Hybridization method, kit, and hybridization device

Abstract

To provide a hybridization method that is useful for utilizing automated equipment and that can use conventional high molecular weight volume exclusion agents. [Solution] A hybridization method for hybridizing a target nucleic acid contained in a biological sample with a probe capable of specifically binding to the target nucleic acid, comprising: a liquid composition application step in which a liquid composition containing at least the probe, an organic solvent, and water is ejected from a liquid ejection portion of a liquid ejection device and applied to the biological sample; a polymer application step in which a polymer selected from the group consisting of nonionic polymers and anionic polymers is applied to the biological sample so that it comes into contact with the liquid composition; a heating step in which the liquid composition and the biological sample are heated; and a cooling step in which the heated biological sample is cooled.

Description

[Technical Field]

[0001] The present invention relates to a hybridization method, a kit, and a hybridization device using an automated device. [Background technology]

[0002] With the recent development of molecular biology, the importance of analyzing substances present in living organisms has increased. For example, many attempts have been made to clarify the distribution and amount of specific gene sequences. Hybridization is a method used to analyze target genes in biological samples such as cells, tissues, and their extracts. This method involves binding a probe, which is made of a nucleic acid with a label attached to it, to form a complex with the target gene. By attaching various labeling molecules, detection is achieved by color development, luminescence, fluorescence, etc. This hybridization method is widely used in pathological diagnosis because it can detect and quantify specific genes associated with diseases, etc.

[0003] Detection by hybridization methods relies on the use of probes at precisely measured concentrations. If the probes are used at incorrectly high or low concentrations, adequate staining intensity may not be obtained.

[0004] The hybridization probes used in this process are expensive. Therefore, there is a demand for minimizing the amount used. There is also a demand for applying a liquid composition containing a hybridization probe only to a location where a specific gene sequence is expected to be present.

[0005] Patent Document 1 describes a hybridization method using a solid support to which a probe is reversibly bound. The method discloses a method for preparing a hybridization solution on a biological sample by first applying a buffer solution containing no probe but an additive such as dextran sulfate to the biological sample, and then applying a solid support thereon.

[0006] To ensure that the minimum amount of probe required for hybridization is accurately applied only to the location where a specific gene sequence is expected to be present, it is necessary to prepare a solid support on which the required amount of probe is placed in an area of ​​the same area and shape as the location. Furthermore, since a step is required to accurately align and cover the area of ​​a pre-applied buffer solution with the area where the probe is placed on the solid support, operator error is likely to occur in determining the location where the probe is applied.

[0007] Patent Document 2 discloses a method for dispensing reagent droplets of about 1 pL to about 50 pL onto a biological sample, and also discloses a method for dispensing the reagent droplets by inkjet printing.

[0008] Such automated devices are programmable and capable of customizing the addition of reagent droplets, thereby depositing the required amount of reagent droplets only at specific locations on the biological sample, reducing the labor and time required for detection and promoting consistency among results.

[0009] To accelerate hybridization, high-molecular-weight dextran sulfate (weight-average molecular weight >500,000) is commonly used as a volume exclusion agent mixed with the probe solution at a concentration of approximately 10 wt%. The probe solution containing dextran sulfate is a highly viscous liquid. Even with mechanically controlled pipette dispensers, the handling of highly viscous solutions impairs the quantitative and rapid dispensing and dispensing speed of automated devices. Even with inkjet printing, quantitative dispensing of the probe solution is difficult due to the high viscosity of the liquid.

[0010] Patent Document 3 discloses a hybridization method using low-molecular-weight dextran sulfate. It states that low-molecular-weight dextran sulfate polymer buffers with lower viscosity are useful for automated hybridization processes. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] Patent No. 3401722 [Patent Document 2] Special Publication No. 2018-517895 [Patent Document 3] Special Publication No. 2005-503762 Summary of the Invention [Problem to be solved by the invention]

[0012] In Patent Document 3, hybridization is performed using low-molecular-weight dextran sulfate. The present inventors ejected a probe solution using the low-molecular-weight dextran sulfate described in Patent Document 3 using an inkjet system, applied it to a biological sample, and performed hybridization. As a result, there were cases where the excluded volume effect was not strong, resulting in insufficient hybridization and making it impossible to detect genes. Therefore, an object of the present invention is to provide a hybridization method that is useful for utilizing automated equipment and that can use conventional high-molecular-weight volume-exclusion agents. [Means for solving the problem]

[0013] In order to solve the above problems, the present invention provides: A hybridization method for hybridizing a target nucleic acid contained in a biological sample with a probe capable of specifically binding to the target nucleic acid, comprising: (1) a liquid composition application step of ejecting a liquid composition containing at least the probe, an organic solvent, and water from a liquid ejection unit of a liquid ejection device and applying the liquid composition to the biological sample; (2) a polymer application step of applying a polymer selected from the group consisting of nonionic polymers and anionic polymers to the biological sample so that the polymer contacts the liquid composition; (3) a heating step of heating the liquid composition and the biological sample; (4) a cooling step of cooling the heated biological sample; The hybridization method is characterized by comprising: The present invention also provides 1. A kit for hybridizing a target nucleic acid contained in a biological sample, comprising: a liquid composition containing at least a probe capable of specifically binding to a target nucleic acid, an organic solvent, and water; A solid support having a nonionic polymer or an anionic polymer on its surface, or a liquid containing the nonionic polymer or the anionic polymer. The kit is characterized by comprising: The present invention also provides A hybridization device for hybridizing a target nucleic acid contained in a biological sample with a probe capable of specifically binding to the target nucleic acid, comprising: a liquid ejection unit capable of ejecting a liquid composition containing at least the probe, an organic solvent, and water toward the biological sample; a polymer applying unit that applies a nonionic polymer or an anionic polymer to the biological sample so that the biological sample contacts the liquid composition; and The hybridization device is characterized by having: [Effects of the Invention]

[0014] A liquid ejection device is utilized to enable hybridization using high molecular weight volume exclusion agents. DETAILED DESCRIPTION OF THE INVENTION

[0015] [First embodiment] The first embodiment is directed to a hybridization method. The hybridization method of the present invention comprises: A hybridization method for hybridizing a target nucleic acid contained in a biological sample with a probe capable of specifically binding to the target nucleic acid, comprising: (1) a liquid composition application step of ejecting a liquid composition containing at least the probe, an organic solvent, and water from a liquid ejection unit of a liquid ejection device and applying the liquid composition to the biological sample; (2) a polymer application step of applying a polymer selected from the group consisting of nonionic polymers and anionic polymers to the biological sample so that the polymer contacts the liquid composition; (3) a heating step of heating the liquid composition and the biological sample; (4) a cooling step of cooling the heated biological sample; The present invention is characterized by comprising:

[0016] It has been difficult to eject liquid compositions containing high-molecular-weight dextran sulfate at concentrations commonly used in hybridization using an inkjet system. On the other hand, liquid compositions containing no dextran sulfate or low-molecular-weight dextran sulfate are easy to eject. The inventors focused on adding high-molecular-weight dextran sulfate to the ejected liquid composition, and arrived at the present invention.

[0017] The hybridization method of the present invention is a hybridization method in which a target nucleic acid contained in a biological sample is hybridized with a probe capable of specifically binding to the target nucleic acid. Each item will be explained below.

[0018] <Liquid composition application step> The hybridization method of the present invention includes a liquid composition application step of ejecting a liquid composition containing at least the probe, an organic solvent, and water from a liquid ejection unit of a liquid ejection device and applying the liquid composition to the biological sample. Preferably, the liquid ejection device and the biological sample are arranged so that the liquid composition ejected from the liquid ejection unit of the liquid ejection device is applied to the biological sample, and the liquid composition is ejected onto the biological sample by setting the desired amount and position in consideration of the region of the biological sample where hybridization is to be performed.

[0019] In the present invention, when the polymer used in the polymer applying step is in the form of a molded article itself or a liquid, the liquid composition applying step may be performed after or before the polymer applying step. The liquid composition and the biological sample used in the liquid composition application step of the present invention may be separately preheated to a temperature higher than Tm, which will be described later.

[0020] <Polymer application step> The hybridization method of the present invention includes a polymer application step in which a polymer selected from the group consisting of nonionic polymers and anionic polymers is applied to the biological sample so that the polymer contacts the liquid composition. When the polymer is in a solid, gel, liquid, sheet, sponge, fiber, or other shaped form, or in the form of a cover glass or solid support to which the polymer is reversibly bound, the polymer may be applied by contact using a tool such as tweezers or an automated device such as a cover glass encapsulation device. When the polymer is in liquid form, the polymer may be applied using the liquid ejection device.

[0021] In the present invention, when the polymer is in the form of the molded article itself or in liquid form, the polymer application step may be carried out after the liquid composition application step or before the liquid composition application step.

[0022] In the polymer application step of the hybridization method of the present invention, it is preferable that the biological sample is coated with a solid support having the polymer on its surface, and that a liquid containing the polymer is applied to the biological sample.

[0023] <Heating process> The hybridization method of the present invention includes a heating step of heating the liquid composition and the biological sample. The liquid composition and the biological sample to which the polymer has been applied, or the biological sample to which the liquid composition has been applied, may be heated. The polymer application step may be carried out after heating the biological sample to which the liquid composition has been applied. The liquid composition and the biological sample to which the polymer has been applied may be heated separately before the liquid composition application step, or the liquid composition and the biological sample may be heated separately.

[0024] The heating step in the present invention may use a heat source device that can be controlled to a constant temperature, or the above device equipped with a temperature variable device that can programmably control the temperature cycle and incubation time.

[0025] <Cooling process> The hybridization method of the present invention includes a cooling step of cooling the heated biological sample. From the viewpoint of efficiently performing hybridization, it is preferable to cool the biological sample that has been subjected to the liquid composition application step, the polymer application step, and the heating step.

[0026] The cooling step in the present invention may use a heat source device that can be controlled to a constant temperature, or the above device equipped with a temperature variable device that can programmably control the temperature cycle and incubation time.

[0027] <Biological samples> The biological sample in the present invention is not particularly limited as long as it may contain the gene of interest. Examples include cultured cells, animal body fluids (e.g., blood, serum, plasma, cerebrospinal fluid, sweat, saliva, urine, and diluted or concentrated solutions thereof), hair, excrement, organs, organ extracts, tissues, tissue extracts, animals and plants themselves, or formalin-fixed, paraffin-embedded samples or dried forms thereof. Other examples include river water, lake water, seawater, water from waterworks and sewage systems, soil, and the like, which contain biological substances and may contain the gene of interest.

[0028] The biological sample may be immobilized on a solid phase, which facilitates the step of removing unhybridized probes after hybridization, such as by washing.

[0029] Examples of the solid phase include, but are not limited to, polystyrene resin particles, nylon resin particles, glass particles, glass plates, polystyrene microplates, latex particles, various magnetic particles, metal particles, metal-coated particles, metal plates, metal-coated plates, various porous bodies, various electrodes, etc. Among these, glass plates such as glass slides are preferably used.

[0030] A solid phase that has been surface-modified to strengthen the bond between the solid phase and a biological sample is preferably used in the present invention. The bond between the solid phase and the biological sample is not particularly limited as long as it does not inhibit hybridization in the biological sample. Examples of surface modifications of the solid phase include the addition of functional groups such as amino groups, carboxy groups, thiol groups, disulfide groups, and hydroxy groups, or the addition of compounds having specific amino acid sequences. Furthermore, a solid phase that has been subjected to a hydrophilization treatment to suppress nonspecific adsorption is also preferably used in the present invention.

[0031] <Target nucleic acid> The target nucleic acid is any nucleic acid, such as DNA or RNA, contained in the biological sample and having a specific target sequence to be detected. For example, the target nucleic acid may be nucleic acid present within a cell, such as in the cell nucleus, outside the cell nucleus, cytoplasm, or organelle; nucleic acid present outside a cell, such as in blood, urine, or extracellular vesicles; or nucleic acid present in the natural environment, such as rivers, lakes, oceans, water supply and sewage systems, or soil.

[0032] The target sequence can be a genomic or subgenomic target sequence, e.g., from a eukaryotic genome, such as the human genome, or a pathogen genome, such as a viral genome, bacterial genome, or fungal genome. The target nucleic acid can be a sequence associated with a disease. The disease or condition-associated target sequence is selected so that detection of the target nucleic acid using hybridization can infer information related to the disease or condition, such as diagnosis, disease, treatment response prediction, prognosis, or prognosis. For example, detection of the target nucleic acid in a biological sample obtained from a subject can provide information related to the subject's disease or condition.

[0033] For example, a target nucleic acid associated with cancer may be selected. In this case, the genomic target sequence may include a gene or chromosomal region associated with cancer. Examples include HER2, c-Myc, MYCN, KRAS, BRAF, FGFR2, ALK, ROS1, IGH, ABL, BCL1, BCL2, BCL6, p53, TOP2A, MET, and genes encoding other receptors or signal transducers. Detection of these target sequences can provide information related to translocation, deletion, duplication, or amplification of the target nucleic acid. HER2, a gene that plays a role in regulating cell growth, is amplified in human breast cancer, ovarian cancer, gastric cancer, and other cancers. Therefore, the HER2 gene can be used as a genomic target nucleic acid sequence.

[0034] <probe> Complementary nucleic acids that have a base sequence complementary to a target sequence bind with high selectivity (hybridization) due to the affinity between the complementary sequences. Therefore, complementary nucleic acids can be used in hybridization as detection reagents (probes) for detecting target nucleic acids. Hybridization can also be performed to detect homology between target nucleic acids and complementary nucleic acids, or to identify the expression level or location of target nucleic acids.

[0035] The probe sequence may be any base sequence that allows the target nucleic acid and the probe to bind to each other to form a double strand, and may be completely complementary to the target sequence or may have a sequence complementary to at least a portion of the target nucleic acid. Probes can be obtained by extraction from a living organism, artificially synthesized, or by amplifying naturally occurring nucleic acids. Probes can also contain any suitable nucleic acid, such as RNA, DNA, LNA, PNA, or a combination thereof.

[0036] To use the complementary nucleic acid as a probe, it is preferable to attach a labeling molecule to it. Examples of the labeling molecule include, but are not limited to, hapten labels such as digoxigenin, biotin labels, fluorescent dyes such as fluorescein isocyanate (FITC) and fluorescent protein labels, enzyme labels such as peroxidase and alkaline phosphatase, and the like. 32 Radioisotope labels such as phosphate with P can be used.

[0037] Alternatively, both the nucleic acid and the complementary nucleic acid can be labeled, such as by labeling a nucleic acid with a fluorescent dye and a complementary nucleic acid with a quencher, and detecting quenching when the two hybridize. After hybridization, detection can also be performed by immunohistochemical staining, utilizing an antigen-antibody reaction using an anti-digoxigenin antibody, anti-FITC antibody, or the like. These can be detected by detecting color development, luminescence, fluorescence, radiation, etc.

[0038] The probes used in the present invention may be a single probe specific to a target nucleic acid, or may be used in combination with a second probe that provides an indication of chromosome number. Probes specific to other target nucleic acids may also be used in combination. By using multiple probes in combination, hybridization to different target sequences in the same biological sample can be simultaneously performed.

[0039] When multiple probes are used, each probe can be labeled with a different labeling molecule to detect each target sequence individually. For example, when a fluorescent dye is used as a labeling molecule, the second probe can be labeled with a fluorescent dye with a different fluorescent wavelength from that of the fluorescent dye labeled on the first probe, allowing the probes to be detected individually by detecting their respective fluorescent wavelengths.

[0040] For example, a probe targeting the centromere of a chromosome can be used as a second probe to provide an index of chromosome number. In detecting the HER2 gene, a HER2 probe for detecting the HER2 gene can be used in combination with a CEP17 probe that specifically binds to the CEP17 gene, which is located at the centromere of chromosome 17.

[0041] Hybridization using these probes makes it possible to evaluate the presence or absence of HER2 gene amplification. For example, in normal cells, such as healthy cells, the ratio of HER2 signal counts to CEP17 signal counts (HER2 / CEP17 ratio) is less than 2, but cancer cells with increased HER2 gene expression show a HER2 / CEP17 ratio of 2 or more. HER2 gene amplification can be determined by calculating the HER2 / CEP17 ratio.

[0042] <Organic solvents> The organic solvent used in the present invention is not particularly limited as long as it has the potential to promote gene denaturation or lower the melting temperature (Tm) of DNA or nucleic acid. Denaturation refers to weakening the base-base interactions within the gene to allow the single-stranded portion to grow. In view of the water-solubility of the target nucleic acid and probe, the organic solvent used in this embodiment is preferably a polar solvent that is miscible with water.

[0043] For gene denaturation, organic solvents with hydrogen acceptor or hydrogen donor properties may be used to weaken hydrogen bonds, which are part of base-base interactions. Furthermore, aprotic polar solvents may be used to weaken hydrophobic interactions. Examples of suitable solvents include formamide, N-methylformamide, N-methylacetamide, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, sulfolane, 1,4-dioxane, ethylene carbonate, γ-butyrolactone, 2-pyrrolidone, and combinations thereof.

[0044] For optimal hybridization, it is preferable to add an organic solvent to a concentration of 1 to 50% (v / v). Formamide can be suitably used to adjust the melting point of hybridization. Tm can be adjusted by the concentration of the organic solvent. For example, 1% formamide typically lowers Tm by about 0.6°C. For reactions at an optimal Tm, it is preferable to add formamide to a concentration of 10 to 50% (v / v).

[0045] <Liquid composition> The liquid composition of the present invention is a liquid composition containing at least the probe, the organic solvent, and water. It is preferable that the liquid composition does not contain nuclease from the viewpoint of the stability of the target nucleic acid and the probe during hybridization. It is preferable that the liquid composition contains a salt or a buffer solution to ensure stable hybridization. The liquid composition of the present invention preferably does not contain a nonionic polymer or anionic polymer in order to maintain low viscosity and improve dischargeability, but may contain a nonionic polymer or anionic polymer.

[0046] The type and concentration of salt are not particularly limited as long as hybridization can be performed. For example, buffer solutions commonly used in hybridization include SSC (saline-sodium citrate) buffer solution consisting of sodium citrate and sodium chloride, and SSPE (saline-sodium phosphate-EDTA) buffer solution consisting of sodium chloride, sodium dihydrogen phosphate, and EDTA. Tm can also be adjusted by the salt concentration.

[0047] Therefore, the preferred salt concentration for hybridization varies depending on the target nucleic acid, the base sequence and length of the probe used, the probe concentration used, etc., but the most common compositions include 1 to 10x SSC (1x SSC composition: 0.015 M sodium citrate and 0.15 M sodium chloride, pH 7.0) and 1 to 20x SSPE (1x SSPE composition: 0.01 M sodium dihydrogen phosphate, 0.15 M sodium chloride and 1 mM EDTA, pH 7.4).

[0048] To enhance the binding strength between the target nucleic acid and the probe, the salt concentration is preferably adjusted to 2 to 5×SSC or 2 to 10×SSPE. The pH is preferably 5.0 to 10.0, and more preferably 6.0 to 8.5.

[0049] To suppress nonspecific binding of the probe, Denhardt's solution, which consists of bovine serum albumin, Ficoll, and polyvinylpyrrolidone, may be included. It is preferable to use 1 to 5x Denhardt's solution (1x Denhardt's solution composition: 0.02% bovine serum albumin, 0.02% Ficoll, 0.02% polyvinylpyrrolidone).

[0050] Furthermore, from the viewpoint of enhancing the specificity of hybridization, a blocking reagent that prevents nonspecific binding of the probe can be used, such as total RNA extracted from yeast, DNA derived from herring sperm, or DNA derived from salmon sperm, which are available in large quantities. The content of the blocking reagent is preferably 0.01 to 10 mg / mL, and more preferably 0.01 to 1 mg / mL.

[0051] A surfactant may be added to enhance the permeability of the probe. The surfactant is preferably a nonionic or anionic surfactant. Examples include anionic surfactants such as sodium alkyl sulfonate, sodium alkyl benzene sulfonate, sodium dialkyl sulfosuccinate, and sodium alkyl carboxylate; nonionic surfactants such as acetylene glycol surfactants, polyoxyethylene alkyl ethers, polyoxyethylene alkyl phenyl ethers, polyoxyethylene alkyl esters, polyoxyethylene polyoxypropylene glycol, sorbitan alkyl esters, polyoxyethylene sorbitan alkyl esters, glycerin alkyl esters, alcohol alkoxylates, and polyoxyethylene hydrogenated castor oil; and amphoteric surfactants such as alkyl betaines, alkyl dimethylamine oxides, and cholate derivatives. The surfactant content is preferably 0.1% by mass or more and 1.0% by mass or less.

[0052] In addition to the above components, the liquid composition of the present invention may contain various additives, such as antifoaming agents, pH adjusters, viscosity adjusters, rust inhibitors, preservatives, antifungal agents, antioxidants, antireducing agents, and chelating agents, as needed.

[0053] <Viscosity> The optimal viscosity of the liquid composition in the present invention depends on the liquid ejection method of the liquid ejection device, but a lower viscosity is preferable. This is because a low-viscosity liquid composition flows easily and is easily extruded or ejected from the liquid ejection portion. This facilitates ejection, allowing the liquid composition to be precisely applied to a biological sample in an accurate amount and at an accurate position on the biological sample. Furthermore, since the liquid composition can be applied quickly, a large number of specimens can be processed in a short period of time.

[0054] Furthermore, carryover of the liquid composition in the liquid ejection section is less likely to occur, and errors between samples can be reduced. For example, in the case of a thermal inkjet liquid ejection device according to the present invention, the viscosity of the liquid composition is preferably 0.3 mPa·s or more and 10 mPa·s or less, and more preferably 0.4 mPa·s or more and 5 mPa·s or less. In the present invention, the viscosity can be confirmed using a viscometer.

[0055] <Hybridization> Hybridization is carried out by heating to a temperature higher than Tm to denature the target nucleic acid and probe, and then incubating at a temperature lower than Tm to allow the target nucleic acid and probe to bind to each other. The hybridization conditions used in the present invention are similar to those used in conventional hybridization.

[0056] The optimal temperature for hybridization, denaturation time, incubation time for binding of the target nucleic acid and the probe, etc. depend on the base sequence and length of the target nucleic acid and the probe used, the probe concentration used, salt concentration, organic solvent concentration, etc. Tm can be determined by measuring the change in absorbance at 260 nm of the target nucleic acid or probe depending on the temperature, or can be determined by theoretical calculation from base sequence information.

[0057] The denaturation temperature can be set depending on the Tm of the target nucleic acid and probe, and is generally carried out in the range of 65 to 100°C. If the target nucleic acid has a stable sequence, it is preferable to carry out the denaturation at a temperature in the range of 80 to 98°C to promote denaturation. The denaturation time is affected by heat propagation in the denaturing container, substrate, etc., but is carried out in the range of 15 seconds to 30 minutes. From the viewpoint of sufficient denaturation, it is preferable to carry out the denaturation for 5 to 20 minutes.

[0058] The incubation temperature for binding between the target nucleic acid and the probe is generally 10 to 30° C. lower than the Tm, and from the viewpoint of efficient binding, it is preferably 30 to 72° C., more preferably 32 to 45° C. The incubation time for binding between the target nucleic acid and the probe is generally about 0.5 to about 96 hours, and from the viewpoint of sufficient binding, it is preferably about 2 to about 16 hours.

[0059] <Liquid discharge device> The method for applying a liquid composition according to the present invention is characterized by comprising a step of discharging the liquid composition from a liquid discharge portion of a liquid discharge device and applying it to a biological sample, and when applying, the amount of the liquid composition to be applied to the biological sample can be changed.

[0060] Examples of liquid ejection devices include manual micropipettes, electric micropipettes, microdispensers, automated microdispensers, and devices that eject droplets from nozzles using energy generating elements, i.e., liquid ejection devices that use the inkjet method. These liquid ejection devices may be devices in which the ejection amount and ejection position can be programmably controlled.

[0061] The liquid ejection method is not particularly limited as long as it is capable of ejecting the liquid composition. Examples of the liquid ejection method include an air replacement method, a forced replacement method, an air syringe method, a tubing method, a plunger method, a positive load method, a hybrid plunger method, an endless piston method, and an inkjet method. In the present invention, the ejection method of the liquid ejection device is preferably an inkjet method, since it can eject minute droplets. Furthermore, among the inkjet methods, a thermal inkjet method and a piezo inkjet method can be suitably used in the present invention, and the inkjet method is preferably a thermal method.

[0062] <Liquid discharge part> Examples of the liquid ejection part include a pipette tip, a nozzle, a needle, and an orifice. It may be connected to or integrated with a tank for storing the liquid. In the inkjet method, a liquid ejection head in which the nozzle and tank are integrated can be used. Examples of liquid ejection heads include a type that ejects the liquid composition by generating film boiling in the liquid composition using an electrothermal converter to form bubbles, a type that ejects the liquid composition using an electromechanical converter, and a type that ejects the liquid composition using static electricity. Among these, from the viewpoint of performing high-speed, high-density printing, a liquid ejection head that uses an electrothermal converter, i.e., a liquid ejection head having a heating element that imparts thermal energy to the liquid composition, is preferred.

[0063] <Solid phase support> In the present invention, the solid support preferably has a nonionic or anionic polymer on its surface, and the solid support preferably has a sheet or plate shape. Here, a plate-like shape refers to a surface that is not flexible, and a sheet-like shape refers to a surface that is not flexible. Examples of solid supports include slide glasses, cover glasses, plastic plates, and plastic sheets. In the present invention, the solid support is preferably a microscope slide or a cover glass.

[0064] In general hybridization, in order to prevent the solvent from drying out due to heating during the process of denaturing the target nucleic acid or probe, the target nucleic acid or probe is covered with a cover glass and the periphery of the cover glass is sealed with a seal such as paper bond before heating. In the present invention, by using a cover glass as a solid support, it is possible to add a nonionic or anionic polymer to the liquid composition applied to the biological sample and simultaneously cover it with a conventional cover glass.

[0065] From the viewpoint that hybridization can be performed with the same number of steps as in general hybridization, it is preferable to use a cover glass as the solid support. In order to place a nonionic or anionic polymer on the solid support, a solid support that has been subjected to hydrophilization treatment such as plasma treatment may be used.

[0066] The solid support of the present invention may contain a reagent necessary for hybridization in addition to the nonionic or anionic polymer. From the viewpoint of improving hybridization in the present invention, a reagent that affects the ejection of the liquid composition may be placed on the solid support.

[0067] The solid support of the present invention may be a lid or a part of a lid of a lidded container containing a solid phase on which a biological sample is immobilized. Preferably, the lid of the lidded container is designed to be in contact with the solid phase placed in the container. This is because by enclosing the solid phase in the container, a nonionic or anionic polymer can be added to the liquid composition applied to the biological sample through contact between the solid phase and the lid.

[0068] <Nonionic or anionic polymer> The nonionic or anionic polymer in the present invention is used mainly for the purpose of obtaining a volume exclusion effect, and is not particularly limited as long as it has the potential to promote hybridization. Examples include polyvinyl alcohol, polyethylene glycol, polysaccharides, DNA, RNA, polyacrylates, polymethacrylates, polystyrene sulfonates, polyvinyl sulfonates, dextran sulfate, chondroitin sulfate A, chondroitin sulfate C, and copolymers, conjugates, and complexes thereof.

[0069] Nonionic or anionic polymers do not adsorb or bind to the target gene or probe due to electrostatic interactions, so they do not inhibit hybridization between the target gene and the probe, and can promote hybridization through the excluded volume effect. Among these, anionic polymers are preferred in the present invention, as they exert electrostatic repulsion on the anionic gene or probe, further promoting hybridization. In the present invention, the anionic polymer is preferably a sulfonic acid polymer, and more preferably a dextran sulfate salt. The chemical compounds of the present invention can be confirmed by nuclear magnetic resonance (NMR), infrared spectroscopy, or pyrolysis GC / MS.

[0070] To obtain the excluded volume effect, the nonionic or anionic polymer preferably has a high molecular weight. This is because a high molecular weight increases the volume occupied by the polymer molecules and the entropy of the system, thereby promoting hybridization between the target gene and the probe. In the present invention, the weight-average molecular weight of the nonionic or anionic polymer is preferably 100,000 or more and 5,000,000 or less, and more preferably 500,000 or more and 5,000,000 or less. In the present invention, the weight-average molecular weight can be confirmed by light scattering measurement or size exclusion chromatography.

[0071] The nonionic or anionic polymer of the present invention may be applied to a biological sample in any form, including solid, gel, liquid, sheet, sponge, or fiber, as long as it is soluble in the liquid composition. In this specification, a film-like polymer, regardless of thickness, is referred to as a sheet. Polymers in these forms may be applied to a biological sample in their entirety. For example, when using a sheet-like polymer molded product, the molded product may be placed on the liquid composition on the biological sample to cover it before being coated with a cover glass or solid support.

[0072] This allows the polymer to dissolve in the liquid composition applied to the biological sample, thereby preparing a liquid composition containing a nonionic or anionic polymer suitable for hybridization. That is, hybridization can be carried out by coating a molded body onto the biological sample onto which the liquid composition has been ejected, further coating a cover glass or solid support, and then heating and cooling the resulting mixture. Alternatively, the biological sample may be coated with a molded body in advance, and the liquid composition may be ejected onto the molded body.

[0073] The biological sample may be applied via contact with a solid support to which a nonionic or anionic polymer is reversibly bound. A solid support may be placed on top of the liquid composition applied to the biological sample, covering the surface to which the polymer is bound, so that the surface comes into contact with the liquid composition. This allows the polymer from the solid support to dissolve or mix with the liquid composition applied to the biological sample. Hybridization can then be carried out by heating and cooling. The solid support itself may also be made of a nonionic or anionic polymer.

[0074] A nonionic or anionic polymer solution can be placed on a solid support by a known application method such as spraying, casting, or coating to obtain a solid support to which a nonionic or anionic polymer is reversibly bound. The solid support on which the nonionic or anionic polymer solution is applied may be used with the solution still on it without drying, or may be used after drying.

[0075] The liquid polymer may be a solution containing a nonionic or anionic polymer. The polymer may be applied to the biological sample by adding a polymer solution to the liquid composition applied to the biological sample and mixing the polymer solution. Alternatively, a polymer solution may be applied to the biological sample in advance, and then the liquid composition may be applied to the polymer solution. The method for adding the polymer solution is not particularly limited, as long as the polymer solution can be dropped or ejected. The liquid ejection device described above may also be used.

[0076] The solvent for the polymer solution may be water, a buffer solution, the organic solvent used in the liquid composition, a mixed solution of the organic solvent with water or a buffer solution, etc. From the viewpoint of uniformly mixing the mixed solution of the liquid composition and the polymer solution, it is preferable to use a polymer solution in which the solvent is a mixed solution of the organic solvent used in the liquid composition and the buffer solution in the same volume ratio.

[0077] The amount of nonionic or anionic polymer used when applying to a biological sample in the present invention depends on the area within the biological sample where hybridization is desired, but is preferably at least 10% by mass of the liquid volume of the liquid composition applied to that area. Since nonionic or anionic polymers are available at lower cost than probes, when using a solid support with an area larger than that area, the nonionic or anionic polymer may be applied to only a portion of the solid support or to the entire solid support, as long as the applied portion of the nonionic or anionic polymer can be brought into contact with the liquid composition.

[0078] Second Embodiment The second embodiment is directed to a kit. The kit of the present invention comprises: 1. A kit for hybridizing a target nucleic acid contained in a biological sample, comprising: a liquid composition containing at least a probe capable of specifically binding to a target nucleic acid, an organic solvent, and water; A solid support having a nonionic polymer or an anionic polymer on its surface, or a liquid containing the nonionic polymer or the anionic polymer. The present invention is characterized by having the following. The items described in the first embodiment are the same as those described in the first embodiment, so the description will be omitted.

[0079] The kit of the present invention preferably has a solid support in the form of a sheet or plate. In the kit of the present invention, the probe capable of specifically binding to the target nucleic acid, the liquid composition containing at least an organic solvent and water may be mixed or separate, and may be contained in a container either individually or mixed together. The kit may also include additional diluents, blocking agents, positive controls, negative controls, instructions, and the like. Examples of positive controls include tissue sections and liquid samples that clearly contain the target nucleic acid to be measured. Furthermore, the kit of the present invention may also include a cartridge container that can be attached to an inkjet ejection head for ejecting the liquid composition.

[0080] Third Embodiment The third embodiment is directed to a hybridization device. The hybridization device of the present invention comprises: A hybridization device for hybridizing a target nucleic acid contained in a biological sample with a probe capable of specifically binding to the target nucleic acid, comprising: a liquid ejection unit capable of ejecting a liquid composition containing at least the probe, an organic solvent, and water toward the biological sample; a polymer applying unit that applies a nonionic polymer or an anionic polymer to the biological sample so that the biological sample contacts the liquid composition; and The present invention is characterized by having the following. The items explained in the first and second embodiments are the same as those explained in the first and second embodiments, so the explanation will be omitted.

[0081] In the device, the ejection head capable of ejecting a liquid composition containing at least a probe capable of specifically binding to a target nucleic acid, an organic solvent, and water may be removable. Multiple ejection heads may be attached so that unique detection can be selected and performed according to various target nucleic acids. The device may also be provided with a stage on which a slide having a biological sample attached thereto is placed. The stage on which a slide having a biological sample attached thereto is placed may be a stage that can accommodate only one slide, or a stage that can accommodate multiple slides arranged side by side. A stage that can accommodate multiple slides arranged side by side is preferred from the viewpoint of being able to simultaneously apply the liquid composition to each of the multiple slides.

[0082] The apparatus may be equipped with a programmable device that can place a solid support having a nonionic or anionic polymer on its surface or a liquid containing a nonionic or anionic polymer on the slide. The recording head may be provided with a second ejection head or a second liquid ejection device capable of dropping or ejecting a polymer solution in which a nonionic or anionic polymer is dissolved.

[0083] A temperature variable device capable of programmably controlling the temperature cycle and incubation time may be provided to enable heating and cooling during hybridization. Alternatively, a thermal block whose temperature is controlled by the temperature variable device may be provided, or the stage on which the slide is placed may be a thermal block whose temperature is controlled. [Example]

[0084] The present invention will be described in more detail below using examples and comparative examples. The present invention is not limited to the following examples in any way as long as the gist of the invention is not exceeded. <Biological samples> The biological samples used were HER2 FISH Posicon slides (PS-17006, Pathology Institute Co., Ltd.) with three types of cultured cell lines affixed to them according to the HER2 gene amplification: negative (no HER2 gene amplification) "0," weakly positive (mild HER2 gene amplification) "1+," and strongly positive (strong HER2 gene amplification) "2+."

[0085] <Buffer> The buffer used was 20×SSC (319-90015, manufactured by Nippon Gene Co., Ltd.) Various liquid compositions were prepared by adding this buffer.

[0086] <probe> The HER2 probe used was SureFISH 17q12 HER2 191kb P20 GR (G110969G-8, Agilent Technologies), which is labeled with the fluorescent dye FITC (excitation wavelength 498nm, emission wavelength 522nm).

[0087] The CEP17 probe used was SureFISH Chr17 CEP 436kb P20 RD (G110291R-8, Agilent Technologies), which is labeled with the fluorescent dye Cy3 (excitation wavelength 550nm, emission wavelength 570nm). These reagents are solutions in which probes are dissolved, and the manufacturer's protocol states that they should be diluted 10-fold before use in hybridization.

[0088] <Organic solvents> As the organic solvent, formamide (000-32645, manufactured by Kishida Chemical Co., Ltd.) and ethylene carbonate (809950-25G, manufactured by Sigma-Aldrich Co.) were used.

[0089] <Blocking reagent> Salmon testis-derived DNA solution (D7656, Sigma-Aldrich) was used as a blocking reagent.

[0090] <Nonionic or anionic polymer> The anionic polymers used were dextran sodium sulfate with a weight-average molecular weight of 5,000 (DB005, manufactured by TdB Labs), dextran sodium sulfate with a weight-average molecular weight of 20,000 (DB013, manufactured by TdB Labs), dextran sodium sulfate with a weight-average molecular weight of 100,000 (DB016, manufactured by TdB Labs), dextran sodium sulfate with a weight-average molecular weight of 500,000 (DB050, manufactured by TdB Labs), and dextran sodium sulfate with a weight-average molecular weight of 2,000,000 (DB054, manufactured by TdB Labs).

[0091] <Solid phase support> As a solid support, a cover glass (C218181, manufactured by Matsunami Glass Industry Co., Ltd.) having a size of 18 mm x 18 mm was used.

[0092] <Preparation of liquid composition and viscosity measurement> The components were mixed to the contents shown in Table 1. Solutions A-1, A-10, and B-1 did not contain dextran sodium sulfate.

[0093] [Table 1]

[0094] <Preparation of solid support> Dextran sodium sulfate solutions (10 μL) containing dextran sodium sulfate with weight-average molecular weights of 100,000, 500,000, and 2,000,000 were dissolved in nuclease-free water to the weight percentages shown in Table 2. The solution was spread over the entire cover glass and then vacuum dried. C-6 does not contain dextran sodium sulfate. C-7 was prepared by spreading a solution (10 μL) of a 10-fold diluted probe reagent in nuclease-free water over the entire cover glass and then vacuum drying.

[0095] [Table 2]

[0096] <Preparation of dextran sulfate sodium sheet> 0.08 mL of a dextran sulfate sodium solution, prepared by dissolving 8.0 mg of dextran sulfate sodium with a weight-average molecular weight of 500,000 in nuclease-free water, was spread over a 20 mm x 20 mm area of ​​a glass plate and dried. The glass plate was then immersed in ethyl acetate and dried. This procedure was repeated twice. The dextran sulfate film on the glass plate was peeled off using tweezers to obtain a dextran sulfate sodium sheet.

[0097] <Preparation of dextran sulfate sodium solution 1> Dextran sulfate sodium solution 1 was prepared by adding 0.4 mL of formamide, 0.1 mL of 20×SSC, and 0.5 mL of nuclease-free water to 340 mg of dextran sulfate sodium having a weight-average molecular weight of 500,000.

[0098] (viscosity measurement) The viscosity of each of the liquid compositions, Liquid Compositions Nos. A-1 to A-10 and B-1 and B-2, was measured using a viscometer (RE-85L, manufactured by Toki Sangyo Co., Ltd.).

[0099] (Evaluation of ejection properties) A thermal jet liquid ejection head with a nozzle diameter of 3 μm was prepared, and a tank connected to it was filled with BC-345Bk ink (Canon). The ejection head was driven by a controller electrically connected to the liquid ejection head, and liquid was ejected from the ejection port at a frequency of 15 kHz and a voltage of 13 volts. The mass of the ejected liquid was measured and taken as 100%.

[0100] Next, liquid compositions A-1 to A-10 and B-1 to B-2 were ejected under the same conditions, and the ejection mass ratio was calculated. This procedure was repeated five times, and the average ejection mass ratio was calculated. The ejection properties of the ejected liquid were evaluated according to the following criteria.

[0101] "Rating A" and "Rating B" indicate a level at which the liquid composition can be continuously and stably ejected, while "Rating C" indicates that ejection of the liquid composition is unstable or difficult. (Evaluation criteria) A: The average value is 90% or more B: The average value is 30% or more and less than 90% C: The average value is less than 30%

[0102] Table 3 shows the evaluation results of the viscosity and ejection properties of Liquid Compositions Nos. A-1 to A-10 and Liquid Compositions Nos. B-1 and B-2.

[0103] [Table 3]

[0104] <Evaluation of dyeing> Example 1 The deparaffinization process of the biological sample, the pretreatment process of the biological sample, hybridization, and observation were carried out as follows. (1) Deparaffinization of biological samples a) The HER2 FISH positive control slide was immersed in a container containing FastSolv (Pharma Co., Ltd.), a xylene substitute, and allowed to stand at room temperature for 10 minutes. This procedure was repeated twice. b) The slide was immersed in a container containing 99.5% ethanol (Kishida Chemical Co., Ltd.) and allowed to stand at room temperature for 5 minutes. This procedure was repeated twice. c) The slide was immersed in a container containing 95% ethanol and allowed to stand at room temperature for 5 minutes. d) The slide was immersed in a container containing 70% ethanol and allowed to stand at room temperature for 5 minutes. e) The slide was immersed in a container of pure water and allowed to stand at room temperature for 1 minute.

[0105] (2) Pretreatment of biological samples a) The slide was immersed in a container containing 0.2 N hydrochloric acid and allowed to stand at room temperature for 20 minutes. b) The slide was immersed in a container of nuclease-free water and allowed to stand at room temperature for 3 minutes. c) The slide was immersed in a container containing 2×SSC heated to 80° C. and allowed to stand at 80° C. for 30 minutes. d) The slide was immersed in a container of nuclease-free water and allowed to stand at room temperature for 2 minutes. This procedure was repeated twice. e) On the slide, 4cm diameter cells were placed around three types of cultured cell lines with different levels of HER2 gene expression. 2 The frame was made using a Pappen (liquid blocker, manufactured by Daido Sangyo Co., Ltd.). f) Pepsin solution (160 μL) was dropped into the frame, and the slide was placed in a humidified box and allowed to stand at 37° C. for 25 minutes. g) The slide was removed from the humidified box, immersed in a container containing 2×SSC, and allowed to stand at room temperature for 5 minutes. This procedure was repeated twice. h) The slides were removed and air dried at 37°C.

[0106] (3) Hybridization a) Liquid composition No. A-1 was applied to a biological sample by ejecting it using a thermal inkjet. Specifically, liquid composition No. A-1 was filled into an ink tank for an inkjet printer (product name: TS-203, manufactured by Canon Inc.) and attached to the print head. The ejection volume of one droplet was measured in advance using an ejection volume evaluation, and then the ejection volume was measured using a 4 cm 2 The liquid composition was ejected into a frame made with a dap pen under conditions set so that the amount was 10 μL per droplet. b) Solid support No. C-4 was coated on the biological sample onto which the liquid composition had been dispensed so that the surface of the solid support coated with dextran sulfate sodium was in contact with the liquid composition. c) The solid support was surrounded by paper glue and air-dried. d) The slide was placed on a thermoblock of a Mastercycler (registered trademark) nexus flat (manufactured by Eppendorf) set to 95°C, and left to stand at 95°C for 20 minutes. e) The slides were transferred to a humidified box and allowed to stand at 37°C for 15 hours.

[0107] (4) Probe cleaning process a) The slide was removed from the humidity chamber, the paper bond was removed, and the cover glass was removed. b) The slide was immersed in a container containing 2×SSC and allowed to stand at room temperature for 5 minutes. c) Posthybridization washing solution (160 μL, manufactured by Leica Microsystems) heated to 48°C was added dropwise to the biological sample, and the sample was allowed to stand at 48°C for 4 minutes. d) The slide was immersed in a container containing 0.1×SSC and allowed to stand at 48° C. for 5 minutes. e) The slide was immersed in a container containing 2×SSC and allowed to stand at 48° C. for 5 minutes.

[0108] (5) Nuclear staining a) 1 μg / mL DAPI solution (160 μL) was added dropwise to the biological sample and allowed to stand at room temperature for 30 minutes. b) The slide was immersed in a container containing 1x PBS and allowed to stand at room temperature for 5 minutes. This procedure was repeated three times. c) A cover glass was placed.

[0109] (6) Fluorescence observation a) Fluorescence images of three different cultured cell lines were acquired using a fluorescence microscope (BZ-X810, Keyence Corporation). Specifically, a 40x objective lens compatible with fluorescence observation and three fluorescence filter units were used: BZ-X Filter DAPI-V OP-88359 (Keyence Corporation, excitation wavelength 395 nm, detection wavelength 460 nm), BZ-X Filter GFP OP-87763 (Keyence Corporation, excitation wavelength 470 nm, detection wavelength 525 nm), and BZ-X Filter TRITC OP-87764 (Keyence Corporation, excitation wavelength 545 nm, detection wavelength 605 nm). Fluorescence images of the biological samples were acquired using each filter. b) A green substitute color was applied to the fluorescent image acquired using the GFP filter, a red substitute color was applied to the fluorescent image acquired using the TRITC filter, and a blue substitute color was applied to the fluorescent image acquired using the DAPI-V filter. The three acquired fluorescent images were superimposed using additive compositing to generate one composite image for each of the three different cultured cell lines. c) The image was observed and the dyeability was evaluated according to the following criteria, resulting in an evaluation of "A".

[0110] (Evaluation criteria) A: For 20 cell nuclei in which both HER2 and CEP17 signals were observed, the HER2 / CEP17 ratio was "0" (less than 2), "1+" and "2+" (2 or higher), with "2+" being higher than "1+." The HER2 gene expression levels of "0," "1+," and "2+" were stained more clearly, making it easy to determine. B: The HER2 / CEP17 ratio was "0" and "1+" were less than 2, and "2+" was greater than 2. "0" and "2+" stained in stages, making it easy to determine whether they were negative or strongly positive, but the difference between "0" and "1+" was unclear, making it difficult to determine whether they were negative or weakly positive. C: The HER2 / CEP17 ratio was less than 2 for "0", "1+", and "2+". Alternatively, there were no cell nuclei in which both HER2 and CEP17 signals were observed. The difference between "0", "1+", and "2+" was unclear and could not be determined at all. D: Dyeing could not be carried out due to difficulty in discharging the liquid composition.

[0111] Example 2 The same procedure as in Example 1 was carried out, except that the solid support was replaced with C-5. The staining property was evaluated and given an "A" rating.

[0112] Example 3 The same procedure as in Example 1 was carried out, except that the solid support was replaced with C-1. The staining evaluation result was a rating of "B."

[0113] Example 4 The same procedure as in Example 1 was carried out, except that the liquid composition was changed to B-1. The dyeability evaluation result was an "A" rating.

[0114] Example 5 The liquid ejection device was replaced with a microdispenser (Model 525, manufactured by Drummond Co.), and the same procedure as in Example 1 was carried out. The dyeability evaluation result was an "A" rating.

[0115] Example 6 The liquid ejection device was replaced with a piezo inkjet device (LaboJet-500, manufactured by Microjet Co., Ltd.), and the same procedure as in Example 1 was carried out. The dyeability evaluation result was an "A" rating.

[0116] Example 7 The same procedure as in Example 1 was carried out, except that the liquid composition was changed to A-4. The dyeability evaluation result was an "A" rating.

[0117] Example 8 The same procedure as in Example 1 was carried out, except that the liquid composition was changed to A-5. The dyeability evaluation result was an "A" rating.

[0118] Example 9 The same procedure as in Example 1 was carried out, except that the liquid composition was changed to A-6 and the solid support to C-3. The dyeing property was evaluated and given an "A" rating.

[0119] Example 10 The same procedure as in Example 1 was carried out, except that the liquid composition was changed to A-7 and the solid support to C-2. The dyeing property was evaluated and given an "A" rating.

[0120] (Comparative Example 1) The same procedure as in Example 1 was carried out, except that the liquid composition was changed to A-1 and the solid support was changed to C-6. The dyeing property was evaluated and given a rating of "C."

[0121] (Comparative Example 2) The same procedure as in Example 1 was carried out, except that the liquid composition was changed to A-2 and the solid support to C-6. The dyeing property was evaluated and given a rating of "C."

[0122] (Comparative Example 3) The same procedure as in Example 1 was carried out, except that the liquid composition was changed to A-3 and the solid support to C-6. The dyeing property was evaluated and given a rating of "C."

[0123] Comparative Example 4 The same procedure as in Example 1 was carried out, except that the liquid composition was changed to A-4 and the solid support to C-6. The dyeing property was evaluated and given a rating of "C."

[0124] (Comparative Example 5) The same procedure as in Example 1 was carried out, except that the liquid composition was changed to A-5 and the solid support to C-6. The dyeing property was evaluated and given a rating of "C."

[0125] (Comparative Example 6) The same procedure as in Example 1 was carried out, except that the liquid composition was changed to A-6 and the solid support to C-6. The dyeing property was evaluated and given a rating of "C."

[0126] (Comparative Example 7) The same procedure as in Example 1 was carried out, except that the liquid composition was changed to A-7 and the solid support to C-6. The dyeing property was evaluated and given a rating of "C."

[0127] (Comparative Example 8) The same procedure as in Example 1 was carried out, except that the liquid composition was changed to A-8 and the solid support to C-6. The dyeing property was evaluated and given a rating of "D".

[0128] Comparative Example 9 The same procedure as in Example 1 was carried out, except that the liquid composition was changed to A-9 and the solid support to C-6. The dyeing property was evaluated and given a rating of "D".

[0129] (Comparative Example 10) The same procedure as in Example 1 was carried out, except that the liquid composition was changed to B-2 and the solid support to C-6. The dyeing property was evaluated and given a rating of "D".

[0130] (Comparative Example 11) The same procedure as in Example 1 was carried out, except that the liquid composition was changed to A-8 and the solid support to C-7. The dyeing property was evaluated and given a rating of "D".

[0131] Tables 4 and 5 show the evaluation results of dyeability for Examples 1 to 10 and Comparative Examples 1 to 11.

[0132] [Table 4]

[0133] [Table 5]

[0134] Example 11 (3) Hybridization was carried out in the same manner as in Example 1, except that step b) was replaced by a step of covering the biological sample onto which the liquid composition had been ejected with a dextran sodium sulfate sheet (1.0 mg, 7 mm × 7 mm) and then with solid support C-6. The staining evaluation resulted in an evaluation of "A."

[0135] Example 12 The same method as in Example 1 was used, except that the liquid composition was changed to A-10, the amount of the ejected liquid was set to 7 μL, and step b) in (3) Hybridization was replaced by adding dextran sulfate sodium solution 1 (3 μL) using a manual micropipette to the biological sample into which the liquid composition had been ejected, thereby coating the solid support No. C-6. The staining evaluation resulted in an evaluation of "A."

[0136] The disclosure of this embodiment includes the following methods and configurations. (Method 1) A hybridization method for hybridizing a target nucleic acid contained in a biological sample with a probe capable of specifically binding to the target nucleic acid, comprising: (1) a liquid composition application step of ejecting a liquid composition containing at least the probe, an organic solvent, and water from a liquid ejection unit of a liquid ejection device and applying the liquid composition to the biological sample; (2) a polymer application step of applying a polymer selected from the group consisting of nonionic polymers and anionic polymers to the biological sample so that the polymer contacts the liquid composition; (3) a heating step of heating the liquid composition and the biological sample; (4) a cooling step of cooling the heated biological sample; A hybridization method comprising: (Method 2) The hybridization method according to Method 1, wherein in the polymer application step, the biological sample is coated with a solid support having the polymer on its surface. (Method 3) The hybridization method according to Method 2, wherein the solid support has a sheet or plate shape. (Method 4) 4. The hybridization method of method 2 or 3, wherein the solid support is a microscope slide or a cover slip. (Method 5) 5. The hybridization method according to any one of Methods 1 to 4, wherein in the polymer applying step, a liquid containing the polymer is applied to the biological sample. (Method 6) The hybridization method according to any one of methods 1 to 5, wherein the polymer is an anionic polymer. (Method 7) The hybridization method of method 6, wherein the anionic polymer is a sulfonic acid polymer. (Method 8) The hybridization method according to method 6 or 7, wherein the anionic polymer is a dextran sulfate salt. (Method 9) 9. The hybridization method according to any one of Methods 1 to 8, wherein the weight average molecular weight of the polymer is 100,000 or more and 5,000,000 or less. (Method 10) 10. The hybridization method according to any one of Methods 1 to 9, wherein the ejection method of the liquid ejection device is an inkjet method. (Method 11) 11. The hybridization method according to Method 10, wherein the ink-jet method is a thermal method. (Method 12) 12. The hybridization method according to any one of Methods 1 to 11, wherein the viscosity of the liquid composition is 0.3 mPa·s or more and 10 mPa·s or less. (Configuration 1) 1. A kit for hybridizing a target nucleic acid contained in a biological sample, comprising: a liquid composition containing at least a probe capable of specifically binding to a target nucleic acid, an organic solvent, and water; A solid support having a nonionic polymer or an anionic polymer on its surface, or a liquid containing the nonionic polymer or the anionic polymer. A kit comprising: (Configuration 2) 2. The kit according to claim 1, wherein the solid support is in the form of a sheet or plate. (Configuration 3) 3. The kit according to configuration 1 or 2, comprising a cartridge container that can be attached to an inkjet ejection head for ejecting the liquid composition. (Configuration 4) A hybridization device for hybridizing a target nucleic acid contained in a biological sample with a probe capable of specifically binding to the target nucleic acid, comprising: a liquid ejection unit capable of ejecting a liquid composition containing at least the probe, an organic solvent, and water toward the biological sample; a polymer applying unit that applies a nonionic polymer or an anionic polymer to the biological sample so that the biological sample contacts the liquid composition; and A hybridization device comprising:

Claims

1. A hybridization method for hybridizing a target nucleic acid contained in a biological sample with a probe capable of specifically binding to the target nucleic acid, comprising: (1) a liquid composition application step of ejecting a liquid composition containing at least the probe, an organic solvent, and water from a liquid ejection unit of a liquid ejection device and applying the liquid composition to the biological sample; (2) a polymer application step of applying a polymer selected from the group consisting of nonionic polymers and anionic polymers to the biological sample so that the polymer contacts the liquid composition; (3) a heating step of heating the liquid composition and the biological sample; (4) a cooling step of cooling the heated biological sample; A hybridization method comprising:

2. 2. The hybridization method according to claim 1, wherein in the polymer application step, the biological sample is coated with a solid support having the polymer on its surface.

3. The hybridization method according to claim 2 , wherein the solid support has a sheet or plate shape.

4. The hybridization method of claim 2, wherein the solid support is a microscope slide or a cover slip.

5. The hybridization method according to claim 1 , wherein in the step of applying the polymer, a liquid containing the polymer is applied to the biological sample.

6. The hybridization method according to claim 1 , wherein the polymer is an anionic polymer.

7. The hybridization method according to claim 6, wherein the anionic polymer is a sulfonic acid polymer.

8. The hybridization method according to claim 6, wherein the anionic polymer is a dextran sulfate salt.

9. 2. The hybridization method according to claim 1, wherein the weight average molecular weight of the polymer is 100,000 or more and 5,000,000 or less.

10. The hybridization method according to claim 1 , wherein the liquid ejection device uses an inkjet method.

11. The hybridization method according to claim 10 , wherein the ink-jet method is a thermal method.

12. 2. The hybridization method according to claim 1, wherein the viscosity of the liquid composition is 0.3 mPa·s or more and 10 mPa·s or less.

13. 1. A kit for hybridizing a target nucleic acid contained in a biological sample, comprising: a liquid composition containing at least a probe capable of specifically binding to a target nucleic acid, an organic solvent, and water; A solid support having a nonionic polymer or an anionic polymer on its surface, or a liquid containing the nonionic polymer or the anionic polymer. A kit comprising:

14. The kit according to claim 13 , wherein the solid support is in the form of a sheet or plate.

15. The kit according to claim 13, further comprising a cartridge container that can be attached to an inkjet ejection head for ejecting the liquid composition.

16. A hybridization device for hybridizing a target nucleic acid contained in a biological sample with a probe capable of specifically binding to the target nucleic acid, comprising: a liquid ejection unit capable of ejecting a liquid composition containing at least the probe, an organic solvent, and water toward the biological sample; a polymer applying unit that applies a nonionic polymer or an anionic polymer to the biological sample so that the biological sample contacts the liquid composition; and A hybridization device comprising:

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