WO2015060417A1 - Dnaの検出方法 - Google Patents
Dnaの検出方法 Download PDFInfo
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- WO2015060417A1 WO2015060417A1 PCT/JP2014/078298 JP2014078298W WO2015060417A1 WO 2015060417 A1 WO2015060417 A1 WO 2015060417A1 JP 2014078298 W JP2014078298 W JP 2014078298W WO 2015060417 A1 WO2015060417 A1 WO 2015060417A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
- G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
- G01N27/4473—Arrangements for investigating the separated zones, e.g. localising zones by electric means
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44756—Apparatus specially adapted therefor
- G01N27/44791—Microapparatus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
- G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
- G01N33/48721—Investigating individual macromolecules, e.g. by translocation through nanopores
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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
Definitions
- the present invention solves the above-mentioned conventional problems, bridges the DNA between the pair of electrodes of the detection device with the stretched DNA, and determines the property of the DNA bridging between the electrodes of the protrusions, whereby the DNA is fluorescent. It is an object of the present invention to provide a method for detecting DNA that can be easily and reliably detected without using a labeling substance such as a body.
- the distance between the electrodes is further changed at a predetermined frequency.
- the DNA bridging between the electrodes is a bundle containing double-stranded DNA molecules.
- the properties of a plurality of DNAs are determined by fixing a plurality of primers to a plurality of electrode pairs.
- a DNA detection method using a detection device comprising at least a pair of electrodes, wherein a primer is fixed to the electrodes and a solution containing a single-stranded DNA circular template
- the electrodes are immersed in the substrate, the circular template is annealed, and a single-stranded DNA product is generated by an RCA method, whereby the electrodes to which a predetermined voltage is applied are bridged by stretched DNA, and the electrodes are bridged. Covering the DNA to be coated with conductive fine particles and confirming the presence of DNA bridging between the electrodes.
- reference numeral 10 denotes a nano forceps device (Nanoweezers) as a detection device in the present embodiment, which is a device manufactured from a silicon substrate by MEMS technology, and is shown in Non-Patent Documents 1 and 2. And has a similar structure.
- the nano forceps device 10 includes a flat plate-like main body portion 11 having a substantially rectangular shape in a plan view, and a pair of parallel arm members 15 protruding from one side of the main body portion 11.
- the arm member 15 includes a movable arm 15a attached to the main body 11 so as to be movable or displaceable, and a fixed arm 15b attached so as not to be movable.
- the movable arm 15 a and the fixed arm 15 b are arranged so as to be aligned on a plane parallel to the surface of the main body 11, and the movable arm 15 a moves on a plane parallel to the surface of the main body 11.
- a moving tip 16a having a sharp shape is formed at the tip of the moving arm 15a, and a fixed tip 16b having a sharp shape is formed at the tip of the fixed arm 15b.
- the moving tip portion 16a and the fixed tip portion 16b face each other.
- tip part 16b integrally it demonstrates as the front-end
- FIG. The tip 16 functions as an electrode, and a predetermined AC voltage is applied.
- the main body 11 has a displacement sensor 18 for measuring the displacement amount of the movable arm 15a.
- the displacement sensor 18 is a capacitive sensor that detects a change in capacitance, and can measure the amount of displacement of the movable arm 15a.
- interval can be measured.
- An arm member terminal 23 for applying an AC voltage to the distal end portion 16 of the member 15 is formed.
- the nano forceps device 10 is used together with the solution storage device 30.
- the solution storage device 30 includes a pair of flat plate members 31 and a minute space 32 formed between the plate members 31.
- the plate member 31 includes an upper plate member 31a disposed on the upper side and a lower plate member 31b disposed on the lower side, and the upper plate member 31a and the lower plate member 31b are separated by a minute distance (for example, They are arranged so as to be parallel to each other with an interval of about 300 [ ⁇ m].
- the upper plate member 31a is preferably made of a transparent material such as glass.
- a solution containing DNA is injected and stored.
- the micro space 32 is open at the front (left side in FIG. 2), but since the distance between the upper plate member 31a and the lower plate member 31b is very small, the contained solution is almost leaked. Or evaporate.
- the surface of the main body portion 11 is substantially horizontal on the flat upper surface of the forceps holding base 41 fixed to the upper surface of the base portion 40 fixed to the floor or the like of the laboratory. It is attached as follows. Therefore, the arm member 15 is disposed so as to be substantially horizontal.
- the solution storage device 30 is attached to a solution holding device 42 fixed to the upper surface of the base portion 40 so as to face the forceps holding table 41.
- the solution holding device 42 includes a movable holding table 42a that can move toward and away from the forceps holding table 41, and the plate member 31 is substantially horizontal on a flat upper surface of the movable holding table 42a.
- the solution storage device 30 is attached.
- the height direction of the movable holding table 42 a is set so that the position of the minute space 32 formed between the plate members 31 corresponds to the position of the arm member 15 of the nano forceps device 10 attached to the forceps holding table 41. The position of is adjusted.
- the distal end portion 16 of the arm member 15 of the nano forceps device 10 is moved into the minute space 32 of the solution storage device 30 by moving the movable holding base 42 a in the horizontal direction. As shown in FIG. 2B, the distal end portion 16 can be immersed in a solution accommodated in the minute space 32.
- FIG. 3 is a photograph showing the positional relationship between the nano forceps device 10 and the solution storage device 30 actually manufactured by the inventor of the present invention.
- the tip 16 of the arm member 15 of the nano forceps device 10 is stored in the solution.
- the state which protruded from the micro space 32 of the apparatus 30 is shown.
- FIG. 4 is a diagram for explaining a method of fixing a primer to the distal end portion of the arm member of the nano forceps device according to the first embodiment of the present invention
- FIG. 5 is a diagram of the nano forceps device according to the first embodiment of the present invention.
- FIG. 6 is a diagram for explaining a method for generating a DNA product at the distal end portion of an arm member.
- FIG. 6 is a diagram for explaining DNA bridging between the distal end portions of the arm member of the nano forceps device according to the first embodiment of the present invention.
- FIG. 7 is a photomicrograph showing DNA bridging between the distal ends of the arm members of the nano forceps device according to the first embodiment of the present invention.
- the distance between the tip portions 16 facing each other is at least about 3 to 5 [ ⁇ m].
- the moving tip portion 16a is located on the left side and the fixed tip portion 16b is located on the right side, but either the moving tip portion 16a or the fixed tip portion 16b may be located on the left side. , Either may be located on the right side.
- the tip 16 is coated with gold on the surface.
- the movable holding base 42a is moved in the left direction in FIG. 2A, and the tip portion 16 is put into the micro space 32 of the solution storage device 30, and the first solution stored in the micro space 32 is obtained.
- the primer 51 is attached to the surface of the tip portion 16 and fixed.
- the first solution is a solution containing the primer 51.
- the annular template 52 is annealed at a constant temperature (isothermal) as shown in FIG.
- the annealing does not require a thermal cycle and can be performed at room temperature.
- a single-stranded DNA product 53 is generated by an RCA method performed in situ as shown in FIG. 5 (b) with the tip 16 immersed in the second solution.
- the RCA method is a powerful DNA amplification method that can be performed isothermally without requiring a thermal cycle, and can be performed at room temperature, and can achieve an amplification factor of about one billion times per hour.
- the inventor of the present invention performs the RCA method for 2 hours in an environment of 30 [° C.] to obtain a very long (for example, 100 kilobase [kB] or more) single-stranded DNA product 53.
- a very long for example, 100 kilobase [kB] or more
- the DNA bridge 54 includes a plurality of single-stranded DNA molecules, and DNA is bundled. Alternatively, the DNA bridge 54 may be a bundle of DNA containing double-stranded DNA molecules.
- the inventor of the present invention applies a high-frequency AC voltage (for example, 1 [MHz], 1 [MV / m]) to the tip portion 16 to generate a strong electric field, stretches the DNA molecule, and performs dielectrophoresis (DEP). : DNAphoresis) and attracted to the left and right tips 16, thereby forming a DNA bridge 54.
- a DNA bridge 54 having both ends fixed to the left and right tip portions 16 a long DNA bridge 54 of 15 [ ⁇ m] or more could be obtained.
- 7 is a photograph of the DNA bridge 54 actually formed when the distance between the left and right tip portions 16 is 6 [ ⁇ m], and the tip portion 16 is outside the minute space 32 of the solution storage device 30. This is a photo taken in
- the nano forceps device 10 as it is, it is possible to determine the properties of the DNA bridging between the tips 16.
- the characterization is performed in real time based on the mechanical properties of the DNA or the electrical properties of the DNA.
- the resonance frequency of the arm member 15 considered that the DNA bridge 54 is formed between the tip portions 16 by the above-described method is measured, and the resonance frequency is not applied to the tip portion 16 as shown in FIG.
- the resonance frequency of the arm member 15 in the initial state that is not attached it can be confirmed in situ whether or not the DNA bridge 54 is actually formed between the distal end portions 16. That is, DNA can be detected in situ.
- the resonance frequency is 1377.75 [Hz]
- single-stranded DNA molecules are bundled between the tip portions 16.
- the resonance frequency is 1669.39 [Hz].
- the DNA bridge 54 is actually formed between the tip portions 16. Whether it is formed or not can be confirmed in-situ.
- FIG. 10 is a first graph showing the electrical conductivity of DNA bridging between the distal ends of the arm members of the nano forceps device according to the first embodiment of the present invention
- FIG. 11 is the first embodiment of the present invention. It is a 2nd graph which shows the electrical conductivity of DNA which bridges between the front-end
- FIG. 4 (a) The current that flows when a voltage is applied between the distal ends 16 of the arm members 15 where the DNA bridge 54 is considered to have been formed between the distal ends 16 by the method described above is shown in FIG. 4 (a). Whether or not a DNA bridge 54 is actually formed between the tip portions 16 by comparing the current flowing when a voltage is applied between the tip portions 16 in the initial state where nothing is attached to the tip portion 16 as shown in FIG. Can be confirmed in-situ. That is, DNA can be detected in situ.
- the current does not increase so much. For example, the current does not increase even if the voltage is increased to 5 [V]. 3 does not reach 3 [pA], but in the state where the DNA bridge 54 in which single-stranded DNA molecules are bundled between the tip portions 16 is formed, if the voltage is increased, the current increases greatly. Is increased to 5 [V], the current exceeds 1.7 [mA].
- a voltage is applied between the tip portions 16 where the DNA bridge 54 is considered to have been formed by the above-described method, and a current is measured. With reference to FIGS. Whether or not 54 is formed can be confirmed in situ.
- the tip portion 16 is covered with gold.
- the bridging between the tips 16 by the stretched DNA is performed at a constant temperature. Therefore, since a temperature cycle operation for changing the temperature of a solution or the like to amplify the DNA as performed by a conventional detection method is not required, the DNA can be easily detected.
- the property determination of the DNA that bridges the tip portions 16 is performed based on the resonance frequency of the DNA that bridges the tip portions 16. More specifically, it is performed by changing the interval between the tip portions 16 at a predetermined frequency. Thereby, detection of DNA can be performed in situ.
- the DNA that bridges the tip 16 is bundled. Furthermore, this DNA is a bundle containing double-stranded DNA molecules.
- the determination of the properties of the DNA that bridges the tip portions 16 is performed based on the conductivity of the DNA that bridges the tip portions 16. Thereby, detection of DNA can be performed in situ.
- the property determination of the DNA that bridges the tip portions 16 is performed by real-time measurement of the DNA that bridges the tip portions 16. Therefore, it is possible to grasp changes in DNA properties over time.
- a plurality of pairs of tip portions 16 are arranged in parallel, for example, in the thickness direction, and different primers 51 are fixed to amplify and bridge DNA from each template interacting with the primer 51. , Characterization can be done.
- FIG. 12 is a diagram for explaining a method for coating gold microparticles with DNA bridging between the tips of the arm members of the nano forceps device according to the second embodiment of the present invention
- FIG. 13 is a diagram illustrating the second embodiment of the present invention.
- FIG. 14 is a micrograph showing a state in which gold particles are coated on DNA bridging between the tips of the arm members of the nano forceps device in the embodiment
- FIG. 14 is an arm member of the nano forceps device in the second embodiment of the present invention. It is a graph which shows the electrical conductivity of the state which coat
- DNA bridging the tips 16 of the arm member 15 of the nano forceps device 10 is coated with fine particles 57 made of gold (Au), thereby The presence of DNA bridging between the tips 16 is confirmed.
- FIG. 12A is the same as FIG. 6A described in the first embodiment.
- the DNA bridge 54 formed between the tip portions 16 is covered with the gold fine particles 57.
- the DNA bridge 54 covered with the gold fine particles 57 is easier to visually recognize and has a higher electric conductivity (lower electric resistance) than the uncoated DNA bridge 54. Therefore, whether or not the DNA bridge 54 is formed between the tip portions 16 by taking a micrograph or measuring a current flowing by applying a voltage between the tip portions 16, that is, between the tip portions 16. The presence of DNA bridging can be easily confirmed.
- FIG. 14 shows a measurement result of a current that flows when a voltage is applied between the tip portions 16 using the nano forceps device 10 actually manufactured by the inventors of the present invention. The voltage applied between the tips 16 of the nano forceps device 10 was changed, and the current value for each voltage was measured.
- the figure shows the measured value of the state of ss DNA bundle, that is, the state in which the DNA bridge 54 in which single-stranded DNA molecules are bundled between the tips 16 is formed, the state of DNA coated with Pd, ie, the tip 16
- the DNA bridge 54 formed between them is covered with the palladium fine particles 57
- the state of rinsed DNA coated with Pd that is, the DNA bridge 54 formed between the tips 16 is covered with the palladium fine particles 57.
- the measurement values are shown in a state where the DNA bridge 54 covered with the fine particles 57 is immersed in pure water and rinsed (washed).
- the horizontal axis indicates voltage [V]
- the vertical axis indicates current [pA].
- FIG. 15 is a conceptual diagram of a microfluidic device according to a third embodiment of the present invention
- FIG. 16 is a photograph of the microfluidic device according to the third embodiment of the present invention
- FIG. 17 is a third embodiment of the present invention.
- the figure which shows the electrode unit of the microfluidic device in a form FIG. 18 is the figure which shows the microchannel unit of the microfluidic device in the 3rd Embodiment of this invention
- FIG. 19 is the micro in the 3rd Embodiment of this invention
- FIG. 20 is a diagram showing a DNA capturing part of a microfluidic device in the third embodiment of the present invention
- FIG. 21 is a third embodiment of the present invention.
- FIG. 16A It is a microscope picture of the DNA capture part of the microfluidic device in FIG. 16A is a photograph showing the entire microfluidic device
- FIG. 16B is a photograph showing the electrode unit
- FIG. 16C is a photograph showing the tip of the electrode
- (b) is the center part enlarged view of an electrode unit
- (a) is a figure which shows the whole microchannel unit
- (b) is the center part enlarged view of a microchannel unit.
- FIG. 19 (a) is a micrograph of the protrusion structure part
- (b) is a photograph in which a black line indicating the outer periphery of the protrusion structure part is added to the photograph of (a), and in FIG.
- DNA is detected using a microfluidic device as shown in FIG.
- the microfluidic device includes an electrode unit 61 as shown in FIG. 17 and a microchannel unit 70 as shown in FIG. 18 integrated with the electrode unit 61. More specifically, in the microfluidic device, a cover glass in which an electrode unit 61 made of gold (Au) is patterned on one surface and the microchannel unit 70 are formed and superimposed on the cover glass.
- Au gold
- the cover glass is a rectangular transparent glass plate having a length of 26 [mm] and a width of 36 [mm].
- the first and second connection pads 62a and 62b are square portions having a length and width of 3 [mm], and the other end is connected to an external power source as shown in FIG. This is a portion to which the tip of the conductive wire is connected.
- the first and second shaft portions 63a and 63b are belt-like portions having a width of 200 [ ⁇ m].
- the first and second tooth portions 65a and 65b are narrow strip-like portions having a narrow width
- the first and second electrode tip portions 66a and 66b are first and second tooth portions 65a, It is a straight belt-like portion narrower than 65b.
- the first and second connecting portions 74a and 74b are square portions having a length and width of 3 [mm], and are connected to the external solution supply port and the solution discharge port as shown in FIG.
- the tip of the tube to which is connected is a portion connected via a pipe-shaped fitting.
- the microchannel 71 is a flow path for flowing a solution such as the first solution, the second solution, the third solution, or pure water, that is, a fluid, and has a width of 70 [ ⁇ m], It is a linear strip-shaped portion having a thickness (depth) of 3 [ ⁇ m].
- the DNA bridge 54 can be formed between the first electrode tip portion 66a and the second electrode tip portion 66b corresponding to each other.
- the DNA bridge 54 Since the solution flows in the microchannel 71, even if the DNA bridge 54 is formed between the corresponding first electrode tip portion 66a and the second electrode tip portion 66b, the DNA bridge 54 is May flow away from the first and second electrode tip portions 66a and 66b. However, since the solution stays in the protrusion structure portion 73, as shown in FIG. 15, between the first electrode tip portion 66a and the second electrode tip portion 66b located at a position corresponding to the protrusion structure portion 73. The formed DNA bridge 54 is not washed away.
- first and second electrode tip portions 66a and 66b can be obtained by changing the solution flowing in the microchannel 71 from, for example, the first solution to the second solution, or by changing to pure water for rinsing.
- the solution or fluid into which the water is immersed can be easily replaced.
- DNA can be bridged between the first electrode tip portion 66a and the second electrode tip portion 66b in the same manner as in the first and second embodiments. Can do.
- the inventor of the present invention can actually make a bridge between the first electrode tip portion 66a and the second electrode tip portion 66b by DNA by conducting a preliminary experiment using the microfluidic device. I confirmed that there was. In the preliminary experiment, DNA was not amplified. Further, in the microfluidic device used in the preliminary experiment, the combination of the electrode unit 61 and the microchannel unit 70 is not perfect, and as shown in FIG. 21A, a pair of first and second electrode tips The portions 66a and 66b and the pair of protruding structure portions 73 do not completely overlap. Further, in the preliminary experiment, for convenience, labeling with a fluorescent dye called YOYO-1 and observing the fluorescence, as shown in the dotted ellipse in FIG. 21 (b). In addition, the presence of DNA bridging between the first electrode tip portion 66a and the second electrode tip portion 66b was confirmed.
- a microfluidic device in which the electrode unit 61 including the pair of first and second electrode tip portions 66a and 66b as a pair of electrodes and the microchannel unit 70 is combined. use. Then, by a method similar to the method described in the first and second embodiments, a bridge between the pair of the first electrode tip portion 66a and the second electrode tip portion 66b is stretched with the DNA, and the DNA Can be determined and the presence of the DNA can be confirmed.
- the present invention relates to a DNA detection method capable of easily and reliably detecting DNA. Therefore, the present invention is based on the detection of mutations and pathogens without using a labeling substance such as a fluorescent substance, genotyping, various DNA interaction factors (for example, cross-linking chemical substances) and physical properties such as irradiation. It can be applied to a wide range of fields up to the determination of the physical properties of sequence-specific DNA corresponding to the physical effect. The physical characterization of sequence-specific DNA can be applied directly to translational research (eg, functional confirmation of cancer therapeutics) and environmental testing. The present invention enables DNA detection and physical property analysis on a single platform.
- a labeling substance such as a fluorescent substance, genotyping, various DNA interaction factors (for example, cross-linking chemical substances) and physical properties such as irradiation. It can be applied to a wide range of fields up to the determination of the physical properties of sequence-specific DNA corresponding to the physical effect.
- the physical characterization of sequence-specific DNA can be applied directly to translational
- the present invention can be applied to a DNA detection method.
Abstract
Description
16 先端部
51 プライマー
52 環状テンプレート
53 単鎖DNA生成物
57 微粒子
Claims (13)
- 少なくとも一対の電極を備える検出装置を使用するDNAの検出方法であって、
(a)前記電極にプライマーを固定し、
(b)単鎖DNAの環状テンプレートを含有する溶液内に前記電極を浸し、前記環状テンプレートをアニールし、RCA法によって単鎖DNA生成物を生成することにより、所定の電圧を印加した前記電極間を伸張したDNAによって橋渡しさせ、
(c)複数の単鎖DNA分子を含む前記電極間を橋渡しするDNAの性質決定を行うこと、
を含むことを特徴とするDNAの検出方法。 - 前記電極の少なくとも一部は金で被覆されている請求項1に記載のDNAの検出方法。
- 前記伸張したDNAによる前記電極間の橋渡しは、定温で行われる請求項1又は2に記載のDNAの検出方法。
- 前記電極間を橋渡しするDNAの性質決定は、前記電極間を橋渡しするDNAの共振周波数に基づいて行われる請求項1~3のいずれか1項に記載のDNAの検出方法。
- 前記電極間の間隔を所定の周波数で変化させる請求項4に記載のDNAの検出方法。
- 前記電極間を橋渡しするDNAは束になっている請求項1~5のいずれか1項に記載のDNAの検出方法。
- 前記電極間を橋渡しするDNAは、二本鎖DNA分子を含む束になっている請求項6に記載のDNAの検出方法。
- 前記電極間を橋渡しするDNAの性質決定は、前記電極間を橋渡しするDNAの導電性に基づいて行われる請求項1~7のいずれか1項に記載のDNAの検出方法。
- 前記電極間を橋渡しするDNAの性質決定は、前記電極間を橋渡しするDNAのリアルタイム計測によって行われる請求項1~8のいずれか1項に記載のDNAの検出方法。
- 単鎖の相補的DNAが生成されるように、対向する電極には異なるプライマーを固定する請求項1~9のいずれか1項に記載のDNAの検出方法。
- 複数の電極対に複数のプライマーを固定することによって、複数のDNAの性質決定を行う請求項1~10のいずれか1項に記載のDNAの検出方法。
- 少なくとも一対の電極を備える検出装置を使用するDNAの検出方法であって、
(a)前記電極にプライマーを固定し、
(b)単鎖DNAの環状テンプレートを含有する溶液内に前記電極を浸し、前記環状テンプレートをアニールし、RCA法によって単鎖DNA生成物を生成することにより、所定の電圧を印加した前記電極間を伸張したDNAによって橋渡しさせ、
(c)前記電極間を橋渡しするDNAに導電性の微粒子を被覆し、
(d)前記電極間を橋渡しするDNAの存在を確認すること、
を含むことを特徴とするDNAの検出方法。 - 複数の電極対に複数のプライマーを固定することによって、複数のDNAの存在を確認する請求項12に記載のDNAの検出方法。
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EP14856487.5A EP3064934B1 (en) | 2013-10-25 | 2014-10-24 | Dna detection method |
JP2015543920A JP6385356B2 (ja) | 2013-10-25 | 2014-10-24 | Dnaの検出方法 |
CA2929042A CA2929042A1 (en) | 2013-10-25 | 2014-10-24 | Dna detection method |
IL24522216A IL245222B (en) | 2013-10-25 | 2016-04-20 | A method for discovering DNA |
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EP (1) | EP3064934B1 (ja) |
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US10481158B2 (en) | 2015-06-01 | 2019-11-19 | California Institute Of Technology | Compositions and methods for screening T cells with antigens for specific populations |
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JP2005520130A (ja) * | 2002-03-08 | 2005-07-07 | インテグレイティッド ナノ−テクノロジーズ エルエルシー | 試料の生物物質の複合検出方法 |
JP2007512810A (ja) * | 2003-11-10 | 2007-05-24 | ジーンオーム サイエンシーズ、インク. | 増加した感度を持つ核酸検出方法 |
JP2008501122A (ja) * | 2004-05-28 | 2008-01-17 | ナノゲン・インコーポレイテッド | ナノスケール電子式検出システムおよびその製造方法 |
JP2013013375A (ja) * | 2011-07-05 | 2013-01-24 | Tosoh Corp | 電極上における核酸の増幅反応を伴う電気化学的検出方法 |
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US9810659B2 (en) * | 2013-02-08 | 2017-11-07 | Board Of Trustees Of Michigan State Univeristy | Nanoparticle-serialized oligonucleotide methods, compositions, and articles |
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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US10481158B2 (en) | 2015-06-01 | 2019-11-19 | California Institute Of Technology | Compositions and methods for screening T cells with antigens for specific populations |
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CA2929042A1 (en) | 2015-04-30 |
IL245222A0 (en) | 2016-06-30 |
IL245222B (en) | 2019-10-31 |
EP3064934A4 (en) | 2017-06-28 |
EP3064934B1 (en) | 2020-06-03 |
US10845333B2 (en) | 2020-11-24 |
JPWO2015060417A1 (ja) | 2017-03-09 |
JP6385356B2 (ja) | 2018-09-05 |
US20170168012A1 (en) | 2017-06-15 |
EP3064934A1 (en) | 2016-09-07 |
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