EP0224995A1
Expired / Lapsed
EP
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Homogeneous assay for specific polynucleotides and kit for performing same
Assignee: Syntex USA LLC
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Filing: 1986-09-05
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Granted: —
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Expires: 2006-09-05
Abstract
Abstract A method for determining the presence of a polynucleotide analyte in a sample suspected of containing the analyte is disclosed. The method comprises combining in an assay medium the sample and first and second polynucleotide reagents complementary to the analyte. Each of the first and second reagents hybridize with a different region of the analyte. The first reagent contains means for rendering the first reagent non-covalently polymerizable. The second reagent contains means for rendering the second reagent detectable. The sample and the first and second reagents are combined in the assay medium under conditions for polymerizing the first reagent wherein the second reagent becomes bound to the polymerized first reagent only when the analyte is present in the sample. A determination is then made as to whether the second reagent has become bound to the polymerized first reagent. The method has broad application for determining the presence of a polynucleotide analyte such as DNA, RNA, the genomes of viruses, bacteria, molds, fungi, and fragments thereof, and the like. Preferred means for rendering the first reagent non-covalently polymerizable includes a repeating oligonucleotide sequence covalently bound to the first reagent.
Inventors
- 👤 Nurith Kurn
- 👤 Chander Bahl
- 👤 Edwin F. Ullman
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-05 10:31
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/70; C12Q1/701; C12Q1/705; C12Q1/68; C12Q1/6813; C12Q1/6832; Y; Y10; Y10S; Y10S435/00; Y10S435/81; Y10S436/00; Y10S436/807; Y10S436/808
Claims Analysis
Total Claims: 19
Claim 1
Independent Claim
127 words
A method for determining the presence of a polynucleotide analyte in a sample suspected of containing said analyte, which method comprises - (a) combining in an assay medium said sample and first and second polynucleotide reagents complementary to said analyte, wherein each of said first and second reagents hybridizes with a different region of said analyte, said first reagent containing means for rendering said first reagent non-covalently polymerizable and said second reagent containing means for rendering said second reagent detectable, wherein, under conditions for polymerizing said first reagent, said second reagent becomes bound to said polymerized first reagent only when said analyte is present in said sample, (b) polymerizing said first reagent, and (c) determining whether said second reagent has become bound to said polymerized first reagent.
Claim 2
Independent Claim
127 words
A method for determining the presence of a polynucleotide analyte in a sample suspected of containing said analyte, which method comprises - (a) combining in an assay medium said sample and first and second polynucleotide reagents complementary to said analyte, wherein each of said first and second reagents hybridizes with a different region of said analyte, said first reagent containing means for rendering said first reagent non-covalently polymerizable and said second reagent containing means for rendering said second reagent detectable, wherein, under conditions for polymerizing said first reagent, said second reagent becomes bound to said polymerized first reagent only when said analyte is present in said sample, (b) polymerizing said first reagent, and (c) determining whether said second reagent has become bound to said polymerized first reagent.
Claim 3
Dependent Claim
24 words
A method of Claim 1 wherein said polynucleotide analyte is DNA, RNA or the genomes of viruses, bacteria, molds, or fungi, or fragments thereof.
Claim 4
Dependent Claim
49 words
A method of claim 1 or claim 2 wherein said means for rendering said first reagent non-covalently polymerizable includes a specific binding pair (sbp) member conjugated to said first reagent and wherein a complementary sbp member having at least two specific binding sites is combined with said assay medium.
Claim 5
Dependent Claim
29 words
A method of claim 1 or claim 2 wherein said means for rendering said first reagent non-covalently polymerizable includes a repeating oligonucleotide sequence covalently bound to said first reagent.
Claim 6
Dependent Claim
33 words
A method of claim 4 wherein said repeating oligonucleotide sequence is a homopolymer having at least 8 nucleotide residues, or a heteropolymer at least eight nucleotides in length consisting of two non-complementary nucleotides.
Claim 7
Dependent Claim
22 words
A method of claim 3 wherein said sbp member and said complementary sbp member are antigen-antibody, avidin-biotin, IgG-protein A, DNA-DNA or DNA-RNA.
Claim 8
Independent Claim
38 words
A method of any one of the preceding claims wherein said means for rendering said second reagent detectable includes a label selected from catalysts, dyes, fluorescers, chemiluminescers, particles, metal sols, carbon particles, polymeric particles, liposomes, and dye aggregates.
Claim 9
Dependent Claim
11 words
A method of claim 7 wherein a second label is employed.
Claim 10
Dependent Claim
59 words
A method of claim 7 wherein said means for rendering said second reagent detectable includes a specific binding pair (sbp) member bound to said second reagent and a complementary sbp member bound to said label wherein the number of sbp member binding sites bound to at least one of said second reagent and said label is limited to one.
Claim 11
Dependent Claim
40 words
A method of claim 9 wherein said sbp member and said complementary sbp member are respectively a ligand, which is biotin and a receptor for said ligand, which is avidin, wherein a single ligand is bound to said second reagent.
Claim 12
Independent Claim
33 words
A method of any one of the preceding claims wherein in Step (c) said polymerized first reagent is examined for the presence of said second reagent by determining the presence of aggregated particles.
Claim 13
Independent Claim
60 words
A method of any one of the preceding claims wherein said first reagent is a polymerization probe capable of hybridizing with said analyte and capable of being non-covalently polymerized, and said second regent is a detection probe capable of hybridizing with said analyte in a region other than that with which said polymerization probe hybridizes and capable of being detected.
Claim 14
Dependent Claim
26 words
The method of claim 12 wherein said polymerization probe comprises an oligonucleotide, complementary to said analyte, conjugated to means for rendering said polymerization probe non-covalently polymerizable.
Claim 15
Dependent Claim
25 words
The method of claim 12 or claim 13 wherein said detection probe comprises an oligonucleotide conjugated to a means for rendering said detection probe detectable.
Claim 16
Dependent Claim
77 words
A method of Claim 12 wherein said polymerization probe comprises a polynucleotide, hybridizable with a region of said analyte conjugated to said means for non-covalently polymerizing said polymerization probe, and said detection probe comprises a polynucleotide, hybridizable with a region of said analyte other than the region recognized by said polymerization probe, conjugated to said means for rendering said detection probe detectable, and including a polymerization agent capable of causing said non-covalent polymerization of said polymerization probe.
Claim 17
Dependent Claim
15 words
A method of claim 1 which comprises employing complementary repeating oligonucleotide sequences to cause agglutination.
Claim 18
Dependent Claim
108 words
A kit useful in a method for determining the presence of a polynucleotide analyte in a sample suspected of containing said analyte, which kit comprises in packaged combination - (a) polymerization probe comprising (i) a polynucleotide, complementary with a region of said analyte, conjugated to (ii) means for rendering said polymerization probe non-covalently polymerizable, (b) a detection probe comprising (i) a polynucleotide, complementary with a region of said analyte other than the region recognized by said polymerization probe, conjugated to (ii) means for rendering said detection probe detectable, (c) a polymerization agent capable of causing the non-covalent polymerization of said polymerization probe, and (d) ancillary materials, as required.
Claim 19
Dependent Claim
108 words
A kit useful in a method for determining the presence of a polynucleotide analyte in a sample suspected of containing said analyte, which kit comprises in packaged combination - (a) polymerization probe comprising (i) a polynucleotide, complementary with a region of said analyte, conjugated to (ii) means for rendering said polymerization probe non-covalently polymerizable, (b) a detection probe comprising (i) a polynucleotide, complementary with a region of said analyte other than the region recognized by said polymerization probe, conjugated to (ii) means for rendering said detection probe detectable, (c) a polymerization agent capable of causing the non-covalent polymerization of said polymerization probe, and (d) ancillary materials, as required.
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EP0154505A2
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