JP2003325199A - Method of assaying nucleic acid and chip for detecting nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To simultaneously amplify two or more kinds of specific nucleic acids in one reaction system without accompanying complex operation such as divided injection of samples and reagents, and detect and assay the amplified nucleic acid without taking out the amplified nucleic acid from the container. <P>SOLUTION: The nucleic acids are amplified and assayed by a method comprising an amplification step of carrying out a polymerase chain reaction of at least two kinds of the specific nucleic acids immobilized at prescribed positions on a detection chip on which one of a primer pair for amplifying the specific nucleic acids by using the one immobilized primer and the other nonimmobilized primer, and a detection step of optically or electrically detecting the nucleic acid amplified on the detection chip. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a nucleic acid analysis method and a nucleic acid detection chip utilizing the polymerase chain reaction.

[0002]

2. Description of the Related Art A chain reaction (Polymerase Chain R) of DNA synthesis by a commonly used DNA polymerase
eaction; hereinafter referred to as "PCR") is a set of complementary sequences to a nucleobase sequence (hereinafter referred to as "specific nucleic acid") of a target gene or gene region (hereinafter referred to as "target gene") to be replicated and amplified. It is performed in a liquid phase using a synthetic oligonucleotide primer (hereinafter referred to as "primer"). Therefore, if you want to duplicate and amplify multiple target genes and determine the success or failure of the amplification reaction for each target gene, prepare reaction reagents and reaction vessels that contain as many primer sets as the number of target genes, and prepare individual reaction vessels. Therefore, it was necessary to dispense reagents and samples. Furthermore, PC for each target gene
In order to determine the success or failure of R amplification, it is necessary to take out a sample from each container and subject each to an analysis operation such as electrophoresis. When the number of target genes to be investigated is large, the operation was extremely complicated. In addition, the amount of reagents and the like to be used also increases in proportion to the number of target genes, which causes an increase in cost.

In order to reduce these burdens, a method in which a microplate capable of simultaneously handling a large number of samples is directly used as a container for PCR reaction, a ready-made electrophoretic gel for facilitating electrophoretic analysis after the reaction, or a microplate Capillary electrophoresis devices have been put into practical use, and the amount of reagents used for individual target genes has been reduced. However, these techniques have not been improved in that they require the handling of a large number of liquid samples, and the labor saving depends solely on the liquid handling robot device, which makes a series of equipment complicated and expensive. Invited.

Also, in recent years, P has been incorporated with a mechanism for irradiating excitation light into the reaction vessel during PCR amplification reaction to detect fluorescence.
A method for observing the progress of the amplification reaction in a reaction vessel was developed by using a temperature controller for CR and a fluorescent dye or reaction reagent devised so that the fluorescence intensity increases with the amplification of the gene by PCR. (Special table 200
0-512138). According to this method, it is not necessary to take out the sample solution from the reaction container after PCR and perform an analysis operation such as electrophoresis, and by using dyes having different fluorescence characteristics for each target gene, a plurality of target genes can be prepared. It makes it possible to carry out the amplification reaction in one reaction vessel.

However, in the method using the fluorescent dye as described above, the kind of the fluorescent dye that does not interfere with the PCR reaction even when added to the reagent of the PCR reaction and is capable of discriminating the fluorescent characteristics is limited. When a plurality of primers are mixed and a plurality of target genes are replicated and amplified, there is a disadvantage that an unexpected replication reaction may occur due to the interaction between the primers and the gene fragments to be amplified. In fact, the number of target genes that can carry out the amplification reaction in one reaction container is limited to about four.

[0006] In addition, an attempt is made to integrate the PCR reaction container, the temperature control mechanism, and the post-PCR analysis electrophoretic device on a minute chip to automate the operation and minimize the amount of reagents mainly in universities in the United States. Can be seen. However, these chips require complicated technologies under development such as a minute valve mechanism, and basically one chip corresponds to one target gene. Therefore, there is no suitable method for processing many genes. It can't be.

On the other hand, for the purpose of simultaneously amplifying a plurality of genes, both sets of primers corresponding to one gene are immobilized on a solid phase, and the amount of DNA bound only to the immobilization site is determined. A quantitative PCR method for measurement has been proposed (JP 2001-299346 A). However, in this method, since both primers are immobilized on the solid phase, a curved DNA duplex is formed during the progress of PCR. Therefore, the length of the gene strand to be amplified needs to be several thousand bases. There is. This is not realistic because it is difficult to set the PCR reaction conditions including the design of the primer and the efficiency and reliability of the actual PCR reaction decrease. Further, in this method, in order to detect the DNA double strand amplified by the PCR reaction, it is necessary to recover the DNA and subject it to the detection step.

[0008]

DISCLOSURE OF THE INVENTION The present invention is capable of simultaneously amplifying plural kinds of specific nucleic acids in one reaction system without complicated operations such as dispensing of samples and reagents, and the amplified nucleic acid from a container. It is an object of the present invention to provide a nucleic acid analysis method and a nucleic acid detection chip capable of detecting and analyzing amplified nucleic acid without taking out the nucleic acid.

[0009]

In order to achieve the above-mentioned object, the nucleic acid analysis method of the present invention provides a method in which one of primer sets for amplifying a specific nucleic acid is immobilized at a predetermined position on a detection chip. An amplification step of performing a polymerase chain reaction on the nucleic acid using the one primer immobilized and the other primer not immobilized, and the nucleic acid amplified on the detection chip Or a detection step of electrically detecting. According to this method, preferably at least two primer sets for amplifying a specific nucleic acid are prepared, and one primer from each primer set is immobilized at a predetermined different position on the detection chip to obtain the specific nucleic acid. Preferably, PCR is performed on at least two kinds of specific nucleic acids, and in the detection step, the amplified nucleic acid amplified at the corresponding position on the detection chip is directly detected optically or electrically. Therefore, it is possible to eliminate complicated work such as dispensing reagents to a plurality of reaction vessels, and to detect a specific nucleic acid without taking out amplified nucleic acid from the reaction vessel,
Moreover, a plurality of specific nucleic acids can be simultaneously and directly detected on one detection chip. Here, the detection step may be performed simultaneously with the amplification step. According to this method, the amplified nucleic acid is simultaneously detected while the specific nucleic acid is being amplified, so that the amount of the nucleic acid amplified in one amplification cycle can be measured in real time.

Further, the nucleic acid detection chip of the present invention comprises an amplification site for amplifying, by a polymerase chain reaction, a specific nucleic acid having one of a primer set for amplifying the specific nucleic acid immobilized at a predetermined position on the detection chip. And a detection site comprising a mechanism or a part of the mechanism capable of optically or electrically detecting the nucleic acid amplified by the amplification site. According to the present invention, the detection chip can have an amplification site to which only one of the primer sets is immobilized, preferably at least two or more. Therefore, a plurality of specific nucleic acids amplified on the measurement chip can be measured. It can be detected directly on the chip optically or electrically.

The measuring chip may also have a heating mechanism inside. In this case, the heating mechanism provided in the detection chip allows the temperature operation when performing PCR to be carried out easily and directly on the detection chip. Further, in addition to the heating mechanism, the measuring chip can also have a temperature measuring mechanism inside thereof. In this case, PCR
It is possible to accurately control the temperature change of the chip necessary for carrying out. The temperature information obtained from the temperature measuring mechanism can also be used to improve the measurement accuracy when detecting amplified nucleic acid, which will be described later. Here, the detection chip can be a measurement chip for measuring surface plasmon. According to this, since the specific nucleic acid is amplified on the measuring chip for measuring the surface plasmon, the amplified nucleic acid can be directly detected by the surface plasmon measurement. In addition, the amplified nucleic acid can be detected as a change in the resonance frequency by setting the amplification site of the specific nucleic acid on the detection chip as a vibrator having a unique resonance frequency. In addition, the amplified nucleic acid can be electrochemically detected by using the amplification site of the specific nucleic acid on the detection chip as an electrode.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below with reference to the accompanying drawings. In the drawings, the same reference numerals indicate the same or corresponding parts. FIG. 1 shows a detection chip 10 of the present invention. Here, an example is shown in which the detection chip 10 is configured by stacking and integrating a measurement plate 12 and a plurality of reaction plates 20.

The measurement plate 12 corresponds to the measurement surface of the measurement chip for plasmon measurement, and as shown in FIG. 2, a metal thin film 16 of gold, silver, or the like is formed on a glass or plastic optical substrate (for example, diffraction). On the surface of a parallel substrate provided with a lattice, or a prism, or a cylindrical lens, or a parallel substrate that is premised to be used in combination with a prism or a cylindrical lens, for example, an ion plating method, a sputtering method, a vapor deposition method, or the like is well known. It is configured by forming a film by using the method described above. Here, as the thin film, not only a metal but also a semiconductor thin film made of, for example, silicon can be used. In the present invention, the measurement chip for measuring surface plasmon resonance means measuring the change in the resonance angle of the surface plasmon resonance (hereinafter referred to as plasmon measurement) to measure the change in the substance density of the target surface. A measuring chip that can be used in a surface plasmon measuring device that detects and measures the presence and amount.

On the metal thin film 16, a plurality of DNA amplification sites 18 for amplifying and detecting the test DNA are arranged on the detection chip at predetermined intervals, five in this embodiment, in a line. The DNA amplification site 18 is a site to which a primer for amplifying a specific DNA is fixed,
50 μm to 3 mm in diameter, preferably 200 μm in diameter
It is a circular region of 1 mm to 1 mm, or an elliptical or polygonal region having a size corresponding thereto. The respective DNA amplification sites are arranged at intervals of 100 μm to 10 mm, preferably 300 μ to 2 mm between the positions of their centers of gravity. Further, although the respective DNA amplification sites are arranged in a line in the present embodiment, the arrangement of the DNA amplification sites in the present invention is not limited to this.

A primer for amplifying a specific DNA is fixed to each DNA amplification site 18. This primer is only one of the primer sets required for carrying out PCR, and the other primer is not fixed. The nucleic acid analysis method described later is carried out using this primer. The primers fixed to the DNA amplification site 18 are prepared by the number of specific DNAs to be amplified.
It can be changed for each NA amplification site 18.

For the immobilization of the primer, a known method for immobilizing the oligonucleotide can be used. For example,
It is a method that utilizes the covalent bond of an organic substance having a sulfur atom to a metal surface such as gold via a sulfur atom.In this case, a solution of a primer having a thiol group introduced at the end of the oligonucleotide during oligonucleotide synthesis Can be dropped on the metal foil film of the washed DNA detection chip. The number of molecules of the primer immobilized on each of the DNA amplification sites 18 is generally P
It only needs to be used for CR reaction, for example, 5 pmo
It is l. The primer may be synthesized on the DNA amplification site 18. In this case, for example, DNA synthesis by a solid phase phosphoamidite method using an ink jet device having a nozzle having a position control function capable of dropping a reagent only to a portion serving as a predetermined DNA detection site is performed. Applicable. That is, the nucleotide at the 3'end of the oligonucleotide serving as a primer is dropped and fixed. Then, following this, DN by the phosphoramidite method
A reaction reagent relating to A synthesis and a protected nucleotide for constituting a primer are sequentially dropped by an inkjet device at a predetermined DNA detection site to synthesize a primer.

The reaction plate 20 comprises a sample plate 22 arranged on the side of the measurement plate 12 and surrounding the sample, and an upper plate 26 for sealing the upper surface of the sample plate 22,
It is also possible to configure these as an integral molded product or processed product.

A sample slot 24 is formed in the sample plate 22. This sample slot 24 is used for the measurement plate 1
A plurality of D's arranged on the measurement plate 12 at positions facing the DNA amplification site 18 when they are superposed on each other.
The NA amplification site 18 is arranged in the sample slot 24.

The upper plate 26 is provided with holes 28 for loading or unloading the sample. Each hole 28 of the upper plate 26
Are arranged at positions which are both ends in the longitudinal direction of the sample slot 24 when they are superposed on the sample plate 22. In addition, although the hole 28 is opened in a column shape in the present embodiment, any shape may be used as long as the sample slot 24 can be filled with the sample when it is superposed on the sample plate 22. In addition, the hole 28 is used as the sample slot 24.
The sample may be introduced from both holes 28 to cause convection in the sample slot 24, or the sample may be introduced from one hole 28 and discharged from the other. Good. Further, although the sample slot 24 has an oblong shape in the present embodiment, it is not limited to the oblong shape as long as it can surround all the measurement sites on the measurement plate 12.

The upper plate 26 and the sample plate 22 which constitute the detection chip are made of a material or a thin plate that has a small effect on heat conduction, and the temperature of the detection chip can be controlled from the outside. As an example of temperature control, the case where a temperature-adjustable heat conductive block 30 is placed is shown. At this time,
The heat conductive block 30 is arranged on the upper surface of the sample slot 24. Measurement plate 12 (optical substrate 14), sample plate 22
And the upper plate 26 is superposed by adhesion with an adhesive or an adhesive tape, self-adhesion by using the sample plate 22 as an elastomer material, ultrasonic welding, heat sealing, or the like.
This may be done by any of the known methods used in the art. Further, the sample plate 22 and the upper plate 26 may be formed as an integral molded product or processed product instead of overlapping.

As a result, when the optical substrate 14, the sample plate 22 and the upper plate 26 are superposed and integrated, the plurality of DNA amplification sites 18 formed on the metal thin film 16 are arranged in the sample slot 24. To be done. One hole 2 of the upper plate 26
When a sample such as a reagent is introduced from 8, the sample slot 24
The inside is filled with the sample and all the DNA amplification sites 18 come into contact with this supplied sample. When the heat conductive block 30 is placed on the upper plate 26, the upper plate 26 and the sample plate 22
The heat from the heat conductive block 30 is transmitted to the sample, and the sample in the sample slot 24 can be adjusted to a predetermined temperature.

Next, the amplified nucleic acid analysis method will be described. In the amplified nucleic acid analysis method of the present invention, the DNA amplification site 1
Only one of the primer sets for amplifying the specific DNA is immobilized on each of the eight. PCR is performed at each of the DNA amplification sites 18 of the detection chip 10 by the immobilized primer, the other free primer for amplifying the specific DNA, the target DNA sequence, and the solution containing the reagents necessary for the PCR reaction. The reaction is carried out. The reagents necessary for the PCR reaction are existing or PCR
It may include everything that seems possible to carry out the reaction.

Here, the non-immobilized primer is provided to the DNA amplification site 18 in order to carry out the PCR reaction. As a providing method, it may be introduced through the hole 28 of the upper plate 26, or The sample slot 2 is previously embedded as an easily soluble solid salt or an embedded body in an easily soluble gel-like substance.
It may be attached on the measuring plate 12 which is inside 4. Examples of the easily soluble solid salt used here include sodium salts of oligonucleotides, and examples of the easily soluble gel-like substance include gelatin and oligosaccharides. When it is attached to the measurement plate 12, it is preferable to attach it to the vicinity of the DNA amplification site 18 on which the corresponding immobilized primer is immobilized. By thus providing the primer integrally with the detection chip, it is possible to save the labor of adding it as a reagent at the time of analysis.

The DNA double strand of the target gene, which is generally extended and generated by PCR, is generally about several hundred base pairs. This is because if there is a length of several hundred bases, this P
This is because it is possible to sufficiently observe the CR product by separating it from the primer and other reaction reagents, and the extension reaction by the PCR reaction can be performed with sufficiently high efficiency and reliability.

When carrying out PCR, the temperature of the heat conductive block 30 is raised or cooled to a predetermined temperature, and the temperature in the sample slot 24 is set to the temperature required for carrying out PCR. Along with this temperature change, elongation of DNA by the polymerase occurs.

As shown in FIG. 3, the target DNA as a template is associated with the immobilized primer, and the DNA chain of the immobilized primer is extended by the polymerase. In the next denaturation step, the target DNA used as a template under predetermined conditions
Is an extended DNA that is continuously extended to an immobilized primer
Separate from the chains. At this time, the extended chain remains fixed to the DNA amplification unit 18 by the immobilized primer. In the annealing step, the free primer binds to the extended DNA strand in the fixed state in the DNA amplification section 18, and the extended DN
DNA is replicated according to the A chain. On the other hand, the target DNA used as a template is associated with another immobilized primer,
Repeat the extension of NA. As described above, in the present invention, since only one of the primers is fixed to the DNA amplification site 18 and the other primer is in a free state, a DNA chain having a length of several hundred base pairs minimizes the influence of steric hindrance. It can be extended directly on the detection chip 10 as long as possible.

In this PCR step, the plurality of DNA amplification sites 18 provided on the detection chip 10 are connected to the sample slot 2
Since all of them are arranged at a predetermined interval so as to be within 4, it is possible to spread the introduced reagents almost simultaneously at a plurality of DNA amplification sites 18 by introducing the reagent once.
Therefore, it is not necessary to add the same reagent to each DNA amplification site 18. It should be noted that the charging and discharging of the reagent can be easily performed using the hole 28 of the upper plate 26.

In addition, D based on the fixed primer
Since the NA chain extends, even when a plurality of types of nucleic acids are amplified in the sample slot 24 based on a plurality of types of primers, the fixed position of the primers can be identified to facilitate the identification of the nucleic acids after amplification. Become. Therefore, the number of specific nucleic acid species that can be amplified by one detection chip 10 is not substantially limited, and it is not necessary to discriminate amplified nucleic acids by type after amplification.

When the DNA is extended at the DNA amplification site 18, the extension of the DNA double strand causes an increase in density near the DNA amplification site 18. At this time, by performing plasmon measurement, D existing at the DNA amplification site 18
The presence and amount of NA is detected. Since the DNA detection based on this plasmon measurement can be performed for each DNA amplification site 18, it is possible to simultaneously detect a plurality of types of DNA.

As a result, a single detection chip 10 is used to amplify a plurality of types of DNA without complicated operations such as adding a reagent or the like for each DNA type, and the amplified DN is amplified.
It can be detected directly and simultaneously without taking out A.

In this embodiment, DNA amplified by PCR is used.
Detection of the amplified DNA by surface plasmon resonance is performed after the completion of a series of PCR steps.
Since the mass change is measured from the outside of the CR reaction system by an optical method, the reaction can be observed in real time during the PCR reaction.

FIG. 4 shows another detection chip 40 capable of observing the DNA amplified during PCR in real time. A heater 42 for heating and a conductor 44 for temperature measurement are provided between the two holes 28 of the upper plate 26 of the detection chip 40. When the upper plate 26 and the reaction plate 20 are integrated, the heating heater 42 and the temperature measuring conductor 44 are
It is arranged at a position above the sample slot 24. The heater 42 for heating and the conductor 44 for temperature measurement are connected to an adjusting mechanism (not shown), and the heater 42 for heating can be turned on and off based on the temperature sensed by the conductor 44 for temperature measurement.

When the amplified nucleic acid analysis is carried out by the detection chip 40, the heater 42 for heating is adjusted to adjust the temperature in the sample slot 24 and double-stranded DNA is formed by PCR. Since the plasmon measurement measures the density of the measurement site, the measured value is strongly affected by the density change due to the thermal expansion due to the temperature change. Therefore, it is necessary to measure under a constant temperature condition, but the temperature change of the detection chip 40 with the PCR cycle is continuously measured by the temperature-measuring conductor 44, and the plasmon detection chip specifies the temperature in the predetermined PCR cycle. By performing the plasmon measurement at a predetermined temperature in the step, for example, at the moment when the temperature reaches a specific temperature on the way from the annealing reaction to the extension reaction, the plasmon measurement value under a constant temperature condition can be continuously accumulated. It will be possible.

This makes it possible to obtain at least one plasmon measurement value per PCR cycle,
The detection of amplification in real time with the PCR cycle can be directly performed. Further, since the heater 42 for heating and the conductor 44 for temperature measurement are arranged on the upper plate 26,
It is possible to detect in real time without increasing the number of plates constituting the chip.

The heater 42 for heating and the conductor 44 for temperature measurement may be arranged separately. FIG. 5 shows a heater 4 for heating.
A modification in which the 2 and the temperature measuring conductor 44 are arranged on different plates is shown. The detection chip 50 is provided with a temperature measuring plate 52 between the sample plate 22 and the upper plate 26, and these constitute the reaction plate 20. This temperature measuring plate 52
A temperature measuring conductor 44 is arranged on the upper side. By disposing the heater 42 for heating and the conductor 44 for temperature measurement on different plates in this way, the temperature in the sample slot 24 can be measured more accurately, and the DNA amplified at the DNA amplification site 18 Plasmon can be measured more accurately.

In the present embodiment, the amplified DNA was detected by the plasmon measurement, but the method for detecting the amplified DNA is not limited to the surface plasmon measurement. For example, the detection chip can be a resonance frequency measurement chip. In this case, a primer is immobilized on an oscillator having a unique resonance frequency, and a mass change caused by extension of DNA immobilized by PCR is detected by using the change in resonance frequency to detect amplified DNA. To do.

Here, as a method of detecting a change in the resonance frequency, a frequency that maximizes the vibration of the vibrator due to resonance is added by sweeping a mechanical vibration wave including the resonance frequency of the vibrator from the outside. There is a method of observing the vibration state of with a laser Doppler sensor, or a method of making an oscillator with a material having a piezo effect and observing it with an amplitude of a potential generated by attaching an electrode thereto. As another method, an oscillator is made of a material having a piezo effect, an electrode is attached to this, an electronic oscillation circuit is added to this, and the oscillator is electrically oscillated at the resonance frequency. The method of measuring can also be mentioned.

Also by these methods, as described above, 1
By using one detection chip, a plurality of types of DNA can be amplified without complicated operations such as adding a reagent or the like for each DNA type, and can be directly and simultaneously detected without taking out the amplified DNA. Further, since it is detected by the change of the resonance frequency, it can be easily converted into an electric signal, and the possibility of realizing a smaller and cheaper device can be expected.

In the present embodiment, detection of amplified DNA is performed by surface plasmon measurement, but electrochemical detection can be used instead. In this case, the amplification site of the specific nucleic acid on the detection chip, that is, the site where each primer is fixed is used as the electrode. And
A gene amplification reaction is performed by adding a drug having an intercalator function to double-stranded DNA such as bisbenzimide H33258 (Hoechst 33258) to the amplification reaction reagent, and improving the electric conductivity of the double-stranded DNA by intercalation. carry out. Alternatively, a drug having an intercalator function such as bisbenzimide H33258 is added after the DNA amplification reaction. When a double strand is formed by the progress of the amplification reaction, the intercalator is incorporated into the gap between the double strands to increase the electrical conductivity between the electrode at the amplification site and the solution. By measuring this change for each electrode to which each primer is immobilized, the success or failure of amplification of each sequence can be determined and the amplification amount can be known. In this method, DNA can be detected by an electric signal, so that it is possible to expect the possibility of realizing a smaller and cheaper device. In this embodiment, the amplified DN
Although A is detected by surface plasmon measurement,
In addition to or instead of this, a fluorescent dye can be used for detection or quantification of double-stranded DNA. In this case, the unfixed primer is labeled with a fluorescent dye in advance and the gene amplification reaction is carried out. When a double strand is formed by the progress of the amplification reaction, the fluorescent dye is incorporated in the amplification step. Then, the free fluorescent dye is removed. By this series of steps, the double strand becomes fluorescent, so that the fluorescence on the detection chip after the amplification reaction is observed, and the primer immobilized at the position where the fluorescence is emitted is specified. This makes it possible to determine the success or failure of amplification of each sequence and to measure the amount of amplification.

Further, the amplified DNA has a function of intercalator for double-stranded DNA such as ethidium bromide and SYBR green, and is reacted with a fluorescent dye which improves the quantum yield by intercalation. You can also Also by this, the amplified DNA can be detected by detecting the fluorescent dye in the same manner as described above.

Although the five DNA amplification sites 18 are arranged in a line in the present embodiment, the present invention is not limited to this. If the immobilized primer is specified, it may be provided at 5 or more positions, or 5 or less positions, or may not be arranged in a line. In addition, in the present embodiment, the sample slot 24
Are arranged so as to correspond to the DNA amplification sites 18 arranged in a line, and two holes 28 are provided to the sample slot 24.
However, the present invention is not limited to this. For example, multiple columns of D
When the NA amplification site 18 is provided, the sample slots 24 may be provided in the number corresponding to each row, or one sample slot 24 may be provided for a plurality of rows. At this time, one hole 28 for performing both charging and discharging may be provided for each sample slot 24, and three or more holes 28 may be provided.
May be provided for one sample slot 24. Further, two holes 28 may be provided for a combination of sample slots 24. An example of each is shown in FIGS.

The nucleic acid analysis method of the present invention can be used not only for detecting the presence and amount of DNA to be amplified in a sample as described above, but also for detecting the presence or absence of single nucleotide mutation. In this case, for example, a mutagenic primer can be used in which the 3'end or within 3 bases from the 3'end is matched with the 1-base mutation part of the target gene.
When PCR is performed using this mutant primer, only the gene containing the target single nucleotide mutation is amplified on the detection chip. A single nucleotide mutation gene can be detected by detecting the amplified DNA in the same manner as above. The mutagenic primer used may be either an immobilized primer or a free primer. Thereby, as in the above, the single nucleotide mutant gene can be directly amplified on the detection chip and directly detected without adding a sample or the like.

[0043]

EXAMPLES Examples of the present invention will be described below. In addition,
The reaction conditions similar to those of conventional PCR generally applied can be applied to the PCR of the present invention. For example, the following examples are given as reaction reagents, but the reagent composition, reagent amount,
The reaction liquid volume is not limited to this. In particular, the thermostable DNA polymerase is not limited to the enzymes currently used in PCR. In addition, the reaction procedure described below is also an example and is not limited.

[0044] (A) Reaction reagent   Reaction volume 20 μl     Thermostable DNA polymerase             (Eg Taq DNA polymerase): 5 units     KCl: 50 mM     MgCl: 1.5 mM     Tris-HCl buffer pH8.3: 10 mM     Bovine serum albumin: 0.1%     dATP: 200 μM     dTTP: 200 μM     dCTP: 200 μM     dGTP: 200 μM   Immobilized primer: 5 pmol for each primer   Non-fixed primer: 5 pmol for each primer   DNA sample containing the target gene: 0.1 μg

(B) Reaction procedure First, heat to 93 ° C. for 5 minutes to obtain a DNA containing the target gene.
After heat denaturing the sample, the following (1) to (3) are repeated tens of times instead of several times. (1) 93 ° C for 1 minute (2) Based on the dissociation temperature (Tm) of the primer, 37 ° C to 60 ° C for 2 minutes (3) 72 ° C for 1 minute

(C) Results According to the above procedure, on the surface plasmon measuring chip,
Specific DNA was amplified. After amplification, the chip for surface plasmon measurement is directly applied to the surface plasmon measurement, and amplification D
The presence and amount of NA was detected.

[0047]

EFFECTS OF THE INVENTION According to the present invention, a plurality of specific nucleic acids can be simultaneously amplified in one reaction system without complicated operations such as dispensing of samples and reagents. Further, the amplified nucleic acid can be detected and analyzed without taking out the amplified nucleic acid from the container.

[Brief description of drawings]

FIG. 1 is an exploded perspective view of a detection chip according to an embodiment of the present invention.

FIG. 2 is a schematic diagram of a detection principle using surface plasmon resonance in the detection chip according to the present embodiment.

FIG. 3 is a conceptual diagram of a PCR process according to this embodiment.

FIG. 4 is an exploded perspective view of a modified example of the detection chip according to the embodiment of the present invention.

FIG. 5 is an exploded perspective view of another modified example of the detection chip according to the embodiment of the present invention.

FIG. 6 is a conceptual diagram showing another example of the sample slot in the detection chip according to the embodiment of the present invention.

FIG. 7 is a conceptual diagram showing another example of the sample slot in the detection chip according to the embodiment of the present invention.

FIG. 8 is a conceptual diagram showing another example of the sample slot in the detection chip according to the embodiment of the present invention.

[Explanation of symbols]

10 Detection chip (nucleic acid analysis chip) 12 Measurement plate (detection site) 14 Optical substrate 16 Metal thin film 18 DNA amplification site (amplification site) 20 Reaction plate 24 sample slots 28 holes 30 Thermal conductive block 42 Heating heater (heating mechanism) 44 Temperature measuring conductor (temperature measuring mechanism)

Claims (7)

[Claims] 1. A specific nucleic acid in which one of a primer set for amplifying a specific nucleic acid is immobilized at a predetermined position on a detection chip, and the immobilized one primer and the non-immobilized primer A nucleic acid analysis method comprising: an amplification step of carrying out a polymerase chain reaction using the other primer; and a detection step of optically or electrically detecting the nucleic acid amplified on the detection chip. 2. The nucleic acid analysis method according to claim 1, wherein the detection chip is a measurement chip for measuring surface plasmon resonance. 3. The nucleic acid analysis method according to claim 1, wherein the detection step is performed simultaneously with the amplification step. 4. An amplification site for amplifying a specific nucleic acid having one of a primer set for amplifying the specific nucleic acid at a predetermined position on a detection chip immobilized by polymerase chain reaction, and amplification at the amplification site. Nucleic acid detection chip comprising a mechanism or a detection site comprising a part of the mechanism capable of optically or electrically detecting the generated nucleic acid. 5. The nucleic acid detection chip according to claim 4, wherein the detection chip is a measurement chip for measuring surface plasmon resonance. 6. The nucleic acid detection chip according to claim 4, wherein the detection chip further includes a heating mechanism. 7. The nucleic acid detection chip according to claim 6, wherein the detection chip further comprises a temperature measuring mechanism.