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WO2008078834A1 - Méthode de détermination de méthylation d'adn - Google Patents

Méthode de détermination de méthylation d'adn Download PDF

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Publication number
WO2008078834A1
WO2008078834A1 PCT/JP2007/075360 JP2007075360W WO2008078834A1 WO 2008078834 A1 WO2008078834 A1 WO 2008078834A1 JP 2007075360 W JP2007075360 W JP 2007075360W WO 2008078834 A1 WO2008078834 A1 WO 2008078834A1
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Prior art keywords
dna
stranded
stranded dna
base sequence
region
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Japanese (ja)
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WO2008078834A9 (fr
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Yoshitaka Tomigahara
Hirokazu Tarui
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Sumitomo Chemical Co Ltd
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Sumitomo Chemical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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

Definitions

  • the present invention relates to a method for measuring the content of methylated DNA in the target DNA region in genomic DNA contained in a biological specimen.
  • methylated DNA in the target DNA region in the genomic DNA is used.
  • this measurement method it is necessary to first extract DNA containing the target DNA region from the DNA sample derived from genomic DNA, and the extraction operation is complicated.
  • PCR polymerase chain reaction
  • a method of amplifying a target region by being subjected to PCR is known. In any of these methods, it takes time to modify DNA for detection of methylation and subsequent purification of the product, preparation of a reaction system for PCR, confirmation of DNA amplification, and the like. Disclosure of the invention
  • 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).
  • the generated free single-stranded DNA is selected by base pairing with the generated single-stranded DNA (positive strand) and the single-stranded immobilized oligonucleotide.
  • Process (second (A) previous process) is selected by base pairing with the generated single-stranded DNA (positive strand) and the single-stranded immobilized oligonucleotide.
  • Double-stranded DNA which is formed by extending DNA in a single-stranded state as a cage, is converted into single-stranded DNA (positive strand) and single-stranded DNA (negative strand).
  • Step A1 The generated single-stranded DNA is formed by base-pairing the generated single-stranded DNA (positive strand) with the single-stranded immobilized oligonucleotide (negative strand).
  • a step A (this step) having a step A 2 that causes the DNA in the selected single-stranded state to be elongated as a double-stranded DNA by extension.
  • each step of the third step is repeated once after the elongated double-stranded DNA obtained in each step is separated into a single-stranded state, and the methyl region in the target DNA region is repeated.
  • the above item is characterized in that base pairing is carried out in a reaction system containing a divalent cation when base-pairing with a single-stranded immobilized oligonucleotide having a base sequence complementary to The method according to 1;
  • the method according to the first step further comprising the following one step as the second step and each main step:
  • Step C 1 DNA in the single-stranded state selected in Step C 1 is used as a saddle type, and the primer is extended once by using the single-stranded oligonucleotide (negative strand) as a primer.
  • Step C (this step) having the second step of making C DNA into a double-stranded DNA that is formed by extension of DNA, which is in a single-stranded state;
  • a part of the 3 ′ end of the single-stranded DNA (positive strand) containing the target DNA region in the post-operation stage of the first pre-step of the third step according to any one of the preceding items 1 to 3 A step of adding a single-stranded oligonucleotide (negative strand) that has a base sequence that is complementary to and in a free state into the reaction system (pre-addition step), and A double-stranded DNA that is formed by extending the single-stranded DNA, which is an undigested product obtained through the above-mentioned steps and the above-mentioned pre-addition step, into a saddle shape (a methylation-sensitive restriction enzyme capable of digesting the single-stranded DNA).
  • it further has the following one process.
  • C (i) The generated single-stranded DNA (positive strand) and the reaction system in the above additional step A first C1 step of selecting DNA that is in the single-stranded state by base pairing with the single-stranded oligonucleotide (negative strand) added therein,
  • a C step (this step) having a C 2 step, wherein the DNA in the single-stranded state is formed into an elongated double-stranded DNA; 6.
  • a method further comprising the following one step as each main step of the third step described in the preceding paragraph 1.
  • Step C (i) The DNA in the single-stranded state selected in the first step C is used as a saddle type, and the primer is extended once by using the single-stranded oligonucleotide (negative strand) as a primer.
  • Step C (this step) having the second step of forming C2 as a double-stranded DN A, which is a single-stranded state of DN A;
  • a part of the 3 ′ end of the single-stranded DNA (positive strand) containing the target DNA region in the post-operation stage of the third pre-step of the third step according to any one of the preceding items 1 to 3 A step of adding a single-stranded oligonucleotide (negative strand) that has a base sequence that is complementary to and in a free state into the reaction system (pre-addition step), and Double-stranded DNA that is an undigested product obtained through the above-mentioned steps and the above-mentioned pre-addition step (the recognition site of the methylation-sensitive restriction enzyme capable of degrading the single-stranded DNA does not contain C p G in the methylated state)
  • a method further comprising the following one step as each main step of the third step described in the preceding paragraph 1.
  • step C1 Base pairing the generated single-stranded DNA (positive strand) with the single-stranded oligonucleotide (negative strand) added to the reaction system in the previous addition step. And step C1 for selecting the DNA in the single-stranded state,
  • Step C 1 The single-stranded DNA selected in Step C 1 is used as a saddle type, and the primer is extended once using the single-stranded oligonucleotide (negative strand) as a primer.
  • 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;
  • the DNA in the target DNA region (the total amount of methylated DNA and unmethylated DNA) is amplified to a detectable amount.
  • a fourth step of quantifying the amount of amplified DNA is performed by performing the third step in any of the methods.
  • a DNA sample derived from genomic DNA contained in a biological sample 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.
  • a DNA sample derived from a genomic DNA contained in a biological specimen is a DNA sample digested with a methylation-sensitive restriction enzyme capable of digesting one or more types of single-stranded DNA 1 A method according to any of -12;
  • a 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 the target DNA region of the genomic DNA contained in the biological specimen.
  • the methylation-sensitive restriction enzyme capable of digesting one or more types of single-stranded DNA is Hhal, which is a methylation-sensitive restriction enzyme.
  • FIG. 1 shows that the sample prepared in Example 1 was subjected to any of the treatments “A (no treatment)”, “B (H pall treatment)”, or “C (Hhal treatment)”.
  • FIG. 22 is a view showing the results obtained by amplifying the methylated DNA in the region consisting of the base sequence shown in 22 by PCR, and subjecting the obtained amplification product to 1.5% DNA gel electrophoresis. From the leftmost lane in the figure, sample with DNA fragment X2 “A” treatment, sample with DNA fragment X2 “B” treatment, DNA fragment X2 with “C” treatment Sample, DNA fragment Y2 sample “A” treated, DNA fragment Y2 sample “B” treated sample, DNA fragment Y2 sample “C” treated sample Is shown.
  • FIG. 2 shows the 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 (Hhal treatment)” treatment.
  • FIG. 5 shows the results of 1.5% fagarose gel electrophoresis of the amplified product obtained by amplifying methylated DNA in the region with PCR.
  • sample 1 with DNA marker “M” and DNA fragment Y2 “A” treatment sample 2 with DNA fragment Y2 “A” treatment
  • sample 2 with DNA fragment Y2 “A” treatment DN A Sample 1 with “B” treatment of fragment Y2
  • Sample 2 with DNA fragment Y2 “B” treatment Sample 1 with DNA fragment X2 “A” treatment
  • DNA fragment X2 “ Sample 2 with "A” treatment DNA fragment X2 “ Sample 2 with "A” treatment
  • Sample 1 with "B” treatment of DNA fragment X2 Sample 2 results are shown.
  • FIG. 3 shows that the sample of DN A fragment X 2 prepared in Example 3 was treated with either “A (no treatment)” or “B (Hhal treatment)” and is represented by SEQ ID NO: 22.
  • FIG. 3 is a diagram showing the results of 1.5% agarose gel electrophoresis of the amplification product obtained by amplifying methylated DNA in a region consisting of the base sequence by PCR. The leftmost lane in the figure shows the DNA marker “M”.
  • FIG. 4 shows that the sample of DN A fragment Y 2 prepared in Example 3 was treated with either “A (no treatment)” or “B (Hhal treatment)” and is represented by SEQ ID NO: 22.
  • FIG. 3 is a diagram showing the results of 1.5% agarose gel electrophoresis of the amplification product obtained by amplifying methylated DNA in a region consisting of the base sequence by PCR. The leftmost one in the figure indicates “M” for DNA DNA.
  • For groups A and B from left, 1 OpgDNA fragment Y2 / mLTE buffer solution sample “1”, lpgDNA fragment Y2 / mLTE buffer solution sample “2”, 0. IpgD NA fragment Y2 / mLTE buffer solution Results are shown for sample “3” and sample “4” for the OpgDNA fragment Y2 / mLTE buffer solution.
  • FIG. 5 shows that the DNA fragment X2 sample prepared in Example 4 was subjected to either “A (no treatment)” or “B (fflial treatment)” treatment, and the base represented by SEQ ID NO: 22.
  • FIG. 5 is a diagram showing the results of 1.5% agarose gel electrophoresis of the amplification product obtained by amplifying methylated DNA in a region consisting of a sequence by PCR. The leftmost lane in the figure shows the DNA marker “M”.
  • FIG. 6 shows that the sample of DN A fragment Y2 prepared in Example 4 was treated with either “A (no treatment)” or “B (Hhal treatment)” and was represented by SEQ ID NO: 22.
  • FIG. 5 is a view showing the results of 1.5% agarose gel electrophoresis of the amplified product obtained by amplifying methylated DNA in a region consisting of a base sequence by PCR. The leftmost lane in the figure shows the DNA marker “M”.
  • FIG. 7 shows that the sample f (I) prepared in Example 5 was added to “A (no treatment)”, “B (Hpall treatment)”, “C (Hhal treatment)” or “D (Hpall and FIG. 7 is a view showing the results of real-time PCR measurement of the amount of methylated DNA in a region consisting of the base sequence represented by SEQ ID NO: 17 after performing any of the processes of “Bial simultaneous treatment)”.
  • the vertical axis in the figure shows the relative value when the amount of DNA in the sample treated with “A” is 1 (3 standard deviations of average values). The theoretical value indicates the calculated value (methylation ratio) expected for the 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 (Hpall treatment)”, “C (Hhal treatment)” or “D (Hpall and 3 ⁇ 4hal).
  • the vertical axis in the figure shows the relative value when the amount of DNA in the sample treated with “A” is 1 (average soil standard deviation of 3 times).
  • the theoretical value indicates the calculated value (methylation ratio) expected for the B, C, and D groups.
  • FIG. 9 shows that the sample “(III)” prepared in Example 5 was added to “A (no treatment)”, “B (Hpall treatment)”, “C (Hhal treatment)” or “D (Hpall and 3 ⁇ 4hal).
  • the results of measuring the amount of methylated DNA in the region consisting of the base sequence shown in SEQ ID NO: 17 by real-time PC is there.
  • the vertical axis in the figure shows the relative value when the amount of DNA in the sample treated with “A” is set to 1 (three standard deviations of standard deviation).
  • the theoretical value indicates the predicted value (methylation ratio) 'for the B, C, and D groups.
  • FIG. 10 shows that in Example 5, the prepared sample “(IV;)” was added to “A (no treatment)”, “B (Hpal treatment)”, “C (Hhal treatment)” or “D ( Result of measurement of the amount of methylated DNA in the region consisting of the base sequence shown in SEQ ID NO: 17 by real-time PCR
  • FIG. The vertical axis in the figure shows the relative value when the amount of DNA in the sample treated with “A” is 1 (average soil standard deviation of 3 times). The theoretical value indicates the calculated value (methylation ratio) expected for Group B, Group C, and Group D.
  • Fig. 1 1 shows that in Example 5, the prepared sample "(V)" was replaced with "A (no treatment)", “B (Hpal treatment)", “C (Hhal treatment)” or “D (Hpal treatment)".
  • the vertical axis in the figure shows the relative value when the amount of DNA in the sample treated with “A” is set to 1 (average soil standard deviation of 3 times).
  • the theoretical value indicates the calculated value (methylation ratio) expected for the B, C, and D groups.
  • biological specimen for example, a cell lysate, a tissue lysate (herein, tissue has a broad meaning including blood, lymph nodes, etc.) or in mammals
  • biological samples such as plasma, serum, lymph fluid, body secretions (urine, milk, etc.), and genomic DNA obtained by extraction from these biological samples.
  • biological specimens include samples derived from microorganisms, viruses, etc.
  • genomic DNA in the measurement method of the present invention means genomic DNA such as microorganisms, viruses, etc. .
  • the mammal-derived specimen is blood, it can be used for regular health checkups and simple tests. Use of the bright measurement method can be expected.
  • 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 (cancer cells such as gastric cancer cells).
  • cancer cells such as gastric cancer cells.
  • there are four types of bases that make up a gene (genomic DNA). Among these bases, it is known that only cytosine is methylated. Such DNA methylation modification is based on the nucleotide sequence represented by 5'-CG-3 '(C represents cytosine).
  • G represents guanine.
  • the base sequence is sometimes referred to as “CpG '.”) It is limited to cytosine.
  • the site that is methylated in cytosine is at position 5.
  • Cytosine is also methylated. Therefore, the DNA methylation state is inherited by two new sets of DNA even after DNA replication.
  • the “methylated DNA” in the present invention means 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 C p G.
  • the “target DNA region” in the present invention (hereinafter sometimes referred to as target region) is a DNA region to be examined for the presence or absence of methylation of cytosine contained in the region, and is at least one kind of DNA region. It has a recognition site for a methylation sensitive restriction enzyme. For example, Lys yl oxidase, H AS-like suppressor, bA305P22.2.
  • Useful proteins in the promoter sequence, untranslated region or translated region (coding region) C examples include DNA regions containing one or more cytosines in the base sequence represented by pG. Specifically, for example, when the useful protein gene is a Lysyl oxidase gene, the base sequence represented by C p G present in the base sequence of the promoter region, untranslated region or translated region (coding region) is identical. Examples of the nucleotide sequence containing two or more include the nucleotide sequence of genomic DNA containing exon 1 of the human Lysyl oxidase gene and the promoter region located 5 'upstream thereof.
  • nucleotide sequence represented by SEQ ID NO: 1 (corresponding to the nucleotide sequence represented by nucleotide numbers 16001 to 18661 of the nucleotide sequence described in Genbank Accession No. AF270645).
  • the ATG codon encoding the amino acid methionine at the amino terminal of the human-derived Lysyl oxidase protein is represented by nucleotide numbers 2031 to 2033.
  • the nucleotide sequence of the above exon 1 is represented by the nucleotide number 1957-2661.
  • Cytosine in G exhibits a high methylation frequency (ie, hypermethylation state) in cancer cells such as gastric cancer cells. More specifically, as cytosine having high methylation frequency in gastric cancer cells, for example, in the base sequence represented by SEQ ID NO: 1, the base numbers 1539, 1560, 1574, 1600, 1623, 1635, 1644, 1654, 1661 1682, 1686, 1696, 1717, -1767, 1774, 1783, 1785, 1787, 1795 and the like.
  • the useful protein gene is the HRAS-1 ike suppressor gene.
  • the base sequence containing one or more base sequences represented by C p G present in the base sequence of the promoter region, non-translation region or translation region (coding region) is HRAS-l derived from human.
  • the base sequence of genomic DNA containing exon 1 of the ike suppres sor gene and a promoter region located 5 'upstream thereof can be raised.
  • the base sequence represented by SEQ ID NO: 2 This corresponds to the nucleotide sequence represented by nucleotide numbers 172001 to 173953 of the nucleotide sequence described in Genbank Accession No. AC068162).
  • the base sequence of exon 1 of the human-derived HRAS-ike suppressor gene is shown in base numbers 1743-1953.
  • Cytosine in the base sequence shown by C p G present in the base sequence shown by SEQ ID NO: 2 especially C in the region where C p G is densely present in the base sequence shown by SEQ ID NO: 2
  • Cytosine in pG exhibits a high methylation frequency (ie, hypermethylation state) in cancer cells such as gastric cancer cells.
  • cytosine having high methylation frequency in gastric cancer cells for example, in the base sequence represented by SEQ ID NO: 2, the base numbers 1316, 1341, 1357, 1359, 1362, 1374, 1390, 1399, 140 5, 1409, 1414, 1416, 1422, 1428, 1434, 1449, 1451, 1454, 1463, 1469, 1477, 1 479, 1483, 1488, 1492, 1494, 1496, 1498, 1 504, 1510, 1513, 1518 And cytosine represented by 1520 etc.
  • the useful protein gene when it is M305P22.2.1 gene, it is represented by C p G present in the base sequence of the promoter region, untranslated region or translated region (coding region).
  • the base sequence containing at least one base sequence include the genomic DNA base sequence containing exon 1 of human-derived bA305P22.2.1 gene and the promoter region located 5 'upstream thereof. More specifically, the base sequence represented by SEQ ID NO: 3 (corresponding to the base sequence represented by base numbers 13001 to 13889 of the base sequence described in Genbank Accession NO. AL121673) can be mentioned. .
  • the ATG codon encoding the methionine at the amino terminal of the human-derived bA305P22.2.1 protein is represented by base numbers 849 to 851, and the base sequence of exon 1 above is shown. Is shown in base numbers 663-889.
  • the salt represented by SEQ ID NO: 3 The cytosine in the base sequence represented by C p G present in the base sequence, particularly the cytosine in C p G present in the region where C p G is densely present in the base sequence represented by SEQ ID NO: 3, For example, it shows a high methylation frequency (ie, hypermethylation state) in cancer cells such as gastric cancer cells.
  • cytosine having high methylation frequency in gastric cancer cells for example, in the base sequence represented by SEQ ID NO: 3, base numbers 329, 335, 337, 351, 363, 373, 405, 424, 427 , 446, 465, 472, 486, and the like. More specifically, for example, when the useful protein gene is a Gamma ⁇ lamin gene, C p G present in the base sequence of the promoter region, untranslated region or translated region (coding region).
  • the base sequence containing one or more base sequences indicated by is the exon 1 of the Gamma fil amin gene derived from human and the base sequence of the genomic DNA that contains the promoter region and the 5 'upstream region.
  • SEQ ID NO: 4 corresponds to the base sequence represented by SEQ ID NO: 4 (complementary sequence of the base sequence represented by base numbers 63528 to 64390 of the base sequence described in Genbank Accessions No. AC074373).
  • the ATG codon encoding the amino terminal methionine of human-derived Gamma fil amin protein is represented by nucleotide numbers 572 to 574, and the nucleotide sequence of exon 1 is Base numbers 463-863. Cytosine in the base sequence shown by C p G present in the base sequence shown by SEQ ID NO: 4, especially C in the region where C p G is densely present in the base sequence shown by SEQ ID NO: 4.
  • Cytosine in pG exhibits a high methylation frequency (ie, hypermethylation state) in cancer cells such as gastric cancer cells. More specifically, as cytosine having a high methylation frequency in gastric cancer cells, for example, in the base sequence represented by SEQ ID NO: 4, base numbers 329, 333, 337, 350, 353, 360, 363, 370, Examples include cytosine represented by 379, 382, 384, 409, 414, 419, 426, 432, 434, 445, 449, 459, 472, 474, 486, 490, 503, 500, etc.
  • the useful protein gene is a HAND1 gene
  • a base sequence including one or more base sequences represented by CG present in the base sequence of the promo region, untranslated region or translation region (coding region), exon 1 of the HAND1 gene derived from human The base sequence of the genomic DNA containing the promoter region located 5 'upstream can be mentioned. More specifically, the base sequence represented by SEQ ID NO: 5 (described in Genbank Accession No. AC026688) It corresponds to a complementary sequence of the base sequence represented by base numbers 24 303 to 26500 of the base sequence.
  • the ATG codon encoding the amino acid at the amino terminal of the HAND1 protein derived from human is shown in base numbers 1656 to 1658, and the base sequence of exon 1 is The number 1400-2198 is shown.
  • the cytosine in G shows a high methylation frequency (ie, hypermethylation state) in cancer cells such as gastric cancer cells.
  • cytosine having high methylation frequency in gastric cancer cells for example, in the base sequence represented by SEQ ID NO: 5, base numbers 1153, 1 160, 1 178, 1 187, 1 193, 1218, 1232 , 1266, 1272, 1292, 1305, 1307, 1316, 1356, 1377, 1399, 1401, 1422, 1434, and the like.
  • the useful protein gene is the homologue of RIKEN 2210016F16 gene
  • the base sequence containing one or more base sequences represented by G is a genomic DNA containing exon 1 of the human-derived homologue of RIKEN 2210016F16 gene and a promoter region located 5 ′ upstream thereof. More specifically, the base sequence represented by SEQ ID NO: 6 (the base sequence represented by base numbers 157056 to 159000 of the base sequence described in Genbank Accessions NO.
  • nucleotide sequence represented by SEQ ID NO: 6 the nucleotide sequence of exon 1 of the human-derived homologue of RIKEN 2210016F16 gene is represented by nucleotide numbers 1392 to 1945. It exists in the base sequence shown in SEQ ID NO: 6. Cytosine in the base sequence represented by C p G, particularly cytosine in C p G present in the region where C p G is densely present in the base sequence represented by SEQ ID NO: 6, such as gastric cancer cells, etc. Shows high methylation frequency (ie, hypermethylation) in 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 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 FU32130 gene, it is represented by C p G present in the base sequence of the promoter region, untranslated region or translated region (coding region).
  • Examples of the base sequence containing one or more base sequences include the base sequence of genomic DNA containing exon 1 of human-derived FLJ32130 gene and the promoter region located 5 'upstream thereof. More specifically, the base sequence represented by SEQ ID NO: 7 (corresponds 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). can give.
  • the ATG codon that encodes the methionine at the amino acid end of the human-derived FU32130 protein is represented by nucleotide numbers 2136 to 2138, and the nucleotide sequence considered to be exon 1 above is , Base numbers 2136 to 2379.
  • Cytosine in CpG exhibits a high methylation frequency (ie, hypermethylation state) in cancer cells such as gastric cancer cells. More specifically, as cytosine having a high methylation frequency in gastric cancer cells, for example, in the base sequence represented by SEQ ID NO: 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.
  • a useful protein gene is PPARG angi opo i et in-rel ated
  • a human base sequence containing one or more base sequences represented by C p G present in the base sequence of a promoter region, untranslated region or translated region (coding region) is human.
  • the base sequence of genomic DNA containing exon 1 of the PPARG angiopoiet in-related protein gene and the promoter region located 5 'upstream thereof can be mentioned.
  • An example is the base sequence represented by SEQ ID NO: 8.
  • the ATG codon encoding the amino acid methionine at the amino terminal of human-derived PPARG angi opoiet i II-related protein protein is represented by base numbers 717 to 719, and the above exon 1
  • the base sequence of the 5 'side of is shown in base numbers 1957 to 2661.
  • the cytosine in it shows a high methylation frequency (ie, hypermethylation state) in cancer cells such as gastric cancer cells.
  • cytosine with 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, Examples include cytosine represented by 143, 184, 194, 223, 227, 236, 251, 258, and the like. More specifically, for example, when the useful protein gene is a Thrombomodul in gene, it is represented by C p G present in the base sequence of the promoter region, untranslated region or translated region (coding region).
  • Examples of the base sequence containing one or more base sequences include the base sequence of genomic DNA containing exon 1 of the Thrombomodulin gene derived from human and a promoter region located 5 'upstream thereof.
  • the base sequence represented by SEQ ID NO: 9 (corresponding to the base sequence represented by base numbers 1 to 6096 of the base sequence described in Genbank Accession No. AF495471) can be mentioned.
  • the ATG codon that encodes the amino terminal methionine of Thrombomodulin protein derived from human is represented by base numbers 2590 to 2592, and the base sequence of the above exon 1 is Base numbers 2048-6096 are shown.
  • Cytosine in the base sequence shown by C p G present in the base sequence shown by SEQ ID NO: 9, especially SEQ ID NO: Cytosine in C p G present in the region where C p G is densely present in the base sequence shown in No. 9 is highly methylated (ie, highly methylated in cancer cells such as gastric cancer cells). (Hype me thyl at i))).
  • 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, Examples include 1601, 1621, 163 2, 1638, 1645, 1648, 1 665, 1667, 1680, 1698, 1710, 1724, 1726, 1756, and the like. More specifically, for example, when the useful protein gene is p53-responsive gene 2 gene, C present in the base sequence of the promoter region, untranslated region or translated region (coding region).
  • genomic DNA containing exon 1 of human-derived p53-responsive gene 2 gene and a promoter region located 5 ′ upstream thereof is used. More specifically, the base sequence represented by SEQ ID NO: 10 (the base sequence represented by base numbers 1 13501 to 116000 of the base sequence described in Genbank Accesion No. AC009471) This is equivalent to the complementary sequence of).
  • SEQ ID NO: 10 the base sequence represented by base numbers 1 13501 to 116000 of the base sequence described in Genbank Accesion No. AC009471
  • the nucleotide sequence of exon 1 of the human-derived p53-responsive gene 2 gene is represented by nucleotide numbers 1558 to 1808.
  • Cytosine in the base sequence represented by C p G present in the base sequence represented by SEQ ID NO: 10 is, for example, a high methylation frequency (i.e., hypermethy status (hypermethy status) in cancer cells such as spleen cancer cells). l at i on)). More specifically, examples of cytosine having a high methylation frequency in vaginal cancer cells include, for example, nucleotide numbers 1282, 1284, 1301, 1308, 1315, 1319, 1349, 1351 in the nucleotide sequence represented by SEQ ID NO: 10. 1357, 1361, 1365, 1378, 1383 and the like.
  • the useful protein gene when it is a fiber in gene, it is indicated by C p G present in the base sequence of the promoter region, untranslated region or translated region (coding region).
  • the nucleotide sequence including one or more nucleotide sequences includes exon 1 of the Fibrill in gene derived from human, and a promoter region located 5 'upstream of ⁇ ". More specifically, the nucleotide sequence of genomic DNA containing the nucleotide sequence shown in SEQ ID NO: 11 (base numbers 118801 to 121000 of the nucleotide sequence described in Genbank Accession No. AC113387) can be mentioned. It corresponds to a complementary sequence of the base sequence shown).
  • nucleotide sequence represented by SEQ ID NO: 11 the nucleotide sequence of exon 1 of the human Fibrill in gene is represented by nucleotide numbers 1091 to 1345.
  • Cytosine in the base sequence represented by C p G present in the base sequence represented by SEQ ID NO: 11 has a high methylation frequency (ie, a hypermethylated state in cancer cells such as S spleen cancer cells). (Hypermethyl at ion)). More specifically, examples of cytosine having a high methylation frequency in spleen cancer cells include, for example, nucleotide numbers 679, 687, 690, 699, 746, 773, 777 in the nucleotide sequence represented by SEQ ID NO: 11.
  • nucleotide sequences that contain one or more nucleotide sequences include those of genomic DNA containing exon 1 of human neuroment 3 gene derived from human and a promoter region located 5 'upstream. More specifically, it corresponds to the base sequence represented by SEQ ID NO: 12 (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 C p G present in the base sequence represented by SEQ ID NO: 1 2 has a high methylation frequency (ie, hypermethylation state ( hyp ermet yl at ion)).
  • cytosine having a high methylation frequency in winning fl 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 can be mentioned.
  • the useful protein gene is a dis integr in and metal loproteinase domain 23 gene
  • the promoter region,, untranslated region or translated region (coding region)
  • the base sequence containing one or more base sequences indicated by C p G present in the base sequence of the gene is located in exon 1 of human-derived disinteggr in and metal loproteinase dom ain 23 gene and 5 ′ upstream thereof.
  • the base sequence of the genomic DNA containing the promoter region can be mentioned, and more specifically, the base sequence represented by SEQ ID NO: 13 (the base sequence of the base sequence described in Genbank Accession No. AC009225) No. 2 It corresponds to the base sequence shown by 1001 to 23300.
  • nucleotide sequence of exon 1 of the disintegrin in and met alloproteinase domain 23 gene derived from chickpea is shown in nucleotide numbers 1194 to 1630 as shown in SEQ ID NO: 13 .
  • cytosine in the base sequence represented by CpG present in the base sequence represented by SEQ ID NO: 13 has a high methylation frequency (ie, hypermethylation state in a cancer cell such as a spleen cancer cell). at i on)).
  • examples of scissin having a high methylation frequency in spleen cancer cells include, for example, base numbers 998, 1003, 1007, 1011, 1016, 1018 in the base sequence represented by SEQ ID NO: 13 , 1020, 1026, 1028, 1031, 1035, 1041, 10 43, 1045, 1051, 1053, 1056, 1060, 1066, 1068, 1070, 1073, 1093, 1096, 1106, 1 112, 1 120, 1 124, 1126
  • the cytosine shown by etc. can be mentioned.
  • the useful protein gene is G protein-coupled receptor 7 gene
  • Cp present in the base sequence of the promoter region, untranslated region or translated region (coding region)
  • the base sequence containing one or more base sequences indicated by G includes genomic DNA containing exon 1 of the human G prote in-coup led receptor 7 gene and a promoter region located 5 ′ upstream thereof.
  • the nucleotide sequence represented by SEQ ID NO: 14 corresponds to a column.
  • the base sequence of exon 1 of the G protein in-coupled receptor 7 gene derived from human is represented by base numbers 1 666 to 2562.
  • the cytosine in the nucleotide sequence shown shows a high methylation frequency (ie, hypermethylation) in cancer cells such as spleen cancer cells.
  • cytosine having a high methylation frequency in a spleen cancer cell includes, for example, the base sequence represented by SEQ ID NO: 14 in the base numbers 1480, 1482, 1485, 1496, 1513, 1526, 154 2, 1560, 1564, 1568, 1570, 1580, 1590, 1603, 1613, 1620, and the like.
  • the useful protein gene is a G-protein coupled somatostatin and angiotensin-1 ike peptide receptor gene, its promoter region, its promoter region, untranslated region or translated region.
  • the base sequence containing one or more base sequences represented by C p G present in the base sequence of (coding region) is an exon of the human G-protein coupled somatostatin and angiotensin-1 ike peptide receptor gene 1 and the 5 ′ upstream promoter region of the genomic DNA can be mentioned. More specifically, the nucleotide sequence represented by SEQ ID NO: 15 (Genbank Accession No. AC008971 In the base sequence of 5700 1 to 60000 of the base sequence described in 1).
  • nucleotide sequence represented by SEQ ID NO: 15 the nucleotide sequence of exon 1 of human-derived G-protein coupled somatostatin an dangiotensin-1 ike peptide receptor gene is represented by nucleotide numbers 776-2632. Cytosine in the base sequence represented by C p G present in the base sequence represented by SEQ ID NO: 15 has a high methylation frequency (i.e., hypermethylation state (hypermethylation state) in cancer cells such as presumptive cancer cells.
  • methylation frequency i.e., hypermethylation state (hypermethylation state
  • cytosine having high methylation frequency in spleen 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, 540, 543, 552, 566, 582, 597, 610, 612 and the like.
  • the useful protein gene is Solute carrier family 6 neurotransmitter transporter noradrenalin member 2 gene
  • a base sequence containing one or more base sequences indicated by CpG present in the base sequence of a single oral motor region, untranslated region or translated region (coding region) human-derived Solute carrier family 6 neurotransmitter transporter is recommended adrenalin
  • the base sequence of genomic DNA containing exon 1 of member 2 gene and the promoter region located 5 ′ upstream thereof can be mentioned. More specifically, the base sequence represented by SEQ ID NO: 16 ( This corresponds to a complementary sequence of the base sequence represented by base numbers 78801 to 81000 of the base sequence described in Genbank Accession No. AC026802.).
  • nucleotide sequence represented by SEQ ID NO: 16 the nucleotide sequence of exon 1 of the human-derived Solute carrier family 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 has a high methylation frequency (ie, hypermethylation state) in cancer cells such as spleen cancer cells. Indicates.
  • cytosine having a high methylation frequency in spleen cancer cells for example, in the base sequence represented by SEQ ID NO: 16, base numbers 1002, 1010, 1019, 1021, 1051, 1056, 1061, 1063, The cytosine shown by 1080, 1099, 1110, 1139, 1141, 1164, 1169, 1184 etc. can be mentioned.
  • “(amplified to a detectable amount of methylated DNA in the target DNA region) and the amount of amplified DNA” means that the genomic DNA contained in the biological sample is The amount after amplification of methylated DNA in the target region possessed, 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 term “after methylated DNA amplification in the target DNA region of the genomic DNA contained in the biological specimen. This is the total of the amount of DNA and the amount after amplification of unmethylated DNA, and means the value obtained by dividing the amount after amplification of 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 single-stranded DNA that is an unmethylated cytosine in one or more C p Gs.
  • the “single-stranded DNA that does not contain CpG in the methylated state at the recognition site of the methylation-sensitive restriction enzyme capable of digesting the single-stranded DNA” means the recognition site of the restriction enzyme in single-stranded DNA. It means single-stranded DNA with cytosine in all CpGs present in it being methylated.
  • the first step of the measurement method of the present invention from a DNA sample derived from genomic DNA contained in a biological sample, a single-stranded DNA containing the target DNA region (positive strand) and the single-stranded DNA 3 'By base-pairing with a single-stranded immobilized oligonucleotide having a base sequence that is complementary to a part of the terminal (excluding the target DNA region), the above-mentioned Select single-stranded 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 a 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 the target DNA region from DNA samples derived from genomic DNA contained in biological specimens.
  • the present immobilized oligonucleotide 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 described later. It may be in a free state so that a single extension reaction that proceeds from the 5 ′ end toward the 3 ′ end by the two previous steps and the second step A 2 is possible.
  • “thing that can be immobilized on a carrier” means a single strand containing the target DNA region.
  • the immobilized oligonucleotide is immobilized on a carrier.
  • the base pair between the single-stranded DNA (positive strand) and the immobilized oligonucleotide may be immobilized by binding of the present immobilized oligonucleotide and the carrier at the pre-combining stage, and (2) the single-stranded DNA (positive strand) and the present immobilized oligonucleotide. It may be immobilized by binding of the present immobilized oligonucleotide and the carrier at the stage after base pairing.
  • a part of the 3 ′ end of a single-stranded DNA (positive strand) containing the target DNA region (however, the target DNA region is not included). If the 5 'end of an oligonucleotide having a complementary base sequence (hereinafter also referred to as this oligonucleotide) is fixed to a carrier according to a normal genetic engineering operation method or a commercially available kit / device, etc. Good (binding to solid phase).
  • a support in which the biotinylated oligonucleotide is coated with streptavidin after the 5 ′ end of the oligonucleotide is biotinylated eg, a PCR tube or streptavidin coated with streptavidin.
  • a method of fixing to magnetic beads coated with eg, a PCR tube or streptavidin coated with streptavidin.
  • a molecule having an active functional group such as an amino group, an aldehyde group, or a thiol group was covalently bonded to the 5 ′ end side of the oligonucleotide, and then the surface was activated with a silane cutting agent or the like.
  • 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' specifically, for example, when the immobilized oligonucleotide is a pyotinylated oligonucleotide,
  • a DNA sample derived from genomic DNA contained in a biological sample is added to an annealing buffer and a pyotinylated oligonucleotide (the single-stranded DNA (positive strand) and the immobilized oligonucleotide).
  • this immobilized oligonucleotide Since it is immobilized by binding to the carrier, a mixture is obtained by adding one that is free at this stage. The resulting mixture is then used at 95 ° C to make the double-stranded DNA containing the desired DNA region present in the DNA sample derived from genomic DNA contained in the biological specimen into a single strand. Heat at C for several minutes.
  • the base pairing between the single-stranded DNA (positive strand) containing the target DNA region and the piotinated oligonucleotide is converted into a piotylated oligonucleotide and a streptogram.
  • either order may be used in this order. That is, for example, by adding a DNA sample derived from genomic DNA contained in a biological specimen to a pyotinylated oligonucleotide immobilized on a support coated with streptavidin, a mixture is obtained, and the resulting mixture is obtained.
  • the solution when using a PCR tube coated with streptavidin, the solution is first removed by pipetting or decantation, and then the volume of the biological sample is approximately the same. Add an amount of TE buffer, then add the TE buffer It can be removed by pipetting or decanting.
  • TE buffer When magnetic beads coated with streptavidin are used, after fixing the beads with a magnet, the solution is first removed by pipetting or decantation, and then the TE buffer is approximately equivalent to the volume of the biological specimen. Add one and then remove the TE buffer by pipetting or decanting.
  • This procedure is important because it removes unimmobilized DNA or DNA suspended in a solution digested with the restriction enzymes described below from the reaction solution. If these operations are insufficient, the DNA floating in the reaction solution will be in the shape of a bowl, and an unexpected amplification product will be obtained in the amplification reaction.
  • a large amount of DNA having a nucleotide sequence that is completely different from the target region eg, rat DNA in the case of human biological samples
  • a single-stranded DNA (positive strand) containing the target DNA region and a part of the 3 ′ end of the single-stranded DNA (however, the purpose When the base pairing is performed with a single-stranded immobilized oligonucleotide having a base sequence that is complementary to), the base pairing is performed in a reaction system containing a divalent cation.
  • a divalent cation is a magnesium ion.
  • reaction system containing a divalent cation means that the single-stranded DNA (positive strand) and the single-stranded immobilized oligonucleotide are mixed in an annealing buffer used for base pairing.
  • 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 produced. And free digest (single-stranded DNA containing one or more amethylated CpG at the recognition site of the methylation-sensitive restriction enzyme capable of digesting the single-stranded DNA).
  • “Methylation-sensitive restriction enzyme” in the second step of the measurement method of the present invention means, for example, that a recognition sequence containing methylated cytosine is not digested, but only a recognition sequence containing unmethylated cytosine is digested. It means a restriction enzyme that can be used. That is, in the case of DNA in which cytosine contained in a recognition sequence that can be originally recognized by a methylation-sensitive restriction enzyme is methylated, even if the methylation-sensitive restriction enzyme acts on the DNA, the DNA DNA is not cut.
  • the methylation-sensitive restriction enzyme can be allowed to act on the DNA. In this case, the DNA is cut.
  • a methylation-sensitive enzyme include Hpal l, BstUK Narl, SacI I, Hhal and the like.
  • the methylation-sensitive restriction enzyme is a double-stranded DNA containing a CpG pair in the hemimethyl state (that is, cytosine on one strand of the CpG pair is methylated).
  • methylation-sensitive restriction enzyme capable of digesting single-stranded DNA refers to a recognition that contains unmethylated cytosine without digesting the recognition sequence containing methylated cytosine in single-stranded DNA. It means a restriction enzyme that can digest only the sequence. That is, some methylation-sensitive restriction enzymes digest single-stranded DNA, such as fflial.
  • a method for examining the presence or absence of digestion with the methylation-sensitive restriction enzyme specifically, for example, using a pair of primers that can amplify DNA containing the cytosine to be analyzed as a recognition sequence using the DNA as a saddle type.
  • a method of examining the presence or absence of DNA amplification (amplification product) by performing PCR is possible.
  • cytosine to be analyzed is methylated, an amplification product is obtained.
  • the scinsin to be analyzed is not methylated, an amplification product cannot be obtained. Compare the amount of amplified DNA in this way By doing so, it is possible to measure the methylated ratio of cytosine to be analyzed.
  • the target DNA region is in a single-stranded state, and the single-stranded immobilized oligonucleotide as a negative strand is not the target DNA region. Since the base pairing is not performed, the single-stranded DNA strand is a single-stranded DNA derived from genomic DNA contained in a biological specimen. Therefore, by utilizing the property that the methylation sensitive restriction enzyme capable of digesting the single-stranded DNA does not cleave the single-stranded DNA in the methyl state, the genomic DNA contained in the biological sample is not affected.
  • cytosine in one or more CpGs present in the recognition site of the methylation-sensitive restriction enzyme capable of digesting the single-stranded DNA is methylated. That is, by subjecting the single-stranded DNA to digestion with a methylation-sensitive restriction enzyme capable of digesting the single-stranded DNA, it is possible to digest the single-stranded DNA in the genomic DNA contained in the biological specimen. If cytosine in one or more CpGs present in the restriction enzyme recognition site is not methylated, the single-stranded DNA is in an amethylated state, and the single-stranded DNA is It is cleaved by a digestible methylation-sensitive restriction enzyme.
  • cytosine in all C p G existing in the recognition site of the methylation sensitive restriction enzyme capable of digesting the single-stranded DNA in the genomic DNA contained in the biological sample is methylated. If so, it is not cleaved by a methylation sensitive restriction enzyme capable of digesting the single-stranded DNA. Therefore, after digestion, as described below, when PCR is performed using a pair of primers that can amplify the DNA region of interest, the genomic DNA contained in the biological sample is If cytosine in one or more CpGs present in the restriction enzyme recognition site is not methylated, PCR-amplified products cannot be obtained, but are included in biological samples. If all cytosines in CpG existing in the recognition site of the methylation-sensitive restriction enzyme capable of digesting the single-stranded DNA in genomic DNA are methylated, PCR amplification The product will be obtained.
  • the single-stranded DNA selected in the first step of the measurement method of the present invention is a single-stranded immobilized oligo. Only the base paired portion with the nucleotide forms a double strand, and most of the target DNA region is in a single-stranded state. Most methylation-sensitive restriction enzymes cannot digest single-stranded DNA even in the unmethylated state, but as mentioned above, some methylation-sensitive restriction enzymes can convert single-stranded DNA in the unmethylated state. Digestible. An example of a methylation-sensitive restriction enzyme that digests single-stranded DNA is Hhal. By using such an enzyme, the presence or absence of methylation of single-stranded DNA can be determined.
  • the C p G cytosine contained in the Hhal recognition site of the single-stranded portion of DNA in the sample base-paired with the single-stranded immobilized oligonucleotide obtained by the above operation is methylated. If so, Hhal cannot digest this DNA, while if it is not methylated, Hhal will digest this DNA. Therefore, after performing the above reaction, when PCR is performed using a pair of primers that can amplify the target DNA region, if the DNA in the sample is unmethylated, an amplification product cannot be obtained. If the DNA in the sample is methylated, an amplification product can be obtained.
  • the second step of the measurement method of the present invention may be carried out as follows, for example, when the immobilized oligonucleotide is a pyotinylated oligonucleotide.
  • the immobilized oligonucleotide is a pyotinylated oligonucleotide.
  • For the single-stranded DNA selected in the first step add 3 L of optimal 10X buffer (330 mM Tris-Acetate H 7.9, 66 OmM K0Ac, lOOmM Mg0Ac2, 5 mM Dithiothrei iol), and 3 mg of 1 mg / mL BSA aqueous solution.
  • methylation-sensitive restriction enzymes Hpall or Hhal 10 U / L
  • sterile ultrapure water to the mixture to a volume of 30 zL, and incubate at 37 for 1 to 3 hours. -Just do it.
  • 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 types of single-stranded DNA, and the resulting free digest (one of the above-mentioned Remove and wash (DNA purification) one or more single-stranded DNA containing CpG in the methylated state at the recognition site of the methylation sensitive restriction enzyme that can digest the strand DNA.
  • the TE buffer when using a PCR tube coated with streptavidin, first remove the solution by pipetting or decantation, then After adding TE buffer in an amount approximately equal to the volume of the biological specimen, the TE buffer may be removed by pipetting or decantation.
  • the TE buffer when using magnetic beads coated with streptavidin, after fixing the beads with a magnet, first remove the solution by pipetting or decanting, and then add TE buffer that is approximately equal to the volume of the biological sample. After that, the TE buffer can be removed by pipetting or decanting.
  • the “DNA sample derived from genomic DNA contained in a biological sample” 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 a restriction enzyme.
  • genomic DNA (cage DNA) contained in a biological sample is selected with immobilized oligonucleotides, short DNA is easier to select, and the target region is amplified by PCR. In this case, it is considered better to use short-type DNA, so a restriction enzyme that does not use the target DNA region as a recognition cleavage site is directly used for DNA samples derived from genomic DNA contained in biological samples. Digestion treatment may be performed. Note that the target DNA region is recognized.
  • a general restriction enzyme treatment method may be used as a method for digestion with a restriction enzyme that is not used as a cleavage site.
  • the “DNA sample derived from genomic DNA contained in a biological sample” is a DNA sample digested with one or more methylation-sensitive restriction enzymes. Can be mentioned.
  • Samples derived from genomic DNA contained in biological samples 3 ⁇ 4 As a method of digestion with a methylation-sensitive restriction enzyme, if the biological sample is genomic DNA itself, a general restriction enzyme treatment method may be used. When the biological specimen is a tissue lysate, cell lysate, etc., a large excess of methylation-sensitive restriction enzyme, for example, 500 times (10 U) or more of methylation with respect to 25 ng of DNA Digestion treatment may be performed using a sensitive restriction enzyme.
  • methylation-sensitive restriction enzyme eg, Hhal
  • Methylation sensitive restriction enzymes capable of digesting eg, HpaI I, BstUI, Narl, SacI I, Hhal, etc.
  • a single-stranded DNA that is an undigested product obtained in the second step methylation sensitivity that can digest the single-stranded DNA is obtained as a pre-step of each of the following steps.
  • Double-stranded DNA that is formed by extension in the second pre-process one that does not contain CpG in the methylated state at the recognition site of the methylation-sensitive restriction enzyme that can digest the single-stranded DNA
  • the process of separating the double-stranded DNA that is elongated from DNA strands in the strand state (DNA) into single-stranded DNA (positive strand) and single-stranded DNA (negative strand) A third previous step) and as this step
  • the generated single-stranded DNA is formed by base-pairing the generated single-stranded DNA (positive strand) with the single-stranded immobilized oligonucleotide (negative strand).
  • 1st step to select the DNA and the single-stranded DNA selected in the 1st step A is used as a template, and the primer is extended once using the single-stranded immobilized oligonucleotide as a primer.
  • a step (this step) having the A2 step of extending and forming the DNA in the selected single-stranded state as a double-stranded DNA,
  • each step of the third step is repeated once after separating the elongated double-stranded DNA obtained in each step into a single-stranded state, and in the target DNA region. Amplify methylated DNA to a detectable amount and quantify the amount of amplified DNA.
  • the third step of the measurement method of the present invention first, the single-stranded DNA that is an undigested product obtained in the second step (the above-mentioned one Remove strand DNA Single-stranded DNA (without DNA-containing CpG) at the recognition site of the methylation-sensitive restriction enzyme that can be converted to single-stranded DNA by separating it from the single-stranded immobilized oligonucleotide. To do.
  • a single-stranded DNA that is an undigested product obtained in the second step contains a CpG in the methyl state.
  • An annealing buffer is added to a DNA sample in which the single-stranded DNA) 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) previous step in the second previous step specifically, for example, it may be carried out according to the first step, and the generated free single-stranded state DN A (positive chain) and The generated single-stranded DNA, which is formed by base pairing with the single-stranded immobilized oligonucleotide, is selected.
  • the present immobilized oligonucleotide is a pyotinylated oligonucleotide
  • it may be carried out as follows.
  • Optimum sterilized ultrapure water is added to the DNA (positive strand), which is the free single-stranded state generated by separation in the first pre-process, and the above-mentioned single-stranded immobilized oligonucleotide, with 17.85 / 2.
  • Add 3 L of 10X buffer (lOOmM Tris-HCl pH 8.3, 500 mM KC1, 15 mM MgCl 2 ), 3 mM of 2 mM dNTP, 6 L of 5 betaine, and then add AmpIiTaq (one type of DNA polymerase: Add 0.15 zL of 5U /// L) to the solution, and incubate at 37 ° C for 2 hours. Then, after removing the incubated solution by pipetting or decanting, add TE buffer approximately equal to the volume of the biological specimen, and then pipetting or decanting the TE buffer. Remove it.
  • the TE buffer can be removed by pipetting or decanting.
  • magnetic beads coated with streptavidin fix the beads with a magnet, and then After removing the solution by pipetting or decanting, after adding TE buffer that is approximately equal to the volume of the biological specimen, the TE buffer can be removed by pipetting or decanting.
  • the DNA in the double-stranded state elongated in the second pre-process is Single-stranded DNA that does not contain C p G in the state of DNA is a double-stranded DNA that has been formed into an eaves-shaped DNA (DNA) and single-stranded DNA (positive strand) and single-stranded Once separated into DNA (negative strand).
  • the second (b) DNA in the double-stranded state obtained in the previous step the methylated CpG in the recognition site of the methylation-sensitive restriction enzyme capable of digesting the single-stranded DNA is added).
  • An annealing buffer is added to a double-stranded DNA that has been elongated from a single-stranded DNA that is not contained in a cage shape to obtain a mixture.
  • the resulting mixture is then heated at 95 ° C. for several minutes.
  • the DNA in the single-stranded state selected in (i) above is used as a saddle type, the single-stranded immobilized oligonucleotide is used as a primer, and the primer is extended once, thereby
  • the DNA in the double-stranded state is elongated and formed as double-stranded DNA (ie, 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 measurement method of the present invention described above.
  • the generated single-stranded DNA (negative strand) is a saddle type, and the single-stranded DNA (negative strand) has a partial base sequence (negative strand). And a base sequence (negative strand) that is complementary to the base sequence (positive strand) of the target DNA region.
  • oligonucleotide reverse primer
  • the oligonucleotide Is elongated once to form the single-stranded DNA as a double-stranded DNA (ie, step B).
  • 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 amplified DNA is quantified.
  • the second (B) in the third step of the measurement method of the present invention described above and the operation method in the previous step, step A and step B, etc. do it.
  • the reaction from the first previous step to the present step can be carried out as a single PCR reaction.
  • PCR 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
  • a methylation sensitive restriction enzyme capable of digesting single-stranded DNA for example, PCR
  • an immobilized oligonucleotide can be used as a primer on one side, so that only the other primer is added and PCR is performed to obtain an amplification product, which is also immobilized. Will be.
  • a primer previously labeled with fluorescence or the like is used and the label is used as an index, the presence or absence of an amplification product can be evaluated without performing a cumbersome operation such as electrophoresis.
  • the PCR reaction solution for example, DNA obtained in the second step of the measurement method of the present invention, 0.15 1 of a 50 zM primer solution, 2.5 1 of 2 mM dNTP, 10X buffer (lOOmM Tris-HCl pH 8.3, 500 mM KC 1, 20 mM MgCl 2 , 0.01% Gelatin) 2.5 ⁇ 1, AmpliTaq Gold (heat resistant MA polymer)
  • 5U / JL is mixed with 0.2 wl, and sterilized ultrapure water is added to this to make a reaction volume of 25 l.
  • the reaction may be carried out by adding an appropriate amount of betaine, DMSO, etc. at times.
  • the above reaction solution is kept at 95 ° C. for 10 minutes, then at 95 for 30 seconds, then at 55 to 65 ° C. for 30 seconds and further at 72 at 30 seconds.
  • One example is the condition for 30 to 40 cycles of heat insulation for one cycle.
  • 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.
  • the real-time PCR method may be used to obtain the amount of amplification product with higher accuracy.
  • the real-time PCR method is a method that monitors PCR. In real time and analyzes the obtained monitoring results by force kinetics. For example, it can detect even a slight difference of about twice the gene amount. This method is known as the quantitative PCR method.
  • Examples of the real-time PCR method include a method using a probe such as a type-dependent nucleic acid polymerase probe, a method using an intermediary method such as Cyber Green, and the like. Commercially available devices and kits for real-time PCR 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 vessel.
  • a biotinylated oligonucleotide having the same base sequence as the single-stranded immobilized oligonucleotide is designed on the 3 ′ end of a new pyotinylated oligonucleotide from one primer or single-stranded immobilized oligonucleotide. It is also possible to amplify the target region using the primer on one side and the primer on the complementary side. In this case, the amplification product obtained is immobilized if there is a support coated with streptavidin.
  • the detection of the amplification product is easy using the labeled primer. If the single-stranded immobilized oligonucleotide is immobilized by a covalent bond or the like, the solution containing the amplification product obtained by PCR is transferred to a container containing a streptavidin-coated support and amplified. It is possible to immobilize the product.
  • the detection may be performed as described above.
  • the complementary primer that amplifies the target region is a primer that can amplify the target region that has one or more recognition sites for methylation-sensitive restriction enzymes capable of digesting single-stranded DNA, and does not include that recognition site. I have to.
  • the reason for this is as follows. Restriction of methylation sensitivity that can eliminate single-stranded DNA at the 3 'end of the DNA strand (new strand) on the immobilized oligonucleotide side of the double-stranded DNA obtained by selection and single extension reaction If only the enzyme recognition site is not methylated, only that part will be digested with a methylation sensitive restriction enzyme capable of digesting single-stranded DNA.
  • the complementary primer contains a recognition site for a methylation sensitive restriction enzyme that can digest this 3 'end single-stranded DNA, several bases on the 3' end of the primer This is because annealing with a few bases at the 3 ′ end of the nascent strand may result in amplification of the target region by PCR.
  • 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, It has a base sequence that is complementary to a part of the 3 ′ end of the single-stranded DNA (positive strand) containing the DNA region to be used (however, it does not contain the DNA region of interest).
  • it includes a modified method that additionally includes a step (pre-addition step) of adding a single-stranded oligonucleotide (negative strand) in a free state into the reaction system.
  • the method according to the third step of the present measuring method further comprising the following one step as the second pre-step and each main step.
  • Step C 1 DNA in the single-stranded state selected in Step C 1 is used as a saddle type, and the primer is extended once by using the single-stranded oligonucleotide (negative strand) as a primer.
  • Step C with Step C 2 (This Step) (Modification 2)
  • Each of the third steps of the measurement method of the present invention further includes the following one step (hereinafter, also referred to as the present methylation ratio measurement method).
  • Step C By extending the primer once by using the single-stranded DNA selected in step C1 as a saddle and using the single-stranded oligonucleotide (negative strand) as a primer, Step C comprising the second step of converting the DNA in the single-stranded state into elongated double-stranded DNA (this step)
  • the method according to the present invention further comprises the following one step as each step of the ⁇ step of the measuring method of the present invention.
  • step C1 Base pairing the generated single-stranded DNA (positive strand) with the single-stranded oligonucleotide (negative strand) added to the reaction system in the previous addition step.
  • Step C having the second step of converting the DNA in the single-stranded state into elongated double-stranded DNA by the second step (this step)
  • a step of adding a single-stranded oligonucleotide (negative strand) in a free state into the reaction system (additional A non-digested double-stranded DNA obtained by the first pre-process and the above-mentioned pre-process (methylation capable of digesting the single-stranded DNA).
  • Sensitivity restriction The process of separating the double-stranded DNA, which is formed by extending single-stranded DNA that does not contain CpG in the methylated state at the enzyme recognition site, into a single-stranded state (additional re-previous step) ), And
  • Each of the third steps of the measurement method of the present invention further includes the following one step (hereinafter, also referred to as the present methylation ratio measurement method).
  • Step C By extending the primer once by using the single-stranded DNA selected in Step C 1 as a saddle and using the single-stranded oligonucleotide (negative strand) as a primer, Step C having Step C 2 to convert the DNA in the single-stranded state into elongated double-stranded DNA (this step)
  • the above-mentioned DNA region of interest is It has a base sequence that is complementary to a part of the 3 ′ end of the contained single-stranded DNA (positive strand) (excluding the target DNA region) and is in a free state
  • the amplification efficiency of the target DNA region in the third step can be easily improved.
  • the single-stranded oligonucleotide (negative strand) added to the reaction system in the pre-addition step is a part of the 3 ′ end of the single-stranded DNA (not including the above-mentioned target DNA region). )) Is a single-stranded oligonucleotide in a free state having a base sequence that is complementary to the single-stranded immobilized oligonucleotide, and whose 5 ′ end is the same as the single-stranded immobilized oligonucleotide.
  • the base sequence may be the same as the single-stranded immobilized oligonucleotide, or may be a short base sequence, or a long base sequence.
  • the reverse primer positive strand
  • the single-stranded oligonucleotide negative strand
  • the immobilized oligonucleotide is used as a primer on one side and only the other primer is added to perform PCR.
  • the analysis method that can compare the amount of each amplification product obtained by PCR is performed, as described above, when a target region is amplified, one (one side) of the immobilized oligonucleotide is used. Instead of using them as primers, PCR may be performed by adding a pair of primers. After performing such PCR, determine the amount of amplification product obtained. '' The third step of the measurement method of the present invention has a repeated step.
  • the generated single-stranded DNA (positive strand)” in the first step A 1 means the first third step This means “generated“ free ”single-stranded DNA (positive strand)” in both the operation of the process and the repetitive operation of the third and subsequent steps.
  • DNA (negative strand) generated in the single-strand state” in Step B means both the first step of the third step and the second and subsequent third steps. It means “DNA (positive chain) that is a“ fixed ”single-stranded state”. However, if the third step has an additional C step, it means “DNA (positive strand) in the generated“ fixed ”single-stranded state” in the first step of the third step. On the other hand, in the second and subsequent third-step repetitive operations, “generated“ fixed ”single-stranded DNA (positive strand)” and “generated“ free ”single-stranded state D It means both “NA (positive chain)”.
  • the “stretched double-stranded DNA” obtained in each step of the third step refers to the “methylation described above” in the first step of the third step.
  • An extension-formed double-stranded DNA that does not contain an amethylated CpG pair at the recognition site of the methylation-sensitive restriction enzyme and "An amethylated CpG pair This means both an elongated double-stranded DNA containing ”.
  • Step B in both the first step, the third step operation, and the second and subsequent third step repeat operations, “all of the above recognition sites for methylation-sensitive restriction enzymes are unanchored. This means a stretched double-stranded DNA NA which is a CpG pair in the methyl state.
  • the third step additionally has a C step.
  • the generated single-stranded DNA (positive strand)” in step C 1 means the first step 3
  • the repetitive operation of the third step from the second time onward “DNA (positive strand) j in the generated“ free ”single-stranded state is meant.
  • a method for measuring a methylation ratio ie, a method for measuring a methylation ratio of the present invention characterized by further comprising the following two steps is included.
  • the measurement method of the present invention After performing the first step of the measurement method of the present invention (including the above-described modified method), the measurement method of the present invention (without the second step of the measurement method of the present invention (including the modified method)) By performing the third step (including the above-mentioned modified method), the DNA of the target DNA region (the total amount of methylated DNA and unmethylated DNA) is amplified to a detectable amount.
  • DNA methylation abnormalities are known to occur in various diseases (eg cancer). Therefore, it is considered that the degree of various diseases can be measured by detecting this DNA methylation abnormality.
  • the measurement method of the present invention or the methylation ratio measurement method of the present invention is performed on the DNA region.
  • the amount of methylated DNA increases, for example, there is a DNA region that is not methylated in genomic DNA contained in a specimen derived from a disease organism. If the inventive methylation ratio measurement method is implemented, the amount of methylated DNA will be close to zero.
  • the amount of methylated DNA is close to 0 in the case of healthy subjects, while in the case of patients with diseases. Since the value is significantly higher than that in the case of a healthy person, the “degree of disease” can be determined based on the difference between the values.
  • the “degree of disease” here has the same meaning as that generally used in the field, and specifically, for example, when the biological specimen is a cell, it means the malignancy of the cell. For example, when the biological specimen is a tissue, it means the abundance of diseased cells in the tissue. Furthermore, when the biological specimen is plasma'serum, it means the probability that the individual has a disease.
  • 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, a primer or a primer that can be used in various methods for measuring the amount of methylated DNA in the target region and measuring the methylation ratio
  • the probe is useful as a reagent in a detection kit.
  • the present invention also provides a detection kit containing these restriction enzymes, primers or probes as reagents, and a detection chip in which these primers or probes are immobilized on a carrier.
  • the scope of rights of the method for measuring methylation ratio of the present invention is: It also includes use in the form of a detection chip as described above that utilizes the substantial principle of the method.
  • Mammal colonic adenocarcinoma cell line Caco-2 (ATCC N0.HT B-37), purchased from ATCC, is confluent in dedicated media for cell lines listed in the ATCC catalog until confluence By culturing, approximately 1 ⁇ 10 7 cells were obtained each. After adding 10 times volume of SEDTA buffer [lOmM Tris-HCl H 8.0, lOmM EDTA pH 8.0, lOOmM NaCl] to the obtained cells, it was homogenized.
  • DNA fragment X 2 a DNA fragment used as a test sample
  • DNA fragment X 2 a DNA fragment used as a test sample
  • SEQ ID NO: 18 a DNA fragment used as a test sample
  • base numbers 76477 to 77002 of the GPR 7 sequence shown in Genbank Accession No. AC009800 etc. Sequence was amplified.
  • PR3 5'-CGATGAGCTTGCACATGAGCT-3 '(SEQ ID NO: 20)
  • PCR reaction solution 2.5 ng of genomic DNA in the form of a cage, and a primer solution consisting of the base sequence represented by SEQ ID NO: 19 prepared in 3 / xM and the base sequence represented by SEQ ID NO: 20
  • Each primer solution 2.5 1 and 2 mM dN TP 2 respectively, 1 OX buffer (lOOmM Tris-HCl pH 8.3, 500 mM KC1, 15 mM MgCl 2 0.01% Gelatin)
  • Thermostable DNA polymerase AmpliTaq Gold, Will
  • the reaction solution is kept at 95 ° C for 10 minutes, then at 95 ° C for 30 seconds, then at 59 ° C for 60 seconds and then at 72 ° C for 45 seconds for one cycle.
  • PCR was performed under the conditions of the vehicle.
  • DNA amplification was confirmed by 1.5% agarose gel electrophoresis, the target DNA fragment (526 bp, DNA fragment X2) was excised and purified using the QIAG EN QIAquick Gel Extraction Kit (QIAGEN). .
  • DNA fragment Y2 A portion of the resulting DNA fragment X2 was treated with methylase S ss I (NEB), and all of the 5, -CG-3 ′ methylated DNA fragment (hereinafter referred to as DNA fragment Y2). Got. Again, as before, confirm amplification by 1.5% capillary electrophoresis, excise the DNA fragment (526 bp, DNA fragment Y2) and use the QIAGEN QIAquick Gel Extraction Kit (QIAGEN). And purified.
  • NEB methylase S ss I
  • an oligonucleotide B 1 (19 bp) having a nucleotide sequence shown in SEQ ID NO: 21 and labeled with a biotin at the 5 ′ end was synthesized.
  • DNA fragment X2 or DNA fragment Y2 a single-stranded DNA containing the target DNA region and the pyotinylated oligonucleotide.
  • the pre-prepared mixture was added as described above, and this was incubated at 37 ° C. for 5 minutes, so that the pyotinylated oligonucleotide was added. It was fixed to a support coated with streptavidin (this corresponds to the first step of the measurement method of the present invention).
  • 100 TE buffer was added, and then the TE buffer was removed by pipetting. This operation was performed twice more. ,
  • the single strand DNA thus obtained was subjected to the following three types of treatment.
  • Group A (untreated group): 1 OX buffer (330 mM Tris-Acetate H 7.9, 660 mM K0Ac, lOOmM Mg0Ac2, 5 mM Dit) suitable for Hpa II and Hha I. othreiiol) and 3X of 10XBSA (Bovine serum albumin lmg / ml) were added, and sterilized ultrapure water was added to the mixture to adjust the volume to 30 ⁇ L.
  • 1 OX buffer 330 mM Tris-Acetate H 7.9, 660 mM K0Ac, lOOmM Mg0Ac2, 5 mM Dit
  • 10XBSA Bovine serum albumin lmg / ml
  • Group B (digested with Hpa II): Single-stranded DNA prepared above, 15U of Hpa II, and 10X buffer (330mM Tris-Acetat optimal for HpaI and Hha I) e Add 3 L of pH 7.9, 660 mM OAc, lOOmM Mg0Ac2, 5 mM Diothothrei tol) and 3 L of 10XB SA (Bovine serum albumin lmg / ml), and then add sterilized ultrapure water to the mixture. The liquid volume was adjusted to 30 ⁇ L.
  • Group B Hha I digestion group: The single-stranded DNA prepared above contains 15 U of Hha I and 10 X buffer (330 mM Tris-Acetat, which is optimal for Hpal I and Hha I, respectively. e Add 3 L of pH 7.9, 660 mM K0Ac, lOOmM MgOAc2, 5 mM Dithothreiiol) and 3 L of 10XB SA (Bovine serum albumin lmg / ml), and add sterilized ultrapure water to the mixture. The amount was 30 ⁇ L.
  • PR4 5'-GCGGAGTTGCCCGCCAGA-3 '(SEQ ID NO: 24)
  • a primer solution consisting of the base sequence shown in SEQ ID NO: 23, prepared in 3 M, is used as a genomic DNA in a cage shape.
  • a primer solution consisting of the base sequence shown in SEQ ID NO: 24, 2.5 X 1 each, 2 mM dNTP 2.5 1 and 10 X buffer (lOOmM Tris-HCl pH 8.3, 500 mM KC1, 15 mM MgCl 2 , 0.01% G elatin) 2.5 1, heat-resistant DNA polymerase (AmpliTaq Gold) 5UZ 1 0.125 1 and 5 N betaine aqueous solution 5 a 1 are mixed, and sterilized ultrapure water is added to the volume. The one with 25 ⁇ 1 was used.
  • reaction solution is incubated at 95 ° C for 10 minutes, and then incubated at 95 ° C for 30 seconds, then at 59 ° C for 60 seconds, and further at 72 ° C for 45 seconds for one cycle for 37 cycles. PCR was performed under conditions.
  • DNA fragment X2 in the case of Group A (untreated group) and Group B (Hpall treated group), amplification was confirmed and the amplification product was obtained. In contrast, in the case of Group C (Hhal treatment group), amplification was not confirmed and the amplification product was not obtained. On the other hand, for DNA fragment Y2, amplification was confirmed and amplification products were obtained in all cases of Group A (no treatment group), Group B (Hpall treatment group), and Group C (Hhal treatment group). It was.
  • Example 2 The following test was conducted. A serum solution was obtained by adding 1 mL of rat serum to a ratio of 1.
  • the resulting mixture was then heated at 95 ° C. for 5 minutes. After that, it was quickly cooled to 50 ° C and kept at that temperature for 5 minutes. This is then incubated at 37 for 5 minutes, then returned to room temperature, and the single-stranded DNA containing the desired DNA region and the piotinylated oligonucleotide are recovered.
  • the single-stranded DNA thus obtained was subjected to the following two treatments.
  • Group A (untreated group): 1 OX buffer (330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM Mg0Ac2, 5 mM) suitable for Hpa II and Hha I to the single-stranded DNA prepared above. Dithothreitol) was added at 3 x L, 10XBSA (Bovine serum albumin lmg / ml) at 3 L, and sterilized ultrapure water was added to the mixture to make a volume of 30 L (preparation of 2 each). ⁇
  • Group B Hha I digestion group: Single-stranded DNA prepared above, 15 U of Hha I and 10 X buffer (330 mM Tris-Acetate optimal for Hpa II and Hha I) Add pH 7.9, 660 mM K0Ac, lOOmM Mg0Ac2, 5 mM Dithothreitol) and 10XBSA (Bovine serum albumin lmg / ral) to 3 and add sterilized ultrapure water to the mixture to a volume of 30 L ( 2 each).
  • Hha I digestion group Single-stranded DNA prepared above, 15 U of Hha I and 10 X buffer (330 mM Tris-Acetate optimal for Hpa II and Hha I) Add pH 7.9, 660 mM K0Ac, lOOmM Mg0Ac2, 5 mM Dithothreitol) and 10XBSA (Bovine serum albumin lmg / ral) to 3 and add sterilized ultrapure water to
  • 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 region corresponding to the base numbers 76669-76835 of the GPR 7 sequence represented by Genbank Accession No. AC009800, etc.) Amplified.
  • PR4 5'-GCGGAGTTGCCCGCCAGA-3 '(SEQ ID NO: 24)
  • 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 3 was added to the genomic DNA in a cage shape 2.
  • 5 / X 1 and 2.5 mM dNTP 2.5 1
  • 10 ⁇ buffer (lOOmM Tris-HCl pH 8,3, 500 mM KC1, 15 mM MgCl 2 , 0.01% Gelatin)
  • Heat resistant DNA polymerase (AmpliTaq Gold) 5U / 1 0.125 1 and 5 N betaine aqueous solution 51 were mixed, and sterilized ultrapure water was added to make the volume 25 a 1.
  • Incubate the reaction solution at 95 ° C for 10 minutes, then perform 95 cycles at 95 ° C for 30 seconds, then at 63 ° C for 60 seconds, and at 72 ° C for 45 seconds for 1 cycle.
  • PCR was performed under conditions.
  • DNA fragment X2 in the case of Group A (untreated group), amplification was confirmed and the amplification product was obtained. On the other hand, in the case of Group B (Hhal treatment group), amplification was not confirmed and the amplification product was not obtained. On the other hand, for DNA fragment Y2, In both cases of Group A (no treatment group) and Group B (Hhal treatment group), amplification was confirmed and the amplification product was obtained. Based on the above, even when a serum solution as a DNA sample derived from genomic DNA contained in a biological sample is used, the single-stranded DNA containing the target DNA region can be obtained in the same manner as in Example 1.
  • Amplification of unmethylated DNA in the target DNA region is possible by using a methylation-sensitive restriction enzyme (Hhal) that can be selected and that can digest a single strand. Without amplification, it was confirmed that only methylated DNA was amplified to a detectable amount, and the amount of amplified DNA could be quantified.
  • Hhal methylation-sensitive restriction enzyme
  • Example 3 The following test was conducted. A pg / 1 OL TE buffer solution (10 mM Tris, ImM EDTA, H 8.0) was prepared.
  • the resulting mixture was then heated at 95 ° C. for 5 minutes. After that, it was quickly cooled to 50 ° C and kept at that temperature for 5 minutes. This is then incubated at 37 ° C for 5 minutes, then returned to room temperature, and a single-stranded DNA (DNA fragment X2 or DNA fragment Y2) containing the target DNA region and piotinylated oligonucleotide are base paired.
  • D To the PCR tube coated with streptavidin, add the pre-prepared mixture as described above, and incubate it at 37 ° C for 5 minutes to The tintin oligonucleotide was immobilized on a support coated with streptavidin (this corresponds to the first step of the measurement method of the present invention).
  • wash buffer 0.05% Tween20-containing phosphate buffer: ImM KH2P04, 3 mM Na2HP0 ⁇ 7H20, 154 NaCK pH 7.4
  • wash buffer 0.05% Tween20-containing phosphate buffer: ImM KH2P04, 3 mM Na2HP0 ⁇ 7H20, 154 NaCK pH 7.4
  • Group A (untreated group): 1 OX buffer (330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM MgOAc2, 5 mM) suitable for Hpa II and Hha I, to the single-stranded DNA prepared above. 3 L of Dithothreitol) and 3 ⁇ L of 10XBSA (Bovine serum albumin lmg / ml) were added, and sterilized ultrapure water was added to the mixture to make the volume 3.
  • 1 OX buffer 330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM MgOAc2, 5 mM
  • Group B Single-stranded DNA prepared above, 15 U of Hha I, and 10 X buffer (330 mM Tris-Acetate pH optimal for Hpa I I and Hha I.
  • Bovine serum albumin lmg / ml was added to 3 / xL, and sterilized ultrapure water was added to the mixture to make the volume 30 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 was added to the genomic DNA to be a cage type.
  • 3 5 mM dNTPs 5 1 each, 10X buffer (lOOmM Tris-HCl pH 8.3, 500 mM KC1, 15 mM MgCl 2 , 0.01% Gela tin) 5 1, thermostable DNA polymerase (AmpliTaq Gold) 5U / 1 was mixed with 0.25 1 and 5 N betaine aqueous solution 101 and sterilized ultrapure water was added to make the volume 50 ⁇ l. Incubate the reaction solution at 95 ° C for 10 minutes, then heat at 32 ° C for 30 seconds, then at 59 ° C for 30 seconds, and then at 72 ° C for 45 seconds for one cycle. PCR was performed under conditions.
  • the single-stranded DNA (positive strand) containing the target DNA region and the 3 ′ end of the single-stranded DNA
  • a single-stranded immobilized oligonucleotide having a base sequence that is complementary to a part (however, the target DNA region is not included)
  • the target DNA region is It is possible to select a single-stranded DNA to be contained, and a non-methylated DN in the target DNA region by treatment with a methylation-sensitive restriction enzyme capable of digesting the single-stranded DNA.
  • Example 4 The following test was conducted. A g / 10 / iL rat serum solution was prepared. Separately add 10 L of biotinylated oligonucleotide B 1 with 1 L, 10 X annealing buffer (330 mM Tris-Acetate ⁇ 7.9, 660 mM K0Ac, lOOmM Mg0Ac2, 5 mM Dithothreitol) and sterilized ultrapure. The mixture was obtained by adding 7 L of water.
  • the resulting mixture was then heated at 95 ° C. for 5 minutes. After that, it was quickly cooled to 50 ° C and kept at that temperature for 5 minutes. This is then incubated at 37 ° C for 5 minutes, then returned to room temperature, and base-paired with single-stranded DNA (DNA fragment X2 or DNA fragment Y2) containing the desired DNA region and biotinylated oligonucleotide.
  • Group A (untreated group): 1 OX buffer (330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM MgOAc2, 5 mM) Di thothreitor) was added with 3 L, 10 XBSA (Bovine serum albumin lmg / ml) with 3 L, and sterilized ultrapure water was added to the mixture to adjust the volume to 30 L.
  • 1 OX buffer 330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM MgOAc2, 5 mM
  • Di thothreitor was added with 3 L
  • 10 XBSA Bovine serum albumin lmg / ml
  • Hha I is added to the single-stranded DNA prepared above.
  • Bovine serum albumin lmg / ml was added to 3 L, and sterilized ultrapure water was added to the mixture to make the volume 30 L.
  • PR4 5'-GCGGAGTTGCCCGCCAGA-3 '(SEQ ID NO: 24)
  • a primer solution consisting of the base sequence shown in SEQ ID NO: 23 prepared in 5 OM is used as a genomic DNA in a cage shape.
  • primer comprising the nucleotide sequence represented by SEQ ID NO: 24, and 5 1 each 2mM of dNTPs, 1 0 X buffer (lOOmM Tris-HCl pH 8.3, 500mM KC1, 15mM MgCl 2, 0.01% Gela tin) 5 1, heat-resistant DNA polymerase (AmpliTaq Gold) 5U // x 1 0.25 1 and 5 N betaine aqueous solution 10 1 are mixed, and sterilized ultrapure water is added to the solution. An amount of 50 1 was used.
  • the reaction solution was incubated at 95 ° C for 10 minutes, and then maintained at 32 ° C for 30 cycles at 95 ° C, 30 seconds at 59 ° C, 30 seconds at 72 ° C, and 45 seconds at 72 ° C. PCR was performed.
  • DNA fragment X2 in the case of group A (untreated group), amplification was confirmed in the lpgZlOL sample and the 10pg / 10L sample, and the amplification product was obtained.
  • Example 5 Mammal breast cancer cell line MCF-7 (ATCC NO. HTB-22), purchased from AT CC, is cultured until confluent in the dedicated medium for the cell line described in the AT CC catalog. As a result, about 1 ⁇ 10 7 cells were obtained. After adding 10 times volume of S EDTA buffer [10 mM Tris-HCl pH 8.0, 10 mM EDTA H 8.0, lOOmM NaCl] to the obtained cells, this was homogenized.
  • S EDTA buffer 10 mM Tris-HCl pH 8.0, 10 mM EDTA H 8.0, lOOmM NaCl
  • TE buffer (10 mM Tris, ImM EDTA, pH 8.0) and add 40 g / m 1 to this: add Nase A (S i gma) and incubate at 37 for 1 hour did.
  • the incubated mixture was subjected to a phenol's black-form extraction process.
  • the aqueous layer was recovered, and NaCl was added to 0.5N, followed by ethanol precipitation to recover the precipitate (genomic DNA).
  • the collected precipitate was rinsed with 70% ethanol to obtain genomic DNA.
  • the obtained genomic DNA was used as a saddle type, and PCR was performed using the following primers and reaction conditions to obtain a sample sample.
  • a sequence corresponding to base numbers 8 to 480 of the LI NE 1 sequence shown in Genbank Accession No. M80343 and the like was amplified.
  • a primer solution consisting of 2 ng of genomic DNA in the form of a cage and a base sequence represented by SEQ ID NO: 26 prepared in 100 pmo1 / ⁇ 1 and a solution thereof are arranged.
  • Primer solution consisting of the base sequence shown in column number 27 0.125 1 each, 2 mM dNTP 2.5 1 and 10 X buffer (lOOmM Tris-HC1 pH 8.3, 500 mM KCl, 15 mM MgCl 2 0.01% Gelatin), 2.51, and thermostable DNA polymerase 51, 0.1251, and sterilized ultrapure water added to make the volume 251, were used.
  • the reaction mixture is kept at 95 ° C for 10 minutes, then kept at 95 ° C for 30 seconds, then at 63 ° C for 60 seconds and then at 72 ° C for 45 seconds for one cycle for 50 cycles.
  • PCR was performed.
  • DNA fragment Y1 A part of the obtained DNA fragment X 1 was treated with methylase S ssi (NEB) to obtain a DNA fragment (hereinafter referred to as DNA fragment Y1) in which all of 5-CG-3 ′ was methylated. .
  • the amplification was confirmed by 1.5% DNA gel electrophoresis, the target DNA fragment (473 bp, DNA fragment Y1) was excised, and the QIAGEN QIAquick Gel Extraction Kit (QIAGEN) ) And purified.
  • DNA fragment Including target MA region Methylation ratio including target DM region
  • Group A (untreated group): About 25 ng of DNA fragment, 2 L of 1 OX buffer (330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM MgOAc2, 5 mM Dithothreitol) suitable for Hpa II and Hha I Then, 2 L of 10XBSA (Bovine serum albumin lmg / ml) was added, and sterilized ultrapure water was further added to the mixture to make the volume 2 OL.
  • 1 OX buffer 330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM MgOAc2, 5 mM Dithothreitol
  • 10XBSA Bovine serum albumin lmg / ml
  • Group B (Hpall treatment group): About 25 ng of DNA fragment, 0.5 U of Hpa II, and 10 X buffer solution suitable for Hpa II and Hha I (330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM MgOAc2, 5 mM Dithothreitol) and 2X of 10XBSA (Bovine serum albumin lmg / ml) were added, and sterilized ultrapure water was added to the mixture to make 2 QL.
  • 10X buffer solution suitable for Hpa II and Hha I 330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM MgOAc2, 5 mM Dithothreitol
  • 10XBSA Bovine serum albumin lmg / ml
  • Group C Hhal treatment group: Approximately 25 ng of DNA fragment, 0.5 U of H ha I, and 10 X buffer solution (330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM Mg0Ac2) 2 mM of 5 mM Dithothreitol) and 2 zL of 10 XBSA (Bovine serum albumin lmg / ml) were added, and sterilized ultrapure water was added to the mixture to make the volume 20.
  • 10 X buffer solution 330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM Mg0Ac2
  • 10 XBSA Bovine serum albumin lmg / ml
  • Group D Hpall and Hhal treatment group: About 25 ng of DNA fragment, 0.5 U of Hpal I and Hha I, and 10 X buffer (330 mM Tris- optimal for H pa II and Hha I, respectively) Add 2 L of Acetate pH7.9, 660 mM K0Ac, lOOmM Mg0Ac2, 5 mM Dithothreitol) and 2 L of 10XBSA (Bovine serum albumin lmg / ml), and add sterilized ultrapure water to the mixture. 20 ⁇ L. Each reaction solution was incubated at 37 for 2 hours, and then sterilized ultrapure water was added to dilute it 100 times.
  • 10XBSA Bovine serum albumin lmg / ml
  • T 1 5'-CGAATATTGCGCTTTTCAGACCGGCTT-3 '(SEQ ID NO: 30)
  • a DNA fragment 62.5 pg in the form of a cage and the nucleotide sequence shown in SEQ ID NO: 28 prepared at 3 pm o ⁇
  • 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.
  • Figure 7 (“I") is a fragment mixture with a methylation rate of 0%, so the theoretical values for Group B, C, and D are "0" and Figure 8 (“1 1") is methyl.
  • the theoretical value in group B, C and D is “0.1” and
  • FIG. 9 (“III”) is a fragment with a methylation rate of 25%.
  • the theoretical values in Group B, Group C and Group D are “0.25” and FIG. 10 (“IV”) is a fragment with a methylation ratio of 50%.
  • the theoretical value in the D group is “0.5”
  • FIG. 11 (“V”) is a fragment with a methylation ratio of 100%, so the theoretical values in the B group, the C group, and the D group are “ 1 ”.
  • FIGS. 7 to 11 in Group D, a value closest to this theoretical value was obtained, and it was revealed that digestion with two or more methylation-sensitive enzymes is preferable. It was. Industrial applicability

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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 Ceased WO2008078834A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2272974A4 (fr) * 2008-03-25 2011-06-29 Sumitomo Chemical Co Procédé servant à déterminer la méthylation de l'adn

Citations (3)

* Cited by examiner, † Cited by third party
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

Patent Citations (3)

* Cited by examiner, † Cited by third party
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

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ALLEN R.C. ET AL.: "Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgen-receptor gene correlates with X chromosome inactivation", AM. J. HUM. GENET., vol. 51, no. 6, 1992, pages 1229 - 1239 *
JANG S.J. ET AL.: "Methylation patterns in human androgen receptor gene and clonality analysis", CANCER RES., vol. 60, no. 4, 2000, pages 864 - 866, XP002467560 *
KASSIM S. ET AL.: "Androgen receptor gene methylation and exon one CAG repeat length in ovarian cancer: differences from breast cancer", IUBMB LIFE, vol. 56, no. 7, 2004, pages 417 - 426 *
SHOFFNER M.A. ET AL.: "Chip PCR. I. Surface passivation of microfabricated silicon-glass chips for PCR", NUCLEIC ACIDS RES., vol. 24, no. 2, 1996, pages 375 - 379, XP000605272, DOI: doi:10.1093/nar/24.2.375 *
WANG S.H. ET AL.: "Identification and characterization of Bacillus anthracis by multiplex PCR on DNA chip", BIOSENS. BIOELECTRON., vol. 20, no. 4, 2004, pages 807 - 813, XP004633829, DOI: doi:10.1016/j.bios.2004.03.019 *
WEN J.K. ET AL.: "A visual DNA chip for simultaneous detection of hepatitis B virus, hepatitis C virus and human immunodeficiency virus type-1", BIOSENS. BIOELECTRON., vol. 19, no. 7, 2004, pages 685 - 692, XP002449305, DOI: doi:10.1016/S0956-5663(03)00264-1 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2272974A4 (fr) * 2008-03-25 2011-06-29 Sumitomo Chemical Co Procédé servant à déterminer la méthylation de l'adn

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