WO2004018668A1

WO2004018668A1 - Method of evaluating degree of canceration of human-origin specimen

Info

Publication number: WO2004018668A1
Application number: PCT/JP2003/010480
Authority: WO
Inventors: Toshikazu Ushijima; Toshio Takada; Kazuaki Miyamoto
Original assignee: Sumitomo Chemical Company, Limited; Japan As Represented By President Of National Cancer Center
Priority date: 2002-08-23
Filing date: 2003-08-20
Publication date: 2004-03-04
Also published as: AU2003257587A1; AU2003257587A8

Abstract

A method of evaluating the degree of canceration of a human-origin specimen characterized by having the following steps: (1) the first step of measuring the methylation frequency in a DNA having the base sequence represented by SEQ ID NO:1 or its equivalent contained in the human-origin specimen by the real time PCR method; and (2) the second step of comparing the methylation frequency thus measured with that of a control and judging the degree of canceration of the specimen based on the difference.

Description

Specification

Method for evaluating the degree of canceration of human-derived specimen

Technical field

The present invention relates to a method for evaluating the degree of canceration of a human-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 and therapeutic 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 after-care such as confirmation of the recurrence of cancer after treatment.

Therefore, a method for evaluating the degree of canceration of human-derived specimens based on the detection of genetic abnormalities, which is suitable for diagnosis methods for early detection of cancer, evaluation of the effectiveness of treatment methods for cancer, confirmation of the presence or absence of cancer recurrence, etc. Development is eagerly awaited. Disclosure of the invention

Under such circumstances, the present inventors have conducted intensive studies and found that the methylation frequency of a specific DNA in a cancer patient sample is significantly higher than that of a sample from a healthy subject by using a real-time PCR method. Heading, the present invention has been reached.

That is, the present invention

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

(1) The frequency of methylation in DNA having the nucleotide sequence shown in SEQ ID NO: 1 or its equivalent (hereinafter sometimes referred to as the present DNA) contained in a human-derived specimen is measured by real-time PCR. The first step, and

(2) The second step of determining the degree of cancer in the sample based on the difference obtained by comparing the measured methyl methane frequency with a control

(Hereinafter, also referred to as the evaluation method of the present invention); 2. The evaluation method according to the above item 1, wherein the human-derived specimen is a cell; 3. The evaluation method described in 1 above, wherein the human-derived sample is a tissue;

4. The evaluation method according to the above 1, wherein the human-derived sample is blood, plasma or serum;

5. The evaluation method according to the above 3, wherein the human-derived specimen is breast tissue, mammary gland tissue or mammary gland epithelial tissue, and the cancer is breast cancer;

6. The methylation frequency of the cytosine in the nucleotide sequence represented by one or more 5'-CG-3 'present in the nucleotide sequence of the DNA, wherein the methylation frequency is cytosine methylation frequency. Evaluation method;

7. The evaluation method as described in 6 above, wherein the human-derived sample is blood, plasma or serum;

8. The evaluation method according to the above item 6, wherein the human-derived specimen is breast tissue, breast tissue or breast epithelial tissue, and the cancer is breast cancer;

9. Use of DNA having the nucleotide sequence of SEQ ID NO: 1 or an equivalent thereof as a cancer marker;

10. The use according to the above item 9, wherein the cancer marker is a breast cancer marker;

1 1. Measuring power of methylation frequency by real-time PCR method S. After contacting DNA prepared from a sample with a reagent that modifies unmethylated cytosine, the DNA is shown as SEQ ID NO. PCR is performed using a pair of primers that can identify the presence or absence of cytosine methylation in the DNA having the base sequence or its equivalent, and the amount of the obtained amplification product is determined by the base sequence represented by SEQ ID NO: 1. The evaluation method according to the above 1, wherein the presence or absence of cytosine methylation in DNA or its equivalent is measured in real time using a probe capable of discriminating;

12. The forward primer is an oligonucleotide having the nucleotide sequence of SEQ ID NO: 2, the reverse primer is an oligonucleotide having the nucleotide sequence of SEQ ID NO: 3, and the probe is a nucleotide having the nucleotide sequence of SEQ ID NO: 6. The evaluation method according to the above item 1, wherein the oligonucleotide is an oligonucleotide having a sequence;

13. The forward primer is an oligonucleotide having the nucleotide sequence of SEQ ID NO: 4, and the reverse primer is an oligonucleotide having the nucleotide sequence of SEQ ID NO: 5. The evaluation method according to the above-mentioned item 1, which is a nucleotide, and wherein the probe is an oligonucleotide having a base sequence represented by SEQ ID NO: 7;

14. A kit for detecting a cancer cell, comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 2, an oligonucleotide having the nucleotide sequence of SEQ ID NO: 3, and a nucleotide sequence of SEQ ID NO: 6 A kit containing an oligonucleotide;

15. A kit for detecting a cancer cell, comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 4, an oligonucleotide having the nucleotide sequence of SEQ ID NO: 5, and a nucleotide sequence of SEQ ID NO: 7 A kit containing an oligonucleotide;

And so on. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail.

The present invention relates to the use of methylated DNA having the nucleotide sequence shown in SEQ ID NO: 1 or an equivalent thereof as a cancer marker (for example, a breast cancer marker or the like).

The present DNA used as a cancer marker in the present invention is a human-derived Heparan sulfate D-Glucosaminyl 3-0-sulfotransferase-2 gene (hereinafter sometimes referred to as 30ST-2 gene) [J. Biol. Chem., 274, a part of the promoter region of 5170- 51 ⁸ 4 (1 "9)] nucleotide sequence is a DNA having a. human-derived 30ST- 2 gene coding region of the (coding region) responsible for the nucleotide sequence of Etason The base sequence of genomic DNA containing the most upstream 5 'exon (hereinafter referred to as exon 1) and the 5, upstream promoter region is described in, for example, Genbank Accession No. HUAC003661. In the nucleotide sequence described in Genbank Accession No. HUAC003661, for example, the ATG codon encoding methionine located at the amino terminus of the amino acid sequence of the human 30ST-2 protein is represented by the base number Nos. 58514 to 58516, and the base sequence of exon 1 is shown in base numbers 58514 to 58999. SEQ ID NO: 1 used in the present invention The present DNA having the nucleotide sequence shown is a DNA having the nucleotide sequence represented by nucleotide numbers 58252 to 58432 located in one region of this promoter. The present DNA includes, in addition to DNA having the above-mentioned known nucleotide sequence, as an equivalent, the nucleotide sequence due to a naturally occurring mutation due to a species difference, an individual difference or a difference between organs or tissues of the organism. DNAs having nucleotide sequences in which deletions, substitutions, or additions have occurred are also included. In mammals, there is a phenomenon in which, of the four types of bases that make up a gene (genomic DNA), only cytosine is methylated. In the human-derived, for example, 30ST-2 gene, some cytosines in the genomic DNA are methylated. The methylation modification of DNA is performed in the base sequence represented by 5′-CG-3 ′ (C represents cytosine, G represents guayun. Hereinafter, the base sequence may be referred to as CpG.) Limited to cytosine. The site of methylation 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 fucked. Therefore, the state of DNA methylation will be inherited by two new sets of DNA even after DNA replication. In the first step of the evaluation method of the present invention, the term “methylation frequency” refers to, for example, the fact that the cytosine is methylated when a plurality of haploids are examined for the presence or absence of cytosine methylation in CpG to be prepared. It is expressed as a percentage of the haploid that exists. More specifically, the amount of methylated DNA originally present in the sample determined by the methylation-specific real-time PCR described later and the amount of methyl originally present in the sample determined by the non-methylation-specific real-time PCR The amount of unmodified DNA is added to the total amount of DNA, and expressed as a ratio obtained by dividing the amount of methylated DNA originally present in the sample by the total amount of DNA. It may also be expressed by the amount of methylated DNA originally present in the sample determined by methylation-specific real-time PCR. Examples of human-derived specimens in the first step of the evaluation method of the present invention include breast cancer cells and the like. Cancer cells or tissues containing the same, and cells that may contain DNA derived from cancer cells such as breast cancer cells, and tissues containing the same (the tissues here include blood, plasma, serum, In a broad sense, it includes body fluids such as lymph fluid, lymph nodes, etc.) or biological samples such as body secretions (urine, milk, etc.). Specifically, for example, when the cancer is breast cancer, breast tissue, mammary gland tissue or mammary gland epithelial tissue collected from a subject can be mentioned. When the human-derived sample is blood, for example, a blood sample collected by a periodic medical examination or a simple test can be used.

These biological samples may be used as they are, or biological samples prepared by various operations such as separation, fractionation, and immobilization from the biological sample may be used as the samples. In the first step of the evaluation method of the present invention, a real-time PCR method is used as a method for measuring the methyl nig frequency in DNA having the nucleotide sequence shown in SEQ ID NO: 1 or its equivalent contained in a human-derived specimen.

Real-time PCR is a method for monitoring and analyzing the production process of amplification products in a PCR in real time using a device that integrates a thermal cycler and a spectrofluorometer. For example, by monitoring the PCR reaction product in real time using a probe such as a 鐯 -dependent nucleic acid polymerase probe (hereinafter sometimes referred to as the present probe) and performing force kinetic analysis, for example, the gene amount to be doubled This is a PCR method that can perform high-precision quantification, such as detecting the difference between the two. Equipment and kits for the real-time PCR method are already commercially available. First, DNA is extracted from a human-derived specimen using, for example, a commercially available DNA extraction kit or the like.

By the way, when blood is used as a specimen, plasma or serum is prepared from blood according to an ordinary method, and free DNA contained in the prepared plasma or serum (DC derived from cancer cells such as breast cancer cells). (Including NA) can be used to analyze DNA from cancer cells such as breast cancer cells while avoiding contamination of blood cell-derived DNA. Next, after contacting the extracted DNA with a reagent that modifies unmethylated cytosine, for example, the DNA having the nucleotide sequence shown in SEQ ID NO: 1 or a methylin of the cytosine to be analyzed in an equivalent thereof is obtained. Real-time PCR is performed using primers and probes that can identify the presence or absence of dangling, and the methylation frequency of the present DNA contained in the sample is examined based on the amount of amplification product obtained.

The region for designing the primers and probes used here can be a part of the base sequence of the promoter region located at 5, 5 upstream of exon 1 of the 30ST_2 gene of human origin. And a base sequence represented by SEQ ID NO: 1 (corresponding to the base sequence represented by base numbers 58252 to 58432 of the base sequence described in Genbank Accession No. HUAC003661). Cytosine in the nucleotide sequence represented by CpG present in the nucleotide sequence represented by SEQ ID NO: 1 has, for example, a high methylation frequency (that is, a hypermethylation state) in cancer cells such as breast cancer cells. Show. As a reagent for modifying unmethylated cytosine, for example, bisulfite such as sodium hydrogen sulfite can be used. Incidentally, in principle, a reagent that specifically modifies only methylated cytosine may be used instead of a reagent that modifies unmethylated cytosine.

To bring the extracted DNA into contact with a reagent that modifies non-methylated cytosine, for example, after denaturing the DNA with an alkaline solution (pH 9 to 14), a bisulfite such as sodium bisulfite is used. (Concentration in the solution: for example, 3M final concentration), etc., for about 10 hours to about 16 hours (overnight) at 55 ° C. To accelerate the reaction, the denaturation at 95 ° C and the reaction at 55 ° 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, for example, when the DNA treated with bisulfite or the like is type III and contains methylated cytosine, the nucleotide sequence [methylated cytosine (cytosine in CpG) remains cytosine The unmethylated cytosine (including cytosine not included in CpG) is a base sequence that has become peracil] and is complementary to such base sequence Real-time PCR using a pair of methylation-specific primers and a probe selected from a specific base sequence (hereinafter, also referred to as methyl-specific specific real-time PCR). Alternatively, for example, the DNA sequence treated with bisulfite or the like is type II, and the cytosine is not methylated (base sequence in which all cytosines are peracil) and the base sequence Real-time PCR using a pair of non-methylation-specific primers and a probe selected from complementary nucleotide sequences (hereinafter, also referred to as non-methyl-specific-specific real-time PCR) is performed.

In the case of real-time PCR using methylation-specific primers and probes (the former), DNA is amplified and detected from DNA in which cytosine to be analyzed is methylated. On the other hand, in the case of real-time PCR using unmethylation-specific primers and probes (the latter), DNA is amplified and detected from DNA in which the cytosine to be analyzed is not methylenzyme. From the amounts of these amplification products, the amounts of methylated DNA and unmethylated DNA originally contained in the sample are determined. Next, using the following formula (amount of methylated DNA) / [(amount of methylated DNA) + (amount of methylated DNA)], the amount of methylated DNA is calculated. The ratio, ie, the methylation frequency, can be calculated. Alternatively, the absolute amount of the obtained DNA amount (that is, the amount of methylated DNA originally contained in the sample) obtained by performing only the real-time PCR specific to methyli-dani may be used as the methyleic frequency. . Here, as a primer and a probe, taking into account that unmethylated cytosine is converted to peracil and methylated cytosine is not converted to peracil, Primers (methylation-specific primers) and probes (methylation-specific probes) that are specific to the base sequence containing cytosine Design an unmethylation-specific primer and a probe (unmethylation-specific probe). Since the design is based on the DNA strand that has been chemically converted by bisulfite treatment and is no longer complementary, the methylation specificity is based on each strand of DNA that was originally double-stranded. Primers and probes and unmethylated specific primers and probes It can also be made. Such primers are preferably designed to include cytosine in CpG near the 3rd end of the primer to increase the specificity of methyl and non-methyl. More specifically, in the case of the present DNA, the primer can be designed as described above, for example, based on a base sequence containing one or more cytosines in CpG present in the base sequence of the present DM . For example, in the base sequence represented by SEQ ID NO: 1, base numbers 1, 9, 20, 30, 52, 63, 71, 80, 100, 121, 130, 133, 142, 146, 151, 153, 159, 167, 172 Alternatively, it can be designed based on a nucleotide sequence containing one or more cytosines represented by 174. More specifically, the following primers are preferred.

Methylation-specific primer>

Ml (forward primer): 5, -CGGTTGTTCGGAGTTTTATC-3 '(SEQ ID NO: 2)

M2 (reverse primer): 5, -GTAACGCTACCACGACCACG-3 '(SEQ ID NO: 3) Unmethylated specific primer>

U1 (Forward primer): 5'-TGGGAGTTTTATTGTTTAGGATT-3 '(SEQ ID NO: 4) U2 (reverse primer): 5, -AAAACTCACATAACACTACCACA-3' (SEQ ID NO: 5) Examples of the probe in the present invention include oligonucleotides Examples thereof include those in which a reporter fluorescent dye and a quencher fluorescent dye described below are bonded. The base sequence of the region on the probe that hybridizes with the amplification product from the present DNA is, for example, based on the base sequence containing one or more cytosines in CpG present in the base sequence of the present DNA. It can be designed as follows. For example, in the base sequence represented by SEQ ID NO: 1, base numbers 1, 9, 20, 30, 52, 63, 71, 80, 100, 121, 130, 133, 142, 146, 151, 153, 159, It can be designed based on a nucleotide sequence containing one or more cytosines represented by 167, 172 or 174. Specifically, a probe having the following nucleotide sequence as a nucleotide sequence of a region that hybridizes with an amplification product is preferable.

MP: 5'-CCCGAAAACAACGACTCCTCGAA-3 '(SEQ ID NO: 6) <Unmethylated specific probe>

UP: 5'-TCCCAAAAACAACAACTCCTCAAAA-3 '(SEQ ID NO: 7) In the DNA and amplification product of type III, the region where the probe hybridizes and the region where the forward primer or reverse primer anneals overlap each other. It is necessary to select a combination of primers and probes so that no In addition, in order to enable the amplification product obtained using the forward primer and the reverse primer to be detectable with the probe, the region where the probe hybridizes in the amplification product is the same as the forward primer and the reverse primer. Select the primer and probe combination so that they are between the regions to be annealed, respectively. The excitation light of the reporter fluorescent dye is not measured while it is absorbed by the quencher, and when the probe changes its structure due to hybridization to the amplification product and is not absorbed by the quencher, the excitation light is not measured. Be measured. For example, the probe used in the TaqMan (Registered Trademark) method is based on fluorescence resonance energy transfer when the reporter fluorochrome is bound to the same probe as the quencher fluorochrome. The fluorescence intensity is suppressed, and the fluorescence intensity is not suppressed in a state where the fluorescence intensity is not bound to the same probe as the quencher-fluorescent dye. As a reporter fluorescent dye, a fluorescein-based fluorescent dye such as FAM (6-potassium fluorescein) is preferable, and as a quencher-fluorescent dye, TAMRA (6-potency oxy-tetramethyl-rhodamine) is used. Such rhodamine-based fluorescent dyes are preferred. These fluorescent dyes are known and can be used since they are included in a commercially available detection kit for real-time PCR. The binding positions of the reporter fluorescent dye and the quencher fluorescent dye are not particularly limited. Usually, the reporter fluorescent dye is bound to one end (preferably, the 5 'end) of the probe, and the quencher fluorescent dye is bound to the other end. In addition, a method for binding a fluorescent dye to an oligonucleotide is known. For example, Noble et al., (1984) Nuc. Acids Res. 12: 3387-3403, Iyer et al., (1990) J. Am. Chem. Soc. 112: 1253-1254. The reaction solution for real-time PCR may be, for example, 50 ng of type III DNA, 200 nM of the above-mentioned forward primer and reverse primer at a final concentration of 200 nM, 200 μM of the above-described probe at a final concentration of ΙΟΟηΜ, and 200 μM of a final concentration of dNTP. μ μ, MgCl ₂ at a final concentration of ½Μ, 1.5 U of thermostable DNA polymerase, and 51 of 10X buffer (100 mM Tris-HC1 pH 8.3, 500raM KC1), and mix them. It is possible to increase the volume of the reaction solution by adding sterile ultrapure water. For the combination of primer and probe, in the case of methyl-specific real-time PCR, use a combination of methyl-specific primer and methylation-specific probe, and in the case of non-methyl-specific real-time PCR. For this, a non-methylation-specific primer and a non-methyl-specific probe are used in combination. As reaction conditions, for example, the above-mentioned reaction solution is kept at 95 ° C for 3 minutes, and then kept at 95 ° C for 15 seconds and then at 60 ° C for 60 seconds as one cycle. Conditions for performing the cycle are given. Performing such a reaction, the fluorescence intensity from the reaction solution is measured in real time. The real-time PCR method itself is known, and devices and kits for the real-time PCR method are commercially available. Therefore, the real-time PCR method can be performed using such commercially available devices and kits. In the reaction, amplification of DNA occurs by PCR using bisulfite-treated DNA as type II. If the DNA has a region that is complementary to the methylidani-specific primer (for example, if the cytosine portion contained in the region that anneals with the methylation-specific primer is completely methylated, If originally present in the sample), the DNA is amplified by the methylidani-specific primer. Furthermore, if the amplified DNA has a region that is complementary to the methylidani-specific probe (for example, the cytosine portion contained in the region that hybridizes with the methionylation-specific probe in the amplified DNA is completely methylmethylated) If the DNA that was originally present in the sample), the methylation-specific probe will hybridize to the amplified DNA in a single-stranded state. Next, for example, in real-time PCR by the TaqMan (registered trademark of Roche Molecular Systems) method, the single-stranded DNA is converted into type I DNA in a state where the methylation-specific probe is completely hybridized to the single-stranded DNA. When extension occurs, DN Due to the exonuclease activity of A polymerase, the methylidani-specific probe hybridized to the single-stranded DNA is hydrolyzed from the 5 ′ terminal side. As a result of this decomposition, the reporter-fluorescent dye and the quencher-fluorescent dye bound to the methylation-specific probe are separated, and suppressed by the fluorescence resonance energy transfer caused by the quencher-fluorescent dye. The fluorescence intensity from the reporter fluorescent dye increases. On the other hand, if the above DNA is not present in the sample, DNA amplification does not occur, and the methyl-specific probe does not hybridize to the DNA, and thus the methylation-specific probe is hydrolyzed by DNA polymerase. Not even. Thus, the fluorescence from the reporter fluorescent dye remains suppressed by the quencher fluorescent dye, and the fluorescent intensity does not increase. Therefore, it is possible to quantify the methylated present DNA present in the sample by measuring the fluorescence intensity. When quantifying the present DNA that has not been methylated, the unmethylated specific primer and probe may be used in place of the unmethylated primer and probe used in the above to obtain methylated DNA. It is possible to quantify this DNA that has not been used. In the evaluation method of the present invention, the fluorescence intensity is measured in real time. That is, the PCR reaction is performed while measuring the fluorescence intensity. The measured fluorescence intensity exceeds the lower detection limit after a certain number of cycles and increases rapidly. Then, as the amount of the target DNA in the sample is larger, the fluorescence intensity increases rapidly with a smaller number of cycles. Therefore, the target DNA in the sample can be quantified by examining the number of cycles after which the sudden increase in the fluorescence intensity starts. More specifically, for example, the threshold value is set to 10 times the standard deviation of the fluorescence intensity blur up to the 10th cycle, for example, where no rapid increase can occur even if 108 target DNAs are contained. Then, by determining the number of cycles in which the fluorescence intensity exceeds this threshold value, it is possible to accurately quantify the methyl-enriched DNA of the sample. That is, when the horizontal axis is the common logarithm of the number of methyl-enriched DNAs in the sample and the vertical axis is the number of cycles when the above threshold is exceeded, the measurement results are almost completely on a straight line. By preparing a calibration curve, it is possible to quantify the methylated DNA of the sample by examining how many cycles the threshold is exceeded. Therefore, according to the evaluation method of the present invention, the conventional PCR As described above, there is no need to perform an operation to check the amplification of DNA by electrophoresis of the reaction solution after PCR, which is very simple. If a probe is used in addition to the primer, the noise can be reduced, and it can be applied to samples derived from elderly people with high accuracy. Using the above method, the methylation frequency of the DNA having the base sequence shown in SEQ ID NO: 1 or its equivalent contained in the human-derived specimen is measured. The measured methylation frequency and, for example, the DNA having the base sequence represented by SEQ ID NO: 1 contained in a healthy human-derived sample that can be diagnosed as having no cancer cells such as breast cancer cells, or an equivalent thereof. And the degree of canceration of the specimen is determined based on the difference obtained by the comparison. If the methylation frequency of the DNA having the nucleotide sequence of SEQ ID NO: 1 or its equivalent contained in a human-derived specimen is higher than that of the control (DNA having the nucleotide sequence of SEQ ID NO: 1 or If the equivalent is in a highly methylated state compared to the control), it can be determined that the specimen has a higher degree of canceration compared to the control.

Here, the term “degree of canceration” has the same meaning as generally used in the art. Specifically, for example, when a human-derived specimen is a cell, it means the degree of malignancy of the cell. Further, for example, when a human-derived specimen is a tissue, it means the abundance of cancer cells in the fibrous tissue or the individual that is the source of the specimen. Incidentally, for example, when a human-derived specimen is blood, plasma, or serum, the “degree of canceration” may be considered as the degree of possibility that the individual from which the specimen originated has cancer. In the evaluation method of the present invention, primers and probes that can be used in the real-time PCR method for measuring the methylation frequency of the DNA having the base sequence represented by SEQ ID NO: 1 or an equivalent thereof are cancer cells such as breast cancer cells. It is useful as a reagent for detecting kits. The present invention also provides a kit for detecting cancer cells such as breast cancer cells containing these primers and probes as reagents, and the scope of the evaluation method of the present invention utilizes the substantial principle of the method. It also includes use in the form of a detection kit as described above. Example

Hereinafter, the power S for describing the present invention in detail by examples, the present invention is not limited to these.

Example 1 (Confirmation test of methylation status of DNA or its equivalent having nucleotide sequence shown in SEQ ID NO: 1 in blood of early-stage breast cancer patient)

2 mL of plasma was obtained from 30 stage I and II breast cancer patients and 35 healthy subjects (informed consent was implemented). DNA in 2 mL of plasma was extracted using QIAamp DNA Blood Midi Kit (QIAGEN). The total amount of DNA obtained by the extraction was determined by Clark et al., Nucl. Acids Res., 22, 2990-2997, 1994; Herman et al., Proc. Natl. Acad. Sci. USA, 93, 9821-9826, 1996. The product was treated with sodium bisulfite according to the method described in (1). That is, the above DNA is dissolved in TE buffer to prepare a 15 / zl genomic DNA solution, about 2 μl of 6M sodium hydroxide is added thereto, and the mixture is left at 37 ° C for 15 minutes. did. 9 μl of lOmM hydroquinone (Sigma) and 120 l of 3.6N sodium bisulfite (Sigma) are added to the mixture, and the cycle is 95 ° C for 30 seconds and 50 ° C for 15 minutes. The incubation was performed for 15 cycles. DNA was purified from the incubated solution using a Wizard DNA clean-up system (Promega). After adding 5 μl of 6M sodium hydroxide to the purified DNA solution (about 501 TE buffer solution), the mixture was left at room temperature for 5 minutes. Then, the precipitate (DNA) was recovered by precipitating the left mixture with ethanol. The collected precipitate was suspended in 10/1 buffer.

The obtained DNA was designated as type III, and the methylation-specific primers Ml and M2 shown below and the 5 'end were labeled with the reporter fluorescent dye FAM (6-potassium fluorescein), and the end was quenched. Real-time PCR was performed using a methylation-specific probe MP labeled with a fluorescent dye, TAMRA (6-carboxy-tetramethyl-rhodamine). In this case, a 172 bp DNA corresponding to the base sequence of base numbers 1 to 172 of the base sequence represented by SEQ ID NO: 1 after bisulfite treatment is amplified. That is, in the base sequence represented by SEQ ID NO: 1, base numbers 1, 9, 20, 63, 71, 80, 153, 159, 167 172 bp DNA is specifically amplified from the present DNA in which all the cytosines represented by 172 and 172 are methylated.

Methylation-specific primers>

Ml (forward primer): 5, -CGGTTGTTCGGAGTTTTATC-3, (SEQ ID NO: 2)

M2 (Repurse primer): 5'-GTAACGCTACCACGACCACG-3 '(SEQ ID NO: 3)

MP: 5'-CCCGAAAACAACGACTCCTCGAA-3 '(SEQ ID NO: 6)

The reaction solution for PCR includes l ^ L for the DNA to be type II, 200 nM each for the above primer at a final concentration, MηΜ for the above probe for a final concentration, and 200 M for the final concentration for dNTP. mixed MgCl ₂ and 4mM final concentration, Platinum Taq DNA polymerase (Platinum is a registered trademark of Invitrogen) and 1. 5U a, 10 X buffer and 5 1 (lOOmM Tris-HCl pH 8. 3 , 500mM KCl) Then, sterilized ultrapure water was added thereto to adjust the liquid volume to 50 // l. After incubating the reaction solution at 95 ° C for 3 minutes, real-time PCR was performed under the conditions that 55 cycles of 95 ° C for 15 seconds and 60 ° C for 60 seconds as one cycle were performed. As a device for real-time PCR, an iCycler iQ Real-Time Detection System (Bio-Rad) was used. In order to confirm that the methylation-specific PCR primer and the methylation-specific probe specifically amplify only the methylated present DNA, normal human mammary epithelial cells (HMEC, Clonetics) were used. genomic DNA in the usual way from: extracting (1 negative control), the portion of the treated genomic DNA by methylation enzyme SSSL (NEB Co.) - C _P G_ all were methylation (2: positive Control). This untreated genomic DNA (1: negative control) and methylated DNA (2: positive control) were subjected to bisulfite treatment in the same manner as described above, and real-time PCR was performed using the obtained DNA as type III. . As a result, no amplification product was obtained with untreated DNA, but only with methylated DNA. Based on this fact, it was confirmed that the methyl-specific primer for PCR and the methyl-specific probe were specifically amplified only for the methylated DNA. Also, use methylated DNA (2: positive control)! /, A calibration curve was created. That is, first, a PCR product obtained by amplifying DNA by PCR was purified. Molecules in PCR reaction solution Was measured using an absorption altimeter, and the number of molecules in the solution was calculated from the predicted molecular weight of the PCR product. Based on the number of molecules, a PCR containing 10, 100, 1000, 10,000, 100,000, 1,000,000, 10,000,000,000, or 100,000,000 copies of type I PCR product A reaction solution was prepared and used for real-time PCR under a fluorescence monitor. In real-time PCR, the number of cycles in which the fluorescence intensity from the PCR reaction solution exceeds the threshold value is checked. The number of cycles at the time was plotted on the vertical axis, and a calibration curve was created. After creating this calibration curve, the samples were subjected to real-time PCR and the number of cycles in which the fluorescence intensity exceeded the threshold was determined. From the obtained results, the amount of the methylated present DNA was calculated using a calibration curve.

Tables 1 and 2 show the results of examining the frequency of methylation in the DNA having the nucleotide sequence shown in SEQ ID NO: 1 or its equivalent contained in a specimen (plasma) derived from a breast cancer patient. 2 As shown in Tables 1 and 2, 9 out of 30 breast cancer patients (30%) were able to quantify the methylated DNA. In healthy subjects, only 1 out of 35 persons could quantitate the methylated DNA, and the amount was 55.3 copies / 0.2 mL plasma equivalent. In other words, if the present DNA contained in the sample is methylated (if amplification products are obtained by methyl-specific real-time PCR), it is highly likely that the individual from which the sample originated has breast cancer. was gotten. In these breast cancer patients, the tumor markers CEA (normal value at 5 ng / mL or less) and CA15-3 (normal value at 28 U / mL or less) were measured. False positives were shown, and all of CA15-3 were negative. Therefore, it was suggested that the evaluation method of the present invention may have higher sensitivity than the currently commonly used tumor markers.

Specimen Age Age Amount of this DNA that has been breached CEA CAI5-3 ヽ (Kohichi ヽ

/0.2 ml blood equivalent) ig / ml) (U / ml)

BBOOl 46 0

BB002 40 0

BB003 58 0

BB004 60 0

BB005 51 4400

BB006 48 248

BB007 56 0

BB008 57 27. 3

BB009 50 4.72

BBOIO 54 0

BBOll 44 0 5.D

BB012 46 0

BB013 59 0

BB014 52 0

BB015 34 0

Table 2

The above results indicate that the subject is highly likely to have breast cancer if the DNA contained in the sample is methylated (if amplification products are obtained by methylich-specific real-time PCR). Was done. Industrial potential

The present invention, and a method for assessing a cancerous state of the specimen from human is possible to provide _t Sequence Listing Free Text

SEQ ID NO: 2 Oligonucleotide primer designed for real-time PCR SEQ ID NO: 3

Oligonucleotide primer designed for real-time PCR SEQ ID NO: 4

Oligonucleotide primers designed for real-time PCR SEQ ID NO: 5

Oligonucleotide primers designed for real-time PCR SEQ ID NO: 6

Oligonucleotide probe designed for real-time PCR SEQ ID NO: 7

Oligonucleotide probes designed for real-time PCR

Claims

The scope of the claims

(1) a first step of measuring the methylation frequency of DNA having the nucleotide sequence shown in SEQ ID NO: 1 or its equivalent contained in a human-derived specimen by real-time PCR, and

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

An evaluation method comprising: 2. The evaluation method according to claim 1, wherein the human-derived specimen is a cell.

3. The evaluation method according to claim 1, wherein the human-derived specimen is a tissue.

4. The evaluation method according to claim 1, wherein the human sample is blood, plasma or serum.

5. The evaluation method according to claim 3, wherein the human-derived specimen is breast tissue, breast tissue or breast epithelial tissue, and the cancer is breast cancer.

6. The methylation frequency is the cytosine methylation frequency in one or more nucleotide sequences represented by 5'-CG-3 'present in the nucleotide sequence of the DNA. Evaluation method described.

7. The evaluation method according to claim 6, wherein the human-derived specimen is blood, plasma, or serum.

8. The evaluation method according to claim 6, wherein the human-derived specimen is a breast tissue, a mammary gland tissue or a mammary gland epithelial tissue, and the cancer is breast cancer.

9. Use of methylated DNA having the nucleotide sequence of SEQ ID NO: 1 or an equivalent thereof as a marker.

10. The use according to claim 9, wherein the cancer marker is a breast cancer marker.

1 1. Ability to measure methylation frequency by real-time PCR After contacting DNA prepared from a sample with a reagent that modifies non-methylated cytosine, the DNA is designated as 铸 and the nucleotide sequence represented by SEQ ID NO: 1 PCR is performed using a pair of primers capable of distinguishing the presence or absence of cytosine methylation in the DNA or its equivalent, and the amount of the resulting amplification product is determined by the DNA having the nucleotide sequence represented by SEQ ID NO: 1 or 2. The evaluation method according to claim 1, wherein the evaluation is performed by measuring in real time using a probe capable of discriminating the presence or absence of cytosine methylation in the equivalent.

12. The forward primer is an oligonucleotide having the nucleotide sequence of SEQ ID NO: 2, the reverse primer is an oligonucleotide having the nucleotide sequence of SEQ ID NO: 3, and the probe is a nucleotide having the nucleotide sequence of SEQ ID NO: 6. 2. The evaluation method according to claim 1, wherein the evaluation method is an oligonucleotide having a sequence.

13. The forward primer is an oligonucleotide having the nucleotide sequence of SEQ ID NO: 4, the reppers primer is an oligonucleotide having the nucleotide sequence of SEQ ID NO: 5, and the probe is represented by SEQ ID NO: 7. 2. The evaluation method according to claim 1, wherein the evaluation method is an oligonucleotide having a base sequence.

14. A kit for detecting a cancer cell, comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 2, an oligonucleotide having the nucleotide sequence of SEQ ID NO: 3, and a nucleotide sequence of SEQ ID NO: 6 A kit comprising an oligonucleotide.

15. A kit for detecting a cancer cell, comprising an oligonucleotide having the nucleotide sequence of SEQ ID NO: 4, an oligonucleotide having the nucleotide sequence of SEQ ID NO: 5, and a nucleotide sequence of SEQ ID NO: 7 A kit comprising an oligonucleotide.