JP6766804B2 - Nucleic acid probe - Google Patents

Description

The present invention is a nucleic acid probe used for FISH (Fluorescence In situ Hybridization) or the like in which a nucleic acid molecule having a reactive site that binds (hybridizes) to a gene on a chromosome and a phosphor-accumulated nanoparticles are bound via a linker. , And a method of performing FISH using this.

FISH, which is one of the methods used in the detection of a specific gene in a tissue section such as gene mapping and detection of chromosomal abnormality, is a probe of a nucleic acid having a base sequence complementary to a specific gene labeled with a fluorescent substance or the like. This is a method of hybridization with a gene of interest and detecting with a fluorescence microscope.

When a normal fluorescent dye is used to label the probe, the problem of reduced quantification due to fluorescent fading may occur. Therefore, fluorescence and luminescence are sensitized by enzymes, but non-specific fluorescence due to non-specific adsorption of enzymes on tissue sections has become a problem. Under such circumstances, phosphor-accumulated nanoparticles have become one of the options for fluorescence sensitization means.

In Patent Document 1, when an antibody or the like is directly modified on the surface of fluorescent nanoparticles, the fluorescence characteristics thereof are changed. Therefore, by introducing a bifunctional PEG linker between the phosphor-accumulated nanoparticles and the antibody, Inventions have been described that allow the desired photophysical properties of fluorophore nanoparticles (such as photostability and quantum yield) to be maintained while preserving the specificity of specific binding moieties such as antibodies. .. However, there is no description that the probe and the fluorescent nanoparticles are bound via avidin or biotin.

Patent Document 2 discloses a nanoparticle-labeled probe characterized by binding nanoparticles to a probe (nucleic acid) via an avidin-biotin interaction or an antigen-antibody reaction. In the document, metal nanoparticles (gold colloidal particles) or semiconductor nanoparticles are exemplified as nanoparticles, and an antigen / antibody or avidin / biotin is interposed instead of directly binding the probe to the nanoparticles. By doing so, the distance between the nanoparticles and the probe is widened, the reaction efficiency is suppressed from dropping by the solid phase method, and the reactivity of the nanoparticles (luminescence efficiency) and the reactivity of the probe (to the target nucleic acid). It has been suggested that the reduction of (hybridation) can be prevented. However, although the examples in this document disclose embodiments in which anti-FITC antibody or streptavidin is bound to colloidal gold particles via PEG (Examples 1 and 3), the probe has a 5'end. It is only disclosed that FITC (antigen) or biotin is bound to the probe (Examples 2 and 4), and an embodiment in which PEG is interposed between the probe and FITC (antigen) or biotin is disclosed. Not. Further, the invention described in the document is mainly assumed to be hybridized in the solid phase method using a substrate-shaped measuring member, and is not described to be used for FISH for tissue sections and the like, and is fluorescently labeled. It is also not described that phosphor-accumulated nanoparticles are used as the body.

The applicant has previously filed an invention relating to a nucleic acid probe in which phosphor-accumulated nanoparticles and a nucleic acid molecule bind to each other (application number: Japanese Patent Application No. 2014-058271, filing date: March 20, 2014). ). The specification according to the application discloses an embodiment in which a phosphor-accumulated nanoparticles and a nucleic acid molecule are bound via a specific bond between streptavidin and biotin, but between the nucleic acid molecule and streptavidin. There is no mention of interposing a linker.

Japanese Patent Publication No. 2008-541015 Japanese Unexamined Patent Publication No. 2008-295326

When the present inventor fluorescently labels the target gene by using a nucleic acid probe to which biotin or the like is directly bound and phosphor-accumulated nanoparticles to which avidin or the like is bound when performing FISH, the labeling efficiency, that is, It was found that the reactivity between the nucleic acid probe and the phosphor-accumulated nanoparticles was not sufficient and there was room for improvement.

In order to solve the above problems, an object of the present invention is to provide a nucleic acid probe having high reactivity with phosphor-accumulated nanoparticles and excellent detection accuracy, and a method for performing FISH using the nucleic acid probe.

The present inventor has found that the above problems can be solved by binding a nucleic acid probe and a ligand such as biotin using a linker having a certain length. That is, the present invention provides the following nucleic acid probe and a method of FISH using the same.

In order to achieve at least one of the above objectives, nucleic acid probes that reflect one aspect of the invention are lengthened by a ligand that specifically binds to a binding agent bound to the phosphor-accumulated nanoparticles. A nucleic acid probe modified via a linker of 10 nm or more and 100 nm or less.

In order to achieve at least one of the above-mentioned purposes, the method of performing FISH reflecting one aspect of the present invention is a hybridization in which the nucleic acid probe specifically binds to a gene on the chromosome of the sample. Process,
The step of washing the sample,
A step of fluorescently labeling a gene on a chromosome by binding a binding substance bound to phosphor-accumulated nanoparticles to a ligand possessed by the nucleic acid probe bound to the gene.
It is a method of performing FISH including.

In order to achieve at least one of the above-mentioned objectives, another method of performing FISH that reflects one aspect of the present invention is to apply a nucleic acid probe bound to a phosphor-accumulated nanoparticles to a gene on the chromosome. A method of performing FISH, which comprises a hybridization step of binding.

By using the nucleic acid probe of the present invention, it is possible to increase the labeling rate of the phosphor-accumulated nanoparticles obtained by accumulating fluorescent substances, thereby making it possible to stably obtain a strong fluorescent signal.

FIG. 1 is a diagram illustrating a state of performing FISH using the nucleic acid probe according to the first embodiment of the present invention. FIG. 2 is a diagram illustrating a state of performing FISH using the nucleic acid probe according to the second embodiment of the present invention. FIG. 3 is a diagram illustrating a state of performing FISH using the nucleic acid probe according to the third embodiment of the present invention. FIG. 4 is a diagram illustrating a state of performing FISH using the nucleic acid probe according to the fourth embodiment of the present invention. FIG. 5 shows FISH using a nucleic acid probe in which a polymer is bound to the phosphor-accumulated nanoparticles and a binding substance and a spacer is interposed between the phosphor-accumulated nanoparticles and the binding substance. It is a figure explaining the state. FIG. 6 is a flowchart showing an example of the entire process of the method for quantifying a biological substance based on FISH according to the present invention. FIG. 7 is a diagram showing an image acquired by a fluorescence microscope when FISH based on Example 3 was performed using the probe prepared in Example 1. FIG. 8 is a diagram showing an image acquired by a fluorescence microscope when FISH based on Example 3 was performed using the probe prepared in Example 2. FIG. 9 is a diagram showing an image acquired by a fluorescence microscope when FISH based on Example 3 was performed using the probe prepared in Comparative Example 1.

Hereinafter, embodiments for carrying out the present invention will be described, but the present invention is not limited thereto.

(Nucleic acid molecule)
A nucleic acid molecule is a nucleic acid molecule having a sequence (probe sequence) containing a part or all of a specific region on a chromosome. Nucleic acid molecules include naturally occurring nucleic acids such as DNA and RNA (mRNA, tRNA, miRNA, siRNA, non-coding RNA, etc.) and PNA, LNA (or BNA: Bridged Nucleic Acid (bridged structure type nucleic acid molecule)). Includes artificial nucleic acids. Therefore, the nucleic acid molecule is not limited as long as it can form a complementary strand with the nucleic acid sequence on the chromosome. The nucleic acid molecule may be a natural nucleic acid, an artificial nucleic acid, or a molecule of a nucleic acid in which a natural nucleic acid and an artificial nucleic acid are linked. Among these nucleic acids, DNA, RNA and PNA are particularly preferable.

(probe)
As the probe, all or part of the nucleic acid sequence on the chromosome related to the detection of a biomarker gene such as HER2 is preferably used. Biomarkers include diagnostic biomarkers, biomarkers for determining disease stages, disease prognosis biomarkers, and monitoring biomarkers for the purpose of observing responses to therapeutic treatment. For example, genes related to cancer growth and response rate of molecular target drugs include HER2, TOP2A, HER3, EGFR, P53, MET and the like. Further, as a gene known as a cancer-related gene, the following can be mentioned. As tyrosine kinase related genes, ALK, FLT3, AXL, FLT4 (VEGFR3, DDR1, FMS (CSF1R), DDR2, EGFR (ERBB1), HER4 (ERBB4), EML4-ALK, IGF1R, EPHA1, INSR, EPHA2, IRR (INSRR) ), EPHA3, KIT, EPHA4, LTK, EPHA5, MER (MERTK), EPHA6, MET, EPHA7, MUSK, EPHA8, NPM1-ALK, EPHB1, PDGFRα (PDGFRA), EPHB2, PDGFRβ (PDGFRB) EPHB3, RET, EPHB4 RON (MST1R), FGFR1, ROS (ROS1), FGFR2, TIE2 (TEK), FGFR3, TRKA (NTRK1), FGFR4, TRKB (NTRK2), FLT1 (VEGFR1), TRKC (NTRK3) are also mentioned. Genes include ATM, BRCA1, BRCA2, BRCA3, CCND1, E-Cadherin, ERBB2, ETV6, FGFR1, HRAS, KRAS, NRAS, NTRK3, p53, PTEN. Genes associated with cartinoid tumors include BCL2, BRD4, CCND1, CDKN1A, CDKN2A, CTNNB1, HES1, MAP2, MEN1, NF1, NOTCH1, NUT, RAF, SDHD, VEGFA. Colorectal cancer-related genes include APC, MSH6, AXIN2, MYH, BMPR1A, p53, DCC. Examples include PMS2, KRAS2 (Ki-ras), PTEN, MLH1, SMAD4, MSH2, STK11, MSH6. Lungoma-related genes include ALK, PTEN, CCND1, RASSF1A, CDKN2A, RB1, EGFR, RET, EML4, ROS1. , KRAS2, TP53, MYC. Examples of liver cancer-related genes include Axin1, MALAT1, b-catenin, p16 INK4A, c-ERBB-2, p53, CTNNB1, RB1, Cyclin D1, SMAD2, EGFR, SMAD4. , IGFR2, TCF1, KRAS. Examples of kidney cancer-related genes include Alpha, PRCC, ASPSCR1, PSF, CLTC, TFE3, p54nrb / NONO, and TFEB. Examples of thyroid cancer-related genes include AKAP10, NTRK1, and AKA. Examples thereof include P9, RET, BRAF, TFG, ELE1, TPM3, H4 / D10S170, and TPR. Examples of ovarian cancer-related genes include AKT2, MDM2, BCL2, MYC, BRCA1, NCOA4, CDKN2A, p53, ERBB2, PIK3CA, GATA4, RB, HERAS, RET, KRAS, and RNASET2. Prostate cancer-related genes include AR, KLK3, BRCA2, MYC, CDKN1B, NKX3.1, EZH2, p53, GSTP1, and PTEN. Bone tumor-related genes include CDH11, COL12A1, CNBP, OMD, COL1A1, THRAP3, COL4A5, and USP6.

When the copy number of a specific gene on a chromosome is detected by FISH, the probe is preferably designed to include a specific sequence in a specific region on the chromosome to be detected, in order to extract the specific sequence. A public database, such as "HD FISH" (http:///www.hdfish.eu./Find_probes.php), can be used for. In addition, it is necessary to design the probe considering the genomic sequence including the intron before splicing. As a method for obtaining a genome sequence containing a gene to be detected, for example, it can be obtained by searching using a publicly available gene database DDBJ (DNA Data Bank of Japan), "Cancer cell lines BACS", or the like. ..

When the copy number of a normal structural gene on a chromosome is detected by FISH, the number of copies of the probe becomes polymorphic, such as indel, VNTR (Variable Number of Tandem Repeat), and microsatellite. It is preferable not to include the gene sequence portion. For example, in a human (2n = 46) cell, the copy number of a normal gene per cell (nucleus) is 1-2, so the copy number estimated from the number of bright spots of the phosphor is 3 or more. In the case of, it can be determined that the chromosomal abnormality in which the gene is amplified has occurred, and conversely, when the copy number is 0, it can be determined that the chromosomal abnormality in which the gene is deleted has occurred. When the sequence of the polymorphic gene as described above is included in the probe, the number of bright spots of the phosphor does not match the copy number of the specific gene of interest, which hinders the detection of the copy number as described above. ..

Regarding the method of obtaining a nucleic acid molecule, if the number of bases of the nucleic acid molecule is several tens of bases, submit the data of the sequence of the nucleic acid molecule including the probe sequence and request a nucleic acid synthesis contract service such as Funakoshi to obtain the nucleic acid molecule. It is preferable to do so. On the other hand, when the number of bases of the nucleic acid molecule is large (for example, when it exceeds 1000 bases), it is possible to synthesize as described above, but it takes time, so the nucleic acid molecule is correctly formed by sequencing the base sequence of DNA. For example, the following may be performed on the premise that it is confirmed.

As one mode, primers are designed and synthesized so as to sandwich the probe sequence portion contained in the genomic DNA of the organism to be detected, and a pfuDNA polymerase having high replication accuracy for genomic DNA or the like is used using this primer set. Perform the PCR method that was used. Next, the PCR reaction solution is separated by electrophoresis, a band corresponding to the length of the target nucleic acid molecule is cut out, and the band is eluted using a nucleic acid purification kit (a kit such as MonoFas (registered trademark) DNA purification kit I). Thereby, the desired nucleic acid molecule can be obtained.

(Linker)
As a polymer for forming a linker that binds a nucleic acid probe and a ligand described later, a hydrophobic polymer or a hydrophilic polymer having a predetermined length, and a combination thereof are preferably used. The linker may be formed from one kind (single) substance, or may be formed from two or more kinds of substances.

Here, the "linker formed from two or more kinds of substances" means that two or more kinds of linkers formed from a single substance are contained.

Further, in the specification of the present application, "single substance" means a compound or a compound group that can be represented by one name and a chemical formula (general formula including repeating units), and the number of repeating units (eg, polyoxyethylene). The number of units) does not matter. For example, even if there are a plurality of variations of polymer compounds marketed under a certain trade name (trademark) having different average molecular weights (number of repeating units), they are regarded as a single substance.

Examples of the hydrophobic polymer include polyamide, saturated hydrocarbon, hydrophobic polyamino acid, polystyrene, polymethacrylic acid ester and the like having a predetermined length described later.

Examples of the hydrophilic polymer are not particularly limited, but are polyethylene glycol, polypropylene glycol, Phycol (registered trademark) (neutral hydrophilic polymer rich in side chains obtained by copolymerizing sucrose with epichlorohydrin), and the like. Polyvinyl alcohol, styrene-maleic anhydride copolymer, divinyl ether-maleic anhydride copolymer, polyvinylpyrrolidone, polyvinylmethyl ether, polyvinylmethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, Examples thereof include polymethaacrylamide, polydimethylacrylamide, polyhydroxypropyl methacrylate, polyhydroxyethyl acrylate, hydroxymethyl cellulose, hydroxyethyl cellulose, polyaspartamide, synthetic polyamino acids, and the like.

Among these linkers, polyethylene glycol (PEG), which is a hydrophilic polymer, is preferable because it can suppress non-specific adsorption and it is easy to set the chain length by the number of oxyethylene units.

Further, by using a plurality of types of linkers, the reactivity between the nucleic acid probe and the phosphor-accumulated nanoparticles can be stabilized. When the particle surface of the phosphor-accumulated nanoparticles is hydrophobic, when only hydrophilic PEG is used as a linker and the nucleic acid molecule is biolabeled via the linker, both the linker PEG and the particle surface come into contact with each other. As a result of the difficulty, it is possible that the amount of avidin bound to the particle surface and the amount of biotin that undergoes a binding reaction may decrease, and therefore, depending on the degree of hydrophobicity on the surface of the phosphor-accumulated nanoparticles, it may be somewhat hydrophobic. It is presumed that it is also effective in mixing polymers.

When using PEG as the linker, for example, purchase a commercially available product such as Thermofisher scientific, or set the number of repeating units of the chemical structure and request the reagent manufacturer to manufacture it. You can get the one of any length by doing it.

As illustrated in FIGS. 1 and 2, the linkers that bind to a nucleic acid probe may all have a single length (see FIG. 1), or may have two or more different lengths. The linker may be used in combination (see FIG. 2).

In particular, when the linker has two or more lengths, the phosphor-accumulated nanoparticles can efficiently bind to the nucleic acid probe without interfering with each other due to binding damage caused by steric hindrance or the like. It is more preferable because there is.

Here, when a polymer compound commercially available under a certain trade name (trademark) is used as a linker, the polymer compound contained in the product sold under the indication of a specific average molecular weight among the products has a molecular weight. Although there is some distribution in (number of repeating units), it is regarded as a linker having a "single length". Therefore, among such products, the product sold under the indication of a certain average molecular weight (number average molecular weight or weight average molecular weight) is used as the first linker and sold under the indication of an average molecular weight different from that. When the product is used as a second linker, it means that the nucleic acid probe is modified with a ligand via a linker having "two kinds of lengths". The molecular length of a polymer compound can be estimated from the distance between atoms related to the repeating unit (for example, the distance between CC and CO in the case of PEG) and the number of repeating units. When an aggregate of polymer compounds having a certain distribution in molecular weight (number of repeating units) is used as a linker, at least a part of the molecules of the aggregate, preferably more than half of the molecules, more preferably most of them. The length of the molecule (80% or more) may be within the predetermined range specified in the present invention. If the length (average molecular length) when the molecular weight of the polymer compound is assumed to be the average molecular weight is within the predetermined range specified in the present invention, as described above, at least a part of the molecules of the aggregate. The length of the molecule, preferably more than half the length of the molecule, can be considered to be within the predetermined range specified in the present invention.

When a linker having a single length is used, the linker used is 10 nm or more and 100 nm or less.

The linker is a linker of 70 nm or more and 90 nm or less, for example, PEG having an average molecular weight of about 10,000 (estimated average molecular length is about about 10,000) from the viewpoint that the nucleic acid probe can be suitably labeled with the phosphor-accumulated nanoparticles. 80 nm) is preferable.

When a linker having two or more kinds of lengths is used, at least one kind of linker has a length of 10 nm or more and 100 nm or less. At this time, from the viewpoint of suppressing steric hindrance in the binding between the phosphor-accumulated nanoparticles and the nucleic acid probe as described above, the length of the other linker is preferably 0.1 nm or more and less than 10 nm, and is 7 nm or more and 9 nm or less. The one is more preferable.

(Binding between linker and ligand)
The bond between the linker and the ligand described later is a bond by an appropriate bonding mode such as covalent bond, ionic bond, hydrogen bond, coordination bond, physical adsorption, chemical adsorption, etc., and amide, especially from the viewpoint of the strength of the bonding force. Covalent bonds such as bonds, ester bonds, imide bonds, and bonds utilizing thiol addition to maleimide groups are preferred.

The linker is preferably one in which, for example, a functional group (for example, a maleimide group, an amino group, a thiol group, etc.) that binds to a nucleic acid probe and a ligand, respectively, is introduced at both ends of the polymer.

Examples of the method for introducing a functional group into the polymer include an amidation reaction and the like, but the method is not particularly limited and can be carried out by a known method. Moreover, you may use the polymer which introduced the commercially available functional group. The above-mentioned functional groups (for example, NHS group, etc.) introduced at both ends of the linker and the reactive groups (for example, amino group, carboxyl group, thiol group, etc.) possessed by each of the ligand and the nucleic acid probe are combined. By sequentially binding in a predetermined reaction mode, a nucleic acid probe to which a ligand is bound via a linker is obtained.

(Method of introducing functional groups)
As a method for introducing the functional group into a nucleic acid probe into which no functional group has been introduced, for example, a substrate used in the PCR method when preparing the nucleic acid probe by the PCR method from a template nucleic acid molecule (BAC clone or the like). The functional group is introduced by using dNTP having a functional group (N is any one of adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U)). Nucleic acid probes can be prepared. For example, when an amino group is introduced, aminoallyl-dNTP is used as the dNTP having a functional group.

The introduction of the functional group may be carried out by a nick translation method using dNTP having a functional group as a substrate for the nucleic acid probe into which the functional group has not been introduced.

(Ligand)
In the present invention, the ligand is bound to the nucleic acid probe via the linker composed of the above polymer, and specifically binds to the binding substance described below which is directly and / or indirectly bound to the phosphor-accumulated nanoparticles described later. By doing so, it is used to bind the phosphor-accumulated nanoparticles to the nucleic acid probe.

The ligand may be a molecule that specifically binds to the binding substance that is bound to the phosphor-accumulated nanoparticles described later, and is, for example, avidin, biotin, dinitrophenol, digoxigenin, streptavidin, neutravidin, and the like. , Biotin, dinitrophenol, digoxigenin are preferably used.

A compound (NHS-PEG-biotin) in which one end of PEG is preliminarily modified with biotin as a ligand and an NHS group that reacts with the amino group of the nucleic acid probe is introduced into the other end is commercially available and is easy. Can be obtained at.

(Binding substance)
The binding substance in the present invention is bound to the phosphor-accumulated nanoparticles and mediates the binding between the phosphor-accumulated nanoparticles and the ligand on the nucleic acid probe. The substance is not particularly limited as long as it can modify the phosphor-accumulated nanomolecule and binds to a ligand, and is, for example, avidin, biotin, anti-dinitrophenol antibody, anti-digoxigenin antibody, streptavidin, neutravidin, etc. Biotin, anti-dinitrophenol antibody, and anti-digoxigenin antibody are preferably used.

The bond between the phosphor-accumulated nanoparticles and the binding substance may be a direct bond or an indirect bond with other molecules intervening. The mode in which the binding substance is directly bonded to the phosphor-accumulated nanoparticles is not particularly limited, and examples thereof include covalent bonds, ionic bonds, hydrogen bonds, coordination bonds, physical adsorption, and chemisorption, which are known. Can be done in a way. From the viewpoint of bond stability, a bond having a strong binding force such as a covalent bond is preferable.

In the case of indirectly binding the binding substance to the phosphor-accumulated nanoparticles, a polymer is bonded to the phosphor-accumulated nanoparticles and the binding substance, and the binder is formed between the phosphor-accumulated nanoparticles and the binding substance. An embodiment in which a spacer is interposed is mentioned.

As the hydrophilic polymer used when indirectly binding the binding substance to the phosphor-accumulated nanoparticles, various molecules similar to the above-mentioned linker, for example, a hydrophilic polymer such as polyethylene glycol may be used. it can. In particular, from the viewpoint of suppressing non-specific adsorption, it is preferable to use polyethylene glycol, which is a hydrophilic polymer.

The binding between the spacer and the binding substance, and the binding between the spacer and the phosphor-accumulated nanoparticles are the same as the binding between the linker and the ligand described above and the binding between the linker and the probe, and the binding according to the appropriate binding mode described above. Can be used. From the viewpoint of the strength of the binding force, a covalent bond such as an amide bond, an ester bond, an imide bond, preferably a bond utilizing thiol addition to a maleimide group is preferable.

(Binding of nucleic acid molecule and phosphor-accumulated nanoparticles)
The binding between the nucleic acid molecule and the phosphor-accumulated nanoparticles takes a method of indirectly binding through the binding between the ligand and the binding substance.

For example, when avidin is used as a binding substance and biotin is used as a ligand, the above-mentioned indirect binding prepares, for example, a biotin-labeled nucleic acid molecule and streptavidin-modified phosphor-accumulated nanoparticles as follows. , Achieved by combining the two.

(Fluorescent material-accumulated nanoparticles)
Fluorescent material-integrated nanoparticles are fluorescee-accumulated nanoparticles. By using such phosphor-accumulated nanoparticles, it is possible to increase the amount of fluorescence emitted per particle, that is, the brightness of the bright spot marking a predetermined biomolecule, as compared with the phosphor itself.

(Fluorescent body)
As used herein, the term "fluorescent substance" refers to a general substance that is excited by being irradiated with X-rays, ultraviolet rays, or visible light from the outside and emits light in the process from the excited state to the ground state. Therefore, the "fluorescent substance" referred to in the present invention is a substance that emits fluorescence in a narrow sense, which is light emission accompanying deactivation from the excited singlet, regardless of the transition mode when returning from the excited state to the ground state. It may be a substance that emits phosphorescence, which is a luminescence associated with deactivation from the triplet.

Further, the "fluorescent body" referred to in the present invention is not limited by the emission lifetime after blocking the excitation light. Therefore, it may be a substance known as a phosphorescent substance such as zinc sulfide and strontium aluminate. Such phosphors can be roughly classified into organic phosphors (fluorescent dyes) and inorganic phosphors.

(Organic phosphor)
Examples of organic phosphors that can be used as phosphors include fluorescein dye molecules, rhodamine dye molecules, Alexa Fluor (registered trademark, Invigen) dye molecules, and BODIPY (registered trademark, Invigen) dyes. Molecules, Cascade (registered trademark, Invitrogen) dye molecules, coumarin dye molecules, NBD (registered trademark) dye molecules, pyrene dye molecules, Texas Red (registered trademark) dye molecules, cyanine dye molecules, perylene type Examples thereof include substances known as organic fluorescent dyes such as dye molecules and oxazine-based dye molecules. Specifically, the dyes and the like exemplified in International Publication WO2012 / 133847 can be mentioned. Any of these organic phosphors may be used alone or in combination of two or more.

(Inorganic phosphor)
Quantum dots can be mentioned as an example of an inorganic phosphor that can be used as a phosphor. The quantum dots include II-VI group compounds, III-V group compounds, and quantum dots containing Group IV elements as components (“II-VI group quantum dots”, “III-V group quantum dots”, and “III-V group quantum dots, respectively”. Any of the group IV quantum dots ") can be used. Specifically, particle dots such as CdSe exemplified in International Publication WO2012 / 13047 can be mentioned. Any of these inorganic phosphors may be used alone or in combination of two or more.

Further, it is also possible to use a quantum dot having the above quantum dot as a core and a shell provided on the core. Hereinafter, as the notation of the quantum dot having a shell, when the core is CdSe and the shell is ZnS, it is described as CdSe / ZnS. Specifically, CdSe / ZnS and the like exemplified in International Publication WO2012 / 133407 can be mentioned, but the present invention is not limited thereto.

If necessary, the quantum dots may be surface-treated with an organic polymer or the like. For example, commercially available CdSe / ZnS having a surface carboxy group (manufactured by Invitrogen), CdSe / ZnS having a surface amino group (manufactured by Invitrogen), and the like can be used.

(Manufacturing method of phosphor-accumulated nanoparticles)
The method for producing the phosphor-accumulated nanoparticles used in the present invention is not particularly limited, and can be produced by a known method. In general, a production method can be used in which the phosphors are put together using resin or silica as a base (the phosphors are immobilized on the inside or the surface of the base). For example, the resin used as the base of the phosphor-accumulated nanoparticles may be a thermosetting resin or a thermoplastic resin. Specific examples of various resins and a method for producing phosphor-accumulated nanoparticles produced using these resins as a base are based on known documents (WO2014 / 136767).

Further, for example, silica nanoparticles encapsulating a fluorescent organic dye can be synthesized with reference to the synthesis of FITC-encapsulated silica particles described in Langmuir Vol. 8, p. 2921 (1992). By using a desired fluorescent organic dye instead of FITC, various fluorescent organic dye-encapsulating silica nanoparticles can be synthesized. Silica nanoparticles encapsulating semiconductor nanoparticles can be synthesized with reference to the synthesis of CdTe-encapsulating silica nanoparticles described in New Journal of Chemistry Vol. 33, p. 561 (2009).

The particle size of the phosphor-accumulated nanoparticles is not particularly limited as long as it is within the range of the average particle size within the fluorescent observation range, but from the viewpoint of preferred fluorescence observation, the average particle size of the phosphor-accumulated nanoparticles is 10 nm or more. It is preferably 500 nm or less, and more preferably 50 nm or more and 200 nm or less.

The average particle size of the produced phosphor-accumulated nanoparticles can be measured by methods known in the art, for example, using a scanning electron microscope (SEM) to obtain a sufficient number (eg, 1000) of fluorescence. After measuring the particle size of the body-accumulated nanoparticles, it is calculated as the arithmetic average.

Preparation of the phosphor-accumulated nanoparticles modified with streptavidin can be performed, for example, as follows. A functional group is introduced by a reagent that introduces a functional group into the phosphor-accumulated nanoparticles and streptavidin, respectively, and the streptavidin and the phosphor-accumulated nanoparticles are bound to each other through the bonding between the functional groups. As described above, a spacer may be interposed between the functional groups. As an example of the combination of functional groups, a combination of NHS ester group-amino group, thiol group-maleimide group and the like can be exemplified. As the spacer, a spacer such as EMCS (N- [epsylon-maleimide caproyloxy] succinimide ester) (manufactured by Thermo Fisher Scientific Co., Ltd.) can be exemplified.

(FISH)
Hereinafter, FISH will be described. The FISH is not particularly limited, and a known method can be used.

(Sample preparation process)
Specimen slides used as specimens can be prepared, for example, by the method used for general histopathological diagnosis of tissues of subjects suspected of having cancer (humans, dogs, cats, etc.). For example, as an example, the subject tissue is fixed and dehydrated, and then paraffin-embedded to prepare a tissue sample, and then the tissue sample is divided into sections of 3 μm or more and 4 μm or less.

(Deparaffin treatment)
The section is dipped in xylene for deparaffinization, then dipped in alcohol to remove xylene, and then further dipped in water to remove the alcohol. Each immersion time is not particularly limited, but is preferably 3 minutes or more and 30 minutes or less, and if necessary, water alcohol or water may be exchanged during each immersion.

(Preprocessing)
Following a known method, pretreatment is performed by immersing the treated sample slides in the pretreatment liquid and heating them under appropriate conditions. A buffer solution may be used for the pretreatment, or an acid may be used. When pretreatment is performed using a buffer solution, citrate buffer solution, EDTA solution, urea, Tris-hydrochloric acid buffer solution, Good's buffer (MES, etc.), etc. are used as the buffer solution, and autoclave, microwave, pressure pan, water, etc. are used. It is carried out by heating at 50 ° C. or higher and 130 ° C. or lower for 5 minutes or longer and 30 minutes or lower using a heating device such as a bath. When the pretreatment is performed using an acid, it can be performed by treating with HCl, washing, and immersing in NaSCN.

Next, the pretreated section is immersed in a container containing PBS (Phosphate Buffered Saline) for washing. The temperature is not particularly limited, but it can be carried out at room temperature. The immersion time is preferably 3 minutes or more and 30 minutes or less. If necessary, PBS may be replaced during immersion.

(Enzyme treatment)
The treated slides are subjected to enzyme treatment in order to decompose proteins in cell membranes and nuclear membranes, particularly collagen. The enzyme treatment follows a known method, and any protease such as proteinase, pepsin, or proteinase K can be used. For example, when pepsin is used as a protease, it is preferable to immerse 100 μg / μL of pepsin (2500-3000 Units / mg) / 10 mM HCL [pH 2.0] at 37 ° C. for 10 minutes or more and 20 minutes or less.

(Dehydration process)
The sample slides that have undergone the above treatment may be dehydrated. The dehydration treatment can be carried out using ethanol or air-drying. When it is carried out using ethanol, it is preferable to immerse the sample slide in 70% ethanol at room temperature for 2 minutes and wash it, and then immerse the sample slide in 100% ethanol at room temperature for 2 minutes to wash.

(Dyeing process)
(Denaturation of DNA on sample slides)
DNA denaturation treatment is performed on the sample slides subjected to the above treatment. The DNA denaturation treatment may be carried out by heat denaturation or by treatment with a denaturation solution such as a formamide solution.

(Hybridization)
Hybridization using a nucleic acid probe, similar to known FISH (eg, "Agilent FISH General Purose Resources Protocol", "Clinical FISH Protocol-Visual Chromosome / Gene Diagnostic Method (Cell Engineering Separate Volume-Experimental Protocol Series", etc.)) Processing can be performed, where the term "hybridization" refers to the binding process of two DNAs or DNA to an RNA complementary strand for the formation of a double-stranded molecule, or the formed double-stranded molecule. means.

Regarding the setting of hybridization conditions, the nucleic acid probe has a chromosome depending on the GC content of the nucleic acid probe, the concentration of monovalent cations present in the hybridization reaction system (M), and the formamide concentration (%) of the reaction system. The accuracy of binding changes when binding to the above sequence. Therefore, the hybridization condition (Tm value) can be appropriately adjusted to adjust the accuracy of binding.

In addition, when setting conditions, refer to Leitch at al. In situ Hybridization: a practical guide, Oxford BIOS Scientific Publishers, Microscopy handbooks V 2 (1994), which describes general conditions for in situ hybridization. it can.

(Addition of fluorescently integrated nanoparticles)
After the hybridization treatment, the cells are sequentially washed with deionized water, phosphate buffered saline (PBS), etc., and have a binding substance that can specifically bind to the ligand portion of the nucleic acid probe via the linker. Particles are added to the reaction system and fluorescently labeled in the reaction system.

(blocking)
Before adding the phosphor-accumulated nanoparticles, it may be preferable to perform a blocking treatment to suppress non-specific adsorption of the protein, if necessary. Blocking is performed using a commercially available reagent.

(Fixed after)
Furthermore, the sample (section) is immobilized with a paraformaldehyde solution or the like because the hybridized probe is less likely to come off the sample (section) and the fluorescence-accumulated nanoparticles are more firmly fixed on the sample (section). It is preferable to do so.

(Nuclear staining)
After the above treatment, usually further nuclear staining is performed to count the number of cells. Although DAPI is generally used as the nuclear staining reagent, Hoechst 33258, Hoechst 33342 or other nuclear staining reagents can be used.

(Encapsulation process)
Specimen slides that have undergone FISH staining and nuclear staining are washed several times with PBS, air-dried or dehydrated, and then an encapsulant is added dropwise onto the tissue section. The encapsulant may be a water-based encapsulant or an oil-based encapsulant. When an oil-based encapsulant is used, the tissue section is permeated with xylene or the like before encapsulation.

(Observation)
The number of bright spots of fluorescence or the emission brightness of the stained section is measured from a wide-field microscope image using a fluorescence microscope. Select an excitation light source and an optical filter for fluorescence detection corresponding to the absorption maximum wavelength and the fluorescence wavelength of the fluorescent substance used. The number of bright spots or the emission brightness can be measured by using commercially available image analysis software, for example, all bright spot automatic measurement software G-Count manufactured by G-Angstrom Co., Ltd. Image analysis itself using a microscope is well known, and for example, the method disclosed in JP-A-9-197290 can be used. The field of view of the microscope image is preferably 3 mm ² or more, more preferably 30 mm ² or more, and even more preferably 300 mm ² or more. The copy number of a particular gene of interest is evaluated based on the number of bright spots measured from the microscopic image and / or the emission brightness. Specifically, for example, if the copy number is 1 to 2, it is normal, and if it is 3 or more, it can be evaluated that an abnormality (proliferation) has occurred.

Hereinafter, the actions and effects of the above-mentioned nucleic acid probe will be described.
[1] Fluorescent Accumulation If the nucleic acid probe is modified via a linker having a length of 10 nm or more and 100 nm or less by a ligand that specifically binds to a binding substance bound to the phosphor-accumulated nanoparticles, the fluorescent substance is integrated. By increasing the labeling rate of nanoparticles, it becomes possible to stably obtain a strong fluorescent signal.
[2] If the nucleic acid probe according to [1] is formed from a single substance, the characteristics (hydrophobicity, hydrophilicity, etc.) of the nucleic acid probe can be changed depending on the type of the linker, and fluorescence The above labeling can be preferably performed by selecting the type of linker according to the characteristics (hydrophobicity, hydrophilicity, etc.) of the particle surface of the body-accumulated nanoparticles. For example, if the particle surface of the phosphor-accumulated nanoparticles is hydrophobic, the use of a hydrophobic linker facilitates labeling. On the contrary, if the particle surface of the phosphor-accumulated nanoparticles is hydrophilic, the use of a hydrophilic linker facilitates labeling.
[3] The ligand is modified via a linker formed from a single substance having two or more lengths, and the length of at least one linker is 10 nm or more and 100 nm or less [1] or. With the nucleic acid probe of [2], the phosphor-accumulated nanoparticles can be efficiently bound to the nucleic acid probe without interfering with each other due to binding failure caused by steric hindrance or the like.
[4] The nucleic acid probe of [1] that is modified with a ligand via a linker formed from two or more substances having a single length, that is, a single length and a single. A nucleic acid probe using two or more types of linker molecules formed of a substance is hydrophilic or hydrophobic according to the particle surface of the phosphor-accumulated nanoparticles and the characteristics of the staining environment (degree of hydrophobicity and hydrophilicity). By combining the linker molecules of the above, the characteristics (hydrophilicity, hydrophobicity) of the nucleic acid probe can be adjusted, and the above labeling and hybridization can be preferably performed.
[5] A linker formed from two or more kinds of substances having two or more kinds of lengths (that is, two or more kinds of linkers formed from a single substance having different lengths for each kind). In the case of the nucleic acid probe of [1], which is modified with the ligand and has a length of at least one kind of linker of 10 nm or more and 100 nm or less, the length of at least one kind of linker is 10 nm or more and 100 nm or less. Since it is as follows, the effect of the above [1] can be obtained. Further, since the length of the linker is different for each type, it is possible to obtain an effect that the phosphor-accumulated nanoparticles are less likely to interfere with each other due to a binding disorder caused by a steric hindrance or the like. Furthermore, since the linker is formed of two or more kinds of substances (there are two or more kinds of linkers formed of a single substance), as described in the above [4], the characteristics of the nucleic acid probe (hydrophilicity). Gender, etc.) can be adjusted.
[6] If the nucleic acid probe according to [3] or [5], wherein at least one of the linkers is in the range of 0.1 nm or more and less than 10 nm, the phosphor-accumulated nanoparticles are bound due to steric hindrance or the like. Particularly preferably, the effect of preventing mutual interference due to obstacles or the like can be obtained.
[7] The nucleic acid probe according to any one of [1] to [6], wherein at least one of the linkers is formed of polyethylene glycol, causes non-specific adsorption of the nucleic acid probe to a section. Be prevented.
[8] The nucleic acid probe according to any one of [1] to [7] selected from the group consisting of DNA, RNA and PNA as the type of nucleic acid used as the nucleic acid probe, for FISH having the above effect. Can be used as a probe for.
[9] The ligand is a substance that specifically binds to avidin, biotin, anti-dinitrophenol antibody, anti-digoxigenin antibody, streptavidin, or neutravidin as the binding substance, and biotin, avidin, dinitrophenol, In the case of the nucleic acid probe according to any one of [1] to [8], which is selected from the group consisting of digoxigenin and digoxigenin, the ligand portion of the nucleic acid probe and the binding substance portion of the fluorescent substance-accumulated nanoparticles. Due to the specific binding with, the nucleic acid probe can be suitably fluorescently labeled not only in the in vitro reaction system but also in the in vivo reaction system.
[10] When the nucleic acid probe according to any one of [1] to [9] is bound to the phosphor-accumulated nanoparticles through the binding between the ligand and the binding substance, the nucleic acid probe is preferably labeled. It is particularly preferable in that it can be used.
[11] The nucleic acid probe according to [10], wherein the average particle size of the phosphor-accumulated nanoparticles is 10 nm or more and 500 nm or less, can be preferably labeled.
[12] A hybridization step of specifically binding the nucleic acid probe according to any one of [1] to [9] to a gene on the chromosome of the sample, a step of washing the sample, and binding to the gene. If it is a method of performing FISH including a step of binding a binding substance bound to a phosphor-accumulated nanoparticles to a ligand possessed by the said nucleic acid probe to fluorescently label a gene on a chromosome, a hybridization step is performed. , The nucleic acid probe specifically binds to the gene on the chromosome, and the reaction system impurities (unreacted substances, etc.) that may interfere with the labeling reaction are removed by the washing step, and the fluorescent labeling step is performed. Since the labeling reaction is carried out, the labeling rate can be increased even in vivo.
[13] Any method for performing FISH, which comprises a hybridization step of binding the nucleic acid probe according to [10] or [11] to a gene on the chromosome of the sample, is preferably labeled in vitro. FISH using the nucleic acid probe is performed.

[Manufacturing Example 1]
(Manufacture of streptavidin-bound Texas red dye-encapsulating melamine resin nanoparticles)
After dissolving 2.5 mg of sulforhodamine 101 (“Sulforhodamine 101”, Sigma-Aldrich, TexasRed dye) in 22.5 mL of pure water, the solution was stirred with a hot stirrer for 20 minutes while maintaining the temperature of the solution at 70 ° C. To the stirred solution, 1.5 g of the melamine resin "Nicarac MX-035" (manufactured by Nippon Carbide Industries Co., Ltd.) was added, and the mixture was further heated and stirred under the same conditions for 5 minutes.

100 μL of formic acid was added to the stirred solution, and the solution was stirred for 20 minutes while maintaining the temperature of the solution at 60 ° C., and then the solution was left to cool to room temperature. The cooled solution is dispensed into multiple centrifuge tubes, centrifuged at 12,000 rpm for 20 minutes, and Texas red pigment-encapsulated melamine resin nanoparticles as phosphor-accumulated nanoparticles contained as a mixture in the solution. (Hereinafter, abbreviated as particle X) was precipitated to remove the supernatant. Then, the precipitated particles X were washed with ethanol and water.

Disperse 0.1 mg of washed particles in 1.5 mL of ethanol, add 2 μL of aminopropyltrimethoxysilane LS-3150 (manufactured by Shin-Etsu Chemical Co., Ltd.), and react at room temperature for 8 hours with stirring to surface aminate. Processing was performed.

The concentration of particles X whose surface was aminated was adjusted to 3 nM using PBS containing 2 mM of EDTA (ethylenediaminetetraacetic acid), and the spacer reagent "SM (PEG) ₁₂ " was used to bring the final concentration to 10 mM in this solution. Thermo Fisher Scientific Co., Ltd., cat. No. 22112) was mixed and reacted at room temperature for 1 hour with stirring.

The reaction solution was centrifuged at 10,000 G for 20 minutes to remove the supernatant, then PBS containing 2 mM of EDTA was added to disperse the precipitate, and the reaction mixture was centrifuged again under the same conditions. By performing the washing according to the same procedure three times, particles X surface-modified with a PEG chain having a maleimide group at the terminal were obtained.

On the other hand, streptavidin having a sulfhydryl group was prepared as follows. First, for 40 μL of streptavidin (manufactured by Wako Pure Chemical Industries, Ltd.) adjusted to 1 mg / mL, N-succinimidyl-S-acetylthioacetate adjusted to 64 mg / mL (N-succinimidyl S-acetylthioacetate, SATA, 70 μL (manufactured by acetone) was reacted at room temperature for 1 hour. That is, a thiol group (-NH-CO-CH _2- S-CO-CH ₃ ) protected against the amino group of streptavidin was introduced.

Then, by a known hydroxylamine treatment, a free thiol group (-SH) was generated from the protected thiol group, and a thiol group (-SH) was added to streptavidin.

This streptavidin solution was desalted with a gel filtration column (Zaba Spin Desalting Colors) to obtain streptavidin capable of binding to particles X surface-modified with a PEG chain having a maleimide group at the end.

Particle X surface-modified with a PEG chain having a maleimide group and the above-mentioned streptavidin having an SH group were mixed in PBS containing 2 mM of EDTA and reacted for 1 hour. 10 mM mercaptoethanol was added to terminate the reaction. After concentrating the obtained solution with a centrifugal filter, unreacted streptavidin and the like are removed using a gel filtration column for purification, and the solution has PEG (33.6 angstrom (= 3.36 nm)) terminally modified with streptavidin. Streptavidin-bound Texas red dye-encapsulating melamine resin nanoparticles were obtained.

[Example 1]
[Preparation of probes with multiple additions of PEG-biotin with a single linker length]
(I) Preparation of Aminoallyl Probe 5 × GoTaq reaction buffer (Promega) 5 μL in 0.2 mL PCR strip tube (Bio-Rad), GoTaq DNA primer (5 U / μL, Promega) 0.25 μL, Human Genetic DNA (100 ng / μL, Takara Bio) 0.25 μL, dATP, dGTP, dCTP (all 10 mM, Takara Bio) 0.5 μL each, dTTP (1 mM, Takara Bio) 3.75 μL, aminoallyl-dUTP (1 mM, Thermo) Fisher Scientific) 1.25 μL, Forward primer (10 μM) 0.5 μL, Reverse primer (10 μM) 0.5 μL, and milliQ 7 μL, for a total of 25 μL. The primers used in the examples and comparative examples in the present specification are human HER2Forward primer and Reverse primer, and their sequences are shown below.
Forward primer: 5'-CGATGTGACTGTCTCCTCCC-3'
Reverse primer: 5'-ATCCTACTCCATCCCAAGCC-3'
After setting the strip tube in a thermal cycler (Chromo4, Bio-Rad), PCR was carried out by the methods from step 1 to step 5 shown below to prepare a PCR product to be an amination probe of about 200 base pairs. .. The PCR product was purified using a PCR purification kit (Qiagen).
Step 1: 95 ° C, 3 minutes Step 2: 95 ° C, 30 seconds Step 3: 60 ° C, 30 seconds Step 4: Return to step 2 (repeat 39 times)
Step 5: 72 ° C., 3 minutes (ii) Preparation of biotin-added probe using NHS-PEG-biotin 1 μg of amination probe and NHS-PEG-biotin so that the molar ratio of the probe to PEG-biotin is 1:10. (Average molecular weight: 10000, Nanox) 1.5 μg was reacted in 200 μL of 0.1 M aqueous sodium hydroxide solution at room temperature for 1 hour. Unreacted NHS-PEG-biotin was removed by PCR purification kit.

For "NHS-PEG-biotin" (average molecular weight (Mw): 10000, Nanox), the average molecular length of PEG is estimated to be about 80 nm by the following calculation, and most of the length of PEG contained therein is estimated. Is estimated to be within the range of 70 nm or more and 90 nm or less.

For the PEG-derived moiety ((CH ₂ CH ₂ O) _n ), the oxyethylene unit “CH ₂ CH ₂ O” having a molecular weight of 44 is a repeating unit. The bond length of C—C is about 0.15 nm, the bond length of C—O is about 0.14 nm, and the actual atomic bond is an atom-atom because it is almost a regular tetrahedron in the sp3 hybrid orbital. The distance between them is multiplied by sin (54.75 ° (half the tetrahedral angle)) = about 0.81 to 0.11 to 0.12 nm. Therefore, the repeating structure (CCO—) of PEG is about 0.35 nm. If the molecular weight of PEG is 10,000, which is the same as the average molecular weight, the number of oxyethylene units (n) contained therein is 227, so the molecular length (average molecular length) is estimated to be 0.35 × 227 = 79.45 nm. To. If n = 200, the molecular length is estimated to be about 70 nm, and if n = 258, the molecular length is estimated to be about 90 nm.

[Example 2]
[Preparation of PEG-biotin-added probe with two linker lengths]
(I) The amination probe was prepared in the same manner as in Example 1.
(Ii) Preparation of biotin-added probe using NHS-PEG-biotin 1 μg of amination probe and NHS-PEG-biotin (average molecular weight: 10000, Nanox Co., Ltd.) so that the molar ratio of the probe to PEG-biotin is 1:10. ) 0.75 μg and 7.5 μg of NHS-PEG-biotin (average molecular weight: 1000, Nanox) were reacted in 200 μL of 0.1 M sodium hydroxide aqueous solution at room temperature for 1 hour. Unreacted NHS-PEG-biotin was removed by PCR purification kit. This NHS-PEG-biotin (average molecular weight: 1000, Nanox) mainly contains a linker molecule of 5 nm or more and 8 nm or less.

According to the above estimation, in the case of MW1000 PEG, the number of oxyethylene units (n) contained therein is 23, and its molecular length (average molecular length) is 8.05 nm.

[Example 3]
[FISH using PEG-biotin-added probe]
(Deparaffin treatment)
Specimen slides of HER2-positive stained control specimens (“HER2 Dual ISH 3-in-1 control slides” manufactured by Roche) were treated in the following order (1) to (5) to perform deparaffinization treatment. (1) Immerse in xylene twice at room temperature for 5 minutes. (2) Immerse the sample slide twice in 100% ethanol at room temperature for 5 minutes. (3) Immerse the sample slides in 70% ethanol at room temperature for 2 minutes. (4) Immerse the sample slides in milliQ water at room temperature for 2 minutes.

(Preprocessing)
In order to improve the reachability of the DNA probe, the sample slides were pretreated in the following order (1) to (2) to remove proteins from the cell membrane and the nuclear membrane. (1) Treatment with MES buffer (HER2 FISH PharmaDx "Dako") at 95 to 99 ° C. for 10 minutes, and then treatment at room temperature for 15 minutes. (2) The sample slide is immersed in a Tris wash buffer (HER2 FISH PharmaDx "Dako") for 3 minutes and washed twice.

(Protease treatment)
The treated sample slides were treated with an enzyme by performing the following treatments (1) to (4) in this order. (1) Take out the pretreated sample slide, attach the lower end of the slide glass to a paper towel, and remove excess wash buffer. (2) Drop 5 to 6 drops of the protease solution onto the sample slide and react for 10 to 60 minutes. This immersion treatment is preferably treated with Pepsin (HER2 FISH PharmaDx "Dako") at room temperature for 10 minutes in order to degrade proteins in cell membranes and nuclear membranes, especially collagen.
(3) Immerse sample slides in a Tris wash buffer (HER2 FISH PharmDx "Dako") for 3 minutes. This operation is repeated twice. (4) Specimen slides are air dried or dried on a slide warmer at 45-50 ° C for 2-5 minutes.

(dehydration)
The sample slides were immersed in 70% ethanol at room temperature for 2 minutes and washed, and then the sample slides were immersed in 100% ethanol at room temperature for 2 minutes and washed to perform dehydration treatment.

(Denaturation and hybridization)
By performing the following treatments on the dehydrated sample slides in this order, 1 μL (10 to 50 ng) of DNA probe prepared as described in Examples 1 and 2 was used for the sample slides. Hybridization treatment was performed. (1) Add 1 μL of the above DNA probe prepared to the hybridization region of the sample slide and 9 μL of IQFISH FFPE Hybridization Buffer (Agilent Technologies), mix by pipetting on the slide, and immediately after mixing by pipetting, a cover glass of 22 mm × 22 mm. Cover the probe and spread the probe evenly. Prevent air bubbles from entering the hybridization region. (2) Seal the cover glass with a paper bond (KOKUYO Co., Ltd.). (3) A sample slide is placed on a hybridizer (Dako), and denaturation and hybridization are performed at 80 ° C. for 10 minutes and 45 ° C. for 1 hour.

(Washing slide glass)
The sample slides were washed by performing the following processes (1) to (6) in this order on the sample slides that had undergone the hybridization treatment. (1) Post-hybridization wash buffer (1 × SSC / 0.1% NP-40) is placed in a coplin jar. Pre-heat in a warm bath until the post-hybridization wash buffer reaches 63 ° C (place in a warm bath at 63 ° C for at least 30 minutes).
(2) Prepare another coplin jar containing a post-hybridization wash buffer and maintain it at room temperature. (3) Remove the paper bond seal with tweezers. Place the sample slides in a coplin jar containing post-hybridization wash buffer maintained at room temperature and remove the cover glass. (4) The sample slide is placed in a post-hybridization washing buffer solution kept at 63 ° C., immersed for 10 minutes, and washed. (5) Immerse in Tris wash buffer (HER2 FISH PharmDx "Dako") at room temperature for 3 minutes and wash twice. (6) Take out the sample slide from the coplin jar and air-dry it under shading (closed drawers, closed cabinet shelves, etc.).

(blocking)
Blocking was performed by immersing in In Situ Hybridization Blocking Solution (Vector) at room temperature for 30 minutes.

(Avidin-biotin reaction)
Streptavidin-labeled phosphor-accumulated nanoparticles (particle size: 150 nm, concentration: 0.1 nM) produced in Production Example 1 were added dropwise to a 100 μL sample slide, and a binding reaction was carried out at room temperature for 60 minutes. Soaked in PBS for 5 minutes and washed 3 times.

(Fixed after)
Post-fixation was performed by immersing in 4% paraformaldehyde / phosphate buffer (Wako) at room temperature for 10 minutes.

(DAPI staining)
DAPI (2- (4-amidinophenyl) indole-6-carboxamidin dihydrochloride) staining was performed as follows. First, 10 μL of DAPI (Molecular Probes: D1306) stain was added to the hybridization region of the sample slides. The reaction was carried out at room temperature for 15 minutes to stain the cell nuclei, sealed with Aquatex (MERCK), covered with a cover glass, and the sample slides were stored in the dark until signal measurement.

(Observation)
Fluorescent images of the sample slides prepared above and subjected to FISH were acquired using a fluorescence microscope BZ-X710 (manufactured by KEYENCE). A cell line (Calu-3) in which HER2 was highly expressed was used for imaging. The magnification of the objective lens was 100 times, and the excitation times at the time of imaging of DAPI and phosphor-integrated nanoparticles were 20 ms and 500 ms, respectively.

[Comparative Example 1]
The procedure was the same as in Example 3 except that the biotin-added DNA probe used for the hybridization treatment on the sample slide was used. The biotin-added DNA probe was prepared by the following method.

5 × GoTaq reaction buffer (Promega) 5 μL in 0.2 mL PCR strip tube (Bio-Rad), GoTaq DNA primer (5 U / μL, Promega) 0.25 μL, Human Generic DNA (100 ng / μL, Takara Bio) ) 0.25 μL, dATP, dGTP, dCTP (all 10 mM, Takara Bio) 0.5 μL each, dTTP (1 mM, Takara Bio) 3.75 μL, biotin-dUTP (1 mM, Takara Bio) 1.25 μL, Forward A total of 25 μL of solution was prepared: 0.5 μL of primer (10 μM), 0.5 μL of Reverse primer (10 μM), and 7 μL of milliQ. A biotin-added probe was prepared by executing the primer sequences and PCR execution conditions described in [Example 1].

Images obtained with a fluorescence microscope when FISH was performed using the probes of Examples 1 and 2 and Comparative Example 1 are shown in FIGS. 7 to 9. It was observed that Example 1 (FIG. 7) and Example 2 (FIG. 8) captured the amplification of the HER2 gene better than Comparative Example 1 (FIG. 9).

The nucleic acid probe according to the present invention and FISH using the same have been described in detail based on the embodiments and examples, but the present invention does not deviate from the gist of the invention described in the claims. , Design changes are allowed.

1 Nucleic acid probe 2a Linker 2b Linker (linker of a substance different from 2a)
3 Fluorescent material-accumulated nanoparticles 4 Spacer A Ligand B Bound substance

SEQ ID NO: 1; human Her2 forward primer
SEQ ID NO: 2; human Her2 reverse primer

Claims (12)

Phosphor integrated by ligand nanoparticles are particles bound specifically to bound binding substance, and a nucleic acid probe a length is modified through a linker is 10nm or more 100nm or less. The nucleic acid probe according to claim 1, wherein the linker is formed from a single substance. The first or second claim, wherein the ligand is modified via a linker formed from a single substance having two or more kinds of lengths, and the length of at least one kind of linker is 10 nm or more and 100 nm or less. Nucleic acid probe. The nucleic acid probe according to claim 1, wherein the nucleic acid probe is modified with a ligand via a linker formed from two or more substances having a single length. Claim 1 in which the ligand is modified via a linker formed from two or more substances having two or more kinds of lengths, and the length of at least one kind of linker is 10 nm or more and 100 nm or less. The nucleic acid probe according to. The nucleic acid probe according to claim 3 or 5, wherein the length of at least one of the linkers is in the range of 0.1 nm or more and less than 10 nm. The nucleic acid probe according to any one of claims 1 to 6, wherein at least one of the linkers is formed of polyethylene glycol. The nucleic acid probe according to any one of claims 1 to 7, wherein the type of nucleic acid used as the nucleic acid probe is selected from the group consisting of DNA, RNA and PNA. The ligand is a substance that specifically binds to avidin, biotin, anti-dinitrophenol antibody, anti-digoxigenin antibody, streptavidin, or neutravidin as the binding substance, and is derived from biotin, avidin, dinitrophenol, and digoxigenin. The nucleic acid probe according to any one of claims 1 to 8, which is selected from the group. The nucleic acid probe according to any one of claims 1 to 9, wherein the average particle size of the phosphor-accumulated nanoparticles is 10 nm or more and 500 nm or less. A method for performing FISH, which comprises a hybridization step of binding the nucleic acid probe according to any one of claims 1 to 10 to a gene on a chromosome possessed by a sample. The step of washing the sample and
The method for performing FISH according to claim 11, further comprising a step of fluorescently labeling the gene on the chromosome.