WO2008123537A1 - Method for measurement of methylation in dna - Google Patents

Abstract

Disclosed is are: a method for measuring the content of methylated DNA in a DNA region of interest in the genomic DNA contained in a organism-derived sample; and others.

Description

 Specification

 DNA Methylation Measurement Method Technical Field ''

 The present invention relates to a method for measuring the content of methylated DNA in a target DNA region in genomic DNA contained in a biological specimen. Background art

 Examples of a method for evaluating the methylation status of DN A ′ in the target DNA region of genomic DNA contained in a biological sample include methylated DNA in the target DNA region of genomic DNA. (For example, see Nucleic Acids Res. 1994 Aug 11; 22 (15): 2990-7, and Proc Natl Acad Sci US A. 1997 Mar 18; 94 (6): 2284-9) ) In the measurement method, first, it is necessary to extract DNA containing a target DNA region from a DNA sample derived from genomic DNA: the extraction operation is complicated.

 As a method for measuring the content of methylated DNA in the target region of the extracted DNA, for example, (1) After modifying the DNA with sulfite, etc., DNA by DNA polymerase A method of amplifying the target region by subjecting it to a polymerase chain reaction (hereinafter sometimes referred to as PCR). (2) After digesting the DNA with a methylation sensitive restriction enzyme, PCR There are known methods for amplifying the target region by subjecting it to the above. Both of these methods require labor for modification of DNA for detection of methylation and subsequent purification of the product, preparation of a reaction system for PCR, confirmation of DNA amplification, and the like. Disclosure of Invention ■

An object of the present invention is to provide a method for easily measuring the content of methylated DNA in a target DNA region in genomic DNA contained in a biological specimen. ' That is, the present invention

 1. A method for measuring the content of methylated DNA in a target DNA region in genomic DNA contained in a biological sample,

 (1) First (A) to separate methylated single-stranded DNA from DNA samples derived from genomic DNA contained in biological samples, and separated in the first (A) step A first step of selecting the above-mentioned single-stranded DNA comprising the first step (B) of binding the single-stranded DNA and the immobilized methylated DNA antibody;

 (2) a second step of digesting the single-stranded DNA selected in the first step with a methylation-sensitive restriction enzyme capable of degrading at least one kind of single-stranded DNA; and

, (3) As a pre-process of each of the following steps, a single-stranded DNA that is an undigested product obtained in the second step (a recognition site of a methylation-sensitive restriction enzyme capable of digesting the single-stranded DNA) A single-stranded DNA containing no CpG in the methyl state is separated from the immobilized methylated DNA antibody into a single-stranded DNA (positive strand) (third (previous A) step:) .When,

 The single-stranded DNA (positive strand) separated in the third (previous A) step is used as a saddle, and the single-stranded DNA (positive strand) has a partial base sequence (positive And complementary to the partial base sequence (positive strand) located further to the 3 ′ end than the 3 ′ end of the base sequence (positive strand) of the target DNA region. By using an extension primer (forward primer) having a certain base sequence (negative strand) as an extension primer, the extension primer is extended once, so that the DNA (positive strand) in the single-stranded state is obtained. The process of extending and forming double-stranded DNA (third (previous B) process) and the double-stranded DNA elongated and formed in the third (previously B) process are converted into single-stranded DNA containing the target DNA region ( Step of separating the DNA into a single-stranded DNA (negative strand) containing the target DNA region (positive strand) (third) Has a pre-C) step), and, as this process

(a) The generated single-stranded DNA (positive strand) containing the target DNA region is used as a cage, and the extension primer is extended once by using the foraging primer as an extension primer. This step of extending and forming single-stranded DNA containing the target DNA region as double-stranded DNA—A,, (b) Using the generated single-stranded DNA (negative strand) containing the target DNA region as a cage, the partial base of the base sequence of the single-stranded DNA (negative strand) containing the target DNA region It is a sequence (negative strand) and is further to the end of the 3 ′ end of the 3 ′ end of the base sequence (negative strand) that is complementary to the base sequence (positive strand) of the target DNA region. By extending the extension primer once with the extension primer (reverse primer) having a partial base sequence (negative strand) and a complementary base sequence (positive strand) as the extension primer. A step B for extending and forming a single-stranded DNA containing the target DNA region as a double-stranded DNA,

 Further, each of the steps is repeated after the elongated double-stranded DNA obtained in each step is once separated into a single-stranded state, and then methylated in the target DNA region. Amplify the amplified DNA to a detectable amount, and amplify the DN

A third step of quantifying the amount of A, which is characterized by the following (hereinafter sometimes referred to as the present measuring method);

 2. The method according to item 1 above, wherein the methylated DNA antibody in the third step is a methylcytosine antibody;

 3. The method according to any one of items 1 to 2 above, wherein the biological specimen is mammalian serum or plasma;

 4. The method according to any one of items 1 to 2 above, wherein the biological specimen is mammalian blood or body fluid;

 5. The method according to any one of items 1 to 2 above, wherein the biological specimen is a cell lysate or a tissue lysate;

 6. A DNA sample derived from a genomic DNA contained in a biological sample is a DNA sample that has been digested in advance with a restriction enzyme that does not use the target DNA region of the genomic DNA as a recognition cleavage site. The method according to any one of items 1 to 5 above, characterized by:

7. A DNA sample derived from genomic DNA contained in a biological sample is a DNA sample digested with at least one methylation-sensitive restriction enzyme. The described method; 8. The method according to any one of 1 to 7 above, wherein the DNA sample derived from genomic DNA contained in the biological specimen is a DNA sample purified in advance;

9. A methylation-sensitive restriction enzyme capable of digesting at least one kind of single-stranded DNA has a recognition cleavage site in the target DNA region of the genomic DNA contained in the biological sample. The method according to any one of 1 to 8 above, which is a restriction enzyme;

 10. The method according to any one of 1 to 9 above, wherein the methylation sensitive restriction enzyme capable of digesting at least one kind of single-stranded DNA is Hhal which is a methylation sensitive restriction enzyme; .

'11. The method according to any one of 1 to 10 above, wherein the third step is carried out without carrying out the quenching treatment with a methylation sensitive restriction enzyme capable of digesting single-stranded DNA in the second step. A method; and

 12. When separating the single-stranded DNA methylated in the first step (A), the counter: the method according to any one of 1 to 11 above, wherein an oligonucleotide is added; Brief Description of Drawings

 FIG. 1 shows the amplification of methylated DNA in the region consisting of the base sequence shown in SEQ ID NO: 21 from the prepared sample in Example 1 by PCR, and 1.5% of the obtained amplification product. It is the figure which showed the result used for agarose gel electrophoresis. From the leftmost lane in the figure, sample “D” prepared from DNA marker “MK”, UBC-M solution D, sample “C”, UBC-M prepared from solution C in UBC-M Sample “B” prepared from solution B, sample “A” prepared from solution A in UBC—M, “A” from DNA fragment Y2, sample “D” prepared from solution D in UBC—U, Results are shown for sample “C” prepared from UBC—U solution C, sample “A” prepared from solution B of UBC—U, and sample “A” prepared from solution A of UBC—U. .

Figure 2 shows that the sample prepared in Example 2 is labeled “A (no treatment)” or “B (ffiial treatment) ”is performed, the methylated DNA in the region consisting of the base sequence shown in SEQ ID NO: 27 is amplified by PCR, and the obtained amplification product 1.5% It is the figure which showed the result for which it used for the mouth-and-mouth gel electrophoresis.

 From leftmost lane in the figure, DNA marker “MK”, partially methylated oligonucleotide not digested with HAL GPR 7—207.9— 2176 / 98mer — M (7) Solution “A” was applied Sample “M”, partially methylated oligonucleotide digested with Hha I GPR 7— 2079-2176 / 98me r -HM (5) Sample “HMJ”, unmethylated with solution “A” treated Rigonucleotide G PR7-2079-2176 / 98me r— Samples treated with “A” in UM solution “U”, partially methylated oligonucleotide not digested with Hha I GPR7— 2 07,9-2176 / 98me r -M (7) Sample “B” treated with solution “B”, partially methylated oligonucleotide digested with Hha I GPR7—2079— 2176 / 98me r -HM (5) “B” of solution Treated sample “HM” ：: Unmethylated oligonucleotide GPR 7 -2079-2176 / 98me r— “B” of UM solution Management is shows the results of a sample "U", which has been subjected to.

 FIG. 3 shows the arrangement of the sample that had been treated with methylated cytosine antibody in Example 3 in advance for either “A (no treatment)” or “B (Hhal treatment)”. FIG. 5 shows the results of amplification of methylated DNA in the region consisting of the base sequence shown by No. 27 with PCR and subjecting the obtained amplification product to 1.5% agarose gel electrophoresis. .

From leftmost lane in the figure, DNA marker “MK:”, partially methylated oligonucleotide that is not digested with Hhal GPR 7 -2079-2176 / 98mer — M (7) Solution “A” was applied. Sample `` M '', partially methylated oligonucleotide digested with Hha I GPR 7-2079-2176 / 98mer-HM (5) Sample `` HM '' treated with solution `` A '', ammethyl Oligonucleotide G PR 7-2079-2176 / 98me r—UM treated sample “U”, partially methylated oligonucleotide not digested with Hha I GPR7— 2 079-2176 / 98me r -M (7) Sample "M" treated with solution "B" ”Partially methylated oligonucleotide digested with Hha I GPR7— 2079 — 2176 / 98me r HM (5) solution treated sample“ Η ”, unmethylated oligonucleotide GPR 7-2079 -2176 / 98me r— Shows the results for the sample “U” with the “&” treatment of the UM solution.

'' Figure 4 shows the amplification of the methylated DNA in the target DNA region consisting of the base sequence represented by SEQ ID NO: 32 from the prepared sample in Example 4 by PCR, and the resulting amplification product. 1. A diagram showing the results of 5% agarose gel electrophoresis. From leftmost lane in the figure, DNA marker “MK:”, methylated DNA fragment MX solution “MD” (negative control), unmethylated DN A fragment X solution “D” (Negative control), methylated DNA fragment MX solution “MC”, unmethylated DNA fragment X solution “C”, methylated DNA fragment MX solution “MB”, methylated Not solution of DNA fragment X “B”, methylated D: NA fragment MX solution “MA”, unmethylated DNA fragment X solution “A”, shows the results.

 FIG. 5 shows the amplification of the methylated DNA in the target DNA region consisting of the base sequence represented by SEQ ID NO: 49 from the prepared sample in Example 5 by PCR. FIG. 5 shows the results of 5% agarose gel electrophoresis. From the leftmost lane in the figure, DNA marker “MK:”, methylated DNA fragment MY solution “MD” (negative control), unmethylated DNA fragment Y solution “D” (negative) Control), methylated DNA fragment MY solution “MC”, unmethylated DNA fragment Y solution “C”, methylated DNA fragment MY solution “B”, unmethylated DNA The results are shown in solution “B” for fragment Y, solution “M′A” for methylated DNA fragment MY, and solution “A” for DNA fragment Y that is not methylated.

FIG. 6 shows the methylated DNA in the target DNA region consisting of the base sequence represented by SEQ ID NO: 57 from the prepared sample in Example 6, using PCR. FIG. 5 is a diagram showing the results of amplification and 1.5% agarose gel electrophoresis of the amplification product obtained. From the leftmost lane in the figure, DNA marker “MK”, methylated DNA fragment MS solution “MC” (negative control), unmethylated DNA fragment S solution “C” ( Negative control), methyl 'methylated DNA fragment MS solution' MB ', unmethylated DNA fragment S solution' B ', methylated DNA fragment MS solution' MA ', methylated Not shown in DNA fragment S solution “A”, with results.

 FIG. 7 shows the amplification obtained in Example 7 by amplifying methylated DNA in the target DNA region consisting of the salt sequence shown in SEQ ID NO: 72 from the prepared sample with PCR. It is the figure which showed the result of 1.5% agarose gel electrophoresis of the product. From the leftmost lane in the figure, DNA marker “MK:”, methylated DNA fragment MT solution “MC” (negative control), methylated: not DNA fragment T solution “Cj (negative) Control solution), methylated DNA fragment MT solution “MB”, unmethylated DNA fragment T solution “B”, methylated DNA fragment MT solution “MA”, methylated The results for the DNA fragment T solution “A”, not shown.

FIG. 8 shows the amplification product obtained by amplifying methylated DNA in the target DNA region consisting of the base sequence represented by SEQ ID NO: 57 from the prepared sample in Example 8 using PCR. 1. HI showing the result of 5% agarose gel electrophoresis. From leftmost lane in the figure, DNA marker “MK”, methylated yeast genomic DNA solution “MD” (negative control), unmethylated yeast genomic DNA solution “D” (negative control) ) Methylated yeast genomic DNA solution "MC", Unmethylated yeast genomic DNA solution "C", Methylated yeast genomic DNA solution "MB", Unmethylated yeast genomic DNA solution Results are shown for solution “B”, methylated yeast genomic DNA solution “MA”, and unmethylated yeast genomic DNA solution “A”. . FIG. 9 shows the amplification product obtained by amplifying the methylated DNA in the target DNA region consisting of the base sequence represented by SEQ ID NO: 72 from the prepared sample in Example 9 by PCR. FIG. 5 shows the results of 5% agarose gel electrophoresis. From leftmost lane in the figure, DNA marker “MK:”, methylated yeast genomic DNA solution “MD” (negative control), unmethylated yeast genomic DNA solution “D” (negative) Control), methylated yeast genomic DNA solution “MC”, unmethylated yeast genomic DNA solution “C”, methylated yeast genomic DNA solution “MB”, unmethylated yeast genomic DNA solution Solution "B", methylated yeast genomic DNA solution "MA", unmethylated yeast 'genomic DNA solution "A", shows the results.

FIG. 10 shows the amplification of the methylated DNA in the target DNA region consisting of the nucleotide sequence shown in SEQ ID NO: 32 from the prepared sample by PCR. FIG. 5 shows the results of 5% agarose gel electrophoresis. From the leftmost lane, “MK:”, methylated DNA fragment MX solution “MD” (negative control), unmethylated DNA fragment X solution “D” (Negative control) Methylated DNA fragment MX solution “MC”, Unmethylated DNA fragment X solution “C”, Methylated DNA fragment MX solution “MB” Results are shown for the unmethylated DNA fragment X solution “B”, the methylated DNA fragment MX solution “MA”, and the unmethylated DNA fragment X solution “A”. . · Figure 11 shows the amplification product obtained in Example 11 by amplifying methylated DNA in the target DNA region consisting of the base sequence represented by SEQ ID NO: 57 from the prepared sample by PCR. 1. A diagram showing the results of 5% agarose gel electrophoresis. From the leftmost lane in the figure, DNA marker “MK”, methylated DNA fragment MS solution “MD” (negative control), unmethylated DNA fragment S solution “D” ( Negative control), methylated DNA fragment MS solution “MC”, unmethylated DN Solution A of fragment S “C”, solution of methylated DNA fragment MS “MB”, solution of DNA fragment S not methylated “B”, solution of methylated DNA fragment MS “MA”, methyl The results for the unfragmentated DNA fragment S solution “A”, are shown.

'' FIG. 12 is obtained by amplifying the methylated DNA in the target DNA region consisting of the base sequence represented by SEQ ID NO: 72 from the prepared sample in Example 12 with PCR. FIG. 5 shows the results of 1.5% agarose gel electrophoresis of amplification products. From the leftmost lane in the figure, DNA marker “MK:”, methylated DNA fragment MT solution. “MD” (negative control), unmethylated DNA fragment T solution “D” ”(Negative control)., Methylated DNA fragment MT solution“ MC ”, unmethylated DNA fragment T solution“ C ”, methylated DNA fragment MT solution Γ MB ”Solution of unmethylated DNA fragment T“ B ”, solution of methylated DNA fragment MT“ MA ”, solution of unmethylated DNA fragment T solution“ A ”, Show.

 FIG. 13 shows the amplification of methylated DNA in the target DNA region consisting of the base sequence shown in SEQ ID NO: 32 from the prepared sample by PCR. FIG. 5 is a diagram showing the results of 5% slag gel electrophoresis. From leftmost lane in the figure, DNA marker “MK:”, methylated human genomic DNA solution “MC” (negative control), unmethylated human genomic DNA solution “C” (negative control), methyl Results for the solution of methylated human genomic DNA “MB”, the solution of unmethylated human genomic DNA “B”, the solution of methylated human genomic DNA “MA”, and the solution of unmethylated human genomic DNA “A” Show.

FIG. 14 shows the results obtained by amplifying methylated DNA in the target DNA region consisting of the base sequence represented by SEQ ID NO: 57 from the prepared sample in Example 14 by PCR, and obtaining the amplified product 1 FIG. 5 is a diagram showing the results of 5% agarose gel electrophoresis. From the leftmost lane in the figure, the DNA marker “MK”, methylated yeast Genomic DNA solution “MD” (negative control), unmethylated yeast genomic DNA solution “D” (negative control), methylated yeast genomic DNA solution “MC”, unmethylated yeast genomic DNA Solution "C", methylated yeast genomic DNA solution "MB", unmethylated yeast genomic DNA solution "B", methylated yeast genomic DNA solution "MA", unmethylated yeast Results are shown for solution “A” of Genomic DN A.

 FIG. 15 shows the amplification obtained in Example 15 by amplifying the methylated DNA in the target DNA region consisting of the base sequence represented by SEQ ID NO: 72 from the prepared sample with PCR. FIG. 2 is a diagram showing the results of electrophoresis of a product with 1.5% agarose gel. From the leftmost lane in the figure, DNA marker “MK:”, methylated yeast genomic DNA solution “MD” (negative control), unmethylated yeast genomic DNA solution “D” (negative control) ), Methylated yeast genomic DNA solution “M (:”), Unmethylated yeast genomic DNA solution “C”, Methylated yeast genomic DNA solution “MB”, Unmethylated yeast genome Results are shown for DNA solution “B”, methylated yeast genomic DNA solution “MA”, and unmethylated yeast genomic DNA solution “A”.

 Examples of the “biological specimen” in the present invention include, for example, a cell lysate, a tissue lysate (herein, tissue has a broad meaning including blood, lymph nodes, etc.), or in mammals, plasma. Examples thereof include biological samples such as serum and lymph, body fluids such as body secretions (urine, milk, etc.), and genomic DNA obtained by extraction from these biological samples. Examples of the biological specimen include samples derived from microorganisms, viruses, and the like. In this case, “genomic DNA” in the measurement method of the present invention means genomic DNAs such as microorganisms and viruses.

 In the case where the sample of the mammal application is blood, the measurement method of the present invention can be expected to be used in periodic health examinations and simple tests.

To obtain genomic DNA from mammalian samples, for example, for commercially available DNA extraction DNA may be extracted using a kit or the like.

 When blood is used as a specimen, plasma or serum is prepared from blood according to a normal method, and the prepared plasma or serum is used as a specimen, and free DNA contained therein (derived from cancer cells such as stomach cancer cells) Analysis of DNA derived from cancer cells such as gastric cancer cells, avoiding blood cell-derived DNA, cancer cells such as gastric cancer cells, tissues containing it, etc. Sensitivity for detection can be improved. Normally, there are four types of bases that make up a gene (genomic DNA). Among these bases, the phenomenon that only cytosine is methylated is known, and such methylation modification of DN'A is based on the base sequence shown by 5'-CG-3 (C is cytosine) G represents guanine, and the base sequence may be referred to as “CpG”. The site that is methylated in cytosine is at position 5. During DNA replication prior to cell division, immediately after replication in the “CpG” of the cage chain: only cytosine is methylated, but immediately in the “CpG” of the nascent chain due to the action of methyltransferase. Cytosine is also methylated. Therefore, the DNA methylation status is inherited by two new DNA groups after DNA replication. In the present invention, “methylated DNA” means DNA produced by such methylation modification.

The “CpG pair” in the present invention means a double-stranded oligonucleotide formed by binding a base sequence represented by CpG and a complementary C pG by base pairing. The “target DNA region” in the present invention (hereinafter sometimes referred to as the target region) is a DNA region to be examined for the presence or absence of methylation of cytosine contained in the region, and includes at least one kind of DNA region. It has a recognition site for a methylation sensitive restriction enzyme. For example, Lysyl oxidase, HRAS-like suppressor ^ bA305P22.2. and metalloproteinase domain 23, G protein-coupled receptor 7, G-protein coupled somatos tat in and angi otens in- 1 ike pept ide receptor, Solute carr ier f ami ly 6 neurotransmit ter transpor ter noradrenal in member 2 etc. A region of DNA containing at least one cytosine in the base sequence indicated by CG existing in the base sequence of the coding region). Specifically, for example, when the useful protein gene is a Lysyl ox i dase gene, it is indicated by C p G present in the base sequence of the promoter region, untranslated region or translated region (coding region). Examples of the nucleotide sequence containing one or more nucleotide sequences include genomic DNA nucleotide sequences containing exon 1 of the Lysyl oxidase gene derived from human ', and its promoter region located upstream of the gene. Specifically, the base sequence represented by SEQ ID NO: 1 (corresponding to the base sequence represented by base numbers 16001 to 18661 of the base sequence described in Genbank Accession No. AF270645) can be mentioned. In the nucleotide sequence shown in sequence number 1, the ATG codon encoding the amino terminal methionine of the Lysyl oxidase protein derived from human is shown in nucleotide numbers 2031 to 2033, and the nucleotide sequence of exon 1 above is shown. Is represented by nucleotide numbers 1957 to 2661. Cytosine in the nucleotide sequence represented by C p G present in the nucleotide sequence represented by SEQ ID NO: 1, particularly C p in the nucleotide sequence represented by SEQ ID NO: 1. Cytosine in C p G present in a region where G is densely present shows a high methylation frequency (ie, hypermethyl ation) in cancer cells such as gastric cancer cells. More specifically, as cytosine having a high methylation frequency in gastric cancer cells, for example, in the base sequence represented by SEQ ID NO: 1, base numbers 1539, 1560, 1574, 1600, 1623, 1635, 1644, 1654, 1661 168 2, 1686, 1696, 1717, 1767, 1774, 1783, 1785, 1787, 1795, etc. More specifically, for example, when the useful protein gene is the HRAS-like suppressor gene Includes a base sequence containing one or more base sequences represented by C p G present in the base sequence of the promoter region, untranslated region or translated region (coding region). For example, the base sequence of genomic DNA containing exon 1 of the HRAS-like suppres sor gene derived from human and the promoter region located 5 'upstream thereof can be mentioned. The nucleotide sequence represented by SEQ ID NO: 2 (corresponds to the nucleotide sequence represented by nucleotide numbers 172001-173953 of the nucleotide sequence described in Genbank Accession No. AC068162). In the base sequence represented by SEQ ID NO: 2, the base sequence of exon 1 of the HRAS-ike suppres sor gene derived from human is represented by base numbers 1'743-1953. Cytosine in the base sequence shown by C p G present in the base sequence shown in SEQ ID NO: 2, especially C p G present in the region where C p G is densely present in the base sequence shown in SEQ ID NO: 2 Cytosine in 'shows a high methylation frequency (ie, hypermethylation state) in cancer cells such as gastric cancer cells. • More specifically, as cytosine having high methylation frequency in gastric cancer cells, for example, in the base sequence represented by SEQ ID NO: 2, base numbers 1316, 1341, 1357, 1359, 1362, 1374, 1390, 1399, 1405, 1409, 1414, 1416, 1422, 1428, 1434, 1449,: 1451, 1454, 1463, 1469, 1477, 1479, 1483, 1488, 1492, 1494, 1496, 1498, 1504, 1510, 1513, 1518, 1520 The cytosine shown by etc. can be mention | raise | lifted. More specifically, for example, when the useful protein gene is the M305P22.2.1 gene, the CpG present in the base sequence of the promoter region, untranslated region or translated region (coding region) Examples of the base sequence including one or more of the base sequences shown include the base sequence of genomic DNA containing exon 1 of human-derived bA305P22.2.1 gene and the promoter region located 5 ′ upstream thereof. More specifically, the base sequence represented by SEQ ID NO: 3 (corresponding to the base sequence represented by base numbers 13001 to 13889 of the base sequence described in Genbank Accession No. AL121673) can be mentioned. It is possible. In the nucleotide sequence shown in SEQ ID NO: 3, the ATG codon encoding the amino terminal methionine of bA305P22.2.1 protein derived from human is shown in nucleotide numbers 849 to 851, and the nucleotide sequence of exon 1 above is shown. Is shown in base numbers 663-889. Cytosine in the base sequence shown by C p G present in the base sequence shown by SEQ ID NO: 3, especially in the region where C p G is densely present in the base sequence shown by SEQ ID NO: 3. The cytosine in CpG shows a high methylation frequency (ie, hypermethylation state) in cancer cells such as gastric cancer cells. More specifically, as cytosine having high methylation frequency in gastric cancer cells, for example, in the base sequence represented by SEQ ID NO: 3, base numbers 329, 335, 337, 351, 363, 373, 405, 424, 427 , '446, 465, 472, 486 etc. can be mentioned cytosine. More specifically, for example, when the useful protein gene is a Gamma fil amin gene, the C p G present in the base sequence of the promoter region, untranslated region or translated region (coding region) The base sequence containing one or more of the base sequences shown is the base sequence of the genomic DNA containing exon 1 of the human-derived Ga thigh afil amin gene, and part 5 of the promoter region located upstream. More specifically, the nucleotide sequence represented by SEQ ID NO: 4 (corresponding to the complementary sequence of the nucleotide sequence represented by nucleotide numbers 63528 to 64390 of the nucleotide sequence described in Genbank Accession No. AC074373) ). In the base sequence shown in SEQ ID NO: 4, the ATG codon encoding the methionine at the amino terminal of human-derived gamma fil amin protein is shown in base numbers 572 to 574, and the base sequence of exon 1 is base Numbers 463-863. Cytosine in the base sequence shown by C p G present in the base sequence shown in SEQ ID NO: 4, especially C p in the region where C p G is densely present in the base sequence shown in SEQ ID NO: 4 The cytosine in G shows a high methylation frequency (ie, hypermethylation state) in cancer cells such as gastric cancer cells. More specifically, as cytosine having a high methylation frequency in gastric cancer cells, for example, in the base sequence represented by SEQ ID NO: 4, base numbers 329, 333, 337, 350, 353, 360., 363, 370 379, 382, 384, 409, 414, 419, 426, 432, 434, 445, 449, 459, 472, 474, 486, 490, 503, 505 and the like.

 '

In addition, for example, when the useful protein gene is a HAND1 gene, the base sequence represented by C p G present in the base sequence of the promoter region, untranslated region or translated region (coding region). The base sequence containing one or more of The base sequence of genomic DNA containing exon 1 of the conventional HAND1 gene and the promo overnight region located 5 'upstream thereof can be given. More specifically, it is represented by SEQ ID NO: 5. And a base sequence (corresponding to a complementary sequence of the base sequence represented by base number 24303 26500 of the base sequence described in Genbank Accession No. AC026688). In the base sequence shown in SEQ ID NO: 5, the ATG codon encoding the amino terminal methionine of the HAND1 protein derived from human is shown in base numbers 1656 to 1658, and the base sequence of the above exon 1 is base The number 1400-2198 is shown. Cytosine in the base sequence shown by C p G present in the base sequence shown by SEQ ID NO: 5, especially C in the region where C p G is densely present in the base sequence shown by SEQ ID NO: 5. Cytosine in p′G shows a high methylation frequency (ie, hypermethylation state) in cancer cells such as gastric cancer cells. More specifically, as cytosine having high methylation frequency in gastric cancer cells, for example, in the base sequence represented by SEQ ID NO: 5, the base numbers 1153, 1160, 1178, 1187, 1193, 1218, 1232, 1266, Examples include scissors represented by 1272, 1292, 1305, 1307, 1316, 1356, 1377, 1399, 1401, 1422, 1434, and the like. Specifically, for example, a useful protein gene is Homologue of RIKEN

 In the case of the 2210016F16 gene, the base sequence containing one or more base sequences indicated by CpG present in the base sequence of the promoter region, untranslated region or translation region (coding region) is a human-derived homologue. of RIKEN 2210016F16 gene can include the base sequence of genomic DNA containing exon 1 and the promoter region located 5 'upstream thereof. More specifically, the base sequence represented by SEQ ID NO: 6 .

(Corresponding to a complementary base sequence of the base sequence represented by base numbers 157056 to 159000 of the base sequence described in Genbank Accession No. AL354733). In the nucleotide sequence represented by SEQ ID NO: 6, the nucleotide sequence of exon 1 of the human-derived Homologue of RIKEN 2210016F16 gene is represented by nucleotide numbers 1392 to 1945. A cytosine in the base sequence shown by CpG that exists in the base sequence shown by SEQ ID NO: 6, especially CpG that exists in a region where C p G is densely present in the base sequence shown by SEQ ID NO: 6 The cytosine in it shows a high methylation frequency (ie, hypermethyl aUon) in cancer cells such as gastric cancer cells. More specifically, as cytosine having a high methylation frequency in the gastric cancer cell J3 capsule, for example, in the base sequence represented by SEQ ID NO: 6, the base numbers 1172, 1175, 1180, 1183, 1189, 1204, 1209 1267, 1271, 1278, 1281, 1313, 1319, 1332, 1334, 1338, 1346, 1352, 1358, 1366, 1378,

The cytosine shown by 1392, 1402, 1433, 1436, 1438 etc. can be mentioned. Specifically, for example, when the useful protein gene is the FLJ32130 gene, the base of the promoter region, the untranslated region or the translated region (coding region) 'the base indicated by C p G As a base sequence including one or more sequences, a base sequence of genomic DNA containing exon 1 of human-derived FU32130 gene and a promoter region located 5 ′ upstream thereof can be mentioned. Specifically, the nucleotide sequence represented by SEQ ID NO: 7 (corresponding to the complementary nucleotide sequence of the nucleotide sequence represented by nucleotide numbers 1 to 2379 in the nucleotide sequence described in Genbank Accession No. AC002310) )). In the base sequence represented by SEQ ID NO: 7, the ATG codon that encodes the amino acid terminal methionine of FLJ32130 protein derived from baboon is represented by base numbers 2136 to 2138, and the base sequence considered to be exon 1 is The base numbers 2136 to 2379 are shown. Cytosine in the base sequence shown by C p G present in the base sequence shown by SEQ ID NO: 7, particularly C p present in the region where C p G is densely present in the base sequence shown by SEQ ID NO: 7. The cytosine in G shows a high methylation frequency (ie, hypermethyl aU on) in cancer cells such as gastric cancer cells. More specifically, as cytosine having high methylation frequency in gastric cancer cells, for example, in the base sequence represented by SEQ ID NO: 7, the base numbers 1714, 1716, 1749, 1753, 1762, 1795, 1814, 1894, The cytosine shown by 1911, 1915, 1925, 1940, 1955, 1968, etc. can be mentioned. Specifically, for example, when the useful protein gene is a PPARG angi opoi et in-related protein gene, its promoter region, untranslated region or translated region ( The base sequence containing one or more base sequences indicated by C p G present in the base sequence of the coding region) includes human-derived PPARG angiopoi et in-related protein gene exon 1 and 5 ′ upstream thereof. The base sequence of genomic DNA containing a region of the promoter located can be mentioned, and more specifically, the base sequence shown by SEQ ID NO: 8 can be mentioned. In the base sequence represented by SEQ ID NO: 8, the ATG codon encoding the amino terminal methionine of the human-derived PPARG angiopoi et in-rel at ed prote in protein is represented by base numbers 717 to 719, and The base sequence of the 5 ′ portion of exon 1 is shown in base numbers 1957 to 2661. Cytosine in the base sequence shown by C p G present in the base sequence shown in SEQ ID NO: 8, especially C in the region where C p G is densely present in the base sequence shown in SEQ ID NO: 8. Cytosine in pG exhibits a high methylation frequency (ie, hypermethylation state) in cancer cells such as gastric cancer cells. More specifically, as cytosine having high methylation frequency in gastric cancer cells, for example, in the base sequence represented by SEQ ID NO: 8, salt ^: group numbers 35, 43, 51, 54, 75, 85, 107, 127 , 129, 143, 184, 194, 223, 227, 236, 251, 258 and the like. Specifically, for example, when the useful protein gene is a Thrombomodulin gene, the base represented by C p G present in the base sequence of the promoter region, untranslated region or translated region (coding region). Examples of the base sequence containing one or more sequences include the base sequence of genomic DNA containing exon 1 of human-derived Thrombomodulin gene and the 5 'upstream promoter region. Specifically, the base sequence represented by SEQ ID NO: 9 (corresponding to the base sequence represented by base numbers 1 to 6096 of the base sequence described in Genbank Accession No. AF495471) can be mentioned. In the base sequence represented by SEQ ID NO: 9, the ATG codon encoding the amino terminal methionine of the human-derived Thrombomodulin protein is represented by base numbers 2590 to 2592. The base sequence of exon 1 is The number 2048-6096 is shown. Cytosine in the base sequence shown by C p G present in the base sequence shown by SEQ ID NO: 9, especially in a region where C p G is densely present in the base sequence shown by SEQ ID NO: 9. The cytosine present in CpG exhibits a high methylation frequency (ie, hypermethylation) in cancer cells such as gastric cancer cells. More specifically, as cytosine with high methylation frequency in gastric cancer cells, for example, in the base sequence represented by SEQ ID NO: 9, base numbers 1539, 1551, 1571, 1579, 1581, 1585, 1595, ', 1598, 1601, 1621, 1632, 1638, 1645, 1648, 1665, 1667, 1680, .1698, 1710,

The cytosine shown by 1724, 1726, Π56, etc. can be mentioned. In addition, for example, when the useful protein gene is p53-responsive gene 2 gene, it is indicated by CpG present in the base sequence of the promoter region, untranslated region or translated region (coding 'region). Examples of the base sequence including one or more base sequences include the genomic DNA base sequence containing exon 1 of human-derived p53-responsive gene 2 gene and a promoter region located 5 'upstream thereof. More specifically, the nucleotide sequence represented by SEQ ID NO: 10 (corresponding to the complementary sequence of the nucleotide sequence represented by nucleotide numbers 113501 to 116000 of the nucleotide sequence described in Genbank Accession! NO. AC009471) is mentioned. It is done. In the base sequence represented by SEQ ID NO: 10, the base sequence of exon 1 of the human-derived p53_responsive gene 2 gene is represented by base numbers 1558 to 1808. Cytosine in the base sequence represented by C p G present in the base sequence represented by SEQ ID NO: 10 has a high methylation frequency (that is, a hypermethylated state (ie, a hypermethylated state ( hypermethylation)). More specifically, cytosine having a high methylation frequency in spleen cancer cells, for example, in the base sequence represented by SEQ ID NO: 10, the base numbers 1282, 1284, 1301, 1308, 131.5, 1319, 1349 1351, 1357, 1361, 1365, 1378, 1383 and the like. More specifically, for example, when the useful protein gene is a Fibrillin gene, it is represented by C p G present in the base sequence of the promoter region, untranslated region or translated region (coding region). The nucleotide sequence containing one or more nucleotide sequences includes exon 1 of the Fibrillin gene derived from human and a promoter located 5 'upstream The base sequence of genomic DNA containing one region can be mentioned, more specifically, the base sequence represented by SEQ ID NO: 11 (base number 118801 of the base sequence described in Genbank Accession No. AC113387) It corresponds to a complementary sequence of the base sequence represented by ~ 121000. In the base sequence represented by SEQ ID NO: 11, the human-derived

The base sequence of exon 1 of the Fibrill in gene is shown in nucleotide numbers 1091 to 1345. Cytosine in the nucleotide sequence represented by C p G present in the nucleotide sequence represented by SEQ ID NO: 11 has a high methylation frequency (that is, a highly methylated state (for example, in cancer cells such as knee cancer cells). hypermethyl at ion)). More specifically, as cytosine having a high methylation frequency in a spleen cancer cell, for example, in the base sequence represented by SEQ ID NO: 11, the base numbers 679, 687, 690, 699, 746, 773, The cytosine shown by 777, 783, 795, 799, 812, 823, 830, 834, 843 etc. can be mentioned. Further, specifically, for example, when the useful protein gene is a neurofilament 3 gene, “C p G present in the base sequence of the promoter region, untranslated region or translated region (coding region)” The base sequence containing one or more of the base sequences indicated by is a genomic DNA base sequence containing exon 1 of a human-derived neurofilaments gene and a promoter region located 5 ′ upstream thereof. More specifically, the nucleotide sequence represented by SEQ ID NO: 12 (the complementary sequence of the nucleotide sequence represented by nucleotide numbers 28001 to 30000 of the nucleotide sequence described in Genbank Accession No. AF106564) In the nucleotide sequence represented by SEQ ID NO: 12, the nucleotide sequence of exon 1 of the human-derived neurofilament 3 gene is represented by nucleotide numbers 614 to 1694. Number 1 in 2 Cytosine in the base sequence shown by C p G present in the base sequence shown has a high methylation frequency (ie, hypermethyl at ion) in cancer cells such as, for example, presumptive cancer cells. More specifically, as a cytosine having a high methylation frequency in knee cancer cells, for example, in the base sequence represented by SEQ ID NO: 12, the base numbers 428, 432, 443, 451, 471, 475, 482, 491, 499, 503, 506, 514, 519, 532, 541, 544, 546, 563, 566, 572, 580 etc. can be raised. Specifically, for example, a useful protein gene is di s integr in and

 In the case of a metal loproteinase domain 23 gene, a nucleotide sequence containing one or more nucleotide sequences indicated by C p G present in the nucleotide sequence of the promoter region, non-translation region or translation region (coding region) As a specific example, the base sequence of genomic DNA containing exon 1 of the human di s integr in and metal loprote inase domain 23 gene and the promoter region located 5 'upstream thereof can be mentioned. Includes a base sequence represented by SEQ ID NO: 13 (corresponding to a base sequence represented by base numbers 21001 to 23300 of the base sequence described in Genbank Accession No. AC009225). In the nucleotide sequence represented by SEQ ID NO: 13, the nucleotide sequence of exon Γ of human-derived lointe inase domain 23 gene is from nucleotide numbers 1194 to

It is shown in 1630. Cytosine in the nucleotide sequence represented by CpG present in the nucleotide sequence represented by SEQ ID NO: 13 is a high methylation frequency (ie, hypermethylated state (ie, hypermethylated state) in cancer cells such as knee cancer cells. at i on)). More specifically, as cytosine having a high methylation frequency in pancreatic cancer cells, for example, in the base sequence represented by SEQ ID NO: 13 base numbers 998, 1003, 1007, 1011, 1016, 1018, 1020, 1026 1028, 1031, 1035, 1041, 1043, 1.045, 1051, 1053, 1056, 1060, 1066, 1068, 1070, 1073, 1093, 1096, 1106, 1112, 1120, 1124, 1 126 etc. Can give. Specifically, for example, when the useful protein gene is a G protein-coupled receptor 7 gene, Cp present in the base sequence of the promoter region, untranslated region or translated region (coding region) The base sequence including one or more base sequences represented by G includes exon 1 of the human G protein-coupled receptor 7 gene and a promoter region located 5 ′ upstream thereof. More specifically, the nucleotide sequence of genomic DNA can be mentioned. More specifically, the nucleotide sequence represented by SEQ ID NO: 14 (the salt represented by nucleotide numbers 75001 to 78000 of the nucleotide sequence described in Genbank Accession No. AC009800) It corresponds to the base sequence. In the base sequence represented by SEQ ID NO: 14 The base sequence of exon 1 of the G protein-coupled receptor 7 gene derived from human is shown in base numbers 1666 to 2652. Cytosine in the base sequence represented by CpG present in the base sequence represented by SEQ ID NO: 14 has a high methylation frequency (ie, hypermethylation state) in cancer cells such as, for example, a premature cancer cell. Indicates. More specifically, as cytosine with high methylation frequency in spleen cancer cells, for example, in the base sequence represented by SEQ ID NO: 14, base numbers 1480, 1482, 1485, 1496, 1513, 1526, 1542, The cytosine shown by 1560, 1564, 1568, 1570, 1580, 1590, 1603, 1613, 1620 etc. can be mentioned. 'More specifically, for example, a useful protein gene is G-protein coupled

In the case of somatostatin and angiotensin-1 ike peptide receptor gene, it is indicated by C p G present in the nucleotide sequence of its promoter region, its promoter region, untranslated region or translated region (coding region) Nucleotide sequence including at least `` base sequence '' ^{: The} sequence includes exon 1 of the human Taki G-protein coupled somatostatin and angiotensin-1 ike peptide receptor gene and a promoter region located 5 'upstream thereof. The base sequence of genomic DNA can be mentioned. More specifically, the base sequence represented by SEQ ID NO: 15 (the base sequence represented by base numbers 57001 to 60000 of the base sequence described in Genbank. Accession No. AC008971) It corresponds to the complementary sequence of the sequence. In the base sequence represented by SEQ ID NO: 15, the base sequence of exon 1 of human-derived G-protein coupled somatostatin and angiotensin-1 ike peptide receptor gene is represented by base numbers 776 to 2632. Cytosine in the base sequence represented by CpG present in the base sequence represented by SEQ ID NO: 15 has a high methylation frequency (ie, hypermethylation state) in cancer cells such as spleen cancer cells. Show. More specifically, cytosine having a high methylation frequency in spleen cancer cells includes, for example, the nucleotide sequence represented by SEQ ID NO: 15 ′, nucleotide numbers 470, 472, 490, 497, 504, 506, 509, 514 , 522, 540, 543, 552, 566, 582, 597, 610, 612 and the like. More specifically, for example, when a useful protein gene is a Solute carrier family 6 neurotransmitter transporter noradrenalin member 2 gene, it exists in the base sequence of the promoter region, untranslated region or translated region (coding region). The base sequence containing one or more base sequences represented by C p G includes human origin 'Solute carrier family 6 neurotransmitter transporter noradrenalin member 2 gene exon 1 and 5' upstream promoter region. The base sequence of the contained genomic DNA can be mentioned, and more specifically, the base sequence represented by SEQ ID NO: 16 (base sequences 78801 to 81000 of the base sequence described in Genbank Accession No. AC026802) It corresponds to the complementary sequence of the sequence. In the base sequence represented by SEQ ID NO: 16, the base sequence of exon 1 of human-derived Solute carrier family 6 neurotransmitter transporter noradrenalin member 2 is shown in base numbers 1479 to 1804. Cytosine in the nucleotide sequence represented by CpG present in the nucleotide sequence represented by SEQ ID NO: 16 is, for example, cancer such as a premature cancer cell: high methylation frequency in the cell (ie, hypermethylation state) Indicates. More specifically, as succicin having high methylation frequency in S spleen cancer cells, for example, in the base sequence represented by SEQ ID NO: 16, base numbers 1002, 1010, 1019, 1021, 1051, 1056, 1061 1063, 1080, 1099, 1110, 1139, 1141, 1164, 1169, 1184, and the like. The “methylated DNA antibody” in the measurement method of the present invention means an antibody that binds with a methylated base in DNA as an antigen. Any antibody having the property of recognizing and binding to cytosine methylated at the 5-position in single-stranded DNA may be used, and more specifically, a methylcytosine antibody may be mentioned. Further, even a commercially available methylated DNA antibody may be used as long as it is capable of specifically recognizing and binding specifically to the methylated DNA described in this patent.

A methylated DNA antibody can be prepared by a conventional method using a methylated base, methylated DNA or the like as an antigen. Specifically, antibodies prepared using 5-methylcytidine, 5-methylcytosine, or DNA containing 5-methylcytosine as an antigen Can be prepared by selecting specific binding to methylcytosine in DNA as an index.

 Antibodies obtained by immunizing animals with antigens include antibodies for the IgG fraction (polyclonal antibodies) and antibodies produced by a single clone (monoclonal antibodies). In this patent, it is desirable to use an antibody capable of specifically recognizing methylated DNA or methylcytosine, so it is desirable to use a monoclonal antibody.

As a method for producing a monoclonal antibody, a cell fusion method can be mentioned. For example, in the cell fusion method, spleen cells (B cells) derived from immunized mice and myeloma cells are fused to produce a hyperidoma, and the antibody produced by the hyperidoma is selected, and a methylcytosine antibody (monoclonal) is produced. Antibody). When producing monoclonal antibodies by the cell fusion method, it is not necessary to purify the antigen. For example, 5-methylcytidine, 5-methylcytosine, or a mixture of DNA containing 5-methylcytosine is used as an antigen. Can be administered to animals used in As the administration method, • 5-methylcytidine, 5-methylcytosine, or DNA containing 5-methylcytosine is directly administered to the mouse producing the antibody. When it is difficult to produce an antibody, immunization may be performed by binding an antigen to a support. In addition, an antigen solution can be obtained by mixing the antigen with an adjuvant solution (for example, liquid paraffin and A race 1 A mixed with dead bacteria of Mycobacterium tuberculosis as an adjuvant) Can increase immunity. Alternatively, add an equal amount of antigen-containing solution and adjuvant solution to make it sufficiently milky, then inject it subcutaneously or intraperitoneally into mice, or mix well with alum water and use pertussis killed as an adjuvant. There is a method of adding. It is also possible to boost the mouse intraperitoneally or intravenously after an appropriate period after the first immunization. When the amount of antigen is small, a solution in which the antigen is suspended may be directly injected into the mouse spleen for immunization. A few days after the final immunization, the spleen is removed and the adipose tissue is removed, and then a spleen cell suspension is prepared. The spleen cells are fused with, for example, HGPRT-deficient myeloma cells, to produce octabridomas. Any cell fusion agent can be used as long as it can efficiently fuse spleen cells (B cells) and myeloma cells. For example, Sendai virus (HV J), poly And a method using ethylene glycol (PEG). In addition, cell fusion may be performed by a method using a high voltage pulse.

After cell fusion, culture in HAT medium, select a hybridoma clone in which splenocytes and myeloma cells are fused, and wait for cells to grow until screening becomes possible. An antigen-antibody reaction system can be used for the antibody detection method and antibody titer measurement method for selecting a hyperidoma producing the desired antibody. Specifically, antibody measurement methods for soluble antigens include radioisotope immunoassay (RIA) and enzyme immunoassay (ELISA). 'If CpG present in the single-stranded DNA is methylated at least at one position, it can bind to the anti-methylated antibody. “Methylated single-stranded DNA” in the measurement method of the present invention means single-stranded DNA in which CpG present in single-stranded DNA is methylated at least at one site. It is not limited to single-stranded DNA that has all methylated C p G's. In the present invention, “(amplified to a detectable amount of methylated DNA in the target DNA region) and the amount of amplified DNA” refers to the genomic DNA contained in the biological sample. It means the amount of methylated DNA after amplification in the target region itself, that is, the amount determined in the third step of the measurement method of the present invention. For example, when the biological specimen is 1 mL of serum, it means the amount of DNA amplified based on the methylated DNA contained in 1 mL of serum. In the present invention, the “single-stranded DNA comprising at least one CpG in the methylated state at the recognition site of the methylation-sensitive restriction enzyme capable of digesting the single-stranded DNA” refers to the restriction enzyme recognition site. Means a single-stranded DNA which is an unmethylated cytosine in at least one C p G present in In addition, “the single-stranded DNA that does not contain CpG in the methylated state at the recognition site of the methylation-sensitive restriction enzyme capable of digesting the single-stranded DNA” described above refers to the restriction enzyme in the single-stranded DNA. This refers to single-stranded DNA in which cytosine in all C p G existing in the recognition site is methylated. In the first step of the measurement method of the present invention, a methylated single-stranded DNA and an immobilized methylated DNA antibody are bound from a DNA sample derived from genomic DNA contained in a biological specimen, The single stranded DNA is selected. Specifically, the first step includes a first step (A) for separating methylated single-stranded DNA from a genomic DNA-derived DNA sample contained in a biological specimen, and a methylated product. The first step (B) involves selecting the single-stranded DNA by binding the single-stranded DNA to the immobilized methyl-DNA DNA antibody. In the first step (A) of the measurement method of the present invention, when “isolating methylated single-stranded DNA from a genomic DNA-derived DNA sample contained in a biological sample”, two: one strand DNA In general, it is sufficient to operate to make a double-stranded DNA. Specifically, a genomic DNA-derived DNA sample contained in a biological sample may be dissolved in an appropriate amount of ultrapure water, heated at 95 ° C for 10 minutes, and then rapidly cooled in ice. The “immobilized methylated DNA antibody” in step (B) of the measurement method of the present invention selects single-stranded DNA methylated from genomic DNA-derived DNA samples contained in biological samples. Used to do. The immobilized methylated DNA antibody may be any antibody as long as it can be immobilized on a support. The term “capable of being immobilized on a support” means that a methylated DNA antibody is directly or indirectly attached to a support. It means that it can be fixed.

In order to immobilize in this way, the methylated DNA antibody may be immobilized on a support according to a normal genetic engineering operation method or a commercially available kit / equipment (binding to a solid phase). Specifically, a support coated with streptavidin on a methylated D'NA antibody obtained by piotination of a methylated D'NA antibody (for example, a PCR tube coated with streptavidin, or coated with streptavidin) And fixing to a magnetic bead). . In addition, for example, a glass having a surface activated with a silane coupling agent or the like after a molecule having an active functional group such as an amino group, a thiol group, or an aldehyde group is covalently bonded to a methylated DNA antibody. There is also a method of covalently bonding to a saccharide derivative, silica gel, the above synthetic resin or the like, or a heat-resistant plastic support. In the case of a covalent bond, for example, a covalent bond may be made by using a spacer such as five triglycerides connected in series, a crosslinker, or the like.

 Furthermore, the methylated DNA antibody may be directly immobilized on the support, or the antibody against the methylated DNA antibody (secondary antibody) is immobilized on the support, and the methylated antibody is bound to the secondary antibody. You may fix to a support body.

In addition, when selecting a single-stranded DNA containing the target DNA region (positive strand), it is sufficient that the immobilized methylated DNA antibody is immobilized on a support. (1) It may be immobilized by the binding of the immobilized methylated DNA antibody and the support in the step before the binding of the double-stranded DNA (positive strand) and the immobilized methylated DNA antibody, and (2 The single-stranded DNA (positive strand) and the immobilized methylated DNA antibody may be immobilized by the binding between the immobilized methylated DNA antibody and the support at the stage after the binding.

In the first step (B) of the measurement method of the present invention, “selecting the single-stranded DNA by binding a methylated single-stranded DNA and an immobilized methylated DNA antibody” Specifically, for example, “Piotin-labeled pyotinylated methylated cytosine antibody” may be used as the immobilized methylated DNA antibody, and the procedure may be carried out as follows.

(a) A mixture obtained by adding an appropriate amount (eg, 0.1 igZ5 OL) of a pyotinylated methylated cytosine antibody to an avidin-coated PCR tube is allowed to stand at room temperature for about 1 hour. Promotes the immobilization of methylated cytosine antibody and streptavidin. After this operation, the remaining solution is removed and washed. The resulting avidin-coated PC cleaned R tube puffer [e.g., 0. 05% T ween 20-containing phosphate buffer one _{(lmM KH, P0 4 3mM Na} 2 HP0 · 7 0, 154mM NaCl pH7.4)) 10 Q fiL / Ju After adding at the rate of the tube, remove the solution from the avidin-coated PCR tube. By repeating this washing operation several times, the pyotinylated methylated cytosine antibody immobilized on the support remains in the avidin-coated PCR tube.

 (b) Double-stranded DNA derived from genomic DNA contained in biological samples is buffered (for example, 33 mM Tris-Acetate pH 7.9, 66 mM K0Ac, lOmM MgOAc2, 0.5 mM

 Dithothreitol) and heat at 95 ° C for several minutes. After that, it is quickly cooled to a temperature of about 0-4 ° C, and kept at that temperature for several minutes to form single-stranded DNA. Then return to room temperature.

 (c) The formed single-chain DN.A is added to an avidin-coated PCR tube on which a pyotinylated methylated cytosine antibody is immobilized, and then left at room temperature for about 1 hour. Promote binding between the cytosine antibody and methylated single-stranded DNA of the single-stranded DNA (formation of a conjugate) (At this stage, single-stranded DNA containing at least a non-methylated DNA region) Does not form a conjugate.) After that, the remaining solution is removed and washed. Add wash buffer [eg, 0.05¾ Tween20-containing phosphate buffer (ImM KH2P04, 3 mM Na2HP0 · 7H20, 15 mM NaCl pH7.)] At a rate of 100 L / tube and remove the solution. Repeat this wash several times to leave the conjugate in the PCR tube (select conjugate). The specific description of the first (A) step and the first (B) step is not limited to the above description. For example, the buffer used in (b) is not limited to the buffer as long as it is suitable for separating a double-stranded DNA derived from a genomic DNA derived from a biological sample into a single-stranded DNA.

In addition, for example, the washing operations in (a) and (c) may be performed by the non-immobilized methylated DNA antibody floating in the solution, or the methylation floating in the solution that is not bound to the methylated DNA antibody. This is important for removing unsuspended single-stranded DNA or DNA suspended in a solution that has been extinguished with the restriction enzyme described below from the reaction solution. The washing buffer only needs to be suitable for removing the above-mentioned free methylated DNA antibody, single-stranded DNA floating in the solution, etc. FIA buffer (PerkinElmer, Tris-HC1 pH 7.8 with Tween 80), TEA buffer, etc. may be used. 'In the second step of the measurement method of the present invention, the single-stranded DNA selected in the first step is digested with a methylation sensitive restriction enzyme capable of digesting at least one kind of single-stranded DNA. The single-stranded DNA selected in the first step is digested with a methylation-sensitive restriction enzyme capable of digesting at least one kind of single-stranded DNA, and then a free digest produced (single strand as described above). What is necessary is to remove at least one single-stranded DNA containing a Cp 0. in the methylated state at the recognition site of a methylation sensitive restriction enzyme capable of digesting DNA.

'' “Methylation-sensitive restriction enzyme” in the second step of the measurement method of the present invention means, for example, that a recognition sequence containing methylated cytosine is not digested, but only a recognition sequence containing unmethylated cytosine is digested. It means a restriction enzyme that can be used. That is, in the case of a DNA in which the saccharin contained in the recognition sequence that can be originally recognized by the methylation sensitive restriction enzyme is methylated, the methylation sensitive restriction enzyme is allowed to act on the DNA. The DNA is not cleaved. In contrast, when cytosine contained in a recognition sequence that can be originally recognized by a methylation-sensitive restriction enzyme is not methylated, the methylation-sensitive restriction enzyme acts on the DNA. If so, the DN A will be disconnected. Specific examples of such a methylation-sensitive enzyme include HpaII, BstUK Narl, SacII, Hhal and the like. The methylation-sensitive restriction enzyme is a double-stranded DNA containing a CpG pair in the hemimethyl state (that is, the cytosine of one strand of the CpG pair is methylated and the cytosine of the other strand is methylated). It does not cleave double-stranded DNA that is not methylated at the syn) and has already been revealed by Gruenbaum et al. (Nucleic Acid Research, 9, 2509-2515).

“Methylation-sensitive restriction enzyme capable of digesting single-stranded DNA” refers to, for example, non-methylated cytosine that does not digest a recognition sequence containing methylated cytosine in single-stranded DNA. It means a restriction enzyme that can digest only the recognition sequence. That is, some methylation-sensitive restriction enzymes digest single-stranded DNA. The For example, Hhal and the like can be mentioned. As a method for examining the presence or absence of digestion with a methylation-sensitive restriction enzyme capable of digesting the single-stranded DNA, specifically, for example, among the above-mentioned methylated single-stranded DNA, the target DNA region PCR is performed using a primer pair that can amplify DNA containing a recognition sequence containing cytosine to be analyzed, and the presence or absence of DNA amplification (amplified product). You can give a way to check. When cytosine to be analyzed is methylated, an amplification product is obtained. On the other hand, if the cytosine to be analyzed is not methylated, an amplification product cannot be obtained. In this way, by comparing the amount of amplified DNA, the ratio of cytosine to be analyzed can be measured.

Incidentally, in the methylated single-stranded DNA selected in the first step, the target DNA region is in a single-stranded state, and the negative-strand oligonucleotide is the target DNA region. Since it does not bind, the single-stranded DNA is a single-stranded DNA derived from genomic DNA contained in a biological specimen. In addition, as described above, in the single-stranded DNA, not all CpGs in the target DNA region are methylated. Therefore, by utilizing the property that the methylation sensitive restriction enzyme capable of digesting the single-stranded DNA does not cleave the single-stranded DNA in the methyl state, in the genomic DNA contained in the biological sample It is possible to distinguish whether or not cytosine in at least one C p G present in the recognition site of the restriction enzyme that can digest the single-stranded DNA is methylated, The content of methylated DNA in the target DNA region can be determined more accurately. In other words, since the DNA region of interest or only a part of CpG other than the region of interest of DNA was methylated, the single-stranded DNA selected by forming a conjugate with the methyl babies antibody Digestion with a methylation-sensitive restriction enzyme capable of digesting single-stranded DNA, making it possible to select only single-stranded DNA containing the target DNA region that is truly methylated. Yes. That is, by digesting with the methylation-sensitive restriction enzyme capable of digesting the single-stranded DNA, it is assumed that the genomic DNA contained in the biological sample is assumed. If the CpG cytosine at the recognition site of the methylation-sensitive restriction enzyme capable of digesting single-stranded DNA contained in the target DNA region of single-stranded DNA derived from nom is methylated A methylated DNΑ antibody is bound to the recognition site of a methylation sensitive restriction enzyme capable of digesting strand DNA, but it is included in the target DNA NA region of the genomic single-stranded DNA. If the CpG cytosine at the recognition site of the methylation-sensitive restriction enzyme capable of digesting strand DNA is not methylated, methylated DNA antibody is not bound, so methylation that can digest single-stranded DNA Digested by sensitive restriction enzymes.

 Therefore, after performing digestion, as described below, when PCR is performed using a pair of primers capable of amplifying the target DNA region, the genomic DNA contained in the biological specimen is If at least one cytosine in the recognition site of the restriction enzyme is not methylated in CpG, no PCR amplification product can be obtained, while in biological samples. If cytosine in all C p G existing in the recognition site of the methylation sensitive restriction enzyme capable of digesting the single-stranded DNA in the genomic DNA contained in An amplified product by PCR will be obtained.

Most methylation-sensitive restriction enzymes cannot digest single-stranded DNA even in the unmethylated state, but as mentioned above, some methylation-sensitive restriction enzymes can digest single-stranded DNA in the unmethylated state. An example of a methylation-sensitive restriction enzyme capable of digesting single-stranded DNA is Hhal. By using such an enzyme, the presence or absence of methylation of single-stranded DNA can be determined. That is, if the C p G cytosine contained in the Hhal recognition site of the single-stranded DNA in the sample bound to the immobilized methylated antibody obtained by the above operation is methylated, Is unable to digest this DNA, and if not methylated, Hhal will digest this DNA. Therefore, after performing the above reaction, when PCR is performed using a pair of primers that can amplify the target DNA region, if the DNA in the sample is in an unmethylated state, an amplification product cannot be obtained. If the DNA in the sample is methylated, an amplification product can be obtained. · Specifically, for example, the second step of the measurement method of the present invention may be performed as follows. 3 L of optimal 10X buffer (330iM Tris- Acetate pH 7.9, 660 mM K0Ac, lOOmM MgOAc2, 5 mM Di thothrei tol), 3 ^ L of lig / mL BSA aqueous solution ' Add 1.5 L each of methylation sensitive restriction enzymes Hhal (10 U / L) capable of digesting single-stranded DNA, then add sterilized ultrapure water to the mixture to a volume of 30 iL for 1 hour at 37 ° C. ~ 3 hours incubation. In this way, the single-stranded DNA selected in the first step is digested with a methylation-sensitive restriction enzyme capable of digesting at least one kind of single-stranded DNA, and then the resulting free digest ■ (as described above) Removal and washing (DNA purification) of at least one single-stranded DNA containing CpG in an ammethyl state at the recognition site of a methylation sensitive restriction enzyme capable of digesting single-stranded DNA.

 'More specifically, for example, when using a PCR tube coated with streptavidin, first remove the solution by pipetting or decantation, and then add TE to the volume of the biological sample. After adding the buffer, the TEA buffer can be removed by pipetting or decanting. When using magnetic beads coated with streptavidin, after fixing the beads with a magnet, first remove the solution by pipetting or decantation, and then use TE buffer that is approximately equal to the volume of the biological sample. After adding the TE buffer, the TE buffer can be removed by pipetting or decanting.

 Subsequently, by performing such an operation several times, removal and washing of digested material (single-stranded DNA containing at least one or more amethylated CpG at the recognition site of the restriction enzyme) (DNA purification) To do.

The above washing operation is not always necessary. For example, all the solutions obtained by the methylation-sensitive restriction enzyme treatment can be used in the third step. The methylation sensitization capable of digesting single-stranded DNA in the second step of the measurement method of the present invention. As a concern in the digestion treatment with sex restriction enzymes, there is a possibility that a recognition sequence containing unmethylated cytosine cannot be completely digested (so-called “DNA residue”). If such a concern becomes a problem, if there are many recognition sites for methylation-sensitive restriction enzymes capable of digesting single-stranded DNA, DNA residue can be minimized. The target DNA region has at least one recognition site for a methylation-sensitive restriction enzyme capable of digesting single-stranded DNA, and the more recognition sites, the better. In the measurement method of the present invention, “a DNA sample derived from genomic DNA contained in a biological sample” is previously digested with a restriction enzyme that does not use the target DNA region of the genomic DNA as a recognition cleavage site. One preferred embodiment is that it is a prepared DNA sample. Here, when selecting genomic DNA (A type DNA) contained in a biological sample with this immobilized oligonucleotide, the short type D: NA is easier to select, and the purpose of PCR When amplifying a region, it is considered better to have a short cage DNA. Therefore, a restriction enzyme that does not use the target DNA region as a recognition cleavage site is added to a DNA sample derived from genomic DNA contained in a biological sample. It may be used directly for digestion. As a method of digesting with a restriction enzyme that does not use the target DNA region as a recognition cleavage site, a general restriction enzyme treatment method may be used.

 In addition, “a DNA sample derived from genomic DNA contained in a biological specimen” is a DNA sample that has been digested with at least one or more methylation-sensitive restriction enzymes. be able to.

These preferred embodiments are because the amount of methylation can be obtained with high accuracy by previously digesting the biological specimen itself with the restriction enzyme as described above. This method is useful for eliminating “DNA fragments” as described above. As a method for digesting a sample derived from genomic DNA contained in a biological sample with a methylation-sensitive restriction enzyme, the same method as described above may be used when the biological sample is genomic DNA itself. If the specimen is tissue lysate, cell lysate, etc. According to the same method as above, T-extinction treatment is performed using a large excess of methylation sensitive restriction enzyme, for example, 500 times (10U) or more methylation sensitive restriction enzyme for 25 ng of DNA. Just do it.

 Genomic DNA basically exists as double-stranded DNA. Therefore, in this operation, not only methylation-sensitive restriction enzymes that can digest single strands (eg, fflial), but also methylation-sensitive restriction enzymes that can digest double-stranded DNA (eg, HpaII, BstUL NarK SacII, fflial etc.) can be used. In the third step of the measurement method of the present invention, as a pre-step of each of the following steps, a single-stranded DNA that is an undigested product obtained in the second step '(the single-stranded DNA can be digested) A single-stranded DNA that does not contain CpG in the methylated state at the recognition site of the methylation-sensitive restriction enzyme is separated from the immobilized methylated DNA antibody into a single-stranded DNA (positive strand). Process (third (previous A) process), and

: Partial base sequence of the base sequence of the single-stranded DNA (positive strand) using the single-stranded DNA (positive strand) separated in the third (previous A) step as a saddle type Complementary to the partial base sequence (positive strand) located on the 3 ′ end side of the 3 ′ end of the base sequence (positive strand) of the target DNA region. By using the extension primer (forward primer) having a certain base sequence (negative strand) as an extension primer, the extension primer is extended once, so that the DNA in the single-stranded state (positive strand) ) As a double-stranded DNA (third (previous B) step) and the third (previously B) double-stranded DNA that has been formed into a single-stranded DNA containing the target DNA region. Step of separating DNA (positive strand) into single-stranded DNA (negative strand) containing the target DNA region (third) (Previous C) step) and as this step

 (a) Using the generated single-stranded DNA (positive strand) containing the target DNA region as a cage, and extending the extension primer once using the forward primer as an extension primer, This process of extending a single-stranded DNA containing a DNA region as a double-stranded DNA A—

(b) The single-stranded DNA (negative strand) that contains the target DNA region is A partial base sequence (negative strand) of the base sequence of the single-stranded DNA (negative strand) containing the target DNA region, and the base sequence (positive strand) of the target DNA region An extension having a partial base sequence (negative strand) that is complementary to the 3 'end of the base sequence (negative strand) that is complementary to the 3' end of the base sequence (negative strand). This step of extending the single-stranded DNA A containing the target A region as a double-stranded DNA by extending the extended primer one time using the primer (reverse primer one) as an extension primer. One B and

Furthermore, each of these steps is once separated into a single-stranded state after the extended double-stranded DNA obtained in each step is repeated, whereby methylation in the target DNA region 'is performed. Amplify the DNA to a detectable amount and quantify the amount of amplified DNA. In the third step of the measurement method of the present invention, first, among the following steps of each of the following steps, as the third (previous ^: A) step, single-stranded DNA that is an undigested product obtained in the second step (described above) Single-stranded DNA that does not contain CpG in the methylated state at the recognition site of the methylation-sensitive restriction enzyme capable of digesting single-stranded DNA) is separated from the immobilized methylated DNA antibody in the single-stranded state. Make it a certain DNA. Specifically, for example, a single-stranded DNA which is an undegraded product obtained in the second step (the recognition site of the methylation-sensitive restriction enzyme capable of digesting the single-stranded DNA does not contain Cp G in the methyl state). Add the annealing buffer to the single-stranded DNA) to obtain a mixture. Next, the resulting mixture is heated at 95 ° C. for several minutes to obtain single-stranded DNA (positive strand). After that, in the third (previous B) step, specifically, for example, the single-stranded DNA (positive strand) obtained in the third (previous A) step and the forward primer are mixed with sterile ultrapure water. L, 3 ^ L of an optimal buffer (e.g. ΙΟΟιη Tris-HCl pH 8.3, 500ra KCK 15 mM MgCl ₂ ), 3 L of 2 mM dNTP, 6 t of 5N ' 1 type of DNA polymerase: 5U / iL) is mixed in a solution made up to a volume of 30L, and incubated at 37 ° C for about 2 hours. Single-stranded DNA containing the target DNA region (Normal strand) is extended to double stranded DNA. In the third (previous C) process, For example, by adding an annealing buffer to the double-stranded DNA elongated in the third (previous B) step, a mixture is obtained, and the resulting mixture is heated at 95 ° C for several minutes. Once separated into single-stranded DNA containing the target DNA region.

 Then, as this process,

'(i) In order to anneal the forward primer to the single-stranded DNA (positive strand) containing the target DNA region, for example, the Tm value of the forward primer is about Cool immediately to a temperature as low as 0 to 20 ° C, and keep at that temperature for several minutes.

 (ii) Then return to room temperature. .

'(iii) The DNA in the single-stranded state annealed in (i) above is used as a saddle type, the primer for extension is used as an extension primer, and the primer is extended once. The single-stranded DNA containing the DNA region to be extended is formed as double-stranded DNA (ie, third step 1 A). Specifically, for example, it may be carried out in accordance with the following explanation or the operation method in the extension reaction in the third (previous B) step of the measurement method of the present invention described above.

 (iv) A partial salt of the base sequence of the single-stranded DNA (negative strand) containing the target DNA region as described above, with the generated single-stranded DNA (negative strand) containing the target DNA region as a saddle type It is a base sequence (negative strand) and is further 3 'end side than the 3' end of the base sequence (negative strand) complementary to the base sequence (positive strand) of the target DNA region. The extension primer (reverse primer) having a base sequence (negative strand) complementary to the partial base sequence (negative strand) located at is used as an extension primer, and the extension primer is extended once. The DNA, which is in the single-stranded state, is used as an extended double-stranded DNA (that is, third step 1B). Specifically, for example, it may be carried out according to the operation method in the extension reaction in the third (previous B) step, as in the third step 1A of (iii) above. '

(V) Further, each of these steps is repeated after separating the double-stranded DNA formed in the extension obtained in each step into a single-stranded state (for example, the third step 1A and the third step). In step B), the methylated DNA in the target DNA region is examined. Amplify until the amount is available and quantify the amount of amplified DNA. Specifically, the third step is the reaction from the third (previous A) step to this step. It can also be carried out as a single PCR reaction. Alternatively, the third (previous A) step to the third (previous C) step can be performed as independent reactions, and only this step can be performed as a PCR reaction. As a method for amplifying a target DNA region (ie, a target region) after digestion with a methylation sensitive restriction enzyme capable of digesting single-stranded DNA, for example, PCR can be used. When a target region is amplified, by using a primer previously labeled with fluorescence or the like as an indicator, the presence or absence of an amplification product can be evaluated without performing cumbersome operations such as electrophoresis. As the PCR reaction solution, for example, the DNA obtained in the second step of the measurement method of the present invention is prepared by adding 0.15 1 of a 50 M primer solution, 2.5 1 of 2 mM dNTP, and 10 × buffer (lOOmM Tris-HCl pH). 8.3, 500 mM KCK 20 mM MgCl ₂ , 0.01% Gelatin) and AmpliTaq Gold (a kind of heat-resistant DNA polymerase: 5U / X 1) are mixed with 0.2 1 and sterilized ultrapure water is added to the solution. The reaction liquid whose volume is 25 1 can be raised.

Since the target DNA region (ie, the target region) has many GC-rich base sequences, the reaction may be carried out by adding an appropriate amount of betaine, DMSO, etc. at times. As the reaction conditions, for example, the above reaction solution is kept at 95 ° C. for 10 minutes, then at 95 ° C. for 30 seconds, then at 55 to 65 ° C. for 30 seconds and further at 72. One example is a condition in which 30 to 40 cycles are performed for one cycle per second. After performing such PCR, the obtained amplification product is detected. For example, when a pre-labeled primer is used, the amplification amount in the PCR reaction can be evaluated by measuring the amount of the fluorescent label after performing the same washing and purification operation as before. In addition, when PCR using a normal unlabeled primer is performed, gold colloid particles, probes labeled with fluorescence, etc. are annealed, and the amount of the probe bound to the target region should be measured. Can be detected. In order to obtain the amount of amplification product more accurately, for example, in real time PCR method may be used. Real-time PCR is a method of monitoring PCR in real time and analyzing the obtained monitoring results by force kinetics. For example, high-precision quantification that can detect even a slight difference of about twice the gene amount. This method is known as the PCR method. Examples of the real-time PCR method include a method using a probe such as a saddle-type dependent nucleic acid polymerase probe and a method using an intermediary such as Cyber Green. Commercially available equipment and kits for real-time PCR may be used. As described above, the detection is not particularly limited, and detection by any known method can be performed. In these methods, the operation up to the detection can be performed without changing the reaction container. In addition, as an aspect of the second step, the third step can be performed without performing a digestion treatment with a methylation sensitive restriction enzyme capable of digesting single-stranded DNA. Objective: If there is no base sequence that can be digested with a methylation-sensitive restriction enzyme capable of digesting single-stranded DNA in the DNA region to be, the third step may be performed without performing the second step.

In addition, in the first step (A), a preferred embodiment for separating methylated single-stranded DNA includes addition of a counter oligonucleotide. The counter oligonucleotide is a short oligonucleotide having the same base sequence as the target DNA region. The length is usually 10 to 100 bases, more preferably 20 to 50 bases. Note that the counter oligonucleotide is not designed on the base sequence that the forward primer or reverse primer binds complementarily to the target DNA region. Counter-oligonucleotide is added in a large excess compared to genomic DNA, and the target DNA region is made single-stranded (positive strand) and then bound to the immobilized methylated DNA antibody. It is added to prevent the complementary strand (negative strand) of the target DNA region and the single strand (positive strand) of the target DNA region from recombining due to complementarity. In the DNA region of interest When measuring DNA methylation frequency or index value correlated therewith, it is easier to bind to the methylated DNA antibody when the target region is single-stranded. is there. In addition, it is desirable that the amount of Kanuta oligonucleotide is at least 10 times, usually 100 times or more, compared to the target DNA region.

Specifically, “adding a counter oligonucleotide when separating methylated single-stranded DNA” refers to methylated DNA samples derived from genomic DNA contained in biological samples. In order to select single-stranded DNA, a DNA sample derived from a genomic DNA contained in a biological specimen is mixed with a counter oligonucleotide, and the complementary strand of the target DNA region and the counter oligonucleotide are mixed together. The double strands may be formed. For example, the DN A and the sample, the counter one Origonuku Reochido, buffer (330mM Tris- Acetate pH 7.9, 660mM K0Ac, lOOmM MgOAc 2, · ■ 5mM Dithiothreitol) and 5 L, M g C 1 ₂ solution 5 LOOmM / L and 5 mg L of lmg / mL BSA solution, add sterilized ultrapure water to the mixture to a volume of 50 L, mix, heat at 95 ° C for 10 minutes, and heat to 70 ° Cool quickly to C, incubate for 10 minutes at that temperature> then cool to 50, incubate for 10 minutes, and then incubate for 10 minutes at 37 ° C, then return to room temperature. It is known that abnormal DNA methylation occurs in various diseases (for example, cancer), and it is considered that the degree of various diseases can be measured by detecting this abnormal DNA methylation.

For example, there is a DNA region that is 100% methylated in genomic DNA contained in a sample derived from a disease patient, and if the measurement method of the present invention is performed on that DNA region, the amount of methylated DNA will increase. I will. In addition, for example, there is a DNA region that is not 100% methylated in genomic DNA contained in a sample derived from a disease patient, and if the measurement method of the present invention is performed on that DNA region, the amount of methylated DNA Will be close to zero. In addition, for example, healthy living organisms DNA region that is hypomethylated in the genomic DNA contained in the sample of origin and hypermethylated in the genomic DNA contained in the biological sample of the patient with the disease If the present invention measurement method is performed for the DNA region, the amount of methylated DNA is close to 0 in the case of healthy subjects, while in the case of patients with diseases. Shows a value significantly higher than the value in the case of a healthy person, and therefore the “degree of disease” can be determined based on the difference between the values. The “degree of disease” here has the same meaning as that generally used in this field. Specifically, for example, when the biological specimen is a cell, it means the malignancy of the cell. In addition, for example, when the biological specimen is a tissue, it means the abundance of disease cells in the tissue. Furthermore, when the biological specimen is plasma / serum, it means the probability that the individual has a disease. Therefore, the measurement method of the present invention makes it possible to diagnose various diseases by examining methylation abnormality.

In the measurement method of the present invention, a restriction enzyme, primer or probe that can be used in various methods for measuring the amount of methylated DNA in the target region is useful as a reagent for a detection kit. is there. The present invention also provides a detection kit containing these restriction enzymes, primers or probes as reagents, and a detection chip in which these primers or probes are immobilized on a support. The scope of rights of the measurement method includes use in the form of a detection chip as described above, which utilizes the substantial principle of the method. Example

 EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. Example 1.

A commercially available methylated cytosine antibody (Aviva Systems Biology) is listed in the catalog using a commercially available biotinylated kit (Biot in Labeling Kit-NH2, manufactured by Dojindo Laboratories). According to the method described, it was labeled with piotin. Solution of the resulting biotin-labeled methylated cytosine antibody [antibody about 0.1 ^ / 100 1 ^ 0.1¾ BSA-containing phosphate buffer (ImM KH ₂ P0 ₄ , 3 mM Na ₂ HP0 · 7H ₂₀ , 15 complement NaCl pH 7.4) The solution was stored refrigerated. '' Add the above-mentioned piotin-labeled methylated succin antibody solution to each streptavidin-coated PCR tube (total of 8 tubes) at a ratio of 10 O ^ L and leave it at room temperature for about 1 hour. Immobilized to. Thereafter, the solution in the PCR tube was removed by pipetting, and then 100 L of washing buffer [0.05 Tween20-containing phosphate buffer (ImM KH ₂ P0 ₄ , 3 mM Na ₂ HP0] was added to the PCR tube. -7H ₂ 0 15 mM 'NaCl pH 7.)] was added. The buffer was then removed by pipetting. This operation was repeated two more times. Separately synthesize methylated oligonucleotides UB C—-M consisting of the base sequence shown in SEQ ID NO: 17 and unmethylated oligonucleotides UBC— U consisting of the base sequence shown in SEQ ID NO: 18 respectively. The following solutions (hereinafter referred to as oligonucleotide solutions) were prepared.

 Solution A: 0. OlpmoL / lOO ^ L TE buffer solution

 Solution B: O.OOlpmoL / lOO L TE buffer solution

 Solution C: 0. OOOlpmoL / lOO L TE buffer solution

 Solution D: OpmoL / lOO ^ L TE buffer solution (negative control solution)

<Methylated oligonucleotide> N represents methylated cytosine.

 UBC-M: 5'-

AGTGACACCATNGAGAATGTCAGATCNGGATCAGAGNGCCATCTAGATGGACATGTCACTGTCTGACTACAACAT CCAGA -3 '(SEQ ID NO: 1 7)'

 <Unmethylated oligonucleotide>

UBC-U: 5 '- CCAGA-3 ′ (SEQ ID NO: 18) Each of the obtained solutions was subjected to the following treatment (prepared one for each solution). ·

100 L of the oligonucleotide solution prepared above was added to the streptavidin-covered PCR tube on which the above-mentioned pyotin-labeled methyl-hycytosine antibody was immobilized, and left at room temperature for 2 hours. Thereafter, the solution in the PCR tube was removed by pipetting, and then 200 washing buffers [0.05% Tween20-containing phosphate buffer (lmM K¾P0 3 mM Na ₂ HP0 · 7H ₂₀ , 154m NaCl pH7 .4)] was added. Next, the buffer in the PCR tube was removed by pipetting. This operation was repeated twice more (this corresponds to the first step of the measurement method of the present invention).

 Next, PCR is performed on the PCR tubes thus obtained using the primer solutions (PF and PR) consisting of the nucleotide sequences shown in SEQ ID NO: 19 and SEQ ID NO: 20 and the following reaction conditions: Thus, the methylated DNA in the target DNA region UBC (SEQ ID NO: 21, methylated cytosine is also represented by C) was amplified.

'One>

 PF: 5'-AGTGACACCATCGAGAATGTCA-3 '(SEQ ID NO: 19)

PR: 5'-TCTGGATGTTGTAGTCAGACAG-3 '(SEQ ID NO: 20)

 <Target DNA region>

UBC: 5 '-AGTGACA

CCAGA-3 '(SEQ ID NO: 21)

As a PCR reaction solution, a DNA solution having a cage shape, a primer solution consisting of the nucleotide sequence represented by SEQ ID NO: 19 and SEQ ID NO: 20 prepared in 50 M each 0.3, each 2 mM dNTP 5 L, Buffer (lOOmM Tris-HCl pH 8.3, 500m KCK 15 mM MgCl ₂ , 0.01% Gelat in) 5 L and heat-resistant DNA polymerase (AmliTaq Gold) \ J / 0.25 L are mixed, and then sterilized ultrapure water is added to the mixture to reduce the volume. What was set to 50 L was used. The reaction solution was kept at 95 ° C for 10 minutes, then at 95 ° C for 30 seconds, then at 59 ° C for 30 seconds, and then at 72 ° C for 45 seconds for 1 cycle. Went.

 After PCR, DNA amplification was confirmed by subjecting to 1.5% agarose gel electrophoresis. The result is shown in FIG. 1 (this corresponds to the third step of the measurement method of the present invention). Amplification of DN A was confirmed in solution A (O.Olpmol), solution B (OOOlpmol), and solution C (0. OOOlpmol) of UBC—M. The amplification product (target DNA 'region: UBC) was gotten. In solution D (Opmol) of UBC-M, amplification of DNA was not confirmed and the amplification product was not obtained. On the other hand, no amplification of DNA was observed in all groups of UBC—U solution A (0,01pmol), solution B (O.OOlpmol), solution C (0. OOOlpmol), and solution D (Opmol). No amplification product was obtained. Based on the above, it is possible to select a single-stranded DNA containing the methylated target DNA region with the immobilized methylated cytosine antibody, and in the target DNA region. It was confirmed that, without amplifying non-methylated DNA, only methylated DNA was amplified to a detectable amount, and the amount of amplified DNA could be quantified. Example 2

A commercially available methylated cytosine antibody (manufactured by Aviva Systems Biology) was labeled using a commercially available pyotinization kit (Biotin Labeling Kit-NH2, manufactured by Dojindo Laboratories) according to the method described in the catalog. The resulting biotin-labeled methylated cytosine antibody in solution [antibody approx. 0 lg / 50 L Ό.1¾ BSA-containing phosphate buffer (ImM K¾P0 3 mM Na ₂ · 7 鹏₂₀ , 154 mM NaCl pH 7.4) The solution was stored refrigerated.

Add the above-mentioned piotin-labeled methylated cytosine antibody solution to each streptavidin-coated PCR tube (total of 6 tubes) at a ratio of 50; L and leave it at room temperature for about 1 hour. And immobilized on a PCR tube. Then, the solution in the PCR tube was removed by pipetting, 1 0 0 L washing buffer [0.05% Tween20-containing phosphate buffer _{(ImM KH 2 P0 4, 3mM} Na 2 HP0 - 7H 2 0, 15 m NaCl pH 7.4)] was added. Next, the buffer in the PCR tube was removed by pipetting. This operation was repeated two more times.

 Partially methylated oligonucleotide that is not digested with HhaI consisting of the base sequence shown in SEQ ID NO: 2 2 GPR 7— 2 0 7 9-2 1 7 6/9 8mer-M (7), SEQ ID NO: 2 Partially methylated oligonucleotide digested with Hha I consisting of the base sequence shown GPR 7—207 9—2.1 7 6 / 98mer—HM (5), unmethylated oligo consisting of the base sequence shown in SEQ ID NO: 24 Nucleotides GPR 7-2 0 7 9-2 1.76 / 98me r UMs were synthesized separately to prepare 0. l pmo 1/50 L TE buffer solutions (hereinafter referred to as oligonucleotide solutions).

• <Partially methylated oligonucleotide not digested with HhaI>

 N represents methylated scissin.

 GPR7-2079-2176 / 98mer-M (7):

 Five'-

GTTGGCCACTGCGGAGTCGNGCNGGGTGGCNGGCCGCACCTACAGNGCCGNGNGNGCGGTGAGCCTGGCCGTGTG GGGGATCGTCACACTCGTCGTGC -3 '(SEQ ID NO: 22)

 <Partially methylated oligonucleotide digested with HhaI>

 N represents methylated cytosine.

 GPR7-2079-2176 / 98mer-HM (5):

 Five' -

GTTGGCCACTGCGGAGTCGCGCCGGGTGGCNGGCCGCACCTACAGNGCCGNGNGNGCGGTGAGCCTGGCCGTGTG GGGGATCGTCACACTCGTCGTGC -3 '(SEQ ID NO: 2 3)

 Guanmethylated oligonucleotide>

GPR7-2079-2176 / 98mer-U: Five' -

GTTGGCCACTGCGGAGTCGCGCCGGGTGGCCGGCCGCACCTACAGCGCCGCGCGCGCGGTGAGCCTGGCCGTGTG GGGGATCGTCACACTCGTCGTGC -3 '(SEQ ID NO: 24)' Each of the obtained solutions was subjected to the following treatment (prepared in duplicate for each solution).

 The oligonucleotide solution 5 OL prepared above was added to the streptavidin-coated PCR tube on which the above-mentioned pyotin-labeled methylated cytosine antibody was immobilized, and left at room temperature for 1 hour. After removing the solution in the PCR tube by pipetting, add 100 L of wash buffer [0.05%

Tween20-containing phosphate buffer (ImM KH ₂ P0 ₄ , 3 mM Na ₂ HP0-7H ₂₀ , 154 mM NaCl pH 7.4)] was added. Next, the buffer of the tube Λ was removed by pipetting. This operation was repeated two more times (or more, the first step of the present measuring ^method: corresponds to). The samples thus obtained were subjected to the following cocoon treatment or cocoon treatment (prepared for each one). .

A-treated group (untreated group): Buffer (330 mM Tris-Acetate ρΗ 7,9, 660 mM KOAc, lOOmM Mg0Ac ₂ , 5 mM Dithiothreitol) IOL and BSA (Bovine serum albumin lmg / mL) ) After adding IOL, sterilized ultrapure water was further added to the mixture to make the volume 100 L. .

B treatment group (Hha I treatment group): The sample prepared above was mixed with buffer solution (330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM Mg0Ac ₂ , 5 mM Dithiothreitol) 1 O ^ L, BSA (Bovine serum albumin l'mg / mL) After adding IOL and 64 U of Hha I, sterilized ultrapure water was further added to the mixture to make the volume 50 0 L. Each reaction was incubated at 37 for 3 hours. Then inside the PCR tube After removing the solution by pipetting, 100 L of washing buffer [0.05% Tween20-containing phosphate buffer (lmM KH ₂ P0 ₄ , 3 mM Na ₂ HP0 · 7H ₂₀ , 154 mM NaCl pH 7.4)] was added . Thereafter, the buffer was removed by pipetting. This operation was repeated twice more (this corresponds to the second step of the measurement method of the present invention). Next, PCR is performed on the PCR tubes thus obtained using the primer solutions (PF2 and PR2) consisting of the nucleotide sequences shown in SEQ ID NO: 25 and SEQ ID NO: 26 and the following reaction conditions. Was used to amplify the methylated DNA in the target DNA region (GPR7-20 79-2176, SEQ ID NO: 27, methylated cytosine is also represented by C).

'One>

 PF2: 5'-GTTGGCCACTGCGGAGTCG-3 '(SEQ ID NO: 25)

PR2: 5'-TCTGGATGTTGTAGTCAGACAG-3 '(SEQ ID NO: 26)

<Target DNA region>

GPR7-2079-2176: 5'- GTTGGCCACTGCGGAGTCGC

GGGGATCGTCACACTCGTCGTGC -3 '(SEQ ID NO: 27)

As PCR reaction solution, the DNA in the vertical form, 5 L each of the primer solution consisting of the nucleotide sequence shown in SEQ ID NO: 25 and SEQ ID NO: 26 prepared in 3, and ea.ch 2 mM dNTP 5 ^ L Buffer solution (lOOmM Tris-HCl pH 8.3, 500iM KCK 15mM MgCl ₂ , 0.01% Gelatin) 5 L, thermostable DNA polymerase (Amp liTaq Gold) 5UZ 0.25 L, 5 N betaine aqueous solution 10 Then, the mixture was further added with sterile ultrapure ice to make the liquid volume 50. The reaction solution was incubated at 95 at 10 minutes, and then PCR was carried out under the conditions of incubation at 95 for 30 seconds, 59 at 30 seconds and 72 at 45 seconds for one cycle. - After PCR, the obtained amplification product was subjected to 1.5% agarose gel electrophoresis to confirm DNA amplification. The result is shown in FIG. 2 (this corresponds to the third step of the measurement method of the present invention). In the case of A-treated group (untreated group), partially methylated oligonucleotide GPR 7— 2079 -2176 / 98me r M (7) that is not digested with Hha and partially methylated oligonucleotide GPR that is digested with Hha I 7 With -2079-2176 / 98mer-HM (5), amplification of DNA was confirmed and the amplification product (target DNA region: GPR7-2079-2176) was obtained. With the unmethylated oligonucleotide GPR 7 -2079-2176 / 98mer-UM, amplification of DNA was not confirmed and the amplification product was not obtained. In the case of the B treatment group (H'ha I treatment group), the partially methylated oligonucleotide GP 7-2079-2176 / 98mer-M (7), which is not digested with Hha I, was confirmed to have amplified DNA. The product (targeted DN A region: GPR7-2079-2176) was obtained, but partially methylated oligonucleotide digested with Hha I GPR 7- 207 ■ 9-2176 / 98mer-HM (5), In addition, amplification of DNA was not confirmed with the unmethylated oligonucleotide G 7-2079 _2176 / 981116 1 "-1; no amplification product was obtained. From the above, immobilized methylated cytosine antibody It is possible to select a single-stranded DNA containing the target DNA region that has been methylated, and to limit methylation sensitivity by treatment with a methylation-sensitive restriction enzyme. The ability to digest the target DNA region where the enzyme recognition site is not methylated, and It is possible to amplify only methylated DNA to a detectable amount without amplifying unmethylated DNA in the target DNA region, and to quantify the amount of amplified DNA. Example 3 was confirmed.

A commercially available methylated cytosine antibody (Aviva Systems Biology) is described in the Kata log using a commercially available pyotinization kit (Biotin Labeling Kit-NH2, manufactured by Dojindo Laboratories). According to the method described, it was labeled with piotin. The resulting biotin-labeled methylated scissin antibody was used as a solution [antibody about 0.1 ^ / 50 ^ 1 ^ 0.1% BSA-containing phosphate buffer (ImM KH ₂ P0 ₄ , '3 mM Na ₂ HP0 · 7H ₂ 0, 154m NaCl pH7 ) Refrigerated as a solution].

 The above-mentioned piotin-labeled ■ methylated cytosine antibody solution was added to each streptavidin-coated PCR tube (total of 6 tubes) at a ratio of 50 tL and allowed to stand at room temperature for about 1 hour to be immobilized on the PCR tube. After removing the solution in the PCR tube by pipetting, add 100 L of washing buffer in the PCR tube.

[0.05% Tween20-containing phosphate buffer (ImM KH ₂ P0 ₄ , 3 mM Na ₂ HP0-7H ₂₀ , 154 mM NaCl pH 7.4)] was added. Thereafter, the buffer in the tube was removed by pipetting. This operation was repeated two more times.

 Partial methylation not digested with Hti a I consisting of the base sequence shown in SEQ ID NO: 22 Oligonucleotide G PR 7-2079-2176 / 98me r -M (7), Hha consisting of the base sequence shown in SEQ ID NO: 23 Partially methylated oligonucleotide digested with I GPR 7-2079-2176 / 98me r -HM (5), unmethylated oligonucleotide GPR 7— 2079-2 1 76 / 98me r — UM was synthesized separately to prepare 0. l pmol / 50 xL TE buffer solution (hereinafter referred to as oligonucleotide solution).

<Partially methylated oligonucleotide not digested with HhaI>

 N represents methylated cytosine.

 GPR7-2079-2176 / 98mer-M (7):

 Five' -

GTTGGCCACTGCGGAGTCGNGCNGGGTGGCNGGCCGCACCTACAGNGCCGNGNGNGCGGTGAGCCTGGCCGTGTG GGGGATCGTCACACTCGTCGTGC -3 '(SEQ ID NO: 22)

 <Partial mesylation oligonucleotide digested with H a I>

 N represents methylated cytosine.

 GPR7-2079-2176 / 98mer-HM (5):

Five' - . GTTGGCCACTGCGGAGTCGCGCCGGGTGGCNGGCCGCACCTACAGNGCCGNGNGNGCGGTGAGCCTGGCCGTGTG GGGGATCGTCACACTCGTCGTGC -3 '(SEQ ID NO: 23)

 <Unmethylated oligonucleotide>

 GPR7-2079-2176 / 98mer-UM:

' Five'-

GGGGATCGTCACACTCGTCGTGC -3 '(SEQ ID NO: 24) The sample thus obtained was subjected to the following A or B treatment (' prepared for each one)）

Α Treatment group (untreated group): Samples prepared above (each l O iL) were mixed with buffer solution (330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM MgOAc ₂ , 5raM Dithiothreitol) 5 ^: and BSA (Bovine serum albumin lmg / mL) 5 L was added, and sterilized ultrapure water was further added to the mixture to make the volume 50 L.

B treatment group (Hha I treatment group): Samples prepared above (10 L each) were mixed with buffer solution (330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM MgOAc ₂ , 5 mM Dithiothreitol) 5 and BSA ( Bovine serum albumin lmg / mL) 5 L and Hhal 64U were added, and then sterilized ultrapure water was added to the mixture to make the volume 50. Each reaction was incubated at 37 ° C for 3 hours. The entire reaction solution was added to the streptavidin-coated PCR tube on which the biotin-labeled methylated cytosine antibody prepared above was immobilized, and then allowed to stand at room temperature for 1 hour. Next, remove the solution in the PCR tube by pipetting, and then wash with 100 / L washing buffer.

[0.05% Tween20-containing phosphate buffer (1 KH ₂ P0 ₄ , 3 mM Na ₂ HP0-7¾0, 15 raM NaCl pH 7.4)] was added. Thereafter, the buffer was removed by pipetting. This operation was repeated two more times (this corresponds to the first step of the measurement method of the present invention). ) Next, PCR is carried out on the PCR tube thus obtained using each primer solution (PF 2 and PR2) consisting of the nucleotide sequences shown in SEQ ID NO: 25 and SEQ ID NO: 26 and the following reaction conditions: Thus, methylated DNA in the target DNA region (GPR7-20 79-2176, SEQ ID NO: 27, methylated cytosine is also represented by C) was amplified.

'―>

'PF2: 5' -GTTGGCCACTGCGGAGTCG-3 '(SEQ ID NO: 25)

 PR2: 5'-TCTGGATGTTGTAGTCAGACAG-3 '(SEQ ID NO: 26)

 <Target DNA region>

 GPR7-2079-2176: 5'-

G TGGCCACTGCGGAGTCGC GGGGATCGTCACACTCGTCGTGC -3 '(SEQ ID NO: 27)

As a PCR reaction solution, each 5 L of a primer solution consisting of the nucleotide sequence shown in SEQ ID NO: 25 and SEQ ID NO: 26 prepared in 3 M, each 2 mM dNTP 5 L, in a vertical DNA, buffer and (lOOmM Tris-HCl pH 8.3, 500mM KC1, 15mM MgCl 2, 0.01% Gelatin) and a 5 L, thermostable DNA polymerase (AmpliTaq Gold) 5 U / iL of 0. 25 ^ L, 5 N betaine aqueous solution After mixing with 10 L, sterilized ultrapure water was further added to the mixture to make the volume 50 L. The reaction solution was incubated at 95 ° C for 10 minutes, then at 95 ° C for 30 seconds, then at 59 ° C for 30 seconds, and further at 72 ° C for 45 seconds for one cycle. PCR was performed at. '

After PCR, the obtained PCR product was subjected to 1.5% agarose gel electrophoresis to confirm DNA amplification. The result is shown in FIG. 2 (this corresponds to the third step of the measurement method of the present invention). In the case of A treatment group (no treatment group), digested with Hh al WO 2008/123537

 Not partially methylated oligonucleotide GPR 7— 2079 -2176 / 98me r M (7) and partially methylated oligonucleotide GPR-2079-2176 / 98 me r -HM (5) digested with Hha I As a result, amplification of the DNA was confirmed and the amplified product (target DNA region: GPR7-2079-2176) was obtained. With the AMP oligonucleotide GPR7-2079-2176 / 98mer-UM, amplification of DNA was not confirmed and the amplification product was not obtained. In the case of the B treatment group (Hha I treatment group), the partially methylated oligonucleotide GP R7—2079—21 76 / 98me r—M (7), which is not digested with Hha I, has been confirmed to have been amplified. The target DNA region: GPR7—2079—2176) was obtained, but the partial methyl oligonucleotides digested with Hh ■ aI. GPR7—2079-21.76 / 98me r -HM (5) and With the unmethylated oligonucleotide GP R 7-20 79-2176 / 98me r UM, amplification of DNA was not confirmed and the amplification product was not obtained. Example 4

A commercially available methylcytosine antibody (manufactured by Aviva Systems Biology) can be used according to the method described in the catalog using a commercially available biotinylated kit (Biotin Labeling Kit-NH ₂ , manufactured by Dojindo Laboratories). Piotin labeled. Refrigerated Piochin labeled methylcytosine antibody obtained as a solution Antibody about 0.25 g / iL 0.1% BSA-containing phosphate buffer _{(ImM KH 2 P0 4, 3mM} Na 2 HP0 · 7Η 2 0, 154mM NaCl pH7.4) solution] saved.

To a PCR tube coated with streptavidin (a total of 8 tubes), add 0.1 jLig / 50 solution of the synthesized biotin-labeled methylcytosine antibody at a ratio of 50 each, and leave it at room temperature for about 1 hour. Immobilized in a tube. Then, the solution was removed by pipetting, and 100 washing buffer [0.05% Tween20-containing phosphate buffer (ImM KH ₂ P0 ₄ , 3 mM Na ₂ HP0'7H ₂₀ , 154raM NaCl pH 7.4)] was added. Thereafter, the buffer was removed by pipetting. This operation was repeated twice more (this corresponds to the preparation of the immobilized methylated DNA antibody used in the measurement method of the present invention). The following sequence for human blood-derived genome DN A purchased from Clontech DN used as a test sample by performing PCR using the oligonucleotide primers (? 1 and? 1) designed for PCR shown in SEQ ID NO: 28 and SEQ ID NO: 29 and reaction conditions A fragment (X, SEQ ID NO: 30, Genbank Accession No. NT — in base numbers 25687390-25687775 shown in 029419 etc.

'Corresponding region) was amplified.

<Oligonucleotide primer designed for PCR>

 PF1: 5'-CTCAGCACCCAGGCGGCC-3 '(SEQ ID NO: 28)

 PR 1: 5'-CTGGCCAAACTGGAGATCGC -3 '(SEQ ID NO: 29)

'<DNA fragment>

 X: 5'-

 AGTTTGGCCAG (SEQ ID NO: 30)

As PCR reaction solutions, 5 ng of genomic DNA in a bowl shape, each 3 1 oligonucleotide primer solution prepared to 5 M, each 2 mM dNTP, and 1 OX buffer (lOOmM Tris-HCl H 8.3, 500 mM KC1, 15 mM MgCl ₂ , 0.01% Gelatin) 5 1 and heat-resistant DNA polymerase (AmpliTaq Gold, AB I) 5U / 1 mixed with 0.25 1 Pure water was added to make the volume 50 1. The reaction solution was incubated at 95 for 10 minutes, and then PCR was performed under the conditions of 40 cycles of incubation at 95 for 30 seconds, then 6: TC for 30 seconds and 72 for 45 seconds.

After PCR, amplification was confirmed by 1.5% agarose gel electrophoresis, and DNA fragment X. was purified using Wizard SV Gel / PCR Kit (PR0MEGA). About a part of the obtained DNA fragment solution, Sssl methylase (manufactured by NEB) 1 1, 10 x NEBuf fer 2 (manufactured by NEB) 1 0 1, S- adenosyl methionine (3.2 mM, NEB) was mixed with 11 and sterilized ultrapure water was added thereto to prepare a solution with a volume of 100 1. The reaction solution was incubated at 37 ° C for 15 to 30 minutes, and further S-adenosyl methionine (3.2 mM, manufactured by NEB) was added to add 1 1 at 37 ° C. Incubated for 0 min. This was purified by Wizard SV Gel / PCR Kit (PROMEGA). Further, these operations were repeated 5 times to obtain methylated DNA fragment (MX, SEQ ID NO: 3 1.).

,

 <DNA fragment> (N is 5-methylcytosine)

 MX: 5 '-

For CTCAGCACCCAGGNGGCNGNGATCATGAGGNGNGAGNGGNGNGNGGGCTGTTGCAGAGTCTTGAGNGGGTGGCAC ACNGNGATGTAGNGGTNGGCTGTCATGACTACCAGCATGTAGGCNGANGCAAACATGCNGAACACCTGCAGGTGC TTCACCANGNGGCACAGCCAGTNGGGGCNGNGGAAGNGGTAGGTGATGTCCCAGCACATTTGNGGCAGCACCTGG AAGAATGCCANGGCCAGGTNGGCCAGGCTGAGGTGTNGGATGAAGAGGTGCATGNGGGANGTCTTGNGNGGNGTC NGGTGCAGAGCCAGCAGTANGCTGCTGTTGCCCAGCANGGCCACNGNGAAAGTCACNGCCAGCANGGNGATCTCC AGTTTGGCCAG -3 '(SEQ ID NO: 3 1) The resulting D NA fragment X, the following solutions were prepared.

 Solution A: lOOpg / lO ^ L TE solution

 Solution B: lOpg / lO ^ L TE solution

 Solution C: lpg / 10 / z L TE solution

 Solution D: TE solution (negative control solution) The following solution was prepared for the obtained DNA fragment MX.

 Solution MA: 100pg / 10 6L TE solution

Solution MB: lOpg / lO ^ L TE solution ' Solution MC: lpg / lO ^ L TE solution

 Solution MD: TE solution (negative control solution) 'In addition, the bases represented by SEQ ID NO: 33 to SEQ ID NO: 44 that can bind by complementarity to the negative strand of the target DNA region consisting of the base sequence represented by SEQ ID NO: 32 A counter-oligonucleotide C1 to C12 consisting of a sequence was synthesized, and a TE buffer solution having a concentration of 0.01 M was prepared.

<Target DNA region> (5-methylcytosine is also represented by C)

'Χ': 5'—

 AGTTTGGCCAG -3 '(SEQ ID NO: 32)

 <Counter oligonucleotide>

 C 1: 5 '-GCCACCGCGAAAGTCACCGCCAGCACGGCG-3, (SEQ ID NO: 33)

 C2: 5'-GCCAGCAGTACGCTGCTGTTGCCCAGCACG-3 '(SEQ ID NO: 34)

 C3: 5'-CGGGACGTCTTGCGCGGCGTCCGGTGCAGA-3 '(SEQ ID NO: 35)

 C4: 5'-AGGCTGAGGTGTCGGATGAAGAGGTGCATG-3 '(SEQ ID NO: 36)

 C 5: 5 '-ACCTGGAAGAATGCCACGGCCAGGTCGGCC -3' (SEQ ID NO: 37)

 C 6: 5'-TAGGTGATGTCCCAGCACATTTGCGGCAGC -3 '(SEQ ID NO: 38)

 C7: 5'-CGGCACAGCCAGTCGGGGCCGCGGAAGCGG-3 '(SEQ ID NO: 39)

 C 8: 5 '-ATGCCGAACACCTGCAGGTGCTTCACCACG -3' (SEQ ID NO: 40)

 C 9: 5 '-ATGACTACCAGCATGTAGGCCGACGCAAAC-3, (SEQ ID NO: 41)

 C 10: 5'-TGGCACACCGCGATGTAGCGGTCGGCTGTC-3 '(SEQ ID NO: 42)

C 1 1: 5'-CGCGCGGGCTGTTGCAGAGTCTTGAGCGGG-3 '(SEQ ID NO: 43) C12: 5′-CAGGCGGCCGCGATCATGAGGCGCGAGCGG-3 ′ (SEQ ID NO: 44) The following treatment was performed on each of the DNA fragment X solution and the methylated DNA fragment MX solution.

In a PCR tube, prepare the DNA fragment solution 1 O ^ L prepared above, 10 L of the counter-oligonucleotide solution prepared above, and buffer solution (330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM MgOAc ₂ , 5 mM Di thiothrei tol) 5 L, lOOmM M 8. 1 ₂ solution 5 was added BSA solution 5 L of lmg / mL, the liquid volume of 5.0 L further added with sterile ultrapure water to the mixture and mixed. Thereafter, the PCR tube was heated at 95 ° C for 10 minutes, rapidly cooled to 70 ° C, and kept at that temperature for 10 minutes. Next, the mixture was cooled to 50 ° C., kept warm for 10 minutes, further kept at 37 ° C. for 10 minutes, and then returned to room temperature (this corresponds to the first step (A) of the measurement method of the present invention). Streptavidin coated with the above-mentioned pyotin-labeled methylcytosine antibody immobilized ■ 50 L of the DNA fragment reaction solution prepared above was added to the PCR tube and allowed to stand at room temperature for 30 minutes. Then, the solution was removed by pipetting, and after adding 100 L of washing buffer [0.05¾ Tween20-containing phosphate buffer (ImM KH ₂ P0 ₄ , 3 mM Na ₂ HP0-7¾0, 154 mM NaCl pH 7.4)] The buffer was removed by pipetting. This operation was repeated twice more (this corresponds to the first step (B) of the measurement method of the present invention).

 Next, PCR is performed on the above PCR tube using each solution of oligonucleotide primers PF 1 and PR 1 consisting of the nucleotide sequences shown in SEQ ID NO: 28 and SEQ ID NO: 29, and the reaction conditions shown below: The methylated DNA in the target DNA region X ′ consisting of the base sequence represented by SEQ ID NO: 32 was amplified. <Oligonucleotide primer designed for PCR>

 PF1: 5'-CTCAGCACCCAGGCGGCC-3 '(SEQ ID NO: 28)

 PR1: 5'-CTGGCCAAACTGGAGATCGC-3 '(SEQ ID NO: 29)

<Target DNA region> (5-methylcytosine is also represented by C) · W

55

X ': 5'-

CTCAGCACCCAGGCGGCCGCGATCATGAGGCGCGAGCGGCGCGCGGGCTGTTGCAGAGTCTTGAGCGGGTGGCAC

 AAGAATGCCACGGCCAGGTCGGCCAGGCTGAGGTGTCGGATGAAGAGGTGCATGCGGGACGTCTTGCGCGGCGTC

AGTTTGGCCAG -3 '(SEQ ID NO: 3 2)

The PCR reaction solution is a type IV DNA, 3 L each of oligonucleotide primer solution prepared in 5 M, 5 L each 2 mM dNTP, buffer solution

(lOOmM Tris-HCl pH 8.3, 500 mM KCK 15 mM MgCl ₂ , 0.01% Gelatin) 5 L and heat-resistant DNA polymerase (AmpliTaq Gold, manufactured by AB I) 5UZ L 0.25 and sterilized Ultra pure water was added to adjust the liquid volume to 50 L. Incubate the reaction solution at 95 ° C for 10 minutes, then heat at 95 ° C for 20 seconds, then at 61 ° C for 30 seconds, and then at 72 ° C for 30 seconds for 25 cycles. PCR was performed under conditions.

 After PCR, amplification was confirmed by 1.5% Agaro gel electrophoresis. The results were as follows (corresponding to the third step of the measurement method of the present invention). The results are shown in FIG. Amplification was confirmed in the methylated DNA fragment MX solutions MA, MB and MC. No increase was confirmed in the negative control solution MD. No amplification was confirmed in solutions A, B, C and D of DNA fragment X which was not methylated.

Based on the above, it is possible to select methylated DNA containing the target DNA region with the immobilized methylcytosine antibody, and amplify the methylated DNA to a detectable amount. It was confirmed that the amplified DNA could be detected with higher sensitivity. Example 5

Using a commercially available methylcytosine antibody (Aviva Systems Biology) and a commercially available piotisylated kit (Biot in Labeling Kit-NH ₂ , manufactured by Dojindo Laboratories) according to the method described in the catalog, Piotin labeled. Obtained Piotin-labeled methylcytosine antibody 'as a solution [antibody 0.25 ug / fl 0.1¾ BSA-containing phosphate buffer (ImM KH ₂ P0 ₄ , 3 mM Na ₂ HP0 • 7H ₂₀ , 154 mM NaCl pH 7.4) solution] Stored refrigerated.

Add a 0.1 g / 50 solution of the synthesized biotin-labeled methylcytosine antibody to each streptavidin-coated PCR tube (total of 8 tubes) at a rate of 50 L and leave it at room temperature for about 1 hour. Immobilized in an R tube. Thereafter, the solution was removed by pipetting, and 100 L of washing buffer [0.05% Tween20-containing phosphate buffer (ImM KH ₂ P0 ₄ , 3 mM Na ₂ HPO-7H ₂₀ , 154 mM NaCl pH 7.4)] was added. Thereafter, the buffer was removed by pipetting. This operation was repeated twice more (this corresponds to the preparation of the immobilized methylated DNA antibody used in the measurement method of the present invention). • For human blood genomic DNA purchased from Clontech, oligonucleotide primer primers (PF 2 and PR 2) designed for the PCR shown in SEQ ID NO: 45 and SEQ ID NO: 46 below and reaction conditions PCR was performed to amplify a DNA fragment (Y, a region corresponding to nucleotide numbers 76606 to 76726 shown in SEQ ID NO: 47, Genbank Accession No. ac009800, etc.) used as a test sample. > Oligonucleotide primers designed for PCR>

 PF2: 5'-TGAGCTCCGTAGGGCGTCC-3 '(SEQ ID NO: 45)

 PR 2: 5'- GCGCCGGGTCCGGGCCC -3 '(SEQ ID NO: 46)

 <DNA Fragment>

 Υ: 5'- '

GCGCCGGGTCCGGGCCCGATGCGTTGGCGGGCCAGGGCTCCGAGAACGAGGCGTTGTCCATCTCAACGAGGGCAG AGGAGCCGGCGACCTGGCGTCCCCCAAGGACGCCCTACGGAGCTCA -3 '(SEQ ID NO: 47) As PCR reaction solution, 5 ng of genomic DNA to form a cage, 3 1 each of oligonucleotide primer solution prepared in 5 M, 5 1 each 2 mM dNTP, 10X buffer (lOOmM Tris-HCl H 8.3, 500 mM KCK 15 mM MgCl ₂ , 0.01% Gelatin) .5 l and heat-resistant DNA polymerase (AmpliTaq Gold, AB I) '511/1 mixed with 0.25 1 Pure water was added to make the volume 50 1. The reaction solution is kept at 95 ° C for 10 minutes, then at 95 ° C for 30 seconds, then at 60 ° C for 30 seconds, and at 72 at 45 seconds for one cycle. I did a PCR.

 After performing PCR, amplification was confirmed by 1.5% agarose gel electrophoresis, and DNA fragment Y was purified using 'Wizard SV Gel / PCR Kit (PR0MEGA).

For a portion of the resulting DNA fragment solution, add Sssl methylase (NEB): 1 1, 10 x NEBuffer2 (NEB) 10 l, S-adenosyl methionine (3.2 mM, NEB) 1 / i 1 was mixed, and sterilized ultrapure water was added thereto to prepare a liquid volume of 100 1. The reaction solution was incubated at 37 ° C. for 15-30 minutes, and further S-adenosyl.methionine (3.2 mM, manufactured by NEB) was added at 11 and incubated at 37 for 15-30 minutes. This was purified by Wizard SV Gel / PCR Kit (PROMEGA). This operation was further repeated 5 times to obtain a methylated DNA fragment (MY, SEQ ID NO: 48).

<DNA fragment> (N is 5-methylcytosine)

 MY: 5'-

GNGCNGGGTCNGGGCCNGATGNGTTGGNGGGCCAGGGCTCNGAGAANGAGGNGTTGTCCATCTCAANGAGGGCAG AGGAGCNGGNGACCTGGNGTCCCCCAAGGANGCCCTANGGAGCTCA -3 '(SEQ ID NO: 48) For the obtained DN A fragment Y, the following solution was prepared

Solution A: lOOpg / lO ^ L TE solution Solution B: lOpg / lO ^ L TE solution

 Solution C: lpg / 10 L TE solution

 Solution D: TE solution (negative control solution) For the obtained DNA fragment MY, the following solutions were prepared.

 Solution MA: 100pg / 10 ^ L TE solution

 Solution MB: 10pg / 10 L TE solution

 Solution MC: lpg / 10 // L TE solution

 Solution MD: TE solution (negative control solution) Further, SEQ ID NO: 50, base sequence 24, and SEQ ID NO: 52 capable of binding to the negative strand of the target DNA region Y ′ consisting of the base sequence represented by SEQ ID NO: 49 by complementarity Counter-oligonucleotides C13, C14, and C15 having the indicated base sequence were synthesized, and a TE buffer solution having a concentration of 0.01 M was prepared.

<Target DNA region> (5-methylcytosine is also expressed as C)

Y ': 5'-GCGCCGGGTC

AGGAGCCGGCGACCTGGCGTCCCCCAAGGACGCCCTACGGAGCTCA -3 '(SEQ ID NO: 49)

<Counter oligonucleotide>

 C13: 5'-GCGTCCCCCAAGGACGCCCTACGGAGCTCA-3 '(SEQ ID NO: 50)

C1: 5'-CTCAACGAGGGCAGAGGAGCCGGCGACCTG-3 '(SEQ ID NO: 5 1)

C 15: 5'-CGCCGGGTCCGGGCCCGATGCGTTGGCGGG-3 '(SEQ ID NO: 52)

The following treatment was applied to each of the liquids.

In a PCR tube, prepare 10 L of the DNA fragment solution prepared above, 10 L of the counter oligonucleotide solution prepared above, and buffer (330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM MgOAc ₂ , 5 mM Di thiothrei tol) Add 5 L, lOOmM M <1 ₂ solution 5 ^, and add lmg / mL BSA solution 5 and sterilize ultrapure Water was added to make the volume 50 L and mixed. Thereafter, this PCR tube was heated at 95 ° C. for 10 minutes, rapidly cooled to 70 ° C., and kept at that temperature for 10 minutes. Next, it was cooled to 50, kept for 10 minutes, further kept at 37 ° C. for 10 minutes, and then returned to room temperature (corresponding to the first step (A) of the measurement method of the present invention). 50 L of the DNA fragment reaction solution prepared above was added to the streptavidin-coated PCR tube on which the biotin-labeled methylcytosine antibody was immobilized, and left at room temperature for 1 hour. Then, the solution was removed by pipetting, and 100'L of washing buffer [0.05% Tween20-containing phosphate buffer (ImM KH ₂ P0 ₄ , 3 mM Na ₂ HP0-7H ₂ 0, 154 mM NaCl pH 7.4)] was added. Thereafter, the buffer was removed by pipetting. This operation was repeated twice more (this corresponds to the first step (B) of the measurement method of the present invention). Next, PCR is performed on the above PCR tube using each solution of oligonucleotide primers PF 2 and PR 2 consisting of the base sequences represented by SEQ ID NO: 45 and SEQ ID NO: 46 and the following reaction conditions: The methylated DNA in the target DNA region Y ′ consisting of the base sequence represented by SEQ ID NO: 49 was amplified. <An oligonucleotide primer designed for PCR>

PF2: 5'-TGAGCTCCGTAGGGCGTCC-3 '(SEQ ID NO: 45)

PR 2: 5 '-GCGCCGGGTCCGGGCCC -3' (SEQ ID NO: 46)

<Target DNA region>

Y ': 5'- GCGCCGGGTCCGGGCCCGATGCGTTGGCGGGCCAGGGCTCCGAGAACGAGGCGTTGTCCATCTCAACGAGGGCAG AGGAGCCGGCGACCTGGCGTCCCCCAAGGACGCCCTACGGAGCTCA -3' (SEQ ID NO: 49)

As a PCR reaction solution, the oligonucleotide prepared in 5 M was added to the vertical DNA. 3 L each of the primer primer solution, 5 L each 2 mM dNTP, 5 L buffer (ΙΟΟπιΜ Tris-HCl pH 8.3, 500 mM KCK 15iM MgCl ₂ , 0.01% Gelatin), heat resistant DNA polymerase (AmpliTaq Gold, manufactured by AB I) was mixed with 0.25 and sterilized ultrapure water was added to make the volume 50 L. The reaction solution is kept at 95 ° C for 10 minutes, then kept at 95 ° C for 20 seconds, then at 60 ° C for 30 seconds, and then at 72 ° C for 30 seconds for 25 cycles. PCR was performed.

 After PCR, amplification was confirmed by 1.5% agarose gel electrophoresis. The results were as follows (this corresponds to the third step of the measurement method of the present invention).

The results are shown in FIG. Amplification was confirmed in the methylated DNA fragment MY solutions MA, MB and MC. No amplification was confirmed in the negative control solution MD. Amplification was not confirmed in solutions A, B ^: , -C and D of unmethylated DNA fragment Y.

 From the above, it is possible to select methylated DNA containing the target DNA region with the immobilized methylcytosine antibody, and to amplify the methylated DNA to a detectable amount. It was confirmed that the amplified DNA could be detected with higher sensitivity. Example 6

A commercially available methylcytosine antibody (Aviva Systems Biology) was converted into a commercially available pyotinized kit (Biot in Labeling Kit-NH ₂ , manufactured by Dojindo Laboratories) according to the method described in the catalog. Piotin labeled. Solution of the obtained Piotin-labeled methylcytosine antibody [Antibody 0.25 ig / fl 0.1% BSA-containing phosphate buffer (ImM KH ₂ P0 ₄ , 3 mM Na ₂ HP0 · 70, 154m Na'Cl pH 7.4) solution] As refrigerated.

Add 0.1 μ / 50 solution of the synthesized biotin-labeled methylcytosine antibody to each streptavidin-coated PCR tube (total of 6 tubes) at a rate of 50 L and leave it at room temperature for about 1 hour. Immobilized in a tube. Then pipette the solution After adding 100 µm of washing buffer [0.05 Tween20-containing phosphate buffer (ImM K¾P0 ₄ , 3 mM Na ₂ HP0-7H ₂₀ , 154 mM NaCl pH7.)], Pipette the buffer. Removed. This operation was repeated twice more (this corresponds to the preparation of the immobilized methylated DNA antibody used in the measurement method of the present invention). · Baker's yeast strain X 2 1 8 0—1A in YPD medium (1¾ Yeast extract, 2% Peptone, 2% Glucose, pH 5.6-6.0) with turbidity of 0D _6Q . The cells were cultured until 0.6-1.0 and centrifuged at 10,000 ^ for 10 minutes to prepare lxlO ⁷ yeast cells. Yeast genomes were obtained from the prepared yeast cells using a general yeast genome preparation method as described in Methods in Yeast Genetics (Cold Spring Harbor Laboratory).

'Suspend the prepared yeast cells in Buffer A (1M sorbitol, 0.1M EDTA, pH 7.4), add 2-mercaptoethanol (final concentration 14 mM) and 100 U zymolase (10 mg / ml). Incubated with agitation at 30 ° C for 1 hour until clear. Protoplasts were collected by centrifugation at 550 for 10 minutes, suspended in buffer B (50 mM Tris-HC1, pH 7.4, 20 mM EDTA), and sodium dodecyl sulfate was added to 1% (w / γ). Thereafter, it was incubated at 65 ° C for 30 minutes. Subsequently, 5M CH ₃ C00K in a volume ratio of 2/5 was added and mixed, and after ice-cooling for 30 minutes, the supernatant was collected by centrifugation at 15,000 for 30 minutes. Add 1/10 volume ratio of 3M C¾C00Na and isopropanol to the collected supernatant, mix well, and centrifuge at 15,000 4 ° C for 30 minutes to rinse the precipitate with 70% ethanol. And recovered. After drying the precipitate, dissolve it in 1 ml of TE buffer (10 mM Tris-HCK pH 8.0, 1 mM EDTA), add RNase A (Sigma) to 40 g / ml, and add 37 ° C. Incubate at C for 1 hour, and then add 500 g / m 1 of proteinase (Sigma) and 1% sodium dodecyl sulfate to the mixture.

After adding (w / v), this was shaken at 55 for about 16 hours. After completion of the shaking, the mixture was subjected to phenol [saturated with 1M Tris-HCl (pH 8.0)] · kokuguchi form extraction. The ice layer was recovered, and NaCl was added to the residue so that the concentration was 0.5N, followed by ethanol precipitation, and the resulting precipitate was recovered. Genomic DNA was obtained by rinsing the collected precipitate with 70% ethanol.

From the obtained genomic DNA, P represented by the following SEQ ID NO: 53 and SEQ ID NO: 54 DNA fragments used as test samples (S, SEQ ID NO: 55, Genbank Accession No. NC) by performing PCR using oligonucleotide primers (PF 3 and PR 3) designed for CR and reaction conditions — The region corresponding to the base number 271743- 272083 of yeast chromosome VII shown in 001139 etc. was amplified.

<Oligonucleotide primers designed for PCR>

 PF3: 5'-AGGTGAGCTACGTGTGTTTGG-3 '(SEQ ID NO: 53)

 PR 3: 5'— AGACATGTGCTCACGTACGGT—3 ′ (SEQ ID NO: 54)

 <DNA fragment>.

· S: 5'-

 CCGTTTCCACACCGTACGTGAGCACATGTCT -3 '(SEQ ID NO: 55)

The PCR reaction solution is 10 ng genomic DNA, and 3 1 each of the oligonucleotide primer solution prepared to 5 M, 5 1 each 2 mM dNTP, 1 OX buffer (LOOmM Tris-HCl pH 8.3, 500 mM KC1, 15 mM MgCl ₂ , 0.01% Gelatin) 5 1 and heat-resistant DNA polymerase (ArapliTaq Gold, AB I) 511/1 mixed with 0.25 1 Then, sterilized ultrapure water was added thereto and the volume was 50 1. The reaction solution is kept at 95 ° C for 10 minutes, and then kept at 95 ° C for 20 seconds, then at 58 ° C for 30 seconds and then at 72 ° C for 30 seconds for one cycle for 40 cycles. PC R was done.

 After PCR, confirm amplification by 1.5% agarose gel electrophoresis,

DNA fragment S was purified using Wizard SV. Gel / PCR Kit (PR0MEGA) For a portion of the resulting DNA fragment solution, add Sssl methylase (NEB) 1 1, 10 x NEBuf fer 2 (NEB) 10 1, and S-adenosyl methionine (3.2 mM, NEB) 1 was mixed, and sterilized ultrapure water was added thereto to prepare a liquid volume of 100 1. Incubate the reaction solution at 37 ° C for 15-30 minutes. ■ Add 1 1 to S-adenosyl methionine (3.2 mM, NEB) and incubate at 37 ° C for 15-30 minutes. . This was purified by Wizard SV Gel / PCR Kit (PROMEGA). Further, this operation was repeated three times to obtain a methylated DNA fragment (MS, SEQ ID NO: 56). 'DNA fragment> (N stands for 5-methylcytosine)

 MS .: 5'-

AGGTGAGCTANGTGTGTTTGGGNGTNGTGCACTGGCTCACTTGTANGNGCAGAAATGGCAGCTTGTANGATTGGT GACCNGCCTTTTNGACACTGGACNGCTATGGANGTGGNGGNGGTGTGGNGGNGGCTCAATGACCTGTGGNGCCNG

: TTTGTGGNGTGNGATAGTNGAGCNGCCTGTCANGTGNGNGGCNGCCCTGCTCNGTTTGANGNGATGCATAGCATG

CNGTTTCCACACNGTANGTGAGCACATGTCT-3 ′ (SEQ ID NO: 56). For the obtained DNA fragment S, the following solution was prepared.

 Solution A 10pg / 10 L TE solution

 Solution B lpg / lO ^ L TE solution

 Solution C TE solution (negative control solution) The following solution was prepared for the obtained DNA fragment MS.

 Solution MA: lGpg / lO ^ L TE solution

 Solution MB: lpg / lO ^ L TE solution

Solution MC: TE solution (negative control solution) In addition, the negative DNA of the target DNA region S, consisting of the base sequence represented by SEQ ID NO: 57 A counter oligonucleotide C16-C25 consisting of the nucleotide sequence shown in SEQ ID NO: 58 to SEQ ID NO: 67 capable of binding to the strand by complementarity was synthesized, and a TE buffer solution having a concentration of 0 · '0 1 M was prepared. Prepared. '<Target DNA region> (methylcytosine is also represented by C)

 S ': 5'-

 CCGTTTCCACACCGTACGTGAGCACATGTCT -3 '(SEQ ID NO: 57)

 <Counter-oligonucleotide>

 C 16: 5'-AGGTGAGCTACGTGTGTTTGG-3 '(SEQ ID NO: 58)

 e 17: 5'-GCGTCGTGCACTGGCTCACTTGTACGCGCA-3 '(SEQ ID NO: 5 9)

 C18: 5'-CTTGTACGATTGGTGACCCGCCTTTTCGAC-3 '(SEQ ID NO: 60)

 C19: 5'-ACTGGACCGCTATGGACGTGGCGGCGGTGT-3 '(SEQ ID NO: 6 1)

 C 20: 5'- GGCGGCGGCTCAATGACCTGTGGCGCCCGT -3 '(SEQ ID NO: 6 2)

 C21: 5'-TTGTGGCGTGCGATAGTCGAGCCGCCTGTC-3 '(SEQ ID NO: 6 3)

 C 22: 5'-ACGTGCGCGGCCGCCCTGCTCCGTT-3 '(SEQ ID NO: 64)

 C23: 5'-TGACGCGATGCATAGCATGCGACCACCCAG-3 '(SEQ ID NO: 6 5)

 C 24: 5'-ACTGCTGACGCTATTGGTCACGTGGTTATG-3 '(SEQ ID NO: 6 6)

 C 25: 5′-CTGCTGTTGACTGCGGTGGCGTCCCGTTTC-3 ′ (SEQ ID NO: 67) The following treatment was performed on each of the DNA fragment S solution and the methylated DNA fragment MS solution.

In a PCR tube, the DNA fragment solution 10 prepared above, 10 L of the counter oligonucleotide solution prepared above, 5 L of buffer (330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM MgOAc ₂ , 5 mM Dithiothreitol) LOOmM M 1 ₂ solution 5 was added B SA solution 5 L of lmg / mL, a liquid volume of 50 zL further added with sterile ultrapure water to the mixture and mixed. The PCR tube was then heated at 95 for 10 minutes, quickly cooled to 70 ° C, and kept at that temperature for 10 minutes. Next, the mixture was cooled to 50 ° C., kept warm for 10 minutes, further kept at 37 ° C. for 10 minutes, and then returned to room temperature (this corresponds to the first step (A) of the measurement method of the present invention).

The above-prepared DNA fragment reaction solution 5 OL was added to the streptavidin-coated PCR tube on which the above-mentioned pyotin-labeled methylcytosine antibody was immobilized, and left at room temperature for 30 minutes. Then, remove the solution by pipetting, and add 100 L of wash buffer [0.05% Tween20-containing phosphate buffer (ImM KH ₂ P0 ₄ , 3 mM 'Na ₂ HPO-7H ₂ 0, 154 mM NaCl pH 7.4]] After the addition, the buffer was removed by pipetting. This operation was repeated twice more (this corresponds to the first step (B) of the measurement method of the present invention).

• Next, PCR was performed in the above PCR tube using each solution of oligonucleotide primers PF3 and PR3 consisting of the nucleotide sequences represented by SEQ ID NO: 53 and SEQ ID NO: 54 and the following reaction conditions: The methylated DNA in the target A region S ′ consisting of the base sequence represented by SEQ ID NO: 57 was amplified.

<Oligonucleotide primers designed for PCR>

 PF3: 5'-AGGTGAGCTACGTGTGTTTGG-3 '(SEQ ID NO: 53)

 PR 3: 5'-AGACATGTGCTCACGTACGGT-3 '(SEQ ID NO: 54)

 Target DNA region> (5-methylcytosine is also represented by C)

S ': 5'-

 TTTGTGGCGTGCGATAGTCGAGCCGCCTGTCACGTGCGCGGCCGCCCTGCTCCGTTTGACGCGATGCATAGCATG

CCGTTTCCACACCGTACGTGAGCACATGTCT -3 '(SEQ ID NO: 5 7) As PCR reaction solution, the DNA in the vertical shape, 3 L each of oligonucleotide primer solution prepared at 5 M, 5 L each 2 mM dNTP, buffer (lOOmM Tris-HCl pH 8.3, 500 mM KC1, Mix 5 L of 15 mM MgCl ₂ , 0.01% Gelatin), and heat resistant DNA polymerase (AmpliTaq Gold, manufactured by AB I) 5 UZ L with 0.25 and add sterilized ultrapure water to the solution. A volume of 50 L was used. The reaction solution is kept at 95 ° C for 10 minutes, and then kept at 95 ° C for 20 seconds, then at 58 ° C for 30 seconds and then at 72 ° C for 30 seconds for one cycle for 25 cycles. PCR was performed.

'After PCR, amplification was confirmed by 1.5% agarose gel electrophoresis. The results were as follows (corresponding to the third step of the measurement method of the present invention). The results are shown in FIG. Amplification was confirmed in the solutions M A,: and MB of the methylated DNA fragment MS. No amplification was confirmed with the negative control solution MC. Amplification was not confirmed in solutions A, B, and C of DNA fragment S that was not methylated.

 Based on the above, it is possible to select DN Α containing the methylated target DNA region with the immobilized methylcytosine, and to detect methylated DN A in an detectable amount. It was confirmed that the amplified DNA could be detected with higher sensitivity. Example 7

Using a commercially available methylcytosine antibody (Aviva Systems Biology) and a commercially available piotinization kit (Biotin Labeling Kit-NH ₂ , manufactured by Dojindo Laboratories) according to the method described in the catalog, Labeled. Piotin-labeled methylcytosine antibody obtained as a solution [Antibody 0.25 i / ll 0.1¾ BSA-containing phosphate buffer (lmM KH ₂ P0 ₄ , 3 mM Na ₂ HP0 • 7H ₂₀ , 15 solution NaCl pH 7.4) solution] Stored refrigerated.

Synthesized bi-streptavidin-coated PCR tubes (total of 6 tubes) A 0.1 μg / O solution of a phototin-labeled methylcytosine antibody was added at a rate of 50 L each, and allowed to stand at room temperature for about 1 hour and immobilized on a PCR tube. Then, the solution was removed by pipetting, and 100 L of washing buffer [0.05% Tween20-containing phosphate buffer (ImM KH ₂ P0 ₄ , 3 mM Na ₂ HP0-7H ₂₀ , 154 mM NaCl pH 7.4)] was added. Thereafter, the buffer was removed by pipetting. This operation was repeated twice more (this corresponds to the preparation of the immobilized methylated DNA antibody used in the measurement method of the present invention). Baker's yeast strain X2180-1A in YPD medium (1% Yeast extract, 2% Peptone, 2% Glucose, pH 5.6-6.0) with a turbidity of 0D ₆ . . Cultured to 0.6-1.0, 10, 000 by centrifugation for 10 minutes to prepare a yeast cell lxlO ^7. From the prepared yeast cells, the yeast genome was obtained using a general yeast genome preparation method as described in Methods' in Yeast Genetics (Cold Spring Harbor Laboratory).

Suspend prepared yeast cells in buffer A (1M sorbitol, 0.1M EDTA, pH 7.4), add 2_mercaptoethanol (final concentration 14mM) and 100ϋ zymolase (10mg / ml) ' Incubated with stirring at 30 ° C for 1 hour. Protoplasts were collected by centrifugation at 550 for 10 minutes, suspended in Buffer B (50 mM Tris-HC1, pH 7.4, 20 mM EDTA), and sodium dodecyl sulfate was added to 1% (w Zv). Thereafter, it was incubated at 65 ° C for 30 minutes. Subsequently, 2/5 volume of 5M C C00K was added and mixed, and after ice cooling for 30 minutes, the mixture was centrifuged at 15,000 for 30 minutes to collect the supernatant. Add 1/10 volume ratio of 3M CH ₃ C00Na and isopropanol to the collected supernatant, mix well, and centrifuge at 15,000 4 ° C for 30 minutes to rinse the precipitate with 70% ethanol. It was collected. After drying the precipitate, dissolve in 1 ml of TE buffer (10 mM Tris-HCK pH 8.0, 1 mM EDTA), add RNase A (Sigma) to 40 g / ml, and add 1 at 37. Incubate for 5 hours, and then add proteinase K (manufactured by Sigma) to 500 g / m 1 and sodium dodecyl sulfate to 1% (wZv), and shake this at 55 for about 16 hours. did. After the completion of the shaking, the mixture was subjected to phenol [saturated with 1M Tris-HCl (pH 8.0)] · black mouth form extraction treatment. The aqueous layer was recovered, and NaCl was added to this to 0.5 N, and then the resulting precipitate was recovered by ethanol precipitation. 70% of the collected precipitate Genomic DNA was obtained by rinsing with Nord.

 From the obtained genomic DNA, PCR was performed using the oligonucleotide primers (PF 4 and PR 4) and reaction conditions shown in SEQ ID NO: 68 and SEQ ID NO: 69 and designed for PCR. A DNA fragment (T, region corresponding to base number 384569-384685 of yeast chromosome VI I shown in SEQ ID NO: 70, Genbank Accession No. NC-001139, etc.) used as a test sample was amplified.

<Oligonucleotide primers designed for PCR>

 PF4: 5'-GGACCTGTGTTTGACGGGTAT-3 '(SEQ ID NO: 68)

'PR4: 5'-AGTACAGATCTGGCGTTCTCG-3' (SEQ ID NO: 69)

 <DNA fragment>

 T: 5 '-

CTTGAGCGCATGTGCCGTTTCCGAGAACGCCAGATCTGTACT -3 '(SEQ ID NO: 70)

The PCR reaction solution includes 10 ng of genomic DNA in a bowl, 3 1 each of oligonucleotide primer solutions prepared at 5 M, 5 1 each 2 mM dNTP, 1 OX buffer (lOOmM Tris- HCl H 8.3, 500 mM KC1, 15iM MgCl ₂ , 0.01% Gelatin) 5 1 and heat-resistant DNA polymerase (AmpliTaq Gold, manufactured by ΓΓ) 5U / 1 mixed with 0.25 1 Pure water was added to make the volume 50 1. The reaction solution is kept at 95 ° C for 10 minutes, and then kept at 95 ° C for 2 Q seconds, then at 58 ° C for 30 seconds, and then at 72 ° C for 30 seconds for one cycle for 40 cycles. PC R was done.

 After performing PCR, confirm amplification by 1.5% agarose gel electrophoresis,

DNA fragment T was purified using Wizard SV Gel / PCR Kit (PR0MEGA)

Sssl methylase (manufactured by NEB) was added to a part of the obtained DNA fragment solution. 1 1 and 10 x NEBuf fer 2 (NEB) 10 1 and S-adenosyl methionine (3.2 mM, NEB) 1 ^ 1 are mixed, and sterilized ultrapure water is added to the solution. 100′1 was prepared. The reaction solution was incubated at 37 ° C for 15-30 minutes, and S-adenosyl methionine (3.2 mM, manufactured by NEB) was added 1 and incubated at 37 ° C for 1'5-30 minutes. . This was purified by Wizard SV Gel / PCR Kit (PRO EGA). This operation was further repeated three times to obtain a methylated DNA fragment (MT, SEQ ID NO: 71).

<D N A fragment> (N represents 5-methylcytosine)

'MT: 5'-

CTTGAGNGCATGTGCNGTTTCNGAGAANGCCAGATCTGTACT -3 '(SEQ ID NO: 71)

^{: For the} obtained DNA fragment T, the following solutions were prepared.

 Solution A: lOpg / lO ^ L TE solution

 Solution B: lpg / lO ^ L TE solution

 Solution C: TE solution (negative control solution) The following solution was prepared for the obtained DNA fragment MT.

 Solution MA: lOpg / lO ^ L TE solution

 Solution MB: lpg / lO ^ L TE solution

Solution MC: TE solution (negative control solution) In addition, as shown in SEQ ID NO: 73 to SEQ ID NO: 76, which can bind more complementarily to the negative strand of the target DNA region T ′ consisting of the base sequence shown in SEQ ID NO: 72 Counter oligonucleotides C 26 to C 29 consisting of the base sequences described above were synthesized, and TE buffer solutions with respective concentrations of 0.01 jM were prepared. <Target DNA region> (Methylcytosine is also represented by C)

 T ': 5'-

CTTGAGCGCATGTGCCGTTTCCGAGAACGCCAGATCTGTACT-3 '(SEQ ID NO: 72)

'Counter oligonucleotides>

 C 26: 5'-GGACCTGTGTTTGACGGGTAT-3 '(SEQ ID NO: 73)

 C27: 5'-AACACTAAGTTGCGCAATTTGCTGT-3 '(SEQ ID NO: 74)

 C28: 5'-ATTGCGAAATCCGCCCGGACGATAT-3 '(SEQ ID NO: 75)

 C29: 5'-CACTCTTGAGCGCATGTGCCGTTTC-3 '(SEQ ID NO: 76)

,

 Each of the DNA fragment T solution and the DNA fragment MT solution was subjected to the following treatment.

In a PCR tube, 10 L of the DNA fragment solution prepared above, 10 L of the counter-oligonucleotide solution prepared above, and buffer (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, lOOmM MgOAc ₂ , 5 mM Di thiothrei tol) Add 5 L, lOOmM M 8 (: 1 ₂ solution 5 and 1 mg / mL BSA solution 5, and add sterile ultrapure water to the mixture to bring the volume to 50 ^ Li. The PCR tube was then heated at 95 ° C for 10 minutes, quickly cooled to 70 ° C, and kept at that temperature for 10 minutes, then cooled to 50 ° C and kept for 10 minutes. The mixture was further incubated at 37 ° C. for 10 minutes, and then returned to room temperature (this corresponds to the first step (A) of the measurement method of the present invention). Add 50 ml of the DNA fragment reaction solution prepared above to the PCR tube. It was left at room temperature for 30 minutes. Thereafter, the solution was removed by pipetting, 10 0 L washing buffer [0.05% Tween20-containing phosphate buffer _{(lmM KH 2 P0 4, 3mM} Na 2 HP0-7H 2 0, 154mM NaCl pH7. 4) After adding '], the buffer was removed by pipetting This operation was repeated twice more (this corresponds to the first step (B) of the measurement method of the present invention). Next, PCR is carried out in the above PCR tube using each solution of oligonucleotide primers PF 4 and PR 4 consisting of the nucleotide sequences represented by SEQ ID NO: 68 and SEQ ID NO: 69, and the reaction conditions described in the following: The methylated DNA in the target DNA region T ′ consisting of the base sequence represented by SEQ ID NO: 72 was amplified.

'

 <An oligonucleotide primer designed for PCR>

 PF4: 5'-GGACCTGTGTTTGACGGGTAT-3 '(SEQ ID NO: 68)

 PR4: 5'-AGTACAGATCTGGCGTTCTCG-3 '(SEQ ID NO: 69)

 <Target DN A region> (5-methylcytosine is also represented by C)

'Τ,: 5'-

CTTGAGCGCATGTGCCGTTTCCGAGAACGCCAGATCTGTACT -3 '(SEQ ID NO: 72)

:-The PCR reaction solution consists of a vertical DNA, 3 / L of each oligonucleotide primer solution prepared to 5 M, each 2 mM (11 ^-?? 5 ^, buffer solution

(lOOmM Tris-HCl pH 8.3, 500 mM KCK 15 mM MgCl ₂ , 0.01% Gelatin) 5 L, heat-resistant DNA polymerase (AmpliTaq Golc ^ AB I) 5U /// L 0 · 25 L The mixture was mixed and sterilized ultrapure water was added to make the volume 50 L. The reaction solution is kept at 95 ° C for 10 minutes, and then kept warm for 28 cycles at 95 ° C for 20 seconds, then at 58 ° C for 30 seconds and then at 72 ° C for 30 seconds. PCR was performed.

After PCR, amplification was confirmed by 1.5% agarose gel electrophoresis. The results were as follows (corresponding to the third step of the measurement method of the present invention). The results are shown in FIG. Amplification was confirmed in the solutions MA and MB of the methylated DNA fragment MT. No amplification was confirmed in the negative control solution MC. Amplification was not confirmed in solutions A, B, and C of DNA fragment T that was not methylated. · Based on the above, it is possible to select methylated DNA containing the target DNA region with the immobilized methylcytosine antibody, and to amplify the methylated DA to a detectable amount. It was confirmed that the amplified DNA could be detected with higher sensitivity.

'

 Example 8

Using a commercially available methylcytosine antibody (Aviva Systems Biology) and a commercially available piotination kit (Biotin Labeling Kit-NH ₂ , manufactured by Dojindo Laboratories) according to the method described in the catalog, Sign. Obtained Piotin-labeled methylcytosine body as a solution [Antibody 0.25 i / fl 0.1% BSA-containing phosphate buffer (lmM K P0 ₄ , 3 mM Na ₂ HP0 • 7H ₂₀ , 154 iM NaCl pH 7.4) solution] Stored refrigerated.

Add 0.1 g / 50 solution of the synthesized biotin-labeled methylcytosine antibody to each streptavidin-coated PCR tube (total 8 tubes) at a ratio of 50 iL, and leave it at room temperature for about 1 hour. Immobilized in an R tube. Then, the solution was removed by pipetting and 100 L of washing buffer [0.05% Tween20-containing phosphate buffer (ImM KH ₂ P0 ₄ , 3 mM Na ₂ HPO-7H ₂₀ , 154 mM NaCl pH 7.4)] was added. Thereafter, the buffer was removed by pipetting. This operation was repeated twice more (this corresponds to the preparation of the immobilized methylated DNA antibody used in the measurement method of the present invention). The baker's yeast strain X 2180-1A in YPD medium (1% Yeast extract, 2% Peptone, 2% Glucose, H 5.6-6.0) has a turbidity of 0D ₆ . . The cells were cultured until 0.6-1.0 and centrifuged at 10,000 for 10 minutes to prepare IxlO ⁷ yeast cells. Yeast genomes were obtained from the prepared yeast cells using a general yeast genome preparation method as described in Methods in Yeast Genetics (Cold Spring Harbor Laboratory).

Suspend the prepared yeast cells in Buffer A (1M sorbi! ^ L, 0.1M EDTA, pH 7.4) and add 2_mercaptoethanol (final concentration 14 mM) and 100 U zymolase (10 mg / ml). The solution was incubated at 30 ° C for 1 hour with stirring until the solution became clear. Protoplasts are collected by centrifugation at 550 for 10 minutes, suspended in buffer B (50 mM Tris-HCK pH 7.20 mM EDTA), and then 1% (w / v) and then incubated at 65 ° C. for 30 minutes. Subsequently, 5M CH ₃ C00K in a volume ratio of 2/5 was added and mixed, and after ice-cooling for 30 minutes, the supernatant was collected by centrifugation at 15,000 for 30 minutes. Add 1/10 volume ratio of 3M CH ₃ C00Na and isopropanol to the collected supernatant, mix well, and centrifuge at 15,000 4 ° C for 30 minutes. Rinse and collect. Allow precipitate to dry, then 1 ml TE buffer

(10 mM Tris-HCK pH 8.0, 1 mM EDTA), add RNase A (Sigma) to 40 g / ml, incubate at 37 for 1 hour, and then add proteinase to the mixture. K (Sigma) 500 g / m 1 and sodium dodecyl sulfate 1%

After adding to (wZv), this was shaken at 55 for about 16 hours. After shaking, the mixture was extracted with phenol [saturated with 1M Tris-HC1 (pH.8.0)]. The aqueous layer was recovered, and NaCl was added to 0.5 N, and then the resulting precipitate was recovered by ethanol precipitation. Genomic DNA was obtained by rinsing the collected precipitate with 70% ethanol. For a portion of the resulting yeast genomic DNA, 1 1 for Sssl methylase (NEB), 10 l for 10 x NEBuffer2 (NEB), 1 for S, adenosyl.methionine (3.2 mM, NEB) n 1 was mixed, and sterilized ultrapure water was added thereto to adjust the volume to 100 1 to prepare. The reaction solution was incubated at 37 ° C for 15-30 minutes, and S-adenosyl methionine (3.2 mM, manufactured by NEB) was added at 1 and incubated at 37 ° C for 15-30 minutes. . This was purified by Wizard SV Gel / PCR Kit (PR0MEGA). This operation was further repeated 3 times to obtain methylated yeast genomic DNA. For the obtained yeast genome DNA, the following solutions were prepared.

 Solution A: 10ng / 5; «L TE solution. Solution B: lng / 5 ^ L TE solution

 Solution C: 0.1 ng / 5 L TE solution

Solution D: TE solution (negative control solution). For the methylated yeast genome DNA obtained, the following solutions were prepared. Solution MA: 1 Ong / 5 L TE solution

 Solution MB: lng / 5 L TE solution

'Solution MC: 0.1ng / 5 ^ L TE solution

 Solution MD: TE solution (negative control solution) The following treatment was performed on each of the above-mentioned yeast genome DNA solution and methylated yeast genome DNA solution. .

'In a PCR tube, add 5 L of the genomic DNA solution prepared above, 5 U of the restriction enzyme X sp I, and 10 x buffer (200 mM Tris-HCl H 8.5, lOOmM MgCl ₂ , lOmM DithiothreitoK lOOOOmM KC1) 21 was mixed, and sterilized ultrapure water was added thereto to prepare a solution having a liquid volume of 20β1. The reaction solution was incubated at 37 ° C for 1 hour.

 DNA fragment S "(Genbank consisting of the base sequence represented by SEQ ID NO: 77

Accession No. NC—A counter consisting of the nucleotide sequence represented by SEQ ID NO: 58 to SEQ ID NO: 67 capable of binding by complementarity to the negative strand of yeast chromosome VII shown in 001139 etc.) One oligonucleotide C16-C25 was synthesized, and a TE buffer solution having a concentration of 0.01 iM was prepared.

<DNA fragment> (methylcytosine is also represented by C). ": 5'-

GTTTGTGGCGTGCGATAGTCGAGCCGCCTGTCACGTGCGCGGCCGCCCTGCTCCGTTTGACGCGATGCATAGCAT GCGACCACCCAGTAATCATACTGCTGACGCTATTGGTCACGTGGTTATGGCAGCTGCTGTTGACTGCGGTGGCGT CCCGTTTCCACACCGTACGTGAGCACATGTCTGGATTGC -3 'array number 2008/056524

75 counter oligonucleotides>

 C 16: 5'-AGGTGAGCTACGTGTGTTTGG-3 '(SEQ ID NO: 58)

 C 17: 5'- GCGTCGTGCACTGGCTCACTTGTACGCGCA-3, (SEQ ID NO: 5 9)

 C 1 8: 5 '-CTTGTACGATTGGTGACCCGCCTTTTCGAC-3, (SEQ ID NO: 60)

 C 19: 5'_ACTGGACCGCTATGGACGTGGCGGCGGTGT-3 '. (SEQ ID NO: 6 1)

 C 2 0: 5'- GGCGGCGGCTCAATGACCTGTGGCGCCCGT -3 '(SEQ ID NO: 6 2)

 C 2 1: 5'- TTGTGGCGTGCGATAGTCGAGCCGCCTGTC -3 '(SEQ ID NO: 6 3)

 C 2 2: 5'-ACGTGCGCGGCCGCCCTGCTCCGTT-3 '(SEQ ID NO: 64)

 C 2 3: 5 '-TGACGCGATGCATAGCATGCGACCACCCAG -3' (SEQ ID NO: 6 5)

'C 2 4: 5'-ACTGCTGACGCTATTGGTCACGTGGTTATG -3 '(SEQ ID NO: 6 6)

 C 2 5: 5'-CTGCTGTTGACTGCGGTGGCGTCCCGTTTC-3 '(SEQ ID NO: 6 7)

 Each reaction solution of the yeast genome DNA and methylated yeast genome DNA was subjected to the following treatment.

■-In a PCR tube, prepare the genomic DNA reaction solution 20 prepared above, 10 L of the counter-oligonucleotide solution prepared above, and buffer solution (330 mM Tris-Acetate H 7.9, 660 mM K0Ac, lOOmM MgOAc ₂ , 5 mM Di and thiothrei tol) 5. L, and Mg C 1 ₂ solution 5 L of lOOmM,. added B SA solution 5 L of lmg / mL, a liquid volume of 50 L further added with sterile ultrapure water to the mixture And mixed. The PCR tube was then heated at 95 ° C for 10 minutes, quickly cooled to 70 ° C, and kept at that temperature for 10 minutes. Next, the mixture was cooled to 50 ° C., kept for 10 minutes, further kept at 37 for 10 minutes, and then returned to room temperature (this corresponds to the first step (A) of the measurement method of the present invention).

50 L of the DNA fragment reaction solution prepared above was added to the streptavidin-coated PCR tube on which the pyotin-labeled methylcytosine antibody was immobilized, and left at room temperature for 30 minutes. Then, the solution was removed by pipetting, and 100 L of washing buffer [0.05% Tween20-containing phosphate buffer (ImM KH ₂ P0 ₄ , 3 mM Na ₂ HPO-7H ₂₀ , 154 mM NaCl pH 7.4)] was added. Thereafter, the buffer was removed by pipetting. This operation was repeated twice more (this corresponds to the first step (B) of the measurement method of the present invention). · Next, PCR is performed on the above PCR tube using each solution of oligonucleotide primers PF3 and PR3 consisting of the nucleotide sequences represented by SEQ ID NO: 53 and SEQ ID NO: 54 and the following reaction conditions: The methylated DNA in the target DNA region S ′ consisting of the base sequence represented by SEQ ID NO: 57 was amplified.

<Oligonucleotide primers designed for PCR>

PF3: 5'-AGGTGAGCTACGTGTGTTTGG-3 '(SEQ ID NO: 53)

PR 3: 5 '-AGACATGTGCTCACGTACGGT-3' (SEQ ID NO: 54)

<Target DN A region> (5-methylcytosine is also represented by C)

S ': 5'-

 CCGTTTCCACACCGTACGTGAGCACATGTCT -3 '(SEQ ID NO: 57)

The PCR reaction solution consists of 铸 -shaped DNA, 3 L each of oligonucleotide primer solution prepared at 5 xM, 5 L each 2 mM dNTP, buffer (lOOmM Tris-HCl pH 8.3, 500 mM KCK). Mix 15 mM MgCl ₂ , 0.01% Gelatin) 5 with heat resistant DNA polymerase (AmpliTaQ Gold, manufactured by AB I) 5 U; L 0.25 L and add sterilized ultrapure water to it. The liquid volume was 50. The reaction mixture was incubated at 95 ° C for 10 minutes, then PCR was carried out under the conditions of incubation for 30 cycles at 95 ° C for 20 seconds, then 58 ° C for 30 seconds, and 72 ° C for 30 seconds. Went.

After PCR, amplification was confirmed by 1.5% agarose gel electrophoresis. The results were as follows (this corresponds to the first step of the measurement method of the present invention). The results are shown in FIG. Amplification was confirmed in the methylated yeast genomic DNA solutions MA, MB and MC. No amplification was confirmed with the negative control solution MD. No amplification was observed in solutions A, B, C and D of the unmethylated yeast genome DNA.

 Based on the above, it is possible to select methylated DNA containing the desired DN A region with the immobilized methylcytosine antibody, and until the amount of methylated DN A is detectable. It was confirmed that the amplified DNA could be detected with higher sensitivity. 'Example 9

A commercially available methylcytosine antibody (manufactured by Aviva Systems Biology Co., Ltd.) was used in accordance with the method described in the catalog using a commercially available biotinylated kit (Biotin Labeling Kit-NH ₂ , manufactured by Dojindo Laboratories). Piotin labeled. The resulting Piochin labeled methylcytosine antibody: A solution [il 0.1¾ BSA-containing phosphate buffer one N antibody about _{0.25 i (ImM KH 2 P0 4} , 3mM Na 2 HP0 · 7Η 2 0, 154mM NaCl pH7.4) solution] As refrigerated.

Add a 0.1 g / 50 solution of the synthesized biotin-labeled methylcytosine antibody to each streptavidin-coated PCR tube (total of 8 tubes) at a rate of 50 L and leave it at room temperature for about 1 hour. Immobilized in an R tube. Then remove the solution by pipetting and add 100 L of wash buffer [0.05% Tween20-containing phosphate buffer (ImM KH ₂ P0 ₄ , 3 mM Na ₂ HP0-7H ₂₀ , 15 mM NaCl pH 7.4)] Then, the buffer was removed by pipetting. This operation was repeated twice more (this corresponds to the preparation of the immobilized methyl DNA antibody used in the measurement method of the present invention). The baker's yeast strain X 2180-1A in YPD medium (1% Yeast extract, 2% Peptone, 2% Glucose, pH 5.6-6.0) has a turbidity of 0D ₆ . . The cells were cultured until 0.6-1.0 and centrifuged at 10,000 ^ for 10 minutes to prepare lxlO ⁷ yeast cells. Yeast genomes were obtained from the prepared yeast cells using a general yeast genome preparation method as described in Methods in Yeast Genetics (Cold Spring Harbor Laboratory).

Prepared yeast cells in Buffer A (1M Sorbi! ^ 1l, 0.1M EDTA, pH 7.4) Suspended, 2-mercaptoethanol (final concentration 14 mM) and 100 U zymolase (10 mg / ml) were added and incubated with stirring at 30 ° C. for 1 hour until the solution became clear. Protoplasts were collected by centrifugation at 550 for 10 minutes, suspended in Buffer B (50 mM Tris-HCK pH 7.4, 20 mM EDTA), and sodium dodecyl sulfate was added to 1% (w'Ζν). Thereafter, it was incubated at 65 ° C for 30 minutes. Subsequently, 2/5 volume ratio of 5M C¾C00K was added and mixed. After ice-cooling for 30 minutes, the supernatant was collected by centrifugation at 15,000 for 30 minutes. Add 1/10 volume ratio of 3M CH ₃ C00Na and isopropanol to the collected supernatant, mix well, and centrifuge at 15,000 4 ° C for 30 minutes. Rinse the precipitate with 70% ethanol. And recovered. After drying the precipitate, dissolve it in 1 ml of TE buffer (10 mM Tris-HCK pH 8.0, 1 mM EDTA) and add RNase A (Sigma) to .40 g / ml. Incubate at ° C for 1 hour, and then add proteinase K (manufactured by Sigma) to the mixture to 500 g / m 1 and sodium dodecyl sulfate to 1% (wZv). Shake for about 16 hours at C. After shaking ■■, the mixture was extracted with phenol [saturated with 1M Tris-HCl (pH 8.0)] · clo mouthform. After recovering the aqueous layer and adding NaCl to 0.5N, the precipitate produced by ethanol precipitation was recovered. Genomic DNA was obtained by rinsing the collected precipitate with 70% ethanol. For some of the resulting yeast genomic DNA, 1 1 for Sssl methylase (NEB), 10 1 for 10 x NEBuffer2 (NEB), and 1 for S-adenosyl methionine (3.2 mM, NEB) 1 1 And sterilized ultrapure water was added thereto to make the volume 100 100. Incubate the reaction solution at 37 ° C for 15-30 minutes ^ ", and add S-adenosyl methionine (3.2 mM, manufactured by NEB) 1 1 at 37 ° C. -Incubated for 30 minutes Purified with Wizard SV Gel / PCR Kit (PR0MEGA) This operation was repeated three times to obtain methylated yeast genomic DNA. Prepared solution Solution: 101½ / 5 ^ TE solution

 Solution B: lng / 5 L TE solution

 Solution C: 0.1 ng / 5 L TE solution

 Solution D: TE solution (negative control solution)

'

 About the obtained methylated yeast genomic DNA, the following solutions were prepared.

 Solution MA: 10ng / 5 ^ L TE solution

 Solution MB: lng / 5 ^ L TE solution

 Solution MC: 0. lng ^ L TE solution

■ Solution MD: TE solution (negative control solution) The following treatment was applied to each of the above-mentioned yeast genome DNA solution and methylated yeast genome DNA solution.

--In a PCR tube, add 5 L of the genomic DNA solution prepared above, 5 U of the restriction enzyme X sp I, and 10 x buffer solution suitable for X sp I (200 mM Tris-HCl ρΗ 8.5, lOOm MgCl ₂ , ΙΟιΜ DithiothreitoKlOOOOmM KC1) was mixed, and sterilized ultrapure water was added thereto to adjust the volume to 201. The reaction was incubated at 37 for 1 hour.

 Complementary binding to the negative strand of DNA fragment T "(region corresponding to base numbers 384523-384766 of yeast chromosome VII shown in Genbank Accession No. NC—001139 etc.) consisting of the base sequence shown in SEQ ID NO: 78 The counter oligonucleotides C26 to C33 consisting of the nucleotide sequences represented by SEQ ID NO: 73 to SEQ ID NO: 76 and SEQ ID NO: 79 to SEQ ID NO: 82 were synthesized and dissolved in TE buffer at a concentration of 0.01 M respectively. DNA fragment> (methylcytosine is also represented by C)

T ": 5'- CCTGGCCACGCCTCTAATC -3 '(SEQ ID NO: 78)

 <Force untigo nucleotide>

'C 26: 5'-GGACCTGTGTTTGACGGGTAT -3' (SEQ ID NO: 73)

 C27: 5'-AACACTAAGTTGCGCAATTTGCTGT-3 '(SEQ ID NO: 74)

 C28: 5'-ATTGCGAAATCCGCCCGGACGATAT-3 '(SEQ ID NO: 75)

 C29: 5'-CACTCTTGAGCGCATGTGCCGTTTC-3 '(SEQ ID NO: 76)

 C30: 5'-AATACATTCCGAGGGCGC.CCGCACAAGGCC-3 '(SEQ ID NO: 79)

, C 31: 5'-GCGATCGCACACGAGGAGACA-3 '(SEQ ID NO: 80)

 C32: 5'-AGCGTCACGTGTTTTGCCATTTTGTACGAC-3 '(SEQ ID NO: 8 1)

 C 33: 5'-AAATGAACCGCCTGGCCACGCCTCTAATC-3 '(SEQ ID NO: 82)

--The following treatment was applied to each reaction solution of the yeast genome DNA and the methylated yeast genome DNA. .

In the PCR tube, 20 L of the genomic DNA reaction solution prepared above, 10 L of the counter-oligonucleotide solution prepared above, and buffer solution (330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM MgOAc ₂ , 5 mM Dithiothreitol) 5 and zL, and M 8_Rei 1 ₂ solution 5 LOOmM, was added BSA solution 5 L of lmg / mL, a liquid volume of 50 further added with sterile ultrapure water to the mixture and mixed. The PCR tube was then heated at 95 for 10 minutes, quickly cooled to 7 O, and kept at that temperature for 10 minutes. Next, it was cooled to 50, kept for 10 minutes, further kept at 37 ° C. for 10 minutes, and then returned to room temperature (this corresponds to the first step (A) of the measurement method of the present invention). 50 L of the DNA fragment reaction solution prepared above was added to the streptavidin-coated PCR tunib immobilized with the above-mentioned pyotin-labeled methylcytosine antibody and left at room temperature for 30 minutes. Then, the solution was removed by pipetting, and after adding 100 tih washing buffer [0.05¾ Tween20-containing phosphate buffer (ImM K P0 ₄ , 3 mM Na ₂ HP0'7H ₂₀ , 154 mM NaCl pH 7.)] Pipette the buffer Removed. This operation was repeated twice more (this corresponds to the first step (B) of the measurement method of the present invention). Next, PCR was performed on the above-mentioned PCR tube using each solution of oligonucleotide primers PF4 and PR4 consisting of the nucleotide sequences represented by SEQ ID NO: 68 and SEQ ID NO: 69 and the following reaction conditions: The methylated DNA in the target DNA region T ′ consisting of the base sequence represented by SEQ ID NO: 72 was amplified.

<Oligonucleotide primers designed for PCR>

, P F 4: 5'- GGACCTGTGTTTGACGGGTAT -3 ', (SEQ ID NO: 68)

 PR4: 5'-AGTACAGATCTGGCGTTCTCG-3 '(SEQ ID NO: 69)

 Target DNA region> (5-methylcytosine is also represented by C)

 T ': 5'-CTTGAGCGCATGTGCCGTTTCCGAGAACGCCAGATCTGTACT-3' (SEQ ID NO: 72)

The PCR reaction solution is a vertical DNA, 3 each of oligonucleotide primer solution prepared to 5 M, and 2 mM each. 5 ^, buffer solution (lOOm Tris-HCl H 8.3, 500 mM KCK 15 mM MgCl in 0.01% Gelat in), 5 L, heat resistant DNA polymerase (AmpliTaq Gold, manufactured by AB I) 5 UZ / L 0 . 25 L was mixed with sterilized ultrapure water to make the volume 50 iL. The reaction solution was incubated at 95 ° C for 10 minutes, then PCR was carried out under conditions of 31 cycles of incubation at 95 ° C for 20 seconds, then 58 for 30 seconds, and 72 ° C for 30 seconds. Went.

After PCR, amplification was confirmed by 1.5% slag gel electrophoresis. The results were as follows (corresponding to the third step of the measurement method of the present invention). The results are shown in FIG. Methylated yeast genomic DNA solution MA, MB and MC Then, amplification was confirmed. No amplification was confirmed with the negative control solution MD. No amplification was observed in solutions A, B, C and D of the unmethylated yeast genome DNA.

 From the above, it is possible to select methylated DNA containing the target DNA 'region with the immobilized methylcytosine antibody, and amplify the methylated DNA to a detectable amount. It was confirmed that the amplified DNA could be detected with higher sensitivity. Example 10

'' A commercially available methylcytosine rod (Aviva Systems Biology) was used according to the method described in the catalog using a commercially available pyotinylated kit (Biotin Labeling Kit-NH ₂ , manufactured by Dojindo Laboratories). Piotin labeled. The obtained piotin-labeled methylcytosine antibody was used as a solution [antibody 0.25 ug / i 0.1¾ BSA-containing phosphate buffer (lmM KH ₂ P0 ₄ , 3 mM Na ₂ HP0: · 70, 154 mM NaCl pH 7.4) solution] Stored refrigerated.

Add 50 g of each 0.1 g / 50 solution of the biotin-labeled methylcytosine antibody obtained by synthesis to a streptavidin-coated PCR tube (total of 8 tubes) and leave it at room temperature for about 1 hour. Immobilized in a tube. Then, the solution was removed by pipetting, and 100 L of washing buffer [0.05% Tween20-containing phosphate buffer (lmM KH ₂ P0 ₄ , 3 mM Na ₂ HPO-7H ₂₀ , 154iM NaCl pH 7.4)] was added. Thereafter, the buffer was removed by pipetting. This operation was repeated twice more (this corresponds to the preparation of the immobilized methylated DNA antibody used in the measurement method of the present invention). For the human blood-derived genomic DNA purchased from Clontech, the oligonucleotide primer (? 1 and? 1) designed for the PCR shown in SEQ ID NO: 28 and SEQ ID NO: 29 below and the reaction conditions The DNA fragment (X, a region corresponding to nucleotide numbers 25687390-25687775 shown in SEQ ID NO: 30, Genbank Accession No. NT 029419, etc.) was amplified by performing PCR using the resulting DNA. <Oligonucleotide primers designed for PCR>

 PF1: 5'-CTCAGCACCCAGGCGGCC-3 '(SEQ ID NO: 28)

 PR 1: 5'-CTGGCCAAACTGGAGATCGC-3 '(SEQ ID NO: 29)

 <DNA Fragment>

 X: 5'-

CTCAGCACCCAGGCGGCCGCGATCATGAGGCGCGAGCGGCGCGCGGGCTGTTGCAGAGTCTTGAGCGGGTGGCAC

 AGTTTGGCCAG (SEQ ID NO: 30)

The PCR reaction solution was prepared as 5 ng of genomic DNA in a vertical form and 5 M: each oligonucleotide primer solution 3 1, each 2 mM dNTP 5 1, 10 X buffer (lOOmM Tris - HC1 pH 8.3, 500mM KCK 15mM MgCl 2, 0.01%

 Gelatin) 5 1 and heat-resistant DNA polymerase (AmpliTaq Gold, manufactured by AB I) 5U / 1 are mixed with 0.25 1 and sterilized ultrapure water is added thereto to make the volume 50 1 We used the following. The reaction solution is kept at 95 ° C for 10 minutes, then at 95 ° C for 30 seconds, then at 6 1 for 30 seconds and further at 72 ° C for 45 seconds. PCR was performed under the condition of 40 cycles.

After PCR, the amplification was confirmed by 1.5% agarose gel electrophoresis, and DNA fragment X was purified using Wizard SV Gel / PCR Kit (PR0MEGA). Sssl methylase (NEB) 1 1, 10 x NEBuf fer 2 (NEB) 1 0 1, and S-adenosyl methionine (3.2 mM, NEB) 1 1 were mixed together. Pure water was added to adjust the liquid volume to 100 ^ 1. The reaction solution was incubated at 37 ° C for 15 to 30 minutes, and further S-adenosyl methionine (3.2 mM, manufactured by NEB) was added at 1 to 37 ° C. Incubated for 5-30 minutes. This was purified by Wizard SV Gel / PCR Kit (PROMEGA). Further, these operations were repeated 5 times to obtain a methylated DNA fragment (MX, SEQ ID NO: 31).

 <DN A fragment> (N indicates 5-methylcytosine)

 MX: 5'-

CTCAGCACCCAGGNGGCNGNGATCATGAGGNGNGAGNGGNGNGNGGGCTGTTGCAGAGTCTTGAGNGGGTGGCAC ACNGNGATGTAGNGGTNGGCTGTCATGACTACCAGCATGTAGGCNGANGCAAACATGCNGAACACCTGCAGGTGCTCTCACCANGNGGCACGNCCAGTNGGGGGCTGTGGG

NGGTGCAGAGCCAGCAGTANGCTGCTGTTGCCCAGCANGGCCACNGNGAAAGTCACNGCCAGCANGGNGATCTCC AGTTTGGCCAG -3 ′ (SEQ ID NO: 31) The following solution was prepared for the obtained DNA fragment X.

 -Solution A: lOOpg / lO ^ L TE solution

 Solution B: lOpg / 10 / zL TE solution

 Solution C: lpg / lO ^ L TE solution

 Solution D: TE solution (negative control solution) The following solution was prepared for the obtained DNA fragment MX.

 Solution MA: lOOpg / lO ^ L TE solution

 Solution MB: 10pg / 10 ^ L TE solution

 Solution MC: lpg / lO ^ L TE solution

Solution MD: TE solution (negative control solution) The nucleotide sequence represented by SEQ ID NO: 33 to SEQ ID NO: 44 that can bind to the negative strand of the target DNA region X ′ consisting of the nucleotide sequence represented by SEQ ID NO: 32 by complementarity Counter oligonucleotides C1 to C12 consisting of the following were synthesized, and a TE buffer solution having a concentration of 0.01 M was prepared. . <Target DNA region> (5-methylcytosine is also represented by C)

X ': 5'_

 AAGAATGCCACGGCCAGGTCGGCCAGGCTGAGGTGTCGGATGAAGAGGTGCATGCGGGACGTCTTGCGCGGCGTC

AGTTTGGCCAG -3 '(SEQ ID NO: 32)

<Ruiun Yuichi 1 year old Rigo nucleotide>

 C1: 5'-GCCACCGCGAAAGTCACCGCCAGCACGGCG-3 '(SEQ ID NO: 33)

 C2: 5'-GCCAGCAGTACGCTGCTGTTGCCCAGCACG-3 '(SEQ ID NO: 34)

 C3: 5'-CGGGACGTCTTGCGCGGCGTCCGGTGCAGA-3 '(SEQ ID NO: 35)

 C4: 5'-AGGCTGAGGTGTCGGATGAAGAGGTGCATG-3 '(SEQ ID NO: 36)

 C 5: 5 '-ACCTGGAAGAATGCCACGGCCAGGTCGGCC -3' (SEQ ID NO: 37)

 C 6: 5'-TAGGTGATGTCCCAGCACATTTGCGGCAGC -3 '(SEQ ID NO: 38)

 C7: 5'-CGGCACAGCCAGTCGGGGCCGCGGAAGCGG-3 '(SEQ ID NO: 39)

 C 8: 5 '-ATGCCGAACACCTGCAGGTGCTTCACCACG -3' (SEQ ID NO: 40)

 C9: 5'-ATGACTACCAGCATGTAGGCCGACGCAAAC-3 '(SEQ ID NO: 41)

 C 10: 5'-TGGCACACCGCGATGTAGCGGTCGGCTGTC -3 (SEQ ID NO: 42)

C 11: 5'- CGCGCGGGCTGTTGCAGAGTCTTGAGCGGG -3 (SEQ ID NO: 43)

C12: 5'-CAGGCGGCCGCGATCATGAGGCGCGAGCGG-3 (SEQ ID NO: 44)

In a PCR tube, prepare 10 L of the DNA fragment solution prepared above, 1 OL of the prepared counter oligonucleotide solution, buffer solution (330 niM Tris-Acetate H 7.9, 660 mM K0Ac, lOOmM MgOAc _z , 5 mM Di thiothrei tol) 5 ^ L , and Mg <1 ₂ solution 5 LOOmM, was added BSA solution 5 L of lmg / mL, a liquid volume of 50 L further added with sterile ultrapure water to the mixture and mixed. Then, this PCR tube Was heated at 95 for 10 minutes, quickly cooled to 70 ° C, and kept at that temperature for 10 minutes. Next, the mixture was cooled to 50 ° C., kept warm for 10 minutes, further kept at 37 ° C. for 10 minutes, and then returned to room temperature (this corresponds to the first step (A) of the measurement method of the present invention).

50 L of the DNA fragment reaction solution prepared above was added to the streptavidin-coated PCR tube on which the biotin-labeled methylcytosine antibody was immobilized, and left at room temperature for 30 minutes. Then, the solution was removed by pipetting, and after adding 100 0 wash buffer [0.05% Tween20-containing phosphate buffer (lmM KH ₂ P0 ₄ , 3 mM Na ₂ HPO'7H ₂₀ , 154 mM NaCl pH 7.4)] The buffer was removed by pipetting. This operation was repeated twice more (this corresponds to the second step of the measurement method of the present invention) ₀

Buffer solution (330mM Tris-Acetate H7.9, 660mM K0Ac, lOOmM Mg0Ac2, 5mM Di thiot rei tol) is added to 5 zL of the streptavidin-coated PCR tube on which the piotine-labeled methylcytosine antibody that has been treated as above is immobilized. And BSA (Bovine serum .. albumin lmg / ml) 5, methylation sensitive restriction enzyme Hhal 16 U, and sterilized ultrapure water added to the mixture to bring the volume to 50 L. The mixture was incubated at 37 for 1 hour. After removing the solution by pipetting, add 100 L of wash buffer [0.05% Tween20-containing phosphate buffer (lmM KH ₂ P0 ₄ , 3 mM Na ₂ HP0-7H ₂₀ , 154 mM NaCl pH 7.4)] The buffer was removed by pipetting. This operation was repeated twice more (this corresponds to the second step of the measurement method of the present invention). Next, PCR is performed on the above PCR tube using each solution of oligonucleotide primers PF1 and PR1 consisting of the nucleotide sequences represented by SEQ ID NO: 28 and SEQ ID NO: 29 and the following reaction conditions: The methylated DNA in the target DNA region X ′ ′ consisting of the base sequence represented by SEQ ID NO: 32 was amplified.

<Oligonucleotide primers designed for PCR>

PF1: 5'-CTCAGCACCCAGGCGGCC-3 '(SEQ ID NO: 28) PR1: 5'-CTGGCCAAACTGGAGATCGC-3 '(SEQ ID NO: 29)

 <Target DNA region> (5-methylcytosine is also represented by C)

X ': 5'-

 AAGAATGCCACGGCCAGGTCGGCCAGGCTGAGGTGTCGGATGAAGAGGTGCATGCGGGACGTCTTGCGCGGCGTC

AGTTTGGCCAG -3 '(SEQ ID NO: 32)

The reaction solution of PCR is a vertical DNA, .3 each of oligonucleotide primer solution prepared to 5 M, each 2 mM (11 ^ 5? 5), buffer (lOOmM Tris-HCl H 8.3, 500 mM KCI, 15 mM MgCl ₂ , 0.01% Gelatin) 5 and heat-resistant DNA polymerase (AmpliTaq Gold, manufactured by AB I) 0.25 L are mixed with sterilized ultrapure water. In addition, the solution volume was 50. The reaction solution was incubated at 95 ° C for 10 minutes, then at 95 ° C for 20 seconds, then at 61 ° C for 30 seconds and further at 72 ° C. PCR was performed under the condition of 25 cycles of heat insulation with one cycle of 30 seconds.

 • After PCR, amplification was confirmed by 1.5% agarose gel electrophoresis. The results were as follows (corresponding to the third step of the measurement method of the present invention). The results are shown in FIG. Amplification was confirmed in the methylated DNA fragment MX solutions MA, MB and MC. No amplification was confirmed with the negative control solution MD. Amplification was not confirmed in solutions A, B, C and D of unmethylated DNA fragment X.

From the above, it is possible to select methylated DNA containing the target DNA region with the immobilized methylcytosine antibody, and to amplify the methylated DNA to a detectable amount. Can detect amplified DNA with higher sensitivity It was confirmed. Example 11

A commercially available methylcytosine antibody (manufactured by Aviva Systems Biology) can be used according to the method described in the catalog using a commercially available pyotinylated kit (Biot in Labeling Kit-NH ₂ , manufactured by Dojindo Laboratories). Piotin labeled. Refrigerate the obtained piotin-labeled methylcytosine antibody as a solution [antibody 0.25 jLg / fl 0.1% BSA-containing phosphate buffer solution (ImM KH ₂ P0 ₄ , 3 mM Na ₂ HP0 • 70, 154 mM NaCl pH 7.4)] saved. .

To a streptavidin-coated P.CR tube (a total of 8 tubes), add a 0.1 g / 50 solution of the biotin-labeled methylcytosine antibody obtained by synthesis at a ratio of 50 / L, and then at room temperature for about 1 hour. It was allowed to stand and immobilized on a PCR tube. Then, the solution was removed by pipetting and 100 L of washing buffer [0.05% Tween20-containing phosphate buffer (ImM KH ₂ P0 ₄ , 3 mM Na ₂ HP0-7H ₂₀ , 154 mM NaCl pH 7.4)] was added. Thereafter, the buffer was removed by pipetting. This operation was repeated twice more (this corresponds to the preparation of the immobilized methylated DNA antibody used in the measurement method of the present invention). Baker's yeast strain X 2180—1? 0 medium (1% Yeast extract, 2% Peptone, 2% Glucose, H 5.6-6.0) with a turbidity of 0D ₆ . . The cells were cultured until 0.6-1.0 and centrifuged at 10,000 ^ for 10 minutes to prepare lxlO ⁷ yeast cells. Yeast genomes were obtained from the prepared yeast cells using a general yeast genome preparation method such as Methods in Yeast Genetics (Cold Spring Harbor Laboratory).

The prepared yeast cells are suspended in Buffer A (1M sorbitol, 0.1M EDTA, pH 7.4), 2-mercaptoethanol (final concentration 14 mM) and 100 U zymolase (10 mg / ml) are added, and the solution is clear. Incubated with stirring at 30 ° C for 1 hour. Protoplasts were collected by centrifugation at 550 for 10 minutes, suspended in buffer B (50 mM Tris-HCK pH 7.4, 20 mM EDTA), sodium dodecyl sulfate was added to 1% (w Zv), and Incubated for 30 minutes at 65 ° C. Subsequently, 2/5 volume ratio of 5M C¾C00K was added and mixed, and after ice cooling for 30 minutes, the mixture was centrifuged at 15,000 g for 30 minutes to collect the supernatant. 1/10 volume ratio of 3M C¾C00Na and isoprop The precipitate obtained by centrifuging at 15,000 4 ° C for 30 minutes was rinsed with 70% ethanol and collected. After drying the precipitate, dissolve in 1 ml of TE buffer (10 mM Tris-HCK pH 8.0, 1 mM EDTA), add RNase A (Sigma) to 40 g / ml and add 37 ° C. Incubate for 1 hour, then add proteinase K (manufactured by Sigma) to the mixture to 500 gZm 1 and sodium dodecyl sulfate to 1% (w / v), and add this at about 55 ° C. Shake for 16 hours. After completion of the shaking, the mixture was subjected to extraction with phenol [1M Tris-HC1 (pH 8.0): 1 · black mouth form extraction. The aqueous layer was recovered, and NaCl was added to this to 0.5 N, and then the resulting precipitate was recovered by ethanol precipitation. The collected precipitate was rinsed with 70% ethanol and genol to obtain genomic DNA.

From the obtained genomic DNA, PCR was performed using oligonucleotide primers (PF 3 and PR 3) designed for the PCR shown in SEQ ID NO: 53 and SEQ ID NO: 54 below and reaction conditions. DNA used as a test sample _: Fragment (S, SEQ ID NO _: 55, region corresponding to nucleotide numbers 271743- 272083 of yeast chromosome VII shown in Genbank Accession No. NC-001139, etc.) was amplified.

<An oligonucleotide primer designed for PCR>

 PF3: 5'-AGGTGAGCTACGTGTGTTTGG-3 '(SEQ ID NO: 53)

 PR 3: 5'-AGACATGTGCTCACGTACGGT-3 '(SEQ ID NO: 54)

 <DNA fragment>

S: 5 '-

 CGACCACCCAGTAATCATACTGCTGACGCTATTGGTCACGTGGTTATGGCAGCTGCTGTTGACTGCGGTGGCGTC CCGTTTCCACACCGTACGTGAGCACATGTCT -3 '(SEQ ID NO: 5 5)

As a PCR reaction solution, 10 ng of genomic DNA, which is a cocoon type, was prepared to 5 M. Each oligonucleotide primer solution 3 1, each 2 mM dNTP 5 U, 1 OX buffer (lOOmM Tris-HCl H 8.3, 500 mM CK 15m MgCl ₂ , 0.01% Gelatin) 5 1, heat resistant DNA polymerase Z (AmpliTaq Gold, manufactured by AB I) 5U / 1 was mixed with 0 .. 25 1 and sterilized ultrapure water was added to make the volume 501, which was used. The reaction solution was kept at 95 ° C for 10 minutes, then at 95: for 20 seconds, then at 58 ° C for 30 seconds, and further at 72 ° C for 30 seconds for one cycle for 40 cycles. PCR was performed.

 After performing PCR, amplification was confirmed by 1.5% agarose gel electrophoresis, and DNA fragment S was purified using Wizard SV Gel / PCR Kit (PR0MEGA).

 For a portion of the resulting DNA fragment solution, add Sssl methylase (NEB) 1 1, 10 x NEBuf fer 2 (NEB) 10 1, and S-adenosyl methionine (3.2 mM, NEB) 1 n 1 was mixed, and sterilized ultrapure water was added thereto to prepare a liquid volume of 1001. The reaction solution was incubated at 37 ° C for 15-30 minutes, and further S-adenosyl methionine (3. mM, manufactured by NEB) was added, and incubated at 37 ° C for 15-30 minutes. . This was purified by Wizard SV Gel / PCR Kit (PR0MEGA). Further, this operation was repeated three times to obtain a methylated DNA fragment (MS, SEQ ID NO: 56). <DNA fragment> (N indicates 5-methylcytosine)

 MS: 5'-

For AGGTGAGCTANGTGTGTTTGGGNGTNGTGCACTGGCTCACTTGTANGNGCAGAAATGGCAGCTTGTANGATTGGT GACCNGCCTTTTNGACACTGGACNGCTATGGANGTGGNGGNGGTGTGGNGGNGGCTCAATGACCTGTGGNGCCNG TTTGTGGNGTGNGATAGTNGAGCNGCCTGTCANGTGNGNGGCNGCCCTGCTCNGTTTGANGNGATGCATAGCATG NGACCACCCAGTAATCATACTGCTGANGCTATTGGTCANGTGGTTATGGCAGCTGCTGTTGACTGNGGTGGNGTC CNGTTTCCACACNGTANGTGAGCACATGTCT -3 '(SEQ ID NO: 56) resulting DN A fragment S, the following solutions were prepared. Solution A lOOpg / lO ^ L TE solution

 Solution B lOpg / lO ^ L TE solution

 Solution C lpg / 10 / iL TE solution

 solution. TE solution (negative control solution) The following solution was prepared for the obtained DNA fragment MS.

 Solution MA! OOpg / lO L TE solution

 Solution MB lOpg / 10 L TE solution

 Solution MC lpg / lO ^ L Τβ solution

 Solution MD TE solution (negative control solution) In addition, the nucleotide sequence represented by SEQ ID NO: 58 to SEQ ID NO: 67 capable of binding by complementarity to the negative strand of the target DNA region S ′ consisting of the nucleotide sequence represented by SEQ ID NO: 57 Counter oligonucleotides C16 to C25 consisting of the following were synthesized, and a TE buffer solution having a concentration of 0.01 M was prepared.

<Target DNA region> (Methylcytosine is also represented by C)

 S ': 5'-

 CCGTTTCCACACCGTACGTGAGCACATGTCT -3 '(SEQ ID NO: 57)

 <Counter oligonucleotide>

 C 16: 5'-AGGTGAGCTACGTGTGTTTGG-3 '(SEQ ID NO: 58)

 C 17: 5'-GCGTCGTGCACTGGCTCACTTGTACGCGCA-3 '(SEQ ID NO: 59)

 C18: 5'-CTTGTACGATTGGTGACCCGCCTTTTCGAC-3 '(SEQ ID NO: 60)

C 19: 5'-ACTGGACCGCTATGGACGTGGCGGCGGTGT-3 '(SEQ ID NO: 61) C 20: 5'- GGCGGCGGCTCAATGACCTGTGGCGCCCGT -3 '(SEQ ID NO: 62)

C21: 5'-TTGTGGCGTGCGATAGTCGAGCCGCCTGTC-3 '(SEQ ID NO: 63)

 C 22: 5'-ACGTGCGCGGCCGCCCTGCTCCGTT-3 '(SEQ ID NO: 64)

 C23: 5'-TGACGCGATGCATAGCATGCGACCACCCAG-3 '(SEQ ID NO: 65)

 C 24: 5 '-ACTGCTGACGCTATTGGTCACGTGGTTATG-3' (SEQ ID NO: 66)

 C 25: 5′-CTGCTGTTGACTGCGGTGGCGTCCCGTTTC-3 ′ (SEQ ID NO: 67) The following treatment was applied to each of the DNA fragment S solution and the methylated DNA fragment MS solution.

, 10 L of the DNA fragment solution prepared above, 10 L of the counter oligonucleotide solution prepared above, and buffer (330 mM Tris-Acetate H 7.9, 660 mM K0Ac, lOOmM MgOAc ₂ , 5 mM Di thiothrei tol ) 5, lOOmM M80 1 ₂ solution 5 // and lmg / mL of BSA solution 5 L were added, and the mixture was mixed: sterilized ultrapure water was added to the product to make the volume 50, and mixed. . The PCR tube was then heated at 95 ° C for 10 minutes, quickly cooled to 70 ° C, and kept at that temperature for 10 minutes. Next, the mixture was cooled to 50 ° C., kept warm for 10 minutes, further kept at 37 ° C. for 10 minutes, and then returned to room temperature (this corresponds to the first step (A) of the measurement method of the present invention). 50 L of the DNA fragment reaction solution prepared above was added to the streptavidin-coated PCR tube on which the piotin-labeled methylcytosine antibody had been immobilized, and left at room temperature for 30 minutes. Then, the solution was removed by pipetting, and 100 L of washing buffer [0.05 Tween20-containing phosphate buffer (ImM KH ₂ P0 ₄ , 3 mM Na ₂ HP0-7H ₂₀ , 154 mM NaCl pH 7.4)] was added. Thereafter, the buffer was removed by pipetting. This operation was repeated twice more (this corresponds to the first step (B) of the measurement method of the present invention).

To the above-mentioned process a streptavidin coated PC R tube Piochin labeled methylcytosine antibody has been immobilized subjected, buffer (330m Tris-Acetate pH 7.9, 660mM K0Ac, lOOmM Mg0Ac 2, 5mM Di thiothrei tol) a 5 L And 5 of BSA (Bovine serum albumin lmg / ml) and 16 U of methylation sensitive restriction enzyme Hhal. Into each mixture, sterilized ultrapure water was added to adjust the volume to 50 L. Each mixture was incubated at 37 for 1 hour. Then, remove the solution by pipetting, add 100 L wash buffer [0.05% Tween20-containing phosphate buffer (ImM KH ₂ P0 ₄ , 3 mM Na ₂ HP0-7H ₂₀ , 154 mM NaCl pH7.)] The buffer was removed by pipetting. This operation was repeated twice more (this corresponds to the second step of the measurement method of the present invention). Next, PCR is performed on the above PCR tube using each solution of oligonucleotide primers PF3 and PR3 consisting of the nucleotide sequences represented by SEQ ID NO: 53 and SEQ ID NO: 54 and the following reaction conditions: The methylated DNA in the target DNA region S ′ consisting of the base sequence represented by SEQ ID NO: 57 was amplified.

<Oligonucleotide primer designed for PCR>

: P F 3: 5 '-AGGTGAGCTACGTGTGTTTGG -3' (SEQ ID NO: 53)

 P R 3: 5 '-AGACATGTGCTCACGTACGGT -3' (SEQ ID NO: 54)

 <Target DNA region> (5-methylcytosine is also represented by C)

 S ': 5'-

 CCGTTTCCACACCGTACGTGAGCACATGTCT -3 '(SEQ ID NO: 57)

The PCR reaction solution consists of と す る -shaped DNA, 3 L each of oligonucleotide primer solution prepared to 5 M, 5 L each 2 mM dNTP, buffer (lOOmM Tris-HCl pH 8.3, 500 mM KCK). Mix 5 L of 15 mM MgCl ₂ and 0.01% Gelatin) and heat-resistant DNA polymerase (AmpliTaq Gold, manufactured by AB I) 5UZ L with 0.25 iL and add sterilized ultrapure water to the solution. A volume of 50 L was used. The reaction The solution is incubated at 95 ° C for 10 minutes, then at 95 ° C for 20 seconds and then at 58 ° C for 30 seconds for an additional 72 seconds. PCR was carried out under conditions of 25 cycles of incubation at C for 30 seconds.

 After PCR, amplification was confirmed by 1.5% gel mouth gel electrophoresis. The results were as follows (corresponding to the third step of the measurement method of the present invention). The results are shown in FIG. Amplification was confirmed in the methylated DNA fragment MS solutions MA, MB and MC. No amplification was confirmed with the negative control solution MD. Amplification was not confirmed in solutions A,, B, C and D of DNA fragment S which was not methylated.

 From the above, it is possible to select methylated DNA containing the target DNA region with the immobilized methylcytosine antibody, and to amplify the methylated DNA to a detectable amount. It was confirmed that the amplified DNA could be detected with higher sensitivity. Example 12

A commercially available methylcytosine antibody (manufactured by Aviva Systems Biology) was labeled with a biotin labeled biotin kit (Biot in Labeling KU_N, manufactured by Dojindo Laboratories) according to the method described in the catalog. did. Refrigerate the obtained biotin-labeled methylcytosine solution as a solution [antibody 0.25 ug / iL 0.1¾ BSA-containing phosphate buffer solution (ImM K P0 ₄ , 3 M Na ₂ HP0 • 7H ₂₀ , 154 mM NaCl pH 7.)] saved.

Add 0.1 _g / 50 solution of the synthesized biotin-labeled methylcytosine antibody to the streptavidin-coated PCR tubes (total of 8 tubes) at a ratio of 50 each, and leave it at room temperature for about 1 hour. Immobilized in a tube. Then remove the solution by pipetting and remove 100 L 'of wash buffer [0.05% Tween20-containing phosphate buffer (ImM KH ₂ P0 ₄ , 3 mM Na ₂ HP0'7H ₂₀ , 154 mM NaCl pH 7.4)]. After the addition, the buffer was removed by pipetting. This operation was repeated twice more (this corresponds to the preparation of the immobilized methylated DNA antibody used in the measurement method of the present invention). Baker's yeast strain X 2180-1 A in YPD medium (1% Yeast extract, 2% Peptone, 2% Glucose, pH 5.6-6.0) with a turbidity of 0D ₆ . . The cells were cultured until 0.6-1.0 and centrifuged at 10,000 ^ for 10 minutes to prepare lxlO ⁷ yeast cells. Yeast genomes were obtained from the prepared yeast cells using a general yeast genome preparation method as described in Methods in Yeast Genetics (Cold Spring Harbor Laboratory).

The prepared yeast cells are suspended in buffer A (1M sorbitol, 0.1M EDTA, pH 7.4), and 2-mercaptoethanol (final concentration 14 mM) and 100 U zymolase (10 mg / ml) are added to clear the solution. Incubated with stirring at 30 ° C for 1 hour. Centrifuge at 550 for 10 minutes, collect the protoplasts, suspend in buffer B (50 mM Tris-, HC1, pH 7.4, 20 mM EDTA), and add sodium dodecyl sulfate to 1% (w Zv). After the addition, it was incubated at 65 for 30 minutes. Subsequently, 5M CH ₃ C00K in a volume ratio of 2/5 was added and mixed, and after ice-cooling for 30 minutes, the supernatant was collected by centrifugation at 15,000 for 30 minutes. To the collected supernatant, add 1/10 volume ratio of 3M CH ₃ C00Na and an equal amount of isopropanol to the mixture, mix well, and centrifuge at 15,000 4 ° C for 30 minutes to precipitate the precipitate with 70% ethanol. Rinse and collect. After drying the precipitate, dissolve in 1 ml of TE buffer (10 mM Tris-HCK pH 8.0, 1 mM EDTA), add RNase A (Sigma) to 40 g / ml and add 37 ° C. Incubate for 1 hour, then add proteinase K (manufactured by Sigma) to the mixture to 500 gZm 1 and sodium dodecyl sulfate to 1% (w / v), and add this at about 55 ° C. Shake for 16 hours. After completion of the shaking, the mixture was subjected to phenol [saturated with 1M Tris-HCl (pH 8.0)] · Kroguchi form extraction. The aqueous layer was recovered, and NaCl was added to this to 0.5 N, and then the resulting precipitate was recovered by ethanol precipitation. Genomic DNA was obtained by rinsing the collected precipitate with 70% ethanol.

The obtained genomic DNA was subjected to PCR using oligonucleotide primers (PF 4 and PR 4) designed for the PCR shown in SEQ ID NO: 68 and SEQ ID NO: 69 below and reaction conditions. A DNA fragment (T, region corresponding to nucleotide number 384569-384685 of yeast chromosome VII shown in SEQ ID NO: 70, Genbank Accession No. NC-001139, etc.) used as a test sample was amplified. . 56524

 96

<Oligonucleotide primers designed for PCR>

 PF4: 5'-GGACCTGTGTTTGACGGGTAT-3 '(SEQ ID NO: 68)

 PR4: 5'-AGTACAGATCTGGCGTTCTCG-3 '(SEQ ID NO: 69)

 <DNA fragment>

 T: 5'-

CTTGAGCGCATGTGCCGTTTCCGAGAACGCCAGATCTGTACT-3 '(SEQ ID NO: 70), the reaction solution for PCR is 10 ng of genomic DNA, and 3 1 each of the oligonucleotide primer solution prepared in 5 M, and each 2 mM dNTP 5 1 10X buffer (lOOmM Tris_HCl ρΗ 8.3, 500m KCK 15mM MgCl ₂ , 0.01% Gelatin) 5 1 and heat-resistant DNA polymerase (AmpliTaq Gold, AB I) 5U / 1 0.25 1 And sterilized ultrapure water was added to make the volume 50 1. The reaction was incubated at 95 ° C for 10 minutes, then PCR was carried out under conditions of 40 cycles of incubation at 95 ° C for 20 seconds, then at 58 for 30 seconds and then at 72 ° C for 30 seconds. I went.

 After conducting PCR, amplification was confirmed by 1.5% slag gel electrophoresis, and DNA fragment T was purified by Wizard SV Gel / PCR Kit (PR0MEGA).

For a portion of the resulting DNA fragment solution, add 1 〃 1 of Sssl methylase (NEB), 10 1 of 10 x NEBufer 2 (NEB), and S_adenosyl methionine (3.2 mM, NEB) 1 n 1 was mixed, and sterilized ultrapure water was added thereto to prepare a liquid volume of 1001. The reaction solution was incubated at 37 for 15-30 minutes, and S-adenosyl methionine '(3.2 mM, NEB) 1 (added 1 and incubated at 37 for 15-30 minutes. Purification was performed using Wizard SV Gel / PCR Kit (PR0MEGA), and this operation was repeated three times to obtain a methylated DNA fragment (MT, SEQ ID NO: 71). <DNA fragment> (N indicates 5-methylcytosine)

 MT: 5'-

GGACCTGTGTTTGANGGGTATAACACTAAGTTGNGCAATTTGCTGTATTGNGAAATCNGCCNGGANGATATCACT

CTTGAGNGCATGTGCNGTTTCNGAGAANGCCAGATCTGTACT-3 ′ (SEQ ID NO: 71) The following solution was prepared for the obtained DNA fragment T.

 Solution A: lOOpg / lO ^ L TE solution

 Solution B: lOpg / lO ^ L TE solution

, Solution C: lpg / lO ^ L TE solution

 Solution D: TE solution (negative control solution) The following solution was prepared for the obtained DNA fragment MT.

 Solution MA: lOOpg / lO L TE solution

 Solution MB: 10pg / 10 L TE solution

 Solution MC: lpg / lO ^ L TE solution

 Solution MD: TE solution (negative control solution) In addition, bases represented by SEQ ID NO: 73 to SEQ ID NO: 76 that can bind to the negative strand of the target DNA region T ′ consisting of the base sequence represented by SEQ ID NO: 72 by complementarity A counter oligonucleotide C 26 to C 29 consisting of the sequence was synthesized, and a TE buffer solution having a concentration of 0.01 M was prepared.

<Target DNA region> (Methylcytosine is also represented by C)

 T,: 5'-,

 GGACCTGTGTTTGACGGGTATAACACTAAGTTGCGCAATTTGCTGTATTGCGAAATCCGCCCGGACGATATCACT

CTTGAGCGCATGTGCCGTTTCCGAGAACGCCAGATCTGTACT-3 '(SEQ ID NO: 72)

<Counter-oligonucleotide>, C 26: 5'-GGACCTGTGTTTGACGGGTAT-3 '(SEQ ID NO: 73)

 C27: 5'-AACACTAAGTTGCGCAATTTGCTGT-3 '(SEQ ID NO: 74)

 C 28: 5'-ATTGCGAAATCCGCCCGGACGATAT -3 '(SEQ ID NO: 75)

 C 29: 5 '-CACTCTTGAGCGCATGTGCCGTTTC -3' (SEQ ID NO: 76)

·

 The following treatment was applied to each of the above-mentioned solution of DNA fragment T and the solution of methylated DNA fragment MT.

In a PCR tube, add 10 L of the DNA fragment solution prepared above, 10 L of the counter oligonucleotide solution prepared above, and buffer solution (330 mM Tris-Acetate, ρΗ 7.9, 660 mM K0Ac, lOOmM MgOAc ₂ , 5 mM Di thiothrei and tol), and M 8_Rei 1 ₂ solution 5 LOOmM, was added BSA solution 5 of lmg / mL, a liquid volume of 50 further added with sterile ultrapure water to the mixture and mixed. The PCR tube was then heated at 95 ° C for 10 minutes, quickly cooled to 70 ° C, and kept at that temperature for 10 minutes. Next, the mixture was cooled to 50 ° C., kept warm for 10 minutes, further kept at 37 ° C. for 10 minutes, and then returned to room temperature (this corresponds to the first step (A) of the measurement method of the present invention). The above-prepared DNA fragment reaction solution 50 was added to a streptavidin-coated PCR tube on which the above-mentioned pyotin-labeled methylcytosine antibody was immobilized, and left at room temperature for 30 minutes. Then, remove the solution by pipetting and add 100 L of wash buffer [0.05% Tween20-containing phosphate buffer (ImM KH ₂ P0 ₄ , 3 mM Na ₂ HP0'7H ₂₀ , 15 mM NaCl pH 7.4)] Then, the buffer was removed by pipetting. This operation was repeated twice more (this corresponds to the first step (B) of the measurement method of the present invention).

A buffer solution (330niM Tris-Acetate H7.9, 660mM K0Ac, lOOmM Mg0Ac2, 5mM Di thi & threi tol) was added to 5 L of a streptavidin-coated PCR tube on which the piotine-labeled methylcytosine antibody subjected to the above treatment was immobilized. Add 5 ^ L of BSA (Bovine serum albumin lmg / ml), 16 U of methylation-sensitive restriction enzyme Hhal, and add sterile ultrapure water to the mixture to make the volume 50 L. Was incubated at 37 for 1 hour. Then remove the solution by pipetting, After adding 100 L of washing buffer [0.05% Tween20-containing phosphate buffer (ImM KH ₂ P0 ₄ , 3 mM Na ₂ HPO-7H ₂₀ 154 mM NaCl pH 7.4)], the buffer was pipetted. Removed. This operation was repeated twice more (this corresponds to the second step of the measurement method of the present invention).

.

 Next, perform PCR in the above PCR tube using each solution of oligonucleotide primers PF4 and PR consisting of the nucleotide sequences shown in SEQ ID NO: 68 and SEQ ID NO: 69 and the following reaction conditions: Thus, methylated DNA in the target DNA region T ′ consisting of the base sequence represented by SEQ ID NO: 72 was amplified.

,

 <Oligonucleotide primers designed for PCR>

 PF4: 5'-GGACCTGTGTTTGACGGGTAT-3 '(SEQ ID NO: 68)

 PR4: 5'-AGTACAGATCTGGCGTTCTCG-3 '(SEQ ID NO: 6.9)

 Target DNA region> (5-methylcytosine is also represented by C)

 T ': 5'-

CTTGAGCGCATGTGCCGTTTCCGAGAACGCCAGATCTGTACT -3 '(SEQ ID NO: 72)

The reaction solution of PCR is the vertical DNA, 3 L of each oligonucleotide primer solution prepared to 5 M, 57 mM of each 2 mM dNTP, buffer (lOOmM Tris-HCl pH 8.3, 500 mM) Mix 5 L of KC1, 15 mM MgCl ₂ , 0.01% Gelatin) with 0.2 5 L of heat-resistant DNA polymerase (AmpliTaq Gold, AB I) 5 U / L, and add sterile ultrapure water to this. In addition, the liquid volume was 50 L. The reaction solution is incubated at 95 ° C for 10 minutes, then at 95 for 20 seconds, then at 58 ° C for 30 seconds, and at 72, 30 seconds is one cycle. PCR was performed under the condition of performing 28 cycles. ''

After PCR, amplification was confirmed by 1.5% agarose gel electrophoresis. The results were as follows (this corresponds to the third step of the measurement method of the present invention). The results are shown in FIG. Amplification was confirmed in the methylated DNA fragments MT solutions MA, MB and MC. No amplification was confirmed with the negative control solution MD. Amplification was not confirmed in solutions A, B, C and D of DNA methylation fragment T that was not methylated.

· From the above, it is possible to select methylated DNA containing the target DNA region with the immobilized methyl succin antibody, and the amount of methylated DNA can be detected. It was confirmed that the amplified DNA could be detected with higher sensitivity. Example 13

Using a commercially available methylcytosine antibody (Aviva Systems Biology) and a commercially available piotinization kit (Biot in Labeling Kit-NH ₂ manufactured by Dojindo Laboratories), according to the method described in the catalog, Labeled. The resulting Piochin labeled methylcytosine antibody: A solution Antibody about 0.25 ug / {l 0.1¾ BSA-containing phosphate buffer _{(ImM KH 2 P0 4, 3mM} Na 2 HP0 · 7Η 2 0, 154mM NaCl pH7.4) solution] As refrigerated.

Add 50 μL each of the 0.1 μg / ^ solution of the synthesized pitin-labeled methylcytosine antibody to the streptavidin-coated PCR tubes (6 tubes in total), and leave it at room temperature for about 1 hour. Immobilized in a tube. Then, remove the solution by pipetting and add 100 L of wash buffer [0.05% Tween20-containing phosphate buffer (ImM KH ₂ P0 ₄ , 3m Na ₂ HPO-7H ₂₀ , 15 mM NaCl pH 7.)] The buffer was removed by pipetting. This operation was repeated twice more (this corresponds to the preparation of the immobilized methylated DNA antibody used in the measurement method of the present invention). For human blood-derived genomic DNA purchased from Clontech, Sssl met ylase (NEB) 1 ^ 1, 10 x NEBuf fer 2 (NEB) 10 ^ 1, S- adenosyl methionine (3.2 mM, NEB) was mixed with 11 and sterilized ultrapure water was added to prepare a liquid volume of 101. The reaction solution was incubated at 37 ° C. for 15-30 minutes, and further S-adenosyl methionine (3.2 mM, manufactured by NEB) was added at 11 and incubated at 37 for 15-30 minutes. This is Wizard SV Purification was performed with Gel / PCR Kit (PROMEGA). This operation was repeated three times to obtain methylated human genomic DNA.

 The following solutions were prepared for genomic DNA derived from human blood purchased from Clontech.

· Solution A: 10ng / 5 ^ L TE solution

 Solution B: lng / 5 L TE solution

 Solution C: TE solution (negative control solution) For the obtained methylated human genomic DNA, the following solutions were prepared.

, Solution MA: 10ng / 5 L TE solution.

 Solution MB: lng / 5 / L TE solution

 Solution M C: TE solution (negative control solution)

The following treatment was applied to each of the above-mentioned human genome DNA solution and methylated human genome DNA solution.

In a PCR tube, add 5 iL of the genomic DNA solution prepared above, 6 U of the restriction enzyme A 1 u I, and 10 x buffer solution suitable for A 1 u I (lOOmM Tris-HCl pH 7.5, lOOmM gCl ₂ , lOmM DithiothreitoK 500 mM NaCl) was mixed, and sterilized ultrapure water was added thereto to adjust the volume to 101. The reaction solution was incubated at 37 ° C for 1 hour.

 SEQ ID NO: 33- that can bind by complementarity to the negative strand of DNA fragment X "(region corresponding to base numbers 25687390-25687775 shown in Genbank Accession No. NT-029419 etc.) consisting of the base sequence shown in SEQ ID NO: 83 Counter oligonucleotides C1 to C12 consisting of the base sequence shown in SEQ ID NO: 44 were synthesized, and TE buffer solutions each having a concentration of 0.01 M were prepared, and DNA fragments> (5-methylcytosine was also used. (C)

X ": 5'-, CTCAGCACCCAGGCGGCCGCGATCATGAGGCGCGAGCGGCGCGCGGGCTGTTGCAGAGTCTTGAGCGGGTGGCAC

 AAGAATGCCACGGCCAGGTCGGCCAGGCTGAGGTGTCGGATGAAGAGGTGCATGCGGGACGTCTTGCGCGGCGTC

AGTTTGGCCAG -3 '(SEQ ID NO: 83)

 <Counter oligonucleotide>

 C1: 5'-GCCACCGCGAAAGTCACCGCCAGCACGGCG-3 '(SEQ ID NO: 3 3)

 C2: 5'-GCCAGCAGTACGCTGCTGTTGCCCAGCACG-3 '(SEQ ID NO: 3 4)

 , C3: 5'-CGGGACGTCTTGCGCGGCGTCCGGTGCAGA-3 '(SEQ ID NO: 3 5)

 C4: 5'-AGGCTGAGGTGTCGGATGAAGAGGTGCATG-3 '(SEQ ID NO: 3 6)

 C 5: 5 '-ACCTGGAAGAATGCCACGGCCAGGTCGGCC -3' (SEQ ID NO: 3 7)

 C6: 5'-TAGGTGATGTCCCAGCACATTTGCGGCAGC-3 '(SEQ ID NO: 3 8)

 C7: 5'-CGGCACAGCCAGTCGGGGCCGCGGAAGCGG-3 '(SEQ ID NO: 3 9)

 C 8: 5 '-ATGCCGAACACCTGCAGGTGCTTCACCACG -3' (SEQ ID NO: 40)

 C9: 5'-ATGACTACCAGCATGTAGGCCGACGCAAAC-3 '(SEQ ID NO: 4 1)

 C I O: 5'-TGGCACACCGCGATGTAGCGGTCGGCTGTC -3 '(SEQ ID NO: 42)

 C 1 1: 5'-CGCGCGGGCTGTTGCAGAGTCTTGAGCGGG-3 '(SEQ ID NO: 43)

 C12: 5'_CAGGCGGCCGCGATCATGAGGCGCGAGCGG-3 '(SEQ ID NO: 44)

: About liquid

The following treatment was performed.

In a PCR tube, add 10 L of the genomic DNA reaction solution prepared above, 10 L of the counter oligonucleotide solution prepared above, and buffer solution (330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM MgOAc ₂ , 5 mM Di thiothrei tol ) 5 L, 5 L of lOOmM MgCl ₂ solution, and 5 L of lmg / mL BSA solution were added, and sterilized ultrapure water was added to the mixture to bring the volume to 50 and mixed. The PCR tube is then heated at 95 for 10 minutes, quickly cooled to 70, and kept at that temperature for 10 minutes. Warm up. Next, it was cooled to 50, kept for 10 minutes, further kept at 37 ° C. for 10 minutes, and then returned to room temperature (this corresponds to the first step (A) of the measurement method of the present invention). 50 L of the DNA fragment reaction solution prepared above was added to the streptavidin-coated PCR tube on which the biotin-labeled methylcytosine antibody was immobilized, and left at room temperature for 30 minutes. Then, the solution was removed by pipetting, and after addition of 100 0 wash buffer [0.05% Tween20-containing phosphate buffer (ImM KH ₂ P0 ₄ , 3 mM Na ₂ HPO'7H ₂₀ , 154 mM NaCl pH 7.4)] The buffer was removed by pipetting. This operation was repeated twice more (this corresponds to the first step (B) of the measurement method of the present invention). . '

, 5 ml of buffer solution (330 mM Tris-Aceiate pH 7.9, 660 mM K0Ac, lOOmM MgOAc ₂ , 5iM Dithiothreitol) is added to the Streb® avidin-coated PC R tube to which the above-treated piotin-labeled methylcytosine antibody is immobilized. Add 5 L of BSA (Bovine serum albumin lmg / ml), 16 U of methylation sensitive restriction enzyme Hhal, and add sterile ultrapure water to the mixture to make the volume 50 L. The mixture was incubated at 37 ° C for 1 hour. Then, the solution was removed by pipetting, and 100 washing buffers [0.05% Tween20-containing phosphate buffer (ImM K P0 ₄ , 3 mM Na ₂ HP0'70, 154 mM NaCl pH 7.4)] were added, and then the buffer was added. One was removed by pipetting. This operation was repeated twice more (this corresponds to the second step of the measurement method of the present invention). Next, PCR is carried out in the above PCR tube using each solution of oligonucleotide primers PF 1 and PR 1 consisting of the nucleotide sequences represented by SEQ ID NO: 28 and SEQ ID NO: 29, and the following reaction conditions. The methylated DNA in the target DNA region X ′ consisting of the base sequence represented by No. 32 was amplified.

 ,

 <Oligonucleotide primers designed for PCR>

 PF1: 5'-CTCAGCACCCAGGCGGCC-3 '(SEQ ID NO: 28)

PR 1: 5'-CTGGCCAAACTGGAGATCGC-3 '(SEQ ID NO: 29). <Target DNA region> (5-methylcytosine is also represented by C)

 X ': 5'-

CTCAGCACCCAGGCGGCCGCGATCATGAGGCGCGAGCGGCGCGCGGGCTGTTGCAGAGTCTTGAGCGGGTGGCAC ACCGCGATGTAGCGGTCGGCTGTCATGACTACCAGCATGTAGGCCGACGCAAACATGCCGAACACCTGCAGGTGC TTCACCACGCGGCACAGCCAGTCGGGGCCGCGGAAGCGGTAGGTGATGTCCCAGCACATTTGCGGCAGCACCTGG AAGAATGCCACGGCCAGGTCGGCCAGGCTGAGGTGTCGGATGAAGAGGTGCATGCGGGACGTCTTGCGCGGCGTC

AGTTTGGCCAG -3 '(SEQ ID NO: 32), PCR reaction solution is a D-shaped DNA, 3 zxL each of oligonucleotide primer solution prepared in 5 M, 5 L each 2 mM dNTP, buffer solution (lOOmM Tris-HCl pH 8.3, 500mM KCK 15mM MgCl 2, 0.01% Gelatin) and a 5 L, heat resistance DNA polymerase (AmpliTaq Gold, AB I Co.) 5UZ L to 0 · 25 L _and: mixing, this Then, sterilized ultrapure water was added to make the volume 50 L. The reaction solution was incubated at 95 ° C for 10 minutes, then PCR was carried out under conditions of 34 cycles of incubation at 95 ° C for 20 seconds, then at 61 ° C for 30 seconds and then at 72 ° C for 30 seconds. Went.

 After PCR, amplification was confirmed by 1.5% agarose gel electrophoresis. The results were as follows (corresponding to the third step of the measurement method of the present invention).

 The results are shown in FIG. Amplification was confirmed in solutions MA and MB of methylated human genomic DNA. In the negative control solution MC, amplification was not confirmed and the amplification product was not obtained. In solutions A, B, and C of unmethylated human genomic DNA, amplification was not confirmed and no amplification product was obtained.

Based on the above, it is possible to select DNA containing the target DNA region that has been methylated with the immobilized methylcysin antibody, and until the amount of methylated DNA is detectable. It was confirmed that the amplified DNA could be detected with higher sensitivity. , 2008/056524

105

Example 14 'A commercially available methylcytosine antibody (available from Aviva Systems Biology) was described in the catalog using a commercially available biotinylated kit (Biot.in Labeling Kit-NH ₂ , manufactured by Dojindo Laboratories). Biotin was labeled according to the method described above. Refrigerate the resulting biotin-labeled methylcytosine antibody as a solution [antibody 0.25 / fl 0.1% BSA-containing phosphate buffer (l M KH ₂ P0 ₄ , 3 mM Na ₂ HP0 • 7H ₂₀ , 154 mM NaCl pH 7.4) solution] saved.

Add P.1 g / 50 solution of the biotin-labeled methylcytosine antibody obtained by synthesis to a streptavidin-coated PCR tube (total of 8 tubes) at a rate of 50 L each and leave at room temperature for about 1 hour. And immobilized on a PCR tube. Then, remove the solution by pipetting and add 100 L wash buffer [0.05% Tween20-containing phosphate buffer (ImM KH ₂ P0 ₄ , 3raM Na ₂ HP0-7H ₂₀ , 154 mM NaCl pH 7.4)] The buffer was removed by pipetting. This operation was repeated twice more (this corresponds to the preparation of the immobilized methylated DNA antibody used in the measurement method of the present invention).

The baker's yeast strain X 2180-1A in YPD medium (1% Yeast extract, 2% Peptone, 2% Glucose, pH 5.6-6.0) has a turbidity of 0D ₆ . . Cultured to 0.6-1.0, 10, 000 by centrifugation for 10 minutes to prepare a yeast cell lxlO ^7. Yeast genomes were obtained from the prepared yeast cells using a general yeast genome preparation method as described in Methods in Yeast Genetics (Cold Spring Harbor Laboratory).

Suspend the prepared yeast cells in buffer A (1 M Sorpitol, 0.1 M EDTA, pH 7.4), add 2-mercaptoethanol (final concentration mM) and 100 U zymolase (10 mg / ml) to make the solution clear. Incubated with stirring at 30 ° C for 1 hour. Protoplasts are collected by centrifugation at 550 for 10 minutes, suspended in Buffer B (50 niM Tris-HCU pH 7.4, 20 mM EDTA), and sodium dodecyl sulfate is added to 1% (w Zv). And incubated at 65 '° C for 30 minutes. Subsequently, 2/5 volume ratio of 5M C¾C00K was added and mixed, and after ice cooling for 30 minutes, the mixture was centrifuged at 15,000 for 30 minutes to collect the supernatant. Add 1/10 volume ratio of 3M CH ₃ C00Na and isopropanol to the recovered supernatant, mix well, and centrifuge at 15,000 4 ° C for 30 minutes for 70% of the precipitate. Rinse with ethanol and collect. After drying the precipitate, dissolve it in 1 ml of TE buffer (10 mM Tris-HC1, pH 8.0, 1 mM EDTA), add RNase A (Sigma) to 40 ig / ml, and 37 ° Incubate at C for 1 hour, and then add proteinase. K (Sigma) to the mixture to 500 g / m 1 and sodium dodecyl sulfate to 1% · (w / v). Shake for about 16 hours at 55 ° C. After completion of the shaking, the mixture was subjected to phenol extraction [saturated with 1M Tris-HCl (pH 8.0)]. The aqueous layer was recovered, and NaCl was added to this to 0.5 N, and then the resulting precipitate was recovered by ethanol precipitation. Genomic DNA was obtained by rinsing the collected precipitate with 70% ethanol.

, Sssl methylase (NEB) 1 1, 10 xNEBuffer 2 (NEB) 10 l, S-adenosyl methionine (3.2 mM, NEB) 1 1 was mixed, and sterilized ultrapure water was added thereto to make the volume 100 1 to prepare. The reaction mixture, 37 ° and incubated for 15-30 minutes at C, _is; et to S- adenosyl methionine (3.2 mM, NEB Inc.) 15 at by adding a 1 n 1 37 ° C - 30 minutes Incubated. This was purified by Wizard SV Gel / PCR Kit (PR0MEGA). This operation was further repeated 3 times to obtain methylated yeast genomic DNA. About the obtained yeast genomic DNA, the following solutions were prepared.

 Solution A: 10ng / 5 L TE solution

 Solution B: lng / 5 ^ L TE solution

 Solution C: 0. lng / 5 ^ L TE solution

 Solution D: TE solution (negative control solution) The following solutions were prepared for the obtained methylated yeast genomic DNA.

 Solution MA: 10ng / 5 ^ L TE solution

 Solution MB: lng / 5 ^ L TE solution

Solution MC: 0.1 ng / 5 L TE solution, Solution MD: TE solution (negative control solution) The following treatment was applied to each of the above-mentioned yeast genome DNA solution and methylated yeast genome DNA solution.

· In a PCR tube, add 5 L of the genomic DNA solution prepared above, 5 U of the restriction enzyme Xs p I, and 1 Ox buffer (200 mM Tris-HCl H 8.5, lOOmM MgCl ₂ , lOmM DithiothreitoK) lOOOmM KC1) 2 1 was mixed, and sterilized ultrapure water was added thereto to prepare a liquid volume of 20 μ1. The reaction solution was incubated at 37 ° C for 1 hour.

, DNA fragment S "(Genbank

Accession No. NC—A counter consisting of the nucleotide sequence shown in SEQ ID NO: 58 to SEQ ID NO: 67 that can bind by complementarity to the negative strand of yeast chromosome VII shown in Accession No. NC—001139 etc. synthesizing an oligonucleotide C 16~C25, _{respectively;} s concentration to prepare a TE buffer first solution is 0. 01 iM.

<DNA fragment> (methylcytosine is also represented by C)

 S ": 5'-

 CCCGTTTCCACACCGTACGTGAGCACATGTCTGGATTGC -3 '(SEQ ID NO: 77)

<Counter oligonucleotide>

 C 16: 5'-AGGTGAGCTACGTGTGTTTGG-3 '(SEQ ID NO: 58)

 C 17: 5'-GCGTCGTGCACTGGCTCACTTGTACGCGCA-3 '(SEQ ID NO: 59)

 C 18: 5'- CTTGTACGATTGGTGACCCGCCTTTTCGAC -3 '(SEQ ID NO: 60)

C 19: 5'-ACTGGACCGCTATGGACGTGGCGGCGGTGT-3 '(SEQ ID NO: 61) C 20: 5'- GGCGGCGGCTCAATGACCTGTGGCGCCCGT -3 '(SEQ ID NO: 62)

C 21: 5'- TTGTGGCGTGCGATAGTCGAGCCGCCTGTC-3, (SEQ ID NO: 63)

 C 22: 5'-ACGTGCGCGGCCGCCCTGCTCCGTT-3 '(SEQ ID NO: 64)

 C23: 5'-TGACGCGATGCATAGCATGCGACCACCCAG-3 '(SEQ ID NO: 65)

· C24: 5'-ACTGCTGACGCTATTGGTCACGTGGTTATG-3 '(SEQ ID NO: 66)

 C 25: 5′-CTGCTGTTGACTGCGGTGGCGTCCCGTTTC-3 ′ (SEQ ID NO: 67) The following treatments were carried out for the respective reaction solutions of the yeast genome DNΑ and the methylated yeast genome DNΑ.

, In the PCR tube, genomic DNA reaction solution 20 prepared above, counter oligonucleotide solution 1 OL prepared above, and buffer solution (330 mM Tris-Acetaie H 7.9, 660 mM K0Ac, lOOmM MgOAc ₂ 5 mM Di thiothrei tol) 5 L, lOOmM Mg <1 ₂ solution 5 and 1 mg / mL BSA solution 5 L were added, and further sterilized ultrapure water was added to the mixture: to a volume of 50 zL and mixed. The PCR tube was then heated at 95 ° C for 10 minutes, quickly cooled to 70 ° C, and kept at that temperature for 10 minutes. Next, it was cooled to 5′0 ° C. and kept for 10 minutes, further kept at 37 ° C. for 10 minutes, and then returned to room temperature (this corresponds to the first step (A) of the measurement method of the present invention).

50 L of the DNA fragment reaction solution prepared above was added to a streptavidin-coated PCR tube on which the piotin-labeled methylcytosine antibody had been immobilized, and left at room temperature for 30 minutes. Then, the solution was removed by pipetting, and 100 L of washing buffer [0.05% Tween20-containing phosphate buffer (lmM KH ₂ P0 ₄ , 3 mM Na ₂ HPO'7H ₂₀ , 154 mM NaCl pH 7.4)] was added Thereafter, the buffer was removed by pipetting. This operation was repeated twice more (this corresponds to the first step (B) of the measurement method of the present invention).

Buffer L (330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM Mg0Ac2, 5 mM Di thiothrei tol) and 5 L were added to a streptavidin-coated PCR tube on which the piotine-labeled methylcytosine antibody was immobilized. Add 5 L of BSA (Bovine serum albumin lmg / ml) and 16 U of the methylation-sensitive restriction enzyme Hhal. Each mixture was incubated at 37 for 1 hour with sterile ultrapure water added to the mixture to a volume of 50 iL. Then, the solution was removed by pipetting, and 100 L of washing buffer [0.05% Tween20-containing phosphate buffer (ImM KH ₂ P0 ₄ , 3 mM Na ₂ HP0-7H ₂₀ , 154 mM NaCl pH 7.);] was added The buffer was removed by pipetting. This operation was repeated twice more (this corresponds to the second step of the measurement method of the present invention). Next, PCR is performed on the above PCR tube using each solution of oligonucleotide primers PF 3 and PR 3 consisting of the nucleotide sequences represented by SEQ ID NO: 53 and SEQ ID NO: 54 and the following reaction conditions: The methylated DNA in the target DNA region S ′ consisting of the base sequence represented by SEQ ID NO: 57 was amplified.

<An oligonucleotide primer designed for PCR>

 : P F 3: 5 '-AGGTGAGCTACGTGTGTTTGG -3' (SEQ ID NO: 53)

 PR 3: 5'-AGACATGTGCTCACGTACGGT-3 '(SEQ ID NO: 54)

 <Target DNA region> (5-methylcytosine is also represented by C)

 S ': 5'-

 CCGTTTCCACACCGTACGTGAGCACATGTCT -3 '(SEQ ID NO: 57)

The PCR reaction solution is a vertical DNA, 3 each of oligonucleotide primer solution prepared at 5 M, 5 L each 2 mM dNTP, buffer (lOOmM Tris-HCl pH 8.3, 500 mM KC1 , 15 mM MgCl in 0.01% Gelatin), 5 and heat-resistant DNA polymerase (AmpliTaq Gold, manufactured by AB I) 5U ^ L and 0.25 L are mixed with sterilized ultrapure water. What was set to 50 L was used. The reaction The solution was incubated at 95 ° C. for 10 minutes, and then PCR was performed under the conditions of 31 cycles of incubation at 95 ° C. for 20 seconds, followed by 58 for 30 seconds and 72 for 30 seconds.

 After PCR, amplification was confirmed by 1.5% agarose gel electrophoresis. The results were as follows (corresponding to the third step of the measurement method of the present invention). The results are shown in FIG. In the solutions of methylated yeast genomic DNA MA, MB and MC, amplification was confirmed and amplification products were obtained. In the negative control solution M D, amplification was not confirmed and no amplification product was obtained. In the unmethylated yeast genomic DNA solutions A, B, C and D, amplification was not confirmed and the amplification product was not obtained.

 Based on the above, it is possible to select DNA containing the target DNA region that has been methylated with the immobilized methylcytosine antibody, and to detect the amount of methylated DN A :. It was confirmed that the amplified DNA could be detected with higher sensitivity. Example 15

A commercially available methylcytosine antibody (manufactured by Aviva Systems Biology) was labeled with a biotin using a commercially available biotinylated kit (Biotin Labeling Kit_N, manufactured by Dojindo Laboratories) according to the method described in the catalog. . As a solution [Phosphate buffer solution (ImM KH ₂ P0 ₄ , 3 mM Na ₂ HP0 • 7H ₂₀ , 154 mM NaCl pH 7.4) containing 0.1% BSA antibody] Stored refrigerated.

Add a 0.1 g / 50 solution of the synthesized biotin-labeled methylcytosine antibody to each streptavidin-coated PCR tube (total of 8 tubes) at a rate of 50 L and leave it at room temperature for about 1 hour. Fixed to R Cube. Then, the solution was removed by pipetting, and 100 L of washing buffer [0.05% Tween20-containing phosphate buffer (ImM KH ₂ P0 ₄ , 3 mM Na ₂ HP0-7H ₂₀ , 15 mM NaCl pH 7.4)] was added. Thereafter, the buffer was removed by pipetting. This operation was repeated twice more ( As described above, this corresponds to the preparation of an immobilized methylated DNA antibody used in the measurement method of the present invention). The baker's yeast strain X 2180-1A in YPD medium (1% Yeast extract, 2% Peptone, 2% Glucose, pH 5.6-6.0) has a turbidity of 0D ₆ . . Cultured to 0.6-1.0, 10, 000 by centrifugation for 10 minutes to prepare a yeast cell lxlO ^7. Yeast genomes were obtained from the prepared yeast cells using a general yeast genome preparation method as described in Methods in Yeast Genetics (Cold Spring Harbor Laboratory).

 The prepared yeast cells are suspended in buffer A (1M sorbi ^ l, 0.1M EDTA, pH 7.4), and 2-mercaptoethanol (final concentration 14 mM) and .100 U zymolase (10 mg / ml) are added. Incubate with stirring for 1 hour at 30 ° C until the solution became clear. Protoplasts are collected by centrifugation at 550 for 10 minutes, suspended in buffer B (50 DiM Tris-HCK pH 7.4, 20 raM EDTA), and sodium dodecyl sulfate is added to 1% (w Xv). Incubated at 65 ° C for 30 minutes. Subsequently, 2/5 volume ratio of 5M C¾C00K was added and mixed, ice-cooled for 30 minutes, centrifuged at 15,000 for 30 minutes, and the supernatant was recovered. Add 1/10 volume ratio of 3M C C00Na and isopropanol to the collected supernatant, mix well, and centrifuge at 15,000 4 ° C for 30 minutes to rinse the precipitate with 70% ethanol. And recovered. After drying the precipitate, dissolve it in 1 ml of TE buffer (10 mM Tris-HCK pH 8.0, 1 mM EDTA), add RNase A (Sigma) to 40 g / ml, and 37 ° Incubate at C for 1 hour, and then add proteinase K (Sigma) to the mixture to 500 ^ g / m 1 and sodium dodecyl sulfate to 1% (w / v). Shake for about 16 hours at ° C. After completion of the shaking, the mixture was subjected to phenol [saturated with 1M Tris-HCl (pH 8.0)] · Kroguchi form extraction. The aqueous layer was recovered, and NaCl was added to this to 0.5 N, followed by ethanol precipitation, and the resulting precipitate was recovered. Genomic DNA was obtained by rinsing the collected precipitate with 70% ethanol.

For a portion of the obtained yeast genomic DNA, 1 1 for Sssl methylase (NEB), 10 1 for 10 x NEBuffer 2 (NEB), 1 for S-adenosyl methionine (3.2 mM, NEB) 1 was mixed, and sterilized ultrapure water was added thereto to prepare a liquid volume of 1001. The reaction solution was incubated 1 5-30 minutes at 37 ^e C, is Furthermore, S-adenosyl methionine (3.2 mM, manufactured by NEB) was added (i 1 was added and incubated at 37 t: for 15 to 30 minutes. This was purified by Wizard SV Gel / PCR Kit (PR0MEGA). This operation was further repeated 3 times to obtain methylated yeast genomic DNA.

·

 For the obtained yeast genome DNA, the following solutions were prepared.

 Solution A: 10ng / 5 / iL TE solution

 Solution B: lng / 5 L TE solution

 Solution C: 0.1 ng / 5 L TE solution

, Solution D: TE solution (negative control solution) The following solutions were prepared for the obtained methylated yeast genomic DNA.

 Solution MA: 10ng / 5 L TE solution

 Solution MB: lng / 5 L TE solution

 Solution MC: 0.1ng / 5 // L TE solution

 Solution MD: TE solution (negative control solution) The following treatment was applied to each of the above-mentioned yeast genome DNA solution and methylated yeast genome DNA solution.

In a PCR tube, add 5 L of the genomic DNA solution prepared above, 5 U of the restriction enzyme X sp I, and 10 x buffer (200 mM Tris-HCl H 8.5, ΙΟΟ Μ MgCl ₂ , lOm DithiothreitoU lOOOmM KC1) 2 l was mixed, and sterilized ultrapure water was added thereto to prepare a liquid volume of 201. The reaction solution was incubated at 37 t: for 1 hour.

 DNA fragment T "(Genbank comprising the nucleotide sequence represented by SEQ ID NO: 78

Accession No. NC—SEQ ID NO: 73 to SEQ ID NO: 76 and SEQ ID NO: 79 to SEQ ID NO: 73 that can bind by complementarity to the negative strand of the yeast chromosome VI I shown in Accession No. NC—001139 etc. Counter oligonucleotide consisting of the base sequence indicated by number 82 Chido C 26 to C 33 were synthesized and TE buffer solutions with respective concentrations of 0.01 were prepared. DNA fragment> (Methylcytosine is also represented by C)

T ": 5'-

 CCTGGCCACGCCTCTAATC -3 '(SEQ ID NO: 78)

, <Counter oligonucleotide>

 C 26: 5'-GGACCTGTGTTTGACGGGTAT-3 '(SEQ ID NO: 73)

 C27: 5'-AACACTAAGTTGCGCAATTTGCTGT-3 '(SEQ ID NO: 74)

 C28: 5'-ATTGCGAAATCCGCCCGGACGATAT-3 '(SEQ ID NO: 75)

; C29: 5'-CACTCTTGAGCGCATGTGCCGTTTC-3 '(SEQ ID NO: 76)

 C 30: 5'- AATACATTCCGAGGGCGCCCGCACAAGGCC -3 '(SEQ ID NO: 79)

 C31: 5'-GCGATCGCACACGAGGAGACA-3 '(SEQ ID NO: 80)

 C32: 5'-AGCGTCACGTGTTTTGCCATTTTGTACGAC-3 '(SEQ ID NO: 81)

 C 33: 5′-AAATGAACCGCCTGGCCACGCCTCTAATC-3 ′ (SEQ ID NO: 82) The following treatments were performed on the respective reaction solutions of the yeast genome DNA and methylated yeast genome DNA.

In the PCR tube, reaction solution 20 of genomic DNA prepared above, 10 L of county oligonucleotide solution prepared above, and buffer (330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM MgOAc ₂ , 5 mM Dithiothreitol) Add 5j L, 5 liters of lOOmM M80 1 ₂ solution, 5 L of lmg / mL BS A solution, and add sterilized ultrapure water to the mixture to make the volume 50 L, and mix . The PCR tube was then heated at 95 ° C for 10 minutes, quickly cooled to 70 ° C, and kept at that temperature for 10 minutes. Next, cool to 50 ° C and incubate for 10 minutes, then incubate at 37 ° C for 10 minutes Then, the temperature was returned to room temperature (this corresponds to the first step (A) of the measurement method of the present invention). The above-prepared DNA fragment reaction solution 5 OL was added to the streptavidin-coated PCR tube on which the above-mentioned pyotin-labeled methylcytosine antibody was immobilized, and left at room temperature for 30 minutes. Then, the solution was removed by pipetting, and 10 '0 L of washing buffer [0.05% Tween20-containing phosphate buffer (ImM KH ₂ P0 ₄ 3mM Na ₂ HP0'70, 15 mM NaCl pH 7.4)] was added. Thereafter, the buffer was removed by pipetting. This operation was repeated twice more (this corresponds to the first step (B) of the measurement method of the present invention).

Buffer (330 mM Tris-Acetate pH 7.9, 6.6 OmM K0Ac, lOOmM Mg0Ac2, 5 mM Di thiothrei iol) is added to a Strept 'avidin-coated PCR tube to which the biotin-informed methylcytosine antibody has been treated. Add 5 L, 5 L of BSA (Bovine serum albumin lmg / ml) and 16 U of methylation sensitive restriction enzyme fflial, and add sterile ultrapure water to the mixture to make the volume 50 L Each mixture was incubated at 37: 1 hour. Then, the solution was removed by pipetting, and after adding 100 j ^ L washing buffer [0.05¾ Tween20-containing phosphate buffer (ImM K¾P0 ₄ , 3 mM Na ₂ HPO-7H ₂₀ , 154 mM NaCl pH 7.4)] The buffer was removed by pipetting. This operation was repeated twice more (this corresponds to the second step of the measurement method of the present invention). Next, in the above-mentioned PCR tube, using each solution of oligonucleotide primers PF 4 and PR 4 consisting of the base sequences shown in SEQ ID NO: 68 and SEQ ID NO: 69, and the reaction conditions shown below, As a result, the methylated DNA in the target DNA region T ′ consisting of the base sequence represented by SEQ ID NO: 72 was amplified. '<Oligonucleotide primer designed for PCR>

 PF4: 5'-GGACCTGTGTTTGACGGGTAT-3 '(SEQ ID NO: 6 8)

P R4: 5'-AGTACAGATCTGGCGTTCTCG-3 '(SEQ ID NO: 6 9) Target DNA region> (5-methylcytosine is also represented by C)

 T ': 5'-

CTTGAGCGCATGTGCCGTTTCCGAGAACGCCAGATCTGTACT-3 '(SEQ ID NO: 72)

'

 The PCR reaction solution is a vertical DNA, 3 L each of an oligonucleotide primer solution prepared at 5 M, 5 L each 2 mM dNTP, buffer (lOOmM Tris-HCl pH 8.3, 500 mM) KC1, 15 mM MgCl (0.01% Gelatin) 5 and heat-resistant DNA polymerase (AmpliTaq Gold, manufactured by AB I) 5UZ L 0.25 L and 'are mixed, and sterilized ultrapure water is added to the solution. A value of 50 was used. The reaction solution is kept at 95 ° C for 10 minutes, and then kept at 95 ° C for 20 seconds, then at 58 ° C for 30 seconds and then at 72 ° C for 30 seconds for 31 cycles. PCR was performed.

 ■ After PCR, 1 .. Amplification was confirmed by 5% agarose gel electrophoresis. The results were as follows (corresponding to the third step of the measurement method of the present invention). The results are shown in FIG. In the solutions of methylated yeast genomic DNA MA, MB and MC, amplification was confirmed and amplification products were obtained. In the negative control solution M D, amplification was not confirmed and no amplification product was obtained. In the unmethylated yeast genomic DNA solutions A, B, C, and D, amplification was not confirmed and the amplification product was not obtained. Based on the above, it is possible to select methylated DNA containing the target DNA region with the immobilized methylcytosine antibody, and amplify the methylated DNA to a detectable amount. As a result, it was confirmed that the amplified DNA could be detected with higher sensitivity. '' Industrial applicability

According to the present invention, a target DN possessed by genomic DNA contained in a biological specimen It is possible to provide a method for easily measuring the content of methylated DNA in the A region. Sequence listing free text

'SEQ ID NO: 17

 Designed oligonucleotide

 SEQ ID NO: 18

 Designed oligonucleotide

 SEQ ID NO: 19

. Oligonucleotide primers designed for PCR

 SEQ ID NO: 20

 An oligonucleotide primer designed for PCR

 SEQ ID NO: 21

 Designed oligonucleotide

 SEQ ID NO: 22

 Designed oligonucleotide

 SEQ ID NO: 23

 Designed oligonucleotide

 SEQ ID NO: 24

 Designed oligonucleotide

 SEQ ID NO: 25

 An oligonucleotide primer designed for PCR

 SEQ ID NO: 26

 An oligonucleotide primer designed for PCR

 SEQ ID NO: 27

 Designed oligonucleotide

 SEQ ID NO: 28

An oligonucleotide primer designed for PCR. 2008/056524

117 SEQ ID NO: 2 9

 Oligonucleotide primers designed for PCR

 SEQ ID NO: 30

 Amplified oligonucleotide consisting of the desired DNA region

 SEQ ID NO: 3 1

 Designed oligonucleotide consisting of the desired DNA region

 SEQ ID NO: 3 2

 Designed oligonucleotide consisting of the desired DNA region

 SEQ ID NO: 3 3

'' Oligonucleotides designed for experiments

 SEQ ID NO: 3 4

 Oligonucleotides designed for experiments

 SEQ ID NO: 3 5

 : Oligonucleotides designed for experiments

 SEQ ID NO: 3 6

 Oligonucleotides designed for experiments

 SEQ ID NO: 3 7

 Oligonucleotides designed for experiments

 SEQ ID NO: 3 8

 Oligonucleotides designed for experiments

 SEQ ID NO: 3 9

 Oligonucleotides designed for experiments

 SEQ ID NO: 4 0

 Oligonucleotides designed for experiments

 SEQ ID NO: 4 1 '

 Oligonucleotides designed for experiments

 SEQ ID NO: 4 2

Oligonucleotide designed for experiments T JP2008 / 056524

118 SEQ ID NO: 4 3

 Oligonucleotides designed for experiments

 SEQ ID NO: 4 4

 Oligonucleotides designed for experiments

'SEQ ID NO: 4 5

 An oligonucleotide primer designed for PCR

 SEQ ID NO: 4 6

 Oligonucleotide primers designed for PCR

 SEQ ID NO: 4 7

'' Amplified oligonucleotide consisting of the desired DNA region

 SEQ ID NO: 4 8

 Designed oligonucleotide consisting of the desired DNA region

 SEQ ID NO: 4 9

 -Designed oligonucleotide consisting of the desired DNA region

 SEQ ID NO: 5 0

 Oligonucleotides designed for experiments

 SEQ ID NO: 5 1.

 Oligonucleotides designed for experiments

 SEQ ID NO: 5 2

 Oligonucleotides designed for experiments

 SEQ ID NO: 5 3

 Oligonucleotide primers designed for PCR

 SEQ ID NO: 5 4

 Oligonucleotide primers designed for PCR

 SEQ ID NO: 5 5 ''

 Amplified oligonucleotide consisting of the desired DNA region

 SEQ ID NO: 5 6

Designed oligonucleotide consisting of the desired DNA region SEQ ID NO: 5 7

 Designed oligonucleotide consisting of the desired DNA region SEQ ID NO: 5 8

 Oligonucleotides designed for experiments

'SEQ ID NO: 5 9

 Oligonucleotides designed for experiments

 SEQ ID NO: 6 0

 Oligonucleotides designed for experiments

 SEQ ID NO: 6 1

 Oligonucleotides designed for experiments

 SEQ ID NO: 6 2

 Oligonucleotides designed for experiments

 SEQ ID NO: 6 3

 • Oligonucleotides designed for experimentation

 SEQ ID NO: 6 4

 Oligonucleotides designed for experiments

 SEQ ID NO: 6 5,

 Oligonucleotides designed for experiments

 SEQ ID NO: 6 6

 Oligonucleotides designed for experiments

 SEQ ID NO: 6 7

 Oligonucleotides designed for experiments

 SEQ ID NO: 6 8

 Oligonucleotide primer designed for PCR SEQ ID NO: 6 9 '

 An oligonucleotide primer designed for PCR SEQ ID NO: 7 0

Amplified oligonucleotide consisting of the desired DNA region SEQ ID NO: 7 1

 Designed oligonucleotide consisting of the desired DNA region 'SEQ ID NO: 7 2

 Designed oligonucleotide consisting of the desired DNA region 'SEQ ID NO: 7 3

 Oligonucleotides designed for experiments

 SEQ ID NO: 7 4

 Oligonucleotides designed for experiments

 SEQ ID NO: 7 5

. Oligonucleotides designed for experimentation

 SEQ ID NO: 6

 Oligonucleotides designed for experiments

 SEQ ID NO: 7 7

 -Designed oligonucleotide consisting of the desired DNA region SEQ ID NO: 7 8

 Designed oligonucleotide consisting of the desired DNA region SEQ ID NO: 7 9

 Oligonucleotides designed for experiments

 SEQ ID NO: 80

 Oligonucleotides designed for experiments

 SEQ ID NO: 8 1

 Oligonucleotides designed for experiments

 SEQ ID NO: 8 2

 Oligonucleotides designed for experiments

 SEQ ID NO: 8 3

 Designed oligonucleotide consisting of the desired DNA region

Claims

The scope of the claims 1. A method for measuring the content of methylated DNA in a target DNA region in genomic DNA contained in a biological specimen,  (1) derived from genomic DNA contained in a biological sample; the first (A) step that separates methylated single-stranded DNA from the DNA sample, and the one that was separated in the first (A) step A first step of selecting the single-stranded DNA described above, comprising a first step (B) for binding the single-stranded DNA and the immobilized methylated DNA antibody;  (2) a second step of digesting the single-stranded DNA selected in the first step with a methylation sensitive restriction enzyme capable of degrading at least one kind of single-stranded DNA; and  (3) As a pre-process of each of the following steps, single-stranded DNA that is an undigested product obtained in the second step (the recognition site of the methylation-sensitive restriction enzyme capable of digesting the single-stranded DNA described above) Single-stranded DNA that does not contain CpG in the methyl state) : Separating from antibody to make single-stranded DNA (positive strand) (third (previous A) step),  The single-stranded DNA (positive strand) separated in the third (previous A) step is used as a saddle, and the single-stranded DNA (positive strand) has a partial base sequence (positive And complementary to a partial base sequence (positive strand) located further to the 3 ′ end side than the 3 ′ end of the base sequence (positive strand) of the target DNA region. By using the extension primer (forward primer) having the base sequence (negative strand) as an extension primer, the extension primer is extended once, so that the pNA (positive strand) in the single-stranded state is added. A single-stranded DNA containing the target DNA region by the step of extending the double-stranded DNA as a double-stranded DNA (third (pre-B) step) and the double-stranded DNA formed by the third (pre-B) step. (Positive strand) and single-stranded DNA (negative strand) containing the target DNA region Has C) step), and, as this process (a) The generated single-stranded DNA (positive strand) containing the target DNA region is used as a saddle, the forward primer is used as an extension primer, and the extension primer is extended once. Double-stranded single-stranded DNA containing DNA region This process A to stretch and form as DN A,  (b) A partial base sequence of the base sequence of the single-stranded DNA (negative strand) containing the target DNA region, with the generated single-stranded DNA (negative strand) containing the target DNA region as a saddle type (Negative strand) and located further 3 'end than the 3' end of the base sequence (negative strand) that is complementary to the base sequence (positive strand ') of the target DNA region. By extending the extension primer once, using an extension primer (reverse primer) having a partial base sequence (negative strand) and a complementary base sequence (positive strand) as an extension primer, A step B for extending and forming a single-stranded DNA containing the target DNA region as a double-stranded DNA,  'Furthermore, each of these steps is repeated by once separating the elongated double-stranded DNA obtained in each of the steps into a single-stranded state and then repeating the methylation in the target DNA region. A third step of amplifying the amplified DNA to a detectable amount and quantifying the amount of amplified DNA. 2. The method according to claim 1, wherein the methylated DNA antibody in the third step is a methylcytosine antibody. 3. The method according to any one of claims 1 to 2, wherein the biological specimen is mammalian serum or plasma. 4. The method according to claim 1, wherein the biological specimen is mammalian blood or body fluid. 5. The method according to any one of claims 1 to 2, wherein the biological specimen is a cell lysate or a tissue lysate. ' 6. A genomic DNA-derived DNA sample contained in a biological sample is digested in advance with a restriction enzyme that does not use the target DNA region of the genomic DNA as a recognition cleavage site. 6. The method according to any one of claims 1 to 5, wherein the sample is a DNA sample. 7. A DNA sample derived from genomic DNA contained in a biological specimen is a DNA sample digested with at least one kind of methylation-sensitive restriction enzyme, according to any one of claims 1 to 6 The method of crab. 8. The method according to claim 1, wherein the DNA sample derived from genomic DNA contained in the biological specimen is a DNA sample purified in advance. '  9. A restriction enzyme capable of digesting at least one kind of single-stranded DNA has a recognition cleavage site in the target DNA region of the genomic DNA contained in the biological sample. The method according to any one of claims 1 to 8, wherein -Method. 10. The method according to any one of claims 1 to 9, wherein the methylation-sensitive restriction enzyme capable of digesting at least one kind of single-stranded DNA is Hhal which is a methylation-sensitive restriction enzyme. . 1 1. The third step is carried out without carrying out an extinguishing treatment with a methylation sensitive restriction enzyme capable of digesting single-stranded DNA in the second step. The method described. 12. The method according to any one of claims 1 to 11, wherein an oligonucleotide is added when separating the single-stranded DNA that has been methylated in the first step (A).