JP6995604B2 - Design method and probe set for single nucleotide polymorphism detection probe - Google Patents

Description

The present invention relates to a method and a probe set for designing a wild type probe corresponding to a wild type of a single nucleotide polymorphism and a mutant probe corresponding to the mutant type of the single nucleotide polymorphism.

Single nucleotide polymorphism (SNP) is a type of gene mutation that exists in the genome of a population of species, and means the diversity of single nucleotide polymorphisms in the base sequence. In particular, it may be referred to as a single nucleotide polymorphism only when a mutation is observed with a frequency of 1% or more in the population, but it can be referred to as a single nucleotide polymorphism even if the frequency is less than 1%. The single nucleotide polymorphism is typically composed of two types consisting of a wild-type allele and one type of mutant allele, but may have two or more types of mutant alleles.

Polymorphisms in a broad sense, including single nucleotide polymorphisms, are related to phenotypes such as the degree of side effects on drugs and susceptibility to diseases. In plants, polymorphisms in a broad sense including single nucleotide polymorphisms are also related to phenotypes such as disease resistance, stress tolerance, and biomass amount. By detecting a single nucleotide polymorphism (also referred to as typing) in this way, it is possible to predict the risk of contracting a disease, the risk of side effects, and the like.

As a method for detecting a single nucleotide polymorphism, various methods have been conventionally known as an SNP typing method. For example, a direct sequence method for amplifying a DNA fragment containing a single nucleotide polymorphism and reading a base sequence, and a method using a probe containing a single nucleotide polymorphism site can be mentioned. Examples of the method using a probe containing a single nucleotide polymorphism site include a cycling probe method to which real-time PCR is applied, a bead array method, and a microarray (DNA chip) method.

In the method using a probe containing a single nucleotide polymorphism site, a wild-type probe corresponding to a wild-type base and a mutant probe corresponding to a mutant-type base are designed for the single nucleotide polymorphism to be detected. In general, the probe is a thermodynamic variable, including the melting temperature with the target DNA, length, intramolecular self-binding formation, sequence homology with other probes, and the location of the target site (3'end side or It is designed in consideration of 5'end side) and the like. Of these, the thermodynamic variable considers the hybridization energy between the probe and the target DNA, and can be used as an index representing the probe characteristics.

As a method for designing these wild-type probes and mutant probes, for example, as described in Patent Document 1, the Tm value when a double strand is formed with an exact match target is a wild-type probe and a mutant probe. There is a way to design them to be equal. Further, Patent Document 2 describes a method of designing the wild-type probe and the mutant-type probe to have the same length.

Japanese Unexamined Patent Publication No. 2010-4782 Special Table 9-507121 Gazette

However, since both wild-type and mutant probes that detect single nucleotide polymorphisms have the same sequence except for one base, non-specific binding (cross-hybridization) other than hybridization with a completely matching target DNA. ) May be formed. When cross-hybridization occurs, the signal value due to cross-hybridization is added to the true signal value due to the binding of the exact target DNA. That is, when cross hybridization occurs, noise is included in the measured value, which is a factor that greatly lowers the reliability of the measured result.

Therefore, in view of such circumstances, the present invention provides wild-type probes and mutant probes for single nucleotide polymorphism detection, which can suppress the occurrence of cross-hybridization and detect perfectly matched targets with high accuracy. It is an object of the present invention to provide a design method and a probe set.

The present invention includes the following.
(1) A method for designing a wild-type probe corresponding to a wild-type of a single-base polymorphism and a mutant probe corresponding to the variant of the single-base polymorphism, which includes a plurality of the above-mentioned single-base polymorphism sites. Candidate wild-type probes and a plurality of candidate mutant probes including the above-mentioned single-base polymorphic site are designed, and the binding rate with wild-type targets that are completely consistent with these candidate wild-type probes, and these candidate wild-type probes The binding rate with a mutant target that has a one-base mismatch at the one-base polymorphism site, the binding rate with a variant target that is an exact match for these candidate mutant probes, and the one-base polymorphism for these candidate mutant probes. The binding rate with the wild-type target that causes a one-base mismatch at the site is calculated, and the value obtained by subtracting the binding rate with the candidate variant probe from the binding rate with the candidate wild-type probe for the wild-type target is greater than or equal to the predetermined value. And with respect to the variant target, one or more of the wild-type probe and the variant probe in which the value obtained by subtracting the binding rate with the candidate wild-type probe from the binding rate with the candidate variant probe is equal to or more than a predetermined value. The combination is selected from the candidate wild-type probe and the candidate variant-type probe, and one or more combinations of the selected wild-type probe and the variant-type probe are used as a wild-type probe for detecting the one-base polymorphism. How to design as a variant probe.

(2) The binding rate is calculated in a predetermined hybridization temperature or a predetermined hybridization temperature range, and a wild-type probe and a mutant probe to be used in the hybridization temperature or the hybridization temperature range are designed. (1) The method described.

(3) When a plurality of combinations of a wild-type probe and a mutant probe are selected, the difference between the binding rate of the wild-type probe to the wild-type target and the binding rate of the mutant probe to the mutant target for the plurality of combinations. Sum the absolute value and 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 select the combination having the lowest total value among the plurality of combinations. The method according to (1), characterized in that it is designed as a wild-type probe and a mutant probe for detecting the one-base polymorphism.

(4) When multiple combinations of wild-type probe and mutant probe are selected, the difference between the binding rate of the wild-type probe to the wild-type target and the binding rate of the mutant probe to the wild-type target is different for the multiple combinations. The wild-type probe for detecting the above-mentioned single-base polymorphism and the combination having the largest difference between the binding rate of the mutant probe to the mutant target and the binding rate of the wild-type probe to the mutant target. The method according to (1), characterized in that it is designed as a mutant probe.

(5) A probe set consisting of a wild type probe corresponding to a wild type of a single base polymorphism and a mutant probe corresponding to the mutant type of the single base polymorphism, and a plurality of probes including the above-mentioned single base polymorphism site. The binding rate of the wild type target to the candidate mutant type probe is selected from the binding rate of the wild type target to the candidate wild type probe. With respect to the mutant target, the value obtained by subtracting the binding rate with the candidate wild type probe from the binding rate with the candidate mutant type probe is equal to or more than the predetermined value. A probe set consisting of a mutant probe for detecting the single-base polymorphism.

(6) A DNA chip comprising a carrier and a wild-type probe and a mutant 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 probe for detecting a single nucleotide polymorphism according to the present invention, the wild-type probe and the mutant probe with excellent detection sensitivity of a target DNA in which the occurrence of cross hybridization is suppressed are suppressed. Can be designed.

In addition, the primer set according to the present invention has excellent detection sensitivity of the target DNA because the occurrence of cross hybridization is suppressed.

The type of candidate wild-type probe is shown in the row, the type of candidate mutant probe is shown in the column, and the value obtained by subtracting the mismatch binding rate in the candidate mutant probe from the full-match binding rate in the candidate wild-type probe is shown in the characteristic diagram for each cell. be. The type of candidate wild-type probe is shown in the row, the type of candidate mutant probe is shown in the column, and the value obtained by subtracting the mismatch binding rate in the candidate mutant probe from the full-match binding rate in the candidate wild-type probe is shown in the characteristic diagram for each cell. be. The type of candidate wild-type probe is shown in the row, the type of candidate mutant probe is shown in the column, and the value obtained by subtracting the mismatch binding rate in the candidate mutant probe from the full-match binding rate in the candidate wild-type probe is shown in the characteristic diagram for each cell. be. The type of candidate wild-type probe is shown in the row, the type of candidate mutant probe is shown in the column, 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 probe is shown in the characteristic diagram for each cell. be. The type of candidate wild-type probe is shown in the row, the type of candidate mutant probe is shown in the column, 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 probe is shown in the characteristic diagram for each cell. be. The type of candidate wild-type probe is shown in the row, the type of candidate mutant probe is shown in the column, 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 probe is shown in the characteristic diagram for each cell. be. It is a characteristic diagram which shows the result of having performed the genotyping experiment using the probe set of Example, the probe set of Comparative Example 1, and the probe set of Comparative Example 2. Based on the results of the genotyping experiment using the probe set of Example, the probe set of Comparative Example 1, and the probe set of Comparative Example 2, a characteristic diagram showing the relationship with the total width and ratio when each probe set is used. Is.

The present invention provides a method for designing a wild-type probe and a mutant probe for single nucleotide polymorphism detection. Here, the single nucleotide polymorphism is a gene mutation existing in the genome of an organism population, and means a single base mutation in a 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). Meaning.

The wild-type probe is a probe that corresponds to the wild-type allele in the single nucleotide polymorphism to be detected and has a base complementary to the genotype of the wild-type allele. The mutant probe is a probe that corresponds to the mutant allele in the single nucleotide polymorphism to be detected and has a base complementary to the genotype of the mutant allele. For example, if a single nucleotide polymorphism in which the genotype of a wild-type allergen is A (adenin) and the genotype of a mutant allergen is G (guanine) is targeted for detection, the wild-type probe corresponds to the single nucleotide polymorphism. The position contains T complementary to A, and the mutant probe contains C complementary to G at that position. A combination of a wild-type probe and a mutant probe designed for a predetermined 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 probe, a plurality of wild-type probe candidates (candidate wild-type probe) and a plurality of mutant probes (candidate mutant probe) are designed. .. Specifically, in this step, a plurality of candidate wild-type probes and a plurality of mutant probes are designed as the base sequences of oligonucleotides containing the single nucleotide polymorphism from the peripheral genomic sequences containing the single nucleotide polymorphism to be detected. do.

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

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 so as to be located substantially at the center of the base sequence to be designed. When the base sequence to be designed consists of an odd number of bases, one base in the center of the 5'end and the 3'end can be the position corresponding to the single nucleotide polymorphism. Further, when the base sequence to be designed consists of an even number of bases, any one of the two bases at the center of the 5'end and the 3'end can be the position corresponding to the single nucleotide polymorphism.

However, in the candidate wild-type probe and the candidate mutant probe to be designed, the base corresponding to the single nucleotide polymorphism to be detected is not limited to the substantially center in the base sequence designed as described above, and is 5'from the substantially center. The position may be biased toward the terminal side or the 3'end side. For example, in the candidate wild-type probe and the candidate mutant probe to be designed, the base corresponding to the single nucleotide polymorphism to be detected is in the range of 1/4 of the total length, preferably 1/5, from the substantially center in the base sequence to be designed. The position can be biased toward the 5'end side or the 3'end side in the range of, more preferably 1/6.

Further, 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 centering on the base corresponding to the single nucleotide polymorphism to be detected. Only candidate wild-type probes and candidate mutant-type probes may be designed in which the base corresponding to the single nucleotide polymorphism to be detected is biased toward the 5'end side or the 3'end side from the substantially center. 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 centered on a base corresponding to the single nucleotide polymorphism to be detected, and a single nucleotide polymorphism to be detected. Candidate wild-type probes and candidate mutant-type probes may be designed in which the bases corresponding to are biased toward the 5'end side or the 3'end side from substantially the center.

Next, in the design method according to the present invention, for the designed candidate wild-type probe and candidate mutant probe, the Tm curve of the target DNA that is an exact match and the Tm curve and the target DNA that is a one-base mismatch are calculated, respectively.

Here, for the candidate wild-type probe, the target DNA that is a perfect match is a wild-type target DNA (referred to as a wild-type target) in which the single nucleotide polymorphism to be detected is a wild-type allyl. For the candidate wild-type probe, the target DNA that causes a single nucleotide 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 probe, the target DNA that is a perfect match is a mutant target in which the single nucleotide polymorphism to be detected is a mutant allele. For the candidate mutant probe, the target DNA that has a single nucleotide mismatch is a wild-type target in which the single nucleotide polymorphism to be detected is a wild-type allele.

The method for calculating the Tm curve is not particularly limited, and examples thereof include the Nearest Neighbor method, the Wallace method, and the GC% method, and in particular, the Nearest Neighbor method. ) Is preferable. Note that Tm is defined as the temperature at which 50% of the probe and the target are hybridized when they dissociate (that is, the temperature when the binding rate is 50%). Factors that affect the Tm value include base composition, salt concentration, oligo chain concentration and modifier (formamide, DMSO, etc.), solvation effect, conjugate group (biotin, digoxigenin, alkaline phosphatase, fluorescent dye, etc.). ).

More specifically, the Tm value can be calculated by the WEB service provided by Integrated DNA Technologies, which includes the base sequence of the probe, the probe concentration, the concentration of the target that hybridizes with the probe, and Na ⁺ in the reaction solution. The Tm curve can also be calculated by setting the K ⁺ concentration, the Mg ²⁺ concentration in the reaction solution and (in some cases) the dNTPs concentration. For example, the Tm curve can be calculated with the probe concentration and target concentration set to 0.002 μM, the Na ⁺ and K ⁺ concentrations set to 195 mM, the Mg ²⁺ concentration set to 0 mM, and the dNTPs concentration set to 0 mM. The calculated Tm curve can also be displayed on a two-dimensional plane in which one axis (for example, the vertical axis) is the coupling ratio and the other axis (for example, the horizontal axis) is heated.

The obtained Tm curve can show the relationship between the temperature and the binding rate with respect to the candidate wild-type probe or the candidate mutant probe having a predetermined base sequence and the wild-type target or the mutant target. That is, based on the obtained Tm curve, the binding rate with the wild-type target (exact match) in the candidate wild-type probe designed as described above can be calculated. Similarly, based on the obtained Tm curve, the binding rate with the mutant target (single nucleotide mismatch) in the candidate wild-type probe can be calculated. Similarly, based on the obtained Tm curve, the binding rate with the mutant target (exact match) in the candidate mutant probe can be calculated. Similarly, based on the obtained Tm curve, the binding rate of the candidate mutant probe to the wild-type target (single nucleotide mismatch) can be calculated.

Next, in the design method according to the present invention, among the candidate wild-type probes and the candidate mutant-type probes, the wild-type probe and the mutant-type probe most suitable for the single nucleotide polymorphism to be detected are selected based on the calculated binding rate. And select. At this time, the bond ratio calculated as described above can be calculated as a bond rate in a predetermined temperature or a predetermined temperature range. For example, in an analysis method using a DNA chip in general, the temperature at which the target DNA is hybridized with the probe is set in the range of 40 to 80 ° C, preferably 50 to 70 ° C, and more preferably 50 to 60 ° C. Therefore, the binding rate within these temperature ranges, for example, at 55 ° C., should be calculated, and the optimum wild-type probe and mutant probe for the single nucleotide polymorphism to be detected should be selected based on the binding rate at 55 ° C. Is preferable.

Specifically, for example, at 55 ° C., the value obtained by subtracting the binding rate with the candidate mutant probe from the binding rate with the candidate wild-type probe for the wild-type target becomes a predetermined value or more, and with respect to the mutant target. Select one or a plurality of combinations of the wild-type probe and the mutant probe in which the value obtained by subtracting the binding rate with the candidate wild-type probe from the binding rate with the candidate mutant probe is equal to or more than a predetermined value.

Here, the binding rate with the candidate wild-type probe for the wild-type target means the binding rate (full-match binding rate) with respect to the target that completely matches the candidate wild-type probe. Further, the binding rate with the candidate mutant probe for the wild-type target means the binding rate (mismatch binding rate) with respect to the target having a single nucleotide polymorphism for the candidate mutant probe. Similarly, the binding rate with a candidate wild-type probe for a mutant target means the binding rate (mismatch binding rate) with respect to a target that differs by one base from the candidate wild-type probe. Further, the binding rate with the candidate mutant probe for the mutant target means the binding rate (full match binding rate) with respect to the target that completely matches the candidate mutant probe.

Here, for the wild-type target, the value obtained by subtracting the binding rate with the candidate wild-type probe (full-match binding rate) and the binding rate with the candidate mutant probe (mismatch binding rate) is, for example, 0.3 (however, binding). The rate can be 0.00 to 1.00 or more), preferably 0.4 or more, and more preferably 0.5 or more.

Further, for the mutant target, the value obtained by subtracting the binding rate with the candidate mutant probe (full-match binding rate) and the binding rate with the candidate wild-type probe (mismatch binding rate) may be, for example, 0.3 or more. It is possible, preferably 0.4 or more, and more preferably 0.5 or more.

As described above, one or a plurality of combinations of the wild-type probe and the mutant probe that meet the above-mentioned conditions can be selected from the candidate wild-type probe and the candidate mutant-type probe based on the binding rate. Then, it can be designed as one or a plurality of combinations of the selected wild-type probe and the mutant probe, and as a wild-type probe and a mutant probe for detecting a single nucleotide polymorphism to be detected. One or more combinations of the selected wild-type probe and the mutant probe are the combinations having the lowest probability of occurrence of cross-hybridization when detecting the single nucleotide polymorphism to be detected, and the single nucleotide polymorphism is used. It can be detected with high accuracy (SNP typing).

In particular, when a plurality of combinations of the wild-type probe and the mutant probe that meet the above-mentioned conditions can be selected, a better combination of the wild-type probe and the mutant probe may be further selected according to the procedure described below. ..

That is, with respect to a plurality of combinations of the selected wild-type probe and the mutant probe, the binding rate of the wild-type probe to the wild-type target (full-match binding rate) and the binding rate of the mutant probe to the mutant target (full-match binding rate). The absolute value of the difference between the above 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) are summed. Then, the combination having the lowest total value is selected from the plurality of combinations. In the combination of the wild type probe and the mutant probe having the lowest total value, the judgment values of the wild type, the heterotype and the mutant type are evenly separated with respect to the single nucleotide polymorphism to be detected, and the single nucleotide polymorphism is more accurately separated. Polymorphism can be detected.

Further, when a plurality of combinations of the wild-type probe and the mutant probe that meet the above-mentioned conditions can be selected, the procedure for further selecting a better combination of the wild-type probe and the mutant probe is not limited to the above-mentioned procedure. For example, any procedure described below may be followed.

That is, with respect to a plurality of combinations of the selected wild-type probe and the mutant probe, the binding rate of the wild-type probe to the wild-type target (full-match binding rate) and the binding rate of the mutant probe to the wild-type target (mismatch binding rate). Select the combination with the largest difference. In the combination of the wild-type probe and the mutant-type probe selected in this way, the determination values of the wild-type (group A) and the heterotype and the mutant type (group B) are largely separated with respect to the single nucleotide polymorphism to be detected. Group A and group B can be detected with higher accuracy.

Alternatively, for multiple combinations of the selected wild-type probe and the mutant probe, the binding rate of the mutant probe to the mutant target (full-match binding rate) and the binding rate of the wild-type probe to the mutant target (mismatch binding rate). Select the combination with the largest difference. In the combination of the wild-type probe and the mutant-type probe selected in this way, the determination values of the wild-type, the heterotype (group C), and the mutant type (group D) are largely separated with respect to the single nucleotide polymorphism to be detected. Group C and group D can be detected with higher accuracy.

From multiple combinations of wild-type probes and mutant probes, as described above, wild-type probes and mutations that can detect wild-type (group A), heterozygous and mutant (group B) with higher accuracy. The combination of type probes is effective, for example, when a single nucleotide polymorphism related to a dominant mutation is to be detected.

After drinking, the highly toxic acetaldehyde produced from alcohol is detoxified by the catalytic action of several enzymes. The enzyme ALDH2 plays the most important role in this process. For the gene encoding this enzyme, it is known that the single nucleotide polymorphism from G to A located on chromosome 12 exon 12 has a significant effect on the enzyme activity. This single nucleotide polymorphism is "dominantly dominant", and it has been clarified that the heterozygotes (ALDH2 * 1) or mutant (ALDH2 * 2) have less than 10% enzyme activity compared to the wild type. ..

When the enzyme activity of ALDH2 is judged by the single nucleotide polymorphism, the combination of the wild type probe and the mutant type probe capable of detecting the above-mentioned wild type (group A), heterozygous type and mutant type (group B) with higher accuracy. It is preferable to use.

By the way, the wild-type probe and the mutant-type probe designed as described above are preferably nucleic acids, and more preferably DNA. The DNA includes both double-stranded and single-stranded DNA, but is preferably single-stranded DNA. Wild-type probes and mutant-type probes can be obtained, for example, by chemically synthesizing them with a nucleic acid synthesizer. As the nucleic acid synthesizer, a device called a DNA synthesizer, a fully automatic nucleic acid synthesizer, an automatic nucleic acid synthesizer, or the like can be used.

Wild-type and mutant probes designed as described above are preferably used in the form of microarrays (as an example, DNA chips) by immobilizing their 5'ends on a carrier. At this time, when there are a plurality of single nucleotide polymorphisms to be detected, the microarray has a mutant probe and a wild-type probe at predetermined positions for each single nucleotide polymorphism.

The microarray according to the present invention can be produced 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 and are not particularly limited. For example, precious metals such as platinum, platinum black, gold, palladium, rhodium, silver, mercury, tungsten and their compounds, and conductive materials such as carbon represented by graphite and carbon fiber; monocrystalline silicon, amorphous. Silicon materials typified by silicon, silicon carbide, silicon oxide, silicon nitride, etc., composite materials of these silicon materials typified by SOI (silicon on insulator); glass, quartz glass, alumina, sapphire, ceramics, fol Inorganic materials such as sterite and photosensitive glass; polyethylene, ethylene, polyprovylene, cyclic polyolefin, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluororesin, polyvinyl chloride, polyvinylidene chloride, polyvinylacetate, 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 such as polysulfone. Materials and the like can be mentioned. The shape of the carrier is also not particularly limited, but is preferably flat.

In the present invention, as the carrier, a carrier having a carbon layer and a chemically modifying group on the surface is preferably used. The carrier having a carbon layer and a chemical modification group on the surface includes a carrier having a carbon layer and a chemical modification group on the surface of the substrate and a carrier having a chemical modification group on the surface of the substrate composed of the carbon layer. As the material of the substrate, those known in the art can be used, and the same materials as those mentioned above as the carrier materials can be used without particular limitation.

In the microarray according to the present invention, a carrier having a fine flat plate-like structure is preferably used. The shape is not limited to rectangular, square and round, but usually 1 to 75 mm square, preferably 1 to 10 mm square, and more preferably 3 to 5 mm square are used. Since it is easy to produce a carrier having a fine flat plate-like structure, it is preferable to use a substrate made of a silicon material or a resin material, and in particular, a carrier having a carbon layer and a chemically modifying group on the surface of a substrate made of single crystal silicon is more preferable. preferable. Single crystal silicon contains a slight change in the orientation of the crystal axis in a partial part (sometimes called a mosaic crystal) or an atomic scale disorder (lattice defect). Is also included.

The carbon layer formed on the substrate in the present invention is not particularly limited, but is limited to synthetic diamond, high-pressure synthetic diamond, natural diamond, soft diamond (for example, diamond-like carbon), amorphous carbon, and carbon-based material (for example, graphite and fullerene). , Carbon nanotubes), a mixture thereof, or a laminate thereof is preferable. Further, carbides such as hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, thorium carbide, titanium carbide, uranium carbide, tungsten carbide, zirconium carbide, molybdenum carbide, chromium carbide, vanadium carbide and the like may be used. Here, soft diamond is a general term for incomplete diamond structures that are a mixture of diamond and carbon, such as so-called diamond-like carbon (DLC: Diamond Like Carbon), and the mixing ratio thereof is not particularly limited. The carbon layer is excellent in chemical stability and can withstand the subsequent reaction in the introduction of chemical modifying groups and the bond with the substance to be analyzed, and the bond is flexible because it is bonded to the substance to be analyzed by electrostatic bonding. It is advantageous in that it has the property, it is transparent to the detection system UV because it does not absorb UV, and it can be energized during electroblotting. It is also advantageous in that there is little non-specific adsorption in the binding reaction with the substance to be analyzed. As described above, a carrier whose substrate itself is made of a carbon layer may be used.

In the present invention, the carbon layer can be formed by a known method. For example, microwave plasma CVD (Chemical vapor deposit) method, ECRCVD (Electric cyclotron resonance chemical vapor deposit) method, ICP (Inductive coupled plasma) method, DC sputtering method, ECR (Electric cyclotron resonance) sputtering method, ionized vapor deposition method, arc. Examples include a formula vapor deposition method, a laser vapor deposition method, an EB (Electron beam) vapor deposition method, and a resistance heating vapor deposition method.

In the high-frequency plasma CVD method, the raw material gas (methane) is decomposed by the glow discharge generated between the electrodes by the high frequency, and a carbon layer is synthesized on the substrate. In the ionization vapor deposition method, thermions generated by the tungsten filament are used to decompose and ionize the raw material gas (benzene), and a carbon layer is formed on the substrate by the bias voltage. A carbon layer may be formed by an ionization vapor deposition method in a mixed gas consisting of 1 to 99% by volume of hydrogen gas and 99 to 1% by volume of remaining methane gas.

In the arc-type vapor deposition method, an arc discharge is generated in a vacuum by applying a DC voltage between a solid graphite material (cathode evaporation source) and a vacuum vessel (anode) to generate carbon atom plasma from the cathode and evaporate source. By applying a more 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 vapor deposition method, for example, a carbon layer can be formed by irradiating a graphite target plate with Nd: YAG laser (pulse oscillation) light to melt it and depositing carbon atoms on a glass substrate.

When a carbon layer is formed on the surface of a substrate, the thickness of the carbon layer is usually about a monolayer to 100 μm, and if it is too thin, the surface of the underlying substrate may be locally exposed, and conversely, it is thick. In this case, the productivity is deteriorated, so it is preferably 2 nm to 1 μm, more preferably 5 nm to 500 nm.

By introducing a chemically modifying 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 chemically modifying 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 rays in ammonia gas or by plasma treatment. Alternatively, it can be carried out by irradiating the carbon layer with ultraviolet rays in chlorine gas to chlorinate it, and then irradiating it with ultraviolet rays in ammonia gas. Alternatively, it can be carried out by reacting a polyvalent amine gas such as methylenediamine or ethylenediamine with a chlorinated carbon layer.

The introduction of the carboxyl group can be carried out, for example, by reacting the aminated carbon layer as described above with an appropriate compound. Examples of the compound used for introducing a carboxyl group are represented by the formula: X-R1-COOH (in the formula, X represents a halogen atom and R1 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; Among them, R2 represents a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms), and dicarboxylic acids such as oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, and phthalic acid; polyacrylic acid. , Polycarboxylic acids such as polymethacrylic acid, trimellitic acid, butanetetracarboxylic acid; formula: R3-CO-R4-COOH (in the formula, R3 is a hydrogen atom or a divalent hydrocarbon group having 1 to 12 carbon atoms. , R4 represents a divalent hydrocarbon group having 1 to 12 carbon atoms); keto acid or aldehyde acid; formula: X-OC-R5-COOH (in the formula, X is a halogen atom, R5 is a single bond or Represents a divalent hydrocarbon group having 1 to 12 carbon atoms.) Monohalides of dicarboxylic acids, such as succinic acid monoclonal acid, malonic acid monoclonal acid; phthalic acid anhydride, succinic acid anhydride, oxalic acid anhydride, maleine anhydride. Examples thereof include acid anhydrides such as acid and butanetetracarboxylic acid anhydride.

The introduction of the epoxy group can be carried out, for example, by reacting the carbon layer aminolated as described above with a suitable polyvalent epoxy compound. Alternatively, it can be obtained by reacting an organic peracid with a carbon-carbon double bond contained in the carbon layer. Examples of the organic peracetic acid include peracetic acid, perbenzoic acid, diperoxyphthalic acid, performic acid, trifluoroperacetic acid and the like.

The introduction of the formyl group can be carried out, for example, by reacting the carbon layer aminolated as described above with glutaraldehyde.

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

The active ester group means an ester group having an electron-withdrawing group having a high acidity on the alcohol side of the ester group and activating the nucleophilic reaction, that is, an ester group having a high reaction activity. It is an ester group that has an electron-withdrawing group on the alcohol side of the ester group and is more activated than the alkyl ester. The active ester group has reactivity with a group such as an amino group, a thiol group and a hydroxyl group. More specifically, active ester groups such as phenol esters, thiophenol esters, N-hydroxyamine esters, cyanomethyl esters, and esters of heterocyclic hydroxy compounds have much higher activity than alkyl esters and the like. Known as. More specifically, examples of the active ester group include p-nitrophenyl group, N-hydroxysuccinimide group, succinimide group, phthalateimide group, 5-norbornen-2,3-dicarboxyimide group and the like. In particular, an N-hydroxysuccinimide group is preferably used.

To introduce the active ester group, for example, the carboxyl group introduced as described above is combined with a dehydration condensing agent such as cyanamide or carbodiimide (for example, 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide) and N-. It can be carried out by active esterification with a compound such as hydroxysuccinimide. By this treatment, a group to which an active ester group such as an N-hydroxysuccinimide group is bonded can be formed at the end of the hydrocarbon group via an amide bond (Japanese Patent Laid-Open No. 2001-139532).

A wild-type probe and a mutant 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 device or the like. By spotting, it is possible to produce a microarray in which a wild-type probe and a mutant probe are immobilized on a carrier. Alternatively, the spotting solution may be manually spotted with a micropipetter.

After spotting, incubation is preferred because the wild-type and mutant probes proceed with the reaction of binding to the carrier. Incubation is usually carried out 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. Incubation is preferably performed in a high humidity atmosphere, for example, in a humidity of 50 to 90%. Following the incubation, it is preferable to wash with a wash solution (eg, 50 mM TBS / 0.05% Tween20, 2 × SSC / 0.2% SDS solution, ultrapure water, etc.) to remove DNA that is not bound to the carrier. ..

By using the microarray configured as described above, it is possible to determine the genotype (wild type, heterotype or mutant type) of the single nucleotide polymorphism to be detected in the diagnosis target person.

Specifically, when determining the genotype of a predetermined single nucleotide polymorphism, a step of extracting DNA from a sample derived from a person to be diagnosed and a region containing the single nucleotide polymorphism using the extracted DNA as a template are used. It includes a step of amplifying and a step of determining the genotype of a single nucleotide polymorphism contained in the amplified nucleic acid using the above-mentioned microarray.

The person to be diagnosed is usually a human, and is not particularly limited to a race or the like, but is particularly a yellow race, preferably an East Asian race, and particularly preferably a Japanese. In addition, the diagnosis target can be a patient suspected of having a myeloproliferative neoplasm.

The sample derived from the person to be diagnosed is not particularly limited. Examples include blood-related samples (blood, serum, plasma, etc.), lymph, feces, cancer cells, crushed tissues or organs and extracts.

First, DNA is extracted from a sample collected from a person 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, an amplification reaction is carried out using the obtained DNA as a template to amplify the region containing the single nucleotide polymorphism to be detected. As the amplification reaction, a polymerase chain reaction (PCR), LAMP (Loop-Mediated Isothermal Amplification), ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids) method and 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, the method for labeling the amplified nucleic acid is not particularly limited, but for example, a method in which a primer used for the amplification reaction is labeled in advance may be used, or a labeled nucleotide is used as a substrate for the amplification reaction. You may use the method of The labeling substance is not particularly limited, but a radioisotope, a fluorescent dye, or an organic compound such as digoxigenin (DIG) or biotin can be used.

In addition, this reaction system includes a buffer required for nucleic acid amplification / labeling, a heat-resistant DNA polymerase, a primer specific to the amplification region, and a labeled nucleotide triphosphate (specifically, a nucleotide triphosphate to which a fluorescent label or the like is added). , Nucleotide triphosphate, magnesium chloride and the like.

The nucleic acid fragment amplified by the primer is not particularly limited as long as it contains a region corresponding to the designed wild-type probe and mutant probe, and is, for example, preferably 1 kbp or less, more preferably 800 bp or less, and more preferably 500 bp or less. More preferably, 350 bp or less is particularly preferable.

By performing a hybridization reaction between the amplified nucleic acid obtained as described above and the wild-type probe and the mutant probe immobilized on the carrier, the hybridization of the amplified nucleic acid to the wild-type probe and the mutant probe is detected. The genotype of the above-mentioned single-base hybridization in the person to be diagnosed can be determined.

For the signal from the label, for example, when a fluorescent label is used, the fluorescent signal can be detected by 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. The stringent condition is a condition in which a specific hybrid is formed and a non-specific hybrid is not formed. For example, after a hybridization reaction at 55 ° C. for 16 hours, 2 × SSC / 0.2% SDS, 25. Refers to the conditions for cleaning at ℃, 10 minutes and 2 × SSC, 25 ℃, 5 minutes. Alternatively, the hybridization temperature 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 chain length of the probe is short. When the chain length is long, it is more preferable to set the hybridization temperature higher than this. Needless to say, the higher the salt concentration, the higher the hybridization temperature having specificity, and conversely, the lower the salt concentration, the lower the hybridization temperature having specificity.

In particular, the hybrids temperature is preferably the hybrids temperature at the binding rate calculated when designing the wild-type probe and the mutant 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 probe are designed based on the binding rate, it is preferable to set the hybrids temperature to 55 ° C.

Further, when a microarray including a mutant probe and a wild-type probe is used, the genotype of the single nucleotide polymorphism can be determined by using the signal intensities from these mutant probe and the wild-type probe. Specifically, the signal intensity in the wild-type probe and the signal intensity in the mutant probe are measured, respectively, and a determination value for evaluating the signa intensity derived from the mutant probe is calculated. As an example of calculating 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 probe]) = judgment value is used. There is a way to do it.

Then, the judgment value calculated by the above formula is compared with a predetermined threshold value (cutoff value), and if the judgment value exceeds the first threshold value, the one-base polymorphism contained in the amplified nucleic acid is a wild type. If the determination value is below the first threshold value and exceeds the second threshold value, it is determined that the one-base polymorphism in the amplified nucleic acid is heteromorphic, and the determination value is below the second threshold value. In some cases, it is determined that the monobasic polymorphism is a variant in the amplified nucleic acid (first threshold> second threshold).

Here, as the first threshold and the second threshold, it is confirmed that the sample in which the single nucleotide polymorphism to be tested is a wild type and the single nucleotide polymorphism to be tested are mutant types. It can be specified based on the judgment value calculated by the above formula using the sample. More specifically, a plurality of judgment values are calculated using a plurality of samples in which it is confirmed that the single nucleotide polymorphism to be tested is a wild type, and the average value + 3σ (σ: standard deviation). The value can be the first threshold. In addition, multiple judgment values are calculated using multiple samples for which it is confirmed that the single nucleotide polymorphism to be tested is a mutant type, and the average value + 3σ (σ: standard deviation) is used as the second value. Can be the threshold of. The value of the average value + 2σ or the average value + σ can also be used as the 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 of the exon 5 single nucleotide polymorphism (CYP2C19 * 2) of the CYP2C19 gene was designed as an example. The targets containing the single nucleotide polymorphism were a wild-type target (TTAAGTAATTTGTTATGGGTTCcGGGAAATAATCAATGATAGTGGG: SEQ ID NO: 1, lowercase polymorphic site) and a mutant target (TTAAGTAATTTGTTATGGGTTCCtGGGAAATAATCAATGATAGTGGG: SEQ ID NO: 2, lowercase polymorphic site).

First, candidate wild-type probes (Table 1) and candidate mutant probes (Table 2) for detecting these wild-type targets were designed. In Tables 1 and 2, the bases corresponding to single nucleotide polymorphisms are shown in lowercase.

Next, for the candidate wild-type and candidate mutant probes shown in Tables 1 and 2, the Tm curves and Tm values for the wild-type and mutant targets were calculated. 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 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.

Table 3 shows the results of calculating the Tm value for the candidate wild-type probe, and Table 4 shows the results of calculating the Tm value for the candidate mutant probe. In Tables 3 and 4, "FM" means the Tm value in hybridization with a target that fully matches, and "MM" means a hybrid with a target that has a single nucleotide mismatch (Miss Match). It means the Tm value in soybeans.

In addition, the bond rate at a temperature of 55 ° C. was calculated based on the obtained Tm curve. Table 5 shows the results of calculating the binding rate for the candidate wild-type probe, and Table 6 shows the results of calculating the binding rate for the candidate mutant probe.

In Table 5, "WP-FM" means hybridization between the candidate wild-type probe and the wild-type target (full match), and "WP-MM" means mutation with the candidate wild-type probe. It means hybridization with a type target (single nucleotide mismatch: Miss Match). Similarly, in Table 6, “VP-FM” means hybridization (full match) between the candidate mutant probe and the mutant target, and “VP-MM” means the candidate mutant probe. It means hybridization with a wild-type target (single nucleotide 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 probe (mismatch binding) with respect to the wild-type target. The value obtained by subtracting the rate) was obtained. The results are shown in FIGS. 1 to 3. The numerical values of each cell shown in FIGS. 1 to 3 are the mismatch binding rate (Table 6) of the candidate mutant probe shown on the horizontal axis from the full-match binding rate (Table 5) of the candidate wild-type probe shown on the vertical axis. Shows the subtracted value. Then, in FIGS. 1 to 3, squares having a subtracted value of 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 probe (full-match binding rate) and the binding rate with the candidate wild-type probe (mismatch binding rate) with respect to the mutant target. ) Was subtracted. The results are shown in FIGS. 4-6. The numerical values of each cell shown in FIGS. 4 to 6 are the mismatch binding rate (Table 5) of the candidate wild-type probe shown on the vertical axis from the full-match binding rate (Table 6) of the candidate mutant probe shown on the horizontal axis. Shows the subtracted value. Then, in FIGS. 4 to 6, squares having a subtracted value of 0.5 or more are shaded.

Further, the cells in which the shades shown in FIGS. 4 to 6 and the shades shown in FIGS. 1 to 3 overlap are surrounded by thick lines. That is, in the mass surrounded by the thick line, at 55 ° C., the value obtained by subtracting the binding rate from the candidate wild-type probe from the binding rate to the candidate wild-type probe is 0.5 or more for the wild-type target, and the mutant type Regarding the target, the combination of the wild-type probe and the mutant probe in which the value obtained by subtracting the binding rate with the candidate wild-type probe from the binding rate with the candidate mutant probe is 0.5 or more is shown.

That is, in this example, the following combinations could be selected as the wild-type probe and the mutant probe for determining the single nucleotide polymorphism of exon 5 single nucleotide polymorphism (CYP2C19 * 2) of the CYP2C19 gene with high accuracy. ..
・ WP_20mer_L and VP_24mer_R
・ WP_21mer and VP_24mer_R
・ WP_22mer_R and VP_24mer_R
・ WP_22mer_L and VP_24mer_R
・ WP_23mer and VP_24mer_R
・ WP_22mer_R and VP_24mer_L
・ WP_22mer_L and VP_24mer_L
・ WP_23mer and VP_24mer_L
・ WP_23mer and VP_25mer
Further, in this 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] Was used to perform a single nucleotide polymorphism genotyping experiment.

In this genotyping experiment, for comparison, a probe set selected from the viewpoint of making the probe lengths equal (Comparative Example 1) and a probe set selected from the viewpoint that the Tm value is close to 55 ° C (Comparative Example 2). Also used. The probe set of Comparative Example 1 is five sets of [WP_22mer_R and VP_22mer_R], [WP_22mer_L and VP_22mer_L], [WP_23mer and VP_23mer], [WP_24mer_R and VP_24mer_R], and [WP_24mer_L and VP_24mer_L]. The probe sets of Comparative Example 2 are five sets of [WP_19mer and VP_23mer], [WP_18mer_L and VP_23mer], [WP_19mer and VP_23mer], [WP_20mer_R and VP_23mer], and [WP_19mer and VP_24mer_R].

A DNA chip (CYP chip) in which the probe set of these examples, the probe set of Comparative Example 1 and the probe set of Comparative Example 2 were arranged was prepared. Then, the wild-type target, hetero-type target or mutant target is reacted at a concentration of 2 μM at a concentration of 2 μM, the CYP chip at 1xSSC, 0.1% SDS, and the hybridization temperature of 55 ° C for 1 hour, and the fluorescence scanner (manufactured by BIOSHOT) is used for 7 seconds. Fluorescence intensity was obtained.

Then, wild-type, hetero-type, and mutant-type determination values were calculated from the fluorescence intensities obtained in the probe set of Example, the probe set of Comparative Example 1, and the probe set of Comparative Example 2, and compared. The results are shown in FIG. Further, based on the results of each probe set shown in FIG. 7, the difference (total width) between the judgment value when the wild-type target was used and the judgment value when the mutant target was used, and the mutant target were used. The value obtained by subtracting the judgment value when the heterotype target is used from the judgment value at the time divided by the judgment value when the heterotype target is used minus the judgment value when the wild type target is used. (Ratio (VH / HW)) is shown in FIG.

In the graph shown in FIG. 8, a probe set having a large total width value and a ratio close to 1 means that cross hybridization is unlikely to occur, and the total width is smaller or the ratio is farther from 1. This means that the single nucleotide polymorphism to be detected can be detected with higher accuracy than the probe set which is the value.

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 that when the probe sets of Comparative Examples 1 and 2 are used. It turns out that it is close to 1. From this result, it was clarified that when the probe set selected in this example was used, the occurrence of cross hybridization was suppressed and the genotype of the single nucleotide polymorphism could be determined with higher accuracy.

Furthermore, for the five types of probe sets selected in this example used in the genotyping experiment, the binding rate of the wild-type probe to the wild-type target (full-match binding rate) and the binding rate of the mutant 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). Totaled. 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 total value. (Total value: 0.20), [WP_23mer and VP_24mer_R] (Total value: 0.21).

As shown in FIG. 8, it can be seen that the probe set with the lower total value has a larger overall width and a ratio closer to 1. From this, when multiple probe sets for detecting single nucleotide polymorphisms are selected as described above, it is better to calculate the above total value and use the probe set having the lower total value. It turns out to be preferable.

Claims (3)

It is a method of designing a wild type probe corresponding to a wild type of a single nucleotide polymorphism and a mutant probe corresponding to the mutant type of the single nucleotide polymorphism.
We designed a plurality of candidate wild-type probes containing the single nucleotide polymorphism site and a plurality of candidate mutant probes containing the single nucleotide polymorphism site.
Binding rates of these candidate wild-type probes to wild-type targets that are in perfect agreement, binding rates of these candidate wild-type probes to mutant targets that have a single nucleotide mismatch at the single nucleotide polymorphism site, and these candidate mutant-type probes. The binding rate with the mutant target that is an exact match and the binding rate of these candidate mutant probes to the wild-type target that is a single nucleotide mismatch at the single nucleotide polymorphism site are calculated.
For wild-type targets, the value obtained by subtracting the binding rate with the candidate mutant probe from the binding rate with the candidate wild-type probe is greater than or equal to the predetermined value, and for the mutant target, the candidate is obtained from the binding rate with the candidate mutant probe. One or more combinations of the wild-type probe and the mutant probe whose value obtained by subtracting the binding rate with the wild-type probe is equal to or more than a predetermined value are selected from the candidate wild-type probe and the candidate mutant probe.
For one or more combinations of the selected wild-type probe and mutant probe, the absolute value of the difference between the binding rate of the wild-type probe to the wild-type target and the binding rate of the mutant probe to the mutant target, and the wild-type target. The absolute value of the difference between the binding rate of the mutant probe to the mutant target and the binding rate of the wild-type probe to the mutant target is summed up, and the combination having the lowest total value among the plurality of combinations is detected as the above-mentioned one-base polymorphism. A method of designing as a wild-type probe and a mutant probe for use. The claim is characterized in that the binding rate is calculated in a predetermined hybridization temperature or a predetermined hybridization temperature range, and a wild-type probe and a mutant probe to be used in the hybridization temperature or the hybridization temperature range are designed. 1 The method described. When multiple combinations of wild-type and mutant probes are selected, the combination with the largest difference between the binding rate of the wild-type probe to the wild-type target and the binding rate of the mutant probe to the wild-type target for multiple combinations. Or, the combination with the largest difference between the binding rate of the mutant probe to the mutant target and the binding rate of the wild-type probe to the mutant target is the wild-type probe and the mutant probe for detecting the above-mentioned single-base polymorphism. The method according to claim 1, wherein the method is designed as follows.

Images