WO2008078834A9 - Méthode de détermination de méthylation d'adn - Google Patents

Méthode de détermination de méthylation d'adn Download PDF

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WO2008078834A9
WO2008078834A9 PCT/JP2007/075360 JP2007075360W WO2008078834A9 WO 2008078834 A9 WO2008078834 A9 WO 2008078834A9 JP 2007075360 W JP2007075360 W JP 2007075360W WO 2008078834 A9 WO2008078834 A9 WO 2008078834A9
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dna
stranded
stranded dna
base sequence
oligonucleotide
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WO2008078834A1 (fr
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冨ヶ原祥隆
樽井弘和
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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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

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  • the present invention relates to a method for measuring the content of methylated DNA in a target DNA region in genomic DNA contained in a biological specimen.
  • the content of methylated DNA in the target DNA region in genomic DNA is determined.
  • There are methods of measuring see, for example, Nucleic Acids Res. 1994 Aug 11; 22 (15): 2990-7, and Proc Natl Acad Sci USA A. 1997 Mar 18; 94 (6): 2284-9).
  • the measurement method first, it is necessary to extract DNA containing a target DNA region from a DNA sample derived from genomic DNA, and the extraction operation is complicated.
  • a DNA synthesis chain reaction by DNA polymerase after the DNA is modified with sulfite or the like Polymerase Chain Rection; hereinafter referred to as PCR.
  • a method of amplifying a target region (2) After digesting the DNA with a methylation sensitive restriction enzyme, and subjecting it to PCR, A method for amplifying a target region is known. In any of these methods, labor is required for modification of DNA for detection of methylation and subsequent purification of the product, preparation of a reaction system for PCR, confirmation of DNA amplification, and the like.
  • An object of the present invention is to provide a method for easily measuring the content of methylated DNA in a target DNA region in genomic DNA contained in a biological specimen. That is, the present invention 1.
  • a method for measuring the content of methylated DNA in a target DNA region possessed by 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 a part of the 3 ′ end of the single-stranded DNA (provided that The first single-stranded DNA is selected by base pairing with a single-stranded immobilized oligonucleotide having a base sequence complementary to the target DNA region).
  • the single-stranded DNA selected in the first step is digested with a methylation-sensitive restriction enzyme capable of digesting one or more types of single-stranded DNA, and then the free digest produced (the single-stranded DNA described above)
  • a second step of removing one or more single-stranded DNA containing CpG in an ammethyl state at a recognition site of a methylation-sensitive restriction enzyme capable of digesting (3)
  • single-stranded DNA that is an undigested product obtained in the second step (the methylation state is present at the recognition site of the methylation-sensitive restriction enzyme capable of digesting the single-stranded DNA).
  • a single-stranded DNA not containing CpG) from the single-stranded immobilized oligonucleotide (first pre-process),
  • the generated free single-stranded DNA is selected by base-pairing the generated single-stranded DNA (positive strand) with the single-stranded immobilized oligonucleotide.
  • the single-stranded immobilized oligonucleotide is prepared using the DNA in the single-stranded state selected in step A1 and the single-stranded DNA selected in step A1 as a template.
  • the primer By rotating extension, the first A step having a first A2 step of extending form said selected a single-stranded state DNA as double-stranded DNA (this step), (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, Using the oligonucleotide (reverse primer) having a complementary base sequence (positive strand) as an extension primer, the oligonucleotide is extended once to extend the generated single
  • a third step of amplifying the amplified DNA to a detectable amount and quantifying the amount of amplified DNA (Hereinafter, also referred to as the measurement method of the present invention); 2.
  • a single-stranded DNA positive strand
  • a part of the 3 ′ end of the single-stranded DNA (however, the target DNA region is not included).
  • a part of the 3 ′ end of the single-stranded DNA (positive strand) containing the target DNA region A step of adding a single-stranded oligonucleotide (negative strand) having a complementary base sequence and a free state into the reaction system (pre-addition step), and The method according to the first step, further comprising the following one step as the second step and each main step: (C) (i) Base-pairing the generated single-stranded DNA (positive strand) with the single-stranded oligonucleotide (negative strand) added to the reaction system in the preceding 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 C step (this step) having a C2 step of converting the DNA in a strand state into a double-stranded DNA formed by extension; 5).
  • 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 C step (this step) having a C2 step of converting the DNA in a strand state into a double-stranded DNA formed by extension; 6).
  • 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 C step (this step) having a C2 step of converting the DNA in a strand state into a double-stranded DNA formed by extension; 7).
  • 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 C step (this step) having a C2 step of converting the DNA in a strand state into a double-stranded DNA formed by extension; 8).
  • the method for measuring a methylation ratio which further comprises the following two steps as a step of the method according to any one of the preceding items 1 to 7; (4) After performing the first step of the method according to any one of the preceding items 1 to 8, without performing the second step of the method according to any one of the preceding items 1 to 7, By performing the third step in the method described in 1. above, the DNA in the target DNA region (the total amount of methylated DNA and non-methylated DNA) is amplified to a detectable amount and amplified.
  • a fourth step of quantifying the amount of DNA and (5) Based on the difference obtained by comparing the amount of DNA quantified by the third step according to any one of 1 to 7 and the amount of DNA quantified by the fourth step, A fifth step of calculating the ratio of methylated DNA in the DNA region to be treated; 9.
  • the method according to any one of 1 to 8 above, wherein the biological specimen is mammalian blood or body fluid; 11. 9.
  • the biological specimen is a cell lysate or a tissue lysate; 12
  • the DNA sample derived from genomic DNA contained in the biological specimen is a DNA sample that has been previously digested with a restriction enzyme that does not use the target DNA region of the genomic DNA as a recognition cleavage site.
  • the methylation-sensitive restriction enzyme capable of digesting one or more types of single-stranded DNA is a restriction enzyme having a recognition cleavage site in a target DNA region of a genomic DNA contained in a biological sample. 15.
  • the methylation sensitive restriction enzyme capable of digesting one or more types of single-stranded DNA is HhaI which is a methylation sensitive restriction enzyme; Etc. are provided.
  • FIG. 1 shows that the sample prepared in Example 1 was subjected to any of the treatments “A (no treatment)”, “B (HpaII treatment)”, or “C (HhaI treatment)”. It is the figure which showed the result of having amplified the methylated DNA in the area
  • FIG. 2 shows a region consisting of the base sequence shown in SEQ ID NO: 22 after the sample prepared in Example 2 was subjected to either “A (no treatment)” or “B (HhaI treatment)” treatment. It is the figure which showed the result of having amplified the methylated DNA in (1) by PCR and performing 1.5% agarose gel electrophoresis of the obtained amplification product.
  • DNA marker “M” sample 1 subjected to “A” treatment of DNA fragment Y2, sample 2 subjected to “A” treatment of DNA fragment Y2, and DNA fragment Y2
  • Sample 1 subjected to “B” treatment sample 2 subjected to “B” treatment of DNA fragment Y2, sample 1 subjected to “A” treatment of DNA fragment X2, and “A” treatment of DNA fragment X2.
  • the results are shown for the sample 2 that was subjected to the treatment, the sample 1 that was subjected to the “B” treatment of the DNA fragment X2, and the sample 2 that was subjected to the “B” treatment of the DNA fragment X2.
  • Example 3 shows that the sample of DNA fragment X2 prepared in Example 3 was subjected to either “A (no treatment)” or “B (HhaI treatment)” treatment, and the base shown in SEQ ID NO: 22 It is the figure which amplified the methylated DNA in the area
  • the leftmost lane in the figure shows the DNA marker “M”.
  • Example 4 shows a sample of DNA fragment Y2 prepared in Example 3, which was subjected to either “A (no treatment)” or “B (HhaI treatment)”, and the base represented by SEQ ID NO: 22 It is the figure which amplified the methylated DNA in the area
  • the leftmost lane in the figure shows the DNA marker “M”.
  • Example 5 shows that the sample of DNA fragment X2 prepared in Example 4 was treated with either “A (no treatment)” or “B (HhaI treatment)”, and the base shown in SEQ ID NO: 22 It is the figure which amplified the methylated DNA in the area
  • the leftmost lane in the figure shows the DNA marker “M”.
  • Example 6 shows that the sample of DNA fragment Y2 prepared in Example 4 was treated with either “A (no treatment)” or “B (HhaI treatment)”, and the base represented by SEQ ID NO: 22 It is the figure which amplified the methylated DNA in the area
  • the leftmost lane in the figure shows the DNA marker “M”.
  • FIG. 7 shows that the sample “(I)” prepared in Example 5 was added to “A (no treatment)”, “B (HpaII treatment)”, “C (HhaI treatment)” or “D (HpaII and HhaI).
  • FIG. 18 is a view showing the result of measuring the amount of methylated DNA in the region consisting of the base sequence represented by SEQ ID NO: 17 by real-time PCR after performing any of the processes of
  • the vertical axis in the figure shows the relative value when the amount of DNA in the sample subjected to the “A” treatment is 1, (average value of three times ⁇ standard deviation). Moreover, the theoretical value has shown the calculated value (methylation ratio) anticipated in B group, C group, and D group.
  • FIG. 8 shows that the sample “(II)” prepared in Example 5 was added to “A (no treatment)”, “B (HpaII treatment)”, “C (HhaI treatment)” or “D (HpaII and HhaI).
  • FIG. 8 shows that the sample “(II)” prepared in Example 5 was added to “A (no treatment)”, “B (HpaII treatment)”, “C (HhaI treatment)” or “D (HpaII and HhaI).
  • FIG. 18 is a view showing the result of measuring the amount of methylated DNA in the region consisting of the base sequence represented by SEQ ID NO: 17 by real-time PCR after performing any of the processes of
  • the vertical axis in the figure shows the relative value when the amount of DNA in the sample subjected to the “A” treatment is 1, (average value of three times ⁇ standard deviation). Moreover, the theoretical value has shown the calculated value (methylation ratio) anticipated in B group, C group, and D group.
  • FIG. 9 shows that the sample “(III)” prepared in Example 5 was added to “A (no treatment)”, “B (HpaII treatment)”, “C (HhaI treatment)” or “D (HpaII and HhaI).
  • FIG. 9 shows that the sample “(III)” prepared in Example 5 was added to “A (no treatment)”, “B (HpaII treatment)”, “C (HhaI treatment)” or “D (HpaII and HhaI).
  • FIG. 18 is a view showing the result of measuring the amount of methylated DNA in the region consisting of the base sequence represented by SEQ ID NO: 17 by real-time PCR after performing any of the processes of
  • the vertical axis in the figure shows the relative value when the amount of DNA in the sample subjected to the “A” treatment is 1, (average value of three times ⁇ standard deviation). Moreover, the theoretical value has shown the calculated value (methylation ratio) anticipated in B group, C group, and D group.
  • FIG. 10 shows that the sample “(IV)” prepared in Example 5 was added to “A (no treatment)”, “B (HpaII treatment)”, “C (HhaI treatment)” or “D (HpaII and HhaI).
  • FIG. 10 shows that the sample “(IV)” prepared in Example 5 was added to “A (no treatment)”, “B (HpaII treatment)”, “C (HhaI treatment)” or “D (HpaII and HhaI).
  • FIG. 18 is a view showing the result of measuring the amount of methylated DNA in the region consisting of the base sequence represented by SEQ ID NO: 17 by real-time PCR after performing any of the processes of
  • the vertical axis in the figure shows the relative value when the amount of DNA in the sample subjected to the “A” treatment is 1, (average value of three times ⁇ standard deviation). Moreover, the theoretical value has shown the calculated value (methylation ratio) anticipated in B group, C group, and D group.
  • FIG. 11 shows that the sample “(V)” prepared in Example 5 was added to “A (no treatment)”, “B (HpaII treatment)”, “C (HhaI treatment)” or “D (HpaII and HhaI).
  • FIG. 11 shows that the sample “(V)” prepared in Example 5 was added to “A (no treatment)”, “B (HpaII treatment)”, “C (HhaI treatment)” or “D (HpaII and HhaI).
  • FIG. 18 is a view showing the result of measuring the amount of methylated DNA in the region consisting of the base sequence represented by SEQ ID NO: 17 by real-time PCR after performing any of the processes of
  • the vertical axis in the figure shows the relative value when the amount of DNA in the sample subjected to the “A” treatment is 1, (average value of three times ⁇ standard deviation).
  • the theoretical value has shown the calculated value (methylation ratio) anticipated in B group, C group, and D group.
  • biological specimen examples include, for example, a cell lysate, a tissue lysate (herein, tissue has a broad meaning including blood, lymph nodes, etc.), or in mammals, plasma. And biological samples such as body fluids such as serum and lymph, body secretions (such as urine and milk), and genomic DNA obtained by extraction from these biological samples.
  • biological specimen examples include samples derived from microorganisms, viruses and the like.
  • genomic DNA in the measurement method of the present invention means genomic DNA such as microorganisms and viruses.
  • DNA may be extracted using a commercially available DNA extraction kit or the like.
  • plasma or serum is prepared from blood according to a normal method, and the prepared plasma or serum is used as a specimen, and free DNA contained therein (derived from cancer cells such as stomach cancer cells) Analysis of cancer cells such as gastric cancer cells, avoiding blood cell-derived DNA, and improving sensitivity to detect cancer cells such as gastric cancer cells and tissues containing them Can be made.
  • the “methylated DNA” in the present invention means a DNA produced by such methylation modification.
  • the “CpG pair” in the present invention means a double-stranded oligonucleotide formed by base pairing of a base sequence represented by CpG and a complementary CpG.
  • a “target DNA region” in the present invention (hereinafter sometimes referred to as a target region) is a DNA region to be examined for the presence or absence of cytosine methylation contained in the region, and includes at least one kind of methyl region. It has a recognition site for a susceptibility restriction enzyme.
  • cytosines in the base sequence indicated by CpG present in the base sequence of the promoter region, non-translation region or translation region (coding region) of a useful protein gene such as r, true carrier family 6 neurotransmitter transporter noradrenalin member 2 Examples thereof include DNA regions.
  • the useful protein gene is a Lysyl oxidase gene
  • Examples of the base sequence to be included include a base sequence of genomic DNA containing exon 1 of a human-derived lysyl oxidase gene and a promoter region located 5 ′ upstream thereof, and more specifically, SEQ ID NO: 1 (Corresponding to the base sequence represented by base numbers 16001 to 18661 of the base sequence described in Genbank Accession No. AF270645).
  • the ATG codon that encodes the amino terminal methionine of Lysyl oxidase protein derived from human is represented by base numbers 2031 to 2033, and the base sequence of exon 1 is represented by the base number 1957-2661.
  • cytosine having high methylation frequency in gastric cancer cells for example, in the base sequence represented by SEQ ID NO: 1, base numbers 1539, 1560, 1574, 1600, 1623, 1635, 1644, 1654, 1661, Examples include cytosine represented by 1682, 1686, 1696, 1717, 1767, 1774, 1783, 1785, 1787, 1795, and the like.
  • the useful protein gene is an HRAS-like suppressor gene
  • the base sequence represented by CpG existing in the base sequence of the promoter region, untranslated region or translated region (coding region) is used.
  • Examples of the base sequence containing one or more include a base sequence of genomic DNA containing exon 1 of a human-derived HRAS-like suppressor gene and a promoter region located 5 ′ upstream thereof. Includes the base sequence represented by SEQ ID NO: 2 (corresponding to the base sequence represented by base numbers 172001 to 173953 of the base sequence described in Genbank Accession No. AC068162). In the base sequence represented by SEQ ID NO: 2, the base sequence of exon 1 of the human-derived HRAS-like suppressor gene is represented by base numbers 1743 to 1953.
  • the cytosine in the base sequence represented by CpG present in the base sequence represented by SEQ ID NO: 2 particularly the cytosine in CpG present in the region where CpG is densely present in the base sequence represented by SEQ ID NO: 2, Shows high methylation frequency (ie, hypermethylation) in cancer cells such as gastric cancer cells.
  • cytosine having high methylation frequency in gastric cancer cells for example, in the base sequence represented by SEQ ID NO: 2, base numbers 1316, 1341, 1357, 1359, 1362, 1374, 1390, 1399, 1405, 1409, 1414, 1416, 1422, 1428, 1434, 1449, 1451, 1454, 1463, 1469, 1477, 1479, 1483, 1488, 1492, 1494, 1496, 1498, 1504, 1510, 1513, 1518, 1520, etc. Can be given cytosine.
  • the base represented by CpG present in the base sequence of the promoter region, untranslated region or translated region (coding region).
  • the base sequence containing one or more sequences include a base sequence of genomic DNA containing exon 1 of human-derived bA305P22.2.1 gene and a promoter region located 5 ′ upstream thereof, and more Specifically, the base sequence represented by SEQ ID NO: 3 (corresponding to the base sequence represented by base numbers 13001 to 13889 of the base sequence described in Genbank Accession No. AL121673) can be mentioned.
  • the ATG codon encoding the amino terminal methionine of the human-derived bA305P22.2.1 protein is represented by base numbers 849 to 851, and the base sequence of the exon 1 is The base numbers 663 to 889 are shown.
  • cytosine having high methylation frequency in gastric cancer cells for example, in the base sequence represented by SEQ ID NO: 3, base numbers 329, 335, 337, 351, 363, 373, 405, 424, 427, And cytosine represented by 446, 465, 472, 486, etc.
  • the useful protein gene is a Gamma filamin gene
  • the base sequence represented by CpG existing in the base sequence of the promoter region, untranslated region or translated region (coding region) is identical.
  • Examples of the base sequence comprising two or more include the base sequence of genomic DNA containing exon 1 of the human-derived Gamma filamin gene and the promoter region located 5 ′ upstream thereof, more specifically, No.
  • the cytosine in the base sequence represented by CpG present in the base sequence represented by SEQ ID NO: 4, particularly the cytosine in CpG present in the region where CpG is densely present in the base sequence represented by SEQ ID NO: 4 is, for example, Shows high methylation frequency (ie, hypermethylation) in cancer cells such as gastric cancer cells.
  • cytosine having high methylation frequency in gastric cancer cells for example, in the base sequence represented by SEQ ID NO: 4, base numbers 329, 333, 337, 350, 353, 360, 363, 370, 379, Examples include cytosine represented by 382, 384, 409, 414, 419, 426, 432, 434, 445, 449, 459, 472, 474, 486, 490, 503, 505, and the like. More specifically, for example, when the useful protein gene is a HAND1 gene, one base sequence represented by CpG existing in the base sequence of the promoter region, untranslated region or translated region (coding region) is used.
  • Examples of the base sequence include the base sequence of genomic DNA containing exon 1 of human-derived HAND1 gene and the promoter region located 5 ′ upstream thereof, and more specifically, SEQ ID NO: 5 (Corresponding to a complementary sequence of the base sequence represented by base numbers 24303 to 26500 of the base sequence described in Genbank Accession No. AC026668).
  • SEQ ID NO: 5 Corresponding to a complementary sequence of the base sequence represented by base numbers 24303 to 26500 of the base sequence described in Genbank Accession No. AC026668.
  • the base sequence represented by SEQ ID NO: 5 the ATG codon encoding the amino terminal methionine of the HAND1 protein derived from human is represented by base numbers 1656 to 1658, and the base sequence of the exon 1 is represented by base number 1400. ⁇ 2198.
  • the cytosine in the base sequence represented by CpG present in the base sequence represented by SEQ ID NO: 5, particularly the cytosine in CpG present in the region where CpG is densely present in the base sequence represented by SEQ ID NO: 5 is, for example, Shows high methylation frequency (ie, hypermethylation) in cancer cells such as gastric cancer cells.
  • cytosine having high methylation frequency in gastric cancer cells for example, in the base sequence represented by SEQ ID NO: 5, base numbers 1153, 1160, 1178, 1187, 1193, 1218, 1232, 1266, 1272, Examples include cytosine represented by 1292, 1305, 1307, 1316, 1356, 1377, 1399, 1401, 1422, 1434, and the like.
  • the base sequence represented by CpG present in the base sequence of the promoter region, untranslated region or translated region (coding region)
  • a base sequence containing one or more of the above a base sequence of genomic DNA containing exon 1 of a human-derived homologue of RIKEN 2210016F16 gene and a promoter region located 5 ′ upstream thereof can be exemplified.
  • SEQ ID NO: 6 corresponding to a complementary base sequence of the base sequence represented by base numbers 157056 to 159000 of the base sequence described in Genbank Accession No. AL354733.
  • the base sequence of exon 1 of the human-derived homologue of RIKEN 2210016F16 gene is represented by base numbers 1392 to 1945.
  • the cytosine in the base sequence represented by CpG present in the base sequence represented by SEQ ID NO: 6, particularly the cytosine in CpG present in the region where CpG is densely present in the base sequence represented by SEQ ID NO: 6 is, for example, Shows high methylation frequency (ie, hypermethylation) in cancer cells such as gastric cancer cells.
  • cytosine having high methylation frequency in gastric cancer cells for example, in the base sequence represented by SEQ ID NO: 6, base numbers 1172, 1175, 1180, 1183, 1189, 1204, 1209, 1267, 1271
  • Examples include cytosine represented by 1278, 1281, 1313, 1319, 1332, 1334, 1338, 1346, 1352, 1358, 1366, 1378, 1392, 1402, 1433, 1436, 1438, and the like.
  • the useful protein gene is the FLJ32130 gene
  • one base sequence represented by CpG existing in the base sequence of the promoter region, untranslated region or translated region (coding region) is used.
  • Examples of the base sequence include the base sequence of genomic DNA containing exon 1 of human-derived FLJ32130 gene and the promoter region located 5 ′ upstream thereof, and more specifically, SEQ ID NO: 7 (Corresponding to a complementary base sequence of the base sequence represented by base numbers 1 to 2379 of the base sequence described in Genbank Accession No. AC002310).
  • SEQ ID NO: 7 Corresponding to a complementary base sequence of the base sequence represented by base numbers 1 to 2379 of the base sequence described in Genbank Accession No. AC002310
  • the base sequence represented by SEQ ID NO: 7 the ATG codon encoding the methionine at the amino acid terminal of human-derived FLJ32130 protein is represented by base numbers 2136 to 2138, and the base sequence considered to be exon 1 is a base sequence Reference numerals 2136 to 2379 are shown.
  • the cytosine in the base sequence represented by CpG present in the base sequence represented by SEQ ID NO: 7, particularly the cytosine in CpG present in the region where CpG is densely present in the base sequence represented by SEQ ID NO: 7, Shows high methylation frequency (ie, hypermethylation) in cancer cells such as gastric cancer cells. More specifically, as cytosine having high methylation frequency in gastric cancer cells, for example, in the base sequence represented by SEQ ID NO: 7, base numbers 1714, 1716, 1749, 1753, 1762, 1795, 1814, 1894, 1911, The cytosine shown by 1915, 1925, 1940, 1955, 1968 etc. can be mentioned.
  • the base represented by CpG present in the base sequence of the promoter region, untranslated region or translated region (coding region).
  • the base sequence containing one or more sequences include a base sequence of genomic DNA containing exon 1 of the human-derived PPARG angiopoietin-related protein gene and a promoter region located 5 ′ upstream thereof, and more Specifically, the base sequence represented by SEQ ID NO: 8 is exemplified.
  • the ATG codon encoding the amino terminal methionine of the human-derived PPARG angiopoietin-related protein protein is represented by base numbers 717 to 719, and the 5 ′ side of exon 1 above.
  • the base sequence of the portion is shown in base numbers 1957 to 2661.
  • cytosine having high methylation frequency in gastric cancer cells for example, in the base sequence represented by SEQ ID NO: 8, base numbers 35, 43, 51, 54, 75, 85, 107, 127, 129, And cytosine represented by 143, 184, 194, 223, 227, 236, 251, 258 and the like.
  • the useful protein gene is a Thrombomodulin gene
  • one base sequence represented by CpG existing in the base sequence of the promoter region, untranslated region or translated region (coding region) is used.
  • Examples of the base sequence described above include a base sequence of genomic DNA containing exon 1 of a human-derived Thrombodyulin gene and a promoter region located 5 ′ upstream thereof, and more specifically, SEQ ID NO: 9 (Corresponding to the base sequence represented by base numbers 1 to 6096 of the base sequence described in Genbank Accession No. AF495471).
  • SEQ ID NO: 9 Corresponding to the base sequence represented by base numbers 1 to 6096 of the base sequence described in Genbank Accession No. AF495471.
  • the ATG codon that encodes the amino terminal methionine of the human-derived thrombomodulin protein is represented by base numbers 2590 to 2592.
  • the base sequence of the exon 1 is represented by the base number 2048. ⁇ 6096.
  • the cytosine in the base sequence represented by CpG present in the base sequence represented by SEQ ID NO: 9, particularly the cytosine in CpG present in the region where CpG is densely present in the base sequence represented by SEQ ID NO: 9 is, for example, Shows high methylation frequency (ie, hypermethylation) in cancer cells such as gastric cancer cells.
  • cytosine having high methylation frequency in gastric cancer cells for example, in the base sequence represented by SEQ ID NO: 9, base numbers 1539, 1551, 1571, 1579, 1581, 1585, 1595, 1598, 1601, Examples include cytosine represented by 1621, 1632, 1638, 1645, 1648, 1665, 1667, 1680, 1698, 1710, 1724, 1726, 1756, and the like. More specifically, for example, when the useful protein gene is the p53-responsive gene 2 gene, the base represented by CpG present in the base sequence of the promoter region, untranslated region or translated region (coding region).
  • Examples of the base sequence containing one or more sequences include a base sequence of genomic DNA containing exon 1 of human-derived p53-responsive gene 2 gene and a promoter region located 5 ′ upstream thereof, and more Specifically, a base sequence represented by SEQ ID NO: 10 (corresponding to a complementary sequence of the base sequence represented by base numbers 113501 to 116000 of the base sequence described in Genbank Accession No. AC009471) can be mentioned. In the base sequence represented by SEQ ID NO: 10, the base sequence of exon 1 of the human-derived p53-responsive gene 2 gene is represented by base numbers 1558 to 1808.
  • Cytosine in the base sequence represented by CpG present in the base sequence represented by SEQ ID NO: 10 exhibits a high methylation frequency (ie, hypermethylation state) in cancer cells such as pancreatic cancer cells, for example. . More specifically, as cytosine having high methylation frequency in pancreatic cancer cells, for example, in the base sequence represented by SEQ ID NO: 10, base numbers 1282, 1284, 1301, 1308, 1315, 1319, 1349, 1351, 1357 , 1361, 1365, 1378, 1383, and the like.
  • the useful protein gene is the Fibrillin 2 gene
  • one base sequence represented by CpG existing in the base sequence of the promoter region, untranslated region or translated region (coding region) is used.
  • the base sequence include the base sequence of genomic DNA containing exon 1 of the human-derived fibrillin 2 gene and the promoter region located 5 ′ upstream thereof, and more specifically, SEQ ID NO: 11 (Corresponding to a complementary sequence of the base sequence represented by base numbers 118801 to 121000 of the base sequence described in Genbank Accession No. AC113387).
  • the base sequence of exon 1 of the human-derived fibrillin 2 gene is represented by base numbers 1091 to 1345.
  • Cytosine in the base sequence represented by CpG present in the base sequence represented by SEQ ID NO: 11 exhibits a high methylation frequency (ie, hypermethylation) in cancer cells such as pancreatic cancer cells. . More specifically, as cytosine having high methylation frequency in pancreatic cancer cells, for example, in the base sequence represented by SEQ ID NO: 11, base numbers 679, 687, 690, 699, 746, 773, 777, 783, 795 799, 812, 823, 830, 834, 843, and the like.
  • the useful protein gene is a Neurofilament 3 gene
  • one base sequence represented by CpG existing in the base sequence of the promoter region, untranslated region or translated region (coding region) is used.
  • the base sequence include the base sequence of genomic DNA containing exon 1 of the human-derived Neurofilament 3 gene and the promoter region located 5 ′ upstream thereof, and more specifically, SEQ ID NO: 12 (Corresponding to a complementary sequence of the base sequence represented by base numbers 28001 to 30000 of the base sequence described in Genbank Accession No. AF106564).
  • SEQ ID NO: 12 Corresponding to a complementary sequence of the base sequence represented by base numbers 28001 to 30000 of the base sequence described in Genbank Accession No. AF106564.
  • the base sequence of exon 1 of the human-derived Neurofilament 3 gene is represented by base numbers 614 to 1694.
  • Cytosine in the base sequence represented by CpG present in the base sequence represented by SEQ ID NO: 12 exhibits a high methylation frequency (ie, hypermethylation state) in cancer cells such as pancreatic cancer cells. . More specifically, as cytosine with high methylation frequency in pancreatic cancer cells, for example, in the base sequence represented by SEQ ID NO: 12, base numbers 428, 432, 443, 451, 471, 475, 482, 491, 499 , 503, 506, 514, 519, 532, 541, 544, 546, 563, 566, 572, 580, and the like.
  • the base represented by CpG present in the base sequence of the promoter region, untranslated region or translated region (coding region).
  • the base sequence including one or more sequences include a base sequence of genomic DNA containing exon 1 of human-derived disintegrin and metalloproteinase domain 23 gene and a promoter region located 5 ′ upstream thereof, and more Specifically, the nucleotide sequence represented by SEQ ID NO: 13 (the nucleotide sequence represented by nucleotide numbers 21001 to 23300 of the nucleotide sequence described in Genbank Accession No. AC009225) Equivalent.), And the like.
  • the base sequence of exon 1 of human-derived disintegrin and metalloproteinase domain 23 gene is represented by base numbers 1194 to 1630.
  • Cytosine in the base sequence represented by CpG present in the base sequence represented by SEQ ID NO: 13 exhibits a high methylation frequency (ie, hypermethylation state) in cancer cells such as pancreatic cancer cells. .
  • cytosine having high methylation frequency in pancreatic cancer cells for example, in the base sequence represented by SEQ ID NO: 13, base numbers 998, 1003, 1007, 1011, 1016, 1018, 1020, 1026, 1028 , 1031, 1035, 1041, 1043, 1045, 1051, 1053, 1056, 1060, 1066, 1068, 1070, 1073, 1093, 1096, 1106, 1112, 1120, 1124, 1126, etc.
  • the useful protein gene is G protein-coupled receptor 7 gene, it is represented by CpG present in the base sequence of the promoter region, untranslated region or translated region (coding region).
  • Examples of the base sequence containing one or more base sequences include the base sequence of genomic DNA containing exon 1 of the human-derived G protein-coupled receptor 7 gene and the promoter region located 5 ′ upstream thereof. More specifically, the base sequence represented by SEQ ID NO: 14 (corresponding to the base sequence represented by base numbers 75001 to 78000 of the base sequence described in Genbank Accession No. AC009800) can be mentioned. In the base sequence represented by SEQ ID NO: 14, the base sequence of exon 1 of the human-derived G protein-coupled receptor 7 gene is represented by base numbers 1666 to 2652.
  • Cytosine in the base sequence represented by CpG present in the base sequence represented by SEQ ID NO: 14 exhibits a high methylation frequency (ie, hypermethylation state) in cancer cells such as pancreatic cancer cells. . More specifically, as cytosine having high methylation frequency in pancreatic cancer cells, for example, in the base sequence represented by SEQ ID NO: 14, base numbers 1480, 1482, 1485, 1496, 1513, 1526, 1542, 1560, 1564 , 1568, 1570, 1580, 1590, 1603, 1613, 1620 and the like.
  • the useful protein gene is a G-protein coupled somatostatin and angiotensin-like peptide receptor gene
  • the promoter region the untranslated region or the translation region (coding region) of the promoter region.
  • the base sequence containing one or more base sequences represented by CpG present in the sequence includes exon 1 of human-derived G-protein coupled somatostatin and angiotensin-like peptide receptor gene, and a promoter region located 5 ′ upstream thereof.
  • the base sequence represented by SEQ ID NO: 15 (Genba nk Accession No. AC008971, which corresponds to the complementary sequence of the base sequence represented by base numbers 57001 to 60,000 of the base sequence described in nk Accession No.
  • cytosine with high methylation frequency in pancreatic cancer cells for example, in the base sequence represented by SEQ ID NO: 15, base numbers 470, 472, 490, 497, 504, 506, 509, 514, 522 Examples thereof include cytosine represented by 540, 543, 552, 566, 582, 597, 610, 612 and the like.
  • the useful protein gene is a single carrier family 6 neurotransmitter transporter noradrenalin member 2 gene, CpG present in the base sequence of the promoter region, untranslated region or translated region (coding region).
  • the base sequence comprising one or more base sequences represented by the following is a base sequence of genomic DNA containing exon 1 of human-derived carrier carrier family 6 neurotransmitter transporter noradrenalin member 2 gene and a promoter region located 5 ′ upstream thereof: More specifically, the base sequence represented by SEQ ID NO: 16 (Genba Corresponding to a complementary sequence to a nucleotide sequence represented by base No. 78801 to 81000 in the nucleotide sequence described in k Accession No.AC026802.) And the like.
  • nucleotide sequence represented by SEQ ID NO: 16 the nucleotide sequence of exon 1 of the human-derived carrier carrier 6 neurotransmitter transporter noradrenalin member 2 gene is represented by nucleotide numbers 1479 to 1804. Cytosine in the base sequence represented by CpG present in the base sequence represented by SEQ ID NO: 16 exhibits a high methylation frequency (ie, hypermethylation state) in cancer cells such as pancreatic cancer cells. .
  • the amount of amplified DNA refers to the purpose of genomic DNA contained in a biological sample. This means the amount of methylated DNA in the region after amplification itself, that is, the amount determined in the third step of the measurement method of the present invention.
  • the biological specimen when the biological specimen is 1 mL of serum, it means the amount of DNA amplified based on the methylated DNA contained in 1 mL of serum.
  • the “methylation ratio” in the present invention is the amount after amplification of methylated DNA in the target DNA region of the genomic DNA contained in the biological sample. It means a value obtained by dividing the amount after amplification of methylated DNA by the total amount with the amount after amplification of non-methylated DNA.
  • the “single-stranded DNA containing one or more CpG in the methylated state at the recognition site of the methylation-sensitive restriction enzyme capable of digesting the single-stranded DNA” is present in the recognition site of the restriction enzyme. It means a single-stranded DNA in which the cytosine in one or more CpGs is an unmethylated cytosine.
  • the “single-stranded DNA containing no CpG in the methylated state at the recognition site of the methylation-sensitive restriction enzyme capable of digesting the single-stranded DNA” is present in the recognition site of the restriction enzyme in the single-stranded DNA. It means single-stranded DNA in which cytosine in all CpGs is methylated.
  • the first step of the measurement method of the present invention 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 3 ′ of the single-stranded DNA
  • a single strand immobilized oligonucleotide having a base sequence that is complementary to a part of the terminal (but not including the target DNA region) is subjected to base pairing, thereby Select strand DNA.
  • the “single-stranded immobilized oligonucleotide” in the first step of the measurement method of the present invention is a part of the 3 ′ end of the single-stranded DNA (positive strand) containing the target DNA region (provided that the above-mentioned purpose 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 to select single-stranded DNA (positive strand) containing a target DNA region from a DNA sample derived from genomic DNA contained in a biological 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 second pre-step and step A2 described later.
  • “what can be immobilized on a carrier” means that the present immobilized oligonucleotide is immobilized on a carrier when selecting a single-stranded DNA (positive strand) containing the intended DNA region.
  • the single-stranded DNA (positive strand) and the immobilized oligonucleotide are immobilized by binding of the immobilized oligonucleotide and the carrier at the stage before base pairing.
  • an oligonucleotide having a base sequence (hereinafter sometimes referred to as the present oligonucleotide) may be fixed to a carrier according to a normal genetic engineering operation method or a commercially available kit / device.
  • 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 first step of the measurement method of the present invention is, for example, when the immobilized oligonucleotide is a biotinylated oligonucleotide
  • A First, to a DNA sample derived from genomic DNA contained in a biological specimen, an annealing buffer and a biotinylated oligonucleotide (after single-paired DNA (positive strand) and base-immobilized oligonucleotide) At this stage, the mixture is obtained by adding a substance which is immobilized by binding of the present immobilized oligonucleotide and the carrier, and is in a free state at this stage). The resulting mixture is then heated at 95 ° C.
  • the base pairing between the single-stranded DNA (positive strand) containing the target DNA region and the biotinylated oligonucleotide is changed between the biotinylated oligonucleotide and the streptoid.
  • the support coated with avidin either of them may be in this order. That is, for example, by adding a DNA sample derived from genomic DNA contained in a biological specimen to a biotinylated oligonucleotide immobilized on a support coated with streptavidin, a mixture is obtained, and the resulting mixture is obtained.
  • 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.
  • a large amount of DNA having a completely different base sequence from the target region for example, rat DNA in the case of a human biological specimen
  • What is necessary is just to add to a biological specimen and to implement said operation.
  • 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 Base pairing in a reaction system containing a divalent cation when base-pairing with a single-stranded immobilized oligonucleotide having a base sequence that is complementary to Can be mentioned. More preferably, the divalent cation is a magnesium ion.
  • reaction system containing a divalent cation means a divalent in an annealing buffer used for base pairing the single-stranded DNA (positive strand) and the single-stranded immobilized oligonucleotide. It means a reaction system containing a cation, and specifically includes, for example, a salt containing magnesium ion as a constituent element (for example, MgOAc2, MgCl2, etc.) at a concentration of 1 mM to 600 mM.
  • a salt containing magnesium ion as a constituent element
  • the single-stranded DNA selected in the first step is digested with a methylation sensitive restriction enzyme capable of digesting one or more kinds of single-stranded DNAs, and then generated free digests.
  • a methylation sensitive restriction enzyme capable of digesting one or more kinds of single-stranded DNAs
  • the “methylation-sensitive restriction enzyme” in the second step of the measurement method of the present invention means, for example, that only a recognition sequence containing unmethylated cytosine is digested without digesting a recognition sequence containing methylated cytosine. It means a restriction enzyme etc.
  • the methylation-sensitive restriction enzyme is a double-stranded DNA containing a CpG pair in a hemimethyl state (that is, cytosine on one strand of the CpG pair is methylated, and cytosine on the other strand is methylated.
  • “Methylation-sensitive restriction enzyme capable of digesting single-stranded DNA” means only a recognition sequence containing unmethylated cytosine without digesting a recognition sequence containing methylated cytosine in single-stranded DNA. It means a restriction enzyme or the like that can digest.
  • some methylation sensitive restriction enzymes digest a single-stranded DNA, such as HhaI.
  • a method for examining the presence or absence of digestion with the methylation sensitive restriction enzyme specifically, for example, PCR is performed using a primer pair capable of amplifying DNA containing the cytosine to be analyzed as a recognition sequence using the DNA as a template. And a method for examining the presence or absence of DNA amplification (amplified product).
  • an amplification product is obtained.
  • the cytosine to be analyzed is not methylated, an amplification product cannot be obtained.
  • the ratio of cytosine to be analyzed can be measured.
  • the target DNA region is in a single-stranded state, and the single-stranded immobilized oligonucleotide as a negative strand has a base pair with the target DNA region. Therefore, the single-stranded DNA strand is a single-stranded DNA derived from genomic DNA contained in a biological specimen.
  • the genomic DNA contained in the biological sample is It can be distinguished whether cytosine in one or more CpGs present in the recognition site of a methylation sensitive restriction enzyme capable of digesting single stranded DNA has been methylated. That is, the restriction on methylation sensitivity that can digest the single-stranded DNA in the genomic DNA contained in the biological specimen is assumed by digesting with the methylation-sensitive restriction enzyme capable of digesting the single-stranded DNA.
  • the single-stranded DNA is in an unmethylated state and can be digested with the single-stranded DNA. Cleaved by a sensitive restriction enzyme.
  • cytosine in all CpGs present in the recognition site of the methylation sensitive restriction enzyme capable of digesting the single-stranded DNA in the genomic DNA contained in the biological specimen is methylated, It is not cleaved by a methylation sensitive restriction enzyme capable of digesting the single-stranded DNA.
  • the restriction enzyme in the genomic DNA contained in the biological sample is detected. If cytosine in one or more CpGs present in the recognition site is not methylated, an amplification product by PCR cannot be obtained, whereas the above-mentioned one in the genomic DNA contained in the biological specimen is not obtained. If cytosine in all CpGs existing in the recognition site of the methylation-sensitive restriction enzyme capable of digesting the double-stranded DNA is methylated, an amplified product by PCR is obtained.
  • the single-stranded DNA selected in the first step of the measurement method of the present invention forms a double strand only at the base-paired portion with the single-stranded immobilized oligonucleotide, Most are single-stranded. Most methylation-sensitive restriction enzymes cannot digest single-stranded DNA even in an unmethylated state, but as described above, some methylation-sensitive restriction enzymes can digest single-stranded DNA in an unmethylated state.
  • An example of a methylation-sensitive restriction enzyme that digests single-stranded DNA is HhaI. By using such an enzyme, the presence or absence of methylation of single-stranded DNA can be determined.
  • HhaI cytosine of CpG contained in the recognition site of HhaI of the single-stranded DNA in the sample base-paired with the single-stranded immobilized oligonucleotide obtained by the above operation is methylated HhaI is unable to digest this DNA, whereas if it is not methylated, HhaI will digest this DNA. Therefore, after performing the above reaction, if a PCR reaction is performed using a pair of primers capable of amplifying the target DNA region, an amplification product cannot be obtained if the DNA in the sample is in an unmethylated state. If the DNA is methylated, an amplification product can be obtained.
  • the second step of the measurement method of the present invention may be performed as follows. 3 ⁇ L of the optimal 10 ⁇ buffer (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc2, 5 mM Dithothreitol), 3 ⁇ L of 1 mg / mL BSA aqueous solution, methylated to the single-stranded DNA selected in the first step Sensitive restriction enzyme HpaII or HhaI (10 U / ⁇ L) or the like is added in an amount of 1.5 ⁇ L, and then the mixture is sterilized with ultrapure water to a volume of 30 ⁇ L, followed by incubation at 37 ° C.
  • the optimal 10 ⁇ buffer 330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc2, 5 mM Dithothreitol
  • 3 ⁇ L of 1 mg / mL BSA aqueous solution
  • the single-stranded DNA selected in the first step in this way is digested with a methylation sensitive restriction enzyme capable of digesting one or more kinds of single-stranded DNAs, and then a free digest (the above-mentioned single-stranded DNA is generated).
  • a methylation sensitive restriction enzyme capable of digesting one or more kinds of single-stranded DNAs is generated.
  • Removal and washing (DNA purification) of one or more amethylated CpGs at the recognition site of a methylation sensitive restriction enzyme capable of digesting DNA are performed. More specifically, for example, when using a PCR tube coated with streptavidin, first remove the solution by pipetting or decantation, and then add TE buffer approximately equal to the volume of the biological specimen. After that, the TE buffer may be removed by pipetting or decantation.
  • the solution was first removed by pipetting or decantation, and TE buffer was added in an amount approximately equal to the volume of the biological specimen. Thereafter, the TE buffer may be removed by pipetting or decantation. Subsequently, by performing such an operation several times, the digest (single-stranded DNA containing one or more CpG in an ammethyl state at the restriction enzyme recognition site) is removed and washed (DNA purification).
  • a recognition sequence containing cytosine that is not methylated cannot be completely digested (so-called “DNA remains uncut”).
  • recognition sites for a methylation sensitive restriction enzyme capable of digesting single-stranded DNA it is possible to minimize “DNA fragmentation”.
  • the DNA region it has one or more recognition sites for methylation sensitive restriction enzymes capable of digesting single-stranded DNA, and it is considered that the more recognition sites, the better.
  • the restriction that the “DNA sample derived from genomic DNA contained in a biological specimen” does not use the target DNA region of the genomic DNA as a recognition cleavage site.
  • One preferred embodiment is a DNA sample that has been previously digested with an enzyme.
  • genomic DNA template DNA
  • a short template DNA is more easily selected, and when a target region is amplified by PCR.
  • digestion is performed by directly using a restriction enzyme that does not use the target DNA region as a recognition cleavage site for genomic DNA-derived DNA samples contained in biological samples. May be.
  • a general restriction enzyme treatment method may be used as a method for digesting with a restriction enzyme that does not use the target DNA region as a recognition cleavage site.
  • the “DNA sample derived from genomic DNA contained in a biological specimen” is a DNA sample digested with one or more types of methylation sensitive restriction enzymes. . These preferred embodiments are because the amount of methylation can be obtained with high precision by digesting the biological specimen itself with the restriction enzymes as described above. This method is useful for eliminating the “DNA residue” as described above.
  • a method for digesting a sample derived from genomic DNA contained in a biological sample with a methylation-sensitive restriction enzyme when the biological sample is genomic DNA itself, a general restriction enzyme treatment method may be used.
  • the derived specimen is tissue lysate, cell lysate, etc.
  • a large excess of methylation sensitive restriction enzyme for example, 500 times (10 U) or more methylation sensitive restriction enzyme for 25 ng of DNA
  • the digestion process may be carried out using this.
  • genomic DNA exists as double-stranded DNA, not only methylation-sensitive restriction enzyme (eg, HhaI) capable of digesting single-stranded DNA but also double-stranded DNA can be digested.
  • a methylation sensitive restriction enzyme for example, HpaII, BstUI, NarI, SacII, HhaI etc.
  • a single-stranded DNA that is an undigested product obtained in the second step a methylation-sensitive restriction that can digest the single-stranded DNA described above
  • the generated free single-stranded DNA (positive strand) and the single-stranded immobilized oligonucleotide are base-paired to select the generated free single-stranded DNA.
  • a process (second (A) pre-process) By using the single-stranded DNA selected in the step (second (A) pre-step) as a template, the single-stranded immobilized oligonucleotide as a primer, and extending the primer once, A step of converting the DNA in a single-stranded state into an elongated double-stranded DNA (second (B) pre-step), Having a step (second pre-step), DNA that is in a double-stranded state extended in the second previous step (in a single-stranded state in which the recognition site of the methylation-sensitive restriction enzyme capable of digesting the DNA that is in the single-stranded state does not contain CpG in an ammethyl state) A step of once separating double-stranded DNA formed by extension using a certain DNA into a single-stranded DNA (positive strand) and a single-stranded DNA (negative strand) (third pre-step) And as this process (A) The generated DNA in the single-strand
  • the oligonucleotide (reverse primer) having a complementary base sequence (positive strand) as an extension primer the oligonucleotide is extended once to extend the generated single-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.
  • the amplified DNA is amplified to a detectable amount, and the amount of amplified DNA is quantified.
  • a single-stranded DNA that is an undigested product obtained in the second step is obtained as the first pre-step among the pre-steps of the following main steps.
  • a single-stranded DNA that does not contain CpG in the methylated state at the recognition site of the methylation-sensitive restriction enzyme capable of digesting DNA is once separated from the above-mentioned single-stranded immobilized oligonucleotide to form a single-stranded DNA.
  • a single-stranded DNA which is an undigested product obtained in the second step (a single-stranded DNA containing no CpG in the methylated state at the recognition site of the methylation-sensitive restriction enzyme capable of digesting the single-stranded DNA)
  • An annealing buffer is added to a DNA sample in which the single-stranded immobilized oligonucleotide and the single-stranded immobilized oligonucleotide are base-paired to obtain a mixture.
  • the resulting mixture is then heated at 95 ° C. for several minutes.
  • the second (A) pre-process in the second pre-process specifically, for example, it may be carried out according to the first process, and the generated free single-stranded DNA (positive strand) and the above-mentioned A DNA in the above-described free single-stranded state formed by base pairing with a single-stranded immobilized oligonucleotide is selected.
  • the P2 step for example, when the present immobilized oligonucleotide is a biotinylated oligonucleotide, it may be carried out as follows.
  • a TE buffer having an amount substantially equal to the volume of the biological specimen is added thereto, and the TE buffer may be removed by pipetting or decanting. More specifically, for example, when using a PCR tube coated with streptavidin, first remove the solution by pipetting or decantation, and then add TE buffer approximately equal to the volume of the biological specimen. After that, the TE buffer may be removed by pipetting or decantation. In addition, when using magnetic beads coated with streptavidin, after fixing the beads with a magnet, the solution was first removed by pipetting or decantation, and TE buffer was added in an amount approximately equal to the volume of the biological specimen.
  • the TE buffer may be removed by pipetting or decantation. Subsequently, the residual solution is removed and washed (DNA purification) by performing such an operation several times.
  • the DNA in the double-stranded state extended in the second pre-process (the CpG in the amethyl state is added to the recognition site of the methylation sensitive restriction enzyme capable of digesting the DNA in the single-strand state).
  • the DNA in the double-stranded state formed by using the DNA in the single-stranded state not included as a template is temporarily converted into the DNA in the single-stranded state (positive strand) and the DNA in the single-stranded state (negative strand). To separate.
  • DNA in the double-stranded state obtained in the second (b) previous step (the recognition site of the methylation-sensitive restriction enzyme capable of digesting the single-stranded DNA does not contain CpG in an ammethyl state)
  • a mixture is obtained by adding an annealing buffer to DNA in a double-stranded state formed by extension using single-stranded DNA as a template. The resulting mixture is then heated at 95 ° C. for several minutes.
  • Step A1 in Step A (Iii)
  • the single-stranded DNA selected in (i) above is used as a template, the single-stranded immobilized oligonucleotide is used as a primer, and the primer is extended once to thereby produce the single-stranded DNA.
  • the DNA in a state is extended and formed as double-stranded DNA (that is, step A2 in step A). Specifically, it may be carried out in accordance with, for example, the following description or the operation method in the extension reaction in the second (B) previous step of the above-described measurement method of the present invention.
  • each step of the third step is repeated after separating the extended double-stranded DNA 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 second (B) previous step, the A step, and the B step in the third step of the measurement method of the present invention described above. do it.
  • the reaction from the first previous step to the present step can be carried out as one PCR reaction.
  • PCR can be used as a method for amplifying a target DNA region (that is, a target region) after digestion with a methylation-sensitive restriction enzyme capable of digesting single-stranded DNA.
  • an immobilized oligonucleotide When amplifying the target region, 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.
  • PCR reaction solution for example, 0.15 ⁇ l of a 50 ⁇ M primer solution, 2.5 ⁇ l of 2 mM dNTP, 10 ⁇ buffer (100 mM Tris-HCl) are added to the DNA obtained in the second step of the measurement method of the present invention. 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 bring the liquid volume to 25 ⁇ l. The reaction solution obtained can be raised.
  • the reaction may be carried out by adding an appropriate amount of betaine, DMSO or the like.
  • the reaction conditions for example, the above-mentioned reaction solution is kept at 95 ° C. for 10 minutes, then at 95 ° C. for 30 seconds, then at 55 to 65 ° C. for 30 seconds, and further at 72 ° C. for 30 seconds for one cycle.
  • the conditions for carrying out the heat insulation for 30 to 40 cycles are mentioned.
  • 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, the detection is performed by annealing the colloidal gold particles, a probe labeled with fluorescence, etc., and measuring the amount of the probe bound to the target region. be able to.
  • a real-time PCR method may be used.
  • 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 single-stranded immobilized oligonucleotide is designed on one side with a primer on one side or a new biotinylated oligonucleotide on the 3 'end side from the single-stranded immobilized oligonucleotide.
  • the target region can also be amplified by using the primer on the complementary side thereof.
  • 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 single-stranded immobilized oligonucleotide is immobilized by covalent bond or the like, the solution containing the amplification product obtained by PCR is transferred to a container where the streptavidin-coated support is present, and the amplification product is It is possible to immobilize.
  • the detection may be performed as described above.
  • the complementary primer that amplifies the target region must be a primer that can amplify the target region having one or more recognition sites for methylation sensitive restriction enzymes capable of digesting single-stranded DNA, and does not include the recognition site. . The reason for this is as follows.
  • methylation sensitive restriction enzyme capable of digesting single-stranded DNA at the 3 'end of the DNA strand (new strand) on the side of immobilized oligonucleotide (new strand) of double-stranded DNA obtained by selection and single extension reaction
  • a methylation sensitive restriction enzyme capable of digesting single-stranded DNA. After the digestion, even if the washing operation is performed as described above, the double-stranded DNA that has lost only a part of the 3 ′ end of the nascent strand is present in an immobilized state.
  • the complementary primer contains a recognition site for a methylation-sensitive restriction enzyme capable of digesting the single-stranded DNA at the most 3 ′ end
  • several bases at the 3 ′ end of the primer are newly born. This is because there is a possibility that the target region is amplified by PCR by annealing with several bases at the 3 ′ end of the strand.
  • the present invention provides the above object in the pre-operation stage or post-operation stage of the first pre-process of the third process of the measurement method of the present invention, or the pre-operation stage or post-operation stage of the third pre-process of the third process.
  • a modified method that additionally includes a step (pre-addition step) of adding a single-stranded oligonucleotide (negative strand) to the reaction system. That is, (Modification 1) In the pre-operation stage of the first pre-process of the third process of the measurement method of the present invention, It has 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).
  • 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 Step C2 (this step) having the step C2 to convert the DNA in a stranded state into an elongated double-stranded DNA (Modification 2)
  • Step C2 (this step) having the step C2 to convert the DNA in a stranded state into an elongated double-stranded DNA (Modification 2)
  • It has 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).
  • pre-addition step a step of adding a single-stranded oligonucleotide (negative strand) in a free state into the reaction system (pre-addition step), and obtained through the first pre-step and the above-described pre-addition step.
  • Double-stranded DNA extended using the single-stranded DNA that is the undigested product as a template one that does not contain CpG in the methylated state at the recognition site of the methylation-sensitive restriction enzyme capable of digesting the single-stranded DNA
  • the method further comprises the following one step (hereinafter, sometimes referred to as the present methylation ratio measurement method).
  • 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 Step C2 (this step) having the step C2 to convert the DNA in a stranded state into an elongated double-stranded DNA (Variation 3)
  • Step C2 (this step) having the step C2 to convert the DNA in a stranded state into an elongated double-stranded DNA (Variation 3)
  • It has 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).
  • 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 Step C2 (this step) having the step C2 to convert the DNA in a stranded state into an elongated double-stranded DNA (Modification 4)
  • Step C2 (this step) having the step C2 to convert the DNA in a stranded state into an elongated double-stranded DNA (Modification 4)
  • It has 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).
  • pre-addition step a step of adding a single-stranded oligonucleotide (negative strand) in a free state into the reaction system (pre-addition step), and obtained through the first pre-step and the above-described pre-addition step.
  • Double stranded DNA that is a non-digested product two strands formed by extension using a single stranded DNA containing no CpG in the methylated state at the recognition site of the methylation sensitive restriction enzyme capable of digesting the single stranded DNA
  • a step of once separating the strand DNA) into a single strand state (additional re-pre-step), and
  • the method further comprises the following one step (hereinafter, sometimes referred to as the present methylation ratio measurement method).
  • 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 Step C2 (this step) having the step C2 to convert the DNA in a stranded state into an elongated double-stranded DNA
  • Step C2 this step having the step C2 to convert the DNA in a stranded state into an elongated double-stranded DNA
  • the amplification efficiency of the target DNA region in the third step can be increased. It can be easily improved.
  • 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 base sequence may be the same as that of the strand-immobilized oligonucleotide, or it may be a short base sequence or a long base sequence.
  • the reverse primer positive strand
  • the single-stranded oligonucleotide negative strand
  • an extension primer is used.
  • the single-stranded oligonucleotide is in a free state that is not available for the reaction to be extended.
  • an immobilized oligonucleotide is used as a primer on one side and PCR is performed by adding only the other primer, but other methods (for example, for example, detection of a target product) Analysis method that can compare the amount of each amplification product obtained by PCR), as described above, when a target region is amplified, the immobilized oligonucleotide is used as one (one side) primer.
  • the PCR may be carried out by adding a pair of primers. After performing such PCR, the amount of amplification product obtained is determined.
  • the third step of the measurement method of the present invention has a repetition step.
  • the “generated single-stranded DNA (positive strand)” in step A1 means the first step of the third step and the first step. This means “generated“ free ”single-stranded DNA (positive strand)” in both operations of the second and subsequent third steps.
  • “generated single-stranded DNA (negative strand)” in Step B means “the first step in the third step and the second and subsequent third steps in both operations. It means the “fixed” single-stranded DNA produced (positive strand) ”.
  • the third step further has a C step, it means “generated (fixed) single-stranded DNA (positive strand)” in the first third step operation.
  • “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 third step means that “the above methylation sensitivity” Means a double-stranded DNA formed by extension that does not contain an ammethyl CpG pair at the restriction enzyme recognition site.
  • the repetitive operations of the third step after the second round And a double-stranded DNA that contains an amethylated CpG pair at the recognition site of the methylation-sensitive restriction enzyme.
  • step B in both operations of the first step of the third step and the second and subsequent steps of the third step, “all of the methylation sensitive restriction enzyme recognition sites are in an amethyl state. It means “extended double-stranded DNA that is a CpG pair”.
  • the third step further includes a C step.
  • the “generated single-stranded DNA (positive strand)” in the C1 step refers to the first step of the third step and the first step. This means “generated“ free ”single-stranded DNA (positive strand)” in both operations of the second and subsequent third steps.
  • the present invention includes a methylation ratio measurement method (that is, the present invention methylation ratio measurement method), which further includes the following two steps as steps of the measurement method of the present invention.
  • the measurement method of the present invention (without performing the second step of the measurement method of the present invention (including the modified method))
  • the third step (including the above-mentioned modified method)
  • the DNA in the target DNA region (the total amount of methylated DNA and non-methylated DNA) is amplified to a detectable amount.
  • Fifth step of calculating the ratio of methylated DNA in the target DNA region of The methylation ratio measurement method may be used in the following situations. It is known that abnormal DNA methylation occurs in various diseases (for example, cancer), and it is considered possible to measure the degree of various diseases by detecting this abnormal DNA methylation.
  • the DNA region is methylated if the measurement method of the present invention or the methylation ratio measurement method of the present invention is performed.
  • the measurement method of the present invention or the methylation ratio measurement method of the present invention As a result, the amount of methylated DNA will be close to zero.
  • the amount of methylated DNA in a healthy person shows a value close to 0, whereas in the case of a diseased patient, Since the value is significantly higher than the value in, the “degree of disease” can be determined based on the difference between the values.
  • the “degree of disease” here is the same as the meaning generally used in the field, and specifically, for example, when the biological specimen is a cell, it means the malignancy of the cell.
  • the measurement method of the present invention or the methylation ratio measurement method of the present invention makes it possible to diagnose various diseases by examining methylation abnormality.
  • a restriction enzyme, primer or probe that can be used in various methods for measuring the amount of methylated DNA in the target region and measuring the methylation ratio Is useful as a reagent 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 rights of the methylation ratio measuring method of the present invention includes use in the form of the detection kit or the detection chip as described above using the substantial principle of the method.
  • Example 1 By culturing a colon adenocarcinoma cell line Caco-2 (ATCC No. HTB-37) derived from a mammal purchased from ATCC in a dedicated medium for the cell line described in the ATCC catalog until confluent. , About 1x10 each 7 Cells were obtained. A 10-fold volume of SEDTA buffer [10 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 100 mM NaCl] was added to the obtained cells, and this was homogenized.
  • Caco-2 ATCC No. HTB-37
  • the recovered precipitate was dissolved in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0), and RNase A (Sigma) was added thereto at 40 ⁇ g / ml and incubated at 37 ° C. for 1 hour. The incubated mixture was extracted with phenol / chloroform. The aqueous layer was recovered, NaCl was added to this to 0.5 N, and this was ethanol precipitated to recover the precipitate (genomic DNA). Genomic DNA was obtained by rinsing the collected precipitate with 70% ethanol.
  • DNA fragment X2 a DNA fragment used as a test sample (hereinafter referred to as DNA fragment X2; the base sequence represented by SEQ ID NO: 18)
  • DNA fragment X2 the base sequence represented by SEQ ID NO: 18
  • PCR reaction solution 2.5 ng of genomic DNA as a template and a primer solution consisting of the base sequence represented by SEQ ID NO: 19 prepared at 3 ⁇ M and a primer solution consisting of the base sequence represented by SEQ ID NO: 20 2.5 ⁇ l each, 2.5 ⁇ l each 2 mM dNTP, 10 ⁇ buffer (100 mM Tris-HCl pH 8.3, 500 mM KCl, 15 mM MgCl 2 , 0.01% Gelatin) and 0.125 ⁇ l of heat-resistant DNA polymerase (AmpliTaq Gold, 5 U / ⁇ l) were mixed, and sterilized ultrapure water was added thereto to make a liquid volume of 25 ⁇ l.
  • 10 ⁇ buffer 100 mM Tris-HCl pH 8.3, 500 mM KCl, 15 mM MgCl 2 , 0.01% Gelatin
  • 0.125 ⁇ l of heat-resistant DNA polymerase AmpliTaq Gold, 5 U / ⁇ l
  • the reaction solution was incubated at 95 ° C. for 10 minutes, and then PCR was performed under the conditions of 50 cycles of incubation at 95 ° C. for 30 seconds, then 59 ° C. for 60 seconds and 72 ° C. for 45 seconds. It was. After performing PCR, DNA amplification was confirmed by 1.5% agarose gel electrophoresis, the target DNA fragment (526 bp, DNA fragment X2) was excised and purified using QIAGEN QIAquick Gel Extraction Kit (QIAGEN). .
  • DNA fragment Y2 A part of the obtained DNA fragment X2 was treated with methylase SssI (NEB) to obtain a DNA fragment (hereinafter referred to as DNA fragment Y2) in which all 5′-CG-3 ′ was methylated. Similarly to the above, amplification was confirmed by 1.5% agarose electrophoresis, and a DNA fragment (526 bp, DNA fragment Y2) was excised and purified using QIAGEN QIAquick Gel Extraction Kit (QIAGEN).
  • QIAGEN QIAquick Gel Extraction Kit
  • an oligonucleotide B1 (19 bp) labeled with biotin at the 5 ′ end having the base sequence shown in SEQ ID NO: 21 was synthesized.
  • ⁇ Biotin-labeled oligonucleotide> Separately each of DNA fragment X2 and DNA fragment Y2 (25 pg / mL aqueous solution, 10 ⁇ L), 1 ⁇ L of 5 ⁇ M biotinylated oligonucleotide B1, and annealing buffer (0.5 M charlinic acid buffer, 7% SDS, 1 mM EDTA aqueous solution) was added to obtain a mixture. The resulting mixture was then heated at 95 ° C. for 5 minutes. Thereafter, it was quickly cooled to 50 ° C. and kept at that temperature for 5 minutes. Subsequently, this was kept at 37 ° C.
  • the bioprepared oligonucleotide is coated with streptavidin by adding the pre-prepared mixture as described above to the PCR tube coated with streptavidin and further incubating it at 37 ° C. for 5 minutes. (This corresponds to the first step of the measurement method of the present invention.) Next, after removing the solution from the PCR tube, 100 ⁇ L of TE buffer was added, and then the TE buffer was removed by pipetting. The operation was further performed twice.
  • Group A (untreated group): To the single-stranded DNA prepared above, 3 ⁇ L of 10 ⁇ buffer (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc2, 5 mM Dithothreitol) optimal for HpaII and HhaI 10 ⁇ BSA (Bovine serum albumin 1 mg / ml) was added to 3 ⁇ L, and sterilized ultrapure water was further added to the mixture to make the volume 30 ⁇ L.
  • 10 ⁇ buffer 330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc2, 5 mM Dithothreitol
  • HpaII and HhaI 10 ⁇ BSA Bovine serum albumin 1 mg / ml
  • Group B (digestion treatment group with HpaII): To the single-stranded DNA prepared above, 15 U of HpaII and 10 ⁇ buffer (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc2) optimal for HpaII and HhaI 3 ⁇ L of 5 mM Dithothreitol) and 3 ⁇ L of 10 ⁇ BSA (Bovine serum albumin 1 mg / ml) were added, and sterilized ultrapure water was further added to the mixture to adjust the liquid volume to 30 ⁇ L.
  • HpaII and 10 ⁇ buffer 330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc2
  • HpaII and HhaI 3 ⁇ L of 5 mM Dithothreitol 3 ⁇ L
  • 10 ⁇ BSA Bovine serum albumin 1 mg / ml
  • Group B (digestion group with HhaI): To the single-stranded DNA prepared above, 15 U of HhaI and 10 ⁇ buffer (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM) optimal for HpaII and HhaI, respectively. 3 ⁇ L of MgOAc2, 5 mM Dithothreitol) and 3 ⁇ L of 10 ⁇ BSA (Bovine serum albumin 1 mg / ml) were added, and further sterilized ultrapure water was added to the mixture to make the volume 30 ⁇ L. After each reaction solution was incubated at 37 ° C.
  • DNA having the base sequence represented by SEQ ID NO: 22; the sequence corresponding to base numbers 76669 to 76835 of the GPR7 sequence represented by Genbank Accession No. AC009800 etc. is amplified.
  • a primer solution consisting of the base sequence shown in SEQ ID NO: 23 and a primer solution consisting of the base sequence shown in SEQ ID NO: 24 prepared in 3 ⁇ M on the genomic DNA used as a template
  • 2.5 ⁇ l each of 2 mM dNTP and 10 ⁇ buffer 100 mM Tris-HCl pH 8.3, 500 mM KCl, 15 mM MgCl 2 , 0.01% Gelatin
  • 5 ⁇ l of 5N betaine aqueous solution are mixed, and sterilized ultrapure water is added to the solution.
  • Example 2 The following test was conducted using the DNA fragment X2 and the DNA fragment Y2 obtained in Example 1.
  • a serum solution was obtained by adding each of DNA fragment X2 and DNA fragment Y2 separately to 1 mL of rat serum at a rate of 25 pg / ml.
  • the resulting mixture was then heated at 95 ° C. for 5 minutes.
  • the bioprepared oligonucleotide is coated with streptavidin by adding the pre-prepared mixture as described above to the PCR tube coated with streptavidin and further incubating it at 37 ° C. for 5 minutes. (The above corresponds to the first step of the measurement method of the present invention).
  • Group A (untreated group): To the single-stranded DNA prepared above, 3 ⁇ L of 10 ⁇ buffer (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc2, 5 mM Dithothreitol) optimal for HpaII and HhaI 10 ⁇ BSA (Bovine serum albumin 1 mg / ml) was added to 3 ⁇ L, and sterilized ultrapure water was further added to the mixture to make a liquid volume of 30 ⁇ L (preparing two each).
  • 10 ⁇ buffer 330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc2, 5 mM Dithothreitol
  • HpaII and HhaI 10 ⁇ BSA Bovine serum albumin 1 mg / ml
  • Group B (digestion treatment group with HhaI): To the single-stranded DNA prepared above, 15 U of HhaI and a 10 ⁇ buffer (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc2) optimal for HpaII and HhaI 3 ⁇ L of 5 mM Dithothreitol) and 3 ⁇ L of 10 ⁇ BSA (Bovine serum albumin 1 mg / ml) were added, and further sterilized ultrapure water was added to the mixture to make 30 ⁇ L (two each). After each reaction solution was incubated at 37 ° C.
  • a 10 ⁇ buffer 330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc2
  • HpaII and HhaI 3 ⁇ L of 5 mM Dithothreitol 3 ⁇ L of 10 ⁇ BSA (Bovine serum albumin 1 mg
  • the DNA (having the base sequence represented by SEQ ID NO: 22; the region corresponding to the base numbers 76669 to 76835 of the GPR7 sequence represented by Genbank Accession No. AC009800) is amplified.
  • a primer solution consisting of the base sequence shown in SEQ ID NO: 23 and a primer solution consisting of the base sequence shown in SEQ ID NO: 24 prepared in 3 ⁇ M on the genomic DNA used as a template, 2.5 ⁇ l each of 2 mM dNTP and 10 ⁇ buffer (100 mM Tris-HCl pH 8.3, 500 mM KCl, 15 mM MgCl 2 , 0.01% Gelatin), 0.125 ⁇ l of heat-resistant DNA polymerase (AmpliTaq Gold) 5U / ⁇ l, and 5 ⁇ l of 5N betaine aqueous solution are mixed, and sterilized ultrapure water is added to the solution.
  • 2 mM dNTP and 10 ⁇ buffer 100 mM Tris-HCl pH 8.3, 500 mM KCl, 15 mM MgCl 2 , 0.01% Gelatin
  • Example 3 The following test was conducted using the DNA fragment X2 and the DNA fragment Y2 obtained in Example 1. A solution of DNA fragment X2 and DNA fragment Y2 in 0, 0.1, 1, and 10 pg / 10 ⁇ L of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) was prepared.
  • TE buffer 10 mM Tris, 1 mM EDTA, pH 8.0
  • the single-stranded DNA (DNA fragment X2 or DNA fragment Y2) containing the target DNA region and the biotinylated oligonucleotide were subjected to base pairing ( 2 pieces each for DNA fragment X2 and DNA fragment Y2).
  • the bioprepared oligonucleotide is coated with streptavidin by adding the pre-prepared mixture as described above to the PCR tube coated with streptavidin and further incubating it at 37 ° C. for 5 minutes.
  • washing buffer 0.05% Tween20-containing phosphate buffer: 1 mM KH2PO4, 3 mM Na2HPO ⁇ 7H2O, 154 mM NaCl, pH 7.4
  • Tween20-containing phosphate buffer 1 mM KH2PO4, 3 mM Na2HPO ⁇ 7H2O, 154 mM NaCl, pH 7.4
  • Group A (untreated group): To the single-stranded DNA prepared above, 3 ⁇ L of 10 ⁇ buffer (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc2, 5 mM Dithothreitol) optimal for HpaII and HhaI 10 ⁇ BSA (Bovine serum albumin 1 mg / ml) was added to 3 ⁇ L, and sterilized ultrapure water was further added to the mixture to make the volume 30 ⁇ L.
  • 10 ⁇ buffer 330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc2, 5 mM Dithothreitol
  • HpaII and HhaI 10 ⁇ BSA Bovine serum albumin 1 mg / ml
  • Group B (digestion treatment group with HhaI): To the single-stranded DNA prepared above, 15 U of HhaI and a 10 ⁇ buffer (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc2) optimal for HpaII and HhaI 3 ⁇ L of 5 mM Dithothreitol) and 3 ⁇ L of 10 ⁇ BSA (Bovine serum albumin 1 mg / ml) were added, and sterilized ultrapure water was further added to the mixture to adjust the liquid volume to 30 ⁇ L. After incubating each reaction solution at 37 ° C.
  • PCR was performed from the obtained undigested product using the following primers and reaction conditions. If the target DNA region is methylated, DNA (having the base sequence represented by SEQ ID NO: 22; the sequence corresponding to base numbers 76669 to 76835 of the GPR7 sequence represented by Genbank Accession No. AC009800 etc.) is amplified.
  • the PCR reaction solution was prepared by adding 0.3 ⁇ l each of a primer solution consisting of the base sequence shown in SEQ ID NO: 23 and a primer solution consisting of the base sequence shown in SEQ ID NO: 24 prepared in 50 ⁇ M to the genomic DNA used as a template.
  • 5 ⁇ l each of 2 mM dNTP and 10 ⁇ buffer 100 mM Tris-HCl pH 8.3, 500 mM KCl, 15 mM MgCl 2 , 0.01% Gelatin
  • 10 ⁇ l of 5N betaine aqueous solution are mixed, and sterilized ultrapure water is added thereto to make the volume.
  • Example 4 The following test was conducted using the DNA fragment X2 and the DNA fragment Y2 obtained in Example 1. 0, 0.1, 1 and 10 pg / 10 ⁇ L rat serum solutions of DNA fragment X2 and DNA fragment Y2 were prepared.
  • the single-stranded DNA (DNA fragment X2 or DNA fragment Y2) containing the target DNA region and the biotinylated oligonucleotide were subjected to base pairing ( 2 pieces each for DNA fragment X2 and DNA fragment Y2).
  • the bioprepared oligonucleotide is coated with streptavidin by adding the pre-prepared mixture as described above to the PCR tube coated with streptavidin and further incubating it at 37 ° C. for 5 minutes.
  • Group A (untreated group): To the single-stranded DNA prepared above, 3 ⁇ L of 10 ⁇ buffer (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc2, 5 mM Dithothreitol) optimal for HpaII and HhaI 10 ⁇ BSA (Bovine serum albumin 1 mg / ml) was added to 3 ⁇ L, and sterilized ultrapure water was further added to the mixture to make the volume 30 ⁇ L.
  • 10 ⁇ buffer 330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc2, 5 mM Dithothreitol
  • HpaII and HhaI 10 ⁇ BSA Bovine serum albumin 1 mg / ml
  • Group B (digestion treatment group with HhaI): To the single-stranded DNA prepared above, 15 U of HhaI and a 10 ⁇ buffer (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc2) optimal for HpaII and HhaI 3 ⁇ L of 5 mM Dithothreitol) and 3 ⁇ L of 10 ⁇ BSA (Bovine serum albumin 1 mg / ml) were added, and sterilized ultrapure water was further added to the mixture to adjust the liquid volume to 30 ⁇ L. After each reaction solution was incubated (digestion treatment) at 37 ° C.
  • DNA having the base sequence represented by SEQ ID NO: 22; the sequence corresponding to base numbers 76669 to 76835 of the GPR7 sequence represented by Genbank Accession No. AC009800 etc. is amplified.
  • the PCR reaction solution was prepared by adding 0.3 ⁇ l each of a primer solution consisting of the base sequence shown in SEQ ID NO: 23 and a primer solution consisting of the base sequence shown in SEQ ID NO: 24 prepared in 50 ⁇ M to the genomic DNA used as a template.
  • 5 ⁇ l each of 2 mM dNTP and 10 ⁇ buffer 100 mM Tris-HCl pH 8.3, 500 mM KCl, 15 mM MgCl 2 , 0.01% Gelatin
  • 10 ⁇ l of 5N betaine aqueous solution are mixed, and sterilized ultrapure water is added thereto to make the volume.
  • the target DNA region is obtained using a serum solution as a DNA sample derived from genomic DNA contained in a biological specimen and a solution containing a divalent cation (magnesium ion) as an annealing buffer.
  • Example 5 By culturing a breast cancer cell line MCF-7 (ATCC NO.
  • HTB-22 HTB-22 derived from a mammal purchased from ATCC in a dedicated medium for the cell line described in the ATCC catalog until confluent, About 1 ⁇ 10 7 Cells were obtained.
  • a 10-fold volume of SEDTA buffer [10 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 100 mM NaCl] was added to the obtained cells, and this was homogenized.
  • proteinase K Sigma
  • the mixture was shaken at 55 ° C. for about 16 hours.
  • the mixture was extracted with phenol [saturated with 1M Tris-HCl, pH 8.0] and chloroform.
  • the aqueous layer was recovered, NaCl was added to this to 0.5 N, and then the resulting precipitate was recovered by ethanol precipitation.
  • the recovered precipitate was dissolved in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0), and RNase A (Sigma) was added thereto at 40 ⁇ g / ml and incubated at 37 ° C. for 1 hour.
  • the incubated mixture was extracted with phenol / chloroform.
  • the aqueous layer was recovered, and NaCl was added to this to 0.5 N, followed by ethanol precipitation to recover the precipitate (genomic DNA).
  • the recovered precipitate was rinsed with 70% ethanol to obtain genomic DNA.
  • the base sequence shown in SEQ ID NO: 17 used as a test sample (LINE1 sequence shown in Genbank Accession No. M80343, etc.) DNA fragment (sequence corresponding to nucleotide numbers 257 to 352) of DNA fragment X1 having the nucleotide sequence represented by SEQ ID NO: 25.
  • DNA fragment numbers 8 to 480 of the LINE1 sequence represented by Genbank Accession No. M80343, etc. Sequence was amplified.
  • PCR reaction solution 2 ng of genomic DNA as a template and a primer solution consisting of the base sequence represented by SEQ ID NO: 26 prepared at 100 pmol / ⁇ l and a primer solution consisting of the base sequence represented by SEQ ID NO: 27 0.125 ⁇ l, 2.5 ⁇ l each 2 mM dNTP, 10 ⁇ buffer (100 mM Tris-HCl pH 8.3, 500 mM KCl, 15 mM MgCl 2 , 0.01% Gelatin) was mixed with 2.5 ⁇ l and heat-resistant DNA polymerase 5 U / ⁇ l was mixed with 0.125 ⁇ l, and sterilized ultrapure water was added thereto to make the volume 25 ⁇ l.
  • the reaction solution was incubated at 95 ° C. for 10 minutes, and then PCR was performed under the conditions of 50 cycles of incubation with 95 ° C. for 30 seconds, then 63 ° C. for 60 seconds and 72 ° C. for 45 seconds. It was. After performing PCR, amplification was confirmed by 1.5% agarose gel electrophoresis, and the target DNA fragment (473 bp, DNA fragment X1) was excised and purified using QIAGEN QIAquick Gel Extraction Kit (QIAGEN). A part of the obtained DNA fragment X1 was treated with methylase SssI (NEB) to obtain a DNA fragment (hereinafter referred to as DNA fragment Y1) in which all of 5-CG-3 ′ was methylated.
  • DNA fragment Y1 DNA fragment in which all of 5-CG-3 ′ was methylated.
  • Group A (untreated group): About 25 ng of DNA fragment was added 2 ⁇ L of 10 ⁇ buffer solution (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc2, 5 mM Dithothreitol), 10 ⁇ BSA (10 mM BSA). 2 ⁇ L of Bovine serum albumin 1 mg / ml) was added, and sterilized ultrapure water was further added to the mixture to make the volume 20 ⁇ L.
  • 10 ⁇ buffer solution 330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc2, 5 mM Dithothreitol
  • 10 ⁇ BSA (10 mM BSA).
  • Bovine serum albumin 1 mg / ml was added, and sterilized ultrapure water was further added to the mixture to make the volume 20 ⁇ L.
  • HpaII treatment group About 25 ng of DNA fragment, 0.5 U of HpaII and 10 ⁇ buffer solution (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc2, 5 mM Dithothreitol) optimal for HpaII and HhaI 2 ⁇ L and 2 ⁇ L of 10 ⁇ BSA (Bovine serum albumin 1 mg / ml) were added, and sterilized ultrapure water was further added to the mixture to make the volume 20 ⁇ L.
  • BSA Bovine serum albumin 1 mg / ml
  • Group C HhaI treatment group: About 25 ng of DNA fragment, 0.5 U of HhaI, and 10 ⁇ buffer (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc2, 5 mM Dithothreitol) optimal for HpaII and HhaI 2 ⁇ L and 2 ⁇ L of 10 ⁇ BSA (Bovine serum albumin 1 mg / ml) were added, and sterilized ultrapure water was further added to the mixture to make the volume 20 ⁇ L.
  • 10 ⁇ buffer 330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc2, 5 mM Dithothreitol
  • 10 ⁇ BSA Bovine serum albumin 1 mg / ml
  • Group D About 25 ng of DNA fragment, 0.5 U of HpaII and HhaI, respectively, and 10 ⁇ buffer (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc2) optimal for HpaII and HhaI 2 ⁇ L of 5 mM Dithothreitol) and 2 ⁇ L of 10 ⁇ BSA (Bovine serum albumin 1 mg / ml) were added, and further sterilized ultrapure water was added to the mixture to make the volume 20 ⁇ L. Each reaction solution was incubated at 37 ° C.
  • PCR reaction solution As a PCR reaction solution, a DNA fragment 62.5 pg used as a template, a primer solution consisting of the base sequence represented by SEQ ID NO: 28 prepared to 3 pmol / ⁇ l, and a primer solution consisting of the base sequence represented by SEQ ID NO: 29 2.5 ⁇ l each of the two types, 2.5 ⁇ L of the probe consisting of the base sequence shown in SEQ ID NO: 30 prepared at 2.5 pmol / ⁇ L, 2.5 ⁇ l of 2 mM dNTP each, 10 ⁇ PCR buffer ( 100 mM Tris-HCl pH 8.3, 500 mM KCl, 15 mM MgCl 2 , 0.01% Gelatin) and heat-resistant DNA polymerase (AmpliTaq Gold) 5 U / ⁇ l mixed with 0.125 ⁇ l, and sterilized ultrapure water was added to make the volume 25 ⁇ l.
  • PCR buffer 100 mM Tris-HCl pH 8.3, 500 mM KCl, 15
  • Real-time PCR was performed using the Gene Amp 5700 Sequence Detection System (Applied Biosystems). In order to amplify a region (DNA) consisting of the base sequences represented by base numbers 1 to 94 in the base sequence represented by SEQ ID NO: 17, the reaction solution was kept at 95 ° C. for 10 minutes, Real-time PCR was performed for 15 seconds at 60 ° C. for 60 seconds as one cycle. The amount of DNA in the region was quantified based on the result of the real-time PCR. Three tests were performed for each biological specimen. The results are shown in FIGS. The amount of DNA in the region in group A was taken as 1, and the amount of DNA in the region in other groups was shown. Since FIG.
  • FIG. 7 (“I”) is a fragment mixture having a methylation ratio of 0%, the theoretical values in the groups B, C, and D are “0”, and FIG. 8 (“II”) is the methylation ratio. Since it is a 10% fragment, the theoretical value in group B, C and D is “0.1”, and FIG. 9 (“III”) is a fragment with a methylation ratio of 25%. Since the theoretical value in the C group and the D group is “0.25” and FIG. 10 (“IV”) is a fragment with a methylation ratio of 50%, the theoretical value in the B group, the C group, and the D group is “ 0.5 ”and FIG.
  • V are fragments with a methylation ratio of 100%, and thus the theoretical values in the B group, the C group, and the D group indicate“ 1 ”.
  • FIGS. 7 to 11 a value closest to the theoretical value was obtained in the group D, and it was revealed that digestion treatment with two or more kinds of methylation sensitive enzymes is preferable.
  • the present invention it is possible to provide a method for easily measuring the content of methylated DNA in a target DNA region possessed by genomic DNA contained in a biological sample.
  • Oligonucleotide primer designed for PCR SEQ ID NO: 19 Oligonucleotide primer designed for PCR SEQ ID NO: 20 Oligonucleotide primer designed for PCR SEQ ID NO: 21 Biotin-labeled oligonucleotide designed for immobilization SEQ ID NO: 23 Oligonucleotide primer designed for PCR SEQ ID NO: 24 Oligonucleotide probe 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 probes designed for real-time PCR [Sequence Listing]

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Abstract

L'invention concerne, entre autres, une méthode de détermination du contenu d'un ADN méthylé dans une région d'intérêt d'un ADN génomique contenu dans un échantillon biologique.
PCT/JP2007/075360 2006-12-25 2007-12-25 Méthode de détermination de méthylation d'adn WO2008078834A1 (fr)

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JP2006347301A JP4940940B2 (ja) 2006-12-25 2006-12-25 Ｄｎａメチル化測定方法
JP2006-347301 2006-12-25

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WO2008078834A1 WO2008078834A1 (fr) 2008-07-03
WO2008078834A9 true WO2008078834A9 (fr) 2010-03-25

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JP5206059B2 (ja) * 2008-03-25 2013-06-12 住友化学株式会社 メチル化されたｄｎａの含量を測定する方法

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Publication number Priority date Publication date Assignee Title
US6605432B1 (en) * 1999-02-05 2003-08-12 Curators Of The University Of Missouri High-throughput methods for detecting DNA methylation
US20050009059A1 (en) * 2003-05-07 2005-01-13 Affymetrix, Inc. Analysis of methylation status using oligonucleotide arrays
WO2006088978A1 (fr) * 2005-02-16 2006-08-24 Epigenomics, Inc. Procede de determination du modele de methylation d'un acide polynucleique

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JP4940940B2 (ja) 2012-05-30
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