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WO2004018668A1 - Procede d'evaluation du degre de cancerisation d'un prelevement d'origine humaine - Google Patents

Procede d'evaluation du degre de cancerisation d'un prelevement d'origine humaine Download PDF

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WO2004018668A1
WO2004018668A1 PCT/JP2003/010480 JP0310480W WO2004018668A1 WO 2004018668 A1 WO2004018668 A1 WO 2004018668A1 JP 0310480 W JP0310480 W JP 0310480W WO 2004018668 A1 WO2004018668 A1 WO 2004018668A1
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dna
seq
nucleotide sequence
oligonucleotide
evaluation method
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Japanese (ja)
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Toshikazu Ushijima
Toshio Takada
Kazuaki Miyamoto
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Sumitomo Chemical Co Ltd
National Cancer Center Japan
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Sumitomo Chemical Co Ltd
National Cancer Center Japan
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • the present invention relates to a method for evaluating the degree of canceration of a human-derived specimen, and the like.
  • 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.
  • 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 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;
  • the human-derived specimen is breast tissue, mammary gland tissue or mammary gland epithelial tissue, and the cancer is breast cancer;
  • 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 wherein the presence or absence of cytosine methylation in DNA or its equivalent is measured in real time using a probe capable of discriminating;
  • 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
  • the probe is a nucleotide having the nucleotide sequence of SEQ ID NO: 6.
  • the forward primer is an oligonucleotide having the nucleotide sequence of SEQ ID NO: 4
  • the reverse primer is an oligonucleotide having the nucleotide sequence of SEQ ID NO: 5.
  • 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 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
  • 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
  • exon 1 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.
  • mammals there is a phenomenon in which, of the four types of bases that make up a gene (genomic DNA), only cytosine is methylated.
  • genomic DNA 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.
  • C represents cytosine
  • G represents guayun.
  • the base sequence may be referred to as CpG.
  • CpG. Limited to cytosine.
  • the site of methylation in cytosine is at position 5.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ -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.
  • 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.
  • 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.
  • 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.
  • a reagent for modifying unmethylated cytosine for example, bisulfite such as sodium hydrogen sulfite can be used.
  • bisulfite such as sodium hydrogen sulfite
  • a reagent that specifically modifies only methylated cytosine may be used instead of a reagent that modifies unmethylated cytosine.
  • 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.
  • 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.
  • the nucleotide sequence [methylated cytosine (cytosine in CpG) remains cytosine
  • the unmethylated cytosine 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).
  • 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.
  • the ratio, ie, the methylation frequency can be calculated.
  • 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.
  • 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).
  • 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 .
  • 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
  • base numbers 1, 9, 20, 30, 52, 63, 71, 80 100, 121, 130, 133, 142, 146, 151, 153, 159, 167, 172
  • the following primers are preferred.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • TAMRA 6-potency oxy-tetramethyl-rhodamine
  • 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.
  • 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 ⁇ M of the above-described probe at a final concentration of ⁇ , and 200 ⁇ M of a final concentration of dNTP.
  • 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.
  • amplification of DNA occurs by PCR using bisulfite-treated DNA as type II.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the noise can be reduced, and it can be applied to samples derived from elderly people with high accuracy.
  • 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 degree of canceration of the specimen is determined based on the difference obtained by the comparison.
  • 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.
  • 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.
  • 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 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)
  • the above DNA is dissolved in TE buffer to prepare a 15 / zl genomic DNA solution, about 2 ⁇ l of 6M sodium hydroxide is added thereto, and the mixture is left at 37 ° C for 15 minutes. did. 9 ⁇ l 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).
  • 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).
  • TAMRA 6-carboxy-tetramethyl-rhodamine
  • 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.
  • 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, M ⁇ 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.
  • 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. .
  • no amplification product was obtained with untreated DNA, but only with methylated DNA.
  • the methyl-specific primer for PCR and the methyl-specific probe were specifically amplified only for the methylated DNA.
  • use methylated DNA (2: positive control)! / A calibration curve was created.
  • 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.
  • 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.
  • 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.
  • the tumor markers CEA normal value at 5 ng / mL or less
  • CA15-3 normal value at 28 U / mL or less
  • 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
  • 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 designed for real-time PCR

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Abstract

L'invention concerne un procédé d'évaluation du degré de cancérisation d'un prélèvement d'origine humaine, qui se caractérise par: 1) une première étape consistant à mesurer, par la méthode d'amplification par la polymérase (PCR), la fréquence de méthylation dans un ADN dont la séquence de bases est représentée par SEQ ID NO: 1 ou son équivalent contenu dans le prélèvement d'origine humaine; et 2) une seconde étape consistant à: comparer la fréquence de méthylation ainsi mesurée à celle d'une régulation, puis déterminer le degré de cancérisation du prélèvement sur la base de la différence constatée.
PCT/JP2003/010480 2002-08-23 2003-08-20 Procede d'evaluation du degre de cancerisation d'un prelevement d'origine humaine Ceased WO2004018668A1 (fr)

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AU2003257587A AU2003257587A1 (en) 2002-08-23 2003-08-20 Method of evaluating degree of canceration of human-origin specimen

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002018632A2 (fr) * 2000-09-01 2002-03-07 Epigenomics Ag Procede de determination du degre de methylation de cytosines determinees d'adn genomique dans le contexte sequentiel 5'-cpg-3'
WO2002018631A2 (fr) * 2000-09-01 2002-03-07 Epigenomics Ag Diagnostic de maladies declarees ou de la predisposition a certaines maladies
WO2002060318A2 (fr) * 2001-01-31 2002-08-08 The Ohio State University Research Foundation Detection de sequences a forte teneur en cpg methyle pour le diagnostic de cellules malignes
WO2003018840A1 (fr) * 2001-08-23 2003-03-06 Sumitomo Chemical Company, Limited Evaluation du niveau de cancerisation d'un prelevement mammalien

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002018632A2 (fr) * 2000-09-01 2002-03-07 Epigenomics Ag Procede de determination du degre de methylation de cytosines determinees d'adn genomique dans le contexte sequentiel 5'-cpg-3'
WO2002018631A2 (fr) * 2000-09-01 2002-03-07 Epigenomics Ag Diagnostic de maladies declarees ou de la predisposition a certaines maladies
WO2002060318A2 (fr) * 2001-01-31 2002-08-08 The Ohio State University Research Foundation Detection de sequences a forte teneur en cpg methyle pour le diagnostic de cellules malignes
WO2003018840A1 (fr) * 2001-08-23 2003-03-06 Sumitomo Chemical Company, Limited Evaluation du niveau de cancerisation d'un prelevement mammalien

Non-Patent Citations (1)

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Title
MIYAMOTO K. ET AL.: "Methylation-associated silencing of heparan sulfate D_glucosaminyl 3-O-sulfotransferase-2 (3-Ost-2) in human breast, colon, lung and pancreatic cancers", ONCOGENE, vol. 22, no. 2, 2003, pages 274 - 280, XP002973975 *

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