US5215899A
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Nucleic acid amplification employing ligatable hairpin probe and transcription
Assignee: Bayer Corp
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Filing: 1990-08-23
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Granted: 1993-06-01
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Expires: 2010-08-23
Abstract
Abstract Specific nucleic acid sequences are amplified through the use of a hairpin probe which, upon hybridization with and ligation to, a target sequence is capable of being transcribed. 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 single stranded probe sequence extending from the 3' end of the hairpin sequence. Upon hybridization with a target sequence complementary to the probe sequence and ligation of the 3' end of the hybridized target sequence to the 5' end of the hairpin probe, the target sequence is rendered transcribable in the presence of a suitable RNA polymerase and appropriate ribonucleoside triphosphate (rNTPs). Amplification is accomplished by hybridizing the desired target nucleic acid sequence with the probe, ligating the target sequence to the probe, adding the RNA polymerase and rNTPs to the separated hybrids, and allowing transcription to proceed until a desired amount of RNA transcription product has accumulated. The amplification method is particularly useful in assays for the detection of particular nucleic acid sequences.
Inventors
- 👤 Nanibhushan Dattagupta
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 16:38
CPC Classifications
C; C12; C12N; C12N15/00; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/682; Y; Y10; Y10S; Y10S435/00; Y10S435/805; Y10S436/00; Y10S436/815
Claims Analysis
Total Claims: 41
Claim 1
Independent Claim
91 words
A nucleic acid probe comprising (1) a single stranded self-complementary sequence capable of forming, under hybridizing conditions, a hairpin structure having a functional promoter region, and (2) a single stranded probe sequence extending from the 3' end of said self-complementary sequence, whereby upon hybridization of a target sequence complementary to said probe sequence and ligation of the resulting hybridized target sequence to the 5' end of the hairpin-forming self-complementary sequence, said target sequence is transcribable in the presence of an RNA polymerase and required ribonucleoside triphosphates under said hairpin-forming hybridizing conditions.
Claim 2
Dependent Claim
91 words
A nucleic acid probe comprising (1) a single stranded self-complementary sequence capable of forming, under hybridizing conditions, a hairpin structure having a functional promoter region, and (2) a single stranded probe sequence extending from the 3' end of said self-complementary sequence, whereby upon hybridization of a target sequence complementary to said probe sequence and ligation of the resulting hybridized target sequence to the 5' end of the hairpin-forming self-complementary sequence, said target sequence is transcribable in the presence of an RNA polymerase and required ribonucleoside triphosphates under said hairpin-forming hybridizing conditions.
Claim 3
Dependent Claim
17 words
The probe of claim 1 composed of DNA and wherein said polymerase is a DNA-dependent RNA polymerase.
Claim 4
Dependent Claim
16 words
The probe of claim 2 wherein said polymerase is T7, T3, or SP6 bacteriophage RNA polymerase.
Claim 5
Dependent Claim
33 words
The probe of claim 3 wherein said promoter region comprises the following phase sequence shown in its hairpin form: ##STR3## wherein T n represents a sequence of from 2 to 50 thymidine residues.
Claim 6
Dependent Claim
101 words
A method for amplifying a particular target nucleic acid sequence, comprising the steps of: (a) hybridizing said target sequence with a probe of claim 1 in a liquid mixture, (b) ligating the resulting hybridized target sequence to the 5' end of said hairpin-forming self-complementary sequence, (c) adding to the ligated hybrids said RNA polymerase and ribonucleoside triphosphates sufficient for transcription of said target sequence in the hybrids, and (d) allowing the resulting transcription of said target sequence to take place for a predetermined period of time to accumulate the resulting complementary RNA transcription product as an amplification of said target sequence.
Claim 7
Dependent Claim
19 words
The method of claim 5 wherein the target sequence is present in a nucleic acid that has been fragmented.
Claim 8
Dependent Claim
16 words
The method of claim 6 wherein such fragmentation has been accomplished with a restriction endonuclease enzyme.
Claim 9
Dependent Claim
21 words
The method of claim 6 wherein the target sequence is present in a nucleic acid found in a biological test sample.
Claim 10
Dependent Claim
19 words
The method of claim 5 wherein the resulting RNA transcription product is further amplified in a second stage amplification.
Claim 11
Dependent Claim
40 words
The method of claim 9 wherein said second stage amplification is accomplished by separating the resulting RNA transcription product from the reaction mixture, hybridizing such separated transcription product with a transcribable probe and transcribing said probe under hairpin-forming hybridizing conditions.
Claim 12
Dependent Claim
45 words
The method of claim 10 wherein the resulting RNA transcription product is separated from the reaction mixture by having transcribed hybridized probe in the presence of one or more rNTPs comprising a bindable ligand and by adding an immobilized form of a ligand binding partner.
Claim 13
Dependent Claim
27 words
The method of claim 11 wherein said bindable ligand is biotin or a hapten and wherein said ligand binding partner is avidin or an anti-hapten antibody, respectively.
Claim 14
Dependent Claim
27 words
The method of claim 10 wherein the resulting RNA transcription product is separated from the reaction mixture by adding an immobilized form of an anti-RNA/DNA antibody reagent.
Claim 15
Dependent Claim
13 words
The method of claim 5 wherein said polymerase is a DNA-dependent RNA polymerase.
Claim 16
Dependent Claim
16 words
The method of claim 10 wherein said polymerase is T7, T3, or SP6 bacteriophage RNA polymerase.
Claim 17
Dependent Claim
33 words
The method of claim 15 wherein said promoter region comprises the following base sequence shown in its hairpin form: ##STR4## wherein T n represents a sequence of from 2 to 50 thymidine residues.
Claim 18
Dependent Claim
109 words
A method for detecting a particular target nucleic acid sequence in a test sample, comprising the steps of: (a) hybridizing said target sequence in the test sample with a probe of claim 1 in a liquid mixture, (b) ligating the resulting hybridized target sequence to the 5' end of said hairpin-forming self-complementary sequence, (c) adding to the ligated sequences said RNA polymerase and ribonucleoside triphosphates sufficient for transcription of said target sequence in the hybrids, (d) allowing the resulting transcription of said target sequence to take place for a predetermined period of time to accumulate the resulting complementary RNA transcription product, and (e) detecting said accumulated RNA transcription product.
Claim 19
Dependent Claim
49 words
The method of claim 17 wherein the accumulated RNA transcription product is detected by transcribing said target sequence in the presence of ribonucleoside triphosphates which comprise a detectable label, separating resulting labeled transcription product from the labeled ribonucleoside triphosphates, and detecting the label in the separated labeled transcription product.
Claim 20
Dependent Claim
28 words
The method of claim 17 wherein the accumulated RNA transcription product is detected by hybridizing such product with a detectable nucleic acid probe and detecting the resulting hybrids.
Claim 21
Dependent Claim
12 words
The method of claim 19 wherein the detectable probe comprises a label.
Claim 22
Dependent Claim
28 words
The method of claim 19 wherein the hybrids formed between the RNA transcription product and the detectable probe are detected by binding of a anti-hybrid selective antibody reagent.
Claim 23
Dependent Claim
21 words
The method of claim 21 wherein said hybrids are DNA/RNA hybrids and said antibody reagent is selective for binding DNA/RNA hybrids.
Claim 24
Dependent Claim
19 words
The method of claim 17 wherein the target sequence is present in a nucleic acid that has been fragmented.
Claim 25
Dependent Claim
16 words
The method of claim 23 wherein such fragmentation has been accomplished with a restriction endonuclease enzyme.
Claim 26
Dependent Claim
21 words
The method of claim 23 wherein the target sequence is present in a nucleic acid found in a biological test sample.
Claim 27
Dependent Claim
19 words
The method of claim 17 wherein the resulting RNA transcription product is further amplified in a second stage amplification.
Claim 28
Dependent Claim
40 words
The method of claim 26 wherein said second stage amplification is accomplished by separating the resulting RNA transcription product from the reaction mixture, hybridizing such separated transcription product with a transcribable probe, and transcribing said probe under hairpin-forming hybridizing conditions.
Claim 29
Dependent Claim
45 words
The method of claim 27 wherein the resulting RNA transcription product is separated from the reaction mixture by having transcribed hybridized probe in the presence of one or more rNTPs comprising a bindable ligand and by adding an immobilized form of a ligand binding partner.
Claim 30
Dependent Claim
27 words
The method of claim 28 wherein said bindable ligand is biotin or a hapten and wherein said ligand binding partner is avidin or an anti-hapten antibody, respectively.
Claim 31
Dependent Claim
27 words
The method of claim 27 wherein the resulting RNA transcription product is separated from the reaction mixture by adding an immobilized form of an anti-RNA/DNA antibody reagent.
Claim 32
Dependent Claim
13 words
The method of claim 17 wherein said polymerase is a DNA-dependent RNA polymerase.
Claim 33
Dependent Claim
16 words
The method of claim 31 wherein said polymerase is T7, T3, or SP6 bacteriophage RNA polymerase.
Claim 34
Dependent Claim
33 words
The method of claim 32 wherein said promoter region comprises the following base sequence shown in its hairpin form: ##STR5## wherein T n represents a sequence of from 2 to 50 thymidine residues.
Claim 35
Dependent Claim
48 words
A reagent kit for use in amplifying a particular nucleic acid sequence, comprising: (1) a nucleic acid probe of claim 1 which, upon hybridization with and ligation to said target sequence, is transcribable in the presence of an RNA polymerase and ribonucleoside triphosphates, and (2) said RNA polymerase.
Claim 36
Dependent Claim
24 words
The kit of claim 34 which additionally comprises the ribonucleoside triphosphates required for transcription of the probe in the presence of the RNA polymerase.
Claim 37
Dependent Claim
21 words
The kit of claim 34 which additionally comprises a restriction endonuclease enzyme capable of fragmenting nucleic acids comprising said target sequence.
Claim 38
Dependent Claim
21 words
The kit of claim 36 wherein the target sequence is present in a nucleic acid found in a biological test sample.
Claim 39
Dependent Claim
13 words
The kit of claim 34 wherein said polymerase is a DNA-dependent RNA polymerase.
Claim 40
Dependent Claim
16 words
The kit of claim 34 wherein said polymerase is T7, T3, or SP6 bacteriophage RNA polymerase.
Claim 41
Dependent Claim
33 words
The kit of claim 39 wherein said promoter region comprises the following base sequence shown in its hairpin form: ##STR6## wherein T n represents a sequence of from 2 to 50 thymidine residues.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1996-05-31 | 1996-11-30 | 1997-06-01 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2000-05-31 | 2000-11-30 | 2001-06-01 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2004-05-31 | 2004-11-30 | 2005-06-01 | $7700.00 | Window Closed / Lapsed |