Method for hybridizing nucleic acids and hybridization apparatus

Abstract

A method for hybridizing a target nucleic acid and a probe nucleic acid that can specifically bind to the target nucleic acid and that is immobilized on a substrate includes a first reaction step of allowing the target nucleic acid contained in a sample solution to react with the probe nucleic acid, a recovery step of recovering the sample solution after the first reaction step, a heating step of heating the recovered sample solution to the denaturation temperature of the target nucleic acid or a higher temperature, and a second reaction step of allowing the target nucleic acid contained in the sample solution after the heating step to react with the probe nucleic acid.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view showing an apparatus suitably used for performing a method of the present invention.

FIGS. 2A to 2C are schematic views showing the outline of a hybridization reaction.

FIG. 3 is a schematic view showing an apparatus suitably used for performing the method of the present invention.

FIG. 4 is a schematic view showing an apparatus suitably used for performing a method of the present invention.

FIG. 5 is a schematic view showing the outline of a detection method of the present invention.

FIG. 6 is a schematic view showing the outline of a detection method of the present invention.

FIG. 7 is a schematic view showing an apparatus suitably used for performing a method of the present invention.

FIG. 8 is a schematic view showing a cartridge suitably used for performing a method of the present invention.

Claims

1. A method for hybridizing a target nucleic acid and a probe nucleic acid that can specifically bind to the target nucleic acid and that is immobilized on a substrate, comprising: a first reaction step of allowing the target nucleic acid contained in a sample solution to react with the probe nucleic acid;a recovery step of recovering the sample solution after the first reaction step;a heating step of heating the recovered sample solution to the denaturation temperature of the target nucleic acid or a higher temperature; anda second reaction step of allowing the target nucleic acid contained in the sample solution after the heating step to react with the probe nucleic acid.

2. The method according to claim 1, wherein the temperature of the sample solution when the sample solution is supplied to the probe nucleic acid is controlled to the hybridization temperature.

3. The method according to claim 1, further comprising: a step of amplifying the target nucleic acid performed between the recovery step and the second reaction step, wherein the sample solution contains an amplification reagent.

4. The method according to claim 1, wherein the first and second reaction steps each comprises a step of controlling the temperature of the sample solution to the hybridization temperature.

5. The method according to claim 1, wherein the substrate is a glass plate.

6. The method according to claim 1, wherein the sample solution comprises a single-stranded nucleic acid complementary to the target nucleic acid.

7. The method according to claim 6, wherein the flow rate for supplying the probe nucleic acid with the sample solution is set so as to satisfy the following relationship: the time required for the sample solution whose temperature is controlled to the hybridization temperature to reach the probe nucleic acid<the time required for the number of single-stranded target nucleic acid molecules in the sample solution to decrease by half.

8. A hybridization apparatus for hybridizing a target nucleic acid and a probe nucleic acid that can specifically bind to the target nucleic acid and that is immobilized on a substrate, comprising: a setting section for setting the substrate;a supply path for supplying the substrate with a sample solution containing the target nucleic acid;a recovery mechanism for recovering the supplied sample solution; anda heater for heating the sample solution recovered by the recovery mechanism to the denaturation temperature or higher,wherein the recovery mechanism is configured so as to again supply the heated sample solution to the substrate.

9. The hybridization apparatus according to claim 8, wherein the recovery mechanism constitutes a circulation path connected to the setting section, and the heater is disposed at a halfway position of the circulation path.

10. The hybridization apparatus according to claim 8, wherein the sample solution comprises an amplification reagent, and the recovery mechanism comprises an amplifier for amplifying the target nucleic acid in the sample solution.

11. The hybridization apparatus according to claim 8, wherein the recovery mechanism comprises a temperature control unit for controlling the temperature of the sample solution to the hybridization temperature.

12. The hybridization apparatus according to claim 11, wherein the flow rate of the sample solution when the sample solution is again supplied by the recovery mechanism is set so as to satisfy the following relationship: the time required for the sample solution whose temperature is controlled to the hybridization temperature to reach the probe nucleic acid<the time required for the number of single-stranded target nucleic acid molecules in the sample solution to decrease by half.

13. The hybridization apparatus according to claim 8, wherein the recovery mechanism comprises a pump and the sample solution is recovered and supplied again by the pump.

14. The hybridization apparatus according to claim 8, wherein the recovery mechanism constitutes a circulation path connected to the setting section, and wherein part of the circulation path has an irregular-shaped cross section to adjust the flow rate of the sample solution.

15. The hybridization apparatus according to claim 8, wherein the recovery mechanism constitutes a circulation path connected to the setting section, and wherein a buffer area is provided in the circulation path to temporarily retain the sample solution.

16. A cartridge in which a probe nucleic acid that can specifically bind to a target nucleic acid is immobilized, comprising: a reaction chamber for accommodating the probe nucleic acid;a supply port for supplying the reaction chamber with a sample solution containing the target nucleic acid; anda recovery section for recovering the sample solution supplied to the reaction chamber.

17. The cartridge according to claim 16, wherein the recovery section constitutes a circulation path for circulating the sample solution in the reaction chamber.

18. A hybridization apparatus for hybridizing the target nucleic acid and the probe nucleic acid using the cartridge according to claim 16, comprising: a setting section for setting the cartridge;a supply mechanism for supplying the sample solution to a supply port of the cartridge;a recovery mechanism for recovering the sample solution supplied to the reaction chamber to the recovery section; anda heater for heating the recovered sample solution to the denaturation temperature or higher,wherein the recovery mechanism is configured so as to again supply the heated sample solution from the recovery section to the reaction chamber.

19. An analysis apparatus for hybridizing a labeled target nucleic acid and a probe nucleic acid that can specifically bind to the target nucleic acid and that is immobilized on a substrate and for detecting a label present on the substrate comprising: a setting section for setting the substrate;a supply path for supplying the substrate with a sample solution containing the target nucleic acid;a recovery mechanism for recovering the sample solution;a heater for heating the sample solution recovered by the recovery mechanism to the denaturation temperature or higher; anda detector for detecting the label,wherein the recovery mechanism is configured so as to again supply the heated sample solution to the substrate.

20. The analysis apparatus according to claim 19, further comprising an amplifier for amplifying the target nucleic acid in the sample solution.