Method of detecting and characterizing a nucleic acid or reactant for the application of this method

• US 4,581,333 A
• Filed: 04/29/1982
• Issued: 04/08/1986
• Est. Priority Date: 04/13/1978
• Status: Expired due to Term

• Current Assignee: Institut Pasteur
• Original Assignee: Institut Pasteur
• Inventors: Guesdon, Jean-Luc, Avrameas, Stratis, Kourilsky, Philippe, Cami nee Contamine, Brigitte
• Primary Examiner(s): Kepplinger, Esther M.
• Application Number:
  US06/373,017
• Time in Patent Office: 1,440 Days
• Field of Search: 435/6, 435/7, 435/172, 435/188, 435/805, 435/810, 435/176, 435/177, 435/5, 435/18
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2563/125 the label being enzymatic, i.e. proteins, and non proteins, such as nucleic acid with enzymatic activity
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 435/975 Kit

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.

Claims

1. 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.

2. The method of claim 1 wherein the chemical modification is performed prior to hybridization.

3. The method of claim 1 wherein the hybridized product is coupled directly to the enzyme.

4. The method of claim 3 wherein the enzyme is modified by avidin.

5. The method of claim 1 wherein the probe is chemically modified.

6. The method of claim 5 wherein to the hybrid there is affixed an anti-hybrid antibody, thereby forming a complex.

7. The method of claim 6 wherein to the complex there is coupled an enzyme.

8. The method of claim 5 wherein the probe is modified by biotin or avidin.

9. The method of claim 8 wherein the probe is modified by biotin through the intermediary of cytochrome C.

10. The method of claim 1 wherein the enzyme has an --SH group.

11. The method of claim 1 wherein the enzyme is beta-galactosidase.

12. The method of claim 11 wherein the substrate for the enzyme is orthonitrophenol galactoside.

13. The method of claim 1 wherein the complementary nucleic acid is selected from the group consisting of DNA and RNA.

14. The method of claim 13 wherein the DNA or RNA is single-stranded.

15. 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.

16. 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.

17. The method of claim 16 wherein the probe is a chemically-modified probe.

18. 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.

19. The composition of matter of claim 18 wherein the substrate is a chromogen.

20. The composition of matter of claim 18 wherein the chemical group is selected from the group consisting of biotin and avidin.

21. The composition of matter of claim 20 wherein the probe is modified with biotin and the enzyme with avidin.

22. 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.

23. The composition of matter of claim 22 wherein the chemical group is selected from the group consisting of biotin and avidin.

24. The composition of matter of claim 22 wherein the hybridized probe is bonded directly to the enzyme.

25. 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.

26. The article of claim 25 wherein the enzyme is modified by a chemical group.

27. The article of claim 25 which comprises a reactant for the lysis of the biological sample.

28. The article of claim 25 wherein the chemical is selected from the group consisting of biotin and avidin.

29. The article of claim 25 wherein the probe is coupled to biotin and the enzyme to avidin.

30. The article of claim 29 wherein the enzyme is beta-galactosidase.

31. 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.

32. The article of manufacture of claim 25 further comprising the nucleic acid to be detected in an amount sufficient for detection.