JPH0994086A - Dna amplifier - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a DNA amplifier capable of efficiently conducting DNA amplification by precisely controlling reaction solution temperature. SOLUTION: This DNA amplifier has a planar reaction cell formed on an inorganic or organic substrate, two flow channels communicating with the reaction cell, two-way valves set up on the respective flow channels so as to sandwich the reaction cell, and heating means provided on the bottom of the reaction cell and/or the reverse face of the substrate whose surface is provided with the reaction cell.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a device for amplifying a small amount of DNA by performing a DNA amplification reaction.

[0002]

2. Description of the Related Art In recent years, a polymerase chain reaction (hereinafter abbreviated as PCR) has been used as a means for amplifying a small amount of DNA.
Is actively used. This method is disclosed in
As disclosed in Japanese Patent Publication No. 292579, first, in a sample containing a target nucleic acid, two kinds of primers containing base sequences at both ends of each strand of the target DNA, heat resistance D
NA synthase and four types of deoxyribonucleoside triphosphates (dATP, dCTP, dGTP and dTT)
P) is added. Next, a step of dissociating the DNA (denaturation), a step of binding the single-stranded DNA generated by the dissociation to the primer (annealing), and a single-stranded DN bound with the primer only by controlling the temperature of the system
Each step of synthesizing (extending) a complementary chain with DNA synthase using A as a template is performed, and this step is repeated once or more.

As described above, PCR can efficiently amplify a small amount of DNA. In addition to this PCR,
Further, attempts have been made to automate the process including pretreatment and posttreatment. For example, in the device disclosed in JP-A-6-327476, first, whole blood is centrifuged and extracted with a phenol and chloroform solution to purify the nucleic acid component in the whole blood (pretreatment step). Next, the purified nucleic acid component is dissolved in a buffer, and the above-mentioned two types of primers, thermostable DNA synthase and four types of deoxyribomucleoside triphosphates are added, and the target nucleic acid is amplified by carrying out a thermal cycle. (PCR step). Finally, the reaction solution containing the amplified nucleic acid is treated by gel electrophoresis to remove impurities such as primers and by-products to obtain the target nucleic acid (post-treatment step).

Further, some attempts have been made on the reaction container itself for carrying out PCR. For example, the apparatus disclosed in Japanese Patent Laid-Open No. 7-75544 discloses a thermocycle required for a DNA synthesis reaction by providing a capillary tube passing through two thermostatic chambers and simply flowing a mixed solution containing a sample into the capillary tube. Has been realized. In this method, since the amplification reaction is carried out in a capillary, there is an advantage that the temperature gradient between the outside and the inside of the liquid flow is reduced, and the temperature in the system can be homogenized more quickly.

[0005]

In the above-mentioned conventional DNA amplification apparatus, the reaction vessel is surrounded by a thermostat or a metal block, and the reaction vessel itself is heated and cooled to carry out the thermal cycle necessary for the DNA amplification reaction. ing.
Therefore, in these devices, the temperature of the reaction solution is not directly controlled, but indirectly controlled through the reaction vessel. In addition, these devices cannot directly measure the temperature of the reaction solution because the reaction solution is in a very small amount. For this reason, the conventional DNA amplification device cannot precisely control the temperature of the reaction solution, and lacks reliability. In the device disclosed in Japanese Patent Laid-Open No. 7-75544, the reaction is carried out in a capillary tube to prevent a temperature gradient from being formed in the reaction solution. However, even with this device, the temperature of the reaction solution cannot be directly measured, and the temperature control is also inaccurate.

Further, the apparatus disclosed in Japanese Patent Laid-Open No. 6-327476 mentioned above realizes full automation of automatically performing all steps including pretreatment and posttreatment, but the movement of the reaction vessel All dispensing operations and the like are done mechanically. Therefore, the device becomes large and the cost becomes very high. Similarly, the device disclosed in Japanese Patent Laid-Open No. 7-75544 also uses a mechanical pump to circulate the reaction solution, which has the drawbacks of increasing the size of the device and increasing the cost.

Therefore, it is an object of the present invention to provide a DNA amplification device capable of precisely controlling the temperature of a reaction solution and efficiently carrying out a DNA amplification reaction.

Another object of the present invention is to provide a DNA amplification device which can reduce mechanical operating parts, thereby making the entire device compact and reducing the cost.

[0009]

According to the present invention, a planar reaction cell formed on an inorganic or organic substrate, two flow channels communicating with the reaction cell, and the reaction in each of the flow channels First DNA amplification comprising two valves provided with a cell sandwiched between them and a heating means provided at least on either the bottom surface of the reaction cell or the back surface of the surface of the substrate on which the reaction cell is formed A device is provided.

According to another aspect of the present invention, a channel provided on an inorganic or organic substrate, two valves provided in the channel, and a bottom surface of the channel between the two valves are provided. The two electrodes provided, at least one temperature detecting means provided on the bottom surface of the flow path between the two electrodes, the flow path bottom surface near each of the at least one temperature detecting means, or the flow path of the substrate. Heating means provided in pair with each of the temperature detecting means on at least one of the rear surface of the surface provided, a voltage control means for controlling the voltage applied to the two electrodes, and the temperature. A second DNA amplification device is provided which comprises heating control means for controlling the heating means based on information from the detection means.

According to another aspect of the present invention,
A channel provided on an inorganic or organic substrate, first to fourth electrodes provided on the bottom surface of the channel, a first valve provided between the first electrode and the second electrode, A sample inlet provided between the first electrode and the first valve, and communicated with an impurity introduction cell located between the sample inlet and the first valve and having a fifth electrode on the bottom surface. First branch flow path, this first
Storage cell having a sixth electrode on the bottom surface, which is located between the first valve and the second electrode, and which is provided in the flow channel between the branch flow channel and the sample inlet. Second communication with
Branch flow path, at least one temperature detection means provided on the bottom surface of the flow path between the second electrode and the third electrode, the flow path bottom surface near each of the at least one temperature detection means, or the substrate. The heating means provided in pair with each of the temperature detecting means on at least one of the back surfaces of the flow paths, and the heating means is provided between the third electrode and the fourth electrode. DNA detecting means, a second valve provided between the DNA detecting means and the fourth electrode, voltage control means for controlling the voltage applied to the first to sixth electrodes, and the temperature detecting means. A third DN comprising heating control means for controlling the heating means based on information from the means
An A amplification device is provided.

[0012]

DETAILED DESCRIPTION OF THE INVENTION A DNA amplification apparatus according to the present invention will be described below with reference to the drawings.

FIG. 1 is a schematic plan view of a specific example of the first DNA amplification apparatus according to the present invention, and FIG. 2 is a schematic sectional view taken along the line AA of the DNA amplification apparatus shown in FIG. In this device, a reaction cell 102 and channels 103 and 104 are formed on the upper surface of an inorganic or organic substrate 101, and a transparent substrate 105 is bonded to the upper surface thereof. Also, the flow path 10
A valve 105 is provided at 3 and a valve 106 is provided at the flow path 104, and a heating element 108 is provided in contact with the back surface of the substrate 101 on which the reaction cell 102 is formed. As the inorganic or organic substrate 101, for example, a silicon wafer substrate,
A glass substrate, an inorganic substrate such as aluminum or stainless steel, and an organic substrate such as fluororesin, epoxy, or polycarbonate are used. Reaction cell 102 and flow path 103
And 104 can be formed by various methods depending on the material of the substrate 101. For example, when a silicon wafer substrate is used as the substrate 101, a groove having a predetermined shape is formed on the silicon wafer by plasma etching or the like. After that, a transparent substrate such as a glass substrate may be anodically bonded. Reaction cell
102 is formed in a planar shape. This reaction cell
Various shapes including the size can be selected according to the required capacity. For example, when the volume is 3 μl, the length may be 5 mm, the width may be 3 mm, and the depth may be 200 μm, but the shallow depth is preferable for more precise control of the temperature of the reaction solution. The heating element 108 is also DNA
It is not particularly limited as long as it can carry out the heat cycle necessary for the amplification reaction, and examples thereof include an electric heater, a Peltier element, an infrared lamp and the like.

Next, the procedure of DNA amplification using this apparatus will be described. First, open valves 106 and 107,
By a delivery means (not shown), a target nucleic acid, two or more kinds of primers, a thermostable DNA synthase and four kinds of deoxyribonucleoside triphosphates (dATP, dCTP,
After introducing a buffer containing dGTP and dTTP) into the reaction cell 102, the valves 106 and 107 are closed.
Next, the heating element 108 is activated to subject the buffer in the reaction cell 102 to thermal cycling to amplify the target nucleic acid. Specifically, first, the heating element 108 is heated to heat the buffer to a temperature (90 to 94 ° C.) at which the nucleic acid in the buffer dissociates, and this temperature is maintained for a predetermined time. Next, the operation of the heating element 108 is stopped, and the buffer is cooled to a temperature (about 37 ° C.) at which the dissociated nucleic acid and the primer bind. After the dissociated nucleic acid is bound to the primer, the heating element 108 is activated again, and the buffer is heated to a temperature (50 to 75 ° C.) at which the activity of the thermostable DNA synthase is maximized and maintained for a predetermined time. As a result, the primer is extended and the nucleic acid is amplified. After the extension of the primer is completed, the heating element 108
And the buffer is heated to a temperature at which the nucleic acid dissociates to dissociate the generated double-stranded nucleic acid. Hereinafter, this cycle may be repeated a desired number of times. After the nucleic acid amplification reaction is completed, the valves 106 and 107 are opened again, and the amplified nucleic acid is recovered from the reaction cell 102 through the flow path 104. At this time, when the amplification reaction is continuously performed, a new buffer is introduced into the reaction cell 102 through the channel 103 at the same time as the recovery, and the same thermal cycle is started.

In this apparatus, as described above, the reaction cell 102
Has a planar shape. Therefore, the heating element 108
The heat generated from the heat can be efficiently transferred to the buffer in the reaction cell, and the efficiency of heating and cooling can be increased.

In the apparatus shown in FIGS. 1 and 2, the heating element 108 is provided only on the back surface of the substrate 101, but the back surface of the substrate 101 and the top surface of the substrate 101, that is, the reaction cell 102.
It can also be provided on both bottom surfaces of the. In this case, the rate of temperature increase / decrease of the buffer in the reaction cell 102 can be increased, thereby reducing the temperature gradient in the buffer and improving the accuracy of the DNA amplification reaction. Further, in the above description, the buffer is cooled by stopping the operation of the heating element 108 and naturally radiating the heat. However, as the heating element 108, it is possible to perform both heating and cooling operations like a Peltier element. By using, it is possible to shorten the cooling time and achieve more precise temperature control.

In the first DNA amplifying apparatus according to the present invention, as shown in FIGS.
The bottom surface of 02 may be provided with one or more temperature detecting means 301 and 302. Here, FIG. 3 is a plan view schematically showing another specific example of the first DNA amplification apparatus according to the present invention, and FIG. 4 is a BB of the apparatus shown in FIG.
It is a figure which shows the cross section along a line typically. In FIGS. 3 and 4, the same members as those in FIGS. 1 and 2 are denoted by the same reference numerals, and in principle, the same reference numerals represent the same members in the following drawings. Temperature detection means 3
As 01 and 302, for example, a thermocouple, a diode or the like can be used.

By thus providing the temperature detecting means 301 and 302 on the bottom surface of the reaction cell 102, it becomes possible to directly measure the temperature of the reaction solution in the reaction cell.
The temperature at the time of heat cycle required for the A amplification reaction can be controlled more precisely. This precise temperature control is particularly important in the nucleic acid dissociation step in the above heat cycle. That is, in the step of dissociating the nucleic acid as described above, the reaction solution in the reaction cell 102 is heated to a high temperature (90 to 94
(° C). At such high temperatures, even thermostable enzymes gradually lose their activity, and at temperatures above 94 ° C, the enzyme denatures and loses its activity completely. There is. In the apparatus shown in FIGS. 3 and 4, the temperature of the reaction solution can be directly measured by the temperature detecting means 301 and 302. Therefore, by feeding back the measurement result to the heating element 108, the efficiency of nucleic acid dissociation is not reduced. It is possible to prevent overheating.

Furthermore, in the first DNA amplification apparatus according to the present invention, as shown in FIGS. 5 and 6, one or more protrusions 501 can be provided on the bottom surface of the reaction cell 102. Here, FIG. 5 is a plan view schematically showing still another specific example of the first DNA amplification apparatus according to the present invention, and FIG. 6 shows a cross section taken along line CC of the apparatus shown in FIG. It is a figure which shows typically. This protrusion 501 may form its shape at the same time that the planar reaction cell 102 is formed, or the planar reaction cell 1
The projections produced in advance may be joined after forming 02, but the same material as that of the substrate 101 is preferable.

By providing one or more protrusions 501 on the bottom surface of the reaction cell 102 in this manner, the surface area of the bottom surface of the reaction cell can be increased, and the heat generation element 108 provided on the back surface of the substrate 101 generated. The heat can be efficiently transferred to the reaction solution in the reaction cell 102. Also, the heating element 108
If the above-mentioned heating and cooling operations are possible, the reaction solution can be cooled more quickly. Therefore, the apparatus shown in FIGS. 5 and 6 can further increase the temperature rising / falling rate of the reaction solution in the reaction cell 102 and can reduce the temperature gradient of the reaction solution. Therefore, by using this device, the time required for the DNA amplification reaction can be shortened and the temperature of the thermal cycle can be controlled more precisely.

The same effect can be achieved by using a reaction cell 701 having a sawtooth or corrugated bottom surface instead of the reaction cell 102 as shown in FIGS. Here, FIG. 7 is a plan view schematically showing still another specific example of the first DNA amplification apparatus according to the present invention, and FIG. 8 shows a cross section taken along line D-D of the apparatus shown in FIG. It is a figure which shows typically. Also in the apparatus shown in FIGS. 7 and 8, the surface area of the bottom surface of the reaction cell is increased,
The heat generated by the heating element 108 provided on the back surface of the substrate 101 can be efficiently transferred to the reaction solution in the reaction cell 701. Therefore, similar to the device shown in FIGS. 5 and 6, it is possible to shorten the time required for the DNA amplification reaction and to control the temperature of the heat cycle more precisely. The bottom surface of the reaction cell 701 having a sawtooth or corrugated cross-sectional shape may be formed at the same time when the reaction cell 701 is formed, as in the apparatus shown in FIGS. Although the reaction cell may be processed into a saw-tooth shape or a wavy shape after the reaction cell is formed, the same material as that of the substrate 101 is preferable.

Furthermore, the first DNA according to the present invention
In the amplification device, as shown in FIGS. 9 and 10, one or more protrusions 901 may be arranged obliquely with respect to the flow direction of the reaction solution on the bottom surface of the reaction cell 102. Here, FIG. 9 is a plan view schematically showing still another specific example of the first DNA amplification apparatus according to the present invention.
10 is a diagram schematically showing a cross section taken along line EE of the device shown in FIG. 9. This protrusion 901 may be formed at the same time when the planar reaction cell 102 is formed, as in the device shown in FIGS. 5 and 6, or after the planar reaction cell 102 is formed. The produced protrusions may be joined, but the same material as that of the substrate 101 is preferable.

As described above, the DNA amplification reaction proceeds in three steps: dissociation of nucleic acid, binding of the dissociated nucleic acid to the primer, and extension of the primer by a thermostable DNA synthase. Among them, the binding between the dissociated nucleic acid and the primer competes with the reaction in which the dissociated nucleic acids are bound again. On the other hand, the nucleic acid gradually increases in the system and the primer gradually decreases due to the repeated amplification reaction. Therefore, as the amplification is repeated, the dissociated nucleic acid is often not bound to the primer but bound to each other by the nucleic acids, and the efficiency of the amplification reaction is reduced. Therefore, in order to minimize the reduction in efficiency, when starting the amplification reaction, that is, when the reaction solution is introduced into the reaction cell 102, the nucleic acid and the primer should be as much as possible in the reaction solution. It is preferable to disperse it uniformly. In the apparatus shown in FIGS. 9 and 10, the protrusion 901 obliquely arranged with respect to the flow direction of the reaction solution serves to more evenly disperse the nucleic acid and the primer in the reaction solution. That is, the reaction solution that has flowed into the reaction cell 102 from the flow path 103 is subjected to resistance by the protrusions 901 to generate a turbulent flow, whereby the reaction solution is sufficiently stirred. Therefore, by using the apparatus shown in FIGS. 9 and 10, the binding reaction between the dissociated nucleic acid and the primer, and thus the DNA amplification reaction, can be efficiently performed, and the accuracy of the reaction can be improved. it can. Also, valve 10
Even when 7 is opened to let the reaction solution flow out from the reaction cell 102, the reaction solution is still stirred by the protrusions 109, so that the subsequent purification and analysis steps can be carried out evenly.

Of course, the protrusion 901 also contributes to increase the surface area of the bottom surface of the reaction cell 102. Therefore,
The apparatus shown in FIGS. 9 and 10 can shorten the time required for the DNA amplification reaction and can control the temperature of the thermal cycle more precisely, like the apparatus shown in FIGS. 5 to 8.

Next, a plan view of a specific example of the second DNA amplification apparatus according to the present invention is shown in FIG. 11 and D shown in FIG.
FIG. 12 schematically shows a cross-sectional view taken along the line FF of the NA amplification device. In this device, a channel 1102 provided with two valves 1104 and 1105 is formed on an inorganic or organic substrate 1101, and a transparent substrate 1103 is bonded to the upper surface thereof. valve
Between 1104 and 1105, three heating elements 1106, 1107 and 1108 are provided in contact with the back surface of the substrate 1101, and a heating element control device 1109 for individually controlling the temperature of each heating element,
Connected to 1110 and 1111. Corresponding to these heating elements 1106, 1107 and 1108, temperature detecting means 1112, 1113 and 1114 for detecting the temperature of the reaction solution are provided on the bottom surface of the channel 1102, respectively. Further, electrodes 1115 and 1116 are provided on the bottom surface of the flow channel 1102 located between the valve 1104 and the heating element 1106 and on the bottom surface of the flow channel 1102 located between the valve 1105 and the heating element 1108, both of which are It is connected to a voltage control device 1117 capable of applying a voltage to the electrodes and capable of inverting the applied voltage. The material of the inorganic or organic substrate 1101, the method of forming the channel 1102, the material of the transparent substrate 1103, the usable heating element, and the usable temperature detecting means are all used in the first DNA amplification device according to the present invention. It is similar to the one.

DNA using this second DNA amplification device
The procedure of the amplification reaction is as follows. First, the valves 1104 and 1105 are opened, and the target nucleic acid, two or more kinds of primers, thermostable DNA synthase, and four kinds of deoxyribonucleoside triphosphates (dATP, dCTP, dGTP).
And a buffer containing dTTP) are introduced into channel 1102, valves 1104 and 1105 are closed. Next, under the control of the heating element control devices 1109, 1110, and 1111, the heating elements 1106, 1107, and 1108 are operated, and the buffers in the channels 1102 at the positions corresponding to the heating elements are brought to a predetermined temperature. Heat and maintain its temperature. At this time, the temperature detecting means
By measuring the temperature of the buffer in the vicinity of each of 1112, 1113 and 1114 and feeding back the information to each heating element control means, it becomes possible to accurately maintain the temperature of the buffer at a predetermined value. The heating temperature of each heating element is different. For example, the heating temperature of the heating element 1106 is set to a temperature (90 to 94 ° C.) at which nucleic acid is dissociated, and the heating element 1107 is heated.
The heating temperature of the heat-resistant DNA synthase is increased, and the temperature of the primer bound to the dissociated nucleic acid is extended by the action of the heat-resistant DNA synthase (50 to 75 ° C). The temperature may be set to a temperature at which the primer binds to the nucleic acid (about 37 ° C). Then, the voltage controller 1117 applies a voltage to the electrodes 1115 and 1116. Nucleic acid, primer, DN contained in buffer
A synthase and deoxyribonucleoside triphosphates have different isoelectric points and show positive or negative polarity depending on the pH of the buffer, but by appropriately selecting the pH of the buffer, all components can be made positive or negative. It can be unified to either polarity. For example, adjust all components in the buffer to have a negative charge, and
When a voltage is applied so that 15 becomes negative and electrode 1116 becomes positive, only the component contained in the buffer starts moving toward electrode 1116. At this time, the buffer does not move. Each component that has started to move first passes through the upper region of the heating element 1106. As described above, this region is heated and maintained at the temperature at which the nucleic acid dissociates, so that only the nucleic acid among the moving components causes the dissociation reaction. Each component including the dissociated nucleic acid continues to move, passes through the upper region of the heating element 1107, and reaches the upper region of the heating element 1108. This region is heated and maintained at a temperature at which the dissociated nucleic acid and the primer cause a binding reaction, and the binding reaction between the dissociated nucleic acid and the primer occurs. Here, the electrodes are controlled by the voltage control device 1117.
Reverse the polarity of 1115 and 1116. As a result, the respective components including the bound body of the nucleic acid and the primer reverse their moving directions and start moving toward the electrode 1115. Each component that starts moving in the opposite direction first passes through the upper region of the heating element 1107, which is heated and maintained at a temperature at which the activity of the thermostable DNA synthase is increased, and the primer bound to the nucleic acid extends. Each component containing the nucleic acid in which the primer is extended and the double strand is formed,
The movement continues further, and the nucleic acid dissociates by reaching the upper region of the heating element 1106 again. Here again both electrodes 1104 and 11
By reversing the polarity of 05, a new thermal cycle is started, and by repeating this cycle, the nucleic acid can be amplified.

As described above, this apparatus moves only the components contained in the buffer and necessary for the DNA amplification reaction by applying a voltage to the electrodes. That is, it is not necessary to heat and cool the buffer having the largest capacity and therefore the largest heat capacity, and the buffer in the upper region of each heating element is always kept at a constant temperature by the action of the heating element control device based on the information of the temperature measuring means. It is kept. The heat capacities of the respective components moving between different temperature regions are small, and when the temperature reaches the different temperature regions, the temperature change is instantly completed. Therefore, the problem of temperature gradient associated with the execution of thermal cycling is solved, and the DNA amplification reaction can be carried out more precisely. Further, since no mechanical buffer sending means is required, the device can be downsized and the cost can be suppressed.

Further, in recent years, the temperature used for the heat cycle has been increased to 2
DN, depending on the type, ie, the temperature at which the nucleic acid dissociates and the temperature at which the thermostable enzyme is most activated and primer extension occurs
Studies have been conducted to carry out the A amplification reaction. This method has the advantage that the time required for thermal cycling can be shortened and the DNA amplification reaction can be performed in a shorter time.
The second DNA amplification device according to the present invention can also be applied to such a method. That is, FIGS. 11 and 12
In the apparatus shown in (1), the heating element 1106 is set to a temperature at which nucleic acid dissociation occurs, and the heating element 1107 is set to a temperature at which primer extension is efficiently performed, and each component in the buffer may be reciprocated between them. When the device is dedicated to this method, the heating element 1108, the heating element control device 1111 and the temperature detecting means 1114 can be omitted.

In the second DNA amplification apparatus according to the present invention, as shown in FIGS. 13 and 14, the inner surface of the flow channel 1102 located above the heating element 1107 maintained at a temperature at which the activity of the thermostable enzyme is increased. , That is, the temperature detecting means of the channel 1102
The thermostable enzyme 1301 can be immobilized on the inner surface near 1113. Here, FIG. 13 is a plan view schematically showing another specific example of the second DNA amplification apparatus according to the present invention, and FIG.
It is a figure which shows typically the cross section along the GG line of the apparatus shown in FIG. The position at which the thermostable enzyme is fixed may be any of the bottom surface, the side surface, and the top surface as long as it is the inner surface of the channel 1102.
The thermostable enzyme is immobilized on the inner surface of the flow channel 1102 by, for example, the flow channel.
If the inner surface of 1102 is treated with a silane coupling agent containing an amino group, it can be carried out by a conventional method such as a carbodiimide method, a glutaraldehyde method, or a sodium periodate method. By using the device in which the thermostable enzyme 1301 is immobilized in this manner, the amplification reaction can be performed by transferring only the deoxyribonucleoside triphosphate to the nucleic acid to be amplified, two types of primers and four types.

As described above, in the apparatus shown in FIGS. 11 and 12, the components necessary for the amplification reaction including the thermostable DNA synthase are moved between regions where the temperature is preset and maintained. Amplification reaction is underway. However, thermostable enzymes are proteins that are thermostable, but they gradually deteriorate when exposed to the temperature at which nucleic acids dissociate. Therefore, the reaction efficiency decreases as the number of thermal cycles increases. That is, the deterioration of the thermostable enzyme is one of the factors that determine the upper limit of the amplification rate in the DNA amplification reaction. In the devices shown in FIGS. 13 and 14, the thermostable enzyme always stays at the site where its activity is highest, and the activity can be maintained for a long time without being deteriorated by high temperature. For this reason, D
The NA amplification reaction can be performed more efficiently over a long period of time, and the amplification rate of DNA can be increased.

Further, in the second DNA amplification apparatus according to the present invention, as shown in FIGS. 15 and 16, between each heating element, that is, between heating elements 1106 and 1107 and between heating elements 1107. A cooling liquid flow path 1501 for circulating a cooling liquid may be provided between the first and second layers 1108 and 1108 in contact with the back surface of the substrate 1101. Here, FIG. 15 shows a second D according to the present invention.
FIG. 16 is a plan view schematically showing still another specific example of the NA amplification device, and FIG. 16 is a view schematically showing a cross section taken along line HH of the device shown in FIG. In the cooling liquid channel 1501, a cooling liquid having a temperature lower than at least a temperature (about 37 ° C.) at which dissociated nucleic acid binds to the primer is circulated by a pump or the like (not shown).

By circulating the cooling liquid between the heating elements in this way, the flow path in the upper region of each heating element is obtained.
The temperature of the reaction solution in 1102 can be controlled more precisely without being affected by the adjacent heating element.

Furthermore, in the second DNA amplification apparatus of the present invention, as shown in FIGS.
A branch channel 1703 communicating with the storage cell 1702 having an electrode 1701 on the bottom surface can be provided between the channel 1102 and the channel 1102, that is, between the temperature detecting means 1113 and 1114 of the channel 1102.
Here, FIG. 17 is a plan view schematically showing still another specific example of the second DNA amplification device according to the present invention, and FIG. 18 is a sectional view taken along line I-I of the device shown in FIG. It is a figure which shows typically. A primer is stored in the storage cell 1702, and an electrode 1701 provided on the bottom surface of the storage cell 1702 is connected to the voltage controller 1117.

As described above, in the DNA amplification reaction, a primer is bound to the dissociated nucleic acid and the primer is extended by the action of the thermostable DNA synthase. Therefore, the primer bound once is consumed as it is as a part of the amplified nucleic acid. Therefore, the number of primers decreases as the amplification reaction is repeated, and when the primers are completely consumed, the amplification reaction also ends. That is, like the deterioration of the thermostable enzyme described above, the decrease of the primer is one factor that determines the upper limit of the amplification rate in the DNA amplification reaction. Figure 17
In the device shown in 18 and 18, it is possible to additionally supply the primer from the storage cell 1702 to the flow channel 1102 through the branch flow channel 1703, and it is possible to solve the problem of insufficient primer as the amplification reaction is repeated. .

DN using the apparatus shown in FIGS. 17 and 18.
The procedure for A amplification is as follows. First, Figures 11 and 12
In the same manner as in the procedure for DNA amplification using the device shown in FIG.
After maintaining the buffer in the channel 1102 in the upper region of 07 and 1108 at a predetermined temperature, a voltage is applied to the electrodes 1115 and 1116 or inverted to move each component in the buffer to carry out an amplification reaction. After repeating the heat cycle an appropriate number of times, a new cycle is started, and the same procedure is performed until the nucleic acid is dissociated by passing through the upper region of the heating element 1106. After that, each component is further moved toward the electrode 1116, and when the appropriate position is reached, the electrode 1701 is moved to the electrode 1115.
Apply a voltage of the same polarity as. This allows the storage cell 1702
The primer stored in the electrode starts moving toward the electrode 1116. The primer that has started moving flows into the flow channel 1102 through the branch flow channel 1703, and joins with each component that has moved through the flow channel 1102 from the upper region of the heating element 1106. After that, the heat cycle may be repeated in the same manner.

As described above, by using the apparatus shown in FIGS. 17 and 18, it is possible to supply additional primers even during the course of repeating the DNA amplification reaction, avoiding the lack of primers, The amplification rate of DNA can be increased. Further, in the DNA amplification reaction, not only the primer but also four kinds of deoxyribonucleoside triphosphates are respectively consumed, which becomes a factor of decreasing the reaction efficiency as the amplification reaction is repeated. By storing four kinds of deoxyribonucleoside triphosphates, it is possible to avoid the deoxyribonucleoside triphosphate deficiency and further increase the amplification rate.

Furthermore, the second DNA according to the present invention
The amplifying apparatus can also be configured such that the apparatus shown in FIGS. 11 to 18 is regarded as one unit and a plurality of units are connected by a flow path having a branch. A specific example in which the devices shown in FIGS. 13 and 14 are regarded as one unit and a plurality of these units are connected is shown in FIG.
Shown in In this figure, units 1901, 1902 and 1903 each having the structure shown in FIGS. 13 and 14 are connected via a flow path 1904. Channel 1904 is a unit
After passing 1901, specifically branching downstream of valve 1905 of unit 1901, one to unit 1902 via valve 1906,
The other is in communication with unit 1903 via valve 1907, respectively.

A DNA amplification reaction using this apparatus is performed according to the following procedure. First, in the unit 1901, a DNA amplification reaction is performed by the procedure described in the DNA amplification reaction using the apparatus shown in FIGS. 11 and 12 described above. After repeating the heat cycle a predetermined number of times, each component including the amplified nucleic acid is passed over the heating element 1908 and collected in the vicinity of the electrode 1909. Then open valves 1905, 1906 and 1907,
The voltage applied to the electrodes 1909 and 1910 is cut off, subsequently, the polarity of the electrode 1909 is reversed, and at the same time, the voltage is applied to the electrodes 1911 and 1912. Specifically, a voltage is applied so that the electrode 1909 is negative and the electrodes 1911 and 1912 are positive. As a result, the components collected near the electrode 1909 move in the flow path 1904 toward the electrodes 1911 and 1912. When each component moved into the units 1902 and 1903, the voltage applied to each electrode was cut off, and valves 1905, 1906 and
1907 closed. Then, in units 1902 and 1903, a DNA amplification reaction is performed according to the procedure described above.

As described above, the deterioration of the thermostable enzyme and the decrease of the primers are the factors that determine the upper limit of the amplification rate in the DNA amplification reaction.
The increase in the concentration of C.sub.c also becomes a factor that influences the upper limit of the amplification factor.
It was previously explained that the binding between the dissociated nucleic acid and the primer is a competitive reaction with the binding between the nucleic acids. The apparatus shown in FIGS. 17 and 18 solves this problem by adding a system capable of additionally supplying a primer during the amplification reaction. However, since the capacity of the flow channel is constant and the nucleic acid is only amplified, the chances of the nucleic acids being associated with each other and binding are increased even if the concentration of the primer is increased. In this device, each component flowing out of the unit 1901 is divided into two substantially equal parts at the branch point of the flow path 1904, and the unit 1902 is divided.
And into 1903. That is, unit 1902 and
The concentration of nucleic acid flowing into 1903 is approximately half the final concentration of nucleic acid in unit 1901. Therefore, the probability that the nucleic acids bind to each other during the amplification reaction is reduced, and the amplification reaction can be performed more efficiently, and as a result, the amplification rate can be increased.

In FIG. 19, the number of units following the unit 1901 is two, but the number is not limited to this, and the flow path 1904 may be further branched and connected to more units. it can. Further, although the amplification reaction is carried out in two stages in FIG. 19, it is also possible to carry out the amplification reaction in more stages by branching the channel 1904 after the units 1902 and 1903 to connect a plurality of units. .

Next, FIG. 20 shows a plan view of a specific example of the third DNA amplification apparatus according to the present invention. In this device, two valves 2003 and 2004 are provided on an inorganic or organic substrate 2001.
A flow path 2002 in which the transparent substrate is provided is formed, and a transparent substrate is bonded to the upper surface of the substrate 2001 in contact therewith. Between the valves 2003 and 2004, the three heating elements 2005, which are in contact with the back surface of the substrate 2001,
2006 and 2007 are provided, and each heating element control device 2008, 2009 and 20 individually controls the temperature of each heating element.
Connected to 10. In addition, these heating elements 2005, 2006
And the bottom of the channel 2002 corresponding to 2007, respectively,
Temperature detecting means 2011, 20 for detecting the temperature of the reaction solution
12 and 2013 are provided. Furthermore, a thermostable DNA synthase 2014 is fixed to the bottom surface of the flow path 2002 at a position corresponding to the heating element 2006. In addition, the bottom surface of the flow path 2002 located between the valve 2003 and the heating element 2005 and the valve 2004 and the heating element 20.
Electrodes 20 are provided on the bottom of the channel 2002 located between 07 and
15 and 2016 are provided, plus electrode 2015 and valve 20
A branch channel 2019 communicating with a storage cell 2018 having an electrode 2017 on the bottom surface is provided between the channel 03 and the port 03. An electrode 2020 is provided on the bottom surface of the flow path 2002 on the upstream side of the valve 2003.
Between the 2020 and the valve 2003, a sample inlet 2021 penetrating a transparent substrate joined to the upper surface of the substrate 2001 is provided.
The sample inlet 2021 has a form in which a sample can be taken into the channel 2002 from the outside. Also, the sample inlet 2021
The gel-like compound 2022 is in the flow path 2002 between the valve and the valve 2003.
A branch flow path which is filled so as to close the 2002 and is connected between the portion filled with the gel compound 2022 and the valve 2003 through a valve 2023 to a waste cell 2025 having an electrode 2024 on the bottom surface.
2026 is provided. Similarly, the flow path downstream of valve 2004
An electrode 2027 is also provided in 2002, and a DNA detecting means 2028 is provided between the electrode 2027 and the valve 2004. Electrode 20
15, 2016, 2017, 2020, 2024 and 2027 are all connected to a voltage control device 2029 capable of applying a voltage to each electrode and capable of reversing the applied voltage. The material of the inorganic or organic substrate 2001, the method of forming the flow path 2002, the material of the transparent substrate, the heat generating element that can be used, the temperature detecting means that can be used, and the like are all used in the first DNA amplification device according to the present invention. It is similar to the one. As the gel-like compound 2022 filled in the flow channel 2002, for example, cellulose, agarose, allylamide or the like can be used. Further, the DNA detecting means 2028 is not particularly limited, and any means usually used for detecting DNA can be used, and examples thereof include an ultraviolet absorption detection method, a refractive index detection method, a fluorescence detection method and an elliptical polarization. Those that use analysis methods can be mentioned.

DNA using this third DNA amplification device
The procedure of the amplification reaction is as follows. First, a sample such as whole blood or serum is introduced into the flow path 2002 filled with the buffer from the sample inlet 2021. Then, after closing valve 2003 and opening valve 2023, electrodes 2020 and 2024 are
A voltage is applied so that 2020 is negative and 2024 is positive, and the sample containing nucleic acid is moved to the electrode 2024. Flow path 2002 with electrode 20
The sample migrating towards 24 passes through the gel compound 2022. At this time, due to the principle of gel electrophoresis, the nucleic acid passes through the gel compound 2022 earlier than the nucleic acid passes through the gel compound 2022, and the branch channel 2026
To reach the waste cell 2025. After this, when the target nucleic acid has finished passing through the gel compound 2022, the valve 2023
Is closed to shut off the voltage applied to the electrode 2024, and then the valve 2003 is opened to apply the voltage to the electrode 2015. As a result, only the target nucleic acid from which impurities have been removed will be stored in the valve 20.
Go through 23. Further, the voltage is applied to the electrode 2015 at the same time as the voltage is applied to the electrode 2015. In the storage cell 2018,
Two kinds of primers and four kinds of deoxyribonucleoside triphosphates (dAT) necessary for the DNA amplification reaction in advance.
P, dCTP, dGTP, and dTTP) are stored, and a voltage is applied to the electrode 2017, and at the same time, the electrode starts moving toward the electrode 2015 and flows through the branch channel 2019 to the channel 2002.
And join the nucleic acid that has passed through the valve 2003. Nucleic acid valve
Passed through 2003, electrode 20 with each component from the storage cell 2018
After gathering in the vicinity of 15, valve 2003 is closed to shut off the voltage applied to electrodes 2017 and 2020, then electrode 2016.
The polarity of the voltage applied to the electrode 2015 is reversed at the same time when the voltage is applied to the electrode. As a result, each component starts moving toward the electrode 2016, and the amplification reaction of DNA is performed by the procedure described in each specific example in the second DNA amplification apparatus described above. After completion of the amplification reaction by performing heat cycles a predetermined number of times, first, each component containing the amplified nucleic acid is placed on the electrode 20.
Collect in the vicinity of 16. Next, the voltage applied to the electrode 2015 is cut off, the valve 2004 is opened, the voltage is applied to the electrode 2027, and at the same time, the polarity of the voltage applied to the electrode 2016 is reversed. As a result, the amplified nucleic acid starts moving toward the electrode 2027. When the nucleic acid moves to the position of the DNA detecting means 2028, the voltage applied to the electrode 2016 and the electrode 2027 is cut off to detect the DNA.

In this apparatus, everything from electric charge of the sample to amplification of the DNA and detection of the amplified DNA is electrical operation, and mechanical operation is not required. Therefore, the device can be made extremely small, which is also very advantageous in reducing the cost. Also, for the same reason, full automation is easy.

In the apparatus shown in FIG. 20, the gel compound is filled only between the sample inlet 2021 and the valve 2003, but the flow path 2002 between the valve 2004 and the DNA detection means 2028 is used.
Can also be filled. By arranging the gel-like compound in this way, it is possible to separate the primer and deoxyribonucleoside triphosphate that remain unreacted even after the DNA amplification and interfere with the detection of the DNA, and further improve the detection accuracy. You can

Further, instead of filling the gel compound 2022 in the flow channel 2002, the same effect can be obtained by setting the diameter of the flow channel in this portion to 150 μm or less. That is, by setting the diameter of the channel to 150 μm or less, the nucleic acid and other impurities can be separated due to the difference in electric affinity with the wall surface.

The DN according to the present invention has been described above with reference to the drawings.
Although the embodiment of the A amplifying apparatus has been described, the present invention is not limited to the above-described embodiment, and various modifications and applications are possible within the scope of the gist of the present invention. here,
The summary of the invention is as follows.

(1) Corresponding to FIGS. 1 to 10 A planar reaction cell formed on an inorganic or organic substrate, two flow channels communicating with the reaction cell, and the reaction cell in each of the flow channels. A DNA amplification apparatus comprising: two valves sandwiched therebetween; and a heating means provided at least on the bottom surface of the reaction cell or the back surface of the surface of the substrate on which the reaction cell is formed.

In this apparatus, the reaction cell has a planar shape, which allows the reaction solution in the reaction cell to be heated and cooled more quickly. Further, when the heating means is provided on both the bottom surface of the reaction cell and the back surface of the substrate, the temperature rising / falling rate of the reaction solution in the reaction cell can be increased. As a result, the temperature gradient in the reaction solution can be reduced, and the accuracy of the DNA amplification reaction can be improved.

(2) Corresponding to FIGS. 11 to 19 A channel provided on an inorganic or organic substrate, two valves provided in the channel, and a bottom surface of the channel between the two valves. Two electrodes, at least one temperature detecting means provided on the bottom surface of the flow path between the two electrodes, a bottom surface of the flow path near each of the at least one temperature detecting means, or a flow path of the substrate are provided. The temperature detecting means, a heating means provided in a pair with each of the temperature detecting means, a voltage controlling means for controlling the voltage applied to the two electrodes, and the temperature detecting means. A DNA amplification apparatus comprising heating control means for controlling the heating means based on information from the above.

In this apparatus, a voltage was applied to the reaction solution filled in the flow path so that only the nucleic acid, the thermostable DNA synthase, the primer and the deoxyribonucleoside triphosphate in the reaction solution were maintained at a predetermined temperature. The DNA amplification reaction is carried out by moving the DNA among a plurality of regions. Therefore, the problem of temperature gradient that occurs during the thermal cycle necessary for the amplification reaction is solved. Moreover, since only the components in the solution are moved by an electric means and the solution is not moved, a mechanical actuating member such as a pump is not required, and the apparatus can be downsized and the cost can be reduced.

(3) Corresponding to FIG. 20: A channel provided on an inorganic or organic substrate, first to fourth electrodes provided on the bottom surface of the channel, and a first electrode and a second electrode. A first valve provided between them, a sample inlet provided between the first electrode and the first valve, a sample inlet located between the sample inlet and the first valve, and a fifth valve on the bottom surface. A first branch channel communicating with the impurity introduction cell having the electrode, a gel-like compound provided in the channel between the first branch channel and the sample inlet, the first valve and the first valve A second branch flow path that is located between the second electrode and communicates with a storage cell having a sixth electrode on the bottom surface, and is provided on the bottom surface of the flow path between the second electrode and the third electrode. At least one temperature detecting means, the bottom surface of the flow path near each of the at least one temperature detecting means or the back surface of the surface of the substrate on which the flow path is provided, In either one of them, heating means provided in pair with each of the temperature detecting means, DNA detecting means provided between the third electrode and the fourth electrode, the DNA detecting means and the fourth means. A second valve provided between the first and sixth electrodes, a voltage control means for controlling the voltage applied to the first to sixth electrodes, and the heating means based on the information from the temperature detecting means. A DNA amplifying apparatus comprising a heating control means for controlling.

In this device, everything from the sample injection to the amplification of the DNA and the detection of the amplified DNA can be carried out by electric operation, and the device can be made very small and low in size. Cost reduction is also possible. Further, since no mechanical member is required, full automation is easy.

The present invention also includes the following aspects.

(4) Corresponding to FIGS. 3 and 4 In the apparatus of (1) above, a DNA amplification apparatus having at least one temperature detecting means on the bottom surface of the reaction cell.

In this apparatus, at least one temperature detecting means provided on the bottom surface of the reaction cell directly measures the temperature of the reaction solution. Therefore, the temperature of the reaction solution heated by the heating means can be controlled more precisely.

(5) Corresponding to FIGS. 5 and 6 The DNA amplifying device according to (1) above, which has at least one protrusion on the bottom surface of the reaction cell.

In this apparatus, by providing at least one protrusion on the bottom surface of the reaction cell, the area where the reaction solution in the reaction cell contacts the bottom surface of the cell is enlarged. as a result,
The heat generated from the heating means can be transferred to the reaction solution more efficiently, and the reaction solution can be heated or cooled more quickly.

(6) Corresponding to FIGS. 7 and 8 The DNA amplifying device according to (1) above, wherein the bottom surface of the reaction cell has a saw-toothed or wavy cross section.

This device is also similar to the device of (5) above.
By making the cross-sectional shape of the bottom surface of the reaction cell into a saw-tooth shape or a wavy shape, the area where the reaction solution in the reaction cell contacts the bottom surface of the cell is further expanded. Therefore, the heat generated from the heating means can be more efficiently transferred to the reaction solution, and the reaction solution can be heated or cooled more quickly.

(7) Corresponding to FIGS. 9 and 10 In the apparatus of (1), one or more protrusions are arranged on the bottom surface of the reaction cell at an angle to the flow direction of the reaction solution.

In this apparatus, the reaction solution flowing into the reaction cell is agitated in the reaction cell due to the resistance of the projections provided on the bottom surface of the cell. As a result, each component is uniformly dispersed in the reaction solution, and the DNA amplification reaction is uniformly generated. Further, when the reaction solution is allowed to flow out from the reaction cell, the reaction solution is still stirred by the protrusions, so that the subsequent purification and analysis steps can be carried out evenly. Further, similarly to the devices (5) and (6), the protrusions provided on the bottom surface of the reaction cell act to increase the area where the reaction solution in the reaction cell contacts the bottom surface of the cell. Therefore, the heat generated from the heating means can be more efficiently transferred to the reaction solution, and the reaction solution can be heated or cooled more quickly.

(8) Corresponding to FIGS. 11 to 19 A channel provided on an inorganic or organic substrate, two valves provided in the channel, and a bottom surface of the channel between the two valves. A DNA amplification device having two electrodes.

In this device, by applying a voltage to the two electrodes provided on the bottom of the flow channel, the nucleic acid in the reaction solution,
Only the primer, thermostable DNA synthase and deoxyribonucleoside triphosphate are transferred. Therefore,
The absence of solution migration eliminates the temperature gradient problem that occurs in the thermal cycling required for DNA amplification reactions. Further, since the solution is not moved, a mechanical actuating member such as a pump is not required, and the device can be downsized and the cost can be reduced.

(9) Corresponding to FIGS. 13 and 14 In the device of (2), a DNA amplification device in which a thermostable DNA synthase is immobilized on the inner surface of the flow path at a position where at least one temperature detecting means is provided. .

As the name implies, thermostable DNA synthase
The activity can be maintained to some extent even at a temperature at which the nucleic acid is dissociated, but the activity decreases with each repetition of the reaction, which is one of the factors determining the upper limit of the amplification rate in the DNA amplification reaction. In this device, the thermostable DNA synthase is immobilized on the inner wall of the channel in the temperature region where the primer bound to the nucleic acid is extended, for example, the bottom surface or the upper surface of the channel. This makes it possible to significantly suppress the deterioration of the enzyme and increase the amplification rate in the DNA amplification reaction without affecting the amplification reaction.

(10) Corresponding to FIGS. 15 and 16 In the apparatus of (2) above, an inorganic or organic substrate having two or more heating means and at a position corresponding to each heating means is provided. A DNA amplification device having a cooling liquid channel provided on the upper surface or the back surface of the substrate in contact with the substrate.

In this apparatus, by circulating the cooling liquid in the cooling liquid flow path, each of the plurality of heating means provided on the substrate can be thermally separated. Therefore, it becomes possible to more precisely control the temperature of the buffer in the flow path heated by each heating means.

(11) Corresponding to FIGS. 17 and 18 In the apparatus of (2) above, there are two or more heating means, and the bottom surface is provided in the flow path at a position corresponding to a predetermined heating means. A DNA amplifying apparatus, wherein a branch flow path communicating with a storage cell having an electrode is provided.

As described above, one of the factors that determines the upper limit of the amplification rate in the DNA amplification reaction is the deterioration of the thermostable DNA synthase, but the other factor is the decrease of the primer. Binding of the primer to the dissociated nucleic acid is a condition for the thermostable enzyme to start the extension of the primer, but since the primer once bound becomes a part of the amplified nucleic acid as it is, the primer is consumed each time the amplification reaction is repeated. Will be done. Therefore, the amplification reaction ends when the primer is completely consumed.

In this apparatus, by storing the primer in the storage cell in advance, the primer can be additionally supplied even during the amplification reaction, and the amplification rate in the DNA amplification reaction can be increased. The supply of the primer from the storage cell to the flow channel can be performed electrically using the electrode provided on the bottom surface of the storage cell.

Further, in this apparatus, not only the primer but also four kinds of deoxyribonucleoside triphosphates may be stored in the storage cell. In this case, deoxyribonucleoside triphosphate deficiency can also be avoided.

(12) Corresponding to FIG. 19 At least three units each having any one of the devices (2), (8), (9), (10) or (11) as one unit are provided and one unit is provided. A DNA amplification device in which another unit is connected to a downstream side of the unit by a flow path having a branch.

Factors that determine the upper limit of the amplification rate in the DNA amplification reaction include not only the deterioration of the thermostable enzyme and the decrease of the primer described above, but also the increase of the DNA concentration resulting from the amplification reaction. As the concentration of DNA increases, the possibility that nucleic acids will bind to each other when the dissociated nucleic acid binds to the primer increases. Since the reaction in which the nucleic acid binds to the primer competes with the reaction in which the nucleic acid binds to each other, increasing the primer concentration relative to the nucleic acid concentration relatively decreases the probability that the nucleic acid binds to each other. However, since the volume of the channel is constant, there is a limit to suppressing the recombination of nucleic acids by increasing the concentration of the primer.

This device has three or more units capable of carrying out a DNA amplification reaction by itself, and another unit is connected downstream of one unit through a branched flow path. There is. In this apparatus, first, the DNA amplification reaction is repeated a predetermined number of times in the upstream unit, then the valve of each unit is opened, and the nucleic acid amplified in the upstream unit is electrically transferred to the downstream unit. At this time, the nucleic acid is divided into approximately equal parts at the branch point of the flow path and introduced into the downstream unit. For example, when the number of units on the downstream side is two, the concentration of the nucleic acid introduced into each unit is about half the final concentration of the nucleic acid on the unit on the upstream side. Therefore, when the nucleic acid and the primer are bound to each other, the probability that the nucleic acids are bound to each other is reduced, and the DNA amplification reaction can be efficiently performed.

In this device, the number of branches of the flow path on the downstream side of one unit may be two or more, and there is no particular limitation. Further, it is possible to further branch the flow path after the branched unit to provide a plurality of units to carry out a multi-step reaction, and the number of steps is not limited.

(13) In the apparatus of (3) above, instead of filling the gel compound in the channel between the first branch channel and the sample inlet, a channel having a diameter of 150 μm or less is provided. DN
A amplification device.

In the device (3), capillary electrophoresis using affinity is used to remove impurities from the sample introduced from the sample inlet. Capillary electrophoresis includes one that utilizes this affinity as well as one that utilizes the electroaffinity in the capillaries. In this device, the diameter of the channel is set to 150 μm or less, and the impurities are separated by using the capillary electrophoresis utilizing this electric affinity. Therefore, a carrier such as a gel-like compound is not necessary, which makes it possible to simplify the device structure and further reduce the cost.

[0078]

As described above, the first D according to the present invention is used.
The NA amplification device can heat and cool the reaction solution more quickly by forming the reaction cell into a planar shape, which causes a temperature gradient generated in the reaction solution, which is a problem in the thermal cycle necessary for the DNA amplification reaction. Can be made smaller. Further, it is possible to perform heating and cooling more promptly by appropriately selecting the portion where the heating means is provided and the material of the heating means.

Further, the second DNA amplification apparatus according to the present invention applies a voltage to the reaction solution filled in the flow channel, so that the nucleic acid, the thermostable DNA synthase, the primer and the deoxyribonucleoside triphosphate in the reaction solution are applied. Only the DNA is moved between a plurality of regions maintained at a predetermined temperature to carry out a DNA amplification reaction. Therefore, it is not necessary to heat and cool the solvent having the largest heat capacity, and the problem of temperature gradient that occurs during the heat cycle can be solved. Moreover, since only the components in the solution are moved by an electric means and the solution is not moved, a mechanical actuating member such as a pump is unnecessary,
It is possible to reduce the size and cost of the device.

Furthermore, in the third DNA device according to the present invention, the DNA is amplified from the input of the sample, and the amplified DN is obtained.
It is possible to perform all of the operations by electric operation until A is detected, the device can be made extremely small, and the cost can be reduced. Further, since no mechanical member is required, full automation is easy.

[Brief description of drawings]

FIG. 1 is a plan view of a specific example of a first DAN amplifier according to the present invention.

FIG. 2 is a sectional view taken along line AA of the device shown in FIG.

FIG. 3 is a plan view of another specific example of the first DNA amplification device according to the present invention.

4 is a cross-sectional view taken along the line BB of the device shown in FIG.

FIG. 5 is a plan view of yet another specific example of the first DNA amplification device according to the present invention.

6 is a cross-sectional view taken along the line CC of the apparatus shown in FIG.

FIG. 7 is a plan view of yet another specific example of the first DNA amplification device according to the present invention.

8 is a cross-sectional view taken along line DD of the device shown in FIG.

FIG. 9 is a plan view of yet another specific example of the first DNA amplification device according to the present invention.

10 is a sectional view taken along line EE of the device shown in FIG.

FIG. 11 is a plan view of a specific example of the second DNA amplification device according to the present invention.

12 is a cross-sectional view of the device shown in FIG. 11 taken along the line FF.

FIG. 13 is a plan view of another specific example of the second DNA amplification device according to the present invention.

14 is a sectional view taken along line GG of the device shown in FIG.

FIG. 15 is a plan view of yet another specific example of the second DNA amplification device according to the present invention.

16 is a cross-sectional view taken along line HH of the device shown in FIG.

FIG. 17 is a plan view of yet another specific example of the second DNA amplification device according to the present invention.

FIG. 18 is a cross-sectional view taken along the line II of the device shown in FIG.

FIG. 19 is a plan view of a device in which a plurality of units, each of which is one of the devices shown in FIGS. 11 to 18, is connected downstream of one unit through a branched flow path.

FIG. 20 is a plan view of a specific example of the third DNA amplification device according to the present invention.

[Explanation of symbols]

101, 1101, 2001 ... Inorganic or organic substrate, 102, 701
… Reaction cells, 103, 104, 1102, 2002… Flow paths, 105, 11
03 ... Transparent substrate, 106, 107, 1104, 1105, 1905, 1906,
1907, 2003, 2004, 2023 ... Valve, 108, 1106, 1107, 110
8, 1908, 2005, 2006, 2007 ... Heating means, 301, 302, 1
112, 1113, 1114, 2011, 2012, 2013 ... Temperature detecting means,
501, 901 ... Protrusion, 1109, 1110, 1111, 2008, 2009, 201
0 ... Heating element control means, 1115, 1116, 1701, 1909, 1910, 1
911, 1912, 2015, 2016, 2017, 2020, 2024, 2027 ... Electrode, 1117, 2029 ... Voltage control means, 1301, 2014 ... Heat resistance D
NA synthase, 1501 ... Coolant flow channel, 1702, 2018 ... Storage cell, 1703, 1904, 2019, 2026 ... Branch flow channel, 1901, 1902,
1903 ... Unit, 2021 ... Sample input port, 2022 ... Gel compound, 2025 ... Impurity cell, 2028 ... DNA detection means.

Claims (3)

[Claims] 1. A planar reaction cell formed on an inorganic or organic substrate, two channels communicating with the reaction cell, and two channels provided in each of the channels sandwiching the reaction cell. A DNA amplification apparatus comprising a valve and at least one heating means provided on the bottom surface of the reaction cell or the back surface of the substrate on which the reaction cell is formed. 2. A channel provided on an inorganic or organic substrate, two valves provided on the channel, two electrodes provided on the bottom surface of the channel between the two valves, and two electrodes At least one temperature detection means provided on the bottom surface of the flow path between the electrodes, at least the bottom surface of the flow path near each of the at least one temperature detection means, or at least the back surface of the surface of the substrate on which the flow path is provided. Either one of the heating means is provided in pair with each of the temperature detecting means, the voltage controlling means for controlling the voltage applied to the two electrodes, and the information based on the information from the temperature detecting means. A DNA amplification device comprising heating control means for controlling heating means. 3. A flow channel provided on an inorganic or organic substrate, first to fourth electrodes provided on the bottom surface of the flow channel,
A first valve provided between the first electrode and the second electrode,
A sample inlet provided between the first electrode and the first valve,
A first branch flow channel located between the sample input port and the first valve and communicating with an impurity introduction cell having a fifth electrode on the bottom surface, the first branch flow channel and the sample input port. A gel-like compound provided in the flow passage between the first valve and the second electrode, the second branch flow passage communicating with the storage cell having the sixth electrode on the bottom surface, At least one temperature detecting means provided on the bottom surface of the flow path between the second electrode and the third electrode,
At least one of the bottom surface of the flow path near each of the at least one temperature detection means or the back surface of the surface of the substrate on which the flow path is provided is provided in pair with each of the temperature detection means. Heating means, DNA detection means provided between the third electrode and the fourth electrode, second valve provided between the DNA detection means and the fourth electrode, the first
To a voltage control means for controlling the voltage applied to the sixth electrode, and a heating control means for controlling the heating means based on the information from the temperature detecting means.