Composition for confirming the presence or absence of nucleic acid methylation and method for confirming 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 confirming the presence or absence of methylation of nucleic acid and a method for confirming the presence or absence of methylation of nucleic acid.

Background Art

[0002] There are methods such as qMSP, MethyLight, and MS-HRM for measuring the methylation level of a DNA sample, and all the methods are constructed to relatively confirm the methylation level of each sample. For example, the qMSP method is a method for confirming the methylation level by measuring the PCR Cq value of the amount of DNA produced by amplifying the DNA under such conditions using a primer that binds only to a methylated (or unmethylated) template strand for the same amount of DNA sample. The Methylight method is a method for enhancing the specificity of measurement by adding a probe to qMSP and binding the DNA specific to the marker to be confirmed among the amplified DNA. Also, the MS-HRM method is a method for confirming the signal of the melting point difference of the amplification product due to temperature increase by using a primer that can bind to the template strand regardless of the methylation state of the DNA sample used and including a certain number of CpGs in the portion of the amplification product excluding the primer binding region.

[0003] qMSP and MethyLight analysis have the advantage of being convenient in experimental design due to fewer limitations on primer design, and can achieve relatively high sensitivity. However, they require additional control group marker analysis to correct for the sample volume used in the analysis, and the quantitative analysis is indirect and limited because the results are used to correct the measured values, resulting in a high frequency of type 2 errors. Furthermore, biased amplification of specific template strands can increase the frequency of non-specific amplification in each DNA sample, leading to a disadvantage of low specificity. In the case of MethyLight, which was developed as a way to compensate for specificity, an additional fluorescent probe that can specifically bind to the amplification product is required, which is a disadvantage as it increases costs and the difficulty of designing analytical tests. In many cases, the specific binding of the probe also reduces the amplification efficiency of PCR, which is a limitation as it makes it difficult to analyze very small sample sizes.

[0004] In MS-HRM analysis, methylated and unmethylated nucleic acids are amplified simultaneously in the same test tube, allowing for a more intuitive quantitative measurement of the methylation level of a DNA sample than MSP, without the need for additional control marker analysis. However, due to design limitations such as the need to either not include CpG in the primer or include nonspecific bases, developing a specific analytical method is not easy. Furthermore, if CpG is absent in the primer, there is a problem of biased binding to unmethylated strands rather than methylated strands, which can lead to type 1 errors due to the unmethylation bias in the validation results for the sample. In experiments requiring sensitive methylation mutation measurement, such as cancer diagnosis, this can result in underestimating the methylation ratio in the actual sample or yielding inaccurate results. [Overview of the project] [Problems that the invention aims to solve]

[0005] One example of this application aims to construct a method for confirming the presence or absence of methylation that complements the shortcomings of conventional experimental verification methods. The method for confirming the presence or absence of methylation according to this example allows for the measurement of the relative methylation ratio in the same test tube without the need for control marker analysis, and has high detection sensitivity because it does not use probes that interfere with amplification efficiency. Furthermore, it is possible to adjust the amplification efficiency of methylated and unmethylated DNA to measure very low methylation levels in biological samples such as blood.

[0006] The composition and method according to one example of this application, by using a mixture of methylated and non-methylated primers, does not generate nonspecific amplification products and can measure the methylation level of a sample more sensitively than existing PCR-based analytical methods, even under conditions where the desired target nucleic acid is present in minute amounts.

[0007] One example of this application aims to provide a composition for confirming the presence or absence of methylation in a target nucleic acid with high sensitivity, and a method for confirming the presence or absence of methylation in a target nucleic acid, in order to complement the shortcomings of conventional methods for measuring the methylation level of a DNA sample.

[0008] Another example of this application is to provide a primer design strategy and optimization method that can be universally applied to a variety of DNA methylation markers.

[0009] Another example of this application is to provide a cancer diagnostic method, a method for providing information for cancer diagnosis, a cancer diagnostic composition, or a cancer diagnostic kit that confirms 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 biological samples, such as blood, through a semi-quantitative method within the sample, and achieves high sensitivity and specificity based on this. [Means for solving the problem]

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

[0011] Another example of this application is a method for determining whether or not a nucleic acid to be confirmed is methylated, comprising the steps of: deforming a target nucleic acid contained in a biological sample so that methylated and unmethylated target nucleic acids are different from each other; and treating the biological sample with the composition to amplify it.

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

[0013] One example of this application relates to a novel method for measuring the methylation level of a target nucleic acid, which complements the shortcomings of conventional experimental verification methods for measuring the methylation level of a target nucleic acid. Specifically, when designing MS-HRM primers, a methylated primer containing a CpG recognition site that recognizes a CpG sequence was used, and a non-methylated primer containing a TpG recognition site that recognizes a TpG sequence used for amplification of the non-methylated template chain was used in combination to prevent only the methylated template chain from being amplified. At this time, in order to prevent the relatively low sensitivity of MS-HRM and biased amplification of primers containing TpG, a method was developed to increase the binding opportunity of primers containing CpG and decrease the binding opportunity of primers containing TpG by adjusting the temperature of the primer annealing step in PCR.

[0014] In this specification, the term "methylation" refers to the addition of a methyl group to a base that makes up DNA. For example, methylation may occur to cytosine at a specific CpG site in a particular gene or nucleic acid.

[0015] In this specification, the terms “presence or absence of methylation” or “methylation status” mean the presence or absence of methylation occurring at a cytosine site at a specific CpG site in a particular gene or nucleic acid. Specifically, it means the presence or absence of 5-methylcytosine at one or more CpG dinucleotides within a base sequence.

[0016] In this specification, the term "methylation level" or "degree of methylation" refers to the amount of methylation present in the base sequence within the nucleic acid being examined.

[0017] In this specification, the terms “CpG site” or “CpG sequence” mean a CpG site present in the base sequence of a particular gene or nucleic acid. The gene is a concept encompassing all the constituent units necessary for expression and operably linked together, and may include, for example, a promoter region, a protein-coding region (open reading frame, ORF), and a terminator region. Therefore, the CpG site may be located 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 located in the promoter region of the gene.

[0018] In this specification, the term “nucleic acid” refers to a nucleotide in polymeric form, specifically a ribonucleotide or deoxyribonucleotide, and includes meanings such as polynucleotide, oligonucleotide, oligomer, oligo, and coding sequence. The term “nucleic acid” can 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, unnatural, or derivatized nucleotide bases.

[0019] Whenever a component is described as "including" in this specification, it means that, unless otherwise stated, it may include other components rather than excluding them.

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

[0021] In this specification, the singular form includes multiple subjects unless the context explicitly indicates otherwise.

[0022] An example of this application relates to a composition for confirming the presence or absence of methylation of a nucleic acid to be confirmed, or a kit 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. The primer set comprises a forward primer and a reverse primer, wherein one of the forward primer and the reverse primer may comprise a methylating primer and a non-methylating primer, and the remaining one may be a common primer. The methylating primer comprises a CpG recognition site that recognizes the CpG sequence of the nucleic acid to be confirmed, and the non-methylating primer comprises a TpG recognition site that recognizes the TpG sequence of the nucleic acid to be confirmed, wherein the TpG sequence may be a modified CpG sequence of the nucleic acid to be confirmed. The common primer means a primer that can specifically recognize and bind to the nucleic acid to be confirmed regardless of whether or not the nucleic acid to be confirmed is methylated.

[0023] Another example of this application relates to a method for determining the presence or absence of methylation in a target nucleic acid contained in a biological sample, comprising the steps of: deforming the methylated and unmethylated target nucleic acids so that they are different from each other; and treating the biological sample with the composition to amplify the results. The deformation step may be performed by treating the biological sample with a formulation that deforms the methylated and unmethylated target nucleic acids so that they are different from each other.

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

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

[0026] In an example of the present application, the target site may include a biomarker existing within a CpG island. The primer set according to an example of the present application includes (1) a methylation forward primer, an unmethylated forward primer, and a common reverse primer; or (2) a common forward primer, a methylation reverse primer, and an unmethylated reverse primer, and can accurately confirm the presence or absence of methylation of the target site even when it is difficult to prepare a common primer, such as a biomarker existing within a CpG island where the sequence complexity is reduced by bisulfite treatment or changes occur together at peripheral sites. Specifically, in the conventional HRM analysis method, it was difficult to analyze because the sequence complexity of gDNA was reduced by bisulfite treatment and non-specific reactions frequently occurred, or particularly in the case of a CpG island, it was difficult to prepare a MIP (Methylation Independent Primer; common primer) that does not contain CpG. However, in the present application, a methylation primer and an unmethylated primer are used in combination with a primer in one direction, so that the freedom in primer design can be ensured, and the presence or absence of methylation of the target site can be accurately confirmed while inducing specific binding to the target.

[0027] The methylation primer, specifically the forward methylation primer or the reverse methylation primer, contains a CpG recognition site that recognizes the CpG sequence of the nucleic acid to be confirmed. The unmethylated primer, specifically the forward unmethylated primer or the reverse unmethylated primer, contains a TpG recognition site that recognizes the TpG sequence obtained by converting the CpG sequence of the nucleic acid to be confirmed. The TpG sequence is obtained by converting the CpG sequence with a preparation that modifies the methylated nucleic acid to be confirmed and the unmethylated nucleic acid to be confirmed to be different from each other. For example, the preparation for modification may be one that converts an unmethylated cytosine residue to thymine. As an example, the preparation for modification may be one or more selected from the group consisting of sulfurous acid, bisulfite, hydrogen sulfite, and disulfite.

[0028] When a preparation that modifies the methylated nucleic acid to be confirmed and the unmethylated nucleic acid to be confirmed to be different from each other is used to treat the template strand, if the CpG cytosine of the template strand is methylated, the CpG cytosine will not be converted to thymine, and if the CpG cytosine is unmethylated, the CpG cytosine will be converted to thymine.

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

[0030] The TpG recognition site of the unmethylated primer can recognize the TpG sequence obtained by converting the CpG sequence of the unmethylated nucleic acid to be confirmed with a preparation that modifies the methylated nucleic acid to be confirmed and the unmethylated nucleic acid to be confirmed to be different from each other. For example, the TpG recognition site may contain a TG sequence.

[0031] The CpG recognition site and the TpG recognition site are located near the 3' end of the methylation primer and the unmethylation primer, respectively, and can specifically recognize the methylated and unmethylated target nucleic acids, respectively, that have been transformed by a formulation that deforms the methylated and unmethylated target nucleic acids in a manner different from each other. For example, the CpG recognition site and the TpG recognition site may 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 methylation primer and the unmethylation primer, or at the 3' end.

[0032] Since the non-methylated primer has a lower Tm value than the methylated primer, a nucleotide can be added to the 5' end of the non-methylated primer to compensate for this and design the Tm values ​​of the methylated and non-methylated primers to be similar. For example, the non-methylated primer may be larger in size than the methylated primer by including an additional nucleotide at its 5' end. For example, the non-methylated primer may contain 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 additional nucleotide at its 5' end compared to the methylated primer. For example, the Tm difference between the methylated and non-methylated primers 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. When the methylation primer and the non-methylation primer are forward primers, the reverse primer is a reverse primer. When the methylation primer and the non-methylation primer are reverse primers, the reverse primer is a forward primer. The reverse primer is a common primer that can bind to all nucleic acids to be methylated and non-methylated. Therefore, the reverse primer (common primer) may not contain CpG binding sites. However, if the structure of the nucleic acid to be confirmed makes it unavoidable for the reverse primer to contain CpG binding sites, it may contain 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or, as an example, 1 CpG binding site at the 5' end. However, even in this case, it is preferable to design the reverse primer to have a Tm similar to that of the methylation primer and the non-methylation primer.

[0034] The methylation primer, the non-methylation primer, and the opposite direction primer may be sized to contain 15-40, 15-35, 15-30, 15-25, 18-40, 18-35, 18-30, 18-25, 20-40, 20-35, 20-30, or 20-25 bases.

[0035] The methylation primer, the non-methylation primer, and the opposite-direction primer may contain one or more, two or more, or three or more Non-CpG cytosines, thereby enabling them to bind to a target nucleic acid converted by a formulation that deforms the methylated target nucleic acid and the non-methylated target nucleic acid in a manner different from each other.

[0036] The Tm of the methylation primer, the non-methylation primer, and the opposite direction primer may be 50-80°C, 50-75°C, 50-70°C, 50-65°C, 55-80°C, 55-75°C, 55-70°C, 55-65°C, 60-80°C, 60-75°C, 60-70°C, or 60-65°C.

[0037] A composition according to an example of this application involves the methylation primer and the non-methylation primer in ratios 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, less than 100:1 to 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, and 100:1 to 4 :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, less than 50:1~1:1, 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 1:1, 5:1~1.3:1, 5:1~1.5:1, 5:1~2:1, 5:1~2.5:1, 5:1~ It may contain the substance in a concentration ratio of 3:1, 5:1-3.5:1, 5:1-4:1, 4.5:1-1:100, 4.5:1-1:50, 4.5:1-1:20, 4.5:1-1:10, 4.5:1-1:5, 4.5:1-1:1, less than 4.5:1-1:1, 4.5:1-1.3:1, 4.5:1-1.5:1, 4.5:1-2:1, 4.5:1-2.5:1, 4.5:1-3:1, 4.5:1-3.5:1, or 4.5:1-4:1.

[0038] For example, the concentration of the non-methylated 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 concentration of the methylated primer, and as an example, it may be 50% or less.

[0039] The composition and method according to one example of this application can accurately confirm the presence or absence of methylation of the target nucleic acid without using a fluorescent probe to detect the amplified product. Therefore, the composition according to one example of this application may not contain a fluorescent probe. Furthermore, the method according to one example of this application may not use a fluorescent probe.

[0040] In the composition and method according to an example of this application, the nucleic acid to be confirmed for the presence or absence of methylation may be one contained in a biological sample. The biological sample 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 example of this application can confirm 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 the biological sample is very low.

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

[0043] For example, the biological sample comprises a methylated target nucleic acid and an unmethylated target nucleic acid, and the concentration of the methylated target nucleic acid 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 target nucleic acid.

[0044] For example, the biological sample may contain methylated and unmethylated nucleic acids, with the methylated nucleic acids being present in 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, based on 100% of the total chain of the methylated and unmethylated nucleic acids.

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

[0046] A method for confirming the presence or absence of methylation of a target nucleic acid according to an example of this application may additionally include a step of quantifying the degree of methylation of the target nucleic acid. The quantification step may involve comparing the AUC (Area under the curve) of the melting curves of the biological sample, a sample in which the target nucleic acid is 100% methylated, and a sample in which the target nucleic acid is 100% unmethylated, in order to quantify the degree of methylation of the biological sample.

[0047] Specifically, the quantification step may include: obtaining a melt curve of the biological sample; obtaining a normalized melt curve of the biological sample for which the methylation ratio of the nucleic acid to be identified is known; 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 identified may be obtained from the content ratio of methylated nucleic acid to unmethylated nucleic acid.

[0048] In another example of this application, the nucleic acid to be confirmed may include a cancer diagnostic biomarker. Specifically, the nucleic acid to be confirmed may include a cancer diagnostic biomarker, such as a marker that is specifically methylated or demethylated in cancer patients, and the target site of the nucleic acid to be confirmed may be the location of the cancer diagnostic biomarker. In this case, the presence or absence of methylation of the nucleic acid to be confirmed can be confirmed by the composition or method according to an example of this application and utilized for cancer diagnosis.

[0049] Therefore, another example of this application relates to a cancer diagnostic composition or cancer diagnostic kit, which includes a composition for confirming the presence or absence of methylation of a nucleic acid to be confirmed according to an example of this application.

[0050] Another example of this application relates to a cancer diagnostic method or a method for providing information for cancer diagnosis, which includes a step of confirming the presence or absence of methylation of a target nucleic acid by a method for confirming the presence or absence of methylation of a target nucleic acid according to an example of this application. The cancer diagnostic method or method for providing information for cancer diagnosis may additionally include a step of comparing the methylation level of the target nucleic acid with the methylation level of a control group.

[0051] The aforementioned control group refers to nucleic acids isolated from biological samples whose origin is already known, and can include all samples derived from subjects without liver cancer (normal control group) or subjects with liver cancer.

[0052] For example, if a sample derived from a subject without liver cancer (normal control group) is used as a control group, and 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, it may provide information for determining, diagnosing, or diagnosing that the organism from which the target nucleic acid or the biological sample originated (e.g., vertebrates, mammals, rodents, goats, deer, pigs, birds, chickens, turkeys, cattle, horses, sheep, fish, primates, and, as an example, humans) has cancer.

[0053] For example, if a sample derived from an organism with liver cancer is used as a control group, and the methylation level of the target nucleic acid is similar to that of the nucleic acid isolated from the control group, or if the biological sample contains a target nucleic acid with a similar methylation level to that of the control group, then it may provide information for determining, diagnosing, or diagnosing that the organism from which the target nucleic acid or the biological sample originated (e.g., vertebrates, mammals, rodents, goats, deer, pigs, birds, chickens, turkeys, cattle, horses, sheep, fish, primates, and, as an example, humans) has cancer.

[0054] The aforementioned cancers may include one or more selected from the group consisting of liver cancer, colorectal 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 hematological cancer.

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

[0056] Another example of this application relates to a method for treating cancer, which includes the step of treating a subject who has been confirmed to have cancer by a cancer diagnostic composition, cancer diagnostic kit, cancer diagnostic method, or cancer diagnostic information provision method according to an example of this application. The treatment step may include administering an effective amount of a therapeutic agent to the subject, chemotherapy, hormone therapy, radiotherapy, surgery, or a combination thereof.

[0057] The aforementioned therapeutic agents include, for example, 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, Garnicertinib, 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. This may include syntilimab, sorafenib, SU6656, sunitinib, sintilimab, spartalizumab, sutant, TACE, tasquinimod, temozolomide, temsirolimus, tislerizumab, tivantinib, tosylate, toripalimab, tremelimumab, vandetanib, vatalanib, XL888, Y90, their pharmaceutically acceptable salts, or combinations thereof. [Effects of the Invention]

[0058] One example of this application is that it is possible to design a new experimental validation method that complements the shortcomings of existing qMSP, MethyLight, and MS-HRM analytical methods, and that DNA methylation markers can be detected through liquid biopsy, and that methylation levels can be quantified with higher sensitivity than existing MS-HRM analytical methods.

[0059] One example of this application is the ability to perform PCR at a more sensitive level to targets present in minute amounts, such as circulating tumor DNA in blood. Experiments can be designed by adjusting the degree of binding of methylated and unmethylated primers to the template strand according to the application by controlling the annealing temperature and primer concentration ratio. [Brief explanation of the drawing]

[0060] [Figure 1a] This diagram shows the results of an experiment using MIP (Methylation Independent Primer) for conventional MS-HRM analysis. [Figure 1b] This diagram shows the results of an experiment using MIP (Methylation Independent Primer) for conventional MS-HRM analysis. [Figure 1c] This diagram shows the results of an experiment using MIP (Methylation Independent Primer) for conventional MS-HRM analysis. [Figure 2a] This diagram shows the results of an attempt to improve PCR amplification using MIP by including CpG at the 5' end of the primer to suppress non-methylation biased amplification. [Figure 2b] This diagram shows the results of an attempt to improve PCR amplification using MIP by including CpG at the 5' end of the primer to suppress non-methylation biased amplification. [Figure 2c] This diagram shows the results of an attempt to improve PCR amplification using MIP by including CpG at the 5' end of the primer to suppress non-methylation biased amplification. [Figure 3a] This diagram illustrates a phenomenon observed when using fluorescent probes, such as those used in methylite analysis, that interferes with marker amplification. [Figure 3b] This diagram illustrates a phenomenon observed when using fluorescent probes, such as those used in methylite analysis, that interferes with marker amplification. [Figure 4a]This figure shows the results of MS-HRM analysis performed with a mixture of methylated and unmethylated primers. [Figure 4b] This figure shows the results of MS-HRM analysis performed with a mixture of methylated and unmethylated primers. [Figure 4c] This figure shows the results of MS-HRM analysis performed with a mixture of methylated and unmethylated primers. [Figure 5a] This figure shows the melting peak distribution of each sample based on its methylation ratio. [Figure 5b] This diagram shows the HRM analysis results for each sample based on their methylation ratio, as determined by melting curve analysis. [Figure 6a] This diagram shows whether quantitative amplification occurs when methylated and unmethylated primers are mixed and used in a single experimental tube. [Figure 6b] This diagram shows the results of verifying the accuracy of methylation ratio measurement using mAUC (melting curve area under curve), which indicates the methylation ratio. [Figure 6c] The diagram shows an example of a method for confirming the presence or absence of methylation according to this application, which distinguishes between methylated DNA concentrations of 1% (3 strands of methylated DNA) and 0% and confirms that 1% methylated DNA can be detected in a sample where unmethylated DNA is present at a concentration of 99%. [Figure 7] This diagram shows the presence or absence of biased amplification of methylated DNA depending on the concentration ratio of methylated and non-methylated primers. [Figure 8] Figures 8a and 8b are diagrams showing preliminary clinical results of diagnosing cancer by applying the methylation detection method according to an example of this application to actual blood. [Modes for carrying out the invention]

[0061] The present application will be described in more detail below with reference to the following embodiments. However, these embodiments are merely illustrative and do not limit the scope of the present application.

[0062] Comparative Example 1. Confirmation of the limitations of the conventional MS-HRM analysis method. As an example of the nucleic acid to be identified, we used a nucleic acid containing the sequence of Sequence ID No. 1 to confirm the limitations of the conventional MS-HRM analysis method. The nucleic acid sequence of Sequence ID No. 1 is a liver cancer biomarker that is specifically methylated in liver cancer cells. DNA isolated from liver cancer cells will contain the methylated Sequence ID No. 1 nucleic acid, while DNA isolated from other cells will contain the unmethylated Sequence ID No. 1 nucleic acid. A variety of samples containing the Sequence ID No. 1 nucleic acid and control group samples were prepared as shown in Table 1 and used in the experiment. Samples No. 2 to 4 were purchased from Qiagen. Sample No. 2 was obtained by sulfite treatment of the gDNA of sample No. 3 using the EpiTect Bisulfite Kit, in which all cytosine residues were converted to thymine. Sample No. 4 was obtained by 100% methylation of the gDNA of sample No. 3 using SssI methylase, followed by sulfite conversion treatment using the EpiTect Bisulfite Kit, in which cytosines other than CpG cytosine were converted to thymine. Samples 5-7 were treated with sulfite before the PCR reaction, which converted unmethylated cytosine to thymine.

[0063] [Table 1]

[0064] Conventional MS-HRM analysis uses methylation-independent primers (MIPs) that do not contain CpG and TpG recognition sites, allowing for non-selective amplification of sulfite-treated methylated and unmethylated nucleic acids. The sequences of the methylation-independent primers used are as follows:

[0065] -Forward direction: gTtgTatTatTtgTTaggggTtgT (Sequence ID 7) -Reverse direction: cccacaAAAcctccaAAcaAtAA (Sequence ID 8)

[0066] MS-HRM Master Mix was prepared by mixing the components shown in Table 2. The volumes listed in Table 2 are the required amounts for one sample; when testing multiple samples, prepare and use Master Mix at the number of samples + 2 levels (22 uL / rxn).

[0067] [Table 2]

[0068] The prepared Master Mix was dispensed in 22 μL portions into each well of a 96-well PCR plate (Hard-Shell® PCR Plates, Biorad). Seven samples from Table 1 were prepared to a concentration of 0.33 ng / uL, and 3 μL portions were dispensed into each well so that the total amount of DNA was 1 ng. The PCR reaction 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 / cooling at 60°C for 30 seconds, and extension at 72°C for 30 seconds. The fluorescence values ​​for each cycle were confirmed by fluorescence measurement after 30 seconds of heating / cooling and before starting the extension temperature step. After the PCR reaction was complete, a 5-minute stabilization period was observed at 72°C. Then, a melting analysis was performed to check the degree of amplification product binding with increasing temperature. The fluorescence intensity was measured at 10-second intervals while increasing the temperature from 65°C to 95°C in 0.2°C increments, and HRM analysis was performed.

[0069] Fluorescence values ​​were confirmed for PCR and HRM analysis, and the characteristics of each sample were confirmed by examining the melting curve or peak. Furthermore, after PCR was completed, electrophoresis was performed to confirm whether the size of the amplified product was accurately produced by the designed primers.

[0070] As shown in Figure 1a, amplification was nearly identical in the sample with 100% methylated target nucleic acid (EpiTect Met; sample number 4) and the sample with 100% unmethylated target nucleic acid (EpiTect Unmet; sample number 2). Furthermore, amplification was observed with similar efficiency in liver cancer cells (Huh-1; sample number 6), kidney cells (Hek293T; sample number 5), and blood cells (PBMC; sample number 7), and the amplification of all methylated and unmethylated target nucleic acids was confirmed by the increase in PCR fluorescence RFU values. However, there was a problem in that nonspecific amplification occurred due to MIP binding even in the untreated sulfite control group (EpiTect gDNA; sample number 3) and the control group without the target nucleic acid (NTC; sample number 1), which should not be amplified.

[0071] As shown in Figure 1b, High Resolution Melting curve (HRM) analysis confirmed that the amplified DNA consisted of methylated markers, non-methylated markers, and non-specific MIP amplified DNA. Furthermore, as shown in the electrophoresis results in Figure 1c, the presence of multiple non-specific amplified DNAs was confirmed.

[0072] Therefore, conventional MS-HRM analysis methods have the problem that genomic DNA sequences are simplified by sulfite treatment, and the simplicity of MIP primer sequences, which consist only of adenine, guanine, and thymine, frequently leads to the generation of nonspecific amplification products, thus limiting the amplification of target nucleic acids.

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

[0074] -Forward direction: TTtCGgTtgTatTatTtgTTaggggT (Sequence ID 9) -Reverse direction: AACGCccccacaAAAcctccaAA (Sequence ID 10)

[0075] As shown in Figure 2a, by adding CpG to the 5' end of MIP, a bias was introduced to allow for more favorable amplification of methylated DNA. As a result, 100% methylated samples (EpiTect Met; sample number 4) and hepatoma cell DNA (Huh-1; sample number 6) were effectively amplified, while unmethylated DNA samples (EpiTect Unmet; sample number 2) and the untreated control group (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 samples. Therefore, when analyzing unmethylated samples under conditions where the bias is weighted by methylation, there was a problem in that very high levels of nonspecific amplification appeared.

[0076] Comparative Example 3. Confirmation of the limitations of the MS-HRM analysis method using a fluorescent probe. The procedure was carried out as in Comparative Example 1, but the PCR amplification efficiency was confirmed when using a fluorescent probe used for methylite analysis, etc. Figure 3a shows the results of MS-HRM performed on 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, and Figure 3b shows the results of performing the same procedure with the composition in Table 2, but replacing 10uL of NFW with a fluorescent probe (ggTTCGTTaCGTtgTTTt; SEQ ID NO: 11). An untreated sulfite sample (EpiTect gDNA; sample number 3) was used as a control group in each experiment.

[0077] Figure 3a shows the amount of amplified DNA measured 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, there was a problem in that the Cq value increased by about 4 when the fluorescent probe was added, resulting in a 10 to 20-fold decrease in amplification efficiency.

[0078] Example 1. Preparation of methylated and non-methylated primers As an example of a nucleic acid to be confirmed, primers were prepared to check for methylation in 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 indicated 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, and other bases are written in lowercase. In the sequence of SEQ ID NO: 1, the CpG sequence is shown in bold.

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

[0080] Considering the sequence of the template strand converted after sulfite treatment, one of the primers, either forward or reverse, was designed to contain a CpG or TpG sequence near the 3' end so that it could specifically bind to one of the methylated or unmethylated template strands. Since the methylated template strand retains CpG cytosine that has not been converted to thymine after sulfite treatment, the methylation primer was designed to contain a CpG recognition site that recognizes the CpG sequence, and the unmethylation 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 compensate for this. The primer in the opposite direction to the methylation and unmethylation primers was designed to bind regardless of the methylation state of the methylated and unmethylated strands. All primers were designed to contain 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 nucleic acids 147–171 of the nucleic acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3, and the reverse primer was designed to bind to nucleic acids 206–229 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 methylating primer that can bind to nucleic acids 147-171 in the nucleic acid sequence of Sequence ID No. 2, and a non-methylating primer that can bind to nucleic acids 206-229 in the nucleic acid sequence of Sequence ID No. 3. At the same time, the reverse primer was a common reverse primer that can bind to all of the sulfite-treated methylation-confirming nucleic acids and sulfite-treated non-methylation-confirming nucleic acids.

[0083] Table 3 shows the sequences of the fabricated primers. The sequences that differed depending on whether the target nucleic acid was methylated or not are underlined in the methylated and non-methylated primer sequences.

[0084] [Table 3]

[0085] Example 2. MS-HRM using methylated and non-methylated primers MS-HRM analysis was performed using the primers prepared in Example 1. The MS-HRM Master Mix was prepared by mixing the compositions shown in Table 4. The volumes listed in Table 4 are the required amounts for one sample; when examining multiple samples, prepare and use Master Mix at the number of samples + 2 levels (22 uL / rxn).

[0086] [Table 4]

[0087] A variety of samples containing the nucleic acid of sequence number 1, as well as 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 22 μL portions into each well of a 96-well PCR plate (Hard-Shell® PCR Plates, Biorad). The six samples listed in Table 5 were prepared to contain a concentration of 0.33 ng / uL, and 3 μL of each sample was dispensed into each well so that the total amount of DNA was 1 ng.

[0090] The PCR reaction was performed using the CFX96 Touch Real-Time PCR Detection System (Biorad) at a denaturation temperature of 95°C for 5 minutes, followed by a cycle of 20 seconds at 95°C, 30 seconds at a heating / cooling temperature of 60°C, and 30 seconds at an extension temperature of 72°C, repeated 50 times. Fluorescence values ​​for each cycle were confirmed via fluorescence measurement after the 30 seconds of heating / cooling and before starting the extension temperature phase. After the PCR reaction was complete, a 5-minute stabilization period was observed at 72°C. Then, a melting analysis was performed to confirm the degree of amplification product binding with increasing temperature. Fluorescence intensity was measured at 10-second intervals as the temperature was increased from 65°C to 95°C in 0.2°C increments, and HRM analysis was performed.

[0091] Fluorescence values ​​were confirmed for PCR and HRM analysis, and the characteristics of each sample were confirmed by examining the melting curve or peak. Furthermore, after PCR completion, electrophoresis was performed to confirm whether the size of the amplified product was accurately generated by the designed primers.

[0092] Methylating and unmethylating primers were added simultaneously to a single sample to amplify the target nucleic acid under conditions where both methylated and unmethylated DNA could be amplified. As shown in Figure 4a, amplification was equally good for both sulfite-treated methylated DNA (EpiTect Met and Huh-1) and unmethylated DNA (EpiTect Unmet and PBMC), while no amplification occurred in the control group samples (NTC and untreated EpiTect gDNA). The HRM analysis results in Figure 4b and the electrophoresis analysis results in Figure 4c confirmed that only specific amplification occurred.

[0093] Example 3. Confirmation of detection limits and quantification of methylation levels To determine the minimum detection limit for 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%, which were then used in the experiments. 3 uL of each methylation ratio sample was dispensed into each well, and PCR reactions and HRR analysis were performed in the same manner as in Example 2. The results are shown in Figures 5a and 5b.

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

[0095] Figure 5b is a diagram showing the HRM analysis results using melting curve analysis. It shows that the slope of the melting curve increases gradually with increasing hepatocyte DNA, and lies between the slope of unmethylated blood cells and the slope of 100% methylated hepatocyte cells. Therefore, by calculating the area under the melting curve, the methylation ratio can be quantified. Specifically, the methylation level of the unknown sample can be quantified by dividing the difference between the melting curve AUC of the unknown sample and the melting curve AUC of the 100% unmethylated sample 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 with respect to temperature, we select temperatures in which the MS-HRM fluorescence change rate is constant within the lower and upper intervals of the melting temperature (Tm). L , T R), the parameters of exponential background noise are estimated from the fluorescence change rate (dF / dT) at each point. The fluorescence data function M(T), which is proportional to the amount of residual dsDNA, is calculated by calculating the exponential background noise value from the estimated parameters and removing it from the total fluorescence value for each temperature point. Maximum-minimum normalization (min-max normalization) is performed on M(T) within the temperature interval [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) is obtained from each sample within the [TL, TR] interval, and then the degree of methylation of the sample can be confirmed based on the S value.

[0097] Example 4. Confirmation of detection sensitivity Since ctDNA is present in blood at very low concentrations, we confirmed the detection limit to determine whether the method for confirming the presence or absence of methylation, as described in this application, can be used in actual blood test-based cancer diagnosis.

[0098] To identify the smallest DNA strand detectable by the methylation detection method described in this application, PBMC and Huh-1 (50 ng / 3 uL) samples were diluted with NFW to prepare Huh-1 samples at 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 concentration sample was dispensed into each well, and PCR reaction and HRR analysis were performed in the same manner as in Example 2.

[0099] MS-HRM analysis was performed on methylated and unmethylated mixed primers using amounts of sulfite-treated liver cancer cell DNA ranging from 0.01 ng to 50 ng, representing a 5,000-fold difference. The amplification of the target nucleic acid was analyzed by measuring the PCR Cq value, as shown in Figure 6a. As shown in Figure 6a, when 0.01 ng (3 strands) of DNA was used, the Cq value started at approximately 38, and as the DNA was increased to 50 ng (15,200 strands), the Cq value decreased in a relationship that precisely corresponded to the increase in DNA amount. This result indicates that mixed MS-HRM analysis of methylated and unmethylated primers effectively amplifies even 0.01 ng of DNA, and that effective amplification is possible over a wide range of 5,000-fold increases.

[0100] Figure 6b shows 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 when analyzing various amounts of DNA. As shown in Figure 6b, it was confirmed that the difference in AUC values ​​was not large depending on the amount of each sample and remained constant. The amplification product of each sample was the target nucleic acid, and the methylation level of each sample could be confirmed. Therefore, it was possible to confirm the presence or absence of methylation of the target nucleic acid even in samples containing a minimum of 3 strands.

[0101] Furthermore, in order to determine the minimum ratio at which the method for confirming the presence or absence of methylation according to one example of this application can detect the target methylated DNA in an environment where unmethylated DNA is present, samples were prepared at 0, 1, 2, 4, 8, 16, 32, and 100% concentrations by serially diluting unmethylated DNA and methylated DNA, and MS-HRM analysis was performed. The correlation between the change in AUC value with respect to the ratio was confirmed and is shown in Figure 6c.

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

[0103] Therefore, we confirmed that the method for confirming the presence or absence of methylation according to the example in this application has high detection sensitivity, capable of detecting 1% methylated DNA even in samples containing 99% unmethylated DNA.

[0104] Example 6. Improvement of detection sensitivity using non-methylated primers To determine whether detection sensitivity was improved by using non-methylated primers, Huh-1 samples of 6.25%, 25.0%, 50.0%, or 100.0% were used. The procedure was carried out as in Example 5, but with the composition shown in Table 3, the volume of the non-methylated forward primer was changed to 1, 0.75, 0.5, or 0.25 μL, thereby changing the NFW volume to 5.25, 5.50, 5.75, or 6 μL.

[0105] We investigated whether biased amplification of methylated DNA occurs depending on the concentration ratio of methylated and unmethylated primers. PCR was performed under conditions where the amount of methylated primer was fixed at 0.4 μM, and the amount of unmethylated primer was decreased to 0.4, 0.3, 0.2, and 0.1 μM.

[0106] As shown in Figure 7, when the ratio of methylated hepatocellular carcinoma DNA was low (25% or less), the sensitivity to methylated DNA detection was higher when the amount of non-methylated primer was 50% or less of that of methylated primer.

[0107] Therefore, the combined use of methylating and non-methylating primers increased detection sensitivity, making 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 the blood. A preliminary clinical trial was conducted to determine whether it was possible to confirm the presence or absence of methylation of target nucleic acids by applying MS-HRM analysis with methylated and non-methylated primers to actual blood samples.

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

[0110] MS-HRM Master Mix was prepared according to Table 3. 22 μL 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 methylation levels of healthy individuals and liver cancer patients were determined by examining the melting curve or peak.

[0111] Figure 8a shows the results of plasma separation from 2 ml of blood samples taken from 92 healthy individuals and 119 liver cancer patients. cfDNA was then separated, treated with sulfur dioxide, and approximately 1 ng of cfDNA was used for primer-mixed MS-HRM analysis. The analysis results from healthy individuals (green) all show unmethylated, low-temperature melting peaks, while the blood samples from liver cancer patients (red) show high-temperature melting peaks.

[0112] Figure 8b shows the melting curve analysis results obtained by experimenting on the target nucleic acid of Sequence ID No. 1 using the MS-HRM method with mixed blood from normal individuals and liver cancer patients. Normal individuals (green) are unmethylated, and the amplified DNA dissolves quickly, resulting in a steep curve slope. In contrast, blood samples from liver cancer patients (red) contain methylated cancer DNA, which has a higher melting point, and show a relatively slower dissolution curve slope. It can be confirmed that over 60% of liver cancer patient cfDNA contains more methylated DNA than normal individuals. Therefore, it is possible to effectively detect trace amounts of methylated marker DNA present in blood by detecting extremely small amounts of nucleic acids, such as methylated cfDNA fragments of about 100 bp.

Claims

1. Includes a primer set for amplifying the target site of the nucleic acid to be identified. The aforementioned primer set is (1) a methylated forward primer, a non-methylated forward primer, and a reverse primer; or (2) comprising a methylated reverse primer, a non-methylated reverse primer, and a reverse primer, The methylation forward primer and the methylation reverse primer include a CpG recognition site that recognizes the CpG sequence of the nucleic acid to be identified. The non-methylated forward primer and the non-methylated reverse primer include a TpG recognition site that recognizes the TpG sequence of the nucleic acid to be identified. The TpG sequence is obtained by converting the CpG sequence of the nucleic acid to be confirmed. A composition for confirming the presence or absence of methylation in target nucleic acids.

2. The composition according to claim 1, wherein the TpG sequence is converted by a formulation that deforms a methylated target nucleic acid and an unmethylated target nucleic acid in a manner different from each other.

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

4. The composition according to 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.

5. The composition according to claim 1, wherein the target site includes a CpG sequence.

6. The composition according to claim 1, wherein the target portion has a size of 50 to 150 bp.

7. The composition according to claim 1, wherein the primer is sized to contain 15 to 40 bases.

8. The composition according to claim 1, wherein the reverse-direction primer contains five or fewer CpG or TpG recognition sites of the nucleic acid to be identified.

9. The composition according to claim 1, wherein the Tm difference between the methylated forward primer and the non-methylated forward primer in (1) above; or the Tm difference between the methylated reverse primer and the non-methylated reverse primer in (2) above is 15°C or less.

10. The methylated forward primer and the non-methylated forward primer of (1) above; or The composition according to claim 1, wherein the methylated reverse primer and the non-methylated reverse primer of (2) are included in a concentration ratio of 100:1 to 1:

100.

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

12. The composition according to claim 11, wherein the biological sample comprises one or more selected from the group consisting of blood, plasma, tissue, cells, feces, and urine.

13. The composition according to claim 11, wherein the biological sample comprises a methylated target nucleic acid and an unmethylated target nucleic acid.

14. The composition according to claim 13, wherein the concentration of the methylated nucleic acid to be identified is 75% or less of the concentration of the unmethylated nucleic acid to be identified.

15. The composition according to claim 1, wherein the nucleic acid to be identified includes a biomarker for cancer diagnosis.

16. The composition according to claim 15, wherein the cancer comprises 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 hematological cancer.

17. The composition according to claim 1, wherein the target site is a biomarker located within a CpG island.

18. A step in which methylated and unmethylated nucleic acids in a biological sample are deformed in such a way that they are different from each other; and The step of treating the biological sample with the composition described in any one of claims 1 to 17 to amplify the target site, A method for checking whether or not a target nucleic acid is methylated.

19. The method according to claim 18, wherein the deformation step is performed by treating the biological sample with a formulation that deforms the methylated target nucleic acid and the unmethylated target nucleic acid in a manner different from each other.

20. The method according to claim 18, wherein the biological sample is one or more selected from the group consisting of blood, plasma, tissue, cells, feces, and urine.

21. The method according to claim 18, further comprising the step of quantifying the degree of methylation of the nucleic acid to be confirmed.

22. The method according to claim 21, wherein the quantification step involves comparing the AUC (Area Under the Curve) of the normalized melting curves of the biological sample, the sample in which the nucleic acid to be identified is 100% methylated, and the sample in which the nucleic acid to be identified is 100% unmethylated, in order to quantify the degree of methylation of the biological sample.

23. The step of obtaining the melting curve of the biological sample; The steps include obtaining a normalized melt curve for a biological sample whose methylation ratio of the nucleic acid to be examined is known; and The method according to claim 18, further comprising the step of quantifying the degree of methylation of the biological sample by comparing the melting curve of the biological sample with the normalized melting curve.

24. The method according to claim 18, wherein the target site includes a biomarker located within a CpG island.

25. A step in which methylated and unmethylated nucleic acids in a biological sample are deformed to be distinct from each other; A step of treating the biological sample with the composition described in any one of claims 1 to 17 to amplify it; and This includes a step of confirming whether or not the nucleic acid to be checked is methylated, The nucleic acids subject to confirmation include cancer diagnostic biomarkers. Methods for providing information for cancer diagnosis.

26. The method according to claim 25, wherein the cancer diagnostic biomarker nucleic acid is methylated or demethylated in cancer patients.

27. The method according to claim 25, further comprising the step of comparing the methylation level of the nucleic acid to be confirmed with the methylation level of a control group.

28. The method according to claim 25, wherein the cancer comprises one or more selected from the group consisting of liver cancer, colorectal 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 hematological malignancy.

29. The method according to claim 25, wherein the cancer diagnostic biomarker is located within a CpG island.

30. A kit for confirming the presence or absence of methylation of a target nucleic acid, comprising the composition described in any one of claims 1 to 17.

31. A cancer diagnostic composition comprising the composition according to any one of claims 1 to 17, wherein the nucleic acid to be identified comprises a cancer biomarker.

32. A cancer diagnostic kit comprising the composition described in claim 31.

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