Methods for detecting colorectal cancer

Abstract

The present invention relates to a method for providing information about the diagnosis of colorectal cancer, a composition for diagnosing colorectal cancer and a kit containing the composition, and more particularly to: a method for providing information about the diagnosis of colorectal cancer by using primers that specifically amplify multiple methylated colorectal cancer marker genes; a composition for diagnosing colorectal cancer; and a kit containing the composition.

Description

Technical Field

[0001] The present invention relates to a method for providing information about diagnosing colorectal cancer, a composition for diagnosing colorectal cancer, and a kit comprising the composition, and more particularly to a method for providing information about diagnosing colorectal cancer using primers that specifically amplify multiple methylated colorectal cancer marker genes, a composition for diagnosing colorectal cancer, and a kit comprising the composition. Background Art

[0002] In the genomic DNA of mammalian cells, in addition to A, C, G and T, there is a fifth base, 5-methylcytosine (5-mC), in which a methyl group is attached to the fifth carbon of the cytosine ring. 5-mC is always attached only to the C of the CG dinucleotide (5'-mCG-3'), and this CG is usually expressed as CpG. Most of the C in CpG is methylated by the methyl group to which it is attached. This methylation of CpG inhibits the expression of repetitive sequences such as Alu or transposons in the genome, and CpG is the most common site of extragenic changes in mammalian cells. The 5-mC of this CpG is naturally converted to T by deamination. Therefore, CpG only appears at a frequency of 1% in the mammalian genome, which is much lower than its normal frequency (1 / 4x1 / 4=6.25%).

[0003] There are regions called CpG sites (CpG islands) where CpG is very dense. CpG sites are 0.2-3kb in length and are highly concentrated regions where the percentage of C and G base distribution is greater than 50% and the percentage of CpG distribution is more than 3.75%. About 45,000 CpG sites appear in the entire human genome and are concentrated in the promoter region that regulates gene expression. In fact, CpG sites appear in the promoters of housekeeping genes, which account for about half of human genes (Cross S. et al., Curr. Opin. Gene Develop., 5:309, 1995). It is known that abnormal DNA methylation mainly occurs in the 5' regulatory region of the corresponding gene, thereby reducing the expression of the corresponding gene.

[0004] On the other hand, in normal human somatic cells, the CpG islands in the promoter regions of these housekeeping genes are not methylated, but the imprinted genes and inactivated genes on the X chromosome are methylated, thereby preventing the expression of the imprinted genes and inactivated genes during development.

[0005] During carcinogenesis, promoter CpG islands become methylated and the expression of the corresponding genes is impaired. In particular, when CpG islands in the regulatory regions of tumor suppressor genes are methylated, the expression and function of these genes are blocked (like coding sequence mutations), thereby promoting the occurrence and development of cancer. The tumor suppressor genes regulate the cell cycle or apoptosis, repair DNA, participate in cell adhesion and cell-to-cell interactions, and inhibit invasion and metastasis. Partial methylation may also occur on CpG islands due to aging.

[0006] Methylation of regulatory regions of tumor-related genes is an important indicator of cancer and can therefore be used in a variety of ways, such as diagnosis and early diagnosis of cancer, prediction of cancer risk, prediction of cancer prognosis, follow-up after treatment, prediction of response to anticancer therapy, etc. In fact, recent efforts have been actively made to study promoter methylation of tumor-related genes in blood, sputum, saliva, feces, urine, etc., and to use the results for the treatment of various types of cancer (Ahlquist, DA et al., Gastroenterol., 119:1219, 2000).

[0007] Against this technical background, the inventors of the present application determined that primers that specifically amplify multiple methylated colorectal cancer marker genes can be used to detect methylation of colorectal cancer marker genes with high detection limits and accuracy, thereby completing the present invention. Summary of the invention

[0008] An object of the present invention is to provide a method for providing information on diagnosing colorectal cancer using primers that specifically amplify a plurality of methylated colorectal cancer marker genes.

[0009] Another object of the present invention is to provide a composition for diagnosing colorectal cancer using primers that specifically amplify multiple methylated colorectal cancer marker genes.

[0010] Still another object of the present invention is to provide a kit for diagnosing colorectal cancer comprising the composition.

[0011] In order to achieve the above-mentioned purpose, the present invention provides a method for providing information about diagnosing colorectal cancer, which comprises (a) treating a sample with at least one reagent, wherein the at least one reagent differently modifies methylated SDC2 (synthetic proteoglycan 2) and ADHFE1 (iron-containing alcohol dehydrogenase 1) genes and unmethylated SDC2 and ADHFE1 genes; and (b) treating the sample with primers that specifically amplify the methylated SDC2 and ADHFE1 genes.

[0012] In addition, the present invention provides a composition for diagnosing colorectal cancer, which comprises at least one reagent that differently modifies methylated SDC2 (syndecan 2) and ADHFE1 (iron-containing alcohol dehydrogenase 1) genes and unmethylated SDC2 and ADHFE1 genes, and primers that specifically amplify the methylated SDC2 and ADHFE1 genes.

[0013] In addition, the present invention provides a kit for diagnosing colorectal cancer comprising the composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The results of genome-wide methylation analysis (MeDIA-CpG microarray) using DNA from cancer tissues and adjacent normal tissues of patients undergoing colorectal cancer surgery are shown;

[0015] Figure 2 The results of amplification of the ADHFE1 gene using methylation-specific primers are shown;

[0016] Figure 3 shows the results of amplification of the ADHFE1 gene using methylation-specific primers; and

[0017] Figure 4 The results of diagnosing colorectal cancer by detecting methylation of the SDC2 gene and methylation of the ADHFE1 gene in normal subjects and colorectal cancer patients are shown. DETAILED DESCRIPTION

[0018] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Generally, the nomenclature used herein is well known and typical in the art.

[0019] The inventors of the present application have determined that the use of primers that specifically amplify multiple methylated colorectal cancer marker genes can improve the sensitivity and specificity of methylated DNA detection compared to the degree of methylation detection based on a single colorectal cancer marker gene. In a specific embodiment according to the present invention, the sensitivity and specificity of colorectal cancer diagnosis are improved by detecting the methylation of the SDC2 gene and the ADHFE1 gene compared to the methylation detection of the SDC2 gene, thereby being highly useful in diagnosing colorectal cancer.

[0020] Therefore, one aspect of the present invention relates to a method for providing information about diagnosing colorectal cancer, the method comprising (a) treating a sample with at least one reagent that differently modifies methylated SDC2 (syndecan 2) and ADHFE1 (ferroalcohol dehydrogenase 1) genes and unmethylated SDC2 and ADHFE1 genes; and (b) treating with primers that specifically amplify the methylated SDC2 and ADHFE1 genes.

[0021] In the present invention, step (a) is to treat the sample with at least one agent that differently modifies the methylated SDC2 and ADHFE1 genes and the unmethylated SDC2 and ADHFE1 genes.

[0022] As used herein, the term "methylation" means modification to 5-methylcytosine (5-mC), in which a methyl group is attached to the fifth carbon of the cytosine base ring, and 5-methylcytosine is always attached only to the C of the CG dinucleotide (5'-mCG-3'), and this CG is generally referred to as CpG. Methylation of CpG inhibits the expression of repetitive sequences such as Alu or transposons in the genome, and CpG is the site where extragenic changes most frequently occur in mammalian cells. The 5-mC of this CpG is naturally converted to T by deamination, and therefore, CpG is present at a frequency of only 1% in the mammalian genome, which is much lower than its normal frequency (1 / 4x1 / 4=6.25%).

[0023] There are regions called CpG islands where CpGs are very dense. CpG islands are 0.2-3kb in length and are highly concentrated sites where the percentage of C and G base distribution is greater than 50% and the percentage of CpG distribution is above 3.75%. About 45,000 CpG islands appear throughout the human genome and are concentrated in promoter regions that regulate gene expression. CpG islands actually appear in the promoters of housekeeping genes, which account for about half of human genes.

[0024] The nucleic acid isolated from the sample is obtained from a biological sample of the sample. In order to diagnose colorectal cancer or the progression of colorectal cancer, nucleic acids must be isolated from colorectal tissue by scraping or biopsy. Such samples can be obtained by various medical procedures known in the art.

[0025] The degree of methylation of nucleic acid of a sample obtained from the sample is measured by comparing the same portion of nucleic acid from a sample without colorectal tissue cell growth abnormalities. Hypermethylation indicates the presence of a methylated allele in at least one nucleic acid. When the same nucleic acid is tested in a sample without colorectal tissue cell growth abnormalities, the methylated allele does not appear.

[0026] A "normal" cell is a cell that does not display abnormal cell morphology or changes in cytological properties. A "tumor" cell is a cancer cell, while a "non-tumor" cell is a cell that is part of diseased tissue but is not a tumor site.

[0027] According to the present invention, by determining the methylation stage of at least one nucleic acid isolated from the sample, an early diagnosis of abnormal cell growth in the colorectal tissue of the sample can be performed. The methylation stage of the at least one nucleic acid can be compared with the methylation stage of at least one nucleic acid isolated from a sample that does not exhibit abnormal colorectal tissue cell growth. Preferably, the nucleic acid is a CpG-containing nucleic acid, such as a CpG island.

[0028] According to the present invention, a predisposition for abnormal cell growth in colorectal tissue of a sample can be diagnosed, comprising determining the methylation of at least one nucleic acid isolated from the sample. The methylation stage of the at least one nucleic acid can be compared with the methylation stage of at least one nucleic acid isolated from a sample without a predisposition for abnormal cell growth in colorectal tissue.

[0029] As used herein, the term "predisposition" refers to the property of being susceptible to the above-mentioned abnormal cell growth. A sample with a predisposition is a sample that has not yet exhibited abnormal cell growth but in which abnormal cell growth exists or in which the possibility of abnormal cell growth occurring is increased.

[0030] The presence of CpG methylation in the target DNA may be an indicator of a disease, and, for example, CpG methylation of any one of a promoter, a 5' untranslated region, and an intron of the target DNA may be measured.

[0031] Genes containing CpG are usually DNA. However, the method of the present invention can be performed using a sample containing, for example, DNA or DNA and RNA (including mRNA), wherein the DNA or RNA can be single-stranded or double-stranded, or a sample containing a DNA-RNA hybrid can be used.

[0032] Nucleic acid mixtures can also be used. As used herein, the term "multiple" includes the situation where there are multiple specific nucleic acid sequence sites to be detected in a gene and the situation where multiple target DNA sequences are included in one tube (single reactor). The specific nucleic acid sequence to be detected can be a part of a macromolecule, or can initially exist in the form of discrete molecules, wherein the specific sequence constitutes the entire nucleic acid sequence. Nucleic acid sequences are not necessarily nucleic acids that exist in pure form, and nucleic acids can be a small part in a complex mixture, such as the one included in the total DNA of a person.

[0033] In particular, the present invention relates to detecting methylation of multiple target DNA sequences in a sample in a single reactor, wherein the sample may include multiple target DNA sequences, and any target DNA can be used without limitation as long as it is a gene and a control gene that affects the occurrence or development of colorectal cancer when its expression is inhibited due to abnormal methylation.

[0034] In the present invention, the sample may be derived from a human body, and the sample may include, for example, colorectal cancer tissue, cells, feces, urine, blood, serum or plasma.

[0035] At least one reagent that differently modifies methylated DNA and non-methylated DNA can be used without limitation, as long as it can distinguish between non-methylated cytosine bases and methylated cytosine bases, and examples of the reagent may include, but are not limited to, bisulfite, acid sulfite, disulfite, and a combination thereof. In particular, when treated with the reagent, methylated cytosine bases are not converted, while non-methylated cytosine bases can be converted to uracil or bases other than cytosine.

[0036] In the present invention, step (b) is to perform treatment using primers that specifically amplify the methylated SDC2 gene and the methylated ADHFE1 gene.

[0037] For example, for primers, forward and reverse primers can be paired and used for PCR at the same time. The methylated "C" of the CpG region must be located at the 3' end of the primer, thereby showing high specificity of methylation detection. The forward primer for specifically amplifying the methylated ADHFE1 gene may include, for example, a sequence of SEQ ID NO: 1 or 4. The reverse primer may include, for example, a sequence of SEQ ID NO: 2 or 5. The primer for specifically amplifying the methylated SDC2 gene may include a sequence of SEQ ID NO: 10 or 11.

[0038] The present invention may further include treating with a probe capable of complementary hybridization with each of the methylated SDC2 gene and the methylated ADHFE1 gene specifically amplified using the primers in step (b).

[0039] In hybridization reactions, the conditions for achieving a specific stringency level vary depending on the characteristics of the nucleic acids to be hybridized. For example, the length of the nucleic acid sites to be hybridized, the degree of homology, the nucleotide sequence composition (e.g., GC / AT ratio), and the nucleic acid type (e.g., RNA, DNA) are considered when selecting hybridization conditions. Another consideration is whether the nucleic acid is fixed on, for example, a filter, etc.

[0040] Examples of very stringent conditions are as follows: 2X SSC / 0.1% SDS at room temperature (hybridization conditions), 0.2XSSC / 0.1% SDS at room temperature (low stringency conditions), 0.2X SSC / 0.1% SDS at 42° (medium stringency conditions), and 0.1X SSC at 68°C (high stringency conditions). The washing process can be performed using any of these conditions, and for example, high stringency conditions or any of the above conditions can be used. These conditions can be applied in the above order for 10 to 15 minutes each time, or all or some of the above conditions can be repeatedly applied. However, as described above, the optimal conditions vary depending on the specific hybridization reaction involved and can be determined experimentally. Typically, high stringency conditions are used for hybridization of the probe of interest.

[0041] The probe capable of complementarily hybridizing to the amplified methylated SDC2 gene may include, for example, the sequence of SEQ ID NO: 3 or 6. The probe capable of complementarily hybridizing to the amplified methylated ADHFE1 gene may include the sequence of SEQ ID NO:12.

[0042] In some cases, the probe can be detectably labeled and can be labeled with, for example, a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelate or an enzyme. Suitable labeling of the probe as described above is a technique well known in the art and can be performed by typical methods.

[0043] The amount of the amplified product can be detected based on the fluorescent signal. The detection method may include an intercalation method using an intercalator that exhibits fluorescence by binding to the double-stranded DNA of the amplified product to which the probe binds, a method using an oligonucleotide in which the 5' end is labeled with a fluorescent material and the 3' end is labeled with a quencher, etc.

[0044] Amplification according to the present invention can be carried out by real-time quantitative amplification (e.g., real-time polymerase chain reaction (PCR)), and in real-time PCR, the amount of PCR amplification product can be detected using a fluorescent signal. As real-time PCR proceeds, the intensity of the fluorescent signal increases proportionally with the increase in the amount of polynucleotides, and an amplification characteristic curve showing the intensity of the fluorescent signal (depending on the number of amplification cycles) is obtained.

[0045] In general, the amplification characteristic curve is divided into: a baseline region, which shows a background fluorescence signal that basically does not reflect the amount of polynucleotides; an exponential region, in which the fluorescence signal increases with the increase in the amount of polynucleotide products; and a platform region, in which PCR reaches saturation and therefore the intensity of the fluorescence signal no longer increases.

[0046] Typically, the fluorescence signal intensity at the transition point from the baseline region to the exponential region (i.e., the point when the amount of PCR amplification product reaches a fluorescently detectable amount) is called the threshold, and the amplification cycle number corresponding to the threshold on the amplification characteristic curve is called the threshold cycle (Ct) value.

[0047] Methylation-specific sensitivity and / or specificity can be determined by measuring Ct values, analyzing a standard curve in which concentrations are determined based on Ct (threshold cycle) values ​​of standard materials, and confirming the concentrations of amplified genes.

[0048] In one embodiment, methylation can be detected using any method selected from the following: PCR, methylation-specific PCR, real-time methylation-specific PCR, PCR using a methylated DNA-specific binding protein, PCR using a methylated DNA-specific binding antibody, quantitative PCR, gene chip, sequencing, sequencing by synthesis, and sequencing by ligation.

[0049] (1) Methylation-specific PCR: For detection by methylation-specific PCR, when treated with bisulfite, the cytosine in the 5'-CpG'-3 region remains cytosine in the case of methylation, and is converted into uracil in the case of non-methylation. Therefore, for the modified nucleotide sequence obtained after treatment with bisulfite, primers corresponding to the region in which the 5'-CpG-3' nucleotide sequence exists can be constructed. When PCR is performed using the primers, in the case of methylation, since primers corresponding to the methylated nucleotide sequence are used, PCR products are generated, and methylation can be confirmed by agarose gel electrophoresis. Here, the methylation detection probe can be TaqMan, a molecular beacon, or a probe having a self-reporting function or an energy transfer labeling function, but is not limited thereto.

[0050] (2) Real-time methylation-specific PCR: Real-time methylation-specific PCR is a real-time measurement method improved from methylation-specific PCR, and includes treating genomic DNA with bisulfite, designing PCR primers corresponding to the methylation status, and using these primers to perform real-time PCR. Here, there are two detection methods: a detection method using a TaqMan probe complementary to the amplified nucleotide sequence and a detection method using SYBR Green. Therefore, real-time methylation-specific PCR is able to selectively and quantitatively analyze only methylated DNA. In this way, a standard curve is created using an in vitro methylated DNA sample, and a gene amplification without a 5'-CpG-3' sequence in the nucleotide sequence is also used as a standardized negative control group, thereby quantitatively analyzing the degree of methylation.

[0051] (3) PCR, quantitative PCR and DNA chip assay using methylated DNA-specific binding protein: In the PCR or DNA chip method using methylated DNA-specific binding protein, after a protein that specifically binds only to methylated DNA is mixed with DNA, the protein specifically binds only to methylated DNA, thereby selectively separating methylated DNA.

[0052] Furthermore, methylation can be measured by quantitative PCR, and methylated DNA separated with a methylated DNA-specific binding protein is labeled with a fluorescent dye and hybridized with a DNA chip integrated with a complementary probe, thereby measuring methylation.

[0053] (4) Detection of differential methylation - bisulfite sequencing method: Another method for detecting methylated CpG-containing nucleic acids includes contacting a nucleic acid sample with an agent that modifies unmethylated cytosine, and amplifying the CpG-containing nucleic acids in the sample using CpG-specific oligonucleotide primers. Here, the oligonucleotide primers can be characterized in that they detect methylated nucleic acids by distinguishing between modified methylated and unmethylated nucleic acids. The amplification step is optional and preferred, but not required. The method relies on a PCR reaction to distinguish between modified (e.g., chemically modified) methylated DNA and unmethylated DNA.

[0054] (5) Bisulfite sequencing method: Another method for detecting methylated CpG-containing nucleic acids includes contacting a nucleic acid-containing sample with an agent that modifies non-methylated cytosine, and amplifying the CpG-containing nucleic acids in the sample using non-methylation-dependent oligonucleotide primers. Here, the oligonucleotide primers may be characterized in that they amplify the nucleic acids without distinguishing between modified methylated and non-methylated nucleic acids. The amplification products have been described in conjunction with bisulfite sequencing for use in detecting methylated nucleic acids by next-generation sequencing methods or sequencing by the Sanger method using sequencing primers.

[0055] (6) Next generation sequencing methods include synthesis sequencing methods and ligation sequencing methods. These methods are characterized by the fact that instead of creating bacterial clones, individual DNA fragments are spatially separated, amplified in situ (clonal amplification), and sequenced. Here, since hundreds of thousands of fragments are read simultaneously, this method is also called a massively parallel sequencing method.

[0056] Basically, a sequencing-by-synthesis method is performed using a method of obtaining a signal by sequentially attaching a mononucleotide or a dinucleotide, and examples thereof may include pyrosequencing, ion torrent, and Solexa methods.

[0057] Examples of NGS devices based on synthetic sequencing methods include Roche's 454 platform, Illumina's HiSeq platform, Life Technology's Ion PGM platform, and Pacific BioSciences' PacBio platform. 454 and IonPGM use emulsion PCR as a clonal amplification method, while HiSeq uses bridge amplification. Synthetic sequencing methods read sequences by detecting phosphates, protons, or pre-attached fluorescence generated when DNA is synthesized by sequentially attaching one nucleotide. In the method of detecting the sequence, 454 uses a pyrophosphate sequencing method using phosphates, while Ion PGM uses proton detection. HiSeq and PacBio detect fluorescence to decode the sequence.

[0058] The ligation sequencing method is a sequencing technique that uses DNA ligase, which recognizes nucleotides at certain positions in a DNA nucleotide sequence. Unlike most sequencing techniques that use polymerases, the ligation sequencing method does not use polymerases, and is characterized in that DNA ligases do not connect mismatched sequences. One example is the SOLiD system. In this technique, two bases are read at intervals, which are independently repeated five times by primer reset, thus increasing accuracy by repeatedly reading each base twice.

[0059] In the sequencing by ligation method, dinucleotide primers corresponding to the nucleotide sequence are sequentially linked in a dinucleotide primer set consisting of 16 combinations, and finally these linked combinations are analyzed to complete the nucleotide sequence of the corresponding DNA.

[0060] Here, the next generation sequencing method can be exemplified by a sequencing by synthesis method or a sequencing by ligation method.The methylated DNA specific binding protein includes but is not limited to MBD2bt, and the antibody is a 5'-methyl-cytosine antibody, but is not limited thereto.

[0061] Regarding the primers used in the present invention, when a reagent such as bisulfite is used in step (a), the cytosine at the 5'-CpG'-3' site remains cytosine in the case of methylation, and is converted into uracil in the case of non-methylation. Therefore, for the modified nucleotide sequence obtained after treatment with a reagent such as bisulfite, a primer corresponding to the region in which the 5'-CpG-3' nucleotide sequence exists can be constructed.

[0062] Primers can be constructed to have "substantially" complementarity to each strand of the locus to be amplified. This means that the primers have sufficient complementarity to hybridize to the corresponding nucleic acid strands under polymerization reaction conditions.

[0063] Another aspect of the present invention relates to a composition for diagnosing colorectal cancer, which comprises at least one reagent that differently modifies methylated cohesin 2 (SDC2) and ADHFE1 genes and unmethylated SDC2 and ADHFE1 genes, and primers that specifically amplify the methylated SDC2 gene and the methylated ADHFE1 gene.

[0064] Since the components contained in the composition according to the present invention overlap with the above-mentioned components, the description of the above-mentioned components also applies here.

[0065] Still another aspect of the present invention relates to a kit for detecting target DNA methylation, comprising the above composition.

[0066] In one embodiment, the kit includes: a compartmentalized carrier device containing a sample therein, a container including reagents, and a container including primers for methylated SDC2 gene and methylated ADHFE1 gene, respectively. In some cases, it may also include a container containing probes for detecting amplification products of methylated SDC2 gene and methylated ADHFE1 gene, respectively.

[0067] The carrier is suitable for accommodating one or more individual containers, such as bottles and tubes, containing individual components for use in the methods of the present invention. With reference to the description of the present invention, one of ordinary skill in the art can easily determine the allocation of the necessary agents among the containers.

[0068] The present invention can be better understood by the following examples. These examples are only used to illustrate the present invention and should not be interpreted as limiting the scope of the present invention, as will be clear to those skilled in the art.

[0069] Example 1. Selection of colorectal cancer-specific methylated gene ADHFE1

[0070] The inventors used the cancer tissues and adjacent normal tissue DNA of 12 patients undergoing colorectal cancer surgery to perform genome-level methylation analysis (MeDIA-CpG microarray) to find genes that are specifically methylated in colorectal cancer, and thus reported 32 methylated genes in colorectal cancer (Oh et al., Journal of Molecular Diagnostics, 2013; 15(4):498-507: Supplementary Table 1). Among these 32 genes, ADHFE1 (NM_144650, iron-containing alcohol dehydrogenase 1) which is highly methylated in colorectal cancer tissue was selected as a colorectal cancer-specific methylated gene ( Figure 1 ).

[0071] Example 2. Validation of primers and probes for ADHFE1 methylation detection

[0072] In order to construct primers for detecting methylation of the ADHFE1 gene, methylation-specific primers and probes were designed using MethPrimer (http: / / www.urogene.org / cgi-bin / methprimer / methprimer.cgi) for the ADHFE1 gene sequence modified with bisulfite (Table 1).

[0073] [Table 1]

[0074] Primer and probe sequences for detecting ADHFE1 gene methylation

[0075]

[0076] In order to confirm the methylation-specific detection ability of the designed primers for the ADHFE1 gene, a quantitative methylation-specific real-time PCR (qMSP) method was performed using methylated and non-methylated control DNA (EpiTect PCR control DNA set (Qiagen, catalog number 59695)). For qMSP, an AB 7500Fast (Thermo Fischer) instrument was used. A total of 30 μl of PCR reaction solution (20 ng / 2 μl template human DNA modified with bisulfite, 6 μl of 5X AptaTaq DNA Master (Roche Diagnostics), 2 μl (2.5 pmol / μl) of methylation-specific PCR primers (IDT, USA), 2 μl (2.5 pmol / μl) of TaqMan probe (IDT, USA) and 14 μl of DW) was prepared, and PCR was performed for a total of 40 cycles at 95°C for 5 minutes, 95°C for 15 seconds and then 65°C for 1 minute. Whether the PCR product was amplified was determined in real time. The COL2A1 gene was amplified as an internal control gene ( Figure 2 ).

[0077] like Figure 2 As shown, both ADHFE1 (Group 1) and the control gene COL2A1 were amplified in the methylated DNA, but the ADHFE1 (Group 1) gene was not amplified and only the control gene COL2A1 was amplified in the non-methylated DNA, indicating that the methylation-specific primers for the ADHFE1 (Group 1) gene worked properly.

[0078] like Figure 3As shown, both ADHFE1 (Group 2) and the control gene COL2A1 were amplified in the methylated DNA, but the ADHFE1 (Group 2) gene was not amplified and only the control gene COL2A1 was amplified in the non-methylated DNA, indicating that the methylation-specific primers for the ADHFE1 (Group 2) gene worked properly.

[0079] Example 3: Increased diagnostic performance of colorectal cancer by combining methylation of SDC2 and ADHFE1 genes in fecal DNA To determine whether the ability to diagnose colorectal cancer is improved after combining methylation of ADHFE1 gene and SDC2 gene (Korean Patent No. 10-1142131), which are conventional methylation biomarkers for early diagnosis of colorectal cancer, methylation of SDC2 gene was measured in 24 normal persons (Yonsei Medical Center Check-Up Center) and in 33 colorectal cancer patients (Yonsei Medical Center, Colorectal and Anal Surgery) after colonoscopy. Figure 4 ) had previously measured SDC2 methylation in these samples and used samples in which positive SDC2 methylation was observed in 1 of 24 normal subjects (4.2%) and in 18 colorectal cancer patients (54.5%) after colonoscopy.

[0080] The methylation measurement in these samples was performed using the quantitative methylation-specific real-time PCR (qMSP) method described in Example 2. For qMSP, an AB 7500Fast (Thermo Fischer) instrument was used, and the EZ Methylation Gold Kit (Cat. No. D5006, Zymo Research, USA) was used for modification with bisulfite. A total of 30 μl of PCR reaction solution (2.0 μg / 10 μl fecal DNA modified with bisulfite, 6 μl of 5X AptaTaq DNA Master (Roche Diagnostics), 2 μl (2.5 pmol / μl) of methylation-specific PCR primers (IDT, USA), 2 μl (2.5 pmol / μl) of TaqMan probe (IDT, USA) and 6 μl of DW) was used to measure the methylation of the SDC2 and ADHFE1 genes in fecal DNA, and the COL2A1 gene as an internal control gene was amplified together. PCR was performed for a total of 40 cycles at 95°C for 5 minutes, 95°C for 15 seconds, and then 65°C for 1 minute. Whether the PCR product was amplified ( Figure 3Methylated and non-methylated control DNA (EpiTect PCR Control DNA Set (Qiagen, Cat. No. 59695)) were used as controls and tested.

[0081] [Table 2]

[0082] Primer and probe sequences for SDC2 gene methylation detection

[0083]

[0084] When methylation of the ADHFE1 gene (Group 2) was measured in the feces of normal persons after colonoscopy, positive methylation was observed only in 1 person (specificity of 95.8%), whereas positive methylation was observed in 26 colorectal cancer patients (sensitivity of 78.8%), indicating that it is effective in detecting colorectal cancer. In addition, after combining the methylation of the ADHFE1 gene (Group 2) and the previously known methylation of the SDC2 gene, a total of 2 normal persons (including 1 additional person) showed positive methylation after colonoscopy, indicating a specificity as high as 91.7%; that is, there was no significant loss in specificity. In addition, a total of 27 colorectal cancer patients (including 9 additional patients) showed positive methylation, indicating a significant increase in sensitivity to 81.8% ( Figure 4 , P < 0.033, Fisher's exact test).

[0085] Practicality

[0086] According to the present invention, primers that specifically amplify multiple methylated colorectal cancer marker genes can be used to detect methylation with high detection sensitivity. The method of the present invention is therefore useful because it can improve the ability to detect intestinal cancer and can accurately and quickly diagnose colorectal cancer compared to detection methods using a single marker gene.

[0087] Although the specific embodiments of the present invention have been disclosed in detail above, it will be apparent to those skilled in the art that the description is merely a preferred exemplary embodiment and should not be interpreted as limiting the scope of the present invention. Therefore, the essential scope of the present invention should be defined by the appended claims and their equivalents.

[0088] Sequence Listing Free Text

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Claims

1. Primers and probes are prepared for use in samples SDC2 (syndecan 2) and ADHFE1 Use of a kit for detecting CpG methylation of the (ferroalcohol dehydrogenase 1) gene, wherein the sample has methylation levels differently modified by at least one reagent SDC2 and ADHFE1 Genes and unmethylated SDC2 and ADHFE1 Gene, wherein the primers and probes comprise: specific amplification of methylated SDC2 Gene primers, and methylation primers that can amplify SDC2 A probe for complementary hybridization of a gene, wherein the sequences of the primers are SEQ ID NOs: 10 and 11, wherein the sequence of the probe is SEQ ID NO: 12; and a specific amplification of methylation ADHFE1 Gene primers, and methylation primers that can amplify ADHFE1 A probe for complementary gene hybridization, wherein the sequences of the primers are SEQ ID NOs: 1 and 2 or SEQ ID NOs: 4 and 5, and the sequence of the probe is SEQ ID NO: 3 or 6.

2. The method according to claim 1, wherein the agent is a bisulfite, a bisulfite, a disulfite or a combination thereof.

3. The use according to claim 1, wherein at least one cytosine base is converted into uracil or a base other than cytosine by treatment with the reagent.

4. The use according to claim 1, wherein methylation is detected using a method selected from the group consisting of PCR, gene chip and sequencing.

5. The use according to claim 1, wherein methylation is detected using a method selected from the group consisting of methylation-specific PCR, quantitative PCR, sequencing by synthesis and sequencing by ligation. The use according to claim 1 , wherein methylation is detected using a method selected from the group consisting of real-time methylation-specific PCR and PCR using a methylated DNA-specific binding protein.

7. The use according to claim 1, wherein the detection of methylation is performed using a method selected from PCR using a methylated DNA-specific binding antibody.

8. The use according to any one of claims 4 to 7, wherein methylation is detected by detecting a material that binds to the probe and exhibits fluorescence.

9. A composition for diagnosing colorectal cancer, comprising: at least one agent that differently modifies methyl groups SDC2 (syndecan 2) and ADHFE1 (iron-containing alcohol dehydrogenase 1) gene and unmethylated SDC2 and ADHFE1 Genes; and Specific amplification of methylation SDC2 Gene primers, and methylation primers that can amplify SDC2 A probe for complementary hybridization of a gene, wherein the sequences of the primers are SEQ ID NOs: 10 and 11, wherein the sequence of the probe is SEQ ID NO: 12; and a specific amplification of methylation ADHFE1 Gene primers, and methylation primers that can amplify ADHFE1 A probe for complementary gene hybridization, wherein the sequences of the primers are SEQ ID NOs: 1 and 2 or SEQ ID NOs: 4 and 5, and the sequence of the probe is SEQ ID NO: 3 or 6.

10. The composition of claim 9, wherein the agent is a bisulfite, a bisulfite, a disulfite, or a combination thereof.

11. The composition according to claim 9, wherein at least one cytosine base is converted into uracil or a base other than cytosine by treatment with the reagent.

12. A kit for diagnosing colorectal cancer, the kit comprising the composition according to any one of claims 9 to 11.

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