Patent EP0427074A2 successfully refreshed from Google Patents.
EP0427074A2
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Nucleic acid amplification employing transcribable hairpin probe
Assignee: Bayer Corp
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Filing: 1990-10-27
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Granted: —
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Expires: 2010-10-27
Abstract
Abstract Specific nucleic acid sequences are amplified through the use of transcribable hairpin probes. The probe comprises a single stranded self-complementary sequence which, under hybridizing conditions, forms a hairpin structure having a functional promoter region, and further comprises a transcribable sequence extending from the 5′ end of the hairpin sequence and a probe sequence which may be comprised in the transcribable 5′ sequence or in a sequence extending from the 3′ end of the hairpin sequence. The hairpin form of the probe is transcribable in the presence of a suitable RNA polymerase and appropriate ribonucleoside triphosphates (rNTPs). Amplification is accomplished by hybridizing the desired target nucleic acid sequence with the transcribable probe, transcribing substantially only probe which has become hybridized to the sequence of interest, and allowing transcription to proceed until a desired amount of RNA transcription product has accumulated. Optional second stage amplification of the RNA transcripts can produce higher levels of amplification. The amplification method is particularly useful in assays for the detection of particular nucleic acid sequences.
Inventors
- 👤 Nanibhushan Dattagupta
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-29 15:23
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/682
Claims Analysis
Total Claims: 13
Claim 1
Independent Claim
164 words
A method for amplifying a particular nucleic acid sequences comprising the steps of: (a) hybridizing said sequence of interest n a liquid mixture with a transcribable probe comprising (1) a single stranded self -complementary sequence capable of forming, under hybridizing conditions, a hairpin structure having a functional promoter region, (2) a transcribable sequence extending from the 5′ end of said self-complementary sequence, which 5′ sequence is transcribable in the presence of an RNA polymerase and required ribonucleoside triphosphates under said hairpin-forming hybridizing conditions, and (3) a single stranded probe sequence substantially complementary to said sequence of interest, (b) transcribing said probe which has become hybridized with said sequence of interest without transcribing substantially any of the unhybridized probe, such transcription taking place under hairpin-forming hybridizing conditions, and (c) allowing the resulting transcription of the probe sequence in said hybridized probe to take place for a predetermined period of time to accumulate the resulting RNA transcription product as an amplification of said sequence of interest.
Claim 2
Independent Claim
164 words
A method for amplifying a particular nucleic acid sequences comprising the steps of: (a) hybridizing said sequence of interest n a liquid mixture with a transcribable probe comprising (1) a single stranded self -complementary sequence capable of forming, under hybridizing conditions, a hairpin structure having a functional promoter region, (2) a transcribable sequence extending from the 5′ end of said self-complementary sequence, which 5′ sequence is transcribable in the presence of an RNA polymerase and required ribonucleoside triphosphates under said hairpin-forming hybridizing conditions, and (3) a single stranded probe sequence substantially complementary to said sequence of interest, (b) transcribing said probe which has become hybridized with said sequence of interest without transcribing substantially any of the unhybridized probe, such transcription taking place under hairpin-forming hybridizing conditions, and (c) allowing the resulting transcription of the probe sequence in said hybridized probe to take place for a predetermined period of time to accumulate the resulting RNA transcription product as an amplification of said sequence of interest.
Claim 3
Dependent Claim
16 words
The method of claim 1 wherein said probe sequence is comprised in said transcribable 5′ sequence.
Claim 4
Dependent Claim
34 words
The method of claim 1 wherein said probe sequence is comprised in a sequence extending from the 3′ end of said self-complementary sequence and which is not substantially complementary to said 5′ transcribable sequence.
Claim 5
Independent Claim
23 words
The method of any of claims 1 to 3 wherein the resulting RNA transcription product is further amplified in a second stage amplification.
Claim 6
Independent Claim
51 words
The method of any of claims 1 to 4 wherein said polymerase is a DNA-dependent RNA polymerase, preferably T7, T3, or SP6 bacteriophage RNA polymerase, and preferably wherein said promoter region comprises the following base sequence shown in its hairpin form: wherein n is an integer from 2 to 50 inclusive.
Claim 7
Independent Claim
175 words
A method for detecting a particular nucleic acid sequence in a test sample, comprising the steps of: (a) hybridizing said sequence of interest in a liquid mixutre with a transcribable probe comprising (1) a single stranded self-complementary sequence capable of forming, under hybridizing conditions, a hairpin structure having a functional promoter region, (2) a transcribable sequence extending from the 5′ end of said self-complementary sequence, which 5′ sequence is transcribable in the presence of an RNA polymerase and required ribonucleoside triphosphates her said hairpin-forming hybridizing conditions, and (3) a single stranded probe sequence substantially complementary to said sequence of interest, (b) transcribing said probe which has become hybridized with said sequence of interest without transcribing substantially any of the unhybridized probe, such transcription taking place under haiprin-forming hybridizing conditions, and (c) allowing the resulting RNA transcription of the probe sequence in said hybridized probe to take place for a predetermined period of time to accumulate the resulting transcription product as an amplification of said sequence of interest and (d) detecting the accumulated RNA transcription product.
Claim 8
Independent Claim
175 words
A method for detecting a particular nucleic acid sequence in a test sample, comprising the steps of: (a) hybridizing said sequence of interest in a liquid mixutre with a transcribable probe comprising (1) a single stranded self-complementary sequence capable of forming, under hybridizing conditions, a hairpin structure having a functional promoter region, (2) a transcribable sequence extending from the 5′ end of said self-complementary sequence, which 5′ sequence is transcribable in the presence of an RNA polymerase and required ribonucleoside triphosphates her said hairpin-forming hybridizing conditions, and (3) a single stranded probe sequence substantially complementary to said sequence of interest, (b) transcribing said probe which has become hybridized with said sequence of interest without transcribing substantially any of the unhybridized probe, such transcription taking place under haiprin-forming hybridizing conditions, and (c) allowing the resulting RNA transcription of the probe sequence in said hybridized probe to take place for a predetermined period of time to accumulate the resulting transcription product as an amplification of said sequence of interest and (d) detecting the accumulated RNA transcription product.
Claim 9
Dependent Claim
16 words
The method of claim 6 wherein said probe sequence is comprised in said transcribable 5′ sequence.
Claim 10
Dependent Claim
34 words
The method of claim 6 wherein said probe sequence is comprised in a sequence extending from the 3′ end of said self-complementary sequence and which is not substantially complementary to said 5′ transcribable sequence.
Claim 11
Independent Claim
23 words
The method o£ any of claims 6 to 8 wherein the resulting RNA transcription product is further amplified in a second stage amplification.
Claim 12
Dependent Claim
92 words
A reagent kit for use in amplifying a particular nucleic acid sequence, comprising: (1) a transcribable hairpin probe comprising (1) a single stranded self-complementary sequence capable of forming, under hybridizing conditions, a hairpin structure having a functional promoter region, (2) a transcribable sequence extending from the 5′ end of said self-complementary sequence, which 5′ sequence is transcribable in the presence of an RNA polymerase and required ribonucleoside triphosphates under said hair-in-forming hybridizing conditions, and (3) a single stranded probe sequence substantially complementary to said sequence of interest, and (2) said RNA polymerase.
Claim 13
Dependent Claim
92 words
A reagent kit for use in amplifying a particular nucleic acid sequence, comprising: (1) a transcribable hairpin probe comprising (1) a single stranded self-complementary sequence capable of forming, under hybridizing conditions, a hairpin structure having a functional promoter region, (2) a transcribable sequence extending from the 5′ end of said self-complementary sequence, which 5′ sequence is transcribable in the presence of an RNA polymerase and required ribonucleoside triphosphates under said hair-in-forming hybridizing conditions, and (3) a single stranded probe sequence substantially complementary to said sequence of interest, and (2) said RNA polymerase.
References Cited (Backward Citations)
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