DNA methylation analysis method and use thereof

Abstract

The present invention relates to a real-time polymerase chain reaction method for determining a degree and pattern of DNA methylation at a specific DNA sequence site and to a use thereof. More specifically, the present invention relates to a method for determining a degree and pattern of methylation by determining respective Ct values obtained from performing qPCR in two buffer systems (buffer 1 and buffer 2), in which differences occur depending on the presence or absence of methylation, using DNA obtained from a biological sample, calculating a difference between the Ct values of the two buffer systems, and more specifically, calculating a relative ratio of copy number calculation values obtained from tests in the two buffer systems, and to a use thereof.

Description

DNA methylation analysis method and its application

[0001] The present invention relates to a real-time polymerase chain reaction method for determining the degree and pattern of DNA methylation on a specific DNA sequence region and its use.

[0002] More specifically, the present invention relates to a method for determining the degree and mode of methylation by performing qPCR in two buffer systems (buffer 1 and buffer 2) that differ depending on the presence and degree of methylation using DNA obtained from a biological sample, determining each Ct value obtained, calculating the difference between the Ct values ​​of the two buffer systems, and more specifically, calculating the relative ratio of the copy number calculation values ​​obtained by the test in the two buffer systems, and its use.

[0003] DNA methylation is one of the most widely studied epigenetic modifications. It is a chemical mechanism in which a methyl group is added to the 5′-C of cytosine, forming 5'-methyl cytosine (5mC). This DNA methylation occurs primarily at CpG dinucleotide sites (hereafter, "CpGs") through the action of DNA methyltransferases such as DNMTI (S.ssI).

[0004] In the 1970s, it was first suggested that 5'-C methylation of CpGs plays a role in gene silencing by changing chromatin structure and DNA stability (Serre D., et al., 2010) without changing the DNA sequence, and that 5mC is a very important epigenetic marker. In the 1980s, it was discovered that CpG sites are distributed heterogeneously within the genome and tend to be clustered at a high density, especially in promoter regions. Since then, regions with high frequency of CpG methylation have been named CpG islands (CGIs).

[0005] Of the approximately 30,000 CGIs known in the human genome, most are distributed in the promoter regions of functional genes and are primarily unmethylated. However, DNA methylation in promoter regions has been shown to alter chromatin structure, thereby blocking transcription initiation and suppressing gene transcription.

[0006] On the other hand, it is known that 20-40% of the approximately 9,000 CGIs located within genes (gene bodies) are methylated. It has been confirmed that methylation of gene body regions in the human genome is very closely related to gene transcription, and methylation is known to affect histone protein modification, alternative splicing, and spurious transcription. Generally, DNA methylation is thought to induce gene silencing, but some genetic studies have reported that transcriptional activation promotes methylation within genes (gene bodies).

[0007] As described above, gene methylation occurs widely in promoters and within genes, and its modalities are highly complex. It is an unknown area that remains to be explored from an ecological, physiological, and genetic perspective. Although the biological functions associated with DNA methylation mechanisms differ somewhat, it has been widely reported in living organisms, spanning not only higher animals like humans but also plants, invertebrates, and microorganisms, making it a crucial mechanism in biogenetics. DNA methylation is a crucial mechanism in epigenetics, implicated in the regulation of fundamental life phenomena such as cell differentiation and development.

[0008] Furthermore, DNA methylation patterns are clinically very important. In particular, changes in DNA methylation of specific genes, namely hypermethylation or hypomethylation, can serve as valuable biomarkers in the clinical diagnosis of diseases such as cancer. Aberrant DNA methylation is reported to play a crucial role in the induction and progression of cancer (carcinogenesis), and therefore, DNA methylation analysis has value as a tumor biomarker.

[0009] Changes in DNA methylation occur in cancers such as breast, pancreatic, colon, and stomach cancer, and these changes could be utilized to develop specific diagnostic tools. DNA methylation changes can be utilized as biomarkers for cancer detection from relatively easy-to-obtain clinical tissues. Changes in methylation patterns have already been demonstrated to be important markers for drug sensitivity, and promoter methylation patterns for several genes, such as MGMT, SHOX2, and SEPT9, have been developed into commercial clinical tests and are being used for clinical evaluation of patients. For example, DNA methylation patterns for the lung cancer gene EGFR have been shown to be a factor in determining drug treatment efficacy (Li, XY, et al., 2013). In cell line tests, the therapeutic agent gefitinib showed enhanced anticancer efficacy by blocking DNA methylation, suggesting a close relationship between cancer-related genes and methylation patterns.

[0010] Therefore, it will be very important for the implementation of precision medicine to identify the methylation pattern of target genes by cell and patient, and to elucidate the causal relationship between metabolism, heredity, development, cell differentiation, cancer, diet, living environment, and other diseases.

[0011] Over the past several decades, numerous methods have been developed to identify DNA methylation status and its quantitative aspects across the genome and within individual genes of interest. These methods can be broadly categorized into bisulfite-based and bisulfite-free methods.

[0012] Bisulfite treatment is the most widely used method for mapping methylated cytosines in DNA (Frommer, M., et al., 1992). This method uses bisulfite to convert cytosine (C) residues to uracil (U) residues, but 5-methylcytosine (5mC) remains unchanged, allowing for the identification of methylation patterns via base pair sequencing (BS-sequencing) or PCR. However, this method suffers from the harsh chemical treatment and temperature conditions that can result in significant nucleic acid loss, with up to 90% of the sample DNA lost (Grunau et al., 2001). This represents a significant barrier to the application of precision diagnostics using small amounts of biological samples, particularly diagnostic techniques that utilize limited quantities of biological samples (e.g., ctDNA, cfDNA), such as liquid biopsy. In addition, since most of the cytosine (C) bases in the DNA sequence are changed to uracil (U) and ultimately to thymine (T), the complexity of the genome sequence is reduced, and it is changed into genomic DNA without C, consisting of three bases (T, A, G), which causes limitations in performing sequence analysis and PCR using it.

[0013] For accurate sequence analysis after bisulfite treatment, a large amount of DNA sample is required, and for PCR of bisulfite-treated DNA sample, there are difficulties in designing primers and probes and performing PCR, i.e., the probability of off-target occurrence that targets a site other than the target gene increases, and the Tm value of primers and probes is low, so the length must be long, etc.

[0014] To overcome the limitations associated with bisulfite conversion, several bisulfite-free methods have been developed. Bisulfite-free enzyme-based DNA conversion methods are representative examples. A representative example is Enzymatic Methyl-seq (EM-seq), developed by New England Biolabs, which uses TET2 and APOBAC enzymes to convert methylated and hydroxylmethylated cytosines to uracil, allowing the identification of modified cytosines (Vaisvila R., et al., 2021). Another technology is the TET-assisted pyridine borane sequencing (TAPS) method, which is particularly useful for analyzing DNA fragments larger than 10 Kb (Liu Y., et al., 2019).

[0015] EM-seq-based methods, such as the above, outperform bisulfite sequencing in various indicators, including DNA damage, conversion efficiency, alignment quality, coverage, and sensitivity (Vaisvila R., et al., 2021). However, this method also requires expensive enzymes and involves multiple reaction steps, resulting in loss of clinical samples. Furthermore, like bisulfite-based methods, this method still suffers from fundamental limitations in converting cytosine to uracil.

[0016] In addition, a technique has been developed that uses a method of using a restriction enzyme that recognizes a CpG-containing base sequence and cuts DNA, such as a methylation-dependent restriction enzyme (e.g., GlaI) or a methylation-sensitive restriction enzyme (e.g., HpaII) that recognizes CpG. Examples include the reduced representation bisulfite sequencing (RRBS) method (Meissner A., ​​et al., 2005), the methyl-sensitive restriction enzymes (MSRE) method (Hashimoto K., et al., 2007), the end-specific PCR (ES-PCR) and helper-dependent chain reaction (HDCR) methods (Rand KN, and Molloy PL, 2010; Rand KN, et al., 2013), the combined bisulfite restriction analysis (COBRA) method (Xiong Z., and Laird PW, 1997), and the digital restriction enzyme analysis of methylation (DREAM) method (Jelinek J., et al., 2012). These methods are applied to more clearly and easily determine the methylation pattern through sequence analysis or PCR analysis of the target gene by performing them before or after bisulfite treatment or before performing PCR or base sequence analysis. These methods selectively select or amplify target DNA through DNA cleavage based on the presence or absence of CpG methylation, thereby significantly reducing the number of base sequences to be analyzed or more clearly showing the methylation pattern through PCR analysis.

[0017] The methylation profile of samples prepared using the above sample preparation techniques must ultimately be confirmed through base sequence analysis or PCR pattern analysis. Base sequence analysis of sample genomes obtained through the various methods mentioned above is typically performed using next-generation sequencing (NGS). Methylation profile information can be obtained by sequencing the entire genome (WGBS, whole genome bisulfite sequencing) or by analyzing the sequence of PCR amplified fragments using specific primers.

[0018] Many methods based on PCR or qPCR have been reported, including MethyLight analysis (Eads CA, et al., 2000), QuARTS (Quantitative Allele-Specific Real-time Target and Signal amplification) (Zou H., et al., 2012), HM (HeavyMethyl) qPCR (Cottrell SE, et al., 2004), and MS-HRM (Methylation-sensitive high-resolution melting) (Wojdacz T, K., and Dobrovic, A., 2007). These methods are technologies that apply CSP (conversion-specific PCR) or MSP (methylation-specific PCR) (Herman JG, et al., 1996), which are representative PCR performance methods. This method designs and uses specific primers to amplify only methylated DNA by targeting cytosine sequences that have not been converted to methylation.

[0019] The technology based on the above CSP or MSP performs PCR using a nucleic acid sample containing a sequence in which unmethylated cytosine is converted to uracil as a template. This technology can determine methylation or unmethylation of the target gene by PCR using specific primers composed of sequences that can distinguish the template generated by the conversion or non-conversion of cytosine to uracil depending on the presence or absence of cytosine methylation at the CpG site of the target region. Therefore, the PCR-based technology developed to date is based on the enzymatic conversion of cytosine to uracil, either bisulfite-free or bisulfite-free, and thus inevitably has technological limitations.

[0020] That is, performing PCR using sequence-converted gDNA, such as through bisulfite treatment, has several limitations. Namely, the template composed of a pseudo-trinucleotide formed by the conversion of cytosine requires a longer sequence length due to the low Tm value during primer design. In addition, in the case of a converted genome composed of a three-base sequence, the sequence diversity is greatly reduced compared to a four-base sequence, making it more difficult to exclude off-target regions that can be non-specifically amplified, and designing PCR primers with sequences that specifically amplify only the target molecule is more difficult. To overcome these drawbacks, nested PCR is sometimes used to achieve amplification specificity with bisulfite-treated template DNA. However, this is not suitable for diagnostic purposes because the DNA amplified in the first PCR is exposed to the clinical laboratory environment.

[0021] The existing technologies mentioned above are numerous technologies that selectively amplify DNA sequences converted through bisulfite or enzyme treatment using PCR and qPCR. While the application of preprocessing techniques for such conversion can help to determine the presence or absence of methylation with relative accuracy, there is a significant risk of sample loss, modification, and contamination during the process, which can lead to distortion of clinical information. Furthermore, this requires increased amounts of clinical samples (e.g., formalin-fixed paraffin-embedded (FFPE) biopsy samples, cell-free (Cf) DNA samples for liquid biopsy) and increased analysis volume, thus reducing economic efficiency. In fact, recovery, conversion efficiency, and conversion specificity vary significantly depending on the type of conversion kit developed by various manufacturers. Therefore, a technology that can distinguish methylated and unmethylated DNA without DNA sample preprocessing would be ideal to overcome these problems.

[0022] To implement this ideal technology, a method must be devised to directly distinguish between methylated and unmethylated DNA by exploiting the differences in their chemical and physical properties.

[0023] Affinity-based capture methods, such as antibody immunoprecipitation using the methyl-CpG binding domain (MDB) protein (Weber M., et al., 2005; Serr D., et al., 2010) and gold nanoparticle-based methods for detecting differences in methylation (Sina AA, et al., 2018), have been reported. These techniques are useful for the enrichment of methylated DNA.

[0024] Another method, EpiDirect, which uses primers designed using intercalating nucleotides (INA) that can distinguish between methylated and unmethylated cytosine by qPCR has been reported (Bendixen KK, et al., 2023). This method uses special oligonucleotides synthesized using intercalating pseudonucleotides (IPN), which affect the π-stacking of the DNA helix and cause changes in the Tm value depending on the template DNA, as primers. Expensive oligonucleotides must be specially manufactured, and this method has a very narrow discrimination range because it evaluates the presence or absence of methylation in a very narrow region corresponding to the sequence to which these primers bind, and also the second repetition (2) of the specially modified primers used nd After the first round, primers must be specially designed and manufactured to address issues such as non-recognition by the polymerase.

[0025] As mentioned above, it is very difficult for DNA polymerase to make a difference in polymerization reaction by recognizing only the difference in the mode of methylation with the same sequence, that is, the difference in 5' methylation of cytosine between methylated and unmethylated DNA.

[0026] Hypermethylated DNA (meDNA) has been reported to undergo structural environmental changes in which the surrounding area is dehydrated, making it more hydrophobic, resulting in a stiffer A form rather than the general B form structure of unmethylated DNA, thereby decreasing the contour length and increasing the persistence length. In addition, it has been reported that cytosine base methylation increases the thermal stability of DNA structure and slightly increases the Tm value compared to unmethylated DNA (Tm value increases by about 4℃ for about 4 CpGs methylated) (Kaur P et al., 2012). It has been reported that changes in the physical properties of DNA depending on the presence or absence of methylation have some effect on PCR.

[0027] It has been reported that changes in the physical properties of DNA depending on the presence or absence of methylation have some differences in the efficiency of PCR under traditional conditions (Kiselev KV, et al., 2014).

[0028] Another report presented a method to compare the methylation pattern according to the deviation of △Ct values ​​by performing qPCR under different denaturation conditions of low and high temperatures targeting the target gene (US2024 / 0002920 A1).

[0029] However, in the case of general PCR, it is very difficult to clearly confirm the influence of these Tm differences on PCR according to the presence or absence of methylation under general PCR buffer and temperature conditions. This is because most high-efficiency PCRs require a denaturation step at a high temperature above 95℃, making it difficult to clearly show the presence or absence of methylation with a slight difference in Tm in the primer region or a difference in Ct value in qPCR. Furthermore, these methods have technical limitations in that they estimate the copy number of the target gene or measure the degree and pattern of methylation that reflects it.

[0030] Furthermore, these conventional methods do not include measurement of the target gene's copy number. Genetic mutations and their associated patterns, such as hereditary diseases, cancer, and metabolic disorders, require a comprehensive review of genetic mutations, copy number, and methylation. Therefore, an economical PCR technology capable of simultaneously measuring copy number and methylation patterns is required.

[0031] [Prior Art Literature]

[0032] [Patent Document]

[0033] US Published Patent US 2024-0002920 A1 (2024.01.04) “METHOD AND KIT FOR DETECTING DNA METHYLATION BASED ON QUANTITATIVE POLYMERASE CHAIN ​​REACTION (qPCR)”

[0034] Korean Patent No. 1845715 (March 30, 2018) "Antifreeze Composition"

[0035] [Non-patent literature]

[0036] Bendixen, K. K et al., (2023). Nature communications, 14(1), 5153.

[0037] Cottrell, S. E et al., (2004). Nucleic acids research, 32(1), e10.

[0038] Eads, C. A et al.,(2000). Nucleic acids research, 28(8), E32.

[0039] Frommer, M et al., (1992). Proceedings of the National Academy of Sciences of the United States of America, 89(5), 1827-1831.

[0040] Hashimoto, K et al.,(2007). Epigenetics, 2(2), 86-91.

[0041] Herman, J. G et al.,(1996). Proceedings of the National Academy of Sciences of the United States of America, 93(18), 9821-9826.

[0042] Jelinek, J et al.,(2012). Epigenetics, 7(12), 1368-1378.

[0043] Kiselev, K. V et al.,(2015). Journal of plant physiology, 175, 59-67.

[0044] Li, X. Y et al.,(2013). Oncology reports, 29(5), 1975-1982.

[0045] Liu, Y et al.,(2019). Nature biotechnology, 37(4), 424-429.

[0046] Meissner, A et al.,(2005). Nucleic acids research, 33(18), 5868-5877.

[0047] Rand, K. N et al., (2010). BioTechniques, 49(4), xiii-xvii.

[0048] Rand, K. N et al., (2013). Nucleic acids research, 41(1), e15.

[0049] Serre, D et al., (2010). Nucleic acids research, 38(2), 391-399.

[0050] Sina, A. A et al., (2018). Nature communications, 9(1), 4915.

[0051] Vaisvila, R et al., (2021). Genome research, 31(7), 1280-1289.

[0052] Weber, M et al., (2005). Nature genetics, 37(8), 853-862.

[0053] Wojdacz, T. K et al., (2007). Nucleic acids research, 35(6), e41.

[0054] Xiong, Z., & Laird, P. W. (1997). Nucleic acids research, 25(12), 2532-2534.

[0055] Zou, H et al., (2012). Clinical chemistry, 58(2), 375-383.

[0056] Accordingly, the purpose of the present invention is to provide a highly efficient and economical DNA methylation identification method, a DNA methylation quantification method, and a kit thereof capable of distinguishing between methylated DNA and unmethylated DNA in a method for quantifying the degree of DNA methylation of a target gene and its use.

[0057] In addition, it is an object of the present invention to provide a method for identifying DNA methylation using a difference in the composition of a PCR buffer solution in a very economical and simple manner without using a specific probe or primer set for differentiation, a method for quantifying methylation, and a kit thereof.

[0058] In addition, it is an object of the present invention to provide a method and kit for simultaneously identifying DNA methylation and the copy number of a target gene by utilizing the difference in the composition of a PCR buffer solution in a very economical and simple manner without using a specific probe or primer set for differentiation.

[0059] In addition, it is an object of the present invention to provide a method and a kit for quantifying DNA methylation of a target gene without causing chemical modification of nucleotides.

[0060] In order to solve the problems of the prior art for determining the degree of DNA methylation, the present inventors sought a method for determining the degree and mode of DNA methylation very economically and simply without using a specific probe or primer set, and more specifically, a qPCR method capable of determining the degree of DNA methylation by utilizing the phenomenon in which the Ct value deviation occurs depending on the mode of DNA methylation in two different qPCR systems with different compositions of buffer solutions.

[0061] In typical PCR, two DNA strands are separated through high-temperature denaturation, then annealed to attach primers, allowing amplification by DNA polymerase. While DNA polymerase cannot distinguish between C and 5mC bases, resulting in a consistent amplification efficiency, the Tm (melting temperature) of DNA varies depending on the degree of methylation.

[0062] As methylation increases, the Tm of DNA increases because, in addition to increased hydrophobicity, methylation also increases π-stacking between bases between or within the same strand, which further stabilizes hydrogen bonds and thus increases the thermal stability of DNA.

[0063] Therefore, altering the DNA environment by changing buffer conditions during PCR can affect PCR efficiency depending on the degree of DNA methylation. More specifically, the hydrophobicity of the buffer conditions can influence the thermal stability of DNA, which in turn affects the degree (type) of methylation.

[0064] Taking this into account, the present inventors sought to develop a method for clearly distinguishing the state of DNA methylation and calculating its degree by simply performing qPCR in at least two contrasting PCR buffer conditions (e.g., buffer 1 and buffer 2).

[0065] In at least two contrasting PCR buffers, buffer 1 may contain betaine (0 to 2 M) and / or DMSO (0 to 10%), which are ingredients that resolve secondary structure formation due to high GC% in general buffers, and buffer 2 may be selected from the group of organic solvents that are water-miscible and do not strongly denature proteins, such as ethanol, methanol, and isopropanol, which can increase hydrophobicity in buffer 1, and the amount thereof may be selected from, for example, 0 to 10%, and preferably from about 1 to 5%. More specifically, an ethanol-containing cryoprotectant composition disclosed in the applicant's patent No. 1845715 may be selected.

[0066] The antifreeze composition of the above patent may specifically include one of 10 to 40% (v / v) ethanol or 10 to 30% (v / v) methanol. More specifically, it may be prepared and used as a 2X solution (40% glycerol, 20% EtOH, 400 mM α-MG).

[0067] A more specific composition of the buffer solution of the present invention is

[0068] a) Buffer 1 can be used with Mg2+ in the concentration range of 1.5 to 5 mM, KCl in the concentration range of 35 to 100 mM, (NH4)2SO4 in the concentration range of 6 to 50 mM, and betaine in the concentration range of 0.5 to 2.5 M. In addition, PCR buffer 1 can additionally contain a composition included in a typical PCR solution, such as BSA (bovine serum albumin) in the concentration range of 10 to 500 ug / mL and a nonionic surfactant, preferably Trinton X-100 in the concentration range of 0.1 to 1%, NP-40 in the concentration range of 0.3 to 0.6%, and more specifically, it can contain 1.3 mM dNTPs, 10 mM Tris-HCl (pH 9.0), 3 mM MgCl2, 60 mM KCl, 10 mM (NH4)2SO4, 1.5 M betaine,

[0069] b) Buffer 2 can be prepared by adding the composition, AFA, described in Korean Patent No. 1845715 to the buffer 1, and can be used in a concentration range of 20 to 60% glycerol, 5 to 40% ethanol, and 50 to 500 mM α-MG. More preferably, it can contain a mixed solution containing 40% glycerol, 20% ethanol, and 20% 400 mM α-MG in an amount of more than 0 and less than 10% (v / v). However, there is no particular limitation on the type and content of specific salts, surfactants, etc. included in buffer 1, and any buffer suitable for performing qPCR can be applied, and an appropriate amount of a nucleic acid secondary structure formation inhibitor can be added thereto to be used as buffer 1.

[0070] A more specific composition of buffer 1 of the present invention may include 1.3 mM dNTPs, 10 mM Tris-HCl (pH 9.0), 3 mM MgCl2, 60 mM KCl, 10 mM (NH4)2SO4, 1.5 M betaine, and buffer 2 is a buffer in which 10% of the 2X above-mentioned cryoprotectant composition solution (40% glycerol, 20% EtOH, 400 mM α-MG) is added to buffer 1. However, this is only an example of the buffer of the present invention, and is not limited to the composition described above. The present invention is not limited by the description of the types of each salt, surfactant, etc. contained in the above buffer and the concentration range thereof, and the concentration range of the composition exemplified above does not limit the scope of the present invention.

[0071] The present invention provides a method for performing qPCR using buffer 2 with the cryoprotectant composition added thereto and buffer 1 without the cryoprotectant composition added thereto, obtaining a cycle threshold (Ct, threshold cycle) value of each PCR, and calculating the degree of DNA methylation using the difference between the obtained Ct values.

[0072] More specifically, in the present invention, a base sequence region in which the Ct value of qPCR does not change under the conditions of buffer 1 and buffer 2 for performing qPCR, specifically, a base sequence region without a CpG island and with a low CG%, can be selected as an internal control (IC) for comparison. It is preferable that this region be selected from the base sequence region of a housekeeping gene. The housekeeping gene can be selected from, for example, CANX, HPRT1, PGK1, TBP, YWHAZ, SDHA, UBC, GUSB, GAPDH, ACTB, TUBA1A ATP5B, PPIA, HNRNPL, IPO8, PUM1, UBC, RPP30, PCBP1, RNaseP, etc., and preferably, RNaseP can be selected.

[0073] It is preferable that the qPCR of the above internal standard (e.g., RNaseP) gene region be selected in a region where the difference in Ct values ​​in buffer 1 and buffer 2 (dCt of IC =│Ct of IC in buffer 1 - Ct of IC in buffer 2│) is small (Fig. 3 and Table 2).

[0074] It is desirable that the qPCR region of the target gene be selected from a region in which the difference in Ct values ​​(dCt of UM = │Ct of UM in buffer 1 - Ct of UM in buffer 2│) is small in qPCR using unmethylated gDNA (UM) and a region in which the difference in Ct values ​​(dCt of M = │Ct of M in buffer 1 - Ct of M in buffer 2│) is large in qPCR using methylated gDNA (M) (Fig. 1, Table 2).

[0075] Additionally, the qPCR region of the target gene can be selected from the promoter of the gene and within the gene, or from the region where a CpG island exists or a region adjacent thereto (Fig. 1, Fig. 14, Fig. 18). Here, the "adjacent region" refers to a region within 1 Kb outside the CpG island, more preferably a region within 300 bp.

[0076] After performing qPCR of the internal standard and the target gene, the copy number of the target gene can be inferred by comparing and calculating the qPCR Ct value of the internal standard and a gene (RNaseP) with a known copy number (CN) in the genome, preferably a copy number of 2. The method for calculating the copy number (CN) value of the target gene is as follows.

[0077] Typical 2 nThe formula for the change in Ct value for the copy number of the qPCR amplification curve amplified by is the conversion formula Ct = S x LN(CN) + I, which can be obtained by using the Ct value of IC(CN 2) and the Ct value (CN 4) obtained by subtracting 1 from this Ct value. Using this formula, the relative CN value (Relative CN) can be calculated based on the qPCR result values ​​(Ct) in buffer 1 and buffer 2 of the target gene (Table 2). The "relative copy number value" or "relative CN value" (Relative CN) refers to the CN value of the target gene calculated based on the qPCR result value (Ct) in a test that is not synchronized with the internal standard. In the above formula Ct = S x LN(CN) + I, under ideal conditions where the efficiency is 100%, S is calculated as -1.44, which becomes the Ct value of CN = 1 measured in the internal standard test. The I value is calculated from the Ct value of the internal standard (IC, e.g., RNaseP) gene of CN2 from the formula, and is equal to the Ct value of the measured IC gene of CN2 plus 1. Using this formula, the relative CN value (CN = EXP((Ct - I) / S)) can be calculated based on the qPCR results of the target gene in Buffer 1 and Buffer 2 (Table 2).

[0078] The ratio of the relative CN values ​​in buffer 1 and buffer 2 obtained in this way (Relative CN Ratio = relative CN of target gene in buffer 1 / relative CN of target gene in buffer 2) can be calculated to determine the degree of methylation of the target gene.

[0079] Depending on the copy number ratio, the degree of methylation of the target gene can be classified as hypermethylation for ≤0.5, hypomethylation for 0.5 to 0.8, and unmethylation for ≥0.8.

[0080] In addition, in one embodiment of the present invention, it was intended to construct a qPCR system capable of simultaneously and accurately assessing the copy number of a target gene as well as determining the degree of methylation by two contrasting qPCR systems having different compositions of the buffers described above.

[0081] First, qPCR of an internal standard (e.g., RNaseP) and the target gene are synchronized. qPCR synchronization minimizes errors and enables precise qPCR performance by simultaneously testing the internal standard and the target gene in a single reaction. To achieve this, the two genes to be tested are physically linked together, and a standard plasmid (control plasmid DNA) with the same copy number can be used to synchronize qPCR. To determine the degree of methylation under synchronized qPCR conditions, a qPCR system containing at least two buffers with different compositions is performed, thereby providing a method for simultaneously determining the degree of methylation and the copy number of the target gene (Tables 7 to 10).

[0082] In addition, the present invention also provides a method for simultaneously determining the methylation degree and copy number of multiple target genes by performing a multiplex qPCR that synchronizes two or more target genes in one test area and compares them with an internal standard, and performing a qPCR system including at least two buffers with different compositions to determine the methylation degree under qPCR conditions (Tables 10, 11 and Figures 16, 17).

[0083]

[0084] According to the present invention, DNA methylation can be identified and quantified and analyzed very efficiently and economically through a method capable of distinguishing between methylated DNA and unmethylated DNA without chemical modification of nucleotides such as cytosine being converted to methylcytosine.

[0085] In addition, according to the present invention, DNA methylation can be identified and quantified using differences in the composition of PCR buffer solutions in a very economical and simple manner without using specific probes or primer sets for differentiation.

[0086] In addition, according to the present invention, it is possible to simultaneously identify not only DNA methylation but also the copy number of a target gene by utilizing the difference in the composition of a PCR buffer solution in a very economical and simple manner without using a specific probe or primer set for differentiation, and furthermore, it is possible to simultaneously identify DNA methylation and copy number of multiple target genes in addition to one target gene.

[0087]

[0088] Figure 1. This diagram shows the CGI and PCR regions surrounding the EGFR promoter and Exon 1. CGIs were analyzed at https: / / www.urogene.org / cgi-bin / methprimer / methprimer.cgi under conditions of Obs / Exp. 0.6 or higher and GC% 50 or higher. CGIs are highlighted in gray.

[0089] Figure 2. qPCR amplification curves for each EGFR Exon 1 region. A, B, and C are qPCR amplification curves using 0.5 M betaine for regions A, B, and C shown in Figure 1.

[0090] Figure 3. RNaseP region for internal control. The underlined sequences are the primer and probe recognition sequences.

[0091] Figure 4. EGFR Exon1 C region qPCR amplification curve. This figure is a representative representation of the qPCR amplification curves of the test numbers indicated by numbers in the tests in Table 2.

[0092] Figure 5. Amplification curves of qPCR of EGFR-Exon1 regions A and C of human gDNA according to methylation status under different buffer conditions. This figure is a representative representation of the qPCR amplification curves of the test numbers indicated by numbers in the tests in Table 3.

[0093] Figure 6. This figure shows the CGI and PCR regions near EGFR Exon 20. CGI was analyzed at https: / / www.urogene.org / cgi-bin / methprimer / methprimer.cgi under conditions of Obs / Exp. 0.6 or higher and GC% 50 or higher. No CGI was detected.

[0094] Figure 7. Amplification curves of qPCR for the EGFR-Exon 20 region of human gDNA according to methylation status under different buffer conditions. This figure represents the qPCR amplification curves of the test numbers indicated by numbers in the tests in Table 4.

[0095] Figure 8. qPCR amplification curves according to the mixing ratio of unmethylated and methylated gDNA under different buffer conditions. This figure represents the qPCR amplification curves for the test numbers indicated in Table 5.

[0096] Figure 9. This graph shows the causal relationship between the mixing ratio of unmethylated gDNA and methylated gDNA and the calculated relative CN ratio. It was created based on the test results in Table 5.

[0097] Figure 10. This is a photograph of a plasmid sample prepared to confirm the degree of CpG methylation reaction of the pTOP-RNaseP-EGFR-Exon1 plasmid, digested with HpaII, and subjected to agarose gel electrophoresis.

[0098] Figure 11. qPCR amplification curves according to the degree of methylation of the plasmid (pTOP-RNaseP-EGFR-Exon1) under different buffer conditions. This figure is a representative representation of the qPCR amplification curves for the test numbers indicated by numbers in the tests in Table 6.

[0099] Figure 12. Amplification curves of qPCR for tests to assess EGFR methylation and copy number. These are qPCR amplification curves for test numbers indicated by numbers in the tests in Table 7.

[0100] Figure 13. This figure shows the Ct values ​​of EGFR qPCR tests and the copy number calculated from them. The qPCR results and calculated EGFR copy number for the test numbers in Table 7 are expressed numerically.

[0101] Figure 14. This figure shows the CGI and PCR regions of SHOX2 exon 1 and adjacent regions. CGI was analyzed at https: / / www.urogene.org / cgi-bin / methprimer / methprimer.cgi under conditions of Obs / Exp. 0.6 or higher and GC% 50 or higher. CGI is highlighted in gray.

[0102] Figure 15. Amplification curves of qPCR for the test to evaluate methylation and copy number of SHOX2. These are the qPCR amplification curves for the test numbers indicated by numbers in the tests in Table 9.

[0103] Figure 16. Amplification curves of multiplex qPCR for the test to assess methylation and copy number of EGFR and SHOX2. These are qPCR amplification curves for the test numbers indicated by numbers in the tests in Table 10.

[0104] Figure 17. This is a plot of the results of a multiplex qPCR test to assess methylation and copy number of EGFR and SHOX2 in clinical samples. It is a graph of the copy number and relative copy number ratio (Relative CN Ratio) for the test numbers indicated by numbers in the tests in Table 11. The numbers in the graph are the average values ​​of three replicate tests.

[0105] Figure 18. This figure shows the CGI and PCR regions near CYP2D6 Exon 2. CGI was analyzed at https: / / www.urogene.org / cgi-bin / methprimer / methprimer.cgi under conditions of Obs / Exp. 0.6 or higher and GC% 50 or higher. CGI is shown in gray.

[0106] Figure 19. qPCR amplification curves for the test to assess methylation and copy number of CYP2D6. These are the qPCR amplification curves for the test numbers indicated by numbers in the tests in Table 12.

[0107] Figure 20. This figure shows the qPCR test Ct values ​​of CYP2D6 and the copy number calculated from them. The qPCR results and calculated CYP2D6 copy numbers for the test numbers in Table 12 are expressed numerically.

[0108] The present invention

[0109] (1) A step of performing real-time quantitative PCR using the subject DNA in two buffers, buffer 1 and buffer 2, each containing an internal standard and each primer and probe that specifically reacts to the target gene sequence region and dNTP and polymerase;

[0110] (2) A step of calculating the relative copy number using the Ct values ​​of qPCR performed in two buffers obtained in step (1);

[0111] (3) a step of calculating the ratio of the relative replication numbers calculated in step (2); and

[0112] (4) A method for analyzing the degree of methylation of target gene DNA, comprising: a step of determining the degree of methylation of the target gene by the ratio value of the relative copy number;

[0113] In addition, the present invention

[0114] (1) A step of synchronizing real-time quantitative PCR by including an internal standard and a target gene sequence region in one standard plasmid;

[0115] (2) A step of performing real-time quantitative PCR using a subject in two buffers, buffer 1 and buffer 2, containing primers and probes and dNTPs that specifically react to an internal standard and a target gene sequence region, and a polymerase;

[0116] (3) A step of calculating the number of copies using the Ct value of real-time quantitative PCR performed in two buffers, buffer 1 and buffer 2, obtained in step (2);

[0117] (4) a step of calculating the ratio of the number of copies from the number of copies calculated in step (3); and

[0118] (5) A method for simultaneously analyzing the methylation degree and copy number of a target gene DNA, including a step of determining the methylation degree of the target gene by the ratio of the copy number obtained in the step (4).

[0119] In addition, the present invention relates to an analysis method in which the subject DNA is DNA extracted from cells, tissues, blood, plasma, saliva, feces, urine, skin or organs of a living organism.

[0120] In addition, the present invention relates to an analysis method in which an internal standard is selected from a sequence region without a CpG island among the base sequences of a host gene.

[0121] In addition, the present invention relates to an analytical method in which the internal standard is selected from a group of genes consisting of CANX, HPRT1, PGK1, TBP, YWHAZ, SDHA, UBC, GUSB, GAPDH, ACTB, TUBA1A ATP5B, PPIA, HNRNPL, IPO8, PUM1, UBC, RPP30, PCBP1, and RNaseP. It will be apparent to a person skilled in the art that these genes are merely examples of internal standards and that internal standards are not necessarily limited to these listed genes.

[0122] In addition, the present invention relates to an analysis method, characterized in that the sequence region of the target gene is selected from a region having a CpG Island and a region adjacent thereto. Here, the adjacent region refers to a region within 1 Kb outside the CpG island, more preferably a region within 300 bp.

[0123] In addition, the present invention relates to an analysis method in which a copy number ratio obtained by the above analysis method is judged as hypermethylation when ≤0.5, hypomethylation when the copy number ratio is 0.5 to 0.8, and unmethylation when the copy number ratio is ≥0.8.

[0124] In addition, the present invention comprises two buffers

[0125] a) Buffer 1 further contains a nucleic acid secondary structure formation inhibitor in a buffer for real-time quantitative PCR,

[0126] A) Buffer 2 relates to an analysis method characterized in that it further includes a water-miscible organic solvent that increases hydrophobicity in the composition of buffer 1.

[0127] In addition, the present invention comprises two buffers

[0128] a) Buffer 1 further contains a nucleic acid secondary structure formation inhibitor in a buffer for real-time quantitative PCR,

[0129] b) Buffer 2 relates to an analysis method characterized in that it further includes a water-miscible organic solvent that increases hydrophobicity and does not denature proteins in the composition of buffer 1.

[0130] In addition, the present invention relates to an analytical method, wherein the nucleic acid secondary structure formation inhibitor is at least one selected from betaine or DMSO. Betaine or DMSO are merely examples of nucleic acid secondary structure formation inhibitors, and any suitable inhibitor that does not affect the qPCR reaction may be used.

[0131] In addition, the present invention relates to an analysis method in which the organic solvent of the buffer solution 2 is at least one selected from ethanol, methanol, and isopropanol.

[0132] In addition, the present invention relates to an analysis method, wherein the buffer solution 2 contains the organic solvent in an amount of more than 0% and less than or equal to 20% (v / v).

[0133] In addition, the present invention relates to an analysis method, wherein the organic solvent of the buffer solution 2 contains at least one of ethanol or methanol, more than 0% and less than 20% (v / v), and more than 0% and less than 40% (v / v) of glycerol.

[0134] In addition, in the present invention

[0135] a) Buffer 1 can be used with Mg2+ in the concentration range of 1.5 to 5 mM, KCl in the concentration range of 35 to 100 mM, (NH4)2SO4 in the concentration range of 6 to 50 mM, and betaine in the concentration range of 0.5 to 2.5 M. In addition, PCR buffer 1 can additionally contain a composition included in a typical PCR solution, such as BSA (bovine serum albumin) in the concentration range of 10 to 500 ug / mL and a nonionic surfactant, preferably Trinton X-100 in the concentration range of 0.1 to 1%, NP-40 in the concentration range of 0.3 to 0.6%, and more specifically, it can contain 1.3 mM dNTPs, 10 mM Tris-HCl (pH 9.0), 3 mM MgCl2, 60 mM KCl, 10 mM (NH4)2SO4, 1.5 M betaine,

[0136] b) Buffer 2 can be used by adding the composition, AFA, described in Korean Patent No. 1845715 to the buffer 1, and containing 20 to 60% glycerol, 5 to 40% ethanol, and 50 to 500 mM α-MG. More preferably, it can contain a mixed solution containing 40% glycerol, 20% ethanol, and 20% 400 mM α-MG in an amount of more than 0 and less than 10% (v / v).

[0137] In addition, the present invention comprises two buffers

[0138] a) Buffer 1 contains 1.3 mM dNTPs, 10 mM Tris-HCl (pH 9.0), 3 mM MgCl2, 60 mM KCl, 10 mM (NH4)2SO4, 1.5 M betaine,

[0139] b) The method relates to an analysis method, wherein buffer 2 contains a mixed solution of buffer 1 containing 40% glycerol, 20% ethanol, and 20% 400 mM α-MG in an amount of more than 0% and less than 10% (v / v).

[0140] In addition, the present invention relates to an analysis method that uses the formula of Ct = S x LN(CN) + I (wherein S = -1.44, I is the Ct value of the copy number CN = 1 calculated by the test of the internal standard) in the calculation of the copy number, and calculates the copy number value by substituting the qPCR Ct value of the target gene into the formula and obtains the calculated copy number (calculated CN) by comparison with the internal standard. The "calculated copy number (calculated CN)" is the CN value of the target gene calculated based on the qPCR result value (Ct) in the test synchronized with the internal standard.

[0141] In addition, the present invention relates to an analysis method in which the formula Ct = S x LN(CN) + I (where S = -1.44, I is the Ct value of the copy number CN = 1 calculated by testing the internal standard) is used in calculating the copy number, the qPCR Ct value of the target gene is substituted into the formula to calculate the copy number value, and the copy number is obtained by comparison with the internal standard, and the ratio of the copy number is obtained as (the copy number of the target gene obtained in the buffer 1 test) / (the copy number of the target gene obtained in the buffer 2 test).

[0142] In addition, the present invention relates to an analysis method including two or more target genes in the step (1).

[0143] In addition, the present invention

[0144] a) Target gene-specific primers and target gene-specific probes;

[0145] b) Reference gene-specific primers and reference gene-specific probes capable of amplifying and identifying the reference gene;

[0146] c) DNA polymerase;

[0147] a) Buffer 1; and

[0148] b) Contains buffer solution 2;

[0149] The above buffer 1 further contains a nucleic acid secondary structure formation inhibitor in a buffer for real-time quantitative PCR,

[0150] The present invention relates to a qPCR kit for analyzing the degree of methylation of target gene DNA, characterized in that the buffer solution 2 further includes a water-miscible organic solvent that increases the hydrophobicity of the buffer solution 1.

[0151] In addition, the present invention relates to a kit characterized in that the buffer solution 2 further includes a water-miscible organic solvent that increases hydrophobicity in the composition of the buffer solution 1 and does not denature proteins.

[0152] Furthermore, the present invention relates to a kit, wherein the nucleic acid secondary structure formation inhibitor is at least one selected from betaine or DMSO. Betaine or DMSO are merely exemplary inhibitors, and any suitable inhibitor that does not affect the qPCR reaction may be used.

[0153] In addition, the present invention relates to a kit in which the organic solvent of the buffer solution 2 is at least one selected from ethanol, methanol, and isopropanol.

[0154] In addition, the present invention relates to a kit, wherein the buffer 2 contains more than 0% (v / v) and less than 20% (v / v) of the organic solvent. If the organic solvent exceeds 20% (v / v), it may have an undesirable effect on the PCR reaction.

[0155] In addition, the present invention relates to a kit, wherein the buffer solution 2 contains more than 0% and less than 20% (v / v) of at least one of ethanol or methanol as the organic solvent and more than 0% and less than 40% (v / v) of glycerol.

[0156] In addition, in the present invention, a) buffer 1 may be used in a concentration range of 1.5 to 5 mM for Mg2+, 35 to 100 mM for KCl, 6 to 50 mM for (NH4)2SO4, and 0.5 to 2.5 M for betaine. In addition, the PCR buffer 1 may additionally include a composition included in a typical PCR solution, such as 10 to 500 ug / mL of BSA (bovine serum albumin) and a nonionic surfactant, preferably 0.1 to 1% of Trinton X-100, 0.3 to 0.6% of NP-40, etc. More specifically, it may include 1.3 mM dNTPs, 10 mM Tris-HCl (pH 9.0), 3 mM MgCl2, 60 mM KCl, 10 mM (NH4)2SO4, 1.5 M betaine,

[0157] b) Buffer 2 can be used by adding the composition, AFA, described in Korean Patent No. 1845715 to the buffer 1, and containing 20 to 60% glycerol, 5 to 40% ethanol, and 50 to 500 mM α-MG. More preferably, it can contain a mixed solution containing 40% glycerol, 20% ethanol, and 20% 400 mM α-MG in an amount of more than 0 and less than 10% (v / v).

[0158] In addition, the present invention relates to a kit, wherein the buffer 1 comprises 1.3 mM dNTPs, 10 mM Tris-HCl (pH 9.0), 3 mM MgCl2, 60 mM KCl, 10 mM (NH4)2SO4, 1.5 M betaine, and the buffer 2 comprises a mixed solution of the buffer 1 containing 40% glycerol, 20% ethanol, and 20% 400 mM α-MG, in an amount of more than 0 and less than 10% (v / v).

[0159]

[0160] Below, the composition of the present invention is described in more detail with specific examples and test examples. However, it will be apparent to those skilled in the art that the scope of the present invention is not limited to the description of the examples and test examples.

[0161]

[0162] Example 1: Construction of a standard plasmid

[0163] Plasmids for the experiment were introduced into the pTOP Blunt V2 vector (Enzynomics Korea) and constructed using fragments amplified using PfuDNA polymerase (Enzynomics, Korea) as described below. Human genomic DNA (Promega) was used as the template.

[0164] As a control gene, the sequence region of RNaseP, a type of housekeeping gene (SEQ ID NO: 23, Figure 3), was cut by KpnI and SpeI using amplification primers to obtain an RNaseP gene fragment, and pTOP-RNaseP was secured. In addition, EGFR exon 1 region (SEQ ID NO: 24, Figure 1), EGFR exon 20 region (SEQ ID NO: 25, Figure 6), SHOX2 exon 1 region (SEQ ID NO: 26, Figure 14), and CYP2D6 exon 1, 2, 3 regions (SEQ ID NO: 27, Figure 18) were amplified and introduced to obtain pTOP-EGFR-Exon1, pTOP-EGFR-Exon20, pTOP-SHOX2-Exon1, and pTOP-EGFR-CYP2D6-100CT plasmids.

[0165] The secured plasmid was constructed by linking the RNaseP gene as follows to match the copy number of the control gene and the target gene. The pTOP-RNaseP vector was digested with KpnI / SpeI to obtain a fragment (456 bp), which was cloned into the plasmid of each gene fragment digested with KpnI / SpeI to obtain pTOP-RNaseP-EGFR-Exon1, pTOP-RNaseP-EGFR-Exon20, pTOP-RNaseP-SHOx2-Exon1, and pTOP-RNaseP-CYP2D6-100CT, which were digested with a restriction enzyme (SpeI), purified, and used for qPCR.

[0166]

[0167] Example 2: Performing qPCR

[0168] qPCR (real-time quantitative PCR) was performed using the CFX Opus 96 Real-Time PCR System. The primers, probes (Table 1) and conditions used for qPCR for each target gene were the same as those in each example, in terms of type, amount, buffer and conditions. Unless otherwise specified, the composition of Buffer 1 is 1.3 mM dNTPs, 10 mM Tris-HCl (pH 9.0), 3 mM MgCl, 60 mM KCl, 10 mM (NH4)2SO4, and 1.5 M betaine. Unless otherwise specified, Buffer 2 is Buffer 1 with 10% addition of 2X "AFA solution" (40% glycerol, 20% EtOH, 400 mM α-MG; hereinafter referred to as "AFA solution").

[0169] The total volume was 20 μl using 2U of Taq DNA polymerase (STexS-Taq, GenoTech Korea) used in the reaction. The Ct value of the performed qPCR was determined at a threshold of 400. At this time, the plasmid was 10 3 copies, 5 ng of human genomic DNA were used. EpiScope Unmethylated HCT116 DKO gDNA (Code No. 3521), EpiScope Methylated HCT116 gDNA (Code No. 3522) (Takara Bio, Japan), and human genomic DNA (Cat. No. G3041) (Promega, USA) were purchased and used. Lung cancer cell line NCI-H854 gDNA (KCLB No. 90854) was donated from the Korea Cell Line Bank, and clinical human gDNA was donated from the Daejeon Biomedical Regulation-Free Special Zone Human Material Bank (IRB file No. CNUH 2022-08-047).

[0170] Unless otherwise stated in the tests of the examples below, the qPCR conditions of Example 2 are followed.

[0171]

[0172] Example 3: CpG methylation of plasmids

[0173] Methylation was performed using the plasmid pTOP-RNaseP-EGFR-Exon1 obtained in Example 1 as a template and CpG methyltransferase (M.SssI) (NEB, UK). 1 μg of DNA template was used to achieve a final concentration of 10 μM, 2.5 μM, and 0.625 μM of S-adenosyl methionine (SAM), and the reaction was terminated. For the remainder, 10 units (u) of CpG methyltransferase (M.SssI) and buffer were used according to the supplier's manual and the procedure was followed. The total reaction volume was 50 μl, and the reaction was performed at 37°C for 1 hour and then heated at 65°C for 20 minutes to terminate the reaction. The methylated DNA was purified, quantified, and then digested with SpeI for use. Table 2 shows the Ct values ​​and copy number values ​​obtained from condition-dependent EGFR Exon1 (C) qPCR using gDNA.

[0174]

[0175] Example 4: qPCR performed on EGFR Exon1 and surrounding regions.

[0176] The EGFR gene has a wide distribution of CpGs, centered around Exon 1 and the promoter region (SEQ ID NO: 20) (Fig. 1). We aimed to establish a qPCR system for the region surrounding the promoter region, where CpGs are concentrated. High GC%s (greater than 60%) posed challenges in primer design. Primers and probes (Table 1) were developed and tested, focusing on the region with a very high CpG content (Fig. 1B), the region above it (Fig. 1A), and the region below it (Fig. 1C). For region A, EGFR-Exon1-F4 (SEQ ID NO: 1), EGFR-Exon1-R4 (SEQ ID NO: 2), and EGFR-Exon1-FAM4 (SEQ ID NO: 3) were used; for region B, EGFR-Exon1-F1 (SEQ ID NO: 4), EGFR-Exon1-R1 (SEQ ID NO: 5), and FAM-EGFR-Exon1 (SEQ ID NO: 6) were used; and for region C, EGFR-Exon1-F3 (SEQ ID NO: 7), EGFR-Exon1-R3 (SEQ ID NO: 8), and EGFR-Exon1-FAM3 (SEQ ID NO: 9) were used. Each primer was used at 20 pmol, and each FAM probe was used at 10 pmol. qPCR was performed as in Example 2 by repeating 95℃, 10 minutes, 95℃, 40 seconds, 60℃, 40 seconds for 45 cycles. In region B, qPCR was not performed under various conditions, and the addition of betaine (0.5 to 2 M), which is used to improve PCR defects due to high GC%, did not improve the results. On the other hand, qPCR in regions A and C was significantly improved by adding a certain amount of betaine (0.5 to 2 M) (Fig. 2).

[0177]

[0178] Example 5: Calculation of Ct values ​​and their relative CN values ​​from condition-dependent qPCR using genomic DNA.

[0179] As in Example 4, the design of primers and probes for general PCR performance is very limited, and it is also very difficult to establish PCR denaturation and annealing conditions for the amplification reaction. In this experiment, qPCR was evaluated under various conditions targeting the test region C (Fig. 1), which is the region around the EGFR promoter and exon 1, where qPCR is relatively smooth. At this time, qPCR was performed using the region of the RNaseP gene (SEQ ID NO: 21) (Fig. 3), which has few CpG sites, no CpG islands, and is reported to exist in two copies in the human genome, as an internal control gene.

[0180] In general, by adding ingredients such as betaine or DMSO to the buffer in a region of high GC% template DNA, such as the Exon 1 sequence region of EGFR (SEQ ID NO: 24) (Fig. 1), the formation of secondary structures due to high GC% can be prevented or the Tm value can be lowered, thereby significantly improving the efficiency of qPCR. In the test of the present invention, the concentration of betaine (0.5 to 2 M) was varied to improve qPCR, and as a result, the PCR due to high GC% was improved when 0.5 M or more was added. The qPCR of regions A and C was significantly improved when betaine was added at the optimal concentration of 1.5 M (Table 2, Fig. 4). The RNaseP gene showed high qPCR efficiency regardless of the addition of betaine, but in the EGFR qPCR, it was confirmed that the addition of betaine greatly improved the qPCR of EGFR, which has a high GC%. However, despite these improvements, methylated gDNA showed higher Ct values ​​(lower amplification efficiency) in qPCR than unmethylated gDNA even with the same template usage (5 ng). In this case, there was no CpG island within or near the qPCR sequence region, and in the case of RNaseP qPCR with low GC%, there was little difference in the Ct values ​​of the two gDNAs. However, in the case of the EGFR gene, methylated DNA still showed higher Ct values ​​than unmethylated DNA (test of buffer 1 in Table 3 and test numbers 15 and 16 in Figure 4). In the present invention, to improve this, AFA, which can increase hydrophobicity in the PCR reaction conditions, was added to buffer 1 and qPCR was performed. As a result, qPCR of methylated gDNA was improved, resulting in a lower Ct value, and similar Ct values ​​were observed in methylated and unmethylated gDNA (buffer 2 test in Table 2 and test numbers 21 and 22 in Figure 3).

[0181] Typical 2 nThe formula for the change in Ct value for the copy number of the qPCR amplification curve amplified by is the conversion formula Ct = S x LN(CN) + I using the Ct value of IC (CN 2) and the Ct value (CN 4) obtained by subtracting 1 from this Ct value, and using this formula, the relative copy number value can be calculated based on the qPCR result values ​​(Ct) in buffer 1 and buffer 2 of the target gene (Table 2). In the above formula Ct = S x LN(CN) + I, under ideal conditions where the efficiency is 100%, S is calculated as -1.44, and I is the Ct value of CN = 1 calculated by the test of the internal standard. The I value is calculated from the Ct value (JOE) of the RNaseP gene of CN 2 from the formula, and is equal to the value obtained by adding 1 to the measured Ct value of the RNaseP gene of CN2. In the experimental example, the relative copy number (relative CN) was inferred using the Ct value tested (Table 2). The relative copy number value in Table 2 is the value calculated using the formula converted from the above formula, Relative CN = EXP((Ct - I) / S)) (where S = -1.44 and I = Ct of RNaseP(IC, CN2) +1) using the formula for the Ct value (FAM) of the target gene (EGFR), which corresponds to the comparative value for RNaseP CN = 2. However, since this test was not performed under synchronized qPCR conditions of the standard gene RNaseP and the target gene EGFR, the exact copy number is not determined, but is a relative value. In particular, in buffer 1 with 1.5 M betaine added, the relative copy number of methylated gDNA was 2.6, which showed a significant difference from 1.3 of methylated gDNA, but in buffer 2 with more AFA added, the relative copy numbers were all calculated as 2.9 (*Note: Of course, this value does not mean the actual copy number of EGFR).The different relative copy numbers calculated in buffers 1 and 2 indicate that PCR efficiency varies depending on the mode of methylation under the two buffer conditions. This difference is due to 2. nd PCR after round 1 has nothing to do with methylation st This is a test result reflecting the methylation pattern of the raw material applied to round PCR.

[0182]

[0183] Example 6: Comparison of the ratio of relative copy number values ​​of methylated DNA and unmethylated DNA.

[0184] In Example 4, appropriate PCR amplification was not achieved in qPCR of EGFR PCR region B, which had a high GC%. However, differences in relative copy number values ​​occurred due to differences in PCR that reflected the methylation patterns in buffers 1 and 2 of Example 5. This characteristic was the same in EGFR PCR regions A and C (Table 3, Fig. 5). The relative copy number ratio (relative CN Ratio = relative CN of buffer 1 / relative of buffer 2) was also similar, 1.09 (region A) and 1.02 (region B) for unmethylated gDNA, and 0.31 (region A) and 0.36 (region B) for methylated gDNA (Table 3). This difference is interpreted as reflecting the influence of methylation not only in the target region for amplification but also in neighboring regions.

[0185] Table 3 shows the differences in qPCR of the EGFR Exon 1 region under different buffer conditions, and the relative copy numbers and ratios thereof.

[0186]

[0187] Example 7: qPCR test under different buffer conditions for the EGFR exon 20 region

[0188] To further clarify the qPCR pattern by methylation, EGFR exon 20 (SEQ ID NO: 25), which is an EGFR region but does not have a CpG island, was used in buffer 1 and buffer 2 as in Examples 5 and 6, and methylated gDNA and unmethylated gDNA were used to compare qPCR using primers, CRC-F52-1 (SEQ ID NO: 10), EXT-R1.2 (SEQ ID NO: 11), and FAM probe, TaqMAN-790-CRC-FAM (SEQ ID NO: 12) as shown in the region of Figure 6. Primers were used at 20 pmol each, and FAM probes were used at 10 pmol each. After a 10-minute reaction at 95℃, 40 seconds at 95℃, 40 seconds at 60℃ were repeated 45 times to perform qPCR as in Example 2. As a result, the relative copy number value of EGFR exon 20 was accurately calculated as a value similar to 2 regardless of methylated gDNA and unmethylated gDNA (*Note: The above PCR conditions were performed under synchronized conditions for the PCR of the reference gene and the target gene), and the ratio of the values ​​of buffer 1 and buffer 2 was confirmed to be 0.98 and 1.01, which are similar to 1 (Table 4). This is presumed to be because the test region was not methylated due to the absence of a CpG island or because the methylation was very low. Based on the results of Example 7, it is clear that the difference in qPCR of the EGFR-exon1 region in buffer 1 and buffer 2 in Examples 5 and 6 reflects the methylation pattern.

[0189] Table 4 shows the results of calculating the difference in qPCR of the EGFR Exon 20 region by buffer condition, the relative copy number, and the ratio.

[0190]

[0191] Example 8: qPCR test according to methylated gDNA mixing ratio

[0192] The purpose was to more clearly confirm the difference according to the methylation mode of Examples 5 and 6 and to confirm that quantification was possible.

[0193] Quantified unmethylated gDNA was mixed with methylated gDNA at a ratio of 0% to 100% and qPCR was performed as in region A of Example 6. As a result, buffer 2 showed similar Ct values ​​regardless of the mixing ratio, but the Ct value in buffer 1 decreased as the mixing ratio decreased, that is, as the methylated gDNA ratio increased (Table 5). The mixing ratio and the ratio of relative copy numbers CN in the calculated buffer 1 and buffer 2 tests were calculated as in Example 6 (Table 5). As a result, the higher the ratio of methylated gDNA, the lower the relative copy number ratio of buffer 1 and buffer 2, and there was a clear causal relationship (R) between the methylation mixing amount and the relative ratio (ratio). 2 It was confirmed that the coefficient of variation (C) was 0.9799 (Fig. 9).

[0194] Table 5 shows the differences in qPCR according to the mixing ratio of unmethylated and methylated gDNA, and the results of calculating the relative copy number and its ratio.

[0195]

[0196] Example 9: qPCR test according to methylation degree

[0197] Similar to Example 8, in order to clearly test the difference according to the degree of methylation, the test was performed using plasmid DNA with a controlled degree of methylation.

[0198] The pTOP-RNaseP-EGFR-Exon1 constructed in Example 1 was reacted with CpG methyltransferase (M.SssI) according to Example 3 at different concentrations of S-Adenosyl methionine (SAM) to prepare plasmids with different degrees of methylation. The degree of methylation of the prepared methylated pTOP-RNaseP-EGFR-Exon1 plasmid was confirmed by 1% agarose electrophoresis after digestion with HpaII, a cytosine methylation-sensitive restriction enzyme (Fig. 10). As a result, in the reaction using a low concentration of 0.625 μM SAM, the plasmid was digested into many fragments, similar to the sample that was not methylated (-methyl), and the plasmid using 10 μM SAM was hardly digested. Looking at these results, it was confirmed that there was a difference in the degree of methylation depending on the amount of SAM of 0.625, 2.5, and 10 μM. Using this, qPCR of EGFR Exon1 region A was performed in Buffer 1 and Buffer 2, which had different Ct values ​​of qPCR depending on the methylation pattern, as in Examples 5 and 6. As a result (Table 6, Fig. 11), for the sample using 10 μM SAM, the relative copy number ratio was calculated to be 0.34, confirming hypermethylation, and for the sample reacted with 2.5 μM SAM, the relative copy number ratio was calculated to be 0.74. In addition, for the sample reacted with 0.62 μM SAM, the relative copy number ratio was calculated to be 0.92, which was similar to the relative copy number ratio value of 0.99 of the unmethylated plasmid DNA that did not undergo methylation. This demonstrates that the results of HpaII treatment electrophoresis (Fig. 10) and the methylation level determination qPCR results of the present invention (Table 6, Fig. 11) are highly consistent. These results confirm that the methylation assessment qPCR system of the present invention is a highly effective and economical method.

[0199] Table 6 shows the differences in qPCR according to the degree of methylation, and the calculation of the relative copy number and its ratio.

[0200]

[0201] Example 10: Establishment of a qPCR assay for simultaneous assessment of EGFR methylation level and copy number.

[0202] In the present invention, we further attempted to construct a qPCR system capable of simultaneously and accurately evaluating the methylation pattern and the copy number of the target gene using the dual buffer system described above. To this end, in this example, we first attempted to synchronize the qPCR of RNaseP, a constitutive gene with only two copies in the human genome as an internal control, and the target gene (EGFR). qPCR synchronization refers to performing precise qPCR by simultaneously testing the internal standard and the target gene in a single reaction tube to minimize errors. To this end, as in Example 1, the two genes to be tested were physically linked into one, and the plasmid pTOP-RNaseP-EGFR-Exon1 (Control plasmid DNA) with the same copy number was used to synchronize the qPCR. For synchronization, PCR conditions were set to minimize (dCt ≤ 0.2) the difference (dCt) in the qPCR Ct between the target gene (labeled with FAM) and the internal standard (labeled with JOE); qPCR was performed with 20 pmol EGFR-Exon1-F4 (SEQ ID NO: 1), 20 pmol EGFR-Exon1-R4 (SEQ ID NO: 2), 20 pmol EGFR-Exon1-FAM4 (SEQ ID NO: 3) and 8 pmol RnaseP-F-2 (SEQ ID NO: 19), 8 pmol RnaseP-R-2 (SEQ ID NO: 20), and 8 pmol JOE-Rnase P-1 (SEQ ID NO: 21).

[0203] Table 7 shows the results of the qPCR test for simultaneous evaluation of methylation and copy number of EGFR.

[0204]

[0205] Under synchronized qPCR conditions, qPCR using unmethylated plasmid DNA in both buffers 1 and 2 resulted in a calculated CN of 2 and a relative copy number ratio of 1.0 (Table 7). When unmethylated gDNA and methylated gDNA were tested under synchronized qPCR conditions, the unmethylated gDNA yielded a calculated copy number of 2.3 (buffer 1) and 2.2 (buffer 2), similar to the internal standard plasmid DNA test, indicating that EGFR was measured as a copy number of 2 on the genome. In addition, the calculated relative copy number ratio was 1.1, indicating that methylation was evaluated as “none.” However, when methylated gDNA was used, in the test of buffer 2, copy number values ​​were measured similarly to those of unmethylated gDNA and internal standard plasmid DNA tests, but in the test of buffer 1, the copy number was calculated to be low at 0.3, and it was confirmed that the relative copy number ratio was 0.3, indicating hypermethylation (Table 7, Fig. 13).

[0206] In this way, it was confirmed that the number of copies and the degree of methylation can be accurately analyzed simultaneously through a qPCR test using buffer 1 and buffer 2 of the present invention.

[0207]

[0208] Example 11: Simultaneous evaluation of methylation level and copy number of EGFR in clinical samples using qPCR test

[0209] The qPCR (EGFR exon 1 region A region) was performed to evaluate the methylation degree and copy number of EGFR present in various samples, including unknown human clinical gDNA (gDNA isolated from human whole blood) samples, under the synchronized conditions described in Example 10. The CN value and its relative ratio were calculated based on the Ct value of the qPCR performed for each sample and buffer condition. For most clinical gDNAs, CN = 2 was evaluated and confirmed to be unmethylated (Table 8). In general, hypermethylation of EGFR is frequently reported in lung cancer patients, but the association with cancer is not known due to restrictions on access to medical information of clinical patients of the clinical samples provided. However, this result evaluated that unmethylation of the EGFR promoter is common.

[0210] Table 8 shows the results of the qPCR test for simultaneous evaluation of EGFR methylation and copy number in clinical samples.

[0211]

[0212] Example 12: Simultaneous evaluation of SHOX2 methylation level and copy number by qPCR test

[0213] Methylation of the SHOX2 promoter is considered a key biomarker for potential cancer diagnosis. CGIs are present around SHOX2 exon 1. Therefore, the SHOX2 exon 1 region was selected as the qPCR region for simultaneous evaluation of methylation and copy number in the SHOX2 promoter region (Fig. 14).

[0214] To measure methylation and copy number of SHOX2 promoter, qPCR was performed under qPCR conditions synchronized with RNaseP as an internal standard {20 pmol SHOX2-B-F1 (SEQ ID NO: 13), 20 pmol SHOX2-B-R2 (SEQ ID NO: 14), 20 pmol SHOX2-B-CalRed610 (SEQ ID NO: 15) and 8 pmol RnaseP-F-2 (SEQ ID NO: 19), 8 pmol RnaseP-R-2 (SEQ ID NO: 20), 8 pmol JOE-Rnase P-1 (SEQ ID NO: 21)} (Table 9, Fig. 15).

[0215] Under synchronized qPCR conditions, CN was 2 in qPCR using unmethylated gDNA in both buffer 1 and buffer 2, and methylated gDNA was also measured as CN=2 under buffer 2 conditions. However, the relative copy number in methylated gDNA was measured as 0.62 in buffer 1, resulting in a relative copy number ratio of 0.26, which was significantly lower than the relative copy number ratio of 0.87 for unmethylated gDNA. Thus, it was confirmed that the above qPCR conditions were suitable for measuring methylation and copy number of the SHOX2 promoter. Table 9 shows the results of the qPCR test for evaluating methylation and copy number of SHOX2.

[0216]

[0217] Example 13: Simultaneous evaluation of methylation levels and copy numbers of EGFR and SHOX2 using multiplex qPCR testing.

[0218] In Examples 10 and 12, EGFR and SHOX2 were tested separately by qPCR, and a multiplex qPCR test was performed to simultaneously evaluate the methylation level and copy number of each gene by performing qPCR on both genes simultaneously in one test group. qPCR conditions for this were; The assay was performed with 20 pmol EGFR-Exon1-F4 (SEQ ID NO: 1), 20 pmol EGFR-Exon1-R4 (SEQ ID NO: 2), 20 pmol EGFR-Exon1-FAM4 (SEQ ID NO: 3) and 20 pmol SHOX2-B-F1 (SEQ ID NO: 13), 20 pmol SHOX2-B-R2 (SEQ ID NO: 14), 20 pmol SHOX2-B-CalRed610 (SEQ ID NO: 15) and 8 pmol RnaseP-F-2 (SEQ ID NO: 19), 8 pmol RnaseP-R-2 (SEQ ID NO: 20), 8 pmol JOE-Rnase P-1 (SEQ ID NO: 21).

[0219] As a result (Table 10 and Fig. 16), even when qPCR was performed in multiplex, the copy number and methylation of each gene were evaluated with values ​​similar to the test results of Examples 10 and 12, respectively. Table 10 shows the results of the multiplex qPCR test for evaluating methylation and copy number of EGFR and SHOX2.

[0220]

[0221] Example 14: Simultaneous evaluation of methylation level and copy number of EGFR and SHOX2 in clinical samples using multiplex qPCR test

[0222] In Example 13, multiplex qPCR was performed to evaluate the methylation degree and copy number of EGFR and SHOX2 in various samples, including unknown human clinical gDNA (gDNA isolated from human whole blood) samples for simultaneous analysis of the two genes, EGFR and SHOX2. The copy number N value and its relative ratio were calculated based on the Ct value of the qPCR performed for each sample and buffer condition. For most clinical gDNAs, both genes were evaluated as CN = 2, confirming that they were not methylated (Table 11 and Figure 17). The results were the same as those of the EGFR single test in Example 11. In this way, it was confirmed that methylation and copy number of each gene can be analyzed by performing qPCR for two or more genes in a single test group.

[0223]

[0224] Example 15: Construction of a qPCR test for simultaneous evaluation of CYP2D6 methylation level and copy number.

[0225] The simultaneous evaluation system for the degree of methylation and copy number constructed in the present invention was performed on the CYP2D6 gene region (SEQ ID NO: 23). CYP2D6 is one of the most important enzymes in drug metabolism. The methylatable CpG islands of this gene are mainly distributed within the gene body of exons 2 and 3 rather than in the promoter region (Fig. 18). First, pTOP-RNaseP-CYP2D6-100CT (Control plasmid DNA) constructed in Example 1 was used for qPCR synchronization of RNaseP as an internal standard and the target gene (CYP2D6). For synchronization, PCR conditions were set to minimize (dCt ≤ 0.2) the difference (dCt) between the qPCR Ct of the target gene (labeled with FAM) and CalRed610 as an internal standard; 20 pmol ST2-CYP2D6-100-F6-2 (SEQ ID NO: 16), 20 pmol CYP2D6-100-R1 (SEQ ID NO: 17), 20 pmol FAM-R-CYP2D6-2 (SEQ ID NO: 18) and 10 pmol RnaseP-F-2 (SEQ ID NO: 19), 10 pmol RnaseP-R-2 (SEQ ID NO: 20), 8 pmol CalRed610-RnaseP-1 (SEQ ID NO: 22) were used. qPCR was performed in buffer 1 and buffer 2 under synchronized qPCR conditions, and the results were compared. Similar to the EGFR qPCR results in Example 10, CN = 2 was calculated for the unmethylated gDNA, as was the control plasmid, and the relative copy number ratio was 0.9, which was similar to 1.0 (Table 12, Fig. 19).

[0226] However, in the case of methylated gDNA, the calculated copy number value of CYPD6 was measured as 3 (Fig. 20), and the relative copy number ratio was 0.5, confirming that methylation occurred at a high level. Table 12 shows the results of the qPCR test for evaluating methylation and copy number of CYP2D6.

[0227]

[0228] Example 16: Simultaneous evaluation of CYP2D6 methylation level and copy number qPCR test

[0229] qPCR was performed to evaluate the methylation degree and copy number of CYP2D6 in various samples, including unknown human clinical gDNA (gDNA isolated from human whole blood), under the synchronized conditions described in Example 15. The copy number and its relative ratio were calculated based on the Ct values ​​of the qPCR performed for each sample and buffer condition. For most clinical gDNAs, the CN ranged from 2 to 5, and unlike EGFR, most were confirmed to be methylated (Table 13). Table 13 shows the results of the qPCR test for evaluating EGFR methylation and copy number in clinical samples.

[0230] TargetNameSequence (Tm, ℃)서열번호EGFR Exon1(A region)EGFR-Exon1-F45’-GAGGTGGGGACCCGAATAAAGGAG (68.5)1EGFR-Exon1-R45’-CGAGCCAAATCTGTGCCAGGGTC (68.3)2EGFR-Exon1-FAM45’-FAM-TGCCATTATCCGACGCTGGCTCTA-BHQ (66.9)3EGFR Exon1(B region)EGFR-Exon1-F15’-GTCCCTCCTCCTCCCGCCCTGC (73.2)4EGFR-Exon1-R15’-GACGCCGACGAGGTGGCCTGTCGTC (73.8)5FAM-EGFR-Exon15’-FAM-CAGCAGCCTCCGCCCCCCGCAC-BHQ (75.2)6EGFR Exon1(C region)EGFR-Exon1-F35’-CGAGACTGCACTGTTTAGGGAAG (64.6)7EGFR-Exon1-R35’-GAGGCGGGGAGGTCCTCTCAG (69)8EGFR-Exon1-FAM35’-FAM-TACAGCCTCCCCTCGGACCC-BHQ (66.6)9EGFR Exon20CRC-F52-15’-GGCAGCCGAAGGGCATGAGC (66.6)10EXT-R1.25’-TAGCGTGGACAACCCCCACGTGTGC (72.3)11TaqMAN-790-CRC-FAM5’-FAM-CGGTGGAGGTGAGGCAGATG-BHQ1 (64.6)12SHOX2 Exon1SHOX2-B-F15’-AGAGCGGGGCTCTGCTGGCAGAG (71.9℃)13SHOX2-B-R25’-CTCTCCAGCACCTCCCGGTACGTG (66.6℃)14SHOX2-B-CalRed6105’-CalRed610-TCGTCTCCAAGTCTTTTGACCAGAAAG-BHQ2 (72.2℃)15CYP2D6ST2-CYP2D6-100-F6-25’-GTGGACCTGATGCACCGACG (64.6)16CYP2D6-100-R15’-CTGGTCGAAGCAGTATGGTGTG (64.2)17FAM-R-CYP2D6-25’-FAM-CAGGTTGCCCAGCCCGGGCAGTG-BHQ1 (70.6)18RNasePRNaseP-F-25’-TCCATGGAGGTAAGCAAGTTCTTC (63.6)16RNaseP-R-25’-GATGACTGTGTTTAGAACAGGTTGAG (64.6)19JOE-RNaseP-15’-JOE-TACAGCCATACCTATTTTTATTGCCCTA-BHQ1 (64.4)20CalRed610-RNaseP-15’-CalRed610-TACAGCCATACCTATTTTTATTGCCCTA-BHQ2 (64.4)21.

[0231] ReactionTest No.gDNA * Ct (FAM)of EGFR Exon1 **Ct (JOE) ofIC (CN2)Calculated Ct ofIC-1 (CN4)Ct = S x LN(CN) + IRelative CN(Avg.)SIno add1Unmethylated gDNA29.7328.5227.52-1.4429.520.9(0.8)229.7328.527.529.500.9329.8428.3527.3529.350.74Methylated gDNA30.4528.6627.6629.660.6(0.5)530.3628.4127.4129.410.5630.3728.3327.3329.330.5+ 1 M Betaine7Unmethylated gDNA29.3128.8827.8829.881.5(1.4)829.2428.6527.6529.651.3929.5728.9327.9329.931.310Methylated gDNA29.8128.7227.7229.720.9(1.0)1130.2129.3628.3630.361.11230.0428.8427.8429.840.9+ 1.5 M Betaine(Buffer 1)13Unmethylated gDNA27.7528.3527.3529.353.0(2.6)1428.0628.3427.3429.342.41528.1628.4327.4329.432.416Methylated gDNA29.3428.5427.5429.541.1(1.3)1729.4229.0428.0430.041.51829.5728.8327.8329.831.2+ 1.5 M Betaine+1X AFA(Buffer 2)19Unmethylated gDNA28.4128.9927.9929.993.0(2.9)2028.0428.3827.3829.382.52128.2728.9827.9829.983.322Methylated gDNA28.2228.5127.5129.512.4(2.9)2328.3329.1928.1930.193.62428.2728.5927.5929.592.5

[0232] *5 ng each of EpiScope Unmethylated HCT116 DKO gDNA (Code No. 3521) and EpiScope Methylated HCT116 gDNA (Code No. 3522) were used. ** This is qPCR of the C region of EGFR Exon1.

[0233] EGFR Exon1 RegionsgDNA*Test No.Reaction ** Ct (FAM) ofEGFR Exon1Ct (JOE) ofIC(CN2)RelativeCNRelative CN RatioAUnmethylated gDNA1Buffer 129.3329.201.881.092Buffer 229.4929.271.72Methylated gDNA3Buffer 130.9729.050.530.314Buffer 229.1528.931.72CUnmethylated gDNA1Buffer 128.6229.082.751.022Buffer 228.9629.392.69Methylated gDNA3Buffer 130.0628.900.900.364Buffer 228.3928.702.48

[0234] * 5 ng each of EpiScope unmethylated HCT116 DKO gDNA (Code No. 3521) and EpiScope methylated HCT116 gDNA (Code No. 3522) were used. ** The reaction buffers are buffer 1 (Taq PCR buffer + 1.5 M betatine) and buffer 2 (buffer 1 + AFA) shown in Table 2.

[0235] gDNA*Test No.ReactionCt (FAM) ofEGFR Exon 20Ct (JOE) ofIC(CN2)RelativeCNRelative CN RatioUM1Buffer 129.4029.281.840.982Buffer 229.4929.391.87M3Buffer 129.3229.181.821.014Buffer 229.3329.181.80

[0236] * UM, EpiScope Unmethylated HCT116 DKO gDNA (Code No.3521) and M, EpiScope Methylated HCT116 gDNA (Code No.3522), each 5 ng was used.

[0237] % Unmethylated gDNA*Test No.ReactionCt (FAM) ofEGFR Exon I(region A)Ct (JOE) ofIC(CN2)RelativeCNRelative CN Ratio1001Buffer 129.5529.471.890.992Buffer 229.4429.361.89703Buffer 129.9329.551.540.694Buffer 229.2529.402.22505Buffer 130.2429.481.180.596Buffer 229.2429.231.99307Buffer 130.5329.531.000.498Buffer 229.1729.212.0609Buffer 131.2029.370.560.2810Buffer 229.3129.301.99

[0238] * 5 ng of EpiScope Unmethylated HCT116 DKO gDNA (Code No.3521) and EpiScope Methylated HCT116 gDNA (Code No.3522) were mixed to the indicated %.

[0239] DNA*Test No.ReactionRelative CNRelative CN RatioNo methylated control plasmid DNA1Buffer 11.980.992Buffer 22.01Methylated Plasmid DNA with 0.625 μM SAM3Buffer 12.060.924Buffer 22.25Methylated Plasmid DNA with 2.5 μM SAM5Buffer 11.730.746Buffer 22.34Methylated Plasmid DNA with 10 μM SAM7Buffer 10.720.348Buffer 22.10

[0240] * pTOP-RNaseP-EGFR-Exon1 was 10 times the plasmid DNA that had undergone CpG methylation using SAM at the concentrations indicated. 3 Copies were used.

[0241] DNA * Test No.ReactionCt (FAM) ofEGFR Exon I(Region A)Ct (JOE) ofIC (CN2)dCt(FAM-JOE)CalibratedCt (CN2)CalculatedCt (CN4)Ct = S x LN(CN) + ICalculated EGFR CNRelative CN RatioSIControl plasmid DNA1Buffer 129.3829.260.1229.3828.38-1.44330.382.01.02Buffer 229.3329.290.0429.3328.3330.332.0Unmethylated gDNA3Buffer 129.8129.91-30.0329.0331.032.31.14Buffer 229.7729.8629.9028.9030.902.2Methylated gDNA5Buffer 131.4629.66-29.7828.7830.780.60.36Buffer 229.5529.7329.7728.7730.772.3

[0242] *Control plasmid DNA (pTOP-RNaseP-EGFR-Exon1) is 10 3 5 ng of copy, unmethylated gDNA (Code No. 3521), and methyl gDNA (Code No. 3522) were used. qPCR was performed targeting region A of EGFR-Exon 1.

[0243] Test No.DNA*Calibrated EGFR CN(Avg. of 2-repeates)Relative CN Ratio(① / ②)Decision①Buffer 1②Buffer 2Methylation**EGFR CN (②)1Control plasmid DNA(pTOP-RNaseP-EGFR-Exon1)2.002.021.0UM221.961.951.0UM232.052.041.0UM24UnMethylated gDNA2.152.290.9UM25Methylated gDNA0.672.160.3M26Promega gDNA1.932.110.9UM27gDNA#61.751.910.9UM28gDNA#71.882.000.9UM29gDNA#81.872.080.9UM210gDNA#121.901.951.0UM211gDNA#22.002.2 10.9UM212gDNA#32.192.181.0UM213gDNA#41.791.990.9UM214gDNA#92.001.971.0UM215gDNA#391.922.130.9UM216gDNA#861.962.200.9UM2

[0244] *pTOP-RNaseP-EGFR-Exon1 is 10 3Copy, Unmethylated gDNA (Code No. 3521), methyl gDNA (Code No. 3522), Promega gDNA, and each clinical sample gDNA (human whole blood DNA) were used at 5 ng. qPCR was performed targeting region A of EGFR-Exon 1.** In methylation determination, UM is unmethylated and M is methylated.

[0245] DNA*Test No.ReactionCt (CFR610)Ct (JOE)CalculatedCNRelative CN RatioSHOX2IC(CN2)Unmethylated gDNA1Buffer 129.9829.922.080.872Buffer 230.2129.952.39Methylated gDNA3Buffer 129.5631.260.620.264Buffer 229.6829.452.35

[0246] * 5 ng each of unmethylated gDNA (Code No. 3521) and methyl gDNA (Code No. 3522) were used.

[0247] DNA*EGFRSHOX2CalculatedCNRelative CN Ratio(① / ②)CountingCalculated CNRelative CN Ratio(③ / ④)CountingBuffer 1①Buffer 2②CNMethylationBuffer 1③Buffer 2④CNMethylation**Unmethylated gDNA1.932.050.942UM1.952.030.962UMMethylatedgDNA0.762.140.362M0.802.040.392M

[0248] * 5 ng each of unmethylated gDNA (Code No. 3521) and methyl gDNA (Code No. 3522) were used.** In methylation determination, UM stands for unmethylated and M stands for methylated.

[0249] EGFRTest No.DNA*Calculated CN (Buffer2)Relative CN RatioCounting1 st 2 nd 3 rd Average1 st 2 nd 3 rd AverageCNMethylation**1Unmethylated gDNA1.921.822.132.00.991.090.931.02 UM2Methylated gDNA1.931.942.132.00.390.390.310.42 M3NCI-H854 gDNA2.712.692.732.71.21.20.971.13 UM4gDNA#62.001.832.192.01.081.150.981.12 UM5gDNA#71.911.852.081.91.121.140.941.12 UM6gDNA#81.842.012.032.01.21.020.961.12 UM7gDNA#121.891.992.142.00.980.890.850.92 UM8gDNA#21.972.182.22.11.060.930.981.02 UMSHOX2No.DNA*Calculated CN (Buffer2)Relative CN RatioCounting1 st 2 nd 3 rd Average1 st 2 nd 3 rdAverageCNMethylation**1Unmethylated gDNA1.931.812.152.01.011.10.871.02 UM2Methylated gDNA1.691.651.721.70.40.480.410.42 M3NCI-H854 gDNA2.262.122.182.20.941.010.941.02 UM4gDNA#61.871.802.061.91.061.131.031.12 UM5gDNA#71.801.902.011.91.031.050.951.02 UM6gDNA#82.021.991.982.011.071.021.02 UM7gDNA#121.781.81.851.80.940.930.950.92 UM8gDNA#21.991.912.082.00.890.990.981.02 UM

[0250] * 5 ng of unmethylated gDNA (Code No. 3521), methyl gDNA (Code No. 3522), gDNA from the cell line NCI-H854, and each clinical sample gDNA (human whole blood DNA) were used. qPCR was repeated three times. ** In methylation determination, UM indicates unmethylated and M indicates methylated.

[0251] DNA*Test No.ReactionCt (FAM)CYPD6Ct (610)IC(CN2)dCt(FAM-610)CalibratedCt (CN2)CalculatedCt (CN4)Ct = S x LN(CN)+ ICalculatedCYPD6 CNRelative CN RatioSIControl plasmid DNA1Buffer 128.7328.650.0828.7327.73-1.44329.732.01.02Buffer 228.4128.230.1828.4127.4129.412.0Unmethylated gDNA3Buffer 128.6428.73-28.8127.8129.812.30.94Buffer 228.2328.3228.5027.5029.502.4Methylated gDNA5Buffer 129.2328.5428.6227.6229.621.30.56Buffer 227.9428.2028.3827.3829.382.7

[0252] * Control plasmid DNA (pTOP-RNaseP-CYP2D6-100CT) is 10 3 5 ng of copy, unmethylated gDNA (Code No. 3521), and methyl gDNA (Code No. 3522) were used. qPCR was performed targeting the CYPD6 region.

[0253] TestNo.DNA*Calibrated CYP2D6 CN(Avg. of 2-repeates)Relative CN Ratio(① / ②)Decision①Buffer 1②Buffer 2Methylation**CYP2D6 CN (②)1Control plasmid DNA(pTOP-RNaseP-CYP2D6-100CT)1.982.021.0UM222.061.961.1UM231.972.031.0UM24UnMethylated DNA2.182.370.9UM25Methylated DNA1.172.570.5M36Promega gDNA1.642.190.7UM27gDNA #61.413.130.4M38gDNA #71.523.080.5M39gDNA #82.665.180.5M510gDNA #121.623.340.5M311gDNA #21.182.070.6M212gDNA #31.052.070.5M213gDNA #41.533.240.5M314gDNA #91.854.160.4M415gDNA #391.653.250.5M316gDNA #862.034.480.5M5

[0254] *pTOP-RNaseP-CYP2D6-100CT is 10 3 5 ng of copy, unmethylated gDNA (Code No. 3521), methyl gDNA (Code No. 3522), promega gDNA, and each clinical sample gDNA (human whole blood DNA) were used. qPCR was performed targeting the CYP2D6 region.** In methylation determination, UM is unmethylated and M is methylated.

[0255]

[0256] The present invention can be widely used in genetic testing of agricultural, livestock, and fishery products, as well as in diagnosis in the medical field.

[0257] Electronic file attached

Claims

1. (1) A step of performing real-time quantitative PCR using the subject DNA in two buffers, buffer 1 and buffer 2, each containing an internal standard and each primer and probe that specifically reacts to the target gene sequence region and dNTP and polymerase; (2) A step of calculating the relative copy number using the Ct values ​​of qPCR performed in two buffers obtained in step (1); (3) a step of calculating the ratio of the relative replication numbers calculated in step (2); and (4) A method for analyzing the degree of methylation of target gene DNA, comprising: a step of determining the degree of methylation of the target gene by the ratio value of the relative copy number; 2.(1) A step of synchronizing real-time quantitative PCR by including an internal standard and a target gene sequence region in one standard plasmid; (2) A step of performing real-time quantitative PCR using a subject in two buffers, buffer 1 and buffer 2, containing primers and probes and dNTPs that specifically react to an internal standard and a target gene sequence region, and a polymerase; (3) A step of calculating the number of copies using the Ct value of real-time quantitative PCR performed in two buffers, buffer 1 and buffer 2, obtained in step (2); (4) a step of calculating the ratio of the number of copies from the number of copies calculated in step (3); and (5) A method for simultaneous analysis of the methylation degree and copy number of target gene DNA, including a step of determining the methylation degree of the target gene by the ratio of copy number obtained in step (4).

3. In claim 1 or claim 2, The above subject DNA is an analysis method in which the subject DNA is DNA extracted from cells, tissues, blood, plasma, saliva, feces, urine, skin or organs of a living organism.

4. In claim 1 or claim 2, An analytical method in which the internal standard is selected from a sequence region without a CpG island among the base sequences of the host gene.

5. In claim 1 or claim 2, The internal standard is selected from the group of genes consisting of CANX, HPRT1, PGK1, TBP, YWHAZ, SDHA, UBC, GUSB, GAPDH, ACTB, TUBA1A ATP5B, PPIA, HNRNPL, IPO8, PUM1, UBC, RPP30, PCBP1 and RNaseP.

6. In claim 1 or claim 2, An analysis method in which the sequence region of the target gene is selected from the region containing the CpG Island and its adjacent region.

7. In claim 1 or claim 2, An analysis method in which a copy number ratio of ≤0.5 indicates hypermethylation, a copy number ratio of 0.5 to 0.8 indicates hypomethylation, and a copy number ratio of ≥0.8 indicates unmethylation.

8. In claim 1 or claim 2, Among the two buffers above a) Buffer 1 further contains a nucleic acid secondary structure formation inhibitor in a buffer for real-time quantitative PCR, A) An analysis method characterized in that buffer solution 2 further includes a water-miscible organic solvent that increases hydrophobicity in the composition of buffer solution 1.

9. In claim 1 or claim 2, Among the two buffers above a) Buffer 1 further contains a nucleic acid secondary structure formation inhibitor in a buffer for real-time quantitative PCR, A) An analysis method characterized in that buffer solution 2 further includes a water-miscible organic solvent that increases hydrophobicity and does not denature proteins in the composition of buffer solution 1.

10. In claim 8, An analysis method wherein the nucleic acid secondary structure formation inhibitor is at least one selected from betaine and DMSO.

11. In claim 8, An analysis method wherein the organic solvent of the above buffer solution 2 is at least one selected from ethanol, methanol, and isopropanol.

12. In claim 8, An analysis method wherein the above buffer solution 2 contains more than 0% and less than 20% (v / v) of the above organic solvent.

13. In claim 8, An analysis method wherein the organic solvent of the buffer solution 2 contains at least one of ethanol or methanol in an amount of more than 0% and less than 20% (v / v) and glycerol in an amount of more than 0% and less than 40% (v / v).

14. In claim 1 or claim 2, Among the two buffers above a) Buffer 1 is Mg 2+ Contains 1.5~5 mM, KCl 35~100 mM, (NH4)2SO4 6~50 mM, and betaine 0.5~2.5 M. b) An analytical method wherein buffer 2 contains a mixed solution containing 20% ​​of 5 to 40% (v / v) ethanol in an amount of more than 0 and less than 10% (v / v).

15. In claim 1 or claim 2, The calculation of the above copy number uses the formula Ct = S x LN(CN) + I (where S = -1.44 and I is the Ct value of CN = 1 calculated by testing the internal standard), and the qPCR Ct value of the target gene is substituted into the formula to calculate the copy number value, thereby obtaining the copy number with respect to the internal standard.

16. In claim 1 or claim 2, The calculation of the above copy number uses the formula Ct = S x LN(CN) + I (where S = -1.44 and I is the Ct value of CN = 1 calculated by testing the internal standard), and the copy number value is calculated by substituting the qPCR Ct value of the target gene into the formula to obtain the copy number with respect to the internal standard. The ratio of the above copy number is obtained as (the copy number of the target gene obtained from the above buffer 1 test) / (the copy number of the target gene obtained from the buffer 2 test), and the analysis method..

17. In claim 1 or 2, An analysis method comprising two or more target genes in the above step (1). 18.a) Target gene-specific primers and target gene-specific probes; b) Reference gene-specific primers and reference gene-specific probes capable of amplifying and identifying the reference gene; c) DNA polymerase; a) Buffer 1; and b) Contains buffer solution 2; The above buffer 1 further contains a nucleic acid secondary structure formation inhibitor in a buffer for real-time quantitative PCR, A qPCR kit for analyzing the degree of methylation of target gene DNA, characterized in that the buffer 2 further contains a water-miscible organic solvent that increases the hydrophobicity of the buffer 1.

19. In claim 18, A kit characterized in that the buffer solution 2 further comprises a water-miscible organic solvent that increases hydrophobicity and does not denature proteins in the composition of the buffer solution 1.

20. In claim 18, A kit wherein the above nucleic acid secondary structure formation inhibitor is at least one selected from betaine and DMSO.

21. In claim 18, The organic solvent of the above buffer solution 2 is at least one selected from ethanol, methanol, and isopropanol.

22. In claim 18, A kit wherein the above buffer solution 2 contains more than 0% and less than 20% (v / v) of the above organic solvent.

23. In claim 18, A kit wherein the organic solvent of the above buffer solution 2 contains at least one of ethanol or methanol in an amount of more than 0% and less than 20% (v / v) and glycerol in an amount of more than 0% and less than 40% (v / v).

24. In claim 18, Among the two buffers above a) Buffer 1 is Mg 2+ Contains 1.5~5 mM, KCl 35~100 mM, (NH4)2SO4 6~50 mM, and betaine 0.5~2.5 M. b) A kit wherein buffer solution 2 contains a mixed solution containing 20% ​​ethanol (5-40%) in an amount of more than 0% but not more than 10% (v / v).

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