WO2007111324A1 - Procédé de détection de la 5-méthylcytosine dans un gène et trousse de détection - Google Patents

Procédé de détection de la 5-méthylcytosine dans un gène et trousse de détection Download PDF

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Publication number
WO2007111324A1
WO2007111324A1 PCT/JP2007/056296 JP2007056296W WO2007111324A1 WO 2007111324 A1 WO2007111324 A1 WO 2007111324A1 JP 2007056296 W JP2007056296 W JP 2007056296W WO 2007111324 A1 WO2007111324 A1 WO 2007111324A1
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methylcytosine
naphthoquinone
target dna
dna
detecting
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PCT/JP2007/056296
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English (en)
Japanese (ja)
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Sei-Ichi Nishimoto
Kazuhito Tanabe
Hisatsugu Yamada
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Kyoto University
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Publication of WO2007111324A1 publication Critical patent/WO2007111324A1/fr

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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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • 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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G01N2333/922—Ribonucleases (RNAses); Deoxyribonucleases (DNAses)

Definitions

  • the present invention relates to a method for detecting 5-methylcytosine in a gene and a detection kit.
  • the existing 5-methylcytosine base detection system includes Maxam-Gilbert chemical modification method, Methylation Specific PCR (MSP) method with sodium bisulfite treatment, and PNA-DNA complex and enzyme treatment method. Have been reported so far (Non-Patent Documents 1 to 4).
  • the Maxam-Gilbert chemical modification method selectively damages the cytosine base of DNA by treating the DNA with hydrazine. At this time, cytosine that has not been methylated is cleaved by hydrazine treatment, whereas cytosine that has been methylated is not cleaved by hydrazine treatment. Therefore, when hydrazine-treated DNA is analyzed by polyacrylamide gel electrophoresis (PAGE), cytosine methyli-Z and non-methyli can be detected by the presence or absence of a band produced by cleavage.
  • PAGE polyacrylamide gel electrophoresis
  • the MSP method utilizes the difference in reactivity between 5-methylcytosine and non-methylcytosine with respect to sodium bisulfite.
  • DNA is treated with sodium bisulfite.
  • non-methylcytosine in DNA is deaminated and converted to uracil.
  • Force to be converted 5-Methylcytosine remains undeaminated as it is less reactive.
  • PCR is performed using appropriate primers, the site that was originally cytosine is amplified as thymine (uracil), while 5-methylcytosine is amplified as cytosine.
  • the amplified sample is sequenced, the portion that was originally 5-methylcytosine is detected as cytosine, and the portion that was non-methylcytosine is detected as thymine. As a result, it is possible to determine cytosine methyli-z Z-nonmethyli.
  • Non-patent Document 5 2-methyl 1,4 naphthoquinone known as vitamin K3 has been reported to function as a photooxidant and to oxidize various nucleobases with one electron. Specifically, in the presence of naphthoquinone, it has been reported that 5-methyl-2'-deoxycytidine is converted to acid complexes such as 5formyl 2'deoxycytidine and 5hydroxymethyl 2'deoxycytidine by UV irradiation. ing.
  • a modified DNA oligomer is synthesized by introducing naphthoquinone into the terminal or central part of a DNA oligomer having a sequence complementary to the DNA to be detected for 5-methylcytosine. To hybridize with the target DNA. Then, the irradiated double-stranded DNA is irradiated with light. At this time, 5-mech Lucitosin is cleaved by naphthoquinone photoacid, while non-methylcytosine is not cleaved. Therefore, the presence of 5-methylcytosine can be detected as a cleavage band by analyzing the double-stranded DNA after light irradiation by PAGE.
  • the Invader method (Tird Wave Technologies, USA) is known as a method that can detect gene mutations quickly and at low cost without the need for gene amplification, and is widely used for the analysis of monobasic polymorphisms. (Patent Document 1, Non-Patent Document 8).
  • Patent Literature 1 Special Table 2001-526526 (published December 18, 2001)
  • Non-Patent Document 1 Church, G. M., Gilbert, W. Proc. Natl. Acad. Sci. USA 1984, 81, 199 1.
  • Non-Patent Document 2 Frommer, M., McDonald, LE, Millar, DS, Collis, CM, Watt, F., Gigge, GW, Molloy, PL, Paul, C. and Proc. Natl. Acad. Sci. USA 1992 , 89, 182 7.
  • Non-Patent Document 3 Herman, H. G “Graff, J. R” Myohanen, S., Nelkin, B. D “Baylin, S. B. Proc. Natl. Acad. Sci. USA 1996, 93, 9821.
  • Non-Patent Document 4 Okamoto, A., Tanabe, K., Satio, I. J. Am. Chem. Soc. 2002, 124, 102 62.
  • Non-Patent Document 5 Douki, T., Cadet, J. Int. J. Radiat. Biol. 1999, 75, 571.
  • Non-Patent Document 6 Yamada, H., Tanabe, K., Nishimoto, S. Bioorganic & Medicinal Chemi stry Letters 2005, 15, 665
  • Non-Patent Document 7 Yamada, H., Tanabe, K., Nishimoto, S. Nucleic Acids Symposium Series 2005, No. 49, 149-150
  • Non-Patent Document 8 Lyamichev, V., et al, Nat. BioltechnoL, 17 (3) 292-296 (1999) Disclosure of the Invention
  • the method for detecting 5-methylcytosine using naphthoquinone developed by the present inventors has to analyze the presence of 5-methylcytosine as a DNA fragment by PAGE.
  • the invader method widely used for detecting gene mutations, particularly single nucleotide polymorphisms cannot detect the presence or absence of a base methyl candy. That is, it is paired with a complementary base regardless of the presence or absence of methyl candy.
  • the present invention has been made in view of the above-mentioned problems, and its purpose is to modify the invader method so that it can be applied to detection of 5-methylcytosine in a gene, and it is simple, short time, low cost, The aim is to realize a highly sensitive 5-methylcytosine detection method.
  • the 5-methylcytosine site of the target DNA containing 5-methylcytosine to be detected is selectively cleaved, and the 5-methylcytosine 5 is detected.
  • a step of obtaining a DNA fragment having a base adjacent to the 3 side at the 3 ′ end (1); a FRET probe having a base sequence capable of hybridizing with the DNA fragment or the 3 ′ end region of the DNA fragment in the terminal region And a step (2) of reacting with a flap endonuclease; and a step (3) of detecting fluorescence of the reaction mixture.
  • the “photofunctional nucleic acid” is a nucleic acid for detecting 5-methylcytosine, and the function that naphthoquinone causes acidic cleavage of 5-methylcytosine by irradiation with external light. It refers to the nucleic acid that it has.
  • the photofunctional nucleic acid has the following general formula (1)
  • a and B each represent a linear nucleotide of 1 or more bases, and Z represents a compound containing naphthoquinone
  • Z represents the following chemical formulas (2) to (6):
  • the above-mentioned photofunctional nucleic acid has a base sequence that can hybridize with the target DNA, and when hybridized with the target DNA, the above-mentioned naphthoquinone is inserted at a position facing 5-methylcytosine to be detected. I like to do it!
  • the kit according to the present invention is a kit for detecting 5-methylcytosine in a gene, and is characterized by comprising the following (a) to (c).
  • a photofunctional nucleic acid comprising a nucleotide sequence capable of hybridizing with a target DNA and having naphthoquinone inserted at a position facing the detection target 5-methylcytosine when hybridized with the target DNA
  • the 3 'end region has a base sequence that can hybridize with the 3' end region of the DNA fragment having the 3 'end base adjacent to the 5' side of the target 5-methylcytosine present in the target DNA.
  • the method according to the present invention can detect the presence of 5-methylcytosine by fluorescence. Therefore, there is an effect that 5-methylcytosine can be detected easily and at a low cost without requiring a complicated operation. In addition, since more fluorescent molecules are released than the amount of DNA fragments generated by cleavage of the target DNA, there is an effect that amplification detection is possible even with a small amount of the target gene in the sample. Further, in the conventional method, several days are required for detection. However, if the method according to the present invention is used, it is possible to detect 5-methylcytosine in about several hours.
  • FIG. 1 is a schematic diagram showing the principle of a method according to the present invention.
  • FIG. 2 shows a method for synthesizing a hydrazine derivative of naphthoquinone.
  • FIG. 3 is a view showing a FRET probe used in Examples.
  • FIG. 4 (A) HPLC profile of a reaction solution subjected to light irradiation and piperidine treatment to confirm that the target 5 ′ fragment was produced from the target DNA (ODNl).
  • FIG. 4 (B) is a MA LDI-TOF-MS chart confirming that the target 5 ′ fragment was generated from the target DNA (ODNl).
  • FIG. 5 is a fluorescence spectrum chart showing that 5-methylcytosine in target DNA was detected by the method according to the present invention.
  • FIG. 6 is a fluorescence spectrum chart showing that in the method according to the present invention, no fluorescence emission is observed when no light irradiation is performed and when piperidine treatment is not performed.
  • FIG. 7 A fluorescence showing that in the method according to the present invention, no fluorescence is observed when a probe oligomer (FRET probe) is used without a complementary sequence of a 5 'fragment generated from a target DNA. It is a spectrum chart.
  • FRET probe probe oligomer
  • FIG. 8 is a fluorescence spectrum chart showing the results of examining the detection limit in the method according to the present invention.
  • FIG. 9 (A) is a fluorescence micrograph showing the result of 0 hours of light irradiation using ODN2 as the target DNA in the method according to the present invention.
  • FIG. 9 (B) is a fluorescence micrograph showing the result of light irradiation for 2 hours using ODN2 as the target DNA in the method according to the present invention.
  • FIG. 9 (C) is a fluorescence micrograph showing the result of 0 hours light irradiation using ODN1 as the target DNA in the method according to the present invention.
  • FIG. 9 (D) is a fluorescence micrograph showing the result of light irradiation for 2 hours using ODN1 as the target DNA in the method according to the present invention, showing that the 5-methylcytosine detection signal can be visualized by a fluorescence microscope. Show.
  • the method for detecting 5-methylcytosine in a gene according to the present invention comprises the following steps (1) to (3): If it includes,
  • Step (1) DNA that selectively cleaves the 5-methylcytosine site of the target DNA containing 5-methylcytosine to be detected, and has a base adjacent to the 5 ′ side of the 5-methylcytosine at the 3 ′ end Get a fragment.
  • Step (2) A FRET probe having 3 base sequences capable of hybridizing with the DNA fragment or the 3 ′ terminal region of the DNA fragment and a flap endonuclease in the terminal region is reacted.
  • Step (3) The fluorescence of the reaction mixture is detected.
  • FIG. 1 An outline of a method for detecting 5-methylcytosine in a gene according to the present invention is shown in FIG. Hereinafter, each step will be described in detail.
  • step (1) the 5-methylcytosine site of the target DNA containing 5-methylcytosine to be detected is selectively cleaved, and the base adjacent to the 5 'side of the 5-methylcytosine is placed at the 3' end.
  • a DNA fragment is obtained.
  • a method for obtaining such a DNA fragment for example, a method using a photofunctional nucleic acid having naphthoquinone inside the polynucleotide chain developed by the present inventors (see Non-Patent Documents 6 and 7), tetraoxide And the like (Okamoto, A., Tainaka, K. Nucleic Acids symposium Series No. 49, 45-4, 2005).
  • the method using a photofunctional nucleic acid having naphthoquinone in the polynucleotide chain developed by the present inventors is preferable because no poisonous or deleterious substance is used as a reaction reagent.
  • the photofunctional nucleic acid has a polynucleotide chain and naphthoquinone inside the polynucleotide chain.
  • the photofunctional nucleic acid can be hybridized with the target DNA containing 5-methylcytosine to be detected by the polynucleotide chain portion.
  • cytosine catalyzed in the vicinity of the photofunctional nucleic acid in the target DNA may be damaged. The strands are broken, but not cytosine If not cut off.
  • photofunctionality has the function of causing oxidative cleavage of 5-methylcytosine by naphthoquinone by irradiating light from the outside as described above. It means that Therefore, in the present specification, “photofunctional nucleic acid” means that naphthoquinone has a function of causing acidic cleavage of 5-methylcytosine by irradiating light from the outside! / Refers to that.
  • polynucleotide is synonymous with “gene”, “nucleic acid”, or “nucleic acid molecule”, and refers to a polymer of nucleotides.
  • base sequence is synonymous with “nucleic acid sequence” or “nucleotide sequence” and is a sequence of deoxyribonucleotides or ribonucleotides (abbreviated as A, G, C and T). As shown.
  • the polynucleotide may be in the form of RNA or DNA. Furthermore, in the case of DNA, it may be either double-stranded or single-stranded. In the polynucleotide, any number of nucleotide bonds may be used as long as it is 2 or more.
  • the polynucleotide may be chemically synthesized or may be extracted from natural environmental forces.
  • flank the polynucleotide chain refers to nucleotides other than the 5, terminal and 3, terminal nucleotides of the polynucleotide chain.
  • naphthoquinone means the following chemical formula:
  • the photofunctional nucleic acid according to the present invention only needs to contain any one of the above naphthoquinone structures.
  • the naphthoquinone contained in the photofunctional nucleic acid is not limited to 1,4 naphthoquinone and 1,2 naphthoquinone, but may be a derivative in which a substituent is introduced into each naphthoquinone.
  • Such derivatives include, for example, 2-methyl 1,4 naphthoquinone, known as vitamin koji. And those obtained by substituting the 2-position methyl group with another alkyl group, alkoxy group, halogen group, nitro group, or amino group.
  • aromatic ring various substituents (such as an alkyl group, an amino group, an alkoxy group, or a hydroxy group as an electron donating group; or a -tro group or a halogen group as an electron withdrawing group) may be introduced on the aromatic ring.
  • substituents such as an alkyl group, an amino group, an alkoxy group, or a hydroxy group as an electron donating group; or a -tro group or a halogen group as an electron withdrawing group
  • naphthoquinone a compound having a naphthoquinone structure containing these derivatives is simply referred to as naphthoquinone.
  • the naphthoquinone may be present at the end (5 'end or 3' end) of the polynucleotide chain, or may be present at a portion other than both ends, that is, "inside the polynucleotide chain". From the viewpoint of limiting the range of naphthoquinone movement and suppressing acid-specific cleavage of non-specific guanine sequence, it is preferable to have naphthoquinone “inside the polynucleotide chain”.
  • the photofunctional nucleic acid may be a compound in which naphthoquinone is added to a nucleotide, or may be one in which a nucleotide is substituted with naphthoquinone.
  • a and B each represent a linear nucleotide of 1 or more bases, and Z represents a compound containing naphthoquinone
  • the compound Z containing naphthoquinone is a nucleotide A 3
  • nucleotide B It is preferable that it is bonded to the phosphate group at the end and the phosphate group at the 5th end of nucleotide B.
  • the method for binding the polynucleotide and naphthoquinone is not particularly limited.
  • Z in the above general formula (1) is represented by the following chemical formula (2)
  • the linker portion that binds the polynucleotide and naphthoquinone is preferably as short as possible. As a result, the structural freedom of a part of the linker is limited, and non-specific acid cleavage by naphthoquinone can be further suppressed.
  • the polynucleotides A and B are required to have a base sequence capable of hybridizing with the target DNA. However, as long as it can be hybridized, it may not have 100% identity with the complementary sequence to the base sequence of the region containing 5-methylcytosine to be detected by the target DNA. Those skilled in the art will readily understand that hybridization is possible without 100% identity. Specifically, taking 5′—A—Z—B—3 ′ in the general formula (1) as an example, assuming that Z is one base, the polynucleotide A—Z—B It preferably has 80% or more identity with a sequence complementary to a partial region containing 5-methylcytosine.
  • the identity is more preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more.
  • step (1) As a specific procedure in step (1), first, a photofunctional nucleic acid and a target DNA are neutralized. In addition, what is necessary is just to perform a hybridization according to a regular method.
  • piperidine is added to the reaction solution for treatment, whereby the 5-methylcytosine site is completely cleaved.
  • piperidine treatment for example, 50 L of 10% piperidine aqueous solution may be added to 10 L of reaction solution and heated at 90 ° C. for 20 minutes. This gives a dry DNA pellet containing the cleaved fragment of the target DNA.
  • the target DNA is cleaved into two fragments in a state where the 5-methylcytosine site is deleted.
  • the fragment necessary for step (2) is a 5 ′ fragment, that is, a DNA fragment having a base adjacent to the 5 ′ side of 5-methylcytosine at the 3 ′ end.
  • the 5 ′ fragment of the target DNA may be isolated and used in the next step (2), but the dried DNA pellet containing the 5 ′ fragment of the target DNA can be used as it is in the step (2).
  • it is preferable that the dried DNA pellet containing the 5 ′ fragment of the target DNA is directly used in step (2).
  • step (2) the 5 ′ fragment of target DNA produced in step (1), a FRET probe and a flap endonuclease (hereinafter sometimes abbreviated as “FEN”) are reacted.
  • FEN a flap endonuclease
  • Examples of the solution for dissolving the dried DNA pellet include a buffer containing 20 mM Tris-HCl (pH 7.4), 15 mM NaCl, 10 XREC TM reaction buffer 12, and 1 X BSA for addition.
  • the present invention is not limited to this as long as it does not inhibit the FEN enzyme reaction.
  • the FRET probe is adjusted so that the FRET probe is 1 ⁇ to 5 ⁇ M and the FEN is 1 unit to 5 units. Preferred to add lobes and FEN! /. However, it is not limited to this, and a suitable addition amount should be set appropriately.
  • reaction conditions a reaction at 30 ° C for 1 minute to 5 minutes may be used as a standard. It has been confirmed by the inventors that if the reaction time is lengthened, the intensity of fluorescence generated including the background increases.
  • reaction solution reaction solution
  • the process immediately proceeds to the next step (3), and the fluorescence of the reaction mixture (reaction solution) can be detected. Therefore, by using the detection method according to the present invention, it is possible to detect the presence of 5-methylcytosine in a gene in a very short time compared to the conventional method.
  • the oligonucleotide include an oligonucleotide having a base sequence ability as shown in FIG. That is, the 3 ′ end region of the FRET probe has a base sequence that can hybridize with the 3 ′ region of the 5 ′ fragment selectively cleaved at the 5-methylcytosine portion of the target DNA (the line in FIG. 3). The nucleotide sequence enclosed in brackets). Therefore, the base sequence of this region of the FRET probe can be appropriately changed according to the target DNA and the detection target 5-methylcytosine.
  • the base sequence may not have 100% identity with the complementary sequence of the 3 'region of the 5' fragment.
  • hybridization is possible without 100% identity. However, it is preferable to have 100% identity.
  • the 5'-most base of the 3 'region of the FRET probe that can hybridize with the 5' fragment of the target DNA is the base adjacent to the 5'-side of the detection target 5-methylcytosine in the target DNA ( It must be the complementary base of the 3 'terminal base of the 5' fragment. This is because this part becomes a recognition site for FEN.
  • the number of bases in the 3 'terminal region of the FRET probe that can hybridize with the 5' fragment of the target DNA is preferably 5 to 20 bases. If it is shorter than 5 bases, it may not be a substrate for the enzyme (FEN). If it is longer than 20 bases, the cleavage efficiency by the enzyme (FEN) may decrease.
  • the number of bases in the 5 ′ end region of the FRET probe constituting the hairpin structure is not particularly limited, but is preferably 25 to 40 bases.
  • the base at the 5 'end of the FRET probe is labeled with a fluorescent dye, and a quencher (taencher) that suppresses the fluorescence intensity is bound to the vicinity of the 5' end.
  • a quencher such as quencher
  • the quencher may be at the 5 ′ end and the fluorescent dye may be in the vicinity thereof.
  • FEN recognizes when the 5 'fragment of the target DNA hybridizes with the FRET probe. The structure is constructed, and as a result, the fluorescent dye at the 5 ′ end is cleaved, so that the presence of 5-methylcytosine can be detected as the fluorescence intensity.
  • fluorescent dye used in the FRET probe examples include FMA (6-carboxy-fluorescein), TAMRA (6-carboxytetramethyl-rhodamine), Cy-3, and Cy-5. Further, as a quencher, 4- (4'-dimethylaminophenylazo) benzoic acid, dabcyl), 4-dimethylaminoazobenzene-4-sulfonic acid (daosyl) can be used.
  • the FRET probe can be synthesized using a commercially available DNA synthesizer (see Examples). Use phosphoramidites labeled with fluorescent dyes and phosphoramidites labeled with quenchers.
  • a general invader method for detecting single nucleotide polymorphisms is a signal probe using two types of unlabeled oligonucleotides commonly called signal probes and invader probes and FEN.
  • the flap arm is cut out, and the FEN recognition site is formed by hybridization of this flap arm and the FRET probe to release the fluorescent dye.
  • SNPs are detected based on whether the hybridized complex has a matching sequence or a mismatch sequence.
  • a target DNA fragment cleaved at a 5-methylcytosine site is prepared in advance, and the obtained 5 ′ fragment of the target DNA is hybridized to the FRET probe ( By invading, the FEN recognition site is constructed and the fluorescent dye is released. That is, the release of the fluorescent dye is controlled by the presence or absence of a target DNA cut fragment, and the presence or absence of methyl candy is detected. In this case, do not use so-called signal probes and invader probes! /.
  • the fluorescence of the reaction mixture may be detected.
  • the method for detecting fluorescence is not particularly limited, and examples thereof include a method for measuring fluorescence intensity using a fluorometer.
  • the reaction mixture can be directly applied to a fluorimeter and measured by the method recommended by the equipment used depending on the fluorescent material used.
  • the presence of the target 5-methylcytosine can be detected more accurately by providing a control and comparing the fluorescence intensity. For example, target DNA And those that do not contain flap endonuclease.
  • a fluorescence intensity of 110% or more is measured as compared with the fluorescence intensity of the control, it is preferable to make a 5-methylcytosine positive determination.
  • a method of observing with a fluorescence microscope can be cited.
  • agarose or the like is added to the reaction mixture to form a gel, a part of which is placed on a slide glass and sandwiched between cover glasses. What is necessary is just to select an excitation wavelength, a detection wavelength, and exposure time according to the used fluorescent substance. As shown in the examples described later, if the entire gel emits fluorescence, it can be determined that 5-methylcytosine is positive.
  • the photofunctional nucleic acid can be synthesized by condensing a naphthoquinone derivative with a polynucleotide chain obtained by abasifying the nucleotide at the position where naphthoquinone is introduced.
  • An example of a specific condensation method is shown below, but various other known methods can be used without being limited thereto.
  • a hydrazine derivative of naphthoquinone is synthesized by introducing a carboxylic acid into the 3-position of 2-methyl-1,4 naphthoquinone and then condensing hydrazine.
  • a and B are nucleotide chains that hybridize with the target DNA described above.
  • Y is an abasic precursor modification part, for example, the following chemical formula
  • the diol part of the cocoon contained in the modified polynucleotide chain is converted into a formyl group with sodium periodate or the like.
  • the photofunctional nucleic acid represented by this can be obtained.
  • the condensation reaction between the formyl group and hydrazine is preferably carried out under acidic conditions, and pH 6 is particularly preferred, and pH 6 is particularly preferred. Further, the reaction temperature is preferably room temperature or less, preferably 10 ° C or less, more preferably 4 ° C. Thereby, naphthoquinone can be introduced into the modified polynucleotide chain. [0082] Further, in the above-described example, the force obtained by binding the modified polynucleotide chain and naphthoquinone by condensation of formyl group and hydrazine is not limited to this.
  • the nucleotide chain may be condensed with a naphthoquinone derivative having a succinimidyl group introduced. An example of this reaction equation is shown in the following reaction equation (8).
  • reaction formula (9) shows an example in which a modified polynucleotide chain having an amino group introduced is condensed with a naphthoquinone derivative having a succinimidyl group introduced.
  • a modified polynucleotide chain having an alkylthiol group introduced may be condensed with a maleimide group or a naphthoquinone derivative having a thiol group introduced.
  • An example of a reaction formula for condensing a modified polynucleotide chain having a thiol group introduced therein and a naphthoquinone having a maleimide group introduced therein is shown in the following reaction formula (10).
  • a modified nucleobase having a naphthoquinone (a single-base modified nucleotide) is synthesized, and a modified polynucleotide chain in which the modified nucleobase is inserted into an arbitrary portion is synthesized by a DNA synthesizer.
  • a naphthoquinone-containing uridine derivative may be synthesized as a modified nucleobase, and a polynucleotide chain containing this may be synthesized by a DNA synthesizer! /.
  • the naphthoquinone-containing uridine derivative is, for example, referred to the method described in “0 kamoto, A. et al. J. Am. Chem. So 126, 4820-4827, 2004”. And can be synthesized.
  • the photofunctional nucleic acid that can be used in the present invention includes various products obtained by the above-mentioned method and other known methods as long as it is a polynucleotide chain containing naphthoquinone.
  • the present invention provides a kit for detecting 5-methylcytosine in a gene, comprising the following (a) to (c).
  • a photofunctional nucleic acid comprising a nucleotide sequence capable of hybridizing with a target DNA and having naphthoquinone inserted at a position facing the detection target 5-methylcytosine when hybridized with the target DNA
  • the 3 'end region has a base sequence that can hybridize with the 3' end region of the DNA fragment having the 3 'end base adjacent to the 5' side of the target 5-methylcytosine present in the target DNA.
  • the method for detecting 5-methylcytosine in the gene according to the present invention can be carried out simply and rapidly.
  • This kit comprises the above (a) to (c) in the form of a solution or a lyophilized product, respectively! / Drowning That's fine.
  • the specific kit configuration other than the above (a) to (c) is not particularly limited, and the necessary kits and kits may be selected by appropriately selecting necessary reagents and instruments. Examples thereof include a reaction buffer, a reaction tube, and a piperidine solution.
  • the term "kit” is intended to include a package with a container (eg, bottle, plate, tube, dish, etc.) containing a particular material.
  • a container eg, bottle, plate, tube, dish, etc.
  • an instruction manual for using the material is provided. Instructions for use may be written or printed on paper or other media, or attached to electronic media such as magnetic tape, computer-readable discs or tapes, CD-ROMs, etc. .
  • kits for detecting 5-methylcytosine in a gene According to the present invention, the description here will be omitted as long as it is in accordance with the embodiment of the method for detecting 5-methylcytosine in a gene described above.
  • DNA synthesis reagents such as Abssic Phosphoramidite, 5-Fluorescein Phosphoramidite, Dabcyl-dT were purchased from Glen Reseach.
  • ODNs oligomers
  • MALDI—TOF matrix-assisted laser desorption ionization time-of-flight
  • Reversed phase HPLC was performed using a 6 ⁇ HPLC system (Shimadzu), D-7000 HPLC system (Hitachi) or L-2400 HPLC system (Hitachi).
  • Reverse phase column Inertsi 1 ODS-3, GL—Sciences Inc., 4.6 mm X 250 mm or ⁇ 10 mm X 15 Omm, or CAPCELL PAR MF SHISEIDO, ⁇ 10 mm X 250 mm
  • Sample solution was injected. Column elution was monitored by UV absorption at 260 nm.
  • Irradiation was performed using a Lourmat TFX-20M transilluminator.
  • T4 polynucleotide kinase (lOunitesZmL) was purchased from -Tubon Gene, respectively.
  • Human flap endonuclease I (Human FEN-1), 10 XREC TM reaction buffer 12 and 10 X BSA for addition were also purchased from Travigen Inc. Pure water (Yamato WR600A) was used for the preparation of all solutions.
  • a fluorometer RF-5300PC manufactured by Shimadzu Corporation was used.
  • oligomers standard oligodeoxynucleotides (hereinafter referred to as “oligomers”) containing modified sites that function as abasic site precursors. Synthesis was performed using the phosphoramidite method. The base sequence of the synthesized oligomer will be described later. After automated synthesis, the resulting oligomer was subjected to reverse phase HPLC (elution with a 0.1M TEAA, pH 7.0 solvent mixture, flow rate 3. 0% to 30% acetonitrile over 60 min in OmLZ min. Purified by a first gradient and a 30% to 100% acetonitrile gradient over 80 minutes).
  • HPLC reverse phase HPLC
  • the dried oligomer was suspended in 200 ⁇ L of 80% acetic acid and incubated at room temperature for 30 minutes. After 30 minutes, an equal volume of deionized water was added to the reaction mixture and incubated for an additional 4 hours at room temperature to remove DMT and ⁇ BDMS groups.
  • Oligomers (100 ⁇ ) with modified sites that function as abasic site precursors This was incubated with 0.5 mM sodium periodate dissolved in MNaOAc (pH 6.0, 80 L) for 1 hour under the light-shielded condition of 4 ° C. To the obtained reaction mixture, the above naphthoquinone hydrazine derivative 3 dissolved in acetonitrile (5 mM, 80 ⁇ L) was added and incubated at ambient temperature for 14 hours. The resulting reaction mixture was then eluted with reverse-phase HPLC (elution with 0.1 M TEAA, pH 7.0 solvent mixture, flow rate 3. 0% to 30% first gradient of acetonitrile over 60 min at OmLZ min and NQ-ODN2 was obtained by purification with a 30% to 100% acetonitrile gradient over 80 minutes.
  • reverse-phase HPLC elution with 0.1 M TEAA, pH 7.0 solvent mixture, flow rate 3. 0% to 30% first gradient of acetonitrile over
  • NQ-ODN The purity and concentration of the obtained NQ-ODN was determined by complete digestion with alkaline phosphatase, nuclease P 1 and phosphodiesterase I at 37 ° C. Whether it was a synthesized oligomer was confirmed by MALDI—TOF MASS analyzer (mZz 6455. 26 (calcd. For [M—H] _ 6456. 22)).
  • the DNA was synthesized with a DNA synthesizer (Applied Biosystems 3400) using phosphoramidite labeled with light and 4- (4'-dimethylaminophenylazo) benzoic acid (dabcyl). After synthesis, purification by reverse phase HPLC (elution with 0.1 M TEAA (pH 7.0) solvent mixture, flow rate 3. first gradient of acetonitrile to 0% force 60% over 60 min at OmLZ min). After complete digestion with alkaline phosphatase, nuclease P1 and phosphodiesterase I, the purity and concentration of the probe oligomers were determined.
  • Target oligomer 50 nM-50 pM in 2-mL-1,4 naphthoquinone-complementary oligomer (100 nM-100 pM) and 2 mM NaC1 buffer (pH 7.0) containing sodium codylate in 10 L Hybridized with.
  • the sample was heated at 90 ° C for 5 minutes using a thermocycler (BIO-RAD, iCycler), and then slowly lowered to 4 ° C to complete the hybridization. Subsequently, the target double-stranded oligomer was irradiated with light of 312 nm at 0 ° C. using a transilluminator.
  • the target sample solution is 500 fmol—50 in a buffer containing 20 mM Tris-HCl (pH 7.4), 15 mM NaCl, 10 X REC TM Reaction Buffer 12, and 1 X BSA for addition.
  • the oligomer with 5-methylcytosine is ODNl (5,-CTGGGAGAGACC m GGCGCACAG-3 ': ⁇ ⁇ ⁇ IJ No. 1) and the oligomer without 5-methynocytosine is ODN2 ( 5.
  • sequence of the oligomer into which naphthoquinone is to be introduced is basically a sequence complementary to ODN1 and ODN2, and only the complementary site of the cytosine base that can be methylated in codon282 of ODN1 and ODN2 is a triol. It was set as the compound which has group. Specifically, 5′—CTGTGCGCCYGTCTCTCCCAG—3 ′, Y is
  • this oligomer was treated with sodium periodate to convert the triol group into a formyl group, and then condensed with 3 (hydrazinocarbo-ruethyl) 2-methyl-1,4 naphthoquinone (naphthoquinone hydrazine derivative 3). Then, a DNA oligomer introduced with naphthoquinone, NQ-ODN, was obtained.
  • the sequence of this NQ— ODN2 is 5, — CTGTGCGCCZGTCTCT CCCAG— 3 ', and Z is
  • Probe Rigomer 1 (SEQ ID NO: 3) has a complementary sequence in the 3 ′ region of the 5 ′ fragment of the target DNA, and probe oligomer 2 (SEQ ID NO: 4) does not have a complementary sequence in that region of the target DNA.
  • the synthesis procedure is as described above.
  • MALDI—TOF—MS confirmed the target probe oligomer (probe oligomer l: calcd. For 13130. 77, found 13 130. 90, probe oligomer 2: calcd. For 13866. 27, found 13867. 10) .
  • a sample containing 50 nM (500 fmol) of ODN1 (target DNA) and NQ—ODNlOOnM (lpmol) was noblized, irradiated with light, and treated with piperidine to obtain a dry pellet.
  • Buffer, probe oligomer 1 and flap endonuclease I were added to the total volume of 15 / zL and incubated at 30 ° C for 5 minutes. This was diluted 40 times and fluorescence was measured.
  • the target DNA was ODN2, the target DNA was not added! /, And the flap endonuclease I was not added.
  • the amount of ODN1 contained in the sample was reduced to 500 fmol, 50 fmol, 5 fmol, and 500 amol, and the enzyme reaction of the probe oligomer with flap endonuclease I was performed under the same conditions as in Experiment 1 above.
  • the target DNA was ODN2.
  • Probe oligomer 1 was used, and flap endonuclease I was added to all samples.
  • FIGS. 9 (A) to (D) show the results when ODN1 is the target DNA.
  • ODN1 having 5-methylcytosine was used as the target DNA and irradiated for 2 hours, intense fluorescence was observed.
  • ODN2 was used as the target DNA when light irradiation was not performed, fluorescence was not observed. This result showed that 5-methylcytosine in DNA could be detected visually.
  • a simple and highly sensitive cytosine determination method for cytosine can be realized. Since the method for determining methyl candy according to the present invention can be applied to a gene having an arbitrary base sequence, it has a wide range of medicine and biology, such as genetic diagnosis, gene analysis, and elucidation of interaction between biomolecules involved in a gene. Can be used in the field.

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Abstract

La présente invention concerne un procédé qui permet de détecter de manière commode la 5-méthylcytosine en un temps très court et à faible coût, avec une haute sensibilité et une trousse de détection. Un procédé invasif classique, au moyen duquel la présence ou l'absence de méthylation d'une base ne peut être détectée est modifié et combiné à un procédé de clivage sélectif d'un site de 5-méthylcytosine dans un ADN cible. De manière plus générale, le site de 5-méthylecytosine de l'ADN cible est sélectivement clivé pour produire un fragment 5'. Ensuite, une réaction enzymatique est conduite au moyen d'une sonde FRET pouvant s'hybrider à ce fragment 5' et à une endonucléase flap, puis la fluorescence est détectée.
PCT/JP2007/056296 2006-03-29 2007-03-27 Procédé de détection de la 5-méthylcytosine dans un gène et trousse de détection WO2007111324A1 (fr)

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WO2014103321A1 (fr) * 2012-12-26 2014-07-03 学校法人北里研究所 Inhibiteur pdk4 et son utilisation
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Cited By (6)

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Publication number Priority date Publication date Assignee Title
WO2012141324A1 (fr) 2011-04-15 2012-10-18 独立行政法人理化学研究所 Procédé et trousse de détection de 5-hydroxyméthylcytosine dans des acides nucléiques
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EP2894158A4 (fr) * 2012-09-04 2016-06-01 Dnaform Kk Composé, acide nucléique, substance de marquage et procédé de détection
JPWO2014038561A1 (ja) * 2012-09-04 2016-08-12 株式会社ダナフォーム 化合物、核酸、標識物質および検出方法
US10294261B2 (en) 2012-09-04 2019-05-21 Kabushiki Kaisha Dnaform Compound, nucleic acid, labeling substance, and detection method
WO2014103321A1 (fr) * 2012-12-26 2014-07-03 学校法人北里研究所 Inhibiteur pdk4 et son utilisation

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