JP2015000047A - Method for measuring mutation rate of target nucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method which can measure mutation rate more reliably in a gene having a mutation.SOLUTION: Method for measuring mutation rate of target nucleic acid comprises a step of preparing a solution containing a nucleic acid-containing sample, a fluorescent probe whose fluorescence wavelength specifically binding to a range comprising a mutation site in a target nucleic acid is in the range of 580-680 nm, a nonluminous nucleic acid probe, a solid phase carrier bound by a probe for recovery, an intercalator whose fluorescence wavelength is outside of the range of 580-680 nm, and forming a complex which comprises a target nucleic acid, the fluorescent probe, the nonluminous nucleic acid probe, and the fluorescent intercalator fixed to a solid-phase carrier through the probe for recovery in the solution; a step of preparing from the solution a sample solution comprising the complex concerned by solid liquid separation treatment; a step of calculating a number of fluorescent probes and a number of the complexes in the prepared sample solution; and a step of calculating a mutation rate on the basis of the number of the calculated fluorescent probes and the number of the complexes.

Description

  The present invention relates to a method for measuring a mutation rate by detecting a mutation type and all genotypes containing the mutation type by separately fluorescently labeling them in a gene having a mutation.

  In gene analysis, when analyzing mutations in nucleotide sequences such as single nucleotide mutations and modifications due to modifications such as methylation, the mutation rate (the ratio of the mutated genes in the total amount of the gene) is important. Become. For this reason, in the analysis of gene mutation, for the same nucleic acid-containing sample, the total amount of the target gene to be analyzed and the amount of the mutant gene having the mutant type to be analyzed among the target genes, It is necessary to measure. In particular, in the case of a nucleic acid-containing sample derived from a living body, the amount of the sample itself is often small. Therefore, a method capable of detecting a very small amount of target nucleic acid with high sensitivity for detection of a target gene and a mutant gene. In particular, a method capable of detecting both the target gene and the mutant gene with high sensitivity from one nucleic acid-containing sample is particularly preferable. Therefore, a method is widely used in which the target gene and the mutant gene are detected by a fluorescence analysis technique using separate fluorescent probes having different fluorescence characteristics. For example, a target nucleic acid in one nucleic acid-containing sample binds to a first fluorescent probe that specifically binds to a specific mutant type and all genotypes, and the first fluorescent probe has a different fluorescence wavelength. Labeling with the second fluorescent probe, the mutation rate can be calculated from the amount of fluorescence from the first fluorescent probe and the amount of fluorescence from the second fluorescent probe.

  On the other hand, with the recent development of optical measurement technology, optical systems that can detect light from a small area in a solution, such as optical systems for confocal microscopes and multiphoton microscopes, and photon counting (one-photon detection) are also possible. It is possible to detect and measure weak light at the level of one photon or one molecule of fluorescence using a highly sensitive light detection technique (single molecule light detection technique). Thus, various apparatuses or methods for detecting intermolecular interactions such as biomolecules or binding / dissociation reactions using such single-molecule photodetection technology have been proposed. In particular, a microscopic region (microscope laser beam) using a confocal microscope optical system such as fluorescence correlation spectroscopy (FCS) and fluorescence-intensity distribution analysis (FIDA) and photon counting technology. According to the method using the fluorescence measurement technique of the focused focal region-confocal volume), the sample required for the measurement may be in a very low concentration and a very small amount compared to the conventional ( The amount used in one measurement is at most about several tens of μL), and the measurement time is greatly shortened (measurement of time on the order of seconds is repeated several times in one measurement).

  Recently, using an optical system that can detect light from a small area in a solution, such as an optical system of a confocal microscope or a multiphoton microscope, the position of the small area that is a light detection area in the sample solution is moved. In addition, a novel optical analysis technique (scanning molecule counting method) that individually detects luminescent particles (particles that emit light) traversing the minute region has been proposed (see, for example, Patent Documents 1 to 3). ). Specifically, the scanning molecule counting method uses the optical system of a confocal microscope or a multiphoton microscope to move the position of the light detection region of the optical system in a sample solution, and emit light in the light detection region. Detects the light emitted from the particles, thereby detecting each individual luminescent particle in the sample solution and obtaining information on the counting of the luminescent particles and the concentration or number density of the luminescent particles in the sample solution It is a technology that makes possible.

  The scanning molecule counting method is configured to detect light from a light detection region of a confocal microscope or a multiphoton microscope, as in the case of optical analysis techniques such as FIDA, for the light detection mechanism itself. Similar to FIDA and the like, the amount of the sample solution may be very small (for example, about several tens of μL), and the measurement time is short. On the other hand, in the scanning molecule counting method, unlike FIDA and the like that require statistical processing such as calculating fluctuations in fluorescence intensity, such statistical processing is not executed. For this reason, the optical analysis technique of the scanning molecule counting method can be applied to a sample solution in which the number density or concentration of particles is significantly lower than the level required for an optical analysis technique such as FIDA. In other words, when the target particles (observation target particles) in the sample solution labeled with the luminescent probe are detected using the scanning molecule counting method, the concentration or number density of the target particles in the sample solution is very low. Even so, the state or characteristics of the target particles can be detected and analyzed (for example, see Patent Document 4). Thus, single molecule emission analysis techniques such as scanning molecule counting and FCS are suitable for analyzing gene mutations because they can detect extremely small amounts of target particles with high sensitivity.

International Publication No. 2011/108369 International Publication No. 2011/108370 International Publication No. 2011/108371 International Publication No. 2012/014778

  When detecting a plurality of fluorescence wavelengths from one sample solution, there is a problem that the detection sensitivity differs depending on the fluorescence wavelength. For example, in the short wavelength side measurement, since the background signal is increased by noise derived from Raman scattering, there is a problem that the sensitivity is lower than that on the long wavelength side. Further, in the long and high wavelength side measurement, there is a tendency that the noise becomes larger depending on the detector of the measuring apparatus. In other words, the fluorescence wavelength range that can be detected with high sensitivity is limited, but when using a plurality of fluorescence wavelengths for detection, either one has insufficient detection sensitivity in order to distinguish and detect each. The fluorescence wavelength must be used.

  The present invention relates to a method for detecting a mutant type and all genotypes containing the mutant type by separately fluorescently labeling them in a gene having a mutation. It is an object of the present invention to provide a method that can detect a mutation rate more reliably and can measure a more reliable mutation rate.

  As a result of diligent research to solve the above problems, the present inventor labeled a target nucleic acid containing a mutant type with a fluorescent probe having a fluorescence wavelength with a high detection sensitivity, and labeled all of the target nucleic acids. By labeling the mold with an intercalator with a fluorescence wavelength with low detection sensitivity, both the mutant type and all genotypes of the target nucleic acid can be detected with high sensitivity. The present inventors have found that a highly reliable mutation rate can be calculated and completed the present invention.

That is, the method for measuring the mutation rate of the target nucleic acid according to the present invention is the following [1] to [7].
[1] (a) a fluorescent probe having a fluorescence wavelength within a range of 580 to 680 nm, which specifically binds to a nucleic acid-containing sample, a region containing a mutation site in the target nucleic acid, the target nucleic acid, and the fluorescent probe A non-luminescent nucleic acid probe that binds at a different position, a solid phase carrier to which a recovery probe that binds at a different position from the target nucleic acid, the fluorescent probe, and the non-luminescent nucleic acid probe, and a fluorescence wavelength of 580 to A solution containing a fluorescent intercalator that is outside the range of 680 nm is prepared, and the target nucleic acid, the fluorescent probe, the non-luminescent nucleic acid probe, and the fluorescent intercalator are contained in the solution, or the target nucleic acid A process comprising the non-luminescent nucleic acid probe and the fluorescent intercalator, and forming a complex fixed to the solid phase carrier via the recovery probe. About
(B) after the step (a), recovering the complex from the solution by solid-liquid separation, and preparing a sample solution containing the complex;
(C) calculating the number of fluorescent probes and the number of complexes including the target nucleic acid, the non-luminescent nucleic acid probe, and the fluorescent intercalator in the sample solution prepared in the step (b) Process,
(D) Based on the number of the fluorescent probes calculated in the step (c) and the number of complexes including the target nucleic acid, the non-luminescent nucleic acid probe, and the fluorescent intercalator, Calculating the abundance ratio of the target nucleic acid containing the mutation site in the target nucleic acid in the sample;
A method for measuring a mutation rate of a target nucleic acid, comprising:
[2] After the step (b) and before the step (c),
(E) a step of dissociating the complex in the sample solution prepared in the step (b) from the solid phase carrier, and then separating and recovering the complex from the solid phase carrier by a solid-liquid separation process;
The method for measuring a mutation rate of a target nucleic acid according to [1], comprising:
[3] In the step (e), when the complex includes the fluorescent probe, the fluorescent probe is also dissociated from the complex, and the target nucleic acid and the non-luminescent nucleic acid are subjected to solid-liquid separation treatment. [2] The method for measuring a mutation rate of a target nucleic acid according to [2] above, wherein a complex containing a probe and the fluorescent intercalator and the fluorescent probe are separated and recovered from the solid phase carrier.
[4] The fluorescent probe in which the step (a) specifically binds to the nucleic acid-containing sample (a ′) and the region containing the mutation site in the target nucleic acid, and the fluorescence wavelength is in the range of 580 to 680 nm; A solid phase carrier in which a non-luminescent nucleic acid probe that binds to a target nucleic acid and the fluorescent probe at different positions, and a recovery probe that binds to the target nucleic acid, the fluorescent probe, and the non-luminescent nucleic acid probe in different positions A solution containing the target nucleic acid, the fluorescent probe, and the non-luminescent nucleic acid probe, or the target nucleic acid and the non-luminescent nucleic acid probe, and through the recovery probe. Forming a complex immobilized on the solid phase carrier,
And the step (b)
(B′1) after the step (a), recovering the complex from the solution by solid-liquid separation, and preparing a sample solution containing the complex;
(B′2) After the step (b′1), a fluorescent intercalator having a fluorescence wavelength outside the range of 580 to 680 nm is added to the sample solution, and inserted between base pairs in the complex. Process,
The method for measuring the mutation rate of the target nucleic acid according to any one of the above [1] to [3].
[5]
In the step (c), calculation of the number of the fluorescent probes and the number of complexes including the target nucleic acid, the non-luminescent nucleic acid probe, and the fluorescent intercalator is performed by scanning molecule counting method, fluorescence correlation spectroscopy. The method for measuring the mutation rate of the target nucleic acid according to any one of [2] to [4], which is carried out by fluorescence intensity distribution analysis or fluorescence polarization intensity distribution analysis.
[6]
In the step (e), the target nucleic acid and the non-luminescent probe can form an aggregate in the sample solution prepared in the step (b), and the target nucleic acid, the fluorescent probe, and the target The target nucleic acid according to any one of [3] to [5], wherein the solid phase carrier and the fluorescent probe are dissociated from the complex by incubating at a temperature at which the nucleic acid and the recovery probe cannot form an aggregate. Mutation rate measurement method.
[7] In the step (a) or the step (a ′), the target nucleic acid, the fluorescent probe, and the non-luminescent nucleic acid probe may bind to the recovery probe and bind to the recovery probe. The method for measuring a mutation rate of a target nucleic acid according to any one of [1] to [6], wherein the solution is prepared by adding a solid phase carrier.

  The method for measuring a mutation rate of a target nucleic acid according to the present invention can detect both a mutant type to be analyzed and a whole type of the target nucleic acid with high sensitivity by fluorescence analysis. it can. For this reason, in the mutation rate measurement method, it is possible to accurately measure both the amount of mutations in one nucleic acid-containing sample and the total amount of all target nucleic acids, and as a result, more reliable. Mutation rate is obtained.

In Example 1, it is the figure which showed the count result of the number of peaks of the sample solution which labeled all the target nucleic acids with the nonluminous nucleic acid probe and SYTOX (trademark) Orange. In Example 1, it is the figure which showed the count result of the peak number of the sample solution which labeled all the target nucleic acids with the TAMRA labeled probe.

  The method for measuring the mutation rate of a target nucleic acid according to the present invention (hereinafter sometimes simply referred to as “method for measuring the mutation rate according to the present invention”) is a method for identifying a target nucleic acid containing a mutant type in one nucleic acid-containing sample. This is a method for measuring the mutation rate of the mutant type. In the present invention and the specification of the present application, the “target nucleic acid” is a nucleic acid including all types including a mutant type, and the “mutation rate” is the total amount of the target nucleic acid (ie, the total amount of the target nucleic acid). Means the abundance ratio of the mutant type to be analyzed. For example, when the target nucleic acid is a nucleic acid derived from a gene having a wild type and one type of mutant, the target nucleic acid includes both the wild type and the mutant type, and the mutation rate is between the wild type and the mutant type. It refers to the abundance ratio of mutants relative to the total amount. When the target nucleic acid is a nucleic acid derived from a gene that has a wild type and multiple types of mutants, and the target nucleic acid is a specific mutant type, the target nucleic acid includes the wild type and all types of mutants. The mutation rate refers to the abundance ratio of the mutant to be detected relative to the total amount of the wild type and all types of mutants. In addition, the mutation type contained in the target nucleic acid may be a mutation in the base sequence such as base substitution, deletion or insertion, or may be a mutation due to modification such as methylation.

  The mutation rate measurement method according to the present invention can utilize a single-molecule fluorescence analysis technique such as a scanning molecule counting method, and thereby can detect a very small amount of fluorescent particles with high sensitivity. For this reason, the mutation rate measurement method according to the present invention can be suitably used in clinical fields that are particularly required to be excellent in both sensitivity and accuracy, and among them, the target nucleic acid is a gene having a gene polymorphism, It is preferably a nucleic acid derived from a gene that can undergo modification such as methylation due to oncogenesis.

  In the mutation rate measurement method according to the present invention, a fluorescence wavelength capable of highly sensitively detecting a mutant type (hereinafter, also referred to as “target mutant nucleic acid”) that is a target for measuring the mutation rate of the target nucleic acid. Intercalators in the fluorescence wavelength range where detection sensitivity is relatively low for all types of target nucleic acids including target mutant nucleic acids (hereinafter sometimes referred to as “total target nucleic acids”). It is characterized by detecting with a label. In the present invention, the amount of all target nucleic acids including the target mutant nucleic acid is determined from the fluorescence amount of the fluorescent intercalator, the amount of target mutant nucleic acid is determined from the fluorescence amount of the fluorescent probe, and the mutation rate is calculated based on these amounts. To do. Since many intercalators can be inserted between base pairs in one target nucleic acid, the fluorescence intensity per one target nucleic acid can be increased compared to the case of using a fluorescent probe, and sensitivity can be increased even when the noise is large. Can be raised. As described above, the method for measuring a mutation rate according to the present invention can detect both of the target mutant nucleic acid and all target nucleic acids with high sensitivity even though they are labeled with fluorescence having different fluorescence wavelengths.

Specifically, the mutation rate measuring method according to the present invention includes the following steps (a) to (d).
(A) a fluorescent probe having a fluorescence wavelength in the range of 580 to 680 nm, which specifically binds to a nucleic acid-containing sample and a region containing a mutation site in the target nucleic acid, and a position where the target nucleic acid and the fluorescent probe are different A solid-phase carrier bound with a non-luminescent nucleic acid probe that binds at a different position, a recovery probe that binds at a position different from the target nucleic acid, the fluorescent probe, and the non-luminescent nucleic acid probe; Preparing a solution containing a fluorescent intercalator that is outside, and containing the target nucleic acid, the fluorescent probe, the non-luminescent nucleic acid probe, and the fluorescent intercalator in the solution, or the target nucleic acid and the non-luminescent Forming a complex immobilized on the solid phase carrier via the recovery probe, comprising a functional nucleic acid probe and the fluorescent intercalator;
(B) after the step (a), recovering the complex from the solution by solid-liquid separation, and preparing a sample solution containing the complex;
(C) calculating the number of fluorescent probes and the number of complexes including the target nucleic acid, the non-luminescent nucleic acid probe, and the fluorescent intercalator in the sample solution prepared in the step (b) Process,
(D) Based on the number of the fluorescent probes calculated in the step (c) and the number of complexes including the target nucleic acid, the non-luminescent nucleic acid probe, and the fluorescent intercalator, Calculating the abundance ratio of the target nucleic acid containing the mutation site in the target nucleic acid in the sample;

  The fluorescent probe used in the present invention is a probe that specifically binds to a region containing a mutation site in a target mutant nucleic acid. When the fluorescent probe specifically binds to the target mutant nucleic acid in the region including the mutation site, the target mutant nucleic acid is labeled with the fluorescence of the fluorescent probe. Here, “specifically binds to a region containing a mutation site in the target mutation nucleic acid” means that the fluorescent probe is significantly more than the target nucleic acid against a nucleic acid in which the region is different from the mutation site. This means that the region binds preferentially, and the region does not have to bind to a substance other than the target mutant nucleic acid that is the mutation site.

  The fluorescent probe used in the present invention has a fluorescence wavelength in the range of 580 to 680 nm. If the fluorescence wavelength is 580 nm or more, the background signal due to noise derived from Raman scattering is small and can be detected with high sensitivity. Also, depending on the type of fluorescence detector, when the fluorescence wavelength is very long, the detection sensitivity may decrease. If the fluorescence wavelength is 680 nm or less, it can be detected with high sensitivity by a detector.

  As the fluorescent probe used in the present invention, when the mutant type of the target mutant nucleic acid is a nucleotide sequence mutation, for example, an oligonucleotide or polynucleotide that hybridizes with a region containing the mutation site in the target mutant nucleic acid ( Hereinafter, it may be referred to as “oligonucleotide or the like.”) And a fluorescent substance bound thereto, or a fluorescent substance bound to a protein or peptide that can specifically bind to the region. When the mutation type of the target mutant nucleic acid is a mutation caused by modification such as methylation, for example, a fluorescent substance is added to a protein or peptide that specifically binds to the mutation site (for example, a methylated DNA-binding protein). Examples thereof include those obtained by binding, and those obtained by binding a fluorescent substance to an oligonucleotide or the like that hybridizes with a region containing the mutation site.

  Such oligonucleotides may be DNA, RNA, artificially amplified, such as cDNA, and nucleotide chains and bases as well as natural nucleobases. A part or all of the nucleic acid analogs capable of forming a pair may be included. 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. More specifically, for example, Bridged Nucleic Acid (BNA), 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.

  When the fluorescent probe is a substance in which a fluorescent substance is bound to an oligonucleotide or the like, the fluorescent substance is not particularly limited as long as the fluorescent wavelength is in the range of 580 to 680 nm, and is widely used in the field of fluorescence analysis. It can be used by appropriately selecting from the fluorescent materials used. “The fluorescence wavelength is in the range of 580 to 680 nm” means that the peak of the fluorescence spectrum is in the range of 580 to 680 nm. Examples of the fluorescent substance having a fluorescence wavelength within the range include Texas Red (590 nm (Ex) / 615 nm (Em)), DsRed (558 nm (Ex) / 583 nm (Em)), TRITC (543 nm (Ex) / 580). (Em)), Alexa Fluor (registered trademark) 647N (650 nm (Ex) / 668 nm (Em)), Cy (registered trademark) 5 (650 nm (Ex) / 670 nm (Em)), and the like.

  The non-luminescent nucleic acid probe used in the present invention is a nucleic acid probe that binds to a target nucleic acid at a position different from that of the fluorescent probe, and is not labeled with a luminescent substance such as a fluorescent substance. In the target nucleic acid other than the region to which the fluorescent probe binds, the nucleotide sequence is the same for all types. That is, the non-luminescent nucleic acid probe binds to all target nucleic acids including the target mutant nucleic acid.

  Examples of the non-luminescent nucleic acid probe used in the present invention include an oligonucleotide that hybridizes with a region other than the region to which the fluorescent probe binds in the target mutant nucleic acid. The oligonucleotide or the like may have a base sequence that is completely complementary to the partial base sequence of the target nucleic acid, and has a base sequence that has a mismatch of one to several bases with the partial base sequence of the target nucleic acid. It may be. The oligonucleotide or the like may be DNA, RNA, artificially amplified, such as cDNA, or nucleic acid-like substance as in the case of the fluorescent probe. May be included in part or in whole.

  By inserting a fluorescent intercalator between the base pairs of the aggregate of the non-luminescent nucleic acid probe and the target nucleic acid, all types of target nucleic acids are labeled by fluorescence from the fluorescent intercalator. Here, the binding amount (insertion amount) of the fluorescent intercalator to the target nucleic acid is proportional to the base pair length of the region where the non-luminescent nucleic acid probe associates with the target nucleic acid. In other words, the longer the region where the non-luminescent nucleic acid probe associates with the target nucleic acid, the greater the amount of fluorescent labeling by the fluorescent intercalator per molecule of the target nucleic acid, and the higher the brightness, the noise (background signal) during fluorescence detection The difference is clearer and easier to detect. For this reason, the length of the region where the non-luminescent nucleic acid probe associates with the target nucleic acid differs from the noise in consideration of the type of fluorescent intercalator used, the method of fluorescence detection, the size of the background signal, etc. Can be determined as appropriate so that the length becomes sufficiently long. For example, the base pair length into which the fluorescent intercalator is inserted is preferably 50 bp or more, more preferably 60 bp or more, and may be 100 bp or more. On the other hand, if the amount of inserted fluorescent intercalator is too large, the background when detecting fluorescence becomes too high, which may affect the detection accuracy. For this reason, the length of the region where the non-luminescent nucleic acid probe is associated with the target nucleic acid is preferably 400 bp or less.

  In addition, since the non-luminescent nucleic acid probe binds to the target nucleic acid in a region different from the fluorescent probe, a complex in which the fluorescent probe and the non-luminescent nucleic acid probe are associated (bound) together is formed on one target nucleic acid. obtain. Therefore, when the target mutant nucleic acid is bound to both the fluorescent probe and the non-luminescent nucleic acid probe during fluorescence detection, the fluorescent intercalator was formed by the association of the target mutant nucleic acid and the non-luminescent nucleic acid probe. It is inserted both between the base pair and between the base pair formed by the association of the target mutant nucleic acid and the fluorescent probe. For this reason, when detecting a complex of a fluorescent probe, a non-luminescent nucleic acid probe, and a target base nucleic acid, the non-luminescent nucleic acid probe is targeted in consideration of the length of the region where the fluorescent probe binds to the target mutant nucleic acid. It is preferred to determine the length of the region associated with the nucleic acid.

  The fluorescence wavelength of the fluorescent intercalator used in the present invention is outside the range of 580 to 680 nm. Fluorescence wavelengths outside this range have low detection sensitivity due to noise or the like, but in the present invention, the fluorescence luminance per molecule of target nucleic acid is increased by binding multiple molecules of fluorescent intercalator per molecule of target nucleic acid. Increasing the detection probability and increasing the sensitivity.

  Among them, the fluorescent intercalator used in the present invention is preferably one having a fluorescence wavelength of less than 580 nm because the fluorescence probe is more clearly distinguished from the fluorescence. Examples of the fluorescent intercalator include ethidium bromide (295 nm (Ex) / 595 nm (Em)), acridine orange (3,6-Bis (dimethylamino) -acidine, hydrochloride) (502 nm (Ex) / 526 nm (Em)). ), PI (Propidium iodide) (488 nm (Ex) / 615 nm (Em)), DAPI (4 ′, 6-Diamino-2-phenylindylene chloride) (345 nm (Ex) / 455 nm (Em)), Hoechst (registered trademark) 33342 (343 nm (Ex) / 483 nm (Em)), SYBR (registered trademark) Green I (498 nm (Ex) / 522 nm (Em)), SYBR (registered trader) ) Green II (492 nm (Ex) / 513 nm (Em)), SYBR (registered trademark) Gold (495 nm (Ex) / 537 nm (Em)), GelGreen (registered trademark) (500 nm (Ex) / 530 nm (Em)), PicoGreen (registered trademark) (502 nm (Ex) / 522 nm (Em)), Ribo Green (registered trademark) (502 nm (Ex) / 522 nm (Em)), OliGreen (registered trademark) (502 nm (Ex) / 522 nm (Em)) ), EvaGreen (registered trademark) (500 nm (Ex) / 525 nm (Em)), SYTOX (registered trademark) Orange (547 nm (Ex) / 570 nm (Em)), SYTOX (registered trademark) Green (504 nm (Ex) / 523) ) Nm (Em)), SYTOX R) Blue (445nm (Ex) / 470nm (Em)), YOYO (R) -1 (491nm (Ex) / 509 (Em)), and the like.

  The recovery probe used in the present invention binds to a target nucleic acid and binds to a solid phase carrier. The recovery probe binds at a position different from the target nucleic acid, the fluorescent probe, and the non-luminescent nucleic acid probe. For this reason, it binds to all target nucleic acids including the target mutant nucleic acid, similarly to the non-luminescent nucleic acid probe.

  When the recovery probe binds to the target nucleic acid, a complex including the target nucleic acid, the fluorescent probe, and the non-luminescent nucleic acid probe is immobilized on the solid phase carrier via the recovery probe. The recovery probe used in the present invention has a portion that binds to a solid phase carrier, and further binds to the solid phase carrier in a state of binding to the target nucleic acid, and the target nucleic acid and the fluorescent probe In addition, the substance is not particularly limited as long as it is a substance that binds or adsorbs specifically or non-specifically independently of the non-luminescent nucleic acid probe, but is immobilized on a solid phase carrier during fluorescence detection. Since it is possible to detect a fluorescent probe or the like in the absence of the target, it is preferable that the binding with the target nucleic acid is a reversible reaction and is more easily dissociated from the target nucleic acid than the non-luminescent nucleic acid probe.

  The recovery probe is, for example, an oligonucleotide that hybridizes with a region other than the region to which the fluorescent probe and the non-luminescent nucleic acid probe bind in the target mutant nucleic acid, and has a portion that binds to a solid phase carrier. Things. The oligonucleotide or the like may have a base sequence that is completely complementary to the partial base sequence of the target nucleic acid, and has a base sequence that has a mismatch of one to several bases with the partial base sequence of the target nucleic acid. It may be. In addition, the oligonucleotide may be DNA, RNA, artificially amplified, such as cDNA, and nucleic acid-like substance as in the fluorescent probe. May be included in part or in whole.

  When the recovery probe is an oligonucleotide or the like that hybridizes with the target nucleic acid, for example, the Tm value of the association between the target nucleic acid and the non-luminescent nucleic acid probe is expressed as the Tm of the association between the target nucleic acid and the recovery probe. By setting the value higher than the value, the recovery probe can be more easily dissociated from the target nucleic acid than the non-luminescent nucleic acid probe. For example, the length of the region hybridized with the recovery probe is shorter than the length of the region hybridized with the non-luminescent nucleic acid probe in the target nucleic acid, or the target nucleic acid and base pair in the non-luminescent nucleic acid probe are shortened. A part or all of the bases forming the base are made to be nucleic acid analogs having a smaller base pairing force than natural base pairs, or part or all of the bases forming base pairs with the target nucleic acid in the non-luminescent nucleic acid probe Is a nucleic acid analog having a greater base pairing force than a natural base pair, so that the Tm value of the association of the target nucleic acid and the non-luminescent nucleic acid probe can be determined from the association of the target nucleic acid and the recovery probe. It can be higher than the Tm value.

  The solid phase carrier used in the present invention is not particularly limited in its shape, material and the like as long as it is a solid having a site that directly or indirectly binds to the recovery probe. For example, particles that can be suspended in water such as beads and can be separated from the liquid by a general solid-liquid separation process, a membrane, a container, a chip substrate, etc. Good.

  The material of the solid phase carrier is not particularly limited as long as it is insoluble in water or a solvent used for measurement and can immobilize a substance that binds to target particles on the surface thereof. For example, natural rubber, polystyrene, polystyrene-divinylbenzene copolymer, styrene-butadiene copolymer, polyacrylic acid ester, polymethacrylic acid ester and other plastics; kaolin, carbon, carbon, activated carbon, glass, silica, alumina, Examples thereof include inorganic substances such as silica-alumina; natural organic polymers such as gelatin, liposomes, and blood cells; magnetic substances.

  The solid phase carrier used in the present invention is preferably a magnetic particle, a silica membrane, a silica filter, a plastic plate, a plastic container, a glass container, and a chip substrate because only the target nucleic acid can be easily isolated from a nucleic acid-containing sample. Magnetic particles are more preferred. Specific examples of the solid phase carrier include, for example, magnetic beads, silica beads, agarose gel beads, polyacrylamide resin beads, latex beads, polystyrene beads, and other plastic beads, ceramic beads, zirconia beads, silica membranes, silica filters, and plastics. A plate etc. are mentioned.

The magnetic particles used as the solid support are not particularly limited as long as they can be easily magnetized by magnetic induction. As magnetic particles, for example, particles made of a metal such as triiron tetroxide (Fe ₃ O ₄ ), iron sesquioxide (γ-Fe ₂ O ₃ ), various ferrites, iron, manganese, nickel, cobalt, chromium, or the like, or Examples thereof include particles made of an alloy containing cobalt, nickel, manganese, and the like. In addition, the magnetic particles may be particles composed of only a magnetic material, or may be particles in which fine particles of a magnetic material are contained inside non-magnetic material particles, and a magnetic material on the surface of the non-magnetic material particles. The particle | grains which fix | immobilized the microparticles | fine-particles may be sufficient. Examples of such non-magnetic particles include latex particles made of a hydrophobic polymer, latex particles made of a crosslinked hydrophilic polymer, gelatin, and liposome.

  Examples of the hydrophobic polymer include polystyrene, polyacrylonitrile, polymethacrylonitrile, polymethyl methacrylate, polycoupleramide, polyethylene terephthalate, and the like. Latex particles made of a copolymer of about 2 to 4 types of monomers may be used. Examples of the crosslinked hydrophilic polymer include polyacrylamide, polymethacrylamide, polyvinyl pyrrolidone, polyvinyl alcohol, poly (2-oxyethyl acrylate), poly (2-oxyethyl methacrylate), poly (2,3- Dioxypropyl acrylate), poly (2,3-dioxypropyl methacrylate), polyethylene glycol methacrylate, dextran, and the like.

  The recovery probe and the solid phase carrier can be bound using two substances that specifically bind to each other, such as biotin and avidin (or streptavidin). For example, when the recovery probe has biotin as a site that binds to the solid phase carrier in addition to the site that binds to the target nucleic acid, a bead or filter with avidin or streptavidin bound to the surface can be used. It can be used as a solid support. In addition, glutathione, DNP (dinitrophenol), digoxigenin, digoxin, sugar chain composed of two or more sugars, polypeptide composed of four or more amino acids, auxin, gibberellin, steroid as a site where the recovery probe binds to the solid phase carrier , Proteins, hydrophilic organic compounds, and analogs thereof, beads or filters in which antibodies, antigens, ligands, or receptors against these substances are bound to the surface are used as solid phase carriers. Can be used as In addition, the recovery probe and the solid phase carrier may form one molecule. Specifically, for example, the recovery probe may be added to the surface of the solid phase carrier by a covalent bond other than the site that binds to the target nucleic acid.

  The nucleic acid-containing sample used in the mutation rate measurement method according to the present invention is not particularly limited as long as it is a sample containing nucleic acid molecules. Examples of the nucleic acid-containing sample include biological samples collected from animals and the like, samples prepared from cultured cells, and reaction solutions after nucleic acid synthesis reaction. A biological sample or the like may be used, or a nucleic acid solution extracted and purified from a biological sample or the like may be used.

  In the mutation rate measuring method according to the present invention, first, as step (a), a nucleic acid-containing sample, the fluorescent probe that specifically binds to the target mutant nucleic acid, and the non-luminescent that specifically binds to all target nucleic acids. A solution is prepared that contains a fluorescent nucleic acid probe, a solid phase carrier to which the recovery probe is bound, and the fluorescent intercalator. When the recovery probe and the solid phase carrier are composed of two molecules that bind to each other, in the preparation of the solution, a recovery probe that has been previously bound to the solid phase carrier may be added. A solid phase carrier capable of binding to each other may be added separately, and both may be bound in the solution.

  In the solution, a target nucleic acid contained in the nucleic acid-containing sample, a non-luminescent nucleic acid probe, and a fluorescent intercalator are formed, and a complex fixed on a solid phase carrier is formed via a recovery probe. To do. The fluorescent probe specifically binds to the target mutant nucleic acid. For this reason, when the target nucleic acid is a target mutant nucleic acid, the complex further includes a fluorescent probe. On the other hand, when the target nucleic acid is other than the target mutant nucleic acid, the complex includes the target nucleic acid, the non-luminescent nucleic acid probe, and the fluorescent intercalator, but does not include the fluorescent probe.

  A solution containing a nucleic acid-containing sample or the like can be prepared using an appropriate solvent. The solvent is not particularly limited as long as it does not damage the target nucleic acid (particularly the target mutant nucleic acid), the fluorescent probe, the fluorescent intercalator and the like. It is typically an aqueous solution, but may be an organic solvent such as formamide or any other liquid. Specifically, the solvent can be appropriately selected from buffers generally used in the technical field. Examples of the buffer include a phosphate buffer such as PBS (phosphate buffered saline, pH 7.4), a Tris buffer, and the like.

  Binding reaction between the non-luminescent nucleic acid probe and the target nucleic acid, binding reaction between the fluorescent intercalator and the target nucleic acid, binding reaction between the recovery probe bound to the solid phase carrier and the target nucleic acid, and the target nucleic acid In this case, the binding reaction between the target mutant nucleic acid and the fluorescent probe may be performed simultaneously, or the respective binding reactions may be performed sequentially. Alternatively, the analysis probe and the solid phase carrier may be separately added to the solution, and both may be bound in the solution. For example, a nucleic acid-containing sample, a fluorescent probe, a non-luminescent nucleic acid probe, a fluorescent intercalator, and a recovery probe bound to a solid phase carrier are added to a solvent, and fixed to the solid phase carrier by performing a binding reaction. Complexed complexes can be formed. In addition, a nucleic acid-containing sample, a fluorescent probe, a non-luminescent nucleic acid probe, a fluorescent intercalator, and a recovery probe are added to a solvent, and a binding reaction is performed to form a complex. The complex may be immobilized on a solid phase carrier via a recovery probe by contacting with the carrier. Note that “contacting a solution with a solid phase carrier” means, for example, when the solid phase carrier is a porous membrane, the solution may be permeated through the porous membrane, and when the solid phase carrier is a container. The solution may be injected into the container, and when the solid support is a chip substrate, the solution may be dropped on the surface of the chip substrate or the chip substrate may be immersed in the solution.

  When the target nucleic acid and the non-luminescent nucleic acid probe are bound, it is preferable to denature the nucleic acid molecule in the solution and then associate the two. Even when the binding site with the target nucleic acid in the fluorescent probe or the recovery probe is an oligonucleotide or the like, it is preferable that the nucleic acid molecule in the solution is denatured and then associated with the target nucleic acid. Note that “denaturing a nucleic acid molecule or nucleic acid-like substance” means dissociating a base pair. For example, it means that a double-stranded nucleic acid molecule is a single-stranded nucleic acid molecule.

  Examples of the denaturation treatment include denaturation by high temperature treatment (thermal denaturation) and denaturation by low salt concentration treatment. Among them, it is preferable to perform heat denaturation because the operation is simple. Specifically, heat denaturation can denature nucleic acid molecules and the like in the solution by treating the solution at a high temperature. Generally, it can be denatured by keeping it at 90 ° C. for DNA and 70 ° C. for RNA for several seconds to 2 minutes, but the denaturation temperature depends on the length of the base in the region forming the aggregate, etc. The temperature is not limited to this temperature as long as the temperature can be modified. On the other hand, denaturation by low salt concentration treatment can be performed by adjusting the salt concentration of the solution to be sufficiently low, for example, by diluting with purified water or the like.

  After denaturation as necessary, the target nucleic acid in the solution, the non-luminescent nucleic acid probe, the fluorescent probe, and the recovery probe are associated with each other to form a complex. When heat denaturation is performed, after the high temperature treatment, the temperature of the solution is lowered to a temperature at which the target nucleic acid and the non-luminescent nucleic acid probe etc. can specifically hybridize (specific association conditions). The two in the solution can be associated as appropriate. Preferably, the temperature of the solution containing the target nucleic acid or the like is lowered to about the temperature of the Tm value ± 3 ° C. of the binding region with each probe in the target nucleic acid. In addition, when denaturation by low salt concentration treatment is performed, the salt concentration of the solution is increased to a concentration at which the target nucleic acid and each probe can specifically hybridize by adding a salt solution or the like. The target nucleic acid in the solution can be associated with each probe as appropriate.

  The temperature at which two single-stranded nucleic acid molecules can specifically hybridize can be determined from the melting curve of the aggregate comprising both. The melting curve can be obtained, for example, by changing the temperature of a solution containing only both from a high temperature to a low temperature and measuring the absorbance and fluorescence intensity of the solution. From the obtained melting curve, the temperature ranges from the temperature at which the two denatured single-stranded nucleic acid molecules started to form an aggregate to the temperature at which almost all became an aggregate. It can be set as the temperature which can be soybean. By changing the salt concentration in the solution from a low concentration to a high concentration instead of the temperature, the melting curve is determined in the same manner, and the concentration at which two single-stranded nucleic acid molecules can specifically hybridize is determined. Can be sought.

  Thus, the specific association conditions differ depending on the type of target nucleic acid and each probe, and are determined experimentally. In general, however, the Tm value (melting temperature) can be substituted. For example, by using commonly used primer / probe design software, the Tm value of the region hybridizing with the target particle (50% of the double-stranded DNA is single-stranded DNA) from the base sequence information of the target particle binding site. Temperature of dissociation) can be calculated. A condition in which the temperature is a value in the vicinity of the Tm value, for example, a condition in which the Tm value is about ± 3 ° C. can be set as the specific association condition. The specific association conditions can be determined in more detail by experimentally obtaining a melting curve in the vicinity of the calculated Tm value.

  Further, in order to suppress non-specific hybridization, it is preferable to lower the temperature of the solution relatively slowly when forming a complex. For example, after denaturing nucleic acid molecules by setting the temperature of the solution to 70 ° C. or higher, the temperature of the solution can be lowered at a rate of temperature decrease of 0.05 ° C./second or higher.

  In order to suppress non-specific hybridization, it is also preferable to add a surfactant, formamide, dimethyl sulfoxide, urea or the like to the solution in advance. 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.

  If the fluorescent probe, non-luminescent nucleic acid probe, and recovery probe can all be combined with the target nucleic acid by coexisting with the target nucleic acid in the same solution, the solution is incubated for a predetermined time as necessary. Alone, it can be bound to the target nucleic acid in the solution. For example, when the binding site with the target nucleic acid in the non-luminescent nucleic acid probe is an oligonucleotide containing a nucleic acid analog such as PNA, even if the target nucleic acid is a double-stranded nucleic acid molecule, a special denaturation treatment is performed. In some cases, the non-luminescent nucleic acid probe and the target nucleic acid can be bound to each other.

  Next, as step (b), after the step (a), the complex is recovered from the solution by solid-liquid separation, and a sample solution containing the complex is prepared. By this solid-liquid separation treatment, the complex containing the target mutant nucleic acid and the fluorescent probe is separated and recovered from the free fluorescent probe that is not bound to the target mutant nucleic acid.

  The solid-liquid separation process is not particularly limited as long as it is a method capable of recovering the solid phase carrier in the solution by separating it from the liquid component. Among the known processes used for the solid-liquid separation process It can be appropriately selected and used. For example, when the solid phase carrier is a particle such as a bead, the solid phase carrier is precipitated and the supernatant is removed by leaving the suspension in a suspension containing the solid phase carrier or performing a centrifugation process. Alternatively, the solution may be filtered using a filter paper or a filter, and the solid support remaining on the surface of the filter paper or the like may be recovered. In the case where the solid phase carrier is a magnetic bead, the magnet is brought close to the container in which the solution is placed, and after the solid phase carrier is converged on the surface of the container closest to the magnet, May be removed. When the container whose inner wall is coated with a substance that binds to the recovery probe is a solid phase carrier, the solution containing the complex is injected into the container and incubated as necessary. Discharge. When the solid phase carrier is a membrane or a filter, the solution containing the complex is allowed to permeate the solid phase carrier, thereby immobilizing the complex to the solid phase carrier and from the fluorescent probe not bound to the target nucleic acid. The complex can be separated and recovered in one operation.

  By adding an appropriate solvent to the recovered solid phase carrier, a solution (suspension) containing the complex bound to the solid phase carrier is prepared. The solvent is not particularly limited as long as it does not inhibit the detection of fluorescence emitted from a fluorescent probe or a fluorescent intercalator in a later step, and is a buffer generally used in the technical field. Can be appropriately selected and used. Examples of the buffer include a phosphate buffer such as PBS (phosphate buffered saline, pH 7.4), a Tris buffer, and the like.

  The recovered solid phase carrier may be washed with an appropriate solvent or the like once to several times before preparing a suspension. The solvent may be washed with a solvent such as water or a buffer. The buffer preferably contains a surfactant in order to enhance the cleaning effect.

  In the present invention, in step (a), a solution containing a nucleic acid-containing sample, a fluorescent probe, a non-luminescent nucleic acid probe, and an analysis probe bound to a solid phase carrier, and not containing a fluorescent intercalator is prepared. After forming the complex immobilized on the solid phase carrier, after recovering the complex separated from the free fluorescent probe and immobilized on the solid phase carrier by solid-liquid separation treatment, the fluorescent intercalator And a fluorescent intercalator may be inserted between the base pairs in the complex. This is because the fluorescent intercalator does not emit fluorescence unless it is inserted between base pairs, and therefore there is no particular problem even if a free fluorescent intercalator is included in the sample solution at the time of fluorescence analysis. In this case, the preparation of the solution containing the nucleic acid-containing sample, the formation of the complex in the solution, and the solid-liquid separation treatment can be performed in the same manner as above except that the fluorescent intercalator is not included. it can.

  The complex thus prepared and immobilized on the solid phase carrier may be subjected to the step (c) as it is, or may be subjected to the step (c) while being separated from the solid phase carrier. The complex containing the target nucleic acid and the non-luminescent nucleic acid probe can be separated from the solid phase carrier and recovered by performing a solid-liquid separation process after dissociating from the solid phase carrier. The solution containing the complex separated and recovered from the solid phase carrier is subjected to step (c) as a sample solution.

  In the present invention, the amount of all target nucleic acids including the target mutant nucleic acid is determined from the fluorescence amount of the fluorescent intercalator, and the amount of the target mutant nucleic acid is determined from the fluorescence amount of the fluorescent probe. When the fluorescence detection in the step (c) is performed by a single molecule fluorescence analysis method such as a scanning molecule counting method, separate molecules are used rather than the fluorescent intercalator and the fluorescent probe emitted from the same molecule. The amount of both can be detected with higher accuracy. Therefore, when the immobilized complex includes a fluorescent probe, the fluorescent probe is also dissociated from the complex, and the target nucleic acid, the non-luminescent nucleic acid probe, and the fluorescent intercalator are separated by solid-liquid separation treatment. And a complex containing the fluorescent probe may be separated and recovered from the solid phase carrier.

  The complex can be dissociated from the solid phase carrier by dissociating the analytical probe from the target nucleic acid, or by decomposing the binding site with the analytical probe or the analytical probe in the solid phase carrier. . When decomposing a binding site with an analysis probe or an analysis probe in a solid phase carrier, it is preferable to use a decomposing enzyme or the like that does not decompose a fluorescent probe or a non-luminescent nucleic acid probe. For example, if a fluorescent probe or a non-luminescent nucleic acid probe has no peptide bond, and the binding site to the analysis probe or the analysis probe in the solid phase carrier contains a peptide bond, the analysis probe is used by proteolytic enzyme. Etc. can be decomposed only. When the analysis probe is a nucleic acid probe, the restriction enzyme site is present in the analysis probe but not present in the non-luminescent nucleic acid probe. Only the analysis probe or the like can be decomposed.

  The analysis probe includes an oligonucleotide or the like that hybridizes with the target nucleic acid, and the Tm value of the association between the analysis probe and the target nucleic acid is calculated from the Tm value of the association between the target nucleic acid and the non-luminescent nucleic acid probe. When designed to be lower than the value, the target nucleic acid and the non-luminescent probe can form an aggregate in the solution containing the solid phase carrier, but the target nucleic acid and the recovery probe form an aggregate. By incubating at a temperature that cannot be performed, the complex containing the target nucleic acid and the non-luminescent probe, or the complex containing the target nucleic acid, the non-luminescent probe and the fluorescent probe can be dissociated from the solid phase carrier. Further, when the Tm value of the aggregate of the fluorescent probe and the target nucleic acid is designed to be lower than the Tm value of the aggregate of the target nucleic acid and the non-luminescent nucleic acid probe, a solution containing the solid phase carrier is used. Incubate at a temperature at which the target nucleic acid and the non-luminescent probe can form an aggregate, but the target nucleic acid and the recovery probe, and the target nucleic acid and the fluorescent probe cannot form an aggregate, and then subject to solid-liquid separation treatment As a result, a solution containing the complex containing the target nucleic acid and the non-luminescent probe and the fluorescent probe in an independent free state is obtained, and this solution is used as a sample solution for step (c).

  Thereafter, as the step (c), the number of fluorescent probes and the number of complexes including the target nucleic acid, the non-luminescent nucleic acid probe, and the fluorescent intercalator in the prepared sample solution are calculated. The number of fluorescent probes is determined based on the amount of fluorescence emitted from the fluorescent probe. The number of complexes containing the target nucleic acid is determined based on the amount of fluorescence emitted from the fluorescent intercalator.

  As the fluorescence amount (fluorescence intensity) of the sample solution, the fluorescence intensity emitted from all the fluorescent molecules in the sample solution may be measured. The fluorescence intensity of the entire sample solution can be measured by a conventional method using a fluorescence spectrophotometer such as a fluorescence plate reader. The fluorescence intensity of the entire sample solution depends on the amount of fluorescent probe and the amount of complex contained in the sample solution. For example, when a fluorescence curve is measured in advance for a fluorescent probe solution with a known concentration, and a calibration curve is created in which the relationship between the fluorescence probe volume and the fluorescence volume in the solution is examined, the calibration curve is based on the calibration curve. Thus, the number (or concentration) of fluorescent probes in the sample solution can be calculated from the amount of fluorescence measured from the sample solution. By measuring the amount of fluorescence in a solution in which a fluorescent intercalator is inserted into an aggregate formed using a non-luminescent nucleic acid probe with a known concentration and a target nucleic acid, and using a calibration curve similarly prepared, the sample The number (or concentration) of complexes in the solution can be calculated.

  When the fluorescent probe or complex in the sample solution is released from the solid phase carrier, in step (c), the fluorescence intensity may be measured for each molecule in the sample solution. Examples of the method for measuring the fluorescence intensity for each molecule in the sample solution include scanning molecule counting method, fluorescence correlation spectroscopy, fluorescence intensity distribution analysis method, and fluorescence polarization intensity distribution analysis method.

  In the present invention, the number of fluorescent probes and the number of complexes are preferably calculated by a scanning molecule counting method. In the scanning molecule counting method, each particle dispersed or dissolved in a solution is individually detected. Therefore, using this information, the particle counting and the concentration of particles in the measurement sample solution are quantitatively determined. Alternatively, it is possible to calculate the number density or obtain information on the concentration or the number density. That is, according to the scanning molecule counting method, the particles passing through the light detection region and the 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 measured, and to determine the concentration or number density of the particles in the measurement sample solution with higher accuracy than before. For example, the detection of free fluorescent probes and complexes in the mutation rate measurement method according to the present invention is performed by scanning molecule counting method, and the particles in the measurement sample solution are individually detected by the fluorescence emitted from each, and the number is counted. When determining the particle concentration, the concentration of the fluorescent probe or complex in the measurement sample solution is much lower than the concentration that can be determined based on the fluorescence intensity measured by a fluorescence spectrophotometer or a plate reader (for example, The fluorescent probe and the complex can be detected even in the femtomolar concentration order or the attomolar concentration order.

  In high-sensitivity measurement such as single-molecule measurement, particularly scanning molecule counting method, the detection accuracy of luminescent particles such as a solid phase carrier that has a relatively slow diffusion movement in a solution may be lowered. In the present invention, even when a solid phase carrier is used in the middle of the process, at the time of measurement by a scanning molecule counting method, a free luminescent probe separated from the solid phase carrier is used as a detection target. Even when a single molecule measurement method is used, it is possible to detect the fluorescent probe and the complex with high accuracy by eliminating the influence of the solid phase carrier.

  In the scanning molecule counting method, specifically, the number of molecules of the fluorescent probe or the complex in the obtained measurement sample solution is determined in the measurement sample solution using the optical system of a confocal microscope or a multiphoton microscope. While moving the position of the light detection region of the optical system, light emitted from the fluorescent probe or the complex in the light detection region is detected, and the number of each molecule is calculated.

  In the present invention and the present specification, 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.

For the calculation of the number of fluorescent probes and complexes, for example, FIDA detects statistical fluctuations in the fluorescence intensity of molecules present in the focal region (photodetection region) in the confocal optical system, and then performs statistical analysis. Thus, the number of each molecule can be calculated.
Moreover, after detecting the polarization of the fluorescence intensity of the molecules existing in the focal region (photodetection region) in the confocal optical system by FIDA-PO, the number of molecules of the fluorescent probe or the complex is determined by performing statistical analysis. Can be calculated.
In addition, the number of molecules in the fluorescent probe or complex is calculated by performing statistical analysis after detecting fluctuations in the fluorescence intensity of the molecules present in the focal region (photodetection region) in the confocal optical system by FCS. can do.
With these measurement methods, even if the concentration of the fluorescent probe or complex in the measurement sample solution is as small as the picomolar concentration order, these can be detected.

  In addition, detection and analysis of the time change of the fluorescence signal of such a molecule | numerator can be performed by a conventional method, for example using well-known single molecule fluorescence analysis systems, such as MF20 (made by Olympus).

Finally, as step (d), the nucleic acid is calculated based on the number of fluorescent probes calculated in step (c) and the number of complexes containing the target nucleic acid, non-luminescent nucleic acid probe, and fluorescent intercalator. The abundance ratio (mutation rate) of the target mutant nucleic acid with respect to all target nucleic acids in the contained sample is calculated.
In the present invention, the number of fluorescent probes and the number of complexes containing a target nucleic acid, a non-luminescent nucleic acid probe, and a fluorescent intercalator are both accurately measured despite two types of fluorescent labels. Since it can be calculated, a highly reliable mutation rate is required.

  EXAMPLES Next, although an Example etc. are shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

[Example 1]
For the target nucleic acids whose wild type and mutant types have the base sequences described in Table 1, the content ratio (mutation rate) of the mutant type relative to the total target nucleic acid amount (total amount of wild type and mutant type) was 0% or 10%. A target nucleic acid solution is prepared, and these mutation rates are detected with a conventional method in which both are labeled with a fluorescent probe capable of specifically binding both, and a mutant probe is specifically labeled with a fluorescent probe capable of specifically binding, It was determined by the mutation rate measuring method according to the present invention in which the wild type was labeled with a fluorescent intercalator.

  First, as a method for measuring a mutation rate according to the present invention, a target nucleic acid of 1 nM (1 nmol / L), a fluorescent probe for mutation detection in which ATTO (registered trademark) 647N is modified at the 5 ′ end of 2 nM, and 2 nM non-luminescent nucleic acid The reaction buffer (10 mM Tris-HCl, 400 mM NaCl, 0.05% by volume Triton) was used to obtain a probe, 2 nM biotinylated probe modified with biotin at the 3 ′ end, and 2 μM SYTOX® Orange. X-100: respective final concentrations). The prepared solution was incubated at 95 ° C. for 5 minutes using a thermal cycler, then cooled to 25 ° C. and incubated for 30 minutes. A control was prepared by adding water instead of the target nucleic acid solution.

  In addition, as a conventional method, a 1 nM (1 nmol / L) target nucleic acid, a fluorescent probe for detection of mutants in which ATTO (registered trademark) 647N is modified at the 5 ′ end of 2 nM, and TAMRA is modified at the 5 ′ end of 2 nM These were dissolved in a reaction buffer so as to become a fluorescent probe for detecting the target nucleic acid and a biotinylated probe in which biotin was modified at the 3 ′ end of 2 nM. The prepared solution was incubated at 95 ° C. for 5 minutes using a thermal cycler, then cooled to 25 ° C. and incubated for 30 minutes. A control was prepared by adding water instead of the target nucleic acid solution.

  200 μL of these solutions prepared by both methods and 10 μg of magnetic beads (dynabead (registered trademark) Myone) washed three times with Wash Buffer (10 mM Tris-HCl, 400 mM NaCl, 0.05 vol% Triton X-100) Were mixed and reacted at room temperature with shaking for 5 minutes. Next, the magnetic beads were washed 5 times with 100 μL of Wash Buffer, 20 μL of Elution Buffer (1 mM Tris-HCl, 0.01 vol% Triton X-100) was added, and heat treatment was performed at 50 ° C. for 1 minute. Thereafter, the magnetic beads were collected on the bottom of the container using a magnet, and the whole supernatant was collected. The supernatant is placed in a 96-well filter plate (Millipore, product name: MAHVN4510) once passed through 100 μL of Elution Buffer, and the filtrate is added to a 96-well glass bottom plate (Whatman, product name: 7706-1365). Dropped. All the plates were centrifuged at 1000 rpm for 5 minutes.

  Subsequently, measurement and analysis were performed by the scanning molecule counting method under the following conditions. Measurement was performed using a single-molecule fluorescence measuring apparatus MF-20 (manufactured by Olympus) equipped with an optical system of a confocal fluorescence microscope and a photon counting system, and time-series light intensity data (photon count data) was obtained. The laser used for excitation is 543 nm and 0.5 mW on the short wavelength side, 642 nm and 0.1 mW are used on the long wavelength side, 560 nm to 620 nm for 543 nm excitation and 650 nm to 690 nm for 642 nm excitation, respectively, using bandpass filters. Fluorescence was detected. The light detection region in the sample solution was moved at a moving speed of 15 mm / second. Further, the BIN TIME was set to 10 μs, and the time series light intensity data obtained by the measurement was smoothed, and then the peak was detected by differentiation. Among the regions regarded as peaks, peak intensities that can be approximated to a Gaussian function and have an intensity of 1 or more were extracted. In each sample solution, the number of molecules emitting fluorescence of ATTO (registered trademark) 647N is the number of mutant target nucleic acids, and the number of molecules emitting fluorescence of SYTOX (registered trademark) Orange or TAMRA is the total number of target nucleic acids. (Total number of wild type and mutant type).

  FIG. 1 is a diagram showing the number of peaks counted from a sample solution in which all target nucleic acids are labeled with a non-luminescent nucleic acid probe and SYTOX (Orange) Orange by the mutation rate measurement method according to the present invention. FIG. 2 is a diagram showing the number of peaks counted from a sample solution in which all target nucleic acids are labeled with a TAMRA-labeled probe by a conventional method. 1 and 2, “No DNA” indicates the result of the sample solution in which water was added instead of the target nucleic acid solution, and “0%” indicates the target nucleic acid solution having a mutation rate of 0% (ie, the wild type target nucleic acid). “10%” indicates the target nucleic acid solution with a mutation rate of 10% (that is, a solution containing 90% of the wild type target nucleic acid and 10% of the target nucleic acid of the mutation type). ) Shows the results of the sample solution to which) was added. The white columns show the counting results of all target nucleic acids, and the black columns show the counting results of mutant target nucleic acids.

  In either method, the peak number of the mutant target nucleic acid (ATTO (registered trademark) 647N) has a clear difference between the mutation rate of 0% and 10%. It was shown that detection is possible. On the other hand, when all target nucleic acids are labeled with a TAMRA-labeled probe, the number of peaks is higher at a mutation rate of 0% and 10% than in the control group not containing the target nucleic acid, but the mutation rate is 10%. In the target nucleic acid solution, the number of peaks was almost the same as that of ATTO (registered trademark) 647N (FIG. 2). In contrast, when all the target nucleic acids were labeled with a non-luminescent nucleic acid probe and SYTOX (registered trademark) Orange, the number of peaks was four times that when labeled with a TAMRA labeled probe (FIG. 1). . This is because, when labeled with a TAMRA-labeled probe, the number of peaks detected buried in noise was reduced, whereas in the mutation rate measurement method according to the present invention, a non-luminescent nucleic acid probe and SYTOX (registered) It is considered that the number of peaks counted in spite of noise is increased by increasing the fluorescence per molecule by using Orange (trademark).

  Since the mutation rate measurement method according to the present invention provides a more reliable mutation rate from a single nucleic acid-containing sample, the detection method according to the present invention is a sample in which the concentration of a target nucleic acid such as gene mutation analysis is a trace amount. It can be particularly suitably used in the field of analysis and inspection.

Claims (7)

(A) a fluorescent probe having a fluorescence wavelength in the range of 580 to 680 nm, which specifically binds to a nucleic acid-containing sample and a region containing a mutation site in the target nucleic acid, and a position where the target nucleic acid and the fluorescent probe are different A solid-phase carrier bound with a non-luminescent nucleic acid probe that binds at a different position, a recovery probe that binds at a position different from the target nucleic acid, the fluorescent probe, and the non-luminescent nucleic acid probe; Preparing a solution containing a fluorescent intercalator that is outside, and containing the target nucleic acid, the fluorescent probe, the non-luminescent nucleic acid probe, and the fluorescent intercalator in the solution, or the target nucleic acid and the non-luminescent Forming a complex immobilized on the solid phase carrier via the recovery probe, comprising a functional nucleic acid probe and the fluorescent intercalator;
(B) after the step (a), recovering the complex from the solution by solid-liquid separation, and preparing a sample solution containing the complex;
(C) calculating the number of fluorescent probes and the number of complexes including the target nucleic acid, the non-luminescent nucleic acid probe, and the fluorescent intercalator in the sample solution prepared in the step (b) Process,
(D) Based on the number of the fluorescent probes calculated in the step (c) and the number of complexes including the target nucleic acid, the non-luminescent nucleic acid probe, and the fluorescent intercalator, Calculating the abundance ratio of the target nucleic acid containing the mutation site in the target nucleic acid in the sample;
A method for measuring a mutation rate of a target nucleic acid, comprising: After the step (b) and before the step (c),
(E) a step of dissociating the complex in the sample solution prepared in the step (b) from the solid phase carrier, and then separating and recovering the complex from the solid phase carrier by a solid-liquid separation process;
The method for measuring a mutation rate of a target nucleic acid according to claim 1, comprising:   In the step (e), when the complex includes the fluorescent probe, the fluorescent probe is also dissociated from the complex, and the target nucleic acid, the non-luminescent nucleic acid probe, and the The method for measuring a mutation rate of a target nucleic acid according to claim 2, wherein the complex containing a fluorescent intercalator and the fluorescent probe are separated and collected from the solid phase carrier. The step (a) (a ′) a nucleic acid-containing sample, a fluorescent probe that specifically binds to a region containing a mutation site in the target nucleic acid and having a fluorescence wavelength in the range of 580 to 680 nm, the target nucleic acid, A non-luminescent nucleic acid probe that binds at a position different from the fluorescent probe; and a solid-phase carrier to which a recovery probe that binds at a position different from the target nucleic acid, the fluorescent probe, and the non-luminescent nucleic acid probe is bound. A solution is prepared, and the target nucleic acid, the fluorescent probe, and the non-luminescent nucleic acid probe are included in the solution, or the target nucleic acid and the non-luminescent nucleic acid probe are included. Forming a complex immobilized on a phase carrier;
And the step (b)
(B′1) after the step (a), recovering the complex from the solution by solid-liquid separation, and preparing a sample solution containing the complex;
(B′2) After the step (b′1), a fluorescent intercalator having a fluorescence wavelength outside the range of 580 to 680 nm is added to the sample solution, and inserted between base pairs in the complex. Process,
The method for measuring a mutation rate of a target nucleic acid according to any one of claims 1 to 3.   In the step (c), calculation of the number of the fluorescent probes and the number of complexes including the target nucleic acid, the non-luminescent nucleic acid probe, and the fluorescent intercalator is performed by scanning molecule counting method, fluorescence correlation spectroscopy. The method for measuring a mutation rate of a target nucleic acid according to any one of claims 2 to 4, which is performed by a fluorescence intensity distribution analysis method or a fluorescence polarization intensity distribution analysis method.   In the step (e), the target nucleic acid and the non-luminescent probe can form an aggregate in the sample solution prepared in the step (b), and the target nucleic acid, the fluorescent probe, and the target The target according to any one of claims 3 to 5, wherein the solid phase carrier and the fluorescent probe are dissociated from the complex by incubating at a temperature at which the nucleic acid and the recovery probe cannot form an aggregate. Nucleic acid mutation rate measurement method.   In the step (a) or step (a ′), a recovery probe that binds at a position different from the target nucleic acid, the fluorescent probe, and the non-luminescent nucleic acid probe, and a solid phase carrier that can bind to the recovery probe The method for measuring a mutation rate of a target nucleic acid according to any one of claims 1 to 6, wherein the solution is prepared by adding.

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