JP2018023334A - Heat cycle device, reaction vessel and nucleic acid amplification method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a heat cycle device which increases speed of heat transfer to a reaction liquid and improves easiness of movement of the reaction liquid in a flow channel, and to provide a reaction vessel and a nucleic acid amplification method.SOLUTION: A heat cycle device 1 comprises: an installation part where a reaction vessel 100 in which a flow channel 160 is formed of walls each having flexibility and a reaction liquid 150 can move between a first region 170 and a second region 180, is installed; a first heating part 210 which heats the first region 170 to a first temperature; a second heating part 220 which heats the second region 180 to a second temperature; a reciprocating mechanism that causes the reaction liquid 150 to perform reciprocating operation between the first region 170 and the second region 180; and a moving mechanism that changes a gap between facing inner walls of the first region 170 by changing the gap between a first heat application part 211 and a second heat application part 212 that the first heating part 210 has, or that changes the gap between facing inner walls of the second region 180 by changing the gap between a third heat application part 221 and a fourth heat application part 222 that the second heating part 220 has.SELECTED DRAWING: Figure 3A

Description

  The present invention relates to a thermocycling device, a reaction vessel, and a nucleic acid amplification method.

  Conventionally, a PCR (Polymerase Chain Reaction) method has been widely used as a method for amplifying nucleic acids at high speed, and the application range of PCR-related technology is expected to continue to expand in the future. The PCR method is a technique for amplifying a target nucleic acid by subjecting a solution (reaction solution) containing a nucleic acid (target nucleic acid) to be amplified and a reagent to thermal cycling. In the PCR method, it is currently required to efficiently amplify a target nucleic acid by shortening the time required for a thermal cycle between each step of thermal denaturation, annealing and extension.

  Patent Document 1 describes a nucleic acid amplification reaction apparatus in which a reaction solution moves in a reaction vessel. The distance between the first inner wall and the second inner wall is such that the reaction solution has a first inner wall and a second inner wall. It is disclosed that the distance contacts both of the inner walls. According to this configuration, the heating amount of the nucleic acid amplification reaction solution can be managed, and the nucleic acid can be stably amplified by suppressing variations in the nucleic acid amplification amount.

JP-A-2015-154722

If the thickness of the flow path of the reaction vessel in which the reaction solution is enclosed, including the reaction vessel of Patent Document 1, is reduced, the movement of the reaction solution is alienated and it is difficult to move at high speed. On the other hand, if the thickness of the flow path of the reaction vessel is increased, the thermal conductivity from the heating part (heat block, etc.) to the reaction solution deteriorates, making it difficult to transfer heat at high speed. .
Accordingly, there has been a demand for a thermal cycle apparatus, a reaction vessel, and a nucleic acid amplification method that can increase the speed of heat transfer to the reaction solution.

  SUMMARY An advantage of some aspects of the invention is to solve at least a part of the problems described above, and the invention can be implemented as the following forms or application examples.

  Application Example 1 The thermal cycler according to this application example is provided with a reaction vessel in which a flow path is formed by a flexible wall and the reaction solution can move between the first region and the second region. A first heating unit having a first heat application unit and a second heat application unit for heating the first region to the first temperature, and a mounting unit. When the reaction vessel is mounted, a second heating unit having a third heat application unit and a fourth heat application unit that heats the second region to the second temperature, and the reaction solution is provided in the first region and the second region. And a reciprocating mechanism for reciprocating between the first heat applying portion and the second heat applying portion by changing a gap between the first portion and the first portion of the inner wall of the first region. And changing the gap between the third heat application part and the fourth heat application part to change the second of the inner wall of the second region wall. A moving mechanism for changing the distance between the divided and portions of the opposed inner walls in the second portion, characterized in that it comprises a.

According to the heat cycle apparatus of this application example, the wall forming the flow path of the reaction vessel has flexibility. When the reaction vessel is mounted on the mounting part, when the first region is heated to the first temperature by the first heating part, the gap between the first heat application part and the second heat application part is changed by the moving mechanism. Then, heating is performed by changing the distance between the first portion of the inner wall of the first region wall and the portion of the inner wall facing the first portion. In addition, when the second region is heated to the second temperature by the second heating unit, the gap between the third heat application unit and the fourth heat application unit is changed by the moving mechanism to change the inner wall of the second region wall. Heating is performed by changing the distance between the second portion and the portion of the inner wall facing the second portion. The reciprocating mechanism moves the reaction solution from the first region to the second region and performs a reciprocating operation for moving the reaction solution from the second region to the first region.
Thereby, when the reaction solution is located in the first region, the interval between the inner walls (the interval between the first portion of the inner wall of the wall of the first region and the portion of the inner wall opposite to the first portion) is reduced. Thus, the reaction solution is compressed and spreads flatly, for example, and the contact area between the reaction solution and the first heat application unit (second heat application unit) is increased, so that heat is transmitted to the reaction solution at a high speed. When the reaction solution is located in the second region, the interval between the inner walls (the interval between the second portion of the inner wall of the second region wall and the portion of the inner wall opposite to the second portion) should be reduced. Thus, the reaction solution is compressed and spreads flatly, for example, and the contact area between the reaction solution and the third heat application unit (fourth heat application unit) increases, so that heat is transferred to the reaction solution at a high speed.
Therefore, it is possible to realize a heat cycle apparatus that can increase the speed of heat transfer to the reaction liquid.

  Application Example 2 In the thermal cycle apparatus according to the application example described above, the reaction vessel has a first inner wall and a second inner wall facing each other, and the distance between the first inner wall and the second inner wall is When the reaction solution is disposed in the flow path, the distance is preferably such that the reaction solution contacts both the first inner wall and the second inner wall facing each other.

  According to the thermal cycle device of this application example, when the reaction liquid is arranged in the flow path, the reaction liquid is set to a distance that contacts both the first inner wall and the second inner wall facing each other. By reducing the distance between the first inner wall and the second inner wall with the moving mechanism, the reaction liquid is compressed and spreads, and the contact area between the reaction liquid and the first heating part (second heating part) is ensured. Can be bigger.

  Application Example 3 In the heat cycle apparatus according to the application example described above, the reciprocating mechanism switches the placement of the mounting portion, the first heating portion, and the second heating portion between the first placement and the second placement, The first arrangement is an arrangement in which the first region is located at the lowermost part of the flow path in the direction in which gravity acts, and the second arrangement is located in the lowermost part of the flow path in the direction in which gravity acts. It is preferable that the arrangement is.

  According to the thermal cycle device of this application example, the reciprocating mechanism causes the first arrangement in which the first region is located at the lowest part of the flow path in the direction in which the gravity acts and the flow in the direction in which the second region acts in the gravity. The second arrangement located at the bottom of the road is switched. By using gravity as in this configuration, for example, the time required for heat denaturation, annealing, and elongation for amplifying a nucleic acid can be shortened.

  Application Example 4 In the heat cycle apparatus according to the application example described above, the reciprocating mechanism preferably includes a moving mechanism, and the reaction liquid is moved by bringing the inner wall of the reaction vessel into close contact with each other to perform a reciprocating operation.

  According to the heat cycle apparatus of this application example, the reciprocating mechanism performs the reciprocating operation by moving the reaction liquid to the non-contact region by bringing the inner wall into close contact. According to this configuration, for example, nucleic acid can be amplified by reciprocating the reaction solution without rotating the reaction vessel.

  Application Example 5 In the heat cycle apparatus according to the application example described above, a first connection region that thermally connects the first heat application unit and the second heat application unit, a third heat application unit, and a fourth heat application. It is preferable to provide the 2nd connection area | region which connects a part thermally.

According to the heat cycle device of this application example, the first heat application unit and the second heat application unit are thermally connected by the first connection region, and the third heat application unit and the fourth heat application are provided by the second connection region. The part is thermally connected. With this configuration, when the first region of the reaction vessel is heated to the first temperature by suppressing temperature variation between the first heat application unit and the second heat application unit, the reaction vessel in contact with the first heating unit ( Stable heat transfer from the wall) to the reaction solution and high speed transfer.
Similarly, when the second region of the reaction vessel is heated to the second temperature by suppressing temperature variation between the third heat application unit and the fourth heat application unit, the reaction vessel (wall) in contact with the second heating unit ) To the reaction liquid, and stable heat transfer and high-speed transfer can be achieved.
Further, for example, when the first connection region is in contact with the reaction vessel and heat transfer is possible, the heat transfer to the reaction solution can be further speeded up. The same applies to the second connection region.

  Application Example 6 In the heat cycle apparatus according to the application example described above, it is preferable that the first heat application unit has a protrusion and the second heat application unit has a recess at a position corresponding to the protrusion.

  According to the thermal cycle device of this application example, the first heat application unit has a convex portion, and the second heat application unit has a concave portion at a position corresponding to the convex portion. With this configuration, the flexible reaction vessel is pressed (held) between the convex portion of the first heat application unit and the concave portion of the second heat application unit, so that the wall The contact area can be increased. Accordingly, the efficiency of heat conduction to the reaction solution can be improved.

  [Application Example 7] A reaction container according to this application example includes a flexible first wall, a flexible second wall disposed relative to the first wall, and a wall. And the distance between the inner wall of the first wall and the inner wall of the second wall is such that when the reaction liquid is disposed in the flow path, It is the distance which contacts both the inner wall of the wall of this, and the inner wall of the 2nd wall.

  According to the reaction container of this application example, since the reaction solution has a distance that contacts both the inner wall of the first wall and the inner wall of the second wall, the heat application unit is in contact with the outer wall of the reaction container. Thus, the heat of the heat application unit can be efficiently transmitted to the reaction solution. Furthermore, the contact area between the reaction solution and the heat application unit can be increased by reducing the distance between both inner walls and pressing the heat application unit, further increasing the efficiency of heat conduction to the reaction solution. Can be improved.

  [Application Example 8] In the nucleic acid amplification method according to this application example, the reaction solution is moved between the first region and the second region of the flow path formed in the reaction vessel having the flexible wall, and thus the nucleic acid. A method for amplifying the nucleic acid, wherein the gap between the first heat application unit and the second heat application unit is changed to change the interval between the inner walls of the walls of the first region, the first heat application unit, Between the step of heating the first region to the first temperature by the second heat application unit, the step of moving the reaction solution from the first region to the second region, and the third heat application unit and the fourth heat application unit. Changing the gap and changing the distance between the inner walls of the walls of the second region, heating the second region to the second temperature by the third heat applying unit and the fourth heat applying unit, and removing the reaction solution from the second region And moving to the first region.

According to the nucleic acid amplification method of this application example, the wall forming the flow path of the reaction vessel has flexibility. When the reaction container is attached to the attachment part, the gap between the first heat application part and the second heat application part is changed to change the interval between the inner walls of the first region of the reaction container. Next, the first region is heated to the first temperature by the first heat application unit and the second heat application unit of the first heating unit. Next, the reaction solution is moved from the first region to the second region. Next, the gap between the inner wall of the second region wall of the reaction vessel is changed by changing the gap between the third heat applying unit and the fourth heat applying unit. Next, the second region is heated to the second temperature by the third heat application unit and the fourth heat application unit of the second heating unit. The process as described above is included.
Thereby, when the reaction solution is located in the first region, the reaction solution is compressed and spread flatly, for example, by reducing the interval between the inner walls, and the reaction solution and the first heat application unit (second heat application unit). ), The heat is transferred to the reaction solution at a high speed. In addition, when the reaction solution is located in the second region, the reaction solution is compressed and spread flatly, for example, by reducing the interval between the inner walls, and the reaction solution and the third heat application unit (fourth heat application unit). Since the contact area with the liquid becomes large, heat is transferred to the reaction solution at high speed.
Accordingly, it is possible to realize a nucleic acid amplification method capable of increasing the speed of heat transfer to the reaction solution.

The side sectional view of the reaction container concerning a 1st embodiment. The plane sectional view of a reaction vessel. FIG. 3 is a schematic cross-sectional view showing a positional relationship between a reaction container and a heating unit in a heat cycle device. Sectional drawing which shows the state which the 1st heat application part moved to the heating position in 1st arrangement | positioning. Sectional drawing which shows the state switched from 1st arrangement | positioning to 2nd arrangement | positioning. Sectional drawing which shows the state which switched to the 2nd arrangement | positioning and the 1st heat application part moved to the initial position. Sectional drawing which shows operation | movement of the reaction liquid at the time of switching to 2nd arrangement | positioning. Sectional drawing which shows the state which the 4th heat application part moved to the heating position in 2nd arrangement | positioning. Sectional drawing which shows the state switched from 2nd arrangement | positioning to 1st arrangement | positioning. Sectional drawing which shows the state which switched to the 1st arrangement | positioning and the 4th heat application part moved to the initial position. The flowchart which shows the process sequence in the case of performing 2 step PCR using a thermal cycle apparatus. The schematic sectional drawing which shows the positional relationship of the reaction container in the thermal cycle apparatus of 2nd Embodiment, a heating part, and a press part. The figure which shows the state which heats the reaction liquid of a 1st area | region. The figure which shows the state which moves the reaction liquid of a 1st area | region to a 2nd area | region. The figure which shows the state which heats the reaction liquid of a 2nd area | region. The figure which shows the state which moves the reaction liquid of a 2nd area | region to a 1st area | region. The schematic sectional drawing which shows the positional relationship of the reaction container and heating part in the heat cycle apparatus of 3rd Embodiment. The perspective view which shows the state which the 1st heat application part of the 1st heating part was located in the initial position. The perspective view which shows the state which the 1st heat application part of the 1st heating part was located in the heating position. The perspective view which shows the state which the 4th heat application part of the 2nd heating part was located in the initial position. The perspective view which shows the state which the 4th heat application part of the 2nd heating part was located in the heating position. Sectional drawing which shows the state which the 1st heat application part moved to the heating position in 1st arrangement | positioning. Sectional drawing which shows the state switched from 1st arrangement | positioning to 2nd arrangement | positioning. Sectional drawing which shows the state which switched to the 2nd arrangement | positioning and the 1st heat application part moved to the initial position. Sectional drawing which shows operation | movement of the reaction liquid at the time of switching to 2nd arrangement | positioning. Sectional drawing which shows the state which the 4th heat application part moved to the heating position in 2nd arrangement | positioning. The schematic sectional drawing which shows the positional relationship of the reaction container and heating part in the thermal cycle apparatus of 4th Embodiment. The perspective view which shows the state which the 1st heat application part of the 1st heating part was located in the initial position. The perspective view which shows the state which the 1st heat application part of the 1st heating part was located in the heating position. The perspective view which shows the state which the 4th heat application part of the 2nd heating part was located in the initial position. The perspective view which shows the state which the 4th heat application part of the 2nd heating part was located in the heating position.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. In the following drawings, the scale of each member is illustrated differently from the actual scale for convenience of explanation.

[First Embodiment]
The thermal cycle apparatus 1 of the present embodiment is configured as an apparatus that amplifies a nucleic acid using a PCR (Polymerase Chain Reaction) method.

  FIG. 1A is a side sectional view of a reaction vessel 100 according to the present embodiment. Specifically, it is a cross-sectional view of the reaction vessel 100 from the third wall 130 side. FIG. 1B is a plan sectional view of the reaction vessel 100. Specifically, it is a cross-sectional view of the reaction vessel 100 from the first wall 110 side.

  As shown in FIGS. 1A and 1B, the reaction vessel 100 has a substantially rectangular parallelepiped shape, and includes a first wall 110, a second wall 120 facing the first wall 110, a first wall 110, and a second wall. It is comprised by the 3rd wall 130 which connects 120. FIG. In addition, the first wall 110, the second wall 120, and the third wall 130 are configured to have flexibility.

  A reaction solution 150 is sealed inside the reaction vessel 100 surrounded by the first wall 110, the second wall 120, and the third wall 130. The reaction liquid 150 according to the present embodiment is located in the lowermost region in the gravity direction of the reaction vessel 100, and the first wall 110, the second wall 120, and the third wall 130 are located at the lowermost region. Are in contact with the respective inner walls (first inner wall 110a, second inner wall 120a, and third inner wall 130a). In other words, the distance between the inner walls of the reaction vessel 100 is a distance at which the reaction solution 150 contacts both the first inner wall 110a and the second inner wall 120a facing each other when the reaction vessel 100 is mounted. With the above-described configuration, the reaction vessel 100 forms a flow channel 160 that is a path through which the reaction solution 150 moves in the longitudinal direction. The reaction solution 150 can move between a first region 170 and a second region 180 described later by the flow channel 160.

  In the thermal cycle apparatus 1 of the present embodiment, as a reaction solution 150, a DNA sample (target nucleic acid) containing a DNA (deoxyribonucleic acid) sequence to be amplified by PCR, a DNA polymerase necessary for amplifying DNA, and a primer (20 bases) An aqueous solution containing a certain degree of oligonucleotide, etc.).

  FIG. 2 is a schematic cross-sectional view showing the positional relationship between the reaction vessel 100 and the heating unit 200 in the heat cycle apparatus 1.

  The heating unit 200 includes a first heating unit 210 and a second heating unit 220. The first heating unit 210 and the second heating unit 220 are members that transmit heat generated from a heater (not shown) to the reaction vessel 100. The first heating unit 210 includes two parts, a first heat application unit 211 and a second heat application unit 212. Similarly, the second heating unit 220 includes two parts, a third heat application unit 221 and a fourth heat application unit 222. In addition, the 1st heat application part 211 and the 2nd heat application part 212 hold | maintain temperature so that it may become 1st temperature. Moreover, the 3rd heat application part 221 and the 4th heat application part 222 hold | maintain temperature so that it may become 2nd temperature.

  In the present embodiment, the first heat application unit 211, the second heat application unit 212, the third heat application unit 221, and the fourth heat application unit 222 are configured as aluminum blocks, so that the reaction vessel 100 can be efficiently used. Can be heated well. Moreover, since the heat conductivity is high, uneven heating is hardly generated in the block, and a highly accurate heat cycle is realized. Further, since the processing is easy, the block can be accurately molded, and the heating accuracy can be improved. Therefore, a more accurate thermal cycle can be realized.

The first heating unit 210 is opposed to a region of one end of the reaction vessel 100 (in other words, one end of the flow channel 160) when the reaction vessel 100 is attached to an attachment portion (not shown). Note that a region at one end of the flow channel 160 is defined as a first region 170.
The second heating unit 220 faces the region of the other end of the reaction vessel 100 (in other words, the other end of the channel 160) when the reaction vessel 100 is attached to the attachment portion (not shown). The region at the other end of the flow channel 160 is referred to as a second region 180.

  The first heat application unit 211 and the second heat application unit 212 of the first heating unit 210 are in contact with the outer walls of the first wall 110 and the second wall 120 of the reaction vessel 100 corresponding to the first region 170, respectively. . During the heat cycle operation, the first heat application unit 211 heats the outer wall of the first wall 110 at the first temperature, and similarly, the second heat application unit 212 heats the outer wall of the second wall 120 at the first temperature. Heat. Details will be described later.

  The third heat application unit 221 and the fourth heat application unit 222 of the second heating unit 220 are in contact with the outer walls of the first wall 110 and the second wall 120 of the reaction vessel 100 corresponding to the second region 180, respectively. . During the heat cycle operation, the third heat application unit 221 heats the outer wall of the first wall 110 at the second temperature, and the fourth heat application unit 222 similarly heats the outer wall of the second wall 120 at the second temperature. Heat. Details will be described later.

  The heat cycle apparatus 1 of this embodiment is provided with a reciprocating mechanism (not shown). The reciprocating mechanism is a mechanism that switches the reaction vessel 100, the heating unit 200 (the first heating unit 210, the second heating unit 220), and a moving mechanism described later between the first arrangement and the second arrangement.

  The reciprocating mechanism includes a motor, a drive shaft (both not shown), and the like. The drive shaft is set perpendicular to the center of the outer wall of the first wall 110. Then, by operating the motor, the reaction vessel 100, the heating unit 200, and the moving mechanism are rotated while maintaining the positional relationship with the drive shaft as the rotation center.

  The first arrangement is an arrangement in which the first area 170 is lower than the second area 180 in the direction in which gravity acts. In other words, the first arrangement is an arrangement in which the first region 170 is located at the lowermost part of the flow path 160 in the direction in which gravity acts. In the first arrangement, the reaction solution 150 is located in the first region 170.

  In addition, the second arrangement is an arrangement in which the second region 180 is lower than the first region 170 in the direction in which gravity acts. In other words, the second arrangement is an arrangement in which the second region 180 is located at the lowermost part of the flow path 160 in the direction in which gravity acts. In the second arrangement, the reaction solution 150 is located in the second region 180.

  The heat cycle apparatus 1 of the present embodiment is provided with a moving mechanism (not shown). The moving mechanism changes the gap between the first heat application unit 211 and the second heat application unit 212 when applying heat to the reaction vessel 100 to change the inner wall of the first region 170 (of the first wall 110). The first portion (the portion of the first inner wall 110a corresponding to the first region 170) of the first inner wall 110a) and the portion of the inner wall (the second inner wall 120a of the second wall 120) opposite to the first portion (first This is a mechanism for changing the distance from the second inner wall 120a corresponding to the region 170). In addition, the movement mechanism changes the gap between the third heat application unit 221 and the fourth heat application unit 222 when applying heat to the reaction vessel 100 to change the inner wall (first wall) of the second region 180. 110 (the first inner wall 110a) of the second portion (the portion of the first inner wall 110a corresponding to the second region 180) and the portion of the inner wall (the second inner wall 120a of the second wall 120) opposite to the second portion ( This is a mechanism for changing the distance from the second inner wall 120 a corresponding to the second region 180.

  The moving mechanism includes a motor, a guide shaft, a guide member (all not shown) that connects the guide shaft and the heat application unit, and the like. In this embodiment, the moving mechanism includes two sets of a motor, a guide shaft, and a guide member.

  In one set of moving mechanisms, the guide shaft is set to be perpendicular to the outer wall of the first wall 110 facing the first region 170. The guide shaft has a spiral groove. When the guide shaft rotates by operating the motor, the guide member guided by the guide shaft moves the first heat application unit 211 in the present embodiment. Thus, the gap between the opposing second heat application units 212 is changed.

  In the moving mechanism that is the other set, the guide shaft is set to be perpendicular to the outer wall of the second wall 120 facing the second region 180. The guide shaft is similarly provided with a spiral groove, and when the guide shaft rotates by operating the motor, the guide member guided by the guide shaft is the fourth heat application unit 222 in this embodiment. To change the gap between the opposing third heat application units 221.

  In the present embodiment, the moving mechanism as one set moves the first heat application unit 211 and changes the gap with the opposing second heat application unit 212. In addition, the moving mechanism that is the other set moves the fourth heat application unit 222 and changes the gap with the third heat application unit 221 facing each other.

  The moving mechanism moves the position of the heat application unit (in this embodiment, the first heat application unit 211 and the fourth heat application unit 222) to the initial position and the heating position. The control unit that moves the moving mechanism to such a position is a control unit (not shown).

  The initial position is a position where the heat application part is in contact with the outer walls of the first wall 110 and the second wall 120, and is an interval position where the opposing heat application parts are initially set. The heating position refers to the first inner wall 110a and the second inner wall 120a facing each other by deforming the flexible outer wall (wall) by pressing either the first wall 110 or the second wall 120. The position is narrowed.

  In this embodiment, the moving mechanism moves the first heat application unit 211 and the fourth heat application unit 222 to the heating position and the initial position in the first arrangement and the second arrangement. In the present embodiment, the positions of the second heat application unit 212 and the third heat application unit 221 are fixed, and the reaction container 100 is sandwiched between the first heat application unit 211 and the fourth heat application unit 222 facing each other. Yes.

FIG. 3A to FIG. 3G are diagrams schematically showing an operation when performing two-step PCR using the thermal cycler 1. Specifically, FIG. 3A is a cross-sectional view illustrating a state where the first heat application unit 211 has moved to the heating position in the first arrangement. FIG. 3B is a cross-sectional view showing a state where the first arrangement is switched to the second arrangement. FIG. 3C is a cross-sectional view illustrating a state in which the first heat application unit 211 is moved to the initial position by switching to the second arrangement. FIG. 3D is a cross-sectional view showing the operation of the reaction solution 150 when switched to the second arrangement. FIG. 3E is a cross-sectional view illustrating a state where the fourth heat application unit 222 has moved to the heating position in the second arrangement. FIG. 3F is a cross-sectional view showing a state where the second arrangement is switched to the first arrangement. FIG. 3G is a cross-sectional view illustrating a state in which the fourth heat application unit 222 is moved to the initial position by switching to the first arrangement. 3A to 3G, the direction of the arrow g (the downward direction in the figure) is the direction in which gravity acts. FIG. 4 is a flowchart showing a processing procedure when performing two-step PCR using the thermal cycler 1.
With reference to FIG. 3A to FIG. 3G and FIG. 4, an operation when performing PCR using the thermal cycler 1 will be described.

  Two-step PCR is a method of amplifying nucleic acid in the reaction solution 150 by repeatedly performing two-stage temperature treatment on the reaction solution 150. In high-temperature treatment, heat denaturation (denaturation (separation) of double-stranded DNA into single strands) is performed. In the low-temperature treatment, annealing (reaction in which the primer binds to single-stranded DNA) and extension reaction (reaction in which a DNA polymerase is reacted to form a complementary strand of DNA) are performed.

  In the two-step PCR, a high temperature (first temperature) at which heat denaturation is performed is, for example, a temperature between about 92 ° C. and 97 ° C., and a low temperature (second temperature) is, for example, a temperature between about 65 ° C. and 72 ° C. Set by. Processing at each temperature is performed for a predetermined time. In the present embodiment, the predetermined time is assumed to be within a few seconds. In addition, since the appropriate temperature, time, and number of cycles differ depending on the type and amount of reagent used, the reaction should be performed after determining an appropriate protocol in consideration of the type of reagent and the amount of reaction solution 150. Is preferred.

  The temperatures of the first heating unit 210 and the second heating unit 220 are controlled by a temperature sensor (not shown) and a control unit. The control unit controls the operation of the reciprocating mechanism. The control unit performs control so as to hold each of the first arrangement and the second arrangement for a predetermined time. The control unit controls the operation of the moving mechanism. For example, the control unit controls the first heat application unit 211 to move to the heating position in the first arrangement and holds it for a predetermined time, and moves the fourth heat application unit 222 to the heating position in the second arrangement for a predetermined time. Control to hold.

  The control unit includes a CPU (Central Processing Unit) that performs various arithmetic processes as a processor. The control unit includes a storage device such as a ROM (Read Only Memory) and a RAM (Random Access Memory). In the storage device, a storage area for storing various programs and data for controlling each of the above operations is set. In addition, the storage device is set with a work area for temporarily storing data being processed and various processing results for the CPU, a storage area that functions as a temporary file, and other various storage areas. Yes.

The operation when performing PCR will be specifically described.
First, the reaction vessel 100 in which the reaction solution 150 is sealed is attached and fixed to the attachment portion (step S101). As a result, as shown in FIG. 2, the first heating unit 210 is in contact with the reaction vessel 100 (the first wall 110 and the second wall 120) at a position facing the first region 170. The second heating unit 220 is in contact with the reaction vessel 100 (the first wall 110 and the second wall 120) at a position facing the second region 180. At this time, the 1st heat application part 211 and the 4th heat application part 222 are located in the initial position.

  The arrangement of the reaction vessel 100, the heating unit 200, and the moving mechanism in step S101 is the first arrangement. As shown in FIG. 2, the first arrangement is an arrangement in which the first region 170 is located at the lowermost part of the flow path 160 in the direction in which gravity acts. Then, the reaction liquid 150 is in a state located in the first region 170.

  In step S102, the first heating unit 210 and the second heating unit 220 are heated to a predetermined temperature while being positioned in the first arrangement. Specifically, the first heating unit 210 is heated to the first temperature, and the second heating unit 220 is heated to the second temperature. In the present embodiment, the first temperature corresponds to a high temperature, and the second temperature corresponds to a low temperature.

  In step S <b> 103, as shown in FIG. 3A, in the first region 170, the moving mechanism moves the first heat application unit 211 to the heating position and sandwiches the reaction vessel 100 with the second heat application unit 212. The position of the second heat application unit 212 is fixed. By this operation, in the first region 170, the reaction vessel 100 presses the second wall 120 against the second heat application unit 212 and is pressed by the first heat application unit 211. The second wall 120 and the third wall 130 are deformed. In other words, in the first region 170, the distance between the first inner wall 110a and the second inner wall 120a is smaller than the distance in the initial shape (the state in which the distance between the first inner wall 110a and the second inner wall 120a is reduced). ) In the nucleic acid amplification method, this operation changes the gap between the first heat application unit 211 and the second heat application unit 212 to change the inner wall (the first inner wall 110a and the second inner wall 120a of the first region 170 of the reaction vessel 100). ) Corresponding to the step of changing the interval.

  In this state, the first heat application unit 211 and the second heat application unit 212 heat the reaction vessel 100 to the first temperature. This operation corresponds to the step of heating the first region 170 to the first temperature by the first heat application unit 211 and the second heat application unit 212 of the first heating unit 210 in the nucleic acid amplification method.

  By reducing the distance between the first inner wall 110a and the second inner wall 120a of the reaction vessel 100, the reaction liquid 150 is compressed and spreads flat. As a result, although the contact area between the reaction solution 150 and the first heat application unit 211 and the second heat application unit 212 is increased through the first wall 110 and the second wall 120, heat is rapidly generated in the reaction solution 150. Will be transmitted.

  At this time, in the second region 180, as shown in FIG. 3A, the position of the third heat application unit 221 is fixed, and the fourth heat application unit 222 is the initial position and is in contact with the reaction vessel 100. It is in a state. In this state, the third heat application unit 221 and the fourth heat application unit 222 heat the reaction vessel 100 to the second temperature.

  Thereby, a temperature gradient in which the temperature gradually changes between the first temperature and the second temperature is formed between the first region 170 and the second region 180. The thermal cycle apparatus 1 of this embodiment is a two-step PCR, the first temperature is a temperature suitable for heat denaturation of double-stranded DNA, and the second temperature is set to a temperature suitable for annealing and extension reaction. .

  The arrangement of the first heating unit 210 and the second heating unit 220 in step S103 is the first arrangement, and the reaction liquid 150 is located in the first region 170. For this reason, the reaction solution 150 is heated to the first temperature. Accordingly, in step S103, thermal denaturation is performed on the reaction solution 150, which is a reaction at the first temperature.

  In step S104, it is determined whether the first time has elapsed. In the present embodiment, the determination is performed by the control unit. The first time is a time for holding the first arrangement, and in the present embodiment, the first time is a time required for heat denaturation.

  If it is determined in step S104 that the first time has elapsed (YES), the process proceeds to step S105. When it is determined that the first time has not elapsed (NO), the reaction liquid 150 is heated with the first heat application unit 211 moved to the heating position, and step S104 is repeated.

  In step S105, the arrangement of the reaction vessel 100, the heating unit 200, and the moving mechanism is switched from the first arrangement to the second arrangement by the reciprocating mechanism of the heat cycle apparatus 1. Specifically, when moving from the first arrangement to the second arrangement, the rotation is performed by 180 °. This operation corresponds to the step of moving the reaction solution 150 from the first region 170 to the second region 180 in the nucleic acid amplification method. As shown to FIG. 3B, 2nd arrangement | positioning is an arrangement | positioning in which the 2nd area | region 180 is located in the lowest part of the flow path 160 in the direction where gravity acts.

  3B, when switching from the first arrangement to the second arrangement, the positional relationship between the first heat application unit 211 and the second heat application unit 212 in the first arrangement is maintained. Therefore, immediately after switching from the first arrangement to the second arrangement, the reaction solution 150 is sandwiched between the first heat application unit 211 and the second heat application unit 212 and is located in the first region 170. Immediately after entering this state, the process proceeds to step S106.

  In step S106, as shown in FIG. 3C, the first heat application unit 211 is moved from the heating position to the initial position by the moving mechanism. By this operation, the reaction vessel 100 is released from the pressure in the first region 170 and returns to the initial shape. In other words, in the first region 170, the distance between the first inner wall 110a and the second inner wall 120a is the distance in the initial shape. When the reaction vessel 100 returns to the initial shape, the reaction solution 150 moves to the second region 180 by gravity as shown in FIG. 3D.

  In step S <b> 107, as shown in FIG. 3E, in the second region 180, the moving mechanism moves the fourth heat application unit 222 to the heating position and sandwiches the reaction vessel 100 with the third heat application unit 221. Note that the position of the third heat applying unit 221 is fixed. By this operation, in the second region 180, the reaction vessel 100 presses the first wall 110 against the third heat application unit 221 and is pressed by the fourth heat application unit 222, so that the first wall 110 and the first wall 110 from the initial shape. The second wall 120 and the third wall 130 are deformed. In other words, in the second region 180, the distance between the first inner wall 110a and the second inner wall 120a is smaller than the distance in the initial shape (the state in which the distance between the first inner wall 110a and the second inner wall 120a is reduced). ) In the nucleic acid amplification method, this operation changes the gap between the third heat application unit 221 and the fourth heat application unit 222 to change the inner wall (the first inner wall 110a and the second inner wall 120a of the second region 180 of the reaction vessel 100). ) Corresponding to the step of changing the interval.

  In this state, the third heat application unit 221 and the fourth heat application unit 222 heat the reaction vessel 100 to the second temperature. This operation corresponds to the step of heating the second region 180 to the second temperature by the third heat application unit 221 and the fourth heat application unit 222 of the second heating unit 220 in the nucleic acid amplification method.

  By reducing the distance between the first inner wall 110a and the second inner wall 120a of the reaction vessel 100, the reaction liquid 150 is compressed and spreads flat. As a result, although the contact area between the reaction solution 150 and the third heat application unit 221 and the fourth heat application unit 222 is increased through the first wall 110 and the second wall 120, heat is rapidly generated in the reaction solution 150. Will be transmitted.

  The arrangement of the first heating unit 210 and the second heating unit 220 in step S107 is the second arrangement, and the reaction liquid 150 is located in the second region 180. For this reason, the reaction liquid 150 is heated to the second temperature. Accordingly, in step S107, the reaction solution 150 is subjected to annealing and extension reaction that are reactions at the second temperature.

  In step S108, it is determined whether the second time has elapsed. In the present embodiment, the determination is performed by the control unit. The second time is a time for holding the second arrangement, and in the present embodiment, the second time is a time required for the annealing and extension reaction.

  If it is determined in step S108 that the second time has elapsed (YES), the process proceeds to step S109. When it is determined that the second time has not elapsed (NO), the reaction liquid 150 is heated with the fourth heat application unit 222 moved to the heating position, and Step S108 is repeated.

  In step S109, as shown in FIG. 3F, the arrangement of the reaction vessel 100, the heating unit 200, and the moving mechanism is switched from the second arrangement to the first arrangement by the reciprocating mechanism of the heat cycle apparatus 1. When switching from the second arrangement to the first arrangement, the positional relationship between the third heat application unit 221 and the fourth heat application unit 222 in the second arrangement is maintained. Therefore, immediately after switching from the second arrangement to the first arrangement, the reaction solution 150 is sandwiched between the third heat application unit 221 and the fourth heat application unit 222 and is located in the second region 180. Immediately after entering this state, the process proceeds to step S110.

  In addition, when moving from the 2nd arrangement to the 1st arrangement, it is performed by rotating 180 degrees in the opposite direction to the case of moving from the 1st arrangement to the 2nd arrangement. Thereby, the twist of the wiring of the apparatus caused by the driving of the reciprocating mechanism can be reduced, and the wiring of the thermal cycle apparatus 1 is not easily damaged, so that the reliability of the thermal cycle is improved. This operation corresponds to the step of moving the reaction solution 150 from the second region 180 to the first region 170 in the nucleic acid amplification method.

  In step S110, as shown in FIG. 3G, the fourth heat application unit 222 is moved from the heating position to the initial position by the moving mechanism. By this operation, the reaction vessel 100 is released from the pressure in the second region 180 and returns to the initial shape. In other words, in the second region 180, the distance between the first inner wall 110a and the second inner wall 120a is the distance in the initial shape. When the reaction vessel 100 returns to the initial shape, the reaction solution 150 moves to the first region 170 by gravity as shown in FIG.

  In step S111, the value of the number of times (the number of cycles) of performing the thermal cycle operation from step S103 to step S110 is incremented by +1. In the present embodiment, when the procedure from step S103 to step S110 is performed once, the reaction solution 150 is subjected to one thermal cycle. Therefore, the number of times the step S103 to step S110 is performed is calculated as the cycle of the thermal cycle. It is a number.

  Next, the process proceeds to step S112. In step S112, it is determined whether or not the number of thermal cycles (procedure from step S103 to step S110) has reached a predetermined number of cycles (for example, 40 times).

  If it is determined in step S112 that the number of thermal cycles has been performed for a predetermined number of cycles (YES), the process is completed (END). When it is determined that the predetermined number of thermal cycles has not been performed (NO), the process proceeds to step S103.

According to the embodiment described above, the following effects can be obtained.
(1) In the heat cycle apparatus 1 of the present embodiment, the walls (the first wall 110, the second wall 120, and the third wall 130) that form the flow path 160 of the reaction vessel 100 have flexibility. When the reaction container 100 is mounted on the mounting unit, when the first heating unit 210 heats the first region 170 to the first temperature, the first heat application unit 211 is moved from the initial position to the heating position by the moving mechanism. As a result, the gap between the second heat application unit 212 is changed and heating is performed while changing the interval between the inner walls of the first region 170 having flexibility (the first inner wall 110a and the second inner wall 120a). When the second region 180 is heated to the second temperature by the second heating unit 220, the fourth heat application unit 222 is moved from the initial position to the heating position by the moving mechanism, so that the third heat application unit 221 and Heating is performed by changing the gap between the inner walls of the flexible wall of the second region 180 (the first inner wall 110a and the second inner wall 120a). The reciprocating mechanism performs a reciprocating operation for moving the reaction solution 150 from the first region 170 to the second region 180 and moving the reaction solution 150 from the second region 180 to the first region 170.
Accordingly, when the reaction liquid 150 is located in the first region 170, the reaction liquid 150 is compressed and flattened by reducing the interval between the inner walls (the first inner wall 110a and the second inner wall 120a). Since the contact area between the liquid 150 and the first heat application unit 211 (second heat application unit 212) is increased, heat is transferred to the reaction liquid 150 at a high speed. When the reaction liquid 150 is located in the second region 180, the reaction liquid 150 is compressed and flattened by reducing the interval between the inner walls (the first inner wall 110a and the second inner wall 120a). Since the contact area between 150 and the third heat application unit 221 (fourth heat application unit 222) increases, heat is transferred to the reaction solution 150 at a high speed.
Therefore, it is possible to realize the heat cycle apparatus 1 that can increase the speed of heat transfer to the reaction liquid 150.

  (2) In the thermal cycle apparatus 1 of the present embodiment, when the reaction vessel 100 is mounted, the distance is set such that the reaction solution 150 contacts both the inner walls (the first inner wall 110a and the second inner wall 120a) facing each other. By reducing the distance between the inner walls (the first inner wall 110a and the second inner wall 120a) by the moving mechanism, the reaction solution 150 is compressed and spreads, and the reaction solution 150 and the first heating unit 210 (second heating unit) are compressed. 220) can be reliably increased.

  (3) In the heat cycle apparatus 1 of the present embodiment, the first region 170 is located at the lowermost part of the flow path 160 in the direction in which gravity acts by the reciprocating mechanism, and the second region 180 is The second arrangement located at the lowermost part of the flow path 160 in the acting direction is switched. By using gravity as in this configuration, the time required for heat denaturation, annealing, and elongation for amplifying a nucleic acid can be shortened.

  (4) The reaction vessel 100 of the present embodiment includes a first wall 110 having flexibility, a second wall 120 having flexibility and disposed relative to the first wall 110, and a first wall. 110 and a flow path 160 formed by being surrounded by the second wall 120. The distance between the first inner wall 110a and the second inner wall 120a is the distance at which the reaction liquid 150 contacts both the first inner wall 110a and the second inner wall 120a when the reaction liquid 150 is disposed in the flow path 160. Therefore, the heat application part (the first heat application part 211, the second heat application part 212, the third heat application part 221 and the fourth heat application part 222) is in contact with the outer wall of the reaction vessel 100, thereby reacting. The heat of the heat application unit can be efficiently transferred to the liquid 150. Furthermore, the contact area between the reaction solution 150 and the heat application unit can be increased by reducing the distance between both inner walls (the first inner wall 110a and the second inner wall 120a) and pressing the heat application unit. Further, the efficiency of heat conduction to the reaction liquid 150 can be improved.

(5) According to the nucleic acid amplification method of the present embodiment, the walls (the first wall 110, the second wall 120, and the third wall 130) forming the flow path 160 of the reaction vessel 100 have flexibility. . When the reaction container 100 is attached to the attachment part, the gap between the first heat application part 211 and the second heat application part 212 is changed by the moving mechanism to change the inner wall of the first region 170 of the reaction container 100 ( The distance between the first inner wall 110a and the second inner wall 120a) is changed. Next, the first region 170 is heated to the first temperature by the first heat application unit 211 and the second heat application unit 212 of the first heating unit 210. Next, the reaction solution 150 is moved from the first region 170 to the second region 180 by a reciprocating mechanism. Next, the gap between the third heat application unit 221 and the fourth heat application unit 222 is changed by the moving mechanism to change the inner wall of the second region 180 of the reaction vessel 100 (the first inner wall 110a and the second inner wall 120a). ). Next, the second region 180 is heated to the second temperature by the third heat application unit 221 and the fourth heat application unit 222 included in the second heating unit 220. Next, the reaction solution 150 is moved from the second region 180 to the first region 170 by a reciprocating mechanism. The process as described above is included.
Accordingly, when the reaction liquid 150 is located in the first region 170, the reaction liquid 150 is compressed and flattened by reducing the interval between the inner walls (the first inner wall 110a and the second inner wall 120a). Since the contact area between the liquid 150 and the first heat application unit 211 (second heat application unit 212) is increased, heat is transferred to the reaction liquid 150 at a high speed. When the reaction liquid 150 is located in the second region 180, the reaction liquid 150 is compressed and flattened by reducing the interval between the inner walls (the first inner wall 110a and the second inner wall 120a). Since the contact area between 150 and the third heat application unit 221 (fourth heat application unit 222) increases, heat is transferred to the reaction solution 150 at a high speed.
Accordingly, it is possible to realize a nucleic acid amplification method that can increase the speed of heat transfer to the reaction solution 150.

[Second Embodiment]
In the thermal cycle apparatus 2 of the present embodiment, the thermal cycle apparatus 1 of the first embodiment rotates the reaction vessel 100 to reciprocate the reaction solution 150 using gravity, whereas the reaction vessel 100 The difference is that the reaction solution 150 is reciprocated without rotating.

  FIG. 5 is a schematic cross-sectional view showing the positional relationship among the reaction vessel 100, the heating unit 200, and the pressing unit 300 in the thermal cycle apparatus 2 of the present embodiment. 6A to 6D are diagrams schematically showing an operation in the case of performing the two-step PCR using the thermal cycle apparatus 2. Specifically, FIG. 6A is a diagram illustrating a state in which the reaction liquid 150 in the first region 170 is heated. FIG. 6B is a diagram illustrating a state in which the reaction solution 150 in the first region 170 is moved to the second region 180. FIG. 6C is a diagram illustrating a state in which the reaction solution 150 in the second region 180 is heated. FIG. 6D is a diagram illustrating a state in which the reaction solution 150 in the second region 180 is moved to the first region 170. In addition, the same code | symbol is attached | subjected to the structure similar to 1st Embodiment.

  The thermal cycle device 2 of the present embodiment does not include the reciprocating mechanism having the configuration used in the first embodiment. The reciprocating mechanism of the present embodiment does not have a function of rotating the reaction vessel 100, and includes a moving mechanism described later, and moves the reaction solution 150 to perform a reciprocating operation. In other words, the reciprocating mechanism of this embodiment is substituted by the moving mechanism of this embodiment.

  The heat cycle apparatus 2 newly includes a pressing unit 300. In the first embodiment, the reciprocating mechanism (moving mechanism) moves the first heat applying unit 211 and the fourth heat applying unit 222 to the initial position and the heating position. The heat application unit 211 and the third heat application unit 221 are moved to the initial position and the heating position, and further moved to the pressing position. Further, the moving mechanism moves the pressing unit 300 between the initial position and the pressing position.

  As shown in FIG. 5, the thermal cycle device 2 of the present embodiment is configured to include a pressing unit 300 between the first heating unit 210 and the second heating unit 220 of the first embodiment. In other words, the pressing unit 300 is installed in a region between the first region 170 and the second region 180 of the reaction vessel 100 so as to be opposed to the reaction vessel 100.

  The pressing part 300 includes a first pressing part 301 and a second pressing part 302, which are opposed to the first wall 110 and the second wall 120, respectively. The first pressing portion 301 is a position that contacts the outer wall of the first wall 110 as an initial position by the moving mechanism, and a position that presses the first wall 110 as a pressing position to closely contact the first inner wall 110a with the second inner wall 120a. Move to two positions. The second pressing portion 302 is fixed in contact with the outer wall of the second wall 120.

The operation of the heat cycle apparatus 2 will be described.
The state shown in FIG. 5 is that the first heating unit 210 (first heat application unit 211), the second heating unit 220 (third heat application unit 221), and the pressing unit 300 (first pressing unit 301) are in the initial positions. It is a state, and shows a state in which the reaction vessel 100 is first attached to the attachment part. Moreover, the heating part 200 is heated in this state. Accordingly, the first heating unit 210 is heated to the first temperature, and the second heating unit 220 is heated to the second temperature.

  As shown in FIG. 6A, the first heat application unit 211 is moved to the heating position by the moving mechanism from the state in which the reaction vessel 100 is mounted on the mounting unit. This state is the same as that in the first embodiment and is efficient. The reaction solution 150 in the first region 170 is heated at a first temperature for a first time. Then, thermal denaturation that is a reaction at the first temperature is performed on the reaction solution 150.

  When the first time has elapsed, the first heat application unit 211 is moved from the heating position to the pressing position by the moving mechanism, and the first pressing unit 301 is also moved from the initial position to the pressing position. By this movement, as shown in FIG. 6B, the first inner wall 110a and the second inner wall 120a are in close contact with each other, so that the reaction solution 150 located in the first region 170 is not in close contact with the second region 180. Moving.

  Next, as illustrated in FIG. 6C, the moving mechanism maintains the state where the first heat application unit 211 and the first pressing unit 301 are moved to the pressing position, and moves the third heat applying unit 221 to the heating position. Let Thereby, the reaction liquid 150 in the second region 180 is efficiently heated at the second temperature for the second time. Then, annealing and extension reaction are performed on the reaction solution 150 as a reaction at the second temperature.

  When the second time elapses, the moving mechanism causes the third heat application unit 221 to move from the heating position to the pressing position, and the first pressing unit 301 moves from the pressing position to the initial position. The part 211 moves from the pressed position to the initial position. By this movement, as shown in FIG. 6D, the first inner wall 110a and the second inner wall 120a in the second region 180 are in close contact with each other, and in the other regions, the first inner wall 110a and the second inner wall 120a are in the initial state. Due to the shape, the reaction solution 150 located in the second region 180 moves to the first region 170.

  In the present embodiment, since the first region 170 is disposed below the flow path 160 in the direction in which gravity acts as compared to the second region 180, the reaction solution 150 is also affected by gravity. Smoothly move from the second area 180 to the first area 170. Next, the third heat application unit 221 is moved from the pressed position to the initial position, thereby returning to the initial state shown in FIG.

  Note that the nucleic acid in the reaction solution 150 can be amplified to a desired amount by repeating the necessary number of cycles with the above-described series of operations as one cycle.

  According to the above-described embodiment, the effect of the first embodiment can be obtained in the same manner, although it is different from the configuration of the first embodiment. In particular, according to the heat cycle device 2, the reciprocating mechanism (moving mechanism) causes the inner wall (the first inner wall 110a and the second inner wall 120a) to come into close contact with each other, thereby moving the reaction solution 150 to a non-contact region. Let the action take place. As in this configuration, the reaction solution 150 can be easily reciprocated without rotating the reaction vessel 100, and nucleic acid can be amplified.

[Third Embodiment]
The heat cycle apparatus 3 of this embodiment differs in the structure of the heating part 200 in the heat cycle apparatus 1 of 1st Embodiment. Other configurations are the same as those in the first embodiment.

  FIG. 7 is a schematic cross-sectional view showing the positional relationship between the reaction vessel 100 and the heating unit 200A in the thermal cycle device 3 of the present embodiment. 8A and 8B are schematic perspective views illustrating the configuration of one first heating unit 210A that constitutes the heating unit 200A. Specifically, FIG. 8A is a perspective view showing a state where the first heat application unit 211A of the first heating unit 210A is located at the initial position. FIG. 8B is a perspective view showing a state where the first heat application unit 211A of the first heating unit 210A is located at the heating position. 9A and 9B are schematic perspective views showing the configuration of the other second heating unit 220A that constitutes the heating unit 200A. Specifically, FIG. 9A is a perspective view illustrating a state where the fourth heat application unit 222A of the second heating unit 220A is located at the initial position. FIG. 9B is a perspective view illustrating a state where the fourth heat application unit 222A of the second heating unit 220A is located at the heating position. 9A and 9B are perspective views of the second heating unit 220A in the second arrangement as viewed from above.

  As shown in FIG. 7, FIG. 8A, FIG. 8B, FIG. 9A, and FIG. 9B, the heating unit 200A of the present embodiment that transfers heat to the reaction vessel 100 includes a first heating unit 210A and a second heating unit 220A. ing.

  As shown in FIGS. 7, 8A, and 8B, the first heating unit 210A includes a first heat application unit 211A, a second heat application unit 212A, and a first connection unit 213. The first connection part 213 is configured to have an L-shaped cross section by being fixed to the second heat application part 212A. The first heat application unit 211A is movably connected to the first connection unit 213 and can slide to the initial position and the heating position. The first connection unit 213 functions as a first connection region that thermally connects the first heat application unit 211A and the second heat application unit 212A.

  The second heating unit 220A is configured similarly to the first heating unit 210A. As shown in FIGS. 7, 9A, and 9B, the second heating unit 220A includes a third heat application unit 221A, a fourth heat application unit 222A, and a second connection unit 223. The second connection part 223 is configured to have an L-shaped cross section by being fixed to the third heat application part 221A. The fourth heat application unit 222A is movably connected to the second connection unit 223, and can slide to the initial position and the heating position. The second connection unit 223 functions as a second connection region that thermally connects the third heat application unit 221A and the fourth heat application unit 222A.

  In the first heating unit 210 of the first embodiment, the heat generated from the heater is branched and transmitted to the first heat application unit 211 and the second heat application unit 212 separately. However, the first heating unit 210A of the present embodiment is configured to transmit the heat generated from the heater to the first connection unit 213 without branching. The heat transferred to the first connection unit 213 can be transferred to the second heat application unit 212A and can be transferred to the movable first heat application unit 211A. Similarly, the second heating unit 220A of the present embodiment is configured to transmit the heat generated from the heater to the second connection unit 223 without branching. The heat transferred to the second connection part 223 can be transferred to the third heat application part 221A and can be transferred to the movable fourth heat application part 222A. In addition, 210 A of 1st heating parts hold | maintain temperature so that it may become 1st temperature. Further, the second heating unit 220A holds the temperature so as to be the second temperature.

  As shown in FIG. 7, the first heating unit 210 </ b> A and the second heating unit 220 </ b> A are configured in the same manner except for the directions at the time of installation. The state shown in FIG. 7 is a state in which the first heating unit 210A (first heat application unit 211A) and the second heating unit 220A (fourth heat application unit 222A) are in the initial positions, and the reaction vessel 100 is first attached. The state where it is attached to the part is shown.

  As shown in FIG. 7, the first region 170 is located in the first heating unit 210A. In the first region 170, the first heat application unit 211A is in contact with the outer wall of the first wall 110, the second heat application unit 212A is in contact with the outer wall of the second wall 120, and the first connection unit 213 is the third wall. It is in contact with the outer wall of the wall 130. Further, as shown in FIG. 7, the second region 180 is located in the second heating unit 220A. In the second region 180, the third heat application unit 221A is in contact with the outer wall of the first wall 110, and the fourth heat application unit 222A is in contact with the outer wall of the second wall 120.

10A to 10E are diagrams schematically showing an operation in the case of performing two-step PCR using the thermal cycler 3. Specifically, FIG. 10A is a cross-sectional view showing a state in which the first heat application unit 211A has moved to the heating position in the first arrangement. FIG. 10B is a cross-sectional view showing a state where the first arrangement is switched to the second arrangement. FIG. 10C is a cross-sectional view showing a state in which the first heat application unit 211A is moved to the initial position by switching to the second arrangement. FIG. 10D is a cross-sectional view showing the operation of the reaction liquid 150 when switched to the second arrangement. FIG. 10E is a cross-sectional view showing a state where the fourth heat application unit 222A has moved to the heating position in the second arrangement. 10A to 10E, the direction of the arrow g (the downward direction in the figure) is the direction in which gravity acts.
With reference to FIG. 7, FIG. 10A-FIG. 10E, the operation | movement at the time of performing PCR using the thermal cycle apparatus 3 is demonstrated easily. The operation of the heat cycle device 3 is the same as that of the heat cycle device 1 of the first embodiment.

  As shown in FIG. 7, in the first arrangement, the reaction vessel 100 is first attached to the attachment part, and the first heating part 210A (first heat application part 211A) and the second heating part 220A (fourth heat application part 222A). ) Heats the heating unit 200A in the initial position. Accordingly, the first heating unit 210A is heated to the first temperature, and the second heating unit 220A is heated to the second temperature.

  From the state where the reaction container 100 is mounted on the mounting portion, as shown in FIG. 10A, the first heat application portion 211A is slid with respect to the first connection portion 213 by the moving mechanism and moved from the initial position to the heating position. This state is the same as in the first embodiment, and the reaction solution 150 in the first region 170 is efficiently heated at the first temperature for the first time. Then, thermal denaturation that is a reaction at the first temperature is performed on the reaction solution 150.

  When the first time has elapsed, the first arrangement is switched to the second arrangement as shown in FIG. 10B by the reciprocating mechanism. In this case, the positional relationship between the first heat application unit 211A and the second heat application unit 212A is maintained.

  Next, as illustrated in FIG. 10C, the first heat application unit 211 </ b> A is slid with respect to the first connection unit 213 by the moving mechanism and moved from the heating position to the initial position. By this operation, the reaction solution 150 in the first region 170 moves to the second region 180 by gravity as shown in FIG. 10D.

  Next, as illustrated in FIG. 10E, in the second region 180, the moving mechanism slides the fourth heat application unit 222 </ b> A with respect to the second connection unit 223 to move from the initial position to the heating position. This state is the same as in the first embodiment, and the reaction solution 150 in the second region 180 is efficiently heated at the second temperature for the second time. Then, annealing and extension reaction are performed on the reaction solution 150 as a reaction at the second temperature.

  Next, although not illustrated, the second arrangement is switched to the first arrangement by the reciprocating mechanism, and the fourth heat application unit 222A is moved from the heating position to the initial position. As a result, the reaction solution 150 in the second region 180 moves to the first region 170 and enters the initial state shown in FIG. By repeating the necessary number of cycles with this series of operations as one cycle, the nucleic acid in the reaction solution 150 can be amplified to a desired amount.

  According to the above-described embodiment, although slightly different from the configuration of the first embodiment, the effects of the first embodiment can be obtained in the same manner, and the following effects can be achieved.

  (1) According to the thermal cycle device 3 of the present embodiment, the first heat application unit 211A and the second heat application unit 212A are thermally connected by the first connection unit 213 (first connection region), and the second The third heat application unit 221A and the fourth heat application unit 222A are thermally connected by the connection unit 223 (second connection region). With this configuration, temperature variation between the first heat application unit 211A and the second heat application unit 212A can be further suppressed as compared to the first embodiment, and the first region 170 of the reaction vessel 100 is heated to the first temperature. In this case, stable heat transfer from the reaction vessel 100 in contact with the first heating unit 210 </ b> A to the reaction solution 150 and high-speed transfer can be achieved. Similarly, the temperature variation between the third heat application unit 221A and the fourth heat application unit 222A can be further suppressed as compared to the first embodiment, and the second region 180 of the reaction vessel 100 is heated to the second temperature. In this case, stable heat transfer from the reaction vessel 100 in contact with the second heating unit 220 </ b> A to the reaction solution 150 and high-speed transfer can be achieved. In addition, when the first connection portion 213 is in contact with the reaction vessel 100 and heat transfer is possible, the heat transfer to the reaction liquid 150 can be further speeded up. The same applies to the second connection portion 223.

  (2) According to the thermal cycle device 3 of the present embodiment, the first heating unit 210A includes the first connection unit 213, and thermally connects the first heat application unit 211A and the second heat application unit 212A. doing. In addition, the 1st heat application part 211 and the 2nd heat application part 212 of 1st Embodiment branch and connect the heat | fever from a heater. However, in this embodiment, the heat from the heater is connected to the first connection portion 213 without branching. Thereby, compared with 1st Embodiment, a heating nonuniformity is hard to produce and heat can be efficiently transmitted to 210 A of 1st heating parts. Moreover, the member concerning a branch can be reduced. The same applies to the second heating unit 220A.

[Fourth Embodiment]
The heat cycle device 4 of the present embodiment is different in the shape of the surface in contact with the reaction vessel 100 of the first heating unit 210B, as compared to the heat cycle device 1 of the first embodiment. The shape of the surface in contact with the reaction vessel 100 of the second heating unit 220B is also different. Other configurations are the same as those of the first embodiment.

  FIG. 11 is a schematic cross-sectional view showing the positional relationship between the reaction vessel 100 and the heating unit 200B in the thermal cycle apparatus 4 of the present embodiment. In addition, FIG. 11 is a cross-sectional view from the third wall 130 side along the longitudinal direction (direction of the flow path 160). 12A and 12B are schematic cross-sectional views illustrating the configuration of one first heating unit 210B that configures the heating unit 200B. 12A and 12B are cross-sectional views showing a state cut in a direction perpendicular to the longitudinal direction. In detail, FIG. 12A is a perspective view showing a state in which the first heat application unit 211B of the first heating unit 210B is located at the initial position. FIG. 12B is a perspective view illustrating a state where the first heat application unit 211B of the first heating unit 210B is located at the heating position.

  13A and 13B are schematic cross-sectional views showing the configuration of the other second heating unit 220B that constitutes the heating unit 200B. 13A and 13B are cross-sectional views showing a state cut in a direction perpendicular to the longitudinal direction. Specifically, FIG. 13A is a perspective view showing a state where the fourth heat application unit 222B of the second heating unit 220B is located at the initial position. FIG. 13B is a perspective view illustrating a state where the fourth heat application unit 222B of the second heating unit 220B is located at the heating position. 13A and 13B show cross-sectional views of the second heating unit 220B as viewed from above in the second arrangement.

  As shown in FIG. 11, FIG. 12A, FIG. 12B, FIG. 13A, and FIG. 13B, the heating unit 200B of the present embodiment that transfers heat to the reaction vessel 100 includes a first heating unit 210B and a second heating unit 220B. ing.

  As shown in FIGS. 12A and 12B, the first heating unit 210B includes a first heat application unit 211B and a second heat application unit 212B. The first heating unit 210B is installed relative to the first region 170, the first heat application unit 211B is installed relative to the first wall 110 of the reaction vessel 100, and the second heat application unit 212B is installed on the second wall. It is installed relative to 120.

  As shown in FIG. 12A, the surface 211S of the first heat application unit 211B (the surface 211S in contact with the first wall 110) facing the first wall 110 of the reaction vessel 100 has a convex part 2111 and a concave part 2112. Yes. In other words, the surface 211S is configured to have an uneven portion. The surface 212S of the second heat application unit 212B facing the second wall 120 of the reaction vessel 100 (the surface 212S in contact with the second wall 120) has a recess 2121 and a projection 2122 as shown in FIG. 12B. Yes. In other words, the surface 212S is configured to have an uneven portion.

  The concave portion 2121 of the second heat application unit 212B is formed to correspond to the convex portion 2111 of the first heat application unit 211B opposed via the reaction vessel 100. In addition, the convex portion 2122 of the second heat application unit 212 </ b> B is formed corresponding to the concave portion 2112 of the first heat application unit 211 </ b> B facing through the reaction vessel 100.

  As shown in FIGS. 13A and 13B, the second heating unit 220B includes a third heat application unit 221B and a fourth heat application unit 222B. The second heating unit 220B is installed relative to the second region 180, the third heat application unit 221B is installed relative to the first wall 110 of the reaction vessel 100, and the fourth heat application unit 222B is installed on the second wall. It is installed relative to 120.

  The surface 221S of the third heat application unit 221B facing the first wall 110 of the reaction vessel 100 (the surface 221S in contact with the first wall 110) has a convex portion 2211 and a concave portion 2212 as shown in FIG. 13A. Yes. In other words, the surface 221S is configured to have an uneven portion. The surface 222S of the fourth heat application unit 222B facing the second wall 120 of the reaction vessel 100 (the surface 222S in contact with the second wall 120) has a concave portion 2221 and a convex portion 2222 as shown in FIG. 13B. Yes. In other words, the surface 222S has a concavo-convex portion.

  The concave portion 2221 of the fourth heat application unit 222B is formed to correspond to the convex portion 2211 of the third heat application unit 221B that is opposed to the fourth heat application unit 222B. Further, the convex part 2222 of the fourth heat application part 222B is formed corresponding to the concave part 2212 of the third heat application part 221B that is opposed via the reaction vessel 100.

  In the initial state (FIG. 11) of the heating unit 200B, as shown in FIGS. 12A and 13A, when viewed from above the heating unit 200B, the shapes of the convex portions 2111 and the concave portions 2112 of the first heat application unit 211B Is substantially the same as the shape of the convex part 2211 and the concave part 2212 of the third heat application part 221B. In addition, the shapes of the concave portion 2121 and the convex portion 2122 of the second heat application unit 212B substantially coincide with the shapes of the concave portion 2221 and the convex portion 2222 of the fourth heat application unit 222B.

  Accordingly, when the first region 170 is heated or the second region 180 is heated, the deformed uneven shape of the reaction vessel 100 can be matched between the first region 170 and the second region 180. With this configuration, twisting due to deformation of the reaction vessel 100 is prevented, and damage to the reaction vessel 100 due to local stress concentration on the reaction vessel 100 is prevented. This is particularly effective when the PCR cycle speed is increased or when the installation distance between the first heating unit 210B and the second heating unit 220B is short.

  In addition, since the operation | movement when performing PCR by the heating part 200B of this embodiment is the same as the operation | movement when performing PCR in 1st Embodiment, detailed description of operation | movement is abbreviate | omitted. Hereinafter, the operation regarding the heating unit 200B different from the first embodiment will be mainly described.

  Description will be made on the assumption that the heat cycle device 4 has the first arrangement and the reaction solution 150 is located in the first region 170. The description will be made on the assumption that the first heating unit 210B (first heat application unit 211B) is heated to the first temperature at the initial position.

  When heating the first region 170, the first heating unit 210B moves the first heat application unit 211B from the initial position to the heating position by the moving mechanism. When the 1st heat application part 211B moves to a heating position, as shown to FIG. 12B, the reaction container 100 becomes the shape of the convex part 2111 of the 1st heat application part 211B, and the recessed part 2121 of the 2nd heat application part 212B. Further, the distance between the inner walls (the first inner wall 110a and the second inner wall 120a) of the reaction vessel 100 is also reduced and sandwiched. Similarly, the reaction vessel 100 is deformed by being sandwiched and deformed along the shapes of the concave portion 2112 of the first heat application unit 211B and the convex portion 2122 of the second heat application unit 212B, and each inner wall of the reaction vessel 100 is further deformed. The distance between the first inner wall 110a and the second inner wall 120a is also reduced.

  By heating the first region 170 in this state, the contact area to be heated can be increased as compared to the first embodiment, so that the reaction liquid 150 is efficiently heated to the first temperature. Thereby, the heat denaturation which becomes the reaction at the first temperature is performed on the reaction liquid 150. In addition, the 1st time as reaction time in heat denaturation can also be made shorter than the 1st time of 1st Embodiment.

  Next, when the second arrangement is achieved by the reciprocating mechanism, the first heat application unit 211B moves to the initial position as in the first embodiment, so that the deformation of the reaction vessel 100 returns, and the reaction solution 150 becomes gravity. To move to the second region 180.

  Next, description will be made on the assumption that the heat cycle apparatus 4 has the second arrangement and the reaction liquid 150 is located in the second region 180. The description will be made on the assumption that the second heating unit 220B (fourth heat application unit 222B) is heated to the second temperature at the initial position.

  When heating the second region 180, the second heating unit 220B moves the fourth heat application unit 222B from the initial position to the heating position by the moving mechanism. When the 4th heat application part 222B moves to a heating position, as shown to FIG. 13B, the reaction container 100 has the convex part 2222 and the recessed part 2221 of the 4th heat application part 222B, and the recessed part 2212 of the 3rd heat application part 221B. Further, it is sandwiched and deformed along the shape of the convex portion 2211, and the distance between the inner walls (the first inner wall 110a and the second inner wall 120a) of the reaction vessel 100 is also reduced and sandwiched.

  By heating the second region 180 in this state, the contact area to be heated can be increased as compared with the first embodiment, so that the reaction solution 150 is efficiently heated to the second temperature. As a result, annealing and extension reaction are performed on the reaction solution 150 as a reaction at the second temperature. It should be noted that the second time as the reaction time in the annealing and extension reaction can also be shorter than the second time in the first embodiment.

  Next, when the first arrangement is made by the reciprocating mechanism, the fourth heat applying unit 222B moves to the initial position as in the first embodiment, so that the deformation of the reaction vessel 100 returns, and the reaction solution 150 becomes gravity. To move to the first area 170.

  The nucleic acid in the reaction solution 150 can be amplified to a desired amount by repeating the necessary number of cycles with the series of operations described above as one cycle.

  According to the above-described embodiment, although slightly different from the configuration of the first embodiment, the effects of the first embodiment can be obtained in the same manner, and the following effects can be achieved.

  (1) According to the thermal cycle device 4 of the present embodiment, the first heat application unit 211B has the convex part 2111 and the concave part 2112, and the second heat application part 212B has the concave part 2121 at a position corresponding to the convex part 2111. And a convex portion 2122 at a position corresponding to the concave portion 2112. With this configuration, the flexible reaction vessel 100 is pressed (held) between the convex portion 2111 of the first heat application unit 211B and the concave portion 2121 of the second heat application unit 212B. Further, the reaction vessel 100 is pressed (held) between the concave portion 2112 of the first heat application unit 211B and the convex portion 2122 of the second heat application unit 212B. Thereby, the contact area of the 1st wall 110 and the 2nd wall 120, and each convex part recessed part can be increased compared with the case where it contacts on a plane. Therefore, the efficiency of heat conduction to the reaction solution 150 can be improved. Moreover, reaction time can be shortened.

  Note that the present invention is not limited to the above-described embodiment, and various modifications and improvements can be added to the above-described embodiment. A modification will be described below.

  (1) In the second embodiment, the reaction vessel 100 is attached to the attachment portion so that the direction of the flow channel 160 is parallel to the direction of gravity. However, the reaction vessel 100 has the horizontal direction of the flow channel 160 in the horizontal direction. It is good also as a structure mounted so that it may become. With this configuration, the reaction solution 150 can be moved back and forth between the first region 170 and the second region 180.

(2) Press configured similarly to the pressing unit 300 (first pressing unit 301, second pressing unit 302) in the second embodiment using the heating unit 200A of the heat cycle device 3 in the third embodiment. Similarly to the second embodiment, the heat cycle device may be configured by installing the unit between the first heating unit 210A and the second heating unit 220A.
By adopting this configuration, similarly to the second embodiment, the reaction solution 150 is brought into an area where the inner wall (the first inner wall 110a and the second inner wall 120a) is brought into close contact with each other by the reciprocating mechanism (moving mechanism). A reciprocating motion can be performed by moving the same. Thereby, the reaction solution 150 can be easily reciprocated without rotating the reaction vessel 100, and the nucleic acid can be amplified.

(3) Press configured similarly to the pressing unit 300 (first pressing unit 301, second pressing unit 302) in the second embodiment, using the heating unit 200B of the heat cycle device 4 in the fourth embodiment. Similarly to the second embodiment, the heat cycle device may be configured by installing the unit between the first heating unit 210B and the second heating unit 220B. In this case, the surface opposite to the outer wall of the reaction vessel 100 of the first pressing portion and the second pressing portion constituting the pressing portion has a shape similar to the shape of the convex portion and the concave portion of each heat application portion. It's okay.
By adopting this configuration, similarly to the second embodiment, the reaction solution 150 is brought into an area where the inner wall (the first inner wall 110a and the second inner wall 120a) is brought into close contact with each other by the reciprocating mechanism (moving mechanism). A reciprocating motion can be performed by moving the same. Thereby, the reaction solution 150 can be easily reciprocated without rotating the reaction vessel 100, and the nucleic acid can be amplified.

  (4) In the first embodiment, when the first time has elapsed, the rotation is driven to the second arrangement, but the period of the first arrangement, the period of rotation from the first arrangement to the second arrangement And the period in which the first heat application unit 211 is not moved to the initial position and the heating position is maintained without being moved to the initial position is the time during which heat denaturation by the first temperature is performed. It is sufficient to appropriately determine the control time and control method in consideration of the time. The same applies to the second time. The same applies to the first time and the second time in the third and fourth embodiments.

  (5) In the first embodiment, the first heat application unit 211 and the fourth heat application unit 222 are moved to the initial position and the heating position, but the present invention is not limited to this. Either the first heat application unit 211 or the second heat application unit 212, or any one of the third heat application unit 221 or the fourth heat application unit 222 may be moved between the initial position and the heating position. . Moreover, both the 1st heat application part 211 and the 2nd heat application part 212, and both the 3rd heat application part 221 and the 4th heat application part 222 may move to an initial position and a heating position. This also applies to the third and fourth embodiments.

  (6) In the first embodiment, the example in which the material of the first heating unit 210 and the second heating unit 220 is aluminum is shown. However, the material of the heating unit 200 is thermal conductivity, heat retention, and ease of processing. It can be selected in consideration of such conditions. For example, a copper alloy may be used and a plurality of materials may be combined. The first heating unit 210 and the second heating unit 220 may be made of different materials. The same applies to the second, third, and fourth embodiments.

  (7) In the first embodiment, the reaction vessel 100 includes the first wall 110, the second wall 120, and the third wall 130 as shown in FIGS. 1A and 1B, and the third wall 130 is thin. It has a rectangular parallelepiped shape. However, it is not limited to this shape. For example, the third wall that is the side surface in the longitudinal direction is eliminated, and the third wall that forms the surface in the vertical direction is configured with one wall that is continuous so that the longitudinal direction connects the outer circumferences of the upper and lower third walls. It is good also as a container. In this case, the side surface in the longitudinal direction is formed by one wall, but in the positional relationship described in the first embodiment, the opposing wall is the first wall, the second wall, or the opposing inner wall is the first inner wall. The second inner wall may be used.

  1, 2, 3, 4 ... Thermal cycle device, 100 ... Reaction vessel, 110 ... First wall, 110a ... First inner wall, 120 ... Second wall, 120a ... Second inner wall, 130 ... Third wall, 130a ... First 3 inner walls, 150 ... reaction liquid, 160 ... channel, 170 ... first region, 180 ... second region, 210, 210A, 210B ... first heating unit, 211, 211A, 211B ... first heat application unit, 212, 212A, 212B: second heat application unit, 220, 220A, 220B ... second heating unit, 213 ... first connection unit, 221, 221A, 221B ... third heat application unit, 222, 222A, 222B ... fourth heat application Part, 223 ... second connection part, 300 ... pressing part, 301 ... first pressing part, 302 ... second pressing part, 2111 ... convex part of the first heat application part, 2121 ... concave part of the second heat application part, 2212 ... Concave part of third heat application part, 2222 The convex portion of the fourth heat application portion.

Claims (8)

A mounting part to which a reaction vessel is formed in which a flow path is formed by a flexible wall and the reaction solution is movable between the first region and the second region;
A first heating part having a first heat application part and a second heat application part for heating the first region to a first temperature when the reaction vessel is attached to the attachment part;
A second heating part having a third heat application part and a fourth heat application part for heating the second region to a second temperature when the reaction container is attached to the attachment part;
A reciprocating mechanism for causing the reaction solution to reciprocate between the first region and the second region;
A gap between the first portion of the inner wall of the wall of the first region and the portion of the inner wall facing the first portion by changing the gap between the first heat applying portion and the second heat applying portion. And changing the gap between the third heat application part and the fourth heat application part to change the inner wall opposite to the second part and the second part of the inner wall of the second region. A moving mechanism that changes the distance from the part,
A thermal cycle apparatus comprising: The thermal cycle device according to claim 1,
The reaction vessel has a first inner wall and a second inner wall facing each other,
The distance between the first inner wall and the second inner wall is such that both the first inner wall and the second inner wall facing the reaction liquid when the reaction liquid is disposed in the flow path. A thermal cycle device characterized in that the distance is in contact with the heat cycle device. The thermal cycle device according to claim 1 or 2, wherein
The reciprocating mechanism switches the placement of the mounting portion, the first heating portion, and the second heating portion between the first placement and the second placement,
The first arrangement is an arrangement in which the first region is located at the lowest part of the flow path in the direction in which gravity acts.
The second arrangement is an arrangement in which the second region is located at a lowermost portion of the flow path in a direction in which gravity acts. The thermal cycle device according to claim 1 or 2, wherein
The reciprocating mechanism includes the moving mechanism, and moves the reaction liquid by closely contacting the inner wall of the reaction vessel to perform the reciprocating operation. It is a heat cycle apparatus as described in any one of Claims 1-4, Comprising:
A first connection region that thermally connects the first heat application unit and the second heat application unit;
A second connection region for thermally connecting the third heat application unit and the fourth heat application unit;
A thermal cycle apparatus comprising: It is a heat cycle apparatus as described in any one of Claims 1-5, Comprising:
The first heat application part has a convex part, and the second heat application part has a concave part at a position corresponding to the convex part. A flexible first wall;
A second wall having flexibility and disposed relative to the first wall;
A channel formed by being surrounded by a wall,
The distance between the inner wall of the first wall and the inner wall of the second wall is such that when the reaction liquid is disposed in the flow path, the reaction liquid is separated from the inner wall of the first wall and the second wall. A reaction vessel characterized by being a distance in contact with both the inner wall of the container. A nucleic acid amplification method for amplifying a nucleic acid by moving a reaction solution between a first region and a second region of a flow path formed in a reaction vessel having a flexible wall,
Changing the gap between the first heat application part and the second heat application part to change the distance between the inner walls of the wall of the first region;
Heating the first region to a first temperature by the first heat application unit and the second heat application unit;
Moving the reaction solution from the first region to the second region;
Changing the gap between the third heat application part and the fourth heat application part to change the interval between the inner walls of the walls of the second region;
Heating the second region to a second temperature by the third heat application unit and the fourth heat application unit;
Moving the reaction solution from the second region to the first region;
A nucleic acid amplification method comprising:

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