
23 words
A process as claimed in claim 9 or 10 wherein the template id MDV-1 and/or the RNA directed RNA polymerase is Q-beta replicase.
Claim 16
Dependent Claim
39 words
A process as claimed in any of claims 9 to 11 further comprising a nucleic acid spacer sequence, not specific to a target nucleic acid, and attached to the 3′ end of the RNA template and the nucleic acid.
Claim 17
Dependent Claim
66 words
A process as claimed in claim 12 further comprising a second nucleic acid specific for a target nucleic acid and having attached thereto release agent means whereby hybridization of the second nucleic acid and the first nucleic acid with the target nucleic acid forms a complex whereby the release agent means acts to cleave the spacer sequence thereby releasing the template from the target nucleic acid.
Claim 18
Dependent Claim
21 words
A process as claimed in claim 13 wherein the release agent means comprises a cofactor for activiting the release agent means.
Claim 19
Dependent Claim
91 words
A process as claimed in any of claims 9 to 14 for detecting the presence or absence of a target nucleic acid sequence, the probe comprising: a first nucleic acid probe sequence at one end, such as the 5′ end, of the RNA template and substantially complementary to the first nucleic acid sequence of the target nucleic acid, and a second nucleic acid probe sequence at the other end, such as the 3′end, of the RNA template and substantially complementary to the second nucleic acid sequence of the target nucleic acid.
Claim 20
Dependent Claim
38 words
A process as claimed in claim 15 wherein the first and second nucleic acid sequences are separated by a sequence comprising 5′-GAAA-3′ and/or the second nucleic acid probe sequence has a length of at least about 4 bases.
Claim 21
Dependent Claim
64 words
A process as claimed in claim 15 or 16 for detecting the presence of a target nucleic acid having a first target specific nucleic acid sequence attached to the 3′ end of the RNA template and a second nucleic acid sequence attached to the 5′ end of the RNA template whereby hybridization of the probe to the target nucleic acid produces a ribozyme structure.
Claim 22
Dependent Claim
20 words
A process as claimed in claim 17 wherein the ribozyme structure cleaves the second nucleic acid from the RNA template.
Claim 23
Dependent Claim
49 words
A process as claimed in any of claims 9 to 18 wherein the nucleic acid is ribonucleic acid and is specific for target organism deoxyribonucleic acid and further comprises RNase H for releasing the ribonucleic acid from the target organism deoxyribonucleic acid when the ribonucleic acid is hybridized thereto.
Claim 24
Dependent Claim
172 words
A process for the preparation of a kit for detecting the presence or absence of the target nucleic acid sequence comprising: a first probe comprising an RNA template having a 3′ end and a 5′ end and replicable by an RNA directed RNA polymerase such as Q-beta replicase, and having a first nucleic acid probe sequence attached to one end, such as the 5′ end, of the RNA template and substantially complementary to a first nucleic acid sequence of the target nucleic acid, and a second probe with a 3′ and a 5′ end comprising a second nucleic acid probe sequence at one end, such as the 3′ end and substantially complementary to a second nucleic acid sequence of the target nucleic acid, and having a third sequence at the other end, such as the 5′ end, substantially complementary to a portion of the first probe; and optionally instructions for using the first and second probes for detecting the target nucleic acid, the process comprising coupling successive nucleotides together or ligating oligonucleotides.
Claim 25
Dependent Claim
172 words
A process for the preparation of a kit for detecting the presence or absence of the target nucleic acid sequence comprising: a first probe comprising an RNA template having a 3′ end and a 5′ end and replicable by an RNA directed RNA polymerase such as Q-beta replicase, and having a first nucleic acid probe sequence attached to one end, such as the 5′ end, of the RNA template and substantially complementary to a first nucleic acid sequence of the target nucleic acid, and a second probe with a 3′ and a 5′ end comprising a second nucleic acid probe sequence at one end, such as the 3′ end and substantially complementary to a second nucleic acid sequence of the target nucleic acid, and having a third sequence at the other end, such as the 5′ end, substantially complementary to a portion of the first probe; and optionally instructions for using the first and second probes for detecting the target nucleic acid, the process comprising coupling successive nucleotides together or ligating oligonucleotides.
Claim 26
Dependent Claim
37 words
A process as claimed in claim 20 wherein the first and second nucleic acid sequences are separated by a sequence comprising 5′-GAAA-3′ and/or the second nucleic acid probe sequence has a length of at least 4 bases.
Claim 27
Independent Claim
106 words
A method of detecting a target nucleic acid sequence, having a first and second nucleic acid sequence, in a sample suspected of comprising the target nucleic acid sequence, the method comprising contacting the sample with a first probe sequence substantially complementary to the first nucleic acid sequence and linked to an RNA template replicable by an RNA directed RNA polymerase through a spacer sequence, and a second probe sequence substantially complementary to the second nucleic acid sequence, linked to a release agent capable of cleaning the spacer sequence, providing hybridizing conditions, activating the release agent, allowing replication of the RNA template and detecting the replicated RNA.
Claim 28
Independent Claim
106 words
A method of detecting a target nucleic acid sequence, having a first and second nucleic acid sequence, in a sample suspected of comprising the target nucleic acid sequence, the method comprising contacting the sample with a first probe sequence substantially complementary to the first nucleic acid sequence and linked to an RNA template replicable by an RNA directed RNA polymerase through a spacer sequence, and a second probe sequence substantially complementary to the second nucleic acid sequence, linked to a release agent capable of cleaning the spacer sequence, providing hybridizing conditions, activating the release agent, allowing replication of the RNA template and detecting the replicated RNA.
References Cited (Backward Citations)
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