Patent Application
20100196209
The present invention relates to a reaction vessel processing apparatus for detecting a genome DNA polymorphism for plants and animals including human beings, particularly an SNP (single-nucleotide polymorphism) using a reaction vessel which is suited for various automatic analyses in medical fields, for example, research of gene analysis or clinic, as well as for chemical reactions. Using the detected gene polymorphism detection result, diagnosis of disease morbidity, diagnosis of the relationship between the type and effect or side effect of a drug administered and the like may be achieved.
A method and apparatus for estimating susceptibility to diseases, etc., by using gene polymorphism have been proposed as follows:
For determining whether a patient is susceptible to sepsis and/or rapidly develops sepsis, a nucleic acid sample is collected from the patient, a pattern 2 allelic gene or a marker gene which is in linkage disequilibrium with a pattern 2 allelic gene in the sample is detected, and if a pattern 2 allelic gene or a marker gene in linkage disequilibrium with a pattern 2 allelic gene is detected, the patient is judged to be susceptible to sepsis (see Patent Literature 1).
For diagnosis of one or more single-nucleotide polymorphisms in the human flt-1 gene, a sequence of one or more positions in human nucleic acid, that is, positions 1953, 3453, 3888 (which are respectively in accordance with numbering in EMBL Accession No. X51602), 519, 786, 1422, 1429 (which are respectively in accordance with numbering in EMBL Accession No. D64016), 454 (in accordance with Sequence No. 3) and 696 (in accordance with Sequence No.: 5) is determined, and by referring to the polymorphism in fl1-1 gene, the constitution of the human is determined (JP-A 2001-299366).
Many methods have been reported on typing, that is, discrimination of bases in SNP sites. A typical example of these methods is as follows:
For carrying out typing several hundred thousand SNP sites with a relatively small amount of genome DNA, a plurality of base sequences containing at least one single-nucleotide polymorphism are amplified simultaneously with a genome DNA and pairs of primer, and a plurality of base sequences thus amplified are used to discriminate bases in single-nucleotide polymorphic sites contained in the base sequences by a typing step. For the typing step, an invader method or TaqMan PCR is used (see Patent Literature 3).
The inventors of the present invention have proposed a reaction vessel suited for automating measurement of a chemical reaction and gene polymorphism detection for the purpose of measurements of a chemical reaction and automatic detection of a gene polymorphism.
The reaction vessel includes at least a reaction part having a plurality of reaction wells for allowing a reaction of a sample. At the time of use, a nonvolatile liquid such as mineral oil having a lower specific gravity than the reaction solution is dispensed to the reaction wells to cover the surface of the reaction solution.
When such a reaction vessel is a gene polymorphism diagnosing reaction vessel, the size of the reaction well is as small as 100 μm to 2 mm in diameter, and 50 μm to 1.5 mm in depth, for example.
An amount of the reaction solution dispensed to such a reaction well is as small as about 0.1 μL to 5 μL, for example. In dispensing such a minute amount of reaction solution with a nozzle, the liquid may not be successfully dispensed to a reaction well because it adheres to a tip end of the nozzle. When the reaction solution is transferred to a reaction well by bringing the tip end of the nozzle into contact with the bottom face of the reaction well, contamination occurs when the tip end of the nozzle comes into contact with the bottom face of the reaction well in the case where a substance relating to the reaction is arranged.
In the reaction well, a nonvolatile liquid such as mineral oil is dispensed so as to prevent a reaction solution from evaporating during reaction. In such a case, the dispensing amount of the nonvolatile liquid is also as small as about 1 μL to 10 for example, and the nonvolatile liquid has high viscosity and is hard to leave the tip end of the nozzle, making it difficult to accurately dispense. When a reaction is carried out in the condition that the top face of the reaction solution in the reaction well cannot be covered with a nonvolatile liquid, and thus the reaction solution is exposed, there arises the problem that the reaction solution dries up during the reaction and measurement with high accuracy is not realized.
Further, when the reaction solution is dispensed first and the nonvolatile liquid is dispensed thereon, contamination occurs when the tip end of the nozzle comes into contact with the reaction solution in dispensing the nonvolatile liquid.
It is an object of the present invention to facilitate dispensing of a reaction solution and a nonvolatile liquid to a reaction well of a reaction vessel.
A dispensing method of the present invention is a dispensing method that sequentially dispenses a reaction solution, and a nonvolatile liquid having a lower specific gravity than the reaction solution, to a reaction well of a reaction vessel which includes at least a reaction part having a plurality of reaction wells for allowing a reaction of a sample, by means of a nozzle. The reaction solution and the nonvolatile liquid may be dispensed in any order.
A dispensing step of a liquid to be dispensed first is a method in which a liquid droplet of the liquid is formed on a tip end of the nozzle, and the liquid droplet is brought into contact with the bottom face or inner wall face of the reaction well to be transferred into the reaction well.
The first method of a dispensing step of a liquid to be dispensed later is a method of pushing in which a tip end of the nozzle is approached to an inner wall face of the reaction well so that the liquid transfers into the reaction well along the inner wall face.
The second method of a dispensing step of the liquid to be dispensed later is a method in which a liquid droplet of the liquid is formed on a tip end of the nozzle, and the liquid droplet is transferred into the reaction well by being in contact with the inner wall face of the reaction well or the surface of the liquid that has been dispensed first to the reaction well.
The reaction solution and the nonvolatile liquid may be dispensed in any order; however, it is preferred to dispense the reaction liquid first for desirably covering the surface of the reaction solution with the nonvolatile liquid.
A preferred example of the reaction vessel is one that integrally has a nonvolatile liquid reservoir reserving a nonvolatile liquid.
A more preferred example of the reaction vessel is a gene polymorphism diagnosing reaction vessel which further includes integrally a typing reagent reservoir reserving a typing reagent, and probe arrangement parts, each individually holding a probe that emits fluorescence in correspondence with each of a plurality of polymorphic sites as a reaction well of the reaction part.
A more preferred example of the reaction vessel is a gene polymorphism diagnosing reaction vessel which further includes integrally a gene amplification reagent reservoir for reserving a gene amplification reagent containing a plurality of primers to bind to a plurality of polymorphic sites by sandwiching each site between the primers, and an amplification reaction part that allows a gene amplification reaction for a mixture solution of the gene amplification reagent and a sample.
A detachable tip is attached to a tip end of a nozzle, and a liquid can be dispensed via the tip. In the present invention, the nozzle having a tip attached to its tip end is referred to as a nozzle including the tip.
As a nonvolatile liquid having a lower specific gravity than a reaction solution, mineral oil, vegetable oil, animal oil, silicone oil, or diphenylether may be used. Mineral oil is a liquid hydrocarbon mixture obtained by distillation from petrolatum, and is also called liquid paraffin, liquid petrolatum, white oil and the like, and includes light oil of low specific gravity. Examples of animal oil include cod-liver oil, halibut oil, herring oil, orange roughy oil, shark liver oil, and the like. Examples of vegetable oil include canola oil, almond oil, cotton seed oil, corn oil, olive oil, peanut oil, safflower oil, sesame oil, soybean oil, and the like.
The reaction vessel processing apparatus of the present invention includes at least a reaction vessel mounting part for mounting a reaction vessel having at least a plurality of reaction wells for allowing a reaction of a sample, a dispenser 112 for conducting liquid transfer of the reaction vessel by moving a nozzle 28 for aspiration and discharge, as shown in
In order to operate the controller 118 externally or display a test result, a personal computer (PC) 122 may be connected to the controller 118.
In the present invention, as to a liquid that is to be dispensed first, a liquid droplet of the liquid is formed on a tip end of a nozzle, and the liquid droplet is transferred into a reaction well while being in contact with a bottom face or inner wall face of the reaction well, and as to a liquid that is to be dispensed later, it is pushed so that the liquid moves into the reaction well along the inner wall face by approaching the tip end of the nozzle to the inner wall face of the reaction well, or a liquid droplet of the liquid is formed on the tip end of the nozzle, and the liquid droplet is transferred into the reaction well while it is in contact with the inner wall face of the reaction well or the surface of the liquid previously dispensed to the reaction well, so that it is possible to dispense a minute amount of the reaction solution accurately and to avoid contamination. Also, it becomes possible to dispense the nonvolatile liquid accurately and avoid contamination at the time of dispensing. As a result, the surface of the reaction solution can be covered with the nonvolatile liquid in the reaction well so that the reaction solution is prevented from drying up during reaction and accurate measurement is realized.
On the same side of a plate-like substrate 10, a reagent reservoir part 14 and a nonvolatile liquid reservoir part 16 are formed as concave portions. As the nonvolatile liquid, mineral oil is used, and hereinafter, the nonvolatile liquid reservoir part is referred to as a mineral oil reservoir part. On the same side of the substrate 10, further formed is a reaction part 18. The reagent reservoir part 14 and the mineral oil reservoir part 16 are sealed with a film 20, and for aspirating the reagent and the mineral oil and transferring them to other locations by a nozzle, they are aspirated by a nozzle after removal of the film 20, or the film 20 that is adapted to be penetrable by a nozzle is penetrated by the nozzle and the reagent and the oil are aspirated by the nozzle.
The surface of the substrate 10 is covered from above the film 20 with a detachable sealing material 22 of the size that covers the reagent reservoir part 14, the mineral oil reservoir part 16 and the reaction part 18.
One example of concrete use of the reaction vessel is a gene polymorphism diagnosing reagent kit in which a sample reaction solution having DNA amplified by a PCR is dispensed and SNP is detected by an invader reaction.
The relationship between the polymorphic sites and primers is as follows: For amplifying one polymorphic site, a pair of primers binding to the polymorphic site by sandwiching it between primers is necessary. A plurality of kinds of polymorphic sites occur in a target biological sample, and when polymorphic sites occur in positions separated from one another, twice as many kinds of primers as kinds of polymorphic sites are necessary. However, when two polymorphic sites are close to each other, amplification thereof can be effected by binding the primers to each of the polymorphic sites by sandwiching each site between the primers or by binding the primers to both sides of a sequence of the two polymorphic sites with no primer between the polymorphic sites. Accordingly, the types of necessary primers are not always twice as many as kinds of polymorphic sites. In the present invention, “a plurality of primers to bind to a plurality of polymorphic sites by sandwiching each site between the primers” is intended to refer to types of primers necessary for amplifying a plurality of polymorphic sites not only in the case where a pair of primers bind to one polymorphic site by sandwiching it between the primers but also in the case where a pair of primers bind to two or more polymorphic sites by sandwiching a series of such polymorphic sites between the primers.
The polymorphism includes mutation, deletion, overlap, transfer etc. A typical example is SNP.
Examples of the biological sample include blood, saliva, and genome DNA
As the amplification step, a PCR method or the like may be used. In such a case, the PCR method is preferably conducted in a condition of a pH of 8.5 to 9.5 at 25° C. In such a case, the gene amplification reagent is a PCR reagent.
For typing of SNP, adjustment of genome DNA is required at the stage of entering the amplification step, which takes labor and cost Taking a PCR method for amplifying DNA into account, a direct PCR method which is conducted on a sample such as blood without conducting a pre-treatment is proposed According to this proposal, in a nucleic acid synthesis technique for amplifying an objective gene in a sample containing genes, a gene conjugate in a sample containing genes or a sample containing genes itself is added to a gene amplification reaction solution, and an objective gene in the sample containing genes is amplified at a pH ranging from 8.5 to 9.5 (25° C.) in the reaction solution after addition (see Patent document 4).
In a typing system already constructed, only a small amount of DNA is collected first because a plurality of SNP sites to be typed are amplified by a PCR method; however, it is necessary to carry out a pre-treatment for extracting DNA in advance from a biological sample prior to amplification by the PCR method. This takes labor and cost for the pre-treatment.
Such an automated system has not been constructed heretofore that amplifies a plurality of SNP sites to be typed simultaneously when a direct PCR method and a typing method are combined.
The typing step may be achieved by an invader method or a TaqMan PCR method. In such a case, the typing reagent is an invader reagent or a TaqMan PCR reagent.
In the PCR step, a PCR reagent 4 is added to a biological sample 2 such as blood, or alternatively, the biological sample 2 is added to the PCR reagent 4. For example, 1 μL of the biological sample 2 is collected, and about 10 μL of the PCR reagent 4 is added thereto. The PCR reagent 4 is adjusted in advance and contains a plurality of primers for SNP sites to be measured, as well as a buffer solution for adjusting pH, four kinds of deoxyribonucleotides, and other essential reagents, and adjusted so that the pH is 8.5 to 9.5 when mixed with the sample 2.
The PCR is caused to occur in a mixture solution of the biological sample 2 and the PCR reagent 4 according to a predetermined temperature cycle. The PCR temperature cycle includes 3 steps, which are denaturation, primer adhesion (annealing) and primer extension, and this cycle is repeated whereby DNA is amplified. In one example of the steps, the denaturation step is carried out at 94° C. for 1 minute, the primer adhesion step at 55° C. for 1 minute, and the primer extension at 72° C. for 1 minute. The sample may be subjected to a genome extraction procedure; however, the one that is not subjected to the genome extraction procedure is used herein. Even with the biological sample not subjected to the genome extraction procedure, DNA is released from blood cells or cells at high temperature in the PCR temperature cycle, and the reagents necessary for the PCR come into contact with the DNA to make the reaction proceed.
After the PCR reaction is finished, an invader reagent 6 is added. A fluorescence-emitting FRET probe and cleavase (structure-specific DNA degradative enzyme) are contained in the invader reagent 6. The FRET probe is a fluorescent-labeled oligo having a sequence completely irrelevant to the genome DNA, and, irrespective of the type of SNP, its sequence is common.
Next, the reaction solution to which the invader reagent 6 has been added is reacted by addition to a plurality of probe arrangement parts 8 of a typing reaction part. At each site of the probe arrangement parts 8, an invader probe and a reporter probe are individually held correspondingly to each of a plurality of SNP sites, and the reaction solution reacts with the invader probe to emit fluorescence if SNP corresponding to the reporter probe is present.
The invader method is described in detail in paragraphs [0032] to [0034] in Patent Literature 3.
Two reporter probes have been prepared depending on each base of SNP and can judge whether the SNP is a homozygote or heterozygote.
The PCR method of the amplification step which may be used in the present invention amplifies a plurality of objective SNP sites simultaneously, and amplifies a plurality of genome DNA containing SNP sites by a direct PCR method from a biological sample not subjected to a nucleic acid extraction procedure. For achieving this, a gene amplification reaction reagent containing a plurality of primers for these SNP sites is caused to act on the biological sample, and the PCR is carried out under the pH condition of 8.5 to 9.5 at 25° C.
The PCR reagent contains a pH buffer solution, salts such as MgCl2 and KCl, primers, deoxyribonucleotides, and a thermostable synthase. Besides the above, other substances such as a surfactant and protein may be added as necessary.
The pH buffer solution may be a combination of tris(hydroxymethyl)aminomethane and a mineral acid such as hydrochloric acid, nitric acid or sulfuric acid, as well as various pH buffer solutions. The buffer solution having adjusted pH is preferably used at a concentration between 10 mM and 100 mM in the PCR reagent. The primer refers to an oligonucleotide acting as a starting point for DNA synthesis by the PCR. The primer may be synthesized or isolated from biological sources.
The synthase is an enzyme for synthesis of DNA by primer addition, and includes chemically synthesized synthases. Suitable synthase includes, but is not limited to, E. coli DNA polymerase I, E. coli DNA polymerase Klenow fragment, T4 DNA polymerase, Taq DNA polymerase, T. litoralis DNA polymerase, Tth DNA polymerase, Pfu DNA polymerase, Hot Start Taq polymerase, KOD DNA polymerase, EX Taq DNA polymerase, and a reverse transcriptase. The term “thermostable” means the property of a compound which maintains its activity even at high temperatures, preferably between 65° C. and 95° C.
The invader method used in the typing step is a method of typing SNP site by hybridizing an allele-specific oligo with DNA containing SNP as an object of typing, wherein DNA containing SNP as an object of typing, two kinds of reporter probes specific to the each allele of SNP as an object of typing, one kind of invader probe, and an enzyme having a special endonuclease activity by which a structure of DNA is recognized and cleaved are used (see Patent Literature 3).
Next, specific explanation will be made for the reaction vessel. Referring to
On the same side of a plate-like substrate 10, a sample applying part 12, a typing reagent reservoir 14 and a mineral oil reservoir 16 are formed as concave portions. On the same side of the substrate 10, a plurality of probe arrangement parts 18 are formed.
A biological sample reaction solution having DNA amplified by a PCR will be injected to the sample injection part 12; however in the condition before use, the sample injection part 12 is provided in an empty state in which a sample is not injected. The typing reagent reservoir part 14 reserves about 10 μL to 300 μL of a typing reagent that is prepared in correspondence with a plurality of polymorphic sites, and the mineral oil reservoir part 18 reserves 20 μL to 300 μL of mineral oil for preventing evaporation of the reaction solution. The typing reagent reservoir part 14 and mineral oil reservoir part 18 are sealed with the film 20 which is penetrable by a nozzle. Such a film 20 is, for example, an aluminum foil or a laminate film with a resin such as aluminum and a PET (polyethylene terephthalate) film, and is bonded by fusion or adhesion so that it will not be readily detached.
Each probe arrangement part 18 individually has a probe that emits fluorescence in correspondence with each of plural polymorphic sites, and is a concave portion capable of holding the mineral oil when it is dispensed from the mineral oil reservoir part 16. Each concave portion of the probe arrangement part 18 is, for example, in the shape of a circle of 100 μm to 2 mm in diameter, and 50 μm to 1.5 mm in depth.
The surface of the substrate 10 is covered from above the film 20 with the detachable sealing material 22 of the size that covers the sample injection part 12, the typing reagent reservoir part 14, the mineral oil reservoir part 16 and the probe arrangement part 18. This sealing material 22 may also be an aluminum foil or a laminate film of aluminum and a resin; however, the bonding strength is smaller than that of the film 20 and is bonded by an adhesive or the like in such a degree that it can be detached.
In order to measure fluorescence from the bottom face side, the substrate 10 is made of a light-permeable resin with a low-spontaneous-fluorescent property (that is, a property of generating little fluorescence from itself), for example, a material such as polycarbonate. The thickness of the substrate 10 is 1 mm to 2 mm.
A method of using the reaction vessel according to the present example will be described.
As shown in
To the sample injection part 12, 2 μL to 20 μL of a sample reaction solution 24 having DNA amplified externally by a PCR reaction is injected with a pipette 26 or the like. Then the reaction vessel is mounted on the detecting apparatus.
In the detecting apparatus, as shown in
Thereafter, 0.5 μL to 4 μL of the reaction solution of the sample reaction solution and the typing reagent is dispensed to each probe arrangement part 18 by the nozzle 28. To each probe arrangement part 18, 0.5 μL to 10 μL of mineral oil is dispensed from the mineral oil reservoir part 18 by the nozzle 28. Dispensing of mineral oil to the probe arrangement part 18 may be conducted before dispensing of the reaction solution to the probe arrangement part 18. In each probe arrangement part 18, the mineral oil covers the surface of the reaction solution to prevent the reaction solution from evaporating during typing reaction time which is associated with heat generation at the typing reaction temperature control part of the detecting apparatus.
In each probe arrangement part 18, the reaction solution and the probe react, and if a predetermined SNP is present, fluorescence is emitted from the probe. Fluorescence is detected upon irradiation with exciting light from the back face side of the substrate 10.
In this reaction vessel, a biological sample not subjected to a nucleic acid extraction procedure is injected as a sample, and both amplification of DNA by a PCR reaction and SNP detection by an invader reaction are conducted. It is to be noted, however, a biological sample not subjected to a nucleic acid extraction procedure may be injected.
On the same side of a plate-like substrate 10a, the sample injection part 12, the typing reagent reservoir part 14, the mineral oil reservoir part 16, and the plurality of probe arrangement parts 18 similar to those in the example of
The gene amplification reagent reservoir part 30 is also formed as a concave portion in the substrate 10a, and holds a gene amplification reagent containing a plurality of primers to bind to a plurality of polymorphic sites by sandwiching each site between the primers. The gene amplification reagent reservoir part 30, the typing reagent reservoir part 14 and the mineral oil reservoir part 16 are sealed with the film 20 which is penetrable by a nozzle. The gene amplification reagent reservoir part 30 reserves 2 μL to 300 μL of a PCR reagent. In the same way as the example shown in
The PCR-finished solution injection part 31 is provided for mixing the reaction solution having finished a PCR reaction in the gene amplification reaction part 32 and the typing reagent, and is formed as a concave portion in the substrate 10a, and provided in an empty state before use.
The gene amplification reaction part 32 allows the mixture solution of the PCR reagent and the sample to proceed a gene amplification reaction.
The gene amplification reaction part 32 has a smaller thickness in the bottom face side of the substrate 10a so as to improve the heat conductivity, as shown in
To the sample injection part 12, a biological sample not subjected to a nucleic acid extraction procedure is injected in the present example; however, it is provided in an empty state where a sample is not injected before use.
In the same way as the reaction vessel shown in
In the same way as the reaction vessel shown in
The surface of the substrate 10a is covered from above the film 20 with the sealing material 22 which can be detached and has such a size that covers the sample injection part 12, the PCR-finished solution injection part 31, the typing reagent reservoir part 14, the mineral oil reservoir part 16, the gene amplification reagent reservoir part 30, the gene amplification reaction part 32 and the probe arrangement part 18. The materials and the manner of bonding the film 20 and the sealing material 22 are as described in the reaction vessel of
In order to also measure fluorescence from the bottom side, the substrate 10a is made of a light-permeable resin with a low-spontaneous-fluorescent property, for example, a material such as polycarbonate. The thickness of the substrate 10 is 1 mm to 2 mm.
The manner of using the reaction vessel according to the present example is shown below.
As shown in
To the sample injection part 12, 0.5 μL to 2 μL of a sample 25 is injected with a pipette 26 or the like. In the reaction vessel of
In the detecting apparatus, as shown in
Next, as shown in
When dispensing the mineral oil 40, a liquid droplet of the mineral oil 40 is formed on a tip end of the nozzle according to the present invention, and the nozzle is moved to approach the ports 34a, 34b, and the liquid droplet of the mineral oil 40 is brought into contact with bottom faces or wall faces of the ports 34a, 34b to achieve dispensing.
Here, the liquid droplet of the mineral oil 40 may be formed on a tip end of the nozzle before making the nozzle approach the ports 34a, 34b to such a degree that the liquid droplet comes into contact with the bottom face or wall face of the ports 34a, 34b, or may be formed after approach of the nozzle to the ports 34a, 34b.
After completion of the PCR reaction, the PCR solution is collected by the nozzle 28, and at this time, mineral oil 40 is injected through one port 34a of the gene amplification reaction part 32 as shown in
The reaction-finished PCR solution 38a collected from the gene amplification reaction part 32 by the nozzle 28 is transferred and injected to the PCR-finished solution injection part 31.
Next the nozzle 28 is inserted into the typing reagent reservoir part 14 through the film 20 and the typing reagent is aspirated, and the typing reagent is transferred and injected to the PCR-finished solution injection part 31 by the nozzle 28. In the PCR-finished solution injection part 31, the PCR solution and the typing reagent are mixed by repetition of aspiration and discharge by the nozzle 28.
Then, 0.5 μL to 4 μL of the reaction solution of the PCR solution and the typing reagent is dispensed to each probe arrangement part 18 by the nozzle 28. To each probe arrangement part 18, 0.5 μL to 10 μL of mineral oil is dispensed by the nozzle 28 from the mineral oil reservoir part 18. Dispensing of mineral oil to the probe arrangement part 18 may be conducted before dispensing of the reaction solution to the probe arrangement part 18. In each probe arrangement part 18, the mineral oil covers the surface of the reaction solution to prevent the reaction solution from evaporating during the period of typing reaction in the typing reaction part of the detecting apparatus, which is associated with heat generation.
In each probe arrangement part 18, the reaction solution and the probe react, and if a predetermined SNP is present, fluorescence is emitted from the probe. Fluorescence is detected upon irradiation with exciting light from the back-face side of the substrate 10.
In the following, the present invention will be described in detail while showing a composition of each reaction reagent however, the technical scope of the present invention is not limited by these examples.
The PCR reagent is known in the art, and a reaction reagent containing a primer, DNA polymerase and TaqStart (available from CLONTECH Laboratories) as described in Patent document 3, paragraph [0046], for example, may be used. Further, AmpDirect (available from SHIMADZU Corporation) may be contained in the PCR reagent. As the primer, for example, SNP. IDs 1 to 20, SEQ No. 1 to 40 described in Table 1 in Patent document 3 may be used.
As the typing reagent, an invader reagent is used. As the invader reagent, an invader-assay kit (available from Third Wave Technology) is used. For example, a signal buffer, an FRET probe, a structure specific DNase and an allele specific probe are prepared in concentrations as described in Patent document 3, paragraph [0046].
As shown in
The upper heat block 62 constituting the typing reaction part has openings 150 only at the positions corresponding to probe arrangement parts, and also the part that constitutes the typing reaction part in the lower heat block 60 has openings 152 only at the positions corresponding to probe arrangement parts. On the heat block 62, a typing reaction part cover 154 is disposed, and the cover 154 is also provided with openings 156 at the positions of the openings 150 of the heat block 62.
Below the heat block 60, a fluorescence detector 64 for detecting fluorescence is disposed, and the fluorescence detector 64 emits exciting light to a probe arrangement part via the opening 152 of the heat block 60 from the bottom face side of the reaction vessel 41, and detects fluorescence from the probe arrangement part via the opening 152 of the heat block 60 on the bottom face side of the reaction vessel 41. The fluorescence detector 64 moves in the direction of the arrow X in
Returning to
The nozzle 28 of the dispenser dispenses a reaction solution to a probe arrangement part via the opening 156 of the cover 154 and the opening 150 of the heat block 62, as shown in
Returning to
When the reaction vessel 41 not having a gene amplification reaction part as in the case of the reaction vessel of
Even when the mineral oil 40 is dispensed first and then the reaction solution 170 is dispensed, the mineral oil 40 covers the surface of the reaction solution 170 owing to its specific gravity.
The detector 64 of
The present invention may be utilized in various types of automatic analyses, for example, in research of gene analysis or clinical field, as well as in measurement of various chemical reactions. For example, the present invention can be used in detecting genome DNA polymorphism for plants and animals including humans, particularly SNP and can further be utilized, not only in diagnosing disease morbidity, the relationship between the type and effect or side effect of a drug administered and so on by using the results of the above detection, but also in judgment of the variety of animal, or plant, diagnosis of injections (judgment of the type of invader) etc.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2005-100258 | Mar 2005 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2006/306735 | 3/30/2006 | WO | 00 | 11/7/2008 |
