WO2012102326A1 - 核酸分子の多型識別方法 - Google Patents
核酸分子の多型識別方法 Download PDFInfo
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- WO2012102326A1 WO2012102326A1 PCT/JP2012/051625 JP2012051625W WO2012102326A1 WO 2012102326 A1 WO2012102326 A1 WO 2012102326A1 JP 2012051625 W JP2012051625 W JP 2012051625W WO 2012102326 A1 WO2012102326 A1 WO 2012102326A1
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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
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
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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
- C12Q1/6813—Hybridisation assays
- C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
Definitions
- the present invention uses an optical system capable of detecting light from a minute region in a solution, such as an optical system of a confocal microscope or a multiphoton microscope, and uses a genetic polymorphism such as a somatic mutation or a single nucleotide polymorphism. And a method for identifying nucleic acids having similar base sequences.
- nucleic acid identification methods using a FRET probe for example, the 5 ′ end side and the 3 ′ end side have complementary base sequences, and both ends use a fluorescent substance and a quenching substance, respectively.
- molecular beacon method that uses a labeled single-stranded nucleic acid (molecular beacon probe) (for example, see Non-Patent Document 1).
- molecular beacon probe for example, see Non-Patent Document 1
- both ends associate to form an intramolecular loop, which is in a quenching state, but the intramolecular loop is resolved by hybridizing with the target nucleic acid molecule. And emits fluorescence.
- a genetic polymorphism can be identified using a molecular beacon that specifically hybridizes with a specific genotype of the genetic polymorphism (see, for example, Non-Patent Document 2).
- Non-Patent Document 3 there is a method for detecting a nucleic acid using FRET using a fluorescent intercalator and a fluorescent probe.
- a probe labeled with a fluorescent substance is hybridized with another single-stranded nucleic acid
- a fluorescent intercalator enters between the base pairs of the formed double-stranded nucleic acid.
- FRET occurring between this fluorescent intercalator and a fluorescent substance that labels the probe, it is possible to distinguish between a fluorescent probe that exists alone and a fluorescent probe that forms a double-stranded nucleic acid. it can.
- a probe that specifically hybridizes with a specific genotype of a gene polymorphism and a probe that specifically hybridizes with another genotype are different from each other in fluorescent substances.
- polymorphism can be identified by detecting FRET occurring between each fluorescent substance and the fluorescent intercalator (see, for example, Non-Patent Document 4).
- Fluorescence intensity distribution analysis FIDA, for example, Patent Document 3
- photon counting histogram Photon Counting Histogram: PCH, for example, Patent Document 4
- Patent Documents 5 and 6 propose a method of detecting a fluorescent substance based on the time lapse of a fluorescence signal of a sample solution measured using an optical system of a confocal microscope.
- Patent Document 7 discloses a method of measuring weak light from fluorescent fine particles circulated in a flow cytometer or fluorescent fine particles fixed on a substrate by using a photon counting technique, and fluorescent fine particles in a flow or on a substrate. A signal arithmetic processing technique for detecting the presence of a signal is proposed.
- the sample required for measurement has an extremely low concentration compared with the conventional method. It can be very small (the amount used in one measurement is about several tens of ⁇ L), and the measurement time is greatly shortened (measurement of time on the order of seconds is repeated several times in one measurement). . Therefore, these technologies are used especially when conducting analysis on rare or expensive samples often used in the field of medical and biological research and development, clinical diagnosis of diseases, screening of physiologically active substances, etc. When the number of specimens is large, it is expected to be a powerful tool capable of performing experiments or tests at a lower cost or faster than conventional biochemical methods.
- the magnitude of the temporal fluctuation of the measured fluorescence intensity is calculated by statistical processing, and the sample solution is based on the magnitude of the fluctuation.
- Various characteristics such as fluorescent molecules entering and exiting the minute region are determined. Therefore, in order to obtain a significant result in the above optical analysis technique, the concentration or number density of the fluorescent molecules to be observed in the sample solution must be as long as one second in the equilibrium state. It is preferable that the number of fluorescent molecules that can be statistically processed within the measurement time is adjusted so as to enter and exit the minute region.
- the concentration or the number density of the above-described fluorescent molecules is preferably prepared so that about one fluorescent molecule or the like is always present in a minute region (typically, confocal Since the volume of the volume is about 1 fL, the concentration of fluorescent molecules and the like is preferably about 1 nM or more. In other words, when the concentration or number density of the particles to be observed in the sample solution is significantly lower than the level capable of statistical processing (for example, significantly lower than 1 nM), the observation target is measured in a minute region. A situation occurs that only rarely enters in time.
- Patent Documents 5 and 6 In the method for detecting a fluorescent substance using the optical system of a confocal microscope described in Patent Documents 5 and 6, the statistical processing relating to the fluctuation of the fluorescence intensity as described above is not performed, and the measurement time over several seconds is used.
- the presence or absence of a fluorescent molecule or the like to be observed in the sample can be specified by the presence or absence of the generation of a significant intensity fluorescent signal. Therefore, it is possible to obtain a correlation between the frequency of the fluorescent signal having a significant intensity and the number of particles such as fluorescent molecules in the sample.
- Patent Document 6 suggests that detection sensitivity is improved when a random flow is generated in a sample solution by stirring.
- Patent Document 7 is to individually detect the presence of fluorescent fine particles in a flow in a flow cytometer or fluorescent fine particles fixed on a substrate, and in a normal state in a sample solution. It is not a technique for detecting particles such as molecules or colloids dissolved or dispersed in the sample, that is, particles moving randomly in the sample solution. Therefore, the concentration or number density of particles dissolved or dispersed in the sample solution cannot be calculated quantitatively.
- Patent Document 7 since the technique of Patent Document 7 includes a process such as measurement in a flow cytometer or immobilization of fluorescent particles on a substrate, the amount of sample required for inspection is optical analysis such as FCS, FIDA, PCH, etc. It is considered to require a complicated and sophisticated operation technique for the practitioner as well as the number of techniques.
- the genomic sample that is the target for detecting the gene polymorphism is often a very small amount, such as a specimen in a clinical test. For this reason, even when the concentration of the nucleic acid to be analyzed in the sample is very low, it is desired to develop a method capable of identifying the polymorphism of the nucleic acid to be analyzed with high sensitivity.
- Non-Patent Document 2 a molecular beacon is used at 300 nM, which cannot be said to be a measurement in a sparse state.
- the base sequence is identified for a relatively high concentration of nucleic acid of about several tens to several hundreds nM.
- polymorphism has not been identified for a nucleic acid sample having a relatively low concentration on the order of pM.
- An object of the present invention is to provide a method for identifying a nucleic acid having a polymorphic sequence such as a gene polymorphism that does not require statistical processing performed by optical analysis techniques such as FCS, FIDA, and PCH. To do.
- optical analysis techniques such as FCS, FIDA, and PCH.
- this nucleic acid molecule polymorphism identification method provided by the present invention, even if the concentration or number density of the observation target particles is lower than the level handled by optical analysis techniques such as FCS, FIDA, and PCH, A novel optical analysis technique capable of detecting the state or characteristics of particles to be observed in a solution is used.
- the present inventors have used a nucleic acid probe that specifically hybridizes with one type of nucleic acid among polymorphic sequences to form an association with the nucleic acid probe.
- the concentration of the nucleic acid molecule to be analyzed in the sample is extremely high by detecting an aggregate including a nucleic acid probe using a scanning molecule counting method. It was found that polymorphisms can be identified with high sensitivity even in the case of a low value.
- the formation reaction of the association between the nucleic acid probe and the nucleic acid molecule in the sample, and the detection of the association by the scanning molecule counting method are complementary to the base sequence of a type other than the one of the polymorphic sequences.
- the present invention was completed by finding that polymorphisms can be identified with high accuracy by carrying out in the presence of an oligonucleotide (decoy nucleic acid) having a simple base sequence.
- the scanning molecule counting method is a novel optical analysis technique proposed by the present applicant in Japanese Patent Application No. 2010-044714.
- the present invention has the following aspects. (1) (a) A sample solution containing a first nucleic acid probe that specifically hybridizes with a single-stranded nucleic acid molecule having a first type base sequence in a polymorphic sequence, and a nucleic acid molecule to be analyzed A sample solution preparation step to be prepared; and (B) an association step for associating nucleic acid molecules in the sample solution prepared in the step (a); (C) After the step (b), a calculation step of calculating the number of molecules of the aggregate containing the first nucleic acid probe in the sample solution prepared in the step (a); (D) an identification step for identifying a polymorphism of the nucleic acid molecule to be analyzed based on the result of the step (c); (E) a detection area moving step of moving the position of the light detection area of the optical system in the sample solution using the optical system of a confocal microscope or a multiphoton microscope in the previous step (c); (F) a fluorescence detection step of detecting
- a method for identifying a polymorphism of a nucleic acid molecule further comprising an oligonucleotide having a typical base sequence.
- the sample solution further includes a fluorescent double-stranded nucleic acid binding substance
- One of the fluorescent substance labeling the first nucleic acid probe and the fluorescent double-stranded nucleic acid binding substance is a fluorescent substance that serves as an energy donor in a fluorescence energy transfer phenomenon, and the other is the fluorescent energy. It is a substance that becomes an energy acceptor in the transfer phenomenon
- the fluorescence emitted from the aggregate containing the first nucleic acid probe is between the fluorescent substance labeled with the first nucleic acid probe and the fluorescent double-stranded nucleic acid binding substance.
- the method for identifying a polymorphism of a nucleic acid molecule according to the above (2) which is fluorescence emitted by a fluorescence energy transfer phenomenon that has occurred in 1).
- fluorescence energy transfer occurs, and in the state where an association with other single-stranded nucleic acid molecules is formed, fluorescence energy transfer does not occur.
- a fluorescent substance that serves as an energy donor and a substance that serves as an energy acceptor are combined.
- the method for identifying a polymorphism of a nucleic acid molecule according to the above (2) wherein the fluorescence emitted from the aggregate containing the nucleic acid probe is fluorescence emitted from the fluorescent substance serving as the energy donor.
- nucleic acid molecule according to any one of (1) to (5), wherein in the step of moving the position of the light detection region, the position of the light detection region is moved at a predetermined speed. Polymorphism identification method. (7) In the step of moving the position of the light detection region, the position of the light detection region is moved at a speed faster than the diffusion movement speed of the aggregate. The method for identifying a polymorphism of a nucleic acid molecule according to any one of the above. (8) Based on the shape of the detected time-series optical signal in the step of individually detecting optical signals from the individual aggregates from the detected light and detecting the aggregates individually.
- step (b) The method for identifying a polymorphism of a nucleic acid molecule according to any one of (1) to (7) above, wherein it is detected that one aggregate has entered the light detection region.
- step (b) The nucleic acid molecule according to any one of (1) to (8), wherein the sample solution contains one or more selected from the group consisting of a surfactant, formamide, dimethyl sulfoxide, and urea. Polymorph identification method.
- step (b) the temperature of the sample solution prepared in step (a) is set to 70 ° C.
- nucleic acid molecule according to any one of (1) to (9), wherein the nucleic acid molecule in the sample solution is associated by lowering the temperature at a rate of temperature decrease of 0.05 ° C./second or more.
- Polymorph identification method (11) From the above (1), wherein the first nucleic acid probe and the second nucleic acid probe are constituted by combining two or more molecules selected from the group consisting of DNA, RNA, and nucleic acid analogs (10) The method for identifying a polymorphism of a nucleic acid molecule according to any one of (10).
- polymorphic sequence according to any one of (1) to (11) above, wherein the polymorphic sequence is a base sequence including a polymorphic site of a gene polymorphism or a base sequence including a mutation site of somatic mutation.
- Nucleic acid molecule polymorphism identification method (13) The method for identifying a polymorphism of a nucleic acid molecule according to the above (12), wherein the somatic mutation is a K-ras gene mutation.
- the nucleic acid molecule polymorphism identification method of the present invention a statistical method for calculating fluctuations in fluorescence intensity is used. There is no need to execute processing. Therefore, according to the method for identifying a polymorphism of a nucleic acid molecule of the present invention, a polymorphism can be identified even if a nucleic acid molecule having a polymorphic sequence to be analyzed is present in a trace amount in a sample. Can do.
- the method for identifying a polymorphism of a nucleic acid molecule of the present invention detection of an aggregate of a nucleic acid probe and a nucleic acid molecule in a sample by a scanning molecule counting method is performed.
- the reaction is performed in the presence of an oligonucleotide having a base sequence complementary to the base sequence.
- the nucleic acid probe is effectively inhibited from forming an association with another type of nucleic acid molecule between the formation of the association and detection, and as a result, the nonspecific association by the nucleic acid probe is suppressed. Formation of coalescence can be effectively suppressed, and polymorphism can be identified with very high accuracy.
- FIG. 1A is a schematic diagram of the internal structure of an optical analyzer for the scanning molecule counting method.
- FIG. 1B is a schematic diagram of a confocal volume (observation region of a confocal microscope).
- FIG. 1C is a schematic diagram of a mechanism for changing the direction of the mirror 7 and moving the position of the light detection region in the sample solution.
- FIGS. 2A and 2B are a schematic diagram for explaining the principle of light detection by an optical analysis technique for the scanning molecule counting method and a schematic diagram of a temporal change in measured light intensity, respectively.
- FIGS. 1A is a schematic diagram of the internal structure of an optical analyzer for the scanning molecule counting method.
- FIG. 1B is a schematic diagram of a confocal volume (observation region of a confocal microscope).
- FIG. 1C is a schematic diagram of a mechanism for changing the direction of the mirror 7 and moving the position of the light detection region in the sample solution.
- 3A and 3B are a model diagram in the case where the observation target particle crosses the light detection region while performing the Brownian motion, and a diagram illustrating an example of a time change of the photon count (light intensity) in that case.
- It is. 4A and 4B show the case where the observation target particle crosses the light detection region by moving the position of the light detection region in the sample solution at a speed faster than the diffusion movement speed of the observation target particle. It is a figure which shows the example of this, and the example of the time change of the photon count (light intensity) in that case.
- FIG. 5 is a flowchart showing a processing procedure for counting particles from a temporal change in photon count (light intensity) measured by the scanning molecule counting method.
- FIG. 6 is a diagram for explaining an example of a signal processing step of a detection signal in a processing procedure for counting particles from a time change of photon count (light intensity) measured by a scanning molecule counting method.
- FIG. 7 shows an actual measurement example (bar graph) of photon count data measured by the scanning molecule counting method, a curve (dotted line) obtained by smoothing the data, and a Gaussian function (solid line) fitted in the peak existence region. Show. In the figure, a signal labeled “noise” is ignored as it is a signal due to noise or foreign matter. It is the figure which showed typically an example of the clustering by the number of molecules of a polymorphism and an aggregate.
- Example 1 it is the figure which showed the value of the peak number counted for every GTT variation
- Example 2 it is the figure which showed the value of the peak number counted for every GTT variation
- FIG. 10A shows the result of the sample solution to which the decoy nucleic acid was not added
- FIG. 10B shows the result of the sample solution to which the decoy nucleic acid was added.
- the vertical axis represents the number of molecules of an aggregate containing a mutant (GTT) molecular beacon probe (the number of mutant (GTT) peaks), and the horizontal axis represents the molecular of an aggregate containing a wild type (GGT) molecular beacon probe. It is the figure which showed the value of the counted peak number for every mutation rate in the nucleic acid molecule of the analysis object added to the sample solution as number (wild type (GGT) peak number).
- the vertical axis represents the number of molecules containing the mutant (GTT) molecule beacon probe (number of mutant (GTT) peaks), and the horizontal axis represents the molecule of the aggregate containing the wild type (GGT) molecular beacon probe.
- Example 6 it is the figure which showed the value of the peak number counted for every GTT variation
- Example 7 it is the figure which showed the value of the number of peaks counted for every variation
- Example 8 for each mutation rate of the nucleic acid molecule to be analyzed added to the sample solution, the value of the number of peaks counted for the aggregate containing the mutant type (GTT) probe and the aggregate containing the wild type (GGT) probe FIG.
- the scanning molecule counting method will be described.
- particles that emit light that moves in a sample solution and moves randomly while traversing the sample solution by the minute region (hereinafter referred to as “luminescent particles”) cross the minute region.
- the light emitted from the luminescent particles in the micro area is detected, whereby each luminescent particle in the sample solution is individually detected to count the luminescent particles and the concentration of the luminescent particles in the sample solution.
- the sample required for measurement may be a very small amount (for example, about several tens of ⁇ L), the measurement time is short, and in the case of the optical analysis technology such as FIDA Compared to the above, it is possible to quantitatively detect the characteristics such as the concentration or number density of the luminescent particles having a lower concentration or number density.
- the luminescent particle means a particle in which a particle to be observed and a luminescent probe are bonded or associated.
- a “luminescent probe” is a substance (usually a molecule or an aggregate thereof) that has a property of binding or associating with a particle to be observed and emits light, and is typically a fluorescent particle. However, it may be a particle that emits light by phosphorescence, chemiluminescence, bioluminescence, light scattering, or the like.
- the first nucleic acid probe and the second nucleic acid probe used in the nucleic acid molecule polymorphism identification method of the present invention correspond to a luminescent probe.
- the “light detection region” of the optical system of the confocal microscope or the multiphoton microscope is a minute region in which light is detected in those microscopes, and illumination light is given from the objective lens. Corresponds to a region where the illumination light is condensed. In the confocal microscope, such a region is determined particularly by the positional relationship between the objective lens and the pinhole.
- the light is sequentially detected while moving the position of the light detection region in the sample solution, that is, while scanning the sample solution with the light detection region. Then, when the moving photodetection region includes a luminescent probe that is bound or associated with a randomly moving particle, the light from the luminescent probe is detected, thereby detecting the presence of one particle. (Depending on the mode of the experiment, the luminescent probe may be dissociated from the particle when it is detected after light is once bound to the particle to be detected). Then, the light signals from the luminescent probes are individually detected in the sequentially detected light, thereby detecting the presence of particles (coupled with the luminescent probe) one by one individually, Various information on the state in the solution is acquired.
- the number of particles detected individually may be counted to count the number of particles detected during movement of the position of the light detection region (particle counting). ).
- information on the number density or concentration of particles in the sample solution can be obtained by combining the number of particles and the amount of movement of the position of the light detection region.
- the total volume of the movement locus of the position of the light detection region is specified by an arbitrary method, for example, by moving the position of the light detection region at a predetermined speed, the number density or concentration of the particles is concrete. Can be calculated automatically.
- the absolute number density value or concentration value is not directly determined, but the relative number density or concentration ratio with respect to a plurality of sample solutions or a standard sample solution serving as a reference for the concentration or number density is calculated. It may be like this.
- the optical detection path is moved by changing the optical path of the optical system, so that the movement of the light detection area is quick and the mechanical movement of the sample solution is performed. Because there is virtually no mechanical vibration or hydrodynamic action, it is possible to measure light in a stable state without the influence of mechanical action on the particles to be detected (in the sample solution).
- the physical properties of the particles may change when vibrations or flows are applied.) Further, since a configuration in which the sample solution is circulated is not necessary, measurement and analysis can be performed with a small amount (about 1 to several tens of ⁇ L) of the sample solution as in the case of FCS, FIDA, and the like.
- a light emitting probe coupled to one particle is detected from sequentially detected optical signals (if one light emitting probe is coupled to one particle, a plurality of light emitting probes are In the case of binding to one particle and the case where it is a luminescent probe dissociated from the particle after binding to one particle according to the experimental mode. This may be done based on the shape of the optical signal detected in time series. In the embodiment, typically, when an optical signal having an intensity greater than a predetermined threshold is detected, it is detected that a luminescent probe bound to one particle has entered the light detection region. Good.
- the moving speed of the position of the light detection region in the sample solution is based on the characteristics of the luminescent probe bound to the particles or the number density or concentration in the sample solution. It may be changed as appropriate. As will be appreciated by those skilled in the art, the manner of light detected from a luminescent probe bound to a particle can vary depending on its properties or number density or concentration in the sample solution. In particular, when the moving speed of the light detection region is increased, the amount of light obtained from the light emitting probe bonded to one particle is reduced, so that light from the light emitting probe bonded to one particle is measured with high accuracy or sensitivity. It is preferable that the moving speed of the light detection region is appropriately changed so that it can be performed.
- the moving speed of the position of the light detection region in the sample solution is preferably set so that the luminescent probe bound to the particles to be detected (that is, of the present invention).
- the nucleic acid molecule polymorphism identification method it is set higher than the diffusion movement speed (average particle movement speed due to Brownian motion) of the aggregate including the first nucleic acid probe and the aggregate including the second nucleic acid probe. Is done.
- the scanning molecule counting method when the photodetection region passes through the position of the luminescent probe bonded to one particle, the light emitted from the luminescent probe is detected, and the luminescent probe is individually detected. To detect.
- the moving speed of the light detection region is set to be higher than the diffusion moving speed of the luminescent probe bound to the particles (specifically, the aggregate including the first nucleic acid probe or the second nucleic acid probe).
- the luminescent probe bound to one particle is made to correspond to one optical signal (representing the presence of the particle). It becomes possible. Since the diffusion movement speed varies depending on the luminescent probe bonded to the particle, as described above, the movement speed of the light detection region is appropriately changed according to the characteristics (particularly the diffusion constant) of the luminescent probe bonded to the particle. It is preferable.
- the change of the optical path of the optical system for moving the position of the light detection area may be performed by an arbitrary method.
- the position of the light detection region may be changed by changing the optical path using a galvanometer mirror employed in a laser scanning optical microscope.
- the movement trajectory of the position of the light detection region may be arbitrarily set, and may be selected from, for example, a circle, an ellipse, a rectangle, a straight line, and a curve.
- the scanning molecule counting method is configured to detect light from the light detection region of the confocal microscope or the multiphoton microscope, as in the case of the optical analysis technology such as FIDA, the light detection mechanism itself, The amount of sample solution may be trace as well.
- the photoanalysis technique of the scanning molecule counting method is necessary for an optical analysis technique in which the number density or concentration of particles is such as FIDA. Applicable to sample solutions that are significantly lower than certain levels.
- the scanning molecule counting method since each particle dispersed or dissolved in the solution is individually detected, the information is used to quantitatively count the particle concentration or the concentration or number density of the particles in the sample solution. It is possible to obtain information on calculation or concentration or number density. That is, according to the scanning molecule counting method, particles passing through the light detection region and detected light signals are detected one by one so that the particles are detected one by one. It is possible to count the particles to be determined, and to determine the concentration or number density of the particles in the sample solution with higher accuracy than before. Actually, according to the nucleic acid molecule polymorphism identification method of the present invention that individually detects aggregates and the like containing the first nucleic acid probe and counts the number thereof to determine the particle concentration, these associations in the sample solution are determined. Polymorphic nucleic acids can be identified even when the concentration of the combination is lower than the concentration that can be determined based on the fluorescence intensity measured by a fluorescence spectrophotometer or a plate reader.
- the sample solution is not affected by mechanical vibration or hydrodynamic action.
- the sample solution is observed in a mechanically stable state. Therefore, for example, when a flow is generated in a sample (when applying a flow, it is difficult to always provide a uniform flow velocity, the apparatus configuration is complicated, and the amount of sample required is greatly increased. , There is a possibility that particles, luminescent probes or conjugates or other substances in the solution may be altered or denatured due to hydrodynamic action due to flow.) The reliability of quantitative detection results is improved. To do.
- measurement can be performed in a state where there is no influence or artifact due to mechanical action on particles to be detected in the sample solution (in the present invention, aggregates including the first nucleic acid probe).
- the scanning molecule counting method is a combination of a confocal microscope optical system capable of executing FCS, FIDA, etc. and a photodetector as schematically illustrated in FIG. It can be realized by using an optical analyzer.
- the above-described optical analyzer will be described with reference to FIG.
- the optical analyzer 1 includes optical systems 2 to 17 and a computer 18 for controlling the operation of each part of the optical system and acquiring and analyzing data.
- the optical system of the optical analyzer 1 may be the same as the optical system of a normal confocal microscope, in which the laser light (Ex) emitted from the light source 2 and propagated through the single mode fiber 3 is a fiber.
- the light is emitted as a divergent light at an angle determined by a specific NA at the outgoing end of the light, becomes parallel light by the collimator 4, is reflected by the dichroic mirror 5, the reflection mirrors 6, 7, and the objective lens 8. Is incident on.
- a microplate 9 in which a sample container or well 10 into which a sample solution of 1 to several tens of ⁇ L is dispensed is typically arranged is emitted from the objective lens 8.
- the laser beam is focused in the sample solution in the sample container or well 10 to form a region (excitation region) having a high light intensity.
- particles to be observed and luminescent probes that bind to the particles typically molecules to which luminescent labels such as fluorescent dyes have been added are dispersed or dissolved, and are bound to the luminescent probes.
- an associated particle a luminescent probe that is once bonded to the particle and then dissociated from the particle depending on the mode of experiment
- the luminescent probe is excited and light is emitted during that time.
- the emitted light (Em) passes through the objective lens 8 and the dichroic mirror 5, is reflected by the mirror 11, is collected by the condenser lens 12, passes through the pinhole 13, and passes through the barrier filter 14.
- the pinhole 13 is disposed at a position conjugate with the focal position of the objective lens 8, and as a result, as shown in FIG. Only the light emitted from the focal region of the laser beam, that is, the excitation region as schematically shown, passes through the pinhole 13, and the light from other than the excitation region is blocked.
- 1B is usually a light detection region in the present optical analyzer having an effective volume of about 1 to 10 fL (typically, the light intensity is in the region). Gaussian distribution or Lorentzian distribution with the vertex at the center.
- the effective volume is the volume of an approximately elliptical sphere bounded by the plane where the light intensity is 1 / e 2), called confocal volume .
- light from a conjugate of one particle and a luminescent probe or light from a luminescent probe for example, faint light from one or several fluorescent dye molecules is detected.
- an ultrasensitive photodetector that can be used for photon counting is used.
- a stage position changing device 17a for moving the horizontal position of the microplate 9 may be provided on a microscope stage (not shown) in order to change the well 10 to be observed.
- the operation of the stage position changing device 17a may be controlled by the computer 18. With this configuration, it is possible to achieve quick measurement even when there are a plurality of specimens.
- the optical path of the optical system is changed to scan the sample solution with the light detection region, that is, the position of the focal region (ie, the light detection region) in the sample solution.
- a mechanism for moving is provided.
- a mechanism for moving the position of the light detection region for example, a mirror deflector 17 that changes the direction of the reflection mirror 7 may be employed as schematically illustrated in FIG.
- the mirror deflector 17 may be the same as a galvanometer mirror device provided in a normal laser scanning microscope. Further, in order to achieve a desired movement pattern of the position of the light detection region, the mirror deflector 17 is driven in cooperation with light detection by the light detector 16 under the control of the computer 18.
- the movement locus of the position of the light detection region may be arbitrarily selected from a circle, an ellipse, a rectangle, a straight line, a curve, or a combination thereof (in the program in the computer 18, various movement patterns can be selected. Good.)
- the position of the light detection region may be moved in the vertical direction by moving the objective lens 8 up and down.
- mechanical vibrations and hydrodynamic actions are substantially caused in the sample solution. It is possible to eliminate the influence of the dynamic action on the observation object and to achieve stable measurement.
- the above optical system is used as a multiphoton microscope. In that case, since there is light emission only in the focal region (light detection region) of the excitation light, the pinhole 13 may be removed.
- the optical systems 2 to 5 for generating the excitation light may be omitted.
- the optical system of the confocal microscope is used as it is.
- the optical analyzer 1 may be provided with a plurality of excitation light sources 2 as shown in the figure, and the wavelength of the excitation light is appropriately selected according to the wavelength of the light that excites the combined particle or luminescent probe or the luminescent probe. You may be able to do it.
- a plurality of photodetectors 16 may be provided, and when a sample includes a combination of a plurality of types of particles having different wavelengths and a combination of luminescent probes or a luminescent probe, the light from them is changed depending on the wavelength. It may be possible to detect them separately.
- a spectroscopic analysis technique such as FIDA is superior to a conventional biochemical analysis technique in that it requires an extremely small amount of sample and can perform inspection quickly.
- the concentration and characteristics of the observation target particle are calculated based on the fluctuation of the fluorescence intensity. Therefore, in order to obtain an accurate measurement result, The concentration or number density of the observation target particles is a level at which about one observation target particle is always present in the light detection region CV during the measurement of the fluorescence intensity, and a significant light intensity (photon count) is always obtained during the measurement time. It is required to be detected.
- the concentration or number density of the observation target particles is lower than that, for example, if the observation target particles are at a level that only occasionally enters the light detection region CV, a significant light intensity (photon count) is measured. It appears only in a part of the time, and it is difficult to calculate the fluctuation of the light intensity with high accuracy. Also, if the concentration of the observation target particle is much lower than the level at which one observation target particle is always present in the light detection region during measurement, the light intensity fluctuation calculation is affected by the background. The measurement time becomes longer in order to obtain significant light intensity data sufficient for calculation. In contrast, the scanning molecule counting method can detect characteristics such as the number density or concentration of particles to be observed even when the concentration of particles to be observed is lower than the level required by spectroscopic techniques such as FIDA. It is.
- light detection schematically illustrated in FIG. 2 is performed.
- a mechanism for moving the position of the light detection region is driven to change the optical path.
- light detection is performed while moving the position of the light detection region CV in the sample solution, that is, while scanning the sample solution by the light detection region CV.
- one particle a fluorescent dye is bound as a luminescent probe in the figure.
- the concentration of the fluorescently labeled particles is measured from the fluorescence intensity measured by a fluorescence spectrophotometer or a plate reader. It is possible to measure to a lower concentration than when doing so.
- the fluorescence intensity is proportional to the concentration of the fluorescently labeled particles.
- the concentration of the fluorescently labeled particles when the concentration of the fluorescently labeled particles is sufficiently low, the amount of noise signal with respect to the amount of signal due to the light emitted from the fluorescently labeled particles increases (deterioration of the S / N ratio), and fluorescence The proportional relationship between the concentration of the labeled particles and the amount of optical signal is broken, and the accuracy of the determined concentration value deteriorates.
- the noise signal in the process of detecting a signal corresponding to each particle from the detected optical signal, the noise signal is excluded from the detection result, and only the signal corresponding to each particle is counted to obtain the concentration. Since the calculation is performed, it is possible to detect the fluorescence intensity with high accuracy even at a lower concentration than when the concentration is detected on the assumption that the fluorescence intensity is proportional to the concentration of the fluorescently labeled particles.
- the fluorescence intensity is fluorescently labeled. Therefore, the measurement accuracy of the particle concentration on the higher particle concentration side is improved as compared with the conventional method of determining the concentration under the assumption that the concentration is proportional to the concentration of the generated particles.
- the luminescent probe relatively binds to the particles when the concentration of the target target particle increases.
- the number of since the fluorescence intensity per observation target particle is reduced, the proportional relationship between the concentration of the fluorescently labeled particles and the light amount is lost, and the accuracy of the determined concentration value is deteriorated.
- the scanning molecule counting method since the signal corresponding to each particle is detected from the detected optical signal, the influence of the reduction of the fluorescence intensity per particle is small, and the concentration is calculated from the number of particles.
- the fluorescence intensity can be detected with high accuracy even at a higher concentration than when the concentration is detected on the assumption that the fluorescence intensity is proportional to the concentration of the fluorescently labeled particles.
- the measurement of the light intensity in the optical analysis of the scanning molecule counting method is that the mirror deflector 17 is driven during the measurement to move the position of the light detection region in the sample solution (scanning in the sample solution). It may be executed in the same manner as the light intensity measurement step in FCS or FIDA. In the operation process, typically, after the sample solution is injected into the well 10 of the microplate 9 and placed on the stage of the microscope, the user inputs an instruction to start measurement to the computer 18.
- the computer 18 stores a program stored in a storage device (not shown) (a procedure for changing the optical path to move the position of the light detection region in the sample solution, and the light detection region during the movement of the position of the light detection region).
- a program stored in a storage device not shown
- irradiation of excitation light and measurement of light intensity in the light detection region in the sample solution are started.
- the mirror deflector 17 drives the mirror 7 (galvanomirror) to move the position of the light detection region in the well 10.
- the photodetector 16 converts the sequentially detected light into an electrical signal and transmits it to the computer 18, and the computer 18 performs time-series light intensity data from the transmitted optical signal in an arbitrary manner. Generate and save.
- the photodetector 16 is an ultra-sensitive photodetector that can detect the arrival of one photon. Therefore, the light detection is performed sequentially for a predetermined unit time over a predetermined time.
- BINTIME is a photon counting executed in a mode in which, for example, the number of photons arriving at the photodetector every 10 ⁇ s is measured, and time-series light intensity data is time-series photon count data. It may be.
- the moving speed of the position of the light detection region during the measurement of the light intensity may be a predetermined speed that is arbitrarily set, for example, experimentally or so as to suit the purpose of analysis.
- the size or volume of the region through which the light detection region has passed is required, so that the moving distance is grasped.
- the movement of the position of the light detection region is executed in the manner.
- the movement speed is basically a constant speed.
- the present invention is not limited to this.
- the moving speed of the position of the light detection region in order to quantitatively and accurately perform individual detection of the observation target particles from the measured time-series light intensity data or counting of the number of observation target particles.
- the moving speed is determined by the random movement of the observation target particle (more precisely, the combination of the particle and the luminescent probe or the luminescent probe which is decomposed and released after the binding to the particle), that is, the moving speed due to the Brownian movement. It is preferable to set a faster value. Since the observation target particle of the photoanalysis technique of the scanning molecule counting method is a particle that is dispersed or dissolved in the solution and moves freely at random, the position moves with time by Brownian motion.
- the particle moves randomly within the region as schematically illustrated in FIG.
- the light intensity changes randomly as shown in FIG. 3B (as already mentioned, the excitation light intensity in the photodetection area decreases outward with the center of the area as the apex), and each individual. It is difficult to specify a significant change in light intensity corresponding to the observation target particle. Therefore, preferably, as depicted in FIG. 4 (A), the particles cross the light detection region in a substantially straight line, so that in the time-series light intensity data, as illustrated in FIG.
- the light intensity change profile corresponding to each individual particle becomes substantially uniform (if the particle passes through the light detection region substantially linearly, the light intensity change profile is substantially the same as the excitation light intensity distribution.
- the movement speed of the position of the light detection region is faster than the average movement speed (diffusion movement speed) due to the Brownian motion of the particles so that the correspondence between the individual observation target particles and the light intensity can be easily identified. Is set.
- an observation target particle having a diffusion coefficient D (more precisely, a conjugate of a particle and a luminescent probe, or a luminescent probe that is decomposed and released after binding to the particle) has a photodetection region having a radius Wo due to Brownian motion.
- the moving speed of the position of the light detection region may be set to a value sufficiently faster than that with reference to the Vdif.
- Wo is about 0.62 ⁇ m
- Vdif is 1.0 ⁇ Since it is 10 ⁇ 3 m / s
- the moving speed of the position of the light detection region may be set to 15 mm / s, which is approximately 10 times that.
- the profile of the change in the light intensity by setting the moving speed of the position of the light detection region in various ways is expected (typically, the excitation light intensity distribution).
- a preliminary experiment for finding a condition that is substantially the same as that described above may be repeatedly performed to determine a suitable moving speed of the position of the light detection region.
- the computer 18 may execute the following light intensity analysis by the processing according to the program stored in the storage device.
- the trajectory when one observation target particle passes through the light detection region is substantially linear as shown in FIG.
- the change in light intensity corresponding to the particle is a profile that reflects the light intensity distribution in the light detection region (determined by the optical system) (typically depicted in FIG. 6A) (normally , Substantially bell-shaped). Therefore, in one method of detecting the observation target particle, when the threshold Io is set for the light intensity and the time width ⁇ in which the light intensity exceeding the threshold continues is within a predetermined range, the profile of the light intensity is It may be determined that one particle has passed through the light detection region, and one observation target particle may be detected.
- the predetermined range for the threshold value Io and the time width ⁇ for the light intensity is a combination of the observation target particle and the luminescent probe that moves relative to the light detection region at a predetermined speed (or is decomposed after the combination with the particle).
- the specific value may be arbitrarily set experimentally, and the binding between the observation target particle and the luminescent probe It may be selectively determined by the properties of the body (or the luminescent probe that has been degraded and released after binding to the particle).
- Counting of observation target particles may be performed by counting the number of particles detected by the above-described detection target particle detection method using an arbitrary method. However, when the number of particles is large, for example, the processing illustrated in FIGS. 5 and 6B may be performed.
- step 100 the time-series optical signal data (photon count data) is acquired (step 100)
- smoothing is performed on the time-series optical signal data (FIG. 6B, “detection result (unprocessed)” in the uppermost stage).
- smoothing processing is performed (step 110, “smoothing” in the upper part of FIG. 6B).
- the light emitted from the combination of the particle and the luminescent probe or the light emitted from the luminescent probe is probabilistically emitted, and data values may be lost in a very short time. It can be ignored.
- the smoothing process may be performed by a moving average method, for example. It should be noted that the parameters for executing the smoothing process, such as the number of data points averaged at a time in the moving average method and the number of moving averages, are the moving speed (scanning speed) of the position of the light detection region at the time of optical signal data acquisition. , BIN TIME may be set as appropriate.
- Step 120 in order to detect a time domain (peak existence area) where a significant signal exists in the time-series optical signal data after the smoothing process, a first-order differential value for the time of the time-series optical signal data after the smoothing process is calculated.
- the time differential value of the time-series optical signal data as illustrated in the lower “time differential” in FIG. 6B, the value change at the time of the change of the signal value becomes large. Therefore, the time differential value is referred to.
- the start point and end point of a significant signal (peak signal) can be advantageously determined.
- a significant signal (peak signal) is sequentially detected on the time-series optical signal data, and it is determined whether or not the detected peak signal is a signal corresponding to the observation target particle.
- peak signal a significant signal
- the start point and the end point of one peak signal are searched and determined, A peak presence region is identified (step 130).
- a bell-shaped function fitting is performed on the smoothed time-series optical signal data in the peak existence area (FIG. 6 (B) lower “bell-shaped function fitting”).
- Parameters such as the peak intensity Imax of the bell-shaped function, the peak width (full width at half maximum) w, and the correlation coefficient (of the least square method) in the fitting are calculated (step 140).
- the bell-shaped function to be fitted is typically a Gaussian function, but may be a Lorentz-type function.
- the calculated bell-shaped function parameter is assumed as a bell-shaped profile parameter drawn by an optical signal detected when a combination of one particle and the light-emitting probe or the light-emitting probe passes through the light detection region. It is determined whether it is within the range, that is, whether the peak intensity, the peak width, and the correlation coefficient are each within a predetermined range (step 150).
- the range that is, whether the peak intensity, the peak width, and the correlation coefficient are each within a predetermined range
- the calculated bell-shaped function parameter is within an expected range in the optical signal corresponding to the combination of one particle and the luminescent probe or the luminescent probe.
- the signal is determined to be a signal corresponding to one observation target particle, whereby one observation target particle is detected and counted as one particle (the number of particles is counted up).
- Step 160 On the other hand, as shown in the right side of FIG. 7, a peak signal whose calculated bell-shaped function parameter is not within the assumed range is ignored as noise.
- the search and determination of the peak signal in the processing of the above steps 130 to 160 is repeatedly performed over the entire time-series optical signal data, and is counted as a particle every time one observation target particle is detected.
- the particle count value obtained so far is set as the number of observation target particles detected in the time series optical signal data.
- (Iii) Determination of the number density or concentration of the observation target particles
- the total volume of the region through which the light detection region passes during acquisition of time-series optical signal data is used.
- the number density or concentration of the particles is determined.
- the effective volume of the light detection region varies depending on the wavelength of the excitation light or detection light, the numerical aperture of the lens, and the adjustment state of the optical system, it is generally difficult to calculate from the design value. Accordingly, it is not easy to calculate the total volume of the region through which the light detection region passes. Therefore, typically, for a solution (reference solution) with a known particle concentration, the light intensity measurement, particle detection, and counting described above are performed under the same conditions as the measurement of the sample solution to be examined.
- the total volume of the region through which the light detection region has passed that is, the relationship between the number of detected particles and the concentration is determined from the number of detected particles and the concentration of the reference solution particles. It's okay.
- the reference solution particles are preferably luminescent labels (fluorescent dyes or the like) having the same wavelength characteristics as the particles formed by the observation target particles and the luminescent probe conjugate (or the luminescent probe released after binding to the observation target particles). It may be.
- the total volume Vt of the region through which the light detection region has passed is expressed by the following equation (5).
- Vt N / C (5) Given by.
- a plurality of solutions having different concentrations are prepared as reference solutions, and measurement is performed on each of the solutions.
- the calculated average value of Vt is adopted as the total volume Vt of the region through which the light detection region has passed. It may be.
- the number density c of the sample solution particles whose particle counting result is n is expressed by the following formula (6).
- c n / Vt (6) Given by.
- the volume of the light detection region and the total volume of the region through which the light detection region has passed are given by any method, for example, using FCS or FIDA, without depending on the above method. It's okay.
- the optical analyzer of the present embodiment information on the relationship between the concentration C of various standard particles and the number N of particles (formula (5)) is assumed for the assumed movement pattern of the light detection region.
- the information stored in advance in the storage device of the computer 18 may be used by the user of the device when the optical analysis is performed.
- the nucleic acid molecule polymorphism identification method of the present invention uses a nucleic acid probe that specifically binds to a specific type (first type) of nucleic acid molecules in a polymorphic sequence, and the nucleic acid probe and the nucleic acid to be analyzed.
- the type of the nucleic acid molecule to be analyzed is discriminated based on the amount of aggregates formed by hybridizing the molecule.
- the association is detected by the scanning molecule counting method described above.
- the scanning molecule counting method is a measurement method capable of measuring particles having fluorescence for each particle in a state where the molecules are discrete, and therefore, even for a nucleic acid molecule having a relatively low concentration of pM order or less. Measurement is possible. Therefore, according to the method for identifying a polymorphism of a nucleic acid molecule of the present invention, formed aggregates can be counted with high sensitivity even when the concentration of the nucleic acid molecule to be analyzed in the sample solution is very low. .
- a polymorphic sequence is a base sequence having two or more similar base sequences, and a nucleic acid molecule having a polymorphic sequence is referred to as a polymorphic nucleic acid.
- that the base sequences are similar means that, for example, 1 to several nucleobases out of 10 to 20 are different by substitution, deletion, insertion, or duplication.
- identifying a polymorphism distinguishes a nucleic acid molecule having a specific type of base sequence in the polymorphic sequence from a nucleic acid molecule having a base sequence of a type other than the specific type in the polymorphic sequence. Means that.
- the polymorphic sequence to be analyzed in the nucleic acid molecule polymorphism identification method of the present invention may be a base sequence on a gene such as a single gene polymorphism (SNP) or the like, or an artificial base sequence. May be.
- SNP single gene polymorphism
- somatic mutations include K-ras gene mutations.
- the nucleic acid probe specifically binds to a single-stranded nucleic acid molecule having a specific type in the polymorphic sequence.
- the nucleic acid probe is a single-stranded nucleic acid molecule having the polymorphic sequence. It means preferentially hybridizing with a single-stranded nucleic acid molecule of a specific type over a single-stranded nucleic acid molecule of a type other than the specific type. For this reason, the nucleic acid probe may have a base sequence that is completely complementary to a specific type of base sequence, and has a base sequence having a mismatch other than a polymorphic site where the bases differ between the polymorphic sequences. It may be.
- a first nucleic acid probe (hereinafter referred to as a first nucleic acid probe) that specifically hybridizes with a single-stranded nucleic acid molecule having a specific type (first type) base sequence in a polymorphic sequence is used.
- first nucleic acid probe By hybridizing the nucleic acid molecule to be analyzed and the first nucleic acid probe and detecting the formed aggregate, it is possible to identify whether the polymorphic sequence in the nucleic acid molecule is the same as the first type. .
- nucleic acid molecule to be analyzed when distinguishing a single-gene polymorphism that is known to have two types, wild type and mutant type, it is possible to determine whether the nucleic acid molecule to be analyzed is mutant type or wild type by setting the mutant type as the first type. Can be identified.
- the nucleic acid molecule to be analyzed is a mutant type, an aggregate containing the first nucleic acid probe is detected from the sample solution.
- the nucleic acid probe such as the first nucleic acid probe used in the present invention may be an oligonucleotide composed of DNA, an oligonucleotide composed of RNA, or a chimeric oligonucleotide composed of DNA and RNA. Alternatively, it may contain a part or all of a nucleic acid analog capable of forming a nucleotide chain or base pair in the same manner as a natural nucleobase.
- Nucleic acid analogues include those in which side chains of natural nucleotides (naturally occurring nucleotides) such as DNA and RNA are modified with functional groups such as amino groups, and labeled with proteins, low molecular compounds, etc. And the like.
- BNA Bridged Nucleic Acid
- nucleotides in which the 4′-position oxygen atom of natural nucleotides is substituted with sulfur atoms, and hydroxyl groups in the 2′-position of natural ribonucleotides are substituted with methoxy groups.
- Nucleotides Hexitol Nucleic Acid (HNA), peptide nucleic acid (PNA) and the like.
- the nucleic acid to be analyzed may be DNA, RNA, or artificially amplified such as cDNA.
- the nucleic acid probe in the scanning molecule counting method, is different from the state in which the nucleic acid probe exists alone and the state in which the nucleic acid probe forms an aggregate with other single-stranded nucleic acid molecules.
- An association formed from a probe and other single-stranded nucleic acid molecules can be distinguished from a nucleic acid probe that does not form an association.
- a nucleic acid probe that does not have a double-stranded structure when present alone by using a fluorescent double-stranded nucleic acid binding substance that specifically binds to a double-stranded structure as a first nucleic acid probe, a nucleic acid probe that does not have a double-stranded structure when present alone.
- the aggregate containing the nucleic acid probe can be detected separately from the nucleic acid probe present alone.
- the nucleic acid probe present alone is not labeled with a fluorescent substance, it does not emit fluorescence.
- the fluorescent double-stranded nucleic acid binding substance binds to the aggregate containing the nucleic acid probe, the fluorescence emitted from the fluorescent double-stranded nucleic acid binding substance is detected.
- nucleic acid probe labeled with a fluorescent substance that causes fluorescence energy transfer (FRET) between the fluorescent double-stranded nucleic acid binding substance an aggregate containing the nucleic acid probe exists alone. It can be detected separately from the nucleic acid probe. That is, one of the fluorescent double-stranded nucleic acid binding substance and the fluorescent substance that labels the nucleic acid probe serves as an FRET energy donor, and the other serves as the FRET energy acceptor. Fluorescence emitted from a fluorescent substance that labels the nucleic acid probe is detected from a nucleic acid probe such as the first nucleic acid probe that exists alone.
- FRET fluorescence energy transfer
- Examples of the fluorescent double-stranded nucleic acid binding substance that specifically binds to the double-stranded structure include a fluorescent intercalator, a groove binder to which a fluorescent substance is bound, and the like. If the amount of the fluorescent intercalator entering between the base pairs of the aggregate is too large, the background when detecting the fluorescence emitted by FRET becomes too high, which may affect the detection accuracy. is there. For this reason, it is preferable to design the nucleic acid probe such as the first nucleic acid probe so that the region forming the double strand in the aggregate is 400 bp or less.
- molecular beacon probes can be used as nucleic acid probes.
- a molecular beacon probe is an oligonucleotide that forms an intramolecular structure in the state of a single-stranded nucleic acid molecule.
- FRET occurs in the state of a single-stranded nucleic acid molecule between a fluorescent substance serving as an energy donor and a substance serving as an energy acceptor (fluorescent substance or quenching substance) in FRET, and other single-stranded nucleic acids. In the state of an aggregate formed by hybridizing with a molecule, it is bound so that FRET does not occur.
- Fluorescence emitted from a fluorescent substance that is an energy donor is detected from the nucleic acid probe that forms an aggregate, whereas a fluorescent substance that is an energy donor is detected from a nucleic acid probe that exists alone
- the fluorescence emitted from is not detected or attenuated. Therefore, by detecting the fluorescence emitted from the fluorescent substance serving as an energy donor, the aggregate including the nucleic acid probe can be detected separately from the nucleic acid probe present alone.
- the molecular beacon probe used as the first nucleic acid probe has a base sequence that can hybridize with a specific type of polymorphic sequence, and is complementary to the 3 ′ end region and the 5 ′ end region. And an oligonucleotide having a simple base sequence.
- a fluorescent substance serving as an energy donor or a substance serving as an energy acceptor is bound to the 3 ′ end side, one remaining on the 5 ′ end side is bound, and a region on the 3 ′ end side And 5 ′ terminal side have complementary base sequences, and base pairs are formed in these base sequences to form an intramolecular structure (so-called stem-loop structure).
- the mutually complementary regions forming the intramolecular base pair of the molecular beacon probe may be present so as to sandwich the region consisting of the base sequence complementary to the target nucleic acid molecule.
- the region on the “terminal side” may be a region including the 3 ′ end or the 5 ′ end, or a region not including it.
- the number of bases and the base sequence of the region forming the base pair are less stable than the stability of the associated base nucleic acid molecule and the base pair is formed under the measurement conditions. It is only necessary to obtain it.
- the fluorescent substance is not particularly limited as long as it emits fluorescence by emitting light of a specific wavelength, and is appropriately selected from fluorescent dyes used in FCS, FIDA, and the like. Can be used.
- the nucleic acid probe can be labeled with a fluorescent substance by a conventional method.
- a fluorescence that is designed to specifically hybridize with a single-stranded nucleic acid molecule containing a first type base sequence in a state adjacent to the first nucleic acid probe and that labels the first nucleic acid probe
- a nucleic acid probe labeled with a fluorescent substance or a quenching substance in which FRET occurs between the substance and the substance the aggregate containing the first nucleic acid probe is detected separately from the first nucleic acid probe present alone. be able to.
- FRET occurs in the aggregate consisting of these three members. For this reason, the aggregate
- the nucleic acid molecule polymorphism identification method of the present invention comprises the following steps.
- B associating nucleic acid molecules in the sample solution prepared in the step (a)
- D A step of identifying a polymorphism of the nucleic acid molecule to be analyzed based on the result of the step (c).
- a sample solution containing the first nucleic acid probe and the nucleic acid molecule to be analyzed is prepared.
- the sample solution is prepared by adding the first nucleic acid probe and the nucleic acid molecule to be analyzed to an appropriate solvent.
- the solvent is not particularly limited as long as it does not inhibit the formation of the aggregate by the first nucleic acid probe and the detection of the aggregate by the scanning molecule counting method, and is generally used in the technical field. It can be used by appropriately selecting from among the existing buffers.
- the buffer include a phosphate buffer such as PBS (phosphate buffered saline, pH 7.4), a Tris buffer, and the like.
- a fluorescent double-stranded nucleic acid binding substance that specifically binds to a double-stranded structure or a nucleic acid probe other than the first nucleic acid probe is used to detect an aggregate containing the first nucleic acid probe, the first nucleic acid probe or the like Similarly to the above, a fluorescent double-stranded nucleic acid binding substance or other nucleic acid probe is added to the sample solution.
- the nucleic acid molecules in the sample solution prepared in the step (a) are associated.
- the nucleic acid molecule in the sample solution is a double-stranded nucleic acid molecule
- it is preferably denatured before associating.
- “denaturing a nucleic acid molecule” means dissociating base pairs.
- a base pair formed by mutually complementary base sequences in a single-stranded nucleic acid molecule is dissociated to release the intramolecular structure into a single-stranded structure, or a double-stranded nucleic acid molecule is converted into a single-stranded nucleic acid.
- the nucleic acid probe is an oligonucleotide containing a nucleic acid-like substance such as PNA, even if the nucleic acid molecule is a double-stranded nucleic acid molecule, the aggregate containing the nucleic acid probe is not subjected to any special denaturation treatment. It may be possible to form.
- thermo denaturation can denature nucleic acid molecules in the sample solution by subjecting the sample solution to high temperature treatment. Generally, it can be denatured by incubating at 90 ° C. for DNA and 70 ° C. for RNA for several seconds to 2 minutes, but the denaturation temperature varies depending on the length of the base of the target nucleic acid molecule. The temperature is not limited to the above as long as it can be modified.
- denaturation by low salt concentration treatment can be performed by adjusting the sample solution to have a sufficiently low salt concentration by, for example, dilution with purified water or the like.
- the nucleic acid molecules in the sample solution are associated.
- the temperature of the sample solution is set to a temperature at which the first nucleic acid probe and the single-stranded nucleic acid molecule having the first type base sequence can specifically hybridize.
- the nucleic acid molecules in the sample solution can be associated with each other as appropriate.
- the salt concentration of the sample solution is changed to the first nucleic acid probe and the single-stranded nucleic acid having the first type base sequence by adding a salt solution or the like.
- the nucleic acid molecules in the sample solution can be associated with each other by raising the concentration to a level at which the molecules can specifically hybridize.
- the temperature at which the first nucleic acid probe and the single-stranded nucleic acid molecule having the first type base sequence can be specifically hybridized is such that the association of the nucleic acid molecule having the polymorphic sequence and the nucleic acid probe melts. It can be obtained from a curve.
- the melting curve is obtained by changing the temperature of a solution containing only the first nucleic acid probe and the single-stranded nucleic acid molecule having the first type base sequence from a high temperature to a low temperature, and changing the absorbance and fluorescence intensity of the solution. It can be determined by measuring.
- the temperature in the range can be a temperature at which the first nucleic acid probe and the single-stranded nucleic acid molecule having the first type base sequence can specifically hybridize.
- the melting curve is similarly determined by changing the salt concentration in the solution from the low concentration to the high concentration instead of the temperature, and the first nucleic acid probe and the first nucleic acid probe having the first type base sequence are determined. The concentration at which the strand nucleic acid molecule can specifically hybridize can be determined.
- the temperature at which the first nucleic acid probe can specifically hybridize with the single-stranded nucleic acid molecule having the first type base sequence can be generally substituted with the Tm value (melting temperature).
- the Tm value of a region that hybridizes with a single-stranded nucleic acid molecule having a first type base sequence can be obtained from the base sequence information of the first nucleic acid probe ( The temperature at which 50% of the double-stranded DNA is dissociated into single-stranded DNA can be calculated.
- the temperature of the sample solution is set such that the Tm value of the region having a base sequence complementary to the single-stranded nucleic acid molecule having the base sequence of the first type in the first nucleic acid probe is ⁇ 3 ° C. Reduce to about temperature.
- the temperature of the sample solution in order to suppress non-specific hybridization, it is preferable to lower the temperature of the sample solution relatively slowly when forming an aggregate. For example, after denaturing nucleic acid molecules by setting the temperature of the sample solution to 70 ° C. or higher, the liquid temperature of the sample solution can be lowered at a temperature decrease rate of 0.05 ° C./second or higher.
- an oligonucleotide having a base sequence complementary to a base sequence of a type other than the first type in the polymorphic sequence is used to detect the aggregate in step (c).
- a decoy nucleic acid of a type other than the first type is included in the sample solution, or after step (b) and before step (c) A decoy nucleic acid of a type other than the first type is added to the sample solution.
- the sample solution is prepared in the step (a).
- the amount of the decoy nucleic acid of a type other than the first type added to the sample solution is not particularly limited, but the amount of the decoy nucleic acid of a type other than the first type in the sample solution is a large amount to be analyzed. For example, it is preferably added so as to be more than about 5 times the amount of the type nucleic acid.
- Non-specific hybridization between the added nucleic acid probe and other types of nucleic acid molecules is achieved by the presence of an excessive amount of oligonucleotides that specifically hybridize with nucleic acid molecules of other types in the sample solution. It can be effectively suppressed.
- a surfactant in order to suppress nonspecific hybridization, it is also preferable to add a surfactant, formamide, dimethyl sulfoxide, urea or the like to the sample solution in advance before step (c).
- a surfactant formamide, dimethyl sulfoxide, urea or the like
- These compounds may be added alone or in combination of two or more. By adding these compounds, nonspecific hybridization can be made difficult to occur in a relatively low temperature environment.
- These compounds may be included in the sample solution when preparing the sample solution in step (a), or may be added to the sample solution after step (b) and before step (c).
- step (c) the number of molecules of the aggregate containing the first nucleic acid probe in the sample solution is calculated by a scanning molecule counting method. Specifically, the sample solution after forming the aggregate is placed in the optical analyzer for the scanning molecule counting method, and the fluorescence emitted from the aggregate is detected and analyzed by the above method. Thus, the number of molecules of the aggregate is calculated.
- step (d) the polymorphism of the nucleic acid molecule to be analyzed is identified based on the result of step (c).
- step (c) an aggregate containing the first nucleic acid probe is detected in step (c).
- the mutant type is the first type
- the mutant probe (Mt probe) that specifically hybridizes with the mutant nucleic acid molecule is the first nucleic acid probe.
- each type in the polymorphic sequence is a first type
- the polymorphic nucleic acid of the present invention is subjected to the polymorphic nucleic acid identification method of the present invention for all types, so that the polymorphic nucleic acid is more accurately obtained.
- Can be identified For example, when a single nucleotide polymorphism is identified, the method for identifying the polymorphism of the nucleic acid molecule of the present invention is performed using the mutant type as the first type, and the nucleic acid molecule of the present invention is further defined using the wild type as the first type.
- a mutant probe that specifically hybridizes with a mutant nucleic acid molecule and an oligonucleotide (wild-type decoy nucleic acid) having a base sequence complementary to the wild-type base sequence are used.
- Steps (a) to (c) are performed, and independently, a wild type probe (Wt probe) that specifically hybridizes with a wild type nucleic acid molecule, and a base sequence complementary to the mutant type base sequence
- Steps (a) to (c) are performed using the oligonucleotide (mutant decoy nucleic acid).
- nucleic acid molecule to be analyzed was a mutant type or a wild type based on the counting result of the number of molecules of the aggregate containing the Mt probe and the counting result of the number of molecules of the aggregate containing the Wt probe Can be identified.
- the nucleic acid molecule to be analyzed is a mutant type, an association is preferentially formed with the Mt probe. Therefore, the number of molecules of the association containing the Mt probe is significantly larger than that of the association containing the Wt probe. .
- Steps (a) to (c) performed using the Mt probe and steps (a) to (c) performed using the Wt probe may be performed separately or substantially simultaneously. For example, after preparing a sample solution to which an Mt probe, a nucleic acid molecule to be analyzed, and a wild-type decoy nucleic acid are added, forming an association of nucleic acid molecules in the sample solution, and counting the formed associations, A sample solution to which a Wt probe, a nucleic acid molecule to be analyzed and a mutant decoy nucleic acid are added may be formed, and an aggregate of nucleic acid molecules in the sample solution may be formed, and the aggregates formed may be counted.
- a nucleic acid molecule to be analyzed a Wt probe, an Mt probe, a mutant decoy nucleic acid, and a wild-type decoy nucleic acid are added to one sample solution to form an aggregate of nucleic acid molecules in the sample solution. Thereafter, the aggregate containing the Wt probe and the aggregate containing the Mt probe can be distinguished and counted.
- a Wt probe formed by adding a Wt probe using a control single-stranded nucleic acid molecule whose polymorphic sequence is known instead of the nucleic acid molecule to be analyzed is used.
- the number of molecules of the aggregate including the probe and the number of molecules of the aggregate including the Mt probe formed when the Mt probe is added are calculated.
- a single-stranded nucleic acid molecule for control As a single-stranded nucleic acid molecule for control, a wild-type nucleic acid molecule, a mutant nucleic acid molecule, or a mixture of a wild-type nucleic acid molecule and a mutant nucleic acid molecule in equimolar amounts, respectively, and a nucleic acid as a negative control are used. Add probe only. Information necessary for clustering the relationship between the polymorphism of the single-stranded nucleic acid molecule for control and the number of molecules of the aggregate can be obtained from the results of each measurement.
- FIG. 8 schematically shows an example of clustering based on the number of molecules of polymorphisms and aggregates.
- the number of molecules of the aggregate containing the Wt probe is clearly larger than the number of molecules of the aggregate containing the Mt probe (in FIG. 8, cluster 2 ).
- the number of molecules of the aggregate containing the Mt probe is clearly larger than the number of molecules of the aggregate containing the Wt probe (cluster 3 in FIG. 8).
- the number of molecules of the aggregate containing the Wt probe and the number of molecules of the aggregate containing the Mt probe are both large. (Cluster 4 in FIG. 8).
- the first nucleic acid probe and the single-stranded nucleic acid molecule containing the first type base sequence specifically hybridize in a region other than the polymorphic sequence region.
- Other nucleic acid probes designed as described above can also be used in combination. Since the second nucleic acid probe designed in this manner binds to a nucleic acid molecule having a polymorphic sequence in a region other than the polymorphic sequence, the nucleic acid molecule having a genotype other than the first type in the polymorphic sequence Together form an aggregate.
- the second nucleic acid probe is added to the sample solution together with the first nucleic acid probe and the nucleic acid molecule to be analyzed, and the temperature in the sample solution is simultaneously increased and then gradually decreased, whereby the nucleic acid to be analyzed is
- the molecule is the first type
- an aggregate including the first nucleic acid probe and the second nucleic acid probe is formed in the sample solution.
- the nucleic acid molecule to be analyzed is a wild type
- an aggregate containing the second nucleic acid probe is formed in the sample solution, but the aggregate containing the first nucleic acid probe and the second nucleic acid probe is Not formed.
- the nucleic acid molecule to be analyzed is the first type, and the second nucleic acid probe includes the first nucleic acid molecule.
- the nucleic acid molecule to be analyzed can be identified as a type other than the first type in the polymorphic sequence.
- the second nucleic acid probe is a single-stranded nucleic acid containing the first type base sequence as described above.
- a single-stranded nucleic acid molecule ie, a polymorphic sequence having a base sequence complementary to a single-stranded nucleic acid molecule containing the first type of base sequence, which can be designed to specifically hybridize with the molecule.
- the second nucleic acid probe can also be designed to specifically hybridize with a single-stranded nucleic acid molecule associated with a single-stranded nucleic acid molecule having Also in this case, the second nucleic acid probe associates with a single-stranded nucleic acid molecule that is a complementary strand of the single-stranded nucleic acid molecule having a polymorphic sequence, in a region where the base sequence is common between the polymorphic nucleic acids.
- the second nucleic acid probe forms an association with a single-stranded nucleic acid molecule that forms a double-stranded nucleic acid molecule by associating with the single-stranded nucleic acid molecule having the polymorphic sequence regardless of the type of the polymorphic sequence.
- the single-stranded nucleic acid molecule containing the first type base sequence exists as a double-stranded nucleic acid molecule (associate) with a single-stranded nucleic acid molecule complementary to the single-stranded nucleic acid molecule.
- the single-stranded nucleic acid molecule is associated with the first nucleic acid probe
- the single-stranded nucleic acid molecule complementary to the single-stranded nucleic acid molecule is associated with the second nucleic acid probe.
- the nucleic acid molecule to be analyzed is a type other than the first type in the polymorphic sequence
- only the aggregate containing the second nucleic acid probe is formed by the same process, and the first nucleic acid probe Aggregates containing are not formed.
- the nucleic acid molecule to be analyzed is the first type, and the second nucleic acid
- the nucleic acid molecule to be analyzed is of a type other than the first type in the polymorphic sequence. Can be identified.
- the second nucleic acid probe is designed to form an aggregate with a single-stranded nucleic acid molecule having a polymorphic sequence or a single-stranded nucleic acid molecule complementary to the single-stranded nucleic acid molecule, regardless of the type of the polymorphic sequence. By doing so, it is possible to calculate the proportion of single-stranded nucleic acid molecules containing the first type base sequence in the polymorphic nucleic acid present in the sample solution.
- the nucleic acid molecule polymorphism identification method of the present invention is applied to a sample solution containing genomic DNA of a subject, wherein a SNP having two types, a wild type and a mutant type, is a polymorphic sequence, the mutant type is a first type.
- the genotype of the subject was a mutant homozygous and the first nucleic acid
- the subject's genotype is heterogeneous and almost all the aggregates containing the first nucleic acid probe are detected. If not, it can be identified that the subject's genotype is wild-type homozygote.
- the second nucleic acid probe forms an aggregate with a single-stranded nucleic acid molecule having a polymorphic sequence or a single-stranded nucleic acid molecule complementary to the single-stranded nucleic acid molecule, regardless of the type of the polymorphic sequence. Therefore, it can be used for accuracy evaluation of the measurement system. For example, when an aggregate containing the second nucleic acid probe is hardly detected, the sample solution used does not contain a sufficient amount of polymorphic nucleic acid for measurement, or the measurement system functions well. This may indicate that the results are not reliable.
- the fluorescence used as an index for detecting the aggregate containing the second nucleic acid probe and the fluorescence used as the index for detecting the aggregate containing the first nucleic acid probe are made different from each other and emitted. By detecting light, the aggregate containing the first nucleic acid probe and the aggregate containing the second nucleic acid probe may be distinguished and detected.
- Example 1 Using one type of molecular beacon probe, it was verified that the codon 12_GTT mutation of the K-ras gene can be identified by scanning molecular counting.
- a mutant (GTT) molecular beacon probe a nucleic acid molecule having the base sequence shown in Table 1 (SEQ ID NO: 1) having TAMRA added at the 5 ′ end and BHQ-2 added at the 3 ′ end was used.
- wild type (GGT) nucleic acid SEQ ID NO: 2 having the base sequence shown in Table 1 (mutation rate: 0%), wild type (GGT) nucleic acid and mutant type (GTT) ) Nucleic acid (SEQ ID NO: 3) mixed at a molar ratio of 9: 1 (mutation rate: 10%), and mutant (GTT) nucleic acid alone (mutation rate: 100%) were used.
- a non-fluorescent labeled probe wild type (GGT) decoy nucleic acid
- SEQ ID NO: 4 having a base sequence complementary to the wild type (GGT) nucleic acid was used.
- oligonucleotides were synthesized at the request of Sigma Genosys.
- Table 1 the right column shows the sequence number.
- bold bases are bases that form base pairs with mutation sites or mutation sites.
- underlined bases in mutant (GTT) molecular beacon probes are regions that hybridize to each other when forming intramolecular structures.
- Tris buffer (GTT) molecular beacon probe nucleic acid molecule to be analyzed, and wild-type decoy nucleic acid are tris buffer solution (concentration of wild type and mutant type) and 500 nM, respectively.
- a sample solution was prepared by dissolving in 10 mM Tris-HCl, 1 mM EDTA, 400 mM NaCl, pH 8.0). The prepared sample solution was denatured by heating at 95 ° C. for 5 minutes, and then the liquid temperature was gradually lowered to 20 ° C. to form an aggregate. Specifically, the cooling rate is 0.1 ° C./second, 90 ° C. for 5 minutes, 80 ° C. for 10 minutes, 70 ° C. for 10 minutes, 60 ° C. for 10 minutes, 50 ° C. for 10 minutes, 40 ° C. for 10 minutes. For 10 minutes at 30 ° C. for 10 minutes.
- the number of molecules of the aggregate in the sample solution after the temperature lowering treatment was counted by a scanning molecule counting method.
- a single molecule fluorescence measurement device MF20 (Olympus Co., Ltd.) equipped with an optical system of a confocal fluorescence microscope and a photon counting system is used. Time-series photon count data was obtained. At that time, the excitation light was irradiated at 300 ⁇ W using 543 nm laser light, and the detection light wavelength was 560 to 620 nm using a bandpass filter.
- the moving speed of the position of the light detection region in the sample solution was 15 mm / second, the BIN TIME was 10 ⁇ sec, and the measurement time was 2 seconds. The measurement was performed 5 times for each sample, and the average and standard deviation were calculated. After the measurement of the light intensity, the optical signals detected in the time series data were counted from the time series photon count data acquired for each sample solution. In the smoothing of the data by the moving average method, the number of data points averaged at a time was set to nine, and the moving average process was repeated five times. In the fitting, a Gaussian function was fitted to the time series data by the least square method, and the peak intensity (in the Gaussian function), the peak width (full width at half maximum), and the correlation coefficient were determined.
- FIG. 9 shows the number of peaks counted for each GTT mutation rate in the nucleic acid molecule to be analyzed added to the sample solution.
- Example 2 By adding a nucleic acid molecule having a base sequence complementary to a polymorphic sequence of a type other than the type to which the nucleic acid probe specifically binds to the sample solution, the detection accuracy of the aggregate containing the nucleic acid probe was examined. . Specifically, the mutant (GTT) molecular beacon probe, the nucleic acid molecule to be analyzed, and the wild type (GGT) decoy nucleic acid used in Example 1 were respectively 100 pM and 100 nM (concentration of the total of the wild type and the mutant type).
- a sample solution containing a wild type (GGT) decoy nucleic acid was prepared by dissolving in Tris buffer (10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, pH 8.0) to 1 ⁇ M.
- Tris buffer 10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, pH 8.0
- a sample solution containing no wild type (GGT) decoy nucleic acid was prepared in the same manner except that no wild type (GGT) decoy nucleic acid was added.
- the liquid temperature was raised and lowered to denature the nucleic acid molecules in the sample solution, and then an aggregate was formed. Further, under the same conditions as in Example 1, The number of molecules of the aggregate containing the mutant (GTT) molecular beacon probe in the sample solution was counted.
- FIG. 10 shows the number of peaks counted for each GTT mutation rate in the nucleic acid molecule to be analyzed added to the sample solution.
- FIG. 10A shows the result of the sample solution to which the wild type (GGT) decoy nucleic acid was not added
- FIG. 10B shows the result of the sample solution to which the wild type (GGT) decoy nucleic acid was added.
- the mutant (GTT) molecular beacon probe hybridized non-specifically to the wild type (GGT) nucleic acid.
- FIG. 10A shows the mutant (GTT) molecular beacon probe hybridized non-specifically to the wild type (GGT) nucleic acid.
- Example 3 Each genotype was identified using the mutant (GTT) molecular beacon probe and wild type (GGT) molecular beacon probe used in Example 1.
- GTT mutant
- GGT wild type molecular beacon probe
- SEQ ID NO: 5 a nucleic acid molecule having the base sequence shown in Table 2 (SEQ ID NO: 5) in which TAMRA was added to the 5 ′ end and BHQ-2 was added to the 3 ′ end was used.
- GTT mutant-type nucleic acid
- GTT mutation rate 100%
- a type (GGT) nucleic acid and a mutant (GTT) nucleic acid mixed at a molar ratio of 1: 1 (GTT mutation rate: 50%) were used.
- the wild-type (GGT) decoy nucleic acid used in Example 1 and a non-fluorescent labeled probe (mutant type) having a base sequence complementary to the mutant type (GTT) nucleic acid (GTT) decoy nucleic acid (SEQ ID NO: 6) was used.
- Table 2 shows the sequence of the mutant (GTT) decoy nucleic acid.
- the right column indicates the SEQ ID No.
- the bold base is the base that forms a base pair with the mutation site or mutation site, and the underlined bases are mutually attached when forming the intramolecular structure. It is a region that hybridizes.
- the oligonucleotides used in this example were synthesized upon request from Sigma Genosys.
- the mutant type (GTT) molecular beacon probe, the nucleic acid molecule to be analyzed, and the wild type (GGT) decoy nucleic acid are 100 pM, 100 nM (concentration of the wild type and the mutant type), and 500 nM, respectively.
- a sample solution containing a mutant (GTT) molecular beacon probe and a wild type (GGT) decoy nucleic acid is prepared by dissolving in Tris buffer (10 mM Tris-HCl, 1 mM EDTA, 400 mM NaCl, pH 8.0). did.
- a control sample solution was prepared in the same manner except that the nucleic acid molecule to be analyzed was not added.
- the wild type (GGT) molecular beacon probe, the nucleic acid molecule to be analyzed, and the mutant (GTT) decoy nucleic acid are 100 pM, 100 nM (concentration of the wild type and mutant), and 500 nM, respectively.
- a sample solution containing a wild type (GGT) molecular beacon probe and a mutant (GTT) decoy nucleic acid was prepared by dissolving in Tris buffer.
- a control sample solution was prepared in the same manner except that the nucleic acid molecule to be analyzed was not added.
- Example 1 For each of these sample solutions, as in Example 1, the liquid temperature was raised and lowered to denature nucleic acid molecules in the sample solution, and then an aggregate was formed. Further, the same as in Example 1 Under the conditions, the number of molecules in the sample solution containing the mutant (GTT) molecular beacon probe and the number of molecules in the aggregate containing the wild type (GGT) molecular beacon probe were counted.
- GTT mutant
- GTT wild type
- the vertical axis represents the number of molecules of an aggregate containing mutant (GTT) molecular beacon probes (mutant type (GTT) peak number) (“peak number (GTT)” in the figure), and the horizontal axis represents wild type ( GGT)
- GTT mutant molecular beacon probes
- wild type (GGT) As the number of molecules of the aggregate containing the beacon probe (number of wild-type (GGT) peaks) (“peak number (GGT)” in the figure)), for each mutation rate in the nucleic acid molecule to be analyzed added to the sample solution Shows the value of the number of peaks counted.
- nucleic acid molecule to be analyzed for each mutation rate by identifying the gene mutation using the nucleic acid molecule polymorphism identification method of the present invention, in particular, single nucleotide polymorphism, etc.
- the genotype of the genomic DNA to be analyzed is a wild type homozygote, a heterozygote or a mutant homozygote.
- Example 4 In the method for identifying a polymorphism of a nucleic acid molecule of the present invention, conditions for forming both an aggregate containing a mutant (GTT) molecular beacon probe and an aggregate containing a wild type (GGT) molecular beacon probe in one sample solution The codon 12_GTT mutation of the K-ras gene was identified.
- ATTO registered trademark
- 647N was added to the 5 ′ end of an oligonucleotide having the same base sequence (SEQ ID NO: 1) as the mutant (GTT) molecular beacon probe described in Table 1. What added BHQ-3 to 3 'terminal was used.
- the wild type (GGT) molecular beacon probe, the nucleic acid molecule to be analyzed, the wild type (GGT) decoy nucleic acid, and the mutant type (GTT) decoy nucleic acid used in Example 3 were used.
- the oligonucleotides used in this example were synthesized upon request from Sigma Genosys. Specifically, a mutant (GTT) molecular beacon probe, a wild type (GGT) molecular beacon probe, a nucleic acid molecule to be analyzed, a wild type (GGT) decoy nucleic acid, and a mutant (GTT) decoy nucleic acid are each 100 pM.
- Example 1 For each of these sample solutions, as in Example 1, the liquid temperature was raised and lowered to denature nucleic acid molecules in the sample solution, and then aggregates were formed. The number of molecules of the aggregate in the sample solution after the temperature lowering treatment was counted by a scanning molecule counting method. Specifically, in order to excite the TAMRA-labeled wild type (GGT) molecular beacon probe, the excitation light is irradiated at 300 ⁇ W using 543 nm laser light, and the detection light wavelength is measured using a bandpass filter. It was 565 to 595 nm.
- TAMRA-labeled wild type (GGT) molecular beacon probe the excitation light is irradiated at 300 ⁇ W using 543 nm laser light, and the detection light wavelength is measured using a bandpass filter. It was 565 to 595 nm.
- the excitation light was irradiated at 300 ⁇ W using a 633 nm laser beam, and the detection light wavelength was 650 to 690 nm using a bandpass filter. Other than that, measurement was performed by the scanning molecule counting method under the same conditions as in Example 3, and the analysis was performed.
- the vertical axis represents the number of molecules of the aggregate containing the mutant (GTT) molecular beacon probe (number of mutant (GTT) peaks) (“peak number (GTT)” in the figure), and the horizontal axis represents the wild type ( GGT) As the number of molecules of the aggregate containing the beacon probe (number of wild-type (GGT) peaks) (“peak number (GGT)” in the figure)), for each mutation rate in the nucleic acid molecule to be analyzed added to the sample solution Shows the value of the number of peaks counted.
- Example 5 In Example 4, it was possible to identify the nucleic acid molecule to be analyzed for each mutation rate, but the distance between the spots of each sample solution in FIG. 12 was different from that in Example 3 shown in FIG. the distance is close. This suggests that the method of Example 4 is slightly inferior to the method of Example 3. Therefore, the length of the decoy nucleic acid was adjusted to improve the discrimination ability of the mutation rate.
- the wild type (GGT) decoy nucleic acid and the mutant (GTT) decoy nucleic acid used in Example 4 were replaced by the wild type (GGT) decoy nucleic acid (SEQ ID NO: 7) described in Table 3 and A sample solution was prepared in the same manner as in Example 4 except that the mutant (GTT) decoy nucleic acid (SEQ ID NO: 8) was used.
- the decoy nucleic acid used in this example is shorter by one base each than that used in Example 4.
- the right column indicates the SEQ ID No., and the base in bold indicates the base that forms a base pair with the mutation site or mutation site.
- Example 4 For these sample solutions, as in Example 4, the liquid temperature was raised and lowered to denature the nucleic acid molecules in the sample solution, and then an aggregate was formed. Further, under the same conditions as in Example 4, The number of molecules in the sample solution containing the mutant (GTT) molecular beacon probe and the number of molecules in the aggregate containing the wild type (GGT) molecular beacon probe were counted.
- the vertical axis represents the number of molecules of the aggregate containing mutant (GTT) molecular beacon probes (mutant type (GTT) peak number) (“peak number (GTT)” in the figure), and the horizontal axis represents wild type ( GGT)
- GTT mutant molecular beacon probes
- wild type (GGT) As the number of molecules of the aggregate containing the beacon probe (number of wild-type (GGT) peaks) (“peak number (GGT)” in the figure)), for each mutation rate in the nucleic acid molecule to be analyzed added to the sample solution Shows the value of the number of peaks counted.
- the distance between the spots of each sample solution was wider than in the case of Example 4 shown in FIG. 12, and the discrimination ability of the GTT mutation rate in the sample solution was found.
- Example 4 The discriminating ability in Example 4 was lower than the discriminating ability in Example 3 because two types of molecular beacon probes and two types of decoy nucleic acids coexisted. This is probably because not only non-specific binding but also specific binding was inhibited. In this example, it is inferred that the ability to discriminate the GTT mutation rate was improved because the specific binding inhibition of the molecular beacon to the target sequence was reduced due to the short length of the decoy nucleic acid. From these results, by devising the design of the decoy nucleic acid, a nucleic acid probe specific for one type in the polymorphic sequence and a nucleic acid probe specific for the other type in the polymorphic sequence are combined.
- a nucleic acid probe having a base sequence different from that of the first nucleic acid probe and not recognizing a polymorphic site consisting of the polymorphic sequence to be analyzed is used as the second nucleic acid probe, and used together with the first nucleic acid probe, The codon 12_GTT mutation of the gene was identified.
- a base sequence (common sequence) (SEQ ID NO: 9) of a region in the K-ras gene that does not include codon 12 and is common to the wild type and the mutant type
- a common molecular beacon probe in which ATTO (registered trademark) 647N was added to the 5 ′ end and BHQ-3 was added to the 3 ′ end was used for the oligonucleotide (SEQ ID NO: 10) that specifically hybridizes.
- the oligonucleotide which consists of the said common sequence was used as a common nucleic acid.
- Table 4 shows the common molecular beacon probe and the base sequence of the common sequence. In Table 4, the right column shows the sequence number.
- the underlined bases in the common molecular beacon probe are regions that hybridize with each other when forming an intramolecular structure.
- mutant (GTT) molecular beacon probe used in Example 1 was used as the first nucleic acid probe.
- wild type (GGT) nucleic acid used in Example 1 mutant rate: 0%
- mutant type (GTT) nucleic acid alone GTT mutation rate: 100%
- wild type (GGT) decoy nucleic acid used in Example 1 was used.
- the oligonucleotides used in this example were synthesized upon request from Sigma Genosys.
- a mutant (GTT) molecular beacon probe, a common molecular beacon probe, a wild type (GGT) nucleic acid or a mutant (GTT) nucleic acid, a common nucleic acid, and a wild type (GGT) decoy nucleic acid are 100 pM and 100 pM, respectively.
- Sample solutions were prepared by dissolving in Tris buffer (10 mM Tris-HCl, 1 mM EDTA, 400 mM NaCl, pH 8.0) so as to be 100 nM, 100 nM, and 500 nM.
- a control sample solution was prepared in the same manner except that neither wild type (GGT) nucleic acid nor mutant type (GTT) nucleic acid was added.
- Example 2 For these sample solutions, in the same manner as in Example 1, the liquid temperature was raised and lowered to denature nucleic acid molecules in the sample solution, and then aggregates were formed. Then, the number of molecules in the sample solution containing the mutant (GTT) molecular beacon probe and the number of molecules in the aggregate containing the wild type (GGT) molecular beacon probe were counted.
- FIG. 14 shows the number of peaks counted for each GTT mutation rate in the nucleic acid molecule to be analyzed added to the sample solution.
- FIG. 14A shows the counting result of the aggregate containing the mutant (GTT) molecular beacon probe
- FIG. 14B shows the counting result of the aggregate containing the common molecular beacon probe.
- the common nucleic acid was added to the sample solution separately from the wild type (GGT) nucleic acid and the mutant type (GTT) nucleic acid.
- the nucleic acid molecule to be analyzed is genomic DNA
- the common sequence and the polymorphic sequence are present on the same single-stranded nucleic acid molecule or the complementary strand of the single-stranded nucleic acid molecule.
- the second nucleic acid probe is a probe that hybridizes to a region common to polymorphic nucleic acids as in this example, the sample solution depends on the presence or absence of an aggregate containing the second nucleic acid probe.
- nucleic acid molecule containing the polymorphic sequence to be analyzed Whether or not a nucleic acid molecule containing the polymorphic sequence to be analyzed is present can be confirmed. Moreover, the amount of nucleic acid molecules due to variation in the amount of PCR amplification can be corrected by the detected amount of the aggregate containing the second nucleic acid probe, and the accuracy of polymorphism identification can be improved.
- Example 7 It was verified that the codon 12_GTT mutation of the K-ras gene can be identified by scanning molecule counting using one type of nucleic acid probe and a fluorescent intercalator.
- a mutant (GTT) probe a nucleic acid molecule having the base sequence shown in Table 5 (SEQ ID NO: 11) in which ROX was added to the 5 ′ end was used.
- the right column indicates the SEQ ID No., and the bold base is a base that forms a base pair with the mutation site or mutation site.
- the wild-type (GGT) nucleic acid and the mutant (GTT) nucleic acid used in Example 1 were used as the nucleic acid molecules to be analyzed.
- a sample solution containing wild type (GGT) decoy nucleic acid was prepared by dissolving in (10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, pH 8.0).
- a sample solution containing no wild type (GGT) decoy nucleic acid was prepared in the same manner except that no wild type (GGT) decoy nucleic acid was added.
- the prepared sample solution was denatured by heating at 94 ° C. for 5 minutes, and then the temperature was lowered at 0.1 ° C./second, followed by a constant temperature treatment at 68.8 ° C. for 5 minutes to form an aggregate.
- a solution obtained by diluting PicoGreen (manufactured by Molecular Probe) 1000 times with the above Tris buffer was added to each of these concentrated sample solutions to prepare a 10-fold dilution of the concentrated sample solution, and these sample solutions were obtained. These sample solutions were allowed to stand for 30 minutes or more after the addition of the PicoGreen diluent.
- the number of molecules of the aggregate in the sample solution was counted by a scanning molecule counting method.
- the optical analysis device a single molecule fluorescence measurement device MF20 (Olympus Co., Ltd.) equipped with an optical system of a confocal fluorescence microscope and a photon counting system is used. Time-series photon count data was obtained.
- the excitation light was irradiated with a laser beam of 488 nm at a rotation speed of 6,000 rpm and 100 ⁇ W, and the detection light wavelength was 560 to 620 nm using a bandpass filter.
- the signal obtained from the avalanche photodiode was BIN TIME of 10 ⁇ s, and the measurement time was 2 seconds.
- the time series data obtained by the measurement was smoothed by the Savinzky-Golay algorithm, and then the peak was detected by differentiation. Of the regions regarded as peaks, a region that can be approximated to a Gaussian function was extracted as a signal. The number of peaks obtained was compared between the sample solution with and without the wild type (GGT) decoy nucleic acid.
- FIG. 15 shows the value of the number of peaks counted for each nucleic acid molecule to be analyzed added to the sample solution.
- Example 8 Using the mutant (GTT) probe, wild-type (GGT) probe, and fluorescent intercalator used in Example 7, the codon 12_GTT mutation of the K-ras gene was identified by scanning molecule counting.
- a wild-type (GGT) probe a nucleic acid molecule having the base sequence (SEQ ID NO: 12) described in Table 6 in which ROX was added to the 5 ′ end was used.
- SEQ ID NO: 12 a nucleic acid molecule having the base sequence (SEQ ID NO: 12) described in Table 6 in which ROX was added to the 5 ′ end was used.
- the right column shows the sequence number, and the base in bold type is the base that forms a base pair with the mutation (polymorphism) site or mutation site.
- a mutant (GTT) probe, a nucleic acid molecule to be analyzed, and a wild-type (GGT) decoy nucleic acid are respectively added to a Tris buffer (10 mM Tris-HCl, 1 mM) so as to be 100 pM, 100 pM, and 1 nM, respectively.
- a sample solution containing a mutant (GTT) probe and a wild type (GGT) decoy nucleic acid was prepared by dissolving in EDTA, 100 mM NaCl, pH 8.0).
- a wild type (GGT) probe, a nucleic acid molecule to be analyzed, and a mutant (GTT) decoy nucleic acid are dissolved in the Tris buffer so as to be 100 pM, 100 pM, and 1 nM, respectively.
- a sample solution containing a probe and a mutant (GTT) decoy nucleic acid was prepared. The prepared sample solution was denatured by heating at 94 ° C. for 5 minutes, and then the temperature was lowered at 0.1 ° C./second, followed by a constant temperature treatment at 68.8 ° C. for 5 minutes to form an aggregate.
- a solution obtained by diluting PicoGreen (manufactured by Molecular Probe) 1000 times with the above Tris buffer was added to each of these concentrated sample solutions to prepare a 10-fold dilution of the concentrated sample solution, and these sample solutions were obtained. These sample solutions were allowed to stand for 30 minutes or more after the addition of the PicoGreen diluent.
- FIG. 16 shows, for each mutation rate of the nucleic acid molecule to be analyzed added to the sample solution, the counted peak number (“GTT probe” in the figure) of the aggregate containing the mutant (GTT) probe and the wild type (GGT). ) The number of counted peaks (“GGT probe” in the figure) of the aggregate containing the probe is shown.
- a sample solution containing a mutant type (GTT) probe and a wild type (GGT) decoy nucleic acid can be obtained as the mutation rate increases (that is, the concentration of the mutant type (GTT) nucleic acid in the sample solution increases). It was found that the number of peaks increased and the amount of aggregates containing mutant (GTT) probes increased. Similarly, in a sample solution containing a wild type (GGT) probe and a mutant (GTT) decoy nucleic acid, the lower the mutation rate (that is, the higher the wild type (GGT) nucleic acid concentration in the sample solution), the more obtainable It was found that the number of peaks increased and the amount of aggregates containing wild type (GGT) probe increased. From these results, it is clear that polymorphisms can be identified by the nucleic acid molecule polymorphism identification method of the present invention, even though they are very similar nucleic acid molecules that differ by only one base.
- Example 9 Using a nucleic acid probe and a fluorescent intercalator, the influence of the base pair length of the double-stranded structure in the aggregate when the aggregate containing the nucleic acid probe was counted by the scanning molecule counting method was examined.
- a plasmid pUC19 manufactured by Takara Bio Inc.
- an oligonucleotide whose 5 ′ end is modified with Rox, an unlabeled oligonucleotide, and AmpliTaq Gold manufactured by Applied Biosystems
- 100 bp, 200 bp, 400 bp PCR products with different chain lengths of 800 bp and 1.5 kbp were prepared.
- PCR products were removed from these PCR products using Wizard V Gel and PCR Clean-Up System (Promega), and the presence and concentration of PCR products were measured by electrophoresis using Bioanalyzer (Agilent). These PCR products (fluorescent labels) are an association of a single-stranded nucleic acid molecule whose 5 ′ end is labeled with Rox and an unlabeled single-stranded nucleic acid molecule.
- An unlabeled single-stranded nucleic acid molecule between the unlabeled single-stranded nucleic acid molecules was used in the same manner as described above, except that an oligonucleotide not modified at the 5 ′ end was used instead of the oligonucleotide modified at the 5 ′ end with Rox.
- a PCR product (unlabeled) as an aggregate was obtained. Thereafter, each PCR product was adjusted to 100 pM using Tris buffer (10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, pH 8.0).
- the prepared PCR product solution was added to PicoGreen (manufactured by Invitrogen) solution diluted 10,000 times with the above Tris buffer at an arbitrary concentration to obtain a sample solution. These sample solutions were allowed to stand for 30 minutes or more after the addition of the PicoGreen diluent.
- nucleic acid molecule polymorphism identification method of the present invention Using the nucleic acid molecule polymorphism identification method of the present invention, a polymorphic nucleic acid present in a very low concentration in a sample solution is identified and scanned using a nucleic acid probe that specifically hybridizes to a specific type. Since it can be counted by the molecular counting method, it can be used in fields such as clinical examinations that identify genetic polymorphisms and somatic mutations.
- Optical analyzer confocal microscope
- Light source 3 Single mode optical fiber
- Collimator lens 5 Dichroic mirror 6, 7, 11 Reflection mirror 8
- Objective lens 9
- Microplate 10 Well (sample solution container)
- DESCRIPTION OF SYMBOLS 12
- Condenser lens 13
- Pinhole 14
- Barrier filter 15
- Multimode optical fiber 16
- Photo detector 17 Mirror deflector 17a
- Stage position change apparatus 18 Computer
Abstract
Description
本願は、2011年1月26日に、日本に出願された特願2011-014064号に基づき優先権を主張し、その内容をここに援用する。
また、非特許文献4においても、数十~数百nM程度の比較的高濃度の核酸について塩基配列の識別が行われている。このように、一般的に、pMオーダーの比較的低濃度の核酸検体に対して多型を識別するには至っていない。
ここで、走査分子計数法は、特願2010-044714に於いて、本願出願人により提案されている新規な光分析技術である。
(1)(a)多型配列中の第1の型の塩基配列を有する1本鎖核酸分子と特異的にハイブリダイズする第1の核酸プローブと、解析対象の核酸分子とを含む試料溶液を調製する試料溶液調製工程と、
(b)前記工程(a)において調製された試料溶液中の核酸分子を会合させる会合工程と、
(c)前記工程(b)の後、前記工程(a)において調製された試料溶液中の、第1の核酸プローブを含む会合体の分子数を算出する算出工程と、
(d)前記工程(c)の結果に基づき、前記解析対象の核酸分子の多型を識別する識別工程と、
(e)前期工程(c)において、共焦点顕微鏡又は多光子顕微鏡の光学系を用いて、前記試料溶液内において前記光学系の光検出領域の位置を移動する検出領域移動工程と、
(f)前期工程(c)において、前記試料溶液内において前記光学系の光検出領域の位置を移動させながら、当該光検出領域中の前記会合体から放出される蛍光を検出する蛍光検出工程と、
(g)前期工程(c)において、前記検出された光から、個々の会合体からの光信号を個別に検出して、会合体を個別に検出する会合体検出工程と、
(h)前期工程(c)において、前記個別に検出された会合体の数を計数して前記光検出領域の位置の移動中に検出された前記粒子の数を計数する粒子計数工程と、を有し、
前記工程(a)における試料溶液、又は前記工程(b)の後、前記工程(c)の前の試料溶液が、前記多型配列のうちの前記第1の型以外の型の塩基配列と相補的な塩基配列を有するオリゴヌクレオチドをさらに含んでいる核酸分子の多型識別方法。
(2)前記第1の核酸プローブが、蛍光物質により標識されている上記(1)に記載の核酸分子の多型識別方法。
(3)前記工程(a)において、前記試料溶液中に、2本鎖構造に特異的に結合する蛍光性2本鎖核酸結合物質がさらに含まれている上記(1)又は(2)記載の核酸分子の多型識別方法。
(4)前記工程(a)において、前記試料溶液中に、蛍光性2本鎖核酸結合物質がさらに含まれており、
前記第1の核酸プローブを標識している蛍光物質と前記蛍光性2本鎖核酸結合物質とのいずれか一方が、蛍光エネルギー移動現象におけるエネルギー・ドナーと成る蛍光物質であり、他方が前記蛍光エネルギー移動現象におけるエネルギー・アクセプターと成る物質であり、
前記工程(c)において、前記第1の核酸プローブを含む会合体から放出される蛍光は、前記第1の核酸プローブを標識している蛍光物質と前記蛍光性2本鎖核酸結合物質との間に起こった蛍光エネルギー移動現象により放出される蛍光であ上記(2)に記載の核酸分子の多型識別方法。
(5)前記第1の核酸プローブは、単独で存在している状態では蛍光エネルギー移動が起こり、かつ他の1本鎖核酸分子と会合体を形成した状態では蛍光エネルギー移動が起こらないように、エネルギー・ドナーと成る蛍光物質とエネルギー・アクセプターと成る物質とが結合されており、
当該核酸プローブを含む会合体から放出される蛍光は、前記エネルギー・ドナーと成る蛍光物質から放出される蛍光である上記(2)に記載の核酸分子の多型識別方法。
(6)前記光検出領域の位置を移動する工程に於いて、前記光検出領域の位置が所定の速度にて移動される上記(1)から(5)のいずれか一つに記載の核酸分子の多型識別方法。
(7)前記光検出領域の位置を移動する工程に於いて、前記光検出領域の位置が、前記会合体の拡散移動速度よりも速い速度にて移動される上記(1)から(6)のいずれか一つに記載の核酸分子の多型識別方法。
(8)前記検出された光から、個々の会合体からの光信号を個別に検出して、会合体を個別に検出する工程に於いて、検出された時系列の光信号の形状に基づいて、1つの会合体が前記光検出領域に入ったことが検出される上記(1)から(7)のいずれか一つに記載の核酸分子の多型識別方法。
(9)前記試料溶液が、界面活性剤、ホルムアミド、ジメチルスルホキシド、及び尿素からなる群より選択される1種以上を含む上記(1)から(8)のいずれか一つに記載の核酸分子の多型識別方法。
(10)前記工程(b)を、前記工程(a)において調製された試料溶液の温度を70℃以上にすることにより、当該試料溶液中の核酸分子を変性させた後、当該試料溶液の液温を0.05℃/秒以上の降温速度で低下させることにより、当該試料溶液中の核酸分子を会合させることにより行う上記(1)から(9)のいずれか一つに記載の核酸分子の多型識別方法。
(11)前記第1の核酸プローブ及び前記第2の核酸プローブが、DNA、RNA、及び核酸類似物質からなる群より選択される2以上の分子が結合して構成されている上記(1)から(10)のいずれか一つに記載の核酸分子の多型識別方法。
(12)前記多型配列が、遺伝子多型の多型部位を含む塩基配列、又は体細胞変異の変異部位を含む塩基配列である上記(1)から(11)のいずれか一つに記載の核酸分子の多型識別方法。
(13)前記体細胞変異がK-ras遺伝子の変異である上記(12)に記載の核酸分子の多型識別方法。
また、本発明の核酸分子の多型識別方法では、核酸プローブと試料中の核酸分子との会合体の走査分子計数法による検出を、前記多型配列のうちの前記1の型以外の型の塩基配列と相補的な塩基配列を有するオリゴヌクレオチドの存在下で行う。そのため、会合体形成反応から検出までの間に核酸プローブが他の型の核酸分子と非特異的に会合体を形成することが効果的に抑制され、その結果、核酸プローブによる非特異的な会合体の形成を効果的に抑制することができ、非常に精度よく多型を識別することができる。
走査分子計数法は、基本的な構成に於いて、図1(A)に模式的に例示されている如き、FCS、FIDA等が実行可能な共焦点顕微鏡の光学系と光検出器とを組み合わせた光分析装置により実現可能である。同図を参照して、上記の光分析装置について説明する。光分析装置1は、光学系2~17と、光学系の各部の作動を制御すると共にデータを取得し解析するためのコンピュータ18とから構成される。光分析装置1の光学系は、通常の共焦点顕微鏡の光学系と同様であってよく、そこに於いて、光源2から放射されシングルモードファイバー3内を伝播したレーザ光(Ex)が、ファイバーの出射端に於いて固有のNAにて決まった角度にて発散する光となって放射され、コリメーター4によって平行光となり、ダイクロイックミラー5、反射ミラー6、7にて反射され、対物レンズ8へ入射される。対物レンズ8の上方には、典型的には、1~数十μLの試料溶液が分注される試料容器又はウェル10が配列されたマイクロプレート9が配置されており、対物レンズ8から出射したレーザ光は、試料容器又はウェル10内の試料溶液中で焦点を結び、光強度の強い領域(励起領域)が形成される。試料溶液中には、観測対象物である粒子と、かかる粒子と結合する発光プローブ、典型的には、蛍光色素等の発光標識が付加された分子が分散又は溶解されており、発光プローブと結合又は会合した粒子(実験の態様によっては、粒子と一旦結合した後に粒子から解離した発光プローブ)が励起領域に進入すると、その間、発光プローブが励起され光が放出される。放出された光(Em)は、対物レンズ8、ダイクロイックミラー5を通過し、ミラー11にて反射してコンデンサーレンズ12にて集光され、ピンホール13を通過し、バリアフィルター14を透過して(ここで、特定の波長帯域の光成分のみが選択される。)、マルチモードファイバー15に導入されて、光検出器16に到達し、時系列の電気信号に変換された後、コンピュータ18へ入力され、後に説明される態様にて光分析のための処理が為される。なお、当業者に於いて知られている如く、上記の構成に於いて、ピンホール13は、対物レンズ8の焦点位置と共役の位置に配置されており、これにより、図1(B)に模式的に示されている如きレーザ光の焦点領域、即ち、励起領域内から発せられた光のみがピンホール13を通過し、励起領域以外からの光は遮断される。図1(B)に例示されたレーザ光の焦点領域は、通常、1~10fL程度の実効体積を有する本光分析装置に於ける光検出領域であり(典型的には、光強度が領域の中心を頂点とするガウス型分布又はローレンツ型分布となる。実効体積は、光強度が1/e2 となる面を境界とする略楕円球体の体積である。)、コンフォーカル・ボリュームと称される。また、走査分子計数法では、1つの粒子及び発光プローブの結合体又は発光プローブからの光、例えば、一個又は数個の蛍光色素分子からの微弱光が検出されるので、光検出器16としては、好適には、フォトンカウンティングに使用可能な超高感度の光検出器が用いられる。また、顕微鏡のステージ(図示せず)には、観察するべきウェル10を変更するべく、マイクロプレート9の水平方向位置を移動するためのステージ位置変更装置17aが設けられていてよい。ステージ位置変更装置17aの作動は、コンピュータ18により制御されてよい。かかる構成により、検体が複数在る場合にも、迅速な計測が達成可能となる。
FIDA等の分光分析技術は、従前の生化学的な分析技術に比して、必要な試料量が極めて少なく、且つ、迅速に検査が実行できる点で優れている。しかしながら、FIDA等の分光分析技術では、原理的に、観測対象粒子の濃度や特性は、蛍光強度のゆらぎに基づいて算定されるので、精度のよい測定結果を得るためには、試料溶液中の観測対象粒子の濃度又は数密度が、蛍光強度の計測中に常に一個程度の観測対象粒子が光検出領域CV内に存在するレベルであり、計測時間中に常に有意な光強度(フォトンカウント)が検出されることが要求される。もし観測対象粒子の濃度又は数密度がそれよりも低い場合、例えば、観測対象粒子がたまにしか光検出領域CV内へ進入しないレベルである場合には、有意な光強度(フォトンカウント)が、計測時間の一部にしか現れず、精度のよい光強度のゆらぎの算定が困難となる。また、観測対象粒子の濃度が計測中に常に一個程度の観測対象粒子が光検出領域内に存在するレベルよりも大幅に低い場合には、光強度のゆらぎの演算において、バックグラウンドの影響を受けやすくなり、演算に十分な量の有意な光強度データを得るために計測時間が長くなる。これに対して、走査分子計数法では、観測対象粒子の濃度がFIDA等の分光分析技術にて要求されるレベルよりも低い場合でも、観測対象粒子の数密度又は濃度等の特性の検出が可能である。
そうすると、例えば、図2(A)の如く、光検出領域CVが移動する間(図中、時間t0~t2)において1つの粒子(図中、発光プローブとして蛍光色素が結合している。)の存在する領域を通過する際(t1)には、図2(B)に描かれている如く有意な光強度(Em)が検出されることとなる。かくして、上記の光検出領域CVの位置の移動と光検出を実行し、その間に出現する図2(B)に例示されている如き有意な光強度を一つずつ検出することによって、発光プローブの結合した粒子が個別に検出され、その数をカウントすることにより、計測された領域内に存在する粒子の数、或いは、濃度若しくは数密度に関する情報が取得できる。かかる走査分子計数法の光分析技術の原理においては、蛍光強度のゆらぎの算出の如き、統計的な演算処理は行われず、粒子が一つずつ検出されるので、FIDA等では十分な精度にて分析ができないほど、観測されるべき粒子の濃度が低い試料溶液でも、粒子の濃度若しくは数密度に関する情報が取得可能であることは理解されるべきである。
走査分子計数法の光分析における光強度の測定は、測定中にミラー偏向器17を駆動して、試料溶液内での光検出領域の位置の移動(試料溶液内の走査)を行う他は、FCS又はFIDAにおける光強度の測定工程と同様の態様にて実行されてよい。操作処理において、典型的には、マイクロプレート9のウェル10に試料溶液を注入して顕微鏡のステージ上に載置した後、使用者がコンピュータ18に対して、測定の開始の指示を入力すると、コンピュータ18は、記憶装置(図示せず)に記憶されたプログラム(試料溶液内において光検出領域の位置を移動するべく光路を変更する手順と、光検出領域の位置の移動中に光検出領域からの光を検出する手順)に従って、試料溶液内の光検出領域における励起光の照射及び光強度の計測が開始される。かかる計測中、コンピュータ18のプログラムに従った処理動作の制御下、ミラー偏向器17は、ミラー7(ガルバノミラー)を駆動して、ウェル10内において光検出領域の位置の移動を実行し、これと同時に光検出器16は、逐次的に検出された光を電気信号に変換してコンピュータ18へ送信し、コンピュータ18では、任意の態様にて、送信された光信号から時系列の光強度データを生成して保存する。なお、典型的には、光検出器16は、一光子の到来を検出できる超高感度光検出器であるので、光の検出は、所定時間に亘って、逐次的に、所定の単位時間毎(BINTIME)に、例えば、10μ秒毎に光検出器に到来するフォトンの数を計測する態様にて実行されるフォトンカウンティングであり、時系列の光強度のデータは、時系列のフォトンカウントデータであってよい。
走査分子計数法の光分析技術の観測対象粒子は、溶液中に分散又は溶解されて自由にランダムに運動する粒子であるので、ブラウン運動によって位置が時間と伴に移動する。従って、光検出領域の位置の移動速度が粒子のブラウン運動による移動に比して遅い場合には、図3(A)に模式的に描かれている如く、粒子が領域内をランダムに移動し、これにより、光強度が図3(B)の如くランダムに変化し(既に触れた如く、光検出領域の励起光強度は、領域の中心を頂点として外方に向かって低減する。)、個々の観測対象粒子に対応する有意な光強度の変化を特定することが困難となる。そこで、好適には、図4(A)に描かれている如く、粒子が光検出領域を略直線に横切り、これにより、時系列の光強度データにおいて、図4(B)に例示の如く、個々の粒子に対応する光強度の変化のプロファイルが略一様となり(粒子が略直線的に光検出領域を通過する場合には、光強度の変化のプロファイルは、励起光強度分布と略同様となる。)、個々の観測対象粒子と光強度との対応が容易に特定できるように、光検出領域の位置の移動速度は、粒子のブラウン運動による平均の移動速度(拡散移動速度)よりも速く設定される。
(2Wo)2 =6D・Δt (1)
から、
Δt=(2Wo)2 /6D (2)
となるので、観測対象粒子がブラウン運動により移動する速度(拡散移動速度)Vdifは、概ね、
Vdif=2Wo/Δt=3D/Wo (3)
となる。そこで、光検出領域の位置の移動速度は、かかるVdifを参照して、それよりも十分に早い値に設定されてよい。例えば、観測対象粒子の拡散係数が、D=2.0×10-10m2 /s程度であると予想される場合には、Woが、0.62μm程度だとすると、Vdifは、1.0×10-3 m/sとなるので、光検出領域の位置の移動速度は、その略10倍の15mm/sと設定されてよい。なお、観測対象粒子の拡散係数が未知の場合には、光検出領域の位置の移動速度を種々設定して光強度の変化のプロファイルが、予想されるプロファイル(典型的には、励起光強度分布と略同様)となる条件を見つけるための予備実験を繰り返し実行して、好適な光検出領域の位置の移動速度が決定されてよい。
上記の処理により試料溶液の時系列の光強度データが得られると、コンピュータ18において、記憶装置に記憶されたプログラムに従った処理により、下記の如き光強度の分析が実行されてよい。
時系列の光強度データにおいて、一つの観測対象粒子の光検出領域を通過する際の軌跡が、図4(A)に示されている如く略直線状である場合、その粒子に対応する光強度の変化は、図6(A)に模式的に描かれている如く、(光学系により決定される)光検出領域の光強度分布を反映したプロファイル(通常、略釣鐘状)を有する。そこで、観測対象粒子の検出の一つの手法において、光強度に対して閾値Ioが設定され、その閾値を超える光強度が継続する時間幅Δτが所定の範囲にあるとき、その光強度のプロファイルが一つの粒子が光検出領域を通過したことに対応すると判定され、一つの観測対象粒子の検出が為されるようになっていてよい。光強度に対する閾値Io及び時間幅Δτに対する所定の範囲は、光検出領域に対して所定の速度にて相対的に移動する観測対象粒子と発光プローブとの結合体(又は粒子との結合後分解され遊離した発光プローブ)から発せられる光の強度として想定されるプロファイルに基づいて定められるところ、具体的な値は、実験的に任意に設定されてよく、また、観測対象粒子と発光プローブとの結合体(又は粒子との結合後分解され遊離した発光プローブ)の特性によって選択的に決定されてよい。
ガウス分布:
I=A・exp(-2t2 /a2 ) (4)
であると仮定できるときには、有意な光強度のプロファイル(バックグラウンドでないと明らかに判断できるプロファイル)に対して式(4)をフィッティングして算出された強度A及び幅aが所定の範囲内にあるとき、その光強度のプロファイルが一つの観測対象粒子が光検出領域を通過したことに対応すると判定され、一つの観測対象粒子の検出が為されてよい。(強度A及び幅aが所定の範囲外にあるときには、ノイズ又は異物として分析において無視されてよい。)
観測対象粒子のカウンティングは、上記の観測対象粒子の検出の手法により検出された粒子の数を、任意の手法により、計数することにより為されてよい。しかしながら、粒子の数が大きい場合には、例えば、図5及び図6(B)に例示された処理により為されてよい。
具体的には、まず、時系列光信号データの時系列の時間微分値データ上にて、逐次的に時間微分値を参照して、一つのピーク信号の始点と終点とが探索され決定され、ピーク存在領域が特定される(ステップ130)。一つのピーク存在領域が特定されると、そのピーク存在領域におけるスムージングされた時系列光信号データに対して、釣鐘型関数のフィッティングが行われ(図6(B)下段「釣鐘型関数フィッティング」)、釣鐘型関数のピーク強度Imax、ピーク幅(半値全幅)w、フィッティングにおける(最小二乗法の)相関係数等のパラメータが算出される(ステップ140)。なお、フィッティングされる釣鐘型関数は、典型的には、ガウス関数であるが、ローレンツ型関数であってもよい。そして、算出された釣鐘型関数のパラメータが、一つの粒子及び発光プローブの結合体又は発光プローブが光検出領域を通過したときに検出される光信号が描く釣鐘型のプロファイルのパラメータについて想定される範囲内にあるか否か、即ち、ピーク強度、ピーク幅、相関係数が、それぞれ、所定範囲内にあるか否か等が判定される(ステップ150)。かくして、図7左に示されている如く、算出された釣鐘型関数のパラメータが一つの粒子及び発光プローブの結合体又は発光プローブに対応する光信号おいて想定される範囲内にあると判定された信号は、一つの観測対象粒子に対応する信号であると判定され、これにより、一つの観測対象粒子が検出されたこととなり、一つの粒子としてカウントされる(粒子数がカウントアップされる。ステップ160)。一方、図7右に示されている如く、算出された釣鐘型関数のパラメータが想定される範囲内になかったピーク信号は、ノイズとして無視される。
観測対象粒子のカウンティングが為されると、時系列光信号データの取得の間に光検出領域の通過した領域の総体積を用いて、観測対象粒子の数密度又は濃度が決定される。しかしながら、光検出領域の実効体積は、励起光又は検出光の波長、レンズの開口数、光学系の調整状態に依存して変動するため、設計値から算定することは、一般に困難である。従って、光検出領域の通過した領域の総体積を算定することも簡単ではない。そこで、典型的には、粒子の濃度が既知の溶液(参照溶液)について、検査されるべき試料溶液の測定と同様の条件にて、上記に説明した光強度の測定、粒子の検出及びカウンティングを行い、検出された粒子の数と参照溶液の粒子の濃度とから、光検出領域の通過した領域の総体積、即ち、観測対象粒子の検出数と濃度との関係が決定されるようになっていてよい。
参照溶液の粒子としては、好ましくは、観測対象粒子が形成する粒子及び発光プローブ結合体(又は観測対象粒子に結合後遊離した発光プローブ)と同様の波長特性を有する発光標識(蛍光色素等)であってよい。具体的には、例えば、粒子の濃度Cの参照溶液について、その粒子の検出数がNであったとすると、光検出領域の通過した領域の総体積Vtは、下記式(5)
Vt=N/C (5)
により与えられる。また、参照溶液として、複数の異なる濃度の溶液が準備され、それぞれについて測定が実行されて、算出されたVtの平均値が光検出領域の通過した領域の総体積Vtとして採用されるようになっていてよい。そして、Vtが与えられると、粒子のカウンティング結果がnの試料溶液の粒子の数密度cは、下記式(6)
c=n/Vt (6)
により与えられる。なお、光検出領域の体積、光検出領域の通過した領域の総体積は、上記の方法によらず、任意の方法にて、例えば、FCS、FIDAを利用するなどして与えられるようになっていてよい。また、本実施形態の光分析装置においては、想定される光検出領域の移動パターンについて、種々の標準的な粒子についての濃度Cと粒子の数Nとの関係(式(5))の情報をコンピュータ18の記憶装置に予め記憶しておき、装置の使用者が光分析を実施する際に適宜記憶された関係の情報を利用できるようになっていてよい。
本発明の核酸分子の多型識別方法は、多型配列のうちの特定の型(第1の型)の核酸分子と特異的に結合する核酸プローブを用いて、当該核酸プローブと解析対象の核酸分子とをハイブリダイズさせ、形成された会合体の量に基づいて解析対象の核酸分子の型を識別する方法である。さらに本発明においては、会合体の検出を、上記の走査分子計数法により測定する。走査分子計数法は、分子が離散的な状況において、蛍光を有する粒子を一粒子毎に測定することができる測定方法であることから、pMオーダー以下の比較的低濃度の核酸分子に対しても測定が可能である。このため、本発明の核酸分子の多型識別方法により、試料溶液中の解析対象の核酸分子の濃度が非常に低い場合であっても、形成された会合体を高感度に計数することができる。
(a)多型配列中の第1の型の塩基配列を有する1本鎖核酸分子と特異的にハイブリダイズする第1の核酸プローブと、解析対象の核酸分子とを含む試料溶液を調製する工程と、(b)前記工程(a)において調製された試料溶液中の核酸分子を会合させる工程と、
(c)前記工程(b)の後、前記工程(a)において調製された試料溶液中の、第1の核酸プローブを含む会合体の分子数を算出する工程と、
(d)前記工程(c)の結果に基づき、前記解析対象の核酸分子の多型を識別する工程。
各測定の結果から、対照用1本鎖核酸分子の多型と会合体の分子数との関係をクラスタリングするために必要な情報を得ることができる。
1種類の分子ビーコンプローブを用いて、走査分子計数法により、K-ras遺伝子のコドン12_GTT変異を識別可能であることを検証した。
変異型(GTT)分子ビーコンプローブとして、5’末端にTAMRAが付加され、3’末端にBHQ-2が付加された表1に記載の塩基配列(配列番号1)を有する核酸分子を用いた。また、解析対象の核酸分子として、同じく表1に記載の塩基配列を有する野生型(GGT)核酸(配列番号2)のみ(変異率:0%)、野生型(GGT)核酸と変異型(GTT)核酸(配列番号3)を9:1のモル比で混合したもの(変異率:10%)、及び変異型(GTT)核酸のみ(変異率:100%)用いた。さらに、非特異的なハイブリダイゼーションを抑制するために、野生型(GGT)核酸と相補的な塩基配列を有する非蛍光標識プローブ(野生型(GGT)デコイ核酸)(配列番号4)を用いた。これらのオリゴヌクレオチドは、シグマジェノシス株式会社に依頼して合成した。表1中、右欄には配列番号を示す。また、表1中、太字の塩基が変異部位又は変異部位と塩基対を形成する塩基である。さらに、変異型(GTT)分子ビーコンプローブ中の下線が付された塩基は、分子内構造体を形成する際に互いにハイブリダイズする領域である。
調製された試料溶液を、95℃で5分間加熱することにより変性させた後、20℃まで徐々に液温を低下させて会合体を形成させた。具体的には、降温速度を0.1℃/秒とし、90℃で5分間、80℃で10分間、70℃で10分間、60℃で10分間、50℃で10分間、40℃で10分間、30℃で10分間の降温処理を行った。
20μ秒<ピーク幅<400μ秒
ピーク強度>1(フォトン/10μ秒)
相関係数>0.95
を満たすピーク信号のみを観測対象の核酸分子に対応する光信号であると判定する一方、上記の条件を満たさないピーク信号はノイズとして無視し、観測対象の核酸分子に対応する光信号であると判定された信号の数を「ピーク数」として計数した。
試料溶液中に、核酸プローブが特異的に結合する型以外の型の多型配列と相補的な塩基配列を有する核酸分子を添加することによる、当該核酸プローブを含む会合体の検出精度を調べた。
具体的には、実施例1で用いた変異型(GTT)分子ビーコンプローブ、解析対象の核酸分子、野生型(GGT)デコイ核酸を、それぞれ、100pM、100nM(野生型と変異型を合計した濃度)、1μMとなるように、トリス緩衝液(10 mM Tris-HCl、1mM EDTA、100mM NaCl、pH8.0)に溶解することにより、野生型(GGT)デコイ核酸を含有する試料溶液を調製した。一方、野生型(GGT)デコイ核酸を添加しない以外は同様にして、野生型(GGT)デコイ核酸を含有しない試料溶液を調製した。
これらの試料溶液に対して、実施例1と同様に、液温を上昇及び下降させて試料溶液中の核酸分子を変性させた後会合体を形成し、さらに、実施例1と同じ条件で、当該試料溶液中の変異型(GTT)分子ビーコンプローブを含む会合体の分子数を計数した。
実施例1で用いた変異型(GTT)分子ビーコンプローブと、野生型(GGT)分子ビーコンプローブとを用いて、各遺伝子型を識別した。
野生型(GGT)分子ビーコンプローブとして、5’末端にTAMRAが付加され、3’末端にBHQ-2が付加された表2に記載の塩基配列(配列番号5)を有する核酸分子を用いた。
表2に、変異型(GTT)デコイ核酸の配列を示す。表2中、右欄には配列番号を示し、太字の塩基が変異部位又は変異部位と塩基対を形成する塩基であり、下線が付された塩基は、分子内構造体を形成する際に互いにハイブリダイズする領域である。なお、本実施例で用いたオリゴヌクレオチドは、シグマジェノシス株式会社に依頼して合成した。
一方、野生型(GGT)分子ビーコンプローブ、解析対象の核酸分子、変異型(GTT)デコイ核酸を、それぞれ、100pM、100nM(野生型と変異型を合計した濃度)、500nMとなるように、前記トリス緩衝液に溶解することにより、野生型(GGT)分子ビーコンプローブと変異型(GTT)デコイ核酸を含有する試料溶液を調製した。また、解析対象の核酸分子を添加しなかった以外は同様にして対照用試料溶液を調製した。
本発明の核酸分子の多型識別方法において、一の試料溶液中で、変異型(GTT)分子ビーコンプローブを含む会合体と野生型(GGT)分子ビーコンプローブを含む会合体の両方を形成させる条件で、K-ras遺伝子のコドン12_GTT変異を識別した。
変異型(GTT)分子ビーコンプローブとして、表1に記載の変異型(GTT)分子ビーコンプローブと同じ塩基配列(配列番号1)からなるオリゴヌクレオチドの5’末端にATTO(登録商標)647Nを付加し、3’末端にBHQ-3を付加したものを用いた。野生型(GGT)分子ビーコンプローブ、解析対象の核酸分子、及び野生型(GGT)デコイ核酸、変異型(GTT)デコイ核酸は、実施例3で用いたものを用いた。なお、本実施例で用いたオリゴヌクレオチドは、シグマジェノシス株式会社に依頼して合成した。
具体的には、変異型(GTT)分子ビーコンプローブ、野生型(GGT)分子ビーコンプローブ、解析対象の核酸分子、野生型(GGT)デコイ核酸、及び変異型(GTT)デコイ核酸を、それぞれ、100pM、100pM、100nM(野生型と変異型を合計した濃度)、500nM、500nMとなるように、トリス緩衝液(10 mM Tris-HCl、1mM EDTA、400mM NaCl、pH8.0)に溶解することにより試料溶液を調製した。また、解析対象の核酸分子を添加しなかった以外は同様にして対照用試料溶液を調製した。
実施例4では、解析対象の核酸分子を変異率ごとに識別可能ではあったが、図12における各試料溶液のスポット間の距離は、図11に示される実施例3の場合よりも、互いの距離が近い。これは、実施例3の方法よりも、実施例4の方法のほうが、識別能がやや劣ることを示唆する。
そこで、デコイ核酸の長さを調整し、変異率の識別能を改善させた。
具体的には、実施例4で用いた野生型(GGT)デコイ核酸及び変異型(GTT)デコイ核酸に代えて、表3に記載されている野生型(GGT)デコイ核酸(配列番号7)及び変異型(GTT)デコイ核酸(配列番号8)を用いた以外は、実施例4と同様にして試料溶液を調製した。なお、本実施例において用いたデコイ核酸は、実施例4で用いたものよりもそれぞれ1塩基だけ短い。また、表3中、右欄は配列番号を示し、太字の塩基が変異部位又は変異部位と塩基対を形成する塩基を示す。
実施例4における識別能が、実施例3における識別能よりも低かったのは、2種類の分子ビーコンプローブと2種類のデコイ核酸とが共存していたため、デコイ核酸が分子ビーコンの標的配列への非特異結合だけでなく、特異的な結合も阻害したためだと考えられる。本実施例においては、デコイ核酸の長さが短いことにより、分子ビーコンの標的配列への特異的結合阻害が小さくなったために、GTT変異率の識別能が改善されたものと推察される。
これらの結果から、デコイ核酸の設計を工夫することにより、多型配列中の一の型に特異的な核酸プローブと、当該多型配列中の他の型に特異的な核酸プローブとを、一の試料溶液中で共存させた状態で、変異型(GTT)分子ビーコンプローブを含む会合体と野生型(GGT)分子ビーコンプローブを含む会合体の両方の会合体の形成・検出を行う場合であっても、非常に高い精度で多型を識別することができることが明らかである。
第1核酸プローブとは異なる塩基配列を有し、かつ、解析対象の多型配列からなる多型部位を認識しない核酸プローブ第2核酸プローブとし、第1核酸プローブと合わせて用いて、K-ras遺伝子のコドン12_GTT変異を識別した。
具体的には、第2核酸プローブとして、K-ras遺伝子中の領域であって、コドン12を含まず、野生型と変異型に共通する領域の塩基配列(共通配列)(配列番号9)と特異的にハイブリダイズするオリゴヌクレオチド(配列番号10)に対して、5’末端にATTO(登録商標)647Nを付加し、3’末端にBHQ-3を付加した共通分子ビーコンプローブを用いた。また、前記共通配列からなるオリゴヌクレオチドを、共通核酸として用いた。表4に、共通分子ビーコンプローブ及び共通配列の塩基配列を示す。表4中、右欄には配列番号を示す。また、共通分子ビーコンプローブ中の下線が付された塩基は、分子内構造体を形成する際に互いにハイブリダイズする領域である。
1種類の核酸プローブと蛍光性インターカレーターを用いて、走査分子計数法により、K-ras遺伝子のコドン12_GTT変異を識別可能であることを検証した。
変異型(GTT)プローブとして、5’末端にROXが付加された表5に記載の塩基配列(配列番号11)を有する核酸分子を用いた。表5中、右欄には配列番号を示し、太字の塩基が変異部位又は変異部位と塩基対を形成する塩基である。さらに、解析対象の核酸分子として、実施例1で用いた野生型(GGT)核酸と変異型(GTT)核酸を、それぞれ用いた。
調製された試料溶液を、94℃で5分間加熱することにより変性させた後、0.1℃/秒で降温させ、68.8℃で5分間恒温処理させて会合体を形成させた。
これらの濃縮試料溶液に、それぞれ、PicoGreen(Molecular Probe社製)を前記トリス緩衝液で1000倍希釈した溶液を添加し、当該濃縮試料溶液の10倍希釈を調製し、これらの試料溶液とした。
これらの試料溶液を、PicoGreen希釈液添加後30分間以上放置した。
測定によって得られた時系列データをSavinzky-Golayのアルゴリズムでスムージングした後、微分によりピークの検出を行った。ピークとみなされた領域のうち、ガウス関数に近似できる領域をシグナルとして抽出した。野生型(GGT)デコイ核酸を加えた試料溶液と加えない試料とで得られるピーク数を比較した。
実施例7で用いた変異型(GTT)プローブと、野生型(GGT)プローブと、蛍光性インターカレーターを用いて、走査分子計数法により、K-ras遺伝子のコドン12_GTT変異を識別した。
野生型(GGT)プローブとして、5’末端にROXが付加された表6に記載の塩基配列(配列番号12)を有する核酸分子を用いた。表6中、右欄には配列番号を示し、太字の塩基が変異(多型)部位又は変異部位と塩基対を形成する塩基である。また、解析対象の核酸分子として、表1に記載の塩基配列を有する野生型(GGT)核酸(配列番号2)のみ(変異率:0%)、野生型(GGT)核酸と変異型(GTT)核酸(配列番号3)を9:1のモル比で混合したもの(変異率:10%)、野生型(GGT)核酸と変異型(GTT)核酸(配列番号3)を1:1のモル比で混合したもの(変異率:50%)、及び変異型(GTT)核酸のみ(変異率:100%)用いた。さらに、非特異的なハイブリダイゼーションを抑制するために、実施例3で用いた変異型(GTT)デコイ核酸を用いた。
調製された試料溶液を、94℃で5分間加熱することにより変性させた後、0.1℃/秒で降温させ、68.8℃で5分間恒温処理させて会合体を形成させた。
これらの濃縮試料溶液に、それぞれ、PicoGreen(Molecular Probe社製)を前記トリス緩衝液で1000倍希釈した溶液を添加し、当該濃縮試料溶液の10倍希釈を調製し、これらの試料溶液とした。
これらの試料溶液を、PicoGreen希釈液添加後30分間以上放置した。
核酸プローブと蛍光性インターカレーターを用いて、当該核酸プローブを含む会合体を走査分子計数法により計数する場合における、会合体中の2本鎖構造の塩基対長が与える影響を調べた。
まず、プラスミドpUC19(タカラバイオ社製)を鋳型とし、5’末端をRoxで修飾したオリゴヌクレオチド、非標識のオリゴヌクレオチド、及びAmpliTaq Gold(アプライドバイオシステムズ社製)を用いて、100bp、200bp、400bp、800bp、及び1.5kbpの鎖長の異なるPCR産物を調製した。これらのPCR産物からWizard V Gel and PCR Clean-Up System (Promega社製)を用いてプライマー除去し、Bioanalyzer(Agilent社製)を用いた電気泳動により、PCR産物の有無及び濃度を測定した。これらのPCR産物(蛍光標識)は、5’末端がRoxで標識された1本鎖核酸分子と、非標識の1本鎖核酸分子との会合体である。
上記で用いた5’末端をRoxで修飾したオリゴヌクレオチドに代えて、5’末端が修飾されていないオリゴヌクレオチドを用いた以外は、上記と同様にして、非標識の1本鎖核酸分子同士の会合体であるPCR産物(非標識)を得た。
その後、トリス緩衝液(10 mM Tris-HCl、1mM EDTA、100mM NaCl、pH8.0)を用いて、各PCR産物が100pMとなるように調製した。この調製されたPCR産物の溶液を、前記トリス緩衝液で10000倍希釈したPicoGreen(invitrogen社製)溶液に任意の濃度で加え、試料溶液とした。
これらの試料溶液を、PicoGreen希釈液添加後30分間以上放置した。
これらの結果より、およそ400bp程度の鎖長以下であれば、充分な非特異シグナルの低減が成されていると見ることができる。
2 光源
3 シングルモードオプティカルファイバー
4 コリメータレンズ
5 ダイクロイックミラー
6、7、11 反射ミラー
8 対物レンズ
9 マイクロプレート
10 ウェル(試料溶液容器)
12 コンデンサーレンズ
13 ピンホール
14 バリアフィルター
15 マルチモードオプティカルファイバー
16 光検出器
17 ミラー偏向器
17a ステージ位置変更装置
18 コンピュータ
Claims (13)
- (a)多型配列中の第1の型の塩基配列を有する1本鎖核酸分子と特異的にハイブリダイズする第1の核酸プローブと、解析対象の核酸分子とを含む試料溶液を調製する試料溶液調製工程と、
(b)前記工程(a)において調製された試料溶液中の核酸分子を会合させる会合工程と、
(c)前記工程(b)の後、前記工程(a)において調製された試料溶液中の、第1の核酸プローブを含む会合体の分子数を算出する算出工程と、
(d)前記工程(c)の結果に基づき、前記解析対象の核酸分子の多型を識別する識別工程と、
(e)前期工程(c)において、共焦点顕微鏡又は多光子顕微鏡の光学系を用いて、前記試料溶液内において前記光学系の光検出領域の位置を移動する検出領域移動工程と、
(f)前期工程(c)において、前記試料溶液内において前記光学系の光検出領域の位置を移動させながら、当該光検出領域中の前記会合体から放出される蛍光を検出する蛍光検出工程と、
(g)前期工程(c)において、前記検出された光から、個々の会合体からの光信号を個別に検出して、会合体を個別に検出する会合体検出工程と、
(h)前期工程(c)において、前記個別に検出された会合体の数を計数して前記光検出領域の位置の移動中に検出された前記粒子の数を計数する粒子計数工程と、を有し、
前記工程(a)における試料溶液、又は前記工程(b)の後、前記工程(c)の前の試料溶液が、前記多型配列のうちの前記第1の型以外の型の塩基配列と相補的な塩基配列を有するオリゴヌクレオチドをさらに含んでいる核酸分子の多型識別方法。 - 前記第1の核酸プローブが、蛍光物質により標識されている請求項1に記載の核酸分子の多型識別方法。
- 前記工程(a)において、前記試料溶液中に、2本鎖構造に特異的に結合する蛍光性2本鎖核酸結合物質がさらに含まれている請求項1又は2に記載の核酸分子の多型識別方法。
- 前記工程(a)において、前記試料溶液中に、蛍光性2本鎖核酸結合物質がさらに含まれており、
前記第1の核酸プローブを標識している蛍光物質と前記蛍光性2本鎖核酸結合物質とのいずれか一方が、蛍光エネルギー移動現象におけるエネルギー・ドナーと成る蛍光物質であり、他方が前記蛍光エネルギー移動現象におけるエネルギー・アクセプターと成る物質であり、
前記工程(c)において、前記第1の核酸プローブを含む会合体から放出される蛍光は、前記第1の核酸プローブを標識している蛍光物質と前記蛍光性2本鎖核酸結合物質との間に起こった蛍光エネルギー移動現象により放出される蛍光である請求項2に記載の核酸分子の多型識別方法。 - 前記第1の核酸プローブは、単独で存在している状態では蛍光エネルギー移動が起こり、かつ他の1本鎖核酸分子と会合体を形成した状態では蛍光エネルギー移動が起こらないように、エネルギー・ドナーと成る蛍光物質とエネルギー・アクセプターと成る物質とが結合されており、
当該核酸プローブを含む会合体から放出される蛍光は、前記エネルギー・ドナーと成る蛍光物質から放出される蛍光である請求項2に記載の核酸分子の多型識別方法。 - 前記光検出領域の位置を移動する工程に於いて、前記光検出領域の位置が所定の速度にて移動される請求項1~5のいずれか一項に記載の核酸分子の多型識別方法。
- 前記光検出領域の位置を移動する工程に於いて、前記光検出領域の位置が、前記会合体の拡散移動速度よりも速い速度にて移動される請求項1~6のいずれか一項に記載の核酸分子の多型識別方法。
- 前記検出された光から、個々の会合体からの光信号を個別に検出して、会合体を個別に検出する工程に於いて、検出された時系列の光信号の形状に基づいて、1つの会合体が前記光検出領域に入ったことが検出される請求項1~7のいずれか一項に記載の核酸分子の多型識別方法。
- 前記試料溶液が、界面活性剤、ホルムアミド、ジメチルスルホキシド、及び尿素からなる群より選択される1種以上を含む請求項1~8のいずれか一項に記載の核酸分子の多型識別方法。
- 前記工程(b)を、前記工程(a)において調製された試料溶液の温度を70℃以上にすることにより、当該試料溶液中の核酸分子を変性させた後、当該試料溶液の液温を0.05℃/秒以上の降温速度で低下させることにより、当該試料溶液中の核酸分子を会合させることにより行う請求項1~9のいずれか一項に記載の核酸分子の多型識別方法。
- 前記第1の核酸プローブ及び前記第2の核酸プローブが、DNA、RNA、及び核酸類似物質からなる群より選択される2以上の分子が結合して構成されている請求項1~10のいずれか一項に記載の核酸分子の多型識別方法。
- 前記多型配列が、遺伝子多型の多型部位を含む塩基配列、又は体細胞変異の変異部位を含む塩基配列である請求項1~11のいずれか一項に記載の核酸分子の多型識別方法。
- 前記体細胞変異がK-ras遺伝子の変異である請求項12に記載の核酸分子の多型識別方法。
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EP2669376A1 (en) | 2013-12-04 |
EP2669376A4 (en) | 2014-09-17 |
JPWO2012102326A1 (ja) | 2014-06-30 |
EP2669376B1 (en) | 2017-08-16 |
CN103339256A (zh) | 2013-10-02 |
US20140004519A1 (en) | 2014-01-02 |
CN103339256B (zh) | 2016-03-16 |
JP6009944B2 (ja) | 2016-10-19 |
US8900812B2 (en) | 2014-12-02 |
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