Method for detecting methylation status of nucleic acid

Abstract

The present application relates to a method for detecting methylation status of a nucleic acid, comprising the steps of: treating a biological sample with an unmethylation-sensitive restriction enzyme; and performing a methylation-sensitive high-resolution melting (MS-HRM) analysis.

Description

How to check the methylation status of nucleic acids

[0001] The present application relates to a method for determining the methylation status of a nucleic acid.

[0002] Cancer is a disease that frequently causes illness and death worldwide, and is caused by a variety of factors. Research into various diagnostic methods is actively underway, and technologies for diagnosing cancer in its early stages are receiving particular attention. While cancer survival rates increase with early detection, early-stage cancer often presents no specific symptoms. Cancer diagnosis is typically performed through blood tests, imaging techniques, and tissue biopsies. However, each method has limitations in sensitivity and specificity, as well as clear risks and side effects. Therefore, developing technologies for early cancer diagnosis is crucial for not only improving medical outcomes but also bringing significant social and economic benefits. Ultimately, these technologies are expected to provide patients with more effective and accurate information and contribute to the efficient use of medical resources.

[0003] Liquid biopsy is a non-invasive or minimally invasive method compared to conventional tissue biopsies, allowing for sample collection with minimal stress on the patient. This significantly reduces the burden on the patient, allows for regular monitoring of health status, and offers the advantage of being able to perform the test without concerns about radiation exposure. Recently, technologies are being developed to detect cancer-specific biomarkers, including genetic and epigenetic features found in circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), using blood-based liquid biopsies. These technological advancements are expected to revolutionize the fields of cancer diagnosis and treatment monitoring. Therefore, the present application aims to develop a method for more sensitively detecting cancer-specific ctDNA methylation levels.

[0004] An example of the present application is to provide a method for determining the methylation status of a nucleic acid by pretreating a biological sample with an unmethylation-sensitive restriction enzyme (uMSRE) to increase the amplification efficiency of a target substance and improve the clinical sensitivity of an MS-HRM analysis method, thereby enabling liquid biopsy-based diagnosis, prognosis prediction, and treatment response monitoring.

[0005] One example of the present application relates to a method for determining the methylation status of a nucleic acid, comprising the steps of treating a biological sample with an unmethylation-sensitive restriction enzyme; and performing methylation-sensitive high-resolution melting (MS-HRM) analysis.

[0006] Another example of the present application relates to a method for improving the sensitivity of a MS-HRM analysis method, comprising the steps of treating a biological sample with an unmethylation-sensitive restriction enzyme; and performing MS-HRM analysis.

[0007] Another example of the present application relates to a method for providing information for predicting the risk of cancer development, comprising the steps of treating a biological sample with an unmethylation-sensitive restriction enzyme; performing methylation-sensitive high-resolution melting (MS-HRM) analysis; and determining the methylation status of a cancer diagnostic biomarker contained in the biological sample.

[0008] Another example of the present application relates to a kit for determining the methylation status of a nucleic acid, comprising an unmethylation-sensitive restriction enzyme; and a methylation-sensitive high-resolution melting (MS-HRM) kit.

[0009] Another example of the present application relates to a cancer diagnostic kit comprising an unmethylation-sensitive restriction enzyme; and a methylation-sensitive high-resolution melting (MS-HRM) kit, wherein the MS-HRM kit determines the methylation status of a cancer diagnostic biomarker.

[0010] Another example of the present application relates to a method for treating cancer, comprising the steps of: treating a biological sample with an unmethylation-sensitive restriction enzyme; performing methylation-sensitive high-resolution melting (MS-HRM) analysis; determining the methylation status of a cancer diagnostic biomarker contained in the biological sample; and treating a subject determined to have a risk of cancer.

[0011]

[0012] Hereinafter, the present application will be described in more detail.

[0013] While PCR-based methods for DNA methylation detection, such as MSP, qMSP, and MS-HRM, have proven their utility in tissues, recent developments in liquid biopsy-based assays have revealed limitations such as low sensitivity and specificity, limiting their practical clinical application. While various methods, such as next-generation sequencing (NGS) and droplet digital PCR (ddPCR), are being developed, they are still considered in the research and development phase, considering their clinical utility and marketability.

[0014] A method for determining the methylation status of a nucleic acid according to an example of the present application can detect methylated DNA in a biological sample by combining unmethylation-sensitive restriction enzyme (uMSRE) and MS-HRM technology, and can more sensitively detect ctDNA present in trace amounts in a liquid biopsy. Specifically, a method for determining the methylation status of a nucleic acid according to an example of the present application achieves a) establishing an optimal uMSRE usage method, b) securing high analytical sensitivity and specificity through uMSRE processing when noise signals are present, c) securing higher analytical sensitivity and specificity for methylation detection, d) cost and efficiency of the test method, and e) securing broad clinical applicability by combining unmethylation-sensitive restriction enzyme and MS-HRM technology.

[0015] The above unmethylation-sensitive restriction enzyme (uMSRE) is a substance that can induce more efficient amplification of methylated DNA, which is a target substance, in PCR, and can specifically recognize and cut nucleic acids in which the target site is unmethylated among nucleic acids contained in a biological sample. For example, the nonmethylation-sensitive restriction enzyme may be at least one selected from the group consisting of AtaII, AciI, AclI, AfeI, AgeI, AsiSI, AvaI, BceAI, BmgBI, BsaAI, BsaHI, BsiEI, BsiWI, BspDI, BssHII, BstBI, BstUI, ClaI, EagI, Esp3I, FauI, FseI, FspI, HaeII, HgalI, HhaI, HinP1I, HpaII, Hpy99I, HpyCH4IV, NaeI, NarI, NgoMIV, NotI, NruI, PaeR7I, PluTI, Pm1I, PvuI, RsrII, SacII, Sa1I, SfoI, SgrAI, SmaI, SnaBI, SrfI, TspMI, and ZraI.

[0016] In this example, the optimal reaction conditions for non-methylation-sensitive restriction enzyme pretreatment were verified, and as a result, the detection sensitivity of methylated DNA varied depending on the concentration of non-methylation-sensitive restriction enzyme treatment in the control DNA. The effect of increasing analytical sensitivity was excellent when the concentration of uMSRE treatment was about 1.25 Units (U) or higher, and the effect was particularly excellent when the concentration of uMSRE treatment was about 2.5 Units (U) or higher. In addition, the effect increase began to saturate when the concentration of uMSRE treatment was about 5 Units (U) or higher. In addition, treatment with uMSRE at the level of 5 Units (U) in liquid biopsy was able to secure the same specificity as the improvement in clinical sensitivity. Accordingly, the non-methylation-sensitive restriction enzyme is present in the biological sample at 0.025 Unit(U) or more, 0.05 Unit(U) or more, 0.1 Unit(U) or more, 0.5 Unit(U) or more, 1 Unit(U) or more, 1.25 Unit(U) or more, 1.5 Unit(U) or more, 2 Unit(U) or more, 2.1 Unit(U) or more, 2.2 Unit(U) or more, 2.3 Unit(U) or more, 2.4 Unit(U) or more, 2.5 Unit(U) or more, 2.6 Unit(U) or more, 2.7 Unit(U) or more, 2.8 Unit(U) or more, 2.9 Unit(U) or more, 3 Unit(U) or more, 3.1 Unit(U) or more, 3.2 Unit(U) or more, 3.3 Unit(U) or more, 3.4 Unit(U) or more, 3.5 It can be processed as Unit(U) or more, 3.6 Unit(U) or more, 3.7 Unit(U) or more, 3.8 Unit(U) or more, 3.9 Unit(U) or more, 4 Unit(U) or more, 4.1 Unit(U) or more, 4.2 Unit(U) or more, 4.3 Unit(U) or more, 4.4 Unit(U) or more, 4.5 Unit(U) or more, 4.6 Unit(U) or more, 4.7 Unit(U) or more, 4.8 Unit(U) or more, 4.9 Unit(U) or more, or 5 Unit(U) or more.At this time, even if the upper limit of the concentration of the non-methylation-sensitive restriction enzyme to be treated on the biological sample is not specified, a person skilled in the art will be able to clearly implement an example of the present application for the purpose of improving analytical sensitivity, but for example, the upper limit of the concentration of the non-methylation-sensitive restriction enzyme to be treated on the biological sample may be 10 Unit(U) or less, 9 Unit(U) or less, 8 Unit(U) or less, 7 Unit(U) or less, 6.5 Unit(U) or less, 6 Unit(U) or less, 5.9 Unit(U) or less, 5.8 Unit(U) or less, 5.7 Unit(U) or less, 5.6 Unit(U) or less, or 5.5 Unit(U) or less.

[0017] The above biological sample may be one or more selected from the group consisting of blood, plasma, tissue, cells, feces, and urine.

[0018] The biological sample may contain methylated nucleic acids at a target site at 50% or less, 40% or less, 30% or less, 29% or less, 28% or less, 27% or less, 26% or less, 25% or less, 20% or less, 15% or less, 9% or less, 8% or less, 7% or less, 6.5% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1.9% or less, 1.8% or less, 1.7% or less, or 1.6% or less, based on 100% of the total nucleic acids contained in the biological sample.

[0019] The biological sample above contains methylated nucleic acid at the target site at 100 ng / μL or less, 90 ng / μL or less, 80 ng / μL or less, 70 ng / μL or less, 60 ng / μL or less, 50 ng / μL or less, 40 ng / μL or less, 30 ng / μL or less, 20 ng / μL or less, 15 ng / μL or less, 10 ng / μL or less, 9 ng / μL or less, 8 ng / μL or less, 7 ng / μL or less, 6 ng / μL or less, 5 ng / μL or less, 4 ng / μL or less, 3 ng / μL or less, 2 ng / μL or less, 1.5 ng / μL or less, 1 ng / μL or less, 0.5 ng / μL or less, 0.4 ng / μL or less, 0.3 It may contain 0.2 ng / μL or less, 0.1 ng / μL or less, 0.05 ng / μL or less, 0.04 ng / μL or less, 0.03 ng / μL or less, 0.025 ng / μL or less, 0.02 ng / μL or less, 0.01 ng / μL or less, 0.009 ng / μL or less, 0.008 ng / μL or less, 0.007 ng / μL or less, 0.0065 ng / μL or less, 0.006 ng / μL or less, 0.005 ng / μL or less, 0.003 ng / μL or less, 0.002 ng / μL or less, or 0.0016 ng / μL or less.

[0020] The above MS-HRM analysis is a method that can semi-quantitatively confirm the methylation level of a sample, and can confirm the methylation status of a specific DNA sequence (target site). Specifically, when a biological sample is treated with an agent that differently modifies a nucleic acid in which the target site is methylated and a nucleic acid in which the target site is unmethylated, unmethylated cytosine (C) is converted to uracil (U), and methylated cytosine (C) remains unconverted, so that the nucleic acid in which the target site is methylated and the nucleic acid in which the target site is unmethylated are differently modified, and when the converted DNA is amplified by PCR, different PCR products are generated because the sequences differ depending on the methylation status. High-resolution melting curve analysis (HRM) is performed to measure the melting temperature (Tm) at which DNA double strands separate into single strands while slowly heating the PCR product. At this time, methylated nucleic acids show a higher Tm due to triple hydrogen bonding between cytosine (C) and guanine (G), and unmethylated nucleic acids show a lower Tm due to double hydrogen bonding between thiamine (T) and adenine (A). By comparing the obtained melting curve of the biological sample with that of a standard methylation sample (e.g., 0% methylation, 50% methylation, 100% methylation, etc.), the methylation level of the biological sample can be quantified.

[0021] Accordingly, the MS-HRM analysis may include at least one selected from the group consisting of a step of differently modifying a nucleic acid in which the target region is methylated and a nucleic acid in which the target region is unmethylated; a step of amplifying the nucleic acid; a step of obtaining a melt curve of the biological sample; a step of comparing the melt curve of the biological sample with a melt curve of a standard methylated sample; and a step of semi-quantitatively quantifying the methylation level of the nucleic acid.

[0022] Another example of the present application relates to a kit for determining the methylation status of a nucleic acid, comprising an unmethylation-sensitive restriction enzyme; and a methylation-sensitive high-resolution melting (MS-HRM) kit.

[0023] The above MS-HRM kit is a kit for performing MS-HRM analysis, and may include materials, reagents, devices, etc. necessary for MS-HRM analysis. For example, the MS-HRM kit may include an agent that differently modifies a nucleic acid having a methylated target region and a nucleic acid having an unmethylated target region; and a primer set for amplifying the nucleic acids. The MS-HRM kit may additionally include a PCR master mix containing a DNA polymerase, a fluorescent reagent (Fluorescent Dye), or a PCR device (e.g., a real-time PCR machine).

[0024] The agent that differently modifies the nucleic acid having a methylated target site and the nucleic acid having an unmethylated target site may be at least one selected from the group consisting of sulfurous acid, bisulfite, hydrogen sulfite, and disulfite.

[0025] In this example, the analytical sensitivity of the MS-HRM assay was verified by the treatment with a non-methylation-sensitive restriction enzyme. As a result, regardless of the concentration of methylated nucleic acids contained in the biological sample, the treatment with uMSRE showed a higher methylation peak, and the analytical sensitivity of MS-HRM was improved. In particular, in biological samples containing methylated nucleic acids at a low concentration of 25% or less among the total nucleic acids, the analytical sensitivity was significantly increased when MS-HRM analysis was performed after treatment with a non-methylation-sensitive restriction enzyme compared to the group not treated with a non-methylation-sensitive restriction enzyme. In particular, in biological samples containing very low methylated nucleic acids of 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, or 2% or less among the total nucleic acids, the sensitivity of MS-HRM analysis was further significantly increased by treatment with a non-methylation-sensitive restriction enzyme. Accordingly, another example of the present application relates to a method for improving the sensitivity of a MS-HRM analysis method, comprising the steps of treating a biological sample with an unmethylation-sensitive restriction enzyme; and performing MS-HRM (methylation-sensitive high-resolution melting) analysis.

[0026] In one example of the present application, the nucleic acid whose methylation status is to be determined may include a cancer diagnostic biomarker. The cancer diagnostic biomarker may be a methylated nucleic acid in a cancer patient. Specifically, the cancer diagnostic biomarker may be a biomarker whose methylation level is higher only in DNA isolated from a biological sample derived from a subject at risk of cancer, compared to the methylation level in DNA isolated from a biological sample derived from a normal subject.

[0027] When the nucleic acid to be confirmed as the target of the methylation status includes a biomarker for cancer diagnosis, if the methylation status of the nucleic acid is confirmed according to the method for confirming the methylation status of the nucleic acid according to an example of the present application, the risk of cancer development of the subject from which the nucleic acid is derived can be predicted based on the methylation status. Accordingly, another example of the present application relates to a method for providing information for predicting the risk of cancer development, the method including the steps of: treating a biological sample with an unmethylation-sensitive restriction enzyme; performing MS-HRM (methylation-sensitive high-resolution melting) analysis; and confirming the methylation status of a cancer diagnostic biomarker included in the biological sample.

[0028] Early diagnosis is paramount in cancer treatment. Many cancers, including liver, breast, lung, and colon cancers, often progress asymptomatically, making early detection difficult, leading to generally poor prognoses. Current clinical diagnostic methods rely heavily on imaging and biochemical markers, but these methods have been reported to have significant limitations in sensitivity and specificity. The method for predicting cancer risk, according to one example of the present application, can identify the methylation status of biomarkers for specific cancer types, which is expected to improve the early diagnosis rate. This, in turn, can lead to appropriate treatment choices for patients, increased survival rates, and the advancement of precision medicine.

[0029] A method for providing information for predicting the risk of cancer development according to an example of the present application may further include a step of comparing the methylation status of the cancer diagnostic biomarker with the methylation status of a normal control group. If the methylation status of the cancer diagnostic biomarker is higher than the methylation level of the normal control group, information may be provided indicating a risk of cancer development.

[0030] Another example of the present application relates to a cancer diagnostic kit comprising an unmethylation-sensitive restriction enzyme; and a methylation-sensitive high-resolution melting (MS-HRM) kit, wherein the MS-HRM kit determines the methylation status of a cancer diagnostic biomarker.

[0031] The cancer may be at least one selected from the group consisting of liver cancer, colon cancer, esophageal cancer, stomach cancer, rectal cancer, colorectal cancer, oral cancer, pharynx cancer, laryngeal cancer, lung cancer, non-small cell lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, biliary tract cancer, bone cancer, connective tissue cancer, skin cancer, melanoma, brain cancer, head and neck cancer, thyroid cancer, leukemia, Hodgkin's disease, lymphoma, urinary tract cancer, and multiple myeloma.

[0032] A method for providing information for predicting the risk of cancer development according to an example of the present application may further include a step of treating the subject. The step of treating may include administering an effective amount of a therapeutic agent to the subject, performing chemotherapy, hormone therapy, radiation therapy, surgery, or a combination thereof. The therapeutic agent may be, for example, a cancer treatment agent.

[0033] The above treatments include, for example, Afatinib, AK105, Anlotinib, Apatinib, Atezolizumab, Avelumab, Axitinib, Bevacizumab, Bosutinib, BSC, Cabozantinib, Cabozantinib-S-Malate, Camrelizumab, Camertinib, Carboplatin, Capecitabine, Celecoxib, CC-122, CF102, Crizotinib, Dasatinib, Docetaxel, Donafenib, Dovitinib (Dovitinib), doxorubicin, durvalumab, EKB-569, entrectinib, epirubicin, erlotinib, etoposide, everolmus, FGF401, FOLFOX 4, fostamatinib, galunisertib, gefitinib, gemcitabine, IBI305, ibrutinib, imatinib, INC280, infigratinib, ipilimumab, irinotecan, lapatinib, leflunomide, lenvatinib (Lenvatinib), LY2875358, Mesylate, Mitomycin c, MSC2156119J, Neratinib, Nilotinib, Nintedanib,Nivolumab, oxaliplatin, palbociclib, panobinostat, pazopanib, PDR001, pembrolizumab, pemigatinib, Pexavec, phosphate, ramucirumab, regorafenib, ruxolitinib, semaxinib, selumetinib, SGO-110, SHR-1210, sintilimab, sorafenib, SU6656, sunitinib, sintilimab, spartalizumab It may include sutent, TACE, Tasquinimod, Temozolomide, Temsirolimus, Tislelizumab, Tivantinib, Tosylate, toripalimab, Tremelimumab, vandetanib, vatalanib, XL888, Y90, a pharmaceutically acceptable salt thereof, or a combination thereof.

[0034] Another example of the present application relates to a method for treating cancer, comprising the steps of treating a biological sample with an unmethylation-sensitive restriction enzyme; performing methylation-sensitive high-resolution melting (MS-HRM) analysis; determining the methylation status of a cancer diagnostic biomarker contained in the biological sample; and treating a subject determined to have a risk of cancer. The step of treating the subject may include administering an effective amount of a therapeutic agent to the subject, performing chemotherapy, hormone therapy, radiation therapy, surgical operation, or a combination thereof.

[0035] A method for determining the methylation status of a nucleic acid according to an example of the present application can be designed to build a highly sensitive test capable of detecting liquid biopsy or minimal residual disease (MRD) through enrichment of methylated target DNA by combining a non-methylation-sensitive restriction enzyme and MS-HRM analysis, and is generally applicable to various types of cancer.

[0036] One example of this application establishes optimal uMSRE reaction conditions for specimen preprocessing and improves analytical sensitivity in MS-HRM assays using uMSRE treatment. Furthermore, one example of this application verifies that uMSRE treatment is unaffected by noise present in each specimen, and that there is no decrease in clinical specificity that may arise from increased amplification efficiency of methylated DNA. Therefore, one example of this application may improve clinical performance in liquid biopsy specimens.

[0037] Figure 1 is a diagram verifying the optimal reaction conditions for non-methylation-sensitive restriction enzyme pretreatment.

[0038] Figure 2 is a diagram verifying analytical sensitivity according to the presence or absence of non-methylation-sensitive restriction enzyme treatment.

[0039] Figure 3 is a drawing verifying the effect of reducing the influence of noise present in a sample and improving the detection stability of a methylated template by treatment with a non-methylation-sensitive restriction enzyme.

[0040] Figure 4 is a drawing verifying that the problem of decreased clinical specificity does not occur due to treatment with a non-methylation-sensitive restriction enzyme.

[0041] Figure 5 is a diagram verifying the effect of improving sensitivity in liquid biopsy specimens by treatment with a nonmethylation-sensitive restriction enzyme.

[0042] Hereinafter, the present application will be described in more detail with reference to the following examples. However, these examples are merely intended to illustrate the present application, and the scope of the present application is not limited by these examples.

[0043]

[0044] Reference Example 1. Liver cancer biomarker

[0045] In order to verify the method for confirming methylation of nucleic acids according to an example of the present application, information on liver cancer biomarkers used in the experiment is listed in Table 1 based on the human genome reference sequence (Reference Genome) GRCh37 version, and information on primers for amplifying the same is listed in Table 2. “Marker 1” and “Marker 2” in Table 1 are biomarkers for liver cancer in which the CpG site at the corresponding position is specifically methylated in liver cancer patients.

[0046] NameChromosomeStartEndMarker 1Chr122181075021810751Marker 2Chr191973940719739408

[0047] NameSeqenceSEQ ID NO.Forward Common Primer for Marker 1gagaaggTtTtggagaTTtTtgtaaTag1Reverse Methylation Primer for Marker 1gagaaggTtTtggagaTTtTtgtaaTag2Reverse Unmethylation Primer for Marker 1tccttAcaAaAcCAACAcCA3Forward Common Primer for Marker 2gtTGgatTagggagtggTttgtaaTt4Reverse Methylation Primer for Marker 2ccCGctctcttcttCGtA5Reverse Unmethylation Primer for Marker 2ccCActctcttcttCAtAt6

[0048] Example 1. Optimal reaction conditions for nonmethylation-sensitive restriction enzyme pretreatment.

[0049] (1) Pretreatment with nonmethylation-sensitive restriction enzymes

[0050] Methylated & Non-methylated DNA was quantified using Qubit 1X dsDNA High Sensitivity Kit (Cat. # Q33230 or Q33231) after diluting 10X using NFW for Human Methylated & Non-methylated DNA Set from Zymo Research (Cat. # D5014). Based on the quantification value of the 10X diluted Methylated & Non-methylated DNA, NFW was used to dilute each Methylated & Non-methylated DNA to a concentration of 2.5 ng / μL. Using 2.5 ng / μL of Methylated & Non-methylated DNA, methylation samples of 0.5, 0.25, and 0.125% were prepared, and a total of four methylation percentages (0, 0.125, 0.25, and 0.5%) were used in the uMSRE reaction.

[0051] By serially diluting 10 U / μL of AciI 2X using NFW, samples of nonmethylation-sensitive restriction enzyme (uMSRE) were prepared at concentrations of 5, 2.5, 1.25, 0.63, and 0.31 U / μL.

[0052] Using different capacities of uMSRE, reagents were mixed for each methylation % sample as shown in Table 3, incubated at 37°C for 1 hour, and then inactivated at 65°C for 20 minutes.

[0053] ReagentConcentrationVolume (μL)FinalAciⅠ0, 0.31, 0.63, 1.25, 2.5, 5 U / μL20, 0.6, 1.25, 2.5, 5, 10 UrCutSmartBuffer10X21XNFW-6-DNA Sample2.5 ng / μL1025 ngTotal-20-

[0054] (2) Sulfur dioxide conversion

[0055] Specimens that had undergone 20-minute inactivation at 65°C were immediately used for sulfite conversion. Sulfite conversion was performed using the Zymo Research EZ DNA Methylation-Lightning Kit, following the manufacturer's recommendations. All 20 μL used in the uMSRE were used for sulfite conversion. However, the elution step was performed using 25 μL of EB Buffer, rather than the 10 μL recommended by the manufacturer. After sulfite conversion, the specimens were stored at -20°C until MS-HRM experiments.

[0056]

[0057] (3) MS-HRM analysis

[0058] Marker 1, listed in Table 1, was used as a nucleic acid to determine methylation status. MS-HRM analysis was performed on samples that had completed uMSRE capacity and sulfurous acid treatment using the MS-HRM primer set of SEQ ID NOs: 1 to 3. Specifically, the PCR mixture for MS-HRM was first mixed as shown in Table 4.

[0059] ReagentConcentrationVolumeFinal ConcentrationMaster Mix2X12.5 μL1XDye20X1.25 μL1XPrimer20X1.25 μL1XSample-10 μL-Total-25 μL-

[0060] After this, the PCR mixture was dispensed into a 96-well, 10 μL of the sample was dispensed, and pre-amplification was performed to amplify the target material as follows:

[0061] a) Initial denaturation 95℃ 5 minutes;

[0062] b) One plate read was performed for each cycle, repeating the following for a total of 40 cycles: denaturation at 95°C for 10 s / annealing at 63°C for 30 s / extension at 72°C for 15 s;

[0063] c) Final extension: 72°C for 5 minutes. After pre-amplification, melt curve analysis was performed by increasing the temperature from 60°C to 95°C in 0.2°C increments, holding at each temperature for 10 seconds, and then performing a plate read.

[0064] Among the MS-HRM analysis results, changes for each sample were confirmed based on the Cycle value confirmed based on RFU 1,000 at the stage of confirming the amplification signal.

[0065] As shown in Fig. 1, Cq increased with uMSRE treatment, and this was due to the action of uMSRE. In particular, the efficiency of uMSRE was excellent when the methylation % of the sample was 1.25 Units or higher, and especially when uMSRE was 2.5 Units or higher under all conditions. In addition, it was confirmed that the efficiency of uMSRE began to saturate when a uMSRE dose of approximately 5 Units was used. Based on these results, the optimal dose of uMSRE pretreatment in liquid biopsy was established as 5 Units.

[0066]

[0067] Example 2. Improvement of analytical sensitivity of MS-HRM assay by treatment with nonmethylation-sensitive restriction enzymes.

[0068] Methylated & Non-methylated DNA was quantified using the Qubit 1X dsDNA High Sensitivity Kit after diluting 10X with nuclease-free water (NFW) for Huma Methylated & Non-methylated DNA from Zymo Research. Based on the quantification value of the 10X diluted Methylated & Non-methylated DNA, the Methylated & Non-methylated DNA was diluted using NFW to a concentration of 0.1 ng / μL. Using 0.1 ng / μL of Methylated & Non-methylated DNA, methylation samples of 1.6, 6.3, and 25% were prepared, and a total of five methylation percentages (0, 1.6, 6.3, 25, and 100%) were used in the uMSRE reaction. As shown in Table 5, the uMSRE reaction reagent and DNA were mixed, incubated at 37°C for 1 h, and then inactivated at 65°C for 20 min. Sulfite conversion was performed in substantially the same manner as (2) of Example 1, and stored at -20°C until performing the MS-HRM experiment.

[0069] ReagentConc. Vol. (μL)FinalAciⅠ10 U / μL0.50, 0.6, 1.25, 2.5, 5, 10 UrCutSmartBuffer10X61XNFW-3.5-Sample0.1 ng / μL505 ngTotal-60-

[0070] Marker 1 and Marker 2 described in Table 1 were used as nucleic acids for confirming the methylation status. Using a primer set for Marker 1 of SEQ ID NOs: 1 to 3 and a primer set for Marker 2 of SEQ ID NOs: 4 to 6, MS-HRM analysis was performed on uMSRE-treated and sulfite-converted DNA in substantially the same manner as in (3) of Example 1. Among the MS-HRM analysis results, a Methylation Peak representing the methylation level was derived, and the level of the Peak according to each Methylation % was confirmed, and the statistical significance of the difference in analytical sensitivity according to the presence or absence of uMSRE was confirmed.

[0071] Figure 2 shows the results of confirming the analytical sensitivity of each methylation % according to the presence or absence of uMSRE treatment at the PCR input level of 1 ng. Based on the results, the case where uMSRE was treated (Tx) showed a higher methylation peak at 1.6, 6.3, and 25% methylation than the case where it was not treated (UTx), and in particular, the sensitivity was further improved by uMSRE treatment in biological samples containing 6.3% methylated DNA (methylated DNA concentration 0.0063 ng / μL) or 1.6% methylated DNA (methylated DNA concentration 0.0016 ng / μL) than in biological samples containing 25% methylated DNA (methylated DNA concentration 0.025 ng / μL). This means that the analytical sensitivity of MS-HRM is improved by uMSRE treatment, and this effect is further maximized in biological samples containing low concentrations of methylated DNA.

[0072]

[0073] Example 3. Improved detection stability by pretreatment with a nonmethylation-sensitive restriction enzyme.

[0074] Among the Huma Methylated & Non-methylated DNA Set from Zymo Research, methylated DNA was diluted 10X using NFW (nuclease-free water), and methylated DNA was quantified using the Qubit 1X dsDNA High Sensitivity Kit. Based on the quantitative value of the 10X diluted methylated DNA, NFW was used to dilute the methylated DNA concentration to 2.4 ng / μL. Then, 0, 5, 10, 20, and 40 ng of Roche's Human Genomic DNA (non-methylated DNA) and 2.4 ng of methylated DNA were mixed to prepare a total volume of 50 μL. Samples that underwent uMSRE pretreatment were prepared according to Table 6, and samples that were not underwent uMSRE pretreatment were prepared according to Table 7. Each sample that was or was not pretreated with uMSRE was used directly for sulfite conversion. Sulfurous acid conversion was performed in substantially the same manner as (2) of Example 1, and stored at -20°C until performing MS-HRM experiments.

[0075] ReagentConc. Vol. (μL)FinalAciⅠ10 U / μL0.55 UrCutSmartBuffer10X61XNFW-3.5-Sample0.1 ng / μL505 ngTotal-60-

[0076] ReagentConc. Vol. (μL)FinalAciⅠ---rCutSmartBuffer10X61XNFW-4-Sample0.1 ng / μL505 ngTotal-60-

[0077] Marker 1 and Marker 2 described in Table 1 were used as nucleic acids for confirming the methylation status. MS-HRM analysis was performed on samples that had been completely treated with sulfurous acid using a primer set for Marker 1 of SEQ ID NOs: 1 to 3 and a primer set for Marker 2 of SEQ ID NOs: 4 to 6 in substantially the same manner as (3) of Example 1. Among the results of MS-HRM analysis, the Methylation Ratio value according to the presence or absence of uMSRE treatment was analyzed to confirm changes according to Roche gDNA (Background or Contamination Level).

[0078] As shown in Fig. 3, in samples not treated with uMSRE, the same level of Methylation Ratio was not confirmed depending on the background or gDNA contamination of the sample, and the Methylation Ratio decreased, resulting in a decrease in analytical sensitivity. On the other hand, in samples treated with uMSRE, it was statistically confirmed that no change in Methylation according to the background or gDNA contamination of the sample was observed. Therefore, uMSRE pretreatment had the effect of reducing the influence of noise present in the sample and amplifying the detection stability of the Methylated Template.

[0079]

[0080] Example 4. Diagnostic specificity verification

[0081] Blood collected from patients at high risk for liver cancer was centrifuged at 1,600 g for 10 minutes, and only the plasma present in the supernatant was separated. LepiMag was used to separate the plasma. TMUsing a Blood Cell-Free DNA Extraction Kit, cfDNA was extracted according to the manufacturer's protocol. For clinical samples, the entire extracted cfDNA was used in the next step without quantification. The extracted samples were subjected to sulfite conversion directly, with or without uMSRE pretreatment. Samples subjected to uMSRE pretreatment were prepared according to Table 8, and samples without uMSRE pretreatment were prepared according to Table 9. Sulfite conversion was performed in substantially the same manner as in (2) of Example 1, and MS-HRM analysis was performed in substantially the same manner as in (3) of Example 1. The distribution of the methylation ratio according to the presence or absence of uMSRE treatment was confirmed, verifying that clinical specificity was not reduced by uMSRE treatment.

[0082] ReagentConc. Vol. (μL)FinalAciⅠ10 U / μL0.55 UrCutSmartBuffer10X61XNFW-3.5-Sample-50-Total-60-

[0083] ReagentConc. Vol. (μL)FinalAciⅠ---rCutSmartBuffer10X61XNFW-4-Sample-50-Total-60-

[0084] As shown in Figure 4, the frequency of false-positive results in the high-risk group for liver cancer was examined according to the presence or absence of uMSRE treatment. The false-positive frequency did not significantly change with uMSRE treatment. Therefore, we verified that the potential problem of decreased clinical specificity, which could arise due to increased methylated DNA amplification efficiency with uMSRE treatment, did not occur.

[0085]

[0086] Example 5. Effect of improved sensitivity in liquid biopsy specimens

[0087] Blood collected from liver cancer patients was centrifuged at 1,600 g for 10 minutes, and only the plasma present in the supernatant was separated. LepiMag was used to isolate the separated plasma. TM Using a Blood Cell-Free DNA Extraction Kit, cfDNA was extracted according to the manufacturer's protocol. For clinical samples, the entire extracted cfDNA was used in the next step without quantification. The extracted samples were subjected to sulfite conversion directly, with or without uMSRE pretreatment. Samples subjected to uMSRE pretreatment were prepared according to Table 8, and samples without uMSRE pretreatment were prepared according to Table 9. Sulfite conversion was performed in substantially the same manner as in Example 3, and MS-HRM analysis was performed in substantially the same manner as (3) of Example 1 using the two primer sets described in Table 2. The distribution of the methylation ratio according to the presence or absence of uMSRE treatment was confirmed, confirming the improvement in clinical sensitivity according to uMSRE treatment.

[0088] As shown in Fig. 5, in the cfDNA of liver cancer patients who did not receive uMSRE treatment, methylated DNA amplification was almost nonexistent, showing a low methylation ratio. On the other hand, in the cfDNA of liver cancer patients who received uMSRE treatment, methylated DNA amplification was more effective, showing a tendency to frequently show a high methylation ratio. Therefore, the beneficial effect of achieving higher clinical sensitivity was achieved by uMSRE treatment.

Claims

1. A step of treating a biological sample with an unmethylation-sensitive restriction enzyme; and Comprising a step of performing MS-HRM (methylation-sensitive high-resolution melting) analysis, A method for determining the methylation status of nucleic acids.

2. In paragraph 1, A method for treating the biological sample with 0.025 Unit (U) or more of the non-methylation-sensitive restriction enzyme.

3. In paragraph 1, The biological sample comprises a nucleic acid comprising a target region, The above MS-HRM analysis, A step of differently modifying a nucleic acid in which the target region is methylated and a nucleic acid in which the target region is unmethylated; A step of amplifying the above nucleic acid; A step of obtaining a melt curve of the above biological sample; Comparing the melting curve of the biological sample with the melting curve of a standard methylated sample; and A method comprising the step of semi-quantitatively quantifying the methylation level of the nucleic acid.

4. In paragraph 1, A method wherein the above non-methylation-sensitive restriction enzyme specifically recognizes and cuts nucleic acids in which the target region is non-methylated among the nucleic acids contained in the biological sample.

5. In paragraph 1, The method according to claim 1, wherein the non-methylation-sensitive restriction enzyme is at least one selected from the group consisting of AtaII, AciI, AclI, AfeI, AgeI, AsiSI, AvaI, BceAI, BmgBI, BsaAI, BsaHI, BsiEI, BsiWI, BspDI, BssHII, BstBI, BstUI, ClaI, EagI, Esp3I, FauI, FseI, FspI, HaeII, HgalI, HhaI, HinP1I, HpaII, Hpy99I, HpyCH4IV, NaeI, NarI, NgoMIV, NotI, NruI, PaeR7I, PluTI, Pm1I, PvuI, RsrII, SacII, Sa1I, SfoI, SgrAI, SmaI, SnaBI, SrfI, TspMI, and ZraI.

6. In paragraph 1, A method wherein the biological sample is at least one selected from the group consisting of blood, plasma, tissue, cells, feces, and urine.

7. In paragraph 1, A method wherein the biological sample contains 100 ng / μL or less of a nucleic acid having a methylated target site.

8. In paragraph 1, A method wherein the biological sample contains 0.01 ng / μL or less of a methylated nucleic acid at the target site.

9. In paragraph 1, A method wherein the nucleic acid comprises a biomarker for cancer diagnosis.

10. In paragraph 9, A method wherein the cancer is at least one selected from the group consisting of liver cancer, colon cancer, esophageal cancer, stomach cancer, rectal cancer, colorectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, non-small cell lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, biliary tract cancer, bone cancer, connective tissue cancer, skin cancer, melanoma, brain cancer, head and neck cancer, thyroid cancer, leukemia, Hodgkin's disease, lymphoma, urinary tract cancer, and multiple myeloma.

11. In paragraph 9, The above cancer diagnostic biomarker is in a methylated state in cancer patients.

12. A step of treating a biological sample with an unmethylation-sensitive restriction enzyme; and Comprising a step of performing MS-HRM (methylation-sensitive high-resolution melting) analysis, A method to improve the sensitivity of MS-HRM analysis.

13. A step of treating a biological sample with an unmethylation-sensitive restriction enzyme; Step of performing MS-HRM (methylation-sensitive high-resolution melting) analysis; and Comprising a step of checking the methylation status of a cancer diagnostic biomarker contained in the above biological sample, A method for providing information for predicting the risk of developing cancer.

14. In paragraph 13, A method further comprising a step of comparing the methylation status of the above cancer diagnostic biomarker with the methylation status of a normal control group.

15. In paragraph 14, A method for providing information that a risk of cancer development exists when the methylation status of the above cancer diagnostic biomarker is higher than the methylation level of a normal control group.

16. Unmethylation-sensitive restriction enzyme; and Including MS-HRM (methylation-sensitive high-resolution melting) kit, Kit for checking the methylation status of nucleic acids.

17. In paragraph 16, The above MS-HRM kit, An agent that differently modifies a nucleic acid whose target site is methylated and a nucleic acid whose target site is unmethylated; and A kit comprising a primer set for amplifying the above nucleic acid.

18. In paragraph 16, A kit wherein the agent that differently modifies the nucleic acid having a methylated target site and the nucleic acid having an unmethylated target site is at least one selected from the group consisting of sulfurous acid, bisulfite, hydrogen sulfite, and disulfite.

19. In paragraph 16, A kit wherein the above non-methylation-sensitive restriction enzyme is treated in a biological sample at a concentration of 0.025 Unit (U) or more.

20. Unmethylation-sensitive restriction enzyme; and Includes MS-HRM (methylation-sensitive high-resolution melting) kit, The above MS-HRM kit is for checking the methylation status of biomarkers for cancer diagnosis. Cancer diagnostic kit.

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