WO2005026352A1 - Method of evaluating cancerization degree - Google Patents

Abstract

A method of evaluating the degree of cancerization of a specimen originating in a mammal characterized by comprising: (1) the first step of measuring the methylation frequency in G-protein coupled somatostatin and angiotensin-like peptide receptor gene contained in the mammal-origin specimen; and (2) the second step of judging the degree of cancerization of the above-described specimen based on the difference obtained by comparing the methylation frequency having been measured above with the data of a control; and so on.

Description

 Specification

 Evaluation method of cancer degree

Technical field

 • The present invention relates to a method for evaluating the degree of canceration of a mammal-derived specimen, and the like. Background art.

 Although it is becoming increasingly clear that cancer is a disease caused by genetic abnormalities, the mortality rate of cancer patients is still high, and currently available diagnostic methods and treatment methods are not always satisfactory. It is shown that. It is clinically important to detect cancer at an early stage, to select an effective treatment method for the detected cancer, and to provide aftercare, such as confirmation of the recurrence of cancer, after treatment.

 Therefore, a method for evaluating the degree of canceration of a mammal-derived specimen based on the detection of a genetic abnormality, which is suitable for a diagnostic method for early detection of cancer, evaluation of the effectiveness of a treatment method for cancer, confirmation of the presence or absence of cancer recurrence, etc. The development of is eagerly awaited. Disclosure of the invention

 Under such circumstances, the present inventors have conducted intensive studies and found that G-protein coupled somatos tat in and angiotens in-like peptide receptor gene (hereinafter, SALPR Has been found to be methylated at a significantly higher frequency than immortalized normal cell lines and normal tissue specimens, leading to the present invention.

1. A method for evaluating the degree of canceration of a mammal-derived specimen,

 (1) the first step of measuring the methylation frequency of the G-protein coupled somatos tat in and angiotens in-like peptide receptor gene contained in a mammal-derived specimen, and

(2) a second step of determining the degree of canceration of the sample based on a difference obtained by comparing the measured methylation frequency with a control

(Hereinafter, also referred to as the evaluation method of the present invention); 2. The method according to item 1, wherein the mammal-derived specimen is a cell;

3. The evaluation method according to the above 1, wherein the mammal-derived specimen is a tissue;

4. The evaluation method according to item 1, wherein the mammal-derived specimen is a cell, and the degree of canceration of the specimen is the malignancy of a mammal-derived cell;

 5. The evaluation method according to item 1, wherein the mammal-derived specimen is a tissue, and the degree of canceration of the specimen is the amount of cancer cells present in the mammal-derived tissue;

6. The evaluation method according to the above item 5, wherein the tissue is a normal tissue and the cancer is victory cancer;

 7. The methylation frequency of a gene is determined by the presence of one or more 5'-CG-3 'nucleotides in the promoter sequence, untranslated region, or translated region of the gene. The evaluation method according to the above 1, characterized in that the methylation frequency is;

 8. The evaluation method according to the above item 7, wherein the tissue is a Ji spleen tissue and the cancer is a renal cancer;

 9.The methylation frequency of a gene is determined by the methylation frequency of cytosine in one or more nucleotide sequences represented by 5'-CG-3 'existing in the nucleotide sequence in the promoter region of the gene. The evaluation method described in 1 above, which is characterized in that:

 10 The methylation frequency of a gene is determined by the methylation of cytosine in the nucleotide sequence represented by one or more 5'-CG-3's present in the nucleotide sequence in the untranslated or translated region of the gene. The evaluation method described in 1 above, wherein the evaluation method is frequent;

 11.The methylation frequency of a gene is the methylation frequency of cytosine in one or more nucleotide sequences represented by -CG-3 'present in the nucleotide sequence represented by SEQ ID NO: 1. Evaluation method described in 1 above;

 1 2. The evaluation method according to 11 above, wherein the tissue is a viscera tissue, and the cancer is a viscera cancer;

1 3. Use of methylated G-protein coupled somatos tat in and angiotens in-like peptide receptor gene as a cancer marker;

1 4. The use according to the above item 13 wherein the cancer marker is a visceral cancer marker. Brief Description of Drawings

 Figure 1 shows human-derived immortalized (normal) 塍 duct epithelial cell lines (HPDE-4 / E6E7 and miPDE6-E6E7 c7) and seven victory cancer cell lines (BXPc3, HPAF-IL Capan-2, Mi aPaCa -2, genomic DNA prepared from Hs766T, PANC-1 and tJ-PAC) and treated with sodium bisulfite were subjected to PCR, and the PCR reaction mixture after PCR was analyzed by agarose gel electrophoresis. It is a figure showing a result. The names of the cells used are indicated at the top. The figure described as HPDEVSssI shows the DNA obtained by treating the genomic DNA of HPDE-4 / E6E7 with the methylase SssI. Lane U, PCR reaction using non-methylated specific primers; Lane M, PCR reaction using methylated specific primers.

 Figure 2 shows genomic DNA prepared from 12 samples of human skeletal cancer tissue and its surrounding skeletal normal tissues [obtained by obtaining informed consent from patients] and treated with sodium bisulfite. FIG. 4 is a diagram showing the results of performing PCR as a template and analyzing the PCR reaction solution after PCR by agarose gel electrophoresis. Case l to Case l 2 showed specimens. Cancer shows the data of the liver cancer tissue, and Normal shows the data of the normal viscera tissue around it. Lane U, PCR reaction using unmethylation-specific primer; PCR reaction using lane methylation-specific primer. BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described in detail.

 The present invention relates to the use of a methylated G-protein coupled somatos tat in and angiotensin-like peptide receptor gene as a cancer marker (for example, a visceral cancer marker).

G_protein coupled soiatos tat in and angiotens in like peptide receptor residues used as a marker gene in the present invention; as children, for example, G-prote in co up led somatos tat in and angiotens in-like peptide receptor A human-derived gene containing a non-translated region and a translated region (coding region) of a gene and a promoter region located 5 ′ upstream thereof can be given. G-protein coupled somat of human origin The base sequence of the ostatin and angiotensin-like peptide receptor gene and the amino acid sequence encoded thereby are described in, for example, Genbank Accession No. NM-016568. In addition, among the exons that carry the untranslated region and the translated region (coding region) of the human G-protein coupled somatostatin and angiotensin-like peptide receptor genes, the exon located at the 5 'upstream most (hereinafter, exon) The base sequence of genomic DNA including the promoter region located 5 ′ upstream of the sequence is described in, for example, Genbank Accession No. AC008971. In the nucleotide sequence described in Genbank Accession No. AC008971, for example, the nucleotide sequence of exon 1 of the human-derived G-protein coupled somatostatin and angiotensin-like peptide receptor gene is represented by nucleotide numbers 57369 to 59225. The G-prote in coupled somatostatin and angiotensin-like peptide receptor gene used in the present invention includes, in addition to the above-mentioned genes having the known nucleotide sequences, It also includes a gene having a base sequence in which a base is deleted, substituted or added due to a naturally occurring mutation due to a difference between tissues or the like. In mammals, there is a phenomenon in which only cytosine is methylated among the four types of bases that make up a gene (genomic DNA). In mammals, for example, in the gene for G-protein coupled somatostatin and angiotensin-like peptide receptor, a part of cytosine in the genomic DNA of the gene is methylated. The methylation modification of DNA is carried out in the base sequence represented by 5′-CG-3 ′ (C represents cytosine, G represents guanine, and the base sequence is sometimes referred to as CpG hereinafter.) Limited to cytosine. The site that is methylated in cytosine is at position 5. During DNA replication prior to cell division, only cytosine in the type I chain CpG is methylated immediately after replication, but cytosine in the nascent chain CpG is immediately activated by the action of methyltransferase. Is also methylated. Therefore, the state of DNA methylation is inherited by two new sets of DNA even after DNA replication. In the first step of the evaluation method of the present invention, “methylation frequency” means, for example, When the presence or absence of cytosine methylation in CpG is examined for a plurality of haploids, it is expressed as a percentage of haploids in which the cytosine is methylated. Samples derived from mammals in the first step of the evaluation method of the present invention may include, for example, DNA derived from cancer cells such as murine cancer cells or tissues containing the same, and cancer cells such as spleen cancer cells. Sexual cells, tissues containing them (tissues in the broad sense include blood, plasma, serum, lymph, body fluids such as serum, lymph nodes, etc.) or body secretions (urine And milk). Specifically, for example, when the cancer is a renal cancer, the extract may include a spleen tissue 01 extracted from a test animal).

 These biological samples may be used as samples as they are, or biological samples prepared by various operations such as separation, fractionation, and immobilization from such biological samples may be used as samples.

 When the mammal-derived specimen is blood, the use of the present invention evaluation method in periodic health examinations and simple tests can be expected. In the first step of the evaluation method of the present invention, a method for measuring the frequency of the G-protein coupled somatostat in and angiotens in-1 ike peptide receptor gene contained in a mammal-derived specimen, The following may be performed. As a first method, after contacting a DNA derived from a sample with a reagent that modifies unmethylated cytosine, the DNA is converted into a type II, and the presence or absence of cytosine methylation to be analyzed can be identified. A method of performing a polymerase chain reaction (hereinafter, referred to as PCR) using a primer and examining the amount of amplification product obtained can be mentioned.

 First, DNA is extracted from a mammal-derived specimen using, for example, a commercially available DNA extraction kit or the like.

When blood is used as a sample, plasma or serum is prepared from the blood according to an ordinary method, and the prepared plasma or serum is used as a sample to prepare free DNA (Tengen Cancer Cell) contained therein. (Including DNA derived from cancer cells such as vesicles), it is possible to analyze DNA derived from cancer cells such as kidney cancer cells while avoiding DNA derived from blood cells. In addition, the sensitivity for detecting a tissue or the like containing the same can be improved.

 Next, after contacting the extracted DNA with a reagent that modifies unmethylated cytosine, the DNA is converted into type 錶 using a primer capable of discriminating the presence or absence of cytosine methylation to be analyzed. Perform PCR to determine the amount of amplification product obtained. The cytosine to be analyzed is one or more CpGs present in the base sequence of the promoter region, untranslated region or translated region (coding region) of the G-protein coupled somatostatin and angiotensin-1 ike peptide recep tor gene. Can be selected from the cytosines in the nucleotide sequence represented by.

Here, the base sequence represented by one or more CpGs present in the base sequence of the promoter region, untranslated region or translated region (coding region) of the G-protein coupled somatostatin and angiotensin-like peptide recep tor gene is as follows. The base sequence of genomic DNA containing exon 1 of the human-derived G-protein coupled somatostatin and angiotensin-like peptide receptor gene and the promoter region located 5 ′ upstream thereof can be increased. Specifically, the base sequence represented by SEQ ID NO: 1 (corresponding to the complementary sequence of the base sequence represented by base numbers 57001 to 60000 of the base sequence described in Gen bank Accession No. AC008971) can be mentioned. The nucleotide sequence of exon 1 of the human-derived G-protein coupled somatostatin and angiotensin-like peptide receptor gene is represented by nucleotide numbers 776 to 2632. Cytosine in the nucleotide sequence represented by CpG present in the nucleotide sequence represented by SEQ ID NO: 1 has a high methylation frequency (ie, a hypermethylation state) in cancer cells such as, for example, cancer cells of the kidney. ). More specifically, cytosine having a high methylation frequency in victory cancer cells includes, for example, nucleotides 470, 472, 490, 497, 504, 506, Cytosine represented by 509, 514, 522, 540, 543, 552, 566, 582, 597, 610, 612, etc. can be mentioned. As a reagent for modifying unmethylated cytosine, for example, sodium bisulfite or the like Bisulfite (bi sul fite) or the like can be used. Incidentally, in principle, a reagent that specifically modifies only methylated cytosine may be used. In order to bring the extracted DNA into contact with a reagent that modifies unmethylated cytosine, for example, the DNA is first denatured with an alkaline solution (pH 9 to 14), and then a bisulfite such as sodium bisulfite (bi sul fite) (concentration in solution: for example, 3M final concentration), etc., and treat at 55 ° C for about 10 to 16 hours (（). To accelerate the reaction, denaturation at 95 and reaction at 50 ° C can be repeated 10-20 times. In this case, unmethylated cytosine is converted to peracyl, while methylated cytosine is not converted to peracyl and remains cytosine.

 Next, the DNA sequence treated with bisulfite or the like was converted to type II, and the base sequence when methylated cytosine was contained [the cytosine at the methylated position (cytosine in CpG) remains cytosine. Yes, non-methylated cytosine (cytosine not included in CpG) is a peracil base sequence and a base sequence complementary to the base sequence, using a pair of methylation-specific primers. PCR (hereinafter also referred to as methylation-specific PCR) and the base sequence in the case where the DNA treated with bisulfite or the like is type II and cytosine is not methylated (all cytosine PCR using a pair of unmethylated specific primers selected from a base sequence complementary to the base sequence and a base sequence complementary to the base sequence (hereinafter referred to as non-methylated Sometimes also referred to as a-specific P C R.) And performing.

In the above PCR, in the case of PCR using methylation-specific primers (the former), DNA in which cytosine to be analyzed is methylated is amplified, while PCR using unmethylation-specific primers is performed. In the latter case (the latter), DNA in which the cytosine to be analyzed is not methylated is amplified. By comparing the amounts of these amplification products, the presence or absence of the target cytosine methylated is examined. In this way, the methylation frequency can be measured. Here, as a primer, unmethylated cytosine is converted to peracyl. PCR primers (methylation-specific primers) specific to nucleotide sequences containing methylated cytosine were designed in consideration of the fact that methylated cytosine would not be converted to peracil. Also, design a PCR primer (unmethylation-specific primer) specific to the base sequence containing unmethylated cytosine. Designing based on DNA chains that have been chemically converted by bisulfite treatment and are no longer complementary, and based on the DNA chains that were originally double-stranded, It is also possible to generate a methylation-specific primer and an unmethylation-specific primer. Such a primer is preferably designed to contain cytosine in CpG near the 3 ′ end of the primer in order to increase the specificity of methyl and non-methyl. Further, one of the primers may be labeled to facilitate the analysis. More specifically, primers for measuring the methylation frequency of the G-prote in coupled somatos tat in and angiotensin-1 ike peptide receptor gene by methylation-specific PCR include, for example, G-protein. protein coupled somatos tat in and angiotensin-like peptide receptor A nucleotide sequence containing at least one cytosine in CpG present in the nucleotide sequence in the promoter region, untranslated region or translated region (coding region) of the gene. Based on this, it can be designed as described above. For example, cytosine in CpG present in the nucleotide sequence represented by SEQ ID NO: 1, specifically, in the nucleotide sequence represented by SEQ ID NO: 1, base numbers 470, 472, 490, 497, 504, 506, 509, 514, 522, 540, 543, 552, 566, 582, 597, 610, 612, etc., and can be designed based on the nucleotide sequence containing one or more cytosines. Examples of such primers are shown below.

<Unmethylated specific primer>

U1: 5'-GTGTGGTTGGGTTTAGTATTTT-3 '(SEQ ID NO: 2)

U2: 5'-TCTCCACAATTATCAACTCCA-3 '(SEQ ID NO: 3)

<Methylation specific primer>

 Ml: 5'-CGCGGTTGGGTTTAGTATTTC-3 '(SEQ ID NO: 4)

M2: 5'-CCGCGATTATCAACTCCG-3 '(SEQ ID NO: 5) Examples of the reaction solution for the methylation-specific PCR include, for example, 50 ng of type I DNA, 1 liter of each primer solution of 1 Opmol / l, 2.5 mM dNTP, 10X buffer ( l OOmM Tris-HC l ρΗ8.3, 500 mM KCK 20 mM MgCl ₂ ) 2.51 and heat-resistant DNA polymerase 5U / 1 0.21 mixed with sterile ultrapure A reaction solution having a liquid volume of 251 by adding water can be used.

 As the reaction conditions, for example, after keeping the above reaction solution at 95 ° C for 10 minutes, the reaction solution is heated at 95 ° C for 30 seconds, then at 55 to 65 ° C for 30 seconds, and further at 72 ° C for 30 seconds. One condition is that the temperature is maintained for 30 to 40 cycles.

After performing such PCR, the amounts of the obtained amplification products are compared. For example, an analytical method (denatured polyacrylamide gel electrophoresis) that can compare the amount of each amplification product obtained by PCR using methylated-specific primers and PCR using unmethylated-specific primers In the case of electrophoresis (agarose gel electrophoresis), the gel after electrophoresis is stained with DNA to detect the band of the amplification product, and the concentrations of the detected bands are compared. Here, instead of DNA staining, a pre-labeled primer can be used to compare the band concentrations using the label as an index. When quantification is needed, real-time monitoring of PCR reaction products and force kinetic analysis can provide high-precision quantification that can detect, for example, a slight difference of about twice the amount of gene. The amount of each product can also be compared using real-time PCR, a possible PCR method. Examples of the method for performing real-time PCR include a method using a probe such as a type-dependent nucleic acid polymerase probe and a method using an intercalator such as Cyber Green. As devices and reagents for the real-time PCR method, commercially available devices and reagent kits can be used. Such a method is generally referred to as methylation-specific PCR, and is a method reported by Herman et al. (Herman et al., Pro atl. Acad. ScI USA, 93, 9821-9826, 1996). This is a method that takes advantage of the difference in chemical properties between cytosine and 5-methylcytosine. As a second method, after contacting the DNA derived from the sample with a reagent that modifies unmethylated cytosine, the DNA containing cytosine to be analyzed is converted into a type III DNA, and the amplified product obtained is amplified by PCR. A method for directly analyzing the nucleotide sequence of First, DNA is extracted from a mammal-derived specimen using, for example, a commercially available DNA extraction kit or the like.

 When blood is used as a specimen, plasma or serum is prepared from the blood according to an ordinary method, and the prepared plasma or serum is used as a specimen to prepare free DNA (cancer such as kidney cancer cells) contained therein. (Including DNA derived from cells), it is possible to analyze DNA derived from cancer cells such as lung cancer cells while avoiding DNA derived from blood cells, and to analyze cancer cells such as S-extracted cancer cells. It is possible to improve the sensitivity for detecting the containing tissue and the like.

 Next, after contacting the extracted DNA with a reagent that modifies unmethylated cytosine, the DNA is used as a template, and the promoter region, untranslated region or translation of the G-protein coupled somatostatin and angiotensin-like peptide receptor gene is determined. PCR is performed using a primer designed as described below based on the nucleotide sequence containing cytosine in the nucleotide sequence represented by one or more CpGs present in the nucleotide sequence of the region (coding region) As a result, DNA containing cytosine to be analyzed is amplified, and the nucleotide sequence of the resulting amplification product is directly analyzed.

Here, the nucleotide sequence represented by one or more CpGs present in the base sequence of the promoter region, untranslated region or translated region (coding region) of the G-protein coupled somatostatin and angiotensin-like peptide receptor gene And the base sequence of genomic DNA containing exon 1 of the human-derived G-protein coupled somatostatin and angioiens in-like peptide receptor gene and the promoter region located 5 ′ upstream thereof. More specifically, the base sequence represented by SEQ ID NO: 1 (corresponding to the complementary sequence of the base sequence represented by base numbers 57001 to 60000 of the base sequence described in Gen bank Accession No. AC008971) can be mentioned. . In the base sequence represented by SEQ ID NO: 1, the nucleotide sequence of exon 1 of the human G-protein coupled somatostatin and angiotensin-like peptide receptor gene is represented by nucleotide numbers 776 to 2632. The sequence in the nucleotide sequence represented by CpG existing in the nucleotide sequence represented by SEQ ID NO: 1 Tosin, especially cytosine in CpG present in the region where CpG is densely present in the nucleotide sequence shown in SEQ ID NO: 1, has a high methylation frequency (ie, hypermethylation) in cancer cells such as kidney cancer cells. State (hyperme thyl at ion)). More specifically, cytosine having a high methylation frequency in a pancreatic cancer cell includes, for example, nucleotides 470, 472, 490, 497, 504, 506, 509, 514 in the nucleotide sequence represented by SEQ ID NO: 1. , 522, 540, 543, 552, 566, 582, 597, 610, 612 and the like. As a primer used in the PCR, a primer pair capable of amplifying a DNA having a base sequence containing the cytosine is designed based on the base sequence 5 ′ upstream and 3 ′ downstream of the cytosine to be analyzed. Good. The base sequence for primer design is selected so as not to contain cytosine in the CpG to be analyzed. If the nucleotide sequence selected for the primer design does not contain any cytosine, the selected nucleotide sequence and the nucleotide sequence complementary to the nucleotide sequence are the same as the primer sequence of the primer. It can be. In addition, when the base sequence selected for the primer design contains cytosines other than those to be analyzed, but the cytosine is not cytosine in CpG, the primers should be considered in consideration that these cytosines are converted to peracil. design. That is, a pair of primers each having a base sequence in which all cytosines are peracyl and a base sequence complementary to the base sequence are designed. Furthermore, when the base sequence selected for primer design contains cytosine other than the target to be analyzed and the cytosine is cytosine in CpG, unmethylated cytosine is converted to peracyl and methylated. Design primers in consideration of the fact that cytosine that has been converted is not converted to peracil. In other words, the base sequence when methylated cytosine is contained [cytosine at the methylated position (cytosine in CpG) remains cytosine, and unmethylated cytosine (cytosine not included in CpG) Is a base sequence that becomes peracil] and a pair of methylation-specific primers respectively selected from base sequences complementary to the base sequence, and a base sequence when cytosine is not methylated (all cytosine Is a base sequence that becomes peracil) and a base sequence complementary to the base sequence A pair of unmethylated specific primers, each with a row, are designed. In this case, in the PCR described above, a methylation-specific primer pair and an unmethylation-specific primer pair are mixed and used in equal amounts. As a reagent for modifying unmethylated cytosine, for example, bisulfite such as sodium bisulfite can be used. Incidentally, in principle, a reagent that modifies only methylated cytosine may be used. In order to bring the extracted DNA into contact with a reagent that modifies unmethylated cytosine, for example, first, the DNA is placed in an alkaline solution (pH 9 to U), such as bisulfite such as sodium bisulfite (bisulfite) ( Concentration in the solution: for example, at a final concentration of 3M), etc., for about 10 to 16 hours (overnight) at 55 ° C. To accelerate the reaction, denaturation at 95 ° C and reaction at 50 ° C can be repeated 10-20 times. In this case, unmethylated cytosine is converted to peracyl, while methylated cytosine is not converted to peracyl and remains cytosine.

Next, the DNA treated with bisulfite or the like is subjected to PCR using a primer designed as described above. The nucleotide sequences of the obtained amplification products are compared, and the methylation frequency can be measured from the comparison. More specifically, primers for measuring the methylation frequency of the G-protein coupled somatostatin and angiotensin-like pepti de receptor gene by direct nucleotide sequence analysis include, for example, G-protein coupled somatostatin and Based on the nucleotide sequence containing one or more cytosines in CpG present in the nucleotide sequence in the promoter region, untranslated region or translated region (coding region) of the angiotensin-like peptide receptor gene, as described above. Can be designed. For example, cytosine in CpG present in the base sequence represented by SEQ ID NO: 1, specifically, base numbers 887, 890, 905, 939, 957, 966, 970, in the base sequence represented by SEQ ID NO: 1 One or more cytosines represented by 975, 977, 985, 987, 1042, 1046 and 1051 can be designed to be analyzed. For example, When the primers B1 and B2 shown below are used, a DNA (214 bp) having a base sequence obtained by performing a bi sul fite treatment on a DNA having a base sequence represented by base numbers 864 to 1077 of SEQ ID NO: 1 is amplified. . The primer pair is a cytosine represented by base numbers 887, 890, 905, 939, 957, 966, 970, 975, 977, 985, 987, 1042, 1046, and 1051 in the nucleotide sequence represented by SEQ ID NO: 1. It can be used as a primer to check the methylation frequency.

<Primer>

 B1: 5'-AGTTTGGTTTGAGAATTTTTGGA-3 '(SEQ ID NO: 6)

 B2: 5'-CCAATTTTATTACCAAACTAACC-3 '(SEQ ID NO: 7).

As a reaction solution in the PCR, for example, 25 ng of type II DNA, 1 liter of each primer solution of 20 pmol / 1, 2 mM (^^ 31, 10X buffer solution (100 mM Tri s-HCl ρΗ 8.3, 500 mM KCK 15 mM MgCl ₂ ) 2.51 and heat-resistant DNA polymerase 5 U / 1 0.21 are mixed, and sterile ultrapure water is added thereto to adjust the volume. Examples of the reaction solution include the reaction solution of 25. As the reaction conditions, for example, the above reaction solution is kept at 95 ° C for 10 minutes, then at 95 ° C for 30 seconds, and then at 55 ° C. Another condition is to carry out 30 to 40 cycles of heat retention at 72 seconds for 30 seconds as one cycle.

 Such: After performing PCR, the base sequences of the obtained amplification products are compared, and the methylation frequency is measured from the comparison.

That is, by directly analyzing the base sequence of the amplification product, it is determined whether the base at the position corresponding to the cytosine to be analyzed is cytosine or thymine (peracyl). By comparing the peak area indicating cytosine detected at the position corresponding to the cytosine to be analyzed with the peak area indicating thymine (peracil) in the peak chart indicating bases in the obtained amplification product, In addition, the frequency of methylation of cytosine to be analyzed can be measured. In addition, as a method for directly analyzing the nucleotide sequence, DNA clones were prepared from a plurality of clones obtained by once-cloning the amplification product obtained by PCR using E. coli or the like as a host. Alternatively, the base sequence of the DNA may be analyzed. Equivalent to cytosine to be analyzed in the sample to be analyzed The frequency of cytosine methylation to be analyzed can also be measured by determining the proportion of the sample in which the base detected at the corresponding position is cytosine. As a third method, after contacting DNA derived from a sample with a reagent that modifies unmethylated cytosine, the DNA is hybridized with a probe capable of discriminating the presence or absence of methylation of cytosine to be analyzed. To check the presence or absence of probe binding.

 First, DNA is extracted from a mammal-derived specimen using, for example, a commercially available DNA extraction kit or the like.

 When blood is used as a specimen, plasma or serum is prepared from the blood according to an ordinary method, and the prepared plasma or serum is used as a specimen to prepare free DNA (cancer such as kidney cancer cells) contained therein. (Including DNA derived from cells), it is possible to analyze DNA derived from cancer cells such as kidney cancer cells while avoiding DNA derived from blood cells, and to analyze cancer cells such as victory cancer cells. It is possible to improve the sensitivity for detecting the containing tissue and the like.

 Next, after contacting the extracted DNA with a reagent that modifies unmethylated cytosine, the DNA is hybridized with a probe capable of discriminating the presence or absence of methylation of cytosine to be analyzed. The presence or absence of binding to the probe is examined. The cytosine to be analyzed is a nucleotide sequence represented by one or more CpGs present in the nucleotide sequence of the promoter region, untranslated region or translated region (coding region) of the G-protein coupled somatostatin and angiotensin-like peptide receptor gene. You can choose from the cytosines inside.

Here, G-protein coupled somatostatin and angiotensin-1 ike peptide The base represented by one or more CpGs present in the base sequence of the promoter region, untranslated region or translated region (coding region) of the peptide recep tor gene. Sequences include the base sequence of genomic DNA containing exon 1 of the human G-protein coupled somatostatin and angiotensin-like peptide receptor gene and the promoter region located 5 'upstream. More specifically, the nucleotide sequence represented by SEQ ID NO: 1 (represented by nucleotide numbers 57001 to 60000 of the nucleotide sequence described in Gen bank Accession No. AC008971) Corresponding to the complementary sequence of the base sequence. ). The nucleotide sequence of exon 1 of the human rice G_protein coupled somatos tat in and angiotens in-like peptide ide receptor gene is shown in SEQ ID NO: 1 as nucleotides 776 to 2632. It is shown. Cytosine in the nucleotide sequence represented by CpG present in the nucleotide sequence represented by SEQ ID NO: 1, particularly cytosine in CpG present in the region where CpG is densely present in the nucleotide sequence represented by SEQ ID NO: 1, For example, it shows a high methylation frequency (ie, hypermethylation state) in cancer cells such as victory cancer cells. More specifically, examples of cytosine having a high methylation frequency in victory cancer cells include, for example, nucleotides 470, 472, 490, 497, 504, 506, 509, 514 in the nucleotide sequence represented by SEQ ID NO: 1. , 522, 540, 543, 552, 566, 582, 597, 610, 612 and the like. In the probe used for the hybridization, unmethylated cytosine is converted into peracyl based on the base sequence containing cytosine to be analyzed, and methylated cytosine is converted to cytosine.設計 It is recommended to design in consideration of not being converted to racile.

In other words, the base sequence when methylated cytosine is contained [cytosine at the methylated position (cytosine in CpG) remains cytosine, and unmethylated cytosine (cytosine not included in CpG) Is a peracil base sequence) or a methylation-specific probe having a base sequence complementary to this base sequence, and a base sequence when cytosine is not methylated (all cytosines are peracil Base sequence) or a non-methylation-specific probe having a base sequence complementary to such base sequence. Such a probe may be used after being labeled to facilitate analysis of the presence or absence of binding between DNA and the probe. Alternatively, the probe may be immobilized on a carrier according to an ordinary method. In this case, DNA extracted from a mammal-derived specimen may be labeled in advance. As a reagent for modifying unmethylated cytosine, for example, sodium bisulfite or the like Bisulfite and the like can be used. Incidentally, in principle, a reagent that specifically modifies only methylated cytosine may be used. To bring the reagent that modifies unmethylated cytosine into contact with the extracted DNA, for example, first denature the DNA with an alkaline solution (pH 9 to 14) and then use bisulfite such as sodium bisulfite. ) (Concentration in solution: 3M final concentration, etc.) and treat at 55 ° C for about 10-16 hours (overnight). To accelerate the reaction, denaturation at 95 ° C and reaction at 50 ° C can be repeated 10-20 times. In this case, unmethylated cytosine is converted to peracyl, while methylated cytosine is not converted to peracyl and remains cytosine.

 If necessary, the DNA may be amplified in advance by performing PCR in the same manner as in the second method using DNA treated with bisulfite or the like as type II.

Next, hybridization is performed between the DNA that has been treated with bisulfite or the like or the DNA that has been amplified in advance by the PCR, and a probe that can identify the presence or absence of cytosine methylation to be analyzed. By comparing the amount of DNA that binds to the methylation-specific probe with the amount of DNA that binds to the unmethylation-specific probe, the frequency of cytosine methylation to be analyzed can be measured. . More specifically, a probe for measuring the methylation frequency of the G-protein coupled somatostatin and angiotensin-like pepti de receptor gene is, for example, G_pro.tein coupled somatostatin and angiotensin-like peptide receptor. It can be designed as described above based on a nucleotide sequence containing one or more cytosines in CpG existing in the nucleotide sequence in the overnight region, untranslated region or translated region (coding region). For example, cytosine in CpG present in the nucleotide sequence represented by SEQ ID NO: 1, specifically, nucleotide numbers 459, 461, 464, 466, 470, 472, 490 in the nucleotide sequence represented by SEQ ID NO: 1 , 497, 504, 506, 509, 514, 522, 540, 543, 552, 566, 582, 597, 610, 612, 620, etc. . Examples of such probes are shown below. W 200

 17

<Set 1>

Unmethylation-specific probe: 5'-TTGTGGTGTGGGTGTGGTTGGGTTTAGTATTTTG-3 '(SEQ ID NO: 8) Methylation-specific probe: 5'-TCGCGGCGCGGGCGCGGTTGGGTTTAGTATTTCG-3' (SEQ ID NO: 9)

<Set 2>

Non-methylation-specific probe: 5'-TTGGAGTTGATAATTGTGGAGAGATGT-3 '(SEQ ID NO: 10) Methylation-specific probe: 5'-TCGGAGTTGATAATCGCGGAGAGACGT-3' (SEQ ID NO: 11) , Maniatis T., Molecular Cloning 2nd edition, published by Cold Spring Harbor Laboratory (Cold Spring Harbor Laboratory pre ss). . Hybridization is usually performed under stringent end conditions. Here, “under stringent conditions” means, for example, that a hybrid is formed at 45 in a solution containing 6XSSC (a solution containing 1.5M NaCl, 0.15M trisodium citrate is 10XSSC). After that, conditions such as washing with 2XSSC at 50 (Molecular Biology, John Wiley & Sons, NY (1989), 6.3. 6.3.6) can be mentioned. The salt concentration in the washing step can be selected, for example, from 2XSSC at 50 ° C (low stringency conditions) to 0.2XSSC up to 50 ° C (high stringency conditions). Wear. The temperature in the washing step can be selected, for example, from room temperature (low stringency conditions) to 65 (high stringency conditions). Also, both salt concentration and temperature can be varied.

After performing such hybridization, the amount of DNA bound to the methylation-specific probe is compared with the amount of DNA bound to the unmethylation-specific probe to be analyzed. The methylation frequency of cytosine (ie, cytosine in CpG contained in the base sequence on which the probe was designed) can be measured. As a fourth method, the DNA derived from the specimen must have cytosine methylation to be analyzed. After a restriction enzyme capable of discriminating nothing is allowed to act, a method of examining the presence or absence of digestion by the restriction enzyme can be used.

 First, DNA is extracted from a mammal-derived specimen using, for example, a commercially available DNA extraction kit or the like.

 When blood is used as a specimen, plasma or serum is prepared from the blood according to an ordinary method, and the prepared plasma or serum is used as a specimen to prepare free DNA (cancer such as kidney cancer cells) contained therein. (Including DNA derived from cells), it is possible to analyze DNA derived from cancer cells such as kidney cancer cells, avoiding DNA derived from blood cells, and to analyze cancer cells such as victory cancer cells. It is possible to improve the sensitivity for detecting the containing tissue and the like.

 Next, a restriction enzyme capable of discriminating the presence or absence of methylation of cytosine to be analyzed is allowed to act on the extracted DNA, and then the presence or absence of digestion by the restriction enzyme is examined. The cytosine to be analyzed is represented by one or more CpGs present in the nucleotide sequence of the promoter region, untranslated region or translated region (coding region) of the G-protein coupled somatostatin and angiotensin-1 ike peptide receptor gene. It can be selected from cytosines in the nucleotide sequence.

Here, the base sequence represented by one or more CpGs present in the base sequence of the promoter region, untranslated region or translated region (coding region) of the G-protein coupled somatostatin and angiotensin-like peptide recep tor gene is as follows. The base sequence of genomic DNA containing exon 1 of the human G-protein coupled somatostatin and angiotensin-like peptide receptor gene and the promoter region located 5 ′ upstream thereof can be increased. Specifically, the base sequence represented by SEQ ID NO: 1 (corresponding to the complementary sequence of the base sequence represented by base numbers 57001 to 60000 of the base sequence described in Gen bank Accession No. AC008971) can be mentioned. The nucleotide sequence of exon 1 of the human G-protein coupled somatostatin and angiotensin-like peptide receptor gene is represented by nucleotide numbers 776 to 2632. . Cytosine in the nucleotide sequence represented by CpG present in the nucleotide sequence represented by SEQ ID NO: 1, particularly cytosine in CpG present in the region where CpG is densely present in the nucleotide sequence represented by SEQ ID NO: 1, For example, high methyl levels in cancer cells such as kidney cancer cells It shows the frequency of oxidation (ie, hypermethylation). More specifically, examples of cytosine having a high methylation frequency in victory cancer cells include, for example, nucleotides 470, 472, 490, 497, 504, 506, 509, 514 in the nucleotide sequence represented by SEQ ID NO: 1. , 522, 540, 543, 552, 566, 582, 597, 610, 612 and the like. The “restriction enzyme capable of discriminating the presence or absence of cytosine methylation” (hereinafter sometimes referred to as a methylation-sensitive restriction enzyme) used in the method refers to digestion of a recognition sequence containing methylated cytosine. And a restriction enzyme capable of digesting recognition sequences containing unmethylated cytosine. In the case of DNA in which the cytosine contained in the recognition sequence is methylated, the DNA is not cleaved by the action of a methylation-sensitive restriction enzyme, while the DNA in which the cytosine contained in the recognition sequence is not methylated is treated. In this case, the DNA is cleaved by the action of a methylation-sensitive restriction enzyme. Specific examples of the methylation-sensitive enzyme include, for example, HpaII, BstUI, Narl, SacII and the like. As a method for examining the presence or absence of digestion by the restriction enzyme, for example, DNA treated with a methylation-sensitive restriction enzyme containing the cytosine to be analyzed in the recognition sequence is type II, and the cytosine to be analyzed is included. A method is described in which PCR is performed using a primer pair capable of amplifying DNA containing a recognition sequence but not containing the recognition sequence of the restriction enzyme other than the recognition sequence, and the presence or absence of DNA amplification (amplification product) is examined. be able to. If the cytosine to be analyzed is methylated, an amplification product is obtained. On the other hand, if the cytosine to be analyzed is not methylated, no amplification product can be obtained. Thus, by comparing the amount of the amplified DNA, the frequency of methylation of cytosine to be analyzed can be measured. When quantification is needed, real-time monitoring of PCR reaction products and analysis of force kinetics enable high-precision quantification that can detect, for example, a slight difference of about twice the amount of gene. The amount of each product can also be compared using real-time PCR, a PCR method. Methods for performing real-time PCR include, for example, type II-dependent nucleic acid polymerase probe Or a method using an intercalator such as Cyber Green. As the apparatus and reagents for the real-time PCR method, commercially available apparatuses and reagent kits can be used.

 For example, in the case of cytosine represented by base numbers 610, 612, 810, 812, 2053, 2055, 2455, and 2457 in the base sequence represented by SEQ ID NO: 1, the cytosine is included in the recognition sequence of SacII. In addition, the methylation frequency of the cytosine can be measured by the above method. Other methods for examining the presence or absence of digestion by the restriction enzyme include, for example, G-protein coupled somatostatin and DNA, which have been subjected to the action of a methylation-sensitive restriction enzyme containing cytosine to be analyzed in the recognition sequence. Angiotensin-like peptide recepio r gene can be used to detect the length of hybridized DNA by performing Southern hybridization using DNA that does not contain the recognition sequence of the restriction enzyme as a probe. . When the cytosine to be analyzed is methylated, a longer DNA is detected than when the cytosine is not methylated. By comparing the amount of long and short DNA detected, the frequency of cytosine methylation to be analyzed can be determined. Using the various methods as described above, the frequency of the methylated G-protein-coupled somatostatin and angiotensin-1 ike peptide receptor gene contained in a specimen derived from a mammal is measured.

The measured methylation frequency and the methylation of G-protein coupled somatostatin and an giotensin-like peptide receptor gene contained in a sample derived from a healthy mammal that can be diagnosed as having no cancer cells such as victory cancer cells The degree of canceration of the specimen is determined based on the difference obtained by the comparison with the transformation frequency (control). For example, if the methylation frequency of the G_protein coupled somatostatin and angiotensin-1 ike peptide receptor gene contained in a specimen derived from a mammal is higher than that of the control (G-protein couple d somatostatin and angiotensin-like peptide receptor gene On comparison with If the sample is hypermethylated), it can be determined that the degree of canceration of the specimen is higher than that of the control.

 Here, the term “degree of canceration” has the same meaning as generally used in the art. Specifically, for example, when a mammal-derived specimen is a cell, it means the degree of malignancy of the cell. In addition, for example, when the specimen derived from a mammal is a tissue, it means the amount of cancer cells present in the tissue. In the evaluation method of the present invention, primers or probes that can be used in various methods for measuring the methylation frequency of the G-protein coupled somatostatin and angiotensin-like peptide receptor gene are used for detecting cancer cells such as ligament cancer cells. Useful as a kit reagent. The present invention provides a kit for detecting cancer cells such as kidney cancer cells containing these primers or probes as reagents, and a cancer such as kidney cancer cells in which these primers or probes are immobilized on a carrier. Also provided is a chip for detecting cells, and the scope of the evaluation method of the present invention covers a detection kit as described above, which utilizes the substantial principle of the method, and a form such as a detection chip. Use of course is included. Example

 Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto. Example 1 (Confirmation test of methylation status of G-protein coupled somatostatin and angioten sin-like peptide receptor gene in knee cancer cell line)

Seven human-derived renal cancer cell lines [BXPc3, HPAF-II, Capan-2, MiaPaCa-2, Hs766T, PANCl-1 and HPAC (all manufactured by ATCC)] were cataloged by ATCC (American Type Culture Collection). described, was cultured to subconfluent E cement dedicated medium for each cell line, they were collected each about LxlO ⁷ cells. Two immortalized (normal). Duct epithelial cell lines (HPDE-4 / E6E7 and PDE6_E6E7c7) obtained by the general immortalization method [Am. J. Patho 1., 148, 1763-1770 (1996)] [These are Dr. Tsao (Ontario Cancer Institute and stored by the Department of Pathology, the University of Toronto, and transferable from the investigator), and a final concentration of 50 lI / mL penicillin and a final concentration of 5 Oug / mL streptomycin. After culturing until subconfluent using the added Kerat inocyte-SFM, liquid (manufactured by Invitrogen) as a medium, about IxlO ⁷ cells were collected from each. To the collected cells, a 10-fold volume of SEDTA buffer [lOmM Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0), lOOmM NaCl] was added, followed by homogenization. To the resulting mixture, proteinase K (Sigma) was added at 500 g / ml and sodium dodecyl sulfate to 1% (w / v), and the mixture was shaken at 55 ° C for about 16 hours. . After completion of the shaking, the mixture was subjected to phenol [saturated with 1 M Tris-HCl (ρΗ80)] · form extraction. The aqueous layer was recovered, and NaCl was added thereto to a concentration of 0.5N, and the precipitate was recovered by ethanol precipitation. The collected precipitate was dissolved in TE buffer (10 mM Tris, ImM EDTA, pH 8.0), RNase A (Sigma) was added thereto to a concentration of 402 g / ml, and the mixture was incubated at 37 ° C for 1 hour. I caught it. The incubated mixture was subjected to phenol-cloth form extraction. The aqueous layer was recovered, and NaCl was added thereto to a concentration of 0.5N, followed by ethanol precipitation to recover a precipitate (genomic DNA). The recovered precipitate was rinsed with 70% ethanol to obtain genomic DNA.

After digesting the obtained genomic DNA with the restriction enzyme BamHI, Clark et al., Nucl. Acids. Res., 22, 2990-2997, 1994; Herman et al., Pro. Natl. Acad. USA, 93, 98 21-9826, 1996, and treated with sodium bisulfite. That is, genomic DNA (about 500 ng) after restriction enzyme treatment was dissolved in distilled water to prepare a 20 ^ 1 genomic DNA solution, and about 1/1 of 6M sodium hydroxide was added thereto ( The final concentration was approximately 0.3M) and the mixture was incubated at 37 for 15 minutes. To this mixture was added 0.6 mM hydroquinone (Sigma) to a final concentration of 0 • 5 mM and sodium bisulfite (Sigma) to a final concentration of 3.1 M, and this was added at 95 ° C for 30 seconds and then at 50 ° C. Insulation was carried out for 15 cycles, with 15 minutes as one cycle. DNA was purified from the incubated solution using a Wizard DNA clean-up system (Promega). The purified DNA was dissolved in 50/1 TE buffer, and sodium hydroxide was added thereto to a final concentration of 0.3 M, and the mixture was left at room temperature for 5 minutes. Then, the precipitate (DNA) was recovered by ethanol precipitation of the left mixture. Recover the collected precipitate to 20 1 T Suspended in E buffer. Using the obtained DNA as type III, PCR was carried out using the following non-methyl-specific primers U1 and U2 or the methylation-specific primers Ml and M2. Unmethylation-specific primers — When U1 and U2 are used, a 149 bp DNA having the nucleotide sequence shown in SEQ ID NO: 1 having a nucleotide sequence represented by nucleotides 469 to 617 after biisulfite treatment is amplified On the other hand, when the methylation-specific primers Ml and M2 are used, the DNA has the nucleotide sequence obtained by bisulfite-treating DNA having the nucleotide sequence represented by nucleotide numbers 470 to 614 of SEQ ID NO: 1. A 145 bp DNA is amplified.

<Non-methylated specific primer>

 U1: 5'-GTGTGGTTGGGTTTAGTATTTT -3 '(SEQ ID NO: 2)

U2: 5'-TCTCCACAATTATCAACTCCA-3 '(SEQ ID NO: 3)

<Methylation specific primer>

Ml: 5'-CGCGGTTGGGTTTAGTATTTC-3 '(SEQ ID NO: 4)

M2: 5'-CCGCGATTATCAACTCCG-3 '(SEQ ID NO: 5)

 To confirm the specificity of the methylation-specific primer and the unmethylation-specific primer, first, genomic DNA was obtained from an immortalized (normal) 塍 duct epithelial cell line (HPDE-4 / E6E7) by the usual method. (DNAl) was extracted and a part of this was treated with the methylating enzyme Sssl (NEB) to methylate all 5'-CG-3 'of genomic DNA (DNA2). Methylation-specific PCR and unmethylation-specific PCR were also performed on these DNAs 1 and 2.

As a reaction solution for PCR, 25 ng of type III DM, 1 liter of each of the above primer solutions of 20 pmol / 1, 2.5 1 of each 2 mM dNTP, 10X buffer (100 mM Tris-HCl pH 8.3, 500 iM KC1, 20 ¾18 (; 1 ₂ ) was mixed with 2.5 1 and heat-resistant DNA polymerase 5U / 1 at 0.1, and sterilized ultrapure water was added to make a liquid volume of 251. If the above-mentioned unmethylated specific primer is used, the reaction solution is incubated at 95 ° C for 10 minutes, then at 95 ° C for 30 seconds, then at 59 ° C for 30 seconds and then at 72 ° C. The PCR was performed under the condition that 32 cycles of incubation were performed at 30 C for 1 cycle for 30 seconds, and the methylation-specific primer described above was used. If used, incubate the reaction solution at 95 ° C for 10 minutes, and then cycle at 95 ° C for 30 seconds, then at 62 ° C for 30 seconds, and at 72 ° C for 30 seconds. PCR was performed under the condition that the incubation was performed for 32 cycles. In each case, after performing the PCR, the PCR reaction solution containing the amplification product was subjected to 2% agarose gel electrophoresis.

 The results are shown in FIG. In the case of human-derived immortalized (normal) 塍 duct epithelial cell lines (HPDE-4 / E6 E7 and HPDE6-E6E7C), the use of unmethylated specific primers (lane U) A band was observed, and no band of the amplified DNA was detected when the methylation-specific primer was used (lane M). Therefore, in the case of human-derived immortalized (normal) 脖 ductal epithelial cell lines (HPDE-4 / E6E7 and HPDE6-E6E7c), at least the nucleotide numbers 470, 472, and 472 of the nucleotide sequence represented by SEQ ID NO: 1 490, 597, one of the seven renal cancer cell lines (BXPc3, HPAF-II, Capan 2, MiaPaCa-2, Hs766T, MNC-1 and HP AC) was immortalized (normal) As in the 塍 duct epithelial cell lines (HPDE-4 / E6E7 and ¾PD E6-E6E7C), it was not methylated. For the other six species (BXPc3, HPAF-II, Capan-2, MiaPaCa-2, Hs766tRO¾ffAC), no amplified MA band was detected when unmethylated specific primers were used (lane U). When a methylation-specific primer was used (lane M), an amplified DNA band was observed. Therefore, under these conditions, the six types of cells (BXPc3, HPAF-II, Capan-2, MiaPaCa-2, Hs766T and ¾MC) have the nucleotide numbers 470, 472, 490, The cytosines indicated at 597, 610 and 612, respectively, were determined to be methylated. Example 2 (Confirmation test of methylation status of G-protein coupled somatostatin and angiotensin-like peptide receptor gene in Teng carcinoma tissue)

Human-derived visceral cancer tissue and normal splanchnic tissue around it [obtained by obtaining informed consent from the patient] 12 specimens (Casel to Casel2) were added to SEDTA buffer [10 Tris-HCl (pH 8.0)] , lOmM EDTA (pH8.0), lOOmM NaCl] was added 10 times by volume and homogenized. After adding 500 g / ml of proteinase K (Sigma) and 1% (w / v) of sodium dodecyl sulfate to the obtained mixture, the mixture was shaken with 55 at about 16 hours. After the shaking, the mixture was subjected to phenol [saturated with 1 M Tris-HCl (pH 8.0)] 'chloroform extraction treatment. The aqueous layer was recovered, and NaCl was added to the aqueous layer to a concentration of 0.5N, and the precipitate was recovered by ethanol precipitation. Dissolve the collected precipitate in TE buffer (10 mM Tris, ImM EDTA, pH 8.0), add RNase A (Sigina) to this at 40 g / ml, and incubate at 37 ° C for 1 hour did. The incubated mixture was subjected to phenol * chloroform extraction. The aqueous layer was recovered, and NaCI was added thereto to a concentration of 0.5N, followed by ethanol precipitation to recover a precipitate (genomic DNA). The collected precipitate was rinsed with 70% ethanol to obtain genomic DNA.

After digesting the obtained genomic MA with the restriction enzyme BamHI, Clark et al., N cl. Acids. Res., 22, 2990-2997, 1994; Herman et al., Pro. Natl. Acad. Sci. USA , 93, 98 21-9826, 1996. That is, genomic DNA (about 50 Ong) after restriction enzyme treatment was dissolved in distilled water to prepare a 201 genomic DNA solution, and about 6 I of 6M sodium hydroxide was added thereto (to a final concentration of about 1 I). 0.3M) and the mixture was incubated at 37 for 15 minutes. To this mixture was added 0.6 hydroquinone (Sigma) to a final concentration of 0.5 mM and sodium bisulfite (Sigma) to a final concentration of 3.1 M, and this was added at 95 ° C for 30 seconds and then at 50 ° C for 15 minutes. Insulation for one cycle was performed for 15 cycles. DNA was purified from the incubated solution using a Wizard DNA clean-up system (Promega). The purified DNA was dissolved in 501 TE buffer, sodium hydroxide was added thereto to a final concentration of 0.3 M, and the mixture was left at room temperature for 5 minutes. Then, the precipitate (DNA) was recovered by ethanol precipitation of the left mixture. The collected precipitate was suspended in TE buffer. Using the obtained MA as type II, PCR was performed using the following unmethylation-specific primers U1 and U2 or the methylation-specific primers Ml and M2. When unmethylation-specific primers U1 and U2 were used, a 149 bp DNA having a base sequence obtained by subjecting a DNA having a base sequence represented by base numbers 469 to 617 of SEQ ID NO: 1 to biisulfite treatment was amplified. On the other hand, when the methylation-specific primers Ml and M2 were used, the base sequence after bisuite treatment of DNA having the base sequence represented by base numbers 470 to 614 of SEQ ID NO: 1 was performed. The resulting 145 bp DNA is amplified.

<Non-methylation specific primer>

U1: 5'-GTGTGGTTGGGTTTAGTATTTT-3 '(SEQ ID NO: 2)

U2: 5'-TCTCCACAATTATCAACTCCA-3 '(SEQ ID NO: 3)

<Methylation specific primer>

 Ml: 5'-CGCGGTTGGGTTTAGTATTTC-3 '(SEQ ID NO: 4)

M2: 5'-CCGCGATTATCAACTCCG-3 '(SEQ ID NO: 5) The specificity of the methylation-specific primer and the unmethylation-specific primer was determined as described in Example 1 in the immortalized (normal) 塍 duct epithelial cell line Genomic DNA (l) of (HPDE-4 / E6E7) and a part of it have been treated with methylase Sssl (NEB) and confirmed in genomic DNA (2).

As a reaction solution for PCR, 25 ng of type II DNA, 1 l of each of the above-mentioned primer solutions of 20 pmol / 1, 2 mM dNTP, and 10 X buffer (100 mM Tris-HCl ρΗ8.3 , 500 mM KC1, 20111¾1¾¾ (1 ₂ ), 2.5 and heat-resistant DNA polymerase 5U / 1, 0.1, mixed with sterile ultrapure water to make the volume When the above-mentioned unmethylation-specific primer was used, the reaction solution was incubated at 95 for 10 minutes, then at 95 for 30 seconds, then at 59 ° C for 30 seconds and further at 72. PCR was performed under the condition that 32 cycles of incubation were performed with 30 seconds as one cycle, and when the above methylation-specific primer was used, the reaction solution was incubated at 95 ° C for 10 minutes. The PCR was performed under the conditions of 30 cycles at 95 ° C., followed by 30 cycles at 62, 30 seconds at, and 30 seconds at, and 32 cycles of incubation. Again, after the PCR, it was subjected to reaction solution of PCR containing the amplification product 2% Agarosugeru electrophoresis.

The results are shown in FIG. In 12 samples of normal human-derived viable tissue, the band of the amplified DNA was observed when the unmethylation-specific primer was used (lane U), and when the methylation-specific primer was used (lane M). In), no band of amplified DNA was detected. Therefore, in the case of human-derived normal skeletal tissue, at least It was determined that cytosine represented by base numbers 470, 472, 490, 597, 610, and 612 of the base sequence represented by SEQ ID NO: 1 was not methylated. Spleen cancer tissue 1 When unmethylated specific primers were used in 8 out of 2 samples (in the case of using methylated specific primers (lane M) in addition to the amplified DNA band in the lane) Therefore, under the conditions, at least the cytosine represented by nucleotide numbers 470, 472, 490, 597, 610, and 612 of the nucleotide sequence represented by SEQ ID NO: 1 was obtained under the above conditions. It was determined that a part of the tissue had become methylated due to its canceration.

 According to the present invention, a method for evaluating the degree of canceration of a specimen derived from a mammal can be provided. Sequence listing free text

SEQ ID NO: 2 SEQ ID NO: 3

 Oligonucleotide primers designed for PCR

SEQ ID NO: 4

 Oligonucleotide primers designed for PCR

SEQ ID NO: 5

 Oligonucleotide primers designed for PCR

SEQ ID NO: 6

 Oligonucleotide primers designed for PCR

SEQ ID NO: 7

 Oligonucleotide primers designed for PCR

SEQ ID NO: 8

 Oligonucleotides designed for probes

SEQ ID NO: 9

Oligonucleotides designed for probes SEQ ID NO: 10

 Oligonucleotide designed for probe SEQ ID NO: 1 1

 Oligonucleotides designed for probes

Claims

The scope of the claims  1. A method for evaluating the degree of canceration of a mammal-derived specimen,  (1) the first step of measuring the methylation frequency of the G-protein coupled somatos tat in and angiotens in-like peptide receptor gene contained in a mammal-derived specimen, and (2) the measured methylation A second step of determining the degree of cancer in the sample based on a difference obtained by comparing the frequency with a control An evaluation method comprising: 2. The method according to claim 1, wherein the mammal-derived specimen is a cell. 3. The evaluation method according to claim 1, wherein the mammal-derived specimen is a tissue. 4. The evaluation method according to claim 1, wherein the mammal-derived specimen is a cell, and the degree of canceration of the specimen is the malignancy of the mammal-derived cell. 5. The evaluation method according to claim 1, wherein the mammal-derived specimen is a tissue, and the degree of canceration of the specimen is the amount of cancer cells present in the mammal-derived tissue. 6. The evaluation method according to claim 5, wherein the tissue is a knee tissue and the cancer is victory cancer. 7. The methylation frequency of a gene is determined by the cytosine in the base sequence represented by one or more 5'-CG-3 'present in the base sequence in the promoter region, untranslated region or translated region of the gene. 2. The method according to claim 1, wherein the frequency is methylation frequency. 8. The method according to claim 7, wherein the tissue is a knee tissue, and the cancer is Teng's cancer. 9.The methylation frequency of a gene is the methylation frequency of cytosine in one or more nucleotide sequences represented by 5'-CG-3 'existing in the base sequence in the promoter region of the gene. The evaluation method according to claim 1, wherein the evaluation method is characterized in that: 10.The methylation frequency of a gene is determined by the methylation of cytosine in the nucleotide sequence represented by one or more 5'-CG-3's present in the nucleotide sequence in the untranslated or translated region of the gene. 2. The evaluation method according to claim 1, wherein the evaluation method is frequent. 11 1.The methylation frequency of the gene is the methylation frequency of cytosine in one or more nucleotide sequences represented by 5'-CG-3 'existing in the nucleotide sequence represented by SEQ ID NO: 1. 2. The evaluation method according to claim 1, wherein the evaluation method is characterized in that: 12. The evaluation method according to claim 11, wherein the tissue is an S-extracted tissue and the cancer is victory cancer. 1 3. Use of methylated G-protein coupled somatostat in and angiotens in-like peptide receptor gene as a cancer marker. 14. Use according to claim 13, wherein the cancer marker is a Teng pancreatic cancer marker.