WO2019004335A1 - Genetic mutation evaluation method, and kit for evaluating genetic mutation - Google Patents

Abstract

For a type of genetic mutation for which there can be a plurality of kinds, the present invention determines the plurality of kinds of genetic mutations. The present invention uses a value calculated by the ratio [signal intensity of first probe]/[signal intensity of shared probe], using an amplified fragment having a label and amplified from a region including a specific location at which a plurality of genetic mutations can be present, a first probe corresponding to a wild-type sequence in the specific location, and a shared probe corresponding to a sequence in the amplified fragment excluding the genetic mutation.

Description

Gene mutation evaluation method, kit for gene mutation evaluation

The present invention relates to a gene mutation evaluation method and a gene mutation evaluation kit for evaluating gene mutations of types in which multiple types may exist.

Gene mutations include so-called innate gene polymorphism polymorphism (also referred to simply as polymorphism) and acquired mutations introduced. These gene mutations have a great influence on the individual's phenotype such as diagnosis, treatment and efficacy in various diseases. Genetic mutations include polymorphisms such as single nucleotide polymorphisms which are differences of one base in a DNA sequence, and microsatellite polymorphisms which are differences in repetitive sequences of a sequence having several bases as one unit, and acquired mutations There are known mutations or the like in which bases of the length are inserted or deleted.

Myeloproliferative tumors can be mentioned as an example in which individual gene mutations are used for diagnosis. Myeloproliferative neoplasms (MPN) are diseases caused by tumorigenesis of myeloid cells. MPN is characterized by marked proliferation of myeloid cells (such as granulocytes, blasts, megakaryocytes and mast cells). In MPN, chronic myelogenous leukemia (CML), chronic neutrophilic leukemia (CNL), erythrocytatosis or polycythemia vera (PV), primary bone marrow fiber Disease (primary myelofibrosis: PMF), essential thrombocythemia (ET), chronic eosinophilic leukemia (CEL), hypereosinolytic syndrome (HES), mastocytosis (hyperosinophilic syndrome (HES)) and myoproliferative neoplasms (unclassifiable: MPN, U).

As described in Non-patent Document 1, diagnosis of MPN is based on clinical parameters, bone marrow morphology and genetic data. The combination of these can be used to diagnose MPNs excluding CML in patients with Philadelphia chromosome negative. As genetic data, specifically, mutation information on three genes: JAK2, CALR and MPL, and additionally, mutation information on ASXL1, EZH2, TET2, IDH1 / IDH2, SRSF2 and SF3B1 are used. In particular, since JAK2, CALR and MPL are considered to be the molecular basis of MPN onset, the presence or absence of mutations in the gene group is an important factor in the definitive diagnosis of MPN.

In addition, non-patent document 2 shows that the JAK2V617F mutation (a substitution mutation of valine at position 617 to phenylalanine) is frequently found in PV, ET and PMF with respect to JAK2, and in a small number of PVs It is disclosed that the insertion / deletion type mutation of is found. JAK2 (Janus activating kinase 2) is a gene encoding a protein that controls the signal of erythropoietin receptor.

Non Patent Literature 2 further discloses that the MPLW515L / K mutant PMF was found in PMF and ET with respect to MPL. MPL is a gene encoding a thrombopoietin receptor.

Further, Non-Patent Document 2 discloses that type 1 mutations of 52 base deletion and type 2 mutations of 5-base insertion are most frequent for CALR, and these mutations are found in ET and PMF. Type 1 mutations are disclosed to be more frequent in PMF and to be associated with conversion to myelofibrosis in ET. CALR is a gene encoding calreticulin, which is one of endoplasmic reticulum molecular chaperones.

Furthermore, Patent Document 1 discloses a JAK2V617F site-specific fluorescently labeled probe as a mutation analysis method of the JAK2 gene. Patent Document 2 discloses a technique for detecting a mutation different from the JAK2V617F mutation, which is found in a patient who is negative for the JAK2V617F mutation and shows a myeloproliferative tumor.

Furthermore, Patent Document 3 discloses a probe set for detecting W515K mutation and W515L mutation in MPL as a probe for detecting MPL gene polymorphism.

Furthermore, Patent Document 4 discloses a technique for identifying a mutation in CALR.

JP 2012-034580 WO2009 / 060804 WO2011 / 052755 Special table 2016-537012

However, in the prior art, there is a problem that, with respect to gene mutations of types in which a plurality of types can exist, even if a mutant type probe designed based on the sequence of gene mutations can be used, these gene mutations can not be determined. The

Therefore, in view of such circumstances, the present invention aims to provide a gene mutation evaluation method and a gene mutation evaluation kit capable of determining a plurality of types of gene mutations in which there may be a plurality of types of gene mutations. I assume.

The present invention includes the following.
(1) A step of amplifying a region including a specific site where a plurality of gene mutations may exist to obtain an amplified fragment having a label, a first probe corresponding to a wild-type sequence at the specific site, and the amplified fragment Contacting a common probe corresponding to the sequence excluding the gene mutation in step with a solution containing the amplified fragment, and detecting a signal based on the label in the first probe and the common probe, and a determination formula: [ A step of calculating a first determination value based on the signal intensity of the first probe / the signal intensity of the common probe, and the first determination value calculated by the above determination formula are compared with a preset cutoff value. And a step of determining the presence or absence of a gene mutation based on the comparison result.
(2) In the step of detecting the signal, a second probe corresponding to a specific gene mutation among the plurality of gene mutations is brought into contact with a solution containing the amplification fragment, and the labeling of the second probe is carried out Detecting the signal based on the gene mutation evaluation method according to (1).
(3) In the step of calculating the first determination value, further determination equation: [signal strength of second probe] / ([signal strength of first probe] + [signal strength of second probe]) In the step of calculating the second determination value by the step of determining the presence or absence of the gene mutation, the second determination value is compared with a preset cutoff value, and the result of the comparison using the first determination value The method for evaluating gene mutation according to (2), wherein the presence or absence of a gene mutation is determined based on the result of comparison using the second determination value and the second determination value.
(4) In the step of calculating the first judgment value, the judgment value is further calculated according to the judgment formula: [signal strength of second probe] / [signal strength of common probe], and the presence or absence of the gene mutation In the step of determining the presence or absence of the gene mutation, the determination value is compared with a preset cutoff value, and the result of the comparison using the first determination value and the result of the comparison using the determination value (2) the method for evaluating gene mutation according to (2).
(5) The gene mutation evaluation method according to (1), wherein the plurality of gene mutations are insertions or deletions of bases.
(6) The specific part is between the 501st and the 579th in the nucleotide sequence of the CALR gene shown in SEQ ID NO: 2, and the plurality of gene mutations are due to the insertion or deletion of bases in the 501st to 579th regions The gene mutation evaluation method according to (1), characterized in that
(7) A gene mutation evaluation kit for determining the presence or absence of a gene mutation using an amplified fragment having a label obtained by amplifying a region including a specific part where a plurality of gene mutations may exist, which is a wild type of the above specific part A kit for evaluating gene mutation, which comprises a first probe corresponding to a sequence and a common probe corresponding to a sequence excluding the gene mutation in the amplified fragment.
(8) The kit for gene mutation evaluation according to (7), further comprising a second probe corresponding to a specific gene mutation among the plurality of gene mutations.
(9) The kit for gene mutation evaluation according to (7), wherein the plurality of gene mutations are insertions or deletions of bases.
(10) The above specific site is between the 501st and the 579th in the nucleotide sequence of the CALR gene shown in SEQ ID NO: 2, and the plurality of gene mutations are due to the insertion or deletion of bases in the 501st to 579th regions The kit for gene mutation evaluation as described in (7) characterized by a certain thing.
(11) The gene mutation evaluation kit according to (7), wherein the common probe is an oligonucleotide containing a sequence of 397 to 659 in the nucleotide sequence of the CALR gene shown in SEQ ID NO: 2.
(12) The gene mutation evaluation kit according to (7), wherein the common probe is an oligonucleotide containing CTCCTCCATCC TCATCTTTGTC (SEQ ID NO: 15) or CTCTCGTCCTGTTTGTC (SEQ ID NO: 31).
(13) The above gene mutation is a 52 base deletion type 1 mutation in which 52 bases from 513 to 564 in the base sequence of the CALR gene shown in SEQ ID NO: 2 is deleted, and the second probe corresponding to the mutation type is A kit for evaluating gene mutation according to (7), which is an oligonucleotide containing TCCTGTGTCTCCTGCTCC (SEQ ID NO: 5).

The present specification includes the disclosure content of Japanese Patent Application No. 2017-125929 based on which the priority of the present application is based.

According to the gene mutation evaluation method and the gene mutation evaluation kit according to the present invention, when there are plural types of gene mutations in specific places, the presence or absence of these gene mutations can be determined.

FIG. 2 schematically shows type 1 mutations and type 2 mutations associated with myeloproliferative tumors in CALR. FIG. 7 is a characteristic diagram in which determination values are plotted for each gene mutation in the sample used in Example 1. FIG. 10 is a characteristic diagram in which first determination values and second determination values are plotted for CALR type 1 mutation in the sample used in Example 1. FIG. 16 is a characteristic diagram showing the relationship between the blocker concentration and the second determination value in the hybridization experiment in the present Example 2. FIG. 16 is a characteristic diagram showing the relationship between the mutation ratio (mutation%) in a mutant sample and the second determination value in the hybridization experiment in the present Example 2.

The method for evaluating gene mutation and the kit for evaluating gene mutation according to the present invention can be applied when there are multiple types of gene mutations at specific places. The presence of a plurality of gene mutations at a specific site means, for example, that a mutation pattern is not one but a plurality of patterns at predetermined positions in genomic DNA. Here, mutation in a plurality of patterns means that, when a gene mutation is an insertion or deletion of a nucleotide sequence, there are a plurality of lengths of the nucleotide sequence to be inserted or deleted, and the gene mutation repeats a predetermined nucleotide sequence. In the case of the number, it means that there are a plurality of patterns of the number of repetitions. In addition, to mutate in a plurality of patterns means that, when the gene mutation is a substitution of 1 to several bases, there are a plurality of patterns of base sequences after substitution.

In addition, gene mutation due to insertion or deletion means that a base of a predetermined length is inserted when looking at the other allele from one allele, conversely, when looking at one allele from the other allele It is a gene mutation whose difference is that a base of the predetermined length is deleted. The type in which the base of a predetermined length is deleted may be a wild type, and the type in which the base of a predetermined length is inserted may be a wild type.

More specifically, as a gene mutation of a type in which a region of a predetermined length is deleted, and other deletion types having different lengths are known, 52 bases in the CALR gene, the details of which will be described later. Mention may be made of deletion type gene mutations (referred to as type 1 mutations). In the CALR gene, mutations similar to type 1 such as 46 base deletion mutation, 34 base deletion mutation and 24 base deletion mutation at the same position in addition to this 52 base deletion type type 1 (these are collectively called type 1 like (Leukemia (2016) 30, 431-438), and at the same position, it has been shown that there are other mutations not classified into these.

In the gene mutation evaluation method and the gene mutation evaluation kit according to the present invention, an amplified fragment (labeled) obtained by amplifying a region including a specific portion where such a gene mutation may exist, and a wild-type probe And use a common probe. That is, in the present invention, the following probe set is designed. That is, a first probe (also referred to as a wild-type probe) corresponding to a wild-type sequence without gene mutation and a common probe corresponding to a sequence excluding the gene mutation in the amplified fragment are designed.

Further, in the method for evaluating gene mutation and the kit for evaluating gene mutation according to the present invention, in addition to the first probe and the common probe, a second probe (also referred to as a mutant probe) corresponding to the sequence of gene mutation is designed. You may. This second probe is designed to correspond to one gene mutation (gene mutation to be detected) among the plurality of types of gene mutations described above. That is, the second probe specifically hybridizes to a nucleic acid fragment having one gene mutation among the plurality of types of gene mutations described above, and a nucleic acid fragment having another gene mutation and a wild-type nucleic acid fragment It is designed to prevent hybridization.

Then, the labeled amplification fragments are brought into contact with these probes, and signals based on the labels in the first probe and the common probe are detected. In the gene mutation evaluation method according to the present invention, a first judgment value is calculated from the obtained signal according to the judgment formula: [signal strength of the first probe] / [signal strength of the common probe], and a preset cutoff is used. The presence or absence of a gene mutation in the obtained amplified fragment is determined by comparison with a value (threshold value). Here, the common probe specifically hybridizes to the amplified nucleic acid regardless of the presence or absence of a gene mutation to be detected in the amplified nucleic acid.

At this time, according to the first determination value, the denominator is compared with the determination value calculated as [signal strength of the first probe] + [signal strength of the second probe], and a plurality of types of higher precision can be obtained. Gene mutations can be detected. Specifically, when there is a gene mutation similar to the gene mutation to be detected, according to the first judgment value, either the gene mutation to be detected or the gene mutation similar to the gene mutation is selected. Having these can be distinguished from wild type which does not have any of these gene mutations. Thus, in the gene mutation evaluation method and the gene mutation evaluation kit according to the present invention, a plurality of genes containing gene mutations to be detected without using probes corresponding to a plurality of gene mutations including gene mutations to be detected Mutations can be detected.

Further, in the method for evaluating gene mutation according to the present invention, in addition to the above-described first determination value, a determination formula: [signal strength of second probe] / ([signal strength of first probe] + [second The second determination value is calculated by the signal intensity of the probe of [1], and the gene mutation in the obtained amplified fragment can be determined with higher accuracy by comparing with the preset cutoff value (threshold value). . That is, specifically, when there is a gene mutation similar to a gene mutation to be detected, having the gene mutation to be detected is similar to the gene mutation to be detected according to the second judgment value Can be distinguished from the wild type which does not have any of these gene mutations and these gene mutations.

Furthermore, different from the first judgment value and the second judgment value, different judgment values are calculated from the obtained signal according to the judgment formula: [signal strength of second probe] / [signal strength of common probe] Also good. That is, the determination value may be used for determination of gene mutation using the first determination value described above and determination using the first determination value and the second determination value.

The gene mutation evaluation method and the gene mutation evaluation kit according to the present invention can be applied, for example, to gene mutations present in CALR among gene mutations associated with myeloproliferative tumors. More specifically, among gene mutations present in CALR, types such as so-called 52 base deletion type gene mutations (type 1), 46 base deletion mutations at the same position, 34 base deletion mutations, 24 base deletion mutations, etc. It can be applied in determining the presence or absence of mutations similar to 1 (type 1 like) and further mutations not classified as these at the same position. In addition, among gene mutations present in CALR, so-called 5-base insertion type gene mutations (type 2), other mutations similar to type 2 in the same position (type 2 like), and others not classified in these in the same position It can be applied when determining the presence or absence of mutation.

Hereinafter, a system for identifying and evaluating gene mutations associated with myeloproliferative tumors, including gene mutations present in CALR, will be described. The kit for evaluating gene mutations associated with myeloproliferative tumors described below relates to gene mutations present in JAK2, CALR and MPL. These gene mutations in JAK2, CALR and MPL are gene mutations used for diagnosis of myeloproliferative tumors in the classification by World Health Organization (WHO) (for example, the 2016 fiscal year version).

The kit for gene mutation evaluation shown as an example includes a probe set for identifying gene mutations present in each of these JAK2, CALR and MPL.

Specifically, the gene mutation of JAK2 means the V617F mutation (the substitution mutation of valine at position 617 to phenylalanine). This mutation contributes to the activation of the JAK-STAT pathway and is a hallmark of polycytemia vera (PV). The V617F mutation is also found at a frequency of 50 to 60% in patients with primary myelofibrosis (PMF) or patients with essential thrombocythemia (ET). The nucleotide sequence encoding wild type JAK2 is shown in SEQ ID NO: 1. When the V617F mutation is present, at the 351st position of the base sequence shown in SEQ ID NO: 1, G is substituted for T.

In addition, CALR gene mutation means type 1 mutation with 52 bases deletion and type 2 mutation with 5 bases insertion. The 52 base deletion and 5 base insertions are located at the C-terminus of the CALR protein. Any of these mutations is found at a frequency of 20-25% in patients with primary myelofibrosis (PMF) or in patients with essential thrombocythemia (ET). Primarily, type 2 mutations are associated with essential thrombocythemia (ET) and type 1 mutations are involved in primary myelofibrosis (PMF). The CALR gene mutation is also a mutation found in myeloproliferative tumors that do not have the above-mentioned gene mutation in JAK2. The nucleotide sequence encoding wild type CALR is shown in SEQ ID NO: 2. If it has a type 1 mutation, 52 bases from 513 to 564 in the base sequence shown in SEQ ID NO: 2 will be deleted. In the case of having a type 2 mutation, TGTTC is inserted between the 568th and 569th in the base sequence shown in SEQ ID NO: 2.

Furthermore, the gene mutation of MPL means the W515K mutation (a substitution mutation of tryptophan at position 515 to lysine) or the W515L mutation (a substitution mutation of tryptophan at position 515 to leucine). This gene mutation of MPL is found in 3 to 5% of patients with essential thrombocythemia (ET) and in 6 to 10% of primary myelofibrosis (PMF). The nucleotide sequence encoding wild type MPL is shown in SEQ ID NO: 3. When the W515K mutation is present, TGs at positions 305 and 306 in the base sequence shown in SEQ ID NO: 3 are subjected to substitution mutation to AA. In the case of having the W515L mutation, G at position 306 in the base sequence shown in SEQ ID NO: 3 is substituted for T.

More specifically, for the V617F mutation of JAK2, for example, an oligonucleotide containing CTCCACAGAaACATACTCC (SEQ ID NO: 4), which corresponds to the substitution mutation in SEQ ID NO: 1, can be used as a variant probe. In the above sequence, the small letter a corresponds to a 351st G to T substitution mutation in the base sequence shown in SEQ ID NO: 1. In addition, when identifying the V617F mutation of JAK2, a wild-type probe corresponding to wild-type JAK2 (a small letter a in the above sequence can be used as c) can also be used. That is, in order to identify the V617F mutation of JAK2, a mutant-type probe containing the base sequence of SEQ ID NO: 4 may be used, or a probe set consisting of the mutant-type probe and a wild-type probe may be used. .

In addition, for type 1 mutation of CALR, a region selected from 52 bases from 513 to 564 in the base sequence shown in SEQ ID NO: 2 is a wild-type probe (first probe) corresponding to wild-type CALR. Use as. As for type 1 mutation of CALR, for example, an oligonucleotide containing TCCTTGT-CCTCTGCTCC (SEQ ID NO: 5) corresponding to the 52 base deletion in SEQ ID NO: 2 can be used as a probe (second probe) . The position of the hyphen "-" in the above sequence is the position of 52 base deletion.

Furthermore, for CALR type 2 mutation, a wild-type probe (first probe designed as a sequence in which the above 5 bases are not inserted, ie, 568th and 569th adjacent sequences in the base sequence shown in SEQ ID NO: 2) Use). As for type 2 mutations of CALR, for example, an oligonucleotide containing, for example, ATCC TCC gacaa TTGTCCT (SEQ ID NO: 6) corresponding to the above-mentioned 5-base insertion in SEQ ID NO: 2 can be used as a probe. In the above sequence, lower case gacaa has a 5-base insertion.

Furthermore, for the W515K mutation of MPL, an oligonucleotide corresponding to the above substitution mutation in SEQ ID NO: 3, for example, containing GAAACTGCttCCTCAGCA (SEQ ID NO: 7) can be used as a variant probe. In the above sequence, lower case tt corresponds to substitution mutation of the 305th and 306th TGs to AA in the base sequence shown in SEQ ID NO: 3. In addition, when identifying the W515K mutation of MPL, a wild-type probe corresponding to wild-type MPL (a sequence in which the lower-case tt in the above sequence is a ca) can also be used. That is, in order to identify the W515K mutation of MPL, a mutant-type probe containing the base sequence of SEQ ID NO: 7 may be used, or a probe set consisting of the mutant-type probe and a wild-type probe may be used. .

Furthermore, for the W515L mutation of MPL, an oligonucleotide corresponding to the above substitution mutation in SEQ ID NO: 5, for example, GGAAACTGCAAaCCTCAG (SEQ ID NO: 8) can be used as a variant probe. The small letter a in the above sequence corresponds to a substitution mutation of the 306th G to the T in the nucleotide sequence shown in SEQ ID NO: 3. In addition, when identifying the W515L mutation of MPL, a wild-type probe corresponding to wild-type MPL (a sequence in which a in the above sequence is a c) can be used. That is, in order to identify the W515L mutation of MPL, a mutant-type probe containing the base sequence of SEQ ID NO: 8 may be used, or a probe set consisting of the mutant-type probe and a wild-type probe may be used. .

As mentioned above, although the mutant-type probe for identifying the gene mutation which exists in JAK2, CALR, and MPL was respectively illustrated, the base sequence of a mutant-type probe is not limited to sequence number 4-8, but sequence number 1 It can design suitably based on the base sequence of JAK2 shown, the base sequence of CALR shown to sequence number 2, and the base sequence of MPL shown to sequence number 3.

The base length of these probes is not particularly limited, but may be, for example, 10 to 30 bases, and preferably 15 to 25 bases. As described above, the probe is a base sequence designed based on a region containing a gene mutation in the base sequence of SEQ ID NO: 1, 3 or 5, and a base sequence added to one or both ends of the base sequence. Can be, for example, 10 to 30 bases in length, preferably 15 to 25 bases in length.

Also, the probe designed as described above is preferably a nucleic acid, more preferably DNA. The DNA may be double stranded or single stranded but is preferably single stranded DNA. The probe can be obtained, for example, by chemical synthesis by a nucleic acid synthesizer. As the nucleic acid synthesizer, an apparatus called a DNA synthesizer, a fully automatic nucleic acid synthesizer, an automatic nucleic acid synthesizer or the like can be used.

The probe designed as described above is preferably used in the form of a microarray (as an example, a DNA chip) by immobilizing its 5 'end on a carrier. At this time, the microarray preferably has a mutant-type probe and a wild-type probe for each of the aforementioned gene mutations. For each gene mutation, not only the presence or absence of the mutation but also the ratio of the mutation can be accurately determined by using the mutant-type probe and the wild-type probe. Here, the length of the mutant-type probe and the wild-type probe are preferably differences within 2 bases, and more preferably the lengths are the same.

The microarray according to the present invention can be prepared by immobilizing the above-described probe on a carrier.

As the material of the carrier, those known in the art can be used without particular limitation. For example, noble metals such as platinum, platinum black, gold, palladium, rhodium, silver, mercury, tungsten and compounds thereof, and conductive materials such as carbon represented by graphite and carbon fiber; single crystal silicon, amorphous Silicon materials represented by silicon, silicon carbide, silicon oxide, silicon nitride etc., and composite materials of these silicon materials represented by SOI (silicon on insulator) etc .; glass, quartz glass, alumina, sapphire, ceramics, foam Stellite, inorganic materials such as photosensitive glass; polyethylene, ethylene, polypropylene, cyclic polyolefin, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol , Polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile / butadiene styrene copolymer, polyphenylene oxide And organic materials such as polysulfone and the like. The shape of the carrier is also not particularly limited, but is preferably flat.

In the present invention, preferably, a carrier having a carbon layer and a chemical modification group on the surface is used as the carrier. Carriers having a carbon layer and a chemical modification group on the surface include those having a carbon layer and a chemical modification group on the surface of the substrate and those having a chemical modification group on the surface of the substrate consisting of a carbon layer. As the material of the substrate, those known in the art can be used without particular limitation, and the same ones as mentioned above as the carrier material can be used.

In the microarray according to the present invention, a carrier having a fine tabular structure is suitably used. The shape is not limited to rectangular, square and round shapes, but usually 1 to 75 mm square, preferably 1 to 10 mm square, more preferably 3 to 5 mm square is used. It is preferable to use a substrate made of a silicon material or a resin material because it is easy to manufacture a carrier having a fine flat plate structure, and in particular, a carrier having a carbon layer and a chemical modification group on the surface of a substrate made of single crystal silicon is more preferable. preferable. Single crystal silicon has a slight change in orientation of crystallographic axis in part (sometimes referred to as a mosaic crystal), or includes disorder on atomic scale (lattice defect) Is also included.

The carbon layer formed on the substrate in the present invention is not particularly limited, but synthetic diamond, high-pressure synthetic diamond, natural diamond, soft diamond (for example, diamond like carbon), amorphous carbon, carbon-based material (for example, graphite, fullerene) It is preferable to use any of carbon nanotubes, mixtures thereof, or laminates thereof. In addition, carbides such as hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, thorium carbide, titanium carbide, uranium carbide, tungsten carbide, zirconium carbide, molybdenum carbide, chromium carbide, vanadium carbide and the like may be used. Here, soft diamond generally refers to an incomplete diamond structure which is a mixture of diamond and carbon such as so-called diamond like carbon (DLC: Diamond Like Carbon), and the mixing ratio is not particularly limited. The carbon layer is excellent in chemical stability and can withstand the subsequent reaction with the introduction of a chemical modification group and the binding to the analyte, and the bond is flexible because it is bound to the analyte by electrostatic binding. It is advantageous in that it has properties, is transparent to the detection system UV due to lack of UV absorption, and can be energized during electroblotting. In addition, it is also advantageous in that nonspecific adsorption is small in the binding reaction with the analyte. As described above, the substrate itself may use a carrier composed of a carbon layer.

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

In the high frequency plasma CVD method, a source gas (methane) is decomposed by glow discharge generated between electrodes by high frequency to synthesize a carbon layer on a substrate. In the ionization vapor deposition method, the source gas (benzene) is decomposed and ionized by using thermoelectrons generated by a tungsten filament, and a carbon layer is formed on a substrate by a bias voltage. The carbon layer may be formed by ionization vapor deposition in a mixed gas consisting of 1 to 99% by volume of hydrogen gas and 99 to 1% by volume of methane gas.

In the arc vapor deposition method, an arc discharge is generated in vacuum by applying a direct current voltage between a solid graphite material (cathode evaporation source) and a vacuum vessel (anode) to generate a plasma of carbon atoms from the cathode and the evaporation source Furthermore, by applying a negative bias voltage to the substrate, carbon ions in the plasma can be accelerated toward the substrate to form a carbon layer.

In the laser deposition method, a carbon layer can be formed by, for example, irradiating a target plate of graphite with Nd: YAG laser (pulse oscillation) light to melt and depositing carbon atoms on a glass substrate.

When a carbon layer is formed on the surface of the substrate, the thickness of the carbon layer is usually from about 1 to 100 μm, and if it is too thin, the surface of the base substrate may be exposed locally. If this is the case, the productivity will deteriorate, so it is preferably 2 nm to 1 μm, more preferably 5 nm to 500 nm.

By introducing a chemical modification group on the surface of the substrate on which the carbon layer is formed, the oligonucleotide probe can be firmly immobilized on the carrier. The chemical modification group to be introduced can be appropriately selected by those skilled in the art and is not particularly limited, and examples thereof include an amino group, a carboxyl group, an epoxy group, a formyl group, a hydroxyl group and an active ester group.

The introduction of the amino group can be carried out, for example, by irradiating the carbon layer with ultraviolet light in ammonia gas or by plasma treatment. Alternatively, the carbon layer may be chlorinated by irradiating ultraviolet light in chlorine gas and further irradiating ultraviolet light in ammonia gas. Alternatively, it can also be carried out by reacting with a chlorinated carbon layer in a polyvalent amines gas such as methylene diamine and ethylene diamine.

The introduction of the carboxyl group can be carried out, for example, by reacting the carbon layer aminated as described above with a suitable compound. The compound used to introduce a carboxyl group is, for example, represented by the formula: X—R 1 —COOH (wherein, X represents a halogen atom, R 1 represents a divalent hydrocarbon group having 10 to 12 carbon atoms) Halocarboxylic acids such as chloroacetic acid, fluoroacetic acid, bromoacetic acid, iodoacetic acid, 2-chloropropionic acid, 3-chloropropionic acid, 3-chloroacrylic acid, 4-chlorobenzoic acid; formula: HOOC-R2-COOH (formula In which R 2 represents a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms), such as oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, phthalic acid; polyacrylic acid And polyvalent carboxylic acids such as polymethacrylic acid, trimellitic acid and butanetetracarboxylic acid; Formula: R3-CO-R4-COOH (wherein, R3 is a hydrogen atom or a divalent hydrocarbon group having 1 to 12 carbon atoms) , R 4 represents a divalent hydrocarbon group having 1 to 12 carbon atoms) Dicarbon represented by the formula: X-OC-R5-COOH (wherein, X is a halogen atom, R 5 represents a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms) Mono halides of acids such as succinic acid monochloride, malonic acid monochloride; acid anhydrides such as phthalic anhydride, succinic anhydride, oxalic anhydride, maleic anhydride, butanetetracarboxylic acid anhydride and the like can be mentioned.

The introduction of the epoxy group can be carried out, for example, by reacting the carbon layer aminated as described above with an appropriate polyvalent epoxy compound. Alternatively, it can be obtained by reacting an organic peroxy acid with the carbon = carbon double bond contained in the carbon layer. Examples of organic peracids include peracetic acid, perbenzoic acid, diperoxyphthalic acid, formic acid, and trifluoroperacetic acid.

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

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

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

The introduction of the active ester group may be carried out, for example, with a dehydrating condensation agent such as cyanamide or carbodiimide (for example, 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide) and N-containing carboxyl group introduced as described above. It can be carried out by active esterification with a compound such as hydroxysuccinimide. By this treatment, it is possible to form a group in which an active ester group such as N-hydroxysuccinimide group is bonded to an end of a hydrocarbon group via an amide bond (Japanese Patent Laid-Open No. 2001-139532).

The probe is dissolved in a buffer for spotting to prepare a spotting solution, which is dispensed into a 96-well or 384-well plastic plate, and the aliquoted solution is spotted on a carrier by a spotter device or the like. Can be immobilized on a carrier. Alternatively, the spotting solution may be spotted manually with a micropipettor.

After spotting, it is preferable to carry out incubation in order to advance the reaction in which the probe is bound to the carrier. Incubation is usually performed at a temperature of −20 to 100 ° C., preferably 0 to 90 ° C., usually for 0.5 to 16 hours, preferably 1 to 2 hours. The incubation is preferably performed under an atmosphere of high humidity, for example, 50 to 90% humidity. Following the incubation, it is preferable to wash using a washing solution (eg, 50 mM TBS / 0.05 % Tween 20, 2 × SSC / 0.2% SDS solution, ultrapure water, etc.) in order to remove DNA not bound to the carrier. .

By using the microarray configured as described above, it is possible to simultaneously determine the presence or absence of each of the aforementioned gene mutations in JAK2, CALR and MPL in a diagnosis subject.

Specifically, when determining the presence or absence of the above gene mutation present in JAK2, CALR and MPL, a step of extracting DNA from a sample derived from a diagnosis subject, and the extracted DNA as a template, the above gene mutation in JAK2 Amplifying the region containing the above gene mutation in CALR and the region containing the above gene mutation in MPL, and present in JAK2, CALR and MPL contained in the amplified nucleic acid using the above-mentioned microarray And d) detecting the presence or absence of the gene mutation.

The subject to be diagnosed is usually a human, and is not particularly limited to the race and the like, but particularly the yellow race, preferably the East Asian race, particularly preferably the Japanese. Moreover, it can be set as the patient who is suspected of myeloproliferative tumor as a diagnostic subject.

The sample from the subject of diagnosis is not particularly limited. For example, blood related samples (blood, serum, plasma, etc.), lymph, feces, cancer cells, tissue or organ fragments and extracts, etc. may be mentioned.

First, DNA is extracted from a sample collected from a subject to be diagnosed. The extraction means is not particularly limited. For example, a DNA extraction method using phenol / chloroform, ethanol, sodium hydroxide, CTAB or the like can be used.

Next, an amplification reaction is carried out using the obtained DNA as a template to amplify a region containing JAK2, a region containing CALR and a region containing MPL. As an amplification reaction, a polymerase chain reaction (PCR), LAMP (Loop-Mediated Isothermal Amplification), ICAN (Isothermal and Chimeric primer-Initiated Amplification of Nucleic acids) method or the like can be applied. In the amplification reaction, it is desirable to add a label so that the region after amplification can be identified. At this time, a method for labeling the amplified nucleic acid is not particularly limited. For example, a method in which a primer used for amplification reaction is previously labeled may be used, or a labeled nucleotide is used as a substrate for amplification reaction. Methods may be used. The labeling substance is not particularly limited, and radioactive isotopes, fluorescent dyes, or organic compounds such as digoxigenin (DIG) and biotin can be used.

In addition, this reaction system includes a buffer necessary for nucleic acid amplification and labeling, a thermostable DNA polymerase, a primer specific to the amplification region, a labeled nucleotide triphosphate (specifically, a nucleotide triphosphate to which a fluorescent label or the like is added) , A nucleotide triphosphate, and a reaction system containing magnesium chloride and the like.

The primer used for the amplification reaction of the region containing the above gene mutation in JAK2 is not particularly limited as long as it can specifically amplify the region containing the above gene mutation, and can be appropriately designed by those skilled in the art. For example,
A set of primers consisting of primer JAK2-F: 5'-GAGCAAGCTTTCTCACAAAGCATTTGG-3 '(SEQ ID NO: 9) and primer JAK2-R: 5'-CTGACACCTAGCTGTGATCCTGAAACTG-3' (SEQ ID NO: 10) can be mentioned.

The primer used for the amplification reaction of the region containing the gene mutation in CALR is not particularly limited as long as it can specifically amplify the region containing the gene mutation, and can be appropriately designed by those skilled in the art. For example,
A set of primers consisting of primer CALR-F: 5'-CGTAAAAAAGGTGAGGCCTGGT-3 '(SEQ ID NO: 11) and primer CALR-R: 5'-GGCCTCTCTAGACTCGTCTTG-3' (SEQ ID NO: 12) can be mentioned.

The primer used for amplification reaction of the region containing the gene mutation in MPL is not particularly limited as long as it can specifically amplify the region containing the gene mutation, and can be appropriately designed by those skilled in the art. For example,
A set of primers consisting of primer MPL-F: 5'- CTCCTAGCCCTGGATCTCCTTGG-3 '(SEQ ID NO: 13) and primer MPL-R: 5'-ACAGAGCGAACCAAGAATGCCTGTTTTAC-3 '(SEQ ID NO: 14) can be mentioned.

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

The hybridization reaction between the amplified nucleic acid obtained as described above and the probe immobilized on the carrier is carried out, and the hybridization of the amplified nucleic acid to the mutant type probe is detected to detect the presence or absence of the above gene mutation in the person to be diagnosed. It can be evaluated. That is, hybridization of the amplified nucleic acid to the mutant-type probe can be measured, for example, by detecting a label. Also, by quantitatively measuring the signal from the label, the amount of nucleic acid hybridized to the mutant probe can be quantified.

For example, when a fluorescent label is used, the signal from the label can be detected as a fluorescent signal using a fluorescent scanner and analyzed by image analysis software to quantify the signal intensity. The amplified nucleic acid hybridized to the mutant-type probe can also be quantified, for example, by preparing a calibration curve using a sample containing a known amount of DNA. The hybridization reaction is preferably carried out under stringent conditions. Stringent conditions are conditions under which a specific hybrid is formed and a nonspecific hybrid is not formed, for example, after hybridization reaction at 50 ° C. for 16 hours, 2 × SSC / 0.2% SDS, 25 The conditions for washing at 10 ° C. for 10 minutes and 2 × SSC at 25 ° C. for 5 minutes are described. Alternatively, the temperature for hybridization can be 45 to 60 ° C. when the salt concentration is 0.5 × SSC, and it is more preferable to lower the hybridization temperature when the length of the probe is short, When the chain length is long, it is more preferable to make the hybridization temperature higher. It goes without saying that the hybridization temperature having specificity increases as the salt concentration increases, and the hybridization temperature having specificity decreases as the salt concentration decreases.

In addition, when using a microarray comprising a mutant-type probe (second probe) and a wild-type probe (first probe) for each of the gene mutations described above, the signal intensities from these mutant-type probes and the wild-type probe are used The presence or absence of the above-mentioned gene mutation can be evaluated. Specifically, the signal intensity in the wild type probe and the signal intensity in the mutant probe are each measured, and the judgment value for evaluating the signal intensity derived from the mutant probe is calculated. As a calculation example of the judgment value, for example, judgment equation: [signal strength derived from mutant probe] / ([signal strength derived from wild type probe] + [signal strength derived from mutant probe]) = second judgment value How to use

Then, the second determination value calculated by the above equation is compared with a predetermined threshold (cut-off value), and when the second determination value exceeds the threshold, the amplified nucleic acid contains the gene mutation. If the second judgment value is below the threshold value, it can be judged that the amplified nucleic acid does not contain the gene mutation. Thus, by using the second determination value, it is possible to determine the presence or absence of each gene mutation in JAK2, CALR (gene mutation of type 2) and MPL described above.

Here, the threshold value is not particularly limited. For example, the above-mentioned test is performed using a sample in which each of the aforementioned gene mutations present in JAK2, CALR (gene mutation of type 2) and MPL is wild type. It can prescribe | regulate based on the 2nd determination value calculated by the type | formula. More specifically, a plurality of second judgment values using a plurality of samples in which each of the aforementioned gene mutations present in JAK2, CALR (type 2 gene mutation) and MPL is determined to be wild type Can be calculated, and the value of the average value + 3σ (σ: standard deviation) can be used as the threshold value. In addition, the value of average value +2 (sigma) or average value + (sigma) can also be used as a threshold value.

By the way, gene mutations that exist in the above-mentioned CALR are similar to type 1 such as 46 base deletion mutation, 34 base deletion mutation, 24 base deletion mutation, etc. In addition to type 1 mutations, these type 1 like mutations are also known to be involved in the disease (Leukemia (2016) 30, 431-438). ). According to Leukemia (2016) 30, 431-438, it is shown that there are other mutations not classified as type 1 or type 1 like sites where 52 bases are deleted. Therefore, if it becomes possible to detect the presence or absence of type 1 gene mutations, type 1 like gene mutations, and other mutations not classified as these as gene mutations present in CALR, it is useful information for definitive diagnosis of MPN. It is considered to be.

By using the second determination value calculated by the above-mentioned equation, type 1 of 52 base deletion can be distinguished from the above-mentioned type 1 like and other mutations described above. That is, when the second determination value exceeds the threshold value, it can be determined to have type 1 of 52 base deletion. In addition, when the second determination value is below the threshold value, it can be determined that it is a wild type without mutation, a type 1 like mutation, or another mutation.

The presence or absence of the type 1 gene mutation present in the above-described CALR is determined using the above-described first determination value. With regard to the type 1 gene mutation present in this CALR, the presence or absence of the gene mutation may be determined only by the first determination value, but the gene variation using the first determination value and the second determination value You may judge the presence or absence of.

Specifically, by using the first determination value, it is correctly determined whether it has any mutation, type 1 with 52 base deletion, type 1 like the above, and other types of mutations not classified as these. It can be determined. As described above, the first determination value is a value obtained by dividing the signal intensity of the wild-type probe by the signal intensity of the common probe different from the mutant-type probe and the wild-type probe. The common probe is a nucleotide consisting of a base sequence complementary to a region commonly present in wild-type amplified nucleic acid and amplified nucleic acid having gene mutation for type 1 gene mutation. That is, the common probe specifically hybridizes to the amplified nucleic acid regardless of the presence or absence of the type 1 gene mutation contained in the amplified nucleic acid.

The common probe is not particularly limited, but can be an oligonucleotide containing the 397th to 659th sequences in the nucleotide sequence of the CALR gene shown in SEQ ID NO: 2. More specifically, as a common probe, there can be mentioned, for example, an oligonucleotide containing CTCCTCCATCC TCATCTTTGTC (SEQ ID NO: 15). In addition, as a common probe, there can be mentioned an oligonucleotide containing CCTC CTCATCC TCAT CTTTGTC (SEQ ID NO: 26) and an oligonucleotide containing CCTC CTTGTCCCTCCTCAT (SEQ ID NO: 27). In addition, as a common probe, an oligonucleotide containing CCTCGTCCTGTTTGTCC (SEQ ID NO: 31) can be mentioned.

Specifically, as a formula for calculating the first determination value, [signal intensity of wild-type probe] / [signal intensity of common probe] can be used. The first judgment value calculated by [signal strength of wild type probe] / [signal strength of common probe] is a value that decreases when there is a mutation (deletion or insertion) at a position corresponding to the wild type probe in the amplified nucleic acid. It is. Then, when the first judgment value is below the threshold value, it is judged that the amplified nucleic acid contains any of the 52 base deletion type 1, the above type 1 like and other mutations not classified into these. In addition, when the obtained first judgment value exceeds the threshold value, it is judged that the amplified nucleic acid does not contain type 1 of 52 base deletion, type 1 like as described above and other mutations not classified into these. In this way, by using the first judgment value, one having any of the above-mentioned CALR type 1 gene mutation, type 1 like gene mutation and other mutations as described above does not have any of these mutations. And can be distinguished.

In addition, the “first determination value” and the “second determination value” described above may be used together to determine the presence or absence of a gene mutation present in the CALR described above. Whether you have a 52 base deletion type 1 mutation or a type 1 like mutation or any other mutation by using the “first judgment value” and “second judgment value” described above The presence or absence of these mutations can be accurately determined. Specifically, when the first determination value is below the threshold value and the second determination value is above the threshold value, it is determined that the amplified nucleic acid contains a type 1 gene mutation. In addition, when the first determination value is below the threshold and the second determination value is below the threshold, it is determined that the amplified nucleic acid contains type 1 like gene mutation or other mutation. In addition, when the first determination value exceeds the threshold value and the second determination value is less than the threshold value, it is determined that the amplified nucleic acid does not contain any of the type 1, type 1 like and other mutations. Thus, by using the first judgment value and the second judgment value, the above-mentioned CALR type 1 gene mutation is identified from type 1 like and other mutations at type 1 mutation sites. Can.

On the other hand, when determining a type 2 mutation among the gene mutations present in the above-described CALR, as in the type 1 mutation, only the above-described “first determination value” may be used, or The judgment value of 1 and the second judgment value may be used together. Mutations similar to type 2 (type 2 like) are also present for type 2 gene mutations described above (Leukemia (2016) 30, 431-438). Where type 2 or type 2 like occurs, it has been shown that there are other mutations not classified into these. Therefore, by using the “first judgment value”, it is distinguished from those having any of these mutations as having any of the type 2 gene mutations, type 2 like gene mutations and other mutations. can do. Further, by using both the “first determination value” and the “second determination value”, the type 2 gene mutation described above can be distinguished from type 2 like and other mutations.

As described above, it is possible to simultaneously identify each gene mutation present in JAK2, CALR and MPL by using a microarray provided with a mutation type probe for identifying each gene mutation present in JAK2, CALR and MPL. it can. In particular, each gene mutation can be identified with high accuracy by using the first determination value described above or by using the first determination value and the second determination value. Information on gene mutations present in JAK2, CALR and MPL can be used, for example, for diagnosis of myeloproliferative tumors in the classification by WHO (2016 version). In particular, according to the classification by WHO, it is one requirement that the above gene mutation in JAK2 be present for the diagnosis of polycythemia vera or polycythemia vera (PV). In addition, according to the classification by WHO, the diagnosis of essential thrombocythemia (ET) requires one of presence of any of gene mutations present in JAK2, CALR and MPL. Furthermore, according to the WHO classification, JAK2, CALR and / or J for the diagnosis of prefibrotic / early primary myelofibrosis (prefibrotic / early PMF) or primary myelofibrosis (PMF). One requirement is that any of the genetic mutations present in MPL be present.

Thus, for example, in the diagnosis of myeloproliferative tumors using classification by WHO (2016 version), using a microarray provided with a mutated probe for identifying each gene mutation present in JAK2, CALR and MPL Can.

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 1
1. Sample Preparation In this example, peripheral blood (a patient sample for which informed consent was obtained from a document) collected by clinical research with the Yamaguchi University Hospital as a main facility was used as a sample. From these samples, DNA as a sample was extracted as follows. Peripheral blood leukocyte genomic DNA was extracted by a conventional method (NaI method).

Predetermined regions of the JAK2 gene, the CALR gene and the MPL gene were amplified by PCR using the DNA samples prepared as described above. The primer set shown in Table 1 was designed for this PCR. In the primer sets shown in Table 1, a fluorescent label (IC5) is added to the forward primer to which “F” is attached.

The primer set designed as described above was mixed so as to have the composition shown in Table 2 to prepare a primer mix.

The PCR reaction solution of the composition shown in Table 3 was prepared using the DNA sample and primer mix which were prepared as mentioned above.

Then, the PCR thermal cycle is performed for 5 minutes at 95 ° C., followed by 40 cycles of 30 seconds at 95 ° C., 30 seconds at 59 ° C. and 45 seconds at 72 ° C., and then 10 minutes at 72 ° C. The final temperature was maintained at 4 ° C.

2. Microarray In this example, mutant probes corresponding to the V617F mutation in the JAK2 gene, type 1 mutations and type 2 mutations in the CALR gene, and W515L / K mutation in the MPL gene, and the corresponding wild-type probes were designed.

Further, in this example, a common probe corresponding to a site which is commonly present in a region amplified by the above primer for the CALR gene regardless of whether or not the mutation of type 1 is present was designed. That is, the common probe specifically hybridizes to the amplified nucleic acid regardless of the presence or absence of the type 1 gene mutation contained in the amplified nucleic acid.
The nucleotide sequences of the designed probes are shown in Table 4.

As for type 1 and type 2 gene mutations in CALR, wild type probe 1 and mutant type probe 1, wild type probe 2 and mutant type probe 2 were designed as shown in FIG. In FIG. 1, 52 base deletion, which is a type 1 gene mutation in CALR, is indicated by “-”. In addition, in FIG. 1, the corresponding wild-type region is indicated by “-” for 5-base insertion, which is a type 2 gene mutation in CALR.

3. Identification of Gene Mutation Hybridization was performed as follows using a chip having the above probe. First, the wet box was placed in a chamber set to a specified temperature (52 ° C.), and the chamber and the wet box were sufficiently preheated. Mix 4 μL of PCR reaction solution and 2 μL of hybridization buffer (2.25 × SSC / 0.23% SDS / 0.2 nM Cy5-labeled oligo DNA (manufactured by Sigma Aldrich)), take 3 μL of this solution, and place on the central convex part of the hybrid cover The solution was dropped onto the chip, covered with a chip, and allowed to react for 1 hour with a hybridization chamber apparatus (made by Toyo Kogyo Co., Ltd.) set at 52 ° C. After completion of the hybridization reaction, the stainless steel holder for washing was immersed in a 0.1 × SSC / 0.1% SDS solution, and the chip from which the hybrid cover was removed was set in the holder. After shaking several times up and down, the holder was immersed in 1 × SSC solution (room temperature) until the fluorescence intensity of the chip was detected.

Immediately before the detection, the chip was covered with a cover film, and the fluorescence intensity of the chip was detected by BIOSHOT (manufactured by Toyo Kogyo Co., Ltd.). The second judgment value was calculated for the gene mutation of JAK2, the gene mutation of CALR and the gene mutation of MPL by the following formula using the fluorescence intensities of the wild type probe and the mutant type probe measured as described above.
Second determination value = [fluorescence intensity of mutant probe] / ([fluorescence intensity of wild-type probe] + [fluorescence intensity of mutant probe])

Moreover, about the gene mutation of type 1 of CALR, the 1st judgment value was computed by the following formula.
First determination value = [fluorescence intensity of wild-type probe] / [fluorescence intensity of common probe]

In this example, the second judgment value is calculated using a sample in which all mutations of JAK2 gene mutation, CALR gene mutation and MPL gene mutation are wild type (n = 4), and the second judgment The mean and standard deviation of the values were determined. Then, a value of average value + 3σ or average + 4σ (σ: standard deviation) was set as a cutoff value (see the following table).

Furthermore, in the present embodiment, the cutoff value is defined as follows for the first determination value. That is, the first judgment value was calculated using a sample confirmed to have no mutation at the type 1 mutation site (n = 40), and the average value and the standard deviation of the first judgment value were obtained. Then, a value of the average value -1.5σ was set as a cutoff value (see the following table).

4. Result The result of having plotted the 2nd judgment value computed as mentioned above about each specimen used by the present Example for every gene mutation was shown in FIG. In FIG. 2, the cutoff value defined as described above is indicated by a broken line. The plots below the dashed line indicate the specimens that are wild type (no gene mutation) in each gene mutation, and the plots above the dashed line indicate specimens with each gene mutation.

As shown in FIG. 2, among the gene mutations of JAK2 and CALR, among the gene mutations of type 2 and MPL, it was revealed that the second judgment value can identify the mutant and the wild type with high accuracy. . However, as shown in FIG. 2, among the CALR gene mutations, there are not a few samples plotted near the cutoff value for type 1, and the second judgment value alone makes it possible to judge the type 1 gene mutation with respect to the judgment accuracy. Could have been low.

Therefore, in this example, the first determination value and the second determination value were used for the type 1 gene mutation determination of CALR. Specifically, for each sample used in the present embodiment, the first determination value and the second determination value calculated for the type 1 of CALR are shown in FIG. And the vertical axis is plotted as a first determination value in a graph. As shown in FIG. 3, the plot of each sample is divided into four regions divided by the cutoff value defined for the first determination value and the cutoff value defined for the second determination value. . When the gene mutation was determined by other methods for each of the plotted samples, the sample which exceeded the cutoff value defined for the second determination value and was less than the cutoff value defined for the first determination value All were type 1 gene mutations of CALR, ie 52 base deletion. On the other hand, all the samples below the cutoff value specified for the second determination value and below the cutoff value specified for the first determination value are 46 bases similar to the type 1 gene mutation of CALR. It was revealed to be deletion, 34 bases deletion or 24 bases deletion.

From the results of this example, type 1 like mutant genes such as 46 base deletion, 34 base deletion and 24 base deletion that could not be distinguished from the wild type only by the second judgment value were wild by using the first judgment value. It can be distinguished from the type.

Example 2
In this example, for each of the JAK2 gene, the CALR gene and the MPL gene, a blocker that specifically hybridizes to the wild-type derived amplification by-product is designed, and the amplification product from the wild-type is nonspecific to the mutant-type probe. By suppressing the hybridization, it was examined whether the detection sensitivity of gene mutation of JAK2 gene, type 1 mutation of CALR gene and gene mutation of MPL gene could be improved. In addition, since the type 2 mutation of the CALR gene has a difference of 5 bases from the wild type and nonspecific hybridization is unlikely to occur in this example, no blocker was designed.

1. Sample Preparation In this example, healthy human peripheral blood leukocyte-derived genomic DNA (purchased from Biochain) diluted to 8 ng / μL with TE buffer was used as a wild-type sample.

In addition, in this example, mutant samples were prepared as follows. In this example, wild-type plasmids and mutant-type plasmids were prepared through the artificial gene synthesis service of FASMAC. As a wild-type plasmid of the JAK2 gene, a plasmid into which a region consisting of 400 bases at positions 151 to 550 in the base sequence shown in SEQ ID NO: 1 was inserted was used. As a wild-type plasmid of the CALR gene, a plasmid into which a region consisting of 389 bases at positions 376 to 764 in SEQ ID NO: 2 was inserted was used. As a wild-type plasmid of the MPL gene, a plasmid into which a region consisting of 399 bases at positions 107 to 505 in SEQ ID NO: 3 was inserted was used. The same region was inserted as a mutant-type plasmid of each gene except that it had the above-mentioned mutation (V617F mutation of JAK2 gene, W515L mutation of MPL gene or W515K mutation and type 1 mutation or type 2 mutation of CALR gene). The plasmid was used. The purchased plasmid DNA was dissolved to about 100 ng / μL with TE buffer and further diluted to about 1 ng / μL with TE buffer.

Then, a plasmid corresponding to the wild type of the JAK2 gene, a plasmid corresponding to the wild type of the MPL gene, and three types of plasmids corresponding to the wild type of the CALR gene are mixed, diluted with TE buffer, and each plasmid concentration is about 200 pg A wild-type plasmid mix was prepared at / μL. In addition, a plasmid corresponding to the V617F mutant of the JAK2 gene, a plasmid corresponding to the W515L mutant of the MPL gene, and a plasmid corresponding to the type 1 mutant of the CALR gene are mixed, diluted with TE buffer, A 100% mutant plasmid mix A was prepared with a plasmid concentration of about 200 pg / μL. Similarly, a plasmid corresponding to the V617F variant of the JAK2 gene, a plasmid corresponding to the W515K variant of the MPL gene, and a plasmid corresponding to the type 2 variant of the CALR gene are mixed and diluted with TE buffer, A 100% mutant plasmid mix B was prepared at a concentration of about 200 pg / μL for each plasmid.

In this example, mutant samples were prepared by mixing these wild-type plasmid mixes with 100% mutant-type plasmid mix A or 100% mutant-type plasmid mix B at a predetermined ratio. After preparation of mutant samples,% mutation (ratio of mutant to total wild type and mutant) for JAK2 gene and MPL gene was quantified by digital PCR, and mutation% for CALR gene was quantified by fragment analysis. Then, the mutant sample was diluted to about 0.16 pg / μL and used for PCR.

In this example, predetermined regions of the JAK2 gene, the CALR gene and the MPL gene were amplified by PCR under the same conditions as in Example 1 using the wild-type sample or the mutant-type sample prepared as described above.

In this example, the microarray used in Example 1 was used, and a hybridization buffer was prepared in the same manner as in Example 1 except that the blocker was included, and hybridization experiments were performed. In this example, the hybridization buffer was prepared such that the blocker for JAK2 gene, the blocker for CALR gene, and the blocker for MPL gene shown in Table 7 had concentrations of 90 to 210 nM, respectively. Then, the PCR reaction solution and the hybridization buffer were mixed at 2: 1 to conduct a hybridization experiment.

In the present example, in the same manner as Example 1, a second determination value was calculated as a result of the hybridization experiment. However, in this example, unlike Example 1, for detection of the W515L mutant of the MPL gene: second determination value = [fluorescence intensity of W515L mutant-type probe] / ([fluorescence intensity of wild-type probe] + According to the formula of [fluorescent intensity of W515L mutant probe] + [fluorescent intensity of W515K mutant probe], for detection of W515K mutant of MPL gene: second judgment value = [fluorescent intensity of W515K mutant probe] The equation of ([fluorescent intensity of wild type probe] + [fluorescent intensity of W515L mutant probe] + [fluorescent intensity of W515K mutant probe]) was followed.

The relationship between the blocker concentration and the second determination value is shown in FIG. FIG. 4 (a) shows the result when detecting the V617F mutant of the JAK2 gene, and FIG. 4 (b) shows the result when the W515L mutant of the MPL gene is detected, and FIG. 4 (c) shows the W515K of the MPL gene. The results when a mutant is detected, the same (d) is the results when a type 1 mutant of the CARL gene is detected, and the same (e) is the results when a type 2 mutant of the CARL gene is detected It is. As shown in FIG. 4, it became clear that, by adding the blocker to the hybridization buffer, it is possible to detect the mutant form of the gene with excellent detection sensitivity even if the mutation rate is 2.6 to 5.8%.

Moreover, in this example, the mixing ratio of the wild-type plasmid mix and the mutant-type plasmid mix A or B in the mutant-type sample is changed to adjust the ratio of the mutant-type of each gene, and the same hybridization experiment is performed. The The relationship between the mutation rate (mutation%) in the mutant-type sample and the second judgment value is shown in FIG. FIG. 5 (a) shows the result when detecting the V617F mutant of the JAK2 gene, and FIG. 5 (b) shows the result when the W515L mutant of the MPL gene is detected, and FIG. 5 (c) shows the W515K of the MPL gene. The results when a mutant is detected, the same (d) is the results when a type 1 mutant of the CARL gene is detected, and the same (e) is the results when a type 2 mutant of the CARL gene is detected It is. As shown in FIG. 5, it became clear that, by adding the blocker to the hybridization buffer, excellent detection sensitivity can be achieved even if the mutation rate in the mutant sample is about 2%. In the hybridization experiments whose results are shown in FIG. 5, the blocker concentration for JAK2 gene was 150 nM, the blocker concentration for CALR gene was 210 nM, and the blocker concentration for MPL gene was 150 nM.

In addition, the result of having measured the actual density | concentration of each mutant with respect to the theoretical value of the mutation rate contained in a mutant sample was shown in Table 8. The actual values shown in Table 8 are the results of quantification of the mutation% for the JAK2 gene and the MPL gene by digital PCR and the mutation% for the CALR gene by fragment analysis.

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

Claims (13)

1. Amplifying a region including a specific site where a plurality of gene mutations may exist to obtain an amplified fragment having a label;
   A first probe corresponding to a wild-type sequence at the specific location and a common probe corresponding to a sequence excluding the gene mutation in the amplified fragment are brought into contact with a solution containing the amplified fragment, the first probe And detecting a signal based on the label in the common probe;
   Judgment formula: A step of calculating a first judgment value by [signal strength of first probe] / [signal strength of common probe],
   And D. comparing the first determination value calculated by the above determination equation with a preset cutoff value, and determining the presence or absence of a gene mutation based on the comparison result.
2. In the step of detecting the signal, a second probe corresponding to a specific gene mutation among the plurality of gene mutations is brought into contact with a solution containing the amplification fragment, and the signal based on the label in the second probe is detected. The method for evaluating gene mutation according to claim 1, which comprises detecting.
3. In the step of calculating the first determination value, the second determination equation further includes: [signal strength of second probe] / ([signal strength of first probe] + [signal strength of second probe]) In the step of calculating the judgment value of and determining the presence or absence of the gene mutation, the second judgment value is compared with a preset cutoff value, and the result of the comparison using the first judgment value is compared with the first judgment value. The method for evaluating gene mutation according to claim 2, wherein the presence or absence of the gene mutation is determined based on the result of comparison using the determination value of 2.
4. In the step of calculating the first determination value, the determination value is further calculated according to the determination formula [signal strength of second probe] / [signal strength of common probe] to determine the presence or absence of the gene mutation. In the step, the determination value is compared with a preset cutoff value, and the presence or absence of gene mutation is determined based on the result of comparison using the first determination value and the result of comparison using the determination value. The method for evaluating gene mutation according to claim 2, wherein the method is as follows.
5. The method for evaluating gene mutation according to claim 1, wherein the plurality of gene mutations are insertions or deletions of bases.
6. The above specific site is between the 501st and 579th in the nucleotide sequence of the CALR gene shown in SEQ ID NO: 2, and the plurality of gene mutations are insertions or deletions of bases in the 501st to 579th regions. The method for evaluating gene mutation according to claim 1 characterized by the above.
7. A kit for gene mutation evaluation which determines the presence or absence of a gene mutation using an amplification fragment having a label obtained by amplifying a region including a specific site where a plurality of gene mutations may exist.
   A kit for gene mutation evaluation, comprising a first probe corresponding to a wild-type sequence at the specific site, and a common probe corresponding to a sequence excluding the gene mutation in the amplified fragment.
8. The kit for gene mutation evaluation according to claim 7, further comprising a second probe corresponding to a specific gene mutation among the plurality of gene mutations.
9. The gene mutation evaluation kit according to claim 7, wherein the plurality of gene mutations are insertions or deletions of bases.
10. The above specific site is between the 501st and 579th in the nucleotide sequence of the CALR gene shown in SEQ ID NO: 2, and the plurality of gene mutations are insertions or deletions of bases in the 501st to 579th regions. The kit for gene mutation evaluation according to claim 7, which is characterized.
11. The gene mutation evaluation kit according to claim 7, wherein the common probe is an oligonucleotide comprising a sequence of 397 to 659 in the nucleotide sequence of the CALR gene shown in SEQ ID NO: 2.
12. The gene mutation evaluation kit according to claim 7, wherein the common probe is an oligonucleotide containing CTCCTCCATCC TCATCTTTGTC (SEQ ID NO: 15) or CTCTCGTCCTGTTTGTC (SEQ ID NO: 31).
13. The above gene mutation is a 52 base deleted type 1 mutation in which 52 bases from 513 to 564 in the base sequence of the CALR gene shown in SEQ ID NO: 2 is deleted, and the second probe corresponding to the mutant is TCCTTGTCCTGCTGCTCC ( The gene mutation evaluation kit according to claim 7, which is an oligonucleotide containing SEQ ID NO: 5).

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