WO2008038833A1

WO2008038833A1 - Method for determination of dna methylation

Info

Publication number: WO2008038833A1
Application number: PCT/JP2007/069414
Authority: WO
Inventors: Yoshitaka Tomigahara; Hirokazu Tarui
Original assignee: Sumitomo Chemical Company, Limited
Priority date: 2006-09-27
Filing date: 2007-09-27
Publication date: 2008-04-03
Also published as: JP2008104443A; JP4924014B2

Abstract

Disclosed are: a method for determining the content of methylated DNA in a DNA region of interest in genomic DNA contained in a biological sample; and others.

Description

P T / JP2007 / 069414

1 Statement

DN A methylation measurement method Technical field

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. Background art

As 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 There is 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 ) In 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.

In addition, as 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

1. A method for measuring the content of methylated DNA in a target DNA region in genomic DNA contained in a biological specimen,

(1) From a DNA sample derived from genomic DNA contained in a biological sample, a single-stranded DNA containing a target DNA region and a part of the 3 ′ end of the single-stranded DNA 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,

(2) 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,

(3) The third step in which the double-stranded DNA formed in the second step is digested with one or more types of methylation-sensitive restriction enzymes, and then the free digest produced is removed, where the digest is A double-stranded DNA containing at least one CpG pair in the ammethyl state at the recognition site of the methylation-sensitive restriction enzyme,

(4) Undigested double-stranded DNA obtained in the third step (extended double-stranded DNA containing no CpG pair in the methylated state at the recognition site of the methylation-sensitive restriction enzyme) The fourth step of separating the single-stranded state, wherein the double-stranded DNA of the undigested product does not contain a CpG pair in the methylation-sensitive restriction enzyme recognition site of the methylation-sensitive restriction enzyme. Double-stranded DNA and

(Five)

(5— a) (i) The generated single-stranded DNA and the single-stranded immobilized oligonucleotide (negative strand) are base-paired to select the single-stranded DNA, ii) Using the selected single-stranded DNA as a saddle, and using the single-stranded immobilized oligonucleotide as a primer, the oligonucleotide is once extended from the primer to form double-stranded DNA. 5—a step,

(5-b) The negative-strand single-stranded DNA that has been generated is And a partial base sequence that is further 3 'from the 3' end of the base sequence that is complementary to the base sequence of the target DNA region. And a 5-b step of forming a double-stranded DNA by extending the oligonucleotide from the primer once by using an oligonucleotide having a complementary base sequence as a primer,

Furthermore, after separating the double-stranded DNA formed in each of the 5-a step and 5-b step into a single-stranded state, by repeating the 5-a step and 5-b step, A fifth step of amplifying the methylated DNA in the target DNA region to a detectable amount and quantifying the amount of amplified DNA;

(Hereinafter, it may be described as the measurement method of the present invention.);

2. In the first step, 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 method according to item 1 above;

3. The method according to item 2 above, wherein the divalent cation is a magnesium ion.

4. Before the fourth step described in the preceding paragraph 1,

A part of the 3 ′ end of a positive single-stranded DNA containing the target DNA region, but not including the target DNA region, and a complementary nucleotide sequence A step of adding a single-stranded oligonucleotide having a free state into the reaction system, and

A method characterized by further comprising the following one step in addition to the fifth-a step and the fifth-b step in the fifth step described in the preceding paragraph 1,

(5-c) (i) The generated single-stranded DNA is base-paired with the added free single-stranded oligonucleotide to select the single-stranded DNA.

(ii) The selected single-stranded DNA is in a saddle shape, and the single-stranded oligonucleotide is As a primer, the fifth _c step of forming a double-stranded DNA by extending the oligonucleotide once from the primer;

5. After the fourth step described in the preceding paragraph 1,

A part of the 3 ′ end of a single-stranded positive-stranded DNA containing the target DNA region, but does not contain the target DNA region, and has a base sequence that is complementary to And a step of adding a single-stranded oligonucleotide in a free state into the reaction system, and

Double-stranded DNA that is an undigested product obtained through the third step and the additional step described above, wherein the double-stranded DNA does not contain the CpG pair in the methylated state at the recognition site of the methylation-sensitive restriction enzyme. A step of separating the double-stranded DNA into a single-stranded state, and

A method characterized by further comprising the following one step in addition to the fifth-a step and the fifth-b step in the fifth step described in the preceding paragraph 1.

(ii) Using the selected single-stranded DNA as a saddle, using the single-stranded oligonucleotide as a primer and extending the oligonucleotide once from the primer, a double-stranded DNA is formed. c step;

6. The method according to any one of 1 to 5 above, further comprising the following two steps: a method for measuring a methylation ratio (hereinafter referred to as the methylation ratio measurement method of the present invention). );

(6) After performing the first step and the second step of the method according to any one of 1 to 5 above, 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) to a detectable amount and quantifying the amount of amplified DNA; and

(7) Based on the difference obtained by comparing the amount of DNA quantified in the fifth step with the amount of DNA quantified in the sixth step, the above-mentioned target DNA region A seventh step of calculating the percentage of methylated DNA in

7. The method according to any one of items 1 to 6 above, wherein the biological specimen is mammalian serum or plasma;

8. The method according to any one of items 1 to 6 above, wherein the biological specimen is mammalian blood or body fluid;

9. The method according to any one of items 1 to 6 above, wherein the biological specimen is a cell lysate or a tissue lysate;

10. 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. The method according to any one of 1 to 9 above

11. Any one of items 1 to 10 above, wherein the DNA sample derived from genomic DNA contained in the biological sample is a DNA sample digested with at least one methylation-sensitive restriction enzyme. The method described in;

12. The method according to any one of 1 to 11 above, wherein the DNA sample derived from genomic DNA contained in the biological sample is a DNA sample purified in advance; 13. One or more types of methylation 13. The method according to any one of 1 to 12 above, wherein the sensitive restriction enzyme is a restriction enzyme having a recognition cleavage site in a target DNA region in genomic DNA contained in a biological specimen; and,

14. The method according to any one of 1 to 13 above, wherein the one or more methylation-sensitive restriction enzymes are Hpa II or Hhal, which are methylation-sensitive restriction enzymes; . Brief Description of Drawings

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. In the figure From leftmost lane, 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 ¾ial). )), 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. From the leftmost lane in the figure, DNA sample “M”, DNA fragment X 2 “A” treated sample, DNA fragment X 2 “B” treated sample, Sample with DNA fragment X2 “C” treatment, sample with DNA fragment X2 “D” treatment, sample with DN A fragment Y2 “A” treatment, DN A fragment Y2 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 ¾ial)” 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. From leftmost lane in the figure, 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

7

After performing either “A (no treatment)” or “B (simultaneous treatment of Hpall and ¾ial)”, the methylated DNA in the region consisting of the base sequence shown in SEQ ID NO: 22 is converted into a PCR. 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. lpgDNA fragment X2 / mLTE buffer solution Sample `` A '' treated with `` A '' of `` A '' treatment of lpgDNA fragment X2 / mLTE buffer solution `` A '' treatment `` 3 '', 1 `` A '' treatment of OpgDNA fragment X2 / mLTE buffer solution The result of sample “4” with, is shown.

Figure 5 shows a sample of the DN A fragment Y 2 prepared in Example 4.

Methylated DN A in the region consisting of the base sequence shown in SEQ ID NO: 22 after either “A (no treatment)” or “; B (simultaneous treatment of Hpall and ¾ ^ 1)” FIG. 2 is a diagram showing the results of 1.5% agarose gel electrophoresis of the amplification product obtained by PCR amplification. From the leftmost lane in the figure, DNA marker “M”, OpgDN A fragment Y2 / mLTE buffer “A” treated sample “1”, lpgDNA fragment Y2 / mLTE buffer “A” Sample "2" treated with "A" treatment of lpgDNA fragment Y2 / mLTE buffer solution "3", Sample "A" treatment of 1 OpgDNA fragment Y2 / mLTE buffer solution Sample "4", shows the results with.

Figure 6 shows the sample of DNA fragment X 2 prepared in Example 5.

Perform either "A (no treatment)" or "B (simultaneous treatment of Hpall and Hhal)" and amplify methylated DNA in the region consisting of the base sequence shown in SEQ ID NO: 22 by PCR FIG. 3 shows the results of 1.5% agarose gel electrophoresis of the obtained amplification product. From the leftmost lane in the figure, 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. From the leftmost lane in the figure, DNA marker “M”, OpgDN A fragment Y 2 / mL sample treated with “A” of rat serum solution “1”, 0.1 μpgDN A fragment Y 2 / Sample `` A '' treated with mL rat serum solution `` 2 '', lpgDN A fragment Y 2 / mL Sample `` A '' treated with rat serum solution `` 3 '', 1 OpgDN A fragment Y 2 / Results are shown in sample “4”, which was treated with “A” of mL rat serum solution.

FIG. 8 shows that the sample “(I)” prepared in Example 6 was added to “A (no treatment)”, “B (Hpall treatment)”, “C (Hhal treatment)” or “D (Hpall and hal). Is a diagram showing 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 PCR. 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 is the calculated value (methylation ratio) expected for the B, C, and D groups.

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 ¾hal). Is a diagram showing the result of measuring the amount of methylated DNA in the region consisting of the base sequence shown in SEQ ID NO: 17 by real-time PCR after performing any of the treatments of “. 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 D¾hal). (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.

Fig. 1 2 shows that in Example 6, the prepared sample "(V)" was replaced with "A (no treatment)", "B (Hpal treatment)", "C (Hhal treatment)" or "D (Hpal treatment)". Figure 1 shows the result of real-time PCR measurement of the amount of methylated DNA in the region consisting of the nucleotide sequence shown in SEQ ID NO: 17 It is. 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. BEST MODE FOR CARRYING OUT THE INVENTION

Examples of the “biological specimen” in the present invention include, for example, a cell lysate, a tissue lysate (herein, tissue has a broad meaning including blood, lymph nodes, etc.), or in mammals, plasma. Examples thereof include 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. Examples of the biological specimen include samples derived from microorganisms, viruses, etc. In this case, “genomic DNA” in the measurement method of the present invention means genomic DNA such as microorganisms, viruses, etc. .

If the mammal-derived specimen is blood, the book is used for periodic health checkups and simple tests. Use of the invention measuring method can be expected.

In order to obtain genomic DNA from a sample derived from a mammal, for example, DNA may be extracted using a commercially available DNA extraction kit or the like.

When blood is used as a specimen, plasma or serum is prepared from blood according to a normal method, and the prepared plasma or serum is used as a specimen, and free DNA contained therein (derived from cancer cells such as stomach cancer cells) Analysis of cancer cells such as gastric cancer cells, avoiding blood cell-derived DNA, and detecting cancer cells such as gastric cancer cells and tissues containing them Sensitivity can be improved. Normally, there are four types of bases that make up a gene (genomic DNA). Among these bases, the phenomenon that only cytosine is methylated is known. Such a methylation modification of DNA is based on the base sequence represented by 5 '— CG—3, where C represents cytosine. , G represents guanine Hereinafter, the base sequence may be referred to as “CpG.”) Limited to cytosine. The site that is methylated in cytosine is at position 5. At the time of DNA replication prior to cell division, immediately after replication, only cytosine in the cage chain “CpG” is methylated. However, the cytosine in the nascent strand “CpG” is immediately activated by methyltransferase. Are 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. K Gamma filamin, HANDK Horaologue of RIKEN 2210016F16, FLJ32130, PPARG angiopoiet in-related protein , Thrombomodul in, p53-responsive gene 2, Fibr ill in2, Neurof i laments, dis int egrin and metal loproteinase domain 23, G protein-coupled receptor 7, G-protein coupled somatostat in and angiotens in-l ike pept ide Represented by C p G present in the base sequence of the promoter region, untranslated region or translated region (coding region) of useful protein genes such as receptor, Solute carr ier family 6 neurotransmiter transporter noradrenal in member 2 Examples include DNA regions containing one or more cytosines in the base sequence. Specifically, for example, when the useful protein gene is a Lysyl oxidase gene, the base sequence indicated 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 include the base sequence of genomic DNA containing exon 1 of the Lysyl oxidase gene derived from human and the promoter region located 5 'upstream thereof. More specifically, And the 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). In the 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. More specifically, as the cytosine with high methylation frequency in gastric cancer cells, for example, in the base sequence represented by SEQ ID NO: 1, base numbers 1539, 1560, 1574, 1600, 1623, 1635, 1644, 1654, 1661 1682, 1686, 1696, 1717, 1767, 1774, 1783, 1785, 1787, 1795 and the like. Specifically, for example, the useful protein gene is the HRAS-1 ike suppressor gene. In some cases, 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. Cytosine in the base sequence shown by C p G present in the base sequence shown in SEQ ID NO: 2, especially C p G present in a region where C p G is densely present in the base sequence shown in SEQ ID NO: 2 The cytosine in the middle shows a high methylation frequency (that is, a 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: 2, base numbers 1316, 1341, 1357, 1359, 1362, 1374, 1390, 1399, 1405, 1409, 1414, 1416, 1422, 1428, 1434, 1449, 1451, 1454, 1463, 1469, 1477, 1479, 1483, 1488, 1492, 1494, 1496, 1498, 1504, 1510, 151 3, 1518, 1520 The cytosine shown by etc. can be mention | raise | lifted. More specifically, for example, when 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) As a base sequence containing one or more of the base sequences shown, the base sequence of genomic DNA containing the exon 1 of human bA305P22.2.1 gene and the promoter region located upstream 5 ' 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 give. In the base sequence shown in SEQ ID NO: 3, the ATG codon encoding the amino terminal methionine of human-derived bA305 2.2.1 protein is shown in base numbers 849 to 851, and the base of exon 1 above is shown. The sequence is shown in base numbers 663-889. Sequence number Cytosine in the base sequence shown by C p G present in the base sequence shown by No. 3, especially in C p G present in the region where CG is densely present in the base sequence shown by SEQ ID NO: 3. This cytosine 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: 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 Π 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 Π lamin gene and the promoter region located 5 'upstream thereof. More specifically, the base sequence represented by SEQ ID NO: 4 (corresponding to the complementary sequence of the base sequence represented by base numbers 63528 to 64390 of the base sequence described in Genbank Accession No. AC074373) ) In the base sequence represented by SEQ ID NO: 4, the ATG codon encoding the methionine at the amino terminal of human-derived gamma fil amin protein is represented by base numbers 572 to 574, and the base sequence of exon 1 is base It is shown in numbers 463-863. Cytosine in the base sequence shown by C p G present in the base sequence shown in SEQ ID NO: 4, especially C p G present in a region where C p G is densely present in the base sequence shown in SEQ ID NO: 4 The cytosine in the middle shows a high methylation frequency (that is, a 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, 379 , 382, 384, 409, 414, 419, 426, 432, 434, 445, 449, 459, 472, 474, 486, 490, 503, 505 and the like. Specifically, for example, when the useful protein gene is a HAND1 gene, TJP2007 / 069414

14 The base sequence containing one or more base sequences represented by C p G present in the base sequence of the promoter region, untranslated region or translation region (coding region) is exon 1 of the HAND1 gene derived from humans. The nucleotide sequence of the genomic DNA containing the 5 'upstream promoter region can be mentioned. More specifically, the nucleotide sequence represented by SEQ ID NO: 5 (described in Genbank Accession NO.AC026688) This corresponds to a complementary sequence of the base sequence represented by base numbers 24303 to 26500 of the base sequence. In the base sequence shown in SEQ ID NO: 5, the ATG codon encoding the amino terminal methionine of the HAND1 protein derived from human is shown in base numbers 1656 to 1658, and the base sequence of the above exon 1 is base The number 1400-2198 is shown. Cytosine in the base sequence shown by CpG present in the base sequence shown by SEQ ID NO: 5, especially in CpG present in the region where CG is densely present in the base sequence shown by SEQ ID NO: 5 For example, cytosine exhibits a high methylation frequency (ie, hypermethylation) in cancer cells such as gastric cancer cells. More specifically, as cytosine having high methylation frequency in gastric cancer cells, for example, in the base sequence represented by SEQ ID NO: 5, base numbers 1153, 1160, 1178, 1187, 1193, 1218, 1232, 1266, Examples include cytosines represented by 1272, 1292, 1305, 1307, 1316, 1356, 1377, 1399, 1401, 1422, 1434 and the like. More specifically, for example, when the useful protein gene is the Homologue of RIKEN 2210016F 16 gene, C p G present in the nucleotide sequence of the promoter region, untranslated region or translated region (coding region). 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) ). In the 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. Base shown in SEQ ID NO: 6 2007/069414

15 Cytosine in the base sequence represented by C p G present in the sequence, especially 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, For example, it shows 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: 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. Specifically, for example, when 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. Examples of the nucleotide sequence containing one or more include the nucleotide sequence of genomic DNA containing exon 1 of the FLJ32130 gene derived from human and the promoter region located 5 'upstream. Specifically, 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. In the base sequence represented by SEQ ID NO: 7, 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. A scissin in the base sequence shown by C p G present in the base sequence shown in SEQ ID NO: 7, particularly in a region where C p G is densely present in the base sequence shown in SEQ ID NO: 7. Cytosine in CpG exhibits a high methylation frequency (ie, hypermethylation state) in cancer cells such as gastric cancer cells. More specifically, as cytosine with high methylation frequency in gastric cancer cells, for example, in the base sequence represented by SEQ ID NO: 7, the base numbers 1714, 1716, 1749, 1753, 1762, 1 795, 1814, 1894, The cytosine shown in 191, 1915, 1925, 1940, 1955, 1968, etc. can be raised. PT / JP2007 / 069414

16 More specifically, for example, when 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. In the nucleotide sequence represented by SEQ ID NO: 8, 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. Cytosine in the base sequence shown by C p G present in the base sequence shown by SEQ ID NO: 8, especially C p G present in the region where C p G is densely present in the base sequence shown by SEQ ID NO: 8 The cytosine in it shows 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: 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. Specifically, 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. In the base sequence represented by SEQ ID NO: 9, 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. Arrangement Cytosine in the base sequence shown by CG present in the base sequence shown in column number 9, especially in C p G in the region where C p G is densely present in the base sequence shown in SEQ ID NO: 9 Cytosine exhibits a high methylation frequency (ie, hypermethylation state) in cancer cells such as gastric cancer cells. More specifically, as cytosine with high methylation frequency in gastric cancer cells, for example, in the base sequence represented by SEQ ID NO: 9, base numbers 1539, 1551, 1571, 1579, 1581, 1585, 1595, 1598, 1601, Examples include cytosine represented by 1621, 1632, 1638, 1645, 1648, 1665, 1667, 1680, 1698, 1710, 1724, 1726, 1756, and the like. In addition, for example, when the useful protein gene is p53-responsive gene 2 gene, C p G present in the nucleotide sequence of the promoter region, untranslated region or translated region (coding region). 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. More specifically, for example, 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. In the nucleotide sequence represented by SEQ ID NO: 11, the nucleotide sequence of exon 1 of the human Fibrill in2 gene is represented by nucleotide numbers 1091 to 1345. The cytosine in the base sequence represented by C p G present in the base sequence represented by SEQ ID NO: 1 1 has a high methylation frequency (that is, a hypermethylated state in cancer cells such as a spleen cancer cell). (Hypermethyl at ion)). More specifically, cytosine having a high methylation frequency in spleen cancer cells, for example, in the base sequence represented by SEQ ID NO: 11, the base numbers 679, 687, 690, 699, 746, 773, 777, The cytosine shown by 783, 795, 799, 812, 823, 830, 834, 843 etc. can be mentioned. Also, specifically, for example, when the useful protein gene is a Neurofilament 3 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 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

19

514, 519, 532, 541, 544, 546, 563, 566, 572, 580, etc. can be raised. More specifically, for example, when the useful protein gene is disintegrin and metalloprotease domain 23 gene, it is indicated by CpG present in the base sequence of the promoter region, untranslated region or translated region (coding region). Examples of the 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. More specifically, the base sequence represented by SEQ ID NO: 13 (corresponding to the base sequence represented by base numbers 21001 to 23300 of the base sequence described in Genbank Accession No. AC009225) can be mentioned. In the base sequence represented by SEQ ID NO: 13, the base sequence of exon 1 of the human-derived disintegrin and metalloproteinase domain 23 gene is represented by base numbers 1194 to 1630. The cytosine in the nucleotide sequence represented by CpG present in the nucleotide sequence represented by SEQ ID NO: 13 has a high methylation frequency (ie, hypermethylation) in cancer cells such as spleen cancer cells. ). More specifically, as 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. In addition, for example, when 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. Specifically, for example, when the useful protein gene is a G-protein coupled somatostatin and angiotensin-like peptide receptor gene, its bromo-region, its promoter region, untranslated region or translated region (coding region) The base sequence containing one or more base sequences represented by C p G present in the base sequence of G-protein coupled somatostatin and angiotensin-1 ike peptide rec eptor gene exon 1 of human origin, and 5 'The base sequence of the genomic DNA containing the promo region located upstream can be raised, more specifically, the base sequence represented by SEQ ID NO: 15 (described in Genbank Accession No. AC008971 This corresponds to a complementary sequence of the base sequence represented by base numbers 57001 to 60000 of the base sequence. In the nucleotide sequence represented by SEQ ID NO: 15, the nucleotide sequence of exon 1 of human-derived G-protein coupled somatostatin and angiotensin-like peptide receptor gene is represented by nucleotide numbers 776 to 2632. The cytosine in the nucleotide sequence represented by CpG present in the nucleotide sequence represented by SEQ ID NO: 15 has a high methylation frequency (ie, hypermethylation state) in cancer cells such as, for example, premature cancer cells. Indicates. More specifically, as cytosine having a high methylation frequency in the premature cancer cell, for example, in the base sequence represented by SEQ ID NO: 15, the base numbers 470, 472, 490, 497, 504, 506, 509, 514 522, 540, 543, 552, 566, 582, 597, 610, 612 etc. You can give Shin. Also, specifically, for example, when the useful protein gene is Solute carrier family 6 neurotransmitter transporter noradrenalin member 2 gene, in the nucleotide sequence of its mouth motor region, untranslated region or translated region (coding region) The base sequence containing one or more base sequences represented by CpG present in human is derived from the human-derived carrier family 6 neurotransmitter transporter noradrenalin member 2 exon 1 and a promoter located 5 ′ upstream. More specifically, the nucleotide sequence of the genomic DNA containing the nucleotide region can be raised, more specifically, the nucleotide sequence represented by SEQ ID NO: 16 (base numbers 78801 to 81000 of the nucleotide sequence described in Genbank Accession No. AC026802) It corresponds to a complementary sequence of the base sequence shown. In 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. More specifically, as cytosine with 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, 1 051, 1056, 1061, Examples include cytosine represented by 1063, 1080, 1099, 1110, 1139, 1141, 1164, 1169, 1184 and the like. In the present invention, “(amplified to a detectable amount of methylated DNA in the target DNA region) and the amount of amplified DNA” means the purpose in the genomic DNA contained in the biological sample. It means the amount of methylated DNA in the region after amplification itself, that is, the amount obtained in the fifth step of the measurement method of the present invention. For example, 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. 2007/069414

22 “Methylation ratio” in the present invention (particularly the method for measuring methylation ratio of the present invention) means that after amplification of methylated DNA in the target DNA region in genomic DNA contained in biological samples. This is the sum of the amount of DNA and the amount after amplification of unmethylated DNA, and the value after dividing the amount after amplification of methylated DNA. In the present invention, the “double-stranded DNA containing at least one ammethyl-state CpG pair at the recognition site of the methylation-sensitive restriction enzyme” is present in the recognition site of the restriction enzyme in double-stranded DNA. Double-stranded DNA in which both cytosines in at least one C p G pair are unmethylated cytosine, i.e. at least 1 present in the restriction enzyme recognition site in double-stranded DNA. In one CpG pair, it means a double-stranded DNA which is a cytosine in which both the positive-strand DNA cytosine and the corresponding negative-strand DNA cytosine are not methylated. In addition, “a double-stranded DNA that does not contain a CpG pair in the methylated state at the recognition site of the methylation-sensitive restriction enzyme” is present in the recognition site of the restriction enzyme in double-stranded DNA. Double-stranded DNA in which one of cytosines in all CpG pairs is methylated and the other is unmethylated cytosine, that is, in the restriction enzyme recognition site in double-stranded DNA. In all existing CpG pairs, either the positive DNA cytosine or the corresponding negative DNA cytosine is methylated, and the other is unmethylated cytosine. Means double-stranded DNA. In 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 positive single-stranded DNA containing the target DNA region and the 3 ′ end of the single-stranded DNA (However, the DNA region of interest is not included.) A single-strand immobilized oligonucleotide having a base sequence complementary to the above-mentioned base region is used for base pairing. Select double-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 the single-stranded DNA (positive strand) containing the intended DNA region (provided that the above-mentioned Eye P2007 / 069414

23 Does not include the target DNA region. ) A single-stranded immobilized oligonucleotide having a base sequence that is complementary to (hereinafter also referred to as the present immobilized oligonucleotide). That is, this immobilized oligonucleotide is a partial base sequence of a single-stranded DNA of a positive strand containing the target DNA region and further 3 ′ from the 3 ′ end of the target DNA region. It has a complementary base sequence. Here, as the “base sequence further 3 ′ from the 3 ′ end of the target DNA region”, 1 to 2 K bases from the 3 ′ end of the target DNA region, preferably, 1 base to: LK base, more preferably 1 base to 200 bases, more preferably 1 base to 100 bases, and can be selected from the 3 ′ base sequence.

This immobilized oligonucleotide is used to select positive single-stranded DNA containing a target DNA region from a DNA sample derived from genomic DNA contained in a biological specimen. The immobilized oligonucleotide preferably has a length of 5 to 50 bases.

The 5 ′ terminal side of the present immobilized oligonucleotide can be immobilized with a carrier, while the 3 ′ terminal side thereof proceeds in the 5 ′ to 3 ′ direction in the second and fifth steps described later. What is necessary is just to be in a free state so that one-time extension reaction is possible. Here, “what can be immobilized on a carrier” means that the present immobilized oligonucleotide is immobilized on a carrier when a single-stranded DNA (positive strand) containing the target DNA region is selected. (1) The single-stranded DNA (positive strand) and the immobilized oligonucleotide are immobilized by the binding of the immobilized oligonucleotide and the carrier at the stage before base pairing. (2) The single-stranded DNA (positive strand) and the immobilized oligonucleotide are base-paired and then immobilized by binding of the immobilized oligonucleotide and the carrier. May be.

In order to obtain such a structure, it is complementary to a part of the 3 ′ end of a single-stranded DNA (positive strand) containing the target DNA region (excluding the target DNA region). If the 5 ′ end of an oligonucleotide having a base sequence (hereinafter sometimes referred to as the present oligonucleotide) is fixed to a carrier according to an ordinary genetic engineering operation method or a commercially available kit / device, etc. Good (binding to solid phase). Specifically, for example, TJP2007 / 069414

24 Piotylated 5 'end of ligonucleotide, and then fixed to the support (eg, PCR tube coated with streptavidin, magnetic beads coated with streptavidin, etc.) with the resulting piotinated oligonucleotide coated with streptavidin Can be mentioned.

In addition, 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. 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. Furthermore, a method of chemically synthesizing directly from the 5 ′ end side of the oligonucleotide on a glass or silicon support is also included. Specifically, the first step of the measurement method of the present invention is, for example, when the immobilized oligonucleotide is a pyotinylated oligonucleotide,

(a) First, a DNA sample derived from a genomic DNA contained in a biological specimen is added to a annealing buffer and a biotinylated oligonucleotide (the base of the single-stranded DNA (positive strand) and the immobilized oligonucleotide). After the pairing, the mixture is obtained by adding oligonucleotides which are immobilized by binding of the present immobilized oligonucleotide and carrier, so that oligonucleotides in a free state at this stage are added. The resulting mixture is then used for several minutes at 95 ° C in order to make the double-stranded DNA containing the desired DNA region in the DNA sample derived from genomic DNA contained in the biological specimen into a single strand. Heat. After that, in order to form a double strand of single-stranded DNA containing the target DNA region and piotinylated oligonucleotide, it is promptly brought to a temperature about 10-20 ° C lower than the Tm value of the piotinylated oligonucleotide. Cool and keep at that temperature for several minutes.

(b) Then return to room temperature.

(c) Add the mixture obtained in (b) above to the support coated with streptavidin, and then incubate the pyotinylated oligonucleotide with streptavidin by incubating it at 37 ° C for several minutes. Secure to the support. As described above, in the above (a) to (c), the base pairing between the single-stranded DNA (positive strand) containing the target DNA region and the biotinylated oligonucleotide is converted into a biotinylated oligonucleotide. Is carried out at a stage prior to the fixation with the support coated with streptavidin, either of which may be in this order. That is, for example, by adding a DNA sample derived from a 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. In order to make double-stranded DNA containing the target DNA region present in DNA samples derived from genomic DNA contained in biological samples into single strands 9 5. Heat at C for a few minutes to form a double strand of a single-stranded DNA containing the desired DNA region and then a piotinylated oligonucleotide, approximately 10 to 2 of the Tm value of the piotinylated oligonucleotide. It may be cooled quickly to a temperature as low as 0 ° C and kept at that temperature for several minutes.

(d) After fixing the pyotinylated oligonucleotide to the support coated with streptavidin in this way, the remaining solution is removed and washed (DNA purification).

More specifically, for example, when using a PCR tube coated with streptavidin, first remove the solution by pipetting or decantation, and then add TE to the volume of the biological sample. Add the buffer, and then remove the TE buffer by pipetting or decanting. Also, when using magnetic beads coated with streptavidin, after fixing the beads with a magnet, first remove the solution by pipetting or decantation, and then add TE that is approximately equal in volume to the biological specimen. Add buffer and then remove the TE buffer by pipetting or decanting.

Subsequently, the remaining solution is removed and washed (DNA purification) by performing such an operation several times.

This operation is important in order to remove DNA NA that has not been immobilized, or DNA NA that has been suspended in a solution digested with the restriction enzymes described below, from the reaction solution. If these operations are inadequate, DNA floating in the reaction solution will be in a bowl shape, and an unexpected amplification product will be obtained in the amplification reaction. Non-specialty between support and DNA in biological samples P2007 / 069414

26 To avoid extraneous binding, add a large amount of DNA having a completely different base sequence from the target region (for example, rat DNA in the case of human biological specimens) to the biological specimen, and add May be performed.

As a preferred embodiment in the first step of the measurement method of the present invention, a single-stranded DN A (normal strand) containing the target DN A region and a part of the 3 ′ end of the single-stranded DN A (provided that the above-mentioned In the reaction system containing a divalent cation when base-pairing with a single-stranded immobilized oligonucleotide having a base sequence complementary to Base pairing can be mentioned. More preferably, the divalent cation is a magnesium ion. Here, the “reaction system containing a divalent cation” refers to an annealing buffer used for base pairing the single-stranded DNA (positive strand) and the single-stranded immobilized oligonucleotide. In particular, for example, a salt containing magnesium ions (eg, MgOAc ₂ , MgCl _2, etc.) at a concentration of lmM to 60 OmM is included. It is good to be. In the second step of the measurement method of the present invention, the single-stranded DNA selected in the first step is used as a cage, the single-stranded immobilized oligonucleotide is used as a primer, and the oligonucleotide is extended once from the primer. To form a double-stranded DNA containing the single-stranded DNA.

The single-stranded DNA selected in the first step is a base complementary to a part of the 3 ′ end of the single-stranded DNA (but not including the target DNA region). By base-pairing with a single-stranded immobilized oligonucleotide having a sequence, only the paired portion has already formed a double strand. In contrast, the parts other than the mating part are in a single-stranded state. However, many normal methylation-sensitive restriction enzymes use double-stranded DNA as a substrate in their digestion reaction, but do not use single-stranded DNA as a substrate. Therefore, before the digestion treatment with the methylation-sensitive restriction enzyme in the third step of the measurement method of the present invention, double-stranded DNA containing the single-stranded DNA is formed.

In the second step of the measurement method of the present invention, the single-stranded DNA selected in the first step is 2007/069414

27. In order to extend the primer once using the single-stranded immobilized oligonucleotide as a primer, an extension reaction may be performed using DNA polymerase. Specifically, the second step of the measurement method of the present invention may be carried out as follows, for example, when the present immobilized oligonucleotide is a pyotinylated oligonucleotide.

To the single-stranded DNA containing the target DNA region selected in the first step, sterilized ultrapure water (17.85 L), optimal 10 X buffer (lOOmM Tris-HCl pH 8.3, 500 mM KC1, 15 mM MgCl ₂ ) 3L, 2mM dNTP 3L, 5N betaine, then add 0.15 AmpliTaq (1 type of DNA polymerase: 5U /// L) to the mixture to bring the volume to 30 iL. Incubate at ° 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 remove the TE buffer by pipetting or decanting. Good.

More specifically, for example, when using a PCR tube coated with streptavidin, first remove the solution by pipetting or decantation, and then add a TE buffer approximately equal to the volume of the biological sample. And then remove the TE buffer by pipetting or decanting. In addition, when using magnetic beads coated with streptavidin, after fixing the beads with a magnet, first remove the solution by pipetting or decantation, and then add TE that is approximately equal to the volume of the biological specimen. Add a buffer and then remove the TE buffer by pipetting or decanting.

Then, by performing such an operation several times, the remaining solution is removed and washed, and then purified (DNA purification). In the third step of the measurement method of the present invention, the double-stranded DNA formed in the second step is digested with one or more types of methylation-sensitive restriction enzymes, and then generated free digest (the above-mentioned methylation sensitivity). Cp G in at least one ammethyl state at the restriction enzyme recognition site JP2007 / 069414

Remove double-stranded DN A) containing 28 pairs.

The “methylation-sensitive restriction enzyme” in the third step of the measurement method of the present invention means that, for example, 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 the recognition sequence that can be originally recognized by the methylation-sensitive restriction enzyme is methylated, the methylation-sensitive restriction enzyme can be allowed to act on the DNA. The DNA is not cleaved. In contrast, when cytosine contained in a recognition sequence that can be originally recognized by a methylation-sensitive restriction enzyme is not methylated, the methylation-sensitive restriction enzyme acts on the DNA. If so, the DN A will be disconnected. Specific examples of such methylation-sensitive enzymes include HpaII, BstUI, Narl, SacII, Hhal and the like. The methylation-sensitive restriction enzyme is a double-stranded DNA containing a CpG pair in the hemimethyl state (that is, the cytosine of one strand of the CpG pair is methylated, and the other strand It does not cleave double-stranded DNA such that cytosine is not methylated, and has already been clarified by Gruenbaum et al. (Nucleic Acid Research, 9, 2509-2515

As a method for examining the presence or absence of digestion with the methylation-sensitive restriction enzyme, specifically, for example, 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). When the cytosine to be analyzed is methylated, an amplification product is obtained. On the other hand, if the cytosine to be analyzed is not methylated, an amplification product cannot be obtained. In this way, by comparing the amount of amplified DNA, the ratio of cytosine to be analyzed can be measured.

Incidentally, in the double-stranded DNA formed in the second step, as described above, cytosine contained in the DNA strand as a negative strand is not methylated and is contained in the DNA strand on the positive strand side. The cytosine is found in the genomic DNA contained in biological samples. Whether or not the state of the double-stranded DNA is an ammethyl state depends on whether the syn is methylated or not. That is, if the genomic DNA contained in a biological sample is methylated, the resulting double-stranded DNA is in a hemimethyl state (a state that is not an ammethyl state. Negative strand: an unmethylated state, a positive strand : If the genomic DNA contained in the biological sample is not methylated, the resulting double-stranded DNA is in an unmethylated state (negative strand: not methylated) State, positive chain: unmethylated state). Accordingly, by utilizing the property that the methylation-sensitive restriction enzyme does not cleave the double-stranded DNA in the hemimethyl state, the methylation-sensitive restriction enzyme is recognized in the genomic DNA contained in the biological specimen. It can be distinguished whether cytosine in at least one C p G pair present in the site was methylated. That is, by digestion with the methylation sensitive restriction enzyme, at least one C p G present in the recognition site of the methylation sensitive restriction enzyme in the genomic DNA contained in the biological specimen. If the cytosine in the pair is not methylated, the double-stranded DNA is in an unmethylated state and is cleaved by the methylation sensitive restriction enzyme. In addition, if cytosine in all C p G pairs existing in the recognition site of the methylation-sensitive restriction enzyme in the genomic DNA contained in the biological sample is methylated, Double-stranded DNA is in a hemimethyl state and is not cleaved by the methylation sensitive restriction enzyme. Therefore, after performing digestion, as described below, PCR is performed using a pair of primers capable of amplifying the target DNA region. If the cytosine in at least one C p G pair present in the restriction enzyme recognition site is not methylated, PCR-amplified products cannot be obtained, while in biological samples, If cytosine in all C p G pairs existing in the recognition site of the above-mentioned methylation-sensitive restriction enzyme in the contained genomic DNA is methylated, PCR amplification products can be obtained. It will be. Specifically, the third step of the measurement method of the present invention is, for example, the present immobilized oligonucleotide. Four

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). Add 3 L, methylation sensitive restriction enzyme Hpal l or Hhal (10 ϋ L), etc. 1.5 ^ L respectively, then add sterilized ultrapure water to the mixture to a volume of 30 2 L, 37 to 1 hour ~ Incubate for 3 hours. 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.

More specifically, for example, when using a PCR tube coated with streptavidin, the solution is first removed by pipetting or decanting, and then a TE buffer having a volume substantially equal to the volume of the biological sample is added thereto. After adding the TE buffer, the TE buffer may be removed by pipetting or decanting. When using magnetic beads coated with streptavidin, after fixing the beads with a magnet, first remove the solution by pipetting or decantation, and then use TE buffer that is approximately equal to the volume of the biological sample. After adding the TE buffer, the TE buffer can be removed by pipetting or decanting.

Then, by performing such an operation several times, the digest (double-stranded DNA containing at least one CpG pair in the ammethyl state at the recognition site of the 5 艮 enzyme) is removed and washed. (DNA purification). As a concern in the digestion treatment with a methylation-sensitive restriction enzyme in the third step of the measurement method of the present invention, a recognition sequence containing unmethylated cytosine cannot be completely digested (so-called “DNA remains uncut”). ]) I can raise concerns. When such a concern becomes a problem, if there are many recognition sites for methylation-sensitive restriction enzymes, DNA residues can be minimized. As an area Has one or more recognition sites for methylation-sensitive restriction enzymes, and the more recognition sites, the better.

Therefore, when the treatment with a plurality of methylation sensitive restriction enzymes is performed in the third step of the measurement method of the present invention, specifically, for example, the following may be performed. To the double-stranded DNA that was formed in the second step, 3 L of an optimal 10X buffer (330 mM Tris-Acetate H 7.9, 660 mM K0Ac, lOOmM Mg0Ac2, 5 mM Dithothreitol), lmg / mL BSA aqueous solution Add 3 L, 1.5 L each of methylation sensitive enzymes Hpall and Hhal (10U / xL), etc., then add sterilized ultrapure water to the mixture to make the volume 30, and at 37 ° C for 1 to 3 hours Incubate. Thereafter, according to the same operation as described above, the remaining solution is removed and washed (DNA purification) by pipetting or decantation. More specifically, for example, when using a PCR tube coated with streptavidin, first remove the solution by pipetting or decanting, and then add TE to the volume of the biologically-derived sample. After adding a buffer, the TE buffer can be removed by pipetting or decanting. When using magnetic beads coated with streptavidin, after fixing the beads with a magnet, first remove the solution by pipetting or decanting, and then use TE buffer that is approximately equal to the volume of the biological specimen. After adding, the TE buffer should be removed by pipetting or decanting.

Subsequently, the remaining solution is removed and washed (DNA purification) by performing such an operation several times. In the measurement method of the present invention or the methylation ratio measurement method described later, 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. One preferred embodiment is that it is a DNA sample that has been digested in advance. Here, when genomic DNA (cage DNA) contained in a biological specimen is selected with an immobilized oligonucleotide, short cDNA is more easily selected, and the target region is increased with PCR. Even if it is wide, it is considered better to have a short cage DNA. Four

A restriction enzyme that does not recognize the region 32 as a recognition cleavage site may be directly used for a DNA sample derived from genomic DNA contained in a biological sample to perform digestion. In addition, as a method for digesting with a restriction enzyme that does not use the target DNA region as a recognition cleavage site, a general restriction enzyme treatment method may be used.

One of the preferred embodiments is that the “DNA sample derived from genomic DNA contained in a biological specimen” is a DNA sample that has been digested with one or more methylation-sensitive restriction enzymes. Can do.

These preferred embodiments are because the amount of methylation can be obtained with high accuracy by digesting the biological specimen itself with the restriction enzymes as described above. This method is useful for eliminating the “DNA residue” as described above. As a method of digesting a sample derived from genomic DNA contained in a biological sample with a methylation-sensitive restriction enzyme, the same method as described above may be used when the biological sample is genomic DNA itself. When the sample is a tissue lysate, cell lysate, etc., in the same manner as described above, a large excess of methylation-sensitive restriction enzyme, eg, 500-fold amount (10 U) for 25 ng of DNA Alternatively, the extinction treatment may be carried out using a methylation sensitive restriction enzyme or more. In the fourth step of the measurement method of the present invention, double-stranded DNA that is an undigested product obtained in the third step (extension formation that does not include the CpG pair in the methylated state at the recognition site of the methylation-sensitive restriction enzyme) The double-stranded DNA) is once separated into a single-stranded state. Then in the fifth step

(5-a) (i) 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). ,

(ii) Using the selected single-stranded DNA as a saddle, using the single-stranded immobilized oligonucleotide as a primer, and extending the oligonucleotide from the primer once, the single-stranded DNA A 5-step a to form a double-stranded DNA containing A, and

(5-b) The generated single-stranded DNA (negative strand) is used as a cage, and the single-stranded DNA (negative 3) of the base sequence (negative strand) which is a partial base sequence (negative strand) of the base sequence possessed by the strand and complementary to the base sequence (positive strand) of the target DNA region. The oligonucleotide having a partial base sequence (negative strand) located 3 'further than the end and a base sequence (positive strand) complementary to is used as an extension primer, and the oligonucleotide is used once from the primer. By performing the 5-step b to form a double-stranded DNA containing the single-stranded DNA by extension,

Furthermore, after separating the double-stranded DNA formed in the 5-a step and 5-b step into a single-stranded state, the 5-a step and 5-b step are repeated, Amplify the methylated DNA in the DNA region of interest to a detectable amount and quantify the amount of amplified DNA. In the fourth step of the measurement method of the present invention, the undigested double-stranded DNA obtained in the third step (extension formation that does not include the CpG pair in the methylated state at the recognition site of the methylation-sensitive restriction enzyme) The resulting double-stranded DNA (NA) is once separated into a single-stranded state. Specifically, for example, the undigested double-stranded DNA obtained in the third step (two double-strand DNAs that do not contain a CpG pair in the methylated state at the recognition site of the methylation-sensitive restriction enzyme). Add the annealing buffer to the strand (DNA) to obtain a mixture. The resulting mixture is then heated at 95 ° C. for several minutes.

Then, as the fifth step,

(i) In order to anneal the generated single-stranded DNA (positive strand) to the single-stranded immobilized oligonucleotide (negative strand), the single-stranded immobilized oligonucleotide (negative strand) Cool immediately to a temperature approximately 10 to 20 ° C lower than the Tm value of the chain, and keep at that temperature for several minutes.

(i i) Then return to room temperature. (5th-Step a (i))

(iii) Using the single-stranded DNA selected in (i) above as a saddle, using the single-stranded immobilized oligonucleotide as a primer, and extending the oligonucleotide once from the primer. To form a double-stranded DNA containing the single-stranded DNA (that is, step 5a-step (ii)). Specifically, for example, the explanation below and the above-mentioned What is necessary is just to carry out according to the operation method etc. in the extension reaction in the second step of the measurement method of the present invention.

(iv) A partial base sequence (negative strand) of the base sequence possessed by the single-stranded DNA (negative strand) using the generated single-stranded DNA (negative strand) as a saddle type, and the purpose and Complementary to the partial base sequence (negative strand) located 3 'from the 3' end of the base sequence (negative strand) that is complementary to the base sequence (positive strand) of the DNA region to be By using 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. Form (ie, step 5-b). Specifically, for example, as in the above (iii), it may be carried out according to the following description or the operation method in the extension reaction in the second step of the above-described measurement method of the present invention. Here, “a partial base sequence (negative) that is further 3 ′ from the 3 ′ end of the base sequence (negative strand) that is complementary to the base sequence (positive strand) of the target DNA region. As for “)”, the base sequence (negative strand) complementary to the base sequence (positive strand) of the target DNA region is further 1 to 2K bases, preferably 1 base to The base sequence can be selected from 1K bases, more preferably 1 base to 200 bases, more preferably 1 bases to 100 bases 3 ′ side. The oligonucleotide used as the extension primer (reverse primer) is preferably 5 to 50 bases in length.

(V) Furthermore, after the double-stranded DNA formed in the 5th-a step and the 5th-b step is once separated into a single-stranded state, the 5th-a step and the 5th-b step are repeated. By amplifying the methylated DNA in the target DNA region to a detectable amount, the amount of the amplified DNA is quantified. Specifically, for example, in the same manner as described above, 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. As 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. Use this immobilized oligonucleotide as a primer on one side when amplifying the target region Therefore, by adding only the other primer and performing PCR, an amplification product can be obtained and the amplification product can be immobilized. At this time, if a primer previously labeled with fluorescence or the like is used and the label is used as an index, the presence or absence of the amplification product can be evaluated without performing a cumbersome operation such as electrophoresis. As the 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. In addition, a reaction liquid with a volume of 25 1 can be raised.

Since the target DNA region (ie, the target region) has many GC-rich base sequences, the reaction may be carried out by adding an appropriate amount of betaine, DMSO, etc. at times. As the reaction conditions, for example, the above reaction solution is kept at 95 ° C. for 10 minutes, then at 95 ° C. for 30 seconds and then at 55 to 65 ° C. for 30 seconds and further to 72 ° C. For example, the temperature can be maintained for 30 to 40 cycles with 30 seconds as one cycle. After performing such PCR, the obtained amplification product is detected. For example, when a pre-labeled primer is used, the amount of the immobilized fluorescent label can be measured after performing the same washing / purification operation as before. In addition, when performing PCR using a normal unlabeled primer, anneal the gold colloid particles, the probe labeled with fluorescence, etc., and measure the amount of the probe bound to the target region. Can be detected. In addition, for example, real-time PCR may be used to obtain the amount of amplification product with higher accuracy. Real-time PCR is a method that monitors PCR in real time and kinetics analyzes the results of the monitoring. For example, it can detect even a slight difference of about 2 times in terms of gene dosage. This method is known as precision quantitative PCR. Examples of the real-time PCR method include a method using a probe such as a saddle type-dependent nucleic acid polymerase probe, and a method using an in-curry dish such as Saipa Green. Commercially available equipment and kits for real-time PCR may be used. As described above, the detection is not particularly limited, and detection by any known method can be performed. With these methods 69414

36 The operation until detection is possible without changing the reaction container.c ₎ Furthermore, the 3 'end side of the primer or immobilized oligonucleotide with the same base sequence as that of the immobilized oligonucleotide. In addition, a new pyotinylated oligonucleotide can be designed and used as a primer on one side, and the target region can be amplified using the primer on the complementary side. In this case, the amplified product obtained will be immobilized if there is a support coated with streptavidin.For example, if PCR is performed in a streptavidin-coated PCR tube, it will be contained in the tube. As described above, the amplified product can be easily detected by using a labeled primer. If the previous immobilized oligonucleotide is immobilized by covalent bond or the like, the solution containing the amplification product obtained by PCR is transferred to the container where the streptavidin-coated support is present, and the amplification product is immobilized. It is possible. The detection may be performed as described above. The complementary primer that amplifies the target region should be a primer that can amplify the target region having one or more recognition sites for methylation-sensitive restriction enzymes and does not include the recognition site. The reason for this is as follows. Only the recognition site of the methylation-sensitive restriction enzyme at the 3 ′ end of the DNA strand (new strand) on the immobilized oligonucleotide side of the double-stranded DNA obtained by the selection and single extension reaction is methylated. If not, only that part will be digested with a methylation sensitive restriction enzyme. After digestion, even if the washing operation is performed as described above, the double-stranded DNA that has lost only the 3 ′ end of the nascent strand remains in an immobilized state. If the primer on the complementary side contains the recognition site for this 3 'end methylation sensitive restriction enzyme, the 3' end of the primer and several bases on the end will be the 3 'end of the nascent strand. This is because annealing with several bases may result in amplification of the target region by PCR. In the present invention, before or after the fourth step of the measurement method of the present invention, a part of the 3 ′ end of the single-stranded DNA (positive strand) containing the intended DNA region (ie, It does not contain the target DNA region, has a base sequence that is complementary to) and is in a free state 14

37 Includes a modification that additionally includes the step of adding a single-stranded oligonucleotide (negative strand) into the reaction system.

That is,

(Variation 1)

Before the fourth step of the measurement method of the present invention,

A nucleotide sequence that is complementary to a part of the 3 ′ end of a single-stranded DNA (positive strand) containing the target DNA region (excluding the target DNA region). And a step of adding a single-stranded oligonucleotide (negative strand) that is in a free state to the reaction system, and

As a fifth step of the measurement method of the present invention, the method further comprises one of the following steps arranged in the fifth-a step and the fifth-b step.

(5-c) (i) Base pairing the generated single-stranded DNA (positive strand) with the free single-stranded oligonucleotide (negative strand) added to the reaction system in the additional step above. To select the single-stranded DNA A

(ii) The selected single-stranded DNA is in a saddle shape, the single-stranded oligonucleotide (negative strand) is used as a primer, and the single-stranded DNA is extended from the primer once to thereby produce the single-stranded DNA. 5th-c ェ king that forms double-stranded DNA containing DN A. (Variation 2)

After the fourth step of the measurement method of the present invention,

A nucleotide sequence that is complementary to a part of the 3 ′ end of a single-stranded DNA (positive strand) containing the target DNA region (not including the target DNA region). An undigested product obtained through the third step and the additional step described above, and additionally comprising a step of adding a single-stranded oligonucleotide (negative strand) that is in a free state to the reaction system. A step of once separating a double-stranded DNA (a double-stranded DNA containing no CpG pair in the methylated state at the recognition site of the methylation-sensitive restriction enzyme) into a single-stranded state, and the present invention As the process of the fifth process of the measurement method, it is arranged in the 5th-a process and the 5th-b process A method further comprising one step as described above

(5-c) (i) Base pairing the generated single-stranded DNA (positive strand) with the free single-stranded oligonucleotide (negative strand) added to the reaction system in the additional step above. To select the single-stranded DNA,

(ii) Using the selected single-stranded DNA as a saddle, using the single-stranded oligonucleotide (negative strand) as a primer, and extending the oligonucleotide from the primer once, the single-stranded DN The 5th-c step of forming double-stranded DNA containing A <= In this modified method, “a part of the 3 ′ end of the single-stranded DNA (positive strand) containing the target DNA region mentioned above” (However, the target DNA region is not included.) A single-stranded oligonucleotide (negative strand) having a complementary base sequence and free state is added to the reaction system. Thus, it becomes possible to easily improve the amplification efficiency of the target DNA region in the fifth step. Here, 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). 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 that of the oligonucleotide immobilized on the present strand, or may be a short base sequence, or a long base sequence. However, in the case of a base sequence longer than the single-stranded immobilized oligonucleotide, the reverse primer (positive strand) is an extension primer, and the single-stranded oligonucleotide (negative strand) is a saddle type. It is important that the single-stranded oligonucleotide is in a free state that cannot be used in the reaction of extending the extension primer. The fifth step of the measurement method of the present invention has a repetition step. For example, “the generated single-stranded DNA in the positive strand” in step 5-a (i) means the operation and the fifth step in the first step. In both operations of the 5th process after the second and subsequent operations, `` generated 'free' 7069414

39 It means “single-stranded DNA (positive strand)”.

“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)”. In addition, the “formed double-stranded DNA” obtained in the fifth step means that in the case of the fifth-a step, the above-mentioned methylation-sensitive restriction enzyme is used in the first step of the fifth step. Means a double-stranded DNA that does not contain a CpG pair in the methyl state at the recognition site of `` Methylation-sensitive restriction enzyme of the above-mentioned methylation-sensitive restriction enzyme ''. “An elongated double-stranded DNA that does not contain an ammethyl-state C pG pair at the recognition site” and “A double-stranded DNA that contains an ammethyl-state CpG pair at the recognition site of the methylation-sensitive restriction enzyme. It means both “DNA”. In the case of the 5th-b step, in both the operation of the 5th step of the first time and the repetitive operation of the 5th step after the 2nd time, “all of the recognition sites for the above-mentioned methylation sensitive restriction enzymes are all This means that the formed double-stranded DNA is a CpG pair in an ammethyl state. The same applies when the fifth step further includes a 5-c step. In addition, in the case where the fifth step has an additional 5-c step, the “generated single-stranded DNA” in the fifth-el step (i) is the same as the first step of the fifth step. It means “generated“ free ”single-stranded DNA (positive strand)” in both the operations and the repeated operations of the fifth step after the second round. P2007 / 069414

40. The present invention also includes a methylation ratio measurement method (that is, the methylation ratio measurement method of the present invention) characterized in that it further comprises the following two steps as steps of the measurement method of the present invention. .

(6) After performing the first step and the second step of the measurement method of the present invention (including the above-mentioned modified method), the present method is performed without performing the third step of the measurement method of the present invention (including the modified method). By performing the fourth and subsequent steps of the invention measurement method (including the above-mentioned modified methods), the DNA of the target DNA region (total amount of methylated and non-methylated DNA) is detected. A sixth step of amplifying to the possible amount and quantifying the amount of amplified DNA; and

(7) Based on the difference obtained by comparing the amount of DNA quantified in the fifth step of the measurement method of the present invention (including the above-mentioned modified method) with the amount of DNA quantified in the sixth step. The seventh step of calculating the percentage of methylated DNA in the target DNA region of. The methylation ratio measurement method may be used in the following situations.

It is known that abnormal DNA methylation occurs in various diseases (for example, cancer), and it is considered possible to measure the degree of various diseases by detecting this abnormal DNA methylation. .

For example, there is a DNA region that is 100% methylated in the genomic DNA contained in a specimen derived from a disease patient, and the measurement method of the present invention or the method of measuring the methylation ratio of the present invention is performed on the DNA region. For example, the amount of methylated DNA will increase. In addition, for example, there is a DNA region that is not 100% methylated in genomic DNA contained in a sample derived from a disease patient, and the present measurement method or the present methylation ratio measurement method is performed on the DNA region. Then, the amount of methylated DNA will be close to zero. In addition, for example, there is a DNA region with a low methylation rate in the genomic DNA contained in the biological sample of a healthy person and a high methylation rate in the genomic DNA contained in the biological sample of the diseased patient. When the measurement method of the present invention or the methylation ratio measurement method of the present invention is carried out, the amount of methylated DNA in a healthy person shows a value close to 0, whereas in the case of a patient with a disease Four

41

Since the value is significantly higher than the value 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 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. In such a measurement method of the present invention or a methylation ratio measurement method of the present invention, a restriction enzyme or primer that can be used in various methods for measuring the amount of methylated DNA in the target region and measuring the methylation ratio Alternatively, the probe is useful as a reagent for a detection kit. The present invention also provides a detection kit containing these restriction enzymes, primers or probes as reagents, and a detection chip in which these primers or probes are immobilized on a carrier. The scope of rights of the method or the methylation ratio measurement method of the present invention includes use in the form of a detection chip or a detection chip as described above using the substantial principle of the method. Example

EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. Example 1

Mammal colon adenocarcinoma cell line Caco-2 (ATCC oH TB-37), purchased from ATCC, is cultured until confluent in a dedicated medium for the cell line described in the ATCC catalog. To obtain about 1 × 10 ⁷ cells. After adding 10 times volume of SEDTA buffer [lOmM Tris-HCl H 8.0, 10 mM EDTA pH 8.0, lOOmM NaCl] to the obtained cells, it was homogenized. Four

42 After adding proteinase K (Sigma) to 500 g / m 1 and sodium dodecyl sulfate to 1% (wZv) to the obtained mixture, the mixture was shaken at 55 ° C. for about 16 hours.

After completion of shaking, the mixture was subjected to phenol [saturated with [1M Tris-HCK H 8.0]] · chloroform extraction. 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 dissolved in TE buffer (10 mM Tris, lmM EDTA, pH 8.0), and RNase A (Sigma) was added to this so as to be 40 g / ml, and incubated at 37 ° C for 1 hour. . The incubated mixture was subjected to phenol'black mouth form extraction treatment. The aqueous layer was recovered, and NaCl was added to 0.5 N, followed by ethanol precipitation to recover the precipitate (genomic DNA). Genomic DNA was obtained by rinsing the collected precipitate with 70% ethanol.

The obtained genomic DNA is used as a saddle type, and PCR is performed using the following primers and reaction conditions to obtain a DNA fragment (hereinafter referred to as DNA fragment X2) used as a test sample. Base represented by SEQ ID NO: 18 A sequence corresponding to base numbers 76477 to 77002 of the GPR 7 sequence shown in Gen bank Accession No. AC009800 and the like was amplified.

PF3: 5'-GTCCGCGGCGACATTGGG-3 '(SEQ ID NO: 1 9)

PR3: 5'-CGATGAGCTTGCACATGAGCT-3 '(SEQ ID NO: 20)

As a 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 to 3 / M and the base sequence represented by SEQ ID NO: 20 were used. Primer solutions 2.5 1 each, 2.5 mM dNTPs 2.5 1 each, 10 X buffer (lOOmM Tris-HC1 pH 8.3, 500 mM KC1, 0.01 mM Gelatin with 15 mM MgCl) 2.5 n 1 And thermostable DNA polymerase (AmpliTaq

Gold, 5U / D was mixed with 0.125 1 and sterilized ultrapure water was added to make the volume 25 1. The reaction solution is kept at 95 ° C for 10 minutes, and then at 95 ° C for 30 minutes. 69414

PCR was performed for 43 seconds, then at 59 for 60 seconds, and at 72 for 45 seconds for 1 cycle.

After PCR, confirm DNA amplification by 1.5% DNA gel electrophoresis, cut out the DNA fragment of interest (526 bp, DNA fragment X2), and QI AGEN QIAquick Gel Extraction Kit (QIAGEN) The product was purified using

A part of the obtained DNA fragment X2 was treated with methylase S ss I (NEB), and 5′-CG-3 ′ was all methylated DNA fragment (hereinafter referred to as DNA fragment Y2). Got. Again, as before, confirm amplification by 1.5% agarose electrolysis, excise the DNA fragment (526 bp, DNA fragment Y2) and use the QIAGEN QIAquick Gel Extraction Kit (QIAGEN). And purified. The oligonucleotide B 1 (19 bp) having the base sequence shown in SEQ ID NO: 21 and labeled with a thiotin at the 5 ′ end was synthesized as an oligonucleotide that was aligned with the DNA fragment X 2 and the DNA fragment Y 2.

<Piotin-labeled oligonucleotide>

B1: 5'-GACAACGCCTCGTTCTCGG-3 '(SEQ ID NO: 21)

1 L of 5 M Pyotinylated Oligonucleotide B 1 and 10 L of an annealing buffer (0.5 M chalcylic acid buffer, 1% SDS, lmM EDTA aqueous solution) A mixture was obtained.

The resulting mixture was then heated at 95 ° C. for 5 minutes. Thereafter, it was quickly cooled to 50 ° C and kept at that temperature for 5 minutes. Next, after incubating at 37 ° C. for 5 minutes, the temperature is returned to room temperature, and the single-stranded DNA (DNA fragment X2 or DNA fragment Y2) containing the target DNA region and the biotinylated oligonucleotide are base-paired. )

Next, the mixture prepared as described above is added to a PCR tube coated with streptavidin, and this is further incubated at 37 ° C. for 5 minutes, whereby the biotinylated oligonucleotide is stretched. It fixed to the support body coat | covered with butavidin (Hereinafter, it corresponds to the 1st process of this invention measuring method.). Next, after removing the solution from the PCR tube, an IOLO TE buffer was added, and then the TE buffer was removed by pipetting. This operation was performed twice more.

To the single-stranded DNA thus selected, 3 L of 10X buffer solution (lOOmM Tris-HCl pH 8.3, 500 mM KCK 15m MgCl ₂ , 0.01% Gelatin), 3 L each of 2 mM dNTP aqueous solution, Add 5 L of 5N betaine aqueous solution, 0.15 L of heat-resistant DNA polymerase (AmpliTaq: 5U / ^ L) and 17.85 L of ultrapure water (total solution volume: 30 / zL), 3 7 ° C The extension reaction was performed once by incubating for 2 hours. After the reaction, the supernatant was removed, 100 L of TE buffer was added, and then the TE buffer was removed by pipetting (this corresponds to the second step of the measurement method of the present invention).

Thereafter, 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.

Group B (digested with Hpa II and Hha I): Double-stranded DN prepared above 07069414

45

A and H pa II and H ha I each with 15 U and 10 X buffer (330 mM Tris-Acetate pH 7.9, 660 m KOAc, lOOmM MgOAc2, 5 mM Dith othreitol) optimal for H pa II and H ha I 10XBSA (Bovine serum albumin lmg / ml) was added to 3 L, and sterile ultrapure water was added to the mixture to make the volume 3 O ^ L.

Incubate each reaction solution at 37 for 1 hour, remove the supernatant, add 100 L of TE buffer, and then remove the TE buffer by pipetting. This corresponds to the third step of the invention measurement method. Next, the obtained undigested product (DNA bound to the tube) was used as a cage, and PCR was performed using the following primers and reaction conditions. If the target DNA region is methylated, DNA (having the base sequence shown by SEQ ID NO: 22; the sequence corresponding to the base numbers 76669-76835 of the GPR7 sequence shown in Genbank Accession No. AC009800 etc.) Amplified. PF4: 5'-GACAACGCCTCGTTCTCGG-3 '(SEQ ID NO: 23)

PR4: 5'-GCGGAGTTGCCCGCCAGA-3 '(SEQ ID NO: 24)

As a PCR reaction solution, each of a primer solution consisting of the base sequence shown in SEQ ID NO: 24 and a primer solution consisting of the base sequence shown in SEQ ID NO: 24 prepared in 3 M in a DNA-bound tube was used. 2.5 1 with 2.5 mM dNTPs each 2.5 ^ 1 and 10 X buffer (lOOmM Tris-HCl pH 8.3, 500 mM KCK 15 mM MgCl ₂ , 0.01% Gelatin) _2.5 1 and heat resistant DNA polymerase (AmpliTaq Gold) 5 UZ ^ l 0.125 zl and 5 Ν betaine aqueous solution 51 were mixed, and sterilized ultrapure water was added to make the volume 25 1. The reaction solution was incubated at 95 ° C for 10 minutes (corresponding to the fourth step of the measurement method of the present invention), and then at 95 for 30 seconds and then at 59 ° C for 60 seconds and further at 72 ° C. PCR was performed under the condition of 37 cycles of incubation with one cycle of 45 seconds.

After PCR, amplification was confirmed by 1.5% agarose gel electrophoresis. So The results were as follows (corresponding to the fifth step of the measurement method of the present invention).

Regarding 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

As for (methylated DNA fragment), amplification was confirmed in both group A (no treatment group) and group B (Hpa II, Hhal treatment group), and amplification products were obtained. From the above, it is possible to select a single-stranded DNA containing the target DNA region, and without amplifying the unmethylated DNA in the target DNA region. It was confirmed that only the methylated DNA was amplified to a detectable amount, and the amount of amplified DNA could be quantified. Example 2

The following test was performed using the DN A fragment X 2 and the DN A fragment Y 2 obtained in Example 1.

In each solution of DNA fragment X2 and DNA fragment Y2 (25 pg / mL aqueous solution, \ 0 1), add 1 liter of 5 / Μ piotinylated oligonucleotide Β 1 and the annealing buffer (0.5M Churchillin) 10 L of acid buffer, 7% SDS, ImM EDTA aqueous solution) was added to obtain a mixture.

The resulting mixture was then heated at 95 ° C. for 5 minutes. Thereafter, it was quickly cooled to 50 ° C and kept at that temperature for 5 minutes. Next, after incubating at 37 ° C. for 5 minutes, the temperature is returned to room temperature, and the single-stranded DNA (DNA fragment X2 or DNA fragment Y2) containing the target DNA region is base-paired with the piotinylated oligonucleotide. The

Next, prepare a PCR tube coated with streptavidin as described above. 07069414

47, and the mixture was incubated at 37 ° C for 5 minutes to immobilize the biotinylated oligonucleotide on the streptavidin-coated support. It corresponds to one process. Next, after removing the solution from the PCR tube, an IOLO TE buffer was added, and then the TE buffer was removed by pipetting. This operation was performed twice more.

The single-stranded DNA thus selected was added with 3 iL of 10X buffer (lOOmM Tris-HCl pH 8.3, 500 mM KCK 15 mM MgCl in 0.01% Gelatin), 3 L of 5 mM dNTP aqueous solution, 5 N Add 6 L of betaine aqueous solution, 0.15 L of thermostable DNA polymerase (AmpliTaq: 5 U / L) and 17.75 L of ultrapure water (total solution volume: 30 ^ L), 37 ° C The extension reaction was performed once by incubating for 2 hours. After incubation, the supernatant was removed, 100 L of TE buffer was added, and then the TE buffer was removed by pipetting (this corresponds to the second step of the measurement method of the present invention). Thereafter, each of the double-stranded DNAs thus formed with extension was subjected to the following four treatments. Group A (untreated group): Double-stranded DNA prepared above and 10 X buffer (330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM Mg0Ac2, 5 mM) optimal for Hpa II and Hha I Add 3 L of Dithothreitol) and 3 II L of 10XBSA (Bovine serum albumin Img / ml), and add sterile ultrapure water to the mixture to a volume of 30 L.

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 C (digested with Hha I): The double-stranded DNA prepared above contains 15 U of Hh a I and 10 X buffer (330 mM Tris- Acetate pH7.9 , 660 mM K 0Ac, lOOmM MgOAc2, 5 mM Dithothreitol) 3 L, 10 XBSA (Bovine serum albumin lmg / ml) 3 L, and add sterilized ultrapure water to the mixture. 30 / L. Group D (Digestion treatment group with Hpa II and HhaI): The double-stranded DNA prepared above is optimal for Hpa II and HhaI with 15 U and Hpa II and Hha I, respectively. 10X buffer (330mM Tris-Acetate pH7.9, 660mM K0Ac, lOOmM Mg0Ac2, 5EM Ditho threitol) and 10XB SA (Bovine serum albumin lmg / ml) and 3L Sterile ultrapure water was added to the mixture to make the volume 30 L.

After incubating each reaction solution at 37 ° C for 1 hour (digestion), removing the supernatant, mixing 100 L of TE buffer, and then removing the TE buffer by pipetting (above) This corresponds to the third step of the measurement method of the present invention. Next, using the obtained undigested material (DNA bound to the tube) as a cage and performing PCR using the following primers and reaction conditions, the target DNA region is methylated. DNA (having the base sequence shown by SEQ ID NO: 22; the sequence corresponding to the base Nos. 76669-76835 of the GPR 7 sequence shown by Genbank Accession No. AC009800 etc.) is amplified. PF4: 5'-GACAACGCCTCGTTCTCGG-3 '(SEQ ID NO: 2 3)

PR4: 5′-GCGGAGTTGCCCGCCAGA-3 ′ (SEQ ID NO: 2 4)

As a PCR reaction solution, use the sequence number prepared in 3 に of the genomic DNA in the 铸 shape. A primer solution consisting of the base sequence shown in No. 23 and a primer solution consisting of the base sequence shown in SEQ ID NO: 24, 2.5 21 each, 2 mM dNTP 2.5 1 and 10X buffer (lOOmM Tris -HCl H 8.3, 500 mM KC1, 15 mM MgCl ₂ , 0.01% Gelatin) _2.5 1, thermostable DNA polymerase (AmpliTaq Gold) 5 U / l 0.125 1 and 5 N betaine aqueous solution 5 1 And sterilized ultrapure water was added to make the volume 25 1. The reaction solution was incubated at 95 ° C for 10 minutes (corresponding to the fourth step of the measurement method of the present invention), and then at 95 for 30 seconds, then at 59 ° C for 60 seconds and further at 72 ° C at 45 ° C. PCR was performed under the condition of 37 cycles of heat insulation with one cycle per second.

After PCR, amplification was confirmed by 1.5% agarose gel electrophoresis. The results were as follows (this corresponds to the fifth step of the measurement method of the present invention).

Regarding DNA fragment X2, in the case of Group A (untreated group), amplification was confirmed and the amplification product was obtained. In contrast, in Group B (Hpall treatment group) and Group C (Hhal treatment group), some amplification was observed and a small amount of amplification product was obtained. In the case of Group D (HpaII, Hhal treatment group), almost no amplification was confirmed and the amplification product was not obtained. On the other hand, with respect to the DNA fragment Y2, amplification was confirmed and the amplification product was obtained in all cases of the A group to the D group. '' From the above, when treating with two or more methylation sensitive enzymes simultaneously, methylation in the target DNA region is more than when treating with only one methylation sensitive enzyme. It was confirmed that the amount of the amplified DNA can be quantified more clearly and accurately. Therefore, it was confirmed that simultaneous treatment with two or more methylation-sensitive enzymes is a preferable method for determining the amount of methylated DNA or the methylation ratio. Example 3 14

50

The following tests were conducted.

A serum solution was obtained by adding 1 mL of rat serum to a ratio of 1. To the serum solution (DN A fragment 25 pg / mL aqueous solution, 10 L), add 1 L of 5 M pyotinylated oligonucleotide B 1 and annealing buffer (0.5 M church phosphate buffer, Ί% SDS, lmM EDTA aqueous solution). By adding 10 L, a mixture was obtained.

The resulting mixture was then heated at 95 ° C. for 5 minutes. Thereafter, it was quickly cooled to 50 ° C and kept at that temperature for 5 minutes. Next, this was incubated at 37 ° C. for 5 minutes and then returned to room temperature, and the single-stranded DNA containing the target DNA region and base-paired oligonucleotide were subjected to base pairing (DNA fragment X 2 and DNA fragment Y 2) 4 pieces each.

Next, to the PCR tube coated with streptavidin, the pre-prepared mixture was added as described above, and this was incubated at 37 ° C for 5 minutes to streak the pyotinylated oligonucleotide. It was fixed on a support coated with avidin (this corresponds to the first step of the measurement method of the present invention). Next, after removing the solution from the PCR tube, an IOOL TE buffer was added, and then the TE buffer was removed by pipetting. This operation was performed twice more.

The single-stranded DNA thus selected was added with 3 L of 10X buffer solution (lOOmM Tris-HCl H8.3, 500 mM KCK 15 mM MgCl 0.01% Gelatin), 3 L each of 2 mM dNTP water solution, 5 N Add 6 L of Bevenin aqueous solution, 0.15 / L of thermostable DNA polymerase (AmpliTaq: Will) and 17.85 L of ultrapure water (total solution volume: 30) and incubate at 37 ° C for 2 hours After the reaction, the supernatant was removed, 100 L of TE buffer was added, and then the TE buffer was removed from the pipe (in the second step of the measurement method of the present invention). Equivalent to. ) TJP2007 / 069414

51

Thereafter, each double-stranded DNA thus obtained was subjected to the following two types of treatment.

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). '

Group B (Digested with Hpa II and Hha I): The above-prepared double-stranded DNA is optimal for H pa II ¾ and 11 ha I, 15 U, and H pa II and H ha I, respectively. Add 1 OX buffer (330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM Mg0Ac2, 5 mM Dith othreiiol) and 3 L of 10XBSA (Bovine serum albumin lmg / ml). Water was added to bring the volume to 30 L (2 bottles each). After incubating each reaction solution at 37 ° C for 1 hour (digestion treatment), the supernatant was removed, 100 TE buffer was added, and then the TE buffer was removed by pipetting. Corresponds to the third step of the method). Next, PCR was performed from the obtained undigested product using the following primers and reaction conditions. DNA if the target DNA region is methylated (has the base sequence shown by SEQ ID NO: 22; the sequence corresponding to the base numbers 76669-76835 of the GPR 7 sequence shown by Genbank Accession No. AC009800, etc.) Is amplified.

PF4: 5'-GACAACGCCTCGTTCTCGG-3 '(SEQ ID NO: 23) Four

52

PR4: 5'-GCGGAGTTGCCCGCCAGA-3 '(SEQ ID NO: 24)

As a PCR reaction solution, a primer solution consisting of the base sequence shown in SEQ ID NO: 23 prepared in 3 M and the base sequence shown in SEQ ID NO: 24 were prepared in a cocoon-shaped genomic DNA (DNA bound to the tube). Each primer solution consisting of 2.5 1 and 2.5 mM dNTP 2.5 1 and 10 X buffer (lOOmM Tris-HC1 pH 8.3, 500 mM KCK 15 mM MgCl ₂ , 0.01% Gelatin) _2.5 1 and thermostable DNA polymerase (AmpliTaq Gold) 5 UZ 1 0.125 1 and 5 N betaine aqueous solution 5 1 are mixed, and sterilized ultrapure water is added to this to make the volume 25 a 1 It was. The reaction solution was incubated at 95 ° C for 10 minutes (corresponding to the fourth step of the measurement method of the present invention), and then at 95 ° C for 30 seconds and then at 63 ° C for 60 seconds and further to 72 ° C. PCR was performed under the condition of 35 cycles of heat insulation with 45 seconds as one cycle.

As for DN A 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, fflial treatment group), amplification was not confirmed and the amplification product was not obtained. On the other hand, with respect to DNA fragment Y2, amplification was confirmed in both group A (no treatment group) and group B (HpaII, fflial treatment group), and an amplification product was obtained. As described above, even when a serum solution as a DNA sample derived from genomic DNA contained in a biological sample is used, a single-stranded DNA containing the target DNA region as in Example 1 above. Amplifying the methylated DNA to a detectable amount without amplifying the unmethylated DNA in the target DNA region. It was confirmed that the amount of amplified DNA could be quantified. Example 4 The following test was conducted. A pg / l 0 buffer solution (10 mM Tris, lmM EDTA, pH 8.0) was prepared. 1 L of each TE buffer solution, 1 L of 5 M piotinylated oligonucleotide B 1 and 2 L of 10 X annealing buffer (330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM MgOAc2, 5 mM Dithothrei tol) A mixture was obtained by adding 7 L of sterile ultrapure water.

The resulting mixture was then heated at 95 ° C. for 5 minutes. Thereafter, it was quickly cooled to 50 ° C and kept at that temperature for 5 minutes. Next, after incubating at 37 ° C. for 5 minutes, the temperature is returned to room temperature, and the single-stranded DNA (DNA fragment X2 or DNA fragment Y2) containing the target DNA region and the biotinylated oligonucleotide are bases. Paired

Next, to the PCR tube coated with streptavidin, add the pre-prepared mixture as described above, and further incubate it for 5 minutes at 37 ° C to strep the pyotinylated oligonucleotide. It was fixed on a support coated with avidin (this corresponds to the first step of the measurement method of the present invention). Next, after removing the solution from the PCR tube, a 100 / L wash buffer (0.05 nween20-containing phosphate buffer (lmM KH2P04, 3 mM Na2HP0-7H20, 154 mM NaCK pH 7.4)) was added. After the addition, the washing buffer was removed by pipetting. This operation was performed twice more.

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

54

: 5U / zL) 0.15 L and ultrapure water 17.85 L (total solution volume: 30) were added and incubated at 37 ° C for 2 hours to carry out an extension reaction once. The supernatant was removed, and 100 iL of the above washing buffer was mixed, and then the washing buffer was removed by pipetting (this corresponds to the second step of the measurement method of the present invention). Each of the extended double-stranded DNAs was subjected to the following two treatments: Group A (untreated group): The double-stranded DN A prepared above was added to Hpa II and Hha I Add 1 OX buffer (330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOiM Mg0Ac2, 5 mM Dithothreitol) and 3 L of 10XBSA (Bovine serum albumin lmg / ml), and sterilize the mixture. Ultra pure water was added to make the liquid volume 30.

Group B (digestion treatment group with Hpa II and HhaI): Optimal for Hpa II and Hha I with 15 U of Hpa II and Hha I, respectively. 1 Add OX buffer (330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM Mg0Ac2, 5 mM Ditho threitol) and 3 L of 10XBSA (Bovine serum albumin lmg / ml) Pure water was added to make the volume 30 L.

After incubating each reaction solution at 37 ° C for 5 hours (digestion treatment), the supernatant was removed, 100 L of the washing buffer was added, and then the washing buffer was removed by pipetting. This corresponds to the third step of the measurement method.

Next, 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 shown by SEQ ID NO: 22; GPR 7 shown by Genbank Accession No. AC009800 etc. 07069414

55 sequences corresponding to base numbers 76669-76835) are amplified.

PF4: 5'-GACAACGCCTCGTTCTCGG-3 '(SEQ ID NO: 23)

PR4: 5'-GCGGAGTTGCCCGCCAGA-3 '(SEQ ID NO: 24)

The PCR reaction solution includes a primer solution consisting of the base sequence shown in SEQ ID NO: 24 and a primer solution consisting of the base sequence shown in SEQ ID NO: 24, prepared in 50 M, on the genomic DNA in the shape of a cage. and 0.3 1, respectively and dNTP to 5 1 of 2 mM, 10 X buffer (lOOmM Tris-HCl H 8.3, 500mM KC1, 15mM MgCl 2, 0.01% Gelatin) and the 5 1, thermostable DNA polymerase (AmpliTaq Gold ) 5U / 1 0.25 1 and 5 N betaine aqueous solution 10 1 were mixed and sterilized ultrapure water was added to make the volume 50 1. The reaction solution was incubated at 95 ° C for 10 minutes (corresponding to the fourth step of the measurement method of the present invention), and then at 95 ° C for 30 seconds and then at 59 ° C for 30 seconds. Furthermore, PCR was performed under the condition that the temperature was maintained for 32 cycles at 72 ° C for 45 seconds.

As for 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.

In contrast, in the case of group B (HpaII, Hhal treatment group), the amplification product was not obtained for all samples. On the other hand, regarding the DNA fragment Y2, amplification was confirmed in the 1 pgZl 0 L sample and the 10 pgZl 0 sample of the A group and the B group, and the amplified product was obtained. From the above, divalent cation (magnesium ion) as an annealing buffer Four

Using a solution containing 56, a single-stranded DNA containing the target DNA region (positive strand) and a part of the 3 ′ end of the single-stranded DNA (provided that the target DNA region is In the same manner as in Examples 1 to 3, the DNA region of interest can be obtained by base pairing with a single-stranded immobilized oligonucleotide having a base sequence complementary to It is possible to select a single-stranded DNA containing DNA and amplify the non-methylated DNA in the target DNA region by treatment with a methylation-sensitive restriction enzyme. It was confirmed that only the methylated DNA was amplified to a detectable amount, and the amount of amplified DNA could be quantified. Example 5 The following test was conducted. A pg / 10 L rat serum solution was prepared. Separately, each of these 10 solutions is prepared with 5 M piotinylated oligonucleotide B 1 in 1 xL, 10 X annealing buffer (3

A mixture was obtained by adding 2 L of 30 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM Mg0Ac2, 5 mM Dithothreitol) and 7 / iL of sterile ultrapure water.

The resulting mixture was then heated at 95 ° C. for 5 minutes. Thereafter, it was quickly cooled to 50 ° C and kept at that temperature for 5 minutes. Next, this was incubated at 37 ° C for 5 minutes, then returned to room temperature, and single-stranded DNA containing the desired DNA region (DNA fragment X

2 or DNA fragment Y 2) and piotinylated oligonucleotide

Next, to the PCR tube coated with streptavidin, the pre-prepared mixture was added as described above. It was fixed to a support coated with vidin (this corresponds to the first step of the measurement method of the present invention). 69414

57 Next, after removing the solution from the above-mentioned PCR tube, 100 L of washing buffer (0.05% Tween20-containing phosphate buffer (ImM KH2P04, 3 mM Na2HP0-7H20 154 mM NaCK pH 7.4) was added, The washing buffer was removed by pipetting The operation was performed twice more.

To the single-stranded DNA thus selected, 3 × iL of 10X buffer solution (lOOmM Tris-HCl pH 8.3, 500 mM KCK 15 mM MgCl 0.01% Gelatin), 2 mM dNTP aqueous solution each, 3 L, 5N Add 6 L of betaine solution, heat resistant DNA polymerase (AmpliTaq: 0.15 / L of 5U / U and 17.85 L of ultrapure water (total solution volume: 30 L), and incubate at 37 ° C for 2 hours. After the reaction, the supernatant was removed, 100 L of the washing buffer was mixed, and then the washing buffer was removed by pipetting. O) corresponding to the second step o After that, each of the extension-formed double-stranded DNA thus obtained was subjected to the following two treatments.

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.

Group B (Digestion treatment group with Hpa II and Hha I): The double-stranded DNA prepared above has 15 U of Hpa II and Hha I, and is optimal for Hpa II and Hha I 1 Add 3 L of OX buffer (330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM Mg0Ac2, 5 mM Ditho threitol) and 3 L of 10XBSA (Bovine serum albumin lmg / ml), and further sterilize the mixture. Pure water was added to bring the liquid volume to 30 XL.

After incubating each reaction solution at 37t for 5 hours (digestion treatment), 14

After removing and adding 100 of the above washing buffer, the washing buffer was removed by pipetting (this corresponds to the third step of the present measuring method).

Next, PCR was performed from the obtained undigested product using the following primers and reaction conditions. If the target DNA region is methylated, the DNA (has the base sequence represented by SEQ ID NO: 22; the sequence corresponding to the base numbers 76669-76835 of the GPR 7 sequence represented by Genbank Accession No. AC009800 etc. ) Is amplified. PF4: 5'-GACAACGCCTCGTTCTCGG-3 '(SEQ ID NO: 23)

PR4: 5'-GCGGAGTTGCCCGCCAGA-3 '(SEQ ID NO: 24)

As PCR reaction solution, add 0.5 ml each of primer solution consisting of the nucleotide sequence shown in SEQ ID NO: 23 and SEQ ID NO: 24 prepared in 50 M and 5 mM dNTP to 5% genomic DNA. 1 and 10 X buffer (lOOmM Tris-HC1 pH 8.3, 500 mM KCK 15 mM MgCl ₂ , 0.01% Gelatin), thermostable DNA polymerase (Am liTaq Gold) 5U / 1, 0.25 ^ 1, A 5 N aqueous solution of Bainbain was mixed with 10 1 and sterilized ultrapure water was added to make the volume 50 1. The reaction solution was incubated at 95 ° C for 10 minutes (corresponding to the fourth step of the measurement method of the present invention;), and then at 95 ° C for 30 seconds and then at 59 ° C for 30 seconds and further at 72 ° C. PCR was carried out under the conditions of 32 cycles of incubation with 45 seconds as one cycle.

Regarding DNA fragment X2, in the case of group A (untreated group), amplification was confirmed in samples of lpgZl OL and 10 pgZlO L, and amplification products were obtained. On the other hand, in the case of Group B (HpaII, Hhal treatment group), the increase was observed in all samples. 69414

59 No bread products were obtained. On the other hand, regarding the DNA fragment Y2, amplification was confirmed in the l pgZl O L and l O pg / 10 L samples of the A group and the B group, and the amplification products were obtained. Based on the above, using a serum solution as a DNA sample derived from genomic DNA contained in a biological specimen, and using a solution containing divalent cations (magnesium ions) as an annealing buffer Complementary to single-stranded DN A (positive strand) containing the DN A region and part of the 3 ′ end of the single-stranded DNA (but not including the target DNA region) By base pairing with a single-stranded immobilized oligonucleotide having a base sequence, it is possible to select a single-stranded DNA containing the target DNA region as in Example 4 above. In addition, treatment with a methylation-sensitive restriction enzyme makes it possible to detect only methylated DNA without amplifying unmethylated DNA in the target DNA region. Amplify until It was confirmed that the quantify the amount of width is the DNA. Example 6 (Reference test example)

By culturing the breast cancer cell line MCF-7 (ATCC No. HTB-22) derived from mammals purchased from ATCC in a dedicated medium for the cell line described in the ATCC catalog until confluent. About 1 x 10 ⁷ cells were obtained. After adding 10 times volume of SEDTA buffer [10 mM Tris-HCl H 8.0, 10 mM EDTA pH 8.0, lOOmM NaC 1] to the obtained cells, this was homogenized. After adding proteinase K (Sigma) to 500 gZm1 and sodium dodecyl sulfate to 1% (w / v) to the obtained mixture, this was shaken at 55 ° C for about 16 hours. After the completion of the shaking, the mixture was subjected to phenol [saturated with [1M Tris-HCl pH 8.0]] · clo mouthform extraction. The aqueous layer was recovered, and NaC 1 was added to 0.5 N to this, and then the resulting precipitate was recovered by ethanol precipitation. Dissolve the collected precipitate in TE buffer (10 mM Tris, ImM EDTA, pH 8.0), add RNase A (Sigma) to 40 ^ g_ / m 1 and add 1 at 37 ° C. Incubated for hours. 69414

60 The incubated mixture was subjected to phenol / chloroform extraction. 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 nom DNA.

Using the obtained genomic DNA as a saddle type and performing PCR using the following primers and reaction conditions, the base sequence represented by SEQ ID NO: 17 used as a test sample (Genbank Accession No. M80343 etc. DNA fragment (sequence corresponding to nucleotide numbers 257 to 352 of the indicated LINE1 sequence) (DNA fragment X 1, having the nucleotide sequence represented by SEQ ID NO: 25. LI NE 1 sequence represented by Genbank Accession NO.M80343, etc. Region corresponding to base numbers 8 to 480) was amplified.

PF1: 5'-GAGCCAAGATGGCCGAATAGG-3 '(SEQ ID NO: 26)

PR1: 5'-CTGCTTTGTTTACCTAAGCAAGC-3 '(SEQ ID NO: 27) As a PCR reaction solution, the base sequence represented by SEQ ID NO: 26 was prepared in 2 ng of genomic DNA in the form of 铸 and lOO pmol / PL 1 A primer solution consisting of a base sequence represented by SEQ ID NO: 27, 0.125 1 each, 2 mM dNTPs 2.5 1 and 10 X buffer (lOOmM Tris-HC1 pH 8.3, 50 OmM KCK 15mM MgCl ₂ , 0.01% Gelatin) _2.5 1 and heat-resistant DNA polymerase 5 17 1 0.125 1 5 n 1 was used. The reaction solution 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.

After PCR, DNA amplification was confirmed by 1.5% agarose gel electrophoresis, the DNA fragment (473 bp, DNA fragment XI) was excised and purified using the QIAGEN Q IAquick Gel Extraction Kit (QIAGEN). .

A part of the obtained DNA fragment XI was treated with methylase S ss I (NEB), and all 5, 1 CG-3 'methylated DNA fragments (hereinafter referred to as DNA) TJP2007 / 069414

61 Marked as Fragment Yl. ) Again, as before, confirm amplification by 1.5% agarose gel electrophoresis, excise the DNA fragment (473 bp, DNA fragment Yl) and use the QIAGEN QIAquick Gel Extraction Kit (QIAGEN). Purified.

Using DNA fragment XI and DNA fragment Yl, a mixture of the following methylated fragment and non-methyl fragment was prepared. table 1

The following four types of solutions were prepared using each of the DNA fragments of I to V.

Group A (untreated group): Approximately 25 ng of DNA fragment, 2 L of 10 X buffer (330 mM Tris-Acetate pH 7.9, 660 mM K0Ac, lOOmM MgOAc2, 5m Dithothreitol) optimal for Hpa II and Hha I Add 2 L of 10XBSA (Bovine serum albumin lmg / ml), and add sterile ultrapure water to the mixture to a volume of 20 L.

Group B (Hpall treatment group): Approximately 25 ng of DNA fragment, 0.5 U of 11 & 11 and 10 X buffer solution suitable for Hpa II and Hha I (330 mM Tris-Acetate pH 7.9, 66 OmM K0Ac, lOOmM MgOAc2, 5mM Dithothreitol) with 2 L, 10XBSA (Bovine 14

62 serum albumin lmg / ml) was added to 2 iL, and sterilized ultrapure water was added to the mixture to a volume of 20 L.

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. 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.

In order to determine the amount of DNA in the region consisting of the base sequence shown in SEQ ID NO: 17 using 5 L of each diluted solution (equivalent amount of DNA fragment 62.5 pg), the following primers PF 2 and PR 2 and 5 ' Probe T 1 labeled with FAM (6-force lupoxy monofluorescein), which is a reporter fluorescent dye at the end, and TAMRA (6 force lupoxy tetramethyl-mouth damine), which is a quencher-one fluorochrome at the end The real-time PC R used was performed. <Primer>

P F 2 (Forward side): 5'-CACCTGGAAAATCGGGTCACT-3 "(SEQ ID NO: 28)

PR 2 (reverse side): 5'-CGAGCCAGGTGTGGGATATA-3 '(SEQ ID NO: 29) <Probe>

T1: 5'-CGAATATTGCGCTTTTCAGACCGGCTT-3 '(SEQ ID NO: 30)

As a PCR reaction solution, a primer solution consisting of 62.5 pg of the DNA fragment of the vertical type and the base sequence shown in SEQ ID NO: 28 prepared in 3 pmo 11 and the base shown in SEQ ID NO: 29 Two primer solutions consisting of two sequences: 2.5 1 each, 2.5 L of a probe consisting of the nucleotide sequence shown in SEQ ID NO: 30 prepared in 2.5 pmo IZL, 2 mM each dNTP 2.5 1 and 10XPCR buffer (lOOmM Tris-HCl H 8.3, 500 mM KCK 15raM MgCl 0.01% Gelatin) 2.5 1 and thermostable DNA polymerase (AmpliTaq Gold), 5U / 1 0.15 II 1 was mixed, and sterilized ultrapure water was added thereto to make the liquid volume 25 1. Real time PCR was performed using the Gene Amp 5700 Sequence Detection System (Applied Biosystems). In order to amplify the region (DNA) consisting of the base sequence represented by base numbers 1 to 94 among the base sequence represented by SEQ ID NO: 17, the reaction solution was incubated at 95 ° C. for 10 minutes, then 95 Real-time PCR was performed at 15 ° C for 15 seconds and 60 ° C for 60 seconds as one cycle. The amount of DNA in the region was quantified based on the result of the real-time PCR. Three tests were conducted for each biological specimen. The results are shown in FIGS. The amount of DNA in the region in group A was taken as 1, and the amount of DNA in the region in other groups was shown. Figure 8 (“I”) is a fragment mixture with a methylation ratio of 0%, so the theoretical value for Group B, Group C, and Group D is “0”, and FIG. 9 (“II”) is methylation. Since it is a fragment with a ratio of 10%, the theoretical value in Group B, Group C and Group D is “0.1”, and FIG. 10 (“III”) is a fragment with a methylation ratio of 25%. The theoretical value in group C, group C and group D is “0.25”, and since FIG. 11 (“IV”) is a fragment with a methylation ratio of 50%, the theoretical value in groups B, C and D Is “0.5” and FIG. 12 (“V”) is a fragment with a methylation rate of 100%, so the theoretical value in Group B, Group C, and Group D is “1”. As a result of the experiment, as shown in FIGS. 8 to 12, the value closest to this theoretical value was obtained in Group D, and it was confirmed that digestion with two or more methylation sensitive enzymes was preferable. . Industrial applicability

According to the present invention, it is possible to provide a method for easily measuring the content of methylated DNA in a target DNA region possessed by genomic DNA contained in a biological specimen. Sequence listing free text

SEQ ID NO: 1 9 '

Oligonucleotide primers designed for PCR

SEQ ID NO: 2 0

Oligonucleotide primers designed for PCR

SEQ ID NO: 2 1

Piotin-labeled oligonucleotide designed for immobilization on a support SEQ ID NO: 2 3

Oligonucleotide primers designed for PCR

SEQ ID NO: 2 4

Oligonucleotide probes designed for PCR

SEQ ID NO: 2 6

Oligonucleotide primers designed for PCR

SEQ ID NO: 2 7

An oligonucleotide primer designed for PCR

SEQ ID NO: 2 8

Oligonucleotide primers designed for PCR

SEQ ID NO: 2 9

An oligonucleotide primer designed for PCR

SEQ ID NO: 30

Oligonucleotide probes designed for real-time PCR

Claims

The scope of the claims

1. A method for measuring the content of methylated DNA in a target DNA region in genomic DNA contained in a biological sample,

(1) From a DNA sample derived from genomic DNA contained in a biological specimen, a positive single-stranded DNA containing the target DNA region and a part of the 3 ′ end of the single-stranded DNA, provided that The first single-stranded DNA is selected by base pairing with a single-stranded immobilized oligonucleotide having a base sequence that is complementary to Process,

(2) The single-stranded DNA selected in the first step is used as a saddle, the single-stranded immobilized oligonucleotide is used as a primer, and the oligonucleotide is extended once from the brimer to form a double-stranded DNA. A second step of forming DNA,

(3) A third step in which the double-stranded DNA formed in the second step is digested with one or more types of methylation-sensitive restriction enzymes, and then the generated free digest is removed. A double-stranded DNA comprising at least one ammethylated C p G pair at the recognition site of the methylation sensitive restriction enzyme of

(4) Double-stranded DNA of the undigested product obtained in the third step (double-stranded DNA formed by extension that does not contain the CpG pair in the methylated state at the recognition site of the methylation-sensitive restriction enzyme) ) In the single-stranded state, where the undigested double-stranded DNA does not contain the amethylated C p G pair at the recognition site of the methylation-sensitive restriction enzyme. Double-stranded DNA, and

(Five)

(5— a) (i) The generated single-stranded DNA and the single-stranded immobilized oligonucleotide (negative strand) are base-paired to select the single-stranded DNA, ii) A double-stranded DNA is formed by extending the oligonucleotide from the primer once using the selected single-stranded DNA as a saddle and using the single-stranded immobilized oligonucleotide as a primer. 5th-Step a

(5-b) The generated negative-strand single-stranded DNA is a saddle type, and is a partial base sequence of the base sequence possessed by the negative-strand single-stranded DNA, and the salt of the target DNA region A partial base sequence located further 3 'from the 3' end of the base sequence complementary to the base sequence, and an oligonucleotide having a base sequence complementary to A 5-step that forms double-stranded DNA by extending the DNA once, and

Furthermore, after separating the double-stranded DNA formed in each of the 5th_a step and the 5th-b step into a single-stranded state, by repeating the 5th-a step and the 5th-b step, A fifth step of amplifying the methylated DNA in the target DNA region to a detectable amount and quantifying the amount of amplified DNA;

A method characterized by comprising:

2. In the first step, a positive single-stranded DNA containing the target DNA region and a part of the 3 ′ end of the single-stranded DNA, provided that the target DNA region is not included. The base pairing is performed in a reaction system containing a divalent cation when base-pairing with a single-stranded immobilized oligonucleotide having a complementary base sequence. the method of.

3. The method according to claim 2, wherein the divalent cation is a magnesium ion. 4. Prior to the fourth step of claim 1,

A part of the 3 ′ end of a single-stranded single-stranded DNA containing the target DNA region, but not including the target DNA region, is complementary to the base sequence An additional step of adding a single-stranded oligonucleotide that is in a free state into the reaction system, and

The method according to claim 1, further comprising the following one step in addition to the fifth-a step and the fifth-b step:

(5-c) (i) The generated single-stranded DNA is base-paired with the added free single-stranded oligonucleotide to select the single-stranded DNA. (ii) Using the selected single-stranded DNA as a saddle, using the single-stranded oligonucleotide as a primer, and extending the oligonucleotide from the primer once to form double-stranded DNA. — C process.

5. After the fourth step of claim 1,

The method according to claim 1, further comprising the following one step in addition to the fifth_a step and the fifth-b step in the fifth step according to claim 1:

(5-c) (i) The generated single-stranded DNA is base-paired with the added free single-stranded oligonucleotide, and the single-stranded DNA is selected ( ii) Using the selected single-stranded DNA as a saddle, using the single-stranded oligonucleotide as a primer and extending the oligonucleotide once from the primer, a double-stranded DNA is formed. Process.

6. The method according to any one of claims 1 to 5, further comprising the following two steps:

(6) After performing the first step and the second step of the method according to any one of claims 1 to 5, without performing the third step, the fourth and subsequent steps are performed, thereby achieving the above object. DNA of DN A region (total amount of methylated DN A and unmethylated DN A) A sixth step of amplifying to a detectable amount and quantifying the amount of amplified DNA; and

(7) Based on the difference obtained by comparing the amount of DNA quantified in the fifth step and the amount of DNA quantified in the sixth step, methylation in the target DN A region was performed. Seventh step of calculating the percentage of DN A.

7. The method according to any one of claims 1 to 6, wherein the biological specimen is mammalian serum or plasma.

8. The method according to any one of claims 1 to 6, wherein the biological specimen is mammalian blood or body fluid.

9. The method according to any one of claims 1 to 6, wherein the biological specimen is a cell lysate or a tissue lysate.

10. 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. The method according to any one of claims 1 to 9.

11. The DNA sample derived from genomic DNA contained in a biological sample is a DNA sample digested with at least one or more methylation sensitive restriction enzymes. The method of crab.

12. The method according to any one of claims 1 to 11, wherein the DNA sample derived from genomic DNA contained in the biological specimen is a DNA sample purified in advance.

13. Genome D containing one or more methylation-sensitive restriction enzymes in biological samples The method according to any one of claims 1 to 12, which is a restriction enzyme having a recognition cleavage site in a target DNA region in NA.

1 4. The method according to any one of claims 1 to 13, wherein the one or more types of methylation-sensitive restriction enzymes are methylation-sensitive restriction enzymes Hpa II or Hhal.