WO2019117192A1 - Method for designing probe for detecting single nucleotide polymorphism and probe set - Google Patents

Method for designing probe for detecting single nucleotide polymorphism and probe set Download PDF

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WO2019117192A1
WO2019117192A1 PCT/JP2018/045641 JP2018045641W WO2019117192A1 WO 2019117192 A1 WO2019117192 A1 WO 2019117192A1 JP 2018045641 W JP2018045641 W JP 2018045641W WO 2019117192 A1 WO2019117192 A1 WO 2019117192A1
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type
wild
mutant
probe
type probe
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PCT/JP2018/045641
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French (fr)
Japanese (ja)
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大場　光芳
山野　博文
稔也 津田
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東洋鋼鈑株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules

Definitions

  • the present invention relates to a method and a probe set for designing a wild-type probe corresponding to a wild-type single nucleotide polymorphism and a mutant-type probe corresponding to a mutant single nucleotide polymorphism.
  • Single nucleotide polymorphism is a type of gene mutation present in the genome of a population of species, and means a variation in which one base in a base sequence is mutated.
  • a single nucleotide polymorphism may be referred to only when mutations are found at a frequency of 1% or more in a population, but a frequency of less than 1% can also be referred to as a single nucleotide polymorphism.
  • single nucleotide polymorphism usually consists of two types which consist of a wild type allele and one type of variant allele in many cases, it may have two or more types of variant alleles.
  • Broad polymorphisms including single nucleotide polymorphisms are associated with phenotypes such as the degree of side effects on drugs and susceptibility to diseases. Further, in plants, phenotypes such as disease resistance, stress tolerance, biomass amount, etc. are associated with broad polymorphisms including single nucleotide polymorphisms. As described above, by detecting a single nucleotide polymorphism (also referred to as typing), it is possible to predict the risk of suffering from a disease, the occurrence risk of side effects, and the like.
  • a SNP typing method as a method of detecting a single nucleotide polymorphism.
  • a direct sequencing method of amplifying a DNA fragment containing single nucleotide polymorphism and reading a nucleotide sequence a method of using a probe including a single nucleotide polymorphism site, and the like can be mentioned.
  • Methods using probes containing single nucleotide polymorphism sites include cycling probe methods to which real-time PCR is applied, bead array methods and microarray (DNA chip) methods.
  • thermodynamic variable including melting temperature with target DNA, length, intramolecular self-binding, presence or absence of sequence homology with other probes, and position of target site (3 'end side or 5 'end side) etc. are considered.
  • the thermodynamic variable is one that considers the hybridization energy between the probe and the target DNA, and can be used as an index representing the probe characteristic.
  • Patent Document 1 As a method for designing these wild-type probes and mutant-type probes, for example, as described in Patent Document 1, the Tm value when a perfect match target and a double strand are formed is different between the wild-type probe and the mutant-type probe. There is a way to design them to be equal between them. Further, Patent Document 2 describes a method for designing a wild-type probe and a mutant-type probe to have the same length.
  • the present invention provides a wild-type probe and a mutant-type probe for single nucleotide polymorphism detection, which can suppress the occurrence of cross hybridization and detect a perfectly matched target with high accuracy. It is an object to provide a design method and a probe set.
  • the present invention includes the following. (1) A method of designing a wild-type probe corresponding to a wild-type single nucleotide polymorphism and a mutant-type probe corresponding to a mutation type of the single nucleotide polymorphism, comprising a plurality of single nucleotide polymorphism sites as described above Of the candidate wild-type probes of the present invention and a plurality of candidate mutant-type probes containing the single nucleotide polymorphism site, and the binding ratio to the wild-type target that is a perfect match for these candidate wild-type probes, The binding rate to a mutant target in which there is a single base mismatch at the single nucleotide polymorphism site, the binding rate to a mutant target in which there is a perfect match for these candidate mutant probes, and the single nucleotide polymorphism for these candidate mutant probes Calculate the binding rate with the wild-type target that causes a single base mismatch at each site, and for the wild-type target, The value
  • the binding rate is calculated at a predetermined hybridization temperature or a predetermined hybridization temperature range, and a wild-type probe and a mutant-type probe to be used at the hybridization temperature or the hybridization temperature range are designed. (1) The method described.
  • the difference between the binding ratio of wild type probe to wild type target and the binding ratio of mutant type probe to mutant target for multiple combinations The absolute value is added to the absolute value of the difference between the binding rate of the mutant probe to the wild-type target and the binding rate of the wild-type probe to the mutant target, and the combination having the lowest total value among the plurality of combinations is
  • the method according to (1) which is designed as a wild-type probe and a mutant-type probe for detecting the single nucleotide polymorphism.
  • the difference between the binding ratio of wild type probe to wild type target and the binding ratio of mutant type probe to wild type target for multiple combinations is A wild-type probe for detecting the single nucleotide polymorphism and a combination having the largest combination or the largest difference between the binding ratio of the mutant probe to the mutant target and the binding ratio of the wild-type probe to the mutant target.
  • a plurality of probe sets comprising a wild-type probe corresponding to a wild-type single nucleotide polymorphism and a mutant-type probe corresponding to a mutant single nucleotide polymorphism, the plurality comprising a plurality of single nucleotide polymorphism sites as described above Selected from among a plurality of candidate mutant-type probes including the single nucleotide polymorphism site and the candidate wild-type target from the ratio of the candidate wild-type probe to the candidate mutant-type probe And a wild-type probe for which the value obtained by subtracting the rate of binding to the candidate wild-type probe from the rate of binding to the candidate mutant-type probe is greater than or equal to the predetermined value.
  • a probe set for detecting the single nucleotide polymorphism which comprises a mutated probe.
  • a DNA chip comprising a carrier, and a wild-type probe and a mutant-type probe contained in the probe set according to (5) immobilized on the carrier.
  • the present specification includes the disclosure content of Japanese Patent Application No. 2017-241070 on which the priority of the present application is based.
  • a wild-type probe and a mutant-type probe for single nucleotide polymorphism detection According to the method for designing a wild-type probe and a mutant-type probe for single nucleotide polymorphism detection according to the present invention, a wild-type probe and a mutant-type probe excellent in detection sensitivity of target DNA in which occurrence of cross hybridization is suppressed Can be designed.
  • the primer set according to the present invention is excellent in the detection sensitivity of target DNA.
  • the characteristic wild type indicates the type of candidate wild-type probe in the row and the type of candidate mutant-type probe in the row, and the value obtained by subtracting the mismatch binding rate in the candidate mutant-type probe from the full match binding rate in the candidate wild-type probe is there.
  • the characteristic wild type indicates the type of candidate wild-type probe in the row and the type of candidate mutant-type probe in the row, and the value obtained by subtracting the mismatch binding rate in the candidate mutant-type probe from the full match binding rate in the candidate wild-type probe is there.
  • the characteristic wild type indicates the type of candidate wild-type probe in the row and the type of candidate mutant-type probe in the row, and the value obtained by subtracting the mismatch binding rate in the candidate mutant-type probe from the full match binding rate in the candidate wild-type probe is there.
  • a characteristic chart showing the type of candidate wild-type probe in the row and the type of candidate mutant-type probe in the row, and the value obtained by subtracting the mismatch binding rate in the candidate wild-type probe from the full match binding rate in the candidate mutant-type probe is there.
  • a characteristic chart showing the type of candidate wild-type probe in the row and the type of candidate mutant-type probe in the row, and the value obtained by subtracting the mismatch binding rate in the candidate wild-type probe from the full match binding rate in the candidate mutant-type probe is there.
  • a characteristic chart showing the type of candidate wild-type probe in the row and the type of candidate mutant-type probe in the row, and the value obtained by subtracting the mismatch binding rate in the candidate wild-type probe from the full match binding rate in the candidate mutant-type probe is there.
  • the present invention provides a method for designing a wild-type probe and a mutant-type probe for single nucleotide polymorphism detection.
  • a single nucleotide polymorphism is a gene mutation present in the genome of a species population, and means a single base mutation in the base sequence.
  • single nucleotide polymorphisms are not limited to mutations found at a frequency of 1% or more in a population, but also include mutations found at a frequency of less than 1% (sometimes referred to as mutations). It is a meaning.
  • a wild-type probe is a probe that corresponds to a wild-type allele in a single nucleotide polymorphism to be detected and has a base complementary to the genotype of the wild-type allele.
  • a mutant-type probe is a probe that corresponds to a mutant-type allele in a single nucleotide polymorphism to be detected and has a base that is complementary to the genotype of the mutant-type allele.
  • the wild type probe corresponds to the single nucleotide polymorphism Contains a T that is complementary to A at the position where the mutant probe contains a C that is complementary to G at that position.
  • the combination of a wild-type probe and a mutant-type probe designed for a given single nucleotide polymorphism is referred to as a probe set.
  • a plurality of wild-type probe candidates (candidate wild-type probe) and a plurality of mutant-type probes (candidate mutant-type probes) are designed. .
  • a plurality of candidate wild-type probes and a plurality of mutant-type probes are designed as nucleotide sequences of oligonucleotides containing single nucleotide polymorphisms, from surrounding genomic sequences containing single nucleotide polymorphisms to be detected.
  • the lengths of candidate wild-type probes and candidate mutant-type probes to be designed are not particularly limited, and can be, for example, 5 to 50 bases, preferably 7 to 40 bases, and 10 to 30 bases in length. More preferably, the length is 11 to 25 bases, and most preferably 11 to 25 bases.
  • the base corresponding to the single nucleotide polymorphism to be detected it is preferable to design the base corresponding to the single nucleotide polymorphism to be detected to be located approximately at the center in the designed nucleotide sequence.
  • the designed nucleotide sequence consists of an odd number of bases, one central base at the 5 'end and the 3' end can be at a position corresponding to the single nucleotide polymorphism.
  • the designed base sequence comprises an even number of bases, any one of the central two bases at the 5 'end and the 3' end can be taken as the position corresponding to the single nucleotide polymorphism.
  • the base corresponding to the single nucleotide polymorphism to be detected is not limited to the approximate center in the nucleotide sequence to be designed as described above.
  • the position may be biased toward the end side or the 3 'end side.
  • the base corresponding to the single nucleotide polymorphism to be detected is in the range of 1 ⁇ 4, preferably 1 ⁇ 5 of the full length from the approximate center in the designed nucleotide sequence.
  • the position may be biased toward the 5 'end or the 3' end in the range of 1, preferably in the range of 1/6.
  • the candidate wild-type probe and the candidate mutant-type probe when designing the candidate wild-type probe and the candidate mutant-type probe, only the candidate wild-type probe and the candidate mutant-type probe may be designed with the base corresponding to the single nucleotide polymorphism to be detected as the approximate center. Only the candidate wild-type probe and the candidate mutant-type probe may be designed with the base corresponding to the single nucleotide polymorphism to be detected at a position shifted from the approximate center to the 5 'end or the 3' end.
  • a candidate wild-type probe and a candidate mutant-type probe when designing a candidate wild-type probe and a candidate mutant-type probe, a candidate wild-type probe and a candidate mutant-type probe substantially centered on a base corresponding to a single nucleotide polymorphism to be detected, and a single nucleotide polymorphism to be detected
  • a candidate wild-type probe and a candidate mutant-type probe may be designed in which the base corresponding to is located approximately at the 5 'end or the 3' end from the center.
  • Tm curves of a target DNA and a Tm curve to be a perfect match and a target DNA to be a single base mismatch are respectively calculated.
  • the target DNA to be a perfect match is a wild-type target DNA (referred to as a wild-type target) whose single nucleotide polymorphism to be detected is a wild-type allele.
  • the target DNA that causes a single base mismatch is a mutant target DNA (referred to as a mutant target) in which the single nucleotide polymorphism to be detected is a mutant allele.
  • the target DNA that is a perfect match is a mutant-type target whose single nucleotide polymorphism to be detected is a mutant allele.
  • the target DNA that causes a single base mismatch is a wild-type target in which a single nucleotide polymorphism to be detected is a wild-type allele.
  • the method of calculating the Tm curve is not particularly limited, and examples thereof include the Nearest Neighbor method, the Wallace method, the GC% method, and the like, and in particular, the Nearest Neighbor method. It is preferable to calculate by.
  • Tm is defined as the temperature at which 50% of the probe and the target are dissociated (ie, the temperature at a binding rate of 50%) when the probe and target are hybridized.
  • factors that affect the Tm value include base composition, salt concentration, concentration of oligo chain, denaturing agent (formamide, DMSO, etc.), solvation effect, conjugate group (biotin, digoxigenin, alkaline phosphatase, fluorescent dye, etc. Can be mentioned.
  • the Tm value can be calculated by the WEB service provided by Integrated DNA Technologies, including the base sequence of the probe, the concentration of the probe, the concentration of the target to be hybridized with the probe, and Na + in the reaction solution.
  • the Tm curve can be calculated by setting the K + concentration, the Mg 2+ concentration in the reaction solution, and the (optionally) dNTPs concentration.
  • Tm curves can be calculated with probe concentration and target concentration of 0.002 ⁇ M, Na + and K + concentrations of 195 mM, Mg 2+ concentration of 0 mM, and dNTPs concentration of 0 mM.
  • the calculated Tm curve can also be displayed in a two-dimensional plane in which one axis (e.g., the vertical axis) is the binding rate and the other axis (e.g., the horizontal axis) is the temperature.
  • the obtained Tm curve can show the relationship between temperature and the binding rate for a candidate wild-type probe or a candidate mutant-type probe having a predetermined base sequence and a wild-type target or a mutant-type target. That is, based on the obtained Tm curve, the binding rate with the wild-type target (perfect match) in the candidate wild-type probe designed as described above can be calculated. Similarly, based on the obtained Tm curve, the binding rate of a candidate wild-type probe to a mutant target (single base mismatch) can be calculated. Similarly, based on the obtained Tm curve, the binding rate of the candidate mutant probe to the mutant target (perfect match) can be calculated. Similarly, based on the obtained Tm curve, it is possible to calculate the binding ratio of the candidate mutant probe to the wild-type target (single base mismatch).
  • the binding rate calculated as described above can be calculated as the binding rate at a predetermined temperature or a predetermined temperature range.
  • the temperature at which target DNA is hybridized with the probe is set in the range of 40 to 80 ° C., preferably 50 to 70 ° C., more preferably 50 to 60 ° C. Therefore, calculate the binding rate at, for example, 55 ° C. included in these temperature ranges, and select a wild-type probe and a mutant-type probe that are optimal for the single nucleotide polymorphism to be detected based on the binding rate at 55 ° C. Is preferred.
  • a value obtained by subtracting the binding rate with a candidate mutant-type probe from the binding rate with a candidate wild-type probe becomes a predetermined value or more.
  • One or more combinations of a wild-type probe and a mutant-type probe are selected such that the value obtained by subtracting the ratio of binding to the candidate wild-type probe from the ratio of binding to the candidate mutant-type probe is greater than a predetermined value.
  • the binding rate to a candidate wild-type probe with respect to a wild-type target means the binding rate (full-match binding rate) to a target that completely matches the candidate wild-type probe.
  • the binding rate with a candidate mutant-type probe with respect to a wild-type target means the binding rate (mismatch binding rate) with respect to a target that differs by one base from the candidate mutant-type probe.
  • the binding rate to a candidate wild-type probe with respect to a mutant target means the binding rate (mismatch binding rate) to a target that differs by one base from the candidate wild-type probe.
  • the binding rate with a candidate mutant probe with respect to a mutant target means the binding rate (full match binding rate) to a target that completely matches the candidate mutant probe.
  • the value obtained by subtracting the binding rate (full match binding rate) with the candidate wild-type probe and the binding rate (mismatch binding rate) with the candidate mutant-type probe is, for example, 0.3 (but with binding
  • the rate can be a value of 0.00 to 1.00) or more, preferably 0.4 or more, and more preferably 0.5 or more.
  • the value obtained by subtracting the binding rate (full match binding rate) with the candidate mutant type probe and the binding rate (mismatch binding rate) with the candidate wild type probe may be, for example, 0.3 or more It is preferably 0.4 or more, more preferably 0.5 or more.
  • one or more combinations of a wild-type probe and a mutant-type probe can be selected from the candidate wild-type probe and the candidate mutant-type probe that meet the above-described conditions. Then, it can be designed as one or more combinations of a selected wild-type probe and a mutant-type probe, a wild-type probe and a mutant-type probe for detecting a single nucleotide polymorphism to be detected.
  • the combination of one or more of the selected wild-type probe and the mutant-type probe is a combination having the lowest probability of occurrence of cross hybridization when detecting a single nucleotide polymorphism to be detected. It can be detected with high accuracy (SNP typing).
  • a combination of better wild-type probes and mutant-type probes may be further selected according to the procedure described below.
  • the binding rate (full match binding rate) of the wild type probe to the wild type target and the binding rate (full match binding rate) of the mutant probe to the variant target for a plurality of combinations of the selected wild type probe and the mutant type probe
  • the absolute value of the difference between the binding rate of the mutant probe to the wild type target (mismatch binding rate) and the binding rate of the wild type probe to the mutant target (mismatch binding rate) is selected from the plurality of combinations.
  • the combination of a wild-type probe and a mutant-type probe with the lowest total value can be obtained by uniformly separating the wild-type, hetero-type and mutant-type determination values with respect to single nucleotide polymorphisms to be detected. Polymorphism can be detected.
  • the binding rate (full match binding rate) of the wild type probe to the wild type target and the binding rate (mismatch binding rate) of the mutant probe to the wild type target for a plurality of combinations of the selected wild type probe and the mutant type probe Choose the combination with the largest difference.
  • judgment values of the wild-type (group A) and the hetero-type and the mutant-type (group B) are largely separated with respect to single nucleotide polymorphisms to be detected. Groups A and B can be detected with higher accuracy.
  • the binding rate of the mutant-type probe to the mutant-type target (full match binding rate) and the binding ratio of the wild-type probe to the mutant-type target (mismatch binding rate) choose the combination with the largest difference.
  • the judgment values of the wild-type and hetero-type (group C) and the mutant-type (group D) are largely separated with respect to the single nucleotide polymorphism to be detected. Groups C and D can be detected more accurately.
  • wild-type probes and mutant-type probes that can detect wild-type (group A), hetero-type and mutant-type (group B) with higher accuracy
  • the combination of type probes is effective, for example, when a single nucleotide polymorphism for a dominant mutation is to be detected.
  • the enzyme ALDH2 plays the most important role in this process.
  • a single nucleotide polymorphism from G to A located in exon 12 of chromosome 12 has a significant effect on the enzyme activity.
  • This single nucleotide polymorphism is "dominant dominant", and heterozygotes (ALDH2 * 1) or variants (ALDH2 * 2) have been shown to have no more than 10% enzymatic activity compared to wild type .
  • the wild-type probe and the mutant-type probe designed as described above are preferably nucleic acids, more preferably DNA.
  • the DNA may be double stranded or single stranded but is preferably single stranded DNA.
  • the wild-type probe and the mutant-type probe can be obtained, for example, by chemical synthesis with a nucleic acid synthesizer.
  • a nucleic acid synthesizer an apparatus called a DNA synthesizer, a fully automatic nucleic acid synthesizer, an automatic nucleic acid synthesizer or the like can be used.
  • the wild-type probe and the mutant-type probe designed as described above are preferably used in the form of a microarray (as an example, a DNA chip) by immobilizing the 5 'end thereof on a carrier. At this time, when there are a plurality of single nucleotide polymorphisms to be detected, the microarray has a mutant-type probe and a wild-type probe at a predetermined position for each single nucleotide polymorphism.
  • the microarray according to the present invention can be prepared by immobilizing the above-mentioned wild-type probe and mutant-type probe on a carrier.
  • noble metals such as platinum, platinum black, gold, palladium, rhodium, silver, mercury, tungsten and compounds thereof, and conductive materials such as carbon represented by graphite and carbon fiber; single crystal silicon, amorphous Silicon materials represented by silicon, silicon carbide, silicon oxide, silicon nitride etc., and composite materials of these silicon materials represented by SOI (silicon on insulator) etc .; glass, quartz glass, alumina, sapphire, ceramics, foam Stellite, inorganic materials such as photosensitive glass; 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, polystyren
  • a carrier having a carbon layer and a chemical modification group on the surface is used as the carrier.
  • Carriers having a carbon layer and a chemical modification group on the surface include 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 the substrate consisting of a carbon layer.
  • the material of the substrate those known in the art can be used without particular limitation, and the same ones as mentioned above as the carrier material can be used.
  • a carrier having a fine tabular structure is suitably used.
  • the shape is not limited to rectangular, square and round shapes, but usually 1 to 75 mm square, preferably 1 to 10 mm square, more preferably 3 to 5 mm square is used. It is preferable to use a substrate made of a silicon material or a resin material because it is easy to manufacture a carrier having a fine flat plate structure, and in particular, a carrier having a carbon layer and a chemical modification group on the surface of a substrate made of single crystal silicon is more preferable. preferable.
  • Single crystal silicon has a slight change in orientation of crystallographic axis in part (sometimes referred to as a mosaic crystal), or includes disorder on atomic scale (lattice defect) Is also included.
  • the carbon layer formed on the substrate in the present invention is not particularly limited, but synthetic diamond, high-pressure synthetic diamond, natural diamond, soft diamond (for example, diamond like carbon), amorphous carbon, carbon-based material (for example, graphite, fullerene) It is preferable to use any of carbon nanotubes, mixtures thereof, or laminates thereof.
  • carbides such as hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, thorium carbide, titanium carbide, uranium carbide, tungsten carbide, zirconium carbide, molybdenum carbide, chromium carbide, vanadium carbide and the like may be used.
  • soft diamond generally refers to an incomplete diamond structure which is a mixture of diamond and carbon such as so-called diamond like carbon (DLC: Diamond Like Carbon), and the mixing ratio is not particularly limited.
  • the carbon layer is excellent in chemical stability and can withstand the subsequent reaction with the introduction of a chemical modification group and the binding to the analyte, and the bond is flexible because it is bound to the analyte by electrostatic binding. It is advantageous in that it has properties, is transparent to the detection system UV due to lack of UV absorption, and can be energized during electroblotting. In addition, it is also advantageous in that nonspecific adsorption is small in the binding reaction with the analyte.
  • the substrate itself may use a carrier composed of a carbon layer.
  • the formation of the carbon layer can be performed by a known method.
  • microwave plasma CVD Chemical vapor deposition
  • ECR CVD Electro cyclotron resonance chemical vapor deposition
  • ICP Inductive coupled plasma
  • DC sputtering ECR (Electric cyclotron resonance)
  • ionization deposition arc Deposition methods, laser deposition methods, electron beam (EB) deposition methods, resistance heating deposition methods, and the like.
  • a source gas (methane) is decomposed by glow discharge generated between electrodes by high frequency to synthesize a carbon layer on a substrate.
  • the source gas (benzene) is decomposed and ionized by using thermoelectrons generated by a tungsten filament, and a carbon layer is formed on a substrate by a bias voltage.
  • the carbon layer may be formed by ionization vapor deposition in a mixed gas consisting of 1 to 99% by volume of hydrogen gas and 99 to 1% by volume of methane gas.
  • an arc discharge is generated in vacuum by applying a direct current voltage between a solid graphite material (cathode evaporation source) and a vacuum vessel (anode) to generate a plasma of carbon atoms from the cathode and the evaporation source Furthermore, by applying a negative bias voltage to the substrate, carbon ions in the plasma can be accelerated toward the substrate to form a carbon layer.
  • a carbon layer can be formed by, for example, irradiating a target plate of graphite with Nd: YAG laser (pulse oscillation) light to melt and depositing carbon atoms on a glass substrate.
  • Nd: YAG laser pulse oscillation
  • the thickness of the carbon layer is usually from about 1 to 100 ⁇ m, and if it is too thin, the surface of the base substrate may be exposed locally. If this is the case, the productivity will deteriorate, so it is preferably 2 nm to 1 ⁇ m, more preferably 5 nm to 500 nm.
  • the oligonucleotide probe By introducing a chemical modification group on the surface of the substrate on which the carbon layer is formed, the oligonucleotide probe can be firmly immobilized on the carrier.
  • the chemical modification group to be introduced can be appropriately selected by those skilled in the art and is not particularly limited, and examples thereof include an amino group, a carboxyl group, an epoxy group, a formyl group, a hydroxyl group and an active ester group.
  • the introduction of the amino group can be carried out, for example, by irradiating the carbon layer with ultraviolet light in ammonia gas or by plasma treatment.
  • the carbon layer may be chlorinated by irradiating ultraviolet light in chlorine gas and further irradiating ultraviolet light in ammonia gas.
  • it can also be carried out by reacting with a chlorinated carbon layer in a polyvalent amines gas such as methylene diamine and ethylene diamine.
  • the introduction of the carboxyl group can be carried out, for example, by reacting the carbon layer aminated as described above with a suitable compound.
  • the compound used to introduce a carboxyl group is, for example, represented by the formula: X—R 1 —COOH (wherein, X represents a halogen atom, R 1 represents a divalent hydrocarbon group having 10 to 12 carbon atoms)
  • Halocarboxylic acids such as chloroacetic acid, fluoroacetic acid, bromoacetic acid, iodoacetic acid, 2-chloropropionic acid, 3-chloropropionic acid, 3-chloroacrylic acid, 4-chlorobenzoic acid; formula: HOOC-R2-COOH (formula In which R 2 represents a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms), such as oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, phthalic acid; polyacrylic acid And polyvalent
  • organic peracids include peracetic acid, perbenzoic acid, diperoxyphthalic acid, formic acid, and trifluoroperacetic acid.
  • the introduction of the formyl group can be carried out, for example, by reacting glutaraldehyde with the carbon layer aminated as described above.
  • the introduction of hydroxyl groups can be carried out, for example, by reacting water with the carbon layer chlorinated as described above.
  • the active ester group means an ester group having an electron withdrawing group with high acidity on the alcohol side of the ester group to activate a nucleophilic reaction, that is, an ester group with high reaction activity. It is an ester group having an electron withdrawing group on the alcohol side of the ester group and activated more than the alkyl ester.
  • the active ester group has reactivity with groups such as amino group, thiol group and hydroxyl group. More specifically, an active ester group in which phenol esters, thiophenol esters, N-hydroxyamine esters, cyanomethyl esters, esters of heterocyclic hydroxy compounds, etc. have much higher activity than alkyl esters etc.
  • examples of the active ester group include p-nitrophenyl group, N-hydroxysuccinimide group, succinimide group, phthalimido group, 5-norbornene-2,3-dicarboximide group and the like.
  • N-hydroxysuccinimide group is preferably used.
  • the introduction of the active ester group may be carried out, for example, with a dehydrating condensation agent such as cyanamide or carbodiimide (for example, 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide) and N-containing carboxyl group introduced as described above. It can be carried out by active esterification with a compound such as hydroxysuccinimide. By this treatment, it is possible to form a group in which an active ester group such as N-hydroxysuccinimide group is bonded to an end of a hydrocarbon group via an amide bond (Japanese Patent Laid-Open No. 2001-139532).
  • a dehydrating condensation agent such as cyanamide or carbodiimide (for example, 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide) and N-containing carboxyl group introduced as described above. It can be carried out by active esterification with a compound such as hydroxy
  • a wild type probe and a mutant type probe are dissolved in a spotting buffer to prepare a spotting solution, which is dispensed into a 96-well or 384-well plastic plate, and the dispensed solution is placed on a carrier by a spotter or the like
  • a spotting solution which is dispensed into a 96-well or 384-well plastic plate, and the dispensed solution is placed on a carrier by a spotter or the like
  • the spotting solution may be spotted manually with a micropipettor.
  • Incubation is usually performed at a temperature of ⁇ 20 to 100 ° C., preferably 0 to 90 ° C., usually for 0.5 to 16 hours, preferably 1 to 2 hours.
  • the incubation is preferably performed under an atmosphere of high humidity, for example, 50 to 90% humidity.
  • a washing solution eg, 50 mM TBS / 0.05% Tween 20, 2 ⁇ SSC / 0.2% SDS solution, ultrapure water, etc.
  • microarray configured as described above, it is possible to determine the genotype (wild-type, hetero-type or mutant-type) of the single nucleotide polymorphism to be detected in the diagnosis subject.
  • a step of extracting DNA from a sample derived from a subject to be diagnosed and a region containing the single nucleotide polymorphism, wherein the extracted DNA is a template The steps of amplifying and determining the genotype of single nucleotide polymorphism contained in the amplified nucleic acid using the above-mentioned microarray are included.
  • the subject to be diagnosed is usually a human, and is not particularly limited to the race and the like, but particularly the yellow race, preferably the East Asian race, particularly preferably the Japanese. Moreover, it can be set as the patient who is suspected of myeloproliferative tumor as a diagnostic subject.
  • the sample from the subject of diagnosis is not particularly limited.
  • blood related samples blood, serum, plasma, etc.
  • DNA is extracted from a sample collected from a subject to be diagnosed.
  • the extraction means is not particularly limited.
  • a DNA extraction method using phenol / chloroform, ethanol, sodium hydroxide, CTAB or the like can be used.
  • amplification reaction is carried out using the obtained DNA as a template to amplify a region containing single nucleotide polymorphism to be detected.
  • a polymerase chain reaction PCR
  • LAMP Long-Mediated Isothermal Amplification
  • ICAN Isothermal and Chimeric primer-Initiated Amplification of Nucleic acids
  • a method for labeling the amplified nucleic acid is not particularly limited.
  • a method in which a primer used for amplification reaction is previously labeled may be used, or a labeled nucleotide is used as a substrate for amplification reaction. Methods may be used.
  • the labeling substance is not particularly limited, and radioactive isotopes, fluorescent dyes, or organic compounds such as digoxigenin (DIG) and biotin can be used.
  • this reaction system includes a buffer necessary for nucleic acid amplification and labeling, a thermostable DNA polymerase, a primer specific to the amplification region, a labeled nucleotide triphosphate (specifically, a nucleotide triphosphate to which a fluorescent label or the like is added) , A nucleotide triphosphate, and a reaction system containing magnesium chloride and the like.
  • the nucleic acid fragment amplified by the primer is not particularly limited as long as it contains the regions corresponding to the designed wild-type probe and mutant-type probe, for example, 1 kbp or less is preferable, 800 bp or less is more preferably 500 bp or less More preferably, 350 bp or less is particularly preferred.
  • the genotype of the single nucleotide polymorphism in the subject can be determined.
  • the signal from the label can be detected as a fluorescent signal using a fluorescent scanner and analyzed by image analysis software to quantify the signal intensity.
  • the amplified nucleic acid hybridized to the wild-type probe and the mutant-type probe can also be quantified, for example, by preparing a calibration curve using a sample containing a known amount of DNA.
  • the hybridization reaction is preferably carried out under stringent conditions. Stringent conditions are conditions under which a specific hybrid is formed and a nonspecific hybrid is not formed, for example, after hybridization reaction at 55 ° C. for 16 hours, 2 ⁇ SSC / 0.2% SDS, 25 The conditions for washing at 10 ° C. for 10 minutes and 2 ⁇ SSC at 25 ° C.
  • the temperature for hybridization can be 40 to 80 ° C. when the salt concentration is 0.5 ⁇ SSC, and it is more preferable to lower the hybridization temperature when the length of the probe is short, When the chain length is long, it is more preferable to make the hybridization temperature higher. It goes without saying that the hybridization temperature having specificity increases as the salt concentration increases, and the hybridization temperature having specificity decreases as the salt concentration decreases.
  • the hybridization temperature is preferably the hybridization temperature at the binding rate calculated when designing the wild-type probe and the mutant-type probe described above.
  • the hybrids temperature it is preferable to set the hybrids temperature to 55 ° C.
  • the signal intensity from the mutant-type probe and the wild-type probe can be used to determine the genotype of the single nucleotide polymorphism.
  • the signal intensity in the wild type probe and the signal intensity in the mutant probe are each measured, and the judgment value for evaluating the signal intensity derived from the mutant probe is calculated.
  • the judgment value calculated by the above equation is compared with a predetermined threshold (cutoff value), and if the judgment value exceeds the first threshold, the single nucleotide polymorphism contained in the amplified nucleic acid is wild type If the judgment value is below the first threshold and above the second threshold, it is judged that the single nucleotide polymorphism is heterozygous in the amplified nucleic acid, and the judgment value is below the second threshold. In the case, it is judged that the single nucleotide polymorphism is a mutation in the amplified nucleic acid (first threshold> second threshold).
  • the first threshold and the second threshold it is determined that the sample whose single nucleotide polymorphism to be tested is determined to be wild type and the single nucleotide polymorphism to be tested are mutant. It can prescribe
  • a plurality of judgment values are calculated using a plurality of samples whose single nucleotide polymorphisms to be tested are determined to be mutant types, and the average value + 3 ⁇ ( ⁇ : standard deviation) is calculated as the second The threshold of In addition, the value of average value +2 (sigma) or average value + (sigma) can also be used as a threshold value.
  • a probe set for detecting the single nucleotide polymorphism was designed, taking a single nucleotide polymorphism of exon 5 of the CYP2C19 gene (CYP2C19 * 2) as an example.
  • the target containing the single nucleotide polymorphism was a wild type target (TTAAGTAATTTGTTATGGGTTCCcGGGAAATAATCAATGATAGTGGG: SEQ ID NO: 1, lower case is a polymorphic site) and a mutant target (TTAAGTAATTTGTTATGGGTTCCtGGGAAATAATCAATGATAGTGGG: lower case is a polymorphic site).
  • candidate wild-type probes (Table 1) and candidate mutant-type probes (Table 2) for detecting these wild-type targets were designed.
  • the bases corresponding to single nucleotide polymorphisms in Table 1 and Table 2 are shown in lower case letters.
  • Tm curves and Tm values for wild-type targets and mutant targets were calculated for the candidate wild-type probes and candidate mutant-type probes shown in Tables 1 and 2.
  • the Tm curve and Tm value were calculated by the WEB service provided by Integrated DNA Technologies.
  • the probe concentration and the target concentration were each set to 0.002 ⁇ M
  • the Na + and K + concentrations were set to 195 mM
  • the Mg 2+ concentration was set to 0 mM
  • the dNTPs concentration was set to 0 mM.
  • the binding rate at a temperature of 55 ° C. was calculated.
  • the results of calculating the binding rates for candidate wild-type probes are shown in Table 5, and the results for calculating the binding rates for candidate variant probes are shown in Table 6.
  • WP-FM means hybridization (perfect match: Full Match) of a candidate wild-type probe and a wild-type target
  • WP-MM means a candidate wild-type probe and a mutation. It means hybridization with single target (single base mismatch: Miss Match).
  • VP-FM means hybridization (full match) between a candidate mutant probe and a mutant target
  • VP-MM means a candidate mutant probe and It means hybridization with a wild-type target (single base mismatch: Miss Match).
  • the following combination could be selected as a wild-type probe and a mutant-type probe for highly accurately determining single nucleotide polymorphism of exon 5 of the CYP2C19 gene (CYP2C19 * 2) .
  • a probe set selected from the viewpoint of equalizing probe lengths
  • a probe set selected from the viewpoint that the Tm value is close to 55 ° C. (comparative example 2) Also used.
  • the probe sets of Comparative Example 1 are five sets of [WP_22 mer_R and VP_22 mer_R], [WP_22 mer_L and VP_22 mer_L], [WP_23 mer and VP_23 mer], [WP_24 mer_R and VP_24 mer_R] and [WP_24 mer_L and VP_24 mer_L].
  • the probe sets of Comparative Example 2 are five sets of [WP_19 mer and VP_23 mer], [WP_18 mer_L and VP_23 mer], [WP_19 mer and VP_23 mer], [WP_20 mer_R and VP_23 mer], and [WP_19 mer and VP_24 mer_R].
  • a DNA chip (CYP chip) was prepared in which the probe sets of these examples, the probe set of Comparative Example 1 and the probe set of Comparative Example 2 were arranged. Then, react wild type target, hetero type target or mutant type target with concentration 2 ⁇ M and CYP chip with 1x SSC, 0.1% SDS at hybridization temperature 55 ° C for 1 hour, and use fluorescence scanner (manufactured by BIOSHOT) for 7 seconds The fluorescence intensity was obtained.
  • a probe set having a large overall width value and a ratio close to 1 means that cross hybridization is unlikely to occur, and the overall width is smaller or the ratio is farther from 1 This means that it is possible to detect the single nucleotide polymorphism to be detected with higher accuracy as compared with the value of the probe set.
  • the overall width is larger and the ratio is larger than when the probe sets of Comparative Examples 1 and 2 are used. It is understood that it is close to 1. From this result, it is clear that when the probe set selected in this example is used, the occurrence of cross hybridization can be suppressed and the genotype of single nucleotide polymorphism can be determined more accurately.
  • the binding rate of the wild-type probe to the wild-type target full match binding rate
  • the binding rate of the mutant-type probe to the mutant target full match
  • the absolute value of the difference from the binding rate), and the absolute value of the difference between the binding rate of the mutant probe to the wild-type target summed up.
  • [WP_22mer_L and VP_24mer_L] total value: 0.06), [WP_23mer and VP_25mer] (total value: 0.07), [WP_22mer_L and VP_24mer_R] (total value: 0.08), [WP_23mer and VP_24mer_L] in ascending order of the total value (Total value: 0.20), [WP_23 mer and VP_24 mer_R] (total value: 0.21).
  • the probe set with a low total value has a larger overall width and a ratio closer to one. From this, when a plurality of probe sets for detecting single nucleotide polymorphisms are selected as described above, the total value is calculated, and it is more preferable to use a probe set having a lower total value. It turns out that it is preferable.

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Abstract

In order to suppress generation of cross-hybridization and to detect an exact match target with high accuracy, design is carried out by calculating the binding percentage for a candidate wild-type probe with a wild-type target or a mutant target and the binding percentage for a candidate mutant probe with a mutant target or wild-type target, and by having the value provided by subtracting, for the wild-type target, the binding percentage with the candidate mutant probe from the binding percentage with the candidate wild-type probe, and the value provided by subtracting, for the mutant target, the binding percentage with the candidate wild-type probe from the binding percentage with the candidate mutant probe, be equal to or greater than a prescribed value.

Description

一塩基多型検出用プローブの設計方法及びプローブセットDesign method and probe set for single nucleotide polymorphism detection probe
　本発明は、一塩基多型の野生型に対応する野生型プローブと当該一塩基多型の変異型に対応する変異型プローブとを設計する方法及びプローブセットに関する。 The present invention relates to a method and a probe set for designing a wild-type probe corresponding to a wild-type single nucleotide polymorphism and a mutant-type probe corresponding to a mutant single nucleotide polymorphism.
　一塩基多型（SNP : Single Nucleotide Polymorphism）とは、生物種集団のゲノム中に存在する遺伝子変異の一種であり、塩基配列における一塩基が変異した多様性を意味する。特に、集団内で１%以上の頻度で変異が見られる場合にのみ一塩基多型と呼称する場合もあるが、１%未満の頻度であっても一塩基多型と称することができる。なお、一塩基多型は、典型的には野生型アレルと一種類の変異型アレルとからなる二種類からなる場合が多いが、二種類以上の変異型アレルを有する場合もある。 Single nucleotide polymorphism (SNP: Single Nucleotide Polymorphism) is a type of gene mutation present in the genome of a population of species, and means a variation in which one base in a base sequence is mutated. In particular, a single nucleotide polymorphism may be referred to only when mutations are found at a frequency of 1% or more in a population, but a frequency of less than 1% can also be referred to as a single nucleotide polymorphism. In addition, although single nucleotide polymorphism usually consists of two types which consist of a wild type allele and one type of variant allele in many cases, it may have two or more types of variant alleles.
　一塩基多型を含む広義の多型は、薬剤に対する副作用の程度、疾患に対する罹りやすさといった表現型に関係している。また、植物においては、病害抵抗性、ストレス耐性、バイオマス量等の表現型にも一塩基多型を含む広義の多型が関係している。このように、一塩基多型を検出する（タイピングとも称する）ことで、疾患に罹患するリスクや、副作用の発生リスク等を予測することができる。 Broad polymorphisms including single nucleotide polymorphisms are associated with phenotypes such as the degree of side effects on drugs and susceptibility to diseases. Further, in plants, phenotypes such as disease resistance, stress tolerance, biomass amount, etc. are associated with broad polymorphisms including single nucleotide polymorphisms. As described above, by detecting a single nucleotide polymorphism (also referred to as typing), it is possible to predict the risk of suffering from a disease, the occurrence risk of side effects, and the like.
　一塩基多型を検出する方法としては、従来、SNPタイピング法として様々な手法が知られている。例えば、一塩基多型を含むDNA断片を増幅して塩基配列を読み取るダイレクトシーケンス法、一塩基多型部位を含むプローブを使用する方法が挙げられる。一塩基多型部位を含むプローブを使用する方法には、リアルタイムPCRを適用したサイクリングプローブ法、ビーズアレイ法やマイクロアレイ（DNAチップ）法が挙げられる。 Conventionally, various methods are known as a SNP typing method as a method of detecting a single nucleotide polymorphism. For example, a direct sequencing method of amplifying a DNA fragment containing single nucleotide polymorphism and reading a nucleotide sequence, a method of using a probe including a single nucleotide polymorphism site, and the like can be mentioned. Methods using probes containing single nucleotide polymorphism sites include cycling probe methods to which real-time PCR is applied, bead array methods and microarray (DNA chip) methods.
　一塩基多型部位を含むプローブを使用する方法では、検出対象の一塩基多型について、野生型の塩基に対応する野生型プローブと、変異型の塩基に対応する変異型プローブとを設計する。一般的に、プローブは、ターゲットDNAとの融解温度を含む熱力学的変数、長さ、分子内自己結合形成、他のプローブとの配列相同性の有無及び標的部位の位置（３’末端側若しくは５’末端側）等を考慮して設計される。これらのうち、熱力学的変数は、プローブとターゲットDNAとのハイブリダイズエネルギーを考慮する物であり、プローブ特性を表す指標とすることができる。 In the method of using a probe containing a single nucleotide polymorphism site, for a single nucleotide polymorphism to be detected, a wild type probe corresponding to a wild type base and a mutant type probe corresponding to a mutant base are designed. Generally, the probe is a thermodynamic variable including melting temperature with target DNA, length, intramolecular self-binding, presence or absence of sequence homology with other probes, and position of target site (3 'end side or 5 'end side) etc. are considered. Among these, the thermodynamic variable is one that considers the hybridization energy between the probe and the target DNA, and can be used as an index representing the probe characteristic.
　これら野生型プローブ及び変異型プローブを設計する方法としては、例えば特許文献１に記載されるように、完全一致ターゲットと二本鎖を形成したときのTm値が野生型プローブと変異型プローブとの間で等しくなるように設計する方法がある。また、特許文献２には、野生型プローブと変異型プローブとが同じ長さになるように設計する方法が記載されている。 As a method for designing these wild-type probes and mutant-type probes, for example, as described in Patent Document 1, the Tm value when a perfect match target and a double strand are formed is different between the wild-type probe and the mutant-type probe. There is a way to design them to be equal between them. Further, Patent Document 2 describes a method for designing a wild-type probe and a mutant-type probe to have the same length.
特開２０１０－４７８２号公報Unexamined-Japanese-Patent No. 2010-4782 特表平９－５０７１２１号公報Japanese Patent Publication No. 9-507121
　ところが、一塩基多型を検出する野生型プローブ及び変異型プローブは、いずれも一塩基を除いて同じ配列を有するため、完全一致するターゲットDNAとハイブリダイゼーション以外に非特異的な結合（クロスハイブリダイゼーションと称す）が形成される虞がある。クロスハイブリダイゼーションが生じた場合、完全一致のターゲットDNAが結合することによる真のシグナル値に、クロスハイブリダイゼーションによるシグナル値が加算される。すなわち、クロスハイブリダイゼーションが生じた場合、測定値にノイズが含まれ、測定結果の信頼性を大きく下げる要因となってしまう。 However, since both wild-type and mutant-type probes that detect single nucleotide polymorphisms have the same sequence except for one base, nonspecific binding (cross-hybridization) is possible other than hybridization with a completely identical target DNA. May be formed. When cross hybridization occurs, the signal value by cross hybridization is added to the true signal value by the binding of a perfectly matched target DNA. That is, when cross hybridization occurs, noise is included in the measurement value, which causes the reliability of the measurement result to be greatly reduced.
　そこで、本発明は、このような実情に鑑み、クロスハイブリダイゼーションの発生を抑え、完全一致のターゲットを高精度に検出することができる、一塩基多型検出用の野生型プローブ及び変異型プローブの設計方法及びプローブセットを提供することを目的とする。 Therefore, in view of such circumstances, the present invention provides a wild-type probe and a mutant-type probe for single nucleotide polymorphism detection, which can suppress the occurrence of cross hybridization and detect a perfectly matched target with high accuracy. It is an object to provide a design method and a probe set.
　本発明は以下を包含する。
　（１）一塩基多型の野生型に対応する野生型プローブと、当該一塩基多型の変異型に対応する変異型プローブとを設計する方法であって、上記一塩基多型部位を含む複数の候補野生型プローブと、上記一塩基多型部位を含む複数の候補変異型プローブとを設計し、これら候補野生型プローブについて完全一致となる野生型ターゲットとの結合率、これら候補野生型プローブについて当該一塩基多型部位において一塩基ミスマッチとなる変異型ターゲットとの結合率、これら候補変異型プローブについて完全一致となる変異型ターゲットとの結合率、及びこれら候補変異型プローブについて当該一塩基多型部位における一塩基ミスマッチとなる野生型ターゲットとの結合率をそれぞれ計算し、野生型ターゲットに関して、候補野生型プローブとの結合率から候補変異型プローブとの結合率を引いた値が所定の値以上となり、且つ、変異型ターゲットに関して、候補変異型プローブとの結合率から候補野生型プローブとの結合率を引いた値が所定の値以上となる野生型プローブと変異型プローブとの１又は複数の組み合わせを、候補野生型プローブ及び候補変異型プローブのなかから選択し、選択した野生型プローブと変異型プローブとの１又は複数の組み合わせを、当該一塩基多型を検出するための野生型プローブ及び変異型プローブとして設計する方法。 The present invention includes the following.
(1) A method of designing a wild-type probe corresponding to a wild-type single nucleotide polymorphism and a mutant-type probe corresponding to a mutation type of the single nucleotide polymorphism, comprising a plurality of single nucleotide polymorphism sites as described above Of the candidate wild-type probes of the present invention and a plurality of candidate mutant-type probes containing the single nucleotide polymorphism site, and the binding ratio to the wild-type target that is a perfect match for these candidate wild-type probes, The binding rate to a mutant target in which there is a single base mismatch at the single nucleotide polymorphism site, the binding rate to a mutant target in which there is a perfect match for these candidate mutant probes, and the single nucleotide polymorphism for these candidate mutant probes Calculate the binding rate with the wild-type target that causes a single base mismatch at each site, and for the wild-type target, The value obtained by subtracting the binding rate to the candidate mutant probe from the binding rate is equal to or higher than a predetermined value, and the value obtained by subtracting the binding rate to the candidate wild-type probe from the binding rate to the candidate mutant probe One or more combinations of a wild-type probe and a mutant-type probe for which the value of 以上 is a predetermined value or more is selected from the candidate wild-type probe and the candidate mutant-type probe, and one selected wild-type probe and the mutant-type probe Alternatively, a method of designing a plurality of combinations as a wild-type probe and a mutant-type probe for detecting the single nucleotide polymorphism.
　（２）上記結合率は、所定のハイブリダイズ温度若しくは所定のハイブリダイズ温度範囲において計算し、当該ハイブリダイズ温度若しくはハイブリダイズ温度範囲において使用する野生型プローブ及び変異型プローブを設計することを特徴とする（１）記載の方法。 (2) The binding rate is calculated at a predetermined hybridization temperature or a predetermined hybridization temperature range, and a wild-type probe and a mutant-type probe to be used at the hybridization temperature or the hybridization temperature range are designed. (1) The method described.
　（３）野生型プローブと変異型プローブとの複数の組み合わせを選択した場合、複数の組み合わせに関して、野生型ターゲットに対する野生型プローブの結合率と変異型ターゲットに対する変異型プローブの結合率との差の絶対値と、野生型ターゲットに対する変異型プローブの結合率と変異型ターゲットに対する野生型プローブの結合率との差の絶対値とを合計し、複数の組み合わせのうち当該合計値が最も低い組み合わせを、上記一塩基多型を検出するための野生型プローブ及び変異型プローブとして設計することを特徴とする（１）記載の方法。 (3) When multiple combinations of wild type probe and mutant type probe are selected, the difference between the binding ratio of wild type probe to wild type target and the binding ratio of mutant type probe to mutant target for multiple combinations The absolute value is added to the absolute value of the difference between the binding rate of the mutant probe to the wild-type target and the binding rate of the wild-type probe to the mutant target, and the combination having the lowest total value among the plurality of combinations is The method according to (1), which is designed as a wild-type probe and a mutant-type probe for detecting the single nucleotide polymorphism.
　（４）野生型プローブと変異型プローブとの複数の組み合わせを選択した場合、複数の組み合わせに関して、野生型ターゲットに対する野生型プローブの結合率と野生型ターゲットに対する変異型プローブの結合率との差が最も大きい組み合わせ、又は、変異型ターゲットに対する変異型プローブの結合率と変異型ターゲットに対する野生型プローブの結合率との差が最も大きい組み合わせを、上記一塩基多型を検出するための野生型プローブ及び変異型プローブとして設計することを特徴とする（１）記載の方法。 (4) When multiple combinations of wild type probe and mutant type probe are selected, the difference between the binding ratio of wild type probe to wild type target and the binding ratio of mutant type probe to wild type target for multiple combinations is A wild-type probe for detecting the single nucleotide polymorphism and a combination having the largest combination or the largest difference between the binding ratio of the mutant probe to the mutant target and the binding ratio of the wild-type probe to the mutant target The method according to (1), wherein the method is designed as a mutant-type probe.
　（５）一塩基多型の野生型に対応する野生型プローブと、当該一塩基多型の変異型に対応する変異型プローブとからなるプローブセットであって、上記一塩基多型部位を含む複数の候補野生型プローブと、上記一塩基多型部位を含む複数の候補変異型プローブとのなかから選択され、野生型ターゲットに関して、候補野生型プローブとの結合率から候補変異型プローブとの結合率を引いた値が所定の値以上となり、且つ、変異型ターゲットに関して、候補変異型プローブとの結合率から候補野生型プローブとの結合率を引いた値が所定の値以上となる野生型プローブと変異型プローブとからなる、当該一塩基多型を検出するためのプローブセット。 (5) A plurality of probe sets comprising a wild-type probe corresponding to a wild-type single nucleotide polymorphism and a mutant-type probe corresponding to a mutant single nucleotide polymorphism, the plurality comprising a plurality of single nucleotide polymorphism sites as described above Selected from among a plurality of candidate mutant-type probes including the single nucleotide polymorphism site and the candidate wild-type target from the ratio of the candidate wild-type probe to the candidate mutant-type probe And a wild-type probe for which the value obtained by subtracting the rate of binding to the candidate wild-type probe from the rate of binding to the candidate mutant-type probe is greater than or equal to the predetermined value. A probe set for detecting the single nucleotide polymorphism, which comprises a mutated probe.
　（６）担体と、当該担体に固定された（５）記載のプローブセットに含まれる野生型プローブ及び変異型プローブとを備えるDNAチップ。 (6) A DNA chip comprising a carrier, and a wild-type probe and a mutant-type probe contained in the probe set according to (5) immobilized on the carrier.
　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2017-241070号の開示内容を包含する。 The present specification includes the disclosure content of Japanese Patent Application No. 2017-241070 on which the priority of the present application is based.
　本発明に係る一塩基多型検出用の野生型プローブ及び変異型プローブの設計方法によれば、クロスハイブリダイゼーションの発生が抑えられた、ターゲットDNAの検出感度に優れた野生型プローブ及び変異型プローブを設計することができる。 According to the method for designing a wild-type probe and a mutant-type probe for single nucleotide polymorphism detection according to the present invention, a wild-type probe and a mutant-type probe excellent in detection sensitivity of target DNA in which occurrence of cross hybridization is suppressed Can be designed.
　また、本発明に罹るプライマーセットは、クロスハイブリダイゼーションの発生が抑えられているため、ターゲットDNAの検出感度に優れたものとなる。 In addition, since the generation of cross hybridization is suppressed, the primer set according to the present invention is excellent in the detection sensitivity of target DNA.
行に候補野生型プローブの種類、列に候補変異型プローブの種類を示し、候補野生型プローブにおけるフルマッチ結合率から、候補変異型プローブにおけるミスマッチ結合率を引いた値を各マスに示す特性図である。The characteristic wild type indicates the type of candidate wild-type probe in the row and the type of candidate mutant-type probe in the row, and the value obtained by subtracting the mismatch binding rate in the candidate mutant-type probe from the full match binding rate in the candidate wild-type probe is there. 行に候補野生型プローブの種類、列に候補変異型プローブの種類を示し、候補野生型プローブにおけるフルマッチ結合率から、候補変異型プローブにおけるミスマッチ結合率を引いた値を各マスに示す特性図である。The characteristic wild type indicates the type of candidate wild-type probe in the row and the type of candidate mutant-type probe in the row, and the value obtained by subtracting the mismatch binding rate in the candidate mutant-type probe from the full match binding rate in the candidate wild-type probe is there. 行に候補野生型プローブの種類、列に候補変異型プローブの種類を示し、候補野生型プローブにおけるフルマッチ結合率から、候補変異型プローブにおけるミスマッチ結合率を引いた値を各マスに示す特性図である。The characteristic wild type indicates the type of candidate wild-type probe in the row and the type of candidate mutant-type probe in the row, and the value obtained by subtracting the mismatch binding rate in the candidate mutant-type probe from the full match binding rate in the candidate wild-type probe is there. 行に候補野生型プローブの種類、列に候補変異型プローブの種類を示し、候補変異型プローブにおけるフルマッチ結合率から、候補野生型プローブにおけるミスマッチ結合率を引いた値を各マスに示す特性図である。A characteristic chart showing the type of candidate wild-type probe in the row and the type of candidate mutant-type probe in the row, and the value obtained by subtracting the mismatch binding rate in the candidate wild-type probe from the full match binding rate in the candidate mutant-type probe is there. 行に候補野生型プローブの種類、列に候補変異型プローブの種類を示し、候補変異型プローブにおけるフルマッチ結合率から、候補野生型プローブにおけるミスマッチ結合率を引いた値を各マスに示す特性図である。A characteristic chart showing the type of candidate wild-type probe in the row and the type of candidate mutant-type probe in the row, and the value obtained by subtracting the mismatch binding rate in the candidate wild-type probe from the full match binding rate in the candidate mutant-type probe is there. 行に候補野生型プローブの種類、列に候補変異型プローブの種類を示し、候補変異型プローブにおけるフルマッチ結合率から、候補野生型プローブにおけるミスマッチ結合率を引いた値を各マスに示す特性図である。A characteristic chart showing the type of candidate wild-type probe in the row and the type of candidate mutant-type probe in the row, and the value obtained by subtracting the mismatch binding rate in the candidate wild-type probe from the full match binding rate in the candidate mutant-type probe is there. 実施例のプローブセット、比較例１のプローブセット及び比較例２のプローブセットを使用して遺伝子型判定実験を行った結果を示す特性図である。It is a characteristic view showing the result of having conducted the genotyping experiment using the probe set of an example, the probe set of comparative example 1, and the probe set of comparative example 2. 実施例のプローブセット、比較例１のプローブセット及び比較例２のプローブセットを使用した遺伝子型判定実験の結果に基づいて、各プローブセットを使用した時の全幅及び比との関係を示す特性図である。Characteristic chart showing the relationship between the total width and the ratio when using each probe set based on the results of the genotyping experiment using the probe set of the example, the probe set of the comparative example 1, and the probe set of the comparative example 2 It is.
　本発明では、一塩基多型検出用の野生型プローブ及び変異型プローブの設計方法を提供する。ここで、一塩基多型とは、生物種集団のゲノム中に存在する遺伝子変異であって、塩基配列における一塩基の変異を意味する。本発明において、一塩基多型とは集団内で１％以上の頻度で見られる変異に限定されず、１％未満の頻度で見られる変異（突然変異と呼称される場合もある）をも含む意味である。 The present invention provides a method for designing a wild-type probe and a mutant-type probe for single nucleotide polymorphism detection. Here, a single nucleotide polymorphism is a gene mutation present in the genome of a species population, and means a single base mutation in the base sequence. In the present invention, single nucleotide polymorphisms are not limited to mutations found at a frequency of 1% or more in a population, but also include mutations found at a frequency of less than 1% (sometimes referred to as mutations). It is a meaning.
　野生型プローブとは、検出対象の一塩基多型における野生型アレルに対応し、野生型アレルの遺伝子型に相補的な塩基を有するプローブである。変異型プローブとは、検出対象の一塩基多型における変異型アレルに対応し、変異型アレルの遺伝子型に相補的な塩基を有するプローブである。例えば、野生型アレルの遺伝子型がA（アデニン）であり、変異型アレルの遺伝子型がG（グアニン）である一塩基多型を検出対象とする場合、野生型プローブは一塩基多型に対応する位置にAに相補的なTを含み、変異型プローブは当該位置にGに相補的なCを含む。なお、所定の一塩基多型について設計された、野生型プローブ及び変異型プローブの組み合わせをプローブセットと称する。 A wild-type probe is a probe that corresponds to a wild-type allele in a single nucleotide polymorphism to be detected and has a base complementary to the genotype of the wild-type allele. A mutant-type probe is a probe that corresponds to a mutant-type allele in a single nucleotide polymorphism to be detected and has a base that is complementary to the genotype of the mutant-type allele. For example, when a single nucleotide polymorphism in which the wild type allele genotype is A (adenine) and the mutant allele genotype is G (guanine) is to be detected, the wild type probe corresponds to the single nucleotide polymorphism Contains a T that is complementary to A at the position where the mutant probe contains a C that is complementary to G at that position. The combination of a wild-type probe and a mutant-type probe designed for a given single nucleotide polymorphism is referred to as a probe set.
　本発明に係る設計方法では、先ず、野生型プローブ及び変異型プローブを選択するため、複数の野生型プローブの候補（候補野生型プローブ）及び複数の変異型プローブ（候補変異型プローブ）を設計する。具体的に、本工程では、検出対象の一塩基多型を含む周辺のゲノム配列から、複数の候補野生型プローブ及び複数の変異型プローブを、一塩基多型を含むオリゴヌクレオチドの塩基配列として設計する。 In the design method according to the present invention, first, in order to select a wild-type probe and a mutant-type probe, a plurality of wild-type probe candidates (candidate wild-type probe) and a plurality of mutant-type probes (candidate mutant-type probes) are designed. . Specifically, in this step, a plurality of candidate wild-type probes and a plurality of mutant-type probes are designed as nucleotide sequences of oligonucleotides containing single nucleotide polymorphisms, from surrounding genomic sequences containing single nucleotide polymorphisms to be detected. Do.
　設計する候補野生型プローブ及び候補変異型プローブの長さとしては、特に限定されないが、例えば５～５０塩基長とすることができ、７～４０塩基長とすることが好ましく、１０～３０塩基長とすることがより好ましく、１１～２５塩基長とすることが最も好ましい。 The lengths of candidate wild-type probes and candidate mutant-type probes to be designed are not particularly limited, and can be, for example, 5 to 50 bases, preferably 7 to 40 bases, and 10 to 30 bases in length. More preferably, the length is 11 to 25 bases, and most preferably 11 to 25 bases.
　また、設計する候補野生型プローブ及び候補変異型プローブにおいて、検出対象の一塩基多型に対応する塩基は、設計する塩基配列における略中心に位置するように設計することが好ましい。設計する塩基配列が奇数個の塩基からなる場合、５’末端と３’末端の中央の１つの塩基を一塩基多型に対応する位置とすることができる。また、設計する塩基配列が偶数個の塩基からなる場合、５’末端と３’末端の中央の２塩基のうちいずれかを一塩基多型に対応する位置とすることができる。 Further, in the candidate wild-type probe and the candidate mutant-type probe to be designed, it is preferable to design the base corresponding to the single nucleotide polymorphism to be detected to be located approximately at the center in the designed nucleotide sequence. When the designed nucleotide sequence consists of an odd number of bases, one central base at the 5 'end and the 3' end can be at a position corresponding to the single nucleotide polymorphism. In addition, when the designed base sequence comprises an even number of bases, any one of the central two bases at the 5 'end and the 3' end can be taken as the position corresponding to the single nucleotide polymorphism.
　ただし、設計する候補野生型プローブ及び候補変異型プローブにおいて、検出対象の一塩基多型に対応する塩基は、上述のように設計する塩基配列における略中心に限定されず、当該略中心から５’末端側又は３’末端側に偏った位置としてもよい。例えば、設計する候補野生型プローブ及び候補変異型プローブにおいて、検出対象の一塩基多型に対応する塩基は、設計する塩基配列における略中心から、全長の１／４の範囲、好ましくは１／５の範囲、より好ましくは１／６の範囲で５’末端側又は３’末端側に偏った位置とすることができる。 However, in the candidate wild-type probe and the candidate mutant-type probe to be designed, the base corresponding to the single nucleotide polymorphism to be detected is not limited to the approximate center in the nucleotide sequence to be designed as described above. The position may be biased toward the end side or the 3 'end side. For example, in the candidate wild-type probe and the candidate mutant-type probe to be designed, the base corresponding to the single nucleotide polymorphism to be detected is in the range of 1⁄4, preferably 1⁄5 of the full length from the approximate center in the designed nucleotide sequence. The position may be biased toward the 5 'end or the 3' end in the range of 1, preferably in the range of 1/6.
　また、候補野生型プローブ及び候補変異型プローブを設計する際、検出対象の一塩基多型に対応する塩基を略中心とした候補野生型プローブ及び候補変異型プローブのみを設計しても良いし、検出対象の一塩基多型に対応する塩基を略中心から５’末端側又は３’末端側に偏った位置とした候補野生型プローブ及び候補変異型プローブをのみを設計しても良い。さらに、候補野生型プローブ及び候補変異型プローブを設計する際、検出対象の一塩基多型に対応する塩基を略中心とした候補野生型プローブ及び候補変異型プローブと、検出対象の一塩基多型に対応する塩基を略中心から５’末端側又は３’末端側に偏った位置とした候補野生型プローブ及び候補変異型プローブを設計しても良い。 Also, when designing the candidate wild-type probe and the candidate mutant-type probe, only the candidate wild-type probe and the candidate mutant-type probe may be designed with the base corresponding to the single nucleotide polymorphism to be detected as the approximate center. Only the candidate wild-type probe and the candidate mutant-type probe may be designed with the base corresponding to the single nucleotide polymorphism to be detected at a position shifted from the approximate center to the 5 'end or the 3' end. Furthermore, when designing a candidate wild-type probe and a candidate mutant-type probe, a candidate wild-type probe and a candidate mutant-type probe substantially centered on a base corresponding to a single nucleotide polymorphism to be detected, and a single nucleotide polymorphism to be detected A candidate wild-type probe and a candidate mutant-type probe may be designed in which the base corresponding to is located approximately at the 5 'end or the 3' end from the center.
　次に、本発明に係る設計方法では、設計した候補野生型プローブ及び候補変異型プローブについて、完全一致となるターゲットDNAとTm曲線及び一塩基ミスマッチとなるターゲットDNAとのTm曲線をそれぞれ計算する。 Next, in the design method according to the present invention, for the designed candidate wild-type probe and candidate mutant-type probe, Tm curves of a target DNA and a Tm curve to be a perfect match and a target DNA to be a single base mismatch are respectively calculated.
　ここで、候補野生型プローブについては、完全一致となるターゲットDNAは、検出対象の一塩基多型が野生型アリルである野生型のターゲットDNA（野生型ターゲットと称する）である。また、候補野生型プローブについては、一塩基ミスマッチとなるターゲットDNAは、検出対象の一塩基多型が変異型アリルである変異型のターゲットDNA（変異型ターゲットと称する）である。一方、候補変異型プローブについては、完全一致となるターゲットDNAは、検出対象の一塩基多型が変異型アリルである変異型ターゲットである。また、候補変異型プローブについては、一塩基ミスマッチとなるターゲットDNAは、検出対象の一塩基多型が野生型アリルである野生型ターゲットである。 Here, for the candidate wild-type probe, the target DNA to be a perfect match is a wild-type target DNA (referred to as a wild-type target) whose single nucleotide polymorphism to be detected is a wild-type allele. In addition, for the candidate wild-type probe, the target DNA that causes a single base mismatch is a mutant target DNA (referred to as a mutant target) in which the single nucleotide polymorphism to be detected is a mutant allele. On the other hand, for the candidate mutant-type probes, the target DNA that is a perfect match is a mutant-type target whose single nucleotide polymorphism to be detected is a mutant allele. In addition, for the candidate mutant-type probe, the target DNA that causes a single base mismatch is a wild-type target in which a single nucleotide polymorphism to be detected is a wild-type allele.
　Tm曲線を計算する方法は、特に限定されないが、例えば、最近接塩基対法(Nearest Neighbor method)、Wallace法及びGC％法等を挙げることができるが、特に最近接塩基対法(Nearest Neighbor method)により計算することが好ましい。なお、Tmとは、プローブとターゲットがハイブリダズしているとき、その５０％が解離する時の温度と定義される（すなわち、結合率５０％の時の温度）。また、Tm値に影響を与える要因としては、塩基組成、塩濃度、オリゴ鎖の濃度や変性剤（ホルムアミド、DMSO等）、溶媒和効果、コンジュゲート基（ビオチン、ジゴキシゲニン、アルカリフォスファターゼ、蛍光色素等）が挙げられる。 The method of calculating the Tm curve is not particularly limited, and examples thereof include the Nearest Neighbor method, the Wallace method, the GC% method, and the like, and in particular, the Nearest Neighbor method. It is preferable to calculate by. Tm is defined as the temperature at which 50% of the probe and the target are dissociated (ie, the temperature at a binding rate of 50%) when the probe and target are hybridized. In addition, factors that affect the Tm value include base composition, salt concentration, concentration of oligo chain, denaturing agent (formamide, DMSO, etc.), solvation effect, conjugate group (biotin, digoxigenin, alkaline phosphatase, fluorescent dye, etc. Can be mentioned.
　より具体的には、Integrated DNA Technologies社が提供するWEBサービスによりTm値を計算することができ、これにはプローブの塩基配列、プローブ濃度、プローブとハイブリダズするターゲットの濃度、反応液中のNa+並びにK+濃度、反応液中のMg2+濃度及び（場合によっては）dNTPs濃度を設定することでTm曲線を計算することができる。例えば、プローブ濃度並びにターゲット濃度をそれぞれ0.002μMとし、Na+並びにK+濃度を195mMとしMg2+濃度を0mMとし、dNTPs濃度を0mMとしてTm曲線を計算することができる。計算したTm曲線は、一方軸（例えば縦軸）を結合率とし、他方軸（例えば横軸）を温度した２次元平面に表示することもできる。 More specifically, the Tm value can be calculated by the WEB service provided by Integrated DNA Technologies, including the base sequence of the probe, the concentration of the probe, the concentration of the target to be hybridized with the probe, and Na + in the reaction solution. The Tm curve can be calculated by setting the K + concentration, the Mg 2+ concentration in the reaction solution, and the (optionally) dNTPs concentration. For example, Tm curves can be calculated with probe concentration and target concentration of 0.002 μM, Na + and K + concentrations of 195 mM, Mg 2+ concentration of 0 mM, and dNTPs concentration of 0 mM. The calculated Tm curve can also be displayed in a two-dimensional plane in which one axis (e.g., the vertical axis) is the binding rate and the other axis (e.g., the horizontal axis) is the temperature.
　得られたTm曲線は、所定の塩基配列を有する候補野生型プローブ又は候補変異型プローブと、野生型ターゲット又は変異型ターゲットとに関して、温度と結合率との関係を示すことができる。すなわち、得られたTm曲線に基づいて、上述のように設計した候補野生型プローブにおける野生型ターゲット（完全一致）との結合率を計算することができる。同様に、得られたTm曲線に基づいて、候補野生型プローブにおける変異型ターゲット（一塩基ミスマッチ）との結合率を計算することができる。同様に、得られたTm曲線に基づいて、候補変異型プローブにおける変異型ターゲット（完全一致）との結合率を計算することができる。同様に、得られたTm曲線に基づいて、候補変異型プローブにおける野生型ターゲット（一塩基ミスマッチ）との結合率を計算することができる。 The obtained Tm curve can show the relationship between temperature and the binding rate for a candidate wild-type probe or a candidate mutant-type probe having a predetermined base sequence and a wild-type target or a mutant-type target. That is, based on the obtained Tm curve, the binding rate with the wild-type target (perfect match) in the candidate wild-type probe designed as described above can be calculated. Similarly, based on the obtained Tm curve, the binding rate of a candidate wild-type probe to a mutant target (single base mismatch) can be calculated. Similarly, based on the obtained Tm curve, the binding rate of the candidate mutant probe to the mutant target (perfect match) can be calculated. Similarly, based on the obtained Tm curve, it is possible to calculate the binding ratio of the candidate mutant probe to the wild-type target (single base mismatch).
　次に、本発明に係る設計方法では、計算された結合率に基づいて、候補野生型プローブ及び候補変異型プローブのなかから、検出対象の一塩基多型に最適な野生型プローブと変異型プローブとを選択する。このとき、上述のように計算された結合率は、所定の温度或いは所定の温度範囲における結合率として計算することができる。例えば、一般的にDNAチップを用いた解析方法では、ターゲットDNAをプローブとハイブリダズさせる温度として４０～８０℃、好ましくは５０～７０℃、より好ましくは５０～６０℃の範囲に設定される。したがって、これら温度範囲に含まれる、例えば５５℃における結合率を計算し、５５℃における結合率に基づいて、検出対象の一塩基多型に最適な野生型プローブと変異型プローブとを選択することが好ましい。 Next, in the design method according to the present invention, a wild-type probe and a mutant-type probe most suitable for a single nucleotide polymorphism to be detected among candidate wild-type probes and candidate mutant-type probes based on the calculated binding rate. And choose. At this time, the binding rate calculated as described above can be calculated as the binding rate at a predetermined temperature or a predetermined temperature range. For example, in the analysis method generally using a DNA chip, the temperature at which target DNA is hybridized with the probe is set in the range of 40 to 80 ° C., preferably 50 to 70 ° C., more preferably 50 to 60 ° C. Therefore, calculate the binding rate at, for example, 55 ° C. included in these temperature ranges, and select a wild-type probe and a mutant-type probe that are optimal for the single nucleotide polymorphism to be detected based on the binding rate at 55 ° C. Is preferred.
　具体的に、例えば、５５℃において、野生型ターゲットに関して、候補野生型プローブとの結合率から候補変異型プローブとの結合率を引いた値が所定の値以上となり、且つ、変異型ターゲットに関して、候補変異型プローブとの結合率から候補野生型プローブとの結合率を引いた値が所定の値以上となる野生型プローブと変異型プローブとの１又は複数の組み合わせを選択する。 Specifically, for example, at 55 ° C., with respect to a wild-type target, a value obtained by subtracting the binding rate with a candidate mutant-type probe from the binding rate with a candidate wild-type probe becomes a predetermined value or more. One or more combinations of a wild-type probe and a mutant-type probe are selected such that the value obtained by subtracting the ratio of binding to the candidate wild-type probe from the ratio of binding to the candidate mutant-type probe is greater than a predetermined value.
　ここで、野生型ターゲットに関して候補野生型プローブとの結合率とは、候補野生型プローブについて完全一致するターゲットに対する結合率（フルマッチ結合率）を意味する。また、野生型ターゲットに関して候補変異型プローブとの結合率とは、候補変異型プローブについて一塩基相違するターゲットに対する結合率（ミスマッチ結合率）を意味する。同様に、変異型ターゲットに関して候補野生型プローブとの結合率とは、候補野生型プローブについて一塩基相違するターゲットに対する結合率（ミスマッチ結合率）を意味する。また、変異型ターゲットに関して候補変異型プローブとの結合率とは、候補変異型プローブについて完全一致するターゲットに対する結合率（フルマッチ結合率）を意味する。 Here, the binding rate to a candidate wild-type probe with respect to a wild-type target means the binding rate (full-match binding rate) to a target that completely matches the candidate wild-type probe. Moreover, the binding rate with a candidate mutant-type probe with respect to a wild-type target means the binding rate (mismatch binding rate) with respect to a target that differs by one base from the candidate mutant-type probe. Similarly, the binding rate to a candidate wild-type probe with respect to a mutant target means the binding rate (mismatch binding rate) to a target that differs by one base from the candidate wild-type probe. In addition, the binding rate with a candidate mutant probe with respect to a mutant target means the binding rate (full match binding rate) to a target that completely matches the candidate mutant probe.
　ここで、野生型ターゲットに関して、候補野生型プローブとの結合率（フルマッチ結合率）と候補変異型プローブとの結合率（ミスマッチ結合率）を引いた値は、例えば、０．３（但し、結合率は０．００～１．００の値を取る）以上とすることができ、０．４以上とすることが好ましく、０．５以上とすることがより好ましい。 Here, for the wild-type target, the value obtained by subtracting the binding rate (full match binding rate) with the candidate wild-type probe and the binding rate (mismatch binding rate) with the candidate mutant-type probe is, for example, 0.3 (but with binding The rate can be a value of 0.00 to 1.00) or more, preferably 0.4 or more, and more preferably 0.5 or more.
　また、変異型ターゲットに関して、候補変異型プローブとの結合率（フルマッチ結合率）と候補野生型プローブとの結合率（ミスマッチ結合率）を引いた値は、例えば、０．３以上とすることができ、０．４以上とすることが好ましく、０．５以上とすることがより好ましい。 In addition, with regard to the mutant target, the value obtained by subtracting the binding rate (full match binding rate) with the candidate mutant type probe and the binding rate (mismatch binding rate) with the candidate wild type probe may be, for example, 0.3 or more It is preferably 0.4 or more, more preferably 0.5 or more.
　このように、結合率に基づいて、候補野生型プローブ及び候補変異型プローブの中から、上述した条件に合致する野生型プローブと変異型プローブとの１又は複数の組み合わせを選択することができる。そして、選択した野生型プローブと変異型プローブとの１又は複数の組み合わせ、検出対象の一塩基多型を検出するための野生型プローブ及び変異型プローブとして設計することができる。選択した野生型プローブと変異型プローブとの１又は複数の組み合わせは、検出対象の一塩基多型を検出する際に、クロスハイブリダイゼーション発生の確率が最も低い組み合わせであり、当該一塩基多型を高精度に検出（SNPタイピング）することができる。 Thus, based on the binding rate, one or more combinations of a wild-type probe and a mutant-type probe can be selected from the candidate wild-type probe and the candidate mutant-type probe that meet the above-described conditions. Then, it can be designed as one or more combinations of a selected wild-type probe and a mutant-type probe, a wild-type probe and a mutant-type probe for detecting a single nucleotide polymorphism to be detected. The combination of one or more of the selected wild-type probe and the mutant-type probe is a combination having the lowest probability of occurrence of cross hybridization when detecting a single nucleotide polymorphism to be detected. It can be detected with high accuracy (SNP typing).
　特に、上述した条件に合致する野生型プローブと変異型プローブとの組み合わせが複数選択できた場合、以下に説明する手順に従ってより優れた野生型プローブと変異型プローブの組み合わせを更に選択しても良い。 In particular, when a plurality of combinations of wild-type probes and mutant-type probes that can meet the conditions described above can be selected, a combination of better wild-type probes and mutant-type probes may be further selected according to the procedure described below. .
　すなわち、選択した野生型プローブと変異型プローブとの複数の組み合わせに関して、野生型ターゲットに対する野生型プローブの結合率（フルマッチ結合率）と変異型ターゲットに対する変異型プローブの結合率（フルマッチ結合率）との差の絶対値と、野生型ターゲットに対する変異型プローブの結合率（ミスマッチ結合率）と変異型ターゲットに対する野生型プローブの結合率（ミスマッチ結合率）との差の絶対値とを合計する。そして、複数の組み合わせのうち当該合計値が最も低い組み合わせを選択する。当該合計値が最も低い野生型プローブと変異型プローブとの組み合わせは、検出対象の一塩基多型に関して、野生型、ヘテロ型及び変異型の判定値が均等に分離され、より高精度に一塩基多型を検出することができる。 That is, the binding rate (full match binding rate) of the wild type probe to the wild type target and the binding rate (full match binding rate) of the mutant probe to the variant target for a plurality of combinations of the selected wild type probe and the mutant type probe And the absolute value of the difference between the binding rate of the mutant probe to the wild type target (mismatch binding rate) and the binding rate of the wild type probe to the mutant target (mismatch binding rate). Then, the combination having the lowest total value is selected from the plurality of combinations. The combination of a wild-type probe and a mutant-type probe with the lowest total value can be obtained by uniformly separating the wild-type, hetero-type and mutant-type determination values with respect to single nucleotide polymorphisms to be detected. Polymorphism can be detected.
　また、上述した条件に合致する野生型プローブと変異型プローブとの組み合わせが複数選択できた場合、より優れた野生型プローブと変異型プローブの組み合わせを更に選択するには上述した手順に限定されず、例えば以下に説明する如何なる手順に従っても良い。 In addition, when a plurality of combinations of the wild-type probe and the mutant-type probe that can meet the above-described conditions can be selected, it is not limited to the above-described procedure to further select the superior combination of the wild-type probe and the mutant-type probe For example, any procedure described below may be followed.
　すなわち、選択した野生型プローブと変異型プローブとの複数の組み合わせに関して、野生型ターゲットに対する野生型プローブの結合率（フルマッチ結合率）と野生型ターゲットに対する変異型プローブの結合率（ミスマッチ結合率）との差が最も大きい組み合わせを選択する。こうして選択した野生型プローブと変異型プローブとの組み合わせは、検出対象の一塩基多型に関して、野生型（A群）と、ヘテロ型と変異型（B群）との判定値が大きく分離され、A群とB群をより高精度に検出することができる。 That is, the binding rate (full match binding rate) of the wild type probe to the wild type target and the binding rate (mismatch binding rate) of the mutant probe to the wild type target for a plurality of combinations of the selected wild type probe and the mutant type probe Choose the combination with the largest difference. In the combination of the wild-type probe and the mutant-type probe thus selected, judgment values of the wild-type (group A) and the hetero-type and the mutant-type (group B) are largely separated with respect to single nucleotide polymorphisms to be detected. Groups A and B can be detected with higher accuracy.
　もしくは、選択した野生型プローブと変異型プローブとの複数の組み合わせに関して、変異型ターゲットに対する変異型プローブの結合率（フルマッチ結合率）と変異型ターゲットに対する野生型プローブの結合率（ミスマッチ結合率）との差が最も大きい組み合わせを選択する。こうして選択した野生型プローブと変異型プローブとの組み合わせは、検出対象の一塩基多型に関して、野生型とヘテロ型（C群）と、変異型（D群）との判定値が大きく分離され、C群とD群をより高精度に検出することができる。 Alternatively, for multiple combinations of the selected wild-type probe and the mutant-type probe, the binding rate of the mutant-type probe to the mutant-type target (full match binding rate) and the binding ratio of the wild-type probe to the mutant-type target (mismatch binding rate) Choose the combination with the largest difference. In the combination of the wild-type probe and the mutant-type probe thus selected, the judgment values of the wild-type and hetero-type (group C) and the mutant-type (group D) are largely separated with respect to the single nucleotide polymorphism to be detected. Groups C and D can be detected more accurately.
　野生型プローブと変異型プローブとの複数の組み合わせのなかから、上述のように、野生型（A群）とヘテロ型と変異型（B群）とをより高精度に検出できる野生型プローブと変異型プローブの組み合わせは、例えば優性変異に関する一塩基多型が検出対象である場合に有効である。 Among multiple combinations of wild-type probes and mutant-type probes, as described above, wild-type probes and mutations that can detect wild-type (group A), hetero-type and mutant-type (group B) with higher accuracy The combination of type probes is effective, for example, when a single nucleotide polymorphism for a dominant mutation is to be detected.
　飲酒後、アルコールから生成した毒性の強いアセトアルデヒドは、いくつかの酵素の触媒作用で解毒される。この過程にはALDH2という酵素が最も重要な役割を果たす。この酵素をコードする遺伝子については、第12番染色体エキソン12に位置するGからAへの一塩基多型が酵素活性に重大な影響を及ぼすことが知られている。この一塩基多型は「優性 dominant」であり、ヘテロ接合体(ALDH2*1)又は変異型(ALDH2*2)では、野生型に比べ10%以下しか酵素活性がないことが明らかになっている。 After drinking, the highly toxic acetaldehyde generated from alcohol is detoxified by the catalysis of several enzymes. The enzyme ALDH2 plays the most important role in this process. For the gene encoding this enzyme, it is known that a single nucleotide polymorphism from G to A located in exon 12 of chromosome 12 has a significant effect on the enzyme activity. This single nucleotide polymorphism is "dominant dominant", and heterozygotes (ALDH2 * 1) or variants (ALDH2 * 2) have been shown to have no more than 10% enzymatic activity compared to wild type .
　ALDH2の酵素活性を当該一塩基多型で判断する場合、上述した野生型（A群）とヘテロ型と変異型（B群）とをより高精度に検出できる野生型プローブと変異型プローブの組み合わせを使用することが好ましい。 When the enzyme activity of ALDH2 is determined by the single nucleotide polymorphism, a combination of a wild-type probe and a mutant-type probe that can detect the wild-type (group A), the hetero-type and the mutant (group B) more accurately It is preferred to use
　ところで、上述のように設計した野生型プローブ及び変異型プローブは、好ましくは核酸であり、より好ましくはDNAである。DNAには二本鎖も一本鎖も含まれるが、好ましくは一本鎖DNAである。野生型プローブ及び変異型プローブは、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。 By the way, the wild-type probe and the mutant-type probe designed as described above are preferably nucleic acids, more preferably DNA. The DNA may be double stranded or single stranded but is preferably single stranded DNA. The wild-type probe and the mutant-type probe can be obtained, for example, by chemical synthesis with a nucleic acid synthesizer. As the nucleic acid synthesizer, an apparatus called a DNA synthesizer, a fully automatic nucleic acid synthesizer, an automatic nucleic acid synthesizer or the like can be used.
　上述のように設計した野生型プローブ及び変異型プローブは、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（一例としてDNAチップ）の形態で用いるのが好ましい。このとき、マイクロアレイは、検出対象の一塩基多型が複数である場合、各一塩基多型について所定の位置に変異型プローブ及び野生型プローブを有する。 The wild-type probe and the mutant-type probe designed as described above are preferably used in the form of a microarray (as an example, a DNA chip) by immobilizing the 5 'end thereof on a carrier. At this time, when there are a plurality of single nucleotide polymorphisms to be detected, the microarray has a mutant-type probe and a wild-type probe at a predetermined position for each single nucleotide polymorphism.
　本発明に係るマイクロアレイは、上述した野生型プローブ及び変異型プローブを担体上に固定することで作製することができる。 The microarray according to the present invention can be prepared by immobilizing the above-mentioned wild-type probe and mutant-type probe on a carrier.
　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－ボンファイバ－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。 As the material of the carrier, those known in the art can be used without particular limitation. For example, noble metals such as platinum, platinum black, gold, palladium, rhodium, silver, mercury, tungsten and compounds thereof, and conductive materials such as carbon represented by graphite and carbon fiber; single crystal silicon, amorphous Silicon materials represented by silicon, silicon carbide, silicon oxide, silicon nitride etc., and composite materials of these silicon materials represented by SOI (silicon on insulator) etc .; glass, quartz glass, alumina, sapphire, ceramics, foam Stellite, inorganic materials such as photosensitive glass; polyethylene, ethylene, polypropylene, cyclic polyolefin, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol , Polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile / butadiene styrene copolymer, polyphenylene oxide And organic materials such as polysulfone and the like. The shape of the carrier is also not particularly limited, but is preferably flat.
　本発明においては、担体として、好ましくは表面にカーボン層と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。 In the present invention, preferably, a carrier having a carbon layer and a chemical modification group on the surface is used as the carrier. Carriers having a carbon layer and a chemical modification group on the surface include 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 the substrate consisting of a carbon layer. As the material of the substrate, those known in the art can be used without particular limitation, and the same ones as mentioned above as the carrier material can be used.
　本発明に係るマイクロアレイにおいては、微細な平板状の構造を有する担体が好適に用いられる。形状は、長方形、正方形および丸形など限定されないが、通常、1～75mm四方のもの、好ましくは1～10mm四方のもの、より好ましくは3～5mm四方のものを用いる。微細な平板状の構造の担体を製造しやすいことから、シリコン材料や樹脂材料からなる基板を用いるのが好ましく、特に単結晶シリコンからなる基板の表面にカーボン層および化学修飾基を有する担体がより好ましい。単結晶シリコンには、部分部分でごくわずかに結晶軸の向きが変わっているものや（モザイク結晶と称される場合もある）、原子的尺度での乱れ(格子欠陥)が含まれているものも包含される。 In the microarray according to the present invention, a carrier having a fine tabular structure is suitably used. The shape is not limited to rectangular, square and round shapes, but usually 1 to 75 mm square, preferably 1 to 10 mm square, more preferably 3 to 5 mm square is used. It is preferable to use a substrate made of a silicon material or a resin material because it is easy to manufacture a carrier having a fine flat plate structure, and in particular, a carrier having a carbon layer and a chemical modification group on the surface of a substrate made of single crystal silicon is more preferable. preferable. Single crystal silicon has a slight change in orientation of crystallographic axis in part (sometimes referred to as a mosaic crystal), or includes disorder on atomic scale (lattice defect) Is also included.
　本発明において基板上に形成させるカーボン層としては、特に制限されないが、合成ダイヤモンド、高圧合成ダイヤモンド、天然ダイヤモンド、軟ダイヤモンド（例えば、ダイヤモンドライクカーボン）、アモルファスカーボン、炭素系物質（例えば、グラファイト、フラーレン、カーボンナノチューブ）のいずれか、それらの混合物、またはそれらを積層させたものを用いることが好ましい。また、炭化ハフニウム、炭化ニオブ、炭化珪素、炭化タンタル、炭化トリウム、炭化チタン、炭化ウラン、炭化タングステン、炭化ジルコニウム、炭化モリブデン、炭化クロム、炭化バナジウム等の炭化物を用いてもよい。ここで、軟ダイヤモンドとは、いわゆるダイヤモンドライクカーボン（DLC：Diamond Like Carbon）等の、ダイヤモンドとカーボンとの混合体である不完全ダイヤモンド構造体を総称し、その混合割合は、特に限定されない。カーボン層は、化学的安定性に優れておりその後の化学修飾基の導入や分析対象物質との結合における反応に耐えることができる点、分析対象物質と静電結合によって結合するためその結合が柔軟性を持っている点、UV吸収がないため検出系UVに対して透明性である点、およびエレクトロブロッティングの際に通電可能な点において有利である。また、分析対象物質との結合反応において、非特異的吸着が少ない点においても有利である。前記のとおり基板自体がカーボン層からなる担体を用いてもよい。 The carbon layer formed on the substrate in the present invention is not particularly limited, but synthetic diamond, high-pressure synthetic diamond, natural diamond, soft diamond (for example, diamond like carbon), amorphous carbon, carbon-based material (for example, graphite, fullerene) It is preferable to use any of carbon nanotubes, mixtures thereof, or laminates thereof. In addition, carbides such as hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, thorium carbide, titanium carbide, uranium carbide, tungsten carbide, zirconium carbide, molybdenum carbide, chromium carbide, vanadium carbide and the like may be used. Here, soft diamond generally refers to an incomplete diamond structure which is a mixture of diamond and carbon such as so-called diamond like carbon (DLC: Diamond Like Carbon), and the mixing ratio is not particularly limited. The carbon layer is excellent in chemical stability and can withstand the subsequent reaction with the introduction of a chemical modification group and the binding to the analyte, and the bond is flexible because it is bound to the analyte by electrostatic binding. It is advantageous in that it has properties, is transparent to the detection system UV due to lack of UV absorption, and can be energized during electroblotting. In addition, it is also advantageous in that nonspecific adsorption is small in the binding reaction with the analyte. As described above, the substrate itself may use a carrier composed of a carbon layer.
　本発明においてカーボン層の形成は公知の方法で行うことができる。例えば、マイクロ波プラズマCVD(Chemical vapor deposit）法、ECRCVD(Electric cyclotron resonance chemical vapor deposit）法、ICP(Inductive coupled plasma）法、直流スパッタリング法、ECR(Electric cyclotron resonance)スパッタリング法、イオン化蒸着法、アーク式蒸着法、レーザ蒸着法、EB(Electron beam)蒸着法、抵抗加熱蒸着法などが挙げられる。 In the present invention, the formation of the carbon layer can be performed by a known method. For example, microwave plasma CVD (Chemical vapor deposition), ECR CVD (Electric cyclotron resonance chemical vapor deposition), ICP (Inductive coupled plasma), DC sputtering, ECR (Electric cyclotron resonance), ionization deposition, arc Deposition methods, laser deposition methods, electron beam (EB) deposition methods, resistance heating deposition methods, and the like.
　高周波プラズマCVD法では、高周波によって電極間に生じるグロー放電により原料ガス（メタン）を分解し、基板上にカーボン層を合成する。イオン化蒸着法では、タングステンフィラメントで生成される熱電子を利用して、原料ガス（ベンゼン）を分解・イオン化し、バイアス電圧によって基板上にカーボン層を形成する。水素ガス1～99体積％と残りメタンガス99～1体積％からなる混合ガス中で、イオン化蒸着法によりカーボン層を形成してもよい。 In the high frequency plasma CVD method, a source gas (methane) is decomposed by glow discharge generated between electrodes by high frequency to synthesize a carbon layer on a substrate. In the ionization vapor deposition method, the source gas (benzene) is decomposed and ionized by using thermoelectrons generated by a tungsten filament, and a carbon layer is formed on a substrate by a bias voltage. The carbon layer may be formed by ionization vapor deposition in a mixed gas consisting of 1 to 99% by volume of hydrogen gas and 99 to 1% by volume of methane gas.
　アーク式蒸着法では、固体のグラファイト材料（陰極蒸発源）と真空容器（陽極）の間に直流電圧を印加することにより真空中でアーク放電を起こして陰極から炭素原子のプラズマを発生させ蒸発源よりもさらに負のバイアス電圧を基板に印加することにより基板に向かってプラズマ中の炭素イオンを加速しカーボン層を形成することができる。 In the arc vapor deposition method, an arc discharge is generated in vacuum by applying a direct current voltage between a solid graphite material (cathode evaporation source) and a vacuum vessel (anode) to generate a plasma of carbon atoms from the cathode and the evaporation source Furthermore, by applying a negative bias voltage to the substrate, carbon ions in the plasma can be accelerated toward the substrate to form a carbon layer.
　レーザ蒸着法では、例えばNd:YAGレーザ（パルス発振）光をグラファイトのターゲット板に照射して溶融させ、ガラス基板上に炭素原子を堆積させることによりカーボン層を形成することができる。 In the laser deposition method, a carbon layer can be formed by, for example, irradiating a target plate of graphite with Nd: YAG laser (pulse oscillation) light to melt and depositing carbon atoms on a glass substrate.
　基板の表面にカーボン層を形成する場合、カーボン層の厚さは、通常、単分子層～100μm程度であり、薄すぎると下地基板の表面が局部的に露出する可能性があり、逆に厚くなると生産性が悪くなるので、好ましくは2nm～1μm、より好ましくは5nm～500nmである。 When a carbon layer is formed on the surface of the substrate, the thickness of the carbon layer is usually from about 1 to 100 μm, and if it is too thin, the surface of the base substrate may be exposed locally. If this is the case, the productivity will deteriorate, so it is preferably 2 nm to 1 μm, more preferably 5 nm to 500 nm.
　カーボン層が形成された基板の表面に化学修飾基を導入することにより、オリゴヌクレオチドプローブを担体に強固に固定化できる。導入する化学修飾基は、当業者であれば適宜選択することができ、特に制限されないが、例えば、アミノ基、カルボキシル基、エポキシ基、ホルミル基、ヒドロキシル基および活性エステル基が挙げられる。 By introducing a chemical modification group on the surface of the substrate on which the carbon layer is formed, the oligonucleotide probe can be firmly immobilized on the carrier. The chemical modification group to be introduced can be appropriately selected by those skilled in the art and is not particularly limited, and examples thereof include an amino group, a carboxyl group, an epoxy group, a formyl group, a hydroxyl group and an active ester group.
　アミノ基の導入は、例えば、カーボン層をアンモニアガス中で紫外線照射することによりまたはプラズマ処理することにより実施できる。または、カーボン層を塩素ガス中で紫外線を照射して塩素化し、さらにアンモニアガス中で紫外線照射することにより実施できる。または、メチレンジアミン、エチレンジアミンで等の多価アミン類ガス中を、塩素化したカーボン層と反応させることによって実施することもできる。 The introduction of the amino group can be carried out, for example, by irradiating the carbon layer with ultraviolet light in ammonia gas or by plasma treatment. Alternatively, the carbon layer may be chlorinated by irradiating ultraviolet light in chlorine gas and further irradiating ultraviolet light in ammonia gas. Alternatively, it can also be carried out by reacting with a chlorinated carbon layer in a polyvalent amines gas such as methylene diamine and ethylene diamine.
　カルボキシル基の導入は、例えば、前記のようにアミノ化したカーボン層に適当な化合物を反応させることにより実施できる。カルボキシル基を導入するために用いられる化合物としては、例えば、式：X-R1-COOH（式中、Xはハロゲン原子、R1は炭素数10～12の2価の炭化水素基を表す）で示されるハロカルボン酸、例えばクロロ酢酸、フルオロ酢酸、ブロモ酢酸、ヨード酢酸、2-クロロプロピオン酸、3-クロロプロピオン酸、3-クロロアクリル酸、4-クロロ安息香酸；式：HOOC-R2-COOH（式中、R2は単結合または炭素数1～12の2価の炭化水素基を表す）で示されるジカルボン酸、例えばシュウ酸、マロン酸、コハク酸、マレイン酸、フマル酸、フタル酸；ポリアクリル酸、ポリメタクリル酸、トリメリット酸、ブタンテトラカルボン酸などの多価カルボン酸；式：R3-CO-R4-COOH（式中、R3は水素原子または炭素数1～12の2価の炭化水素基、R4は炭素数1～12の2価の炭化水素基を表す）で示されるケト酸またはアルデヒド酸；式：X-OC-R5-COOH（式中、Xはハロゲン原子、R5は単結合または炭素数1～12の2価の炭化水素基を表す。）で示されるジカルボン酸のモノハライド、例えばコハク酸モノクロリド、マロン酸モノクロリド；無水フタル酸、無水コハク酸、無水シュウ酸、無水マレイン酸、無水ブタンテトラカルボン酸などの酸無水物が挙げられる。 The introduction of the carboxyl group can be carried out, for example, by reacting the carbon layer aminated as described above with a suitable compound. The compound used to introduce a carboxyl group is, for example, represented by the formula: X—R 1 —COOH (wherein, X represents a halogen atom, R 1 represents a divalent hydrocarbon group having 10 to 12 carbon atoms) Halocarboxylic acids such as chloroacetic acid, fluoroacetic acid, bromoacetic acid, iodoacetic acid, 2-chloropropionic acid, 3-chloropropionic acid, 3-chloroacrylic acid, 4-chlorobenzoic acid; formula: HOOC-R2-COOH (formula In which R 2 represents a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms), such as oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, phthalic acid; polyacrylic acid And polyvalent carboxylic acids such as polymethacrylic acid, trimellitic acid and butanetetracarboxylic acid; Formula: R3-CO-R4-COOH (wherein, R3 is a hydrogen atom or a divalent hydrocarbon group having 1 to 12 carbon atoms) , R 4 represents a divalent hydrocarbon group having 1 to 12 carbon atoms) Dicarbon represented by the formula: X-OC-R5-COOH (wherein, X is a halogen atom, R 5 represents a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms) Mono halides of acids such as succinic acid monochloride, malonic acid monochloride; acid anhydrides such as phthalic anhydride, succinic anhydride, oxalic anhydride, maleic anhydride, butanetetracarboxylic acid anhydride and the like can be mentioned.
　エポキシ基の導入は、例えば、前記のようにアミノ化したカーボン層に適当な多価エポキシ化合物を反応させることによって実施できる。あるいは、カーボン層が含有する炭素＝炭素２重結合に有機過酸を反応させることにより得ることができる。有機過酸としては、過酢酸、過安息香酸、ジペルオキシフタル酸、過ギ酸、トリフルオロ過酢酸などが挙げられる。 The introduction of the epoxy group can be carried out, for example, by reacting the carbon layer aminated as described above with an appropriate polyvalent epoxy compound. Alternatively, it can be obtained by reacting an organic peroxy acid with the carbon = carbon double bond contained in the carbon layer. Examples of organic peracids include peracetic acid, perbenzoic acid, diperoxyphthalic acid, formic acid, and trifluoroperacetic acid.
　ホルミル基の導入は、例えば、前記のようにアミノ化したカーボン層に、グルタルアルデヒドを反応させることにより実施できる。 The introduction of the formyl group can be carried out, for example, by reacting glutaraldehyde with the carbon layer aminated as described above.
　ヒドロキシル基の導入は、例えば、前記のように塩素化したカーボン層に、水を反応させることにより実施できる。 The introduction of hydroxyl groups can be carried out, for example, by reacting water with the carbon layer chlorinated as described above.
　活性エステル基は、エステル基のアルコール側に酸性度の高い電子求引性基を有して求核反応を活性化するエステル群、すなわち反応活性の高いエステル基を意味する。エステル基のアルコール側に、電子求引性の基を有し、アルキルエステルよりも活性化されたエステル基である。活性エステル基は、アミノ基、チオール基、水酸基等の基に対する反応性を有する。さらに具体的には、フェノールエステル類、チオフェノールエステル類、N-ヒドロキシアミンエステル類、シアノメチルエステル、複素環ヒドロキシ化合物のエステル類等がアルキルエステル等に比べてはるかに高い活性を有する活性エステル基として知られている。より具体的には、活性エステル基としては、たとえばp-ニトロフェニル基、N-ヒドロキシスクシンイミド基、コハク酸イミド基、フタル酸イミド基、5-ノルボルネン-2,3-ジカルボキシイミド基等が挙げられ、特に、N-ヒドロキシスクシンイミド基が好ましく用いられる。 The active ester group means an ester group having an electron withdrawing group with high acidity on the alcohol side of the ester group to activate a nucleophilic reaction, that is, an ester group with high reaction activity. It is an ester group having an electron withdrawing group on the alcohol side of the ester group and activated more than the alkyl ester. The active ester group has reactivity with groups such as amino group, thiol group and hydroxyl group. More specifically, an active ester group in which phenol esters, thiophenol esters, N-hydroxyamine esters, cyanomethyl esters, esters of heterocyclic hydroxy compounds, etc. have much higher activity than alkyl esters etc. Known as More specifically, examples of the active ester group include p-nitrophenyl group, N-hydroxysuccinimide group, succinimide group, phthalimido group, 5-norbornene-2,3-dicarboximide group and the like. In particular, N-hydroxysuccinimide group is preferably used.
　活性エステル基の導入は、例えば、前記のように導入したカルボキシル基を、シアナミドやカルボジイミド（例えば、1-[3-(ジメチルアミノ)プロピル]-3-エチルカルボジイミド)などの脱水縮合剤とN-ヒドロキシスクシンイミドなどの化合物で活性エステル化することにより実施できる。この処理により、アミド結合を介して炭化水素基の末端に、N-ヒドロキシスクシンイミド基等の活性エステル基が結合した基を形成することができる（特開2001-139532）。 The introduction of the active ester group may be carried out, for example, with a dehydrating condensation agent such as cyanamide or carbodiimide (for example, 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide) and N-containing carboxyl group introduced as described above. It can be carried out by active esterification with a compound such as hydroxysuccinimide. By this treatment, it is possible to form a group in which an active ester group such as N-hydroxysuccinimide group is bonded to an end of a hydrocarbon group via an amide bond (Japanese Patent Laid-Open No. 2001-139532).
　野生型プローブ及び変異型プローブを、スポッティング用バッファーに溶解してスポッティング用溶液を調製し、これを96穴もしくは384穴プラスチックプレートに分注し、分注した溶液をスポッター装置等によって担体上にスポッティングすることにより、野生型プローブ及び変異型プローブが担体に固定化されたマイクロアレイを製造することができる。または、スポッティング溶液をマイクロピペッターにて手動でスポッティングしてもよい。 A wild type probe and a mutant type probe are dissolved in a spotting buffer to prepare a spotting solution, which is dispensed into a 96-well or 384-well plastic plate, and the dispensed solution is placed on a carrier by a spotter or the like By spotting, it is possible to produce a microarray in which a wild-type probe and a mutant-type probe are immobilized on a carrier. Alternatively, the spotting solution may be spotted manually with a micropipettor.
　スポッティング後、野生型プローブ及び変異型プローブが担体に結合する反応を進行させるため、インキュベーションを行うことが好ましい。インキュベーションは、通常－20～100℃、好ましくは0～90℃の温度で、通常0.5～16時間、好ましくは1～2時間にわたって行う。インキュベーションは、高湿度の雰囲気下、例えば、湿度50～90％の条件で行うのが望ましい。インキュベーションに続き、担体に結合していないDNAを除去するため、洗浄液（例えば、50mM TBS/0.05% Tween20、2×SSC/0.2%SDS溶液、超純水など）を用いて洗浄を行うことが好ましい。 After spotting, it is preferable to carry out incubation in order to allow the reaction in which the wild-type probe and the mutant-type probe are bound to the carrier to proceed. Incubation is usually performed at a temperature of −20 to 100 ° C., preferably 0 to 90 ° C., usually for 0.5 to 16 hours, preferably 1 to 2 hours. The incubation is preferably performed under an atmosphere of high humidity, for example, 50 to 90% humidity. Following the incubation, it is preferable to wash using a washing solution (eg, 50 mM TBS / 0.05% Tween 20, 2 × SSC / 0.2% SDS solution, ultrapure water, etc.) in order to remove DNA not bound to the carrier. .
　以上のように構成されたマイクロアレイを用いることで、診断対象者における検出対象の一塩基多型について遺伝子型を判定する（野生型、ヘテロ型或いは変異型）ことができる。 By using the microarray configured as described above, it is possible to determine the genotype (wild-type, hetero-type or mutant-type) of the single nucleotide polymorphism to be detected in the diagnosis subject.
　具体的に、所定の一塩基多型について遺伝子型を判定する際には、診断対象者由来の試料からDNAを抽出する工程と、抽出したDNAを鋳型とし、当該一塩基多型を含む領域を増幅する工程と、上述したマイクロアレイを用いて、増幅された核酸に含まれる一塩基多型の遺伝子型を判定する工程とを含む。 Specifically, when determining the genotype for a predetermined single nucleotide polymorphism, a step of extracting DNA from a sample derived from a subject to be diagnosed and a region containing the single nucleotide polymorphism, wherein the extracted DNA is a template The steps of amplifying and determining the genotype of single nucleotide polymorphism contained in the amplified nucleic acid using the above-mentioned microarray are included.
　診断対象者は通常ヒトであり、人種等には特に限定されないが、特に、黄色人種、好適には東アジア人種、特に好適には日本人とする。また、診断対象者としては、骨髄増殖性腫瘍が疑われる患者とすることができる。 The subject to be diagnosed is usually a human, and is not particularly limited to the race and the like, but particularly the yellow race, preferably the East Asian race, particularly preferably the Japanese. Moreover, it can be set as the patient who is suspected of myeloproliferative tumor as a diagnostic subject.
　診断対象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 sample from the subject of diagnosis is not particularly limited. For example, blood related samples (blood, serum, plasma, etc.), lymph, feces, cancer cells, tissue or organ fragments and extracts, etc. may be mentioned.
　まず、診断対象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 to be diagnosed. The extraction means is not particularly limited. For example, a DNA extraction method using phenol / chloroform, ethanol, sodium hydroxide, CTAB or the like can be used.
　次に、得られたDNAを鋳型として用いて増幅反応を行い、検出対象の一塩基多型を含む領域を増幅する。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, amplification reaction is carried out using the obtained DNA as a template to amplify a region containing single nucleotide polymorphism to be detected. As an amplification reaction, a polymerase chain reaction (PCR), LAMP (Loop-Mediated Isothermal Amplification), ICAN (Isothermal and Chimeric primer-Initiated Amplification of Nucleic acids) method or the like can be applied. In the amplification reaction, it is desirable to add a label so that the region after amplification can be identified. At this time, a method for labeling the amplified nucleic acid is not particularly limited. For example, a method in which a primer used for amplification reaction is previously labeled may be used, or a labeled nucleotide is used as a substrate for amplification reaction. Methods may be used. The labeling substance is not particularly limited, and radioactive isotopes, fluorescent dyes, or organic compounds such as digoxigenin (DIG) and biotin can be used.
　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、増幅領域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。 In addition, this reaction system includes a buffer necessary for nucleic acid amplification and labeling, a thermostable DNA polymerase, a primer specific to the amplification region, a labeled nucleotide triphosphate (specifically, a nucleotide triphosphate to which a fluorescent label or the like is added) , A nucleotide triphosphate, and a reaction system containing magnesium chloride and the like.
　また、プライマーにより増幅される核酸断片は、設計した野生型プローブ及び変異型プローブに対応する領域を含んでいれば特に限定されず、例えば1kbp以下が好ましく、800bp以下がより好ましくは、500bp以下が更に好ましく、350bp以下が特に好ましい。 The nucleic acid fragment amplified by the primer is not particularly limited as long as it contains the regions corresponding to the designed wild-type probe and mutant-type probe, for example, 1 kbp or less is preferable, 800 bp or less is more preferably 500 bp or less More preferably, 350 bp or less is particularly preferred.
　上記のようにして得られた増幅核酸と、担体に固定された野生型プローブ及び変異型プローブとのハイブリダイゼーション反応を行い、野生型プローブ及び変異型プローブに対する増幅核酸のハイブリダイズを検出することで診断対象者における上記一塩基多型の遺伝子型を判定することができる。 By performing a hybridization reaction between the amplified nucleic acid obtained as described above and a wild-type probe and a mutant-type probe immobilized on a carrier, by detecting hybridization of the amplified nucleic acid to the wild-type probe and the mutant-type probe The genotype of the single nucleotide polymorphism in the subject can be determined.
　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、野生型プローブ及び変異型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。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、55℃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。或いは、ハイブリダイズする温度としては、塩濃度が0.5×SSCのとき、40～80℃とすることができ、プローブの鎖長が短い場合にはハイブリダイズ温度をこれより低くすることがより好ましく、鎖長が長い場合にはハイブリダイズ温度をこれより高くとすることがより好ましい。塩濃度が高くなると特異性を有するハイブリダイズ温度は高くなり、逆に塩濃度が低くなると特異性を有するハイブリダイズ温度は低くなることはいうまでもない。 For example, when a fluorescent label is used, the signal from the label can be detected as a fluorescent signal using a fluorescent scanner and analyzed by image analysis software to quantify the signal intensity. Alternatively, the amplified nucleic acid hybridized to the wild-type probe and the mutant-type probe can also be quantified, for example, by preparing a calibration curve using a sample containing a known amount of DNA. The hybridization reaction is preferably carried out under stringent conditions. Stringent conditions are conditions under which a specific hybrid is formed and a nonspecific hybrid is not formed, for example, after hybridization reaction at 55 ° C. for 16 hours, 2 × SSC / 0.2% SDS, 25 The conditions for washing at 10 ° C. for 10 minutes and 2 × SSC at 25 ° C. for 5 minutes are described. Alternatively, the temperature for hybridization can be 40 to 80 ° C. when the salt concentration is 0.5 × SSC, and it is more preferable to lower the hybridization temperature when the length of the probe is short, When the chain length is long, it is more preferable to make the hybridization temperature higher. It goes without saying that the hybridization temperature having specificity increases as the salt concentration increases, and the hybridization temperature having specificity decreases as the salt concentration decreases.
　特に、ハイブリダズ温度は、上述した野生型プローブ及び変異型プローブを設計する際に計算した結合率におけるハイブリダズ温度とすることが好ましい。例えば、温度５５℃における結合率を計算し、当該結合率に基づいて野生型プローブ及び変異型プローブを設計した場合には、ハイブリダズ温度を５５℃とすることが好ましい。 In particular, the hybridization temperature is preferably the hybridization temperature at the binding rate calculated when designing the wild-type probe and the mutant-type probe described above. For example, when the binding rate at a temperature of 55 ° C. is calculated, and the wild-type probe and the mutant-type probe are designed based on the binding rate, it is preferable to set the hybrids temperature to 55 ° C.
　また、変異型プローブと野生型プローブとを備えるマイクロアレイを使用する場合、これら変異型プローブ及び野生型プローブからのシグナル強度を用いて上記一塩基多型の遺伝子型を判定することができる。具体的には、野生型プローブにおけるシグナル強度及び変異型プローブにおけるシグナル強度をそれぞれ測定し、変異型プローブに由来するシグナ強度を評価するための判定値を算出する。判定値の算出例としては、例えば、上述した判定式：[野生型プローブ由来のシグナル強度]/([野生型プローブ由来のシグナル強度]+[変異型プローブ由来シグナル強度])＝判定値を使用する方法が挙げられる。 In addition, when using a microarray comprising a mutant-type probe and a wild-type probe, the signal intensity from the mutant-type probe and the wild-type probe can be used to determine the genotype of the single nucleotide polymorphism. Specifically, the signal intensity in the wild type probe and the signal intensity in the mutant probe are each measured, and the judgment value for evaluating the signal intensity derived from the mutant probe is calculated. As an example of calculation of the judgment value, for example, the above-mentioned judgment formula: [signal strength derived from wild type probe] / ([signal strength derived from wild type probe] + [signal strength derived from mutant type probe)] = judgment value is used Methods are included.
　そして、上記式にて算出される判定値と予め定めた閾値（カットオフ値）とを比較し、判定値が第１の閾値を上回る場合には増幅核酸に含まれる一塩基多型が野生型であると判断し、判定値が第１の閾値を下回り且つ第２の閾値を上回る場合には増幅核酸に一塩基多型がヘテロ型であると判断し、判定値が第２の閾値を下回る場合には増幅核酸に一塩基多型が変異型であると判断する（第１の閾値＞第２の閾値）。 Then, the judgment value calculated by the above equation is compared with a predetermined threshold (cutoff value), and if the judgment value exceeds the first threshold, the single nucleotide polymorphism contained in the amplified nucleic acid is wild type If the judgment value is below the first threshold and above the second threshold, it is judged that the single nucleotide polymorphism is heterozygous in the amplified nucleic acid, and the judgment value is below the second threshold. In the case, it is judged that the single nucleotide polymorphism is a mutation in the amplified nucleic acid (first threshold> second threshold).
　ここで、第１の閾値及び第２の閾値としては、検査対象の一塩基多型が野生型であることが確定している検体及び検査対象の一塩基多型が変異型であることが確定している検体を用いて上記式により算出した判定値に基づいて規定することができる。より具体的には、検査対象の一塩基多型が野生型であることが確定している複数の検体を用いて複数の判定値を算出し、その平均値+３σ（σ：標準偏差）の値を第１の閾値とすることができる。また、検査対象の一塩基多型が変異型であることが確定している複数の検体を用いて複数の判定値を算出し、その平均値+３σ（σ：標準偏差）の値を第２の閾値とすることができる。なお、平均値+２σや平均値+σの値を閾値とすることもできる。 Here, as the first threshold and the second threshold, it is determined that the sample whose single nucleotide polymorphism to be tested is determined to be wild type and the single nucleotide polymorphism to be tested are mutant. It can prescribe | regulate based on the determination value calculated by said Formula using the sample currently done. More specifically, a plurality of judgment values are calculated using a plurality of samples in which the single nucleotide polymorphism to be tested is determined to be wild type, and the average value + 3σ (σ: standard deviation) The value may be a first threshold. In addition, a plurality of judgment values are calculated using a plurality of samples whose single nucleotide polymorphisms to be tested are determined to be mutant types, and the average value + 3σ (σ: standard deviation) is calculated as the second The threshold of In addition, the value of average value +2 (sigma) or average value + (sigma) can also be used as a threshold value.
　以下、実施例により本発明を更に詳細に説明するが、本発明の技術的範囲はこれに限定されるものではない。 Hereinafter, the present invention will be described in more detail by way of examples, but the technical scope of the present invention is not limited thereto.
　［実施例］
　本実施例では、CYP2C19遺伝子のエキソン5の一塩基置換（CYP2C19*2）の一塩基多型を例として、当該一塩基多型を検出するためのプローブセットを設計した。なお、当該一塩基多型を含むターゲットは、野生型のターゲット（TTAAGTAATTTGTTATGGGTTCCcGGGAAATAATCAATGATAGTGGG：配列番号１、小文字が多型部位）と、変異型ターゲット（TTAAGTAATTTGTTATGGGTTCCtGGGAAATAATCAATGATAGTGGG：配列番号２、小文字が多型部位）とした。 [Example]
In this example, a probe set for detecting the single nucleotide polymorphism was designed, taking a single nucleotide polymorphism of exon 5 of the CYP2C19 gene (CYP2C19 * 2) as an example. The target containing the single nucleotide polymorphism was a wild type target (TTAAGTAATTTGTTATGGGTTCCcGGGAAATAATCAATGATAGTGGG: SEQ ID NO: 1, lower case is a polymorphic site) and a mutant target (TTAAGTAATTTGTTATGGGTTCCtGGGAAATAATCAATGATAGTGGG: lower case is a polymorphic site).
　先ず、これら野生型ターゲットを検出するための候補野生型プローブ（表１）及び候補変異型プローブ（表２）を設計した。表１及び表２において一塩基多型に対応する塩基を小文字で表記した。 First, candidate wild-type probes (Table 1) and candidate mutant-type probes (Table 2) for detecting these wild-type targets were designed. The bases corresponding to single nucleotide polymorphisms in Table 1 and Table 2 are shown in lower case letters.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　次に、表１及び２に示した候補野生型プローブ及び候補変異型プローブに関して、野生型ターゲット及び変異型ターゲットについてのTm曲線及びTm値を計算した。Tm曲線及びTm値は、Integrated DNA Technologies社が提供するWEBサービスにより算出した。このとき、プローブ濃度並びにターゲット濃度をそれぞれ0.002μMとし、Na+並びにK+濃度を195mMとしMg2+濃度を0mMとし、dNTPs濃度を0mMとし設定した。 Next, Tm curves and Tm values for wild-type targets and mutant targets were calculated for the candidate wild-type probes and candidate mutant-type probes shown in Tables 1 and 2. The Tm curve and Tm value were calculated by the WEB service provided by Integrated DNA Technologies. At this time, the probe concentration and the target concentration were each set to 0.002 μM, the Na + and K + concentrations were set to 195 mM, the Mg 2+ concentration was set to 0 mM, and the dNTPs concentration was set to 0 mM.
　候補野生型プローブに関してTm値を計算した結果を表３に示し、候補変異型プローブに関してTm 値を計算した結果を表４に示した。なお、表３及び４において「FM」とは、完全一致（Full Match）するターゲットとのハイブリダイズにおけるTm値を意味し、「MM」とは、一塩基ミスマッチ（Miss Match）するターゲットとのハイブリダイズにおけるTm値を意味する。 The results of calculating Tm values for candidate wild-type probes are shown in Table 3, and the results of calculating Tm values for candidate mutant probes are shown in Table 4. In Tables 3 and 4, “FM” means the Tm value in the hybridization with the target for a full match, and “MM” means a hybrid with the target for a single base mismatch (Miss Match). It means Tm value in soybean.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　また、得られたTm曲線に基づいて、温度55℃における結合率を計算した。候補野生型プローブに関して結合率を計算した結果を表５に示し、候補変異型プローブに関して結合率を計算した結果を表６に示した。 In addition, based on the obtained Tm curve, the binding rate at a temperature of 55 ° C. was calculated. The results of calculating the binding rates for candidate wild-type probes are shown in Table 5, and the results for calculating the binding rates for candidate variant probes are shown in Table 6.
　なお、表５において「WP-FM」とは、候補野生型プローブと野生型ターゲットとのハイブリダイズ（完全一致：Full Match）を意味し、「WP-MM」とは、候補野生型プローブと変異型ターゲットとのハイブリダイズ（一塩基ミスマッチ：Miss Match）を意味している。同様に、表６において「VP-FM」とは、候補変異型プローブと変異型ターゲットとのハイブリダイズ（完全一致：Full Match）を意味し、「VP-MM」とは、候補変異型プローブと野生型ターゲットとのハイブリダイズ（一塩基ミスマッチ：Miss Match）を意味している。 In addition, in Table 5, "WP-FM" means hybridization (perfect match: Full Match) of a candidate wild-type probe and a wild-type target, and "WP-MM" means a candidate wild-type probe and a mutation. It means hybridization with single target (single base mismatch: Miss Match). Similarly, in Table 6, "VP-FM" means hybridization (full match) between a candidate mutant probe and a mutant target, and "VP-MM" means a candidate mutant probe and It means hybridization with a wild-type target (single base mismatch: Miss Match).
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　次に、表５及び６に示した、55℃における結合率の値から、野生型ターゲットに関して、候補野生型プローブとの結合率（フルマッチ結合率）と候補変異型プローブとの結合率（ミスマッチ結合率）を引いた値を求めた。結果を図１～３に示した。図１～３に示した各マスの数値は、縦軸に示した候補野生型プローブにおけるフルマッチ結合率（表５）から、横軸に示した候補変異型プローブにおけるミスマッチ結合率（表６）を引いた値を示している。そして、図１～３には、この引いた値が０．５以上の場合に下線を付した。 Next, from the values of the binding rate at 55 ° C. shown in Tables 5 and 6, the binding rate with the candidate wild-type probe (full match binding rate) and the binding rate with the candidate mutant-type probe (mismatch binding) The value obtained by subtracting the rate) was obtained. The results are shown in FIGS. The numerical value of each mass shown in FIGS. 1 to 3 is based on the full match binding ratio (Table 5) of the candidate wild type probe shown on the vertical axis, and the mismatch binding ratio (Table 6) of the candidate mutant type probe shown on the horizontal axis. It shows the subtracted value. Then, in FIGS. 1 to 3, the underlined value is added when the subtracted value is 0.5 or more.
　また、表５及び６に示した、55℃における結合率の値から、変異型ターゲットに関して、候補変異型プローブとの結合率（フルマッチ結合率）と候補野生型プローブとの結合率（ミスマッチ結合率）を引いた値を求めた。結果を図４～６に示した。図４～６に示した各マスの数値は、横軸に示した候補変異型プローブにおけるフルマッチ結合率（表６）から、縦軸に示した候補野生型プローブにおけるミスマッチ結合率（表５）を引いた値を示している。そして、図４～６には、この引いた値が０．５以上の場合に下線を付した。 Further, from the values of the binding rate at 55 ° C. shown in Tables 5 and 6, the binding rate with the candidate mutant-type probe (full match binding rate) and the binding rate with the candidate wild-type probe (mismatch binding rate) The value obtained by subtracting) was obtained. The results are shown in FIGS. The numerical value of each mass shown in FIGS. 4 to 6 is based on the full match binding ratio (Table 6) of the candidate mutant type probe shown on the horizontal axis, and the mismatch binding ratio (Table 5) on the candidate wild type probe shown on the vertical axis. It shows the subtracted value. And, in FIGS. 4 to 6, underlines are given when the subtracted value is 0.5 or more.
　また、図４～６に示した下線部と、図１～３に示した下線部とが重複するマスを太線で囲った。すなわち、太線で囲ったマスは、55℃において、野生型ターゲットに関して、候補野生型プローブとの結合率から候補変異型プローブとの結合率を引いた値が０．５以上となり、且つ、変異型ターゲットに関して、候補変異型プローブとの結合率から候補野生型プローブとの結合率を引いた値が０．５以上となる野生型プローブと変異型プローブとの組み合わせを示している。 In addition, squares in which the underlined portions shown in FIGS. 4 to 6 and the underlined portions shown in FIGS. 1 to 3 overlap are surrounded by thick lines. That is, at the temperature of 55 ° C., for the wild type target, the value obtained by subtracting the binding rate with the candidate mutant-type probe from the binding rate with the candidate wild-type probe is 0.5 or more at 55 ° C. As for the target, a combination of a wild-type probe and a mutant-type probe is shown in which the value obtained by subtracting the ratio of binding to the candidate wild-type probe from the ratio of binding to the candidate mutant-type probe is 0.5 or more.
　すなわち、本実施例では、CYP2C19遺伝子のエキソン5の一塩基置換（CYP2C19*2）の一塩基多型を高精度に判定する野生型プローブ及び変異型プローブとして以下の組み合わせを選択することができた。
・WP_20mer_LとVP_24mer_R
・WP_21merとVP_24mer_R
・WP_22mer_RとVP_24mer_R
・WP_22mer_LとVP_24mer_R
・WP_23merとVP_24mer_R
・WP_22mer_RとVP_24mer_L
・WP_22mer_LとVP_24mer_L
・WP_23merとVP_24mer_L
・WP_23merとVP_25mer
　また、本実施例では、以上のように選択したプローブセットのうち、５つのセット[WP_22mer_L及びVP_24mer_R]、[WP_23mer及びVP_24mer_R]、[WP_22mer_L及びVP_24mer_L]、[WP_23mer及びVP_24mer_L]並びに[WP_23mer及びVP_25mer]を用いて一塩基多型の遺伝子型判定実験を行った。 That is, in this example, the following combination could be selected as a wild-type probe and a mutant-type probe for highly accurately determining single nucleotide polymorphism of exon 5 of the CYP2C19 gene (CYP2C19 * 2) .
・ WP_20mer_L and VP_24mer_R
・ WP_21 mer and VP_24 mer_R
・ WP_22mer_R and VP_24mer_R
・ WP_22mer_L and VP_24mer_R
・ WP_23 mer and VP_24 mer_R
・ WP_22mer_R and VP_24mer_L
・ WP_22mer_L and VP_24mer_L
・ WP_23 mer and VP_24 mer_L
・ WP_23 mer and VP_25 mer
Further, in the present embodiment, among the probe sets selected as described above, five sets [WP_22mer_L and VP_24mer_R], [WP_23mer and VP_24mer_R], [WP_22mer_L and VP_24mer_L], [WP_23mer and VP_24mer_L], and [WP_23mer and VP_25mer]. The genotyping experiment of single nucleotide polymorphism was performed using.
　また、この遺伝子型判定実験には、比較のため、プローブ長を等しくする観点で選択したプローブセット（比較例１）、Tm値が55℃に近いという観点で選択したプローブセット（比較例２）も使用した。比較例１のプローブセットは、[WP_22mer_R及びVP_22mer_R]、[WP_22mer_L及びVP_22mer_L]、[WP_23mer及びVP_23mer]、[WP_24mer_R及びVP_24mer_R]並びに[WP_24mer_L及びVP_24mer_L]の５つのセットである。比較例２のプローブセットは、[WP_19mer及びVP_23mer]、[WP_18mer_L及びVP_23mer]、[WP_19mer及びVP_23mer]、[WP_20mer_R及びVP_23mer]並びに[WP_19mer及びVP_24mer_R]の５つのセットである。 In addition, in this genotyping experiment, for comparison, a probe set (comparative example 1) selected from the viewpoint of equalizing probe lengths, a probe set selected from the viewpoint that the Tm value is close to 55 ° C. (comparative example 2) Also used. The probe sets of Comparative Example 1 are five sets of [WP_22 mer_R and VP_22 mer_R], [WP_22 mer_L and VP_22 mer_L], [WP_23 mer and VP_23 mer], [WP_24 mer_R and VP_24 mer_R] and [WP_24 mer_L and VP_24 mer_L]. The probe sets of Comparative Example 2 are five sets of [WP_19 mer and VP_23 mer], [WP_18 mer_L and VP_23 mer], [WP_19 mer and VP_23 mer], [WP_20 mer_R and VP_23 mer], and [WP_19 mer and VP_24 mer_R].
　これら実施例のプローブセット、比較例１のプローブセット及び比較例２のプローブセットを配置したDNAチップ（CYPチップ）を作製した。そして、野生型ターゲット、ヘテロ型ターゲット或いは変異型ターゲットを濃度2μMとCYPチップを1xSSC、0.1%SDS、ハイブリダイズ温度55℃で1時間反応させ、蛍光スキャナー（BIOSHOT社製）を用いて7秒より蛍光強度を得た。 A DNA chip (CYP chip) was prepared in which the probe sets of these examples, the probe set of Comparative Example 1 and the probe set of Comparative Example 2 were arranged. Then, react wild type target, hetero type target or mutant type target with concentration 2μM and CYP chip with 1x SSC, 0.1% SDS at hybridization temperature 55 ° C for 1 hour, and use fluorescence scanner (manufactured by BIOSHOT) for 7 seconds The fluorescence intensity was obtained.
　そして、実施例のプローブセット、比較例１のプローブセット及び比較例２のプローブセットにおいて得られた蛍光強度より、野生型、ヘテロ型及び変異型判定値を算出し、比較した。結果を図７に示した。また、図７に示した各プローブセットの結果に基づいて、野生型ターゲットを用いた時の判定値と変異型ターゲットを用いた時の判定値との差（全幅）、変異型ターゲットを用いた時の判定値からヘテロ型ターゲットを用いた時の判定値を引いた値を、ヘテロ型ターゲットを用いた時の判定値から野生型ターゲットを用いた時の判定値を引いた値で割った値（比(V-H/H-W)）を図８に示した。 And wild type, a hetero type, and a mutation type judgment value were computed from the fluorescence intensity obtained in the probe set of an Example, the probe set of the comparative example 1, and the probe set of the comparative example 2, and it compared. The results are shown in FIG. Also, based on the results of each probe set shown in FIG. 7, the difference (full width) between the judgment value when using a wild-type target and the judgment value when using a mutant-type target, and using a mutation-type target A value obtained by subtracting the judgment value when using a hetero-type target from the judgment value when using a hetero-type target, divided by the value obtained by subtracting the judgment value when using a wild-type target from the judgment value when using a hetero-type target The (ratio (VH / HW)) is shown in FIG.
　なお、図８に示したグラフにおいて、全幅の値が大きく、比が１に近い値であるプローブセットは、クロスハイブリダイゼーションが起こりにくいことを意味し、全幅がより小さいか比が１からより遠い値であるプローブセットと比較して、検出対象の一塩基多型をより高精度に検出できることが意味している。 In the graph shown in FIG. 8, a probe set having a large overall width value and a ratio close to 1 means that cross hybridization is unlikely to occur, and the overall width is smaller or the ratio is farther from 1 This means that it is possible to detect the single nucleotide polymorphism to be detected with higher accuracy as compared with the value of the probe set.
　図７及び図８に示すように、本実施例で選択したプローブセットを使用した場合には、比較例１及び２のプローブセットを使用した場合と比較して、全幅がより大きく、且つ比が１に近いことが分かる。この結果より、本実施例で選択したプローブセットを使用した場合には、クロスハイブリダイゼーションの発生を抑え、より高精度に一塩基多型の遺伝子型を判定できることが明らかとなった。 As shown in FIGS. 7 and 8, when the probe set selected in this example is used, the overall width is larger and the ratio is larger than when the probe sets of Comparative Examples 1 and 2 are used. It is understood that it is close to 1. From this result, it is clear that when the probe set selected in this example is used, the occurrence of cross hybridization can be suppressed and the genotype of single nucleotide polymorphism can be determined more accurately.
　さらに、遺伝子型判定実験に使用した本実施例で選択したプローブセットの５種類について、野生型ターゲットに対する野生型プローブの結合率（フルマッチ結合率）と変異型ターゲットに対する変異型プローブの結合率（フルマッチ結合率）との差の絶対値と、野生型ターゲットに対する変異型プローブの結合率（ミスマッチ結合率）と変異型ターゲットに対する野生型プローブの結合率（ミスマッチ結合率）との差の絶対値とを合計した。その結果、合計値の低い順に、[WP_22mer_L及びVP_24mer_L]（合計値：0.06）、[WP_23mer及びVP_25mer]（合計値：0.07）、[WP_22mer_L及びVP_24mer_R]（合計値：0.08）、[WP_23mer及びVP_24mer_L]（合計値：0.20）、[WP_23mer及びVP_24mer_R]（合計値：0.21）であった。 Furthermore, for five types of probe sets selected in the present example used in genotyping experiments, the binding rate of the wild-type probe to the wild-type target (full match binding rate) and the binding rate of the mutant-type probe to the mutant target (full match) The absolute value of the difference from the binding rate), and the absolute value of the difference between the binding rate of the mutant probe to the wild-type target (mismatch binding rate) and the binding rate of the wild-type probe to the mutant target (mismatch binding rate) Summed up. As a result, [WP_22mer_L and VP_24mer_L] (total value: 0.06), [WP_23mer and VP_25mer] (total value: 0.07), [WP_22mer_L and VP_24mer_R] (total value: 0.08), [WP_23mer and VP_24mer_L] in ascending order of the total value (Total value: 0.20), [WP_23 mer and VP_24 mer_R] (total value: 0.21).
　図８に示すように、当該合計値が低いプローブセットは、全幅がより大きく、且つ比が１により近いことが分かる。このことから、上述のようにして一塩基多型を検出するためのプローブセットが複数選択された場合には、上記合計値を計算し、当該合計値がより低いプローブセットを使用することがより好ましいことが分かる。 As shown in FIG. 8, it can be seen that the probe set with a low total value has a larger overall width and a ratio closer to one. From this, when a plurality of probe sets for detecting single nucleotide polymorphisms are selected as described above, the total value is calculated, and it is more preferable to use a probe set having a lower total value. It turns out that it is preferable.
　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。 All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims (6)

  1. 　一塩基多型の野生型に対応する野生型プローブと、当該一塩基多型の変異型に対応する変異型プローブとを設計する方法であって、
    　上記一塩基多型部位を含む複数の候補野生型プローブと、上記一塩基多型部位を含む複数の候補変異型プローブとを設計し、
    　これら候補野生型プローブについて完全一致となる野生型ターゲットとの結合率、これら候補野生型プローブについて当該一塩基多型部位において一塩基ミスマッチとなる変異型ターゲットとの結合率、これら候補変異型プローブについて完全一致となる変異型ターゲットとの結合率、及びこれら候補変異型プローブについて当該一塩基多型部位における一塩基ミスマッチとなる野生型ターゲットとの結合率をそれぞれ計算し、
    　野生型ターゲットに関して、候補野生型プローブとの結合率から候補変異型プローブとの結合率を引いた値が所定の値以上となり、且つ、変異型ターゲットに関して、候補変異型プローブとの結合率から候補野生型プローブとの結合率を引いた値が所定の値以上となる野生型プローブと変異型プローブとの１又は複数の組み合わせを、候補野生型プローブ及び候補変異型プローブのなかから選択し、
    　選択した野生型プローブと変異型プローブとの１又は複数の組み合わせを、当該一塩基多型を検出するための野生型プローブ及び変異型プローブとして設計する方法。     A method of designing a wild-type probe corresponding to a wild-type of a single nucleotide polymorphism and a mutant-type probe corresponding to a mutant of the single nucleotide polymorphism,
    Designing a plurality of candidate wild-type probes including the single nucleotide polymorphism site and a plurality of candidate mutant-type probes including the single nucleotide polymorphism site,
    For these candidate wild-type probes, the binding rate with the wild-type target that is a perfect match, for these candidate wild-type probes, the binding rate with a mutant-type target that results in a single base mismatch at the single nucleotide polymorphism site, The percentage of binding to a mutant target that completely matches, and the percentage of binding to a wild-type target that is a single base mismatch at the single nucleotide polymorphism site for these candidate mutant probes are calculated,
    With respect to wild-type targets, the value obtained by subtracting the binding rate with candidate mutant-type probes from the binding rate with candidate wild-type probes is equal to or greater than a predetermined value, and with respect to mutant-type targets, the binding rate with candidate mutant-type probes is a candidate One or more combinations of a wild-type probe and a mutant-type probe for which the value obtained by subtracting the binding rate to the wild-type probe is a predetermined value or more is selected from the candidate wild-type probe and the candidate mutant-type probe,
    A method of designing one or more combinations of a selected wild-type probe and a mutant-type probe as a wild-type probe and a mutant-type probe for detecting the single nucleotide polymorphism.
  2. 　上記結合率は、所定のハイブリダイズ温度若しくは所定のハイブリダイズ温度範囲において計算し、当該ハイブリダイズ温度若しくはハイブリダイズ温度範囲において使用する野生型プローブ及び変異型プローブを設計することを特徴とする請求項１記載の方法。 The binding rate is calculated at a predetermined hybridization temperature or a predetermined hybridization temperature range, and wild-type probes and mutant-type probes to be used at the hybridization temperature or hybridization temperature range are designed. Method 1 described.
  3. 　野生型プローブと変異型プローブとの複数の組み合わせを選択した場合、複数の組み合わせに関して、野生型ターゲットに対する野生型プローブの結合率と変異型ターゲットに対する変異型プローブの結合率との差の絶対値と、野生型ターゲットに対する変異型プローブの結合率と変異型ターゲットに対する野生型プローブの結合率との差の絶対値とを合計し、複数の組み合わせのうち当該合計値が最も低い組み合わせを、上記一塩基多型を検出するための野生型プローブ及び変異型プローブとして設計することを特徴とする請求項１記載の方法。 When multiple combinations of a wild-type probe and a mutant-type probe are selected, the absolute value of the difference between the binding ratio of the wild-type probe to the wild-type target and the binding ratio of the mutant-type probe to the mutant target for multiple combinations And adding the absolute value of the difference between the binding rate of the mutant-type probe to the wild-type target and the binding rate of the wild-type probe to the mutant-type target, and combining the combination having the lowest total value among the plurality of combinations, The method according to claim 1, wherein the method is designed as a wild-type probe and a mutant-type probe for detecting a polymorphism.
  4. 　野生型プローブと変異型プローブとの複数の組み合わせを選択した場合、複数の組み合わせに関して、野生型ターゲットに対する野生型プローブの結合率と野生型ターゲットに対する変異型プローブの結合率との差が最も大きい組み合わせ、又は、変異型ターゲットに対する変異型プローブの結合率と変異型ターゲットに対する野生型プローブの結合率との差が最も大きい組み合わせを、上記一塩基多型を検出するための野生型プローブ及び変異型プローブとして設計することを特徴とする請求項１記載の方法。 When multiple combinations of wild-type probe and mutant-type probe are selected, the combination of wild-type-target binding ratio and wild-type-target-type binding ratio has the largest difference for multiple combinations. Alternatively, a wild-type probe and a mutant-type probe for detecting the single nucleotide polymorphism may be a combination having the largest difference between the binding ratio of the mutant-type probe to the mutant-type target and the binding ratio of the wild-type probe to the mutant-type target. A method according to claim 1, characterized in that it is designed as
  5. 　一塩基多型の野生型に対応する野生型プローブと、当該一塩基多型の変異型に対応する変異型プローブとからなるプローブセットであって、
    　上記一塩基多型部位を含む複数の候補野生型プローブと、上記一塩基多型部位を含む複数の候補変異型プローブとのなかから選択され、
    　野生型ターゲットに関して、候補野生型プローブとの結合率から候補変異型プローブとの結合率を引いた値が所定の値以上となり、且つ、変異型ターゲットに関して、候補変異型プローブとの結合率から候補野生型プローブとの結合率を引いた値が所定の値以上となる野生型プローブと変異型プローブとからなる、当該一塩基多型を検出するためのプローブセット。     A probe set comprising: a wild-type probe corresponding to a wild-type of a single nucleotide polymorphism, and a mutant-type probe corresponding to a mutation of the single nucleotide polymorphism,
    A plurality of candidate wild-type probes including the single nucleotide polymorphism site, and a plurality of candidate mutant-type probes including the single nucleotide polymorphism site;
    With respect to wild-type targets, the value obtained by subtracting the binding rate with candidate mutant-type probes from the binding rate with candidate wild-type probes is equal to or greater than a predetermined value, and with respect to mutant-type targets, the binding rate with candidate mutant-type probes A probe set for detecting the single nucleotide polymorphism, which comprises a wild-type probe and a mutant-type probe in which the value obtained by subtracting the binding rate to the wild-type probe is a predetermined value or more.
  6. 　担体と、当該担体に固定された請求項５記載のプローブセットに含まれる野生型プローブ及び変異型プローブとを備えるDNAチップ。 A DNA chip comprising a carrier, and a wild-type probe and a mutant-type probe contained in the probe set according to claim 5 immobilized on the carrier.
PCT/JP2018/045641 2017-12-15 2018-12-12 Method for designing probe for detecting single nucleotide polymorphism and probe set WO2019117192A1 (en)

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