Composition for determining the presence or absence of nucleic acid methylation and method for determining the presence or absence of nucleic acid methylation

Abstract

The present application relates to a composition for determining the presence or absence of methylation of a nucleic acid and a method for determining the presence or absence of methylation of a nucleic acid.

Description

[Technical field]

[0001] The present application relates to a composition for determining the presence or absence of methylation of a nucleic acid and a method for determining the presence or absence of methylation of a nucleic acid. [Background technology]

[0002] There are methods for measuring the methylation level of a DNA sample, such as qMSP, MethyLight, and MS-HRM, and all of these methods are designed to check the methylation level of each sample relatively. For example, the qMSP method uses a primer that binds only to the methylated (or unmethylated) template strand for the same amount of DNA sample, amplifies the DNA under the corresponding conditions in the sample, and measures the PCR Cq value to check the methylation level of the DNA produced. The Methylight method is a method that enhances the specificity of the measurement by adding a probe to qMSP to bind specific DNA to the marker to be checked among the amplified DNA. In addition, the MS-HRM method uses a primer that can bind to the template strand regardless of the methylation state of the DNA sample used, and includes a certain number of CpGs in the part of the amplified product excluding the primer binding region, and checks the signal of the melting point difference of the amplified product due to the increase in temperature.

[0003] The qMSP and MethyLight assays have the advantage of being convenient in experimental design because there are no major limitations in primer design, and can achieve relatively high sensitivity. However, because an additional control marker analysis is required to correct the amount of sample used in the analysis, and the results are used to correct the measured values, the quantitative analysis is indirect and limited, and the frequency of type 2 errors is high. In addition, biased amplification of a specific template strand can increase the frequency of non-specific amplification of each DNA sample, resulting in low specificity. In the case of MethyLight, which was developed as a method to complement specificity, an additional fluorescent probe that can specifically bind to the amplified product is required, which is a disadvantage in terms of increased costs and increased difficulty in designing analytical tests. In addition, in many cases, the specific binding of the probe reduces the amplification efficiency of PCR, making it difficult to analyze very small amounts of sample.

[0004] In the case of MS-HRM analysis, methylated and unmethylated nucleic acids are amplified simultaneously in the same test tube, so the methylation level of a DNA sample can be measured quantitatively more intuitively than with MSP without the need for additional control marker analysis. However, due to design restrictions that require primer design to contain either CpG or non-specific bases, it is not easy to develop a specific analysis method. In addition, if there is no CpG in the primer, there is a problem that it will bind more to the unmethylated strand than to the methylated strand, and type 1 error may occur due to the unmethylation bias of the verification results for the sample. In experiments that require sensitive measurement of methylation mutations such as cancer diagnosis, there is a problem that the methylation ratio in the actual sample is measured low or inaccurate results are obtained. Summary of the Invention [Problem to be solved by the invention]

[0005] One example of the present application aims to develop a method for determining the presence or absence of methylation that compensates for the shortcomings of conventional experimental verification methods. The method for determining the presence or absence of methylation according to one example of the present application is capable of measuring the relative methylation ratio in the same test tube without the analysis of a control marker, and has high detection sensitivity since it does not use a probe that interferes with the amplification efficiency. In addition, it is possible to adjust the amplification efficiency of methylated and unmethylated DNA in order to measure a very low methylation level in a biological sample such as blood.

[0006] The composition and method according to one embodiment of the present application uses a combination of methylated and unmethylated primers to prevent the generation of non-specific amplification products, and can measure the methylation level of a sample more sensitively than existing PCR-based analytical methods even in conditions where a desired target nucleic acid is present in extremely small amounts.

[0007] One example of the present application is to provide a composition for confirming the presence or absence of methylation of a target nucleic acid with high sensitivity, and a method for confirming the presence or absence of methylation of a target nucleic acid, by compensating for the shortcomings of conventional methods for measuring the methylation level of a DNA sample.

[0008] Another embodiment of the present application is to provide a primer design strategy and optimization method that can be generally applied to various DNA methylation markers.

[0009] Another example of the present application is to provide a method for diagnosing cancer, a method for providing information for diagnosing cancer, a composition for diagnosing cancer, or a kit for diagnosing cancer, which achieves high sensitivity and specificity based on confirming the DNA methylation level of ctDNA (circulating tumor DNA), which is present in small amounts in cancer patients among cfDNA (cell-free DNA) present in a biological sample, for example, blood, through an in-sample semi-quantitative method. [Means for solving the problem]

[0010] One example of the present application relates to a composition for confirming the presence or absence of methylation of a nucleic acid to be confirmed, comprising a primer set for amplifying a target site of the nucleic acid to be confirmed.

[0011] Another example of the present application relates to a method for confirming the presence or absence of methylation of a nucleic acid to be confirmed, the method comprising the steps of: modifying a nucleic acid to be confirmed contained in a biological sample so that a methylated nucleic acid to be confirmed and an unmethylated nucleic acid to be confirmed are different from each other; and treating the biological sample with the composition to amplify the nucleic acid to be confirmed.

[0012] The present application will now be described in more detail.

[0013] One example of the present application relates to a novel method for measuring the methylation level of a nucleic acid to be confirmed, which compensates for the shortcomings of the conventional experimental verification method for measuring the methylation level of a nucleic acid to be confirmed. Specifically, when designing MS-HRM primers, a methylated primer containing a CpG recognition site that recognizes a CpG sequence in the primer is used, and a non-methylated primer containing a TpG recognition site that recognizes a TpG sequence used for amplifying a non-methylated template strand is used in combination to prevent only the methylated template strand from being amplified. In this case, in order to prevent the relatively low sensitivity of MS-HRM and biased amplification of primers containing TpG, a method was developed in which the temperature of the primer annealing step of PCR is adjusted to increase the binding opportunity of primers containing CpG and decrease the binding opportunity of primers containing TpG.

[0014] As used herein, the term "methylation" refers to the addition of a methyl group to a base that constitutes DNA. For example, methylation may occur at a cytosine at a particular CpG site in a particular gene or nucleic acid.

[0015] As used herein, the term "presence or absence of methylation" or "methylation status" refers to the presence or absence of methylation at a cytosine at a specific CpG site in a specific gene or nucleic acid, specifically, the presence or absence of 5-methyl-cytosine at one or more CpG dinucleotides within a base sequence.

[0016] As used herein, the term "methylation level" or "degree of methylation" refers to the amount of methylation present in a base sequence within a nucleic acid to be confirmed.

[0017] As used herein, the term "CpG site" or "CpG sequence" refers to a CpG site present in a base sequence of a specific gene or nucleic acid. The gene is a concept including all of a series of constituent units necessary for expression and operably linked to each other, and may include, for example, a promoter region, a protein coding region (open reading frame, ORF), and a terminator region. Thus, the CpG site may be present in the promoter region, protein coding region (open reading frame, ORF), or terminator region of the gene. For example, it may be a CpG site present in the promoter region of the gene.

[0018] As used herein, the term "nucleic acid" refers to a polymeric form of nucleotide, either ribonucleotides or deoxyribonucleotides, and includes polynucleotides, oligonucleotides, oligomers, oligos, coding sequences, etc. The nucleic acid may be used to include single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers having purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0019] In this specification, when an element is described as "comprising," this means that it can further include other elements, but not excluding other elements, unless otherwise specified.

[0020] Unless otherwise indicated herein, nucleic acids are written from left to right and 5' to 3', respectively.

[0021] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0022] One example of the present application relates to a composition for confirming the presence or absence of methylation of a nucleic acid to be confirmed, comprising a primer set for amplifying a target site of the nucleic acid to be confirmed, or a kit for confirming the presence or absence of methylation of a nucleic acid to be confirmed, comprising the composition. The primer set comprises a forward primer and a reverse primer, one of the forward primer and the reverse primer may comprise a methylation primer and an unmethylation primer, and the remaining one may be a common primer. The methylation primer comprises a CpG recognition site that recognizes a CpG sequence of the nucleic acid to be confirmed, and the unmethylation primer comprises a TpG recognition site that recognizes a TpG sequence of the nucleic acid to be confirmed, and the TpG sequence may be a converted CpG sequence of the nucleic acid to be confirmed. The common primer refers to a primer that can specifically recognize and bind to the nucleic acid to be confirmed regardless of the presence or absence of methylation of the nucleic acid to be confirmed.

[0023] Another example of the present application relates to a method for confirming the presence or absence of methylation of a nucleic acid to be confirmed, the method comprising the steps of: modifying a nucleic acid to be confirmed contained in a biological sample so that a methylated nucleic acid to be confirmed and an unmethylated nucleic acid to be confirmed are different from each other; and treating the biological sample with the composition to amplify the nucleic acid. The modifying step may be performed by treating the biological sample with a preparation that modifies a methylated nucleic acid to be confirmed and an unmethylated nucleic acid to be confirmed so that they are different from each other.

[0024] The target site is a site of interest for which the presence or absence of methylation of the nucleic acid to be confirmed is to be confirmed, and may include at least one or more CpG sequences. The size of the target site may be 50 to 150 bp, 50 to 140 bp, 50 to 130 bp, 50 to 120 bp, 50 to 110 bp, 50 to 100 bp, 60 to 150 bp, 60 to 140 bp, 60 to 130 bp, 60 to 120 bp, 60 to 110 bp, or 60 to 100 bp.

[0025] The target site may include, for example, a biomarker. Specifically, the biomarker may be a methylation biomarker. The methylation biomarker refers to a biomarker that indicates association with a specific disease depending on the presence or absence of methylation of the biomarker. More specifically, the methylation biomarker refers to a biomarker that indicates association with a specific disease due to an epigenetic change that occurs when a methyl group (CH3) is added to C (cytosine) of CG in the base sequence of the biomarker.

[0026] In one embodiment of the present application, the target site may include a biomarker present in a CpG island. The primer set according to one embodiment of the present application includes (1) a methylated forward primer, an unmethylated forward primer, and a common reverse primer; or (2) a common forward primer, a methylated reverse primer, and an unmethylated reverse primer, and can accurately confirm the presence or absence of methylation at the target site even when it is difficult to prepare a common primer, such as a biomarker present in a CpG island where sequence complexity is reduced by sulfite treatment or where changes occur simultaneously at surrounding sites. Specifically, in the conventional HRM analysis method, analysis is difficult when non-specific reactions frequently occur due to the reduction in sequence complexity of gDNA due to sulfite treatment, or when it is difficult to prepare a MIP (Methylation Independent Primer; common primer) that does not contain CpG, particularly in the case of a CpG island. However, the present application can ensure freedom in primer design by using a methylated primer and an unmethylated primer in combination as a one-way primer, and can accurately confirm the presence or absence of methylation at the target site while inducing specific binding of the target.

[0027] The methylated primer, specifically the methylated forward primer or the methylated reverse primer, includes a CpG recognition site that recognizes the CpG sequence of the nucleic acid to be confirmed, and the unmethylated primer, specifically the unmethylated forward primer or the unmethylated reverse primer, includes a TpG recognition site that recognizes a TpG sequence obtained by converting the CpG sequence of the nucleic acid to be confirmed. The TpG sequence is obtained by converting a CpG sequence using a preparation that converts the methylated nucleic acid to be confirmed and the unmethylated nucleic acid to be confirmed differently. For example, the preparation that converts the unmethylated cytosine residue to thymine. For example, the preparation that converts the unmethylated cytosine residue to thymine may be one or more selected from the group consisting of sulfite, bisulfite, hydrogen sulfite, and disulfite.

[0028] When a template strand is treated with a preparation that modifies the methylated and unmethylated target nucleic acids to be confirmed differently, if the CpG cytosine in the template strand is in a methylated state, the CpG cytosine is not converted to thymine, and if the CpG cytosine is in an unmethylated state, the CpG cytosine is converted to thymine.

[0029] The CpG recognition site of the methylation primer can recognize a CpG sequence of the methylated nucleic acid to be confirmed. For example, the CpG recognition site can include a CG sequence.

[0030] The TpG recognition site of the unmethylated primer can recognize a TpG sequence obtained by converting the CpG sequence of the unmethylated nucleic acid to be confirmed by a compound that changes the methylated nucleic acid to be confirmed and the unmethylated nucleic acid to be confirmed differently from each other. For example, the TpG recognition site can include a TG sequence.

[0031] The CpG recognition site and the TpG recognition site are located near the 3' end of the methylated primer and the unmethylated primer, respectively, and can specifically recognize the methylated nucleic acid to be confirmed and the unmethylated nucleic acid to be confirmed that have been converted by a preparation that changes the methylated nucleic acid to be confirmed and the unmethylated nucleic acid to be confirmed differently from each other. For example, the CpG recognition site and the TpG recognition site can be located within 40, 35, 30, 25, 20, 10, 9, 8, 7, 6, 5, 4, 3, or 2 bases from the 3' end of the methylated primer and the unmethylated primer, or at the 3' end.

[0032] The unmethylated primer has a lower Tm value than the methylated primer, so that in order to complement this, a nucleotide may be added to the 5' end of the unmethylated primer to design the methylated primer and the unmethylated primer to have similar Tm. For example, the unmethylated primer may have a larger size than the methylated primer by including an additional nucleotide at the 5' end. For example, the unmethylated primer may include 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 additional nucleotide at the 5' end compared to the methylated primer. For example, the Tm difference between the methylated primer and the unmethylated primer may be 15°C or less, 14°C or less, 13°C or less, 12°C or less, 11°C or less, 10°C or less, 9°C or less, 8°C or less, 7°C or less, 6°C or less, 5°C or less, 4°C or less, 3°C or less, 2°C or less, or 1.5°C or less.

[0033] The primer set includes a forward primer and a reverse primer, and when the methylated primer and the unmethylated primer are forward primers, the reverse primer is a reverse primer, and when the methylated primer and the unmethylated primer are reverse primers, the reverse primer is a forward primer. The reverse primer is a common primer that can bind to both the nucleic acid to be confirmed for methylation and the nucleic acid to be confirmed for unmethylation. Thus, the reverse primer (common primer) may not contain a CpG binding site, but when it is unavoidable that the reverse primer contains a CpG binding site due to the structure of the nucleic acid to be confirmed, the 5'-end portion may contain 5 or less, 4 or less, 3 or less, 2 or less, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or one CpG binding site, for example. However, even in this case, it is preferable to design the reverse primer to have a Tm similar to that of the methylated primer and the unmethylated primer.

[0034] The methylated primer, the unmethylated primer, and the opposite primer may be in a size range of 15 to 40, 15 to 35, 15 to 30, 15 to 25, 18 to 40, 18 to 35, 18 to 30, 18 to 25, 20 to 40, 20 to 35, 20 to 30, or 20 to 25 bases.

[0035] The methylated primer, the unmethylated primer, and the opposite primer may contain one or more, two or more, or three or more non-CpG cytosines, and thus be capable of binding to a nucleic acid to be confirmed that has been converted by an agent that differentially modifies the methylated and unmethylated nucleic acids to be confirmed.

[0036] The Tm of the methylated primer, the unmethylated primer, and the opposite primer can be 50 to 80°C, 50 to 75°C, 50 to 70°C, 50 to 65°C, 55 to 80°C, 55 to 75°C, 55 to 70°C, 55 to 65°C, 60 to 80°C, 60 to 75°C, 60 to 70°C, or 60 to 65°C.

[0037] The composition according to one embodiment of the present application may be a composition in which the methylated primer and the unmethylated primer are mixed at a ratio of 100:1 to 1:100, 100:1 to 1:50, 100:1 to 1:20, 100:1 to 1:10, 100:1 to 1:5, 100:1 to 1:1, 100:1 to less than 1:1, 100:1 to 1.3:1, 100:1 to 1.5:1, 100:1 to 2:1, 100:1 to 2.5:1, 100:1 to 3:1, 100:1 to 3.5:1, 100:1 to 4:1, :1, 50:1-1:100, 50:1-1:50, 50:1-1:20, 50:1-1:10, 50:1-1:5, 50:1-1:1, 50:1-less than 1:1, 50:1-1.3:1, 50:1-1.5:1, :1-2:1, 50:1-2.5:1, 50:1-3:1, 50:1-3.5:1, 50:1-4:1, 10:1-1:100, 10:1-1:50, 10:1-1:20, 10:1-1:10, 10:1-1:5, 10 :1-1:1, 10:1-less than 1:1, 10:1-1.3:1, 10:1-1.5:1, 10:1-2:1, 10:1-2.5:1, 10:1-3:1, 10:1-3.5:1, 10:1-4:1, 5:1-1:100, 5:1-1:50, 5:1-1:20, 5:1-1:10, 5:1-1:5, 5:1-1:1, 5:1-less than 1:15:1-1.3:1, 5:1-1.5:1, 5:1-2:1, 5:1-2.5:1, 5:1- The concentration ratio may be 3:1, 5:1 to 3.5:1, 5:1 to 4:1, 4.5:1 to 1:100, 4.5:1 to 1:50, 4.5:1 to 1:20, 4.5:1 to 1:10, 4.5:1 to 1:5, 4.5:1 to 1:1, 4.5:1 to less than 1:1, 4.5:1 to 1.3:1, 4.5:1 to 1.5:1, 4.5:1 to 2:1, 4.5:1 to 2.5:1, 4.5:1 to 3:1, 4.5:1 to 3.5:1, or 4.5:1 to 4:1.

[0038] For example, the concentration of the unmethylated primer may be 100% or less, less than 100%, 99.99% or less, 99.95% or less, 99.9% or less, 99.5% or less, 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 40% or less, 30% or less, or 25% or less of the methylated primer concentration, and as an example, may be 50% or less.

[0039] The composition and method according to one embodiment of the present application can accurately confirm the presence or absence of methylation of a nucleic acid to be confirmed without using a fluorescent probe to detect an amplification product. Thus, the composition according to one embodiment of the present application may not include a fluorescent probe. Also, the method according to one embodiment of the present application may not use a fluorescent probe.

[0040] In the composition and method according to an embodiment of the present application, the nucleic acid to be confirmed for the presence or absence of methylation may be contained in a biological sample, which may include one or more selected from the group consisting of blood, plasma, tissue, cells, feces, and urine.

[0041] The composition and method according to one embodiment of the present application can determine the presence or absence of methylation of a target nucleic acid with high sensitivity even when the concentration of the methylated target nucleic acid in a biological sample is very low.

[0042] For example, the concentration of the methylated nucleic acid to be confirmed in the biological sample is 100ng / ul or less, 90ng / ul or less, 80ng / ul or less, 70ng / ul or less, 60ng / ul or less, 50ng / ul or less, 40ng / ul or less, 30ng / ul or less, 20ng / ul or less, 15ng / ul or less, 10ng / ul or less, 9ng / ul or less, 8ng / ul or less, 7ng / ul or less, 6ng / ul or less, 5ng / ul or less, 4ng / ul or less, 3ng / ul or less, or 2ng / ul or less. , 1.5ng / ul or less, 1ng / ul or less, 0.5ng / ul or less, 0.4ng / ul or less, 0.3ng / ul or less, 0.2ng / ul or less, 0.1ng / ul or less, 0.05ng / ul or less, 0.04ng / ul or less, 0.03ng / ul or less, 0.02ng / ul or less, 0.01ng / ul or less, 0.009ng / ul or less, 0.008ng / ul or less, 0.007ng / ul or less, 0.006ng / ul or less, or 0.005ng / ul or less.

[0043] For example, the biological sample may include methylated nucleic acids to be confirmed and unmethylated nucleic acids to be confirmed, and the concentration of the methylated nucleic acids to be confirmed may be 75% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 25% or less, 20% or less, less than 19%, 18% or less, 15% or less, 10% or less, 5% or less, 4.5% or less, 4% or less, 3.5% or less, 3% or less, 2.5% or less, 2% or less, 1.5% or less, 1.4% or less, 1.3% or less, 1.2% or less, or 1.1% or less of the concentration of the unmethylated nucleic acids to be confirmed.

[0044] For example, the biological sample may contain a methylated nucleic acid to be confirmed and an unmethylated nucleic acid to be confirmed, and may contain the methylated nucleic acid to be confirmed in an amount of 100% or less, less than 100%, 99% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, less than 10%, 5% or less, less than 5%, 4.5% or less, 4% or less, 3.5% or less, 3% or less, 2.5% or less, 2% or less, 1.5% or less, 1.4% or less, 1.3% or less, 1.2% or less, 1.1% or less, or 1% or less of the methylated nucleic acid to be confirmed based on the entire strands of the methylated nucleic acid to be confirmed and the unmethylated nucleic acid to be confirmed.

[0045] For example, the biological sample may contain up to 20,000 strands, up to 15,000 strands, up to 10,000 strands, up to 5,000 strands, up to 4,000 strands, up to 3,000 strands, up to 2,000 strands, up to 1,000 strands, up to 500 strands, up to 400 strands, up to 300 strands, up to 200 strands, up to 150 strands, up to 100 strands, up to 90 strands, up to 80 strands, up to 70 strands, up to 60 strands, up to 50 strands, up to 40 strands, up to 30 strands, up to 20 strands, up to 10 strands, up to 9 strands, up to 8 strands, up to 7 strands, up to 6 strands, up to 5 ... strands, or up to 3 strands of the methylated nucleic acid to be confirmed.

[0046] A method for confirming the presence or absence of methylation of a nucleic acid to be confirmed according to an embodiment of the present application may further include a step of quantifying the degree of methylation of the nucleic acid to be confirmed, which may involve quantifying the degree of methylation of the biological sample by comparing the area under the curve (AUC) of melting curves of the biological sample, a sample in which the nucleic acid to be confirmed is 100% methylated, and a sample in which the nucleic acid to be confirmed is 100% unmethylated.

[0047] Specifically, the quantifying step may include the steps of: obtaining a melt curve of the biological sample; obtaining a normalized melt curve of the biological sample having a known methylation ratio of the nucleic acid to be confirmed; and quantifying the degree of methylation of the biological sample by comparing the melt curve of the biological sample and the normalized melt curve. The melt curve of the biological sample may be normalized. The melt curve of the biological sample may be obtained through HRM analysis. The methylation ratio of the nucleic acid to be confirmed may be obtained from the content ratio of the methylated nucleic acid to be confirmed and the unmethylated nucleic acid to be confirmed.

[0048] In another embodiment of the present application, the nucleic acid to be confirmed may include a biomarker for cancer diagnosis. Specifically, the nucleic acid to be confirmed may include a biomarker for cancer diagnosis, for example, a marker that is specifically methylated or unmethylated in a cancer patient, and the target site of the nucleic acid to be confirmed may be the position of the biomarker for cancer diagnosis. In this case, the presence or absence of methylation of the nucleic acid to be confirmed may be confirmed by a composition or method according to an embodiment of the present application, and the composition or method may be used for cancer diagnosis.

[0049] Thus, another example of the present application relates to a composition or kit for diagnosing cancer, comprising a composition for confirming the presence or absence of methylation of a target nucleic acid according to an example of the present application.

[0050] Another example of the present application relates to a method for diagnosing cancer or a method for providing information for cancer diagnosis, comprising a step of confirming the presence or absence of methylation of a nucleic acid to be confirmed by a method for confirming the presence or absence of methylation of a nucleic acid to be confirmed according to an example of the present application. The method for diagnosing cancer or the method for providing information for cancer diagnosis may further comprise a step of comparing the methylation level of the nucleic acid to be confirmed with the methylation level of a control group.

[0051] The control group refers to nucleic acid isolated from a biological sample whose subject of origin is already known, and can be any sample derived from subjects without liver cancer (normal control group) or subjects with liver cancer.

[0052] For example, when a sample derived from a subject without liver cancer (normal control group) is used as a control group, if the methylation level of the target nucleic acid is higher than the methylation level of the nucleic acid isolated from the control group, or if the biological sample contains a target nucleic acid with a higher methylation level than the control group, this may provide information for determining, diagnosing, or diagnosing that the organism (e.g., vertebrate, mammal, rodent, goat, deer, pig, bird, chicken, turkey, cow, horse, sheep, fish, primate, such as a human) from which the target nucleic acid or biological sample was derived has cancer.

[0053] For example, when a sample derived from a subject with liver cancer is used as a control, if the methylation level of the nucleic acid to be confirmed is similar to the methylation level of a nucleic acid isolated from the control, or if a biological sample contains a nucleic acid to be confirmed that has a methylation level similar to that of the control, this may provide information for determining, diagnosing, or diagnosing that the organism from which the nucleic acid to be confirmed or the biological sample was derived (e.g., a vertebrate, mammal, rodent, goat, deer, pig, bird, chicken, turkey, cow, horse, sheep, fish, primate, including, for example, a human) has cancer.

[0054] The cancer may include one or more selected from the group consisting of liver cancer, colorectal cancer, esophageal cancer, gastric cancer, rectal cancer, colon cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, non-small cell lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, biliary tract cancer, bone cancer, connective tissue cancer, skin cancer, melanoma, brain cancer, head and neck cancer, thyroid cancer, leukemia, Hodgkin's disease, lymphoma, urinary tract cancer, and multiple myeloma blood cancer.

[0055] The liver cancer may be one or more types selected from the group consisting of hepatocellular carcinoma, hepatocellular carcinoma, cholangiocarcinoma, intrahepatic cholangiocarcinoma, hepatoblastoma, hepatocarcinoma, hepatic angiosarcoma or metastatic liver cancer, vaguely nodular type HCC, and low vascular liver cancer.

[0056] Another example of the present application relates to a method for treating cancer, comprising treating a subject identified as having cancer by a cancer diagnostic composition, a cancer diagnostic kit, a cancer diagnostic method, or a method for providing information for cancer diagnosis according to an example of the present application, wherein the treating step may include administering an effective amount of a therapeutic agent, chemotherapy, hormone therapy, radiation therapy, surgery, or a combination thereof to the subject.

[0057] Examples of the therapeutic agent include apatinib, AK105, anlotinib, apatinib, atezolizumab, avelumab, axitinib, bevacizumab, bosutinib, BSC, carbozantinib, carbozantinib-S-malate, camrelizumab, canertinib, carboplatin, and capecitabine. (capecitabine), celecoxib, CC-122, CF102, crizotinib, dasatinib, docetaxel, donafenib, dovitinib, doxorubicin, durvalumab, EKB-569, entrectinib, epirubicin, erlotinib, etoposide, everolimus, FGF401, FOLFOX 4, fostamatinib, galunisertib, gefitinib, gemcitabine, IBI305, ibrutinib, imatinib, INC280, infigratinib, ipilimumab, irinotecan, lapatinib, leflunomide, lenvatinib, LY2875358, mesylate, mitomycin cc), MSC2156119J, neratinib, nilotinib, nintedanib, nivolumab, oxaliplatin, palbociclib, panobinostat, pazopanib, PDR001, pembrolizumab, pemigatinib, pexavec, phosphate, ramucirumab, regorafenib, ruxolitinib, semaxinib, selumetinib, SGO-110, SHR-12 10, Sintilimab, Sorafenib, SU6656, Sunitinib, Sintilimab, Spartalizumab, sutent, TACE, Tasquinimod, Temozolomide, Temsirolimus, Tislelizumab, Tivantinib, Tosylate, Toripalimab, Tremelimumab, Vandetanib, Vatalanib, XL888, Y90, a pharma- ceutical acceptable salt thereof, or a combination thereof. Effect of the Invention

[0058] One example of the present application is capable of constructing a new experimental validation design that complements the shortcomings of existing qMSP, MethyLight, and MS-HRM analytical methods, and is capable of detecting DNA methylation markers through liquid biopsy, thereby enabling quantification of methylation levels with higher sensitivity than existing MS-HRM analytical methods.

[0059] One example of the present application is that PCR can be performed at a more sensitive level for targets present in extremely small amounts, such as circulating tumor DNA in blood, and experiments can be designed by adjusting the degree of binding of methylated and unmethylated primers to the template strand according to the application, by adjusting the annealing temperature and primer concentration ratio. [Brief description of the drawings]

[0060] [Figure 1a] 1 shows the results of an experiment using MIP (Methylation Independent Primer) for conventional MS-HRM analysis. [Figure 1b] 1 shows the results of an experiment using MIP (Methylation Independent Primer) for conventional MS-HRM analysis. [Figure 1c] 1 shows the results of an experiment using MIP (Methylation Independent Primer) for conventional MS-HRM analysis. [Figure 2a] FIG. 1 shows the results of an attempt to improve the amplification by including CpG at the 5′-end of a primer in order to suppress non-methylation biased amplification that occurs during PCR amplification using MIPs. [Figure 2b] FIG. 1 shows the results of an attempt to improve the amplification by including CpG at the 5′-end of a primer in order to suppress non-methylation biased amplification that occurs during PCR amplification using MIPs. [Figure 2c] FIG. 1 shows the results of an attempt to improve the amplification by including CpG at the 5′-end of a primer in order to suppress non-methylation biased amplification that occurs during PCR amplification using MIPs. [Figure 3a] This is a diagram confirming the phenomenon that the amplification of a marker is hindered when a fluorescent probe used in methylite analysis is used. [Figure 3b] This is a diagram confirming the phenomenon that the amplification of a marker is hindered when a fluorescent probe used in methylite analysis is used. [Figure 4a]FIG. 1 shows the results of MS-HRM analysis using a mixture of methylated and unmethylated primers. [Figure 4b] FIG. 1 shows the results of MS-HRM analysis using a mixture of methylated and unmethylated primers. [Figure 4c] FIG. 1 shows the results of MS-HRM analysis using a mixture of methylated and unmethylated primers. [Figure 5a] 1 is a graph showing the distribution of melting peaks of each sample according to the methylation ratio of the sample. [Figure 5b] 1 shows melting curve analysis of the results of HRM analysis of each sample according to the methylation ratio of the sample. [Figure 6a] This is a diagram confirming whether quantitative amplification occurs when a mixture of methylated and unmethylated primers is used in one experimental tube. [Figure 6b] 1 is a diagram showing the results of confirming the accuracy of methylation ratio measurement using mAUC (melting curve area under curve), which indicates the methylation ratio. [Figure 6c] This figure shows that a method for confirming the presence or absence of methylation according to one example of the present application distinguishes between a 1% level of methylated DNA concentration (three strands of methylated DNA) and a 0% concentration, and confirms that 1% methylated DNA can be detected in a sample containing 99% unmethylated DNA. [Figure 7] 1 is a graph showing whether or not biased amplification of methylated DNA occurs depending on the concentration ratio of methylated primer to unmethylated primer. [Figure 8] 8a and 8b are diagrams showing preliminary clinical results of diagnosing cancer by applying the method for determining the presence or absence of methylation according to one embodiment of the present application to actual blood. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] The present application will be described in more detail below with reference to the following examples, but these examples are merely for the purpose of illustrating the present application and are not intended to limit the scope of the present application.

[0062] Comparative Example 1. Confirmation of the limitations of conventional MS-HRM analysis methods The limitations of conventional MS-HRM analysis methods were confirmed using a nucleic acid having the sequence of SEQ ID NO: 1 as an example of a nucleic acid to be confirmed. The nucleic acid sequence of SEQ ID NO: 1 is a liver cancer biomarker that is specifically methylated in liver cancer cells, and DNA isolated from liver cancer cells will contain the methylated nucleic acid of SEQ ID NO: 1, while DNA isolated from other cells will contain the unmethylated nucleic acid of SEQ ID NO: 1. Various samples containing the nucleic acid of SEQ ID NO: 1 and control samples were prepared as shown in Table 1 and used in the experiment. Samples Nos. 2 to 4 were purchased from Qiagen and used. Sample No. 2 was obtained by converting all cytosine residues to thymine by sulfite treatment of gDNA of Sample No. 3 using EpiTect Bisulfite Kit, and Sample No. 4 was obtained by 100% methylation of gDNA of Sample No. 3 using SssI methylase, followed by sulfite conversion treatment using EpiTect Bisulfite Kit to convert cytosines other than CpG cytosines to thymine. Samples Nos. 5 to 7 were subjected to sulfite treatment prior to PCR reaction to convert unmethylated cytosine to thymine.

[0063] [Table 1]

[0064] Conventional MS-HRM analysis uses methylation independent primers (MIPs) that do not contain CpG or TpG recognition sites so that sulfite-treated methylated and unmethylated nucleic acids can be nonselectively amplified. The sequences of the methylation independent primers used are as follows:

[0065] - Forward: gTtgTatTatTtgTTaggggTtgT (SEQ ID NO: 7) - Reverse: ccacaAAAcctccaAAcaAtAA (SEQ ID NO: 8)

[0066] MS-HRM Master Mix was prepared by mixing the components shown in Table 2. The volume shown in Table 2 is the amount required for one sample. When multiple samples are to be tested, prepare and use Master Mix at a level equal to the number of samples + 2 (22uL / rxn).

[0067] [Table 2]

[0068] The prepared Master Mix was dispensed in 22uL portions into each well of a 96-Well PCR plate (Hard-Shell® PCR Plates, Biorad). Seven samples according to Table 1 were prepared to have a concentration of 0.33ng / uL, and 3uL portions were dispensed into each well so that the total amount of DNA was 1ng. PCR reactions were performed using a CFX96 Touch Real-Time PCR Detection System (Biorad) at a denaturation temperature of 95°C for 5 minutes once, and 50 cycles of a denaturation temperature of 95°C for 20 seconds, a heating / cooling temperature of 60°C for 30 seconds, and an extension temperature of 72°C for 30 seconds were repeated. In addition, after 30 seconds of heating / cooling, the fluorescence value of each cycle was confirmed through fluorescence measurement before starting the extension temperature step. After the PCR reaction was completed and a stabilization period of 5 minutes at 72°C was performed, a melting analysis was performed to confirm the degree of binding of the amplified products due to the increase in temperature, and the fluorescence intensity was measured at 10-second intervals from 65°C to 95°C by increasing the temperature by 0.2°C., and then HRM analysis was performed.

[0069] The fluorescence values ​​for PCR and HRM analysis were confirmed, and the melting curve or peak was confirmed to confirm the characteristics of each sample. After PCR was completed, electrophoresis was performed to confirm whether the size of the amplified product was generated correctly by the designed primers.

[0070] As shown in Figure 1a, the amplification of the sample in which the target nucleic acid was 100% methylated (EpiTect Met; sample number 4) and the sample in which the target nucleic acid was 100% unmethylated (EpiTect Unmet; sample number 2) was almost equal. Furthermore, the amplification was equally efficient in liver cancer cells (Huh-1; sample number 6), kidney cells (Hek293T; sample number 5), and blood cells (PBMC; sample number 7), and it was confirmed by the increase in the PCR fluorescence RFU value that all of the target nucleic acids, both methylated and unmethylated, were amplified. However, there was a problem in that nonspecific amplification occurred due to MIP binding even in the control group that was not treated with sulfite (EpiTect gDNA; sample number 3) and the control group that did not contain the target nucleic acid (NTC; sample number 1), which should not be amplified.

[0071] As shown in Figure 1b, the amplified DNA was confirmed to be methylated markers, non-methylated markers, and non-specific MIP-amplified DNA through High Resolution Melting Curve (HRM) analysis. In addition, as shown in Figure 1c, the electrophoresis results confirmed the presence of multiple non-specific amplified DNAs.

[0072] Therefore, conventional MS-HRM analysis methods have problems in that the genomic DNA sequence is simplified by sulfite treatment, and the simple MIP primer sequence, which consists only of adenine, guanine, and thymine, frequently generates non-specific amplification products, limiting the amplification of target nucleic acids.

[0073] Comparative Example 2. Confirmation of the limitations of the MS-HRM analysis method using primers with added CpG recognition sites to prevent biased amplification of unmethylated nucleic acids In order to suppress non-methylation biased amplification that occurs during amplification of PCDR using methylation-independent primers, we attempted to improve the method by including a CpG sequence at the 5'-end of the primer. The MIP primer used in Example 1 was changed to the following primer, and PCR was performed in the same manner for sample numbers 2, 3, 4, and 6 in Table 1, followed by HRM analysis and electrophoresis. The sequences of the primers used are as follows:

[0074] - Forward: TTtCGgTtgTatTatTtgTTaggggT (SEQ ID NO: 9) - Reverse: AACGccccacaAAAcctccaAA (SEQ ID NO: 10)

[0075] As shown in Figure 2a, MIP was biased to amplify methylated DNA more favorably by adding CpG to the 5' end. As a result, 100% methylated sample (EpiTect Met; sample number 4) and hepatoma cell DNA (Huh-1; sample number 6) were effectively amplified, while unmethylated DNA sample (EpiTect Unmet; sample number 2) and the control group not treated with sulfite (EpiTect gDNA; sample number 3) were not amplified. However, as shown in the HRM analysis results in Figure 2b and the electrophoresis analysis results in Figure 2c, nonspecific amplification occurred in the unmethylated sample. Therefore, when unmethylated samples were analyzed under biased conditions due to methylation, there was a problem that nonspecific amplification was very high.

[0076] Comparative Example 3. Confirmation of the Limitations of MS-HRM Analysis Method Using Fluorescent Probes The PCR amplification efficiency was confirmed when a fluorescent probe used in methylite analysis was used, as in Comparative Example 1. The results of MS-HRM for a liver cancer sample (Huh-1; sample number 2) and a 100% methylated sample (EpiTect Met; sample number 1) using MIP as in Comparative Example 1 are shown in FIG. 3a, and the results of the same experiment were shown in FIG. 3b, except that 10 uL of NFW was replaced with a fluorescent probe (ggTTCGTTaCGTtgTTTt; sequence number 11) in the composition of Table 2. A sample not treated with sulfite (EpiTect gDNA; sample number 3) was used as a control in each experiment.

[0077] Figure 3a shows the results of measuring the amount of amplified DNA by PCR Cq value when no fluorescent probe that specifically binds to the nucleic acid to be methylated was added, and Figure 3b shows the results when the fluorescent probe was added. As shown in Figure 3b, the fluorescent probe increased the Cq value by about 4, resulting in a problem of a 10-20-fold decrease in amplification efficiency.

[0078] Example 1. Preparation of methylated and unmethylated primers As an example of a nucleic acid to be confirmed, a primer was prepared to confirm the presence or absence of methylation of a nucleic acid having the sequence of SEQ ID NO: 1. The nucleic acid sequence of SEQ ID NO: 1 is shown in Table 3, with the target site shown in italics and the primer binding site underlined. In the sequence of SEQ ID NO: 1, CpG and non-CpG cytosines are written in uppercase letters, and other bases are written in lowercase letters. In the sequence of SEQ ID NO: 1, the CpG sequence is shown in bold.

[0079] The nucleic acid sequence of the methylated nucleic acid sequence of SEQ ID NO: 1 after sulfite treatment is shown in SEQ ID NO: 2, and the nucleic acid sequence of the unmethylated nucleic acid sequence of SEQ ID NO: 1 after sulfite treatment is shown in SEQ ID NO: 3. In SEQ ID NO: 2 and SEQ ID NO: 3, bases that have differences due to sulfite treatment depending on whether or not CpG cytosine is methylated are shown in bold.

[0080] Considering the sequence of the template strand to be converted after sulfite treatment, one of the forward or reverse primers was designed to contain a CpG or TpG sequence near the 3'-end so that it could specifically bind to either the methylated or unmethylated template strand. Since the CpG cytosine remains in the methylated template strand after sulfite treatment without being converted to thymine, the methylated primer was designed to contain a CpG recognition site that recognizes the CpG sequence, and the unmethylated primer was designed to contain a TpG recognition site that recognizes the TpG sequence. In this case, since the primer that specifically binds to the unmethylated template strand has a lower Tm than the primer that specifically binds to the methylated template strand, a nucleotide can be added to the 5'-end to complement this. The primers in the opposite direction to the methylated and unmethylated primers were designed to be able to bind regardless of the methylation state of the methylated and unmethylated strands. All primers contained two or more non-CpG cytosines so that they could specifically bind to the sulfite-converted DNA strand.

[0081] The forward primer was designed to bind to the 147th to 171st nucleic acid of the nucleic acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3, and the reverse primer was designed to bind to the 206th to 229th nucleic acid of the nucleic acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3. The primer binding sites of SEQ ID NO: 2 or SEQ ID NO: 3 are underlined.

[0082] Two types of forward primers were prepared: a methylated primer capable of binding to the 147th to 171st nucleic acids in the nucleic acid sequence of SEQ ID NO: 2, and an unmethylated primer capable of binding to the 206th to 229th nucleic acids in the nucleic acid sequence of SEQ ID NO: 3. In this case, the reverse primer was a common reverse primer capable of binding to both the sulfite-treated nucleic acid to be confirmed for methylation and the sulfite-treated unmethylated nucleic acid to be confirmed.

[0083] The sequences of the prepared primers are shown in Table 3. The sequences that differ between the methylated and unmethylated primers depending on the presence or absence of methylation of the target nucleic acid are underlined.

[0084] [Table 3]

[0085] Example 2. MS-HRM using methylated and unmethylated primers MS-HRM analysis was carried out using the primers prepared in Example 1. MS-HRM Master Mix was prepared by mixing the components shown in Table 4. The volumes shown in Table 4 are the amounts required for one sample. When multiple samples are to be tested, the Master Mix should be prepared to be equal to the number of samples + 2 (22 uL / rxn).

[0086] [Table 4]

[0087] Various samples containing the nucleic acid of SEQ ID NO: 1 and control samples were prepared as shown in Table 5 and used in the experiment.

[0088] [Table 5]

[0089] The prepared Master Mix was dispensed in 22uL portions into each well of a 96-well PCR plate (Hard-Shell® PCR Plates, Biorad). Six samples shown in Table 5 were prepared to contain a concentration of 0.33ng / uL, and 3uL of each sample was dispensed into each well so that the total amount of DNA was 1ng.

[0090] PCR was performed using a CFX96 Touch Real-Time PCR Detection System (Biorad) at a denaturation temperature of 95°C for 5 minutes, followed by 50 cycles of denaturation at 95°C for 20 seconds, heating and cooling at 60°C for 30 seconds, and extension at 72°C for 30 seconds. After 30 seconds of heating and cooling, the fluorescence value of each cycle was measured by fluorescence measurement before starting the extension temperature step. After the PCR reaction was completed and a stabilization period at 72°C for 5 minutes, a melting analysis was performed to check the degree of binding of the amplified products due to the increase in temperature, increasing the temperature from 65°C to 95°C in 0.2°C increments, measuring the fluorescence intensity at 10-second intervals, and then performing HRM analysis.

[0091] The fluorescence values ​​for PCR and HRM analysis were confirmed, and the melting curve or peak was confirmed to confirm the characteristics of each sample. After PCR was completed, electrophoresis was performed to confirm whether the size of the amplified product was generated correctly by the designed primers.

[0092] Methylated and unmethylated primers were added simultaneously to one sample, and the target nucleic acid was amplified under conditions where both methylated and unmethylated DNA could be amplified. As shown in Figure 4a, amplification occurred equally well in the methylated DNA (EpiTect Met and Huh-1) and unmethylated DNA (EpiTect Unmet and PBMC) treated with sulfite, while no amplification occurred in the control samples (NTC and EpiTect gDNA not treated with sulfite). The results of HRM analysis in Figure 4b and electrophoresis analysis in Figure 4c confirmed that only specific amplification occurred.

[0093] Example 3. Confirmation of detection limit and quantification of methylation levels To confirm the minimum detection limit of the methylation ratio of the samples, PBMC (0.33 ng / uL) and Huh-1 (0.33 ng / uL) were mixed to prepare samples with methylation ratios of 50%, 25%, 12.5%, and 6.25% for use in the experiment. 3uL of each sample with a methylation ratio was dispensed into each well, and PCR and HRR analysis were performed in the same manner as in Example 2. The results are shown in Figure 5a and Figure 5b.

[0094] Figure 5a shows the distribution of melting peaks in each sample after mixing sulfite-treated hepatoma cell DNA with sulfite-treated blood cell DNA at the indicated ratios and analyzing 1 ng of DNA using the primer-mixed MS-HRM method. It can be seen that the size and ratio of the melting peaks of methylated DNA and unmethylated DNA change gradually depending on the ratio of methylated Huh-1 DNA.

[0095] Figure 5b shows the melting curve analysis of the HRM analysis results. As the amount of hepatoma cell DNA increases, the slope of the melting curve gradually increases and is between the slope of unmethylated blood cells and the slope of 100% methylated hepatoma cells. Therefore, the methylation ratio can be quantified by calculating the area under the melting curve. Specifically, the methylation level of the unknown sample can be quantified as the percentage of the difference between the melting curve AUC of the unknown sample and the melting curve AUC of the 100% unmethylated sample divided by the difference between the melting curve AUC of the 100% methylated sample and the melting curve AUC of the 100% unmethylated sample.

[0096] More specifically, in order to remove exponential background noise from the total fluorescence value by temperature, temperatures at which the MS-HRM fluorescence change rate is constant within the upper and lower ranges of the melting temperature (Tm) were selected (each T L , T R), and estimate the exponential background noise parameters from the fluorescence change rate (dF / dT) at each point. The exponential background noise value is calculated from the estimated parameters and removed from the total fluorescence value at each temperature point to calculate the fluorescence data function M(T) proportional to the amount of residual dsDNA. M(T) is subjected to min-max normalization within the temperature range [TL, TR] to calculate the residual dsDNA ratio function M1(T) with increasing temperature. To derive the methylation level, the area under the curve (AUC) value S of M1(T) within the [TL, TR] range is calculated from each sample, and the degree of methylation of the sample can be confirmed based on the S value.

[0097] Example 4. Confirmation of detection sensitivity Since ctDNA exists in very low concentrations in blood, the detection limit was confirmed to confirm whether the method for confirming the presence or absence of methylation according to one example of the present application can actually be used for blood test-based cancer diagnosis.

[0098] In order to confirm the minimum DNA strand that can be confirmed by the method for confirming the presence or absence of methylation according to one example of the present application, PBMC and Huh-1 (50 ng / 3 uL) samples were diluted with NFW to prepare Huh-1 samples with concentrations of 50, 30, 10, 5, 3, 1, 0.5, 0.3, 0.1, 0.05, or 0.01 ng / 3 uL. 3 uL of each sample was dispensed into each well, and PCR reaction and HRR analysis were performed in the same manner as in Example 2.

[0099] Methylated and unmethylated mixed primer MS-HRM analysis was performed using sulfite-treated hepatoma cell DNA in amounts ranging from 0.01ng to 50ng, a difference of 5,000-fold, and the amount of amplified nucleic acid to be confirmed was analyzed by measuring the PCR Cq value, as shown in Figure 6a. As shown in Figure 6a, when 0.01ng (3 strands) of DNA was used, the amplification started with a Cq value of about 38 and increased the amount of DNA to 50ng (15,200 strands), resulting in a decrease in the Cq value that was in exact agreement with the increase in DNA amount. As a result, it was found that the methylated and unmethylated primer mixed MS-HRM analysis effectively amplified 0.01ng of DNA, and was capable of effective amplification over a wide range of 5,000-fold.

[0100] When analyzing various amounts of DNA, the results of an analysis using mAUC (melting curve area under curve), which indicates the methylation ratio, to determine whether accurate methylation ratio measurement is possible, are shown in Figure 6b. As shown in Figure 6b, it was confirmed that the AUC value was consistent and did not vary significantly depending on the amount of each sample, and the amplified product of each sample was the nucleic acid to be confirmed, allowing the methylation level of each sample to be confirmed. Therefore, it was possible to confirm the presence or absence of methylation of the nucleic acid to be confirmed even in samples where the nucleic acid to be confirmed contains at least three strands.

[0101] In addition, in order to confirm the minimum ratio at which the method for confirming the presence or absence of methylation according to one embodiment of the present application can confirm the target methylated DNA in the presence of unmethylated DNA, unmethylated DNA and methylated DNA were serially diluted to prepare samples of 0, 1, 2, 4, 8, 16, 32, and 100%, and MS-HRM analysis was performed to confirm the correlation between the change in AUC value and the ratio, which is shown in FIG. 6c.

[0102] As shown in Figure 6c, the minimum detectable ratio in the presence of unmethylated DNA was confirmed, and it was found that there was a difference between the 1% methylated DNA concentration level (3 strands of methylated DNA) and the 0% concentration. A high R value was also confirmed in the correlation analysis between the AUC value and the ratio of methylated DNA.

[0103] Therefore, it was confirmed that the method for determining the presence or absence of methylation according to one embodiment of the present application has high detection sensitivity capable of detecting 1% methylated DNA even in a sample containing 99% unmethylated DNA.

[0104] Example 6. Improved detection sensitivity using unmethylated primers To confirm whether unmethylated primers improve detection sensitivity, samples containing 6.25, 25.0, 50.0, or 100.0% Huh-1 were used. The procedure was the same as in Example 5, except that the volume of the unmethylated forward primer in the composition of Table 3 was changed to 1, 0.75, 0.5, or 0.25 uL, and the NFW volume was changed to 5.25, 5.50, 5.75, or 6 uL.

[0105] We confirmed whether biased amplification of methylated DNA occurs depending on the concentration ratio of methylated primer to unmethylated primer. PCR was performed under the condition that the amount of methylated primer was fixed at 0.4uM and the amount of unmethylated primer was decreased to 0.4, 0.3, 0.2, and 0.1uM.

[0106] As shown in FIG. 7, when the DNA ratio of methylated hepatoma cells was low (25% or less), the sensitivity of detecting methylated DNA was higher when the amount of unmethylated primer was 50% or less of the methylated primer.

[0107] Therefore, the combined use of methylated and unmethylated primers increased detection sensitivity and made it possible to confirm the presence or absence of methylation in low concentrations of methylated DNA, such as cfDNA.

[0108] Example 7. Cancer diagnosis by measuring the presence or absence of methylation of cancer biomarkers in blood A preliminary clinical trial was conducted to determine whether or not the methylation of the target nucleic acid could be confirmed by applying the mixed MS-HRM analysis of methylated and unmethylated primers to actual blood.

[0109] After extracting 10 mL of blood from healthy subjects and liver cancer patients, plasma was separated using a centrifuge (1,900 g, 15 min). cfDNA was extracted from the separated plasma using the MagListo™ cfDNA Extraction Kit (Bioneer) according to the method provided by the manufacturer. cfDNA extracted using the EZ DNA Methylation-Lightning Kits (Zymo Research) according to the method provided by the manufacturer was subjected to sulfite conversion for MS-HRM analysis. The sulfite converted cfDNA was subjected to MS-HRM analysis through the following process.

[0110] MS-HRM Master Mix was prepared according to Table 3, and 22 uL of the prepared Master Mix was dispensed into each well of a 96-well PCR plate (Hard-Shell® PCR Plates, Biorad), and PCR reaction and HRR analysis were performed in the same manner as in Example 2. Fluorescence values ​​for PCR and HRM analysis were confirmed, and the melting curve or peak was confirmed to confirm the methylation levels of healthy subjects and liver cancer patients.

[0111] Figure 8a shows the results of taking 2ml of blood from 92 healthy subjects and 119 liver cancer patients, isolating plasma, isolating cfDNA from this, treating it with sulfite, and then performing mixed primer MS-HRM using approximately 1ng of cfDNA. The analysis results for the healthy subjects (green) all show a non-methylated low-temperature melting peak, while the liver cancer patient blood samples (red) show a high-temperature melting peak.

[0112] Figure 8b shows the melting curve analysis results of an experiment using MS-HRM method for the nucleic acid of SEQ ID NO: 1, which was confirmed by mixing blood from a normal person and a liver cancer patient. The normal person (green) is unmethylated, and the amplified DNA melts quickly, showing a steep curve slope, whereas the liver cancer patient blood sample (red) contains cancer DNA with a high melting point due to the amplification of methylated cancer DNA, showing a relatively slow melting curve slope. It was confirmed that more than 60% of liver cancer patient cfDNA contains more highly methylated DNA than normal person cfDNA, and therefore it is possible to effectively detect trace amounts of methylation marker DNA present in blood by detecting extremely small amounts of nucleic acid such as cfDNA, for example, methylated cfDNA that is very small and broken down to about 100 bp.

Claims

1. A primer set for amplifying a target site of a nucleic acid to be confirmed, The primer set includes: (1) a methylated forward primer, an unmethylated forward primer, and a common reverse primer; or (2) a common forward primer, a methylated reverse primer, and an unmethylated reverse primer; the methylated forward primer and the methylated reverse primer each contain a CpG recognition site that recognizes a CpG sequence in the nucleic acid to be confirmed; the unmethylated forward primer and the unmethylated reverse primer each contain a TpG recognition site that recognizes a TpG sequence in the nucleic acid to be confirmed; The TpG sequence is a sequence obtained by converting a CpG sequence of the nucleic acid to be confirmed. A composition for confirming the presence or absence of methylation of a target nucleic acid.

2. The composition of claim 1 , wherein the TpG sequence is altered by an agent that alters methylated and unmethylated target nucleic acids to be confirmed differently from each other.

3. The composition of claim 2 , wherein the modifying agent converts unmethylated cytosine residues to thymine.

4. 3. The composition of claim 2, wherein the agent to be transformed is one or more selected from the group consisting of sulfite, bisulfite, hydrogen sulfite, and disulfite.

5. 2. The composition of claim 1, wherein the CpG recognition site or the TpG recognition site is located within 40 bases from the 3' end of the methylated forward primer, the methylated reverse primer, the unmethylated forward primer, or the unmethylated reverse primer.

6. The composition of claim 1 , wherein the target site comprises a CpG sequence.

7. The composition of claim 1, wherein the target site is between 50 and 150 bp in size.

8. 2. The composition of claim 1, wherein the primers are between 15 and 40 bases in size.

9. The composition of claim 1 , wherein the common reverse primer or the common forward primer contains 5 or less CpG or TpG recognition sites of the nucleic acid to be confirmed.

10. The composition of claim 1, wherein the Tm difference between the methylated forward primer and the unmethylated forward primer in (1); or the Tm difference between the methylated reverse primer and the unmethylated reverse primer in (2) is 15° C. or less.

11. The methylated forward primer and the unmethylated forward primer according to (1); or The composition according to claim 1, wherein the methylated reverse primer and the unmethylated reverse primer (2) are contained in a concentration ratio of 100:1 to 1:

100.

12. The composition according to claim 1 , wherein the nucleic acid to be confirmed is contained in a biological sample.

13. The composition of claim 12, wherein the biological sample comprises one or more selected from the group consisting of blood, plasma, tissue, cells, stool and urine.

14. The composition of claim 12, wherein the biological sample comprises a methylated nucleic acid to be confirmed and an unmethylated nucleic acid to be confirmed.

15. The composition of claim 14, wherein the concentration of the methylated nucleic acid to be confirmed is 75% or less than the concentration of the unmethylated nucleic acid to be confirmed.

16. The composition according to claim 1 , wherein the nucleic acid to be confirmed comprises a biomarker for diagnosing cancer.

17. The composition of claim 16, wherein the cancer comprises one or more selected from the group consisting of liver cancer, colon cancer, esophageal cancer, gastric cancer, rectal cancer, colorectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, non-small cell lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, biliary tract cancer, bone cancer, connective tissue cancer, skin cancer, melanoma, brain cancer, head and neck cancer, thyroid cancer, leukemia, Hodgkin's disease, lymphoma, urinary tract cancer, and multiple myeloma blood cancer.

18. The composition of claim 1 , wherein the target site is a biomarker located within a CpG island.

19. modifying nucleic acids to be confirmed contained in a biological sample so that methylated nucleic acids to be confirmed and unmethylated nucleic acids to be confirmed are different from each other; and 20. The method of claim 1, further comprising the step of treating said biological sample with a composition according to any one of claims 1 to 18 to amplify the target site. A method for confirming the presence or absence of methylation of a target nucleic acid.

20. The method according to claim 19, wherein the modifying step is carried out by treating the biological sample with a preparation that modifies methylated and unmethylated target nucleic acids to be confirmed differently from each other.

21. 20. The method of claim 19, wherein the biological sample is one or more selected from the group consisting of blood, plasma, tissue, cells, stool and urine.

22. The method according to claim 19, further comprising a step of quantifying the degree of methylation of the nucleic acid to be confirmed.

23. The method according to claim 22, wherein the quantifying step comprises comparing the AUC (area under the curve) of normalized melting curves of the biological sample, a sample in which the nucleic acid to be confirmed is 100% methylated, and a sample in which the nucleic acid to be confirmed is 100% unmethylated, to quantify the degree of methylation of the biological sample.

24. obtaining a melt curve for said biological sample; Obtaining a normalized melt curve of a biological sample having a known methylation ratio of the nucleic acid to be identified; and 20. The method of claim 19, further comprising the step of comparing the melting curve of the biological sample and the normalized melting curve to quantify the degree of methylation of the biological sample.

25. 20. The method of claim 19, wherein the target site comprises a biomarker located within a CpG island.

26. modifying nucleic acids to be confirmed contained in a biological sample so that methylated nucleic acids to be confirmed and unmethylated nucleic acids to be confirmed are different from each other; Treating the biological sample with a composition according to any one of claims 1 to 18 to amplify it; and confirming whether or not the nucleic acid to be confirmed is methylated; The nucleic acid to be confirmed includes a biomarker for diagnosing cancer. A method for providing information for cancer diagnosis.

27. 27. The method of claim 26, wherein the cancer diagnostic biomarker nucleic acid is methylated or unmethylated in a cancer patient.

28. 27. The method of claim 26, further comprising the step of comparing the methylation level of the nucleic acid to be confirmed with the methylation level of a control group.

29. The method of claim 26, wherein the cancer comprises one or more selected from the group consisting of liver cancer, colon cancer, esophageal cancer, gastric cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's disease, lymphoma, and multiple myeloma blood cancer.

30. 27. The method of claim 26, wherein the cancer diagnostic biomarker is located within a CpG island.

31. A kit for confirming the presence or absence of methylation of a target nucleic acid, comprising the composition according to any one of claims 1 to 18.

32. A composition for cancer diagnosis comprising the composition according to any one of claims 1 to 18, wherein the nucleic acid to be confirmed comprises a cancer biomarker.

33. A kit for diagnosing cancer comprising the composition of claim 30.

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