JP6235284B2 - Nucleic acid amplification method and nucleic acid amplification microchip - Google Patents

Description

  The present invention relates to a nucleic acid amplification method and a nucleic acid amplification microchip. The present invention is useful for nucleic acid amplification reaction using a trace amount sample.

  A microchip capable of handling a small amount of liquid sample is known. For example, a microchannel for transporting a liquid sample or the like is formed in a substrate (chip) that can be easily handled by hand. If necessary, the sample introduction part, reagent holding part, reaction A microchip or a microfluidic device provided with a tank or the like is known (for example, Patent Documents 1 and 2).

  Nucleic acid amplification reactions such as polymerase chain reaction (PCR) can be performed using a microchip. By using the microchip, the nucleic acid amplification reaction can be easily performed even with a small amount of liquid sample.

  The nucleic acid amplification reaction by the PCR method repeats a denaturation step for denaturing (dissociating) nucleic acids, an annealing step for annealing nucleic acids, and an extension step for extending nucleic acids. In general, the denaturation step is performed at a high temperature of about 94 ° C. to 96 ° C., while the annealing step and the elongation step are performed at a lower temperature, for example, about 55 ° C. to 75 ° C. Therefore, in the nucleic acid amplification reaction, the step of exposing the reaction solution to at least two kinds of temperatures is repeated alternately. That is, the reaction solution is repeatedly raised and lowered.

  As a measure for repeating the temperature increase and the temperature decrease of the reaction solution, a plurality of regions maintained at different predetermined temperatures are set, and the reaction solution is moved or reciprocated between these regions. According to this measure, the reaction solution can be exposed to a desired temperature in a shorter time than when the temperature of one reaction tank is raised and lowered, and the time required for nucleic acid amplification can be shortened.

  A technique for moving or reciprocating a reaction solution between different temperature ranges is known. For example, Patent Document 3 discloses a DNA amplification device in which a reciprocating piston is installed in a cylindrical reaction vessel made of a glass capillary, and the DNA amplification reaction solution in the reaction vessel is reciprocated by reciprocating the piston. Is disclosed. In this apparatus, the reaction solution can be reciprocated between three types of temperature regions. Patent Document 4 discloses a configuration in which a reaction solution sequentially passes through different temperature regions by flowing the reaction solution in one direction through a zigzag channel formed in the microfluidic device.

JP 2012-132879 A JP 2012-215535 A Japanese Patent Laid-Open No. 7-303469 JP 2005-192554 A

However, the technique described in Patent Document 3 reciprocates the reaction liquid using a piston, and the configuration of the apparatus is complicated. Also in the technique described in Patent Document 4, the reaction liquid is fed using a separately prepared micro syringe pump, which cannot be said to be a simple configuration.
Accordingly, an object of the present invention is to provide a technique capable of repeatedly raising and lowering the temperature of a reaction solution in a nucleic acid amplification reaction with a simpler configuration.

One aspect for solving the aforementioned problems is a method for amplifying a nucleic acid repeating cooling and heating of the reaction solution, and a high temperature region kept at a predetermined temperature, lower than the predetermined temperature of the high temperature region predetermined A low-temperature region maintained at a temperature is set, and the reaction solution is reciprocated between the high-temperature region and the low-temperature region to repeatedly raise and lower the temperature of the reaction solution. A nucleic acid characterized in that the reaction solution is reciprocated by alternately applying a step of applying a pressure by a gas generated from a generating agent to the reaction solution and moving the reaction solution from one region to the other region. This is an amplification method.

The present invention relates to a method for amplifying a nucleic acid by repeatedly raising and lowering the temperature of a reaction solution. In the nucleic acid amplification method of the present invention, a high temperature region and a low temperature region maintained at different predetermined temperatures are set, and the reaction solution is reciprocated between these regions to repeatedly raise and lower the reaction solution.
And in this invention, when reciprocating a reaction liquid, the pressure of the gas which generate | occur | produced from the gas generating agent of photoresponsiveness or a heat-responsive property is utilized. That is, by applying the pressure of the gas to the reaction solution, the reaction solution is moved from one region to the other region.
In this invention, since the pressure of the gas generated from the photoresponsive or thermally responsive gas generating agent is used, the reaction solution can be reciprocated with a simpler configuration. Therefore, the nucleic acid amplification reaction can be performed simply and efficiently. The nucleic acid amplification method of the present invention is particularly useful for nucleic acid amplification reactions using a trace amount of sample.

Similar modal phase of the present invention to solve the problems, the nucleic acid amplification reaction of repeating Atsushi Nobori and temperature drop of the reaction solution to a nucleic acid amplification method carried out in a microchip, the microchip is kept at a predetermined temperature A high-temperature reaction tank, a low-temperature reaction tank maintained at a predetermined temperature lower than a predetermined temperature of the high-temperature reaction tank, a connection path connecting the high-temperature reaction tank and the low-temperature reaction tank, and each predetermined temperature The reaction liquid is reciprocated between the maintained high-temperature reaction tank and the low-temperature reaction tank and returned to the original reaction tank, thereby repeatedly raising and lowering the temperature of the reaction liquid. Alternatively, it further includes a heat-responsive gas generating agent, supplying a gas generated from the gas generating agent to one reaction tank to apply pressure by the gas to the reaction liquid, and supplying the reaction liquid to the other reaction tank. By repeating the transfer process alternately A method for amplifying a nucleic acid, characterized in that shuttling the reaction solution.

In the nucleic acid amplification method of the present invention, a nucleic acid amplification reaction in which the temperature of a reaction solution is raised and lowered is repeated in a microchip. In the present invention, the microchip has a high-temperature reaction tank and a low-temperature reaction tank that are maintained at different predetermined temperatures, and the reaction liquid is reciprocated between these reaction tanks to raise and lower the reaction liquid. repeat.
In the present invention, the microchip has a photo-responsive or heat-responsive gas generating agent, and the pressure of the gas generated from the gas generating agent is used when the reaction solution is reciprocated. That is, the reaction liquid is moved from one reaction tank to the other reaction tank by applying the pressure of the gas to the reaction liquid in the reaction tank.
In the present invention, since the gas generating agent is provided in the microchip, the nucleic acid amplification reaction using the microchip can be performed with a simple configuration. For example, it is not necessary to prepare a separate gas generator or the like, and the entire system can be reduced in size, made disposable, and improved in maintainability.

  Preferably, the connection path is provided with a narrow tube that connects the connection path and the outside of the system, and prevents the reaction liquid from flowing into the narrow tube while reciprocating the reaction liquid, To escape.

In this preferable aspect, the thin tube is provided in the connection path which connects between reaction tanks. And the structure which escapes gas from the said thin tube at the time of the reciprocation of a reaction liquid is employ | adopted. With this configuration, the reaction solution can be moved smoothly.
A configuration in which the gas is allowed to escape from the narrow tube while preventing the reaction solution from flowing into the narrow tube can be realized by, for example, a liquid stop valve mechanism using a Laplace pressure. The mechanism is described in, for example, Japanese Patent Application Laid-Open Nos. 2008-64748, 2009-128049, and 2009-168487.

Another aspect of the present invention is a nucleic acid amplification method in which a nucleic acid amplification reaction in which a reaction solution is repeatedly heated and cooled is repeated in a microchip, the microchip comprising a high-temperature reaction tank maintained at a predetermined temperature, A low-temperature reaction tank maintained at a predetermined temperature lower than a predetermined temperature of the high-temperature reaction tank; a high-temperature reaction tank having a connection path connecting the high-temperature reaction tank and the low-temperature reaction tank; The reaction liquid is reciprocated between the low-temperature reaction tank and the reaction liquid is repeatedly heated and lowered, and the microchip further includes a photoresponsive or heat-responsive gas generating agent, and the gas The reaction solution is reciprocated by alternately repeating the steps of supplying the gas generated from the generating agent to one reaction vessel, applying pressure to the reaction solution by the gas, and transferring the reaction solution to the other reaction vessel. The connecting path Is provided with a thin tube for connecting the connection path and the outside of the system, and when the reaction solution is reciprocated, gas is allowed to escape from the thin tube while preventing the reaction solution from flowing into the thin tube. This is a nucleic acid amplification method.

  Preferably, the predetermined temperature at which the high temperature region or the high temperature reaction vessel is maintained is 93 ° C to 97 ° C.

  This aspect assumes a mode in which a nucleic acid denaturation step is performed in a high temperature region or a high temperature reaction tank.

  Preferably, the predetermined temperature at which the low temperature region or the low temperature reaction vessel is maintained is 55 ° C to 75 ° C.

  This aspect assumes a mode in which an annealing process is performed in a low temperature region or a low temperature reaction tank. In the region or reaction vessel, an extension step can be performed in addition to the annealing step.

  Preferably, the gas generating agent is an azo or azide-based gas generating agent.

  Preferably, the gas generating agent is contained in an adhesive binder resin.

  Preferably, the gas generating agent has a film shape or a tape shape.

Another aspect of the present invention is a nucleic acid amplification microchip for use in a nucleic acid amplification reaction that repeatedly raises and lowers the temperature of a reaction solution, a high-temperature reaction tank maintained at a predetermined temperature, and a predetermined temperature of the high-temperature reaction tank A low-temperature reaction tank maintained at a predetermined temperature lower than that, a connecting path connecting the high-temperature reaction tank and the low-temperature reaction tank, and a photoresponsive or heat-responsive gas generant, which is generated from the gas generant. Gas can be supplied to the high temperature reaction tank and the low temperature reaction tank, the gas is supplied to one reaction tank to apply pressure to the reaction liquid in the reaction tank, and the reaction liquid is supplied to the other reaction tank. the step of transferring is repeated alternately, the reaction solution Ri can der able to shuttle, to the connecting passage, capillary connecting the said coupling passage and systems external is provided, the reciprocating of the reaction solution Sometimes prevent the reaction solution from flowing into the capillary tube. One is a nucleic acid amplification microchip, characterized in that it is possible to escape the gas from the capillary.

The present invention relates to a nucleic acid amplification microchip used for a nucleic acid amplification reaction in which a reaction solution is repeatedly heated and lowered. The microchip of the present invention has a high-temperature reaction tank and a low-temperature reaction tank that are maintained at different predetermined temperatures, and the reaction liquid is heated and lowered by reciprocating the reaction liquid between these reaction tanks. Can be repeated.
The microchip of the present invention has a photoresponsive or heat responsive gas generating agent. And it is set as the structure which can supply the gas which generate | occur | produced from the gas generating agent to a high temperature reaction tank and a low temperature reaction tank, The pressure by gas can be provided to the reaction liquid in a reaction tank, and a reaction liquid can be moved.
Since the microchip of the present invention has a gas generating agent, the nucleic acid amplification reaction using the microchip can be performed with a simple configuration. For example, it is not necessary to prepare a separate gas generator or the like, and the entire system can be reduced in size, made disposable, and maintainability can be improved.

In the present invention, the connecting path is provided with a narrow tube that connects the connecting path and the outside of the system, and prevents the reaction liquid from flowing into the thin tube while the reaction liquid reciprocates. It is possible to escape the gas from.

  With this configuration, the reaction solution can be moved smoothly. A configuration in which gas is allowed to escape from the narrow tube while preventing the reaction solution from flowing into the narrow tube can be realized, for example, by a liquid stop valve mechanism using the Laplace pressure described above.

Another aspect of the present invention is a nucleic acid amplification microchip for use in a nucleic acid amplification reaction that repeatedly raises and lowers the temperature of a reaction solution, a high-temperature reaction tank maintained at a predetermined temperature, and a predetermined temperature of the high-temperature reaction tank A low-temperature reaction tank maintained at a predetermined temperature lower than that, a connecting path connecting the high-temperature reaction tank and the low-temperature reaction tank, and a photoresponsive or heat-responsive gas generant, which is generated from the gas generant. Gas can be supplied to the high temperature reaction tank and the low temperature reaction tank, the gas is supplied to one reaction tank to apply pressure to the reaction liquid in the reaction tank, and the reaction liquid is supplied to the other reaction tank. It is possible to reciprocate the reaction solution by alternately repeating the steps of transferring the gas, and the gas generating agent is provided independently for each reaction tank. It is.

  Preferably, the gas generating agent is provided independently for each reaction tank.

  With this configuration, it is possible to efficiently supply gas to each reaction tank.

  Preferably, the gas generating agent is an azo or azide-based gas generating agent.

  Preferably, the gas generating agent is contained in an adhesive binder resin.

  Preferably, the gas generating agent has a film shape or a tape shape.

  According to the nucleic acid amplification method of the present invention, the nucleic acid amplification reaction in which the temperature of the reaction solution is repeatedly raised and lowered can be performed with a simpler configuration. In particular, according to the configuration using a microchip, it is possible to realize downsizing, disposable, and maintenance performance of the entire system necessary for the nucleic acid amplification reaction.

  The same applies to the nucleic acid amplification microchip of the present invention, and the nucleic acid amplification reaction in which the temperature of the reaction solution is repeatedly raised and lowered can be performed with a simpler configuration. Furthermore, the entire system can be reduced in size, made disposable, and maintainability can be improved.

(A) is a top view which represents typically the reaction tank of the microchip which concerns on 1st embodiment of this invention, (b) is AA sectional drawing of (a). It is sectional drawing showing an example of a structure of a reaction tank and a gas generation film. It is sectional drawing showing the other example of a structure of a reaction tank and a gas generation film. It is explanatory drawing explaining the method of reciprocating a reaction liquid between the reaction tanks of FIG. 1, (a) is the state in which the reaction liquid is accommodated in the 1st reaction tank, (b) is the reaction liquid in the 1st reaction tank. (C) shows a state in which the reaction liquid is accommodated in the second reaction tank, and (d) shows a state in which the reaction liquid has moved from the second reaction tank to the first reaction tank. (E) shows the state which the reaction liquid returned to the 1st reaction tank. (A) is a top view which represents typically the reaction tank of the microchip which concerns on 2nd embodiment of this invention, (b) is AA sectional drawing of (a). In 2nd embodiment of this invention, it is explanatory drawing explaining the method of reciprocating a reaction liquid between reaction tanks, (a) is the state in which the reaction liquid is accommodated in the 1st reaction tank, (b) is reaction. The state in which the liquid is moving from the first reaction tank to the second reaction tank, (c) is the state in which the reaction liquid is accommodated in the second reaction tank, and (d) is the state in which the reaction liquid is first from the second reaction tank. The state which has moved to the reaction tank, (e) represents the state where the reaction liquid has returned to the first reaction tank. It is explanatory drawing which expanded the mode of the thin tube vicinity of FIG.6 (b) and FIG.6 (d). It is a top view which represents typically the reaction tank of the microchip which concerns on 3rd embodiment of this invention. It is a top view which represents typically the reaction tank of the microchip which concerns on 4th embodiment of this invention. It is a top view which represents typically the reaction tank of the microchip of the modification of 2nd embodiment of this invention.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. In order to facilitate understanding of the invention, in each drawing, the size and thickness of each member are partially exaggerated and may not necessarily match the actual size and ratio. Of course, the present invention is not limited to these embodiments.

The microchip (microchip for nucleic acid amplification) 1 according to the first embodiment of the present invention shown in FIG. 1 has a basic structure in which the overall shape is a plate shape, and a protective member 3 is laminated on a plate-like substrate 2. Have. In the microchip 1, a first reaction tank (high temperature region, high temperature reaction tank) 5, a second reaction tank (low temperature region, low temperature reaction tank) 6, and a connection path 7 are formed.
In plan view, the first reaction tank 5 and the second reaction tank 6 are adjacent to each other, and both are connected by a connecting path 7.

The first reaction tank 5 and the second reaction tank 6 are both sealable rooms into which the reaction liquid 10 is introduced and accommodated. The first reaction tank 5 and the second reaction tank 6 are configured by a space surrounded by a recess formed in the substrate 2 and the protective member 3. The size and shape of the first reaction tank 5 and the second reaction tank 6 are substantially the same. The volume of the first reaction tank 5 and the second reaction tank 6 is about several to several hundred mm ³ (μL).
The first reaction tank 5 and the second reaction tank 6 are provided with a mechanism (not shown) for letting gas escape. This mechanism works only when the reaction liquid 10 described later moves.
Further, both the first reaction tank 5 and the second reaction tank 6 can be heated by an external heater. That is, by bringing an external heater into contact with the first reaction tank 5 or the second reaction tank 6, the first reaction tank 5 and the second reaction tank 6 can be maintained at a predetermined temperature.

  The connection path 7 is a flow path that connects the first reaction tank 5 and the second reaction tank 6. The connection path 7 is configured by a cylindrical space surrounded by a linear groove formed in the base material 2 and the protection member 3. The first reaction tank 5 and the second reaction tank 6 communicate with each other via a connection path 7. The reaction solution 10 for nucleic acid amplification introduced into the microchip 1 can reciprocate between the first reaction tank 5 and the second reaction tank 6 via the connection path 7.

  The connection path 7 is constituted by a so-called micro flow path. The micro flow path is a fine flow path formed in a shape and dimension in which a so-called micro effect appears in the liquid flowing in the flow path. Specifically, a fine channel that is formed in a shape and dimension in which the liquid flowing through the channel is strongly affected by surface tension and capillary action and exhibits a behavior different from that of a liquid flowing through a normal size channel. It is. The inner diameter of the microchannel is generally about 0.02 μm to 2 mm.

  When the affinity between the wall surface of the connection path 7 and the reaction solution is optimized, the reaction solution moves more smoothly. If the affinity between the wall of the connection path 7 and the reaction solution is low, the movement of the reaction solution is likely to be inhibited. If the affinity between the wall surface of the connection path 7 and the reaction solution is high, an unintended reaction due to capillary action. Liquid movement occurs, which is not preferable. For example, when the reaction solution is an aqueous solution, the contact angle of the wall surface of the connection path 7 is preferably 40 to 60 ° at an air temperature of 23 ° C.

  The microchip 1 further has a gas generating film (gas generating agent) 8. And it is set as the structure which can supply the gas generated from the gas generation film 8 to the 1st reaction tank 5 and the 2nd reaction tank 6, and the micropump is formed. Here, the gas generating film 8 is composed of a photoresponsive or heat responsive gas generating agent. Therefore, gas can be generated by applying an external stimulus composed of light and heat to the gas generating film 8.

  Examples of the configuration capable of supplying the gas generated from the gas generating film 8 to the first reaction tank 5 and the second reaction tank 6 include those shown in FIGS. 2 and 3. 2 and 3 show only the first reaction tank 5, the same configuration can be adopted for the second reaction tank 6.

In the configuration shown in FIG. 2, the gas generating film 8 is provided on the lower side (back surface) of the substrate 2. Further, a gas supply port 12 and a gas supply channel 13 extending from the gas supply port 12 to the first reaction tank 5 are provided on the back surface of the substrate 2. And the gas generation film 8 is affixed on the back surface of the base material 2 so that the gas supply port 12 whole may be plugged up. That is, one surface of the gas generating film 8 is exposed toward the inside of the gas supply channel 13.
When supplying gas to the 1st reaction tank 5, in the state shown in FIG. 2, light is irradiated from the light source 16 toward the gas generating film 8 from the back surface side of the base material 2, for example. Or give heat. As a result, gas is generated from the gas generating film 8 and supplied to the gas supply port 12. The supplied gas is introduced into the first reaction tank 5 through the gas supply channel 13.
A backflow prevention means 15 such as a check valve is provided in the middle of the gas supply flow path 13. The backflow prevention means 15 prevents the gas and the reaction liquid 10 from flowing back from the first reaction tank 5 toward the gas supply port 12.

  On the other hand, in the configuration shown in FIG. 3, the gas generating film 8 is provided on the upper side of the first reaction tank 5. Specifically, the gas generating film 8 is provided between the base material 2 and the protective member 3 and at a position corresponding to the first reaction tank 5, and constitutes the top surface of the first reaction tank 5. . That is, the lower surface of the gas generating film 8 is exposed toward the inside of the first reaction tank 5. Therefore, by applying an external stimulus composed of light and heat to the gas generating film 8, the gas can be generated and supplied to the inside of the first reaction tank 5. Note that, when the protective member 3 is made of a light transmissive material, the light can be irradiated from the upper side of the microchip 1 using the light source 16 so that the light can reach the gas generating film 8.

  A procedure for performing a nucleic acid amplification reaction using the microchip 1 of the present embodiment will be described with reference to FIGS. In the present embodiment, a nucleic acid amplification reaction is performed by a PCR method. A denaturation process is performed in a first reaction tank (high temperature reaction tank) 5, and an annealing process and an extension process are performed in a second reaction tank (low temperature reaction tank) 6. (2-step method). Moreover, in this embodiment, the gas supply mechanism using the photoresponsive or heat-responsive gas generating film 8 as illustrated in FIG. 2 and FIG. 3 in each of the first reaction tank 5 and the second reaction tank 6. (Micropump) is provided. 4A to 4E, the reaction solution 10 is hatched.

  First, as a preliminary preparation, a reaction solution 10 containing components (template DNA, primers, heat-resistant DNA polymerase, substrate, etc.) necessary for nucleic acid amplification reaction is prepared. On the other hand, using an external heater, the first reaction tank 5 of the microchip 1 is 93 ° C. to 97 ° C., preferably 94 ° C. to 96 ° C., and the second reaction tank 6 is 55 ° C. to 75 ° C., preferably 66 ° C. to Each predetermined temperature of 70 ° C. is maintained.

  First, the reaction solution 10 is introduced into the first reaction tank 5, and the temperature of the reaction solution 10 is raised (FIG. 4A). Then, it is left as it is for a predetermined time (for example, about 30 seconds to 1 minute) (denaturing step). Thereby, the reaction solution 10 is exposed to a temperature of 94 ° C. to 96 ° C., for example, and the template DNA is denatured (dissociated).

  Next, the gas generating film 8 corresponding to the first reaction tank 5 is irradiated with light to generate gas. Thereby, the generated gas is supplied to the first reaction tank 5, and pressure by the gas is applied to the reaction solution 10. Then, the reaction liquid 10 starts moving toward the second reaction tank 6 through the connection path 7 (FIG. 4B). And the reaction liquid 10 is transferred to the 2nd reaction tank 6 (FIG.4 (c)).

  When the transfer to the second reaction tank 6 is completed (FIG. 4C), the reaction solution 10 is cooled to 55 ° C. to 75 ° C., which is the maintenance temperature of the second reaction tank 6. It is left as it is for a predetermined time (for example, about 1 to 2 minutes) (annealing step and extension step). Thereby, annealing and subsequent DNA synthesis reaction are performed.

Next, the gas generating film 8 corresponding to the second reaction tank 6 is irradiated with light to generate gas. Thereby, the generated gas is supplied to the second reaction tank 6, and pressure by the gas is applied to the reaction solution 10. Then, the reaction liquid 10 starts to move toward the first reaction tank 5 through the connection path 7 (FIG. 4D). And the reaction liquid 10 is transferred to the 1st reaction tank 5 (FIG.4 (e)). That is, the process returns to the first reaction tank 5. This cycle of temperature increase and decrease is one cycle.
By repeating this cycle about 30 times, the nucleic acid is amplified in the reaction solution 10.

Next, a second embodiment of the present invention will be described. Similar to the first embodiment, the microchip 21 according to the second embodiment has a basic structure in which the overall shape is a plate shape, and the protective member 3 is laminated on the plate-like base material 2. And the 1st reaction tank (high temperature area | region, high temperature reaction tank) 5, the 2nd reaction tank (low temperature area | region, low temperature reaction tank) 6, and the connection path 7 are formed.
In the microchip 21 according to the second embodiment, a thin tube 22 is provided in the middle of the connection path 7 as shown in FIG. The narrow tube 22 branches in a direction perpendicular to the connection path 7 and connects the connection path 7 and the outside of the system.

The narrow tube 22 is narrower than the connecting path 7. The flow path area of the narrow tube 22 is formed so that the liquid flowing through the connecting channel 7 does not flow into the narrow tube 22 due to the Laplace pressure. Therefore, the thin tube 22 is configured to act as a liquid stop valve mechanism using Laplace pressure. That is, the reaction solution 10 passing through the connection path 7 does not flow into the narrow tube 22, but the gas can escape to the outside of the system through the narrow tube 22.
Specifically, the flow channel area of the narrow tube 22 is preferably ½ or less of the flow channel area of the connection path 7. The flow passage area of the narrow tube 22 is preferably 1/10 or more of the flow passage area of the connection path 7. More specifically, when the connecting path 7 has a rectangular cross section, the height and width may be on the order of 100 μm. On the other hand, when the thin tube 22 has a rectangular cross section, the height and width may be about 1 to 50 μm, and particularly preferably about 5 to 25 μm. The height of the connection path 7 may be about 1 to 10 times the height of the narrow tube 22 and is preferably about 1 to 5 times.
Moreover, when compared with the ratio of the side circumference of the cross section relative to the cross-sectional area of the flow path, the ratio of the narrow tube 22 is preferably 2 to 10 times the ratio of the connection path 7.
The configuration of the microchip 1 and the microchip 21 is the same except that the thin tube 22 is provided.

The procedure for performing the nucleic acid amplification reaction using the microchip 21 of the second embodiment is basically the same as that of using the microchip 1 of the first embodiment.
First, as a preliminary preparation, the reaction liquid 10 is prepared and the first reaction tank 5 and the second reaction tank 6 are heated.

  The reaction liquid 10 is introduce | transduced into the 1st reaction tank 5, and the temperature of the reaction liquid 10 is raised (FIG. 6 (a)). Then, it is left as it is for a predetermined time (for example, about 30 seconds to 1 minute) (denaturing step). Thereby, the reaction solution 10 is exposed to a temperature of 94 ° C. to 96 ° C., for example, and the template DNA is denatured (dissociated).

Next, the gas generating film 8 corresponding to the first reaction tank 5 is irradiated with light to generate gas. Thereby, the reaction liquid 10 starts moving toward the second reaction tank 6 through the connection path 7 (FIG. 6B). At this time, the reaction liquid 10 flowing through the connection path 7 does not flow into the narrow tube 22, and only gas escapes from the narrow tube 22. Therefore, the reaction liquid 10 can move smoothly. Furthermore, a part of the reaction solution 10 is not lost during the movement process. The state in the vicinity of the narrow tube 22 in this process is as shown in FIG. 7, and the gas-liquid interface of the reaction solution 10 does not substantially enter the narrow tube 22, and all the reaction solution 10 moves to the second reaction tank 6 side. To do.
And the reaction liquid 10 is transferred to the 2nd reaction tank 6 (FIG.6 (c)).

  When the transfer to the second reaction tank 6 is completed (FIG. 6C), the temperature of the reaction liquid 10 is lowered to 55 ° C. to 75 ° C., which is the maintenance temperature of the second reaction tank 6. It is left as it is for a predetermined time (for example, about 1 to 2 minutes) (annealing step and extension step). Thereby, annealing and subsequent DNA synthesis reaction are performed.

Next, the gas generating film 8 corresponding to the second reaction tank 6 is irradiated with light to generate gas. Thereby, the reaction liquid 10 starts moving toward the first reaction tank 5 through the connection path 7 (FIG. 6D). Also at this time, the reaction liquid 10 flowing through the connection path 7 does not flow into the narrow tube 22, but only gas escapes from the narrow tube 22 (FIG. 7).
And the reaction liquid 10 is transferred to the 1st reaction tank 5 (FIG.6 (e)). The above is one cycle.
By repeating this cycle about 30 times, the nucleic acid is amplified in the reaction solution 10.

  In the above-described embodiment, a two-step nucleic acid amplification reaction using the first reaction tank 5 and the second reaction tank 6 was performed. In the third embodiment to be described next, the third reaction tank 23 is further set to enable a three-step nucleic acid amplification reaction. The third reaction tank 23 is set to a predetermined temperature different from that of the first reaction tank 5 and the second reaction tank 6.

In this embodiment, as shown in FIG. 8, the 1st reaction tank 5, the 2nd reaction tank 6, and the 3rd reaction tank 23 are connected in series via the connection paths 7a and 7b in this order. The reaction solution can reciprocate between the first reaction tank 5 and the second reaction tank 6 and between the second reaction tank 6 and the third reaction tank 23. It can be said that the reaction solution can reciprocate between the first reaction tank 5 and the third reaction tank 23 via the second reaction tank 6.
The configuration of the third reaction tank 23 is the same as that of the first reaction tank 5 and the second reaction tank 6. For example, the gas generated from the gas generating film 8 can be supplied to the third reaction tank 23 as shown in FIGS.

  When nucleic acid amplification reaction such as PCR method is performed in the present embodiment, for example, a denaturation step (eg, 94 ° C. to 96 ° C.) in the first reaction tank 5 and an annealing step (eg, 55 ° C. to 65 ° C.) in the second reaction tank 6 ), An extension step (for example, 60 ° C. to 75 ° C.) can be performed in the third reaction tank 23.

The transfer of the reaction liquid 10 can also be performed as shown in FIGS. That is, after subjecting the reaction solution to the denaturation step in the first reaction tank 5, the gas generated from the gas generating film is supplied to the first reaction tank 5, and the reaction solution is transferred to the second reaction tank 6. After the reaction solution is subjected to the annealing step in the second reaction tank 6, the gas generated from the gas generating film is supplied to the second reaction tank 6, and the reaction solution is transferred to the third reaction tank 23. After subjecting the reaction liquid to the extension step in the third reaction tank 23, the gas generated from the gas generating film is supplied to the third reaction tank 23, and the reaction liquid is supplied to the first reaction tank via the second reaction tank 6. Transfer to 5. This is one cycle.
In addition, although it will pass through the 2nd reaction tank 6 at the time of transfer from the 3rd reaction tank 23 to the 1st reaction tank 5, if a reaction liquid is transferred rapidly, a problem will not arise.
In addition, when the gas is supplied to the second reaction tank 6, the moving direction of the reaction liquid can be limited to only a desired direction by providing a channel opening / closing mechanism in the connection paths 7a and 7b.

  It is good also as a structure which supplies gas only to the 1st reaction tank 5 and the 3rd reaction tank 23, ie, only the reaction tank of both ends. In this case, the reaction liquid can be transferred to the second reaction tank 6 and the third reaction tank 23 by supplying gas to the first reaction tank 5. Similarly, the reaction liquid can be transferred to the second reaction tank 6 and the first reaction tank 5 by supplying gas to the third reaction tank 23.

  In the present embodiment, the third reaction tank 23 functions as a low temperature reaction tank (low temperature region) in relation to the first reaction tank 5, and functions as a high temperature reaction tank (high temperature region) in relation to the second reaction tank 6. .

  Also in this embodiment, the thin tubes 22 having the above-described configuration can be provided in the connection paths 7a and 7b.

As a modification of the third embodiment, a configuration as shown in FIG. 9 is also possible. In 4th embodiment shown in FIG. 9, the 1st reaction tank 5, the 2nd reaction tank 6, and the 3rd reaction tank 23 are connected cyclically | annularly via connection path 7a, 7b, 7c. And said temperature cycle is implement | achieved by moving (circulating) a reaction liquid to one direction. For example, the reaction liquid is allowed to flow in one direction from the first reaction tank 5 to the second reaction tank 6, from the second reaction tank 6 to the third reaction tank 23, and from the third reaction tank 23 to the first reaction tank 5.
In the present embodiment, the reaction liquid is transferred from the first reaction tank 5 to the second reaction tank 6 and returns to the first reaction tank via the third reaction tank 23, so that the reaction liquid is in the first reaction tank. It can be said that it is reciprocating between 5 and the second reaction tank 6. Similarly, it can be said that the reaction liquid reciprocates between the second reaction tank 6 and the third reaction tank 23, and further reciprocates between the first reaction tank 5 and the third reaction tank 23. That is, in the present embodiment, the flow path for the reaction liquid and the return flow path are separate.

  Also in this embodiment, the thin tubes 22 having the above-described configuration can be provided in the connection paths 7a, 7b, and 7c.

  In addition, the movement of the reaction liquid 10 can be made smoother by making the shape of each reaction tank 5,6,23 mentioned above and the connection path 7,7a-7c into a thing without a corner | angular. For example, in the case of the second embodiment described above, if the shape is substantially U-shaped as shown in FIG. I can move.

Hereinafter, materials and the like constituting each member will be described.
The base material 2 can be comprised by resin, ceramics, glass, etc., for example. As resin which comprises the base material 2, an organic siloxane compound, polymethacrylate resin, cyclic polyolefin resin etc. are mentioned, for example. Specific examples of the organic siloxane compound include polydimethylsiloxane (PDMS) and polymethylhydrogensiloxane.

  The protective member 3 is made of a light transmissive material such as polyethylene terephthalate (PET).

Examples of the material constituting the gas generating film (gas generating agent) 8 include azo compounds, azide compounds, polyoxyalkylene resins, and the like.
Specific examples of the azo compound include, for example, 2,2′-azobis (N-cyclohexyl-2-methylpropionamide), 2,2′-azobis [N- (2-methylpropyl) -2-methylpropionamide]. Etc.
Examples of the azide compound include compounds having a sulfonyl azide group or an azidomethyl group. Specific examples of the compound having an azidomethyl group include a glycidyl azide polymer.
Further, a photoacid generator (sulfonic acid onium salt, benzylsulfonic acid ester, halogenated isocyanurate, bisarylsulfonyldiazomethane, etc.) and an acid stimulating gas generator (described in International Publication No. 2009/113666) Combinations of carbonates and / or bicarbonates), photobase generators (cobaltamine complexes, o-nitrobenzyl carbamate, oxime esters, carbamoyloxyimino group-containing compounds, etc.) and base proliferators (Flu3, Flu4, etc.) ) And the like can be used as a material constituting the gas generating agent.

  The gas generating agent can be used by mixing with an adhesive binder resin. Specific examples of binder resins preferably used include acrylic resins and glycidyl azide polymers. The gas generating agent may contain a photosensitizer. Specific examples of photosensitizers that are preferably used include benzophenone, diethylthioxanthone, anthraquinone, benzoin, and acridine derivatives.

  As an example of the light source for irradiating the gas generating film 8 with light, a light source composed of a light emitting diode (LED) can be cited. The output (wattage) of the light source can be appropriately selected according to the required gas generation amount and heating time.

  In the above-described embodiment, the PCR method has been described as an example of the nucleic acid amplification reaction. However, the present invention is not limited to this, and any nucleic acid amplification reaction in which the temperature of the reaction solution is repeatedly raised and lowered can be applied in addition to the PCR method.

1 Microchip (microchip for nucleic acid amplification)
5 First reaction tank (high temperature region, high temperature reaction tank)
6 Second reaction tank (low temperature region, low temperature reaction tank)
7, 7a-7c Connection 8 Gas generating film (gas generating agent)
10 reaction solution 21 microchip (microchip for nucleic acid amplification)
22 Narrow tube 23 3rd reaction tank (high temperature area, high temperature reaction tank, low temperature area, low temperature reaction tank)

Claims (14)

A nucleic acid amplification method in which a nucleic acid amplification reaction in which a temperature rise and a temperature drop of a reaction solution are repeated in a microchip,
The microchip includes a high temperature reaction tank maintained at a predetermined temperature, a low temperature reaction tank maintained at a predetermined temperature lower than a predetermined temperature of the high temperature reaction tank, and a connection connecting the high temperature reaction tank and the low temperature reaction tank. Have a road,
By reciprocating the reaction liquid between the high-temperature reaction tank and the low-temperature reaction tank maintained at each predetermined temperature and returning it to the original reaction tank , the reaction liquid is repeatedly heated and lowered,
The microchip further includes a photoresponsive or heat responsive gas generant,
The reaction solution is produced by alternately repeating the steps of supplying a gas generated from the gas generating agent to one reaction vessel, applying a pressure by the gas to the reaction solution, and transferring the reaction solution to the other reaction vessel. A method for amplifying a nucleic acid, wherein The connection path is provided with a narrow tube that connects the connection path and the outside of the system, and allows gas to escape from the narrow tube while preventing the reaction liquid from flowing into the narrow tube during the reciprocation of the reaction liquid. The method for amplifying a nucleic acid according to claim 1 . A nucleic acid amplification method in which a nucleic acid amplification reaction in which a temperature rise and a temperature drop of a reaction solution are repeated in a microchip,
The microchip includes a high temperature reaction tank maintained at a predetermined temperature, a low temperature reaction tank maintained at a predetermined temperature lower than a predetermined temperature of the high temperature reaction tank, and a connection connecting the high temperature reaction tank and the low temperature reaction tank. Have a road,
The reaction liquid is reciprocated between a high temperature reaction tank and a low temperature reaction tank maintained at each predetermined temperature, and the temperature increase and decrease of the reaction liquid are repeated.
The microchip further includes a photoresponsive or heat responsive gas generant,
The reaction solution is produced by alternately repeating the steps of supplying a gas generated from the gas generating agent to one reaction vessel, applying a pressure by the gas to the reaction solution, and transferring the reaction solution to the other reaction vessel. Are reciprocating,
The connection path is provided with a narrow tube that connects the connection path and the outside of the system, and allows gas to escape from the narrow tube while preventing the reaction liquid from flowing into the narrow tube during the reciprocation of the reaction liquid. A method for amplifying a nucleic acid characterized by the above.   The nucleic acid amplification method according to any one of claims 1 to 3, wherein a predetermined temperature at which the high temperature region or the high temperature reaction tank is maintained is 93 ° C to 97 ° C.   The nucleic acid amplification method according to any one of claims 1 to 4, wherein the predetermined temperature at which the low temperature region or the low temperature reaction tank is maintained is 55 ° C to 75 ° C.   The nucleic acid amplification method according to claim 1, wherein the gas generating agent is an azo or azide-based gas generating agent.   The nucleic acid amplification method according to claim 1, wherein the gas generating agent is contained in an adhesive binder resin.   The nucleic acid amplification method according to claim 1, wherein the gas generating agent is in the form of a film or a tape. A nucleic acid amplification microchip used in a nucleic acid amplification reaction that repeatedly raises and lowers the temperature of a reaction solution,
A high-temperature reaction tank maintained at a predetermined temperature; a low-temperature reaction tank maintained at a predetermined temperature lower than a predetermined temperature of the high-temperature reaction tank; a connection path connecting the high-temperature reaction tank and the low-temperature reaction tank; Or having a heat-responsive gas generant,
The gas generated from the gas generating agent can be supplied to the high temperature reaction tank and the low temperature reaction tank,
Supplying the gas to one reaction tank, applying pressure to the reaction liquid in the reaction tank, and alternately transferring the reaction liquid to the other reaction tank, and reciprocating the reaction liquid possible der is,
The connection path is provided with a narrow tube that connects the connection path and the outside of the system, and allows gas to escape from the narrow tube while preventing the reaction liquid from flowing into the narrow tube during the reciprocation of the reaction liquid. nucleic acid amplification microchip, characterized in that it is possible. The nucleic acid amplification microchip according to claim 9 , wherein the gas generating agent is provided independently for each reaction tank. A nucleic acid amplification microchip used in a nucleic acid amplification reaction that repeatedly raises and lowers the temperature of a reaction solution,
A high-temperature reaction tank maintained at a predetermined temperature; a low-temperature reaction tank maintained at a predetermined temperature lower than a predetermined temperature of the high-temperature reaction tank; a connection path connecting the high-temperature reaction tank and the low-temperature reaction tank; Or having a heat-responsive gas generant,
The gas generated from the gas generating agent can be supplied to the high temperature reaction tank and the low temperature reaction tank,
Supplying the gas to one reaction tank, applying pressure to the reaction liquid in the reaction tank, and alternately transferring the reaction liquid to the other reaction tank, and reciprocating the reaction liquid Is possible,
The gas generating agent is provided independently for each reaction tank, and the nucleic acid amplification microchip.   The nucleic acid amplification microchip according to any one of claims 9 to 11, wherein the gas generating agent is an azo or azide-based gas generating agent.   The nucleic acid amplification microchip according to claim 9, wherein the gas generating agent is contained in an adhesive binder resin.   The nucleic acid amplification microchip according to claim 9, wherein the gas generating agent is in the form of a film or a tape.

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