WO2017217530A1 - Procédé de mesure d'un acide nucléique cible comprenant une base d'acide nucléique modifiée - Google Patents

Procédé de mesure d'un acide nucléique cible comprenant une base d'acide nucléique modifiée 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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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

La présente invention concerne un procédé de mesure, à l'aide de sondes de capture, des quantités de bases modifiées comprises dans des acides nucléiques cibles dans un échantillon d'acides nucléiques qui comprend d'innombrables acides nucléiques non cibles et les acides nucléiques cibles comprenant les bases d'acide nucléique modifiées, la présente invention concernant un procédé de mesure spécifique des quantités de bases d'acide nucléique modifiées comprises dans les acides nucléiques cibles. Plus particulièrement, la présente invention concerne un procédé de mesure d'acides nucléiques cibles comprenant des bases d'acide nucléique modifiées, comportant : (1) la fixation, dans un échantillon d'acides nucléiques qui comprend d'innombrables acides nucléiques non cibles et les acides nucléiques cibles comprenant les bases d'acide nucléique modifiées, des acides nucléiques cibles à plusieurs reprises dans une phase solide à l'aide de multiples types de sondes de capture différentes pour les acides nucléiques cibles; et (2) la mesure des quantités des bases d'acide nucléique modifiées comprises dans les acides nucléiques cibles.
PCT/JP2017/022280 2016-06-17 2017-06-16 Procédé de mesure d'un acide nucléique cible comprenant une base d'acide nucléique modifiée WO2017217530A1 (fr)

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