EP0373960A2
Expired / Lapsed
EP
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Self-sustained, sequence replication system
Assignee: Akzo Nobel NV
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Filing: 1989-12-15
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Granted: —
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Expires: 2009-12-15
Abstract
Abstract The present disclosure provides details of an invention comprising an amplification system for the detection of target nucleic acids, most particularly target nucleic acid sequences, in an isothermal setting wherein all of the reagents necessary to conduct the amplification are present and the reactions are self-sustaining and continuous. Featured is a selective enzymatic digestion of a RNA/DNA duplex, formed by hybridization of a DNA primer with target nucleic acid, thus freeing the DNA strand for further hybridization followed by primer extension to provide a DNA duplex that can serve as a template for production of a plurality of transcripts that can be recycled and/or detected as such for deduced presence of target nucleic acid sequence.
Inventors
- 👤 Thomas Raymond Gingeras
- 👤 John C. Guatelli
- 👤 Kristina Marie Whitfield
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-01 19:32
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/70; C12Q1/701; C12Q1/702; C12Q1/703; C12N; C12N9/00; C12N9/10; C12N9/12; C12Q1/68; C12Q1/6844; C12Q1/6865
Claims Analysis
Total Claims: 35
Claim 1
Independent Claim
188 words
A method of preparing a double-stranded DNA encoding a sequence corresponding to a target nucleic acid sequence and having an operative polymerase promoter, comprising: a) providing a first DNA primer containing a RNA polymerase promoter sequence operatively associated with a sequence that is a complement of a segment of a target nucleic acid sequence, b) contacting under suitable hybridizing conditions said first DNA primer with a nucleic acid sample that may contain said target nucleic acid sequence, c) permitting primer extension of any hybridization product of said first DNA primer with said target nucleic acid sequence in a DNA polymerase extension reaction to form a corresponding RNA/DNA duplex nucleic acid, d) selectively digesting enzymatically the RNA strand of said RNA/DNA duplex nucleic acid, e) permitting hybridization to the freed promoter containing cDNA strand under suitable hybridization conditions of a second nucleic acid primer, said second nucleic acid primer being a product of said selective digestion or being an extrinsically derived oligonucleotide primer optionally bearing operatively a promoter sequence, and f) permitting primer extension of hybridization product of primer with said DNA strand in a DNA polymerase extension reaction.
Claim 2
Independent Claim
188 words
A method of preparing a double-stranded DNA encoding a sequence corresponding to a target nucleic acid sequence and having an operative polymerase promoter, comprising: a) providing a first DNA primer containing a RNA polymerase promoter sequence operatively associated with a sequence that is a complement of a segment of a target nucleic acid sequence, b) contacting under suitable hybridizing conditions said first DNA primer with a nucleic acid sample that may contain said target nucleic acid sequence, c) permitting primer extension of any hybridization product of said first DNA primer with said target nucleic acid sequence in a DNA polymerase extension reaction to form a corresponding RNA/DNA duplex nucleic acid, d) selectively digesting enzymatically the RNA strand of said RNA/DNA duplex nucleic acid, e) permitting hybridization to the freed promoter containing cDNA strand under suitable hybridization conditions of a second nucleic acid primer, said second nucleic acid primer being a product of said selective digestion or being an extrinsically derived oligonucleotide primer optionally bearing operatively a promoter sequence, and f) permitting primer extension of hybridization product of primer with said DNA strand in a DNA polymerase extension reaction.
Claim 3
Dependent Claim
65 words
A method useful for the detection of at least one specific target nucleic acid sequence in a nucleic acid sample that may contain said nucleic acid target sequence, comprising: g) employing the prepared double-stranded DNA of Claim 1 as a double-stranded DNA template for the preparation of a plurality of RNA transcripts therefrom, each bearing a RNA sequence corresponding to said target nucleic acid sequence.
Claim 4
Dependent Claim
21 words
The method according to Claim 2 comprising the additional step of detecting and optionally measuring the presence of said RNA sequence.
Claim 5
Independent Claim
84 words
The method according to any one of Claims 1, 2, or 3 wherein said target nucleic acid sequence in said nucleic acid sample is present intrinsically as such or is a corresponding DNA target sequence extrapolation product prepared by transcription from a double-stranded DNA template prepared by polymerase based primer extension of a primer hybridized to a separated DNA strand operatively bearing a promoter sequence prepared from a hybridization/primer extension product of said DNA target sequence with a primer operatively bearing a promoter sequence.
Claim 6
Dependent Claim
178 words
The method according to Claim 2 comprising the additional steps of h) permitting hybridization of said RNA transcripts under suitable hybridization conditions with a DNA primer containing a promoter sequence operatively associated with a sequence that is a complement of a segment of said RNA transcript sequence, i) permitting primer extension of hybridized product of step h) in a DNA polymerase extension reaction to form a corresponding RNA/DNA duplex nucleic acid, j) selectively digesting enzymatically the RNA strand of said RNA/DNA duplex nucleic acid of step i), k) permitting hybridization of the freed promoter containing DNA strand product of step j) under suitable hybridization conditions with a nucleic acid primer, said nucleic acid primer being a product of said selective digestion of step j) or being an extrinsically derived oligonucleotide primer optionally bearing operatively a promoter sequence, l) permitting primer extension of hybridization product of step k) in a DNA polymerase extension reaction, and m) employing the double-stranded DNA product of step 1) as a double-stranded DNA template for the preparation of a plurality of RNA transcripts therefrom.
Claim 7
Dependent Claim
22 words
The method according to Claim 5 wherein the RNA transcript products have a sense opposite those RNA transcript products of Claim 2.
Claim 8
Dependent Claim
52 words
The method according to Claim 5 permitted to proceed continuously and spontaneously by presence in the reaction milieu of enzyme activities provided by: 1) a reverse transcriptase, 2) an enzyme having RNase H activity, 3) a DNA-dependent RNA polymerase and 4) at least one oligonucleotide primer sequence operatively bearing a promoter sequence.
Claim 9
Dependent Claim
9 words
The method according to Claim 7 conducted substantially isothermally.
Claim 10
Dependent Claim
66 words
A method comprising employing the double-stranded nucleic acid according to Claim 1 or step 1) of Claim 5 as a template for the preparation of a plurality of RNA transcripts therefrom in a reaction catalyzed by a polymerase that recognizes the promoter thereof, each bearing a RNA sequence corresponding to said target nucleic acid sequence, and detecting and optionally measuring the presence of said RNA transcripts.
Claim 11
Dependent Claim
25 words
The method according to Claim 2 or 9 wherein said transcripts contain RNA replicase recognition site for replication of said transcripts by RNA replicase induction.
Claim 12
Dependent Claim
50 words
The method according to Claim 2, 9, or 10 wherein the detected RNA sequence of said RNA transcripts is measured in a standardized manner so as to measure the amount of target nucleic acid sequence contained in a sample of nucleic acid used in preparing the double-stranded nucleic acid template.
Claim 13
Dependent Claim
38 words
The method according to Claim 11 wherein the detected RNA sequence of said RNA transcripts is measured in a manner internally standardized with the presence of a known copy number of nucleic acid also contained in said sample.
Claim 14
Dependent Claim
25 words
The method according to Claim 1 wherein said target nucleic acid sequence is associated with the characteristics of a genetic or pathogenic disease or condition.
Claim 15
Dependent Claim
19 words
The method according to Claim 1 wherein said target nucleic acid sequence is associated with a human immunodeficiency virus.
Claim 16
Dependent Claim
18 words
The method according to Claim 1 wherein said target nucleic acid sequence is associated with a defective gene.
Claim 17
Dependent Claim
26 words
The method according to Claim 1 or 5 wherein the promoter is a bacteriophage T7 promoter and the RNA transcripts are produced using T7 RNA polymerase.
Claim 18
Dependent Claim
18 words
The method according to Claim 1 or 5 wherein the selective digestion is conducted with RNase H enzyme.
Claim 19
Dependent Claim
20 words
The method according to Claim 1 or 5 wherein the extension reaction is catalyzed by E. coli DNA polymerase I.
Claim 20
Dependent Claim
23 words
The method according to Claim 1 or 5 wherein the extension reaction is catalyzed by Klenow fragment of E. coli DNA polymerase I.
Claim 21
Dependent Claim
18 words
The method according to Claim 1 or 5 wherein the extension reaction is catalyzed by T7 DNA polymerase.
Claim 22
Dependent Claim
17 words
The method according to Claim 1 or 5 wherein the extension reaction is catalyzed by reverse transcriptase.
Claim 23
Dependent Claim
17 words
The method according to Claim 2 or 5 wherein said RNA transcripts are labelled prior to detection.
Claim 24
Dependent Claim
12 words
The method according to Claim 22 wherein said RNA transcripts are radio-labelled.
Claim 25
Dependent Claim
13 words
The method according to Claim 22 wherein said RNA transcripts are chromophore labelled.
Claim 26
Dependent Claim
31 words
The product of the process which comprises treating a RNA/DNA duplex RNA with an enzyme having RNase H activity, wherein the DNA strand has at its 5′-end an operative promoter sequence.
Claim 27
Dependent Claim
31 words
The product of the process which comprises treating a RNA/DNA duplex RNA with an enzyme having RNase H activity, wherein the DNA strand has at its 5′-end an operative promoter sequence.
Claim 28
Dependent Claim
12 words
The product according to Claim 25 wherein said enzyme is RNase H.
Claim 29
Dependent Claim
158 words
In a method of amplifying a target nucleic acid sequence, generated as such or as an extrapolation product from a corresponding target DNA sequence, which method comprises the steps of hybridization with a DNA primer having a promoter sequence operatively associated therewith followed by polymerase primer extension to give a corresponding RNA/DNA duplex and hybridization of the said DNA extension product strand bearing the promoter sequence of said duplex with a second primer followed by polymerase primer extension to form a double-stranded DNA template useful for preparation of optionally replicatable RNA transcripts therefrom for detection as such or for further use in reactions as defined above, the improvement wherein said DNA extension product strand bearing the promoter sequence of the said RNA/DNA duplex is made available for the following hybridization thereof with said second primer by the selective digestion enzymatically of the RNA strand of said RNA/DNA duplex with an enzyme having the activity of RNase H enzyme.
Claim 30
Dependent Claim
158 words
In a method of amplifying a target nucleic acid sequence, generated as such or as an extrapolation product from a corresponding target DNA sequence, which method comprises the steps of hybridization with a DNA primer having a promoter sequence operatively associated therewith followed by polymerase primer extension to give a corresponding RNA/DNA duplex and hybridization of the said DNA extension product strand bearing the promoter sequence of said duplex with a second primer followed by polymerase primer extension to form a double-stranded DNA template useful for preparation of optionally replicatable RNA transcripts therefrom for detection as such or for further use in reactions as defined above, the improvement wherein said DNA extension product strand bearing the promoter sequence of the said RNA/DNA duplex is made available for the following hybridization thereof with said second primer by the selective digestion enzymatically of the RNA strand of said RNA/DNA duplex with an enzyme having the activity of RNase H enzyme.
Claim 31
Dependent Claim
12 words
The improvement according to Claim 27 wherein said enzyme is RNase H.
Claim 32
Dependent Claim
98 words
A kit useful for the detection of at least one specific nucleic acid target sequence in a RNA sample that may contain said nucleic acid target sequence, comprising a reaction mixture comprising a reverse transcriptase, an enzyme having RNase H activity, a DNA-dependent RNA polymerase and at least one DNA primer containing a promoter sequence operatively associated with a sequence complementary to a segment of a target nucleic acid sequence or its complement and means whereby upon contact of said mixture with sample for detection of target nucleic acid sequence, target nucleic acid sequence is detected after amplification.
Claim 33
Dependent Claim
98 words
A kit useful for the detection of at least one specific nucleic acid target sequence in a RNA sample that may contain said nucleic acid target sequence, comprising a reaction mixture comprising a reverse transcriptase, an enzyme having RNase H activity, a DNA-dependent RNA polymerase and at least one DNA primer containing a promoter sequence operatively associated with a sequence complementary to a segment of a target nucleic acid sequence or its complement and means whereby upon contact of said mixture with sample for detection of target nucleic acid sequence, target nucleic acid sequence is detected after amplification.
Claim 34
Dependent Claim
17 words
The kit according to Claim 29 having means to conduct reactions involving its components at isothermal conditions.
Claim 35
Dependent Claim
16 words
The kit according to Claim 30 wherein said enzyme having RNase H activity is RNase H.
References Cited (Backward Citations)
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