Patent EP0192168A2 successfully refreshed from Google Patents.
EP0192168A2
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Solution-phase dual hybridization assay for detecting polynucleotide sequences
Assignee: Molecular Diagnostics Inc
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Filing: 1986-02-11
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Granted: —
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Expires: 2006-02-11
Abstract
Abstract A dual hybridization assay method for detecting the presence of a particular polynucleotide sequence in a test sample wherein hybridization is accomplished in a solution phase. Two soluble polynucleotide probes are employed which hybridize to mutually exclusive portions of the sequence to be detected. The first, or separation, probe comprises a reactive site for a reaction partner, preferably a binding site (e.g., biotin) for a binding substance (e.g., avidin). After hybridization, the resulting solution is contacted with an immobilized form of the reaction partner whereby dual hybrids comprising the sequence to be detected and the detection and separation probes become immobilized. The immobilized fraction is then separated and the detection probe determined in one of the separated fractions. Preferably, the detection probe is labeled such as with a specifically bindable ligand, e.g., biotin or a hapten, and is detectable by binding of a labeled binding partner, e.g., enzyme-labeled avidin or antibody. The assay method has the advantages of a dual hybridization technique plus the kinetic and manipulative advantages of a solution-phase assay.
Inventors
- 👤 Nanibhushan Dattagupta
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-25 08:56
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813
Claims Analysis
Total Claims: 13
Claim 1
Independent Claim
147 words
A method of detecting the presence of a particular polynucleotide sequence in a test sample, comprising the steps of contacting the test sample with a first, separation nucleic acid probe and a second, detection nucleic acid probe, said probes each comprising at least one single stranded base sequence that is hybridizable with a mutually exclusive portion of the sequence to be detected, the separation probe comprising additionally a reactive site capable of forming a stable bond to a reaction partner, such contact between the test sample and probes being effected in solution under hybridizing conditions, contacting the resulting solution with an immobilized form of the reaction partner and forming said stable bond with the reactive site in the separation probe separating the resulting immobilized fraction from the remaining solution, and determining the presence of the detection probe in the separated immobilized fraction or in the remaining solution.
Claim 2
Independent Claim
147 words
A method of detecting the presence of a particular polynucleotide sequence in a test sample, comprising the steps of contacting the test sample with a first, separation nucleic acid probe and a second, detection nucleic acid probe, said probes each comprising at least one single stranded base sequence that is hybridizable with a mutually exclusive portion of the sequence to be detected, the separation probe comprising additionally a reactive site capable of forming a stable bond to a reaction partner, such contact between the test sample and probes being effected in solution under hybridizing conditions, contacting the resulting solution with an immobilized form of the reaction partner and forming said stable bond with the reactive site in the separation probe separating the resulting immobilized fraction from the remaining solution, and determining the presence of the detection probe in the separated immobilized fraction or in the remaining solution.
Claim 3
Dependent Claim
32 words
The method of Claim 1 wherein the immobilized form of the reaction partner for the reactive site in the separation probe comprises a solid support to which the reaction partner is attached.
Claim 4
Independent Claim
36 words
The method of any of Claims 1 and 2 wherein the reactive site in the separation probe is a binding site capable of specific noncovalent binding witn a binding substance which serves as the reaction partner.
Claim 5
Independent Claim
36 words
The method of any of Claims 1 to 3 wherein the binding site in the spearation probe is a biotin or a hapten and the binding substance tneretor is avidin or an anti-hapten antibody reagent respectively.
Claim 6
Independent Claim
41 words
The method of any of Claims 1 to 4 wherein the detection probe comprises a detectable label and wherein the presence of the detection probe in the separated immobilized fraction or the remaining solution is determined by measuring such label therein.
Claim 7
Independent Claim
43 words
The method of any of Claims 1 to 5 wherein the detectable label is an enzymatically active group, a fluorescer, a chromophore, a luminescer, a radioisotope or a specifically bindable ligand which can be determined by binding with a labeled binding partner therefore.
Claim 8
Independent Claim
48 words
The method of any of Claims 1 to 6 wherein the binding partner is labeled with a detectable chemical group, wherein the labeled binding partner is contacted with the solution resulting from the hybridization step or with the separation immobilized fraction, and the resulting immobilized label is measured.
Claim 9
Independent Claim
72 words
The method of any of Claims 1 to 7 wherein the hybrid formed between the polynucleotide sequence of interest and the detection probe is antigenically distinct from its individual strands and wherein the presence of the detection probe in the separated immobilized fraction is determined by binding with an antibody reagent that is selective for binding said hybrid and measurement of the antibody reagent that becomes bound to such separated immobilized fraction.
Claim 10
Dependent Claim
73 words
A reagent combination for detecting the presence of a particular polynucleotide sequence in a test sample, comprising (1) a first, separation probe (2) a second detection probe, said probes each comprising at least one single stranded base sequence that is hybridizable with a mutually exclusive portion of the sequence to be detected, the separation probe comprising additionally a reactive site for a reaction partner, and (3) an immobilized form of said reaction partner.
Claim 11
Dependent Claim
73 words
A reagent combination for detecting the presence of a particular polynucleotide sequence in a test sample, comprising (1) a first, separation probe (2) a second detection probe, said probes each comprising at least one single stranded base sequence that is hybridizable with a mutually exclusive portion of the sequence to be detected, the separation probe comprising additionally a reactive site for a reaction partner, and (3) an immobilized form of said reaction partner.
Claim 12
Dependent Claim
79 words
Use of a reagent combination for detecting the presence of a particular polynucleotide sequence in a test sample, comprising (1) a first, separation probe (2) a second detection probe, said probes each comprising at least one single stranded base sequence that is hybridizable with a mutually exclusive protion of the sequence to be detected, the separation probe comprising additionally a reactive site for a reaction partner, and (3) an immobilized form of said reaction partner in a hybridization assay.
Claim 13
Dependent Claim
79 words
Use of a reagent combination for detecting the presence of a particular polynucleotide sequence in a test sample, comprising (1) a first, separation probe (2) a second detection probe, said probes each comprising at least one single stranded base sequence that is hybridizable with a mutually exclusive protion of the sequence to be detected, the separation probe comprising additionally a reactive site for a reaction partner, and (3) an immobilized form of said reaction partner in a hybridization assay.
References Cited (Backward Citations)
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