WO2005054458A1

WO2005054458A1 - Method for analyzing nucleic acid, cell for analyzing nucleic acid, and analyzer for nucleic acid

Info

Publication number: WO2005054458A1
Application number: PCT/JP2003/015490
Authority: WO
Inventors: Tomoharu Kajiyama; Satoshi Takahashi
Original assignee: Hitachi High-Technologies Corporation
Priority date: 2003-12-03
Filing date: 2003-12-03
Publication date: 2005-06-16
Also published as: JP4426528B2; JPWO2005054458A1

Abstract

A convenient, inexpensive and highly accurate method for analyzing expression of a gene in which amplification and individual inspection are performed simultaneously for a plurality of kinds of target in a single sample. A plurality of sections capable of controlling temperature conditions individually are provided in a chip and utilized for amplification and detection of nucleic acid thus performing amplification and detection simultaneously in a single chip. Since a plurality of amplifying sections and detecting sections are provided and temperature conditions can be determined independently, a plurality of kinds of target in a single sample can be amplified and detected collectively. Since amplification and inspection can be performed simultaneously, the analyzer is utilized conveniently while contributing to cost reduction. Furthermore, detection sensibility is enhanced because a sample including a plurality of targets is not required to be subdivided.

Description

Description Nucleic acid analysis method, nucleic acid analysis cell, and nucleic acid analyzer

The present invention relates to a method for analyzing nucleic acids (polynucleotides) such as DNA, mRNA, and the like, a cell for nucleic acid analysis, and a nucleic acid analyzer. Background art

As a technique for measuring the type of DNA or mRNA present in a sample, a DNA chip is known. DNA chips use various single-stranded oligonucleotides to bind (capture) and synthesize nucleic acids of interest (target nucleic acids) such as genes in a complementary manner through hybridization (complementary strand binding). Probes are aligned and fixed on a substrate at high density. A typical example is a DNA chip from Affimetrix (USA) that uses oligonucleotides to align and immobilize oligonucleotide probes at a high density to capture target nucleic acids on a semiconductor chip using photolithography technology. Is well known. In addition, as a technique for quantitatively measuring the expression level of an interested mRNA, a real-time PCR method, a TaQMan probe method, and the like are well known.

'Furthermore, as disclosed in Japanese Patent Application Laid-Open No. 5-310730, a conventional biochemical reactor has a large number of holes (champers) arranged on a two-dimensional plane. (Micro Multi-Champer) discloses a technology in which a temperature control function is incorporated in each of the champers so that the temperature can be independently controlled for each champer.

Also, the publication discloses that a nucleic acid amplification reaction by PCR (polymerase chain reaction: PCR) is performed using such a micro multi-champer. The technology is described. In this technology, a small amount of various PCR reaction liquids (biochemical samples) are supplied to each chamber, and temperature cycling control according to the reaction liquid is performed for each chamber. Enables PCR. For example, silicon is used as a chamber base material, and a hole serving as a chamber is dug by anisotropic etching, and a semiconductor Peltier element is formed in the champ as a temperature control means.

On the other hand, in Japanese Unexamined Patent Application Publication No. 2000-342264, Japanese Unexamined Patent Application Publication No. 2000-25469 and Japanese Unexamined Patent Application Publication No. Discloses a technique in which a plurality of independent temperature conditions can be set on one substrate (sections). The purpose of this conventional technology is to detect (capture) nucleic acids (polynucleotides) such as DNA and mRNA by hybridization. For multiple compartments where independent temperature control is possible, different oligonucleotide probes (probes for nucleic acid detection) are fixed for each compartment, and the type of sample solution containing the target nucleic acid is the sample common to each compartment. It states that the sample is added to the substrate (chip surface) and that each compartment is set at a temperature suitable for the hybridization of each nucleic acid detection probe.

As described above, the conventional techniques include a DNA chip that arranges and fixes various single-stranded oligonucleotide probes on a substrate at high density, and a multi-chamber and many samples on a single substrate. And a technology for immobilizing nucleic acid detection probes (oligonucleotide probes) for each type on a single substrate in an independently temperature-controllable compartment. ing. These techniques are disclosed as being used exclusively for amplifying or detecting nucleic acids of interest (target nucleic acids) such as DNA and mRNA. However, a large number of nucleic acids _g Amplification · No technology for detection is disclosed.

By the way, as gene expression becomes important in the field of drug discovery and the like, it is required to quantitatively evaluate the expression level of 10 to 20 to 100 types of limited genes of interest. In the conventional technology, it was possible to evaluate the increase and decrease of expression by using a DNA chip.However, the quantitativeness was poor, and the target and nucleic acid amplification procedures were performed externally, which was complicated. There was a problem. In addition, in nucleic acid amplification, there is a difference in the type of optimal temperature control, so that the number of types that can be amplified at one time was limited.

On the other hand, the real-time PCR and TaqMan methods, which are techniques for quantitatively evaluating the expression level, can be detected simultaneously with amplification, so that the detection accuracy is good and simple. However, since only one type of gene can be examined in one tube, if there are multiple genes of interest, the reaction increases accordingly and the cost and complexity increase dramatically. Furthermore, it is necessary to subdivide the measurement sample by dispensing, and the amount of sample per reaction decreases. In general, most samples examined for gene expression are in very small amounts, and a decrease in detectability due to subdivision is a major problem. Disclosure of the invention

An object of the present invention is to provide a method for simultaneously producing target nucleic acids (DNA, mRNA, etc.) even when about 10 to 20 to 100 kinds of nucleic acids (target nucleic acids) such as genes of interest are present. The present invention relates to the provision of a nucleic acid analysis method capable of amplifying and collectively performing a quantitative assay. .

The present invention is basically configured as follows. At least one surface that forms a space (reaction layer) for accommodating solutions such as samples and reagents, a compartment for generating nucleic acid by amplification (compartment for nucleic acid amplification), and the generated amplified nucleic acid after separation Specific capture by hybridization And a compartment (nucleic acid detection compartment) for controlling temperature independently of each other. In the nucleic acid amplification section, temperature cycle control for nucleic acid amplification is performed, and in the nucleic acid detection section, set temperature control suitable for specific capture is performed, so that nucleic acid amplification and nucleic acid amplification can be performed in a single reaction layer. Perform detection. Each compartment has its own set of temperature sensors and heaters, each of which can be controlled independently.

The nucleic acid amplification compartment is provided with a nucleic acid amplification probe, and is formed with at least one or more. For example, when the target nucleic acid is mRNA, a probe of a poly-T sequence, which is complementary to the poly-A sequence, which is a characteristic sequence at the end of the mRNA, is immobilized on the surface of the nucleic acid amplification compartment. I have.

A plurality of nucleic acid detection compartments are formed, and each nucleic acid detection compartment contains two or more detection probes (oligonucleotide probes) so that two or more target nucleic acids of interest can be captured by hybridization. Is fixed to each nucleic acid detection compartment for each species.

In each nucleic acid detection compartment, the hybridization temperature is determined for each type of nucleic acid detection probe in order to guarantee the thermal stability of the hybridization between the nucleic acid detection probe and the polynucleotide that binds complementarily thereto. Is set. An amplified product using a labeled primer is used for nucleic acid detection.

Furthermore, we propose a device that uses an evanescent light, which is a phenomenon in which an excitation laser is incident on the plate above the chip and detects the interface of the excitation light in nucleic acid detection.

Hereinafter, the above and other novel features and effects of the present invention will be described with reference to the drawings. The drawings are for explanation only, and do not limit the scope of the present invention. g JP2003 / 015490 Brief description of drawings

FIG. 1 is a top view of a chip (substrate) in a nucleic acid amplification / detection cell (for example, a detection cell for gene expression analysis) used in a first embodiment of the present invention. FIG. 2 is a detailed structural diagram showing one of the temperature controllable sections provided on the chip of the first embodiment. FIG. 3 is a diagram showing the temperature controllable sections of FIG. 2 in an equivalent circuit. FIG. 4 is a basic circuit diagram showing a temperature control system of the section shown in FIG. FIG. 5 is a diagram showing an example of temperature characteristics of a temperature detecting diode provided in the section shown in FIG. 2; FIG. 6 is an overall configuration diagram of a temperature control system according to the present invention. FIG. 7 is a cross-sectional view of a nucleic acid amplification and detection cell (for example, a detection cell for gene expression analysis) according to the first embodiment of the present invention. FIG. 8 is a schematic diagram of a nucleic acid amplification / detection cell and a detection system used in the first embodiment of the present invention. FIG. 9 shows a flow of a detection procedure in the first embodiment of the present invention. FIG. 10 is a diagram showing temperature conditions in a detection procedure in the first embodiment of the present invention. FIG. 11 is a diagram for explaining a nucleic acid amplification / detection method in the first embodiment of the present invention, and shows an initial state. FIG. 12 is a diagram for explaining a method for detecting and detecting nucleic acid amplification in the first embodiment of the present invention, in which mRNA is hybridized to an amplification poly-T probe. FIG. 13 is a diagram for explaining the nucleic acid amplification / detection method in the first embodiment of the present invention, showing a state in which a complementary chain extension of mRNA is synthesized by a reverse transcription reaction (RT reaction). I have. FIG. 14 is a diagram illustrating a method for amplifying and detecting a nucleic acid in the first embodiment of the present invention, and shows a single-stranded state of an extended product of the complementary chain of mRNA. FIG. 15 is a diagram for explaining the nucleic acid amplification / detection method in the first embodiment of the present invention, in which the amplification primer is hybridized to one strand of the complementary chain extension of mRNA. Is shown. FIG. 16 is a diagram for explaining the nucleic acid amplification / detection method in the first embodiment of the present invention, showing a state in which a fluorescently labeled detection amplification product is synthesized by extension of the amplification primer. _

6 shows. FIG. 17 is a diagram for explaining the nucleic acid amplification / detection method in the first embodiment of the present invention, wherein the fluorescently labeled detection amplification product specifically hybridizes to the probe in the detection compartment. The state is shown. FIG. 18 is a schematic view (cross-sectional view) of a nucleic acid amplification / detection cell (for example, a detection cell for gene expression analysis) used in the second embodiment of the present invention. FIG. 19 is a schematic diagram showing an example in which the cell of the second embodiment of the present invention utilizes excitation by evanescent light. FIG. 20 shows that in the initial state of the nucleic acid amplification / detection chip used in the third embodiment of the present invention, a reverse primer specific to a target nucleic acid (target gene) is immobilized in the nucleic acid amplification compartment. Bird's-eye view showing the example being performed. FIG. 21 is a bird's-eye view showing an example in which mRNA is hybridized to a nucleic acid amplification primer (a reverse primer) in the third embodiment of the present invention, and complementary strand extension synthesis is performed. FIG. 22 is a bird's-eye view showing an example in which a forward primer with a fluorescent label is hybridized to an extended portion of the reverse primer in the third embodiment of the present invention. FIG. 23 is a diagram showing an example in which the fluorescent-labeled forward primer is extended to become a fluorescent-labeled amplification product for detection in the third example of the present invention. FIG. 24 is a top view of a chip (substrate) in a nucleic acid amplification / detection cell (for example, a detection cell for gene expression analysis) used in a fifth embodiment of the present invention. BEST MODE FOR CARRYING OUT THE INVENTION

(First embodiment).

As a first example of the present invention, an example in which gene expression analysis was performed is described below.

FIG. 1 is a top view of a chip (substrate) in a nucleic acid amplification / detection cell (for example, a detection cell for gene expression analysis) used in the present example.

The chip (substrate) 11 in this embodiment is used for dissolving a sample, ^ It has a space for accommodating liquid (hereinafter referred to as "reaction layer") 12, and has an inlet 3-1 and an outlet 13-2 for the solution.

On one surface of the reaction layer 12 of the chip 11, a plurality of sections 14 and 15-1 to 15-8 are formed. Each compartment is equipped with a set of independently operating temperature sensors and heaters. An example of the temperature sensor and the heater will be described later with reference to FIG.

Of the plurality of compartments, some function as nucleic acid amplification compartments, and some function as nucleic acid detection. In FIG. 1, in chip 11, section 14 is a section for nucleic acid amplification, and sections 15-1 to 15-8 are sections for nucleic acid detection. In this example, by disposing a plurality of nucleic acid detection compartments 15-1 to 15-8 on both sides or around the nucleic acid amplification compartment 14, the nucleic acid detection compartments 15-1 to 15 — All eight are equidistant relative to nucleic acid amplification compartment 14. Although FIG. 1 illustrates one nucleic acid amplification section 14 and eight nucleic acid detection sections 15, the number is not limited to this number. The number of commercial lots is in the order of tens or hundreds.

FIG. 2 is a diagram illustrating the structure of one of the sections shown in FIG. Each section has a similar structure. Each section is fabricated using semiconductor manufacturing technology.

The temperature sensor that detects the temperature is a diode formed by the junction of the Ρ-type diffusion layer 21 and the Ν-type diffusion layer 22, and is used as a sensor by using the temperature dependence of the resistance value. In this diode, the ダイオード -type diffusion layer 23 is used as a protective layer to control the potential of the diode. The chip substrate 24 of the entire section uses a Ν-shaped substrate, and its potential is determined by the Ν-shaped diffusion layer 25.

The potential of the chip 24, that is, the potential of the 拡散 -type diffusion layer 25 is increased by the Ρ-type _{g By} making the potential of the diffusion layer 21 equal to that, even if the protection layer 23 and the chip substrate 24 are PN junctions, it is possible to prevent the current from flowing out of the temperature sensor to the outside of the protection layer 23 from the temperature sensor. Becomes possible. Further, when viewed from the chip substrate 24 side, since the chip substrate 24 and the protective layer 23 form an NP junction, it is possible to prevent a noise current from flowing into the temperature sensor 1 from outside the protective layer.

In this embodiment, the heater is formed of the N diffusion layer 26. The N-type diffusion layer 26 is surrounded by a protective layer 27 of a P-type diffusion layer. The potential of the protection layer 27 is determined by the P-type diffusion layer 28.

If one end of the N-type diffusion layer 26 has the same potential (1) as the protection layer 27 and the other end has a positive electrode (+), the N-type diffusion layer 26 and the protection layer 27 have an NP junction. Current does not leak to the protective layer. Therefore, the N-type diffusion layer 26 is equivalent to the structure of the heating wire. By controlling the current flowing through the heater 26, the heating can be controlled. For the connection between the sensor and the circuit, the positive side is represented by S (ten), and the negative side is represented by S (—). For the connection between the heater and the circuit, the positive side is represented by R (ten) and the negative side is represented by R (-). If these structures are represented by an equivalent circuit, the result is shown in Figure 3. The temperature sensor is a diode 31 and the heater is represented by 32.

Figure 4 shows an outline of the basic circuit that controls the compartment temperature and the connection to the compartment on the chip. This is an example of a circuit that detects temperature and controls heating for one section.

A constant current circuit is connected to diode 31 of the temperature sensor. This circuit is a constant current circuit in which almost constant current determined by the resistance 41 flows when the resistance of the diode 41 is negligibly large. The voltage drop in the diode 31 is measured by a voltmeter 42, and the value is sent to the control unit 43. The y control unit 43 compares a preset voltage value (set value V s) with a measured value (V x) of the voltmeter 42, and when V s <VX, sets the gate 44 to 0 N, V s> In the case of VX, control is performed so that the gate 44 is set to 〇FF. In the heater one-side circuit, a voltage 45 is applied to both ends when the gate is turned on, and the voltage is cut off when the gate is turned off. Control using this basic circuit will be described below.

Figure 5 shows the relationship between the voltage drop across diode 31 and the chip section temperature (one example). Hereinafter, description will be made using this example. As shown in Figure 5, the chip temperature (T [degrees Celsius]) and the voltage drop (Vx CmV]) show linearity, with a slope of about 1-2 mVZ in this example. Therefore, it is represented by the following approximate expression (1).

Vx =-2 T + 560-(1) In other words, if the chip section temperature rises once, the voltage drop will decrease by about 2 mV. By measuring this relational expression in advance, the chip temperature can be calculated from the voltage drop. Note that the value of the slope differs depending on the measurement conditions and the like. Although not described in this patent, it is needless to say that the temperature dependency of the resistance value of the diode can be obtained by using a constant voltage condition or other conditions to obtain a relational expression.

As shown in FIG. 5, in this example, the temperature rise is observed by a decrease in the voltage drop. Therefore, it is possible to control the temperature of any section in the chip as follows.

For example, a case will be described in which a certain section is controlled at 50 degrees Celsius. If the temperature of the compartment is lower than the desired temperature condition of 50 degrees Celsius, the voltage drop at the sensor is greater than the voltage drop at 50 degrees Celsius of 450 mV. Assuming that the set value Vs is Vs = 460, the condition of VX> Vs is satisfied, so that the gate 44 is controlled by 〇N, and the circuit is operated until VX <Vs. ^ Causes current to flow through the heater circuit, heating the compartment. When the temperature of the compartment rises due to heating and Vs is reached, the gate is turned off and heating stops, so no further temperature rise occurs. When the temperature of the partition drops due to thermal diffusion and VX> Vs again, the gate returns to 0 N and heating resumes. This control keeps the compartment temperature at 50 degrees Celsius.

In this embodiment, it is possible to control the temperature of each section independently by connecting this basic circuit to all sections of the chip, one circuit at a time, and controlling each set value independently. Become. That is, a single chip (substrate) 11 has a section 14 for amplifying nucleic acid and a section 15 for specifically capturing the amplified nucleic acid by hybridization. ~ 15-8 are formed with heaters whose temperature can be controlled independently of each other. FIG. 6 is a diagram illustrating the entire system of the present embodiment. 6 1 and 6 2-1 to 6 2-8 indicate the control units of the basic circuit connected to section 14 and section 15-1 to; I 5 _ 8. The computer 63 controls the entire system and performs pre-programmed temperature control. The interface 6.4 controls the computer 63 and all the control units 61, 62-1 to 62-8. Contact 9 '

In the computer 163, a set temperature for each time of each section or a set voltage value corresponding thereto is programmed in advance. In accordance with the program, the computer 63 sends the set temperature or the corresponding voltage value to the control units 61, 62-1 to 62-8 of the respective basic circuits via the interface 64. Enter as the set value Vs for each time. As a result, each section can faithfully implement the programmed temperature sequence. The above is the outline of the structure of the partition and the circuit and system connected to the partition in the chip of the present invention. Next, the structure of the chip will be described. In Figure 1 ', on the surface of the nucleic acid amplification compartment 14, a poly-T probe that captures the expressed gene (target nucleic acid, eg, mRNA) in the sample is immobilized with its sequence end 5' facing the compartment surface. Have been. The poly T probe is an oligonucleotide probe having the sequence represented by SEQ ID NO: 1, and has a function of specifically hybridizing to the poly A region at the end of the expressed gene. Here, a sequence length of 2 Omer was adopted. However, as the poly T probe, a poly T oligonucleotide having a length in the range of 8 to 20 Omer can be used. Items shorter than 8 mer lack stability during hybridization. Further, in the case of an object longer than 200 mer, the function is sufficient, but there is a disadvantage that the manufacturing cost is high. In some cases, a region other than T is optionally added to the 5 'end of the poly T oligo. This has the effect of increasing the distance from the solid phase surface, increasing the efficiency of the hybridization.

5, — TTTTTTTTTTTTTTTTTTTT-3, (SEQ ID NO: 1) Nucleic acid detection compartments 15—1 to 15—8 can specifically bind to the amplified product corresponding to the target gene on its surface Oligonucleotide probe is immobilized. 'For this probe, the probe with the optimal sequence is selected for each gene of interest. In this example, an attempt was made to measure the expression levels of two types of mRNA, GAPDH and P53. The sequences of the probes for detecting GAP DH and P53 mRNA are SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

5 '-GC GTCAAAGGTGGAGGAGTGGGTGT- 3'

(SEQ ID NO: 2)

5 '-C GCTGTGC CGAAAAGTCTGC CTGTC- 3'

(SEQ ID NO: 3) ^ Assume that these .arrays are fixed to block 15-1 and block 15-5, respectively. The most convenient method for immobilization is to use 1,4-Phenylene Diisothiocyanate as a linker and immobilize the above oligo probe whose 3 'end is amino-modified to the surface of the aminosilane-treated chip. It is. Here, it is particularly important to fix the 3 'side to the chip surface. As a result, no extension reaction by the polymerase occurs in this probe. For the same purpose, it is also effective to chemically treat the 3 ′ terminal base of the probe so that the extension reaction does not occur when the 5 ′ terminal side is used as the fixed side.

Since the amplified product corresponding to the target gene hybridizes in these nucleic acid detection compartments, the amount of the target gene can be determined by detecting the amount. The details will be described later.

As shown in FIG. 7, the nucleic acid amplification detection cell used in the present invention comprises a chip 11 having the nucleic acid amplification compartment 14 and the nucleic acid detection compartments 15-1 to 15-8 described above, A transparent sheet (upper plate) 72 arranged on the surface of the chip 11 so as to face the chip surface, and the sample is placed in the space 12 sandwiched between the chip 11 and the upper plate 72 (Solution of sample, reagent, etc.) 72 Here, a combination of the chip 11 and the upper plate 72 is referred to as a “cell”.

In FIG. 1, the chip 11 is viewed with the upper plate 72 removed, and FIG. 7 is a cross-sectional view of FIG. 1 passing through the sections 15-1 and 15-5. .

The layered space between the chip (substrate) 11 and the upper plate 72 becomes the reaction layer 12 for accommodating the sample, and is formed by processing the substrate 17 by lithography. In addition, the chip 11 and the upper part 72 are formed by a sheet, and a spacer is interposed therebetween to form the reaction layer 12. Is also possible. '

The thickness of the upper plate 72 is optimally 0.01 mm to 1 mm. Glass, various plastics, etc. can be used for the material, but it is important that the inside does not contain fluorescent substances as much as possible. Although it is possible to use a material with a thickness exceeding 1 mm, it is difficult to maintain the temperature independence in the chip, which is the object of the present invention, because the upper plate acts as a heat transfer material. Become. On the other hand, if it is less than 0.01 mm, there is a problem in strength. The layer 73 of the sample is preferably between 0.05 mm and 1 mm. If it exceeds 1 mm, heat convection occurs in the sample solution in the chip, so that the heat independence deteriorates.

Between the nucleic acid amplification compartment 14 and the nucleic acid detection compartments 15-1-1 to 15-8 and between the nucleic acid detection compartments, it is important to have a thin-film structure to maintain heat independence. It is industrially effective to use a silicon oxide film, but it is also possible to use other organic material films, such as plastic and polyimide films. Also, since this chip is planar, it has high heat diffusion efficiency in the up and down method. Therefore, when the detected temperature is high, it is possible to lower the temperature to a predetermined temperature without providing a cooling function by simply turning off the heater.

The amount of the nucleic acid amplification product with a fluorescence label captured in the nucleic acid detection sections 15-1 to 15-8 on the chip surface is detected as the amount of fluorescence (the generation of the amplification product with a fluorescence label will be described later). Figure 8 shows a state in which the detection system is added to the cell.

In this embodiment, the fluorescence detection system has the same configuration as the confocal microscope. Other detection methods will be described later in embodiments.

In the fluorescence detection system, rather than using the lens 81, the photodetector 82 and the excitation laser 83 are in a confocal relationship with the cell surface 84 and the laser 83 The fluorescent label (fluorescence amount) in each detection compartment in the excited reaction layer 12 is measured by the photodetector 8'2.

The detector 82, laser 83, and lens 81 are assembled as a single unit, and they move in the horizontal direction (indicated by the arrow in the figure) as a whole to scan the cell surface.

Next, the pretreatment of nucleic acid amplification using the chip of this example, the procedure of nucleic acid amplification and detection, and the transition of the product formed on the chip (within the tube) are described in FIGS. This will be described using FIG.

The sample is, for example, total RNA extracted from somatic cells. In addition, the target nucleic acids (targets) are two types of mRNA, GAPDH and P53.

FIG. 9 summarizes the operation procedure of the present embodiment. FIG. 10 summarizes the temperature control conditions of the nucleic acid amplification section and the nucleic acid detection section 15-1 in each procedure.

FIG. 11 is a bird's-eye view schematically illustrating the surface of the chip 11. As shown in Fig. 11, in the initial state of the chip surface, the poly-T probe 11 (SEQ ID NO: 1) is placed on the surface of the nucleic acid amplification section 14 and the nucleic acid detection section 15-1 for GAPDH is The probe 112 for GAPDH (SEQ ID NO: 2) is immobilized.

A sample solution containing all RNA is injected into the reaction layer 12 of the chip 11, and then set to the temperature condition (1) shown in FIG. The temperature conditions are 35 ° C. where the amplification section 14 is suitable for hybridization, and 75 ° C., which is much higher than that of the nucleic acid amplification section 15 -1 to 15 -8. As a result, mRNA in the sample is fixed by hybridizing with the poly T probe 111 of the nucleic acid amplification section 14.

Fig. 12 shows that mRNA is transferred to the nucleic acid amplification compartment 14 by poly T probe 1 1 1 FIG. 4 is a bird's-eye view schematically showing a state captured by the camera. Here, all mRNAs are captured, regardless of type. For example, mRNA 122 of GAPDH, mRNA 122 of p53 and other mRNA 123 are captured. On the other hand, hybridization does not occur in the nucleic acid detection compartment 155-1 because of the high temperature condition.

Next, unnecessary substances are washed, and a reverse transcription reagent (RT reagent) is introduced, and under the temperature condition (2) shown in FIG. 10, reverse transcription occurs in the nucleic acid amplification compartment. At this time, the temperature of the amplification section 14 is set to 42 ° C. suitable for reverse transcription. The nucleic acid detection compartment 15-1 is maintained at a high temperature of 75 ° C.

FIG. 3 is a diagram showing a state in which the poly-T probe 111 has been extended by reverse transcription. Here, it is important that the extended probe faithfully reproduces the mRNA abundance. In other words, here, there are an extension 13 1 of GAPDH mRNA, an extension 13 of P53 mRNA, and an extension 13 of other mRNA.

Next, when the temperature condition (3) in Fig. 10 was applied and the inside of the chip was washed, all mRNA was discharged, and the expanded probe (reverse transcription product) was placed in the nucleic acid amplification compartment 14 as shown in Fig. 14. There is only a single strand of 13 1 to 13 3.

The above process is the pretreatment for forming a reverse probe for amplifying the target nucleic acid. In this pretreatment, the probe for target nucleic acid detection fixed in the nucleic acid detection compartment is under pretreatment as described above. The temperature is controlled to a temperature that does not cause the operation of the hybrid (for example, a high temperature condition of 75 ° C).

Next, a nucleic acid amplification reagent is introduced into the reaction layer 12, and the temperature condition is set to the temperature of (4) in FIG. The temperature condition at this time is 60 for amplification section 14 and 65 degrees for detection section 15-1.

Under this temperature condition, the operation shown in FIG. 15 is performed. That is, nuclear Among the acid amplification reagents, there are a primer for GAPDH with a fluorescent label at the 5 'end and a primer for p53. It can hybridize to a predetermined sequence of the main strand. Here, GAPDH primer 15 1 hybridizes to GAPDH mRNA extension 131, and P53 primer 15 2 generates P53 mRNA extension 13 2 Hybridize to

Here, assuming that the temperature condition (5) is a thermal cycle in the amplification section 14, in the amplification section, amplification products with fluorescent labels are obtained by linear amplification from the GAPDH and P53 extension products. 2 is generated.

FIG. 16 shows a state in which the respective fluorescent-labeled amplification products 161 and 162 were synthesized. On the other hand, the nucleic acid detection compartments 15-1 and 15-5 are set to optimal temperature conditions for the respective probes to hybridize. Therefore, the linearly amplified product specifically hybridizes to each probe (Fig. 17). If this amount is detected in real time in parallel with amplification, a signal of 2 N times the original mRNA amount can be detected for N amplification cycles.

In this example, the detection of two types of mRNA was described for the sake of simplicity, but in the present invention, more than two types of mRNA are simultaneously measured by the number of the nucleic acid detection compartments. It is possible.

As described above, according to the present example, amplification and detection can be performed in parallel in one chip, and the expression levels of multiple types of mRNA in one sample can be simultaneously, rapidly and easily determined. It is possible to measure.

(Second embodiment)

As a second embodiment, a nucleus with further improved detection performance of the first embodiment The cell for “acid amplification” detection will be described with reference to FIG. 18 and FIG. FIG. 18 is a schematic diagram (cross-sectional view) in which a reaction layer (sample layer) 73 is interposed between the chip 11 and the upper plate 18 2 opposed thereto. The chip 11 is provided with a nucleic acid amplification compartment and a nucleic acid detection compartment as in the first embodiment, but here, for convenience of illustration, one of the nucleic acid detection compartments 15 — Only one is exaggerated.

In this embodiment, the probe 18 1 for nucleic acid detection (corresponding to the probe 1 12 in the first embodiment) is not on the surface of the nucleic acid detection compartment but on the opposite surface, that is, the upper plate portion (Transparent sheet) Fixed to 18 2. The upper plate 18 2 corresponds to the upper plate 72 of the first embodiment. In other words, the detection probe 181, which captures the target nucleic acid in the nucleic acid detection compartment, is fixed to a surface facing the heater surface such as a temperature-controllable compartment 151-1-1 ... 151-n. I have.

That is, the nucleic acid detection compartment in the present embodiment is formed with functions separated on the opposing surface between the substrate 11 and the sheet 18 2, and the temperature cycle control of the nucleic acid detection compartment is provided on the opposing surface on the substrate 11 A heater is provided for each section, and a nucleic acid detection function is provided by fixing a nucleic acid detection probe 181 to the opposite surface of the sheet 18 2 side.

In the cell of the present embodiment, under the condition that the thickness of the reaction layer 73 is less than or equal to 1.0 mm, the temperature of the section (15 1—1... Surface temperatures are about equal.

Therefore, even in the structure of this embodiment, it is possible to control the temperature of the probe fixing region under each hybridization condition. Therefore, the measurement object hybridized to the probe 18 1 is fixed to the surface of the upper plate.

Amplification products (probes for amplification) As shown in Fig. 19, the upper plate 182 has a laser light source 191, which is located on the side of the upper plate 182. The excitation laser 192 is incident laterally from. FIG. 19 schematically shows a case where the excitation laser 1992 is incident on the upper plate 182.

The excitation laser travels while totally reflecting inside the upper plate 182, and is controlled to an angle that is not emitted to the outside of the upper plate 182. Then, only in the vicinity of the upper plate 18 2, leakage of the excitation laser 19 2 occurs as evanescent light. As a result, only the amplification product 193 with the fluorescent label present on the upper plate surface is excited, and the amplified product 194 with the fluorescent label floating in the middle of the sample layer is not excited. Therefore, according to this embodiment, in addition to the effect of the first embodiment, the primer present in the reaction solution does not contribute to the emission of fluorescence, so that the background of fluorescence detection is significantly reduced, and the measurement S / N is improved.

(Third embodiment)

Next, a third embodiment of the present invention will be described with reference to FIGS. The difference between this example and the first example is the sequence structure of the oligonucleotide probe immobilized on the nucleic acid amplification section 14. The nucleic acid detection compartment does not change.

In this example, an example is shown in which a reverse primer that captures the target mRNA in a complementary and specific manner without performing pretreatment as in the first example is used as a probe to be fixed to the nucleic acid amplification compartment. As in the previous and examples, two types of target RNAs, GAPDH and P53, are described as examples. Sequences 4 to 7 are prepared as primers specific to these two types of mRNA.

GTCT C CTCTGACTTCAACAGC (SEQ ID NO: 4) 丄

AAAGTGGTC GTTGAGGGCA (SEQ ID NO: 5)

C C GTATGCTGAGTATCTGGAC (SEQ ID NO: 6)

GAC CTCAGGTGGCTCATAC (SEQ ID NO: 7) Here, Sequence 4 and Sequence 5 are forward and reverse for GAPDH, and Sequence 6 and Sequence 7 are forward and reverse for P53. The reverse primer is fixed to the surface of the nucleic acid amplification section as indicated by reference numerals 201 and 202. On the other hand, the forward primer has a fluorescent label at the 5 ′ end. Using these, nucleic acid amplification is performed. The reaction in the nucleic acid amplification compartment is described below.

FIG. 20 is a bird's-eye view of the inside of the chip. When total RNA is injected into the reaction layer of chip 11 as a sample, only those specific to fixed reverse primer 201 and primer 102, namely GAPDH and P53, correspond Hybridize to the reverse primer (Figure 21). By the extension reaction, the reverse primers 211 and 212 are extended to have a sequence complementary to the mRNA. Due to the thermal cycle in this nucleic acid amplification, the dissociated mRNA re-hybridizes with another reverse primer. On the other hand, the free forward primer complementarily binds to the extended reverse primer (eight hybridizes) and extends (Fig. 22).

By repeating the above procedure, the fixed extended reverse primer and the extended free forward primer (with a fluorescent label) are generated at 2 N times the initial mRNA amount (Fig. 23). Since the extended free forward primer has a sequence complementary to the primer in the nucleic acid detection compartment, it is detected in the detection compartment as in the first and second embodiments. Since the amplification rate of this method is extremely large, it is possible to detect a trace amount of target. In this example, it is necessary to set the temperature cycle of the two types of primer sets to optimal conditions. Therefore, it is better to set the annealing temperature individually for each array. In this case, if a plurality of nucleic acid amplification compartments are prepared, it is possible to simultaneously implement a plurality of types of temperature cycles in the chip. That is, a compartment for nucleic acid amplification for GAPDH and a compartment for nucleic acid amplification for P53 are separately provided. In this example, since each reparse primer is fixed on the surface of the compartment, each amplification reaction can be generated only in the corresponding compartment.

(Fourth embodiment)

In each of the above examples, nucleic acid amplification and detection were performed simultaneously. Therefore, as in the first embodiment, the background may be high due to the presence of free primer in the sample. In this case, the detection at the same time as the amplification is an approximate evaluation of the signal amount, and in the precision evaluation, it is effective to perform the process of washing the free component to improve the accuracy.

(Fifth embodiment)

FIG. 24 shows an embodiment in which two compartments for nucleic acid amplification, 2241-1 and 241-2, are provided in one reaction layer. As for other configurations, any one of the first to fourth embodiments is employed. 2 4 2— :! 2242 1 n is a nucleic acid detection compartment.

Optimum conditions for the nucleic acid amplification temperature cycle differ depending on the sequence of the specified nucleic acid amplification primers. In the first embodiment, as shown in FIG. 10, a temperature cycle (95 ° C .: 5 seconds—55: 15 seconds, at −72: 15 seconds) was used. However, when the GC rate of the primer is large, it may be effective to raise the primer's eight-fold temperature to 60, which is 5 degrees higher than that of the primer, because there is a fake synthesis due to pseudo-hybridization. Also, the base length of the product is If it is long, it is necessary to make the extension synthesis time 15 seconds longer. In order to improve the accuracy of nucleic acid analysis, which is an object of the present invention, it is effective to optimize these temperature cycles for each target nucleic acid. To achieve this, it is necessary to provide a plurality of nucleic acid amplification zones in the reaction layer and control them at different temperature cycles. The configuration shown in Fig. 24 is effective.

According to the above embodiments, a simple, inexpensive, and highly accurate gene expression analysis method is provided by simultaneously performing amplification and individual detection of a plurality of types of evening targets in a single sample. be able to. Industrial applicability

According to the present invention, for example, when there are about 10 to 20 to 100 kinds of nucleic acids (for example, genes) of interest, those genes can be simultaneously amplified and quantitatively assayed at once. In addition, since the reaction is performed in a single reaction layer, there is no problem of miniaturization due to subdivision of samples, and detection with higher sensitivity than before can be performed.

Claims

Im Claims

1. At least one surface that forms a space for accommodating solutions such as samples and reagents (hereinafter referred to as a “reaction layer”) is provided with a compartment for generating nucleic acids by amplification (hereinafter referred to as a “nucleic acid amplification compartment”). ) And the generated amplified nucleic acid

And a compartment (hereinafter, referred to as a “nucleic acid detection compartment”) for specifically capturing by the hybridization after the separation of 5 from each other so that temperature control can be performed independently of each other. In this section, amplification and detection of nucleic acids are performed in a single reaction layer by controlling the temperature cycle for nucleic acid amplification, and performing the set temperature control suitable for specific capture in the nucleic acid detection section. 10 Nucleic acid analysis method.

2. The nucleic acid analysis method according to claim 1, wherein amplification and detection of the nucleic acid are performed in the reaction layer in parallel. '

3. The method according to claim 1, wherein the nucleic acid amplification section is formed with at least one or more, and each nucleic acid amplification section is provided with a nucleic acid amplification probe.

15 A plurality of the nucleic acid detection compartments are formed, and in each nucleic acid detection compartment, a nucleic acid detection probe for fixing a target nucleic acid of interest for each type by hybridization is fixed,

In each of the nucleic acid detection compartments, a hybridization temperature is set for each type of the nucleic acid detection probe in accordance with the thermal stability of hybridization.

20 Nucleic acid analysis method to be set.

4. The method according to claim 1, wherein in the nucleic acid amplification section, a pretreatment for forming a reverse probe for amplifying a target nucleic acid is performed under a predetermined set temperature control,

In the nucleic acid detection compartment, a nucleic acid detection probe 'for immobilizing a target nucleic acid by hybridization 25 is fixed.

-During the pretreatment, the nucleic acid detection probe A nucleic acid analysis method in which the temperature of the nucleic acid detection compartment is controlled so as not to cause cropping.

5. The nucleic acid detection probe according to claim 1, wherein a nucleic acid detection probe for capturing a nucleic acid amplification product with a fluorescent label deviating from the nucleic acid amplification compartment is fixed to the nucleic acid detection compartment. A nucleic acid analysis method using evanescent light as excitation light for detecting an amplification product with a fluorescent label captured by the probe.

6. It has a space for accommodating solutions such as samples and reagents (hereinafter referred to as “reaction layer”). At least one surface of the reaction layer forms a compartment for generating nucleic acids by amplification. Hereinafter, a “compartment for nucleic acid amplification” and a compartment for specifically capturing generated nucleic acid by hybridization after separation (hereinafter, referred to as a “compartment for nucleic acid detection”) are independent of each other. A cell for nucleic acid analysis formed so as to enable controlled temperature.

7. The cell for nucleic acid analysis according to claim 6, wherein a probe of a poly T sequence having a sequence complementary to the poly A sequence of mRNA is fixed on the surface of the nucleic acid amplification section.

8. The nucleic acid analysis cell according to claim 6, wherein a nucleic acid amplification probe capable of specifically hybridizing an mRNA of interest is fixed on a surface of the nucleic acid amplification section.

9. The cell according to claim 6, wherein the reaction layer is formed between a substrate and a light-transmissive sheet disposed to face the substrate, and the nucleic acid amplification section and the nucleic acid detection section At least one cell for nuclear acid analysis provided on one side of the substrate.

10. The method according to claim 6, wherein the reaction layer is formed between a substrate and a light-transmitting sheet disposed opposite the substrate, and the reaction layer has a thickness of 0.05 mm to 1 mm. A cell for nucleic acid analysis which satisfies at least one condition that the thickness of the sheet and the thickness of the sheet is from 0.01 mm to 1 mm.

11. The method according to claim 6, wherein the reaction layer is formed between the substrate and a light-transmitting sheet disposed opposite to the substrate.

The nucleic acid amplification section is formed on one surface of the substrate,

The nucleic acid detection compartment is formed with a function divided on an opposing surface between the substrate and the sheet, and a heater function for controlling the temperature cycle of the nucleic acid detection compartment for each compartment is provided on the opposing surface on the substrate side. A nucleic acid analysis cell, wherein a nucleic acid detection probe is fixed to the opposite surface on the sheet side to provide a nucleic acid detection function.

12. The nucleic acid analysis cell according to claim 6, wherein a plurality of the nucleic acid detection compartments are arranged on both sides or around the nucleic acid amplification compartment.

13. The nucleic acid analysis cell according to claim 6, wherein all of the nucleic acid detection compartments are arranged at an equal distance from the nucleic acid amplification compartment.

1. There is a space (hereinafter referred to as “reaction layer”) for storing solutions such as samples and reagents, and at least one side of the reaction layer is formed with a compartment for generating nucleic acids by amplification. Hereinafter, a “compartment for nucleic acid amplification” and a compartment for specifically capturing the generated amplified nucleic acid by hybridization after separation (hereinafter, referred to as a “compartment for nucleic acid detection”) are interchangeable. Cells formed so that independent temperature control is possible,

Temperature control means for performing a temperature cycle control for nucleic acid amplification of the nucleic acid amplification compartment,

In the nucleic acid detection compartment, a temperature control means for performing a set temperature control suitable for specific capture by hybridization.

A nucleic acid analyzer comprising:

15. The method according to claim 14, wherein the reaction layer is formed between the substrate and a light-transmitting sheet disposed to face the substrate.

The nucleic acid detection compartment has a function on the opposite surface of the substrate and the sheet. A heater function for controlling the temperature cycle of the nucleic acid detection section for each section is provided on the opposed surface on the substrate side, and a nucleic acid detection probe is fixed on the opposed surface on the sheet side. Provided with a detection function,

A nucleic acid analyzer using an evanescent light set so that a nucleic acid amplification product with a fluorescent label is captured by the nucleic acid detection probe, and a means for detecting the amount of fluorescence is set to leak out to the inner surface of the sheet.

16. The nucleic acid analyzer according to claim 14, wherein the cell has a plurality of nucleic acid amplification sections, and performs different temperature cycle control in each of the nucleic acid amplification sections.