JP2019106907A - One base polymorphism detection probe design method and probe set - Google Patents

Abstract

To suppress generation of cross hybridization and accurately detect a target of perfect matching.SOLUTION: The invention is configured to calculate a coupling ratio with a wild target or variant target of a candidate wild probe, and a coupling ratio with a variant target or wild target of a candidate variant probe, then design so that, a value obtained by subtracting the coupling ratio with the candidate variant probe from the coupling ratio with the candidate wild probe, about the wild target, and a value obtained by subtracting the coupling ratio with the candidate wild probe from the coupling ratio with the candidate variant probe about the variant target, become a prescribed value or greater value, respectively.SELECTED DRAWING: None

Description

  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 (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.

  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.

  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.

  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.

  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.

Unexamined-Japanese-Patent No. 2010-4782 Japanese Patent Publication No. 9-507121

  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 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.

  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.

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) 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) 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) 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) 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) 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.

  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.

  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.

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. 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. 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.

  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.

  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.

  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.

  The lengths of the candidate wild-type probe and the candidate mutant-type probe to be designed are not particularly limited, but can be, for example, 5 to 50 bases, preferably 7 to 40 bases, and preferably 10 to 30 bases It is more preferable to set the length to 11 to 25 bases, and most preferable.

  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.

  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.

  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.

  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.

  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.

  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.

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, a Tm curve can be calculated with a probe concentration and a target concentration of 0.002 μM, Na ^{+ and} K ⁺ concentrations of 195 mM, an Mg ²⁺ concentration of 0 mM, and a 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).

  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 for hybridizing the target DNA 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.

  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.

  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.

  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 ratio may be 0.00 to 1.00) or more, preferably 0.4 or more, and more preferably 0.5 or more.

  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.

  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).

  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. .

  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.

  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.

  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.

  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.

  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.

  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 .

  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

  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.

  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.

  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.

  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.

  In the microarray according to the present invention, a carrier having a fine tabular structure is suitably used. The shape is not limited such as rectangular, square and round, 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 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. 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.

  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.

  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 deposition in a mixed gas consisting of 1 to 99% by volume of hydrogen gas and 99 to 1% by volume of methane gas.

  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.

  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.

  When a carbon layer is formed on the surface of a substrate, the thickness of the carbon layer is usually about a monomolecular layer to about 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 is degraded, so the thickness is preferably 2 nm to 1 μm, more preferably 5 nm to 500 nm.

  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. 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.

  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: R 3 -CO-R 4 -COOH (wherein R 3 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 represents a halogen atom, R 5 represents a single bond or a C 1-12 divalent hydrocarbon group); 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.

  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.

  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. 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.

  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 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.

  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. The incubation is carried out usually at a temperature of -20 to 100 ° C, preferably 0 to 90 ° C, for usually 0.5 to 16 hours, preferably 1 to 2 hours. Incubation is desirably 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. .

  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.

  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.

  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. For example, blood related samples (blood, serum, plasma, etc.), lymph, feces, cancer cells, tissue or organ fragments and extracts, etc. may be mentioned.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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).

  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.

  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.

[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).

  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.

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.

  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.

  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 rate for the candidate wild-type probe are shown in Table 5, and the results for calculating the binding rate for the candidate mutant-type probe are shown in Table 6.

  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).

  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 rate (Table 5) of the candidate wild type probe shown on the vertical axis, and the mismatch binding rate (Table 6) of the candidate mutant type probe shown on the horizontal axis. It shows the subtracted value. And, in FIGS. 1 to 3, the squares in which the subtracted value is 0.5 or more are shaded.

  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 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. 4-6, the value which this value drew was shaded by 0.5 or more.

  Moreover, the squares in which the hatching shown in FIGS. 4 to 6 and the hatching shown in FIGS. 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.

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.

  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, and WP 24 mer and L 24 mer 24 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 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.

  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.

  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.

  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.

  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).

  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.

Claims (6)

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.   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.   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.   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 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 is a candidate 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.   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.

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