WO2018194280A1 - Method for detecting methylation of syndecan 2 (sdc2) gene - Google Patents

Abstract

The present invention relates to: a method for detecting the methylation of a CpG island region of a Syndecan 2 (SDC2) gene; a composition for detecting the methylation of a CpG island region of an SDC2 gene; and a kit for detecting the methylation of a CpG island region of an SDC2 gene and, more specifically, to: a method for detecting the methylation of a CpG island region of an SDC2 gene by amplifying methylated SDC2 DNA by using a forward primer and/or a reverse primer, which complementarily binds to methylated SDC2 DNA and comprises at least one CG and/or GC; a composition for detecting the methylation of a CpG island region of an SDC2 gene, containing the primer; and a kit for detecting the methylation of a CpG island region of an SDC2 gene, containing the primer.

Description

Method for detecting methylation of SDC2 (Syndecan 2) gene

The present invention relates to a method for detecting CpG islet methylation of SDC2 gene, a composition for detecting CpG islet methylation of SDC2 gene, and a kit for detecting CpG islet methylation of SDC2 gene. Method for amplifying methylated SDC2 DNA using forward and / or reverse primer comprising CG or GC to detect CpG island region methylation of SDC2 gene, Composition for detecting CpG island region methylation of SDC2 gene comprising said primer It relates to a kit for detecting the CpG island region methylation of the SDC2 gene comprising the primer.

The genomic DNA of mammalian cells contains a fifth base in addition to A, C, G, and T, which is 5-methylcytosine (5-mC) having a methyl group attached to the fifth carbon of the cytosine ring. 5-mC always appears in C of CG dinucleotides (5'-mCG-3 '), and this CG is often referred to as CpG. Most of C of CpG is methylated because a methyl group is attached. This methylation of CpG inhibits the expression of repetitive sequences in the genome, such as Alu or transposon, and is the site where extragenic changes occur most frequently in mammalian cells. The 5-mC of this CpG is naturally deaminoated and converted to T, thus the CpG in the mammalian genome is 1% less than the frequency at which it should normally appear (1/4 × 1/4 = 6.25%). Indicates only.

There are exceptionally densely packed CpGs, which are called CpG islands. CpG islands are 0.2 ~ 3kb in length, the distribution percentage of C and G bases is over 50%, and the distribution percentage of CpG is more than 3.75%. About 45,000 CpG islands appear in the entire human genome, particularly in promoter regions that regulate gene expression. Indeed, CpG islands appear in promoters of housekeeping genes, which make up about half of human genes (Cross, S. et al., Curr. Opin. Gene Develop ., 5: 309, 1995).

Abnormal DNA methylation is known to occur mainly in the 5 'regulatory region of the gene, thereby reducing the expression of the gene. Here, the 5 'expression control region of the gene includes a promoter, an enhancer, and a 5'-untranslated region. In recent years, attempts have been actively made to examine methylation of 5 'expression-regulated sites of tumor-related genes in blood, sputum, saliva, feces, and urine, and to use them for diagnosis of various cancers. In addition, DNA leaked by processes such as apoptosis or necrosis flows into the blood, and methylated DNA fragments are also present in cell-free tumor DNA in serum or plasma. It is known that these abnormally present DNA methylation fragments are used as targets for cancer diagnosis. In addition, since tumor cells are exfoliated and fall out in the form of body fluids such as feces, urine, sputum, and bronchial lavage fluid, the methylation of DNA in cells present in them is used as a target for diagnosing cancer.

The SDC2 (syndecane-2) gene is a gene involved in cell migration, differentiation and proliferation, and was first identified by the inventors of the methylation in colorectal cancer (Oh et al., J. Mol. Diag. 2013). Candidate genes hypermethylated in colorectal cancer tissues compared to normal tissues by separating methylated DNA from colorectal cancer tissues and normal tissue DNAs of 12 colorectal cancer patients from stage I to stage IV through DNA microarray analysis Excavated them. After a series of validation procedures, SDC2 was identified as a promising methylation biomarker for early diagnosis of colorectal cancer. Clinical validation using 139 colorectal cancer tissues showed higher levels of methylation compared to normal tissue in 97.8% of colorectal cancer tissues regardless of stage. In 131 patients with colorectal cancer from stage I to stage IV and 125 normal serum, the sensitivity of the colon cancer diagnosis was 87% and specificity 95.2%. In particular, the sensitivity of stage I was 92.3%, confirming the usefulness of early diagnosis of colorectal cancer.

Under this technical background, the inventors of the present application can efficiently methylate SDC2 (Syndecan 2) gene CpG island region with high sensitivity and accuracy when using complementary binding to methylated SDC2 DNA and comprising one or more CG. It was confirmed that it can detect, and the present invention was completed.

Summary of the Invention

The main object of the present invention is to provide a method for effectively detecting the CpG island region methylation of SDC2 (Syndecan 2) gene.

Another object of the present invention is to provide a composition and kit for detecting methylation of CpG island region of SDC2 gene.

In order to achieve the above object, the present invention comprises the steps of (a) processing a reagent for differently modifying the methylated DNA and unmethylated DNA in the DNA isolated from the clinical sample; (b) amplifying methylated SDC2 DNA using forward and / or reverse primers complementarily binding to methylated SDC2 DNA and comprising one or more CpG dinucleotides; And (c) provides a method for detecting the methylation of the CpG island region of the SDC2 (Syndecan 2) gene comprising the step of confirming the production of the product amplified by the primer.

The present invention also provides a composition for detecting methylation of a CpG island region of an SDC2 gene comprising a forward and / or reverse primer comprising at least one CpG dinucleotide, which can complement and amplify methylated SDC2 DNA. do.

The present invention furthermore detects methylation of the CpG island region of the SDC2 (Syndecan 2) gene comprising forward and / or reverse primers that can complement and amplify methylated SDC2 DNA and comprise one or more CpG dinucleotides. Provide a kit for

1 is a schematic of the detection rate of detecting SDC2 gene methylation in a variety of specimens using 808 sets of primers and probes used in the method according to the present invention.

Figure 2 is a measure of the degree of methylation of the SDC2 gene in stool samples of normal and colorectal cancer patients using the 808 sets of primers and probes used in the method according to the present invention.

Figure 3 measures the methylation of the SDC2 gene in serum samples of normal and colorectal cancer patients using the 808 sets of primers and probes used in the method according to the present invention.

Detailed Description of the Invention and Specific Embodiments

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 this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In the present invention, we designed 808 sets of methylation specific primers and probes (Table 1) complementary to the methylated SDC2 sequence after conversion to bisulfite for methylation detection of the SDC2 gene to analyze the methylation of the SDC2 gene. SDC2 gene methylation detection ability of these primers and probes was confirmed by PCR, and it was confirmed that methylation of SDC2 gene was normally detected in all 808 sets of primers and probes tested (Table 2).

Thus, in one aspect, the present invention relates to a method for detecting methylation of an SDC2 (Syndecan 2) gene CpG island region comprising the following steps:

(a) treating the DNA isolated from the clinical sample with a reagent that differently modifies the methylated and unmethylated DNA;

(b) amplifying methylated SDC2 DNA using forward and / or reverse primers comprising one or more CG or GC dinucleotides; And

(c) confirming whether or not the product amplified by the primer is produced.

"Methylation" of the present invention means that the methyl group attached to the fifth carbon of the cytosine (cytocine) base ring is modified to 5-methylcytosine (5-mC), 5-methylcytosine is always C only of CG dinucleotide (5'-mCG-3 '), this CG is often referred to as CpG. This methylation of CpG inhibits the expression of repeating sequences in the genome, such as alu and transposon, and is the site where extragenic changes occur most frequently in mammalian cells. The 5-mC of this CpG is naturally deaminoated and converted to T, so that the CpG in the mammalian genome shows only 1% of the frequency, much lower than the frequency that should normally appear (1/4 x 1/4 = 6.25%).

Among CpGs, there are exceptionally dense ones, which are called CpG islands. CpG islands are 0.2-3kb in length, and the distribution percentage of C and G bases is over 50%, and the distribution percentage of CpG is concentrated to 3.75% or more. About 45,000 CpG islands appear in the entire human genome, particularly in promoter regions that regulate gene expression. Indeed, CpG islands appear in the promoters of housekeeping genes, which make up about half of human genes.

The presence of CpG methylation in the target DNA can be indicative of disease, for example measuring the CpG methylation of any one of a promoter, 5 ′ untranslated region, intron and exon of the target DNA.

CpG-containing genes are typically DNA. However, the method of the present invention may apply for example a sample containing DNA or RNA comprising DNA and mRNA, wherein the DNA or RNA may be single stranded or double stranded, or DNA-RNA hybrids may be used. It may be characterized by the contained sample.

Nucleic acid mixtures may also be used. As used herein, "multiple" includes both cases where there are a plurality of specific nucleic acid sequence sites to be detected in a kind of gene and a case where a plurality of target DNAs are included in one tube (single reactor). The specific nucleic acid sequence to be detected may be a fraction of a large molecule, and from the outset the specific sequence may exist in the form of isolated molecules that make up the entire nucleic acid sequence. The nucleic acid sequence need not be nucleic acid present in pure form, and the nucleic acid may be a small fraction in a complex mixture, such as containing whole human DNA.

Specifically, the present invention is to detect the methylation of a plurality of target DNA in a sample in a single reactor, the sample may include a plurality of multiple target DNA, the target DNA, as well as the control gene, abnormally methylated expression When inhibited, any gene that affects the development or progression of cancer can be used without limitation.

In the present invention, the sample may be derived from the human body, for example, liver cancer, glioblastoma, ovarian cancer, colon cancer, head and neck cancer, bladder cancer, kidney cell cancer, gastric cancer, breast cancer, metastatic cancer, prostate Cancer, pancreatic cancer or lung cancer patients may be derived, and the sample may use solid or liquid tissue, cells, feces, urine, blood, serum or plasma.

As used herein, “sample,” “clinical sample,” or sample, includes a wide range of all biological fluids obtained from an individual, body fluid, cell line, tissue culture, etc., depending on the type of assay being performed, for example, Cells, tissues, biopsies, paraffin tissue, and microneedle aspirates may also be included, including feces, blood, serum, plasma, and urine, and flowed out of the lavage of cells, feces, urine, sputum, and bronchi. For example, the clinical sample may be used to wash tissue, biopsy, paraffin tissue, feces, blood, serum, plasma, microneedle aspirate, urine, cells, feces, urine, sputum and bronchus, for example, from a suspected cancer patient or a diagnosis target. It may be selected from the group consisting of cells flowed out of one wash solution and combinations thereof, but is not limited thereto. It is popularly known as.

DNA is separated from the clinical sample, and DNA separation may be performed by inducing magnetic particles to bind with DNA in clinical samples, for example, using magnetic particles, and then applying an external magnetic field to the specimen. Magnetic particles used for DNA separation may have a particle size of about 50-2000 nm. Separation of DNA from the clinical sample may be performed using any one of various DNA separation kits or similar DNA separation reagents that are commercially available.

For example, reagents that are bisulfite, hydrogen sulfite, disulfite, or a combination thereof can be treated to modify the methylated and unmethylated DNA differently. The genomic DNA sequence of the CpG island region where methylation may occur in the SDC2 gene is shown in SEQ ID NO: 1, and the reagent for modifying the DNA sequence differently from methylated DNA and unmethylated DNA, for example, When artificially modified by treatment with sulfite, the cytosine bases methylated by the reagent are not converted, and the unmethylated cytosine bases can be converted to bases other than uracil or cytosine. Specifically, the nucleotide sequence corresponding to the methylated SDC2 DNA is shown in SEQ ID NO: 2.

In the present invention, primers are, for example, 10mer to 40mer oligonucleotides capable of complementarily binding to methylated SDC2 DNA to amplify methylated SDC2 DNA, wherein the primers are "substituted" with each strand of the locus to be amplified in the target DNA. It can be manufactured to have complementarity. This means that the primers have sufficient complementarity to hybridize with the corresponding nucleic acid strands under the conditions for carrying out the polymerization.

The primers comprise forward and / or reverse primers, and the forward and / or reverse primers comprise one or more CG or GC.

Specifically, the forward primer may bind to a sequence complementary to the sequence of SEQ ID NO: 2 to specifically amplify a sequence complementary to the sequence of SEQ ID NO: 2, and may include cytosine (C) at the 3 'end. For example, the sequence of converting the methylated sense strand of the SDC2 gene into bisulfite is described in SEQ ID NO: 2, and the forward primer binds to a sequence complementary to the sequence of SEQ ID NO: 2, at the 3 'end. It can be designed to further distinguish between methylated ("C") and unmethylated ("U", "T") SDC2 genes by ending with "C" in CG.

The reverse primer may bind to the sequence of SEQ ID NO: 2, amplify the sequence of SEQ ID NO: 2, and include guanine (G) at the 3 'end. For example, it may be designed to bind complementary to SEQ ID NO: 2 and to make guanine (G) at the 3 'end by directing the orientation of the primer to the opposite of the forward primer.

The reverse primer primarily binds to the template of SEQ ID NO: 2, and amplification occurs. At this time, the forward primer is secondarily bound to the amplified sequence, thereby enabling amplification.

Specifically, the primer may include a pair of primers including forward and reverse primers. For example, the primers may be, for example, SEQ ID NOs: 3, 4, 6-67, 69-100, 102-153, 155-216, 218-279, 281-342, 344-395, 397-448, 450 At least 80% homology with one or more sequences selected from the group consisting of -511, 513-574, 576-637, 639-700, 702-763, 765-826, 828-841, specifically 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% It may include a sequence showing the above homology, but is not limited thereto. The primers may be selected from the group consisting of CH 3 (methyl), -OCH 3, -NH 2, -F (fluorine), -O-2-methoxyethyl -O-propyl, By substitution with -O-2-methylthioethyl, -O-3-aminopropyl, -O-3-dimethylaminopropyl, -ON-methylacetamido or -O-dimethylamidooxyethyl transform; Modification in which oxygen in a sugar structure in nucleotides is replaced with sulfur; Modification of internucleotide linkages to phosphorothioate or boranophosphophate, methyl phosphonate linkages, or combinations of one or two or more, or combinations of peptide nucleic acid (PNA), LNA nucleotides modified in the form of (locked nucleic acid), UNA (unlocked nucleic acid) or inosine; It may include one or more nucleotides modified in the form of 2'-5 'phosphodiester linkage.

Said primers are for example SEQ ID NOs: 3, 4, 6-67, 69-100, 102-153, 155-216, 218-279, 281-342, 344-395, 397-448, 450-511, 513 One or more sequences selected from the group consisting of -574, 576-637, 639-700, 702-763, 765-826, 828-841. Specifically, the primer may include, for example, the forward and reverse primers described in Table 2.

In some cases, the method may include detecting methylation of the target DNA using a self-reporting function or using an energy transfer labeled primer.

The term "self-reporting" as used herein is often referred to as an "energy transfer label" and may be used interchangeably herein. In the present specification, a universal primer having a self-reporting function may be mixed with an energy transfer labeled primer.

Self-reporting or energy transfer labeling function does not fluoresce by self-quenching at a stage where amplification does not occur, but self-quenching or self probing (self-quenching) where quenching is released when amplification occurs. -probing) (Nazarenko IA et al., Nulceic Acid Res, 1997, 25 (12): 2516-2521; Whitcombe D et al., Nat Biotechnol , 1999, 17 (8): 804- 807; Myakishev MV et al., Genome Res , 2001, 11: 163-169; Nazarenko IA et al., Nucleic Acid Res , 2002, 30 (9): 2089-2195; Bengra C et al., Clin Chem, 2002 , 48 (12): 2131-2140; Murray JL et al., J Mol Diag, 2014, 16 (6): 627-638; Gao L et al., Mol Cell Probes , 2015, 29 (6): 438- 441). A material having a self-reporting function or a material having an energy transfer labeling function may include, but is not limited to, a TaqMan probe, a fluorophore, or a molecular beacon.

In some cases, it may further include a probe capable of hybridizing with the methylated SDC2 DNA to determine whether a product amplified with a primer is present.

The methylation may be, for example, PCR, methylation specific PCR, real time methylation specific PCR, PCR using methylated DNA specific binding antibody or aptamer, quantitative PCR, nucleic acid chip, sequencing, The method may be performed by a method selected from the group consisting of sequencing by synthesis and sequencing by ligation, but is not limited thereto.

(1) Methylation specific PCR: When detected by the methylation specific PCR, bisulfite treatment results in cytosine at the 5'-CpG'-3 site as it is methylated, and remains unmethylated. If so, it turns into uracil. Therefore, a primer corresponding to the site where the 5'-CpG-3 'nucleotide sequence exists can be prepared for the converted nucleotide sequence after bisulfite treatment. When PCR is carried out using a primer, the PCR product is made from the primer corresponding to the methylated nucleotide sequence, and methylation can be confirmed by agarose gel electrophoresis. Here, the methylation detection probe may be a TaqMan, a Molecular Beacon, a probe having a self-reporting function or an energy transfer labeling function, but is not limited thereto.

(2) Real time methylation specific PCR (real time methylation specific PCR): The real time methylation specific PCR is a conversion of the methylation specific PCR method to a real-time measurement method, in the case of methylation after treating bisulfite to genomic DNA The PCR primers are designed and real-time PCR is performed using these primers. At this time, there are two methods of detection using a TaqMan probe complementary to the amplified base sequence, and two methods of detection using Sybergreen. Thus, real-time methylation specific PCR can selectively quantitate only methylated DNA. In this case, a standard curve may be prepared using an in vitro methylated DNA sample, and for standardization, a gene without a 5'-CpG-3 'sequence in the base sequence may be amplified with a negative control to quantitatively analyze the degree of methylation.

In addition, the real-time methylation-specific PCR is a conversion of the methylation-specific PCR method to a real-time measurement method, after treating bisulfite in the genomic DNA, designing a methylation independent PCR primers, these primers To perform real-time PCR using. At this time, the amplified nucleotide sequence includes both methylated and unmethylated DNA, and there is a method of detecting using a probe capable of complementarily binding to methylated DNA. Thus, real-time methylation specific PCR can selectively quantitate only methylated DNA.

(3) Detection of differential methylation-bisulfite sequencing method: Another method of detecting nucleic acid containing methylated CpG comprises contacting a sample containing nucleic acid with an agent that modifies unmethylated cytosine and a CpG-specific oligonucleotide Amplifying the CpG-containing nucleic acid of the sample using a primer. Here, the oligonucleotide primer may be characterized by detecting the methylated nucleic acid by distinguishing the modified methylated and unmethylated nucleic acid. The amplification step is optional and desirable but not necessary. The method relies on a PCR reaction that distinguishes between modified (eg, chemically modified) methylated and unmethylated DNA.

(4) Bisulfite sequencing method: Another method for detecting nucleic acid containing methylated CpG is contacting a sample containing nucleic acid with an agent that modifies non-methylated cytosine and methylation independent oligonucleotide primer. Amplifying the CpG-containing nucleic acid in the sample. Here, the oligonucleotide primer may be characterized by amplifying the nucleic acid without distinguishing the modified methylated and unmethylated nucleic acid. The amplified products have been described in connection with bisulfite sequencing for the detection of methylated nucleic acids by sequencing by Sanger method using sequencing primers or by next generation sequencing method.

(5) Here, the next generation sequencing method may be characterized by sequencing by synthesis and sequencing by ligation. The unique feature of this method is that instead of producing bacterial clones, a single DNA fragment is spatially separated, amplified in situ, and sequenced. In this case, since several hundreds of thousands of fragments are read at the same time, it is also called a massively parallel sequencing method.

Basically, it is a sequencing by synthesis method, and a method of obtaining a signal by attaching mono or dinucleotides sequentially includes pyro sequencing, ion torrent, and Solexa methods.

NGS equipment based on sequencing by synthesis includes Roche's 454 platform, Illumina's HiSeq platform, Life Technology's Ion PGM platform, and finally PacBio platform on Pacific BioSciences. have. 454 and Ion PGM use emersion PCR as a clonal amplification method, and HiSeq uses bridge amplification. The sequencing by synthesis method reads a sequence by detecting phosphate, hydrogen ions, or pre-attached fluorescence generated when DNA is synthesized by sequentially attaching one nucleotide. In the method of detecting the sequence, 454 uses a pyroseqeuncing method using phosphoric acid, and Ion PGM uses hydrogen ion detection. HiSeq and PacBio decode sequences by detecting fluorescence.

Sequencing by ligation is a sequencing technique that uses DNA ligase to identify nucleotides at specific positions in the DNA sequence. Unlike most sequencing techniques that use polymerases, they do not use polymerases and take advantage of the feature that DNA ligase does not ligation mismatch sequences. This is the case with SOLiD systems. In this technique, two bases are read at intervals, which are repeated five times independently through a primer reset, so that each base is read twice twice to increase accuracy.

In the case of sequencing by ligation, of 16 sets of dinucleotide primer sets, dinucleotide primers corresponding to the corresponding sequencing are sequentially ligated, and the combination of these ligations is analyzed and finally The base sequence of the DNA is completed.

Here, the next generation sequencing method may be characterized by a sequencing by synthesis or a sequencing by ligation method.

The product amplified by the primer may be used as a probe without limitation as long as it can hybridize with the target DNA and detect methylation, for example, but may include, for example, one or more CpG dinucleotides, or the probe may include SEQ ID NOs: 5 and 68 At least 80% homology with one or more sequences selected from the group consisting of 101, 154, 217, 280, 343, 396, 449, 512, 575, 638, 701, 764, 827 and 842, specifically 81%, 82 %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, It may include, but is not limited to, sequences that exhibit 99% or more homology. The probe may comprise one or more sequences selected from the group consisting of SEQ ID NOs: 5, 68, 101, 154, 217, 280, 343, 396, 449, 512, 575, 638, 701, 764, 827 and 842. . The probe may include, for example, the probes described in Table 2.

When the reverse primer is primarily bound to the template of SEQ ID NO: 2, amplification occurs. At this time, the forward primer and the probe are secondarily bound to the amplified sequence, whereby amplification proceeds to release a signal by a probe or fluorescent dye. Can be.

In nucleic acid hybridization (hybridization) reactions, the conditions used to achieve stringent specific levels vary depending on the nature of the nucleic acid being hybridized. For example, the length of the nucleic acid region to be hybridized, degree of homology, nucleotide sequence composition (eg, GC / AT composition ratio), and nucleic acid type (eg, RNA, DNA) select hybridization conditions. Is considered. Further considerations are whether the nucleic acid is immobilized, for example, in a filter or the like.

Examples of very stringent conditions are as follows: 2X SSC / 0.1% SDS at room temperature (hybridization conditions); 0.2X SSC / 0.1% SDS at room temperature (low stringency conditions); 0.2X SSC / 0.1% SDS at 42 ° C. (conditions with moderate stringency); 0.1X SSC at 68 ° C. conditions with high stringency. The washing process can be carried out using one of these conditions, for example a condition with high stringency, or each of the above conditions, each of 10-15 minutes in the order described above, all or all of the conditions described above. Some iterations can be done. However, as described above, the optimum conditions vary with the particular hybridization reaction involved and can be determined experimentally. In general, conditions of high stringency are used for hybridization of critical probes.

In some cases, the probe may be characterized in that the reporter (quencher) or the reporter (quencher) is coupled to the sock end, the reporter is FAM (6-carboxyfluorescein), Texas red, HEX (2 ', 4 ', 5', 7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE, CY3 and CY5 may be one or more selected from the group consisting of, the quencher TAMRA (6- carboxytetramethyl-rhodamine), BHQ1, BHQ2 and Dabcyl can be characterized in that at least one selected from the group consisting of.

In another aspect, the present invention, a composition for detecting methylation of the CpG island region of the SDC2 gene comprising a forward and / or reverse primer comprising at least one CpG dinucleotide and can amplify by binding to a methylated SDC2 DNA It is about.

In the composition according to the invention the description of the composition overlapping with the composition in the method described above applies equally.

In another aspect, the present invention provides a method for binding to amplified methylated SDC2 DNA and complementing the CpG island region of an SDC2 (Syndecan 2) gene comprising forward and / or reverse primers comprising at least one CpG dinucleotide. It relates to a kit for detecting methylation.

In one embodiment, the kit may comprise a compartment containing carrier means for containing a sample, a container comprising a reagent, a container comprising a primer capable of amplifying the methylated SDC2 gene. In addition, it may also include a container containing a probe for detecting the amplification product.

The carrier means is suitable for containing one or more containers, such as bottles, tubes, each container containing independent components used in the method of the invention. In the context of the present invention, one of ordinary skill in the art can readily dispense the required formulation in the container.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1.SDC2 Gene Methylation Detection Using Methylated and Unmethylated Genomic DNA

For methylation detection of the SDC2 gene, 808 sets of methylation specific primers and probes complementary to SEQ ID NO: 2 corresponding to the methylated SDC2 sequence after conversion to bisulfite were designed (Table 1). EpiTect PCR Control DNA set (Qiagen, Cat. No. 59695) was used to test the ability of these primers and probes to detect SDC2 gene methylation. The EpiTect PCR Control DNA set is a DNA set that converts methylated and unmethylated human genomic DNA into bisulfite. Methylation specific real time PCR (qMSP) was performed using the 808 sets of methylation specific primers and probes using these genomic DNAs. qMSP was used Rotor-Gene Q PCR equipment (Qiagen). 20 μl total PCR reaction solution (template DNA, 2 μl; 5X AptaTaq DNA Master (Roche Diagnostics), 4 μl; PCR primer, 2 μl (2 pmole / μl), TaqMan probe, 2 μl (2 pmole / μl); DW 10 μl) was prepared, and PCR conditions were performed 40 times with 95 ° C. for 5 minutes, 15 seconds at 95 ° C., and 1 minute at an annealing temperature. PCR product amplification was confirmed by measuring the cycle threshold (CT) value. SDC2 gene methylation degree of each primer and probe pair was measured by CT value. If CT value is detected in methylated genomic DNA, methylation is detected normally. Determined. It was confirmed that methylation of the SDC2 gene was normally detected in all 808 sets of primers and probes tested (Table 2).

FIG. 1 shows an amplification curve of qMSP using 12 sets of primers and probes as an example of 808 sets. The amplification curve is shown in methylated DNA, but amplified in non-methylated DNA and distilled water (DW) as a template. You can see that the curve does not appear at all.

N.D: not detected

Example 2 SDC2 Gene Methylation Detection Results in Cell Line, Stool and Serum Sample DNA

Using the primers and probes for the detection of SDC2 gene methylation, the ability to detect SDC2 gene methylation was examined in various samples. For this, set 808, the lowest CT value of methylated DNA in the methylation assay using methylated and unmethylated DNA, was selected as an example. To determine if SDC2 gene methylation was detected in a variety of samples, 20 ng of genomic DNA of MRC-5 (KCLB No. 10171), unmethylated SDC2 gene, and 1.0 mL of unserialized human serum from SDC2 gene 2.0 ug of isolated DNA and non-methylated human stool DNA were spiked from 20 ng to 0.01 ng of genomic DNA of SDC2 methylated colorectal cancer cell line HCT116 (ATCC, CCL247), followed by 24 repeated qMSPs. The methylation detection rate of the SDC2 gene was confirmed by the same method as in Example 1 (FIG. 2). Table 3 shows the methylation detection rate of the SDC2 gene in each sample. For cell line DNA, the detection rate was 100% from 20 ng to 0.05 ng, and 96% and 88% at 0.02 g and 0.01 ng, respectively. Stool DNA showed a detection rate of 100% from 20 ng to 0.1 ng and 96%, 92% and 54% at 0.05 ng, 0.02 ng and 0.01 ng, respectively. In the case of serum DNA, the detection rate was 100% from 20 ng to 0.1 ng and 96%, 71% and 33% at 0.05 ng, 0.02 ng and 0.01 ng, respectively. In conclusion, SDC2 gene methylation can be detected in various samples such as cell lines, feces and serum.

Example 3 Results of SDC2 Gene Methylation Detection in Fecal and Serum Clinical Sample DNA

SDC2 gene methylation detection primers and probes were used to reconfirm the ability to detect SDC2 gene methylation in various samples. For this, set 808 was selected as the best CT value of 23.7 in methylated DNA in methylation assay using methylated and unmethylated DNA. To determine if SDC2 gene methylation was detected in stool clinical samples, genomic DNA was isolated from 47 stool patients and 16 normal stool patients. The isolated genomic DNA, 2.0 ug, was converted to bisulfite using the EZ DNA Methylation Gold kit (Zymo Research) according to the manufacturer's instructions and eluted with final 10 uL of distilled water. Methylation specific real time PCR (qMSP) was performed using the set 808 methylation specific primers and probes using these genomic DNAs. qMSP was used Rotor-Gene Q PCR equipment (Qiagen). 20 μl total PCR reaction solution (template DNA, 2 μl; 5X AptaTaq DNA Master (Roche Diagnostics), 4 μl; PCR primer, 2 μl (2 pmole / μl), TaqMan probe, 2 μl (2 pmole / μl); DW 10 μl) was prepared and PCR conditions were performed 40 times with 95 ° C. for 5 minutes, 15 seconds at 95 ° C., and 1 minute at 60 ° C. annealing temperature. Amplification of the PCR product was confirmed by measuring a cycle threshold (CT) value.

CT values were used to assess the sensitivity and specificity for the diagnosis of colorectal cancer through ROC analysis (MedCalc program, Valgye). As a result, the sensitivity for diagnosing colorectal cancer was 89.4% (42/47) and the specificity was 93.8% (1/16) (FIG. 2).

In addition, genomic DNA was isolated from the sera of 13 colorectal cancer patients and 6 normal subjects to determine whether SDC2 gene methylation was detected in serum samples of colorectal cancer patients. The isolated genomic DNA was converted to bisulfite using the EZ DNA Methylation Gold kit (Zymo Research) according to the manufacturer's instructions and eluted with final 10 uL of distilled water. Using these genomic DNAs, methylation was measured in the same manner as above using the methylation specific primers and probes of set 808. CT values were used to assess sensitivity and specificity for the diagnosis of colorectal cancer through ROC analysis (MedCalc program, Belgium). As a result, the sensitivity for the diagnosis of colorectal cancer was excellent at 84.6% (11/13) and specificity at 100% (0/6) (FIG. 3).

According to the methylation detection method of the SDC2 gene CpG island region according to the present invention, it is possible to detect methylation of the SDC2 gene CpG island region with a high detection rate in clinical samples, and to accurately and quickly and efficiently efficiently methylate the SDC2 gene CpG island region. Can be detected.

As described above in detail specific parts of the present invention, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. will be. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Electronic file attached.

Claims (26)

A method for detecting methylation of the CpG island region of the SDC2 (Syndecan 2) gene, comprising the following steps: (a) treating the DNA isolated from the clinical sample with a reagent that differently modifies the methylated and unmethylated DNA; (b) amplifying methylated SDC2 DNA using forward and / or reverse primers comprising one or more CG or GC dinucleotides; And (c) confirming whether or not the product amplified by the primer is produced. The method of claim 1, wherein the reagent for differently modifying the methylated DNA and the unmethylated DNA is bisulfite, hydrogen sulfite, disulfite, or a combination thereof. The method of claim 1, wherein the reagent-treated SDC2 gene is characterized in that represented by SEQ ID NO: 2. The method of claim 1, wherein the methylation detection is performed by PCR, methylation specific PCR, real time methylation specific PCR, quantitative PCR, nucleic acid chip, sequencing, sequencing by synthesis and sequencing by ligation. Characterized in that performed by a method selected from the group consisting of. The method of claim 1, wherein the forward primer binds to a sequence complementary to the sequence of SEQ ID NO: 2 to specifically amplify a sequence complementary to the sequence of SEQ ID NO: 2, and comprises a cytosine (C) at the 3 'end How to feature. The method of claim 1, wherein the reverse primer binds to the sequence of SEQ ID NO: 2, amplifies the sequence of SEQ ID NO: 2, and comprises guanine (G) at the 3 'end. The method of claim 1, wherein the primers are SEQ ID NOs: 3, 4, 6-67, 69-100, 102-153, 155-216, 218-279, 281-342, 344-395, 397-448, 450-511 , 513-574, 576-637, 639-700, 702-763, 765-826, 828-841. A method comprising a sequence having at least 80% homology with at least one sequence selected from the group consisting of . The method of claim 1, wherein step (b) further comprises a probe capable of hybridizing with the methylated SDC2 DNA. The method of claim 8, wherein the probe is one or more selected from the group consisting of SEQ ID NOs: 5, 68, 101, 154, 217, 280, 343, 396, 449, 512, 575, 638, 701, 764, 827 and 842 And a sequence having at least 80% homology with the sequence. The method of claim 8, wherein the probe further comprises a reporter or quencher. The method of claim 10, wherein the reporter is FAM (6-carboxyfluorescein), Texas red, HEX (2 ', 4', 5 ', 7', -tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE, CY3 And one or more selected from the group consisting of CY5. The method of claim 10, wherein the quencher is at least one selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 and Dabcyl. A composition for detecting methylation of a CpG island region of an SDC2 gene comprising a forward and / or reverse primer comprising at least one CG or GC dinucleotide and capable of complementarily binding to methylated SDC2 DNA. The method of claim 13, wherein the forward primer binds to a sequence complementary to the sequence of SEQ ID NO: 2 to specifically amplify a sequence complementary to the sequence of SEQ ID NO: 2, and comprises a cytosine (C) at the 3 'end Characterized in that the composition. The composition of claim 13, wherein the reverse primer binds to the sequence of SEQ ID NO: 2, amplifies the sequence of SEQ ID NO: 2, and comprises guanine (G) at the 3 'end. The method of claim 13, wherein the primers are SEQ ID NOs: 3, 4, 6-67, 69-100, 102-153, 155-216, 218-279, 281-342, 344-395, 397-448, 450-511 , 513-574, 576-637, 639-700, 702-763, 765-826, 828-841. A composition comprising a sequence having at least 80% homology with at least one sequence selected from the group consisting of . The method according to claim 14, wherein the methylation is PCR, methylation specific PCR, real time methylation specific PCR, PCR using methylated DNA specific binding antibody or aptamer, quantitative PCR, nucleic acid chip And sequencing, sequencing by synthesis, and sequencing by ligation. The composition of claim 13, further comprising a probe capable of hybridizing with the amplified methylated SDC2 DNA. The method of claim 18, wherein the probe is one or more selected from the group consisting of SEQ ID NOs: 5, 68, 101, 154, 217, 280, 343, 396, 449, 512, 575, 638, 701, 764, 827 and 842 A composition, characterized by the sequence. A kit for detecting methylation of a CpG island region of an SDC2 (Syndecan 2) gene comprising a forward and / or reverse primer that can complement and amplify methylated SDC2 DNA and complement one or more CG or GC dinucleotides. The method of claim 20, wherein the forward primer binds to a sequence complementary to the sequence of SEQ ID NO: 2 to specifically amplify a sequence complementary to the sequence of SEQ ID NO: 2, comprising a cytosine (C) at the 3 'end Kit characterized by the above. The kit of claim 20, wherein the reverse primer binds to the sequence of SEQ ID NO: 2, amplifies the sequence of SEQ ID NO: 2, and comprises guanine (G) at the 3 ′ end. The method of claim 20, wherein the primers are SEQ ID NOs: 3, 4, 6-67, 69-100, 102-153, 155-216, 218-279, 281-342, 344-395, 397-448, 450-511 , 513-574, 576-637, 639-700, 702-763, 765-826, 828-841 A kit comprising at least 80% homology with at least one sequence selected from the group consisting of . The method of claim 20, wherein the methylation is PCR, methylation specific PCR, real time methylation specific PCR, PCR using methylated DNA specific binding antibody or aptamer, quantitative PCR, nucleic acid chip. And detecting by a method selected from the group consisting of sequencing, sequencing by synthesis and sequencing by ligation. 21. The kit of claim 20, further comprising a probe capable of hybridizing with said amplified methylated SDC2 DNA. The method of claim 25, wherein the probe is one or more selected from the group consisting of SEQ ID NOs: 5, 68, 101, 154, 217, 280, 343, 396, 449, 512, 575, 638, 701, 764, 827 and 842 A kit comprising a sequence having at least 80% homology with the sequence.

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