JP2007175003A - Genetic analyzer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To shorten a whole analyzer by starting measurement of the following reactor vessel without waiting until typing reaction temperature control part is cooled. <P>SOLUTION: The temperature control part constituting a typing reaction temperature control part is composed of a lower temperature control unit 60a located in the lower part of a probe-arranging part in a state in which the reactor vessel 41 is mounted on a mounting part and the upper temperature control unit 62a which can be located in the upper part of a probe-arranging part. The temperature of the upper temperature control unit 62a is previously controlled and the upper temperature control unit 62a is movably supported in a state slid in the horizontal direction between a position covering the upper part of the probe-arranging part and a position deviated from the position. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a genetic analysis apparatus using a reaction vessel suitable for performing various types of automatic analysis, for example, genetic analysis research and clinical practice, for example, polymorphisms of genomic DNA of animals and plants including humans. For example, the present invention relates to a genetic analyzer that handles a reaction vessel for detecting SNP (single nucleotide polymorphism) and the like.

The followings have been proposed as methods or apparatuses for predicting the susceptibility of diseases using genetic polymorphism.
In order to determine whether a patient is susceptible to sepsis and / or is likely to progress rapidly to sepsis, a nucleic acid sample is taken from the patient and the pattern 2 allele in the sample, or the pattern 2 allele When a marker gene that is linkage disequilibrium is detected and a marker gene that is linkage disequilibrium with the pattern 2 allele is detected, it is determined that the patient is susceptible to sepsis (see Patent Document 1). ).

  For diagnosis of one or more single nucleotide polymorphisms in the human flt-1 gene, one or more positions of human nucleic acids: 1953, 3453, 3888 (each position in EMBL accession number X51602) ), 519, 786, 1422, 1429 (according to the position in EMBL accession number D64016, respectively), 454 (according to SEQ ID NO: 3) and 696 (according to SEQ ID NO: 5). The human constitution is determined by referring to the formality (see Patent Document 2).

Many methods have been reported for so-called typing for discriminating the base of an SNP site. A typical one is the following method.
In order to perform typing on hundreds of thousands of SNP sites using a relatively small amount of nucleic acid, a plurality of base sequences including at least one single nucleotide polymorphism site can be simultaneously obtained using a nucleic acid and a plurality of pairs of primers. Using a plurality of amplified base sequences, the bases of single nucleotide polymorphic sites contained in the base sequences are discriminated by a typing process. As the typing process, an invader method or a Tuckman PCR (polymerase chain reaction) method is used (see Patent Document 3).

  The present inventors have proposed a reaction container for genetic polymorphism diagnosis provided with a plurality of probe placement portions each holding a fluorescent probe corresponding to each of a plurality of polymorphic sites. There, the size of the probe placement portion is, for example, a diameter of 100 μm to 2 mm and a depth of 50 μm to 1.5 mm.

In addition, the reaction vessel is provided with an amplification reaction section that handles a small amount of reaction solution. The gene amplification reaction region of the amplification reaction part is as thin as 0.1 to 0.5 mm, for example, so as to facilitate heat exchange. The amount of the amplification reaction solution handled in the amplification reaction unit is a very small amount, for example, about 0.1 μL to 5 μL.
Japanese translation of PCT publication No. 2002-533096 JP 2001-299366 A Japanese Patent Laid-Open No. 2002-300894 Japanese Patent No. 3542717 Hsu TM, Law S. M, Duan S, Neri BP, Kwok PY, "Genotyping single-nucleotide polymorphisms by the invader assay with dual-color fluorescence polarization detection", Clin. Chem., 2001 Aug; 47 (8): 1373 -7

  In order to react the reaction solution of the nucleic acid and the typing reagent with the probe of the probe placement unit, a typing reaction temperature control unit for controlling the temperature of the probe placement unit is required. Usually, the reaction vessel is arranged in the typing reaction temperature adjusting unit, and after the reaction solution is dispensed into the probe arrangement unit of the reaction vessel, the temperature rise in the typing reaction temperature adjusting unit is started.

  After the analysis is complete, the temperature of the typing reaction temperature controller is unlikely to drop.If the temperature remains high, the reaction solution evaporates during dispensing when the next reaction vessel is installed and the reaction solution is dispensed. If you wait until it cools, the problem will be that the waiting time until the next analysis becomes longer.

  Therefore, the object of the present invention is to enable the measurement of the next reaction vessel to be started without waiting for the typing reaction temperature controller to be cooled, thereby shortening the entire analysis time. To do.

  The genetic analysis device of the present invention is a mounting part for mounting a reaction container for diagnosing a genetic polymorphism having a plurality of probe placement parts each individually holding a fluorescent probe corresponding to each of a plurality of polymorphic sites; A typing reaction temperature control unit for controlling the temperature of the probe placement unit in order to cause the reaction solution of the genomic DNA and the typing reagent to react with the probe of the probe placement unit; a dispensing unit for transferring the reaction solution to each probe placement unit; A fluorescence detection device that detects fluorescence by irradiating each probe placement unit with excitation light, a temperature control of the typing reaction temperature adjustment unit, a dispensing operation of the dispensing unit, and a control unit that controls the detection operation of the fluorescence detection device; And at least a part of the typing reaction temperature control unit is located at a position where the temperature of the probe placement unit is adjusted in a state where the reaction container is mounted on the mounting unit. It is movably supported between the off-position position.

In a preferred form, the movable part of the temperature adjustment unit is pre-temperature adjusted before being moved to a position for temperature adjustment of the probe arrangement.
In another preferred embodiment, the typing reaction temperature control unit includes a lower temperature control unit positioned below the probe placement unit and an upper temperature control unit positioned above the probe placement unit. Or both are movable. More preferably, the one that can be moved by the temperature control unit is supported so as to be slidable in the horizontal direction, engages with a part of the dispensing unit, and is slid by movement of the dispensing unit.

A preferred example of the reaction vessel is a substrate provided with a probe placement portion, further comprising a non-volatile liquid storage portion formed as a recess, containing the non-volatile liquid and sealed with a film.
A more preferable example of a reaction vessel is a substrate on which a probe placement unit is provided, which contains a gene amplification reagent that includes a plurality of primers that are formed as recesses and that are bonded to each other across a plurality of polymorphic sites, and is sealed with a film And a gene amplification reagent storage unit.

  A more preferable example of the reaction vessel is a substrate provided with a probe placement portion, and a typing reagent storage portion formed as a recess, containing a typing reagent and sealed with a film.

  In the genetic analyzer of the present invention, the upper temperature control unit of the typing reaction temperature control unit is supported so as to be movable between a position covering the top of the probe placement unit and a position deviating from that position. It can be used to move the upper temperature control unit to the probe placement part after dispensing to each probe placement part of the reaction vessel, and the reaction liquid evaporates during dispensing even if the upper temperature control unit is not cooled. Therefore, the waiting time until the next analysis can be shortened.

  When trying to raise the temperature of the upper temperature control unit from the cooled state, it takes about several minutes and the overall analysis time becomes longer, but the upper temperature control unit can be moved to a position that covers the upper part of the probe placement unit. If the temperature is adjusted in advance, the temperature of the probe placement unit can be raised immediately after the upper temperature control unit is moved to the top of the probe placement unit, thereby shortening the overall analysis time. Can do.

  If the upper temperature control unit is supported so as to be slidable in the horizontal direction and engages with a part of the dispensing part and is slid by moving the dispensing part, the upper temperature control unit can be moved. A separate drive mechanism becomes unnecessary, the mechanism can be simplified, and a small and inexpensive device can be supplied.

FIG. 1 schematically shows an example of a gene analysis apparatus to which the present invention is applied.
Although not shown in the figure, it has a mounting part for mounting a reaction vessel having an amplification reaction part for performing a gene amplification reaction. Further, as shown in FIG. 1, a dispensing unit 112 that moves the nozzle 28 for suction and discharge to transfer the liquid in the reaction vessel, and a control unit that controls at least the dispensing operation of the dispensing unit 112. 118 at least.

  The reaction container is a genetic polymorphism diagnosis reaction container, and as a reagent storage part, a gene amplification reagent storage part containing a gene amplification reagent including a plurality of primers that bind across a plurality of polymorphic sites, and a typing reagent And a reaction reagent that includes an amplification reaction part that performs a gene amplification reaction on the mixture of the gene amplification reagent and the sample, and further provides fluorescence corresponding to each of the plurality of polymorphic sites. Are provided with a plurality of probe placement portions that individually hold probes that emit light.

  As shown in FIG. 1, the gene analysis apparatus includes an amplification reaction temperature control unit 120 that controls the temperature of an amplification reaction unit to a temperature for gene amplification that amplifies DNA in a reaction solution of a sample and a gene amplification reagent. And a typing reaction temperature control unit 110 that controls the temperature of the probe placement unit to a temperature at which the reaction solution of the sample genomic DNA and the typing reagent reacts with the probe of the probe placement unit, and excitation light is irradiated to each probe placement unit. And a fluorescence detection unit 64 that detects fluorescence. The control unit 118 also performs temperature control of the amplification reaction temperature control unit 120, temperature control of the typing reaction temperature control unit 110, and detection operation of the fluorescence detection unit 64.

When the invader reaction is used as the typing reaction, the typing reaction temperature control unit 110 serves as a temperature adjusting unit for the invader reaction.
When a PCR reaction is used as the gene amplification reaction, the amplification reaction temperature control unit 120 is a temperature control unit for a temperature cycle for the PCR reaction.
A personal computer (PC) 122 may be connected to the control unit 118 in order to operate the control unit 118 from the outside or display the inspection result.

The target of polymorphism detection according to the present invention is not particularly limited, and examples include cDNA, genes of various organisms including humans, genes of various microorganisms including viruses and bacteria, and the like. Accordingly, those having the nucleic acid sequence can be used as a template nucleic acid for nucleic acid amplification. Moreover, it does not ask | require whether it is a gene of DNA or a gene of RNA as a gene used as the object of these polymorphism detection.
A sample containing these genes is not particularly limited. That is, polymorphism detection can be performed on biological samples, biological samples, extracted nucleic acid samples, and the like. Here, a biological sample or a biological sample is a sample in a form in which a nucleic acid inclusion body (for example, a membrane structure containing nucleic acid inside, such as cells, fungi, bacteria, and viruses) is maintained without performing a nucleic acid extraction operation. is there. Examples of biological samples include organs, tissues, body fluids, excreta and the like. Body fluids include blood samples, spinal fluid, saliva, milk and the like. Blood samples include whole blood, plasma, serum and the like. Excrements include urine and feces. A biological sample is a nucleic acid inclusion body recovered from a biological sample. The method for recovering the nucleic acid inclusions is not particularly limited, and examples thereof include centrifugation and ultracentrifugation, methods using a coprecipitation agent such as polyethylene glycol, and an adsorption carrier. The extracted nucleic acid sample is a sample subjected to nucleic acid extraction operation, and may be a sample obtained by further purifying nucleic acid after extraction. Examples of the extraction method include a method using an enzyme, a surfactant, a chaotropic agent, and the like. Examples of the purification method include a method using phenol / chloroform, an ion exchange resin, a glass filter, glass beads, magnetic beads, a reagent having a protein aggregation action, and the like.
The polymorphisms that can be detected in the present invention are not particularly limited, and include both SNPs and polymorphisms that span multiple nucleotide sequences. In the present invention, the polymorphism further includes a mutation. Specifically, examples of the polymorphism that can be detected in the present invention include SNP, insertion polymorphism, deletion polymorphism, and repeat sequence polymorphism.
Here, when the relationship between the polymorphic site and the primer is shown, in order to amplify one polymorphic site, a pair of primers that bind across the polymorphic site are required. Since there are multiple types of polymorphic sites in the target biological sample, if these polymorphic sites are located at a distance from each other, twice as many types of primers as the types of polymorphic sites are required. become. However, when two polymorphic sites are close to each other, amplification can be performed by binding a primer between each of the polymorphic sites, or between the two polymorphic sites. Amplification can also be performed by binding primers only on both sides of the sequences of the two polymorphic sites without binding. Therefore, the number of necessary primers is not necessarily twice the number of types of polymorphic sites. In the present invention, “a plurality of primers that bind each of a plurality of polymorphic sites” refers not only to a case where a pair of primers binds to a single polymorphic site but also two or more polymorphic sites. It is used to mean the type of primer necessary to amplify multiple polymorphic sites, including when binding between.
An example of a gene amplification reagent is a PCR reaction reagent.

  For SNP typing, it is essential to adjust genomic DNA at the stage of entering the amplification process, which requires labor and cost. If attention is paid only to the PCR method for amplifying nucleic acids, a method of directly performing a PCR reaction from a sample such as blood without pretreatment has also been proposed. In the nucleic acid synthesis method for amplifying a target gene in a sample, a gene inclusion in the sample containing the gene, that is, a biological sample, or a sample containing the gene itself, that is, the biological sample is added to the amplification reaction solution. The target gene in the sample containing the gene is amplified when the pH of the reaction solution after addition is 8.5 to 9.5 (25 ° C.) (see Patent Document 4).

  The typing system that has already been constructed uses a PCR method to amplify a plurality of SNP regions to be typed by the PCR method. However, the amount of nucleic acid to be collected at first can be reduced. It is necessary to perform a pre-processing for extracting the. Therefore, it takes time and labor for the preprocessing.

Until now, no automated system has been constructed that simultaneously amplifies a plurality of SNP sites intended for typing when the direct PCR method and the typing method are combined.
The invader method or the Tuckman PCR method can be used for the typing process. In that case, the typing reagent is an invader reagent or a Tuckman PCR reagent.

FIG. 18 schematically shows a detection method for detecting a genetic polymorphism using the reaction container used in the present invention as a genetic polymorphism diagnostic reagent kit. Here, it is assumed that the PCR method is used for the amplification process and the invader method is used for the typing process.
In the PCR process, the PCR reaction reagent 4 is added to the biological sample 2 such as blood, or conversely, the biological sample 2 is added to the PCR reaction reagent 4.

The PCR reaction reagent 4 is prepared in advance and includes a plurality of primers for the SNP site to be measured, pH buffer for adjusting the pH, four types of deoxyribonucleotides, and thermostable synthesis. Necessary reagents such as enzymes and salts such as MgCl ₂ and KCl are added. In addition, substances such as surfactants and proteins can be added as necessary. The PCR method of the amplification step that may be used in the present invention is to simultaneously amplify a plurality of target SNP sites. The biological sample may be subjected to a nucleic acid extraction operation or may not be subjected to a nucleic acid extraction operation. Here, the biological sample that has not been subjected to the nucleic acid extraction operation is the biological sample, the biological sample, and the biological sample or biological body in which the membrane structure of the nucleic acid inclusion body is destroyed by an operation such as heat treatment or freezing treatment. Includes derived samples. When a plurality of genomic DNAs containing those SNP sites are directly amplified by PCR from a biological sample that has not been subjected to nucleic acid extraction operation, a gene amplification reaction reagent containing a plurality of primers for those SNP sites is used. The PCR reaction is allowed to occur under conditions such that when mixed with sample 2, the pH at 25 ° C. is 8.5-9.5.

As the pH buffer solution, various pH buffer solutions can be used in addition to a combination of tris (hydroxymethyl) aminomethane and a mineral acid such as hydrochloric acid, nitric acid and sulfuric acid. The pH-adjusted buffer is preferably used at a concentration between 10 mM and 100 mM in the PCR reaction reagent.
A primer refers to an oligonucleotide that serves as a starting point for DNA synthesis by a PCR reaction. The primer may be synthesized or may be isolated from the living world.

  Synthetic enzymes are enzymes for DNA synthesis by primer addition and include chemical synthesis systems. Suitable synthases include E. coli. DNA polymerase I, E. coli Klenow fragment of DNA polymerase of E. coli, T4 DNA polymerase, Taq DNA polymerase, T. Examples include, but are not limited to, litoralis DNA polymerase, Tth DNA polymerase, Pfu DNA polymerase, Hot Start Taq polymerase, KOD DNA polymerase, EX Taq DNA polymerase, and reverse transcriptase. “Thermal stability” means the property of a compound that retains its activity at elevated temperatures, preferably at 65-95 ° C.

  In the PCR step, a PCR reaction is performed on the mixture of the biological sample 2 and the PCR reaction reagent 4 according to a predetermined temperature cycle. The PCR temperature cycle includes three steps of denaturation, primer attachment (annealing), and primer extension, and DNA is amplified by repeating the cycle. As an example of each step, the denaturation step is 94 ° C. for 1 minute, the primer attachment step is 55 ° C. for 1 minute, and the primer extension is 72 ° C. for 1 minute. The biological sample may have been subjected to a nucleic acid extraction operation, but here, a biological sample that has not been subjected to a nucleic acid extraction operation is used. Even in the case of a biological sample that has not been subjected to nucleic acid extraction operation, the nucleic acid is released from blood cells and cells at a high temperature in the PCR temperature cycle, and the reaction proceeds when a reagent necessary for the PCR reaction contacts the nucleic acid.

  After the PCR reaction, the invader reagent 6 is added as a typing reagent. The invader reagent 6 includes a fluorescent (FRET) probe and a chestnut (Cleavase: structure-specific DNA degrading enzyme). A fret probe is a fluorescently labeled oligo having a sequence completely unrelated to genomic DNA, and the sequence is often common regardless of the type of SNP.

  Next, the reaction solution to which the invader reagent 6 is added is added to the plurality of probe placement units 8 to cause a reaction. In each probe placement section 8, an invader probe and a reporter probe are individually held corresponding to each of the plurality of SNP sites, and the reaction solution reacts with the invader probe, and the SNP corresponding to the reporter probe exists. Emits fluorescence.

The invader method is described in detail in paragraphs [0032] to [0034] of Patent Document 3.
If two types of reporter probes are prepared according to the corresponding SNP bases, it can be determined whether the SNP is a homozygote or a heterozygote.

  The invader method used in the typing step is a method of typing an SNP site by hybridizing an allele-specific oligo and a DNA containing the SNP to be typed. The DNA containing the SNP to be typed and the SNP to be typed Using two types of reporter probes and one type of invader probe specific to each of the alleles and an enzyme having a special endonuclease activity that recognizes and cleaves the DNA structure (see Patent Document 3). .)

FIG. 2 is an example of a reaction vessel used in the gene analysis apparatus of the present invention. (A) is a front view, (B) is a plan view.
On the same side of the flat substrate 10, a reagent container 14 and a non-volatile liquid container 16 having a specific gravity lower than that of the reaction liquid are formed as recesses. A reaction portion 18 is also formed on the same side of the substrate 10. The reagent container 14 and the non-volatile liquid container 16 are sealed with a film 20, and when the reagent and the non-volatile liquid are sucked with a nozzle and transferred to another place, the film 20 is removed and the nozzle 20 Inhalation is performed, or the film 20 is made to be piercable by the nozzle, and the nozzle is pierced and sucked by the nozzle.
The surface of the substrate 10 is covered with a peelable sealing material 22 having a size covering the reagent storage unit 14, the non-volatile liquid storage unit 16, and the reaction unit 18 from the film 20.

As the non-volatile liquid having a specific gravity lower than that of the reaction liquid, mineral oil (mineral oil), vegetable oil, animal oil, silicone oil, diphenyl ether, or the like can be used. Mineral oil is a liquid hydrocarbon mixture obtained by distillation from petrolatum and is also called liquid paraffin, liquid petrolatum, white oil, etc., and also includes low specific gravity light oil. Examples of animal oils include cod liver oil, halibut oil, herring oil, orange luffy oil, and shark liver oil. As the vegetable oil, canola oil, tonsil oil, cottonseed oil, corn oil, olive oil, peanut oil, safflower oil, sesame oil, soybean oil, and the like can be used.
In the embodiment, mineral oil is used as the non-volatile liquid, and hereinafter, the non-volatile liquid container is referred to as a mineral oil container.

  An example of a specific use of this reaction vessel is a genetic polymorphism diagnostic reagent kit for detecting SNP by invader reaction after amplifying DNA from a sample by PCR reaction in the reaction vessel. With reference to FIG. 2, the Example as the reagent kit for a genetic polymorphism diagnosis is demonstrated in detail.

  On the same side of the flat substrate 10a, a sample injection part 12, a PCR end liquid injection part 31, a gene amplification reagent storage part 30, a typing reagent storage part 14, and a mineral oil storage part 16 are formed as recesses. An amplification reaction portion 32 and a plurality of probe placement portions 18 are further formed on the same side of the substrate 10a.

  The sample injection unit 12 is for injecting a biological sample that has not been subjected to the nucleic acid extraction operation or a biological sample that has been subjected to the nucleic acid extraction operation, and is provided in an empty state in which the sample is not yet injected before use. The PCR end solution injection unit 31 is used to mix the reaction solution that has completed the PCR reaction in the amplification reaction unit 32 and the typing reagent, and is provided in an empty state before use.

The gene amplification reagent storage unit 30 stores 2 to 300 μL of a PCR reaction reagent as a gene amplification reagent including a plurality of primers that are combined with each other across a plurality of polymorphic sites. The typing reagent storage unit 14 stores about 10 to 300 μL of a typing reagent prepared corresponding to a plurality of polymorphic sites. The mineral oil storage unit 16 stores 20 to 300 μL of mineral oil for preventing the reaction liquid from evaporating. The gene amplification reagent container 30, the typing reagent container 14, and the mineral oil container 16 are sealed with a film 20 that can be penetrated by a nozzle. Such a film 20 is, for example, an aluminum foil, a laminated film of aluminum and a resin such as a PET (polyethylene terephthalate) film, and is attached by fusion or adhesion so as not to be easily peeled off.
The amplification reaction unit 32 performs a gene amplification reaction on the mixture of the PCR reaction reagent and the sample.

  Each probe placement unit 18 individually holds a fluorescent probe corresponding to each of a plurality of polymorphic sites, and holds the mineral oil when the mineral oil from the mineral oil storage unit 16 is dispensed. It is a recess that can be made. The size of the concave portion of each probe placement portion 18 is, for example, a circle having a diameter of 100 μm to 2 mm and a depth of 50 μm to 1.5 mm.

  The surface of the substrate 10 is formed on the film 20 from the sample injection unit 12, the PCR end solution injection unit 31, the gene amplification reagent storage unit 30, the typing reagent storage unit 14, the mineral oil storage unit 16, the amplification reaction unit 32, and the probe placement unit. 18 is covered with a peelable sealing material 22 having a size covering 18. The sealing material 22 is also an aluminum foil, a laminated film of aluminum and resin, etc., but the bonding strength is weaker than that of the film 20 and is bonded to such an extent that it can be peeled off with an adhesive or the like.

  In order to measure fluorescence from the bottom surface side, the substrate 10 is formed of a material such as a resin having a low autofluorescence property (a property of generating less fluorescence from itself) and a light transmitting resin, for example, polycarbonate. The thickness of the substrate 10 is 0.3 to 4 mm, preferably 1 to 2 mm. From the viewpoint of low autofluorescence, the substrate 10 is preferably as thin as possible.

  An example of the amplification reaction unit 32 is shown in FIG. 3 is a cross-sectional view taken along the line YY in FIG. 2, and FIG. 2 also shows two upper and lower temperature control units 62 and 60 sandwiching the amplification reaction section 32-1. The upper temperature control unit 62 is supported so as to descend and contact the amplification reaction unit 32, and the lower temperature control unit 60 is supported so as to rise and contact the amplification reaction unit 32.

  The amplification reaction section 32-1 includes a flow path 33 serving as a reaction chamber formed in the substrate 10a and at least two ports 34a and 34b for taking in and out the liquid into and from the reaction chamber at both ends thereof. The ports 34a and 34b have openings 36a and 36b opened upward corresponding to the shape of the tip of the nozzle 28, and are made of an elastic material such as PDMS (polydimethylsiloxane) or silicone rubber so as to be in close contact with the tip of the nozzle 28. It is configured.

In order that the amplification reaction part 32-1 may improve thermal conductivity, the thickness of the lower part side of the board | substrate 10a of the part is thin. The thickness of the portion is, for example, 1.2 to 0.5 mm.
The amplification reaction unit 32-1 includes a flow channel-shaped reaction chamber 33 formed in the substrate, and ports 34a and 34b that are connected to both ends of the flow channel in which the reaction chamber 33 is formed and open upward. Yes.

FIG. 4A shows a first form 33a of the reaction chamber 33 having a flow path shape, and the flow path constituting the reaction chamber 33a extends linearly between the ports 34a and 34b.
FIG. 4B shows a second form 33b of the flow channel-like reaction chamber 33, and the flow channel constituting the reaction chamber 33b is bent in a “U” shape between the ports 34a and 34b. Yes.
Temperature control units 37a and 37b come into contact with the planar portion of the amplification reaction section 32 from above and below to control the temperature of the reaction solution in the reaction chambers 33a and 33b, thereby causing an amplification reaction.

  Returning to FIG. 3, the lower temperature adjustment unit 60 includes a Peltier element 120 as a heating / cooling element, and is provided on one side of the Peltier element 120 to be in contact with the bottom surface of the amplification reaction section 32-1. Is made of a metal having good thermal conductivity, for example, aluminum, and the shape of the portion in contact with the bottom surface of the amplification reaction section 32-1 is formed to have a convex portion that matches the bottom surface of the amplification reaction section 32-1. Yes. A heat radiating plate 124 made of a metal having good thermal conductivity, such as aluminum, is in contact with the opposite surface of the Peltier element 120.

A Peltier element 130 is also arranged as a heating / cooling element on the surface side of the amplification reaction section 32-1, and the shape of the portion where the heat block 132 provided on one surface thereof contacts the surface of the amplification reaction section 32-1 is amplified. It is formed in a shape that matches the surface of the reaction section 32-1. A heat radiating plate 134 is in contact with the opposite surface of the Peltier element 130. The heat block 132 and the heat radiating plate 134 are also made of a metal having good thermal conductivity, such as aluminum.
In FIG. 3 (B), the reaction liquid in the reaction part 32-2 becomes a predetermined temperature cycle by the Peltier elements 130 and 120 in a state where the amplification reaction part 32-1 is sandwiched between the upper and lower temperature control units 62, 60. Heating and cooling are repeated.

An example of a reaction vessel provided with another form of amplification reaction unit is shown in FIG. FIG. 5 shows another example of the reaction vessel. (A) is a front view, (B) is a plan view, and (C) is a perspective view. Since the configuration other than the gene amplification reaction unit 32-2 is the same as that of the reaction container shown in FIG.
The amplification reaction part 32-2 has a concave well 35 serving as a reaction chamber formed in the substrate 10 and a convex part 37a that enters the concave part of the well 35 and occupies a part of the well 35. It consists of a detachable lid 37. FIG. 5C shows a state in which the lid 37 is removed from the well 33 and placed at a specific location on the substrate 10.

  The gene amplification reaction part 32-2 is specifically shown in FIG. The well 35 may be an integral type integrally formed with the substrate 10 of the reaction vessel, or may be bonded or fused to the substrate 10, but this embodiment has another form. Indicates. The well 35 is formed separately from the substrate 10 of the reaction vessel. The well 35 has a disc shape, and a recess serving as a reaction chamber for containing the reaction solution is formed at the center thereof. The thickness of the concave portion of the well 35 is thinly formed in order to improve the heat conduction efficiency, and the thickness is, for example, 0.1 to 0.5 mm. A flange 35a having a large thickness is provided around the well 35. A circular hole for attaching the well 35 is formed in the substrate 10, and a claw portion 11 a rising from the surface of the substrate 10 is formed around the hole. The well 35 is fitted into the hole of the substrate 10 so that the concave portion of the well faces the front side of the substrate 10, and the flange 35a is fixed to the substrate 10 by engaging with the claw portion 11a.

  The convex portion 37a provided on the lid 37 occupies a part of the space of the concave portion of the well 35, and is formed so that the outer diameter of the convex portion 37a coincides with the inner diameter of the concave portion of the well 35, The lid 37 is detachably attached to the concave portion of the well 35 by the convex portion 37a. The inside of the convex part 37a of the lid 37 is opened, and a protrusion 37b serving as an engaging part is formed by forming a notch at the edge of the opening. At the time of heat treatment by the reaction vessel, the convex portion of the heat block is inserted inside the convex portion 37a and is brought into contact therewith for heat conduction.

  When a predetermined amount of the reaction solution 38 is put into the well 35 and the well 35 is closed with the lid 37, a part of the convex portion 37a comes into contact with the reaction solution 38, or the space of the concave portion of the well 35 is made as small as possible. The dimension of the well 35 and the dimension of the convex part 37a are set.

  FIG. 6B shows a state in which the reaction solution 38 is placed in the well 35 and closed with the lid 37, and temperature control is performed by PCR reaction or the like. The heat block 60 is brought into contact with the lower side of the reaction unit 32-2, and the heat block 62 is brought into contact with the upper side of the reaction unit 32-2.

  The lower temperature control unit 60 includes a Peltier element 120 as a heating / cooling element, and a heat block 122 provided on one side of the Peltier element 120 and in contact with the bottom surface of the well 35 is made of a metal having good thermal conductivity, such as aluminum. In addition, the shape of the portion in contact with the bottom surface of the well 35 is formed into a shape having a recess that matches the bottom surface of the well. A heat radiating plate 124 made of a metal having good thermal conductivity, such as aluminum, is in contact with the opposite surface of the Peltier element 120.

A convex portion in which a Peltier element 130 is arranged as a heating / cooling element on the lid 37 side of the reaction unit 32-2 and the shape of the portion where the heat block 132 provided on one surface thereof contacts the lid 37 coincides with the lid 37. It is formed in the shape with. A heat radiating plate 134 is in contact with the opposite surface of the Peltier element 130. The heat block 132 and the heat radiating plate 137 are also made of a metal having good thermal conductivity, such as aluminum.
In the state of FIG. 6B, heating and cooling are repeated by the Peltier elements 130 and 120 so that the reaction liquid in the reaction unit 32-2 has a predetermined temperature cycle.

FIG. 7 shows an embodiment in which the present invention is applied to a simple genetic analyzer for detecting SNP in a biological sample using the above reaction container as a reagent kit.
A pair of temperature control units 60 and 62 are arranged vertically as heaters in the apparatus. The reaction vessel mounting portion is configured by forming a guide portion on which the reaction vessel 41 shown in FIG. 2 slides and is positioned at a predetermined position on the lower temperature adjustment unit 60. In the reaction vessel mounting portion, five pieces of samples injected into the reaction vessel 41 are arranged in parallel. These temperature control units 60 and 62 can move in the Y direction indicated by arrows.

  The upper and lower temperature control units 60 and 62 constitute an amplification reaction temperature control unit that controls the temperature of the amplification reaction unit 32 to be in a predetermined temperature cycle. Moreover, both temperature control units 60 and 62 also constitute a typing reaction temperature control unit that controls the temperature of the probe placement unit 18 to a temperature at which the DNA and the probe are reacted. The amplification reaction temperature control unit and the typing reaction temperature control unit are denoted by reference numerals 120 and 110 in FIG. 1, respectively. The temperature of the amplification reaction temperature control unit is set so that, for example, it is changed in three stages of 94 ° C., 55 ° C., and 72 ° C. in that order, and the cycle is repeated. The temperature of the typing reaction temperature control unit is set to 63 ° C., for example.

  The temperature control units 60 and 62 constituting the amplification reaction temperature controller are shown in detail in FIGS. In this embodiment, five reaction vessels 10 are mounted, and a set of temperature control units 60 and 62 is provided for each reaction vessel 10. The upper temperature control unit 62 is supported by a member different from the reaction vessel mounting portion so as to be movable up and down, and the lower temperature control unit 60 is supported by the reaction vessel mounting portion so as to be movable up and down.

In order to lower the five upper temperature control units 62 at the same time, the shafts 144 are in contact with the upper surfaces of the upper temperature control units 62, and the upper ends of the five shafts 144 are driven to the upper surface of the substrate 143. The lower end of the bar screw 146 for use is in contact.
The lower temperature control unit 60 is supported on the front end side of a common lever 141 by a joint 142 that can rotate on the lower surface side. The lever 141 is rotatably supported by a shaft 140, and a lower end portion of a connecting member integrated with a substrate 143 for lowering the upper temperature adjustment unit 62 is in contact with a base end portion of the lever 141.

  When the bar screw 146 is raised, the temperature adjustment unit 60 is lowered to a predetermined position by the biasing means or its own weight so that the temperature adjustment units 60 and 62 are separated from the amplification reaction part of the reaction vessel 10, and the temperature adjustment unit 62 Is raised to a predetermined position. When the bar screw 146 is driven and lowered, the substrate 143 is pushed down, and accordingly, the upper temperature adjustment unit 62 is lowered, and at the same time, the lower temperature adjustment unit 60 is raised. Since the lower temperature control unit 60 is supported by the lever 141 by a rotatable joint 142, the lower temperature control unit 60 can be in good contact with the amplification reaction part of the reaction vessel 10.

  As shown in FIG. 10, among the temperature control units 60 and 62, the temperature control unit constituting the typing reaction temperature control unit is a lower part positioned below the probe placement unit with the reaction vessel 41 mounted on the mounting unit. It comprises a temperature adjustment unit 60a and an upper temperature adjustment unit 62a that can be positioned above the probe placement portion. The position of the lower temperature adjustment unit 60a is fixed, but the upper temperature adjustment unit 62a slides in a horizontal direction between a position (the state shown in FIG. 10) that covers the upper portion of the probe placement portion and a position that deviates from that position. It is supported so that it can move.

  The upper temperature adjustment unit 62a is temperature-controlled in advance before being moved to a position that covers the upper portion of the probe placement portion. The temperature is a temperature necessary for setting the temperature of the probe placement portion to a temperature necessary for the reaction solution of the genomic DNA and the typing reagent to react with the probe of the probe placement portion.

A recess 161 is formed on the surface of the upper temperature control unit 62a, and a pin 160 that can be engaged with the recess 161 is provided on the liquid feeding arm 66 of the dispensing unit. By engaging the pin 160 with the concave portion 161 and moving the liquid feeding arm 66 in the horizontal direction, the upper temperature adjustment unit 62a can be slid.
The lower temperature adjustment unit 60a has an opening 152 only at a position corresponding to the probe placement portion.

  When dispensing the reaction solution and the typing reagent to the probe placement part of the reaction container 41, the upper temperature control unit 62a is moved to a position removed from the position covering the upper part of the probe placement part, and the upper temperature is adjusted after dispensing. The adjustment unit 62a is moved to a position that covers the top of the probe placement portion.

  Returning to FIG. 7, in order to transfer, suck, and discharge the liquid by the nozzle 28, a liquid feeding arm 66 that moves in the X direction, the Y direction, and the Z direction is provided as a dispensing unit. The arm 66 includes a nozzle 28. The dispensing part is indicated by reference numeral 112 in FIG.

FIG. 11 shows the tip of the nozzle 28. A disposable dispensing tip 70 is detachably attached to the tip of the nozzle 28. (A) shows a state where the dispensing tip 70 is not attached to the nozzle 28, and (B) shows a state where the dispensing tip 70 is attached to the nozzle 28. The nozzle 28 sucks and discharges the liquid into the dispensing tip 70. Therefore, the nozzle 28 has a mounting portion 28a having an outer dimension for mounting the dispensing tip 70 at the tip, and a length at the tip of the mounting portion 28a that is not in contact with the liquid to be dispensed and is more external than the mounting portion 28a. And a projection 28b having a small size.
The nozzle 28 has a dispensing tip 70 attached to the tip when sucking and discharging the liquid.

  As shown in FIG. 10, a fluorescence detection unit 64 that performs fluorescence detection is disposed below the temperature adjustment unit 60 a, and the fluorescence detection unit 64 opens the opening 152 of the temperature adjustment unit 60 a from the lower surface side of the reaction vessel 41. Then, the probe placement portion is irradiated with excitation light, and fluorescence from the probe placement portion is detected on the lower surface side of the reaction vessel 41 through the opening 152 of the temperature adjustment unit 60a. The fluorescence detection unit 64 moves in the direction of the arrow X in FIG. Fluorescence detection is performed on each probe by moving the probe placement unit 18 in the Y direction by the reaction vessel mounting unit and moving the fluorescence detection unit 64 in the X direction.

  Returning to FIG. 7, in order to control the operation of the temperature adjustment units 60 and 62, the fluorescence detection unit 64, and the liquid feeding arm 66, a control unit 118 is disposed near them. The control unit 118 includes a CPU and holds a program for operation. The control unit 118 controls the temperature of the typing reaction temperature control unit 110 and the amplification reaction temperature control unit 120 realized by the temperature adjustment units 60 and 62, the detection operation of the fluorescence detection unit 64, and the liquid feeding arm 66 of the dispensing unit 112. The dispensing operation and the recovery operation of the amplification reaction completion liquid are controlled.

  FIG. 12 shows the fluorescence detection unit 64 in detail. The fluorescence detection unit 64 includes a laser diode (LD) or a light emitting diode (LED) 92 that emits a laser beam of 473 nm as an excitation light source, and a pair of the laser beam is focused and irradiated on the bottom surface of the probe placement unit of the reaction vessel 41. Lenses 94 and 96 are provided. The lens 94 condenses the laser light from the laser diode 92 into parallel light, and the lens 96 is an objective lens that converges and irradiates the collimated laser light on the bottom surface of the reaction vessel 41. The objective lens 96 also functions as a lens that collects the fluorescence generated from the reaction vessel 41. A dichroic mirror 98 is provided between the pair of lenses 94 and 96, and the dichroic mirror 98 has wavelength characteristics set so as to transmit excitation light and reflect fluorescence. A dichroic mirror 100 is further arranged on the optical path of the reflected light (fluorescence) of the dichroic mirror 98. The dichroic mirror 100 has a wavelength characteristic that reflects 525 nm light and transmits 605 nm light. A lens 102 and a light detector 104 are arranged on the optical path of reflected light by the dichroic mirror 100 so as to detect fluorescence of 525 nm, and a lens so as to detect fluorescence of 605 nm on the optical path of transmitted light by the dichroic mirror 100. 106 and a photodetector 108 are arranged. The presence or absence of a SNP corresponding to an invader probe fixed at each probe placement position and whether the SNP is a homozygote or a heterozygote by two types of fluorescence detection by the two photodetectors 104 and 108. Is detected. As the labeling phosphor, for example, FAM, ROX, VIC, TAMRA, Redmond Red, or the like can be used.

  Although the detector 64 of FIG. 13 is configured to be excited by excitation light from one light source and measure fluorescence of two wavelengths, the detector 64 can be excited with different excitation wavelengths for fluorescence measurement of two wavelengths. As described above, two light sources may be used.

The usage method of the reaction container in this Example is shown. Here, a case where the amplification reaction unit 32 shown in FIG. 3 is used is shown.
As shown in FIG. 13, the sealing material 22 is peeled off during use. The film 20 that seals the typing reagent container 14, the mineral oil container 18, and the gene amplification reagent container 30 remains without being peeled off.

The sample 25 is injected into the sample injection unit 12 by 0.5 to 2 μL, for example, 1 μL, using a pipette 26 or the like. The sample is a biological sample that has not been subjected to a nucleic acid extraction operation, such as blood. The sample may be a biological sample subjected to a nucleic acid extraction operation. After sample injection, this reaction vessel is attached to the gene analyzer.
A disposable dispensing tip is set in the gene analyzer.

In the gene analyzer, the dispensing device attaches a disposable dispensing tip to the nozzle 28. The nozzle 28 performs a dispensing operation with a dispensing tip attached to its tip.
The nozzle 28 penetrates the film 20 and is inserted into the gene amplification reagent storage unit 30 to suck in the PCR reaction reagent. The PCR reaction reagent is transferred to the sample injection unit 12 by 2 to 20 μL through the nozzle 28. In the sample injection unit 12, the suction and discharge by the nozzle 28 are repeated, whereby the sample reaction solution and the PCR reaction reagent are mixed to form a PCR reaction solution.

  Next, the PCR reaction liquid is sucked by the nozzle 28. At this time, as shown in FIG. 14, the mineral oil 39, the PCR reaction liquid 38, and the mineral oil 39 are sucked into the dispensing tip in this order and dispensed. In the chip, the mineral oil 39 is formed in the upper layer, the PCR reaction solution 38 is formed in the intermediate layer, and the mineral oil is formed in the lower layer in three layers.

  Next, it is injected into the amplification reaction section 32. That is, as shown in FIG. 15 (A), the nozzle 28 is inserted into one port 34a of the amplification reaction section 32, and three layers of liquid in the dispensing tip are injected as it is. It will be in the state shown by 15 (B). This state is a state in which the injected PCR reaction solution 38 is disposed only in the gene amplification reaction temperature control region of the amplifying unit, and the control device controls the mineral oil 39 in the dispensing device so as to be in such a state. The dispensing amount of the PCR reaction solution 38 and the mineral oil 39 is controlled. In addition, this state is a state in which both ends of the PCR reaction solution 38 are closed with mineral oil 39 in order to prevent the PCR reaction solution 38 from evaporating during the reaction in the amplification reaction section 32.

After completion of the PCR reaction, the PCR reaction end solution 38a is recovered from the port 34a or 34a by the nozzle 28. At this time, it is recovered in the three layers of mineral oil 39 / PCR reaction end solution 38a / mineral oil 39 or in the two layers of mineral oil 39 / PCR reaction end solution 38a. Here, in order to facilitate recovery, mineral oil may be injected from one port 34a of the amplification reaction section 32 as shown in FIGS. 16 (A) and 16 (B). The PCR reaction end solution 38a is pushed to the other port 34b. Therefore, the nozzle 28 is inserted into the port 34b, and the PCR reaction end solution 38a is sucked into the nozzle 28. When collecting the PCR reaction end solution 38a, three layers of mineral oil 39 / PCR reaction end solution 38a / mineral oil 39 may be sucked into the nozzle 28, or two layers of mineral oil 39 / PCR reaction end solution 38a. May be inhaled. The ports 34a and 34b have openings 36a and 36b formed in accordance with the shape of the nozzle 28, and are made of an elastic material, so that the nozzle 28 is in close contact with the ports 34a and 34b to prevent liquid leakage, and PCR. The reaction liquid injection and recovery operations are easy.
Here, the liquid injected from the port 34a is not limited to the mineral oil 40 but may be other liquids. Preferably, the specific gravity is the same as or lower than that of the reaction liquid.

  The PCR reaction completion liquid 38a after completion of the reaction collected from the amplification reaction section 32 by the nozzle 28 is transferred to the PCR completion liquid injection section 31 and injected. At this time, the mineral oil 39 layer of the recovered liquid may be discarded in the PCR end liquid injection unit 31 and only the PCR reaction end liquid 38a may be dispensed, or the entire mineral oil 39 may be dispensed as it is. The mineral oil 39 does not inhibit the reaction in the next step.

  Next, as shown in FIG. 17, the nozzle 28 penetrates the film 20 and is inserted into the typing reagent container 14 to suck in the typing reagent, and the typing reagent is transferred to the PCR end solution injection unit 31 by the nozzle 28. Being injected. In the PCR end liquid injecting unit 31, the PCR reaction end liquid and the typing reagent are mixed by repeating suction and discharge by the nozzle 28.

Thereafter, the reaction solution of the PCR reaction solution and the typing reagent is dispensed by the nozzle 28 to each probe placement unit 18 by 0.5 to 4 μL. Mineral oil is dispensed into each probe placement portion 18 by 0.5 to 10 μL from the mineral oil storage portion 16 by the nozzle 28. The dispensing of the mineral oil to the probe placement unit 18 may be before the reaction solution is dispensed to the probe placement unit 18. In each probe arrangement unit 18, mineral oil covers the surface of the reaction solution, and prevents evaporation of the reaction solution during the typing reaction time accompanied by heating in the typing reaction temperature control unit of the detection device.
In each probe placement unit 18, if the reaction solution reacts with the probe and there is a predetermined SNP, fluorescence is emitted from the probe. Fluorescence is detected by irradiating excitation light from the back side of the substrate 10.

Hereinafter, although the composition of each reaction reagent is shown and the present invention is described in detail, the technical scope of the present invention is not limited to these reaction vessels.
PCR reaction reagents are known, and for example, reaction reagents including primers, DNA polymerase, and TaqStart (manufactured by CLONTECH Laboratories) as described in paragraph [0046] of Patent Document 3 can be used. In addition, AmpDirect (manufactured by Shimadzu Corporation) may be mixed in the PCR reaction reagent. As the primer, for example, SNP IDs 1 to 20 described in Table 1 of Patent Document 3 and SEQ ID NOs: 1 to 40 can be used.

  An invader reagent is used as a typing reagent. As the invader reagent, an invader assay kit (manufactured by Third Wave Technology) is used. For example, a signal buffer, a fret probe, a structure-specific DNA degrading enzyme, and an allele-specific probe are prepared at concentrations as described in paragraph [0046] of Patent Document 3.

  In addition to measurement of various chemical reactions, the present invention can be used for various automatic analyzes in, for example, genetic analysis research and clinical fields. For example, polymorphisms of genomic DNA of animals and plants including humans, In particular, SNPs can be detected, and the results are used to diagnose disease prevalence, diagnose the relationship between the type of drug administered, effects and side effects, animal and plant breeding, and infection diagnosis It can also be used for (type determination of infecting bacteria).

It is a block diagram which shows roughly the gene-analysis apparatus to which this invention is applied. It is a figure which shows an example of reaction container, (A) is a front view, (B) is a top view, (C) is an expanded sectional view in the XX line position of (B). It is an expanded sectional view which shows an example of the amplification reaction part in the same reaction container with the upper and lower temperature control units in the YY line position of FIG. 2 (B). (A), (B) is a perspective view which shows the example of the amplification reaction part of FIG. 3 as a perspective view, respectively. It is a figure which shows the other example of the reaction container, (A) is a front view, (B) is a top view, (C) is a perspective view. It is a figure which shows an example of the gene amplification reaction part in the reaction container, (A) is sectional drawing which shows the state by which the lid | cover was removed and the reaction liquid was inject | poured, (B) is a lid | cover closed after that, and PCR reaction is carried out It is sectional drawing which shows the state to perform with a temperature control mechanism. It is a schematic perspective view which shows one Example of the gene-analysis apparatus of this invention. It is a perspective view which shows the moving mechanism of the temperature control unit in the Example. It is a side view which shows the movement mechanism. It is sectional drawing which shows the typing reaction temperature control part in the Example. It is the front view showing the nozzle front-end | tip part in the Example, (A) represents the state in which the dispensing tip is not attached to the nozzle, and (B) represents the state in which the dispensing tip is attached to the nozzle. It is a schematic block diagram which shows the fluorescence detection part in the Example. It is a figure which shows the first half part of the process of the SNP detection method using the reaction container in one Example, (A) is a front view, (B) is a top view. It is process drawing which shows the suction | inhalation method of the PCR reaction liquid of the liquid to the dispensing tip of a dispensing nozzle front-end | tip. (A), (B) is front sectional drawing which shows the injection | pouring method of the liquid from a dispensing nozzle to an amplification reaction part. (A), (B) is front sectional drawing which shows the collection | recovery method of the PCR reaction completion | finish liquid from an amplification reaction part to a dispensing nozzle. It is a figure which shows the latter half part of the process of the SNP detection method using the reaction container, (A) is a front view, (B) is a top view. FIG. 2 is a flow chart diagram schematically illustrating a SNP detection method to which the present invention may relate.

Explanation of symbols

2 Sample 4 PCR reaction reagent 6 Invader reagent 8 Probe placement part 10a Substrate 12 Sample injection part 14 Typing reagent storage part 16 Mineral oil storage part 18 Probe placement part 20 Film 22 Sealing material 28 Dispensing nozzle 30 Gene amplification reagent storage part 31 PCR End solution injection unit 32 Amplification reaction unit 34a, 34b Port 36a, 36b Port opening 38 PCR reaction solution 38a PCR reaction end solution 41 Reaction vessel 60, 62 Temperature control unit 60a, 62a Temperature control unit of typing reaction temperature control unit 64 Fluorescence Detection unit 66 Liquid feeding arm 70 Dispensing tip 110 Typing reaction temperature control unit 112 Dispensing unit 118 Control unit

Claims (7)

A mounting part for mounting a reaction container for genetic polymorphism diagnosis provided with a plurality of probe placement parts each holding a probe emitting fluorescence corresponding to each of a plurality of polymorphic sites;
A typing reaction temperature control unit for controlling the temperature of the probe placement unit in order to react the reaction solution of the nucleic acid and the typing reagent with the probe of the probe placement unit;
A dispensing part for transferring the reaction solution to each probe placement part;
A fluorescence detection device for detecting fluorescence by irradiating each probe placement portion with excitation light;
A temperature control of the typing reaction temperature control unit, a dispensing operation of the dispensing unit, and a control unit for controlling the detection operation of the fluorescence detection device,
At least a part of the typing reaction temperature adjustment unit is supported so as to be movable between a position where the temperature of the probe placement unit is adjusted and a position outside the position when the reaction container is mounted on the mounting unit. Gene analysis device.   2. The gene analyzing apparatus according to claim 1, wherein the movable part of the temperature adjustment unit is temperature-controlled in advance before the probe placement unit is moved to a temperature-adjusting position.   The typing reaction temperature control unit includes a lower temperature control unit positioned below the probe placement unit and an upper temperature control unit positioned above the probe placement unit, and one or both of both units are The gene analysis apparatus according to claim 1 or 2, which is movable.   The gene analysis according to claim 3, wherein the movable unit by the temperature control unit is supported so as to be slidable in the horizontal direction, engages with a part of the dispensing unit, and is slid by movement of the dispensing unit. apparatus.   The gene analysis according to any one of claims 1 to 4, wherein the reaction vessel further includes a non-volatile liquid storage portion formed as a recess in the substrate and containing a non-volatile liquid and sealed with a film. apparatus.   The reaction container further includes a gene amplification reagent storage unit that stores a gene amplification reagent that includes a plurality of primers that are formed as recesses and bind to each of the plurality of polymorphic sites, and is sealed with a film. The gene analysis apparatus according to any one of claims 1 to 5, wherein 7. The genetic polymorphism diagnosis reaction container provided with a typing reagent storage part formed as a recess in the substrate and storing a typing reagent and sealed with a film. 8. Genetic analysis device.

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