JPH04325080A - Amplifier of deoxyribonucleic acid - Google Patents

Abstract

PURPOSE:To obtain the title device constituted so as to carry out PCR method in such state as to hold both ends of reaction liquid put into a capillary by air, etc., made operation such as removal of mineral oil and sealing by combustion gas unnecessary and capable of carrying out the PCR method in a short time. CONSTITUTION:A capillary 1 is put into a heating medium 21 kept at a temperature for modifying a reaction liquid 11 containing a deoxyribonucleic acid to be amplified and then a reaction liquid feed port 2, the capillary 1 and reaction liquid discharge port 4 communicate with operating three-way valve and stop valve 7. Then the reaction liquid 11 is introduced from the feed port 2 into the capillary 1 and then a gas feed port 3 communicates with the capillary 1 to feed the gas so as to carry the reaction liquid 11 at prescribed position in the capillary 1. The capillary 1 is put into a heating medium 22 of annealing temperature at a point when the reaction liquid 11 attained heat distortion temperature and then put into the heating medium 23 kept to polymerization temperature at a point when the reaction liquid 11 attained annealing temperature. The above-mentioned operation is repeated to carry out PCR method and then a gas is fed from the gas feed port 3 and the reaction liquid 11 is discharged from the discharge port 4.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus for amplifying deoxyribonucleic acid using the PCR method, and more particularly to an apparatus for amplifying deoxyribonucleic acid using a thin tube such as a capillary tube as a container for a reaction solution.

0002

2. Description of the Related Art Japanese Patent Application Laid-Open No. 62-240862 discloses a prior art device for amplifying deoxyribonucleic acid using the PCR method. In addition, a method using a capillary tube as a container for the reaction solution is used in Analytical Biochemistry, 1.
86 (1990), pp. 328-331 (Analy
tical Biochemistry 186 (1
990) pp328-331).

0003

However, the prior art disclosed in JP-A No. 62-240862 uses a disposable plastic container with a lid as a container for the reaction solution.
For example, assuming the use of a 0.5 ml microfuge tube (for example, a 0.5 ml microfuge tube), approximately 0.1 ml of the reaction solution is placed in the above-mentioned disposable plastic container with a lid. Mineral oil is superimposed to prevent this, and the temperature is changed repeatedly several dozen times in the following order: heat denaturation temperature of about 95 degrees Celsius, annealing temperature of about 55 degrees Celsius, and polymerization temperature of about 70 degrees Celsius.
Although the R method is used to amplify deoxyribonucleic acid, it has the disadvantage that a mineral oil removal step is required after the amplification reaction is completed. In addition, disposable plastic containers with lids are
PCR has a large heat capacity and poor heat transfer to the reaction solution.
The disadvantage is that it takes a long time to perform the dharma. On the other hand, the above-mentioned Analytical Biochemistry, 186 (1990) No. 3
The prior art disclosed on pages 28 to 331 uses a capillary tube as a container for the reaction liquid, and after putting the reaction liquid into the capillary tube, both ends of the capillary tube are sealed with combustion gas.
In addition to preventing evaporation of water in the reaction solution, the heat capacity of the container is reduced, and heat transfer to the reaction solution is improved to improve PCR performance.
Although the time required for performing the method is shortened, there are disadvantages in that it is necessary to seal both ends of the capillary tube with combustion gas, and to take out the reaction liquid from the sealed capillary tube after the amplification reaction is completed. In addition, DNA amplification using the PCR method is a step such as gene analysis and genetic diagnosis, and before and after it there are steps such as extraction of target DNA and sequence reaction, so it is important to check the continuity between these steps. Despite the importance of this process, conventional techniques do not consider continuity with previous and subsequent processes.

[0004] The object of the present invention was made in view of the drawbacks of the prior art.
It shortens the time required to perform the R method, eliminates the need for sealing both ends of the capillary tube with combustion gas, and removing the reaction solution from the sealed capillary tube.
The object of the present invention is to provide a deoxyribonucleic acid amplification device that takes into consideration continuity with the steps before and after the amplification step A.

[0005]

[Means for Solving the Problems] The above object is achieved by focusing on the fact that water evaporation from the gas-liquid interface of a reaction liquid can be substantially prevented by sealing the reaction liquid in a capillary with a fluid. This is achieved by placing the reaction solution in a thin tube such as a capillary tube, sandwiching both ends of the reaction solution with air or other gas, and performing the PCR method.

[0006]

[Operation] In order to perform the PCR method, the reaction solution should be approximately 95°C.
The temperature is usually changed several dozen times in the following order: thermal denaturation temperature of approximately 55°C, annealing temperature of approximately 55°C, and polymerization temperature of approximately 70°C. At this time, when water evaporates, the composition of the reaction solution changes and PCR The law cannot be carried out as intended. However, since water evaporation from the reaction liquid occurs at the interface between the reaction liquid and the fluid, if the area of the interface is made sufficiently small, water evaporation can be sufficiently reduced, and the reaction liquid can be kept at a level that does not cause problems when performing PCR. The composition can be changed. Furthermore, since the transfer of the reaction solution can be controlled using the fluid, P
It is possible to easily realize a deoxyribonucleic acid amplification device that takes into consideration the continuity between the steps before and after the DNA amplification step using the CR method.

[0007]

[Example] Fig. 1 is a configuration diagram showing an example, in which 1 is a capillary tube with an inner diameter of about 1 mm, 2 is a reaction liquid supply port, 3 is a gas supply port, 4 is a reaction liquid outlet, and 6 is a three-way valve. , 7 is a stop valve. 8 is a capillary support, and 9 is a capillary moving mechanism, which controls the position of the capillary support 8. The details of both will be omitted, but the point is that any configuration can be used as long as the movement can be controlled smoothly. 21a, 22a, and 23a are containers, respectively. Reference numerals 21, 22, and 23 are heat carriers contained in containers 21a, 22a, and 23a, respectively. Each heat medium is maintained at the denaturation temperature, annealing temperature, and polymerization temperature of the reaction solution. Reference numeral 11 denotes a reaction liquid, which is sealed in a capillary tube with gas. The operation will be explained below according to FIG. In advance, the inside of the capillary tube 1 or the three-way valve 6 stop valve 7
Contamination of the reaction liquid is prevented by flowing a cleaning liquid instead of the reaction liquid through the part through which the reaction liquid passes, and the capillary 1 is moved by the capillary support 8 by the capillary movement mechanism 9 into the heating medium 2.
1, the three-way valve 6 and the stop valve 7 are operated to connect the reaction liquid supply port 2 and the capillary tube 1, and the capillary tube 1 and the reaction liquid discharge port 4, respectively. After putting the reaction liquid 11 into the capillary tube 1 from the reaction liquid supply port 2, the three-way valve 6 is operated to communicate the gas supply port 3 and the capillary tube 1, so that the reaction liquid 11 flows into the capillary tube 1.
Gas is supplied from the gas supply port 3 so as to reach a predetermined position within the gas supply port 3. When the reaction liquid 11 reaches the heat denaturation temperature after a predetermined period of time, the capillary moving mechanism 9 connected to the capillary support 8 is operated to place the capillary 1 into the heat medium 22. When the reaction liquid 11 reaches the annealing temperature after a predetermined period of time, the capillary moving mechanism 9 connected to the capillary support 8 is operated to place the capillary tube 1 into the heat medium 23. When the reaction liquid 11 reaches the polymerization temperature after a predetermined time, the capillary moving mechanism 9 connected to the capillary support 8 is operated to move the capillary 1 to the heat medium 21.
put it inside. Hereinafter, the movement of the capillary tube 1 is repeated in the same order a predetermined number of times at the heat denaturation temperature, annealing temperature,
After carrying out the PCR method by changing the temperature of the reaction solution in the order of the polymerization temperature, gas is supplied from the gas supply port 3 to form the reaction solution 11.
is discharged from the reaction liquid outlet 4. Among the above operations,
When operating the three-way valve 6 to connect the gas supply port 3 and the capillary tube 1 and supplying gas from the gas supply port 3 so that the reaction liquid 11 comes to a predetermined position in the capillary tube 1, the stop valve 7 is operated. It is also possible to apply an appropriate internal pressure to the capillary tube 1 by manipulating the capillary tube 1. In this way, even if a small amount of gas such as air is mixed into the reaction liquid 11, it is possible to prevent the reaction liquid from being divided due to expansion of the gas due to a temperature change of the reaction liquid.

[0008] As an example, the inner diameter is 1 mm and the outer diameter is 2 mm.
Using a plastic capillary tube, the temperature change of the reaction solution was determined by numerical calculation when the reaction solution was placed in hot water with a heat denaturation temperature of 95 degrees Celsius and an annealing temperature of 55 degrees Celsius. Figure 2 shows the change in the average temperature of the reaction solution over time. It can be seen from this figure that the temperature of the reaction solution changes from the thermal denaturation temperature of 95° C. to the annealing temperature of approximately 55° C. in about 15 seconds. In other words, even if you include the capillary movement time, the time required for a series of temperature changes of heat denaturation temperature, annealing temperature, and polymerization temperature is about 1 minute, and even if you repeat this process about 30 times, it will take about 30 minutes to carry out the PCR method. can. Although calculation results are not shown, the inner and outer diameters are each 1/
If it is set to 2, the PCR method can be carried out in about 15 minutes.

FIG. 3 is a configuration diagram showing another embodiment, in which 1 is a capillary tube, 2 is a reaction liquid supply port, 3 and 5 are gas supply and exhaust ports, 4 is a reaction liquid discharge port, 61 and 62 are three-way valves, 21, 22, 23
21a, 22a, and 23a are containers, and the heating media contained therein are maintained at a heat denaturation temperature, an annealing temperature, and a polymerization temperature, respectively. 11 is a reaction solution. The embodiment shown in FIG. 3 is different from that shown in FIG. 1 in that the reaction liquid 11 itself is moved instead of the capillary tube 1 being moved. The operation will be explained below according to FIG. After preventing contamination of the reaction liquid by flowing a cleaning liquid instead of the reaction liquid through the capillary tube 1 and the three-way valves 61 and 62, which pass through the reaction liquid, the three-way valves 61 and 62 are operated to remove the reaction liquid. The supply port 2 and the capillary tube 1 are communicated with each other, and the capillary tube 1 and the gas supply/exhaust port 5 are communicated with each other. After introducing the reaction liquid 11 into the capillary tube 1 from the reaction liquid supply port 2, the three-way valve 61 is operated to connect the gas supply/exhaust port 3 and the capillary tube 1, and the capillary tube in which the reaction liquid 11 is immersed in the heat medium 21 is opened. Gas is supplied from the gas supply/exhaust port 3 so as to reach a predetermined position within the gas supply/exhaust port 1. After a predetermined time, when the reaction liquid 11 reaches the heat denaturation temperature, gas is supplied from the gas supply/exhaust port 3 so that the reaction liquid 11 comes to a predetermined position in the capillary tube 1 immersed in the heat medium 22. After a predetermined time, when the reaction liquid 11 reaches the annealing temperature, gas is supplied from the gas supply/exhaust port 3 so that the reaction liquid 11 comes to a predetermined position in the capillary tube 1 immersed in the heat medium 23. When the reaction liquid 11 reaches the polymerization temperature after a predetermined period of time, gas is supplied from the gas supply/exhaust port 5 so that the reaction liquid 11 comes to a predetermined position in the capillary tube 1 immersed in the heat medium 21 . Hereinafter, the movement of the reaction liquid 11 in the capillary tube 1 is repeated a predetermined number of times, changing the temperature of the reaction liquid in the order of heat denaturation temperature, annealing temperature, and polymerization temperature.
After carrying out the CR method, the three-way valve 62 is operated to connect the capillary tube 1 and the reaction liquid discharge port 4, gas is supplied from the gas supply/exhaust port 3, and the reaction liquid 11 is discharged from the reaction liquid discharge port 4. Of course, when the reaction solution is sent back for repetition, it goes without saying that it should be controlled so that it returns in a short time so that there is no influence due to this back delivery. Further, when the reaction liquid 11 is repeatedly moved within the capillary tube 1 by supplying gas from the gas supply/exhaust ports 3 and 5, an appropriate internal pressure may be applied within the capillary tube 1. This effect is similar to that of the first embodiment. In this example, if a capillary tube with the same dimensions as in the first example is used, the capillary tube is already at the target temperature, so the time required for the temperature of the reaction solution to reach the target temperature is the same as the first example. It can be easily inferred that it is shorter than the example. Further, in the present invention, since the movement of the reaction liquid is performed by supplying and exhausting gas, there are almost no moving parts, and there is an advantage that the apparatus can be made inexpensive and highly reliable.

FIG. 4 is a block diagram showing still another embodiment.
is spirally wound in a capillary tube. 2 is a reaction liquid supply port,
3 is a gas supply port, 4 is a reaction liquid outlet, and 6 is a three-way valve.

[0011] Reference numerals 31, 32, and 33 are heat blocks that are maintained at a heat denaturation temperature, an annealing temperature, and a polymerization temperature, respectively, and the capillary tube 1 is wound in a spiral shape while being in sufficient thermal contact with these blocks. 11 is a reaction solution. The number of spiral turns of the capillary tube 1 is set to be greater than the number of times required for the PCR method, in which temperature changes are repeated in the order of heat denaturation temperature, annealing temperature, and polymerization temperature. The operation will be explained below according to FIG. Contamination of the reaction liquid is prevented by flowing a washing liquid instead of the reaction liquid through the capillary tube 1 and the three-way valve 6 through which the reaction liquid passes. communicate with. Reaction liquid 11 is transferred from reaction liquid supply port 2 to capillary tube 1.
After putting the reaction liquid 11 into the heat block 31, the three-way valve 6 is operated to connect the gas supply port 3 and the capillary tube 1.
Gas is supplied from the gas supply port 3 so that the gas is at a predetermined position. When the reaction liquid 11 reaches the heat denaturation temperature after a predetermined time, gas is supplied from the gas supply port 3 so that the reaction liquid 11 comes to a predetermined position of the heat block 32. When the reaction liquid 11 reaches the annealing temperature after a predetermined time, the reaction liquid 11
Gas is supplied from the gas supply port 3 so that the heat block 33 is at a predetermined position. After repeating the same operation a predetermined number of times and changing the temperature of the reaction solution in the order of heat denaturation temperature, annealing temperature, and polymerization temperature to carry out the PCR method, the gas supply rate from the gas supply port 3 was changed to the above-mentioned rate. Continuously supply the reaction liquid 11 to the reaction liquid outlet 4.
Emit more. If the gas supply rate from the air supply port 3 is appropriately controlled so that the reaction liquid 11 reaches the target temperature during movement during the above-described operation, the PCR method can be carried out even if the gas is continuously supplied. can. In addition, fluid resistance elements such as throttles are provided at appropriate positions before and after the reaction liquid outlet 4 to connect the capillary tube 1.
Appropriate internal pressure may be applied inside. This effect is similar to that of the first embodiment. In this example, if a capillary tube with the same dimensions as in the first example is used, the capillary tube is already at the target temperature, so the time required for the temperature of the reaction solution to reach the target temperature is the same as the first example. It can be easily inferred that it is shorter than the example. In addition, in this example, since the PCR method is performed by moving the reaction liquid in one direction, it is easy to control the gas supply, and there is no need to send the reaction liquid back to a place where the temperature is inappropriate. Since there is no Furthermore, there are almost no moving parts, which has the effect of making the device inexpensive and highly reliable.

The amount of evaporation of the reaction liquid at the interface is estimated as follows.

[0013] When the reaction solution is poured into the plastic container with a lid, the area of the interface is approximately 35 mm2;
In a capillary tube with an inner diameter of 1 mm, the total area of both ends is about 1.6 mm2, so the area of the interface is less than 1/20. If necessary, the area of the interface can be easily made even smaller by using a capillary tube with a smaller inner diameter. The following fact also proves that water evaporation can be sufficiently reduced by reducing the area of the interface. Using a plastic capillary tube with an inner diameter of 1 mm, the reaction solution is put into the capillary tube, and even if the reaction solution is placed in a constant temperature room at 95°C for 10 minutes with both ends sandwiched with air, no moisture remains. The amount of evaporation was about 0.1%. It is obvious that the lower the temperature, the smaller the amount of water evaporation. That is, if a capillary tube with an inner diameter of about 1 mm or less is used as a container for the reaction liquid, the reaction liquid is put into the capillary tube, and both ends of the reaction liquid are sandwiched between air or other gas, the conventional technique can be used. It is not necessary to use the mineral oil that is currently used, and the mineral oil used in
186 (1990) pp328-331)
As proposed in 2006, the desired PCR method can be performed without sealing the front and rear of the capillary tube with a change in the composition of the reaction solution that does not cause any trouble when performing the PCR method.

[0014]

As explained above, according to the present invention, the process of removing mineral oil, which is a drawback of the prior art, or
It is not necessary to seal both ends of the capillary tube with combustion gas and to take out the reaction liquid from the sealed capillary tube, and
Not only is it possible to realize a PCR method that can be processed in a short time, but
The supply and discharge of the reaction solution are the pretreatment steps of the PCR method, respectively.
Since it is designed to be continuous with the post-processing process, PC
It is possible to realize a deoxyribonucleic acid amplification device that has continuity between the steps before and after the DNA amplification step using the R method.

[Brief explanation of the drawing]

FIG. 1 is a configuration diagram showing an embodiment of the present invention.

FIG. 2 is a diagram showing an example of an average temperature change of a reaction solution.

FIG. 3 is a configuration diagram showing another embodiment of the present invention.

FIG. 4 is a configuration diagram showing another embodiment of the present invention.

[Explanation of symbols]

1... Capillary tube, 2... Reaction liquid supply port, 3... Gas supply port, 4...
Reaction liquid discharge port, 5... Gas supply/exhaust port, 6, 61, 62... Three-way valve, 7 is a stop valve, 21, 22, 23... Heat medium, 31,
32, 33...Heat block

Claims (5)

[Claims] 1. A thin tube containing a reaction solution containing deoxyribonucleic acid to be amplified, a device for feeding the reaction solution into the thin tube, and a device for controlling the temperature of the thin tube at a predetermined temperature. 1. A deoxyribonucleic acid amplification device, characterized in that a reaction liquid within the tube is sealed at a predetermined position within the tube with a fluid for transporting the reaction liquid, and is transferred to a predetermined position. [Claim 2] A reaction solution containing a mixture of deoxyribonucleic acid to be amplified, a DNA primer complementary to a portion of the deoxyribonucleic acid, deoxyribonucleotides such as adenine, thymine, guanine, and cytosine, and DNA polymerase is heated with air or other gas. The single-stranded deoxyribonucleic acid and the DNA primer are combined with a means for achieving a heat denaturation temperature for separating the double-stranded deoxyribonucleic acid into single-stranded deoxyribonucleic acid by placing the two ends in a tubule with the two ends sandwiched between them. a means for achieving an annealing temperature that causes the deoxyribonucleic acid to polymerize, and a means for realizing a polymerization temperature that causes the deoxyribonucleotide complementary to the single-stranded deoxyribonucleic acid to polymerize to the DNA primer bound to the single-stranded deoxyribonucleic acid by the action of DNA polymerase, 1. A deoxyribonucleic acid amplification device characterized by automating a PCR method for amplifying deoxyribonucleic acid by repeatedly changing the temperature a predetermined number of times. [Claim 3] By providing means for creating temperature conditions of a heat denaturation temperature, an annealing temperature, and a polymerization temperature in the pipe direction of the thin tube, and supplying or removing air or other gas from one side of the thin tube. A claim characterized in that the reaction solution in the capillary is moved to a position corresponding to each of the temperature conditions, and the temperature of the reaction solution is repeatedly changed a predetermined number of times in the order of heat denaturation temperature, annealing temperature, and polymerization temperature to perform the PCR method. Item 2. The deoxyribonucleic acid amplification device according to item 2. 4. The thin tube is wound spirally a necessary number of times, and means for creating temperature conditions of a heat denaturation temperature, an annealing temperature, and a polymerization temperature are arranged in a predetermined order in the circumferential direction of the spiral thin tube. , the reaction liquid inside the tube is moved in one direction by supplying air or other gas intermittently or continuously at a predetermined rate from one side of the tube, and the reaction solution is heated to a temperature of denaturation temperature, annealing temperature, and polymerization temperature. 3. The deoxyribonucleic acid amplification apparatus according to claim 2, wherein the PCR method is performed by sequentially changing the temperature and is repeated a predetermined number of times. 5. The repetition is performed by using air or air from one side of the capillary so that the reaction solution moves at high speed so that the temperature change of the reaction solution does not reach the above-mentioned heat denaturation temperature, annealing temperature, or polymerization temperature. By supplying other gases,
5. The deoxyribonucleic acid amplification device according to claim 4, wherein the reaction solution is controlled to return to its initial position.