JP2009092420A - Microarray processing apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for speedily and efficiently performing a hybridization reaction and detecting it in a DNA microarray. <P>SOLUTION: A microarray processing apparatus for detecting a hybridization reaction between the microarray in which DNA is immobilized on a carrier having an active ester group and a nucleic acid to be tested is provided with: a reaction part, provided with both a housing part for housing a sample liquid and a temperature regulating part for regulating the temperature of the sample liquid, for processing a nucleic acid amplification reaction and a hybridization reaction; a cleaning part for cleaning the microarray; a detection part for processing the detection of the hybridization reaction; a microarray mounting part; and a moving means for moving the microarray mounting part or the housing part in such a way as to immerse the microarray in the sample liquid in the housing part. The microarray mounting part is provided with a moving means for moving the microarray to the reaction part, the cleaning part, or the detection part to perform the processings. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a microarray processing apparatus for performing a hybridization reaction in a DNA microarray and detecting the hybridization reaction.

  Currently, a method is widely used in which biomolecules such as nucleic acids, sugar chains, and proteins are immobilized on a carrier, and a target biomolecule is reacted therewith to study the interaction between both molecules. A carrier on which DNA is immobilized is called a DNA microarray, and is produced by aligning and immobilizing DNA on a carrier at a density of several hundred to several hundred thousand per square centimeter. When all the gene sequences of a certain species have been elucidated, the entire expression information of the sample can be obtained by a single hybridization reaction by preparing a DNA microarray based on the information. In other words, thousands of different types of DNA probes with known sequences are aligned and immobilized in an array, and an unknown sample is added thereto to perform a hybridization reaction, thereby simultaneously identifying thousands of genes. it can. In the field of basic research, DNA microarrays are used to estimate the function of newly discovered genes by comparing them with the expression patterns of known genes, to identify important substances in signal transduction pathways, and to identify new genes Used for research. It is also an important technology in fields that require high throughput, such as discovery of new target molecules for therapeutic drugs, disease diagnosis, genomic toxicology, and disease-related gene search.

  The measurement work using the DNA microarray as described above requires repetition of simple work due to the large number of samples, requires a large installation area because the equipment used in each process is independent, reaction temperature Because it is necessary to manage the process, it takes a long time to proceed with the reaction, and it is necessary to handle a very small amount of sample with high accuracy. It has long been desired to do.

  However, when a sample-derived amplified nucleic acid is hybridized to a DNA probe on a DNA microarray, a purification step is required after the amplification reaction. The purification process is generally carried out by filter filtration using a centrifuge, but if such a purification process is to be incorporated into an automated apparatus, the apparatus becomes considerably complicated. As one of the methods actually performed, there is a method in which the reaction solution after the amplification reaction is filtered with a centrifuge, and the amplified nucleic acid on the filter is washed and dissolved again in the buffer. However, if a mechanism for actually carrying out such a purification process is incorporated in an automated apparatus, the apparatus becomes very large and the production cost of the apparatus increases.

  In addition, the method of detecting the fluorescence intensity is mainly used to detect the interaction in the DNA microarray, but this detection method detects the fluorescence by scanning the entire surface of the microarray at intervals of several μm to several tens of μm. It uses a device to do. Since such a fluorescence detection apparatus is provided with an optical system and a drive part, the apparatus is very large, and it is difficult to connect the process related to the hybridization reaction to an automated apparatus.

  An object of the present invention is to provide means for carrying out a hybridization reaction and its detection quickly and efficiently in a DNA microarray.

  The present inventors have found that by using a microarray in which DNA is immobilized on a carrier having an active ester group, the hybridization reaction can be carried out and detected without purifying the reaction solution after the nucleic acid amplification reaction. Therefore, the hybridization reaction can be carried out by directly immersing the microarray in the reaction solution after the amplification reaction. As a result, the microarray processing is performed automatically by mechanizing each step of the amplification reaction, the hybridization reaction, and the detection of the hybridization reaction. The present inventors have found that the apparatus can be miniaturized and completed the present invention.

That is, the present invention includes the following inventions.
(1) A microarray processing apparatus for subjecting a microarray having DNA immobilized on a carrier having an active ester group to a hybridization reaction with a test nucleic acid, and detecting the hybridization reaction,
A reaction section for performing a nucleic acid amplification reaction process and a hybridization reaction process, comprising a storage section for storing a sample liquid and a temperature adjustment section for adjusting the temperature of the sample liquid in the storage section;
A cleaning section for cleaning the microarray;
A detection unit for detecting and processing a hybridization reaction in the microarray;
A microarray mounting portion for mounting the microarray;
Moving means for moving the microarray mounting part or the container so that the microarray can be immersed in the sample solution in the container;
With
The apparatus, wherein the microarray mounting unit includes a moving unit for moving the microarray to the reaction unit, the cleaning unit, or the detection unit to perform the processing.
(2) The apparatus according to (1), wherein the microarray mounting unit has a swivel arm that swivels the microarray, and the reaction unit, the cleaning unit, and the detection unit are arranged on a circumference around which the microarray is swung.
(3) The apparatus according to (1) or (2), wherein the detection unit irradiates the microarray with excitation light and detects the intensity of fluorescence obtained.
(4) The microarray mounting part is configured so that a plurality of microarrays can be mounted, and the reaction part and the cleaning part can simultaneously perform the above-described processing on the plurality of microarrays attached to the mounting part. The apparatus according to any one of (1) to (3), configured as described above.
(5) The device according to any one of (1) to (4), wherein the microarray attachment unit is configured to function as a lid of the storage unit.
(6) The apparatus according to any one of (1) to (5), wherein the microarray is a flat plate having a dimension of 10 mm or less.
(7) A method of detecting a hybridization reaction by subjecting a microarray in which DNA is immobilized on a carrier having an active ester group to a hybridization reaction with a test nucleic acid,
Performing a nucleic acid amplification reaction of nucleic acid extracted from a sample;
A step of performing a hybridization reaction between the DNA on the microarray and the amplified nucleic acid by immersing the microarray in a sample solution containing the amplified nucleic acid;
Washing the microarray, and detecting a hybridization reaction in the microarray,
The method described above, wherein the hybridization reaction is carried out without purifying the sample solution after the nucleic acid amplification reaction.
(8) The method according to (7), wherein the hybridization reaction is detected after washing the microarray without drying.
(9) The method according to (7) or (8), wherein the microarray is washed with a cleaning solution containing a surfactant and NaAc, or a cleaning solution containing a surfactant and MgCl ₂ .

  The present invention provides a means for rapidly and efficiently carrying out a hybridization reaction and its detection in a DNA microarray.

  The present invention provides a microarray processing apparatus for subjecting a microarray to a hybridization reaction with a test nucleic acid and detecting the hybridization reaction, wherein the microarray comprises a DNA immobilized on a carrier having an active ester group. The invention relates to an apparatus for processing.

  A microarray in which DNA is immobilized on a carrier having an active ester group can be prepared by spotting DNA on a carrier into which an active ester group has been introduced as a chemically modifying group, and the DNA has a covalent bond with the active ester group. It is immobilized on the formed carrier.

  The active ester group means an ester group having an electron-withdrawing group having high acidity on the alcohol side of the ester group and activating the nucleophilic reaction, that is, an ester group having high reaction activity. An ester group activated more than an alkyl ester. The active ester group has reactivity with groups such as amino group, thiol group, and hydroxyl group. More specifically, an active ester group in which phenol esters, thiophenol esters, N-hydroxyamine esters, cyanomethyl esters, esters of heterocyclic hydroxy compounds, etc. have much higher activity than alkyl esters etc. Known as. More specifically, examples of the active ester group include a p-nitrophenyl group, an N-hydroxysuccinimide group, a succinimide group, a phthalimide group, and a 5-norbornene-2,3-dicarboximide group. In particular, an N-hydroxysuccinimide group is preferred.

  For example, the introduction of the active ester group is carried out by introducing a carboxyl group onto the carrier, followed by a dehydrating condensing agent such as cyanamide or carbodiimide (for example, 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide) and N- It can be carried out by esterifying the carboxyl group with a compound such as hydroxysuccinimide. By this treatment, a group in which an active ester group such as an N-hydroxysuccinimide group is bonded to the end of the hydrocarbon group via an amide bond can be formed (Japanese Patent Laid-Open No. 2001-139532).

The introduction of the carboxyl group may be carried out on an aminated or chlorinated support by, for example, formula: X—R ¹ —COOH (wherein X is a halogen atom, R ¹ is a divalent hydrocarbon group having 10 to 12 carbon atoms). Chlorocarboxylic acid, such as chloroacetic acid, fluoroacetic acid, bromoacetic acid, iodoacetic acid, 2-chloropropionic acid, 3-chloropropionic acid, 3-chloroacrylic acid, 4-chlorobenzoic acid; formula: HOOC-R A dicarboxylic acid represented by ^2- COOH (wherein R ² represents a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms), such as oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid phthalic acid; polyacrylic acid, polymethacrylic acid, trimellitic acid, polycarboxylic acids such as butane tetracarboxylic acid; ^{formula: R 3 -CO-R 4 -COOH} ( wherein, ^{R 3} is Atom or a divalent hydrocarbon group having 1 to 12 carbon atoms, ^{R 4} is keto acid or aldehyde acids represented by representing) a divalent hydrocarbon group having 1 to 12 carbon atoms; wherein:. X-OC- A monohalide of a dicarboxylic acid represented by R ⁵ —COOH (wherein X represents a halogen atom, R ⁵ represents a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms), such as succinic acid monochloride, Malonic acid monochloride: It can be carried out by reacting acid anhydrides such as phthalic anhydride, succinic anhydride, oxalic anhydride, maleic anhydride, and butanetetracarboxylic anhydride.

  As the material for the carrier into which the active ester group is introduced, those known in the art can be used and are not particularly limited. For example, noble metals such as platinum, platinum black, gold, palladium, rhodium, silver, mercury, tungsten and their compounds, and conductor materials such as graphite and carbon typified by carbon fiber; single crystal silicon, amorphous Silicon materials represented by silicon, silicon carbide, silicon oxide, silicon nitride, etc., composite materials of these silicon materials represented by SOI (silicon on insulator), etc .; glass, quartz glass, alumina, sapphire, ceramics, Inorganic materials such as stellite and photosensitive glass; polyethylene, ethylene, polypropylene, cyclic polyolefin, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl Lucol, polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile / butadiene styrene copolymer, polyphenylene oxide and Examples thereof include organic materials such as polysulfone.

  The apparatus of the present invention is suitably used for processing a microarray comprising a carrier having a fine flat plate structure. The shape of the carrier is not limited to a rectangle, square, or round shape, but a flat carrier having a size of 1 to 75 mm, preferably 1 to 10 mm, more preferably 1 to 3 mm is usually used. The dimension refers to the longest length of the flat plate. Alternatively, a flat carrier of 1 to 75 mm square, preferably 1 to 10 mm square, more preferably 1 to 3 mm square is used. Since it is easy to produce a fine flat carrier, it is preferable to use a carrier made of a silicon material or a resin material, more preferably a carrier made of single crystal silicon into which an active ester group is introduced. Single crystal silicon has a slightly different orientation of the crystal axis in some parts (sometimes referred to as mosaic crystal), or includes disorder on the atomic scale (lattice defects) Are also included. In particular, it is preferable to use a silicon substrate used in the field of semiconductors or the like as a carrier.

  By using a microarray obtained using a carrier having a relatively small size as described above, the entire apparatus for carrying out the hybridization reaction and its detection can be miniaturized. Further, even when the amount of the sample solution is small, the entire microarray can be immersed in the sample solution and the hybridization reaction can be performed, so that the apparatus structure can be prevented from becoming complicated.

  In the present invention, an active ester group is preferably introduced into a carrier having a carbon layer on the surface. The carbon layer is not particularly limited, but any of synthetic diamond, high-pressure synthetic diamond, natural diamond, soft diamond (for example, diamond-like carbon), amorphous carbon, carbon-based material (for example, graphite, fullerene, carbon nanotube), A mixture thereof or a laminate of them can be mentioned. Further, a layer of a carbide such as hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, thorium carbide, titanium carbide, uranium carbide, tungsten carbide, zirconium carbide, molybdenum carbide, chromium carbide, vanadium carbide, or the like may be used. A carbon layer can be formed by reacting these substances with the carrier as described above. Soft diamond is a generic term for an incomplete diamond structure that is a mixture of diamond and carbon, such as so-called diamond-like carbon (DLC), and the mixing ratio is not particularly limited. The carbon layer is excellent in chemical stability, can withstand subsequent reaction in the introduction of an active ester group and binding to the analyte, and is transparent to the detection system UV because there is no UV absorption. This is advantageous. In addition, it is advantageous in that nonspecific adsorption is small in a hybridization reaction with a test nucleic acid. The formation of the carbon layer can be performed by a known method. For example, a microwave plasma CVD (Chemical vapor deposition) method, an ECRCVD (Electrical cyclotron resonance) method, an ICP (Inductive coupled plasma) method, a direct current sputtering method, an ICP (Inductive coupled plasma) method, a direct current sputtering method, an ECR method. Examples thereof include a vapor deposition method, a laser vapor deposition method, an EB (Electron beam) vapor deposition method, and a resistance heating vapor deposition method.

  In the present invention, it is particularly preferable that a DLC layer formed on a silicon substrate and an active ester group introduced into the DLC layer is used as a carrier for a microarray.

  A DNA microarray can be produced by dissolving a DNA probe in a spotting buffer to prepare a spotting solution, and spotting it on the carrier as described above using a spotter apparatus or the like.

  The apparatus of the present invention includes a reaction part for performing a nucleic acid amplification reaction process and a hybridization reaction process, comprising a storage part for storing a sample liquid and a temperature adjustment part for adjusting the temperature of the sample liquid in the storage part. Is provided. The temperature adjustment unit adjusts the sample solution in the storage unit to a temperature suitable for the nucleic acid amplification reaction and the hybridization reaction. The storage unit stores a nucleic acid such as DNA or RNA extracted from a sample such as a cell or tissue, and a nucleic acid amplification reagent (for example, nucleic acid synthase, nucleotide mix, labeling substance, etc.) added thereto. . And a nucleic acid amplification reaction is implemented in an accommodating part by changing temperature in a temperature control part. The nucleic acid amplification reaction may be performed with the microarray immersed in a sample solution. Subsequently, the microarray is immersed in the sample solution containing the amplified nucleic acid, and the temperature of the sample solution is adjusted to a temperature suitable for the hybridization reaction by the temperature adjustment unit, whereby the DNA probe on the microarray and the test nucleic acid (ie, the sample) Hybridization reaction with an amplified nucleic acid prepared from the nucleic acid of origin.

  By using a microarray in which DNA is immobilized on a carrier having an active ester group, the hybridization reaction can be carried out and detected without purifying the sample solution after the nucleic acid amplification reaction. Therefore, after carrying out the amplification reaction in the storage section, the hybridization reaction can be carried out by immersing the microarray as it is in the sample solution. As a result, it is not necessary to incorporate an apparatus for purifying the amplified nucleic acid, and a microarray processing apparatus that automatically performs each process of amplification reaction, hybridization reaction, and detection of hybridization reaction can be miniaturized. .

  The accommodating part is not particularly limited as long as it can accommodate the microarray. The accommodating part preferably has a plurality of compartments that can accommodate a plurality of microarrays. In addition, as a material for the housing portion, a material that can withstand temperature changes in a nucleic acid amplification reaction or a hybridization reaction is used.

  The reaction unit may have means for swinging the storage unit. The reaction can be efficiently carried out by swinging the container during the nucleic acid amplification reaction and the hybridization reaction.

  The temperature adjusting unit may be any unit that can adjust the temperature of the sample solution in the storage unit for the nucleic acid amplification reaction or the hybridization reaction. In the case where the temperature adjustment is only heating and the natural cooling is sufficient for cooling, the temperature adjusting unit may be configured to cover the housing portion with an electric heater. In addition, when cooling is also necessary, a structure may be adopted in which a jacket structure is provided around the housing portion and a heat medium heated or cooled to a predetermined temperature is circulated. A device having a Peltier element that can easily reverse heating / cooling by reversing the polarity of the power source may be used as the temperature adjustment unit.

  The reaction unit preferably further includes a promoter supply unit for supplying a promoter for promoting the hybridization reaction. The accelerator contains a salt (such as SSC) and a surfactant (such as SDS), and has a function of promoting a hybridization reaction in the accommodation unit. The promoter supply unit is a promoter bottle filled with a promoter, a nozzle for sucking the promoter and supplying it to the storage unit, a nozzle arm, a pump for feeding the promoter, and a promoter. A moving means for injecting the promoter into the accommodating portion by moving the nozzle may be further provided. For example, the nozzle arm can be arbitrarily moved in the horizontal and vertical directions with respect to the housing portion by moving means such as screw feed, belt feed or actuator. An electromagnetic cylinder, piston, or the like may be used as long as the vertical movement is repeated at regular intervals.

  The apparatus of the present invention includes a cleaning unit for cleaning the microarray. In the washing section, the microarray after the hybridization reaction is washed to remove nucleic acids and the like that have not hybridized to the DNA probe. In the washing section, the microarray before the hybridization reaction may be washed. The cleaning unit may have any structure as long as it can clean the microarray. For example, the cleaning unit may include a cleaning container for storing a cleaning liquid and a microarray to perform a cleaning process, and may be configured to be cleaned by immersing the microarray in a cleaning liquid in the container. In that case, the cleaning section may have means for swinging the cleaning liquid stored in the cleaning container. By swinging the cleaning liquid, the cleaning can be performed efficiently. The cleaning container preferably has a plurality of compartments in order to accommodate a plurality of cleaning liquids. Alternatively, the cleaning unit may be configured to perform cleaning by jetting cleaning liquid from the cleaning nozzle onto the microarray.

  The cleaning unit supplies the cleaning liquid bottle filled with the cleaning liquid, the nozzle for supplying the cleaning liquid, the nozzle for sucking and discarding the used cleaning liquid, the nozzle arm, the pump for feeding the cleaning liquid, and the cleaning liquid. It may further include a moving means for moving the nozzle for supplying the cleaning liquid. In the case where the cleaning unit has a cleaning container and the microarray is immersed in the cleaning unit, the nozzle arm is placed horizontally with respect to the cleaning container by moving means such as screw feeding, belt feeding, or an actuator. It is configured to be arbitrarily movable in the vertical direction. An electromagnetic cylinder, piston, or the like may be used as long as the vertical movement is repeated at regular intervals.

The cleaning unit may further include a drying unit for drying the microarray. In the drying section, the washed microarray is dried. The drying unit can be configured to dry the microarray by, for example, air blow drying, N ₂ blow drying, hot air drying, vacuum drying, and centrifugal drying.

The reason why the drying step is necessary is that, for example, in the detection of the optical system, when the material is naturally dried, a salt contained in the cleaning solution is precipitated and causes noise. Measurement can be performed without drying by placing the chip in a transparent solution in a transparent container, or by covering with a transparent cover glass to prevent evaporation of the moisture on the chip. Possible methods. However, there is a concern that the structure of the apparatus becomes complicated. Therefore, the present inventors have found that the hybridization reaction can be detected even if the measurement is performed without drying after the washing treatment after the hybridization reaction by devising the composition of the washing solution to prevent natural drying. It was. Therefore, the cleaning unit in the apparatus of the present invention does not need to include a drying unit, and as a result, the entire apparatus can be further downsized. In the case where drying is not performed after the cleaning, the cleaning liquid for cleaning in the cleaning section includes a cleaning liquid containing NaAc (sodium acetate) and a surfactant (for example, Tween 20), and MgCl ₂ and a surfactant (for example, Tween 20). It is preferable to use a cleaning liquid containing the same. Washing may be carried out using one of these washing liquids or both, but is preferably carried out using both. Accordingly, the device of the present invention preferably comprises a cleaning liquid bottle for filling a cleaning liquid comprising NaAc (sodium acetate) and a surfactant (eg Tween 20), and / or MgCl ₂ and a surfactant (eg Tween 20). A cleaning liquid bottle is provided for filling the cleaning liquid.

  The apparatus of the present invention includes a detection unit for detecting a hybridization reaction in a microarray. The detection unit preferably detects the intensity of fluorescence obtained by irradiating the microarray with excitation light using a laser or the like. In the nucleic acid amplification reaction in the reaction part, the fluorescent label is incorporated into the amplified nucleic acid, the obtained amplified nucleic acid is hybridized to the DNA probe on the microarray as the test nucleic acid, washed or washed and dried, and then the detection part is put into the microarray. When the excitation light is irradiated, fluorescence derived from the test nucleic acid hybridized to the DNA probe is detected. By detecting this, a hybridization reaction can be detected. In the apparatus of the present invention, when a microarray having a relatively small size, for example, a microarray having a dimension of 10 mm or less, preferably 5 mm or less, more preferably 3 mm or less, most preferably 1 to 5 mm square is used at a time. Since the fluorescence can be detected by irradiating the entire microarray with a laser, it is not necessary to scan the entire surface of the microarray with a laser, so that the detection can be performed in a short time and the apparatus can be miniaturized.

  One embodiment of the detection unit in the apparatus of the present invention is shown in FIG. In this embodiment, a CCD camera (11) is used for the light receiving portion, and one microarray can be detected by one shot. When the time for irradiating the excitation light becomes long (1 s or longer), it is preferable to use a cooled CCD camera. As the laser (12) for irradiating the excitation light, it is preferable to use a laser having a large φ so that the entire microarray can be irradiated. The laser beam having a large φ may be adjusted so as to be irradiated to the entire microarray through the slit (15). Further, by irradiating the laser beam obliquely to the microarray (14), it is possible to capture weak light from the fluorescent label while cutting the specular reflection light, and realize a higher S / N value. Can do. The fluorescence (16) passes through the fluorescence filter (17) and is detected by the CCD element (18). The diffuse reflected light is cut in the fluorescent filter.

  The apparatus of the present invention includes a microarray mounting portion for mounting the microarray. The microarray attachment unit includes a moving means for moving the microarray to the reaction unit, the cleaning unit, or the detection unit for processing. The method for attaching the microarray to the microarray attachment portion may be any method as long as the microarray can be attached and removed. You may hold | grip a microarray by the microarray attachment part which has a holding tool. You may attach a microarray to a microarray attachment part with an adhesive tape. When using an adhesive tape, one that does not inhibit the hybridization reaction and does not melt or lose its adhesiveness even under temperature conditions in the reaction part is used.

  A top view of one embodiment of the present invention is shown in FIG. In this embodiment, the microarray mounting part (21) has a turning arm (22) for turning the microarray (14), and the reaction part (23), the washing part (24) and the detection part (25) are: The microarray is arranged on a circle around which the microarray rotates. After the nucleic acid amplification reaction and the hybridization reaction in the reaction part (23), the microarray is moved to the washing part by the turning of the turning arm (22). After performing the cleaning process of the microarray in the cleaning unit, the microarray is moved to the detection unit by the swing of the swing arm (22). The reaction unit (23), the cleaning unit (24), and the detection unit (25) are arranged on the circumference around which the microarray rotates. When the microarray is moved by the rotation of the rotation arm, the reaction unit (23). In the cleaning unit (24) and the detection unit (25), the microarray reaction process, the cleaning process, and the detection process are respectively arranged at positions where the process can be performed.

  In one embodiment, the microarray mounting part is configured to be able to mount a plurality of microarrays, and the reaction part and the cleaning part simultaneously react and process the plurality of microarrays attached to the mounting part. It is configured to be able to perform processing. For example, the reaction unit has storage units corresponding to the number of microarrays, and the cleaning unit has cleaning containers or cleaning nozzles corresponding to the number of microarrays.

  The cleaning unit (24) is preferably configured to be movable in the horizontal direction with respect to the microarray mounting unit. In one embodiment, the cleaning part (24) is arranged on the moving means (26), and when the microarray has been moved to the cleaning part by the pivot arm, the moving means (26) is in a horizontal direction with respect to the microarray mounting part. By moving, the microarray can be immersed in the cleaning containers (28) in order or the drying section (27) can be moved so that the microarray can be dried. It is preferable that the detection unit (25) is also configured to be movable in the horizontal direction with respect to the microarray mounting unit. In one embodiment, the detector (25) is disposed on the moving means (26), and when the microarray has been moved to the detector by the pivot arm (22), the moving means (26) is relative to the microarray mounting part. By moving in the horizontal direction, it is possible to sequentially detect the hybridization reaction by sequentially irradiating a plurality of microarrays with excitation light. In the embodiment as described above, the detection unit and the cleaning unit may be arranged on the same moving unit, or may be arranged on different moving units.

  Further, instead of moving the cleaning unit and the detection unit in the horizontal direction with respect to the microarray mounting unit, the microarray mounting unit may be moved in the horizontal direction with respect to the cleaning unit and the detection unit.

  Furthermore, the apparatus of the present invention includes a moving means for moving the microarray mounting part or the container so that the microarray can be immersed in the sample solution in the container. The immersion of the microarray in the sample solution in the storage section may be performed by moving the microarray attachment section perpendicular to the storage section, or by moving the storage section perpendicular to the microarray attachment section. It is good also as a structure to implement. For example, the microarray mounting part or the housing part can be arbitrarily moved in the horizontal and vertical directions by moving means such as screw feed, belt feed or actuator. An electromagnetic cylinder, piston, or the like may be used as long as the vertical movement is repeated at regular intervals.

  In one embodiment, the microarray mounting part is configured to function as a lid of the accommodating part (31) in the reaction part (23) (FIG. 3). When the microarray is immersed in the sample solution (32) in the container, the microarray mounting part (21) functions as a lid for the container (31), so that temperature adjustment can be performed in a short time in the hybridization reaction. Soybean reaction can be carried out quickly and efficiently. The microarray mounting part (21) is preferably configured to function also as a lid of the cleaning container of the cleaning part.

  Further, as shown in FIG. 3, the microarray attachment part (21) preferably has an insertion part (33), and is preferably configured so that the microarray can be attached to the insertion part. When the microarray mounting part (21) has the insertion part (33), the insertion part is immersed in the sample liquid together with the microarray, and the water level of the sample liquid rises. Therefore, even when the sample solution is small, the microarray can be immersed in the sample solution.

  In the apparatus of the present invention, each reaction process of the nucleic acid amplification reaction and the hybridization reaction in the reaction part, the washing process in the washing part, the detection process in the detection part, and the microarray attachment so that the microarray can be immersed in the sample solution in the storage part It is preferable that the process of moving the part or the storage part and the process of moving the microarray to the reaction part, the washing part or the detection part are automated by the control means. The apparatus of the present invention can be automated by connecting each component to the apparatus as a computer as a control means.

The operation and processing of the apparatus in the embodiment of the present invention shown in FIG. 2 will be described below.
(i) The microarray (14) is fixed to the microarray mounting part (21) having the swivel arm (22) with an adhesive tape. At this time, the microarray mounting part (21) is located at the position A, that is, immediately above the reaction part.
(ii) The microarray attachment part is lowered, the microarray is immersed in the accommodation part in which the sample liquid is accommodated, and the microarray attachment part functions as a lid of the accommodation part, and the accommodation part is sealed. The PCR reaction is performed by controlling the temperature with the temperature controller.
(iii) After the PCR reaction, the temperature of the sample solution is lowered, and then the microarray attachment part (21) is raised. By reducing the temperature of the sample solution to about 30 ° C., it is possible to prevent the sample solution from evaporating after the PCR reaction and at the same time prevent the microarray from drying.
(iv) The accelerator is injected into the sample liquid by the nozzle for supplying the accelerator and optionally pipetting. When the microarray mounting portion is lowered again, the microarray is immersed again in the storage portion in which the sample liquid is stored, the microarray mounting portion functions as a lid of the storage portion, and the storage portion is sealed. The hybridization reaction is performed by controlling the temperature with the temperature controller. At that time, the accommodating portion is swung by the swinging mechanism. After the hybridization reaction, the temperature of the sample solution is lowered to about 30 ° C., thereby preventing the microarray from being dried simultaneously with the evaporation of the sample solution.
(v) The microarray mounting part (21) is raised and the turning arm (22) is turned to the right, whereby the microarray mounting part is moved directly above the cleaning part. The microarray mounting part (21) is lowered, the microarray is immersed in a cleaning container containing a cleaning solution, and the microarray mounting part functions as a lid of the container, thereby sealing the container. At that time, the container is rocked by the rocking mechanism. When the cleaning in the first row of the cleaning containers is finished, the moving means (26) in which the cleaning section is arranged moves to the left, and when the next cleaning container row moves below the microarray mounting portion, the microarray The mounting portion is lowered again, and the microarray is immersed in the next row of cleaning containers.
(vi) When the cleaning process is finished, the microarray mounting part moves up, the moving means (26) moves to the left, and when the drying part (27) moves below the microarray mounting part, the microarray mounting part descends again. Then, a drying process by air blow is performed. Drying is performed as necessary. After the drying process is finished, the microarray mounting part (21) is raised, the turning arm (22) is turned to the right, and the microarray mounting part (21) is moved onto the detection part (25).
(vii) The CCD camera and laser of the detection unit (25) face the direction of the microarray, where the microarray is irradiated with laser light and fluorescence is detected by the CCD camera. At that time, the moving means (26) in which the detection unit is arranged moves to the left whenever the detection of one microarray is completed, and the detection of the hybridization reaction in the plurality of microarrays is sequentially performed.

The present invention also relates to a method for detecting a hybridization reaction by subjecting a microarray in which DNA is immobilized on a carrier having an active ester group to a hybridization reaction with a test nucleic acid. The method of the present invention comprises:
Performing a nucleic acid amplification reaction of nucleic acid extracted from a sample;
A step of performing a hybridization reaction between the DNA on the microarray and the amplified nucleic acid by immersing the microarray in a sample solution containing the amplified nucleic acid;
Washing the microarray, and detecting a hybridization reaction in the microarray,
The hybridization reaction is carried out without purifying the sample solution after the nucleic acid amplification reaction.

  By using a microarray in which DNA is immobilized on a carrier having an active ester group, it is possible to carry out and detect a hybridization reaction without purifying the reaction solution after the nucleic acid amplification reaction.

In the step of washing the microarray, it is preferable to wash with a cleaning solution containing a surfactant and NaAc, or a cleaning solution containing a surfactant and MgCl ₂ . By using such a washing solution, natural drying can be prevented, and the hybridization reaction can be detected even if the measurement is performed without drying after the washing treatment after the hybridization reaction. That is, in the method of the present invention, the hybridization reaction can also be detected without drying after washing the microarray.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to the range of an Example.

(Example 1) Preparation of DNA microarray A 3 mm square silicon substrate was coated with a diamond-like carbon layer by ionization deposition, modified with a carboxyl group, and then 1- [3- (dimethylamino) propyl] -3-ethyl. Active esterification was performed with carbodiimide and N-hydroxysuccinimide to prepare a carrier having a DLC layer and an N-hydroxysuccinimide group on the surface of the silicon substrate.

  An amino group was introduced onto the silicon substrate having the DLC layer by ammonia plasma to produce a carrier having a DLC layer and an amino group on the surface of the silicon substrate.

  Oligonucleotide (Hokkaido System Science Co., Ltd.) was adjusted to 100 μM with sterilized water. 4 times concentrated Sol. 6 (manufactured by Toyo Kohan Co., Ltd.) to obtain a DNA solution having a final concentration of 5 μM. The DNA solution was spotted (64 points / carrier) on the carrier prepared above with SPBIO2000 (manufactured by Hitachi Soft Engineering).

  The carrier spotted with the DNA solution was placed in a baking chamber at 80 ° C. and allowed to stand for 1 hour to immobilize the DNA. Subsequently, the substrate was washed with a room temperature washing solution (2 × SSC / 0.2% SDS) for 15 minutes. Washing was performed for 5 minutes with a washing solution (2 × SSC / 0.2% SDS) heated to about 70 ° C. Rinsing was performed three times with ultrapure water. At this time, the active ester was inactivated by dipping in a heated cleaning solution. Then, a microarray in which DNA was immobilized on a carrier having an active ester group was produced by blowing off water with a centrifuge.

  The amino group-introduced carrier is spotted with a DNA solution, soaked in BSA (bovine serum albumin 3%) for 15 minutes to block the amino group, and then washed in a room temperature washing solution (2 × SSC / 0.2% SDS). For 15 minutes, and then washed 3 times with ultrapure water. Then, a microarray in which DNA was immobilized on a carrier having an amino group was produced by blowing off water with a centrifuge.

Example 2 DNA extraction was performed using a nucleic acid extraction system Quick Gene 800 (manufactured by Fuji Film Co., Ltd.). A cell lysate was added to various cells to dissolve the cells, and the lysate was added to the apparatus cartridge to extract DNA. A PCR solution was prepared using TAKARA Ex Taq (manufactured by Takara Bio Inc.), and PCR was performed. In addition, Cy5 was incorporated into the PCR-amplified nucleic acid by using Cy5-labeled dCTP (GE Healthcare).

  Without purifying the Cy5-labeled amplified nucleic acid prepared above, an accelerator (5 × SSC / 0.5% SDS) was added thereto, and the DNA microarray prepared in Example 1 was immersed therein, and the hybridization reaction was performed while rocking. I let you. The reaction was carried out at 50 ° C. for 1 hour.

After the hybridization reaction, washing was performed using a washing solution at room temperature (2 × SSC / 0.2% SDS, 2 × SSC) while shaking for 30 s × 3 times and dried by N ₂ blow.

  The FLA-8000 (manufactured by Hitachi Software) detector detected fluorescence derived from Cy5-labeled amplified nucleic acid hybridized to DNA on the microarray (FIG. 4).

  As a result, in the microarray in which DNA is immobilized on a carrier having an amino group, a lot of unnecessary fluorescence was observed, and the spot of the hybridized amplified nucleic acid could not be confirmed, but it had an active ester group. In the microarray in which the DNA was immobilized on the carrier, the hybridized amplified nucleic acid spots could be clearly confirmed. That is, it was found that the hybridization reaction can be detected well without purifying the reaction solution after the amplification reaction.

(Example 3) Detection in an automated apparatus Using a microarray in which DNA is immobilized on a carrier having an active ester group, the same process as in Example 2 was performed using the automated microarray processing apparatus shown in Fig. 2. , Hybridization reaction was detected.

  The exposure time of the cooled CCD was 30 s. The laser was irradiated at an angle. A fluorescent filter for Cy5 was used as the fluorescent filter.

  The detected fluorescence image is shown in FIG. An image with a very clear spot was obtained. Thus, it was confirmed that a series of steps up to nucleic acid amplification reaction, hybridization reaction, and detection can be automatically performed.

Example 4
DNA was extracted using an automatic nucleic acid extraction system Quick Gene 800 (Fuji Film). A cell lysate was added to various cells to dissolve the cells, and the lysate was added to the apparatus cartridge to extract DNA. A PCR solution was prepared using TAKARA Ex Taq (manufactured by Takara Bio Inc.), and PCR was performed. In addition, Cy5 was incorporated into the PCR-amplified nucleic acid by using Cy5-labeled dCTP (GE Healthcare).

  A promoter (0.5N NaAc / 0.5% Tween 20) was added to the Cy5-labeled amplified nucleic acid prepared above without purification, and the DNA on the carrier having an active ester group prepared in Example 1 was added thereto. The microarray on which was immobilized was immersed and allowed to hybridize while rocking. The reaction was carried out at 50 ° C. for 1 hour.

After the hybridization reaction, washing was performed using a washing solution at room temperature (1N NaAc / 0.5% Tween 20) for 1 minute × 4 times, and further washing at room temperature (1N MgCl ₂ /0.5% Tween 20). Was washed with rocking once for 1 minute × 1. Drying with N ₂ blow was not performed.

  The FLA-8000 (manufactured by Hitachi Software) detector detected fluorescence derived from Cy5-labeled amplified nucleic acid hybridized to DNA on the microarray (FIG. 6).

  From this, it was found that the fluorescence can be detected even if the measurement is performed without drying after the washing treatment after the hybridization reaction.

An embodiment of the detection part in the apparatus of this invention is shown. FIG. 2 shows a top view of one embodiment of the apparatus of the present invention. 1 shows one embodiment of a microarray attachment part, a microarray and a reaction part in the present invention. For a microarray (41) in which DNA is immobilized on a carrier having an active ester group and a microarray (42) in which DNA is immobilized on a carrier having an amino group, a Cy5-labeled amplified nucleic acid hybridized with a DNA probe The result of having detected the fluorescence derived from is shown. FIG. 2 shows the result of detecting a hybridization reaction using a microarray in which DNA is immobilized on a carrier having an active ester group, using the automated microarray processing apparatus shown in FIG. 2, and performing the same steps as in Example 2. . Result of detecting fluorescence derived from Cy5-labeled amplified nucleic acid hybridized to DNA probe without drying after washing treatment after hybridization reaction for microarray in which DNA is immobilized on carrier having active ester group Indicates.

Explanation of symbols

DESCRIPTION OF SYMBOLS 11 CCD camera 12 Laser 13 Lens 14 Microarray 15 Slit 16 Fluorescence 17 Fluorescence filter 18 CCD element 21 Microarray attachment part 22 Turning arm 23 Reaction part 24 Washing part 25 Detection part 26 Transfer means 27 Drying part 28 Washing container 31 Storage part 32 Sample Liquid 33 Insertion part 41 Microarray in which DNA is immobilized on a carrier having an active ester group 42 Microarray in which DNA is immobilized on a carrier having an amino group

Claims (9)

A microarray processing apparatus for subjecting a microarray having DNA immobilized on a carrier having an active ester group to a hybridization reaction with a test nucleic acid, and detecting the hybridization reaction,
A reaction section for performing a nucleic acid amplification reaction process and a hybridization reaction process, comprising a storage section for storing a sample liquid and a temperature adjustment section for adjusting the temperature of the sample liquid in the storage section;
A cleaning section for cleaning the microarray;
A detection unit for detecting and processing a hybridization reaction in the microarray;
A microarray mounting portion for mounting the microarray;
Moving means for moving the microarray mounting part or the container so that the microarray can be immersed in the sample solution in the container;
With
The apparatus, wherein the microarray mounting unit includes a moving unit for moving the microarray to the reaction unit, the cleaning unit, or the detection unit to perform the processing.   The apparatus according to claim 1, wherein the microarray attachment unit has a swivel arm that swivels the microarray, and the reaction unit, the cleaning unit, and the detection unit are arranged on a circumference around which the microarray swivels.   The apparatus according to claim 1 or 2, wherein the detection unit irradiates the microarray with excitation light and detects the intensity of fluorescence obtained.   The microarray mounting part is configured so that a plurality of microarrays can be attached, so that the reaction part and the washing part can simultaneously perform the above processing on the plurality of microarrays attached to the mounting part. The device according to claim 1, wherein the device is configured.   The apparatus according to any one of claims 1 to 4, wherein the microarray mounting part is configured to function as a lid of the housing part.   The device according to any one of claims 1 to 5, wherein the microarray has a flat plate shape having a dimension of 10 mm or less. A method for detecting a hybridization reaction by subjecting a microarray in which DNA is immobilized on a carrier having an active ester group to a hybridization reaction with a test nucleic acid,
Performing a nucleic acid amplification reaction of nucleic acid extracted from a sample;
A step of performing a hybridization reaction between the DNA on the microarray and the amplified nucleic acid by immersing the microarray in a sample solution containing the amplified nucleic acid;
Washing the microarray, and detecting a hybridization reaction in the microarray,
The method described above, wherein the hybridization reaction is carried out without purifying the sample solution after the nucleic acid amplification reaction.   The method according to claim 7, wherein the hybridization reaction is detected after washing the microarray without drying. The method according to claim 7 or 8, wherein the microarray is washed with a cleaning solution containing a surfactant and NaAc or a cleaning solution containing a surfactant and MgCl ₂ .

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