DNA methylation cytosine detection method and detection kit

Abstract

This invention provides a method and kit for detecting DNA methylated cytosine. The method combines an oxidative enzyme and a glycosylation enzyme. First, methylated cytosine is oxidized to 5-hydroxymethylcytosine (5-hmC), and then further oxidized to 5-formylcytosine (5-fC) and 5-carboxycytosine (5-caC). The glycosylation enzyme then labels the oxidized cytosine with glucose, or removes 5-fC and 5-caC from the DNA double strand, generating apurinic / apyrimidinic or abasic site (AP site). The presence of this AP site indicates the presence of methylated cytosine at that location. The detection and localization of methylated cytosine are achieved by comparing amplification efficiency or by processing the AP site to create a 3'-OH, 3'-aldehyde, and 5'-phosphate group fragment, or a fragmented target fragment, after further processing.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology and provides a method and kit for detecting DNA methylated cytosine. Background Technology

[0002] DNA methylation is an epigenetic mechanism in which DNA methyltransferases add a methyl group at the C-5 position of cytosine in dinucleotides, causing transcriptional activation or repression of different genes and regulating cellular function. DNA methylation is a form of DNA epigenetic modification that can alter genetic expression without changing the DNA sequence, thus regulating gene expression. The DNA sequences before and after the gene promoter contain positive and negative regulatory units for gene expression. Generally, methylation of functional unit sequences inhibits the function of the original functional unit, but can promote tumors. Genes that play an important role in tumor development include oncogenes (promoting tumor cell growth), tumor suppressor genes (inhibiting tumor cell growth), growth-promoting genes, and immune-regulating genes. Gene methylation detection is increasingly used for the early diagnosis of tumors. Generally, its specificity and sensitivity are significantly higher than protein markers, allowing for earlier detection of tumors. DNA methylation targets are increasingly being used to screen, diagnose, and monitor the progression and prognosis of tumors and other diseases.

[0003] Currently, there are several methods for detecting methylated cytosine.

[0004] The first method is the bisulfite conversion method: By treating DNA with bisulfite, unmethylated cytosine is converted to uracil, while methylated cytosine remains unchanged, thus distinguishing methylated DNA from unmethylated DNA. Then, PCR, NGS, and other methods are used to analyze which methylation sites in the DNA sequence have been methylated. Therefore, this technology helps researchers differentiate between methylated and unmethylated cytosine, providing single-nucleotide resolution information for DNA methylation regions.

[0005] The second method is enzymatic transformation: Methylated cytosine is glycosylated using TET and T4-BGT enzymes. Unmethylated cytosine is then deaminated by APOBEC enzyme to become uracil (U), which is recognized as thymine (T) during PCR or sequencing. Glycosylated cytosine remains unaffected (retaining its C value). This difference distinguishes between methylated and unmethylated cytosine in subsequent sequencing detection. The results are then detected by sequencing and PCR.

[0006] The third method involves directly detecting methylation of cytosine in the sequence using third-generation sequencing technology—nanopore sequencing. A DNA strand passes through a nanopore driven by motor proteins or voltage. Each base (A, T, C, G) characteristically blocks the ionic current as it passes through, generating a corresponding current signal. When a cytosine with a methyl group (-CH3) (5mC) passes through the nanopore, this extra methyl group interferes with the ionic current in a different way, producing a current signal distinctly different from that of unmodified cytosine (C).

[0007] The fourth method involves detecting the presence of methylated cytosine with the assistance of endonucleases sensitive to methylated cytosine. These endonucleases have sequence-specific cleavage sites. If the cytosine C at the cleavage site is methylated, it affects the endonuclease's function, preventing complete cleavage and keeping the DNA double strand continuous at that position. Conversely, if the C at that position is not methylated, the endonuclease will cleave the site, causing a break in the DNA double strand, which cannot be further amplified by PCR.

[0008] However, the above method also has obvious drawbacks, as follows:

[0009] Regarding the first method, the sulfite conversion method for detecting DNA methylation modification, its most significant drawback is that sulfite treatment extensively damages DNA, reportedly destroying over 80% of it. This significantly reduces the sensitivity and accuracy of the detection, especially with limited sample availability. Furthermore, standard sulfite treatment is time-consuming, typically requiring 3-4 hours, while rapid conversion kits also take 1.5 hours. Considering the sulfite conversion process, nucleic acid extraction, and PCR amplification, methylation detection results generally take at least until the second business day. Sequencing based on sulfite conversion typically yields results after 3 days.

[0010] The second enzymatic transformation method has the advantage of being relatively mild and causing less damage to the sample. However, it also has significant disadvantages. First, the enzymatic process is relatively slow, requiring one working day for sample preparation alone, and even longer when combined with sequencing and other detection processes. Second, the combination of enzymatic transformation and sequencing increases the detection cost, and the sequencing information requires bioinformatics analysis, which places certain demands on computing power, further increasing the detection cost.

[0011] Regarding the third method, which directly uses third-generation sequencing technology—nanopore sequencing—nanopore sequencing directly reads natural DNA, avoiding DNA degradation and sequencing bias caused by pretreatment. It also offers longer read lengths and allows for real-time analysis. However, nanopore sequencing also has significant drawbacks, such as a higher error rate, requiring improvements in accuracy and precision, and placing high demands on sample size, bioinformatics analysis capabilities, and computational power.

[0012] Regarding the fourth method, the detection of methylated cytosine is performed using endonucleases sensitive to methylated cytosine. The advantage of this method is its simplicity and convenience, but its disadvantages are significant: firstly, the location of the restriction enzyme site can have a substantial impact on subsequent detection, potentially even preventing detection altogether; secondly, for an unknown quantity of DNA sample, even if the amount of endonuclease used is sufficient, the digestion time is adequate, and the activity of the endonuclease is normal, it still cannot guarantee that all sites will be completely recognized and cleaved by the endonuclease. If unrecognized and untreated non-methylated cytosine sites are missed during the digestion reaction (sites that should be cleaved are not), the subsequent PCR reaction has a huge amplification effect, easily leading to false positive results. Guaranteeing 100% complete cleavage is unreliable in reality, making this defect fatal. Conversely, if the added enzyme is unstable, or if it becomes insensitive to methylated C and cleaves methylated sites, false negative results may also occur, which is equally disastrous. Summary of the Invention

[0013] This invention addresses the aforementioned problems by providing a novel method for detecting DNA cytosine methylation, aiming to overcome the technical shortcomings of existing methods, such as long detection cycles and high error rates. This method not only labels methylated cytosine through enzymatic action but also locates the methylated cytosine sites (determining their position on the DNA sequence) by recognizing DNA sequence characteristics. Combined with specific detection techniques, this technology achieves single-nucleotide resolution and allows for simultaneous detection of multiple genes and multiple sites (achieving high-throughput detection).

[0014] The core technology for detecting DNA methylated cytosine in this invention is as follows: An oxidative enzyme and a glycosylation enzyme are used in combination. First, methylated cytosine is oxidized to 5-hydroxymethylcytosine (5-hmC), and then further oxidized to 5-formylcytosine (5-fC) and 5-carboxycytosine (5-caC). Then, the glycosylation enzyme labels glucose onto the oxidized cytosine, or removes 5-fC and 5-caC from the DNA double strand, generating apurinic / apyrimidinic or abasic site (AP site). The presence of this AP site indicates the presence of methylated cytosine at that location; therefore, labeling and detecting this AP site is equivalent to labeling and detecting methylated cytosine. Thus, the presence of this type of AP site is a crucial structural basis for subsequent detection.

[0015] Among them, the oxidase for methylated cytosine is selected from TET isoenzymes, including any one of TET1 or TET1-CD, TET2 or TET2-CD, TET3 or TET3-CD, TET-S, NgTET or NgTET-CD, and CciTET.

[0016] Glycosylation tool enzymes include β-glucosyltransferase, TDG, UNG or UDG, SMUG1 or MBD4. Among them, β-glucosyltransferase is used to label glucose onto methylated cytosine; TDG, UNG or UDG, SMUG1 or MBD4 are tool enzymes that can cleave oxidized or modified bases and form AP sites at the methylated cytosine sites.

[0017] Based on the generated AP sites, this invention provides two approaches to the detection of methylated cytosine:

[0018] Method 1: Based on the significant structural differences between 5-fC, 5-caC, oxidized glycosyl-labeled cytosine, and AP sites and the original or methylated cytosine, a high-fidelity polymerase amplification experiment is used to compare whether the amplification is successful or whether there are significant changes in amplification efficiency, thus intuitively determining whether there is methylation modification at the relevant sites.

[0019] Method 2: The AP site is cut or physically treated to generate a 3'-OH, a 3'-aldehyde group, and a 5'-phosphate group. Based on these breaks or the resulting fragments, further processing is performed before detection.

[0020] Preferably, in Method 1, different samples are compared using high-fidelity polymerase amplification experiments depending on the glycosylation enzyme used, resulting in two detection methods:

[0021] Method 1-1: Using TET (Ten-eleven translocation enzyme), methylated cytosine is oxidized by TET under the action of ferrous ions, α-ketoglutarate, and oxygen. The methylated C is first converted to 5-hydroxymethylcytosine (5-hmC), then further oxidized to 5-formylcytosine (5-fC), and finally to 5-carboxycytosine (5-caC). After the methyl group on the DNA is oxidized by TET, glucose is labeled onto the methylated cytosine by T4 phage β-glucosyltransferase. The products of the oxidation of the methylated cytosine, 5-fC and 5-caC, as well as the cytosine labeled with a glycosyl group after oxidation, have undergone significant structural changes compared to the original cytosine or the methylated cytosine. When using high-fidelity polymerases (such as enzymes with 3'-5' exonuclease calibration functions) such as Pfu, vent, Q5, and Phusion for PCR amplification or other amplification experiments, the amplification efficiency will be greatly reduced or even impossible. Therefore, by selecting and applying high-fidelity polymerases to amplify fragments containing methylated cytosine oxidation products and glycosylation modification products, and comparing the amplification efficiency with control samples (samples without methylation sites), it is possible to determine whether methylation modification exists at the relevant sites.

[0022] Method 1-2 involves oxidizing methylated cytosine with TET enzyme, followed by removing 5-fC and 5-caC from the DNA double strand using TDG enzyme (or other potentially effective glycosylation enzymes, including UNG, SMUG1, MBD4, etc.), resulting in apurinine / pyrimidine-free AP sites. After the original methylated cytosine sites are transformed into AP sites, amplification efficiency is significantly reduced or even impossible when using high-fidelity polymerase for PCR or other amplification experiments. Therefore, based on the TET oxidation effect and corresponding labeling effect, high-fidelity polymerase amplification experiments can be used to determine whether methylation modification exists at the relevant sites by comparing the success of amplification or significant changes in amplification efficiency.

[0023] Preferably, in method two, the AP site is cleaved or treated under physical conditions, mainly producing a 3'-OH, a 3'-aldehyde group or a 5'-phosphate group, as well as denatured and dissociated sequence fragments.

[0024] The methods for generating a 3'-OH group, a 3'-aldehyde group, or a 5'-phosphate group are as follows:

[0025] (a) Using APE1 enzyme to cut the DNA strand at the AP site: APE1 enzyme will cut the phosphodiester bond at the 5' end to the left of the AP site, causing a single-strand break in the DNA. -OH will be generated at the 3' end at the break, and a deoxyribose phosphate group will be generated at the 5' end. The latter can be further cleaved by DNA polymerase β to produce a 5' phosphate group.

[0026] Enzymes such as APE1 (Apurinic / apyrimidinic endonuclease 1) cleave the DNA strand at the AP site. The APE1 enzyme will cut the phosphodiester bond to the left of the AP site (5' end), causing a single-strand break in the DNA. A -OH group will be generated at the 3' end at the break, and a deoxyribose phosphate group will be generated at the 5' end. The latter can be further cleaved by DNA polymerase β and other enzymes to produce a 5' phosphate group.

[0027] The -OH at the 3' end can be joined by using tools such as TdT (terminal deoxynucleotidyl transferase) or nick transferase (E. coli DNA polymerase I or its large fragment Klenow fragment) to polymerize labeled or unlabeled nucleotides at the -OH position at the 3' end, thus attaching the relevant nucleotides.

[0028] (b) The AP site is cleaved using a tool enzyme with β-elimination reaction or β,δ-elimination effect to generate a break with a 3' aldehyde group.

[0029] The tool enzymes with β-elimination effects are selected from NTH1 and T4 PDG; the tool enzymes with β,δ-elimination effects are selected from OGG1, Fpg, NEIL1, NEIL2, and NEIL3. Among them, T4 PDG, OGG1, Fpg, NTH1, NEIL1, NEIL2, and NEIL3 are also bifunctional glycosylation enzymes.

[0030] The 3' end of the β-elimination group has an unsaturated aldehyde group, while the 5' end produces a phosphate-containing terminus. Additionally, the 3' end of the β-elimination group can be pruned by APE1 to produce a -OH group at the 3' end. Conversely, the 3' end of the β,δ-elimination group can be pruned by PNKP enzyme, producing a -OH group at the 3' end and a phosphate-containing terminus at the 5' end.

[0031] Therefore, the structures generated at the 3' and 5' ends after the AP site is cleaved by relevant enzyme tools can serve as characteristic structures for subsequent labeling and detection. These groups that can be used for labeling and detection are mainly divided into three types: 3'-OH, 3'-aldehyde, and 5'-phosphate groups.

[0032] Furthermore, based on the generated 3'-OH, 3'-aldehyde, or 5'-phosphate groups, as well as the free fragments, several methods for detecting methylated cytosine have been integrated:

[0033] (1) The -OH at the 3' end is captured by TdT enzyme or nick transferase (E. coli DNA polymerase I or its large fragment Klenow fragment) through sequence complementary pairing or by the capture structure or molecule of the labeled nucleotide. The methylated cytosine is detected by directly detecting the labeling signal of the labeled nucleotide, or by detecting whether complementary pairing is achieved, or by detecting the labeling signal on the paired nucleotide chain.

[0034] Specifically, depending on the functional characteristics of different enzyme tools, various types of nucleotides are attached: these can be deoxyribonucleotides, dideoxyribonucleotides, ribonucleotides, BrdU and EdU, or even artificially synthesized nucleotide analogs. The attached nucleotides can be labeled in various ways, including fluorescent labeling, biotin labeling, digoxigenin labeling, and bromine labeling. For example, FITC-dUTP can be generated by adding polyFITC-dUTP to the end of TdT. FITC emits fluorescence upon excitation, which can be detected using equipment capable of detecting fluorescence.

[0035] Labeled nucleotides can be detected by fluorescence, or by subsequent reactions and detection using anti-digoxigenin antibodies, anti-BrdU antibodies, and azides. If nucleotides are biotin-labeled, they can bind efficiently to labeled avidin (or streptavidin). Labeled avidin may be fluorescent, a pigment, luciferase, an enzyme promoting chemiluminescence, or a substance that causes changes in electrical properties, Raman scattering, magnetic or magnetoresistance properties, or other factors that alter physicochemical properties. Detection can then be performed using fluorescence, chemiluminescence, electrical signals, magnetic signals, Raman scattering, or other similar instruments. The labeled or modified nucleotides can be a single type of nucleotide or a mixture of multiple nucleotides.

[0036] Furthermore, due to the high density of methylated cytosine in the promoter regions of certain important genes, numerous nicks are generated in these regions. If a TdT enzyme is used, a template is not required; the TdT enzyme forms new fragments at the nick sites. These newly generated fragments, or individual nucleotides, can be configured in length, nucleotide type, and labeling method according to different reaction conditions. The newly generated fragments may contain capture or detection markers, and the upstream sequence of the newly generated fragment—that is, the upstream sequence information in the 5' direction of the AP site nick—can serve as localization information, locating the position of the AP site on a specific gene. This sequence can be detected through sequence complementation.

[0037] Besides passive complementation capture, it can also be captured by protein complexes containing nucleic acid fragments with complementary sequences. Examples include dCas9 bound to gRNA (complementary to the target fragment, whose sequence information can be used for localization on related genes), or Argonaute proteins bound to guiding DNA (complementary to the target fragment) that have lost their enzymatic activity. These complementary sequences, or protein complexes bound to nucleic acids with complementary sequences, can be fixed at a specific location by gRNA, guiding DNA, or complementary fragments, or they may not be fixed. The target fragment contains labeled or modified nucleotides, which can be detected and analyzed by relevant detection systems. For example, biotin (or digoxigenin, or fluorescein, or other groups, etc.) labeled fragments can bind to avidin-labeled markers (for digoxigenin, the binding can be a digoxigenin antibody). The marker itself can be an enzyme (catalyzing chemiluminescence, fluorescence, or pigment chromatography, etc.), or a characteristic that can be successfully detected, such as superparamagnetic nanoparticles (detectable by magnetoresistive sensors), Raman tags (detectable by Raman scattering), methylene blue (detectable by electrochemical methods, etc.), quantum dot labeling (detectable by laser or fluorescence, etc.). The marker itself has signal release and / or amplification effects.

[0038] (2) As a special method of linking nucleotides, nick transferases, mainly E. coli DNA polymerase I or its large fragment Klenow fragment, are used to extend free nucleotides from the -OH at the nick site towards the 3' end, using the complementary strand as a template, until the next AP site is reached. The nucleotide modifications that can be added to E. coli DNA polymerase I or its large fragment Klenow fragment can be BrdU, fluorescent groups, or other functional groups. The newly synthesized fragments using E. coli DNA polymerase I or its large fragment Klenow fragment have corresponding sequence information (which can be used for location determination, etc.), and can also be captured or detected by the nucleotide characteristics on the newly synthesized fragments (for example, nucleotides with fluorescent groups can be detected by fluorescence; other functional group labeling methods can be used for detection or capture using corresponding techniques).

[0039] (3) Based on the generation of the aldehyde-containing end, the aldehyde reactive probe (ARP) labeled with the labeling molecule is used to bind to the site, and then the signal is amplified by the labeling molecule-binding molecule system to realize the detection and localization of methylated cytosine.

[0040] Preferably, an acylhydrazide probe is selected for the reaction. This probe can also be labeled with biotin or the like, and then the signal is amplified through the biotin-avidin system.

[0041] (4) Based on the generation of a 5' phosphate group, detection can be performed in the following two ways: (4-1) After the reaction of carbodiimide with the 5' phosphate group, a phosphoramide bond is formed with a substance containing a primary amine. Based on the biomarkers (such as dye markers, biotin, or certain enzymes, such as peroxidase) on these substances containing primary amino groups, DNA fragments can be captured or detected by fluorescence, biotin-avidin systems, enzyme reaction systems, etc.; (4-2) The target gene is cut with TtArgonaute and guiding DNA, and the resulting new fragment is used as new guiding DNA to guide TtArgonaute to cut the fluorescent probe, while detecting changes in fluorescence signal.

[0042] Preferably, when using the Argonaute system for detection, the DNA sample to be tested is first treated with alkaline phosphatase at 37°C for 20 min, then inactivated at 65°C for 15 min. The treated sample is then treated with a combination of oxidative enzyme, glycosylation enzyme, and AP site cleavage enzyme at 37°C for 1 h to produce a break with a 5' phosphate group.

[0043] Argonaute proteins include Pf Argonaute, Tt Argonaute, Ttd Argonaute, and Tc Argonaute proteins. These Ago proteins all rely on single-stranded DNA with a phosphate group at the 5' end as guiding DNA, and other similar AGOs also require this.

[0044] (5) The method for detecting methylated cytosine based on the generated fragmented target fragment is as follows: the target fragment is captured by using a base complementary DNA sequence or a nucleic acid protein complex containing a complementary sequence nucleic acid fragment through base complementary pairing. Based on the biomarker modification of the complementary sequence or the change in electrical properties brought about by the binding of the target fragment and the captured fragment, the detection and localization of methylated cytosine is achieved.

[0045] Different enzymes can be used to cleave AP sites, resulting in different sequence fragments. Target sequences between adjacent AP sites will denature and dissociate under suitable conditions (physical, chemical, or biological methods), binding to the capture sequence via base complementation or the aforementioned nucleic acid-protein complex method. The detached target fragment (whose sequence information can be used for gene localization) can be bound to or captured by a base-complementary DNA sequence. After the target fragment is captured, the corresponding complementary fragment, or the bound gRNA, guiding DNA, or even dCas and Argonuate proteins, can carry characteristic modifications, such as luciferase, enzyme-catalyzed reactions, vitamins, digoxigenin, etc. These modifications can be used as detection targets.

[0046] Alternatively, other detection methods can be used. For example, the binding of the target fragment and the captured fragment itself can cause changes in electrical properties, which can be detected using electrochemical or field-effect transistor devices. Sequencing can also be used to analyze the sequence and determine the presence of AP sites. Sequencing methods include first-generation sequencing, second-generation sequencing, and third-generation sequencing. Sequence information can indicate the location of the cleavage site, which in turn indicates the location of methylated cytosine.

[0047] Furthermore, the DNA methylation cytosine detection method provided by this invention also includes a pretreatment step for the test sample: before detection, the 3'-OH of the sample is blocked with dideoxynucleotides (such as ddUTP) or cordycepin triphosphate, or the 3'-OH is modified by thiolation. Since other nucleotides cannot be added, the non-specific detection signal caused by the already existing AP site and the 3'-OH at the DNA end is avoided.

[0048] In addition, to accurately locate the sequence containing the methylation site, the sample can be pre-processed by enzyme digestion or mechanical fragmentation, or by introducing a CRISPR or Argonuate system. Guiding RNA or guiding DNA can be designed to accurately locate the sequence position to be examined before cutting the DNA.

[0049] Based on the above-mentioned method for detecting methylated cytosine, in another aspect, the present invention provides a DNA methylated cytosine detection kit, comprising the following reagents: 3'-OH blocking reagent, DNA pre-cutting reagent, methylated cytosine oxidation reagent, post-processing reagent, high-fidelity polymerase amplification reagent, AP site cutting reagent, and methylated cytosine detection reagent.

[0050] The 3'-OH blocking reagent is selected from dideoxynucleotides, cordycepin triphosphate, or thiol modification reagents.

[0051] DNA pre-cleavage reagents are selected from restriction endonucleases or guiding RNA or guiding DNA;

[0052] Methylation cytosine oxidizing agents include TET enzymes, ferrous ions, and α-ketoglutarate;

[0053] Post-treatment reagents are selected from β-glucosyltransferase, TDG, UNG or UDG, SMUG1 or MBD4;

[0054] The high-fidelity polymerase amplification reagent is selected from amplification reagents containing VENT enzyme;

[0055] AP site cleavage reagents are selected from APE1 enzyme, β-elimination reaction or β,δ-elimination effect tool enzymes and DNA polymerase β;

[0056] The methylated cytosine detection reagent is selected from any one of TdT enzyme, nick transferase, aldehyde reactive probe (ARP), or carbodiimide reagent.

[0057] This kit can be used directly for the detection of methylated cytosine in samples.

[0058] In a third aspect, this invention provides the application of the aforementioned DNA methylation cytosine detection method in the detection of DNA methylation in tumor samples. In a specific embodiment of this invention, a liver cancer sample is used as an example to illustrate the detection process, but the method is also applicable to the methylation detection of other solid tumor samples such as gastric cancer, breast cancer, colorectal cancer, pancreatic cancer, lung cancer, and cervical cancer, as well as normal tissues.

[0059] The role and effect of invention

[0060] (1) Regarding the detection time, this invention uses a combination of TET enzyme and tool enzyme that generates AP sites for sample enzymatic treatment and combined with marker detection. Since TET enzyme is not the main factor, the enzyme treatment time is 1 hour. With the addition of detection time, experimental results can be obtained within 2 hours. Compared with the existing enzyme method dominated by TET enzyme, which requires more than 24 hours to obtain sequencing data, the detection time is greatly saved.

[0061] (2) Regarding sample quantity requirements, the technology of this invention has significant advantages over PCR or sequencing technologies after sulfite conversion: sulfite conversion of DNA requires treatment at high temperatures in an acidic environment, which can severely damage DNA samples and lead to serious DNA fragmentation; while the technology of this invention can preserve long DNA fragments with very little DNA damage. The detection method of this invention, when performing PCR amplification using high-fidelity polymerase, requires approximately 80% less sample quantity compared to methylation-specific PCR technology using sulfite treatment. Based on the AP site cleavage and labeling detection technology of this invention, new detection technologies can be integrated, achieving near-single-molecule detection technology, and minimizing the sample quantity requirements.

[0062] (3) In terms of accuracy, the basic principle of methylation-specific PCR technology is ARMS detection technology. ARMS technology relies on the strict pairing standard of polymerase at the 3' end of DNA, but it is not absolutely reliable in actual experiments and non-specific amplification can still occur. The detection technology based on AP site cutting and labeling in this invention avoids the shortcomings of ARMS technology.

[0063] (4) In the detection method of the present invention, after the AP site is cut, a 5' phosphate group that is not common in the ordinary system is generated. Carbodiimide can be used to react with the 5' phosphate to form a phosphoramide bond. Based on the biomarkers on these substances containing primary amino groups, DNA fragments can be captured or detected. At the same time, this phosphate group is exactly what Argonaute protein is required to recognize guiding DNA. Argonaute protein and guiding DNA are used to cut the target gene, and the generated new fragment is used as a new guiding DNA to guide Argonaute to cut the fluorescent probe. At the same time, the change in fluorescence signal can be detected, which can also realize the detection of methylated DNA. Attached Figure Description

[0064] Figure 1 The comparison shows the VENT enzyme amplification performance of TET-treated tumor tissue DNA and untreated tissue DNA;

[0065] Figure 2 The comparison of VENT enzyme amplification performance between TET+TDG treated tumor tissue DNA and untreated tissue DNA is shown.

[0066] Figure 3 The comparison of VENT enzyme amplification performance between tumor tissue DNA that was not treated with TET+TDG+APE1 and untreated tissue DNA is shown.

[0067] Figure 4 The results show a comparison of DNA detection in tumor tissues treated with TET+TDG+APE1 and labeled with methylated cytosine, DNA in tumor tissues treated with TET+TDG+APE1 but not labeled with methylated cytosine, and DNA from normal control tissue samples.

[0068] Figure 5 The dynamic changes in fluorescence catalyzed by peroxidase after methylation site labeling are shown.

[0069] Figure 6 The APE1 incision and the new fragment cut from Tt Argonaute are shown to guide the cutting fluorescent probe to generate a fluorescent signal. Detailed Implementation

[0070] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0071] The following examples use liver cancer samples to demonstrate the detection results of different detection methods, but the detection methods are also applicable to other samples; at the same time, the tool enzymes used in the examples are not limited to these, and tool enzymes with the same or similar functions are also applicable.

[0072] Example 1

[0073] The sample in this example was DNA extracted from clinical liver cancer samples at Renji Hospital, affiliated with Shanghai Jiao Tong University School of Medicine. After sulfite conversion, methylation at a certain site in the VIM gene was confirmed by methylation-specific PCR. The sample DNA was then treated with TET at 37°C for 1 hour, and the fragment was amplified using a pair of primers corresponding to the aforementioned methylation-specific PCR amplicons (the corresponding sequences were the original sequences without sulfite conversion) with the VENT enzyme (using Quantgene 9600). The results are as follows... Figure 1As shown, curve B represents tumor tissue DNA without TET treatment, exhibiting a typical amplification curve; curve A represents tumor tissue DNA samples treated with TET for 1 hour, showing no typical amplification curve. This suggests that after methylated cytosine is oxidized by TET enzymes, the DNA contains AP sites and nicks, preventing efficient amplification.

[0074] Example 2

[0075] This example uses DNA extracted from clinical liver cancer samples at Renji Hospital, affiliated with Shanghai Jiao Tong University School of Medicine. After sulfite conversion, methylation at a specific site in the VIM gene was confirmed by methylation-specific PCR. The sample DNA was first treated with TET+TDG enzyme at 37°C for 1 hour, and then the fragment was amplified using a pair of primers corresponding to the aforementioned methylation-specific PCR amplicons (the corresponding sequences were the original sequences without sulfite conversion) with the VENT enzyme (using Quantgene 9600). The results are as follows... Figure 1 Curve B represents tumor tissue DNA without TET+TDG treatment, showing a typical amplification curve; curve A represents tumor tissue DNA samples treated with TET+TDG for 1 hour, and no typical high-efficiency amplification curve appears. This suggests that after enzyme treatment, the DNA contains AP sites and nicks, preventing high-efficiency amplification.

[0076] Example 3

[0077] The sample in this example was DNA extracted from clinical liver cancer samples at Renji Hospital, affiliated with Shanghai Jiao Tong University School of Medicine. After sulfite conversion, methylation at a certain site of the VIM gene was confirmed by methylation-specific PCR. The sample DNA was first treated with TET+TDG+APE1 at 37℃ for 1 hour to cleave the AP site; then, a pair of primers corresponding to the above methylation-specific PCR amplicons (the corresponding sequences were the original sequences without sulfite conversion) were used to amplify the fragment with VENT enzyme (using Quantgene 9600).

[0078] The results are as follows Figure 3 As shown, curve A represents tumor tissue DNA that has not been treated with TET+TDG+APE1, exhibiting a typical amplification curve; curve B represents tumor tissue DNA samples treated with TET+TDG+APE1 for 1 hour, showing no typical amplification curve. This suggests that after enzyme treatment, the DNA contains AP sites and nicks, preventing amplification.

[0079] Example 4

[0080] The samples used in this example were DNA samples from hypermethylated and hypomethylated cell lines from Nanjing Kebai Biotechnology Co., Ltd. The sample DNA was first treated with TET+TDG+APE1 at 37℃ for 1 hour, then labeled with TdT enzyme and ThermoScientific luciferin-12-dUTP for 30 minutes. After labeling, fluorescence values ​​were detected using a qRI analyzer. The results are as follows: Figure 4 As shown in the figure, the black curve represents hypomethylated DNA samples, the green curve represents hypermethylated DNA that has not been treated with TET+TDG+APE1 and has not been labeled with methylated cytosine, and the red curve represents hypermethylated cellular DNA that has been treated with TET+TDG+APE1 and labeled with methylated cytosine. The fluorescence values ​​of the black and green curves tend to be consistent, while the fluorescence value of the red curve is significantly higher than that of the black and green curves, indicating that TdT is effectively linked to the fluorescein-labeled fluorescein-12-dUTP at the 3-OH position where the AP site is cleaved by APE1.

[0081] Example 5

[0082] The samples in this example were DNA extracted from hypermethylated cell line samples (C, D) and hypomethylated cell line samples (A, B) from Nanjing Kebai Biotechnology Co., Ltd. DNA from samples A and C was first treated with TET+TDG+APE1 at 37℃ for 1 h, followed by blocking the 3' end "-OH" with TdT enzyme and dd-ATP for 30 min. Then, samples A, B, C, and D were all treated with TdT enzyme and Biotin-ATP for 30 min, followed by reaction with peroxidase-labeled avidin for 30 min. After washing three times, hydrogen peroxide probe ADHP (10-acetyl-3,7-dihydroxyphenazine) and reaction solution were added, and fluorescence dynamics were immediately detected using a Quantgene 9600 ROX channel.

[0083] The results are as follows Figure 5 As shown, the fluorescence value of hypomethylated cellular DNA did not change much dynamically, regardless of whether the "-OH" at the 3' end of the DNA was blocked; the fluorescence value of hypermethylated cellular DNA did not change much dynamically after blocking, while the fluorescence value of unblocked DNA increased rapidly, indicating the presence of a large number of methylation sites.

[0084] Example 6

[0085] The sample in this example was DNA extracted from clinical liver cancer samples at Renji Hospital, affiliated with Shanghai Jiao Tong University School of Medicine. The sample DNA (200 ng) was first treated with alkaline phosphatase at 37°C for 20 min, then inactivated at 65°C for 15 min. After treatment, the sample was further treated with TET+TDG+APE1 at 37°C for 1 h. The target gene VIM was then cleaved using Tt Argonaute and guiding DNA. The resulting fragment served as the new guiding DNA to guide the Tt Argonaute cleavage of the fluorescent probe. Simultaneously, changes in fluorescence signal were detected using a Quantgene 9600 PCR instrument. The detection system is shown in Table 1.

[0086] The results are as follows Figure 6 As shown, curve 1 represents the sample without TET+TDG+APE1 treatment, which was directly detected using fluorescent probe P (0.2ul) + Guiding DNAv2 (0.5ul) + Buffer (4ul) + TTAgo (3ul), and no enhanced fluorescence signal was detected; curve 2 represents the sample treated with TET+TDG+APE1 first, and then detected using fluorescent probe P (0.2ul) + Guiding DNAv2 (0.5ul) + Buffer (4ul) + TTAgo (3ul), and the results showed a significantly enhanced fluorescence signal; curve 3 is the positive control, where the fluorescent probe was directly cleaved by TTAgo and guiding DNA.

[0087] The probe sequence is: 5'-FAM-CCTCGTAGAGGTCCCCCAGGCGCGACTT-BHQ1-3' (SEQ ID NO.1);

[0088] The VIM target sequence is: 5'-AAGGGCCAAGGCAAGTCmGCmGCCTGGGGGACCTCTA

[0089] CmGAGGAGGAGATG-3' (SEQ ID NO. 2);

[0090] In curves 1 and 2, the sequence of Guiding DNAv2 is: 5'-TCCTCCTCGTAGAGGT-3' (SEQ ID NO. 3);

[0091] The guiding DNA sequence in curve 3 is: 5'-GCCTGGGGGACCTCTA-3' (SEQ ID NO.4).

[0092] Table 1. Detection System of Argonaute System

[0093]

[0094] The undescribed parts of this invention are the same as or implemented using existing technology. The applicant declares that this invention is illustrated through the above specific embodiments, but the invention is not limited to the above detailed methods, i.e., it does not mean that the invention must rely on the above detailed methods to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product of this invention, additions of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

Claims

1. A method for detecting DNA methylated cytosine, characterized in that, Includes the following steps: A. Oxidation and post-treatment of methylated cytosine The combined application of oxidative and glycosylation enzymes first oxidizes methylated cytosine to 5-hydroxymethylcytosine (5-hmC), and then further oxidizes it to 5-formylcytosine (5-fC) and 5-carboxycytosine (5-caC). Then, the glycosylation enzyme labels glucose onto the oxidized cytosine, or removes 5-fC and 5-caC from the DNA double strand, producing apurinol and / or pyrimidine AP sites. B. Detection of methylated cytosine Use any of the following methods for detection: Method 1: Based on the significant structural differences between 5-fC, 5-caC, cytosine labeled with glycosyl groups after oxidation, and AP sites and the original or methylated cytosine, amplification experiments are conducted to compare whether the amplification is successful or whether there are significant changes in amplification efficiency, thereby determining whether methylation modification exists at the relevant sites. Method 2: The AP site is cut or physically treated to generate a 3'-OH, a 3'-aldehyde group, and a 5'-phosphate group. Based on these breaks or the resulting fragments, further processing is performed before detection.

2. The method for detecting DNA methylated cytosine according to claim 1, characterized in that, In step A, the oxidative tool enzyme is selected from TET isoenzymes, including any one of TET1 or TET1-CD, TET2 or TET2-CD, TET3 or TET3-CD, TET-S, NgTET or NgTET-CD, and CciTET. The glycosylation tool enzymes include β-glucosyltransferase, TDG, UNG or UDG, SMUG1 or MBD4. The combined application of oxidative enzymes and glycosylation enzymes resulted in a treatment time of 37℃ for 0.8–1.5 h.

3. The method for detecting DNA methylated cytosine according to claim 1, characterized in that, In step B, method one involves using a high-fidelity polymerase to amplify fragments containing methylated cytosine oxidation products and glycosylation-modified products, and comparing the amplification efficiency with a control sample (sample without methylation sites) to determine whether methylation modification exists at the relevant sites. Alternatively, after applying appropriate labeling effects to the AP sites, a high-fidelity polymerase amplification experiment can be used to compare whether the amplification was successful or whether there was a significant change in amplification efficiency, thus determining whether methylation modification exists at the relevant sites.

4. The method for detecting DNA methylated cytosine according to claim 1, characterized in that, In step B, method two, the method for generating a 3'-OH, a 3'-aldehyde, or a 5'-phosphate group after cleaving the AP site or undergoing physical treatment is as follows: (a) Cutting the DNA strand at the AP site using the APE1 enzyme: The APE1 enzyme will cut the DNA strand at the 5' end of the AP site. Ester bond cleavage causes DNA single-strand breaks. At the break, a -OH group will be generated at the 3' end and a deoxyribose phosphate group will be generated at the 5' end. The latter can be further cleaved by DNA polymerase β to produce a 5' phosphate group. (b) The AP site is cleaved using a tool enzyme with β-elimination reaction or β,δ-elimination effect to generate a break with a 3' aldehyde group.

5. The method for detecting DNA methylated cytosine according to claim 1, characterized in that, In step B, method two, the method for detecting methylated cytosine based on the generated 3'-OH, 3'-aldehyde, or 5'-phosphate group is selected from any of the following: (1) Based on the -OH at the 3' end, the newly linked nucleotide is captured by sequence complementary pairing or by the capture structure or molecule of the labeled nucleotide. The methylated cytosine is detected by directly detecting the labeling signal of the labeled nucleotide, or by detecting whether complementary pairing is achieved, or by detecting the labeling signal on the paired nucleotide chain. (2) Based on the generation of aldehyde-containing ends, aldehyde reactive probes (ARPs) labeled with labeled molecules are used to bind to the site, and then the signal is amplified through the labeled molecule-binding molecule system to realize the detection and localization of methylated cytosine; (3) Based on the generation of a 5' phosphate group, the following methods can be used for detection: (3-1) After the reaction of carbodiimide with the 5' phosphate group, a phosphoramide bond is formed with a substance containing a primary amine. Based on the biomarkers on these substances containing primary amino groups, the DNA fragment is captured or detected; (3-2) The target gene is cut using Argonaute protein and guiding DNA. The newly generated fragment is used as a new guiding DNA to guide the Argonaute cutting fluorescent probe, and the change in fluorescence signal is detected at the same time.

6. The method for detecting DNA methylated cytosine according to claim 5, characterized in that, in, In method (3-2), the DNA sample to be tested is first treated with alkaline phosphatase at 37°C for 20 min, then inactivated at 65°C for 15 min. The treated sample is then treated with a combination of oxidative enzymes, glycosylation enzymes, and AP site cleavage enzymes at 37°C for 1 h to produce a 5' phosphate group. Argonaute proteins include Pf Argonaute protein, Tt Argonaute protein, Ttd Argonaute protein, and Tc Argonaute protein.

7. The method for detecting DNA methylated cytosine according to claim 1, characterized in that, Step B In Method 2, the method for detecting methylated cytosine based on the generated fragmented target fragment is as follows: The method of complementary base pairing uses complementary DNA sequences or nucleic acid protein complexes containing complementary nucleic acid fragments to capture detached target fragments. Based on the biomarker modification of the complementary sequence or the change in electrical properties brought about by the binding of the target fragment and the captured fragment, the detection and localization of methylated cytosine can be achieved.

8. The method for detecting DNA methylated cytosine according to claim 1, characterized in that, Before testing, the 3'-OH of the sample to be tested is blocked with dideoxynucleotides or cordycepin triphosphate, or the 3'-OH is modified by thiolation to avoid the non-specific detection signal brought by the AP site and the 3'-OH at the end of the DNA. To accurately locate the sequence containing the methylation site, the sample can be pre-processed by enzyme digestion or mechanical fragmentation, or by introducing a CRISPR or Argonuate system. Guiding RNA or guiding DNA can be designed to precisely locate the sequence position to be examined before cutting the DNA.

9. A DNA methylation cytosine detection kit, characterized in that, The reagents include: 3'-OH blocking reagent, DNA pre-cleavage reagent, methylated cytosine oxidation reagent, post-processing reagent, high-fidelity polymerase amplification reagent, AP site cleavage reagent, and methylated cytosine detection reagent. The 3'-OH blocking reagent is selected from dideoxynucleotides, cordycepin triphosphate, or thiol modification reagents; The DNA pre-cleavage reagent is selected from restriction endonucleases or guiding RNA or guiding DNA; Methylation cytosine oxidizing agents include TET enzymes, ferrous ions, and α-ketoglutarate; Post-treatment reagents are selected from β-glucosyltransferase, TDG, UNG or UDG, SMUG1 or MBD4; The high-fidelity polymerase amplification reagent is selected from amplification reagents containing VENT enzyme; AP site cleavage reagents are selected from APE1 enzyme, β-elimination reaction or β,δ-elimination effect tool enzymes and DNA polymerase β; The methylated cytosine detection reagent is selected from any one of TdT enzyme, nick transferase, aldehyde reactive probe (ARP), or carbodiimide reagent.

10. The application of the DNA methylation cytosine detection method according to any one of claims 1 to 8 in the detection of DNA methylation in tumor samples.

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