JP7123593B2 - Microsatellite detection microarray and microsatellite detection method using the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a microsatellite detection method for detecting microsatellites consisting of repeating unit sequences consisting of one or more bases, and a method for detecting microsatellites using the microarray.

A microsatellite means a repetition of a unit sequence consisting of one or more bases present in the genome, and the number of repetitions is regarded as a genotype and used as a polymorphic marker. Microsatellites are also known to have different numbers of repeats between normal and tumor tissues (microsatellite instability (MSI)). That is, microsatellite instability means a phenomenon in which the number of microsatellite repeats in tumor tissue is different from that in non-tumor (normal) tissue due to dysfunction of the DNA repair mechanism in tumor tissue.

Microsatellite instability has recently been recommended for detection in the diagnosis of Lynch syndrome and sporadic colorectal cancer. Specifically, as an example, the repetition numbers of five types of microsatellites consisting of BAT25, BAT26, MONO27, NR21 and NR24 are measured in normal tissue and tumor tissue, respectively. Then, when a decrease in the number of repetitions is detected, it is determined to be MSI positive, and if one of the five types of microsatellites is MSI positive, it is low-frequency microsatellite instability (MSI-L), and two or more types are MSI. Classify microsatellite instability high (MSI-H) if positive and microsatellite stable (MSS) if no MSI positive.

At this time, in conventional techniques, a region containing microsatellites is amplified, and the number of repetitions of microsatellites is determined by so-called fragment analysis. For example, in Patent Document 1, a set of at least three genes, a mononucleotide microsatellite region and a tetranucleotide microsatellite region, are simultaneously amplified by a multiplex amplification reaction, and the amplified DNA fragments are separated based on size. A method of parsing is disclosed. In addition, US Pat. No. 6,200,001 discloses a method of amplifying a given microsatellite locus and assessing tumor-associated microsatellite instability based on the size of the DNA amplification product.

In addition to microsatellite instability, when detecting microsatellites as polymorphic markers, regions containing microsatellite polymorphisms are amplified and polymorphisms are determined based on the size of the amplified DNA fragment.

Japanese Patent Publication No. 2004-533241 Japanese Patent Publication No. 2005-518798

As mentioned above, for the analysis of microsatellite instability and microsatellite polymorphisms, after amplifying the region containing the microsatellites, size-based analysis (capillary electrophoresis) and sequence analysis (sequencing) can be used to determine the number of microsatellite repeats. was judging However, fragment analysis such as size-based analysis and sequence analysis has the problem that the number of microsatellite repeats cannot be determined with high accuracy.

SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide a microsatellite detection microarray and a microsatellite detection method using the microsatellite detection microarray that can determine the number of repetitions of microsatellites with high accuracy.

In order to achieve the above-described object, the present inventors have made intensive studies and found that when designing probes for each of a plurality of types with different number of repetitions for a given microsatellite, the probe length of each probe is set within a predetermined range. The present inventors have found that by providing a region for aligning with the substrate on the 5'-end side of the substrate, these multiple types of microsatellites can be detected with high precision using a microarray, and have completed the present invention.

The present invention includes the following.
(1) For microsatellites to be detected, a plurality of probes corresponding to a plurality of types with different numbers of repetitions, and a substrate for fixing the plurality of probes,
A microsatellite detection microarray, wherein the probe includes a region corresponding to the microsatellite and a region for adjusting probe length at the 5' end, and is immobilized on the substrate at the 5' end.
(2) The plurality of probes consist of a wild-type probe corresponding to the wild-type of the microsatellite to be detected and a mutant probe corresponding to the mutant of the microsatellite to be detected, and the mutant is a unit The microsatellite detection microarray according to (1), wherein the number of sequence repeats is smaller than that of the wild type.
(3) The microsatellite detection microarray according to (2), wherein a plurality of the mutant probes are provided according to a plurality of mutation types having different repetition numbers of unit sequences.
(4) The microsatellite detection microarray according to (1), wherein the plurality of probes have a difference in probe length of 12 bases or less.

(5) A step of amplifying a nucleic acid containing a microsatellite to be detected using DNA derived from a subject as a template;
For the microsatellites to be detected, a plurality of probes corresponding to a plurality of types with different repetition numbers and a substrate for fixing the plurality of probes are provided, and the probes include regions corresponding to the microsatellites and 5'. A step of contacting the nucleic acid amplified in the above step with a microsatellite detection microarray that includes a probe length adjusting region at the end and is immobilized on the substrate at the 5′ end;
detecting a signal based on hybridization with the amplified nucleic acid for each of the plurality of probes;
A microsatellite detection method, comprising analyzing microsatellites in the subject based on signals detected with the plurality of probes.
(6) Genomic DNA derived from normal tissue and genomic DNA derived from tumor tissue are used as the DNA derived from the subject, and the number of repetitions varies between microsatellites in normal tissue and the same microsatellites in tumor tissue. The microsatellite detection method according to (5), characterized in that microsatellite instability is analyzed.
(7) The plurality of probes consist of a wild-type probe corresponding to a wild-type microsatellite and a plurality of mutant probes corresponding to a plurality of mutation types having different unit sequence repetition numbers in mutant microsatellites. , the signal ratio obtained by normalizing the signal measured for each of the plurality of mutant probes with the signal measured for the wild-type probe is obtained for each of the normal tissue and the tumor tissue, and for each of the plurality of mutant probes The difference between the signal ratio in tumor tissue and the signal ratio in normal tissue is obtained as a judgment value, and the total value of the judgment values obtained for the plurality of mutant probes is compared with a predetermined threshold to reduce the microsatellite anxiety. The microsatellite detection method according to (6), characterized in that qualitative analysis is performed.

In the microsatellite detection microarray according to the present invention, the microsatellite to be detected can be detected with high accuracy by using a probe having a region for adjusting the probe length at the 5' end fixed to the substrate. Thus, by using the microsatellite detection microarray according to the present invention, genotyping of microsatellite polymorphisms, analysis of microsatellite instability, and the like can be performed with high accuracy.

In the microsatellite detection method according to the present invention, by using a microsatellite detection microarray having a probe having a region for adjusting the probe length at the 5' end fixed to the substrate, the microsatellite to be detected is detected with high accuracy. be able to. Thus, by applying the microsatellite detection method according to the present invention, genotyping of microsatellite polymorphisms, analysis of microsatellite instability, and the like can be performed with high accuracy.

FIG. 2 is a characteristic diagram showing base sequences and the like of microsatellites employed in this example. FIG. 4 is a characteristic diagram showing the results of measuring the fluorescence intensity of probes 3 and 7 designed in Experiment 1 when using an MSI-negative sample and an MSI-positive sample, respectively. 1 is a characteristic diagram showing the measurement results of the probe designed in Experiment 1 when using an MSI-negative sample and an MSI-positive sample, with the horizontal axis representing the number of microsatellite repetitions of the probe and the vertical axis representing the fluorescence intensity ratio. FIG. 10 is a characteristic diagram showing the results of microsatellite instability analysis of MSI-negative and MSI-positive samples using probes designed for NR21, NR24, BAT25 and BAT26 designed in Experiment 2;

Hereinafter, the microsatellite detection microarray and the microsatellite detection method using the same according to the present invention will be described in detail.

A microsatellite detection microarray according to the present invention comprises a plurality of probes corresponding to a plurality of types with different repetition numbers for a given microsatellite, and a substrate on which the plurality of probes are immobilized.

Here, the microsatellite means a region in which a unit sequence consisting of one base or multiple bases existing in the genome is repeated. A microsatellite that repeats a unit sequence consisting of one base, in other words, a region in which A, G, C or T is continuous (5'-(N) _n -3': N is A, G, C or T and n is the number of bases). In microsatellites, the unit sequence consisting of a plurality of bases is not particularly limited, but for example, a unit sequence consisting of 2 to 10 bases, preferably 2 to 4 bases, can be mentioned. Furthermore, the number of repetitions of the unit array in the microsatellite is not particularly limited, and can be, for example, 2 to 100 times, preferably 4 to 100 times.

Regarding microsatellites, for example, a phenomenon (microsatellite instability) is known in which the number of repetitions of a given microsatellite unit sequence differs between normal tissue and tumor tissue. Microsatellite instability refers to a phenomenon in which the number of microsatellite repeats varies due to a decline in the ability to repair mismatches during genome replication.

The microsatellite detection microarray according to the present invention comprises wild-type probes corresponding to microsatellites in normal tissue and mutant probes corresponding to microsatellites with different repeat numbers in tumor tissue, thereby evaluating microsatellite instability. can be used on occasion.

Specifically, Lynch syndrome (Hereditary Nonpolyposis Colon Cancer (HNPCC)) is known to be caused by germline mutations in the mismatch repair genes MLH1, MSH2, MSH6, and PMS2. It is Therefore, tumor cells having the mutated gene show characteristics such as variations in the number of microsatellite repeats compared to normal cells. Thus, one example of microsatellite instability is variation in the number of microsatellite repeats due to mutations in these mismatch repair genes in Lynch syndrome.

More specifically, in the Lynch syndrome test, the number of repeats of five microsatellites consisting of BAT25, BAT26, MONO27, NR21 and NR24 is measured. BAT25 is a 25T repeat specifically located in intron 16 of c-kit. BAT26 is a 26T repeat located in intron 5 of hMSH2. MONO27 is specifically a 27T repeat located in the MAP4K3 gene (cDNA sequence GenBank AC007684). NR21 is a 21T repeat specifically identified in the 5' untranslated region of the SLC7A8 gene (cDNA sequence GenBank XM_033393). NR24 is a 24T repeat specifically identified in the 3' untranslated region of the zinc finger-2 gene (cDNA sequence GenBank X60152). Additionally, microinstability is known to have a characteristic molecular and clinicopathological profile in colon and gastric tumors and is often associated with favorable prognosis. Evidence also suggests that colorectal cancer patients with microunstable tumors show a favorable survival benefit from 5FU-based chemotherapy. Microinstability is therefore considered to be a useful molecular predictive marker for response to this type of adjuvant therapy.

In addition, with regard to microsatellites, a large number of alleles (major alleles and minor alleles) having different unit sequence repeat counts may exist among individuals. Such microsatellites can be used as polymorphic markers whose genotype is the number of repeats. That is, the microsatellite detection microarray according to the present invention includes wild-type probes corresponding to major alleles and mutant probes corresponding to minor alleles, and can be used to identify (typing) microsatellite polymorphisms. .

More specifically, an example of using microsatellites as polymorphic markers in genes associated with asthma (International Publication No. WO1999/037809), microsatellites as a useful diagnostic tool for predictive evaluation of human hepatocellular carcinoma disease (HCC). Examples of using microsatellites as polymorphic markers (International Publication No. WO1998/045478), and examples of using microsatellites as polymorphic markers in a method for diagnosing the possibility that a mammary gland tumor sample obtained from a non-human subject is a malignant tumor (especially JP 2013-039070), an example of using microsatellites as polymorphic markers for cancer diagnosis and treatment (International Publication WO2004/043387), an example of using microsatellites as polymorphic markers for cancer diagnosis ( International Publication WO2008/090930), an example of using microsatellites as polymorphic markers for the diagnosis of cancer associated with OBCAM and NTM genes (Japanese Unexamined Patent Publication No. 2009-165473), using microsatellites for purity control of rice seeds, etc. An example of utilization can be given (JP-A-11-206374).

As described above, the microsatellite detection microarray according to the present invention includes a plurality of probes (one wild-type probe and one , one or more mutant probes). For example, when using the microsatellite detection microarray according to the present invention to detect microsatellites that can be used as polymorphic markers, probes are designed for each of a plurality of repetitions present in the microsatellite, that is, for each genotype. Further, for example, when evaluating microsatellite instability using the microsatellite detection microarray according to the present invention, wild-type probes corresponding to the microsatellites in normal cells and microsatellites with a lower number of repetitions than the microsatellites in normal cells Mutant probes corresponding to satellites (one or more) are designed.

Here, the probes of the microsatellite detection microarray according to the present invention have a region corresponding to the microsatellite to be detected and a region for adjusting probe length. The probe has a region corresponding to the microsatellite to be detected and a region for adjusting the probe length in this order from the 3' end to the 5' end. fixed to the board.

The region corresponding to the microsatellite to be detected is a nucleotide chain having a sequence complementary to the microsatellite to be detected. For example, when the microsatellite to be detected is 5'-(CA) _n -3', the region corresponding to the microsatellite to be detected is 5'-(TG) _n -3'. Note that n is the number of repetitions of microsatellites. In addition, the region corresponding to the microsatellite to be detected may have a sequence complementary to the microsatellite to be detected and a region following the 3'-end and/or 5'-end of the sequence. The regions following the 3′ end and 5′ end of the sequence complementary to the microsatellite to be detected are sequences complementary to the regions contiguous to the 5′ end and 3′ end of the microsatellite to be detected, respectively. . The region following the 3'-end and 5'-end of the sequence complementary to the microsatellite to be detected can be, for example, a total length of 10 to 40 bases on both sides, preferably 15 to 30 bases. .

As described above, regions corresponding to microsatellites to be detected have different sequences and lengths for each probe depending on the number of microsatellite repeats. Hypothetically, when designing multiple probes, if only the regions corresponding to the target microsatellites are different and the other regions have the same sequence, the shorter the region corresponding to the target microsatellites (the number of repetitions is the less), the shorter the probe length. Therefore, when designing probes for each of a plurality of types with different numbers of repetitions for a given microsatellite, a region for adjusting the probe length is designed so that the probe length between probes is within a predetermined range. do. More specifically, the shorter the region corresponding to the microsatellite to be detected (the smaller the number of repetitions), the longer the region for adjusting the probe length, thereby suppressing the variation in probe length for multiple probes, It can be within a predetermined range.

Here, the probe is designed so that the region for adjusting the probe length is located on the 5' end side of the region corresponding to the microsatellite to be detected. Therefore, the region for adjusting the probe length is designed as a sequence complementary to the region continuous from the microsatellite to be detected to the 3'-end side.

As described above, the probes of the microsatellite detection microarray according to the present invention are designed to have a region corresponding to the microsatellite to be detected and a region for adjusting the probe length. It may have an area other than the area. For example, the probe has a region for adjusting the probe length at the 5' end, a region of several bases at the 3' end, and a region corresponding to microsatellites between these regions. may be Alternatively, the probe may be configured to have a linker sequence for fixing to the substrate on the 5'-end side of the region for adjusting the probe length.

Further, by providing a region for adjusting the probe length, the difference in base length between a plurality of probes is within a predetermined range, for example, preferably within 12 bases, more preferably within 11 bases, It is more preferably within 10 bases, more preferably within 9 bases, further preferably within 8 bases, further preferably within 7 bases, further preferably within 6 bases, It is more preferably 5 bases or less, more preferably 4 bases or less, still more preferably 3 bases or less, still more preferably 2 bases or less, and even more preferably 1 base or less.

Furthermore, by providing a region for adjusting probe length, the difference in base length between a plurality of probes is preferably within 40% of the length of the shortest probe.

On the other hand, when designing a probe having a region corresponding to a microsatellite to be detected and a region for adjusting probe length, it is preferable to set the Tm value between probes within a predetermined range. The Tm values of multiple probes designed for a given microsatellite are preferably within a range of ±5°C. By setting the Tm values in this range for the plurality of probes, it is possible to more accurately detect a plurality of microsatellites to be detected that differ in the number of repetitions.

Here, the method for calculating the Tm value is not particularly limited, and examples thereof include the Nearest Neighbor method, the Wallace method and the GC% method. It is preferable to calculate by Nearest Neighbor method). The Tm value is defined as the temperature at which 50% of the hybridized probe and target dissociate (that is, the temperature at which the binding rate is 50%). Factors affecting the Tm value include base composition, salt concentration, concentration of oligo chain, denaturant (formamide, DMSO, etc.), solvation effect, conjugate group (biotin, digoxigenin, alkaline phosphatase, fluorescent dye, etc.). ).

More specifically, the Tm value can be calculated by a web service provided by Integrated DNA Technologies, which includes the base sequence of the probe, the concentration of the probe, the concentration of the target that hybridizes with the probe, and Na ⁺ in the reaction solution. and the Tm value can be calculated by setting the K ⁺ concentration, the Mg ²⁺ concentration in the reaction and (optionally) the dNTPs concentration. The Tm values of a plurality of probes designed as described above are calculated by fixing parameters other than the base sequences of the probes to predetermined conditions.

With respect to the microsatellite to be detected, which is designed as described above, a plurality of probes corresponding to different repetition numbers are immobilized on a carrier at their 5' ends, and used in the form of a microarray (a DNA chip as an example). be done.

Materials known in the art can be used as the carrier material, and are not particularly limited. For example, noble metals such as platinum, platinum black, gold, palladium, rhodium, silver, mercury, tungsten and their compounds, and conductive materials such as carbon represented by graphite and carbon fiber; single crystal 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, foil Inorganic materials such as stellite and 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, phenolic resin, urea resin, epoxy resin, melamine resin, styrene-acrylonitrile copolymer, acrylonitrile-butadiene-styrene copolymer, polyphenylene oxide and polysulfone. materials and the like. Although the shape of the carrier is not particularly limited, it is preferably flat.

In the present invention, a carrier having a carbon layer and chemical modification groups on its surface is preferably used as the carrier. Carriers having a carbon layer and chemically modifying groups on the surface include those having a carbon layer and chemically modifying groups on the surface of a substrate and those having a substrate consisting of a carbon layer and chemically modifying groups on the surface. As the material for the substrate, those known in the art can be used without particular limitation, and the same materials as those listed above as the carrier material can be used.

In the microarray according to the present invention, a carrier having a fine tabular structure is preferably used. The shape is not limited to rectangular, square, round, etc., but usually 1 to 75 mm square, preferably 1 to 10 mm square, more preferably 3 to 5 mm square. 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 plate-like structure. preferable. In single crystal silicon, the direction of the crystal axis changes slightly in some parts (sometimes called mosaic crystals), and the disorder (lattice defect) on an atomic scale is included. 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 (e.g., diamond-like carbon), amorphous carbon, carbon-based substances (e.g., graphite, fullerene , carbon nanotubes), mixtures thereof, or laminates thereof. Carbides such as hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, thorium carbide, titanium carbide, uranium carbide, tungsten carbide, zirconium carbide, molybdenum carbide, chromium carbide, and vanadium carbide may also be used. Here, soft diamond is a general term for imperfect diamond structures, such as so-called diamond-like carbon (DLC), which are mixtures of diamond and carbon, and the mixing ratio is not particularly limited. The carbon layer has excellent chemical stability and can withstand the subsequent introduction of chemical modification groups and reactions during bonding with the analyte, and the bond is flexible because it bonds with the analyte through electrostatic bonding. It is advantageous in that it is transparent to the detection system UV because it does not absorb UV, and that it can be energized during electroblotting. It is also advantageous in that non-specific adsorption is less in the binding reaction with the substance to be analyzed. As described above, a carrier may be used in which the substrate itself is a carbon layer.

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

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

In the arc vapor deposition method, a direct current voltage is applied between a solid graphite material (cathode evaporation source) and a vacuum container (anode) to cause arc discharge in vacuum, generating a plasma of carbon atoms from the cathode to form an evaporation source. 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 vapor deposition method, for example, a carbon layer can be formed by irradiating a graphite target plate with Nd:YAG laser (pulsed oscillation) light to melt it and deposit carbon atoms on a glass substrate.

When forming a carbon layer on the surface of a substrate, the thickness of the carbon layer is usually from a monomolecular layer to about 100 μm. The thickness is preferably from 2 nm to 1 μm, more preferably from 5 nm to 500 nm, because the productivity deteriorates.

By introducing a chemical modification group to 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, but examples thereof include amino group, carboxyl group, epoxy group, formyl group, hydroxyl group and active ester group.

Amino groups can be introduced, for example, by irradiating the carbon layer with ultraviolet rays in ammonia gas or by plasma treatment. Alternatively, it can be carried out by chlorinating the carbon layer by irradiating it with ultraviolet rays in chlorine gas and then irradiating it with ultraviolet rays in ammonia gas. Alternatively, it can be carried out by reacting a chlorinated carbon layer in a polyvalent amine gas such as methylenediamine or ethylenediamine.

The introduction of carboxyl groups can be carried out, for example, by reacting the above-aminated carbon layer with a suitable compound. Compounds used to introduce a carboxyl group include, for example, the formula: X-R1-COOH (wherein X is 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; formula: HOOC-R2-COOH (formula in which R2 represents a single bond or a divalent hydrocarbon group with 1 to 12 carbon atoms), such as oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, phthalic acid; polyacrylic acid , polymethacrylic acid, trimellitic acid, butanetetracarboxylic acid, and other polycarboxylic acids; formula: R3-CO-R4-COOH (wherein R3 is a hydrogen atom or a divalent hydrocarbon group , R4 represents a divalent hydrocarbon group having 1 to 12 carbon atoms); formula: X-OC-R5-COOH (wherein X is a halogen atom, R5 is a single bond or Represents a divalent hydrocarbon group having 1 to 12 carbon atoms.), such as succinic acid monochloride, malonic acid monochloride; phthalic anhydride, succinic anhydride, oxalic anhydride, maleic anhydride Examples include acids and acid anhydrides such as butanetetracarboxylic anhydride.

Epoxy groups can be introduced, for example, by reacting the aminated carbon layer with an appropriate polyepoxy compound. Alternatively, it can be obtained by reacting the carbon=carbon double bond contained in the carbon layer with an organic peracid. Organic peracids include peracetic acid, perbenzoic acid, diperoxyphthalic acid, performic acid, trifluoroperacetic acid and the like.

Formyl groups can be introduced, for example, by reacting the aminated carbon layer with glutaraldehyde.

Hydroxyl groups can be introduced, for example, by reacting the carbon layer chlorinated as described above with water.

The active ester group means an ester group that has an electron-withdrawing group with high acidity on the alcohol side of the ester group and activates a nucleophilic reaction, that is, an ester group with 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. Active ester groups have reactivity with groups such as amino groups, thiol groups, and hydroxyl groups. More specifically, phenol esters, thiophenol esters, N-hydroxyamine esters, cyanomethyl esters, esters of heterocyclic hydroxy compounds, and the like have active ester groups with much higher activity than alkyl esters and the like. known as More specifically, active ester groups include, for example, p-nitrophenyl group, N-hydroxysuccinimide group, succinimide group, phthalimide group, 5-norbornene-2,3-dicarboximide group, and the like. In particular, an N-hydroxysuccinimide group is preferably used.

Introduction of an active ester group, for example, the carboxyl group introduced as described above, a dehydration condensation agent such as cyanamide or carbodiimide (e.g., 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide) and N- It can be carried out by active esterification with a compound such as hydroxysuccinimide. This treatment can form a group in which an active ester group such as an N-hydroxysuccinimide group is bonded to the end of a hydrocarbon group via an amide bond (JP-A-2001-139532).

A probe is dissolved in a spotting buffer to prepare a spotting solution, this is dispensed into a 96-well or 384-well plastic plate, and the dispensed solution is spotted on a carrier using a spotter device or the like to obtain a probe. is immobilized on a carrier. Alternatively, the spotting solution may be spotted manually with a micropipettor.

After spotting, incubation is preferably performed in order to advance the reaction in which the probe binds to the carrier. Incubation is usually carried out at a temperature of -20 to 100°C, preferably 0 to 90°C, for a period of usually 0.5 to 16 hours, preferably 1 to 2 hours. Incubation is desirably carried out in an atmosphere of high humidity, for example, at a humidity of 50-90%. Following incubation, it is preferable to wash with a washing solution (e.g., 50 mM TBS/0.05% Tween20, 2×SSC/0.2% SDS solution, ultrapure water, etc.) 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, heterozygous type, or mutant type) of the microsatellite to be detected in the subject, or to evaluate the microsatellite instability. be able to.

Specifically, when determining the genotype for a given microsatellite polymorphism, there are steps of extracting DNA from a sample derived from a subject, and using the extracted DNA as a template to determine a region containing the microsatellite polymorphism. A step of amplifying, and a step of determining the genotype (number of repetitions) of the microsatellite polymorphism contained in the amplified nucleic acid using the microarray described above. Further, specifically, when evaluating the instability of a given microsatellite, a step of extracting DNA from each of the normal tissue and the tumor tissue of the subject, and using the extracted DNA as a template, including the microsatellite Amplifying the region and determining the number of microsatellite repeats contained in the amplified nucleic acid using the microarray described above.

Subjects are usually humans, and although there are no particular restrictions on their race and the like, they are particularly of yellow race, preferably of East Asian race, and particularly preferably of Japanese. Subject-derived samples are not particularly limited. Examples thereof include blood-related samples (blood, serum, plasma, etc.), lymph, feces, cancer cells, tissue or organ lysates and extracts, and the like. In addition, when evaluating microsatellite instability, the subject can be a patient suspected of having Lynch syndrome or sporadic colorectal cancer.

The extraction means for extracting DNA from a sample collected from a subject is not particularly limited. For example, DNA extraction methods using phenol/chloroform, ethanol, sodium hydroxide, CTAB and the like can be used.

Next, an amplification reaction is performed using the obtained DNA as a template to amplify the region containing the single nucleotide polymorphism to be detected. As the amplification reaction, 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 an amplification reaction, it is desirable to add a label so that the amplified region can be identified. At this time, the method for labeling the amplified nucleic acid is not particularly limited. For example, a method of labeling the primers used in the amplification reaction in advance may be used, or a labeled nucleotide may be used as a substrate in the amplification reaction. method may be used. Labeling substances are not particularly limited, but radioisotopes, fluorescent dyes, or organic compounds such as digoxigenin (DIG) and biotin can be used.

In addition, this reaction system contains a buffer necessary for nucleic acid amplification and labeling, a heat-stable DNA polymerase, a primer specific to the amplification region, a labeled nucleotide triphosphate (specifically, a nucleotide triphosphate with a fluorescent label, etc.) , nucleotide triphosphates and magnesium chloride.

In addition, the nucleic acid fragment amplified by the primers is not particularly limited as long as it contains the microsatellite to be detected. .

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, and detecting the hybridization of the amplified nucleic acid to the wild-type probe and the mutant probe. It is possible to determine the number of repetitions of unit arrays in the microsatellites in the person to be diagnosed.

For the signal from the label, for example, when a fluorescent label is used, the fluorescence signal is detected using a fluorescence scanner, and the signal intensity can be quantified by analyzing it with image analysis software. Amplified nucleic acids hybridized to wild-type probes and mutant-type probes can also be quantified, for example, by preparing a standard curve using samples containing known amounts of DNA. Hybridization reactions are preferably performed under stringent conditions. Stringent conditions refer to conditions under which specific hybrids are formed and non-specific hybrids are not formed. °C for 10 minutes and 2xSSC at 25°C for 5 minutes. Alternatively, the hybridization temperature can be 40 to 80° C. when the salt concentration is 0.5×SSC, and if the probe chain length is short, the hybridization temperature is preferably lower than this, When the chain length is long, it is more preferable to use a higher hybridization temperature. Needless to say, the higher the salt concentration, the higher the specific hybridization temperature, and conversely, the lower the salt concentration, the lower the specific hybridization temperature.

By the way, when evaluating microsatellite instability using the microarray according to the present invention, hybridization of amplified nucleic acids to wild-type probes and mutant-type probes is detected, and determination can be made as follows. When evaluating microsatellite instability, a plurality of mutant probes (for example, mutant probes 1 to 3) are designed with gradually decreasing number of repetitions. The fluorescence intensities detected with the mutant probes 1 to 3 are normalized with respect to the fluorescence intensities detected with the wild-type probes for each of the normal tissue and the tumor tissue. That is, fluorescence intensity ratios 1 to 3 are calculated for normal tissue and tumor tissue according to the following formulas for mutant probes 1 to 3, respectively.
Tumor Tissue_Fluorescence Intensity Ratio 1 = Tumor Tissue_Mutant Probe 1/Tumor Tissue_Wild Type Probe Normal Tissue_Fluorescence Intensity Ratio 1 = Normal Tissue_Mutant Probe 1/Normal Tissue_Wild Type Probe Tumor Tissue_Fluorescence Intensity Ratio 2 = Tumor Tissue_Mutant Probe 2/Tumor Tissue_Wild Type Probe Normal Tissue_Fluorescence Intensity Ratio 2 = Normal Tissue_Mutant Probe 2/Normal Tissue_Wild Type Probe Tumor Tissue_Fluorescence Intensity Ratio 3 = Tumor Tissue_Mutation type probe 3/tumor tissue_wild-type probe normal tissue_fluorescence intensity ratio 3 = normal tissue_mutant probe 3/normal tissue_wild-type probe

Then, according to the following formula, the fluorescence intensity ratio calculated for the normal tissue is subtracted from the fluorescence intensity ratio calculated for the tumor tissue to calculate judgment values 1 to 3 for each of the mutant probes 1 to 3.
Judgment value 1 = tumor tissue_fluorescence intensity ratio 1 - normal tissue_fluorescence intensity ratio 1
Judgment value 2 = tumor tissue_fluorescence intensity ratio 2 - normal tissue_fluorescence intensity ratio 2
Judgment value 3 = tumor tissue_fluorescence intensity ratio 3 - normal tissue_fluorescence intensity ratio 3

Finally, the sum of the calculated determination values 1 to 3 is obtained. Based on the total values determined, microsatellite instability in tumor tissue can be assessed. For example, if the sum of judgment values 1-3 is higher than a predetermined threshold value, it can be evaluated that there is microsatellite instability in the tumor tissue. Further, when the total value of judgment values 1 to 3 is equal to or lower than a predetermined threshold value, it can be evaluated that there is no microsatellite instability in the tumor tissue.

In the above description, three types of mutant probes 1 to 3 are illustrated for convenience, but the types of mutant probes are not limited to this, and can be appropriately set for each microsatellite. For example, all mutation type probes may be prepared for each mutation type so as to correspond to all mutation types (number of repetitions) that microsatellites to be detected can take. Further, among all possible mutation types (number of repetitions) of the microsatellite to be detected, a mutation type probe corresponding to a mutation type with a high frequency of occurrence may be prepared. In other words, among all mutation types (number of repetitions) that can be taken by the microsatellite to be detected, a mutation type with a low frequency of appearance, for example, a mutation type with an appearance frequency of 1% or less, preferably an appearance frequency of 0.5% Mutant probes may be prepared for the following mutation types, more preferably mutation types with an appearance frequency of 0.1% or less, and more preferably mutation types excluding mutation types whose detection cases are not known. Further, mutation probes may be prepared for mutation types whose frequency of appearance is included in the upper ⅓ range of all mutation types (number of repetitions) that can be taken by the microsatellite to be detected. Alternatively, for all mutation types (number of repetitions) that can be taken by the microsatellite to be detected, a mutant probe corresponding to every other repetition number, a mutant probe corresponding to every second repetition number, or every third Mutant probes corresponding to the number of repetitions may be prepared.

For example, in the case of NR24, which is a wild-type microsatellite sequence in which mononucleotides are repeated 24 times, a mutation type in which the number of mononucleotide repeats is less than 24 has been reported. In this case, mutant probes can optionally be designed that detect fewer repetitive sequences than wild-type probes. Moreover, in order to specifically detect a signal derived from the wild type, a probe having a sequence about 5 bases longer than the wild type probe can be arbitrarily designed.

In particular, the microarray according to the present invention comprises probes having a region corresponding to the microsatellite to be detected and a region for adjusting the probe length on the 5'-end side immobilized on the carrier. By using the probe configured in this manner, variations in the number of repetitions in the microsatellites to be detected can be detected with high accuracy. In comparison, when configured to have a region corresponding to the microsatellite to be detected on the 5′ end side fixed to the carrier and a region for adjusting the probe length on the 3′ end side, particularly As a result of non-specific hybridization in the mutant probes corresponding to the mutant microsatellites, it is not possible to detect variations in the number of repeats in the microsatellites to be detected with high accuracy.

Therefore, according to the microarray of the present invention having probes designed as described above, genotyping of microsatellite polymorphisms and evaluation of microsatellite instability can be performed with high accuracy.

As described above, when evaluating microsatellite instability, the signal measured for each of a plurality of mutant probes using the microarray according to the present invention is normalized by the signal measured for the wild-type probe, The difference between the signal ratio in tumor tissue and the signal ratio in normal tissue is obtained as a judgment value, and the total value of the judgment values obtained for a plurality of mutant probes is used. Thus, since the above judgment value is used, it is not essential to have mutant probes corresponding to all mutation types (number of repetitions) that can be taken by the microsatellite to be detected, and some mutant probes are used as described above. It can be used to fully assess microsatellite instability. In general, when detecting polymorphisms using macroarrays, single nucleotide polymorphisms can be detected with high accuracy, but microsatellite polymorphisms are known to have low detection sensitivity. This is because non-specific hybridization of probes tends to occur when only the number of repetitions of the same base is different. However, as described above, instead of using the signal values per se of multiple mutant probes corresponding to microsatellites, microsatellite instability can be determined with high accuracy by using the above judgment values calculated from the signal values. can be evaluated to

EXAMPLES The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to the following examples.

In this example, microsatellites used for diagnosis of Lynch syndrome and sporadic colorectal cancer were analyzed. Examples of such microsatellites include five types of microsatellites (NR21, NR24, BAT25, BAT26 and MONO27). FIG. 1 shows the nucleotide sequences of the regions containing each microsatellite (SEQ ID NOS: 1-5). In FIG. 1, the boxed region is a microsatellite, and the pair of underlined parts in each base sequence indicates a pair of primers (Table 1) for amplifying the region containing the microsatellite.

To amplify a region containing microsatellites, first, a primer mixture having the composition shown in Table 2 was prepared.

Next, a PCR reaction solution having the composition shown in Table 3 was prepared.

Temperature conditions for PCR were set as shown in Table 4.

Then, a hybridization reaction was performed using a microsatellite instability analysis chip (MSI chip) prepared in advance. Specifically, first, the hybridization oven was set to 60° C., and a Tupperware filled with 30 mL of water was placed for 1 hour or longer. Hybridization buffer (2.25×SSC, 0.23% SDS) and PCR products were then removed from the freezer and allowed to cool to room temperature. 4 μl of PCR product and 2 μl of hybridization buffer were mixed. After adding 3 μL of the mixed solution to the chip cover, it was set on the chip. The temperature condition in the hybridization reaction was set at 60°C.

Then, a washing solution (0.1×SSC/0.1% SDS solution) was prepared, and the chip after the hybridization reaction was placed in a stainless steel holder, washed by moving up and down in the washing solution 10 times, and allowed to stand for 5 minutes. After washing, the stainless steel holder holding the chip was placed in the 1×SSC solution until the fluorescence intensity was detected.
Then, the moisture was wiped off, a cover film was placed on the surface, and measurement was performed using the fluorescence intensity estimation method with BIOHSHOT.

[Experiment 1]
In Experiment 1, the design of probes for detecting microsatellites was examined. Specifically, condition 1 in which probe sets with the same sequence are used except for the region corresponding to the microsatellite region, and the region corresponding to the microsatellite region and the same region having a region for adjusting the probe length on the 3′ end side. An MSI chip was prepared under condition 2 using a probe set of the sequence and condition 3 using a probe set of the same sequence having a region corresponding to the microsatellite region and a region for adjusting the probe length on the 5' end side. . Here, the region for adjusting the probe length is a region in which bases are added according to the decrease in the number of bases in the microsatellite region. The probe sets designed under Conditions 1-3 are shown in Tables 5-7, respectively.

In this experiment, the PCR described above was performed using the RKO cell line as a sample positive for microsatellite instability, and the PCR described above was performed using the SW948 cell line as a sample negative for microsatellite instability. In the sample negative for microsatellite instability, the number of repeated microsatellites tested was 24 bases (corresponding to probe 3). In addition, although not described in detail, as a result of fragment analysis after performing the above PCR using the MSI-positive RKO cell line, in samples positive for microsatellite instability, the number of repeated microsatellites tested was 18 bases. (corresponding to probe 7).

Fluorescence intensity of probe 3 (corresponding to MSI-negative microsatellites) when using microsatellite instability-negative SW948 cell line under conditions 1, 2 and 3, microsatellite instability-positive RKO cell line and measured with probe 7 (corresponding to MSI-positive microsatellites) are shown in FIG. As shown in FIG. 2, under conditions 2 and 3, both probe 3 in the MSI-negative sample and probe 7 in the MSI-positive sample were detected at higher fluorescence intensities than under condition 1.

Also, from the obtained fluorescence intensity, a fluorescence intensity ratio was calculated according to the following formula and normalized.
Fluorescence intensity ratio = (mutant probe/wild-type probe (probe 3))

FIG. 3 shows a characteristic diagram in which the horizontal axis represents the number of microsatellite repetitions of the probe and the vertical axis represents the fluorescence intensity ratio. In FIG. 3, (a) to (c) correspond to conditions 1 to 3, respectively. From the results of the fragment analysis described above, the fluorescence intensity ratio of probe 3 was the highest when using the SW948 cell line, which is negative for microsatellite instability, and the RKO cell line, which is positive for microsatellite instability, was used. At that time, the fluorescence intensity ratio of probe 7 should be the highest. In FIG. 3, the fluorescence intensity ratio of probe 3 in the characteristics diagram when using the SW948 cell line and the fluorescence intensity ratio of probe 7 when using the RKO cell line are enclosed in squares.

As shown in FIG. 3, under conditions 1 and 3, the fluorescence intensity ratio was high for the probes that should have the highest fluorescence intensity ratio (probe 3 for SW948, probe 7 for RKO), as expected. That is, the probes designed under conditions 1 and 3 were generally consistent with the results of fragment analysis. However, the probes designed under condition 2 did not match the results of fragment analysis, and when using the SW948 cell line, which is negative for microsatellite instability, probes with a short number of repetitions (e.g., probes 8 to 12) A high fluorescence intensity was detected. From this result, it was clarified that the probe designed under Condition 2 has poor determination performance when analyzing the number of microsatellite repetitions.

From the results of this experiment, it was revealed that condition 3, that is, probes designed to have a region for adjusting the probe length at the 5′ end fixed to the substrate are excellent in both fluorescence intensity and determination performance.

[Experiment 2]
In this experiment 2, the determination performance was confirmed whether the positive (+) and negative (-) of microsatellite instability could be separated using actual specimens. In this experiment, four types of microsatellites, NR21, NR24, BAT25 and BAT26, were designed according to the probe design method for which excellent effects were confirmed in Experiment 1. The probes designed for NR21, NR24, BAT25 and BAT26 are shown in Tables 8-11, respectively.

In this experiment, 8 actual samples (normal tissue and cancer tissue) with known mutations, MSI positive (+): 3 samples, MSI negative (-): 5 samples were used. Tissue-derived DNA was extracted and used. In addition, the number of tests was set to n=4 for each specimen.

Fluorescence intensity was measured for probes designed for NR21, NR24, BAT25 and BAT26 (Tables 8-11) as described above. Then, the fluorescence intensity ratio was calculated (normalized) using the wild-type probe of each tissue site as a reference. Specifically, the fluorescence intensity ratio was obtained according to the following formula.
Fluorescence intensity ratio 1 = same tissue_mutant probe 1/same tissue_wild type probe Fluorescence intensity ratio 2 = same tissue_mutant probe 2/same tissue_wild type probe Fluorescence intensity ratio 3 = same tissue_mutant probe 3 / same tissue_wild-type probe (hereafter, the fluorescence intensity ratio is calculated in the same way after 4)

Next, a determination value is calculated by subtracting the fluorescence intensity ratio of normal tissue from the fluorescence intensity ratio of tumor tissue. Specifically, the determination value was obtained according to the following formula.
Judgment value 1 = tumor tissue_fluorescence intensity ratio 1 - normal tissue_fluorescence intensity ratio 1
Judgment value 2 = tumor tissue_fluorescence intensity ratio 2 - normal tissue_fluorescence intensity ratio 2
Judgment value 3 = tumor tissue_fluorescence intensity ratio 3 - normal tissue_fluorescence intensity ratio 3
(Hereafter, judgment value 4 and later are calculated in the same way)

Next, the sum of positive judgment values is obtained. The discrimination of MSI (+) and (-) was confirmed from the obtained judgment total value. The results are shown in FIG. As shown in Fig. 4, for the four types of microsatellites tested, the total determination value for MSI-positive samples (indicated by (+) in the figure) and the determination total value for MSI-negative samples (indicated by (-) in the figure) was clearly separated. From these results, it was found that the NR21, NR24, BAT25 and BAT26 probes designed in this experiment are excellent in determination performance for MSI positive and negative.

Claims (3)

For the microsatellite to be detected, a wild-type probe corresponding to the wild-type, a plurality of mutant probes corresponding to a plurality of mutation types having different numbers of unit sequence repeats in the microsatellite , these wild-type probes and a plurality of mutations a substrate for fixing the mold probe,
The mutant probe includes a region corresponding to the microsatellite and a region that adjusts the probe length at the 5' end, and is fixed to the substrate at the 5' end , and the mutation type is a repeating unit sequence. A microsatellite detection microarray characterized in that the number of times is small compared to the wild type . 2. The microsatellite detection microarray according to claim 1, wherein the plurality of mutant probes have a difference in probe length of 12 bases or less. A microsatellite detection method for analyzing microsatellite instability in which the number of repetitions varies between microsatellites in normal tissue and the same microsatellites in tumor tissue,
A step of amplifying a nucleic acid containing a microsatellite to be detected using genomic DNA derived from normal tissue and genomic DNA derived from tumor tissue in a subject, respectively , as templates;
For the microsatellite to be detected, a wild-type probe corresponding to the wild-type, a mutant probe corresponding to a plurality of mutation types having different numbers of unit sequence repeats in the microsatellite , these wild-type probes and a plurality of mutant types a substrate for immobilizing the probe, wherein the mutant probe includes a region corresponding to the microsatellite and a region for adjusting probe length at the 5' end, and is immobilized on the substrate at the 5' end. , the step of contacting the nucleic acid amplified in the step with a microsatellite detection microarray in which the mutation type has a smaller number of repetitions of the unit sequence than the wild type ;
detecting a signal based on hybridization with the amplified nucleic acid for each of the wild-type probe and the plurality of mutant probes;
Based on the signals detected for the wild-type probe and a plurality of mutant probes , the signals measured for each of the plurality of mutant probes are normalized by the signals measured for the wild-type probes. Obtained for each of the tumor tissues, obtained as a judgment value the difference between the signal ratio in the tumor tissue and the signal ratio in the normal tissue for each of the plurality of mutant probes, and the judgment value obtained for the plurality of mutant probes A method for detecting microsatellites, comprising analyzing the microsatellites in the subject by comparing the total value of .

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