Method for detecting a target nucleic acid by using an interaction of two kinds of reagents

• US 5,705,346 A
• Filed: 06/25/1996
• Issued: 01/06/1998
• Est. Priority Date: 11/27/1992
• Status: Expired due to Term

• Current Assignee: Canon Kabushiki Kaisha (Canon Inc.)
• Original Assignee: Canon Kabushiki Kaisha (Canon Inc.)
• Inventors: Kawaguchi, Masahiro, Tomida, Yoshinori, Okamoto, Tadashi, Makino, Keisuke, Murakami, Akira, Yamamoto, Nobuko
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/671,829
• Time in Patent Office: 560 Days
• Field of Search: 435/6, 435/810, 436/501, 536/23.1, 536/24.1, 536/24.3-24.33, 935/77, 935/78
• US Class Current: 435/6.14
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2537/155 Cyclic reactions
• CPC Class Codes: C12Q 2563/113 the label being electroactive, e.g. redox labels
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

A method for detecting a target nucleic acid comprises the steps of reacting a sample with a probe in the presence of two or more kinds of reagents capable of being made an irreversible change capable of being detected and accumulating by an interaction through a double helix structure under a condition enabling the replication of the formation and dissociation of a hybrid composed of the target nucleic acid in the sample and the probe, accumulating the irreversible change caused by the interaction of the reagents, and then detecting the accumulated change.

Claims

1. A method for detecting a target nucleic acid which comprises:

(a) providing a sample containing a single stranded target nucleic acid having a specific base sequence;

(b) providing a probe having a base sequence complementary to the specific base sequence;

(c) forming a double-stranded hybrid between the target nucleic acid and the probe in the presence of two kinds of reagents both of which coexisting in the sample and being capable of interacting with each other through the double-stranded hybrid without direct physical contact between the two kinds of reagents to cause an irreversible detectable change in at least one of the reagents;

(d) causing repeated formation and dissociation of the double stranded hybrid to generate the interaction between the two kinds of reagents which results in accumulations of irreversibly changed reagent which provides the detectable change; and

(e) detecting the change provided from the accumulated irreversibly changed reagent to identify the target nucleic acid in the sample.

2. The method according to claim 1, wherein the step (d) is carried out by holding the sample under a condition that a temperature is fluctuated between points above and below a melting point of the target nucleic acid and probe.

3. The method according to claim 2, wherein the temperature is fluctuated within ±

5°

C. of the melting point.

4. The method according to claim 1, wherein the step (d) is carried out with a forward reaction of that in the double stranded hybrid is produced from the target and the probe, and with a back reaction of the double stranded hybrid is denatured to the target and the probe, being in equilibrium.

5. The method according to claim 4, wherein the step (d) is carried out by holding the sample under a predetermined temperature within ±

5°

C. of the melting point of the target nucleic acid and probe in the sample.

6. The method according to claim 5, wherein the predetermined temperature is the melting point.

7. The method for detecting a target nucleic acid according to claim 1, wherein the interaction between the two kinds of the reagents is accelerated by locating, through predetermined formation of the hybrid of the target nucleic acid and the probe, the two kinds of the reagents at a position where they are capable of interacting with each other.

8. The method for detecting a target nucleic acid according to claim 1, wherein the change by the interaction between the two kinds of the reagents is mediated by another material.

9. The method for detecting a target nucleic acid according to claim 1, wherein the irreversible change of the reagent is chemically detected.

10. The method for detecting a target nucleic acid according to claim 1, wherein the irreversible change of the reagent is optically detected.

11. The method for detecting a target nucleic acid according to claim 1, wherein the irreversible change by the interaction between the two kinds of reagents is based on a charge transfer between the reagents.

12. The method for detecting a target nucleic acid according to claim 11, wherein one of the two kinds of the reagents is an electron donor and the other is an electron acceptor.

13. The method for detecting a target nucleic acid according to claim 11, wherein the charge transfer is conducted through the stacking of the base pairs of the double helix of the nucleic acids formed as the result of the formation of the hybrid of the target nucleic acid and the probe.

14. The method for detecting a target nucleic acid according to claim 11, wherein the irreversible change is detected by the change in an electron spin resonance spectrum based on the donation or acceptance of electrons.

15. The method for detecting a target nucleic acid according to claim 1, wherein at least one of the two kinds of the reagents is an intercalator to the bases of the nucleic acid forming the double helix structure.

16. The method for detecting a target nucleic acid according to claim 1, wherein at least one of two kinds of the reagents is covalently bonded to the probe.

17. A kit for detecting a single stranded target nucleic acid, comprising

a probe having a complementary base sequence to that of the target nucleic acid to form a double-stranded hybrid;

first and second reagents, both of which being capable of interacting with each other through the hybrid without direct physical contact between the reagents to cause an irreversible, detectable change in at least one of the reagents; and

a reactor for hybridizing the target with the probe, the reactor being capable of controlling a temperature of a sample containing the target, the probe and the first and the second reagents so as to cause repeated formation and dissociation of the double-stranded hybrid to generate an interaction between the two reagents through the hybrid which results in accumulation of irreversibly changed reagent which provides the detectable change.

18. A method for detecting a target nucleic acid which comprises:

(a) providing a sample containing a single stranded target nucleic acid having a specific base sequence;

(b) providing a probe having a base sequence complementary to the specific base sequence and having a first and a second reagent bound to both ends of the probe, the reagents being capable of interacting with each other through a double-stranded nucleic acid which is formed between the probe and the target nucleic acid;

(c) forming a double-stranded hybrid between the target nucleic acid and the probe to interact the reagents with each other and causing an irreversible detectable change in at least one of the reagents by the interaction;

(d) causing repeated formation and dissociation of the double stranded hybrid to generate the interaction between the two kinds of reagents which results in accumulations of irreversibly changed reagent which provides the detectable change; and

(e) detecting the change provided from the accumulated irreversibly changed reagent to ascertain nucleic acid in the sample.

19. The method according to claim 18, wherein the first reagent is an electron donor and the second reagent is an electron acceptor.

20. The method according to claim 19, wherein the electron acceptor is a spin labeling agent containing spins which are decoupled irreversibly when the spin labelling agent receives charges.

21. The method according to claim 20, wherein the step (e) is conducted by detecting a signal representing a quantity of the spin labeling agent whose spins are decoupled.

22. The method according to claim 20, wherein the spin labeling agent is 4-hydroxy-2,2,6,6-tetramethylpiperidine.

23. The method according to claim 19, wherein the electron donor is fluorescein.

24. The method according to claim 19, wherein the probe has at least an 8-base length.

25. A method for detecting a target nucleic acid comprising the steps of:

(a) providing a sample containing a single stranded target nucleic acid having a specific base sequence;

(b) providing a probe having a base sequence complementary to the specific base sequence and having a first reagent bound to the probe;

(c) forming a double-stranded hybrid between the target nucleic acid and the probe in a presence of a second reagent capable of interacting with the first reagent through the double-stranded hybrid without direct physical contact between the two kinds of reagents, to interact the reagents with each other and causing an irreversible detectable change in at least one of the reagents by the interaction;

(d) causing repeated formation and dissociation of the double stranded hybrid to generate the interaction between the reagents which results in accumulations of irreversibly changed reagent which provides the detectable change; and

(e) detecting the change provided from the accumulated irreversibly changed reagent to ascertain the target nucleic acid in the sample.

26. The method according to claim 25, wherein the first reagent is an electron donor and the second reagent is an electron acceptor.

27. The method according to claim 26, wherein the electron acceptor is a spin labeling agent containing spins which are decoupled irreversibly when the spin labelling agent receives charges.

28. The method according to claim 27, wherein the step (e) is conducted by detecting a signal representing a quantity of the spin labeling agent whose spins are decoupled.

29. The method according to claim 27, wherein the spin labeling agent is 4-hydroxy-2,2,6,6-tetramethylpiperidine.

30. The method according to claim 26, wherein the electron donor is fluorescein.