Nucleic acid amplification method and thermal cycler

Abstract

The object of the present invention is to provide a method for amplifying nucleic acids using a large volume PCR reaction solution of 30 mL or more, and an apparatus for amplifying nucleic acids using a large volume PCR reaction solution of 30 mL or more. [Solution] A method for amplifying nucleic acids by performing a polymerase chain reaction (PCR) by controlling the temperature of one or more reaction vessels containing a reaction solution containing DNA polymerase, deoxyribonucleotide triphosphate (dNTP), template DNA, forward primer, reverse primer, and buffer, wherein the reaction solution is 30 mL or more, and the polymerase chain reaction is performed while stirring the reaction solution.

Description

[Technical Field]

[0001] This invention relates to a method for amplifying nucleic acids using polymerase chain reaction (PCR), and to a thermal cycler. [Background technology]

[0002] In nucleic acid analysis, polymerase chain reaction (PCR) is widely used as a method for amplifying nucleic acids by replicating the target nucleic acid (see Patent Document 1). PCR is widely used in disease diagnosis, nucleic acid drugs, microbial analysis, genetic engineering research, DNA analysis, and other applications because the process is simple, and it can be performed in a relatively short time using small, simple devices.

[0003] Typical PCR efficiently amplifies DNA by repeating a thermal cycle consisting of a series of steps 25 to 40 times: (1) thermal denaturation of double-stranded template DNA, (2) primer annealing, and (3) extension by DNA polymerase. Temperature control at each step is crucial in PCR, and various techniques have been developed. For example, a nucleic acid amplification method (see Patent Document 2) is disclosed that includes a step of adjusting the temperature of the reaction mixture in each sample bath using a Peltier element as a cooling element that is in thermal contact with the liquid, and the liquid is stirred with a stirring rod. Another disclosed device (see Patent Document 3) is a temperature control device for a microchip having a microchannel, which includes a Peltier element with opposing first and second surfaces, and controls the temperature of the microchip by driving a heat dissipation fan during the heating and cooling periods to achieve rapid heating and cooling operations and prevent temperature unevenness on the heating surface of the Peltier element.

[0004] Until now, PCR has typically involved replicating nucleic acids in units of a few ng to several hundred ng in a μL PCR reaction solution. However, recent vaccine development and other applications have created a need to replicate DNA and RNA in large quantities. With conventional PCR techniques, replicating large quantities of RNA requires repeating the PCR process dozens to hundreds of times, which is time-consuming, costly, and labor-intensive.

[0005] In this context, devices for performing large-volume PCR have also been developed. For example, a thermal cycler has been disclosed that includes a pair of temperature control units arranged symmetrically above and below a bag-shaped member containing a reaction solution, and connected via a connecting means so as to be slidable in the vertical direction (see Patent Document 4). In this invention, a bag-shaped member made of polypropylene is used, and the device is configured to adjust the distance between the pair of metal plates even when the reaction solution is sealed inside the bag-shaped member and the container is inflated. Furthermore, a Peltier element is used as the temperature control unit.

[0006] Furthermore, methods have been disclosed for performing emulsion PCR by placing the PCR reaction solution in a flexible bag and bringing the flexible bag into contact with an opposing thermal cycler (see Patent Document 5), and for amplifying nucleic acids in a cuvette, wherein the cuvette may be shaken to mix the components of the reaction solution contained within it (see Patent Document 6). [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Special Publication No. 4-67957 [Patent Document 2] Special Publication No. 2009-537152 [Patent Document 3] Japanese Patent Publication No. 2017-063778 [Patent Document 4] Japanese Patent Publication No. 2018-139505 [Patent Document 5] U.S. Patent Publication No. 2010 / 0261230 [Patent Document 6] U.S. Patent Registration No. 5229297 [Overview of the project] [Problems that the invention aims to solve]

[0008] In order to amplify nucleic acids while maintaining specificity in PCR, it is necessary to accurately control the temperature of the reaction vessel at each step of the thermal cycle shown in Figure 1. However, when the reaction solution volume exceeds 30 mL, the time required for the temperature to rise and fall to reach each set temperature in the PCR thermal cycle becomes longer. While short cycles were possible with small volumes, with large volumes, conditions are needed that can withstand not only temperature non-uniformity but also prolonged temperature rise and fall cycles, enabling accurate DNA synthesis. The reason why conventional PCR reactions are generally performed with 50 μL is thought to be because it was not possible to achieve a combination of reaction buffer and DNA polymerase that could withstand prolonged temperature control while enabling accurate PCR DNA synthesis, as well as temperature uniformity. Therefore, the object of the present invention is to provide a method for amplifying nucleic acids using a large volume of PCR reaction solution of 30 mL or more, and an apparatus for amplifying nucleic acids using a large volume of PCR reaction solution of 30 mL or more. [Means for solving the problem]

[0009] As a result of diligent research to solve the above problems, the inventors of the present invention have found that by devising the composition of the PCR reaction solution and stirring the reaction solution, it is possible to specifically amplify nucleic acids even when using a large volume of PCR reaction solution of 30 mL or more, and have completed the present invention.

[0010] In other words, the present invention is as follows: [1] A method for amplifying nucleic acids by performing a polymerase chain reaction (PCR) by controlling the temperature of one or more reaction vessels containing a reaction solution containing DNA polymerase, deoxyribonucleotide triphosphate (dNTP), template DNA, forward primer, reverse primer, and buffer, characterized in that the reaction solution is 30 mL or more, and the polymerase chain reaction is performed while stirring the reaction solution. [2] The method according to [1] above, characterized in that the concentration of the buffer solution is 0.4 to 1.5 M. [3] The method according to [1] or [2] above, characterized in that the pH of the reaction solution is 8.5 to 10. [4] The method according to any one of the above [1] to [3], characterized in that the reaction solution is stirred by shaking the reaction vessel. [5] The method according to any one of the above [1] to [4], characterized in that the reaction vessel is made of an expandable material. [6] The method according to any one of the above [1] to [5], characterized in that the reaction vessel contains 1 to 90% gas. [7] The method according to any one of the above [1] to [6], characterized in that the temperature of the outer surface of the reaction vessel or the temperature of the reaction solution is measured by a temperature sensor, and the temperature of the thermal cycle in the polymerase chain reaction is controlled based on the measured temperature. [8] The method according to [7] above, characterized in that the temperature of the surface of a heat source adhering to the reaction vessel is measured by a temperature sensor, and the temperature of the thermal cycle in the polymerase chain reaction is controlled based on the temperature of the outer surface of the reaction vessel or the temperature of the reaction liquid and the temperature of the surface of the heat source adhering to the reaction vessel. [9] A thermal cycler comprising one or more reaction vessels made of a thermally conductive material containing 30 mL or more of reaction solution, a temperature control unit which is attached to the reaction solution to adjust the temperature of the reaction solution, and a stirring unit for stirring the reaction solution, wherein the temperature control unit comprises a heat source, a reaction vessel support plate which is in contact with the upper surface of the reaction vessel to support the reaction vessel, and a temperature sensor which measures the temperature of the outer surface of the reaction vessel or the temperature of the reaction solution, and a thermal cycler for use in the nucleic acid amplification method described in any one of [1] to [8] above.

[10] The thermal cycler according to [9] above, characterized in that the heat source in the temperature control unit is a Peltier element, and it comprises a heat conductive plate that is bonded to the Peltier element and can come into contact with the reaction vessel, and a temperature control unit connected to the Peltier element.

[11] The thermal cycler according to [9] or

[10] above, characterized in that it controls the temperature of the thermal cycle in a polymerase chain reaction based on the temperature of the outer surface of the reaction vessel or the temperature of the reaction solution measured by a temperature sensor.

[12] A thermal cycler according to any one of [9] to

[11] above, characterized in that a recess or through hole is provided in the thermal conductive plate, the temperature sensing part of a temperature sensor is positioned in the recess or through hole without contacting the thermal conductive plate, and the bottom surface of the reaction vessel and the temperature sensing part of the temperature sensor can come into contact when the reaction vessel is placed on the thermal conductive plate.

[13] A thermal cycler according to any one of [9] to

[12] above, characterized by comprising a temperature control unit for thermal denaturation, a temperature control unit for annealing and stretching reactions, and a preheating or cooling temperature control unit. [Effects of the Invention]

[0011] The nucleic acid amplification method of the present invention makes it possible to amplify nucleic acids even when using large volumes of PCR reaction solution of 30 mL or more. The thermal cycler of the present invention makes it possible to control the temperature of the reaction vessel or reaction solution so that nucleic acids are amplified even when using large volumes of PCR reaction solution of 30 mL or more. [Brief explanation of the drawing]

[0012] [Figure 1] It is a diagram showing an image of a thermal cycle in general PCR. [Figure 2] It is a diagram showing the result of analyzing the DNA polymerase Pfu solution by SDS-PAGE in Example 1. [Figure 3] It is a diagram showing the result of agarose electrophoresis of the PCR product in Example 2. [Figure 4] It is a diagram showing the result of agarose electrophoresis of the PCR product in Example 3. [Figure 5] It is a diagram showing the result of agarose electrophoresis of the PCR product in Example 4. [Figure 6] It is a diagram showing the result of agarose electrophoresis of the PCR product in Example 5. [Figure 7] It is a diagram showing the result of agarose electrophoresis of the PCR product in Example 6. [Figure 8A] Figure 8A is a schematic diagram of the temperature control unit of the thermal cycler used in Example 7. [Figure 8B] Figure 8B is a diagram showing the arrangement of the autoclave bag placed on the thermal cycler of Figure 8A and the Peltier element and the aluminum plate. [Figure 8C] Figure 8C is a plan view of the state where a 200 μL reaction solution bag is arranged on the lower autoclave bag. [Figure 9] The upper part of Figure 9 is a diagram showing the result of agarose electrophoresis of the PCR product in Example 7. The lower part of Figure 9 is a diagram showing the result of examining the temperature of the thermal cycle of PCR in the 200 μL reaction solution bag at the position 2 shown in Figure 8C. [Figure 10A] Figure 10A is a bottom perspective view of the state where a temperature sensor is attached to the center of the bottom surface of the reaction solution bag in Example 8. [Figure 10B] Figure 10B is a top perspective view of the state where the temperature sensing part of the temperature sensor is arranged in the through hole provided in the aluminum plate. [Figure 11A]The upper panel of Figure 11A shows the results of agarose electrophoresis of PCR products when the reaction solution volume was 100 mL in Example 8. The lower panel of Figure 11A shows the results of investigating the thermal cycling of PCR when the reaction solution volume was 100 mL. [Figure 11B] The upper panel of Figure 11B shows the results of agarose electrophoresis of PCR products when the reaction solution volume was 200 mL in Example 8. The lower panel of Figure 11B shows the results of the thermal cycling of PCR when the reaction solution volume was 200 mL. [Figure 11C] The upper panel of Figure 11C shows the results of agarose electrophoresis of the model PCR product when the reaction solution volume was 300 mL in Example 8. The lower panel of Figure 11C shows the results of investigating the thermal cycling of the model PCR when the reaction solution volume was 300 mL. [Figure 11D] Figure 11D shows the results of examining the thermal cycling of PCR without shaking in Example 8. [Figure 12] This shows the results of investigating the thermal cycling of PCR when different shaking methods were used in Example 9. [Figure 13] In Example 10, the upper left of Figure 13 shows the results of investigating the thermal cycle of PCR when PCR was performed with temperature control only from below. The upper right of Figure 13 shows the results of investigating the thermal cycle of PCR when PCR was performed with temperature control from both above and below. The lower left of Figure 13 shows the results of investigating the thermal cycle of PCR when PCR was performed using a 200 mL reaction solution model. The lower center of Figure 13 shows the results of investigating the thermal cycle of PCR when PCR was performed using a 300 mL reaction solution model. The lower right of Figure 13 shows the results of investigating the thermal cycle of PCR when PCR was performed using a 400 mL reaction solution model. [Figure 14] The upper part of Figure 14 shows the results of agarose electrophoresis of PCR products in the 600 mL reaction solution model in Example 11. The lower part of Figure 14 shows the results of investigating the thermal cycling of PCR in the 600 mL reaction solution model. [Figure 15]The upper part of Figure 15 shows the results of agarose electrophoresis of PCR products in the 1000 mL reaction solution model in Example 11. The lower part of Figure 15 shows the results of investigating the thermal cycling of PCR in the 1000 mL reaction solution model. [Figure 16] This is an overall front view of the first example of a thermal cycler. [Figure 17A] Figure 17A is a front view of the temperature control unit in the first example of a thermal cycler. [Figure 17B] Figure 17B is a right-side view of the temperature control unit in the first example of a thermal cycler. [Figure 18] This is a plan view of the copper plate in the first example of a thermal cycler. [Figure 19A] Figure 19A shows the positions of the copper plate and temperature sensor in the first example of a thermal cycler. [Figure 19B] Figure 19B is a plan view of the first example of a thermal cycler, showing a reaction vessel with a temperature sensor attached to a copper plate. [Figure 20A] Figure 20A is a front view of the reaction vessel support plate. [Figure 20B] Figure 20B is a plan view of the reaction vessel support plate. [Figure 20C] Figure 20C shows a side view of the reaction vessel support plate. [Figure 21A] Figure 21A shows the state when water vapor is generated and expands inside the reaction vessel. [Figure 21B] Figure 21B shows the reaction vessel support plate fixed in such a way that a gap is created between the reaction vessel and the reaction vessel support plate. [Figure 22A] Figure 22A is a front view of the temperature control unit in a second example of a thermal cycler. [Figure 22B] Figure 22B is a plan view of the reaction vessel support plate in the second example of a thermal cycler. [Figure 23] This is a front view of the third example of a thermal cycler. [Figure 24A]This is a diagram showing the positional relationship between a copper plate with a 1-cm diameter through-hole in the center for installing a temperature sensor and Peltier elements, in a mode where 8 Peltier elements are used on the lower surface of a thermal cycler. [Figure 24B] This is a diagram showing the positional relationship between a copper plate with a hollow part of 3 cm in length and 3 cm in width in the center and a diameter 1-cm through-hole for installing a temperature sensor near the edge, and Peltier elements, in a mode where 8 Peltier elements are used on the lower surface of a thermal cycler. [Figure 25] This is a front view when a temperature probe is used as a temperature sensor in a temperature control unit.

Mode for Carrying Out the Invention

[0013] The nucleic acid amplification method of the present invention is a nucleic acid amplification method for performing a polymerase chain reaction (PCR) by controlling the temperature of one or more reaction vessels containing a reaction solution containing a DNA polymerase, deoxyribonucleotide triphosphates (dNTPs), template DNA, a forward primer, a reverse primer, and a buffer solution, wherein the reaction solution is 30 mL or more, and the polymerase chain reaction is performed while stirring the reaction solution, which is also hereinafter referred to as "the nucleic acid amplification method of the present case".

[0014] Further, the thermal cycler of the present invention is a thermal cycler comprising one or more reaction vessels made of a heat-conductive material containing 30 mL or more of a reaction solution, an adhesive temperature control unit for adjusting the temperature of the reaction solution, and a stirring unit for stirring the reaction solution, wherein the temperature control unit comprises a heat source, a reaction vessel support plate that abuts on the upper surface of the reaction vessel to support the reaction vessel, and a temperature sensor for measuring the temperature of the outer surface of the reaction vessel or the temperature of the reaction solution, and is a thermal cycler for use in the nucleic acid amplification method of the present case (hereinafter also referred to as "the thermal cycler of the present case").

[0015] <PCR Reaction> In this specification, polymerase chain reaction is a method for amplifying template DNA by repeating a thermal cycle consisting of a series of steps—thermal denaturation, annealing (primer binding), and DNA extension—for 20 to 50 cycles in the presence of DNA polymerase, deoxyribonucleotide triphosphate (dNTPs), template DNA, forward primers, and reverse primers. Such polymerase chain reactions include quantitative polymerase chain reaction (qPCR), reverse transcription PCR (RT-PCR), single nucleotide extension (SBE), multiple single nucleotide extension (MSBE), and ligase-chain polymerase chain reaction (LCR-PCR).

[0016] In this specification, nucleic acids may be DNA or RNA. DNA may be single-stranded DNA or double-stranded DNA.

[0017] (DNA polymerase) In this specification, the DNA polymerase is preferably a heat-resistant DNA polymerase. A heat-resistant DNA polymerase means a DNA polymerase derived from thermophilic bacteria, and for example, it means that even after heat treatment at 70°C for 1 minute or more, the DNA polymerase activity is maintained at more than half.The DNA polymerase used can be either polI or α type, but specifically, it can be Thermus aquaticus (Taq), Thermus thermophilus, Thermococcus litoralis, Thermococcus gorgonarius, Thermococcus kodakaraensis KOD1, Thermococcus cleftensis, Thermococcus eurythermalis, Thermococcus paralvinellae, Thermococcus sibiricus, or Bacillus stearothermophilus. stearothermophilus), Methanothermus fervidus, Pyrococcus woesei, Pyrococcus furiosus (PFU), Pyrococcus sp. GB-D, Pyrococcus kukulkanii, Pyrococcus yayanosii, Pyrolobus fumarii, Aeropyrum pernix, Aquifex aeolicus, Sulfolobus tokodaii, or Methanopyrus candrelli Examples of DNA polymerases include Kandleri, and these may be used in combination. Furthermore, such DNA polymerases may be expressed genetically engineered in microorganisms such as E. coli, or commercially available products may be used.Furthermore, such DNA polymerase may be artificially synthesized through genetic engineering or may be of biological origin. The DNA polymerase may be expressed in microorganisms such as E. coli using genetic engineering technology, extracted, and purified by ion chromatography or the like as needed.

[0018] As for the concentration of DNA polymerase in the reaction solution, when the ability to synthesize DNA from 10 ng of DNA per 1 μL of DNA polymerase (DNA in which the polynucleotide encoding EMrfp in PSRLaP-EMrfp described in Nakamura et al.'s literature (Molecular Biotechnology 60, pages 912-923 (2018)) is replaced with a 711 bp polynucleotide encoding EGFP consisting of the base sequence shown in SEQ ID NO: 1) is defined as 1 U / μL, a concentration of 2000 to 10000 U / μL, preferably 3000 to 8000 U / μL, can be given.

[0019] (Deoxyribonucleotide triphosphate (dNTP)) In this specification, the deoxyribonucleotide triphosphate (dNTP) may be any combination of dATP, dCTP, dGTP, and dTTP, and dUTP may be included instead of dTTP. The concentration of dNTP can be appropriately adjusted depending on the length of the DNA to be amplified, the amount of DNA, and the type of sequence, but the concentrations of dATP, dCTP, dGTP, and dTTP are preferably 0.01 to 0.6 mM each, with lower limits of 0.02 mM, 0.05 mM, 0.1 mM, and 0.2 mM, and upper limits of 0.55 mM, 0.5 mM, and 0.45 mM.

[0020] (Template DNA) In this specification, template DNA can include genomic DNA, cDNA, and plasmid DNA. The length of such template DNA can range from 100 to 20,000 bp, with lower limits of 200 bp, 300 bp, or 500 bp, and upper limits of 10,000 bp, 5,000 bp, or 3,000 bp. Furthermore, the template DNA may exist alone or be incorporated into a vector such as a plasmid vector or a viral vector.

[0021] (Primer) In this specification, forward primers and reverse primers may be oligonucleotides that have complementarity with the template DNA and can hybridize to the region to be amplified in the template DNA or to an upstream sequence adjacent to that region, thereby serving as a starting point for nucleic acid synthesis in PCR.

[0022] The lengths of the forward and reverse primers can be appropriately adjusted depending on the length, amount, and type of DNA to be amplified, but can range from 8 to 100 mers. The lower limit may be 12 mers, 15 mers, 20 mers, or 25 mers, and the upper limit may be 80 mers, 60 mers, 50 mers, 45 mers, or 40 mers.

[0023] The forward primer and reverse primer may be modified, for example, they may be labeled with avidin, a fluorescent substance, or phosphorothioate-modified or phosphoramidite-modified oligonucleotide derivatives.

[0024] (buffer) In this specification, the buffer is not particularly limited as long as it is a buffer used in PCR reactions, but examples include Tris-HCl buffer, Tris-acetic acid buffer, Bistris buffer, HEPES buffer, MOPS buffer, etc. Such buffers may have their pH adjusted with hydrochloric acid or the like as needed, or may contain EDTA or Mg 2+ or K +Metal ions such as those listed above may be added. The concentration of the buffer solution can be adjusted as appropriate depending on the length of the DNA to be amplified, the number of thermal cycles, etc., but preferably it can be 0.02M to 1.5M, with the lower limit being 0.05M, 0.1M, 0.15M, 0.17M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, or 0.7M, and the upper limit being 1.2M, 1M, 0.9M, or 0.8M. For example, the concentration of the buffer solution can be 0.4 to 1.5M. In this specification, the concentration of the buffer solution refers to the concentration (M) of the buffering component contained in the buffer solution. Specifically, in the case of Tris-HCl buffer, it refers to the concentration of Tris-HCl.

[0025] The pH of the buffer solution is preferably 7.0 to 10.0, with a lower limit of 7.5, 8.0, 8.5, or 9.0, and an upper limit of 9.5 or 9.3. For example, the pH of the buffer solution can be 8.5 to 10.0, preferably 9.0 to 9.3.

[0026] (Reaction solution) In this specification, the amount of reaction solution is not particularly limited, but it can be 30 mL or more, and the lower limit may be 50 mL, 100 mL, 150 mL, 180 mL, 200 mL, 220 mL, 250 mL, or 300 mL, and the upper limit may be 10 L, 5 L, 4 L, 3 L, or 2 L. Note that the amount of reaction solution here refers to the total amount of reaction solution used to perform nucleic acid amplification reactions by PCR in the same thermal cycle. That is, such reaction solution may be contained in a single reaction vessel, but it may also be contained in two or more reaction vessels, as long as the thermal cycle of each reaction vessel can be controlled under approximately the same temperature conditions. For example, the temperature of a reaction vessel may be controlled by placing a single reaction vessel containing 30 mL or more of reaction solution on a heat conductive plate in contact with a heat source, or three reaction vessels containing 10 mL of reaction solution each may be prepared and placed on a heat conductive plate so that the bottom surface of each of the three reaction vessels is in contact with the heat conductive plate, thereby controlling the temperature inside each reaction vessel under the same conditions. Furthermore, the reaction solution may contain dimethyl sulfoxide, bovine serum albumin, glycerol, heparin, trehalose, nonionic surfactants, ammonium sulfate, betaine, etc., to the extent that they do not inhibit nucleic acid amplification by PCR. A probe may be included in the reaction solution to detect the amplified product. Examples of probes include TaqMan probes and cycling probes.

[0027] Furthermore, the reaction solution may contain labels such as dyes that bind to the phosphate groups on the DNA surface or dyes that intercalate between bases for the detection and monitoring of the amplification product. Examples of dyes that bind to the phosphate groups on the DNA surface or intercalate between bases include ethidium bromide, cyanine dyes, SYBR® Green, fluorescent coumarin, ruthenium, and ethidium bromide.

[0028] (Polymerase chain reaction) The polymerase chain reaction may be a two-step cycle in which annealing and extension reactions are carried out at the same temperature after thermal denaturation, or a three-step cycle in which annealing and extension reactions are carried out at different temperatures after thermal denaturation.

[0029] In this specification, the thermal denaturation temperature in the polymerase chain reaction can be 84 to 98°C, with the lower limit being 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, or 94°C, and the upper limit being 97°C, 96°C, or 95°C. The thermal denaturation time can be 1 to 90 seconds, with the lower limit being 5 seconds, 7 seconds, or 10 seconds, and the upper limit being 60 seconds or 30 seconds.

[0030] In this specification, the annealing temperature in the polymerase chain reaction can be 50 to 75°C, with a lower limit of 55°C, 57°C, or 60°C, and an upper limit of 72°C, 70°C, or 68°C. The annealing time can be 10 to 60 seconds, with a lower limit of 15 seconds, 20 seconds, or 25 seconds, and an upper limit of 50 seconds, 45 seconds, or 40 seconds.

[0031] In this specification, the temperature for the extension reaction in the polymerase chain reaction can be 50 to 80°C, with a lower limit of 55°C, 60°C, or 62°C, and an upper limit of 75°C, 70°C, or 68°C. The duration of the extension reaction can be 10 seconds to 20 minutes, with a lower limit of 30 seconds, 1 minute, or 2 minutes, and an upper limit of 15 minutes, 10 minutes, 5 minutes, or 3 minutes.

[0032] In this specification, when a polymerase chain reaction is carried out by a two-step cycle, the temperature for the annealing and extension reactions can be 55 to 80°C, with the lower limit being 55°C, 60°C, or 62°C, and the upper limit being 72°C, 70°C, or 68°C. The time for the annealing and extension reactions can be 20 seconds to 20 minutes, with the lower limit being 30 seconds, 1 minute, or 2 minutes, and the upper limit being 15 minutes, 10 minutes, 5 minutes, or 3 minutes.

[0033] Furthermore, the thermal cycles for the thermal denaturation, annealing, and extension reactions can range from 20 to 50 cycles, with a lower limit of 22 or 24 cycles and an upper limit of 40, 38, or 36 cycles. The duration of one cycle, i.e., the time for each step in one cycle of the thermal denaturation, annealing, and extension reactions, as well as the temperature control time, can range from 3 to 30 minutes, with a lower limit of 5, 7, or 10 minutes and an upper limit of 25, 20, or 15 minutes.

[0034] In the nucleic acid amplification method of the present invention, a typical two-step example of a polymerase chain reaction is as follows. (1) Initial thermal denaturation: 90°C for 10 seconds ↓ (2) Thermal denaturation 90 or 92°C for 10 or 30 seconds (3) Annealing and stretching reaction: 60 or 65°C for 2.5 minutes (4)(2) and (3) for 30 cycles

[0035] In the nucleic acid amplification method of the present invention, a typical three-step polymerase chain reaction is as follows: (1) Initial thermal denaturation: 90°C for 10 seconds ↓ (2) Thermal denaturation 90 or 92°C for 10 or 30 seconds (3) Annealing 65℃ 30 seconds (4) Extension reaction: 72°C, 2.5 minutes (5)(2)~(4) Repeat for 30 cycles

[0036] (Reaction vessel) The reaction vessel only needs to be able to contain the reaction liquid, and can take any shape such as a bag, box, cylinder, or flat plate, but a bag shape with a flat surface is preferable from the viewpoint of heat conduction efficiency. The reaction vessel may be able to seal the reaction liquid completely or it may be partially open. Furthermore, the reaction vessel may be provided with an opening for the reaction liquid to be placed inside, and after the reaction liquid is placed inside, it may be possible to close the opening to prevent leakage. Methods for closing the opening include heat sealing and bonding with adhesive.

[0037] The reaction vessel material should have thermal conductivity that allows heat from the heat source to be conducted to the reaction solution through the vessel. Examples include polyolefin resins such as polyethylene and polypropylene, polyester resins such as polyethylene terephthalate, styrene resins such as polystyrene, and metals such as aluminum, copper, silver, and gold. The thickness of the reaction vessel can be 0.01 to 1.5 mm for resins such as polyolefin resins, polyester resins, or styrene resins, and 50 μm to 3 cm for metals. In the case of resins, multiple types of resins may be laminated. In the case of metals, metals with a thermal conductivity of 50 W / m·K or higher can be used. When using two or more reaction vessels simultaneously, it is preferable to use reaction vessels of the same material from the viewpoint of uniformly controlling the reaction solution.

[0038] The reaction vessel preferably has both thermal conductivity and elasticity. Elasticity improves the adhesion between the reaction vessel and the heat source or a heat-conducting plate attached to the heat source, thereby increasing thermal conductivity. Furthermore, elasticity allows the reaction vessel to expand when steam is generated during the thermal denaturation stage, and then contract during the annealing and stretching reaction stages. Since the temperature can exceed 95°C during the thermal denaturation stage, the reaction vessel is preferably made of a material resistant to high temperatures of 95°C, preferably 100°C, that is, a material that prevents leakage of the reaction liquid due to damage to the reaction vessel at high temperatures.

[0039] The reaction vessel may be provided with a temperature sensor holder to facilitate the measurement of the temperature of the outer surface of the reaction vessel or the temperature of the reaction solution by holding a temperature sensor. An example of such a temperature sensor holder is one that is formed in a pocket shape on the outer surface of the reaction vessel, with the temperature measuring part housed within the pocket and thus enclosed within the reaction vessel.

[0040] (Stirring of the reaction solution) In this specification, there are no particular limitations on the method of stirring the reaction solution, but examples include shaking the reaction vessel itself, installing a stirrer such as a propeller-type stirrer or a jet-type stirrer inside the reaction vessel, or irradiating the reaction solution with ultrasonic waves.

[0041] One method for oscillating the reaction vessel is to use a shaker. Another method is to fix or install the reaction vessel directly or indirectly above the shaker so that the shaking motion generated by the shaker is transmitted to the reaction vessel. There are no particular restrictions on the method of fixing or installing the reaction vessel; for example, the reaction vessel can be fixed or installed on the shaking plate of the shaker, or on a shaking shaft fixed to the shaking plate and on which the shaking motion is transmitted. Furthermore, to oscillate the reaction vessel, the shaker may use rotational motion or reciprocating motion, and the direction of motion may be horizontal, vertical, or a combination of these. In the case of rotational motion, the rotational speed may be, for example, 10 to 200 rpm, or 50 to 100 rpm. Other units may be placed between the reaction vessel and the shaking plate as long as they do not hinder the shaking of the reaction vessel; for example, a heat source, a heat conduction plate, etc., described later may be placed between the reaction vessel and the shaking plate.

[0042] In the method of installing a stirrer such as a propeller-type stirrer or a jet-type stirrer inside the reaction vessel, the stirrer can be a propeller-type stirrer or a jet-type stirrer. In this case, the propeller-type stirrer or jet-type stirrer should be positioned inside the reaction vessel while keeping it sealed so that the reaction liquid does not leak out.

[0043] Another method for stirring the reaction mixture is to place a magnetic stirring bar inside the reaction vessel and use a magnetic stirrer to stir the mixture using magnetic force from outside the vessel. The magnetic stirring bar can be anything magnetic, and may be rod-shaped or elliptical, and may be coated with fluororesin or the like. The reaction mixture can be stirred by the rotation or sliding of the magnetic stirring bar due to the magnetic force from outside the reaction vessel.

[0044] When stirring the reaction solution by oscillating the reaction vessel, it is preferable to contain a predetermined amount or more of the reaction vessel inside the reaction vessel to improve stirring efficiency. The amount of reaction solution can be adjusted as appropriate according to the bottom area of ​​the reaction vessel, but it is preferable that the height of the reaction solution inside the reaction vessel, i.e., the vertical thickness, be 2 mm or more when the reaction vessel is standing still, and it may be 3 mm or more, 4 mm or more, or 5 mm or more.

[0045] When using a sealed container as the reaction vessel, it is preferable that the reaction vessel contains 1-90% gas before the PCR reaction is started. The lower limit may be 3%, 5%, 7%, or 10%, and the upper limit may be 80%, 70%, 60%, 50%, 40%, 30%, or 20%. Note that the gas content in the reaction vessel here refers to the content before the PCR reaction is performed, when the reaction solution is placed in the reaction vessel and the vessel is closed. Considering heat transfer efficiency, it is considered preferable for the vessel to be sealed with the reaction solution without containing gas. However, if the reaction vessel is filled with a large volume of reaction solution, the mixing efficiency is poor, for example, even if the reaction vessel itself is shaken to agitate the reaction solution. Therefore, by containing a predetermined amount of gas in the reaction vessel, the gas moves quickly within the reaction vessel when the vessel is shaken, and as a result, the mixing efficiency of the reaction solution is improved. In addition, the presence of gas makes it easier to suppress leakage of the reaction solution when removing the reaction solution from the reaction vessel after the PCR reaction. The gas mentioned above is not particularly limited as long as it does not inhibit the PCR reaction, and examples include air, carbon dioxide, nitrogen, etc. Alternatively, instead of directly including the gas, a bag containing the gas and / or liquid may be included in the reaction vessel. Furthermore, to improve the stirring efficiency of the reaction solution, stirring aids that can move freely in the reaction solution, such as beads, may be included in the reaction vessel.

[0046] (Temperature control of the reaction vessel) One method for controlling the temperature of the reaction vessel is to use a thermal cycler equipped with a temperature control unit that contacts at least a portion of the reaction vessel and is capable of controlling the temperature of the reaction vessel. It is preferable to use the thermal cycler of the present invention. The following describes the temperature control of the reaction vessel in the nucleic acid amplification method of the present invention, and the main components of the temperature control unit in the thermal cycler of the present invention.

[0047] The temperature control unit includes a heat source, a reaction vessel support plate that contacts the top surface of the reaction vessel to support it, and a reaction vessel temperature sensor that measures the temperature of the outer surface of the reaction vessel or the temperature of the reaction solution. To control the temperature of the reaction vessel, one method is to heat or cool the reaction solution by directly or indirectly contacting the reaction vessel with the heat source via a heat conductive plate. The heat source may be in contact with one or more locations on the bottom, side, or top surface of the reaction vessel, or only the bottom surface, or the bottom and top surface, or all or part of the bottom and side surfaces. When the heat source is in contact with the top surface, for example, the heat source may be provided symmetrically above and below the reaction vessel, as described in Patent Document 4 above. In the PCR reaction, water vapor is generated in the PCR reaction solution during the thermal denaturation stage, and gas may accumulate in the upper part of the reaction vessel. Therefore, from the viewpoint of temperature control of the PCR reaction solution or heat conduction efficiency, it is preferable that the heat source be in contact with at least the bottom surface of the reaction vessel, or it may be in contact with only the bottom surface.

[0048] Preferably, the reaction vessel is supported on its upper surface, that is, the surface symmetrical to its bottom surface, by a reaction vessel support plate. The material of the reaction vessel support plate is not particularly limited, but examples include metals such as aluminum and copper, or plastic. Alternatively, two heat sources may be provided, with one heat source serving as the reaction vessel support plate, so that the reaction vessel is sandwiched between the heat sources on the top and bottom surfaces.

[0049] Furthermore, the reaction vessel support plate may be made of an insulating material or a heater such as an electric heater. In the case of a heater, for example, by controlling the temperature to be near the temperature of the annealing stage, it is possible to easily control the temperature of the thermal denaturation, annealing, and extension reactions, and shorten the time of the thermal cycle.

[0050] There are no particular restrictions on the heat source, but it should contain a heat source capable of both endothermic and heating. Examples of such heat sources include heaters such as Peltier elements and electric heaters. The number of heat sources can be adjusted as appropriate according to the volume of the PCR reaction solution, and one or more heat sources may be provided, for example, two or more, three or more, five or more, eight or more, ten or more, or twenty or more. In other words, a heat source may be provided only on the bottom surface of a single reaction vessel, but if the surface area of ​​the reaction vessel is large, multiple heat sources may be placed horizontally side by side to enable endothermic and heating of the outer surface of the reaction vessel in a shorter time.

[0051] Furthermore, while the heat source, such as the Peltier element or heater, may be in direct contact with the reaction vessel, a heat conductive plate may be provided on the surface in contact with the reaction vessel. The heat released from the heat source is then transferred to the reaction liquid through the reaction vessel via this heat conductive plate. In addition, from the viewpoint of thermal conductivity, it is preferable that the Peltier element and the heat conductive plate are bonded together with a heat conductive filler such as heat conductive grease.

[0052] The material of the heat conduction plate can be a material with high thermal conductivity, such as aluminum, copper, silver, or gold. Furthermore, while the heat conduction plate is preferably plate-shaped, the surface that adheres to the reaction vessel may have protrusions, indentations, unevenness, fan shapes, or inclines to improve the stirring efficiency of the reaction vessel and the contact area with the reaction vessel. The thickness of the heat conduction plate can be adjusted as appropriate depending on the material, but can be 0.1 mm to 5 cm, 0.5 mm to 3 cm, or 1 mm to 1 cm. The length and width of the heat conduction plate can be adjusted as appropriate according to the length and width of the reaction vessel and the number of heat sources, and one heat conduction plate may be provided for one heat source, or one heat conduction plate may be provided for multiple heat sources. The shape of the heat conduction plate can be a square, rectangle, or ellipse. For temperature control, it is preferable that the heat conduction plate is in contact with 50% or more of the bottom surface of the reaction vessel, and may be 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more. Furthermore, since the reaction liquid is agitated, there may be areas where the heat conductive plate is not in contact with the reaction liquid via the reaction vessel, which may be 5% or more, 10% or more, 15% or more, 20% or more, or 30% or more of the bottom surface of the reaction vessel.

[0053] Further, it is preferable that the heat conduction plate is provided with a recess or a through hole for preventing the heat of the heat source from directly being transmitted when a temperature detection part of a temperature sensor for measuring the temperature of the outer surface of the reaction vessel is arranged thereon. The shape of the recess or the through hole is not particularly limited, such as a circle, a square, a rectangle, an ellipse, a conical shape, etc. The size of the recess or the through hole (the minor diameter in the case of a rectangle or an ellipse) may be such that the temperature detection part of the temperature sensor does not touch the heat conduction plate. For example, it is preferable that the shortest distance between the temperature detection part of the temperature sensor and the heat conduction plate is kept at 0.5 mm or more, 1 mm or more, 2 mm or more, 3 mm or more, 4 mm or more, 5 mm or more, 7 mm or more, 10 mm or more, 15 mm or more. In other words, the radius or the minor diameter of the recess or the through hole can be 0.5 mm or more, 1 mm or more, 2 mm or more, 3 mm or more, 4 mm or more, 5 mm or more, 7 mm or more, 10 mm or more, 15 mm or more. The position of the recess or the through hole may be any position on the heat conduction plate, but it is preferably near the center. Further, instead of providing the recess or the through hole, a heat insulating sheet such as a sheet made of silica aerogel may be attached to the heat conduction plate, and the temperature detection part of the temperature sensor may be arranged on such a heat insulating sheet, or the temperature detection part of the temperature sensor may be attached to the reaction vessel using the above heat insulating sheet.

[0054] Furthermore, the heat conduction plate may be provided with a hollow part for improving the heat radiation efficiency from the reaction vessel. The area of such a hollow part is not particularly limited. For example, it is 1 cm 2 ~9 cm 2 There is, and as the lower limit, it is 1.5 cm 2 、2 cm 2 As the upper limit, it is 8 cm 2 、5 cm 2 Or 4 cm 2 It may be. In addition, in order to prevent the reaction vessel from sinking, the hollow part may be in a mesh shape, a lattice shape, or a linear hollow shape as necessary.

[0055] The Peltier element described above may be equipped with a heat sink, fins, fan, and / or water cooling unit on the side opposite to the side in contact with the reaction vessel. Preferably, the Peltier element and the heat sink are in close contact with thermal conductive grease.

[0056] The temperature control unit is connected to a power control unit, a calculation unit, a current control unit equipped with a power supply unit, and a temperature sensor.

[0057] Examples of temperature sensors include a reaction vessel temperature sensor that measures the temperature of the outer surface of the reaction vessel, a reaction liquid temperature sensor in which a temperature sensing unit is located inside the reaction vessel and the temperature sensing unit is in direct contact with the reaction liquid to measure the temperature of the reaction liquid, and a heat conduction plate temperature sensor that measures the temperature of a heat source or heat conduction plate in contact with the reaction vessel. Any one of the above temperature sensors may be used, or any combination of the above may be used, but it is preferable to have at least a reaction vessel temperature sensor or a reaction liquid temperature sensor.

[0058] Examples of reaction vessel temperature sensors for measuring the temperature of the outer surface of a reaction vessel include thin temperature sensors that can be attached to the outer surface of the reaction vessel, probe-type temperature sensors equipped with a temperature sensing element at the tip, and radiation temperature sensors such as infrared temperature sensors capable of measuring the temperature of the outer surface of the reaction vessel. As for probe-type temperature sensors, temperature sensors that can be extended and retracted by springs or the like may be used. If the heat conduction plate has through holes, the temperature sensing element of the probe-type temperature sensor should be positioned in the through holes so that when the reaction vessel is placed on the heat conduction plate, the temperature of the outer surface of the reaction vessel and the temperature sensing element come into contact, thereby enabling measurement of the temperature of the outer surface of the reaction vessel. When using sterilized reaction solutions, or when it is desirable to maintain the sterility of the reaction solution, it is preferable to use a reaction vessel temperature sensor or a heat conduction plate temperature sensor.

[0059] Furthermore, examples of reaction liquid temperature sensors, in which the temperature sensing unit is located inside the reaction vessel and measures the temperature of the reaction liquid by directly contacting it, include wireless temperature sensors and rod-shaped temperature sensors that can be positioned so that the temperature sensing unit penetrates the reaction vessel and is located inside the reaction liquid. When using a wireless temperature sensor, one method is to place the wireless temperature sensor in the reaction liquid as is, or after waterproofing and / or heat-resistant treatment.

[0060] Furthermore, the number of temperature sensors may be one or more. In addition, when using the reaction vessel temperature sensor, the position in contact with the reaction vessel may be the outer surface of the reaction vessel, and any position that contacts the reaction solution through the reaction vessel is acceptable, with the bottom and / or side being preferred. In the case of the bottom, the position is not particularly limited, whether it is in the center or the periphery. Conventional temperature control in PCR has mainly been performed based on the temperature measured by a heat conduction plate temperature sensor. This is thought to be because, conventionally, the reaction solution is about 10 to 50 μL, so the heat from the heat source or heat conduction plate in contact with the reaction vessel is easily conducted throughout the reaction solution. However, when the reaction solution is 30 mL or more, the temperature of the reaction solution and the temperature of the heat source or heat conduction plate tend to diverge. Therefore, in order to make it easier to control the temperature of the reaction solution, it is preferable to measure the temperature of the outer surface of the reaction vessel or the temperature of the reaction solution.

[0061] The temperature information detected by the temperature sensor is sent to the current control unit, where the calculation unit determines the current to be supplied from the power control unit to the heat source, such as a Peltier element. In other words, the current control unit functions as a power source for the heat source, such as a Peltier element, and controls the temperature of the heat source or the heat conductive plate by adjusting the current supplied to the heat source, such as a Peltier element, based on the temperature data of the outer surface and / or heat conductive plate of the reaction vessel detected by the temperature sensor. To suppress overshoot or undershoot in temperature control, it is preferable to use PID control or the like.

[0062] In the calculation unit, for example, the thermal cycle conditions such as temperature, time, and number of thermal cycles for each of the thermal denaturation, annealing, and extension reactions are set in advance, and the current is controlled so that heating occurs if the temperature of the outer surface of the reaction vessel or the reaction solution is lower than the set temperature, and cooling occurs if it is higher than the set temperature. However, in large-volume PCR, temperature variations in the reaction solution tend to occur. Unless the reaction temperature and reaction time of each step in the thermal cycle shown in Figure 1 are accurately controlled, specific nucleic acid amplification cannot be achieved. In the thermal denaturation stage, if the temperature is lower than the set temperature, the template DNA cannot be denatured, and if the temperature is higher than the set temperature for a long time, components contained in the reaction solution, such as DNA polymerase, will be denatured. Also, in annealing, if the temperature is lower than the set temperature, nonspecific binding increases, and if the temperature is higher, the primers will detach from the template DNA. Furthermore, in the extension reaction, if the temperature is lower or higher than the set temperature, the function of DNA polymerase will decrease. Regarding reaction time, the longer the time per thermal cycle, the more likely DNA polymerase is to be deactivated. Therefore, it is extremely important to control the reaction solution to the set temperature quickly and accurately.

[0063] Furthermore, if the temperature control unit is equipped with a reaction vessel temperature sensor and a heat conduction plate temperature sensor, it is preferable to control the temperature so that the temperature of the outer surface of the reaction vessel measured by the reaction vessel temperature sensor meets the conditions for each stage of the thermal cycle, while also controlling the temperature of the heat source or heat conduction plate measured by the heat conduction plate temperature sensor so that it does not become excessively high or low above the set temperature, i.e., so that no overshoot or undershoot occurs during temperature rise or fall. Conventional thermal cyclers generally performed nucleic acid amplification with reaction solutions of 50 μL or less in which heat conduction occurs rapidly, so it was common practice to control the temperature of the reaction vessel by measuring the temperature of the heat source or heat conduction plate in contact with the reaction vessel, rather than the reaction vessel itself. However, when the volume of reaction solution increases, a difference tends to occur between the temperature of the heat source or heat conduction plate and the temperature of the outer surface of the reaction vessel. Therefore, by using the two sensors mentioned above, it becomes possible to control the temperature to more closely approximate the temperature of the reaction solution.

[0064] In the polymerase chain reaction, during the transition from thermal denaturation to the annealing reaction, the transition from annealing to the extension reaction, and / or the transition from the extension reaction to thermal denaturation, the temperature rise or fall of the outer surface of the reaction vessel or the reaction solution can be controlled to 0.05°C / second or more. Such temperature rise or fall can be appropriately adjusted depending on the size of the reaction vessel, the amount of reaction solution, etc. For example, the temperature rise can be 0.06 to 6°C / second, with the lower limit being 0.08, 0.1, 0.2, 0.3, 0.4, or 0.5°C / second, and the upper limit being 5, 4, 3, 2, 1.5, 1.2, 1, 0.8, 0.5, 0.2, or 0.1°C / second, but not particularly limited. The temperature decrease can be, for example, 0.1 to 6°C / second, with a lower limit of 0.2, 0.3, 0.4, or 0.5°C / second, and an upper limit of 5, 4, 3, 2, 1.5, 1.2, 1, 0.8, or 0.7°C / second, but is not particularly limited. Furthermore, it is preferable that the rate of temperature decrease is faster than the rate of temperature increase, specifically, the rate of temperature decrease may be 1.2 times, 1.5 times, 1.8 times, or 2 times faster than the rate of temperature increase.

[0065] Multiple temperature control units may be provided for each temperature set in the PCR thermal cycle. For example, a temperature control unit may be provided for each step of the reaction: thermal denaturation, annealing, and extension, and temperature control may be performed by the temperature control unit for each step. Specifically, when performing a PCR reaction in three steps, four units can be provided: temperature control unit A for thermal denaturation, temperature control unit B for annealing, temperature control unit C for the extension reaction, and preheating or cooling temperature control unit D to raise or lower the temperature of the reaction vessel in a shorter time so that it reaches the set temperature for the next step in a shorter time. The temperature can then be controlled in the following cycle.

[0066] 1. The thermal denaturation is performed for a predetermined period of time in a temperature control unit A for thermal denaturation, which is adjusted to the set temperature for thermal denaturation. 2. Transfer the reaction vessel to a preheating or cooling temperature control unit D that has been preheated to a temperature below the annealing temperature, for example, 0°C. 3. When the temperature of the reaction vessel approaches the set temperature for the next step, namely annealing, move the reaction vessel to annealing temperature control unit B, which is adjusted to the annealing temperature. 4. Anneal for the specified time using the annealing temperature control unit B. 5. Transfer the reaction vessel to a preheating or cooling temperature control unit D that has been adjusted to a temperature above the set temperature for the extension reaction, for example, 90°C. 6. When the temperature of the reaction vessel approaches the set temperature for the next step, i.e., the extension reaction, move the reaction vessel to the extension reaction temperature control unit C, which has been adjusted to the extension reaction temperature. 7. The stretch reaction is performed for a predetermined time using the stretch reaction temperature control unit C. 8. Transfer the reaction vessel to a preheating or cooling temperature control unit D that has been adjusted to a temperature above the set temperature for thermal denaturation, for example, 98°C. 9. When the temperature of the reaction vessel approaches the next step, namely the thermal denaturation temperature, the reaction vessel is moved to thermal denaturation temperature control unit A, which is adjusted to the thermal denaturation temperature.

[0067] By performing steps 1-9 above as one cycle, and completing a predetermined cycle, it becomes possible to perform the PCR reaction while controlling the temperature more quickly and accurately. Note that since heating can control the temperature of the reaction vessel in a shorter time than cooling, step 8 above may be omitted, and the process may proceed to step 9' "Transfer the reaction vessel to the thermal denaturation temperature control unit A, which has been adjusted to the thermal denaturation temperature" instead of step 9. Also, since the set temperatures for the annealing and extension reactions are close, step 5 above may be omitted, and the process may proceed to step 6' "Transfer the reaction vessel to the extension reaction temperature control unit C, which has been adjusted to the extension reaction temperature" instead of step 6.

[0068] Since the temperature rise or fall between each step can be handled quickly by using a preheating or cooling temperature control unit, the overall reaction time of the PCR reaction can be shortened, and the degradation of the function of components contained in the reaction solution, such as DNA polymerase, can be suppressed. The reaction solution may be stirred by providing a support stand to support all temperature control units and rotating or reciprocating such a stand using a shaker or motor, or by using a stirrer, agitator, shaking mechanism, or ultrasound for each temperature control unit.

[0069] Similarly, when performing a PCR reaction in two steps, a method can be used in which three temperature control units are provided: a temperature control unit A for thermal denaturation, a temperature control unit B for annealing and extension reactions, and a preheating or cooling temperature control unit D, and the temperature is controlled in the following cycle. 1. The thermal denaturation is performed for a predetermined period of time in a temperature control unit A for thermal denaturation, which is adjusted to the set temperature for thermal denaturation. 2. Transfer the reaction vessel to a preheating or cooling temperature control unit D that has been preheated to a temperature below the annealing and extension reaction temperature, for example, 0°C. 3. When the temperature of the reaction vessel approaches the set temperature for the next step, namely the annealing and stretching reaction, move the reaction vessel to the annealing and stretching reaction temperature control unit B, which has been adjusted to the temperature of the annealing and stretching reaction. 4. The annealing and stretching reaction is carried out for a predetermined time in the temperature control unit B for the annealing and stretching reaction. 5. Transfer the reaction vessel to a preheating or cooling temperature control unit D that has been adjusted to a temperature above the set temperature for thermal denaturation, for example, 98°C. 6. When the temperature of the reaction vessel approaches the next step, namely the thermal denaturation temperature, the reaction vessel is moved to the thermal denaturation temperature control unit A, which is adjusted to the thermal denaturation temperature. By performing steps 1-6 above as one cycle, and completing a predetermined cycle, it becomes possible to perform the PCR reaction while controlling the temperature more quickly and accurately. Note that since heating can control the temperature of the reaction vessel in a shorter time compared to cooling, step 5 above may be omitted, and the process may proceed to step 6', "Transfer the reaction vessel to the thermal denaturation temperature control unit A, which has been adjusted to the thermal denaturation temperature," instead of step 6.

[0070] (Thermal cycler) In this thermal cycler, the temperature control unit is as described above, but a more detailed description will be given. First, the temperature control unit of the reaction vessel comprises a heat source, a reaction vessel support plate that contacts the upper surface of the reaction vessel and supports it, and a reaction vessel temperature sensor that measures the temperature of the outer surface of the reaction vessel or the temperature of the reaction liquid. In a typical example, multiple heat sources are arranged in parallel horizontally, and a heat conductive plate on which the reaction vessel is placed is bonded above them via heat conductive grease. A certain space for housing the reaction vessel is provided above the heat conductive plate, and the reaction vessel support plate is positioned above this space. A slip prevention plate may be installed vertically at the end of the heat conductive plate to prevent the reaction vessel from shifting due to the oscillation of the reaction vessel.

[0071] The reaction vessel support plate is mounted horizontally above the reaction vessel, and its distance from the heat source or a heat conductive plate attached to the heat source can be appropriately adjusted according to the size and capacity of the reaction vessel used. The reaction vessel support plate may be supported by its own weight so as to abut the reaction vessel, or it may be fixed or maintained in a position in contact with the reaction vessel using screw jacks or wires. Alternatively, a shaft supporting the reaction vessel support plate may be provided, and the reaction vessel support plate may be fixed or maintained by an extendable mechanism such as an air cylinder or robotic cylinder, or a spring. Furthermore, the reaction vessel support plate may be positioned so as to allow a predetermined pressure to be applied to the reaction vessel in order to improve the airtightness between the reaction vessel and the heat source or heat conductive plate.

[0072] Furthermore, the reaction vessel support plate may be equipped with fixing or maintaining means that can fix or maintain it in a position that leaves a predetermined gap between it and the reaction vessel, for example, a gap of 1 cm or more, 2 cm or more, 3 cm or more, 4 cm or more, 5 cm or more, 7 cm or more, or 10 cm or more. With such a configuration, for example, a gap may be provided between the reaction vessel support plate and the reaction vessel during the temperature decrease step in the thermal cycle, i.e., the step between thermal denaturation and annealing. By providing such a gap during the temperature decrease step, the upper surface of the reaction vessel comes into contact with the air, which makes heat dissipation from the reaction vessel more efficient and shortens the time per thermal cycle. There are no particular limitations on the means for fixing or maintaining the reaction vessel support plate above the reaction vessel with a predetermined gap in between, but examples include using a screw jack or wire that can be electrically controlled to automatically raise and lower in accordance with the thermal cycle, using a cylinder such as an air cylinder or robot cylinder, or using a cam mechanism. Alternatively, when the reaction vessel support plate is supported by its own weight via a support shaft or when wires are used, a stopper such as a set collar can be provided on the support shaft of the reaction vessel support plate, and the position of the stopper can be adjusted so that a gap is created between the top surface of the reaction vessel and the reaction vessel support plate when the reaction vessel has contracted during cooling.

[0073] From the viewpoint of heat transfer efficiency to the reaction vessel, it is preferable that the heat source and the heat conduction plate are connected by screws or the like. Furthermore, the heat source is supported by a heat source support shaft, but if the heat conduction plate and the heat source are connected, only the heat conduction plate may be supported by the heat source support shaft. Additionally, the upper reaction vessel support plate may be supported by such a heat source support shaft.

[0074] Next, as a stirring unit, one that can oscillate the reaction vessel can be used, for example, a shaker. By fixing the temperature control unit to the shaker so that the shaking of the shaker is transmitted to the reaction vessel, it becomes possible to oscillate the reaction vessel. As for the shaker, one that can perform rotational shaking or reciprocating shaking can be used. In the case of rotational shaking, the rotation speed is as described in the "Agitation of the reaction solution" section above.

[0075] Furthermore, if a stirring bar is placed inside the reaction vessel as described above, a stirrer can be used as the stirring unit. In this case, a space is provided in the temperature control unit between the stirrer and the stirring bar inside the reaction vessel that does not interfere with magnetism. Specifically, when multiple heat sources are placed side by side horizontally, a space is provided between each heat source, and the stirring bar is operated by the stirrer through that space.

[0076] Furthermore, an ultrasonic device can also be used as a stirring unit. By irradiating the reaction vessel with ultrasonic waves, the reaction liquid inside the vessel is stirred. In this case, a space is provided between the ultrasonic device and at least a portion of the reaction vessel that does not obstruct the ultrasonic waves. Specifically, when multiple heat sources are installed in parallel horizontally, a space is provided between each heat source, and ultrasonic waves are irradiated into the reaction vessel by the ultrasonic device through this space.

[0077] The connection between the temperature control unit and the stirring unit is not particularly limited, but for example, if a shaker is used as the stirring unit, a method can be given in which a vertical shaking axis is provided on the shaking platform and the temperature control unit is fixed to this shaking axis. If a Peltier element is used as the heat source and a cooling fan is provided, a predetermined width, preferably 3 cm or more, can be left between the cooling fan and the shaking platform so that the cooling fan does not come into close contact with the shaking platform. [Examples]

[0078] The present invention will be described more specifically below with reference to examples, but the technical scope of the present invention is not limited to these examples.

[0079] [Example 1] Preparation of DNA polymerase Pfu In the following examples, DNA polymerases used were DNA polymerase Pfu expressed in E. coli, and commercially available PrimeSTAR® GXL (Takara Bio Inc.) and KOD Fxneo (Toyobo Co., Ltd.). DNA polymerase Pfu expressed in E. coli was prepared by the following method.

[0080] First, a plasmid containing the base sequence encoding DNA polymerase Pfu (SEQ ID NO: 2) was mixed with E. coli HB101 competent cells (Takara Bio Inc.). After 10 minutes in an ice bath and 5 seconds at 40°C, 50 μL of sterile water was added, and the cells were incubated at 37°C for 24 hours on a plate medium containing yeast extract, peptone, and ampicillin to obtain transformant colonies. These colonies were added to the yeast extract, peptone, and ampicillin medium and incubated at 200 rpm for 24 hours. 500 μL of the cultured medium was taken into a tube, centrifuged, and the supernatant was discarded. Then, a buffer containing 1% Triton X-100 was added and incubated. Further centrifugation was performed to obtain the supernatant as DNA polymerase Pfu solution (hereinafter simply referred to as "Pfu solution"). For SDS-PAGE, the supernatant was obtained by centrifugation, mixed with 6× sample buffer, and allowed to stand at 97°C for 5 minutes. Figure 2 shows the results of analyzing 50 μL of the DNA polymerase Pfu solution by SDS-PAGE. As is clear from Figure 2, a protein with a molecular weight of 90 kDa, the same as that of the Pfu protein, was obtained.

[0081] [Example 2] Investigation of reaction solution volume and time The volume of the reaction solution and the DNA amplification efficiency, as well as the reaction time and DNA amplification efficiency, were investigated. PCR was performed using the Pfu solution prepared in Example 1, and commercially available PrimeSTAR® GXL (Takara Bio Inc.) and KOD Fxneo (Toyobo Co., Ltd.) as DNA polymerases.

[0082] The reaction solution was added to a 200 μL microtube in amounts of 10, 25, 50, 75, 100, 125, 150, or 200 μL, and PCR was performed. As the template DNA, a plasmid containing a 711 bp DNA encoding EGFP consisting of the nucleotide sequence shown in SEQ ID NO: 1 was used. The primers were a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 3 and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 4, which were designed to amplify the 711 bp DNA encoding the above EGFP. The composition (final concentration) of the PCR reaction solution is shown below.

[0083] <PCR Reaction Solution Composition> For PrimeSTAR GXL and KOD Fxneo, the following PCR reaction solutions were prepared according to the protocols of their respective products, based on the attached buffers, dNTPs, etc. ■PCR Reaction Solution Containing PrimeSTAR GXL Template DNA 0.1 ng / μL dNTP 0.2 mM Forward Primer 0.3 μM Reverse Primer 0.3 μM 5×PrimeSTAR GXL Buffer 0.2 μL / 10 μL PrimeSTAR GXL DNA Polymerase 1.0 μL / 10 μL

[0084] ■PCR Reaction Solution Containing KOD Fxneo Template DNA 0.1 ng / μL dNTP 0.4 mM<00004…​​​​​​​​​​​​​​​​Forward primer 0.5 μM Reverse primer 0.5 μM Tris-HCl buffer 0.15M DNA polymerase Pfu 0.2 μL / 10 μL MgSO4 2.5mM DMSO 5%

[0086] The PCR reaction was performed using a C1000Touch thermal cycler (manufactured by BIO-RAD) under the following conditions. (1) Initial thermal denaturation: 98°C for 10 seconds (2) Thermal denaturation: 98°C for 10 seconds, 30 seconds, 60 seconds, or 120 seconds (3) Annealing at 60°C for 10, 30, 60, or 120 seconds (4) Extension reaction: 68°C for 10 seconds, 30 seconds, 60 seconds, or 120 seconds (5) Repeat (2) to (4) for 30 cycles.

[0087] Figure 3 shows the results of agarose electrophoresis analysis of the obtained PCR products. As is clear from Figure 3, with any enzyme, almost no nucleic acid amplification was observed when the reaction solution volume exceeded 150 μL. Furthermore, by extending the time of the denaturation, annealing, and extension reactions, a slight amplification was observed at 200 μL. However, when the time of the denaturation, annealing, and extension reactions was extended for a long time, smear bands (indistinct bands that extend continuously up and down) were observed, confirming that nonspecific nucleic acid amplification was occurring. Most PCRs widely performed to date are carried out in volumes of 50 μL or less, which is thought to be based on the fact that amplification efficiency and specificity decrease when the volume exceeds 50 μL, especially above 150 μL.

[0088] [Example 3] Investigation of the pH of the PCR reaction solution Next, the pH of the PCR reaction solution was examined. The PCR reaction solution was prepared to have the same composition as the Pfu-containing PCR reaction solution in Example 2, except that the pH was adjusted to 7.70, 7.90, 8.10, 8.30, 8.52, 8.81, 9.26, or 9.86. 10 μL of the PCR reaction solution was used, and the PCR reaction was performed using the following five methods. All cycles were 30. (a) Thermal denaturation 98°C 2 min, annealing 60°C 2 min, extension reaction 68°C 2 min (b) Thermal denaturation: 98°C for 10 seconds, annealing and extension reaction: 72°C for 10 seconds (c) Thermal denaturation: 98°C for 10 seconds, annealing and extension reaction: 72°C for 2 minutes (d) Thermal denaturation: 98°C for 2 minutes, annealing and extension reaction: 72°C for 10 seconds (e) Thermal denaturation 98°C for 2 minutes, annealing and extension reaction 72°C for 2 minutes

[0089] Figure 4 shows the agarose electrophoresis results for each PCR product. As shown in Figure 4, it was found that nucleic acid amplification efficiency was higher in alkaline conditions with a pH of 7.9 or higher, and that nucleic acid amplification efficiency was higher when the annealing and extension reactions were performed at 72°C.

[0090] [Example 4] Investigation of pH and buffer concentration of PCR reaction solution Focusing on the buffer concentration of the PCR reaction solution, PCR was performed using the same Pfu-containing PCR reaction solution as in Example 2, except that the concentration of Tris-HCl buffer was set to 150 mM or 300 mM and the pH was the same as in Example 3. The PCR reaction solution was 10 μL, and the PCR reaction was performed as follows. The number of cycles was 30. Thermal denaturation at 98°C for 2 minutes, followed by annealing and extension reaction at 72°C for 2 minutes.

[0091] Figure 5 shows the agarose electrophoresis results for each PCR product. As shown in Figure 5, it was revealed that increasing the buffer concentration resulted in clearer bands and reduced nonspecific amplification, and that amplification efficiency was better on the alkaline side at pH 8.3 or higher.

[0092] [Example 5] Investigation of PCR reaction time and buffer concentration - 1 Based on the results of Examples 3 and 4 described above, the relationship between PCR reaction time and buffer concentration was investigated. PCR was performed using the same Pfu-containing PCR reaction mixture as in Example 2, except for the buffer concentration. The PCR reaction mixture was 10 μL, and the reaction temperature, time, and buffer concentration were varied to the eight types listed in Table 1 below.

[0093] [Table 1]

[0094] Figure 6 shows the agarose electrophoresis results for each PCR product. As shown in Figure 6, when the buffer concentration is 0.15 M, it was confirmed that the nucleic acid amplification efficiency decreases significantly when the thermal denaturation time or the annealing and extension reaction time reaches 60 seconds, as shown in (e) or (g). On the other hand, when the buffer concentration is 0.6 M, although the nucleic acid amplification efficiency is poor when the annealing and extension reaction time is short, as shown in (b), it was confirmed that the nucleic acid amplification efficiency improves when the annealing and extension reaction time is 30 seconds or longer, as shown in (d), (f), and (h). When the reaction solution volume is large, it is unavoidable that the annealing and extension reaction time will be longer compared to the case of normal reaction solution volume due to temperature control in the PCR thermal cycle. From the above results, it was found that large-volume PCR is difficult with the currently widely used buffer concentration of 0.15 M, but the problem unique to large volumes, where the annealing and extension reaction time is extended, can be solved by increasing the buffer concentration.

[0095] [Example 6] Investigation of PCR reaction time and buffer concentration - 2 Based on the results of Examples 3 to 5 above, the relationship between PCR reaction time and buffer concentration was further investigated. PCR was performed using the same Pfu-containing PCR reaction mixture as in Example 2, except that the Tris-HCl buffer concentration was 0.15, 0.3, 0.45, 0.6, 0.65, 0.7, 0.75, or 0.9 M. The reaction mixture was 10 μL, and the reaction time and temperature were 10 seconds at 90°C for thermal denaturation, and 1 minute, 2 minutes, 3 minutes, 5 minutes, 10 minutes, or 20 minutes at 65°C for annealing and extension reactions.

[0096] Figure 7 shows the agarose electrophoresis results for each PCR product. As shown in Figure 7, when the buffer solution was 0.15 M, the nucleic acid amplification efficiency decreased when the annealing and extension reaction time exceeded 5 minutes, and the bands became smears, with nonspecific amplification also observed. After 20 minutes, nucleic acids were not amplified. By increasing the buffer solution to 0.3 M or higher, nucleic acids were amplified even when the annealing and extension reaction time exceeded 20 minutes, and nonspecific amplification decreased as the buffer concentration increased.

[0097] When performing large-volume PCR, conditions that can accommodate long reaction times are necessary from the perspective of temperature control. Considering the above examples 3 to 5, it was confirmed that when performing large-volume PCR, the control of buffer concentration and pH is extremely important in terms of nucleic acid amplification efficiency and specific amplification.

[0098] [Example 7] Up to Example 6 described above, microtubes were used as reaction vessels in the PCR reaction to investigate various reaction solutions and reaction conditions. However, microtubes are not suitable for large volumes of reaction solution. Therefore, to perform large-volume PCR, a plastic bag was used as the reaction vessel.

[0099] As a model for a reaction vessel containing 50 mL of PCR reaction solution, two polypropylene autoclave bags (35 μm thick, 22.5 cm long, 10 cm wide: Bioremo Co.) were prepared by filling them with 50 mL of water and sealing them with heat to prevent air from entering. These two bags were stacked in two layers, and a polypropylene autoclave bag (35 μm thick, 3 cm long, 3 cm wide, hereinafter also referred to as the "200 μL reaction solution bag") containing 200 μL of Pfu reaction solution was placed between them and set in a thermal cycler. For PCR, as shown in Figure 8A, a thermal cycler 1, which is an improved version of the thermal cycler described in Patent Document 4, was used. In short, the temperature control unit of the thermal cycler 1 consists of an air-cooled Peltier element 13 (two on the bottom and two on the top) equipped with a heat sink 11 and a cooling fan 12, an aluminum plate 14 with a short axis of 10 cm and a long axis of 22.5 cm fixed to the Peltier element 13 with screws, and a temperature control unit (not shown). The temperature control unit includes a current control unit, a calculation unit, a set temperature input unit, and a power supply unit. As shown in Figure 8B, the 200 μL reaction solution bag 16 is sandwiched between the two layers of autoclave bags 15 described above, which are stacked so that they are sandwiched between the upper and lower Peltier elements 13 and the 3 mm thick aluminum plate 14 that is bonded to them, and this is placed on the thermal cycler 1. The 200 μL reaction solution bag 16 is sandwiched on the upper surface of the lower autoclave bag 15 so that it is positioned at five locations 1 to 5 in Figure 8C. A thin, adhesive-type thermocouple temperature sensor 17 (Rika Kogyo Co., Ltd.) with a temperature sensing element 18 was attached to the bottom surface of the 200 μL reaction solution bag 16 at position No. 2. The temperature information of the outer surface of the 200 μL reaction solution bag detected was transmitted to the current control unit, which controlled the current to the Peltier element 13 to adjust the temperature. A GL840 (Graphtec Corporation) temperature data logger was used. Temperature measurement conditions were a temperature measurement range of 0 to 500°C and a sampling interval of 0.5 seconds.

[0100] The PCR reaction was performed as follows. (1) Thermal denaturation: 90°C for 10 seconds (2) Thermal denaturation 90°C for 30 seconds (3) Annealing and stretching reaction 65°C 2.5 minutes (4) Repeat (2) and (3) for 30 cycles

[0101] The Pfu-containing PCR reaction mixture was prepared to have the following composition (final concentration). Furthermore, instead of the polynucleotide encoding EGFP, the template DNA used was the polynucleotide encoding HGF-BGH shown in SEQ ID NO: 5. The nucleotide sequences of the forward and reverse primers are shown in SEQ ID NOs: 6 and 7, respectively. The PCR amplification product is 3167 bp, as shown in SEQ ID NO: 8. ■Pfu-containing PCR reaction solution Template DNA (Human HGF-BGH 1 ng / μL) 0.1 ng / μL dNTP 0.4mM Forward primer 0.5 μM Reverse primer 0.5 μM Tris-HCl buffer, pH 9.23, 0.6M Pfu DNA polymerase 0.3 μL / 10 μL MgSO4 2.5mM

[0102] The results of agarose electrophoresis of each PCR product are shown in the upper panel of Figure 9. In Figure 9, C represents the result of applying 1 μL of PCR reaction solution from a 200 μL microtube containing 10 μL of reaction solution as a control for the conventional method, while 1-5 represent the results of applying 1 μL of PCR reaction solution from 200 μL reaction solution bags placed at the positions shown in Figure 8C. As shown in Figure 9, nucleic acids were amplified in the 200 μL reaction solution bags at positions 2-4 shown in Figure 8C, but not in the 200 μL reaction solution bags at positions 1 or 5, confirming that the temperature inside the reaction solution bag was non-uniform during the thermal cycle. Furthermore, the results of investigating the PCR cycle temperature in the 200 μL reaction solution bag at position 2 shown in Figure 8C are shown in the lower panel of Figure 9. In the lower panel of Figure 9, the horizontal axis represents the elapsed time (seconds) from the start of the PCR reaction (start of heating for initial thermal denaturation), and the vertical axis represents the temperature of the outer surface of the reaction solution bag measured by a temperature sensor attached to the 200 μL reaction solution bag. As shown in the lower part of Figure 9, the thermal denaturation temperature, annealing temperature, and extension reaction temperature fluctuated within a range of 63-69°C, indicating that the set temperature could not be maintained and temperature variations were observed. From these results, it was concluded that when the volume was as large as 50 mL, fluctuations occurred in the liquid temperature, resulting in a decrease in the efficiency of thermal denaturation, annealing, and extension reactions.

[0103] [Example 8] Based on Example 7 described above, it is anticipated that when the amount of PCR reaction solution increases further, it will be necessary to homogenize the PCR reaction solution within the PCR reaction solution bag and adjust the temperature to the set temperature during the thermal denaturation, annealing, and extension reaction stages. Therefore, we attempted to see if the above problem could be solved by stirring the PCR reaction solution.

[0104] In Example 7, two layers of bags containing 50 mL of water were stacked. However, in this example, (1) 100 mL of PCR reaction solution was placed in one reaction solution bag, (2) 200 mL was placed in one reaction solution bag, and (3) as a model for 300 mL of reaction solution, 300 mL of water was placed in one reaction solution bag, and a plastic bag containing 200 μL of PCR reaction solution was placed inside the above reaction solution bag. (1) to (3) were prepared, and air was added to each of the reaction solution bags (1) to (3) so that the volume of air was 5 to 10%. These reaction solution bags were placed in the thermal cycler described in Example 7, and the thermal cycler itself was placed on the shaking platform of a shaker (rotary shaker SR-300: Shimadzu Corporation). The shaking platform and the thermal cycler were then fixed so that the rotational shaking from the shaker was transmitted to the reaction bags placed in the thermal cycler. PCR was performed while stirring the reaction mixture by rotating and shaking the reaction vessel at 100 rpm. As a control without shaking, one bag containing 50 mL of reaction mixture was placed in the thermal cycler and PCR was performed without shaking. The composition of the PCR reaction mixture and the PCR thermal cycle were the same as in Example 7. As shown in Figure 10A, a thin thermocouple temperature sensor 17 was attached to the center of the bottom surface of the reaction mixture bag 16. Furthermore, as shown in Figure 10B, the reaction mixture bag 16 was placed on the aluminum plate 14, which is a heat conductive plate, so that the temperature sensing part 18 of the temperature sensor was positioned in the center of the through hole 19 provided in the aluminum plate 14. Note that in the case of 300 mL of reaction mixture in the thermal cycler, a copper plate was used instead of the aluminum plate 14.

[0105] The results for a 100 mL reaction solution are shown in Figure 11A, the results for a 200 mL reaction solution are shown in Figure 11B, the results for a 300 mL reaction solution are shown in Figure 11C, and the control without shaking is shown in Figure 11D. In Figures 11A-C, the 10 μL lane on the left represents the control, where PCR was performed without shaking using a 200 μL microtube containing 10 μL of reaction solution. The upper panel in Figures 11A-C shows the results of agarose electrophoresis, and the lower panel in Figures 11A-C and Figure 11D show the results of the thermal cycle temperature. The horizontal axis represents the time (seconds) from the start of the PCR reaction, and the vertical axis represents the temperature. As the volume of the reaction solution increased, the time required for one reaction cycle increased, even though the reaction time and temperature settings were the same. The time required for 5 cycles was 1897 seconds, 2202 seconds, and 2105 seconds for 100 mL, 200 mL, and 300 mL, respectively. While there were concerns that longer reaction times would lead to a decrease in nucleic acid amplification efficiency, it was confirmed that nucleic acids were amplified even with large reaction volumes of 100-300 mL by stirring the reaction solution. Regarding reaction temperature, in the case of the no-shaking control shown in Figure 11D, there was variation in temperature during thermal denaturation, annealing, and extension reactions. However, in the 100 mL, 200 mL, and 300 mL samples with shaking, the temperature was controlled to be close to the set temperature.

[0106] [Example 9] In Example 8, rotational shaking was used, but other shaking methods were also investigated. PCR was performed in the same manner as in Example 8, except that no shaking, rotational shaking at 100 rpm, reciprocating shaking in the long axis direction at 50 rpm or 100 rpm, and reciprocating shaking in the short axis direction at 50 rpm or 100 rpm were used, and a reaction bag containing 100 mL of reaction solution was used. The results are shown in Figure 12. In Figure 12, the horizontal axis represents the time (seconds) from the start of the PCR reaction, and the vertical axis represents the temperature. As shown in Figure 12, the time per 5 cycles was short at 1711 seconds with rotational shaking at 100 rpm. Furthermore, it was confirmed that the temperature was controlled closer to the set temperature with rotational shaking at 100 rpm or short-axis shaking at 100 rpm.

[0107] [Example 10] Examples 8 and 9 above confirmed that shaking the reaction solution bag allows for temperature control to more closely approximate the set temperature. However, when the PCR reaction solution is 100 mL or more, water vapor may be generated inside the bag during and around the thermal denaturation stage, causing the bag to expand and air to accumulate at the top. In this case, even if a heat source is placed above the reaction vessel, temperature control from above becomes inefficient as heat is transferred to the PCR reaction solution via the air. Therefore, taking into account the simplification of the thermal cycler structure and the reduction of overall temperature variation by agitation of the PCR reaction solution by shaking, we attempted to see if nucleic acids could be amplified by temperature control only at the bottom of the bag containing the PCR reaction solution.

[0108] PCR was performed in the same manner as in Example 7, using a reaction bag containing 200 mL of Pfu-containing PCR reaction solution and rotating and shaking at 100 rpm. In the thermal cycler shown in Figure 8A, the temperature control of the upper Peltier element was stopped, and a bag containing only air (air thickness 2-5 cm) was placed above the reaction bag to provide insulation between it and the upper Peltier element. In other words, the heat source was only the bottom surface of the reaction bag. As a control, the same 100 rpm rotation and shaking was performed, and the temperature was controlled at both the upper and lower Peltier elements. A copper plate was used as the heat conductive plate instead of an aluminum plate.

[0109] The results of measuring the temperature of the PCR reaction are shown in the upper panel of Figure 13. The upper left of Figure 13 shows the results of investigating the thermal cycling of PCR when temperature control is performed only from below, and the upper right of Figure 13 shows the results when temperature control is performed from both above and below. It was confirmed that even with only temperature control from below and no temperature control from above, temperature control of almost the same degree as control (when temperature control is performed from both above and below) can be achieved by rotational shaking. Furthermore, as a model of 200 mL, 300 mL, or 400 mL reaction solutions, the results of agarose electrophoresis when PCR was performed by placing a plastic bag containing 100 μL of reaction solution inside a bag containing 200 mL, 300 mL, or 400 mL of water are shown in the lower panel of Figure 13. Even when using 200-400 mL reaction models, it was confirmed that nucleic acids could be amplified with temperature control from below only.

[0110] [Example 11] Since it was confirmed in Example 10 above that nucleic acids could be amplified even with 400 mL, we investigated whether nucleic acid amplification was possible with even larger volumes.

[0111] As a model for a 600 mL or 1000 mL reaction solution, a plastic bag containing 100 μL of Pfu-containing PCR reaction solution was placed inside a 32 cm x 32 cm autoclave bag (containing air so that the air volume was 5-10%) containing 600 mL or 1000 mL of water, and PCR was performed in the same manner as in Example 10 by rotating and shaking at 100 rpm.

[0112] The thermal cycler used was the first example shown in Figure 16 below. An air-filled bag (air thickness 2-5 cm) was placed above the reaction solution bag for insulation between it and the Peltier element. In other words, only the bottom surface of the reaction solution bag was in contact with the heat conductive plate acting as a heat source. A copper plate shown in Figure 24A below was used as the heat conductive plate, and approximately eight Peltier elements were fixed to the copper plate in a U-shape on the sides, excluding the center, and arranged in parallel horizontally. The concentration of Tris-HCl buffer in the reaction solution was 0.6 M or 0.65 M for the 600 mL reaction solution model, and 0.6, 0.65, or 0.7 M for the 1000 mL reaction solution model. Furthermore, the concentration of template DNA was 0.1 ng / μL (final concentration) for the 600 mL reaction solution model, and 0.1 ng / μL (final concentration) or 1 ng / μL (final concentration) for the 1000 mL reaction solution model.

[0113] Figures 14 and 15 show the results of agarose electrophoresis and thermal cycling when PCR was performed on 600 mL and 1000 mL reaction solution models, respectively. In both models, the target nucleic acid was amplified, confirming that nucleic acid amplification is possible even with large volumes of 600 mL or 1000 mL using the nucleic acid amplification method of the present invention.

[0114] In the case of a 1000 mL reaction solution model, the temperature rise in the second cycle was 0.06 °C / second, and the temperature decrease was 0 / 16 °C / second. In conventional PCR methods, temperature rise and decrease are generally 1 to 6 °C / second, but in the nucleic acid amplification method of the present invention, amplification products can be obtained even with a slow temperature rise of 0.06 °C / second, which is extremely surprising for nucleic acid amplification technology, which has always required a fast temperature rise rate. Furthermore, as shown in the above examples, by controlling the concentration and pH of the buffer solution, it was possible to amplify the target nucleic acid even when the time required for 30 cycles was 160 minutes or more, 200 minutes or more, 250 minutes or more, or 360 minutes or more.

[0115] [Example 12] (Example of a thermal cycler) Next, an example of the first thermal cycler 2 usable in the nucleic acid amplification method of the present invention will be described with reference to Figures 16 and 17. Figure 16 is an overall front view of the first thermal cycler 2, Figure 17A is a front view of the temperature control unit 31, and Figure 17B is a right side view of the temperature control unit 31. The temperature control unit 31 is fixed on the shaking platform 33 of the shaking unit 32 by four temperature control unit fixing shafts 34 having spiral grooves on their outer circumference. Due to this fixing, the shaking platform 33 of the shaking unit 32 rotates and shakes, causing the reaction vessel 35 to rotate and shake, and the reaction liquid (not shown) inside the reaction vessel 35 to be stirred.

[0116] The vibration unit 32 is equipped with a vibration on / off switch 36, a rotation speed adjustment dial 37, and a rotation speed display unit 38, allowing the vibration to be controlled by adjusting it to a predetermined rotation speed. A motor 39 is installed at the center of the vibration unit 32 so that its rotation axis is vertical, and it is perpendicularly connected to the vibration platform 33. Rotational vibration is possible using the power of this motor 39.

[0117] The temperature control unit 31 has four Peltier elements 13 arranged in a 2x2 parallel row on a horizontal surface, and a 3mm thick copper plate 40 fixed with screws so that it is in close contact with the entire upper surface of the Peltier elements 13. A reaction vessel 35 containing the PCR reaction solution is placed on the copper plate 40, and the heat from the Peltier elements 13 is transferred to the reaction vessel 35 via the copper plate 40. A stainless steel reaction vessel support plate 41 is placed on the top surface of the reaction vessel 35 by its own weight. In addition, anti-slip plates 42 are installed on the sides of the copper plate 40 to prevent the reaction vessel from shifting due to oscillation.

[0118] The Peltier element 13 is equipped with a heat sink 11 and a cooling fan 12 on the side opposite to the side in contact with the reaction vessel 35. The lower end of the cooling fan 12 is positioned at a predetermined height from the base end of the temperature control unit fixing shaft 34, and space is provided to prevent convection of the air released by the cooling fan 12. The Peltier element 13 is also connected to a temperature control unit, which includes a power control unit, a calculation unit, a power supply unit, a temperature display unit, and a set temperature input unit (not shown).

[0119] Figure 18 shows a plan view of the copper plate 40. It has a through hole 19 with a diameter of 10 mm in the center. At both ends are insertion holes 44a for inserting into a linear shaft 43 that is provided so that the reaction vessel support plate 41 can move freely in the vertical direction. At the four corners are screw holes 45a for connecting to the temperature control unit fixing shaft 34 with screws, and four sets of four screw holes 45b for connecting to the Peltier element 13 with screws. The copper plate 40 and the Peltier element 13 are fixed by screws via the screw holes 45a for connecting to the Peltier element 13. In addition, as shown in Figure 17A, the copper plate 40 is fixed to the temperature control unit fixing shaft 34 by its own weight by inserting the screw holes of the copper plate 40 into the temperature control unit fixing shaft 34, which is equipped with nuts 46 as stoppers in predetermined positions.

[0120] Next, the arrangement of the temperature sensor will be explained with reference to Figures 10A and 19A,B. A thin, adhesive-type thermocouple temperature sensor 17 is attached to the center of the outer surface of the reaction vessel 35, and the temperature sensing part 18 of the temperature sensor 17 is positioned so as not to come into contact with the copper plate 40 through a through hole 19 in the center of the copper plate 40, as shown in Figure 19A (the reaction vessel is not shown). Figure 19B is a plan view (the reaction vessel support plate is not shown) of the reaction vessel 35, which contains the PCR reaction solution and 10% air 51, placed on the copper plate 40, with the temperature sensing part 18 of the temperature sensor 17 located in the center. The temperature detection result from the temperature sensor 17 is sent to a temperature control unit (not shown), and then the calculation unit determines the power to be supplied from the power control unit to the Peltier element 13. That is, the temperature control unit has the function of controlling the temperature of the copper plate 40 by adjusting the power supplied to the Peltier element 13 based on the temperature data of the reaction vessel 35 detected by the temperature sensor 17.

[0121] In the temperature setting input section, the operator inputs information necessary for the PCR thermal cycle, such as reaction temperatures for initial thermal denaturation, thermal denaturation, annealing, and extension reactions, reaction time, and the number of cycles. In addition, the temperature measured by the temperature sensor 17 is displayed in the temperature display section.

[0122] The reaction vessel support plate 41 is explained with reference to Figures 20A-C and 21A and 21B. Figures 20A-C show the front view, top view, and side view of the reaction vessel support plate 41, respectively. Figure 21A shows the state when water vapor 54 is generated in the reaction vessel and air accumulates and expands above the reaction vessel 35. Both ends of the reaction vessel support plate 41 are equipped with support plate holding parts 52 having insertion holes 44b for insertion into the linear shaft 43. By inserting it into the linear shaft 43, the reaction vessel support plate 41 can move up and down vertically relative to the linear shaft 43 and is in contact with the upper surface of the reaction vessel 35 by its own weight. With this configuration, when water vapor is generated from the PCR reaction solution in the reaction vessel and the height of the upper surface of the reaction vessel increases, or when the water vapor in the reaction vessel decreases and the height of the upper surface of the reaction vessel decreases, it is possible to hold down and support the reaction vessel without adjusting the height of the reaction vessel support plate. Furthermore, this configuration allows the reaction vessel support plate 41 to contact the top surface of the reaction vessel 35 according to its height, while also maintaining close contact between the bottom surface of the reaction vessel 35 and the copper plate 40. Additionally, by supporting the Peltier element 13 with the temperature control unit fixed shaft 34 and the reaction vessel support plate 41 with the linear shaft 43, the conduction of heat from the copper plate 40 to the reaction vessel support plate 41 can be suppressed, enabling more precise temperature control.

[0123] Furthermore, in Figure 21B, the reaction vessel support plate 41 is equipped with fixing means that can be fixed in a position that leaves a predetermined gap between it and the reaction vessel 35. In the process of cooling the reaction solution after thermal denaturation for annealing, as shown in Figure 21B, a gap for air is provided between the reaction vessel 35 and the reaction vessel support plate 41, improving the heat dissipation efficiency of the reaction solution and accelerating the temperature decrease.

[0124] [Example 13] (Second example of a thermal cycler)

[0125] Next, an example of a second thermal cycler usable in the nucleic acid amplification method described above will be explained with reference to Figures 22A and 22B. In Figure 22A, the stirring unit is omitted. In the above example 10, the Peltier element 13 was air-cooled by a cooling fan 12, whereas the Peltier element 13 shown in the second example is water-cooled. A pair of water-cooled cooling units using the Peltier element 13 are provided vertically (the Peltier element 13 is located inside the cooling unit 60 and is therefore not shown), and a solvent inlet 61 and a solvent outlet 62 for cooling are provided. The upper copper plate 40 also serves as a reaction vessel support plate. There is no reaction vessel support plate fixing shaft, and two cooling unit support plates 63 are provided parallel to the temperature control unit fixing shaft 34, and the Peltier element 13 and cooling unit 60 are supported by the cooling unit support plates 63. The cooling unit support plate 63 has through-holes 64 at both ends that are inserted onto the temperature control unit fixing shaft 34, and is fixed to the temperature control unit fixing shaft 34 from above by wing nuts 65.

[0126] [Example 14] (Third example of a thermal cycler) Furthermore, an example of a third thermal cycler usable in the above nucleic acid amplification method will be explained with reference to Figure 23. In order to speed up the heating and cooling of large volumes of reaction solution, the system is equipped with three temperature control units 31 arranged vertically: a temperature control unit 31a for thermal denaturation, a temperature control unit 31b for annealing and extension reactions, and a preheating or cooling temperature control unit 31c for rapidly raising or lowering the temperature so that the reaction vessel reaches the set temperature for the next step in a short time. Above each of the temperature control units 31a to c, an air-cooled Peltier element 13 equipped with a heat sink 11 and a cooling fan 12 can be raised and lowered vertically by a lifting cylinder 71. When controlling the temperature of the reaction vessel 35, the Peltier element lowers and contacts the top surface of the reaction vessel 35 to fix the reaction vessel 35 and strengthen the adhesion with the copper plate 40, thereby increasing the heat conduction efficiency. Also, the upper parts of the temperature control units 31a to c are raised when the reaction vessel 35 is in the other temperature control unit position. The temperature control unit at the top may simply be a heater capable of controlling the temperature to a constant level, or it may not have a heat source at all.

[0127] Each of the temperature control units 31a to 31c is equipped with a thin thermocouple temperature sensor to measure the temperature of the outer surface of the bottom of the reaction vessel 35, and a thin thermocouple temperature sensor to measure the temperature of a copper plate measuring 20 cm in length, 10 cm in width, and 5 mm in thickness (not shown). The measured temperature information is sent to a current control unit (not shown) to perform temperature control. In the third example of this thermal cycler, the temperature rise and fall is rapid, making overshoot or undershoot likely, so the temperature is controlled by PID.

[0128] The temperature control units 31a to 31c are fixed via a temperature control unit fixing shaft 34 onto a shaking platform 33 of a shaker equipped with a motor 39. By shaking the shaking platform 33, the reaction vessel 35 can be oscillated. Alternatively, instead of using the shaking unit 32, the reaction vessel 35 may be oscillated by shaking at least one temperature control unit 31, either as a whole or a part thereof, by sandwiching the reaction vessel between an upper and lower pair of temperature control units 31 and shaking the entire temperature control unit or the copper plate 40.

[0129] The reaction vessel 35 is placed on a mobile platform 72 made of a thermally conductive sheet, and is movable between temperature control units 31a to 31c along a travel rail 73.

[0130] Next, we will explain the operation of the third example of this thermal cycler when performing a thermal cycle. First, the reaction vessel 35 is placed in the temperature control unit 31a for thermal denaturation, which is adjusted to the set temperature for thermal denaturation. The upper temperature control unit is lowered by the lifting cylinder 71 so that the top and bottom surfaces of the reaction vessel 35 come into contact with the copper plate 40. In this state, once the temperature of the outer surface of the reaction vessel 35 reaches the thermal denaturation temperature, thermal denaturation is performed for a predetermined time. Next, once the thermal denaturation is complete, the upper part of the temperature control unit 31a rises, and the moving platform 72 is moved from side to side to shake the reaction vessel 35 and stir the reaction liquid. After that, the reaction vessel 35 is moved to the preheating or cooling temperature control unit 31c, which is set to 0°C in advance, and the upper part of the cooling temperature control unit is lowered to sandwich the reaction vessel 35, rapidly cooling the reaction vessel 35. Once the outer surface of the reaction vessel 35 has cooled to near the set temperature for the annealing and extension reaction, the upper part of the temperature control unit 31c rises, and the transfer platform 72 moves, transferring the reaction vessel 35 to the temperature control unit 31b for the annealing and extension reaction. Then, the upper part of the temperature control unit 31b for the annealing and extension reaction lowers, sandwiching the reaction vessel 35, and annealing is performed for a predetermined time. Next, once the annealing and extension reaction is complete, the temperature control unit above the temperature control unit 31b for the annealing and extension reaction rises, and the transfer platform 72 moves, transferring the reaction vessel 35 to the thermal denaturation temperature control unit 31a, where thermal denaturation is performed for a predetermined time. By repeating the above steps in a thermal cycle, rapid heating and cooling can be achieved, shortening the PCR reaction time and improving the efficiency of nucleic acid amplification.

[0131] [Example 15] (Position of copper plate and Peltier element) Furthermore, Figure 24A illustrates an embodiment of a thermal cycler usable in the above nucleic acid amplification method in which eight Peltier elements 13 are used on the underside of the copper plate 40. Figure 24A is a plan view of the copper plate 40 in the first example of thermal cycler 2, in which approximately eight Peltier elements 13 (positions indicated by dotted lines in the figure) are fixed under the copper plate 40 in a square shape. The copper plate 40 is 32 cm in both length and width and 3 mm thick. The copper plate 40 has a through hole 19 with a diameter of 1 cm in the center. No Peltier elements 13 are installed in the center, and this space can be used to install a stirrer, ultrasonic device, etc.

[0132] Furthermore, the central area may have a hollow section 20 measuring 3 cm vertically and 3 cm horizontally, as shown in Figure 24B. With this configuration, the reaction vessel 35 is more easily exposed to air during the step of lowering the temperature of the reaction liquid, improving heat dissipation efficiency. In addition, if the hollow section 20 has a mesh, grid, or linear hollow shape, it is possible to improve the heat dissipation effect by utilizing the airflow generated by the movement of the fan of the Peltier element 13. To further improve heat dissipation efficiency, a heat pipe may be provided below the hollow section 20, and the cooling efficiency of the heat pipe may be increased by utilizing the convection generated near the Peltier element 13 by the fan of the Peltier element 13. Furthermore, to improve the heat dissipation effect, the thickness of the copper plate near the center may be thinner than that of the surrounding area, without providing a hollow section.

[0133] [Example 16] Figure 25 shows an embodiment of a temperature control unit in which a temperature probe 81 is used as a temperature sensor. The Peltier element 13, heat sink, and cooling fan are omitted from the description in order to show the position of the temperature probe 81. The temperature probe 81, which is extendable and retractable by a spring, is equipped with a temperature sensing part 82 at its tip. The temperature sensing part 82 protrudes from the through hole 19 without touching the copper plate 40, and when the reaction vessel 35 is placed on the copper plate 40, the outer surface of the reaction vessel 35 comes into contact with the temperature sensing part 82, making it possible to measure the temperature of the outer surface of the reaction vessel 35. Note that the upper end of the temperature sensing part 82 only needs to be able to touch the reaction vessel 35, and depending on the material of the reaction vessel 35, it does not need to protrude from the through hole 19. [Industrial applicability]

[0134] This invention enables high-volume PCR, which can be used for the production of DNA vaccines, PCR testing, and the like. [Explanation of symbols]

[0135] 1…Thermal cycler 2…The first thermal cycler 11… Heatsink 12…Cooling fan 13…Peltier element 14…Aluminum plate 15… Autoclave bag 16…Reaction solution bag 17…Temperature sensor 18...Temperature sensor temperature measuring part 19…Through hole 20...Hollow part 31, 31a~c...Temperature control unit 32... Shaking Unit 33…Shaking table 34...Temperature control unit fixed shaft 35…Reaction vessel 36…On / Off switch 37... Rotation speed adjustment dial 38... Rotation speed display 39...motor 40...Copper plate 41…Reaction vessel support plate 42... Anti-slip plate 43…Linear shaft 44a, 44b... Through holes 45a, 45b... screw holes 46... Nut 51...Air 52...Support plate holding part 53…PCR reaction solution 54...Water vapor 60…Cooling unit 61... Solvent inlet 62... Solvent outlet 63...Cooling unit support plate 64…Through hole 65... Wing nut 71... Lifting cylinder 72... Mobile platform 73… Rails 81...Temperature probe 82...Temperature screening section

Claims

1. A thermal cycler for carrying out a polymerase chain reaction using a reaction vessel containing a reaction solution, The system includes a temperature control unit that contacts the reaction vessel to adjust the temperature of the reaction solution, and a stirring unit for stirring the reaction solution. The reaction vessel is made of a thermally conductive material. The temperature control unit includes a heat source in contact with the reaction vessel, and A temperature sensor selected from the following: a reaction vessel temperature sensor that measures the temperature of the outer surface of the reaction vessel; a reaction liquid temperature sensor in which a temperature sensing unit is located inside the reaction vessel and the temperature sensing unit is in direct contact with the reaction liquid to measure the temperature of the reaction liquid; and a heat conduction plate temperature sensor that measures the temperature of a heat source or heat conduction plate in contact with the reaction vessel. A thermal cycler characterized by being equipped with [a certain feature].

2. The thermal cycler according to claim 1, characterized in that the heat source is in contact with at least the bottom surface of the reaction vessel.

3. The thermal cycler according to claim 1 or 2, wherein the stirring unit is a shaker or a stirrer.

4. The thermal cycler according to any one of claims 1 to 3, characterized in that the temperature control unit comprises a reaction vessel support plate disposed on the upper surface of the reaction vessel and supporting the reaction vessel.

5. The thermal cycler according to claim 4, characterized in that the reaction vessel support plate has a structure that supports the reaction vessel by its own weight so as to abut it against the reaction vessel, a structure that fixes or maintains the reaction vessel support plate in a position that abuts the reaction vessel, or a structure in which the reaction vessel support plate is fixed or maintained by a retractable mechanism.

6. The thermal cycler according to any one of claims 1 to 5, characterized in that the temperature sensor is either a reaction vessel temperature sensor that measures the temperature of the outer surface of the reaction vessel, or a reaction liquid temperature sensor in which a temperature sensing unit is located inside the reaction vessel and the temperature sensing unit directly contacts the reaction liquid to measure the temperature of the reaction liquid.

7. The thermal cycler according to claim 6, further comprising a control unit that controls the temperature of the thermal cycle in the polymerase chain reaction based on the temperature of the outer surface of the reaction vessel measured by the reaction vessel temperature sensor, or the temperature of the reaction solution measured by the reaction solution temperature sensor.

8. The heat source is in contact with the reaction vessel via a heat conductive plate. The temperature sensor is a reaction vessel temperature sensor that measures the temperature of the outer surface of the reaction vessel, The thermal cycler according to claim 6 or 7, characterized in that the heat conduction plate is provided with a recess or through hole, the temperature sensing part of the reaction vessel temperature sensor is positioned in the recess or through hole without contacting the heat conduction plate, and the bottom surface of the reaction vessel and the temperature sensing part of the reaction vessel temperature sensor can come into contact when the reaction vessel is placed on the heat conduction plate.

9. A thermal cycler according to any one of claims 1 to 8, characterized by comprising a temperature control unit for thermal denaturation, a temperature control unit for annealing and stretching reactions, and a preheating or cooling temperature control unit.

10. The thermal cycler according to any one of claims 1 to 9, characterized in that the temperature control unit includes a control unit that controls the temperature rise of the outer surface of the reaction vessel or the reaction liquid to 0.05 to 1°C / second during the steps of transitioning from thermal denaturation to an annealing reaction in the polymerase chain reaction, transitioning from an annealing reaction to an extension reaction, and / or transitioning from an extension reaction to thermal denaturation.

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