Method for detecting gene mutations

Abstract

Analyze gene mutations including missense mutations and / or nonsense mutations and silent mutations with high precision. 【Solution means】Detect signals from a first probe corresponding to a missense mutation or nonsense mutation in a gene mutation, a second probe corresponding to a silent mutation in the gene mutation, and a wild-type probe corresponding to the wild type, and determine the signal from the second probe as the wild type in the gene mutation.

Description

Technical Field

[0001] The present invention relates to a method for detecting gene mutations including missense mutations and / or nonsense mutations and silent mutations.

Background Art

[0002] Gene mutation generally means a state in which a gene has some kind of abnormality either congenitally or acquiredly. Examples of gene mutations include states in which bases constituting DNA are substituted, deleted, or added. Here, a gene is composed of a coding region encoding a protein and non-coding regions such as an expression control region. Gene mutations exist in both the coding region and the non-coding region. Among them, gene mutations existing in the coding region include missense mutations in which the encoded amino acid changes, nonsense mutations in which a codon encoding a predetermined amino acid becomes a stop codon, and silent mutations in which the encoded amino acid does not change.

[0003] Gene mutations are the cause of genetic diseases and cancer, and are also related to the efficacy of specific drugs. In addition, gene mutations are related to physical constitutions such as obesity. Therefore, identifying a specific gene mutation (also referred to as genotyping) is an essential technique for diagnosing genetic diseases and knowing the efficacy of drugs.

[0004] As genotyping methods, for example, in addition to DNA sequencing, there are the SSCP (Single Strand Conformation Polymorphism) method, the RFLP (Restriction Fragment Length Polymorphism) method, the PCR (Polymerase Chain Reaction) method, the AFLP (Amplified Fragment Length Polymorphism) method, the ASO (Allele Specific Oligonucleotide) probe method, and methods for detecting binding to DNA microarrays and DNA beads. Among these techniques, in the method using a DNA microarray, the DNA microarray usually has mutant probes and wild-type probes fixed on a carrier for the gene mutations to be detected.

[0005] In the method using a DNA microarray, first, a region having a gene mutation to be detected is amplified by a nucleic acid amplification reaction using a primer having a fluorescent label. Then, a hybridization reaction is performed between the nucleic acid fragment having the fluorescent label and the mutant probe and the wild-type probe. When the amplified nucleic acid fragment contains a mutant type, fluorescence is observed from the mutant probe. Therefore, genotyping of gene mutations can be performed by observing the fluorescence from the mutant probe and the wild-type probe.

[0006] On the other hand, as a technique for determining the disease onset risk by genotyping, Patent Document 1 can be cited. According to Patent Document 1, it is disclosed that the onset risk of primary central nervous system lymphoma is determined based on gene mutations in GRB2 and / or MYD88. Further, Patent Document 2 discloses determining whether to use a BTK inhibitor such as ibrutinib for the treatment of diffuse large B-cell lymphoma (DLBCL) based on modifications (gene mutations) to the 198th amino acid or the 265th amino acid in MYD88 and a modification (gene mutation) to the 196th amino acid in CD79B.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0008] However, a method for accurately analyzing gene mutations including missense mutations and / or nonsense mutations and silent mutations has not been established. Therefore, an object of the present invention is to provide a new detection method for gene mutations including missense mutations and / or nonsense mutations and silent mutations.

Means for Solving the Problems

[0009] The method for detecting gene mutations according to the present invention that achieves the above object includes the following.

[0010] (1) A method for detecting a gene mutation including a missense mutation and / or a nonsense mutation and a silent mutation, the method comprising: a step of amplifying a nucleic acid region containing the gene mutation; a step of hybridizing a nucleic acid fragment containing the gene mutation, a first probe corresponding to the missense mutation or nonsense mutation in the gene mutation, a second probe corresponding to the silent mutation in the gene mutation, and a wild-type probe corresponding to the wild type; and a step of detecting signals from the first probe, the second probe, and the wild-type probe, wherein a signal from the second probe is determined as the wild type in the gene mutation. A method for detecting a gene mutation, characterized by the above.

[0011] (2) Quadratic formula: Calculate the determination value by the formula: determination value = [signal value from the second probe] / [signal value from the wild-type probe + signal value from the second probe], and when the calculated determination value exceeds a preset cut-off value, determine that the gene mutation is wild-type as described in (1).

[0012] (3) Cubic formula: Calculate the determination value by the formula: determination value = [signal value from the first probe] / [signal value from the wild-type probe + signal value from the first probe], and when the calculated determination value exceeds a preset cut-off value, determine that there is a missense mutation and / or nonsense mutation for the gene mutation as described in (1).

[0013] (4) For a plurality of missense mutations and / or nonsense mutations in the above gene mutation, calculate the determination value for each by the above formula, and determine the existence for each of the plurality of missense mutations and / or nonsense mutations as described in (3).

[0014] (5) When the determination values calculated for each of the plurality of missense mutations and / or nonsense mutations are all below the cut-off value, determine that the gene mutation is wild-type as described in (4).

[0015] (6) The gene mutation is a missense mutation and / or nonsense mutation that occurs in a codon encoding a predetermined amino acid, and the first probe corresponds to a missense mutation and / or nonsense mutation having a base substitution at the same position as the base substitution that results in a silent mutation for the amino acid as described in (1).

[0016] (7) The gene mutation is a missense mutation and / or nonsense mutation that occurs in a codon encoding a predetermined amino acid, and the first probe corresponds to a missense mutation and / or nonsense mutation having a base substitution in the codon in which a base substitution resulting in a silent mutation for the amino acid has occurred as described in (1).

[0017] Using a plurality of first probes for all missense mutations and / or nonsense mutations having a base substitution in a codon in which a base substitution resulting in a silent mutation occurs for the above amino acid, the method according to (7).

[0018] (9) The method according to (1), wherein the gene mutation includes a missense mutation and / or a nonsense mutation of the 196th tyrosine residue in the CD79B gene and a silent mutation of the tyrosine residue.

[0019] (10) The missense mutation in the CD79B gene is at least one mutation selected from the group consisting of 589T>C, 589T>A, 589T>G, 590A>C, 590A>G, and 590A>T, the nonsense mutation in the CD79B gene is 591C>G or 591C>A, and the silent mutation in the CD79B gene is 591C>T, the method according to (9).

[0020] (11) The first probe corresponding to the missense mutation and the nonsense mutation in the CD79B gene, the second probe corresponding to the silent mutation in the CD79B gene, and the wild-type probe corresponding to the wild type corresponding to these missense mutations, nonsense mutations, and silent mutations include the base sequences shown in Table 1, the method according to (10).

Table 1

Advantages of the Invention

[0021] In the method for detecting a gene mutation according to the present invention, the signal from the probe corresponding to the silent mutation and the signal from the probe corresponding to the wild type are detected together as the wild type. Therefore, according to the method for detecting a gene mutation according to the present invention, missense mutations and / or nonsense mutations can be detected with high sensitivity.

Brief Description of the Drawings

[0022]

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Mode for Carrying Out the Invention

[0023] Hereinafter, the present invention will be described in detail. In the method for detecting gene mutations according to the present invention, gene mutations including silent mutations are the detection targets. Gene mutations including silent mutations are mutations in the bases constituting the codon encoding a predetermined amino acid, and include both a mutant form that becomes a silent mutation where the amino acid does not change, a mutant form that becomes a missense mutation where the amino acid changes, and / or a mutant form that becomes a nonsense mutation where a stop codon appears.

[0024] Such gene mutations are not particularly limited, and examples thereof include gene mutations related to diseases, gene mutations related to drug efficacy, gene mutations related to constitution, and the like. However, the gene mutations related to diseases, drug efficacy, or constitution only need to have the possibility of being related to diseases, drug efficacy, or constitution, and do not necessarily have to be scientifically proven to be related to diseases, drug efficacy, or constitution. For example, gene mutations related to genetic diseases or hereditary diseases can be targeted for detection. Note that genetic diseases or hereditary diseases refer to all diseases caused by mutations in chromosomes or genes. Among these genetic diseases or hereditary diseases, some develop diseases by having a predetermined gene mutation either congenitally or acquiredly. Therefore, detecting the gene mutation can contribute to the diagnosis of genetic diseases or hereditary diseases or assist in the diagnosis.

[0025] Whether such gene mutations include silent mutations can be determined by using known databases. For example, for gene mutations related to cancer, by using (COSMIC: The Catalogue Of Somatic Mutations In Cancer), which is a catalog of somatic mutations in cancer, it is possible to determine whether there are silent mutations in the gene mutation, or to identify gene mutations related to cancer that have silent mutations.

[0026] As an example, mutations in the CD79B gene related to diffuse large B-cell lymphoma (DLBCL), mutations in the BRAF gene related to malignant melanoma or lung cancer, mutations in the GNAS gene related to intraductal papillary mucinous neoplasm of the pancreas, and the like can be mentioned.

[0027] Specifically, in the CD79B gene, gene mutations are known for the tyrosine located at the 196th position from the N-terminus. As a result of a base substitution in the codon (TAC) encoding the tyrosine, three types of gene mutations are included: a missense mutation resulting in a different amino acid, a nonsense mutation resulting in a stop codon, and a silent mutation where the encoded amino acid remains tyrosine.

[0028] Here, the missense mutations in the CD79B gene are mutations in which the 589th thymine from the 5’ end of the region encoding CD79B is substituted with cytosine (this mutation is described as 589T>C. Hereinafter, A: adenine, G: guanine, C: cytosine, T: thymine, and the same shall apply), 589T>A, 589T>G, 590A>C, 590A>G, and 590A>T. The nonsense mutations in the CD79B gene are 591C>G and 591C>A. Furthermore, the silent mutation in the CD79B gene is 591C>T.

[0029] The mutations for the 196th thymine in these CD79B genes are summarized in Table 2. In Table 2, for the wild-type sequence, it is denoted as W in the type column, M1 - M6 in the same column are missense mutations, M7 and M8 are nonsense mutations, and M9 is a silent mutation.

[0030]

Table 2

[0031] Specifically, in the BRAF gene, gene mutations are known for the valine located at the 600th position from the N-terminus. As a result of a base substitution in the codon (GTG) encoding the valine, two types of gene mutations are included: a missense mutation resulting in a different amino acid and a silent mutation where the encoded amino acid remains valine.

[0032] Examples of the missense mutations in the BRAF gene include 1799T>A, 1799T>G, 1799T>C, 1798G>A, and 1798G>C. Examples of the silent mutations in the BRAF gene include 1800G>A and 1800G>T.

[0033] Specifically, in the GNAS gene, gene mutations are known for the arginine located at the 844th position from the N-terminus. As a result of a base substitution in the codon (CGT) encoding the arginine, two types of gene mutations are included: a missense mutation that results in a different amino acid and a silent mutation in which the encoded amino acid remains arginine.

[0034] The method for detecting gene mutations according to the present invention is not limited to the gene mutations contained in these CD79B gene, the gene mutations contained in the BRAF gene, and the gene mutations contained in the GNAS gene, and can be widely applied to gene mutations including missense mutations and / or nonsense mutations and silent mutations.

[0035] In addition, in the method for examining a gene according to the present invention, in the gene mutation to be examined, missense mutations and / or nonsense mutations for a predetermined amino acid are the detection targets. In other words, among the base mutations (for example, substitution mutations) that occur in the codon encoding a predetermined amino acid in the gene mutation to be examined, missense mutations and / or nonsense mutations are the detection targets. In particular, the method for examining a gene according to the present invention is characterized in that when a silent mutation is detected among the base mutations (for example, substitution mutations) that occur in the codon encoding a predetermined amino acid, it is determined as the wild type.

[0036] For example, among missense mutations and / or nonsense mutations occurring in a codon encoding a predetermined amino acid in a gene mutation to be examined, missense mutations and / or nonsense mutations having a base substitution at the same position as the base substitution that results in a silent mutation for the amino acid can be targeted for detection. Specifically, in the gene mutation in the CD79B gene described above, nonsense mutations 591C>G and 591C>A (M7 and M8 in Table 2), which are at the same position as the silent mutation 591C>T, can be targeted for detection.

[0037] Also, for example, among missense mutations and / or nonsense mutations occurring in a codon encoding a predetermined amino acid in a gene mutation to be examined, missense mutations and / or nonsense mutations having a base substitution in the codon in which a base substitution that results in a silent mutation for the amino acid has occurred can be targeted for detection. Specifically, in the gene mutation in the CD79B gene described above, one or more mutations selected from six missense mutations consisting of 589T>C, 589T>A, 589T>G, 590A>C, 590A>G, and 590A>T (M1 to M6 in Table 2) and two nonsense mutations consisting of 591C>G and 591C>A (M7 and M8 in Table 2), which are codons containing the silent mutation 591C>T, can be targeted for detection. Note that the missense mutations and nonsense mutations to be detected may be all of the missense mutations of 589T>C, 589T>A, 589T>G, 590A>C, 590A>G, and 590A>T and the nonsense mutations of 591C>G and 591C>A (M1 to M8 in Table 2).

[0038] In the method for detecting gene mutations according to the present invention, silent mutations, missense mutations, and / or nonsense mutations in these gene mutations to be examined are detected using probes. That is, a nucleic acid region containing the gene mutation to be examined is amplified, and the presence of silent mutations, missense mutations, and / or nonsense mutations is confirmed based on the hybridization between the obtained nucleic acid fragment and the probe. When it is confirmed that none of these silent mutations, missense mutations, and nonsense mutations are present, the gene mutation contained in the amplified fragment is identified as wild type.

[0039] Here, a probe for detecting missense mutations and / or nonsense mutations in the gene mutation to be examined is referred to as the first probe, and a probe for detecting silent mutations in the same gene mutation is referred to as the second probe. In addition, a probe for detecting the wild type in the same gene mutation is referred to as the wild type probe. These first probes, second probes, and wild type probes can be appropriately designed based on the base sequences of missense mutations and / or nonsense mutations, the base sequences of silent mutations, and the base sequences of the wild type in the gene mutation to be examined.

[0040] The base length of these probes is not particularly limited, but for example, it can be 10 to 30 base lengths, and preferably 15 to 25 base lengths. The probe is preferably a nucleic acid, more preferably DNA. DNA includes both double-stranded and single-stranded, but preferably single-stranded DNA. The probe can be obtained, for example, by chemically synthesizing it with a nucleic acid synthesizer. As the nucleic acid synthesizer, devices called DNA synthesizers, fully automatic nucleic acid synthesizers, nucleic acid automatic synthesizers, etc. can be used.

[0041] The probe designed as described above is preferably used in the form of a microarray (for example, a DNA chip) by immobilizing its 5'-end on a carrier. At this time, the microarray has a first probe, a second probe, and a wild-type probe for the gene mutation to be examined. Note that the microarray may have a first probe, a second probe, and a wild-type probe for each of a plurality of types of gene mutations. The microarray can be produced by immobilizing the above-described first probe, second probe, and wild-type probe on a carrier.

[0042] As the material of the carrier, those known in the art can be used and are not particularly limited. For example, noble metals such as platinum, platinum black, gold, palladium, rhodium, silver, mercury, tungsten, and their compounds, and conductive materials such as carbon represented by graphite and carbon fiber; silicon materials represented by single crystal silicon, amorphous silicon, silicon carbide, silicon oxide, silicon nitride, and composite materials of these silicon materials represented by SOI (silicon-on-insulator); inorganic materials such as glass, quartz glass, alumina, sapphire, ceramics, forsterite, and photosensitive glass; organic materials such as polyethylene, ethylene, polypropylene, cyclic polyolefin, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene-acrylonitrile copolymer, acrylonitrile-butadiene-styrene copolymer, polyphenylene oxide, and polysulfone. The shape of the carrier is not particularly limited, but is preferably flat.

[0043] As the carrier, preferably a carrier having a carbon layer and a chemically modified group on its surface is used. Carriers having a carbon layer and a chemically modified group on their surface include those having a carbon layer and a chemically modified group on the surface of a substrate, and those having a chemically modified group on the surface of a substrate made of a carbon layer. As the material of the substrate, those known in the art can be used, and there is no particular limitation, and the same materials as those listed as the above carrier materials can be used.

[0044] In a microarray, a carrier having a fine flat plate-like structure is preferably used. The shape is not limited, such as rectangular, square, and round, but usually a square one with a side length of 1 to 75 mm, preferably a square one with a side length of 1 to 10 mm, and more preferably a square one with a side length of 3 to 5 mm is used. Since it is easy to manufacture a carrier having a fine flat plate-like structure, it is preferable to use a substrate made of a silicon material or a resin material, and particularly a carrier having a carbon layer and a chemically modified group on the surface of a substrate made of single crystal silicon is more preferable. Single crystal silicon includes those in which the direction of the crystal axis changes slightly in some parts (sometimes referred to as mosaic crystals), and those containing atomic-scale disorder (lattice defects).

[0045] The carbon layer to be formed on the substrate is not particularly limited, but it is preferably synthetic diamond, high-pressure synthetic diamond, natural diamond, soft diamond (e.g., diamond-like carbon), amorphous carbon, any of carbon-based substances (e.g., graphite, fullerene, carbon nanotube), their mixtures, or those obtained by laminating them. Carbides such as hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, thorium carbide, titanium carbide, uranium carbide, tungsten carbide, zirconium carbide, molybdenum carbide, chromium carbide, vanadium carbide, etc. may also be used. Here, soft diamond generally refers to an imperfect diamond structure that is a mixture of diamond and carbon, such as so-called diamond-like carbon (DLC), and its mixing ratio is not particularly limited. The carbon layer is advantageous in that it has excellent chemical stability and can withstand reactions in subsequent introduction of chemical modification groups and binding to the analyte, the binding is flexible because it binds to the analyte by electrostatic interaction, it is transparent to the detection system UV because it has no UV absorption, and it can be energized during electroblotting. It is also advantageous in that there is little non-specific adsorption in the binding reaction with the analyte. As described above, a carrier in which the substrate itself is made of a carbon layer may be used.

[0046] The carbon layer can be formed by a known method. For example, microwave plasma CVD (Chemical vapor deposit) method, ECRCVD (Electric cyclotron resonance chemical vapor deposit) method, ICP (Inductive coupled plasma) method, DC sputtering method, ECR (Electric cyclotron resonance) sputtering method, ionized evaporation method, arc evaporation method, laser evaporation method, EB (Electron beam) evaporation method, resistance heating evaporation method, etc. can be mentioned.

[0047] In the high-frequency plasma CVD method, the raw material gas (methane) is decomposed by glow discharge generated between electrodes by high frequency, and a carbon layer is synthesized on the substrate. In the ionization deposition method, using thermoelectrons generated by a tungsten filament, the raw material gas (benzene) is decomposed and ionized, and a carbon layer is formed on the substrate by a bias voltage. A carbon layer may be formed by the ionization deposition method in a mixed gas composed of 1 to 99% by volume of hydrogen gas and the remaining 99 to 1% by volume of methane gas.

[0048] In the arc evaporation method, an arc discharge is caused in a vacuum by applying a DC voltage between a solid graphite material (cathode evaporation source) and a vacuum vessel (anode), generating a plasma of carbon atoms from the cathode, and applying a bias voltage more negative than the evaporation source to the substrate to accelerate carbon ions in the plasma toward the substrate, thereby forming a carbon layer.

[0049] In the laser evaporation method, for example, a carbon layer can be formed by irradiating a graphite target plate with Nd:YAG laser (pulse oscillation) light to melt it and depositing carbon atoms on a glass substrate.

[0050] When forming a carbon layer on the surface of the substrate, the thickness of the carbon layer is usually about a monolayer to 100 μm. If it is too thin, the surface of the underlying substrate may be locally exposed. Conversely, if it is too thick, the productivity deteriorates. Therefore, it is preferably 2 nm to 1 μm, more preferably 5 nm to 500 nm.

[0051] By introducing a chemical modification group on the surface of the substrate on which the carbon layer is formed, the probe can be firmly immobilized on the carrier. The chemical modification group to be introduced can be appropriately selected by those skilled in the art and is not particularly limited. Examples include an amino group, a carboxyl group, an epoxy group, a formyl group, a hydroxyl group, and an active ester group.

[0052] The introduction of an amino group can be carried out, for example, by irradiating the carbon layer with ultraviolet rays in ammonia gas or by plasma treatment. Or it can be carried out by irradiating the carbon layer with ultraviolet rays in chlorine gas for chlorination and then irradiating with ultraviolet rays in ammonia gas. Or it can also be carried out by reacting the carbon layer chlorinated with polyamines such as methylenediamine and ethylenediamine in a gas.

[0053] The introduction of a carboxyl group can be carried out, for example, by reacting a suitable compound with the carbon layer aminated as described above. Examples of the compounds used for introducing a carboxyl group include, for example, halocarboxylic acids represented by the formula: X-R1-COOH (wherein X represents a halogen atom and R1 represents a divalent hydrocarbon group having 10 to 12 carbon atoms), such as chloroacetic acid, fluoroacetic acid, bromoacetic acid, iodoacetic acid, 2-chloropropionic acid, 3-chloropropionic acid, 3-chloroacrylic acid, 4-chlorobenzoic acid; dicarboxylic acids represented by the formula: HOOC-R2-COOH (wherein R2 represents a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms), such as oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, phthalic acid; polycarboxylic acids such as polyacrylic acid, polymethacrylic acid, trimellitic acid, butanetetracarboxylic acid; keto acids or aldehyde acids represented by the formula: R3-CO-R4-COOH (wherein R3 represents a hydrogen atom or a divalent hydrocarbon group having 1 to 12 carbon atoms and R4 represents a divalent hydrocarbon group having 1 to 12 carbon atoms); monohalides of dicarboxylic acids represented by the formula: X-OC-R5-COOH (wherein X represents a halogen atom and R5 represents a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms), such as succinic acid monochloride, malonic acid monochloride; acid anhydrides such as phthalic anhydride, succinic anhydride, oxalic anhydride, maleic anhydride, butanetetracarboxylic anhydride.

[0054] The introduction of epoxy groups can be carried out, for example, by reacting a suitable polyvalent epoxy compound with the carbon layer aminated as described above. Alternatively, it can be obtained by reacting an organic peracid with the carbon-carbon double bond contained in the carbon layer. Examples of the organic peracid include peracetic acid, perbenzoic acid, diperoxyphthalic acid, performic acid, trifluoroperacetic acid, and the like.

[0055] The introduction of formyl groups can be carried out, for example, by reacting glutaraldehyde with the carbon layer aminated as described above.

[0056] The introduction of hydroxyl groups can be carried out, for example, by reacting water with the carbon layer chlorinated as described above.

[0057] The active ester group means an ester group having an electron-withdrawing group with high acidity on the alcohol side of the ester group to activate the nucleophilic reaction, that is, an ester group with high reaction activity. It is an ester group having an electron-withdrawing group on the alcohol side of the ester group and is more activated than an alkyl ester. The active ester group has reactivity with groups such as amino groups, thiol groups, and hydroxyl groups. More specifically, phenolic esters, thiophenolic esters, N-hydroxyamine esters, cyanomethyl esters, esters of heterocyclic hydroxy compounds, etc. are known as active ester groups having much higher activity than alkyl esters and the like. More specifically, examples of the active ester group include, for example, p-nitrophenyl group, N-hydroxysuccinimide group, succinimide group, phthalimide group, 5-norbornene-2,3-dicarboximide group, etc. In particular, the N-hydroxysuccinimide group is preferably used.

[0058] The introduction of the active ester group can be carried out, for example, by subjecting the carboxyl group introduced as described above to active esterification with a dehydrating condensing agent such as cyanamide or carbodiimide (e.g., 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide) and a compound such as N-hydroxysuccinimide. By this treatment, a group in which an active ester group such as an N-hydroxysuccinimide group is bonded to the terminal of the hydrocarbon group via an amide bond can be formed (Japanese Patent Laid-Open No. 2001-139532).

[0059] The probe is dissolved in a spotting buffer to prepare a spotting solution, which is dispensed into a 96-well or 384-well plastic plate, and the dispensed solution is spotted onto the carrier by a spotter device or the like, whereby a microarray in which the probe is immobilized on the carrier can be manufactured. Alternatively, the spotting solution may be manually spotted with a micropipettor.

[0060] After spotting, it is preferable to perform incubation in order to allow the reaction of the probe with the carrier to proceed. The incubation is usually carried out at a temperature of -20 to 100°C, preferably 0 to 90°C, and usually for 0.5 to 16 hours, preferably 1 to 2 hours. The incubation is desirably carried out in an atmosphere of high humidity, for example, under conditions of a humidity of 50 to 90%. Subsequently to the incubation, in order to remove the DNA not bound to the carrier, it is preferable to perform washing using a washing solution (e.g., 50 mM TBS / 0.05% Tween 20, 2×SSC / 0.2% SDS solution, ultrapure water, etc.).

[0061] By using the microarray configured as described above, for example, it is possible to determine whether a diagnostic subject has a missense mutation and / or a nonsense mutation or is wild type with respect to a gene mutation.

[0062] When specifically examining gene mutations, it includes the steps of extracting DNA from a sample derived from a subject to be diagnosed, using the extracted DNA as a template to amplify the region containing the gene mutation to be examined, hybridizing the amplified nucleic acid fragment with the first probe, the second probe, and the wild-type probe using the above-described microarray, and detecting signals from the first probe, the second probe, and the wild-type probe.

[0063] The subject to be diagnosed is usually a human, and the race and the like are not particularly limited. In particular, it is preferably a Mongoloid, more preferably an East Asian race, and most preferably a Japanese. Also, as the subject to be diagnosed, it can be a patient suspected of having a disease related to the gene mutation to be examined.

[0064] The sample derived from the subject to be diagnosed is not particularly limited. For example, it includes blood-related samples (blood, serum, plasma, etc.), lymph fluid, feces, cancer cells, crushed products and extracts of tissues or organs, and the like.

[0065] First, DNA is extracted from the sample collected from the subject to be diagnosed. The extraction means is not particularly limited. For example, a DNA extraction method using phenol / chloroform, ethanol, sodium hydroxide, CTAB, or the like can be used.

[0066] Next, an amplification reaction is performed using the obtained DNA as a template to amplify the region containing the gene mutation to be tested. As the amplification reaction, polymerase chain reaction (PCR), LAMP (Loop-Mediated Isothermal Amplification), ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids) method, etc. can be applied. In the amplification reaction, it is desirable to add a label so that the amplified region can be identified. At this time, the method for labeling the amplified nucleic acid is not particularly limited. For example, a method of pre-labeling the primers used in the amplification reaction may be used, or a method of using labeled nucleotides as substrates in the amplification reaction may be used. The labeling substance is not particularly limited, and radioactive isotopes, fluorescent dyes, or organic compounds such as digoxigenin (DIG) and biotin can be used.

[0067] This reaction system is a reaction system containing a buffer, a heat-resistant DNA polymerase, primers specific to the amplification region, labeled nucleotide triphosphates (specifically, nucleotide triphosphates with a fluorescent label, etc.), nucleotide triphosphates, magnesium chloride, etc., which are necessary for nucleic acid amplification and labeling.

[0068] Also, the nucleic acid fragment amplified by the primer is not particularly limited as long as it contains the region corresponding to the designed probe. For example, it is preferably 1 kbp or less, more preferably 800 bp or less, still more preferably 500 bp or less, and particularly preferably 350 bp or less.

[0069] A hybridization reaction is performed between the amplified nucleic acid obtained as described above and a probe immobilized on a carrier, and the gene mutation in the subject to be diagnosed can be examined by detecting the hybridization of the amplified nucleic acid to the first probe, the second probe, and the wild-type probe. That is, the hybridization of the amplified nucleic acid to the first probe, the second probe, and the wild-type probe can be measured, for example, by detecting a label. In particular, in the present invention, when it is detected by the second probe that the gene mutation is a silent mutation, it is determined that the gene mutation is wild-type.

[0070] The signal from the label can be quantified by detecting the fluorescence signal using a fluorescence scanner and analyzing it with image analysis software, for example, when a fluorescent label is used. The hybridization reaction is preferably carried out under stringent conditions. Stringent conditions refer to conditions under which specific hybrids are formed and non-specific hybrids are not formed. For example, after a hybridization reaction is carried out at 47 to 52 °C (specifically 51.5 °C) for 15 to 90 minutes (specifically 30 minutes), it refers to the conditions of washing 30 times under the conditions of 0.1×SSC / 0.1% SDS, 25 °C, and then washing 80 times under the conditions of 1×SSC, 25 °C. When the chain length of the probe is short, it is more preferable to lower the hybridization temperature, and when the chain length is long, it is more preferable to raise the hybridization temperature. It goes without saying that the hybridization temperature with specificity increases as the salt concentration increases, and conversely, the hybridization temperature with specificity decreases as the salt concentration decreases.

[0071] In addition, when using a microarray comprising a first probe, a second probe, and a wild-type probe for the above-described gene mutation, the gene mutation can be examined using the signal intensities from these first probe, second probe, and wild-type probe. Specifically, the signal intensities in the first probe, second probe, and wild-type probe are measured respectively, and a determination value for evaluating the signal intensity derived from the first probe or the second probe is calculated. As an example of calculating the determination value, for example, Equation 1: [Signal intensity derived from the first probe] / ([Signal intensity derived from the wild-type probe] + [Signal intensity derived from the first probe]) is used to evaluate the missense mutation and / or nonsense mutation corresponding to the first probe in the amplified nucleic acid, and Equation 2: [Signal intensity derived from the second probe] / ([Signal intensity derived from the wild-type probe] + [Signal intensity derived from the second probe]) is used to evaluate the silent mutation corresponding to the second probe.

[0072] Then, the determination value calculated by the above Equation 1 is compared with a predetermined threshold value (cutoff value). When the determination value exceeds the threshold value, it is determined that the amplified nucleic acid contains the missense mutation and / or nonsense mutation corresponding to the first probe for the above gene mutation. When the determination value is below the threshold value, it is determined that the amplified nucleic acid does not contain the missense mutation and / or nonsense mutation. Also, the determination value calculated by the above Equation 2 is compared with a predetermined threshold value (cutoff value). When the determination value exceeds the threshold value, it is determined that the amplified nucleic acid contains the silent mutation corresponding to the second probe for the above gene mutation. When the determination value is below the threshold value, it is determined that the amplified nucleic acid does not contain the silent mutation.

[0073] Then, when it is determined that a silent mutation is included based on the above Equation 2, it is determined that the gene mutation to be examined is wild type. Also, when it is determined that no missense mutation and / or nonsense mutation is included based on the above Equation 1 and no silent mutation is included based on the above Equation 2, it is determined that the gene mutation to be examined is wild type.

[0074] Here, the threshold value is not particularly limited. For example, it can be defined based on the determination value calculated by the above formula (1) or formula (2) using a specimen for which it has been confirmed that the gene mutation is wild type. More specifically, a plurality of determination values can be calculated using a plurality of specimens for which it has been confirmed that the gene mutation is wild type, and the value of the average value + 3σ (σ: standard deviation) can be used as the threshold value. Note that the value of the average value + 2σ or the average value + σ can also be used as the threshold value.

[0075] As described above, by using a microarray including a first probe, a second probe, and a wild-type probe for detecting a predetermined gene mutation, it is possible to accurately identify whether the gene mutation is a mutant type of missense mutation and / or nonsense mutation or a wild type. For example, by detecting the mutant types M1 to M8 (M1 to M6 are missense mutations, M7 and M8 are nonsense mutations) shown in Table 2 in the CD79B gene and determining that it is wild type when it is M9, it is possible to accurately diagnose diffuse large B-cell lymphoma (DLBCL) associated with the above gene mutation in the CD79B gene.

[0076] Although not particularly limited, the wild-type probe, the first probe, and the second probe designed to detect the wild type, the mutant types M1 to M8, and the silent mutation M9 shown in Table 2 in the CD79B gene are summarized in Table 3. In the probe base sequences described in Table 3, the portion corresponding to the codon encoding the 196th amino acid from the N-terminus is underlined.

[0077]

Table 3

[0078] Using the wild-type probe, the first probe (M1 to M8), and the second probe (M8) shown in Table 2, when [signal intensity derived from the second probe] / ([signal intensity derived from the wild-type probe] + [signal intensity derived from the second probe]) exceeds the threshold value, it can be determined as wild-type assuming that there is a silent mutation in the codon encoding the 196th amino acid from the N-terminus. Thereby, when the gene mutation in the CD79B gene has a missense mutation and / or a nonsense mutation, it is possible to identify the case with high precision, and it is possible to diagnose diffuse large B-cell lymphoma (DLBCL) with high precision.

Example

[0079] Hereinafter, the present invention will be described in more detail by way of examples. However, the technical scope of the present invention is not limited to the following examples.

[0080] <Array design> In this example, for the gene mutation in the MYD88 gene and the gene mutation in the CD79B gene, a primer set for amplifying the region containing the gene mutation, a probe for detecting the wild type or mutant type contained in the nucleic acid fragment amplified by the primer set, and a blocking nucleic acid corresponding to the wild type of the gene mutation of the MYD88 gene and the gene mutation of the CD79B gene were designed.

[0081] The probes corresponding to the gene mutations of the MYD88 gene designed in this example are shown in Table 4. In the "wild type (W) / mutant type (M)" column of Table 4, W is described for the probe corresponding to the wild type, and M is described for the probe corresponding to the mutant type.

[0082]

Table 4

[0083] The probes corresponding to the gene mutations of the CD79B gene designed in this example are shown in Tables 5 and 6. In the "Wild type (W) / Mutant type (M)" columns of Tables 5 and 6, W is described for the probes corresponding to the wild type, and M1 to M9 are described for the probes corresponding to the mutant type. The notations of M1 to M9 follow Table 3.

[0084]

Table 5

[0085]

Table 6

[0086] In addition, a primer set for amplifying the region containing the gene mutation of the MYD88 gene designed in this example is shown in Table 7.

[0087]

Table 7

[0088] Furthermore, a primer set for amplifying the region containing the gene mutation of the CD79B gene designed in this example is shown in Table 8.

[0089]

Table 8

[0090] Moreover, the blocking nucleic acids corresponding to the wild type in the gene mutations of the MYD88 gene designed in this example are shown in Table 9.

[0091]

Table 9

[0092] Moreover, the blocking nucleic acids corresponding to the wild type in the gene mutations of the CD79B gene designed in this example are shown in Table 10.

[0093]

Table 10

[0094] <Examination of Optimal Variant Probes> Regarding the probes described in Tables 4 to 6, the fluorescence intensity was measured using a 100% mutation model specimen or a 5% mutation model specimen. Specifically, a DNA chip immobilized with each of the above-mentioned various probes was set in a gene analysis device BIOSHOT HT-32 (manufactured by Toyo Kohan Co., Ltd.) to perform a hybridization reaction with the model specimen. A DNA chip needle for fixing the DNA chip, and further, after the hybridization reaction of the DNA chip, a washing solution (0.1×SSC / 0.1% SDS solution, room temperature), a rinsing solution (1×SSC solution, room temperature), and a detection solution for fluorescence detection (1×SSC solution, room temperature) for washing the excess reagent were set in the BIOSHOT HT-32, and the operation was started.

[0095] By automatic operation, with the reaction solution in the designated PCR tube heated to 51.5 °C, the DNA chip needle was inserted and the hybridization reaction was carried out for 30 minutes. After the hybridization reaction, the DNA chip needle was quickly immersed in the washing solution tank, and stirred 30 times so that the DNA chip entered and exited the washing solution surface. The DNA chip needle after washing was immersed in the rinsing solution tank, and stirred 80 times so that the DNA chip entered and exited the rinsing solution surface.

[0096] The DNA chip needle after rinsing was slowly immersed in the detection solution tank, and by irradiating with a single-wavelength laser of 640 nm, the excitation light was captured by the CCD camera for 2 seconds. Then, a determination value was calculated from the measured fluorescence intensity value according to the following formula. [Determination value] = [Fluorescence intensity of variant probe] / ([Fluorescence intensity of wild-type probe] + [Fluorescence intensity of variant probe]) Note that the 100% mutation model specimen means a sample in which the ratio of the variant type to the wild type in the gene mutation to be detected is 100:0. Also, the 5% mutation model specimen means a sample in which the ratio of the variant type to the wild type in the gene mutation to be detected is 5:95.

[0097] The results of measuring the fluorescence intensity values when using the 100% mutant model specimen are shown in Figure 1, the results of measuring the fluorescence intensity values when using the 5% mutant model specimen are shown in Figure 2, and the results of measuring the determination values from the results shown in Figure 2 are shown in Figure 3. In addition, in Figures 1 to 3, for comparison, the results of calculating the fluorescence intensity values from each probe using a wild-type sample in which the ratio of the mutant type to the wild-type in the gene mutation to be detected is 0:100 are also shown.

[0098] The specificities of the mutant probe and the wild-type probe were confirmed from the fluorescence intensity values shown in Figure 1. As a result, it was found that excellent fluorescence intensity values were obtained for all the tested mutant probes and wild-type probes. In addition, for all the tested mutant probes and wild-type probes, the cross-reaction was suppressed to about 1 / 2 or less of the specific fluorescence intensity value, and it was judged that the target gene mutation (mutant type, wild-type) could be distinguished.

[0099] Also, from the results shown in Figure 1, regarding the silent mutation 591C>T represented by M9 among the gene mutations of the CD79B gene, a strong fluorescence intensity value (47125) was detected in the M8 probe (CD79B_M8_v3-2). If detection using a mutant probe for the silent mutation M9 is not attempted, there is a possibility of misidentifying a sample having the silent mutation of M9 as the M8 mutation. Therefore, by surely identifying the silent mutation using a mutant probe corresponding to the silent mutation M9 when identifying the gene mutation of the CD79B gene, misjudgment can be prevented. At this time, when the silent mutation is identified, it is determined as "wild type". Thereby, it is possible to precisely identify whether the gene mutation of the CD79B gene is of the mutant type or the wild type, and very effective information can be provided regarding diseases related to the gene mutation of the CD79B gene.

[0100] On the one hand, FIG. 2 shows the fluorescence intensity values of mutant probes and wild-type probes when using samples with a low mutation rate. By obtaining fluorescence intensity values equal to or higher than the desired value even for samples with a low mutation rate, the target gene mutation can be detected with high precision. For example, assuming that a fluorescence intensity value of 10,000 or more is preferable, it can be seen from the results shown in FIG. 2 that sufficient fluorescence intensity values have not been obtained for CD79B_M8_v3-3 and CD79B_M8_v4-3.

[0101] In addition, FIG. 3 shows the determination values calculated according to the above formula from the fluorescence intensity values shown in FIG. 2. As can be understood from the formula for calculating the determination value, it shows that the higher the value of the determination value, the better the separation ability by the mutant probe and the wild-type probe. Also, it can be said that the lower the determination value calculated for the mutant probe when using a wild-type sample, the better the separation ability. For example, assuming that the determination value calculated for the mutant probe when using a wild-type sample is 0.05 or less as a criterion, it can be seen from the results shown in FIG. 3 that CD79B_M4_v3-2, CD79B_M4_v4-4, and CD79B_M6-v4-3 do not have sufficient separation ability.

[0102] As a result of comprehensively considering the above-described results, it was found that it is preferable to adopt MYD88_M_v2-1 as the mutant probe for the gene mutation of the MYD88 gene. Also, for the gene mutations (9 types of M1 to M9) of the CD79B gene, it was found that it is preferable to adopt CD79B_M1_v2-2, CD79B_M2_v3-1, CD79B_M3-v2-2, CD79B_M4_v4-3, CD79B_M5_v5-3, CD79B_M6_v2-2, CD79B_M7_v2-1, CD79B_M8_v3-2, and CD79B_M9-1.

[0103] <Examination of Optimal Wild-Type Probe> First, wild-type probes for detecting wild-type in gene mutations of the MYD88 gene were examined. Fluorescence intensity values and determination values were calculated from the wild-type probe and the mutant probe (v2-1) when using the tested wild-type samples and the 5% mutant model specimens. Also, fluorescence intensity values and determination values were calculated in the same manner using genomic DNA (gDNA-1) instead of the wild-type samples and the 5% mutant model specimens.

[0104] The results are shown in Fig. 4. When selecting a wild-type probe, it is better that the fluorescence intensity value is higher when using genomic DNA, the resolution is high (that is, the difference between the mutant determination value and the wild-type determination value is large), and the determination value calculated from the fluorescence intensity value when using the wild-type sample is low, which was set as the conditions required for the wild-type probe. Specifically, when using genomic DNA, the fluorescence intensity value from the wild-type probe is 10,000 or more, the resolution is the highest among the tested wild-type probes (the difference between the mutant determination value and the wild-type determination value is the largest), and the determination value when using the wild-type sample is 0.05 or less. These were comprehensively evaluated. As a result, v2-1 with the highest resolution could be selected as the optimal wild-type probe.

[0105] Next, wild-type probes for detecting wild-type in gene mutations of the CD79B gene were examined. The examination method and evaluation criteria were in accordance with the method for selecting wild-type probes in gene mutations of the MYD88 gene described above. As the mutant probes, CD79B_M1_v2-2, CD79B_M2_v3-1, CD79B_M3-v2-2, CD79B_M4_v4-3, CD79B_M5_v5-3, CD79B_M6_v2-2, CD79B_M7_v2-1, CD79B_M8_v3-2, and CD79B_M9-1 selected above were used.

[0106] The results of measuring the fluorescence intensity values are shown in Fig. 5, and the results of calculating the determination values from the fluorescence intensity values are shown in Fig. 6. As can be seen from Figs. 5 and 6, considering comprehensively that the fluorescence intensity value is high when using genomic DNA, the separation ability is high, and the determination value is low when using the wild-type sample, v3-1 could be selected as the optimal wild-type probe.

[0107] <Performance Evaluation of the Selected Mutant Probes and Wild-Type Probes> Using the mutant probes (MYD88_M_v2-1) and wild-type probes (MYD88_W_v2-1) selected for the gene mutation of the MYD88 gene, and the mutant probes (CD79B_M1_v2-2, CD79B_M2_v3-1, CD79B_M3-v2-2, CD79B_M4_v4-3, CD79B_M5_v5-3, CD79B_M6_v2-2, CD79B_M7_v2-1, CD79B_M8_v3-2, and CD79B_M9-1) and wild-type probes (CD79B_W_v3-1) selected for the gene mutation of the CD79B gene, the same gene mutations in genomic DNA were identified. As genomic DNA, three types: gDNA-1, gDNA-2, and gDNA-3 were used. gDNA-1 is genomic DNA derived from a healthy individual. gDNA-2 is genomic DNA derived from a non-Hodgkin lymphoma patient. gDNA-3 is genomic DNA derived from lymphoplasmacytic lymphoma.

[0108] The results of identifying each gene mutation using these mutant probes and wild-type probes are shown in Fig. 7. As shown in Fig. 7, for gDNA-1, it was identified that the gene mutation of the MYD88 gene is not mutant and the gene mutation of the CD79B gene is not mutant. Also, for gDNA-2, it was identified that the gene mutation of the MYD88 gene is mutant (L265P) and the gene mutation of the CD79B gene is mutant (Y196D, M3). Also, for gDNA-3, it was identified that the gene mutation of the MYD88 gene is mutant (L265P) and the gene mutation of the CD79B gene is not mutant.

[0109] To verify the results when using the mutant probe and wild-type probe shown in Fig. 7, for the three types of genomic DNAs (gDNA-1, gDNA-2, and gDNA-3) tested, gene mutations in the MYD88 gene and gene mutations in the CD79B gene were confirmed by the direct sequencing method. As a result, it was confirmed that both the gene mutation in the MYD88 gene and the gene mutation in the CD79B gene in gDNA-1 were wild-type. Also, it was confirmed that the gene mutation in the MYD88 gene in gDNA-1 was a mutant type of L265P, and the gene mutation in the CD79B gene was a mutant type of Y196D (M3). Furthermore, it was confirmed that the gene mutation in the MYD88 gene in gDNA-3 was a mutant type of L265P, and the gene mutation in the CD79B gene was wild-type.

[0110] The identification results of each gene mutation using the mutant probe and wild-type probe selected as described above were completely consistent with the results confirmed by the direct sequencing method. From this, it was shown that the mutant probe and wild-type probe selected as described above can accurately detect each gene mutation with high precision even in actual use scenarios.

[0111] <Primer Set Design> Regarding the primer set for amplifying the region containing the gene mutation of the MYD88 gene shown in Tables 7 and 8 and the primer set for amplifying the region containing the gene mutation of the CD79B gene, seven combinations ([1] to [7]) shown in Fig. 8 were tested, and the optimal primer set was selected based on the amplification amount of the target nucleic acid fragment and the amplification amount of non-specific nucleic acid amplification.

[0112] First, a reaction solution was prepared using a wild-type model specimen or genomic DNA (gDNA) as a template. Using the prepared PCR reaction solution, the thermal cycle of PCR was carried out at 95°C for 5 minutes, followed by 30 seconds at 95°C, 30 seconds at a predetermined annealing temperature (e.g., 59°C), and 45 seconds at 72°C as one cycle. Then, it was carried out at 72°C for 10 minutes, and finally maintained at 4°C. The annealing temperature was 56°C, 58°C, 60°C, 62°C, or 64°C.

[0113] The relationship between the nucleic acid amplification amount and the annealing temperature is shown in FIG. 9. As can be seen from FIG. 9, in order to amplify the region containing the gene mutation of the MYD88 gene and the region containing the gene mutation of the CD79B gene in the same manner for both the wild-type model specimen and genomic DNA (multiplex amplification), it was the combination of [5], [6], and [7].

[0114] Also, FIG. 10 shows the results of confirming by electrophoresis the nucleic acid fragments contained in the reaction solution after PCR carried out with the combination of [5], [6], and [7]. As shown in FIG. 10, when the combination of [7] was used, a plurality of nucleic acid fragments due to non-specific amplification other than the target nucleic acid fragment could be confirmed. From this result, it was found that the combination of [5] and [6] is superior to the combination of [7].

[0115] Next, a reaction solution was prepared using a 5% mutant model specimen or genomic DNA (gDNA-1) derived from a healthy subject as a template. PCR was similarly carried out using the reaction solution, and the fluorescence intensity based on the amplified fragment was measured. The results are shown in FIG. 11. As shown in FIG. 11, when the combination of [5] was used, it was found that the fluorescence intensity from the mutant probe corresponding to the mutant type in the gene mutation of the MYD88 gene was lower compared to other combinations.

[0116] Then, regarding the combination of [6] and [7], when the mixing ratio of the labeled primer and the unlabeled primer was changed, the change in the fluorescence intensity value was examined. The results are shown in Fig. 12. As shown in Fig. 12, in the case of the combination of [6], the fluorescence intensity increased as the mixing ratio of the labeled primer increased, and the fluorescence intensity value corresponding to the ratio of the labeled primer was obtained. On the other hand, in the case of the combination of [7], the tendency of the change in the ratio of the labeled primer and the fluorescence intensity value did not match. This was considered to be due to the non-specific amplification observed when the combination of [7] was used. From these results, it became clear that it is preferable to multiplex-amplify the region containing the gene mutation of the MYD88 gene and the region containing the gene mutation of the CD79B gene by the combination of [6].

[0117] <Examination of primer ratio (labeled vs unlabeled)> In each of the primer sets for amplifying the region containing the gene mutation of the MYD88 gene and the primer set for amplifying the region containing the gene mutation of the CD79B gene, the ratio of the labeled primer and the unlabeled primer was changed, and how the fluorescence intensity value fluctuated was examined, and the optimal ratio of the labeled primer and the unlabeled primer was examined. In this example, in the primer set for amplifying the region containing the gene mutation of the CD79B gene, the forward primer is the labeled primer (CD79B_Fw-4), and the reverse primer is the unlabeled primer (CD79B_Rv-4). Also, in the primer set for amplifying the region containing the gene mutation of the MYD88 gene, the forward primer is the unlabeled primer (MYD88_Fw-5-3), and the reverse primer is the labeled primer (MYD88_Rv-5).

[0118] In this example, as shown in Fig. 13, the final concentrations of the labeled primers (CD79B_Fw-4, MYD88_Rv-5) and unlabeled primers (CD79B_Rv-4, MYD88_Fw-5-3) contained in the PCR reaction solution were adjusted, and PCR was similarly performed. Note that as the template, a mutant 5% model specimen and genomic DNA were used. Fig. 14 shows the results when a primer set for amplifying the region containing the gene mutation of the MYD88 gene was used, and Fig. 15 shows the results when a primer set for amplifying the region containing the gene mutation of the CD79B gene was used.

[0119] As can be seen from Fig. 14, excellent fluorescence intensity values were achieved in the range where the final concentration of the labeled primer for amplifying the region containing the gene mutation of the MYD88 gene was 2 to 6 times (i.e., labeled primer:unlabeled primer = 20:10) compared to the unlabeled primer. In particular, even more excellent fluorescence intensity values were achieved in the range of 3 to 5 times, and the most excellent fluorescence intensity value was achieved at 4 times.

[0120] Also, as can be seen from Fig. 15, excellent fluorescence intensity values were achieved in the range where the final concentration of the labeled primer for amplifying the region containing the gene mutation of the CD79B gene was 5 to 10 times compared to the unlabeled primer. In particular, even more excellent fluorescence intensity values were achieved in the range of 6 to 7 times, and the most excellent fluorescence intensity value was achieved at 7 times.

[0121] <Examination of Optimal Blocking Nucleic Acid> Based on the function of the blocking nucleic acids related to the gene mutations of the MYD88 gene shown in Table 9 and the blocking nucleic acids related to the gene mutations of the CD79B gene shown in Table 10, the optimal blocking nucleic acids were selected. The blocking nucleic acid has the function of specifically hybridizing to the wild-type nucleic acid fragment among the nucleic acid fragments amplified from the region containing the gene mutation and preventing the wild-type nucleic acid fragment from non-specifically hybridizing to the mutant probe. Therefore, when selecting the optimal blocking nucleic acid, the non-specific hybridization between the wild-type nucleic acid fragment and the mutant probe in the presence of the blocking nucleic acid can be judged based on the fluorescence intensity of the mutant probe. Also, whether the blocking nucleic acid inhibits the specific hybridization between the mutant nucleic acid fragment and the mutant probe in the presence of the blocking nucleic acid can be judged based on the fluorescence intensity of the mutant probe.

[0122] Regarding the blocking nucleic acids related to the gene mutations of the MYD88 gene shown in Table 9, hybridization was performed between the wild-type model specimen and the mutant probe (MYD88_M_v2-1) in the presence of the blocking nucleic acid (125 nM or 500 nM), and the fluorescence intensity of the mutant probe was measured. Also, hybridization was performed between the 100% mutant model specimen and the mutant probe (MYD88_M_v2-1) in the presence of the blocking nucleic acid, and the fluorescence intensity of the mutant probe was measured. The results are shown in Figure 16.

[0123] As can be seen from Figure 16, non-specific hybridization between the wild-type model specimen and the mutant probe could be suppressed in all the tested blocking nucleic acids. However, for v1 and v2-1 among the blocking nucleic acids, it was revealed that they inhibited the specific hybridization between the 100% mutant model specimen and the mutant probe. Therefore, using the 5% mutant model specimen, it was verified which of v2-2 and v2-3 functions better as a blocking nucleic acid.

[0124] That is, in the presence of v2-2 or v2-3 (125 nM or 500 nM), hybridization was performed between the 5% mutation model sample and the wild-type probe (MYD88_W_v2-1) and the mutant probe (MYD88_M_v2-1), and the fluorescence intensities of the wild-type probe and the mutant probe were measured. Further, based on the above formula, a determination value was calculated from the measured fluorescence intensity values. The results of the measured fluorescence intensities are shown in Fig. 17, and the results of the calculated determination values are shown in Fig. 18.

[0125] As can be seen from Figs. 17 and 18, when v2-2 was used, the fluorescence intensity of the wild-type probe was lower compared to the case when v2-3 was used, and it was revealed that v2-2 was superior in function as a blocking nucleic acid compared to v2-3. From these results, among those shown in Table 9, v2-2 was selected as the optimal blocking nucleic acid for the gene mutation of the MYD88 gene. Also, from these results, it was found that even when no blocking nucleic acid for the gene mutation of the MYD88 gene was used (i.e., in the case of 0 nM), the determination value when using the wild-type model sample and the determination value when using the 5% mutation model sample were sufficiently separated. From the above results, it was found that the concentration of the blocking nucleic acid for the gene mutation of the MYD88 gene is preferably 0 to 500 nM.

[0126] Next, regarding the blocking nucleic acids for the gene mutations of the CD79B gene shown in Table 10, hybridization was performed between the wild-type model sample or the 5% mutation model sample and the wild-type probe (CD79B_W_v3-1) and the mutant probes (CD79B_M1_v2-2, CD79B_M2_v3-1, CD79B_M3-v2-2, CD79B_M4_v4-3, CD79B_M5_v5-3, CD79B_M6_v2-2, CD79B_M7_v2-1, CD79B_M8_v3-2, and CD79B_M9-2) in the presence of the blocking nucleic acid, the fluorescence intensities from the wild-type probe and the mutant probes were measured, and the determination value was calculated according to the above formula. The results are shown in Fig. 19.

[0127] As shown in Fig. 19, when using a wild-type model specimen, those with a determination value of 0.05 or less in all mutant probes were only when using the blocking nucleic acids of v4-1 and v4-2. For the blocking nucleic acids of v4-1 and v4-2, the determination values and fluorescence intensity values in each mutant probe calculated using a wild-type model specimen or a mutant 5% model specimen are shown in Figs. 20 and 21, respectively. As can be seen from Figs. 20 and 21, when comparing the determination values for v4-1 and v4-2, both were almost equivalent, but when comparing the fluorescence intensity values for v4-1 and v4-2, it was found that v4-2 showed a higher value. From this result, among those shown in Table 10, v4-2 was selected as the optimal blocking nucleic acid as the blocking nucleic acid for gene mutations of the CD79B gene.

[0128] <Examination of Blocking Nucleic Acid Concentration> Regarding v4-2 selected as the blocking nucleic acid for gene mutations of the CD79B gene, hybridization was performed with a wild-type model specimen or a mutant 5% model specimen and wild-type probes (CD79B_W_v3-1) and mutant probes (CD79B_M1_v2-2, CD79B_M2_v3-1, CD79B_M3-v2-2, CD79B_M4_v4-3, CD79B_M5_v5-3, CD79B_M6_v2-2, CD79B_M7_v2-1, CD79B_M8_v3-2, and CD79B_M9-2) in the presence of blocking nucleic acids of various concentrations, the fluorescence intensity from the wild-type probe and the mutant probe was measured, and the determination value was calculated according to the above formula. The results are shown in Fig. 22.

[0129] As shown in FIG. 22, when the concentration of the blocking nucleic acid was 125 nM, there were multiple mutant probes with a determination value exceeding 0.05 when using the wild-type model specimen. From this result, it was shown that the concentration of the blocking nucleic acid is preferably higher than 125 nM. Further, the concentration of the blocking nucleic acid was set to 500 nM, 750 nM, or 1000 nM, and the results of measuring the fluorescence intensity from each mutant probe when using the 5% mutant model specimen are shown in FIG. 23. From this result, it was found that when the concentration of the blocking nucleic acid was 1000 nM, the fluorescence intensity value was lower compared to the cases where the concentration was 500 nM or 750 nM. From these results, it was suggested that it is preferable to set the concentration of the blocking nucleic acid to 500 nM or 750 nM.

[0130] To confirm the above results, the determination values of each mutant probe when using the wild-type model specimen or the 5% mutant model specimen were calculated when the concentration of the blocking nucleic acid was 500 nM or 750 nM. The results are shown in FIG. 24. As can be seen from FIG. 24, in either case where the concentration of the blocking nucleic acid was 500 nM or 750 nM, the determination value when using the wild-type model specimen and the determination value when using the 5% mutant model specimen were sufficiently separated, and it was found that the wild-type and each mutant type could be accurately detected. In particular, when the concentration of the blocking nucleic acid was 750 nM, the determination value when using the wild-type model specimen and the determination value when using the 5% mutant model specimen were more greatly separated compared to the case where the concentration was 500 nM.

[0131] From the above results, it was found that the concentration of the blocking nucleic acid can be in the range of 125 nM to 1000 nM, and it is preferably 500 to 750 nM. As an example, it was found that the concentration of the blocking nucleic acid is most preferably 750 nM.

Claims

1. A method for detecting a gene mutation including a missense mutation and / or a nonsense mutation and a silent mutation, comprising: a step of amplifying a nucleic acid region including the gene mutation; a step of hybridizing a nucleic acid fragment including the gene mutation, a first probe corresponding to the missense mutation or nonsense mutation in the gene mutation, a second probe corresponding to the silent mutation in the gene mutation, and a wild-type probe corresponding to the wild type; a step of detecting signals from the first probe, the second probe, and the wild-type probe, wherein a signal from the second probe is determined as the wild type in the gene mutation.

2. The following formula: determination value = [signal value from the second probe] / [signal value from the wild-type probe + signal value from the second probe] The method according to claim 1, wherein the determination value is calculated by the formula, and when the calculated determination value exceeds a preset cut-off value, the gene mutation is determined as the wild type.

3. The following formula: determination value = [signal value from the first probe] / [signal value from the wild-type probe + signal value from the first probe] The method according to claim 1, wherein the determination value is calculated by the formula, and when the calculated determination value exceeds a preset cut-off value, it is determined that a missense mutation and / or a nonsense mutation exists in the gene mutation.

4. For a plurality of missense mutations and / or nonsense mutations in the gene mutation, the determination value is calculated by the above formula for each of them, and the existence of each of the plurality of missense mutations and / or nonsense mutations is determined.

5. The method according to claim 4, wherein when all of the determination values calculated for each of the plurality of missense mutations and / or nonsense mutations are below the cut-off value, the gene mutation is determined to be a wild type.

6. The gene mutation is a missense mutation and / or nonsense mutation occurring in a codon encoding a predetermined amino acid, and the first probe corresponds to a missense mutation and / or nonsense mutation having a base substitution at the same position as the base substitution that results in a silent mutation for the amino acid. The method according to claim 1.

7. The gene mutation is a missense mutation and / or nonsense mutation occurring in a codon encoding a predetermined amino acid, and the first probe corresponds to a missense mutation and / or nonsense mutation having a base substitution in the codon in which a base substitution resulting in a silent mutation for the amino acid has occurred. The method according to claim 1.

8. The method according to claim 7, characterized in that a plurality of first probes for all missense mutations and / or nonsense mutations having a base substitution in the codon in which a base substitution resulting in a silent mutation for the amino acid has occurred are used.

9. The gene mutation includes a missense mutation and / or nonsense mutation of the 196th tyrosine residue in the CD79B gene and a silent mutation of the tyrosine residue. The method according to claim 1.

10. The missense mutation in the CD79B gene is at least one mutation selected from the group consisting of 589T>C, 589T>A, 589T>G, 590A>C, 590A>G, and 590A>T. The nonsense mutation in the CD79B gene is 591C>G or 591C>A. The silent mutation in the CD79B gene is 591C>T. The method according to claim 9.

11. The method according to claim 10, wherein the first probe corresponding to the missense mutation and the nonsense mutation in the CD79B gene, the second probe corresponding to the silent mutation in the CD79B gene, and the wild-type probe corresponding to the wild type corresponding to these missense mutations, nonsense mutations, and silent mutations contain the nucleotide sequences shown in Table 1. [Table 1]

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