JP2008237132A - Method and device for amplifying nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To conduct the amplification reaction of a nucleic acid in efficiency higher than that in the reaction in a liquid phase. <P>SOLUTION: The amplification reaction of the nucleic acid is conducted in a vapor phase. The reaction is carried out by adding a primer 12, a target nucleic acid 1, a DNA polymerase 2 and deoxynucleoside triphosphate 3 to a fine drop of water, and releasing the resultant drop to the vapor phase. The temperature control by pressurizing is enabled because of the vapor phase to enable high-efficiency rapid temperature control. The primer 12, the target nucleic acid 1, the DNA polymerase 2 and the deoxynucleoside triphosphate 3 are released to the vapor phase by a finely subdividing means 5, and each thereof reacts and amplified. The temperature control at the amplification is carried out by utilizing a syringe 6 by the change of the pressure. The detection is carried out on the real time by utilizing fluorescence reaction. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method and apparatus for efficiently amplifying nucleic acid by releasing and reacting it in the gas phase.

Conventionally, this type of nucleic acid amplification method has been performed in a liquid phase including real-time polymerase chain reaction (see, for example, Non-Patent Document 1 and Patent Document 1). This is a technique in which primers, nucleic acids to be detected, DNA polymerase, and deoxynucleoside triphosphates are added and reacted as chemicals related to amplification in the microtube, and the temperature in the microtube is adjusted by using a warmer. It is what makes it react. There is a time difference between the temperature in the microtube and the warming of the warmer, and because the temperature is not enough, the temperature in the microtube is grasped in advance and a pre-evaluation such as reacting at the set temperature location is performed. It was necessary. In addition, since the temperature of the polymerase chain reaction changes drastically, the reaction has been carried out by processing the material of the warmer with aluminum having high thermal conductivity.
Anal Biochem. 1996 Nov 1; 242 (1): 84-9 JP 2005-522981

  Such conventional nucleic acid amplification methods and devices are inefficient due to the principle of amplification reaction, and the setting burden on the device side is large. Moreover, in the case of a liquid phase reaction, there exists a subject that reaction becomes slow from the problem of thermal conductivity. In the case of a liquid phase reaction, there is a problem that the increase or decrease in the temperature in the liquid may be insufficient or excessive, and the reaction may not be promoted. Therefore, a nucleic acid amplification method and apparatus with stable reaction conditions are required.

  The present invention solves such conventional problems, and is a means that enables a highly efficient reaction by carrying out a nucleic acid amplification reaction in a gas phase. In addition, by heating or compressing the entire container and adjusting the temperature of the entire gas phase, the reaction is accelerated compared to the liquid phase, and a stable reaction is possible. As a result, an object of the present invention is to provide a nucleic acid amplification method and apparatus that can accelerate the reaction.

  In order to achieve the above object, the method and apparatus for nucleic acid amplification in the gas phase of the present invention sprays fine water droplets, and the primer, nucleic acid to be detected, DNA polymerase, and deoxynucleoside triphosphate are all contained in the fine water droplets. It is characterized in that it can be reacted by containing one or more.

  The nucleic acid amplification method and apparatus in the gas phase according to the present invention is characterized by utilizing ultrasonic vibration as a means for micronized water droplets.

  The nucleic acid amplification method and apparatus in the gas phase of the present invention is characterized by using humidified steam as a means for atomizing water droplets.

  The nucleic acid amplification method and apparatus in the gas phase of the present invention is characterized by utilizing dispersion caused by collision of water molecules.

  The method and apparatus for nucleic acid amplification in the gas phase of the present invention is characterized in that the object to be heated is any one or more or all of a primer, a nucleic acid to be detected, a DNA polymerase, and deoxynucleoside triphosphate. .

  The nucleic acid amplification method and apparatus in the gas phase of the present invention is characterized in that at least the reaction vessel is made of a material that can be heated from 30 ° C. to 100 ° C.

  The nucleic acid amplification method and apparatus in the gas phase of the present invention is characterized in that a metal having high thermal conductivity is used as a heatable material.

  The nucleic acid amplification method and apparatus in the gas phase of the present invention is characterized in that a reaction vessel can be pressurized.

  The nucleic acid amplification method and apparatus in the gas phase of the present invention is characterized in that the temperature of the reaction vessel can be adjusted by increasing or decreasing the internal volume.

  The nucleic acid amplification method and apparatus in the gas phase according to the present invention is characterized in that the pressure in the reaction vessel can be adjusted with a pressure regulating valve.

  The method and apparatus for amplifying nucleic acid in the gas phase of the present invention is characterized in that the reaction vessel is a light-transmitting vessel and the fluorescence emission in the vessel can be measured.

  According to the present invention, nucleic acid amplification can be carried out in a gas phase by carrying out the nucleic acid amplification reaction in a gas phase so that the heating and lowering of the temperature in the reaction vessel can be carried out quickly and stably, resulting in a highly efficient and stable reaction. Methods and apparatus can be provided. Further, since it is in the gas phase, the temperature control becomes finer than the reaction in the liquid phase, the stable reaction becomes possible, and the reaction is accelerated. In addition, by reacting by adding finely differentiated water, even a small amount of nucleic acid to be detected can be efficiently contacted with DNA polymerase and deoxynucleoside triphosphate, the reaction is promoted, and the reaction can be stably performed. Further, the reaction can be performed by increasing or decreasing the gas phase temperature in the reaction vessel, and the temperature can be easily adjusted as compared with the liquid phase. In addition, by adjusting the internal pressure of the reaction vessel and controlling the temperature, it is possible to provide a device that can be heated with low energy and can be easily and quickly performed by adjusting the pressure even when the temperature is lowered. As soon as possible.

  According to the first aspect of the present invention, the primer, the nucleic acid to be detected, the DNA polymerase, and deoxynucleoside triphosphate are all contained in the gas phase in the reaction vessel, or fine water droplets containing one or more are sprayed. In addition, by performing nucleic acid amplification in the gas phase, the detection target nucleic acid and each reagent can be brought into contact with each other with high efficiency and reacted.

  Further, the invention described in claim 2 has an effect that heat is not generated by using ultrasonic vibration as a fine differentiation method, and a certain fine water droplet can be sprayed.

  In addition, the invention described in claim 3 has an effect that a certain fine water droplet can be sprayed with low energy by using heated steam as a fine differentiation method.

  Further, the invention described in claim 4 has the effect that no heat is generated by using dispersion due to collision of water molecules as a fine differentiation method, and that a certain fine water droplet can be sprayed.

  In addition, the invention according to claim 5 leads to maintenance of the activity of an enzyme having a large influence by heating by using any one or all of a primer, a nucleic acid to be detected, a DNA polymerase, and deoxynucleoside triphosphate as a heating target. It has the action.

  Further, the invention according to claim 6 has the effect that the reaction vessel can be heated and lowered by making the reaction vessel a material that can be heated from 30 ° C. to 100 ° C., leading to higher efficiency of the reaction. Have

  The invention according to claim 7 has the effect that the reaction vessel temperature can be adjusted more quickly by making the material of the reaction vessel a metal having high thermal conductivity.

  Further, the invention described in claim 8 has an effect that the temperature can be quickly adjusted by pressurization / depressurization by using a pressure vessel capable of pressurizing the reaction vessel.

  The invention according to claim 9 has the effect that the temperature can be easily adjusted by increasing or decreasing the internal volume of the reaction vessel.

  The invention according to claim 10 has an effect that the temperature can be adjusted by releasing the pressure of the pressure regulating valve by making the pressure in the reaction vessel adjustable by the pressure regulating valve.

  The invention described in claim 11 has the effect that the nucleic acid amplification reaction can be detected in real time with a fluorescent label or the like by using a reaction container as a light-transmitting container and having a structure capable of detecting fluorescence in the container.

  Embodiments of the present invention will be described below.

(Embodiment 1)
One technique for detecting the type of nucleic acid present in a sample is the polymerase chain reaction. The polymerase chain reaction specifically amplifies a specific nucleic acid sequence derived from a specific organism and identifies the specific organism from the increase. In conventional polymerase chain reaction, the reaction in the solution is carried out in a microtube, the heat is increased or decreased by a warmer, the reaction is performed, and the reaction is short, and the reaction can be performed in about 2 hours. However, depending on the heating position and heating method, the reaction does not proceed sufficiently, and since it is a reaction in solution, it is difficult to lower the temperature. To lower the temperature, it is necessary to lower the temperature of the heater, but it consumes a lot of energy. Furthermore, when the amount of nucleic acid is very small, there is a concern that the reaction may not proceed well depending on the amount of liquid in the microtube.

  Therefore, it is expected to develop a method and an apparatus that can perform a stable reaction and can rapidly carry out overall warming and low temperature. For example, improvement of contact efficiency. In order to increase the efficiency of the reaction with the target nucleic acid to be detected, the reaction solution needs to be made in a very small amount. In addition, when the temperature in the reaction solution is lowered once, it is difficult to lower the temperature unless excessive energy is provided from the outside. In particular, in the case of polymerase chain reaction, temperature control is particularly important because a reaction that raises the temperature to 90 degrees or more is assumed. Therefore, it is utilized that the target nucleic acid to be detected is released into the gas phase, and that temperature control and contactability can be improved by adding a primer, DNA polymerase, and deoxynucleoside triphosphate.

  For example, spray fine water droplets under high humidity where the fine water droplets in the reaction vessel drift and exist stably, and all of the primer, nucleic acid to be detected, DNA polymerase, and deoxynucleoside triphosphate are contained in the fine water droplets. Or contain one or more and react in the gas phase.

  Inclusion of a nucleic acid, primer, DNA polymerase, and deoxynucleoside triphosphate in a fine water droplet increases the probability of contact and promotes the reaction. Therefore, nucleic acids and primers to be detected, primers, DNA polymerase, and deoxynucleoside triphosphate released in the gas phase are held in fine water droplets in the gas phase so that each can be contacted efficiently and at a high concentration. Can do. As a result, the reaction can be accelerated. It is effective to mix the nucleic acid, primer, DNA polymerase, and deoxynucleoside triphosphate to be detected at the beginning, or spray separately. It is possible to prevent thermal inactivation of the enzyme.

  The spraying conditions are conditions that float in the gas phase of the reaction vessel, but the reaction can be promoted again by collecting and respraying. Examples of the fine water droplet means include ultrasonic vibration, humidified steam, and dispersion due to collision of water molecules. Ultrasonic vibration can always provide a certain amount and size of water molecules. Further, it is possible to spray the water droplets once accumulated in the lower part again. Although humidified steam is heated, it can always provide a certain amount and size of water molecules with low energy. Dispersion due to collision of water molecules makes the size of water molecules unstable, but does not apply temperature and is effective in maintaining the activity of DNA polymerase.

  In the nucleic acid amplification reaction such as the polymerase chain reaction, temperature control is an important factor, and the target to be heated in this case may be any or all of the nucleic acid to be detected, DNA polymerase, and deoxynucleoside triphosphate before the reaction. Heating can also be performed for each reaction vessel. Therefore, a heat resistant material is desirable for the warming reaction vessel. Specifically, when the whole reaction vessel is heated, a metal having high thermal conductivity is effective. On the other hand, when the reaction is carried out only in the reaction vessel, since it is a gas phase reaction, the temperature can be controlled by pressurization. In the case of pressurization, the reaction vessel needs to be a pressure-resistant and heat-resistant vessel. The temperature control by pressurization can adjust the internal volume and the temperature by using the pressure valve. Heat resistant glass or pressure resistant glass is effective. Moreover, the fluorescence emission in a container can be measured by making a reaction container into a light transmissive container. Thereby, it becomes possible to confirm the reaction state in real time.

  Examples of the present invention will be described below.

  A highly efficient reaction is performed by nucleic acid amplification in the gas phase. This technology consists of a reaction vessel for performing a polymerase chain reaction, a temperature control unit, and a fine differentiation unit. In order to perform the polymerase chain reaction, a means for releasing the target nucleic acid, DNA polymerase, and deoxynucleoside triphosphate into the gas phase is required. The target nucleic acid, DNA polymerase, and deoxynucleoside triphosphate are contained in a minute water droplet for reaction in the gas phase, released into the gas phase, and reacted in a container. Specific examples of vapor phase release means by fine differentiation include ultrasonic vibration, humidified steam, collision of water molecules, and the like. The size of the minute water droplet is a size that does not drop due to condensation or the like, exceeding the amount of the water droplet containing one molecule of the target nucleic acid, DNA polymerase, deoxynucleoside triphosphate. Since the reaction is performed in minute water droplets, it is also effective to stir the reaction gas phase. In order to control the temperature of the polymerase chain reaction in the reaction gas phase, it is required to heat the entire reaction vessel or the inside thereof.

  For heating each reaction vessel, it is effective to use a Peltier element or to lower the temperature. Effective materials for the reaction vessel include metals having high thermal conductivity such as aluminum, copper, silver, and gold, containers having heat resistance within the reaction temperature such as polycarbonate and polystyrene, heat resistant glass, and pressure resistant glass. On the other hand, temperature control by pressurization is effective such as increase / decrease of internal pressure, temperature rise by maintaining a sealed state accompanying heating. In particular, temperature control by pressurization can cope with not only heating but also low temperatures. That is, the internal pressure is improved during heating, and the temperature is lowered by lowering the internal pressure. A pressure-resistant and heat-resistant container is effective. In addition, the intercalating fluorescent reagent used for the real-time polymerase chain reaction is simultaneously released into the gas phase, thereby promoting the reaction and enabling real-time detection. In the case of the liquid phase, a reaction time of about 2 hours is required to capture the fluorescence emission of the intercalating fluorescent reagent. However, in the case of the gas phase, the reaction efficiency is high, so that the speed can be increased. By using a light-transmitting reaction container, measurement with an optical instrument equipped with the detector 9 becomes possible.

  FIG. 1 is a conceptual diagram showing one embodiment of a nucleic acid amplification apparatus for suitably performing a nucleic acid amplification method in a gas phase.

  As shown in FIG. 1, a target vessel 4 containing a target nucleic acid 1, DNA polymerase 2, and deoxynucleoside triphosphate 3 is released into the gas phase by a fine differentiation means 5. The target nucleic acid 1, DNA polymerase 2, and deoxynucleoside triphosphate 3 released into the gas phase are contacted in the gas phase. On the other hand, the reaction vessel 4 is provided with a syringe 6 capable of adjusting the pressure, and the internal pressure can be adjusted. If necessary, the fine differentiation means 5 has a structure in which the aggregated nucleic acid 1, DNA polymerase 2, and deoxynucleoside triphosphate 3 can be re-vaporized again.

  After the reaction or during the reaction, an intercalating type fluorescent reagent 7 such as SYBR GREEN is introduced by the same fine differentiation means 5 to detect the reacted nucleic acid. The reaction vessel 4 is made of a light-transmitting material, and recognizes the emitted light that has reacted to the excitation light emitted from the excitation light source 8 such as a mercury lamp as the total emission luminance.

  FIG. 2 shows an external heating means 10 such as a heater as a temperature adjusting means for the reaction vessel 4.

  In addition, by introducing only one molecule of the target nucleic acid into the fine water droplets, it is possible to react with the intercalating fluorescent reagent and to count the luminescent fine water droplets individually. Thereby, it is possible to grasp the number of target nucleic acids included in the initial stage.

  The intercalating fluorescent reagent is not limited to SYBR GREEN, but DAPI, PI, SYTO series, etc. are also effective.

  In the amplification of nucleic acid, the recovered reaction solution can be detected by a conventional electrophoresis method or the like without using fluorescence emission.

  The fluorescence emission detection can be performed not only by excitation light irradiation from the outside of the container but also by excitation light irradiation inside the container. FIG. 3 shows the reaction vessel 4 and the excitation light source 8.

  It is also possible to make fine water droplets by ejecting nozzles.

  By using the nucleic acid amplification method and apparatus in the gas phase of the present invention, the nucleic acid amplification reaction in the gas phase can be performed and the reaction can be performed with high efficiency. Moreover, by performing in the gas phase, the increase and decrease of the temperature inside the reaction vessel can be efficiently adjusted by adjusting the temperature from the outside. Furthermore, heating and lowering of the temperature can be performed with high efficiency by adjusting the internal pressure, and a stable reaction is possible. Since it is a highly efficient reaction in the gas phase, a stable reaction is possible even with a very small amount of sample, and it is a very effective method for rapid detection and stable reaction.

1 is a conceptual diagram showing an embodiment of a nucleic acid amplification apparatus including a method for amplifying nucleic acid in a gas phase according to Example 1 of the present invention. The figure which shows the reaction container containing an external heating means The figure which shows the reaction container which installed the same excitation light source

Explanation of symbols

1 Nucleic acid 2 DNA polymerase 3 Deoxynucleoside triphosphate 4 Reaction vessel 5 Fine differentiation means 6 Syringe 7 Intercalating fluorescent reagent 8 Excitation light source 9 Detector 10 External heating means 12 Primer

Claims (11)

A nucleic acid amplification method in which fine water droplets are sprayed, and the primer, nucleic acid to be detected, DNA polymerase, deoxynucleoside triphosphate is contained in the fine water droplets, or one or more of them are contained and reacted in the gas phase. The nucleic acid amplification method according to claim 1, wherein ultrasonic vibration is used. The nucleic acid amplification method according to claim 1, wherein warming steam is used. The nucleic acid amplification method according to claim 1, wherein dispersion by collision of water molecules is used. The nucleic acid amplification method according to any one of claims 1 to 4, wherein the heating target is one or more or all of a primer, a nucleic acid to be detected, a DNA polymerase, and deoxynucleoside triphosphate. The nucleic acid amplification device according to claim 1, further comprising a reaction vessel made of a material that can be heated and cooled from 30 to 100 ° C. The nucleic acid amplification device according to claim 6, wherein the heatable material is a metal having high thermal conductivity. The nucleic acid amplification apparatus according to claim 1, further comprising a reaction vessel capable of adjusting pressure. The nucleic acid amplification device according to claim 8, wherein the reaction vessel has a structure in which the pressure and temperature can be adjusted by increasing or decreasing the internal volume. The nucleic acid amplification device according to claim 8, wherein the pressure in the reaction vessel can be adjusted with a pressure regulating valve. The nucleic acid amplification device according to any one of claims 6 to 10, wherein a reaction vessel for nucleic acid amplification is a light-transmitting vessel and has a structure capable of detecting fluorescence in the vessel.

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