JP5303981B2 - DNA methylation measurement method - Google Patents

Abstract

Disclosed is are: a method for measuring the content of methylated DNA in a DNA region of interest in the genomic DNA contained in a organism-derived sample; and others.

Description

  The present invention relates to a method for measuring the content of methylated DNA in a target DNA region of genomic DNA contained in a biological specimen.

As a method for evaluating the methylation state of DNA in the target DNA region of the genomic DNA contained in the biological specimen, for example, in the target DNA region of the genomic DNA contained in the biological specimen There are methods for measuring the content of methylated DNA (see, for example, Non-Patent Documents 1 and 2).
In this measurement method, first, it is necessary to extract DNA containing a target DNA region from a DNA sample derived from genomic DNA contained in a biological specimen. As the extraction method, for example, gel filtration, a silica support, an extraction method using an organic solvent, or the like is known, but any of the extraction methods is complicated.
Next, 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 or the like, the DNA synthesis chain reaction by DNA polymerase (Polymerase Chain Reaction; hereinafter sometimes referred to as PCR) to amplify the target region, (2) by digesting the DNA with a methylation sensitive restriction enzyme and then subjecting it to PCR, A method for amplifying a target region can be exemplified.

Clark SJ, Harrison J, Paul CL, Frommer M., High sensitivity mapping of methylated cytosines.Nucleic Acids Res. 1994 Aug 11; 22 (15): 2990-7. Ushijima T, Morimura K, Hosoya Y, Okonogi H, Tatematsu M, Sugimura T, Nagao M., Establishment of methylation-sensitive- representational difference analysis and isolation of hypo- and hypermethylated genomic fragments in mouse liver tumors.Proc Natl Acad Sci US A. 1997 Mar 18; 94 (6): 2284-9.

In any of the methods described above, operations (specifically, for example, modification of DNA for detection of methylation and subsequent purification of the product, etc.) are very time consuming and labor intensive before being subjected to PCR. Because PCR is carried out in a liquid phase using a pair of oligonucleotide primers complementary to the base sequence of the DNA of the target region to be amplified (hereinafter also referred to as a primer pair). An operation for purifying the amplified DNA fragment and transferring it to a reaction vessel for PCR, an operation for adding a reaction reagent containing the primer pair for amplifying a target region to the reaction system, etc. Is also necessary. Furthermore, in order to confirm the amplification of the DNA of the target region to be amplified, it is necessary to use it for an analytical operation such as electrophoresis, and the operation is extremely complicated.
As described above, a series of operations for measuring the content of methylated DNA in the target DNA region possessed by the genomic DNA contained in the biological specimen had a lot of trouble.

As a result of intensive studies under such circumstances, the present inventors have reached the present invention.
That is, the present invention
1. A method for measuring the content of methylated DNA in a target DNA region possessed by genomic DNA contained in a biological specimen,
(1) Obtaining methylated single-stranded DNA from a genomic DNA-derived DNA sample contained in a biological specimen, and binding the single-stranded DNA with an immobilized methylated DNA antibody A first step of selecting DNA,
(2) a second step of digesting the single-stranded DNA selected in the first step with a methylation sensitive restriction enzyme capable of digesting at least one kind of single-stranded DNA; and
(3) As a pre-process of each of the following steps, single-stranded DNA that is an undigested product obtained in the second step (the methylation state is present at the recognition site of the methylation-sensitive restriction enzyme capable of digesting the single-stranded DNA). A single-stranded DNA containing no CpG is separated from the immobilized methylated DNA antibody into a single-stranded DNA (positive strand) (third (previous A) third (previous A) ) Process)
Using the DNA (positive strand) in the single-stranded state separated in the third (previous A) step as a template, the partial base sequence (positive strand) of the base sequence of the DNA (positive strand) in the single-stranded state And a base sequence (complementary to a partial base sequence (positive strand) located further 3 ′ end than the 3 ′ end of the base sequence (positive strand) of the target DNA region ( The extension primer (forward primer) having a negative strand) is used as an extension primer, and the extension primer is extended once to extend the single-stranded DNA (positive strand) as a double-stranded DNA. A step (third (previous B) step)
The double-stranded DNA extended and formed in the third (previous B) step contains a single-stranded DNA (positive strand) containing the target DNA region and a base sequence that is complementary to the target DNA region. A single-stranded DNA (positive strand) having a step (third (previous C) step) of once separating into single-stranded DNA (negative strand) and including (a) the generated target DNA region as this step ) As a template, the forward primer as an extension primer, and the extension primer is extended once, thereby extending the single-stranded DNA containing the target DNA region as a double-stranded DNA. A and
(B) Using the generated single-stranded DNA (negative strand) containing a base sequence complementary to the target DNA region as a template, the base sequence complementary to the target DNA region is A base sequence that is a partial base sequence (negative strand) of the base sequence of the contained single-stranded DNA (negative strand) and that is complementary to the base sequence (positive strand) of the target DNA region An extension primer (reverse primer) having a base sequence (positive strand) that is complementary to the partial base sequence (negative strand) located further 3 'end than the 3' end of (negative strand) As the step-B, the extension primer is extended once to form a single-stranded DNA containing the target DNA region as a double-stranded DNA.
Furthermore, each of these steps was once methylated in the target DNA region by repeating the extension-formed double-stranded DNA obtained in each of the steps once after separating it into a single-stranded state. A method comprising a third step of amplifying the DNA to a detectable amount and quantifying the amount of the amplified DNA (hereinafter sometimes referred to as the measurement method of the present invention);
2. The content of methylated DNA in the target DNA region of the genomic DNA contained in the biological specimen according to claim 1, wherein the methylated DNA antibody in the third step according to claim 1 is a methylcytosine antibody. How to measure;
3. The method according to claim 1, wherein the biological specimen is mammalian serum or plasma;
4). The method according to any one of claims 1 to 2, wherein the biological specimen is mammalian blood or body fluid.
5. The method according to any one of claims 1 to 2, wherein the biological specimen is a cell lysate or a tissue lysate;
6). 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 5;
7). The DNA sample derived from genomic DNA contained in a biological specimen is a DNA sample obtained by digestion with at least one methylation sensitive restriction enzyme. Described method;
8). The method according to any one of claims 1 to 7, wherein the DNA sample derived from genomic DNA contained in the biological specimen is a DNA sample purified in advance;
9. The methylation-sensitive restriction enzyme capable of digesting at least one kind of single-stranded DNA is a restriction enzyme having a recognition cleavage site in a target DNA region of genomic DNA contained in a biological sample. A method according to any one of claims 1 to 8;
10. The method according to any one of claims 1 to 9, wherein the methylation sensitive restriction enzyme capable of digesting at least one kind of single-stranded DNA is HhaI which is a methylation sensitive restriction enzyme;
11. The method according to any one of the preceding items 1 to 10, wherein the third step is performed without performing a digestion treatment with a methylation sensitive restriction enzyme capable of digesting single-stranded DNA in the second step;
12 A first step (A) for separating methylated single-stranded DNA from a DNA sample derived from genomic DNA contained in a biological specimen;
The first (B) step of binding the single-stranded DNA separated in the first (A) step and the immobilized methylated DNA antibody,
The method according to any one of 1 to 11 above, wherein a counter oligonucleotide is added when separating the methylated single-stranded DNA in the first step (A);
Etc. are provided.

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

The present invention is described in detail below.
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. And biological samples such as body fluids such as serum and lymph, body secretions (such as urine and milk), and genomic DNA obtained by extraction from these biological samples. 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 of microorganisms and viruses. .
When the mammal-derived specimen is blood, it can be expected that the measurement method of the present invention will be used in periodic health examinations and simple tests.
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.
Incidentally, 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 (cancer cells such as gastric cancer cells). Analysis of cancer cells such as gastric cancer cells, avoiding blood cell-derived DNA, and sensitivity for detecting cancer cells such as gastric cancer cells and tissues containing them Can be improved.

The “methylated DNA” in the present invention means the following DNA. Usually, there are four types of bases constituting a gene (genomic DNA). Among these bases, the phenomenon that only cytosine is methylated is known, and such methylation modification of DNA is a base sequence represented by 5′-CG-3 ′ (C represents cytosine, G represents guanine.Hereinafter, the base sequence may be referred to as “CpG”.) The site that is methylated in cytosine is at position 5. During DNA replication prior to cell division, only cytosine in the template strand “CpG” is methylated immediately after replication, but the cytosine in the nascent strand “CpG” is also immediately activated by the action of the methyltransferase. Methylated. Therefore, the DNA methylation state is inherited as it is by two new sets of DNA even after DNA replication. It means DNA produced by such methylation modification.
The “CpG pair” in the present invention means a double-stranded oligonucleotide formed by binding a base sequence represented by CpG and CpG complementary thereto.

  A “target DNA region” in the present invention (hereinafter sometimes referred to as a target region) is a DNA region to be examined for the presence or absence of methylation of cytosine contained in the region, and is at least one kind of methylation. It has a recognition site for a sensitive restriction enzyme, such as Lysyl oxidase, HRAS-like suppressor, bA305P22.2.1, Gamma filamin, HAND1, Homologue of RIKEN 2210016F16, FLJ32130, PPARG angiopoietin-related protein, Thrombomodulin, p53-responsive gene 2, Promoter region of useful protein genes such as Fibrillin2, Neurofilament 3, disintegrin and metalloproteinase domain 23, G protein-coupled receptor 7, G-protein coupled somatostatin and angiotensin-like peptide receptor, Solute carrier family 6 neurotransmitter transporter noradrenalin member 2, etc. A salt represented by one or more CpGs present in the base sequence of the translation region or translation region (coding region) Examples thereof include a DNA region containing cytosine in the base sequence.

  Specifically, for example, when the useful protein gene is a Lysyl oxidase gene, the nucleotide sequence represented by one or more CpGs present in the nucleotide sequence of the promoter region, untranslated region or translated region (coding region) As an example, a base sequence of genomic DNA containing exon 1 of a human-derived Lysyl oxidase gene and a promoter region located 5 ′ upstream thereof can be mentioned, and more specifically, represented by SEQ ID NO: 1. And a base sequence (corresponding to the base sequence represented by base numbers 16001 to 18661 of the base sequence described in Genbank Accession No. AF270645). In the base 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 base numbers 2031 to 2033, and the base sequence of exon 1 is the base number 1957-2661. Cytosine in the base sequence represented by CpG present in the base sequence represented by SEQ ID NO: 1, particularly cytosine in CpG present in the region where CpG is densely present in the base sequence represented by SEQ ID NO: 1, In cancer cells such as gastric cancer cells, a high methylation frequency (ie, hypermethylation state) is exhibited. More specifically, as cytosine having high methylation frequency in gastric cancer cells, for example, in the base sequence represented by SEQ ID NO: 1, base numbers 1539, 1560, 1574, 1600, 1623, 1635, 1644, 1654, 1661, The cytosine which is a base number shown by 1682, 1686, 1696, 1717, 1767, 1774, 1783, 1785, 1787, 1795 etc. can be mentioned.

  More specifically, for example, when the useful protein gene is an HRAS-like suppressor gene, it is represented by one or more CpGs present in the base sequence of the promoter region, untranslated region or translated region (coding region). Examples of the nucleotide sequence that can be mentioned include the nucleotide sequence of genomic DNA containing exon 1 of the human-derived HRAS-like suppressor gene and the promoter region located 5 ′ upstream thereof. 2 (corresponding to the nucleotide sequence represented by nucleotide numbers 172001 to 173953 of the nucleotide sequence described in Genbank Accession No. AC068162). In the base sequence represented by SEQ ID NO: 2, the base sequence of exon 1 of the human-derived HRAS-like suppressor gene is represented by base numbers 1743 to 1953. The cytosine in the base sequence represented by CpG present in the base sequence represented by SEQ ID NO: 2, particularly the cytosine in CpG present in the region where CpG is densely present in the base sequence represented by SEQ ID NO: 2, In cancer cells such as gastric cancer cells, a high methylation frequency (ie, hypermethylation state) is exhibited. 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, 1513, 1518, 1520 etc. Cytosine, which is the base number to be generated.

  More specifically, for example, when the useful protein gene is the bA305P22.2.1 gene, it is represented by one or more CpGs present in the base sequence of the promoter region, untranslated region or translated region (coding region). Examples of the nucleotide sequence include a nucleotide sequence of genomic DNA containing exon 1 of the human-derived bA305P22.2.1 gene and a promoter region located 5 ′ upstream thereof, and more specifically, 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). In the base sequence represented by SEQ ID NO: 3, the ATG codon encoding the amino terminal methionine of the human-derived bA305P22.2.1 protein is represented by base numbers 849 to 851, and the base sequence of the exon 1 is a base sequence. Nos. 663-889. The cytosine in the base sequence represented by CpG present in the base sequence represented by SEQ ID NO: 3, particularly the cytosine in CpG present in the region where CpG is densely present in the base sequence represented by SEQ ID NO: 3, In cancer cells such as gastric cancer cells, a high methylation frequency (ie, hypermethylation state) is exhibited. 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, The cytosine which is a base number shown by 446, 465, 472, 486 etc. can be mention | raise | lifted.

  More specifically, for example, when the useful protein gene is a Gamma filamin gene, it is represented by one or more CpGs present in the base sequence of the promoter region, untranslated region, or translated region (coding region). Examples of the base sequence include a genomic DNA base sequence containing exon 1 of a human-derived Gamma filamin gene and a promoter region located 5 ′ upstream thereof. The base sequence shown (corresponding to the complementary sequence of the base sequence shown by base numbers 63528 to 64390 of the base sequence described in Genbank Accession No. AC074373). In the base sequence shown in SEQ ID NO: 4, the ATG codon encoding the amino terminal methionine of human-derived Gamma filamin protein is shown in base numbers 572 to 574, and the base sequence of exon 1 is the base number 463-863. The cytosine in the base sequence represented by CpG present in the base sequence represented by SEQ ID NO: 4, particularly the cytosine in CpG present in the region where CpG is densely present in the base sequence represented by SEQ ID NO: 4 is, for example, In cancer cells such as gastric cancer cells, a high methylation frequency (ie, hypermethylation state) is exhibited. More specifically, as cytosine having high methylation frequency in gastric cancer cells, for example, in the base sequence represented by SEQ ID NO: 4, base numbers 329, 333, 337, 350, 353, 360, 363, 370, 379, Examples thereof include cytosine having base numbers represented by 382, 384, 409, 414, 419, 426, 432, 434, 445, 449, 459, 472, 474, 486, 490, 503, 505, and the like.

  More specifically, for example, when the useful protein gene is a HAND1 gene, one or more bases represented by one or more CpGs present in the base sequence of the promoter region, untranslated region or translated region (coding region). Examples of the sequence include the base sequence of genomic DNA containing exon 1 of human-derived HAND1 gene and a promoter region located 5 ′ upstream thereof, and more specifically, represented by SEQ ID NO: 5. And a base sequence (corresponding to a complementary sequence of the base sequence represented by base numbers 24303 to 26500 of the base sequence described in Genbank Accession No. AC026688). In the base sequence shown in SEQ ID NO: 5, the ATG codon encoding the amino terminal methionine of the human-derived HAND1 protein is shown in base numbers 1656 to 1658, and the base sequence of the exon 1 is base number 1400. ~ 2198. The cytosine in the base sequence represented by CpG present in the base sequence represented by SEQ ID NO: 5, particularly the cytosine in CpG present in the region where CpG is densely present in the base sequence represented by SEQ ID NO: 5 is, for example, In cancer cells such as gastric cancer cells, a high methylation frequency (ie, hypermethylation state) is exhibited. 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, 1272, Examples include cytosine having base numbers represented by 1292, 1305, 1307, 1316, 1356, 1377, 1399, 1401, 1422, 1434 and the like.

  Further, specifically, for example, when the useful protein gene is a homologue of RIKEN 2210016F16 gene, one or more CpGs present in the base sequence of the promoter region, untranslated region or translated region (coding region). Examples of the base sequence shown include a base sequence of genomic DNA containing exon 1 of the human-derived Homologue of RIKEN 2210016F16 gene and a promoter region located 5 ′ upstream thereof, and more specifically, And 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 base sequence represented by SEQ ID NO: 6, the base sequence of exon 1 of the human-derived homologue of RIKEN 2210016F16 protein is represented by base numbers 1392 to 1945. The cytosine in the base sequence represented by CpG present in the base sequence represented by SEQ ID NO: 6, particularly the cytosine in CpG present in the region where CpG is densely present in the base sequence represented by SEQ ID NO: 6 is, for example, In cancer cells such as gastric cancer cells, a high methylation frequency (ie, hypermethylation state) is exhibited. 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, 1175, 1180, 1183, 1189, 1204, 1209, 1267, 1271, Examples include cytosine having base numbers represented by 1278, 1281, 1313, 1319, 1332, 1334, 1338, 1346, 1352, 1358, 1366, 1378, 1392, 1402, 1433, 1436, 1438, and the like.

  More specifically, for example, when the useful protein gene is the FLJ32130 gene, one or more bases represented by one or more CpGs present in the base sequence of the promoter region, untranslated region or translated region (coding region). Examples of the sequence include a genomic DNA base sequence containing exon 1 of human-derived FLJ32130 gene and a promoter region located 5 ′ upstream thereof, and more specifically, represented by SEQ ID NO: 7. Examples thereof include base sequences (corresponding to complementary base sequences of base sequences represented by base numbers 1 to 2379 of base sequences described in Genbank Accession No. AC002310). In the base sequence shown in SEQ ID NO: 7, the ATG codon encoding the methionine at the amino acid terminal of human-derived FLJ32130 protein is shown in base numbers 2136 to 2138, and the base sequence considered to be exon 1 is a base sequence The number 2136-2379 is shown. The cytosine in the base sequence represented by CpG present in the base sequence represented by SEQ ID NO: 7, particularly the cytosine in CpG present in the region where CpG is densely present in the base sequence represented by SEQ ID NO: 7, In cancer cells such as gastric cancer cells, a high methylation frequency (ie, hypermethylation state) is exhibited. More specifically, as cytosine having high methylation frequency in gastric cancer cells, for example, in the base sequence represented by SEQ ID NO: 7, base numbers 1714, 1716, 1749, 1753, 1762, 1795, 1814, 1894, 1911, Examples thereof include cytosine having base numbers represented by 1915, 1925, 1940, 1955, 1968 and the like.

  More specifically, for example, when the useful protein gene is a PPARG angiopoietin-related protein gene, one or more CpGs present in the base sequence of the promoter region, untranslated region or translated region (coding region). The base sequence represented by can include the base sequence of genomic DNA containing exon 1 of human-derived PPARG angiopoietin-related protein gene and a promoter region located 5 ′ upstream thereof, and more specifically Is the base sequence represented by SEQ ID NO: 8. In the base sequence represented by SEQ ID NO: 8, the ATG codon encoding the amino terminal methionine of the human-derived PPARG angiopoietin-related protein protein is represented by base numbers 717 to 719, and the 5 ′ side of exon 1 above. The base sequence of the portion is shown in base numbers 1957 to 2661. The cytosine in the base sequence represented by CpG present in the base sequence represented by SEQ ID NO: 8, particularly the cytosine in CpG present in the region where CpG is densely present in the base sequence represented by SEQ ID NO: 8, In cancer cells such as gastric cancer cells, a high methylation frequency (ie, hypermethylation state) is exhibited. More specifically, as cytosine having a high methylation frequency in gastric cancer cells, for example, in the base sequence represented by SEQ ID NO: 8, base numbers 35, 43, 51, 54, 75, 85, 107, 127, 129, Examples thereof include cytosine having base numbers represented by 143, 184, 194, 223, 227, 236, 251 and 258.

  Further, specifically, for example, when the useful protein gene is a Thrombomodulin gene, one or more bases represented by one or more CpGs present in the base sequence of the promoter region, untranslated region or translated region (coding region). Examples of the sequence include a genomic DNA base sequence containing exon 1 of a human-derived Thrombomodulin gene and a promoter region located 5 ′ upstream thereof. More specifically, the sequence is represented by SEQ ID NO: 9. And a base sequence (corresponding to the base sequence represented by base numbers 1 to 6096 of the base sequence described in Genbank Accession No. AF495471). In the base sequence shown in SEQ ID NO: 9, the ATG codon encoding the amino terminal methionine of human-derived Thrombomodulin protein is shown in base numbers 2590 to 2592. The base sequence of exon 1 is base number 2048. ~ 6096. The cytosine in the base sequence represented by CpG present in the base sequence represented by SEQ ID NO: 9, particularly the cytosine in CpG present in the region where CpG is densely present in the base sequence represented by SEQ ID NO: 9 is, for example, In cancer cells such as gastric cancer cells, a high methylation frequency (ie, hypermethylation state) is exhibited. More specifically, as cytosine having high methylation frequency in gastric cancer cells, for example, in the base sequence represented by SEQ ID NO: 9, base numbers 1539, 1551, 1571, 1579, 1581, 1585, 1595, 1598, 1601, Examples include cytosine having base numbers represented by 1621, 1632, 1638, 1645, 1648, 1665, 1667, 1680, 1698, 1710, 1724, 1726, 1756, and the like.

  More specifically, for example, when the useful protein gene is p53-responsive gene 2 gene, one or more CpGs present in the base sequence of the promoter region, untranslated region or translated region (coding region). Examples of the base sequence shown by can include a genomic DNA base sequence containing exon 1 of human-derived p53-responsive gene 2 gene and a promoter region located 5 ′ upstream thereof, and more specifically, Is a base sequence represented by SEQ ID NO: 10 (corresponding to a complementary sequence of the base sequence represented by base numbers 113501 to 116000 of the base sequence described in Genbank Accession No. AC009471). In the base sequence represented by SEQ ID NO: 10, the base sequence of exon 1 of human-derived p53-responsive gene 2 gene is represented by base numbers 1558 to 1808. Cytosine in the base sequence represented by CpG present in the base sequence represented by SEQ ID NO: 10 exhibits a high methylation frequency (ie, hypermethylation state) in cancer cells such as pancreatic cancer cells. . More specifically, as cytosine having high methylation frequency in pancreatic cancer cells, for example, in the base sequence represented by SEQ ID NO: 10, base numbers 1282, 1284, 1301, 1308, 1315, 1319, 1349, 1351, 1357 , 1361, 1365, 1378, 1383, etc. can be mentioned cytosine.

  More specifically, for example, when the useful protein gene is Fibrillin2 gene, one or more bases represented by CpG present in the base sequence of the promoter region, untranslated region or translated region (coding region). Examples of the sequence include a base sequence of genomic DNA containing exon 1 of the Fibrillin 2 gene derived from human and a promoter region located 5 ′ upstream thereof. More specifically, the sequence is represented by SEQ ID NO: 11. And a base sequence (corresponding to a complementary sequence of the base sequence represented by base numbers 118801 to 121000 of the base sequence described in Genbank Accession No. AC113387). In the base sequence represented by SEQ ID NO: 11, the base sequence of exon 1 of the human-derived Fibrillin2 gene is represented by base numbers 1091 to 1345. Cytosine in the base sequence represented by CpG present in the base sequence represented by SEQ ID NO: 11 exhibits a high methylation frequency (ie, hypermethylation state) in cancer cells such as pancreatic cancer cells. . More specifically, as cytosine having high methylation frequency in pancreatic cancer cells, for example, in the base sequence represented by SEQ ID NO: 11, base numbers 679, 687, 690, 699, 746, 773, 777, 783, 795 , 799, 812, 823, 830, 834, 843, and the like.

  More specifically, for example, when the useful protein gene is a Neurofilament 3 gene, the base represented by one or more CpGs present in the base sequence of the promoter region, untranslated region or translated region (coding region). Examples of the sequence include a base sequence of genomic DNA containing exon 1 of a human-derived Neurofilament 3 gene and a promoter region located 5 ′ upstream thereof, and more specifically, represented by SEQ ID NO: 12. And a base sequence (corresponding to a complementary sequence of the base sequence represented by base numbers 28001 to 30000 of the base sequence described in Genbank Accession No. AF106564). In the base sequence represented by SEQ ID NO: 12, the base sequence of exon 1 of the human-derived Neurofilament 3 gene is represented by base numbers 614 to 1694. Cytosine in the base sequence represented by CpG present in the base sequence represented by SEQ ID NO: 12 exhibits high methylation frequency (ie, hypermethylation state) in cancer cells such as pancreatic cancer cells. . More specifically, as cytosine having high methylation frequency in pancreatic cancer cells, for example, in the base sequence represented by SEQ ID NO: 12, base numbers 428, 432, 443, 451, 471, 475, 482, 491, 499 , 503, 506, 514, 519, 532, 541, 544, 546, 563, 566, 572, 580, and the like, can be mentioned cytosine.

  More specifically, for example, when the useful protein gene is disintegrin and metalloproteinase domain 23 gene, one or more CpGs present in the base sequence of the promoter region, untranslated region or translated region (coding region). The base sequence represented by can include a base sequence of genomic DNA containing exon 1 of human-derived disintegrin and metalloproteinase domain 23 gene and a promoter region located 5 ′ upstream thereof, and more specifically Is 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). 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. Cytosine in the base sequence represented by CpG present in the base sequence represented by SEQ ID NO: 13 exhibits high methylation frequency (ie, hypermethylation state) in cancer cells such as pancreatic cancer cells. . More specifically, as cytosine having high methylation frequency in pancreatic cancer cells, for example, in the base sequence represented by SEQ ID NO: 13, base numbers 998, 1003, 1007, 1011, 1016, 1018, 1020, 1026, 1028 , 1031, 1035, 1041, 1043, 1045, 1051, 1053, 1056, 1060, 1066, 1068, 1070, 1073, 1093, 1096, 1106, 1112, 1120, 1124, 1126 etc. I can give you.

  Also, specifically, for example, when the useful protein gene is a G protein-coupled receptor 7 gene, one or more existing in the base sequence of the promoter region, untranslated region or translated region (coding region) Examples of the base sequence represented by CpG include a base sequence of genomic DNA containing exon 1 of a human-derived G protein-coupled receptor 7 gene and a promoter region located 5 ′ upstream thereof. Specifically, the base sequence represented by SEQ ID NO: 14 (corresponding to the base sequence represented by base numbers 75001 to 78000 of the base sequence described in Genbank Accession No. AC009800) can be mentioned. In the base sequence represented by SEQ ID NO: 14, the base sequence of exon 1 of human-derived G protein-coupled receptor 7 gene is represented by base numbers 1666 to 2652. Cytosine in the base sequence represented by CpG present in the base sequence represented by SEQ ID NO: 14 exhibits high methylation frequency (ie, hypermethylation state) in cancer cells such as pancreatic cancer cells. . More specifically, as cytosine having high methylation frequency in pancreatic cancer cells, for example, in the base sequence represented by SEQ ID NO: 14, base numbers 1480, 1482, 1485, 1496, 1513, 1526, 1542, 1560, 1564 , 1568, 1570, 1580, 1590, 1603, 1613, 1620, etc.

  Specifically, for example, when the useful protein gene is a G-protein coupled somatostatin and angiotensin-like peptide receptor gene, the base of the promoter region, untranslated region or translated region (coding region) of the promoter region The base sequence represented by one or more CpGs present in the sequence includes exon 1 of human-derived G-protein coupled somatostatin and angiotensin-like peptide receptor gene and a promoter region located 5 ′ upstream thereof. More specifically, the base sequence of the base sequence represented by SEQ ID NO: 15 (the base sequence represented by base numbers 5701 to 60000 of the base sequence described in Genbank Accession No. AC008971) can be mentioned. Corresponding to a complementary sequence). In the base sequence represented by SEQ ID NO: 15, the base sequence of exon 1 of human-derived G-protein coupled somatostatin and angiotensin-like peptide receptor gene is represented by base numbers 776 to 2632. Cytosine in the base sequence represented by CpG present in the base sequence represented by SEQ ID NO: 15 exhibits a high methylation frequency (ie, hypermethylation state) in cancer cells such as pancreatic cancer cells. . More specifically, as cytosine with high methylation frequency in pancreatic cancer cells, for example, in the base sequence represented by SEQ ID NO: 15, base numbers 470, 472, 490, 497, 504, 506, 509, 514, 522 , 540, 543, 552, 566, 582, 597, 610, 612 and the like.

  More specifically, for example, when the useful protein gene is the Solute carrier family 6 neurotransmitter transporter noradrenalin member 2 gene, it is present in the nucleotide sequence of the promoter region, untranslated region or translated region (coding region). Examples of the base sequence represented by one or more CpG include the base sequence of genomic DNA containing exon 1 of the human-derived Solute carrier family 6 neurotransmitter transporter noradrenalin member 2 gene and the promoter region located 5 ′ upstream thereof. More specifically, the base sequence represented by SEQ ID NO: 16 (corresponding to the complementary sequence of the base sequence represented by base numbers 78801 to 81000 of the base sequence described in Genbank Accession No. AC026802) Can be given. In the nucleotide sequence represented by SEQ ID NO: 16, the nucleotide sequence of exon 1 of the human-derived Solute carrier family 6 neurotransmitter transporter noradrenalin member 2 gene is represented by nucleotide numbers 1479 to 1804. Cytosine in the base sequence represented by CpG present in the base sequence represented by SEQ ID NO: 16 exhibits a high methylation frequency (ie, hypermethylation state) in cancer cells such as pancreatic cancer cells. . More specifically, as cytosine having high methylation frequency in pancreatic cancer cells, for example, in the base sequence represented by SEQ ID NO: 16, base numbers 1002, 1010, 1019, 1021, 1051, 1056, 1061, 1063, 1080 , 1099, 1110, 1139, 1141, 1164, 1169, 1184, etc.

The “methylated DNA antibody” in the measurement method of the present invention means an antibody that binds with a methylated base in DNA as an antigen. A specific example is a methylcytosine antibody, which has the property of recognizing and binding to cytosine methylated at the 5-position in single-stranded DNA. Further, even a commercially available methylated DNA antibody may be an antibody that can specifically recognize and specifically bind to the methylated DNA described in this patent.
A methylated DNA antibody can be prepared by a conventional method using a methylated base, methylated DNA or the like as an antigen. Specifically, in order to prepare a methylcytosine antibody, specificity from 5-methylcytidine, 5-methylcytosine, or an antibody prepared using DNA containing 5-methylcytosine as an antigen to methylcytosine in DNA It can produce by selecting as a parameter | index a simple coupling | bonding.
As an antibody obtained by immunizing an animal with an antigen, a method using an antibody (polyclonal antibody) of an IgG fraction after immunization with a purified antigen and an antibody (monoclonal antibody) producing a single clone are used. There is a way to do it. In this patent, it is desirable that the antibody is capable of specifically recognizing methylated DNA or methylcytosine, and therefore it is desirable to use a monoclonal antibody.
As a method for producing a monoclonal antibody, a cell fusion method can be mentioned. For example, in the cell fusion method, a hybridoma is prepared by cell fusion of a spleen cell (B cell) derived from an immunized mouse and a myeloma cell, an antibody produced by the hybridoma is selected, and a methylcytosine antibody (monoclonal antibody) is selected. Can be produced. When producing a monoclonal antibody by the cell fusion method, it is not necessary to purify the antigen. For example, 5-methylcytidine, 5-methylcytosine, or a mixture of DNA containing 5-methylcytosine is used as an antigen for immunization. It can be administered to the animals used. As an administration method, 5-methylcytidine, 5-methylcytosine, or DNA containing 5-methylcytosine is directly administered to a mouse producing an antibody. When it is difficult to produce an antibody, immunization may be performed by binding an antigen to a support. In addition, by mixing well with an adjuvant solution (for example, liquid paraffin and Aracel A mixed with Mycobacterium tuberculosis killed as an adjuvant) and immunizing by incorporating into a liposome, the immunity of the antigen Can be raised. Alternatively, add an equal amount of an antigen-containing solution and an adjuvant solution to make it sufficiently emulsion, then inject it subcutaneously or intraperitoneally into mice, or mix well with alum water and add pertussis bacteria as an adjuvant There is a way to do it. It is also possible to boost the mouse intraperitoneally or intravenously after an appropriate period after the initial immunization. When the amount of the antigen is small, a solution in which the antigen is suspended may be directly injected into the mouse spleen for immunization.
A few days after the final immunization, the spleen is removed and the adipose tissue is removed, and then a spleen cell suspension is prepared. The spleen cells are fused with, for example, HGPRT-deficient myeloma cells to produce a hybridoma. Any cell fusion agent may be used as long as it can efficiently fuse spleen cells (B cells) and myeloma cells. Examples thereof include a method using Sendai virus (HVJ) and polyethylene glycol (PEG). Further, cell fusion may be performed by a method using a high voltage pulse.
After the cell fusion operation, the cells are cultured in HAT medium, a hybridoma clone in which spleen cells and myeloma cells are fused is selected, and the cells are allowed to grow until screening becomes possible. An antigen-antibody reaction system can be used for an antibody detection method or antibody titer measurement method for selecting a hybridoma producing the target antibody. Specific examples of antibody measurement methods for soluble antigens include radioisotope immunoassay (RIA) and enzyme immunoassay (ELISA).

  If CpG present in the single-stranded DNA is methylated at least at one site, it can bind to the anti-methylated antibody. Therefore, the “methylated single-stranded DNA” in the measurement method of the present invention means a single-stranded DNA in which CpG present in the single-stranded DNA is methylated in at least one site. This is not limited to single-stranded DNA in which all CpGs present in the double-stranded DNA are methylated.

  In the present invention, “the amount of amplified DNA (amplified to a detectable amount of methylated DNA in the target DNA region)” refers to the purpose of genomic DNA contained in a biological sample. This means the amount of methylated DNA in the region after amplification itself, that is, the amount determined in the third step of the measurement method of the present invention. 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.

  In the present invention, the “single-stranded DNA containing at least one CpG in the methylated state at the recognition site of the methylation-sensitive restriction enzyme capable of digesting the single-stranded DNA” includes the recognition site of the restriction enzyme. It means single-stranded DNA in which cytosine in at least one or more existing CpGs is unmethylated cytosine. The “single-stranded DNA containing no CpG in the methylated state at the recognition site of the methylation-sensitive restriction enzyme capable of digesting the single-stranded DNA” is present in the recognition site of the restriction enzyme in the single-stranded DNA. It means single-stranded DNA in which cytosine in all CpGs is methylated.

“Binding by complementarity (complementary (by base pairing) binding)” means forming a double-stranded DNA by base pairing by hydrogen bonding between bases. For example, the bases constituting each single-stranded DNA of the double-stranded DNA constituting the DNA form a double strand by purine and pyrimidine base pairing, more specifically, a plurality of consecutive, This means that double-stranded DNA is formed by base bonding by hydrogen bonding between thymine and cytosine and guanine and adenine. Binding by complementarity is sometimes referred to as “complementary binding”. “Complementary binding” may also be expressed as “complementary base pairing” or “binding by complementarity”. In addition, base sequences that can bind complementarily may be expressed as “complementary” [complementary] to each other. It also means that inosine contained in an artificially prepared oligonucleotide binds to cytosine, adenine, and thymine by hydrogen bonding.
“Single-stranded DNA containing the target DNA region (base sequence complementary to the target DNA)” forms a conjugate (double-stranded) with the single-stranded DNA containing the target DNA region. This means that it is a base sequence necessary for this purpose, that is, a base sequence containing a base sequence complementary to a part of the base sequence of the target DNA region, and may be expressed as a “complementary base sequence”. A complementary base sequence may be expressed as “complementary”.

In the first step of the measurement method of the present invention, methylated single-stranded DNA is obtained from a genomic DNA-derived DNA sample contained in a biological specimen, the single-stranded DNA, an immobilized methylated DNA antibody, To select single-stranded DNA. In the first step, a first (A) step of separating methylated single-stranded DNA from a genomic DNA-derived DNA sample contained in a biological sample, and methylated single-stranded DNA This comprises the first step (B) of selecting the single-stranded DNA by binding with an immobilized methylated DNA antibody.
In order to obtain single-stranded DNA, the double-stranded DNA may be obtained by separating into single-stranded DNA, or the single-stranded DNA originally contained in the sample may be obtained as it is. In order to separate the double-stranded DNA into single-stranded DNA, a normal technique can be used.

  In the first (A) step of the measurement method of the present invention, when “isolating methylated single-stranded DNA from a genomic DNA-derived DNA sample contained in a biological sample”, one double-stranded DNA What is necessary is just to perform general operation for making into strand DNA. Specifically, a genomic DNA-derived DNA sample contained in a biological specimen may be dissolved in an appropriate amount of ultrapure water, heated at 95 ° C. for 10 minutes, and rapidly cooled in ice.

The “immobilized methylated DNA antibody” in the first (B) step of the measurement method of the present invention is used to select methylated single-stranded DNA from a genomic DNA-derived DNA sample contained in a biological specimen. It is done. The immobilized methylated DNA antibody may be any antibody that can be immobilized on a support, and “an antibody that can be immobilized on a support” means that a methylated DNA antibody is directly or indirectly immobilized on a support. Means you can.
In order to be immobilized in this way, the methylated DNA antibody may be immobilized on a support according to a normal genetic engineering operation method or a commercially available kit / device (binding to a solid phase). Specifically, a support in which a biotinylated methylated DNA antibody obtained by biotinylating a methylated DNA antibody is coated with streptavidin (for example, a PCR tube coated with streptavidin, a magnetic bead coated with streptavidin, etc.) The method of fixing to can be mentioned.
Further, for example, a glass having a surface in which a molecule having an active functional group such as an amino group, a thiol group, or an aldehyde group is covalently bonded to a methylated DNA antibody, and the surface thereof is activated with a silane coupling agent or the like. There is also a method of covalently bonding to a saccharide derivative, silica gel, the above synthetic resin or the like, or a heat-resistant plastic support. In the case of covalent bonding, for example, the covalent bonding may be performed using a spacer, a cross linker, or the like formed by connecting five triglycerides in series.
Furthermore, the methylated DNA antibody may be directly immobilized on the support, and the antibody against the methylated DNA antibody (secondary antibody) is immobilized on the support and the methylated antibody is bound to the secondary antibody. It may be fixed to the body.
In selecting the single-stranded DNA (positive strand) containing the target DNA region, the immobilized methylated DNA antibody may be immobilized on a support, and (1) the single-stranded DNA It may be immobilized by the binding of the immobilized methylated DNA antibody and the support at the stage before the binding of the strand DNA (positive strand) and the immobilized methylated DNA antibody, and (2) It may be immobilized by the binding of the immobilized methylated DNA antibody and the support at the stage after the binding of the single-stranded DNA (positive strand) and the immobilized methylated DNA antibody.

In the first step (B) of the measurement method of the present invention, “selecting the single-stranded DNA by binding a methylated single-stranded DNA and an immobilized methylated DNA antibody” Specifically, for example, a “biotinylated biotinylated methylcytosine antibody” may be used as an immobilized methylated DNA antibody as follows.
(A) A mixture obtained by adding an appropriate amount (for example, 0.1 μg / 50 μL) of a biotinylated methylcytosine antibody to an avidin-coated PCR tube is allowed to stand at room temperature for about 1 hour, whereby a biotinylated methylcytosine antibody is obtained. And immobilization of streptavidin. After the operation, the remaining solution is removed and washed. Wash buffer [for example, 0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO · 7H ₂ O, 154 mM NaCl pH 7.4)] is added to the obtained avidin-coated PCR tube at 100 μL / tube. After adding in proportion, remove the solution from the avidin-coated PCR tube. By repeating this washing operation several times, the biotinylated methylcytosine antibody immobilized on the support remains in the avidin-coated PCR tube.
(B) A double-stranded DNA derived from genomic DNA contained in a biological sample is mixed with a buffer (for example, 33 mM Tris-Acetate pH 7.9, 66 mM KOAc, 10 mM MgOAc2, 0.5 mM Dithothreitol), and the number is 95 ° C. Heat for minutes. Thereafter, it is quickly cooled to a temperature of about 0 to 4 ° C., and kept at that temperature for several minutes to form single-stranded DNA. Then return to room temperature.
(C) The formed single-stranded DNA is added to an avidin-coated PCR tube on which a biotinylated methylcytosine antibody is immobilized, and then allowed to stand at room temperature for about 1 hour to obtain a biotinylated methylcytosine antibody, Facilitates binding with methylated single-stranded DNA of single-stranded DNA (formation of a conjugate) (At this stage, single-stranded DNA containing at least an unmethylated DNA region forms a conjugate. do not do.). Thereafter, the remaining solution is removed and washed. Washing buffer [eg, 0.05% Tween20-containing phosphate buffer (1 mM KH2PO4, 3 mM Na2HPO · 7H2O, 154 mM NaCl pH 7.4)] is added at a rate of 100 μL / tube, and the solution is removed. This washing operation is repeated several times to leave the conjugate in the PCR tube (selection of conjugate).

The specific description of the first step (B) is not limited to the above description. For example, the buffer used in (b) is not limited to the buffer as long as it is suitable for separating double-stranded DNA derived from a genomic DNA derived from a biological sample into single-stranded DNA.
In addition, for example, the washing operations in (a) and (c) are performed by the non-immobilized methylated DNA antibody floating in the solution or the methylation floating in the solution that did not bind to the methylated DNA antibody. This is important in order to remove single-stranded DNA that has not been removed or DNA floating in a solution digested with a restriction enzyme described later from the reaction solution. The washing buffer only needs to be suitable for removing the above-mentioned free methylated DNA antibody, single-stranded DNA floating in the solution, etc., and is not limited to the washing buffer. Tris-HCl pH 7.8 with Tween 80), TE buffer, etc. may be used.

In the second step of the measurement method of the present invention, the single-stranded DNA selected in the first step is digested with a methylation sensitive restriction enzyme capable of digesting at least one kind of single-stranded DNA. The single-stranded DNA selected in the first step is digested with a methylation-sensitive restriction enzyme capable of digesting at least one kind of single-stranded DNA, and then the free digest produced (the single-stranded DNA described above). The single-stranded DNA containing at least one CpG in the ammethyl state at the recognition site of the methylation-sensitive restriction enzyme capable of digesting.
The “methylation-sensitive restriction enzyme” in the second step of the measurement method of the present invention means, for example, that only a recognition sequence containing unmethylated cytosine is digested without digesting a recognition sequence containing methylated cytosine. It means a restriction enzyme etc. That is, in the case of DNA in which cytosine contained in a recognition sequence that can be originally recognized by a methylation-sensitive restriction enzyme is methylated, even if the methylation-sensitive restriction enzyme is allowed to act on the DNA, the DNA is Not cut. On the other hand, in the case where the cytosine contained in the recognition sequence that can be originally recognized by the methylation-sensitive restriction enzyme is not methylated, if the methylation-sensitive restriction enzyme acts on the DNA, The DNA is cleaved. Specific examples of such a methylation-sensitive enzyme include HpaII, BstUI, NarI, SacII, HhaI and the like. The methylation-sensitive restriction enzyme is a double-stranded DNA containing a CpG pair in a hemimethyl state (that is, cytosine on one strand of the CpG pair is methylated, and cytosine on the other strand is methylated. (Not double-stranded DNA), which has not been cleaved, and has already been clarified by Gruenbaum et al. (Nucleic Acid Research, 9, 2509-2515).
The “methylation-sensitive restriction enzyme capable of digesting single-stranded DNA” includes, for example, non-methylated cytosine that does not digest a recognition sequence containing methylated cytosine in single-stranded DNA. It means a restriction enzyme that can digest only the recognition sequence. That is, some methylation sensitive restriction enzymes digest single-stranded DNA. For example, HhaI etc. can be mentioned.

Specific examples of the method for examining the presence or absence of digestion with a methylation-sensitive restriction enzyme capable of digesting the single-stranded DNA include, for example, one containing the target DNA region in the methylated single-stranded DNA. A method of examining the presence or absence of DNA amplification (amplified product) by performing PCR using a primer pair capable of amplifying DNA containing cytosine to be analyzed as a recognition sequence using the double-stranded DNA as a template can be mentioned. When cytosine to be analyzed is methylated, an amplification product is obtained. On the other hand, when the cytosine to be analyzed is not methylated, an amplification product cannot be obtained. In this way, by comparing the amounts of amplified DNA, the ratio of cytosine to be analyzed can be measured.
Incidentally, in the methylated single-stranded DNA selected in the first step, the target DNA region is in a single-stranded state, and the oligonucleotide as a negative strand does not bind to the target DNA region. The single-stranded DNA is a single strand derived from genomic DNA contained in a biological specimen. Further, as described above, not all CpGs in the target DNA region are methylated in the single-stranded DNA. Therefore, by utilizing the property that the methylation sensitive restriction enzyme capable of digesting the single-stranded DNA does not cleave the single-stranded DNA in the methyl state, the genomic DNA contained in the biological sample is It is possible to distinguish whether or not cytosine in at least one CpG existing in the recognition site of a methylation sensitive restriction enzyme capable of digesting single-stranded DNA is methylated, and the target DNA region The content of methylated DNA in can be determined with higher accuracy. That is, since the target DNA region or only a part of the CpG other than the target DNA region is methylated, the single-stranded DNA selected from the single-stranded DNA that forms a conjugate with the methylated antibody is selected. It is possible to select only single-stranded DNA containing a target DNA region which is digested by a digestion treatment with a methylation sensitive restriction enzyme capable of digesting DNA. That is, the single-stranded DNA contained in the target DNA region of the genomic-derived single-stranded DNA contained in the biological specimen is obtained by digestion with the methylation-sensitive restriction enzyme capable of digesting the single-stranded DNA. When the CpG cytosine at the recognition site of the methylation sensitive restriction enzyme capable of digesting DNA is methylated, the methylated DNA antibody binds to the recognition site of the methylation sensitive restriction enzyme capable of digesting single-stranded DNA. However, when the cytosine of CpG at the recognition site of the methylation sensitive restriction enzyme capable of digesting the single-stranded DNA contained in the target DNA region of the single-stranded DNA derived from the genome is not methylated, Since the DNA antibody is not bound, it is digested by a methylation sensitive restriction enzyme capable of digesting single-stranded DNA.
Therefore, after the digestion treatment, as described below, when PCR is performed using a pair of primers capable of amplifying the target DNA region, the restriction enzyme in the genomic DNA contained in the biological sample is detected. If cytosine in at least one or more CpGs present in the recognition site is not methylated, an amplification product by PCR cannot be obtained, while the genomic DNA contained in the biological sample is not If cytosine in all CpGs existing in the recognition site of the methylation-sensitive restriction enzyme capable of digesting single-stranded DNA is methylated, an amplified product by PCR is obtained.
Most methylation-sensitive restriction enzymes cannot digest single-stranded DNA even in an unmethylated state, but as described above, some methylation-sensitive restriction enzymes can digest single-stranded DNA in an unmethylated state. An example of a methylation-sensitive restriction enzyme capable of digesting single-stranded DNA is HhaI. By using such an enzyme, the presence or absence of methylation of single-stranded DNA can be determined. That is, if CpG cytosine contained in the recognition site of HhaI in the single-stranded DNA in the sample bound to the immobilized methylated antibody obtained by the above operation is methylated, If this DNA cannot be digested and is not methylated, HhaI will digest this DNA. Therefore, after performing the above reaction, if a PCR reaction is performed using a pair of primers capable of amplifying the target DNA region, an amplification product cannot be obtained if the DNA in the sample is in an unmethylated state. If the DNA is methylated, an amplification product will be obtained.

  Specifically, for example, the second step of the measurement method of the present invention may be performed as follows. For the single-stranded DNA selected in the first step, 3 μL of the optimal 10 × buffer (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc2, 5 mM Dithothreitol), 3 μL of 1 mg / mL BSA aqueous solution, single-stranded DNA Methylation-sensitive restriction enzyme HhaI (10 U / μL) capable of digesting can be added to each mixture, and then sterilized ultrapure water is added to the mixture to a volume of 30 μL, followed by incubation at 37 ° C. for 1 to 3 hours.

The single-stranded DNA selected in the first step in this way is digested with a methylation-sensitive restriction enzyme capable of digesting at least one kind of single-stranded DNA, and then the free digest produced (the one Removal and washing (DNA purification) of at least one single-stranded DNA containing CpG in an ammethyl state at the recognition site of a methylation sensitive restriction enzyme capable of digesting the strand DNA.
More specifically, for example, when using a PCR tube coated with streptavidin, first remove the solution by pipetting or decantation, and then add TE buffer approximately equal to the volume of the biological specimen. After that, the TE buffer may be removed by pipetting or decantation. In addition, when using magnetic beads coated with streptavidin, after fixing the beads with a magnet, the solution was first removed by pipetting or decantation, and then TE buffer was added in an amount approximately equal to the volume of the biological specimen. Thereafter, the TE buffer may be removed by pipetting or decantation.
Subsequently, by performing such an operation several times, the digest (a single-stranded DNA containing at least one or more amethylated CpG at the recognition site of the restriction enzyme) is removed and washed (DNA purification).
The above washing operation is not always necessary. For example, all the solutions obtained by the methylation sensitive restriction enzyme treatment can be used in the third step.

  As a concern in the digestion treatment with a methylation-sensitive restriction enzyme capable of digesting single-stranded DNA in the second step of the measurement method of the present invention, a recognition sequence containing cytosine that is not methylated cannot be completely digested (so-called “ There is a concern that "DNA remains uncut"). When such a concern becomes a problem, if there are many recognition sites for a methylation sensitive restriction enzyme capable of digesting single-stranded DNA, it is possible to minimize “DNA fragmentation”. As the DNA region, it has at least one recognition site for a methylation sensitive restriction enzyme capable of digesting single-stranded DNA, and it is considered that the more recognition sites, the better.

In the measurement method of the present invention, “a DNA sample derived from genomic DNA contained in a biological sample” is pre-digested with a restriction enzyme that does not use the target DNA region of the genomic DNA as a recognition cleavage site. One preferred embodiment is that it is a DNA sample. Here, when genomic DNA (template DNA) contained in a biological sample is selected with the present immobilized oligonucleotide, a short template DNA is more easily selected, and a target region is amplified by PCR. In addition, since it is considered better to have a shorter template DNA, digestion is performed by directly using a restriction enzyme that does not recognize the target DNA region as a recognition cleavage site for genomic DNA-derived DNA samples contained in biological samples. May be. A general restriction enzyme treatment method may be used as a method for digesting with a restriction enzyme that does not use the target DNA region as a recognition cleavage site.
Moreover, it is mentioned as one of the preferable embodiments that the “DNA sample derived from genomic DNA contained in a biological specimen” is a DNA sample digested with at least one kind of methylation sensitive restriction enzyme. it can.
These preferred embodiments are because the amount of methylation can be obtained with high precision by digesting the biological specimen itself with the restriction enzymes as described above. This method is useful for eliminating the “DNA residue” as described above.
As a method for digesting a genomic DNA-derived sample contained in a biological sample with a methylation-sensitive restriction enzyme, when the biological sample is genomic DNA itself, the same method as described above may be used. In the case of a lysate, a cell lysate, etc., in accordance with the same method as described above, a large excess of methylation sensitive restriction enzyme, for example, 500 times (10 U) or more of 25 ng of DNA Digestion treatment may be performed using a methylation sensitive restriction enzyme.
Genomic DNA basically exists as double-stranded DNA. Therefore, in this operation, not only a methylation sensitive restriction enzyme capable of digesting a single strand (eg, HhaI) but also a methylation sensitive restriction enzyme capable of digesting a double stranded DNA (eg, HpaII, BstUI, NarI, SacII, HhaI etc.) can be used.

In the third step of the measurement method of the present invention, a single-stranded DNA that is an undigested product obtained in the second step (a methylation-sensitive restriction that can digest the single-stranded DNA described above) A step of separating single-stranded DNA containing no CpG in the methylated state at the enzyme recognition site from the immobilized methylated DNA antibody into a single-stranded DNA (positive strand) (third (previous) A) Step)
Using the DNA (positive strand) in the single-stranded state separated in the third (previous A) step as a template, the partial base sequence (positive strand) of the base sequence of the DNA (positive strand) in the single-stranded state And a base sequence (complementary to a partial base sequence (positive strand) located further 3 ′ end than the 3 ′ end of the base sequence (positive strand) of the target DNA region ( The extension primer (forward primer) having a negative strand) is used as an extension primer, and the extension primer is extended once to extend the single-stranded DNA (positive strand) as a double-stranded DNA. A step (third (previous B) step)
The double-stranded DNA extended and formed in the third (previous B) step contains a single-stranded DNA (positive strand) containing the target DNA region and a base sequence that is complementary to the target DNA region. A single-stranded DNA (positive strand) having a step (third (previous C) step) of once separating into single-stranded DNA (negative strand) and including (a) the generated target DNA region as this step ) As a template, the forward primer as an extension primer, and the extension primer is extended once, thereby extending the single-stranded DNA containing the target DNA region as a double-stranded DNA. A and
(B) Using the generated single-stranded DNA (negative strand) containing a base sequence complementary to the target DNA region as a template, the base sequence complementary to the target DNA region is A base sequence that is a partial base sequence (negative strand) of the base sequence of the contained single-stranded DNA (negative strand) and that is complementary to the base sequence (positive strand) of the target DNA region An extension primer (reverse primer) having a base sequence (positive strand) that is complementary to the partial base sequence (negative strand) located further 3 'end than the 3' end of (negative strand) As the step-B, the extension primer is extended once to form a single-stranded DNA containing the target DNA region as a double-stranded DNA.
Furthermore, each of these steps was once methylated in the target DNA region by repeating the extension-formed double-stranded DNA obtained in each of the steps once after separating it into a single-stranded state. Amplify the DNA to a detectable amount and quantify the amount of amplified DNA.

In the third step of the measurement method of the present invention, first, a single-stranded DNA that is an undigested product obtained in the second step as the third (previous A) step among the following steps of each of the following steps (described above) DNA that is in a single-stranded state is separated from the immobilized methylated DNA antibody by separating a single-stranded DNA that does not contain CpG in the methylated state at the recognition site of a methylation-sensitive restriction enzyme capable of digesting single-stranded DNA ( A positive strand). Specifically, for example, a single-stranded DNA which is an undigested product obtained in the second step (a single-stranded DNA containing no CpG in the methylated state at the recognition site of the methylation-sensitive restriction enzyme capable of digesting the single-stranded DNA) An annealing buffer is added to the strand DNA) to obtain a mixture. Next, the obtained mixture is heated at 95 ° C. for several minutes to obtain DNA (positive strand) in a single-stranded state. Thereafter, in the third (front B) step, specifically, for example, the single-stranded DNA (forward strand) obtained in the third (previous A) step and the forward primer are mixed with 17.85 μL of sterilized ultrapure water. Add 3 μL of optimal buffer (eg, 100 mM Tris-HCl pH 8.3, 500 mM KCl, 15 mM MgCl ₂ ), 3 μL of 2 mM dNTP, 6 μL of 5N betaine, and then add AmpliTaq (1 type of DNA polymerase: 5 U / μL) is mixed in a solution with a volume of 30 μL and incubated at 37 ° C. for about 2 hours to extend the single-stranded DNA (positive strand) containing the target DNA region into double-stranded DNA. Let it form. In the third (previous C) step, specifically, for example, a mixture is obtained by adding an annealing buffer to the double-stranded DNA formed by extension in the third (previous B) step. Is heated to 95 ° C. for several minutes to be once separated into single-stranded DNA containing the target DNA region.
Then, as this process,
(i) In order to anneal the forward primer to the single-stranded DNA (positive strand) containing the target DNA region that has been generated, for example, the Tm value of the forward primer is about 0 to 20 ° C. lower Cool quickly to temperature and keep at that temperature for several minutes.
(ii) Then, return to room temperature.
(iii) The DNA of interest is obtained by extending the primer once using the DNA in the single-stranded state annealed in (i) above as a template and the forward primer as an extension primer. The single-stranded DNA containing the region is elongated as a double-stranded DNA (ie, the third step-A). Specifically, it may be carried out in accordance with, for example, the following description or the operation method in the extension reaction in the third (previous B) step of the above-described measurement method of the present invention.
(iv) Using the generated single-stranded DNA (negative strand) containing a base sequence complementary to the target DNA region as a template, the base sequence complementary to the target DNA region is A base sequence that is a partial base sequence (negative strand) of the base sequence of the contained single-stranded DNA (negative strand) and that is complementary to the base sequence (positive strand) of the target DNA region An extension primer (reverse primer) having a base sequence (positive strand) that is complementary to the partial base sequence (negative strand) located further 3 'end than the 3' end of (negative strand) As described above, by extending the extension primer once, the single-stranded DNA is converted into an extended double-stranded DNA (ie, the third step-B). Specifically, for example, it may be carried out according to the operation method in the extension reaction in the third (previous B) step, as in the third step-A in (iii) above.
(v) Further, each of these steps is repeated after separating the double-stranded DNA formed in the extension obtained in each of the steps into a single-stranded state (for example, the third step-A and the third step). By step-B), the methylated DNA in the target DNA region is amplified to a detectable amount, and the amount of the amplified DNA is quantified.

  Specifically, in the third step, the reaction from the third (previous A) step to the present step can be performed as one PCR reaction. Further, the third (previous A) step to the third (previous C) step can be performed as independent reactions, and only this step can be performed as a PCR reaction.

As a method for amplifying a target DNA region (that is, a target region) after digestion with a methylation-sensitive restriction enzyme capable of digesting single-stranded DNA, for example, PCR can be used. When a target region is amplified, if a primer previously labeled with fluorescence or the like is used and the label is used as an index, the presence or absence of an amplification product can be evaluated without performing a cumbersome operation such as electrophoresis. Examples of the PCR reaction solution include DNA obtained in the second step of the measurement method of the present invention, 0.15 μl of a 50 μM primer solution, 2.5 μl of 2 mM dNTP, 10 × buffer solution (100 mM Tris-HCl pH 8.3). , 500 mM KCl, 20 mM MgCl ₂ , 0.01% Gelatin) 2.5 μl and AmpliTaq Gold (a kind of heat-resistant DNA polymerase: 5 U / μl) are mixed with 0.2 μl, and sterilized ultrapure water is added to the solution. The reaction solution containing 25 μl of can be used.
Since the target DNA region (that is, the target region) has many GC-rich base sequences, the reaction may be carried out by adding an appropriate amount of betaine, DMSO or the like. 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, then at 55 to 65 ° C. for 30 seconds, and further at 72 ° C. for 30 seconds for one cycle. The conditions for performing the heat insulation for 30 to 40 cycles are mentioned. After performing such PCR, the obtained amplification product is detected. For example, when a pre-labeled primer is used, the amplification amount in the PCR reaction can be evaluated by measuring the amount of the fluorescent label after performing the same washing / purification operation as before. In addition, when PCR is performed using ordinary unlabeled primers, detection is performed by annealing the colloidal gold particles, probes labeled with fluorescence, etc., and measuring the amount of the probe bound to the target region. can do. Further, in order to obtain the amount of the amplification product more accurately, for example, a real-time PCR method may be used. The real-time PCR method is a method for monitoring PCR in real time and kinetics analysis of the obtained monitoring results. For example, a high-precision quantitative PCR capable of detecting even a slight difference of about 2 times in terms of gene amount. It is a method known as law. Examples of the real-time PCR method include a method using a probe such as a template-dependent nucleic acid polymerase probe and a method using an intercalator such as Cyber Green. Commercially available devices and kits for the real-time PCR method may be used. As described above, detection is not particularly limited, and detection by any known method can be performed. In these methods, operations up to detection can be performed without changing the reaction container.

  In addition, examples of the second step include performing the third step without performing a digestion treatment with a methylation sensitive restriction enzyme capable of digesting single-stranded DNA. If the target DNA region does not have a base sequence that can be digested with a methylation-sensitive restriction enzyme capable of digesting single-stranded DNA, the third step may be performed without performing the second step.

  In addition, in the first step (A), a preferred embodiment for separating methylated single-stranded DNA includes addition of a counter oligonucleotide. The counter oligonucleotide is obtained by dividing the same base sequence as the target DNA region into short oligonucleotides. The length is usually 10 to 100 bases, more preferably 20 to 50 bases. The counter oligonucleotide is not designed on the base sequence that the forward primer or reverse primer binds complementarily to the target DNA region. The counter oligonucleotide is added in a large excess compared to genomic DNA, and the target DNA region is converted into a single strand (positive strand) and then combined with the immobilized methylated DNA antibody. The complementary strand (negative strand) of the region and the target DNA region are added to prevent the single strand (positive strand) from recombining due to complementarity. When a methylated DNA antibody is bound to the target DNA region and the DNA methylation frequency or an index value correlated therewith is measured, the target region that is single-stranded binds to the methylated DNA antibody. Because it is easy to do. The counter oligonucleotide is preferably added in an amount of at least 10 times, usually 100 times or more, as compared with the target DNA region.

Specifically, “adding a counter oligonucleotide when separating methylated single-stranded DNA” specifically refers to a methylated single DNA from a DNA sample derived from genomic DNA contained in a biological sample. In order to select a strand DNA, a DNA sample derived from a genomic DNA contained in a biological specimen is mixed with a counter oligonucleotide, and a double strand of a complementary strand of the target DNA region and the counter oligonucleotide is obtained. What is necessary is just to form. For example, the DNA sample and the counter oligonucleotide are mixed with 5 μL of a buffer solution (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc ₂ , 5 mM Dithiothreitol), 5 μL of 100 mM MgCl ₂ solution, and 1 mg / mL BSA solution. Add 5 μL, add sterilized ultrapure water to the mixture to make 50 μL, mix, heat at 95 ° C. for 10 minutes, quickly cool to 70 ° C., keep at that temperature for 10 minutes, Then, it is cooled to 50 ° C., kept at temperature for 10 minutes, further kept at 37 ° C. for 10 minutes, and then returned to room temperature.

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 genomic DNA contained in a biological specimen derived from a disease. If the measurement method of the present invention is carried out for that DNA region, the amount of methylated DNA is large. For example, if there is a DNA region that is not 100% methylated in genomic DNA contained in a biological specimen derived from a disease, and the measurement method of the present invention is carried out for that DNA region, the amount of methylated DNA Will be close to zero. In addition, for example, hypomethylation is present in the genomic DNA contained in the biological sample from a healthy person, and hypermethylation is present in the genomic DNA contained in the biological sample from the disease patient. If there is a DNA region and the measurement method of the present invention is performed on the DNA region, the amount of methylated DNA is close to 0 in the case of a healthy person, whereas in the case of a patient with a disease, 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 is the same as the meaning generally used in the field, and specifically, for example, when the biological specimen is a cell, it means the malignancy of the cell. 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 makes it possible to diagnose various diseases by examining methylation abnormality.

  In such a measurement method of the present invention, a restriction enzyme, primer or probe that can be used in various methods for measuring the amount of methylated DNA in a target region 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 support. The scope of right includes, of course, the use in the form of the detection kit or the detection chip as described above using the substantial principle of the method.

  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
A commercially available methylcytosine antibody (Aviva Systems Biology) was labeled with biotin using a commercially available biotinylation kit (Biotin Labeling Kit-NH2, manufactured by Dojindo Laboratories) according to the method described in the catalog. Refrigerate the obtained biotin-labeled methylcytosine antibody as a solution [antibody 0.1 μg / 100 μL 0.1% BSA-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO · 7H ₂ O, 154 mM NaCl pH 7.4) solution] saved.

The biotin-labeled methylcytosine antibody solution was added at a ratio of 100 μL to each of the streptavidin-coated PCR tubes (total of 8 tubes), and the mixture was allowed to stand at room temperature for about 1 hour to be immobilized on the PCR tubes. Thereafter, the solution in the PCR tube was removed by pipetting, and then 100 μL of washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO · 7H ₂ O, 154 mM) was added to the PCR tube. NaCl pH 7.4)] was added. The buffer was then removed by pipetting. This operation was repeated two more times.

The methylated oligonucleotide UBC-M consisting of the base sequence shown by SEQ ID NO: 17 and the unmethylated oligonucleotide UBC-U consisting of the base sequence shown by SEQ ID NO: 18 were separately synthesized, This is referred to as an oligonucleotide solution).
Solution A: 0.01 pmoL / 100 μL TE buffer solution Solution B: 0.001 pmoL / 100 μL TE buffer solution
Solution C: 0.0001 pmoL / 100 μL TE buffer solution Solution D: 0 pmoL / 100 μL TE buffer solution (negative control solution)

<Methylated oligonucleotide> N represents 5-methylcytosine.
UBC-M: 5'-
AGTGACACCATNGAGAATGTCAGATCNGGATCAGAGNGCCATCTAGATGGACATGTCACTGTCTGACTACAACATCCAGA-3 '(SEQ ID NO: 17)
<Unmethylated oligonucleotide>
UBC-U: 5'-AGTGACACCATCGAGAATGTCAGATCCGGATCAGAGCGCCATCTAGATGGACATGTCACTGTCTGACTACAACATCCAGA-3 '(SEQ ID NO: 18)

  Each of the obtained solutions was subjected to the following treatment (prepared one for each solution).

To the PCR tube coated with streptavidin on which the biotin-labeled methylated cytosine antibody was immobilized, 100 μL of the oligonucleotide solution prepared above was added and left at room temperature for 2 hours. Then, after removing the solution in the PCR tube by pipetting, 200 μL of the washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO · 7H ₂ O, 154 mM NaCl] pH 7.4)] was added. Next, the buffer in the PCR tube was removed by pipetting. This operation was further repeated twice (this corresponds to the first step of the measurement method of the present invention).

  Next, PCR is performed on the PCR tube thus obtained using each solution (PF and PR) of the primer consisting of the base sequences shown in SEQ ID NO: 19 and SEQ ID NO: 20 and the following reaction conditions. The DNA methylated in the target DNA region UBC (SEQ ID NO: 21, methylated cytosine is also represented by C) was amplified.

<Oligonucleotide primers designed for PCR>
PF: 5'-AGTGACACCATCGAGAATGTCA-3 '(SEQ ID NO: 19)
PR: 5'-TCTGGATGTTGTAGTCAGACAG-3 '(SEQ ID NO: 20)

<Target DNA region>
UBC: 5'-AGTGACACCATCGAGAATGTCAGATCCGGATCAGAGCGCCATCTAGATGGACATGTCACTGTCTGACTACAACATCCAGA-3 '(SEQ ID NO: 21)

As a PCR reaction solution, a template solution was prepared by adding 0.3 μL each of a primer solution consisting of the nucleotide sequence shown in SEQ ID NO: 19 and SEQ ID NO: 20 prepared in 50 μM, each 2 mM dNTP 5 μL, and a buffer solution (100 mM). Tris-HCl pH 8.3, 500 mM KCl, 15 mM MgCl ₂ , 0.01% Gelatin) 5 μL, heat-resistant DNA polymerase (AmpliTaq Gold) 5 U / μL, 0.25 μL were mixed, and then sterilized ultrapure water was added to the mixture. In addition, the liquid volume was 50 μL. The reaction solution was incubated at 95 ° C. for 10 minutes, and then subjected to PCR under the conditions of 22 cycles of incubation at 95 ° C. for 30 seconds, then 59 ° C. for 30 seconds and 72 ° C. for 45 seconds. It was.
After PCR, amplification of DNA was confirmed by subjecting to 1.5% agarose gel electrophoresis. The result is shown in FIG. 1 (corresponding to the third step of the measurement method of the present invention). DNA amplification was confirmed in UBC-M solution A (0.01 pmol), solution B (0001 pmol), and solution C (0.0001 pmol), and an amplification product (target DNA region: UBC) was obtained. In UBC-M solution D (0 pmol), amplification of DNA was not confirmed and the amplification product was not obtained. On the other hand, in all groups of UBC-U solution A (0,01 pmol), solution B (0.001 pmol), solution C (0.0001 pmol), and solution D (0 pmol), DNA amplification was not confirmed. No product was obtained.

  From the above, it is possible to select a single-stranded DNA containing a target DNA region that has been methylated with an immobilized methylated cytosine antibody, and methylation in the target DNA region. It was confirmed that the amount of the amplified DNA can be quantified by amplifying only the methylated DNA to a detectable amount without amplifying the untreated DNA.

Example 2
A commercially available methylcytosine antibody (Aviva Systems Biology) was labeled with biotin using a commercially available biotinylation kit (Biotin Labeling Kit-NH2, manufactured by Dojindo Laboratories) according to the method described in the catalog. The obtained biotin-labeled methylcytosine antibody is refrigerated as a solution [antibody 0.1 μg / 50 μL 0.1% BSA-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO · 7H ₂ O, 154 mM NaCl pH 7.4) solution] saved.

The above biotin-labeled methylcytosine antibody solution was added at a ratio of 50 μL to each of the streptavidin-coated PCR tubes (total of 6 tubes) and allowed to stand at room temperature for about 1 hour to be immobilized on the PCR tubes. Thereafter, the solution in the PCR tube is removed by pipetting, and 100 μL of a washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO · 7H ₂ O, 154 mM NaCl pH 7.4)] Added. Next, the buffer in the PCR tube was removed by pipetting. This operation was repeated two more times.

  Partially methylated oligonucleotide GPR7-2079-2176 / 98mer-M (7) not digested with HhaI consisting of the base sequence shown in SEQ ID NO: 22, partial methylation digested with HhaI consisting of the base sequence shown in SEQ ID NO: 23 Oligonucleotide GPR7-2079-2176 / 98mer-HM (5) and an unmethylated oligonucleotide GPR7-2079-2176 / 98mer-UM consisting of the base sequence shown in SEQ ID NO: 24 were synthesized separately, and 0.1 pmol / A 50 μL TE buffer solution (hereinafter referred to as an oligonucleotide solution) was prepared.

<Partially methylated oligonucleotide not digested with HhaI>
N represents 5-methylcytosine.
GPR7-2079-2176 / 98mer-M (7):
5'- GTTGGCCACTGCGGAGTCGNGCNGGGTGGCNGGCCGCACCTACAGNGCCGNGNGNGCGGTGAGCCTGGCCGTGTGGGGGATCGTCACACTCGTCGTGC -3 '(SEQ ID NO: 22)
<Partially methylated oligonucleotide digested with HhaI>
N represents 5-methylcytosine.
GPR7-2079-2176 / 98mer-HM (5):
5'- GTTGGCCACTGCGGAGTCGCGCCGGGTGGCNGGCCGCACCTACAGNGCCGNGNGNGCGGTGAGCCTGGCCGTGTGGGGGATCGTCACACTCGTCGTGC -3 '(SEQ ID NO: 23)
<Unmethylated oligonucleotide>
GPR7-2079-2176 / 98mer-UM:
5'- GTTGGCCACTGCGGAGTCGCGCCGGGTGGCCGGCCGCACCTACAGCGCCGCGCGCGCGGTGAGCCTGGCCGTGTGGGGGATCGTCACACTCGTCGTGC -3 '(SEQ ID NO: 24)

  Each of the obtained solutions was subjected to the following treatment (prepared two for each solution).

50 μL of the oligonucleotide solution prepared above was added to the PCR tube coated with streptavidin on which the biotin-labeled methylcytosine antibody was immobilized, and left at room temperature for 1 hour. Thereafter, the solution in the PCR tube was removed by pipetting, and then 100 μL of the washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO · 7H ₂ O, 154 mM NaCl pH 7.4)] was added. Next, the buffer in the tube was removed by pipetting. This operation was further repeated twice (this corresponds to the first step of the measurement method of the present invention).

  The samples obtained in this manner were subjected to the following A treatment or B treatment (prepared for each one).

A treatment group (non-treatment group): 10 μL of buffer solution (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc ₂ , 5 mM Dithiothreitol) and 10 μL of BSA (Bovine serum albumin 1 mg / mL) were prepared. Then, sterilized ultrapure water was further added to the mixture to make the liquid volume 100 μL.

B treatment group (HhaI treatment group): The sample prepared above was mixed with 10 μL of buffer solution (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc ₂ , 5 mM Dithiothreitol) and 10 μL of BSA (Bovine serum albumin 1 mg / mL). After adding 64 U of HhaI, sterilized ultrapure water was further added to the mixture to make the volume 50 μL.

Each reaction was incubated at 37 ° C. for 3 hours. Next, the solution in the PCR tube was removed by pipetting, and then 100 μL of washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO · 7H ₂ O, 154 mM NaCl pH 7.4)] Was added. Thereafter, the buffer was removed by pipetting. This operation was repeated twice more (this corresponds to the second step of the measurement method of the present invention).

  Next, PCR is performed on the PCR tubes thus obtained using the primer solutions (PF2 and PR2) consisting of the nucleotide sequences represented by SEQ ID NO: 25 and SEQ ID NO: 26 and the following reaction conditions: Was used to amplify methylated DNA in the target DNA region (GPR7-2079-2176, SEQ ID NO: 27, methylated cytosine is also represented by C).

<Oligonucleotide primers designed for PCR>
PF2: 5'-GTTGGCCACTGCGGAGTCG-3 '(SEQ ID NO: 25)
PR2: 5'-TCTGGATGTTGTAGTCAGACAG-3 '(SEQ ID NO: 26)

<Target DNA region>
GPR7-2079-2176: 5'- GTTGGCCACTGCGGAGTCGCGCCGGGTGGCCGGCCGCACCTACAGCGCCGCGCGCGCGGTGAGCCTGGCCGTGTGGGGGATCGTCACACTCGTCGTGC -3 '(SEQ ID NO: 27)

As a PCR reaction solution, 5 μL each of a primer solution consisting of the nucleotide sequence shown in SEQ ID NO: 25 and SEQ ID NO: 26 prepared in 3 μM, 5 μL each 2 mM dNTP, and buffer (100 mM Tris- HCl pH 8.3, 500 mM KCl, 15 mM MgCl ₂ , 0.01% Gelatin) 5 μL, heat-resistant DNA polymerase (AmpliTaq Gold) 5 U / μL, 0.25 μL, and 5 N betaine aqueous solution 10 μL were mixed, and the mixture was further mixed. Sterile ultrapure water was added to make the volume 50 μL. The reaction solution was incubated at 95 ° C. for 10 minutes, and then PCR was carried out under the conditions of 18 cycles of incubation at 95 ° C. for 30 seconds, then 59 ° C. for 30 seconds and 72 ° C. for 45 seconds. It was.
After performing PCR, amplification of DNA was confirmed by subjecting the obtained amplification product to 1.5% agarose gel electrophoresis. The result is shown in FIG. 2 (this corresponds to the third step of the measurement method of the present invention). In the case of the A treatment group (non-treatment group), a partially methylated oligonucleotide GPR7-2079-2176 / 98mer-M (7) not digested with HhaI and a partially methylated oligonucleotide GPR7-2079-2176 / digested with HhaI With 98mer-HM (5), amplification of DNA was confirmed and the amplification product (target DNA region: GPR7-2079-2176) was obtained. In the unmethylated oligonucleotide GPR7-2079-2176 / 98mer-UM, amplification of DNA was not confirmed and the amplification product was not obtained. In the case of the B treatment group (HhaI treatment group), in the partially methylated oligonucleotide GPR7-2079-2176 / 98mer-M (7) that is not digested with HhaI, amplification of DNA was confirmed and the amplification product (target DNA region: GPR7-2079-2176) was obtained, but partially methylated oligonucleotide GPR7-2079-2176 / 98mer-HM (5) digested with HhaI and unmethylated oligonucleotide GPR7-2079-2176 / 98mer-UM Then, amplification of DNA was not confirmed and the amplification product was not obtained.

  From the above, it is possible to select a single-stranded DNA containing a methylated target DNA region with an immobilized methylcytosine antibody, and by treating with a methylation-sensitive restriction enzyme, A methylation sensitive restriction enzyme recognition site can digest a target DNA region that is not methylated, and only a methylated DNA without amplifying a non-methylated DNA in the target DNA region. It was confirmed that the amount of amplified DNA could be quantified and so on.

Example 3
A commercially available methylcytosine antibody (Aviva Systems Biology) was labeled with biotin using a commercially available biotinylation kit (Biotin Labeling Kit-NH2, manufactured by Dojindo Laboratories) according to the method described in the catalog. The obtained biotin-labeled methylcytosine antibody is refrigerated as a solution [antibody 0.1 μg / 50 μL 0.1% BSA-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO · 7H ₂ O, 154 mM NaCl pH 7.4) solution] saved.

The above biotin-labeled methylcytosine antibody solution was added to each streptavidin-coated PCR tube (total of 6 tubes) at a ratio of 50 μL, and the mixture was allowed to stand at room temperature for about 1 hour to be immobilized on the PCR tube. Next, after removing the solution in the PCR tube by pipetting, 100 μL of the washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO · 7H ₂ O, 154 mM) was added to the PCR tube. NaCl pH 7.4)] was added. Thereafter, the buffer in the tube was removed by pipetting. This operation was repeated two more times.

  Partially methylated oligonucleotide GPR7-2079-2176 / 98mer-M (7) not digested with HhaI consisting of the base sequence shown in SEQ ID NO: 22, partial methylation digested with HhaI consisting of the base sequence shown in SEQ ID NO: 23 Oligonucleotide GPR7-2079-2176 / 98mer-HM (5) and an unmethylated oligonucleotide GPR7-2079-2176 / 98mer-UM consisting of the base sequence shown in SEQ ID NO: 24 were synthesized separately, and 0.1 pmol / A 50 μL TE buffer solution (hereinafter referred to as an oligonucleotide solution) was prepared.

<Partially methylated oligonucleotide not digested with HhaI>
N represents 5-methylcytosine.
GPR7-2079-2176 / 98mer-M (7):
5'- GTTGGCCACTGCGGAGTCGNGCNGGGTGGCNGGCCGCACCTACAGNGCCGNGNGNGCGGTGAGCCTGGCCGTGTGGGGGATCGTCACACTCGTCGTGC -3 '(SEQ ID NO: 22)
<Partially methylated oligonucleotide digested with HhaI>
N represents 5-methylcytosine.
GPR7-2079-2176 / 98mer-HM (5):
5'- GTTGGCCACTGCGGAGTCGCGCCGGGTGGCNGGCCGCACCTACAGNGCCGNGNGNGCGGTGAGCCTGGCCGTGTGGGGGATCGTCACACTCGTCGTGC -3 '(SEQ ID NO: 23)
<Unmethylated oligonucleotide>
GPR7-2079-2176 / 98mer-UM:
5'- GTTGGCCACTGCGGAGTCGCGCCGGGTGGCCGGCCGCACCTACAGCGCCGCGCGCGCGGTGAGCCTGGCCGTGTGGGGGATCGTCACACTCGTCGTGC -3 '(SEQ ID NO: 24)

  The sample thus obtained was subjected to the following A treatment or B treatment (prepared for each one).

A-treated group (untreated group): The sample prepared above (each 10 μL) was mixed with 5 μL of a buffer solution (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc ₂ , 5 mM Dithiothreitol) and BSA (Bovine serum albumin 1 mg). After adding 5 μL, sterilized ultrapure water was further added to the mixture to make the volume 50 μL.

B treatment group (HhaI treatment group): The sample prepared above (each 10 μL) was mixed with 5 μL of a buffer solution (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc ₂ , 5 mM Dithiothreitol) and BSA (Bovine serum albumin 1 mg). / mL) 5 μL and 64 U of HhaI were added, and sterilized ultrapure water was further added to the mixture to make the volume 50 μL.

Each reaction was incubated at 37 ° C. for 3 hours. All the reaction solutions were added to the PCR tube coated with streptavidin to which the biotin-labeled methylcytosine antibody prepared above was immobilized, and then allowed to stand at room temperature for 1 hour. Next, the solution in the PCR tube was removed by pipetting, and then 100 μL of washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO · 7H ₂ O, 154 mM NaCl pH 7.4)] Was added. Thereafter, the buffer was removed by pipetting. This operation was further repeated twice (this corresponds to the first step of the measurement method of the present invention).

  Next, PCR is performed on the PCR tubes thus obtained using the primer solutions (PF2 and PR2) consisting of the nucleotide sequences represented by SEQ ID NO: 25 and SEQ ID NO: 26 and the following reaction conditions: Was used to amplify methylated DNA in the target DNA region (GPR7-2079-2176, SEQ ID NO: 27, methylated cytosine is also represented by C).

<Oligonucleotide primers designed for PCR>
PF2: 5'-GTTGGCCACTGCGGAGTCG-3 '(SEQ ID NO: 25)
PR2: 5'-TCTGGATGTTGTAGTCAGACAG-3 '(SEQ ID NO: 26)

<Target DNA region>
GPR7-2079-2176: 5'- GTTGGCCACTGCGGAGTCGCGCCGGGTGGCCGGCCGCACCTACAGCGCCGCGCGCGCGGTGAGCCTGGCCGTGTGGGGGATCGTCACACTCGTCGTGC -3 '(SEQ ID NO: 27)

As a PCR reaction solution, 5 μL each of a primer solution consisting of the nucleotide sequence shown in SEQ ID NO: 25 and SEQ ID NO: 26 prepared in 3 μM, 5 μL each 2 mM dNTP, and buffer (100 mM Tris- 5 μL of HCl pH 8.3, 500 mM KCl, 15 mM MgCl ₂ , 0.01% Gelatin), 0.25 μL of thermostable DNA polymerase (AmpliTaq Gold) 5 U / μL, and 10 μL of 5N betaine aqueous solution were mixed. Sterile ultrapure water was added to make the volume 50 μL. The reaction solution was incubated at 95 ° C. for 10 minutes, and then PCR was carried out under the conditions of 18 cycles of incubation at 95 ° C. for 30 seconds, then 59 ° C. for 30 seconds and 72 ° C. for 45 seconds. It was.
After PCR, the obtained PCR product was subjected to 1.5% agarose gel electrophoresis to confirm DNA amplification. The result is shown in FIG. 2 (this corresponds to the third step of the measurement method of the present invention). In the case of the A treatment group (non-treatment group), a partially methylated oligonucleotide GPR7-2079-2176 / 98mer-M (7) not digested with HhaI and a partially methylated oligonucleotide GPR7-2079-2176 / digested with HhaI With 98mer-HM (5), amplification of DNA was confirmed and the amplification product (target DNA region: GPR7-2079-2176) was obtained. In the unmethylated oligonucleotide GPR7-2079-2176 / 98mer-UM, amplification of DNA was not confirmed and the amplification product was not obtained. In the case of the B treatment group (HhaI treatment group), in the partially methylated oligonucleotide GPR7-2079-2176 / 98mer-M (7) that is not digested with HhaI, amplification was confirmed and the amplification product (target DNA region: GPR7- 2079-2176) was obtained, but with HhaI digested partially methylated oligonucleotide GPR7-2079-2176 / 98mer-HM (5) and unmethylated oligonucleotide GPR7-2079-2176 / 98mer-UM, No amplification of DNA was confirmed, and no amplification product was obtained.

Example 4
A commercially available methylcytosine antibody (Aviva Systems Biology) was labeled with biotin using a commercially available biotinylation kit (Biotin Labeling Kit-NH ₂ , manufactured by Dojindo Laboratories) according to the method described in the catalog. The obtained biotin-labeled methylcytosine antibody was refrigerated and stored as a solution [antibody about 0.25 μg / μL 0.1% BSA-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4) solution].

50 µL each of 0.1 µg / 50 µL solution of biotin-labeled methylcytosine antibody obtained by synthesis was added to the streptavidin-coated PCR tubes (total of 8 tubes) and left at room temperature for about 1 hour. Immobilized to. Thereafter, the solution was removed by pipetting, and 100 μL of a washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4)] was added, and then the buffer was pipetted. Removed. This operation was repeated twice more (this corresponds to the preparation of the immobilized methylated DNA antibody used in the measurement method of the present invention).

  For human blood-derived genomic DNA purchased from Clontech, PCR is performed using oligonucleotide primer primers (PF1 and PR1) and reaction conditions designed for PCR shown in SEQ ID NO: 28 and SEQ ID NO: 29 below. As a result, a DNA fragment (region corresponding to base number 25687390-25687775 shown in X, SEQ ID NO: 30, Genbank Accession No. NT — 029419, etc.) used as a test sample was amplified.

<Oligonucleotide primers designed for PCR>
PF1: 5′-CTCAGCACCCAGGCGGCC -3 ′ (SEQ ID NO: 28)
PR1: 5′-CTGGCCAAACTGGAGATCGC -3 ′ (SEQ ID NO: 29)

<DNA fragment>
X: 5'- CTCAGCACCCAGGCGGCCGCGATCATGAGGCGCGAGCGGCGCGCGGGCTGTTGCAGAGTCTTGAGCGGGTGGCACACCGCGATGTAGCGGTCGGCTGTCATGACTACCAGCATGTAGGCCGACGCAAACATGCCGAACACCTGCAGGTGCTTCACCACGCGGCACAGCCAGTCGGGGCCGCGGAAGCGGTAGGTGATGTCCCAGCACATTTGCGGCAGCACCTGGAAGAATGCCACGGCCAGGTCGGCCAGGCTGAGGTGTCGGATGAAGAGGTGCATGCGGGACGTCTTGCGCGGCGTCCGGTGCAGAGCCAGCAGTACGCTGCTGTTGCCCAGCACGGCCACCGCGAAAGTCACCGCCAGCACGGCGATCTCCAGTTTGGCCAG -3 '(SEQ ID NO: 30)

As PCR reaction solution, 5 ng of genomic DNA as a template, 3 μl each of oligonucleotide primer solution prepared to 5 μM, 5 μl each 2 mM dNTP, 10 × buffer (100 mM Tris-HCl pH 8.3, 500 mM KCl) , 15 mM MgCl ₂ , 0.01% Gelatin) and 5 μl of thermostable DNA polymerase (AmpliTaq Gold, manufactured by ABI) 5 U / μl are mixed, and sterilized ultrapure water is added thereto to bring the volume to 50 μl. What was used was used. The reaction solution was incubated at 95 ° C. for 10 minutes, and then PCR was performed under the conditions of 40 cycles of incubation at 95 ° C. for 30 seconds, then at 61 ° C. for 30 seconds, and further at 72 ° C. for 45 seconds. It was.
After performing PCR, amplification was confirmed by 1.5% agarose gel electrophoresis, and DNA fragment X was purified by Wizard SV Gel / PCR Kit (PROMEGA).

  For a part of the obtained DNA fragment solution, 1 μl of SssI methylase (NEB), 10 μl of 10 × NEBuffer2 (NEB) and 1 μl of S-adenosyl methionine (3.2 mM, NEB) are mixed. Then, sterilized ultrapure water was added thereto to prepare a liquid volume of 100 μl. The reaction solution was incubated at 37 ° C. for 15-30 minutes, and 1 μl of S-adenosyl methionine (3.2 mM, manufactured by NEB) was further added and incubated at 37 ° C. for 15-30 minutes. This was purified by Wizard SV Gel / PCR Kit (PROMEGA). Furthermore, these operations were repeated 5 times to obtain a methylated DNA fragment (MX, SEQ ID NO: 31).

<DNA fragment> (N represents 5-methylcytosine)
MX: 5'- CTCAGCACCCAGGNGGCNGNGATCATGAGGNGNGAGNGGNGNGNGGGCTGTTGCAGAGTCTTGAGNGGGTGGCACACNGNGATGTAGNGGTNGGCTGTCATGACTACCAGCATGTAGGCNGANGCAAACATGCNGAACACCTGCAGGTGCTTCACCANGNGGCACAGCCAGTNGGGGCNGNGGAAGNGGTAGGTGATGTCCCAGCACATTTGNGGCAGCACCTGGAAGAATGCCANGGCCAGGTNGGCCAGGCTGAGGTGTNGGATGAAGAGGTGCATGNGGGANGTCTTGNGNGGNGTCNGGTGCAGAGCCAGCAGTANGCTGCTGTTGCCCAGCANGGCCACNGNGAAAGTCACNGCCAGCANGGNGATCTCCAGTTTGGCCAG -3 '(SEQ ID NO: 31)

For the obtained DNA fragment X, the following solution was prepared.
Solution A: 100 pg / 10 μL TE solution Solution B: 10 pg / 10 μL TE solution Solution C: 1 pg / 10 μL TE solution Solution D: TE solution (negative control solution)

The following solution was prepared for the obtained DNA fragment MX.
Solution MA: 100 pg / 10 μL TE solution Solution MB: 10 pg / 10 μL TE solution Solution MC: 1 pg / 10 μL TE solution Solution MD: TE solution (negative control solution)

  In addition, counter oligonucleotides C1 to C12 having the base sequence represented by SEQ ID NO: 33 to SEQ ID NO: 44 capable of binding by complementarity to the negative strand of the target DNA region comprising the base sequence represented by SEQ ID NO: 32 were synthesized. A TE buffer solution having a concentration of 0.01 μM was prepared.

<Target DNA region>
X ': 5'- CTCAGCACCCAGGCGGCCGCGATCATGAGGCGCGAGCGGCGCGCGGGCTGTTGCAGAGTCTTGAGCGGGTGGCACACCGCGATGTAGCGGTCGGCTGTCATGACTACCAGCATGTAGGCCGACGCAAACATGCCGAACACCTGCAGGTGCTTCACCACGCGGCACAGCCAGTCGGGGCCGCGGAAGCGGTAGGTGATGTCCCAGCACATTTGCGGCAGCACCTGGAAGAATGCCACGGCCAGGTCGGCCAGGCTGAGGTGTCGGATGAAGAGGTGCATGCGGGACGTCTTGCGCGGCGTCCGGTGCAGAGCCAGCAGTACGCTGCTGTTGCCCAGCACGGCCACCGCGAAAGTCACCGCCAGCACGGCGATCTCCAGTTTGGCCAG -3' (SEQ ID NO: 32)

<Counter oligonucleotide>
C1: 5'- GCCACCGCGAAAGTCACCGCCAGCACGGCG-3 '(SEQ ID NO: 33)
C2: 5'-GCCAGCAGTACGCTGCTGTTGCCCAGCACG-3 '(SEQ ID NO: 34)
C3: 5′-CGGGACGTCTTGCGCGGCGTCCGGTGCAGA -3 ′ (SEQ ID NO: 35)
C4: 5'-AGGCTGAGGTGTCGGATGAAGAGGTGCATG-3 '(SEQ ID NO: 36)
C5: 5'-ACCTGGAAGAATGCCACGGCCAGGTCGGCC-3 '(SEQ ID NO: 37)
C6: 5′-TAGGTGATGTCCCAGCACATTTGCGGCAGC -3 ′ (SEQ ID NO: 38)
C7: 5'-CGGCACAGCCAGTCGGGGCCGCGGAAGCGG-3 '(SEQ ID NO: 39)
C8: 5'- ATGCCGAACACCTGCAGGTGCTTCACCACG-3 '(SEQ ID NO: 40)
C9: 5'- ATGACTACCAGCATGTAGGCCGACGCAAAC-3 '(SEQ ID NO: 41)
C10: 5'-TGGCACACCGCGATGTAGCGGTCGGCTGTC-3 '(SEQ ID NO: 42)
C11: 5′-CGCGCGGGCTGTTGCAGAGTCTTGAGCGGG-3 ′ (SEQ ID NO: 43)
C12: 5'-CAGGCGGCCGCGATCATGAGGCGCGAGCGG-3 '(SEQ ID NO: 44)

  The following treatment was applied to each of the DNA fragment X solution and the methylated DNA fragment MX solution.

In a PCR tube, 10 μL of the DNA fragment solution prepared above, 10 μL of the counter oligonucleotide solution prepared above, 5 μL of a buffer (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc ₂ , 5 mM Dithiothreitol), 100 mM 5 μL of MgCl ₂ solution and 5 μL of 1 mg / mL BSA solution were added, and further sterilized ultrapure water was added to the mixture to make the volume 50 μL and mixed. Thereafter, the PCR tube was heated at 95 ° C. for 10 minutes, quickly cooled to 70 ° C., and kept at that temperature for 10 minutes. Next, the mixture was cooled to 50 ° C., kept for 10 minutes, further kept at 37 ° C. for 10 minutes, and then returned to room temperature (this corresponds to the first step (A) of the measurement method of the present invention).

50 μL of the DNA fragment reaction solution prepared above was added to the streptavidin-coated PCR tube on which the biotin-labeled methylcytosine antibody was immobilized, and left at room temperature for 30 minutes. Thereafter, the solution was removed by pipetting, and 100 μL of a washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4)] was added, and then the buffer was pipetted. Removed. This operation was further repeated twice (this corresponds to the first step (B) of the measurement method of the present invention).

  Next, PCR is performed on the above PCR tube using each solution of oligonucleotide primers PF1 and PR1 consisting of the nucleotide sequences represented by SEQ ID NO: 28 and SEQ ID NO: 29, and the following reaction conditions, whereby SEQ ID NO: 32 The methylated DNA in the target DNA region X ′ consisting of the base sequence shown in FIG.

<Oligonucleotide primers designed for PCR>
PF1: 5'-CTCAGCACCCAGGCGGCC-3 '(SEQ ID NO: 28)
PR1: 5′-CTGGCCAAACTGGAGATCGC -3 ′ (SEQ ID NO: 29)

<Target DNA region>
X ': 5'- CTCAGCACCCAGGCGGCCGCGATCATGAGGCGCGAGCGGCGCGCGGGCTGTTGCAGAGTCTTGAGCGGGTGGCACACCGCGATGTAGCGGTCGGCTGTCATGACTACCAGCATGTAGGCCGACGCAAACATGCCGAACACCTGCAGGTGCTTCACCACGCGGCACAGCCAGTCGGGGCCGCGGAAGCGGTAGGTGATGTCCCAGCACATTTGCGGCAGCACCTGGAAGAATGCCACGGCCAGGTCGGCCAGGCTGAGGTGTCGGATGAAGAGGTGCATGCGGGACGTCTTGCGCGGCGTCCGGTGCAGAGCCAGCAGTACGCTGCTGTTGCCCAGCACGGCCACCGCGAAAGTCACCGCCAGCACGGCGATCTCCAGTTTGGCCAG -3' (SEQ ID NO: 32)

As a PCR reaction solution, 3 μL each of oligonucleotide primer solution prepared to 5 μM, 5 μL each 2 mM dNTP, and buffer solution (100 mM Tris-HCl pH 8.3, 500 mM KCl, 15 mM MgCl ₂ , 0.01% Gelatin) is mixed with 5 μL of heat-resistant DNA polymerase (AmpliTaq Gold, manufactured by ABI) 5 U / μL with 0.25 μL, and sterilized ultrapure water is added to make the volume 50 μL. It was. The reaction solution was incubated at 95 ° C. for 10 minutes, and then PCR was performed under the conditions of 25 cycles of incubation at 95 ° C. for 20 seconds, then at 61 ° C. for 30 seconds, and further at 72 ° C. for 30 seconds. It was.
After PCR, amplification was confirmed by 1.5% agarose gel electrophoresis. The results were as follows (corresponding to the third step of the measurement method of the present invention).

  The results are shown in FIG. Amplification was confirmed in the solutions MA, MB and MC of the methylated DNA fragment MX. No amplification was confirmed in the negative control solution MD. No amplification was confirmed in solutions A, B, C and D of DNA fragment X which was not methylated.

  From the above, it is possible to select a DNA containing a target DNA region that has been methylated with an immobilized methylcytosine antibody, and to amplify the methylated DNA to a detectable amount. It was confirmed that the amplified DNA can be detected with higher sensitivity.

Example 5
A commercially available methylcytosine antibody (Aviva Systems Biology) was labeled with biotin using a commercially available biotinylation kit (Biotin Labeling Kit-NH ₂ , manufactured by Dojindo Laboratories) according to the method described in the catalog. The obtained biotin-labeled methylcytosine antibody was refrigerated and stored as a solution [antibody about 0.25 μg / μL 0.1% BSA-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4) solution].

50 µL each of 0.1 µg / 50 µL solution of biotin-labeled methylcytosine antibody obtained by synthesis was added to the streptavidin-coated PCR tubes (total of 8 tubes) and left at room temperature for about 1 hour. Immobilized to. Thereafter, the solution was removed by pipetting, and 100 μL of a washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4)] was added, and then the buffer was pipetted. Removed. This operation was repeated twice more (this corresponds to the preparation of the immobilized methylated DNA antibody used in the measurement method of the present invention).

  For human blood-derived genomic DNA purchased from Clontech, PCR is performed using oligonucleotide primer primers (PF2 and PR2) and reaction conditions designed for PCR shown in SEQ ID NO: 45 and SEQ ID NO: 46 below. Thus, a DNA fragment (region corresponding to base number 76606-76726 shown in Y, SEQ ID NO: 47, Genbank Accession No. ac009800, etc.) used as a test sample was amplified.

<Oligonucleotide primers designed for PCR>
PF2: 5′-TGAGCTCCGTAGGGCGTCC -3 ′ (SEQ ID NO: 45)
PR2: 5'- GCGCCGGGTCCGGGCCC -3 '(SEQ ID NO: 46)

<DNA fragment>
Y: 5'- GCGCCGGGTCCGGGCCCGATGCGTTGGCGGGCCAGGGCTCCGAGAACGAGGCGTTGTCCATCTCAACGAGGGCAGAGGAGCCGGCGACCTGGCGTCCCCCAAGGACGCCCTACGGAGCTCA -3 '(SEQ ID NO: 47)

As PCR reaction solution, 5 ng of genomic DNA as a template, 3 μl each of oligonucleotide primer solution prepared to 5 μM, 5 μl each 2 mM dNTP, 10 × buffer (100 mM Tris-HCl pH 8.3, 500 mM KCl) , 15 mM MgCl ₂ , 0.01% Gelatin) and 5 μl of thermostable DNA polymerase (AmpliTaq Gold, manufactured by ABI) 5 U / μl are mixed, and sterilized ultrapure water is added thereto to bring the volume to 50 μl. What was used was used. The reaction solution was incubated at 95 ° C. for 10 minutes, and then PCR was performed under the conditions of 50 cycles of incubation at 95 ° C. for 30 seconds, then 60 ° C. for 30 seconds and 72 ° C. for 45 seconds. It was.
After performing PCR, amplification was confirmed by 1.5% agarose gel electrophoresis, and DNA fragment Y was purified by Wizard SV Gel / PCR Kit (PROMEGA).

  For a part of the obtained DNA fragment solution, 1 μl of SssI methylase (NEB), 10 μl of 10 × NEBuffer2 (NEB) and 1 μl of S-adenosyl methionine (3.2 mM, NEB) are mixed. Then, sterilized ultrapure water was added thereto to prepare a liquid volume of 100 μl. The reaction solution was incubated at 37 ° C. for 15-30 minutes, and 1 μl of S-adenosyl methionine (3.2 mM, manufactured by NEB) was further added and incubated at 37 ° C. for 15-30 minutes. This was purified by Wizard SV Gel / PCR Kit (PROMEGA). This operation was further repeated 5 times to obtain a methylated DNA fragment (MY, SEQ ID NO: 48).

<DNA fragment> (N represents 5-methylcytosine)
MY: 5'- GNGCNGGGTCNGGGCCNGATGNGTTGGNGGGCCAGGGCTCNGAGAANGAGGNGTTGTCCATCTCAANGAGGGCAGAGGAGCNGGNGACCTGGNGTCCCCCAAGGANGCCCTANGGAGCTCA -3 '(SEQ ID NO: 48)

For the obtained DNA fragment Y, the following solution was prepared.
Solution A: 100 pg / 10 μL TE solution Solution B: 10 pg / 10 μL TE solution Solution C: 1 pg / 10 μL TE solution Solution D: TE solution (negative control solution)

The following solution was prepared for the obtained DNA fragment MY.
Solution MA: 100 pg / 10 μL TE solution Solution MB: 10 pg / 10 μL TE solution Solution MC: 1 pg / 10 μL TE solution Solution MD: TE solution (negative control solution)

  Further, a counter oligonucleotide consisting of the base sequences represented by SEQ ID NO: 50, base sequence 24 and SEQ ID NO: 52, which can bind to the negative strand of the target DNA region Y ′ comprising the base sequence represented by SEQ ID NO: 49 by complementarity. C13, C14, and C15 were synthesized, and a TE buffer solution having a concentration of 0.01 μM was prepared.

<Target DNA region>
Y ': 5'- GCGCCGGGTCCGGGCCCGATGCGTTGGCGGGCCAGGGCTCCGAGAACGAGGCGTTGTCCATCTCAACGAGGGCAGAGGAGCCGGCGACCTGGCGTCCCCCAAGGACGCCCTACGGAGCTCA -3' (SEQ ID NO: 49)

<Counter oligonucleotide>
C13: 5'-GCGTCCCCCAAGGACGCCCTACGGAGCTCA-3 '(SEQ ID NO: 50)
C14: 5′-CTCAACGAGGGCAGAGGAGCCGGCGACCTG -3 ′ (SEQ ID NO: 51)
C15: 5'-CGCCGGGTCCGGGCCCGATGCGTTGGCGGG-3 '(SEQ ID NO: 52)

The following treatment was performed on each of the DNA fragment Y solution and the methylated DNA fragment MY solution.

In a PCR tube, 10 μL of the DNA fragment solution prepared above, 10 μL of the counter oligonucleotide solution prepared above, 5 μL of a buffer (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc ₂ , 5 mM Dithiothreitol), 100 mM 5 μL of MgCl ₂ solution and 5 μL of 1 mg / mL BSA solution were added, and further sterilized ultrapure water was added to the mixture to make the volume 50 μL and mixed. Thereafter, the PCR tube was heated at 95 ° C. for 10 minutes, quickly cooled to 70 ° C., and kept at that temperature for 10 minutes. Next, the mixture was cooled to 50 ° C., kept for 10 minutes, further kept at 37 ° C. for 10 minutes, and then returned to room temperature (this corresponds to the first step (A) of the measurement method of the present invention).

50 μL of the DNA fragment reaction solution prepared above was added to the streptavidin-coated PCR tube on which the biotin-labeled methylcytosine antibody was immobilized, and left at room temperature for 1 hour. Thereafter, the solution was removed by pipetting, and 100 μL of a washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4)] was added, and then the buffer was pipetted. Removed. This operation was further repeated twice (this corresponds to the first step (B) of the measurement method of the present invention).

  Next, PCR is carried out on the above PCR tube using each solution of oligonucleotide primers PF2 and PR2 consisting of the nucleotide sequences represented by SEQ ID NO: 45 and SEQ ID NO: 46, and the following reaction conditions, whereby SEQ ID NO: 49 The methylated DNA in the target DNA region Y ′ consisting of the base sequence shown in FIG.

<Oligonucleotide primers designed for PCR>
PF2: 5′-TGAGCTCCGTAGGGCGTCC -3 ′ (SEQ ID NO: 45)
PR2: 5'- GCGCCGGGTCCGGGCCC -3 '(SEQ ID NO: 46)

<Target DNA region>
Y ': 5'- GCGCCGGGTCCGGGCCCGATGCGTTGGCGGGCCAGGGCTCCGAGAACGAGGCGTTGTCCATCTCAACGAGGGCAGAGGAGCCGGCGACCTGGCGTCCCCCAAGGACGCCCTACGGAGCTCA -3' (SEQ ID NO: 49)

As a PCR reaction solution, 3 μL each of oligonucleotide primer solution prepared to 5 μM, 5 μL each 2 mM dNTP, and buffer solution (100 mM Tris-HCl pH 8.3, 500 mM KCl, 15 mM MgCl ₂ , 0.01% Gelatin) is mixed with 5 μL of heat-resistant DNA polymerase (AmpliTaq Gold, manufactured by ABI) 5 U / μL with 0.25 μL, and sterilized ultrapure water is added to make the volume 50 μL. It was. The reaction solution was incubated at 95 ° C. for 10 minutes, and then PCR was performed under the conditions of 25 cycles of incubation at 95 ° C. for 20 seconds, then 60 ° C. for 30 seconds and 72 ° C. for 30 seconds. It was.
After PCR, amplification was confirmed by 1.5% agarose gel electrophoresis. The results were as follows (corresponding to the third step of the measurement method of the present invention).

  The results are shown in FIG. Amplification was confirmed in the solutions MA, MB and MC of the methylated DNA fragment MY. No amplification was confirmed in the negative control solution MD. No amplification was confirmed in solutions A, B, C and D of DNA fragment Y which was not methylated.

  From the above, it is possible to select a DNA containing a target DNA region that has been methylated with an immobilized methylcytosine antibody, and to amplify the methylated DNA to a detectable amount. It was confirmed that the amplified DNA can be detected with higher sensitivity.

Example 6
A commercially available methylcytosine antibody (Aviva Systems Biology) was labeled with biotin using a commercially available biotinylation kit (Biotin Labeling Kit-NH ₂ , manufactured by Dojindo Laboratories) according to the method described in the catalog. The obtained biotin-labeled methylcytosine antibody was refrigerated and stored as a solution [antibody about 0.25 μg / μL 0.1% BSA-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4) solution].

50 µL each of 0.1 µg / 50 µL solution of biotin-labeled methylcytosine antibody obtained by synthesis was added to the streptavidin-coated PCR tubes (total of 6 tubes) and left at room temperature for about 1 hour. Immobilized to. Thereafter, the solution was removed by pipetting, and 100 μL of a washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4)] was added, and then the buffer was pipetted. Removed. This operation was repeated twice more (this corresponds to the preparation of the immobilized methylated DNA antibody used in the measurement method of the present invention).

Baker's yeast strain X2180-1A is cultured in YPD medium (1% Yeast extract, 2% Peptone, 2% Glucose, pH 5.6-6.0) until the turbidity is OD ₆₀₀ 0.6-1.0, and 10,000 g for 10 minutes Centrifugation was performed to prepare 1 × 10 ⁷ yeast cells. The yeast genome was obtained from the prepared yeast cells using a general yeast genome preparation method as described in Methods in Yeast Genetics (Cold Spring Harbor Laboratory).
The prepared yeast cells are suspended in buffer A (1M sorbitol, 0.1M EDTA, pH 7.4), 2-mercaptoethanol (final concentration 14 mM) and 100 U zymolase (10 mg / ml) are added, and the solution becomes clear. Incubated with stirring at 30 ° C for 1 hour until complete. Protoplasts were collected by centrifugation at 550 g for 10 minutes, suspended in buffer B (50 mM Tris-HCl, pH 7.4, 20 mM EDTA), and then sodium dodecyl sulfate was added to 1% (w / v). Thereafter, it was incubated at 65 ° C. for 30 minutes. Subsequently, 5M CH ₃ COOK in a volume ratio of 2/5 was added and mixed, and after ice cooling for 30 minutes, the supernatant was recovered by centrifugation at 15,000 g for 30 minutes. Add 1/10 volume ratio of 3M CH ₃ COONa and an equal amount of isopropanol to the collected supernatant, mix well, and centrifuge at 15,000g for 30 minutes at 4 ° C to rinse the precipitate with 70% ethanol. And recovered. The precipitate is dried, dissolved in 1 ml of TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA), RNase A (manufactured by Sigma) is added to a concentration of 40 μg / ml, and 1 at 37 ° C. After incubating for a time, proteinase K (manufactured by Sigma) was added to the mixture at 500 μg / ml and sodium dodecyl sulfate at 1% (w / v), and this was shaken at 55 ° C. for about 16 hours. That ’s it. After completion of the shaking, the mixture was extracted with phenol [saturated with 1M Tris-HCl (pH 8.0)] and chloroform. The aqueous layer was recovered, and NaCl was added to this to 0.5 N, and then the resulting precipitate was recovered by ethanol precipitation. Genomic DNA was obtained by rinsing the collected precipitate with 70% ethanol.
From the obtained genomic DNA, PCR was carried out using oligonucleotide primers (PF3 and PR3) and reaction conditions designed for PCR shown in SEQ ID NO: 53 and SEQ ID NO: 54 below, as test samples. The DNA fragment used (S, SEQ ID NO: 55, region corresponding to base number 271743-272083 of yeast chromosome VII shown in Genbank Accession No. NC — 001139, etc.) was amplified.

<Oligonucleotide primers designed for PCR>
PF3: 5'-AGGTGAGCTACGTGTGTTTGG-3 '(SEQ ID NO: 53)
PR3: 5'-AGACATGTGCTCACGTACGGT-3 '(SEQ ID NO: 54)

<DNA fragment>
S: 5'- AGGTGAGCTACGTGTGTTTGGGCGTCGTGCACTGGCTCACTTGTACGCGCAGAAATGGCAGCTTGTACGATTGGTGACCCGCCTTTTCGACACTGGACCGCTATGGACGTGGCGGCGGTGTGGCGGCGGCTCAATGACCTGTGGCGCCCGTTTGTGGCGTGCGATAGTCGAGCCGCCTGTCACGTGCGCGGCCGCCCTGCTCCGTTTGACGCGATGCATAGCATGCGACCACCCAGTAATCATACTGCTGACGCTATTGGTCACGTGGTTATGGCAGCTGCTGTTGACTGCGGTGGCGTCCCGTTTCCACACCGTACGTGAGCACATGTCT -3 '(SEQ ID NO: 55)

As PCR reaction solution, 10 ng of genomic DNA as a template, 3 μl each of oligonucleotide primer solution prepared to 5 μM, 5 μl each 2 mM dNTP, 10 × buffer (100 mM Tris-HCl pH 8.3, 500 mM KCl) , 15 mM MgCl ₂ , 0.01% Gelatin) and 5 μl of thermostable DNA polymerase (AmpliTaq Gold, manufactured by ABI) 5 U / μl are mixed, and sterilized ultrapure water is added thereto to bring the volume to 50 μl. What was used was used. The reaction solution was incubated at 95 ° C. for 10 minutes, and then PCR was performed under the conditions of 40 cycles of incubation at 95 ° C. for 20 seconds, then at 58 ° C. for 30 seconds and further at 72 ° C. for 30 seconds. It was.
After performing PCR, amplification was confirmed by 1.5% agarose gel electrophoresis, and DNA fragment S was purified by Wizard SV Gel / PCR Kit (PROMEGA).

  For a part of the obtained DNA fragment solution, 1 μl of SssI methylase (NEB), 10 μl of 10 × NEBuffer2 (NEB) and 1 μl of S-adenosyl methionine (3.2 mM, NEB) are mixed. Then, sterilized ultrapure water was added thereto to prepare a liquid volume of 100 μl. The reaction solution was incubated at 37 ° C. for 15-30 minutes, and 1 μl of S-adenosyl methionine (3.2 mM, manufactured by NEB) was further added and incubated at 37 ° C. for 15-30 minutes. This was purified by Wizard SV Gel / PCR Kit (PROMEGA). This operation was further repeated three times to obtain a methylated DNA fragment (MS, SEQ ID NO: 56).

<DNA fragment> (N represents 5-methylcytosine)
MS: 5'- AGGTGAGCTANGTGTGTTTGGGNGTNGTGCACTGGCTCACTTGTANGNGCAGAAATGGCAGCTTGTANGATTGGTGACCNGCCTTTTNGACACTGGACNGCTATGGANGTGGNGGNGGTGTGGNGGNGGCTCAATGACCTGTGGNGCCNGTTTGTGGNGTGNGATAGTNGAGCNGCCTGTCANGTGNGNGGCNGCCCTGCTCNGTTTGANGNGATGCATAGCATGNGACCACCCAGTAATCATACTGCTGANGCTATTGGTCANGTGGTTATGGCAGCTGCTGTTGACTGNGGTGGNGTCCNGTTTCCACACNGTANGTGAGCACATGTCT -3 '(SEQ ID NO: 56)

For the obtained DNA fragment S, the following solution was prepared.
Solution A: 10 pg / 10 μL TE solution Solution B: 1 pg / 10 μL TE solution Solution C: TE solution (negative control solution)

For the obtained DNA fragment MS, the following solution was prepared.
Solution MA: 10 pg / 10 μL TE solution Solution MB: 1 pg / 10 μL TE solution Solution MC: TE solution (negative control solution)

  In addition, counter oligonucleotides C16 to C25 having the base sequence represented by SEQ ID NO: 58 to SEQ ID NO: 67 capable of binding by complementarity to the negative strand of the target DNA region S ′ comprising the base sequence represented by SEQ ID NO: 57 A TE buffer solution having a concentration of 0.01 μM was prepared.

<Target DNA region>
S ': 5'- AGGTGAGCTACGTGTGTTTGGGCGTCGTGCACTGGCTCACTTGTACGCGCAGAAATGGCAGCTTGTACGATTGGTGACCCGCCTTTTCGACACTGGACCGCTATGGACGTGGCGGCGGTGTGGCGGCGGCTCAATGACCTGTGGCGCCCGTTTGTGGCGTGCGATAGTCGAGCCGCCTGTCACGTGCGCGGCCGCCCTGCTCCGTTTGACGCGATGCATAGCATGCGACCACCCAGTAATCATACTGCTGACGCTATTGGTCACGTGGTTATGGCAGCTGCTGTTGACTGCGGTGGCGTCCCGTTTCCACACCGTACGTGAGCACATGTCT -3' (SEQ ID NO: 57)

<Counter oligonucleotide>
C16: 5'-AGGTGAGCTACGTGTGTTTGG-3 '(SEQ ID NO: 58)
C17: 5'- GCGTCGTGCACTGGCTCACTTGTACGCGCA-3 '(SEQ ID NO: 59)
C18: 5'-CTTGTACGATTGGTGACCCGCCTTTTCGAC-3 '(SEQ ID NO: 60)
C19: 5'-ACTGGACCGCTATGGACGTGGCGGCGGTGT-3 '(SEQ ID NO: 61)
C20: 5'- GGCGGCGGCTCAATGACCTGTGGCGCCCGT-3 '(SEQ ID NO: 62)
C21: 5'-TTGTGGCGTGCGATAGTCGAGCCGCCTGTC-3 '(SEQ ID NO: 63)
C22: 5′-ACGTGCGCGGCCGCCCTGCTCCGTT -3 ′ (SEQ ID NO: 64)
C23: 5'-TGACGCGATGCATAGCATGCGACCACCCAG-3 '(SEQ ID NO: 65)
C24: 5′-ACTGCTGACGCTATTGGTCACGTGGTTATG-3 ′ (SEQ ID NO: 66)
C25: 5'-CTGCTGTTGACTGCGGTGGCGTCCCGTTTC-3 '(SEQ ID NO: 67)

  The following treatment was performed on each of the DNA fragment S solution and the methylated DNA fragment MS solution.

In a PCR tube, 10 μL of the DNA fragment solution prepared above, 10 μL of the counter oligonucleotide solution prepared above, 5 μL of a buffer (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc ₂ , 5 mM Dithiothreitol), 100 mM 5 μL of MgCl ₂ solution and 5 μL of 1 mg / mL BSA solution were added, and further sterilized ultrapure water was added to the mixture to make the volume 50 μL and mixed. Thereafter, the PCR tube was heated at 95 ° C. for 10 minutes, quickly cooled to 70 ° C., and kept at that temperature for 10 minutes. Next, the mixture was cooled to 50 ° C., kept for 10 minutes, further kept at 37 ° C. for 10 minutes, and then returned to room temperature (this corresponds to the first step (A) of the measurement method of the present invention).

50 μL of the DNA fragment reaction solution prepared above was added to the streptavidin-coated PCR tube on which the biotin-labeled methylcytosine antibody was immobilized, and left at room temperature for 30 minutes. Thereafter, the solution was removed by pipetting, and 100 μL of a washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4)] was added, and then the buffer was pipetted. Removed. This operation was further repeated twice (this corresponds to the first step (B) of the measurement method of the present invention).

  Next, PCR is carried out on the above PCR tube using each solution of oligonucleotide primers PF3 and PR3 consisting of the nucleotide sequences represented by SEQ ID NO: 53 and SEQ ID NO: 54 and the following reaction conditions, so that SEQ ID NO: 57 The methylated DNA in the target DNA region S ′ consisting of the base sequence shown in FIG.

<Oligonucleotide primers designed for PCR>
PF3: 5'-AGGTGAGCTACGTGTGTTTGG-3 '(SEQ ID NO: 53)
PR3: 5′-AGACATGTGCTCACGTACGGT-3 ′ (SEQ ID NO: 54)

<Target DNA region>
S ': 5'- AGGTGAGCTACGTGTGTTTGGGCGTCGTGCACTGGCTCACTTGTACGCGCAGAAATGGCAGCTTGTACGATTGGTGACCCGCCTTTTCGACACTGGACCGCTATGGACGTGGCGGCGGTGTGGCGGCGGCTCAATGACCTGTGGCGCCCGTTTGTGGCGTGCGATAGTCGAGCCGCCTGTCACGTGCGCGGCCGCCCTGCTCCGTTTGACGCGATGCATAGCATGCGACCACCCAGTAATCATACTGCTGACGCTATTGGTCACGTGGTTATGGCAGCTGCTGTTGACTGCGGTGGCGTCCCGTTTCCACACCGTACGTGAGCACATGTCT -3' (SEQ ID NO: 57)

As a PCR reaction solution, 3 μL each of oligonucleotide primer solution prepared to 5 μM, 5 μL each 2 mM dNTP, and buffer solution (100 mM Tris-HCl pH 8.3, 500 mM KCl, 15 mM MgCl ₂ , 0.01% Gelatin) is mixed with 5 μL of heat-resistant DNA polymerase (AmpliTaq Gold, manufactured by ABI) 5 U / μL with 0.25 μL, and sterilized ultrapure water is added to make the volume 50 μL. It was. The reaction solution was incubated at 95 ° C. for 10 minutes, and then subjected to PCR under the conditions of 25 cycles of incubation at 95 ° C. for 20 seconds, then at 58 ° C. for 30 seconds and further at 72 ° C. for 30 seconds. It was.
After PCR, amplification was confirmed by 1.5% agarose gel electrophoresis. The results were as follows (corresponding to the third step of the measurement method of the present invention).

  The results are shown in FIG. Amplification was confirmed in the solutions MA and MB of the methylated DNA fragment MS. No amplification was confirmed in the negative control solution MC. Amplification was not confirmed in solutions A, B, and C of DNA fragment S that was not methylated.

  From the above, it is possible to select a DNA containing a target DNA region that has been methylated with an immobilized methylcytosine antibody, and to amplify the methylated DNA to a detectable amount. It was confirmed that the amplified DNA can be detected with higher sensitivity.

Example 7
A commercially available methylcytosine antibody (Aviva Systems Biology) was labeled with biotin using a commercially available biotinylation kit (Biotin Labeling Kit-NH ₂ , manufactured by Dojindo Laboratories) according to the method described in the catalog. The obtained biotin-labeled methylcytosine antibody was refrigerated and stored as a solution [antibody about 0.25 μg / μL 0.1% BSA-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4) solution].

50 µL each of 0.1 µg / 50 µL solution of biotin-labeled methylcytosine antibody obtained by synthesis was added to the streptavidin-coated PCR tubes (total of 6 tubes) and left at room temperature for about 1 hour. Immobilized to. Thereafter, the solution was removed by pipetting, and 100 μL of a washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4)] was added, and then the buffer was pipetted. Removed. This operation was repeated twice more (this corresponds to the preparation of the immobilized methylated DNA antibody used in the measurement method of the present invention).

Baker's yeast strain X2180-1A is cultured in YPD medium (1% Yeast extract, 2% Peptone, 2% Glucose, pH 5.6-6.0) until the turbidity is OD ₆₀₀ 0.6-1.0, and 10,000 g for 10 minutes Centrifugation was performed to prepare 1 × 10 ⁷ yeast cells. The yeast genome was obtained from the prepared yeast cells using a general yeast genome preparation method as described in Methods in Yeast Genetics (Cold Spring Harbor Laboratory).
The prepared yeast cells are suspended in buffer A (1M sorbitol, 0.1M EDTA, pH 7.4), 2-mercaptoethanol (final concentration 14 mM) and 100 U zymolase (10 mg / ml) are added, and the solution becomes clear. Incubated with stirring at 30 ° C for 1 hour until complete. Protoplasts were collected by centrifugation at 550 g for 10 minutes, suspended in buffer B (50 mM Tris-HCl, pH 7.4, 20 mM EDTA), and then sodium dodecyl sulfate was added to 1% (w / v). Thereafter, it was incubated at 65 ° C. for 30 minutes. Subsequently, 5M CH ₃ COOK in a volume ratio of 2/5 was added and mixed, and after ice cooling for 30 minutes, the supernatant was recovered by centrifugation at 15,000 g for 30 minutes. Add 1/10 volume ratio of 3M CH ₃ COONa and an equal amount of isopropanol to the collected supernatant, mix well, and centrifuge at 15,000g for 30 minutes at 4 ° C to rinse the precipitate with 70% ethanol. And recovered. The precipitate is dried, dissolved in 1 ml of TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA), RNase A (manufactured by Sigma) is added to a concentration of 40 μg / ml, and 1 at 37 ° C. After incubating for a time, proteinase K (manufactured by Sigma) was added to the mixture at 500 μg / ml and sodium dodecyl sulfate at 1% (w / v), and this was shaken at 55 ° C. for about 16 hours. That ’s it. After completion of the shaking, the mixture was extracted with phenol [saturated with 1M Tris-HCl (pH 8.0)] and chloroform. The aqueous layer was recovered, and NaCl was added to this to 0.5 N, and then the resulting precipitate was recovered by ethanol precipitation. Genomic DNA was obtained by rinsing the collected precipitate with 70% ethanol.
From the obtained genomic DNA, PCR was performed using the oligonucleotide primers (PF4 and PR4) and reaction conditions designed for PCR shown in SEQ ID NO: 68 and SEQ ID NO: 69 below, as test samples. The DNA fragment to be used (T, the region corresponding to nucleotide number 384569-384685 of yeast chromosome VII shown in SEQ ID NO: 70, Genbank Accession No. NC — 001139, etc.) was amplified.

<Oligonucleotide primers designed for PCR>
PF4: 5'-GGACCTGTGTTTGACGGGTAT-3 '(SEQ ID NO: 68)
PR4: 5'-AGTACAGATCTGGCGTTCTCG-3 '(SEQ ID NO: 69)

<DNA fragment>
T: 5'-GGACCTGTGTTTGACGGGTATAACACTAAGTTGCGCAATTTGCTGTATTGCGAAATCCGCCCGGACGATATCACTCTTGAGCGCATGTGCCGTTTCCGAGAACGCCAGATCTGTACT-3 '(SEQ ID NO: 70)

As PCR reaction solution, 10 ng of genomic DNA as a template, 3 μl each of oligonucleotide primer solution prepared to 5 μM, 5 μl each 2 mM dNTP, 10 × buffer (100 mM Tris-HCl pH 8.3, 500 mM KCl) , 15 mM MgCl ₂ , 0.01% Gelatin) and 5 μl of thermostable DNA polymerase (AmpliTaq Gold, manufactured by ABI) 5 U / μl are mixed, and sterilized ultrapure water is added thereto to bring the volume to 50 μl. What was used was used. The reaction solution was incubated at 95 ° C. for 10 minutes, and then PCR was performed under the conditions of 40 cycles of incubation at 95 ° C. for 20 seconds, then at 58 ° C. for 30 seconds and further at 72 ° C. for 30 seconds. It was.
After performing PCR, amplification was confirmed by 1.5% agarose gel electrophoresis, and DNA fragment T was purified by Wizard SV Gel / PCR Kit (PROMEGA).

  For a part of the obtained DNA fragment solution, 1 μl of SssI methylase (NEB), 10 μl of 10 × NEBuffer2 (NEB) and 1 μl of S-adenosyl methionine (3.2 mM, NEB) are mixed. Then, sterilized ultrapure water was added thereto to prepare a liquid volume of 100 μl. The reaction solution was incubated at 37 ° C. for 15-30 minutes, and 1 μl of S-adenosyl methionine (3.2 mM, manufactured by NEB) was further added and incubated at 37 ° C. for 15-30 minutes. This was purified by Wizard SV Gel / PCR Kit (PROMEGA). This operation was further repeated three times to obtain a methylated DNA fragment (MT, SEQ ID NO: 71).

<DNA fragment> (N represents 5-methylcytosine)
MT: 5'-GGACCTGTGTTTGANGGGTATAACACTAAGTTGNGCAATTTGCTGTATTGNGAAATCNGCCNGGANGATATCACTCTTGAGNGCATGTGCNGTTTCNGAGAANGCCAGATCTGTACT-3 '(SEQ ID NO: 71)

For the DNA fragment T obtained, the following solution was prepared.
Solution A: 10 pg / 10 μL TE solution Solution B: 1 pg / 10 μL TE solution Solution C: TE solution (negative control solution)

For the obtained DNA fragment MT, the following solution was prepared.
Solution MA: 10 pg / 10 μL TE solution Solution MB: 1 pg / 10 μL TE solution Solution MC: TE solution (negative control solution)

  In addition, counter oligonucleotides C26 to C29 consisting of the base sequence represented by SEQ ID NO: 73 to SEQ ID NO: 76 capable of binding by complementarity to the negative strand of the target DNA region T ′ comprising the base sequence represented by SEQ ID NO: 72 A TE buffer solution having a concentration of 0.01 μM was prepared.

<Target DNA region>
T ′: 5′-GGACCTGTGTTTGACGGGTATAACACTAAGTTGCGCAATTTGCTGTATTGCGAAATCCGCCCGGACGATATCACTCTTGAGCGCATGTGCCGTTTCCGAGAACGCCAGATCTGTACT-3 ′ (SEQ ID NO: 72)

<Counter oligonucleotide>
C26: 5'-GGACCTGTGTTTGACGGGTAT-3 '(SEQ ID NO: 73)
C27: 5'-AACACTAAGTTGCGCAATTTGCTGT-3 '(SEQ ID NO: 74)
C28: 5'-ATTGCGAAATCCGCCCGGACGATAT-3 '(SEQ ID NO: 75)
C29: 5'-CACTCTTGAGCGCATGTGCCGTTTC-3 '(SEQ ID NO: 76)

  Each of the solution of the DNA fragment T and the solution of the methylated DNA fragment MT was subjected to the following treatment.

In a PCR tube, 10 μL of the DNA fragment solution prepared above, 10 μL of the counter oligonucleotide solution prepared above, 5 μL of a buffer (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc ₂ , 5 mM Dithiothreitol), 100 mM 5 μL of MgCl ₂ solution and 5 μL of 1 mg / mL BSA solution were added, and further sterilized ultrapure water was added to the mixture to make the volume 50 μL and mixed. Thereafter, the PCR tube was heated at 95 ° C. for 10 minutes, quickly cooled to 70 ° C., and kept at that temperature for 10 minutes. Next, the mixture was cooled to 50 ° C., kept for 10 minutes, further kept at 37 ° C. for 10 minutes, and then returned to room temperature (this corresponds to the first step (A) of the measurement method of the present invention).

50 μL of the DNA fragment reaction solution prepared above was added to the streptavidin-coated PCR tube on which the biotin-labeled methylcytosine antibody was immobilized, and left at room temperature for 30 minutes. Thereafter, the solution was removed by pipetting, and 100 μL of a washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4)] was added, and then the buffer was pipetted. Removed. This operation was further repeated twice (this corresponds to the first step (B) of the measurement method of the present invention).

  Next, PCR is performed on the above PCR tube using each solution of oligonucleotide primers PF4 and PR4 consisting of the nucleotide sequences represented by SEQ ID NO: 68 and SEQ ID NO: 69, and the following reaction conditions, whereby SEQ ID NO: 72 The methylated DNA in the target DNA region T ′ consisting of the base sequence shown in FIG.

<Oligonucleotide primers designed for PCR>
PF4: 5'-GGACCTGTGTTTGACGGGTAT-3 '(SEQ ID NO: 68)
PR4: 5'-AGTACAGATCTGGCGTTCTCG-3 '(SEQ ID NO: 69)

<Target DNA region>
T ': 5'-GGACCTGTGTTTGACGGGTATAACACTAAGTTGCGCAATTTGCTGTATTGCGAAATCCGCCCGGACGATATCACTCTTGAGCGCATGTGCCGTTTCCGAGAACGCCAGATCTGTACT-3' (SEQ ID NO: 72)

As a PCR reaction solution, 3 μL each of oligonucleotide primer solution prepared to 5 μM, 5 μL each 2 mM dNTP, and buffer solution (100 mM Tris-HCl pH 8.3, 500 mM KCl, 15 mM MgCl ₂ , 0.01% Gelatin) is mixed with 5 μL of heat-resistant DNA polymerase (AmpliTaq Gold, manufactured by ABI) 5 U / μL with 0.25 μL, and sterilized ultrapure water is added to make the volume 50 μL. It was. The reaction solution was incubated at 95 ° C. for 10 minutes, and then PCR was performed under the conditions of performing incubation for 28 cycles of 95 ° C. for 20 seconds, then 58 ° C. for 30 seconds and 72 ° C. for 30 seconds. It was.
After PCR, amplification was confirmed by 1.5% agarose gel electrophoresis. The results were as follows (corresponding to the third step of the measurement method of the present invention).

  The results are shown in FIG. Amplification was confirmed in the solutions MA and MB of the methylated DNA fragment MT. No amplification was confirmed in the negative control solution MC. Amplification was not confirmed in solutions A, B, and C of DNA fragment T that was not methylated.

  From the above, it is possible to select a DNA containing a target DNA region that has been methylated with an immobilized methylcytosine antibody, and to amplify the methylated DNA to a detectable amount. It was confirmed that the amplified DNA can be detected with higher sensitivity.

Example 8
A commercially available methylcytosine antibody (Aviva Systems Biology) was labeled with biotin using a commercially available biotinylation kit (Biotin Labeling Kit-NH ₂ , manufactured by Dojindo Laboratories) according to the method described in the catalog. The obtained biotin-labeled methylcytosine antibody was refrigerated and stored as a solution [antibody about 0.25 μg / μL 0.1% BSA-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4) solution].

50 µL each of 0.1 µg / 50 µL solution of biotin-labeled methylcytosine antibody obtained by synthesis was added to the streptavidin-coated PCR tubes (total of 8 tubes) and left at room temperature for about 1 hour. Immobilized to. Thereafter, the solution was removed by pipetting, and 100 μL of a washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4)] was added, and then the buffer was pipetted. Removed. This operation was repeated twice more (this corresponds to the preparation of the immobilized methylated DNA antibody used in the measurement method of the present invention).

Baker's yeast strain X2180-1A is cultured in YPD medium (1% Yeast extract, 2% Peptone, 2% Glucose, pH 5.6-6.0) until the turbidity is OD ₆₀₀ 0.6-1.0, and 10,000 g for 10 minutes Centrifugation was performed to prepare 1 × 10 ⁷ yeast cells. The yeast genome was obtained from the prepared yeast cells using a general yeast genome preparation method as described in Methods in Yeast Genetics (Cold Spring Harbor Laboratory).
The prepared yeast cells are suspended in buffer A (1M sorbitol, 0.1M EDTA, pH 7.4), 2-mercaptoethanol (final concentration 14 mM) and 100 U zymolase (10 mg / ml) are added, and the solution becomes clear. Incubated with stirring at 30 ° C for 1 hour until complete. Protoplasts were collected by centrifugation at 550 g for 10 minutes, suspended in buffer B (50 mM Tris-HCl, pH 7.4, 20 mM EDTA), and then sodium dodecyl sulfate was added to 1% (w / v). Thereafter, it was incubated at 65 ° C. for 30 minutes. Subsequently, 5M CH ₃ COOK in a volume ratio of 2/5 was added and mixed, and after ice cooling for 30 minutes, the supernatant was recovered by centrifugation at 15,000 g for 30 minutes. Add 1/10 volume ratio of 3M CH ₃ COONa and an equal amount of isopropanol to the collected supernatant, mix well, and centrifuge at 15,000g for 30 minutes at 4 ° C to rinse the precipitate with 70% ethanol. And recovered. The precipitate is dried, dissolved in 1 ml of TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA), RNase A (manufactured by Sigma) is added to a concentration of 40 μg / ml, and 1 at 37 ° C. After incubating for a time, proteinase K (manufactured by Sigma) was added to the mixture at 500 μg / ml and sodium dodecyl sulfate at 1% (w / v), and this was shaken at 55 ° C. for about 16 hours. That ’s it. After completion of the shaking, the mixture was extracted with phenol [saturated with 1M Tris-HCl (pH 8.0)] and chloroform. The aqueous layer was recovered, and NaCl was added to this to 0.5 N, and then the resulting precipitate was recovered by ethanol precipitation. Genomic DNA was obtained by rinsing the collected precipitate with 70% ethanol.

  Part of the obtained yeast genomic DNA was mixed with 1 μl of SssI methylase (manufactured by NEB), 10 μl of 10 × NEBuffer2 (manufactured by NEB), and 1 μl of S-adenosyl methionine (3.2 mM, manufactured by NEB). Then, sterilized ultrapure water was added thereto to prepare a liquid volume of 100 μl. The reaction solution was incubated at 37 ° C. for 15-30 minutes, and 1 μl of S-adenosyl methionine (3.2 mM, manufactured by NEB) was further added and incubated at 37 ° C. for 15-30 minutes. This was purified by Wizard SV Gel / PCR Kit (PROMEGA). Further, this operation was repeated three times to obtain methylated yeast genomic DNA.

About the obtained yeast genomic DNA, the following solutions were prepared.
Solution A: 10 ng / 5 μL TE solution Solution B: 1 ng / 5 μL TE solution Solution C: 0.1 ng / 5 μL TE solution Solution D: TE solution (negative control solution)

About the obtained methylated yeast genomic DNA, the following solutions were prepared.
Solution MA: 10 ng / 5 μL TE solution Solution MB: 1 ng / 5 μL TE solution Solution MC: 0.1 ng / 5 μL TE solution Solution MD: TE solution (negative control solution)

  The following treatment was applied to each of the above-described yeast genomic DNA solution and methylated yeast genomic DNA solution.

In a PCR tube, mix 5 μL of the genomic DNA solution prepared above, 5 U of restriction enzyme XspI, and 2 μl of 10 × buffer (200 mM Tris-HCl pH 8.5, 100 mM MgCl ₂ , 10 mM Dithiothreitol, 1000 mM KCl) optimal for XspI. Then, sterilized ultrapure water was added thereto to prepare a liquid volume of 20 μl. The reaction solution was incubated at 37 ° C. for 1 hour.

  SEQ ID NO: that can bind by complementarity to the negative strand of DNA fragment S ″ (the region corresponding to base numbers 271742-272080 of yeast chromosome VII shown in Genbank Accession No. NC_001139 etc.) consisting of the base sequence shown in SEQ ID NO: 77 Counter oligonucleotides C16 to C25 having a nucleotide sequence represented by 58 to SEQ ID NO: 67 were synthesized, and TE buffer solutions having respective concentrations of 0.01 μM were prepared.

<DNA fragment>
S: 5'- TAGGTGAGCTACGTGTGTTTGGGCGTCGTGCACTGGCTCACTTGTACGCGCAGAAATGGCAGCTTGTACGATTGGTGACCCGCCTTTTCGACACTGGACCGCTATGGACGTGGCGGCGGTGTGGCGGCGGCTCAATGACCTGTGGCGCCCGTTTGTGGCGTGCGATAGTCGAGCCGCCTGTCACGTGCGCGGCCGCCCTGCTCCGTTTGACGCGATGCATAGCATGCGACCACCCAGTAATCATACTGCTGACGCTATTGGTCACGTGGTTATGGCAGCTGCTGTTGACTGCGGTGGCGTCCCGTTTCCACACCGTACGTGAGCACATGTCTGGATTGC -3 '(SEQ ID NO: 77)

<Counter oligonucleotide>
C16: 5'-AGGTGAGCTACGTGTGTTTGG-3 '(SEQ ID NO: 58)
C17: 5'- GCGTCGTGCACTGGCTCACTTGTACGCGCA-3 '(SEQ ID NO: 59)
C18: 5'-CTTGTACGATTGGTGACCCGCCTTTTCGAC-3 '(SEQ ID NO: 60)
C19: 5'-ACTGGACCGCTATGGACGTGGCGGCGGTGT-3 '(SEQ ID NO: 61)
C20: 5'- GGCGGCGGCTCAATGACCTGTGGCGCCCGT-3 '(SEQ ID NO: 62)
C21: 5'-TTGTGGCGTGCGATAGTCGAGCCGCCTGTC-3 '(SEQ ID NO: 63)
C22: 5′-ACGTGCGCGGCCGCCCTGCTCCGTT -3 ′ (SEQ ID NO: 64)
C23: 5'-TGACGCGATGCATAGCATGCGACCACCCAG-3 '(SEQ ID NO: 65)
C24: 5′-ACTGCTGACGCTATTGGTCACGTGGTTATG-3 ′ (SEQ ID NO: 66)
C25: 5'-CTGCTGTTGACTGCGGTGGCGTCCCGTTTC-3 '(SEQ ID NO: 67)

  Each reaction solution of the yeast genomic DNA and methylated yeast genomic DNA was subjected to the following treatment.

In a PCR tube, 20 μL of the genomic DNA reaction solution prepared above, 10 μL of the counter oligonucleotide solution prepared above, 5 μL of buffer solution (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc ₂ , 5 mM Dithiothreitol), 100 mM MgCl ₂ solution (5 μL) and 1 mg / mL BSA solution (5 μL) were added, and sterilized ultrapure water was added to the mixture to make a volume of 50 μL and mixed. Thereafter, the PCR tube was heated at 95 ° C. for 10 minutes, quickly cooled to 70 ° C., and kept at that temperature for 10 minutes. Next, the mixture was cooled to 50 ° C., kept for 10 minutes, further kept at 37 ° C. for 10 minutes, and then returned to room temperature (this corresponds to the first step (A) of the measurement method of the present invention).

50 μL of the DNA fragment reaction solution prepared above was added to the streptavidin-coated PCR tube on which the biotin-labeled methylcytosine antibody was immobilized, and left at room temperature for 30 minutes. Thereafter, the solution was removed by pipetting, and 100 μL of a washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4)] was added, and then the buffer was pipetted. Removed. This operation was further repeated twice (this corresponds to the first step (B) of the measurement method of the present invention).

  Next, PCR is carried out on the above PCR tube using each solution of oligonucleotide primers PF3 and PR3 consisting of the nucleotide sequences represented by SEQ ID NO: 53 and SEQ ID NO: 54 and the following reaction conditions, so that SEQ ID NO: 57 The methylated DNA in the target DNA region S ′ consisting of the base sequence shown in FIG.

<Oligonucleotide primers designed for PCR>
PF3: 5'-AGGTGAGCTACGTGTGTTTGG-3 '(SEQ ID NO: 53)
PR3: 5′-AGACATGTGCTCACGTACGGT-3 ′ (SEQ ID NO: 54)

<Target DNA region>
S ': 5'- AGGTGAGCTACGTGTGTTTGGGCGTCGTGCACTGGCTCACTTGTACGCGCAGAAATGGCAGCTTGTACGATTGGTGACCCGCCTTTTCGACACTGGACCGCTATGGACGTGGCGGCGGTGTGGCGGCGGCTCAATGACCTGTGGCGCCCGTTTGTGGCGTGCGATAGTCGAGCCGCCTGTCACGTGCGCGGCCGCCCTGCTCCGTTTGACGCGATGCATAGCATGCGACCACCCAGTAATCATACTGCTGACGCTATTGGTCACGTGGTTATGGCAGCTGCTGTTGACTGCGGTGGCGTCCCGTTTCCACACCGTACGTGAGCACATGTCT -3' (SEQ ID NO: 57)

As a PCR reaction solution, 3 μL each of oligonucleotide primer solution prepared to 5 μM, 5 μL each 2 mM dNTP, and buffer solution (100 mM Tris-HCl pH 8.3, 500 mM KCl, 15 mM MgCl ₂ , 0.01% Gelatin) is mixed with 5 μL of heat-resistant DNA polymerase (AmpliTaq Gold, manufactured by ABI) 5 U / μL with 0.25 μL, and sterilized ultrapure water is added to make the volume 50 μL. It was. The reaction solution was incubated at 95 ° C. for 10 minutes, and then PCR was carried out under the conditions of 31 cycles of incubation at 95 ° C. for 20 seconds, then at 58 ° C. for 30 seconds and further at 72 ° C. for 30 seconds. It was.
After PCR, amplification was confirmed by 1.5% agarose gel electrophoresis. The results were as follows (corresponding to the third step of the measurement method of the present invention).

  The results are shown in FIG. Amplification was confirmed in the solutions MA, MB and MC of methylated yeast genomic DNA. No amplification was confirmed in the negative control solution MD. No amplification was confirmed in the unmethylated yeast genomic DNA solutions A, B, C and D.

  From the above, it is possible to select a DNA containing a target DNA region that has been methylated with an immobilized methylcytosine antibody, and to amplify the methylated DNA to a detectable amount. It was confirmed that the amplified DNA can be detected with higher sensitivity.

Example 9
A commercially available methylcytosine antibody (Aviva Systems Biology) was labeled with biotin using a commercially available biotinylation kit (Biotin Labeling Kit-NH ₂ , manufactured by Dojindo Laboratories) according to the method described in the catalog. The obtained biotin-labeled methylcytosine antibody was refrigerated and stored as a solution [antibody about 0.25 μg / μL 0.1% BSA-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4) solution].

50 µL each of 0.1 µg / 50 µL solution of biotin-labeled methylcytosine antibody obtained by synthesis was added to the streptavidin-coated PCR tubes (total of 8 tubes) and left at room temperature for about 1 hour. Immobilized to. Thereafter, the solution was removed by pipetting, and 100 μL of a washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4)] was added, and then the buffer was pipetted. Removed. This operation was repeated twice more (this corresponds to the preparation of the immobilized methylated DNA antibody used in the measurement method of the present invention).

Baker's yeast strain X2180-1A is cultured in YPD medium (1% Yeast extract, 2% Peptone, 2% Glucose, pH 5.6-6.0) until the turbidity is OD ₆₀₀ 0.6-1.0, and 10,000 g for 10 minutes Centrifugation was performed to prepare 1 × 10 ⁷ yeast cells. The yeast genome was obtained from the prepared yeast cells using a general yeast genome preparation method as described in Methods in Yeast Genetics (Cold Spring Harbor Laboratory).
The prepared yeast cells are suspended in buffer A (1M sorbitol, 0.1M EDTA, pH 7.4), 2-mercaptoethanol (final concentration 14 mM) and 100 U zymolase (10 mg / ml) are added, and the solution becomes clear. Incubated with stirring at 30 ° C for 1 hour until complete. Protoplasts were collected by centrifugation at 550 g for 10 minutes, suspended in buffer B (50 mM Tris-HCl, pH 7.4, 20 mM EDTA), and then sodium dodecyl sulfate was added to 1% (w / v). Thereafter, it was incubated at 65 ° C. for 30 minutes. Subsequently, 5M CH ₃ COOK in a volume ratio of 2/5 was added and mixed, and after ice cooling for 30 minutes, the supernatant was recovered by centrifugation at 15,000 g for 30 minutes. Add 1/10 volume ratio of 3M CH ₃ COONa and an equal amount of isopropanol to the collected supernatant, mix well, and centrifuge at 15,000g for 30 minutes at 4 ° C to rinse the precipitate with 70% ethanol. And recovered. The precipitate is dried, dissolved in 1 ml of TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA), RNase A (manufactured by Sigma) is added to a concentration of 40 μg / ml, and 1 at 37 ° C. After incubating for a time, proteinase K (manufactured by Sigma) was added to the mixture at 500 μg / ml and sodium dodecyl sulfate at 1% (w / v), and this was shaken at 55 ° C. for about 16 hours. That ’s it. After completion of the shaking, the mixture was extracted with phenol [saturated with 1M Tris-HCl (pH 8.0)] and chloroform. The aqueous layer was recovered, and NaCl was added to this to 0.5 N, and then the resulting precipitate was recovered by ethanol precipitation. Genomic DNA was obtained by rinsing the collected precipitate with 70% ethanol.

  Part of the obtained yeast genomic DNA was mixed with 1 μl of SssI methylase (manufactured by NEB), 10 μl of 10 × NEBuffer2 (manufactured by NEB), and 1 μl of S-adenosyl methionine (3.2 mM, manufactured by NEB). Then, sterilized ultrapure water was added thereto to prepare a liquid volume of 100 μl. The reaction solution was incubated at 37 ° C. for 15-30 minutes, and 1 μl of S-adenosyl methionine (3.2 mM, manufactured by NEB) was further added and incubated at 37 ° C. for 15-30 minutes. This was purified by Wizard SV Gel / PCR Kit (PROMEGA). Further, this operation was repeated three times to obtain methylated yeast genomic DNA.

About the obtained yeast genomic DNA, the following solutions were prepared.
Solution A: 10 ng / 5 μL TE solution Solution B: 1 ng / 5 μL TE solution Solution C: 0.1 ng / 5 μL TE solution Solution D: TE solution (negative control solution)

About the obtained methylated yeast genomic DNA, the following solutions were prepared.
Solution MA: 10 ng / 5 μL TE solution Solution MB: 1 ng / 5 μL TE solution Solution MC: 0.1 ng / 5 μL TE solution Solution MD: TE solution (negative control solution)

The following treatment was applied to each of the above-described yeast genomic DNA solution and methylated yeast genomic DNA solution.

In a PCR tube, mix 5 μL of the genomic DNA solution prepared above, 5 U of restriction enzyme XspI, and 2 μl of 10 × buffer (200 mM Tris-HCl pH 8.5, 100 mM MgCl ₂ , 10 mM Dithiothreitol, 1000 mM KCl) optimal for XspI. Then, sterilized ultrapure water was added thereto to prepare a liquid volume of 20 μl. The reaction solution was incubated at 37 ° C. for 1 hour.

  SEQ ID NO: that can bind by complementarity to the negative strand of DNA fragment T ″ (the region corresponding to base numbers 384523-384766 of yeast chromosome VII shown in Genbank Accession No. NC — 001139 etc.) consisting of the base sequence shown in SEQ ID NO: 78 Counter oligonucleotides C26 to C33 consisting of the nucleotide sequences represented by 73 to SEQ ID NO: 76 and SEQ ID NO: 79 to SEQ ID NO: 82 were synthesized, and TE buffer solutions each having a concentration of 0.01 μM were prepared.

<DNA fragment>
T: 5'-TAGGAAATACATTCCGAGGGCGCCCGCACAAGGCCTATTATTAGAGGGACCTGTGTTTGACGGGTATAACACTAAGTTGCGCAATTTGCTGTATTGCGAAATCCGCCCGGACGATATCACTCTTGAGCGCATGTGCCGTTTCCGAGAACGCCAGATCTGCATGTGTGTTGCGCGCTG

<Counter oligonucleotide>
C26: 5'-GGACCTGTGTTTGACGGGTAT-3 '(SEQ ID NO: 73)
C27: 5'-AACACTAAGTTGCGCAATTTGCTGT-3 '(SEQ ID NO: 74)
C28: 5'-ATTGCGAAATCCGCCCGGACGATAT-3 '(SEQ ID NO: 75)
C29: 5'-CACTCTTGAGCGCATGTGCCGTTTC-3 '(SEQ ID NO: 76)
C30: 5′-AATACATTCCGAGGGCGCCCGCACAAGGCC -3 ′ (SEQ ID NO: 79)
C31: 5′-GCGATCGCACACGAGGAGACA -3 ′ (SEQ ID NO: 80)
C32: 5'-AGCGTCACGTGTTTTGCCATTTTGTACGAC-3 '(SEQ ID NO: 81)
C33: 5'-AAATGAACCGCCTGGCCACGCCTCTAATC-3 '(SEQ ID NO: 82)

Each reaction solution of the yeast genomic DNA and methylated yeast genomic DNA was subjected to the following treatment.

In a PCR tube, 20 μL of the genomic DNA reaction solution prepared above, 10 μL of the counter oligonucleotide solution prepared above, 5 μL of a buffer solution (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc ₂ , 5 mM Dithiothreitol), 5 μL of 100 mM MgCl ₂ solution and 5 μL of 1 mg / mL BSA solution were added, and further sterilized ultrapure water was added to the mixture to adjust the volume to 50 μL and mixed. Thereafter, the PCR tube was heated at 95 ° C. for 10 minutes, quickly cooled to 70 ° C., and kept at that temperature for 10 minutes. Next, the mixture was cooled to 50 ° C., kept for 10 minutes, further kept at 37 ° C. for 10 minutes, and then returned to room temperature (this corresponds to the first step (A) of the measurement method of the present invention).

50 μL of the DNA fragment reaction solution prepared above was added to the streptavidin-coated PCR tube on which the biotin-labeled methylcytosine antibody was immobilized, and left at room temperature for 30 minutes. Thereafter, the solution was removed by pipetting, and 100 μL of a washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4)] was added, and then the buffer was pipetted. Removed. This operation was further repeated twice (this corresponds to the first step (B) of the measurement method of the present invention).

  Next, PCR is performed on the above PCR tube using each solution of oligonucleotide primers PF4 and PR4 consisting of the nucleotide sequences represented by SEQ ID NO: 68 and SEQ ID NO: 69, and the following reaction conditions, whereby SEQ ID NO: 72 The methylated DNA in the target DNA region T ′ consisting of the base sequence shown in FIG.

<Oligonucleotide primers designed for PCR>
PF4: 5'-GGACCTGTGTTTGACGGGTAT-3 '(SEQ ID NO: 68)
PR4: 5'-AGTACAGATCTGGCGTTCTCG-3 '(SEQ ID NO: 69)

<Target DNA region>
T ': 5'-GGACCTGTGTTTGACGGGTATAACACTAAGTTGCGCAATTTGCTGTATTGCGAAATCCGCCCGGACGATATCACTCTTGAGCGCATGTGCCGTTTCCGAGAACGCCAGATCTGTACT-3' (SEQ ID NO: 72)

As a PCR reaction solution, 3 μL each of oligonucleotide primer solution prepared to 5 μM, 5 μL each 2 mM dNTP, and buffer solution (100 mM Tris-HCl pH 8.3, 500 mM KCl, 15 mM MgCl ₂ , 0.01% Gelatin) is mixed with 5 μL of heat-resistant DNA polymerase (AmpliTaq Gold, manufactured by ABI) 5 U / μL with 0.25 μL, and sterilized ultrapure water is added to make the volume 50 μL. It was. The reaction solution was incubated at 95 ° C. for 10 minutes, and then PCR was carried out under the conditions of 31 cycles of incubation at 95 ° C. for 20 seconds, then at 58 ° C. for 30 seconds and further at 72 ° C. for 30 seconds. It was.
After PCR, amplification was confirmed by 1.5% agarose gel electrophoresis. The results were as follows (corresponding to the third step of the measurement method of the present invention).

  The results are shown in FIG. Amplification was confirmed in the solutions MA, MB and MC of methylated yeast genomic DNA. No amplification was confirmed in the negative control solution MD. No amplification was confirmed in the unmethylated yeast genomic DNA solutions A, B, C and D.

  From the above, it is possible to select a DNA containing a target DNA region that has been methylated with an immobilized methylcytosine antibody, and to amplify the methylated DNA to a detectable amount. It was confirmed that the amplified DNA can be detected with higher sensitivity.

Example 10
A commercially available methylcytosine antibody (Aviva Systems Biology) was labeled with biotin using a commercially available biotinylation kit (Biotin Labeling Kit-NH ₂ , manufactured by Dojindo Laboratories) according to the method described in the catalog. The obtained biotin-labeled methylcytosine antibody was refrigerated and stored as a solution [antibody about 0.25 μg / μL 0.1% BSA-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4) solution].

50 µL each of 0.1 µg / 50 µL solution of biotin-labeled methylcytosine antibody obtained by synthesis was added to the streptavidin-coated PCR tubes (total of 8 tubes) and left at room temperature for about 1 hour. Immobilized to. Thereafter, the solution was removed by pipetting, and 100 μL of a washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4)] was added, and then the buffer was pipetted. Removed. This operation was repeated twice more (this corresponds to the preparation of the immobilized methylated DNA antibody used in the measurement method of the present invention).

  For human blood-derived genomic DNA purchased from Clontech, PCR is performed using oligonucleotide primer primers (PF1 and PR1) and reaction conditions designed for PCR shown in SEQ ID NO: 28 and SEQ ID NO: 29 below. As a result, a DNA fragment (region corresponding to base number 25687390-25687775 shown in X, SEQ ID NO: 30, Genbank Accession No. NT — 029419, etc.) used as a test sample was amplified.

<Oligonucleotide primers designed for PCR>
PF1: 5′-CTCAGCACCCAGGCGGCC -3 ′ (SEQ ID NO: 28)
PR1: 5′-CTGGCCAAACTGGAGATCGC -3 ′ (SEQ ID NO: 29)

<DNA fragment>
X: 5'- CTCAGCACCCAGGCGGCCGCGATCATGAGGCGCGAGCGGCGCGCGGGCTGTTGCAGAGTCTTGAGCGGGTGGCACACCGCGATGTAGCGGTCGGCTGTCATGACTACCAGCATGTAGGCCGACGCAAACATGCCGAACACCTGCAGGTGCTTCACCACGCGGCACAGCCAGTCGGGGCCGCGGAAGCGGTAGGTGATGTCCCAGCACATTTGCGGCAGCACCTGGAAGAATGCCACGGCCAGGTCGGCCAGGCTGAGGTGTCGGATGAAGAGGTGCATGCGGGACGTCTTGCGCGGCGTCCGGTGCAGAGCCAGCAGTACGCTGCTGTTGCCCAGCACGGCCACCGCGAAAGTCACCGCCAGCACGGCGATCTCCAGTTTGGCCAG -3 '(SEQ ID NO: 30)

As PCR reaction solution, 5 ng of genomic DNA as a template, 3 μl each of oligonucleotide primer solution prepared to 5 μM, 5 μl each 2 mM dNTP, 10 × buffer (100 mM Tris-HCl pH 8.3, 500 mM KCl) , 15 mM MgCl ₂ , 0.01% Gelatin) and 5 μl of thermostable DNA polymerase (AmpliTaq Gold, manufactured by ABI) 5 U / μl are mixed, and sterilized ultrapure water is added thereto to bring the volume to 50 μl. What was used was used. The reaction solution was incubated at 95 ° C. for 10 minutes, and then PCR was performed under the conditions of 40 cycles of incubation at 95 ° C. for 30 seconds, then at 61 ° C. for 30 seconds, and further at 72 ° C. for 45 seconds. It was.
After performing PCR, amplification was confirmed by 1.5% agarose gel electrophoresis, and DNA fragment X was purified by Wizard SV Gel / PCR Kit (PROMEGA).

  For a part of the obtained DNA fragment solution, 1 μl of SssI methylase (NEB), 10 μl of 10 × NEBuffer2 (NEB) and 1 μl of S-adenosyl methionine (3.2 mM, NEB) are mixed. Then, sterilized ultrapure water was added thereto to prepare a liquid volume of 100 μl. The reaction solution was incubated at 37 ° C. for 15-30 minutes, and 1 μl of S-adenosyl methionine (3.2 mM, manufactured by NEB) was further added and incubated at 37 ° C. for 15-30 minutes. This was purified by Wizard SV Gel / PCR Kit (PROMEGA). Furthermore, these operations were repeated 5 times to obtain a methylated DNA fragment (MX, SEQ ID NO: 31).

<DNA fragment> (N represents 5-methylcytosine)
MX: 5'- CTCAGCACCCAGGNGGCNGNGATCATGAGGNGNGAGNGGNGNGNGGGCTGTTGCAGAGTCTTGAGNGGGTGGCACACNGNGATGTAGNGGTNGGCTGTCATGACTACCAGCATGTAGGCNGANGCAAACATGCNGAACACCTGCAGGTGCTTCACCANGNGGCACAGCCAGTNGGGGCNGNGGAAGNGGTAGGTGATGTCCCAGCACATTTGNGGCAGCACCTGGAAGAATGCCANGGCCAGGTNGGCCAGGCTGAGGTGTNGGATGAAGAGGTGCATGNGGGANGTCTTGNGNGGNGTCNGGTGCAGAGCCAGCAGTANGCTGCTGTTGCCCAGCANGGCCACNGNGAAAGTCACNGCCAGCANGGNGATCTCCAGTTTGGCCAG -3 '(SEQ ID NO: 31)

For the obtained DNA fragment X, the following solution was prepared.
Solution A: 100 pg / 10 μL TE solution Solution B: 10 pg / 10 μL TE solution Solution C: 1 pg / 10 μL TE solution Solution D: TE solution (negative control solution)

The following solution was prepared for the obtained DNA fragment MX.
Solution MA: 100 pg / 10 μL TE solution Solution MB: 10 pg / 10 μL TE solution Solution MC: 1 pg / 10 μL TE solution Solution MD: TE solution (negative control solution)

  In addition, counter oligonucleotides C1 to C12 having the base sequence represented by SEQ ID NO: 33 to SEQ ID NO: 44, which can bind to the negative strand of the target DNA region X ′ comprising the base sequence represented by SEQ ID NO: 32 by complementarity, are synthesized Then, TE buffer solutions each having a concentration of 0.01 μM were prepared.

<Target DNA region>
X ': 5'- CTCAGCACCCAGGCGGCCGCGATCATGAGGCGCGAGCGGCGCGCGGGCTGTTGCAGAGTCTTGAGCGGGTGGCACACCGCGATGTAGCGGTCGGCTGTCATGACTACCAGCATGTAGGCCGACGCAAACATGCCGAACACCTGCAGGTGCTTCACCACGCGGCACAGCCAGTCGGGGCCGCGGAAGCGGTAGGTGATGTCCCAGCACATTTGCGGCAGCACCTGGAAGAATGCCACGGCCAGGTCGGCCAGGCTGAGGTGTCGGATGAAGAGGTGCATGCGGGACGTCTTGCGCGGCGTCCGGTGCAGAGCCAGCAGTACGCTGCTGTTGCCCAGCACGGCCACCGCGAAAGTCACCGCCAGCACGGCGATCTCCAGTTTGGCCAG -3' (SEQ ID NO: 32)

<Counter oligonucleotide>
C1: 5'- GCCACCGCGAAAGTCACCGCCAGCACGGCG-3 '(SEQ ID NO: 33)
C2: 5'-GCCAGCAGTACGCTGCTGTTGCCCAGCACG-3 '(SEQ ID NO: 34)
C3: 5′-CGGGACGTCTTGCGCGGCGTCCGGTGCAGA -3 ′ (SEQ ID NO: 35)
C4: 5'-AGGCTGAGGTGTCGGATGAAGAGGTGCATG-3 '(SEQ ID NO: 36)
C5: 5'-ACCTGGAAGAATGCCACGGCCAGGTCGGCC-3 '(SEQ ID NO: 37)
C6: 5′-TAGGTGATGTCCCAGCACATTTGCGGCAGC -3 ′ (SEQ ID NO: 38)
C7: 5'-CGGCACAGCCAGTCGGGGCCGCGGAAGCGG-3 '(SEQ ID NO: 39)
C8: 5'- ATGCCGAACACCTGCAGGTGCTTCACCACG-3 '(SEQ ID NO: 40)
C9: 5'- ATGACTACCAGCATGTAGGCCGACGCAAAC-3 '(SEQ ID NO: 41)
C10: 5'-TGGCACACCGCGATGTAGCGGTCGGCTGTC-3 '(SEQ ID NO: 42)
C11: 5′-CGCGCGGGCTGTTGCAGAGTCTTGAGCGGG-3 ′ (SEQ ID NO: 43)
C12: 5'-CAGGCGGCCGCGATCATGAGGCGCGAGCGG-3 '(SEQ ID NO: 44)

In the PCR tube, 10 μL of the DNA fragment solution prepared above, 10 μL of the prepared counter oligonucleotide solution, 5 μL of buffer solution (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc ₂ , 5 mM Dithiothreitol), 100 mM MgCl ₂ solution 5 μL and 5 μL of 1 mg / mL BSA solution were added, and sterilized ultrapure water was further added to the mixture to make a liquid volume of 50 μL and mixed. Thereafter, the PCR tube was heated at 95 ° C. for 10 minutes, quickly cooled to 70 ° C., and kept at that temperature for 10 minutes. Next, the mixture was cooled to 50 ° C., kept for 10 minutes, further kept at 37 ° C. for 10 minutes, and then returned to room temperature (this corresponds to the first step (A) of the measurement method of the present invention).

50 μL of the DNA fragment reaction solution prepared above was added to the streptavidin-coated PCR tube on which the biotin-labeled methylcytosine antibody was immobilized, and left at room temperature for 30 minutes. Thereafter, the solution was removed by pipetting, and 100 μL of a washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4)] was added, and then the buffer was pipetted. Removed. This operation was repeated twice more (this corresponds to the second step of the measurement method of the present invention).

A buffer solution (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc2, 5 mM Dithiothreitol) and 5 μL of BSA (Bovine) were added to the PCR tube coated with streptavidin to which the biotin-labeled methylcytosine antibody subjected to the above treatment was immobilized. 5 μL of serum albumin (1 mg / ml) and 16 U of methylation sensitive restriction enzyme HhaI were added, and each mixture was further incubated at 37 ° C. for 1 hour by adding sterile ultrapure water to the mixture to a volume of 50 μL. . Thereafter, the solution was removed by pipetting, and 100 μL of a washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4)] was added, and then the buffer was pipetted. Removed. This operation was repeated twice more (this corresponds to the second step of the measurement method of the present invention).

  Next, PCR is performed on the above PCR tube using each solution of oligonucleotide primers PF1 and PR1 consisting of the nucleotide sequences represented by SEQ ID NO: 28 and SEQ ID NO: 29, and the following reaction conditions, whereby SEQ ID NO: 32 The methylated DNA in the target DNA region X ′ consisting of the base sequence shown in FIG.

<Oligonucleotide primers designed for PCR>
PF1: 5'-CTCAGCACCCAGGCGGCC-3 '(SEQ ID NO: 28)
PR1: 5′-CTGGCCAAACTGGAGATCGC -3 ′ (SEQ ID NO: 29)

<Target DNA region>
X ': 5'- CTCAGCACCCAGGCGGCCGCGATCATGAGGCGCGAGCGGCGCGCGGGCTGTTGCAGAGTCTTGAGCGGGTGGCACACCGCGATGTAGCGGTCGGCTGTCATGACTACCAGCATGTAGGCCGACGCAAACATGCCGAACACCTGCAGGTGCTTCACCACGCGGCACAGCCAGTCGGGGCCGCGGAAGCGGTAGGTGATGTCCCAGCACATTTGCGGCAGCACCTGGAAGAATGCCACGGCCAGGTCGGCCAGGCTGAGGTGTCGGATGAAGAGGTGCATGCGGGACGTCTTGCGCGGCGTCCGGTGCAGAGCCAGCAGTACGCTGCTGTTGCCCAGCACGGCCACCGCGAAAGTCACCGCCAGCACGGCGATCTCCAGTTTGGCCAG -3' (SEQ ID NO: 32)

As a PCR reaction solution, 3 μL each of oligonucleotide primer solution prepared to 5 μM, 5 μL each 2 mM dNTP, and buffer solution (100 mM Tris-HCl pH 8.3, 500 mM KCl, 15 mM MgCl ₂ , 0.01% Gelatin) is mixed with 5 μL of heat-resistant DNA polymerase (AmpliTaq Gold, manufactured by ABI) 5 U / μL with 0.25 μL, and sterilized ultrapure water is added to make the volume 50 μL. It was. The reaction solution was incubated at 95 ° C. for 10 minutes, and then PCR was performed under the conditions of 25 cycles of incubation at 95 ° C. for 20 seconds, then at 61 ° C. for 30 seconds, and further at 72 ° C. for 30 seconds. It was.
After PCR, amplification was confirmed by 1.5% agarose gel electrophoresis. The results were as follows (corresponding to the third step of the measurement method of the present invention).

  The results are shown in FIG. Amplification was confirmed in the solutions MA, MB and MC of the methylated DNA fragment MX. No amplification was confirmed in the negative control solution MD. No amplification was confirmed in solutions A, B, C and D of DNA fragment X which was not methylated.

  From the above, it is possible to select a DNA containing a target DNA region that has been methylated with an immobilized methylcytosine antibody, and to amplify the methylated DNA to a detectable amount. It was confirmed that the amplified DNA can be detected with higher sensitivity.

Example 11
A commercially available methylcytosine antibody (Aviva Systems Biology) was labeled with biotin using a commercially available biotinylation kit (Biotin Labeling Kit-NH ₂ , manufactured by Dojindo Laboratories) according to the method described in the catalog. The obtained biotin-labeled methylcytosine antibody was refrigerated and stored as a solution [antibody about 0.25 μg / μL 0.1% BSA-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4) solution].

50 µL each of 0.1 µg / 50 µL solution of biotin-labeled methylcytosine antibody obtained by synthesis was added to the streptavidin-coated PCR tubes (total of 8 tubes) and left at room temperature for about 1 hour. Immobilized to. Thereafter, the solution was removed by pipetting, and 100 μL of a washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4)] was added, and then the buffer was pipetted. Removed. This operation was repeated twice more (this corresponds to the preparation of the immobilized methylated DNA antibody used in the measurement method of the present invention).

Baker's yeast strain X2180-1A is cultured in YPD medium (1% Yeast extract, 2% Peptone, 2% Glucose, pH 5.6-6.0) until the turbidity is OD ₆₀₀ 0.6-1.0, and 10,000 g for 10 minutes Centrifugation was performed to prepare 1 × 10 ⁷ yeast cells. The yeast genome was obtained from the prepared yeast cells using a general yeast genome preparation method as described in Methods in Yeast Genetics (Cold Spring Harbor Laboratory).
The prepared yeast cells are suspended in buffer A (1M sorbitol, 0.1M EDTA, pH 7.4), 2-mercaptoethanol (final concentration 14 mM) and 100 U zymolase (10 mg / ml) are added, and the solution becomes clear. Incubated with stirring at 30 ° C for 1 hour until complete. Protoplasts were collected by centrifugation at 550 g for 10 minutes, suspended in buffer B (50 mM Tris-HCl, pH 7.4, 20 mM EDTA), and then sodium dodecyl sulfate was added to 1% (w / v). Thereafter, it was incubated at 65 ° C. for 30 minutes. Subsequently, 5M CH ₃ COOK in a volume ratio of 2/5 was added and mixed, and after ice cooling for 30 minutes, the supernatant was recovered by centrifugation at 15,000 g for 30 minutes. Add 1/10 volume ratio of 3M CH ₃ COONa and an equal amount of isopropanol to the collected supernatant, mix well, and centrifuge at 15,000g for 30 minutes at 4 ° C to rinse the precipitate with 70% ethanol. And recovered. The precipitate is dried, dissolved in 1 ml of TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA), RNase A (manufactured by Sigma) is added to a concentration of 40 μg / ml, and 1 at 37 ° C. After incubating for a time, proteinase K (manufactured by Sigma) was added to the mixture at 500 μg / ml and sodium dodecyl sulfate at 1% (w / v), and this was shaken at 55 ° C. for about 16 hours. That ’s it. After completion of the shaking, the mixture was extracted with phenol [saturated with 1M Tris-HCl (pH 8.0)] and chloroform. The aqueous layer was recovered, and NaCl was added to this to 0.5 N, and then the resulting precipitate was recovered by ethanol precipitation. Genomic DNA was obtained by rinsing the collected precipitate with 70% ethanol.
From the obtained genomic DNA, PCR was carried out using oligonucleotide primers (PF3 and PR3) and reaction conditions designed for PCR shown in SEQ ID NO: 53 and SEQ ID NO: 54 below, as test samples. The DNA fragment used (S, SEQ ID NO: 55, region corresponding to base number 271743-272083 of yeast chromosome VII shown in Genbank Accession No. NC — 001139, etc.) was amplified.

<Oligonucleotide primers designed for PCR>
PF3: 5'-AGGTGAGCTACGTGTGTTTGG-3 '(SEQ ID NO: 53)
PR3: 5'-AGACATGTGCTCACGTACGGT-3 '(SEQ ID NO: 54)

<DNA fragment>
S: 5'- AGGTGAGCTACGTGTGTTTGGGCGTCGTGCACTGGCTCACTTGTACGCGCAGAAATGGCAGCTTGTACGATTGGTGACCCGCCTTTTCGACACTGGACCGCTATGGACGTGGCGGCGGTGTGGCGGCGGCTCAATGACCTGTGGCGCCCGTTTGTGGCGTGCGATAGTCGAGCCGCCTGTCACGTGCGCGGCCGCCCTGCTCCGTTTGACGCGATGCATAGCATGCGACCACCCAGTAATCATACTGCTGACGCTATTGGTCACGTGGTTATGGCAGCTGCTGTTGACTGCGGTGGCGTCCCGTTTCCACACCGTACGTGAGCACATGTCT -3 '(SEQ ID NO: 55)

As PCR reaction solution, 10 ng of genomic DNA as a template, 3 μl each of oligonucleotide primer solution prepared to 5 μM, 5 μl each 2 mM dNTP, 10 × buffer (100 mM Tris-HCl pH 8.3, 500 mM KCl) , 15 mM MgCl ₂ , 0.01% Gelatin) and 5 μl of thermostable DNA polymerase (AmpliTaq Gold, manufactured by ABI) 5 U / μl are mixed, and sterilized ultrapure water is added thereto to bring the volume to 50 μl. What was used was used. The reaction solution was incubated at 95 ° C. for 10 minutes, and then PCR was performed under the conditions of 40 cycles of incubation at 95 ° C. for 20 seconds, then at 58 ° C. for 30 seconds and further at 72 ° C. for 30 seconds. It was.
After performing PCR, amplification was confirmed by 1.5% agarose gel electrophoresis, and DNA fragment S was purified by Wizard SV Gel / PCR Kit (PROMEGA).

  For a part of the obtained DNA fragment solution, 1 μl of SssI methylase (NEB), 10 μl of 10 × NEBuffer2 (NEB) and 1 μl of S-adenosyl methionine (3.2 mM, NEB) are mixed. Then, sterilized ultrapure water was added thereto to prepare a liquid volume of 100 μl. The reaction solution was incubated at 37 ° C. for 15-30 minutes, and 1 μl of S-adenosyl methionine (3.2 mM, manufactured by NEB) was further added and incubated at 37 ° C. for 15-30 minutes. This was purified by Wizard SV Gel / PCR Kit (PROMEGA). This operation was further repeated three times to obtain a methylated DNA fragment (MS, SEQ ID NO: 56).

<DNA fragment> (N represents 5-methylcytosine)
MS: 5'- AGGTGAGCTANGTGTGTTTGGGNGTNGTGCACTGGCTCACTTGTANGNGCAGAAATGGCAGCTTGTANGATTGGTGACCNGCCTTTTNGACACTGGACNGCTATGGANGTGGNGGNGGTGTGGNGGNGGCTCAATGACCTGTGGNGCCNGTTTGTGGNGTGNGATAGTNGAGCNGCCTGTCANGTGNGNGGCNGCCCTGCTCNGTTTGANGNGATGCATAGCATGNGACCACCCAGTAATCATACTGCTGANGCTATTGGTCANGTGGTTATGGCAGCTGCTGTTGACTGNGGTGGNGTCCNGTTTCCACACNGTANGTGAGCACATGTCT -3 '(SEQ ID NO: 56)

For the obtained DNA fragment S, the following solution was prepared.
Solution A: 100 pg / 10 μL TE solution Solution B: 10 pg / 10 μL TE solution Solution C: 1 pg / 10 μL TE solution Solution D: TE solution (negative control solution)

For the obtained DNA fragment MS, the following solution was prepared.
Solution MA: 100 pg / 10 μL TE solution Solution MB: 10 pg / 10 μL TE solution Solution MC: 1 pg / 10 μL TE solution Solution MD: TE solution (negative control solution)

  In addition, counter oligonucleotides C16 to C25 having the base sequence represented by SEQ ID NO: 58 to SEQ ID NO: 67 capable of binding by complementarity to the negative strand of the target DNA region S ′ comprising the base sequence represented by SEQ ID NO: 57 A TE buffer solution having a concentration of 0.01 μM was prepared.

<Target DNA region>
S ': 5'- AGGTGAGCTACGTGTGTTTGGGCGTCGTGCACTGGCTCACTTGTACGCGCAGAAATGGCAGCTTGTACGATTGGTGACCCGCCTTTTCGACACTGGACCGCTATGGACGTGGCGGCGGTGTGGCGGCGGCTCAATGACCTGTGGCGCCCGTTTGTGGCGTGCGATAGTCGAGCCGCCTGTCACGTGCGCGGCCGCCCTGCTCCGTTTGACGCGATGCATAGCATGCGACCACCCAGTAATCATACTGCTGACGCTATTGGTCACGTGGTTATGGCAGCTGCTGTTGACTGCGGTGGCGTCCCGTTTCCACACCGTACGTGAGCACATGTCT -3' (SEQ ID NO: 57)

<Counter oligonucleotide>
C16: 5'-AGGTGAGCTACGTGTGTTTGG-3 '(SEQ ID NO: 58)
C17: 5'- GCGTCGTGCACTGGCTCACTTGTACGCGCA-3 '(SEQ ID NO: 59)
C18: 5'-CTTGTACGATTGGTGACCCGCCTTTTCGAC-3 '(SEQ ID NO: 60)
C19: 5'-ACTGGACCGCTATGGACGTGGCGGCGGTGT-3 '(SEQ ID NO: 61)
C20: 5'- GGCGGCGGCTCAATGACCTGTGGCGCCCGT-3 '(SEQ ID NO: 62)
C21: 5'-TTGTGGCGTGCGATAGTCGAGCCGCCTGTC-3 '(SEQ ID NO: 63)
C22: 5′-ACGTGCGCGGCCGCCCTGCTCCGTT -3 ′ (SEQ ID NO: 64)
C23: 5'-TGACGCGATGCATAGCATGCGACCACCCAG-3 '(SEQ ID NO: 65)
C24: 5′-ACTGCTGACGCTATTGGTCACGTGGTTATG-3 ′ (SEQ ID NO: 66)
C25: 5'-CTGCTGTTGACTGCGGTGGCGTCCCGTTTC-3 '(SEQ ID NO: 67)

  The following treatment was performed on each of the DNA fragment S solution and the methylated DNA fragment MS solution.

In a PCR tube, 10 μL of the DNA fragment solution prepared above, 10 μL of the counter oligonucleotide solution prepared above, 5 μL of a buffer (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc ₂ , 5 mM Dithiothreitol), 100 mM 5 μL of MgCl ₂ solution and 5 μL of 1 mg / mL BSA solution were added, and further sterilized ultrapure water was added to the mixture to make the volume 50 μL and mixed. Thereafter, the PCR tube was heated at 95 ° C. for 10 minutes, quickly cooled to 70 ° C., and kept at that temperature for 10 minutes. Next, the mixture was cooled to 50 ° C., kept for 10 minutes, further kept at 37 ° C. for 10 minutes, and then returned to room temperature (this corresponds to the first step (A) of the measurement method of the present invention).

50 μL of the DNA fragment reaction solution prepared above was added to the streptavidin-coated PCR tube on which the biotin-labeled methylcytosine antibody was immobilized, and left at room temperature for 30 minutes. Thereafter, the solution was removed by pipetting, and 100 μL of a washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4)] was added, and then the buffer was pipetted. Removed. This operation was further repeated twice (this corresponds to the first step (B) of the measurement method of the present invention).

To the PCR tube coated with the biotin-labeled methylcytosine antibody subjected to the above treatment and coated with streptavidin, 5 μL of a buffer (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc ₂ , 5 mM Dithiothreitol) and BSA ( Bovine serum albumin (1 mg / ml), 5 μL, methylation sensitive restriction enzyme HhaI, 16 U, and sterilized ultrapure water were added to the mixture to bring the volume to 50 μL. did. Thereafter, the solution was removed by pipetting, and 100 μL of a washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4)] was added, and then the buffer was pipetted. Removed. This operation was repeated twice more (this corresponds to the second step of the measurement method of the present invention).

  Next, PCR is carried out on the above PCR tube using each solution of oligonucleotide primers PF3 and PR3 consisting of the nucleotide sequences represented by SEQ ID NO: 53 and SEQ ID NO: 54 and the following reaction conditions, so that SEQ ID NO: 57 The methylated DNA in the target DNA region S ′ consisting of the base sequence shown in FIG.

<Oligonucleotide primers designed for PCR>
PF3: 5'-AGGTGAGCTACGTGTGTTTGG-3 '(SEQ ID NO: 53)
PR3: 5′-AGACATGTGCTCACGTACGGT-3 ′ (SEQ ID NO: 54)

<Target DNA region>
S ': 5'- AGGTGAGCTACGTGTGTTTGGGCGTCGTGCACTGGCTCACTTGTACGCGCAGAAATGGCAGCTTGTACGATTGGTGACCCGCCTTTTCGACACTGGACCGCTATGGACGTGGCGGCGGTGTGGCGGCGGCTCAATGACCTGTGGCGCCCGTTTGTGGCGTGCGATAGTCGAGCCGCCTGTCACGTGCGCGGCCGCCCTGCTCCGTTTGACGCGATGCATAGCATGCGACCACCCAGTAATCATACTGCTGACGCTATTGGTCACGTGGTTATGGCAGCTGCTGTTGACTGCGGTGGCGTCCCGTTTCCACACCGTACGTGAGCACATGTCT -3' (SEQ ID NO: 57)

As a PCR reaction solution, 3 μL each of oligonucleotide primer solution prepared to 5 μM, 5 μL each 2 mM dNTP, and buffer solution (100 mM Tris-HCl pH 8.3, 500 mM KCl, 15 mM MgCl ₂ , 0.01% Gelatin) is mixed with 5 μL of heat-resistant DNA polymerase (AmpliTaq Gold, manufactured by ABI) 5 U / μL with 0.25 μL, and sterilized ultrapure water is added to make the volume 50 μL. It was. The reaction solution was incubated at 95 ° C. for 10 minutes, and then subjected to PCR under the conditions of 25 cycles of incubation at 95 ° C. for 20 seconds, then at 58 ° C. for 30 seconds and further at 72 ° C. for 30 seconds. It was.
After PCR, amplification was confirmed by 1.5% agarose gel electrophoresis. The results were as follows (corresponding to the third step of the measurement method of the present invention).

  The results are shown in FIG. Amplification was confirmed in the solutions MA, MB and MC of the methylated DNA fragment MS. No amplification was confirmed in the negative control solution MD. No amplification was confirmed in solutions A, B, C and D of DNA fragment S which was not methylated.

  From the above, it is possible to select a DNA containing a target DNA region that has been methylated with an immobilized methylcytosine antibody, and to amplify the methylated DNA to a detectable amount. It was confirmed that the amplified DNA can be detected with higher sensitivity.

Example 12
A commercially available methylcytosine antibody (Aviva Systems Biology) was labeled with biotin using a commercially available biotinylation kit (Biotin Labeling Kit-NH ₂ , manufactured by Dojindo Laboratories) according to the method described in the catalog. The obtained biotin-labeled methylcytosine antibody was refrigerated and stored as a solution [antibody about 0.25 μg / μL 0.1% BSA-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4) solution].

50 µL each of 0.1 µg / 50 µL solution of biotin-labeled methylcytosine antibody obtained by synthesis was added to the streptavidin-coated PCR tubes (total of 8 tubes) and left at room temperature for about 1 hour. Immobilized to. Thereafter, the solution was removed by pipetting, and 100 μL of a washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4)] was added, and then the buffer was pipetted. Removed. This operation was repeated twice more (this corresponds to the preparation of the immobilized methylated DNA antibody used in the measurement method of the present invention).

Baker's yeast strain X2180-1A is cultured in YPD medium (1% Yeast extract, 2% Peptone, 2% Glucose, pH 5.6-6.0) until the turbidity is OD ₆₀₀ 0.6-1.0, and 10,000 g for 10 minutes Centrifugation was performed to prepare 1 × 10 ⁷ yeast cells. The yeast genome was obtained from the prepared yeast cells using a general yeast genome preparation method as described in Methods in Yeast Genetics (Cold Spring Harbor Laboratory).
The prepared yeast cells are suspended in buffer A (1M sorbitol, 0.1M EDTA, pH 7.4), 2-mercaptoethanol (final concentration 14 mM) and 100 U zymolase (10 mg / ml) are added, and the solution becomes clear. Incubated with stirring at 30 ° C for 1 hour until complete. Protoplasts were collected by centrifugation at 550 g for 10 minutes, suspended in buffer B (50 mM Tris-HCl, pH 7.4, 20 mM EDTA), and then sodium dodecyl sulfate was added to 1% (w / v). Thereafter, it was incubated at 65 ° C. for 30 minutes. Subsequently, 5M CH ₃ COOK in a volume ratio of 2/5 was added and mixed, and after ice cooling for 30 minutes, the supernatant was recovered by centrifugation at 15,000 g for 30 minutes. Add 1/10 volume ratio of 3M CH ₃ COONa and an equal amount of isopropanol to the collected supernatant, mix well, and centrifuge at 15,000g for 30 minutes at 4 ° C to rinse the precipitate with 70% ethanol. And recovered. The precipitate is dried, dissolved in 1 ml of TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA), RNase A (manufactured by Sigma) is added to a concentration of 40 μg / ml, and 1 at 37 ° C. After incubating for a time, proteinase K (manufactured by Sigma) was added to the mixture at 500 μg / ml and sodium dodecyl sulfate at 1% (w / v), and this was shaken at 55 ° C. for about 16 hours. That ’s it. After completion of the shaking, the mixture was extracted with phenol [saturated with 1M Tris-HCl (pH 8.0)] and chloroform. The aqueous layer was recovered, and NaCl was added to this to 0.5 N, and then the resulting precipitate was recovered by ethanol precipitation. Genomic DNA was obtained by rinsing the collected precipitate with 70% ethanol.
From the obtained genomic DNA, PCR was performed using the oligonucleotide primers (PF4 and PR4) and reaction conditions designed for PCR shown in SEQ ID NO: 68 and SEQ ID NO: 69 below, as test samples. The DNA fragment to be used (T, the region corresponding to nucleotide number 384569-384685 of yeast chromosome VII shown in SEQ ID NO: 70, Genbank Accession No. NC — 001139, etc.) was amplified.

<Oligonucleotide primers designed for PCR>
PF4: 5'-GGACCTGTGTTTGACGGGTAT-3 '(SEQ ID NO: 68)
PR4: 5'-AGTACAGATCTGGCGTTCTCG-3 '(SEQ ID NO: 69)

<DNA fragment>
T: 5'-GGACCTGTGTTTGACGGGTATAACACTAAGTTGCGCAATTTGCTGTATTGCGAAATCCGCCCGGACGATATCACTCTTGAGCGCATGTGCCGTTTCCGAGAACGCCAGATCTGTACT-3 '(SEQ ID NO: 70)

As PCR reaction solution, 10 ng of genomic DNA as a template, 3 μl each of oligonucleotide primer solution prepared to 5 μM, 5 μl each 2 mM dNTP, 10 × buffer (100 mM Tris-HCl pH 8.3, 500 mM KCl) , 15 mM MgCl ₂ , 0.01% Gelatin) and 5 μl of thermostable DNA polymerase (AmpliTaq Gold, manufactured by ABI) 5 U / μl are mixed, and sterilized ultrapure water is added thereto to bring the volume to 50 μl. What was used was used. The reaction solution was incubated at 95 ° C. for 10 minutes, and then PCR was performed under the conditions of 40 cycles of incubation at 95 ° C. for 20 seconds, then at 58 ° C. for 30 seconds and further at 72 ° C. for 30 seconds. It was.
After performing PCR, amplification was confirmed by 1.5% agarose gel electrophoresis, and DNA fragment T was purified by Wizard SV Gel / PCR Kit (PROMEGA).

  For a part of the obtained DNA fragment solution, 1 μl of SssI methylase (NEB), 10 μl of 10 × NEBuffer2 (NEB) and 1 μl of S-adenosyl methionine (3.2 mM, NEB) are mixed. Then, sterilized ultrapure water was added thereto to prepare a liquid volume of 100 μl. The reaction solution was incubated at 37 ° C. for 15-30 minutes, and 1 μl of S-adenosyl methionine (3.2 mM, manufactured by NEB) was further added and incubated at 37 ° C. for 15-30 minutes. This was purified by Wizard SV Gel / PCR Kit (PROMEGA). This operation was further repeated three times to obtain a methylated DNA fragment (MT, SEQ ID NO: 71).

<DNA fragment> (N represents 5-methylcytosine)
MT: 5'-GGACCTGTGTTTGANGGGTATAACACTAAGTTGNGCAATTTGCTGTATTGNGAAATCNGCCNGGANGATATCACTCTTGAGNGCATGTGCNGTTTCNGAGAANGCCAGATCTGTACT-3 '(SEQ ID NO: 71)

For the DNA fragment T obtained, the following solution was prepared.
Solution A: 100 pg / 10 μL TE solution Solution B: 10 pg / 10 μL TE solution Solution C: 1 pg / 10 μL TE solution Solution D: TE solution (negative control solution)

For the obtained DNA fragment MT, the following solution was prepared.
Solution MA: 100 pg / 10 μL TE solution Solution MB: 10 pg / 10 μL TE solution Solution MC: 1 pg / 10 μL TE solution Solution MD: TE solution (negative control solution)

  In addition, counter oligonucleotides C26 to C29 consisting of the base sequence represented by SEQ ID NO: 73 to SEQ ID NO: 76 capable of binding by complementarity to the negative strand of the target DNA region T ′ comprising the base sequence represented by SEQ ID NO: 72 A TE buffer solution having a concentration of 0.01 μM was prepared.

<Target DNA region>
T ′: 5′-GGACCTGTGTTTGACGGGTATAACACTAAGTTGCGCAATTTGCTGTATTGCGAAATCCGCCCGGACGATATCACTCTTGAGCGCATGTGCCGTTTCCGAGAACGCCAGATCTGTACT-3 ′ (SEQ ID NO: 72)

<Counter oligonucleotide>
C26: 5'-GGACCTGTGTTTGACGGGTAT-3 '(SEQ ID NO: 73)
C27: 5'-AACACTAAGTTGCGCAATTTGCTGT-3 '(SEQ ID NO: 74)
C28: 5'-ATTGCGAAATCCGCCCGGACGATAT-3 '(SEQ ID NO: 75)
C29: 5'-CACTCTTGAGCGCATGTGCCGTTTC-3 '(SEQ ID NO: 76)

  Each of the solution of the DNA fragment T and the solution of the methylated DNA fragment MT was subjected to the following treatment.

In a PCR tube, 10 μL of the DNA fragment solution prepared above, 10 μL of the counter oligonucleotide solution prepared above, 5 μL of a buffer (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc ₂ , 5 mM Dithiothreitol), 100 mM 5 μL of MgCl ₂ solution and 5 μL of 1 mg / mL BSA solution were added, and further sterilized ultrapure water was added to the mixture to make the volume 50 μL and mixed. Thereafter, the PCR tube was heated at 95 ° C. for 10 minutes, quickly cooled to 70 ° C., and kept at that temperature for 10 minutes. Next, the mixture was cooled to 50 ° C., kept for 10 minutes, further kept at 37 ° C. for 10 minutes, and then returned to room temperature (this corresponds to the first step (A) of the measurement method of the present invention).

50 μL of the DNA fragment reaction solution prepared above was added to the streptavidin-coated PCR tube on which the biotin-labeled methylcytosine antibody was immobilized, and left at room temperature for 30 minutes. Thereafter, the solution was removed by pipetting, and 100 μL of a washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4)] was added, and then the buffer was pipetted. Removed. This operation was further repeated twice (this corresponds to the first step (B) of the measurement method of the present invention).

A buffer solution (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc2, 5 mM Dithiothreitol) and 5 μL of BSA (Bovine) were added to the PCR tube coated with streptavidin to which the biotin-labeled methylcytosine antibody subjected to the above treatment was immobilized. 5 μL of serum albumin (1 mg / ml) and 16 U of methylation sensitive restriction enzyme HhaI were added, and each mixture was further incubated at 37 ° C. for 1 hour by adding sterile ultrapure water to the mixture to a volume of 50 μL. . Thereafter, the solution was removed by pipetting, and 100 μL of a washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4)] was added, and then the buffer was pipetted. Removed. This operation was repeated twice more (this corresponds to the second step of the measurement method of the present invention).

  Next, PCR is performed on the above PCR tube using each solution of oligonucleotide primers PF4 and PR4 consisting of the nucleotide sequences represented by SEQ ID NO: 68 and SEQ ID NO: 69, and the following reaction conditions, whereby SEQ ID NO: 72 The methylated DNA in the target DNA region T ′ consisting of the base sequence shown in FIG.

<Oligonucleotide primers designed for PCR>
PF4: 5'-GGACCTGTGTTTGACGGGTAT-3 '(SEQ ID NO: 68)
PR4: 5'-AGTACAGATCTGGCGTTCTCG-3 '(SEQ ID NO: 69)

<Target DNA region>
T ': 5'-GGACCTGTGTTTGACGGGTATAACACTAAGTTGCGCAATTTGCTGTATTGCGAAATCCGCCCGGACGATATCACTCTTGAGCGCATGTGCCGTTTCCGAGAACGCCAGATCTGTACT-3' (SEQ ID NO: 72)

As a PCR reaction solution, 3 μL each of oligonucleotide primer solution prepared to 5 μM, 5 μL each 2 mM dNTP, and buffer solution (100 mM Tris-HCl pH 8.3, 500 mM KCl, 15 mM MgCl ₂ , 0.01% Gelatin) is mixed with 5 μL of heat-resistant DNA polymerase (AmpliTaq Gold, manufactured by ABI) 5 U / μL with 0.25 μL, and sterilized ultrapure water is added to make the volume 50 μL. It was. The reaction solution was incubated at 95 ° C. for 10 minutes, and then PCR was performed under the conditions of performing incubation for 28 cycles of 95 ° C. for 20 seconds, then 58 ° C. for 30 seconds and 72 ° C. for 30 seconds. It was.
After PCR, amplification was confirmed by 1.5% agarose gel electrophoresis. The results were as follows (corresponding to the third step of the measurement method of the present invention).

  The results are shown in FIG. Amplification was confirmed in the solutions MA, MB and MC of the methylated DNA fragment MT. No amplification was confirmed in the negative control solution MD. No amplification was confirmed in solutions A, B, C and D of DNA fragment T which was not methylated.

  From the above, it is possible to select a DNA containing a target DNA region that has been methylated with an immobilized methylcytosine antibody, and to amplify the methylated DNA to a detectable amount. It was confirmed that the amplified DNA can be detected with higher sensitivity.

Example 13
A commercially available methylcytosine antibody (Aviva Systems Biology) was labeled with biotin using a commercially available biotinylation kit (Biotin Labeling Kit-NH ₂ , manufactured by Dojindo Laboratories) according to the method described in the catalog. The obtained biotin-labeled methylcytosine antibody was refrigerated and stored as a solution [antibody about 0.25 μg / μL 0.1% BSA-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4) solution].

50 µL each of 0.1 µg / 50 µL solution of biotin-labeled methylcytosine antibody obtained by synthesis was added to the streptavidin-coated PCR tubes (total of 6 tubes) and left at room temperature for about 1 hour. Immobilized to. Thereafter, the solution was removed by pipetting, and 100 μL of a washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4)] was added, and then the buffer was pipetted. Removed. This operation was repeated twice more (this corresponds to the preparation of the immobilized methylated DNA antibody used in the measurement method of the present invention).

  For human blood-derived genomic DNA purchased from Clontech, 1 μl of SssI methylase (NEB), 10 μl of 10 × NEBuffer2 (NEB), and 1 μl of S-adenosyl methionine (3.2 mM, NEB) The mixture was mixed, and sterilized ultrapure water was added thereto to prepare a liquid volume of 100 μl. The reaction solution was incubated at 37 ° C. for 15-30 minutes, and 1 μl of S-adenosyl methionine (3.2 mM, manufactured by NEB) was further added and incubated at 37 ° C. for 15-30 minutes. This was purified by Wizard SV Gel / PCR Kit (PROMEGA). This operation was further repeated three times to obtain methylated human genomic DNA.

For human blood-derived genomic DNA purchased from Clontech, the following solution was prepared.
Solution A: 10ng / 5μL TE solution Solution B: 1ng / 5μL TE solution Solution C: TE solution (negative control solution)

The following solutions were prepared for the obtained methylated human genomic DNA.
Solution MA: 10 ng / 5 μL TE solution Solution MB: 1 ng / 5 μL TE solution Solution MC: TE solution (negative control solution)

  The following treatment was applied to each of the above-described human genomic DNA solution and methylated human genomic DNA solution.

In a PCR tube, mix 5 μL of the genomic DNA solution prepared above, 6 U of restriction enzyme AluI, and 1 μl of 10 × buffer solution (100 mM Tris-HCl pH 7.5, 100 mM MgCl ₂ , 10 mM Dithiothreitol, 500 mM NaCl) optimal for AluI. Then, sterilized ultrapure water was added thereto to prepare a liquid volume of 10 μl. The reaction solution was incubated at 37 ° C. for 1 hour.

  SEQ ID NO: 33 to SEQ ID NO: that can bind by complementarity to the negative strand of DNA fragment X ″ (region corresponding to base numbers 25687390-25687775 shown in Genbank Accession No. NT_029419 etc.) consisting of the base sequence shown in SEQ ID NO: 83 Counter oligonucleotides C1 to C12 having a base sequence represented by 44 were synthesized, and TE buffer solutions having a concentration of 0.01 μM were prepared.

<DNA fragment>
X ": 5'- CTCAGCACCCAGGCGGCCGCGATCATGAGGCGCGAGCGGCGCGCGGGCTGTTGCAGAGTCTTGAGCGGGTGGCACACCGCGATGTAGCGGTCGGCTGTCATGACTACCAGCATGTAGGCCGACGCAAACATGCCGAACACCTGCAGGTGCTTCACCACGCGGCACAGCCAGTCGGGGCCGCGGAAGCGGTAGGTGATGTCCCAGCACATTTGCGGCAGCACCTGGAAGAATGCCACGGCCAGGTCGGCCAGGCTGAGGTGTCGGATGAAGAGGTGCATGCGGGACGTCTTGCGCGGCGTCCGGTGCAGAGCCAGCAGTACGCTGCTGTTGCCCAGCACGGCCACCGCGAAAGTCACCGCCAGCACGGCGATCTCCAGTTTGGCCAG -3 '(SEQ ID NO: 83)

<Counter oligonucleotide>
C1: 5'- GCCACCGCGAAAGTCACCGCCAGCACGGCG-3 '(SEQ ID NO: 33)
C2: 5'-GCCAGCAGTACGCTGCTGTTGCCCAGCACG-3 '(SEQ ID NO: 34)
C3: 5′-CGGGACGTCTTGCGCGGCGTCCGGTGCAGA -3 ′ (SEQ ID NO: 35)
C4: 5'-AGGCTGAGGTGTCGGATGAAGAGGTGCATG-3 '(SEQ ID NO: 36)
C5: 5'-ACCTGGAAGAATGCCACGGCCAGGTCGGCC-3 '(SEQ ID NO: 37)
C6: 5′-TAGGTGATGTCCCAGCACATTTGCGGCAGC -3 ′ (SEQ ID NO: 38)
C7: 5'-CGGCACAGCCAGTCGGGGCCGCGGAAGCGG-3 '(SEQ ID NO: 39)
C8: 5'- ATGCCGAACACCTGCAGGTGCTTCACCACG-3 '(SEQ ID NO: 40)
C9: 5'- ATGACTACCAGCATGTAGGCCGACGCAAAC-3 '(SEQ ID NO: 41)
C10: 5'-TGGCACACCGCGATGTAGCGGTCGGCTGTC-3 '(SEQ ID NO: 42)
C11: 5′-CGCGCGGGCTGTTGCAGAGTCTTGAGCGGG-3 ′ (SEQ ID NO: 43)
C12: 5'-CAGGCGGCCGCGATCATGAGGCGCGAGCGG-3 '(SEQ ID NO: 44)

  Each reaction solution of the above human genomic DNA and methylated human genomic DNA was subjected to the following treatment.

In a PCR tube, 10 μL of the genomic DNA reaction solution prepared above, 10 μL of the counter oligonucleotide solution prepared above, 5 μL of a buffer solution (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc ₂ , 5 mM Dithiothreitol), 5 μL of 100 mM MgCl ₂ solution and 5 μL of 1 mg / mL BSA solution were added, and further sterilized ultrapure water was added to the mixture to adjust the volume to 50 μL and mixed. Thereafter, the PCR tube was heated at 95 ° C. for 10 minutes, quickly cooled to 70 ° C., and kept at that temperature for 10 minutes. Next, the mixture was cooled to 50 ° C., kept for 10 minutes, further kept at 37 ° C. for 10 minutes, and then returned to room temperature (this corresponds to the first step (A) of the measurement method of the present invention).

50 μL of the DNA fragment reaction solution prepared above was added to the streptavidin-coated PCR tube on which the biotin-labeled methylcytosine antibody was immobilized, and left at room temperature for 30 minutes. Thereafter, the solution was removed by pipetting, and 100 μL of a washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4)] was added, and then the buffer was pipetted. Removed. This operation was further repeated twice (this corresponds to the first step (B) of the measurement method of the present invention).

To the PCR tube coated with the biotin-labeled methylcytosine antibody subjected to the above treatment and coated with streptavidin, 5 μL of a buffer (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc ₂ , 5 mM Dithiothreitol) and BSA ( Bovine serum albumin (1 mg / ml), 5 μL, methylation sensitive restriction enzyme HhaI, 16 U, and sterilized ultrapure water were added to the mixture to bring the volume to 50 μL. did. Thereafter, the solution was removed by pipetting, and 100 μL of a washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4)] was added, and then the buffer was pipetted. Removed. This operation was repeated twice more (this corresponds to the second step of the measurement method of the present invention).

  Next, PCR is performed on the above PCR tube using each solution of oligonucleotide primers PF1 and PR1 consisting of the nucleotide sequences represented by SEQ ID NO: 28 and SEQ ID NO: 29, and the following reaction conditions, whereby SEQ ID NO: 32 The methylated DNA in the target DNA region X ′ consisting of the base sequence shown in FIG.

<Oligonucleotide primers designed for PCR>
PF1: 5'-CTCAGCACCCAGGCGGCC-3 '(SEQ ID NO: 28)
PR1: 5′-CTGGCCAAACTGGAGATCGC -3 ′ (SEQ ID NO: 29)

<Target DNA region>
X ': 5'- CTCAGCACCCAGGCGGCCGCGATCATGAGGCGCGAGCGGCGCGCGGGCTGTTGCAGAGTCTTGAGCGGGTGGCACACCGCGATGTAGCGGTCGGCTGTCATGACTACCAGCATGTAGGCCGACGCAAACATGCCGAACACCTGCAGGTGCTTCACCACGCGGCACAGCCAGTCGGGGCCGCGGAAGCGGTAGGTGATGTCCCAGCACATTTGCGGCAGCACCTGGAAGAATGCCACGGCCAGGTCGGCCAGGCTGAGGTGTCGGATGAAGAGGTGCATGCGGGACGTCTTGCGCGGCGTCCGGTGCAGAGCCAGCAGTACGCTGCTGTTGCCCAGCACGGCCACCGCGAAAGTCACCGCCAGCACGGCGATCTCCAGTTTGGCCAG -3' (SEQ ID NO: 32)

As a PCR reaction solution, 3 μL each of oligonucleotide primer solution prepared to 5 μM, 5 μL each 2 mM dNTP, and buffer solution (100 mM Tris-HCl pH 8.3, 500 mM KCl, 15 mM MgCl ₂ , 0.01% Gelatin) is mixed with 5 μL of heat-resistant DNA polymerase (AmpliTaq Gold, manufactured by ABI) 5 U / μL with 0.25 μL, and sterilized ultrapure water is added to make the volume 50 μL. It was. The reaction solution was incubated at 95 ° C. for 10 minutes, and then PCR was performed under the conditions of 34 cycles of incubation at 95 ° C. for 20 seconds, then at 61 ° C. for 30 seconds, and further at 72 ° C. for 30 seconds. It was.
After PCR, amplification was confirmed by 1.5% agarose gel electrophoresis. The results were as follows (corresponding to the third step of the measurement method of the present invention).

  The results are shown in FIG. Amplification was confirmed in the solutions MA and MB of methylated human genomic DNA. In the negative control solution MC, amplification was not confirmed and the amplification product was not obtained. In solutions A, B, and C of human genomic DNA that was not methylated, amplification was not confirmed and no amplification product was obtained.

  From the above, it is possible to select a DNA containing a target DNA region that has been methylated with an immobilized methylcytosine antibody, and to amplify the methylated DNA to a detectable amount. It was confirmed that the amplified DNA can be detected with higher sensitivity.

Example 14
A commercially available methylcytosine antibody (Aviva Systems Biology) was labeled with biotin using a commercially available biotinylation kit (Biotin Labeling Kit-NH ₂ , manufactured by Dojindo Laboratories) according to the method described in the catalog. The obtained biotin-labeled methylcytosine antibody was refrigerated and stored as a solution [antibody about 0.25 μg / μL 0.1% BSA-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4) solution].

50 µL each of 0.1 µg / 50 µL solution of biotin-labeled methylcytosine antibody obtained by synthesis was added to the streptavidin-coated PCR tubes (total of 8 tubes) and left at room temperature for about 1 hour. Immobilized to. Thereafter, the solution was removed by pipetting, and 100 μL of a washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4)] was added, and then the buffer was pipetted. Removed. This operation was repeated twice more (this corresponds to the preparation of the immobilized methylated DNA antibody used in the measurement method of the present invention).

Baker's yeast strain X2180-1A is cultured in YPD medium (1% Yeast extract, 2% Peptone, 2% Glucose, pH 5.6-6.0) until the turbidity is OD ₆₀₀ 0.6-1.0, and 10,000 g for 10 minutes Centrifugation was performed to prepare 1 × 10 ⁷ yeast cells. The yeast genome was obtained from the prepared yeast cells using a general yeast genome preparation method as described in Methods in Yeast Genetics (Cold Spring Harbor Laboratory).
The prepared yeast cells are suspended in buffer A (1M sorbitol, 0.1M EDTA, pH 7.4), 2-mercaptoethanol (final concentration 14 mM) and 100 U zymolase (10 mg / ml) are added, and the solution becomes clear. Incubated with stirring at 30 ° C for 1 hour until complete. Protoplasts were collected by centrifugation at 550 g for 10 minutes, suspended in buffer B (50 mM Tris-HCl, pH 7.4, 20 mM EDTA), and then sodium dodecyl sulfate was added to 1% (w / v). Thereafter, it was incubated at 65 ° C. for 30 minutes. Subsequently, 5M CH ₃ COOK in a volume ratio of 2/5 was added and mixed, and after ice cooling for 30 minutes, the supernatant was recovered by centrifugation at 15,000 g for 30 minutes. Add 1/10 volume ratio of 3M CH ₃ COONa and an equal amount of isopropanol to the collected supernatant, mix well, and centrifuge at 15,000g for 30 minutes at 4 ° C to rinse the precipitate with 70% ethanol. And recovered. The precipitate is dried, dissolved in 1 ml of TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA), RNase A (manufactured by Sigma) is added to a concentration of 40 μg / ml, and 1 at 37 ° C. After incubating for a time, proteinase K (manufactured by Sigma) was added to the mixture at 500 μg / ml and sodium dodecyl sulfate at 1% (w / v), and this was shaken at 55 ° C. for about 16 hours. That ’s it. After completion of the shaking, the mixture was extracted with phenol [saturated with 1M Tris-HCl (pH 8.0)] and chloroform. The aqueous layer was recovered, and NaCl was added to this to 0.5 N, and then the resulting precipitate was recovered by ethanol precipitation. Genomic DNA was obtained by rinsing the collected precipitate with 70% ethanol.

  Part of the obtained yeast genomic DNA was mixed with 1 μl of SssI methylase (manufactured by NEB), 10 μl of 10 × NEBuffer2 (manufactured by NEB), and 1 μl of S-adenosyl methionine (3.2 mM, manufactured by NEB). Then, sterilized ultrapure water was added thereto to prepare a liquid volume of 100 μl. The reaction solution was incubated at 37 ° C. for 15-30 minutes, and 1 μl of S-adenosyl methionine (3.2 mM, manufactured by NEB) was further added and incubated at 37 ° C. for 15-30 minutes. This was purified by Wizard SV Gel / PCR Kit (PROMEGA). Further, this operation was repeated three times to obtain methylated yeast genomic DNA.

About the obtained yeast genomic DNA, the following solutions were prepared.
Solution A: 10 ng / 5 μL TE solution Solution B: 1 ng / 5 μL TE solution Solution C: 0.1 ng / 5 μL TE solution Solution D: TE solution (negative control solution)

About the obtained methylated yeast genomic DNA, the following solutions were prepared.
Solution MA: 10 ng / 5 μL TE solution Solution MB: 1 ng / 5 μL TE solution Solution MC: 0.1 ng / 5 μL TE solution Solution MD: TE solution (negative control solution)

  The following treatment was applied to each of the above-described yeast genomic DNA solution and methylated yeast genomic DNA solution.

In a PCR tube, mix 5 μL of the genomic DNA solution prepared above, 5 U of restriction enzyme XspI, and 2 μl of 10 × buffer (200 mM Tris-HCl pH 8.5, 100 mM MgCl ₂ , 10 mM Dithiothreitol, 1000 mM KCl) optimal for XspI. Then, sterilized ultrapure water was added thereto to prepare a liquid volume of 20 μl. The reaction solution was incubated at 37 ° C. for 1 hour.

  SEQ ID NO: that can bind by complementarity to the negative strand of DNA fragment S ″ (the region corresponding to base numbers 271742-272080 of yeast chromosome VII shown in Genbank Accession No. NC_001139 etc.) consisting of the base sequence shown in SEQ ID NO: 77 Counter oligonucleotides C16 to C25 having a nucleotide sequence represented by 58 to SEQ ID NO: 67 were synthesized, and TE buffer solutions having respective concentrations of 0.01 μM were prepared.

<DNA fragment>
S: 5'- TAGGTGAGCTACGTGTGTTTGGGCGTCGTGCACTGGCTCACTTGTACGCGCAGAAATGGCAGCTTGTACGATTGGTGACCCGCCTTTTCGACACTGGACCGCTATGGACGTGGCGGCGGTGTGGCGGCGGCTCAATGACCTGTGGCGCCCGTTTGTGGCGTGCGATAGTCGAGCCGCCTGTCACGTGCGCGGCCGCCCTGCTCCGTTTGACGCGATGCATAGCATGCGACCACCCAGTAATCATACTGCTGACGCTATTGGTCACGTGGTTATGGCAGCTGCTGTTGACTGCGGTGGCGTCCCGTTTCCACACCGTACGTGAGCACATGTCTGGATTGC -3 '(SEQ ID NO: 77)

<Counter oligonucleotide>
C16: 5'-AGGTGAGCTACGTGTGTTTGG-3 '(SEQ ID NO: 58)
C17: 5'- GCGTCGTGCACTGGCTCACTTGTACGCGCA-3 '(SEQ ID NO: 59)
C18: 5'-CTTGTACGATTGGTGACCCGCCTTTTCGAC-3 '(SEQ ID NO: 60)
C19: 5'-ACTGGACCGCTATGGACGTGGCGGCGGTGT-3 '(SEQ ID NO: 61)
C20: 5'- GGCGGCGGCTCAATGACCTGTGGCGCCCGT-3 '(SEQ ID NO: 62)
C21: 5'-TTGTGGCGTGCGATAGTCGAGCCGCCTGTC-3 '(SEQ ID NO: 63)
C22: 5′-ACGTGCGCGGCCGCCCTGCTCCGTT -3 ′ (SEQ ID NO: 64)
C23: 5'-TGACGCGATGCATAGCATGCGACCACCCAG-3 '(SEQ ID NO: 65)
C24: 5′-ACTGCTGACGCTATTGGTCACGTGGTTATG-3 ′ (SEQ ID NO: 66)
C25: 5'-CTGCTGTTGACTGCGGTGGCGTCCCGTTTC-3 '(SEQ ID NO: 67)

  Each reaction solution of the yeast genomic DNA and methylated yeast genomic DNA was subjected to the following treatment.

In a PCR tube, 20 μL of the genomic DNA reaction solution prepared above, 10 μL of the counter oligonucleotide solution prepared above, 5 μL of buffer solution (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc ₂ , 5 mM Dithiothreitol), 100 mM MgCl ₂ solution (5 μL) and 1 mg / mL BSA solution (5 μL) were added, and sterilized ultrapure water was added to the mixture to make a volume of 50 μL and mixed. Thereafter, the PCR tube was heated at 95 ° C. for 10 minutes, quickly cooled to 70 ° C., and kept at that temperature for 10 minutes. Next, the mixture was cooled to 50 ° C., kept for 10 minutes, further kept at 37 ° C. for 10 minutes, and then returned to room temperature (this corresponds to the first step (A) of the measurement method of the present invention).

50 μL of the DNA fragment reaction solution prepared above was added to the streptavidin-coated PCR tube on which the biotin-labeled methylcytosine antibody was immobilized, and left at room temperature for 30 minutes. Thereafter, the solution was removed by pipetting, and 100 μL of a washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4)] was added, and then the buffer was pipetted. Removed. This operation was further repeated twice (this corresponds to the first step (B) of the measurement method of the present invention).

A buffer solution (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc2, 5 mM Dithiothreitol) and 5 μL of BSA (Bovine) were added to the PCR tube coated with streptavidin to which the biotin-labeled methylcytosine antibody subjected to the above treatment was immobilized. 5 μL of serum albumin (1 mg / ml) and 16 U of methylation sensitive restriction enzyme HhaI were added, and each mixture was further incubated at 37 ° C. for 1 hour by adding sterile ultrapure water to the mixture to a volume of 50 μL. . Thereafter, the solution was removed by pipetting, and 100 μL of a washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4)] was added, and then the buffer was pipetted. Removed. This operation was repeated twice more (this corresponds to the second step of the measurement method of the present invention).

  Next, PCR is carried out on the above PCR tube using each solution of oligonucleotide primers PF3 and PR3 consisting of the nucleotide sequences represented by SEQ ID NO: 53 and SEQ ID NO: 54 and the following reaction conditions, so that SEQ ID NO: 57 The methylated DNA in the target DNA region S ′ consisting of the base sequence shown in FIG.

<Oligonucleotide primers designed for PCR>
PF3: 5'-AGGTGAGCTACGTGTGTTTGG-3 '(SEQ ID NO: 53)
PR3: 5′-AGACATGTGCTCACGTACGGT-3 ′ (SEQ ID NO: 54)

<Target DNA region>
S ': 5'- AGGTGAGCTACGTGTGTTTGGGCGTCGTGCACTGGCTCACTTGTACGCGCAGAAATGGCAGCTTGTACGATTGGTGACCCGCCTTTTCGACACTGGACCGCTATGGACGTGGCGGCGGTGTGGCGGCGGCTCAATGACCTGTGGCGCCCGTTTGTGGCGTGCGATAGTCGAGCCGCCTGTCACGTGCGCGGCCGCCCTGCTCCGTTTGACGCGATGCATAGCATGCGACCACCCAGTAATCATACTGCTGACGCTATTGGTCACGTGGTTATGGCAGCTGCTGTTGACTGCGGTGGCGTCCCGTTTCCACACCGTACGTGAGCACATGTCT -3' (SEQ ID NO: 57)

As a PCR reaction solution, 3 μL each of oligonucleotide primer solution prepared to 5 μM, 5 μL each 2 mM dNTP, and buffer solution (100 mM Tris-HCl pH 8.3, 500 mM KCl, 15 mM MgCl ₂ , 0.01% Gelatin) is mixed with 5 μL of heat-resistant DNA polymerase (AmpliTaq Gold, manufactured by ABI) 5 U / μL with 0.25 μL, and sterilized ultrapure water is added to make the volume 50 μL. It was. The reaction solution was incubated at 95 ° C. for 10 minutes, and then PCR was carried out under the conditions of 31 cycles of incubation at 95 ° C. for 20 seconds, then at 58 ° C. for 30 seconds and further at 72 ° C. for 30 seconds. It was.
After PCR, amplification was confirmed by 1.5% agarose gel electrophoresis. The results were as follows (corresponding to the third step of the measurement method of the present invention).

  The results are shown in FIG. In the solutions of methylated yeast genomic DNA MA, MB and MC, amplification was confirmed and amplification products were obtained. In the negative control solution MD, amplification was not confirmed and the amplification product was not obtained. In the unmethylated yeast genomic DNA solutions A, B, C and D, amplification was not confirmed and no amplification product was obtained.

  From the above, it is possible to select a DNA containing a target DNA region that has been methylated with an immobilized methylcytosine antibody, and to amplify the methylated DNA to a detectable amount. It was confirmed that the amplified DNA can be detected with higher sensitivity.

Example 15
A commercially available methylcytosine antibody (Aviva Systems Biology) was labeled with biotin using a commercially available biotinylation kit (Biotin Labeling Kit-NH ₂ , manufactured by Dojindo Laboratories) according to the method described in the catalog. The obtained biotin-labeled methylcytosine antibody was refrigerated and stored as a solution [antibody about 0.25 μg / μL 0.1% BSA-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4) solution].

50 µL each of 0.1 µg / 50 µL solution of biotin-labeled methylcytosine antibody obtained by synthesis was added to the streptavidin-coated PCR tubes (total of 8 tubes) and left at room temperature for about 1 hour. Immobilized to. Thereafter, the solution was removed by pipetting, and 100 μL of a washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4)] was added, and then the buffer was pipetted. Removed. This operation was repeated twice more (this corresponds to the preparation of the immobilized methylated DNA antibody used in the measurement method of the present invention).

Baker's yeast strain X2180-1A is cultured in YPD medium (1% Yeast extract, 2% Peptone, 2% Glucose, pH 5.6-6.0) until the turbidity is OD ₆₀₀ 0.6-1.0, and 10,000 g for 10 minutes Centrifugation was performed to prepare 1 × 10 ⁷ yeast cells. The yeast genome was obtained from the prepared yeast cells using a general yeast genome preparation method as described in Methods in Yeast Genetics (Cold Spring Harbor Laboratory).
The prepared yeast cells are suspended in buffer A (1M sorbitol, 0.1M EDTA, pH 7.4), 2-mercaptoethanol (final concentration 14 mM) and 100 U zymolase (10 mg / ml) are added, and the solution becomes clear. Incubated with stirring at 30 ° C for 1 hour until complete. Protoplasts were collected by centrifugation at 550 g for 10 minutes, suspended in buffer B (50 mM Tris-HCl, pH 7.4, 20 mM EDTA), and then sodium dodecyl sulfate was added to 1% (w / v). Thereafter, it was incubated at 65 ° C. for 30 minutes. Subsequently, 5M CH ₃ COOK in a volume ratio of 2/5 was added and mixed, and after ice cooling for 30 minutes, the supernatant was recovered by centrifugation at 15,000 g for 30 minutes. Add 1/10 volume ratio of 3M CH ₃ COONa and an equal amount of isopropanol to the collected supernatant, mix well, and centrifuge at 15,000g for 30 minutes at 4 ° C to rinse the precipitate with 70% ethanol. And recovered. The precipitate is dried, dissolved in 1 ml of TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA), RNase A (manufactured by Sigma) is added to a concentration of 40 μg / ml, and 1 at 37 ° C. After incubating for a time, proteinase K (manufactured by Sigma) was added to the mixture at 500 μg / ml and sodium dodecyl sulfate at 1% (w / v), and this was shaken at 55 ° C. for about 16 hours. That ’s it. After completion of the shaking, the mixture was extracted with phenol [saturated with 1M Tris-HCl (pH 8.0)] and chloroform. The aqueous layer was recovered, and NaCl was added to this to 0.5 N, and then the resulting precipitate was recovered by ethanol precipitation. Genomic DNA was obtained by rinsing the collected precipitate with 70% ethanol.

  Part of the obtained yeast genomic DNA was mixed with 1 μl of SssI methylase (manufactured by NEB), 10 μl of 10 × NEBuffer2 (manufactured by NEB), and 1 μl of S-adenosyl methionine (3.2 mM, manufactured by NEB). Then, sterilized ultrapure water was added thereto to prepare a liquid volume of 100 μl. The reaction solution was incubated at 37 ° C. for 15-30 minutes, and 1 μl of S-adenosyl methionine (3.2 mM, manufactured by NEB) was further added and incubated at 37 ° C. for 15-30 minutes. This was purified by Wizard SV Gel / PCR Kit (PROMEGA). Further, this operation was repeated three times to obtain methylated yeast genomic DNA.

About the obtained yeast genomic DNA, the following solutions were prepared.
Solution A: 10 ng / 5 μL TE solution Solution B: 1 ng / 5 μL TE solution Solution C: 0.1 ng / 5 μL TE solution Solution D: TE solution (negative control solution)

About the obtained methylated yeast genomic DNA, the following solutions were prepared.
Solution MA: 10 ng / 5 μL TE solution Solution MB: 1 ng / 5 μL TE solution Solution MC: 0.1 ng / 5 μL TE solution Solution MD: TE solution (negative control solution)

  The following treatment was applied to each of the above-described yeast genomic DNA solution and methylated yeast genomic DNA solution.

In a PCR tube, mix 5 μL of the genomic DNA solution prepared above, 5 U of restriction enzyme XspI, and 2 μl of 10 × buffer (200 mM Tris-HCl pH 8.5, 100 mM MgCl ₂ , 10 mM Dithiothreitol, 1000 mM KCl) optimal for XspI. Then, sterilized ultrapure water was added thereto to prepare a liquid volume of 20 μl. The reaction solution was incubated at 37 ° C. for 1 hour.

  SEQ ID NO: that can bind by complementarity to the negative strand of DNA fragment T ″ (the region corresponding to base numbers 384523-384766 of yeast chromosome VII shown in Genbank Accession No. NC — 001139 etc.) consisting of the base sequence shown in SEQ ID NO: 78 Counter oligonucleotides C26 to C33 consisting of the nucleotide sequences represented by 73 to SEQ ID NO: 76 and SEQ ID NO: 79 to SEQ ID NO: 82 were synthesized, and TE buffer solutions each having a concentration of 0.01 μM were prepared.

<DNA fragment>
T: 5'-TAGGAAATACATTCCGAGGGCGCCCGCACAAGGCCTATTATTAGAGGGACCTGTGTTTGACGGGTATAACACTAAGTTGCGCAATTTGCTGTATTGCGAAATCCGCCCGGACGATATCACTCTTGAGCGCATGTGCCGTTTCCGAGAACGCCAGATCTGCATGTGTGTTGCGCGCTG

<Counter oligonucleotide>
C26: 5'-GGACCTGTGTTTGACGGGTAT-3 '(SEQ ID NO: 73)
C27: 5'-AACACTAAGTTGCGCAATTTGCTGT-3 '(SEQ ID NO: 74)
C28: 5'-ATTGCGAAATCCGCCCGGACGATAT-3 '(SEQ ID NO: 75)
C29: 5'-CACTCTTGAGCGCATGTGCCGTTTC-3 '(SEQ ID NO: 76)
C30: 5′-AATACATTCCGAGGGCGCCCGCACAAGGCC -3 ′ (SEQ ID NO: 79)
C31: 5′-GCGATCGCACACGAGGAGACA -3 ′ (SEQ ID NO: 80)
C32: 5'-AGCGTCACGTGTTTTGCCATTTTGTACGAC-3 '(SEQ ID NO: 81)
C33: 5'-AAATGAACCGCCTGGCCACGCCTCTAATC-3 '(SEQ ID NO: 82)

  Each reaction solution of the yeast genomic DNA and methylated yeast genomic DNA was subjected to the following treatment.

In a PCR tube, 20 μL of the genomic DNA reaction solution prepared above, 10 μL of the counter oligonucleotide solution prepared above, 5 μL of a buffer solution (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc ₂ , 5 mM Dithiothreitol), 5 μL of 100 mM MgCl ₂ solution and 5 μL of 1 mg / mL BSA solution were added, and further sterilized ultrapure water was added to the mixture to adjust the volume to 50 μL and mixed. Thereafter, the PCR tube was heated at 95 ° C. for 10 minutes, quickly cooled to 70 ° C., and kept at that temperature for 10 minutes. Next, the mixture was cooled to 50 ° C., kept for 10 minutes, further kept at 37 ° C. for 10 minutes, and then returned to room temperature (this corresponds to the first step (A) of the measurement method of the present invention).

50 μL of the DNA fragment reaction solution prepared above was added to the streptavidin-coated PCR tube on which the biotin-labeled methylcytosine antibody was immobilized, and left at room temperature for 30 minutes. Thereafter, the solution was removed by pipetting, and 100 μL of a washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4)] was added, and then the buffer was pipetted. Removed. This operation was further repeated twice (this corresponds to the first step (B) of the measurement method of the present invention).

A buffer solution (330 mM Tris-Acetate pH 7.9, 660 mM KOAc, 100 mM MgOAc2, 5 mM Dithiothreitol) and 5 μL of BSA (Bovine) were added to the PCR tube coated with streptavidin to which the biotin-labeled methylcytosine antibody subjected to the above treatment was immobilized. 5 μL of serum albumin (1 mg / ml) and 16 U of methylation sensitive restriction enzyme HhaI were added, and each mixture was further incubated at 37 ° C. for 1 hour by adding sterile ultrapure water to the mixture to a volume of 50 μL. . Thereafter, the solution was removed by pipetting, and 100 μL of a washing buffer [0.05% Tween20-containing phosphate buffer (1 mM KH ₂ PO ₄ , 3 mM Na ₂ HPO 7H ₂ O, 154 mM NaCl pH 7.4)] was added, and then the buffer was pipetted. Removed. This operation was repeated twice more (this corresponds to the second step of the measurement method of the present invention).

  Next, PCR is performed on the above PCR tube using each solution of oligonucleotide primers PF4 and PR4 consisting of the nucleotide sequences represented by SEQ ID NO: 68 and SEQ ID NO: 69, and the following reaction conditions, whereby SEQ ID NO: 72 The methylated DNA in the target DNA region T ′ consisting of the base sequence shown in FIG.

<Oligonucleotide primers designed for PCR>
PF4: 5'-GGACCTGTGTTTGACGGGTAT-3 '(SEQ ID NO: 68)
PR4: 5'-AGTACAGATCTGGCGTTCTCG-3 '(SEQ ID NO: 69)

<Target DNA region>
T ': 5'-GGACCTGTGTTTGACGGGTATAACACTAAGTTGCGCAATTTGCTGTATTGCGAAATCCGCCCGGACGATATCACTCTTGAGCGCATGTGCCGTTTCCGAGAACGCCAGATCTGTACT-3' (SEQ ID NO: 72)

As a PCR reaction solution, 3 μL each of oligonucleotide primer solution prepared to 5 μM, 5 μL each 2 mM dNTP, and buffer solution (100 mM Tris-HCl pH 8.3, 500 mM KCl, 15 mM MgCl ₂ , 0.01% Gelatin) is mixed with 5 μL of heat-resistant DNA polymerase (AmpliTaq Gold, manufactured by ABI) 5 U / μL with 0.25 μL, and sterilized ultrapure water is added to make the volume 50 μL. It was. The reaction solution was incubated at 95 ° C. for 10 minutes, and then PCR was carried out under the conditions of 31 cycles of incubation at 95 ° C. for 20 seconds, then at 58 ° C. for 30 seconds and further at 72 ° C. for 30 seconds. It was.
After PCR, amplification was confirmed by 1.5% agarose gel electrophoresis. The results were as follows (corresponding to the third step of the measurement method of the present invention).

  The results are shown in FIG. In the solutions of methylated yeast genomic DNA MA, MB and MC, amplification was confirmed and amplification products were obtained. In the negative control solution MD, amplification was not confirmed and the amplification product was not obtained. In the unmethylated yeast genomic DNA solutions A, B, C and D, amplification was not confirmed and no amplification product was obtained.

  From the above, it is possible to select a DNA containing a target DNA region that has been methylated with an immobilized methylcytosine antibody, and to amplify the methylated DNA to a detectable amount. It was confirmed that the amplified DNA can be detected with higher sensitivity.

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

FIG. 1 shows that in Example 1, methylated DNA in a region consisting of the base sequence represented by SEQ ID NO: 21 was amplified from a prepared sample by PCR, and the obtained amplification product was converted to 1.5% agarose. It is the figure which showed the result used for gel electrophoresis. From the leftmost lane of the figure, DNA marker “MK”, sample “D” prepared from UBC-M solution D, sample “C” prepared from UBC-M solution C, UBC-M Sample “B” prepared from solution B, sample “A” prepared from solution A of UBC-M, “A” of DNA fragment Y2, sample “D” prepared from solution D of UBC-U, UBC -Shows results for sample "C" prepared from solution C of U, sample "B" prepared from solution B of UBC-U, sample "A" prepared from solution A of UBC-U . FIG. 2 shows a region consisting of the base sequence shown in SEQ ID NO: 27 after the sample prepared in Example 2 was subjected to either “A (no treatment)” or “B (HhaI treatment)” treatment. It is the figure which showed the result of having amplified the methylated DNA in <1> by PCR and having used the obtained amplified product for 1.5% agarose gel electrophoresis. From the leftmost lane in the figure, DNA marker “MK”, sample “M” treated with “A” treatment of partially methylated oligonucleotide GPR7-2079-2176 / 98mer-M (7) solution not digested with HhaI “A” -treated sample “HM” of partially methylated oligonucleotide GPR7-2079-2176 / 98mer-HM (5) digested with HhaI, unmethylated oligonucleotide GPR7-2079-2176 / 98mer -Sample "U" treated with "A" treatment of UM solution, "B" treatment of partially methylated oligonucleotide GPR7-2079-2176 / 98mer-M (7) solution not digested with HhaI " M ", partially methylated oligonucleotide GPR7-2079- digested with HhaI Sample “HM” treated with “B” treatment of 2176 / 98mer-HM (5) solution, sample “U” treated with “B” treatment of unmethylated oligonucleotide GPR7-2079-2176 / 98mer-UM solution , Shows the results. FIG. 3 shows the sequence number of the sample treated with methylcytosine antibody in Example 3 for a sample that has been previously treated with either “A (no treatment)” or “B (HhaI treatment)”. It is the figure which showed the result of having amplified the methylated DNA in the area | region which consists of a base sequence shown by 27 by PCR, and having used the obtained amplified product for 1.5% agarose gel electrophoresis. From the leftmost lane in the figure, DNA marker “MK”, sample “M” treated with “A” treatment of partially methylated oligonucleotide GPR7-2079-2176 / 98mer-M (7) solution not digested with HhaI “A” -treated sample “HM” of partially methylated oligonucleotide GPR7-2079-2176 / 98mer-HM (5) digested with HhaI, unmethylated oligonucleotide GPR7-2079-2176 / 98mer -Sample "U" treated with "A" treatment of UM solution, "B" treatment of partially methylated oligonucleotide GPR7-2079-2176 / 98mer-M (7) solution not digested with HhaI " M ", partially methylated oligonucleotide GPR7-2079- digested with HhaI Sample “HM” treated with “B” treatment of 2176 / 98mer-HM (5) solution, sample “U” treated with “B” treatment of unmethylated oligonucleotide GPR7-2079-2176 / 98mer-UM solution , Shows the results. 4 shows, in Example 4, the methylated DNA in the target DNA region consisting of the base sequence represented by SEQ ID NO: 32 was amplified from the prepared sample by PCR, and the obtained amplification product was 1. It is the figure which showed the result of 5% agarose gel electrophoresis. From the leftmost lane in the figure, DNA marker “MK”, methylated DNA fragment MX solution “MD” (negative control), unmethylated DNA fragment X solution “D” (negative control) , A solution “MC” of a methylated DNA fragment MX, a solution “C” of an unmethylated DNA fragment X, a solution “MB” of a methylated DNA fragment MX, a solution of an unmethylated DNA fragment X The results for solution “B”, solution “MA” of methylated DNA fragment MX, and solution “A” of unmethylated DNA fragment X are shown.

5 shows, in Example 5, the methylated DNA in the target DNA region consisting of the base sequence represented by SEQ ID NO: 49 was amplified from the prepared sample by PCR. It is the figure which showed the result of 5% agarose gel electrophoresis. From the leftmost lane in the figure, DNA marker “MK”, methylated DNA fragment MY solution “MD” (negative control), unmethylated DNA fragment Y solution “D” (negative control) , Solution “MC” of methylated DNA fragment MY, solution “C” of unmethylated DNA fragment Y, solution “MB” of methylated DNA fragment MY, solution of unmethylated DNA fragment Y The results are shown for solution “B”, solution “MA” of methylated DNA fragment MY, and solution “A” of unmethylated DNA fragment Y.

6 shows, in Example 6, the methylated DNA in the target DNA region consisting of the base sequence represented by SEQ ID NO: 57 was amplified from the prepared sample by PCR. It is the figure which showed the result of 5% agarose gel electrophoresis. From the leftmost lane in the figure, DNA marker “MK”, methylated DNA fragment MS solution “MC” (negative control), unmethylated DNA fragment S solution “C” (negative control) , Solution “MB” of methylated DNA fragment MS, solution “B” of unmethylated DNA fragment S, solution “MA” of methylated DNA fragment MS, solution of unmethylated DNA fragment S The results with solution “A” are shown.

7 shows, in Example 7, the methylated DNA in the target DNA region consisting of the base sequence represented by SEQ ID NO: 72 was amplified from the prepared sample by PCR, and the obtained amplification product was 1. It is the figure which showed the result of 5% agarose gel electrophoresis. From the leftmost lane in the figure, DNA marker “MK”, methylated DNA fragment MT solution “MC” (negative control), unmethylated DNA fragment T solution “C” (negative control) A solution “MB” of a methylated DNA fragment MT, a solution “B” of an unmethylated DNA fragment T, a solution “MA” of a methylated DNA fragment MT, a solution of an unmethylated DNA fragment T The results with solution “A” are shown.

8 shows, in Example 8, the methylated DNA in the target DNA region consisting of the base sequence represented by SEQ ID NO: 57 was amplified from the prepared sample by PCR, and the obtained amplification product was 1. It is the figure which showed the result of 5% agarose gel electrophoresis. From the leftmost lane in the figure, DNA marker “MK”, methylated yeast genomic DNA solution “MD” (negative control), unmethylated yeast genomic DNA solution “D” (negative control), methyl Yeast genomic DNA solution “MC”, Unmethylated yeast genomic DNA solution “C”, Methylated yeast genomic DNA solution “MB”, Unmethylated yeast genomic DNA solution “B”, Methyl The results are shown for the solution “MA” of the yeast yeast genomic DNA and the solution “A” of the unmethylated yeast genomic DNA.

9 shows, in Example 9, the methylated DNA in the target DNA region consisting of the base sequence represented by SEQ ID NO: 72 from the prepared sample was amplified by PCR, and the obtained amplification product was 1. It is the figure which showed the result of 5% agarose gel electrophoresis. From the leftmost lane in the figure, DNA marker “MK”, methylated yeast genomic DNA solution “MD” (negative control), unmethylated yeast genomic DNA solution “D” (negative control), methyl Yeast genomic DNA solution “MC”, Unmethylated yeast genomic DNA solution “C”, Methylated yeast genomic DNA solution “MB”, Unmethylated yeast genomic DNA solution “B”, Methyl The results are shown for the solution “MA” of the yeast yeast genomic DNA and the solution “A” of the unmethylated yeast genomic DNA.

10 shows, in Example 10, the methylated DNA in the target DNA region consisting of the base sequence represented by SEQ ID NO: 32 was amplified from the prepared sample by PCR, and the obtained amplification product was 1. It is the figure which showed the result of 5% agarose gel electrophoresis. From the leftmost lane in the figure, DNA marker “MK”, methylated DNA fragment MX solution “MD” (negative control), unmethylated DNA fragment X solution “D” (negative control) , A solution “MC” of a methylated DNA fragment MX, a solution “C” of an unmethylated DNA fragment X, a solution “MB” of a methylated DNA fragment MX, a solution of an unmethylated DNA fragment X The results for solution “B”, solution “MA” of methylated DNA fragment MX, and solution “A” of unmethylated DNA fragment X are shown.

11 shows, in Example 11, the methylated DNA in the target DNA region consisting of the base sequence represented by SEQ ID NO: 57 was amplified from the prepared sample by PCR. It is the figure which showed the result of 5% agarose gel electrophoresis. From the leftmost lane in the figure, DNA marker “MK”, methylated DNA fragment MS solution “MD” (negative control), unmethylated DNA fragment S solution “D” (negative control) A solution “MC” of a methylated DNA fragment MS, a solution “C” of an unmethylated DNA fragment S, a solution “MB” of a methylated DNA fragment MS, a solution of an unmethylated DNA fragment S The results for solution “B”, solution “MA” of methylated DNA fragment MS and solution “A” of unmethylated DNA fragment S are shown.

FIG. 12 shows, in Example 12, the methylated DNA in the target DNA region consisting of the base sequence represented by SEQ ID NO: 72 from the prepared sample was amplified by PCR, and the obtained amplification product was 1. It is the figure which showed the result of 5% agarose gel electrophoresis. From the leftmost lane in the figure, DNA marker “MK”, methylated DNA fragment MT solution “MD” (negative control), unmethylated DNA fragment T solution “D” (negative control) A solution “MC” of a methylated DNA fragment MT, a solution “C” of an unmethylated DNA fragment T, a solution “MB” of a methylated DNA fragment MT, a solution of an unmethylated DNA fragment T The results for solution “B”, solution “MA” of methylated DNA fragment MT, and solution “A” of unmethylated DNA fragment T are shown.

FIG. 13 shows, in Example 13, the methylated DNA in the target DNA region consisting of the base sequence represented by SEQ ID NO: 32 from the prepared sample was amplified by PCR. It is the figure which showed the result of 5% agarose gel electrophoresis. From the leftmost lane in the figure, DNA marker “MK”, methylated human genomic DNA solution “MC” (negative control), unmethylated human genomic DNA solution “C” (negative control), methylated human genome Results are shown for DNA solution “MB”, unmethylated human genomic DNA solution “B”, methylated human genomic DNA solution “MA”, and unmethylated human genomic DNA solution “A”. .

14 shows, in Example 14, the methylated DNA in the target DNA region consisting of the base sequence represented by SEQ ID NO: 57 was amplified from the prepared sample by PCR, and the obtained amplification product was 1. It is the figure which showed the result of 5% agarose gel electrophoresis. From the leftmost lane in the figure, DNA marker “MK”, methylated yeast genomic DNA solution “MD” (negative control), unmethylated yeast genomic DNA solution “D” (negative control), methyl Yeast genomic DNA solution “MC”, Unmethylated yeast genomic DNA solution “C”, Methylated yeast genomic DNA solution “MB”, Unmethylated yeast genomic DNA solution “B”, Methyl The results are shown for the solution “MA” of the yeast yeast genomic DNA and the solution “A” of the unmethylated yeast genomic DNA.

15 shows, in Example 15, the methylated DNA in the target DNA region consisting of the base sequence represented by SEQ ID NO: 72 from the prepared sample was amplified by PCR. It is the figure which showed the result of 5% agarose gel electrophoresis. From the leftmost lane in the figure, DNA marker “MK”, methylated yeast genomic DNA solution “MD” (negative control), unmethylated yeast genomic DNA solution “D” (negative control), methyl Yeast genomic DNA solution “MC”, Unmethylated yeast genomic DNA solution “C”, Methylated yeast genomic DNA solution “MB”, Unmethylated yeast genomic DNA solution “B”, Methyl The results are shown for the solution “MA” of the yeast yeast genomic DNA and the solution “A” of the unmethylated yeast genomic DNA.

SEQ ID NO: 17
Oligonucleotide designed for experiment SEQ ID NO: 18
Oligonucleotide designed for experiment SEQ ID NO: 19
Oligonucleotide primer designed for PCR SEQ ID NO: 20
Oligonucleotide primer designed for PCR SEQ ID NO: 21
Oligonucleotide designed for experiment SEQ ID NO: 22
Oligonucleotide designed for experiment SEQ ID NO: 23
Oligonucleotide designed for experiment SEQ ID NO: 24
Oligonucleotide designed for experiment SEQ ID NO: 25
Oligonucleotide primer designed for PCR SEQ ID NO: 26
Oligonucleotide primer designed for PCR SEQ ID NO: 27
Oligonucleotide designed for experiment SEQ ID NO: 28
Oligonucleotide primer designed for experiment SEQ ID NO: 29
Oligonucleotide primer designed for experiment SEQ ID NO: 30
DNA fragment SEQ ID NO: 31 designed for the experiment
Methylated DNA fragment designed for experiment SEQ ID NO: 32
Oligonucleotide SEQ ID NO: 33 consisting of the desired DNA region
Counter oligonucleotide SEQ ID NO: 34 designed for experiments
Counter oligonucleotide SEQ ID NO: 35 designed for experiment
Counter oligonucleotide SEQ ID NO: 36 designed for experiments
Counter oligonucleotide SEQ ID NO: 37 designed for experiments
Counter oligonucleotide SEQ ID NO: 38 designed for experiments
Counter oligonucleotide SEQ ID NO: 39 designed for experiment
Counter oligonucleotide SEQ ID NO: 40 designed for experiments
Counter oligonucleotide SEQ ID NO: 41 designed for experiment
Counter oligonucleotide designed for experiment SEQ ID NO: 42
Counter oligonucleotide designed for experiment SEQ ID NO: 43
Counter oligonucleotide SEQ ID NO: 44 designed for experiments
Counter oligonucleotide SEQ ID NO: 45 designed for experiments
Oligonucleotide primer designed for experiment SEQ ID NO: 46
Oligonucleotide primer designed for experiment SEQ ID NO: 47
DNA fragment designed for experiment SEQ ID NO: 48
Methylated DNA fragment designed for experiment SEQ ID NO: 49
Oligonucleotide SEQ ID NO: 50 consisting of the desired DNA region
Counter oligonucleotide SEQ ID NO: 51 designed for experiments
Counter oligonucleotide SEQ ID NO: 52 designed for experiments
Counter oligonucleotide designed for experiments SEQ ID NO: 53
Oligonucleotide primer designed for experiment SEQ ID NO: 54
Oligonucleotide primer designed for experiment SEQ ID NO: 55
DNA fragment designed for experiment SEQ ID NO: 56
Methylated DNA fragment designed for experiment SEQ ID NO: 57
Oligonucleotide SEQ ID NO: 58 consisting of the desired DNA region
Counter oligonucleotide SEQ ID NO: 59 designed for experiment
Counter oligonucleotide SEQ ID NO: 60 designed for experiments
Counter oligonucleotide SEQ ID NO: 61 designed for experiments
Counter oligonucleotide SEQ ID NO: 62 designed for experiment
Counter oligonucleotide designed for experiment SEQ ID NO: 63
Counter oligonucleotide SEQ ID NO: 64 designed for experiment
Counter oligonucleotide SEQ ID NO: 65 designed for experiments
Counter oligonucleotide SEQ ID NO: 66 designed for experiment
Counter oligonucleotide SEQ ID NO: 67 designed for experiments
Counter oligonucleotide SEQ ID NO: 68 designed for experiments
Oligonucleotide primer designed for experiment SEQ ID NO: 69
Oligonucleotide primer designed for experiment SEQ ID NO: 70
DNA fragment designed for experiments SEQ ID NO: 71
Methylated DNA fragment designed for experiment SEQ ID NO: 72
Oligonucleotide SEQ ID NO: 73 consisting of the desired DNA region
Counter oligonucleotide SEQ ID NO: 74 designed for experiments Counter oligonucleotide SEQ ID NO: 75 designed for experiments Counter oligonucleotide SEQ ID NO: 76 designed for experiments
Counter oligonucleotide designed for experiment SEQ ID NO: 77
DNA fragment designed for experiment SEQ ID NO: 78
DNA fragment designed for experiment SEQ ID NO: 79
Counter oligonucleotide SEQ ID NO: 80 designed for experiments
Counter oligonucleotide SEQ ID NO: 81 designed for experiment
Counter oligonucleotide SEQ ID NO: 82 designed for experiments
Counter oligonucleotide SEQ ID NO: 83 designed for experiments
DNA fragments designed for experiments

Claims (11)

A method for measuring the content of methylated DNA in a target DNA region possessed by genomic DNA contained in a biological specimen,
(1) Obtaining methylated single-stranded DNA from a genomic DNA-derived DNA sample contained in a biological specimen, and binding the single-stranded DNA with an immobilized methylated DNA antibody A first step of selecting DNA,
(2) a second step of digesting the single-stranded DNA selected in the first step with a methylation sensitive restriction enzyme capable of digesting at least one kind of single-stranded DNA; and
(3) As a pre-process of each of the following steps, single-stranded DNA that is an undigested product obtained in the second step (the methylation state is present at the recognition site of the methylation-sensitive restriction enzyme capable of digesting the single-stranded DNA). A single-stranded DNA not containing CpG) is separated from the immobilized methylated DNA antibody into a single-stranded DNA (positive strand) (third (pre-A) step);
Using the DNA (positive strand) in the single-stranded state separated in the third (previous A) step as a template, the partial base sequence (positive strand) of the base sequence of the DNA (positive strand) in the single-stranded state And a base sequence (complementary to the partial base sequence (positive strand) located further 3 ′ end than the 3 ′ end of the base sequence (positive strand) of the target DNA region ( as the extension primer an extension primer (primer for full O word over de) having a negative strand), by extending the extended primer once, a single-stranded state of the DNA (plus strand) of the double-stranded DNA And a step of forming an extension (third (previous B) step),
The double-stranded DNA extended and formed in the third (previous B) step includes a single-stranded DNA (positive strand) including the target DNA region and a base sequence that is complementary to the target DNA region. A single-stranded DNA (positive strand) having a step (third (previous C) step) of once separating into single-stranded DNA (negative strand) and including (a) the generated target DNA region as this step ) As a template, the forward primer as an extension primer, and the extension primer is extended once, thereby extending the single-stranded DNA containing the target DNA region as a double-stranded DNA. A and
(B) a single-stranded DNA containing a base sequence Ru complementary der against DNA region of the generated object (minus strand) as a template, which is complementary to a DNA region of the target base A partial base sequence (negative strand) of the base sequence of a single-stranded DNA (negative strand) containing the sequence, and complementary to the base sequence (positive strand) of the target DNA region Extension primer (primer for reverse) having a base sequence (positive strand) that is complementary to a partial base sequence (negative strand) located further 3 'end than the 3' end of the base sequence (negative strand) And a step B of extending the single-stranded DNA containing the target DNA region as a double-stranded DNA by extending the extended primer once. Each main process is changed to each main work. The elongated double-stranded DNA obtained in step 1 is once separated into a single-stranded state and then repeated to amplify the methylated DNA in the target DNA region to a detectable amount. And a third step of quantifying the amount of amplified DNA.   The content of methylated DNA in the target DNA region of the genomic DNA contained in the biological specimen according to claim 1, wherein the methylated DNA antibody in the third step according to claim 1 is a methylcytosine antibody. How to measure.   The method according to claim 1, wherein the biological specimen is mammalian serum or plasma.   3. The method according to claim 1, wherein the biological specimen is mammalian blood or body fluid.   The method according to claim 1, wherein the biological specimen is a cell lysate or a tissue lysate.   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 5.   The DNA sample derived from genomic DNA contained in a biological specimen is a DNA sample obtained by digestion with at least one methylation sensitive restriction enzyme. The method described.   The method according to any one of claims 1 to 7, wherein the DNA sample derived from genomic DNA contained in the biological specimen is a DNA sample purified in advance.   The methylation-sensitive restriction enzyme capable of digesting at least one kind of single-stranded DNA is a restriction enzyme having a recognition cleavage site in a target DNA region of genomic DNA contained in a biological sample. The method according to any one of claims 1 to 8.   The method according to any one of claims 1 to 9, wherein the methylation sensitive restriction enzyme capable of digesting at least one kind of single-stranded DNA is HhaI which is a methylation sensitive restriction enzyme. The first step is
A first (A) step of separating methylated single-stranded DNA from a genomic DNA-derived DNA sample contained in a biological specimen;
The first (B) step of binding the single-stranded DNA separated in the first (A) step and the immobilized methylated DNA antibody,
The method according to any one of claims 1 to 10 , wherein a counter oligonucleotide is added when the single-stranded DNA methylated in the first step (A) is separated.

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