WO2008038833A1 - procÉdÉ de dÉtermination de la mÉthylation de l'adn - Google Patents

procÉdÉ de dÉtermination de la mÉthylation de l'adn Download PDF

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WO2008038833A1
WO2008038833A1 PCT/JP2007/069414 JP2007069414W WO2008038833A1 WO 2008038833 A1 WO2008038833 A1 WO 2008038833A1 JP 2007069414 W JP2007069414 W JP 2007069414W WO 2008038833 A1 WO2008038833 A1 WO 2008038833A1
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dna
stranded
base sequence
region
stranded dna
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PCT/JP2007/069414
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English (en)
Japanese (ja)
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Yoshitaka Tomigahara
Hirokazu Tarui
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Sumitomo Chemical Company, Limited
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Publication of WO2008038833A1 publication Critical patent/WO2008038833A1/fr

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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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification

Definitions

  • the present invention relates to a method for measuring the content of methylated DNA in a target DNA region in genomic DNA contained in a biological specimen.
  • a method for evaluating the methylation status of DN A in the target DN A region of genomic DNA contained in a biological sample for example, methylated DNA in the target DNA region of genomic DNA
  • a method to measure the content of urine see, for example, ucleic Acids Res. 1994 Aug 11; 22 (15): 2990-7 and Proc Natl Acad Sci US A. 1997 Mar 18; 94 (6): 2284-9 )
  • this measurement method first, it is necessary to extract DNA containing the target DNA region from the DNA sample derived from genomic DNA, and the extraction operation is complicated.
  • a method for measuring the content of methylated DNA in the target region of the extracted DNA for example, (1) After modifying the DNA with sulfite, etc., DN A polymerase DNA A method of amplifying the target region by subjecting it to a polymerase chain reaction (hereinafter sometimes referred to as PCR). (2) After digesting the DNA with a methylation sensitive restriction enzyme, PCR There are known methods for amplifying the target region by subjecting it to the above. Both of these methods require labor for modification of DNA for detection of methylation and subsequent purification of the product, preparation of a reaction system for PCR, confirmation of DNA amplification, and the like. Disclosure of the invention
  • An object of the present invention is to provide a method for easily measuring the content of methylated DNA in the target DNA region in genomic DNA contained in a biological sample. That is, the present invention
  • a single-stranded DNA containing a target DNA region and a part of the 3 ′ end of the single-stranded DNA is selected by base-pairing with a single-stranded immobilized oligonucleotide that does not contain the target DNA region and has a base sequence that is complementary to First step,
  • the single-stranded DNA selected in the first step is used as a saddle, and the single-stranded immobilized oligonucleotide is used as a primer.
  • a second step of forming A is used.
  • a positive single-stranded DNA containing the target DNA region and a part of the 3 ′ end of the single-stranded DNA, but not including the target DNA region It is characterized by base pairing in a reaction system containing a divalent cation when base-pairing with a single-stranded immobilized oligonucleotide having a base sequence complementary to,.
  • the fourth step and the subsequent steps are performed without performing the third step.
  • Amplifying the region's DNA total amount of methylated and unmethylated DNA
  • FIG. 1 shows that in Example 1, the prepared sample was subjected to either “A (no treatment)” or “B (simultaneous treatment of Hpall and Bial)”, and the base shown in SEQ ID NO: 22
  • FIG. 2 is a diagram showing the results of 1.5% agarose gel electrophoresis of the amplified product obtained by PCR amplification of DNA that was methylated in a region consisting of a sequence.
  • DN A fragment X 2 “A” treated sample DNA fragment X 2 “B” treated sample
  • DNA fragment Y 2 “A” treated Sample DN A fragment Y 2 treated with “B” treatment.
  • Fig. 2 shows that the sample prepared in Example 2 was treated with "A (no treatment)", “B (H pall treatment)", “C (Hhal treatment)” or “D (simultaneous treatment of Hpall and 3 ⁇ 4ial). )),
  • the methylated DNA in the region consisting of the base sequence shown in SEQ ID NO: 22 was amplified with PCR, and the resulting amplification product was 1.5% agarose gel It is the figure which showed the result of having electrophoresed.
  • the results are shown for a sample that has been “B” treated, a sample that has been “C” treated with DNA fragment Y2, and a sample that has been “D” treated with DNA fragment Y2.
  • FIG. 3 shows that the sample prepared in Example 3 was subjected to either “A (no treatment)” or “B (simultaneous treatment of Hpall and 3 ⁇ 4ial)” and represented by SEQ ID NO: 22.
  • FIG. 5 is a diagram 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.
  • sample 1 with DNA marker “M” and DNA fragment Y2 “A” treatment sample 2 with DNA fragment Y2 “A” treatment, DNA fragment Sample 1 with Y2 “B” treatment, Sample 2 with DNA fragment Y2 “B” treatment, Sample 1 with DNA fragment X2 “A” treatment, DN A fragment X2 Sample 2 with ⁇ A '' treatment, sample 1 with ⁇ B '' treatment of DNA fragment X2, sample 2 with ⁇ B '' treatment of DNA fragment X2 Yes.
  • Figure 4 shows the sample of DNA fragment X 2 prepared in Example 4, 69414
  • FIG. 5 is a diagram showing the results of 1.5% agarose gel electrophoresis of the amplification product obtained after amplification. From the leftmost lane in the figure, DNA sample “M”, OpgDN A fragment X2 / mLTE buffer solution sample “1”, 0.
  • Figure 5 shows a sample of the DN A fragment Y 2 prepared in Example 4.
  • Figure 6 shows the sample of DNA fragment X 2 prepared in Example 5.
  • FIG. 3 shows the results of 1.5% agarose gel electrophoresis of the obtained amplification product.
  • DNA marker "M” OpgDN A fragment 'A sample of X2 / mL rat serum solution "A” treated sample "1", lpgDNA fragment X2 / mL rat serum Sample ⁇ 2 '' treated with solution ⁇ A '', Sample ⁇ 3 '' treated with ⁇ A '' of lpgDNA fragment X2 / mL rat serum solution, ⁇ 1 '' of OpgDN A fragment X2 / mL rat serum solution A ” Sample "4", shows the results with.
  • FIG. 7 shows the arrangement of the sample of DN A fragment Y 2 prepared in Example 5 after either “A (no treatment)” or “B (simultaneous treatment of Hpall and / Hhal)”.
  • FIG. 5 is a view showing the results of 1.5% agarose gel electrophoresis of the amplification product obtained by amplifying methylated DNA in the region consisting of the base sequence shown by No. 22 by PCR.
  • FIG. 9 shows that the sample “(II)” prepared in Example 6 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 Group B, Group C, and Group D.
  • FIG. 10 shows that the sample “(III)” prepared in Example 6 was added to “A (no treatment)”, “B (Hpall treatment)”, “C (Hhal treatment)” or “D (Hpall and D3 ⁇ 4hal). (Simultaneous treatment of)) "and the base sequence shown in SEQ ID NO: 17
  • FIG. 5 shows the results of measuring the amount of methylated DNA in a region by real-time PCR.
  • 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 Group B, Group C, and Group D.
  • FIG. 11 shows that in Example 6, the prepared sample “(IV)” was added to “A (no treatment)”, “B (Hpal treatment)”, “C (Hhal treatment)” or “D (The amount of methylated DNA in the region consisting of the base sequence shown in SEQ ID NO: 17 was measured by real-time PCR. It is the figure which showed the result.
  • 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).
  • the theoretical value shows the predicted value (methylation ratio) for the B, C, and D groups.
  • biological specimen examples include, for example, a cell lysate, a tissue lysate (herein, tissue has a broad meaning including blood, lymph nodes, etc.), or in mammals, plasma.
  • tissue has a broad meaning including blood, lymph nodes, etc.
  • biological samples such as serum and lymph, body fluids such as body secretions (urine, milk, etc.), and genomic DNA obtained by extraction from these biological samples.
  • biological specimen examples include samples derived from microorganisms, viruses, etc.
  • genomic DNA in the measurement method of the present invention means genomic DNA such as microorganisms, viruses, etc. .
  • the book is used for periodic health checkups and simple tests. Use of the invention measuring method can be expected.
  • DNA may be extracted using a commercially available DNA extraction kit or the like.
  • G represents guanine
  • the base sequence may be referred to as “CpG.”) Limited to cytosine.
  • the site that is methylated in cytosine is at position 5.
  • cytosine in the cage chain “CpG” is methylated.
  • the cytosine in the nascent strand “CpG” is immediately activated by methyltransferase.
  • methyltransferase 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 a DNA produced by such methylation modification.
  • the “CpG pair” in the present invention means a double-stranded oligonucleotide formed by base pairing of a base sequence represented by CpG and a complementary C pG.
  • the “target DNA region” in the present invention (hereinafter sometimes referred to as the target region) is a DNA region to be examined for the presence or absence of methylation of cytosine contained in the region, and includes at least one kind of methyl region. It has a recognition site for a susceptibility restriction enzyme. For example, Lysyl oxidase ⁇ HRAS-like suppressor, bA305P22.2.
  • nucleotide sequence represented by SEQ ID NO: 1 the ATG codon encoding the amino terminal methionine of Lysyl oxidase protein derived from human is represented by nucleotide numbers 2031 to 2033, and the nucleotide sequence of exon 1 above is shown. Is shown in base numbers 1957 to 2661. Cytosine in the base sequence shown by C p G present in the base sequence shown in SEQ ID NO: 1, particularly C in the region where C p G is densely present in the base sequence shown in SEQ ID NO: 1. Cytosine in pG exhibits a high methylation frequency (ie, hypermethylation) in cancer cells such as gastric cancer cells.
  • a high methylation frequency ie, hypermethylation
  • the useful protein gene is the HRAS-1 ike suppressor gene.
  • a human-derived base sequence including 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 The base sequence of genomic DNA containing exon 1 of the HRAS-ike suppressor gene and its 5, promoter region located upstream can be raised. More specifically, the base sequence represented by SEQ ID NO: 2 (Corresponding to the base sequence represented by base numbers 172001 to 173953 of the base sequence described in Genbank Accession No. AC06 8162). In the base sequence represented by SEQ ID NO: 2, the base sequence of exon 1 of the human-derived HRAS-like suppressor gene is represented by base numbers 1743-1953.
  • the useful protein gene is a bA305P22.2.1 gene
  • the C p G present in the base sequence of the promoter region, untranslated region or translated region (coding region)
  • the base sequence of genomic DNA containing the exon 1 of human bA305P22.2.1 gene and the promoter region located upstream 5 '
  • 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 give.
  • 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.
  • the useful protein gene is a Gamma ⁇ lamin gene
  • the C p G present in the nucleotide sequence of the promoter region, untranslated region or translated region (coding region)
  • Examples of the base sequence containing one or more of the base sequences shown include the base sequence of genomic DNA containing exon 1 of the human-derived Gamma ⁇ lamin gene and the promoter region located 5 'upstream thereof.
  • the cytosine in the middle shows a high methylation frequency (that is, a hypermethylation state) in cancer cells such as gastric cancer cells.
  • cytosine having a high methylation frequency in gastric cancer cells for example, in the base sequence represented by SEQ ID NO: 4, base numbers 329, 333, 337, 350, 353, 360, 363, 370, 379 , 382, 384, 409, 414, 419, 426, 432, 434, 445, 449, 459, 472, 474, 486, 490, 503, 505 and the like.
  • the useful protein gene is a HAND1 gene, TJP2007 / 069414
  • the nucleotide sequence comprising at least one nucleotide sequence represented by is the nucleotide sequence of genomic DNA containing exon 1 of the human-derived Homologue of RIKEN 2210016F16 gene and the promoter region located 5 'upstream thereof. More specifically, the nucleotide sequence represented by SEQ ID NO: 6 (corresponding to the complementary nucleotide sequence of the nucleotide sequence represented by nucleotide numbers 157056 to 159000 of the nucleotide sequence described in Genbank Accession No. AL354733) ).
  • cytosine having high methylation frequency in gastric cancer cells for example, in the base sequence represented by SEQ ID NO: 6, base numbers 1172, 1 175, 1 180, 1183, 1 189, 1204, 1209 1267, 1271, 1278, 128 1, 1313, 1319, 1332, 1334, 1338, 1346, 1352, 1358, 1366, 1378, 1392, 1402, 1433, 1436, 1438 and the like.
  • the useful protein gene is FLJ32130 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 used.
  • nucleotide sequence containing one or more examples include the nucleotide sequence of genomic DNA containing exon 1 of the FLJ32130 gene derived from human and the promoter region located 5 'upstream.
  • the base sequence represented by SEQ ID NO: 7 (corresponding to the 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 encoding the amino acid terminal methionine of human FLJ 32130 protein is represented by base numbers 2136 to 2138, and the base sequence considered to be exon 1 is The base numbers 2136 to 2379 are shown.
  • the useful protein gene is a PPARG angiopoiet in-related protein gene
  • C present in the nucleotide sequence of the promoter region, untranslated region or translated region (coding region).
  • the base sequence containing at least one base sequence represented by pG is a genomic DNA base sequence containing exon 1 of human-derived ITARG angiopoiet in-related protein gene and its 5, promoter region located upstream. More specifically, the base sequence represented by SEQ ID NO: 8 can be mentioned.
  • the ATG codon encoding the amino acid methionine at the amino terminal of the human-derived PPARG ang iopoiet in-related protein protein is represented by nucleotide numbers 717 to 719.
  • the nucleotide sequence of the side portion is shown in nucleotide 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 having high methylation frequency in gastric cancer cells for example, in the base sequence represented by SEQ ID NO: 8, base numbers 35, 43, 51, 54, 75, 85, 107, 127, 129 143, 184, 194, 223, 227, 236, 251, 258 and the like. More specifically, for example, when the useful protein gene is a Thrombomodulin gene, the nucleotide sequence represented by C p G present in the nucleotide sequence of the promoter region, untranslated region or translated region (coding region).
  • Examples of the base sequence containing one or more of the above include the base sequence of genomic DNA containing exon 1 of a human-derived Thrombomodulin gene 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 encoding the amino terminal methionine of the human-derived Thrombomodulin protein is represented by base numbers 2590 to 2592.
  • the base sequence of exon 1 is The number 2048-6096 is shown.
  • cytosine with high methylation frequency in gastric cancer cells for example, in the base sequence represented by SEQ ID NO: 9, base numbers 1539, 1551, 1571, 1579, 1581, 1585, 1595, 1598, 1601, Examples include cytosine represented by 1621, 1632, 1638, 1645, 1648, 1665, 1667, 1680, 1698, 1710, 1724, 1726, 1756, and the like.
  • C p G present in the nucleotide sequence of the promoter region, untranslated region or translated region (coding region).
  • nucleotide sequence As a base sequence including one or more base sequences represented by (1), the base sequence of 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 of the base sequence described in Genbank Accession No. AC009471, base numbers 1 13501 to 116000, complementary to the base sequence Corresponding to the target sequence. In the nucleotide sequence represented by SEQ ID NO: 10, the nucleotide sequence of exon 1 of the human-derived p53-responsive gene 2 gene is represented by nucleotide numbers 1558 to 1808.
  • the cytosine in the base sequence represented by C p G present in the base sequence represented by SEQ ID NO: 10 has a high methylation frequency (ie, hypermethylation state (hyperme state) in cancer cells such as, for example, a premature cancer cell). thyl at i on)). More specifically, as cytosine having high methylation frequency in knee cancer cells, for example, in the base sequence represented by SEQ ID NO: 10, base numbers 1282, 1284, 1301, 1308, 1315, 1319, 1349, The cytosine shown by 1351, 1357, 1361, 1365, 1378, 1383 etc. can be mentioned.
  • the useful protein gene is a fibrillin gene
  • the promoter region, untranslated region, or translated region (coding region) salt thereof when the useful protein gene is a fibrillin gene, the promoter region, untranslated region, or translated region (coding region) salt thereof.
  • the base sequence containing one or more base sequences represented by CG in the base sequence includes a genome containing exon 1 of the human Fibrill in2 gene and a promoter region located 5 'upstream.
  • the base sequence of DNA can be mentioned. More specifically, the base sequence represented by SEQ ID NO: 11 (the base sequence represented by base numbers 118801 to 121000 of the base sequence described in Genbank Accession No. AC113387) It corresponds to the complementary sequence.
  • Examples of a base sequence containing one or more base sequences include a base sequence of genomic DNA containing exon 1 of a human-derived neurofilaments gene and a promoter region located upstream of 'the 5'. More specifically, the base sequence represented by SEQ ID NO: 12 (corresponding to the complementary sequence of the base sequence represented by base numbers 28001 to 30000 of the base sequence described in Genbank Accession No. AF106564) )). In the base sequence represented by SEQ ID NO: 12, the base sequence of exon 1 of human-derived eurofil amen 13 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, hypermethylat state in a cancer cell such as a spleen cancer cell). ion))). More specifically, as cytosine having a high methylation frequency in spleen 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 TJP2007 / 069414
  • nucleotide sequence including one or more nucleotide sequences include the nucleotide sequence of genomic DNA containing exon 1 of human-derived disintegrin and metalloproteinase domain 23 gene and a promoter region located 5 'upstream.
  • cytosine having high methylation frequency in knee cancer cells for example, in the base sequence represented by SEQ ID NO: 13, base numbers 998, 1003, 1007, 1011, 1016, 1018, 1020, 1026, 1028, 1031, 1035, 1041, 1043, 1045, 1051, 1053, 1056, 1060, 1066, 1068, 1070, 1073, 1 093, 1096, 1106, 1112, 1120, 1124, 1126, etc. I can do it.
  • the useful protein gene is a G protein-coupled receptor 7 gene, it exists in the base sequence of the promoter region, untranslated region or translated region (coding region).
  • the base sequence containing one or more base sequences represented by CpG is the base sequence of genomic DNA containing exon 1 of the human G protein-coupled receptor 7 gene and the promoter region located 5 'upstream thereof. More specifically, the nucleotide sequence represented by SEQ ID NO: 14 (Genbank This corresponds to the base sequence represented by base numbers 75001 to 78000 of the base sequence described in Accession No. AC009800. ). In the nucleotide sequence represented by SEQ ID NO: 14, the nucleotide sequence of exon 1 of the G protein-coupled receptor 7 gene derived from human is represented by nucleotide numbers 1666 to 2652.
  • Cytosine in the base sequence represented by CpG present in the base sequence represented by SEQ ID NO: 14 has a high methylation frequency (ie, hypermethylation state in cancer cells such as rod cancer cells). )) More specifically, cytosine having a high methylation frequency in spleen cancer cells, for example, in the base sequence represented by SEQ ID NO: 14, the base numbers 1480, 1482, 1485, 1496, 1513, 1526, 1542 1560, 1564, 1568, 1570, 1580, 1590, 1603, 1613, 1620, and the like.
  • AC026802 It corresponds to a complementary sequence of the base sequence shown.
  • the nucleotide sequence represented by SEQ ID NO: 16 the nucleotide sequence of exon 1 of the human-derived Solute carrier family 6 neurotran smitter 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.
  • a molecule having an active functional group such as an amino group, an aldehyde group, or a thiol group is covalently bonded to the 5 ′ end side of the oligonucleotide, and then the surface is activated with a silane force coupling agent or the like.
  • a silane force coupling agent or the like for example, a method of covalently bonding to a support made of glass, silica, or heat-resistant plastic via a spacer such as five triglycerides connected in series, a cross-linker, or the like.
  • a method of chemically synthesizing directly from the 5 ′ end side of the oligonucleotide on a glass or silicon support is also included.
  • the first step of the measurement method of the present invention is, for example, when the immobilized oligonucleotide is a pyotinylated oligonucleotide,
  • a pair of primers capable of amplifying DNA containing a recognition sequence containing cytosine to be analyzed is used.
  • PCR can be used to examine the presence or absence of DNA amplification (amplified product).
  • an amplification product is obtained.
  • the ratio of cytosine to be analyzed can be measured.
  • the third step of the measurement method of the present invention is, for example, the present immobilized oligonucleotide.
  • 30 When 30 is a piotinylated oligonucleotide, it may be carried out as follows.
  • the double-stranded DNA purified in the second step is mixed with 3 L, lmg / mL BSA aqueous solution of 10 X buffer (330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOraM Mg0Ac2, 5 mM Di thothrei tol).
  • the double-stranded DNA formed in the second step in this way is digested with one or more types of methylation-sensitive restriction enzymes, and then the resulting free digest (in the recognition site of the methylation-sensitive restriction enzyme). Remove and wash (DNA purification) double-stranded DNA (NA) containing at least one ammethyl CpG pair.
  • a “DNA sample derived from genomic DNA contained in a biological specimen” is a restriction enzyme that does not use the target DNA region in the genomic DNA as a recognition cleavage site.
  • a DNA sample is a DNA sample that has been digested in advance.
  • the single-stranded DNA thus produced is selected by base pairing with the single-stranded DNA (positive strand) produced and the single-stranded immobilized oligonucleotide (negative strand).
  • an oligonucleotide having a base sequence (normal strand) as an extension primer (reverse primer) and extending the oligonucleotide from the primer once double-stranded DNA containing the single-stranded DNA can be obtained.
  • the 5th-a step and the 5th-b step are repeated.
  • the amount of the amplified DNA is quantified.
  • it may be carried out according to the following description or the operation method in the fourth step and the fifth step of the measurement method of the present invention described above.
  • a method for amplifying a target DNA region (that is, a target region) after digestion with a methylation-sensitive restriction enzyme for example, PCR can be used.
  • PCR reaction solution for example, DNA obtained in the third step of the measurement method of the present invention, 0.15 1 of the primer solution, 2.5 1 of 2 mM dNTP, 10X buffer (lOOmM Tris-HCl H 8.3, Mix 500 mM KC1, 20 mM MgCi 2 , 0.01% Gelatin) 2.5 ⁇ 1, and AmpliTaq Gold (a type of heat-resistant DNA polymerase: 5 U / l) 0.2 1 with sterilized ultrapure water.
  • a reaction liquid with a volume of 25 1 can be raised.
  • the single-stranded oligonucleotide (negative strand) added to the reaction system here is a part of the 3 ′ end of the single-stranded DNA (however, it does not include the target DNA region).
  • “Generated negative-strand single-stranded DNA” in step 5-b means “production” in both the first step of the fifth step and the second and subsequent fifth steps. “Fixed” single-stranded DNA (positive strand) ”. However, if the fifth step has an additional 5-c step, the “generated” (fixed) single-stranded DNA (positive strand) in the first step of the fifth step operation (positive strand) On the other hand, in the repetitive operations of the 5th step from the second time onwards, “generated (fixed) single-stranded DNA (positive strand)” and “generated“ free ”single-stranded state It means both “DNA (positive strand)”.
  • the “degree of disease” can be determined based on the difference between the values.
  • the “degree of disease” here has the same meaning as that generally used in this field. Specifically, for example, when the biological specimen is a cell, the malignancy of the cell is determined. For example, when the biological specimen is a tissue, it means the abundance of disease cells in the tissue. Furthermore, when the biological specimen is plasma / serum, it means the probability that the individual has a disease. Therefore, the measurement method of the present invention or the methylation ratio measurement method of the present invention makes it possible to diagnose various diseases by examining methylation abnormality.
  • PR3 5'-CGATGAGCTTGCACATGAGCT-3 '(SEQ ID NO: 20)
  • each of the elongated double-stranded DNAs 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, lOOiM Mg0Ac2, 5 mM Dithothreitol) optimal for Hpa II and Hha I. ) 3 L, 10 XBSA (Bovine serum albumin lmg / ml) 3 nL, and sterilized ultrapure water was added to the mixture to a volume of 30 L.
  • 1 OX buffer 330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOiM Mg0Ac2, 5 mM Dithothreitol
  • 3 L 3 L
  • 10 XBSA Bovine serum albumin lmg / ml
  • DNA fragment X2 in the case of Group A (untreated group), amplification was confirmed and the amplification product was obtained. In contrast, in the case of Group B (HpaII, Hhal treatment group), amplification was not confirmed and the amplification product was not obtained. Meanwhile, DNA fragment Y 2
  • Group B (Digestion treatment group with Hpa II): Hp a 1 1 to 1511 and 10 X buffer (330 mM Tris-Acetate pH 7.9, optimal for Hpa II) 6 60 mM K0Ac, lOOmM Mg0Ac2, 5m Dithothreitol) 3 iL, 10XBSA (Bovin e serum albumin lmg / ml) was added to 3 / L, and sterilized ultrapure water was added to the mixture to make the volume 30 L. ,
  • Group A (untreated group): 1 OX buffer (330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM MgOAc2, 5 mM Dithothreitol) optimal for Hpa II and Hha I ) And 3 L of 10XBSA (Bovine serum albumin lmg / ml) were added, and sterilized ultrapure water was further added to the mixture to make a volume of 30 L (preparation of 2 each). '
  • the single-stranded DNA thus selected is added with 3 L of 10X buffer solution (lOOmM Tris-HCl pH 8.3, 500 mM KCK 15 mM MgCl 0.01% Gelatin), 3 L each of 2 mM dNTP aqueous solution, 5 N Betaine aqueous solution 6 L, thermostable DNA polymerase (AmpliTaq 69414
  • DNA fragment X2 in the case of group A (untreated group), amplification was confirmed in samples of l pg / 10 zL and 10 pg / 10, and amplification products were obtained.
  • Group A (untreated group): 1 OX buffer (330mM Tris-Acetate pH 7.9, 660m K0Ac, lOOmM Mg0Ac2, 5mM Dithothreitol suitable for Hpa II and Hha I, to the double-stranded DNA prepared above ) 3 zL, 10 XBSA (Bovine serum albumin lmg / ml) 3 L, and sterilized ultrapure water was added to the mixture to a volume of 30 L.
  • 1 OX buffer 330mM Tris-Acetate pH 7.9, 660m K0Ac, lOOmM Mg0Ac2, 5mM Dithothreitol suitable for Hpa II and Hha I, to the double-stranded DNA prepared above
  • 10 XBSA Bovine serum albumin lmg / ml
  • Group C (fflial treatment group): About 25 ng of DNA fragment, 0.5 U of Hh a I, and 10 X buffer solution suitable for Hpa II and Hha I (330 mM Tris-Acetate H 7.9, 660 mM K0Ac, 2 L of lOOmM MgOAc2, 5 mM Dithothrei tol) and 2 L of 10XBSA (Bovine seruni albumin lmg / ml) were added, and sterilized ultrapure water was added to the mixture to make a volume of 20 L.
  • 10XBSA Bovine seruni albumin lmg / ml
  • Group D (Hpall and fflial treatment group): About 25 ng of DNA fragment, 0.5 pa of H pa II and Hh a I, and 10 X buffer (330 mM Tris-Acetate optimal for Hp a II and Hha I) Add 2 x L of pH7.9, 660 mM K0Ac, lOOmM Mg0Ac2, 5iM Dithothreitol) and 2 L of 10XBSA (Bovine serum albumin lmg / ml), and add sterilized ultrapure water to the mixture. Was set to 20 ⁇ L. Each reaction solution was incubated at 37 ° C for 2 hours (digestion treatment), and then sterilized ultrapure water was added thereto to dilute 100 times.
  • XBSA Bovine serum albumin lmg / ml
  • Oligonucleotide probes designed for real-time PCR designed for real-time PCR

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Abstract

La présente invention a pour objet un procédé de détermination de la teneur en ADN méthylé dans une région d'ADN d'intérêt au sein de l'ADN génomique contenu dans un échantillon biologique ; et autres.
PCT/JP2007/069414 2006-09-27 2007-09-27 procÉdÉ de dÉtermination de la mÉthylation de l'adn WO2008038833A1 (fr)

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JP2006262132 2006-09-27
JP2006-262132 2006-09-27
JP2006347302A JP4924014B2 (ja) 2006-09-27 2006-12-25 Ｄｎａメチル化測定方法
JP2006-347302 2006-12-25

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008123537A1 (fr) * 2007-03-26 2008-10-16 Sumitomo Chemical Company, Limited Procédé de mesure de méthylation dans un adn
WO2008123538A1 (fr) * 2007-03-26 2008-10-16 Sumitomo Chemical Company, Limited Procédé de mesure de méthylation dans un adn
WO2009123367A1 (fr) * 2008-04-04 2009-10-08 住友化学株式会社 Procédé de détermination de la méthylation de l'adn
EP3942068A4 (fr) * 2019-03-18 2023-01-25 Nucleix Ltd. Procédés et systèmes de détection de changements de méthylation dans des échantillons d'adn

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5996513B2 (ja) * 2013-11-27 2016-09-21 京セラドキュメントソリューションズ株式会社 電子機器

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008123537A1 (fr) * 2007-03-26 2008-10-16 Sumitomo Chemical Company, Limited Procédé de mesure de méthylation dans un adn
WO2008123538A1 (fr) * 2007-03-26 2008-10-16 Sumitomo Chemical Company, Limited Procédé de mesure de méthylation dans un adn
WO2009123367A1 (fr) * 2008-04-04 2009-10-08 住友化学株式会社 Procédé de détermination de la méthylation de l'adn
JP2009247260A (ja) * 2008-04-04 2009-10-29 Sumitomo Chemical Co Ltd Ｄｎａメチル化測定方法
EP3942068A4 (fr) * 2019-03-18 2023-01-25 Nucleix Ltd. Procédés et systèmes de détection de changements de méthylation dans des échantillons d'adn

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