JP3743090B2 - Nucleic acid base sequence replication method, replication apparatus, nucleic acid base sequence analysis sample production method, nucleic acid base sequence analysis method, and analysis apparatus - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a nucleic acid base sequence replication method, a replication apparatus, a nucleic acid base sequence analysis sample production method, a nucleic acid base sequence analysis method, and an analysis apparatus. More specifically, the reaction solution is continuously heated and cooled. A nucleic acid base sequence replication method and replication apparatus that replicates the base sequence of a nucleic acid molecule that is DNA or RNA by a polyamylase chain reaction (PCR) repeated a predetermined number of times, and a reaction product replicated by this replication method or replication apparatus The present invention relates to an analysis method and an analysis apparatus for analyzing a nucleic acid base sequence by electrophoresis, and a method for producing a nucleic acid base sequence analysis sample for producing an analysis sample for analysis.
[0002]
[Prior art]
As a method for exponentially amplifying DNA or RNA having a specific nucleic acid sequence, polymirase chain reaction (PCR) is known (Japanese Patent Laid-Open No. 62-281). According to this method, it has been demonstrated that a target nucleic acid sequence can be effectively amplified from a very small amount of sample by using a nucleic acid chain synthesis reagent composed of a primer and a thermostable enzyme. Yes.
[0003]
In this method, a reaction solution containing a nucleic acid as a template, four kinds of nucleoside triphosphates (dNTP), at least one oligonucleotide primer, and a thermostable enzyme is generated. Then, by heating this reaction solution, the nucleic acid serving as a template is denatured by heat to form a single-stranded molecule. Thereafter, the oligonucleotide primer is cooled to a temperature suitable for binding to the complementary sequence portion of the denatured nucleic acid, held at this temperature for a predetermined time, and then set to a temperature at which the thermostable enzyme is effective. Thereby, starting from the bound oligonucleotide primer, a thermostable enzyme synthesizes a new nucleic acid molecule using four types of nucleoside triphosphates and a template nucleic acid as a substrate. By this series of temperature operations, a nucleic acid molecule having the same sequence as the template nucleic acid molecule can be duplicated. By repeating such heating and cooling, a base sequence similar to the base nucleic acid sequence can be obtained. Nucleic acid molecules can be replicated exponentially.
[0004]
Further, since the nucleic acid molecule synthesized by this method is limited to a portion having a sequence complementary to the oligonucleotide primer, it is used for determining a specific base sequence of the nucleic acid molecule (sequence).
[0005]
In the above replication method, it is necessary to strictly control the temperature setting and the temperature holding time in the reaction stage. For this reason, an apparatus for automatically controlling the repetition of heating and cooling of the reaction solution is described in Japanese Patent Application Laid-Open No. 62-240862. This apparatus is composed of a heat pump for heating and cooling the metal block and a computer for controlling the temperature of the heat pump, and realizes a temperature cycle necessary for the reaction.
[0006]
In the reaction by this apparatus, a plastic container in which a reaction solution is placed in a metal block formed so as to surround the plastic container is inserted, and the reaction solution in the plastic container is heated and cooled. Is heated and cooled.
[0007]
During heating, mineral oil is often used to prevent evaporation of the reaction solution inside the plastic container, but it may be necessary to remove the mineral oil from the sample solution with phenol or the like after the reaction. Therefore, a wax that prevents evaporation of a reaction solution that can be used in place of mineral oil is sold as AmpliWAX (trade name) by Perkin-Elmers, USA.
[0008]
The nucleic acid molecule generated by the above apparatus is taken out from the reaction vessel using a pipette or the like as an aqueous solution. In many cases, the nucleic acid molecules thus extracted are measured for molecular weight by electrophoresis, and the generated nucleic acid molecules are determined. At that time, in order to prevent sample loss and contamination with other samples, the experiment worker needs to pay attention to the transfer of the solution and the dropping onto the gel for electrophoresis. In addition, when the solution is dropped onto the gel for electrophoresis, it is necessary to add glycerin, saccharose or the like to the solution in order to increase the specific gravity of the solution and make it easier to drop into the electrophoresis tank.
[0009]
[Problems to be solved by the invention]
In a nucleic acid base sequence replication method that requires heating and cooling of a solution such as PCR, the time required for the reaction can be shortened by increasing the temperature rise and fall of the reaction solution. For this purpose, it is desirable to increase the thermal conductivity between the reaction solution and the heater and between the reaction solution and the cooler, and to reduce the heat capacity of the reaction solution and the portion including the peripheral portion.
[0010]
However, in the above conventional method, the reaction solution is put in a plastic or glass container, and the container is surrounded by a metal block, so that the reaction time is long due to the influence of the heat capacity of the metal block or the like. There's a problem.
[0011]
The present invention has been made to solve the above problems, and a first object of the present invention is to provide a nucleic acid base sequence replication method and replication apparatus that shorten the time required for the reaction.
[0012]
In addition, since the reaction solution may be heated to 90 ° C. or higher during the reaction, it is necessary to prevent evaporation of the solution during the reaction using mineral oil, wax, a sealed container or humidified air. In the conventional technique, since the solution after the reaction is taken out from the reaction container using a pipette or the like, there is a problem that the recovery of the solution after the reaction requires time and the recovery efficiency may be lowered. . There is also a problem that there is a risk of causing contamination with other samples during the collection operation.
[0013]
A second object of the present invention is to provide a method for producing a nucleic acid base sequence analysis sample that simplifies the recovery of a solution after reaction, has high recovery efficiency, and eliminates contamination.
[0014]
In many cases, the molecular weight of the product after the reaction is measured by electrophoresis. However, in this case, there is a problem that it takes time to drop the solution after the reaction onto the gel for electrophoresis. In addition, there is a problem that solution loss occurs during this operation.
[0015]
The present invention has been made to solve the above-mentioned problems. It is a first object of the present invention to provide a nucleic acid base sequence analysis method and analyzer that can easily measure the total amount of a product and its molecular weight by electrophoresis. The purpose of 3.
[0016]
[Means for Solving the Problems]
In order to achieve the first object, the nucleic acid base sequence replication method of the present invention comprises a reaction solution containing a nucleic acid. The surface is coated with a hydrophilic material and a hydrophobic material surrounding the hydrophilic material. Dropping on the heating resistor, supplying power to the heating resistor to heat the dropped reaction solution, and stopping the power supply to the heating resistor to cool the dropped reaction solution And the base sequence of the nucleic acid contained in the dropped reaction solution is replicated.
[0017]
Further, the nucleic acid base sequence replication apparatus of the present invention comprises: The surface is coated with a hydrophilic material and a hydrophobic material surrounding the hydrophilic material. A reactor comprising a heating resistor for dropping the reaction solution and a substrate supporting the heating resistor, a power supply device for supplying power to the heating resistor of the reactor, and power to the heating resistor And a control device that repeatedly controls the supply of power to the heating resistor and the stop of the power supply to the heating resistor.
[0018]
In the nucleic acid base sequence replication method and replication apparatus described above, in the case of replicating a nucleic acid base sequence by repeatedly heating and cooling a reaction solution, such as PCR, first, template DNA, oligonucleotide primers, A reaction solution comprising dNTP, a heat-resistant DNA polymirase, and a buffer solution prepared to an ion concentration suitable for the reaction is generated. And this reaction solution The surface is coated with a hydrophilic material and a hydrophobic material surrounding the hydrophilic material. By dropping on a heating resistor, preferably a planar heating resistor, heating the reaction solution by the heat generated when power is supplied to the heating resistor, and by stopping the heat generation when power supply to the heating resistor is stopped The reaction solution is reacted by cooling by heat radiation from the heating resistor.
[0019]
According to the nucleic acid base sequence replication method and replication apparatus of the present invention, since the reaction is performed in a state where the reaction solution is in direct contact with the surface of the heating resistor, the thermal resistance between the reaction solution and the heating resistor is Low, the temperature of the reaction solution can be raised and lowered quickly, and the reaction rate can be shortened.
[0020]
In order to prevent the reaction solution from evaporating during the reaction, the heating resistor to which the reaction solution is dropped and the oily material having a lighter specific gravity than the reaction solution are both put in a container, and heating and cooling of the dropped reaction solution are repeated. . As the oily material having a lighter specific gravity than the reaction solution, mineral oil or wax of trade name AmpliWAX manufactured by Perkin-Elmers, USA can be used.
[0021]
The heating resistor is a substance having electrical resistance and generates heat when power is supplied. For example, a thick film resistor that is printed on a plate-shaped ceramic substrate with a paste mixed with metal particles or the like to be a resistor and then fired to form a resistor can be used as a planar heating resistor. . With such a thick film resistor, a heating resistor can be produced in any form on the substrate, depending on the pattern during paste printing.
[0022]
As another example of the planar heating resistor, there is one in which a metal or a metal compound is deposited on a glass substrate or a silicon wafer by vacuum deposition or sputtering. In this example, a minute heating resistor suitable for handling a very small amount of reaction solution can be produced. According to these planar heating resistors, a plurality of heating resistors including wiring can be manufactured on the same substrate. Therefore, a plurality of independent reactions can be performed on the heating resistor formed on one substrate at a time. In addition, if the substrate is rectangular and the heating resistor part protrudes from the long side part of the substrate, the heating resistor in which the reaction solution is dropped and a container for containing an oily material having a specific gravity lower than that of the reaction solution ( Since a plurality of heating resistors can be operated at the same time when being inserted into and removed from the reaction vessel) and inserted into the electrophoresis gel when analyzing the base sequence of the nucleic acid, the operation efficiency is improved.
[0023]
The surface of the heating resistor can be coated with a hydrophilic material and a hydrophobic material surrounding the hydrophilic material.
[0024]
To coat the material showing hydrophilicity, when using the above-mentioned method for producing a heating resistor with a thick film resistor, print the paste containing the glass particles after printing the paste that becomes the resistor. Then, a method of forming a glass layer on the surface of the heating resistor can be used by firing. Since glass is known to have a hydrophilic surface, surface processing for coating the heating resistor and the hydrophilic material can be made by a single firing. Further, a resin can be used as a material for forming a hydrophilic surface. As the resin, a polyimide resin is the most suitable resin because it is excellent in hydrophilicity, workability and heat resistance.
[0025]
Since the surface of the part where the reaction solution of the heating resistor is dropped is subjected to processing that exhibits hydrophilicity, and the portion other than the surface that is processed to exhibit hydrophilicity is subjected to processing that exhibits hydrophobicity, Thus, the planar heating resistor can be held in a limited manner on the surface treated to show hydrophilicity, and scattering of the reaction solution and contamination with other reaction solutions can be prevented.
[0026]
The reaction vessel has a structure in which the back surface of the heating resistor and the inner surface of the reaction vessel are in contact with each other in order to improve the heat dissipation of the heating resistor when the reaction solution is cooled, and is in contact with the back surface of the heating resistor of the reaction vessel. It is effective if the portion is made of a metal having good thermal conductivity. By comprising such a reaction vessel and a heating resistor, the temperature drop time of the reaction solution during cooling can be shortened.
[0027]
The duplication device of the present invention is provided with a temperature sensor for measuring the temperature of the reaction solution during the reaction, and the control device controls the temperature of the reaction solution to a predetermined temperature based on the temperature sensor output. be able to. As temperature control, feedback control based on the temperature profile for the reaction and the temperature measurement result can be used.
[0028]
As the temperature sensor, a known temperature sensor such as a thermocouple, a metal film, or a semiconductor temperature sensor can be used. Further, a method of measuring the temperature of the heating resistor from the resistance value using a heating resistor having a thermal gradient in the electrical resistance can be adopted. According to this method, since the heating resistor also serves as the temperature sensor, the temperature sensor and the wiring can be omitted.
[0029]
In order to achieve the second object described above, the method for producing a nucleic acid base sequence analysis sample of the present invention comprises dropping a reaction solution containing a nucleic acid onto a heating resistor, Putting wax together in a container, supplying power to the heating resistor to heat the dropped reaction solution, and stopping the power supply to the heating resistor to cool the dropped reaction solution. The base sequence of the nucleic acid contained in the reaction solution dropped repeatedly is solidified, the wax is solidified, the reaction solution, the heating resistor and the wax are integrated and removed from the container, and the base sequence of the nucleic acid that is the reaction product is determined. An analysis sample in which a reaction solution for analysis, a heating resistor, and wax are integrated is manufactured.
[0030]
In this method for producing a nucleic acid base sequence analysis sample, the heating resistor to which the reaction solution is dropped is heated and cooled in the reaction container together with the wax together with the reaction solution while the reaction solution is dropped. Since the wax is preferably cured at room temperature and melted during the reaction, a wax having a melting temperature in the range of 30 ° C. to 50 ° C. can be used. The most manageable wax has a melting temperature of 35 ° C to 45 ° C.
[0031]
Since both the heating resistor and the wax to which the reaction solution is dropped are placed in a container and repeatedly heated and cooled to react, the wax melts due to the heat of the heating resistor during the reaction and covers the surface of the reaction solution. Evaporation of the reaction solution during the reaction can be prevented.
[0032]
In this method for producing a nucleic acid base sequence analysis sample, the reaction solution is in direct contact with the surface of the heating resistor as in the case of duplicating the nucleic acid base sequence. Since the resistance is low, the temperature of the reaction solution can be quickly raised and lowered, the reaction rate can be shortened, and the analysis sample can be quickly produced.
[0033]
In addition, since the wax is solidified, the wax is solidified so as to surround the heating resistor, and the reaction solution, the heating resistor, and the analytical sample in which the solidified wax is integrated are manufactured. It is sealed between the solidified wax and the heating resistor, and does not scatter or mix with other reaction solutions, which makes it possible to easily recover the solution after the reaction, and to collect other samples during the recovery operation. There will be no contamination.
[0034]
In order to achieve the third object, the nucleic acid base sequence analysis method of the present invention includes the analysis sample produced by the nucleic acid base sequence analysis sample production method in a gel of an electrophoresis tank, By supplying power to the heating resistor and heating, the wax is peeled from the heating resistor to release the reaction solution into the gel, and the nucleic acid base sequence that is a reaction product contained in the released reaction solution Are analyzed by electrophoresis.
[0035]
In addition, the nucleic acid base sequence analyzer of the present invention is an electrophoresis which analyzes the base sequence of a nucleic acid contained in a reaction solution in an analysis sample produced by the above method for producing a nucleic acid base sequence analysis sample by electrophoresis. A tank, a peeling device for peeling the wax from the heating resistor and releasing the reaction solution into the gel by supplying power to the heating resistor and heating, and measuring the potential around the heating resistor And a holding device that holds the potential of the heating resistor at a potential lower than the surrounding potential.
[0036]
When analyzing by electrophoresis, a voltage is applied to the electrodes provided at both ends of the electrophoresis tank so that an electric field is applied in the direction opposite to the direction of nucleic acid migration. The voltage at this time is determined by the length of the nucleic acid to be measured, the size of the electrophoresis tank, and the gel concentration. At that time, for example, the potential Ew around the heating resistor is measured by the measuring device with reference to the cathode of the electrophoresis tank, for example.
[0037]
The holding resistor holds the potential of the heating resistor lower than the surrounding potential Ew, that is, a negative potential, while heating the heating resistor to peel off the wax surrounding the reaction solution from the heating element, Release the solution into the gel. At this time, the nucleic acid, which is a product contained in the released reaction solution, is guided into the electrophoresis gel by the electric field in the electrophoresis tank. For example, when the potential Ew is measured with reference to the cathode of the electrophoresis tank, a voltage that is the voltage Eh is applied to the cathode of the electrophoresis tank. The voltage Eh at this time is applied so that the potential of the heating resistor is lower than the surrounding potential, and the voltage Eh ranges from Ew to Ew-100 [V] (for example, when Ew = 20V, 20V to -80V range). By applying this voltage, separation of nucleic acid molecules from the heating resistor can be promoted. Further, if the voltage Eh is larger than the potential Ew and is a negative voltage, the electric field distribution of the entire electrophoresis tank becomes non-uniform, and the electric field distribution in the gel may be disturbed to curb the electrophoresis direction of the nucleic acid. The voltage Eh is suitably in the range of Ew to Ew-20 [V] (for example, the range of 20 V to 0 V when Ew = 20 V).
[0038]
When electrophoresis proceeds and the reaction product nucleic acid sufficiently migrates to a predetermined distance in the gel, voltage application to the electrophoresis tank electrode, power supply to the heating resistor, and voltage application are all stopped.
[0039]
In the nucleic acid base sequence analysis method described above, an analysis sample in which the reaction solution, the heating resistor and the solidified wax are integrated is used, and the solution after the reaction is recovered using a pipette or the like. Since there is no need to drop the reaction solution on the gel, the solution after the reaction can be easily recovered, and no loss of solution occurs.
[0040]
Thus, the length of the nucleic acid molecule, which is a reaction product guided to the electrophoresis gel, can be measured by electrophoresis. Further, by cutting out a gel having a predetermined migration distance, only nucleic acid molecules having a specific length can be separated and recovered. In addition, immediately after the wax is peeled off and the reaction solution is released into the electrophoresis tank, the applied electric field to the electrophoresis tank is zeroed, and the gel in the vicinity of the heating resistor is recovered to recover all lengths of DNA. You can also.
[0041]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. FIG. 1 shows a reactor in the present embodiment. The reactor 1 includes a support substrate 12 made of alumina formed by projecting a plurality (six in this embodiment) of substantially rectangular support portions 12A to 12F at predetermined intervals from one long side of a substantially rectangular substrate 12G. And a plurality of planar heating resistors 11A to 11F formed on the surfaces of the support portions 12A to 12F of the support substrate 12, respectively.
[0042]
The heating resistors 11A to 11F are formed by attaching a resistor paste mainly composed of silver, lead, and glass powder on the surface of each of the support portions 12A to 12F, followed by firing. . In addition to silver and lead, the component of the metal powder mixed in the resistor paste may be any one of gold, copper, tungsten, nickel, or a combination thereof.
[0043]
The support substrate 12 may be made of a material such as ceramics made of silicon carbide or aluminum nitride in addition to alumina. In particular, the use of silicon carbide is preferable because the thermal conductivity is good, so that the reaction solution can be quickly cooled during the reaction and the reaction time can be shortened.
[0044]
The power supply wirings 21 </ b> A to 21 </ b> F and 22 </ b> A to 22 </ b> F for supplying power to the heating resistors 11 </ b> A to 11 </ b> F are configured by pattern wirings printed on the support substrate 12. The power supply wires 21A to 21F and 22A to 22F are connected to the connector 14, and the connector 14 is connected to a controller device 51 described later.
[0045]
Next, the heating resistor portion will be described in detail. Since all the heating resistor portions have the same structure, only one heating resistor portion will be described, and description of the other heating resistor portions will be omitted. 2 is an enlarged view of the portion of the heating resistor 11A shown in FIG. 1, and FIG. 3 is a cross-sectional view taken along the line AA ′ of FIG.
[0046]
The heating resistor 11A is formed in a planar shape on the support portion 12A so as to be connected to the power supply wirings 21A and 22A. The surface of the heating resistor 11A is covered with a hydrophilic surface material 23 made of glass. The hydrophilic surface material 23 is composed of a glass layer formed on the surface of the heating resistor by printing a paste containing glass particles after printing a resistor paste that forms the heating resistor, and baking it. ing.
[0047]
As the hydrophilic surface material 23, in addition to glass, a hydrophilic resin can be used. As such a resin, polyimide which is resistant to the heat generation of the heating resistor is suitable. The hydrophilic surface material 23 is located on the surface of the heating resistor 11 </ b> A and holds the dropped reaction solution 31.
[0048]
Around the hydrophilic surface material 23, the hydrophobic surface material 24 is formed so as to cover the entire heating resistor 11 </ b> A and the support portion 12 </ b> A except for the portion where the reaction solution 31 of the hydrophilic surface material 23 is dropped. Has been. By covering the hydrophobic surface material 24 in this way, the reaction solution 31 can be held in a portion limited to the exposed portion of the hydrophilic surface material 23. As the hydrophobic surface material 24, a resin exhibiting hydrophobicity after curing can be used. As such a resin, a silicone-based coating agent is an optimum material excellent in processability, heat resistance and hydrophobicity. .
[0049]
The heating resistor 11A generates heat when power is supplied via the power supply wirings 21A and 22A on the support substrate 12. A temperature sensor 27A for measuring the temperature of the reaction solution 31 dropped at that time and performing feedback control is disposed on the surface of the hydrophilic surface material 23. Lead wires from the temperature sensor 27A are connected to wirings 28A and 29A on the support substrate.
[0050]
In the present embodiment, any shape and processing method may be used for the surface of the heating resistor 11A as long as the hydrophilic surface material 23 is surrounded by the hydrophobic surface material 24. FIG. 4 shows an example of such a shape.
[0051]
4A shows a hydrophilic surface material 23 in the case where an appropriate amount of a viscous silicone-based coating agent is dropped as a hydrophobic surface material 24 along the dotted line B around the heating resistor 11A. This shows the shape of the exposed portion of the. Although the shape of the exposed portion is uneven and distorted due to the surface roughness and inclination of the heating resistor 11A, there is no significant functional problem.
[0052]
FIG. 4B shows a shape in which a plurality of circular hydrophilic surface materials 23 are scattered in the hydrophobic surface material 24. In this shape, the area occupied by each hydrophilic surface is reduced, and the amount of the reaction solution accumulated in the hydrophilic surface material 23 is reduced. Therefore, the temperature of the reaction solution can be rapidly increased and decreased, so that the reaction time Can be shortened.
[0053]
The same effect can be obtained even when the shape when the hydrophilic surface material 23 is dotted is a rectangular shape as shown in FIG.
[0054]
4A and 4B can be formed by a method of masking a portion of the hydrophilic surface material 23 to be exposed with a photoresist or the like and coating a material to be the hydrophobic surface material 24. . Alternatively, a seal made of a fluororesin can be used as the hydrophobic surface material 24, and a hole can be formed in a portion corresponding to the portion where the hydrophilic surface material 23 is exposed, and the seal can be attached.
[0055]
FIG. 5 is a diagram showing the connection between the reactor 1 and the controller device 51. In the figure, reference numerals 13A to 13F denote power supply wirings 21A to 21F and 22A to 22F in FIG. 1, and reference numerals 52A to 52F denote wirings to which lead wires from the temperature sensors 27A to 27F are connected (see FIG. 2). Indicates only wirings 28A and 29A).
[0056]
The six heating resistors 11A to 11F are respectively connected to one ends of power supply wirings 13A to 13F on the support substrate 12 that supplies power for heat generation. In addition, signals from the temperature sensors 27 </ b> A to 27 </ b> F provided in the respective heating resistors are connected to one ends of sensor wires 52 </ b> A to 52 </ b> F on the support substrate 12. The other ends of the power supply wirings 13 </ b> A to 13 </ b> F and the sensor wirings 52 </ b> A to 52 </ b> F are connected to the connector 14 of the support substrate 12, and this connector 14 is connected to a connector 53 for connection to the controller device 51.
[0057]
As shown in the block diagram of FIG. 6, the controller device 51 includes amplifiers 52 </ b> A to 52 </ b> F that are connected to the heating resistors 11 </ b> A to 11 </ b> F and supply power for heating. Each of the amplifiers 52A to 52F is connected to a computer 58 including a CPU, a ROM, and a RAM via interface circuits 54A to 54F and a bus line.
[0058]
The temperature sensors 27A to 27F are connected to the computer 58 via A / D converters 56A to 56F that convert analog signals into digital signals, and bus lines.
[0059]
FIG. 7 is a circuit diagram of the interface portion of the controller device when the temperature gradient of the electrical resistance of the heating resistor is utilized and the heating resistor also serves as a temperature sensor. Since each circuit has the same configuration, only the portion connected to the heating resistor 11F is shown in FIG. As shown in the figure, the heating resistor 11F is connected to a computer 58 via an amplifier 52F, an interface circuit 54F, and a bus line. The heating resistor 11F is connected to the computer 58 via a resistor 61, A / D converters 62F and 63F, and a bus line.
[0060]
The resistor 61 has a resistance value sufficiently lower than the resistance value of the heating resistor 11F. As a result, the voltage across the resistor 61 indicates the current Ir flowing through the heating resistor 11F. This value is converted into digital data by the A / D converter 62F and input to the computer through the bus line. The A / D converter 63F converts the voltage Er applied to the heating resistor 11F into digital data and inputs it to the computer through the bus line. The computer calculates Rr = Er / Ir and calculates the temperature of the heating resistor 11F from the value of Rr.
[0061]
If the material of the heating resistor 11F is composed mainly of a metal, it is known that the electrical resistance decreases with increasing temperature. Therefore, the temperature of the heating resistor 11F can be obtained from the value of Rr. it can. In this case, the sensitivity of temperature measurement can be improved by using platinum, nickel, copper or the like as the main component of the heating resistor. In particular, platinum is a material having excellent temperature measurement stability.
[0062]
According to this circuit, since the heating resistor also serves as the temperature sensor, the temperature sensor can be omitted.
[0063]
Next, the reaction container for reacting the reaction solution will be described. As shown in FIGS. 9 and 10, the reaction vessel 72 is made of a plastic having a heat resistance of 100 ° C. or higher such as polypropylene, and a plurality of substantially rectangular parallelepiped holes opened on the upper surface and the side surface are independently drilled. A main body 74 is provided. A plate-like heat radiating member 75 formed of a material having good thermal conductivity such as aluminum, copper, silver, or the like is attached to a side surface having an opening of the main body 74, that is, the back surface. As a result, a plurality of reaction chambers 71 opened only on the upper surface are formed. The heat dissipating member 75 serves as a heat dissipating mechanism when the reactor is cooled, and a cooling device may be attached to the outer surface of the heat dissipating member 75 for further efficient cooling.
[0064]
Next, with reference to FIG. 12 and FIG. 13, an analysis apparatus for analyzing nucleic acid molecules such as DNA, which is a reaction product, by electrophoresis is described. As shown in the figure, this analyzer is provided with an electrophoresis tank 101, and the cathode 121 and the anode 122 are arranged so as to face each other at positions in the vicinity of both side walls inside the electrophoresis tank 101. . A power source 64 is connected to the cathode 121 and the anode 122 so that an electric field is applied in the direction opposite to the direction of migration of nucleic acids. A gel 102 is accommodated in the electrophoresis tank 101. Since the voltage of the power source 64 during electrophoresis is determined by the length of the nucleic acid to be measured, the size of the electrophoresis tank, and the gel concentration, a variable power source whose voltage can be controlled by a computer is preferable.
[0065]
In addition, a potential measurement electrode 104 is disposed below the portion where the reactor is inserted during analysis of the electrophoresis tank 101. A voltmeter 68 is connected between the potential measuring electrode 104 and the cathode 121, and the potential Ew around the heating resistor of the reactor inserted by the voltmeter 68 is measured. Further, a power source 66 for applying a voltage Eh with reference to the potential of the cathode 121 is connected between the cathode 121 and the reactor 1. The voltage Eh is applied so that the potential of the heating resistor is negative with respect to the surrounding potential Ew, and ranges from Ew to Ew-100 [V] (for example, a range from 20 to -80 [V]). ). If the potential of the heating resistor is set to a negative value larger than the surrounding potential Ew, the electric field distribution in the entire electrophoresis tank becomes non-uniform, and the electrophoresis direction of the nucleic acid may be curved, so Eh is Ew to Ew A range of −20 [V] (for example, a range of 20 to 0 [V]) is a preferable range.
[0066]
In this analyzer, when electrophoresis proceeds and the nucleic acid as a reaction product is sufficiently migrated to a predetermined distance in the gel, the voltage application from the power source and the power supply to the heating resistor are all stopped.
[0067]
In the reactor of FIG. 1, six sets of heating resistors are formed on a supporting substrate, and the shape of the supporting substrate is rectangular as a whole as shown in FIG. The support portion for supporting the projection has a protruding shape. By adopting such a shape, a shape suitable for inserting only the portion of the heating resistor of the reactor 1 into the reaction chamber 71 of the reaction vessel 72 or transferring it from the reaction vessel 72 to the electrophoresis tank 101. It becomes. In addition, by providing a plurality of sets of heating resistors in the same reactor, a plurality of reactions that are the same or different can be performed at a time. Furthermore, since the temperature of the heating resistor can be controlled independently, reactions under different conditions can be performed.
[0068]
Next, a method for replicating the base sequence of the nucleic acid contained in the reaction solution using the above reactor and a method for analyzing the base sequence of the replicated nucleic acid by electrophoresis will be described. First, a reaction comprising a template DNA, an oligonucleotide primer, dNTPs, a heat-resistant DNA polymirase, and a buffer solution prepared at an ion concentration suitable for the reaction, on the exposed portion of the hydrophilic surface material 23 on the surface of the heating resistor to be used. The solution 31 is dropped on a heating resistor that causes the reaction with a pipette or the like. As shown in FIG. 9, the reaction solution 31 adheres to the hydrophilic surface material 23 in the form of water droplets.
[0069]
With the reaction solution attached, each portion of the reactor 1 where the heating resistor is provided is inserted into each of the reaction chambers 71 of the reaction vessel 72. In this case, the surface of the reactor 1 opposite to the surface on which the heat generating resistor is provided is in contact with the heat radiating member 75, that is, the surface to which the reaction solution of the reactor 1 is attached is the arrow in FIG. Insert to face the D direction. At this time, the reaction solution 31 is inserted with care so as not to contact the inner wall surface of the reaction chamber 71.
[0070]
Thereafter, the inside of each reaction chamber 71 is inserted with a portion where a reaction solution is attached to small granular wax having a melting temperature of 45 ° C. or wax 73 melted by heating to 50 ° C. Insert into.
[0071]
Next, the controller device performs temperature control for repeatedly supplying power to the heating resistor to heat the dropped reaction solution and stopping the power supply to the heating resistor to cool the dropped reaction solution. The reaction solution is reacted according to 51.
[0072]
The temperature control by the controller device 51 will be described with reference to FIG. First, a heating resistor whose temperature is to be controlled is designated in advance from a keyboard (not shown). In this case, if all the heating resistors are used for reaction, specify all the heating resistors, and if the temperature control conditions differ for each heating resistor to be temperature controlled, temperature control Specify the conditions at the same time.
[0073]
In step 100, the measured value measured by each temperature sensor provided in the heating resistor to be temperature-controlled is taken, and in step 102, a first set temperature (for example, 94) at the time of heating control according to the temperature control condition. ° C), second set temperature during heating control (eg, 72 ° C.), set temperature during cooling control (eg, 55 ° C.), and set temperature during temperature holding control (eg, 68 ° C.) Is read.
[0074]
The set temperature and the repeated cycle of heating and cooling are stored in advance in a storage device such as a ROM or RAM of the computer 58 and read out according to the specified temperature condition. May be.
[0075]
In the next step 104, electric power is supplied and the temperature of the reaction solution is heated for a predetermined time (for example, 1 minute) based on the measured value measured by each temperature sensor and the first set temperature at the time of heating control. Feedback control is performed so as to coincide with the second set temperature at the time of control. After performing the heating control for a predetermined time, in the next step 106, based on the measured value measured by each temperature sensor and the set temperature at the time of cooling control, the supply of electric power is stopped and the temperature of the reaction solution is set to the predetermined time. Feedback control is performed so as to coincide with the set temperature during cooling control (for example, 4 minutes). In this case, since the surface of the reactor 1 opposite to the surface on which the heating resistor is provided is in contact with the heat radiating member 75, it can be quickly cooled. In addition, when a cooling device is attached to the outer surface of the heat radiating member 75, the cooling control time can be further shortened by simultaneously controlling the cooling device during the cooling control. In step 107, electric power is supplied based on the measured value measured by each temperature sensor and the second set temperature at the time of heating control, and the temperature of the reaction solution is heated for a predetermined time (for example, 1 minute). Feedback control is performed so as to coincide with the second set temperature at the time of control.
That is, the computer sends an electric signal corresponding to the heat generation amount corresponding to the set temperature to the amplifier via the interface circuit according to the profile of the set temperature and time for performing the reaction stored in advance in the storage device, and the amplifier Converts this electrical signal into electric power, heats the heating resistor, and heats the reaction solution. As an electric signal supplied to the amplifier, an analog signal in which the amount of generated power is expressed as a voltage or current, or a digital signal in which the amount of generated power is expressed by the number of pulses, pulse width, or the like can be used. In the case of cooling control, power supply is stopped. This operation is programmed to be controlled independently by a temperature vs. time profile set independently for each heating resistor.
[0076]
In the next step 108, it is determined whether or not the heating control and the cooling control are repeated for a predetermined cycle. If the heating control and the cooling control are not repeated, the above heating control and cooling control are repeated. When cycle heating control and cooling control are repeated, temperature holding control is performed so that the temperature of the reaction solution is maintained at a set temperature for a predetermined time (for example, 7 minutes) by feedback control in step 110, and temperature holding control is performed. This routine is terminated when is finished.
[0077]
As shown in FIG. 10, during the reaction, the wax 73 inserted into the reaction chamber 71 is dissolved to surround the reaction solution 31 attached to the hydrophilic surface material 23 of the reactor 1. Prevent evaporation. In this state, the controller device 51 controls the temperature of the reaction solution 31 according to the temperature versus time profile as described above. After the reaction, the power supply to the heating element is stopped, and the temperature inside the reaction chamber 71 is lowered.
[0078]
FIG. 11 is a view showing a state where the reactor 1 is taken out from the reaction container after the PCR reaction. Since the wax 73 inside the reaction chamber 71 is solidified by lowering the temperature inside the reaction chamber 71, the reaction solution and the heating resistor are surrounded by the reaction solution 31 in each heating resistor portion of the reactor 1. And the analysis sample in which the wax is integrated can be taken out of the reaction vessel. Then, the base sequence of the nucleic acid is analyzed using the analysis sample taken out as a unit.
[0079]
According to the present embodiment, the wax is solidified so as to surround the heat generating resistor, and the reaction solution, the heat generating resistor, and the solidified wax are analyzed integrally, so that the reaction solution is solidified with the wax and the heat generating resistance. It is sealed between the body and does not scatter or mix with other reaction solutions. This makes it possible to easily recover the solution after the reaction and cause contamination with other samples during the recovery operation. Nor.
[0080]
FIG. 12 is a view showing a state in which the reactor 1 is taken out from the reaction vessel 72 and transferred to the horizontal type electrophoresis tank 101. In the state of FIG. 11, the portion of the heating resistor of the reactor 1 is transferred into the electrophoresis tank 101, and the electrophoresis analysis of the reaction product by electrophoresis of the reaction solution 31 is performed.
[0081]
FIG. 13 is a diagram showing the inside of the electrophoresis tank 101. The portion of the reactor 1 where the heating resistor is provided is inserted into each square hole 103 provided in the gel 102. In this state, power is supplied to each heating resistor in a state where the potential of the heating resistor is held at a negative potential lower than the surrounding potential. As a result, the attached wax 73 is melted and peeled off by heat, and floats above the square hole 103. Simultaneously with the rising of the wax, the reaction solution 31 dissolves into the square hole 103.
[0082]
FIG. 14 is a diagram showing the relationship between the voltage applied to the heating resistor of the reactor 1 and the voltage applied to the electrode of the electrophoresis tank. In electrophoresis, nucleic acid molecules such as DNA move in the direction of arrow M because the negative electrode 121 is placed from the cathode 121 toward the anode 122, that is, in the direction corresponding to the back surface of the reactor 1. To do. At this time, in a state where the reactor 1 is not inserted into the electrophoresis tank 101, the potential Ew is measured by the potential measuring electrode 104 with reference to the cathode 121 at the position where the reactor 1 is inserted in the electrophoresis tank.
[0083]
By applying a voltage Eh between the reactor 1 and the cathode 121, each heating resistor is supplied with electric power for melting and peeling the wax, and Ew based on the potential of the cathode 121. A voltage Eh set in a range of ~ Ew-20 [V] is applied. Since the strength of the electric field for migrating nucleic acid molecules by applying voltage to such a heating resistor increases, dissolution of the nucleic acid molecules contained in the reaction solution 31 in the gel 102 can be promoted. The nucleic acid molecule can be dissolved in the gel 102 before the surface of the surface. For this reason, it is not necessary to prevent nucleic acid molecules from floating by increasing the specific gravity of the reaction solution by mixing glycerin, saccharose or the like into the reaction solution, as in the prior art.
[0084]
Subsequent operations are the same as in normal electrophoresis, and the length of DNA, which is a nucleic acid, is measured, or DNA having a desired length is cut out from the gel and collected.
[0085]
【The invention's effect】
As described above, according to the nucleic acid base sequence replication method and nucleic acid base sequence replication apparatus of the present invention, the reaction is performed in a state where the reaction solution is in direct contact with the surface of the heat generating resistor. The thermal resistance between the resistor and the resistor is low, and it is possible to quickly increase and decrease the temperature of the reaction solution and shorten the reaction rate.
[0086]
Moreover, if both the exothermic resistor to which the reaction solution is dropped and the oily material having a specific gravity lighter than that of the reaction solution are put in a container and reacted, an effect that evaporation of the reaction solution can be prevented can be obtained.
[0087]
The surface of the heating resistor is coated with a hydrophilic material and a hydrophobic material surrounding the hydrophilic material to make the reaction solution into water droplets, thereby showing the hydrophilicity of the heating resistor. The effect can be obtained that it can be limitedly held in the processed portion, and scattering of the reaction solution and contamination with other reaction solutions can be prevented.
[0088]
According to the method for producing a nucleic acid base sequence analysis sample of the present invention, an analysis sample in which a reaction solution, a heating resistor and a solidified wax are integrated is manufactured. It is sealed between the body and does not scatter or mix with other reaction solutions. This makes it easy to recover the solution after the reaction, and does not cause contamination with other samples during the recovery operation. The effect that it can be made is acquired.
[0089]
Then, according to the nucleic acid base sequence analysis method and analysis apparatus of the present invention, an analysis sample in which a reaction solution, a heating resistor and a solidified wax are integrated is used, and the solution after the reaction is used using a pipette or the like. Since there is no need to recover or drop the reaction solution onto the gel for electrophoresis, it is possible to easily recover the solution after the reaction and there is no loss of solution. It is done.
[Brief description of the drawings]
FIG. 1 is a perspective view showing a reactor according to an embodiment of the present invention.
FIG. 2 is an enlarged view of a portion of a heating resistor of the reactor.
3 is a cross-sectional view taken along line AA ′ of FIG.
FIG. 4 is a plan view showing another example of the shape of the hydrophilic surface material and the hydrophobic surface material on the surface of the heating resistor.
FIG. 5 is a diagram showing a state of connection between a reactor and a controller device.
FIG. 6 is a block diagram showing details of the controller device.
FIG. 7 is a circuit diagram of an interface portion of a controller device when a heating resistor also serves as a temperature sensor.
FIG. 8 is a flowchart showing a temperature control routine for heating and cooling the heating resistor.
FIG. 9 is a perspective view showing a reaction vessel and a reactor dropped with reaction droplets.
FIG. 10 is a view showing a state in which a reactor is inserted into a reaction vessel to perform a reaction.
FIG. 11 is a view when the reactor is taken out of the reaction vessel after the reaction.
FIG. 12 is a diagram showing how the reactor is transferred from the reaction vessel to a horizontal type electrophoresis tank.
FIG. 13 is a view when the reaction solution is recovered from the reactor inside the electrophoresis tank.
FIG. 14 is a diagram showing the relationship between the voltage applied to the heating resistor and the voltage between the electrodes of the electrophoresis tank.
[Explanation of symbols]
1 reactor
11A-11F Heating resistor
12 Support substrate
14 Reactor connector
23 Hydrophilic surface material
24 Hydrophobic surface material
27A-27F Temperature sensor
51 Controller device
52A to 52F Temperature sensor wiring
53 Controller device connector
72 reaction vessel
101 electrophoresis tank
102 Gel used for electrophoresis
103 Square hole in the gel
104 Potential measurement electrode
121 Electrophoresis bath cathode
122 Anode of electrophoresis tank

Claims (10)

A reaction solution containing nucleic acid is dropped on a heating resistor coated with a hydrophilic material on the surface and a hydrophobic material surrounding the hydrophilic material ,
Repeatedly supplying power to the heating resistor to heat the dropped reaction solution and stopping the power supply to the heating resistor to cool the dropped reaction solution,
A method for replicating a nucleic acid base sequence that replicates the base sequence of a nucleic acid contained in a dropped reaction solution.   The nucleic acid base sequence replication method according to claim 1, wherein both the exothermic resistor to which the reaction solution is dropped and the oily material having a specific gravity lighter than that of the reaction solution are placed in a container and heating and cooling of the dropped reaction solution are repeated. Reaction comprising a heating resistor for dropping a reaction solution coated with a hydrophilic material on the surface and a hydrophobic material surrounding the hydrophilic material and a substrate supporting the heating resistor And
A power supply device for supplying power to the heating resistor of the reactor;
A control device that repeatedly controls the supply of power to the heating resistor and the stop of power supply to the heating resistor;
Nucleic acid base sequence replicating apparatus comprising:   4. The nucleic acid base sequence replicating apparatus according to claim 3, further comprising: a heating resistor into which the reaction solution is dropped and a container for storing an oily material having a lighter specific gravity than the reaction solution.   The nucleic acid base sequence according to claim 3 or 4, further comprising a temperature sensor for measuring the temperature of the reaction solution, wherein the control device controls the temperature of the reaction solution to a predetermined temperature based on the output of the temperature sensor. Replication device.   The nucleic acid base sequence replicating apparatus according to claim 5, wherein the heating resistor also serves as a temperature sensor.   A reaction solution containing nucleic acid is dropped on the heating resistor,
  Put the exothermic resistor and wax with the reaction solution dripped into the container,
  The reaction solution dropped by repeatedly supplying power to the heating resistor to heat the dropped reaction solution and stopping the power supply to the heating resistor and cooling the dropped reaction solution Duplicates the base sequence of the nucleic acid contained in
  Solidify the wax and unify the reaction solution, heating resistor and wax from the container,
  A method for producing a nucleic acid base sequence analysis sample for producing an analysis sample in which a reaction solution for analyzing a nucleic acid base sequence as a reaction product, a heating resistor and a wax are integrated.   The analysis sample produced by the method for producing a nucleic acid base sequence analysis sample of claim 7 is placed in a gel of an electrophoresis tank,
  By supplying electric power to the heating resistor and heating, the wax is peeled from the heating resistor to release the reaction solution into the gel,
  Analyze the nucleotide sequence of nucleic acid, a reaction product contained in the released reaction solution, by electrophoresis
  Nucleic acid base sequence analysis method.   The nucleic acid base sequence analysis method according to claim 8, wherein, when the wax is peeled from the heating resistor, the potential of the heating resistor is maintained at a potential lower than the surrounding potential.   An electrophoresis tank for analyzing the base sequence of a nucleic acid contained in a reaction solution in an analysis sample produced by the method for producing a nucleic acid base sequence analysis sample of claim 7, by electrophoresis;
  A peeling device for peeling the wax from the heating resistor and releasing the reaction solution into the gel by supplying power to the heating resistor and heating;
  A measuring device for measuring the potential around the heating resistor;
  A holding device for holding the potential of the heating resistor at a potential lower than the surrounding potential;
  Nucleic acid base sequence analyzer comprising:

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