JP2016034247A - Hybridization reaction apparatus, nucleic acid microarray, and method for inspecting gene using them - Google Patents

Abstract

PROBLEM TO BE SOLVED: To shorten the step of dispensing a given amount of buffer for hybridization reaction and then adding it to a reaction liquid after nucleic acid amplification reaction when conducting hybridization reaction; or the step of taking an amplified DNA out of the reaction liquid after nucleic acid amplification reaction, drying it, and adding it to the buffer.SOLUTION: A component immobilization site is formed which comprises at least a salt to be used for hybridization reaction, in a portion that comes into contact with a reaction liquid after nucleic acid amplification reaction.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a hybridization reaction apparatus, a nucleic acid microarray (also referred to as a nucleic acid chip), and a genetic testing method using them.

  Advances in genome analysis have elucidated biological molecules involved in the physiological responses of various organisms. These biological molecules include nucleic acids such as DNA, proteins, sugar chains, etc. Among them, those whose functions and structures have been elucidated are used in various industrial applications such as drug discovery, clinical testing, food testing, and environmental testing. Used for

  In tests such as clinical tests, the following methods are commonly used. That is, when a specimen is brought into contact with a device in which a probe molecule (hereinafter referred to as a ligand) that specifically binds to a biological molecule to be detected (hereinafter referred to as an analyte) is immobilized on a carrier, the analyte is attached to the specimen. Is present, the analyte is trapped on the support by binding to the ligand. The above-described inspection is performed by detecting the analyte trapped on the carrier.

  Also in the inspection methods as described above, in recent years, speeding up and automation are demanded, and a detection method for comprehensively measuring hundreds to tens of thousands of biologically relevant molecules simultaneously is desired. A device design using an integration technology for immobilizing biological molecules, so-called MEMS (Micro Electro Mechanical Systems) technology, is now possible, and so-called microarrays are used for comprehensive analysis in drug discovery research and bio research. In particular, genetic testing using a so-called DNA microarray (also referred to as a DNA chip) in which nucleic acid molecules, such as DNA, are immobilized on a carrier has been put into practical use.

  In analysis using a DNA microarray, first, a solution containing DNA previously fluorescently labeled with a fluorescent substance is brought into contact with the DNA microarray, for example, and the probe DNA immobilized on the carrier and the fluorescently labeled DNA are contacted with each other. Hybridize specifically. Thereafter, the DNA microarray is washed, and the fluorescent signal emitted from the fluorescent substance is detected and measured to identify or quantify the DNA.

  A DNA microarray is usually a slide glass-like size, and a sample is dropped on it and covered with a preparation for reaction. For example, as shown in FIG. 13, a DNA microarray 102 is placed in a recessed area 101 of a substantially flat plate 100, a solution 103 is dropped in this state, and the solution is held by a DNA microarray cover 104. is there.

  An example of a genetic testing apparatus using a DNA microarray is Patent Document 1. The genetic testing device disclosed in Patent Document 1 includes a temperature adjustment unit for a DNA microarray, a detection unit that detects an optical signal from the DNA microarray, a fluid transport unit that supplies and discharges fluid to and from the DNA microarray, and an overall operation. A control unit for controlling is provided.

  For example, as shown in FIG. 14, the genetic test apparatus includes a DNA microarray 201 and a carrier support member 202 that fixes the DNA microarray 201, and the DNA microarray 201 fixed to the carrier support is movable. Further, as shown in FIG. 14, the genetic test apparatus includes a nucleic acid amplification reaction unit 203 that amplifies a predetermined region by a polymerase chain reaction (PCR) or the like as a nucleic acid extracted from a test target organism, and a nucleic acid amplification reaction such as PCR. A hybridization reaction unit 204 for performing a hybridization reaction by adding a hybridization reaction buffer to the subsequent reaction solution, a washing unit 205 for washing the DNA microarray 201 after the hybridization reaction, and a DNA microarray 201 after washing And a detection unit 206 for detecting fluorescence from the.

JP 2004-28775 A

  By the way, in genetic testing, in order to reduce electrostatic repulsion between negatively charged nucleic acid molecules, the nucleic acid and the nucleic acid are electrostatically charged into a reaction solution after the nucleic acid amplification reaction (hereinafter referred to as a nucleic acid amplification reaction solution). It was necessary to add a certain amount of a buffer containing a monovalent cation (for example, sodium ion) that interacts, and then perform a hybridization reaction. In other words, genetic testing requires a buffer for preparing a solution for hybridization reaction and a mechanism for dispensing the buffer, leading to a long lead time for the entire test, and an increased size of the test apparatus. There was a problem such as.

  In view of the above circumstances, an object of the present invention is to provide a hybridization reaction apparatus, a nucleic acid microarray, and a gene testing method that can easily prepare a solution for a hybridization reaction.

The present invention that has achieved the above-described object includes the following.
(1) A reaction unit that performs a hybridization reaction using a nucleic acid microarray and a component solid phase immobilization unit that includes at least a salt used in the hybridization reaction, and the reaction solution after the nucleic acid amplification reaction is subjected to the component solid phase immobilization A hybridizing reaction apparatus characterized by contacting a part.
(2) The hybridization reaction apparatus according to (1), wherein the component solid phase immobilization part is formed in the reaction part.
(3) The hybridization reaction apparatus according to (1), further comprising a jig on which the nucleic acid microarray can be mounted.
(4) The hybridization reaction apparatus according to (3), wherein the component immobilization part is formed on the jig.
(5) The reaction unit includes a recess for placing the nucleic acid microarray and a channel connected to the recess, and supplies the solution to the recess via the channel (1). ) The hybridization reaction apparatus described.
(6) The hybridization reaction apparatus according to (5), wherein the component solid phase immobilization part is formed in the recess or the channel for supplying a solution to the recess.
(7) The hybridization reaction apparatus according to (1), wherein the reaction unit includes a recess for mounting the nucleic acid microarray and a cover for covering the nucleic acid microarray mounted on the recess.
(8) The hybridization reaction apparatus according to (7), wherein the component solid phase immobilization part is formed in the recess or the cover.
(9) The hybridization reaction apparatus according to (1), wherein the component solid phase immobilization part is formed in a dispensing chip for dispensing a reaction solution after a nucleic acid amplification reaction.
(10) The hybridization reaction apparatus according to (1), wherein the component solid phase immobilization part is formed in a stirring means for stirring the reaction solution after the nucleic acid amplification reaction.
(11) The hybridization according to (1), wherein the reaction unit includes a container that can store a solution and a temperature adjustment unit that can adjust the solution stored in the container to a desired temperature. Reactor.
(12) The hybridization reaction apparatus according to (1), wherein the component solid phase immobilization part further includes a DNA fragment that specifically hybridizes with the position spot DNA of the nucleic acid microarray.
(13) It further includes a DNA fragment immobilization part containing a DNA fragment that specifically hybridizes with the position spot DNA of the nucleic acid microarray, and the reaction solution after the nucleic acid amplification reaction is the component immobilization part and the DNA The hybridization reaction apparatus according to (1), wherein the hybridization reaction apparatus is in contact with a fragment-immobilized part.
(14) A nucleic acid microarray comprising a substrate, a probe molecule fixed to one principal surface of the substrate, and a component solid phase immobilization part containing at least a salt used for the hybridization reaction on one principal surface of the substrate.
(15) The nucleic acid according to (14), wherein the probe molecule includes a position spot DNA, and the component solid phase immobilization part further includes a DNA fragment that specifically hybridizes with the position spot DNA. Microarray.
(16) The probe molecule further includes a DNA fragment solid-phased portion containing a DNA for position spot and a DNA fragment specifically hybridizing with the DNA for position spot on one main surface of the substrate. The nucleic acid microarray according to (14).
(17) A genetic testing method including a hybridization reaction using a nucleic acid microarray, wherein the reaction solution after the nucleic acid amplification reaction is mixed with a component-immobilized part containing a salt used for the hybridization reaction before the hybridization reaction A genetic test method comprising a step of contacting the sample.
(18) The gene testing method according to (17), wherein the component-immobilized part further comprises a DNA fragment that specifically hybridizes with the position spot DNA of the nucleic acid microarray.
(19) In the step of bringing the reaction solution after the nucleic acid amplification reaction into contact with the component solid phase immobilization part, the DNA specifically hybridizes with the position spot DNA of the nucleic acid microarray in the reaction solution after the nucleic acid amplification reaction The genetic testing method according to (17), wherein the method comprises contacting a DNA fragment-immobilized part containing a fragment.

  According to the hybridization reaction apparatus, the nucleic acid microarray, or the gene testing method according to the present invention, the nucleic acid amplification reaction solution can be very easily made into a solution suitable for the hybridization reaction. Therefore, by using the hybridization reaction apparatus, nucleic acid microarray, or genetic testing method according to the present invention, the apparatus can be reduced in size and cost.

It is a top view of a nucleic acid microarray. It is the principal part front view and principal part side view of the jig | tool which mount the nucleic acid microarray in a hybridization reaction apparatus. It is principal part sectional drawing which shows the state immersed in the nucleic acid amplification reaction liquid which put the nucleic acid microarray mounted in the jig | tool in the container. It is a principal part perspective view of the form of the reaction part which mounted the nucleic acid microarray in a hybridization reaction apparatus. It is a disassembled perspective view of a hybridization reaction apparatus provided with a plate and a cover. It is the top view and sectional drawing of a plate of a hybridization reaction apparatus provided with a plate and a cover. It is a perspective view, a top view, a sectional view, and a principal part sectional view of a cover of a hybridization reaction device provided with a plate and a cover. It is principal part sectional drawing of the state which attached the cover to the plate of the hybridization reaction apparatus provided with a plate and a cover. FIG. 2 is a cross-sectional view of a nucleic acid amplification reaction liquid transfer apparatus including a pipette device that sucks up a nucleic acid amplification reaction liquid in a hybridization reaction apparatus and a tip attached to the tip of the pipette apparatus. It is principal part sectional drawing of the stirring means at the time of performing the hybridization reaction in a hybridization reaction apparatus. It is a top view of the nucleic acid microarray used in the comparative example and Example of this invention. In the comparative example and Example of this invention, it is the image which measured the fluorescence after hybridization reaction implementation. It is the flowchart explaining the process of dripping a reaction liquid on the conventional nucleic acid microarray, and covering a cover. It is a schematic block diagram of the conventional genetic testing apparatus.

  A hybridization reaction apparatus to which the present invention is applied performs a genetic test using a nucleic acid microarray, and includes a reaction part that performs a hybridization reaction using a nucleic acid microarray, and at least a salt used for the hybridization reaction. A component-immobilized part, and the nucleic acid amplification reaction solution contacts the component-immobilized part before the hybridization reaction.

  When the component-immobilized part comes into contact with the nucleic acid amplification reaction solution, it is dissolved in the solution. Thus, the nucleic acid amplification reaction solution becomes a solution that can be used for the hybridization reaction.

  Therefore, between the nucleic acid amplification reaction and the hybridization reaction, (1) a step of adding a buffer containing a predetermined salt to the nucleic acid amplification reaction solution, or (2) taking out the amplified nucleic acid from the nucleic acid amplification reaction solution, After drying, a solution for the hybridization reaction can be easily prepared without performing the step of adding to the buffer.

  The nucleic acid microarray 1 that can be used in the hybridization reaction apparatus is not particularly limited. For example, as shown in FIG. 1, the nucleic acid microarray 1 includes a substrate 2 and a probe spot 3 having probe molecules fixed on one main surface of the substrate 2. Yes. Further, the nucleic acid microarray 1 may include a position spot 4 formed by fixing a position spot DNA at a predetermined position on the substrate 2.

  Here, the probe spot 3 indicates a nucleotide having a base sequence that specifically hybridizes to a gene to be examined, that is, a region where a probe molecule is immobilized. Further, the position spot 4 indicates a region in which a nucleotide that specifically hybridizes to a DNA fragment having an arbitrary base sequence, that is, a position spot DNA is fixed.

  The position spot 4 emits fluorescence when a fluorescently labeled DNA fragment specifically hybridizes. Therefore, the position of the probe spot 3 on the nucleic acid microarray 1 can be specified by referring to the fluorescence of the position spot 4. Moreover, the quality of the hybridization reaction can also be confirmed.

  The probe molecule is preferably a nucleic acid, more preferably DNA. The DNA includes both double strands and single strands, but single strand DNA is preferred. The probe molecule is preferably an oligonucleotide having a total length of 17 to 23 bases, particularly an oligonucleotide having a length of 21 to 23 bases.

  The probe molecule can be obtained, for example, by chemically synthesizing with a nucleic acid synthesizer. As the nucleic acid synthesizer, an apparatus called a DNA synthesizer, a fully automatic nucleic acid synthesizer, an automatic nucleic acid synthesizer, or the like can be used.

  The substrate 2 used in the nucleic acid microarray 1 preferably has a fine flat plate structure. The shape is not limited to a rectangle, square, or round shape, but is usually 1 to 75 mm square, particularly preferably 1 to 10 mm square, and more preferably 3 to 5 mm square.

  As a material of the substrate 2, 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 alcohol , 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 organic materials such as polysulfone. In particular, it is preferable to use a substrate made of a silicon material or a resin material. Note that single crystal silicon includes crystal parts that have slightly changed the orientation of the crystal axis (sometimes referred to as mosaic crystals) and atomic scale disturbances (lattice defects). What is included is also included.

  It is preferable that the board | substrate 2 comprised by said material further has a carbon layer and a chemical modification group on the surface. For example, as the substrate 2, a substrate having a carbon layer and a chemical modification group on the surface, or a substrate having a chemical modification group on the substrate surface made of a carbon layer is used. In particular, it is preferable to use the substrate 2 having a carbon layer and a chemical modification group on the surface of the substrate made of single crystal silicon.

  By introducing a chemical modifying group into the surface of the substrate 2 on which the carbon layer is formed, the probe molecules can be firmly immobilized on the carrier. The chemical modification group to be introduced can be appropriately selected by those skilled in the art and is not particularly limited, and examples thereof include an amino group, a carboxyl group, an epoxy group, a formyl group, a hydroxyl group, and an active ester group.

  Probe spots 3 are formed by immobilizing probe molecules on the substrate 2 described above, and the nucleic acid microarray 1 is manufactured. For example, a probe molecule is dissolved in a spotting buffer to prepare a spotting solution, which is dispensed into a 96-hole or 384-hole plastic plate, and the dispensed solution is spotted on the substrate 2 by a spotter device or the like. The nucleic acid microarray 1 is manufactured by forming the probe spot 3. Alternatively, the spotting solution may be spotted manually with a micropipette.

  After spotting, incubation is preferably performed in order to advance a reaction in which the probe molecule binds to the substrate surface. Incubation is usually performed at a temperature of −20 to 100 ° C., preferably 4 to 90 ° C., usually for 0.5 to 16 hours, preferably for 1 to 2 hours. Incubation is preferably performed under a high humidity atmosphere, for example, under conditions of a humidity of 50 to 90%. Subsequent to incubation, in order to remove DNA not bound to the substrate, washing with a washing solution (for example, 50 mM TBS / 0.05% Tween 20, 2 × SSC / 0.2% SDS solution, ultrapure water, etc.) is performed. Preferably it is done.

  In the hybridization reaction apparatus to which the present invention is applied, for example, a jig 5 equipped with a nucleic acid microarray 1 as shown in FIG. 2 can be used. The jig 5 has a recess 6 in which the nucleic acid microarray 1 can be mounted. The recess 6 is formed by an inclined surface 8 and a bottom surface 7. The nucleic acid microarray 1 is mounted on the bottom surface 7. The inclined surface 8 and the bottom surface 7 of the recess 6 form an angle 9. The rising angle 9 of the inclined surface 8 from the bottom surface 7 is an angle between the extended line of the bottom surface 7 (broken line shown in FIG. 2B) and the inclined surface 8. The angle 9 is preferably 45 ° to 75 °, for example.

  The component solid phase immobilization part 10 can be formed on the inclined surface 8 below the nucleic acid microarray 1, for example. The component immobilization part 10 is formed by immobilizing a salt used for a hybridization reaction using the nucleic acid microarray 1 on the inclined surface 8.

The salt used for the hybridization reaction is not particularly limited as long as it can generate a monovalent cation that dissociates in a solution and reduces the electrostatic repulsion between DNAs. Examples of the salt used for the hybridization reaction include sodium chloride, sarinsodium citrate (SSC; 3M sodium chloride / 0.3M sodium citrate), SSPE (including NaCl, NaH ₂ PO ₄ .H ₂ O and EDTA). Can be mentioned.

  Here, solidifying the salt used for the hybridization reaction includes, for example, mixing the salt used for the hybridization reaction with a solvent, and adding a predetermined amount of the resulting mixed solution to the portion forming the component solid phase immobilization part 10. After feeding, the solvent is removed. It is preferable to use a volatile solvent as the solvent. As the solvent, water, alcohol, or the like can be used, but the solvent is not limited thereto. The solvent may dissolve or not dissolve the salt used in the hybridization reaction. Examples of the method for supplying the mixed solution include, but are not limited to, a spray method, a dip coating method, a spin coating method, and a blade coating method. You may make it solid-phase by dripping a mixed solution and drying a solvent.

  Here, the amount of the salt to be immobilized is preferably such an amount that the salt concentration can be dissolved in the nucleic acid amplification reaction solution and hybridized. For example, the amount of salt to be immobilized can be adjusted according to the volume of the nucleic acid amplification reaction solution. For example, the amount of salt to be immobilized is preferably adjusted so that the concentration of the salt in the reaction solution is 1 mmol / l to 1000 mmol / l, preferably 75 mmol / l to 600 mmol / l during the hybridization reaction.

  The component solid phase immobilization unit 10 may solidify other components in addition to the salt used in the above-described hybridization reaction. Other components include components other than salts used in the hybridization reaction, such as sodium dodecyl sulfate (SDS), a surfactant that reduces surface tension, formamide, a component that lowers the melting temperature Tm, and non-specific components. Examples thereof include a blocking agent that minimizes mechanical binding. Blocking agents include Denhardt's reagent, fragmented salmon sperm DNA, tRNA, and Cotl DNA. Where cDNA probe molecules are used, poly (A) + RNA or poly dA nucleotides that bind to T-rich sequences are also included. Further, as other components, there are high-molecular sugars (such as dextran) having an effect of increasing the apparent probe molecule concentration in the solution used for the hybridization reaction.

  Furthermore, when the nucleic acid microarray 1 has the position spot 4, the component solid phase immobilization unit 10 may have a DNA fragment that specifically hybridizes to the position spot DNA. This DNA fragment can be dissolved or dispersed in a solvent used for immobilizing the salt used in the above-described hybridization reaction, and solid-phased with the salt used in the above-described hybridization reaction.

  The DNA fragment that specifically hybridizes to the position spot DNA can also be immobilized on a portion different from the component-immobilized portion 10. That is, although not shown, the hybridization reaction apparatus may further include a DNA fragment immobilization unit that immobilizes a DNA fragment that specifically hybridizes to the position spot DNA.

  In the hybridization reaction apparatus, the jig 5 configured as described above is immersed in a nucleic acid amplification reaction solution placed in a container as shown in FIG. 3 with the nucleic acid microarray 1 mounted thereon. When the jig 5 is immersed in the nucleic acid amplification reaction solution in the container 11, the component solid phase immobilization part 10 comes into contact with the nucleic acid amplification reaction solution, and the salt used for the hybridization reaction is dissolved. Thereby, the nucleic acid amplification reaction solution can be made into a solution having a salt concentration suitable for the hybridization reaction.

  In the example shown in FIG. 2, the component solid phase immobilization part 10 is formed on the inclined surface 8, but the position of the component solid phase immobilization part 10 is not limited to this. Moreover, the component solid-phase immobilization part 10 may be formed in one place, or may be formed in two or more places. The component solid-phase immobilization unit 10 may be a position where the jig 5 can be brought into contact with the nucleic acid amplification reaction solution in a state where the jig 5 is immersed in the nucleic acid amplification reaction solution in the container 11. For example, the component solid phase immobilization unit 10 may be at any position such as the bottom surface 7, the tip of the jig 5, the inner surface of the container 11, and the like.

  In the hybridization reaction apparatus to which the present invention is applied, the hybridization reaction can be carried out using the solution for hybridization reaction prepared as described above. Although the temperature conditions of a hybridization reaction are not specifically limited, For example, it can be 45-65 degreeC.

  After the hybridization reaction, the probe molecules on the probe spot 3 of the nucleic acid microarray 1 and the nucleic acid that has not hybridized with the position spot DNA on the position spot 4 are washed and removed using a washing solution. After the nucleic acid microarray 1 is washed or washed and dried, the test nucleic acid hybridized to the probe molecule is analyzed by a detector.

  As a detector, for example, there is a detector that irradiates the nucleic acid microarray 1 with excitation light using a laser or the like and detects the intensity of fluorescence obtained. In the nucleic acid amplification reaction, the fluorescent label is incorporated into the amplified nucleic acid, and the obtained amplified nucleic acid is hybridized to the probe molecule on the nucleic acid microarray 1 as a test nucleic acid. After washing or washing and drying, the nucleic acid microarray 1 is irradiated with excitation light. Upon irradiation, fluorescence derived from the test nucleic acid hybridized to the probe molecule is detected. By detecting this, a hybridization reaction can be detected.

  As described above, by using the hybridization reaction apparatus having the jig 5 as shown in FIGS. 2 and 3 to which the present invention is applied, (1) a predetermined salt between the nucleic acid amplification reaction and the hybridization reaction. Without adding a step of adding a buffer containing a nucleic acid to a nucleic acid amplification reaction solution, or (2) removing a nucleic acid amplified from a nucleic acid amplification reaction solution, drying, and adding to a buffer. Solutions can be easily prepared.

  By the way, the hybridization reaction apparatus to which the present invention is applied is not limited to the one shown in FIGS. 2 and 3, and is composed of, for example, a reaction unit 12 on which the nucleic acid microarray 1 is placed as shown in FIG. 4. The reaction unit 12 includes a recess 13 on which the nucleic acid microarray 1 is placed, and flow paths 14 and 15 connected to the recess 13.

  The component solid phase immobilization part 10 can be formed, for example, on the inner wall of the recess 13. The component-immobilized portion 10 can be formed on the inner wall of the recess 13 under the same conditions as the conditions in which the salt used for the hybridization reaction using the nucleic acid microarray 1 is immobilized in the jig 5.

  In the reaction unit 12, the nucleic acid amplification reaction solution is injected from the flow path 14 into the recess 13. When the nucleic acid amplification reaction solution is injected into the recess 13, the discharge of the nucleic acid amplification reaction solution from the flow path 15 is suppressed. When the nucleic acid amplification reaction solution is injected to fill the recess 13, the component solid phase immobilization unit 10 comes into contact with the nucleic acid amplification reaction solution, and the salt used for the hybridization reaction is dissolved. At this time, according to the spatial volume formed by the flow paths 14 and 15, the nucleic acid microarray 1, and the recess 13, the component solid phase immobilization unit 10 is used so that the nucleic acid amplification reaction solution is a solution having a salt concentration suitable for the hybridization reaction. It is preferable to adjust the amount of the salt.

  In the example shown in FIG. 4, the component solid phase immobilization part 10 is formed on the inner wall of the recess 13, but the position of the component solid phase immobilization part 10 is not limited to this. Moreover, the component solid-phase immobilization part 10 may be formed in one place, or may be formed in two or more places. The component solid-phase immobilization unit 10 may be any position that can contact the nucleic acid amplification reaction solution in a state where the recess 13 is filled with the nucleic acid amplification reaction solution. For example, the component solid-phase immobilization unit 10 may be at any position such as a channel 14 for supplying a solution to the recess 13 and a channel 15 for discharging the solution from the recess 13. Since the nucleic acid amplification reaction solution is injected from the flow path 14, the component solid phase immobilization unit 10 is preferably formed in the recess 13 or the flow path 14.

  As described above, by using the hybridization reaction apparatus having the reaction section 12 as shown in FIG. 4 to which the present invention is applied, (1) a predetermined salt is included between the nucleic acid amplification reaction and the hybridization reaction. Without performing the step of adding a buffer to the nucleic acid amplification reaction solution, or (2) removing the amplified nucleic acid from the nucleic acid amplification reaction solution, drying it, and adding it to the buffer, the solution for the hybridization reaction is added. Easy to prepare.

  As another form of the hybridization reaction apparatus shown in FIGS. 2 to 4, there is a hybridization reaction apparatus 16 shown in FIGS. The hybridization reaction apparatus 16 includes a plate 18 having a recess 20 on which the nucleic acid microarray 1 is placed, a cover 19 that covers the nucleic acid microarray 1, and a component solid phase immobilization unit that includes the salt used for the hybridization reaction formed on the cover 19. 10.

  As shown in FIG. 6A, the plate 18 is a substantially rectangular flat plate and has a substantially circular recess 20 at a substantially central portion of the one main surface 17. The nucleic acid microarray 1 is detachably mounted in the recess 20. The recess 20 on which the nucleic acid microarray 1 is placed may have a guide corresponding to the outer shape of the nucleic acid microarray 1 formed on the bottom surface of the recess 20.

  In FIG. 6, as shown in a cross-sectional view (shown in FIG. 6B) when the pair of notches 21 of the plate 18 on which the nucleic acid microarray 1 is placed is cut, the thickness of the nucleic acid microarray 1 and the recess 20 The depth is the same. The thickness of the nucleic acid microarray 1 and the depth of the recess 20 are such that a predetermined interval is formed between one main surface of the nucleic acid microarray 1 and the cover 19 in a state where a cover 19 shown in FIG. If it is done, there is no limit. For example, the thickness of the nucleic acid microarray 1 can be made thicker or thinner than the depth of the recess 20.

  The plate 18 preferably has a pair of notches 21 formed on both side surfaces in the longitudinal direction in order to position and attach a cover 19 which will be described in detail later. In FIG. 6A, the pair of notches 21 are shown as positioning means, but the positioning means is not limited to such a configuration.

  Note that the plate 18 has a shape in which one end in the longitudinal direction becomes narrower toward the tip. Thereby, the direction of the nucleic acid microarray 1 placed on the plate 18 can be specified, and thereby the location of the positioning spot spotted at a specific position on the surface of the nucleic acid microarray 1 can be specified.

  Next, the cover 19 will be described. As shown in FIGS. 7A and 7B, the cover 19 has a recess 23 that forms a space with the plate 18 in a posture attached to the plate 18, and a recess 23 that is in the recess 23. A convex portion 22 formed at a position facing the nucleic acid microarray 1 in an attached posture and having a plane substantially the same shape as one main surface of the nucleic acid microarray 1, and a component solid phase immobilization portion 10 formed on the convex portion 22 I have. The cover 19 accommodates the nucleic acid microarray 1 in a space formed in a posture attached to the plate 18 (a space formed by the recess 23 and the recess 20).

  The height of the convex portion 22 can be designed in consideration of the distance from one main surface of the nucleic acid microarray 1 attached to the plate 18 in the posture in which the cover 19 is attached to the plate 18. As shown in FIGS. 7C and 7D, the convex portion 22 is designed to be lower than the contact surface 24 of the cover 19 with the plate 18 in the posture in which the cover 19 is attached to the plate 18. Yes. Thereby, when one main surface of the nucleic acid microarray 1 is at the same height as the one main surface 17 of the plate 18, a predetermined interval (D in FIG. 8) is formed between the convex portion 22 and the nucleic acid microarray 1. be able to. In other words, if a predetermined interval can be formed between the convex portion 22 and the nucleic acid microarray 1, the height of the convex portion 22 is designed to be the same as or higher than the contact surface 24 with the plate 2. You may do it.

  The cover 19 preferably has positioning means for positioning and attaching to the plate 18 described above. As the positioning means, as shown in FIGS. 7A and 7B, a pair of fitting convex portions 26 formed on convex edge portions 25 formed on both ends of the plate member can be used. . The pair of fitting protrusions 26 have positions and shapes corresponding to the pair of notches 21 formed on the plate 18 described above. Positioning means formed on the cover 19 is not limited to such a pair of fitting convex portions 26, and can be changed according to the positioning means formed on the plate 18 described above.

  The component solid phase immobilization part 10 is formed on the convex part 22 of the cover 19 described above. The component solid phase immobilization part 10 can be formed under the same conditions as the conditions in which the salt used for the hybridization reaction using the nucleic acid microarray 1 is solid phased in the jig 5.

  In the hybridization reaction apparatus 16 configured as described above, the nucleic acid amplification reaction solution is held as follows.

  First, the nucleic acid amplification reaction solution is dropped on the convex portion 22 of the cover 19. Thereafter, the cover 19 is immediately attached to the plate 18 in order to prevent the nucleic acid amplification reaction solution from evaporating. A state in which the cover 19 is attached to the plate 18 is shown in FIG. As shown in FIG. 8, when the cover 19 is attached to the plate 18, the one main surface 17 of the plate 18 and the contact surface 24 of the cover 19 are in close contact, and the one main surface of the nucleic acid microarray 1 and the convex portion 22 of the cover 19 A predetermined interval D (hereinafter, interval D) is formed between them. As a result, a space is formed by the recess 20 of the plate 18 on which the nucleic acid microarray 1 is placed, the recess 23 of the cover 19, and the interval D. The nucleic acid amplification reaction solution dropped on the convex portion 22 in the cover 19 is first held in the space formed by the interval D. Thereafter, the nucleic acid amplification reaction liquid having a space volume or more formed by the interval D is then formed in the space part formed by the outside of the convex part 22, that is, the concave part 20 of the plate 18 on which the nucleic acid microarray 1 is placed. To flow.

  Since the component solid phase immobilization part 10 is formed on the cover 19, the salt used for the hybridization reaction is dissolved in the nucleic acid amplification reaction liquid when the nucleic acid amplification reaction liquid is dropped onto the convex part 22 of the cover 19. As a result, the nucleic acid amplification reaction solution becomes a solution having a salt concentration suitable for the hybridization reaction.

  In holding the nucleic acid amplification reaction solution, the cover 19 may be attached after the nucleic acid amplification reaction solution is dropped onto the nucleic acid microarray 1. In this case, the component solid phase immobilization unit 10 comes into contact with the nucleic acid amplification reaction solution when the cover 19 is attached to the plate 18, and the salt used for the hybridization reaction is dissolved in the nucleic acid amplification reaction solution.

  Further, as described above, since the component-immobilized part 10 contacts and dissolves the nucleic acid amplification reaction solution immediately after the cover 19 is attached to the plate 18, the thickness of the component-immobilized part 10 is not particularly limited. Absent. The thickness of the component solid phase immobilization part 10 is preferably not more than the interval D.

  In the example shown in FIGS. 5 to 8, the component solid phase immobilization part 10 is formed on the convex portion 22 of the cover 19, but the position of the component solid phase immobilization part 10 is not limited to this. Moreover, the component solid-phase immobilization part 10 may be formed in one place, or may be formed in two or more places. The component solid phase immobilization unit 10 may be in a position where the cover 19 is attached to the plate 18 on which the nucleic acid microarray 1 is placed and can come into contact with the nucleic acid amplification reaction solution. It can be formed in a space formed by the recess 20 of the plate 18 on which the microarray 1 is placed, the recess 23 of the cover 19, and the distance D. For example, the component solid-phase immobilization unit 10 may be at any position such as the recess 20 of the plate 18 on which the nucleic acid microarray 1 is placed, the recess 23 of the cover 19, and the like.

  Although not shown, the nucleic acid microarray 1 is placed on the plate 18 without providing the concave portion 20, the concave portion is provided on the cover 19 to form the component solid phase immobilization portion 10, and nucleic acid amplification is performed in the concave portion of the cover 19. It is also possible to perform a hybridization reaction by dropping the reaction solution.

  As described above, by using the hybridization reaction apparatus 16 as shown in FIGS. 5 to 8 to which the present invention is applied, (1) a buffer containing a predetermined salt is provided between the nucleic acid amplification reaction and the hybridization reaction. Without adding the step of adding to the nucleic acid amplification reaction solution, or (2) removing the amplified nucleic acid from the nucleic acid amplification reaction solution, drying it, and adding it to the buffer. Can be prepared.

  In addition to the apparatuses described with reference to FIGS. 2 to 8, the hybridization reaction apparatus to which the present invention is applied may have, for example, the solution transfer apparatus shown in FIG. 9 or the stirring means shown in FIG.

  FIG. 9 shows a solution transfer device including a pipette device 27 for sucking up a nucleic acid amplification reaction solution and a dispensing tip 28 attached to the tip of the pipette device 27. The component solid phase immobilization part 10 is formed on the inner wall surface of the dispensing tip 28, for example.

  FIG. 10 shows a stirring means when performing the hybridization reaction. FIG. 10A shows a stirring means having a mechanism including a stirring blade 30 and a shaft 29 attached to the stirring blade 30. The component solid phase immobilization part 10 is formed in the stirring blade 30, for example. FIG. 10B shows a stirring means of a mechanism including a stirrer 31 made of a magnetic material and a stirrer body 32 having a magnetic material capable of rotating inside. The component solid phase immobilization part 10 is formed in the stirring bar 31, for example. (C) of FIG. 10 is a stirring means of a mechanism comprising a pipe 33 for introducing a gas such as argon gas or nitrogen and a bubble generating unit 34 connected to the pipe 33. The component solid phase immobilization unit 10 is formed, for example, on the wall surface of the bubble generation unit 34 on the side in contact with the nucleic acid amplification reaction solution. (D) of FIG. 10 is a stirring means of a mechanism including a pipette device 35 and a tip 36 attached to the tip of the pipette device 35. The component solid phase immobilization unit 10 is formed in a portion that comes into contact with the nucleic acid amplification reaction solution inside and / or outside the tip of the chip 36, for example.

  In addition, the component solid-phase immobilization part 10 shown in FIG.9 and FIG.10 forms the salt used for the hybridization reaction using the nucleic acid microarray 1 on the conditions similar to the conditions solidified in the said jig | tool 5. FIG. be able to.

  Moreover, the component solid-phase immobilization part 10 is not limited to the members and positions shown in FIGS. 9 and 10, and may be any members and positions as long as they can contact the nucleic acid amplification reaction solution. The component solid phase immobilization part 10 may be formed in one place, or may be formed in two or more places.

  From the above, by using a hybridization reaction apparatus having the apparatus and means as shown in FIGS. 9 and 10 to which the present invention is applied, (1) a predetermined value between the nucleic acid amplification reaction and the hybridization reaction can be obtained. For a hybridization reaction without performing a step of adding a buffer containing a salt to the nucleic acid amplification reaction solution, or (2) removing the amplified nucleic acid from the nucleic acid amplification reaction solution, drying it and adding it to the buffer. A simple solution can be prepared.

  In the above description, the component immobilization unit 10 is formed at a position in contact with the nucleic acid amplification reaction solution except for the nucleic acid microarray 1 in the hybridization reaction apparatus. It can also be formed.

  That is, the nucleic acid microarray to which the present invention is applied comprises a substrate, a probe molecule immobilized on one principal surface of the substrate, and at least a component solid phase comprising a salt used for the hybridization reaction on one principal surface of the substrate A nucleic acid microarray comprising a portion.

  The nucleic acid microarray for forming the component-immobilized portion is not particularly limited, and the nucleic acid microarray described in the hybridization reaction apparatus can be used. For example, the nucleic acid microarray 1 shown in FIG. 1 can be used.

  The component-immobilized part is formed on the surface where the probe molecules of the nucleic acid microarray are spotted and / or on the back surface thereof. The component solid phase immobilization part can be formed under the same conditions as the conditions in which the salt used for the hybridization reaction using the nucleic acid microarray is solid phased in the jig 5. Moreover, the component solid phase immobilization part may be formed in one place, or may be formed in two or more places.

  When the nucleic acid microarray is immersed in the nucleic acid amplification reaction solution, the component-immobilized portion comes into contact with the nucleic acid amplification reaction solution, and the salt used for the hybridization reaction is dissolved. Thereby, the nucleic acid amplification reaction solution can be made into a solution having a salt concentration suitable for the hybridization reaction.

  As described above, by using the nucleic acid microarray to which the present invention is applied, (1) a buffer containing a predetermined salt is added between the nucleic acid amplification reaction and the hybridization reaction, regardless of the hybridization reaction apparatus used. A solution for a hybridization reaction is easily prepared without performing the step of adding to the amplification reaction solution, or (2) removing the amplified nucleic acid from the nucleic acid amplification reaction solution, drying and adding it to the buffer. be able to.

  By using the hybridization reaction apparatus or nucleic acid microarray described above, the genetic testing method to which the present invention is applied can be performed.

  That is, a method for examining a gene containing a hybridization reaction using a nucleic acid microarray, wherein the nucleic acid amplification reaction solution is contacted with a component-immobilized portion containing a salt used for the hybridization reaction before the hybridization reaction A genetic testing method comprising

  Between the nucleic acid amplification reaction and the hybridization reaction, the salt used for the hybridization reaction is dissolved in the nucleic acid amplification reaction solution by contacting the nucleic acid amplification reaction solution with the component immobilization part containing the salt used for the hybridization reaction. Thus, the solution can be used for the hybridization reaction.

  Therefore, between the nucleic acid amplification reaction and the hybridization reaction, (1) a step of adding a buffer containing a predetermined salt to the nucleic acid amplification reaction solution, or (2) taking out the amplified nucleic acid from the nucleic acid amplification reaction solution, The solution for hybridization reaction can be easily prepared without performing the step of adding to the buffer after drying, and the lead time of the entire test can be shortened.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to a following example.

Preparation for Experiment 1) Preparation of Nucleic Acid Microarrays Gene DNA (manufactured by Toyo Kohan Co., Ltd.) was spotted with oligo DNA (concentration: 5 μM) shown below (SPBIO2000 manufactured by Hitachi Soft) at the spot position shown in FIG. Then, it is heated by an incubator (80 ° C. × 60 minutes), gene silicon and oligo DNA are amide-bonded, and the unbound oligo DNA is washed in 2 × SSC / 0.2% SDS at 60 ° C. for 10 minutes. Nucleic acid microarrays were prepared by washing and removing with ultrapure water in 2 × SSC at room temperature for 10 minutes, followed by drying.
The sequences of the six types of probe molecules are as shown in Table 1.

  The prepared nucleic acid microarray was set in a hybridization reactor using double-sided tape. The hybridization reaction apparatus used in the examples is of the type having the configuration shown in FIGS.

2) Preparation of a cover having a component-immobilized part The following two types of solutions (i) or (ii) 3.0 μL were dropped on the convex part of the cover, and left to stand for 1 hour to dry. A cover in which a solid phase part was formed was produced.
(I) 0.75 × SSC / 0.075% SDS solution (ii) Mixed solution of 0.75 × SSC / 0.075% SDS solution and Cy5 fluorescently labeled oligo DNA complementary to the probe molecule (Cy5 fluorescent labeling) Oligo DNA concentration 0.04 μM)
The three covers used for the evaluation are shown below.

Cover A: Cover without immobilization.
Cover B: (i) A cover in which 3.0 μL of the solution was dropped to solidify the salt.
Cover C: (ii) A cover obtained by dropping 3.0 μL of the solution and solidifying a composition containing salt.

3) Preparation of hybridization solution The following three types of hybridization solutions were prepared and prepared.
(I) A mixed solution of 6 kinds of Cy5 fluorescently labeled oligo DNA complementary to the probe molecule and 0.75 × SSC / 0.075% SDS solution (Cy5 fluorescently labeled oligo DNA 6 kinds concentration 0.04 μM)
(II) A solution containing only 6 types of Cy5 fluorescently labeled oligo DNA complementary to the probe molecule (Cy5 fluorescently labeled oligo DNA 6 types concentration 0.04 μM)
(III) Ultrapure water

Reaction experiment An experiment was conducted according to the following reaction procedure.

Comparative Example 1 (conventional method)
(1) 2 μL of the solution of (I) prepared in 3) and 1 μL of 2.25 × SSC / 0.225% SDS solution were mixed.
(2) 3 μL of the solution of (1) was placed on the convex part of the cover A produced in 2).
(3) The nucleic acid microarray set in the hybridization reaction apparatus was covered with the cover A of (2), and the hybridization reaction was performed at 52 ° C. for 60 minutes with an incubator.
(4) The hybridization reaction apparatus was taken out of the incubator, and the cover A was removed. The nucleic acid microarray was immersed and washed (5 minutes) with a washing solution (room temperature, 2 × SSC / 0.2% SDS solution), and then lightly and manually washed with a rinse solution (room temperature, 2 × SSC solution) and dried. .
(5) The analysis was performed using FLA-8000 (Fuji Film scanner, PM = 50%).

Comparative Example 2
(1) 2 μL of the solution of (II) prepared in 3) and 1 μL of water were mixed.
(2) 3 μL of the solution of (1) was placed on the convex part of the cover A produced in 2).
(3) The nucleic acid microarray set in the hybridization reaction apparatus was covered with the cover A of (2), and the hybridization reaction was performed at 52 ° C. for 60 minutes with an incubator.
(4) The hybridization reaction apparatus was taken out of the incubator, and the cover A was removed. The nucleic acid microarray was immersed and washed (5 minutes) with a washing solution (room temperature, 2 × SSC / 0.2% SDS solution), and then lightly and manually washed with a rinse solution (room temperature, 2 × SSC solution) and dried. .
(5) The analysis was performed using FLA-8000 (Fuji Film scanner, PM = 50%).

Comparative Example 3
(1) 3 μL of the ultrapure water of (III) prepared in 3) was placed on the convex part of the cover A produced in 2).
(2) The cover A of (1) was placed on the nucleic acid microarray set in the hybridization reaction apparatus, and the hybridization reaction was performed at 52 ° C. for 60 minutes by an incubator.
(3) The hybridization reaction apparatus was taken out from the incubator, and the cover A was removed. The nucleic acid microarray was immersed and washed (5 minutes) with a washing solution (room temperature, 2 × SSC / 0.2% SDS solution), and then lightly and manually washed with a rinse solution (room temperature, 2 × SSC solution) and dried. .
(4) Analysis was performed with FLA-8000 (Fuji Film scanner, PM = 50%).

Comparative Example 4
(1) 3 μL of the ultrapure water of (III) prepared in 3) was placed on the convex part of the cover B produced in 2).
(2) The cover B of (1) was placed on the nucleic acid microarray set in the hybridization reaction apparatus, and the hybridization reaction was performed at 52 ° C. for 60 minutes using an incubator.
(3) The hybridization reaction apparatus was removed from the incubator, and the cover B was removed. The nucleic acid microarray was immersed and washed (5 minutes) with a washing solution (room temperature, 2 × SSC / 0.2% SDS solution), and then lightly and manually washed with a rinse solution (room temperature, 2 × SSC solution) and dried. .
(4) Analysis was performed with FLA-8000 (Fuji Film scanner, PM = 50%).

Example 1
(1) 2 μL of the solution of (II) prepared in 3) and 1 μL of water were mixed.
(2) 3 μL of the solution of (1) was placed on the convex part of the cover B produced in 2).
(3) The nucleic acid microarray set in the hybridization reaction apparatus was covered with the cover B of (2), and the hybridization reaction was performed at 52 ° C. for 60 minutes with an incubator.
(4) The hybridization reaction apparatus was taken out of the incubator, and the cover B was removed. The nucleic acid microarray was immersed and washed (5 minutes) with a washing solution (room temperature, 2 × SSC / 0.2% SDS solution), and then lightly and manually washed with a rinse solution (room temperature, 2 × SSC solution) and dried. .
(5) The analysis was performed using FLA-8000 (Fuji Film scanner, PM = 50%).

Example 2
(1) 3 μL of the ultrapure water of (III) prepared in 3) was placed on the convex part of the cover C produced in 2).
(2) The cover C of (1) was placed on the nucleic acid microarray set in the hybridization reaction apparatus, and hybridization reaction was performed at 52 ° C. for 60 minutes by an incubator.
(3) The hybridization reaction apparatus was taken out from the incubator, and the cover C was removed. The nucleic acid microarray was immersed and washed (5 minutes) with a washing solution (room temperature, 2 × SSC / 0.2% SDS solution), and then lightly and manually washed with a rinse solution (room temperature, 2 × SSC solution) and dried. .
(4) Analysis was performed with FLA-8000 (Fuji Film scanner, PM = 50%).

The results of Comparative Examples 1 to 4 and Examples 1 and 2 are shown in FIG.
As shown in FIG. 12, it was confirmed that Examples 1 and 2 showed the same color of fluorescence as compared with Comparative Example 1 which is a conventional method. From the above, it was found that even if SSC / SDS and oligo DNA were immobilized, the hybridization reaction was not affected.

  Further, it was confirmed that Comparative Example 2 in which no SSC / SDS salt was present at the time of the hybridization reaction showed almost no color of fluorescence as compared with Comparative Example 1 and Examples 1 and 2. From the above, it was found that the cation such as SSC / SDS has an influence on the hybridization reaction.

DESCRIPTION OF SYMBOLS 1 ... Nucleic acid microarray, 2 ... Board | substrate, 3 ... Probe spot, 4 ... Position spot, 5 ... Jig, 6 ... Recessed part, 7 ... Bottom surface, 8 ... Inclined surface, 9 ... Angle, 10 ... Component solid-phased part, 11 DESCRIPTION OF SYMBOLS ... Container, 12 ... Reaction part, 13 ... Recess, 14 ... Flow path, 15 ... Flow path, 16 ... Hybridization reaction apparatus, 17 ... Main surface of plate, 18 ... Plate, 19 ... Cover, 20 ... Recess, 21 DESCRIPTION OF SYMBOLS ... Notch part, 22 ... Convex part, 23 ... Concave part, 24 ... Contact surface, 25 ... Edge part, 26 ... Fitting convex part, 27 ... Pipetting device, 28 ... Dispensing tip, 29 ... Shaft, 30 ... Stirring blade 31 ... Stirrer, 32 ... Stirrer body, 33 ... Pipe, 34 ... Bubble generating part, 35 ... Pipette device, 36 ... Tip

Claims (19)

A reaction part for performing a hybridization reaction using a nucleic acid microarray;
At least a component-immobilized part containing a salt used in the hybridization reaction,
A hybridization reaction apparatus, wherein a reaction solution after a nucleic acid amplification reaction is in contact with the component solid phase immobilization part.   The hybridization reaction apparatus according to claim 1, wherein the component solid phase immobilization part is formed in the reaction part.   The hybridization reaction apparatus according to claim 1, further comprising a jig on which the nucleic acid microarray can be mounted.   The hybridization reaction apparatus according to claim 3, wherein the component solid phase immobilization part is formed on the jig.   The said reaction part is equipped with the recessed part which mounts a nucleic acid microarray, and the flow path connected with the said recessed part, The solution is supplied with respect to the said recessed part through the said flow path. Hybridization reactor.   The hybridization reaction apparatus according to claim 5, wherein the component solid phase immobilization part is formed in the flow path for supplying a solution to the concave part or the concave part.   The hybridization reaction apparatus according to claim 1, wherein the reaction unit includes a recess for mounting the nucleic acid microarray and a cover for covering the nucleic acid microarray mounted on the recess.   The hybridization reaction apparatus according to claim 7, wherein the component solid phase immobilization part is formed in the recess or the cover.   The hybridization reaction apparatus according to claim 1, wherein the component solid phase immobilization part is formed in a dispensing chip for dispensing a reaction solution after a nucleic acid amplification reaction.   2. The hybridization reaction apparatus according to claim 1, wherein the component solid phase immobilization part is formed in a stirring means for stirring the reaction solution after the nucleic acid amplification reaction.   The hybridization reaction apparatus according to claim 1, wherein the reaction unit includes a container that can store a solution and a temperature adjustment unit that can adjust the solution stored in the container to a desired temperature.   The hybridization reaction apparatus according to claim 1, wherein the component solid phase immobilization part further includes a DNA fragment that specifically hybridizes with the position spot DNA of the nucleic acid microarray.   A DNA fragment immobilization part containing a DNA fragment that specifically hybridizes with the position spot DNA of the nucleic acid microarray; and a reaction solution after the nucleic acid amplification reaction is the component immobilization part and the DNA fragment solid phase The hybridization reaction apparatus according to claim 1, wherein the hybridization reaction apparatus is in contact with the crystallization unit. A substrate,
Probe molecules immobilized on one principal surface of the substrate;
A nucleic acid microarray comprising at least a component-immobilized part containing at least one salt used for the hybridization reaction on one main surface of the substrate. The probe molecule includes a position spot DNA,
15. The nucleic acid microarray according to claim 14, wherein the component-immobilized part further comprises a DNA fragment that specifically hybridizes with the position spot DNA. The probe molecule includes a position spot DNA,
15. The nucleic acid microarray according to claim 14, further comprising a DNA fragment solid phase immobilization part comprising a DNA fragment specifically hybridizing with the position spot DNA on one main surface of the substrate. A genetic test method including a hybridization reaction using a nucleic acid microarray,
A genetic test method comprising a step of bringing a reaction solution after a nucleic acid amplification reaction into contact with a component-immobilized part containing a salt used for a hybridization reaction before the hybridization reaction.   The gene testing method according to claim 17, wherein the component-immobilized part further comprises a DNA fragment that specifically hybridizes to the position spot DNA of the nucleic acid microarray.   In the step of bringing the reaction solution after the nucleic acid amplification reaction into contact with the component solid phase immobilization section, the reaction solution after the nucleic acid amplification reaction includes a DNA fragment that specifically hybridizes with the position spot DNA of the nucleic acid microarray. The genetic testing method according to claim 17, wherein the method is brought into contact with a DNA fragment immobilization part.

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