WO2024048608A1 - Myelodysplastic syndrome test kit - Google Patents

Myelodysplastic syndrome test kit Download PDF

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WO2024048608A1
WO2024048608A1 PCT/JP2023/031323 JP2023031323W WO2024048608A1 WO 2024048608 A1 WO2024048608 A1 WO 2024048608A1 JP 2023031323 W JP2023031323 W JP 2023031323W WO 2024048608 A1 WO2024048608 A1 WO 2024048608A1
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nucleic acid
bases
mutation
base
probe
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PCT/JP2023/031323
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French (fr)
Japanese (ja)
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惇一 森弘
恵美 高光
俊昭 湯尻
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東洋鋼鈑株式会社
国立大学法人山口大学
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • C12Q1/6834Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844Nucleic acid amplification reactions
    • C12Q1/686Polymerase chain reaction [PCR]
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to a myelodysplastic syndrome test kit for testing gene mutations related to myelodysplastic syndromes (MDS).
  • MDS myelodysplastic syndromes
  • MDS Myelodysplastic syndromes
  • FAB French-American-British
  • WHO World Health Organization
  • MDS is diagnosed.
  • SF3B1 gene mutations are extremely important information for predicting the prognosis of MDS.
  • the mutation sites are adjacent or close to each other, so when detecting a nucleic acid fragment (target nucleic acid) amplified to include these multiple mutation sites with a nucleic acid probe, the detection efficiency of the target nucleic acid decreases. There was a problem with doing so. Therefore, in view of the above-mentioned actual situation, the present invention aims to detect SF3B1 gene mutations in which mutation sites are adjacent to each other with high accuracy.
  • the present inventors have developed a technology that can improve the detection efficiency when detecting adjacent mutations in the SF3B1 gene using probe nucleic acids. They succeeded in doing so and completed the present invention.
  • the present invention includes the following.
  • a primer set that amplifies a region containing the plurality of mutation sites, a plurality of mutant probes corresponding to each of the target bases to be detected at the plurality of mutation sites; Among the target nucleic acids containing the plurality of mutation sites described above and the non-target nucleic acids containing a plurality of non-detection target bases corresponding to each of the detection target bases at the plurality of mutation sites, which are amplified with the above primer set, and a blocking nucleic acid containing a complementary base sequence.
  • the base to be detected is a base encoding a mutation selected from the group consisting of E622D, R625C, R625H, R625L, H662Q, K666N, K666T, K666R, K666E, K666Q, and K666M in the SF3B1 protein encoded by the SF3B1 gene.
  • the plurality of non-detection target bases include a base encoding the 622nd E (glutamic acid), a base encoding the 625th R (arginine), a base encoding the 662nd H (histidine), and The kit for evaluating genetic mutations according to (1), characterized in that it consists of a base encoding K (lysine) at position 666.
  • the blocking nucleic acid contains a base sequence complementary to the base encoding the 622nd E (glutamic acid) and the 625th R (arginine) in the SF3B1 protein and/or the SF3B1 protein
  • the kit for genetic mutation evaluation according to (2) characterized in that it contains a base sequence complementary to the base encoding H (histidine) at position 662 and the base encoding K (lysine) at position 666.
  • the bases to be detected are bases encoding K700E and G742D in the SF3B1 protein, and include a mutant probe for detecting the bases and a primer set for amplifying the region containing these mutations.
  • the above-mentioned blocking nucleic acid is characterized in that the base corresponding to the plurality of non-detection target bases is located inside two bases from the 5' side and inside three bases from the 3' side (1 ) Kit for gene mutation evaluation.
  • the genetic mutation evaluation kit according to (1) comprising a microarray in which the plurality of mutant probes are immobilized on a substrate.
  • the kit for genetic mutation evaluation according to (1) wherein the mutant probe has a region corresponding to the plurality of detection target bases repeated multiple times.
  • the genetic mutation evaluation kit further comprises a wild-type probe corresponding to the plurality of mutant probes, The signal intensity from the above mutant probe and the signal intensity from the above wild type probe were measured, and the formula: [Signal intensity of mutant probe]/([Signal intensity of wild type probe] + [Signal intensity of mutant probe] ) to calculate the judgment value for each genetic mutation,
  • the data analysis method according to (8) wherein it is determined that a mutation is present when the determination value exceeds a predefined cutoff value.
  • the blocking nucleic acid can suppress non-specific hybridization of the mutant probe and the non-target nucleic acid amplified at the same time as the target nucleic acid. Therefore, according to the gene mutation evaluation kit according to the present invention, target nucleic acids containing detection target bases at multiple mutation sites associated with myelodysplastic syndrome contained in the SF3B1 gene can be detected with high accuracy using a mutant probe. Can be done.
  • the blocking nucleic acid can suppress non-specific hybridization of the mutant probe and the non-target nucleic acid amplified at the same time as the target nucleic acid. Therefore, in the data analysis method for diagnosing myelodysplastic syndrome according to the present invention, target nucleic acids containing detection target bases at multiple mutation sites associated with myelodysplastic syndrome contained in the SF3B1 gene are detected with high precision using a mutant probe. can be detected, allowing accurate analysis for the diagnosis of myelodysplastic syndromes.
  • FIG. 2 is a characteristic diagram showing the results of measuring the fluorescence intensities of a wild-type probe and a mutant probe when a blocking nucleic acid containing a plurality of bases corresponding to a plurality of non-detection target bases or a conventional blocking nucleic acid is used.
  • FIG. 2 is a characteristic diagram showing the results of measuring the fluorescence intensities of a wild-type probe and a mutant probe when the positions of a plurality of bases in a blocking nucleic acid containing a plurality of bases corresponding to a plurality of non-detection target bases are changed.
  • FIG. 2 is a characteristic diagram showing the results of examining the effect of improving detection sensitivity using probe DNA (tandem probe) in which regions corresponding to the bases to be detected are repeatedly connected.
  • A Characteristic diagram showing the fluorescence intensities of the wild type probe and mutant probe in the wild type model specimen
  • B Characteristic diagram showing the fluorescence intensities of the wild type probe and the mutant probe in the mutant 5% model specimen
  • C It is a characteristic diagram showing the determination value calculated for each mutation.
  • a mutation present in the SF3B1 gene is targeted for detection among genetic mutations listed as supplementary criteria in the diagnosis of myelodysplastic syndrome (MDS).
  • MDS myelodysplastic syndrome
  • detecting a mutation means identifying the base in the mutation (typing).
  • a base that specifies a predetermined mutation can be referred to as a base to be detected, and other bases can also be referred to as bases not to be detected.
  • the mutation site to be detected is a single base substitution mutation that can be A (adenine) or C (cytosine), either one of the bases, ie, A (adenine), can be the base to be detected.
  • substitution mutations present in the SF3B1 gene are known to be a favorable prognostic factor in MDS. Specifically, when the SF3B1 protein encoded by the SF3B1 gene has the following substitution mutations, it is determined that the prognosis is good. Substitution mutations showing good prognosis include E622D, R625C, R625H, R625L, H662Q, K666N, K666T, K666R, K666E, K666Q, K666M, K700E and G742D.
  • E glutamic acid
  • R arginine
  • H (histidine) at position 662 is mutated to Q (glutamine), or K (lysine) at position 666 is mutated to N (asparagine), T (tyrosine), R (arginine), E (glutamic acid), Q (glutamine), or
  • the prognosis for MDS is good if it mutates to M (methionine), K (lysine) at position 700 mutates to E (glutamic acid), or G (glycine) at position 742 mutates to D (aspartic acid). It can be determined that
  • bases (codons) encoding amino acids after mutation in these substitution mutations can be used as detection target bases.
  • a target nucleic acid containing a plurality of bases to be detected is amplified by a nucleic acid amplification reaction using a primer set, and each base to be detected contained in the target nucleic acid is detected using a nucleic acid probe.
  • a target nucleic acid containing a codon encoding the 622nd amino acid and a codon encoding the 625th amino acid in the SF3B1 protein encoded by the SF3B1 gene can be amplified using a primer set.
  • a target nucleic acid containing a codon encoding the 662nd amino acid and a codon encoding the 666th amino acid in the SF3B1 protein can be amplified using a primer set. Furthermore, a target nucleic acid containing the codon encoding the 622nd amino acid to the 666th amino acid codon in the SF3B1 protein can also be amplified using a primer set.
  • a target nucleic acid containing a codon encoding the 700th amino acid and a codon encoding the 742nd amino acid in the SF3B1 protein among the above substitution mutations may be amplified using a primer set.
  • the present invention provides a target containing a codon encoding the 622nd amino acid, a codon encoding the 625th amino acid, a codon encoding the 662nd amino acid, and a codon encoding the 666th amino acid in the SF3B1 protein.
  • Nucleic acids Two types of target nucleic acids containing a codon encoding the 700th amino acid and a codon encoding the 742nd amino acid in the SF3B1 protein are amplified.
  • the target nucleic acid may be cDNA obtained by reverse transcription reaction from transcripts collected from individual organisms, tissues, and cells.
  • the base length of the target nucleic acid is not particularly limited, but can be, for example, 60 to 1000 bases, preferably 60 to 500 bases, and more preferably 60 to 400 bases.
  • a nucleic acid molecule (nucleic acid fragment) containing a non-detection target base corresponding to the detection target base is referred to as a non-target nucleic acid.
  • bases other than the base to be detected are set as bases not to be detected. More specifically, when the base at the mutation site where a single base is substituted can be A (adenine) or C (cytosine), if A (adenine) is the base to be detected, C (cytosine) is the base to be detected. It turns out that.
  • a target nucleic acid containing a detection target base is obtained using a pair of primer sets as described above. obtained at the same time.
  • a target nucleic acid is obtained by a nucleic acid amplification reaction such as a polymerase chain reaction, if one allele is a non-detection target base, the non-target nucleic acid will be amplified together with the target nucleic acid.
  • a nucleic acid probe having a base sequence complementary to at least a region containing the base to be detected is used in the target nucleic acid.
  • the nucleic acid probe is not particularly limited, it can be, for example, 10 to 30 bases long, and preferably 15 to 26 bases long.
  • the base complementary to the base to be detected is preferably positioned at the center of the character string when the bases constituting the nucleic acid probe are viewed as a character string. Note that the center of the character string includes the case where it is shifted by one toward the 5' end or 3' end for nucleic acid probes consisting of an even number of bases.
  • the target nucleic acid contains multiple mutation sites.
  • the bases to be detected at these multiple mutation sites are detected by different nucleic acid probes. That is, a nucleic acid probe corresponding to the base to be detected is prepared for each mutation site, and the base to be detected is detected by these nucleic acid probes.
  • the nucleic acid probe preferably includes a base sequence in which the region corresponding to the base to be detected is repeated multiple times. That is, the nucleic acid probe preferably includes a plurality of regions that can hybridize with the target nucleic acid.
  • the number of repetitions of the region corresponding to the base to be detected is not particularly limited, and can be 2 to 10 times, preferably 2 to 5 times, and more preferably 2 to 3 times. .
  • a blocking nucleic acid is used to prevent non-specific hybridization between a non-target nucleic acid and a nucleic acid probe.
  • the blocking nucleic acid has a base sequence complementary to a region containing a plurality of non-detection target bases in the non-target nucleic acid. Therefore, the blocking nucleic acid hybridizes with a non-target nucleic acid containing a plurality of non-detection target bases under conditions where a target nucleic acid having a plurality of detection target bases and a nucleic acid probe corresponding to each detection target nucleic acid can hybridize. be able to.
  • a base sequence that is complementary to a region containing multiple non-detection target bases in a non-target nucleic acid is a base sequence that is complementary to a codon encoding the 622nd glutamic acid in the wild-type SF3B1 protein.
  • a nucleotide sequence complementary to the codon encoding arginine at position 625, a nucleotide sequence complementary to the codon encoding histidine at position 662, and a codon encoding lysine at position 666 The meaning includes two or more base sequences selected from the group consisting of complementary base sequences.
  • a blocking nucleic acid comprising a base sequence complementary to the 622nd glutamic acid-encoding codon and the 625th arginine-encoding codon in the wild-type SF3B1 protein;
  • a blocking nucleic acid containing a complementary base sequence to a codon encoding histidine at position 662 and a codon encoding lysine at position 666 can be used.
  • the positions of the bases corresponding to the plurality of non-detection target bases are not particularly limited.
  • the base corresponding to the non-detection target base is preferably located two bases inward from the 5' side and three bases inward from the 3' side in the blocking nucleic acid.
  • a region containing multiple non-detection target bases includes a codon encoding the 622nd glutamic acid and a codon encoding the 625th arginine in the wild type SF3B1 protein, and a codon encoding the 625th arginine in the wild type SF3B1 protein. It exists within 5 or more and 15 or less bases of each of the codon encoding histidine at position 662 and the codon encoding lysine at position 666 in type SF3B1 protein.
  • the blocking nucleic acid By preparing a blocking nucleic acid corresponding to a non-target nucleic acid containing a plurality of non-detection target bases in close positions in this way, the blocking nucleic acid can be accurately hybridized to the non-target nucleic acid.
  • the blocking nucleic acid is not particularly limited, but preferably has a length of 60% or more of the base length of the nucleic acid probe. Further, it is preferable that the blocking nucleic acid has a length shorter than the base length of the nucleic acid probe. For example, if the length of the nucleic acid probe is 25 bases, the base length of the blocking nucleic acid is preferably 14 to 24 bases.
  • the concentration of the blocking nucleic acid is not particularly limited, but can be appropriately set depending on the primer concentration, for example, depending on the concentration of the non-target nucleic acid and/or the concentration of the target nucleic acid.
  • the concentration of the blocking nucleic acid in the composition can be 0.01 to 1.0 ⁇ M, preferably 0.05 to 1.0 ⁇ M, and preferably 0.125 to 1.0 ⁇ M. is more preferable.
  • the blocking nucleic acid since the above-mentioned blocking nucleic acid is used when detecting a mutation contained in the SF3B1 gene, non-specific hybridization between a non-target nucleic acid and a nucleic acid probe can be suppressed. , specific hybridization between the target nucleic acid and the nucleic acid probe can be prevented from being inhibited. Moreover, since the blocking nucleic acid has a base corresponding to a plurality of non-detection target bases, it can be prevented from hybridizing with any of the plurality of nucleic acid probes corresponding to a plurality of detection target bases. Therefore, by applying the present invention, even when each of a plurality of bases to be detected in a target nucleic acid is detected using a nucleic acid probe, detection can be performed with high accuracy.
  • the blocking nucleic acids described above can be used in any system as long as it involves hybridization, which means complementary binding between nucleic acid molecules. That is, the above-mentioned blocking nucleic acid can be used for Southern hybridization, Northern hybridization, and in situ hybridization.
  • the hybridization buffer composition according to the present invention immobilizes a nucleic acid probe on a carrier (including a substrate, a hollow fiber, and a microparticle), and detects a target nucleic acid (including qualitative and quantitative methods) using the immobilized nucleic acid probe.
  • the hybridization buffer composition according to the present invention is most preferably used when detecting a target nucleic acid using a DNA microarray (DNA chip) in which nucleic acid probes are immobilized on a substrate.
  • blocking nucleic acids in addition to those corresponding to the non-target nucleic acids containing multiple non-detection target bases mentioned above, it is also compatible with non-target nucleic acids containing the base sequence encoding the 700th lysine of the wild-type SF3B1 protein.
  • a blocking nucleic acid corresponding to a non-target nucleic acid containing a base sequence encoding the 742nd glycine of the wild-type SF3B1 protein may be used.
  • nucleic acid probe and the blocking nucleic acid are more preferably single-stranded DNA.
  • Nucleic acid probes and blocking nucleic acids can be obtained by chemically synthesizing them using a nucleic acid synthesizer, for example.
  • a nucleic acid synthesizer for example.
  • devices called DNA synthesizers, fully automatic nucleic acid synthesis devices, automatic nucleic acid synthesis devices, etc. can be used.
  • the nucleic acid probes are preferably used in the form of a microarray by immobilizing their 5' ends on a carrier.
  • Materials for the carrier may be those known in the art and are not particularly limited.
  • conductive materials such as noble metals such as platinum, platinum black, gold, palladium, rhodium, silver, mercury, tungsten and their compounds, and carbon such as graphite and carbon fiber; single crystal silicon, amorphous silicon, and carbide.
  • Silicon materials such as silicon, silicon oxide, and silicon nitride; composite materials of these silicon materials such as SOI (silicon on insulator); glass, quartz glass, alumina, sapphire, ceramics, forsterite, photosensitive materials
  • Inorganic 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, phenolic resin, urea resin, epoxy resin, melamine resin, styrene-acrylonitrile copolymer, acrylonitrile-butadiene styrene copolymer, polyphenylene oxide, polysulfone, and other organic materials. Can be
  • the carrier preferably has a carbon layer such as diamond-like carbon (DLC) on the surface and a chemically modified group such as an amino group, a carboxyl group, an epoxy group, a formyl group, a hydroxyl group, and an active ester group.
  • a carrier Use a carrier.
  • the carrier having a carbon layer and a chemical modification group on its surface includes those having a carbon layer and a chemical modification group on the surface of the substrate, and those having a chemical modification group on the surface of a substrate consisting of a carbon layer.
  • materials known in the art can be used, and there are no particular limitations, and materials similar to those listed as the above-mentioned carrier material can be used.
  • the target nucleic acid in a subject can be detected using the DNA microarray prepared in this way. This involves a step of extracting DNA from a sample derived from a subject, a step of amplifying the target nucleic acid (and non-target nucleic acid) using the extracted DNA as a template, and a step of detecting the amplified nucleic acid using a DNA microarray. and a step of doing so.
  • the subject is usually a human, and can include a patient suffering from myelodysplastic syndrome.
  • the sample derived from the subject is not particularly limited. Examples include blood-related samples (blood, serum, plasma, bone marrow fluid, etc.), lymph fluid, feces, cancer cells, crushed materials and extracts of tissues or organs, and the like.
  • DNA is extracted from a sample collected from a subject.
  • the extraction means is not particularly limited.
  • a DNA extraction method using phenol/chloroform, ethanol, sodium hydroxide, CTAB, etc. can be used.
  • an amplification reaction is performed using the obtained DNA as a template to amplify the target nucleic acid (and non-target nucleic acid).
  • amplification reactions include polymerase chain reaction (PCR), LAMP (Loop-Mediated Isothermal Amplification), and ICAN (Isothermal and Chimeric primer-initiated Amplification). ification of nuclear acids) method, etc. can be applied.
  • PCR polymerase chain reaction
  • LAMP Loop-Mediated Isothermal Amplification
  • ICAN Isothermal and Chimeric primer-initiated Amplification
  • the method of labeling the amplified nucleic acid is not particularly limited, but for example, a method may be used in which the primers used in the amplification reaction are labeled in advance, or a method in which a labeled nucleotide is used as a substrate in the amplification reaction. You may also use the method.
  • the labeling substance is not particularly limited, but a radioactive isotope, a fluorescent dye, or an organic compound such as digoxigenin (DIG) or biotin can be used.
  • This reaction system also includes a buffer necessary for nucleic acid amplification and labeling, a thermostable DNA polymerase, a primer specific to the SF3B1 gene, and a labeled nucleotide triphosphate (specifically, a nucleotide triphosphate with a fluorescent label added). , nucleotide triphosphate, magnesium chloride, etc.
  • the amplified nucleic acids obtained as described above include target nucleic acids and non-target nucleic acids.
  • a hybridization reaction between a nucleic acid probe and a target nucleic acid is performed, and the amount of nucleic acid hybridized to the nucleic acid probe can be measured, for example, by detecting a label.
  • the signal from the label can be quantified by detecting the fluorescent signal using a fluorescent scanner and analyzing it using image analysis software.
  • the amplified nucleic acid hybridized to the nucleic acid probe can also be quantified, for example, by creating a standard curve using a sample containing a known amount of DNA.
  • a hybridization reaction using a hybridization buffer composition is preferably performed 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, 2x SSC/0.2% SDS , 25°C, 10 minutes and 2x SSC, 25°C, 5 minutes. That is, the hybridization buffer composition according to the present invention may contain a salt necessary for the hybridization reaction, such as SSC, and a known blocking agent, such as SDS.
  • reaction solution containing the target nucleic acid and the non-target nucleic acid after the amplification reaction was mixed in advance with the above-mentioned composition containing the blocking nucleic acid to perform specific hybridization between the non-target nucleic acid and the blocking nucleic acid. Thereafter, the reaction solution may be brought into contact with a DNA microarray to allow the hybridization reaction between the target nucleic acid and the nucleic acid probe to proceed.
  • a reaction solution containing a target nucleic acid and a non-target nucleic acid after the amplification reaction and a composition containing the above-mentioned blocking nucleic acid may be mixed on a DNA microarray to generate a specific hybrid between the non-target nucleic acid and the blocking nucleic acid. Specific hybridization of soybean, target nucleic acid, and nucleic acid probe may proceed simultaneously.
  • mutations associated with the prognosis of MDS present in the SF3B1 gene can be identified with high accuracy.
  • the genomic DNA of the subject MDS patient
  • at least one selected from the group consisting of E622D, R625C, R625H, R625L, H662Q, K666N, K666T, K666R, K666E, K666Q, K666M, K700E, and G742D If one mutation is identified, it can be determined that the subject has a good prognosis for MDS.
  • a nucleic acid probe (sometimes referred to as a mutant probe) corresponding to the detection target base nucleic acid contained in the target nucleic acid described above and a nucleic acid probe corresponding to the non-detection target base (sometimes referred to as a wild-type probe), and the determination is made based on the signal intensity from the mutant probe and the signal intensity from the wild-type probe. More specifically, a determination value is calculated using the following formula, and when the determination value exceeds a predefined cutoff value, it can be determined that the mutation is present.
  • Judgment value [Signal intensity of mutant probe]/([Signal intensity of wild type probe] + [Signal intensity of mutant probe]) This determination value can be calculated for each mutation mentioned above, each mutation can be identified, and the diagnosis and prognosis of MDS in the subject can be determined based on the results.
  • Example 1 Sample Preparation In this example, plasmid DNA carrying the SF3B1 gene sequence was used as wild type and mutant model specimens.
  • the 5% mutant model sample is a mixture of wild type plasmid and mutant plasmid at a ratio of 95:5.
  • the target region containing the genetic mutations was amplified.
  • the primers shown in Table 2 were designed to amplify the target regions shown in Table 1.
  • the target region of the SF3B1 gene was amplified by PCR.
  • the model sample serving as a template was set at 2 pg/ ⁇ L. Table 3 shows the reaction solution composition.
  • PCR thermal cycles were performed at 95°C for 5 minutes, followed by 40 cycles of 95°C for 30 seconds, 56°C for 30 seconds, and 72°C for 90 seconds, and then at 72°C for 10 minutes. Finally, the temperature was maintained at 4°C.
  • mutant probes corresponding to the 1986C>G and 1997A>G mutations in the SF3B1 gene shown in Table 1 and wild-type probes corresponding thereto were designed.
  • the base sequences of each probe are summarized in Table 4.
  • Hybridization was performed as follows using a chip containing the above probe. First, a wet box was placed in a chamber set at a specified temperature (52° C.), and the chamber and the wet box were sufficiently preheated. Mix 4 ⁇ L of PCR reaction solution and 2 ⁇ L of hybridization buffer (2.25 ⁇ SSC/0.23%SDS/0.2 nM IC5-labeled oligo DNA (manufactured by Life Technologies Japan)), take 3 ⁇ L of this solution, and place it on the central convex part of the hybrid cover.
  • the chip was placed in a preheated wet box, and the wet box was reacted for 1 hour in a hybridization oven set at 52°C. After the hybridization reaction was completed, the chip from which the hybrid cover was removed was washed in a 0.1 ⁇ SSC/0.1% SDS solution by shaking it up and down several times. Then, the chip was immersed in 1 ⁇ SSC solution (room temperature) until the fluorescence intensity was detected.
  • blocker oligo DNA (nucleic acid for blocking) shown in Table 5 was added to the hybridization buffer.
  • Blocker oligo DNA is added for the purpose of suppressing non-specific hybridization of mutation detection probes, so that sufficient detection sensitivity can be obtained even when the mutation rate of the target gene is small. It is designed to specifically hybridize with the amplification product derived from the type.
  • a method was used in which one type of blocker was added for one mutation, but in this example, a blocker oligo DNA was designed with a sequence containing multiple mutation sites in close proximity. That is, we attempted to improve sensitivity by adding one type of blocker oligo DNA to multiple mutation sites.
  • Table 6 shows blocker sequences designed by the conventional method of adding blocker oligo DNA designed for each of a plurality of mutation sites.
  • the chip was covered with a cover film, and the fluorescence intensity of the chip was detected using BIOSHOT (manufactured by Toyo Kohan).
  • Example 2 In this example, PCR was carried out in the same manner as in Example 1 using the model specimen used in Example 1 and the primers designed in Example 1. In this example, mutant probes corresponding to the 1866G>T and 1874G>T mutations in the SF3B1 gene shown in Table 1 and wild type probes corresponding thereto were designed. The base sequences of each probe are summarized in Table 7.
  • Hybridization reaction and detection of chip fluorescence intensity were performed.
  • the blocker oligo DNA shown in Table 8 was added to the hybridization buffer.
  • Table 9 also shows blocker oligo DNAs designed so that the bases corresponding to the non-detection target bases are located near the ends.
  • Example 2 As in Example 1, it can be seen that when the blocker oligo DNA shown in Table 9 is used, the specific signal of the mutant probe decreases as the blocker concentration increases.
  • the sequence of the blocker oligo DNA shown in Table 9 includes both mutations 1866 and 1874, and the mutant product has a single base mismatch. However, because the base corresponding to the non-detection target base is near the end (one base from the end), specificity is low, and it is thought that it binds to the mutant product.
  • specific signals were maintained even when the blocker concentration increased for both 1866 and 1874, confirming that the common blockers were effective.
  • Example 3 In this example, PCR was performed in the same manner as in Example 1 using the model specimen used in Example 1 (only the mutant 5% model specimen) and the primers designed in Example 1.
  • a probe DNA in which the gene region to be detected is repeatedly linked together was designed and mounted on a DNA chip together with a regular probe that does not repeat the sequence.
  • the probe sequences designed in this example are summarized in Table 10. Further, the hybridization reaction and detection of chip fluorescence intensity were carried out using the same procedure as in Example 3.
  • Example 4 PCR was performed using the model specimen used in Example 1 and the primers designed in Example 1.
  • Table 11 shows the reaction solution composition of the PCR carried out in this example.
  • the thermal cycle of PCR was performed at 95°C for 5 minutes, followed by 40 cycles of 30 seconds at 95°C, 30 seconds at 60°C, and 90 seconds at 72°C, and then 40 cycles at 72°C. The temperature was maintained at 4°C for 10 minutes.
  • an automated detection device For detection of hybridization reaction and chip fluorescence intensity, an automated detection device (HySHOT HT-32 or BIOSHOT HT-32 (medical device notification number: 13B3X10232HT3201), manufactured by Toyo Kohan) was used. 30 ⁇ l of the PCR product and 15 ⁇ l of hybridization buffer were mixed and set in an automated detection device. A cleaning solution (0.1 ⁇ SSC/0.1% SDS solution), a rinsing solution, and a detection solution (1 ⁇ SSC) were prepared, and the mixed solution, cleaning solution, and rinsing solution were set in the automatic detection device according to the device instruction manual. The measurement program was set as shown in Table 13.
  • judgment values were calculated for the SF3B1 gene mutations shown in Table 1 using the following formula.
  • Judgment value [Fluorescence intensity of mutant probe]/([Fluorescence intensity of wild type probe] + [Fluorescence intensity of mutant probe])
  • the blocker oligo DNA mix shown in Table 14 was added to the hybridization buffer.

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Abstract

In the present invention, an SF3B1 gene mutation associated with MDS prognosis prediction is detected at high accuracy. A blocking nucleic acid that contains a base sequence complementary to a non-target nucleic acid containing a plurality of bases not to be detected that correspond to bases to be detected at a plurality of mutation locations associated with MDS in the SF3B1 gene is used.

Description

骨髄異形成症候群検査キットMyelodysplastic syndrome test kit
 本発明は、骨髄異形成症候群(myelodysplastic syndromes:MDS)に関連する遺伝子変異を検査するための骨髄異形成症候群検査キットに関する。 The present invention relates to a myelodysplastic syndrome test kit for testing gene mutations related to myelodysplastic syndromes (MDS).
 骨髄異形成症候群(myelodysplastic syndromes:MDS)は、異形成を伴う造血細胞の異常な増殖とアポトーシスによる細胞死によって特徴づけられる造血器腫瘍である。MDSの病態解明が進むにつれ、French-American-British(FAB)グループによる分類や、World Health Organization(WHO)による分類が改訂されている。 Myelodysplastic syndromes (MDS) are hematopoietic tumors characterized by abnormal proliferation of hematopoietic cells accompanied by dysplasia and cell death due to apoptosis. As the pathology of MDS progresses in understanding, the classification by the French-American-British (FAB) group and the World Health Organization (WHO) have been revised.
 現在、臨床所見として慢性貧血を主とするが、ときに出血傾向及び発熱を認める場合、末梢血で1血球系以上の持続的な血球減少を認める等のMDSが疑われる場合、以下の基準によりMDSが診断される。
A.必須基準
1)末梢血と骨髄の芽球比率が30%未満(WHO分類では20%未満)
2)血球減少や異形成の原因となる他の造血器あるいは非造血器疾患が除外できる。
3)末梢血の単球数が1×10/L未満
4)t(8;21)(q22;q22), t(15;17)(q22;q12), inv(16)(p13q22)又はt(16;16)(p13;q22)の染色体異常を認めない。
B.決定的基準
1)骨髄塗抹標本において異形成が、異形成の程度の区分でLOW以上である。
2)分染法又はfluorescence in situ hybridization(FISH)法で骨髄異形成症候群が推測される染色体異常を認める。
C.補助基準
1)骨髄異形成症候群で認められる遺伝子変異が証明できる。(例、TET2遺伝子変異、DNMT3A遺伝子変異、ASXL1遺伝子変異、SF3B1遺伝子変異、TP53遺伝子変異など)
2)網羅的ゲノム解析で、ゲノム変異が証明できる。
3)フローサイトメトリーで異常な形質を有する骨髄系細胞が証明できる。
Currently, the main clinical finding is chronic anemia, but if MDS is suspected, such as when a bleeding tendency and fever are observed, or when persistent cytopenias of one or more blood cell types are observed in the peripheral blood, the following criteria are used: MDS is diagnosed.
A. Essential criteria 1) Blast ratio in peripheral blood and bone marrow is less than 30% (less than 20% according to WHO classification)
2) Other hematopoietic or non-hematopoietic diseases that cause cytopenias or dysplasia can be excluded.
3) Peripheral blood monocyte count less than 1×10 9 /L 4) t(8;21)(q22;q22), t(15;17)(q22;q12), inv(16)(p13q22) or No chromosomal abnormality of t(16;16)(p13;q22) was observed.
B. Definitive Criteria 1) Dysplasia in the bone marrow smear is graded as LOW or higher in the dysplasia grade category.
2) Chromosomal abnormalities suggestive of myelodysplastic syndrome are detected by differential staining or fluorescence in situ hybridization (FISH).
C. Supplementary criteria 1) Genetic mutations observed in myelodysplastic syndromes can be demonstrated. (For example, TET2 gene mutation, DNMT3A gene mutation, ASXL1 gene mutation, SF3B1 gene mutation, TP53 gene mutation, etc.)
2) Genomic mutations can be proven through comprehensive genome analysis.
3) Myeloid cells with abnormal traits can be demonstrated by flow cytometry.
 以上のように、MDSの診断において補助的基準として幾つかの遺伝子変異が挙げられている。なかでも、SF3B1遺伝子変異については、MDSの予後予測に非常に重要な情報となる。しかしながら、SF3B1遺伝子変異については変異箇所が隣接、近接しているため、これら複数の変異部位を含むように増幅した核酸断片(ターゲット核酸)を核酸プローブで検出する際、ターゲット核酸の検出効率が低下するといった問題があった。そこで、本発明は、上述した実情に鑑み、変異箇所が隣接して存在するSF3B1遺伝子変異を高精度に検出することを目的としている。 As mentioned above, several genetic mutations have been cited as supplementary criteria in the diagnosis of MDS. Among these, SF3B1 gene mutations are extremely important information for predicting the prognosis of MDS. However, with regard to SF3B1 gene mutations, the mutation sites are adjacent or close to each other, so when detecting a nucleic acid fragment (target nucleic acid) amplified to include these multiple mutation sites with a nucleic acid probe, the detection efficiency of the target nucleic acid decreases. There was a problem with doing so. Therefore, in view of the above-mentioned actual situation, the present invention aims to detect SF3B1 gene mutations in which mutation sites are adjacent to each other with high accuracy.
 本発明者らは、上述した目的を達成するために鋭意検討した結果、プローブ核酸を用いて、SF3B1遺伝子における隣接して存在する検出対象の変異を検出する際の検出効率を向上できる技術を開発することに成功し、本発明を完成するに至った。本発明は以下を包含する。 As a result of intensive studies to achieve the above-mentioned objectives, the present inventors have developed a technology that can improve the detection efficiency when detecting adjacent mutations in the SF3B1 gene using probe nucleic acids. They succeeded in doing so and completed the present invention. The present invention includes the following.
 (1)SF3B1遺伝子における骨髄異形成症候群と関連する複数の変異箇所を検出する遺伝子変異検出キットにおいて、
 上記複数の変異箇所を含む領域を増幅するプライマーセットと、
 上記複数の変異箇所における検出対象塩基それぞれに対応する複数の変異型プローブと、
 上記プライマーセットで増幅される、上記複数の変異箇所を含むターゲット核酸及び上記複数の変異箇所における検出対象塩基それぞれに対応する複数の非検出対象塩基を含む非ターゲット核酸のうち、非ターゲット核酸に対して相補的な塩基配列を含むブロッキング用核酸とを含む、骨髄異形成症候群に関連する遺伝子変異評価用キット。
 (2)上記検出対象塩基は、SF3B1遺伝子がコードするSF3B1タンパク質におけるE622D、R625C、R625H、R625L、H662Q、K666N、K666T、K666R、K666E、K666Q及びK666Mからなる群から選ばれる変異をコードする塩基であり、上記複数の非検出対象塩基は、SF3B1タンパク質における622番目のE(グルタミン酸)をコードする塩基、625番目のR(アルギニン)をコードする塩基、662番目のH(ヒスチジン)をコードする塩基及び666番目のK(リシン)をコードする塩基からなることを特徴とする(1)記載の遺伝子変異評価用キット。
 (3)前記ブロッキング用核酸は、SF3B1タンパク質における622番目のE(グルタミン酸)をコードする塩基及び625番目のR(アルギニン)をコードする塩基に対して相補的な塩基配列を含む及び/又はSF3B1タンパク質における662番目のH(ヒスチジン)をコードする塩基及び666番目のK(リシン)をコードする塩基に対して相補的な塩基配列を含むことを特徴とする(2)記載の遺伝子変異評価用キット。
 (4)上記検出対象塩基は、更に、SF3B1タンパク質におけるK700E、G742Dをコードする塩基であり、当該塩基を検出する変異型プローブと、これら変異箇所を含む領域を増幅するプライマーセットを含むことを特徴とする(2)記載の遺伝子変異評価用キット。
 (5)上記ブロッキング用核酸は、前記複数の非検出対象塩基に対応する塩基が5’側から2塩基より内側、且つ、3’側から3塩基より内側に位置することを特徴とする(1)記載の遺伝子変異評価用キット。
 (6)上記複数の変異型プローブが基板上に固定されてなるマイクロアレイを含むことを特徴とする(1)記載の遺伝子変異評価用キット。
 (7)上記変異型プローブは、上記複数の検出対象塩基に対応する領域を複数回繰り返して有することを特徴とする(1)記載の遺伝子変異評価用キット。
 (8)上記(1)~(7)いずれか記載の遺伝子変異評価用キットを用い、診断対象者について、SF3B1遺伝子における骨髄異形成症候群と関連する複数の変異箇所における検出対象塩基を同定する、骨髄異形成症候群の診断に関するデータ分析方法。
 (9)上記遺伝子変異評価用キットは、上記複数の変異型プローブに対応する野生型プローブを更に備えており、
 上記変異型プローブからのシグナル強度及び上記野生型プローブからのシグナル強度を測定し、式:[変異型プローブのシグナル強度]/([野生型プローブのシグナル強度]+[変異型プローブのシグナル強度])により各遺伝子変異について判定値を算出し、
 判定値が予め規定したカットオフ値を上回る場合に変異を有すると判定する(8)記載のデータ分析方法。
 本明細書は本願の優先権の基礎となる日本国特許出願番号2022-139528号の開示内容を包含する。
(1) In a gene mutation detection kit that detects multiple mutation sites associated with myelodysplastic syndrome in the SF3B1 gene,
a primer set that amplifies a region containing the plurality of mutation sites,
a plurality of mutant probes corresponding to each of the target bases to be detected at the plurality of mutation sites;
Among the target nucleic acids containing the plurality of mutation sites described above and the non-target nucleic acids containing a plurality of non-detection target bases corresponding to each of the detection target bases at the plurality of mutation sites, which are amplified with the above primer set, and a blocking nucleic acid containing a complementary base sequence.
(2) The base to be detected is a base encoding a mutation selected from the group consisting of E622D, R625C, R625H, R625L, H662Q, K666N, K666T, K666R, K666E, K666Q, and K666M in the SF3B1 protein encoded by the SF3B1 gene. The plurality of non-detection target bases include a base encoding the 622nd E (glutamic acid), a base encoding the 625th R (arginine), a base encoding the 662nd H (histidine), and The kit for evaluating genetic mutations according to (1), characterized in that it consists of a base encoding K (lysine) at position 666.
(3) The blocking nucleic acid contains a base sequence complementary to the base encoding the 622nd E (glutamic acid) and the 625th R (arginine) in the SF3B1 protein and/or the SF3B1 protein The kit for genetic mutation evaluation according to (2), characterized in that it contains a base sequence complementary to the base encoding H (histidine) at position 662 and the base encoding K (lysine) at position 666.
(4) The bases to be detected are bases encoding K700E and G742D in the SF3B1 protein, and include a mutant probe for detecting the bases and a primer set for amplifying the region containing these mutations. The genetic mutation evaluation kit according to (2).
(5) The above-mentioned blocking nucleic acid is characterized in that the base corresponding to the plurality of non-detection target bases is located inside two bases from the 5' side and inside three bases from the 3' side (1 ) Kit for gene mutation evaluation.
(6) The genetic mutation evaluation kit according to (1), comprising a microarray in which the plurality of mutant probes are immobilized on a substrate.
(7) The kit for genetic mutation evaluation according to (1), wherein the mutant probe has a region corresponding to the plurality of detection target bases repeated multiple times.
(8) Identifying bases to be detected at multiple mutation sites associated with myelodysplastic syndrome in the SF3B1 gene in a diagnostic subject using the genetic mutation evaluation kit described in any of (1) to (7) above; Data analysis methods for the diagnosis of myelodysplastic syndromes.
(9) The genetic mutation evaluation kit further comprises a wild-type probe corresponding to the plurality of mutant probes,
The signal intensity from the above mutant probe and the signal intensity from the above wild type probe were measured, and the formula: [Signal intensity of mutant probe]/([Signal intensity of wild type probe] + [Signal intensity of mutant probe] ) to calculate the judgment value for each genetic mutation,
The data analysis method according to (8), wherein it is determined that a mutation is present when the determination value exceeds a predefined cutoff value.
This specification includes the disclosure content of Japanese Patent Application No. 2022-139528, which is the basis of the priority of this application.
 本発明に係る遺伝子変異評価用キットによれば、ブロッキング用核酸により、ターゲット核酸と同時に増幅された非ターゲット核酸と変異型プローブとの非特異的ハイブリダイズを抑制することができる。よって、本発明に係る遺伝子変異評価用キットによれば、SF3B1遺伝子に含まれる骨髄異形成症候群と関連する複数の変異箇所における検出対象塩基を含むターゲット核酸を変異型プローブにより高精度に検出することができる。 According to the gene mutation evaluation kit according to the present invention, the blocking nucleic acid can suppress non-specific hybridization of the mutant probe and the non-target nucleic acid amplified at the same time as the target nucleic acid. Therefore, according to the gene mutation evaluation kit according to the present invention, target nucleic acids containing detection target bases at multiple mutation sites associated with myelodysplastic syndrome contained in the SF3B1 gene can be detected with high accuracy using a mutant probe. Can be done.
 本発明に係る骨髄異形成症候群の診断に関するデータ分析方法では、ブロッキング用核酸により、ターゲット核酸と同時に増幅された非ターゲット核酸と変異型プローブとの非特異的ハイブリダイズを抑制することができる。よって、本発明に係る骨髄異形成症候群の診断に関するデータ分析方法では、SF3B1遺伝子に含まれる骨髄異形成症候群と関連する複数の変異箇所における検出対象塩基を含むターゲット核酸を変異型プローブにより高精度に検出することができ、骨髄異形成症候群の診断に関して正確な分析が可能となる。 In the data analysis method for diagnosis of myelodysplastic syndrome according to the present invention, the blocking nucleic acid can suppress non-specific hybridization of the mutant probe and the non-target nucleic acid amplified at the same time as the target nucleic acid. Therefore, in the data analysis method for diagnosing myelodysplastic syndrome according to the present invention, target nucleic acids containing detection target bases at multiple mutation sites associated with myelodysplastic syndrome contained in the SF3B1 gene are detected with high precision using a mutant probe. can be detected, allowing accurate analysis for the diagnosis of myelodysplastic syndromes.
複数の非検出対象塩基に対応する複数の塩基を含むブロッキング用核酸又は従来のブロッキング用核酸を使用したときの野生型プローブ及び変異型プローブにおける蛍光強度を測定した結果を示す特性図である。FIG. 2 is a characteristic diagram showing the results of measuring the fluorescence intensities of a wild-type probe and a mutant probe when a blocking nucleic acid containing a plurality of bases corresponding to a plurality of non-detection target bases or a conventional blocking nucleic acid is used. 複数の非検出対象塩基に対応する複数の塩基を含むブロッキング用核酸における当該複数の塩基の位置を代えたときの野生型プローブ及び変異型プローブにおける蛍光強度を測定した結果を示す特性図である。FIG. 2 is a characteristic diagram showing the results of measuring the fluorescence intensities of a wild-type probe and a mutant probe when the positions of a plurality of bases in a blocking nucleic acid containing a plurality of bases corresponding to a plurality of non-detection target bases are changed. 検出対象塩基に対応する領域を繰り返して連結したプローブDNA(タンデムプローブ)による検出感度の向上効果を検討した結果を示す特性図である。FIG. 2 is a characteristic diagram showing the results of examining the effect of improving detection sensitivity using probe DNA (tandem probe) in which regions corresponding to the bases to be detected are repeatedly connected. (A)野生型モデル検体における野生型プローブ及び変異型プローブの蛍光強度を示す特性図、(B)変異型5%モデル検体における野生型プローブ及び変異型プローブの蛍光強度を示す特性図、及び(C)各変異について算出した判定値を示す特性図である。(A) Characteristic diagram showing the fluorescence intensities of the wild type probe and mutant probe in the wild type model specimen, (B) Characteristic diagram showing the fluorescence intensities of the wild type probe and the mutant probe in the mutant 5% model specimen, and ( C) It is a characteristic diagram showing the determination value calculated for each mutation.
 以下、本発明を詳細に説明する。
 本発明では、骨髄異形成症候群(myelodysplastic syndromes:MDS)の診断において補助的基準として挙げられている遺伝子変異のうち、SF3B1遺伝子に存在する変異を検出対象としている。本明細書において、変異を検出するとは、当該変異における塩基を特定すること(タイピング)を意味する。
The present invention will be explained in detail below.
In the present invention, a mutation present in the SF3B1 gene is targeted for detection among genetic mutations listed as supplementary criteria in the diagnosis of myelodysplastic syndrome (MDS). As used herein, detecting a mutation means identifying the base in the mutation (typing).
 本発明では、特に検出対象とする複数の変異箇所において取りうる塩基のうち、所定の塩基を検出対象塩基と称し、その他の塩基を非検出対象塩基と称する。特に、本発明では、検出対象とする変異箇所において、所定の変異を特定する塩基を検出対象塩基と称し、その他の塩基を非検出対象塩基と称することもできる。例えば、検出対象の変異箇所がA(アデニン)又はC(シトシン)を取りうる一塩基の置換変異である場合、いずれか一方の塩基、すなわちA(アデニン)を検出対象塩基とすることができる。 In the present invention, among the bases that can be found at a plurality of mutation sites to be detected, certain bases are referred to as detection target bases, and other bases are referred to as non-detection target bases. In particular, in the present invention, at a mutation site to be detected, a base that specifies a predetermined mutation can be referred to as a base to be detected, and other bases can also be referred to as bases not to be detected. For example, when the mutation site to be detected is a single base substitution mutation that can be A (adenine) or C (cytosine), either one of the bases, ie, A (adenine), can be the base to be detected.
 なお、SF3B1遺伝子に存在する変異については、MDSにおける予後良好因子であることを示すことが知られている。具体的には、SF3B1遺伝子がコードするSF3B1タンパク質において、以下の置換変異を有する場合に予後良好と判定される。予後良好を示す置換変異としては、E622D、R625C、R625H、R625L、H662Q、K666N、K666T、K666R、K666E、K666Q、K666M、K700E及びG742Dを挙げることができる。すなわち、SF3B1タンパク質における622番目のE(グルタミン酸)がD(アスパラギン酸)に変異するか、625番目のR(アルギニン)がC(システイン)、H(ヒスチジン)又はL(ロイシン)に変異するか、662番目のH(ヒスチジン)がQ(グルタミン)に変異するか、666番目のK(リシン)がN(アスパラギン)、T(チロシン)、R(アルギニン)、E(グルタミン酸)、Q(グルタミン)又はM(メチオニン)に変異するか、700番目のK(リシン)がE(グルタミン酸)に変異するか、742番目のG(グリシン)がD(アスパラギン酸)に変異した場合、MDSにおける予後良好であると判断できる。 Note that mutations present in the SF3B1 gene are known to be a favorable prognostic factor in MDS. Specifically, when the SF3B1 protein encoded by the SF3B1 gene has the following substitution mutations, it is determined that the prognosis is good. Substitution mutations showing good prognosis include E622D, R625C, R625H, R625L, H662Q, K666N, K666T, K666R, K666E, K666Q, K666M, K700E and G742D. That is, whether E (glutamic acid) at position 622 in the SF3B1 protein is mutated to D (aspartic acid), or R (arginine) at position 625 is mutated to C (cysteine), H (histidine), or L (leucine). H (histidine) at position 662 is mutated to Q (glutamine), or K (lysine) at position 666 is mutated to N (asparagine), T (tyrosine), R (arginine), E (glutamic acid), Q (glutamine), or The prognosis for MDS is good if it mutates to M (methionine), K (lysine) at position 700 mutates to E (glutamic acid), or G (glycine) at position 742 mutates to D (aspartic acid). It can be determined that
 すなわち、本発明においては、これら置換変異における変異後のアミノ酸をコードする塩基(コドン)を検出対象塩基とすることができる。特に、本発明では、プライマーセットを用いて複数の検出対象塩基を含むターゲット核酸を核酸増幅反応により増幅し、当該ターゲット核酸に含まれる検出対象塩基をそれぞれ核酸プローブで検出する。例えば、SF3B1遺伝子がコードするSF3B1タンパク質における622番目のアミノ酸をコードするコドン及び625番目のアミノ酸をコードするコドンを含むターゲット核酸をプライマーセットにより増幅することができる。また、SF3B1タンパク質における662番目のアミノ酸をコードするコドン及び666番目のアミノ酸をコードするコドンを含むターゲット核酸をプライマーセットにより増幅することができる。また、SF3B1タンパク質における622番目のアミノ酸をコードするコドンから666番目のアミノ酸をコードするコドンまでを含むターゲット核酸をプライマーセットにより増幅することもできる。 That is, in the present invention, bases (codons) encoding amino acids after mutation in these substitution mutations can be used as detection target bases. In particular, in the present invention, a target nucleic acid containing a plurality of bases to be detected is amplified by a nucleic acid amplification reaction using a primer set, and each base to be detected contained in the target nucleic acid is detected using a nucleic acid probe. For example, a target nucleic acid containing a codon encoding the 622nd amino acid and a codon encoding the 625th amino acid in the SF3B1 protein encoded by the SF3B1 gene can be amplified using a primer set. Furthermore, a target nucleic acid containing a codon encoding the 662nd amino acid and a codon encoding the 666th amino acid in the SF3B1 protein can be amplified using a primer set. Furthermore, a target nucleic acid containing the codon encoding the 622nd amino acid to the 666th amino acid codon in the SF3B1 protein can also be amplified using a primer set.
 また、本発明においては、上記置換変異のうち、SF3B1タンパク質における700番目のアミノ酸をコードするコドン及び742番目のアミノ酸をコードするコドンを含むターゲット核酸をプライマーセットにより増幅しても良い。 Furthermore, in the present invention, a target nucleic acid containing a codon encoding the 700th amino acid and a codon encoding the 742nd amino acid in the SF3B1 protein among the above substitution mutations may be amplified using a primer set.
 以上のように、本発明では、SF3B1タンパク質における622番目のアミノ酸をコードするコドン、625番目のアミノ酸をコードするコドン、662番目のアミノ酸をコードするコドン及び666番目のアミノ酸をコードするコドンを含むターゲット核酸;SF3B1タンパク質における700番目のアミノ酸をコードするコドン及び742番目のアミノ酸をコードするコドンを含むターゲット核酸からなる2種類のターゲット核酸を増幅する。 As described above, the present invention provides a target containing a codon encoding the 622nd amino acid, a codon encoding the 625th amino acid, a codon encoding the 662nd amino acid, and a codon encoding the 666th amino acid in the SF3B1 protein. Nucleic acids: Two types of target nucleic acids containing a codon encoding the 700th amino acid and a codon encoding the 742nd amino acid in the SF3B1 protein are amplified.
 なお、ターゲット核酸としては、生物個体、組織及び細胞採取から採取した転写産物から逆転写反応により得られるcDNAとしても良い。ターゲット核酸の塩基長としては、特に限定されないが、例えば60~1000塩基とすることができ、60~500塩基とすることが好ましく、60~400塩基とすることがより好ましい。 Note that the target nucleic acid may be cDNA obtained by reverse transcription reaction from transcripts collected from individual organisms, tissues, and cells. The base length of the target nucleic acid is not particularly limited, but can be, for example, 60 to 1000 bases, preferably 60 to 500 bases, and more preferably 60 to 400 bases.
 なお、検出対象塩基を含むターゲット核酸に対して、当該検出対象塩基に対応する非検出対象塩基を含む核酸分子(核酸断片)を非ターゲット核酸と称する。例えば、上記検出対象とする複数の変異において取りうる塩基のうち、1つの塩基を検出対象塩基とした場合、検出対象塩基以外の塩基を非検出対象塩基とする。より具体的に、一塩基が置換変異する変異箇所における塩基がA(アデニン)又はC(シトシン)を取りうる場合、A(アデニン)を検出対象塩基とすると、C(シトシン)が非検出対象塩基ということになる。 Note that in contrast to a target nucleic acid containing a detection target base, a nucleic acid molecule (nucleic acid fragment) containing a non-detection target base corresponding to the detection target base is referred to as a non-target nucleic acid. For example, when one base is set as a base to be detected among the bases that can be taken in the plurality of mutations to be detected, bases other than the base to be detected are set as bases not to be detected. More specifically, when the base at the mutation site where a single base is substituted can be A (adenine) or C (cytosine), if A (adenine) is the base to be detected, C (cytosine) is the base to be detected. It turns out that.
 非検出対象塩基を含む非ターゲット核酸は、当該変異をコードする領域に非検出対象塩基が存在する場合、上述のように一対のプライマーセットを用いて検出対象塩基を含むターゲット核酸を取得する際に同時に取得される。例えば、ターゲット核酸をポリメラーゼ連鎖反応等の核酸増幅反応により取得する場合、一方のアレルが非検出対象塩基であれば、ターゲット核酸とともに非ターゲット核酸が増幅されることとなる。 When a non-target nucleic acid containing a non-detection target base is present in the region encoding the mutation, a target nucleic acid containing a detection target base is obtained using a pair of primer sets as described above. obtained at the same time. For example, when a target nucleic acid is obtained by a nucleic acid amplification reaction such as a polymerase chain reaction, if one allele is a non-detection target base, the non-target nucleic acid will be amplified together with the target nucleic acid.
 検出対象塩基を含むターゲット核酸を検出するには、ターゲット核酸において、少なくとも検出対象塩基を含む領域に対して相補的な塩基配列を有する核酸プローブを使用する。核酸プローブは、特に限定されないが、例えば10~30塩基長とすることができ、15~26塩基長とすることが好ましい。また、検出対象塩基に相補的な塩基は、核酸プローブを構成する塩基を文字列として見たときに、当該文字列の中心となる位置とすることが好ましい。なお、文字列の中心とは、偶数個の塩基からなる核酸プローブについては5’末端又は3’末端方向に1つずれている場合を含む意味である。 To detect a target nucleic acid containing a base to be detected, a nucleic acid probe having a base sequence complementary to at least a region containing the base to be detected is used in the target nucleic acid. Although the nucleic acid probe is not particularly limited, it can be, for example, 10 to 30 bases long, and preferably 15 to 26 bases long. Furthermore, the base complementary to the base to be detected is preferably positioned at the center of the character string when the bases constituting the nucleic acid probe are viewed as a character string. Note that the center of the character string includes the case where it is shifted by one toward the 5' end or 3' end for nucleic acid probes consisting of an even number of bases.
 なお、本発明においては、ターゲット核酸には複数の変異箇所が含まれている。これら複数の変異箇所における検出対象塩基は、それぞれ異なる核酸プローブによって検出される。すなわち、変異箇所毎に検出対象塩基に対応する核酸プローブを準備し、これら核酸プローブによって検出対象塩基を検出する。 Note that in the present invention, the target nucleic acid contains multiple mutation sites. The bases to be detected at these multiple mutation sites are detected by different nucleic acid probes. That is, a nucleic acid probe corresponding to the base to be detected is prepared for each mutation site, and the base to be detected is detected by these nucleic acid probes.
 また、核酸プローブは、検出対象塩基に対応する領域が複数繰り返した塩基配列を含むことが好ましい。すなわち、核酸プローブは、ターゲット核酸とハイブリダイズできる領域を複数備えることが好ましい。ここで、検出対象塩基に対応する領域の繰り返し回数は、特に限定されず、2~10回とすることができ、2~5回とすることが好ましく、2~3回とすることがより好ましい。核酸プローブをこのように構成することによって、ターゲット核酸の検出感度を向上させることができる。 Furthermore, the nucleic acid probe preferably includes a base sequence in which the region corresponding to the base to be detected is repeated multiple times. That is, the nucleic acid probe preferably includes a plurality of regions that can hybridize with the target nucleic acid. Here, the number of repetitions of the region corresponding to the base to be detected is not particularly limited, and can be 2 to 10 times, preferably 2 to 5 times, and more preferably 2 to 3 times. . By configuring the nucleic acid probe in this way, the detection sensitivity of the target nucleic acid can be improved.
 特に、本発明においては、非ターゲット核酸と核酸プローブとの非特異的なハイブリダイズを防止するため、ブロッキング用核酸を使用する。ブロッキング用核酸は、非ターゲット核酸における複数の非検出対象塩基を含む領域に対して相補的な塩基配列を有する。よって、ブロッキング用核酸は、複数の検出対象塩基を有するターゲット核酸と各検出対象核酸に対応する核酸プローブとがハイブリダイズできる条件下において、複数の非検出対象塩基を含む非ターゲット核酸とハイブリダイズすることができる。 In particular, in the present invention, a blocking nucleic acid is used to prevent non-specific hybridization between a non-target nucleic acid and a nucleic acid probe. The blocking nucleic acid has a base sequence complementary to a region containing a plurality of non-detection target bases in the non-target nucleic acid. Therefore, the blocking nucleic acid hybridizes with a non-target nucleic acid containing a plurality of non-detection target bases under conditions where a target nucleic acid having a plurality of detection target bases and a nucleic acid probe corresponding to each detection target nucleic acid can hybridize. be able to.
 具体的に、ブロッキング用核酸において、非ターゲット核酸における複数の非検出対象塩基を含む領域に対して相補的な塩基配列とは、野生型SF3B1タンパク質における622番目のグルタミン酸をコードするコドンに対して相補的な塩基配列、625番目のアルギニンをコードするコドンに対して相補的な塩基配列、662番目のヒスチジンをコードするコドンに対して相補的な塩基配列、及び666番目のリシンをコードするコドンに対して相補的な塩基配列からなる群から選ばれる2以上の塩基配列を含む意味である。具体的に、本発明では、野生型SF3B1タンパク質における622番目のグルタミン酸をコードするコドンと625番目のアルギニンをコードするコドンに対して相補的な塩基配列を含むブロッキング用核酸と、野生型SF3B1タンパク質における662番目のヒスチジンをコードするコドンと666番目のリシンをコードするコドンに対して相補的な塩基配列を含むブロッキング用核酸とを使用することができる。 Specifically, in the blocking nucleic acid, a base sequence that is complementary to a region containing multiple non-detection target bases in a non-target nucleic acid is a base sequence that is complementary to a codon encoding the 622nd glutamic acid in the wild-type SF3B1 protein. a nucleotide sequence complementary to the codon encoding arginine at position 625, a nucleotide sequence complementary to the codon encoding histidine at position 662, and a codon encoding lysine at position 666 The meaning includes two or more base sequences selected from the group consisting of complementary base sequences. Specifically, in the present invention, a blocking nucleic acid comprising a base sequence complementary to the 622nd glutamic acid-encoding codon and the 625th arginine-encoding codon in the wild-type SF3B1 protein; A blocking nucleic acid containing a complementary base sequence to a codon encoding histidine at position 662 and a codon encoding lysine at position 666 can be used.
 ブロッキング用核酸において、複数の非検出対象塩基に対応する塩基の位置は特に限定されない。特に、非検出対象塩基に対応する塩基は、ブロッキング用核酸における5’側から2塩基より内側、且つ、3’側から3塩基より内側に位置することが好ましい。ブロッキング用核酸において非検出対象塩基に対応する塩基の位置を上記範囲とすることによって、ターゲット核酸と核酸プローブとがハイブリダイズできる条件下において、非ターゲット核酸とブロッキング用核酸とを確実にハイブリダイズさせることができる。 In the blocking nucleic acid, the positions of the bases corresponding to the plurality of non-detection target bases are not particularly limited. In particular, the base corresponding to the non-detection target base is preferably located two bases inward from the 5' side and three bases inward from the 3' side in the blocking nucleic acid. By setting the position of the base corresponding to the non-detection target base in the blocking nucleic acid within the above range, the non-target nucleic acid and the blocking nucleic acid can be reliably hybridized under conditions where the target nucleic acid and the nucleic acid probe can hybridize. be able to.
 上記SF3B1遺伝子の変異検出に用いるブロッキング用核酸においては、複数の非検出対象塩基を含む領域が、野生型SF3B1タンパク質における622番目のグルタミン酸をコードするコドンと625番目のアルギニンをコードするコドン、及び野生型SF3B1タンパク質における662番目のヒスチジンをコードするコドンと666番目のリシンをコードするコドンのそれぞれにおいて5塩基以上15塩基以内の近傍に存在している。このように近接した位置に複数の非検出対象塩基を含む非ターゲット核酸に対応するブロッキング用核酸を用意することで、非ターゲット核酸に対してブロッキング用核酸を精度よくハイブリダイズさせることができる。 In the blocking nucleic acid used for mutation detection of the SF3B1 gene, a region containing multiple non-detection target bases includes a codon encoding the 622nd glutamic acid and a codon encoding the 625th arginine in the wild type SF3B1 protein, and a codon encoding the 625th arginine in the wild type SF3B1 protein. It exists within 5 or more and 15 or less bases of each of the codon encoding histidine at position 662 and the codon encoding lysine at position 666 in type SF3B1 protein. By preparing a blocking nucleic acid corresponding to a non-target nucleic acid containing a plurality of non-detection target bases in close positions in this way, the blocking nucleic acid can be accurately hybridized to the non-target nucleic acid.
 また、ブロッキング用核酸としては、特に限定されないが、核酸プローブの塩基長に対して60%以上の長さであることが好ましい。また、ブロッキング用核酸は、核酸プローブの塩基長よりも短い長さであることが好ましい。例えば核酸プローブの長さが25塩基長とすると、ブロッキング用核酸の塩基長は14~24塩基長であることが好ましい。 Further, the blocking nucleic acid is not particularly limited, but preferably has a length of 60% or more of the base length of the nucleic acid probe. Further, it is preferable that the blocking nucleic acid has a length shorter than the base length of the nucleic acid probe. For example, if the length of the nucleic acid probe is 25 bases, the base length of the blocking nucleic acid is preferably 14 to 24 bases.
 さらにまた、本発明において、ブロッキング用核酸の濃度は、特に限定されないが、例えば、非ターゲット核酸の濃度及び/又はターゲット核酸の濃度に応じて、プライマー濃度に応じて適宜設定することができる。具体的にブロッキング用核酸の組成物中の濃度は、0.01~1.0μMとすることができ、0.05~1.0μMとすることが好ましく、0.125~1.0μMとすることがより好ましい。 Furthermore, in the present invention, the concentration of the blocking nucleic acid is not particularly limited, but can be appropriately set depending on the primer concentration, for example, depending on the concentration of the non-target nucleic acid and/or the concentration of the target nucleic acid. Specifically, the concentration of the blocking nucleic acid in the composition can be 0.01 to 1.0 μM, preferably 0.05 to 1.0 μM, and preferably 0.125 to 1.0 μM. is more preferable.
 以上のように、本発明では、SF3B1遺伝子に含まれる変異を検出する際に上述したブロッキング用核酸を使用するため、非ターゲット核酸と核酸プローブとの非特異的なハイブリダイズを抑制することができ、ターゲット核酸と核酸プローブとの特異的なハイブリダイズが阻害されることを防止できる。また、ブロッキング用核酸は、複数の非検出対象塩基に対応する塩基を有するため、複数の検出対象塩基に対応する複数の核酸プローブのいずれともハイブリダイズすることを防止することができる。このため、本発明を適用することによって、ターゲット核酸における複数の検出対象塩基のそれぞれを核酸プローブで検出する場合であっても高精度に検出することができる。 As described above, in the present invention, since the above-mentioned blocking nucleic acid is used when detecting a mutation contained in the SF3B1 gene, non-specific hybridization between a non-target nucleic acid and a nucleic acid probe can be suppressed. , specific hybridization between the target nucleic acid and the nucleic acid probe can be prevented from being inhibited. Moreover, since the blocking nucleic acid has a base corresponding to a plurality of non-detection target bases, it can be prevented from hybridizing with any of the plurality of nucleic acid probes corresponding to a plurality of detection target bases. Therefore, by applying the present invention, even when each of a plurality of bases to be detected in a target nucleic acid is detected using a nucleic acid probe, detection can be performed with high accuracy.
 上述したブロッキング用核酸は、核酸分子同士の相補的な結合を意味するハイブリダイゼーションを含むならば、如何なる系にも使用することができる。すなわち、上述したブロッキング用核酸は、サザンハイブリダイゼーション、ノーザンハイブリダイゼーション、in situ ハイブリダイゼーションに使用することができる。特に、本発明に係るハイブリダイゼーション用バッファー組成物は、担体(基板、中空繊維、微粒子を含む)に核酸プローブを固定し、固定化した核酸プローブを用いてターゲット核酸を検出(定性、定量を含む)する系に使用することが好ましい。より具体的に、本発明に係るハイブリダイゼーション用バッファー組成物は、核酸プローブを基板に固定したDNAマイクロアレイ(DNAチップ)を用いてターゲット核酸を検出する際に使用することが最も好ましい。 The blocking nucleic acids described above can be used in any system as long as it involves hybridization, which means complementary binding between nucleic acid molecules. That is, the above-mentioned blocking nucleic acid can be used for Southern hybridization, Northern hybridization, and in situ hybridization. In particular, the hybridization buffer composition according to the present invention immobilizes a nucleic acid probe on a carrier (including a substrate, a hollow fiber, and a microparticle), and detects a target nucleic acid (including qualitative and quantitative methods) using the immobilized nucleic acid probe. ) is preferably used in systems where More specifically, the hybridization buffer composition according to the present invention is most preferably used when detecting a target nucleic acid using a DNA microarray (DNA chip) in which nucleic acid probes are immobilized on a substrate.
 なお、ブロッキング用核酸としては、上述した複数の非検出対象塩基を含む非ターゲット核酸に対応するものに加えて、野生型SF3B1タンパク質の700番目のリシンをコードする塩基配列を含む非ターゲット核酸に対応するブロッキング用核酸及び/又は野生型SF3B1タンパク質の742番目のグリシンをコードする塩基配列を含む非ターゲット核酸に対応するブロッキング用核酸を使用してもよい。 In addition, as blocking nucleic acids, in addition to those corresponding to the non-target nucleic acids containing multiple non-detection target bases mentioned above, it is also compatible with non-target nucleic acids containing the base sequence encoding the 700th lysine of the wild-type SF3B1 protein. A blocking nucleic acid corresponding to a non-target nucleic acid containing a base sequence encoding the 742nd glycine of the wild-type SF3B1 protein may be used.
 なお、核酸プローブ及びブロッキング用核酸は、より好ましくは一本鎖DNAである。核酸プローブ及びブロッキング用核酸は、例えば、核酸合成装置によって化学的に合成することで取得することができる。核酸合成装置としては、DNAシンセサイザー、全自動核酸合成装置、核酸自動合成装置等と呼ばれる装置を使用することができる。 Note that the nucleic acid probe and the blocking nucleic acid are more preferably single-stranded DNA. Nucleic acid probes and blocking nucleic acids can be obtained by chemically synthesizing them using a nucleic acid synthesizer, for example. As the nucleic acid synthesis device, devices called DNA synthesizers, fully automatic nucleic acid synthesis devices, automatic nucleic acid synthesis devices, etc. can be used.
 本例において、核酸プローブは、その5’末端を担体上に固定化することにより、マイクロアレイの形態で用いるのが好ましい。担体の材料としては、当技術分野で公知のものを使用でき、特に制限されない。例えば、白金、白金黒、金、パラジウム、ロジウム、銀、水銀、タングステンおよびそれらの化合物などの貴金属、およびグラファイト、カーボンファイバーに代表される炭素などの導電体材料;単結晶シリコン、アモルファスシリコン、炭化ケイ素、酸化ケイ素、窒化ケイ素などに代表されるシリコン材料、SOI(シリコン・オン・インシュレータ)などに代表されるこれらシリコン材料の複合素材;ガラス、石英ガラス、アルミナ、サファイア、セラミクス、フォルステライト、感光性ガラスなどの無機材料;ポリエチレン、エチレン、ポリプロビレン、環状ポリオレフィン、ポリイソブチレン、ポリエチレンテレフタレート、不飽和ポリエステル、含フッ素樹脂、ポリ塩化ビニル、ポリ塩化ビニリデン、ポリ酢酸ビニル、ポリビニルアルコール、ポリビニルアセタール、アクリル樹脂、ポリアクリロニトリル、ポリスチレン、アセタール樹脂、ポリカーボネート、ポリアミド、フェノール樹脂、ユリア樹脂、エポキシ樹脂、メラミン樹脂、スチレン・アクリロニトリル共重合体、アクリロニトリル・ブタジエンスチレン共重合体、ポリフェニレンオキサイドおよびポリスルホンなどの有機材料等が挙げられる。担体の形状も特に制限されないが、好ましくは平板状である。 In this example, the nucleic acid probes are preferably used in the form of a microarray by immobilizing their 5' ends on a carrier. Materials for the carrier may be those known in the art and are not particularly limited. For example, conductive materials such as noble metals such as platinum, platinum black, gold, palladium, rhodium, silver, mercury, tungsten and their compounds, and carbon such as graphite and carbon fiber; single crystal silicon, amorphous silicon, and carbide. Silicon materials such as silicon, silicon oxide, and silicon nitride; composite materials of these silicon materials such as SOI (silicon on insulator); glass, quartz glass, alumina, sapphire, ceramics, forsterite, photosensitive materials Inorganic 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, phenolic resin, urea resin, epoxy resin, melamine resin, styrene-acrylonitrile copolymer, acrylonitrile-butadiene styrene copolymer, polyphenylene oxide, polysulfone, and other organic materials. Can be mentioned. Although the shape of the carrier is not particularly limited, it is preferably flat.
 なお担体として、好ましくは表面にダイヤモンドライクカーボン(DLC:Diamond Like Carbon)等のカーボン層と、アミノ基、カルボキシル基、エポキシ基、ホルミル基、ヒドロキシル基及び活性エステル基等の化学修飾基とを有する担体を用いる。表面にカーボン層と化学修飾基とを有する担体には、基板の表面にカーボン層と化学修飾基とを有するもの、およびカーボン層からなる基板の表面に化学修飾基を有するものが包含される。基板の材料としては、当技術分野で公知のものを使用でき、特に制限されず、上述の担体材料として挙げたものと同様のものを使用できる。 The carrier preferably has a carbon layer such as diamond-like carbon (DLC) on the surface and a chemically modified group such as an amino group, a carboxyl group, an epoxy group, a formyl group, a hydroxyl group, and an active ester group. Use a carrier. The carrier having a carbon layer and a chemical modification group on its surface includes those having a carbon layer and a chemical modification group on the surface of the substrate, and those having a chemical modification group on the surface of a substrate consisting of a carbon layer. As the material for the substrate, materials known in the art can be used, and there are no particular limitations, and materials similar to those listed as the above-mentioned carrier material can be used.
 このように作製したDNAマイクロアレイを用いて被検者における、ターゲット核酸を検出することができる。これには、被検者由来の試料からDNAを抽出する工程と、抽出したDNAを鋳型とし、ターゲット核酸(及び非ターゲット核酸)を増幅する工程と、DNAマイクロアレイを用いて増幅された核酸を検出する工程とを含む。 The target nucleic acid in a subject can be detected using the DNA microarray prepared in this way. This involves a step of extracting DNA from a sample derived from a subject, a step of amplifying the target nucleic acid (and non-target nucleic acid) using the extracted DNA as a template, and a step of detecting the amplified nucleic acid using a DNA microarray. and a step of doing so.
 被検者は通常ヒトであり、骨髄異形成症候群に罹患した患者を挙げることができる。被検者由来の試料は特に制限されない。例えば、血液関連試料(血液、血清、血漿、骨髄液など)、リンパ液、糞便、がん細胞、組織または臓器の破砕物および抽出物などが挙げられる。 The subject is usually a human, and can include a patient suffering from myelodysplastic syndrome. The sample derived from the subject is not particularly limited. Examples include blood-related samples (blood, serum, plasma, bone marrow fluid, etc.), lymph fluid, feces, cancer cells, crushed materials and extracts of tissues or organs, and the like.
 まず、被検者から採取した試料からDNAを抽出する。抽出手段としては、特に限定されない。例えばフェノール/クロロホルム、エタノール、水酸化ナトリウム、CTABなどを用いたDNA抽出法を用いることができる。 First, DNA is extracted from a sample collected from a subject. The extraction means is not particularly limited. For example, a DNA extraction method using phenol/chloroform, ethanol, sodium hydroxide, CTAB, etc. can be used.
 次に、得られたDNAを鋳型として用いて増幅反応を行い、ターゲット核酸(及び非ターゲット核酸)を増幅する。増幅反応としては、ポリメラーゼ連鎖反応(PCR)、LAMP(Loop-Mediated Isothermal Amplification)、ICAN(Isothermal and Chimeric primer-initiated Amplification of Nucleic acids)法等を適用することができる。増幅反応においては、増幅後の領域を識別できるように標識を付加することが望ましい。このとき、増幅された核酸を標識する方法としては、特に限定されないが、例えば増幅反応に使用するプライマーをあらかじめ標識しておく方法を使用してもよいし、増幅反応に標識ヌクレオチドを基質として使用する方法を使用してもよい。標識物質としては、特に限定されないが、放射性同位元素や蛍光色素、あるいはジゴキシゲニン(DIG)やビオチンなどの有機化合物などを使用することができる。 Next, an amplification reaction is performed using the obtained DNA as a template to amplify the target nucleic acid (and non-target nucleic acid). Examples of amplification reactions include polymerase chain reaction (PCR), LAMP (Loop-Mediated Isothermal Amplification), and ICAN (Isothermal and Chimeric primer-initiated Amplification). ification of nuclear acids) method, etc. can be applied. In an amplification reaction, it is desirable to add a label so that the region after amplification can be identified. At this time, the method of labeling the amplified nucleic acid is not particularly limited, but for example, a method may be used in which the primers used in the amplification reaction are labeled in advance, or a method in which a labeled nucleotide is used as a substrate in the amplification reaction. You may also use the method. The labeling substance is not particularly limited, but a radioactive isotope, a fluorescent dye, or an organic compound such as digoxigenin (DIG) or biotin can be used.
 またこの反応系は、核酸増幅・標識に必要な緩衝剤、耐熱性DNAポリメラーゼ、SF3B1遺伝子に特異的なプライマー、標識ヌクレオチド三リン酸(具体的には蛍光標識等を付加したヌクレオチド三リン酸)、ヌクレオチド三リン酸および塩化マグネシウム等を含む反応系である。 This reaction system also includes a buffer necessary for nucleic acid amplification and labeling, a thermostable DNA polymerase, a primer specific to the SF3B1 gene, and a labeled nucleotide triphosphate (specifically, a nucleotide triphosphate with a fluorescent label added). , nucleotide triphosphate, magnesium chloride, etc.
 上記のようにして得られた増幅核酸には、ターゲット核酸及び非ターゲット核酸が含まれる。核酸プローブとターゲット核酸のハイブリダイゼーション反応を行い、核酸プローブにハイブリダイズした核酸の量を、例えば標識を検出することにより測定できる。標識からのシグナルは、例えば、蛍光標識を用いた場合は、蛍光スキャナを用いて蛍光シグナル検出し、これを画像解析ソフトによって解析することによりシグナル強度を数値化することができる。また、核酸プローブにハイブリダイズした増幅核酸は、例えば、既知量のDNAを含む試料を用いて検量線を作成することにより、定量することもできる。 The amplified nucleic acids obtained as described above include target nucleic acids and non-target nucleic acids. A hybridization reaction between a nucleic acid probe and a target nucleic acid is performed, and the amount of nucleic acid hybridized to the nucleic acid probe can be measured, for example, by detecting a label. For example, when a fluorescent label is used, the signal from the label can be quantified by detecting the fluorescent signal using a fluorescent scanner and analyzing it using image analysis software. Furthermore, the amplified nucleic acid hybridized to the nucleic acid probe can also be quantified, for example, by creating a standard curve using a sample containing a known amount of DNA.
 このとき、上述した本発明では、ブロッキング用核酸により非ターゲット核酸と核酸プローブとの非特異的なハイブリダイズを抑制することができる。ハイブリダイゼーション用バッファー組成物を用いたハイブリダイゼーション反応は、好ましくはストリンジェントな条件下で実施する。ストリンジェントな条件とは、特異的なハイブリッドが形成され、非特異的なハイブリッドが形成されない条件をいい、例えば、50℃で16時間ハイブリダイズ反応させた後、2×SSC/0.2%SDS、25℃、10分および2×SSC、25℃、5分の条件で洗浄する条件をさす。すなわち、本発明に係るハイブリダイゼーション用バッファー組成物には、ハイブリダイゼーション反応に必要な塩、例えばSSCや、公知のブロッキング剤、例えばSDSが含まれていても良い。 At this time, in the present invention described above, non-specific hybridization between the non-target nucleic acid and the nucleic acid probe can be suppressed by the blocking nucleic acid. A hybridization reaction using a hybridization buffer composition is preferably performed 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, 2x SSC/0.2% SDS , 25°C, 10 minutes and 2x SSC, 25°C, 5 minutes. That is, the hybridization buffer composition according to the present invention may contain a salt necessary for the hybridization reaction, such as SSC, and a known blocking agent, such as SDS.
 また、増幅反応後のターゲット核酸及び非ターゲット核酸を含む反応液と上述したブロッキング用核酸を含む組成物とを予め混合して、非ターゲット核酸とブロッキング用核酸との特異的なハイブリダイズを行った後、反応液をDNAマイクロアレイに接触させてターゲット核酸と核酸プローブとのハイブリダイゼーション反応を進行させても良い。或いは、増幅反応後のターゲット核酸及び非ターゲット核酸を含む反応液と上述したブロッキング用核酸を含む組成物とをDNAマイクロアレイ上にて混合して、非ターゲット核酸とブロッキング用核酸との特異的なハイブリダイズ並びにターゲット核酸と核酸プローブとの特異的なハイブリダイズを同時に進行させても良い。 Further, the reaction solution containing the target nucleic acid and the non-target nucleic acid after the amplification reaction was mixed in advance with the above-mentioned composition containing the blocking nucleic acid to perform specific hybridization between the non-target nucleic acid and the blocking nucleic acid. Thereafter, the reaction solution may be brought into contact with a DNA microarray to allow the hybridization reaction between the target nucleic acid and the nucleic acid probe to proceed. Alternatively, a reaction solution containing a target nucleic acid and a non-target nucleic acid after the amplification reaction and a composition containing the above-mentioned blocking nucleic acid may be mixed on a DNA microarray to generate a specific hybrid between the non-target nucleic acid and the blocking nucleic acid. Specific hybridization of soybean, target nucleic acid, and nucleic acid probe may proceed simultaneously.
 以上のように、上述したブロッキング用核酸を使用することで、SF3B1遺伝子に存在するMDSの予後に関連する変異を高精度に同定することができる。具体的には、被検者(MDS患者)のゲノムDNAにおいて、E622D、R625C、R625H、R625L、H662Q、K666N、K666T、K666R、K666E、K666Q、K666M、K700E及びG742Dからなる群から選ばれる少なくとも1つの変異が同定された場合、当該被検者におけるMDSの予後は良好であると判断することができる。 As described above, by using the blocking nucleic acid described above, mutations associated with the prognosis of MDS present in the SF3B1 gene can be identified with high accuracy. Specifically, in the genomic DNA of the subject (MDS patient), at least one selected from the group consisting of E622D, R625C, R625H, R625L, H662Q, K666N, K666T, K666R, K666E, K666Q, K666M, K700E, and G742D. If one mutation is identified, it can be determined that the subject has a good prognosis for MDS.
 ここで、変異を同定する方法の一例としては、上述したターゲット核酸に含まれる検出対象塩基核酸に対応する核酸プローブ(変異型プローブと称する場合もある)と、非検出対象塩基に対応する核酸プローブ(野生型プローブと称する場合もある)とを用い、変異型プローブからのシグナル強度及び上記野生型プローブからのシグナル強度に基づいて判断する方法が挙げられる。より具体的には下記式により判定値を算出し、判定値が予め規定したカットオフ値を上回る場合に変異を有すると判定することができる。
式:判定値=[変異型プローブのシグナル強度]/([野生型プローブのシグナル強度]+[変異型プローブのシグナル強度])
 この判定値を上述した各変異について算出し、各変異の同定することができ、その結果に基づいて被検者におけるMDSの診断や予後を判断することができる。
Here, as an example of a method for identifying a mutation, a nucleic acid probe (sometimes referred to as a mutant probe) corresponding to the detection target base nucleic acid contained in the target nucleic acid described above and a nucleic acid probe corresponding to the non-detection target base (sometimes referred to as a wild-type probe), and the determination is made based on the signal intensity from the mutant probe and the signal intensity from the wild-type probe. More specifically, a determination value is calculated using the following formula, and when the determination value exceeds a predefined cutoff value, it can be determined that the mutation is present.
Formula: Judgment value = [Signal intensity of mutant probe]/([Signal intensity of wild type probe] + [Signal intensity of mutant probe])
This determination value can be calculated for each mutation mentioned above, each mutation can be identified, and the diagnosis and prognosis of MDS in the subject can be determined based on the results.
 以下、実施例により本発明をより詳細に説明するが、本発明の技術的範囲は以下の実施例に限定されるものではない。 Hereinafter, the present invention will be explained in more detail with reference to Examples, but the technical scope of the present invention is not limited to the following Examples.
[実施例1]
1.サンプル調製
 本実施例では、野生型及び変異型モデル検体として、SF3B1遺伝子配列を搭載したプラスミドDNAを用いた。5%変異型モデル検体は野生型プラスミドと変異型プラスミドを95:5の割合で混合したものである。
[Example 1]
1. Sample Preparation In this example, plasmid DNA carrying the SF3B1 gene sequence was used as wild type and mutant model specimens. The 5% mutant model sample is a mixture of wild type plasmid and mutant plasmid at a ratio of 95:5.
 本実施例では、表1に示した遺伝子変異を検出するため、当該遺伝子変異を含む対象領域を増幅した。 In this example, in order to detect the genetic mutations shown in Table 1, the target region containing the genetic mutations was amplified.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
 本実施例では、表1に示した対象領域を増幅するため、表2に示したプライマーを設計した。 In this example, the primers shown in Table 2 were designed to amplify the target regions shown in Table 1.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
 以上のように調製したDNAサンプルを用いて、SF3B1遺伝子について対象領域をPCRにより増幅した。なお、PCRでは鋳型となるモデル検体を2pg/μLとした。反応液組成を表3に示した。 Using the DNA sample prepared as above, the target region of the SF3B1 gene was amplified by PCR. In addition, in PCR, the model sample serving as a template was set at 2 pg/μL. Table 3 shows the reaction solution composition.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
 そして、PCRのサーマルサイクルを、95℃で5分間の後、95℃で30秒、56℃で30秒及び72℃で90秒を1サイクルとして40サイクル行い、その後、72℃で10分間とし、最終的に4℃を維持した。 Then, PCR thermal cycles were performed at 95°C for 5 minutes, followed by 40 cycles of 95°C for 30 seconds, 56°C for 30 seconds, and 72°C for 90 seconds, and then at 72°C for 10 minutes. Finally, the temperature was maintained at 4°C.
 本実施例では、表1に示したSF3B1遺伝子における1986C>G、1997A>G変異に対応する変異型プローブとこれに対応する野生型プローブを設計した。各プローブの塩基配列を表4にまとめて示した。 In this example, mutant probes corresponding to the 1986C>G and 1997A>G mutations in the SF3B1 gene shown in Table 1 and wild-type probes corresponding thereto were designed. The base sequences of each probe are summarized in Table 4.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
2.遺伝子変異の検出
 上記プローブを有するチップを用いて以下のようにハイブリダイズを行った。先ず、規定温度(52℃)に設定したチャンバー内に湿箱を載置し、チャンバー及び湿箱を十分予熱しておいた。PCR反応液4μLとハイブリダイズ緩衝液(2.25×SSC/0.23%SDS/0.2 nM IC5標識オリゴDNA(ライフテクノロジーズジャパン社製))2μLを混合し、この溶液を3μLとり、ハイブリカバーの中央凸部の上に滴下して、これをチップに被せた後、チップを予熱しておいた湿箱に入れ、湿箱ごと52℃に設定したハイブリダイズオーブン内で1時間反応させた。ハイブリダイズ反応終了後、ハイブリカバーをはずしたチップを0.1×SSC/0.1% SDS溶液中で上下に数回振とうして洗浄した。その後、チップの蛍光強度を検出するまで1×SSC溶液(室温)に浸した。
2. Detection of Genetic Mutation Hybridization was performed as follows using a chip containing the above probe. First, a wet box was placed in a chamber set at a specified temperature (52° C.), and the chamber and the wet box were sufficiently preheated. Mix 4 μL of PCR reaction solution and 2 μL of hybridization buffer (2.25×SSC/0.23%SDS/0.2 nM IC5-labeled oligo DNA (manufactured by Life Technologies Japan)), take 3 μL of this solution, and place it on the central convex part of the hybrid cover. After the chip was covered with it, the chip was placed in a preheated wet box, and the wet box was reacted for 1 hour in a hybridization oven set at 52°C. After the hybridization reaction was completed, the chip from which the hybrid cover was removed was washed in a 0.1×SSC/0.1% SDS solution by shaking it up and down several times. Then, the chip was immersed in 1× SSC solution (room temperature) until the fluorescence intensity was detected.
 また、本実施例では、ハイブリダイズ緩衝液に表5に示したブロッカーオリゴDNA(ブロッキング用核酸)を添加した。ブロッカーオリゴDNAは、解析対象遺伝子の変異割合が小さい場合でも十分な検出感度が得られるように、変異検出用プローブの非特異的なハイブリダイズを抑制することを目的として添加されるもので、野生型由来の増幅産物と特異的にハイブリダイズするように設計されている。従来は一つの変異に対して1種類のブロッカーを添加する方法をとっていたが、本実施例では、近接する複数の変異箇所を含む配列のブロッカーオリゴDNAを設計した。すなわち、複数の変異箇所に対して1種類のブロッカーオリゴDNAを添加することで感度向上を図った。比較例として、複数の変異箇所それぞれに対して設計したブロッカーオリゴDNAを添加する従来手法で設計されたブロッカー配列を表6に示した。 Furthermore, in this example, blocker oligo DNA (nucleic acid for blocking) shown in Table 5 was added to the hybridization buffer. Blocker oligo DNA is added for the purpose of suppressing non-specific hybridization of mutation detection probes, so that sufficient detection sensitivity can be obtained even when the mutation rate of the target gene is small. It is designed to specifically hybridize with the amplification product derived from the type. Conventionally, a method was used in which one type of blocker was added for one mutation, but in this example, a blocker oligo DNA was designed with a sequence containing multiple mutation sites in close proximity. That is, we attempted to improve sensitivity by adding one type of blocker oligo DNA to multiple mutation sites. As a comparative example, Table 6 shows blocker sequences designed by the conventional method of adding blocker oligo DNA designed for each of a plurality of mutation sites.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
 検出直前にチップにカバーフィルムを被せ、BIOSHOT(東洋鋼鈑製)でチップの蛍光強度を検出した。 Immediately before detection, the chip was covered with a cover film, and the fluorescence intensity of the chip was detected using BIOSHOT (manufactured by Toyo Kohan).
 結果を図1に示した。1986は、従来のブロッカー(1986W-3B)を添加したとき、ブロッカー濃度の上昇に伴い変異型プローブの特異的シグナルが減少していることが分かる。これは、近接する1997のブロッカー(1997W-4B)の配列が変異型プローブの配列と一部重なっていることから、1986を含む変異産物と結合したことが要因と考えられる。一方、共通ブロッカー(1986_1997W-4)を使用した場合には、ブロッカー濃度が上昇しても変異型プローブの特異的シグナルが維持されていることが分かる。複数の変異部位を含むブロッカーを使用することで、変異型産物への意図しない結合が抑えられ、特異性が高まったと考えられる。1997でも同様に、従来のブロッカー(1997W-4B)ではブロッカー濃度が上昇するとともに変異プローブの特異的シグナルが減少する傾向にあるが、共通ブロッカー(1986_1997W-4)ではブロッカー濃度が上昇しても変異型プローブの特異的シグナルが維持されていることが確認された。 The results are shown in Figure 1. 1986 shows that when a conventional blocker (1986W-3B) is added, the specific signal of the mutant probe decreases as the blocker concentration increases. This is thought to be due to the fact that the sequence of the adjacent 1997 blocker (1997W-4B) partially overlaps with the sequence of the mutant probe, so it binds to the mutant product containing 1986. On the other hand, it can be seen that when the common blocker (1986_1997W-4) was used, the specific signal of the mutant probe was maintained even if the blocker concentration increased. It is thought that the use of a blocker containing multiple mutation sites suppressed unintended binding to the mutant product and increased specificity. Similarly, in 1997, with the conventional blocker (1997W-4B), the specific signal of the mutation probe tends to decrease as the blocker concentration increases, but with the common blocker (1986_1997W-4), even if the blocker concentration increases, the mutation probe's specific signal tends to decrease. It was confirmed that the specific signal of the type probe was maintained.
[実施例2]
 本実施例では、実施例1で使用したモデル検体、実施例1で設計したプライマーを用いて実施例1と同様の手順によりPCRを実施した。本実施例では、表1に示したSF3B1遺伝子における1866G>T、1874G>T変異に対応する変異型プローブとこれに対応する野生型プローブを設計した。各プローブの塩基配列を表7にまとめて示した。
[Example 2]
In this example, PCR was carried out in the same manner as in Example 1 using the model specimen used in Example 1 and the primers designed in Example 1. In this example, mutant probes corresponding to the 1866G>T and 1874G>T mutations in the SF3B1 gene shown in Table 1 and wild type probes corresponding thereto were designed. The base sequences of each probe are summarized in Table 7.
 ハイブリダイズ反応およびチップ蛍光強度の検出を実施した。なお、本実施例では、ハイブリダイズ緩衝液に表8に示したブロッカーオリゴDNAを添加した。また、非検出対象塩基に対応する塩基が端部近傍に位置するように設計したブロッカーオリゴDNAを表9に示した。 Hybridization reaction and detection of chip fluorescence intensity were performed. In this example, the blocker oligo DNA shown in Table 8 was added to the hybridization buffer. Table 9 also shows blocker oligo DNAs designed so that the bases corresponding to the non-detection target bases are located near the ends.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
 結果を図2に示した。実施例1と同様に、表9に示したブロッカーオリゴDNAを使用した場合、ブロッカー濃度の上昇に伴い変異型プローブの特異的シグナルが減少していることが分かる。表9に示したブロッカーオリゴDNAの配列には、1866、1874の両方の変異が含まれており、変異型産物は1塩基のミスマッチが存在する。しかし、非検出対象塩基に対応する塩基が端部近傍(末端から1塩基)であるため特異性が低く、変異型産物と結合したと考えられる。一方、表8に示した共通ブロッカーでは、1866、1874共にブロッカー濃度が上昇しても特異的シグナルが維持されており、共通ブロッカーが有効に作用していることが確認された。 The results are shown in Figure 2. As in Example 1, it can be seen that when the blocker oligo DNA shown in Table 9 is used, the specific signal of the mutant probe decreases as the blocker concentration increases. The sequence of the blocker oligo DNA shown in Table 9 includes both mutations 1866 and 1874, and the mutant product has a single base mismatch. However, because the base corresponding to the non-detection target base is near the end (one base from the end), specificity is low, and it is thought that it binds to the mutant product. On the other hand, for the common blockers shown in Table 8, specific signals were maintained even when the blocker concentration increased for both 1866 and 1874, confirming that the common blockers were effective.
[実施例3]
 本実施例では、実施例1で使用したモデル検体(変異型5%モデル検体のみ)、実施例1で設計したプライマーを用いて実施例1と同様の手順によりPCRを実施した。また、ハイブリダイズ反応に用いるプローブDNAにおいて、検出対象の遺伝子領域を繰り返して連結したプローブDNA(タンデムプローブ)を設計し、配列を繰り返さない通常プローブと共にDNAチップに搭載した。
[Example 3]
In this example, PCR was performed in the same manner as in Example 1 using the model specimen used in Example 1 (only the mutant 5% model specimen) and the primers designed in Example 1. In addition, for the probe DNA used in the hybridization reaction, a probe DNA in which the gene region to be detected is repeatedly linked together (tandem probe) was designed and mounted on a DNA chip together with a regular probe that does not repeat the sequence.
 本実施例で設計した各プローブ配列は表10にまとめて示した。また、ハイブリダイズ反応およびチップ蛍光強度の検出については、実施例3と同様の手順により実施した。 The probe sequences designed in this example are summarized in Table 10. Further, the hybridization reaction and detection of chip fluorescence intensity were carried out using the same procedure as in Example 3.
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
 結果を図3に示す。図3に示したように、通常プローブと比較し、配列を2回繰り返すタンデムプローブを使用した場合には、蛍光強度が1.5~2倍に増強し、その結果、検出感度が向上することが確認された。 The results are shown in Figure 3. As shown in Figure 3, when using a tandem probe that repeats the sequence twice compared to a normal probe, the fluorescence intensity was increased by 1.5 to 2 times, and as a result, detection sensitivity was confirmed to be improved. It was done.
[実施例4]
 本実施例では、実施例1で使用したモデル検体、実施例1で設計したプライマーを用いてPCRを実施した。本実施例で実施したPCRの反応液組成を表11に示した。また、本実施例では、PCRのサーマルサイクルを、95℃で5分間の後、95℃で30秒、60℃で30秒及び72℃で90秒を1サイクルとして40サイクル行い、その後、72℃で10分間とし、最終的に4℃を維持した。
[Example 4]
In this example, PCR was performed using the model specimen used in Example 1 and the primers designed in Example 1. Table 11 shows the reaction solution composition of the PCR carried out in this example. In this example, the thermal cycle of PCR was performed at 95°C for 5 minutes, followed by 40 cycles of 30 seconds at 95°C, 30 seconds at 60°C, and 90 seconds at 72°C, and then 40 cycles at 72°C. The temperature was maintained at 4°C for 10 minutes.
 本実施例では、表1に示したSF3B1遺伝子における変異に対応する変異型プローブとこれに対応する野生型プローブを設計した。各プローブの塩基配列を表12にまとめて示した。表12中、Iはデオキシイノシンを意味している。 In this example, a mutant probe corresponding to the mutation in the SF3B1 gene shown in Table 1 and a corresponding wild type probe were designed. The base sequences of each probe are summarized in Table 12. In Table 12, I means deoxyinosine.
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000011
 なお、ハイブリダイズ反応およびチップ蛍光強度の検出については、自動化検出装置(HySHOT HT-32またはBIOSHOT HT-32(医療機器届出番号:13B3X10232HT3201)、東洋鋼鈑製)を使用した。PCR産物30μlとハイブリダイズ緩衝液15μlを混合し、自動化検出装置にセットした。洗浄液(0.1×SSC/0.1%SDS溶液)、リンス液及び検出液(1×SSC)を調製し、装置の取り扱い説明書に従い、混合液、洗浄液、リンス液を自動検出装置にセットした。測定プログラムは表13のように設定した。 For detection of hybridization reaction and chip fluorescence intensity, an automated detection device (HySHOT HT-32 or BIOSHOT HT-32 (medical device notification number: 13B3X10232HT3201), manufactured by Toyo Kohan) was used. 30 μl of the PCR product and 15 μl of hybridization buffer were mixed and set in an automated detection device. A cleaning solution (0.1×SSC/0.1% SDS solution), a rinsing solution, and a detection solution (1×SSC) were prepared, and the mixed solution, cleaning solution, and rinsing solution were set in the automatic detection device according to the device instruction manual. The measurement program was set as shown in Table 13.
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000013
Figure JPOXMLDOC01-appb-T000013
 以上のように測定した野生型プローブ及び変異型プローブにおける蛍光強度を用い、表1に示したSF3B1遺伝子変異について下記式によって判定値を算出した。
判定値=[変異型プローブの蛍光強度]/([野生型プローブの蛍光強度]+[変異型プローブの蛍光強度])
 本実施例では、ハイブリダイズ緩衝液に表14に示したブロッカーオリゴDNA mixを添加した。
Using the fluorescence intensities of the wild type probe and mutant probe measured as described above, judgment values were calculated for the SF3B1 gene mutations shown in Table 1 using the following formula.
Judgment value = [Fluorescence intensity of mutant probe]/([Fluorescence intensity of wild type probe] + [Fluorescence intensity of mutant probe])
In this example, the blocker oligo DNA mix shown in Table 14 was added to the hybridization buffer.
Figure JPOXMLDOC01-appb-T000014
Figure JPOXMLDOC01-appb-T000014
 結果を図4に示した。表1で示した遺伝子変異について、野生型モデル検体における各野生型プローブの蛍光強度はすべて10000以上であり、判定に十分な強度を得られた。また、変異型プローブへの非特異的な結合は4000以下であった。次に、変異型5%モデル検体における各プローブの蛍光強度はすべて10000以上と判定に十分な強度を得られた。また、判定値は、変異型モデル検体と野生型モデル検体の差が全て0.2以上であり、良好な分離であった。以上より、野生型モデル検体と変異型5%モデル検体を明確に区別して同時に検出できることが明らかとなった。
 
 本明細書で引用した全ての刊行物、特許及び特許出願はそのまま引用により本明細書に組み入れられるものとする。
The results are shown in Figure 4. Regarding the genetic mutations shown in Table 1, the fluorescence intensity of each wild-type probe in the wild-type model specimen was all 10,000 or more, which was sufficient for determination. In addition, non-specific binding to the mutant probe was less than 4000. Next, the fluorescence intensity of each probe in the mutant 5% model sample was all 10,000 or higher, which was sufficient for determination. Furthermore, as for the judgment values, the differences between the mutant model specimen and the wild type model specimen were all 0.2 or more, indicating good separation. From the above, it became clear that wild-type model specimens and mutant 5% model specimens could be clearly distinguished and detected simultaneously.

All publications, patents, and patent applications cited herein are incorporated by reference in their entirety.

Claims (9)

  1.  SF3B1遺伝子における骨髄異形成症候群と関連する複数の変異箇所を検出する遺伝子変異検出キットにおいて、
     上記複数の変異箇所を含む領域を増幅するプライマーセットと、
     上記複数の変異箇所における検出対象塩基それぞれに対応する複数の変異型プローブと、
     上記プライマーセットで増幅される、上記複数の変異箇所を含むターゲット核酸及び上記複数の変異箇所における検出対象塩基それぞれに対応する複数の非検出対象塩基を含む非ターゲット核酸のうち、非ターゲット核酸に対して相補的な塩基配列を含むブロッキング用核酸とを含む、骨髄異形成症候群に関連する遺伝子変異評価用キット。
    In a gene mutation detection kit that detects multiple mutation sites associated with myelodysplastic syndrome in the SF3B1 gene,
    a primer set that amplifies a region containing the plurality of mutation sites,
    a plurality of mutant probes corresponding to each of the target bases to be detected at the plurality of mutation sites;
    Among the target nucleic acids containing the plurality of mutation sites described above and the non-target nucleic acids containing a plurality of non-detection target bases corresponding to each of the detection target bases at the plurality of mutation sites, which are amplified with the above primer set, and a blocking nucleic acid containing a complementary base sequence.
  2.  上記検出対象塩基は、SF3B1遺伝子がコードするSF3B1タンパク質におけるE622D、R625C、R625H、R625L、H662Q、K666N、K666T、K666R、K666E、K666Q及びK666Mからなる群から選ばれる変異をコードする塩基であり、上記複数の非検出対象塩基は、SF3B1タンパク質における622番目のE(グルタミン酸)をコードする塩基、625番目のR(アルギニン)をコードする塩基、662番目のH(ヒスチジン)をコードする塩基及び666番目のK(リシン)をコードする塩基からなることを特徴とする請求項1記載の遺伝子変異評価用キット。 The base to be detected is a base encoding a mutation selected from the group consisting of E622D, R625C, R625H, R625L, H662Q, K666N, K666T, K666R, K666E, K666Q, and K666M in the SF3B1 protein encoded by the SF3B1 gene, and The multiple non-detection target bases include the base encoding E (glutamic acid) at position 622, the base encoding R (arginine) at position 625, the base encoding H (histidine) at position 662, and the base encoding position 666 in SF3B1 protein. The kit for genetic mutation evaluation according to claim 1, characterized in that it consists of a base encoding K (lysine).
  3.  上記ブロッキング用核酸は、SF3B1タンパク質における622番目のE(グルタミン酸)をコードする塩基及び625番目のR(アルギニン)をコードする塩基に対して相補的な塩基配列を含む及び/又はSF3B1タンパク質における662番目のH(ヒスチジン)をコードする塩基及び666番目のK(リシン)をコードする塩基に対して相補的な塩基配列を含むことを特徴とする請求項2記載の遺伝子変異評価用キット。 The blocking nucleic acid contains a base sequence complementary to the 622nd base encoding E (glutamic acid) and the 625th base encoding R (arginine) in the SF3B1 protein, and/or the 662nd base in the SF3B1 protein. 3. The genetic mutation evaluation kit according to claim 2, comprising a base sequence complementary to the base encoding H (histidine) and the base encoding K (lysine) at position 666.
  4.  上記検出対象塩基は、更に、SF3B1タンパク質におけるK700E、G742Dをコードする塩基であり、当該塩基を検出する変異型プローブと、これら変異箇所を含む領域を増幅するプライマーセットを含むことを特徴とする請求項2記載の遺伝子変異評価用キット。 The above-mentioned bases to be detected are bases encoding K700E and G742D in SF3B1 protein, and include a mutant probe for detecting the bases and a primer set for amplifying a region including these mutation sites. The kit for genetic mutation evaluation according to item 2.
  5.  上記ブロッキング用核酸は、前記複数の非検出対象塩基に対応する塩基が5’側から2塩基より内側、且つ、3’側から3塩基より内側に位置することを特徴とする請求項1記載の遺伝子変異評価用キット。 2. The blocking nucleic acid according to claim 1, wherein the base corresponding to the plurality of non-detection target bases is located within two bases from the 5' side and within three bases from the 3' side. Gene mutation evaluation kit.
  6.  上記複数の変異型プローブが基板上に固定されてなるマイクロアレイを含むことを特徴とする請求項1記載の遺伝子変異評価用キット。 The genetic mutation evaluation kit according to claim 1, comprising a microarray in which the plurality of mutant probes are immobilized on a substrate.
  7.  上記変異型プローブは、上記複数の検出対象塩基に対応する領域を複数回繰り返して有することを特徴とする請求項1記載の遺伝子変異評価用キット。 The genetic mutation evaluation kit according to claim 1, wherein the mutant probe has a region corresponding to the plurality of detection target bases repeated multiple times.
  8.  請求項1乃至7いずれか一項記載の遺伝子変異評価用キットを用い、診断対象者について、SF3B1遺伝子における骨髄異形成症候群と関連する複数の変異箇所における検出対象塩基を同定する、骨髄異形成症候群の診断に関するデータ分析方法。 Myelodysplastic syndrome, which identifies bases to be detected at multiple mutation sites associated with myelodysplastic syndrome in the SF3B1 gene in a diagnostic subject using the kit for evaluating genetic mutations according to any one of claims 1 to 7. Data analysis methods for diagnosis.
  9.  上記遺伝子変異評価用キットは、上記複数の変異型プローブに対応する野生型プローブを更に備えており、
     上記変異型プローブからのシグナル強度及び上記野生型プローブからのシグナル強度を測定し、式:[変異型プローブのシグナル強度]/([野生型プローブのシグナル強度]+[変異型プローブのシグナル強度])により各遺伝子変異について判定値を算出し、
     判定値が予め規定したカットオフ値を上回る場合に変異を有すると判定する請求項8記載のデータ分析方法。
    The genetic mutation evaluation kit further includes a wild type probe corresponding to the plurality of mutant probes,
    The signal intensity from the above mutant probe and the signal intensity from the above wild type probe were measured, and the formula: [Signal intensity of mutant probe]/([Signal intensity of wild type probe] + [Signal intensity of mutant probe] ) to calculate the judgment value for each gene mutation,
    9. The data analysis method according to claim 8, wherein it is determined that the mutation is present when the determination value exceeds a predefined cutoff value.
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