Kit for evaluating gene mutations related to prognosis prediction of papillary thyroid carcinoma

Abstract

To inspect simultaneously BRAF V600E gene mutation, TERT 228C>T gene mutation and TERT 250C>T gene mutation.MEANS FOR SOLVING THE PROBLEM: A kit contains BRAF V600E mutation probe, TERT 228C>T mutation probe, TERT 250C>T mutation probe, BRAF primer set, and TERT primer set.SELECTED DRAWING: Figure 6

Description

Technical Field

[0001] The present invention relates to a gene mutation evaluation kit capable of evaluating gene mutations related to the prognosis of papillary thyroid cancer, and a data analysis method for predicting the prognosis of papillary thyroid cancer using the gene mutation evaluation kit.

Background Art

[0002] The annual incidence of thyroid cancer has been increasing year by year. Among thyroid cancers, papillary thyroid cancer accounts for about 90% of thyroid cancers, and its recurrence rate is about 5 - 20%. In recent years, as indicators for predicting the prognosis of papillary thyroid cancer, specific mutations in the BRAF (v-raf murine sarcoma viral oncogene homolog B1) protein, namely the mutation where valine at position 600 becomes glutamic acid (V600E gene mutation), and mutations where cytosine at position 228 becomes thymine (228C>T gene mutation) and cytosine at position 250 becomes thymine (250C>T gene mutation) in the promoter region of the TERT (telomerase reverse transcriptase) gene have attracted attention (Non-Patent Document 1).

[0003] That is, for papillary thyroid cancer patients having the V600E gene mutation in the BRAF protein, the 228C>T gene mutation and the 250C>T gene mutation in the TERT promoter region, it is evaluated that the possibility of recurrence is relatively high, and an appropriate treatment policy can be determined based on this evaluation.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, in order to identify these V600E gene mutations in BRAF proteins, 228C>T gene mutations and 250C>T gene mutations in the TERT promoter region, it is necessary to conduct independent tests respectively, and there are problems such as the cost required for the tests and the inability to make a quick judgment.

[0006] Therefore, in view of the above-mentioned actual situation, the present invention aims to provide a gene mutation evaluation kit capable of simultaneously detecting these V600E gene mutations in BRAF proteins, 228C>T gene mutations and 250C>T gene mutations in the TERT promoter region, and a data analysis method for prognosis prediction of papillary thyroid carcinoma using the same.

Means for Solving the Problems

[0007] The present invention includes the following.

[0008] (1) A BRAF V600E mutant probe that specifically hybridizes to the V600E gene mutation (1799T>A) in BRAF (v-raf murine sarcoma viral oncogene homolog B1), A TERT 228C>T mutant probe that specifically hybridizes to the 228C>T gene mutation in the TERT (telomerase reverse transcriptase) promoter region, and a TERT 250C>T mutant probe that specifically hybridizes to the 250C>T gene mutation in the TERT promoter region, and a primer set for BRAF that amplifies a region containing the V600E gene mutation in the BRAF gene, and a primer set for TERT that amplifies a region containing the 228C>T gene mutation and the 250C>T gene mutation in the TERT gene A kit for evaluating gene mutations related to predicting the prognosis of papillary thyroid carcinoma, comprising

[0009] (2) The primer set for BRAF comprises a forward primer consisting of the nucleotide sequence of SEQ ID NO: 1 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 2. The kit for evaluating gene mutations according to (1) is characterized in that.

[0010] (3) The primer set for TERT comprises a forward primer consisting of the nucleotide sequence of SEQ ID NO: 3 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 4. The kit for evaluating gene mutations according to (1) is characterized in that.

[0011] (4) The primer set for BRAF comprises a forward primer consisting of the nucleotide sequence of SEQ ID NO: 1 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 2, the primer set for TERT comprises a forward primer consisting of the nucleotide sequence of SEQ ID NO: 3 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 4, The kit for evaluating gene mutations according to (1) is characterized in that the concentration of the primer set for TERT is 0.2 μM or more and 0.6 μM or less.

[0012] (5) The gene mutation evaluation kit according to (4), characterized in that the concentration ratio of the above-mentioned BRAF primer set and the above-mentioned TERT primer set is (BRAF primer set concentration):(TERT primer set concentration)=4:1 to 2:1.

[0013] (6) The gene mutation evaluation kit according to (1), further comprising a nucleic acid fragment for blocking that hybridizes to a gene mutation other than V600E for valine (V600) at the 600th position in BRAF.

[0014] (7) The gene mutation evaluation kit according to (6), characterized in that the gene mutation other than the above-mentioned V600E is at least one gene mutation selected from the group consisting of V600D gene mutation, V600K gene mutation, V600R gene mutation, V600G gene mutation, and V600M gene mutation.

[0015] (8) The gene mutation evaluation kit according to (6), characterized in that the above-mentioned nucleic acid fragment for blocking specifically hybridizes to a V600D gene mutation or a V600K gene mutation.

[0016] (9) The gene mutation evaluation kit according to (1), further comprising a common probe for BRAF that hybridizes to both the wild-type BRAF fragment amplified by the above-mentioned BRAF primer set and the mutant BRAF fragment having a mutation at the position corresponding to valine at the 600th position.

[0017] (10) The gene mutation evaluation kit according to (1), further comprising a common probe for TERT that hybridizes to both the wild-type TERT fragment amplified by the above-mentioned TERT primer set and the mutant TERT fragment having a mutation at the position corresponding to cytosine at the 228th position and / or cytosine at the 250th position.

[0018] The gene mutation evaluation kit according to (1), comprising a microarray in which the above-mentioned BRAF V600E mutant probe, the above-mentioned TERT 228C>T mutant probe, and the above-mentioned TERT 250C>T mutant probe are immobilized on a carrier.

[0019] (12) A hybridization buffer composition for a target nucleic acid having a predetermined gene mutation and a mutation detection probe that specifically hybridizes to the target nucleic acid, The hybridization buffer composition contains a blocking nucleic acid fragment that preferentially hybridizes to a non-target nucleic acid having a gene mutation other than the predetermined gene mutation in the target nucleic acid as compared to the target nucleic acid.

[0020] (13) The hybridization buffer composition according to (12), wherein the blocking nucleic acid fragment has a base sequence complementary to the non-target nucleic acid.

[0021] (14) The target nucleic acid is a nucleic acid fragment containing the V600E gene mutation (1799T>A) in BRAF (v-raf murine sarcoma viral oncogene homolog B1), The mutation detection probe is a BRAF V600E mutant probe that specifically hybridizes to the nucleic acid fragment, The non-target nucleic acid is a nucleic acid fragment containing a V600D gene mutation or a V600K gene mutation, The hybridization buffer composition according to (12), wherein the blocking nucleic acid fragment preferentially hybridizes to a nucleic acid fragment containing a V600D gene mutation or a nucleic acid fragment containing a V600K gene mutation.

[0022] (15) Using the gene mutation evaluation kit according to any one of (1) to (11) above, for a subject to be diagnosed, simultaneously identify the V600E gene mutation in BRAF (v-raf murine sarcoma viral oncogene homolog B1), the 228C>T gene mutation and the 250C>T gene mutation in the TERT (telomerase reverse transcriptase) promoter region, a data analysis method for predicting the prognosis of papillary thyroid cancer.

[0023] (16) The gene mutation evaluation kit is a microarray comprising a mutant probe related to each of the above gene mutations and a common probe that hybridizes to both the mutant type having the gene mutation and the wild type having no mutation. Using the microarray, measure the signals derived from the mutant probe and the common probe, and calculate a determination value for each gene mutation according to the formula: [signal intensity of the mutant probe] / [signal intensity of the common probe]. When the determination value exceeds a preset cut-off value, it is determined that the above gene mutation is present. The data analysis method according to (15).

Advantages of the Invention

[0024] According to the gene mutation evaluation kit according to the present invention, three gene mutations related to the prognosis prediction of papillary thyroid cancer can be simultaneously examined. Therefore, by using the gene mutation evaluation kit according to the present invention, the prognosis prediction of papillary thyroid cancer can be performed at low cost and quickly.

[0025] In addition, the data analysis method for predicting the prognosis of papillary thyroid cancer according to the present invention uses a gene mutation evaluation kit that can simultaneously examine three gene mutations related to the prognosis prediction of papillary thyroid cancer, so that the prognosis prediction of papillary thyroid cancer can be performed at low cost and quickly.

Brief Description of the Drawings

[0026]

Figure 1

Figure 2

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Figure 8

Embodiments for Carrying Out the Invention

[0027] The kit for gene mutation evaluation for prognosis prediction of papillary thyroid carcinoma according to the present invention relates to the V600E gene mutation in the BRAF protein, the 228C>T gene mutation and the 250C>T gene mutation in the TERT promoter region. These gene mutations in BRAF and TERT are gene mutations that are said to be related to the prognosis (recurrence) of papillary thyroid carcinoma in Scientific Reports | 7:41752 | DOI: 10.1038 / srep41752 (2017).

[0028] The kit for gene mutation evaluation according to the present invention includes a BRAF V600E mutant probe for identifying the V600E gene mutation in the BRAF protein, a TERT 228C>T mutant probe for identifying the 228C>T gene mutation in the TERT promoter region, and a TERT 250C>T mutant probe for identifying the 250C>T gene mutation in the TERT promoter region. Further, the kit for gene mutation evaluation according to the present invention includes a primer set for BRAF that amplifies a predetermined region containing the V600E gene mutation, and a primer set for TERT that amplifies a predetermined region containing the 228C>T gene mutation and the 250C>T gene mutation. That is, when using the kit for gene mutation evaluation according to the present invention, the V600E gene mutation contained in the nucleic acid fragment amplified using the primer set for BRAF can be identified with the BRAF V600E mutant probe. Also, when using the kit for gene mutation evaluation according to the present invention, the 228C>T gene mutation and the 250C>T gene mutation contained in the nucleic acid fragment amplified using the primer set for TERT can be identified with the TERT 228C>T mutant probe and the TERT 250C>T mutant probe, respectively.

[0029] Figure 1 shows a part of the BRAF gene (SEQ ID NO: 5) containing the V600E gene mutation. As shown in Figure 1, in the V600E gene mutation, with the first A of the start codon (ATG) (not shown in Figure 1), the 1799th T becomes A, resulting in the 600th valine from the N-terminus of the BRAF protein mutating to glutamic acid. Therefore, the BRAF primer set is designed as a forward primer and a reverse primer to amplify the region containing this 1799th base. As an example of the BRAF primer set, a forward primer consisting of the nucleotide sequence of SEQ ID NO: 1 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 2 can be mentioned.

[0030] However, the BRAF primer set is not limited to the set consisting of the forward primer consisting of the nucleotide sequence of SEQ ID NO: 1 and the reverse primer consisting of the nucleotide sequence of SEQ ID NO: 2, and can be appropriately designed based on the part of the BRAF gene shown in Figure 1. For example, as the forward primer, in the part of the BRAF gene shown in Figure 1, the forward primer can be designed at a position shifted several bases, for example 20 bases, preferably 10 bases, more preferably 5 bases, in the 5' side (upstream) direction from the nucleotide sequence of SEQ ID NO: 1. Similarly, as the reverse primer, in the part of the BRAF gene shown in Figure 1, the reverse primer can be designed at a position shifted several bases, for example 20 bases, preferably 10 bases, more preferably 5 bases, in the 5' side (upstream) or 3' side (downstream) direction from the nucleotide sequence of SEQ ID NO: 2.

[0031] In addition, FIG. 2 shows a part of the TERT promoter region (SEQ ID NO: 6) containing the 228C>T gene mutation and the 250C>T gene mutation. As shown in FIG. 2, the 228C>T gene mutation and the 250C>T gene mutation are mutations in which the 228th C is changed to T and the 250th C is changed to T when counted from the transcription start point of the TERT gene towards the upstream on the 5'-terminal side. Therefore, the TERT primer set is designed as a forward primer and a reverse primer so as to amplify the region including these 228th and 250th positions. As an example of the TERT primer set, a forward primer consisting of the nucleotide sequence of SEQ ID NO: 3 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 4 can be mentioned.

[0032] However, the TERT primer set is not limited to a set consisting of a forward primer consisting of the nucleotide sequence of SEQ ID NO: 3 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 4, and can be appropriately designed based on a part of the TERT promoter region shown in FIG. 2. For example, as the forward primer, in a part of the TERT promoter region shown in FIG. 2, the forward primer may be designed at a position shifted by several bases, for example, 10 bases, preferably 5 bases, in the 5'-side (upstream) or 3'-side (downstream) direction from the nucleotide sequence of SEQ ID NO: 3. Similarly, as the reverse primer, in a part of the TERT promoter region shown in FIG. 2, the reverse primer may be designed at a position shifted by several bases, for example, 10 bases, preferably 5 bases, in the 5'-side (upstream) or 3'-side (downstream) direction from the nucleotide sequence of SEQ ID NO: 4.

[0033] The BRAF primer set and the TERT primer set according to the present invention designed as described above can simultaneously amplify the target nucleic acid fragments by a nucleic acid amplification reaction using the genomic DNA of the subject as a template.

[0034] In addition, the nucleic acid fragments amplified by the BRAF primer set and the TERT primer set according to the present invention designed as described above can be detected by a probe (common probe) designed to hybridize regardless of the presence or absence of gene mutations to be detected. More specifically, the nucleic acid fragments amplified by the BRAF primer set and the TERT primer set can be detected based on the fluorescence intensity in the common probe by binding a fluorescent labeling compound to either the forward primer or the reverse primer.

[0035] Here, as the common probe for BRAF for detecting the nucleic acid fragment amplified by the BRAF primer set, it is designed to hybridize to a region that does not contain the codon encoding valine at position 600 in the BRAF protein. As an example, in a part of the BRAF gene shown in FIG. 1, GGTCCCATCAGTTTGAAC (SEQ ID NO: 7) can be designed as the common probe for BRAF from the region sandwiched between the forward primer consisting of the nucleotide sequence of SEQ ID NO: 1 and the reverse primer consisting of the nucleotide sequence of SEQ ID NO: 2.

[0036] As the common probe for TERT for detecting the nucleic acid fragment amplified by the TERT primer set, it is designed to hybridize to a region that does not contain the 228th and 250th bases in the TERT promoter region. As an example, in a part of the TERT promoter region shown in FIG. 2, ACGGGGCGGGGTCCG (SEQ ID NO: 8) can be designed as the common probe for TERT from the region sandwiched between the forward primer consisting of the nucleotide sequence of SEQ ID NO: 3 and the reverse primer consisting of the nucleotide sequence of SEQ ID NO: 4.

[0037] In particular, in the kit for gene mutation evaluation according to the present invention, the concentration of the primer set for TERT is preferably 0.2 μM to 0.6 μM, and more preferably 0.4 μM. By performing a nucleic acid amplification reaction with the primer set for TERT at a concentration within this range together with the primer set for BRAF, the obtained nucleic acid fragment can be detected with high precision without variation in fluorescence intensity by the above-described common probe for BRAF and common probe for TERT.

[0038] In particular, it is preferable that the concentration of the primer set for TERT is within the above range, and the concentration ratio of the primer set for BRAF and the primer set for TERT is (concentration of primer set for BRAF):(concentration of primer set for TERT) = 4:1 to 2:1. By setting the concentration of the primer set for TERT within the above range and setting the concentration ratio of the primer set for BRAF and the primer set for TERT within this range, the variation in fluorescence intensity of the amplified fragment obtained by the nucleic acid amplification reaction can be further suppressed by the above-described common probe for BRAF and common probe for TERT, and it can be detected with even higher precision.

[0039] On the other hand, in the kit for gene mutation evaluation according to the present invention, as the BRAF V600E mutant probe that specifically hybridizes to the V600E gene mutation (1799T>A), it can be designed as a probe having a base sequence for detecting the 1799th base A described above. In other words, the BRAF V600E mutant probe is designed to hybridize with the amplified nucleic acid when the base to be detected (the 1799th base) in the amplified fragment amplified by the above-described primer set for BRAF is A.

[0040] In addition, in the kit for gene mutation evaluation according to the present invention, as the TERT 228C>T mutant probe that specifically hybridizes to the 228C>T gene mutation, it can be designed as a probe having a base sequence that detects the 228th base T described above. In other words, the TERT 228C>T mutant probe is designed to hybridize with the amplified nucleic acid when the base to be detected (the 228th base) in the amplified fragment amplified by the above-described primer set for TERT is T.

[0041] Furthermore, in the kit for gene mutation evaluation according to the present invention, as the TERT 250C>T mutant probe that specifically hybridizes to the 250C>T gene mutation, it can be designed as a probe having a base sequence that detects the 250th base T described above. In other words, the TERT 250C>T mutant probe is designed to hybridize with the amplified nucleic acid when the base to be detected (the 250th base) in the amplified fragment amplified by the above-described primer set for TERT is T.

[0042] The base length of these probes is not particularly limited, but for example, it can be 10 to 40 bases long, and preferably 15 to 35 bases long. Note that, as described above, the probe is the sum of the base sequence designed based on the region containing the gene mutation for detection and the base sequence added to one or both ends of the base sequence, and can be, for example, 10 to 40 bases long, and preferably 20 to 35 bases long.

[0043] In addition, the probe designed as described above 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 using 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.

[0044] Incidentally, the kit for gene mutation evaluation according to the present invention preferably further includes a blocking nucleic acid fragment that hybridizes to gene mutations other than V600E for valine (V600) at position 600 in BRAF. The blocking nucleic acid has a base sequence complementary to the base sequence of a nucleic acid fragment having a gene mutation other than V600E among the nucleic acid fragments amplified by the BRAF primer set. Examples of gene mutations other than V600E include V600D gene mutation, V600K gene mutation, V600R gene mutation, V600G gene mutation, and V600M gene mutation.

[0045] As the blocking nucleic acid fragment, a plurality of types may be prepared to correspond to all of these gene mutations other than V600E, or a plurality of types may be prepared to correspond to some of the gene mutations selected from these gene mutations other than V600E. In particular, as the blocking nucleic acid fragment, it is preferable to use a blocking nucleic acid fragment corresponding to the V600D gene mutation and / or a blocking nucleic acid fragment corresponding to the V600K gene mutation.

[0046] As described above, since the kit for gene mutation evaluation according to the present invention includes a blocking nucleic acid corresponding to a gene mutation other than V600E, even when an amplified nucleic acid having a gene mutation other than V600E is obtained by the BRAF primer set, non-specific hybridization between the amplified nucleic acid having a gene mutation other than V600E and the BRAF V600E mutant probe can be suppressed, and it is possible to prevent the specific hybridization between the amplified nucleic acid having the V600E gene mutation and the BRAF V600E mutant probe from being inhibited. Therefore, by using the blocking nucleic acid corresponding to the gene mutation other than V600E, the presence or absence of the V600E gene mutation can be detected with high accuracy.

[0047] In the kit for gene mutation evaluation according to the present invention, as described above, the BRAF V600E mutant probe, TERT 228C>T mutant probe, and TERT 250C>T mutant probe designed as such are preferably used in the form of a microarray (for example, a DNA chip) by immobilizing their 5'-ends on a carrier.

[0048] At this time, the microarray preferably has a mutant probe and the above-described common probe for each of the above-described gene mutations. By using the mutant probe and the common probe for each gene mutation, it is possible to accurately determine not only the presence or absence of the mutation but also the ratio of the mutation.

[0049] 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.

[0050] In the present invention, as the carrier, a carrier preferably having a carbon layer and a chemical modification group on its surface is used. Carriers having a carbon layer and a chemical modification group on their surface include those having a carbon layer and a chemical modification group on the surface of a substrate, and those having a chemical modification 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-mentioned carrier materials can be used.

[0051] In the microarray according to the present invention, 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, those with a size of 1 to 75 mm square, preferably 1 to 10 mm square, and more preferably 3 to 5 mm square are 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 preferable is a carrier having a carbon layer and a chemical modification group on the surface of a substrate made of single crystal silicon. 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).

[0052] In the present invention, the carbon layer formed on the substrate is not particularly limited, but 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), a mixture thereof, or a laminate thereof is preferably used. Further, 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 may 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 the mixing ratio thereof is not particularly limited. The carbon layer is excellent in chemical stability and can withstand reactions in the 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 advantageous in that it is transparent to the detection system UV because it has no UV absorption, and it can be energized during electroblotting. Further, 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.

[0053] In the present invention, 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, ionization evaporation method, arc evaporation method, laser evaporation method, EB (Electron beam) evaporation method, resistance heating evaporation method, and the like can be mentioned.

[0054] 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.

[0055] 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) to generate a plasma of carbon atoms from the cathode, and a carbon layer can be formed by applying a bias voltage more negative than the evaporation source to the substrate to accelerate carbon ions in the plasma toward the substrate.

[0056] 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.

[0057] 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. On the contrary, if it is too thick, the productivity deteriorates. Therefore, it is preferably 2 nm to 1 μm, more preferably 5 nm to 500 nm.

[0058] By introducing a chemical modification group onto the surface of the substrate on which the carbon layer is formed, an oligonucleotide 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.

[0059] 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 of polyamines.

[0060] 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 compound used for introducing 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, 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, and 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 and malonic acid monochloride; and acid anhydrides such as phthalic anhydride, succinic anhydride, oxalic anhydride, maleic anhydride, and butanetetracarboxylic anhydride.

[0061] 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.

[0062] The introduction of formyl groups can be carried out, for example, by reacting glutaraldehyde with the carbon layer aminated as described above.

[0063] The introduction of hydroxyl groups can be carried out, for example, by reacting water with the carbon layer chlorinated as described above.

[0064] 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 being more activated than alkyl esters. 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.

[0065] 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 can be formed via an amide bond (Japanese Patent Application Laid-Open No. 2001-139532).

[0066] 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 produced. Alternatively, the spotting solution may be manually spotted with a micropipettor.

[0067] After spotting, it is preferable to perform incubation in order to allow the reaction in which the probe binds to 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 under an atmosphere of high humidity, for example, under conditions of a humidity of 50 to 90%. Subsequently to the incubation, 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.) to remove the DNA that has not bound to the carrier.

[0068] By using the microarray configured as described above, it is possible to simultaneously determine the presence or absence of each of the gene mutations present in BRAF and TERT in a subject to be diagnosed.

[0069] Specifically, when determining the presence or absence of the above gene mutations in BRAF and TERT, it includes a step of extracting DNA from a sample derived from a subject to be diagnosed, a step of using the extracted DNA as a template and amplifying a partial region of BRAF containing the above gene mutation and a TERT promoter partial region using a primer set for BRAF and a primer set for TERT, respectively, and a step of detecting the presence or absence of the above gene mutations in BRAF and TERT contained in the amplified nucleic acid using the above-described microarray, respectively.

[0070] The subject to be diagnosed is a normal human and is not particularly limited by race or the like. In particular, it is a yellow race, preferably an East Asian race, and particularly preferably a Japanese. Also, the subject to be diagnosed can be a patient suspected of having papillary thyroid carcinoma.

[0071] The measurement sample derived from the subject to be diagnosed uses thyroid tissue.

[0072] 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.

[0073] Next, an amplification reaction is performed using the obtained DNA as a template to amplify the region containing the V600E gene mutation, the 228C>T gene mutation and the 250C>T gene mutation in the TERT promoter region. 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.

[0074] This reaction system is a reaction system containing a buffer, a heat-resistant DNA polymerase, the above-described primer sets for BRAF and TERT, primers specific to the amplification region, labeled nucleotide triphosphates (specifically, nucleotide triphosphates with a fluorescent label, etc.), nucleotide triphosphates, and magnesium chloride, etc., which are necessary for nucleic acid amplification and labeling.

[0075] A hybridization reaction is performed between the amplified nucleic acid obtained as described above and the BRAF V600E mutant probe, TERT 228C>T mutant probe, and TERT 250C>T mutant probe immobilized on the carrier, and the presence or absence of the above gene mutations in the subject to be diagnosed can be evaluated by detecting the hybridization of the amplified nucleic acid to the mutant probe. That is, the hybridization of the amplified nucleic acid to the mutant probe can be measured, for example, by detecting a label.

[0076] The signal from the label can be quantified by detecting the fluorescence signal using a fluorescence scanner and analyzing it with image analysis software when, for example, 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 at 50°C for 16 hours, washing is carried out under the conditions of 2×SSC / 0.2% SDS, 25°C, 10 minutes and 2×SSC, 25°C, 5 minutes. Alternatively, the hybridization temperature can be 45 to 60°C when the salt concentration is 0.75×SSC. When the probe has a short chain length, it is more preferable to set the hybridization temperature lower than this, and when the chain length is long, it is more preferable to set the hybridization temperature higher than this. It goes without saying that as the salt concentration increases, the hybridization temperature with specificity increases, and conversely, as the salt concentration decreases, the hybridization temperature with specificity decreases.

[0077] Also, when using a microarray equipped with a mutant probe and a common probe for each of the above-described gene mutations, the presence or absence of the above gene mutations can be evaluated using the signal intensities from these mutant probes and common probes. Specifically, the signal intensity of the common probe and the signal intensity of the mutant probe are measured respectively, and a determination value for evaluating the signal intensity derived from the mutant probe is calculated. As an example of calculating the determination value, for example, a method using the formula: [signal intensity derived from the mutant probe] / [signal intensity derived from the common probe] can be mentioned.

[0078] Then, the determination value calculated by the above formula is compared with a predetermined threshold value (cut-off value). When the determination value exceeds the threshold value, it is determined that the amplified nucleic acid contains the above gene mutation, and when the determination value is below the threshold value, it is determined that the amplified nucleic acid does not contain the above gene mutation. By using the determination value in this way, the presence or absence of each gene mutation in the above-described BRAF and TERT promoter regions can be determined.

[0079] Here, the threshold value is not particularly limited. For example, it can be defined based on the determination value calculated by the above formula using a specimen that has been confirmed to be a wild type without the V600E gene mutation, the 228C>T gene mutation, and the 250C>T gene mutation. More specifically, a plurality of determination values can be calculated using a plurality of specimens that have been confirmed to be wild types without the V600E gene mutation, the 228C>T gene mutation, and the 250C>T gene mutation, and the value of the average value + 6σ (σ: standard deviation) can be used as the threshold value. In addition, the values of the average value + 3σ, the average value + 2σ, or the average value + σ can also be used as the threshold value.

[0080] As described above, by simultaneously detecting the V600E gene mutation in BRAF, the 228C>T gene mutation in the TERT promoter region, and the 250C>T gene mutation, it is possible to obtain data that can be used for predicting the prognosis of papillary thyroid cancer. That is, when all of these V600E gene mutation in BRAF, 228C>T gene mutation in the TERT promoter region, and 250C>T gene mutation are present, it can be determined that the possibility of recurrence of papillary thyroid cancer is relatively high.

[0081] Incidentally, as described above, when identifying the V600E gene mutation in BRAF, it was explained that the use of a blocking nucleic acid can detect the gene mutation with high precision. This is because it suppresses the non-specific hybridization between a nucleic acid fragment having another gene mutation that is very similar in nucleotide sequence, such as the V600D gene mutation or the V600K gene mutation, which is different from the V600E gene mutation, and the BRAF V600E mutant probe.

[0082] That is, for example, when there are multiple types of mutations at a predetermined position in a protein and a specific mutation is to be detected, it can be said that it is preferable to use a blocking nucleic acid fragment corresponding to a mutation other than the specific mutation. Therefore, in a hybridization buffer composition of a target nucleic acid having a predetermined gene mutation and a mutation detection probe that specifically hybridizes to the target nucleic acid, a blocking nucleic acid fragment that preferentially hybridizes to a non-target nucleic acid having a gene mutation other than the predetermined gene mutation in the target nucleic acid, as compared with the target nucleic acid, is preferably included.

[0083] Such a blocking nucleic acid fragment is designed to have a base sequence complementary to the non-target nucleic acid. In addition, it can be designed based on the same concept as in the case of designing a probe. For example, the base length can be 10 to 40 bases, preferably 15 to 35 bases.

[0084] In addition, other gene mutations other than the predetermined gene mutation to be detected may be present in the nucleic acid fragment amplified for detecting the predetermined gene mutation. When designing a probe for detecting a predetermined gene mutation, it is useful to use a blocking nucleic acid fragment when other gene mutations may be present within the range where the probe hybridizes. In particular, it is highly useful when the base sequences of the predetermined gene mutation and other gene mutations partially overlap. Further, when detecting the presence or absence of a gene mutation at a lesion site, it is preferable to position the wild type corresponding to the gene mutation as a non-target nucleic acid and include a blocking nucleic acid fragment for the wild type so as to obtain high detection sensitivity even when the mutation ratio is small.

[0085] Thus, by using a hybridization buffer composition containing a blocking nucleic acid fragment, non-specific hybridization between the non-target nucleic acid and the mutation detection probe can be prevented, and the target nucleic acid can be detected with high precision based on specific hybridization between the target nucleic acid and the mutation detection probe.

[0086] Here, in the hybridization buffer composition according to the present invention, the concentration of the blocking nucleic acid is not particularly limited, but it is preferably 1-fold or more the nucleic acid concentration of the nucleic acid mixture composed of the amplified target nucleic acid and non-target nucleic acids. For example, when obtaining a target nucleic acid containing a base to be detected by a nucleic acid amplification reaction such as a polymerase chain reaction, it is preferable to adjust the amount of the blocking nucleic acid so that the concentration is 1-fold or more the concentration of the nucleic acid mixture composed of the amplified target nucleic acid and non-target nucleic acids.

[0087] By setting the concentration of the blocking nucleic acid within the above range, non-specific hybridization between the non-target nucleic acid and the mutation detection probe can be more effectively suppressed, and specific hybridization between the target nucleic acid and the mutation detection probe can be detected with high sensitivity.

[0088] On the other hand, in the hybridization buffer composition according to the present invention, the upper limit of the concentration range of the blocking nucleic acid is not particularly limited, but for example, it is preferably not more than the concentration of the added primer set.

[0089] The nucleic acid concentration of the nucleic acid mixture can be measured according to a conventional method. For example, after purifying the reaction solution after the nucleic acid amplification reaction, the absorbance at a wavelength of 260 nm is measured using a spectrophotometer, and the measured value is converted into the nucleic acid concentration. Thereby, the nucleic acid concentration of the nucleic acid mixture obtained by the nucleic acid amplification reaction can be measured. Also, the nucleic acid concentration of the nucleic acid mixture can be measured by intercalating the amplification product with a fluorescent dye such as SYBR Gold or Pico Green and measuring the absorbance near 600 nm. Alternatively, the nucleic acid concentration of the nucleic acid mixture can be measured by detecting the electrophoresis band of the amplification product by electrophoresis and comparing it with the electrophoresis band of a nucleic acid with a known concentration.

[0090] As described above, since the hybridization buffer composition according to the present invention contains a blocking nucleic acid corresponding to a gene mutation different from the gene mutation to be detected, it is possible to suppress non-specific hybridization between a non-target nucleic acid containing a gene mutation different from the gene mutation to be detected and a nucleic acid probe, and prevent the specific hybridization between a target nucleic acid containing the gene mutation to be detected and the nucleic acid probe from being inhibited. Therefore, by using the hybridization buffer composition according to the present invention, even when there are multiple types of mutations at a predetermined position, for example, a target nucleic acid having a mutation for detection purposes can be detected with high accuracy by a nucleic acid probe.

[0091] Specifically, the gene mutation having multiple types of mutations at a predetermined position is not limited to the substitution mutation of the 600th valine in BRAF described above. For example, substitution mutations of the 12th glycine in KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog), such as G12A, G12C, G12D, G12R, G12S, G12V, etc. can be mentioned.

Example

[0092] Hereinafter, the present invention will be described in more detail by way of examples, but the technical scope of the present invention is not limited thereto.

[0093] [Example 1] In this example, primers were designed that can simultaneously amplify the region having the V600E gene mutation in BRAF and the regions having the 228C>T gene mutation and the 250C>T gene mutation in the TERT promoter region. The nucleic acid amplification reaction for amplifying each of these regions and the method of detecting the nucleic acid fragments obtained by the nucleic acid amplification reaction with a DNA chip are as described in the [Experiment] section below.

[0094] In particular, in this example, as shown in Table 1, the following primer sets A to E were designed to amplify the region having the V600E gene mutation in BRAF, and as shown in Table 2, the following primer sets F to I were designed to amplify the regions having the 228C>T gene mutation and the 250C>T gene mutation in the TERT promoter region.

[0095]

Table 1

[0096]

Table 2

[0097] First, the results of performing nucleic acid amplification reactions using the primer sets A to E and the primer sets F to I alone are shown in Figure 3. As shown in Figure 3, all of the primer sets A to E were able to amplify the target nucleic acid fragment, and among the primer sets F to I, only the primer sets H and I were able to amplify the target nucleic acid fragment.

[0098] Next, the results of performing nucleic acid amplification reactions using the primer sets A to E and the primer sets H and I alone, and detecting the amplified nucleic acid fragments with a common probe for BRAF and a common probe for TERT are shown in Figure 4. As shown in Figure 4, the most excellent fluorescence intensity was observed when using primer set B for BRAF and primer set H for the TERT promoter region. From these results, it became clear that it is preferable to amplify the region having the V600E gene mutation in BRAF by the nucleic acid amplification reaction using primer set B, and to amplify the regions having the 228C>T gene mutation and the 250C>T gene mutation in the TERT promoter region by the nucleic acid amplification reaction using primer set H.

[0099] Next, in this example, the concentrations of the primer sets were examined to highly accurately detect two target regions by simultaneous nucleic acid amplification reactions using these primer sets B and H. The results are shown in FIG. 5. FIG. 5 shows the respective concentrations of primer set B and primer set H in the nucleic acid amplification reaction. As shown in FIG. 5, by setting the concentration of primer set H to 0.4 μM or more and 0.6 μM or less, and setting the concentration ratio of primer set B and primer set H to (primer set B):(primer set H) = 4:1 to 2:1, it became clear that the two regions can be amplified simultaneously in a well-balanced manner to approximately the same extent.

[0100] [Example 2] In this example, using primer sets B and H designed in Example 1, it was examined whether a sample containing only wild type (wild type sample) and a sample containing 5% gene mutation (5% mutation sample) could be detected with high accuracy. Also in this example, the nucleic acid amplification reaction and the method of detecting the nucleic acid fragments obtained by the nucleic acid amplification reaction with a DNA chip are as described in the [Experiment] section below.

[0101] In this example, the wild type sample and the 5% mutation sample were prepared as follows. For the wild type sample, wild type genomic DNA derived from a cell line was purchased, its concentration was measured with a fluorometer, and then it was prepared to 2 ng / μL with TE buffer (pH 8.0, Tris-EDTA) and used. The 5% mutation sample for BRAF was prepared by purchasing 50% V600E mutant genomic DNA derived from a cell line, preparing it to 2 ng / μL in the same manner as above, and further mixing it with the wild type sample for BRAF (2 ng / μL) at a ratio of 9:1 (wild type genomic DNA:50% V600E mutant genomic DNA).

[0102] For the TERT 5% mutant sample, artificial genes (plasmid DNA) with BRAF wild-type sequences, plasmid DNA with TERT wild-type sequences, and plasmid DNA with TERT mutant sequences (including both 228C>T gene mutations and 250C>T gene mutations) were purchased separately. After measuring the concentration with a fluorometer, they were adjusted to 1320 copies / μL (equivalent to 4 ng / μL of genomic DNA) with TE buffer (pH 8.0, Tris-EDTA). Furthermore, a solution was prepared by mixing plasmid DNA with TERT wild-type sequences and plasmid DNA with TERT mutant sequences at a ratio of 95:5, and mixing it in equal amounts with plasmid DNA with BRAF wild-type sequences (1320 copies / μL) (660 copies / μL for each target gene (equivalent to 2 ng / μL of genomic DNA)).

[0103] The results are shown in Figure 6. As shown in Figure 6, it was clarified that by using the primer sets B and H designed in Example 1, wild-type samples and 5% mutant samples can be clearly distinguished and detected with high precision.

[0104] [Example 3] In this example, samples containing gene mutations other than the V600E gene mutation in BRAF were prepared, and the effectiveness of nucleic acid fragments for blocking corresponding to these other gene mutations was evaluated. Also in this example, the nucleic acid amplification reaction and the method of detecting the nucleic acid fragments obtained by the nucleic acid amplification reaction with a DNA chip are as described in the [Experiment] section below.

[0105] In this example, wild-type samples and mutant samples were prepared as follows. In this example, as mutant samples, samples containing 5% V600E gene mutation (5% V600E), samples containing 50% V600R gene mutation (50% V600R), samples containing 50% V600K gene mutation (50% V600K), samples containing 50% V600G gene mutation (50% V600G), samples containing 50% V600M gene mutation (50% V600M), and samples containing 50% V600D gene mutation (50% V600D) were prepared.

[0106] For the wild-type specimen and the 5% V600E mutant specimen, those prepared in Example 2 were used. For the 50% V600R mutant specimen, 50% V600K mutant specimen, 50% V600G mutant specimen, and 50% V600M mutant specimen, each 50% mutant genomic DNA derived from cell lines was purchased individually. After measuring the concentration with a fluorometer, those prepared to 2 ng / μL with TE buffer (pH 8.0, Tris-EDTA) were used respectively.

[0107] For the 50% V600D mutant specimen, plasmid DNA having the BRAF wild-type sequence, plasmid DNA having the V600D mutant sequence, and plasmid DNA having the TERT wild-type sequence were purchased individually. After measuring the concentration with a fluorometer, they were prepared to 1320 copies / μL (equivalent to 4 ng / μL of genomic DNA) with TE buffer (pH 8.0, Tris-EDTA). Further, a solution in which plasmid DNA having the BRAF wild-type sequence and plasmid DNA having the V600D mutant sequence were mixed at a ratio of 1:1 was mixed in equal amounts with plasmid DNA having the TERT wild-type sequence (1320 copies / μL) (660 copies / μL for each target gene (equivalent to 2 ng / μL of genomic DNA)).

[0108] First, the calculation results of the determination values when only the nucleic acid fragment for blocking corresponding to the wild type was added to the reaction solution after the nucleic acid amplification reaction are shown in FIG. 7. As shown in FIG. 7, among the different gene mutations at the same position as the V600E gene mutation, specimens containing the V600D gene mutation and the V600K hereditary mutation were determined to be positive. This was considered to be the result of non-specific hybridization of the amplified nucleic acid containing the V600D gene mutation or the V600K hereditary mutation with the BRAF V600E mutant probe.

[0109] Therefore, in addition to the nucleic acid fragment for blocking corresponding to the wild type, a nucleic acid fragment for blocking corresponding to the V600D gene mutation and a nucleic acid fragment for blocking corresponding to the V600K hereditary mutation were used to calculate the determination value in the same manner for the reaction solution after the nucleic acid amplification reaction. The results are shown in FIG. 8. As shown in FIG. 8, non-specific hybridization between the amplified nucleic acid containing the V600D gene mutation or the V600K hereditary mutation and the BRAF V600E mutant probe was prevented by the nucleic acid fragment for blocking, and as a result, it was clarified that a sample containing the V600E gene mutation could be detected with high accuracy.

[0110] [Experiment] The reaction solution composition of the nucleic acid amplification reaction is shown in Table 3, and the conditions of the nucleic acid amplification reaction are shown in Table 4.

[0111]

Table 3

[0112]

Table 4

[0113] The amplification product obtained by the nucleic acid amplification reaction and the hybridization buffer were mixed at a volume ratio of 2:1 respectively. As the hybridization buffer, a solution prepared to have a concentration of 2.25×SSC / 0.225% SDS was used.

[0114] Approximately 4 μL of the obtained mixed solution was dropped onto the hybridization cover and covered with the DNA chip. Here, the probes immobilized on the DNA chip are summarized in Tables 5 and 6. Here, regarding the probes for TERT, the 228C>T mutant probe 1 corresponds to a sequence having only the 228C>T gene mutation, and the 250C>T mutant probe 1 corresponds to a sequence having only the 250C>T gene mutation. For each of the detection target mutations of the 228C>T gene mutation and the 250C>T gene mutation, there are mutations that are not detection targets in the vicinity, and (unlike BRAF), there may be cases where both the detection target mutation and the non-detection target mutation are present. When the detection target mutation and the non-detection target mutation coexist, hybridization with the above probes is less likely to occur and accurate detection cannot be achieved. Therefore, for the 228C>T mutant probe 2 and the 250C>T mutant probe 2, the base sequences are adjusted so that the detection target mutation can be detected even if there is a non-detection target mutation within the same amplification product.

[0115]

Table 5

[0116]

Table 6

[0117] Thereafter, it was placed in a preheated bath for the DNA chip, transferred to a hybridization oven, and a hybridization reaction was carried out at 58°C for 60 minutes. After the reaction was completed, the hybridization cover was quickly removed, and the DNA chip was washed with a washing solution (1×SSC / 0.1% SDS solution, solution temperature: 20 - 30°C) for 5 minutes. Thereafter, it was rinsed with a 1×SSC solution.

[0118] The fluorescence intensity on the DNA chip was measured using a fluorescence detector, and a determination value was calculated based on the obtained fluorescence intensity. Determination value = Fluorescence intensity of mutant probe DNA / Fluorescence intensity of common probe DNA Fluorescence intensity = Average value of (fluorescence intensity of each spot - background value) (n = 2) When the obtained determination value was greater than the cut-off value, it was determined as mutation positive. Cut-off value = Average value of determination values of wild-type specimens + coefficient × standard deviation (n = 20 or more) Regarding TERT, a mutation was determined to be positive when at least one of Probe 1 or Probe 2 was greater than the cut-off value.

[0119] When using a nucleic acid fragment for blocking, in the hybridization buffer, the blocker for BRAF wild type, the blocker for V600D, and the blocker for V600K were added at 225 μM, and the TERT wild type blockers (C228T) and TERT wild type blockers (C250T) were added at 300 μM. The nucleic acid fragments used for blocking are summarized in Table 7.

[0120]

Table 7

Claims

1. For BRAF (v-raf murine sarcoma viral oncogene homolog B1), a probe set for BRAF comprising a common probe for BRAF consisting of the nucleotide sequence of SEQ ID NO: 23 and a V600E mutant probe consisting of the nucleotide sequence of SEQ ID NO: 24 that specifically hybridizes to the V600E gene mutation (1799T>A), For the TERT (telomerase reverse transcriptase) promoter region, a common probe for TERT consisting of the nucleotide sequence of SEQ ID NO: 25, a 228C>T mutant probe 1 consisting of the nucleotide sequence of SEQ ID NO: 26 that specifically hybridizes to the 228C>T gene mutation, a 228C>T mutant probe 2 consisting of the nucleotide sequence of SEQ ID NO: 27, a 250C>T mutant probe 1 consisting of the nucleotide sequence of SEQ ID NO: 28 that specifically hybridizes to the 250C>T gene mutation, and a 250C>T mutant probe 2 consisting of the nucleotide sequence of SEQ ID NO: 29, a probe set for TERT, A primer set for BRAF consisting of a forward primer consisting of the nucleotide sequence of SEQ ID NO: 1 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 2, which amplifies a region containing the V600E gene mutation in the BRAF gene, A primer set for TERT consisting of a forward primer consisting of the nucleotide sequence of SEQ ID NO: 3 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 4, which amplifies a region containing the 228C>T gene mutation and the 250C>T gene mutation in the TERT gene, A blocking nucleic acid fragment comprising a blocker for BRAF wild type consisting of the nucleotide sequence of SEQ ID NO: 30, a blocker for V600D gene mutation consisting of the nucleotide sequence of SEQ ID NO: 31, a blocker for V600K gene mutation consisting of the nucleotide sequence of SEQ ID NO: 32, a TERT wild type blocker (C228T) consisting of the nucleotide sequence of SEQ ID NO: 33, and a TERT wild type blocker (C250T) consisting of the nucleotide sequence of SEQ ID NO: 34 and wherein the concentration of the primer set for TERT is 0.2 μM or more and 0.6 μM or less, and the concentration ratio of the primer set for BRAF and the primer set for TERT is (concentration of primer set for BRAF):(concentration of primer set for TERT)=4:1 to 2:1, a kit for evaluating gene mutations related to prognosis prediction of papillary thyroid carcinoma.

2. A data analysis method for assisting in predicting the prognosis of papillary thyroid carcinoma, which uses the kit for gene mutation evaluation according to Claim 1 to simultaneously identify the V600E gene mutation in BRAF (v-raf murine sarcoma viral oncogene homolog B1), the 228C>T gene mutation and the 250C>T gene mutation in the TERT (telomerase reverse transcriptase) promoter region for a subject to be diagnosed.

3. The kit for gene mutation evaluation is a microarray comprising the probe set for BRAF and the probe set for TERT. Using the microarray, signals derived from mutant probes and common probes included in each of the probe set for BRAF and the probe set for TERT are measured, and a determination value for each gene mutation is calculated by the formula: [signal intensity of mutant probe] / [signal intensity of common probe]. When the determination value exceeds a pre-specified cut-off value, it is determined that the gene mutation is present. The data analysis method according to Claim 2.

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