Reagent and method for the detection of a nucleotide sequence with signal amplification

• US 6,017,707 A
• Filed: 06/06/1997
• Issued: 01/25/2000
• Est. Priority Date: 09/15/1993
• Status: Expired due to Term

• Current Assignee: Bio Merieux
• Original Assignee: Bio Merieux
• Inventors: Delair, Thierry, Erout, Marie-Noelle, Mandrand, Bernard, Pichot, Christian, Charles, Marie-Helene, Cros, Philippe
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US08/870,730
• Time in Patent Office: 963 Days
• Field of Search: 435/6, 536/22.1, 536/23.1, 536/24.3, 935/77, 935/78
• US Class Current: 435/6.12
• CPC Class Codes: C08G 81/02 at least one of the polymers being obtained by reactions involving only carbon-to-carbon unsaturated bonds
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/682 Signal amplification

Abstract

Methods for the detection of a nucleotide sequence of interest comprising at least one nucleotide probe marked with a tracer. The methods include a reagent essentially comprising a linear backbone copolymer having lateral substituents, whose chain consists of a first type of repetitive unit and at least one other type of repetitive unit, in which at least one part of the units of the first type have a lateral substituent comprising a nucleotide unit, such a lateral substituent not being present on the other types of units. Each of said nucleotide units, all of which are identical, comprise at least one nucleotide sequence capable of hybridizing with said sequence of interest and nucleotide sequence capable of hybridizing with a probe, the reagent containing on average more than two of said nucleotide units, in molar equivalents, per mole of polymer. Such a reagent enables signal amplification to be obtained, and therefor lowers the sensitivity threshold. Application, in particular, in the production of tests for the detection of pathogenic organisms, or in the diagnosis of genetic diseases.

Claims

1. A kit for detecting at least one nucleic acid sequence of interest, with signal amplification, said kit having in suitable containers:

a reagent; and

a single species of tracer-labelled oligonucleotide probe reactive with said reagent;

wherein said reagent comprises at least one linear backbone polymer molecule having a backbone chain formed by carbon-to-carbon covalent bonds, said at least one polymer molecule including at least one monomeric unit having a lateral substituent, said lateral substituent comprising an oligonucleotide unit that hybridizes with said sequence of interest and with said probe, said reagent being soluble in aqueous media and said at least one polymer molecule including at least two said oligonucleotide units per said polymer molecule.

2. A kit according to claim 1, wherein said oligonucleotide unit contains different hybridizing sequences that do not overlap for hybridizing with said sequence of interest and for hybridizing with said probe.

3. A kit according to claim 1, wherein said oligonucleotide unit contains different hybridizing sequences that do overlap for hybridizing with said sequence of interest and for hybridizing with said probe.

4. A kit according to claim 1, wherein said reagent contains from about 3 to 100 of the oligonucleotide units, per said polymer molecule.

5. A kit according to claim 1, wherein said at least one polymer molecule is formed by polymerization of unsaturated monomers.

6. A kit according to claim 1, wherein a precursor of said at least one monomeric unit carries a reactive function which is capable of reacting, either directly or by way of a bifunctional coupling agent, with a precursor of the oligonucleotide unit in order to form said lateral substituent.

7. A kit according to claim 3, wherein said sequence of interest and said probe competitively hybridize with a hybridizing sequence included in said oligonucleotide unit.

8. A kit according to claim 3, wherein said sequence of interest and said probe competitively and coextensively hybridize with a hybridizing sequence included in said oligonucleotide unit and said reagent includes at least three of said oligonucleotide units per said polymer molecule.

9. A kit according to claim 6, wherein said reactive function is selected from the group consisting of amine, imide, aldehyde, epoxy, thiol, halogenoalkyl and carboxylic acid functions, with the carboxylic acid function being selected from the group consisting of carboxylic acid and carboxylic acid activated in at least one form selected from the group consisting of acid halide form, anhydride form and ester form.

10. A kit according to claim 5, wherein said unsaturated monomers comprise a monomer selected from the group consisting of N-vinylpyrrolidone, methyl vinyl ether, ethylene, propylene and styrene.

11. A kit according to claim 1, wherein said oligonucleotide units are identical.

12. A kit for detecting at least one nucleic acid sequence of interest, with signal amplification, said kit having in suitable containers:

a reagent; and

a single species of tracer-labelled oligonucleotide probe reactive with said reagent;

wherein said reagent comprises at least one linear backbone copolymer molecule having a backbone chain formed by carbon-to-carbon covalent bonds, said at least one copolymer molecule comprising a first type of monomeric unit and a second type of monomeric unit, wherein at least one of said first type of monomeric unit carries a lateral substituent, said lateral substituent comprising an oligonucleotide unit that hybridizes with said sequence of interest and with said probe, said reagent being soluble in aqueous media and said at least one copolymer molecule including at least two said oligonucleotide units per said copolymer molecule.

13. A kit according to claim 12, wherein said copolymer molecule is formed by polymerization of a first unsaturated monomer and a second unsaturated monomer.

14. A kit according to claim 12, wherein said at least one lateral substituent is not present on said second type of monomeric unit.

15. A kit according to claim 12, wherein said oligonucleotide unit contains different hybridizing sequences that do not overlap for hybridizing with said sequence of interest and for hybridizing with said probe.

16. A kit according to claim 12, wherein said oligonucleotide unit contains different hybridizing sequences that do overlap for hybridizing with said sequence of interest and for hybridizing with said probe.

17. A kit according to claim 16, wherein said sequence of interest and said probe competitively hybridize with a hybridizing sequence included in said oligonucleotide unit.

18. A kit according to claim 16, wherein said sequence of interest and said probe competitively and coextensively hybridize with a hybridizing sequence included in said oligonucleotide unit and said at least one copolymer molecule includes at least three of said oligonucleotide units per said copolymer molecule.

19. A kit according to claim 12, wherein said at least one copolymer molecule contains from 3 to 100 of the oligonucleotide units per said copolymer molecule.

20. A kit according to claim 12, wherein said at least one copolymer molecule is a bipolymer.

21. A kit according to claim 13, wherein said first monomer carries a reactive function which is capable of reacting, either directly or by way of a bifunctional coupling agent, with a precursor of the oligonucleotide unit in order to form said lateral substituent.

22. A kit according to claim 21, wherein said reactive function is selected from the group consisting of amine, imide, aldehyde, epoxy, thiol, halogenoalkyl and carboxylic acid functions, with the carboxylic acid function being selected from the group consisting of carboxylic acid and carboxylic acid activated in at least one form selected from the group consisting of acid halide form, anhydride form and ester form.

23. A kit according to claim 21, wherein said first monomer is a derivative of an unsaturated carboxylic acid.

24. A kit according to claims 23, wherein said acid is selected from the group consisting of acrylic, methacrylic and maleic acids.

25. A kit according to claim 23, wherein said derivative is selected from the group consisting of anhydrides, acid halides and esters.

26. A kit according to claim 23, wherein said second monomer is selected form the group consisting of N-vinylpyrrolidone, methyl vinyl ether, ethylene, propylene and styrene.

27. A kit according to claim 12, wherein said oligonucleotide units are identical.