Abstract:
A substance amplification reaction apparatus includes a mounting section mountable with a substance amplification reaction container filled with reaction liquid and liquid and having a channel in which a first channel and a second channel are connected via a bent section and the reaction liquid moves, a first to third heating sections respectively capable of heating a first region, which is a region of an end portion on the first channel side of the channel, a second region, which is a region of the bent section, and a third region, which is a region at an end portion on the second channel side of the channel, and a driving mechanism configured to switch first arrangement, second arrangement, and third arrangement in this order. Each of the first to third arrangements is arrangement in which each of the first to third regions is below the other regions.

Description:
BACKGROUND 
       [0001]    1. Technical Field 
         [0002]    The present invention relates to a substance amplification reaction apparatus and a method of amplifying a substance. 
         [0003]    2. Related Art 
         [0004]    As a method of amplifying nucleic acid at high speed, there is known a nucleic acid amplification reaction apparatus that injects oil and a small amount of nucleic acid amplification reaction liquid into a nucleic acid amplification reaction container, maintains one end of the container at high temperature and maintains the other end at low temperature, and causes a heat cycle in the temperature of the nucleic acid amplification reaction liquid at high speed by rotating the container to alternately switch a state in which the one end is placed lower in the vertical direction to locate the reaction liquid in the high-temperature oil and a state in which the other end is placed lower in the vertical direction to locate the reaction liquid in the low-temperature oil (see, for example, JP-A-2012-115208 (Patent Literature 1)). 
         [0005]    The nucleic acid amplification reaction apparatus described in Patent Literature 1 is based on two-step PCR. It is likely that deterioration in amplification efficiency under a specific reaction condition occurs. When a primer shorter than 20 nucleotides is used, amplification is difficult. 
       SUMMARY 
       [0006]    An advantage of some aspects of the invention is to provide a substance amplification reaction apparatus for efficiently amplifying a substance. 
         [0007]    An aspect of the invention is directed to a substance amplification reaction apparatus including: a mounting section mountable with a substance amplification reaction container filled with reaction liquid and liquid that has specific gravity smaller than the specific gravity of the reaction liquid and does not mix with the reaction liquid and having a channel in which the reaction liquid moves, the channel including a first channel and a second channel, the first channel and the second channel being connected via a bent section, and an axis of the first channel and an axis of the second channel forming an angle smaller than 90 degrees; a first heating section capable of heating a first region, which is a region of an end portion on the first channel side of the channel; a second heating section capable of heating a second region, which is a region of the bent section; a third heating section capable of heating a third region, which is a region at an end portion on the second channel side of the channel; a driving mechanism configured to switch first arrangement, second arrangement, and third arrangement in this order; and a control section configured to control the driving mechanism to maintain the first arrangement for a predetermined time, thereafter maintain the second arrangement for the predetermined time, and thereafter maintain the third arrangement for the predetermined time. The first arrangement is arrangement in which the first region is below the second region and the third region in a direction in which the gravity acts. The second arrangement is arrangement in which the second region is below the first region and the third region in the direction in which the gravity acts. The third arrangement is arrangement in which the third region is below the first region and the second region in the direction in which the gravity acts. The first heating section may include a first hole. The first region may be insertable into the first hole. The second heating section may include a second hole. The second region may be insertable into the second hole. The third heating section may include a third hole. The third region may be insertable into the third hole. The first to third holes may be the mounting section. 
         [0008]    Another embodiment of the invention is directed to a method of amplifying a substance using a substance amplification reaction apparatus, the substance amplification reaction apparatus including: a mounting section mountable with a substance amplification reaction container filled with reaction liquid and liquid that has specific gravity smaller than the specific gravity of the reaction liquid and does not mix with the reaction liquid and having a channel in which the reaction liquid moves, the channel including a first channel and a second channel, the first channel and the second channel being connected via a bent section, and an axis of the first channel and an axis of the second channel forming an angle smaller than 90 degrees; a first heating section capable of heating a first region, which is a region of an end portion on the first channel side of the channel; a second heating section into which a second region, which is a region of the bent section, is insertable; and a third heating section capable of heating a third region, which is a region at an end portion on the second channel side of the channel. The method includes maintaining, for a predetermined time, first arrangement in which the first region is below the second region and the third region in a direction in which the gravity acts, thereafter, transitioning to second arrangement in which the second region is below the first region and the third region in the direction in which the gravity acts and maintaining the second arrangement for the predetermined time, and thereafter transitioning to third arrangement in which the third region is below the first region and the second region in the direction in which the gravity acts and maintaining the third arrangement for the predetermined time. 
         [0009]    In both the aspects, the substance may be nucleic acid. 
         [0010]    According to the aspects of the invention, it is possible to provide the substance amplification reaction apparatus for efficiently amplifying the substance. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0011]    The invention will be described with reference to the accompanying drawings, wherein like numbers reference like elements. 
           [0012]      FIG. 1  is a schematic diagram of a substance reaction container and a heater in a substance amplification reaction apparatus according to an embodiment. 
           [0013]      FIGS. 2A to 2C  are schematic diagrams showing a method of amplifying a substance using the substance amplification reaction apparatus according to the embodiment. 
           [0014]      FIG. 3  is a flowchart for explaining a procedure of processing for amplifying a substance using the substance amplification reaction apparatus according to the embodiment. 
       
    
    
     DESCRIPTION OF EXEMPLARY EMBODIMENTS 
       [0015]    Preferred embodiments of the invention are explained below with reference to the drawings. Note that the embodiments explained below do not unduly limit contents of the invention described in the appended claims. Not all of components explained below are essential constituent elements of the invention. 
       1. Substance Amplification Reaction Apparatus According to an Embodiment 
       [0016]    In an embodiment, a substance amplification reaction apparatus includes three heating sections on which a substance amplification reaction container is mountable and a driving mechanism capable of switching the arrangement of the heating sections. 
         [0017]      FIG. 1  is a schematic sectional view showing a state in which a substance amplification reaction container  10  is fixed to a first heating section  200 , a second heating section  300 , and a third heating section  400  in the substance amplification reaction apparatus according to this embodiment. 
         [0018]    As shown in  FIG. 1 , the substance amplification reaction container  10  includes a first channel  16  and a second channel  18 . An axis  16   a  of the first channel  16  and an axis  18   a  of the second channel  18  form an angle “a”. The angle “a” is smaller than 90 degrees. 
         [0019]    Liquid  15  and reaction liquid  14  are filled in the substance amplification reaction container  10 . The liquid  15  does not mix with the reaction liquid  14 , that is, does not dissolve in the reaction liquid  14 . Therefore, as shown in  FIG. 1 , the reaction liquid  14  is retained in the liquid  15  in a state of a droplet. Since the reaction liquid  14  has specific gravity larger than the specific gravity of the liquid  15 , the reaction liquid  14  is located in a bottom region in the gravity direction of the substance amplification reaction container  10 . As the liquid  15 , for example, dimethyl silicone oil or paraffin oil can be used. The reaction liquid  14  is liquid  15  containing a component necessary for reaction. When the reaction is PCR, the reaction liquid  14  includes DNA (target nucleic acid) amplified by the PCR, DNA polymerase necessary for amplifying the DNA, and a primer. For example, when the PCR is performed using oil as the liquid  15 , the reaction liquid  14  is preferably an aqueous solution containing the components described above. 
         [0020]    The first heating section  200 , the second heating section  300 , and the third heating section  400  are members that transmit heat generated from a not-shown heater to the substance amplification reaction container  10 . In this embodiment, the first heating section  200 , the second heating section  300 , and the third heating section  400  are heat blocks made of aluminum. Since the heat blocks are made of aluminum, it is possible efficiently heat the substance amplification reaction container  10 . Since heating unevenness less easily occurs in the heat blocks, it is possible to realize a highly accurate heat cycle. Since machining is easy, it is possible to accurately mold the heat blocks and improve accuracy of the heating. Therefore, it is possible to realize a more accurate heat cycle. 
         [0021]    The first heating section  200 , the second heating section  300 , and the third heating section  400  respectively include holes. A first region  20 , a second region  30 , and a third region  40  of the substance amplification reaction container  10  are respectively inserted into the holes of the first heating section  200 , the second heating section  300 , and the third heating section  400  as shown in  FIG. 1 . The first region  20  is a region at an end portion of the substance amplification reaction container  10  and an end portion of the first channel  16 . The second region  30  is a region located between the first channel  16  and the second channel  18  of the substance amplification reaction container  10 . The third region  40  is a region at an end portion of the substance amplification reaction container  10  and an end portion of the second channel  18 . 
         [0022]    The first region  20  of the substance amplification reaction container  10  is inserted into the hole of the first heating section  200 . During heat cycle treatment, the first region  20  of the substance amplification reaction container  10  is heated by the first heating section  200  to raise the temperature of oil in the first region  20  to a first temperature. The first temperature is, for example, 92 to 97° C. 
         [0023]    The second region  30  of the substance amplification reaction container  10  is inserted into the hole of the second heating section  300 . During the heat cycle treatment, the second region  30  of the substance amplification reaction container  10  is heated by the second heating section  300  to raise the temperature of the oil in the second region  30  to a second temperature. The second temperature is 55 to 72° C. 
         [0024]    The third region  40  of the substance amplification reaction container  10  is inserted into the hole of the third heating section  400 . During the heat cycle treatment, the third region  40  of the substance amplification reaction container  10  is heated by the third heating section  400  to raise the temperature of the oil in the third region  40  to a third temperature. The third temperature is 65 to 80° C. 
         [0025]    The temperatures of the first heating section  200 , the second heating section  300 , and the third heating section  400  are controlled by a temperature sensor and a control section. 
         [0026]    A driving mechanism is provided in the substance amplification reaction apparatus in this embodiment. The driving mechanism is a mechanism for switching the three heating sections to first arrangement, second arrangement, and third arrangement. The driving mechanism includes a motor and a driving shaft. The driving shaft is provided perpendicularly to the second heating section  300 . When the motor is operated, the first heating section  200 , the second heating section  300 , and the third heating section  400  rotate with the driving shaft as an axis of rotation while maintaining a positional relation thereof. 
         [0027]    The substance amplification reaction apparatus in this embodiment includes the control section. The control section controls the operation of the driving mechanism to thereby control the first heating section  200 , the second heating section  300 , and the third heating section  400  to be retained in predetermined arrangement for a predetermined time. 
         [0028]    The control section in the substance amplification reaction apparatus may perform electronic control and control all the items. The control section includes a processor such as a CPU and storage devices such as a ROM (Read Only Memory) and a RAM (Random Access Memory) not shown in the figure. Various computer programs, data, and the like for controlling the items are stored in the storage devices. The storage devices include work areas where data being processed, a processing result, and the like of various kinds of processing are temporarily stored. 
         [0000]    2. Heat Cycle Treatment Performed using the Substance Amplification Reaction Apparatus 
         [0029]      FIGS. 2A to 2C  are diagrams schematically showing three-step PCR performed using the substance amplification reaction apparatus according to this embodiment.  FIG. 2A  shows the first arrangement,  FIG. 2B  shows the second arrangement, and  FIG. 2C  shows the third arrangement. In  FIGS. 2A to 2C , a direction of an arrow g (the downward direction in the figures) is a direction in which the gravity acts.  FIG. 3  is a flowchart for explaining a procedure of the processing. 
         [0030]    Note that, in this specification, concerning two points, “above in the direction in which the gravity acts” and “below in the direction in which the gravity acts” only have to mean that there is a difference between the two points only about the direction in which the gravity acts. A straight line connecting the two points may be parallel to or may not be parallel to the direction in which the gravity acts. 
         [0031]    The three-step PCR is a method of repeatedly applying temperature treatment in three stages to reaction liquid to thereby amplify nucleic acid in the reaction liquid. In treatment at high temperature, dissociation of double-stranded DNA is performed. In treatment at low temperature, annealing (a reaction in which the primer combines with single-stranded DNA) and an extension reaction (a reaction in which a complementary chain of DNA is formed starting from the primer) are performed. 
         [0032]    In the three-step PCR, the high temperature is temperature between approximately 92° C. and 97° C., the low temperature is temperature between approximately 55° C. and 72° C., and intermediate temperature is temperature between approximately 65° C. and 80° C. The kinds of treatment at the temperatures are performed for a predetermined time. Appropriate times, temperatures, and the number of cycles are different depending on a type and an amount of a reagent in use. Therefore, it is preferable to perform the reaction after determining an appropriate protocol taking into account a type of a reagent and an amount of the reaction liquid  14 . 
         [0033]    First, the substance amplification reaction container  10  filled with the liquid  15  and the reaction liquid  14  is fixed to the first heating section  200 , the second heating section  300 , and the third heating section  400  (step S 101 ). The first heating section  200 , the second heating section  300 , and the third heating section  400  are in contact with the substance amplification reaction container  10  in positions respectively including the first region  20 , the second region  30 , and the third region  40 . 
         [0034]    In this embodiment, the arrangement of the first heating section  200 , the second heating section  300 , and the third heating section  400  in step S 101  is the first arrangement. As shown in  FIG. 2A , the first arrangement is arrangement in which the first region  20  is below the second region  30  and the third region  40  in the direction in which the gravity acts. In the first arrangement, the first region  20  is located in the bottom of the channel  16  in the direction in which the gravity acts. The reaction liquid  14  having specific gravity larger than the specific gravity of the liquid  15  is located in the first region  20 . 
         [0035]    In step S 102 , the substance amplification reaction container  10  is heated by the first heating section  200 , the second heating section  300 , and the third heating section  400 . The first heating section  200 , the second heating section  300 , and the third heating section  400  heat different regions of the substance amplification reaction container  10  to different temperatures. That is, the first heating section  200  heats the first region  20  to the first temperature, the second heating section  300  heats the second region  30  to the second temperature, and the third heating section  400  heats the third region  40  to the third temperature. Consequently, a temperature gradient in which temperature gradually changes among the first temperature, the second temperature, and the third temperature is formed among the first region  20 , the second region  30 , and the third region  40 . The heat cycle treatment in this embodiment is the three-step PCR. Therefore, it is preferable to set the first temperature to temperature suitable for dissociation of double-stranded DNA, set the second temperature to temperature suitable for annealing, and set the third temperature to temperature suitable for an extension reaction. 
         [0036]    The arrangement of the first heating section  200 , the second heating section  300 , and the third heating section  400  in step S 102  is the first arrangement. Therefore, when the substance amplification reaction container  10  is heated in step S 102 , the reaction liquid  14  is heated to the first temperature. Therefore, in step S 102 , the reaction liquid  14  is caused to react at the first temperature. 
         [0037]    In step S 103 , it is determined whether the first time has elapsed in the first arrangement. In this embodiment, the determination is performed by the not-shown control section. The first time is time in which the first arrangement is retained. In this embodiment, when step S 103  is performed following step S 101 , that is, when step S 103  is performed for the first time, it is determined whether time after the substance amplification reaction apparatus is actuated has reached the first time. In the first arrangement, the reaction liquid  14  is heated to the first temperature. Therefore, the first time is preferably set to time in which the reaction liquid  14  is caused to react at the first temperature in a target reaction. In this embodiment, the first time is preferably set to time necessary for dissociation of double-stranded DNA. 
         [0038]    When it is determined in step S 103  that the first time has elapsed (YES), the processing proceeds to step S 104 . When it is determined in step S 103  that the first time has not elapsed (NO), step S 103  is repeated. 
         [0039]    In step S 104 , the arrangement of the first heating section  200 , the second heating section  300 , and the third heating section  400  is switched from the first arrangement to the second arrangement by the driving mechanism of the substance amplification reaction apparatus. The second arrangement is arrangement in which the second region  30  is below the first region  20  and the third region  40  in the direction in which the gravity acts ( FIG. 2B ). In step S 104  in this embodiment, the arrangement of the first heating section  200 , the second heating section  300 , and the third heating section  400  is switched from the state shown in  FIG. 2A  to the state shown in  FIG. 2B . 
         [0040]    When the arrangement is switched from the first arrangement to the second arrangement in step S 104 , the reaction liquid  14  moves from the first region  20  to the second region  30  according to the action of the gravity. When the arrangement of the first heating section  200 , the second heating section  300 , and the third heating section  400  reaches the second arrangement, step S 105  is started. In step S 105 , it is determined whether the second time has elapsed in the second arrangement. The second time is time in which the first heating section  200  and the second heating section  300  are retained in the second arrangement. In this embodiment, the second region  30  is heated to the second temperature in step S 102 . Therefore, in step S 105  in this embodiment, it is determined whether time after the arrangement reaches the second arrangement has reached the second time. In the second arrangement, since the reaction liquid  14  is retained in the second region  30 , the reaction liquid  14  is heated to the second temperature in time in which the first heating section  200  and the second heating section  300  are retained in the second arrangement. Therefore, the second time is preferably set to time in which the reaction liquid  14  is heated to the second temperature in the target reaction. In this embodiment, the second time is preferably set to time necessary for annealing. 
         [0041]    When it is determined in step S 105  that the second time has elapsed (YES), the processing proceeds to step S 106 . When it is determined in step S 105  that the second time has not elapsed (NO), step S 105  is repeated. 
         [0042]    In step S 106 , the arrangement of the first heating section  200 , the second heating section  300 , and the third heating section  400  is switched from the second arrangement to the third arrangement by the driving mechanism of the substance amplification reaction apparatus. The third arrangement is arrangement in which the third region  40  is below the first region  20  and the second region  30  in the direction in which the gravity acts ( FIG. 2C ). 
         [0043]    When the arrangement is switched from the second arrangement to the third arrangement in step S 106 , the reaction liquid  14  moves from the second region  30  to the third region  40  according to the action of the gravity. When the arrangement of the first heating section  200 , the second heating section  300 , and the third heating section  400  reaches the third arrangement, step S 107  is started. In step S 107 , it is determined whether the third time has elapsed in the third arrangement. The third time is time in which the third arrangement is retained. In this embodiment, the third region  40  is heated to the third temperature in step S 102 . Therefore, in step S 107  in this embodiment, it is determined whether time after the arrangement reaches the third arrangement has reached the third time. In the third arrangement, the reaction liquid  14  is retained in the third region  40 . Therefore, in time in which the arrangement of the first heating section  200 , the second heating section  300 , and the third heating section  400  are retained in the third arrangement, the reaction liquid  14  is heated to the third temperature. Therefore, the third time is preferably set to time in which the reaction liquid  14  is heated to the third temperature in the target reaction. In this embodiment, the third time is preferably set to time necessary for an extension reaction. 
         [0044]    When it is determined in step S 107  that the third time has elapsed (YES), the processing proceeds to step S 108 . When it is determined in step S 107  that the third time has not elapsed (NO), step S 107  is repeated. 
         [0045]    In this embodiment, the substance amplification reaction container  10  includes a bent section and includes, on both sides of the bent section, the first channel  16  and the second channel  18  in which the reaction liquid  14  moves. The axis  16   a  of the first channel  16  and the axis  18   a  of the second channel  18  form an angle smaller than 90 degrees. The first region  20 , the second region  30 , and the third region  40  can be maintained at the temperatures. Therefore, it is possible to amplify a substance at high speed by moving the reaction liquid  14  in the oil in the substance amplification reaction container  10 . 
         [0046]    In step S 108 , it is determined whether the number of times of the heat cycle reaches a predetermined number of cycles. Specifically, it is determined whether the procedure of step S 103  to step S 107  has been completed a predetermined number of times. In this embodiment, the number of times step S 103  to step S 107  are completed is determined according to the number of times it is determined “YES” in step S 103 , step S 105 , and step S 107 . When step S 103  to step S 107  are performed once, the heat cycle is applied to the reaction liquid  14  by one cycle. Therefore, the number of times step S 103  to step S 107  are performed can be set as the number of cycles of the heat cycle. Therefore, according to step S 108 , it is possible to determine whether the heat cycle has been applied to the reaction liquid  14  the number of times necessary for the target reaction. 
         [0047]    When it is determined in step S 108  that the heat cycle has been performed the predetermined number of cycles (YES), the processing is completed (END). When it is determined in step S 108  that the heat cycle has not been performed the predetermined number of cycles (NO), the processing shifts to step S 109 . 
         [0048]    In step S 109 , the arrangement is switched from the third arrangement to the first arrangement. When the arrangement reaches the first arrangement, step S 103  is started. 
         [0049]    When step S 103  is performed following step S 109 , that is, when step S 103  is performed for the second and subsequent times, it is determined whether time after the arrangement reaches the first arrangement has reached the first time. 
         [0050]    The invention is not limited to the embodiment explained above and various modifications of the embodiment are possible. For example, the invention includes components substantially the same as the components explained in the embodiment (for example, components having functions, methods, and results same as the functions, the methods, and the results of the components explained in the embodiment or components having objects and effects same as the objects and the effects of the components explained in the embodiment). The invention includes components in which unessential portions of the components explained in the embodiment are replaced. The invention includes components that attain action and effects same as the action and effects of the components explained in the embodiment or components that can attain objects same as the objects of the components explained in the embodiment. The invention includes components obtained by adding publicly-known techniques to the components explained in the embodiment. 
         [0051]    The entire disclosure of Japanese Patent Application No. 2014-110427, filed May 28, 2014 is expressly incorporated by reference herein.