JP2006271216A - System for judging presence or absence of nucleic acid having specific nucleic acid sequence and method of the judgement - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an analytical member capable of judging whether there is a nucleic acid having a specific nucleic acid sequence in a specimen or not, quickly and accurately without requiring complex operations, and a method for judging the presence or absence of the nucleic acid having the specific nucleic acid sequence in the specimen by using the analytical member. <P>SOLUTION: This analytical member in which a nucleic acid-amplifying part for performing amplification and a probe part having a porous layer immobilized with a probe for hybridizing a target base sequence are joined by a flow passage is provided. By using the member, the method for judging the presence or absence of the nucleic acid having the specific base sequence in the specimen is performed by flowing a solution containing the specimen, a primer and a DNA polymerase from the nucleic acid-amplifying part to the probe part in this order and detecting the presence or absence of the nucleic acid amplification in the nucleic acid-amplifying part by the hybridization at the probe part. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a system and a determination method for determining whether or not a nucleic acid having a specific base sequence exists in a specimen.

  Many methods for detecting and determining nucleic acids having a specific base sequence in a specimen are known, and examples thereof include a hybridization method. In order to increase the efficiency of hybridization and increase the detection sensitivity, a method is disclosed in which a specimen (nucleic acid) is heated at 70-100 ° C. for 2-10 minutes prior to hybridization and then rapidly cooled to 5 ° C. or less (see FIG. For example, see Patent Document 1). However, when the volume of the sample solution is large, rapid cooling is difficult and a sufficient effect may not be obtained. Further, since the heating and cooling operation takes a long time, it is not suitable for quick determination.

  On the other hand, a method using a microfluidic device is known as a method focusing on rapid hybridization. For example, there is a method in which a hybridizing part in which a DNA probe is fixed is provided in the flow path of a microfluidic device, and the specimen is fed and brought into contact with the hybridizing part for hybridization (see, for example, Patent Document 2). ). Although this method is excellent in rapidity, there are cases in which hybridization efficiency is poor and detection sensitivity is low, and therefore accurate determination is difficult.

Japanese Patent Laid-Open No. 2004-28620 Japanese Patent Laid-Open No. 2002-300877

  The problem to be solved by the present invention is to provide a system and a determination method capable of detecting and determining a nucleic acid having a specific base sequence present in a sample quickly and with high sensitivity without requiring a complicated operation. It is to be.

  As a result of intensive studies, the present inventors inject a specimen containing a nucleic acid into a microfluidic device having a capillary channel and having a probe fixed to a part of the capillary channel. Nucleic acids are thermally denatured while moving inside, then cooled to 10 ° C or lower, and then contacted with an immobilized probe to rapidly and efficiently hybridize nucleic acids having a specific base sequence, resulting in high sensitivity detection. We have found what we can do and have arrived at the present invention.

That is, the present invention includes a microfluidic device (I) having a capillary channel for flowing a solution containing a nucleic acid, and a probe fixed to a part of the capillary channel;
Temperature adjusting means (a) for heating the solution containing the nucleic acid to a temperature at which the nucleic acid is thermally denatured; and temperature adjusting means (b) for cooling the solution containing the nucleic acid to a set temperature of 10 ° C. or lower.
A temperature control device (II) having
The microfluidic device (I), the temperature adjusting means (a) and the temperature so that the nucleic acid flowing in the capillary channel is heated and cooled in the order of the temperature adjusting means (a) and the temperature adjusting means (b). A system for determining the presence or absence of a nucleic acid having a specific base sequence (hereinafter simply referred to as “the system of the present invention”), in which a temperature adjusting device (II) having adjusting means (b) is arranged, is provided.

  The present invention also provides a specific base sequence in a specimen based on a hybridization method using a microfluidic device having a capillary channel and having a probe fixed to a part of the capillary channel. A method for determining the presence or absence of nucleic acid having: a) a step of thermally denaturing nucleic acid by moving a solution containing the nucleic acid in the capillary channel; and b) keeping the heat-denatured nucleic acid at 10 ° C. or lower. A method for determining the presence or absence of a nucleic acid having a specific base sequence (hereinafter, simply referred to as “determination method of the present invention”) comprising a cooling step and c) a step of hybridizing the cooled nucleic acid with a probe in this order. To do.

  According to the system and determination method of the present invention, heat denaturation, cooling, and contact with a probe of a nucleic acid contained in a specimen are performed in a capillary channel of a microfluidic device, so that rapid and highly efficient hybridization is possible. As a result, it is possible to quickly determine the presence or absence of a nucleic acid having a specific base sequence with high sensitivity and accuracy.

(1) System The system of the present invention includes a microfluidic device (I) having a capillary channel for flowing a solution containing a nucleic acid, and a probe fixed to a part of the capillary channel;
Temperature adjusting means (a) for heating the solution containing the nucleic acid to a temperature at which the nucleic acid is thermally denatured; and temperature adjusting means (b) for cooling the solution containing the nucleic acid to a set temperature of 10 ° C. or lower.
A temperature control device (II) having
The microfluidic device (I), the temperature adjusting means (a) and the temperature so that the nucleic acid flowing in the capillary channel is heated and cooled in the order of the temperature adjusting means (a) and the temperature adjusting means (b). A temperature adjustment device (II) having adjustment means (b) is arranged to determine the presence or absence of a nucleic acid having a specific base sequence. This will be described below.

(1.1) Microfluidic device (I)
(1.1.1) Definition of microfluidic device In the present invention, a microfluidic device is a microfluidic device, a microfabricated device, a lab-on-chip, or a micrototal analytical device. -This system is also called a system (μ-TAS). In the flow path, the mechanism in which the fluid undergoes a temperature change, the mechanism in which the concentration is adjusted, the mechanism in which a chemical reaction occurs, the flow velocity of the flow, the branching of the flow, mixing, or separation It is a device having a capillary channel provided with a mechanism for controlling the above or a mechanism for receiving an electrical or optical measurement. The flow path may communicate with the outside of the microfluidic device, or may not communicate with the outside, and other structures in the device, such as a liquid storage tank, a waste liquid absorption unit, a pressure tank, and a vacuum tank Etc. may be provided to contact them.

  The external shape and material of the microfluidic device used in the present invention are arbitrary, as long as the microfluidic device has a capillary channel and a probe is fixed to a part of the capillary channel. The capillary channel may be formed inside a lump or plate-shaped molded product, and a microfluidic device having a capillary channel formed in a thin plate or sheet substrate is preferably used. The substrate may be, for example, a glass, a crystal such as quartz, a semiconductor such as silicon, a ceramic, carbon, an organic polymer (such as polydimethylsiloxane, which contains an inorganic element. Hereinafter, simply referred to as “polymer”. Or a foam thereof. In particular, when a polymer is used as a material, it can be manufactured in large quantities at a low cost, so that it is possible to dispose of each specimen, prevent contamination of other specimens, and easily incinerate, so that contamination from specimens can occur. A polymer is preferred as a material because it can suppress infection and infection.

(1.1.2) Capillary channel The thickness of the capillary channel is not particularly limited as long as the effect of the present invention is not impaired, but the cross-sectional area is 1 × 10 ⁻¹² m ² to 1 × 10. It is preferable to have a capillary channel of ⁻⁶ m ² , preferably 1 × 10 ⁻¹⁰ m ^{2 to} 2.5 × 10 ⁻⁵ m ² . Both the width and height of the cross section of the flow channel are preferably 1 μm to 1000 μm, and more preferably 10 μm to 500 μm. Manufacture is easy when the cross section of the flow path is larger than 10 μm. In addition, when the flow path is smaller than 1000 μm, there are advantages as a microfluidic device, that is, shortening of reaction / analysis time, synthesis / analysis with a small amount of sample, small waste, improvement of temperature control accuracy, etc. Since it is exhibited, it is preferable.

  The width / height ratio of the channel cross section can be arbitrarily set according to the application and purpose, but generally 0.5 to 10 is preferable, and 0.7 to 5 is more preferable. The cross-sectional shape of the channel is arbitrary such as a rectangle (including a rectangle with rounded corners; the same applies hereinafter), a trapezoid, a circle, a semicircle, and a slit. The width of the channel need not be constant.

(1.1.3) Probe part In the microfluidic device used in the present invention, a probe is fixed to at least a part of the inner wall surface of the capillary channel. Since the average distance to the inner wall facing the probe is in the range of 1 to 100 μm, an effective affinity is generated between the probe and the nucleic acid moving in the capillary to which the probe is fixed, so that the probe hybridizes effectively. It becomes possible. The probe is not particularly limited as long as it has a specific affinity for a nucleic acid having a target base sequence. For example, a nucleic acid having a base sequence substantially complementary to the target base sequence is preferably used. The probe may be single-stranded or double-stranded, and is preferably used physically or chemically immobilized on at least a part of the inner wall surface. For fixing, any known and commonly used method can be used. For example, the end of the probe is modified with an amino group or the like, and a porous layer having an aldehyde group or the like is provided on at least a part of the inner wall surface. When chemically bonded, the probe is firmly bonded to the porous layer and can be fixed to the inner wall surface.

  When the immobilized probe is a single-stranded nucleic acid, it can be used as it is. In the case of a double-stranded nucleic acid, it is preferably used after heat-denaturing to make a single-stranded nucleic acid.

  The porous layer means a layer having a large number of pores that communicate with the surface and open to the surface, that is, communicating pores. The shape of the pores is arbitrary, and may be, for example, a three-dimensional network shape (sponge shape), an aggregated particle shape, a well shape, or a fiber-like shape of a nonwoven fabric or a woven fabric. The porous layer may be formed on the entire cross section of the flow path or may be formed on a part thereof. In particular, when the cross section of the flow path is square, it is preferable that the flow path of the porous layer is only one surface that is easy to manufacture as long as a sufficient amount of probe fixation is obtained with the porous layer of only one surface. The thickness of the porous layer is preferably 0.5 μm to 30 μm, more preferably 1 μm to 20 μm, and most preferably 2 μm to 10 μm. By setting it to 0.5 μm or more, a sufficient amount of probes can be fixed to the inner surface of the flow path, and by setting it to 30 μm or less, the time required for separation and analysis can be shortened. Although the pore diameter of the pores of the porous layer is also arbitrary, it is preferably 0.05 μm to 3 μm, more preferably 0.1 μm to 1 μm. By setting the thickness to 0.05 μm or more, a probe that hybridizes a sufficient amount of the target base sequence can be immobilized. In addition, the said pore diameter is a hole diameter of the pore which exists in large quantities, and is not necessarily necessarily an average diameter.

(1.1.4) Nucleic Acid Amplification Portion The microfluidic device used in the present invention may have a portion having means for amplifying nucleic acid in a specimen (hereinafter simply referred to as “nucleic acid amplification portion”), It is used as a more preferred form. Details of this aspect will be described later.

(1.2) Temperature control device (II)
(1.2.1) Temperature adjusting means (a) for heating to a temperature at which nucleic acid is thermally denatured
The temperature adjusting device (II) used in the system of the present invention has at least one temperature adjusting means (a) for heating the solution containing the nucleic acid flowing in the capillary channel to a temperature at which the nucleic acid is thermally denatured. The temperature adjusting means (a) is arbitrary. For example, even if a microfluidic device itself is provided with a heater to heat a predetermined portion, the microfluidic device is brought into contact with a temperature-controlled heating apparatus to heat the predetermined portion. You may do it. As an example of a temperature-adjusted heating device, a heat storage body made of metal or ceramic is heated with a heater, and if it is lower than the set temperature with a temperature controller, it is heated when it is higher than the set temperature. An apparatus that adjusts to a certain temperature by cooling or the like may be used. The temperature at which the nucleic acid is thermally denatured may vary depending on the type of nucleic acid to be detected, for example, the length of the nucleic acid and the GC content, but it is usually 90 to 96 ° C. The nucleic acid in the sample is denatured from a double-stranded nucleic acid to a single-stranded nucleic acid by being heated to the heat denaturation temperature by the temperature adjusting means (a).

(1.2.2) Temperature adjusting means for cooling to a set temperature of 10 ° C. or lower (b)
The temperature adjusting device (II) used in the system of the present invention has at least one temperature adjusting means (b) for cooling a solution containing a nucleic acid flowing in the capillary channel to an arbitrary set temperature of 10 ° C. or lower. . The temperature adjusting means (b) is provided in the downstream portion of the capillary channel with respect to the temperature adjusting means (a). The temperature adjusting means (b) is optional. For example, even if a microfluidic device itself is provided with a cooling device to cool a predetermined portion, the microfluidic device is brought into contact with the temperature-controlled cooling device to cool the predetermined portion. You may do it. As an example of such a cooling device, a heat storage body made of metal or ceramic is cooled with a refrigerant or a Peltier element, and when it is higher than a set temperature by a temperature controller, it is cooled when it is lower than a set temperature. May be a device that adjusts to a certain temperature by heating or the like. It is considered that the nucleic acid in the specimen is kept in a single-strand dissociation state by being rapidly denatured by the temperature adjusting means (b) after heat denaturation. The temperature adjusting means (b) is preferably 10 ° C. or lower so that the nucleic acid can maintain a single-strand dissociation state, and any temperature of −10 ° C. to 10 ° C. so that the liquid can flow without freezing in the capillary. More preferably.

(1.2.3) Temperature adjusting means of probe section (c)
The temperature adjusting device (II) used in the system of the present invention preferably includes temperature adjusting means (c) that can adjust the temperature of the probe section. The temperature adjusting means (c) is optional. For example, even if the microfluidic device itself is provided with a heating or cooling device to adjust the temperature of the probe part, the microfluidic device is added to the temperature adjusting device adjusted to an arbitrary temperature. May be contacted to adjust the temperature of the probe portion. As an example of a temperature adjustment device that has been temperature adjusted, a heat storage body made of metal or ceramic is heated by a heater, and if it is higher than the set temperature by the temperature controller, it is cooled, and conversely if it is lower than the set temperature May be a device that can be maintained at a constant temperature by heating or the like. The temperature of the probe varies depending on the method of use. When nucleic acids are hybridized to the probe, it is desirable that substantially non-complementary nucleic acids, primers, by-products, etc. are not hybridized, and the probe chain length and GC content. For example, it is preferable to maintain the temperature within the range of 45 to 68 ° C.

  When the nucleic acid hybridized to the probe is dissociated, it is preferable that the temperature adjusting means (c) has a function of increasing the temperature continuously or stepwise as necessary. In this case, the temperature may be increased from the hybridized temperature to the Tm value of the probe or more, and at a constant rate, for example, a temperature increase rate of 5 to 20 ° C. per minute, the Tm value or more, for example, 60 to 96 ° C. The temperature may be raised to.

(1.2.4) Temperature adjusting means for nucleic acid amplification (d)
Furthermore, the temperature control device (II) used in the system of the present invention may be provided with a means for amplifying nucleic acid, and a series of detection operations can be simplified, which is more preferable as an embodiment of the present invention. A method for amplifying a nucleic acid is arbitrary, and for example, a PCR method (polymerase chain reaction method) can be used. When a desired nucleic acid is amplified in the capillary channel of the microfluidic device based on the PCR method, the temperature adjusting device (II) used in the system of the present invention is capable of adjusting the heat denaturation temperature, annealing temperature, and extension reaction temperature of the nucleic acid. It is preferable to have temperature adjusting means (d) for holding each. More specifically, a capillary channel (hereinafter referred to as a “nucleic acid amplification part”) that provides a reaction field for nucleic acid amplification is provided in a part of the microfluidic device, and the temperature of the entire nucleic acid amplification part is required for PCR. The method of repeatedly controlling the denaturation, annealing, and elongation reaction temperature, and the nucleic acid amplification part are provided with three temperature zones, namely, denaturation, annealing, and elongation reaction temperature, and a solution containing nucleic acid is passed through those temperature zones. The so-called flow-through PCR method can be mentioned. In the former case, a certain amount of time is required to reach the target temperature, and in addition, in the latter case, the temperature is set in advance. Therefore, the time required for temperature increase / decrease is not required, and the time required for PCR is greatly reduced, which is a more preferable mode.

  The temperature adjusting means (d) may be incorporated in the microfluidic device itself to control the temperature of the nucleic acid amplification part, or may not be incorporated in the microfluidic device itself and may be placed on the device surface corresponding to the nucleic acid amplification part. The temperature adjusting means (d) may be arranged so as to be in contact with each other to control the temperature of the nucleic acid amplification part.

  For example, as a method of providing three temperature zones of denaturation, annealing, and extension reaction temperature in the nucleic acid amplification part, three external heaters having three different temperatures are placed in parallel so that they are parallel to the plane constituted by the external heaters. For example, there may be mentioned a method in which a single capillary channel is bent several times so as to reciprocate on the heater, and is brought into contact as a nucleic acid amplification part.

  The temperature adjustment means (a) for heating to a temperature at which the nucleic acid is thermally denatured and the temperature adjustment means (d) for maintaining the denaturation temperature used in the nucleic acid amplification means such as the PCR method may be common.

(1.3) Nucleic Acid Detection Unit The system of the present invention includes a nucleic acid detection unit as necessary in order to determine the presence or absence of a nucleic acid hybridized to a probe as part of its configuration. In the system of the present invention, when the presence or absence of the nucleic acid hybridized to the probe can be visually detected, it is not necessary to have a special nucleic acid detection means. It has conventionally known nucleic acid detection means such as a plate reader, a fluorometer, an absorptiometer, an ionization chamber, a GM counter, and a scintillation detector.

(1.4) Transfer Means The system of the present invention preferably has a transfer means for transferring the specimen in the capillary channel. The transfer means is arbitrary, and it is preferable that the specimen can be transferred at a constant speed. For example, use of a pump capable of feeding a small amount of liquid such as a microsyringe pump is preferably used. In this case, it may be injected from the injection port of the microfluidic device, or may be sucked from the discharge port. Of course, injection and suction may be performed simultaneously.

(1.5) Arrangement The system of the present invention is arranged so that the nucleic acid flowing in the capillary channel is heated and cooled in the order of the temperature adjusting means (a) and the temperature adjusting means (b). I) and a temperature adjusting device (II) having a temperature adjusting means (a) and a temperature adjusting means (b) are arranged. Under the present circumstances, it passes the inside of a capillary channel on the temperature control apparatus which has the temperature adjustment means (a) which heats to the temperature which nucleic acid denatures, and the temperature adjustment means (b) which cools to the preset temperature of 10 degrees C or less. The microfluidic device is preferably arranged so that the specimen is heated and cooled in the order of the temperature adjusting means (a) and the temperature adjusting means (b).

  In addition, when the temperature adjusting device (II) used in the system of the present invention further has a temperature adjusting means (c), the system of the present invention is configured such that the nucleic acid flowing in the capillary channel is a temperature adjusting means ( a), the microfluidic device (I), the temperature adjusting means (a), the temperature adjusting means (b), and the temperature adjustment so that they are heated and cooled in the order of the temperature adjusting means (b) and the temperature adjusting means (c). A temperature control device (II) having means (c) is arranged. At this time, the specimen passing through the capillary channel on the temperature adjusting device (II) having the temperature adjusting means (a), the temperature adjusting means (b), and the temperature adjusting means (c) is temperature adjusting means (a). The microfluidic device is preferably arranged so that it is heated and cooled in the order of the temperature adjusting means (b) and the temperature adjusting means (c). In that case, a probe part is arrange | positioned so that a temperature control means (c) may be touched. Nucleic acid detection so that a nucleic acid having a specific base sequence can be detected in a hybridized state with the probe part, or alternatively, the nucleic acid once hybridized can be dissociated from the probe part and detected in the downstream part. Means (3) is installed as necessary.

For example, when a microfluidic device having a nucleic acid amplification portion is used as a more preferable form, the system of the present invention includes, for example, a temperature adjustment means (d), a temperature adjustment means (a), a temperature adjustment means ( b) and on the temperature adjustment device (II) having the temperature adjustment means (c), the specimen passing through the capillary channel is a nucleic acid amplification means, temperature adjustment means (a), temperature adjustment means (b), temperature adjustment The microfluidic device is arranged to be heated or cooled in the order of means (c). Other arrangements and configurations are the same as described above.
Further, a positioning device for fixing the microfluidic device (I) and the temperature adjusting device (II) at a predetermined position may be provided for the purpose of assisting the above arrangement.

(1.6) Method of Use The sample liquid is injected from the injection port into the microfluidic device of the system arranged as described above. As a method for transferring the injected specimen liquid, for example, a syringe pump or the like is connected to the injection port and the liquid is sent toward the discharge port. The flow rate of the liquid can be adjusted as appropriate depending on the width and depth of the flow path and the temperature history that you want to receive, but if it is too slow, problems such as evaporation and diffusion will occur, and if it is too fast, the problem will be that temperature adjustment will not be successful. From this, it is preferable to set in the range of 0.1 to 100 μl / min.

  The nucleic acid in the sample liquid (amplified in the process of passing through the nucleic acid amplification part in the system having the nucleic acid amplification part) becomes a single strand when passing over the temperature adjusting means (a) that is heated to a temperature at which the nucleic acid is thermally denatured. By dissociating and then passing over the temperature adjusting means (b), the single-stranded state is maintained. When passing through the probe portion, only the nucleic acid having a sequence substantially complementary to the probe hybridizes.

  Subsequent to the sample solution, a buffer solution may be sent for the purpose of washing the non-specifically adsorbed nucleic acid on the probe part. Examples of the buffer solution include SSC.

  The hybridized nucleic acid can be detected visually or by a nucleic acid detection means as necessary. The detection can also be performed by observing the probe part or by observing the downstream part through which the probe part dissociated by thermal denaturation passes. Thereby, it can be determined whether or not a nucleic acid having a specific base sequence exists in the sample liquid.

(2) Determination method The determination method of the present invention uses a microfluidic device having a capillary channel, and a probe is fixed to a part of the capillary channel, in a sample based on the hybridization method. A method for determining the presence or absence of a nucleic acid having a specific base sequence of: a) a step of thermally denaturing a nucleic acid by moving a solution containing the nucleic acid in the capillary channel; and b) heat denatured. A step of cooling the nucleic acid to 10 ° C. or lower, and c) a step of hybridizing the cooled nucleic acid with the probe in this order. Details will be described below.

(2.1) Hybridization Method Using Microfluidic Device (2.1.1) Sample In the determination method of the present invention, first, a solution containing a sample is introduced into the capillary channel of the microfluidic device.
Samples include nucleic acids and nucleic acid-containing samples, whole blood, serum, stool, urine and other living organisms, or animal, plant, virus-derived cell tissues, cell cultures, and beverages General foods including processed foods such as foods and canned foods. A sample obtained by previously amplifying a nucleic acid or a sample containing the nucleic acid by a nucleic acid amplification method can also be used as a specimen. For example, when a sample contains a small amount of nucleic acid or the amount of nucleic acid contained in the sample is unknown, the nucleic acid can be amplified in advance using a thermal cycler or the like and used as the sample.

(2.1.2) Amplification of nucleic acid using a microfluidic device When a nucleic acid in a specimen is amplified, if a microfluidic device having a nucleic acid amplification part is used, amplification to detection can be performed continuously. This is more preferable because the risk of contamination due to complicated operations and sample movement is reduced.

When amplifying nucleic acid with a microfluidic device, for example, a solution containing a sample, a primer, and a DNA polymerase is injected from the injection port of the microfluidic device, and sent by a syringe pump or the like, so that the nucleic acid amplification part in the capillary channel is removed. Just let it pass. In the process of passing, for example, flow-through PCR is performed, and the nucleic acid in the specimen is amplified.
In the case of flow-through PCR, for example, the metal blocks whose temperatures are adjusted to around 90 ° C., around 72 ° C., around 55 ° C. are arranged side by side, and a device having a channel designed to repeatedly pass through these is in close contact, What is necessary is just to inject | pour the solution containing a test substance, a primer, and DNA polymerase from an injection port, and to send.

  The DNA polymerase used in the present invention is arbitrary, but preferably has thermal stability.

The primer used in the present invention is arbitrary, and a primer prepared based on known knowledge can be used according to the base sequence to be amplified. The primer may be labeled, and usable labels include fluorescent substances, radioisotopes, biotin, enzymes, luminophores, antigens and antibodies. More specifically, FITC, Cy3, Cy5, TET, Texas Red, etc. as fluorescent substances, ³² P, ¹⁴ C, etc. as radioactive isotopes, alkaline phosphatase, peroxidase, etc. as enzymes, Examples include ruthenium.

  The nucleic acid amplification reaction solution may contain a metal salt such as deoxyribonucleoside triphosphate and magnesium chloride, and a buffer such as Tris.

(2.1.3) Heat denaturation of nucleic acid (a)
The determination method of the present invention includes a step of allowing a sample solution containing nucleic acid to be introduced into the capillary channel of the microfluidic device and then passing through the heated channel portion to a temperature at which the nucleic acid is thermally denatured while being fed. . As a result, the nucleic acid in the specimen is thermally denatured to become a single strand. The heat denaturation temperature of a nucleic acid can vary depending on the type of nucleic acid to be determined, for example, the length of the nucleic acid and the GC content, but is usually 90 to 96 ° C.

(2.1.4) Step (b) of cooling the heat-denatured nucleic acid to 10 ° C. or lower
Furthermore, the determination method of the present invention includes a step of passing the cooled channel portion while feeding the heat-denatured single-stranded nucleic acid. This rapid cooling operation suppresses the return to the double strand and maintains the single strand state. The cooling temperature should just be 10 degrees C or less, and it is preferable that it is arbitrary temperature of -10-10 degreeC so that a liquid can flow without freezing in a capillary tube.

(2.1.5) Step of hybridizing cooled nucleic acid with probe (c)
In the determination method of the present invention, the nucleic acid heat-denatured in the step (a) and cooled in the subsequent step (b) is transferred to the probe portion and hybridized with the probe by contacting with the probe fixed to the inner wall of the capillary tube. Process. The temperature condition for hybridization may vary depending on the length of nucleic acid, GC content, etc., but is preferably set to an optimum temperature in the range of 45 to 68 ° C., for example.

(2.2) Method for determining presence / absence of nucleic acid by hybridization detection The presence / absence of a nucleic acid having a specific base sequence in a sample is determined based on the detection result of the nucleic acid hybridized to the probe. If a hybridized nucleic acid is detected, it is determined that a nucleic acid having a specific base sequence is present in the sample.
As a method for detecting the hybridized nucleic acid, there are a method of directly observing the hybridized probe portion and a method of dissociating the hybridized nucleic acid by heating and observing it in the downstream portion in the flow path. The detection means is arbitrary. For example, when the nucleic acid is fluorescently labeled, the fluorescence intensity may be measured by a fluorescence microscope or the like. For example, when the nucleic acid is labeled with a radioisotope, a GM counter or a scintillation counter is used. What is necessary is just to measure by. Further, for example, when the nucleic acid is not labeled, a fluorescent intercalator or the like is allowed to act on the nucleic acid that is hybridized with the probe, and the fluorescence intensity may be measured. In any of the method means, in order to reduce background and noise, it is preferable to wash the hybridized portion with a buffer solution or the like prior to detection after hybridization.

(2.3) Mechanism of action of cooling effect Nucleic acid that can be hybridized to the probe is a single-stranded nucleic acid, and a heat denaturation step is taken to increase the ratio. When it gradually decreases, it will return to the original double strand even after annealing with the complementary strand. Therefore, in order to efficiently hybridize with the probe, it is necessary to contact the probe in a single strand state before annealing with the complementary strand. When a microfluidic device is used, the dissociated single-stranded nucleic acid reaches the probe part in a relatively short time and can come into contact with the probe, but the rate at which the single-stranded nucleic acid anneals with the complementary strand is considerably high. Fast and efficient hybridization is difficult using microfluidic devices.

  The reason why the nucleic acid can be efficiently hybridized by providing a cooling part between the heat-denatured part of the nucleic acid on the microfluidic device and the probe part is not necessarily clear, but the microfluidic device is excellent in heat transfer This is presumably because it can be rapidly cooled and kept in a dissociated state in a very short time. The reason why the dissociated state can be maintained is also not clear, but the three-dimensional structure of the nucleic acid changes due to cooling, and the complementary strands are difficult to anneal, or the nucleic acid anneals nonspecifically. By doing so, it is presumed that complementary strands cannot be annealed. In the latter case, many non-specifically annealed ones are considered to have a binding region shorter than the probe chain length. In this case, they are dissociated by the heat of the probe part to become single-stranded nucleic acids and hybridize to the probe. It becomes easy to soy.

  Also, the nucleic acid heat denaturing part and the probe on the microfluidic device are often set at a relatively close distance in order to make the device lighter and smaller. In particular, if the temperature adjustment means of the probe section needs to be strictly temperature controlled, but there is no cooling section between the heat-denaturing section and the probe section, it is closer to the probe section than the probe section. Since there is a heat-denatured part having a temperature as high as 30 to 60 ° C. or more, the probe part receives disturbance due to the inflow of heat from the heat-denatured part, and precise control becomes difficult. On the other hand, when there is a cooling body between the heat denaturing part and the probe part, the heat of the heat denaturing part flows to the cooling part, and the cooling part plays a role as a buffer, so It is considered that the fact that temperature control is possible is one of the reasons for good hybridization.

  From the above, the system and determination method of the present invention perform rapid denaturation, cooling, and contact with a probe in a capillary channel of a microfluidic device, so that rapid and highly efficient hybridization can be achieved. Soybean can be produced, and as a result, the presence or absence of a nucleic acid having a specific base sequence can be determined quickly, with high sensitivity and accurately. In addition, since a series of operations can be performed in the microfluidic device, a complicated operation is not required, and not only a detection operation can be easily performed, but also the sample can be reliably transported to the probe portion, so that the sample sample Prevention of contamination (contamination) associated with the movement of the device, determination mistakes, sample mistakes, infection to the operator, and human error can be prevented in advance, which is particularly effective for clinical use.

(3) Applications The system and detection method of the present invention can be used for various applications and purposes such as gene expression analysis and DNA diagnosis in the medical field, pharmaceutical field, agricultural field and the like. For example, in the field of agriculture, elucidation of the relationship between a base sequence and a phenotype for the purpose of cultivar identification or species improvement is mentioned, and in the pharmacy field, prediction of patient drug sensitivity is mentioned. In the medical field, analysis of infectious disease-causing microorganisms, typing of disease-related genes, diagnosis of cancer, compatibility analysis of living transplantation, and the like can be mentioned. In addition, it can be used for legal judgment such as single person determination, parent-child determination, gender determination, and use in archaeological research.

  EXAMPLES Hereinafter, although this invention is demonstrated in more detail using an Example, this invention is not limited to the range of a following example. Ultraviolet irradiation and fluorescence characteristic measurement in this example were performed by the following methods.

(Irradiation with ultraviolet lamp 1)
Using a UE031-353CHC type UV irradiation device manufactured by Eye Graphics Co., Ltd. using a 3000 W metal halide lamp as a light source, ultraviolet rays having an ultraviolet intensity at 365 nm of 40 mW / cm ² were irradiated in a nitrogen atmosphere at room temperature unless otherwise specified.

(Irradiation with ultraviolet lamp 2)
Using a light source unit for multi-light 250W series exposure apparatus manufactured by USHIO INC., Which uses a 250W high-pressure mercury lamp as a light source, ultraviolet light with an ultraviolet intensity at 365 nm of 50 mW / cm ² is used in a nitrogen atmosphere at room temperature unless otherwise specified. Irradiated.

(Fluorescence intensity measurement method)
Measurement was performed using a confocal laser microscope TCS-NT manufactured by Leica Corporation. The measurement conditions are PMT sensitivity 450V and pinhole 1.00.

Example 1
[Preparation of energy ray curable composition (i)]
72 parts by mass of trifunctional urethane acrylate oligomer having an average molecular weight of 2000 (“Unidic V-4263” manufactured by Dainippon Ink and Chemicals, Inc.), dicyclopentanyl diacrylate (“R-684” manufactured by Nippon Kayaku Co., Ltd.) ) 18 parts by mass, 10 parts by mass of glycidyl methacrylate (manufactured by Wako Pure Chemical Industries, Ltd.), 150 parts by mass of methyl decanoate (manufactured by Wako Pure Chemical Industries, Ltd.), 10 parts by mass of acetone as a volatile good solvent, A composition (i) was prepared by uniformly mixing 3 parts by mass of 1-hydroxycyclohexyl phenyl ketone (“Irgacure 184” manufactured by Ciba Geigy Co., Ltd.) as an ultraviolet polymerization initiator.

[Preparation of energy beam curable composition (ii)]
As an active energy ray crosslinkable polymerizable compound, 80 parts by mass of “Unidic V-4263” and 20 parts by mass of 1,6-hexanediol diacrylate (“New Frontier HDDA” manufactured by Daiichi Kogyo Seiyaku Co., Ltd.), A composition (ii) was prepared by uniformly mixing 5 parts by mass of “Irgacure 184” as a photopolymerization initiator.

[Preparation of energy beam curable composition (iii)]
As an active energy ray crosslinkable polymerizable compound, “Unidic V-4263” is 60 parts by mass, “New Frontier HDDA” is 40 parts by mass, “Irgacure 184” is 5 parts by mass as a photopolymerization initiator, and 2, 2, Composition (iii) was prepared by uniformly mixing 0.5 parts by mass of 4-diphenyl-4-methyl-1-pentene (manufactured by Kanto Chemical Co., Inc.).

[Formation of a recess (flow path) having a porous layer at the bottom]
The composition (ii) was applied to a 7.5 cm × 2.5 cm × 1 mm plate made of polyacrylate (“Acrylite L 000” manufactured by Mitsubishi Rayon Co., Ltd.) and irradiated with ultraviolet rays from the ultraviolet lamp 1 for 3 seconds. . The composition (i) is applied onto the semi-cured coating film, and the composition (i) is cured by irradiating the composition (i) with ultraviolet rays for 40 seconds using an ultraviolet lamp 1, and the composition (i) is cured with n-hexane. Methyl decanoate was washed away to form a porous coating film.

  The porous coating film is impregnated with the composition (iii) to form an uncured coating film of the composition (iii), and irradiated with ultraviolet rays from the ultraviolet lamp 2 through a predetermined photomask for 120 seconds. A semi-cured coating film of the product (iii) is formed, and the uncured composition (iii) in the non-irradiated portion is removed with ethanol to form a coating film in which the porous coating film is partially present on the support. did. The composition (iii) is applied onto the coating film, an uncured coating film of the composition (iii) is formed, and the composition is subjected to ultraviolet irradiation with an ultraviolet lamp 2 through a predetermined photomask for 120 seconds. The semi-cured coating film of (iii) is formed, the uncured portion of the uncured composition (iii) is removed with ethanol, and a concave coating film in which the porous coating film is exposed on a part of the bottom surface is obtained. Formed on a support.

[Fixing the probe to the porous coating]
A 10% polyallylamine solution (PAA-10C manufactured by Nittobo) was diluted with pure water to prepare a 5% by mass polyallylamine aqueous solution. Next, the support having a recess produced in the above step is brought into contact with the 5% by mass polyallylamine aqueous solution and reacted at 50 ° C. for 2 hours, whereby some amino groups in the polyallylamine are made porous. It was made to react with the epoxy group in a coating film. Then, the amino group was introduce | transduced into the partial porous coating film for probe fixation which hybridizes this target base sequence by wash | cleaning for 30 minutes with running water.

  25% by mass glutaraldehyde solution (25% glutaraldehyde solution manufactured by Wako Pure Chemical Industries, Ltd.) in phosphate buffer (“phosphate buffer powder” manufactured by Wako Pure Chemical Industries, dissolved in 1 liter of sterile water) Was diluted 5 times to prepare a 5 mass% glutaraldehyde aqueous solution. Next, the support having a concave portion into which an amino group has been introduced in the above step is placed in the 5% by mass glutaraldehyde aqueous solution, reacted at 50 ° C. for 2 hours, and introduced into the porous coating film. All amino groups were reacted with one aldehyde group in the glutaraldehyde. Thereafter, the membrane was washed with running water for 5 minutes, and an aldehyde group was introduced into the probe-imparted partially porous coating film that hybridizes the target base sequence.

  1 μl of nucleic acid A (500 μmol / l; SEQ ID NO: 1; nucleic acid A) modified with an amino group at the 5 ′ end in a PCR sample tube, and nucleic acid B complementary to nucleic acid A (500 μmol / l; SEQ ID NO: 2; nucleic acid) 1 μl of B), 1 μl of 10 × buffer solution for PCR (10 × Ex Taq Buffer manufactured by Takara Shuzo Co., Ltd.) and 7 μl of sterilized water were added. Using a thermal cycler, “95 ° C., 2 minutes → 80 ° C., 2 minutes → 70 ° C., 2 minutes → 60 ° C., 2 minutes → 50 ° C., 2 minutes → 40 ° C., 2 minutes → 30 ° C., 2 minutes → By applying a temperature change of “4 ° C., 2 minutes”, nucleic acids A and B were annealed to form double-stranded nucleic acid, and a nucleic acid solution (C) was prepared.

  5 μl of the nucleic acid solution (C) is dropped on the porous coating film into which the aldehyde group has been introduced in the above step and incubated at 100% humidity and 50 ° C. for 15 hours, and the terminal amino group of the nucleic acid is converted into the aldehyde of the porous layer. Covalently bonded to the group. Thereafter, the porous coating film is immersed in a 0.2% by mass sodium tetrahydroborate aqueous solution, subjected to a reduction reaction for 5 minutes, and then rinsed with a 0.2 × SSC / 0.1% SDS solution. After rinsing with 2 × SSC, the double-stranded nucleic acid (C) was immobilized on the partially porous coating film for probe immobilization.

  A 10 × TBE buffer solution (a solution of “10 × TBE powder” manufactured by Wako Pure Chemical Industries, Ltd.) dissolved in sterilized water was multiplied by 100 with sterilized water to prepare a 0.1 × TBE buffer solution. Next, the support having a concave portion into which the nucleic acid solution (C) has been introduced in the above step is immersed in the 0.1 × TBE buffer, boiled at 95 ° C. for 5 minutes, and a partially porous coating film for probe fixation. The nucleic acid (B) was dissociated from the nucleic acid (C) immobilized on the substrate to obtain a support having a recess in which only the nucleic acid (A) was immobilized on the partial porous coating film for probe immobilization.

[Fixing the lid]
A surface of the composition (ii) subjected to corona discharge treatment of a 15 cm × 5 cm × 30 μm polypropylene film (“biaxially stretched polypropylene film“ Taiko ”FOR No. 30” manufactured by Nimura Chemical Co., Ltd.) as a temporary support. The coating was carried out using a 128 μm applicator. The uncured coating film of the composition (ii) is irradiated with ultraviolet rays by an ultraviolet lamp 1 for 3 seconds to form a semi-cured coating film of the composition, which is bonded to the recesses produced in the above process, and the ultraviolet lamp 1 After being completely cured by irradiation with ultraviolet rays for 40 seconds, the polypropylene film as the temporary support was peeled off to form a flow path with the porous layer exposed on the bottom surface. Thereafter, the composition (i) is applied onto the coating film that has been completely cured by laminating on the recesses in the above-mentioned step, and a coating film is formed thereon. Further, the upper substrate is made of the same polyacrylate as the lower substrate. A 7.5 cm x 2.5 cm x 1 mm plate ("Acrylite L 000" manufactured by Mitsubishi Rayon Co., Ltd.) is layered, and 50 mW / cm ² ultraviolet rays are irradiated on the entire surface of the coating film in a nitrogen atmosphere for 40 seconds. The coating was cured.

  Next, holes with a diameter of 1 mm that pass through the upper substrate and communicate with the flow path are formed at both ends of the flow path by using a drill, and a polyvinyl chloride tube with an inner diameter of 3 mm and a height of 5 mm is formed in each of these two holes. The microfluidic device (1 ′) shown in FIGS. 2 and 3 was manufactured by bonding to form an inlet (12) and an outlet (13).

[PCR (thermal cycler)]
Genetack (manufactured by Nippon Gene Co., Ltd.) was used as the DNA polymerase, and the PCR buffer solution and dNTP mixed solution attached to the enzyme were used. In a sample tube for PCR of 200 μl volume, 5 μl of 10 × PCR buffer, 4 μl of dNTP mixture (dATP, dGTP, dCTP, dTTP each 2.5 mmol / l), 2.5 μl of primer 1 (FITC labeled, 10 μmol) / L; SEQ ID NO: 3; primer 1), 2.5 μl of primer 2 (10 μmol / l; SEQ ID NO: 4; primer 2), 0.25 μl of gene tack (5 units / μl), 0.5 μl of ras as template Mutant Set c-Ki-ras codon 12 Gly (DNA 1 ng; manufactured by Takara Bio Inc .; SEQ ID NO: 5) was added, and further sterilized water was added to make 50 μl. Moreover, what used the plasmid pUC119 solution (DNA1ng) instead of ras Mutant Set c-Ki-ras codon12 Gly was also prepared. These were set in a thermal cycler, and nucleic acid was amplified by performing a cycle of “94 ° C., 30 seconds → 55 ° C., 30 seconds → 72 ° C. 10 seconds” 30 times. These PCR products were subjected to the following tests as sample solutions.

[Hybridization]
The microfluidic device (1 ′) was fixed on a temperature adjusting device composed of three metal blocks shown in FIG. The metal blocks (A) and (21) were set at 90 ° C, the metal blocks (B) and (22) at 1 ° C, and the metal blocks (C) and (23) at 55 ° C. 20 μl of each sample solution prepared above was injected into the injection port (12) of the microfluidic device (1 ′), and a micro syringe pump (IC3200 manufactured by kd Scientific) was further connected to the injection port (12). The liquid was sent toward the discharge port (13) in minutes, and then the liquid was switched to 0.2 × SSC. The nucleic acid in the sample liquid is denatured and single-stranded by passing it over the metal block (A) (21) in the process of liquid feeding, and single-stranded by passing over the metal block (B) (22). The state was maintained, and the nucleic acid subsequently single-stranded on the metal block (C) (23) was hybridized with the probe.

[Detection of nucleic acid]
The probe portion (9 ′) was observed with a confocal laser microscope TCS-NT manufactured by Leica Corporation to detect the hybridized nucleic acid. In the test group using ras Mutant Set c-Ki-ras codon 12 Gly as a template, the fluorescence intensity of the probe portion (9 ′) greatly increased about 3 minutes after the sample liquid injection, and the fluorescence intensity at that time was 200. there were. On the other hand, the fluorescence intensity in the test section using pUC119 was 0, and the difference in fluorescence intensity between the two was large. As a result, base sequence-specific detection of a nucleic acid having a specific base sequence in a sample, that is, whether or not a nucleic acid having a specific base sequence is contained in the sample can be determined quickly and accurately.

(Example 2)
Using a 12 cm × 3 cm × 1 mm plate made of polyacrylate (Mitsubishi Rayon “Acrylite L 000”) as the substrate and changing the photomask during exposure, the same operation as in Example 1, The microfluidic device (1 ″) shown in FIGS. 5 and 6 was obtained.

[Flow-through PCR]
The microfluidic device (1 ″) was fixed on a temperature control device consisting of five metal blocks shown in FIG. Metal block (A) (31) is 90 ° C, Metal block (B) (32) is 72 ° C, Metal block (C) (33) is 55 ° C, Metal block (D) (34) is 1 ° C, Metal block (E) (35) was set to 50 ° C.

  Genetack (manufactured by Nippon Gene Co., Ltd.) was used as the DNA polymerase, and the PCR buffer solution and dNTP mixed solution attached to the enzyme were used. In a 200 μl volume of PCR sample tube, 5 μl of 10 × PCR buffer, 4 μl of dNTP mixture (dATP, dGTP, dCTP, dTTP each 2.5 mmol / l), 2.5 μl of primer 1 (FITC labeled, 10 μmol) / L; SEQ ID NO: 3; primer 1), 2.5 μl of primer 2 (10 μmol / l; SEQ ID NO: 4; primer 2), 0.25 μl of gene tack (5 units / μl), 0.5 μl of ras as template Mutant Set c-Ki-ras codon 12 Gly (DNA 1 ng; manufactured by Takara Bio Inc .; SEQ ID NO: 5) 10 μl of 25% Tween 80 aqueous solution and 5 μl of glycerol were added, and sterilized water was added to make 50 μl. Moreover, what used the plasmid pUC119 solution (DNA1ng) instead of ras Mutant Set c-Ki-ras codon12 Gly was also prepared. These were used for the following tests as specimen solutions.

  20 μl of each sample solution is injected into the injection port (12 ′) of the microfluidic device (20), and a micro syringe pump (IC3200 manufactured by kd Scientific) is connected to the injection port (12 ′) at a flow rate of 5 μl / min. Liquid was fed toward the discharge port (13 ″). Subsequently, it switched to 0.2xSSC and liquid-fed. As a result, the sample liquid becomes “metal block (A) (31) → (B) (32) → (C) (33) → (B) (32) → (A) (31) → (B) (32). → (C) (33)... The nucleic acid elongation reaction is performed on (B) (32) when moving from (C) (33) to (B) (32).

[Hybridization]
After passing through the nucleic acid amplification (flow-through PCR) section, the sample liquid passes over the metal blocks (A) (31) → (D) (34), (E) (35) and is discharged (13 ″) Discharged from. On the metal block (A) (31), the nucleic acid is denatured to be single-stranded, and the single-stranded state is maintained on the metal block (D) (34). On the subsequent metal block (E) (35) Hybridized with probe.

[Detection of nucleic acid]
By observing the probe portion (9 ″) with a confocal laser microscope TCS-NT manufactured by Leica Corporation, the hybridized nucleic acid was detected. In the test group using ras Mutant Set c-Ki-ras codon 12 Gly as a template, the fluorescence intensity on the surface of the probe portion (9 ″) greatly increased about 20 minutes after the injection of the sample liquid. 170. On the other hand, the fluorescence intensity in the test section using pUC119 was 0, and the difference in fluorescence intensity between the two was large. As a result, base sequence-specific detection of a nucleic acid having a specific base sequence in a sample, that is, whether or not a nucleic acid having a specific base sequence is contained in the sample can be determined quickly and accurately.

(Comparative Example 1)
In Example 1, detection was performed in the same manner as in Example 1 except that the temperature adjusting means (b) shown in FIG.

  In the test group using ras Mutant Set c-Ki-ras codon 12 Gly as a template, the fluorescence intensity of the probe portion (9 ″) slightly increased about 3 minutes after the sample liquid injection. The fluorescence intensity at that time was 30. On the other hand, the fluorescence intensity in the test section using pUC119 was 0, and the difference in fluorescence intensity between the two was slight. Therefore, it has been difficult to perform base sequence-specific detection of a nucleic acid having a specific base sequence in a sample, that is, to determine whether or not a nucleic acid having a specific base sequence is contained in the sample.

(Comparative Example 2)
As the temperature adjusting device, a temperature adjusting device comprising four metal blocks shown in FIG. 8 is used. The metal block (A) (41) is 90 ° C., the metal block (B) (42) is 72 ° C., and the metal block (C). Detection was carried out in the same manner using the microfluidic device obtained in the same manner as in Example 2 except that (43) was fixed at 55 ° C. and the metal block (D) (44) was set at 50 ° C. .

  In the test group using ras Mutant Set c-Ki-ras codon 12 Gly as a template, the fluorescence intensity of the probe portion (9 ″) slightly increased about 20 minutes after the sample liquid injection. The fluorescence intensity at that time was 30. On the other hand, the fluorescence intensity in the test section using pUC119 was 0, and the difference in fluorescence intensity between the two was slight. Therefore, it has been difficult to perform base sequence-specific detection of a nucleic acid having a specific base sequence in a sample, that is, to determine whether or not a nucleic acid having a specific base sequence is contained in the sample.

It is a schematic diagram of the system of this invention. 2 is a plan view of a schematic diagram of a microfluidic device used in Example 1 and Comparative Example 1. FIG. It is a side view of the schematic diagram of the microfluidic device used in Example 1 and Comparative Example 1. It is a top view of the schematic diagram of the system which has the microfluidic device and temperature control apparatus which were used in Example 1 and Comparative Example 1. FIG. It is a top view of the schematic diagram of the microfluidic device used in Example 2 and Comparative Example 2. It is a side view of the schematic diagram of the microfluidic device used in Example 2 and Comparative Example 2. It is a top view of the schematic diagram of the system which has the microfluidic device and temperature control apparatus which were used in Example 2. FIG. It is a top view of the schematic diagram of the system which has the microfluidic device and temperature control apparatus which were used in the comparative example 2.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Microfluidic device, 1 '... Microfluidic device used in Example 1, 1''... Microfluidic device used in Example 2 and the comparative example 2 ... Temperature adjusting device, 3 ... Temperature adjusting means (a), DESCRIPTION OF SYMBOLS 4 ... Temperature adjustment means (b), 5 ... Temperature adjustment means (c), 6 ... Pump, 7 ... Waste liquid, 8 ... Temperature controller 9 ... Probe part, 9 '... Probe of the microfluidic device used in Example 1 Part,
DESCRIPTION OF SYMBOLS 10 ... Nucleic acid detection means 11 ... Capillary channel, 11 '... Capillary channel of the microfluidic device used in Example 1 and Comparative Example 1, 11 "... Microfluidic used in Example 2 and Comparative Example 2 Capillary channel of device, 12... Microfluidic device inlet used in Example 1 and Comparative Example 1, 12 ′... Microfluidic device inlet used in Example 2 and Comparative Example 2, 13. 1 and the discharge port of the microfluidic device used in Comparative Example 1, 13 '... the discharge port of the microfluidic device used in Example 2 and Comparative Example 2 ... Metal block (A), 22 ... Metal block (B), 23 ... Metal block (C),
24 ... Device installation base, 25 ... Device positioning support 31 ... Metal block (A), 32 ... Metal block (B), 33 ... Metal block (C),
34 ... Metal block (D), 35 ... Metal block (E)
41 ... Metal block (A), 42 ... Metal block (B), 43 ... Metal block (C),
44 ... Metal block (D).

Claims (10)

A microfluidic device (I) having a capillary channel for flowing a solution containing a nucleic acid, and a probe fixed to a part of the capillary channel;
Temperature adjusting means (a) for heating the solution containing the nucleic acid to a temperature at which the nucleic acid is thermally denatured; and temperature adjusting means (b) for cooling the solution containing the nucleic acid to a set temperature of 10 ° C. or lower.
A temperature control device (II) having
The microfluidic device (I), the temperature adjusting means (a) and the temperature so that the nucleic acid flowing in the capillary channel is heated and cooled in the order of the temperature adjusting means (a) and the temperature adjusting means (b). A system for determining the presence or absence of a nucleic acid having a specific base sequence, wherein a temperature adjusting device (II) having an adjusting means (b) is arranged.   Furthermore, the system which determines the presence or absence of the nucleic acid which has a specific base sequence of Claim 1 which has a nucleic acid detection means (III).   The temperature adjusting device (II) further comprises temperature adjusting means (c) for heating or cooling the capillary channel in order to heat or cool the probe fixed in the capillary channel. Or the system which determines the presence or absence of the nucleic acid which has the specific base sequence of 2.   The temperature adjusting device (II) further maintains the solution containing the nucleic acid at a heat denaturation temperature, an annealing temperature, and an extension reaction temperature in order to amplify a desired nucleic acid in the capillary channel based on the PCR method. The system for determining the presence or absence of a nucleic acid having a specific base sequence according to any one of claims 1 to 3, further comprising temperature adjusting means (d) for heating or cooling the capillary channel. .   Using a microfluidic device that has a capillary channel and a probe is fixed to a part of the capillary channel, the presence or absence of a nucleic acid having a specific base sequence is determined based on the hybridization method A) a step of thermally denaturing the nucleic acid by moving a solution containing the nucleic acid in the capillary channel, b) a step of cooling the heat-denatured nucleic acid to 10 ° C. or lower, c) A method for determining the presence or absence of a nucleic acid having a specific base sequence, comprising the step of hybridizing a cooled nucleic acid with a probe in this order.   A method for determining the presence or absence of a nucleic acid having a specific base sequence according to claim 5 using the system according to claim 1.   The method for determining the presence or absence of a nucleic acid having a specific base sequence according to claim 5, wherein the solution is a solution containing a nucleic acid obtained by a nucleic acid amplification reaction.   The method for determining the presence or absence of a nucleic acid having a specific base sequence according to claim 7, wherein the nucleic acid amplification reaction is a PCR method.   The method for determining the presence or absence of a nucleic acid having a specific base sequence according to claim 8, wherein a desired nucleic acid is amplified based on a PCR method in a capillary channel of a microfluidic device using the system according to claim 4. 6. The method for determining the presence or absence of a nucleic acid having a specific base sequence according to claim 5, wherein the detection is performed while changing the probe temperature continuously or stepwise using the system according to claim 3.

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