WO2013047910A1 - Procédé d'évaluation du degré de cancérisation - Google Patents

Procédé d'évaluation du degré de cancérisation Download PDF

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WO2013047910A1
WO2013047910A1 PCT/JP2012/075879 JP2012075879W WO2013047910A1 WO 2013047910 A1 WO2013047910 A1 WO 2013047910A1 JP 2012075879 W JP2012075879 W JP 2012075879W WO 2013047910 A1 WO2013047910 A1 WO 2013047910A1
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seq
base sequence
homology
represented
complementary
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PCT/JP2012/075879
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Japanese (ja)
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祥隆 冨ケ原
佐藤　日出夫
弘和 樽井
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住友化学株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • the present invention relates to a method for evaluating the degree of canceration of a mammal-derived specimen.
  • the present invention relates to a DNA region represented by any one of SEQ ID NOs: 1 to 19 in a DNA present in a cancer tissue specimen or a biological sample such as blood, serum, plasma, body fluid, body secretion, feces and urine derived from a cancer patient.
  • a DNA present in a cancer tissue specimen or a biological sample such as blood, serum, plasma, body fluid, body secretion, feces and urine derived from a cancer patient.
  • biological samples such as immortalized normal cell lines or normal tissue samples, or blood, serum, plasma, body fluids, body secretions, manure, etc. Based on the knowledge that it is methylated at a significantly high frequency. That is, the present invention 1.
  • a method for evaluating the degree of canceration of a mammal-derived specimen (1) a first step of measuring a methylation frequency of one or more DNAs having a base sequence selected from the following base sequences contained in a mammal-derived specimen or an index value correlated therewith, and (2) measurement Evaluation method comprising a second step of determining the degree of canceration of the specimen based on a difference obtained by comparing the control value with a methylation frequency or an index value correlated therewith (hereinafter, evaluation of the present invention) (It may be described as a method.): (A) a base sequence represented by SEQ ID NO: 1 or a base sequence having 80% or more homology with the base sequence represented by SEQ ID NO: 1, (B) a nucleotide sequence complementary to SEQ ID NO: 1 or a nucleotide sequence having 80% or more homology with a nucleotide sequence complementary to SEQ ID NO: 1, (C) a base sequence represented by SEQ ID NO: 2 or a base sequence
  • the evaluation method according to 1 above, wherein the mammal-derived specimen is a cell; 3. 2. The evaluation method according to 1 above, wherein the mammal-derived specimen is a tissue; 4). The evaluation method according to 1 above, wherein the mammal-derived specimen is a biological sample; 5.
  • a method for evaluating the degree of canceration of a mammal-derived specimen (1) a first step of measuring the methylation frequency of one or more DNAs having a base sequence selected from the following base sequences contained in a mammal-derived specimen; and (2) the measured methylation frequency;
  • An evaluation method comprising a second step of determining the degree of canceration of the specimen based on a difference obtained by comparing with a control: (A) a base sequence represented by SEQ ID NO: 1 or a base sequence having 80% or more homology with the base sequence represented by SEQ ID NO: 1, (B) a nucleotide sequence complementary to SEQ ID NO: 1 or a nucleotide sequence having 80% or more homology with a nucleotide sequence complementary to SEQ ID NO: 1, (C) a base sequence represented by SEQ ID NO: 2 or a base sequence having 80% or more homology with the base sequence represented by SEQ ID NO: 2, (D) a nucleotide sequence complementary to SEQ ID NO:
  • the evaluation method according to 7 above, wherein the tissue is lung tissue; 11. 8. The evaluation method according to 7 above, wherein the tissue is mammary gland tissue; 12 9. The evaluation method according to 8 above, wherein the biological sample is any one of blood, serum, plasma, body fluid, body secretion, and excreta; 13.
  • the methylation frequency of the DNA is the methylation frequency of cytosine in one or more base sequences represented by 5′-CG-3 ′ present in the base sequence of the DNA.
  • Evaluation method of 14 A method for evaluating the degree of canceration of a mammal-derived specimen, (1) a first step of measuring an index value correlated with the methylation frequency of one or more DNAs having a base sequence selected from the following base sequences contained in a mammal-derived specimen; and (2) measured And a second step of determining the degree of canceration of the specimen based on a difference obtained by comparing the methylation frequency or an index value correlated therewith with a control: (A) a base sequence represented by SEQ ID NO: 1 or a base sequence having 80% or more homology with the base sequence represented by SEQ ID NO: 1, (B) a nucleotide sequence complementary to SEQ ID NO: 1 or a nucleotide sequence having 80% or more homology with a nucleotide sequence complementary to SEQ ID NO: 1, (C) a base sequence represented by SEQ ID NO: 2 or a base sequence having 80% or more homology with the base sequence represented by SEQ ID NO: 2, (D) a nu
  • the correlated index value is the amount of any expression product of a gene existing downstream of at least one DNA selected from the base sequence; 16. 16. The evaluation method according to 15 above, wherein the amount of the gene expression product is the amount of the gene transcription product;
  • FIG. 1 shows the methylation in the target DNA region consisting of the nucleotide sequence shown in SEQ ID NO: 1 for human breast gland healthy tissue genomic DNA samples (N01 and N02) and human breast cancer tissue genomic DNA samples (C01, C02, C03 and C04).
  • Target02_3_CpG in the figure and Target02_3 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 1
  • the scale on the upper side of the bar indicates the base number in SEQ ID NO: 1
  • the scale on the lower side of the bar indicates The position of cytosine that can be methylated in the base sequence represented by SEQ ID NO: 1 is shown.
  • Target02_3_CpG_1, Target02_3_CpG_3, Target02_3_CpG_4, Target02_3_CpG_5, Target02_3_CpG_8, Target02_3_CpG_9, Target02_3_CpG_10 and Target02_3_CpG_11 respectively cytosines represented by base No. 109,163,176,218,247,257,286 and 391 in the nucleotide sequence represented by SEQ ID NO: 1 The measurement result of a methylation ratio is shown.
  • Target02 — 3_CpG — 6.7 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 226 and 228 in the base sequence represented by SEQ ID NO: 1.
  • the percentage of CG methylation in human breast cancer tissue genomic DNA samples (C01, C02, C03 and C04) was higher than that in human breast healthy tissue genomic DNA (N01 and N02).
  • FIG. 2 shows the methylation in the target DNA region consisting of the base sequence shown in SEQ ID NO: 2 for human breast gland healthy tissue genomic DNA samples (N01 and N02) and human breast cancer tissue genomic DNA samples (C01, C02, C03 and C04). The result of having measured the ratio of oxydized DNA by MassARRAY analysis is shown.
  • Target03_9_CpG in the figure and Target03_9 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 2, the scale on the upper side of the bar indicates the base number in SEQ ID NO: 2, and the scale on the lower side of the bar indicates The position of cytosine that can be methylated in the base sequence shown in SEQ ID NO: 2 is shown.
  • Target03_9_CpG_6 and Target03_9_CpG_7 indicate the measurement results of the cytosine methylation ratios indicated by base numbers 383 and 391 in the base sequence indicated by SEQ ID NO: 2, respectively.
  • Target03_9_CpG_1.2 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 55 and 60 in the base sequence represented by SEQ ID NO: 2.
  • the percentage of CG methylation in human breast cancer tissue genomic DNA samples (C01, C02, C03 and C04) was higher than that in human breast healthy tissue genomic DNA (N01 and N02).
  • FIG. 3 shows the methylation in the target DNA region consisting of the base sequence shown in SEQ ID NO: 3 for human breast gland healthy tissue genomic DNA samples (N01 and N02) and human breast cancer tissue genomic DNA samples (C01, C02, C03 and C04). The result of having measured the ratio of oxydized DNA by MassARRAY analysis is shown.
  • Target 04_19_CpG in the figure and Target 04_19 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 3, the scale on the upper side of the bar indicates the base number in SEQ ID NO: 3, and the scale on the lower side of the bar indicates The position of cytosine that can be methylated in the base sequence shown in SEQ ID NO: 3 is shown.
  • Target04_19_CpG_4 and Target04_19_CpG_5 show the measurement results of the cytosine methylation ratios shown in base numbers 133 and 138 in the base sequence shown in SEQ ID NO: 3, respectively.
  • Target04_19_CpG_2.3 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 113 and 119 in the base sequence represented by SEQ ID NO: 3.
  • the percentage of CG methylation in human breast cancer tissue genomic DNA samples (C01, C02, C03 and C04) was higher than that in human breast healthy tissue genomic DNA (N01 and N02).
  • FIG. 4 shows the methylation in the target DNA region consisting of the base sequence shown in SEQ ID NO: 6 for human breast gland healthy tissue genomic DNA samples (N01 and N02) and human breast cancer tissue genomic DNA samples (C01, C02, C03 and C04). The result of having measured the ratio of oxydized DNA by MassARRAY analysis is shown.
  • Target09_15_CpG in the figure and Target09_15 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 6, the scale above the bar indicates the base number in SEQ ID NO: 6, and the scale below the bar indicates The position of cytosine that can be methylated in the base sequence represented by SEQ ID NO: 6 is shown.
  • Target09_15_CpG_1, Target09_15_CpG_2, Target09_15_CpG_6 and Target09_15_CpG_14 indicate the measurement results of the cytosine methylation ratios indicated by base numbers 27, 43, 118 and 220 in the base sequence indicated by SEQ ID NO: 6, respectively.
  • Target09_15_CpG_4.5 indicates the measurement result of the average methylation ratio of two cytosines represented by base numbers 97 and 102 in the base sequence represented by SEQ ID NO: 6.
  • Target09_15_CpG_7.8 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 131 and 138 in the base sequence represented by SEQ ID NO: 6.
  • Target09_15_CpG_9.10 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 152 and 157 in the base sequence represented by SEQ ID NO: 6.
  • Target09_15_CpG_12.13 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 213 and 216 in the base sequence represented by SEQ ID NO: 6.
  • Target09_15_CpG_15.16 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 229 and 234 in the base sequence represented by SEQ ID NO: 6.
  • the percentage of CG methylation in human breast cancer tissue genomic DNA samples (C01, C02, C03 and C04) was higher than that in human breast healthy tissue genomic DNA (N01 and N02).
  • Target 10_9_CpG in the figure and Target 10_9 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 7, the scale on the upper side of the bar indicates the base number in SEQ ID NO: 7, and the scale on the lower side of the bar indicates The position of cytosine that can be methylated in the base sequence represented by SEQ ID NO: 7 is shown.
  • Target10_9_CpG_3, Target10_9_CpG_6, Target10_9_CpG_8, and Target10_9_CpG_9 indicate the measurement results of the cytosine methylation ratios indicated by base numbers 106, 140, 174, and 237 in the base sequence indicated by SEQ ID NO: 7, respectively.
  • Target10_9_CpG_4.5 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 116 and 118 in the base sequence represented by SEQ ID NO: 7.
  • the percentage of CG methylation in human breast cancer tissue genomic DNA samples (C01, C02, C03 and C04) was higher than that in human breast healthy tissue genomic DNA (N01 and N02).
  • Target 11 — 3_CpG in the figure and Target 11 — 3 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 8, the scale on the upper side of the bar indicates the base number in SEQ ID NO: 8, and the scale on the lower side of the bar indicates: The position of cytosine that can be methylated in the base sequence represented by SEQ ID NO: 8 is shown.
  • Target11_3_CpG_1, Target11_3_CpG_5, Target11_3_CpG_6, and Target11_3_CpG_8 indicate the measurement results of the cytosine methylation ratios represented by base numbers 54, 240, 295, and 420 in the base sequence represented by SEQ ID NO: 8, respectively.
  • Target 11 — 3_CpG — 3.4 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 169 and 172 in the base sequence represented by SEQ ID NO: 8.
  • the percentage of CG methylation in human breast cancer tissue genomic DNA samples (C01, C02, C03 and C04) was higher than that in human breast healthy tissue genomic DNA (N01 and N02).
  • Target12_3_CpG in the figure and Target12_3 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 9, the scale on the upper side of the bar indicates the base number in SEQ ID NO: 9, and the scale on the lower side of the bar indicates The position of cytosine that can be methylated in the base sequence represented by SEQ ID NO: 9 is shown.
  • Target12_3_CpG_2, Target12_3_CpG_4, Target12_3_CpG_8, Target12_3_CpG_10, and Target12_3_CpG_12 have the base numbers 233, 326, 408, 453, and the results of the base numbers indicated by SEQ ID NO: 9.
  • Target12_3_CpG_5.6 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 378 and 383 in the base sequence represented by SEQ ID NO: 9.
  • the percentage of CG methylation in human breast cancer tissue genomic DNA samples (C01, C02, C03 and C04) was higher than that in human breast healthy tissue genomic DNA (N01 and N02).
  • Target 15_4_CpG in the figure and Target 15_4 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 10, the scale on the upper side of the bar indicates the base number in SEQ ID NO: 10, and the scale on the lower side of the bar indicates The position of cytosine that can be methylated in the base sequence represented by SEQ ID NO: 10 is shown.
  • Target15_4_CpG_3, Target15_4_CpG_4, Target15_4_CpG_10, Target15_4_CpG_11 and Target15_4_CpG_12 have the base numbers 78, 129, 264, 297 and 335, respectively, in the base sequence indicated by SEQ ID NO.
  • Target 15 — 4_CpG — 1.2 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 69 and 72 in the base sequence represented by SEQ ID NO: 10.
  • Target 15 — 4_CpG — 6.7 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 159 and 161 in the base sequence represented by SEQ ID NO: 10.
  • Target 15 — 4_CpG — 8.9 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 232 and 235 in the base sequence represented by SEQ ID NO: 10.
  • the percentage of CG methylation in human breast cancer tissue genomic DNA samples (C01, C02, C03 and C04) was higher than that in human breast healthy tissue genomic DNA (N01 and N02).
  • FIG. 9 shows the methylation in the target DNA region consisting of the base sequence shown in SEQ ID NO: 11 for human breast gland healthy tissue genomic DNA samples (N01 and N02) and human breast cancer tissue genomic DNA samples (C01, C02, C03 and C04). The result of having measured the ratio of oxydized DNA by MassARRAY analysis is shown.
  • Target 18_1_CpG in the figure and Target 18_1 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 11, the scale on the upper side of the bar indicates the base number in SEQ ID NO: 11, and the scale on the lower side of the bar indicates: The position of cytosine that can be methylated in the base sequence represented by SEQ ID NO: 11 is shown.
  • Target18_1_CpG_5.6 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 102 and 104 in the base sequence represented by SEQ ID NO: 11.
  • Target18_1_CpG — 9.10.11 shows the measurement result of the average methylation ratio of three cytosines represented by base numbers 164, 170 and 173 in the base sequence represented by SEQ ID NO: 11.
  • Target18_1_CpG — 12.13 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 198 and 201 in the base sequence represented by SEQ ID NO: 11.
  • Target18_1_CpG — 15.16 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 274 and 277 in the base sequence represented by SEQ ID NO: 11.
  • Target18_1_CpG — 17.18 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 296 and 307 in the base sequence represented by SEQ ID NO: 11.
  • Target18_1_CpG — 24.25 indicates the measurement result of the average methylation ratio of two cytosines represented by base numbers 401 and 403 in the base sequence represented by SEQ ID NO: 11.
  • FIG. 10 shows methyls in the target DNA region consisting of the base sequence shown in SEQ ID NO: 12 for human breast gland healthy tissue genomic DNA samples (N01 and N02) and human breast cancer tissue genomic DNA samples (C01, C02, C03 and C04). The result of having measured the ratio of oxydized DNA by MassARRAY analysis is shown.
  • Target 19_2_CpG in the figure and Target 19_2 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 12, the scale above the bar indicates the base number in SEQ ID NO: 12, and the scale below the bar indicates: The position of cytosine that can be methylated in the base sequence represented by SEQ ID NO: 12 is shown.
  • Target19_2_CpG_1.2 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 55 and 58 in the base sequence represented by SEQ ID NO: 12.
  • Target19_2_CpG_3.4 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 77 and 88 in the base sequence represented by SEQ ID NO: 12.
  • Target 19_2_CpG — 10.11 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 182 and 184 in the base sequence represented by SEQ ID NO: 12.
  • Target 19_2_CpG — 19.20.21 shows the measurement result of the average methylation ratio of three cytosines represented by base numbers 359, 362 and 368 in the base sequence represented by SEQ ID NO: 12.
  • the percentage of CG methylation in human breast cancer tissue genomic DNA samples (C01, C02, C03 and C04) was higher than that in human breast healthy tissue genomic DNA (N01 and N02).
  • FIG. 11 shows the methylation in the target DNA region consisting of the nucleotide sequence shown in SEQ ID NO: 13 for human breast gland healthy tissue genomic DNA samples (N01 and N02) and human breast cancer tissue genomic DNA samples (C01, C02, C03 and C04). The result of having measured the ratio of oxydized DNA by MassARRAY analysis is shown.
  • Target 21_13_CpG in the figure and Target 21_13 in the lower bar indicate DNA consisting of the base sequence shown in SEQ ID NO: 13, the scale on the upper side of the bar indicates the base number in SEQ ID NO: 13, and the scale on the lower side of the bar indicates The position of cytosine that can be methylated in the base sequence represented by SEQ ID NO: 13 is shown.
  • Target 21 — 13_CpG — 1.2 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 37 and 44 in the base sequence represented by SEQ ID NO: 13.
  • Target 21 — 13_CpG — 6.7 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 172 and 174 in the base sequence represented by SEQ ID NO: 13.
  • Target 21 — 13_CpG — 13.14 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 253 and 261 in the base sequence represented by SEQ ID NO: 13.
  • FIG. 12 shows methyls in the target DNA region consisting of the base sequence represented by SEQ ID NO: 14 for human breast gland healthy tissue genomic DNA samples (N01 and N02) and human breast cancer tissue genomic DNA samples (C01, C02, C03 and C04). The result of having measured the ratio of oxydized DNA by MassARRAY analysis is shown.
  • Target 23 — 11_CpG in the figure and Target 23 — 11 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 14, the scale on the upper side of the bar indicates the base number in SEQ ID NO: 14, and the scale on the lower side of the bar indicates: The position of cytosine that can be methylated in the base sequence represented by SEQ ID NO: 14 is shown.
  • Target23_11_CpG_6 and Target23_11_CpG_8 indicate the measurement results of the cytosine methylation ratios indicated by base numbers 116 and 257 in the base sequence indicated by SEQ ID NO: 14, respectively.
  • Target 23 — 11_CpG — 1.2 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 27 and 32 in the base sequence represented by SEQ ID NO: 14.
  • Target 23 — 11_CpG — 4.5 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 109 and 112 in the base sequence represented by SEQ ID NO: 14.
  • the percentage of CG methylation in human breast cancer tissue genomic DNA samples (C01, C02, C03 and C04) was higher than that in human breast healthy tissue genomic DNA (N01 and N02).
  • Target 40_18_CpG in the figure and Target 40_18 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 16, the scale on the upper side of the bar indicates the base number in SEQ ID NO: 16, and the scale on the lower side of the bar indicates The position of cytosine that can be methylated in the base sequence represented by SEQ ID NO: 16 is shown.
  • Target40_18_CpG_3 and Target40_18_CpG_5 indicate the measurement results of the cytosine methylation ratios indicated by base numbers 148 and 243 in the base sequence indicated by SEQ ID NO: 16, respectively.
  • the percentage of CG methylation in human breast cancer tissue genomic DNA samples (C01, C02, C03 and C04) was higher than that in human breast healthy tissue genomic DNA (N01 and N02).
  • FIG. 14 is intended to consist of the base sequences represented by SEQ ID NO: 17 and SEQ ID NO: 18 for human breast healthy tissue genomic DNA samples (N01 and N02) and human breast cancer tissue genomic DNA samples (C01, C02, C03 and C04).
  • Target 41_10_CpG in the figure and Target 41_10 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 17, the scale on the upper side of the bar indicates the base number in SEQ ID NO: 17, and the scale on the lower side of the bar indicates The position of cytosine that can be methylated in the base sequence represented by SEQ ID NO: 17 is shown.
  • Target41_10_CpG_1, Target41_10_CpG3, and Target41_10_CpG_6 indicate the measurement results of the cytosine methylation ratios indicated by base numbers 42, 72, and 185 in the base sequence indicated by SEQ ID NO: 17, respectively.
  • Target 41 — 10_CpG — 7.8 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 206 and 211 in the base sequence represented by SEQ ID NO: 17.
  • Target 41_17 represents DNA having the base sequence represented by SEQ ID NO: 18, the scale above the bar represents the base number in SEQ ID NO: 18, and the scale below the bar represents methyl in the base sequence represented by SEQ ID NO: 18. The position of cytosine that can be converted is shown.
  • Target41_17_CpG_2, Target41_17_CpG_6, Target41_17_CpG_7, Target41_17_CpG_8, and Target41_17_CpG_9 represent the base numbers 49, 254, 277, 305, and 333, respectively, in the base sequence indicated by SEQ ID NO: 18.
  • Target41_17_CpG_3.4 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 58 and 61 in the base sequence represented by SEQ ID NO: 18. The percentage of CG methylation in human breast cancer tissue genomic DNA samples (C01, C02, C03 and C04) was higher than that in human breast healthy tissue genomic DNA (N01 and N02).
  • Target 44_9_CpG in the figure and Target 44_9 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 19, the scale on the upper side of the bar indicates the base number in SEQ ID NO: 19, and the scale on the lower side of the bar indicates The position of cytosine that can be methylated in the base sequence represented by SEQ ID NO: 19 is shown.
  • Target 44 — 9_CpG — 13.14 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 254 and 258 in the base sequence represented by SEQ ID NO: 19.
  • Target 44 — 9_CpG — 16.17 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 286 and 288 in the base sequence represented by SEQ ID NO: 19.
  • Target 44_9_CpG — 18.19 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 297 and 303 in the base sequence represented by SEQ ID NO: 19.
  • Target44_9_CpG_22.23 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 333 and 335 in the base sequence represented by SEQ ID NO: 19.
  • Target 44 — 9_CpG — 25.26 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 389 and 392 in the base sequence represented by SEQ ID NO: 19.
  • Target44_9_CpG_29.30 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 435 and 438 in the base sequence represented by SEQ ID NO: 19.
  • Target 44_9_CpG — 32.33 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 453 and 456 in the base sequence represented by SEQ ID NO: 19.
  • the percentage of CG methylation in human breast cancer tissue genomic DNA samples (C01, C02, C03 and C04) was higher than that in human breast healthy tissue genomic DNA (N01 and N02).
  • FIG. 16 shows methylated DNA in a target DNA region comprising the base sequence represented by SEQ ID NO: 1 for human lung healthy tissue genomic DNA samples (N01 and N02) and human lung cancer tissue genomic DNA samples (C01, C02 and C03). The result of having measured the ratio by MassARRAY analysis is shown.
  • Target02_3_CpG in the figure and Target02_3 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 1, the scale on the upper side of the bar indicates the base number in SEQ ID NO: 1, and the scale on the lower side of the bar indicates The position of cytosine that can be methylated in the base sequence represented by SEQ ID NO: 1 is shown.
  • Target02_3_CpG_1, Target02_3_CpG_3, Target02_3_CpG_4, Target02_3_CpG_5, Target02_3_CpG_8, Target02_3_CpG_9, Target02_3_CpG_10 and Target02_3_CpG_11 respectively cytosines represented by base No.
  • Target02 — 3_CpG — 6.7 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 226 and 228 in the base sequence represented by SEQ ID NO: 1.
  • the CG methylation rate in human lung cancer tissue genomic DNA samples (C01, C02 and C03) was higher than that in human lung healthy tissue genomic DNA (N01 and N02).
  • Target 04_19_CpG in the figure and Target 04_19 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 3, the scale on the upper side of the bar indicates the base number in SEQ ID NO: 3, and the scale on the lower side of the bar indicates The position of cytosine that can be methylated in the base sequence shown in SEQ ID NO: 3 is shown.
  • Target04_19_CpG_4 and Target04_19_CpG_5 show the measurement results of the cytosine methylation ratios shown in base numbers 133 and 138 in the base sequence shown in SEQ ID NO: 3, respectively.
  • Target04_19_CpG_2.3 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 113 and 119 in the base sequence represented by SEQ ID NO: 3.
  • the CG methylation rate in human lung cancer tissue genomic DNA samples (C01, C02 and C03) was higher than that in human lung healthy tissue genomic DNA (N01 and N02).
  • Target06_21_CpG in the figure and Target06_21 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 4, the scale above the bar indicates the base number in SEQ ID NO: 4, and the scale below the bar indicates The position of cytosine that can be methylated in the base sequence shown in SEQ ID NO: 4 is shown.
  • Target06_21_CpG_1, Target06_21_CpG_3 and Target06_21_CpG_6 indicate the measurement results of the cytosine methylation ratios indicated by base numbers 69, 113 and 265 in the base sequence indicated by SEQ ID NO: 4, respectively.
  • the CG methylation rate in human lung cancer tissue genomic DNA samples (C01, C02 and C03) was higher than that in human lung healthy tissue genomic DNA (N01 and N02).
  • FIG. 19 shows methylated DNA in a target DNA region comprising the base sequence represented by SEQ ID NO: 6 for human lung healthy tissue genomic DNA samples (N01 and N02) and human lung cancer tissue genomic DNA samples (C01, C02 and C03). The result of having measured the ratio by MassARRAY analysis is shown.
  • Target09_15_CpG below the bar Target09_15 indicates the DNA comprising the base sequence shown in SEQ ID NO: 6, the scale above the bar indicates the base number in SEQ ID NO: 6, and the scale below the bar indicates the sequence The position of cytosine that can be methylated in the base sequence shown by No. 6 is shown.
  • Target09_15_CpG_1, Target09_15_CpG_2, Target09_15_CpG_6 and Target09_15_CpG_14 indicate the measurement results of the cytosine methylation ratios indicated by base numbers 27, 43, 118 and 220 in the base sequence indicated by SEQ ID NO: 6, respectively.
  • Target09_15_CpG_4.5 indicates the measurement result of the average methylation ratio of two cytosines represented by base numbers 97 and 102 in the base sequence represented by SEQ ID NO: 6.
  • Target09_15_CpG_7.8 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 131 and 138 in the base sequence represented by SEQ ID NO: 6.
  • Target09_15_CpG_9.10 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 152 and 157 in the base sequence represented by SEQ ID NO: 6.
  • Target09_15_CpG_12.13 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 213 and 216 in the base sequence represented by SEQ ID NO: 6.
  • Target09_15_CpG_15.16 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 229 and 234 in the base sequence represented by SEQ ID NO: 6.
  • the CG methylation rate in human lung cancer tissue genomic DNA samples (C01, C02 and C03) was higher than that in human lung healthy tissue genomic DNA (N01 and N02).
  • Target 11 — 3_CpG in the figure and Target 11 — 3 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 8, the scale on the upper side of the bar indicates the base number in SEQ ID NO: 8, and the scale on the lower side of the bar indicates: The position of cytosine that can be methylated in the base sequence represented by SEQ ID NO: 8 is shown.
  • Target11_3_CpG_1, Target11_3_CpG_5, Target11_3_CpG_6, and Target11_3_CpG_8 indicate the measurement results of the cytosine methylation ratios represented by base numbers 54, 240, 295, and 420 in the base sequence represented by SEQ ID NO: 8, respectively.
  • Target 11 — 3_CpG — 3.4 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 169 and 172 in the base sequence represented by SEQ ID NO: 8.
  • the CG methylation rate in human lung cancer tissue genomic DNA samples (C01, C02 and C03) was higher than that in human lung healthy tissue genomic DNA (N01 and N02).
  • Target 15_4_CpG in the figure and Target 15_4 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 10, the scale on the upper side of the bar indicates the base number in SEQ ID NO: 10, and the scale on the lower side of the bar indicates The position of cytosine that can be methylated in the base sequence represented by SEQ ID NO: 10 is shown.
  • Target15_4_CpG_3, Target15_4_CpG_4, Target15_4_CpG_10, Target15_4_CpG_11 and Target15_4_CpG_12 have the base numbers 78, 129, 264, 297 and 335, respectively, in the base sequence indicated by SEQ ID NO.
  • Target 15 — 4_CpG — 1.2 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 69 and 72 in the base sequence represented by SEQ ID NO: 10.
  • Target 15 — 4_CpG — 6.7 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 159 and 161 in the base sequence represented by SEQ ID NO: 10.
  • Target 15 — 4_CpG — 8.9 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 232 and 235 in the base sequence represented by SEQ ID NO: 10.
  • the CG methylation rate in human lung cancer tissue genomic DNA samples (C01, C02 and C03) was higher than that in human lung healthy tissue genomic DNA (N01 and N02).
  • FIG. 22 shows methylated DNA in a target DNA region comprising the base sequence represented by SEQ ID NO: 11 for human lung healthy tissue genomic DNA samples (N01 and N02) and human lung cancer tissue genomic DNA samples (C01, C02 and C03). The result of having measured the ratio by MassARRAY analysis is shown.
  • Target 18_1_CpG in the figure and Target 18_1 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 11, the scale on the upper side of the bar indicates the base number in SEQ ID NO: 11, and the scale on the lower side of the bar indicates: The position of cytosine that can be methylated in the base sequence represented by SEQ ID NO: 11 is shown.
  • Target18_1_CpG_5.6 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 102 and 104 in the base sequence represented by SEQ ID NO: 11.
  • Target18_1_CpG — 9.10.11 shows the measurement result of the average methylation ratio of three cytosines represented by base numbers 164, 170 and 173 in the base sequence represented by SEQ ID NO: 11.
  • Target18_1_CpG — 12.13 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 198 and 201 in the base sequence represented by SEQ ID NO: 11.
  • Target18_1_CpG — 15.16 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 274 and 277 in the base sequence represented by SEQ ID NO: 11.
  • Target18_1_CpG — 17.18 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 296 and 307 in the base sequence represented by SEQ ID NO: 11.
  • Target18_1_CpG — 24.25 indicates the measurement result of the average methylation ratio of two cytosines represented by base numbers 401 and 403 in the base sequence represented by SEQ ID NO: 11.
  • FIG. 23 shows methylated DNA in a target DNA region comprising the base sequence represented by SEQ ID NO: 12 for human lung healthy tissue genomic DNA samples (N01 and N02) and human lung cancer tissue genomic DNA samples (C01, C02 and C03). The result of having measured the ratio by MassARRAY analysis is shown.
  • Target 19_2_CpG in the figure and Target 19_2 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 12, the scale above the bar indicates the base number in SEQ ID NO: 12, and the scale below the bar indicates: The position of cytosine that can be methylated in the base sequence represented by SEQ ID NO: 12 is shown.
  • Target19_2_CpG_1.2 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 55 and 58 in the base sequence represented by SEQ ID NO: 12.
  • Target19_2_CpG_3.4 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 77 and 88 in the base sequence represented by SEQ ID NO: 12.
  • Target 19_2_CpG — 10.11 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 182 and 184 in the base sequence represented by SEQ ID NO: 12.
  • Target 19_2_CpG — 19.20.21 shows the measurement result of the average methylation ratio of three cytosines represented by base numbers 359, 362 and 368 in the base sequence represented by SEQ ID NO: 12.
  • the CG methylation rate in human lung cancer tissue genomic DNA samples (C01, C02 and C03) was higher than that in human lung healthy tissue genomic DNA (N01 and N02).
  • FIG. 24 shows methylated DNA in a target DNA region comprising the base sequence represented by SEQ ID NO: 13 for human lung healthy tissue genomic DNA samples (N01 and N02) and human lung cancer tissue genomic DNA samples (C01, C02 and C03). The result of having measured the ratio by MassARRAY analysis is shown.
  • Target 21_13_CpG in the figure and Target 21_13 in the lower bar indicate DNA consisting of the base sequence shown in SEQ ID NO: 13, the scale on the upper side of the bar indicates the base number in SEQ ID NO: 13, and the scale on the lower side of the bar indicates The position of cytosine that can be methylated in the base sequence represented by SEQ ID NO: 13 is shown.
  • Target 21 — 13_CpG — 1.2 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 37 and 44 in the base sequence represented by SEQ ID NO: 13.
  • Target 21 — 13_CpG — 6.7 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 172 and 174 in the base sequence represented by SEQ ID NO: 13.
  • Target 21 — 13_CpG — 13.14 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 253 and 261 in the base sequence represented by SEQ ID NO: 13.
  • FIG. 25 shows methylated DNA in a target DNA region comprising the base sequence represented by SEQ ID NO: 15 for human lung healthy tissue genomic DNA samples (N01 and N02) and human lung cancer tissue genomic DNA samples (C01, C02 and C03). The result of having measured the ratio by MassARRAY analysis is shown.
  • Target 33_9_CpG in the figure and Target 39_9 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 15, the scale above the bar indicates the base number in SEQ ID NO: 15, and the scale below the bar indicates The position of cytosine that can be methylated in the base sequence represented by SEQ ID NO: 15 is shown.
  • Target33_9_CpG_4.5.6.7 shows the measurement result of the average methylation ratio of four cytosines represented by base numbers 65, 67, 71 and 74 in the base sequence represented by SEQ ID NO: 15.
  • the CG methylation rate in human lung cancer tissue genomic DNA samples (C01, C02 and C03) was higher than that in human lung healthy tissue genomic DNA (N01 and N02).
  • FIG. 26 shows methylated DNA in a target DNA region comprising the base sequence represented by SEQ ID NO: 16 for human lung healthy tissue genomic DNA samples (N01 and N02) and human lung cancer tissue genomic DNA samples (C01, C02 and C03). The result of having measured the ratio by MassARRAY analysis is shown.
  • Target 40_18_CpG in the figure and Target 40_18 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 16, the scale on the upper side of the bar indicates the base number in SEQ ID NO: 16, and the scale on the lower side of the bar indicates The position of cytosine that can be methylated in the base sequence represented by SEQ ID NO: 16 is shown.
  • Target40_18_CpG_3 and Target40_18_CpG_5 indicate the measurement results of the cytosine methylation ratios indicated by base numbers 148 and 243 in the base sequence indicated by SEQ ID NO: 16, respectively.
  • FIG. 27 shows methylated DNA in a target DNA region comprising the base sequence represented by SEQ ID NO: 19 for human lung healthy tissue genomic DNA samples (N01 and N02) and human lung cancer tissue genomic DNA samples (C01, C02 and C03). The result of having measured the ratio by MassARRAY analysis is shown.
  • Target 44_9_CpG in the figure and Target 44_9 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 19, the scale on the upper side of the bar indicates the base number in SEQ ID NO: 19, and the scale on the lower side of the bar indicates The position of cytosine that can be methylated in the base sequence represented by SEQ ID NO: 19 is shown.
  • Target 44 — 9_CpG — 13.14 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 254 and 258 in the base sequence represented by SEQ ID NO: 19.
  • Target 44 — 9_CpG — 16.17 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 286 and 288 in the base sequence represented by SEQ ID NO: 19.
  • Target 44_9_CpG — 18.19 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 297 and 303 in the base sequence represented by SEQ ID NO: 19.
  • Target44_9_CpG_22.23 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 333 and 335 in the base sequence represented by SEQ ID NO: 19.
  • Target 44 — 9_CpG — 25.26 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 389 and 392 in the base sequence represented by SEQ ID NO: 19.
  • Target44_9_CpG_29.30 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 435 and 438 in the base sequence represented by SEQ ID NO: 19.
  • Target 44_9_CpG — 32.33 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 453 and 456 in the base sequence represented by SEQ ID NO: 19.
  • the CG methylation rate in human lung cancer tissue genomic DNA samples (C01, C02 and C03) was higher than that in human lung healthy tissue genomic DNA (N01 and N02).
  • FIG. 28 shows a target DNA region consisting of the base sequence represented by SEQ ID NO: 1 for human colon healthy tissue genomic DNA samples (N01 and N02) and human colon cancer tissue genomic DNA samples (C01, C02, C03 and C04). The result of having measured the ratio of methylated DNA in is by MassARRAY analysis is shown.
  • Target02_3_CpG in the figure and Target02_3 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 1, the scale on the upper side of the bar indicates the base number in SEQ ID NO: 1, and the scale on the lower side of the bar indicates The position of cytosine that can be methylated in the base sequence represented by SEQ ID NO: 1 is shown.
  • Target02_3_CpG_1, Target02_3_CpG_3, Target02_3_CpG_4, Target02_3_CpG_5, Target02_3_CpG_8, Target02_3_CpG_9, Target02_3_CpG_10 and Target02_3_CpG_11 respectively cytosines represented by base No.
  • Target02 — 3_CpG — 6.7 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 226 and 228 in the base sequence represented by SEQ ID NO: 1.
  • the CG methylation rate in human colon cancer tissue genomic DNA samples (C01, C02, C03 and C04) was higher than that in human colon healthy tissue genomic DNA (N01 and N02).
  • Target 04_19_CpG in the figure and Target 04_19 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 3, the scale on the upper side of the bar indicates the base number in SEQ ID NO: 3, and the scale on the lower side of the bar indicates The position of cytosine that can be methylated in the base sequence shown in SEQ ID NO: 3 is shown.
  • Target04_19_CpG_4 and Target04_19_CpG_5 show the measurement results of the cytosine methylation ratios shown in base numbers 133 and 138 in the base sequence shown in SEQ ID NO: 3, respectively.
  • Target04_19_CpG_2.3 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 113 and 119 in the base sequence represented by SEQ ID NO: 3.
  • the CG methylation rate in human colon cancer tissue genomic DNA samples (C01, C02, C03 and C04) was higher than that in human colon healthy tissue genomic DNA (N01 and N02).
  • Target08_6_CpG in the figure and Target08_6 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 5, the scale on the upper side of the bar indicates the base number in SEQ ID NO: 5, and the scale on the lower side of the bar indicates The position of cytosine that can be methylated in the base sequence shown in SEQ ID NO: 5 is shown.
  • Target08_6_CpG_1, Target08_6_CpG_2, Target08_6_CpG_3, and Target08_6_CpG_5 represent the cytosine methylation ratio measurement results represented by base numbers 184, 212, 263, and 24 in the base sequence represented by SEQ ID NO: 5, respectively.
  • the CG methylation rate in human colon cancer tissue genomic DNA samples (C01, C02, C03 and C04) was higher than that in human colon healthy tissue genomic DNA (N01 and N02).
  • FIG. 31 shows a target DNA region comprising the base sequence represented by SEQ ID NO: 6 for human colon healthy tissue genomic DNA samples (N01 and N02) and human colon cancer tissue genomic DNA samples (C01, C02, C03 and C04).
  • Target09_15_CpG in the figure and Target09_15 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 6, the scale above the bar indicates the base number in SEQ ID NO: 6, and the scale below the bar indicates The position of cytosine that can be methylated in the base sequence represented by SEQ ID NO: 6 is shown.
  • Target09_15_CpG_1, Target09_15_CpG_2, Target09_15_CpG_6 and Target09_15_CpG_14 indicate the measurement results of the cytosine methylation ratios indicated by base numbers 27, 43, 118 and 220 in the base sequence indicated by SEQ ID NO: 6, respectively.
  • Target09_15_CpG_4.5 indicates the measurement result of the average methylation ratio of two cytosines represented by base numbers 97 and 102 in the base sequence represented by SEQ ID NO: 6.
  • Target09_15_CpG_7.8 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 131 and 138 in the base sequence represented by SEQ ID NO: 6.
  • Target09_15_CpG_9.10 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 152 and 157 in the base sequence represented by SEQ ID NO: 6.
  • Target09_15_CpG_12.13 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 213 and 216 in the base sequence represented by SEQ ID NO: 6.
  • Target09_15_CpG_15.16 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 229 and 234 in the base sequence represented by SEQ ID NO: 6.
  • the CG methylation rate in human colon cancer tissue genomic DNA samples (C01, C02, C03 and C04) was higher than that in human colon healthy tissue genomic DNA (N01 and N02).
  • Target 10_9_CpG in the figure and Target 10_9 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 7, the scale on the upper side of the bar indicates the base number in SEQ ID NO: 7, and the scale on the lower side of the bar indicates The position of cytosine that can be methylated in the base sequence represented by SEQ ID NO: 7 is shown.
  • Target10_9_CpG_3, Target10_9_CpG_6, Target10_9_CpG_8, and Target10_9_CpG_9 indicate the measurement results of the cytosine methylation ratios indicated by base numbers 106, 140, 174, and 237 in the base sequence indicated by SEQ ID NO: 7, respectively.
  • Target10_9_CpG_4.5 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 116 and 118 in the base sequence represented by SEQ ID NO: 7.
  • the CG methylation rate in human colon cancer tissue genomic DNA samples (C01, C02, C03 and C04) was higher than that in human colon healthy tissue genomic DNA (N01 and N02).
  • Target12_3_CpG in the figure and Target12_3 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 9, the scale on the upper side of the bar indicates the base number in SEQ ID NO: 9, and the scale on the lower side of the bar indicates The position of cytosine that can be methylated in the base sequence represented by SEQ ID NO: 9 is shown.
  • Target12_3_CpG_2, Target12_3_CpG_4, Target12_3_CpG_8, Target12_3_CpG_10, and Target12_3_CpG_12 have the base numbers 233, 326, 408, 453, and the results of the base numbers indicated by SEQ ID NO: 9.
  • Target12_3_CpG_5.6 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 378 and 383 in the base sequence represented by SEQ ID NO: 9.
  • the CG methylation rate in human colon cancer tissue genomic DNA samples (C01, C02, C03 and C04) was higher than that in human colon healthy tissue genomic DNA (N01 and N02).
  • Target 15_4_CpG in the figure and Target 15_4 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 10, the scale on the upper side of the bar indicates the base number in SEQ ID NO: 10, and the scale on the lower side of the bar indicates The position of cytosine that can be methylated in the base sequence represented by SEQ ID NO: 10 is shown.
  • Target15_4_CpG_3, Target15_4_CpG_4, Target15_4_CpG_10, Target15_4_CpG_11 and Target15_4_CpG_12 have the base numbers 78, 129, 264, 297 and 335, respectively, in the base sequence indicated by SEQ ID NO.
  • Target 15 — 4_CpG — 1.2 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 69 and 72 in the base sequence represented by SEQ ID NO: 10.
  • Target 15 — 4_CpG — 6.7 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 159 and 161 in the base sequence represented by SEQ ID NO: 10.
  • Target 15 — 4_CpG — 8.9 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 232 and 235 in the base sequence represented by SEQ ID NO: 10.
  • the CG methylation rate in human colon cancer tissue genomic DNA samples (C01, C02, C03 and C04) was higher than that in human colon healthy tissue genomic DNA (N01 and N02).
  • FIG. 35 shows a target DNA region comprising the base sequence shown in SEQ ID NO: 11 for human colon healthy tissue genomic DNA samples (N01 and N02) and human colon cancer tissue genomic DNA samples (C01, C02, C03 and C04). The result of having measured the ratio of methylated DNA in is by MassARRAY analysis is shown.
  • Target 18_1_CpG in the figure and Target 18_1 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 11, the scale on the upper side of the bar indicates the base number in SEQ ID NO: 11, and the scale on the lower side of the bar indicates: The position of cytosine that can be methylated in the base sequence represented by SEQ ID NO: 11 is shown.
  • Target18_1_CpG_5.6 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 102 and 104 in the base sequence represented by SEQ ID NO: 11.
  • Target18_1_CpG — 9.10.11 shows the measurement result of the average methylation ratio of three cytosines represented by base numbers 164, 170 and 173 in the base sequence represented by SEQ ID NO: 11.
  • Target18_1_CpG — 12.13 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 198 and 201 in the base sequence represented by SEQ ID NO: 11.
  • Target18_1_CpG — 15.16 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 274 and 277 in the base sequence represented by SEQ ID NO: 11.
  • Target18_1_CpG — 17.18 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 296 and 307 in the base sequence represented by SEQ ID NO: 11.
  • Target18_1_CpG — 24.25 indicates the measurement result of the average methylation ratio of two cytosines represented by base numbers 401 and 403 in the base sequence represented by SEQ ID NO: 11.
  • FIG. 36 shows a target DNA region consisting of the nucleotide sequence shown in SEQ ID NO: 12 for human colon healthy tissue genomic DNA samples (N01 and N02) and human colon cancer tissue genomic DNA samples (C01, C02, C03 and C04). The result of having measured the ratio of methylated DNA in is by MassARRAY analysis is shown.
  • Target 19_2_CpG in the figure and Target 19_2 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 12, the scale above the bar indicates the base number in SEQ ID NO: 12, and the scale below the bar indicates: The position of cytosine that can be methylated in the base sequence represented by SEQ ID NO: 12 is shown.
  • Target19_2_CpG_1.2 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 55 and 58 in the base sequence represented by SEQ ID NO: 12.
  • Target19_2_CpG_3.4 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 77 and 88 in the base sequence represented by SEQ ID NO: 12.
  • Target 19_2_CpG — 10.11 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 182 and 184 in the base sequence represented by SEQ ID NO: 12.
  • Target 19_2_CpG — 19.20.21 shows the measurement result of the average methylation ratio of three cytosines represented by base numbers 359, 362 and 368 in the base sequence represented by SEQ ID NO: 12.
  • the CG methylation rate in human colon cancer tissue genomic DNA samples (C01, C02, C03 and C04) was higher than that in human colon healthy tissue genomic DNA (N01 and N02).
  • FIG. 37 shows a target DNA region comprising the base sequence represented by SEQ ID NO: 13 for human colon healthy tissue genomic DNA samples (N01 and N02) and human colon cancer tissue genomic DNA samples (C01, C02, C03 and C04).
  • the result of having measured the ratio of methylated DNA in is by MassARRAY analysis is shown.
  • Target 21_13_CpG in the figure and Target 21_13 in the lower bar indicate DNA consisting of the base sequence shown in SEQ ID NO: 13, the scale on the upper side of the bar indicates the base number in SEQ ID NO: 13, and the scale on the lower side of the bar indicates The position of cytosine that can be methylated in the base sequence represented by SEQ ID NO: 13 is shown.
  • Target 21 — 13_CpG — 1.2 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 37 and 44 in the base sequence represented by SEQ ID NO: 13.
  • Target 21 — 13_CpG — 6.7 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 172 and 174 in the base sequence represented by SEQ ID NO: 13.
  • Target 21 — 13_CpG — 13.14 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 253 and 261 in the base sequence represented by SEQ ID NO: 13.
  • FIG. 38 shows a target DNA region consisting of the nucleotide sequence shown in SEQ ID NO: 14 for human colon healthy tissue genomic DNA samples (N01 and N02) and human colon cancer tissue genomic DNA samples (C01, C02, C03 and C04). The result of having measured the ratio of methylated DNA in is by MassARRAY analysis is shown.
  • Target 23 — 11_CpG in the figure and Target 23 — 11 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 14, the scale on the upper side of the bar indicates the base number in SEQ ID NO: 14, and the scale on the lower side of the bar indicates: The position of cytosine that can be methylated in the base sequence represented by SEQ ID NO: 14 is shown.
  • Target23_11_CpG_6 and Target23_11_CpG_8 indicate the measurement results of the cytosine methylation ratios indicated by base numbers 116 and 257 in the base sequence indicated by SEQ ID NO: 14, respectively.
  • Target 23 — 11_CpG — 1.2 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 27 and 32 in the base sequence represented by SEQ ID NO: 14.
  • Target 23 — 11_CpG — 4.5 shows the measurement result of the average methylation ratio of two cytosines represented by base numbers 109 and 112 in the base sequence represented by SEQ ID NO: 14.
  • the CG methylation rate in human colon cancer tissue genomic DNA samples (C01, C02, C03 and C04) was higher than that in human colon healthy tissue genomic DNA (N01 and N02).
  • Target 33_9_CpG in the figure and Target 33_9 in the lower bar indicate DNA consisting of the base sequence represented by SEQ ID NO: 15, the scale on the upper side of the bar indicates the base number in SEQ ID NO: 15, and the scale on the lower side of the bar indicates The position of cytosine that can be methylated in the base sequence represented by SEQ ID NO: 15 is shown.
  • Target33_9_CpG_4.5.6.7 shows the measurement result of the average methylation ratio of four cytosines represented by base numbers 65, 67, 71 and 74 in the base sequence represented by SEQ ID NO: 15.
  • the CG methylation rate in human colon cancer tissue genomic DNA samples (C01, C02, C03 and C04) was higher than that in human colon healthy tissue genomic DNA (N01 and N02).
  • the present invention relates to the use of methylated DNA as a cancer marker (eg, colon cancer marker, breast cancer marker, lung cancer marker, etc.).
  • “Cancer” in the present invention includes, for example, lung cancer (non-small cell lung cancer, small cell lung cancer), esophageal cancer, stomach cancer, duodenal cancer, colon cancer, rectal cancer, liver cancer (hepatocellular carcinoma, cholangiocellular carcinoma), gallbladder cancer, Bile duct cancer, pancreatic cancer, colon cancer, anal cancer, breast cancer, cervical cancer, endometrial cancer, uterine cancer, ovarian cancer, vulvar cancer, vaginal cancer, prostate cancer, kidney cancer, ureteral cancer, bladder cancer, prostate cancer, penis Cancer, testicular cancer, maxillary cancer, tongue cancer, (upper, middle, lower) pharyngeal cancer, laryngeal cancer, acute myeloid leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia
  • a subject who develops cancer is described as a “cancer patient”, a subject who does not develop cancer is described as a “non-cancer patient”, and a non-cancer site in human tissue or a subject who does not develop cancer
  • the tissue collected from the above is referred to as “normal tissue”
  • the blood collected from the subject who has not developed cancer is referred to as “normal blood”.
  • the “cancer marker” in the present invention include a tissue in which cancer occurs in a mammal and an index that can indirectly grasp the degree of canceration.
  • a colorectal cancer marker there is an index made of a biological substance that can indirectly grasp the presence or absence of colorectal cancer, the degree of canceration of colorectal cancer, the nature of cancer described as benign or malignant, etc. be able to.
  • the DNA used as the marker DNA in the present invention include one or more DNAs (hereinafter sometimes referred to as the present DNA) having a base sequence selected from the following base sequences.
  • the nucleotide sequence represented by SEQ ID NO: 1 Genbank Accession No.
  • NT 022171.15, 12175690-12176178, Homoapiens
  • SEQ ID NO: 1 B
  • C the nucleotide sequence represented by SEQ ID NO: 2 (Genbank Accession No.
  • NT 005403.17, 75486912-75487393, Homoapiens
  • SEQ ID NO: 2 a nucleotide sequence complementary to SEQ ID NO: 2 or a nucleotide sequence having 80% or more homology with a nucleotide sequence complementary to SEQ ID NO: 2
  • E a nucleotide sequence represented by SEQ ID NO: 3 (Genbank Accession No.
  • NT_005612.16, 5357894-53557166, Homoapiens or a nucleotide sequence having 80% or more homology with the nucleotide sequence represented by SEQ ID NO: 3
  • F a nucleotide sequence complementary to SEQ ID NO: 3 or a nucleotide sequence having 80% or more homology with a nucleotide sequence complementary to SEQ ID NO: 3
  • G The nucleotide sequence shown in SEQ ID NO: 4 (Genbank Accession No.
  • NT 006576.16, 14316619-14316186, Homoapiens
  • nucleotide sequence having 80% or more homology with the nucleotide sequence shown in SEQ ID NO: 4 H
  • nucleotide sequence complementary to SEQ ID NO: 4 or a nucleotide sequence having 80% or more homology with a nucleotide sequence complementary to SEQ ID NO: 4
  • nucleotide sequence represented by SEQ ID NO: 5 Genbank Accession No.
  • NT 029289.11, 10354292-10354661, Homoapiens
  • a nucleotide sequence having 80% or more homology with the nucleotide sequence represented by SEQ ID NO: 5 J
  • a nucleotide sequence complementary to SEQ ID NO: 5 or a nucleotide sequence having 80% or more homology with a nucleotide sequence complementary to SEQ ID NO: 5
  • K the nucleotide sequence represented by SEQ ID NO: 6 (Genbank Accession No.
  • NT 007592.15, 26671779-26661515, Homoapiens
  • nucleotide sequence having 80% or more homology with the nucleotide sequence represented by SEQ ID NO: 6 L
  • nucleotide sequence complementary to SEQ ID NO: 6 or a nucleotide sequence having 80% or more homology with a nucleotide sequence complementary to SEQ ID NO: 6
  • M a nucleotide sequence represented by SEQ ID NO: 7 (Genbank Accession No.
  • nucleotide sequence represented by SEQ ID NO: 7 N
  • SEQ ID NO: 7 O
  • SEQ ID NO: 8 The nucleotide sequence represented by SEQ ID NO: 8 (Genbank Accession No.
  • NT_0075922, 42159778-421160229, Homoapiens or the nucleotide sequence having 80% or more homology with the nucleotide sequence represented by SEQ ID NO: 8 (P) a nucleotide sequence complementary to SEQ ID NO: 8 or a nucleotide sequence having 80% or more homology with a nucleotide sequence complementary to SEQ ID NO: 8 (Q) SEQ ID NO: 9 (Genbank Accession No.
  • SEQ ID NO: 9 a nucleotide sequence complementary to SEQ ID NO: 9 or a nucleotide sequence having a homology of 80% or more with a nucleotide sequence complementary to SEQ ID NO: 9 (S)
  • SEQ ID NO: 10 The nucleotide sequence represented by SEQ ID NO: 10 (Genbank Accession No.
  • NT 030059.13, 30491593-30491957, Homoapiens) or the nucleotide sequence having 80% or more homology with the nucleotide sequence represented by SEQ ID NO: 10.
  • T a nucleotide sequence complementary to SEQ ID NO: 10 or a nucleotide sequence having 80% or more homology with a nucleotide sequence complementary to SEQ ID NO: 10
  • U a nucleotide sequence represented by SEQ ID NO: 11 (Genbank Accession No.
  • NT 0338999.8, 28298052-28298521, Homoapiens
  • nucleotide sequence having 80% or more homology with the nucleotide sequence represented by SEQ ID NO: 12 (X) a base sequence complementary to SEQ ID NO: 12 or a base sequence having 80% or more homology with a base sequence complementary to SEQ ID NO: 12 (Y) the nucleotide sequence represented by SEQ ID NO: 13 (Genbank Accession No.
  • NT_011519.10, 2547951-2548291, Homoapiens) or the nucleotide sequence having 80% or more homology with the nucleotide sequence shown in SEQ ID NO: 14 (Ab) a nucleotide sequence complementary to SEQ ID NO: 14 or a nucleotide sequence having 80% or more homology with a nucleotide sequence complementary to SEQ ID NO: 14 (Ac) The nucleotide sequence represented by SEQ ID NO: 15 (Genbank Accession No.
  • NT_004350.19, 1161852-1216153, Homoapiens or the nucleotide sequence having 80% or more homology with the nucleotide sequence represented by SEQ ID NO: 15 (Ad) a nucleotide sequence complementary to SEQ ID NO: 15 or a nucleotide sequence having 80% or more homology with a nucleotide sequence complementary to SEQ ID NO: 15 (Ae)
  • SEQ ID NO: 16 Genbank Accession No.
  • SEQ ID NO: 16 a base sequence complementary to SEQ ID NO: 16 or a base sequence having 80% or more homology with a base sequence complementary to SEQ ID NO: 16 (Ag)
  • SEQ ID NO: 17 Genbank Accession No.
  • NT 006576.16, 17509792-17510071, Homoapiens
  • nucleotide sequence having 80% or more homology with the nucleotide sequence shown in SEQ ID NO: 17 (Ah) a base sequence complementary to SEQ ID NO: 17 or a base sequence having 80% or more homology with a base sequence complementary to SEQ ID NO: 17 (Ai)
  • SEQ ID NO: 18 Genbank Accession No.
  • nucleotide sequence shown in SEQ ID NO: 18 A nucleotide sequence complementary to SEQ ID NO: 18 or a nucleotide sequence having 80% or more homology with a nucleotide sequence complementary to SEQ ID NO: 18 (Ak) The nucleotide sequence shown in SEQ ID NO: 19 (Genbank Accession No.
  • nucleotide sequences represented by SEQ ID NOs: 1 to 19 are nucleotide sequences registered in NCBI (National Center for Biotechnology Information), which are NCBI WEB pages (URL; http: //www.ncbi.nlm.
  • the base sequence having 80% or more homology with the base sequences (a) to (al) is preferably 90% or more, more preferably 95%, 98% or 99% or more.
  • the base sequence also includes DNA having a base sequence in which deletion, substitution, or addition of a base is caused by a naturally occurring mutation due to species difference, individual difference, organ, or tissue difference of an organism.
  • base sequence having 80% or more homology with the base sequence complementary to the base sequences (a) to (al) in the present DNA preferably 90% or more, more preferably 95%
  • examples thereof include base sequences having 98% or 99% sequence homology.
  • the base sequence also includes DNA having a base sequence in which deletion, substitution, or addition of a base is caused by a naturally occurring mutation due to species difference, individual difference, organ, or tissue difference of an organism.
  • complementary base sequence refers to a base sequence that can form a base pair with the original base sequence
  • base pair refers to adenine (A) among nucleic acid bases.
  • cytosine This refers to thymine (T), guanine (G) and cytosine (C) paired by hydrogen bonding.
  • T thymine
  • G guanine
  • C cytosine
  • mammals there is a phenomenon in which only cytosine is methylated out of four types of bases constituting a gene (genomic DNA).
  • genomic DNA having a base sequence shown in SEQ ID NO: 1 (Genbank Accession No. NT_022171.15, 12175690-12176178, Homoapiens) on a genome derived from a mammal, a part of cytosine of the DNA is methyl. It has become.
  • the DNA methylation modification is a base sequence represented by 5′-CG-3 ′ (C represents cytosine and G represents guanine.
  • the base sequence may be referred to as CpG).
  • CpG Limited to cytosine.
  • the site that is methylated in cytosine is at position 5.
  • cytosine in CpG of the template strand is methylated immediately after replication, but cytosine in CpG of the nascent strand is also methylated immediately by the action of methyltransferase. . Therefore, the DNA methylation state is inherited as it is by two new sets of DNA even after DNA replication.
  • methylation frequency means, for example, that cytosine is methylated when the presence or absence of cytosine methylation in CpG to be investigated is examined for a plurality of haploids. Expressed as a percentage of haploid.
  • the “index value correlated with (methylation frequency)” is represented by, for example, SEQ ID NO: 1 (Genbank Accession No. NT — 02171.115, 12175690-12176178, Homo sapiens).
  • the expression level is decreased by the amount of the expression product of the downstream gene of the DNA having the base sequence to be detected (more specifically, the amount of the transcription product of the gene) or the methylation of the DNA having any one of SEQ ID NOs: 1 to 19
  • the amount of the expression product of the gene to be treated can be raised. In the case of the amount of such an expression product, there is a negative correlation that decreases as the methylation frequency increases.
  • a biological sample may be used as it is, and it was prepared from such a biological sample by various operations such as separation, fractionation, and immobilization.
  • a biological sample may be used as a specimen.
  • specimens include (a) mammal-derived blood, body fluid, urine, body secretion, cell lysate or tissue lysate, and (b) mammal-derived blood, body fluid, urine, body secretion.
  • DNA extracted from one selected from the group consisting of cell lysate and tissue lysate (c) extracted from one selected from the group consisting of mammal-derived tissue, cells, tissue lysate and cell lysate Examples thereof include DNA prepared using RNA as a template.
  • the tissue has a broad meaning including blood, lymph nodes, and the like, the body fluid means plasma, serum, lymph, and the like, and the body secretion means urine, milk, and the like.
  • cancer colorectal cancer
  • breast cancer the breast tissue extract
  • lung cancer the lung tissue etc. which were extract
  • the mammal-derived specimen is blood, body fluid, body secretion, or the like, it is possible to use a sample collected by a periodic health examination or a simple examination.
  • the “mammal” in the present invention include all animals belonging to mammals.
  • An animal belonging to a mammal is a general term for animals classified into the animal kingdom Chordate vertebrate submammal class (Mammalia).
  • examples include humans, monkeys, marmosets, guinea pigs, rats, mice, cows, sheep, dogs, cats and the like.
  • Examples of the “body fluid” in the present invention include a liquid existing between cells constituting a solid, such as plasma and interstitial fluid (in many cases, a function of maintaining the homeostasis of the solid). .
  • lymph fluid tissue fluid (tissue fluid, intercellular fluid, interstitial fluid), body cavity fluid, serous cavity fluid, pleural effusion, ascites, pericardial fluid, cerebrospinal fluid (spinal fluid), joint fluid (synovial fluid) ), Aqueous humor (aqueous humor), cerebrospinal fluid, intrauterine exudate, and the like.
  • body secretion include secretions from exocrine glands. Specific examples include saliva, gastric juice, bile, intestinal fluid, sweat, tears, runny nose, semen, vaginal fluid, amniotic fluid, and milk.
  • the “cell lysate” in the present invention includes, for example, an intracellular fluid obtained by disrupting cells cultured on a 10 cm plate for cell culture or the like (ie, cell lines, primary cultured cells, blood cells, etc.).
  • a lysis solution can be mentioned.
  • examples of the method for destroying the cell membrane include a method using ultrasonic waves, a method using a surfactant, and a method using an alkaline solution.
  • Various commercially available kits may be used to lyse the cells.
  • the culture solution is discarded, and 0.6 mL of RIPA buffer (1 ⁇ TBS, 1% nonidet P-40, 0.5% sodium deo ⁇ ysholate, 0.1% SDS, 0.004% sodium azide) is added to a 10 cm plate.
  • RIPA buffer (1 ⁇ TBS, 1% nonidet P-40, 0.5% sodium deo ⁇ ysholate, 0.1% SDS, 0.004% sodium azide
  • adherent cells on the 10 cm plate are peeled off using a scraper or the like, and the lysate on the plate is transferred to a microtube. After adding 1/10 volume of 10 mg / mL PMSF of the lysate, leave on ice for 30-60 minutes.
  • tissue lysate in the present invention include a lysate containing intracellular fluid obtained by destroying cells in tissue collected from animals such as mammals. Specifically, for example, after measuring the weight of a tissue obtained from a mammal, the tissue is cut into small pieces using a razor or the like. When slicing frozen tissue, it is necessary to make smaller pieces. After cutting, ice-cold RIPA buffer (protease inhibitor, phosphatase inhibitor, etc.
  • a method for measuring the methylation frequency of the present DNA contained in a mammal-derived specimen or an index value correlated therewith is performed, for example, as follows.
  • the target DNA is treated with bisulfite such as sodium bisulfite, and then amplified by PCR using a primer that can identify the presence or absence of cytosine methylation to be analyzed.
  • a method for examining the amount of amplification product can be mentioned.
  • DNA is extracted from a mammal-derived specimen using, for example, a commercially available DNA extraction kit.
  • plasma or serum is prepared from the blood according to a normal method, and the prepared plasma or serum is used as a specimen for free DNA (derived from cancer cells such as colon cancer cells). Analysis of cancer cells such as colon cancer cells, avoiding blood cell-derived DNA, and sensitivity to detect cancer cells such as colon cancer cells and tissues containing them Can be improved.
  • a reagent that modifies unmethylated cytosine one or more CpGs present in the nucleotide sequence of the promoter region, untranslated region or translated region (coding region) of this DNA Amplification product obtained by amplifying DNA containing cytosine in the base sequence shown by the polymerase chain reaction (hereinafter referred to as PCR) using a primer capable of discriminating the presence or absence of cytosine to be analyzed. Find out the amount of.
  • a reagent that modifies unmethylated cytosine ie, a reagent that selectively modifies unmethylated cytosine without modifying methylated cytosine
  • the difference in chemical properties between cytosine and 5-methylcytosine is used.
  • any reagent that modifies unmethylated cytosine may be used.
  • bisulfite such as sodium bisulfite can be used.
  • a reagent that specifically modifies only methylated cytosine may be used. In order to bring the extracted genomic DNA sample into contact with a reagent that modifies unmethylated cytosine as uniformly as possible, it is necessary to denature the genomic DNA.
  • the DNA is first denatured with an alkaline solution (pH 9 to 14), and then bisulfite (bisulfite) such as sodium bisulfite (concentration in the solution: for example, final concentration of 3M) or the like for about 10 to 16 hours (one time). Treat at 55 ° C for the evening.
  • bisulfite bisulfite
  • the modification at 95 ° C. and the reaction at 50 ° C. can be repeated 10-20 times.
  • unmethylated cytosine is converted to uracil, while methylated cytosine is not converted to uracil and remains cytosine (Furichi et al., Biochem. Biophys. Res. Commun. 41: 1185. ⁇ 1191, 1970).
  • DNA containing cytosine in the base sequence indicated by one or more CpGs present in the base sequence is identified for the presence or absence of cytosine methylation to be analyzed
  • Amplification is performed by PCR using possible primers, and the amount of amplification product obtained is examined. DNA sequence treated with bisulfite, etc.
  • cytosine methylated As a template and containing cytosine methylated [the cytosine at the position to be methylated (cytosine in CpG) remains cytosine and is methylated Unsuccessed cytosine (cytosine not included in CpG) is a uracil base sequence] and PCR using a pair of methylation-specific primers each selected from a base sequence complementary to such base sequence (hereinafter, methyl Base sequence when DNA treated with bisulfite or the like is used as a template and cytosine is not methylated (base sequence in which all cytosines are converted to uracil) ) And a pair of unmethylated specific primers selected from base sequences complementary to the base sequence (hereinafter referred to as non-methylated primers).
  • the methylation specific primer is a cytosine that has undergone methylation, considering that cytosine that has not been methylated is converted to uracil, and cytosine that has undergone methylation is not converted to uracil.
  • Design a PCR primer (methylation specific primer) specific to the nucleotide sequence containing, and a PCR primer (non-methylation specific primer) specific to the nucleotide sequence containing unmethylated cytosine To do. Since the design is based on DNA strands that have been chemically converted by bisulfite treatment and are no longer complementary, based on each strand of DNA that was originally double-stranded, a methylation specific primer and Unmethylated specific primers can also be made.
  • Such a primer is preferably designed to contain cytosine in CpG in the vicinity of the 3 ′ end of the primer in order to increase the specificity of methyl and non-methyl. Further, in order to facilitate analysis, one of the primers may be labeled.
  • reaction solution in methylation-specific PCR for example, 50 ng of DNA as a template, 1 ⁇ l of each primer solution of 10 pmol / ⁇ l, 4 ⁇ l of 2.5 mM dNTP, 10 ⁇ buffer solution (100 mM Tris-HCl) pH 8.3, 500 mM KCl, 20 mM MgCl 2 ) Is mixed with 2.5 ⁇ l and heat-resistant DNA polymerase 5 U / ⁇ l 0.2 ⁇ l, and sterilized ultrapure water is added thereto to make the reaction volume 25 ⁇ l.
  • the reaction conditions for example, the above-mentioned reaction solution is kept at 95 ° C. for 10 minutes, then at 95 ° C.
  • the amount of amplification product obtained is compared.
  • an analytical method denaturing polyacrylamide gel electrophoresis or agarose gel that can compare the amount of each amplification product obtained by PCR using a methylation specific primer and PCR using an unmethylated specific primer.
  • electrophoresis the gel after electrophoresis is stained with DNA to detect the band of the amplification product, and the concentration of the detected band is compared.
  • a pre-labeled primer can be used to compare the band concentrations using the label as an index.
  • high-precision quantification that can detect even a slight difference of about twice as much as the gene amount can be performed by monitoring PCR reaction products in real time and performing kinetic analysis.
  • Real-time PCR a possible PCR method, can also be used to compare the amount of each product. Examples of a method for performing real-time PCR include a method using a probe such as a template-dependent nucleic acid polymerase probe or a method using an intercalator such as Cyber Green. Equipment and kits for real-time PCR are already commercially available. As a second method, fluorescence-based real-time PCR (US Pat.
  • methylated DNA is amplified by real-time quantitative PCR based on fluorescence using a position-specific PCR primer having a fluorescent reporter dye at the 5 ′ end and a quenching dye at the 3 ′ end. Since the fluorescent reporter dye is released by the enzyme during the PCR reaction, the fluorescence intensity increases in proportion to the amount of PCR product.
  • fluorescence proportional to the degree of methylation can be continuously detected in an automated nucleotide sequencer device.
  • a method of sequencing after treatment with bisulfite such as sodium bisulfite can also be mentioned.
  • dsDNA is obtained by primer extension and further amplified by PCR techniques (Clark et al., Nucl. Acids Res. 22: 2990-2997, 1994).
  • the PCR product is sequenced by a standard DNA sequencing method to detect cytosine (corresponding to methylcytosine before treatment with bisulfite).
  • the sequencing method not only the dideoxy method but also a pyro sequencing method (SOLiD system) or the like may be used as long as it is a method for determining a base sequence.
  • a pyro sequencing method SOLiD system
  • individual clones can be sequenced, but it is also possible to provide a methylation map of a single DNA molecule.
  • Several variations are known for determining methylcytosine by sequencing (Radlinska & Skowronek, Acta Microbiol. Pol. 47: 327-334, 1998).
  • a reaction solution in PCR for example, a total solution containing 20 ng or 80 ng of DNA in a DNA solution treated with sodium bisulfite as a template An amount of 50 ⁇ L of reaction solution is prepared and used. Specifically, a DNA solution treated with sodium bisulfite as a template and each oligonucleotide primer solution prepared to 5 ⁇ M are each 3/5 of the total volume, and GeneAmpR dNTPPMi ⁇ (2 mMeach) is the total volume.
  • the obtained DNA fragment is cloned.
  • TOPO TA Cloning® Kit For Sequencing (Invitrogen) is used for cloning.
  • Salt Solution 0.4 ⁇ L, TOPO vector 0.4 ⁇ L, and the PCR amplification product 1.6 ⁇ L are mixed on ice and allowed to stand at room temperature for 5 minutes.
  • 2 ⁇ L of the ligation reaction solution and 50 ⁇ L of the competent cell are mixed and left on ice for 30 minutes. Incubate at 42 ° C. for 30 seconds and store in ice.
  • a plasmid solution can be obtained by extracting a plasmid from the obtained Escherichia coli using a plasmid extraction device (PI-50, KURABO). 2 ⁇ L of the plasmid solution, 1 ⁇ L of BigDyeRT terminator v3.1 Cycle Sequencing RR-100 (ABI), and BigDyeRT terminator v1.1 / v3.1 Sequencing Buffer (5 ⁇ ) (2 for L 1 ⁇ L of a 3.2 ⁇ M solution of the oligonucleotide primer (M13R) designed in (1) and 4 ⁇ L of sterile ultrapure water are mixed. The reaction solution was kept at 96 ° C.
  • reaction solution in PCR for example, 25 ng of DNA as a template, 1 ⁇ l of each primer solution of 20 pmol / ⁇ l, 3 ⁇ l of 2 mM dNTP, 10 ⁇ buffer (100 mM Tris-HCl pH 8.3, 500 mM KCl) 15 mM MgCl 2 ) Is mixed with 2.5 ⁇ l and heat-resistant DNA polymerase 5 U / ⁇ l 0.2 ⁇ l, and sterilized ultrapure water is added thereto to make the reaction volume 25 ⁇ l.
  • reaction condition for example, after the above reaction liquid is kept at 95 ° C. for 10 minutes, one cycle is 95 ° C. for 30 seconds, then 53 ° C. for 30 seconds, and further at 72 ° C. for 30 seconds.
  • a condition for performing the heat retention for 30 to 40 cycles is mentioned.
  • the base sequences of the amplification products obtained are compared, and the methylation frequency is measured from the comparison. That is, by directly analyzing the base sequence of the amplification product, it is determined whether the base at the position corresponding to the cytosine to be analyzed is cytosine or thymine (uracil).
  • each cloned DNA is prepared from a plurality of clones obtained by cloning the amplification product obtained by PCR once using Escherichia coli or the like as a host.
  • the base sequence may be analyzed.
  • the frequency of cytosine methylation to be analyzed can also be measured by obtaining the ratio of the sample whose base detected at the position corresponding to the cytosine to be analyzed in the sample to be analyzed is cytosine.
  • a probe capable of discriminating the presence or absence of methylation of cytosine to be analyzed from DNA containing cytosine in the base sequence represented by one or more CpGs present in the base sequence of the target DNA And a method for examining the presence or absence of binding between the DNA and the probe.
  • a genomic DNA extracted from a specimen is allowed to act on a reagent that modifies unmethylated cytosine, and then a probe that can identify the presence or absence of cytosine methylation is hybridized.
  • cytosine that has not been methylated is converted into uracil based on the base sequence containing cytosine to be analyzed, and cytosine that has been methylated is not converted into uracil. It is better to design in consideration of this.
  • a probe may be used after being labeled in order to facilitate analysis of the presence or absence of binding between the DNA and the probe.
  • the probe may be used by being immobilized on a carrier according to a usual method.
  • DNA extracted from a mammal-derived specimen may be labeled in advance.
  • a reagent for modifying unmethylated cytosine for example, bisulfite such as sodium bisulfite can be used.
  • a reagent that specifically modifies only methylated cytosine may be used.
  • the DNA is first denatured with an alkaline solution (pH 9 to 14), and then bisulfite (bisulfite) such as sodium bisulfite (solution Medium concentration: For example, the final concentration is 3 M) and the like, and the treatment is performed at 55 ° C. for about 10 to 16 hours (overnight).
  • bisulfite such as sodium bisulfite (solution Medium concentration: For example, the final concentration is 3 M) and the like
  • the treatment is performed at 55 ° C. for about 10 to 16 hours (overnight).
  • the modification at 95 ° C. and the reaction at 50 ° C. can be repeated 10-20 times.
  • unmethylated cytosine is converted to uracil, while methylated cytosine is not converted to uracil and remains cytosine.
  • the DNA may be amplified in advance by performing PCR in the same manner as in the second method using DNA treated with bisulfite or the like as a template.
  • hybridization between DNA treated with bisulfite or the like or DNA previously amplified by PCR and a probe capable of identifying the presence or absence of methylation of cytosine to be analyzed is performed.
  • the frequency of cytosine methylation to be analyzed can be measured. Hybridization is described, for example, in Sambrook J. et al. Frisch E .; F. Maniatis T.
  • stringent conditions means, for example, a hybrid at 45 ° C. in a solution containing 6 ⁇ SSC (a solution containing 1.5M NaCl and 0.15M trisodium citrate is 10 ⁇ SSC).
  • the conditions Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6
  • the salt concentration in the washing step can be selected from, for example, conditions of 2 ⁇ SSC at 50 ° C.
  • the temperature in the washing step can be selected, for example, from room temperature (low stringency conditions) to 65 ° C. (high stringency conditions). It is also possible to change both the salt concentration and the temperature.
  • the amount of DNA bound to the methylation-specific probe is compared with the amount of DNA bound to the non-methylation-specific probe, so that the cytosine to be analyzed (that is, the probe)
  • the frequency of methylation of cytosine in CpG contained in the base sequence on which the design was based can be measured.
  • a restriction enzyme capable of discriminating the presence or absence of methylation of cytosine to be analyzed in the base sequence of the target DNA there is a method for examining the presence or absence of digestion by the restriction enzyme. You can also.
  • the “restriction enzyme capable of distinguishing the presence or absence of cytosine methylation” (hereinafter sometimes referred to as a methylation-sensitive restriction enzyme) used in the method means that a recognition sequence containing methylated cytosine is not digested.
  • the DNA in which cytosine contained in a “recognition sequence” that can be originally recognized by a methylation-sensitive restriction enzyme is methylated, the DNA is not cleaved even when a methylation-sensitive restriction enzyme is allowed to act.
  • the cytosine contained in the “recognition sequence” that can be originally recognized by the methylation-sensitive restriction enzyme is DNA that is not methylated, the DNA is cleaved by the action of the methylation-sensitive restriction enzyme.
  • Specific examples of such a methylation-sensitive enzyme include HpaII, BstUI, NarI, SacII and the like (see, for example, Nucleic Acid Research, 9, 2509-2515).
  • the DNA is used as a template, the cytosine to be analyzed is included in the recognition sequence, and the DNA not containing the restriction enzyme recognition sequence other than the recognition sequence is amplified
  • amplification product DNA amplification product
  • cytosine to be analyzed is methylated, an amplification product is obtained.
  • the cytosine to be analyzed is not methylated, an amplification product cannot be obtained.
  • the frequency of cytosine methylation to be analyzed can be measured.
  • the methylation-sensitive restriction enzyme does not cleave DNA in a methylated state. It is possible to distinguish whether cytosine in at least one CpG pair existing in the recognition site of the methylation sensitive restriction enzyme in the genomic DNA contained in is methylated. In other words, by digesting with the methylation-sensitive restriction enzyme, at least one CpG present in the recognition site of the methylation-sensitive restriction enzyme in the genomic DNA contained in the mammal-derived specimen. If the cytosine in the pair is not methylated, it is cleaved by the methylation sensitive restriction enzyme.
  • cytosine in all CpG pairs existing in the recognition site of the methylation sensitive restriction enzyme in genomic DNA contained in a mammal-derived specimen is methylated, the methylation sensitivity Not cleaved by restriction enzymes. Therefore, after the digestion treatment, as described later, by performing PCR using a pair of primers capable of amplifying the target DNA region, the restriction on the genomic DNA contained in the mammal-derived specimen is performed.
  • cytosine in at least one CpG pair existing in the enzyme recognition site is not methylated, an amplification product by PCR cannot be obtained, whereas the genome contained in the mammal-derived specimen If cytosine in all CpG pairs existing in the recognition site of the methylation-sensitive restriction enzyme in DNA is methylated, an amplification product by PCR is obtained.
  • quantification is required, high-accuracy quantification is possible by detecting PCR reaction products in real time and performing kinetic analysis, for example, to detect even a slight difference of about twice the gene amount. The amount of each product can also be compared using real-time PCR which is a PCR method.
  • Examples of a method for performing real-time PCR include a method using a probe such as a template-dependent nucleic acid polymerase probe or a method using an intercalator such as Cyber Green. Equipment and kits for real-time PCR are already commercially available.
  • a method for examining the presence or absence of digestion with the restriction enzyme for example, derived from the Arginine vasopressin receptor 1A gene on DNA subjected to a methylation-sensitive restriction enzyme containing cytosine to be analyzed as a recognition sequence
  • a method of examining the length of the hybridized DNA by performing Southern hybridization using a DNA that does not contain the recognition sequence of the restriction enzyme as a probe can be mentioned.
  • the cytosine to be analyzed When the cytosine to be analyzed is methylated, longer DNA is detected than when the cytosine is not methylated. By comparing the amount of detected long DNA and the amount of short DNA, the frequency of cytosine methylation to be analyzed can be measured.
  • the methylation rate of cytosine contained in the target DNA may be measured quantitatively using the MassARRAY system of SEQUENOM. Specifically, a reagent for modifying unmethylated cytosine is allowed to act on genomic DNA extracted from a specimen, and then a target DNA region is amplified by a PCR method. Next, the amplified DNA region is transcribed into RNA by RNA polymerase.
  • the obtained transcription product is treated with RNase and subjected to mass spectrometry by MASS.
  • This method is a method for quantifying methylcytosine contained in DNA in a specimen based on the change in molecular weight caused by the action of a reagent that modifies unmethylated cytosine. More specifically, the following method may be used in accordance with the outline of EpiTYPER for quantitative DNA methylation analysis using the MassARRAY system shown in the SEQUENOM application note.
  • the following primer system is designed for methylation analysis. To obtain a product suitable for in vitro transcription, a reverse primer with a T7 promoter added is used. Insert an 8 bp insert to prevent cycling failure. In order to balance PCR, a forward primer with a 10-mer tag is used.
  • Bisulfite processing For the Bisulfite conversion treatment of the sample genomic DNA, EZ-96 DNA Methylation Kit or EZ DNA Methylation Kit of Zymo Research is used. After the initial incubation of this protocol, the cycle reaction is performed as follows. 45 cycles with 95 ° C for 30 minutes and then 50 ° C for 15 minutes (1) Step 1: Amplification Amplify 1 ⁇ L of DNA in a total volume of 5 ⁇ L using a 385-microtiter format (use 10 ng / ⁇ L or more of DNA in an amount of 1.00 ⁇ L or more to achieve a final concentration of 2 ng / ⁇ L per reaction).
  • Step 2 Dephosphorylation 2 ⁇ L of shrimp-derived alkaline phosphatase (SAP) enzyme is added to 5 ⁇ L of each PCR reaction solution to dephosphorylate dNTPs that have not been incorporated into PCR.
  • SAP shrimp-derived alkaline phosphatase
  • Step 3 In vitro transcription and RNase cleavage Prepare a transcription / RNase A cocktail for each cleavage reaction (T and C). The standard setup prepares one transcription / RNase A cocktail per plate. Add 5 ⁇ L of transcription / RNase A cocktail and 2 ⁇ L of PCR / SAP sample to a new microtiter plate that has not been cycled. The plate is centrifuged for 1 minute and then the plate is incubated at 37 ° C. for 3 hours. (4) Step 4: Sample conditioning Add 20 ⁇ L of ddH20 to each sample in the 384-well plate. Add 6 mg of Clean Resin to each well using a resin plate.
  • Step 5 Sample movement Dispense 10-15 nL of EpiTYPE reaction product into 384 well SpectroCHIP.
  • Step 6 Sample analysis Using the MassARRAY system, spectra of two types of cleavage reactions are obtained.
  • Step 6 Analysis software The result is analyzed with EpiTYPER software, and the methylation rate of the target DNA is measured. Using the various methods as described above, the methylation frequency of the present DNA contained in a mammal-derived specimen is measured.
  • the measured methylation frequency and, for example, the methylation frequency of a target DNA contained in a sample derived from a healthy mammal that can be diagnosed as having no cancer cells such as colon cancer cells, lung cancer cells, breast cancer cells control
  • the degree of canceration of the specimen is determined based on the difference obtained by the comparison. If the methylation frequency of the DNA contained in a mammal-derived specimen is higher than that of the control (if the DNA is highly methylated in comparison with the control), the degree of canceration of the specimen is increased. It can be determined to be higher than the control.
  • the “degree of canceration” has the same meaning as that generally used in the art.
  • a mammal-derived specimen when a mammal-derived specimen is a cell, the malignancy of the cell or cancer
  • a mammal-derived specimen when a mammal-derived specimen is a tissue, it means the abundance of cancer cells in the tissue.
  • the content of methylated DNA in this DNA can be measured by the following methylated DNA content measurement method.
  • the methylated DNA content measurement method is a method for measuring the content of methylated DNA in a target DNA region possessed by the base sequence of the target DNA region contained in a mammal-derived specimen, (1) a first step of digesting a genomic DNA-derived DNA sample contained in a mammal-derived specimen with a methylation-sensitive restriction enzyme; (2) Obtaining methylated single-stranded DNA from the digested DNA sample obtained in the first step, and binding the single-stranded DNA with an immobilized methylated DNA antibody A second step of selecting a double-stranded DNA; and (3) A step of separating the single-stranded DNA selected in the second step as a pre-step of each of the following steps from the immobilized methylated DNA antibody into a single-stranded DNA (positive strand) ( First pre-processing),
  • the DNA derived from the genome (positive strand) that was made into a single strand state in the first pre-process is a partial base sequence (positive strand) of the base sequence of the
  • extension primer reverse primer having a base sequence (positive strand) that is complementary to the base sequence (negative strand) as an extension primer
  • the extension primer is extended once to achieve the above-mentioned purpose.
  • a step B (this step) of extending a single-stranded DNA containing a DNA region as a double-stranded DNA
  • each step of the third step is repeated after separating the double-stranded DNA formed by extension obtained in each of the steps into a single-stranded state, and then repeating the above steps.
  • a third step of amplifying the amplified DNA to a detectable amount and quantifying the amount of the amplified DNA.
  • “Complementary” in the methylated DNA content measurement method means that double-stranded DNA is formed by base pairing by hydrogen bonding between bases.
  • the bases constituting each single-stranded DNA of the double-stranded DNAs form a double strand by base pairing of purine and pyrimidine, specifically, for example, a plurality of consecutive This means that a double-stranded DNA is formed by a base bond by a hydrogen bond between thymine and cytosine or a base bond by a hydrogen bond between guanine and adenine.
  • Binding by complementarity may be described as “complementary binding”.
  • “Complementary binding” may also be described as “complementary base pairing” or “binding by complementarity”.
  • base sequences that can be complementarily bonded may be described as “complementary to each other” and “bonded by complementarity (complementary (by base pairing) bond)”.
  • the term “complementary” also includes that inosine contained in an artificially prepared oligonucleotide binds to cytosine, adenine or thymine by hydrogen bonding.
  • “single-stranded DNA containing the target DNA region (negative strand)” forms a conjugate (double-stranded) with the single-stranded DNA containing the target DNA region.
  • methylated DNA and “methylated DNA” refer to a base sequence represented by 5′-CG-3 ′ in a DNA base sequence (hereinafter, the base sequence is referred to as “ CpG ”)) means DNA in which the 5-position of cytosine is methylated.
  • CpG DNA base sequence represented by 5′-CG-3 ′ in a DNA base sequence
  • “Methylation frequency” is represented, for example, by the ratio of haploids in which cytosine is methylated when the presence or absence of cytosine methylation in CpG to be investigated is examined for a plurality of haploids.
  • Examples of the “index value correlated with (methylation frequency)” include, for example, the amount of the expression product of the present DNA (more specifically, the amount of the transcription product of the present DNA and the amount of the translation product of the present DNA). Etc. In the case of the amount of such an expression product, there is a negative correlation that decreases as the methylation frequency increases.
  • Examples of the “immobilized methylated DNA antibody” in the method for measuring methylated DNA content include methylcytosine antibody.
  • the immobilized methylated DNA antibody may be any antibody that can be immobilized on a support, and “an antibody that can be immobilized on a support” means that the immobilized methylated DNA antibody is directly or indirectly attached to the support. It can be fixed to.
  • the immobilized methylated DNA antibody is immobilized on a support (binding to a solid phase) according to a normal genetic engineering operation method or a commercially available kit / device.
  • a biotinylated immobilized methylated DNA antibody obtained by biotinylating an immobilized methylated DNA antibody is coated with streptavidin (eg, a PCR tube coated with streptavidin, or coated with streptavidin.
  • the immobilized methylated DNA antibody is obtained by covalently bonding a molecule having an active functional group such as an amino group, a thiol group, or an aldehyde group, and then activating the surface with a silane coupling agent or the like, a polysaccharide derivative,
  • a silane coupling agent or the like a polysaccharide derivative
  • covalent bond examples include, for example, a method in which five triglycerides are linked in series using a spacer, a crosslinker, or the like.
  • the immobilized methylated DNA antibody may be directly immobilized on the support, or the antibody against the immobilized 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 by making it. It may be immobilized by the binding of the immobilized methylated DNA antibody and the support at the stage before the binding of the single stranded DNA and the immobilized methylated DNA antibody, or the single stranded DNA may be immobilized. It may be immobilized by binding of the immobilized methylated DNA antibody and the support at the stage after binding with the methylated DNA antibody.
  • the “methylation-sensitive restriction enzyme” (specifically, a restriction enzyme that can identify the presence or absence of methylation of cytosine) used in the method for measuring methylated DNA content has the same meaning as in the evaluation method of the present invention. It means a restriction enzyme that can digest a recognition sequence containing unmethylated cytosine without digesting a recognition sequence containing methylated cytosine. That is, in the case of DNA in which cytosine contained in a “recognition sequence” that can be originally recognized by a methylation-sensitive restriction enzyme is methylated, the DNA is not cleaved even when a methylation-sensitive restriction enzyme is allowed to act.
  • the DNA is cleaved by the action of the methylation-sensitive restriction enzyme.
  • a methylation-sensitive enzyme include HpaII, BstUI, NarI, SacII and the like (see, for example, Nucleic Acid Research, 9, 2509-2515).
  • the “methylation-sensitive restriction enzyme” in the method for measuring methylated DNA content include a restriction enzyme having a recognition cleavage site in the DNA region intended for the base sequence of the present DNA, HhaI, and the like.
  • the second step is performed without performing the digestion treatment with the methylation sensitive restriction enzyme in the first step in the method.
  • the second step of the method for measuring methylated DNA content the methylated double-stranded DNA contained in the digested DNA sample obtained in the first step is separated into methylated single-stranded DNA.
  • Step A ie, Step A in Step 2
  • Step B ie, Step 2 of binding the methylated single-stranded DNA obtained in Step A and the immobilized methylated DNA antibody.
  • the methylated double-stranded DNA contained in the digested DNA sample obtained in the first step is separated into methylated single-stranded DNA.
  • a general operation for converting double-stranded DNA into single-stranded DNA is performed. Specifically, for example, a DNA sample derived from genomic DNA contained in a mammal-derived specimen is dissolved in an appropriate amount of ultrapure water, heated at 95 ° C. for 10 minutes, and rapidly cooled in ice.
  • the second step of the method for measuring the content of methylated DNA in order to select single-stranded DNA by binding the methylated single-stranded DNA separated as described above and an immobilized methylated DNA antibody, as described in the description of “immobilized methylated DNA antibody”, specifically, for example, “biotinylated methylcytosine antibody labeled with biotin” is used as the immobilized methylated DNA antibody as follows. carry out.
  • a biotinylated methylcytosine antibody is added to an appropriate amount (for example, 0.1 ⁇ g / 50 ⁇ L) of an avidin-coated PCR tube, and then allowed to stand at room temperature for about 1 hour, whereby biotinylated methylcytosine antibody and streptocyte Encourage immobilization with avidin.
  • the washing buffer for example, phosphate buffer containing 0.05% Tween 20 (1 mM KH 2 PO 4 3 mM Na 2 HPO 7H 2 O, 154 mM NaCl pH 7.4)] is added at a rate of 100 ⁇ L / tube.
  • Double-stranded DNA derived from genomic DNA contained in a mammal-derived sample and a buffer for example, 33 mM Tris-Acetate pH 7.9, 66 mM KOAc, 10 mM Mg (OAc) 2 , 0.5 mM Dithothreitol
  • a buffer for example, 33 mM Tris-Acetate pH 7.9, 66 mM KOAc, 10 mM Mg (OAc) 2 , 0.5 mM Dithothreitol
  • the resulting mixture is allowed to return to room temperature.
  • C The formed single-stranded DNA is added to an avidin-coated PCR tube on which a biotinylated methylcytosine antibody is immobilized, and the resulting mixture is allowed to stand at room temperature for about 1 hour, whereby biotinylated methyl Encourage binding between 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 is bound) Does not form a body.) Thereafter, the remaining solution is removed from the PCR tube and washed.
  • Wash buffer [eg, 0.05% Tween 20-containing phosphate buffer (1 mM KH 2 PO 4 3 mM Na 2 HPO 7H 2 O, 15 mM NaCl pH 7.4)] is added at a rate of 100 ⁇ L / tube, and then the solution is removed from the PCR tube. By repeating the washing operation several times, the washed conjugate is left in the PCR tube (selection of conjugate).
  • the buffer used in the above (b) is not limited to the buffer as long as it is suitable for separating double-stranded DNA derived from a biological sample-derived genomic DNA into single-stranded DNA.
  • the cells are suspended in a solution that has not been immobilized to the immobilized methylated DNA antibody that has not been immobilized, or that has not been bound to the immobilized methylated DNA antibody.
  • This is an important operation for removing unmethylated single-stranded DNA or DNA floating in a solution digested with a restriction enzyme described later from the reaction solution.
  • the washing buffer is not limited to the washing buffer as long as it is suitable for removing the above-mentioned free immobilized methylated DNA antibody, single-stranded DNA floating in the solution, and the like, but the DELFIA buffer (PerkinElmer) Manufactured by Tris-HCl pH 7.8 with Tween 80), TE buffer, or the like.
  • a preferred embodiment for separating methylated single-stranded DNA includes, for example, adding a counter oligonucleotide.
  • the counter oligonucleotide include those obtained by dividing the same base sequence as the target DNA region into short oligonucleotides.
  • Preferred examples include those usually designed to have a length of 10 to 100 bases, more preferably 20 to 50 bases.
  • the counter oligonucleotide is not designed on the base sequence that the forward primer or reverse primer binds complementarily to the target DNA region.
  • the counter oligonucleotide is added in a large excess compared to genomic DNA, and the target DNA region is converted into a single strand (positive strand) and then combined with the immobilized methylated DNA antibody.
  • the complementary strand (negative strand) of the region and the target DNA region are added to prevent the single strand (positive strand) from recombining due to complementarity.
  • the counter oligonucleotide is preferably added in an amount of at least 10 times, usually 100 times or more, as compared with the target DNA region.
  • “adding a counter oligonucleotide (when separating methylated single-stranded DNA)” specifically refers to a DNA sample derived from genomic DNA contained in a mammal-derived specimen.
  • a DNA sample derived from genomic DNA contained in a mammal-derived specimen is mixed with a counter oligonucleotide, and the complementary strand of the target DNA region and the counter oligonucleotide are mixed with each other. To form a double strand.
  • a buffer solution (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc is added to the mixture of the DNA sample and the counter oligonucleotide.
  • 2 5 mM Dithiothreitol) 5 ⁇ L and 100 mM MgCl 2 Add 5 ⁇ L of the solution and 5 ⁇ L of 1 mg / mL BSA solution, add sterilized ultrapure water to the mixture to make 50 ⁇ L, mix, heat at 95 ° C. for 10 minutes, and quickly cool to 70 ° C. The temperature is kept at that temperature for 10 minutes, then cooled to 50 ° C., kept at temperature for 10 minutes, further kept at 37 ° C.
  • the third step of the method for measuring methylated DNA content includes the following steps: (1) Pre-process ((i) first pre-process, (ii) second pre-process, (iii) third pre-process) (2) This step ((i) Step A, (ii) Step B) and (3) Repetition step ((i) amplification step, (ii) quantification step).
  • Pre-process in the third process (I) First pre-process in the pre-process in the third process
  • the first pre-process is a process of separating the single-stranded DNA selected in the second process from the immobilized methylated DNA antibody into a free single-stranded DNA.
  • an annealing buffer is added to the single-stranded DNA selected in the second step to obtain a mixture.
  • the obtained mixture is heated at 95 ° C. for several minutes to obtain DNA (positive strand) in a single-stranded state.
  • Second pre-process in the pre-process in the third process In the second pre-process, the genome-derived DNA (positive strand) that was made free single-stranded in the first pre-process and the partial base sequence of the base sequence of the single-stranded DNA (positive strand) ( Complementary to the partial base sequence (positive strand) which is located on the 3 ′ end side further from the 3 ′ end of the base sequence (positive strand) of the target DNA region.
  • extension primer having a certain base sequence (negative strand) as an extension primer
  • the extension primer is extended once, thereby free double-stranded DNA (positive strand).
  • This is a step of extending and forming DNA.
  • the single-stranded DNA (positive strand) obtained in the first pre-process and the forward primer are mixed with 17.85 ⁇ L of sterilized ultrapure water and an optimal buffer (for example, 100 mM Tris-HCl pH).
  • the double-stranded DNA extended in the second pre-process is complementary to the single-stranded DNA (positive strand) containing the target DNA region and the target DNA region.
  • This is a step of once separating into single-stranded DNA (negative strand) containing a sequence. Specifically, for example, by adding an annealing buffer to the double-stranded DNA formed by extension in the second previous step, a mixture is obtained, and the resulting mixture is heated at 95 ° C. for several minutes to achieve the purpose. Once separated into single-stranded DNA containing the DNA region to be treated.
  • Step A in this step in the third step Step A comprises the step of extending the extension primer once using the generated single-stranded DNA (positive strand) containing the target DNA region as a template and the forward primer as an extension primer.
  • a single-stranded DNA containing the DNA region is extended as a double-stranded DNA.
  • the Tm value of the forward primer is about 0 to 20 Immediately cool to a lower temperature and keep at that temperature for several minutes; (B) then return to room temperature; and (C) Using the DNA in the single-stranded state annealed in (c) above as a template, the forward primer as an extension primer, and extending the primer once to achieve the above object A single-stranded DNA containing a base sequence complementary to the DNA region is extended as a double-stranded DNA.
  • Step B in this step in the third step a single-stranded DNA (negative strand) containing a base sequence that is complementary to the generated target DNA region is used as a template, and the base is complementary to the target DNA region. It is a partial base sequence (negative strand) of the base sequence of single-stranded DNA (negative strand) containing the sequence, and is complementary to the base sequence (positive strand) of the target DNA region.
  • An extension primer (reverse primer) having a base sequence (positive strand) that is complementary to a partial base sequence (negative strand) located further 3 'end than the 3' end of the base sequence (negative strand)
  • This is a step of extending a single-stranded DNA containing the target DNA region as a double-stranded DNA by extending the extended primer once as an extended primer.
  • the amplification step is performed by repeating each step of the third step after separating the elongated double-stranded DNA obtained in each step into a single-stranded state, and then repeating the process. Amplifying the methylated DNA in the region to a detectable amount. Specifically, for example, it is carried out according to the same operation method as in the step A and the step B in the third step (2).
  • Quantification step in the repetition step in the third step The quantification step is a step of quantifying the amount of DNA amplified by the amplification step in the repetition step of the third step.
  • the reaction from the first previous step in the previous step to the present step and the repetition step can be carried out as one PCR reaction. Moreover, it can also carry out as an independent reaction from the 1st pre-process in the pre-process to the 3rd pre-process, respectively, and can also implement only this process as PCR reaction then.
  • PCR can be used as a method for amplifying a target DNA region (ie, a target region) contained in the selected single-stranded DNA.
  • a primer previously labeled with fluorescence or the like is used and the label is used as an index
  • the presence or absence of an amplification product can be evaluated without performing a cumbersome operation such as electrophoresis.
  • a PCR reaction solution for example, 0.15 ⁇ l of a 50 ⁇ M primer solution, 2.5 ⁇ l of 2 mM dNTP, 10 ⁇ buffer solution (100 mM Tris-HCl pH 8) are added to the DNA obtained in the second step of this method.
  • the reaction may be carried out by adding an appropriate amount of betaine, DMSO or the like.
  • 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.
  • the conditions for performing the heat retention for 30 to 40 cycles can be given.
  • the obtained amplification product is detected.
  • the amount of the amplification product in the PCR reaction can be measured by measuring the amount of the fluorescent label after performing the same washing / purifying operation as described above.
  • the amount of the probe bound to the target region is measured after annealing gold colloid particles, a probe labeled with fluorescence, and the like.
  • a real-time PCR method is used.
  • the “real-time PCR method” is a method for monitoring PCR in real time and analyzing the obtained monitoring result by kinetic analysis, and can detect even a slight difference of about twice as much as the amount of gene, for example.
  • This method is known as a high-precision quantitative PCR method.
  • the real-time PCR method include a method using a probe such as a template-dependent nucleic acid polymerase probe, a method using an intercalator such as Cyber Green, and the like.
  • Commercially available devices and kits for the real-time PCR method may be used.
  • detection is not particularly limited, and detection by any known method can be performed. In these methods, operations up to detection can be performed without changing the reaction container.
  • Method 1 includes the following steps: The first step, the second step, the third step, and Fourth step (previous step, main step ((i) A step ((i1) step A1 step, (i2) step A2 step), (ii) step B)), repeating step ((i) amplification step, ( ii) Quantitative process)).
  • method 1 is a method for measuring the content of methylated DNA in a target DNA region as a target DNA region, which is the base sequence of the present DNA contained in a mammal-derived specimen, (1) a first step of digesting a genomic DNA-derived DNA sample contained in a mammal-derived specimen with a methylation-sensitive restriction enzyme; (2) Obtaining a single-stranded DNA (positive strand) containing a target DNA region from the digested DNA sample obtained in the first step, the single-stranded DNA (positive strand), By binding a single-stranded immobilized oligonucleotide having a complementary base sequence to a part of the 3 ′ end of the single-stranded DNA (however, the DNA region of interest is not included).
  • a second step of selecting the single-stranded DNA (3) Using the single-stranded DNA selected in the second step as a template, using the single-stranded immobilized oligonucleotide as a primer, and extending the primer once, the single-stranded DNA is converted into a double-stranded DNA.
  • the process has a process (pre-process) for once separating the elongated double-stranded DNA obtained in the third process into a single-stranded state.
  • the single-stranded immobilized oligonucleotide as a primer, and extending the primer once, thereby double-extracting the single-stranded DNA.
  • a step A2 (this step) having an A2 step of extending and forming as a double-stranded DNA; (B) using the generated single-stranded DNA (negative strand) as a template, the single-stranded DNA (negative strand) having a partial base sequence (negative strand), and Complementary to the partial base sequence (negative strand) located 3 'end further than the 3' end of the base sequence (negative strand) complementary to the base sequence (positive strand) of the target DNA region
  • a primer reverse primer
  • the extension primer is extended once, thereby extending the single-stranded DNA as a double-stranded DNA.
  • Method 2 includes the following steps: The first step, the second step, the third step, and Fourth step (previous step, main step ((i) A step ((i1) step A1 step, (i2) step A2 step)), (ii) step B), repetition step ((i) amplification step, (Ii) Quantitative process)
  • the method 2 comprises a single-stranded DNA (positive strand) containing the target DNA region in the second step and a part of the 3 ′ end of the single-stranded DNA (provided that the target DNA region In a reaction system containing a divalent cation when binding to a single-stranded immobilized oligonucleotide having a base sequence complementary to Is the method.
  • Method 3 includes the following steps: The first step, the second step, the third step, and Fourth step (previous step, main step ((i) A step ((i1) step A1 step, (i2) step A2 step), (ii) step B)), repeating step ((i) amplification step, ( ii) Quantitative process)).
  • Method 3 is the method described in Method 2 in which the divalent cation is a magnesium ion.
  • Method 4 includes the following steps: The first step, the second step, the third step, and Fourth step (pre-step (including pre-addition step), main step ((i) step A ((i1) step A1, step (i2) step A2), (ii) step B, (iii) step Step C ((iii1) Step C1, Step (iii2) Step C2)), Repeat step ((i) Amplification step, (ii) Quantification step)).
  • the method further includes the following one step: (C) (i) by binding the generated single-stranded DNA (positive strand) to the single-stranded oligonucleotide (negative strand) added in the reaction system in the previous step, Step C1 for selecting DNA in a single-stranded state; (Ii) using the single-stranded DNA selected in step C1 as a template, using the single-stranded oligonucleotide (negative strand) as a primer, and extending the primer
  • Method 5 includes the following steps: The first step, the second step, the third step, and Fourth step (pre-step (including pre-addition step and pre-addition step), main step ((i) Step A ((i1) Step A1, Step (i2) Step A2), (ii) Step B (Iii) Step C), repetitive step ((i) amplification step, (ii) quantitative step)).
  • the method 5 in the post-operation stage before the fourth step a part of the 3 ′ end of the single-stranded DNA (positive strand) containing the target DNA region (provided that the target is set)
  • a single-stranded oligonucleotide (negative strand) that has a complementary nucleotide sequence to the reaction system and is free to the reaction system pre-addition step
  • a step of once separating the single-stranded DNA) into a single-stranded state (additional re-preceding step), and
  • the method further includes the following one step: (C) (i) by binding the generated single-stranded DNA (positive strand) to the single-
  • Method 6 is a method for measuring a methylation ratio, which further includes the following two steps in addition to the steps of the method described in any one of the methods 1 to 5: (5) Without performing the first step of the method according to any one of the methods 1 to 5, by performing the second step to the fourth step in the method according to any one of the inventions 1 to 5, A fifth step of amplifying the DNA of the target DNA region (total amount of methylated DNA and non-methylated DNA) to a detectable amount, and quantifying the amount of amplified DNA; and (6) Based on the difference obtained by comparing the amount of DNA quantified by the fourth step according to any one of the methods 1 to 5 with the amount of DNA quantified by the fifth step, A sixth step of calculating the ratio of methylated DNA in the DNA region.
  • Method 7 is a method in which the second step is performed without performing the digestion treatment with the methylation-sensitive restriction enzyme in the first step described in the methods 1 to 6 above.
  • a single-stranded DNA (positive strand) containing a target DNA region is obtained from the digested DNA sample obtained in the first step,
  • the single-stranded DNA is selected by binding to an immobilized oligonucleotide.
  • the “single-stranded immobilized oligonucleotide” described in the above methods 1 to 7 is a part of the 3 ′ end of a single-stranded DNA (positive strand) containing the target DNA region (provided that the target is used)
  • a single-stranded immobilized oligonucleotide having a base sequence that is complementary to the DNA region (hereinafter also referred to as the present immobilized oligonucleotide).
  • This immobilized oligonucleotide is used for selecting single-stranded DNA (positive strand) containing a target DNA region from a DNA sample derived from genomic DNA contained in a mammal-derived specimen.
  • the immobilized oligonucleotide preferably has a length of 5 to 50 bases.
  • the 5 ′ end side of the present immobilized oligonucleotide can be immobilized with a carrier, while the 3 ′ end side thereof is directed from the 5 ′ end to the 3 ′ end by the third step and step A2 described later. It may be in a free state so that a single extension reaction that proceeds is possible.
  • the present immobilized oligonucleotide may be immobilized at its 5 ′ or 3 ′ end with a carrier.
  • the “can be immobilized with a carrier” described in the above methods 1 to 7 means that the present immobilized oligonucleotide is used as a carrier when selecting a single-stranded DNA (positive strand) containing the target DNA region.
  • the immobilized oligonucleotide and the carrier are immobilized. There may be.
  • an oligonucleotide having a base sequence (hereinafter sometimes referred to as the present oligonucleotide) is immobilized on a carrier according to a normal genetic engineering operation method or a commercially available kit / device (solid phase To).
  • the obtained biotinylated oligonucleotide is coated with streptavidin (eg, a PCR tube coated with streptavidin, or coated with streptavidin. And fixing to magnetic beads).
  • streptavidin eg, a PCR tube coated with streptavidin, or coated with streptavidin. And fixing to magnetic beads.
  • a method of covalently bonding to a support made of silica or a heat-resistant plastic via a spacer, a crosslinker, or the like, such as a structure in which five triglycerides are connected in series may be mentioned.
  • a method of chemically synthesizing directly from the 5 ′ end side of the present oligonucleotide on a glass or silicon support is also included.
  • the present immobilized oligonucleotide is a biotinylated oligonucleotide
  • A First, a DNA sample derived from genomic DNA contained in a mammal-derived specimen is subjected to an annealing buffer and a biotinylated oligonucleotide (the step after binding of the single-stranded DNA (positive strand) and the present immobilized oligonucleotide)
  • the mixture is obtained by adding the present immobilized oligonucleotide and the carrier, which are immobilized by binding to the carrier, at this stage.
  • the resulting mixture is then used for several minutes at 95 ° C.
  • the binding between the single-stranded DNA (positive strand) containing the target DNA region and the biotinylated oligonucleotide is coated with the biotinylated oligonucleotide and streptavidin.
  • the order may be either. That is, for example, a mixture is obtained by adding a DNA sample derived from a genomic DNA contained in a mammal-derived specimen to a biotinylated oligonucleotide immobilized on a support coated with streptavidin, and the resulting mixture Is heated at 95 ° C.
  • biotinylated oligo is converted to single-stranded double-stranded DNA containing a target DNA region present in a genomic DNA-derived DNA sample contained in a mammal-derived specimen.
  • the biotinylated oligonucleotide may be rapidly cooled to a temperature about 10 to 20 ° C. lower than the Tm value of the biotinylated oligonucleotide, and kept at that temperature for several minutes.
  • D After fixing the biotinylated oligonucleotide to the support coated with streptavidin in this way, the remaining solution is removed and washed (DNA purification).
  • the solution when using a PCR tube coated with streptavidin, the solution is first removed by pipetting or decantation, and then a TE buffer having a volume approximately equal to the volume of the mammal-derived specimen is added thereto. Add, then remove the TE buffer by pipetting or decanting.
  • a TE buffer having a volume approximately equal to the volume of the mammal-derived specimen is added thereto. Add, then remove the TE buffer by pipetting or decanting.
  • magnetic beads coated with streptavidin after fixing the beads with a magnet, first remove the solution by pipetting or decantation, and then add TE buffer that is approximately equal to the volume of the mammal-derived specimen. Add, then remove the TE buffer by pipetting or decanting. Subsequently, the residual solution is removed and washed (DNA purification) by performing such an operation several times.
  • This operation is important for removing unimmobilized DNA or DNA floating in a solution digested with a restriction enzyme described later from the reaction solution. If these operations are insufficient, the DNA floating in the reaction solution becomes a template, and an unexpected amplification product is obtained in the amplification reaction.
  • DNA having a completely different nucleotide sequence from the target region for example, rat DNA in the case of a human mammal-derived specimen
  • a large amount is added to the mammal-derived specimen, and the above operation is performed.
  • a single-stranded DNA (positive strand) containing the target DNA region and a part of the 3 ′ end of the single-stranded DNA (provided that When a single-stranded immobilized oligonucleotide having a base sequence that is complementary to the target DNA region is not bound), it is bound in a reaction system containing a divalent cation.
  • a divalent cation is a magnesium ion.
  • the “reaction system containing a divalent cation” means a divalent cation in an annealing buffer used for binding the single-stranded DNA (positive strand) and the single-stranded immobilized oligonucleotide.
  • a salt containing magnesium ion as a constituent element for example, Mg (OAc)) 2 MgCl 2 Etc.
  • Mg (OAc) MgCl 2 Etc.
  • the single-stranded DNA is elongated as double-stranded DNA.
  • an extension reaction is performed using DNA polymerase.
  • the third step described in the methods 1 to 7 is carried out as follows when, for example, the present immobilized oligonucleotide is a biotinylated oligonucleotide.
  • the incubated solution is removed by pipetting or decanting, and then a TE buffer having an amount substantially equal to the volume of the mammal-derived specimen is added thereto, and the TE buffer is removed by pipetting or decanting. More specifically, for example, when using a PCR tube coated with streptavidin, the solution is first removed by pipetting or decantation, and then a TE buffer having a volume approximately equal to the volume of the mammal-derived specimen is added thereto. Add, then remove the TE buffer by pipetting or decanting.
  • the third step described in the methods 1 to 7 includes a step of separating the single-stranded DNA selected in the second step from the immobilized oligonucleotide and once separating it into a single-stranded state.
  • the forward primer having a complementary base sequence (negative strand) is used as an extension primer, and the primer is extended once to form the single-stranded DNA as a double-stranded DNA.
  • heating is performed at 95 ° C. for several minutes in order to make double-stranded DNA into a single strand.
  • the forward primer is quickly cooled to a temperature about 10-20 ° C.
  • a double strand of DNA (positive strand) and a forward primer is formed.
  • An optimal 10 ⁇ buffer solution 100 mM Tris-HCl pH 8.3, 500 mM KCl, 15 mM MgCl was added to the resulting double-stranded DNA solution.
  • the third step may be performed independently of the fourth step, or may be performed continuously with the PCR reaction performed in the fourth step.
  • the double-stranded DNA formed in the third step (recognized by the recognition site of the methylation sensitive restriction enzyme)
  • the amplified DNA is amplified to a detectable amount and the amount of amplified DNA is quantified.
  • an elongated double-stranded DNA that is an undigested product obtained in the third step (the methylation) The double-stranded DNA formed by extension containing no CpG pair in the ammethyl state at the recognition site of the sensitive restriction enzyme is once separated into a single-stranded state.
  • an extended double-stranded DNA that is an undigested product obtained in the third step (a two-strand DNA that does not contain a CpG pair in an methylated state at the recognition site of the methylation-sensitive restriction enzyme).
  • An annealing buffer is added to the double-stranded DNA) to obtain a mixture.
  • the resulting mixture is then heated at 95 ° C. for several minutes.
  • Step A1 in Step A By using the single-stranded DNA selected in (i) above as a template and the single-stranded immobilized oligonucleotide as a primer, and extending the primer once, A certain DNA is extended and formed as double-stranded DNA (that is, step A2 in step A). Specifically, for example, it is carried out according to the following explanation or the operation method in the extension reaction in the second step described in the above methods 1 to 7.
  • extension primer reverse primer having a complementary base sequence (positive strand) as an extension primer (reverse primer)
  • extension primer reverse primer
  • the extension primer is extended once, so that the DNA in the single-stranded state is obtained. It is formed as a double-stranded DNA by extension (ie, step B).
  • each step of the fourth step is repeated after separating the double-stranded DNA formed by extension obtained in each step into a single-stranded state (for example, step A and step A).
  • step B the methylated DNA in the target DNA region is amplified to a detectable amount, and the amount of the amplified DNA is quantified.
  • the operation is performed according to the following description and the operation method in the previous step, the A step and the B step in the fourth step described in the methods 1 to 7 described above.
  • PCR As a method for amplifying a target DNA region (that is, a target region) after digestion with a methylation-sensitive restriction enzyme described in the methods 1 to 7, for example, PCR can be used.
  • an immobilized oligonucleotide can be used as a primer on one side, so by adding only the other primer and performing PCR, an amplification product is obtained, and the amplification product is also immobilized.
  • the Rukoto At this time, if a primer previously labeled with fluorescence or the like is used and the label is used as an index, the presence or absence of an amplification product can be evaluated without performing a troublesome operation such as electrophoresis.
  • a PCR reaction solution for example, 0.15 ⁇ l of a 50 ⁇ M primer solution, 2.5 ⁇ l of 2 mM dNTP, 10 ⁇ buffer solution (100 mM) are added to the DNA obtained in the third step described in the above methods 1 to 7.
  • Tris-HCl pH 8.3, 500 mM KCl, 20 mM MgCl 2 , 0.01% Gelatin) is mixed with 2.5 ⁇ l and AmpliTaq Gold (a kind of heat-resistant DNA polymerase: 5 U / ⁇ l) is mixed with 0.2 ⁇ l, and sterilized ultrapure water is added thereto to a volume of 25 ⁇ l.
  • AmpliTaq Gold a kind of heat-resistant DNA polymerase: 5 U / ⁇ l
  • the reaction may be carried out by adding an appropriate amount of betaine, DMSO or the like.
  • betaine for example, after the reaction solution is kept at 95 ° C. for 10 minutes as described above, one cycle is 95 ° C. for 30 seconds, 55 to 65 ° C. for 30 seconds, and 72 ° C. for 30 seconds.
  • the condition for performing the heat insulation for 30 to 40 cycles can be given.
  • the obtained amplification product is detected.
  • the amount of the fluorescent label immobilized can be measured after performing the same washing / purifying operation as before.
  • PCR when PCR is performed using a normal unlabeled primer, detection is performed by annealing gold colloid particles, a probe labeled with fluorescence, etc., and measuring the amount of the probe bound to the target region. be able to.
  • real-time PCR method is used, for example.
  • the real-time PCR method is a method for monitoring PCR in real time and kinetics analysis of the obtained monitoring results. For example, a high-precision quantitative PCR capable of detecting even a slight difference of about twice as much as the gene amount. It is a method known as law.
  • Examples of the real-time PCR method include a method using a probe such as a template-dependent nucleic acid polymerase probe and a method using an intercalator such as Cyber Green. Commercially available devices and kits for the real-time PCR method may be used. As described above, detection is not particularly limited, and detection by any known method can be performed. In these methods, operations up to detection can be performed without changing the reaction container.
  • a biotinylated oligonucleotide having the same base sequence as the immobilized oligonucleotide described in the above methods 1 to 7 is designed with a primer on one side or a new biotinylated oligonucleotide on the 3 ′ end side from the immobilized oligonucleotide. It can also be used as a primer on one side, and the target region can be amplified using the complementary primer.
  • the obtained amplification product is immobilized if there is a support coated with streptavidin, for example, when PCR is performed in a streptavidin-coated PCR tube, it is immobilized in the tube.
  • the use of labeled primers makes it easy to detect amplification products. If the previous immobilized oligonucleotide is immobilized by covalent bond or the like, the solution containing the amplification product obtained by PCR is transferred to the container where the streptavidin-coated support is present, and the amplification product is immobilized. It is possible. The detection is performed as described above.
  • the complementary primer for amplifying the target region must be a primer that can amplify a target region having one or more recognition sites for methylation sensitive restriction enzymes and does not include the recognition site. The reason for this is as follows.
  • the complementary primer contains the recognition site for the methylation-sensitive restriction enzyme on the most 3 ′ end
  • several bases on the 3 ′ end of the primer are the number of 3 ′ ends of the nascent strand. This is because the target region may be amplified by PCR as a result of annealing with the base.
  • one of the 3 ′ ends of the single-stranded DNA (positive strand) containing the target DNA region is used.
  • a step of adding a single-stranded oligonucleotide (negative strand) having a base sequence that is complementary to a portion (excluding the target DNA region) and in a free state into the reaction system Variants that additionally have a pre-addition step.
  • (Modification 1) Variant 1 is a pre-operation stage of the pre-step of the fourth step of the method described in methods 1 to 7, Having a base sequence that is complementary to a part of the 3 ′ end of a single-stranded DNA (positive strand) containing the target DNA region (excluding the target DNA region); Additionally having a step of adding a single-stranded oligonucleotide (negative strand) in a free state into the reaction system (pre-addition step), and As the main step of the fourth step of the method according to the method 1 to 7, the method further includes the following one step: (C) (i) By binding the generated single-stranded DNA (positive strand) and the single-stranded oligonucleotide (negative strand) added in the reaction system in the above-mentioned pre-addition step, Step C1 for selecting the DNA in the single-stranded state; (Ii) using the single-stranded DNA selected in step C1 as a template, using the single
  • Modification 2 is a post-operation stage before the fourth step of the method described in methods 1 to 7, Having a base sequence that is complementary to a part of the 3 ′ end of a single-stranded DNA (positive strand) containing the target DNA region (excluding the target DNA region); It additionally has a step (pre-addition step) of adding a single-stranded oligonucleotide (negative strand) that is in a free state into the reaction system, and the unprocessed product obtained through the third step and the above-mentioned pre-addition step.
  • Step C1 By binding the generated single-stranded DNA (positive strand) and the single-stranded oligonucleotide (negative strand) added in the reaction system in the above-mentioned pre-addition step, Step C1 for selecting the DNA in the single-stranded state; (Ii) using the single-stranded DNA selected in step C1 as a template, using the single-stranded oligonucleotide (negative strand) as a primer, and extending the primer once to thereby form the single-stranded state A C step (this step) having a C2 step of extending and forming the DNA as a double-stranded DNA.
  • a part of the 3 ′ end of the single-stranded DNA (positive strand) including the target DNA region (the positive strand) (excluding the target The DNA sequence is not included.
  • the single-stranded oligonucleotide (negative strand) added to the reaction system in the pre-addition step is part of the 3 ′ end of the single-stranded DNA (however, it does not include the target DNA region).
  • the single-stranded oligonucleotide is a single-stranded oligonucleotide in a free state having a complementary base sequence to the 5 ′ end and having the same base sequence as the single-stranded immobilized oligonucleotide, It may be the same base sequence as the immobilized oligonucleotide, a short base sequence, or a long sequence. However, when the sequence is longer than the single-stranded immobilized oligonucleotide, the extension primer is extended using the reverse primer (positive strand) as an extension primer and the single-stranded oligonucleotide (negative strand) as a template.
  • the single-stranded oligonucleotide must be in a free state that is not available for the reaction to be performed.
  • an immobilized oligonucleotide is used as a primer on one side, and only the other primer is added to perform PCR. If other methods (for example, analytical methods that can compare the amount of each amplification product obtained by PCR) are performed for product detection, as described above, when the target region is amplified, Alternatively, PCR may be carried out by adding a pair of primers without using the immobilized oligonucleotide as one (one side) primer. After performing such PCR, the amount of amplification product obtained is determined.
  • the fourth step has a repetition step.
  • the “generated single-stranded DNA (positive strand)” in the step A1 is the first step. In both the operation of the fourth step and the repetition operation of the fourth step after the second time, this means “generated DNA in a“ free ”single-stranded state (positive strand)”.
  • the “generated single-stranded DNA (negative strand)” in the step B means “the first step of the fourth step and the second and subsequent steps of the fourth step repeated” It means the “fixed” single-stranded DNA produced (positive strand) ”.
  • the fourth step further has a C step, it means “generated (fixed) single-stranded DNA (positive strand)” in the first operation of the fourth step.
  • “generated“ fixed ”single-stranded DNA (positive strand)” and “generated“ free ”single-stranded DNA ( "Positive chain)” means both.
  • the “extension-formed double-stranded DNA” obtained in each step of the fourth step is the first fourth step.
  • the CpG in the methylation sensitive restriction enzyme recognition site is all in the methylated state. It means “extension-formed double-stranded DNA that is a pair”.
  • the fourth step further includes a C step.
  • the “generated single-stranded DNA (positive strand)” in the C1 step is In both the first step of the fourth step and the second and subsequent steps of the fourth step, this means “generated“ free ”single-stranded DNA (positive strand)”.
  • the method for measuring a methylation rate additionally having the following two steps May be: (5) After performing the first step and the second step of the method described in the methods 1 to 7 (including the modified method), the method described in the methods 1 to 7 (including the modified method) ) Without performing the third step, the fourth step described in the methods 1 to 7 (including the modified method) is carried out, so that the DNA of the target DNA region (methylated DNA and methyl A fifth step of amplifying the total amount of DNA not detected) to a detectable amount and quantifying the amount of amplified DNA; and (6) It is obtained by comparing the amount of DNA quantified by the fourth step of the method described in the above methods 1 to 7 (including the modified method) with the amount of DNA quantified by the fifth step.
  • the measurement of the amount of methylated DNA in the target DNA region and the measurement of the methylation ratio are performed using the base sequence of the present DNA as the target DNA region.
  • Restriction enzymes, primers or probes that can be used in various methods for performing are useful as reagents for detection kits.
  • 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 carrier.
  • the scope of the right of the method for measuring the methylation ratio includes use in the form of a detection kit or a detection chip using the substantial principle of the method.
  • Examples of other embodiments when “measuring methylation frequency or index value correlated therewith” in the evaluation method of the present invention include, for example, methods 8 to 15 shown below.
  • Method 8 includes the following steps: The first step, the second step, and Third process (first pre-process, second pre-process ((i) second (A) pre-process, (ii) second (B) pre-process), third pre-process, main process ((i) first A Step ((i1) Step A1, (i2) Step A2), (ii) Step B), Repeat Step ((i) Amplification Step, (ii) Quantification Step)).
  • method 8 is a method for measuring the content of methylated DNA in a target DNA region of genomic DNA contained in a biological specimen, (1) From a DNA sample derived from genomic DNA contained in a biological specimen, a single-stranded DNA (positive strand) containing the target DNA region and the complementary DNA region of the single-stranded DNA A single-stranded immobilized oligonucleotide having a base sequence that is a sexual base, to select the single-stranded DNA, and the selected single-stranded DNA and the single-stranded immobilized oligonucleotide A first step of binding and forming a double-stranded DNA formed by binding; (2) After the double-stranded DNA formed in the first step is digested with at least one methylation sensitive restriction enzyme, the resulting free digest (in the recognition site of the methylation sensitive restriction enzyme) A second step of removing at least one double-stranded DNA comprising a CpG pair in an ammethyl state; and (3) As a pre-process
  • a step (third pre-step) for once separating the DNA (positive strand) and the DNA (negative strand) in a single-stranded state and as this step (A) The DNA in the single-stranded state is selected by binding the generated single-stranded DNA (positive strand) to the single-stranded immobilized oligonucleotide (negative strand).
  • the DNA in the single-stranded state selected in Step A1 and Step A1 is used as a template, the single-stranded immobilized oligonucleotide is used as a primer, and the primer is extended once, whereby the single strand A step A (this step) having a step A2 of extending the DNA in a strand state as a double-stranded DNA; (B) using the generated single-stranded DNA (negative strand) as a template, a partial base sequence (negative strand) of the base sequence of the single-stranded DNA (negative strand), and , A partial base sequence (negative strand) located further to the 3 ′ end side than the 3 ′ end of the base sequence (negative strand) that is complementary to the base sequence (positive strand) of the target DNA region, An extension primer (reverse primer) that has a complementary base sequence (positive strand) and cannot be used for an extension reaction using the above-mentioned single-stranded immobilized oligonucleotide as
  • a step B (this step), in which the DNA in the single-stranded state is converted into a double-stranded DNA that has been formed by extending the primer once, and each step of the third step is further performed.
  • the stretch formed in each of the above steps The double-stranded DNA is once separated into a single-stranded state and then repeated to amplify the methylated DNA in the target DNA region to a detectable amount, and the amount of amplified DNA
  • Method 9 includes the following steps: The first step, the second step, and Third process (first pre-process, second pre-process ((i) second (A) pre-process, (ii) second (B) pre-process), third pre-process, main process ((i) first A Step ((i1) Step A1, (i2) Step A2), (ii) Step B), Repeat Step ((i) Amplification Step, (ii) Quantification Step)).
  • a single-stranded DNA (positive strand) containing the target DNA region and a base sequence that is complementary to the target DNA region of the single-stranded DNA
  • the method according to the method 8 wherein the binding is performed in a reaction system containing a divalent cation when the single-stranded immobilized oligonucleotide having a divalent cation is bound.
  • Method 10 includes the following steps: The first step, the second step, and Third process (first pre-process, second pre-process ((i) second (A) pre-process, (ii) second (B) pre-process)), third pre-process, main process ((i) first Step A ((i1) Step A1, Step (i2) Step A2), (ii) Step B), Repeat Step ((i) Amplification Step, (ii) Quantification Step)).
  • Method 10 is the method described in Method 9, wherein the divalent cation is a magnesium ion.
  • Method 11 includes the following steps: The first step, the second step, and Third step (first pre-step (including pre-addition step), second pre-step ((i) second (A) pre-step, (ii) second (B) pre-step), third pre-step, book Step ((i) Step A ((i1) Step A1, Step (i2) Step A2), (ii) Step B, (iii) Step C ((iii1) Step C1, Step (iii) Step C2 Step)), repetition step ((i) amplification step, (ii) quantification step)).
  • the method 11 is a pre-operation stage of the first pre-process of the third process described in any of the methods 8 to 10,
  • a single-stranded oligonucleotide (negative strand) having a base sequence complementary to a part of the 3 ′ end of the single-stranded DNA (positive strand) containing the target DNA region and in a free state Is additionally added to the reaction system (pre-addition step), and
  • the method further includes the following one step: (C) (i) By binding the generated single-stranded DNA (positive strand) and the single-stranded oligonucleotide (negative strand) added in the reaction system in the above-mentioned pre-addition step, Step C1 for selecting the DNA in the single-stranded state, (Ii) Using the single-stranded DNA selected in step C1 as a template, the single-stranded oligonucleotide (negative strand)
  • Method 12 includes the following steps: The first step, the second step, and Third process (first pre-process (including pre-addition process and additional re-pre-process), second pre-process ((i) second (A) pre-process, (ii) second (B) pre-process), first Three previous processes, this process ((i) A process ((i1) A1 process, (i2) A2 process), (ii) B process, (iii) C process ((iii1) C1 process, (Iii2) Step C2)), repeat step ((i) amplification step, (ii) quantitative step)).
  • first pre-process including pre-addition process and additional re-pre-process
  • second pre-process ((i) second (A) pre-process, (ii) second (B) pre-process)
  • first Three previous processes this process ((i) A process ((i1) A1 process, (i2) A2 process), (ii) B process, (iii) C process ((iii1) C1 process, (I
  • the method 12 is a post-operation stage of the first pre-process of the third process described in any of the methods 8 to 10,
  • a single-stranded oligonucleotide (negative strand) having a base sequence complementary to a part of the 3 ′ end of the single-stranded DNA (positive strand) containing the target DNA region and in a free state A double-stranded DNA that is an undigested product obtained through the second step and the above-described pre-addition step (the methylation described above).
  • the method further includes the following one step: (C) (i) By binding the generated single-stranded DNA (positive strand) and the single-stranded oligonucleotide (negative strand) added in the reaction system in the above-mentioned pre-addition step, Step C1 for selecting the DNA in the single-stranded state, (Ii) Using the single-stranded DNA selected in step C1 as a template, the single-stranded oligonucleotide (negative strand) as a primer, and extending the primer once, thereby A second step C2 in which a DNA in a strand state is formed into an extended double-stranded DNA; C process (this process) which has.
  • Method 13 includes the following steps: The first step, the second step, and Third step (first pre-step (including pre-addition step), second pre-step ((i) second (A) pre-step, (ii) second (B) pre-step), third pre-step, book Step ((i) Step A ((i1) Step A1, Step (i2) Step A2), (ii) Step B, (iii) Step C ((iii1) Step C1, Step (iii) Step C2 Step)), repetition step ((i) amplification step, (ii) quantification step)).
  • the method 13 is a pre-operation stage of the third pre-process of the third process described in any of the methods 8 to 10,
  • a single-stranded oligonucleotide (negative strand) having a base sequence complementary to a part of the 3 ′ end of the single-stranded DNA (positive strand) containing the target DNA region and in a free state Is additionally added to the reaction system (pre-addition step), and
  • the method further includes the following one step: (C) (i) By binding the generated single-stranded DNA (positive strand) and the single-stranded oligonucleotide (negative strand) added in the reaction system in the above-mentioned pre-addition step, Step C1 for selecting the DNA in the single-stranded state, (Ii) Using the single-stranded DNA selected in step C1 as a template, the single-stranded oligonucleotide (negative strand)
  • Method 14 includes the following steps: The first step, the second step, and Third process (first pre-process (including pre-addition process and additional re-pre-process), second pre-process ((i) second (A) pre-process, (ii) second (B) pre-process), first Three previous processes, this process ((i) A process ((i1) A1 process, (i2) A2 process), (ii) B process, (iii) C process ((iii1) C1 process, (Iii2) Step C2)), repeat step ((i) amplification step, (ii) quantitative step)).
  • first pre-process including pre-addition process and additional re-pre-process
  • second pre-process ((i) second (A) pre-process, (ii) second (B) pre-process)
  • first Three previous processes this process ((i) A process ((i1) A1 process, (i2) A2 process), (ii) B process, (iii) C process ((iii1) C1 process, (I
  • the method 14 is a post-operation stage of the third pre-process of the third process described in any of the methods 8 to 10,
  • a single-stranded oligonucleotide (negative strand) having a base sequence complementary to a part of the 3 ′ end of the single-stranded DNA (positive strand) containing the target DNA region and in a free state A double-stranded DNA that is an undigested product obtained through the second step and the above-described pre-addition step (the methylation described above).
  • the method further includes the following one step: (C) (i) By binding the generated single-stranded DNA (positive strand) and the single-stranded oligonucleotide (negative strand) added in the reaction system in the above-mentioned pre-addition step, Step C1 for selecting the DNA in the single-stranded state, (Ii) Using the single-stranded DNA selected in step C1 as a template, the single-stranded oligonucleotide (negative strand) as a primer, and extending the primer once, thereby A second step C2 in which a DNA in a strand state is formed into an extended double-stranded DNA; C process (this process) which has.
  • Method 15 includes the following steps: First step, Third process (first pre-process, second pre-process ((i) second (A) pre-process, (ii) second (B) pre-process), third pre-process, main process ((i) first A Step ((i1) Step A1, (i2) Step A2), (ii) Step B), The fourth step ((i) amplification step, (ii) quantification step) and 5th process.
  • the method 15 is a method for measuring a methylation ratio, which further includes the following two steps as the steps of the method described in any of the methods 8 to 14: (4) After performing the first step of the method described in any of the methods 8 to 14, the method 8 without performing the second step of the method described in any of the methods 8 to 14
  • the DNA of the target DNA region becomes a detectable amount
  • a fourth step of amplifying and quantifying the amount of amplified DNA becomes a detectable amount
  • (5) Based on the difference obtained by comparing the amount of DNA quantified by the third step according to any one of the methods 8 to 14 and the amount of DNA quantified by the fourth step, And a fifth step of calculating the ratio of methylated DNA in the DNA region.
  • the method for measuring an index value correlated with the methylation frequency of the present DNA contained in a mammal-derived specimen, for example, transcription of a gene present downstream of the present DNA
  • the method include measuring the amount of mRNA that is a product and the amount of mRNA that is a transcription product of a gene whose expression level decreases due to methylation of the present DNA.
  • real-time PCR method Northern blot method [Molecular Cloning, Cold Spring Harbor Laboratory (1989)]
  • in situ RT-PCR method Nucleic Acids Res.
  • samples containing mRNA, which is a transcription product of a gene present downstream of the DNA contained in a mammal-derived specimen, or mRNA, which is a transcription product of a gene whose expression level decreases due to methylation of the DNA should be Similarly, it is prepared by extraction, purification, etc. from the sample. When Northern blotting is used to measure the amount of mRNA contained in the prepared sample, the expression level of the detection probe decreases due to the gene existing downstream of the DNA or the methylation of the DNA.
  • Genes or a part of them (a restriction enzyme digest of a gene present downstream of this DNA, an oligonucleotide of about 100 bases to about 1000 bases chemically synthesized according to the base sequence of the gene present downstream of this DNA, etc.)
  • a restriction enzyme digest of a gene present downstream of this DNA an oligonucleotide of about 100 bases to about 1000 bases chemically synthesized according to the base sequence of the gene present downstream of this DNA, etc.
  • the expression level of the primer used is the gene present downstream of the DNA or the methylation of the DNA. Any gene can be used as long as it can specifically amplify only the gene to be reduced, and the region to be amplified and the base length are not particularly limited.
  • the first step of the evaluation method of the present invention as another method of measuring an index value correlated with the methylation frequency of the present DNA contained in a mammal-derived specimen, for example, a gene present downstream of the present DNA or the method of measuring the quantity of the protein which is the translation product of the gene whose expression level reduces by methylation of this DNA can also be mention
  • ELISA method indirect competitive inhibition method
  • a specific antibody against a protein can be produced according to a conventional immunological method using the protein as an immunizing antigen.
  • an index value correlated with the methylation frequency of the target DNA contained in the mammal-derived specimen is measured.
  • An index value (control) having a correlation is compared, and the degree of canceration of the specimen is determined based on a difference obtained by the comparison.
  • the primer, probe or specific antibody that can be used in various methods for measuring the target DNA methylation frequency or an index value correlated therewith is used for cancer cells such as colon cancer cells. It is useful as a reagent for detection kits.
  • the present invention relates to a kit for detecting cancer cells such as colorectal cancer cells containing these primers, probes or specific antibodies as reagents, and a colon where these primers, probes or specific antibodies are immobilized on a carrier.
  • a chip for detecting cancer cells such as cancer cells is also provided, and the scope of rights of the evaluation method of the present invention is such as the detection kit and the detection chip as described above using the substantial principle of the method. Including use in various forms.
  • Example 1 Measurement of methylation rate of cytosine
  • Human tissue-derived genomic DNA shown in the table below was obtained from Biochain, and MassARRAY analysis was performed using the MassARRAY system.
  • amplicon 1-19 which is an oligonucleotide consisting of the base sequence shown in any one of SEQ ID NOs: 1 to 19, is prepared according to the method according to the outline of EpiTYPER for quantitative DNA methylation analysis shown in the SEQUENOM application note. The methylation rate of cytosine contained was measured. Amplicon 1-19 is shown below.
  • the 19 primer sets designed to amplify DNA obtained by Bisulfite treatment of the base sequence represented by the above 19 amplicons are shown below.
  • the primer sets for amplicon 1 are primer F1 and primer R1.
  • Primer sets for amplicon 2 are primer F2 and primer R2.
  • the primer sets for amplicon 3 are primer F3 and primer R3.
  • Primer sets for amplicon 4 are primer F4 and primer R4.
  • Primer sets for amplicon 5 are primer F5 and primer R5.
  • Primer sets for amplicon 6 are primer F6 and primer R6.
  • Primer sets for amplicon 7 are primer F7 and primer R7.
  • Primer sets for amplicon 8 are primer F8 and primer R8.
  • Primer sets for amplicon 9 are primer F9 and primer R9.
  • Primer sets for amplicon 10 are primer F10 and primer R10.
  • Primer sets for amplicon 11 are primer F11 and primer R11.
  • Primer sets for amplicon 12 are primer F12 and primer R12.
  • Primer sets for amplicon 13 are primer F13 and primer R13.
  • Primer sets for amplicon 14 are primer F14 and primer R14.
  • Primer sets for amplicon 15 are primer F15 and primer R15.
  • Primer sets for amplicon 16 are primer F16 and primer R16.
  • Primer sets for amplicon 17 are primer F17 and primer R17.
  • Primer sets for amplicon 18 are primer F18 and primer R18.
  • Primer sets for amplicon 19 are primer F19 and primer R19.
  • Primer design The following primer system is designed for methylation analysis. To obtain a product suitable for in vitro transcription, a reverse primer with a T7 promoter added is used. Insert an 8 bp insert to prevent cycling failure. In order to balance PCR, a forward primer with a 10-mer tag is used.
  • Bisulfite processing For the Bisulfite conversion treatment of the sample genomic DNA, EZ-96 DNA Methylation Kit or EZ DNA Methylation Kit of Zymo Research is used. After the initial incubation of this protocol, the cycle reaction is performed as follows. 45 cycles of 95 ° C. for 30 minutes and then 50 ° C.
  • Step 1 Amplify 1 ⁇ L of DNA in a total volume of 5 ⁇ L using amplification 385-microtiter format (final concentration of 2 ng / ⁇ L per reaction) In order to achieve this, use 1.00 ⁇ L or more of DNA of 10 ng / ⁇ L or more.
  • Each reaction solution is divided into two types of cleavage reactions (T cleavage reaction and C cleavage reaction). Seal the plate and perform the cycle reaction as follows. After incubating at 94 ° C. for 15 minutes, 45 cycles of incubating at 94 ° C. for 20 seconds, 56 ° C. for 30 seconds, and then at 72 ° C. for 1 minute are performed for 45 cycles, and then at 72 ° C. for 3 minutes.
  • Step 2 Dephosphorylation Add 2 ⁇ L of shrimp-derived alkaline phosphatase (SAP) enzyme to 5 ⁇ L of each PCR reaction solution to dephosphorylate dNTPs that have not been incorporated into PCR. The plate is incubated for 20 minutes at 37 ° C and then for 5 minutes at 85 ° C.
  • Step 3 In vitro transcription and RNase cleavage Prepare a transcription / RNase A cocktail for each cleavage reaction (T and C). The standard setup prepares one transcription / RNase A cocktail per plate. Add 5 ⁇ L of transcription / RNase A cocktail and 2 ⁇ L of PCR / SAP sample to a new microtiter plate that has not been cycled.
  • SAP shrimp-derived alkaline phosphatase
  • Step 4 Sample conditioning Add 20 ⁇ L of ddH20 to each sample in the 384-well plate. Add 6 mg of Clean Resin to each well using a resin plate. Stir for 10 minutes and spin down at 3,200 xg for 5 minutes.
  • Step 5 Transfer of sample Disperse 10-15 nL of EpiTYPE reaction product into 384-well SpectroCHIP.
  • Step 6 Sample analysis MassARRAY system is used to obtain spectra of two types of cleavage reactions.
  • Step 7 Analyze the analysis software result with EpiTYPER software.
  • FIGS. 1 and C02 human mammary gland healthy tissue genomic DNA samples (N01 and N02) and human breast cancer tissue genomic DNA samples (C01, C02, C03 and C04):
  • FIG. 1 shows the measurement results of the cytosine methylation ratios shown by base numbers 109, 163, 176, 218, 226, 228, 247, 257, 286 and 391 in the base sequence shown by SEQ ID NO: 1;
  • FIG. 2 shows the measurement results of the cytosine methylation ratio represented by base numbers 55, 60, 383 and 391 in the base sequence represented by SEQ ID NO: 2;
  • FIG. 1 shows the measurement results of the cytosine methylation ratios shown by base numbers 109, 163, 176, 218, 226, 228, 247, 257, 286 and 391 in the base sequence shown by SEQ ID NO: 1
  • FIG. 2 shows the measurement results of the cytosine methylation ratio represented by base numbers 55, 60, 383 and 391 in the base sequence represented by SEQ
  • FIG. 3 shows the measurement results of the cytosine methylation ratios represented by base numbers 113, 119, 133 and 138 in the base sequence represented by SEQ ID NO: 3;
  • the measurement result of the cytosine methylation ratio shown by base numbers 27, 43, 97, 102, 118, 131, 138, 152, 157, 213, 216, 220, 229 and 234 in the base sequence shown by SEQ ID NO: 6 Shown in FIG. 4;
  • FIG. 5 shows the measurement results of the methylation ratio of cytosine represented by base numbers 106, 116, 118, 140, 174 and 237 in the base sequence represented by SEQ ID NO: 7;
  • FIG. 6 shows the measurement results of the cytosine methylation ratios shown by base numbers 54, 169, 172, 240, 295 and 420 in the base sequence shown by SEQ ID NO: 8;
  • FIG. 7 shows the measurement results of the cytosine methylation ratios shown by base numbers 233, 326, 378, 383, 408, 429 and 453 in the base sequence shown by SEQ ID NO: 9;
  • FIG. 9 shows the measurement results of the cytosine methylation ratios shown by base numbers 233, 326, 378, 383, 408, 429 and 453 in the base sequence shown by SEQ ID NO: 9;
  • base numbers 37, 52, 64, 80, 102, 104, 117, 164, 170, 173, 198, 201, 216, 274, 277, 296, 307, 314, 327, 379 , 401, 403 and 411 show the measurement results of the methylation rate of cytosine shown in FIG.
  • FIG. 10 shows the measurement results of the methylation rate of cytosine; Cytosine represented by base numbers 37, 44, 72, 136, 172, 174, 181, 193, 207, 227, 241, 253, 261, 285, 305, 338, 359 and 384 in the base sequence represented by SEQ ID NO: 13.
  • FIG. 11 shows the measurement results of the methylation ratio of FIG.
  • FIG. 12 shows the measurement results of the methylation ratio of cytosine represented by base numbers 27, 32, 109, 112, 116 and 257 in the base sequence represented by SEQ ID NO: 14;
  • FIG. 13 shows the measurement results of the cytosine methylation ratios shown by base numbers 148 and 243 in the base sequence shown by SEQ ID NO: 16;
  • the measurement results of the cytosine methylation ratios shown by H.254, 277, 305 and 333 are shown in FIG.
  • FIG. 16 shows the results of measurement of the methylation ratio of cytosine represented by base numbers 109, 163, 176, 218, 226, 228, 247, 257, 286 and 391 in the base sequence represented by SEQ ID NO: 1;
  • FIG. 17 shows the measurement results of the methylation ratio of cytosine represented by base numbers 113, 119, 133 and 138 in the base sequence represented by SEQ ID NO: 3;
  • FIG. 18 shows the measurement results of the methylation ratio of cytosine represented by base numbers 69, 113 and 265 in the base sequence represented by SEQ ID NO: 4;
  • FIG. 20 shows the measurement results of the cytosine methylation ratios shown by base numbers 54, 169, 172, 240, 295 and 420 in the base sequence shown by SEQ ID NO: 8;
  • base numbers 37, 52, 64, 80, 102, 104, 117, 164, 170, 173, 198, 201, 216, 274, 277, 296, 307, 314, 327, 379 , 401, 403 and 411 show the measurement results of the methylation rate of cytosine shown in FIG.
  • FIG. 28 shows the measurement results of the cytosine methylation ratios shown by base numbers 109, 163, 176, 218, 226, 228, 247, 257, 286 and 391 in the base sequence shown by SEQ ID NO: 1;
  • FIG. 29 shows the measurement results of the cytosine methylation ratio represented by base numbers 113, 119, 133 and 138 in the base sequence represented by SEQ ID NO: 3;
  • FIG. 29 shows the measurement results of the cytosine methylation ratio represented by base numbers 113, 119, 133 and 138 in the base sequence represented by SEQ ID NO: 3;
  • FIG. 30 shows the measurement results of the cytosine methylation ratio represented by base numbers 184, 212, 263 and 324 in the base sequence represented by SEQ ID NO: 5;
  • FIG. 32 shows the measurement results of the methylation ratio of cytosine represented by base numbers 106, 116, 118, 140, 174 and 237 in the base sequence represented by SEQ ID NO: 7;
  • FIG. 33 shows the measurement results of the cytosine methylation ratios shown by base numbers 233, 326, 378, 383, 408, 429 and 453 in the base sequence shown by SEQ ID NO: 9;
  • FIG. 34 shows the measurement results of the cytosine methylation ratios shown by base numbers 69, 72, 78, 129, 159, 161, 232, 235, 264, 297 and 335 in the base sequence shown by SEQ ID NO: 10;
  • base numbers 37, 52, 64, 80, 102, 104, 117, 164, 170, 173, 198, 201, 216, 274, 277, 296, 307, 314, 327, 379 , 401, 403 and 411 show the measurement results of the methylation rate of cytosine shown in FIG.
  • the methylation rate of human breast cancer tissue genomic DNA is higher than the methylation rate of human breast gland healthy tissue genomic DNA. It was shown that the methylation rate of human lung cancer tissue genomic DNA is higher than the methylation rate of human lung healthy tissue genomic DNA. From the above, (1) by measuring the methylation rate of human colon tissue genomic DNA, whether or not colon cancer cells are present, (2) by measuring the methylation rate of human mammary tissue genomic DNA, It was confirmed that the presence or absence of lung cancer cells can be evaluated by measuring the presence or absence of breast cancer cells or (3) the methylation ratio of human lung tissue genomic DNA.
  • the content of methylated DNA in a target DNA region possessed by genomic DNA contained in a biological specimen is measured, and whether colon cancer cells are present or whether breast cancer cells are present.
  • by using the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 19 described in the present invention it is possible to easily detect cancer, detection of cancer at an extremely early stage, and recurrence of cancer.
  • the method and kit of the present invention are useful in combination with other inspection methods such as endoscopy, and can obtain more precise inspection results.
  • Oligonucleotide primer designed for PCR SEQ ID NO: 20 Oligonucleotide primer designed for PCR SEQ ID NO: 21
  • Oligonucleotide primer designed for PCR SEQ ID NO: 22 Oligonucleotide primer designed for PCR SEQ ID NO: 23
  • Oligonucleotide primer designed for PCR SEQ ID NO: 24 Oligonucleotide primer designed for PCR SEQ ID NO: 25
  • Oligonucleotide primer designed for PCR SEQ ID NO: 26 Oligonucleotide primer designed for PCR SEQ ID NO: 27
  • Oligonucleotide primer designed for PCR SEQ ID NO: 28 Oligonucleotide primer designed for PCR SEQ ID NO: 29
  • Oligonucleotide primer designed for PCR SEQ ID NO: 30 Oligonucleotide primer designed for PCR SEQ ID NO: 31
  • Oligonucleotide primer designed for PCR SEQ ID NO: 32 Oligonucleotide primer

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Abstract

La présente invention concerne un procédé d'évaluation du degré de cancérisation d'un prélèvement provenant d'un mammifère, le procédé comportant : (1) une première étape de mesure de la fréquence de méthylation ou d'une indication corrélée avec celle-ci, d'au moins un ADN ayant une séquence de base choisie parmi des séquences de base représentées par SEQ ID NO 1-19, comprises dans le prélèvement provenant d'un mammifère ; (2) une seconde étape de détermination du degré de cancérisation du prélèvement sur la base de la différence obtenue par la comparaison de la fréquence de méthylation mesurée, ou de l'indication corrélée avec celle-ci, et d'une valeur témoin.
PCT/JP2012/075879 2011-09-30 2012-09-28 Procédé d'évaluation du degré de cancérisation WO2013047910A1 (fr)

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JP2003144157A (ja) * 2001-08-23 2003-05-20 National Cancer Center-Japan 哺乳動物由来の検体の癌化度を評価する方法
JP2004135661A (ja) * 2002-09-26 2004-05-13 National Cancer Center-Japan 哺乳動物由来の検体の癌化度を評価する方法
JP2010516234A (ja) * 2007-01-19 2010-05-20 エピゲノミクス アーゲー 細胞増殖性障害の検出のための方法及び核酸

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JP2003144157A (ja) * 2001-08-23 2003-05-20 National Cancer Center-Japan 哺乳動物由来の検体の癌化度を評価する方法
JP2004135661A (ja) * 2002-09-26 2004-05-13 National Cancer Center-Japan 哺乳動物由来の検体の癌化度を評価する方法
JP2010516234A (ja) * 2007-01-19 2010-05-20 エピゲノミクス アーゲー 細胞増殖性障害の検出のための方法及び核酸

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