WO2004104582A1

WO2004104582A1 - Method of measuring dna methylation ratio

Info

Publication number: WO2004104582A1
Application number: PCT/JP2003/014873
Authority: WO
Inventors: Naomi Yamakawa
Original assignee: Naomi Yamakawa
Priority date: 2003-05-23
Filing date: 2003-11-21
Publication date: 2004-12-02
Also published as: JP2004347508A; JP3854943B2

Abstract

An immunochemical method of measuring 5-methylcytosine of DNA strand accurately through simple means. An antibody capable of specific binding with 5-methylcytosine is brought into contact with single-strand DNA, and while any antibody having undergone monovalent binding is separated from the DNA strand, the amount of antibody having undergone bivalent bonding is measured, thereby specifying close-packed region of 5-methylcytosine. Further, there is provided a method of measuring the density of 5-methylcytosine in DNA strand.

Description

Specification

Method for measuring DNA methylation rate

The invention of this application relates to a method for measuring a DNA methylation ratio. More specifically, the invention of this application relates to a method for measuring DNA methylation that is useful for evaluating the degree of malignancy of cancer cells, or for evaluating the state of differentiation of cells in vitro. Background art

The correct expression of genetic information is essential for cell differentiation and functional expression in multicellular organisms, but its expression is controlled not only by a network of transcriptional regulators, but also by DNA methylation and chromatin dynamics. Emphasis is placed on the involvement of the epidemi- netic mechanism, such as the change in In particular, it is known that cytosine methylation in genomic DNA negatively regulates gene expression. In addition, differences in methylation patterns on the genome indicate a relationship with genomic imprinting プリン X chromosome inactivation phenomena, as well as cancer and GF GF (immunodeficiency, centromeric instability and facial anomalies). It has been reported that it is also involved in diseases such as syndrome, Rett syndrome, and fragile X syndrome (for example, see Non-Patent Document 1).

Methods for measuring methylcytosine in DNA chains include methods for comparing fragments cut by methylation-sensitive restriction enzymes, bisulfite method, methylation-specific PCR, and methods using high-performance liquid chromatography (HPLC). (See Non-Patent Document 1). Patent Literature 1 discloses a method for specifically PCR-amplifying a DNA strand containing a CG continuous sequence (CpG island) to be methylated. Patent Literature 2 specifically describes a method for specifically amplifying a DNA strand containing methylcytosine. A method using a labeled DNA fragment that hybridizes is disclosed.

[Patent Document 1] Japanese Patent Publication No. 11-511776

[Patent Document 2]

JP 2002-535998A

[Non-Patent Document 1]

Namihira Masaru et al. Experimental Medicine 20 (7): 1_19-1024, 2002 DISCLOSURE OF THE INVENTION As described above, methylation of DNA strands is an important indicator of various diseases including cancer, and control of gene expression. Therefore, it can be used as an index for grasping the degree of cell division, for example, and various measurement methods have been studied so far.

—On the other hand, when measuring DNA methylation in the medical field, for example, it is required to be able to obtain judgment results quickly and accurately. However, from this point of view, the conventional methods are not always preferable methods. For example, in the case of a method using a restriction enzyme, fragment comparison procedures such as Southern plotting are indispensable, and the bisulfite method and the method of Patent Document 1 require cumbersome work such as PCR and DNA sequence analysis of the product. And In particular, the conventional method has a problem that it takes a great deal of time and effort to obtain a judgment result because necessary processing is required for a test sample: a DNA strand.

The invention of this application has been made in view of the circumstances described above, and has as its object to provide a method for simply and accurately measuring the degree of DNA methylation. The present invention solves the above-mentioned problem by contacting an antibody that specifically binds to 5-methylcytosine with single-stranded DNA, and measuring the amount of the antibody bound to the DNA chain. Provided is a method for measuring a methylation rate.

In the method of the present invention, the monovalently bound antibody is separated from the DNA chain, One of the preferable embodiments is to specify the dense region of 5-methylcytosine by measuring the amount of the bound antibody.

In another preferred embodiment of the method of the present invention, a region other than an arbitrary region of the single-stranded DNA is double-stranded, and the DNA methylation rate is measured for the single-stranded region.

In the present invention, the “DNA chain” refers to a molecule in which a phosphoric acid ester of a nucleoside (dATP, dGTP, dCTP, dTTP) in which a purine or pyrimidine is bonded to a sugar with a -Ν-glycosidic bond is a phosphodiester bond. Means chain. Other terms and concepts in the present invention are defined in detail in the description of the embodiments of the present invention and examples. ⁽¹⁾ Various techniques used for carrying out the present invention can be easily and reliably implemented by those skilled in the art based on known documents and the like, except for the technique for which the source is clearly indicated. For example, genetic engineering and molecular biology techniques are described in Sambrook and Maniatis, Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989; Ausubel, FM et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1995 and the like. Hereinafter, embodiments of each invention will be described in detail. As described above, in the invention of this application, an antibody that specifically binds to 5-methylcytosine is brought into contact with single-stranded DNA, the amount of the antibody bound to the DNA chain is measured, and the DNA methyl efficiency is determined based on the amount of the antibody. It is characterized by doing.

An antibody that specifically binds to 5-methylcytosine (hereinafter sometimes referred to as “anti-5-methylcytosine antibody”) is a polyclonal or monoclonal antibody that can bind to 5-methylcytosine Molecules, and Fab, F (ab ') ₂ , Fv fragments, etc., and particularly preferably the whole molecule of the monoclonal antibody. Monoclonal anti-5-methylcytosine antibody can be prepared by using known methods for producing monoclonal antibodies using 5-methylcytosine as an immunogen (for example, “Monoclonal antibody”, written by Kamei Nagamune and Hiroshi Terada, Hiro Kawashoten, 1990; "Monoclonal Antibody" James W. Goding, third edition, Academic Press, 1996). Monoclonal antibodies that specifically recognize 5-methylcytosine are described in the literature (eg, Reynaud C. et al., Cancer Lett, 1992 Jan 31; 61 (3): 255-62; Mizugaki M. et al, Biol. Pharm Bull. 1996 Dec; 19 (12): 1537-40; Podesta A. et al., Int J Biochem, 1993 Jun; 25 (6): 929-33) and the like, and these should be used. You can also.

The amount of the antibody bound to the DNA chain (that is, bound to 5-methylcytosine in the DNA chain) is measured, for example, by labeling an anti-5-methylcytosine antibody and measuring the amount of the labeled signal. be able to. The secondary antibody that binds to the antibody (e.g. anti-I _g G antibodies) labeled, to bind the second antibody to the anti-5-methylcytosine antibody bound to the DNA strand (first antibody), measuring the labeled signal (The so-called “San German law”). Labels can use enzymes, radioisotopes or fluorescent dyes. There are no particular restrictions on the enzyme as long as it meets the conditions such as a large turnover number, stability even when bound to the antibody, and specific coloring of the substrate. Enzymes, such as ぺ ^ / oxidase, β-galatatosidase, anorecaliphosphatase, gnorecosoxidase, acetylcholinesterase, glucose 16-phosphorylation dehydrogenase, and malate dehydrogenase. it can. In addition, enzyme inhibitors, coenzymes, and the like can also be used. The binding between the enzyme and the antibody can be performed by a known method using a crosslinking agent such as a maleimide compound. As the substrate, a known substance can be used depending on the type of the enzyme to be used. For example, when peroxidase is used as an enzyme, 3,3 ', 5,5'-tetramethylbenzicin can be used.When alkaline phosphatase is used as an enzyme, paranitrophenol can be used. . As the radioisotope, those used in normal RIA such as { ²⁵ } and ³ } can be used. Fluorescent dyes such as fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) are commonly used in fluorescent antibody methods. Can be used. In the measurement of such a label signal, when an enzyme is used as the label, the enzyme activity is determined by adding a substrate that develops color by the action of the enzyme and optically measuring the amount of decomposition of the substrate. This is converted to the amount of bound antibody, and the amount of antibody is calculated by comparison with a standard value. When using radioactive isotopes, measure the radiation dose emitted by the radioisotope using a scintillation counter or the like. When a fluorescent dye is used, the amount of fluorescence may be measured by a measuring device combined with a fluorescence microscope.

The method of the present invention can be performed in a liquid phase system or a solid phase system, but is preferably performed in a solid phase system in order to obtain stable measured values. That is, a DNA chain as a test sample is immobilized on a solid phase, and the immobilized DNA chain is reacted with an anti-5-methylcytosine antibody. In order to immobilize a DNA chain (particularly genomic DNA) on a solid phase, for example, a double-stranded linker DNA is immobilized on the solid phase, and the double-stranded strand cut with a restriction enzyme so as to match the end of the linker. A method of linking genomic DNA using ligase can be employed. Immobilization of the linker DNA on the solid phase can be carried out by making one end of the linker DNA biotinylated by a known method and immobilizing it on an avidin-coated solid phase. Alternatively, a method of synthesizing linker DNA into which a functional group has been introduced, spotting the linker DNA on the surface-treated surface of the solid support, and covalently binding the linker DNA (eg, Lamture, JB et al. Nucl. Acids Res. 22 : 2121-2125, 1994; Guo, Z. et al. Nucl. Acids Res. 22: 5456-5465, 1994). Furthermore, the double-stranded DNA immobilized by the above method can be converted into single-stranded DNA by denaturing with, for example, a hydrochloric acid solution or the like.

The method of the present invention also provides a method for separating a monovalently bound anti-5-methylcytosine antibody from a DNA chain and measuring the amount of the divalently bound antibody to specify a dense region of 5-methylcytosine. This is one of the preferred embodiments. That is, an antibody (IgG) molecule is composed of one Fc part and two Fab parts, and the tip of the Fab part binds to the target peptide. Therefore, as shown in Figure 1, the 5-methyl In the case of one cytosine, one Fab portion of the anti-5-methylcytosine antibody binds (monovalent binding: FIG. 1B), while two 5-methylcytosines exist at an appropriate distance. In, two Fabs bind to each 5-methylcytosine (bivalent binding: FIG. 1A). The divalent binding of the antibody is known to have about 103 (Mi) times higher affinity than the monovalent binding, and the affinity of the divalent antibody is removed. By measuring, a region where 5-methylcytosine is concentrated can be detected with high accuracy.

Here we discuss the molecular size of antibody and DNA molecules. The IgG molecular structure has a structure similar to the letter T in the alphabet, and it has been revealed that the distance between the two ends of the two Fabs, that is, the distance between the two antigen-binding sites is about 142 nm (Sarma VR et al., J. Bio. Chem., vol. 246, pp3753-3759, 1971). In the case of B-type DNA with a double helix structure proposed by Watson and Crick, the DNA is arranged at intervals of 0.34 nm for each base pair, and the pitch at which the helix of the DNA makes one rotation is 3.4ηηι. When converted from this, about 14 nm of the two-point antigen-binding site present in the IgG molecule corresponds to a distance of about 42 bases in terms of the number of bases. Therefore, an anti-5-methylcytosine antibody can bind divalently to DNA if the distance between two different 5-methylcytosines present on the same DNA is within about 40 bases.

As shown in Examples 4 and 5 described below, when the content of 5-methylcytosine in single-stranded DNA is, for example, about 4.4%, the anti-5-methylcytosine antibody may contain a plurality of 5-methylcytosine antibodies. It becomes possible to bind divalently to single-stranded DNA containing lucitosin, which is presumed that the average distance between two different 5-methylcytosine molecules on the same single-stranded DNA is about 14 mn or less.

Using this principle, anti-5-methylcytosine antibodies can be used as a nano-scale-rod, ie, a “nano-scale” that measures approximately 14 nm. Specifically, when measuring the distance between two different 5-methylcytosine molecules on the same single-stranded DNA molecule, if the antibody (IgG) can only make a monovalent bond, the distance between the 5-methylcytosine molecules is approximately It can be said that it is within 14nm if it can be more than 14nm and can form a divalent bond. This Using this, it is possible to determine the content of 5-methylcytosine in DNA. In order to remove the monovalent binding antibody, for example, a method in which an alkaline or acidic buffer solution or a buffer solution with a high salt concentration is applied, or a method in which an excess amount of an antigen (5-methylcytosine) is used for antagonism Alternatively, a method of increasing the solution temperature can be adopted. Further, in the method of the present invention, after reacting an anti-5-methylcytosine antibody and binding each of the monovalent and divalent antibodies to the DNA chain, the monovalent binding antibody is obtained by the above method. May be removed (dissociated), or only the divalent antibody may be bound to the DNA chain using the above-mentioned buffer or the like.

Further, in the method of the present invention, another preferred embodiment is that a region other than an arbitrary region of the single-stranded DNA is double-stranded and the DNA methylation rate is measured for the single-stranded region (see FIG. 3). This method is particularly preferable when measuring the abundance of 5-methylcytosine and its density only in a region (eg, a gene expression control region or the like) where DNA methylation is important. In order to make the region other than the region to be measured double-stranded, a method of annealing a single-stranded oligonucleotide fragment complementary to the DNA sequence of the region to be double-stranded can be adopted.

Hereinafter, the invention of this application will be described in more detail and specifically with reference to examples, but the invention is not limited to the following examples. Example 1: Preparation of antibody

5-methylcytidine-KLH (KLH = keyhole limpet hemosinin) conjugate (100μ _§ ) was administered to mouse paws together with FCA (Freund 'Complete' adjuvant), lymph nodes were removed on day 9 and mouse myeloma was removed. It was fused with cells (SP2 / 0). The obtained fused cells were seeded on three 96-well culture plates, and hybridomas were selectively grown using HAT medium according to a conventional method. About 10 days to 2 weeks after the start of the culture, antibody activity was screened for the cultured sperm of the hybridoma that had been grown. Specifically, the antigen [5-methylcytidine binds to BSA (bovine serum albumin) 5-methylcytidine-BSA conjugate] was dissolved in PBS at a concentration of lO g / ml, and this was coated on a 96-well ELISA plate, and then blocked with BSA according to a conventional method. The culture supernatant was allowed to react to obtain a hydrideoma exhibiting antibody reaction positivity. In addition, the antibody reactivity against the cytidine-BSA conjugate in which cytidine was conjugated to BSA was measured in the same manner, and a hybridoma producing no or low cross-activity with cytidine-BSA was selected. First, an anti-5-methylcytosine antibody was obtained from the hybridoma according to a conventional method. Example 2: Examination of antibody specificity (1)

The following experiment was performed to examine the reaction specificity of the anti-5-methylcytosine antibody prepared in Example 1.

A 225-base DNA fragment (SEQ ID NO: 1) corresponding to nucleotides 1966 to 2190 in the cDNA base sequence of mouse focal adhesion kinase (GenBank M95408) was amplified by PCR. At this time, oligo DNA having a 5′-position biotinylated was used as a PCR sense strand primer. Mouse brain cDNA was used as type I DNA, and PCR was performed under the following conditions.

The double-stranded DNA, which is the PCR product, contains 50 cytosine bases in the sense strand of the double-stranded DNA, but 20 bases out of 46 bases excluding the PCR primer are randomly replaced with 5-methylcytosine. As shown in Figure 3, 5-methyl-2'-deoxycytidine-5'-triphosphate (5m_dCTP) was mixed into the PCR reaction solution. Specifically, a PGR reaction was performed using a solution obtained by mixing the following deoxynucleotide stock solution A and stock B solution at a ratio of 26:20.

PCR primers:

Sense primer: 5'-Biotin-CGTGAAGCCTTTTCAAGGAG-3 '(SEQ ID NO: 2) Antisense primer: 5, -TCCATCCTCATCCGTTCTTC-3' (SEQ ID NO: 3) Enzyme used:

Expand High-FideHty PCR System (Roche's Diagnostics) Deoxynucleotides:

Stock A solution (0.5 mM clATP, 0.5 mM dTTP, 0.5 mM dGTP, 0.5 mM dCTP) Stock B solution (0.5 mM dATP, 0.5 mM dTTP, 0.5 mM dGTP, 0.5 mM 5m-dCTP) Deoxynucleotide stock solution above Was added to 50 μL of the PGR reaction solution so that the total of the solution and the solution was 5 /. Each ratio can be arbitrarily set depending on the ratio of 5-methylcytosine incorporated into the PCR product to be produced. Reaction conditions:

(1) 94.5, 2min x l cycle

(2) [94.5, 30sec / 58 ° C, 30sec / 72 ° C, 40sec] x 28 cycles

(3) 72 ° C, 8min x 1 cycle

The PCR reaction was performed using Thermal Cycler MP (Takara Shuzo).

After the PCR reaction, DNA was purified from the PCR product according to a conventional method and immobilized on an avidin-coated 96-well microtiter plate. Next, the double-stranded DNA was treated with 50 mM hydrochloric acid for 2 minutes to be single-stranded. After this treatment, the well was washed several times with PBS / lmM EDTA, and only the single-stranded DNA captured by avidin was immobilized on the well.

To this immobilized single-stranded DNA, the anti-5-methylcytosine antibody (primary antibody) prepared in Example 1 and horseradish peroxidase (HRP) -labeled anti-mouse IgG antibody (secondary antibody) ELISA was carried out according to a standard method using. However, the microtiter plate is usually washed with a buffer containing 10 mM ris-HCl (pH 7.6) /0.15 M NaCl / 0.05% Teen20, and the substrate is paranitrophenyl phosphate (Sigma, N After the reaction with the substrate, the absorbance at 405 nm was measured after a certain period of time.

The content of 5-methylcytosine contained in the single-stranded DNA immobilized on the well was changed arbitrarily, and the reactivity of the anti-5-methylcytosine antibody at that time was examined. Fig. 4 shows the results of the ELISA measurement.

As shown in FIG. 4, the antibody binding to the anti-5-methylcytosine antibody increased as the content of 5-methylcytosine in the DNA immobilized on the gel increased. By hydrochloric acid treatment If the double-stranded DNA is immobilized on a well without being single-stranded, the total amount of the double-stranded DNA is about 43, which is a double-stranded DNA containing 5-methylcytosine. Anti-5-methylcytosine antibody did not bind to the single-stranded DNA. That is, it was confirmed that the anti-5-methylcytosine antibody did not bind to double-stranded DNA containing 5-methylcytosine, but specifically bound only to single-stranded DNA containing 5-methylcytosine.

Example 3: Examination of antibody specificity (2)

After reacting the anti-5-methylcytosine antibody (primary antibody) on the DNA strand having 20 5-methylcytosine randomly, treat it with a washing buffer containing different concentrations of NaCl for 10 minutes. ELISA was performed in the same manner as in Example 2 except for the above. In addition, after the primary antibody reaction, the plate was treated for 10 minutes with a buffer [10 mM Tris-HCl (pH7.6) /0.15M NaCl / 0.05% Tween20] used for normal cell washing containing 0.15M NaCl. Was used as a comparison control (100%).

The results are as shown in FIG. The binding of anti-5-methylcytosine antibody to methylcytosine is inhibited depending on the concentration of NaCl, but it was confirmed that antigen-antibody binding was maintained to some extent even with 0.5 to 1 M NaCl treatment (10 minutes). .

Example 4: Examination of antibody specificity (3)

The sensitivity to NaCl treatment when the content of 5-methylcytosine per single-stranded DNA immobilized on a 96-well microtiter plate was changed was measured. Example 3 except that after binding the primary antibody, the wells were treated for 10 minutes with a well washing buffer containing 0.5N NaCl (10 mM Tris-HCl (pH7.6) /0.5M NaCl / 0.05% Tween20) for 10 minutes. Was performed in the same manner as described above.

The results are as shown in FIG. Instead of the treatment using lOmM Tris-HCl (pH7.6) /0.5M NaCl / 0.05% Teen20, a normal well washing buffer (lOmM Tris-HCl (pH7.6) /0.15M NaCl / 0.05 % Teen20) for 10 minutes as a control.

As shown in Fig. 6, anti-5-methylcytosine antibody was used in a 96-well microtiter plate. If single-stranded DNA (225 bases) immobilized on a single well contains an average of four 5-methylcytosines, the antigen-antibody reaction is easily dissociated by treatment with 0.5 M NaCl, but The presence of an average of 10 5-methylcytosines in the single-stranded DNAC225 base immobilized on the DNA) stabilizes the binding of the antibody to the antigen and sharply increases the resistance to 0.5M NaCl washing. It was confirmed that.

Example 5: Examination of antibody specificity (4)

The anti-5-methylcytosine antibody prepared in Example 1 was a mouse IgG2a, κ chain antibody.A Fab fragment of this antibody was prepared, and the binding resistance of the Fab fragment and the whole IgG molecule to NaCl treatment was compared and examined. . The ELISA protocol followed Example 4.

The results are as shown in FIG. Generally, the I _g G molecules with binding sites of the antigen 2 power plant, the Fab fragments binding sites 1 Chikarasho, Abuiniti one antigen and antibody (equilibrium constant: Mi) opens approximately 1000-fold IgG molecules capable of bivalent binding to an antigen have stronger affinity. As is clear from FIG. 7, IgG molecules of the anti-5-methylcytosine antibody are more resistant to treatment with a high salt concentration (NaCl) than Fab molecules derived from this IgG. That is, when compared at the same NaCl concentration, it was confirmed that the Fab fragment dissociated from the antigen faster than the IgG molecule.

The above results, together with the results of Example 4, are considered as follows. In Example 4, when the content of 5-methylcytosine in the single-stranded DNA was about 1.8%, the treatment with a high concentration of NaCl (here, 0.5 M NaCl) dissociated most of the antigen-antibody reaction. However, when the content of 5-methylcytosine in single-stranded DNA reaches approximately 44%, the proportion of IgG molecules that maintain antigen-antibody binding increases rapidly even with high-concentration NaCl treatment. Was.

That is, as is clear from the test results using the Fab fragment of Example 5, the 5-methylcytosine content in the single-stranded DNA was about 1.8% to about 4.4% in the specific region of the DNA molecule. %, The anti-5-methylcytosine antibody (IgG Molecule) changes from a monovalent bond to a divalent bond. In other words, the presence of the antigen (5-methylcytosine) at a certain density or higher allows one molecule of anti-5-methylcytosine antibody (IgG) to bind to multiple 5-methylcytosine bases present on the same DNA molecule. It means that valence bonding can be performed.

Example 6: Example of measuring 5-methylcytosine content in specific region

Utilizing the fact that an anti-5-methylcytosine antibody does not react with double-stranded DNA, a specific region in the double-stranded DNA is limited to a single strand, and this single strand is used as the measurement target area for 5-methylcytosine. The content was measured.

According to the method of Example 2, the sense strand of the double-stranded DNA whose nucleotide sequence is shown in FIG. 3 (SEQ ID NO: 1) should contain 20 bases of 5-methylcytosine in the sense strand DNA. Synthesized. The PCR primers were the same as in Example 2.

Next, about 500 ng of the double-stranded DNA was added to a denaturing buffer [10 mM Tris-HCK pH 8.l) / 50 mM KCl / 1.5 mM MgCl ₂ ], and the synthetic oligo DNA of SOpmole (three types in FIG. 8; , 5 and 6) were added to make a total volume of 50 / l, which was placed in a PCR tube (200 jl volume) and set in a PCR thermal cycler. This reaction solution was subjected to a denaturation treatment at 98 ° C for 2 minutes, and then immediately treated at 65 ° C for 30 minutes to induce DNA reassociation. As a result, a portion of the double-stranded DNA is stabilized in a single-stranded state in a limited region due to the interference of the added synthetic oligo DNA insert. That is, the synthetic oligo DNA insert is stabilized while being sandwiched by the double-stranded DNA. The obtained PCR product was immobilized on a substrate of an avidin-coated 96-well microtiter plate while the oligo insert was sandwiched. Here, 51 out of 50 μl of the total reaction solution in the PCR tube was added to 1 well of a 96-well microtiter plate and immobilized. Subsequent operations were performed according to the same ELISA protocol as in Example 4 described above. However, HRP-labeled anti-mouse IgG antibody was used as the secondary antibody, and tetramethylbenzidine (SIGMA ο & ΐ # ΊΗ) 440) was used as the substrate. Add 20μ1 of 0.3M sulfuric acid The reaction was stopped by measuring the absorbance at 450 mn.

The results are as shown in FIG. The subject double-stranded DNA (225 bp) contains about 20 bases of 5-methylcytosine per sense strand. The highest absorbance is obtained when using oligo insert # 1, which hybridizes to the region near the end of the double-stranded DNA, and decreases as the region where the oligosinate hybridizes enters the central region of the double-stranded DNA. Keep going. All oligo inserts used are 30raer. Therefore, at this time, when oligo inserts # 1, # 2, and # 3 bind to double-stranded DNA, cytosine of 3, 6, and 11 bases, respectively, is exposed as a single strand, About 50% of these cytosines are replaced by 5-methylcytosine. The absorbance of oligoinsert # 1 with a small number of cytosines exposed from double-stranded DNA is higher, and the absorbance of oligoinserts # 2 and # 3 with a larger number of cytosines exposed is lower. In other words, this means that the position where oligosinate hybridizes on the double-stranded DNA is more stable at the end of the double-stranded DNA, and is more likely to be eliminated from the double-stranded DNA toward the center. . As a method of solving this, there are single-stranded RNA forces that bind more tightly than DNA bonds, or peptide nucleic acids that also bind more tightly than DNA bonds (unlike phosphate bonds, unlike DNA and RNA). Oligo-DNA that forms a backbone with peptide bonds, abbreviated as PNA), to efficiently and locally perform single-strand ligating even at the center of the target double-strand. Can be. The present invention is not limited to these, and if it is a substance that is stronger than the bond between DNAs and hybridizes with DNA, the same results as in the case of the above-described oligosert can be obtained. BRIEF DESCRIPTION OF THE FIGURES

【Figure 1】

(A) shows divalent binding of the anti-5-methylcytosine antibody to single-stranded DNA containing 5-methylcytosine, and (B) shows monovalent binding. 【Figure 2】

FIG. 4 shows the binding state of an anti-5-methylcytosine antibody to 5-methylcytosine of a partially single-stranded DNA chain.

[Figure 3]

It is a base sequence of a DNA chain used in Examples. Cytosine enclosed in a box is randomly substituted with 5-methylcytosine at an arbitrary ratio.

[Fig. 4]

5 is a graph showing the relationship between the number of 5-methylcytosine contained in a DNA chain and the degree of binding of an anti-5-methylcytosine antibody.

[Figure 5]

5 is a graph showing the relationship between the NaCl concentration and the degree of binding of an anti-5: methylcytosine antibody when NaCl treatment is performed after a primary antibody (5-methylcytosine antibody) reaction.

[Fig. 6]

Primary antibody (5-methylcytosine antibody) Relationship between the number of 5-methylcytosine in the DNA chain and the degree of binding of the anti-5-methylcytosine antibody when treated with 0.15M or 0.5M NaCl after the reaction FIG.

[Fig. 7]

It is the graph which showed the 5-methyl cytosine binding state of the 5-methyl cytosine antibody (IgG molecule) and the Fab molecule | numerator at the time of processing by NaCl of each density | concentration.

[Fig. 8]

FIG. 6 shows the base sequences of synthetic oligo DNA inserts # 1 to # 3 used to form a single-stranded DNA region in the examples, and the base sequences of DNA strands to which these inserts hybridize. The hybridized region of Insert is indicated by a box.

[Fig. 9]

4 is a graph showing the degree of binding of an anti-5-methylcytosine antibody to double-stranded DNA hybridized with synthetic oligo DNA inserts # 1 to # 3. Industrial applicability

As described in detail above, the description of this application makes it possible to easily and accurately measure 5-methylcytosine in a DNA chain, and provides a new means for diagnosing, for example, malignancy of cancer.

Claims

The scope of the claims

1. A method for measuring the rate of DNA methylation, which comprises contacting an antibody that specifically binds to 5-methylcytosine with single-stranded DNA, and measuring the amount of the antibody bound to the DNA strand.

2. The method according to claim 1, wherein the monovalently bound antibody is separated from the DNA chain, and the amount of the divalently bound antibody is measured to identify the dense region of 5-methylcytosine.

3. The method according to claim 1, wherein a region other than an arbitrary region of the single-stranded DNA is double-stranded, and the DNA methylation ratio is measured for the single-stranded region.