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

• US 5,695,936 A
• Filed: 06/14/1995
• Issued: 12/09/1997
• Est. Priority Date: 09/15/1993
• Status: Expired due to Term

• Current Assignee: Bio Merieux
• Original Assignee: Bio Merieux
• Inventors: Erout, Marie-Noelle, Mandrand, Bernard, Delair, Thierry, Pichot, Christian, Charles, Marie-Helene, Cros, Philippe
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Atzel, Amy
• Application Number:
  US08/433,505
• Time in Patent Office: 909 Days
• Field of Search: 435/6, 536/24.3, 935/19, 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 comprise use of 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 method for assaying at least one nucleic acid sequence of interest in a nucleic acid target, with signal amplification, comprising:

contacting a sample containing said nucleic acid target with

(i) a linear backbone polymer reagent which provides signal amplification,

wherein said reagent is soluble in aqueous media and comprises monomeric units forming a backbone chain of carbon-to-carbon covalent bonds wherein at least one monomeric unit has an oligonucleotide lateral substituent that hybridizes with said sequence of interest and hybridizes with a tracer-labeled probe, and wherein said reagent has at least two oligonucleotide lateral substituents per polymer molecule,

(ii) a single species of tracer-labeled probe that hybridizes to said oligonucleotide lateral substituent on said reagent,

(iii) a solid support, and

(iv) means for immobilizing said nucleic acid target on said solid support, under conditions that permit hybridization;

thereby forming a complex comprising, in order, said solid support, said nucleic acid target, said reagent, and said tracer-labeled probe; and

assaying for presence of said tracer-labeled probe immobilized on said solid support.

2. The method according to claim 1, wherein the reagent and the probe are each contacted with the sample without an intermediate washing step.

3. The method according to claim 1, wherein said sample is contacted with said reagent before said probe is contacted with said reagent.

4. The method according to claim 1, wherein said probe is contacted with said reagent before said sample is contacted with said reagent.

5. The method according to claim 1, wherein said sample is contacted with said solid support before said sample is contacted with said reagent.

6. The method according to claim 1, wherein said sample is contacted with said reagent before said sample is contacted with said solid support.

7. The method according to claim 1, wherein said oligonucleotide lateral substituents are identical.

8. A method for assaying at least one nucleic acid sequence of interest in a nucleic acid target, with signal amplification, comprising:

contacting a sample containing said nucleic acid target with

(i) a linear backbone copolymer reagent which provides signal amplification,

wherein said reagent is soluble in aqueous media and comprises a backbone chain of carbon-to-carbon covalent bonds formed by a first type of monomeric unit and a second type of monomeric unit, wherein said first type of monomeric unit has an oligonucleotide lateral substituent that hybridizes with said sequence of interest and hybridizes with a tracer-labeled probe, and wherein said reagent has at least two oligonucleotide lateral substituents per copolymer molecule,

ii) a single species of tracer-labeled probe that hybridizes to said oligonucleotide lateral substituent on said reagent,

iii) a solid support, and

iv) means for immobilizing said nucleic acid target on said solid support, under conditions that permit hybridization;

thereby forming a complex comprising, in order, said solid support, said nucleic acid target, said reagent, and said tracer-labeled probe; and

assaying for presence of said tracer-labeled probe immobilized on said solid support.

9. The method according to claim 8, wherein the reagent and the probe are each contacted with the sample without an intermediate washing step.

10. The method according to claim 8, wherein said sample is contacted with said reagent before said probe is contacted with said reagent.

11. The method according to claim 8, wherein said probe is contacted with said reagent before said sample is contacted with said reagent.

12. The method according to claim 8, wherein said sample is contacted with said solid support before said sample is contacted with said reagent.

13. The method according to claim 8, wherein said sample is contacted with said reagent before said sample is contacted with said solid support.

14. The method according to claim 8, wherein said oligonucleotide lateral substituents are identical.