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WO2008123537A1 - Procédé de mesure de méthylation dans un adn - Google Patents

Procédé de mesure de méthylation dans un adn Download PDF

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Publication number
WO2008123537A1
WO2008123537A1 PCT/JP2008/056524 JP2008056524W WO2008123537A1 WO 2008123537 A1 WO2008123537 A1 WO 2008123537A1 JP 2008056524 W JP2008056524 W JP 2008056524W WO 2008123537 A1 WO2008123537 A1 WO 2008123537A1
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Prior art keywords
dna
solution
seq
methylated
region
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Japanese (ja)
Inventor
Yoshitaka Tomigahara
Hirokazu Tarui
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Sumitomo Chemical Co Ltd
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Sumitomo Chemical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]

Definitions

  • 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.
  • 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) )
  • 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.
  • PCR polymerase chain reaction
  • 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
  • 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:) .
  • 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.
  • an extension primer forward 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
  • 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.
  • extension primer 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.
  • reverse primer having a partial base sequence (negative strand) and a complementary base sequence (positive strand) as the extension primer.
  • 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);
  • methylated DNA antibody in the third step is a methylcytosine antibody
  • 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.
  • 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;
  • 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.
  • methylation sensitive restriction enzyme capable of digesting at least one kind of single-stranded DNA is Hhal which is a methylation sensitive restriction enzyme; .
  • 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.
  • 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.
  • 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. .
  • FIG. 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.
  • FIG. 1 A diagram showing the results of 5% agarose gel electrophoresis.
  • 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.
  • 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. 1 shows 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) ) Meth
  • 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”. .
  • FIG. 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.
  • 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.
  • 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.
  • 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.
  • 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”.
  • biological specimen examples include, for example, a cell lysate, a tissue lysate (herein, tissue has a broad meaning including blood, lymph nodes, etc.), or in mammals, plasma.
  • tissue has a broad meaning including blood, lymph nodes, etc.
  • 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.
  • biological specimen examples include samples derived from microorganisms, viruses, and the like.
  • genomic DNA in the measurement method of the present invention means genomic DNAs such as microorganisms and viruses.
  • the measurement method of the present invention can be expected to be used in periodic health examinations and simple tests.
  • DNA extraction DNA may be extracted using a kit or the like.
  • 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).
  • 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.
  • 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).
  • nucleotide sequence containing one or more nucleotide sequences examples 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.
  • genomic DNA nucleotide sequences containing exon 1 of the Lysyl oxidase gene derived from human ' and its promoter region located upstream of the gene.
  • 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
  • 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.
  • a high methylation frequency ie, hypermethyl ation
  • 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.
  • 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).
  • 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).
  • 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 ' shows a high methylation frequency (ie, hypermethylation state) in cancer cells such as gastric cancer cells.
  • 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
  • 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.
  • 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.
  • a high methylation frequency ie, hypermethylation state
  • 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.
  • 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) ).
  • 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.
  • the cytosine in G shows a high methylation frequency (ie, hypermethylation state) in cancer cells such as gastric cancer cells.
  • 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.
  • 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.
  • 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).
  • 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 p′G shows a high methylation frequency (ie, hypermethylation state) in cancer cells such as gastric cancer cells.
  • 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.
  • a useful protein gene is Homologue of RIKEN
  • 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.
  • 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 .
  • nucleotide sequence represented by SEQ ID NO: 6 corresponds 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.
  • nucleotide sequence represented by SEQ ID NO: 6 corresponds 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.
  • 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.
  • the cytosine in it shows a high methylation frequency (ie, hypermethyl aUon) in cancer cells such as gastric cancer cells.
  • 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.
  • the useful protein gene is the FLJ32130 gene
  • 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.
  • 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.
  • 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.
  • the cytosine in G shows a high methylation frequency (ie, hypermethyl aU on) in cancer cells such as gastric cancer cells.
  • 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.
  • the useful protein gene is a PPARG angi opoi et in-related protein gene
  • its promoter region, untranslated region or translated 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.
  • 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 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.
  • 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.
  • 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.
  • 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, ⁇ 56, etc. can be mentioned.
  • 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).
  • 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.
  • 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.
  • 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)).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a useful protein gene is di s integr in and
  • 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 ⁇ of human-derived lointe inase domain 23 gene is from nucleotide numbers 1194 to
  • 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)).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a useful protein gene is G-protein coupled
  • 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.
  • 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.
  • 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.
  • SEQ ID NO: 16 base sequences 78801 to 81000 of the base sequence described in Genbank Accession No. AC026802
  • 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).
  • polyclonal antibodies antibodies for the IgG fraction
  • monoclonal antibodies antibodies produced by a single clone
  • a cell fusion method As a method for producing a monoclonal antibody, a cell fusion method can be mentioned.
  • 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.
  • 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.
  • 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.
  • a method of adding It is also possible to boost the mouse intraperitoneally or intravenously after an appropriate period after the first immunization.
  • a solution in which the antigen is suspended may be directly injected into the mouse spleen for 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.
  • Sendai virus (HV J) poly And a method using ethylene glycol (PEG).
  • cell fusion may be performed by a method using a high voltage pulse.
  • 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.
  • 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.
  • “(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.
  • the biological specimen 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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). .
  • 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.
  • a method of covalently bonding to a saccharide derivative, silica gel, the above synthetic resin or the like, or a heat-resistant plastic support 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.
  • 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.
  • 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.
  • the immobilized methylated DNA antibody is immobilized on a support.
  • 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.
  • 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.
  • 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 Dithothreitol
  • 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.
  • wash buffer eg, 0.053 ⁇ 4 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.
  • 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.
  • 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.
  • 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.
  • Methods of 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.
  • methylation-sensitive restriction enzyme acts on the DNA. If so, the DN A will be disconnected.
  • 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).
  • Methods 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 DN ⁇ 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.
  • 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.
  • 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.
  • 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.
  • Hhal a methylation-sensitive restriction enzyme capable of digesting single-stranded DNA
  • the second step of the measurement method of the present invention may be performed as follows.
  • 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.
  • the TE buffer can be removed by pipetting or decanting.
  • digested material single-stranded DNA containing at least one or more amethylated CpG at the recognition site of the restriction enzyme
  • DNA purification DNA purification
  • the above washing operation is not always necessary.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a general restriction enzyme treatment method may be used as a method of digesting with a restriction enzyme that does not use the target DNA region as a recognition cleavage site.
  • 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.
  • 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.
  • 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.
  • 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.
  • methylation-sensitive restriction enzymes that can digest single strands
  • double-stranded DNA eg, HpaII, BstUL NarK SacII, fflial etc.
  • 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).
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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).
  • the resulting mixture is heated at 95 ° C. for several minutes to obtain single-stranded DNA (positive strand).
  • 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.
  • 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.
  • 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.
  • 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. '
  • 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.
  • 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.
  • 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.
  • PCR 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.
  • 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.
  • 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).
  • the reaction may be carried out by adding an appropriate amount of betaine, DMSO, etc. at times.
  • 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.
  • 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.
  • 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.
  • the real-time PCR method examples 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.
  • 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.
  • 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.
  • 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.
  • the amount of Kanuta oligonucleotide is at least 10 times, usually 100 times or more, compared to the target DNA region.
  • “adding a counter oligonucleotide when separating methylated single-stranded DNA” refers to methylated DNA samples derived from genomic DNA contained in biological samples.
  • 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.
  • 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.
  • 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.
  • DNA region that is 100% methylated in genomic DNA contained in a sample derived from a disease patient 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.
  • 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.
  • 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
  • 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.
  • the measurement method of the present invention makes it possible to diagnose various diseases by examining methylation abnormality.
  • 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.
  • 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.
  • 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 ⁇ 0.13 ⁇ 4 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.
  • oligonucleotide solutions 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.
  • oligonucleotide solutions were prepared.
  • N represents methylated cytosine.
  • 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:
  • PF and PR primer solutions
  • the methylated DNA in the target DNA region UBC SEQ ID NO: 21, methylated cytosine is also represented by C
  • 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 ⁇ .13 ⁇ 4 BSA-containing phosphate buffer (ImM K3 ⁇ 4P0 3 mM Na 2 ⁇ 7 ⁇ 20 , 154 mM NaCl pH 7.4)
  • the solution was stored refrigerated.
  • oligonucleotide solutions 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).
  • N represents methylated scissin.
  • N represents methylated cytosine
  • 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 ⁇ 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 ⁇ 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. .
  • Buffer 330 mM Tris-Acetate ⁇ 7,9, 660 mM KOAc, lOOmM Mg0Ac 2 , 5 mM Dithiothreitol
  • BSA Bovine serum albumin lmg / mL
  • 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.
  • buffer solution 330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM Mg0Ac 2 , 5 mM Dithiothreitol
  • BSA Bovine serum albumin l'mg / mL
  • PR2 5'-TCTGGATGTTGTAGTCAGACAG-3 '(SEQ ID NO: 26)
  • GPR7-2079-2176 5'- GTTGGCCACTGCGGAGTCGC
  • 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).
  • 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
  • GPR7-2079-2176 partially methylated oligonucleotide digested with Hha I GPR 7- 207 ⁇ 9-2176 / 98mer-HM (5)
  • amplification of DNA was not confirmed with the unmethylated oligonucleotide G 7-2079 _2176 / 981116 1 "-1; no amplification product was obtained.
  • 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.
  • 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].
  • 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.
  • oligonucleotide solution 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).
  • N represents methylated cytosine
  • N represents methylated cytosine
  • 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.
  • buffer solution 330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM MgOAc 2 , 5raM Dithiothreitol
  • BSA Bovine serum albumin lmg / mL
  • 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.
  • buffer solution 330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM MgOAc 2 , 5 mM Dithiothreitol
  • BSA Bovine serum albumin lmg / mL
  • 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.
  • PR2 5'-TCTGGATGTTGTAGTCAGACAG-3 '(SEQ ID NO: 26)
  • 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.
  • 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 ⁇ 7 ⁇ 2 0, 154mM NaCl pH7.4) solution] saved.
  • PR 1 5'-CTGGCCAAACTGGAGATCGC -3 '(SEQ ID NO: 29)
  • 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.
  • 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.
  • Sssl methylase manufactured by NEB
  • 10 x NEBuf fer 2 manufactured by NEB
  • S- adenosyl methionine 3.2 mM, NEB
  • 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.).
  • Solution B lOpg / lO ⁇ 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.
  • 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.
  • 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.
  • 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.
  • PR1 5'-CTGGCCAAACTGGAGATCGC-3 '(SEQ ID NO: 29)
  • 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
  • Piotin labeled 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 0.13 ⁇ 4 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.
  • 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.
  • PR 2 5'- GCGCCGGGTCCGGGCCC -3 '(SEQ ID NO: 46)
  • 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
  • Solution A lOOpg / lO ⁇ L TE solution
  • Solution B lOpg / lO ⁇ L TE solution
  • Solution D TE solution (negative control solution)
  • TE solution negative control solution
  • Solution MA 100pg / 10 ⁇ L TE solution
  • Solution MB 10pg / 10 L TE solution
  • Solution MD TE solution (negative control solution)
  • 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.
  • 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.
  • buffer 330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM MgOAc 2 , 5 mM Di thiothrei tol
  • 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.
  • PR 2 5 '-GCGCCGGGTCCGGGCCC -3' (SEQ ID NO: 46)
  • 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 ( ⁇ 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.
  • methylcytosine antibody 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.
  • PR 3 5'— AGACATGTGCTCACGTACGGT—3 ′ (SEQ ID NO: 54)
  • 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.
  • 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)
  • Solution MC TE solution (negative control solution)
  • 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.
  • ' ⁇ Target DNA region> methylcytosine is also represented by C
  • 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.
  • 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).
  • 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.
  • Target DNA region> (5-methylcytosine is also represented by C)
  • PCR reaction solution 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.
  • Piotin-labeled methylcytosine antibody obtained as a solution [Antibody 0.25 i / ll 0.13 ⁇ 4 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.
  • 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.
  • 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 ⁇ ) 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.
  • 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. .
  • 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 MC TE solution (negative control solution)
  • 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> Metalcytosine is also represented by C
  • Each of the DNA fragment T solution and the DNA fragment MT solution was subjected to the following treatment.
  • 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).
  • 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.
  • PR4 5'-AGTACAGATCTGGCGTTCTCG-3 '(SEQ ID NO: 69)
  • 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
  • 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).
  • Solution A 10ng / 5; «L TE solution.
  • Solution B lng / 5 ⁇ L TE solution
  • Solution D TE solution (negative control solution).
  • Solution MA 1 Ong / 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. .
  • NC Genetic Codon Sequence Commission 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.)
  • 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.
  • ⁇ -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,.
  • 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.
  • 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).
  • 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.
  • PR 3 5 '-AGACATGTGCTCACGTACGGT-3' (SEQ ID NO: 54)
  • the PCR reaction solution consists of ⁇ -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.
  • methylcytosine antibody manufactured by Aviva Systems Biology Co., Ltd.
  • Biotin Labeling Kit-NH 2 manufactured by Dojindo Laboratories
  • Piotin labeled The resulting Piochin labeled methylcytosine antibody: A solution [il 0.13 ⁇ 4 BSA-containing phosphate buffer one N antibody about 0.25 i (ImM KH 2 P0 4 , 3mM Na 2 HP0 ⁇ 7 ⁇ 2 0, 154mM NaCl pH7.4) solution] As refrigerated.
  • 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).
  • Solution B lng / 5 L TE solution
  • Solution D TE solution (negative control 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.
  • 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.
  • buffer solution 330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM MgOAc 2 , 5 mM Dithiothreitol
  • M 8_Rei 1 2 solution 5 LOOmM was added BSA solution 5 L of lmg /
  • 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.
  • PR4 5'-AGTACAGATCTGGCGTTCTCG-3 '(SEQ ID NO: 69)
  • Target DNA region> (5-methylcytosine is also represented by C)
  • 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.
  • 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 0.13 ⁇ 4 BSA-containing phosphate buffer (lmM KH 2 P0 4 , 3 mM Na 2 HP0: ⁇ 70, 154 mM NaCl pH 7.4) solution] Stored refrigerated.
  • 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.
  • PR 1 5'-CTGGCCAAACTGGAGATCGC-3 '(SEQ ID NO: 29)
  • 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%
  • 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).
  • Solution B lOpg / 10 / zL TE solution
  • Solution D TE solution (negative control solution) The following solution was prepared for the obtained DNA fragment MX.
  • 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)
  • 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).
  • buffer solution 330 niM Tris-Acetate
  • 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.
  • BSA Bovine serum .. albumin lmg / ml
  • Hhal 16 U methylation sensitive restriction enzyme
  • 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).
  • 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.
  • 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.
  • 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. .
  • 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.
  • Buffer A 1M sorbitol, 0.1M EDTA, pH 7.4
  • 2-mercaptoethanol final concentration 14 mM
  • 100 U zymolase 10 mg / ml
  • 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.
  • 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.
  • C 19 5'-ACTGGACCGCTATGGACGTGGCGGCGGTGT-3 '(SEQ ID NO: 61)
  • C 20 5'- GGCGGCGGCTCAATGACCTGTGGCGCCCGT -3 '(SEQ ID NO: 62)
  • 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).
  • 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).
  • 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.
  • the PCR reaction solution consists of ⁇ ⁇ ⁇ -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.
  • 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.
  • buffer A 1M sorbitol, 0.1M EDTA, pH 7.4
  • 2-mercaptoethanol final concentration 14 mM
  • 100 U zymolase 10 mg / ml
  • 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.
  • oligonucleotide primers PF 4 and PR 4
  • 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
  • PR4 5'-AGTACAGATCTGGCGTTCTCG-3 '(SEQ ID NO: 69)
  • 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 ⁇ 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.
  • DNA fragment T was purified by Wizard SV Gel / PCR Kit (PR0MEGA).
  • CTTGAGNGCATGTGCNGTTTCNGAGAANGCCAGATCTGTACT-3 ′ (SEQ ID NO: 71) The following solution was prepared for the obtained DNA fragment T.
  • Solution B lOpg / lO ⁇ L TE solution
  • Solution D TE solution (negative control solution) The following solution was prepared for the obtained DNA fragment MT.
  • Solution MB 10pg / 10 L TE solution
  • Solution MD TE solution (negative control solution)
  • 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.
  • a PCR tube 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, ⁇ 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.
  • buffer solution 330 mM Tris-Acetate, ⁇ 7.9, 660 mM K0Ac, lOOmM MgOAc 2 , 5 mM Di thiothrei and tol
  • 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.
  • 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.
  • BSA Bovine serum albumin lmg / ml
  • Hhal methylation-sensitive restriction enzyme
  • PR4 5'-AGTACAGATCTGGCGTTCTCG-3 '(SEQ ID NO: 6.9)
  • Target DNA region> (5-methylcytosine is also represented by C)
  • 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. ''
  • Solution B lng / 5 L TE solution
  • Solution C TE solution (negative control solution)
  • TE solution negative control solution
  • Solution MA 10ng / 5 L TE solution.
  • Solution M C TE solution (negative control solution)
  • 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.
  • a PCR tube 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • Solution A 10ng / 5 L TE solution
  • Solution D TE solution (negative control solution)
  • TE solution negative control 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.
  • 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.
  • C 19 5'-ACTGGACCGCTATGGACGTGGCGGCGGTGT-3 '(SEQ ID NO: 61)
  • C 20 5'- GGCGGCGGCTCAATGACCTGTGGCGCCCGT -3 '(SEQ ID NO: 62)
  • 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).
  • 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.
  • BSA Bovine serum albumin lmg / ml
  • Hhal methylation-sensitive restriction enzyme
  • 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).
  • 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.
  • 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.
  • 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. .
  • Biotin Labeling Kit_N Biotin Labeling Kit_N, manufactured by Dojindo Laboratories
  • 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.
  • 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.
  • yeast genome 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 D TE solution (negative control solution)
  • TE solution negative control 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.
  • DNA fragment T "(Genbank comprising the nucleotide sequence represented by SEQ ID NO: 78
  • 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)
  • buffer 330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM MgOAc 2 , 5 mM Dithiothreitol
  • 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.
  • 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.
  • BSA Bovine serum albumin lmg / ml
  • fflial methylation sensitive restriction enzyme
  • 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.
  • 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
  • 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

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  • Crystallography & Structural Chemistry (AREA)
  • Plant Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé pour mesurer le contenu d'un ADN méthylé dans une région d'ADN d'intérêt dans l'ADN génomique contenu dans un échantillon dérivé d'un organisme.
PCT/JP2008/056524 2007-03-26 2008-03-26 Procédé de mesure de méthylation dans un adn Ceased WO2008123537A1 (fr)

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JP2007-078659 2007-03-26
JP2007078659 2007-03-26

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WO2008123537A1 true WO2008123537A1 (fr) 2008-10-16

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
EP2272975A4 (fr) * 2008-03-25 2011-06-29 Sumitomo Chemical Co Procédé servant à déterminer la méthylation de l'adn
CN109234388A (zh) * 2017-07-04 2019-01-18 深圳华大基因研究院 用于dna高甲基化区域富集的试剂、富集方法及应用

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Publication number Priority date Publication date Assignee Title
JP5832760B2 (ja) 2011-02-28 2015-12-16 シスメックス株式会社 試料中のメチル化ｄｎａを検出する方法

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WO2006056478A1 (fr) * 2004-11-29 2006-06-01 Klinikum Der Universität Regensburg Trousses et procedes pour la detection d'adn methyle
WO2008038833A1 (fr) * 2006-09-27 2008-04-03 Sumitomo Chemical Company, Limited procÉdÉ de dÉtermination de la mÉthylation de l'adn

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WO2006056478A1 (fr) * 2004-11-29 2006-06-01 Klinikum Der Universität Regensburg Trousses et procedes pour la detection d'adn methyle
WO2008038833A1 (fr) * 2006-09-27 2008-04-03 Sumitomo Chemical Company, Limited procÉdÉ de dÉtermination de la mÉthylation de l'adn

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XU H.D. ET AL.: "Parentally imprinted allele typing at a short tandem repeat locus in intron 1a of imprinted gene KCNQ1", LEG. MED., vol. 8, 2006, pages 139 - 143, XP005458068 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2272975A4 (fr) * 2008-03-25 2011-06-29 Sumitomo Chemical Co Procédé servant à déterminer la méthylation de l'adn
CN109234388A (zh) * 2017-07-04 2019-01-18 深圳华大基因研究院 用于dna高甲基化区域富集的试剂、富集方法及应用
CN109234388B (zh) * 2017-07-04 2021-09-14 深圳华大生命科学研究院 用于dna高甲基化区域富集的试剂、富集方法及应用

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JP2008263961A (ja) 2008-11-06
JP5303981B2 (ja) 2013-10-02

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