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US4581333A
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Method of detecting and characterizing a nucleic acid or reactant for the application of this method
Assignee: Institut Pasteur
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Filing: 1982-04-29
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Granted: 1986-04-08
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Expires: 2002-04-29
Abstract
Abstract Method for detecting the possible presence of a DNA fragment, notably of a gene, in the midst of a complex sample of nucleic acids. It comprises the hybridization of the sought fragment with a RNA probe, this being, prior or subsequent to the hybridization reaction, modified by an enzyme. Application to seeking of particular genes or DNA fragments in the midst of a biological sample.
Inventors
- 👤 Philippe Kourilsky
- 👤 Stratis Avrameas
- 👤 Brigitte Cami nee Contamine
- 👤 Jean-Luc Guesdon
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-24 09:15
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; Y; Y10; Y10S; Y10S435/00; Y10S435/81; Y10S435/975
Claims Analysis
Total Claims: 37
Claim 1
Independent Claim
171 words
A detection method for detecting the presence or absence of a nucleic acid in a biological medium, the medium containing also other nucleic acids which are not sought to be detected, by means of a probe containing a nucleic acid complementary of said nucleic acid sought to be detected and by means of measurement on a substrate specific to an enzyme, which method comprises: hybridizing the nucleic acid sought to be detected with said probe having a complementary nucleic acid, thereby forming a hybridized product, separating excess non-hybridized probe from said hybridized product and the other nucleic acids, coupling the hybridized product with an enzyme, thereby forming an enzyme-bonded product, wherein either said probe is modified by a chemical group for coupling with the enzyme of said enzyme is modified by a chemical group for coupling with said probe, measuring the activity of the enzyme-bonded product on the subtrate specific to the enzyme in the presence of the other nucleic acids, thereby detecting whether or not the nucleic acid is present.
Claim 2
Dependent Claim
171 words
A detection method for detecting the presence or absence of a nucleic acid in a biological medium, the medium containing also other nucleic acids which are not sought to be detected, by means of a probe containing a nucleic acid complementary of said nucleic acid sought to be detected and by means of measurement on a substrate specific to an enzyme, which method comprises: hybridizing the nucleic acid sought to be detected with said probe having a complementary nucleic acid, thereby forming a hybridized product, separating excess non-hybridized probe from said hybridized product and the other nucleic acids, coupling the hybridized product with an enzyme, thereby forming an enzyme-bonded product, wherein either said probe is modified by a chemical group for coupling with the enzyme of said enzyme is modified by a chemical group for coupling with said probe, measuring the activity of the enzyme-bonded product on the subtrate specific to the enzyme in the presence of the other nucleic acids, thereby detecting whether or not the nucleic acid is present.
Claim 3
Dependent Claim
14 words
The method of claim 1 wherein the chemical modification is performed prior to hybridization.
Claim 4
Dependent Claim
15 words
The method of claim 1 wherein the hybridized product is coupled directly to the enzyme.
Claim 5
Dependent Claim
12 words
The method of claim 3 wherein the enzyme is modified by avidin.
Claim 6
Dependent Claim
11 words
The method of claim 1 wherein the probe is chemically modified.
Claim 7
Dependent Claim
19 words
The method of claim 5 wherein to the hybrid there is affixed an anti-hybrid antibody, thereby forming a complex.
Claim 8
Dependent Claim
14 words
The method of claim 6 wherein to the complex there is coupled an enzyme.
Claim 9
Dependent Claim
14 words
The method of claim 5 wherein the probe is modified by biotin or avidin.
Claim 10
Dependent Claim
18 words
The method of claim 8 wherein the probe is modified by biotin through the intermediary of cytochrome C.
Claim 11
Dependent Claim
12 words
The method of claim 1 wherein the enzyme has an --SH group.
Claim 12
Dependent Claim
10 words
The method of claim 1 wherein the enzyme is beta-galactosidase.
Claim 13
Dependent Claim
14 words
The method of claim 11 wherein the substrate for the enzyme is orthonitrophenol galactoside.
Claim 14
Dependent Claim
20 words
The method of claim 1 wherein the complementary nucleic acid is selected from the group consisting of DNA and RNA.
Claim 15
Dependent Claim
12 words
The method of claim 13 wherein the DNA or RNA is single-stranded.
Claim 16
Dependent Claim
25 words
The method of claim 1 wherein the step of separating the non-hybridized nucleic acid is separated after the bonding but prior to the measuring step.
Claim 17
Dependent Claim
138 words
A detection method for detecting the presence or absence of a nucleic acid in a biological medium, the medium containing also other nucleic acids which are not sought to be detected, by means of measurement on a substrate specific to a chemically-modified enzyme, which comprises: hybridizing the nucleic acid sought to be detected with a probe having a complementary nucleic acid, thereby forming a hybridized product, separating excess non-hybridized probe from said hybridized product and the other nucleic acids, coupling the hybridized product with an enzyme which is chemically-modified to bind to the probe of the hybridized product, thereby forming a chemically modified enzyme-coupled product, measuring the activity of the chemically-modified enzyme-coupled product on the substrate specific to the enzyme in the presence of the other nucleic acids, thereby detecting whether or not the nucleic acid is present.
Claim 18
Dependent Claim
138 words
A detection method for detecting the presence or absence of a nucleic acid in a biological medium, the medium containing also other nucleic acids which are not sought to be detected, by means of measurement on a substrate specific to a chemically-modified enzyme, which comprises: hybridizing the nucleic acid sought to be detected with a probe having a complementary nucleic acid, thereby forming a hybridized product, separating excess non-hybridized probe from said hybridized product and the other nucleic acids, coupling the hybridized product with an enzyme which is chemically-modified to bind to the probe of the hybridized product, thereby forming a chemically modified enzyme-coupled product, measuring the activity of the chemically-modified enzyme-coupled product on the substrate specific to the enzyme in the presence of the other nucleic acids, thereby detecting whether or not the nucleic acid is present.
Claim 19
Dependent Claim
12 words
The method of claim 16 wherein the probe is a chemically-modified probe.
Claim 20
Dependent Claim
97 words
A composition of matter for detecting the presence or absence of a nucleic acid in a biological sample containing said nucleic acid and other nucleic acids, the presence or absence of which is not sought to be detected, said composition of matter comprising an enzyme, a probe modified by a chemical group for coupling with said enzyme and comprising a complementary nucleic acid sequence which is hybridizable with the nucleic acid sought to be detected to thereby form a hybridized product-enzyme complex, and a substrate specific to the enzyme, for the detection of said hybridized product-enzyme complex.
Claim 21
Dependent Claim
97 words
A composition of matter for detecting the presence or absence of a nucleic acid in a biological sample containing said nucleic acid and other nucleic acids, the presence or absence of which is not sought to be detected, said composition of matter comprising an enzyme, a probe modified by a chemical group for coupling with said enzyme and comprising a complementary nucleic acid sequence which is hybridizable with the nucleic acid sought to be detected to thereby form a hybridized product-enzyme complex, and a substrate specific to the enzyme, for the detection of said hybridized product-enzyme complex.
Claim 22
Dependent Claim
13 words
The composition of matter of claim 18 wherein the substrate is a chromogen.
Claim 23
Dependent Claim
21 words
The composition of matter of claim 18 wherein the chemical group is selected from the group consisting of biotin and avidin.
Claim 24
Dependent Claim
19 words
The composition of matter of claim 20 wherein the probe is modified with biotin and the enzyme with avidin.
Claim 25
Dependent Claim
90 words
A composition of matter which comprises a biological sample containing a nucleic acid sought to be detected and other nucleic acids, a probe which comprises a nucleic acid sequence complementary to the nucleic acid sought to be detected and hybridized therewith, which composition is substantially free of unhybridized probe and in which composition the hybridized probe is modified by a chemical group and said probe is bonded to an enzyme through said chemical group and which composition of matter is capable of acting on a substrate specific to the enzyme.
Claim 26
Dependent Claim
90 words
A composition of matter which comprises a biological sample containing a nucleic acid sought to be detected and other nucleic acids, a probe which comprises a nucleic acid sequence complementary to the nucleic acid sought to be detected and hybridized therewith, which composition is substantially free of unhybridized probe and in which composition the hybridized probe is modified by a chemical group and said probe is bonded to an enzyme through said chemical group and which composition of matter is capable of acting on a substrate specific to the enzyme.
Claim 27
Dependent Claim
21 words
The composition of matter of claim 22 wherein the chemical group is selected from the group consisting of biotin and avidin.
Claim 28
Dependent Claim
17 words
The composition of matter of claim 22 wherein the hybridized probe is bonded directly to the enzyme.
Claim 29
Dependent Claim
96 words
An article of manufacture for detecting the presence or absence of a nucleic acid in a biological sample containing other nucleic acids the presence or absence of which is not sought to be detected, which article comprises a probe which comprises a complementary nucleic acid sequence which is hybridizable with the nucleic acid sought to be detected, an enzyme and a substrate specific for the enzyme, and wherein either said probe is modified by a chemical group for coupling with the enzyme or said enzyme is modified by a chemical group for coupling with said probe.
Claim 30
Dependent Claim
96 words
An article of manufacture for detecting the presence or absence of a nucleic acid in a biological sample containing other nucleic acids the presence or absence of which is not sought to be detected, which article comprises a probe which comprises a complementary nucleic acid sequence which is hybridizable with the nucleic acid sought to be detected, an enzyme and a substrate specific for the enzyme, and wherein either said probe is modified by a chemical group for coupling with the enzyme or said enzyme is modified by a chemical group for coupling with said probe.
Claim 31
Dependent Claim
14 words
The article of claim 25 wherein the enzyme is modified by a chemical group.
Claim 32
Dependent Claim
16 words
The article of claim 25 which comprises a reactant for the lysis of the biological sample.
Claim 33
Dependent Claim
18 words
The article of claim 25 wherein the chemical is selected from the group consisting of biotin and avidin.
Claim 34
Dependent Claim
17 words
The article of claim 25 wherein the probe is coupled to biotin and the enzyme to avidin.
Claim 35
Dependent Claim
10 words
The article of claim 29 wherein the enzyme is beta-galactosidase.
Claim 36
Dependent Claim
28 words
The article of claim 25 wherein the chemical is selected from the group consisting of biotin and avidin, the enzyme is beta-galactosidase and the substrate is orthonitrophenol galactoside.
Claim 37
Dependent Claim
21 words
The article of manufacture of claim 25 further comprising the nucleic acid to be detected in an amount sufficient for detection.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 1989-04-07 | 1989-10-07 | 1990-04-08 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1993-04-07 | 1993-10-07 | 1994-04-08 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1997-04-07 | 1997-10-07 | 1998-04-08 | $7700.00 | Window Closed / Lapsed |