Nucleic acid amplification method and thermal cycler

Abstract

The present invention addresses the problem of providing: a method for amplifying nucleic acids using at least 30 mL of a large-capacity PCR reaction solution; and a device that amplifies nucleic acids using at least 30 mL of a large-capacity PCR reaction solution.　The problem is solved by a nucleic acid amplification method in which a polymerase chain reaction is performed by controlling the temperature of one or more reaction vessels accommodating a reaction solution that contains DNA polymerase, deoxyribonucleotide triphosphate (dNTP), a template DNA, a forward primer, a reverse primer, and a buffer solution, wherein the reaction solution is at least 30 mL, and the reaction solution is stirred while the polymerase chain reaction is carried out.

Description

[Technical Field]

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

[0002] In the analysis of nucleic acids, a method of amplifying nucleic acids by replicating the target nucleic acid using the polymerase chain reaction (PCR) method is widely used (see Patent Document 1). Because PCR is a simple process and can be performed in a relatively short time using a small, simple-mechanism device, it is widely used in disease diagnosis, nucleic acid medicine, analysis of microorganisms, research related to genetic engineering, DNA identification, etc.

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

[0004] Until now, PCR has typically involved replicating nucleic acids in the order of several ng to several hundred ng in a PCR reaction solution in the μL range. However, recent developments such as vaccine development have created a need for replicating large amounts of DNA and RNA. To replicate large amounts of DNA and RNA using PCR, conventional techniques require repeating PCR tens to hundreds of times, which requires effort, time, and cost.

[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 that are symmetrically arranged above and below a bag-shaped member containing a reaction solution and are connected via a connecting means so that they can slide vertically (see Patent Document 4). This invention uses a bag-shaped member made of polypropylene, and is configured to adjust the distance between a pair of metal plates even when the bag-shaped member is filled with reaction solution and the container is in an expanded state. In addition, a Peltier element is used as the temperature control unit.

[0006] Also disclosed is a method of carrying out emulsion PCR by placing a PCR reaction solution in a flexible bag and bringing the flexible bag into contact with an opposing thermal cycler (see Patent Document 5), and a method of amplifying nucleic acids in a cuvette in which the cuvette may be shaken to mix the components of the reaction solution contained in the cuvette (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 Application Publication No. 2017-063778 [Patent Document 4] Japanese Patent Application Publication No. 2018-139505 [Patent Document 5] U.S. Patent Publication No. 2010 / 0261230 [Patent Document 6] U.S. Patent Registration No. 5,229,297 Summary of the Invention [Problem to be solved by the invention]

[0008] 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 volume is 30 mL or larger, the time required to reach each set temperature in the PCR thermal cycle becomes longer. While short cycles are possible with small volumes, large volumes require conditions that can withstand temperature nonuniformity and long temperature rise and fall cycles while still allowing accurate DNA synthesis. The reason why conventional PCR reactions are typically performed in 50 μL is believed to be because it has been difficult to achieve a combination of reaction buffer and DNA polymerase that can withstand long-term temperature control and enable accurate PCR DNA synthesis, as well as temperature uniformity. Therefore, the present invention aims to provide a method for amplifying nucleic acids using a large volume of PCR reaction solution (30 mL or larger) and an apparatus for amplifying nucleic acids using a large volume of PCR reaction solution (30 mL or larger). [Means for solving the problem]

[0009] As a result of extensive research to solve the above problems, the present inventors discovered 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 thus completed the present invention.

[0010] That is, 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 a DNA polymerase, deoxyribonucleotide triphosphate (dNTP), template DNA, a forward primer, a reverse primer, and a buffer solution, 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, wherein the concentration of the buffer solution is 0.4 to 1.5 M. [3] The method according to [1] or [2] above, wherein the pH of the reaction solution is 8.5 to 10. [4] The method according to any one of [1] to [3] above, wherein the reaction solution is stirred by rocking the reaction vessel. [5] The method according to any one of the above [1] to [4], wherein the reaction vessel is made of an elastic material. [6] The method according to any one of the above [1] to [5], wherein the reaction vessel contains 1 to 90% gas. [7] The method according to any one of [1] to [6] above, 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 described in [7] above, characterized in that the temperature of the surface of the heat source attached 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 solution, and the temperature of the surface of the heat source attached to the reaction vessel. [9] A thermal cycler comprising a temperature control unit attached to one or more reaction vessels made of a thermally conductive material and containing 30 mL or more of reaction liquid to control the temperature of the reaction liquid, and an agitation unit for agitating the reaction liquid, wherein the temperature control unit comprises a heat source, a reaction vessel support plate that abuts against the top surface of the reaction vessel to support the reaction vessel, and a temperature sensor that measures the temperature of the outer surface of the reaction vessel or the temperature of the reaction liquid, and is for use in the nucleic acid amplification method described in any one of [1] to [8] above.

[10] The thermal cycler described in [9] above, characterized in that the heat source in the temperature control unit is a Peltier element, and the thermal cycler is equipped with a heat conduction plate that is adhered to the Peltier element and can abut against 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 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 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, and the temperature measuring part of the temperature sensor is arranged in the recess or through-hole without contacting the thermal conductive plate, so that when a reaction vessel is placed on the thermal conductive plate, the bottom surface of the reaction vessel and the temperature measuring part of the temperature sensor can abut.

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

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

[0011] The nucleic acid amplification method of the present invention enables nucleic acid amplification even when a large volume of PCR reaction solution of 30 mL or more is used. The thermal cycler of the present invention makes it possible to control the temperature of the reaction vessel or reaction solution so that nucleic acid can be amplified even when a large volume of PCR reaction solution of 30 mL or more is used. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a diagram showing an image of thermal cycles in general PCR. [Figure 2] FIG. 1 shows the results of SDS-PAGE analysis of a DNA polymerase Pfu solution in Example 1. [Figure 3] FIG. 1 shows the results of agarose electrophoresis of PCR products in Example 2. [Figure 4] FIG. 1 shows the results of agarose electrophoresis of PCR products in Example 3. [Figure 5] FIG. 1 shows the results of agarose electrophoresis of PCR products in Example 4. [Figure 6] FIG. 10 shows the results of agarose electrophoresis of PCR products in Example 5. [Figure 7] FIG. 10 shows the results of agarose electrophoresis of PCR products in Example 6. [Figure 8A] FIG. 8A is a schematic diagram of the temperature control unit of the thermal cycler used in Example 7. [Figure 8B] FIG. 8B is a diagram showing the autoclave bag placed on the thermal cycler of FIG. 8A, and the arrangement of the Peltier element and aluminum plate. [Figure 8C] FIG. 8C is a plan view showing the 200 μL reaction solution bag placed on the underlying autoclave bag. [Figure 9] The upper panel of Figure 9 shows the results of agarose electrophoresis of PCR products in Example 7. The lower panel of Figure 9 shows the results of investigating the temperature of the PCR thermal cycle in the 200 μL reaction solution bag at position 2 shown in Figure 8C. [Figure 10A] FIG. 10A is a bottom perspective view of the reaction solution bag in Example 8, with a temperature sensor attached to the center of the bottom surface thereof. [Figure 10B] FIG. 10B is a top perspective view of the state in which the temperature measuring portion 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 the PCR product when the reaction solution was 100 mL in Example 8. The lower panel of Figure 11A shows the results of examining the thermal cycle of PCR when the reaction solution was 100 mL. [Figure 11B] The upper panel of Figure 11B shows the results of agarose electrophoresis of the PCR product when the reaction solution was 200 mL in Example 8. The lower panel of Figure 11B shows the results of examining the thermal cycle of PCR when the reaction solution 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 was 300 mL in Example 8. The lower panel of Figure 11C shows the results of examining the thermal cycle of the model PCR when the reaction solution was 300 mL. [Figure 11D] FIG. 11D shows the results of investigating the PCR thermal cycle without shaking as a control in Example 8. [Figure 12] 10 shows the results of investigating the thermal cycle of PCR when shaking was performed using different shaking methods in Example 9. [Figure 13] In Example 10, the top left of Figure 13 shows the results of investigating the thermal cycle of PCR when PCR was performed with only downward temperature control. The top right of Figure 13 shows the results of investigating the thermal cycle of PCR when PCR was performed with both upward and downward temperature control. The bottom left of Figure 13 shows the results of investigating the thermal cycle of PCR when PCR was performed with a model 200 mL of reaction solution. The bottom center of Figure 13 shows the results of investigating the thermal cycle of PCR when PCR was performed with a model 300 mL of reaction solution. The bottom right of Figure 13 shows the results of investigating the thermal cycle of PCR when PCR was performed with a model 400 mL of reaction solution. [Figure 14] The upper part of Fig. 14 shows the results of agarose electrophoresis of PCR products in the case of a model with a 600 mL reaction solution in Example 11. The lower part of Fig. 14 shows the results of examining the PCR thermal cycle in the case of a model with a 600 mL reaction solution. [Figure 15]The upper part of Figure 15 shows the results of agarose electrophoresis of PCR products in the case of a model with a 1000 mL reaction solution in Example 11. The lower part of Figure 15 shows the results of examining the PCR thermal cycle in the case of a model with a 1000 mL reaction solution. [Figure 16] FIG. 1 is a front view of the entire first example of a thermal cycler. [Figure 17A] FIG. 17A is a front view of the temperature adjustment unit in the first example of the thermal cycler. [Figure 17B] FIG. 17B is a right side view of the temperature adjustment unit in the first example of the thermal cycler. [Figure 18] FIG. 1 is a plan view of a copper plate in a first example of a thermal cycler. [Figure 19A] FIG. 19A is a diagram showing the positions of the copper plate and the temperature sensor in the first example of the thermal cycler. [Figure 19B] FIG. 19B is a plan view of the first example of the thermal cycler, in which a reaction vessel with a temperature sensor attached thereto is placed on a copper plate. [Figure 20A] FIG. 20A is a front view of the reaction vessel support plate. [Figure 20B] FIG. 20B is a plan view of the reaction vessel support plate. [Figure 20C] FIG. 20C shows a side view of the reactor support plate. [Figure 21A] FIG. 21A is a diagram showing a state in which water vapor is generated and expands inside the reaction vessel. [Figure 21B] FIG. 21B is a view showing a state in which the reaction vessel support plate is fixed so as to provide a gap between the reaction vessel and the reaction vessel support plate. [Figure 22A] FIG. 22A is a front view of the temperature adjustment unit in the second example of the thermal cycler. [Figure 22B] FIG. 22B is a plan view of the reaction vessel support plate of the second example thermal cycler. [Figure 23] FIG. 10 is a front view of a third example thermal cycler. [Figure 24A]This is a diagram showing the positional relationship between a copper plate having a 1-cm diameter through-hole for installing a temperature sensor at the center and a Peltier element, in a mode where 8 Peltier elements are used on the lower surface in a thermal cycler. [Figure 24B] This is a diagram showing the positional relationship between a copper plate having a hollow portion with a length of 3 cm and a width of 3 cm at the center and a 1-cm diameter through-hole for installing a temperature sensor near the edge, and a Peltier element, in a mode where 8 Peltier elements are used on the lower surface in 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 triphosphate (dNTP), template DNA, forward primer, reverse primer, and buffer solution, wherein the reaction solution is 30 mL or more, and the polymerase chain reaction is performed while stirring the reaction solution. This method is also hereinafter referred to as "the nucleic acid amplification method of the present case".

[0014] Furthermore, 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, a temperature control unit that adheres to the reaction vessels to adjust the temperature of the reaction solution, and a stirring unit for stirring the reaction solution. The temperature control unit includes 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 that measures 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> As used herein, the term "polymerase chain reaction" refers to a method for amplifying template DNA by repeating 20 to 50 thermal cycles, including a series of steps consisting of thermal denaturation, annealing (primer binding), and DNA extension, in the presence of DNA polymerase, deoxyribonucleotide triphosphates (dNTPs), template DNA, forward primers, and reverse primers. Examples of polymerase chain reactions include quantitative polymerase chain reaction (qPCR), reverse transcription-polymerase chain reaction (RT-PCR), single-base extension reaction (SBE), multiplex single-base extension reaction (MSBE), and ligase chain reaction (LCR-PCR).

[0016] As used herein, nucleic acids include DNA and RNA. DNA may be single-stranded or double-stranded.

[0017] (DNA polymerase) The DNA polymerase used herein is preferably a thermostable DNA polymerase. A thermostable DNA polymerase refers to a DNA polymerase derived from a thermophilic bacterium, which retains at least half of its DNA polymerase activity even after heat treatment at 70°C for 1 minute or more.Such DNA polymerase may be of the pol I type or α type, and specifically includes those from Thermus aquaticus (Taq), Thermus thermophilus, Thermococcus litoralis, Thermococcus gorgonarius, Thermococcus kodakaraensis KOD1, Thermococcus cleftensis, Thermococcus eurythermalis, Thermococcus paralvinellae, Thermococcus sibiricus, Bacillus stearothermophilus, and the like. 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 Examples of suitable DNA polymerases include DNA polymerases such as E. kandleri, and a mixture of these may be used. Such DNA polymerases may be expressed in microorganisms such as Escherichia coli by genetic engineering, or may be commercially available products.Such DNA polymerases may be artificially synthesized by genetic engineering or derived from living organisms. The DNA polymerase may be expressed in a microorganism such as Escherichia coli by genetic recombination technology, extracted, and purified by ion chromatography or the like, if necessary, before use.

[0018] The concentration of DNA polymerase in the reaction solution can be 2000 to 10,000 U / μL, preferably 3000 to 8,000 U / μL, assuming that the ability to synthesize from 10 ng of DNA (DNA in which the polynucleotide encoding EMrfp in PSRLaP-EMrfp described in Nakamura et al. (Molecular Biotechnology 60, pages 912-923 (2018)) has been replaced with a 711-bp polynucleotide encoding EGFP consisting of the base sequence shown in SEQ ID NO: 1) per μL of DNA polymerase is 1 U / μL.

[0019] (deoxyribonucleotide triphosphate (dNTP)) The deoxyribonucleotide triphosphates (dNTPs) used herein may be a combination of dATP, dCTP, dGTP, and dTTP, and may contain dUTP instead of dTTP. The concentration of dNTPs can be adjusted appropriately depending on the length, amount, and sequence of the DNA to be amplified, but the concentrations of dATP, dCTP, dGTP, and dTTP are preferably each 0.01 to 0.6 mM, with lower limits of 0.02 mM, 0.05 mM, 0.1 mM, or 0.2 mM, and upper limits of 0.55 mM, 0.5 mM, or 0.45 mM.

[0020] (template DNA) As used herein, template DNA includes genomic DNA, cDNA, and plasmid DNA. The length of such template DNA can be 100 to 20,000 bp, with a lower limit of 200 bp, 300 bp, or 500 bp and an upper limit of 10,000 bp, 5,000 bp, or 3,000 bp. The template DNA may exist alone or may be incorporated into a vector such as a plasmid vector or a viral vector.

[0021] (Primer) In this specification, the forward primer and reverse primer may be any oligonucleotide that has a complementary property that allows it to hybridize with the template DNA and anneals to the region to be amplified in the template DNA or an upstream sequence adjacent to that region, thereby serving as a starting point for nucleic acid synthesis in PCR.

[0022] The length of the forward primer and reverse primer can be adjusted appropriately depending on the length of the DNA to be amplified, the amount of DNA, and the type of sequence, but can be 8 to 100 mer, with the lower limit being 12 mer, 15 mer, 20 mer, or 25 mer, and the upper limit being 80 mer, 60 mer, 50 mer, 45 mer, or 40 mer.

[0023] The forward primer and reverse primer may be modified, for example, labeled with avidin, a fluorescent substance, or the like, or may be a phosphorothioated or phosphoramidite derivative oligonucleotide.

[0024] (buffer) In this specification, the buffer solution is not particularly limited as long as it is a buffer solution that can be used in PCR reactions, and examples thereof include Tris-HCl buffer, Tris-acetate buffer, Bis-Tris buffer, HEPES buffer, MOPS buffer, etc. Such buffer solutions may be pH adjusted with hydrochloric acid or the like, or may contain EDTA or Mg 2+ or K +Metal ions such as HCl, HCl, HCl- ...

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

[0026] (Reaction solution) In this specification, the volume of the reaction solution is not particularly limited, but can be 30 mL or more, with a lower limit of 50 mL, 100 mL, 150 mL, 180 mL, 200 mL, 220 mL, 250 mL, or 300 mL, and an upper limit of 10 L, 5 L, 4 L, 3 L, or 2 L. Note that the volume of the reaction solution here refers solely to the total volume of reaction solution used to carry out a nucleic acid amplification reaction by PCR using the same thermal cycle. That is, the reaction solution may be contained in a single reaction vessel, or may be contained in two or more reaction vessels as long as the thermal cycle for each reaction vessel can be controlled under approximately the same temperature conditions. For example, the temperature of a single reaction vessel containing 30 mL or more of reaction solution may be controlled by placing it on a heat-conducting plate in contact with a heat source. Alternatively, three reaction vessels containing 10 mL of reaction solution may be prepared and placed on the heat-conducting plate so that the bottom of each of the three reaction vessels is attached to the heat-conducting plate, and the temperature in each reaction vessel may be controlled under the same conditions. The reaction solution may further contain dimethyl sulfoxide, bovine serum albumin, glycerol, heparin, trehalose, nonionic surfactants, ammonium sulfate, betaine, etc., to the extent that they do not inhibit the amplification of nucleic acids by PCR. The reaction solution may also contain a probe for detecting the amplification product. Examples of the probe include a TaqMan probe and a cycling probe.

[0027] Furthermore, the reaction solution may contain labels such as dyes that bind to phosphate groups on the DNA surface or dyes that intercalate between bases for the detection and monitoring of amplification products. Examples of dyes that bind to phosphate groups on the DNA surface or dyes that 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 heat denaturation is followed by annealing and extension reactions at the same temperature, or a three-step cycle in which heat denaturation is followed by annealing and extension reactions at different temperatures.

[0029] In this specification, the temperature for thermal denaturation in the polymerase chain reaction may 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 time for thermal denaturation may 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 may 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 may 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] Herein, the temperature for the extension reaction in the polymerase chain reaction may 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 time for the extension reaction may 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] Herein, the temperature for the annealing and extension reaction when carrying out a polymerase chain reaction in a two-step cycle can be 55 to 80° C., with a lower limit of 55, 60, or 62° C. and an upper limit of 72, 70, or 68° C. The time for the annealing and extension reaction can be 20 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.

[0033] The thermal cycles for thermal denaturation, annealing, and extension reactions can be 20 to 50 cycles, with a lower limit of 22 or 24 cycles and an upper limit of 40, 38, or 36. The time for one cycle, i.e., the time for each step in one cycle of thermal denaturation, annealing, and extension reactions and the temperature adjustment time, can be 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 example of two steps of the polymerase chain reaction is as follows. (1) Initial heat denaturation: 90°C for 10 seconds ↓ (2) Heat denaturation: 90 or 92°C for 10 or 30 seconds (3) Annealing and extension reaction: 60 or 65°C, 2.5 minutes (4) (2) and (3) for 30 cycles

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

[0036] (Reaction vessel) The reaction vessel may be any shape, such as a bag, box, cylinder, or plate, as long as it can accommodate the reaction liquid. However, a flat, bag-like shape is preferred from the viewpoint of heat conduction efficiency. The reaction vessel may be sealed or partially open. Furthermore, the reaction vessel may be provided with an opening for introducing the reaction liquid, and after the reaction liquid is introduced, the opening may be closable to prevent leakage of the reaction liquid. Examples of methods for closure include heat sealing and bonding with an adhesive.

[0037] The material of the reaction vessel may be any material having thermal conductivity that allows heat from the heat source to be conducted to the reaction solution through the reaction vessel. Examples of such materials 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 may 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 may be used. When using two or more reaction vessels simultaneously, it is preferable to use reaction vessels made of the same material in order to uniformly control the reaction solution.

[0038] The reaction vessel preferably has elasticity in addition to the above-mentioned thermal conductivity. Elasticity improves adhesion between the reaction vessel and the heat source or the heat-conducting plate attached to the heat source, thereby increasing heat conduction efficiency. Elasticity also allows the reaction vessel to expand when water vapor is generated during the thermal denaturation stage, and allows the expanded reaction vessel to contract during the annealing and elongation reaction stages. Because the temperature during the thermal denaturation stage can reach 95°C or higher, the reaction vessel is preferably made of a material that can withstand high temperatures of 95°C, preferably 100°C, i.e., a material that will not cause the reaction solution to leak due to damage to the reaction vessel caused by high temperatures.

[0039] The reaction vessel may be provided with a temperature sensor holder for holding a temperature sensor to facilitate measurement of the temperature of the outer surface of the reaction vessel or the temperature of the reaction solution. Examples of such a temperature sensor holder include a pocket-shaped holder formed on the outer surface of the reaction vessel, with a temperature measuring part housed in the pocket and capable of being enclosed by the reaction vessel.

[0040] (Stirring of reaction solution) In the present specification, the method for stirring the reaction solution is not particularly limited, but examples thereof include a method of shaking the reaction vessel itself, a method of providing a stirrer such as a propeller stirrer or a jet stirrer in the reaction vessel, and a method of irradiating the reaction solution with ultrasound.

[0041] Examples of methods for shaking a reaction vessel include those using a shaker. Examples include fixing or placing the reaction vessel directly or indirectly above the shaker so that the shaking motion generated by the shaker is transmitted to the reaction vessel. The method for fixing or placing the reaction vessel is not particularly limited, and examples include fixing or placing the reaction vessel on the shaker plate of the shaker or on a shaking shaft fixed to the shaker plate and transmitting the shaking motion. Furthermore, the reaction vessel can be shaken by rotational or reciprocating motion in the shaker, and the direction of motion can be horizontal or vertical, or a combination of these. In the case of rotational motion, the rotation speed can be, for example, 10 to 200 rpm, or 50 to 100 rpm. Note that other units may be placed between the reaction vessel and the shaker plate as long as they do not interfere with the shaking of the reaction vessel. For example, a heat source, a heat conduction plate, or the like, described below, may be placed between the reaction vessel and the shaker plate.

[0042] In the method of providing an agitator such as a propeller agitator or a jet agitator in a reaction vessel, examples of the agitator include a propeller agitator or a jet agitator. In this case, the propeller agitator or the jet agitator may be positioned in the reaction vessel while sealing the reaction vessel to prevent the reaction liquid from leaking out of the reaction vessel.

[0043] Another method for stirring the reaction solution is to place a magnetic stirrer in the reaction vessel and use a magnetic force from outside the reaction vessel to stir the solution. The magnetic stirrer may be rod-shaped or elliptical, and may be coated with a fluororesin or the like, as long as it is magnetic. The magnetic stirrer rotates or slides due to the magnetic force from outside the reaction vessel, thereby stirring the reaction solution.

[0044] When stirring the reaction solution by rocking the reaction vessel, it is preferable to contain a predetermined amount of reaction solution in the reaction vessel from the viewpoint of improving stirring efficiency. The amount of reaction solution can be adjusted appropriately depending on the bottom area of ​​the reaction vessel, but the height of the reaction solution in the reaction vessel when the reaction vessel is left standing, i.e., the thickness in the vertical direction, is preferably 2 mm or more, and may be 3 mm or more, 4 mm or more, or 5 mm or more.

[0045] When using a sealed reaction vessel as a reaction vessel, it is preferable that the reaction vessel contain 1 to 90% gas before the PCR reaction begins. 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, when the reaction vessel is filled with reaction solution and closed. Considering heat transfer efficiency, it is preferable for the reaction vessel to be sealed with reaction solution without containing gas. However, if a large volume of reaction solution is filled in the reaction vessel, stirring the reaction solution by shaking the reaction vessel itself, for example, is inefficient. Therefore, by containing a predetermined amount of gas in the reaction vessel, shaking the reaction vessel will move the gas more quickly within the reaction vessel, resulting in improved stirring efficiency of the reaction solution. Furthermore, containing gas makes it easier to prevent leakage of reaction solution when removing the reaction solution from the reaction vessel after the PCR reaction. The gas is not particularly limited as long as it does not inhibit the PCR reaction, and examples include air, carbon dioxide, nitrogen, etc. Instead of directly containing a gas, a bag containing a gas and / or liquid may be contained in the reaction vessel. Furthermore, to improve the stirring efficiency of the reaction solution, a stirring aid that moves freely in the reaction solution, such as beads, may be contained in the reaction vessel.

[0046] (Reaction vessel temperature control) The temperature of the reaction vessel can be controlled by using a thermal cycler equipped with a temperature control unit that can control the temperature of the reaction vessel by contacting at least a part of the reaction vessel, and it is preferable to use the thermal cycler of the present invention. Below, we will explain 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 supports the reaction vessel by contacting the top surface of the reaction vessel, 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, the reaction vessel may be contacted with the heat source directly or indirectly via a heat conduction plate to heat or cool the reaction solution. The heat source may be contacted at one or more locations on the bottom, side, or top surface of the reaction vessel, or on the bottom surface only, the bottom and top surfaces, or all or part of the bottom and side surfaces. When the heat source is contacted with the top surface, the heat source may be provided symmetrically above and below the reaction vessel, as described in Patent Document 4, for example. During a PCR reaction, water vapor may be generated in the PCR reaction solution during the thermal denaturation stage, causing gas to accumulate in the upper part of the reaction vessel. Therefore, from the viewpoint of temperature control of the PCR reaction solution and heat conduction efficiency, it is preferable that the heat source be contacted with at least the bottom surface of the reaction vessel, but it may also be contacted with the bottom surface only.

[0048] The reaction vessel is preferably supported by a reaction vessel support plate on the top surface of the reaction vessel, i.e., the surface opposite the bottom surface of the reaction vessel. The material of the reaction vessel support plate is not particularly limited, but examples include metals such as aluminum and copper, and plastics. Alternatively, two heat sources may be provided, one of which serves as the reaction vessel support plate, and the reaction vessel may be sandwiched between the top and bottom heat sources.

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

[0050] The heat source is not particularly limited, as long as it contains a heat source capable of absorbing heat and generating heat, and examples of such a heat source include heaters such as Peltier elements and electric heaters. The heat source can be adjusted appropriately depending on the amount of PCR reaction solution, and there may be one or more, 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 one reaction vessel, but if the reaction vessel has a large area, multiple heat sources may be arranged horizontally side by side so that the outer surface of the reaction vessel can absorb heat and heat in a shorter time.

[0051] Furthermore, the heat source such as the Peltier element or heater may be in direct contact with the reaction vessel, or a heat conductive plate may be provided on the surface in contact with the reaction vessel, and heat emitted from the heat source is transferred to the reaction solution via the reaction vessel via the heat conductive plate. From the viewpoint of thermal conductivity, it is preferable that the Peltier element and the heat conductive plate are bonded with a heat conductive filler such as heat conductive grease.

[0052] The heat conduction plate may be made of a material with high thermal conductivity, such as aluminum, copper, silver, or gold. Furthermore, the heat conduction plate is preferably plate-shaped. However, to improve the stirring efficiency of the reaction vessel and the contact area with the reaction vessel, the surface that contacts the reaction vessel may have protrusions, depressions, irregularities, a fan-like shape, or a slope. The thickness of the heat conduction plate can be adjusted appropriately depending on the material, but may be 0.1 mm to 5 cm, or may be 0.5 mm to 3 cm, or 1 mm to 1 cm. The length and width of the heat conduction plate can be adjusted appropriately depending on the length and width of the reaction vessel and the number of heat sources. 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 may be square, rectangular, or elliptical. From the standpoint of temperature control, the heat conduction plate preferably contacts at least 50% of the bottom surface of the reaction vessel, and may be at least 60%, 70%, 80%, 90%, or 95%. Since the reaction liquid is agitated, the area where the heat conduction plate is not in contact with the reaction liquid through the reaction vessel 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] The heat conduction plate preferably has a recess or through-hole to prevent direct heat transfer from the heat source when a temperature sensor measuring the temperature of the outer surface of the reaction vessel is placed thereon. The shape of the recess or through-hole may be circular, square, rectangular, elliptical, conical, or other shapes. The size of the recess or through-hole (or its minor axis in the case of a rectangular or elliptical shape) is not particularly limited as long as the temperature sensor's temperature sensor does not come into contact with the heat conduction plate. For example, the recess or through-hole is preferably sized so that the shortest distance between the temperature sensor's temperature sensor and the heat conduction plate is 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, or 15 mm or more. In other words, the radius or minor axis of the recess or through-hole may 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, or 15 mm or more. The recess or through-hole may be located anywhere on the heat conduction plate, but is preferably located near the center. Furthermore, instead of providing a recess or a through hole, an insulating sheet such as a sheet made of silica aerogel may be attached to the heat conductive plate, and the temperature measuring part of the temperature sensor may be placed on the insulating sheet, or the above insulating sheet may be used to attach the temperature measuring part of the temperature sensor to the reaction vessel.

[0054] Furthermore, the heat conducting plate may be provided with a hollow portion to improve the efficiency of heat radiation from the reaction vessel. The area of ​​such a hollow portion is not particularly limited, but for example, it may be 1 cm 2 ~9cm 2 Yes, with a lower limit of 1.5cm 2 , 2cm 2 The upper limit is 8cm 2 , 5cm 2 or 4 cm 2 In order to prevent the reaction vessel from sinking, the hollow portion may have a mesh-like, lattice-like or linear hollow shape as required.

[0055] The Peltier element may be provided with a heat sink, fins, a fan, and / or a water cooling unit on the surface opposite to the surface in contact with the reaction vessel. It is preferable that the Peltier element and the heat sink are in close contact with each other using thermally conductive grease.

[0056] The temperature adjustment unit is connected to a power control unit, a calculation unit, a current control unit including 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 measuring part is located inside the reaction vessel and the temperature measuring part 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 a 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 that measure the temperature of the outer surface of a reaction vessel include thin temperature sensors that can be attached to the outer surface of a reaction vessel, probe-type temperature sensors with a temperature measuring part at the tip, and radiation temperature sensors such as infrared temperature sensors that can measure the outer surface of a reaction vessel. Probe-type temperature sensors may also be used that are flexible and retractable using a spring or the like. When a through-hole is provided in the heat conduction plate, the temperature measuring part of the probe-type temperature sensor is positioned in the through-hole, and when the reaction vessel is placed on the heat conduction plate, the temperature of the outer surface of the reaction vessel can be measured by bringing the temperature measuring part into contact with the outer surface of the reaction vessel. Note that when using a sterilized reaction solution, or when maintaining the sterility of the reaction solution, it is preferable to use a reaction vessel temperature sensor or a heat conduction plate temperature sensor.

[0059] Examples of reaction solution temperature sensors that measure the temperature of the reaction solution by directly contacting the reaction solution with a temperature measuring part located inside the reaction vessel include wireless temperature sensors and rod-shaped temperature sensors that can be placed so that the temperature measuring part penetrates the reaction vessel and is located inside the reaction solution. When using a wireless temperature sensor, the wireless temperature sensor can be placed in the reaction solution as is or after being treated to be waterproof and / or heat resistant.

[0060] The number of temperature sensors may be one or more. Furthermore, when using the reaction vessel temperature sensor, the position in contact with the reaction vessel may be on the outer surface of the reaction vessel, as long as it is in contact with the reaction solution via the reaction vessel, and the bottom and / or side surfaces are preferred. In the case of the bottom, the position is not particularly limited, and may be the center or periphery. Conventional PCR temperature control has mainly been performed based on the temperature measured by a heat conduction plate temperature sensor. This is thought to be because, since the reaction solution is typically approximately 10 to 50 μL, heat from the heat source or heat conduction plate in contact with the reaction vessel is easily conducted to the entire 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, to more easily 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] Information on the temperature detected by the temperature sensor is sent to the current control unit, and then the current to be supplied to the heat source such as a Peltier element from the power control unit is calculated in the calculation unit. That is, 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 such as a Peltier element or the heat conduction plate by adjusting the current supplied to the heat source such as a Peltier element based on data on the temperature of the outer surface of the reaction vessel and / or the heat conduction plate detected by the temperature sensor. In order to suppress overshoot or undershoot in temperature control, it is preferable to perform control using PID control or the like.

[0062] The calculation unit pre-sets the thermal cycle conditions, such as the temperature, time, and number of thermal cycles for each of the thermal denaturation, annealing, and extension reactions. The current is then controlled to heat the reaction solution when the temperature of the outer surface of the reaction vessel or the reaction solution is lower than the set temperature, and cool it when it is higher than the set temperature. Large-volume PCR is prone to temperature variations in the reaction solution. Specific nucleic acid amplification is impossible without precise control of the reaction temperature and reaction time for each step of the thermal cycle shown in Figure 1. During the thermal denaturation step, if the temperature is lower than the set temperature, the template DNA cannot be denatured. If the temperature is higher than the set temperature for a long period of time, components contained in the reaction solution, such as DNA polymerase, will denature. Furthermore, during annealing, if the temperature is lower than the set temperature, nonspecific binding will increase, and if the temperature is higher, the primers will be dissociated from the template DNA. Furthermore, if the temperature is lower or higher than the set temperature during the extension reaction, the function of the DNA polymerase will be impaired. Regarding reaction time, the longer the time per thermal cycle, the more likely DNA polymerase will be inactivated. Therefore, it is extremely important to accurately control the reaction solution to the set temperature in a short time.

[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 satisfies each condition 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 is excessively higher or lower than the set temperature, i.e., so that overshoot or undershoot does not occur when the temperature rises or falls. Conventional thermal cyclers generally perform nucleic acid amplification using reaction solutions of 50 μL or less, where heat conduction occurs rapidly, and therefore reaction vessel temperature control has been widely performed 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 reaction solution is large, differences in temperature between the heat source or heat conduction plate and the outer surface of the reaction vessel are likely to occur. Therefore, using the above two sensors makes it possible to control the temperature closer to the temperature of the reaction solution.

[0064] In the polymerase chain reaction, during the transition from thermal denaturation to annealing, during the transition from annealing to extension, and / or during the transition from extension to thermal denaturation, the temperature increase or decrease of the outer surface of the reaction vessel or the reaction solution can be controlled to 0.05°C / second or more. Such temperature increase or decrease can be appropriately adjusted depending on the size of the reaction vessel, the amount of the reaction solution, etc. The temperature increase rate can be, for example, 0.06 to 6°C / second, with a lower limit of 0.08, 0.1, 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, 0.5, 0.2, or 0.1°C / second, but is not particularly limited. The temperature decrease rate is, for example, 0.1 to 6°C / sec, and the lower limit may be 0.2, 0.3, 0.4, or 0.5°C / sec, and the upper limit may be 5, 4, 3, 2, 1.5, 1.2, 1, 0.8, or 0.7°C / sec, but is not particularly limited. Furthermore, the rate of temperature decrease is preferably faster than the rate of temperature increase, and 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] The temperature control unit may be provided in multiple units, one for each temperature set in the PCR thermal cycle. For example, a temperature control unit may be provided for each step of thermal denaturation, annealing, and extension reaction, and temperature control may be performed by the temperature control unit for each step. Specifically, when a PCR reaction is performed in three steps, four units may be provided: a temperature control unit A for thermal denaturation, a temperature control unit B for annealing, a temperature control unit C for extension reaction, and a preheating or cooling temperature control unit D for increasing or decreasing the temperature of the reaction vessel to the set temperature for the next step in a short period of time, and the temperature may be controlled in the following cycle:

[0066] 1. Heat denaturation for a specified time in the temperature control unit A for heat denaturation, which is adjusted to the set temperature for heat denaturation. 2. The reaction vessel is transferred to a preheating or cooling temperature control unit D, which has been adjusted to a temperature lower than the annealing temperature, for example, 0°C. 3. When the temperature of the reaction vessel approaches the set temperature for the next step, i.e., annealing, the reaction vessel is moved to annealing temperature control unit B, which is adjusted to the annealing temperature. 4. Anneal for the specified time in annealing temperature control unit B 5. The reaction vessel is transferred to a preheating or cooling temperature control unit D adjusted to a temperature higher than 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, the reaction vessel is transferred to the extension reaction temperature control unit C, which is adjusted to the temperature of the extension reaction. 7.Extension reaction in temperature control unit C for the specified time. 8. Transfer the reaction vessel to a preheating or cooling temperature control unit D, which is 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, i.e., the thermal denaturation temperature, the reaction vessel is transferred to the thermal denaturation temperature control unit A, which is adjusted to the thermal denaturation temperature.

[0067] By performing a predetermined cycle consisting of the above steps 1 to 9 as one cycle, it becomes possible to carry out a PCR reaction while controlling the temperature more quickly and accurately. Since heating can control the temperature of the reaction vessel in a shorter time than cooling, step 8 above may be omitted and step 9 may be replaced with step 9', "transferring the reaction vessel to a temperature control unit A for thermal denaturation, which is adjusted to the temperature for thermal denaturation." Furthermore, since the set temperatures for the annealing and extension reactions are close, step 5 above may be omitted and step 6 may be replaced with step 6', "transferring the reaction vessel to a temperature control unit C for extension reaction, which is adjusted to the temperature for the extension reaction."

[0068] The temperature between each step can be increased or decreased in a short time by using a preheating or cooling temperature control unit, which not only shortens the overall reaction time of the PCR reaction but also makes it possible to suppress the deterioration of the functionality of components contained in the reaction solution, such as DNA polymerase. The reaction solution can be stirred by providing a support base that supports all the temperature control units and rotating or reciprocating the support base using a shaker or motor or the like, or by using an agitator, stirrer, shaking mechanism, ultrasonic wave, or the like for each temperature control unit.

[0069] Similarly to the above, when performing a two-step PCR reaction, three units can be 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 can be controlled in the following cycle: 1. Heat denaturation for a specified time in the temperature control unit A for heat denaturation, which is adjusted to the set temperature for heat denaturation. 2. The reaction vessel is transferred to a preheating or cooling temperature control unit D, which has been adjusted to a temperature lower than 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, i.e., the annealing and extension reaction, the reaction vessel is transferred to the annealing and extension reaction temperature control unit B, which is adjusted to the temperature of the annealing and extension reaction. 4. Annealing and extension reactions are carried out for a predetermined time in the temperature control unit B for annealing and extension reactions. 5. Transfer the reaction vessel to a preheating or cooling temperature control unit D, which is 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, i.e., the thermal denaturation temperature, the reaction vessel is transferred to the thermal denaturation temperature control unit A, which is adjusted to the thermal denaturation temperature. By performing a predetermined cycle consisting of the above steps 1 to 6 as one cycle, it becomes possible to carry out a PCR reaction while controlling the temperature more quickly and accurately. Note that, because heating can control the temperature of the reaction vessel in a shorter time than cooling, step 5 above may be omitted, and instead of step 6, the process may proceed to step 6', "transferring the reaction vessel to a temperature control unit A for thermal denaturation adjusted to the temperature for thermal denaturation."

[0070] (thermal cycler) The temperature control unit of the present thermal cycler has been described above, but a more detailed description will now be given. First, the reaction vessel temperature control unit includes a heat source, a reaction vessel support plate that abuts against the top surface of the reaction vessel to support the reaction vessel, 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. A typical example is one in which multiple heat sources are arranged in parallel horizontally, and a heat conduction plate on which the reaction vessel is placed is attached above them with thermally conductive grease. A certain space is provided above the heat conduction plate to accommodate the reaction vessel, and the reaction vessel support plate is arranged above this space. A vertical anti-slip plate may be installed at the edge of the heat conduction plate to prevent the reaction vessel from shifting due to rocking of the reaction vessel.

[0071] The reaction vessel support plate is provided horizontally above the reaction vessel, and the distance from the heat source or the heat conductive plate adhered to the heat source can be adjusted appropriately depending on the size and capacity of the reaction vessel used. The reaction vessel support plate may be structured to be supported so as to abut against the reaction vessel by its own weight, or may be fixed or maintained in a position abutting the reaction vessel using a screw jack, wire, or the like. Alternatively, a shaft may be provided to support the reaction vessel support plate, and the shaft may be fixed or maintained by an expandable mechanism such as a cylinder, spring, or the like, such as an air cylinder or a robot cylinder. Furthermore, the reaction vessel support plate may be positioned so that a predetermined pressure can be applied to the reaction vessel to improve adhesion between the reaction vessel and the heat source or the heat conductive plate.

[0072] The reaction vessel support plate may also be equipped with a fixing or maintaining means capable of fixing or maintaining the reaction vessel support plate at a predetermined distance from the reaction vessel, for example, a distance of at least 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 7 cm, or 10 cm. With this configuration, a gap may be provided between the reaction vessel support plate and the reaction vessel, for example, during the temperature drop step of the thermal cycle, i.e., the step between thermal denaturation and annealing. By providing such a gap during the temperature drop step, the upper surfaces of the reaction vessels are exposed to air, thereby improving the efficiency of heat dissipation from the reaction vessels and shortening the time per thermal cycle. The means for fixing or maintaining the reaction vessel support plate above the reaction vessels at a predetermined distance are not particularly limited, but examples include a screw jack or wire that can be automatically raised and lowered in accordance with the thermal cycle, a cylinder such as an air cylinder or a robotic cylinder, or a cam mechanism. Alternatively, when the reaction vessel support plate is supported by its own weight using a shaft supporting the plate or when a wire is used, a stopper such as a set collar can be provided on the shaft supporting 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 contracts during cooling.

[0073] From the viewpoint of heat conduction efficiency to the reaction vessel, the heat source and the heat conduction plate are preferably connected by screws or the like. 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. Furthermore, the reaction vessel support plate above may be supported by such a heat source support shaft.

[0074] Next, examples of the stirring unit include those capable of shaking the reaction vessel, and for example, a shaker can be used. 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 shake the reaction vessel. Examples of the shaker include shakers capable of rotary shaking and reciprocating shaking. In the case of rotary shaking, the rotation speed is as described in the above section "Agitation of the reaction solution."

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

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

[0077] There are no particular limitations on the connection between the temperature control unit and the stirring unit, but for example, when a shaker is used as the stirring unit, a method can be used in which a vertical shaking shaft is provided on the shaking table and the temperature control unit is fixed to the shaking shaft.If a Peltier element is used as the heat source and a cooling fan is provided, a predetermined distance, preferably 3 cm or more, can be left between the cooling fan and the shaking table so that the cooling fan does not come into close contact with the shaking table. [Example]

[0078] The present invention will be explained in more detail 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, the DNA polymerase used was Pfu DNA polymerase expressed in Escherichia coli, and commercially available products PrimeSTAR (registered trademark) GXL (Takara Bio Inc.) and KOD Fxneo (Toyobo Co., Ltd.). The Pfu DNA polymerase expressed in Escherichia coli was prepared by the following method.

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

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

[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 with the nucleotide sequence shown in SEQ ID NO: 1 was used, and 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 Forward Primer 0.3 μM Reverse Primer 0.3 μM 2×KOD FX neo Buffer 5.0 μL / 10 μL KOD FX neo DNA Polymerase 0.2 μL / 10 μL

[0085] ■PCR Reaction Solution Containing Pfu Template DNA 0.1 ng / μL dNTP 0.4 mM Forward primer 0.5 μM Reverse primer 0.5 μM Tris-HCl buffer 0.15M DNA polymerase Pfu 0.2μL / 10μL MgSO4 2.5mM DMSO 5%

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

[0087] The results of agarose electrophoresis of the obtained PCR products are shown in Figure 3. As is clear from Figure 3, almost no nucleic acid amplification was observed when the reaction solution exceeded 150 μL for either enzyme. Furthermore, by extending the thermal denaturation, annealing, and extension reaction times, slight amplification was observed at 200 μL. However, when the thermal denaturation, annealing, and extension reaction times were extended, smeared bands (unclear bands that continuously extend up and down) were observed, confirming the occurrence of nonspecific nucleic acid amplification. Most widely used PCRs to date have been performed using a maximum of 50 μL, which is thought to be due to the fact that amplification efficiency and specificity decrease when volumes exceed 50 μL, especially 150 μL.

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

[0089] The results of agarose electrophoresis of each PCR product are shown in Figure 4. As shown in Figure 4, it was clear that in all cases, the nucleic acid amplification efficiency was higher at alkaline temperatures of pH 7.9 or higher, and that the nucleic acid amplification efficiency was higher when the annealing and extension reactions were performed at 72°C.

[0090] [Example 4] Examination 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 Tris-HCl buffer concentration was 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: 30 cycles were performed. Heat denaturation: 98℃ 2 minutes, annealing and extension reaction: 72℃ 2 minutes

[0091] The results of agarose electrophoresis of each PCR product are shown in Figure 5. As shown in Figure 5, when the buffer concentration was increased, the bands became clearer and nonspecific amplification was reduced, and it was clear that amplification efficiency was good on the alkaline side of 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, the relationship between PCR reaction time and buffer concentration was investigated. PCR was performed using the same Pfu-containing PCR reaction solution as in Example 2, except for the buffer concentration. The PCR reaction solution was 10 μL, and the reaction temperature, time, and buffer concentration were varied at the eight different concentrations listed in Table 1 below.

[0093] [Table 1]

[0094] The results of agarose gel electrophoresis of each PCR product are shown in Figure 6. As shown in Figure 6, when the buffer concentration was 0.15 M, the nucleic acid amplification efficiency was significantly reduced when the thermal denaturation time or annealing and extension reaction time was 60 seconds, as shown in (e) or (g). On the other hand, when the buffer concentration was 0.6 M, the nucleic acid amplification efficiency was poor when the annealing and extension reaction time was as short as 10 seconds, as shown in (b). However, the nucleic acid amplification efficiency improved when the annealing and extension reaction time was 30 seconds or longer, as shown in (d), (f), and (h). When the reaction solution was large, the annealing and extension reaction times inevitably became longer than those for normal reaction solution volumes due to temperature control in the PCR thermal cycle. From these results, we found that although large-volume PCR was difficult with the currently widely used 0.15 M buffer concentration, increasing the buffer concentration could solve the problem of long annealing and extension reaction times, which is unique to large volumes.

[0095] [Example 6] Investigation of PCR reaction time and buffer concentration - 2 Based on the results of Examples 3 to 5, the relationship between PCR reaction time and buffer concentration was further investigated. PCR was performed using the same Pfu-containing PCR reaction solution 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 solution was 10 μL, and the reaction times and temperatures were 90°C for 10 seconds for heat denaturation, and 65°C for annealing and extension reactions for 1, 2, 3, 5, 10, or 20 minutes.

[0096] The results of agarose electrophoresis of each PCR product are shown in Figure 7. 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 was 5 minutes or longer, and the bands became smeared and nonspecific amplification was observed, and nucleic acid amplification did not occur after 20 minutes or longer. By using a buffer solution of 0.3 M or higher, nucleic acid was amplified even when the annealing and extension reaction time was 20 minutes or longer, and nonspecific amplification decreased as the buffer concentration increased.

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

[0098] [Example 7] Up until Example 6 above, microtubes were used as reaction vessels to investigate various reaction solutions or reaction conditions in PCR reactions. However, microtubes cannot accommodate large volumes of reaction solution. Therefore, in order to perform large-volume PCR, plastic bags were used as reaction vessels.

[0099] As a model reaction vessel containing 50 mL of PCR reaction solution, two polypropylene autoclave bags (35 μm thick, 22.5 cm long, 10 cm wide: Bioramo) were prepared by filling them with 50 mL of water and heat-sealing them to prevent air from entering. These two bags were stacked on top of each other, and a 35 μm thick, 3 cm long, 3 cm wide polypropylene autoclave bag (hereinafter referred to as the "200 μL reaction solution bag") containing 200 μL of Pfu reaction solution was sandwiched between them and placed in a thermal cycler. PCR was performed using Thermal Cycler 1, an improved version of the thermal cycler described in Patent Document 4, as shown in Figure 8A. Briefly, the temperature control unit of the thermal cycler 1 comprises air-cooled Peltier elements 13 (two on the bottom and two on the top) equipped with heat sinks 11 and cooling fans 12, an aluminum plate 14 with a 10 cm short axis and a 22.5 cm long axis secured to the Peltier elements 13 by 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 two-layered autoclave bags 15 were sandwiched between a pair of upper and lower Peltier elements 13 and 3 mm thick aluminum plates 14, and the resulting bag was placed on the thermal cycler 1. The 200 μL reaction solution bags 16 were sandwiched between the upper and lower autoclave bags 15 at five locations indicated by 1 to 5 in Figure 8C. The temperature measuring part 18 of an adhesive-type thin thermocouple temperature sensor 17 (Rika Kogyo Co., Ltd.) was attached to the bottom of the 200 μL reaction solution bag 16 at position No. 2, and the detected temperature information of the outer surface of the 200 μL reaction solution bag was sent to the current control part, which controlled the current to the Peltier element 13 to adjust the temperature. A GL840 (Graphtec Co., Ltd.) temperature data logger was used. The 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 carried out as follows. (1) Heat denaturation: 90℃ for 10 seconds (2) Heat denaturation: 90°C for 30 seconds (3) Annealing and extension reaction: 65°C, 2.5 minutes (4) Repeat steps (2) and (3) for 30 cycles.

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

[0102] The results of agarose gel electrophoresis of each PCR product are shown in the upper panel of Figure 9. In Figure 9, C represents 1 μL of PCR reaction solution when PCR was performed in a 200 μL microtube containing 10 μL of reaction solution as a control for the conventional method. 1 to 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 acid was amplified in the 200 μL reaction solution bags placed at positions 2 to 4 shown in Figure 8C, but not in the 200 μL reaction solution bags placed at positions 1 and 5. This confirmed that the temperature within the reaction solution bag was not uniform during the thermal cycle. Furthermore, the results of examining the PCR cycle temperature for the 200 μL reaction solution bag placed 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 (the 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 panel of Figure 9, the thermal denaturation temperature, annealing temperature, and extension reaction temperature fluctuated between 63 and 69°C, indicating that the set temperature could not be maintained and the temperatures varied. These results suggest that when a large amount (50 mL) is used, the liquid temperature fluctuates, resulting in reduced efficiency of thermal denaturation and annealing and extension reactions.

[0103] [Example 8] Based on the results of Example 7, it is expected that if the volume of PCR reaction solution is increased, it will be necessary to homogenize the PCR reaction solution in the PCR reaction solution bag and adjust the set temperatures in the thermal denaturation, annealing, and extension reaction stages. Therefore, we attempted to determine whether stirring the PCR reaction solution would solve the above problem.

[0104] In Example 7, two bags containing 50 mL of water were stacked. In this example, however, (1) 100 mL of PCR reaction solution was stored in one reaction solution bag, (2) 200 mL of PCR reaction solution was stored in one reaction solution bag, and (3) 300 mL of PCR reaction solution was stored in a single reaction solution bag. (1) To (3), 300 mL of water was stored in one reaction solution bag, and a plastic bag containing 200 μL of PCR reaction solution was placed inside the reaction solution bag. Air was added to each of the reaction solution bags (1) to (3) so that the air volume 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 table of a shaker (Rotary Shaker SR-300: Shimadzu Corporation). The shaking table and the thermal cycler were then secured so that the rotational shaking of the shaker was transmitted to the reaction bags placed in the thermal cycler. PCR was performed while stirring the reaction solution by rotating the reaction vessel at 100 rpm. As a control without shaking, one bag containing 50 mL of reaction solution was placed in the thermal cycler and PCR was performed without shaking. The composition of the PCR reaction solution 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 of the reaction solution bag 16. Furthermore, as shown in Figure 10B, the reaction solution bag 16 was placed on an aluminum plate 14, which is a heat-conducting plate, so that the temperature measuring part 18 of the temperature sensor was positioned at the center of a through-hole 19 provided in the aluminum plate 14. In the thermal cycler, a copper plate was used instead of the aluminum plate 14 only when the reaction solution was 300 mL.

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

[0106] [Example 9] Although rotary shaking was used in Example 8, other types of shaking were also investigated. PCR was performed in the same manner as in Example 8, except that no shaking, rotary shaking at 100 rpm, reciprocal shaking in the long axis direction at 50 rpm or 100 rpm, and reciprocal shaking in the short axis direction at 50 rpm or 100 rpm were used, and a reaction solution 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, with rotary shaking at 100 rpm, the time per 5 cycles was short at 1711 seconds. Furthermore, it was confirmed that rotary shaking at 100 rpm or short axis shaking at 100 rpm allowed the temperature to be controlled closer to the set temperature.

[0107] [Example 10] As shown in Examples 8 and 9, shaking the reaction solution bag enabled temperature control closer to the set temperature. Here, when the PCR reaction solution contains 100 mL or more, water vapor may be generated within the reaction solution bag during the thermal denaturation stage and before and after, causing the reaction solution bag to expand and air to accumulate above. Even if a heat source is installed above the reaction vessel, temperature control from above is inefficient because heat is transferred to the PCR reaction solution via the air. Therefore, considering the simplification of the thermal cycler structure and the reduction of overall temperature variation by agitating the PCR reaction solution through shaking, we attempted to determine whether nucleic acid amplification could be achieved by temperature control solely via 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 solution bag containing 200 mL of Pfu-containing PCR reaction solution, with rotary shaking at 100 rpm. The thermal cycler used was the same as shown in Figure 8A, except that 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 solution bag to insulate it from the upper Peltier element. In other words, the heat source was limited to the bottom of the reaction solution bag. As a control, rotary shaking at 100 rpm was performed, and temperature control was performed using both the upper and lower Peltier elements. Note that a copper plate was used as the heat-conducting plate instead of an aluminum plate.

[0109] The results of measuring the PCR reaction temperature are shown in the upper panel of Figure 13. The upper left panel of Figure 13 shows the results of investigating the thermal cycle of PCR when PCR was performed with only bottom temperature control, and the upper right panel of Figure 13 shows the results of investigating the thermal cycle of PCR when PCR was performed with both top and bottom temperature control. Even when only bottom temperature control was performed without top temperature control, it was confirmed that temperature control by rotary shaking was almost equivalent to the control (when temperature control was performed from both top and bottom). Furthermore, as a model for 200 mL, 300 mL, or 400 mL of reaction solution, a plastic bag containing 100 μL of reaction solution was placed inside a bag containing 200 mL, 300 mL, or 400 mL of water. The lower panel of Figure 13 shows the results of agarose electrophoresis when PCR was performed using a 200-mL, 300 mL, or 400 mL reaction solution model. It was confirmed that nucleic acids were amplified by bottom-only temperature control, even when using the 200-mL to 400-mL reaction model.

[0110] [Example 11] Since it was confirmed in Example 10 above that nucleic acids could be amplified even in 400 mL, it was investigated whether nucleic acid amplification was possible in 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 in an autoclave bag (32 cm long and 32 cm wide) containing 600 mL or 1000 mL of water (air was added so that the air volume was 5-10%), and PCR was performed in the same manner as in Example 10 by rotary shaking at 100 rpm.

[0112] The thermal cycler used was the first example of the thermal cycler shown in Figure 16 (described below). A bag containing only air (2–5 cm thick) was placed above the reaction bag to insulate it from the Peltier element. In other words, only the bottom of the reaction bag was in contact with the heat-conducting plate, which served as the heat source. A copper plate (see Figure 24A) was used as the heat-conducting plate, and approximately eight Peltier elements were fixed to the copper plate in a square-shaped configuration, parallel to the horizontal plane, on the side of the bag (excluding the center). The Tris-HCl buffer concentration 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. The template DNA concentration was 0.1 ng / μL (final concentration) for the 600 mL reaction solution model and 0.1 ng / μL or 1 ng / μL (final concentration) for the 1000 mL reaction solution model.

[0113] The results of agarose electrophoresis and thermal cycle analysis when PCR was performed using models with 600 mL and 1000 mL of reaction solution are shown in Figures 14 and 15. The target nucleic acid was amplified in both models, confirming that nucleic acids can be amplified by the nucleic acid amplification method of the present invention even in large volumes such as 600 mL or 1000 mL.

[0114] In the case of a 1000 mL model reaction solution, the temperature increase and decrease in the second cycle were 0.06°C / sec and 0.16°C / sec, respectively. In conventional PCR methods, the temperature increase and decrease are typically 1-6°C / sec. However, in the nucleic acid amplification method of the present invention, amplification products can be obtained even with a slow temperature increase rate of 0.06°C / sec. This was extremely surprising for nucleic acid amplification technologies that have always required a fast temperature increase rate. Furthermore, as shown in the above examples, by controlling the buffer concentration and pH, 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 even 360 minutes or more.

[0115] [Example 12] (Thermal cycler first example) Next, an example of a first thermal cycler 2 that can be used in the nucleic acid amplification method of the present invention will be described with reference to Figures 16 and 17. Figure 16 is a front view of the entire first thermal cycler 2, Figure 17A is a front view of a 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 to the top of a shaking table 33 of a shaking unit 32 by four temperature control unit fixing shafts 34 that have spiral grooves on their outer periphery. With this fixing, the shaking table 33 of the shaking unit 32 rotates and shakes the reaction vessel 35, thereby agitating the reaction solution (not shown) in the reaction vessel 35.

[0116] The shaking unit 32 is equipped with a shaking on / off switch 36, a rotation speed adjustment dial 37, and a rotation speed display 38, and shaking can be controlled by adjusting the rotation speed to a predetermined value. A motor 39 is installed in the center of the body of the shaking unit 32 so that its rotation axis is vertical, and is joined perpendicularly to the shaking table 33. Rotational shaking is possible by the power of the motor 39.

[0117] The temperature control unit 31 has four Peltier elements 13 arranged in two rows by two rows in parallel on a horizontal plane, and a 3 mm thick copper plate 40 fixed with screws so that the entire top surfaces of the Peltier elements 13 are in close contact. A reaction vessel 35 containing a PCR reaction solution is placed on the copper plate 40, and 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 rests on the top surface of the reaction vessel 35 under its own weight. In addition, a slippage prevention plate 42 is attached to the side of the copper plate 40 to prevent the reaction vessel from slipping due to shaking.

[0118] A heat sink 11 and a cooling fan 12 are provided on the surface of the Peltier element 13 opposite to the surface that contacts 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 a space is provided to prevent convection of the air discharged by the cooling fan 12. The Peltier element 13 is also connected to a temperature control unit that 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] FIG. 18 shows a plan view of copper plate 40. It has a through-hole 19 with a diameter of 10 mm in the center. At both ends, it has insertion holes 44a for inserting linear shafts 43, which are provided to allow reaction vessel support plate 41 to move freely in the vertical direction. At the four corners, it has screw holes 45a for connecting to temperature control unit fixing shaft 34 with screws, and four sets of four screw holes 45b for connecting to Peltier element 13 with screws. Copper plate 40 and Peltier element 13 are fixed to temperature control unit fixing shaft 34 with screws via screw holes 45a for connecting to Peltier element 13. Furthermore, as shown in FIG. 17A, the screw holes in copper plate 40 are inserted into temperature control unit fixing shaft 34, which has nuts 46 as stoppers at predetermined positions, so that copper plate 40 is fixed to temperature control unit fixing shaft 34 by its own weight.

[0120] Next, the arrangement of the temperature sensor will be described with reference to FIGS. 10A and 19A and B. A thin, adhesive-type thermocouple temperature sensor 17 is attached to the center of the outer surface of a reaction vessel 35. The temperature-measuring 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 FIG. 19A (the reaction vessel is not shown). FIG. 19B is a plan view (not showing the reaction vessel support plate) of a reaction vessel 35 containing a PCR reaction solution and 10% air 51, placed on the copper plate 40, with the temperature-measuring part 18 of the temperature sensor 17 positioned at the center. The temperature detection result from the temperature sensor 17 is sent to a temperature control unit (not shown), which then calculates the power to be supplied to the Peltier element 13 from the power control unit in the calculation unit. 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 set temperature input section, the operator inputs information necessary for the PCR thermal cycle, such as the reaction temperature, reaction time, and number of cycles for initial thermal denaturation, thermal denaturation, annealing, extension reaction, etc. The temperature measured by the temperature sensor 17 is displayed in the temperature display section.

[0122] The reaction vessel support plate 41 will be described with reference to Figures 20A-C and Figures 21A-B. Figures 20A-C show, respectively, a front view, a plan view, and a side view of the reaction vessel support plate 41. Figure 21A shows the state when water vapor 54 is generated in the reaction vessels, causing air to accumulate and expand above the reaction vessels 35. Both ends of the reaction vessel support plate 41 are provided with support plate holders 52 having insertion holes 44b for inserting linear shafts 43. When inserted into the linear shafts 43, the reaction vessel support plate 41 can be raised and lowered vertically along the linear shafts 43 and abuts against the upper surfaces of the reaction vessels 35 under its own weight. With this configuration, when water vapor is generated from the PCR reaction solution in the reaction vessels and the height of the upper surfaces of the reaction vessels increases, or when the water vapor in the reaction vessels decreases and the height of the upper surfaces of the reaction vessels decreases, the reaction vessels can be held down and supported without adjusting the height of the reaction vessel support plate. Furthermore, this configuration allows the reaction vessel support plate 41 to come into contact with the upper surface of the reaction vessel 35 depending on the height of the upper surface of the reaction vessel 35, while also maintaining close contact between the bottom surface of the reaction vessel 35 and the copper plate 40. Note that by using a structure in which the temperature control unit fixing shaft 34 supports the Peltier element 13 and the linear shaft 43 supports the reaction vessel support plate 41, it is possible to prevent heat from the copper plate 40 from being conducted to the reaction vessel support plate 41, thereby enabling more precise temperature control.

[0123] 21B, the reaction vessel support plate 41 is provided with a fixing means that can fix it in a position that leaves a predetermined gap between it and the reaction vessel 35. After thermal denaturation, in the process of cooling the reaction solution for annealing, a gap that allows air to enter is provided between the reaction vessel 35 and the reaction vessel support plate 41 as shown in FIG. 21B, which improves the heat dissipation efficiency of the reaction solution and speeds up the temperature drop.

[0124] [Example 13] (Thermal cycler second example)

[0125] Next, a second example of a thermal cycler that can be used in the nucleic acid amplification method will be described with reference to Figures 22A and 22B. The stirring unit is omitted in Figure 22A. While the Peltier element 13 in Example 10 was air-cooled using a cooling fan 12, the Peltier element 13 in the second example is water-cooled. A pair of water-cooled cooling units using Peltier elements 13 are provided above and below (the Peltier elements 13 are not shown because they are provided inside the cooling unit 60), 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; instead, two cooling unit support plates 63 are provided parallel to the temperature control unit fixing shaft 34, and the cooling unit support plates 63 support the Peltier element 13 and the cooling unit 60. The cooling unit support plate 63 has insertion holes 64 at both ends through which the temperature adjustment unit fixing shaft 34 is inserted, and is fixed to the temperature adjustment unit fixing shaft 34 from above with thumb screws 65 .

[0126] [Example 14] (Thermal cycler third example) A third example of a thermal cycler that can be used in the nucleic acid amplification method is described with reference to FIG. 23. To accelerate the heating and cooling of large volumes of reaction solution, the thermal cycler includes three upper and lower temperature control units 31: 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 quickly raising or lowering the temperature of the reaction vessel to the set temperature for the next step. Above each of the temperature control units 31a-c, an air-cooled Peltier element 13 equipped with a heat sink 11 and a cooling fan 12 can be raised or lowered vertically by a lift cylinder 71. When controlling the temperature of the reaction vessel 35, the Peltier element 13 lowers to abut against the top surface of the reaction vessel 35, securing the reaction vessel 35 and strengthening adhesion to the copper plate 40 to improve heat conduction efficiency. The upper parts of the temperature control units 31a-c are raised when the reaction vessel 35 is in another temperature control unit. The upper temperature adjustment unit may simply be a heater that can be controlled to a constant temperature, or may not have a heat source.

[0127] Each of the temperature control units 31a-c is equipped with a thin thermocouple temperature sensor that measures the temperature of the outer surface of the bottom of the reaction vessel 35, and a thin thermocouple temperature sensor that measures the temperature of a copper plate that is 20 cm long, 10 cm wide, and 5 mm thick (not shown), and information about each measured temperature is sent to a current control unit (not shown) to control the temperature. In the third example of this thermal cycler, the temperature rises and falls quickly, making it prone to overshoot or undershoot, so the temperature is controlled by a PID.

[0128] The temperature control units 31a to 31c are fixed onto a shaking table 33 of a shaker equipped with a motor 39 via a temperature control unit fixing shaft 34. The reaction vessel 35 can be shaken by shaking the shaking table 33. It is also possible to shake the reaction vessel 35 by shaking all or part of at least one temperature control unit 31, without using the shaking unit 32, for example, by sandwiching the reaction vessel between a pair of upper and lower 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 movable stage 72 made of a heat-conductive sheet, and is movable along a traveling rail 73 between the temperature adjustment units 31a to 31c.

[0130] Next, we will explain the operation of a third example of this thermal cycler when performing thermal cycling. 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, and the top and bottom of the reaction vessel 35 abut against the copper plate 40. In this state, when the temperature of the outer surface of the reaction vessel 35 reaches the temperature for thermal denaturation, thermal denaturation is performed for a predetermined period of time. Next, when thermal denaturation is complete, the upper part of the temperature control unit 31a rises, and the moving platform 72 moves left and right to shake the reaction vessel 35 and stir the reaction solution. Then, the reaction vessel 35 is moved to the preheating or cooling temperature control unit 31c, which is preset to 0°C, and the upper part of the cooling temperature control unit is lowered to clamp the reaction vessel 35 and rapidly cool 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 reactions, the upper part of the temperature control unit 31c rises, and the movable stage 72 is moved to move the reaction vessel 35 to the temperature control unit 31b for the annealing and extension reactions. The upper part of the temperature control unit 31b for the annealing and extension reactions then descends, sandwiching the reaction vessel 35, and annealing is performed for a predetermined period of time. Next, once the annealing and extension reactions are complete, the upper temperature control unit for the temperature control unit 31b for the annealing and extension reactions rises, and the movable stage 72 is moved to move the reaction vessel 35 to the thermal denaturation temperature control unit 31a, where thermal denaturation is performed for a predetermined period of time. Repeating the above steps in the thermal cycle achieves rapid heating and cooling, shortening the PCR reaction time and improving the efficiency of nucleic acid amplification.

[0131] [Example 15] (Position of copper plate and Peltier element) Furthermore, an embodiment in which eight Peltier elements 13 are used on the underside of copper plate 40 in a thermal cycler that can be used in the above-described nucleic acid amplification method is described with reference to Figure 24A. Figure 24A is a plan view of copper plate 40 in the first example of thermal cycler 2, with eight Peltier elements 13 (positions indicated by dotted lines in the figure) fixed in a square shape under copper plate 40. Copper plate 40 is 32 cm long and wide and 3 mm thick. A through-hole 19 with a diameter of 1 cm is provided in the center of copper plate 40. No Peltier element 13 is installed in the center, leaving space for a stirrer, ultrasonic device, etc.

[0132] 24B, a hollow portion 20 measuring 3 cm in length and 3 cm in width may be provided near the center. This configuration allows the reaction vessel 35 to be more easily exposed to air during the step of lowering the temperature of the reaction solution, improving heat dissipation efficiency. Furthermore, if the hollow portion 20 is formed in a mesh, lattice, or linear hollow shape, it is possible to improve the heat dissipation effect by utilizing the wind generated by the movement of the fan of the Peltier element 13. To improve the heat dissipation efficiency, a heat pipe may be provided below the hollow portion 20, and the cooling efficiency of the heat pipe may be improved 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 made thinner than that of the peripheral portion without providing a hollow portion.

[0133] [Example 16] FIG. 25 shows an embodiment in which a temperature probe 81 is used as a temperature sensor in a temperature control unit. The Peltier element 13, heat sink, and cooling fan are omitted to show the position of the temperature probe 81. The spring-operated temperature probe 81 is provided with a temperature measuring part 82 at its tip, which protrudes from the through-hole 19 without touching the copper plate 40. 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 measuring part 82, enabling the temperature of the outer surface of the reaction vessel 35 to be measured. Note that the upper end of the temperature measuring part 82 only needs to be able to come into contact with the reaction vessel 35; depending on the material of the reaction vessel 35, it may not need to protrude from the through-hole 19. [Industrial Applicability]

[0134] The present invention enables large-scale PCR and can be used for the production of DNA vaccines, PCR testing, etc. [Explanation of symbols]

[0135] 1. Thermal cycler 2. First thermal cycler 11...Heat sink 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 fixing shaft 35...Reaction vessel 36...On / Off switch 37...Rotation speed adjustment dial 38... RPM display 39...Motor 40...Copper plate 41...Reaction vessel support plate 42...Slip prevention plate 43...Linear shaft 44a, 44b...Through holes 45a, 45b...Screw holes 46...Nat 51...Air 52...Support plate holding part 53...PCR reaction mixture 54...Water vapor 60...Cooling unit 61...Solvent inlet 62...Solvent outlet 63...Cooling unit support plate 64...Through hole 65...Thumb screw 71...Lifting cylinder 72...Mobile stand 73...Running rail 81...Temperature probe 82...Temperature measurement section

Claims

1. A method for amplifying nucleic acids by carrying out a polymerase chain reaction (PCR) including a step of thermally denaturing double-stranded template DNA 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, comprising: The reaction solution contained in one reaction vessel is 30 mL or more and 10 L or less, the pH of the buffer solution is 8.0 to 10.0; The concentration of the buffer solution is 0.3 to 1.5 M; The material of the reaction vessel has thermal conductivity and elasticity, and at least the bottom surface of the reaction vessel is bonded to a heat source; measuring the temperature of the outer surface of the reaction vessel or the temperature of the reaction solution with a temperature sensor, and controlling the temperature of the thermal cycle in the polymerase chain reaction based on the measured temperature; The method is characterized in that the polymerase chain reaction is carried out while stirring the reaction solution by a method of shaking the reaction vessel itself, a method of providing an agitator in the reaction vessel, or a method of placing a magnetic stirrer in the reaction vessel and stirring using magnetic force from outside the reaction vessel with a magnetic stirrer.

2. 2. The method according to claim 1, wherein the concentration of the buffer solution is 0.4 to 1.5M.

3. 3. The method according to claim 1, wherein the reaction solution is stirred by rocking the reaction vessel.

4. 4. The method according to claim 1, wherein the reaction vessel is bag-shaped.

5. 5. The method according to claim 1, wherein the reaction vessel contains 1 to 90% gas.

6. A method as described in claim 5, characterized in that the temperature of at least the surface of the heat source adhered to the bottom of 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 adhered to the bottom of the reaction vessel.

7. A thermal cycler for use in the nucleic acid amplification method described in any one of claims 1 to 6, comprising: a temperature control unit made of a thermally conductive material and attached to one or more reaction vessels containing 30 mL to 10 L of reaction solution, the temperature control unit controlling the temperature of the reaction solution; and an agitation unit for agitating the reaction solution, wherein the temperature control unit comprises a heat source, a reaction vessel support plate that abuts against the upper surface of the reaction vessel to support the reaction vessel, and a temperature sensor that measures the temperature of the outer surface of the reaction vessel or the temperature of the reaction solution.

8. The thermal cycler of claim 7, characterized in that the heat source in the temperature control unit is a Peltier element, and the thermal cycler is equipped with a heat conduction plate that is adhered to the Peltier element and can abut against the reaction vessel, and a temperature control unit connected to the Peltier element.

9. 9. The thermal cycler according to claim 7, wherein 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 solution measured by a temperature sensor.

10. A thermal cycler described in any one of claims 7 to 9, characterized in that a recess or a through hole is provided in the heat conduction plate, and the temperature measuring part of the temperature sensor is arranged in the recess or through hole without contacting the heat conduction plate, so that when a reaction vessel is placed on the heat conduction plate, the bottom of the reaction vessel and the temperature measuring part of the temperature sensor can abut.

11. 11. The thermal cycler according to claim 7, further comprising a temperature control unit for thermal denaturation, a temperature control unit for annealing and extension reactions, and a preheating or cooling temperature control unit.

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