JP2014103904A - Oligonucleotide probe for detecting mutation in k-ras - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide detection of K-ras mutation with high accuracy.SOLUTION: An oligonucleotide probe for detecting K-ras mutation according to the invention includes a region comprising 6 bases corresponding to codon 12 and codon 13 of K-ras, and comprises 19 bases in its entirety.

Description

  The present invention relates to a mutation detection oligonucleotide probe for detecting a specific mutation in K-ras, a microarray provided with the probe, and a method for detecting a K-ras mutation in a subject using the probe.

  K-ras is a gene symbol of v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog. Specific mutations in K-ras, particularly mutations involving amino acid substitutions at codons 12 and 13, are used as predictors of the effects of anti-EGFR antibody drugs on unresectable advanced / recurrent colorectal cancer. Specifically, anti-EGFR antibody drugs bind to EGFR and inhibit ligands such as EGF from binding to EGFR. Thereby, downstream signaling of EGFR including K-ras is suppressed. However, when there is a mutation accompanied by amino acid substitution in codon 12 or 13 of the K-ras gene, K-ras is constantly activated, and thus the effect of the anti-EGFR antibody drug cannot be expected. Therefore, upon administration of an anti-EGFR antibody drug, it is required to confirm in advance the presence or absence of a mutation involving amino acid substitution at codon 12 or 13 of the K-ras gene.

  As a technique for detecting a mutation of K-ras, for example, as shown in Patent Document 1, there is a method of amplifying a region including a mutation site to be detected using a pair of primers and analyzing the Tm of the amplified nucleic acid. Can be mentioned. Patent Document 2 discloses a method of using a microarray provided with an oligonucleotide probe designed together with a mutation site to be detected as a technique for detecting a mutation in K-ras. Patent Document 2 discloses that an oligonucleotide probe consisting of 21 bases including codons 12 and 13 was designed, and that a K-ras mutation in a clinical sample was detected using a microarray equipped with these oligonucleotide probes. Have been described.

WO 2011-071046 Special table 2007-534331

  However, when the K-ras mutation is detected using the oligonucleotide probe disclosed in Patent Document 2, there is a problem that the accuracy of separating and detecting the wild type and the mutant type is not sufficient. In other words, even with conventional oligonucleotide probes designed to detect mutants, wild-type K-ras is detected at a certain rate, and the reliability of K-ras mutation detection is low. was there.

  Therefore, in view of the actual situation as described above, the present invention can detect a K-ras mutation with high accuracy, and has excellent reliability, an oligonucleotide probe, a microarray provided with the probe, and a target using the probe. The purpose is to provide a method for detecting a mutation in the K-ras gene of the examiner.

  As a result of intensive studies to achieve the above-described object, the present inventors have designed a region corresponding to the codon 13 of K-ras to be detected at a predetermined position, having a specific base length, The present inventors have found that mutant K-ras can be detected with high accuracy and have completed the present invention.

The present invention includes the following.
(1) An oligonucleotide probe for detecting a K-ras mutation comprising a region consisting of 6 bases corresponding to codons 12 and 13 of K-ras, and a total of 19 bases.
(2) The oligonucleotide probe for detecting a K-ras mutation according to (1), wherein the region comprises a GGTGAC sequence corresponding to a codon 12 of a wild type and a codon 13 of a G13D variant.
(3) The oligonucleotide probe for detecting a K-ras mutation according to (1), wherein the region comprises a GGTGGC sequence corresponding to wild-type codon 12 and wild-type codon 13.
(4) The oligonucleotide probe for detecting a K-ras mutation according to (1), wherein the region comprises a GATGGC sequence corresponding to a codon 12 of G12D mutant type and a codon 13 of wild type.
(5) The oligonucleotide probe for detecting a K-ras mutation according to any one of (1) to (4), wherein the 5 ′ terminal side of the region is 2 to 11 bases.
(6) The oligonucleotide probe for detecting a K-ras mutation according to any one of (1) to (4), wherein the 5 ′ terminal side of the region is 5 to 10 bases.
(7) The oligonucleotide probe for detecting a K-ras mutation according to any one of (1) to (4), wherein the 5 ′ terminal side of the region is 7 to 10 bases.
(8) The oligonucleotide probe for detecting a K-ras mutation according to (2), comprising one base sequence selected from the group consisting of the following (a) to (n).
(A) GGTGACGTAGGCAAGAGTG: SEQ ID NO: 1
(B) TGGTGACGTAGGCAAGAGT: SEQ ID NO: 2
(C) CTGGTGACGTAGGCAAGAG: SEQ ID NO: 3
(D) GCTGGTGACGTAGGCAAGA: SEQ ID NO: 4
(E) AGCTGGTGACGTAGGCAAG: SEQ ID NO: 5
(F) GAGCTGGTGACGTAGGCAA: SEQ ID NO: 6
(G) GGAGCTGGTGACGTAGGCA: SEQ ID NO: 7
(H) TGGAGCTGGTGACGTAGGC: SEQ ID NO: 8
(I) TTGGAGCTGGTGACGTAGG: SEQ ID NO: 9
(J) GTTGGAGCTGGTGACGTAG: SEQ ID NO: 10
(K) AGTTGGAGCTGGTGACGTA: SEQ ID NO: 11
(L) TAGTTGGAGCTGGTGACGT: SEQ ID NO: 12
(M) GTAGTTGGAGCTGGTGACG: SEQ ID NO: 13
(N) GGTAGTTGGAGCTGGTGAC: SEQ ID NO: 14
(9) A microarray comprising a substrate and the oligonucleotide probe for detecting a K-ras mutation according to any one of (1) to (8) fixed to the substrate.
(10) The microarray according to (9), wherein the 5 ′ end side of the oligonucleotide probe for detecting a K-ras mutation is fixed to the substrate.
(11) A region containing codon 12 and codon 13 of K-ras is amplified from a biological sample collected from a subject, and the amplified nucleic acid and the oligo for K-ras mutation detection according to any one of (1) to (8) above A method for detecting a mutation in K-ras, which detects hybridization with a nucleotide probe.
(12) The K-ras described in (11), wherein when one of codon 12 and codon 13 of K-ras has a mutation, it is determined that the therapeutic effect of the anti-EGFR antibody on the subject is low. ras mutation detection method.
(13) The K-ras mutation detection method according to (11), wherein the subject is a patient with advanced / recurrent colorectal cancer that cannot be cured and excised.
(14) The method for detecting a mutation of K-ras according to (12), wherein the anti-EGFR antibody drug is cetuximab or panitumumab.

  The oligonucleotide probe for detecting a K-ras mutation according to the present invention can detect the presence or absence of a mutation in codon 12 and / or codon 13 of K-ras with high accuracy. Therefore, by using the oligonucleotide probe for detecting a K-ras mutation according to the present invention, whether or not the codon 12 and / or codon 13 of K-ras in a subject is a wild type or a mutant type, excellent reliability Can be judged.

  The oligonucleotide probe for detecting a K-ras mutation according to the present invention comprises a region consisting of 6 bases corresponding to codons 12 and 13 of K-ras, and the whole consists of 19 bases. The oligonucleotide probe for detecting a K-ras mutation according to the present invention is characterized in that the detection sensitivity is significantly superior compared to a probe designed to have a base length of 18 or 20 bases as a whole. Here, the detection sensitivity can be said to be accuracy in detecting whether the codon 13 of K-ras is a wild type or a mutant type.

  K-ras is a gene symbol of v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog. Therefore, there are cases where they are generally called K-ras and K-ras genes, but both can be treated as the same meaning. Further, human K-ras is stored in the database as GenBank accession number NG_007524, and information on the nucleotide sequence of K-ras and information on the coding region on the genome can be obtained.

  For K-ras, anti-EGFR (Epidermal Growth Factor Receptor) such as cetuximab or panitumumab is used in patients with mutant K-ras with amino acid substitution at the 12th codon (codon 12) and / or the 13th codon (codon 13). : Epidermal growth factor receptor) It is known that the effect of administration of antibody drugs is not observed. In wild-type K-ras, codon 12 and codon 13 both encode glycine. As mutant K-ras, G12C, G12A, G12D, G12R, G12S and G12V mutations are known for codon 12. As mutant K-ras, G13C and G13D for codon 13 are known.

  In addition, the anti-EGFR antibody drug mentioned above has a medicinal effect on colorectal cancer including colon cancer and rectal cancer, head and neck cancer, and non-small cell lung cancer. In Japan, anti-EGFR antibody drugs are approved for the treatment of EGFR-positive advanced / recurrent colorectal cancer. Therefore, the oligonucleotide probe for detecting a K-ras mutation according to the present invention is preferably used for determining the efficacy of an anti-EGFR antibody drug in a patient suffering from colorectal cancer, head and neck cancer and non-small cell lung cancer, In particular, it is more preferably used for determining the efficacy of an anti-EGFR antibody drug in a patient suffering from EGFR-positive advanced / recurrent colorectal cancer.

  In particular, the oligonucleotide probe for detecting a K-ras mutation according to the present invention consists of 19 bases as a whole, and there are 6 bases corresponding to codon 12 and codon 13 in any position. good. That is, the oligonucleotide probe for detecting a K-ras mutation according to the present invention may be designed such that 6 bases corresponding to codon 12 and codon 13 are located in the first to sixth positions from the 5 ′ end, It may be designed to be located at the 14th to 19th positions from the 5 ′ end.

  For example, in the oligonucleotide probe for detecting a K-ras mutation according to the present invention, the 5 ′ end side of the 6 base region corresponding to codon 12 and codon 13 is 2 to 11 bases, and the 3 ′ end side of the region is 3 ′ end side. It is preferable to design to have 11 to 2 bases. In the oligonucleotide probe for detecting a K-ras mutation according to the present invention, the 5 ′ end side of the 6-base region corresponding to codon 12 and codon 13 is 5 to 10 bases, and the 3 ′ end side of the region is the 3 ′ end side. It is more preferable to design to have 8 to 3 bases. Furthermore, in the oligonucleotide probe for detecting a K-ras mutation according to the present invention, the 5 ′ end side of the 6 base region corresponding to codon 12 and codon 13 is 7 to 10 bases, and the 3 ′ end side of the region is the 3 ′ end side. Even more preferably, it is designed to have 6 to 3 bases. Thus, by setting the base length on the 5 ′ end side (and 3 ′ end side) of the region consisting of 6 bases corresponding to codon 12 and codon 13 within the above range, detection sensitivity can be further enhanced.

  The oligonucleotide probe for detecting a K-ras mutation according to the present invention detects one or both of the above-mentioned codon 12 and codon 13 mutations. That is, the region consisting of 6 bases corresponding to codon 12 and codon 13 can be a GGTGAC sequence corresponding to wild-type codon 12 and G13D mutant codon 13. Further, the region consisting of 6 bases corresponding to codon 12 and codon 13 can be a GGTGGC sequence corresponding to wild-type codon 12 and wild-type codon 13. Furthermore, the 6-base region corresponding to codon 12 and codon 13 can be a GATGGC sequence corresponding to G12D mutant codon 12 and wild-type codon 13.

  In particular, it is preferable that the oligonucleotide probe for detecting a K-ras mutation according to the present invention detects a mutation of codon 13 among codon 12 and codon 13 described above, particularly G13D mutant codon 13. That is, in the oligonucleotide probe for detecting a K-ras mutation according to the present invention, the region consisting of 6 bases corresponding to codon 12 and codon 13 has a GGTGAC sequence corresponding to codon 12 of wild type and codon 13 of G13D mutant. It is preferable to do.

  When a region consisting of 6 bases corresponding to codon 12 and codon 13 is a GGTGAC sequence, the oligonucleotide probe for detecting a K-ras mutation according to the present invention specifically has the following base sequences (a) to (n): It can be.

(A) GGTGACGTAGGCAAGAGTG: SEQ ID NO: 1
(B) TGGTGACGTAGGCAAGAGT: SEQ ID NO: 2
(C) CTGGTGACGTAGGCAAGAG: SEQ ID NO: 3
(D) GCTGGTGACGTAGGCAAGA: SEQ ID NO: 4
(E) AGCTGGTGACGTAGGCAAG: SEQ ID NO: 5
(F) GAGCTGGTGACGTAGGCAA: SEQ ID NO: 6
(G) GGAGCTGGTGACGTAGGCA: SEQ ID NO: 7
(H) TGGAGCTGGTGACGTAGGC: SEQ ID NO: 8
(I) TTGGAGCTGGTGACGTAGG: SEQ ID NO: 9
(J) GTTGGAGCTGGTGACGTAG: SEQ ID NO: 10
(K) AGTTGGAGCTGGTGACGTA: SEQ ID NO: 11
(L) TAGTTGGAGCTGGTGACGT: SEQ ID NO: 12
(M) GTAGTTGGAGCTGGTGACG: SEQ ID NO: 13
(N) GGTAGTTGGAGCTGGTGAC: SEQ ID NO: 14

  Among the 14 types of oligonucleotide probes for detecting K-ras mutations comprising the base sequences shown in (a) to (n), the 5 ′ terminal side of the GGTGAC sequence has 2 to 11 bases (c) to (l) Preferably, the base sequence is selected from the base sequences selected from (f) to (k), wherein the 5 ′ end of the GGTGAC sequence is 5 to 10 bases. . Oligonucleotide probes for detecting K-ras mutations having these specific base sequences show very good detection sensitivity. Furthermore, among 14 types of oligonucleotide probes for detecting a K-ras mutation comprising the base sequences shown in (a) to (n), the oligonucleotide probe for detecting a K-ras mutation comprising a base sequence shown in (k) is: The best detection sensitivity can be achieved.

  The oligonucleotide probe for detecting a K-ras mutation according to the present invention is preferably a nucleic acid, more preferably a DNA. Although DNA includes both double strands and single strands, the oligonucleotide probe for detecting a K-ras mutation according to the present invention is preferably a single strand DNA.

  The oligonucleotide probe for detecting a K-ras mutation according to the present invention can be obtained by, for example, chemically synthesizing with a nucleic acid synthesizer. As the nucleic acid synthesizer, an apparatus called a DNA synthesizer, a fully automatic nucleic acid synthesizer, an automatic nucleic acid synthesizer or the like can be used.

  The oligonucleotide probe for detecting a K-ras mutation according to the present invention is preferably used in the form of a microarray by immobilizing its 5 ′ end on a carrier. As the material for the carrier, those known in the art can be used and are not particularly limited. For example, noble metals such as platinum, platinum black, gold, palladium, rhodium, silver, mercury, tungsten and their compounds, and conductor materials such as graphite and carbon typified by carbon fiber; single crystal silicon, amorphous Silicon materials represented by silicon, silicon carbide, silicon oxide, silicon nitride, etc., composite materials of these silicon materials represented by SOI (silicon on insulator), etc .; glass, quartz glass, alumina, sapphire, ceramics, 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, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile / butadiene styrene copolymer, polyphenylene oxide And organic materials such as polysulfone. The shape of the carrier is not particularly limited, but is preferably a flat plate shape.

  In the present invention, a carrier having a carbon layer and a chemical modification group on the surface is preferably 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 made of the carbon layer. As the material for the substrate, those known in the art can be used, and there is no particular limitation, and the same materials as those mentioned above as the carrier material can be used.

  In the microarray according to the present invention, a carrier having a fine flat plate structure is preferably used. The shape is not limited to a rectangle, a square, or a round shape, but a shape of 1 to 75 mm square, preferably 1 to 10 mm square, more preferably 3 to 5 mm square is usually used. Since it is easy to produce a carrier having a fine flat plate structure, it is preferable to use a substrate made of a silicon material or a resin material. Particularly, 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. Single crystal silicon has a slightly different orientation of the crystal axis in some parts (sometimes called a mosaic crystal), or includes atomic scale disturbances (lattice defects) Are also included.

  In the present invention, the carbon layer formed on the substrate 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) , Carbon nanotubes), a mixture thereof, or a laminate of them is preferably used. Further, carbides such as hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, thorium carbide, titanium carbide, uranium carbide, tungsten carbide, zirconium carbide, molybdenum carbide, chromium carbide, and vanadium carbide may be used. Here, the soft diamond is a generic term for an incomplete diamond structure that is a mixture of diamond and carbon, such as so-called diamond-like carbon (DLC), and the mixing ratio is not particularly limited. The carbon layer has excellent chemical stability, can withstand subsequent reactions in the introduction of chemical modification groups and binding to the analyte, and the binding is flexible because of the electrostatic binding to the analyte. It is advantageous in that it has the property of being transparent, it is transparent to the detection system UV because there is no UV absorption, and it can be energized during electroblotting. Further, it is advantageous in that nonspecific adsorption is small in the binding reaction with the analyte. As described above, a carrier whose substrate itself is made of a carbon layer may be used.

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

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

  In the arc evaporation method, an arc discharge is generated in a vacuum by applying a DC voltage between a solid graphite material (cathode evaporation source) and a vacuum vessel (anode), and a plasma of carbon atoms is generated from the cathode to generate an evaporation source. Further, 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 (pulse oscillation) light and melting it, and depositing carbon atoms on a glass substrate.

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

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

  An amino group can be introduced, for example, by irradiating the carbon layer with ultraviolet light in ammonia gas or by plasma treatment. Alternatively, the carbon layer can be chlorinated by irradiation with ultraviolet rays in chlorine gas, and further irradiated with ultraviolet rays in ammonia gas. Alternatively, it can also be carried out by reacting a polyvalent amine gas such as methylenediamine or ethylenediamine with a chlorinated carbon layer.

  The introduction of the carboxyl group can be carried out, for example, by reacting an appropriate compound with the carbon layer aminated as described above. As a compound used for introducing a carboxyl group, for example, represented by the formula: X-R1-COOH (wherein X represents a halogen atom and R1 represents a divalent hydrocarbon group having 10 to 12 carbon atoms) Halocarboxylic acids such as chloroacetic acid, fluoroacetic acid, bromoacetic acid, iodoacetic acid, 2-chloropropionic acid, 3-chloropropionic acid, 3-chloroacrylic acid, 4-chlorobenzoic acid; formula: HOOC-R2-COOH (formula R2 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 Polycarboxylic acid such as polymethacrylic acid, trimellitic acid, butanetetracarboxylic acid; Formula: R3-CO-R4-COOH (wherein R3 is a hydrogen atom or a divalent hydrocarbon group having 1 to 12 carbon atoms) , R4 represents a divalent hydrocarbon group having 1 to 12 carbon atoms) A dicarboxylic acid represented by the formula: X-OC-R5-COOH (wherein X represents a halogen atom, R5 represents a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms). Acid monohalides such as succinic acid monochloride, malonic acid monochloride; acid anhydrides such as phthalic anhydride, succinic anhydride, oxalic anhydride, maleic anhydride, and butanetetracarboxylic anhydride.

  Introduction of an epoxy group can be carried out, for example, by reacting a suitable polyvalent epoxy compound with the carbon layer aminated as described above. Or it can obtain by making an organic peracid react with the carbon = carbon double bond which a carbon layer contains. Examples of the organic peracid include peracetic acid, perbenzoic acid, diperoxyphthalic acid, performic acid, and trifluoroperacetic acid.

Introduction of the formyl group can be carried out, for example, by reacting glutaraldehyde with the carbon layer aminated as described above.
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 having high acidity on the alcohol side of the ester group and activating the nucleophilic reaction, that is, an ester group having high reaction activity. The ester group has an electron-withdrawing group on the alcohol side of the ester group and is 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 a p-nitrophenyl group, an N-hydroxysuccinimide group, a succinimide group, a phthalimide group, and a 5-norbornene-2,3-dicarboximide group. In particular, an N-hydroxysuccinimide group is preferably used.

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

  The K-ras mutation detection oligonucleotide probe according to the present invention is dissolved in a spotting buffer to prepare a spotting solution, which is dispensed into a 96-well or 384-well plastic plate, and the dispensed solution is added to the spotter. By spotting on a carrier with an apparatus or the like, a microarray in which oligonucleotide probes are immobilized on the carrier can be produced. Alternatively, the spotting solution may be spotted manually with a micropipette.

  Incubation is preferably performed after the spotting in order to advance the reaction of the oligonucleotide probe for detecting a K-ras mutation binding 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. Incubation is preferably performed under a high humidity atmosphere, for example, under conditions of a humidity of 50 to 90%. Following the incubation, it is preferable to perform washing using a washing solution (for example, 50 mM TBS / 0.05% Tween20, 2 × SSC / 0.2% SDS solution, ultrapure water, etc.) to remove DNA not bound to the carrier. .

  The present invention also relates to a method for detecting a K-ras mutation in a subject using the oligonucleotide probe for detecting a K-ras mutation or a microarray. The method of the present invention includes a step of extracting DNA from a sample derived from a subject, a step of amplifying a region containing codons 12 and 13 in K-ras using the extracted DNA as a template, and a K- and a step of detecting the amplified nucleic acid using an oligonucleotide probe for detecting ras mutation or a microarray.

  The subject is usually a human and can include patients suffering from colorectal cancer, including colon and rectal cancer, head and neck cancer, or non-small cell lung cancer. A healthy person who does not suffer from these cancers may be the subject. Furthermore, the subject can be a patient suffering from EGFR-positive advanced / recurrent colorectal cancer.

  The sample derived from the subject is not particularly limited. For example, blood-related samples (blood, serum, plasma, etc.), lymph fluid, feces, cancer cells, tissue or organ crushed materials and extracts can be mentioned.

  First, DNA is extracted from a sample collected from a subject. 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 performed using the obtained DNA as a template to amplify a nucleic acid encoding the K-RAS gene, preferably DNA. As the amplification reaction, 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 amplified region can be identified. At this time, the method for labeling the amplified nucleic acid is not particularly limited. For example, a method in which a primer used in the amplification reaction is labeled in advance may be used, or a labeled nucleotide is used as a substrate in the amplification reaction. You may use the method to do. The labeling substance is not particularly limited, and radioisotopes, fluorescent dyes, or organic compounds such as digoxigenin (DIG) and biotin can be used.

  In addition, this reaction system consists of buffers necessary for nucleic acid amplification and labeling, heat-resistant DNA polymerase, primers specific to the K-RAS gene, labeled nucleotide triphosphates (specifically, nucleotide triphosphates added with a fluorescent label or the like). Acid), nucleotide triphosphate, magnesium chloride and the like.

The primer used in the amplification reaction is not particularly limited as long as it can specifically amplify the region containing codon 12 and codon 13 of K-ras, and can be appropriately designed by those skilled in the art. For example,
Primer 1: 5′-gtgtgacatgttctaatatagtcac-3 ′ (SEQ ID NO: 15) and Primer 2: 5′-gaatggtcctgcaccagtaa-3 ′ (SEQ ID NO: 16)
A set of primers consisting of

  Performing a hybridization reaction between the amplified nucleic acid obtained as described above and the oligonucleotide probe for detecting a K-ras mutation according to the present invention, the amount of nucleic acid hybridized to the oligonucleotide probe for detecting a K-ras mutation, For example, it can be measured by detecting a label. For example, when a fluorescent label is used, the signal intensity from the label can be quantified by detecting the fluorescent signal using a fluorescent scanner and analyzing the detected signal with image analysis software. The amplified nucleic acid hybridized with the oligonucleotide probe for detecting a K-ras mutation 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 refer to conditions in which specific hybrids are formed and non-specific hybrids are not formed.For example, after hybridization at 50 ° C. for 16 hours, 2 × SSC / 0.2% SDS, 25 This refers to the conditions for washing at 5 ° C for 10 minutes and 2 x SSC at 25 ° C.

  In the above hybridization reaction, a method is preferred in which a microarray in which the oligonucleotide probe for detecting a K-ras mutation according to the present invention is immobilized on a carrier is used and a solution containing an amplified nucleic acid is applied to the microarray.

  The mutation detection method for K-ras according to the present invention is carried out by comparing the amount of the amplified nucleic acid hybridized with the oligonucleotide probe for detection of K-ras mutation. That is, specifically, based on the amount of nucleic acid hybridized to the oligonucleotide probe for detecting a K-ras mutation, the codon 12 and / or codon 13 in the subject K-ras is a wild type. Or mutated. And when one or both of the codon 12 and codon 13 in K-ras of a subject is a mutant type, it can be judged that the effect of an anti-EGFR antibody drug cannot be expected for the subject.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, the technical scope of this invention is not limited to a following example.

In this example, six types of 19-base probes shown in the following table for detecting a codon 13 mutation (particularly a G13D mutation) in K-ras were designed as Examples 1 to 6, respectively. Moreover, in the present Example, the 20 base or 21 base probe which detects the same mutation was designed as Comparative Examples 1-6. As a control, a 19-base probe corresponding to wild-type codon 13 was designed. In the base sequences shown in the following table, the underlined portions are sequences corresponding to codon 12 and codon 13.

  In this example, LoVo strain (G13D), blood DNA (W1), RKO strain (W2), and SW480 strain (G12V) were used as biological samples. W1 and W2 mean that both codons 12 and 13 of K-ras are wild type.

The equipment and reagents used in this example are as follows: Eppendorf: MasterCycler ep gradient pro S
・ Toyo Kohan: Hybridization heater ・ Toyo Kohan: BIOSHOT (ver.1,133 provisional version)
Gilson: Pipetteman Invitrogen: Ultrapure DNA / RNAse free distillated water, dNTP (4 types)
・ Roche Diagnostics: Fast taq polymerase Hot start, 10 × BUFFER
・ GE Healthcare: Cy5-dCTP
・ Promega: 20 x SSC, 10% SDS

  In this example, the region containing K-ras codons 12 and 13 was amplified by PCR using the genomic DNA extracted from the above-described biological sample as a template, the obtained PCR product and a hybrid buffer were mixed, and the reaction temperature Was evaluated by reacting the probe of Table 1 designed on the condition of 54 degrees with the spotted chip.

(1) PCR amplification
The composition for performing PCR is shown below. The primer mix was prepared from a 10 μM stock (Fasmac) as shown in the table below.

The dNTP mix was adjusted with the following composition.

The composition of the PCR premix is shown below.

  The obtained PCR premix was divided into 40 μL, and 5 μL of each primer mix was added. 5 μL of the premix was dispensed into a PCR tube (assist), and 15 μL of each genome solution was added.

PCR temperature conditions are as follows.

  Using the solution containing the PCR product after the completion of PCR as described above and the above-described chip, a hybridization reaction was performed as follows. First, after completion of PCR, PCR products having the same DNA concentration are mixed. Take 18 μL of the solution, 9 μL of hybridization buffer (3 × citrate buffer (SSC, Promega) /0.3% sodium dodecyl sulfate solution (SDS, Promega) /0.4 nM Cy5 labeled oligo DNA (Sigma Genosys)) Was added.

  3 μL of this solution was taken and dropped onto the chip contact surface of the hybrid cover, and this was placed on the chip and reacted for 1 hour in a hybridization chamber apparatus (Toyo Kohan). At this time, the temperature was 54 degrees.

After the hybridization reaction, the washing stainless steel holder was immersed in 1 × SSC / 0.1% SDS solution, and the chip from which the hybrid cover was removed was set in the holder. The holder was moved in the following order, washed by shaking up and down 15 times, and then allowed to stand for a predetermined time for washing.

After completion of the cleaning operation, the holder was left in a 1 × SSC solution (room temperature) until the fluorescence intensity of the chip was detected.
After the chip is soaked in 1 x SSC, the cover glass and the chip are brought into close contact with each other, and then using BIOSHOT (Toyo Kohan) and its control software (Shot Analyzer Ver 1.133 provisional version 2, Toyo Kohan) The fluorescence intensity on the chip was detected under the conditions.
○ Spot diameter (pix): 16
○ Photographing conditions: Exposure time 20.0 seconds, temperature 10.0 ℃

The fluorescence intensity varies from experiment to experiment due to PCR and hybridization errors. Therefore, in order to correct the fluorescence intensity, since the specimen always contains wild-type DNA, evaluation was performed by dividing the fluorescence intensity of the G13D probe of each sequence by the fluorescence intensity of the control probe. As an evaluation standard, detection was possible at 0.1 or more. The results are shown in Table 7.

  As shown in Table 7, when the DNA of the G13D mutant specimen (LoVo strain) was hybridized, the ratio of fluorescence intensity was higher than 0.1 for all the probes in the examples and comparative examples. From this, it was determined that the G13D mutation could be detected with all the probes examined this time.

  The fluorescence intensity when hybridizing wild type (blood, RKO) or G12V DNA is preferably a lower value because it is a probe designed for G13D detection. From this point of view, as shown in Table 7, in the 19-base probes shown in Examples 1 to 6, the ratios of fluorescence intensities derived from hybridization to W1, W2 and G12V DNA were all lower than 0.1. The value was sufficiently low. In contrast, the 20- or 21-base probes shown in Comparative Examples 1 to 6 showed a ratio of fluorescence intensities derived from hybridization to W1, W2 and G12V DNAs higher than 0.1. From these results, it was revealed that the 19-base probes shown in Examples 1 to 6 can detect G13D with higher accuracy.

Claims (14)

  An oligonucleotide probe for detecting a K-ras mutation comprising a region consisting of 6 bases corresponding to codons 12 and 13 of K-ras, and a total of 19 bases.   2. The oligonucleotide probe for detecting a K-ras mutation according to claim 1, wherein the region comprises a GGTGAC sequence corresponding to a codon 12 of a wild type and a codon 13 of a G13D mutant.   2. The oligonucleotide probe for detecting a K-ras mutation according to claim 1, wherein the region comprises a GGTGGC sequence corresponding to wild-type codon 12 and wild-type codon 13.   2. The oligonucleotide probe for detecting a K-ras mutation according to claim 1, wherein the region comprises a GATGGC sequence corresponding to the codon 12 of the G12D mutant type and the codon 13 of the wild type.   The oligonucleotide probe for detecting a K-ras mutation according to any one of claims 1 to 4, wherein the 5 'terminal side of the region is 2 to 11 bases.   The oligonucleotide probe for detecting a K-ras mutation according to any one of claims 1 to 4, wherein the 5 'terminal side of the region is 5 to 10 bases.   The oligonucleotide probe for detecting a K-ras mutation according to any one of claims 1 to 4, wherein the 5 'terminal side of the region is 7 to 10 bases. The oligonucleotide probe for detecting a K-ras mutation according to claim 2, comprising a single base sequence selected from the group consisting of the following (a) to (n).
(A) GGTGACGTAGGCAAGAGTG: SEQ ID NO: 1
(B) TGGTGACGTAGGCAAGAGT: SEQ ID NO: 2
(C) CTGGTGACGTAGGCAAGAG: SEQ ID NO: 3
(D) GCTGGTGACGTAGGCAAGA: SEQ ID NO: 4
(E) AGCTGGTGACGTAGGCAAG: SEQ ID NO: 5
(F) GAGCTGGTGACGTAGGCAA: SEQ ID NO: 6
(G) GGAGCTGGTGACGTAGGCA: SEQ ID NO: 7
(H) TGGAGCTGGTGACGTAGGC: SEQ ID NO: 8
(I) TTGGAGCTGGTGACGTAGG: SEQ ID NO: 9
(J) GTTGGAGCTGGTGACGTAG: SEQ ID NO: 10
(K) AGTTGGAGCTGGTGACGTA: SEQ ID NO: 11
(L) TAGTTGGAGCTGGTGACGT: SEQ ID NO: 12
(M) GTAGTTGGAGCTGGTGACG: SEQ ID NO: 13
(N) GGTAGTTGGAGCTGGTGAC: SEQ ID NO: 14   A microarray comprising a substrate and the oligonucleotide probe for detecting a K-ras mutation according to any one of claims 1 to 8, which is fixed to the substrate.   The microarray according to claim 9, wherein the 5 'end side of the oligonucleotide probe for detecting K-ras mutation is fixed to the substrate.   A region containing codons 12 and 13 of K-ras is amplified from a biological sample collected from a subject, and the hybridized nucleic acid and the oligonucleotide probe for detecting a K-ras mutation according to any one of claims 1 to 8. A method for detecting a mutation in K-ras, which detects soybean.   The mutation of K-ras according to claim 11, wherein when one of codon 12 and codon 13 of K-ras has a mutation, it is judged that the therapeutic effect of the anti-EGFR antibody drug on the subject is low. Detection method.   12. The method for detecting a mutation in K-ras according to claim 11, wherein the subject is a patient with advanced / recurrent colorectal cancer that cannot be cured and excised.   13. The method for detecting a mutation in K-ras according to claim 12, wherein the anti-EGFR antibody drug is cetuximab or panitumumab.