WO2017217530A1 - Method for measuring target nucleic acid containing modified nucleic acid base - Google Patents

Method for measuring target nucleic acid containing modified nucleic acid base Download PDF

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WO2017217530A1
WO2017217530A1 PCT/JP2017/022280 JP2017022280W WO2017217530A1 WO 2017217530 A1 WO2017217530 A1 WO 2017217530A1 JP 2017022280 W JP2017022280 W JP 2017022280W WO 2017217530 A1 WO2017217530 A1 WO 2017217530A1
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nucleic acid
target nucleic
solid phase
solution
capture probe
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PCT/JP2017/022280
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French (fr)
Japanese (ja)
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達樹 松野
公一 阿部
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富士レビオ株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention relates to a method for measuring a target nucleic acid containing a modified nucleobase.
  • modified nucleobase eg, methylcytosine
  • a method involving amplification of the target nucleic acid eg, bisulfite pyrosequencing method
  • a method not involving amplification of the target nucleic acid eg, modified nucleic acid
  • Immunoassays using antibodies against bases are known.
  • the modified nucleic acid base is measured after capturing the target nucleic acid on the solid phase using one nucleic acid probe (capture probe) used to capture the target nucleic acid on the solid phase.
  • the innumerable non-target nucleic acids have various nucleotide sequences, and therefore, non-target nucleic acids having a partial nucleotide sequence complementary to the nucleotide sequence of the capture probe are also included. It was considered that one of the causes of the high background signal was that the modified nucleobase included in the captured non-target nucleic acid was captured on the solid phase and the modified nucleobase was also measured. It was also found that the presence of non-target nucleic acid adsorbed nonspecifically on the solid phase contributed to a high background signal (Reference Example 3). Therefore, in such a nucleic acid sample, in order to measure the modified nucleobase contained in the target nucleic acid well, it was considered necessary to suppress the background signal derived from the non-target nucleic acid.
  • the present inventors have found that the above-mentioned problems can be solved by using a plurality of different capture probes, and the present invention is completed. It came to.
  • a method for measuring a target nucleic acid containing a modified nucleobase including: (1) immobilizing a target nucleic acid on a solid phase multiple times using a plurality of different capture probes for the target nucleic acid in a target nucleic acid containing a modified nucleobase and a non-target nucleic acid; and (2) a target nucleic acid Measuring the modified nucleobase contained in [2]
  • the method according to [1] wherein the nucleic acid sample is a genomic DNA sample.
  • nucleic acid sample contains the target nucleic acid in an amount of less than 20 amol of target nucleic acid per ⁇ g of non-target nucleic acid.
  • a capture probe having the ability to hybridize to the 5 ′ end region of the target nucleic acid and a capture probe having the ability to hybridize to the 3 ′ end region of the target nucleic acid are used in combination as the capture probe. [1] Any one of [3] methods.
  • [5] The method according to any one of [1] to [4], wherein (1) is performed by a method comprising: (I) capture of a target nucleic acid in a liquid phase onto a solid phase using a first capture probe for the target nucleic acid; (Ii) release of the target nucleic acid from the solid phase to the liquid phase; and (iii) capture of the target nucleic acid in the liquid phase to the solid phase using a second capture probe for the target nucleic acid.
  • [6] The method according to any one of [1] to [4], wherein (1) is performed by a method comprising: (I ′) capture of the target nucleic acid in the first liquid phase to the first solid phase using the first capture probe for the target nucleic acid; (Ii ′) exchange of the first liquid phase to the second liquid phase; (Iii ′) release of the target nucleic acid from the first solid phase to the second liquid phase; (Iv ′) exchange of the first solid phase to the second solid phase; (V ′) capture of the target nucleic acid in the second liquid phase to the second solid phase using the second capture probe for the target nucleic acid; and (vi ′) exchange of the second liquid phase to the third liquid phase.
  • [7] The method according to any one of [1] to [4], wherein (1) is performed by a method comprising: (1-1) (a) a target nucleic acid contained in a nucleic acid sample, (b) a first capture probe for the target nucleic acid labeled with a first affinity substance, and (c) specific to the first affinity substance
  • a first solid phase labeled with a substance having an ability to bind is reacted in a solution, and a first solution including a first solid phase to which a first nucleic acid hybrid including a target nucleic acid and the first capture probe is immobilized is prepared.
  • the first affinity substance is the same as the second affinity substance, and the first solid phase labeled with a substance capable of specifically binding to the first affinity substance is the second affinity substance.
  • the method of the present invention comprising immobilizing a target nucleic acid multiple times on a solid phase using a plurality of different capture probes for the target nucleic acid is a conventional method for measuring a modified nucleobase contained in a target nucleic acid using a single capture probe.
  • the modified nucleobase contained in the target nucleic acid can be measured more specifically.
  • the method of the present invention is excellent in that the modified nucleobase contained in the target nucleic acid can be specifically measured even when the relative amount of the target nucleic acid relative to the non-target nucleic acid is small in the nucleic acid sample.
  • the modified nucleobase contained in the target nucleic acid can be specifically measured without requiring amplification of the target nucleic acid. Therefore, the method of the present invention can avoid the use of equipment necessary for the amplification of the target nucleic acid, and has the advantage that the measurement time can be shortened in that the amplification of the target nucleic acid is not required.
  • FIG. 1 is a diagram showing a comparison of emission counts with and without a capture probe.
  • FIG. 2 shows a comparison of the effect of the conventional method using one capture probe and the method of the present invention using two different capture probes on the reduction of non-target nucleic acid derived signal (background signal).
  • FIG. 3 is a diagram showing a comparison between the amount of nucleic acid adsorbed on a solid phase by one capture (conventional method) and the amount of nucleic acid adsorbed on a solid phase (particle) by two captures (invention).
  • FIG. 4 is a diagram showing a hybridization site of each capture probe to a target nucleic acid in a combination of two capture probes.
  • FIG. 5 is a diagram showing detection of a modified nucleobase contained in a target nucleic acid in a mixed nucleic acid sample by a conventional method using one capture probe.
  • FIG. 6 shows the detection of modified nucleobases contained in target nucleic acids in a mixed nucleic acid sample by the method of the present invention using two different capture probes.
  • FIG. 7 is a diagram showing detection of a modified nucleobase contained in a target nucleic acid in an extracted nucleic acid sample by a conventional method using one capture probe.
  • Line graph measurement result by bisulfite pyrosequencing method (vertical axis: methylation rate); bar graph: measurement result by conventional method (vertical axis: luminescence count).
  • FIG. 8 is a diagram showing detection of modified nucleobases contained in a target nucleic acid in an extracted nucleic acid sample by the method of the present invention using two different capture probes. Details are the same as in FIG.
  • the present invention provides a method for measuring a target nucleic acid containing a modified nucleobase.
  • a target nucleic acid containing a modified nucleobase may be simply referred to as a target nucleic acid for omission.
  • the target nucleic acid is a natural or artificial nucleic acid in which nucleotide units are polymerized.
  • examples of the target nucleic acid include DNA and RNA, and DNA is preferable.
  • the target nucleic acid may also be a non-coding region (eg, transcriptional regulatory region) or a region containing it.
  • the number of nucleotide residues constituting the target nucleic acid is not particularly limited as long as it can hybridize with the capture probe. For example, 30 or more, 40 or more, 50 or more, 60 or more 70 or more, 80 or more, 90 or more, or 100 or more.
  • the number of nucleotides constituting the target nucleic acid may be any number that may be generated by fragmentation processing of genomic DNA.
  • the number of nucleotides constituting the target nucleic acid may be 10,000 or less, 5000 or less, 2000 or less, 1000 or less, 500 or less, or 200 or less.
  • the modified nucleobase is modified with respect to a normal nucleobase selected from the group consisting of adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U).
  • a nucleobase having a structure includes, for example, adenine (A), guanine (G), cytosine (C) and thymine (T) when the target nucleic acid is DNA. It is done.
  • the target nucleic acid when the target nucleic acid is RNA, examples thereof include adenine (A), guanine (G), cytosine (C), and uracil (U).
  • the nucleobase is preferably cytosine (C).
  • Modifications include, for example, introduction of substituents into normal nucleobases, elimination of groups (eg, amino group, oxo group, methyl group) possessed by ordinary nucleobases, substituents of groups possessed by ordinary nucleobases The exchange to is mentioned.
  • the substituent is not particularly limited as long as it can be possessed by a naturally occurring nucleobase.
  • the position of modification such as substitution is not particularly limited, but in the case of a nucleobase having a pyrimidine ring (C, T or U), for example, it is at the 2-position, 4- to 6-position, preferably at the 5-position, In the case of a nucleobase (A or G) having a purine ring, they are, for example, the 2nd, 6th and 8th positions. *
  • the modified nucleobase is not particularly limited as long as it can exist in nature.
  • Administrative Instructions under the Patent Cooperation Treaty (enforced on January 1, 2009), Annex C, Appendix 2, Table 2: Examples thereof include modified nucleobases possessed by modified nucleotides described in Modified Nucleotides.
  • the modified nucleotides described in this document may be the same as the modified nucleotides described in Appendix 2, Table 2: Modified Base Table of the above guidelines. Accordingly, reference can also be made to the above guidelines for modified nucleobases.
  • the modified nucleobase is methylcytosine (eg, 5-methylcytosine), hydroxymethylcytosine (eg, 5-hydroxymethylcytosine), carboxyl cytosine (eg, 5-carboxyl cytosine).
  • a modified nucleobase is known to cause a change in the function of the nucleic acid (eg, a change in the transcriptional regulatory ability of a given gene).
  • the method of the present invention includes: (1) immobilizing a target nucleic acid multiple times on a solid phase using a plurality of different capture probes for the target nucleic acid in a target nucleic acid containing a modified nucleobase and a non-target nucleic acid; and (2) a target Measuring modified nucleobases in nucleic acids.
  • the target nucleic acid is immobilized on the solid phase a plurality of times using a plurality of different capture probes for the target nucleic acid.
  • a non-target nucleic acid particularly a non-target nucleic acid containing a modified nucleobase that hinders measurement of the target nucleic acid (eg, a partial nucleotide sequence complementary to the nucleotide sequence of some of the plurality of capture probes)
  • Non-target nucleic acids containing modified nucleobases can be excluded.
  • a nucleic acid sample is any nucleic acid sample containing a target nucleic acid containing a modified nucleobase as described above and a non-target nucleic acid.
  • Non-target nucleic acids are natural or artificial nucleic acids in which nucleotide units are polymerized.
  • Non-target nucleic acids include, for example, non-target nucleic acids that include modified nucleobases and non-target nucleic acids that do not include modified nucleobases.
  • Non-target nucleic acids also include, for example, DNA and RNA, with DNA being preferred.
  • the method of the present invention is contained in the target nucleic acid even when the relative amount of the target nucleic acid relative to the non-target nucleic acid is small in the nucleic acid sample. It has been confirmed that modified nucleobases can be measured well. Therefore, the method of the present invention is particularly effective when the relative amount of the target nucleic acid with respect to the non-target nucleic acid is small.
  • the relative amount of the target nucleic acid relative to the non-target nucleic acid for which the method of the present invention is particularly effective is 20 amol (1000 amol target nucleic acid / 50 ⁇ g non-target nucleic acid; Example 3 and FIG. 5). Less).
  • a relative amount is an amount of 10 amol or less, 5 amol or less, 2 amol or less, 1 amol or less, or 0.5 amol or less as the number of moles of target nucleic acid per ⁇ g of non-target nucleic acid.
  • the number of moles of target DNA per 1 ⁇ g of non-target nucleic acid is about 0.5 amol.
  • nucleic acid samples include biological biological samples, environmental samples, and synthetic nucleic acid mixed samples.
  • organisms from which biological samples are derived include animals such as mammals (eg, humans, monkeys, mice, rats, rabbits, cows, pigs, horses, goats, sheep), birds (eg, chickens), insects, and the like. , Microorganisms, plants, fungi, and fish.
  • a biological sample can also be a blood-related sample (eg, whole blood, serum, plasma), saliva, urine, milk, tissue or cell extract, or a mixture thereof, which is the blood itself or a sample derived from blood. Also good.
  • the biological sample may further be derived from a mammal suffering from a disease (eg, cancer, leukemia) or a mammal potentially having a disease.
  • a disease eg, cancer, leukemia
  • environmental samples include samples derived from soil, seawater, and fresh water that may contain nucleic acids.
  • the mixed sample of synthetic nucleic acids include target nucleic acids (eg, DNA, RNA) containing artificially synthesized modified nucleobases, for which calibration of modified nucleobases is desired.
  • the nucleic acid sample is a genomic DNA sample.
  • the nucleic acid sample may be subjected to other treatment before the step (1).
  • treatment include extraction of nucleic acid (eg, DNA such as genomic DNA, RNA) and fragmentation (eg, treatment with an enzyme such as a restriction enzyme).
  • the methods of the invention may further comprise extracting nucleic acid from the nucleic acid sample and / or fragmenting the nucleic acid.
  • the method of the present invention may further comprise processing the sample by operations such as centrifugation, extraction, filtration, precipitation, heating, freezing, refrigeration, stirring and the like.
  • the capture probe used in the present invention refers to a nucleic acid probe that has the ability to hybridize with a target nucleic acid to form a nucleic acid hybrid and can be immobilized on a solid phase or is immobilized on a solid phase. .
  • the capture probe can be composed of the same type and / or different type of nucleic acid with respect to the target nucleic acid.
  • the term “same species” means that the capture probe has the same main-chain structure as the main-chain structure of the target nucleic acid (structure composed of a sugar moiety and a phosphate moiety) as a whole of the main-chain structure.
  • the term “heterologous” means that the capture probe has a main chain structure that is different from the main chain structure of the target nucleic acid (structure composed of a sugar moiety and a phosphate moiety) as a part or the whole of the main chain structure. means. Therefore, the type of capture probe may be determined according to the type of target nucleic acid.
  • a DNA probe can be used as a capture probe for homologous nucleic acids, and a nucleic acid probe other than a DNA probe can be used as a capture probe for heterologous nucleic acids.
  • a normal RNA probe composed of the same type of RNA as the natural RNA can be used as the same type of nucleic acid capture probe, and a normal RNA probe as a heterologous nucleic acid capture probe.
  • Other nucleic acid probes can be used.
  • the capture probe may be composed of a nucleic acid heterologous to the target nucleic acid.
  • capture probes include DNA probes, RNA probes, peptide nucleic acid (PNA) probes, lock nucleic acid (LNA) probes or cross-linked nucleic acid (BNA) probes, phosphorothioate (S) nucleic acid probes, and two or more such probes.
  • PNA peptide nucleic acid
  • LNA lock nucleic acid
  • BNA cross-linked nucleic acid
  • S phosphorothioate
  • a nucleic acid probe linked to each other a chimeric nucleic acid probe necessarily includes a nucleic acid heterologous to a target nucleic acid.
  • the capture probe is an RNA probe.
  • the RNA probe include a normal RNA probe composed of a natural ribonucleotide having a hydroxyl group at the 2 ′ position, and a modified RNA probe composed of a ribonucleotide modified with a hydroxyl group at the 2 ′ position.
  • the RNA probe is a modified RNA probe.
  • a ribonuclease resistant RNA probe may be used as the modified RNA probe.
  • modified RNA probes include 2'-O-alkylated RNA probes.
  • the 2'-O-alkylated RNA probe is preferably a 2'-O-C1-C6 alkylated RNA probe.
  • a C1-C6 alkyl group for C1-C6 alkylation is a linear, branched or cyclic alkyl group of 1-6 carbon atoms such as methyl, ethyl, propyl groups (eg, n-propyl, iso-propyl), butyl group (eg, n-butyl, iso-butyl, sec-butyl, tert-butyl), pentyl group, hexyl group and the like.
  • the 2'-O-C1-C6 alkylated RNA probe is a 2'-O-methylated RNA probe.
  • the number of nucleotide residues constituting the capture probe (that is, the length of the capture probe) is not particularly limited as long as it is long enough to hybridize with the target nucleic acid. For example, 12 or more, preferably 15 Or more, preferably 18 or more, more preferably 20 or more.
  • the number of nucleotides constituting the capture probe may also be, for example, 100 or less, 80 or less, 60 or less, or 50 or less.
  • Capture probes can be prepared by probe synthesis methods known in the art.
  • the capture probe When the capture probe is used in the step (1), it can be used in a free form or a form fixed on a solid phase.
  • the capture probe may be labeled with a substance or group that allows specific binding to the solid phase. Labeling can be done, for example, at either the 5 'end or the 3' end of the capture probe.
  • Substances or groups that enable specific binding to the solid phase include, for example, groups or substances that allow specific covalent binding to the solid phase, and specific affinity binding to the solid phase. Affinity substances to be mentioned.
  • Examples of the group or substance that enables specific covalent bonding to the solid phase include, for example, a thiol group or a substance having a thiol group (such a thiol group introduced into the capture probe is a maleimide group on the solid phase). ), An amino group, or a substance having an amino group (such an amino group introduced into a capture probe can bind to maleic anhydride on a solid phase).
  • Affinity substances that enable specific affinity binding to the solid phase include, for example, streptavidin, biotin, digoxigenin, dinitrophenol, fluorescein, fluorescein isothiocyanate, complementary sense strand and antisense strand One strand of a double-stranded nucleic acid molecule is mentioned.
  • the capture probe may be immobilized on a solid phase after hybridization.
  • the solid phase examples include a solid phase that can contain or mount a liquid phase (eg, a support such as a plate, a membrane, or a test tube, and a container such as a well plate, a microchannel, a glass capillary, a nanopillar, or a monolith column).
  • a liquid phase eg, a support such as a plate, a membrane, or a test tube, and a container such as a well plate, a microchannel, a glass capillary, a nanopillar, or a monolith column.
  • solid phases eg, particles
  • the solid phase material include glass, plastic, metal, and carbon.
  • a non-magnetic material or a magnetic material can also be used as the solid phase material, but a magnetic material is preferred from the viewpoint of ease of operation.
  • the solid phase is preferably a solid phase that can be dispersed in a liquid phase, more preferably particles, and even more preferably magnetic particles.
  • the number of capture probes used in the method of the present invention is not particularly limited as long as it is 2 or more, but is preferably 2, 3, 4, or 5, more preferably, 2 or 3 The number is particularly preferably two.
  • a plurality of capture probes may be used in combination with a capture probe capable of hybridizing to the 5 ′ end region of the target nucleic acid and a capture probe capable of hybridizing to the 3 ′ end region of the target nucleic acid. it can. By such combined use, the target nucleic acid can be measured more specifically.
  • the 5 ′ terminal region of the target nucleic acid is n by counting from the 5 ′ terminal nucleotide residue (first) in the target nucleic acid in the 5 ′ ⁇ 3 ′ direction when the total length of the target nucleic acid consists of n nucleotide residues. / Refers to the region up to the third nucleotide residue.
  • the 5 ′ terminal region of the target nucleic acid is n / 4, n / 5, and n / 6 from the 5 ′ terminal nucleotide residue (first) in the target nucleic acid in the 5 ′ ⁇ 3 ′ direction.
  • n / 3, n / 4, n / 5, n / 6, n / 7, n / 8, n / 9, or n / 10 has a value after the decimal point, the value after the decimal point is not considered .
  • the 5 ′ terminal region of the target nucleic acid is calculated from the 5 ′ terminal nucleotide residue (first) in the target nucleic acid in the 5 ′ ⁇ 3 ′ direction.
  • a region up to the 33rd nucleotide residue preferably a region up to the 25th, 20th, 16th, 14th, 12th, 11th, or 10th nucleotide residue.
  • the capture probe having the ability to hybridize to the 5 ′ end region of the target nucleic acid can hybridize to all or a part of the 5 ′ end region as described above to form a nucleic acid hybrid
  • the capture probe may further hybridize to the 3 'adjacent region of the 5' end region as long as it hybridizes to the whole or a part of the 5 'end region as described above.
  • the capture probe capable of hybridizing to the 5 ′ end region of the target nucleic acid hybridizes to all or only part of the 5 ′ end region and does not hybridize to the 3 ′ adjacent region of the 5 ′ end region. It may be a capture probe.
  • the 3 ′ terminal region of the target nucleic acid is the n / 3rd number from the nth nucleotide residue in the target nucleic acid in the 3 ′ ⁇ 5 ′ direction when the total length of the target nucleic acid consists of n nucleotide residues. Refers to the region up to nucleotide residues.
  • the 3 ′ terminal region of the target nucleic acid is n / 4, n / 5, and n / 6 from the 3 ′ terminal nucleotide residue (nth) in the target nucleic acid in the 3 ′ ⁇ 5 ′ direction.
  • n / 3, n / 4, n / 5, n / 6, n / 7, n / 8, n / 9, or n / 10 has a value after the decimal point, the value after the decimal point is not considered .
  • the 3 ′ terminal region of the target nucleic acid is calculated in the 3 ′ ⁇ 5 ′ direction from the 3 ′ terminal nucleotide residue (100th) in the target nucleic acid.
  • the region up to the 33rd nucleotide residue (in other words, the region consisting of the 68th to 100th nucleotide residues from the 5 ′ terminal nucleotide residue (first) in the target nucleic acid in the 5 ′ ⁇ 3 ′ direction)
  • the region up to the 25th, 20, 20, 14, 12, 11, or 10th nucleotide residue (in other words, the 5 ′ terminal nucleotide residue in the target nucleic acid ( From 1st) to 5 ' ⁇ 3', 76th, 81st, 85th, 87th, 89th, 90th, or 91st to 100th A region) consisting of a nucleotide residues.
  • the capture probe having the ability to hybridize to the 3 ′ terminal region of the target nucleic acid can hybridize to all or a part of the 3 ′ terminal region as described above to form a nucleic acid hybrid
  • the capture probe may further hybridize to the 5 'adjacent region of the 3' end region as long as it hybridizes to all or a part of the 3 'end region as described above.
  • a capture probe capable of hybridizing to the 3 ′ end region of the target nucleic acid hybridizes to all or only part of the 3 ′ end region and does not hybridize to the 5 ′ adjacent region of the 3 ′ end region. It may be a capture probe.
  • the capture of the target nucleic acid on the solid phase can be repeated twice or more depending on the number of different capture probes used. For example, if the number of different capture probes used is X, (capture target nucleic acid to solid phase) X times and (release target nucleic acid from solid phase to liquid phase) X-1 times (or X times, the Xth time is optional).
  • the method of the invention can be performed by a method comprising: (I) capture of a target nucleic acid in a liquid phase onto a solid phase using a first capture probe for the target nucleic acid; (Ii) release of the target nucleic acid from the solid phase to the liquid phase; and (iii) capture of the target nucleic acid in the liquid phase to the solid phase using a second capture probe for the target nucleic acid.
  • liquid phase water or an aqueous solvent such as a buffer solution (eg, Tris buffer solution or phosphate buffer solution) can be used.
  • a buffer solution eg, Tris buffer solution or phosphate buffer solution
  • a target nucleic acid containing a modified nucleobase and a nucleic acid sample containing a non-target nucleic acid can be used.
  • Capturing of a target nucleic acid in a liquid phase using a capture probe for the target nucleic acid is performed under any condition that can form a hybrid of the target nucleic acid and the capture probe.
  • any condition that can form a hybrid of the target nucleic acid and the capture probe.
  • by incubating the solid phase if the capture probe is used in free form not immobilized on the solid phase
  • Such conditions eg, salt concentration of solution, incubation temperature, incubation time
  • Such conditions eg, WO 2010/091870, WO 2006/121888, WO 2015/025862; International Publication 2015/025863; International Publication No. 2015/025864; International Publication No. 2015/108177).
  • Release of the target nucleic acid from the solid phase to the liquid phase can be performed by processing under any condition that allows the target nucleic acid to be transferred from the solid phase to the liquid phase.
  • treatments include, for example, pH adjusters (eg, acidic substances, alkaline substances, or solutions containing these substances), heating, competitors (eg, specific affinity binding to solid phases) Affinity substance, complementary nucleic acid of target nucleic acid), treatment with light.
  • the target nucleic acid is released in the form of a hybrid with the capture probe or in the form of the target nucleic acid alone, depending on factors such as the type of means for fixing the capture probe to the solid phase and the type of release treatment.
  • the first capture using the first capture probe not only the target nucleic acid but also a non-target nucleic acid having a partial nucleotide sequence complementary to the nucleotide sequence of the first capture probe can be captured on the solid phase.
  • the second capture using a second capture probe different from the first capture probe can capture only the target nucleic acid and avoid such non-target nucleic acid capture, thereby eliminating the non-target nucleic acid. it can.
  • the target nucleic acid captured for the second time may be released from the solid phase before measurement of the modified nucleobase contained in the target nucleic acid.
  • the method of the invention can be performed by a method comprising: (I ′) capture of the target nucleic acid in the first liquid phase to the first solid phase using the first capture probe for the target nucleic acid; (Ii ′) exchange of the first liquid phase to the second liquid phase; (Iii ′) release of the target nucleic acid from the first solid phase to the second liquid phase; (Iv ′) exchange of the first solid phase to the second solid phase; (V ′) capture of the target nucleic acid in the second liquid phase to the second solid phase using the second capture probe for the target nucleic acid; and (vi ′) exchange of the second liquid phase to the third liquid phase.
  • the first liquid phase is exchanged for the second liquid phase.
  • the first solid phase in which the target nucleic acid is captured is transferred from the first liquid phase to the second liquid phase.
  • the non-target nucleic acid contained in the first liquid phase that has not been captured by the first solid phase can be removed from the system.
  • the first solid phase is exchanged for the second solid phase.
  • the first solid phase is removed from the system and the second solid phase is charged to the system.
  • the non-target nucleic acid adsorbed nonspecifically on the first solid phase can be removed from the system.
  • the second liquid phase is exchanged with the third liquid phase.
  • the second solid phase in which the target nucleic acid is captured is transferred from the second liquid phase to the third liquid phase.
  • the non-target nucleic acid contained in the second liquid phase that was not captured by the second solid phase for example, the first solid phase that may have a partial nucleotide sequence complementary to the nucleotide sequence of the first capture probe
  • aqueous solvent such as a buffer solution (eg, Tris buffer solution or phosphate buffer solution) can be used as the liquid phase.
  • a target nucleic acid containing a modified nucleobase and a nucleic acid sample containing a non-target nucleic acid can be used as the liquid phase in (i ′).
  • a system including the second solid phase and the third liquid phase capturing the target nucleic acid is obtained.
  • the modified nucleobase contained in the target nucleic acid can be measured.
  • the target nucleic acid captured by the second solid phase may be released from the solid phase before measurement of the modified nucleobase contained in the target nucleic acid.
  • the modified nucleobase is measured in the target nucleic acid in the state captured by the second solid phase or released from the second solid phase (eg, in the form of a hybrid with the capture probe, or in the form of the target nucleic acid alone). be able to.
  • (1) can be performed by a method comprising: (1-1) (a) a target nucleic acid contained in a nucleic acid sample, (b) a first capture probe for the target nucleic acid labeled with a first affinity substance, and (c) specific to the first affinity substance A first solid phase labeled with a substance having an ability to bind is reacted in a solution, and a first solution including a first solid phase to which a first nucleic acid hybrid including a target nucleic acid and the first capture probe is immobilized is prepared.
  • the reaction can be performed by incubating (a) to (c) simultaneously or separately. Incubation is as described above.
  • a first solution including a first solid phase to which a first nucleic acid hybrid including a target nucleic acid and a first capture probe is fixed is obtained.
  • the first affinity substance and the substance having the ability to specifically bind to the first affinity substance the above-mentioned affinity substances can be used.
  • the reaction can be performed by preparing a solution containing (a), (b) and (c) and then incubating.
  • the reactions are performed separately, first, (a) and (b) are reacted to form a first nucleic acid hybrid, and then the formed first nucleic acid hybrid is reacted with (c) to form a target.
  • a first solid phase can be prepared on which a first nucleic acid hybrid comprising a nucleic acid and a first capture probe is immobilized.
  • first, (b) and (c) are reacted to form a first solid phase on which the capture probe is immobilized, and then this first solid phase is reacted with (a) to obtain the target nucleic acid and the first nucleic acid.
  • a first solid phase on which a first nucleic acid hybrid containing one capture probe is immobilized may be prepared.
  • the first solid phase on which the first nucleic acid hybrid including the target nucleic acid and the first capture probe is immobilized is transferred from the first solution to the second solution.
  • the non-target nucleic acid non-target nucleic acid contained in the first solution
  • such a first solid phase may be washed before transferring to the second solution.
  • the non-target nucleic acid contained in the 1st solution adhering to the 1st solid phase can further be removed. Washing can be performed one or more times (eg, 1 to 3 times) with a buffer solution (eg, Tris buffer solution, phosphate buffer solution) containing an appropriate surfactant.
  • a buffer solution eg, Tris buffer solution, phosphate buffer solution
  • the target nucleic acid can be released into the second solution by the treatment as described above.
  • the target nucleic acid is released into the second solution in the form of a hybrid with the capture probe or in a single form. Thereby, a first release solution containing the target nucleic acid and the first solid phase is obtained.
  • the first solid phase is removed from the first release solution.
  • a second release solution containing the target nucleic acid and not containing the first solid phase is obtained.
  • the non-target nucleic acid adsorbed on the first solid phase can be removed. Removal of the first solid phase from the first release solution can be performed in any manner that allows separation of the first release solution and the first solid phase.
  • the reaction can be performed by incubating (a ′) to (c ′) simultaneously or separately.
  • the details for carrying out the reaction simultaneously or separately are the same as in (1-1). Incubation is as described above.
  • a third solution containing the second solid phase on which the second nucleic acid hybrid containing the target nucleic acid and the second capture probe is immobilized is obtained.
  • the affinity substance as described above can be used as the second affinity substance and the substance having the ability to specifically bind to the second affinity substance.
  • the target nucleic acid can be specifically captured on the solid phase by using a capture probe different from the first capture probe as the second capture probe.
  • a capture probe different from the first capture probe as the second capture probe.
  • a capture probe having the ability to hybridize to the 5 ′ end region of the target nucleic acid and a capture probe having the ability to hybridize to the 3 ′ end region of the target nucleic acid By using in combination, the target nucleic acid can be specifically captured.
  • the second solid phase on which the second nucleic acid hybrid is immobilized is transferred from the third solution to the fourth solution.
  • the first solid phase which may have a partial nucleotide sequence complementary to the nucleotide sequence of the first capture probe (eg, the non-target nucleic acid not captured by the second solid phase) contained in the third solution.
  • Non-target nucleic acid) captured in the phase can be removed.
  • the second solid phase is taken out from the third solution, such a second solid phase may be washed before being transferred to the fourth solution.
  • the non-target nucleic acid contained in the 3rd solution adhering to the 2nd solid phase can further be removed. Washing can be performed one or more times (eg, 1 to 3 times) with a buffer solution (eg, Tris buffer solution, phosphate buffer solution) containing an appropriate surfactant.
  • a buffer solution eg, Tris buffer solution, phosphate buffer solution
  • aqueous solvent such as a buffer solution (eg, Tris buffer solution, phosphate buffer solution) can be used.
  • a target nucleic acid containing a modified nucleobase and a nucleic acid sample containing a non-target nucleic acid can be used.
  • a fourth solution containing the second solid phase on which the second nucleic acid hybrid is immobilized is obtained.
  • the modified nucleobase contained in the target nucleic acid can be measured by subjecting the fourth solution to the step (2) described later.
  • the target nucleic acid captured by the second solid phase may be released from the solid phase before measurement of the modified nucleobase contained in the target nucleic acid.
  • the modified nucleobase is measured in the target nucleic acid in the state captured by the second solid phase or released from the second solid phase (eg, in the form of a hybrid with the capture probe, or in the form of the target nucleic acid alone). be able to.
  • the modified nucleobase contained in the target nucleic acid can be measured by any method known in the art.
  • the measurement can be performed by a nucleic acid non-amplification analysis method or a nucleic acid amplification analysis method.
  • the nucleic acid non-amplification analysis method is an arbitrary analysis method that does not involve an amplification step of the target nucleic acid and does not increase the absolute amount of the target nucleic acid.
  • the non-amplification analysis method of nucleic acid include, for example, immunoassay (eg, International Publication No. 2015/025862; International Publication No. 2015/025863; International Publication No. 2015/025864; International Publication No.
  • the modified nucleobase contained in the target nucleic acid can be measured using an anti-modified nucleobase antibody.
  • the immunoassay can be performed by any immunological method known in the art. Specifically, as such a method, for example, enzyme immunoassay (EIA) (eg, chemiluminescence EIA (CLEIA), ELISA), fluorescence immunoassay, chemiluminescence immunoassay, electrochemiluminescence immunoassay Method, agglutination method, immunostaining, flowmetry method, biolayer interferometry, In Situ PLA method, chemically amplified luminescence proximity homogenous assay, line blot method, Western blot method.
  • EIA enzyme immunoassay
  • CLIA chemiluminescence EIA
  • ELISA enzyme immunoassay
  • fluorescence immunoassay eg, chemiluminescence EIA (CLEIA), ELISA
  • fluorescence immunoassay
  • the target nucleic acid is degraded by a nuclease (eg, endonuclease, exonuclease), and a monomer unit that is a degradation product of the target nucleic acid
  • a nuclease eg, endonuclease, exonuclease
  • a monomer unit that is a degradation product of the target nucleic acid
  • the modified nucleobase When the modified nucleobase is analyzed by nanopore analysis or micropore analysis, the target nucleic acid is released from the solid phase to obtain a solution containing the target nucleic acid, and then this solution is subjected to nanopore analysis or micropore analysis.
  • the modified nucleobase contained in the nucleic acid can be measured.
  • the nucleic acid amplification analysis method is an arbitrary analysis method capable of increasing the absolute amount of the target nucleic acid by the target nucleic acid amplification step. For example, by using one or more primers, the absolute amount of the target nucleic acid is expressed as an exponential function. This is a method of amplifying in an exponential or non-exponential manner.
  • nucleic acid amplification analysis methods include bisulfite sequencing, bisulfite pyrosequencing, methylation-specific PCR, single nucleotide primer extension (SNuPE), methylite, COBRA, and methylation. Specific MLPA method is mentioned. These methods are well known in the art (for example, JP 2012-090555 A, JP 2014-036672 A, International Publication No. 2009/037635).
  • the measurement can be performed by a non-amplifying analysis method of the target nucleic acid.
  • the non-target nucleic acid containing the modified nucleobase is specifically removed by (1), the target nucleic acid containing the modified nucleobase can be accurately identified without requiring amplification of the target nucleic acid. This is because it can be measured.
  • the method of the present invention can be carried out without requiring any target nucleic acid amplification step.
  • the present invention takes into account the amount and proportion of the desired nucleic acid in the nucleic acid sample (eg, the amount of target nucleic acid in the nucleic acid sample, or the amount of nucleic acid in the nucleic acid sample and the predicted target nucleic acid relative to the amount of nucleic acid).
  • the modification frequency of the target nucleic acid by the modified nucleobase may be evaluated.
  • Reference Example 1 Preparation of target nucleic acid containing methylcytosine Target nucleic acid was prepared by the following procedure.
  • the enzyme for PCR is KOD Plus (product number: KOD-201) manufactured by Toyobo Co., Ltd.
  • the two primers for nucleic acid amplification are artificially synthesized by Hokkaido System Science Co., Ltd.
  • Forward primer: 5′-TAG AAC GCT TTG CGT CCC GAC-3 ′ (SEQ ID NO: 1) and reverse primer: 5′-CTG CAG GAC CAC TCG AGG CTG-3 ′ (SEQ ID NO: 2) were used.
  • PCR amplification protocol after heating at 94 ° C. for 2 minutes, 94 ° C. for 15 seconds, 55 ° C. for 30 seconds, 68 ° C. for 1 minute, and 30 cycles.
  • Nucleic acid artificially synthesized by Hokkaido System Science Nucleotide sequence: 5'-TAG AAC GCT TTG CGT CCC GAC GCC CGC AGG TCC TCG CGG TGG GCG CCG TTT GCG ACT G
  • CTC TCC CTC CTC GGG ACG GTG GCA GCC TCG AGT GGT CCT GCA-3 ′ SEQ ID NO: 3
  • Capture probe 1 which is a nucleic acid probe for capturing a target nucleic acid is 5′-UGC AGG ACC ACU CGA GGC UGC CAC-3 ′ (SEQ ID NO: 4) (The main chain of the nucleic acid is 2′-O-methylated RNA, and the 5 ′ end is labeled with biotin), which was artificially synthesized by Hokkaido System Science. Salmon semen-derived genomic DNA (manufactured by Invitrogen) was used as a non-target nucleic acid.
  • Example 1 Reduction of signal derived from non-target nucleic acid by specific detection of modified nucleobase contained in target nucleic acid
  • the method of the present invention using two different capture probes as well as the conventional using one capture probe The method was evaluated for its effect on reducing non-target nucleic acid-derived signals (background signal).
  • Capture probe 1 is 5′-UGC AGG ACC ACU CGA GGC UGC CAC-3 ′ (sequence No. 4) (the main chain of the nucleic acid is 2′-O-methylated RNA, the 5 ′ end is labeled with biotin), the capture probe 2 is 5′-GUC GGG ACG CAA AGC GUU CUA-3 ′ (SEQ ID NO: 5) (nucleic acid No. 5)
  • the main chain was 2′-O-methylated RNA and the 3 ′ end was labeled with biotin, and was artificially synthesized by Hokkaido System Science.
  • the target nucleic acid containing 5-methylcytosine was prepared in Reference Example 1. Salmon semen-derived genomic DNA (manufactured by Invitrogen) was used as a non-target nucleic acid.
  • a target nucleic acid (100 amol) containing 5-methylcytosine or a genomic DNA derived from salmon semen (200 ⁇ g) and a capture probe 2 (1 pmol) for capturing the target nucleic acid are buffered (100 mM Tris-Cl, 1.5 M imidazole, 50 mM EDTA ⁇ 2Na) was dissolved in 100 ⁇ L. After reacting at 95 ° C. for 5 minutes, it was reacted at 37 ° C. for 30 minutes. Further, a solution not containing both the target nucleic acid containing 5-methylcytosine and the genomic DNA derived from salmon semen was prepared, and the same operation was performed.
  • Capture probe 1 is 5′-UGC AGG ACC ACU CGA GGC UGC CAC-3 ′ (SEQ ID NO: 4)
  • the main chain was 2′-O-methylated RNA and the 5 ′ end was labeled with biotin), which was artificially synthesized by Hokkaido System Science.
  • the target nucleic acid containing 5-methylcytosine was prepared in Reference Example 1. Salmon semen-derived genomic DNA (manufactured by Invitrogen) was used as a non-target nucleic acid.
  • target nucleic acid 100 amol
  • capture probe 1 1 pmol
  • 100 ⁇ L of buffer 100 mM Tris-Cl, 1.5 M imidazole, 50 mM EDTA ⁇ 2Na
  • a solution not containing both the target nucleic acid containing 5-methylcytosine and the genomic DNA derived from salmon semen was prepared, and the same operation was performed.
  • the method of the present invention using two different capture probes can specifically reduce the background signal derived from a non-target nucleic acid as compared with the conventional method using one capture probe.
  • Reference Example 3 Evaluation of Nucleic Acid Adsorption to the Solid Phase
  • the methodology of the present invention that captures the target nucleic acid twice on the solid phase (due to the use of two different capture probes) and the target nucleic acid to the solid phase
  • the amount of nucleic acid adsorbed to the solid phase was evaluated using a conventional methodology (captured by using one capture probe).
  • Evaluation of the amount of nucleic acid adsorbed to the solid phase by the methodology of the present invention was performed by measuring the amount of nucleic acid released from the solid phase after the second capture.
  • Evaluation of the amount of DNA adsorbed to the solid phase by a conventional methodology was performed by measuring the amount of nucleic acid released from the solid phase after the first capture.
  • the nucleic acid for examination used the one prepared in Reference Example 1.
  • the nucleic acid for study (100 amol) was dissolved in 100 ⁇ L of a buffer solution (100 mM Tris-Cl, 1.5 M imidazole, 50 mM EDTA ⁇ 2Na).
  • a buffer solution 100 mM Tris-Cl, 1.5 M imidazole, 50 mM EDTA ⁇ 2Na.
  • 50 ⁇ L of magnetic particles coated with 375 ⁇ g / mL streptavidin Magneticnussphere MS300 / Streptavidin, JSR
  • the reaction supernatant is neutralized by adding an equal amount of 50 mM HCl aqueous solution, and then real-time PCR amplification is performed using a Primer set designed to amplify the nucleic acid for study.
  • the amount of non-specifically adsorbed DNA on the magnetic particles was measured (evaluation of the amount of adsorbed DNA on the solid phase by a conventional methodology for capturing the target nucleic acid once on the solid phase).
  • 15 ⁇ L of the reaction supernatant was added to 85 ⁇ L of a buffer solution (100 mM Tris-Cl, 1.5 M imidazole, 50 mM EDTA ⁇ 2Na).
  • a buffer solution 100 mM Tris-Cl, 1.5 M imidazole, 50 mM EDTA ⁇ 2Na.
  • 50 ⁇ L of magnetic particles coated with 375 ⁇ g / mL streptavidin Magneticnussphere MS300 / Streptavidin, JSR
  • the reaction supernatant is neutralized by adding an equal amount of 50 mM HCl aqueous solution, and then real-time PCR amplification is performed using a Primer set designed to amplify the nucleic acid for study.
  • the amount of non-specifically adsorbed DNA on the magnetic particles was measured (evaluation of the amount of nucleic acid adsorbed to the solid phase by the methodology of the present invention in which the target nucleic acid was captured twice on the solid phase).
  • Premix PCR reagent KOD SYBR qPCR Mix: manufactured by TOYOBO
  • Forward Primer 10 ⁇ M
  • Reverse Primer 10 ⁇ M
  • 0.5 ⁇ L 50x ROX reference dye 0.05 ⁇ L DNA sample adsorbed on magnetic particles: 2 ⁇ L Total: 25 ⁇ L
  • the Primer set designed to amplify the nucleic acid to be studied has a Forward Primer nucleotide sequence of 5'-TAG AAC GCT TTG CGT CCC GAC-3 '(SEQ ID NO: 1), and a Reverse Primer nucleotide sequence of 5'- GAG AGC TCC GCA CTC TTC C-3 ′ (SEQ ID NO: 6), which was artificially synthesized by Hokkaido System Science, was used.
  • reaction steps (2) to (3) were repeated 50 cycles.
  • the methodology of the present invention in which the target nucleic acid is captured twice on the solid phase is compared with the methodology of the present invention in which the target nucleic acid is captured only once on the solid phase, and the amount of nucleic acid adsorbed nonspecifically on the solid phase is reduced. It was shown that it can be greatly reduced.
  • Capture probe 1 is 5′-UGC AGG ACC ACU CGA GGC UGC CAC-3 ′ (SEQ ID NO: 4) (Nucleic acid 2′-O-methylated RNA, 5 ′ end is labeled with biotin), and capture probe 2 is 5′-GUC GGG ACG CAA AGC GUU CUA-3 ′ (SEQ ID NO: 5) (SEQ ID NO: 5) '-O-methylated RNA, 3' end is labeled with biotin), capture probe 3 is 5'-ACC CAG ACA CUC ACC AAG UC-3 '(SEQ ID NO: 7) (nucleic acid main chain is 2'-O-methyl RNA, 5′-end is biotin-labeled) and was artificially synthesized by Hokkaido System Science.
  • the target nucleic acid containing 5-methylcytosine was
  • a target nucleic acid (100 amol or 1 fmol) containing 5-methylcytosine or salmon semen-derived genomic DNA (200 ⁇ g) and a capture probe (1 pmol; capture probe 2 or capture probe 3) are added to a buffer solution (100 mM Tris-Cl, 1.5 M).
  • a buffer solution 100 mM Tris-Cl, 1.5 M.
  • Imidazole, 50 mM EDTA ⁇ 2Na was dissolved in 100 ⁇ L. After reacting at 95 ° C. for 5 minutes, it was reacted at 37 ° C. for 30 minutes. Further, a solution not containing both the target nucleic acid containing 5-methylcytosine and the genomic DNA derived from salmon semen was prepared, and the same operation was performed.
  • the entire amount of the reaction supernatant was added to 85 ⁇ L of a buffer solution (100 mM Tris-Cl, 1.5 M imidazole, 50 mM EDTA ⁇ 2Na) containing a capture probe (1 pmol; capture probe 1 or capture probe 3). added.
  • a buffer solution 100 mM Tris-Cl, 1.5 M imidazole, 50 mM EDTA ⁇ 2Na
  • This reaction solution was reacted at 95 ° C. for 5 minutes and then reacted at 37 ° C. for 30 minutes.
  • 50 ⁇ L of magnetic particles coated with 250 ⁇ g / mL streptavidin (in-house preparation) were added to the solution after the reaction and reacted at 37 ° C. for 10 minutes to immobilize the capture probe on the magnetic particles.
  • the luminescence count was different depending on the combination of two different capture probes, and the luminescence count was the highest when the two capture probes were designed to hybridize to both end regions of the target nucleic acid (Table 4, FIG. 4). .
  • Example 3 Specific Detection of Modified Nucleobases Included in Target Nucleic Acids in Mixed Nucleic Acid Samples of Target and Non-Target Nucleic Acids
  • Two different nucleic acid samples obtained by mixing target and non-target nucleic acids It was investigated whether the method of the present invention using a capture probe can specifically detect a modified nucleobase contained in a target nucleic acid.
  • Capture probe 1 is 5′-UGC AGG ACC ACU CGA GGC UGC CAC-3 ′ (SEQ ID NO: 4) (nucleic acid main chain is 2′-O-methylated RNA, 5 ′ end is labeled with biotin), capture probe 2 is 5'-GUC GGG ACG CAA AGC GUU CUA-3 '(SEQ ID NO: 5) (nucleic acid main chain is 2'-O-methylated RNA, 3' end is labeled with biotin), artificially synthesized by Hokkaido System Science We used what was done.
  • the target nucleic acid containing 5-methylcytosine was prepared in Reference Example 1. Salmon semen-derived genomic DNA (manufactured by Invitrogen) was used as a non-target nucleic acid.
  • target nucleic acid (10 amol, 100 amol, or 1 fmol) containing 5-methylcytosine and / or salmon semen-derived genomic DNA (50 ⁇ g) and capture probe 2 (1 pmol) are added to a buffer (100 mM Tris-Cl, 1.5 M imidazole, 50 mM EDTA ⁇ 2Na) was dissolved in 100 ⁇ L. After reacting at 95 ° C. for 5 minutes, it was reacted at 37 ° C. for 30 minutes. Further, a solution not containing both the target nucleic acid containing 5-methylcytosine and the genomic DNA derived from salmon semen was prepared, and the same operation was performed.
  • a buffer 100 mM Tris-Cl, 1.5 M imidazole, 50 mM EDTA ⁇ 2Na
  • the entire amount of the reaction supernatant was added to 85 ⁇ L of a buffer solution (100 mM Tris-Cl, 1.5 M imidazole, 50 mM EDTA ⁇ 2Na) containing capture probe 1 (1 pmol).
  • a buffer solution 100 mM Tris-Cl, 1.5 M imidazole, 50 mM EDTA ⁇ 2Na
  • capture probe 1 1 pmol.
  • This reaction solution was reacted at 95 ° C. for 5 minutes and then reacted at 37 ° C. for 30 minutes.
  • 50 ⁇ L of magnetic particles coated with 250 ⁇ g / mL streptavidin (in-house preparation) were added to the solution after the reaction and reacted at 37 ° C. for 10 minutes to immobilize the capture probe 1 on the magnetic particles.
  • the modified nucleobase contained in the target nucleic acid was also measured by a conventional method using one capture probe.
  • target nucleic acid (10 amol, 100 amol, or 1 fmol) and / or salmon semen-derived genomic DNA (50 ⁇ g) containing 5-methylcytosine and capture probe 1 (1 pmol) are added to a buffer (100 mM Tris-Cl , 1.5 M imidazole, 50 mM EDTA ⁇ 2Na) in 100 ⁇ L. After reacting at 95 ° C. for 5 minutes, it was reacted at 37 ° C. for 30 minutes.
  • a buffer 100 mM Tris-Cl , 1.5 M imidazole, 50 mM EDTA ⁇ 2Na
  • a solution not containing both the target nucleic acid containing 5-methylcytosine and the genomic DNA derived from salmon semen was prepared, and the same operation was performed. 50 ⁇ L of magnetic particles coated with 250 ⁇ g / mL streptavidin (in-house preparation) were added to the solution after the reaction and reacted at 37 ° C. for 10 minutes to immobilize the capture probe 1 on the magnetic particles.
  • the luminescence count measured in the mixed nucleic acid sample of the target nucleic acid and the non-target nucleic acid is higher than that measured in the nucleic acid sample of the target nucleic acid not containing the non-target nucleic acid, and the target against the non-target nucleic acid
  • the luminescence count tended to increase significantly (Table 5, FIG. 5).
  • the luminescence count measured in the mixed nucleic acid sample of the target nucleic acid and the non-target nucleic acid is the nucleic acid sample of the target nucleic acid not containing the non-target nucleic acid regardless of the relative amount of the target nucleic acid with respect to the non-target nucleic acid. (Table 5, FIG. 6).
  • the background signal of the non-target nucleic acid is greatly reduced, It was shown that the modified nucleobase contained in can be detected.
  • the process of the present invention has been shown to be particularly superior.
  • Example 4 Specific Detection of Modified Nucleobases Included in Target Nucleic Acids in Natural Biological Samples
  • a nucleic acid sample containing target and non-target nucleic acids extracted from cells that are natural biological samples It was investigated whether the method of the present invention using two different capture probes could specifically detect the modified nucleobase contained in the target nucleic acid.
  • the method of the present invention and the conventional method using one capture probe were compared with a known method, bisulfite pyrosequencing method (analysis method involving amplification of target nucleic acid).
  • Capture probe 1 is 5′-UGC AGG ACC ACU CGA GGC UGC CAC-3 ′ (SEQ ID NO: 4) (nucleic acid main chain is 2′-O-methylated RNA, 5 ′ end is labeled with biotin), capture probe 2 is 5'-GUC GGG ACG CAA AGC GUU CUA-3 '(SEQ ID NO: 5) (nucleic acid main chain is 2'-O-methylated RNA, 3' end is labeled with biotin), artificially synthesized by Hokkaido System Science We used what was done.
  • genomic DNA derived from cultured cells corresponding to 3E + 7 copies, 12 units of restriction enzyme PstI (manufactured by Takara Bio Inc.), and 48 units of restriction enzyme XspI (manufactured by Takara Bio Inc.) were reacted with Buffer (20 mM Tris-HCl (pH 8.5)). (10 mM MgCl 2, 1 mM DTT, 100 mM KCl) dissolved in 80 ⁇ L, reacted at 37 ° C. for 24 hours, and then reacted at 80 ° C. for 10 minutes to obtain genomic DNA cleaved by the restriction enzyme.
  • restriction enzyme-cut genomic DNA and capture probe 2 (1 pmol) for capturing the target nucleic acid were dissolved in 160 ⁇ L of a buffer solution (100 mM Tris-Cl, 1.5 M imidazole, 50 mM EDTA ⁇ 2Na). After reacting at 95 ° C. for 5 minutes, it was reacted at 37 ° C. for 30 minutes. A reaction solution not containing genomic DNA was also prepared and the same operation was performed. 50 ⁇ L of magnetic particles (JSR Magnosphere MS300 / Streptavidin) coated with 375 ⁇ g / mL streptavidin are added to the solution after the reaction and reacted at 37 ° C. for 10 minutes to immobilize the capture probe 2 on the magnetic particles. did.
  • a buffer solution 100 mM Tris-Cl, 1.5 M imidazole, 50 mM EDTA ⁇ 2Na
  • the modified nucleobase contained in the target nucleic acid was also measured by a conventional method using one capture probe.
  • restriction enzyme-cut genomic DNA and capture probe 1 (1 pmol) for capturing the target nucleic acid were dissolved in 160 ⁇ L of a buffer (100 mM Tris-Cl, 1.5 M imidazole, 50 mM EDTA ⁇ 2Na). After reacting at 95 ° C. for 5 minutes, it was reacted at 37 ° C. for 30 minutes. A reaction solution not containing genomic DNA was also prepared and the same operation was performed. 50 ⁇ L of magnetic particles coated with 250 ⁇ g / mL streptavidin (in-house preparation) were added to the solution after the reaction and reacted at 37 ° C. for 10 minutes to immobilize the capture probe 1 on the magnetic particles.
  • a buffer 100 mM Tris-Cl, 1.5 M imidazole, 50 mM EDTA ⁇ 2Na
  • the bisulfite pyrosequencing method was performed according to a previously reported method (Science 1998, 281, 363-365, Electrophoresis 2002, 23, 24, 4072-4079).
  • the luminescence count measured by the conventional method did not necessarily correlate with the measurement result of the bisulfite pyrosequencing method (Table 6, FIG. 7).
  • the method of the present invention correlated with the measurement result of the bisulfite pyrosequencing method (Table 6, FIG. 8).
  • the method of the present invention using two different capture probes does not involve amplification of the target nucleic acid, and at least the same analysis accuracy as the method involving amplification of the target nucleic acid, the target nucleic acid in the natural biological sample It was shown that the modified nucleobase contained in can be measured.
  • the method of the present invention can be used in fields such as diagnosis and research.

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Abstract

In a method for measuring, by using capture probes, the amounts of modified bases contained in target nucleic acids in a nucleic acid sample that contains countless non-target nucleic acids and the target nucleic acids containing the modified nucleic acid bases, the present invention relates to a method for specifically measuring the amounts of the modified nucleic acid bases contained in the target nucleic acids. In more detail, the present invention relates to a method for measuring target nucleic acids containing modified nucleic acid bases, including: (1) securing, in a nucleic acid sample that contains countless non-target nucleic acids and the target nucleic acids containing the modified nucleic acid bases, the target nucleic acids multiple times in a solid phase by using multiple types of different capture probes for the target nucleic acids; and (2) measuring the amounts of the modified nucleic acid bases contained in the target nucleic acids.

Description

修飾核酸塩基を含む標的核酸の測定方法Method for measuring target nucleic acid containing modified nucleobase
　本発明は、修飾核酸塩基を含む標的核酸の測定方法に関する。 The present invention relates to a method for measuring a target nucleic acid containing a modified nucleobase.
　標的核酸に含まれる修飾核酸塩基（例、メチルシトシン）の測定として、標的核酸の増幅を伴う方法（例、バイサルファイトパイロシーケンシング法）、および標的核酸の増幅を伴わない方法（例、修飾核酸塩基に対する抗体を用いるイムノアッセイ）が知られている。標的核酸の増幅を伴わない従来法では、標的核酸を固相に捕捉するために用いられる核酸プローブ（捕捉プローブ）を１つ用いて固相に標的核酸を捕捉した後に、修飾核酸塩基が測定されている（特許文献１、２、非特許文献１～３）。 As a measurement of modified nucleobase (eg, methylcytosine) contained in the target nucleic acid, a method involving amplification of the target nucleic acid (eg, bisulfite pyrosequencing method) and a method not involving amplification of the target nucleic acid (eg, modified nucleic acid) Immunoassays using antibodies against bases) are known. In the conventional method that does not involve amplification of the target nucleic acid, the modified nucleic acid base is measured after capturing the target nucleic acid on the solid phase using one nucleic acid probe (capture probe) used to capture the target nucleic acid on the solid phase. (Patent Documents 1 and 2, Non-Patent Documents 1 to 3).
特開２０１２－２３００１９号公報JP2012-230019A 特開２０１４－１７６３３０号公報JP 2014-176330 A
　本発明者らは、修飾核酸塩基を含む標的核酸、および非標的核酸を含む核酸サンプルにおいて、１つの捕捉プローブを用いて標的核酸に含まれる修飾核酸塩基を測定する方法では、捕捉プローブの非標的核酸への結合によりバックグランドシグナルが高くなり、標的核酸に含まれる修飾核酸塩基を良好に測定し得ないという課題があることを見出した（参考例２）。具体的には、核酸サンプルに無数の非標的核酸が含まれる場合、無数の非標的核酸は多様なヌクレオチド配列を有するため、捕捉プローブのヌクレオチド配列と相補的な部分ヌクレオチド配列を有する非標的核酸も固相に捕捉され、この捕捉された非標的核酸に含まれる修飾核酸塩基も測定されてしまうことが高いバックグランドシグナルの一因と考えられた。
　また、固相上に非特異的に吸着した非標的核酸の存在が高いバックグラウンドシグナルの一因であることも見出された（参考例３）。
　したがって、このような核酸サンプルにおいて、標的核酸に含まれる修飾核酸塩基を良好に測定するためには、非標的核酸由来のバックグラウンドシグナルを抑制する必要があると考えられた。 In the method of measuring a modified nucleobase contained in a target nucleic acid using one capture probe in a target nucleic acid containing a modified nucleobase and a nucleic acid sample containing a non-target nucleic acid, the present inventors It has been found that there is a problem that the background signal increases due to binding to the nucleic acid, and the modified nucleobase contained in the target nucleic acid cannot be measured well (Reference Example 2). Specifically, when the nucleic acid sample contains an infinite number of non-target nucleic acids, the innumerable non-target nucleic acids have various nucleotide sequences, and therefore, non-target nucleic acids having a partial nucleotide sequence complementary to the nucleotide sequence of the capture probe are also included. It was considered that one of the causes of the high background signal was that the modified nucleobase included in the captured non-target nucleic acid was captured on the solid phase and the modified nucleobase was also measured.
It was also found that the presence of non-target nucleic acid adsorbed nonspecifically on the solid phase contributed to a high background signal (Reference Example 3).
Therefore, in such a nucleic acid sample, in order to measure the modified nucleobase contained in the target nucleic acid well, it was considered necessary to suppress the background signal derived from the non-target nucleic acid.
　本発明者らは、本発明者らにより見出された上記課題を解決するため鋭意検討した結果、複数の異なる捕捉プローブを用いることなどにより、上記課題を解決できることを見出し、本発明を完成するに至った。 As a result of intensive studies to solve the above-mentioned problems found by the present inventors, the present inventors have found that the above-mentioned problems can be solved by using a plurality of different capture probes, and the present invention is completed. It came to.
　すなわち、本発明は、以下のとおりである。
〔１〕以下を含む、修飾核酸塩基を含む標的核酸の測定方法：
（１）修飾核酸塩基を含む標的核酸、および非標的核酸を含む核酸サンプルにおいて、標的核酸に対する複数の異なる捕捉プローブを用いて標的核酸を固相に複数回固定すること；ならびに
（２）標的核酸に含まれる修飾核酸塩基を測定すること。
〔２〕前記核酸サンプルがゲノムＤＮＡサンプルである、〔１〕の方法。
〔３〕前記核酸サンプルが、非標的核酸１μｇ当たりの標的核酸モル数が２０ａｍｏｌ未満の量において標的核酸を含む、〔１〕または〔２〕の方法。
〔４〕前記捕捉プローブとして、標的核酸の５’末端領域にハイブリダイズする能力を有する捕捉プローブ、および標的核酸の３’末端領域にハイブリダイズする能力を有する捕捉プローブが併用される、〔１〕～〔３〕のいずれかの方法。
〔５〕以下を含む方法により（１）が行われる、〔１〕～〔４〕のいずれかの方法：
（ｉ）標的核酸に対する第１捕捉プローブを用いた液相中の標的核酸の固相への捕捉；
（ｉｉ）固相から液相への標的核酸の放出；および
（ｉｉｉ）標的核酸に対する第２捕捉プローブを用いた液相中の標的核酸の固相への捕捉。
〔６〕以下を含む方法により（１）が行われる、〔１〕～〔４〕のいずれかの方法：
（ｉ’）標的核酸に対する第１捕捉プローブを用いた第１液相中の標的核酸の第１固相への捕捉；
（ｉｉ’）第１液相の第２液相への交換；
（ｉｉｉ’）第１固相から第２液相への標的核酸の放出；
（ｉｖ’）第１固相の第２固相への交換；
（ｖ’）標的核酸に対する第２捕捉プローブを用いた第２液相中の標的核酸の第２固相への捕捉；および
（ｖｉ’）第２液相の第３液相への交換。
〔７〕以下を含む方法により（１）が行われる、〔１〕～〔４〕のいずれかの方法：
（１－１）（ａ）核酸サンプルに含まれる標的核酸、（ｂ）第１親和性物質で標識された、標的核酸に対する第１捕捉プローブ、および（ｃ）第１親和性物質と特異的に結合する能力を有する物質で標識された第１固相を溶液中で反応させて、標的核酸および前記第１捕捉プローブを含む第１核酸ハイブリッドが固定された第１固相を含む第１溶液を得ること；
（１－２）前記第１核酸ハイブリッドが固定された第１固相を、第１溶液から第２溶液に移すこと；
（１－３）前記第１核酸ハイブリッドが固定された第１固相から標的核酸を第２溶液中に放出させて、標的核酸および第１固相を含む第１放出溶液を得ること；
（１－４）第１固相を第１放出溶液から除去して、標的核酸を含み、かつ第１固相を含まない第２放出溶液を得ること；ならびに
（１－５）（ａ’）前記第２放出溶液に含まれる標的核酸、（ｂ’）第２親和性物質で標識された、標的核酸に対する第２捕捉プローブ、および（ｃ’）第２親和性物質と特異的に結合する能力を有する物質で標識された第２固相を反応させて、標的核酸および前記第２捕捉プローブを含む第２核酸ハイブリッドが固定された第２固相を含む第３溶液を得ること；
（１－６）前記第２核酸ハイブリッドが固定された第２固相を、前記第３溶液から第４溶液に移すこと。
〔８〕以下をさらに含む、〔７〕の方法：
（１ａ）（１－２）において、前記第１固相を前記第１溶液から第２溶液に移すときに、前記第１核酸ハイブリッドが固定された第１固相を洗浄すること；
（１ｂ）（１－６）において、前記第２固相を前記第３溶液から第４溶液に移すときに、前記第２核酸ハイブリッドが固定された第２固相を洗浄すること；
（１ｃ）（１ａ）および（１ｂ）の双方を行うこと。
〔９〕第１親和性物質が第２親和性物質と同じであり、かつ、第１親和性物質と特異的に結合する能力を有する物質で標識された第１固相が第２親和性物質と特異的に結合する能力を有する物質で標識された第２固相と同じである、〔７〕または〔８〕の方法。
〔１０〕固相が磁性粒子である、〔１〕～〔９〕のいずれかの方法。
〔１１〕修飾核酸塩基がメチルシトシンである、〔１〕～〔１０〕のいずれかの方法。
〔１２〕前記測定が標的核酸の非増幅的解析方法により行われる、〔１〕～〔１１〕のいずれかの方法。
〔１３〕前記非増幅的解析方法が、イムノアッセイ、質量分析、電気化学分析、高速液体クロマトグラフィー分析、ナノポア分析またはマイクロポア分析である、〔１１〕の方法。 That is, the present invention is as follows.
[1] A method for measuring a target nucleic acid containing a modified nucleobase, including:
(1) immobilizing a target nucleic acid on a solid phase multiple times using a plurality of different capture probes for the target nucleic acid in a target nucleic acid containing a modified nucleobase and a non-target nucleic acid; and (2) a target nucleic acid Measuring the modified nucleobase contained in
[2] The method according to [1], wherein the nucleic acid sample is a genomic DNA sample.
[3] The method according to [1] or [2], wherein the nucleic acid sample contains the target nucleic acid in an amount of less than 20 amol of target nucleic acid per μg of non-target nucleic acid.
[4] A capture probe having the ability to hybridize to the 5 ′ end region of the target nucleic acid and a capture probe having the ability to hybridize to the 3 ′ end region of the target nucleic acid are used in combination as the capture probe. [1] Any one of [3] methods.
[5] The method according to any one of [1] to [4], wherein (1) is performed by a method comprising:
(I) capture of a target nucleic acid in a liquid phase onto a solid phase using a first capture probe for the target nucleic acid;
(Ii) release of the target nucleic acid from the solid phase to the liquid phase; and (iii) capture of the target nucleic acid in the liquid phase to the solid phase using a second capture probe for the target nucleic acid.
[6] The method according to any one of [1] to [4], wherein (1) is performed by a method comprising:
(I ′) capture of the target nucleic acid in the first liquid phase to the first solid phase using the first capture probe for the target nucleic acid;
(Ii ′) exchange of the first liquid phase to the second liquid phase;
(Iii ′) release of the target nucleic acid from the first solid phase to the second liquid phase;
(Iv ′) exchange of the first solid phase to the second solid phase;
(V ′) capture of the target nucleic acid in the second liquid phase to the second solid phase using the second capture probe for the target nucleic acid; and (vi ′) exchange of the second liquid phase to the third liquid phase.
[7] The method according to any one of [1] to [4], wherein (1) is performed by a method comprising:
(1-1) (a) a target nucleic acid contained in a nucleic acid sample, (b) a first capture probe for the target nucleic acid labeled with a first affinity substance, and (c) specific to the first affinity substance A first solid phase labeled with a substance having an ability to bind is reacted in a solution, and a first solution including a first solid phase to which a first nucleic acid hybrid including a target nucleic acid and the first capture probe is immobilized is prepared. Getting;
(1-2) transferring the first solid phase on which the first nucleic acid hybrid is immobilized from the first solution to the second solution;
(1-3) obtaining a first release solution containing the target nucleic acid and the first solid phase by releasing the target nucleic acid into the second solution from the first solid phase to which the first nucleic acid hybrid is fixed;
(1-4) removing the first solid phase from the first release solution to obtain a second release solution containing the target nucleic acid and not containing the first solid phase; and (1-5) (a ′) A target nucleic acid contained in the second release solution; (b ′) a second capture probe for the target nucleic acid labeled with a second affinity substance; and (c ′) an ability to specifically bind to the second affinity substance. Reacting a second solid phase labeled with a substance having: to obtain a third solution comprising a second solid phase to which a second nucleic acid hybrid comprising a target nucleic acid and the second capture probe is immobilized;
(1-6) Transfer the second solid phase on which the second nucleic acid hybrid is immobilized from the third solution to the fourth solution.
[8] The method of [7], further comprising:
(1a) In (1-2), when transferring the first solid phase from the first solution to the second solution, washing the first solid phase to which the first nucleic acid hybrid is immobilized;
(1b) In (1-6), when transferring the second solid phase from the third solution to the fourth solution, washing the second solid phase to which the second nucleic acid hybrid is immobilized;
(1c) Perform both (1a) and (1b).
[9] The first affinity substance is the same as the second affinity substance, and the first solid phase labeled with a substance capable of specifically binding to the first affinity substance is the second affinity substance. The method according to [7] or [8], which is the same as the second solid phase labeled with a substance having the ability to specifically bind to.
[10] The method according to any one of [1] to [9], wherein the solid phase is a magnetic particle.
[11] The method according to any one of [1] to [10], wherein the modified nucleobase is methylcytosine.
[12] The method according to any one of [1] to [11], wherein the measurement is performed by a non-amplifying analysis method of the target nucleic acid.
[13] The method according to [11], wherein the non-amplification analysis method is immunoassay, mass spectrometry, electrochemical analysis, high performance liquid chromatography analysis, nanopore analysis, or micropore analysis.
　標的核酸に対する複数の異なる捕捉プローブを用いて標的核酸を固相に複数回固定することを含む本発明の方法は、１つの捕捉プローブを用いて標的核酸に含まれる修飾核酸塩基を測定する従来法に比し、標的核酸に含まれる修飾核酸塩基をより特異的に測定することができる。特に、本発明の方法は、核酸サンプルにおいて非標的核酸に対する標的核酸の相対量が少ない場合であっても、標的核酸に含まれる修飾核酸塩基を特異的に測定することができる点で優れる。
　また、本発明の方法によれば、標的核酸の増幅を要することなく、標的核酸に含まれる修飾核酸塩基を特異的に測定することができる。したがって、本発明の方法は、標的核酸の増幅に必要な機器の使用を回避でき、また、標的核酸の増幅を要しない点で測定時間を短縮できるという利点も有する。 The method of the present invention comprising immobilizing a target nucleic acid multiple times on a solid phase using a plurality of different capture probes for the target nucleic acid is a conventional method for measuring a modified nucleobase contained in a target nucleic acid using a single capture probe. Compared to the above, the modified nucleobase contained in the target nucleic acid can be measured more specifically. In particular, the method of the present invention is excellent in that the modified nucleobase contained in the target nucleic acid can be specifically measured even when the relative amount of the target nucleic acid relative to the non-target nucleic acid is small in the nucleic acid sample.
Moreover, according to the method of the present invention, the modified nucleobase contained in the target nucleic acid can be specifically measured without requiring amplification of the target nucleic acid. Therefore, the method of the present invention can avoid the use of equipment necessary for the amplification of the target nucleic acid, and has the advantage that the measurement time can be shortened in that the amplification of the target nucleic acid is not required.
図１は、捕捉プローブの有無における発光カウントの比較を示す図である。FIG. 1 is a diagram showing a comparison of emission counts with and without a capture probe. 図２は、非標的核酸由来シグナル（バックグラウンドシグナル）の低減に対する、１つの捕捉プローブを用いる従来法、および２つの異なる捕捉プローブを用いる本発明の方法の効果の比較を示す図である。FIG. 2 shows a comparison of the effect of the conventional method using one capture probe and the method of the present invention using two different capture probes on the reduction of non-target nucleic acid derived signal (background signal). 図３は、１回の捕捉により固相に吸着した核酸量（従来法）、および２回の捕捉により固相（粒子）に吸着した核酸量（本発明）の比較を示す図である。FIG. 3 is a diagram showing a comparison between the amount of nucleic acid adsorbed on a solid phase by one capture (conventional method) and the amount of nucleic acid adsorbed on a solid phase (particle) by two captures (invention). 図４は、２つの捕捉プローブの組み合わせにおける、標的核酸に対する各捕捉プローブのハイブリダイズ部位を示す図である。FIG. 4 is a diagram showing a hybridization site of each capture probe to a target nucleic acid in a combination of two capture probes. 図５は、１つの捕捉プローブを用いる従来法による、混合核酸サンプル中の標的核酸に含まれる修飾核酸塩基の検出を示す図である。FIG. 5 is a diagram showing detection of a modified nucleobase contained in a target nucleic acid in a mixed nucleic acid sample by a conventional method using one capture probe. 図６は、２つの異なる捕捉プローブを用いる本発明の方法による、混合核酸サンプル中の標的核酸に含まれる修飾核酸塩基の検出を示す図である。FIG. 6 shows the detection of modified nucleobases contained in target nucleic acids in a mixed nucleic acid sample by the method of the present invention using two different capture probes. 図７は、１つの捕捉プローブを用いる従来法による、抽出核酸サンプル中の標的核酸に含まれる修飾核酸塩基の検出を示す図である。折れ線グラフ：バイサルファイトパイロシーケンシング法による測定結果（縦軸：メチル化率）；棒グラフ：従来法による測定結果（縦軸：発光カウント）。Ａ：培養細胞Ａ１７２；Ｕ：培養細胞Ｕ８７－ＭＧ；Ｔ：培養細胞Ｔ４７Ｄ；ＤＷ：蒸留水（ネガティブコントロール）FIG. 7 is a diagram showing detection of a modified nucleobase contained in a target nucleic acid in an extracted nucleic acid sample by a conventional method using one capture probe. Line graph: measurement result by bisulfite pyrosequencing method (vertical axis: methylation rate); bar graph: measurement result by conventional method (vertical axis: luminescence count). A: cultured cell A172; U: cultured cell U87-MG; T: cultured cell T47D; DW: distilled water (negative control) 図８は、２つの異なる捕捉プローブを用いる本発明の方法による、抽出核酸サンプル中の標的核酸に含まれる修飾核酸塩基の検出を示す図である。詳細は、図７と同様である。FIG. 8 is a diagram showing detection of modified nucleobases contained in a target nucleic acid in an extracted nucleic acid sample by the method of the present invention using two different capture probes. Details are the same as in FIG.
　本発明は、修飾核酸塩基を含む標的核酸の測定方法を提供する。以下、修飾核酸塩基を含む標的核酸を、省略のため単に標的核酸と呼ぶことがある。 The present invention provides a method for measuring a target nucleic acid containing a modified nucleobase. Hereinafter, a target nucleic acid containing a modified nucleobase may be simply referred to as a target nucleic acid for omission.
　標的核酸は、ヌクレオチド単位が重合した天然または人工の核酸である。標的核酸としては、例えば、ＤＮＡ、およびＲＮＡが挙げられるが、ＤＮＡが好ましい。標的核酸はまた、非コーディング領域（例、転写調節領域）、またはそれを含む領域であってもよい。標的核酸を構成するヌクレオチド残基の個数（即ち、標的核酸の長さ）は、捕捉プローブとハイブリダイズし得る限り特に限定されないが、例えば３０個以上、４０個以上、５０個以上、６０個以上、７０個以上、８０個以上、９０個以上、または１００個以上であってもよい。標的核酸を構成するヌクレオチドの個数はまた、ゲノムＤＮＡの断片化処理によって生ずる可能性のある任意の個数であってもよい。例えば、標的核酸を構成するヌクレオチドの個数は、１００００個以下、５０００個以下、２０００個以下、１０００個以下、５００個以下、または２００個または以下であってもよい。 The target nucleic acid is a natural or artificial nucleic acid in which nucleotide units are polymerized. Examples of the target nucleic acid include DNA and RNA, and DNA is preferable. The target nucleic acid may also be a non-coding region (eg, transcriptional regulatory region) or a region containing it. The number of nucleotide residues constituting the target nucleic acid (that is, the length of the target nucleic acid) is not particularly limited as long as it can hybridize with the capture probe. For example, 30 or more, 40 or more, 50 or more, 60 or more 70 or more, 80 or more, 90 or more, or 100 or more. The number of nucleotides constituting the target nucleic acid may be any number that may be generated by fragmentation processing of genomic DNA. For example, the number of nucleotides constituting the target nucleic acid may be 10,000 or less, 5000 or less, 2000 or less, 1000 or less, 500 or less, or 200 or less.
　本発明において、修飾核酸塩基とは、アデニン（Ａ）、グアニン（Ｇ）、シトシン（Ｃ）、チミン（Ｔ）およびウラシル（Ｕ）からなる群より選ばれる通常の核酸塩基に対して修飾された構造を有する核酸塩基をいう。例えば、表現「修飾核酸塩基」中の用語「核酸塩基」としては、標的核酸がＤＮＡの場合には、例えば、アデニン（Ａ）、グアニン（Ｇ）、シトシン（Ｃ）およびチミン（Ｔ）が挙げられる。一方、標的核酸がＲＮＡの場合には、例えば、アデニン（Ａ）、グアニン（Ｇ）、シトシン（Ｃ）およびウラシル（Ｕ）が挙げられる。核酸塩基は、好ましくはシトシン（Ｃ）である。修飾としては、例えば、通常の核酸塩基への置換基の導入、通常の核酸塩基が有する基（例、アミノ基、オキソ基、メチル基）の脱離、通常の核酸塩基が有する基の置換基への交換が挙げられる。置換基としては、天然に存在する核酸塩基が保有し得るものである限り特に限定されないが、例えば、Ａｄｍｉｎｉｓｔｒａｔｉｖｅ　Ｉｎｓｔｒｕｃｔｉｏｎｓ　ｕｎｄｅｒ　ｔｈｅ　Ｐａｔｅｎｔ　Ｃｏｏｐｅｒａｔｉｏｎ　Ｔｒｅａｔｙ（２００９年１月１日施行版），Ａｎｎｅｘ　Ｃ，Ａｐｐｅｎｄｉｘ　２，Ｔａｂｌｅ　２：Ｌｉｓｔ　ｏｆ　Ｍｏｄｉｆｉｅｄ　Ｎｕｃｌｅｏｔｉｄｅｓに記載される修飾ヌクレオチド中の修飾核酸塩基が保有する置換基が挙げられる。本資料中に記載される修飾ヌクレオチドは、日本特許庁により公開されている「塩基配列又はアミノ酸配列を含む明細書等の作成のためのガイドライン（平成１４年７月）または（平成２１年１２月）」の附属書２，表２：修飾塩基表に記載される修飾ヌクレオチドと同一であり得る。したがって、修飾核酸塩基については、上記ガイドラインもまた参照できる。置換基は、好ましくは、メチル、ヒドロキシメチル、カルボキシルである。置換等の修飾の位置は、特に限定されないが、ピリミジン環を有する核酸塩基（Ｃ、ＴまたはＵ）の場合には、例えば、２位、４～６位であり、好ましくは５位であり、プリン環を有する核酸塩基（ＡまたはＧ）の場合には、例えば、２位、６位、８位である。　 In the present invention, the modified nucleobase is modified with respect to a normal nucleobase selected from the group consisting of adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U). A nucleobase having a structure. For example, the term “nucleobase” in the expression “modified nucleobase” includes, for example, adenine (A), guanine (G), cytosine (C) and thymine (T) when the target nucleic acid is DNA. It is done. On the other hand, when the target nucleic acid is RNA, examples thereof include adenine (A), guanine (G), cytosine (C), and uracil (U). The nucleobase is preferably cytosine (C). Modifications include, for example, introduction of substituents into normal nucleobases, elimination of groups (eg, amino group, oxo group, methyl group) possessed by ordinary nucleobases, substituents of groups possessed by ordinary nucleobases The exchange to is mentioned. The substituent is not particularly limited as long as it can be possessed by a naturally occurring nucleobase. For example, Administrative Instructions under the Patent Cooperative Treaty (enforced on January 1, 2009), Annex C, Appendix 2, Examples include substituents possessed by modified nucleobases in modified nucleotides described in Table 2: List of Modified Nucleotides. The modified nucleotides described in this document are the “Guidelines (July 2002) or (December 2009) for preparation of a description including a base sequence or amino acid sequence” published by the Japan Patent Office. ) ", Annex 2, Table 2: Can be the same as the modified nucleotide described in the modified base table. Accordingly, reference can also be made to the above guidelines for modified nucleobases. The substituent is preferably methyl, hydroxymethyl, or carboxyl. The position of modification such as substitution is not particularly limited, but in the case of a nucleobase having a pyrimidine ring (C, T or U), for example, it is at the 2-position, 4- to 6-position, preferably at the 5-position, In the case of a nucleobase (A or G) having a purine ring, they are, for example, the 2nd, 6th and 8th positions. *
　修飾核酸塩基は、天然に存在し得るものである限り特に限定されないが、例えば、Ａｄｍｉｎｉｓｔｒａｔｉｖｅ　Ｉｎｓｔｒｕｃｔｉｏｎｓ　ｕｎｄｅｒ　ｔｈｅ　Ｐａｔｅｎｔ　Ｃｏｏｐｅｒａｔｉｏｎ　Ｔｒｅａｔｙ（２００９年１月１日施行版），Ａｎｎｅｘ　Ｃ，Ａｐｐｅｎｄｉｘ　２，Ｔａｂｌｅ　２：Ｌｉｓｔ　ｏｆ　Ｍｏｄｉｆｉｅｄ　Ｎｕｃｌｅｏｔｉｄｅｓに記載される修飾ヌクレオチドが保有する修飾核酸塩基が挙げられる。本資料中に記載される修飾ヌクレオチドは、上記ガイドラインの附属書２，表２：修飾塩基表に記載される修飾ヌクレオチドと同一であり得る。したがって、修飾核酸塩基については、上記ガイドラインもまた参照できる。好ましくは、修飾核酸塩基は、メチルシトシン（例、５－メチルシトシン）、ヒドロキシメチルシトシン（例、５－ヒドロキシメチルシトシン）、カルボキシルシトシン（例、５－カルボキシルシトシン）である。修飾核酸塩基は、核酸の機能の変化をもたらすこと（例、所定の遺伝子の転写調節能の変化）が知られている。　 The modified nucleobase is not particularly limited as long as it can exist in nature. For example, Administrative Instructions under the Patent Cooperation Treaty (enforced on January 1, 2009), Annex C, Appendix 2, Table 2: Examples thereof include modified nucleobases possessed by modified nucleotides described in Modified Nucleotides. The modified nucleotides described in this document may be the same as the modified nucleotides described in Appendix 2, Table 2: Modified Base Table of the above guidelines. Accordingly, reference can also be made to the above guidelines for modified nucleobases. Preferably, the modified nucleobase is methylcytosine (eg, 5-methylcytosine), hydroxymethylcytosine (eg, 5-hydroxymethylcytosine), carboxyl cytosine (eg, 5-carboxyl cytosine). A modified nucleobase is known to cause a change in the function of the nucleic acid (eg, a change in the transcriptional regulatory ability of a given gene). *
　本発明の方法は、以下を含む：
（１）修飾核酸塩基を含む標的核酸、および非標的核酸を含む核酸サンプルにおいて、標的核酸に対する複数の異なる捕捉プローブを用いて、標的核酸を固相に複数回固定すること；ならびに
（２）標的核酸に含まれる修飾核酸塩基を測定すること。 The method of the present invention includes:
(1) immobilizing a target nucleic acid multiple times on a solid phase using a plurality of different capture probes for the target nucleic acid in a target nucleic acid containing a modified nucleobase and a non-target nucleic acid; and (2) a target Measuring modified nucleobases in nucleic acids.
　（１）では、核酸サンプルにおいて、標的核酸に対する複数の異なる捕捉プローブを用いて、標的核酸が固相に複数回固定される。これにより、非標的核酸、特に、標的核酸の測定の支障となる修飾核酸塩基を含む非標的核酸（例、複数の捕捉プローブのうちの一部の捕捉プローブのヌクレオチド配列と相補的な部分ヌクレオチド配列を有する、修飾核酸塩基を含む非標的核酸）を排除することができる。 (1) In the nucleic acid sample, the target nucleic acid is immobilized on the solid phase a plurality of times using a plurality of different capture probes for the target nucleic acid. Thereby, a non-target nucleic acid, particularly a non-target nucleic acid containing a modified nucleobase that hinders measurement of the target nucleic acid (eg, a partial nucleotide sequence complementary to the nucleotide sequence of some of the plurality of capture probes) Non-target nucleic acids containing modified nucleobases) can be excluded.
　核酸サンプルは、上述したような修飾核酸塩基を含む標的核酸、および非標的核酸を含む任意の核酸サンプルである。非標的核酸は、ヌクレオチド単位が重合した天然または人工の核酸である。非標的核酸としては、例えば、修飾核酸塩基を含む非標的核酸、および修飾核酸塩基を含まない非標的核酸が挙げられる。非標的核酸としてはまた、例えば、ＤＮＡ、およびＲＮＡが挙げられるが、ＤＮＡが好ましい。 A nucleic acid sample is any nucleic acid sample containing a target nucleic acid containing a modified nucleobase as described above and a non-target nucleic acid. Non-target nucleic acids are natural or artificial nucleic acids in which nucleotide units are polymerized. Non-target nucleic acids include, for example, non-target nucleic acids that include modified nucleobases and non-target nucleic acids that do not include modified nucleobases. Non-target nucleic acids also include, for example, DNA and RNA, with DNA being preferred.
　核酸サンプルに含まれる標的核酸および非標的核酸の量は特に限定されないが、本発明の方法は、核酸サンプルにおいて非標的核酸に対する標的核酸の相対量が少ない場合であっても、標的核酸に含まれる修飾核酸塩基を良好に測定できることが確認されている。したがって、本発明の方法は、非標的核酸に対する前記標的核酸の相対量が少ない場合に特に有効である。本発明の方法が特に有効である、非標的核酸に対する標的核酸の相対量は、非標的核酸１μｇ当たりの標的核酸モル数として、２０ａｍｏｌ（１０００ａｍｏｌ標的核酸／５０μｇ非標的核酸；実施例３および図５を参照）未満の量である。好ましくは、このような相対量は、非標的核酸１μｇ当たりの標的核酸モル数として、１０ａｍｏｌ以下、５ａｍｏｌ以下、２ａｍｏｌ以下、１ａｍｏｌ以下、または０．５ａｍｏｌ以下の量である。例えば、ヒトゲノムＤＮＡ中の１つの標的ＤＮＡのみを測定する場合、非標的核酸（ヒトゲノムＤＮＡ）１μｇ当たりの標的ＤＮＡモル数は、約０．５ａｍｏｌである。 Although the amount of target nucleic acid and non-target nucleic acid contained in the nucleic acid sample is not particularly limited, the method of the present invention is contained in the target nucleic acid even when the relative amount of the target nucleic acid relative to the non-target nucleic acid is small in the nucleic acid sample. It has been confirmed that modified nucleobases can be measured well. Therefore, the method of the present invention is particularly effective when the relative amount of the target nucleic acid with respect to the non-target nucleic acid is small. The relative amount of the target nucleic acid relative to the non-target nucleic acid for which the method of the present invention is particularly effective is 20 amol (1000 amol target nucleic acid / 50 μg non-target nucleic acid; Example 3 and FIG. 5). Less). Preferably, such a relative amount is an amount of 10 amol or less, 5 amol or less, 2 amol or less, 1 amol or less, or 0.5 amol or less as the number of moles of target nucleic acid per μg of non-target nucleic acid. For example, when only one target DNA in human genomic DNA is measured, the number of moles of target DNA per 1 μg of non-target nucleic acid (human genomic DNA) is about 0.5 amol.
　核酸サンプルとしては、例えば、生物由来の生物学的サンプル、環境サンプル、および合成核酸の混合サンプルが挙げられる。生物学的サンプルが由来する生物としては、例えば、哺乳動物（例、ヒト、サル、マウス、ラット、ウサギ、ウシ、ブタ、ウマ、ヤギ、ヒツジ）、鳥類（例、ニワトリ）等の動物、昆虫、微生物、植物、菌類、魚類が挙げられる。生物学的サンプルはまた、血液自体または血液に由来するサンプルである血液関連サンプル（例、全血、血清、血漿）、唾液、尿、乳汁、組織または細胞抽出液、あるいはこれらの混合物であってもよい。生物学的サンプルはさらに、疾患（例、癌、白血病）に罹患している哺乳動物、または疾患に罹患している可能性がある哺乳動物に由来するものであってもよい。環境サンプルとしては、例えば、核酸を含む可能性がある土壌、海水、淡水由来のサンプルが挙げられる。合成核酸の混合サンプルとしては、例えば、人工的に合成された修飾核酸塩基を含む標的核酸（例、ＤＮＡ、ＲＮＡ）であって、修飾核酸塩基の検量が所望されるものが挙げられる。 Examples of nucleic acid samples include biological biological samples, environmental samples, and synthetic nucleic acid mixed samples. Examples of organisms from which biological samples are derived include animals such as mammals (eg, humans, monkeys, mice, rats, rabbits, cows, pigs, horses, goats, sheep), birds (eg, chickens), insects, and the like. , Microorganisms, plants, fungi, and fish. A biological sample can also be a blood-related sample (eg, whole blood, serum, plasma), saliva, urine, milk, tissue or cell extract, or a mixture thereof, which is the blood itself or a sample derived from blood. Also good. The biological sample may further be derived from a mammal suffering from a disease (eg, cancer, leukemia) or a mammal potentially having a disease. Examples of environmental samples include samples derived from soil, seawater, and fresh water that may contain nucleic acids. Examples of the mixed sample of synthetic nucleic acids include target nucleic acids (eg, DNA, RNA) containing artificially synthesized modified nucleobases, for which calibration of modified nucleobases is desired.
　好ましくは、核酸サンプルは、ゲノムＤＮＡサンプルである。 Preferably, the nucleic acid sample is a genomic DNA sample.
　核酸サンプルは、上記（１）の工程の前に、他の処理に付されてもよい。このような処理としては、例えば、核酸（例、ゲノムＤＮＡ等のＤＮＡ、ＲＮＡ）の抽出および断片化（例、制限酵素等の酵素による処理）が挙げられる。したがって、本発明の方法は、核酸サンプルから核酸を抽出すること、および／または核酸を断片化することをさらに含んでいてもよい。本発明の方法はまた、遠心分離、抽出、ろ過、沈殿、加熱、凍結、冷蔵、攪拌等の操作によりサンプルを処理することをさらに含んでいてもよい。 The nucleic acid sample may be subjected to other treatment before the step (1). Examples of such treatment include extraction of nucleic acid (eg, DNA such as genomic DNA, RNA) and fragmentation (eg, treatment with an enzyme such as a restriction enzyme). Thus, the methods of the invention may further comprise extracting nucleic acid from the nucleic acid sample and / or fragmenting the nucleic acid. The method of the present invention may further comprise processing the sample by operations such as centrifugation, extraction, filtration, precipitation, heating, freezing, refrigeration, stirring and the like.
　本発明で用いられる捕捉プローブとは、標的核酸とハイブリダイズして核酸ハイブリッドを形成する能力を有し、かつ、固相に固定可能であるか、または固相に固定されている核酸プローブをいう。 The capture probe used in the present invention refers to a nucleic acid probe that has the ability to hybridize with a target nucleic acid to form a nucleic acid hybrid and can be immobilized on a solid phase or is immobilized on a solid phase. .
　捕捉プローブは、標的核酸に対して同種および／または異種の核酸から構成され得る。ここで、用語「同種」とは、標的核酸の主鎖構造（糖部分およびリン酸部分から構成される構造）と同じ主鎖構造を、主鎖構造の全体として捕捉プローブが有することを意味する。一方、用語「異種」とは、標的核酸の主鎖構造（糖部分およびリン酸部分から構成される構造）と異なる主鎖構造を、主鎖構造の一部または全体として捕捉プローブが有することを意味する。したがって、捕捉プローブの種類は、標的核酸の種類に応じて決定されてもよい。例えば、標的核酸がＤＮＡである場合、同種核酸の捕捉プローブとしては、ＤＮＡプローブを用いることができ、異種核酸の捕捉プローブとしては、ＤＮＡプローブ以外の核酸プローブを用いることができる。一方、標的核酸が天然ＲＮＡである場合、同種核酸の捕捉プローブとしては、当該天然ＲＮＡと同種のＲＮＡから構成されるノーマルＲＮＡプローブを用いることができ、異種核酸の捕捉プローブとしては、ノーマルＲＮＡプローブ以外の核酸プローブを用いることができる。好ましくは、捕捉プローブは、標的核酸に対して異種の核酸から構成されていてもよい。 The capture probe can be composed of the same type and / or different type of nucleic acid with respect to the target nucleic acid. Here, the term “same species” means that the capture probe has the same main-chain structure as the main-chain structure of the target nucleic acid (structure composed of a sugar moiety and a phosphate moiety) as a whole of the main-chain structure. . On the other hand, the term “heterologous” means that the capture probe has a main chain structure that is different from the main chain structure of the target nucleic acid (structure composed of a sugar moiety and a phosphate moiety) as a part or the whole of the main chain structure. means. Therefore, the type of capture probe may be determined according to the type of target nucleic acid. For example, when the target nucleic acid is DNA, a DNA probe can be used as a capture probe for homologous nucleic acids, and a nucleic acid probe other than a DNA probe can be used as a capture probe for heterologous nucleic acids. On the other hand, when the target nucleic acid is a natural RNA, a normal RNA probe composed of the same type of RNA as the natural RNA can be used as the same type of nucleic acid capture probe, and a normal RNA probe as a heterologous nucleic acid capture probe. Other nucleic acid probes can be used. Preferably, the capture probe may be composed of a nucleic acid heterologous to the target nucleic acid.
　捕捉プローブとしては、例えば、ＤＮＡプローブ、ＲＮＡプローブ、ペプチド核酸（ＰＮＡ）プローブ、ロック型核酸（ＬＮＡ）プローブまたは架橋型核酸（ＢＮＡ）プローブ、ホスホロチオエート（Ｓ化）核酸プローブ、およびこのような２以上の核酸プローブが連結したキメラ型核酸プローブ（キメラ型核酸プローブは、必然的に、標的核酸に対して異種の核酸を含む）が挙げられる。 Examples of capture probes include DNA probes, RNA probes, peptide nucleic acid (PNA) probes, lock nucleic acid (LNA) probes or cross-linked nucleic acid (BNA) probes, phosphorothioate (S) nucleic acid probes, and two or more such probes. And a nucleic acid probe linked to each other (a chimeric nucleic acid probe necessarily includes a nucleic acid heterologous to a target nucleic acid).
　一実施形態では、捕捉プローブは、ＲＮＡプローブである。ＲＮＡプローブとしては、例えば、２’位にヒドロキシル基を有する天然リボヌクレオチドから構成されるノーマルＲＮＡプローブ、および２’位のヒドロキシル基が修飾されているリボヌクレオチドから構成される修飾ＲＮＡプローブが挙げられる。好ましくは、ＲＮＡプローブは、修飾ＲＮＡプローブである。修飾ＲＮＡプローブとしては、リボヌクレアーゼ耐性のＲＮＡプローブを用いてもよい。修飾ＲＮＡプローブとしては、例えば、２’－Ｏ－アルキル化ＲＮＡプローブが挙げられる。２’－Ｏ－アルキル化ＲＮＡプローブは、好ましくは、２’－Ｏ－Ｃ１～Ｃ６アルキル化ＲＮＡプローブである。Ｃ１～Ｃ６アルキル化についてのＣ１～Ｃ６アルキル基は、直鎖、分岐鎖または環状の炭素原子数１～６個のアルキル基であり、例えば、メチル、エチル、プロピル基（例、ｎ－プロピル、ｉｓｏ－プロピル）、ブチル基（例、ｎ－ブチル、ｉｓｏ－ブチル、ｓｅｃ－ブチル、ｔｅｒｔ－ブチル）、ペンチル基、ヘキシル基などが挙げられる。製造・入手の容易性等の観点から、２’－Ｏ－Ｃ１～Ｃ６アルキル化ＲＮＡプローブは、２’－Ｏ－メチル化ＲＮＡプローブである。 In one embodiment, the capture probe is an RNA probe. Examples of the RNA probe include a normal RNA probe composed of a natural ribonucleotide having a hydroxyl group at the 2 ′ position, and a modified RNA probe composed of a ribonucleotide modified with a hydroxyl group at the 2 ′ position. . Preferably, the RNA probe is a modified RNA probe. A ribonuclease resistant RNA probe may be used as the modified RNA probe. Examples of modified RNA probes include 2'-O-alkylated RNA probes. The 2'-O-alkylated RNA probe is preferably a 2'-O-C1-C6 alkylated RNA probe. A C1-C6 alkyl group for C1-C6 alkylation is a linear, branched or cyclic alkyl group of 1-6 carbon atoms such as methyl, ethyl, propyl groups (eg, n-propyl, iso-propyl), butyl group (eg, n-butyl, iso-butyl, sec-butyl, tert-butyl), pentyl group, hexyl group and the like. From the viewpoint of ease of production and availability, the 2'-O-C1-C6 alkylated RNA probe is a 2'-O-methylated RNA probe.
　捕捉プローブを構成するヌクレオチド残基の個数（即ち、捕捉プローブの長さ）は、標的核酸とハイブリダイズし得る程度に十分な長さである限り特に限定されないが、例えば１２個以上、好ましくは１５個以上、好ましくは１８個以上、より好ましくは２０個以上であってもよい。捕捉プローブを構成するヌクレオチドの個数はまた、例えば、１００個以下、８０個以下、６０個以下または５０個以下であってもよい。捕捉プローブは、当該分野で公知のプローブ合成法により調製できる。 The number of nucleotide residues constituting the capture probe (that is, the length of the capture probe) is not particularly limited as long as it is long enough to hybridize with the target nucleic acid. For example, 12 or more, preferably 15 Or more, preferably 18 or more, more preferably 20 or more. The number of nucleotides constituting the capture probe may also be, for example, 100 or less, 80 or less, 60 or less, or 50 or less. Capture probes can be prepared by probe synthesis methods known in the art.
　捕捉プローブは、工程（１）で用いられる場合、遊離の形態、または固相に固定された形態で用いることができる。したがって、捕捉プローブは、固相への特異的な結合を可能にする物質または基で標識されていてもよい。標識は、例えば、捕捉プローブの５’末端または３’末端の一方にて行うことができる。固相への特異的な結合を可能にする物質または基としては、例えば、固相への特異的な共有結合を可能にする基または物質、および固相への特異的な親和性結合を可能にする親和性物質が挙げられる。固相への特異的な共有結合を可能にする基または物質としては、例えば、チオール基、またはチオール基を有する物質（捕捉プローブに導入されたこのようなチオール基は、固相上のマレイミド基と結合できる）、アミノ基、またはアミノ基を有する物質（捕捉プローブに導入されたこのようなアミノ基は、固相上の無水マレイン酸と結合できる）が挙げられる。固相への特異的な親和性結合を可能にする親和性物質としては、例えば、ストレプトアビジン、ビオチン、ジゴキシゲニン、ジニトロフェノール、フルオレセイン、フルオレセインイソチオシアネート、互いに相補的なセンス鎖およびアンチセンス鎖を含む二本鎖核酸分子のうちの一方の鎖が挙げられる。この場合、固相としては、捕捉プローブが有する物質または基と特異的に結合する能力を有する物質または基でコーティングされたものを用いることができる。捕捉プローブは、工程（１）において遊離の形態で用いられる場合、ハイブリッド形成後に、固相に固定されてもよい。 When the capture probe is used in the step (1), it can be used in a free form or a form fixed on a solid phase. Thus, the capture probe may be labeled with a substance or group that allows specific binding to the solid phase. Labeling can be done, for example, at either the 5 'end or the 3' end of the capture probe. Substances or groups that enable specific binding to the solid phase include, for example, groups or substances that allow specific covalent binding to the solid phase, and specific affinity binding to the solid phase. Affinity substances to be mentioned. Examples of the group or substance that enables specific covalent bonding to the solid phase include, for example, a thiol group or a substance having a thiol group (such a thiol group introduced into the capture probe is a maleimide group on the solid phase). ), An amino group, or a substance having an amino group (such an amino group introduced into a capture probe can bind to maleic anhydride on a solid phase). Affinity substances that enable specific affinity binding to the solid phase include, for example, streptavidin, biotin, digoxigenin, dinitrophenol, fluorescein, fluorescein isothiocyanate, complementary sense strand and antisense strand One strand of a double-stranded nucleic acid molecule is mentioned. In this case, as the solid phase, one coated with a substance or group having the ability to specifically bind to the substance or group possessed by the capture probe can be used. When used in the free form in step (1), the capture probe may be immobilized on a solid phase after hybridization.
　固相としては、例えば、液相を収容または搭載可能な固相（例、プレート、メンブレン、試験管等の支持体、およびウェルプレート、マイクロ流路、ガラスキャピラリー、ナノピラー、モノリスカラム等の容器）、ならびに液相中に分散可能な固相（例、粒子）が挙げられる。固相の材料としては、例えば、ガラス、プラスチック、金属、カーボンが挙げられる。固相の材料としてはまた、非磁性材料または磁性材料を用いることができるが、操作の簡便性等の観点から、磁性材料が好ましい。固相は、好ましくは液相中に分散可能な固相であり、より好ましくは粒子であり、さらにより好ましくは磁性粒子である。捕捉プローブが固定される固相として、液相中に分散可能な固相が用いられる場合、当然のことながら、液相を収容または搭載可能な固相を併用することができる。 Examples of the solid phase include a solid phase that can contain or mount a liquid phase (eg, a support such as a plate, a membrane, or a test tube, and a container such as a well plate, a microchannel, a glass capillary, a nanopillar, or a monolith column). As well as solid phases (eg, particles) dispersible in the liquid phase. Examples of the solid phase material include glass, plastic, metal, and carbon. A non-magnetic material or a magnetic material can also be used as the solid phase material, but a magnetic material is preferred from the viewpoint of ease of operation. The solid phase is preferably a solid phase that can be dispersed in a liquid phase, more preferably particles, and even more preferably magnetic particles. When a solid phase that can be dispersed in a liquid phase is used as the solid phase to which the capture probe is fixed, it is needless to say that a solid phase that can accommodate or mount the liquid phase can be used in combination.
　本発明の方法で用いられる捕捉プローブの数は、２個以上である限り特に限定されないが、好ましくは、２個、３個、４個、または５個であり、より好ましくは、２個または３個であり、特に好ましくは２個である。 The number of capture probes used in the method of the present invention is not particularly limited as long as it is 2 or more, but is preferably 2, 3, 4, or 5, more preferably, 2 or 3 The number is particularly preferably two.
　好ましい実施形態では、複数の捕捉プローブとして、標的核酸の５’末端領域にハイブリダイズする能力を有する捕捉プローブ、および標的核酸の３’末端領域にハイブリダイズする能力を有する捕捉プローブを併用することができる。このような併用により、標的核酸をより特異的に測定することができる。 In a preferred embodiment, a plurality of capture probes may be used in combination with a capture probe capable of hybridizing to the 5 ′ end region of the target nucleic acid and a capture probe capable of hybridizing to the 3 ′ end region of the target nucleic acid. it can. By such combined use, the target nucleic acid can be measured more specifically.
　標的核酸の５’末端領域とは、標的核酸の全長がｎ個のヌクレオチド残基からなる場合、標的核酸における５’末端ヌクレオチド残基（１番目）から５’→３’方向に起算してｎ／３番目のヌクレオチド残基までの領域をいう。好ましくは、標的核酸の５’末端領域は、標的核酸における５’末端ヌクレオチド残基（１番目）から５’→３’方向に起算してｎ／４番目、ｎ／５番目、ｎ／６番目、ｎ／７番目、ｎ／８番目、ｎ／９番目、またはｎ／１０番目のヌクレオチド残基までの領域である。ｎ／３、ｎ／４、ｎ／５、ｎ／６、ｎ／７、ｎ／８、ｎ／９、またはｎ／１０の値が小数点以下の値を有する場合、小数点以下の値は考慮されない。例えば、標的核酸の全長が１００個のヌクレオチド残基からなる場合、標的核酸の５’末端領域は、標的核酸における５’末端ヌクレオチド残基（１番目）から５’→３’方向に起算して３３番目のヌクレオチド残基までの領域であり、好ましくは、２５番目、２０番目、１６番目、１４番目、１２番目、１１番目、または１０番目のヌクレオチド残基までの領域である。 The 5 ′ terminal region of the target nucleic acid is n by counting from the 5 ′ terminal nucleotide residue (first) in the target nucleic acid in the 5 ′ → 3 ′ direction when the total length of the target nucleic acid consists of n nucleotide residues. / Refers to the region up to the third nucleotide residue. Preferably, the 5 ′ terminal region of the target nucleic acid is n / 4, n / 5, and n / 6 from the 5 ′ terminal nucleotide residue (first) in the target nucleic acid in the 5 ′ → 3 ′ direction. , N / 7th, n / 8th, n / 9th, or n / 10th nucleotide region. If a value of n / 3, n / 4, n / 5, n / 6, n / 7, n / 8, n / 9, or n / 10 has a value after the decimal point, the value after the decimal point is not considered . For example, when the total length of the target nucleic acid consists of 100 nucleotide residues, the 5 ′ terminal region of the target nucleic acid is calculated from the 5 ′ terminal nucleotide residue (first) in the target nucleic acid in the 5 ′ → 3 ′ direction. A region up to the 33rd nucleotide residue, preferably a region up to the 25th, 20th, 16th, 14th, 12th, 11th, or 10th nucleotide residue.
　標的核酸の５’末端領域にハイブリダイズする能力を有する捕捉プローブは、上記のような５’末端領域の全体または一部にハイブリダイズして核酸ハイブリッドを形成できる限り、標的核酸の５’末端領域にハイブリダイズする能力を有するものと解される。したがって、捕捉プローブは、上記のような５’末端領域の全体または一部にハイブリダイズするものであれば、さらに５’末端領域の３’隣接領域にハイブリダイズするものであってもよい。好ましくは、標的核酸の５’末端領域にハイブリダイズする能力を有する捕捉プローブは、５’末端領域の全体または一部のみにハイブリダイズし、５’末端領域の３’隣接領域にはハイブリダイズしない捕捉プローブであってもよい。 As long as the capture probe having the ability to hybridize to the 5 ′ end region of the target nucleic acid can hybridize to all or a part of the 5 ′ end region as described above to form a nucleic acid hybrid, the 5 ′ end region of the target nucleic acid It is understood that it has the ability to hybridize to. Therefore, the capture probe may further hybridize to the 3 'adjacent region of the 5' end region as long as it hybridizes to the whole or a part of the 5 'end region as described above. Preferably, the capture probe capable of hybridizing to the 5 ′ end region of the target nucleic acid hybridizes to all or only part of the 5 ′ end region and does not hybridize to the 3 ′ adjacent region of the 5 ′ end region. It may be a capture probe.
　標的核酸の３’末端領域とは、標的核酸の全長がｎ個のヌクレオチド残基からなる場合、標的核酸におけるｎ番目のヌクレオチド残基から３’→５’方向に起算してｎ／３番目のヌクレオチド残基までの領域をいう。好ましくは、標的核酸の３’末端領域は、標的核酸における３’末端ヌクレオチド残基（ｎ番目）から３’→５’方向に起算してｎ／４番目、ｎ／５番目、ｎ／６番目、ｎ／７番目、ｎ／８番目、ｎ／９番目、またはｎ／１０番目のヌクレオチド残基までの領域である。ｎ／３、ｎ／４、ｎ／５、ｎ／６、ｎ／７、ｎ／８、ｎ／９、またはｎ／１０の値が小数点以下の値を有する場合、小数点以下の値は考慮されない。例えば、標的核酸の全長が１００個のヌクレオチド残基からなる場合、標的核酸の３’末端領域は、標的核酸における３’末端ヌクレオチド残基（１００番目）から３’→５’方向に起算して３３番目のヌクレオチド残基までの領域（換言すれば、標的核酸における５’末端ヌクレオチド残基（１番目）から５’→３’方向に起算すると、６８番目～１００番目のヌクレオチド残基からなる領域）であり、好ましくは、２５番目、２０番目、１６番目、１４番目、１２番目、１１番目、または１０番目のヌクレオチド残基までの領域（換言すれば、標的核酸における５’末端ヌクレオチド残基（１番目）から５’→３’方向に起算すると、７６番目、８１番目、８５番目、８７番目、８９番目、９０番目、または９１番目から１００番目までのヌクレオチド残基からなる領域）である。 The 3 ′ terminal region of the target nucleic acid is the n / 3rd number from the nth nucleotide residue in the target nucleic acid in the 3 ′ → 5 ′ direction when the total length of the target nucleic acid consists of n nucleotide residues. Refers to the region up to nucleotide residues. Preferably, the 3 ′ terminal region of the target nucleic acid is n / 4, n / 5, and n / 6 from the 3 ′ terminal nucleotide residue (nth) in the target nucleic acid in the 3 ′ → 5 ′ direction. , N / 7th, n / 8th, n / 9th, or n / 10th nucleotide region. If a value of n / 3, n / 4, n / 5, n / 6, n / 7, n / 8, n / 9, or n / 10 has a value after the decimal point, the value after the decimal point is not considered . For example, when the total length of the target nucleic acid consists of 100 nucleotide residues, the 3 ′ terminal region of the target nucleic acid is calculated in the 3 ′ → 5 ′ direction from the 3 ′ terminal nucleotide residue (100th) in the target nucleic acid. The region up to the 33rd nucleotide residue (in other words, the region consisting of the 68th to 100th nucleotide residues from the 5 ′ terminal nucleotide residue (first) in the target nucleic acid in the 5 ′ → 3 ′ direction) Preferably, the region up to the 25th, 20, 20, 14, 12, 11, or 10th nucleotide residue (in other words, the 5 ′ terminal nucleotide residue in the target nucleic acid ( From 1st) to 5 '→ 3', 76th, 81st, 85th, 87th, 89th, 90th, or 91st to 100th A region) consisting of a nucleotide residues.
　標的核酸の３’末端領域にハイブリダイズする能力を有する捕捉プローブは、上記のような３’末端領域の全体または一部にハイブリダイズして核酸ハイブリッドを形成できる限り、標的核酸の３’末端領域にハイブリダイズする能力を有するものと解される。したがって、捕捉プローブは、上記のような３’末端領域の全体または一部にハイブリダイズするものであれば、さらに３’末端領域の５’隣接領域にハイブリダイズするものであってもよい。好ましくは、標的核酸の３’末端領域にハイブリダイズする能力を有する捕捉プローブは、３’末端領域の全体または一部のみにハイブリダイズし、３’末端領域の５’隣接領域にはハイブリダイズしない捕捉プローブであってもよい。 As long as the capture probe having the ability to hybridize to the 3 ′ terminal region of the target nucleic acid can hybridize to all or a part of the 3 ′ terminal region as described above to form a nucleic acid hybrid, the 3 ′ terminal region of the target nucleic acid It is understood that it has the ability to hybridize to. Therefore, the capture probe may further hybridize to the 5 'adjacent region of the 3' end region as long as it hybridizes to all or a part of the 3 'end region as described above. Preferably, a capture probe capable of hybridizing to the 3 ′ end region of the target nucleic acid hybridizes to all or only part of the 3 ′ end region and does not hybridize to the 5 ′ adjacent region of the 3 ′ end region. It may be a capture probe.
　本発明の方法では、用いられる異なる捕捉プローブの数に応じて、標的核酸の固相への捕捉を２回以上繰り返し行うことができる。例えば、用いられる異なる捕捉プローブの数がＸである場合、（標的核酸の固相への捕捉）をＸ回、および（固相から液相への標的核酸の放出）をＸ－１回（またはＸ回。Ｘ回目は任意）行うことができる。具体的には、２つの異なる捕捉プローブが用いられる場合、本発明の方法は、以下を含む方法により行うことができる：
（ｉ）標的核酸に対する第１捕捉プローブを用いた液相中の標的核酸の固相への捕捉；
（ｉｉ）固相から液相への標的核酸の放出；および
（ｉｉｉ）標的核酸に対する第２捕捉プローブを用いた液相中の標的核酸の固相への捕捉。 In the method of the present invention, the capture of the target nucleic acid on the solid phase can be repeated twice or more depending on the number of different capture probes used. For example, if the number of different capture probes used is X, (capture target nucleic acid to solid phase) X times and (release target nucleic acid from solid phase to liquid phase) X-1 times (or X times, the Xth time is optional). Specifically, when two different capture probes are used, the method of the invention can be performed by a method comprising:
(I) capture of a target nucleic acid in a liquid phase onto a solid phase using a first capture probe for the target nucleic acid;
(Ii) release of the target nucleic acid from the solid phase to the liquid phase; and (iii) capture of the target nucleic acid in the liquid phase to the solid phase using a second capture probe for the target nucleic acid.
　（ｉ）～（ｉｉｉ）において、液相としては、水、または緩衝液（例、Ｔｒｉｓ緩衝液、リン酸緩衝液）等の水溶系溶媒を用いることができる。また、（ｉ）における液相としては、修飾核酸塩基を含む標的核酸、および非標的核酸を含む核酸サンプルを利用することができる。 In (i) to (iii), as the liquid phase, water or an aqueous solvent such as a buffer solution (eg, Tris buffer solution or phosphate buffer solution) can be used. In addition, as the liquid phase in (i), a target nucleic acid containing a modified nucleobase and a nucleic acid sample containing a non-target nucleic acid can be used.
　標的核酸に対する捕捉プローブを用いた液相中の標的核酸の固相への捕捉は、標的核酸および捕捉プローブのハイブリッドを形成し得る任意の条件下で、標的核酸、および捕捉プローブ（固相に固定されていてもよい）、ならびに必要に応じて固相（捕捉プローブが固相に固定されていない遊離の形態で用いられる場合）を溶液中でインキュベートすることにより行うことができる。このような条件（例、溶液の塩濃度、インキュベーション温度、インキュベーション時間）は周知である（例、国際公開第２０１０／０９１８７０号、国際公開第２００６／１２１８８８号、国際公開第２０１５／０２５８６２号；国際公開第２０１５／０２５８６３号；国際公開第２０１５／０２５８６４号；国際公開第２０１５／１０８１７７号を参照）。 Capturing of a target nucleic acid in a liquid phase using a capture probe for the target nucleic acid is performed under any condition that can form a hybrid of the target nucleic acid and the capture probe. As well as, optionally, by incubating the solid phase (if the capture probe is used in free form not immobilized on the solid phase) in solution. Such conditions (eg, salt concentration of solution, incubation temperature, incubation time) are well known (eg, WO 2010/091870, WO 2006/121888, WO 2015/025862; International Publication 2015/025863; International Publication No. 2015/025864; International Publication No. 2015/108177).
　固相から液相への標的核酸の放出は、標的核酸を固相から液相中に移行させ得る任意の条件下で処理することにより行うことができる。このような処理としては、例えば、ｐＨ調節剤（例、酸性物質、アルカリ性物質、またはこれらの物質を含む溶液）、加熱、競合剤（例、固相への特異的な親和性結合を可能にする親和性物質、標的核酸の相補核酸）、光による処理が挙げられる。標的核酸は、固相への捕捉プローブの固定手段の種類および放出処理の種類等の因子に応じて、捕捉プローブとのハイブリッドの形態、または標的核酸単独の形態で放出される。 Release of the target nucleic acid from the solid phase to the liquid phase can be performed by processing under any condition that allows the target nucleic acid to be transferred from the solid phase to the liquid phase. Such treatments include, for example, pH adjusters (eg, acidic substances, alkaline substances, or solutions containing these substances), heating, competitors (eg, specific affinity binding to solid phases) Affinity substance, complementary nucleic acid of target nucleic acid), treatment with light. The target nucleic acid is released in the form of a hybrid with the capture probe or in the form of the target nucleic acid alone, depending on factors such as the type of means for fixing the capture probe to the solid phase and the type of release treatment.
　第１捕捉プローブを用いた１回目の捕捉では、標的核酸のみならず、第１捕捉プローブのヌクレオチド配列と相補的な部分ヌクレオチド配列を有する非標的核酸も固相に捕捉され得る。しかし、第１捕捉プローブとは異なる第２捕捉プローブを用いた２回目の捕捉により、標的核酸のみ捕捉し、このような非標的核酸の捕捉を回避することで、非標的核酸を排除することができる。２回目に捕捉された標的核酸は、標的核酸に含まれる修飾核酸塩基の測定前に、固相から放出されてもよい。 In the first capture using the first capture probe, not only the target nucleic acid but also a non-target nucleic acid having a partial nucleotide sequence complementary to the nucleotide sequence of the first capture probe can be captured on the solid phase. However, the second capture using a second capture probe different from the first capture probe can capture only the target nucleic acid and avoid such non-target nucleic acid capture, thereby eliminating the non-target nucleic acid. it can. The target nucleic acid captured for the second time may be released from the solid phase before measurement of the modified nucleobase contained in the target nucleic acid.
　より具体的には、２つの異なる捕捉プローブが用いられる場合、本発明の方法は、以下を含む方法により行うことができる：
（ｉ’）標的核酸に対する第１捕捉プローブを用いた第１液相中の標的核酸の第１固相への捕捉；
（ｉｉ’）第１液相の第２液相への交換；
（ｉｉｉ’）第１固相から第２液相への標的核酸の放出；
（ｉｖ’）第１固相の第２固相への交換；
（ｖ’）標的核酸に対する第２捕捉プローブを用いた第２液相中の標的核酸の第２固相への捕捉；および
（ｖｉ’）第２液相の第３液相への交換。 More specifically, when two different capture probes are used, the method of the invention can be performed by a method comprising:
(I ′) capture of the target nucleic acid in the first liquid phase to the first solid phase using the first capture probe for the target nucleic acid;
(Ii ′) exchange of the first liquid phase to the second liquid phase;
(Iii ′) release of the target nucleic acid from the first solid phase to the second liquid phase;
(Iv ′) exchange of the first solid phase to the second solid phase;
(V ′) capture of the target nucleic acid in the second liquid phase to the second solid phase using the second capture probe for the target nucleic acid; and (vi ′) exchange of the second liquid phase to the third liquid phase.
　（ｉ’）、（ｉｉｉ’）および（ｖ’）はそれぞれ、（ｉ）、（ｉｉ）および（ｉｉｉ）と同様にして行うことができる。 (I ′), (iii ′) and (v ′) can be performed in the same manner as (i), (ii) and (iii), respectively.
　（ｉｉ’）では、第１液相が第２液相に交換される。換言すれば、標的核酸が捕捉された第１固相が、第１液相から第２液相に移される。これにより、第１固相に捕捉されなかった、第１液相中に含まれる非標的核酸を系から除去することができる。 (Ii ′), the first liquid phase is exchanged for the second liquid phase. In other words, the first solid phase in which the target nucleic acid is captured is transferred from the first liquid phase to the second liquid phase. Thereby, the non-target nucleic acid contained in the first liquid phase that has not been captured by the first solid phase can be removed from the system.
　（ｉｖ’）では、第１固相が第２固相に交換される。換言すれば、第１固相が系から除去され、第２固相が系に投入される。これにより、第１固相に非特異的に吸着した非標的核酸を系から除去することができる。 In (iv '), the first solid phase is exchanged for the second solid phase. In other words, the first solid phase is removed from the system and the second solid phase is charged to the system. Thereby, the non-target nucleic acid adsorbed nonspecifically on the first solid phase can be removed from the system.
　（ｖｉ’）では、第２液相が第３液相に交換される。換言すれば、標的核酸が捕捉された第２固相が、第２液相から第３液相に移される。これにより、第２固相に捕捉されなかった、第２液相中に含まれる非標的核酸（例、第１捕捉プローブのヌクレオチド配列と相補的な部分ヌクレオチド配列を有し得る、第１固相に捕捉されていた非標的核酸）を系から除去することができる。 (Vi ′), the second liquid phase is exchanged with the third liquid phase. In other words, the second solid phase in which the target nucleic acid is captured is transferred from the second liquid phase to the third liquid phase. Thereby, the non-target nucleic acid contained in the second liquid phase that was not captured by the second solid phase (for example, the first solid phase that may have a partial nucleotide sequence complementary to the nucleotide sequence of the first capture probe) Can be removed from the system.
　（ｉ’）～（ｖｉ’）において、液相としては、水、または緩衝液（例、Ｔｒｉｓ緩衝液、リン酸緩衝液）等の水溶系溶媒を用いることができる。また、（ｉ’）における液相としては、修飾核酸塩基を含む標的核酸、および非標的核酸を含む核酸サンプルを利用することができる。 In (i ′) to (vi ′), water or an aqueous solvent such as a buffer solution (eg, Tris buffer solution or phosphate buffer solution) can be used as the liquid phase. In addition, as the liquid phase in (i ′), a target nucleic acid containing a modified nucleobase and a nucleic acid sample containing a non-target nucleic acid can be used.
　（ｖｉ’）の結果、標的核酸を捕捉した第２固相、および第３液相を含む系が得られる。この系を、後述する工程（２）に付すことにより、標的核酸に含まれる修飾核酸塩基を測定することができる。第２固相に捕捉された標的核酸は、標的核酸に含まれる修飾核酸塩基の測定前に、固相から放出されてもよい。第２固相に捕捉された状態、または第２固相から放出された状態（例、捕捉プローブとのハイブリッドの形態、または標的核酸単独の形態）にある標的核酸において、修飾核酸塩基を測定することができる。 As a result of (vi ′), a system including the second solid phase and the third liquid phase capturing the target nucleic acid is obtained. By subjecting this system to the step (2) described later, the modified nucleobase contained in the target nucleic acid can be measured. The target nucleic acid captured by the second solid phase may be released from the solid phase before measurement of the modified nucleobase contained in the target nucleic acid. The modified nucleobase is measured in the target nucleic acid in the state captured by the second solid phase or released from the second solid phase (eg, in the form of a hybrid with the capture probe, or in the form of the target nucleic acid alone). be able to.
　好ましい実施形態では、（１）は、以下を含む方法により行うことができる：
（１－１）（ａ）核酸サンプルに含まれる標的核酸、（ｂ）第１親和性物質で標識された、標的核酸に対する第１捕捉プローブ、および（ｃ）第１親和性物質と特異的に結合する能力を有する物質で標識された第１固相を溶液中で反応させて、標的核酸および前記第１捕捉プローブを含む第１核酸ハイブリッドが固定された第１固相を含む第１溶液を得ること；
（１－２）前記第１核酸ハイブリッドが固定された第１固相を、第１溶液から第２溶液に移すこと；
（１－３）前記第１核酸ハイブリッドが固定された第１固相から標的核酸を第２溶液中に放出させて、標的核酸および第１固相を含む第１放出溶液を得ること；
（１－４）第１固相を第１放出溶液から除去して、標的核酸を含み、かつ第１固相を含まない第２放出溶液を得ること；ならびに
（１－５）（ａ’）前記第２放出溶液に含まれる標的核酸、（ｂ’）第２親和性物質で標識された、標的核酸に対する第２捕捉プローブ、および（ｃ’）第２親和性物質と特異的に結合する能力を有する物質で標識された第２固相を反応させて、標的核酸および前記第２捕捉プローブを含む第２核酸ハイブリッドが固定された第２固相を含む第３溶液を得ること；
（１－６）前記第２核酸ハイブリッドが固定された第２固相を、前記第３溶液から第４溶液に移すこと。 In a preferred embodiment, (1) can be performed by a method comprising:
(1-1) (a) a target nucleic acid contained in a nucleic acid sample, (b) a first capture probe for the target nucleic acid labeled with a first affinity substance, and (c) specific to the first affinity substance A first solid phase labeled with a substance having an ability to bind is reacted in a solution, and a first solution including a first solid phase to which a first nucleic acid hybrid including a target nucleic acid and the first capture probe is immobilized is prepared. Getting;
(1-2) transferring the first solid phase on which the first nucleic acid hybrid is immobilized from the first solution to the second solution;
(1-3) obtaining a first release solution containing the target nucleic acid and the first solid phase by releasing the target nucleic acid into the second solution from the first solid phase to which the first nucleic acid hybrid is fixed;
(1-4) removing the first solid phase from the first release solution to obtain a second release solution containing the target nucleic acid and not containing the first solid phase; and (1-5) (a ′) A target nucleic acid contained in the second release solution; (b ′) a second capture probe for the target nucleic acid labeled with a second affinity substance; and (c ′) an ability to specifically bind to the second affinity substance. Reacting a second solid phase labeled with a substance having: to obtain a third solution comprising a second solid phase to which a second nucleic acid hybrid comprising a target nucleic acid and the second capture probe is immobilized;
(1-6) Transfer the second solid phase on which the second nucleic acid hybrid is immobilized from the third solution to the fourth solution.
　（１－１）において、反応は、（ａ）～（ｃ）を同時または別々にインキュベーションすることにより行うことができる。インキュベーションは、上述したとおりである。このような反応により、標的核酸および第１捕捉プローブを含む第１核酸ハイブリッドが固定された第１固相を含む第１溶液が得られる。第１親和性物質、および第１親和性物質と特異的に結合する能力を有する物質としては、上述したような親和性物質を用いることができる。 In (1-1), the reaction can be performed by incubating (a) to (c) simultaneously or separately. Incubation is as described above. By such a reaction, a first solution including a first solid phase to which a first nucleic acid hybrid including a target nucleic acid and a first capture probe is fixed is obtained. As the first affinity substance and the substance having the ability to specifically bind to the first affinity substance, the above-mentioned affinity substances can be used.
　反応が同時に行われる場合、（ａ）、（ｂ）および（ｃ）を含む溶液を調製し、次いでインキュベートすることにより、反応を行うことができる。一方、反応が別々に行われる場合、先ず、（ａ）および（ｂ）を反応させて第１核酸ハイブリッドを形成し、次いで、形成された第１核酸ハイブリッドを（ｃ）と反応させて、標的核酸および第１捕捉プローブを含む第１核酸ハイブリッドが固定された第１固相を調製することができる。あるいは、先ず、（ｂ）および（ｃ）を反応させて、捕捉プローブが固定された第１固相を形成し、次いで、この第１固相を（ａ）と反応させて、標的核酸および第１捕捉プローブを含む第１核酸ハイブリッドが固定された第１固相を調製してもよい。 When the reaction is performed simultaneously, the reaction can be performed by preparing a solution containing (a), (b) and (c) and then incubating. On the other hand, when the reactions are performed separately, first, (a) and (b) are reacted to form a first nucleic acid hybrid, and then the formed first nucleic acid hybrid is reacted with (c) to form a target. A first solid phase can be prepared on which a first nucleic acid hybrid comprising a nucleic acid and a first capture probe is immobilized. Alternatively, first, (b) and (c) are reacted to form a first solid phase on which the capture probe is immobilized, and then this first solid phase is reacted with (a) to obtain the target nucleic acid and the first nucleic acid. A first solid phase on which a first nucleic acid hybrid containing one capture probe is immobilized may be prepared.
　（１－２）において、標的核酸および第１捕捉プローブを含む第１核酸ハイブリッドが固定された第１固相が、第１溶液から第２溶液に移される。これにより、第１溶液中に含まれていた、第１固相に捕捉されなかった非標的核酸（第１溶液中に含まれていた非標的核酸）を除去することができる。第１溶液から第１固相を取り出した後、第２溶液に移す前に、このような第１固相を洗浄してもよい。これにより、第１固相に付着している第１溶液中に含まれる非標的核酸をさらに除去することができる。洗浄は、適切な界面活性剤を含む緩衝液（例、Ｔｒｉｓ緩衝液、リン酸緩衝液）で１回以上（例、１～３回）行うことができる。 In (1-2), the first solid phase on which the first nucleic acid hybrid including the target nucleic acid and the first capture probe is immobilized is transferred from the first solution to the second solution. Thereby, the non-target nucleic acid (non-target nucleic acid contained in the first solution) that was not captured by the first solid phase and contained in the first solution can be removed. After removing the first solid phase from the first solution, such a first solid phase may be washed before transferring to the second solution. Thereby, the non-target nucleic acid contained in the 1st solution adhering to the 1st solid phase can further be removed. Washing can be performed one or more times (eg, 1 to 3 times) with a buffer solution (eg, Tris buffer solution, phosphate buffer solution) containing an appropriate surfactant.
　（１－３）において、標的核酸の第２溶液中への放出は、上述したような処理により行うことができる。標的核酸は、捕捉プローブとのハイブリッドの形態、または単独の形態で第２溶液中に放出される。これにより、標的核酸および第１固相を含む第１放出溶液が得られる。 In (1-3), the target nucleic acid can be released into the second solution by the treatment as described above. The target nucleic acid is released into the second solution in the form of a hybrid with the capture probe or in a single form. Thereby, a first release solution containing the target nucleic acid and the first solid phase is obtained.
　（１－４）において、第１固相が第１放出溶液から除去される。これにより、標的核酸を含み、かつ第１固相を含まない第２放出溶液が得られる。これにより、第１固相に吸着した非標的核酸を除去することができる。第１放出溶液からの第１固相の除去は、第１放出溶液と第１固相との分離を可能にする任意の様式において行うことができる。 (1-4), the first solid phase is removed from the first release solution. Thereby, a second release solution containing the target nucleic acid and not containing the first solid phase is obtained. Thereby, the non-target nucleic acid adsorbed on the first solid phase can be removed. Removal of the first solid phase from the first release solution can be performed in any manner that allows separation of the first release solution and the first solid phase.
　（１－５）において、反応は、（ａ’）～（ｃ’）を同時または別々にインキュベーションすることにより行うことができる。反応を同時または別々に行う場合の詳細は、（１－１）と同様である。インキュベーションは、上述したとおりである。このような反応により、標的核酸および前記第２捕捉プローブを含む第２核酸ハイブリッドが固定された第２固相を含む第３溶液が得られる。第２親和性物質、および第２親和性物質と特異的に結合する能力を有する物質としては、上述したような親和性物質を用いることができる。 In (1-5), the reaction can be performed by incubating (a ′) to (c ′) simultaneously or separately. The details for carrying out the reaction simultaneously or separately are the same as in (1-1). Incubation is as described above. By such a reaction, a third solution containing the second solid phase on which the second nucleic acid hybrid containing the target nucleic acid and the second capture probe is immobilized is obtained. As the second affinity substance and the substance having the ability to specifically bind to the second affinity substance, the affinity substance as described above can be used.
　（１－５）において、第２捕捉プローブとして、第１捕捉プローブと異なる捕捉プローブを用いることで、標的核酸を特異的に固相に捕捉することができる。特に、第１捕捉プローブおよび第２捕捉プローブの組み合わせとして、標的核酸の５’末端領域にハイブリダイズする能力を有する捕捉プローブ、および標的核酸の３’末端領域にハイブリダイズする能力を有する捕捉プローブを併用することにより、標的核酸を特異的に捕捉することができる。 In (1-5), the target nucleic acid can be specifically captured on the solid phase by using a capture probe different from the first capture probe as the second capture probe. In particular, as a combination of the first capture probe and the second capture probe, a capture probe having the ability to hybridize to the 5 ′ end region of the target nucleic acid and a capture probe having the ability to hybridize to the 3 ′ end region of the target nucleic acid By using in combination, the target nucleic acid can be specifically captured.
　（１－６）において、第２核酸ハイブリッドが固定された第２固相が、第３溶液から第４溶液に移される。これにより、第３溶液中に含まれていた、第２固相に捕捉されなかった非標的核酸（例、第１捕捉プローブのヌクレオチド配列と相補的な部分ヌクレオチド配列を有し得る、第１固相に捕捉されていた非標的核酸）を除去することができる。第３溶液から第２固相を取り出した後、第４溶液に移す前に、このような第２固相を洗浄してもよい。これにより、第２固相に付着している第３溶液中に含まれる非標的核酸をさらに除去することができる。洗浄は、適切な界面活性剤を含む緩衝液（例、Ｔｒｉｓ緩衝液、リン酸緩衝液）で１回以上（例、１～３回）行うことができる。 (1-6), the second solid phase on which the second nucleic acid hybrid is immobilized is transferred from the third solution to the fourth solution. This allows the first solid phase, which may have a partial nucleotide sequence complementary to the nucleotide sequence of the first capture probe (eg, the non-target nucleic acid not captured by the second solid phase) contained in the third solution. Non-target nucleic acid) captured in the phase can be removed. After the second solid phase is taken out from the third solution, such a second solid phase may be washed before being transferred to the fourth solution. Thereby, the non-target nucleic acid contained in the 3rd solution adhering to the 2nd solid phase can further be removed. Washing can be performed one or more times (eg, 1 to 3 times) with a buffer solution (eg, Tris buffer solution, phosphate buffer solution) containing an appropriate surfactant.
　（１－１）～（１－６）において、溶液としては、水、または緩衝液（例、Ｔｒｉｓ緩衝液、リン酸緩衝液）等の水溶系溶媒を用いることができる。また、（１－１）における溶液としては、修飾核酸塩基を含む標的核酸、および非標的核酸を含む核酸サンプルを利用することができる。 In (1-1) to (1-6), as the solution, water or an aqueous solvent such as a buffer solution (eg, Tris buffer solution, phosphate buffer solution) can be used. As the solution in (1-1), a target nucleic acid containing a modified nucleobase and a nucleic acid sample containing a non-target nucleic acid can be used.
　（１－６）の結果、第２核酸ハイブリッドが固定された第２固相を含む第４溶液が得られる。この第４溶液を、後述する工程（２）に付すことにより、標的核酸に含まれる修飾核酸塩基を測定することができる。第２固相に捕捉された標的核酸は、標的核酸に含まれる修飾核酸塩基の測定前に、固相から放出されてもよい。第２固相に捕捉された状態、または第２固相から放出された状態（例、捕捉プローブとのハイブリッドの形態、または標的核酸単独の形態）にある標的核酸において、修飾核酸塩基を測定することができる。 (1-6) As a result, a fourth solution containing the second solid phase on which the second nucleic acid hybrid is immobilized is obtained. The modified nucleobase contained in the target nucleic acid can be measured by subjecting the fourth solution to the step (2) described later. The target nucleic acid captured by the second solid phase may be released from the solid phase before measurement of the modified nucleobase contained in the target nucleic acid. The modified nucleobase is measured in the target nucleic acid in the state captured by the second solid phase or released from the second solid phase (eg, in the form of a hybrid with the capture probe, or in the form of the target nucleic acid alone). be able to.
　（２）では、標的核酸に含まれる修飾核酸塩基の測定は、当該分野で公知である任意の方法により行うことができる。例えば、測定は、核酸の非増幅的解析方法、または核酸の増幅的解析方法により行うことができる。 In (2), the modified nucleobase contained in the target nucleic acid can be measured by any method known in the art. For example, the measurement can be performed by a nucleic acid non-amplification analysis method or a nucleic acid amplification analysis method.
　核酸の非増幅的解析方法は、標的核酸の増幅工程を伴わない、標的核酸の絶対量を増加させない任意の解析方法である。核酸の非増幅的解析方法としては、例えば、イムノアッセイ（例、国際公開第２０１５／０２５８６２号；国際公開第２０１５／０２５８６３号；国際公開第２０１５／０２５８６４号；国際公開第２０１５／１０８１７７号；ＤＮＡ　Ｒｅｓｅａｒｃｈ　１３，３７－４２（２００６））、質量分析（例、Ａｎａｌｙｔｉｃａｌ　Ｃｈｅｍｉｓｔｒｙ　７７（２），５０４－５１０（２００５））、電気化学分析（例、Ａｎａｌｙｔｉｃａｌ　Ｃｈｅｍｉｓｔｒｙ　８３，７５９５－７５９９（２０１１））、高速液体クロマトグラフィー分析（例、Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ　Ｒｅｓｅａｒｃｈ　３４（８），ｅ６１（２００６））、ナノポア分析またはマイクロポア分析（例、Ｓｃｉｅｎｔｉｆｉｃ　Ｒｅｐｏｒｔｓ　５０１（２），ｓｒｅｐ００５０１（２０１２））が挙げられる。 The nucleic acid non-amplification analysis method is an arbitrary analysis method that does not involve an amplification step of the target nucleic acid and does not increase the absolute amount of the target nucleic acid. Examples of the non-amplification analysis method of nucleic acid include, for example, immunoassay (eg, International Publication No. 2015/025862; International Publication No. 2015/025863; International Publication No. 2015/025864; International Publication No. 2015/108177; DNA Research) 13, 37-42 (2006)), mass spectrometry (eg, Analytical Chemistry 77 (2), 504-510 (2005)), electrochemical analysis (eg, Analytical Chemistry 83, 7595-7599 (2011)), high-speed liquid Chromatographic analysis (eg, Nucleic Acids Research 34 (8), e61 (2006)), nanopore analysis or micropore analysis (eg, Scientific Reports 501 (2) srep00501 (2012)), and the like.
　イムノアッセイにより修飾核酸塩基が測定される場合、抗修飾核酸塩基抗体を用いて標的核酸に含まれる修飾核酸塩基を測定できる。イムノアッセイは、当該分野において公知である任意の免疫学的方法により行うことができる。具体的には、このような方法としては、例えば、酵素免疫測定法（ＥＩＡ）（例、化学発光ＥＩＡ（ＣＬＥＩＡ）、ＥＬＩＳＡ）、蛍光免疫測定法、化学発光免疫測定法、電気化学発光免疫測定法、凝集法、免疫染色、フローメトリー法、バイオレイヤー干渉法、Ｉｎ　Ｓｉｔｕ　ＰＬＡ法、化学増幅型ルミネッセンス・プロキシミティ・ホモジニアス・アッセイ、ラインブロット法、ウエスタンブロット法が挙げられる。 When the modified nucleobase is measured by immunoassay, the modified nucleobase contained in the target nucleic acid can be measured using an anti-modified nucleobase antibody. The immunoassay can be performed by any immunological method known in the art. Specifically, as such a method, for example, enzyme immunoassay (EIA) (eg, chemiluminescence EIA (CLEIA), ELISA), fluorescence immunoassay, chemiluminescence immunoassay, electrochemiluminescence immunoassay Method, agglutination method, immunostaining, flowmetry method, biolayer interferometry, In Situ PLA method, chemically amplified luminescence proximity homogenous assay, line blot method, Western blot method.
　質量分析、電気化学分析、または高速液体クロマトグラフィー分析により修飾核酸塩基が測定される場合、標的核酸をヌクレアーゼ（例、エンドヌクレアーゼ、エキソヌクレアーゼ）により分解して、標的核酸の分解産物であるモノマー単位（ヌクレオチド）を含む溶液を回収した後、この溶液をこのような技術に付すことで、標的核酸に含まれる修飾核酸塩基を測定できる。 When a modified nucleobase is measured by mass spectrometry, electrochemical analysis, or high performance liquid chromatography analysis, the target nucleic acid is degraded by a nuclease (eg, endonuclease, exonuclease), and a monomer unit that is a degradation product of the target nucleic acid After collecting the solution containing (nucleotide), the modified nucleobase contained in the target nucleic acid can be measured by subjecting this solution to such a technique.
　ナノポア分析またはマイクロポア分析により修飾核酸塩基が解析される場合、標的核酸を固相から放出させて標的核酸を含む溶液を得た後、この溶液をナノポア分析またはマイクロポア分析に付すことで、標的核酸に含まれる修飾核酸塩基を測定できる。 When the modified nucleobase is analyzed by nanopore analysis or micropore analysis, the target nucleic acid is released from the solid phase to obtain a solution containing the target nucleic acid, and then this solution is subjected to nanopore analysis or micropore analysis. The modified nucleobase contained in the nucleic acid can be measured.
　核酸の増幅的解析方法は、標的核酸の増幅工程により標的核酸の絶対量を増加することができる任意の解析方法であり、例えば、１以上のプライマーを用いることにより標的核酸の絶対量を指数関数的または非指数関数的に増幅する方法である。核酸の増幅的解析方法としては、例えば、バイサルファイトシーケンシング法、バイサルファイトパイロシーケンシング法、メチル化特異的ＰＣＲ法、単一ヌクレオチド・プライマー伸長（ＳＮｕＰＥ）法、メチライト法、ＣＯＢＲＡ法、メチル化特異的ＭＬＰＡ法が挙げられる。これらの方法は、当該分野において周知である（例、特開２０１２－０９０５５５号公報、特開２０１４－０３６６７２号公報、国際公開第２００９／０３７６３５号）。 The nucleic acid amplification analysis method is an arbitrary analysis method capable of increasing the absolute amount of the target nucleic acid by the target nucleic acid amplification step. For example, by using one or more primers, the absolute amount of the target nucleic acid is expressed as an exponential function. This is a method of amplifying in an exponential or non-exponential manner. Examples of nucleic acid amplification analysis methods include bisulfite sequencing, bisulfite pyrosequencing, methylation-specific PCR, single nucleotide primer extension (SNuPE), methylite, COBRA, and methylation. Specific MLPA method is mentioned. These methods are well known in the art (for example, JP 2012-090555 A, JP 2014-036672 A, International Publication No. 2009/037635).
　好ましくは、測定は、標的核酸の非増幅的解析方法により行うことができる。本発明の方法によれば、（１）により、修飾核酸塩基を含む非標的核酸が特異的に除去されていることから、標的核酸の増幅を要することなく、修飾核酸塩基を含む標的核酸を正確に測定できるためである。この場合、本発明の方法は、標的核酸の増幅工程を一切要せずに行うことができる。 Preferably, the measurement can be performed by a non-amplifying analysis method of the target nucleic acid. According to the method of the present invention, since the non-target nucleic acid containing the modified nucleobase is specifically removed by (1), the target nucleic acid containing the modified nucleobase can be accurately identified without requiring amplification of the target nucleic acid. This is because it can be measured. In this case, the method of the present invention can be carried out without requiring any target nucleic acid amplification step.
　本発明では、核酸サンプル中の所望の核酸の量及び割合（例、核酸サンプル中の標的核酸量、または核酸サンプル中の核酸量および核酸量に対して予測される標的核酸の割合）を考慮して、修飾核酸塩基による標的核酸の修飾頻度を評価してもよい。　 The present invention takes into account the amount and proportion of the desired nucleic acid in the nucleic acid sample (eg, the amount of target nucleic acid in the nucleic acid sample, or the amount of nucleic acid in the nucleic acid sample and the predicted target nucleic acid relative to the amount of nucleic acid). Thus, the modification frequency of the target nucleic acid by the modified nucleobase may be evaluated. *
　以下、本発明を実施例により詳細に説明するが、本発明は、これらの実施例に限定されるものではない。 Hereinafter, the present invention will be described in detail by way of examples. However, the present invention is not limited to these examples.
参考例１：メチルシトシンを含む標的核酸の調製
　標的核酸は下記に示す手順で調製した。標的核酸の調製には、ポリメラーゼ・チェーン・リアクション（ＰＣＲ）法を使用した。ＰＣＲ用の酵素としては東洋紡績社製ＫＯＤ　Ｐｌｕｓ（商品番号：ＫＯＤ－２０１）、核酸増幅用の２種類のプライマーとしては北海道システムサイエンス社により人工合成された、フォワードプライマー：５’－ＴＡＧ　ＡＡＣ　ＧＣＴ　ＴＴＧ　ＣＧＴ　ＣＣＣ　ＧＡＣ－３’（配列番号１）、リバースプライマー：５’－ＣＴＧ　ＣＡＧ　ＧＡＣ　ＣＡＣ　ＴＣＧ　ＡＧＧ　ＣＴＧ－３’（配列番号２）を使用した。ＰＣＲ増幅のプロトコールは、９４℃で２分間加熱した後、９４℃を１５秒、５５℃を３０秒、６８℃を１分、を１セットとして３０サイクルとした。 Reference Example 1: Preparation of target nucleic acid containing methylcytosine Target nucleic acid was prepared by the following procedure. For the preparation of the target nucleic acid, the polymerase chain reaction (PCR) method was used. The enzyme for PCR is KOD Plus (product number: KOD-201) manufactured by Toyobo Co., Ltd. The two primers for nucleic acid amplification are artificially synthesized by Hokkaido System Science Co., Ltd. Forward primer: 5′-TAG AAC GCT TTG CGT CCC GAC-3 ′ (SEQ ID NO: 1) and reverse primer: 5′-CTG CAG GAC CAC TCG AGG CTG-3 ′ (SEQ ID NO: 2) were used. In the PCR amplification protocol, after heating at 94 ° C. for 2 minutes, 94 ° C. for 15 seconds, 55 ° C. for 30 seconds, 68 ° C. for 1 minute, and 30 cycles.
　北海道システムサイエンス社により人工合成された核酸（ヌクレオチド配列：５’－ＴＡＧ　ＡＡＣ　ＧＣＴ　ＴＴＧ　ＣＧＴ　ＣＣＣ　ＧＡＣ　ＧＣＣ　ＣＧＣ　ＡＧＧ　ＴＣＣ　ＴＣＧ　ＣＧＧ　ＴＧＣ　ＧＣＡ　ＣＣＧ　ＴＴＴ　ＧＣＧ　ＡＣＴ　ＴＧＧ　ＴＧＡ　ＧＴＧ　ＴＣＴ　ＧＧＧ　ＴＣＧ　ＣＣＴ　ＣＧＣ　ＴＣＣ　ＣＧＧ　ＡＡＧ　ＡＧＴ　ＧＣＧ　ＧＡＧ　ＣＴＣ　ＴＣＣ　ＣＴＣ　ＧＧＧ　ＡＣＧ　ＧＴＧ　ＧＣＡ　ＧＣＣ　ＴＣＧ　ＡＧＴ　ＧＧＴ　ＣＣＴ　ＧＣＡ－３’（配列番号３））を鋳型として用いてＰＣＲ増幅を行った後、キアゲン社ＱＩＡｑｕｉｃｋ　ＰＣＲ　Ｐｕｒｉｆｉｃａｔｉｏｎ　Ｋｉｔを使用して精製することで、１３８塩基対の核酸を調製した。 Nucleic acid artificially synthesized by Hokkaido System Science (Nucleotide sequence: 5'-TAG AAC GCT TTG CGT CCC GAC GCC CGC AGG TCC TCG CGG TGG GCG CCG TTT GCG ACT G By performing PCR amplification using CTC TCC CTC CTC GGG ACG GTG GCA GCC TCG AGT GGT CCT GCA-3 ′ (SEQ ID NO: 3) as a template and then purifying using Qiagen QIAquick PCR Purification Kit 138 Base pair nucleic acids were prepared.
　上記で調製した核酸中のＣｐＧのシトシンをメチル化するため、Ｔｈｅｒｍｏ　Ｓｃｉｅｎｔｉｆｉｃ社ＣｐＧ　Ｍｅｔｈｙｌｔｒａｎｓｆｅｒａｓｅ（Ｍ．ＳｓｓＩ）（商品番号：ＥＭ０８２１）で処理を行った。反応溶液は添付文書に従って調製した。３７℃で２０分間反応させた後、６５℃でさらに２０分間反応させた後、キアゲン社ＱＩＡｑｕｉｃｋ　Ｎｕｃｌｅｏｔｉｄｅ　Ｒｅｍｏｖａｌ　Ｋｉｔを使用して精製することで、５－メチルシトシンを含む標的核酸を得た。 In order to methylate CpG cytosine in the nucleic acid prepared above, treatment was performed with Thermo Scientific CpG Methyltransferase (M. SssI) (product number: EM0821). The reaction solution was prepared according to the package insert. After reacting at 37 ° C. for 20 minutes and further reacting at 65 ° C. for 20 minutes, purification was performed using Qiagen QIAquick Nucleotide Removal Kit to obtain a target nucleic acid containing 5-methylcytosine.
参考例２：捕捉プローブによる非標的核酸の非特異的な捕捉
　標的核酸を捕捉するための核酸プローブである捕捉プローブ１は５’－ＵＧＣ　ＡＧＧ　ＡＣＣ　ＡＣＵ　ＣＧＡ　ＧＧＣ　ＵＧＣ　ＣＡＣ－３’（配列番号４）（核酸の主鎖は２’－Ｏ－メチル化ＲＮＡ、５’末端はビオチン標識）であり、北海道システムサイエンス社により人工合成されたものを使用した。非標的核酸として、サケ精液由来ゲノムＤＮＡ（インビトロジェン社製）を使用した。 Reference Example 2: Non-specific capture of non-target nucleic acid by capture probe Capture probe 1 which is a nucleic acid probe for capturing a target nucleic acid is 5′-UGC AGG ACC ACU CGA GGC UGC CAC-3 ′ (SEQ ID NO: 4) (The main chain of the nucleic acid is 2′-O-methylated RNA, and the 5 ′ end is labeled with biotin), which was artificially synthesized by Hokkaido System Science. Salmon semen-derived genomic DNA (manufactured by Invitrogen) was used as a non-target nucleic acid.
　まず、サケ精液由来ゲノムＤＮＡ（５０μｇ）と捕捉プローブ（１ｐｍｏｌ）を緩衝液（１００ｍＭＴｒｉｓ－Ｃｌ、１．５Ｍイミダゾール、５０ｍＭＥＤＴＡ・２Ｎａ）１００μＬ中に溶解させた。また、捕捉プローブを含まない溶液も同様に調製した。９５℃で５分間反応させた後、３７℃で３０分間反応させた。反応後の溶液に、２５０μｇ／ｍＬのストレプトアビジンでコートされた磁性粒子（自社調製品）を５０μＬ加え、３７℃で１０分反応させることで、磁性粒子上に捕捉プローブ１を固定化した。２５０μＬのＴＢＳ－Ｔで３回洗浄し、１００ｎｇ／ｍＬのアルカリフォスファターゼ標識抗メチルシトシン抗体（ニッポンジーン社製抗メチルシトシン抗体Ｃｌｏｎｅ３３Ｄ３を同仁化学社製Ａｌｋａｌｉｎｅ　Ｐｈｏｓｐｈａｔａｓｅ　Ｌａｂｅｌｉｎｇ　Ｋｉｔ－ＳＨ（商品番号：ＬＫ１３）を用いてアルカリフォスファターゼ標識）を１００μＬずつ加え、３７℃で１時間反応させた。２５０μＬのＴＢＳ－Ｔで６回洗浄した後、化学発光基質ＡＭＰＰＤ溶液を１００μＬずつ加え、３７℃で５分反応させた。その後、マイクロプレートリーダー（ベルトールド社製Ｃｅｎｔｒｏ　ＬＢ９６０）により発光カウントを測定した。 First, salmon semen-derived genomic DNA (50 μg) and a capture probe (1 pmol) were dissolved in 100 μL of a buffer solution (100 mM Tris-Cl, 1.5 M imidazole, 50 mM EDTA · 2Na). A solution containing no capture probe was prepared in the same manner. After reacting at 95 ° C. for 5 minutes, it was reacted at 37 ° C. for 30 minutes. 50 μL of magnetic particles coated with 250 μg / mL streptavidin (in-house preparation) were added to the solution after the reaction and reacted at 37 ° C. for 10 minutes to immobilize the capture probe 1 on the magnetic particles. Wash 3 times with 250 μL of TBS-T and use 100 ng / mL alkaline phosphatase-labeled anti-methylcytosine antibody (Nippon Gene anti-methylcytosine antibody Clone33D3, Dokindo Chemicals Alkaline Phosphatase Labeling Kit-SH (product number: LK13)) 100 μL of alkaline phosphatase label) was added and reacted at 37 ° C. for 1 hour. After washing 6 times with 250 μL of TBS-T, 100 μL of the chemiluminescent substrate AMPPD solution was added and reacted at 37 ° C. for 5 minutes. Thereafter, the luminescence count was measured with a microplate reader (Centro LB960 manufactured by Bertoled).
　その結果、捕捉プローブが存在する場合、捕捉プローブが存在しない場合と比較して発光カウントが高かった（表１、図１）。 As a result, when the capture probe was present, the luminescence count was higher than when the capture probe was not present (Table 1, FIG. 1).
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　以上より、捕捉プローブが非標的核酸を非特異的に捕捉することにより、非標的核酸由来のバックグラウンドシグナルが上昇すると考えられた。 From the above, it was considered that the background signal derived from the non-target nucleic acid was increased when the capture probe captured the non-target nucleic acid non-specifically.
実施例１：標的核酸に含まれる修飾核酸塩基の特異的な検出による非標的核酸由来シグナルの低減
　本実施例では、２つの異なる捕捉プローブを用いる本発明の方法、ならびに１つの捕捉プローブを用いる従来法について、非標的核酸由来シグナル（バックグラウンドシグナル）の低減に対する効果を評価した。 Example 1: Reduction of signal derived from non-target nucleic acid by specific detection of modified nucleobase contained in target nucleic acid In this example, the method of the present invention using two different capture probes as well as the conventional using one capture probe The method was evaluated for its effect on reducing non-target nucleic acid-derived signals (background signal).
（１－１）２つの異なる捕捉プローブを用いる本発明の方法による、標的核酸に含まれるメチルシトシンの特異的な検出
　捕捉プローブ１は５’－ＵＧＣ　ＡＧＧ　ＡＣＣ　ＡＣＵ　ＣＧＡ　ＧＧＣ　ＵＧＣ　ＣＡＣ－３’（配列番号４）（核酸の主鎖は２’－Ｏ－メチル化ＲＮＡ、５’末端はビオチン標識）、捕捉プローブ２は５’－ＧＵＣ　ＧＧＧ　ＡＣＧ　ＣＡＡ　ＡＧＣ　ＧＵＵ　ＣＵＡ－３’（配列番号５）（核酸の主鎖は２’－Ｏ－メチル化ＲＮＡ、３’末端はビオチン標識）であり、北海道システムサイエンス社により人工合成されたものを使用した。５－メチルシトシンを含む標的核酸は参考例１で調製したものを使用した。非標的核酸として、サケ精液由来ゲノムＤＮＡ（インビトロジェン社製）を使用した。 (1-1) Specific detection of methylcytosine contained in a target nucleic acid by the method of the present invention using two different capture probes Capture probe 1 is 5′-UGC AGG ACC ACU CGA GGC UGC CAC-3 ′ (sequence No. 4) (the main chain of the nucleic acid is 2′-O-methylated RNA, the 5 ′ end is labeled with biotin), the capture probe 2 is 5′-GUC GGG ACG CAA AGC GUU CUA-3 ′ (SEQ ID NO: 5) (nucleic acid No. 5) The main chain was 2′-O-methylated RNA and the 3 ′ end was labeled with biotin, and was artificially synthesized by Hokkaido System Science. The target nucleic acid containing 5-methylcytosine was prepared in Reference Example 1. Salmon semen-derived genomic DNA (manufactured by Invitrogen) was used as a non-target nucleic acid.
　まず、５－メチルシトシンを含む標的核酸（１００ａｍｏｌ）またはサケ精液由来ゲノムＤＮＡ（２００μｇ）と、標的核酸を捕捉するための捕捉プローブ２（１ｐｍｏｌ）を緩衝液（１００ｍＭＴｒｉｓ－Ｃｌ、１．５Ｍイミダゾール、５０ｍＭＥＤＴＡ・２Ｎａ）１００μＬ中に溶解させた。９５℃で５分間反応させた後、３７℃で３０分間反応させた。また、５－メチルシトシンを含む標的核酸およびサケ精液由来ゲノムＤＮＡを共に含まない溶液も調製し、同様の操作を行った。反応後の溶液に、３７５μｇ／ｍＬのストレプトアビジンでコートされた磁性粒子（ＪＳＲ社Ｍａｇｎｏｓｐｈｅｒｅ　ＭＳ３００／Ｓｔｒｅｐｔａｖｉｄｉｎ）を５０μＬ加え、３７℃で１０分反応させることで、磁性粒子上に捕捉プローブ２を固定化した。２００μＬのＴＢＳ－Ｔで２回洗浄した後、５０ｍＭ　ＮａＯＨ水溶液を２０μＬ加え、室温で５分間インキュベートすることで、捕捉プローブとハイブリッドを形成していた核酸を反応溶液中に放出させた。磁性粒子を集磁して除いた後、全量の反応上清を、標的核酸を捕捉するための捕捉プローブ１（１ｐｍｏｌ）を含む緩衝液（１００ｍＭＴｒｉｓ－Ｃｌ、１．５Ｍイミダゾール、５０ｍＭＥＤＴＡ・２Ｎａ）８５μＬに加えた。この反応液を９５℃で５分間反応させた後、３７℃で３０分間反応させた。反応後の溶液に、２５０μｇ／ｍＬのストレプトアビジンでコートされた磁性粒子（自社調製品）を５０μＬ加え、３７℃で１０分反応させることで、磁性粒子上に捕捉プローブ１を固定化した。２５０μＬのＴＢＳ－Ｔで３回洗浄し、１００ｎｇ／ｍＬのアルカリフォスファターゼ標識抗メチルシトシン抗体（ニッポンジーン社製抗メチルシトシン抗体Ｃｌｏｎｅ３３Ｄ３を同仁化学社製Ａｌｋａｌｉｎｅ　Ｐｈｏｓｐｈａｔａｓｅ　Ｌａｂｅｌｉｎｇ　Ｋｉｔ－ＳＨ（商品番号：ＬＫ１３）を用いてアルカリフォスファターゼ標識）を１００μＬずつ加え、３７℃で１時間反応させた。２５０μＬのＴＢＳ－Ｔで６回洗浄した後、化学発光基質ＡＭＰＰＤ溶液を１００μＬずつ加え、３７℃で５分反応させた。その後、マイクロプレートリーダー（ベルトールド社製Ｃｅｎｔｒｏ　ＬＢ９６０）により発光カウントを測定した。 First, a target nucleic acid (100 amol) containing 5-methylcytosine or a genomic DNA derived from salmon semen (200 μg) and a capture probe 2 (1 pmol) for capturing the target nucleic acid are buffered (100 mM Tris-Cl, 1.5 M imidazole, 50 mM EDTA · 2Na) was dissolved in 100 μL. After reacting at 95 ° C. for 5 minutes, it was reacted at 37 ° C. for 30 minutes. Further, a solution not containing both the target nucleic acid containing 5-methylcytosine and the genomic DNA derived from salmon semen was prepared, and the same operation was performed. 50 μL of magnetic particles (JSR Magnosphere MS300 / Streptavidin) coated with 375 μg / mL streptavidin are added to the solution after the reaction and reacted at 37 ° C. for 10 minutes to immobilize the capture probe 2 on the magnetic particles. did. After washing twice with 200 μL of TBS-T, 20 μL of 50 mM NaOH aqueous solution was added and incubated at room temperature for 5 minutes to release the nucleic acid that had formed a hybrid with the capture probe into the reaction solution. After collecting and removing the magnetic particles, 85 μL of a buffer solution (100 mM Tris-Cl, 1.5 M imidazole, 50 mM EDTA · 2Na) containing the capture probe 1 (1 pmol) for capturing the target nucleic acid is collected from the entire reaction supernatant. Added to. This reaction solution was reacted at 95 ° C. for 5 minutes and then reacted at 37 ° C. for 30 minutes. 50 μL of magnetic particles coated with 250 μg / mL streptavidin (in-house preparation) were added to the solution after the reaction and reacted at 37 ° C. for 10 minutes to immobilize the capture probe 1 on the magnetic particles. Wash 3 times with 250 μL of TBS-T and use 100 ng / mL alkaline phosphatase-labeled anti-methylcytosine antibody (Nippon Gene anti-methylcytosine antibody Clone33D3, Dokindo Chemicals Alkaline Phosphatase Labeling Kit-SH (product number: LK13)) 100 μL of alkaline phosphatase label) was added and reacted at 37 ° C. for 1 hour. After washing 6 times with 250 μL of TBS-T, 100 μL of the chemiluminescent substrate AMPPD solution was added and reacted at 37 ° C. for 5 minutes. Thereafter, the luminescence count was measured with a microplate reader (Centro LB960 manufactured by Bertoled).
（１－２）１つの捕捉プローブを用いる従来法による、標的核酸に含まれるメチルシトシンの検出
　捕捉プローブ１は５’－ＵＧＣ　ＡＧＧ　ＡＣＣ　ＡＣＵ　ＣＧＡ　ＧＧＣ　ＵＧＣ　ＣＡＣ－３’（配列番号４）（核酸の主鎖は２’－Ｏ－メチル化ＲＮＡ、５’末端はビオチン標識）であり、北海道システムサイエンス社により人工合成されたものを使用した。５－メチルシトシンを含む標的核酸は参考例１で調製したものを使用した。非標的核酸として、サケ精液由来ゲノムＤＮＡ（インビトロジェン社製）を使用した。 (1-2) Detection of methylcytosine contained in a target nucleic acid by a conventional method using one capture probe Capture probe 1 is 5′-UGC AGG ACC ACU CGA GGC UGC CAC-3 ′ (SEQ ID NO: 4) The main chain was 2′-O-methylated RNA and the 5 ′ end was labeled with biotin), which was artificially synthesized by Hokkaido System Science. The target nucleic acid containing 5-methylcytosine was prepared in Reference Example 1. Salmon semen-derived genomic DNA (manufactured by Invitrogen) was used as a non-target nucleic acid.
　まず、５－メチルシトシンを含む標的核酸（１００ａｍｏｌ）またはサケ精液由来ゲノムＤＮＡ（２００μｇ）と、捕捉プローブ１（１ｐｍｏｌ）を緩衝液（１００ｍＭＴｒｉｓ－Ｃｌ、１．５Ｍイミダゾール、５０ｍＭＥＤＴＡ・２Ｎａ）１００μＬ中に溶解させた。９５℃で５分間反応させた後、３７℃で３０分間反応させた。また、５－メチルシトシンを含む標的核酸およびサケ精液由来ゲノムＤＮＡを共に含まない溶液も調製し、同様の操作を行った。反応後の溶液に、２５０μｇ／ｍＬのストレプトアビジンでコートされた磁性粒子（自社調製品）を５０μＬ加え、３７℃で１０分反応させることで、磁性粒子上に捕捉プローブ１を固定化した。２５０μＬのＴＢＳ－Ｔで３回洗浄し、１００ｎｇ／ｍＬのアルカリフォスファターゼ標識抗メチルシトシン抗体（ニッポンジーン社製抗メチルシトシン抗体Ｃｌｏｎｅ３３Ｄ３を同仁化学社製Ａｌｋａｌｉｎｅ　Ｐｈｏｓｐｈａｔａｓｅ　Ｌａｂｅｌｉｎｇ　Ｋｉｔ－ＳＨ（商品番号：ＬＫ１３）を用いてアルカリフォスファターゼ標識）を１００μＬずつ加え、３７℃で１時間反応させた。２５０μＬのＴＢＳ－Ｔで６回洗浄した後、化学発光基質ＡＭＰＰＤ溶液を１００μＬずつ加え、３７℃で５分反応させた。その後、マイクロプレートリーダー（ベルトールド社製Ｃｅｎｔｒｏ　ＬＢ９６０）により発光カウントを測定した。 First, target nucleic acid (100 amol) containing 5-methylcytosine or genomic DNA derived from salmon semen (200 μg) and capture probe 1 (1 pmol) in 100 μL of buffer (100 mM Tris-Cl, 1.5 M imidazole, 50 mM EDTA · 2Na) Dissolved. After reacting at 95 ° C. for 5 minutes, it was reacted at 37 ° C. for 30 minutes. Further, a solution not containing both the target nucleic acid containing 5-methylcytosine and the genomic DNA derived from salmon semen was prepared, and the same operation was performed. 50 μL of magnetic particles coated with 250 μg / mL streptavidin (in-house preparation) were added to the solution after the reaction and reacted at 37 ° C. for 10 minutes to immobilize the capture probe 1 on the magnetic particles. Wash 3 times with 250 μL of TBS-T and use 100 ng / mL alkaline phosphatase-labeled anti-methylcytosine antibody (Nippon Gene anti-methylcytosine antibody Clone33D3, Dokindo Chemicals Alkaline Phosphatase Labeling Kit-SH (product number: LK13)) 100 μL of alkaline phosphatase label) was added and reacted at 37 ° C. for 1 hour. After washing 6 times with 250 μL of TBS-T, 100 μL of the chemiluminescent substrate AMPPD solution was added and reacted at 37 ° C. for 5 minutes. Thereafter, the luminescence count was measured with a microplate reader (Centro LB960 manufactured by Bertoled).
（１－３）結果
　従来法では、標的核酸と非標的核酸で発光カウントに差はなかった（表２、図２）。一方、本発明の方法では、非標的核酸は緩衝液のみのサンプルと同等の低い発光カウントしか示さず、標的核酸のみ高い発光カウントを示した（表２、図２）。 (1-3) Results In the conventional method, there was no difference in the luminescence count between the target nucleic acid and the non-target nucleic acid (Table 2, FIG. 2). On the other hand, in the method of the present invention, the non-target nucleic acid showed only a low luminescence count equivalent to that of the buffer-only sample, and only the target nucleic acid showed a high luminescence count (Table 2, FIG. 2).
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　以上より、２つの異なる捕捉プローブを用いる本発明の方法は、１つの捕捉プローブを用いる従来法に比し、非標的核酸由来のバックグラウンドシグナルを特異的に低減できることが示された。 From the above, it was shown that the method of the present invention using two different capture probes can specifically reduce the background signal derived from a non-target nucleic acid as compared with the conventional method using one capture probe.
参考例３：固相への核酸の吸着の評価
　本参考例では、標的核酸を固相に２回捕捉する本発明の方法論（２つの異なる捕捉プローブの使用に起因）、ならびに標的核酸を固相に１回捕捉する従来の方法論（１つの捕捉プローブの使用に起因）について、固相への核酸吸着量を評価した。本発明の方法論による固相への核酸吸着量の評価は、２回目の捕捉後に固相から放出された核酸の量を測定することにより行った。従来の方法論による固相へのＤＮＡ吸着量の評価は、１回目の捕捉後に固相から放出された核酸の量を測定することにより行った。 Reference Example 3: Evaluation of Nucleic Acid Adsorption to the Solid Phase In this reference example, the methodology of the present invention that captures the target nucleic acid twice on the solid phase (due to the use of two different capture probes) and the target nucleic acid to the solid phase The amount of nucleic acid adsorbed to the solid phase was evaluated using a conventional methodology (captured by using one capture probe). Evaluation of the amount of nucleic acid adsorbed to the solid phase by the methodology of the present invention was performed by measuring the amount of nucleic acid released from the solid phase after the second capture. Evaluation of the amount of DNA adsorbed to the solid phase by a conventional methodology was performed by measuring the amount of nucleic acid released from the solid phase after the first capture.
　検討用核酸は参考例１で調製したものを使用した。 The nucleic acid for examination used the one prepared in Reference Example 1.
　まず、検討用核酸（１００ａｍｏｌ）を緩衝液（１００ｍＭＴｒｉｓ－Ｃｌ、１．５Ｍイミダゾール、５０ｍＭＥＤＴＡ・２Ｎａ）１００μＬ中に溶解させた。この溶液に、３７５μｇ／ｍＬのストレプトアビジンでコートされた磁性粒子（ＪＳＲ社Ｍａｇｎｏｓｐｈｅｒｅ　ＭＳ３００／Ｓｔｒｅｐｔａｖｉｄｉｎ）を５０μＬ加え、３７℃で３０分反応させた。２００μＬのＴＢＳ－Ｔで２回洗浄した後、５０ｍＭ　ＮａＯＨ水溶液を２０μＬ加え、室温で５分間インキュベートすることで、磁性粒子上に非特異的に吸着した検討用核酸を反応溶液中に放出させた。 First, the nucleic acid for study (100 amol) was dissolved in 100 μL of a buffer solution (100 mM Tris-Cl, 1.5 M imidazole, 50 mM EDTA · 2Na). To this solution, 50 μL of magnetic particles coated with 375 μg / mL streptavidin (Magnussphere MS300 / Streptavidin, JSR) were added and reacted at 37 ° C. for 30 minutes. After washing twice with 200 μL of TBS-T, 20 μL of 50 mM NaOH aqueous solution was added and incubated at room temperature for 5 minutes to release the nucleic acid for examination adsorbed nonspecifically on the magnetic particles into the reaction solution.
　磁性粒子を集磁して除いた後、反応上清に等量の５０ｍＭ　ＨＣｌ水溶液を加えて中和した後、検討用核酸を増幅可能な位置に設計されたＰｒｉｍｅｒセットを使用したリアルタイムＰＣＲ増幅することにより、磁性粒子上に非特異的に吸着したＤＮＡ量を測定した（標的核酸を固相に１回捕捉する従来の方法論による固相へのＤＮＡ吸着量の評価）。 After collecting and removing the magnetic particles, the reaction supernatant is neutralized by adding an equal amount of 50 mM HCl aqueous solution, and then real-time PCR amplification is performed using a Primer set designed to amplify the nucleic acid for study. Thus, the amount of non-specifically adsorbed DNA on the magnetic particles was measured (evaluation of the amount of adsorbed DNA on the solid phase by a conventional methodology for capturing the target nucleic acid once on the solid phase).
　また、磁性粒子を集磁して除いた後、１５μＬの反応上清を緩衝液（１００ｍＭＴｒｉｓ－Ｃｌ、１．５Ｍイミダゾール、５０ｍＭＥＤＴＡ・２Ｎａ）８５μＬに加えた。この溶液に、３７５μｇ／ｍＬのストレプトアビジンでコートされた磁性粒子（ＪＳＲ社Ｍａｇｎｏｓｐｈｅｒｅ　ＭＳ３００／Ｓｔｒｅｐｔａｖｉｄｉｎ）を５０μＬ加え、３７℃で３０分反応させた。２００μＬのＴＢＳ－Ｔで２回洗浄した後、５０ｍＭ　ＮａＯＨ水溶液を２０μＬ加え、室温で５分間インキュベートすることで、磁性粒子上に非特異的に吸着した検討用核酸を反応溶液中に放出させた。磁性粒子を集磁して除いた後、反応上清に等量の５０ｍＭ　ＨＣｌ水溶液を加えて中和した後、検討用核酸を増幅可能な位置に設計されたＰｒｉｍｅｒセットを使用したリアルタイムＰＣＲ増幅することにより、磁性粒子上に非特異的に吸着したＤＮＡ量を測定した（標的核酸を固相に２回捕捉する本発明の方法論による固相への核酸吸着量の評価）。 Further, after collecting and removing the magnetic particles, 15 μL of the reaction supernatant was added to 85 μL of a buffer solution (100 mM Tris-Cl, 1.5 M imidazole, 50 mM EDTA · 2Na). To this solution, 50 μL of magnetic particles coated with 375 μg / mL streptavidin (Magnussphere MS300 / Streptavidin, JSR) were added and reacted at 37 ° C. for 30 minutes. After washing twice with 200 μL of TBS-T, 20 μL of 50 mM NaOH aqueous solution was added and incubated at room temperature for 5 minutes to release the nucleic acid for examination adsorbed nonspecifically on the magnetic particles into the reaction solution. After collecting and removing the magnetic particles, the reaction supernatant is neutralized by adding an equal amount of 50 mM HCl aqueous solution, and then real-time PCR amplification is performed using a Primer set designed to amplify the nucleic acid for study. Thus, the amount of non-specifically adsorbed DNA on the magnetic particles was measured (evaluation of the amount of nucleic acid adsorbed to the solid phase by the methodology of the present invention in which the target nucleic acid was captured twice on the solid phase).
　リアルタイムＰＣＲ増幅の詳細を以下に示す。
Ｐｒｅｍｉｘ　ＰＣＲ試薬（ＫＯＤ　ＳＹＢＲ　ｑＰＣＲ　Ｍｉｘ：ＴＯＹＯＢＯ社製）：１２．５μＬ
Ｆｏｒｗａｒｄ　Ｐｒｉｍｅｒ（１０μＭ）：０．５μＬ
Ｒｅｖｅｒｓｅ　Ｐｒｉｍｅｒ（１０μＭ）：０．５μＬ
５０ｘ　ＲＯＸ　ｒｅｆｅｒｅｎｃｅ　ｄｙｅ：０．０５μＬ
磁性粒子に吸着したＤＮＡサンプル：２μＬ
Ｔｏｔａｌ：２５μＬ Details of real-time PCR amplification are shown below.
Premix PCR reagent (KOD SYBR qPCR Mix: manufactured by TOYOBO): 12.5 μL
Forward Primer (10 μM): 0.5 μL
Reverse Primer (10 μM): 0.5 μL
50x ROX reference dye: 0.05 μL
DNA sample adsorbed on magnetic particles: 2 μL
Total: 25 μL
　検討用核酸を増幅可能な位置に設計されたＰｒｉｍｅｒセットは、Ｆｏｒｗａｒｄ　Ｐｒｉｍｅｒのヌクレオチド配列は５’－ＴＡＧ　ＡＡＣ　ＧＣＴ　ＴＴＧ　ＣＧＴ　ＣＣＣ　ＧＡＣ－３’（配列番号１）、Ｒｅｖｅｒｓｅ　Ｐｒｉｍｅｒのヌクレオチド配列は５’－ＧＡＧ　ＡＧＣ　ＴＣＣ　ＧＣＡ　ＣＴＣ　ＴＴＣ　Ｃ－３’（配列番号６）であり、北海道システムサイエンス社により人工合成されたものを使用した。 The Primer set designed to amplify the nucleic acid to be studied has a Forward Primer nucleotide sequence of 5'-TAG AAC GCT TTG CGT CCC GAC-3 '(SEQ ID NO: 1), and a Reverse Primer nucleotide sequence of 5'- GAG AGC TCC GCA CTC TTC C-3 ′ (SEQ ID NO: 6), which was artificially synthesized by Hokkaido System Science, was used.
　上記組成の反応溶液を以下のプロトコールに従って、増幅反応を行った。
（１）９８℃、２分
（２）９８℃、１０秒
（３）６８℃、１分 An amplification reaction was performed on the reaction solution having the above composition according to the following protocol.
(1) 98 ° C, 2 minutes (2) 98 ° C, 10 seconds (3) 68 ° C, 1 minute
　反応ステップ（１）を行ってから、反応ステップ（２）～（３）を５０サイクル繰り返した。 After performing reaction step (1), reaction steps (2) to (3) were repeated 50 cycles.
　その結果、２回捕捉した場合に固相に吸着した核酸量は、１回捕捉した場合のものに比し、非常に少なかった（表３、図３）。 As a result, the amount of nucleic acid adsorbed on the solid phase when captured twice was very small compared to the amount captured once (Table 3, FIG. 3).
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　以上より、標的核酸を固相に２回捕捉する本発明の方法論は、標的核酸を固相に１回しか捕捉しない本発明の方法論に比し、固相に非特異的に吸着する核酸量を大幅に低減できることが示された。 From the above, the methodology of the present invention in which the target nucleic acid is captured twice on the solid phase is compared with the methodology of the present invention in which the target nucleic acid is captured only once on the solid phase, and the amount of nucleic acid adsorbed nonspecifically on the solid phase is reduced. It was shown that it can be greatly reduced.
実施例２：標的核酸に対する２つの捕捉プローブのハイブリダイズ部位による標的核酸の特異的な検出の評価
　捕捉プローブ１は５’－ＵＧＣ　ＡＧＧ　ＡＣＣ　ＡＣＵ　ＣＧＡ　ＧＧＣ　ＵＧＣ　ＣＡＣ－３’（配列番号４）（核酸の主鎖は２’－Ｏ－メチル化ＲＮＡ、５’末端はビオチン標識）、捕捉プローブ２は５’－ＧＵＣ　ＧＧＧ　ＡＣＧ　ＣＡＡ　ＡＧＣ　ＧＵＵ　ＣＵＡ－３’（配列番号５）（核酸の主鎖は２’－Ｏ－メチル化ＲＮＡ、３’末端はビオチン標識）、捕捉プローブ３は５’－ＡＣＣ　ＣＡＧ　ＡＣＡ　ＣＵＣ　ＡＣＣ　ＡＡＧ　ＵＣ－３’（配列番号７）（核酸の主鎖は２’－Ｏ－メチル化ＲＮＡ、５’末端はビオチン標識）であり、北海道システムサイエンス社により人工合成されたものを使用した。５－メチルシトシンを含む標的核酸は参考例１で調製したものを使用した。非標的核酸として、サケ精液由来ゲノムＤＮＡ（インビトロジェン社製）を使用した。 Example 2 Evaluation of Specific Detection of Target Nucleic Acid by Hybridization Site of Two Capture Probes to Target Nucleic Acid Capture probe 1 is 5′-UGC AGG ACC ACU CGA GGC UGC CAC-3 ′ (SEQ ID NO: 4) (Nucleic acid 2′-O-methylated RNA, 5 ′ end is labeled with biotin), and capture probe 2 is 5′-GUC GGG ACG CAA AGC GUU CUA-3 ′ (SEQ ID NO: 5) (SEQ ID NO: 5) '-O-methylated RNA, 3' end is labeled with biotin), capture probe 3 is 5'-ACC CAG ACA CUC ACC AAG UC-3 '(SEQ ID NO: 7) (nucleic acid main chain is 2'-O-methyl RNA, 5′-end is biotin-labeled) and was artificially synthesized by Hokkaido System Science. The target nucleic acid containing 5-methylcytosine was prepared in Reference Example 1. Salmon semen-derived genomic DNA (manufactured by Invitrogen) was used as a non-target nucleic acid.
　まず、５－メチルシトシンを含む標的核酸（１００ａｍｏｌまたは１ｆｍｏｌ）またはサケ精液由来ゲノムＤＮＡ（２００μｇ）と、捕捉プローブ（１ｐｍｏｌ；捕捉プローブ２または捕捉プローブ３）を緩衝液（１００ｍＭＴｒｉｓ－Ｃｌ、１．５Ｍイミダゾール、５０ｍＭＥＤＴＡ・２Ｎａ）１００μＬ中に溶解させた。９５℃で５分間反応させた後、３７℃で３０分間反応させた。また、５－メチルシトシンを含む標的核酸およびサケ精液由来ゲノムＤＮＡを共に含まない溶液も調製し、同様の操作を行った。反応後の溶液に、３７５μｇ／ｍＬのストレプトアビジンでコートされた磁性粒子（ＪＳＲ社Ｍａｇｎｏｓｐｈｅｒｅ　ＭＳ３００／Ｓｔｒｅｐｔａｖｉｄｉｎ）を５０μＬ加え、３７℃で１０分反応させることで、磁性粒子上に捕捉プローブを固定化した。２００μＬのＴＢＳ－Ｔで２回洗浄した後、５０ｍＭ　ＮａＯＨ水溶液を２０μＬ加え、室温で５分間インキュベートすることで、捕捉プローブとハイブリッドを形成していた核酸を反応溶液中に放出させた。磁性粒子を集磁して除いた後、全量の反応上清を捕捉プローブ（１ｐｍｏｌ；捕捉プローブ１または捕捉プローブ３）を含む緩衝液（１００ｍＭＴｒｉｓ－Ｃｌ、１．５Ｍイミダゾール、５０ｍＭＥＤＴＡ・２Ｎａ）８５μＬに加えた。この反応液を９５℃で５分間反応させた後、３７℃で３０分間反応させた。反応後の溶液に、２５０μｇ／ｍＬのストレプトアビジンでコートされた磁性粒子（自社調製品）を５０μＬ加え、３７℃で１０分反応させることで、磁性粒子上に捕捉プローブを固定化した。２５０μＬのＴＢＳ－Ｔで３回洗浄し、１００ｎｇ／ｍＬのアルカリフォスファターゼ標識抗メチルシトシン抗体（ニッポンジーン社製抗メチルシトシン抗体Ｃｌｏｎｅ３３Ｄ３を同仁化学社製Ａｌｋａｌｉｎｅ　Ｐｈｏｓｐｈａｔａｓｅ　Ｌａｂｅｌｉｎｇ　Ｋｉｔ－ＳＨ（商品番号：ＬＫ１３）を用いてアルカリフォスファターゼ標識）を１００μＬずつ加え、３７℃で１時間反応させた。２５０μＬのＴＢＳ－Ｔで６回洗浄した後、化学発光基質ＡＭＰＰＤ溶液を１００μＬずつ加え、３７℃で５分反応させた。その後、マイクロプレートリーダー（ベルトールド社製Ｃｅｎｔｒｏ　ＬＢ９６０）により発光カウントを測定した。 First, a target nucleic acid (100 amol or 1 fmol) containing 5-methylcytosine or salmon semen-derived genomic DNA (200 μg) and a capture probe (1 pmol; capture probe 2 or capture probe 3) are added to a buffer solution (100 mM Tris-Cl, 1.5 M). (Imidazole, 50 mM EDTA · 2Na) was dissolved in 100 μL. After reacting at 95 ° C. for 5 minutes, it was reacted at 37 ° C. for 30 minutes. Further, a solution not containing both the target nucleic acid containing 5-methylcytosine and the genomic DNA derived from salmon semen was prepared, and the same operation was performed. 50 μL of magnetic particles coated with 375 μg / mL streptavidin (JSR Magnosphere MS300 / Streptavidin) were added to the solution after the reaction and reacted at 37 ° C. for 10 minutes to immobilize the capture probe on the magnetic particles. . After washing twice with 200 μL of TBS-T, 20 μL of 50 mM NaOH aqueous solution was added and incubated at room temperature for 5 minutes to release the nucleic acid that had formed a hybrid with the capture probe into the reaction solution. After collecting and removing the magnetic particles, the entire amount of the reaction supernatant was added to 85 μL of a buffer solution (100 mM Tris-Cl, 1.5 M imidazole, 50 mM EDTA · 2Na) containing a capture probe (1 pmol; capture probe 1 or capture probe 3). added. This reaction solution was reacted at 95 ° C. for 5 minutes and then reacted at 37 ° C. for 30 minutes. 50 μL of magnetic particles coated with 250 μg / mL streptavidin (in-house preparation) were added to the solution after the reaction and reacted at 37 ° C. for 10 minutes to immobilize the capture probe on the magnetic particles. Wash 3 times with 250 μL of TBS-T and use 100 ng / mL alkaline phosphatase-labeled anti-methylcytosine antibody (Nippon Gene anti-methylcytosine antibody Clone33D3, Dokindo Chemicals Alkaline Phosphatase Labeling Kit-SH (product number: LK13)) 100 μL of alkaline phosphatase label) was added and reacted at 37 ° C. for 1 hour. After washing 6 times with 250 μL of TBS-T, 100 μL of the chemiluminescent substrate AMPPD solution was added and reacted at 37 ° C. for 5 minutes. Thereafter, the luminescence count was measured with a microplate reader (Centro LB960 manufactured by Bertoled).
　その結果、異なる２つの捕捉プローブの組み合わせにより発光カウントが異なり、２つの捕捉プローブを標的核酸の両末端領域にハイブリダイズできるように設計した場合、発光カウントが最も高かった（表４、図４）。 As a result, the luminescence count was different depending on the combination of two different capture probes, and the luminescence count was the highest when the two capture probes were designed to hybridize to both end regions of the target nucleic acid (Table 4, FIG. 4). .
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　以上より、標的核酸に対する２つの捕捉プローブを標的核酸の両末端領域にハイブリダイズできるように設計した場合、標的核酸の特異的な検出に優れることが示された。 From the above, it has been shown that when two capture probes for a target nucleic acid are designed to be hybridizable to both end regions of the target nucleic acid, it is excellent in specific detection of the target nucleic acid.
実施例３：標的核酸および非標的核酸の混合核酸サンプルにおける、標的核酸に含まれる修飾核酸塩基の特異的な検出
　標的核酸および非標的核酸を混合することにより得られた核酸サンプルにおいて、２つの異なる捕捉プローブを用いる本発明の方法が標的核酸に含まれる修飾核酸塩基を特異的に検出できるか検討した。 Example 3 Specific Detection of Modified Nucleobases Included in Target Nucleic Acids in Mixed Nucleic Acid Samples of Target and Non-Target Nucleic Acids Two different nucleic acid samples obtained by mixing target and non-target nucleic acids It was investigated whether the method of the present invention using a capture probe can specifically detect a modified nucleobase contained in a target nucleic acid.
　捕捉プローブ１は５’－ＵＧＣ　ＡＧＧ　ＡＣＣ　ＡＣＵ　ＣＧＡ　ＧＧＣ　ＵＧＣ　ＣＡＣ－３’（配列番号４）（核酸の主鎖は２’－Ｏ－メチル化ＲＮＡ、５’末端はビオチン標識）、捕捉プローブ２は５’－ＧＵＣ　ＧＧＧ　ＡＣＧ　ＣＡＡ　ＡＧＣ　ＧＵＵ　ＣＵＡ－３’（配列番号５）（核酸の主鎖は２’－Ｏ－メチル化ＲＮＡ、３’末端はビオチン標識）であり、北海道システムサイエンス社により人工合成されたものを使用した。５－メチルシトシンを含む標的核酸は参考例１で調製したものを使用した。非標的核酸として、サケ精液由来ゲノムＤＮＡ（インビトロジェン社製）を使用した。 Capture probe 1 is 5′-UGC AGG ACC ACU CGA GGC UGC CAC-3 ′ (SEQ ID NO: 4) (nucleic acid main chain is 2′-O-methylated RNA, 5 ′ end is labeled with biotin), capture probe 2 is 5'-GUC GGG ACG CAA AGC GUU CUA-3 '(SEQ ID NO: 5) (nucleic acid main chain is 2'-O-methylated RNA, 3' end is labeled with biotin), artificially synthesized by Hokkaido System Science We used what was done. The target nucleic acid containing 5-methylcytosine was prepared in Reference Example 1. Salmon semen-derived genomic DNA (manufactured by Invitrogen) was used as a non-target nucleic acid.
　まず、５－メチルシトシンを含む標的核酸（１０ａｍｏｌ、１００ａｍｏｌ、または１ｆｍｏｌ）および／またはサケ精液由来ゲノムＤＮＡ（５０μｇ）と、捕捉プローブ２（１ｐｍｏｌ）を緩衝液（１００ｍＭＴｒｉｓ－Ｃｌ、１．５Ｍイミダゾール、５０ｍＭＥＤＴＡ・２Ｎａ）１００μＬ中に溶解させた。９５℃で５分間反応させた後、３７℃で３０分間反応させた。また、５－メチルシトシンを含む標的核酸およびサケ精液由来ゲノムＤＮＡを共に含まない溶液も調製し、同様の操作を行った。反応後の溶液に、３７５μｇ／ｍＬのストレプトアビジンでコートされた磁性粒子（ＪＳＲ社Ｍａｇｎｏｓｐｈｅｒｅ　ＭＳ３００／Ｓｔｒｅｐｔａｖｉｄｉｎ）を５０μＬ加え、３７℃で１０分反応させることで、磁性粒子上に捕捉プローブ２を固定化した。２００μＬのＴＢＳ－Ｔで２回洗浄した後、５０ｍＭ　ＮａＯＨ水溶液を２０μＬ加え、室温で５分間インキュベートすることで、捕捉プローブとハイブリッドを形成していた核酸を反応溶液中に放出させた。磁性粒子を集磁して除いた後、全量の反応上清を捕捉プローブ１（１ｐｍｏｌ）を含む緩衝液（１００ｍＭＴｒｉｓ－Ｃｌ、１．５Ｍイミダゾール、５０ｍＭＥＤＴＡ・２Ｎａ）８５μＬに加えた。この反応液を９５℃で５分間反応させた後、３７℃で３０分間反応させた。反応後の溶液に、２５０μｇ／ｍＬのストレプトアビジンでコートされた磁性粒子（自社調製品）を５０μＬ加え、３７℃で１０分反応させることで、磁性粒子上に捕捉プローブ１を固定化した。２５０μＬのＴＢＳ－Ｔで３回洗浄し、１００ｎｇ／ｍＬのアルカリフォスファターゼ標識抗メチルシトシン抗体（ニッポンジーン社製抗メチルシトシン抗体Ｃｌｏｎｅ３３Ｄ３を同仁化学社製Ａｌｋａｌｉｎｅ　Ｐｈｏｓｐｈａｔａｓｅ　Ｌａｂｅｌｉｎｇ　Ｋｉｔ－ＳＨ（商品番号：ＬＫ１３）を用いてアルカリフォスファターゼ標識）を１００μＬずつ加え、３７℃で１時間反応させた。２５０μＬのＴＢＳ－Ｔで６回洗浄した後、化学発光基質ＡＭＰＰＤ溶液を１００μＬずつ加え、３７℃で５分反応させた。その後、マイクロプレートリーダー（ベルトールド社製Ｃｅｎｔｒｏ　ＬＢ９６０）により発光カウントを測定した。 First, target nucleic acid (10 amol, 100 amol, or 1 fmol) containing 5-methylcytosine and / or salmon semen-derived genomic DNA (50 μg) and capture probe 2 (1 pmol) are added to a buffer (100 mM Tris-Cl, 1.5 M imidazole, 50 mM EDTA · 2Na) was dissolved in 100 μL. After reacting at 95 ° C. for 5 minutes, it was reacted at 37 ° C. for 30 minutes. Further, a solution not containing both the target nucleic acid containing 5-methylcytosine and the genomic DNA derived from salmon semen was prepared, and the same operation was performed. 50 μL of magnetic particles (JSR Magnosphere MS300 / Streptavidin) coated with 375 μg / mL streptavidin are added to the solution after the reaction and reacted at 37 ° C. for 10 minutes to immobilize the capture probe 2 on the magnetic particles. did. After washing twice with 200 μL of TBS-T, 20 μL of 50 mM NaOH aqueous solution was added and incubated at room temperature for 5 minutes to release the nucleic acid that had formed a hybrid with the capture probe into the reaction solution. After collecting and removing the magnetic particles, the entire amount of the reaction supernatant was added to 85 μL of a buffer solution (100 mM Tris-Cl, 1.5 M imidazole, 50 mM EDTA · 2Na) containing capture probe 1 (1 pmol). This reaction solution was reacted at 95 ° C. for 5 minutes and then reacted at 37 ° C. for 30 minutes. 50 μL of magnetic particles coated with 250 μg / mL streptavidin (in-house preparation) were added to the solution after the reaction and reacted at 37 ° C. for 10 minutes to immobilize the capture probe 1 on the magnetic particles. Wash 3 times with 250 μL of TBS-T and use 100 ng / mL alkaline phosphatase-labeled anti-methylcytosine antibody (Nippon Gene anti-methylcytosine antibody Clone33D3, Dokindo Chemicals Alkaline Phosphatase Labeling Kit-SH (product number: LK13)) 100 μL of alkaline phosphatase label) was added and reacted at 37 ° C. for 1 hour. After washing 6 times with 250 μL of TBS-T, 100 μL of the chemiluminescent substrate AMPPD solution was added and reacted at 37 ° C. for 5 minutes. Thereafter, the luminescence count was measured with a microplate reader (Centro LB960 manufactured by Bertoled).
　比較のため、標的核酸に含まれる修飾核酸塩基を、１つの捕捉プローブを用いる従来法によっても測定した。従来法による測定は、まず、５－メチルシトシンを含む標的核酸（１０ａｍｏｌ、１００ａｍｏｌ、または１ｆｍｏｌ）および／またはサケ精液由来ゲノムＤＮＡ（５０μｇ）と、捕捉プローブ１（１ｐｍｏｌ）を緩衝液（１００ｍＭＴｒｉｓ－Ｃｌ、１．５Ｍイミダゾール、５０ｍＭＥＤＴＡ・２Ｎａ）１００μＬ中に溶解させた。９５℃で５分間反応させた後、３７℃で３０分間反応させた。また、５－メチルシトシンを含む標的核酸およびサケ精液由来ゲノムＤＮＡを共に含まない溶液も調製し、同様の操作を行った。反応後の溶液に、２５０μｇ／ｍＬのストレプトアビジンでコートされた磁性粒子（自社調製品）を５０μＬ加え、３７℃で１０分反応させることで、磁性粒子上に捕捉プローブ１を固定化した。２５０μＬのＴＢＳ－Ｔで３回洗浄し、１００ｎｇ／ｍＬのアルカリフォスファターゼ標識抗メチルシトシン抗体（ニッポンジーン社製抗メチルシトシン抗体Ｃｌｏｎｅ３３Ｄ３を同仁化学社製Ａｌｋａｌｉｎｅ　Ｐｈｏｓｐｈａｔａｓｅ　Ｌａｂｅｌｉｎｇ　Ｋｉｔ－ＳＨ（商品番号：ＬＫ１３）を用いてアルカリフォスファターゼ標識）を１００μＬずつ加え、３７℃で１時間反応させた。２５０μＬのＴＢＳ－Ｔで６回洗浄した後、化学発光基質ＡＭＰＰＤ溶液を１００μＬずつ加え、３７℃で５分反応させた。その後、マイクロプレートリーダー（ベルトールド社製Ｃｅｎｔｒｏ　ＬＢ９６０）により発光カウントを測定した。 For comparison, the modified nucleobase contained in the target nucleic acid was also measured by a conventional method using one capture probe. In the measurement by the conventional method, first, target nucleic acid (10 amol, 100 amol, or 1 fmol) and / or salmon semen-derived genomic DNA (50 μg) containing 5-methylcytosine and capture probe 1 (1 pmol) are added to a buffer (100 mM Tris-Cl , 1.5 M imidazole, 50 mM EDTA · 2Na) in 100 μL. After reacting at 95 ° C. for 5 minutes, it was reacted at 37 ° C. for 30 minutes. Further, a solution not containing both the target nucleic acid containing 5-methylcytosine and the genomic DNA derived from salmon semen was prepared, and the same operation was performed. 50 μL of magnetic particles coated with 250 μg / mL streptavidin (in-house preparation) were added to the solution after the reaction and reacted at 37 ° C. for 10 minutes to immobilize the capture probe 1 on the magnetic particles. Wash 3 times with 250 μL of TBS-T and use 100 ng / mL alkaline phosphatase-labeled anti-methylcytosine antibody (Nippon Gene anti-methylcytosine antibody Clone33D3, Dokindo Chemicals Alkaline Phosphatase Labeling Kit-SH (product number: LK13)) 100 μL of alkaline phosphatase label) was added and reacted at 37 ° C. for 1 hour. After washing 6 times with 250 μL of TBS-T, 100 μL of the chemiluminescent substrate AMPPD solution was added and reacted at 37 ° C. for 5 minutes. Thereafter, the luminescence count was measured with a microplate reader (Centro LB960 manufactured by Bertoled).
　その結果、従来法では、標的核酸および非標的核酸の混合核酸サンプルにおいて測定された発光カウントは、非標的核酸を含まない標的核酸の核酸サンプルにおいて測定されたものよりも高く、非標的核酸に対する標的核酸の相対量が低い場合に発光カウントが顕著に高くなる傾向が認められた（表５、図５）。一方、本発明の方法では、標的核酸および非標的核酸の混合核酸サンプルにおいて測定された発光カウントは、非標的核酸に対する標的核酸の相対量にかかわらず、非標的核酸を含まない標的核酸の核酸サンプルにおいて測定されたものと同等であった（表５、図６）。 As a result, in the conventional method, the luminescence count measured in the mixed nucleic acid sample of the target nucleic acid and the non-target nucleic acid is higher than that measured in the nucleic acid sample of the target nucleic acid not containing the non-target nucleic acid, and the target against the non-target nucleic acid When the relative amount of nucleic acid was low, the luminescence count tended to increase significantly (Table 5, FIG. 5). On the other hand, in the method of the present invention, the luminescence count measured in the mixed nucleic acid sample of the target nucleic acid and the non-target nucleic acid is the nucleic acid sample of the target nucleic acid not containing the non-target nucleic acid regardless of the relative amount of the target nucleic acid with respect to the non-target nucleic acid. (Table 5, FIG. 6).
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　以上より、２つの異なる捕捉プローブを用いる本発明の方法は、標的核酸および非標的核酸の混合核酸サンプル（ゲノムＤＮＡ含有サンプル）において、非標的核酸のバックグランドシグナルを大幅に低減しつつ、標的核酸に含まれる修飾核酸塩基を検出できることが示された。また、標的核酸および非標的核酸の混合核酸サンプルにおいて非標的核酸に対する標的核酸の相対量が低い場合（例、非標的核酸１μｇ当たりの標的核酸モル数が２０ａｍｏｌ未満の量において標的核酸を含む核酸サンプルを用いる場合）、本発明の方法が特に優れることが示された。 As described above, in the method of the present invention using two different capture probes, in the mixed nucleic acid sample (sample containing genomic DNA) of the target nucleic acid and the non-target nucleic acid, the background signal of the non-target nucleic acid is greatly reduced, It was shown that the modified nucleobase contained in can be detected. In addition, when the relative amount of the target nucleic acid with respect to the non-target nucleic acid is low in the mixed nucleic acid sample of the target nucleic acid and the non-target nucleic acid (eg, the nucleic acid sample containing the target nucleic acid in an amount of less than 20 amol of target nucleic acid per 1 μg of non-target nucleic acid) The process of the present invention has been shown to be particularly superior.
実施例４：天然の生物学的サンプル中の標的核酸に含まれる修飾核酸塩基の特異的な検出
　天然の生物学的サンプルである細胞から抽出された、標的核酸および非標的核酸を含む核酸サンプルにおいて、２つの異なる捕捉プローブを用いる本発明の方法が標的核酸に含まれる修飾核酸塩基を特異的に検出できるか検討した。本検討では、本発明の方法、および１つの捕捉プローブを用いる従来法を、既知手法であるバイサルファイトパイロシーケンシング法（標的核酸の増幅を伴う解析方法）と比較することにより行った。 Example 4: Specific Detection of Modified Nucleobases Included in Target Nucleic Acids in Natural Biological Samples In a nucleic acid sample containing target and non-target nucleic acids extracted from cells that are natural biological samples It was investigated whether the method of the present invention using two different capture probes could specifically detect the modified nucleobase contained in the target nucleic acid. In this study, the method of the present invention and the conventional method using one capture probe were compared with a known method, bisulfite pyrosequencing method (analysis method involving amplification of target nucleic acid).
　培養細胞Ａ１７２、Ｕ８７－ＭＧ、Ｔ４７Ｄは、ＤＳファーマバイオメディカル社より購入したものを培養後、Ｂｌｏｏｄ＆Ｃｅｌｌ　Ｃｕｌｔｕｒｅ　ＤＮＡ　Ｍａｘｉ　Ｋｉｔ（ＱＩＡＧＥＮ社、ＰＮ１３３６２）を用いてゲノムＤＮＡの抽出を行った。 Cultured cells A172, U87-MG, and T47D were cultured from those purchased from DS Pharma Biomedical, and then genomic DNA was extracted using Blood & Cell Culture DNA Maxi Kit (QIAGEN, PN13362).
　捕捉プローブ１は５’－ＵＧＣ　ＡＧＧ　ＡＣＣ　ＡＣＵ　ＣＧＡ　ＧＧＣ　ＵＧＣ　ＣＡＣ－３’（配列番号４）（核酸の主鎖は２’－Ｏ－メチル化ＲＮＡ、５’末端はビオチン標識）、捕捉プローブ２は５’－ＧＵＣ　ＧＧＧ　ＡＣＧ　ＣＡＡ　ＡＧＣ　ＧＵＵ　ＣＵＡ－３’（配列番号５）（核酸の主鎖は２’－Ｏ－メチル化ＲＮＡ、３’末端はビオチン標識）であり、北海道システムサイエンス社により人工合成されたものを使用した。 Capture probe 1 is 5′-UGC AGG ACC ACU CGA GGC UGC CAC-3 ′ (SEQ ID NO: 4) (nucleic acid main chain is 2′-O-methylated RNA, 5 ′ end is labeled with biotin), capture probe 2 is 5'-GUC GGG ACG CAA AGC GUU CUA-3 '(SEQ ID NO: 5) (nucleic acid main chain is 2'-O-methylated RNA, 3' end is labeled with biotin), artificially synthesized by Hokkaido System Science We used what was done.
　まず、３Ｅ＋７コピー相当の培養細胞由来ゲノムＤＮＡと１２ｕｎｉｔｓの制限酵素ＰｓｔＩ（タカラバイオ社製）、４８ｕｎｉｔｓの制限酵素ＸｓｐＩ（タカラバイオ社製）を、反応Ｂｕｆｆｅｒ（２０ｍＭＴｒｉｓ－ＨＣｌ（ｐＨ＝８．５）、１０ｍＭＭｇＣｌ２、１ｍＭＤＴＴ、１００ｍＭＫＣｌ）８０μＬ中に溶解させ、３７℃で２４時間反応させた後、８０℃で１０分間反応させることで、制限酵素により切断されたゲノムＤＮＡを得た。 First, genomic DNA derived from cultured cells corresponding to 3E + 7 copies, 12 units of restriction enzyme PstI (manufactured by Takara Bio Inc.), and 48 units of restriction enzyme XspI (manufactured by Takara Bio Inc.) were reacted with Buffer (20 mM Tris-HCl (pH = 8.5)). (10 mM MgCl 2, 1 mM DTT, 100 mM KCl) dissolved in 80 μL, reacted at 37 ° C. for 24 hours, and then reacted at 80 ° C. for 10 minutes to obtain genomic DNA cleaved by the restriction enzyme.
　次に、制限酵素切断済みのゲノムＤＮＡと、標的核酸を捕捉するための捕捉プローブ２（１ｐｍｏｌ）を緩衝液（１００ｍＭＴｒｉｓ－Ｃｌ、１．５Ｍイミダゾール、５０ｍＭＥＤＴＡ・２Ｎａ）１６０μＬ中に溶解させた。９５℃で５分間反応させた後、３７℃で３０分間反応させた。また、ゲノムＤＮＡを含まない反応溶液も調製し、同様の操作を行った。反応後の溶液に、３７５μｇ／ｍＬのストレプトアビジンでコートされた磁性粒子（ＪＳＲ社Ｍａｇｎｏｓｐｈｅｒｅ　ＭＳ３００／Ｓｔｒｅｐｔａｖｉｄｉｎ）を５０μＬ加え、３７℃で１０分反応させることで、磁性粒子上に捕捉プローブ２を固定化した。２００μＬのＴＢＳ－Ｔで２回洗浄した後、５０ｍＭ　ＮａＯＨ水溶液を２０μＬ加え、室温で５分間インキュベートすることで、捕捉プローブとハイブリッドを形成していた核酸を反応溶液中に放出させた。磁性粒子を集磁して除いた後、全量の反応上清を捕捉プローブ１（１ｐｍｏｌ）を含む緩衝液（１００ｍＭＴｒｉｓ－Ｃｌ、１．５Ｍイミダゾール、５０ｍＭＥＤＴＡ・２Ｎａ）８５μＬに加えた。この反応液を９５℃で５分間反応させた後、３７℃で３０分間反応させた。反応後の溶液に、２５０μｇ／ｍＬのストレプトアビジンでコートされた磁性粒子（自社調製品）を５０μＬ加え、３７℃で１０分反応させることで、磁性粒子上に捕捉プローブ１を固定化した。２５０μＬのＴＢＳ－Ｔで３回洗浄し、１００ｎｇ／ｍＬのアルカリフォスファターゼ標識抗メチルシトシン抗体（ニッポンジーン社製抗メチルシトシン抗体Ｃｌｏｎｅ３３Ｄ３を同仁化学社製Ａｌｋａｌｉｎｅ　Ｐｈｏｓｐｈａｔａｓｅ　Ｌａｂｅｌｉｎｇ　Ｋｉｔ－ＳＨ（商品番号：ＬＫ１３）を用いてアルカリフォスファターゼ標識）を１００μＬずつ加え、３７℃で１時間反応させた。２５０μＬのＴＢＳ－Ｔで６回洗浄した後、化学発光基質ＡＭＰＰＤ溶液を１００μＬずつ加え、３７℃で５分反応させた。その後、マイクロプレートリーダー（ベルトールド社製Ｃｅｎｔｒｏ　ＬＢ９６０）により発光カウントを測定した。 Next, restriction enzyme-cut genomic DNA and capture probe 2 (1 pmol) for capturing the target nucleic acid were dissolved in 160 μL of a buffer solution (100 mM Tris-Cl, 1.5 M imidazole, 50 mM EDTA · 2Na). After reacting at 95 ° C. for 5 minutes, it was reacted at 37 ° C. for 30 minutes. A reaction solution not containing genomic DNA was also prepared and the same operation was performed. 50 μL of magnetic particles (JSR Magnosphere MS300 / Streptavidin) coated with 375 μg / mL streptavidin are added to the solution after the reaction and reacted at 37 ° C. for 10 minutes to immobilize the capture probe 2 on the magnetic particles. did. After washing twice with 200 μL of TBS-T, 20 μL of 50 mM NaOH aqueous solution was added and incubated at room temperature for 5 minutes to release the nucleic acid that had formed a hybrid with the capture probe into the reaction solution. After collecting and removing the magnetic particles, the entire amount of the reaction supernatant was added to 85 μL of a buffer solution (100 mM Tris-Cl, 1.5 M imidazole, 50 mM EDTA · 2Na) containing capture probe 1 (1 pmol). This reaction solution was reacted at 95 ° C. for 5 minutes and then reacted at 37 ° C. for 30 minutes. 50 μL of magnetic particles coated with 250 μg / mL streptavidin (in-house preparation) were added to the solution after the reaction and reacted at 37 ° C. for 10 minutes to immobilize the capture probe 1 on the magnetic particles. Wash 3 times with 250 μL of TBS-T and use 100 ng / mL alkaline phosphatase-labeled anti-methylcytosine antibody (Nippon Gene anti-methylcytosine antibody Clone33D3, Dokindo Chemicals Alkaline Phosphatase Labeling Kit-SH (product number: LK13)) 100 μL of alkaline phosphatase label) was added and reacted at 37 ° C. for 1 hour. After washing 6 times with 250 μL of TBS-T, 100 μL of the chemiluminescent substrate AMPPD solution was added and reacted at 37 ° C. for 5 minutes. Thereafter, the luminescence count was measured with a microplate reader (Centro LB960 manufactured by Bertoled).
　次に、標的核酸に含まれる修飾核酸塩基を、１つの捕捉プローブを用いる従来法によっても測定した。従来法による測定は、まず、３Ｅ＋７コピー相当の培養細胞由来ゲノムＤＮＡと１２ｕｎｉｔｓの制限酵素ＰｓｔＩ（タカラバイオ社製）、４８ｕｎｉｔｓの制限酵素ＸｓｐＩ（タカラバイオ社製）を、反応Ｂｕｆｆｅｒ（２０ｍＭＴｒｉｓ－ＨＣｌ（ｐＨ＝８．５）、１０ｍＭＭｇＣｌ２、１ｍＭＤＴＴ、１００ｍＭＫＣｌ）８０μＬ中に溶解させ、３７℃で２４時間反応させた後、８０℃で１０分間反応させることで、制限酵素により切断されたゲノムＤＮＡを得た。
　次に、制限酵素切断済みのゲノムＤＮＡと、標的核酸を捕捉するための捕捉プローブ１（１ｐｍｏｌ）を緩衝液（１００ｍＭＴｒｉｓ－Ｃｌ、１．５Ｍイミダゾール、５０ｍＭＥＤＴＡ・２Ｎａ）１６０μＬ中に溶解させた。９５℃で５分間反応させた後、３７℃で３０分間反応させた。また、ゲノムＤＮＡを含まない反応溶液も調製し、同様の操作を行った。反応後の溶液に、２５０μｇ／ｍＬのストレプトアビジンでコートされた磁性粒子（自社調製品）を５０μＬ加え、３７℃で１０分反応させることで、磁性粒子上に捕捉プローブ１を固定化した。２５０μＬのＴＢＳ－Ｔで３回洗浄し、１００ｎｇ／ｍＬのアルカリフォスファターゼ標識抗メチルシトシン抗体（ニッポンジーン社製抗メチルシトシン抗体Ｃｌｏｎｅ３３Ｄ３を同仁化学社製Ａｌｋａｌｉｎｅ　Ｐｈｏｓｐｈａｔａｓｅ　Ｌａｂｅｌｉｎｇ　Ｋｉｔ－ＳＨ（商品番号：ＬＫ１３）を用いてアルカリフォスファターゼ標識）を１００μＬずつ加え、３７℃で１時間反応させた。２５０μＬのＴＢＳ－Ｔで６回洗浄した後、化学発光基質ＡＭＰＰＤ溶液を１００μＬずつ加え、３７℃で５分反応させた。その後、マイクロプレートリーダー（ベルトールド社製Ｃｅｎｔｒｏ　ＬＢ９６０）により発光カウントを測定した。 Next, the modified nucleobase contained in the target nucleic acid was also measured by a conventional method using one capture probe. In the measurement by the conventional method, first, genomic DNA derived from 3E + 7 copies of cultured cells, 12 units of restriction enzyme PstI (manufactured by Takara Bio Inc.) and 48 units of restriction enzyme XspI (manufactured by Takara Bio Inc.) pH = 8.5), 10 mM MgCl 2, 1 mM DTT, 100 mM KCl) dissolved in 80 μL, reacted at 37 ° C. for 24 hours, and then reacted at 80 ° C. for 10 minutes to obtain genomic DNA cleaved by the restriction enzyme .
Next, restriction enzyme-cut genomic DNA and capture probe 1 (1 pmol) for capturing the target nucleic acid were dissolved in 160 μL of a buffer (100 mM Tris-Cl, 1.5 M imidazole, 50 mM EDTA · 2Na). After reacting at 95 ° C. for 5 minutes, it was reacted at 37 ° C. for 30 minutes. A reaction solution not containing genomic DNA was also prepared and the same operation was performed. 50 μL of magnetic particles coated with 250 μg / mL streptavidin (in-house preparation) were added to the solution after the reaction and reacted at 37 ° C. for 10 minutes to immobilize the capture probe 1 on the magnetic particles. Washed 3 times with 250 μL of TBS-T, using 100 ng / mL alkaline phosphatase-labeled anti-methylcytosine antibody (Nippon Gene anti-methylcytosine antibody Clone33D3, Dokindo Chemical Co., Ltd. Alkaline Phosphatase Labeling Kit-SH (product number: LK13)) 100 μL of alkaline phosphatase label) was added and reacted at 37 ° C. for 1 hour. After washing 6 times with 250 μL of TBS-T, 100 μL of the chemiluminescent substrate AMPPD solution was added and reacted at 37 ° C. for 5 minutes. Thereafter, the luminescence count was measured with a microplate reader (Centro LB960 manufactured by Bertoled).
　バイサルファイトパイロシーケンシング法は、既報の方法（Ｓｃｉｅｎｃｅ　１９９８，２８１，３６３－３６５、Ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ　２００２，２３，２４，４０７２－４０７９）にしたがい行った。 The bisulfite pyrosequencing method was performed according to a previously reported method (Science 1998, 281, 363-365, Electrophoresis 2002, 23, 24, 4072-4079).
　その結果、従来法で測定された発光カウントは、バイサルファイトパイロシーケンシング法の測定結果と必ずしも相関しなかった（表６、図７）。一方、本発明の方法は、バイサルファイトパイロシーケンシング法の測定結果と相関した（表６、図８）。 As a result, the luminescence count measured by the conventional method did not necessarily correlate with the measurement result of the bisulfite pyrosequencing method (Table 6, FIG. 7). On the other hand, the method of the present invention correlated with the measurement result of the bisulfite pyrosequencing method (Table 6, FIG. 8).
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　以上より、２つの異なる捕捉プローブを用いる本発明の方法は、標的核酸の増幅を伴うことなく、標的核酸の増幅を伴う方法と少なくとも同等の解析精度で、天然の生物学的サンプル中の標的核酸に含まれる修飾核酸塩基を測定できることが示された。 From the above, the method of the present invention using two different capture probes does not involve amplification of the target nucleic acid, and at least the same analysis accuracy as the method involving amplification of the target nucleic acid, the target nucleic acid in the natural biological sample It was shown that the modified nucleobase contained in can be measured.
　本発明の方法は、診断、研究等の分野で利用することができる。 The method of the present invention can be used in fields such as diagnosis and research.

Claims (13)

  1. 　以下を含む、修飾核酸塩基を含む標的核酸の測定方法：
    （１）修飾核酸塩基を含む標的核酸、および非標的核酸を含む核酸サンプルにおいて、標的核酸に対する複数の異なる捕捉プローブを用いて標的核酸を固相に複数回固定すること；ならびに
    （２）標的核酸に含まれる修飾核酸塩基を測定すること。     A method for measuring a target nucleic acid containing a modified nucleobase, comprising:
    (1) immobilizing a target nucleic acid on a solid phase multiple times using a plurality of different capture probes for the target nucleic acid in a target nucleic acid containing a modified nucleobase and a non-target nucleic acid; and (2) a target nucleic acid Measuring the modified nucleobase contained in
  2. 　前記核酸サンプルがゲノムＤＮＡサンプルである、請求項１記載の方法。 The method according to claim 1, wherein the nucleic acid sample is a genomic DNA sample.
  3. 　前記核酸サンプルが、非標的核酸１μｇ当たりの標的核酸モル数が２０ａｍｏｌ未満の量において標的核酸を含む、請求項１または２記載の方法。 The method according to claim 1 or 2, wherein the nucleic acid sample contains the target nucleic acid in an amount of less than 20 amol of target nucleic acid per 1 g of non-target nucleic acid.
  4. 　前記捕捉プローブとして、標的核酸の５’末端領域にハイブリダイズする能力を有する捕捉プローブ、および標的核酸の３’末端領域にハイブリダイズする能力を有する捕捉プローブが併用される、請求項１～３のいずれか一項記載の方法。 The capture probe having the ability to hybridize to the 5 ′ end region of the target nucleic acid and the capture probe having the ability to hybridize to the 3 ′ end region of the target nucleic acid are used in combination as the capture probe. The method according to any one of the above.
  5. 　以下を含む方法により（１）が行われる、請求項１～４のいずれか一項記載の方法：
    （ｉ）標的核酸に対する第１捕捉プローブを用いた液相中の標的核酸の固相への捕捉；
    （ｉｉ）固相から液相への標的核酸の放出；および
    （ｉｉｉ）標的核酸に対する第２捕捉プローブを用いた液相中の標的核酸の固相への捕捉。     The method according to any one of claims 1 to 4, wherein (1) is carried out by a method comprising:
    (I) capture of a target nucleic acid in a liquid phase onto a solid phase using a first capture probe for the target nucleic acid;
    (Ii) release of the target nucleic acid from the solid phase to the liquid phase; and (iii) capture of the target nucleic acid in the liquid phase to the solid phase using a second capture probe for the target nucleic acid.
  6. 　以下を含む方法により（１）が行われる、請求項１～４のいずれか一項記載の方法：
    （ｉ’）標的核酸に対する第１捕捉プローブを用いた第１液相中の標的核酸の第１固相への捕捉；
    （ｉｉ’）第１液相の第２液相への交換；
    （ｉｉｉ’）第１固相から第２液相への標的核酸の放出；
    （ｉｖ’）第１固相の第２固相への交換；
    （ｖ’）標的核酸に対する第２捕捉プローブを用いた第２液相中の標的核酸の第２固相への捕捉；および
    （ｖｉ’）第２液相の第３液相への交換。     The method according to any one of claims 1 to 4, wherein (1) is carried out by a method comprising:
    (I ′) capture of the target nucleic acid in the first liquid phase to the first solid phase using the first capture probe for the target nucleic acid;
    (Ii ′) exchange of the first liquid phase to the second liquid phase;
    (Iii ′) release of the target nucleic acid from the first solid phase to the second liquid phase;
    (Iv ′) exchange of the first solid phase to the second solid phase;
    (V ′) capture of the target nucleic acid in the second liquid phase to the second solid phase using the second capture probe for the target nucleic acid; and (vi ′) exchange of the second liquid phase to the third liquid phase.
  7. 　以下を含む方法により（１）が行われる、請求項１～４のいずれか一項記載の方法：
    （１－１）（ａ）核酸サンプルに含まれる標的核酸、（ｂ）第１親和性物質で標識された、標的核酸に対する第１捕捉プローブ、および（ｃ）第１親和性物質と特異的に結合する能力を有する物質で標識された第１固相を溶液中で反応させて、標的核酸および前記第１捕捉プローブを含む第１核酸ハイブリッドが固定された第１固相を含む第１溶液を得ること；
    （１－２）前記第１核酸ハイブリッドが固定された第１固相を、第１溶液から第２溶液に移すこと；
    （１－３）前記第１核酸ハイブリッドが固定された第１固相から標的核酸を第２溶液中に放出させて、標的核酸および第１固相を含む第１放出溶液を得ること；
    （１－４）第１固相を第１放出溶液から除去して、標的核酸を含み、かつ第１固相を含まない第２放出溶液を得ること；ならびに
    （１－５）（ａ’）前記第２放出溶液に含まれる標的核酸、（ｂ’）第２親和性物質で標識された、標的核酸に対する第２捕捉プローブ、および（ｃ’）第２親和性物質と特異的に結合する能力を有する物質で標識された第２固相を反応させて、標的核酸および前記第２捕捉プローブを含む第２核酸ハイブリッドが固定された第２固相を含む第３溶液を得ること；
    （１－６）前記第２核酸ハイブリッドが固定された第２固相を、前記第３溶液から第４溶液に移すこと。     The method according to any one of claims 1 to 4, wherein (1) is carried out by a method comprising:
    (1-1) (a) a target nucleic acid contained in a nucleic acid sample, (b) a first capture probe for the target nucleic acid labeled with a first affinity substance, and (c) specific to the first affinity substance A first solid phase labeled with a substance having an ability to bind is reacted in a solution, and a first solution including a first solid phase to which a first nucleic acid hybrid including a target nucleic acid and the first capture probe is immobilized is prepared. Getting;
    (1-2) transferring the first solid phase on which the first nucleic acid hybrid is immobilized from the first solution to the second solution;
    (1-3) obtaining a first release solution containing the target nucleic acid and the first solid phase by releasing the target nucleic acid into the second solution from the first solid phase to which the first nucleic acid hybrid is fixed;
    (1-4) removing the first solid phase from the first release solution to obtain a second release solution containing the target nucleic acid and not containing the first solid phase; and (1-5) (a ′) A target nucleic acid contained in the second release solution; (b ′) a second capture probe for the target nucleic acid labeled with a second affinity substance; and (c ′) an ability to specifically bind to the second affinity substance. Reacting a second solid phase labeled with a substance having: to obtain a third solution comprising a second solid phase to which a second nucleic acid hybrid comprising a target nucleic acid and the second capture probe is immobilized;
    (1-6) Transfer the second solid phase on which the second nucleic acid hybrid is immobilized from the third solution to the fourth solution.
  8. 　以下をさらに含む、請求項７記載の方法：
    （１ａ）（１－２）において、前記第１固相を前記第１溶液から第２溶液に移すときに、前記第１核酸ハイブリッドが固定された第１固相を洗浄すること；
    （１ｂ）（１－６）において、前記第２固相を前記第３溶液から第４溶液に移すときに、前記第２核酸ハイブリッドが固定された第２固相を洗浄すること；
    （１ｃ）（１ａ）および（１ｂ）の双方を行うこと。     The method of claim 7 further comprising:
    (1a) In (1-2), when transferring the first solid phase from the first solution to the second solution, washing the first solid phase to which the first nucleic acid hybrid is immobilized;
    (1b) In (1-6), when transferring the second solid phase from the third solution to the fourth solution, washing the second solid phase to which the second nucleic acid hybrid is immobilized;
    (1c) Perform both (1a) and (1b).
  9. 　第１親和性物質が第２親和性物質と同じであり、かつ、第１親和性物質と特異的に結合する能力を有する物質で標識された第１固相が第２親和性物質と特異的に結合する能力を有する物質で標識された第２固相と同じである、請求項７または８記載の方法。 The first solid phase labeled with a substance having the same affinity as the second affinity substance and capable of specifically binding to the first affinity substance is specific to the second affinity substance. The method according to claim 7 or 8, wherein the method is the same as the second solid phase labeled with a substance capable of binding to.
  10. 　固相が磁性粒子である、請求項１～９のいずれか一項記載の方法。 The method according to any one of claims 1 to 9, wherein the solid phase is a magnetic particle.
  11. 　修飾核酸塩基がメチルシトシンである、請求項１～１０のいずれか一項記載の方法。 The method according to any one of claims 1 to 10, wherein the modified nucleobase is methylcytosine.
  12. 　前記測定が標的核酸の非増幅的解析方法により行われる、請求項１～１１のいずれか一項記載の方法。 The method according to any one of claims 1 to 11, wherein the measurement is performed by a non-amplifying analysis method of the target nucleic acid.
  13. 　前記非増幅的解析方法が、イムノアッセイ、質量分析、電気化学分析、高速液体クロマトグラフィー分析、ナノポア分析またはマイクロポア分析である、請求項１１記載の方法。 The method according to claim 11, wherein the non-amplification analysis method is an immunoassay, mass spectrometry, electrochemical analysis, high performance liquid chromatography analysis, nanopore analysis or micropore analysis.
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