Patent Publication Number: US-2017349935-A1

Title: Biochip

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a divisional of U.S. patent application Ser. No. 14/669,438 filed on Mar. 26, 2015, which claims priority to Japanese Patent Application No. 2014-066849 filed Mar. 27, 2014, both of which are hereby expressly incorporated by reference herein in their entireties. 
    
    
     BACKGROUND 
     1. Technical Field 
     The present invention relates to a biochip. 
     2. Related Art 
     In recent years, as a result of development of technologies utilizing genes, medical treatments utilizing genes such as gene diagnosis and gene therapy are drawing attention. In addition, many methods utilizing genes in determination of breed varieties or breed improvement have also been developed in agricultural and livestock industries. As technologies for utilizing genes, nucleic acid amplification technologies such as a PCR (Polymerase Chain Reaction) method are widely used. Nowadays, a PCR method has become an indispensable technology for elucidation of information on biological materials. 
     In the PCR method, a method in which a reaction is performed using a vessel for performing a biochemical reaction called a tube or a chip (hereinafter referred to as “biochip”) is generally used. However, in the method of the related art, there are problems that a large amount of a reagent or the like is needed, an apparatus is complicated in order to realize a thermal cycle required for the reaction, and it takes time to perform the reaction. Therefore, a biochip or a reaction apparatus for performing PCR in a short time using a very small amount of a reagent or a specimen has been demanded. 
     In order to solve such a problem, JP-A-2009-136250 (PTL 1) discloses a biochip and an apparatus for performing a reaction through a thermal cycle by allowing a reaction mixture contained in the form of a liquid droplet to move reciprocatingly in a tube filled with a liquid (such as a mineral oil) which is immiscible with the reaction mixture and has a different specific gravity from that of the reaction mixture. 
     However, in the case where the biochip disclosed in PTL 1 is used in an application in which an amplification product is detected by measuring fluorescence from the outside of a vessel, it is necessary to form the vessel from a transparent material. As the transparent material, a resin or a heat-resistant glass can be used, however, these materials are easily charged with electricity by friction or the like. It is possible to prevent static electricity by performing a hydrophilic treatment on an inner surface of the vessel, however, the reaction mixture which is an aqueous solution adheres to the vessel to hinder the movement of the reaction mixture. Due to this, it was difficult to adopt a hydrophilic treatment for a biochip. 
     On the other hand, as the liquid which is immiscible with the reaction mixture and has a different specific gravity from that of the reaction mixture, a silicone oil or a mineral oil can be used from the viewpoint of stability against heat or the reaction mixture. However, these oils are generally insulating materials, and therefore, a liquid droplet of the reaction mixture introduced into the oil is easily polarized. Due to this, when a vessel formed from a transparent material is filled with such an oil and the reaction mixture is introduced into the oil, an electric field is generated between the reaction mixture and the vessel, and the reaction mixture is attracted and adheres to the inner wall of the vessel or floats in the oil due to repulsion in some cases. If PCR of a system in which the reaction mixture is allowed to move by the gravitational force in the biochip as described in PTL 1 (hereinafter, in this specification, this system is referred to as “elevating-type system”) is performed in such a state, the reaction mixture does not appropriately move, and therefore, a desired thermal cycle cannot be performed in some cases. 
     Therefore, JP-A-2012-125169 (PTL 2) discloses a biochip which is configured such that when a vessel filled with a liquid immiscible with a reaction mixture is used, the volume resistivity of the liquid is set to more than 0 Ω·cm and 5×10 13  Ω·cm or less in order to eliminate the electric charge generated in the biochip and subject the reaction mixture to a stable thermal cycle. 
     However, even if static electricity is prevented as described in PTL 2, a liquid droplet still adheres to the vessel, and therefore, the liquid droplet cannot appropriately move in the oil in some cases. 
     SUMMARY 
     An advantage of some aspects of the invention is to provide a biochip in which a reaction mixture is prevented from adhering to the inner wall of a vessel so that the reaction mixture can appropriately move in the vessel. 
     The invention can be implemented as the following aspects. 
     A biochip according to an aspect of the invention is a biochip in which a reaction mixture containing a surfactant moves in the longitudinal direction of a vessel, and includes: a vessel; a liquid which has a different specific gravity from that of the reaction mixture and is immiscible with the reaction mixture; and an additive containing, as a principal component, a carbinol-modified silicone resin, a carboxyl-modified silicone resin, an amino-modified silicone resin, a polyether-modified silicone resin, a silanol-modified silicone resin, or a fluoro-modified silicone resin. The liquid may be a mineral oil or a silicone oil. The additive may be any of X-22-160AS, X-22-3701E, KF-857, KF-859, KF-862, KF-867, KF-6017, KF-8005 (Shin-Etsu Silicone Co., Ltd.), SR1000, SS4230, SS4267, YR3370, XS66-C1191, TSF4703, TSF4708, XF42-05196, and XF42-05197 (Momentive Performance Materials, Inc.). The reaction mixture can move in the longitudinal direction of the vessel in the form of a liquid droplet. The reaction mixture may contain a reagent for a nucleic acid amplification reaction. The concentration of the additive may be 1% (v/v) or more and 50% (v/v) or less. The vessel may be made of polypropylene. The surfactant may be NP-40, Triton X-100, or Tween 20. 
     A nucleic acid amplification apparatus according to another aspect of the invention includes any one of the above-described biochips, which is fitted therein. 
     According to the aspects of the invention, a biochip in which a reaction mixture is prevented from adhering to the inner wall of a vessel so that the reaction mixture can appropriately move in the vessel can be provided. 
    
    
     
       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 cross-sectional view of a nucleic acid amplification reaction chip according to an embodiment of the invention. The arrow g indicates the direction of the gravitational force. 
         FIGS. 2A and 2B  are perspective views of an elevating-type PCR apparatus according to one embodiment of the invention.  FIG. 2A  shows a state in which a lid is closed, and  FIG. 2B  shows a state in which the lid is opened. 
         FIG. 3  is an exploded perspective view of a main body of the elevating-type PCR apparatus according to one embodiment of the invention. 
         FIGS. 4A and 4B  are cross-sectional views schematically showing the cross section taken along the line A-A in  FIG. 2A  of the main body of the elevating-type PCR apparatus according to one embodiment of the invention.  FIG. 4A  shows a first arrangement, and  FIG. 4B  shows a second arrangement. 
     
    
    
     DESCRIPTION OF EXEMPLARY EMBODIMENTS 
     The object, characteristics, and advantages of the invention as well as the idea thereof will be apparent to those skilled in the art from the description given herein, and the invention can be easily reproduced by those skilled in the art based on the description given herein. It is to be understood that the embodiments, specific examples, etc. of the invention described below are to be taken as preferred embodiments of the invention, and are presented for illustrative or explanatory purposes and are not intended to limit the invention. It is further apparent to those skilled in the art that various changes and modifications may be made based on the description given herein within the intent and scope of the invention disclosed herein. 
     (1) Biochip 
     The biochip according to the invention is a biochip in which a reaction mixture containing a surfactant moves in the longitudinal direction of a vessel, and includes: a vessel; a liquid which has a different specific gravity from that of the reaction mixture and is immiscible with the reaction mixture; and an additive containing, as a principal component, a carbinol-modified silicone resin, a carboxyl-modified silicone resin, an amino-modified silicone resin, a polyether-modified silicone resin, a silanol-modified silicone resin, or a fluoro-modified silicone resin. In this biochip, a risk that the reaction mixture in the form of a liquid droplet adheres to the inner wall of the vessel is reduced, so that the reaction mixture easily moves in the longitudinal direction of the vessel. Hereinafter, the configuration of the biochip will be described in detail. 
       FIG. 1  is a cross-sectional view of a biochip  100 .  FIG. 1  shows a state in which a reaction mixture is placed in the biochip. 
     The biochip  100  according to the invention is configured to include a vessel  150  and a sealing section  120 . The size and shape of the biochip  100  are not particularly limited, but for example, the biochip may be designed in consideration of at least one of the amount of an oil  130  which is immiscible with a reaction mixture  140 , the thermal conductivity of the oil  130 , the shape of the vessel  150  and the sealing section  120 , and the ease of handling. 
     The vessel  150  of the biochip  100  can be formed from a transparent material. According to this, the movement of the reaction mixture  140  in the vessel  150  can be observed from the outside of the biochip  100 , or the biochip  100  can be used in an application in which the measurement or the like is performed from the outside of the vessel  150  such as real-time PCR. The term “transparent” as used herein refers to a condition in which the visibility to such an extent that the reaction mixture  140  in the vessel  150  can be observed from the outside of the vessel  150  can be ensured, and it is not necessary that the entire biochip  100  should be transparent as long as this condition is met. 
     The application of the biochip  100  is not particularly limited, however, for example, in the case where the biochip  100  is used in an application with a fluorescence measurement such as real-time PCR, the vessel  150  is desirably formed from a material with a low autofluorescence. The vessel  150  is preferably formed from a material which can withstand heating in PCR. Further, the material of the vessel  150  is preferably a material, on which nucleic acids or proteins are less adsorbed, and which does not inhibit the enzymatic reaction by a polymerase or the like. The material which satisfies these conditions is not particularly limited, and for example, polypropylene, polyethylene, a cycloolefin polymer (for example, ZEONEX (registered trademark) 480R), a heat-resistant glass (for example, PYREX (registered trademark) glass), or the like, or a composite material thereof may be used, however, polypropylene is preferred. 
     In the biochip  100  shown in  FIG. 1 , the vessel  150  is formed into a cylindrical shape, and the direction of the center axis (the vertical direction in  FIG. 1 ) coincides with the longitudinal direction. The vessel  150  used here is preferably a tube and may be a tube for a microcentrifuge or a tube designed for PCR. Since the vessel  150  has a shape with a longitudinal direction, in other words, an elongated shape, for example, in the case where the temperature of the biochip  100  is controlled so that regions having different temperatures are formed in the oil  130  in the vessel  150  using an elevating-type thermal cycler, which will be described later, the distance between the regions having different temperatures is easily increased, According to this, it becomes easy to control the temperature of the oil  130  to be different from region to region in the vessel  150 , and therefore, a thermal cycle suitable for PCR can be realized. The “elevating-type thermal cycler” is an apparatus which performs a thermal cycle by forming at least two temperature regions in an oil filled in the vessel  150  and allowing the reaction mixture  140  which is phase-separated from the oil to move reciprocatingly between these temperature regions. 
     The shape of the vessel  150  is not particularly limited as long as it has a longitudinal direction, however, in the case where the vessel  150  is used for elevating-type PCR, it is preferred that the shape is a substantially cylindrical shape and the ratio of the inner diameter D to the length L in the longitudinal direction is in the range of 1:5 to 5:20. It is more preferred that the inner diameter D is from 1.5 to 2 mm, and the length L is from 10 to 20 mm. 
     The vessel  150  has an opening section and the sealing section  120  which seals the opening section, and in the vessel  150 , the reaction mixture  140  and the liquid  130  which has a different specific gravity from that of the reaction mixture  140  and is phase-separated from the reaction mixture  140  are contained. It is preferred that in the case where the opening section is sealed by the sealing section  120 , air does not remain in the vessel  150 . It is because if an air bubble remains in the vessel  150 , the movement of the reaction mixture  140  may be hindered. The sealing section  120  can be formed from the same material as that of the vessel  150 . The structure of the sealing section  120  may be any as long as it can hermetically seal the vessel  150 , and can be a structure of, for example, a screw cap, a plug, an inlay, or the like. In  FIG. 1 , the sealing section  120  has a structure of a screw cap. 
     The reaction to which the reaction mixture  140  is subjected is not particularly limited, however, the reaction mixture  140  may be subjected to a nucleic acid amplification reaction and may contain a reagent for a nucleic acid amplification reaction and may also contain a target nucleic acid to be amplified. Examples of the target nucleic acid include a DNA prepared from a specimen such as blood, urine, saliva, spinal fluid, or a tissue and a cDNA obtained by reverse transcription of an RNA prepared from any of the above specimens. The reagent for a nucleic acid amplification reaction may contain a primer for amplifying a target nucleic acid, a buffer, a polymerase, dNTPs, MgCl 2 , a fluorescent probe for detecting an amplification product of the target nucleic acid, and the like. The DNA polymerase is not particularly limited, but is preferably a heat-resistant enzyme or an enzyme for use in PCR. There are a great number of commercially available products, for example, Taq polymerase, Tfi polymerase, Tth polymerase, modified forms thereof, and the like, however, a DNA polymerase capable of performing hot start PCR is preferred. The concentration of dNTPs or a salt may be set to a concentration suitable for the enzyme to be used, however, the concentration of dNTPs may be set to generally 10 to 1000 μM, preferably 100 to 500 μM, the concentration of Mg 2+  may be set to generally 1 to 100 mM, preferably 5 to 10 mM, and the concentration of Cl −  may be set to 1 to 2000 mM, preferably 200 to 700 mM. The total ion concentration is not particularly limited, but may be higher than 50 mM, and is preferably higher than 100 mM, more preferably higher than 120 mM, further more preferably higher than 150 mM, still further more preferably higher than 200 mM. The upper limit thereof is preferably 500 mM or less, more preferably 300 mM or less, further more preferably 200 mM or less. Each oligonucleotide for the primer is used at 0.1 to 10 μM, preferably 0.1 to 1 μM. 
     The reaction mixture  140  may further contain a surfactant. The surfactant is not particularly limited, however, examples thereof include NP-40, Triton X-100, and Tween 20. The concentration of the surfactant is not particularly limited, but is preferably a concentration which does not inhibit the nucleic acid amplification reaction, and may be from 0.001% to 0.1%, and is preferably from 0.002% to 0.02%, most preferably from 0.005% to 0.01%. The surfactant may be a carry-over from a stock solution of the enzyme described above, however, a surfactant solution may be added to the reaction mixture  140  separately therefrom. 
     By using a liquid which is immiscible with the reaction mixture  140  as the liquid  130 , when the reaction mixture  140  is placed in the vessel  150 , the reaction mixture  140  and the liquid  130  are phase-separated from each other, and therefore, the reaction mixture  140  can be formed into a liquid droplet in the liquid  130 . In this manner, the reaction mixture  140  is maintained in the form of a liquid droplet in the liquid  130 . 
     The liquid  130  is preferably a liquid having a specific gravity smaller than that of the reaction mixture  140 . In this case, when the reaction mixture  140  is placed in the liquid  130 , the liquid droplet of the reaction mixture  140  has a specific gravity larger than that of the liquid  130 , and therefore moves in the direction in which the gravitational force acts by the action of the gravity. Further, the liquid  130  may be a liquid having a specific gravity larger than that of the reaction mixture  140 . In this case, the liquid droplet of the reaction mixture  140  has a specific gravity smaller than that of the liquid  130 , and therefore moves in the direction opposite to the direction in which the gravitational force acts by the action of the gravity. 
     The liquid  130  preferably contains an oil, and for example, a silicone oil or a mineral oil can be used. Here, the “silicone” means an oiligomer or a polymer having a siloxane bond as a main skeleton. In this specification, among silicones, a silicone in the form of a liquid in a temperature range in which the silicone is used in a thermal cycling process is particularly referred to as “silicone oil”. Further, in this specification, an oil which is purified from petroleum and is in the form of a liquid in a temperature range in which the oil is used in a thermal cycling process is referred to as “mineral oil”. These oils have high stability against heat, and for example, products having a viscosity of 5×10 3  Nsm −2  or less are also easily available, and therefore, these oils are preferred for use in elevating-type PCR. 
     Examples of the silicone oil include dimethyl silicone oils such as KF-96L-0.65cs, KF-96L-1cs, KF-96L-2cs, KF-96L-5cs (Shin-Etsu Silicone Co., Ltd.) , SH200 C FLUID 5 CS (Dow Corning Toray Co., Ltd.), TSF451-5A, and TSF451-10 (Momentive Performance Materials Japan LLC Company Ltd.). Examples of the mineral oil include oils containing alkane having about 14 to 20 carbon atoms as a principal component, and specific examples thereof include n-tetradecane, n-pentadecane, n-hexadecane, n-heptadecane, n-octadecane, n-nonadecane, and n-tetracosane. 
     The liquid  130  contains an additive. The additive may be any as long as it is an additive containing, as a principal component, a carbinol-modified silicone resin, a carboxyl-modified silicone resin, an amino-modified silicone resin, a polyether-modified silicone resin, a silanol-modified silicone resin, or a fluoro-modified silicone resin, and for example, a modified silicone oil such as X-22-160AS, X-22-3701E, KF-857, KF-859, KF-862, KF-867, KF-6017, or KF-8005 (Shin-Etsu Silicone Co., Ltd.), a silicone resin such as SR1000, SS4230, SS4267, or YR3370 (Momentive Performance Materials, Inc.), a fluoro-modified silicone resin such as XS66-C1191 (Momentive Performance Materials, Inc.), or the like, and other than these, a modified silicone oil such as TSF4703, TSF4708, XF42-05196, or XF42-05197 (Momentive Performance Materials, Inc.) can be used. The concentration of the additive is not particularly limited, but can be determined in consideration of the structure, material, shape, or the like of the vessel. For example, the concentration thereof is set to preferably 1% (v/v) or more and 50% (v/v) or less, more preferably 2% (v/v) or more and 20% (v/v) or less, further more preferably 5% (v/v). 
     (2) Structure of Elevating-Type Nucleic Acid Amplification Reaction Apparatus 
     In this embodiment, as the biochip to be used for performing a nucleic acid amplification reaction, a nucleic acid amplification reaction tube  100  in the form of a tube is used. Hereinafter, one example of an elevating-type nucleic acid amplification reaction apparatus (hereinafter also referred to as “elevating-type PCR apparatus”) suitable for the nucleic acid amplification reaction tube  100  will be described in detail. 
       FIGS. 2A and 2B  show one example of an elevating-type PCR apparatus  1 .  FIG. 2A  shows a state in which a lid  50  of the elevating-type PCR apparatus  1  is closed, and  FIG. 2B  shows a state in which the lid  50  of the elevating-type PCR apparatus  1  is opened and the nucleic acid amplification reaction tube  100  is fitted in a fitting section  11 .  FIG. 3  is an exploded perspective view of a main body  10  of the elevating-type PCR apparatus  1  according to the embodiment.  FIG. 4A  is a cross-sectional view schematically showing the cross section taken along the line A-A in  FIG. 2A  of the main body  10  of the elevating-type PCR apparatus  1  according to the embodiment. 
     This elevating-type PCR apparatus  1  includes the main body  10  and a driving mechanism  20  as shown in  FIG. 2A . As shown in  FIG. 3 , the main body  10  includes the fitting section  11 , a first heating section  12 , and a second heating section  13 . A spacer  14  is provided between the first heating section  12  and the second heating section  13 . In the main body  10  of this embodiment, the first heating section  12  is disposed on the side of a bottom plate  17 , and the second heating section  13  is disposed on the side of the lid  50 . In the main body  10  of this embodiment, the first heating section  12 , the second heating section  13 , and the spacer  14  are fixed by a flange  16 , the bottom plate  17 , and a fixing plate  19 . 
     The fitting section  11  is configured such that the nucleic acid amplification reaction tube  100 , which will be described later, is fitted therein. As shown in  FIG. 2B  and  FIG. 3 , the fitting section  11  of this embodiment has a slot structure in which the nucleic acid amplification reaction tube  100  is inserted and fitted, and is configured such that the nucleic acid amplification reaction tube  100  is inserted into a hole penetrating a first heat block  12   b  of the first heating section  12 , the spacer  14 , and a second heat block  13   b  of the second heating section  13 . The number of the fitting sections  11  may be more than one, and in the example shown in  FIG. 2B , twenty fitting sections  11  are provided for the main body  10 . 
     This elevating-type PCR apparatus  1  includes a structure in which the nucleic acid amplification reaction tube  100  is held at a predetermined position with respect to the first heating section  12  and the second heating section  13 . More specifically, as shown in  FIGS. 4A and 4B , in a flow channel  110  constituting the nucleic acid amplification reaction tube  100 , which will be described later, a first region  111  can be heated by the first heating section  12  and a second region  112  can be heated by the second heating section  13 . In this embodiment, a structure that defines the position of the nucleic acid amplification reaction tube  100  is the bottom plate  17 , and as shown in  FIG. 4A , by inserting the nucleic acid amplification reaction tube  100  to a position in contact with the bottom plate  17 , the nucleic acid amplification reaction tube  100  can be held at a predetermined position with respect to the first heating section  12  and the second heating section  13 . 
     When the nucleic acid amplification reaction tube  100  is fitted in the fitting section  11 , the first heating section  12  heats the first region  111  of the nucleic acid amplification reaction tube  100 , which will be described later, to a first temperature. In the example shown in  FIG. 4A , in the main body  10 , the first heating section  12  is disposed at a position so as to heat the first region  111  of the nucleic acid amplification reaction tube  100 . 
     The first heating section  12  may include a mechanism that generates heat and a member that transfers the generated heat to the nucleic acid amplification reaction tube  100 . In the example shown in  FIG. 3 , the first heating section  12  includes a first heater  12   a  and a first heat block  12   b.  In this embodiment, the first heater  12   a  is a cartridge heater, and is connected to an external power source (not shown) through a conductive wire  15 . The first heater  12   a  is inserted into the first heat block  12   b,  and the first heat block  12   b  is heated by heat generated by the first heater  12   a.  The first heat block  12   b  is a member that transfers heat generated by the first heater  12   a  to the nucleic acid amplification reaction tube  100 . In this embodiment, the first heat block  12   b  is a block made of aluminum. 
     When the nucleic acid amplification reaction tube  100  is fitted in the fitting section  11 , the second heating section  13  heats the second region  112  of the nucleic acid amplification reaction tube  100  to a second temperature different from the first temperature. In the example shown in  FIG. 4A , in the main body  10 , the second heating section  13  is disposed at a position so as to heat the second region  112  of the nucleic acid amplification reaction tube  100 . As shown in  FIG. 2 , the second heating section  13  includes a second heater  13   a  and the second heat block  13   b.  The second heating section  13  is configured in the same manner as the first heating section  12  except that the region of the nucleic acid amplification reaction tube  100  to be heated and the heating temperature are different from those for the first heating section  12 . 
     In this embodiment, the temperatures of the first heating section  12  and the second heating section  13  are controlled by a temperature sensor (not shown) and a control section (not shown), which will be described later. The temperatures of the first heating section  12  and the second heating section  13  are preferably set so as to heat the nucleic acid amplification reaction tube  100  to a desired temperature. In this embodiment, by controlling the first heating section  12  at the first temperature and the second heating section  13  at the second temperature, the first region  111  of the nucleic acid amplification reaction tube  100  can be heated to the first temperature, and the second region  112  can be heated to the second temperature. The temperature sensor in this embodiment is a thermocouple. 
     The driving mechanism  20  is a mechanism that drives the fitting section  11 , the first heating section  12 , and the second heating section  13 . In this embodiment, the driving mechanism  20  includes a motor (not shown) and a drive shaft (not shown), and the drive shaft is connected to the flange  16  of the main body  10 . The drive shaft in this embodiment is provided perpendicular to the longitudinal direction of the fitting section  11 , and when the motor is activated, the main body  10  is rotated about the drive shaft as the axis of rotation. 
     The elevating-type PCR apparatus  1  of this embodiment includes the control section (not shown). The control section controls at least one of the first temperature, the second temperature, a first period, a second period, and the number of thermal cycles, which will be described later. In the case where the control section controls the first period or the second period, the control section controls the operation of the driving mechanism  20 , thereby controlling the period in which the fitting section  11 , the first heating section  12 , and the second heating section  13  are held in a predetermined arrangement. The control section may have mechanisms different from item to item to be controlled, or may control all items collectively. However, the control section in the elevating-type PCR apparatus  1  of this embodiment is an electronic control system and controls all of the above-described items. The control section of this embodiment includes a processor such as CPU (not shown) and a storage device such as an ROM (Read Only Memory) or an RAM (Random Access Memory). In the storage device, various programs, data, etc. for controlling the above-described respective items are stored. Further, the storage device has a work area for temporarily storing data in processing, processing results, etc. of various processes. 
     As shown in the example of  FIG. 3  and  FIG. 4A , in the main body  10  of this embodiment, the spacer  14  is provided between the first heating section  12  and the second heating section  13 . The spacer  14  of this embodiment is a member that holds the first heating section  12  or the second heating section  13 . In this embodiment, the spacer  14  is a heat insulating material, and in the example shown in  FIG. 4A , the fitting section  11  penetrates the spacer  14 . 
     The main body  10  of this embodiment includes the fixing plate  19 . The fixing plate  19  is a member that holds the fitting section  11 , the first heating section  12 , and the second heating section  13 . In the example shown in  FIG. 2B  and  FIG. 3 , two fixing plates  19  are fitted in the flanges  16 , and the first heating section  12 , the second heating section  13 , and the bottom plate  17  are fixed by the fixing plates  19 . 
     The elevating-type PCR apparatus  1  of this embodiment includes the lid  50 . In the example shown in  FIG. 2A  and  FIG. 4A , the fitting section  11  is covered with the lid  50 . The lid  50  may be fixed to the main body  10  by a fixing section  51 . In this embodiment, the fixing section  51  is a magnet. As shown in the example of  FIG. 2B  and  FIG. 3 , a magnet is provided on a surface of the main body  10  which comes into contact with the lid  50 . Although not shown in  FIG. 2B  and  FIG. 3 , a magnet is provided also for the lid  50  at a place with which the magnet of the main body  10  comes into contact. When the fitting section  11  is covered with the lid  50 , the lid  50  is fixed to the main body  10  by a magnetic force. 
     It is preferred that the fixing plate  19 , the bottom plate  17 , the lid  50 , and the flange  16  are formed using a heat insulating material. 
     (3) Thermal Cycling Process Using Elevating-Type PCR Apparatus 
       FIGS. 4A and 4B  are cross-sectional views schematically showing the cross section taken along the line A-A in  FIG. 2A  of the elevating-type PCR apparatus  1 . FIGS.  4 A and  4 B show a state in which the nucleic acid amplification reaction tube  100  is fitted in the elevating-type PCR apparatus  1 .  FIG. 4A  shows a first arrangement, and  FIG. 4B  shows a second arrangement. Hereinafter, first, the nucleic acid amplification reaction tube  100  according to the embodiment will be described, and thereafter, the thermal cycling process using the elevating-type PCR apparatus  1  according to the embodiment in the case of using the nucleic acid amplification reaction tube  100  will be described. 
     As shown in the example of  FIG. 1 , the nucleic acid amplification reaction tube  100  according to the embodiment includes a flow channel  110  and a sealing section  120 . The flow channel  110  is filled with a reaction mixture  140  and an oil  130  which has a specific gravity smaller than that of the reaction mixture  140  and is immiscible with the reaction mixture  140 , and sealed with the sealing section  120 . 
     The flow channel  110  is formed such that the reaction mixture  140  moves in close proximity to opposed inner walls. Here, the term “opposed inner walls” of the flow channel  110  refers to two regions of a wall surface of the flow channel  110  having an opposed positional relationship. The phrase “in close proximity to” refers to a state in which the distance between the reaction mixture  140  and the wall surface of the flow channel  110  is close, and includes a case where the reaction mixture  140  is in contact with the wall surface of the flow channel  110 . Therefore, the phrase “the reaction mixture  140  moves in close proximity to opposed inner walls” refers to that “the reaction mixture  140  moves in a state of being close in distance to both of the two regions of a wall surface of the flow channel  110  having an opposed positional relationship”, that is, the reaction mixture  140  moves along the opposed inner walls. 
     In the example shown in  FIG. 1 , the outer shape of the nucleic acid amplification reaction tube  100  is a cylindrical shape, and the flow channel  110  is formed in the direction of the center axis (the vertical direction in  FIG. 1 ) therein. The shape of the flow channel  110  is a cylindrical shape having a circular cross section in the direction perpendicular to the longitudinal direction of the flow channel  110 , that is, in the direction perpendicular to the direction of the movement of the reaction mixture  140  in a region in the flow channel  110  (this cross section is defined as the “cross section” of the flow channel  110 ). Therefore, in the nucleic acid amplification reaction tube  100  of this embodiment, the opposed inner walls of the flow channel  110  are regions including two points on the wall surface of the flow channel  110  constituting the diameter of the cross section of the flow channel  110 , and the reaction mixture  140  moves in the longitudinal direction of the flow channel  110  along the opposed inner walls. 
     The first region  111  of the nucleic acid amplification reaction tube  100  is a partial region of the flow channel  110  which is heated to the first temperature by the first heating section  12 . The second region  112  is a partial region of the flow channel  110  which is different from the first region  111  and is heated to the second temperature by the second heating section  13 . In the nucleic acid amplification reaction tube  100  of this embodiment, the first region  111  is a region including one end portion in the longitudinal direction of the flow channel  110 , and the second region  112  is a region including the other end portion in the longitudinal direction of the flow channel  110 . In the example shown in  FIGS. 4A and 4B , a region surrounded by the dotted line including an end portion on the proximal side of the sealing section  120  of the flow channel  110  is the second region  112 , and a region surrounded by the dotted line including an end portion on the distal side of the sealing section  120  is the first region  111 . 
     As shown in  FIG. 1 , the flow channel  110  contains the oil  130  and a liquid droplet of the reaction mixture  140 . The oil  130  and the reaction mixture  140  are prepared according to the description of the above (1) Biochip. 
     Hereinafter, with reference to  FIGS. 4A and 4B , the thermal cycling process using a thermal cycler  1  according to the embodiment will be described. In  FIGS. 4A and 4B , the direction indicated by the arrow g (in the downward direction in the drawing) is the gravitational direction. In this embodiment, a case where shuttle PCR (two-stage temperature PCR) is performed will be described as an example of the thermal cycling process. The respective steps described below are shown as an example of the thermal cycling process, and according to need, the order of the steps may be changed, two or more steps may be performed continuously or concurrently, or a step may be added. 
     The shuttle PCR is a method of amplifying a nucleic acid in a reaction mixture by subjecting the reaction mixture to a two-stage temperature process at a high temperature and a low temperature repeatedly. In the process at a high temperature, denaturation of a double-stranded DNA occurs and in the process at a low temperature, annealing (a reaction in which a primer is bound to a single-stranded DNA) and elongation (a reaction in which a complementary strand to the DNA is formed by using the primer as a starting point) occur. 
     In general, in shuttle PCR, the high temperature is a temperature between 80° C. and 100° C. and the low temperature is a temperature between 50° C. and 70° C. The processes at the respective temperatures are performed for a predetermined period, and a period of maintaining the reaction mixture at a high temperature is generally shorter than a period of maintaining the reaction mixture at a low temperature. For example, the period for the process at a high temperature may be set to about 1 to 10 seconds, and the period for the process at a low temperature may be set to about 10 to 60 seconds, or a period longer than this range may be adopted depending on the condition of the reaction. 
     Since the appropriate period, temperature, number of cycles (number of times of repetition of the process at a high temperature and the process at a low temperature) varies depending on the type or amount of a reagent to be used, it is preferred to determine an appropriate protocol in consideration of the type of a reagent or the amount of the reaction mixture  140  before performing the reaction. 
     First, the nucleic acid amplification reaction tube  100  is fitted in the fitting section  11 . In this embodiment, after the reaction mixture  140  is introduced into the flow channel  110  previously filled with the oil  130 , the flow channel  110  is sealed with the sealing section  120 , and then, the nucleic acid amplification reaction tube  100  is fitted in the fitting section  11 . The introduction of the reaction mixture  140  can be performed using a micropipette, an ink-jet dispenser, or the like. In a state in which the nucleic acid amplification reaction tube  100  is fitted in the fitting section  11 , the first heating section  12  is in contact with the nucleic acid amplification reaction tube  100  at a place including the first region  111  and the second heating section  13  is in contact with the nucleic acid amplification reaction tube  100  at a place including the second region  112 . 
     Here, the arrangement of the fitting section  11 , the first heating section  12 , and the second heating section  13  is the first arrangement. As shown in  FIG. 4A , in the first arrangement, the first region  111  of the nucleic acid amplification reaction tube  100  is located in a lowermost portion of the flow channel  110  with respect to the gravitational direction. In the first arrangement, the first region  111  is located in a lowermost portion of the flow channel  110  with respect to the gravitational direction, and therefore, the reaction mixture  140  having a specific gravity larger than that of the oil  130  is located in the first region  111 . In this embodiment, after the nucleic acid amplification reaction tube  100  is fitted in the fitting section  11 , the fitting section  11  is covered with the lid  50 , and then the elevating-type PCR apparatus  1  is activated. 
     Subsequently, the nucleic acid amplification reaction tube  100  is heated by the first heating section  12  and the second heating section  13 . The first heating section  12  and the second heating section  13  heat different regions of the nucleic acid amplification reaction tube  100  to different temperatures. That is, the first heating section  12  heats the first region  111  to the first temperature, and the second heating section  13  heats the second region  112  to the second temperature. Accordingly, a temperature gradient in which the temperature gradually changes between the first temperature and the second temperature is formed between the first region  111  and the second region  112  of the flow channel  110 . Here, a temperature gradient in which the temperature decreases from the first region  111  to the second region  112  is formed. The thermal cycling process of this embodiment is shuttle PCR, and therefore, the first temperature is set to a temperature suitable for the denaturation of a double-stranded DNA, and the second temperature is set to a temperature suitable for the annealing and elongation. 
     Since the arrangement of the fitting section  11 , the first heating section  12 , and the second heating section  13  is the first arrangement, when the nucleic acid amplification reaction tube  100  is heated, the reaction mixture  140  is heated to the first temperature. When the first period has elapsed, the main body  10  is driven by the driving mechanism  20 , and the arrangement of the fitting section  11 , the first heating section  12 , and the second heating section  13  is switched over from the first arrangement to the second arrangement. The second arrangement is an arrangement in which the second region  112  is located in a lowermost portion of the flow channel  110  with respect to the gravitational direction. In other words, the second region  112  is a region located in a lowermost portion of the flow channel  110  with respect to the gravitational direction when the fitting section  11 , the first heating section  12 , and the second heating section  13  are in a predetermined arrangement different from the first arrangement. In the thermal cycler  1  of this embodiment, by the control of the control section, the driving mechanism  20  rotatively drives the main body  10 . When the flanges  16  are rotatively driven by the motor by using the drive shaft as the axis of rotation, the fitting section  11 , the first heating section  12 , and the second heating section  13  which are fixed to the flanges  16  are rotated. Since the drive shaft is a shaft extending in the direction perpendicular to the longitudinal direction of the fitting section  11 , when the drive shaft is rotated by the activation of the motor, the fitting section  11 , the first heating section  12 , and the second heating section  13  are rotated. In the example shown in  FIGS. 4A and 4B , the main body  10  is rotated at 180°. By doing this, the arrangement of the fitting section  11 , the first heating section  12 , and the second heating section  13  is switched over from the first arrangement to the second arrangement. 
     Here, the positional relationship between the first region  111  and the second region  112  with respect to the gravitational direction is opposite from that of the first arrangement, and therefore, the reaction mixture  140  moves from the first region  111  to the second region  112  by the gravitational force. When the operation of the driving mechanism  20  is stopped after the arrangement of the fitting section  11 , the first heating section  12 , and the second heating section  13  has reached the second arrangement, the fitting section  11 , the first heating section  12 , and the second heating section  13  are held in the second arrangement. When the second period has elapsed in the second arrangement, the main body  10  is rotated again. A nucleic acid amplification reaction is performed by rotating the main body  10  while switching over between the first arrangement and the second arrangement in this manner until the number of thermal cycles has reached a predetermined number of cycles. 
     EXAMPLES 
     Hereinafter, the invention will be described in more detail by showing Examples, however, the invention is not limited thereto. 
     (1) Effect of Surfactant 
     In this example, with reference to the concentration of a surfactant contained in a commercially available enzyme (DNA polymerase), surfactant solutions having various concentrations were prepared, and the adhesion of each of the solutions to a tube was observed. 
     First, a polypropylene tube for an elevating-type PCR apparatus was filled with an oil, and then, 1.6 μL of each of the surfactant solutions was added thereto to form a liquid droplet of the surfactant solution. Then, the tube was laid on its side and left to stand for 5 minutes in a state where the liquid droplet was not in contact with the bottom. Thereafter, the tube was made to stand upright, and thereby the surfactant solution was made to move to the bottom of the tube. One minute after the surfactant solution reached the bottom, the tube was turned upside down, and the state of adhesion of the surfactant solution to the bottom of the tube was evaluated. A case where the surfactant solution immediately fell down was evaluated as “good”, and a case where even the slightest sign of adhesion of the surfactant solution to the bottom of the tube was observed, ranging from a case where the surfactant solution fell down when the tube was shaken to a case where the surfactant solution did not fall down even if the tube was shaken, was evaluated as “bad”. As the oil, KF-96L-2cs which is a dimethyl silicone oil with high purity was used. 
     As a result, when the concentration of the surfactant was a given value or higher, the surfactant solution adhered to the bottom of the tube. In Table 1, with respect to the tube used, the highest concentration at which the surfactant solution did not adhere to the bottom of the tube is shown. That is, when the concentration of the surfactant was equal to or lower than the concentration shown in Table 1, the reaction mixture did not adhere to the inner wall, however, when the concentration of the surfactant was higher than the concentration shown in Table 1, the adhesion of the reaction mixture to the inner wall was observed. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 1 
               
               
                   
                   
               
             
            
               
                   
                 NP-40 
                 0.002 
               
               
                   
                 Triton X-100 
                 0.01 
               
               
                   
                 Tween 20 
                 0.1 
               
               
                   
                   
               
               
                   
                 Unit: % (v/v) 
               
            
           
         
       
     
     When a surfactant is contained in the reaction mixture at a given concentration or higher in this manner, a phenomenon in which the reaction mixture is adhered to the inner wall of the tube and does not fall down even if the tube is turned upside down occurs. 
     (2) Effect of Additive 
     In this Example, each of the additives shown in Table 2 was added at 5% (v/v) to KF-96L-2cs, which is a dimethyl silicone oil with high purity, followed by mixing, and the resulting mixture was used as the oil. Further, the reaction mixture was prepared as follows. 
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 Takara Ex Taq HS (x50) 
                 0.25 
               
               
                   
                 10 × Ex Taq Buffer 
                 5.0 
               
               
                   
                 dNTP Mixture (2.5 mM each) 
                 4.0 
               
               
                   
                 InfA forward primer (20 μM) 
                 2.0 
               
               
                   
                 InfA reverse primer (20 μM) 
                 2.0 
               
               
                   
                 InfA probe (10 μM) 
                 1.0 
               
               
                   
                 plasmid 
                 5.0 
               
               
                   
                 DW 
                 up to 50.0 (unit: μL) 
               
               
                   
                   
               
            
           
         
       
     
     The commercially available Takara Ex Taq HS contains 0.5% NP-40 and 0.5% Tween 20, and therefore, the thus prepared reaction mixture contained 0.02% NP-40 and 0.02% Tween 20. By using these oils and the reaction mixture, the state of adhesion of the reaction mixture to the tube was observed. The state of adhesion of the reaction mixture to the tube was evaluated in the same manner as in the above (1). 
     
       
         
           
               
               
               
             
               
                 TABLE 2 
               
               
                   
               
               
                 Additive 
                 Type of additive 
                 Evaluation 
               
               
                   
               
             
            
               
                 X-22-160AS 
                 carbinol-modified silicone oil 
                 good 
               
               
                 X-22-3701E 
                 carboxyl-modified silicone oil 
                 good 
               
               
                 KF-857 
                 amino-modified silicone oil 
                 good 
               
               
                 KF-859 
                 amino-modified silicone oil 
                 good 
               
               
                 KF-862 
                 amino-modified silicone oil 
                 good 
               
               
                 KF-867 
                 amino-modified silicone oil 
                 good 
               
               
                 KF-6017 
                 polyether-modified silicone oil 
                 good 
               
               
                 KF-8005 
                 amino-modified silicone oil 
                 good 
               
               
                 SR1000 
                 silanol-modified silicone resin 
                 good 
               
               
                 SS4230 
                 silanol-modified silicone resin 
                 good 
               
               
                 SS4267 
                 silanol-modified silicone resin 
                 good 
               
               
                 TSF4703 
                 amino-modified silicone oil 
                 good 
               
               
                 TSF4708 
                 amino-modified silicone oil 
                 good 
               
               
                 YR3370 
                 silanol-modified silicone resin 
                 good 
               
               
                 XF42-C5196 
                 amino-modified silicone oil 
                 good 
               
               
                 XF42-C5197 
                 amino-modified silicone oil 
                 good 
               
               
                 XS66-C1191 
                 fluoro-modified silicone resin 
                 good 
               
               
                 non 
                 — 
                 bad 
               
               
                   
               
            
           
         
       
     
     In this manner, by adding any of the additives shown in Table 2 at about 5% (v/v), the adhesion of the reaction mixture to the tube can be prevented.