Patent Publication Number: US-2015087026-A1

Title: Thermal cycler and control method of thermal cycler

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This is a continuation patent application of U.S. application Ser. No. 13/796,498 filed Mar. 12, 2013 which claims priority to Japanese Patent Application No. 2012-079766 filed Mar. 30, 2012, all of which are incorporated by reference herein in their entireties. 
    
    
     BACKGROUND 
     1. Technical Field 
     The present invention relates to a thermal cycler and a control method of the thermal cycler. 
     2. Related Art 
     Recently, with development of utilization technologies of genes, medical treatment utilizing genes such as gene diagnoses and gene therapies has attracted attention, and many techniques using genes for breed identification and breed improvement have been developed in agriculture and livestock fields. As technologies for utilizing genes, a technology such as a PCR (Polymerase Chain Reaction) method has been widespread. Today, the PCR method is an essential technology in elucidation of information of biological materials. 
     The PCR method is a technique of amplifying target nucleic acid by applying thermal cycling to a solution containing nucleic acid as a target of amplification (target nucleic acid) and reagent (reaction solution). The thermal cycling is processing of periodically applying two or more steps of temperatures to the reaction solution. In the PCR method, generally, thermal cycling of two or three steps is applied. 
     In the PCR method, generally, a container for biochemical reaction called a tube or a chip for biological sample reaction (biochip) is used. However, in the technique of related art, there have been problems that large amounts of reagent etc. are necessary, equipment becomes complex for realization of thermal cycling necessary for reaction, and the reaction takes time. Accordingly, biochips and reactors for performing PCR with high accuracy in short time using extremely small amounts of reagent and specimen have been required. 
     In order to solve the problem, Patent Document 1 (JP-A-2009-136250) has disclosed a biological sample reactor of performing thermal cycling by rotating a chip for biological sample reaction filled with a reaction solution and a liquid being immiscible with the reaction solution and having a lower specific gravity than that of the reaction solution around a rotation axis in the horizontal direction to move the reaction solution. 
     Further, for improvement in accuracy of amplification in PCR, PCR including a step of hot start of activating enzyme used for PCR (PCR enzyme) with heat has been known. 
     The equipment disclosed in Patent Document 1 has applied thermal cycling to a reaction solution by continuously rotating a biochip. However, additional ideas have been required for holding the reaction solution at a desired temperature in a desired period and realizing PCR including a step of hot start or the like different from those of thermal cycling of normal PCR because the reaction solution moves within a channel of the biochip with the rotation. 
     SUMMARY 
     An advantage of some aspects of the invention is to provide a thermal cycler and a control method of the thermal cycler suitable for PCR including hot start. 
     (1) A thermal cycler according to an aspect of the invention includes an attachment unit for attachment of a reaction container including a channel filled with a reaction solution containing hot start PCR enzyme and a liquid having a specific gravity different from that of the reaction solution and being immiscible with the reaction solution, the reaction solution moving close to opposed inner walls, a first heating unit that heats a first region of the channel when the reaction container is attached to the attachment unit, a second heating unit that heats a second region of the channel different from the first region when the reaction container is attached to the attachment unit, a drive mechanism that switches arrangement of the attachment unit, the first heating unit, and the second heating unit between a first arrangement in which a lowermost position of the channel in a direction in which gravity acts is located within the first region and a second arrangement in which the lowermost position of the channel in the direction in which the gravity acts is located within the second region when the reaction container is attached to the attachment unit, and a control unit that controls the drive mechanism, the first heating unit, and the second heating unit, wherein the control unit performs first processing of controlling the first heating unit at a first temperature, second processing of controlling the second heating unit at a second temperature higher than the first temperature, third processing of allowing a first period to elapse with the arrangement of the attachment unit, the first heating unit, and the second heating unit being the second arrangement after the second processing, and fourth processing of controlling the drive mechanism to switch the arrangement of the attachment unit, the first heating unit, and the second heating unit from the second arrangement to the first arrangement if a second period has elapsed with the arrangement of the attachment unit, the first heating unit, and the second heating unit being the second arrangement after the first processing and the third processing. 
     According to the aspect of the invention, the state in which the reaction container is held in the first arrangement and the state in which the reaction container is held in the second arrangement may be switched by switching the arrangement of the attachment unit, the first heating unit, and the second heating unit. The first arrangement is the arrangement in which the first region of the channel forming the reaction container is located in the lowermost part of the channel in the direction in which the gravity acts. The second arrangement is the arrangement in which the second region of the channel forming the reaction container is located in the lowermost part of the channel in the direction in which the gravity acts. That is, when the specific gravity of the reaction solution is relatively large, the reaction solution may be held in the first region in the first arrangement and the reaction solution may be held in the second region in the second arrangement by the action of the gravity. The first region is heated by the first heating unit and the second region is heated by the second heating unit, and thereby, the first region and the second region may be set at different temperatures. Therefore, the reaction solution may be held at a predetermined temperature while the reaction container is held in the first arrangement or the second arrangement, and the thermal cycler that can easily control the heating period may be provided. Further, the reaction solution is held at the second temperature in the third processing and the fourth processing, and the reaction solution is held at the first temperature lower than the second temperature after the fourth processing. When the thermal cycler is applied to PCR, the first temperature corresponds to an annealing and elongation temperature and the second temperature corresponds to a denaturation temperature of DNA. Generally, the temperature at which the PCR enzyme is activated is nearly equal to the denaturation temperature. Therefore, by performing the third processing, thermal cycling that enables hot start of PCR may be realized in addition to the thermal cycling of normal PCR. 
     (2) In the above described thermal cycler, the control unit may perform fifth processing of controlling the drive mechanism to switch the arrangement of the attachment unit, the first heating unit, and the second heating unit from the first arrangement to the second arrangement and the forth processing repeatedly at a predetermined number of times if a third period has elapsed with the arrangement of the attachment unit, the first heating unit, and the second heating unit being the first arrangement after the fourth processing. 
     The reaction solution is held at the second temperature until the second period has elapsed in the second arrangement in the fourth processing, and the reaction solution is held at the first temperature until the third period has elapsed in the first arrangement in the fifth processing. Therefore, thermal cycling suitable for PCR may be performed repeatedly at a predetermined number of times. 
     (3) In the above described thermal cycler, the control unit may further perform sixth processing of controlling the first heating unit at a third temperature lower than the first temperature and allowing a fourth period to elapse with the arrangement of the attachment unit, the first heating unit, and the second heating unit being the first arrangement, seventh processing of controlling the drive mechanism to switch the arrangement of the attachment unit, the first heating unit, and the second heating unit from the first arrangement to the second arrangement after the sixth processing and the second processing, and third processing after the seventh processing. 
     The reaction solution is held at the third temperature lower than the first temperature in the seventh processing. The third temperature may be set to a temperature at which reverse transcription reaction progresses in RT-PCR (reverse transcription polymerase chain reaction). Therefore, by performing the seventh processing prior to the third processing, the reverse transcription reaction may be performed before PCR, and thus, the thermal cycler suitable for RT-PCR may be realized. 
     (4) A control method of a thermal cycler according to an aspect of the invention is a control method of a thermal cycler, and the thermal cycler includes an attachment unit for attachment of a reaction container including a channel filled with a reaction solution containing hot start PCR enzyme and a liquid having a specific gravity different from that of the reaction solution and being immiscible with the reaction solution, the reaction solution moving close to opposed inner walls, a first heating unit that heats a first region of the channel when the reaction container is attached to the attachment unit, a second heating unit that heats a second region of the channel different from the first region when the reaction container is attached to the attachment unit, and a drive mechanism that switches arrangement of the attachment unit, the first heating unit, and the second heating unit between a first arrangement in which a lowermost position of the channel in a direction in which gravity acts is located within the first region and a second arrangement in which the lowermost position of the channel in the direction in which the gravity acts is located within the second region when the reaction container is attached to the attachment unit, and the control method includes performing first processing of controlling the first heating unit at a first temperature, performing second processing of controlling the second heating unit at a second temperature higher than the first temperature, performing third processing of allowing a first period to elapse with the arrangement of the attachment unit, the first heating unit, and the second heating unit being the second arrangement, and performing fourth processing of controlling the drive mechanism to switch the arrangement of the attachment unit, the first heating unit, and the second heating unit from the second arrangement to the first arrangement if a second period has elapsed with the arrangement of the attachment unit, the first heating unit, and the second heating unit being the second arrangement after the third processing. 
     According to the aspect of the invention, the state in which the reaction container is held in the first arrangement and the state in which the reaction container is held in the second arrangement may be switched by switching the arrangement of the attachment unit, the first heating unit, and the second heating unit. The first arrangement is the arrangement in which the first region of the channel forming the reaction container is located in the lowermost part of the channel in the direction in which the gravity acts. The second arrangement is the arrangement in which the second region of the channel forming the reaction container is located in the lowermost part of the channel in the direction in which the gravity acts. That is, when the specific gravity of the reaction solution is relatively large, the reaction solution may be held in the first region in the first arrangement and the reaction solution may be held in the second region in the second arrangement by the action of the gravity. The first region is heated by the first heating unit and the second region is heated by the second heating unit, and thereby, the first region and the second region may be set at different temperatures. Therefore, the reaction solution may be held at a predetermined temperature while the reaction container is held in the first arrangement or the second arrangement, and the control method of the thermal cycler that can easily control the heating period may be provided. Further, the reaction solution is held at the second temperature in the third processing and the fourth processing, and the reaction solution is held at the first temperature lower than the second temperature after the fourth processing. When the thermal cycler is applied to PCR, the first temperature corresponds to the annealing and elongation temperature and the second temperature corresponds to the denaturation temperature of DNA. Generally, the temperature at which the PCR enzyme is activated is nearly equal to the denaturation temperature. Therefore, by performing the third processing, thermal cycling that enables hot start of PCR may be realized in addition to the thermal cycling of normal PCR. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The invention will be described with reference to the accompanying drawings, wherein like numbers reference like elements. 
         FIG. 1  is a perspective view of a thermal cycler  1  according to an embodiment. 
         FIG. 2  is an exploded perspective view of a main body  10  of the thermal cycler  1  according to the embodiment. 
         FIG. 3  is a vertical sectional view along A-A line in  FIG. 1 . 
         FIG. 4  is a sectional view showing a configuration of a reaction container  100  to be attached to the thermal cycler  1  according to the embodiment. 
         FIG. 5  is a functional block diagram of the thermal cycler  1  according to the embodiment. 
         FIG. 6A  is a sectional view schematically showing a section in a plane passing through the A-A line of  FIG. 1A  and perpendicular to a rotation axis R in a first arrangement, and  FIG. 6B  is a sectional view schematically showing a section in the plane passing through the A-A line of  FIG. 1A  and perpendicular to the rotation axis R in a second arrangement. 
         FIG. 7  is a flowchart for explanation of a first specific example of a control method of the thermal cycler  1  according to the embodiment. 
         FIG. 8  is a flowchart for explanation of a second specific example of a control method of the thermal cycler  1  according to the embodiment. 
         FIG. 9  is a table showing a composition of a reaction solution  140  in a first working example. 
         FIG. 10  is a table showing base sequences of forward primers (F primers), reverse primers (R primers), and probes. 
         FIG. 11  is a graph showing relationships between the number of cycles of thermal cycling processing and measured brightness in the first working example. 
         FIG. 12  is a table showing a composition of the reaction solution  140  in a second working example. 
         FIG. 13  is a graph showing relationships between the number of cycles of thermal cycling processing and measured brightness in the second working example. 
     
    
    
     DESCRIPTION OF EXEMPLARY EMBODIMENTS 
     As below, preferred embodiments of the invention will be explained in detail using the drawings. Note that the embodiments to be explained do not unduly limit the invention described in the appended claims. Further, not all of the configurations to be explained are essential component elements of the invention. 
     1. Overall Configuration of Thermal Cycler According to Embodiment 
       FIG. 1  is a perspective view of a thermal cycler  1  according to an embodiment.  FIG. 2  is an exploded perspective view of a main body  10  of the thermal cycler  1  according to the embodiment.  FIG. 3  is a vertical sectional view along A-A line in  FIG. 1 . In  FIG. 3 , arrow g indicates a direction in which gravity acts. 
     The thermal cycler  1  according to the embodiment includes an attachment unit  15  for attachment of a reaction container  100  including a channel  110  filled with a reaction solution  140  containing hot start PCR enzyme and a liquid  130  having a specific gravity different from that of the reaction solution  140  and being immiscible with the reaction solution  140 , the reaction solution  140  moving close to opposed inner walls (the details will be described later in section of “2. Configuration of Reaction Container attached to Thermal Cycler according to Embodiment”), a first heating unit  21  that heats a first region  111  of the channel  110  when the reaction container  100  is attached to the attachment unit  15 , a second heating unit  22  that heats a second region  112  of the channel  110  different from the first region  111  when the reaction container  100  is attached to the attachment unit  15 , a drive mechanism  30  that switches arrangement of the attachment unit  15 , the first heating unit  21 , and the second heating unit  22  between a first arrangement in which the lowermost position of the channel  110  in a direction in which gravity acts is located within the first region  111  and a second arrangement in which the lowermost position of the channel  110  in the direction in which the gravity acts is located within the second region  112  when the reaction container  100  is attached to the attachment unit  15 , and a control unit  40  that controls the drive mechanism  30 , the first heating unit  21 , and the second heating unit  22 . 
     In the example shown in  FIG. 1 , the thermal cycler  1  includes the main body  10  and the drive mechanism  30 . As shown in  FIG. 2 , the main body  10  includes the attachment unit  15 , the first heating unit  21 , and the second heating unit  22 . 
     The attachment unit  15  has a structure to which the reaction container  100  is attached. In the example shown in  FIGS. 1 and 2 , the attachment unit  15  of the thermal cycler  1  has a slot structure with an insertion opening  151  into which the reaction container  100  is attached by insertion from the insertion opening  151 . In the example shown in  FIG. 2 , the attachment unit  15  has a structure in which the reaction container  100  is inserted into a hole penetrating a first heat block  21   b  of the first heating unit  21  and a second heat block  22   b  of the second heating unit  22 . The first heat block  21   b  and the second heat block  22   b  will be described later. A plurality of the attachment units  15  may be provided in the main body  10 , and ten attachment units  15  are provided in the main body  10  in the example shown in  FIGS. 1 and 2 . Further, in the example shown in  FIGS. 2 and 3 , the attachment unit  15  is formed as a part of the first heating unit  21  and the second heating unit  22 , however, the attachment unit  15  and the first heating unit  21  and the second heating unit  22  may be formed as separate members as long as the positional relationship between them may not change when the drive mechanism  30  is operated. 
     Note that, in the embodiment, the example in which the attachment unit  15  has the slot structure has been shown, however, the attachment unit  15  has any structure as long as it may hold the reaction container  100 . For example, a structure of fitting the reaction container  100  in a recess that conforms to the shape of the reaction container  100  or a structure of sandwiching and holding the reaction container  100  may be employed. 
     The first heating unit  21  heats the first region  111  of the channel  110  of the reaction container  100  when the reaction container  100  is attached to the attachment unit  15 . In the example shown in  FIG. 3 , the first heating unit  21  is located in a position for heating the first region  111  of the reaction container  100  in the main body  10 . 
     The first heating unit  21  may include a mechanism of generating heat and a member of transmitting the generated heat to the reaction container  100 . In the example shown in  FIG. 2 , the first heating unit  21  includes a first heater  21   a  as a mechanism of generating heat and the first heat block  21   b  as a member of transmitting the generated heat to the reaction container  100 . 
     In the thermal cycler  1 , the first heater  21   a  is a cartridge heater and connected to an external power supply (not shown) by a conducting wire  19 . The first heater  21   a  is not limited but includes a carbon heater, a sheet heater, an IH heater (electromagnetic induction heater), a Peltier device, a heating liquid, a heating gas, etc. The first heater  21   a  is inserted into the first heat block  21   b  and the first heater  21   a  generates heat to heat the first heat block  21   b . The first heat block  21   b  is a member of transmitting the heat generated from the first heater  21   a  to the reaction container  100 . In the thermal cycler  1 , the first heat block  21   b  is an aluminum block. The cartridge heater is easily temperature-controlled, and, with the cartridge heater for the first heater  21   a , the temperature of the first heating unit  21  may be easily stabilized. Therefore, more accurate thermal cycling may be realized. 
     The material of the heat block may be appropriately selected in consideration of conditions of coefficient of thermal conductivity, heat retaining characteristics, ease of working, etc. For example, aluminum has a high coefficient of thermal conductivity, and, by forming the first heat block  21   b  using aluminum, the reaction container  100  may be efficiently heated. Further, unevenness in heating is hard to be produced in the heat block, and the thermal cycling with high accuracy may be realized. Furthermore, working is easy, and the first heat block  21   b  may be molded with high accuracy and the heating accuracy may be improved. Therefore, more accurate thermal cycling may be realized. Note that, for the material of the heat block, for example, copper alloy may be used or several materials may be combined. 
     It is preferable that the first heating unit  21  is in contact with the reaction container  100  when the attachment unit  15  is attached to the reaction container  100 . Thereby, when the reaction container  100  is heated by the first heating unit  21 , the heat of the first heating unit  21  may be transmitted to the reaction container  100  more stably than in the configuration in which the first heating unit  21  is not in contact with the reaction container  100 , and thus, the temperature of the reaction container  100  may be stabilized. When the attachment unit  15  is formed as the part of the first heating unit  21  like in the embodiment, it is preferable that the attachment unit  15  is in contact with the reaction container  100 . Thereby, the heat of the first heating unit  21  may be stably transmitted to the reaction container  100 , and the reaction container  100  may be efficiently heated. 
     The second heating unit  22  heats the second region  112  of the channel  110  of the reaction container  100  nearer the insertion opening  151  than the first region  111  to a second temperature different from the first temperature when the attachment unit  15  is attached to the reaction container  100 . In the example shown in  FIG. 3 , the second heating unit  22  is located in a position for heating the second region  112  of the reaction container  100  in the main body  10 . The second heating unit  22  includes a second heater  22   a  and a second heat block  22   b . The configuration of the second heating unit  22  in the embodiment is the same as that of the first heating unit  21  except that the region of the reaction container  100  to be heated and the temperature of heating are different from those of the first heating unit  21 . Note that different heating mechanisms may be employed in the first heating unit  21  and the second heating unit  22 . Further, the materials of the first heat block  21   b  and the second heat block  22   b  may be different. 
     The first heating unit  21  and the second heating unit  22  function as a temperature gradient forming section of forming a temperature gradient in a direction in which the reaction solution  140  moves for the channel  110  when the attachment unit  15  is attached to the reaction container  100 . Here, “forming a temperature gradient” refers to forming a state in which a temperature changes along a predetermined direction. Therefore, “forming a temperature gradient in a direction in which the reaction solution  140  moves” refers to forming a state in which a temperature changes in a direction in which the reaction solution  140  moves. “A state in which a temperature changes along a predetermined direction” may refer to a state in which a temperature monotonically becomes higher or lower along a predetermined direction, or a state in which a temperature change is changed in the middle from the change to be higher to the change to be lower or from the change to be lower to the change to be higher along a predetermined direction. In the main body  10  of the thermal cycler  1 , the first heating unit  21  is located at the side farther from the insertion opening  151  of the attachment unit  15  and the second heating unit  22  is located at the side nearer the insertion opening  151  of the attachment unit  15 . 
     Further, the first heating unit  21  and the second heating unit  22  are provided separately from each other in the main body  10 . Thereby, the first heating unit  21  and the second heating unit  22  controlled at the different temperatures from each other are hard to affect each other, and the temperatures of the first heating unit  21  and the second heating unit  22  may be easily stabilized. A spacer may be provided between the first heating unit  21  and the second heating unit  22 . In the main body  10  of the thermal cycler  1 , the first heating unit  21  and the second heating unit  22  are fixed on their peripheries by a fixing member  16 , a flange  17 , and a flange  18 . The flange  18  is supported by a bearing  31 . Note that the number of heating units may be an arbitrary number equal to or more than two as long as the temperature gradient is formed to a degree that may secure desired reaction accuracy. 
     The temperatures of the first heating unit  21  and the second heating unit  22  may be controlled by a temperature sensor (not shown) and the control unit  40  to be described later. It is preferable that the temperatures of the first heating unit  21  and the second heating unit  22  are set so that the reaction container  100  may be heated to a desired temperature. The details of the control of the temperatures of the first heating unit  21  and the second heating unit  22  will be described in the section of “3. Control Example of Thermal Cycler”. Note that it is only necessary that the temperatures of the first heating unit  21  and the second heating unit  22  are controlled so that the first region  111  and the second region  112  of the reaction container  100  may be heated to desired temperatures. For example, in consideration of the material and the size of the reaction container  100 , the temperatures of the first region  111  and the second region  112  may be heated to the desired temperatures more accurately. In the embodiment, the temperatures of the first heating unit  21  and the second heating unit  22  are measured by a temperature sensor. The temperature sensor of the embodiment is a thermocouple. Note that the temperature sensor is not limited but may include a temperature sensing resistor or a thermistor, for example. 
     The drive mechanism  30  switches the arrangement of the attachment unit  15 , the first heating unit  21 , and the second heating unit  22  between the first arrangement in which the lowermost position of the channel  110  in the direction in which the gravity acts is located within the first region  111  and the second arrangement in which the lowermost position of the channel  110  in the direction in which the gravity acts is located within the second region  112  when the reaction container  100  is attached to the attachment unit  15 . In the embodiment, the drive mechanism  30  is a mechanism of rotating the attachment unit  15 , the first heating unit  21 , and the second heating unit  22  around the rotation axis R having a component perpendicular to the direction in which the gravity acts and a component perpendicular to the direction in which the reaction solution  140  moves in the channel  110  when the attachment unit  15  is attached to the reaction container  100 . 
     The direction “having a component perpendicular to the direction in which the gravity acts” refers to a direction having a component perpendicular to the direction in which the gravity acts when the direction is expressed by a vector sum of “a component in parallel to the direction in which the gravity acts” and “a component perpendicular to the direction in which the gravity acts”. 
     The direction “having a component perpendicular to the direction in which the reaction solution  140  moves in the channel  110 ” refers to a direction having a component perpendicular to the direction in which the reaction solution  140  moves in the channel  110  when the direction is expressed by a vector sum of “a component in parallel to the direction in which the reaction solution  140  moves in the channel  110 ” and “a component perpendicular to the direction in which the reaction solution  140  moves in the channel  110 ”. 
     In the thermal cycler  1  of the embodiment, the drive mechanism  30  rotates the attachment unit  15 , the first heating unit  21 , and the second heating unit  22  around the same rotation axis R. Further, in the embodiment, the drive mechanism  30  includes a motor and a drive shaft (not shown), and the drive shaft and the flange  17  of the main body  10  are connected. When the motor of the drive mechanism  30  is operated, the main body  10  is rotated around the drive axis as the rotation axis R. In the embodiment, ten attachment units  15  are provided along the direction of the rotation axis R. Note that, as the drive mechanism  30 , not limited to the motor, but, for example, a handle, a spiral spring, or the like may be employed. 
     The thermal cycler  1  may include a measurement unit  50 . The measurement unit  50  measures intensity of light having a predetermined wavelength. In the embodiment, a fluorescence detector is employed as the measurement unit  50 . Thereby, for example, if a fluorescent probe that changes intensity of light having a predetermined wavelength by complementary binding to specific DNA is contained in the reaction solution  140 , the thermal cycler  1  may be used for application with fluorescence measurement such as real-time PCR. The number of measurement units  50  is arbitrary as long as the measurement may be performed without difficulty. In the example shown in  FIG. 1 , the fluorescence measurement is performed while one measurement unit  50  is moved along a slide  52 . 
     It is more preferable that the measurement unit  50  is located at the side nearer the first heating unit  21  than at the side nearer the second heating unit  22 . Thereby, the measurement unit hardly becomes an obstacle to the operation when the attachment unit  15  is attached to the reaction container  100 . Further, the measurement unit  50  may be provided to measure light from a region containing the first region  111  of the reaction container  100 . When the temperature of the first heating unit  21  is set to an annealing and elongation temperature (a temperature at which annealing and elongation reaction progresses) of PCR, the intensity of the light having the predetermined wavelength correlated with an amount of specific DNA may be measured more accurately. Therefore, appropriate fluorescence measurement may be performed in real-time PCR. Furthermore, when a reaction container  100  with a lid (sealing part  120 ) to be described later is used, more appropriate fluorescence measurement may be performed in the first region  111  at the side farther from the lid than in the second region  112  at the side nearer the lid because there are less members between the measurement unit  50  and the reaction solution  140 . 
     As described above, when the thermal cycler  1  is used for real-time PCR, in a period in which thermal cycling necessary for PCR is applied to the reaction solution  140 , it is preferable that the measurement unit  50  is provided at the side nearer the first heating unit  21  and the first heating unit  21  is set to the annealing and elongation temperature of PCR (about 50° C. to 75° C.). In this case, the second heating unit  22  nearer the insertion opening  151  is set to a thermal denaturation temperature (about 90° C. to 100° C.) higher than the annealing and elongation temperature of PCR. 
     The thermal cycler  1  includes the control unit  40 . The control unit  40  controls the first heating unit  21 , the second heating unit  22 , and the drive mechanism  30 . The control unit  40  may further control the measurement unit  50 . A control example by the control unit  40  will be described in detail in the section of “3. Control Example of Thermal Cycler”. The control unit  40  may be adapted to be realized by a dedicated circuit and perform the control to be described later. Further, the control unit  40  may be adapted to function as a computer using a CPU (Central Processing Unit), for example, by executing control programs stored in a memory device such as a ROM (Read Only Memory) or a RAM (Random Access Memory) and perform the control to be described later. In this case, the memory device may have a work area that temporarily stores intermediate data and control results with the control. Further, the control unit  40  may have a timer for measuring time. Furthermore, the control unit  40  may control the first heating unit  21  and the second heating unit  22  to desired temperatures based on the output of the above described temperature sensor (not shown). 
     It is preferable that the thermal cycler  1  includes a structure of holding the reaction container  100  in a predetermined position with respect to the first heating unit  21  and the second heating unit  22 . Thereby, a predetermined regions of the reaction container  100  may be heated by the first heating unit  21  and the second heating unit  22 . More specifically, the first region  111  and the second region  112  of the channel  110  forming the reaction container  100  may be heated by the first heating unit  21  and the second heating unit  22 , respectively. In the embodiment, by appropriately setting the sizes of through holes provided in the first heat block  21   b  and the second heat block  22   b  (the diameter of the attachment unit  15 ), the reaction container  100  may be held in a predetermined position with respect to the first heating unit  21  and the second heating unit  22 . 
     The first heat block  21   b  may have a structure with fins  210 . Thereby, the surface area of the first heating unit becomes larger and the time taken for changing the temperature of the first heating unit  21  from the higher temperature to the lower temperature becomes shorter. 
     The thermal cycler  1  may include a fan  500  that blows air to the first heating unit  21  and the second heating unit  22 . By blowing air, the heat transfer between the first heating unit  21  and the second heating unit  22  may be suppressed. Therefore, the first heating unit  21  and the second heating unit  22  controlled at the different temperatures from each other become harder to affect each other, and thus, the temperatures of the first heating unit  21  and the second heating unit  22  may be easily stabilized. 
     2. Configuration of Reaction Container Attached to Thermal Cycler According to Embodiment 
       FIG. 4  is a sectional view showing a configuration of the reaction container  100  attached to the thermal cycler  1  according to the embodiment. In  FIG. 4 , arrow g indicates a direction in which gravity acts. 
     The reaction container  100  includes the channel  110  filled with the reaction solution  140  containing hot start PCR enzyme and a liquid  130  having a different specific gravity from that of the reaction solution  140  and being immiscible with the reaction solution  140  (hereinafter, referred to as “liquid  130 ”), in which the reaction solution  140  moves along the opposed inner walls. In the embodiment, the liquid  130  is a liquid having a lower specific gravity than that of the reaction solution  140  and being immiscible with the reaction solution  140 . The hot start PCR enzyme refers to PCR enzyme having enzyme activity increased (activated) in a predetermined temperature environment. Generally, the temperature at which the hot start enzyme is activated is higher than the room temperature and nearly equal to the denaturation temperature of DNA. For the hot start PCR enzyme, known enzyme may be used and, for example, Taq polymerase (“Taq” is a registered trademark) for hot start and hot start PCR enzyme having activity suppressed by antibody or the like may be used. Note that, as the liquid  130 , for example, a liquid being immiscible with the reaction solution  140  and having a higher specific gravity than that of the reaction solution  140  may be employed. In the example shown in  FIG. 4 , the reaction container  100  includes the channel  110  and the sealing part  120 . The channel  110  is filled with the reaction solution  140  and the liquid  130 , and sealed by the sealing part  120 . 
     The channel  110  is formed so that the reaction solution  140  may move along the opposed inner walls. Here, “opposed inner walls” of the channel  110  refer to two regions having an opposed positional relationship on the wall surfaces of the channel  110 . “Along” refers to a state in which a distance from the reaction solution  140  to the wall surface of the channel  110  is short, and includes a state in which the reaction solution  140  is in contact with the wall surface of the channel  110 . Therefore, “the reaction solution  140  moves along the opposed inner walls” refers to “the reaction solution  140  moves in a state in which the distances from the wall surface of the channel  110  to both two regions in the opposed positional relationship are short”. In other words, the distance between the opposed two inner walls of the channel  110  is a distance to a degree that the reaction solution  140  moves along the inner walls. 
     When the channel  110  of the reaction container  100  has the above described shape, the direction in which the reaction solution  140  moves within the channel  110  may be regulated, and thus, the path in which the reaction solution  140  moves within the channel  110  may be defined to some degree. Thereby, the time taken for the reaction solution  140  to move within the channel  110  may be restricted within a certain range. Therefore, it is preferable that the distance between the opposed two inner walls of the channel  110  is a distance to a degree at which variations in thermal cycling conditions applied to the reaction solution  140  produced by variations in time for the reaction solution  140  to move within the channel  110  may satisfy desired accuracy, i.e., a degree at which the reaction result may satisfy desired accuracy. More specifically, it is desirable that the distance in the direction perpendicular to the direction in which the reaction solution  140  between the opposed two inner walls of the channel  110  moves is a distance to a degree not exceeding two or more droplets of the reaction solution  140 . 
     In the example shown in  FIG. 4 , the outer shape of the reaction container  100  is a circular truncated cone shape, and the channel  110  in the direction along the center axis (the vertical direction in  FIG. 4 ) as the longitudinal direction is formed. The shape of the channel  110  is a circular truncated cone shape with a section in the direction perpendicular to the longitudinal direction of the channel  110 , i.e., a section perpendicular to the direction in which the reaction solution  140  moves in a certain region of the channel  110  (this refers to “section” of the channel  110 ) in a circular shape. Therefore, in the reaction container  100 , the opposed inner walls of the channel  110  are regions containing two points on the wall surface of the channel  110  opposed with the center of the section of the channel  110  in between. Further, “the direction in which the reaction solution  140  moves” is the longitudinal direction of the channel  110 . 
     Note that the shape of the channel  110  is not limited to the truncated cone shape, but may be a columnar shape, for example. Further, the section shape of the channel  110  is not limited to the circular shape, but may be any of a polygonal shape or an oval shape as long as the reaction solution  140  may move along the opposed inner walls. For example, when the section of the channel  110  of the reaction container  100  has a polygonal shape, if a channel having a circular section inscribed in the channel  110  is assumed, “opposed inner walls” are opposed inner walls of the channel. That is, it is only necessary that the channel  110  is formed so that the reaction solution  140  may move along opposed inner walls of a virtual channel having a circular section inscribed in the channel  110 . Thereby, even when the section of the channel  110  has a polygonal shape, a path in which the reaction solution  140  moves between the first region  111  and the second region  112  may be defined to some degree. Therefore, the time taken for the reaction solution  140  to move between the first region  111  and the second region  112  may be restricted within a certain range. 
     The first region  111  of the reaction container  100  is a partial region of the channel  110  to be heated by the first heating unit  21 . The second region  112  is a partial region of the channel  110  different from the first region  111  to be heated by the second heating unit  22 . In the example shown in  FIG. 4 , the first region  111  is a region containing one end part in the longitudinal direction of the channel  110 , and the second region  112  is a region containing the other end part in the longitudinal direction of the channel  110 . In the example shown in  FIG. 4 , the region surrounded by a dotted line containing the end part at the side farther from the sealing part  120  of the channel  110  is the first region  111 , and the region surrounded by a dotted line containing the end part at the side nearer the sealing part  120  of the channel  110  is the second region  112 . In the thermal cycler  1  according to the embodiment, the first heating unit  21  heats the first region  111  of the reaction container  100  and the second heating unit  22  heats the second region  112  of the reaction container  100 , and thereby, a temperature gradient is formed in the direction in which the reaction solution  140  moves with respect to the channel  110  of the reaction container  100 . 
     The channel  110  is filled with the liquid  130  and the reaction solution  140 . The liquid  130  has a property of being immiscible, i.e., unmixed with the reaction solution  140 , and the reaction solution  140  is held in droplets in the liquid  130  as shown in  FIG. 4 . The reaction solution  140  has the higher specific gravity than that of the liquid  130  and is located in the lowermost region of the channel  110  in the direction in which the gravity acts. As the liquid  130 , for example, dimethyl silicone oil or paraffin oil may be used. The reaction solution  140  is a liquid containing components necessary for reaction. For example, when the reaction is PCR including hot start, the reaction solution  140  contains DNA to be amplified, hot start DNA polymerase necessary for amplification of the DNA (hot start PCR enzyme), primer, a fluorescent probe that changes intensity of light having a predetermined wavelength by complementary binding to specific DNA, etc. Further, for example, when the reaction is RT-PCR including hot start, the reaction solution  140  contains reverse transcriptase enzyme, RNA as a template of reverse transcription, hot start DNA polymerase necessary for amplification of the reverse transcribed cDNA (hot start PCR enzyme), primer, a fluorescent probe that changes intensity of light having a predetermined wavelength by complementary binding to specific DNA, etc. For example, when PCR is performed using an oil as the liquid  130 , it is preferable that the reaction solution  140  is a solution containing the above described components. 
     3. Control Example of Thermal Cycler 
       FIG. 5  is a functional block diagram of the thermal cycler  1  according to the embodiment. The control unit  40  controls the temperature of the first heating unit  21  by outputting a control signal S 1  to the first heating unit  21 . The control unit  40  controls the temperature of the second heating unit  22  by outputting a control signal S 2  to the second heating unit  22 . The control unit  40  controls the drive mechanism  30  by outputting a control signal S 3  to the drive mechanism  30 . The control unit  40  controls the measurement unit  50  by outputting a control signal S 4  to the measurement unit  50 . 
     Next, a control example of the thermal cycler  1  according to the embodiment will be explained. As below, control of rotating the attachment unit  15 , the first heating unit  21 , and the second heating unit  22  between the first arrangement in which the lowermost position of the channel  110  in the direction in which the gravity acts is located within the first region  111  and the second arrangement in which the lowermost position of the channel  110  in the direction in which the gravity acts is located within the second region  112  when the reaction container  100  is attached to the attachment unit  15  will be explained as an example. 
       FIG. 6A  is a sectional view schematically showing a section in a plane passing through the A-A line of  FIG. 1A  and perpendicular to a rotation axis R in the first arrangement, and  FIG. 6B  is a sectional view schematically showing a section in the plane passing through the A-A line of  FIG. 1A  and perpendicular to the rotation axis R in the second arrangement. In  FIGS. 6A and 6B , white arrows indicate rotation directions of the main body  10  and arrows g indicate the direction in which the gravity acts. 
     As shown in  FIG. 6A , the first arrangement is an arrangement in which, when the attachment unit  15  is attached to the reaction container  100 , the first region  111  is located in the lowermost part of the channel  110  in the direction in which the gravity acts. In the example shown in  FIG. 6A , in the first arrangement, the reaction solution  140  having the higher specific gravity than that of the liquid  130  exists in the first region  111 . Further, as shown in  FIG. 6B , the second arrangement is an arrangement in which, when the attachment unit  15  is attached to the reaction container  100 , the second region  112  is located in the lowermost part of the channel  110  in the direction in which the gravity acts. In the example shown in  FIG. 6B , in the second arrangement, the reaction solution  140  having the higher specific gravity than that of the liquid  130  exists in the second region  112 . 
     In this manner, the drive mechanism  30  rotates the attachment unit  15 , the first heating unit  21 , and the second heating unit  22  between the first arrangement and the second arrangement different from the first arrangement, and thereby, thermal cycling may be applied to the reaction solution  140 . 
     According to the embodiment, by switching the arrangement of the attachment unit  15 , the first heating unit  21 , and the second heating unit  22 , the state in which the reaction container  100  is held in the first arrangement and the state in which the reaction container  100  is held in the second arrangement may be switched. The first arrangement is the arrangement in which the first region  111  of the channel  110  forming the reaction container  100  is located in the lowermost part of the channel  110  in a direction in which the gravity acts. The second arrangement is the arrangement in which the second region  112  of the channel  110  forming the reaction container  100  is located in the lowermost part of the channel  110  in the direction in which the gravity acts. That is, when the specific gravity of the reaction solution  140  is larger than that of the liquid  130 , the reaction solution  140  may be held in the first region  111  in the first arrangement and the reaction solution  140  may be held in the second region  112  in the second arrangement by the action of the gravity. The first region  111  is heated by the first heating unit  21  and the second region  112  is heated by the second heating unit  22 , and thereby, the first region  111  and the second region  112  may be set at different temperatures. Therefore, while the reaction container  100  is held in the first arrangement or the second arrangement, the reaction solution  140  may be held at a predetermined temperature, and thus, the thermal cycler  1  that can easily control the heating period may be provided. 
     The drive mechanism  30  may rotate the attachment unit  15 , the first heating unit  21 , and the second heating unit  22  in opposite directions when rotating them from the first arrangement to the second arrangement and when rotating them from the second arrangement to the first arrangement. Thereby, a special mechanism for reducing twisting of wires such as the conducting wire  19  caused by rotation is unnecessary. Therefore, the thermal cycler  1  suitable for downsizing may be realized. Further, it is preferable that the number of rotations for rotation from the first arrangement to the second arrangement and the number of rotations for rotation from the second arrangement to the first arrangement are less than one (the rotation angle is less than 360°). Thereby, the degree of twisting of the wires may be reduced. Alternately, as shown in  FIGS. 1 and 2 , the configuration in which the flange  18  can take up the conducting wire  19  may be employed. 
     3-1. First Specific Example of Control Method of Thermal Cycler 
     Next, a first specific example of a control method of the thermal cycler  1  will be explained by taking real-time measurement in two-step temperature PCR including a hot start step as an example. Note that the reaction solution  140  contains hot start PCR enzyme and a fluorescent probe that changes intensity of light having a predetermined wavelength by complementary binding to specific DNA.  FIG. 7  is a flowchart for explanation of the first specific example of the control method of the thermal cycler  1  according to the embodiment. 
     In  FIG. 7 , first, the control unit  40  controls the temperature of the first heating unit  21  at a first temperature (first processing), and controls the temperature of the second heating unit  22  at a second temperature higher than the first temperature (second processing) (step S 100 ). In the specific example, the first temperature is the annealing and elongation temperature in PCR. “Annealing and elongation temperature in PCR” refers to a temperature depending on the type of enzyme for amplification of nucleic acid, and generally within a range from 50° C. to 70° C. In the specific example, the second temperature is the thermal denaturation temperature in PCR. “Thermal denaturation temperature in PCR” is a temperature depending on the type of enzyme for amplification of nucleic acid, and generally within a range from 90° C. to 100° C. 
     After step S 100 , the control unit  40  controls the drive mechanism  30  to switch the arrangement of the attachment unit  15 , the first heating unit  21 , and the second heating unit  22  from the first arrangement to the second arrangement (step S 102 ). In the thermal cycler  1  shown in  FIG. 1 , immediately after the reaction container  100  is attached to the attachment unit  15 , the arrangement of the attachment unit  15 , the first heating unit  21 , and the second heating unit  22  is the first arrangement and, by performing step S 102 , the arrangement of the attachment unit  15 , the first heating unit  21 , and the second heating unit  22  is switched to the second arrangement. 
     Note that the reaction container  100  may be attached to the attachment unit  15  after step S 100  and before step S 102 . Further, in the case of the configuration in which the attachment of the reaction container  100  to the attachment unit  15  is performed when the arrangement of the attachment unit  15 , the first heating unit  21 , and the second heating unit  22  is the second arrangement, step S 102  may be unnecessary. When the arrangement of the attachment unit  15 , the first heating unit  21 , and the second heating unit  22  is the second arrangement, the reaction solution  140  is held in the second region  112 . That is, the reaction solution  140  is held at the second temperature. 
     After step S 102 , the control unit  40  performs third processing of allowing a first period to elapse with the arrangement of the attachment unit  15 , the first heating unit  21 , and the second heating unit  22  being the second arrangement. Note that it is only necessary that the first heating unit  21  is at the first temperature in the third processing. That is, the third processing may be performed before the second processing or at the same time with the second processing as long as it is performed after the first processing. 
     More specifically, after step S 102 , the control unit  40  determines whether or not the first period has elapsed after step S 102  is ended (step S 104 ). In the specific example, the first period is a period necessary for activation of PCR enzyme. If the control unit  40  determines that the first period has not elapsed (if NO at step S 104 ), the control unit  40  repeats step S 104 . 
     If the control unit  40  determines that the first period has elapsed (if YES at step S 104 ), the control unit  40  performs fourth processing of controlling the drive mechanism  30  to switch the arrangement of the attachment unit  15 , the first heating unit  21 , and the second heating unit  22  from the second arrangement to the first arrangement if a second period has elapsed with the arrangement of the attachment unit  15 , the first heating unit  21 , and the second heating unit  22  being the second arrangement. 
     More specifically, first, the control unit  40  determines whether or not the second period has elapsed after step S 104  is ended (step S 106 ). In the specific example, the second period is a period necessary for denaturation in PCR. If the control unit  40  determines that the second period has not elapsed (if NO at step S 106 ), the control unit  40  repeats step S 106 . If the control unit  40  determines that the second period has elapsed (if YES at step S 106 ), the control unit controls the drive mechanism  30  to switch the arrangement of the attachment unit  15 , the first heating unit  21 , and the second heating unit  22  from the second arrangement to the first arrangement (step S 108 ). When the arrangement of the attachment unit  15 , the first heating unit  21 , and the second heating unit  22  is the first arrangement, the reaction solution  140  is held in the first region  111 . That is, the reaction solution  140  is held at the first temperature. 
     The reaction solution  140  is held at the second temperature in the third processing and the fourth processing, and the reaction solution  140  is held at the first temperature lower than the second temperature after the fourth processing. When the thermal cycler  1  is applied to PCR, the first temperature corresponds to the annealing and elongation temperature and the second temperature corresponds to the denaturation temperature of DNA. Generally, the temperature at which the PCR enzyme is activated is nearly equal to the denaturation temperature. Therefore, by performing the third processing, thermal cycling that enables hot start of PCR may be realized in addition to the thermal cycling of normal PCR. Further, by performing the third processing before the second processing (allowing the first time to elapse), thermal cycling including hot start may be realized without affecting the second period of the second processing. 
     After the third processing, the control unit  40  may control the measurement unit  50  to measure the intensity of the light having the predetermined wavelength. More specifically, after step S 106 , the measurement unit  50  starts fluorescence measurement (step S 110 ). The fluorescence measurement with respect to plural reaction containers  100  may be performed by moving the measurement unit  50  on the slide  52 . 
     By controlling the measurement unit  50  to measure the intensity of the light having the predetermined wavelength after the third processing, the intensity of the light having the predetermined wavelength emitted by the fluorescent probe binding to the DNA sequence may be measured in the period in which the reaction solution  140  is held at the annealing and elongation temperature. Therefore, the thermal cycler  1  suitable for real-time PCR may be realized. 
     After the fourth processing, the control unit  40  may perform fifth processing of controlling the drive mechanism  30  to switch the arrangement of the attachment unit  15 , the first heating unit  21 , and the second heating unit  22  from the first arrangement to the second arrangement and the forth processing repeatedly at a predetermined number of times if a third period has elapsed with the arrangement of the attachment unit  15 , the first heating unit  21 , and the second heating unit  22  being the first arrangement. 
     More specifically, first, after step S 110 , the control unit  40  determines whether or not the third period has elapsed after step S 108  is ended (step S 112 ). In the specific example, the third period is a period necessary for annealing and elongation in PCR. If the control unit  40  determines that the third period has not elapsed (if NO at step S 112 ), the control unit  40  repeats step S 112 . If the control unit  40  determines that the third period has elapsed (if YES at step S 112 ), the control unit  40  determines whether or not a predetermined number of cycles has been reached (step S 114 ). 
     If the control unit  40  determines that the predetermined number of cycles has not been reached (if NO at step S 114 ), the control unit  40  controls the drive mechanism  30  to switch the arrangement of the attachment unit  15 , the first heating unit  21 , and the second heating unit  22  from the first arrangement to the second arrangement (step S 116 ). After step S 116 , steps S 106  to S 114  are repeated. If the control unit  40  determines that the predetermined number of cycles has been reached (if YES at step S 114 ), the processing is ended. 
     The reaction solution  140  is held at the second temperature until the second period has elapsed in the second arrangement in the fourth processing, and the reaction solution  140  is held at the first temperature until the third period has elapsed in the first arrangement in the fifth processing. In this manner, by repeating the fifth processing and the fourth processing (more specifically, step S 116  and steps S 106  to S 114 ), thermal cycling suitable for PCR may be performed repeatedly at a predetermined number of times. 
     3-2. Second Specific Example of Control Method of Thermal Cycler 
     Next, a second specific example of the control method of the thermal cycler  1  will be explained by taking real-time measurement in RT-PCR including a hot start step as an example.  FIG. 8  is a flowchart for explanation of the second specific example of the control method of the thermal cycler  1  according to the embodiment. Note that the same steps as those in the first specific example of the control method of the thermal cycler  1  shown in  FIG. 7  have the same signs, and their detailed explanation will be omitted. 
     In the second specific example of the control method of the thermal cycler  1 , the control unit  40  performs sixth processing of controlling the first heating unit  21  at a third temperature lower than the first temperature and allowing a fourth period to elapse with the arrangement of the attachment unit  15 , the first heating unit  21 , and the second heating unit  22  being the first arrangement, performs seventh processing of controlling the first heating unit  21  at the first temperature and controlling the drive mechanism  30  to switch the arrangement of the attachment unit  15 , the first heating unit  21 , and the second heating unit  22  from the first arrangement to the second arrangement after the sixth processing and the second processing, and performs third processing after the seventh processing. 
     More specifically, first, the control unit  40  controls the temperature of the first heating unit  21  at the third temperature (step S 200 ). In the specific example, the third temperature is a temperature at which reverse transcription reaction progresses by the reverse transcriptase enzyme. “The temperature at which the reverse transcription reaction progresses by the reverse transcriptase enzyme” is a temperature depending on the type of the reverse transcriptase enzyme and generally within a range from 20° C. to 70° C., and the temperature at which the reverse transcription reaction especially progresses is generally within a range from 40° C. to 50° C. Further, in the specific example, the arrangement of the attachment unit  15 , the first heating unit  21 , and the second heating unit  22  at the initial operation is the first arrangement. Therefore, the reaction solution  140  is held in the first region  111 . That is, the reaction solution  140  is held at the third temperature. 
     Note that, at step S 200 , the control unit  40  may control the temperature of the second heating unit  22  at a temperature at which the reverse transcriptase enzyme is not deactivated. “The temperature at which the reverse transcriptase enzyme is not deactivated” is a temperature depending on the type of the reverse transcriptase enzyme, and generally within a range from 20° C. to 70° C. Further, generally, at a temperature exceeding 70° C., the reverse transcriptase enzyme is easily deactivated and deteriorated. By controlling the temperature of the second heating unit  22  at the temperature at which the reverse transcriptase enzyme is not deactivated, when the reaction container  100  is attached to the attachment unit  15 , the reaction solution  140  is not subjected to a high temperature at which the reverse transcriptase enzyme is deactivated. 
     After step S 200 , the control unit  40  determines whether or not a fourth period has elapsed after step S 200  is ended (step S 202 ). In the specific example, the fourth period is a period necessary for reverse transcription reaction. If the control unit  40  determines that the fourth period has not elapsed (if NO at step S 202 ), the control unit  40  repeats step S 202 . If the control unit  40  determines that the fourth period has elapsed (if YES at step S 202 ), the control unit  40  controls the temperature of the first heating unit  21  at the first temperature and controls the temperature of the second heating unit  22  at the second temperature (step S 204 ). The first temperature and the second temperature are the same as those in the first specific example of the control method of the thermal cycler  1  explained using Fit.  7 . 
     After step S 204 , the control unit  40  controls the drive mechanism  30  to switch the arrangement of the attachment unit  15 , the first heating unit  21 , and the second heating unit  22  from the first arrangement to the second arrangement (step S 206 ). Therefore, the reaction solution  140  is held in the second region  112 . That is, the reaction solution  140  is held at the second temperature. 
     After step S 206 , the control unit  40  performs step S 104 , and the subsequent process is the same as that of the first specific example of the control method of the thermal cycler  1  explained using  FIG. 7 . 
     In this manner, by performing the seventh processing prior to the fifth processing, the reverse transcription reaction may be performed before PCR, and thus, the thermal cycler  1  suitable for RT-PCR may be realized. 
     Further, like the first specific example of the control method of the thermal cycler  1  shown in  FIG. 7 , by performing the third processing, thermal cycling that enables hot start of PCR may be realized in addition to the thermal cycling of normal PCR. Furthermore, by performing the third processing before the second processing (allowing the first period to elapse), the thermal cycling including hot start may be realized without affecting the second period of the second processing. 
     In addition, like the first specific example of the control method of the thermal cycler  1  shown in  FIG. 7 , by repeating the fifth processing and the fourth processing (more specifically, step S 116  and steps S 106  to S 114 ), thermal cycling suitable for PCR may be performed repeatedly at a predetermined number of times. 
     Further, like the first specific example of the control method of the thermal cycler  1  shown in  FIG. 7 , by controlling the measurement unit  50  to measure the intensity of the light having the predetermined wavelength after the third processing, the intensity of the light having the predetermined wavelength correlated with amount of specific DNA may be measured in the period in which the reaction solution  140  is held at the annealing and elongation temperature. Therefore, the thermal cycler  1  suitable for real-time PCR may be realized. 
     4. Working Examples 
     As below, the invention will be more specifically explained using working examples, however, the invention is not limited to the working examples. 
     4-1. First Working Example 
     In the first working example, an example of performing two-step temperature real-time PCR including hot start using the thermal cycler  1  will be explained. 
       FIG. 9  is a table showing a composition of the reaction solution  140  in the first working example. In  FIG. 9 , “SuperScript III Platinum” refers to “SuperScript III Platinum One-Step Quantitative RT-PCR System with ROX (“Platinum” is a registered trademark)”, and contains PCR enzyme. Regarding the plasmid, samples having known copy numbers were produced by subcloning of PCR reaction products obtained using the primers shown in  FIG. 10  in advance.  105  plasmids were added for Sample A,  104  plasmids were added for Sample B,  103  plasmids were added for Sample C, and  102  plasmids were added for Sample D. 
       FIG. 10  is a table showing base sequences of forward primers (F primers), reverse primers (R primers), and probes corresponding to influenza A virus (InfA), swine influenza A virus (SW InfA), and swine influenza H1 virus (SW H1), ribonuclease P (RNase P). All of them are the same as base sequences described in “CDC protocol of realtime RTPCR for swine influenza A (H1N1)” (World Health Organization, Revised First Edition, Apr. 30, 2009). In all of the four types of probes shown in  FIG. 10 , fluorescent brightness to be measured increases with amplification of nucleic acid. 
     The experimental procedure was as shown in the flowcharts in  FIG. 7 , and the first temperature was 58° C., the second temperature was 98° C., the first period was five seconds, the second period was ten seconds, the fourth period was 30 seconds, and the number of cycles of the thermal cycling processing was 50. Further, the number of reaction containers  100  attached to the attachment unit  15  was four (Sample A to Sample D). 
       FIG. 11  is a graph showing relationships between the number of cycles of thermal cycling processing and measured brightness in the first working example. The horizontal axis of  FIG. 11  indicates the number of cycles of the thermal cycling processing and the vertical axis indicates the relative value of brightness. 
     As shown in  FIG. 11 , it is known that, regarding all of Sample A to Sample D, the brightness significantly rose as the number of cycles of the thermal cycling processing was about 20 to 35. Thereby, it is confirmed that DNA has been amplified. Further, from  FIG. 11 , it is confirmed that the brightness rises more significantly at the less number of cycles in the samples having the larger copy numbers of plasmid, and the number of cycles at which the brightness rises is larger as the concentration of the plasmid contained in the reaction solution  140  is higher. 
     As described above, it is confirmed that two-step temperature real-time PCR including hot start may be performed using the thermal cycler  1  according to the embodiment. 
     4-2. Second Working Example 
     In the second working example, an example of performing RT-PCR including hot start using the thermal cycler  1  will be explained. 
       FIG. 12  is a table showing a composition of the reaction solution  140  in the second working example. In  FIG. 12 , “SuperScript III Platinum” refers to “SuperScript III Platinum One-Step Quantitative RT-PCR System with ROX (“Platinum” is a registered trademark)”, and contains PCR enzyme and reverse transcriptase enzyme. As RNA, RNA extracted from a human nasal cavity swab (human sample) was used. Note that, regarding the human sample, immuno chromatography was performed using a commercially available kit (“ESPLINE Influenza A&amp;B-N) (ESPLINE is a registered trademark)”, manufactured by FUJIREBIO), and the sample was positive for influenza A virus. Note that “A virus positive” in immuno chromatography does not specifically determine the influenza A virus (InfA). The base sequences of the forward primers (F primers), reverse primers (R primers), probes (Probes) in  FIG. 12  are the same as the base sequences shown in  FIG. 10 . 
     The experimental procedure was as shown in the flowcharts in  FIG. 8 , and the first temperature was 58° C., the second temperature was 98° C., the third temperature was 45° C., the first period was five seconds, the second period was ten seconds, the third period was 60 seconds, the fourth period was 30 seconds, and the number of cycles of the thermal cycling processing was 50. Further, the number of reaction containers  100  attached to the attachment unit  15  was four (Sample E to Sample H). 
     Sample E contains a forward primer, a reverse primer, and a fluorescent probe corresponding to influenza A virus (InfA). Sample F contains a forward primer, a reverse primer, and a fluorescent probe corresponding to swine influenza A virus (SW InfA). Sample G contains a forward primer, a reverse primer, and a fluorescent probe corresponding to swine influenza H1 virus (SW H1). Sample H contains a forward primer, a reverse primer, and a fluorescent probe corresponding to ribonuclease P (RNase P). 
       FIG. 13  is a graph showing relationships between the number of cycles of thermal cycling processing and measured brightness in the second working example. The horizontal axis of  FIG. 13  indicates the number of cycles of the thermal cycling processing and the vertical axis indicates the relative value of brightness. 
     As shown in  FIG. 13 , it is known that, regarding all of Sample E to Sample H, the brightness significantly rose as the number of cycles of the thermal cycling processing was about 20 to 30. Thereby, it is known that reverse-transcribed cDNA with RNA as the template has been amplified. Sample H was for an experiment of endogenous control, and it is confirmed that DNA (cDNA) derived from the human sample has been amplified because the brightness rose in Sample H. Further, it is known that all RNAs of InfA, SW InfA, SW H1 have been contained in the human sample because cDNA has been amplified in Sample E to Sample H. The result agrees with the result of immuno chromatography. Therefore, it has been confirmed that 1 step RT-PCR including hot start may be performed using the thermal cycler  1  according to the embodiment. 
     Note that the above described embodiment and working example are just examples, and not limited to those. For example, some of the respective embodiments and the respective examples may be appropriately combined. 
     The invention is not limited to the above described embodiment and example, but other various modifications may be made. For example, the invention includes substantially the same configuration as the configuration explained in the embodiment (for example, a configuration having the same function, method, and result, or a configuration having the same purpose and advantage). Further, the invention includes a configuration in which an insubstantial part of the configuration explained in the embodiment is replaced. Furthermore, the invention includes a configuration that exerts the same effect or a configuration that may achieve the same purpose as that of the configuration explained in the embodiment. In addition, the invention includes a configuration formed by adding a known technology to the configuration explained in the embodiment. 
     SEQ ID NO: 1 refers to the sequence of the forward primer of InfA. 
     SEQ ID NO: 2 refers to the sequence of the reverse primer of InfA. 
     SEQ ID NO: 3 refers to the sequence of the fluorescent probe of InfA. 
     SEQ ID NO: 4 refers to the sequence of the forward primer of SW InfA. 
     SEQ ID NO: 5 refers to the sequence of the reverse primer of SW InfA. 
     SEQ ID NO: 6 refers to the sequence of the fluorescent probe of SW InfA. 
     SEQ ID NO: 7 refers to the sequence of the forward primer of SW H1. 
     SEQ ID NO: 8 refers to the sequence of the reverse primer of SW H1. 
     SEQ ID NO: 9 refers to the sequence of the fluorescent probe of SW H1. 
     SEQ ID NO: 10 refers to the sequence of the forward primer of RNase P. 
     SEQ ID NO: 11 refers to the sequence of the reverse primer of RNase P. 
     SEQ ID NO: 12 refers to the sequence of the fluorescent probe of RNase P.