Genetic mutation detection method

Abstract

The purpose of the present invention is to analyze genetic mutations including silent mutations as well as missense mutations and / or nonsense mutations with a high degree of accuracy.　In the present invention, signals from a first probe that corresponds to missense or nonsense mutations among the genetic mutations, a second probe that corresponds to silent mutations among the genetic mutations, and a wild-type probe that corresponds to the wild-type gene are detected, and, among the genetic mutations, signals from the second probe are assessed as being wild type.

Description

How to detect genetic mutations

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

[0002] In a broad sense, a gene mutation refers to a state in which a gene has undergone some kind of congenital or acquired abnormality. Examples of gene mutations include substitution, deletion, or addition of bases constituting DNA. Here, a gene is composed of a coding region that encodes a protein and a non-coding region such as an expression control region. Gene mutations can occur in both coding and non-coding regions. In particular, gene mutations occurring in coding regions include missense mutations that change the encoded amino acid, nonsense mutations that convert a codon encoding a specific amino acid into a stop codon, and silent mutations that do not change the encoded amino acid.

[0003] Gene mutations are the cause of hereditary diseases and cancer, and are also related to the efficacy of certain drugs. Gene mutations are also related to physical conditions such as obesity. Therefore, identifying specific gene mutations (also known as genotyping) is an essential technique for diagnosing hereditary diseases and understanding the efficacy of drugs.

[0004] Genotyping methods include, for example, DNA sequencing, SSCP (Single Strand Conformation Polymorphism), RFLP (Restriction Fragment Length Polymorphism), PCR (Polymerase Chain Reaction), AFLP (Amplified Fragment Length Polymorphism), ASO (Allele Specific Oligonucleotide) probes, and methods that detect binding to DNA microarrays or DNA beads. Among these techniques, methods that use DNA microarrays typically include mutant and wild-type probes immobilized on a carrier for the gene mutation to be detected.

[0005] In the method using a DNA microarray, first, a region containing a gene mutation to be detected is amplified by a nucleic acid amplification reaction using fluorescently labeled primers. Then, a hybridization reaction is carried out between the fluorescently labeled nucleic acid fragment and a mutant probe and a wild-type probe. If the amplified nucleic acid fragment contains a mutant, fluorescence is observed from the mutant probe. Therefore, by observing the fluorescence from the mutant probe and the wild-type probe, gene mutations can be genotyped.

[0006] Meanwhile, Patent Document 1 discloses a technique for determining the risk of developing a disease by genotyping. Patent Document 1 discloses a method for determining the risk of developing primary central nervous system lymphoma based on genetic mutations in GRB2 and / or MYD88. Patent Document 2 discloses a method for determining whether to use a BTK inhibitor such as ibrutinib for the treatment of diffuse large B-cell lymphoma (DLBCL) based on a modification (genetic mutation) of the 198th or 265th amino acid in MYD88 and a modification (genetic mutation) of the 196th amino acid in CD79B.

[0007] WO2016 / 098873 Special Publication No. 2017-523188

[0008] However, no method has been established for analyzing genetic mutations containing missense mutations and / or nonsense mutations and silent mutations with high accuracy. Therefore, an object of the present invention is to provide a new method for detecting genetic mutations containing missense mutations and / or nonsense mutations and silent mutations.

[0009] The method for detecting a gene mutation according to the present invention, which achieves the above-mentioned object, includes the following.

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

[0011] (2) The method according to (1), characterized in that the judgment value is calculated using the following formula: judgment value = [signal value from the second probe] / [signal value from the wild-type probe + signal value from the second probe], and when the calculated judgment value exceeds a predetermined cutoff value, the gene mutation is judged to be wild-type.

[0012] (3) The method according to (1), characterized in that a judgment value is calculated using the following formula: judgment value = [signal value from the first probe] / [signal value from the wild-type probe + signal value from the first probe], and if the calculated judgment value exceeds a predetermined cutoff value, it is determined that the gene mutation contains a missense mutation and / or a nonsense mutation.

[0013] (4) The method according to (3), characterized in that a judgment value is calculated for each of multiple missense mutations and / or nonsense mutations in the gene mutation using the above formula, and the presence of each of the multiple missense mutations and / or nonsense mutations is judged.

[0014] (5) The method according to (4), characterized in that if the judgment values ​​calculated for each of the multiple missense mutations and / or nonsense mutations are all below the cutoff value, the genetic mutation is judged to be wild type.

[0015] (6) The method according to (1), characterized in that the genetic mutation is a missense mutation and / or a nonsense mutation occurring in a codon encoding a specific amino acid, and the first probe corresponds to a missense mutation and / or a nonsense mutation having a base substitution at the same position as a base substitution that results in a silent mutation for the amino acid.

[0016] (7) The method according to (1), characterized in that the genetic mutation is a missense mutation and / or a nonsense mutation occurring in a codon encoding a specific amino acid, and the first probe corresponds to a missense mutation and / or a nonsense mutation having a base substitution in a codon in which a base substitution resulting in a silent mutation for the amino acid has occurred.

[0017] (8) The method described in (7), characterized in that a plurality of first probes for all missense mutations and / or nonsense mutations having base substitutions in codons in which base substitutions resulting in silent mutations for the above amino acids have occurred are used.

[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 method according to (9), wherein 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.

[0020] (11) The method according to (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 mutation, nonsense mutation, and silent mutation contain the nucleotide sequences shown in Table 1. This specification includes the disclosure of Japanese Patent Application No. 2023-216580, from which the present application claims priority.

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

[0022] 1 is a characteristic diagram showing the results of measuring the fluorescence intensity values ​​of various probes designed in the examples when a 100% mutation model specimen was used. FIG. 2 is a characteristic diagram showing the results of measuring the fluorescence intensity values ​​of various probes designed in the examples when a 5% mutation model specimen was used. FIG. 3 is a characteristic diagram showing the determination values ​​calculated from the fluorescence intensity values ​​of various probes designed in the examples when a 5% mutation model specimen was used. FIG. 4 is a characteristic diagram showing the results of measuring the fluorescence intensity values ​​of a wild-type probe (gene mutation of MYD88 gene) designed in the examples when a wild-type sample, a 5% mutation model specimen, or genomic DNA was used, and calculating the determination value. FIG. 5 is a characteristic diagram showing the results of measuring the fluorescence intensity values ​​of a wild-type probe (gene mutation of CD79B gene) designed in the examples when a wild-type sample, a 5% mutation model specimen, or genomic DNA was used. FIG. 6 is a characteristic diagram showing the determination values ​​calculated from the fluorescence intensity values ​​of a wild-type probe (gene mutation of CD79B gene) designed in the examples when a wild-type sample, a 5% mutation model specimen, or genomic DNA was used. FIG. 7 is a characteristic diagram showing the results (determination values) of detecting a gene mutation in genomic DNA using various probes selected in the examples. 1 is a characteristic diagram showing the combinations of a primer set for amplifying a region containing a gene mutation in the CD79B gene and a primer set for amplifying a region containing a gene mutation in the MYD88 gene, which were used in the Examples.

[0034] FIG. 1 is a characteristic diagram showing the relationship between the amount of nucleic acid amplified and the annealing temperature for each primer set combination.

[0035] FIG. 1 is a photograph showing the results of electrophoresis of nucleic acid fragments contained in the reaction solution for primer set combinations [5], [6], and [7].

[0036] FIG. 1 is a characteristic diagram showing the results of PCR performed with primer set combinations [5], [6], and [7], and detecting the amplified fragments by the fluorescence intensity of the wild-type probe and the mutant-type probe.

[0037] FIG. 1 is a characteristic diagram showing the results of measuring fluorescence intensity for primer set combinations [6] and [7], changing the mixing ratio of labeled primers to unlabeled primers.

[0038] FIG. 1 is a characteristic diagram showing the concentration ratio of the forward primer and the reverse primer contained in the primer sets used in the Examples.1 is a characteristic diagram showing the relationship between the concentration ratio of the forward primer and the reverse primer contained in the primer set (MYD88 gene) tested in the Examples and the fluorescence intensity value. FIG. 2 is a characteristic diagram showing the relationship between the concentration ratio of the forward primer and the reverse primer contained in the primer set (CD79B gene) tested in the Examples and the fluorescence intensity value. FIG. 3 is a characteristic diagram showing the results of measuring the fluorescence intensity from the mutant probe when using the blocking nucleic acid (MYD88 gene) designed in the Examples. FIG. 4 is a characteristic diagram showing the results of measuring the fluorescence intensity from the wild-type probe and the mutant probe when using v2-2 or v2-3 of the blocking nucleic acids (MYD88 gene) designed in the Examples. FIG. 5 is a characteristic diagram showing the determination value calculated from the fluorescence intensity measured with the wild-type probe and the mutant probe when using v2-2 or v2-3 of the blocking nucleic acids (MYD88 gene) designed in the Examples. FIG. 6 is a characteristic diagram showing the determination value calculated from the fluorescence intensity measured with the mutant probe when using the blocking nucleic acid (CD79B gene) designed in the Examples. 1 is a characteristic diagram showing the determination values ​​for each mutant probe calculated using v4-1 and v4-2 of the blocking nucleic acids (CD79B gene) designed in the Examples and using a wild-type model specimen or a 5% mutation model specimen. FIG. 2 is a characteristic diagram showing the fluorescence intensity values ​​for each mutant probe calculated using v4-1 and v4-2 of the blocking nucleic acids (CD79B gene) designed in the Examples and using a wild-type model specimen or a 5% mutation model specimen. FIG. 3 is a characteristic diagram showing the results of calculating the determination values ​​for the blocking nucleic acid (CD79B gene) v4-2 designed in the Examples by changing its concentration. FIG. 4 is a characteristic diagram showing the results of measuring the fluorescence intensity from each mutant probe when a 5% mutation model specimen is used and the concentration of the blocking nucleic acid (CD79B gene) v4-2 designed in the Examples is set to 500 nM, 750 nM, or 1000 nM. This is a characteristic diagram showing the results of calculating the judgment values ​​of each mutant probe when using a wild-type model sample or a 5% mutant model sample, when the concentration of the blocking nucleic acid (CD79B gene) v4-2 designed in the examples was 500 nM or 750 nM.

[0023] The present invention will be described in detail below. The method for detecting a genetic mutation according to the present invention targets genetic mutations, including silent mutations. Genetic mutations including silent mutations refer to mutations in bases that constitute codons encoding specific amino acids, and include both silent mutations that do not result in a change in the amino acid, and missense mutations that result in a change in the amino acid and / or nonsense mutations that result in a stop codon.

[0024] Such genetic mutations are not particularly limited, but include genetic mutations associated with diseases, genetic mutations associated with pharmacological efficacy, genetic mutations associated with constitutions, and the like. Genetic mutations associated with diseases, pharmacological efficacy, or constitutions may be related to the disease, pharmacological efficacy, or constitution, and do not necessarily have to be scientifically proven to be related to the disease, pharmacological efficacy, or constitution. For example, genetic mutations associated with genetic diseases or hereditary diseases can be detected. Genetic diseases or hereditary diseases encompass all diseases caused by mutations in chromosomes or genes. Some genetic diseases or hereditary diseases develop due to the presence of specific genetic mutations, either congenital or acquired. Therefore, detecting such genetic mutations can diagnose genetic diseases or hereditary diseases, or contribute to the diagnosis of such diseases.

[0025] Whether such genetic mutations include silent mutations can be determined by using known databases. For example, for cancer-related genetic mutations, the Catalogue of Somatic Mutations in Cancer (COSMIC), a catalog of somatic mutations in cancer, can be used to determine whether the genetic mutations include silent mutations or to identify genetic mutations associated with cancer that have silent mutations.

[0026] Examples include mutations in the CD79B gene associated with diffuse large B-cell lymphoma (DLBCL), mutations in the BRAF gene associated with malignant melanoma and lung cancer, and mutations in the GNAS gene associated with intraductal papillary mucinous neoplasms of the pancreas.

[0027] Specifically, genetic mutations are known for the tyrosine at the 196th position from the N-terminus in the CD79B gene. These mutations include three types: missense mutations, which result in a base substitution in the codon (TAC) encoding the tyrosine, resulting in a different amino acid; nonsense mutations, which result in a stop codon; and silent mutations, which leave the encoded amino acid as tyrosine.

[0028] Here, the missense mutations in the CD79B gene are a mutation in which thymine at position 589 from the 5' end of the CD79B coding region is replaced with cytosine (this mutation is referred to as 589T>C; hereinafter, the same description applies, with A: adenine, G: guanine, C: cytosine, and T: thymine), 589T>A, 589T>G, 590A>C, 590A>G, and 590A>T. Furthermore, the nonsense mutations in the CD79B gene are 591C>G and 591C>A. Furthermore, a silent mutation in the CD79B gene is 591C>T.

[0029] These mutations at thymine 196 in the CD79B gene are summarized in Table 2. In Table 2, the wild-type sequence is marked with W in the type column, and M1 to M6 in the same column are missense mutations, M7 and M8 are nonsense mutations, and M9 is a silent mutation.

[0030]

[0031] Specifically, genetic mutations are known for the valine at the 600th position from the N-terminus of the BRAF gene. These mutations include two types: missense mutations, which result in a base substitution in the codon (GTG) that encodes the valine, resulting in a different amino acid, and silent mutations, which leave the encoded amino acid as valine.

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

[0033] Specifically, genetic mutations are known for the arginine at the 844th position from the N-terminus of the GNAS gene. These mutations include two types: missense mutations, which result in a base substitution in the codon (CGT) that encodes the arginine, resulting in a different amino acid, and silent mutations, which leave the encoded amino acid as arginine.

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

[0035] Furthermore, in the genetic testing method of the present invention, missense mutations and / or nonsense mutations in a predetermined amino acid are detected in the genetic mutation to be tested. In other words, among the base mutations (e.g., substitution mutations) occurring in the codon encoding the predetermined amino acid in the genetic mutation to be tested, missense mutations and / or nonsense mutations are detected. In particular, the genetic testing method of the present invention is characterized in that, among the base mutations (e.g., substitution mutations) occurring in the codon encoding the predetermined amino acid, if a silent mutation is detected, the genetic mutation is determined to be wild-type.

[0036] For example, among the missense and / or nonsense mutations occurring in the codon encoding a specific amino acid in the genetic mutation to be tested, the missense and / or nonsense mutations having a base substitution at the same position as the base substitution resulting in a silent mutation for that amino acid can be detected. Specifically, in the genetic mutation in the CD79B gene described above, the 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 detected.

[0037] Furthermore, for example, among missense and / or nonsense mutations occurring in codons encoding a specific amino acid in the genetic mutations being tested, missense and / or nonsense mutations having a base substitution in a codon that results in a silent mutation for that amino acid can be detected. Specifically, for the genetic 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), which are codons containing the silent mutation 591C>T, and two nonsense mutations consisting of 591C>G and 591C>A (M7 and M8 in Table 2) can be detected. The missense and nonsense mutations to be detected may include all of the missense mutations 589T>C, 589T>A, 589T>G, 590A>C, 590A>G, and 590A>T, and the nonsense mutations 591C>G and 591C>A (M1 to M8 in Table 2).

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

[0039] Here, a probe for detecting a missense mutation and / or a nonsense mutation in a genetic mutation to be tested is referred to as a first probe, and a probe for detecting a silent mutation in the genetic mutation is referred to as a second probe. A probe for detecting a wild-type form of the genetic mutation is referred to as a wild-type probe. These first probe, second probe, and wild-type probe can be appropriately designed based on the base sequence of the missense mutation and / or nonsense mutation in the genetic mutation to be tested, the base sequence of the silent mutation in the genetic mutation, and the wild-type base sequence.

[0040] The base length of these probes is not particularly limited, but can be, for example, 10 to 30 bases, preferably 15 to 25 bases. Furthermore, the probes are preferably nucleic acids, more preferably DNA. DNA includes both double-stranded and single-stranded DNA, but single-stranded DNA is preferred. Probes can be obtained, for example, by chemical synthesis using a nucleic acid synthesizer. Examples of nucleic acid synthesizers that can be used include DNA synthesizers, fully automated nucleic acid synthesizers, and automated nucleic acid synthesizers.

[0041] The probes designed as described above are preferably used in the form of a microarray (e.g., a DNA chip) by immobilizing their 5' ends on a carrier. In this case, the microarray has a first probe, a second probe, and a wild-type probe for the genetic mutation to be tested. Note that the microarray may have a first probe, a second probe, and a wild-type probe for each of multiple types of genetic mutations. The microarray can be prepared by immobilizing the above-mentioned first probe, second probe, and wild-type probe on a carrier.

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

[0043] As the support, a support having a carbon layer and a chemically modified group on its surface is preferably used. Supports 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, any material known in the art can be used, and there is no particular limitation, and the same materials as those listed as the support materials above can be used.

[0044] In microarrays, carriers with a fine, flat structure are preferably used. Shapes include, but are not limited to, rectangular, square, and round. Typically, carriers measuring 1 to 75 mm square, preferably 1 to 10 mm square, and more preferably 3 to 5 mm square are used. Because carriers with a fine, flat structure are easily manufactured, substrates made of silicon or resin materials are preferred. Carriers with a carbon layer and chemically modified groups on the surface of a substrate made of single-crystal silicon are particularly preferred. Single-crystal silicon also includes those with slight variations in the orientation of the crystal axis in parts (sometimes called mosaic crystals) and those containing atomic-scale irregularities (lattice defects).

[0045] As the carbon layer that is formed on substrate, there is no particular limitation, but preferably use synthetic diamond, high-pressure synthetic diamond, natural diamond, soft diamond (for example, diamond-like carbon), amorphous carbon, carbon-based material (for example, graphite, fullerene, carbon nanotube), any of their mixtures, or their laminated material.In addition, can also use carbide 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.Here, soft diamond is a general term for the incomplete diamond structure that is the mixture of diamond and carbon, such as so-called diamond-like carbon (DLC: Diamond Like Carbon), and its mixture ratio is not particularly limited. Carbon layers are advantageous in that they have excellent chemical stability and can withstand subsequent introduction of chemical modification groups and reactions involving binding with the analyte; they bind to the analyte via electrostatic bonding, which allows for flexibility; they do not absorb UV light, making them transparent to the UV light of the detection system; and they can be electrified during electroblotting. Another advantage is that they exhibit minimal nonspecific adsorption during the binding reaction with the analyte. As mentioned above, a carrier in which the substrate itself is made of a carbon layer may also be used.

[0046] The carbon layer can be formed by a known method, such as microwave plasma CVD (chemical vapor deposition), ECRCVD (electric cyclotron resonance chemical vapor deposition), ICP (inductive coupled plasma), DC sputtering, ECR (electric cyclotron resonance) sputtering, ionization deposition, arc deposition, laser deposition, EB (electron beam) deposition, and resistance heating deposition.

[0047] In radio-frequency plasma CVD, the source gas (methane) is decomposed by glow discharge generated between electrodes using radio frequency waves, and a carbon layer is synthesized on the substrate. In ionized vapor deposition, the source gas (benzene) is decomposed and ionized using thermal electrons generated by a tungsten filament, and a carbon layer is formed on the substrate using a bias voltage. The carbon layer can also be formed by ionized vapor deposition in a mixed gas consisting of 1-99% by volume of hydrogen gas and the remaining 99-1% by volume of methane gas.

[0048] In the arc evaporation method, a DC voltage is applied between a solid graphite material (cathode evaporation source) and a vacuum chamber (anode), causing an arc discharge in a vacuum to generate a plasma of carbon atoms from the cathode, and a bias voltage more negative than that of the evaporation source is applied to the substrate, accelerating the carbon ions in the plasma toward the substrate and forming a carbon layer.

[0049] In the laser deposition method, for example, a graphite target plate is irradiated with light from an Nd:YAG laser (pulsed oscillation) to melt it, and carbon atoms are deposited on a glass substrate, thereby forming a carbon layer.

[0050] When a carbon layer is formed on the surface of a substrate, the thickness of the carbon layer is usually about a monolayer to 100 μm. If the carbon layer is too thin, the surface of the base substrate may be locally exposed, while if the carbon layer is too thick, productivity will decrease. Therefore, the thickness is preferably 2 nm to 1 μm, and more preferably 5 nm to 500 nm.

[0051] By introducing a chemical modifying group onto the surface of the substrate on which the carbon layer is formed, the probe can be firmly immobilized on the carrier. The chemical modifying group to be introduced can be appropriately selected by those skilled in the art and is not particularly limited, but examples thereof 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 amino groups can be carried out, for example, by irradiating the carbon layer with ultraviolet light in ammonia gas or by plasma treatment. Alternatively, the carbon layer can be chlorinated by irradiating it with ultraviolet light in chlorine gas, and then further irradiating it with ultraviolet light in ammonia gas. Alternatively, the introduction can be carried out by reacting the chlorinated carbon layer with a polyamine gas such as methylenediamine or ethylenediamine.

[0053] The introduction of a carboxyl group can be carried out, for example, by reacting an appropriate compound with the aminated carbon layer as described above. Examples of compounds that can be used to introduce a carboxyl group include 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, and 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, and phthalic acid; polyacrylic acid; and polymethacrylic 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 and malonic acid monochloride; and acid anhydrides such as phthalic anhydride, succinic acid anhydride, oxalic acid anhydride, maleic acid anhydride, and butanetetracarboxylic acid anhydride.

[0054] Epoxy groups can be introduced by, for example, reacting an appropriate polyepoxy compound with the aminated carbon layer as described above. Alternatively, they can be obtained by reacting an organic peracid with the carbon-carbon double bond contained in the carbon layer. Examples of organic peracids include peracetic acid, perbenzoic acid, diperoxyphthalic acid, performic acid, and trifluoroperacetic acid.

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

[0056] The introduction of a hydroxyl group can be carried out, for example, by reacting the chlorinated carbon layer with water.

[0057] The term "activated ester group" refers to an ester group that has a highly acidic electron-withdrawing group on the alcohol side of the ester group, activating nucleophilic reactions, i.e., a highly reactive ester group. The ester group has an electron-withdrawing group on the alcohol side of the ester group, making it more activated than alkyl esters. The activated ester group is reactive with groups such as amino groups, thiol groups, and hydroxyl groups. More specifically, phenol esters, thiophenol esters, N-hydroxyamine esters, cyanomethyl esters, and esters of heterocyclic hydroxy compounds are known to be activated ester groups with much higher activity than alkyl esters. More specifically, examples of activated ester groups include p-nitrophenyl, N-hydroxysuccinimide, succinimide, phthalimide, and 5-norbornene-2,3-dicarboximide groups, with the N-hydroxysuccinimide group being particularly preferred.

[0058] The introduction of an active ester group can be carried out, for example, by converting the carboxyl group introduced as described above into an active ester using a dehydration condensation agent such as cyanamide or a carbodiimide (e.g., 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide) and a compound such as N-hydroxysuccinimide. This treatment allows the formation of a group in which an active ester group such as an N-hydroxysuccinimide group is bound to the end of a hydrocarbon group via an amide bond (JP 2001-139532 A).

[0059] The probes are 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 using a spotter device, etc., to produce a microarray in which the probes are immobilized on the carrier. Alternatively, the spotting solution may be spotted manually using a micropipette.

[0060] After spotting, incubation is preferably performed to promote the reaction of binding of the probe to the carrier. Incubation is typically performed at temperatures between -20 and 100°C, preferably between 0 and 90°C, for typically 0.5 to 16 hours, preferably 1 to 2 hours. Incubation is preferably performed in a high humidity atmosphere, for example, at a humidity of 50 to 90%. Following incubation, washing with a washing solution (e.g., 50 mM TBS / 0.05% Tween 20, 2x SSC / 0.2% SDS solution, ultrapure water, etc.) is preferably performed to remove DNA not bound to the carrier.

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

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

[0063] The subject of diagnosis is usually a human, and although there are no particular limitations on race, the subject of diagnosis is preferably a person of Asian race, more preferably an East Asian race, and most preferably a Japanese person. The subject of diagnosis may also be a patient suspected of having a disease associated with the gene mutation being tested.

[0064] The sample derived from the subject to be diagnosed is not particularly limited, and examples thereof include blood-related samples (blood, serum, plasma, etc.), lymph, feces, cancer cells, tissue or organ homogenates and extracts, etc.

[0065] First, DNA is extracted from a sample collected from a subject. The extraction method is not particularly limited. For example, DNA extraction methods using phenol / chloroform, ethanol, sodium hydroxide, CTAB, etc. 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. Examples of applicable amplification reactions include polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN). It is desirable to add a label to the amplification reaction so that the amplified region can be identified. The method for labeling the amplified nucleic acid is not particularly limited, but may include, for example, pre-labeling the primers used in the amplification reaction, or using labeled nucleotides as substrates in the amplification reaction. The labeling substance may include, but is not limited to, radioisotopes, fluorescent dyes, or organic compounds such as digoxigenin (DIG) and biotin.

[0067] This reaction system also contains a buffer necessary for nucleic acid amplification and labeling, a heat-stable DNA polymerase, a primer specific to the amplified region, a labeled nucleotide triphosphate (specifically, a nucleotide triphosphate labeled with a fluorescent label, etc.), a nucleotide triphosphate, magnesium chloride, etc.

[0068] Furthermore, the nucleic acid fragment amplified by the primers is not particularly limited as long as it contains a region corresponding to the designed probe, and is preferably 1 kbp or less, more preferably 800 bp or less, even more preferably 500 bp or less, and particularly preferably 350 bp or less.

[0069] The genetic mutation in a subject can be tested by hybridizing the amplified nucleic acid obtained as described above with a probe immobilized on a carrier and detecting hybridization of the amplified nucleic acid with the first probe, the second probe, and the wild-type probe. That is, hybridization of the amplified nucleic acid with the first probe, the second probe, and the wild-type probe can be determined, for example, by detecting the label. In particular, in the present invention, if the second probe detects that the genetic mutation is a silent mutation, the genetic mutation is determined to be wild-type.

[0070] For example, when a fluorescent label is used, the signal from the label can be detected using a fluorescent scanner and analyzed using image analysis software to quantify the signal intensity. The hybridization reaction is preferably performed under stringent conditions. Stringent conditions refer to conditions under which specific hybrids are formed and nonspecific hybrids are not formed, such as hybridization at 47-52°C (specifically, 51.5°C) for 15-90 minutes (specifically, 30 minutes), followed by 30 washes in 0.1xSSC / 0.1% SDS at 25°C, and then 80 washes in 1xSSC at 25°C. It should be noted that, if the probe chain length is short, it is more preferable to use a lower hybridization temperature, and, if the chain length is long, it is more preferable to use a higher hybridization temperature. It goes without saying that the hybridization temperature for specificity increases with increasing salt concentration, and conversely, the hybridization temperature for specificity decreases with decreasing salt concentration.

[0071] Furthermore, when a microarray comprising a first probe, a second probe, and a wild-type probe is used for the above-mentioned genetic mutation, the genetic mutation can be tested using the signal intensities from these first probe, second probe, and wild-type probe. Specifically, the signal intensities of the first probe, second probe, and wild-type probe are measured, respectively, and a judgment value for evaluating the signal intensity derived from the first probe or the second probe is calculated. Examples of calculation methods for the judgment value include, for example, using Formula 1: [signal intensity derived from the first probe] / ([signal intensity derived from the wild-type probe]+[signal intensity derived from the first probe]) to evaluate missense mutations and / or nonsense mutations corresponding to the first probe, and using Formula 2: [signal intensity derived from the second probe] / ([signal intensity derived from the wild-type probe]+[signal intensity derived from the second probe]) to evaluate silent mutations corresponding to the second probe.

[0072] The judgment value calculated by the above formula 1 is then compared with a predetermined threshold (cutoff value), and if the judgment value exceeds the threshold, it is determined that the amplified nucleic acid contains a missense mutation and / or a nonsense mutation corresponding to the first probe for the above genetic mutation, and if the judgment value is below the threshold, it is determined that the amplified nucleic acid does not contain the missense mutation and / or nonsense mutation.Furthermore, the judgment value calculated by the above formula 2 is compared with a predetermined threshold (cutoff value), and if the judgment value exceeds the threshold, it is determined that the amplified nucleic acid contains a silent mutation corresponding to the second probe for the above genetic mutation, and if the judgment value is below the threshold, it is determined that the amplified nucleic acid does not contain the silent mutation.

[0073] The genetic mutation to be tested is determined to be wild-type if it is determined to contain a silent mutation based on the above formula 2. The genetic mutation to be tested is also determined to be wild-type if it is determined to contain no missense mutation and / or nonsense mutation based on the above formula 1 and no silent mutation based on the above formula 2.

[0074] Here, the threshold value is not particularly limited, but can be defined, for example, based on the judgment value calculated by the above-mentioned formula 1 or 2 using a sample in which the genetic mutation has been confirmed to be wild-type. More specifically, a plurality of judgment values ​​can be calculated using a plurality of samples in which the genetic mutation has been confirmed to be wild-type, and the value of the average value + 3σ (σ: standard deviation) can be used as the threshold. Note that the value of the average value + 2σ or the average value + σ can also be used as the threshold.

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

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

[0077]

[0078] Using the wild-type probe, first probes (M1 to M8), and second probe (M8) shown in Table 2, if [signal intensity derived from the second probe] / ([signal intensity derived from the wild-type probe]+[signal intensity derived from the second probe]) exceeds a threshold, it is determined that a silent mutation is present in the codon encoding the 196th amino acid from the N-terminus, and the sample can be determined to be wild-type. This allows for highly accurate identification of cases in which the above-mentioned genetic mutation in the CD79B gene is a missense mutation and / or a nonsense mutation, enabling highly accurate diagnosis of diffuse large B-cell lymphoma (DLBCL).

[0079] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to the following examples.

[0080] <Sequence design> In this example, for gene mutations in the MYD88 gene and gene mutations in the CD79B gene, a primer set was designed to amplify the region containing the gene mutation, a probe was designed to detect the wild-type or mutant type contained in the nucleic acid fragment amplified with the primer set, and blocking nucleic acids were designed to correspond to the wild-type gene mutations in the MYD88 gene and the CD79B gene.

[0081] The probes designed in this example that correspond to gene mutations in the MYD88 gene are shown in Table 4. In the "Wild type (W) / Mutant type (M)" column in Table 4, W is written for probes that correspond to the wild type, and M is written for probes that correspond to the mutant type.

[0082]

[0083] The probes designed in this example that correspond to gene mutations in the CD79B gene are shown in Tables 5 and 6. In the "Wild type (W) / Mutant type (M)" column in Tables 5 and 6, probes corresponding to the wild type are marked with W, and probes corresponding to the mutant types are marked with M1 to M9. The designations M1 to M9 are in accordance with Table 3.

[0084]

[0085]

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

[0087]

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

[0089]

[0090] Furthermore, Table 9 shows blocking nucleic acids corresponding to the wild type in the genetic mutation of the MYD88 gene designed in this example.

[0091]

[0092] Furthermore, Table 10 shows blocking nucleic acids corresponding to the wild type in the gene mutation of the CD79B gene designed in this example.

[0093]

[0094] <Investigation of optimal mutant probes> Fluorescence intensities were measured for the probes listed in Tables 4 to 6 using a 100% mutation model sample or a 5% mutation model sample. Specifically, DNA chips with the above-mentioned probes immobilized were placed in a BioShot HT-32 genetic analyzer (manufactured by Toyo Kohan Co., Ltd.) and hybridization reactions with the model samples were performed. The DNA chip needle with the immobilized DNA chip, along with a cleaning solution (0.1x SSC / 0.1% SDS solution, room temperature) for washing off excess reagent after the hybridization reaction of the DNA chip, a rinse solution (1x SSC solution, room temperature), and a detection solution for fluorescence detection (1x SSC solution, room temperature) were placed in the BioShot HT-32 and operation was started.

[0095] The reaction solution in the designated PCR tube was heated to 51.5°C by automatic operation, and 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 a washing solution bath and agitated 30 times so that the DNA chip went in and out of the washing solution surface. After washing, the DNA chip needle was immersed in a rinse solution bath and agitated 80 times so that the DNA chip went in and out of the rinse solution surface.

[0096] The rinsed DNA chip needle was slowly immersed in the detection solution tank, and a 640 nm single-wavelength laser was irradiated, capturing excitation light for 2 seconds on a CCD camera. The measured fluorescence intensity was then used to calculate the evaluation value according to the following formula: [Evaluation value] = [Fluorescence intensity of mutant probe] / ([Fluorescence intensity of wild-type probe] + [Fluorescence intensity of mutant probe]). The 100% mutation model sample refers to a sample in which the ratio of mutant to wild-type in the gene mutation to be detected is 100:0. The 5% mutation model sample refers to a sample in which the ratio of mutant to wild-type in the gene mutation to be detected is 5:95.

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

[0098] The specificity of the mutant and wild-type probes was confirmed from the fluorescence intensity values ​​shown in Figure 1. The results showed that excellent fluorescence intensity values ​​were obtained for all mutant and wild-type probes tested. Furthermore, cross-reactivity was suppressed to approximately half of the specific fluorescence intensity value for all mutant and wild-type probes tested, indicating that it was possible to distinguish between the target gene mutations (mutant and wild-type).

[0099] Furthermore, as shown in Figure 1, the silent mutation 591C>T, designated M9, in the CD79B gene was detected with a strong fluorescence intensity value (47125) using the M8 probe (CD79B_M8_v3-2). Failure to detect the silent M9 mutation using a mutant probe could result in samples with the M9 silent mutation being erroneously identified as the M8 mutation. Therefore, using a mutant probe corresponding to the silent M9 mutation when identifying CD79B gene mutations can reliably identify the silent mutation and prevent erroneous identification. Furthermore, if a silent mutation is identified, the sample is classified as "wild type." This allows precise identification of whether the CD79B gene mutation is mutant or wild type, providing highly useful information about diseases associated with CD79B gene mutations.

[0100] On the other hand, Figure 2 shows the fluorescence intensity values ​​for the mutant and wild-type probes when a sample with a low mutation rate was used. Even with a sample with a low mutation rate, obtaining a fluorescence intensity value equal to or greater than the desired value allows for highly accurate detection of the target gene mutation. For example, assuming that a fluorescence intensity value of 10,000 or greater is desirable, the results shown in Figure 2 indicate that sufficient fluorescence intensity values ​​were not obtained for CD79B_M8_v3-3 and CD79B_M8_v4-3.

[0101] Figure 3 also shows the judgment values ​​calculated from the fluorescence intensity values ​​shown in Figure 2 according to the above formula. As can be seen from the formula for calculating the judgment value, a higher judgment value indicates better resolution between the mutant probe and the wild-type probe. It can also be said that a lower judgment value calculated for the mutant probe when a wild-type sample is used indicates better resolution. For example, if a judgment value of 0.05 or less calculated for the mutant probe when a wild-type sample is used is used as the standard, the results shown in Figure 3 indicate that CD79B_M4_v3-2, CD79B_M4_v4-4, and CD79B_M6-v4-3 do not have sufficient resolution.

[0102] Taking the above results into consideration, it was found that for genetic mutations in the MYD88 gene, it is preferable to use MYD88_M_v2-1 as the mutant probe, and for genetic mutations in the CD79B gene (nine types, M1 to M9), it is preferable to use 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] <Investigation of the optimal wild-type probe> First, we investigated a wild-type probe for detecting wild-type MYD88 gene mutations. Fluorescence intensity values ​​and judgement values ​​from the wild-type probe and mutant probe (v2-1) were calculated using the wild-type sample and a 5% mutant model specimen. Furthermore, fluorescence intensity values ​​and judgement values ​​were calculated in the same way using genomic DNA (gDNA-1) instead of the wild-type sample and the 5% mutant model specimen.

[0104] The results are shown in Figure 4. The wild-type probes were selected based on the following criteria: a higher fluorescence intensity value when using genomic DNA, a higher resolution (i.e., a larger difference between the mutant and wild-type scores), and a lower score calculated using the above formula from the fluorescence intensity value when using the wild-type sample. Specifically, the wild-type probes were evaluated comprehensively based on the following criteria: a fluorescence intensity value of 10,000 or greater when using genomic DNA, the highest resolution among the wild-type probes tested (the largest difference between the mutant and wild-type scores), and a score of 0.05 or less when using the wild-type sample. As a result, v2-1, which had the highest resolution, was selected as the optimal wild-type probe.

[0105] Next, we investigated wild-type probes for detecting wild-type CD79B gene mutations. The investigation method and evaluation criteria were similar to those used to select wild-type probes for MYD88 gene mutations described above. The mutant probes used were 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.

[0106] The results of measuring the fluorescence intensity values ​​are shown in Figure 5, and the results of calculating the judgment values ​​from the fluorescence intensity values ​​are shown in Figure 6. As can be seen from Figures 5 and 6, v3-1 was selected as the optimal wild-type probe, taking into consideration the high fluorescence intensity values ​​and high resolution when genomic DNA was used, and the low judgment value when a wild-type sample was used.

[0107] <Performance Evaluation of Selected Mutant and Wild-Type Probes> The mutant probe (MYD88_M_v2-1) and wild-type probe (MYD88_W_v2-1) were selected for MYD88 gene mutations, 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 probe (CD79B_W_v3-1) were selected for CD79B gene mutations to identify the same gene mutations in genomic DNA. Three types of genomic DNA were used: gDNA-1, gDNA-2, and gDNA-3. gDNA-1 is genomic DNA from a healthy individual, gDNA-2 is genomic DNA from a patient with non-Hodgkin's lymphoma, and gDNA-3 is genomic DNA from a patient with lymphoplasmacytic lymphoma.

[0108] The results of identifying each gene mutation using these mutant and wild-type probes are shown in Figure 7. As shown in Figure 7, for gDNA-1, the MYD88 gene mutation was identified as not being a mutant type, and the CD79B gene mutation was also identified as not being a mutant type. For gDNA-2, the MYD88 gene mutation was identified as being a mutant type (L265P) and the CD79B gene mutation was identified as being a mutant type (Y196D, M3). For gDNA-3, the MYD88 gene mutation was identified as being a mutant type (L265P) and the CD79B gene mutation was also identified as not being a mutant type.

[0109] To verify the results using the mutant and wild-type probes shown in Figure 7, we used direct sequencing to confirm the MYD88 and CD79B gene mutations for the three genomic DNAs tested (gDNA-1, gDNA-2, and gDNA-3). The results confirmed that both the MYD88 and CD79B mutations in gDNA-1 were wild-type. Furthermore, the MYD88 mutation in gDNA-1 was the L265P mutation, and the CD79B mutation was the Y196D mutation (M3). Furthermore, the MYD88 mutation in gDNA-3 was the L265P mutation, and the CD79B mutation was wild-type.

[0110] The results of identifying each gene mutation using the mutant and wild-type probes selected as described above were completely consistent with the results confirmed by direct sequencing, demonstrating that the mutant and wild-type probes selected as described above can accurately detect each gene mutation with high precision even in practical situations.

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

[0112] First, a reaction mixture was prepared using a wild-type model specimen or genomic DNA (gDNA) as a template. The PCR reaction mixture was then subjected to a thermal cycle consisting of 5 minutes at 95°C, 30 seconds at 95°C, 30 seconds at a predetermined annealing temperature (e.g., 59°C), and 45 seconds at 72°C. This was followed by 10 minutes at 72°C, with a final temperature of 4°C. The annealing temperatures were 56°C, 58°C, 60°C, 62°C, or 64°C.

[0113] The relationship between the amount of nucleic acid amplification and the annealing temperature is shown in Figure 9. As can be seen from Figure 9, the combinations [5], [6], and [7] were sufficient to amplify the regions containing the MYD88 gene mutation and the CD79B gene mutation in the wild-type model specimen and genomic DNA (multiplex amplification was possible).

[0114] Furthermore, the results of electrophoresis of the nucleic acid fragments contained in the reaction solution after PCR performed using combinations [5], [6], and [7] are shown in Figure 10. As shown in Figure 10, when combination [7] was used, multiple nucleic acid fragments due to non-specific amplification other than the target nucleic acid fragment were confirmed. This result shows that the combination of [5] and [6] is superior to combination [7].

[0115] Next, a reaction mixture was prepared using genomic DNA (gDNA-1) derived from a 5% mutation model specimen or a healthy individual as a template. PCR was performed using the reaction mixture in the same manner, and the fluorescence intensity of the amplified fragments was measured. The results are shown in Figure 11. As shown in Figure 11, when combination [5] was used, the fluorescence intensity from the mutant probe corresponding to the mutation in the MYD88 gene mutation was found to be lower than that of the other combinations.

[0116] We then investigated the effect of varying the ratio of labeled primers to unlabeled primers on the fluorescence intensity of combinations [6] and [7]. The results are shown in Figure 12. As shown in Figure 12, for combination [6], the fluorescence intensity increased with increasing ratio of labeled primers, and the fluorescence intensity values ​​were proportional to the ratio of labeled primers. In contrast, for combination [7], the trend in the change in fluorescence intensity did not match the ratio of labeled primers. This was likely due to the nonspecific amplification observed when combination [7] was used. These results demonstrate that combination [6] is preferable for multiplex amplification of regions containing mutations in the MYD88 gene and CD79B gene.

[0117] <Examination of Primer Ratio (Labeled vs. Unlabeled)> The ratio of labeled primers to unlabeled primers was varied for each primer set amplifying the region containing the MYD88 gene mutation and the region containing the CD79B gene mutation, and the changes in fluorescence intensity were examined to determine the optimal ratio of labeled primers to unlabeled primers. In this example, the primer set amplifying the region containing the CD79B gene mutation was a labeled primer (CD79B_Fw-4) as the forward primer and an unlabeled primer (CD79B_Rv-4) as the reverse primer. The primer set amplifying the region containing the MYD88 gene mutation was an unlabeled primer (MYD88_Fw-5-3) as the forward primer and a labeled primer (MYD88_Rv-5) as the reverse primer.

[0118] In this example, as shown in Figure 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 performed in the same manner. A 5% mutation model specimen and genomic DNA were used as templates. The results obtained using a primer set that amplifies the region containing the MYD88 gene mutation are shown in Figure 14, and the results obtained using a primer set that amplifies the region containing the CD79B gene mutation are shown in Figure 15.

[0119] As can be seen from Figure 14, excellent fluorescence intensity values ​​were achieved when the final concentration of the labeled primers amplifying the region containing the gene mutation in the MYD88 gene was between 2-fold (i.e., labeled primer:unlabeled primer = 20:10) and 6-fold higher than that of the unlabeled primers, and even better fluorescence intensity values ​​were achieved in the range of 3-fold to 5-fold, with the best fluorescence intensity value being achieved at 4-fold.

[0120] Furthermore, as can be seen from Figure 15, excellent fluorescence intensity values ​​were achieved when the final concentration of the labeled primers used to amplify the region containing the gene mutation in the CD79B gene was 5 to 10 times higher than that of the unlabeled primers, and even better fluorescence intensity values ​​were achieved when the final concentration was 6 to 7 times higher, with the best fluorescence intensity value being achieved when the final concentration was 7 times higher.

[0121] <Consideration of Optimal Blocking Nucleic Acids> Optimal blocking nucleic acids were selected based on their function as blocking nucleic acids for the MYD88 gene mutations shown in Table 9 and the CD79B gene mutations shown in Table 10. Blocking nucleic acids specifically hybridize to wild-type nucleic acid fragments among nucleic acid fragments obtained by amplifying a region containing a gene mutation, thereby preventing nonspecific hybridization of the wild-type nucleic acid fragment to a mutant probe. Therefore, when selecting an optimal blocking nucleic acid, nonspecific hybridization between a wild-type nucleic acid fragment and a mutant probe in the presence of the blocking nucleic acid can be determined based on the fluorescence intensity of the mutant probe. Furthermore, whether the blocking nucleic acid inhibits specific hybridization between a mutant nucleic acid fragment and a mutant probe in the presence of the blocking nucleic acid can be determined based on the fluorescence intensity of the mutant probe.

[0122] For the blocking nucleic acids related to the gene mutations of the MYD88 gene shown in Table 9, a wild-type model specimen was hybridized with a 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, a 100% mutant model specimen was hybridized with a 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, all of the blocking nucleic acids tested were able to suppress nonspecific hybridization between the wild-type model specimen and the mutant probe, but it was revealed that among the blocking nucleic acids, v1 and v2-1 inhibited specific hybridization between the 100% mutant model specimen and the mutant probe. Therefore, using the 5% mutant model specimen, we examined which of v2-2 and v2-3 functioned as a better blocking nucleic acid.

[0124] Specifically, a 5% mutant model sample was hybridized with a wild-type probe (MYD88_W_v2-1) and a mutant probe (MYD88_M_v2-1) in the presence of v2-2 or v2-3 (125 nM or 500 nM), and the fluorescence intensities of the wild-type and mutant probes were measured. Furthermore, a judgement value was calculated from the measured fluorescence intensity values ​​using the above formula. The results of the fluorescence intensity measurements are shown in Figure 17, and the results of the calculated judgement values ​​are shown in Figure 18.

[0125] As can be seen from Figures 17 and 18, the fluorescence intensity of the wild-type probe was lower when v2-2 was used compared to when v2-3 was used, demonstrating superior blocking nucleic acid functionality compared to v2-3. Based on these results, v2-2 was selected as the optimal blocking nucleic acid for MYD88 gene mutations among those listed in Table 9. Furthermore, these results indicated that even when no blocking nucleic acid for MYD88 gene mutations was used (i.e., at 0 nM), there was sufficient separation between the values ​​determined using the wild-type model sample and the 5% mutant model sample. These results suggest that a concentration of 0 to 500 nM is preferred for blocking nucleic acids for MYD88 gene mutations.

[0126] Next, for the blocking nucleic acids related to the gene mutations of the CD79B gene shown in Table 10, wild-type model samples or 5% mutant model samples were hybridized with the wild-type probe (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 the blocking nucleic acid, and the fluorescence intensity from the wild-type probe and mutant probe was measured. The judged value was calculated according to the above formula. The results are shown in Figure 19.

[0127] As shown in Figure 19, when a wild-type model sample was used, the only cases in which the judgment value for all mutant probes was 0.05 or less were those in which the v4-1 and v4-2 blocking nucleic acids were used. The judgment values ​​and fluorescence intensity values ​​for each mutant probe calculated using the wild-type model sample or the 5% mutant model sample for the v4-1 and v4-2 blocking nucleic acids are shown in Figures 20 and 21, respectively. As can be seen from Figures 20 and 21, when the judgment values ​​for v4-1 and v4-2 were compared, they were nearly equivalent, but when the fluorescence intensity values ​​for v4-1 and v4-2 were compared, it was found that v4-2 exhibited a higher value. Based on these results, v4-2 was selected as the optimal blocking nucleic acid for gene mutations in the CD79B gene from among those listed in Table 10.

[0128] <Investigation of Blocking Nucleic Acid Concentration> For v4-2, selected as a blocking nucleic acid for gene mutations in the CD79B gene, wild-type model samples or 5% mutant model samples were hybridized with the wild-type probe (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 various concentrations of blocking nucleic acid, and the fluorescence intensity from the wild-type probe and mutant probe was measured, and the judged value was calculated according to the above formula. The results are shown in Figure 22.

[0129] As shown in Figure 22, when the blocking nucleic acid concentration was 125 nM, there were several mutant probes whose judgement value exceeded 0.05 when using a wild-type model sample. These results indicated that a blocking nucleic acid concentration higher than 125 nM is preferable. Furthermore, Figure 23 shows the results of measuring the fluorescence intensity from each mutant probe when using a 5% mutant model sample with a blocking nucleic acid concentration of 500 nM, 750 nM, or 1000 nM. These results indicated that the fluorescence intensity value was lower when the blocking nucleic acid concentration was 1000 nM compared to when the concentration was 500 nM or 750 nM. These results suggest that a blocking nucleic acid concentration of 500 nM or 750 nM is preferable.

[0130] To confirm the above results, the judgment values ​​of each mutant probe when using a wild-type model sample or a 5% mutant model sample at a blocking nucleic acid concentration of 500 nM or 750 nM were calculated. The results are shown in Figure 24. As can be seen from Figure 24, when the blocking nucleic acid concentration was either 500 nM or 750 nM, the judgment values ​​when using the wild-type model sample and the judgment values ​​when using the 5% mutant model sample were sufficiently separated, indicating that the wild-type and each mutant could be accurately detected. In particular, when the blocking nucleic acid concentration was 750 nM, the judgment values ​​when using the wild-type model sample and the judgment values ​​when using the 5% mutant model sample were more widely separated than when the blocking nucleic acid 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, with 500 to 750 nM being preferred. As an example, it was found that the most preferred concentration of the blocking nucleic acid is 750 nM. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

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

2. The method according to claim 1, characterized in that the judgment value is calculated according to the following formula: judgment value = [signal value from the second probe] / [signal value from the wild-type probe + signal value from the second probe], and the gene mutation is judged to be wild-type when the calculated judgment value exceeds a predetermined cutoff value.

3. The method according to claim 1, characterized in that the judgment value is calculated according to the following formula: judgment value = [signal value from the first probe] / [signal value from the wild-type probe + signal value from the first probe], and when the calculated judgment value exceeds a predetermined cutoff value, it is judged that the gene mutation contains a missense mutation and / or a nonsense mutation.

4. The method according to claim 3, characterized in that a judgment value is calculated for each of multiple missense mutations and / or nonsense mutations in the gene mutation using the above formula, and the presence of each of the multiple missense mutations and / or nonsense mutations is judged.

5. The method according to claim 4, characterized in that the genetic mutation is judged to be wild type when the judgment values ​​calculated for each of the multiple missense mutations and / or nonsense mutations are all below the cutoff value.

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

7. The method described in claim 1, characterized in that the genetic mutation is a missense mutation and / or a nonsense mutation occurring in a codon that codes for a specific amino acid, and the first probe corresponds to a missense mutation and / or a nonsense mutation having a base substitution in a codon in which a base substitution resulting in a silent mutation for that amino acid has occurred.

8. The method according to claim 7, characterized in that a plurality of first probes are used 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 for the above amino acid has occurred.

9. The method according to claim 1, characterized in that the genetic mutation comprises a missense mutation and / or a nonsense mutation at tyrosine residue 196 in the CD79B gene and a silent mutation at said tyrosine residue.

10. The method of claim 9, wherein 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.

11. The method according to claim 10, characterized in that 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 mutation, nonsense mutation and silent mutation comprise the base sequences shown in Table 1.

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