WO2005054458A1 - Methode d'analyse d'acide nucleique, cellule d'analyse d'acide nucleique et analyseur d'acide nucleique - Google Patents

Methode d'analyse d'acide nucleique, cellule d'analyse d'acide nucleique et analyseur d'acide nucleique Download PDF

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Publication number
WO2005054458A1
WO2005054458A1 PCT/JP2003/015490 JP0315490W WO2005054458A1 WO 2005054458 A1 WO2005054458 A1 WO 2005054458A1 JP 0315490 W JP0315490 W JP 0315490W WO 2005054458 A1 WO2005054458 A1 WO 2005054458A1
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Prior art keywords
nucleic acid
compartment
amplification
detection
substrate
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PCT/JP2003/015490
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English (en)
Japanese (ja)
Inventor
Tomoharu Kajiyama
Satoshi Takahashi
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Hitachi High-Technologies Corporation
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Priority to JP2005511265A priority Critical patent/JP4426528B2/ja
Priority to PCT/JP2003/015490 priority patent/WO2005054458A1/fr
Publication of WO2005054458A1 publication Critical patent/WO2005054458A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/54—Heating or cooling apparatus; Heat insulating devices using spatial temperature gradients

Definitions

  • 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.
  • 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.
  • a real-time PCR method, a TaQMan probe method, and the like are well known.
  • a conventional biochemical reactor has a large number of holes (champers) arranged on a two-dimensional plane.
  • champers holes
  • 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.
  • PCR polymerase chain reaction
  • 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.
  • nucleic acids polynucleotides
  • different oligonucleotide probes probes for nucleic acid detection
  • 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.
  • 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.
  • 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.
  • the reaction increases accordingly and the cost and complexity increase dramatically.
  • 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.
  • a compartment for controlling temperature independently of each other.
  • temperature cycle control for nucleic acid amplification is performed
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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. 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
  • 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. _
  • 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. 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.
  • 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.
  • a nucleic acid amplification / detection cell for example, a detection cell for gene expression analysis
  • 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.
  • reaction layer a space for accommodating liquid
  • 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.
  • section 14 is a section for nucleic acid amplification
  • sections 15-1 to 15-8 are sections for nucleic acid detection.
  • 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 ⁇ -type diffusion layer 21 and the ⁇ -type diffusion layer 22, and is used as a sensor by using the temperature dependence of the resistance value.
  • the ⁇ ⁇ ⁇ ⁇ ⁇ -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 ⁇ -shaped substrate, and its potential is determined by the ⁇ -shaped diffusion layer 25.
  • the potential of the chip 24, that is, the potential of the ⁇ ⁇ -type diffusion layer 25 is increased by the ⁇ -type g
  • the potential of the diffusion layer 21 is increased by the ⁇ -type g
  • 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.
  • the N-type diffusion layer 26 is equivalent to the structure of the heating wire.
  • the heating can be controlled.
  • S (ten) the positive side is represented by S (ten)
  • R (ten) 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 ⁇ FF.
  • 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).
  • the chip temperature T [degrees Celsius]
  • the voltage drop Vx CmV]
  • the voltage drop Vx CmV]
  • Vx -2 T + 560-(1)
  • 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.
  • 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.
  • 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.
  • a set temperature for each time of each section or a set voltage value corresponding thereto is programmed in advance.
  • 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.
  • each section can faithfully implement the programmed temperature sequence.
  • 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.
  • 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.
  • a sequence length of 2 Omer was adopted.
  • a poly T oligonucleotide having a length in the range of 8 to 20 Omer can be used as the poly T probe. Items shorter than 8 mer lack stability during hybridization.
  • 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.
  • the amount of the target gene can be determined by detecting the amount. The details will be described later.
  • 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
  • a combination of the chip 11 and the upper plate 72 is referred to as a “cell”.
  • 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.
  • 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.
  • nucleic acid amplification compartment 14 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.
  • Figure 8 shows a state in which the detection system is added to the cell.
  • the fluorescence detection system has the same configuration as the confocal microscope. Other detection methods will be described later in embodiments.
  • 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.
  • the sample is, for example, total RNA extracted from somatic cells.
  • target nucleic acids 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.
  • the poly-T probe 11 SEQ ID NO: 1
  • the nucleic acid detection section 15-1 for GAPDH 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.
  • 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.
  • all mRNAs are captured, regardless of type.
  • mRNA 122 of GAPDH, mRNA 122 of p53 and other mRNA 123 are captured.
  • hybridization does not occur in the nucleic acid detection compartment 155-1 because of the high temperature condition.
  • 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.
  • 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.
  • the extended probe faithfully reproduces the mRNA abundance.
  • the above process is the pretreatment for forming a reverse probe for amplifying the target nucleic acid.
  • 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).
  • 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.
  • 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.
  • GAPDH primer 15 1 hybridizes to GAPDH mRNA extension 131
  • P53 primer 15 2 generates P53 mRNA extension 13 2 Hybridize to
  • the temperature condition (5) is a thermal cycle in the amplification section 14
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the temperature of the section (15 1—1... Surface temperatures are about equal.
  • 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.
  • 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.
  • target RNAs GAPDH and P53
  • Sequences 4 to 7 are prepared as primers specific to these two types of mRNA.
  • AAAGTGGTC GTTGAGGGCA (SEQ ID NO: 5)
  • Sequence 4 and Sequence 5 are forward and reverse for GAPDH
  • 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.
  • 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.
  • 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.
  • the free forward primer complementarily binds to the extended reverse primer (eight hybridizes) and extends (Fig. 22).
  • the fixed extended reverse primer and the extended free forward primer 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.
  • a compartment for nucleic acid amplification for GAPDH and a compartment for nucleic acid amplification for P53 are separately provided.
  • each reparse primer is fixed on the surface of the compartment, each amplification reaction can be generated only in the corresponding compartment.
  • 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.
  • 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.
  • 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.
  • a temperature cycle 95 ° C .: 5 seconds—55: 15 seconds, at ⁇ 72: 15 seconds
  • 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.
  • the base length of the product is If it is long, it is necessary to make the extension synthesis time 15 seconds longer.
  • 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.
  • nucleic acids for example, genes
  • those genes can be simultaneously amplified and quantitatively assayed at once.
  • the reaction 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.

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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne une méthode pratique, économique et hautement précise d'analyse de l'expression d'un gène dans laquelle une amplification et une inspection individuelle sont mises en oeuvre simultanément pour une pluralité de types de cibles dans un échantillon unique. Une pluralité de sections permettant de contrôler des conditions de température individuellement sont présentes dans une puce et utilisées pour l'amplification et la détection d'acide nucléique, ce qui permet la mise en oeuvre de l'amplification et de la détection simultanément dans une puce unique. Etant donné qu'une pluralité de sections d'amplification et de sections de détection sont présentes et que des conditions de température peuvent être déterminées indépendamment, une pluralité de types de cibles contenues dans un échantillon unique peut être amplifiée et détectée collectivement. Etant donné que l'amplification et l'inspection peuvent être mises en oeuvre simultanément, l'analyseur est utilisé de manière appropriée tout en contribuant à la réduction des coûts. En outre, la sensibilité de détection est améliorée étant donné qu'un échantillon comprenant une pluralité de cibles ne doit pas être subdivisé.
PCT/JP2003/015490 2003-12-03 2003-12-03 Methode d'analyse d'acide nucleique, cellule d'analyse d'acide nucleique et analyseur d'acide nucleique WO2005054458A1 (fr)

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JP2005511265A JP4426528B2 (ja) 2003-12-03 2003-12-03 核酸分析方法、核酸分析用セル、および核酸分析装置
PCT/JP2003/015490 WO2005054458A1 (fr) 2003-12-03 2003-12-03 Methode d'analyse d'acide nucleique, cellule d'analyse d'acide nucleique et analyseur d'acide nucleique

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PCT/JP2003/015490 WO2005054458A1 (fr) 2003-12-03 2003-12-03 Methode d'analyse d'acide nucleique, cellule d'analyse d'acide nucleique et analyseur d'acide nucleique

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JP2009254259A (ja) * 2008-04-15 2009-11-05 Sony Corp 反応処理装置
JP2011237454A (ja) * 2006-03-21 2011-11-24 Koninklijke Philips Electronics Nv 加熱アレイを有するマイクロエレクトロニクスデバイス
WO2013021958A1 (fr) * 2011-08-05 2013-02-14 株式会社 東芝 Instrument de réaction d'amplification d'acide nucléique multiple
WO2013035867A1 (fr) * 2011-09-08 2013-03-14 株式会社 東芝 Récipient de réaction pour multiples acides nucléiques ainsi que procédé de détection mettant en oeuvre celui-ci
JP2016086689A (ja) * 2014-10-31 2016-05-23 セイコーエプソン株式会社 核酸増幅反応装置及び核酸検出方法
EP2553473A4 (fr) * 2010-03-30 2016-08-10 Advanced Liquid Logic Inc Plateforme pour opérations sur des gouttelettes
US9475051B2 (en) 2007-02-27 2016-10-25 Sony Corporation Nucleic acid amplifier
JP2017042112A (ja) * 2015-08-27 2017-03-02 株式会社理研ジェネシス 核酸増幅基板、該基板を用いた核酸増幅方法、及び核酸検出キット

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WO2013035867A1 (fr) * 2011-09-08 2013-03-14 株式会社 東芝 Récipient de réaction pour multiples acides nucléiques ainsi que procédé de détection mettant en oeuvre celui-ci
JP2016086689A (ja) * 2014-10-31 2016-05-23 セイコーエプソン株式会社 核酸増幅反応装置及び核酸検出方法
JP2017042112A (ja) * 2015-08-27 2017-03-02 株式会社理研ジェネシス 核酸増幅基板、該基板を用いた核酸増幅方法、及び核酸検出キット

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