Method for detecting methylation marker of DNA

Abstract

The present invention relates to a method for detecting a methylation marker of DNA and, more specifically, to: a method in which a product obtained by binding an adapter to DNA obtained from a sample and performing primary amplification is treated with a methylation restriction enzyme to remove an unmethylated region, and then a product having undergone secondary amplification using primers specific to a target region is analyzed, thereby detecting a methylation state of the target region; and a disease diagnosis method using same. The method for detecting methylated DNA, according to the present invention, can secure a quantitatively and qualitatively methylated DNA enrichment pool, and can detect methylation levels by real-time polymerase chain reaction using target methylation marker-specific primers / probes in the methylated DNA enrichment pool, thus having excellent detection sensitivity, thereby enabling the detection of methylated DNA with higher sensitivity and specificity than existing methods, and enabling analysis regardless of the type and number of methylation restriction enzymes, which is therefore commercially useful.

Description

Method for detecting methylation markers of nucleic acids

[0001] The present invention relates to a method for detecting a methylation marker of a nucleic acid, and more specifically, to a method for detecting the methylation status of a target region by first amplifying a product by binding an adapter to a nucleic acid obtained from a sample, treating the product with a methylation restriction enzyme to remove an unmethylated region, and then analyzing the product by second amplifying the product using a target region-specific primer, and a disease diagnosis method using the same.

[0002]

[0003] DNA methylation is an epigenetic change that occurs in the genomic DNA of mammalian cells. When cytosine and guanine (CG) are arranged consecutively in the DNA chain of A (adenine), T (thymine), G (guanine), and C (cytosine), it is called CpG, and when a methyl group is attached to the 5th carbon of C, it is called CpG methylation. CpG methylation is generally concentrated in the promoter region that regulates gene expression, and the region with GC% 50% or more and CpG% 3.75% or more within a length of 0.2-3 kb is called a CpG island. In fact, CpG islands appear in the promoters of housekeeping genes in the human body (Cross, S. et al., Curr. Opin. Gene Develop., 5:309, 1995). Methylation of promoter CpG islands is known to be highly correlated with the development of various diseases, including cancer, by reducing and disrupting the expression of the corresponding gene. In particular, methylation in CpG islands, which regulate the expression of tumor suppressor genes, blocks gene expression and function, promoting cancer development and progression. This phenomenon of DNA methylation has garnered attention as a biomarker for early cancer diagnosis due to the fact that abnormal mutations occur from the early stages of cancer development and persist throughout the precancerous stage, its high cancer-specificity, and its high stability among various biobased molecules (DNA, RNA, proteins, etc.).

[0004] In this way, DNA methylation information specifically observed in cancer cells flows into body fluids (blood, sputum, saliva, feces, urine, etc.) due to phenomena such as apoptosis or necrosis of cancer cells and exists as cell-free DNA (cfDNA). In particular, it is observed in circulating tumor DNA (ctDNA) released from cancer cells, and various DNA methylation biomarker selection and detection methods are being developed to confirm the presence of abnormal DNA methylation in this ctDNA (Republic of Korea Patent No. 2701682, Republic of Korea Patent No. 2701683).

[0005] Early DNA methylation detection methods ranged from methylation-specific PCR (MSP), which relied on the chemical property of sodium bisulfite treatment to convert unmethylated cytosine to uracil, to next-generation sequencing (NGS) analysis, which can measure methylation at the whole genome level. However, bisulfite treatment is known to be a strong chemical conversion process that causes DNA damage / loss, which limits the sensitivity of methylation detection, especially in cell-free DNA. Furthermore, NGS-based methylation detection methods still suffer from the drawback of being expensive.

[0006] On the other hand, the MSRE-qPCR method, which removes unmethylated regions by methylation-sensitive restriction enzyme (MSRE) treatment and then detects target methylated regions by real-time polymerase chain reaction (Real-time PCR), has been used for a long time (Hofner M. et al., Epigenetics Methods, Vol. 8, pp. 181-212, 2020). However, the analyzable markers are limited due to the limited types and combinations of MSREs, and there are limitations in detecting cell-free DNA, which exists at very low levels, with the level of real-time polymerase chain reaction technology.

[0007]

[0008] Accordingly, the present inventors have made great efforts to detect methylation markers with high sensitivity and accuracy, and have confirmed that when an adapter is attached to the obtained nucleic acid to perform a first amplification, then a methylation restriction enzyme is treated to remove the unmethylated region, and then a second amplification reaction is performed using a target region-specific primer to analyze the amplified product, the methylation status of the target region can be detected with high sensitivity and accuracy, thereby completing the present invention.

[0009]

[0010] Summary of the invention

[0011] The purpose of the present invention is to provide a method for detecting methylation of nucleic acids.

[0012] Another object of the present invention is to provide a disease diagnosis method, a disease diagnosis composition, and a kit using the above method.

[0013] In order to achieve the above object, the present invention provides a method for producing a nucleic acid, comprising: (a) obtaining a nucleic acid from a biological sample; (b) binding an adapter to the obtained nucleic acid; (c) treating the nucleic acid to which the adapter is bound with one or more methylation restriction enzymes (MRE); (d) amplifying the nucleic acid treated with the restriction enzyme using a primer specific to the adapter; (e) performing a polymerase chain reaction (PCR) reaction on the amplification product obtained in step (d) using a primer capable of amplifying the target region to amplify the target region; and (f) determining whether the amplification product obtained in step (e) exists, and determining that the target region is methylated if the amplification product exists.

[0014] A method for detecting the methylation status of a nucleic acid including

[0015] The present invention also provides a method for providing information for disease diagnosis, including a step of determining that a disease exists when a target region is methylated by the above method.

[0016] The present invention also provides a disease diagnosis method including a step of determining that a disease is present when a target region is methylated by the above method.

[0017] The present invention also provides the use of primers capable of amplifying the target region for disease diagnosis.

[0018] The present invention also provides the use of primers capable of amplifying the target region for the manufacture of a disease diagnostic kit.

[0019] The present invention also provides a composition and kit for diagnosing a disease, comprising a primer capable of amplifying the target region.

[0020]

[0021] FIG. 1 is a conceptual diagram schematically illustrating a methylated DNA enrichment process and a methylated DNA detection method according to one embodiment of the present invention.

[0022] FIG. 2 is a conceptual diagram schematically illustrating the principle of position selection of a target region-specific primer in a detection method according to one embodiment of the present invention.

[0023] FIG. 3 is a conceptual diagram schematically illustrating a method for selecting the location of a target region-specific primer in a detection method according to one embodiment of the present invention.

[0024] Figure 4 is a diagram illustrating the results of selecting a specific primer position for the SDC2 gene, a colon cancer marker, in a detection method according to one embodiment of the present invention.

[0025] Figure 5 shows the positions of primers and probes specific to the SDC2 gene, a colon cancer marker, in a detection method according to one embodiment of the present invention.

[0026] Figure 6 is a conceptual diagram comparing a detection method according to one embodiment of the present invention with a conventional MSRE-qPCR method.

[0027] FIG. 7 is a comparison of the results of detecting DNA methylation status using a detection method according to one embodiment of the present invention and a conventional MSRE-qPCR method, with Ct values. (A) is the result of detecting the methylation status of unmethylated DNA (0% Unme gDNA) and methylated DNA (1% Me gDNA) using a conventional method, (B) is the result using the method of the present invention, (C) is the result of detecting the methylation status of a normal person (NOR cfDNA) and a colon cancer sample (CRC cfDNA) using a conventional method, and (D) is the result using the method of the present invention.

[0028] FIG. 8a is a table showing various MSRE combinations performed in a detection method according to one embodiment of the present invention, and FIG. 8b is a conceptual diagram schematically showing the positions of CMRs and CMRFs that can be generated according to the number of MSRE recognition sequences around a target CpG region.

[0029] FIG. 9 is a diagram illustrating the position of CMRF derived to select a primer position for each lung cancer-specific marker in a detection method according to one embodiment of the present invention, wherein the square in the center left of each drawing represents a target CpG, 9a shows the position of CMRF derived for the cg06065125 marker in a lung adenocarcinoma (ADC) sample, 9b shows the position of CMRF derived for the cg17757602 marker in a lung adenocarcinoma (ADC) sample, 9c shows the position of CMRF derived for the cg00959431 marker in a lung adenocarcinoma (ADC) sample, 9d shows the position of CMRF derived for the cg00959431 marker in a lung squamous cell carcinoma (SCC) sample, and 9e shows the position of CMRF derived for the cg00959431 marker in a lung squamous cell carcinoma (SCC) sample, The location of CMRF derived for the cg22694818 marker in squamous cell carcinoma (SCC) samples is shown.

[0030] Figure 10 is a diagram illustrating the scope of application and performance of a detection method according to one embodiment of the present invention.

[0031] FIG. 11 shows the detection results according to the position of the probe in the detection of the colon cancer marker performed according to the detection method according to one embodiment of the present invention. 11a is a comparison of the positions of the primers and probes of the CRC02 marker (upper panel) and the degree of complete digestion according to the enzyme combination in a normal sample based on Ct=45 (lower panel), 11b is a comparison of the positions of the primers and probes of the CRC03 marker (upper panel) and the degree of complete digestion according to the enzyme combination in a normal sample based on Ct=45 (lower panel), and 11c is a comparison of the positions of the primers and probes of the CRC04 marker (upper panel) and the degree of complete digestion according to the enzyme combination in a normal sample based on Ct=45 (lower panel).

[0032]

[0033] Detailed description of the invention and preferred embodiments

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.

[0035] In the present invention, in order to detect a methylation marker of a nucleic acid, it was confirmed that when an adapter is bound to a nucleic acid, and then a methylation restriction enzyme is treated, and then adapter PCR and marker-specific PCR are performed, a methylation marker can be detected with high sensitivity and accuracy.

[0036] That is, in one embodiment of the present invention, when an adapter is bound to a nucleic acid, then treated with a methylation restriction enzyme, adapter PCR is performed, and then the methylation status is detected using a primer designed to specifically bind to the target region, it was confirmed that the methylation status of the target region can be detected with high sensitivity and accuracy, and this was named the EMFLiMax platform (Enrichment of Methylated cfDNA Fragment by Ligation & MultiMSRE-mediated amplification reaction) (Fig. 1).

[0037] Therefore, the present invention, from one point of view,

[0038] A method for detecting the methylation status of a nucleic acid comprising the following steps:

[0039] (a) a step of obtaining nucleic acid from a biological sample;

[0040] (b) a step of binding an adapter to the obtained nucleic acid;

[0041] (c) a step of treating the nucleic acid to which the adapter is bound with one or more methylation restriction enzymes (MRE);

[0042] (d) a step of amplifying the nucleic acid treated with the above restriction enzyme using a primer specific to the adapter;

[0043] (e) a step of performing a polymerase chain reaction (PCR) reaction using a primer capable of amplifying the target region on the amplification product obtained in step (d) to amplify the target region; and

[0044] (f) a step of determining whether the amplification product obtained in step (e) exists, and if the amplification product exists, determining that the target region is methylated;

[0045]

[0046] In the present invention, the nucleic acid may be used without limitation as long as it is DNA extracted from a biological sample, but is preferably a fragment of cell-free nucleic acid or intracellular nucleic acid, but is not limited thereto.

[0047] In the present invention, the biological sample means any material, biological fluid, tissue or cell obtained from or derived from an individual, for example, whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, blood (including plasma and serum), sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, pelvic fluids, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple It may include, but is not limited to, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, organ secretions, cells, cell extracts, semen, hair, saliva, urine, buccal cells, placental cells, cerebrospinal fluid, and mixtures thereof.

[0048]

[0049] In the present invention, the adapter may be characterized in that it does not include a methylation restriction enzyme recognition sequence.

[0050]

[0051] In the present invention, the MRE can be used without limitation as long as it is a restriction enzyme capable of decomposing methylated nucleic acids, and preferably, it can be a methylation sensitive restriction enzyme (MSRE) or a methylation dependent restriction enzyme (MDRE), and more preferably, it can be characterized as being MSRE.

[0052] In the present invention, the MDRE may be characterized by at least one selected from the group consisting of AbaSI, AoxI, BisI, BlsI, Dpnl, FspEI, Glal, Glul, Krol, LpnPI, Mall, MspJI, Mtel, Pcsl, PkrI, and Sgel, but is not limited thereto.

[0053] In the present invention, the MSRE can be used without limitation as long as it is a restriction enzyme that cuts if the site recognized by the restriction enzyme is not methylated and does not cut if it is methylated, and preferably Aatll, Acc65I, AccI, Acil, Acll, Afel, Agel, Agel-HF®, Ahdl, Alel-v2, Apal, ApaLl ApeKl, Asci, AsiSl, Aval, Avail, Bael, Banl, BbvCI, BceAI,, Bcgl, BcoDI, BfuAI, Bgll, BmgBI, BsaAI, BsaBI, BsaHI, BsaI-HF®v2, BseYI, BsiE, BsiWI, BsiWI-HF®, BslI, BsmAI, BsmBI-v2, BsmFI, BspDI, BspEI, BsrBI, BsrFI-v2, BssHII, BstAPI, BstBI, BstUI, BstZ17I-HF®, BtgZI, Cac8I, Cfr10I, Clal, Dpnl, Dralll-HF®, DrdI, Eael, Eagl-HF®, Earl, Ecil, Eco53kl, ​​EcoRI, EcoRI-HF®,EcoRV, EcoRV-HF®, Esp3I, Faul, Fnu4HI, FokI, Fsel, FspI, Haell, Hgal, Hhal, HinPlI, Hindi, Hinfl, Hpal, Hpall, Hpyl66II, Hpyl88III, Hpy99I, HpyAV, HpyCH4IV, KasI, Mbol, Mini, MluI-HF®, Mmel, MspAlI, Mwol, Nad, Narl, Neil, NgoMIV, Nhd-HF®, NlalV, Notl, Notl-HF®, Nrul, NruI-HF®, Nt.BbvCI, Nt.BsmAI, Nt.CviPII, PaeR7I, PaqCI, Pld, PluTI, PmaCI.It may be at least one selected from the group consisting of Pmd, Pmll, PshAI, PspOMI, PspXI, Pvul, PvuI-HF®, Rsal, RsrII, Sad-HF®, SacII, Sall, Sall-HF®, Sau3AI, Sau96I, ScrFI, SfaNI, Sfil, Sfol, SgrAI, Smal, SnaBI, Srfl, StyD4I, Tfil, Tsd, TspMI, Xhol, Xmal and Zral, and more preferably AciI, BstUI, HpaII, HhaI, HpyC, AatII, BspEI, AfeI, BstBI, BssHII, Cfr10I, ClaI, Eco52I, HaeII, MluI, NaeI, NruI, NsbI, PmaCI, Psp1406I, PvuI, SacII, SalI, It may be characterized by at least one selected from the group consisting of SmaI, SnaB I, RsrII and NotI, and most preferably at least two selected from the group consisting of AciI, HaeII, HhaI and HpaII, but is not limited thereto.

[0054]

[0055] In the present invention, when there are two or more restriction enzymes in step (c), it may be characterized in that they are processed at the same or different ratios.

[0056] In the present invention, the ratio may be, for example, 1:5 to 5:1 in volume ratio when there are two restriction enzymes, and when there are three restriction enzymes, the ratio may be 1:1:1 to 5, 1:1 to 5:1, 1 to 5:1:1, 1 to 5:1 to 5:1, 1:1 to 5:1 to 5, 1 to 5:1:1 to 5, etc., but is not limited thereto.

[0057] In the present invention, when there are two or more restriction enzymes, the restriction enzymes may be characterized by being processed simultaneously or sequentially.

[0058]

[0059] In the present invention, the target region can be used without limitation as long as it is a region that can distinguish between a non-diseased sample and a diseased sample, and preferably, it can be characterized by a difference of 25% or more in DMR (Differentially methylated region) between a non-diseased sample and a diseased sample, or a statistical significance (p-value) of 0.05 or less, but is not limited thereto.

[0060] In the present invention, the term “non-diseased sample” means all samples without a disease to be analyzed, including both normal samples and control samples, a normal sample means a sample obtained from a subject who is completely healthy and has no disease other than the disease to be analyzed, and a control sample means a sample obtained from a subject who has a pathological condition other than normal but has not been confirmed to have a disease to be analyzed.

[0061] For example, in the case of colon cancer samples, a normal sample is a sample obtained from a subject without polyps or cancerous lesions, a control sample is a sample obtained from a subject with a benign lesion such as a hyperplastic polyp, and a diseased sample is a sample obtained from a subject confirmed to have colon cancer or a high-risk adenoma.

[0062] For lung cancer samples, a normal sample is a sample obtained from a completely normal subject without lung nodules, a control sample is a sample obtained from a subject with lung nodules but confirmed as benign, and a diseased sample is a sample obtained from a subject confirmed as having lung cancer.

[0063] For breast cancer samples, a normal sample means a sample obtained from a subject without breast lesions, a control sample means a sample obtained from a subject with fibrocystic changes or a benign mass, and a diseased sample means a sample obtained from a subject confirmed to have breast cancer (invasive or non-invasive).

[0064]

[0065] In the present invention, if the disease is colon cancer, the target region can be used without limitation as long as it is a region that shows a methylation status that can distinguish colon cancer samples from non-disease samples, and preferably, it can be at least one of the SEP9 and SDC2 genes and the markers described in Korean Patent No. 10-2024-0059797, but is not limited thereto.

[0066] In the present invention, if the disease is lung cancer, the target region can be used without limitation as long as it is a region that shows a methylation status that can distinguish between lung cancer samples and non-disease samples, and preferably, it can be any one or more of the markers described in Korean Patent No. 10-2024-0059797, but is not limited thereto. In the present invention, if the disease is liver cancer, the target region can be used without limitation as long as it is a region that shows a methylation status that can distinguish between liver cancer samples and normal samples, and preferably, it can be any one or more of the markers described in Korean Patent No. 10-2023-0148519, but is not limited thereto.

[0067] In the present invention, the target region may be used without limitation as long as it is a region that shows a methylation status that can distinguish between the cancer sample and the non-disease sample when the disease is esophageal cancer or stomach cancer, and preferably, it may be any one or more of the markers described in Korean Patent No. 10-2023-0169650, but is not limited thereto.

[0068] In the present invention, if the disease is breast cancer, the target region can be used without limitation as long as it is a region that shows a methylation status that can distinguish between a breast cancer sample and a non-disease sample, and preferably, it can be any one or more of the markers described in BRCA1, DAPK1, MSH2, CDKN2A, PGR, PRKCDBP, RANKL, OTP, LINE-1, BRCA1, BRCA2, U.S. Patent No. 11,542,559, U.S. Patent No. 11,352,672, U.S. Patent No. 11,242,568, and U.S. Patent No. 10,975,443, but is not limited thereto.

[0069] In the present invention, if the disease is prostate cancer, the target region can be used without limitation as long as it is a region that shows a methylation status that can distinguish between a prostate cancer sample and a non-disease sample, and preferably, it can be any one or more of the markers described in APC, CRIP3, HOXD3, TGFB2, GSTP1, SFRP2, IGFBP3, IGFBP7, PTGS2, RARB, RASSF1, SERPINB9, FLOT1, GAS6, ITPRIPL1, U.S. Patent No. 12071672, U.S. Patent No. 11920200, U.S. Patent No. 11427874, and U.S. Patent No. 9540697, but is not limited thereto.

[0070] In the present invention, if the disease is a neurodegenerative disease, the target region can be used without limitation as long as it is a region that shows a methylation status that can distinguish between a neurodegenerative disease and a non-disease sample, and preferably, it can be at least one selected from the group consisting of APP, PSEN1, SNCA, CDKL4, C2orf78, SNORD115-44, CYSLTR2, SNORA67, KRTAP5-2, LCE1F, LOC642826, SNAR-A14, SNAR-A3, SNAR-A6, SNAR-A4, SNAR-A9, SNAR-A10, SNAR-A7, SNAR-A11, SNAR-A5, SNAR-A8, NBPF14, MIR572 and IFNK, but is not limited thereto.

[0071] In the present invention, if the disease is an autoimmune disease, the target region can be used without limitation as long as it is a region that shows a methylation status that can distinguish between autoimmune disease and non-disease samples, and preferably, it can be at least one of the markers described in INFG, LTA, CYP2E1, DUSP22, IFI44L, IFNK, PCDHB14, U.S. Patent No. 1,059,0475, and U.S. Patent No. 1,087,6163, but is not limited thereto.

[0072] In the present invention, if the disease is a metabolic disease, the target region can be used without limitation as long as it is a region that shows a methylation status that can distinguish between a metabolic disease and a non-disease sample, and preferably, it can be at least one of the markers described in PPARGC1A GLUT4, PPARγ2, POMC, PGC-1α, PHOSPHO1, CDKN2B, P4HA3, CDKN1C, EPHA1, CASP10, HLA, NID1, RXRA, ABCG1, TACSTD2, NFATC2IP, CPT1A, ADRB3, LPL, TXNIP, CP3, PDK4, SNX20, LZTS3, PEX26, CMIP, CYP2E1, AKR1E2, U.S. Patent No. 10,508,308, and U.S. Patent No. 10,221,457, but is not limited thereto.

[0073] In the present invention, if the disease is a cardiovascular disease, the target region can be used without limitation as long as it is a region that shows a methylation status that can distinguish between a cardiovascular disease sample and a non-disease sample, and preferably, it can be any one or more of NOS3, LINE-1, TEAD1, ZNF438, F2RL3, PTPRN2, TRIM59, EDARADD, TBX5, ZBTB16, HOXC4, and markers described in U.S. Patent No. 8,241,855, but is not limited thereto.

[0074] In the present invention, if the disease is a rare disease, the target region can be used without limitation as long as it is a region that shows a methylation status that can distinguish between a rare disease sample and a non-disease sample, and preferably, it can be at least one selected from the group consisting of GSDMD, ECEL1P2, PRDM16, ABCD1P4, TNNT3, COL18A1, and ANK1, but is not limited thereto.

[0075] In the present invention, if the disease is depression, the target region can be used without limitation as long as it is a region that shows a methylation status that can distinguish between depressed and non-diseased samples, and preferably, it can be any one or more selected from the group consisting of HOTAIRM1, NLGN2, ACSF3, HOXA1, KLHDC7B, BDNF, SLC6A4, HTR1B, TPH2, FKBP5, NR3C1, NR3C2, CRH, CRHR1, CRHR2, NR3C1 and MORC1, but is not limited thereto.

[0076] In the present invention, if the disease is PTSD, the target region can be used without limitation as long as it is a region that shows a methylation status that can distinguish between PTSD and non-disease samples, and preferably, it can be at least one selected from the group consisting of TPR, CLEC9A, APC5, ANXA2, TLR8, H19, IL18, ZDHHC11, CSMD2, COL9A3, PDCD6IP, TBC1D24, FAM164A, and AIM2, but is not limited thereto.

[0077]

[0078] In the present invention, the position where the primer binds may be selected by a method including the following steps:

[0079] (i) a step of setting a 100 to 500 bp region in the 5' direction and 3' direction of the target region as a target CpG region;

[0080] (ii) a step of determining the location of a region (CMR, Consecutive MSRE CpG Region) starting from the first MSRE recognition site based on the 5' terminal region in the target CpG region to one or more consecutive MSRE recognition sites including the MSRE recognition site;

[0081] (iii) a step of calculating the detectable fragment fraction (DFF) for each CMR and selecting CMRs with different DFF values ​​between normal and diseased samples as continuous MSRE-resistant fragments (CMRF); and

[0082] (iv) A step of selecting the region with the largest difference in DFF in the selected CMRF as the primer binding position.

[0083]

[0084] In the present invention, the target CpG region can be set by extending 100 to 500 bp in the 5' direction and 100 to 500 bp in the 3' direction of the selected target region, more preferably by extending 100 to 300 bp in the 5' direction and 100 to 300 bp in the 3' direction, and most preferably by extending 150 bp in the 5' direction and 150 bp in the 3' direction to set a 300 bp target CpG region.

[0085] In the present invention, the location of the CMR may vary depending on the type and combination of restriction enzymes. As described in Fig. 4, when the location of the CMR is determined as a region up to five consecutive restriction enzyme recognition sites by setting the restriction enzymes to AciI, HpaII, and HhaI in a 300 bp target CpG region, the region including five consecutive restriction enzyme recognition sites can be determined as the CMR regardless of the type of restriction enzyme.

[0086] That is, the CMR indicated as (1) Mean diff = 0.042 in Fig. 4 includes five sites: an AciI recognition site located at the 18th base sequence from the 5' end, an AciI recognition site located at the 20th base sequence, a HapII recognition site located at the 72nd base sequence, an HhaI recognition site located at the 123rd base sequence, and an AciI recognition site located at the 124th base sequence.

[0087]

[0088] As described in FIG. 8b of the present invention, the CMR of the present invention can be set in various ways depending on the number of consecutive restriction enzyme recognition sites. For example, if the CMR indicated as (1) Mean diff = 0.042 in FIG. 4 is replaced with 1CMR having 1 consecutive recognition site, it can be converted into 5 CMRs, if replaced with 2CMR having 2 consecutive recognition sites, it is converted into 2 CMRs, and if replaced with 3CMR and 4CMR having 3 consecutive recognition sites and 4 consecutive recognition sites, it is replaced with 1 CMR each, and the remaining recognition sites are configured as part of other 3CMR and 4CMR.

[0089] That is, even if it is the same target CpG region, the position and number of CMRs may vary depending on the type of restriction enzyme and the number of consecutive restriction enzyme recognition sites. As described in Fig. 8b, when there are 10 restriction enzyme recognition sites from the 5' end, the position and number of CMRs may be determined differently depending on the number of consecutive restriction enzyme recognition sites set from 1 to 5, and the recognition sites may also vary depending on the type and number of restriction enzymes, and thus may be determined differently.

[0090]

[0091] In the present invention, the DFF may be characterized by being calculated by a method including the following steps:

[0092] (1) A step of counting the total number of reads aligned to each CMR in a database capable of confirming the methylation level;

[0093] (2) a step of counting the number of reads (DF, Detectable Fragment) in which the MSRE recognition site of each CMR is all methylated; and

[0094] (3) A step of calculating DFF by dividing the number of DFs by the total number of leads aligned to CMR.

[0095]

[0096] In the present invention, the CMRF can select all areas where the calculated DFF value of the diseased sample is different from the DFF value of the non-diseased sample, and preferably, it can be the mean difference or AUC value of the DFF values ​​of the diseased sample and the non-diseased sample, but is not limited thereto.

[0097] In the present invention, the mean difference means calculating the difference after calculating the average value of DFF in each of the diseased sample and the non-diseased sample.

[0098] In the present invention, the CMRF can select a region in which the proportion of values ​​calculated from non-diseased samples that are 0 is greater than or equal to a reference value, and the AUC value that can distinguish between non-diseased samples and diseased samples is greater than or equal to a reference value as the CMRF.

[0099] In the present invention, the reference value of the ratio of the calculated value of 0 in the non-diseased sample may be preferably 10 to 100%, more preferably 30 to 90%, and most preferably 60 to 80%, but is not limited thereto.

[0100] In the present invention, the reference value of the AUC can be used without limitation as long as it is a value that can distinguish between a non-diseased sample and a diseased sample, and is preferably 0.1 to 1.0, more preferably 0.5 to 0.9, and most preferably 0.6 to 0.8, but is not limited thereto.

[0101] In the present invention, the two methods for selecting the CMRF are substantially the same methods for distinguishing the difference in methylation status between a non-diseased sample and a diseased sample based on the DFF value, and only differ in the type of database that can confirm the methylation status.

[0102]

[0103] In the present invention, the database capable of confirming the methylation level may be characterized by being created using any one method selected from the group consisting of pyrosequencing, bisulfite sequencing, and methylation next-generation base sequence sequencing.

[0104] In the present invention, the database can be used without limitation as long as it is a database capable of confirming continuous methylation levels, and preferably, it can be a self-produced NGS-based database or an NGS-based database registered in NCBI GEO (https: / www.ncbi.nlm.nih.gov / geo / ), but is not limited thereto.

[0105] In the present invention, the methylation next generation base sequence sequencing (Methylation NGS) may be characterized by at least one selected from the group consisting of BS-Seq (bisulfite sequencing), MeDIP-Seq (methylated DNA immunoprecipitation sequencing), RRBS-Seq (reduced representation bisulfite sequencing), WGBS (whole genome bisulfite sequencing), MethylCap-Seq (methylation capture sequencing), MBD-Seq (methyl-CpG binding domain sequencing), oxBS-Seq (oxidative bisulfite sequencing), TAB-Seq (TET-associated bisulfite sequencing), BSAS (bisulfite amplicon sequencing), WGEM-seq (whole genome enzymatic methylation sequencing), and targeted EM-seq (target enriched enzymatic methyl sequencing).

[0106]

[0107] In the present invention, the next-generation sequencer can be used with any sequencing method known in the art. Sequencing of nucleic acids isolated by the selection method is typically performed using next-generation sequencing (NGS). Next-generation sequencing includes any sequencing method that determines the nucleotide sequence of an individual nucleic acid molecule or a clonally expanded proxy for an individual nucleic acid molecule in a highly similar manner (e.g., more than 105 molecules are sequenced simultaneously). In one embodiment, the relative abundance of a nucleic acid species in a library can be estimated by counting the relative occurrence of its cognate sequence in data generated by a sequencing experiment. Next-generation sequencing methods are well known in the art and are described, for example, in Metzker, M. (2010) Nature Biotechnology Reviews 11:31-46, which is incorporated herein by reference.

[0108] In one embodiment, next-generation sequencing is used to determine the nucleotide sequence of individual nucleic acid molecules (e.g., the HeliScope Gene Sequencing system from Helicos BioSciences and the PacBio RS system from Pacific BioSciences). In another embodiment, a method of determining the nucleotide sequence of a clonally expanded proxy for an individual nucleic acid molecule is used, for example, by a massively parallel short-read sequencing method (e.g., the Solexa sequencer from Illumina Inc., San Diego, Calif.), which produces more bases of sequence per sequencing unit than other sequencing methods that produce fewer but longer reads (e.g., the Solexa sequencer from Illumina Inc., San Diego, Calif.; 454 Life Sciences, Branford, Conn., and Ion Torrent). Other methods or machines for next-generation sequencing include, but are not limited to, 454 Life Sciences (Branford, CT), Applied Biosystems (Foster City, CA; SOLiD sequencer), Helicos Biosciences Corporation (Cambridge, MA), and emulsion and microfluidic sequencing techniques nanodroplets (e.g., GnuBio drops).

[0109] Platforms for next-generation sequencing include, but are not limited to, the Roche / 454 Genome Sequencer (GS) FLX system, the Illumina / Solexa Genome Analyzer (GA), the Life / APG Support Oligonucleotide Ligation Detection (SOLiD) system, the Polonator G.007 system, Helicos BioSciences' HeliScope Gene Sequencing system, the Oxford Nanopore Technologies PromethION, GriION, and MinION systems, and the Pacific Biosciences PacBio RS system.

[0110]

[0111] In the present invention, the step of determining the presence or absence of the amplification product in step (f) may be characterized by using a nucleic acid-binding dye or probe capable of binding to the amplification product.

[0112] In the present invention, the nucleic acid binding dye may be characterized in that it is selected from the group consisting of ethidium bromide, SYBR® Green I, SYBR® Gold, EvaGreen, YO-PRO-1, SYTO, BEBO, and BEXTO.

[0113] In the present invention, the probe capable of binding to the amplification product may be characterized in that it is selected from a group consisting of one or more combinations of oligonucleotides, LNAs, and PNAs.

[0114] In the present invention, the probe capable of binding to the amplification product may be characterized by including one or more MSRE recognition sequences.

[0115] In the present invention, a method characterized in that a probe capable of binding to the amplification product has a reporter and a quencher connected to both ends.

[0116] In the present invention, the reporter may be characterized by being at least one fluorescent substance selected from the group consisting of fluorescein, fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethylrhodamine, FITC, Oregon green, Alexa Fluor, FAM, JOE, ROX, HEX, Texas Red, TET, TRITC, TAMRA, cyanine series dyes, and thiadicarbocyanine dyes.

[0117] In the present invention, the quencher may be characterized by being at least one selected from the group consisting of Dabcyl, TAMRA, Eclipse, DDQ, QSY, Blackberry Quencher, Black Hole Quencher, Qxl, Iowa black FQ, Iowa black RQ, and IRDye QC-1.

[0118] In a preferred embodiment of the present invention, the detection of the amplification product through the nucleic acid polymerase is performed through real-time polymerase chain reaction (real-time PCR), and at this time, an amplification curve according to the increase of the amplification product is obtained and the Ct (threshold cycle) value is measured, but is not limited thereto. In the case of the method using the Ct value, the principle is used that the earlier the amplification product is generated and increases due to the presence of a target region in the sample, the earlier the amount of signal generated by the detection probe increases, and the number of cycles reaching the threshold is reduced, and the Ct value is measured less.

[0119]

[0120] The term "amplification" in the present invention refers to a reaction for amplifying a nucleic acid molecule. Various amplification reactions have been reported in the art, including polymerase chain reaction (hereinafter referred to as PCR) (U.S. Patent Nos. 4,683,195, 4,683,202, and 4,800,159), reverse transcription-polymerase chain reaction (hereinafter referred to as RT-PCR) (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed.Cold Spring Harbor Press (2001)), WO 89 / 06700 and EP 329,822 methods, ligase chain reaction (LCR, WO 90 / 01069), repair chain reaction (EP 439,182), transcription-mediated amplification (MA, WO 88 / 10315), self-sustained sequence replication (WO 90 / 06995), selective amplification of target polynucleotide sequences (US Pat. No. 6,410,276), consensus sequence primed polymerase chain reaction (CP-PCR, US Pat. No. 4,437,975), arbitrarily primed polymerase chain reaction (AP-PCR, Nos. 5,413,909 and 5,861,245), nucleic acid sequence based amplification (NASBA, U.S. Pat. Nos. 5,130,238, 5,409,818, 5,554,517, and 6,063,603), strand displacement amplification, and loop-mediated isothermal amplification (LAMP).

[0121] Other available amplification methods are described in U.S. Patent Nos. 5,242,794, 5,494,810, 4,988,617, and U.S. Patent No. 09 / 854,317.

[0122] PCR is the most well-known nucleic acid amplification method, and many variations and applications have been developed. For example, touchdown PCR, hot start PCR, nested PCR, and booster PCR have been developed by modifying the traditional PCR procedure to enhance the specificity or sensitivity of PCR. Furthermore, real-time PCR, differential display PCR (D-PCR), rapid amplification of cDNA ends (RACE), DL-PCR (PC), inverse polymerase chain reaction (IPCR), vectorette PCR, and thermal asymmetric interlaced PCR (TAIL-PCR) have been developed for specific applications. For more information on PCR, see McPherson, MJ, and Moller, SG PCR. BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, NY (2000), the teachings of which are incorporated herein by reference.

[0123] In the present invention, PCR can be used without limitation as long as it is a known PCR method other than the method described above, and is preferably characterized by being a real-time PCR, but is not limited thereto.

[0124]

[0125] In the present invention, the step (e) may be characterized by being performed by additionally including a primer (DC, Digestion Control) capable of amplifying a region that is completely digested by a methylation restriction enzyme (complete digestion region); and a primer (IC, Internal Control) capable of amplifying a region that is not cut by a methylation restriction enzyme.

[0126] In the present invention, the region completely digested by the methylation restriction enzyme can be set according to the type of restriction enzyme, and the IC may be a promoter region of a housekeeping gene, but is not limited thereto.

[0127]

[0128] From another perspective, the present invention relates to a method for providing information for diagnosing a disease, including a step of determining that a disease exists when a target region is methylated by the above method.

[0129] In another aspect, the present invention relates to a disease diagnosis method including a step of determining that a disease is present when a target region is methylated by the above method.

[0130] In another aspect, the present invention relates to the use of primers capable of amplifying the target region for disease diagnosis.

[0131] In another aspect, the present invention relates to the use of primers capable of amplifying the target region for the manufacture of a kit for diagnosing a disease.

[0132] In another aspect, the present invention relates to a composition for diagnosing a disease, comprising a primer capable of amplifying the target region.

[0133] In another aspect, the present invention relates to a disease diagnostic kit comprising the composition.

[0134] In the present invention, the kit may optionally include reagents necessary for performing a nucleic acid amplification reaction (e.g., polymerase chain reaction), such as a buffer, DNA polymerase, a DNA polymerase cofactor, and deoxyribonucleotide-5-triphosphate (dNTP). Optionally, the kit of the present invention may also include various oligonucleotide molecules, reverse transcriptase, various buffers and reagents, and antibodies that inhibit DNA polymerase activity. Furthermore, the optimal amount of reagents used in a specific reaction of the kit can be readily determined by one skilled in the art having learned the teachings of the present specification. Typically, the device of the present invention may be manufactured as a separate package or compartment containing the aforementioned components.

[0135] In one embodiment, the kit may comprise a compartmented carrier means for containing a sample, a container containing a reagent, and a container containing a primer or probe.

[0136] The carrier means is suitable for containing one or more containers, such as bottles or tubes, each containing independent components used in the method of the present invention. Given the present disclosure, one skilled in the art can readily dispense the required formulations within the containers.

[0137] Additionally, the kit may include a user guide. A user guide is a printed document that explains how to use the kit, such as how to prepare a buffer solution, the reaction conditions provided, etc. User guides may include pamphlets or leaflets, labels attached to the kit, and descriptions on the packaging containing the kit. User guides also include information disclosed or provided through electronic media, such as the Internet.

[0138]

[0139] In the present invention, the disease can be used without limitation as long as it is a disease that can be distinguished from a non-disease state by the nucleic acid methylation status, and is preferably characterized by being selected from the group consisting of cancer, neurodegenerative disease, autoimmune disease, metabolic disease, cardiovascular disease, rare disease, and other diseases, but is not limited thereto.

[0140] In the present invention, the cancer is ovarian cancer, soft tissue sarcoma, peripheral T-cell cancer, colon cancer, intrahepatic cholangiocarcinoma, glioblastoma, esophageal cancer, cutaneous T-cell lymphoma, non-Hodgkin's lymphoma, urinary tract cancer, basal cell carcinoma, epithelioid sarcoma, pancreatic cancer, non-small cell lung cancer, Hodgkin's lymphoma, renal cell cancer, mesothelioma, metastatic uveal melanoma, kidney cancer, blood cancer, HER2-expressing cancer, non-melanoma skin cancer, liposarcoma, hepatocellular carcinoma, small lymphocytic lymphoma, prostate cancer, breast cancer, anal cancer, marginal zone lymphoma, cutaneous squamous cell carcinoma, thyroid cancer, medullary thyroid cancer, triple-negative breast cancer, neuroendocrine prostate cancer, bladder cancer, paraganglioma, medulloblastoma, superficial basal cell carcinoma, head and neck squamous cell carcinoma, blood cancer, melanoma, B-cell lymphoma, relapsed / refractory acute myeloid leukemia, hemangiosarcoma, osteosarcoma, refractory Cervical cancer, cholangiocarcinoma, gastroesophageal adenocarcinoma, rhabdomyosarcoma, carcinoma, non-muscle-invasive bladder cancer, uveal melanoma, small cell lung cancer, cervical cancer, primary open-angle glaucoma, follicular lymphoma, synovial sarcoma, liver cancer, carcinosarcoma, leptomeningeal brain tumor, T-cell lymphoma, lymphoma, small cell lung cancer, mantle cell lymphoma, B-cell malignancies, endometrial cancer, mucinous / round cell liposarcoma, metastatic Merkel cell carcinoma, neuroblastoma, chronic lymphocytic leukemia, tendon sheath giant cell tumor, sarcoma, acute myeloid leukemia, skin cancer, nasopharyngeal cancer, relapsed / refractory Ewing sarcoma, bone cancer, glioma, salivary gland carcinoma, stomach cancer, benign tumor, low-grade serous ovarian cancer, metastatic breast cancer, multiple myeloma, diffuse large B-cell lymphoma, relapsed / refractory lymphoma, metastatic colorectal cancer, advanced malignancies and acute lymphoblastic leukemia.

[0141] In the present invention, the cancer may be characterized as being any one selected from the group consisting of colon cancer, lung cancer, stomach cancer, breast cancer, prostate cancer, and liver cancer.

[0142] In the present invention, the neurodegenerative disease may be characterized as being any one selected from the group consisting of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, frontotemporal dementia, Lewy dementia, corticobasal degeneration, multiple systemic encephalopathy, progressive supranuclear palsy, Huntington's disease, and Charcot-Marie-Tooth disease.

[0143] In the present invention, the autoimmune disease may be characterized as being any one selected from the group consisting of Kawasaki disease, ankylosing spondylitis, psoriasis, psoriatic arthritis, erythema nodosum, Goodpasture syndrome, ulcerative colitis, Graves' disease, Guillain-Barré syndrome, rheumatoid arthritis, and systemic lupus erythematosus.

[0144] In the present invention, the metabolic disease may be characterized as being any one selected from the group consisting of type 2 diabetes, hyperlipidemia, obesity, hypothyroidism, and chronic kidney disease.

[0145] In the present invention, the cardiovascular disease may be characterized as being any one selected from the group consisting of hypertension, ischemic heart disease, heart failure, stroke, coronary artery disease, arrhythmia, and arteriosclerosis.

[0146] In the present invention, the rare disease may be characterized as being any one selected from the group consisting of Prader-Willi syndrome, Angelman syndrome, developmental and epileptic encephalopathies, and hemispheric hypertrophy.

[0147] In the present invention, the other disease may be characterized as depression or post-traumatic stress disorder (PTSD).

[0148]

[0149] Example

[0150] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0151]

[0152] Example 1. Materials and Methods

[0153] 1.1 Nucleic acid extraction and purification

[0154] The standard sample was purchased as fully methylated and completely unmethylated genomic DNA (gDNA) of the human colon cancer cell line HCT116 (human Methylated & Non-methylated (WGA) DNA set, D5013, ZYMO RESEARCH, USA). Then, to mimic the fragmentation state of cell-free DNA, the genomic DNA was fragmented into 200 bp pieces using an ultrasonicator (Q800R3, QSONICA, USA). Unmethylated gDNA (Unme) was prepared by mixing 99% non-methylated gDNA with 1% methylated gDNA.

[0155] cfDNA was isolated from 1 ml of serum from normal individuals, colon cancer patients, and lung cancer patients using the Mag-Bind cfDNA kit (Omega BIO-TEK, USA) applied to the KingFisher Plex (Thermofisher, USA) automated equipment. The extraction process was performed according to the general principle of pretreating the sample with Proteinase K included in the extraction kit, attaching nucleic acids to the magnetic beads included in the extraction kit, removing impurities through repeated washing processes, and then obtaining nucleic acids in the elution buffer.

[0156] The cell-free DNA obtained by the above method was subjected to quality verification and concentration quantification using the Cell-free DNA ScreenTape (Agilent, USA) of the 4200TapeStation equipment. The quality of the extracted cell-free DNA was 190 bp on average, and the total concentration range was 2-15 ng, with a large variation among samples. The proportion of cell-free DNA among the total nucleic acids was approximately 80%.

[0157]

[0158] 1.2 Adapter ligation and library creation

[0159] Adapter ligation of cell-free DNA was performed using the xGEN cfDNA / FFPE kit (IDT, USA) according to the user manual. That is, the nucleic acid concentration was quantified to 4 ng / 50 ul, the cell-free DNA ends extracted with the “End Repair module” were evenly aligned, the 3’ adapter was first ligated with the “Ligation 1 module”, and then the 5’ adapter was ligated with the “Ligation 2 module”. After washing with 2.5x magnetic beads (AMPure XP, BECKMAN COULTER, USA), the final eluted with 21 ul Low TE buffer to prepare an adapter-ligated library. The kit applies adapters applicable to Illumina’s Tru-seq sequencing, so in this example, TruSeq TM - A compatible full-length adapter was custom-made and used (IDT, USA). The above adapter base sequence was confirmed to not contain recognition sites for the AciI, HhaI, and HpaII methylation restriction enzymes used in this technology, confirming that it does not affect the subsequent MSRE processing and methyl amplification steps.

[0160]

[0161] 1.3 MSRE processing and purification

[0162] MSRE used the following three products and buffer solutions: Acil (R0551S, NEB), HhaI (R0139S, NEB), HpaII (R0171S, NEB), and rCutSmart buffer (B6004S, NEB).

[0163] In colon cancer / lung cancer samples and normal samples, the MSRE reaction was performed in a total volume of 60 ul, and 20 ul of the library with the adapter attached was added with 6 ul of 1x rCutSmart buffer, 1.5 ul, 0.8 ul, and 0.8 ul of Acil, HhaI, and HpaII, respectively, and incubated at 37˚C for 3 hours, washed with 1.8x Magnetic beads (AMPure XP, BECKMAN COULTER), and finally eluted with 21 ul of Low TE buffer to obtain the restriction enzyme-treated product.

[0164] In lung cancer samples and normal samples, a total volume of 60 ul was used, and 6 ul of 1x rCutSmart buffer, 1.5 ul of Acil, and 0.8 ul of HhaI were added to 20 ul of the library with the adapter attached, and the same process was performed to obtain restriction enzyme-treated products.

[0165]

[0166] 1.4 Adapter PCR

[0167] PCR using primers that specifically bind to the adapters used the 2x HiFi PCR Premix included in the xGEN cfDNA / FFPE kit, and the adapter primers were compatible with the TruSeq library system of the xGEN cfDNA / FFPE kit. TM -Compatible full-length adapters were custom-made by IDT (IDT, USA).

[0168]

[0169] A total of 50 ul reaction was prepared containing 20 ul of MSRE restriction enzyme treatment product, 1x HiFi PCR Premix, and 1x adapter primer, and then the reaction was performed in a SimpliAmp Thermal Cycler (ABI, USA) at 98˚C for 45 sec, followed by 9 cycles of 98˚C, 15 sec > 60˚C, 30 sec > 72˚C, 30 sec. The amplified product was washed with 1.3x Magnetic beads (AMPure XP, BECKMAN COULTER), and finally eluted with 51 ul Low TE buffer to obtain the methylation-specific amplified product.

[0170] At this stage, the adapter ligation and adapter PCR reaction process were evaluated based on the size and concentration (sample intensity, FU) of the polymerase chain reaction product of 1 ul of the above methylation-specific amplification product on the D1000 ScreenTape (Agilent, USA) of TapeStation. That is, a quality assessment was performed to determine whether a product of an average of 330 bp including the adapter length in the average cell-free DNA size was generated and whether the sample intensity was at least 1,000, and then the next process was performed.

[0171]

[0172] 1.5 Selection of primer positions for colon cancer markers

[0173] 1.5.1 Selection of CpG markers and target regions

[0174] (1) The SEPT9 and SDC2 genes were selected as colorectal cancer standard markers, and the target region for selecting each primer position was set to the region described in Wasserkort, R. et al., BMC Cancer 13, 398 (2013) for the SEPT9 gene, and to the PCR amplicon region described in Oh TJ. Et al., Clin Epigenettics, 9:126 (2017) for the SDC2 gene, and extended by 100 bp in both 5' and 3' directions to set the target region to approximately 300 bp.

[0175] (2) The colorectal cancer test markers were selected from the 50 hypermethylated markers of the first candidate group selected in Korean Patent No. 10-2024-0059797 as target CpGs. The target CpG region was expanded by 150 bp in both the 5' and 3' directions from the established target CpGs, thereby establishing a 300 bp region.

[0176]

[0177] 1.5.2 Selecting MSRE and Setting CMR Location

[0178] In this patent, the DNA region where each MSRE cut site is continuously connected based on the first MSRE cut site on the 5' side of the target CpG region is defined as a CMR (Consecutive MSRE CpG Fragment). In normal specimens, all MSRE cut sites in the CMR are unmethylated and thus cleaved, while in colon cancer, all are methylated and thus not cleaved to detect the methylation level by real-time PCR. Therefore, an appropriate number of MSREs must be present within the target marker, and therefore, MSRE selection is a very important factor in the Amplimax platform.

[0179] In the case of colorectal cancer, three methylation-sensitive restriction enzymes (MSREs), AciI (C^CGC), HhaI (GCG^C), and HpaII (C^CGG), were selected based on the probability of appearing in the genome and the frequency of appearing in the human genome. This combination of three MSREs has the highest CpG coverage rate of 32.4% of the entire human genome among the four-cutter methylation-sensitive restriction enzymes that can appear once per 256 bp (44 = 256) on average in the genome composed of A, T, G, and C bases (Epigenetics Method, 2022 (18) 181-212). A high CpG coverage rate means that it is likely to exist in various target CpG regions, so it can effectively work depending on the methylation level of the target marker to distinguish between normal and target cancer.

[0180] In the case of colon cancer, CMR was defined as a DNA region connecting five consecutive MSRE cut sites from the first MSRE cut site on the 5' side of the target CpG region among the three types of MSREs, Acil, HhaI, and HpaII (Figure 4).

[0181]

[0182] 1.5.3 Calculating DFF and Checking CMRF Area

[0183] (1) In the case of the colorectal cancer standard marker, the Whole genome Enzymatic Methylation sequencing (WGEM-seq) method described in Example 4 of Korean Patent No. 10-2024-0059797 was used to calculate the DFF of the CpG region, and the methylation status results for 92 normal samples, 6 colorectal cancer tissue samples, 26 colorectal cancer cfDNA samples, and 6 colorectal cancer adenoma cfDNA samples were obtained.

[0184] That is, for blood samples, after collection, only the plasma portion was centrifuged for the first time under the conditions of 3000 rpm, 25℃, 10 minutes, and then the plasma from the first centrifugation was centrifuged for the second time under the conditions of 16000g, 25℃, 10 minutes to separate the plasma, and then 400 ul of the buffy coat was separated and genomic DNA was extracted using the QIAmp DNA mini Kit (Qiagen, Germany). For tissue samples, 10-30 ng was disrupted using FastPrep-24 and then genomic DNA was extracted using the QIAmp DNA mini Kit (Qiagen, Germany). The concentration of extracted genomic DNA was measured using the Qubit DS DNA HS assay Kit (Thermo Fisher Scientific, USA), and the purity was measured using Nanodrop. 200 ng of DNA (50 ul) was sheared to 240-290 bp using Covaris, and then the DNA size was confirmed using D1000 screen tape & reagent (Agilent, USA) on a Tapestation 4200 (Agilent, USA).

[0185] After that, 200ng sheared DNA was converted to methylation by substituting unmethylated cytosine with uracil using ten-eleven translocation dioxygenase 2 (TET2) and APOBEC, and a library was created using enzymatic methyl-seq (NEB Kit). The concentration and size of the created DNA library were measured using Qubit DS DNA HS assay Kit (Thermo Fisher Scientific, USA) and TapeStation 4200 (Agilent, USA), respectively, and then sequencing was performed using Novaseq 6000 (Illumina) in 150 paired-end mode at a final concentration of 2nM, producing approximately 600 million reads per sample. EM-Seq data is converted through enzyme treatment, and DNA methylation can be confirmed through modification of cytosine bases.

[0186] After that, in order to increase the sequencing depth of each sample, the sample was pooled and the DFF was calculated. First, the total number of reads containing each 5CMR in the WGEM-seq data set (Total read) was calculated, and the reads in which all MSRE recognition sites existing in each 5CMR were methylated were counted as detectable fragments (DF, Detectable Fragment). Then, the detectable fragments were divided by the total number of reads to calculate the detectable fragment fraction (DFF, Detectable Fragment Fraction) for each 5CMR.

[0187] In particular, the DFF value is calculated only when at least one DF is detected in the target disease, colorectal cancer sample, and if there is a difference in DFF from the normal sample, the fragment is defined as a continuous MSRE-resistant CpG fragment (CMRF). One and three CMRFs (fragments 1, 2, and 15) were detected in the target CpG regions of SEPT9 and SDC2, respectively (Fig. 4). A larger DFF difference means a lower DFF value in the normal sample and a higher DFF value in the colorectal cancer sample, which means that it is a marker position with high potential to distinguish between normal and colorectal cancer. Therefore, among the three CMRFs detected in SDC2, position (15) with the largest DFF difference was selected as the first-priority CMRF region.

[0188] Because the range of total read values ​​varies greatly depending on the sequencing depth of the methylation sequencing dataset, absolute comparisons of DFF differences cannot be made by applying a fixed cutoff value. In this example, when multiple CMRFs were detected, the CMRF with the highest DFF difference was selected as the primer position.

[0189] For the CRC01, CRC02, and CRC03 markers, as described in Table 3 below, a single CMRF was detected and this region was selected as the primer location. For the CRC04 marker, two CMRFs were detected and the region with the highest DFF difference was selected as the primer location.

[0190]

[0191] (2) In addition, in the case of colon cancer test markers, in order to calculate the DFF of the target CpG region of 50 colon cancer-specific hypermethylation markers of Korean Patent No. 10-2024-0059797, data of 16 normal cfDNAs and 15 cfDNAs of colon cancer patients were obtained and utilized using the CRC Targeted EM-Seq method described in Example 8 of the patent.

[0192] That is, after collecting blood from 15 patients with colon cancer and 16 normal people, only the plasma portion was centrifuged for the first time under the conditions of 3000 rpm, 25℃, 10 minutes, and then the first centrifuged plasma was centrifuged for the second time under the conditions of 16000g, 25℃, 10 minutes to separate the plasma, and cell-free DNA was extracted using the Mag-bind cfDNA kit (Omega). The extracted cfDNA concentration was measured using the Qubit DS DNA HS assay Kit (Thermo Fisher Scientific, USA), and the cfDNA size was confirmed using the Tapestation 4200 (Agilent, USA).

[0193] The maximum amount of extracted cfDNA was used, and a library was prepared by performing methylation conversion through the process of substituting unmethylated cytosine with uracil using ten-eleven translocation dioxygenase 2 (TET2) and APOBEC using enzymatic methyl-seq (NEB Kit). Then, the concentration and size of the prepared DNA library were measured using Qubit DS DNA HS assay Kit (Thermo Fisher Scientific, USA) and Tapestation 4200 (Agilent, USA), respectively. Then, 250 ng of the library was pooled into groups of 8 samples, hybridization was performed, and the captured sample was concentrated using Qubit DS DNA HS assay Kit (Thermo Fisher Scientific, USA), and the captured DNA size was confirmed using High sensitivity D1000 screen tape & Reagent (Agilent, USA) with Tapestation 4200 (Agilent, USA). Subsequently, sequencing was performed using a Miseq Dx (Illumina) instrument in 150 paired-end mode with a final concentration of 11 pM. As a result, 500X depth was produced per sample. EM-Seq data is converted through enzyme treatment, and DNA methylation can be confirmed through modification of cytosine bases.

[0194] In the dataset of 16 normal subjects and 15 colorectal cancer patients, the total number of reads containing 5 CMRs of each marker (total reads), detectable fragments (DF, Detectable Fragment), and detectable fragment fraction (DFF, Detectable Fragment Fraction) were calculated. Unlike the WGEM-seq results that calculate DFF by pooling normal and colorectal cancer samples, the Target-seq results have a high sequencing depth, so the DFF of each marker was calculated for each sample. In order to select the region where the DFF value is low in normal samples and high in colorectal cancer samples, the proportion of normal samples with DFF = 0 was 75% or more (Normal third quartile = 0), and the AUC that distinguishes normal samples from colorectal cancer samples was calculated, and 381 CMRFs with an AUC of 0.700 or more were identified.

[0195] These were sorted by each CpG marker, and 13 CpG markers were selected in order of high AUC. This included a total of 96 CMRs, and the AUC range was 0.717-0.921. Among the 13 CpGs, cg17528648 and cg18412834, which were identified as CpGs in the promoter region of OSMR (Nassar et al., Clin Epigenetics, 2021, 13(1) 111), which was reported as a colorectal cancer methylation marker in blood, and NKAIN4 (Zhang et al., Front Oncol. 2023, 12:907464), which was reported as a methylation marker in colorectal cancer adenoma, were selected as CRC01 and CRC02, respectively.

[0196]

[0197]

[0198]

[0199]

[0200] 1.6 Primer and probe design

[0201] Based on the primer positions selected in Example 1-5, primers and probes were designed according to the following criteria. First, the polymerase chain product size was set to 150 bp or less to facilitate amplification and multiplex assay configuration. Second, the probe was designed to have at least one MSRE recognition site. Third, the direction of the probe was designed so that at least one MSRE restriction enzyme recognition site could be placed between the probe and the hydrolysis primer, if possible. That is, in the case of SDC2, when the probe was positioned at a location containing two HhaIs, the probe was designed to be positioned on the plus strand of the DNA when a primer that could have an MSRE recognition site between the primer and the probe was used as a hydrolysis primer (Fig. 5).

[0202] In addition, an internal control IC (Internal Control) was created to verify DNA concentration and quality in marker-specific PCR reactions without being affected by MSRE treatment, targeting regions where MSRE recognition sites do not exist in the promoter regions of housekeeping genes in the human genome. In addition, markers containing one MSRE recognition site each were created as DC (Digestion Control) targeting regions that are always unmethylated in the human genome to confirm complete digestion of MSRE.

[0203] The locations of the IC and DC markers are shown in Table 5 below.

[0204]

[0205] 1.7 Real-time PCR and result analysis

[0206] The base concentrations of primers and probes for each marker were 0.8 / 0.25 pmole / ul, respectively, and only the SDC2 probe was applied at 0.4 pmole / ul. All assays were manufactured as 25x stocks and used as a final 1x. Real-time PCR reactions were prepared as 1x each of 2.5 ul of the product amplified by adapter PCR, 2X TaqMan Multiplex MasterMix (ABI, USA), and 25X Multiplex assay in a total of 50 ul reaction.

[0207] All reactions were performed in duplicate on a Quanta Studio 5 (ABI, USA) instrument, with UNG reaction at 50˚C for 3 minutes, followed by reaction at 95˚C for 10 minutes, followed by 45 cycles of 95˚C, 15 seconds > 60˚C, 1 minute (fluorescence detection).

[0208] After the reaction was completed, the Ct value of each reaction was checked using the software provided by the manufacturer, Quanta Studio 5.

[0209]

[0210] 1.8 Performing MSRE-qPCR

[0211] Nucleic acids were extracted from the standard sample, normal person, and colon cancer sample of Example 1-1 using the method 1-1, and then treated with Acil, HhaI, and HpaII restriction enzymes using the method described in 1-3, and then real-time PCR was performed using the method 1.7.

[0212]

[0213] Example 2. Confirmation of the effectiveness of the AmpliMax platform in colon cancer samples.

[0214] 2-1. Nucleic acid extraction, adapter ligation, MSRE processing, and adapter PCR

[0215] 0% Unme gDNA, 1% Me gDNA, normal cfDNA, and colon cancer cfDNA were extracted using the method described in Examples 1.1 to 1.4, adapters were combined to produce a library, and then adapter PCR was performed by treating three types of MSRE.

[0216]

[0217] 2-2. Selecting the primer location

[0218] SDC2, SEPT9, and CRC01-CRC04, which were selected as regions with the largest DFF differences based on WGEM-seq, had 1-2 CMRFs per target marker, whereas CRC01 and CRC02, explored using Targeted-seq-based AUC values, had 2-20 CMRFs. This difference in the number of CMRFs can be inferred to be related to the sequencing depth of the search database, and can also be thought to be a case where MSRE recognition sites are densely concentrated in the target CpG region.

[0219] If MSREs are densely packed, the 5MSRE CMR size becomes smaller, the GC% is high, and the MSREs exist continuously, which increases the primer binding Tm value, and thus cannot be a primer position. In fact, the average sizes of 20 and 10 CMRFs of CRC01 and CRC02, respectively, were small at 48.5 bp and 43.2 bp, respectively. For this reason, the lower-ranked CRMF that met the 1.6 primer and probe design criteria, rather than the CMRF with the highest AUC, was selected as the primer position, and the CMRF region for each marker is organized as shown in Table 7 below.

[0220]

[0221] 2-3. Primer and probe production and assay configuration

[0222] Primers and probes for the CMRF region selected in Example 2-1 were designed as shown in Table 8 below using the method described in Example 1.6, and were manufactured by order from Bionics (Korea) and then used.

[0223]

[0224] In addition, a Duplex Assay and a Triplex Assay including a DC control were configured to include an IC control marker for each colorectal cancer standard marker, as shown in Table 9.

[0225]

[0226] 2-4. Performing amplification reaction and comparing results with MSRE-qPCR

[0227] Real-time PCR was performed using the primers and probe described in Example 2-3 by the method of Example 1.7, and the Ct value of the existing MSRE-qPCR performed by the method of Example 1.8 was compared.

[0228] In the Amplimax platform, we expected a quantitative and qualitative increase in methylated DNA through cfDNA → adapter ligation → MSRE treatment → PreAmp methyl-specific amplification → Real-time PCR method. In fact, as shown in Table 10, the dCt value of the IC of the Amplimax platform was faster from 2.71 to 4.54 compared to the existing method in both methylated standard gDNA and normal and colon cancer cfDNA samples, confirming a quantitative increase in target DNA. In addition, the dCt values ​​of 0% and 1% Me DNA were faster than 10 in the Amplimax platform compared to the existing method, confirming a qualitative increase in methylated DNA.

[0229]

[0230]

[0231] As described in Tables 10 and 11 above, the range of the average Ct value of the internal control (IC) that can confirm the DNA input amount and Real-time PCR reactivity was compared. While the range of the average value of the MSRE-qPCR internal control (IC) was Ct=32.74-34.31, it was 28.94-31.21 in the Amplimex platform, which was 2.7-4.5 faster on a dCt (delta Ct) basis. This means that 23(=8) times more DNA was input in the Real-time PCR reaction, and it can be confirmed that the quantitative increase of the target DNA was achieved with the Amplimex platform. On average, the Ct value of the internal control (IC) was 32 or less when 4 ng input DNA was applied in the Amplimex platform.

[0232] In addition, as described in Fig. 7, the AmpliMax platform showed a greater shortening of the Ct value in 1% Me DNA compared to 0%, and when this was calculated as dCt (0% & 1%Me), in the case of MSRE-qPCR, it showed a variety of ranges of 0-3.4 in gDNA and 2.38-7.93 in cfDNA, whereas in the AmpliMax platform, it was confirmed that dCt values ​​of 10 or more were mostly shown in gDNA and cfDNA. In other words, when applying the same amount of input DNA, it was confirmed that the IC standard Ct value of the real-time PCR reaction in the AmpliMax platform was faster by 3 or more compared to the MSRE-qPCR, and that methyl-specific amplification was occurring due to a qualitative increase in methylated DNA as well as a quantitative increase in DNA.

[0233] In addition, the large difference in Ct values ​​between 0% (methylation level of normal person) and 1%Me sample (methylation level of cancer patient) of the AmpliMax platform can be very advantageously utilized in the diagnostic field that determines positive and negative using the methylation level of normal person as a cutoff, providing a high level of accuracy and convenience. In addition, it was confirmed that the deviation of the double-repeat Real-time PCR reaction was also very low in the AmpliMax platform compared to MSRE-qPCR, and in the AmpliMax platform, both 0% and normal samples showed Ct=45, that is, a value closer to complete digestion, compared to MSRE-qPCR, confirming that the noise of normal samples is low.

[0234] In addition, in this example, the final products of AmpliMax and MSRE-qPCR are 50 ul and 20 ul, respectively, and the maximum number of PCR reactions that can be used in the marker-specific real-time PCR reaction in which 2.5 ul template is used in 2 replicates is 10 and 4, which shows a large difference between the two platforms. Therefore, when configuring a Multiplex assay of 4 types of fluorescence, AmpliMax can simultaneously confirm up to 40 methylation markers and MSRE-qPCR can simultaneously confirm up to 16 methylation markers. Therefore, the AmpliMax platform can apply a much wider variety of markers to a single cancer type, which can improve the sensitivity of cancer detection. Although the volume of the final product of MSRE-qPCR can be increased from the current 20 ul to 50 ul, which is the same as AmpliMax, to attempt the same number of reactions, it is thought that it will be difficult to obtain reliable results because the Ct value level of MSRE-qPCR is much higher than that of AmpliMax and the deviation is large even under the current conditions.

[0235] In addition, in the Amplimax platform, where the uncut methylated DNA is amplified by Adapter PCR after MSRE treatment to create a methylated DNA enrichment pool, if complete digestion of MSRE is not guaranteed, it is impossible to be sure whether the DNA detected in the real-time PCR reaction is methylated DNA that has not been cut or unmethylated DNA remaining due to incomplete digestion of MSRE. For this reason, in the Amplimax platform, to verify the completeness of MSRE treatment, which is a key variable in the detection of methylated DNA, the MSRE Digestion control (DC) marker was applied to a region that is always unmethylated in the human genome to increase the reliability of the results.

[0236] In the above example, the acceptance criterion for the methylation-sensitive restriction enzyme complete digestion control (DC) of the AmpliMax platform was set to the IC average Ct plus the complete digestion reference Ct value of 7 (e.g., 30 + 7 = 37). In the case where 1% Me gDNA is included, digestion is not performed because fully methylated gDNA is included, and thus the Ct value of the DC is detected to be 30 or higher.

[0237]

[0238] Example 3. Confirmation of the efficacy of the AmpliMax platform in clinical samples.

[0239] First, the methylation level of each marker for approximately 60 normal samples before application to cancer samples was detected using the method of Example 2, and the cutoff was derived as the average of the four fastest Ct values ​​based on the Ct values ​​for the five colon cancer markers detected in duplicate in all normal samples.

[0240] Afterwards, the method of Example 2 was performed with cfDNA extracted from 1 ml of plasma of 7 normal specimens and 1 colon cancer specimen, and the Ct value was confirmed by real-time PCR reaction.

[0241]

[0242] As a result, as described in Table 12, the internal control (IC) and MSRE DC Ct values ​​were 30 or less and 45, respectively, confirming the amount of DNA introduced into the experiment, the real-time PCR reaction, and the completeness of MSRE digestion.

[0243] In addition, it was confirmed that most of the 7 normal samples had Ct=45 compared to the cutoff standard, which means that the methylation level was 0% or very low. CRC03 was detected in 5 and 7 normal samples, but the Ct values ​​were significantly higher (35.1, 35.5) than the cutoff (34.2), confirming that the methylation level was lower than the normal. On the other hand, in the colon cancer samples, all 5 markers were detected with Ct values ​​faster than the cutoff, confirming that they were methylated at a higher level than the normal.

[0244]

[0245] Example 4. Confirmation of the methylation status detection effect according to primer position.

[0246] The Amplimax platform systematically screens target CpG regions and selects primer regions based on DFF values ​​for regions with low methylation levels in normal and high methylation levels in target cancer. In fact, three CMRFs were detected in the SDC2 marker region (regions 1, 2, and 15), and the DFF differences between normal and colorectal cancer were 0.042, 0.053, and 0.161 in regions (1), (2), and (15), respectively (Table 4). Regions (1) and (2) are very closely overlapping regions, so an assay was designed to detect them with a single primer / probe (1&2 SDC2) and compared with region (15) with a high DFF difference (15 SDC2). A library was prepared by combining adapters with cfDNA of seven normal individuals using the method described in Examples 1.1 to 1.4, then adapter PCR was performed by treating three types of MSRE, and real-time PCR was performed using the primers and probes used in Examples 2-4 using (1&2) SDC2 and (15) SDC2 assays.

[0247] As a result of calculating the probability of Ct=45, which means complete digestion, among 14 Ct values ​​in 2 replicates of a total of 7 samples, as a %, the values ​​were 64.3% and 100% in (1&2) SDC2 and (15) SDC2, respectively, as shown in Table 13. This means that the (15)SDC2 region with a large DFF difference is methylated at a higher rate in normal samples. This confirmed that the region with a large DFF difference is a primer region that can more clearly distinguish between normal and colon cancer.

[0248]

[0249] Example 5. Confirmation of the methylation status detection effect according to the MSRE processing order.

[0250] The AmpliMax platform was constructed by recombining the components of each process of the existing target DNA amplification method, adapter ligation & adapter PCR, and the methylated DNA detection method, MSRE-qPCR, into the process of cfDNA > adapter ligation > MSRE > Adapter PCR > Real-time PCR. In order to prove the effectiveness of methylated DNA-specific amplification according to the methodological recombination of the existing technology, it was compared with the general method of analyzing methylated DNA through the adapter ligation method and the MSRE treatment followed by sequencing (Tanaka et al., Analytical Biochemistry, 609, 15, (2020).

[0251] The comparative experiment was conducted in the order of cfDNA > MSRE > adapter ligation > adapter PCR > Real-time PCR, and the main difference from the Amplimax platform is that MSRE is directly applied to cfDNA to cut and remove the unmethylated region, and then adapter ligation / adapter PCR is used to amplify the methylated DNA region on the DNA fragment preserved by methylation.

[0252] The normal and colon cancer samples used in this comparative experiment were identical, and since they used the same MSRE combination and primer / probe as the Amplimax platform, the methylated DNA fragments of each target marker detected on the Amplimax platform would be identically preserved and amplified, and thus would also be detected in the real-time PCR of the comparative experiment. The experimental process used normal and colon cancer DNA prepared according to method 1.1, performed the same MSRE treatment and purification process as in 1.3, and performed the adapter ligation and library construction process as in 1.2. Afterwards, a real-time PCR reaction was performed using the same primers used in Example 2-4, and the Ct values ​​were compared.

[0253]

[0254] As a result, as described in Table 14, the average value of the internal control (IC) in both normal and colon cancer samples was less than 30, confirming the amount of DNA introduced into the experiment and the integrity of the Real-time PCR reaction. However, while the MSRE DC value was mostly close to Ct=45 on the Amplimax platform, the average value in the comparative experiment was high at 32.2. This is because the complete digestion condition for the MSRE treatment and non-treatment group in Real-time PCR is generally defined as dCt>7 between the two test groups (Beikircher et al, 1708, Methods in Molecular Biology, 2018), so DC 32.2 can be judged to be almost a complete digestion condition (IC average 25.94+7 = 32.94).

[0255] In the comparative experimental group, normal and colon cancer samples were hardly distinguished, whereas in the AmpliMax platform, most normal samples were completely digested and detected with Ct=45, and high methyl efficiency was detected in colon cancer samples, confirming the efficiency of methyl DNA specific amplification and detection of the AmpliMax platform, which is a reconfigured MSRE-qPCR with the existing adapter ligation / adapter PCR method.

[0256]

[0257] Example 6. Confirmation of the methylation status detection effect according to the presence or absence of the MSRE recognition sequence in the probe.

[0258] In Example 1.6, at least one MSRE recognition site was placed in the probe. In order to confirm the effect according to the presence or absence of the MSRE recognition sequence in the probe, the degree of complete digestion was compared and analyzed based on Ct=45 when treating three types of MSRE (Acil, HhaI, HpaII, AHP) and two types of MSRE (A / H, & A / P) in the MSRE processing process of the Amplimax platform using normal samples and 1% methylated samples for CRC 02, 03, and 04 markers.

[0259] As a result, as described in Fig. 11a, in the case of the CRC02 marker, since it contains two types of MSREs, Acil and HpaII, at the probe position, when both Acil or HpaII are included (A / H / P, A / H, A / P), it shows a Ct=45 value with almost no deviation, showing a high complete digestion tendency, whereas when only HhaI and HpaII without Acil are present, it shows a deviation of Ct=40, confirming that the complete digestion efficiency is low.

[0260] In addition, as described in Fig. 11b, in the case of the CRC03 marker, the Acil recognition sequence is located in the probe, and all cases containing Acil (A / H / P, A / H, A / P) showed a high complete digestion tendency with Ct=45, but under the H / P MSRE treatment condition without Acil, it was confirmed that digestion was hardly performed and detected at the level of Ct=35.

[0261] However, in the case of the CRC04 marker (Fig. 11c), the MSRE recognition site was very limited, so MSRE was not positioned on the probe, and as a result, an overall unstable complete digestion tendency was confirmed under all MSRE treatment conditions. In addition, although there are two recognition sites for Acil and HpaII, they were located too close to each other, making it difficult to expect a double digestion effect due to the physical simultaneous binding of the MSRE protein.

[0262]

[0263] Example 7. Scalability of the AmpliMax platform in lung cancer samples.

[0264] 7-1. Marker selection and CpG region setting

[0265] The lung cancer test markers were set as target CpGs based on 130 hypermethylated markers selected from Korean Patent No. 10-2023-0148520. Using the Targeted EM-Seq method described in Patent Example 9-1, methylation status results were obtained from 56 normal samples, 79 lung adenocarcinoma (ADC) samples, 36 lung squamous cell carcinoma (SCC) samples, and 7 lung small cell lung cancer (SCLC) samples.

[0266] That is, after collecting the blood of the above patients, only the plasma portion was centrifuged for the first time under the conditions of 3000 rpm, 25℃ for 10 minutes, and then the plasma separated for the first time was centrifuged for the second time under the conditions of 16000g, 25℃ for 10 minutes to isolate the plasma supernatant excluding the precipitate, and cell-free DNA was extracted from the separated plasma using the Mag-bind cfDNA kit, and the concentration was measured using the Qubit DS DNA HS assay Kit (Thermo Fisher Scientific, USA). Using the maximum amount of extracted cfDNA, methylation conversion was performed by substituting unmethylated cytosine into uracil using ten-eleven translocation dioxygenase 2 (TET2) and APOBEC, and then a library was created using enzymatic methyl-seq (NEB Kit).

[0267] The concentration and size of the constructed DNA library were measured using the Qubit DS DNA HS assay Kit (Thermo Fisher Scientific, USA) and Tapestation 4200 (Agilent, USA), respectively. 200 ng of the library was pooled into groups of 8 samples, hybridization was performed, and the captured sample was concentrated using High sensitivity D1000 screen tape & Reagent (Agilent, USA) on Tapestation 4200 (Agilent, USA). Sequencing was performed using a Miseq Dx (Illumina) device in 150 paired-end mode with a final concentration of 11 pM, producing a depth of 700X per sample. EM-Seq data is converted through enzyme treatment, and DNA methylation can be confirmed through modification of cytosine bases. The degree of methylation can be confirmed for each region, and methylation status information was obtained using the beta value, which represents the degree of methylation.

[0268] Among the 130 hypermethylated regions above, regions with a minimum depth of 20 or more for all samples were used, and regions were selected in which the mean difference in beta value between each lung cancer group and the normal group according to lung cancer subtype was 0.01 or more, the mean beta value of the normal group was less than 0.2, and the standard deviation (SD) was less than 0.05. Next, the AUC that distinguishes each lung cancer group and the normal group according to subtype using beta value was calculated, and 30 regions with high AUC were selected. As a result, as shown in Table 15, a total of 64 markers were selected as target CpGs.

[0269] The target CpG region was a 300 bp region extended 150 bp in the 5' and 3' directions from the target CpG marker as described in 1.5.2.

[0270]

[0271]

[0272]

[0273] 7-2. MSRE combination and CMR position setting

[0274] For lung cancer, 27 representative commercially known MSREs were selected to confirm the CMR for various MSRE combinations. These included five 4-cutter MSREs (AciI, BstUI, HpaII, HhaI, HpyC), 20 6-cutter MSREs (AatII, BspEI, AfeI, BstBI, BssHII, Cfr10I, ClaI, Eco52I, HaeII, MluI, NaeI, NruI, NsbI, PmaCI, Psp1406I, PvuI, SacII, SalI, SmaI, SnaB I), one 6-cutter (RsrII), and one 8-cutter (NotI) to include as many MSREs as possible.

[0275] First, for 27 types of MSRE, the count ratio that generally matches the CpG of the human genome was confirmed, and at the same time, the number and size of DNA fragments (CMR) that sequentially connect 2, 3, 4, and 5 MSRE recognition sites were confirmed based on the DNA methylation marker for lung cancer diagnosis selected from Korean Patent No. 10-2023-0148520. In Example 2-2, if there were too many or too few MSREs in the target CpG region, it was not suitable for the PCR amplicon size, so the size of 60-250 bp was filtered to determine which MSRE was most suitable for selecting the primer position of the lung cancer diagnosis marker. The above results are summarized in Table 16 below.

[0276]

[0277] Among the 27 MSREs mentioned above, three 4-cutters (Acil, HpaII, HhaI) and one 6-cutter (HaeII) with high human genome coverage and the highest number of CMRs in lung cancer markers were selected, and a total of seven combinations were constructed by MSRE number and MSRE type (Fig. 8a). For a total of seven MSRE combinations, the 1-5 CMR positions that sequentially connect one, two, three, four, and five MSRE recognition sites were identified.

[0278]

[0279] 7-3. DFF calculation and CMRF area verification

[0280] For lung cancer test markers, the Targeted EM-Seq results of Example 7-1 were utilized to calculate the DFF of 64 lung cancer-specific hypermethylated CpG marker regions described in 7.1 and presented in Table 15. DFF values ​​were calculated in various CMR regions generated from the combination of ①-⑦ MSREs for the normal group and three lung cancer subtypes (ADC, SCC, SCLC), and then the AUC that distinguishes each lung cancer group from the normal group by DFF value according to subtype while having DFF=0 (Normal third quartile=0) in 75% of the normal samples was calculated, and 35 CMRFs with high AUC were selected.

[0281] The results are as described in Table 17, where combination ② HhaI and combination ⑥ HhaI + HaeII are combinations of 4 cutters that commonly recognize GCGC, HhaI (GCGC) and 6 cutter HaeII (RGCGCY), and the CMRF analysis results were the same, so only the results of combination ② were included in the Top 35 count.

[0282]

[0283] Here, ① means AciI, ② means HhaI, ③ means HaeII, ④ means the combination of AcilI and HhaI, ⑤ means the combination of HhaI and HpaII, ⑥ means the combination of HhaI and HaeII, and ⑦ means the combination of Acil, HhaI, and HpaII, which is the same as the colon cancer marker.

[0284] As a result of analyzing the above 105 CMRF regions, as described in Figures 9a to 9e and Tables 18 to 20, it was confirmed that 105 CMRFs were concentrated around 8, 6, and 5 target CpG markers in ADC, SCC, and SCLC, respectively, by lung cancer subtype.

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294] Among the multiple CMRFs for each CpG marker, the CMRF with the highest AUC was selected as the primer region, and a total of four test markers were selected, three from ADC and two from SCC (one of which was common: LU03) that met the primer and probe design conditions of Example 1-6. They were designated LU01, LU02, LU03, and LU04 in that order, and among them, LU03 was a marker commonly detected in different subtypes of ADC and SCC and was designated LU03-1 and LU03-2 in that order. The positions of 30 CMRFs (LU01-9, LU02-5, LU03-3, LU03-12, Lu04-1 = 30) distributed around the CpGs of these four markers are indicated in Figure 9.

[0295] The final CMRF areas selected for each marker are summarized in Table 21 below.

[0296]

[0297] 7-2. Primer and Probe Preparation and Assay Configuration

[0298] Primers and probes for the CMRF region selected in Example 2-1 were designed as shown in Table 22 below using the method described in Example 1.6, and were manufactured by order from Bionics (Korea) and then used.

[0299]

[0300] In addition, a Duplex Assay and a Triplex Assay including a DC control were configured to include an IC control marker for each lung cancer target marker, as shown in Table 23.

[0301] IC and DC used the same primers and probes as described in Example 2.

[0302]

[0303] 7-3. Check the methylation status detection results by marker

[0304] cfDNA of 7 normal specimens and 1 lung cancer specimen was extracted using the method described in Examples 1.1 to 1.4, adapters were combined to create a library, and then adapter PCR was performed by processing two types of MSRE, and real-time PCR was performed using the primers and probe of Example 6-2, and then the Ct value was confirmed, and the cutoff was set based on the average of the three lowest Ct values ​​of the 7 normal specimens.

[0305] As a result, as described in Table 24 below, the internal control (IC) and MSRE DC Ct values ​​were 30 or less and 45, respectively, confirming that the amount of DNA introduced into the experiment and the Real-time PCR reaction and MSRE digestion occurred completely. In normal samples, most had Ct values ​​of 45 or higher than the cutoff, indicating that the methylation level was lower than the reference in normal. In lung cancer samples, except for the LU01 marker, the Ct values ​​were detected faster than the cutoff in the other four markers, indicating that the methylation level was higher than the normal.

[0306] In addition, it was confirmed that the methylation level detected in lung cancer was stable within the real-time PCR detection range of up to 45 cycles, with Ct=33.8-35.6.

[0307]

[0308] That is, as described in FIG. 10, the method of the present invention can be applied to various DNA methylation-related diseases such as lung cancer and colon cancer, and presents a method for selecting primer binding sites with low methylation levels in normal samples and high methylation levels in target diseases based on DFF that is not limited to MSRE combinations and the number of consecutive MSREs, and it was confirmed that it is a technology that has value as a platform for detecting very small amounts of methylated DNA because it has superior quantitative and qualitative amplification effects of methylated DNA compared to typical MSRE-qPCR methods.

[0309]

[0310] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0311]

[0312] The method for detecting methylated DNA according to the present invention can secure a pool enriched in methylated DNA both quantitatively and qualitatively, and detect the methylation level in the methylated DNA enriched pool by real-time polymerase chain reaction using target methylation marker specific primers / probes, so that the detection sensitivity is excellent, and methylated DNA can be detected with higher sensitivity and specificity than existing methods, and analysis can be performed regardless of the type and number of methylation restriction enzymes, so that it is commercially useful.

[0313]

[0314] Electronic file attached.

Claims

1. A method for detecting the methylation status of a nucleic acid comprising the following steps: (a) a step of obtaining nucleic acid from a biological sample; (b) a step of binding an adapter to the obtained nucleic acid; (c) a step of treating the nucleic acid to which the adapter is bound with one or more methylation restriction enzymes (MRE); (d) a step of amplifying the nucleic acid treated with the above restriction enzyme using a primer specific to the adapter; (e) a step of performing a polymerase chain reaction (PCR) reaction using a primer capable of amplifying the target region on the amplification product obtained in step (d) to amplify the target region; and (f) a step of determining whether the amplification product obtained in step (e) exists, and if the amplification product exists, determining that the target region is methylated; 2. A method according to claim 1, characterized in that the MRE is a methylation sensitive restriction enzyme (MSRE) or a methylation dependent restriction enzyme (MDRE).

3. A method according to claim 2, characterized in that the MDRE is at least one selected from the group consisting of AbaSI, AoxI, BisI, BlsI, Dpnl, FspEI, Glal, Glul, Krol, LpnPI, Mall, MspJI, Mtel, Pcsl, PkrI, and Sgel. 4.제2항에 있어서, 상기 MSRE는 Aatll, Acc65I, AccI, Acil, Acll, Afel, Agel, Agel-HF®, Ahdl, Alel-v2, Apal, ApaLl ApeKl, Asci, Asi, Baal, Aval, Aval, Banil BbvCI, BceAI,, Bcgl, BcoDI, BfuAI, Bgll, BmgBI, BsaAI, BsaBI, BsaHI, BsaI-HF®v2, BseYI, BsiE, BsiWI, BsiWI-HF®, BslI, BsmAI, BsmBI-v2, BsmFI, BsmFI, BspBIE, BspBIE BsrFI-v2, BssHII, BstAPI, BstBI, BstUI, BstZ17I-HF®, BtgZI, Cac8I, Cfr10I, Clal, Dpnl, Dralll-HF®, DrdI, Eael, Eagl-HF®, Earl, Ecil, EcoRI5,3kl EcoRI-HF®,EcoRV, EcoRV-HF®, Esp3I, Faul, Fnu4HI, FokI, Fsel, FspI, Haell, Hgal, Hhal, HinPlI, Hindi, Hinfl, Hpal, Hpall, Hpyl66II, Hpyl88III, Hpy99I, HpyCH4, Hpyl66II, Hpyl88III, Hpy99I, HpyCH4, Hpyl, MIV, Misbol, M.I. MluI-HF®, Mmel, MspAlI, Mwol, Nad, Narl, Neil, NgoMIV, Nhd-HF®, NlalV, Notl, Notl-HF®, Nrul, NruI-HF®, Nt.BbvCI, Nt.BsmAI, Nt.CviPII, PaeR7I, PCIld, PCI, PCI.A method characterized in that at least one selected from the group consisting of Pmd, Pmll, PshAI, PspOMI, PspXI, Pvul, PvuI-HF®, Rsal, RsrII, Sad-HF®, SacII, Sall, Sall-HF®, Sau3AI, Sau96I, ScrFI, SfaNI, Sfil, Sfol, SgrAI, Smal, SnaBI, Srfl, StyD4I, Tfil, Tsd, TspMI, Xhol, Xmal and Zral.

5. A method according to claim 4, wherein the MSRE is at least one selected from the group consisting of AciI, BstUI, HpaII, HhaI, HpyC, AatII, BspEI, AfeI, BstBI, BssHII, Cfr10I, ClaI, Eco52I, HaeII, MluI, NaeI, NruI, NsbI, PmaCI, Psp1406I, PvuI, SacII, SalI, SmaI, SnaB I, RsrII, and NotI.

6. A method according to claim 1, characterized in that step (c) processes MSRE.

7. A method characterized in that, in the first paragraph, when there are two or more restriction enzymes in step (c), they are processed at the same or different ratios.

8. A method characterized in that, in the first paragraph, the position where the primer binds is selected by a method including the following steps. (i) a step of setting a 100 to 500 bp region in the 5' direction and 3' direction of the target region as a target CpG region; (ii) a step of determining the position of a region (CMR, Consecutive MSRE CpG Fragment) starting from the first MSRE recognition site based on the 5'-terminal region in the target CpG region to one or more consecutive MSRE recognition sites including the MSRE recognition site; (iii) a step of calculating the detectable fragment fraction (DFF) for each CMR and selecting CMRs with different DFF values between non-diseased and diseased samples as continuous MSRE-resistant fragments (CMRF); and (iv) A step of selecting the region with the largest difference in DFF in the selected CMRF as the primer binding position.

9. In the 8th paragraph, the DFF is characterized in that the method is calculated by a method including the following steps: (1) A step of counting the total number of reads aligned to each CMR in a database capable of confirming the methylation level; (2) a step of counting the number of reads (DF, Detectable Fragment) in which the MSRE recognition site of each CMR is all methylated; and (3) A step of calculating DFF by dividing the number of DFs by the total number of leads aligned to CMR.

10. A method according to claim 9, characterized in that the database capable of confirming the methylation level is created using any one method selected from the group consisting of pyrosequencing, bisulfite sequencing, and methylation next-generation base sequence sequencing.

11. A method according to claim 1, characterized in that the step of determining the presence or absence of the amplification product in step (f) uses a nucleic acid-binding dye or probe capable of binding to the amplification product.

12. A method according to claim 11, wherein the nucleic acid binding dye is selected from the group consisting of ethidium bromide, SYBR® Green I, SYBR® Gold, EvaGreen, YO-PRO-1, SYTO, BEBO, and BEXTO.

13. A method according to claim 11, characterized in that the probe capable of binding to the amplification product is selected from the group consisting of one or more combinations of oligonucleotides, LNAs, and PNAs.

14. A method according to claim 11, wherein the probe capable of binding to the amplification product comprises at least one MSRE recognition sequence.

15. A method according to claim 11, wherein the probe capable of binding to the amplification product has a reporter and a quencher connected to both ends.

16. A method according to claim 15, wherein the reporter is at least one fluorescent substance selected from the group consisting of fluorescein, fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethylrhodamine, FITC, Oregon green, Alexa Fluor, FAM, JOE, ROX, HEX, Texas Red, TET, TRITC, TAMRA, cyanine series dyes, and thiadicarbocyanine dyes.

17. A method according to claim 15, wherein the quencher is at least one selected from the group consisting of Dabcyl, TAMRA, Eclipse, DDQ, QSY, Blackberry Quencher, Black Hole Quencher, Qxl, Iowa black FQ, Iowa black RQ, and IRDye QC-1.

18. A method according to claim 1, characterized in that the step (e) is performed by additionally including a primer (DC, Digestion Control) capable of amplifying a region that is completely digested by a methylation restriction enzyme (complete digestion region); and a primer (IC, Internal Control) capable of amplifying a region that is not cut by a methylation restriction enzyme.

19. A method according to claim 1, characterized in that the adapter does not contain a methylation restriction enzyme recognition sequence.

20. A method for providing information for disease diagnosis, comprising a step of determining that a disease is present when a target region is methylated by any one of the methods of paragraphs 1 to 19.

21. A method according to claim 20, wherein the disease is selected from the group consisting of cancer, neurodegenerative disease, autoimmune disease, metabolic disease, cardiovascular disease, rare disease, and other diseases.

22. In claim 21, the cancer is ovarian cancer, soft tissue sarcoma, peripheral T-cell cancer, colon cancer, intrahepatic cholangiocarcinoma, glioblastoma, esophageal cancer, cutaneous T-cell lymphoma, non-Hodgkin's lymphoma, urinary tract cancer, basal cell carcinoma, epithelioid sarcoma, pancreatic cancer, non-small cell lung cancer, Hodgkin's lymphoma, renal cell cancer, mesothelioma, metastatic uveal melanoma, kidney cancer, blood cancer, HER2-expressing cancer, non-melanoma skin cancer, liposarcoma, hepatocellular carcinoma, small lymphocytic lymphoma, prostate cancer, breast cancer, anal cancer, marginal zone lymphoma, cutaneous squamous cell carcinoma, thyroid cancer, medullary thyroid cancer, triple-negative breast cancer, neuroendocrine prostate cancer, bladder cancer, paraganglioma, medulloblastoma, superficial basal cell carcinoma, head and neck squamous cell carcinoma, blood cancer, melanoma, B-cell lymphoma, relapsed / refractory acute myeloid leukemia, angiosarcoma, Osteosarcoma, refractory cervical cancer, cholangiocarcinoma, gastroesophageal adenocarcinoma, rhabdomyosarcoma, carcinoma, non-muscle-invasive bladder cancer, uveal melanoma, small cell lung cancer, cervical cancer, primary open-angle glaucoma, follicular lymphoma, synovial sarcoma, liver cancer, carcinosarcoma, leptomeningeal brain tumor, T-cell lymphoma, lymphoma, small cell lung cancer, mantle cell lymphoma, B-cell malignancy, endometrial cancer, mucinous / round cell liposarcoma, metastatic Merkel cell carcinoma, neuroblastoma, chronic lymphocytic leukemia, tendon sheath giant cell tumor, sarcoma, acute myeloid leukemia, skin cancer, nasopharyngeal cancer, relapsed / refractory Ewing sarcoma, bone cancer, glioma, salivary gland carcinoma, stomach cancer, benign tumor, low-grade serous ovarian cancer, metastatic breast cancer, multiple myeloma, diffuse large B-cell lymphoma, relapsed / refractory lymphoma, metastatic A method characterized in that any one of the following is selected from the group consisting of colorectal cancer, advanced malignant tumor, and acute lymphoblastic leukemia.

23. A method according to claim 22, characterized in that the cancer is any one selected from the group consisting of colon cancer, lung cancer, stomach cancer, breast cancer, prostate cancer, and liver cancer.

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