# US 8,273,529 B2 · Method for hybridizing nucleic acids and hybridization apparatus

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- Number: US 8,273,529 B2
- Document ID: US8273529B2
- Status: Granted
- Kind: B2
- Application number: 11/702,594
- Filed: 2007-02-06
- Published: 2007-08-09
- Granted: 2012-09-25
- Earliest claimed: 2006-02-08
- Assignees: Canon Kabushiki Kaisha
- Inventors: Koji Nozato
- Web page: https://patentgrep.com/patent/US8273529B2

## CPC

- B01F 25/51: in which the mixture is circulated through a set of tubes, e.g. with gradual introduction of a component into the circulating flow
- B01F 33/30: Micromixers
- B01L 3/5027: {by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip}
- C12Q 1/6837: using probe arrays or probe chips (C12Q1/6874 takes precedence)
- B01J 2219/00608: DNA chips
- B01J 2219/00612: the surface being inorganic
- B01J 2219/00659: Two-dimensional arrays
- B01J 2219/00722: Nucleotides
- B01L 2300/0822: Slides
- B01L 2300/0877: Flow chambers
- B01L 2300/1805: Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
- B01L 2400/0487: fluid pressure, pneumatics
- B01F: MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING (mixing paints B44D3/06)
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT 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.

## Claims

### Claim 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 by circulating a sample solution containing the target nucleic acid, comprising:
a reaction step of allowing the target nucleic acid contained in the sample solution to react with the probe nucleic acid, wherein the reaction step is performed in a first region;
a recovery step of recovering the sample solution after the reaction step;
a heating step of heating the recovered sample solution to the denaturation temperature of the target nucleic acid or a higher temperature;
an adjustment step of adjusting a temperature of the heated sample solution to an optimum temperature for hybridization, wherein the adjustment step is performed in a second region different from the first region, and the temperature of the heated sample solution is adjusted directly from the temperature obtained in the heating step; and
a supplying step of supplying the sample solution containing the target nucleic acid in a denatured state resulting from the adjustment step to the first region to react with the probe nucleic acid,
wherein the sample solution comprises a single-stranded nucleic acid complementary to the target nucleic acid, and
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.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 1)

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

### Claim 4

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; 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,
wherein the sample solution comprises a single-stranded nucleic acid complementary to the target nucleic acid, and
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.

### Claim 5 (depends on 1)

The method according to claim 1, wherein all of the respective steps are performed by circulating the sample solution in a circulation path formed on a substrate.