Solid-phase chemical enzyme method for enriching and identifying glycosylated DNA (Deoxyribose Nucleic Acid) and sites thereof

Abstract

The invention discloses a solid-phase chemical enzyme method for enriching and identifying glycosylated DNA (Deoxyribose Nucleic Acid) and sites thereof. The efficient enrichment of glycosylated deoxynuclear nucleic acid (DNA) fragments is realized through the condensation reaction of aldehyde groups and hydrazide and the specific action of enzyme. The method aims at filling the blank of the prior art, and further reveals the potential effect of DNA glycosylation in the life process and the disease mechanism through deep research on DNA glycosylation. The method can be simultaneously suitable for various types of biological samples, including paraffin embedded tissues, fresh tissues and blood / cell samples, has wide applicability, and can be used for analyzing DNA glycosylation sites and sequences in multiple steps. According to the method, complete DNA glycosylation site information can be obtained, an important technical means is provided for researching DNA epigenetic modification, and the method has important application value in the fields of tumor marker screening, disease diagnosis, drug research and development and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of biomolecule analysis, and in particular relates to a solid phase chemical enzyme method for enriching and identifying DNA glycosylation. Background Art

[0002] Glycosylation modification, as an important post-translational modification, is closely related to the occurrence and development of many diseases. Protein glycosylation has been widely studied, and its glycosylation not only affects the structure and function of biological molecules, but also points out new targets for early screening and treatment of diseases. In other words, glycosylation is closely related to the occurrence and development of diseases, and analyzing disease-related glycosylation provides targets for early screening and treatment of diseases. Since 2021, RNA glycosylation has attracted great attention, and the existence and biological significance of RNA glycosylation were reported for the first time in 2021, and related technologies have been developed to a certain extent. However, despite significant progress in glycosylation research in the fields of protein and RNA, there is still a lack of relevant discoveries and reports on DNA glycosylation.

[0003] DNA modifications, or epigenetics, refer to changes in gene expression without altering the underlying DNA sequence. These modifications, such as DNA methylation and histone modifications, affect how genes are accessed and used. DNA methylation involves the addition of methyl groups to DNA, typically on cytosine bases, which can repress gene expression. Histone modifications, on the other hand, alter the structure of chromatin, a complex of DNA and proteins that packages DNA in the cell nucleus. These modifications can either promote or repress gene expression, depending on the specific modification and its location. Epigenetic modifications play crucial roles in a variety of biological processes, including development, cell differentiation, and disease.

[0004] However, there is no research on how to identify DNA glycosylation and its possible glycosylation sites. This application establishes a chemical enzyme method to achieve specific identification of DNA glycosylation. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the object of the present invention is to overcome the deficiencies in the prior art and provide a solid phase chemical enzyme method for enriching and identifying glycosylated DNA and its sites.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions: comprising:

[0009] Extract DNA from the sample to be identified, and perform fragmentation to obtain DNA fragments;

[0010] The DNA fragments are first oxidized, and then the oxidized DNA fragments are coupled with super-linked hydrazide magnetic beads to obtain a sample solution to be identified;

[0011] Add endoglycosidase H (Endo H) and proteinase F (PNGase F) to the sample solution to be identified, and replace the DEPC water in the reaction system with 18 O-labeled water to obtain analytical solution I, in which the N-glycosylated DNA fragments and their glycosylation sites were analyzed by secondary mass spectrometry and NGS sequencing;

[0012] O-glycosidase was then added to the analytical solution I, and the DEPC water in the reaction system was replaced with heavy water to obtain analytical solution II, and the O-glycosylated DNA fragments and their glycosylation sites therein were analyzed by secondary mass spectrometry analysis and NGS sequencing.

[0013] As a preferred embodiment of the solid phase chemoenzymatic method for enriching and identifying glycosylated DNA and its sites of the present invention, the length of the DNA fragment is 100 to 300 bp.

[0014] As a preferred embodiment of the solid phase chemoenzymatic method for enriching and identifying glycosylated DNA and its sites of the present invention, the fragmentation treatment method includes:

[0015] Take a total of 2 μg of DNA sample, dilute to 40 μl and put it into a low-adsorption 0.2 ml crushing tube. The ultrasonic crushing parameters are set to ON / OFF: 30 seconds / 30 seconds. After every 5 cycles, the reaction tube needs to be taken out, vortexed and briefly centrifuged. A total of 13 cycles are performed to fragment the complete genomic DNA into 100-300 bp.

[0016] As a preferred embodiment of the solid phase chemical enzyme method for enriching and identifying glycosylated DNA and its sites of the present invention, the oxidant for the oxidation treatment is sodium periodate.

[0017] As a preferred embodiment of the solid phase chemical enzyme method for enriching and identifying glycosylated DNA and its sites of the present invention, the oxidation treatment method comprises:

[0018] Dissolve the 2 μg DNA fragment after spin drying in 200 μl of 100 mmol sodium acetate solution, add 1 / 10 of the final volume of 200 mmol sodium periodate, and gently shake for 2 hours at room temperature in the dark for oxidation reaction. After the reaction is completed, dilute the sample to 600 μl with 1×PBS or other appropriate solution to obtain the oxidized DNA fragment.

[0019] As a preferred embodiment of the solid phase chemoenzymatic method for identifying glycosylated DNA and its sites of the present invention, the method of coupling reaction between super-linked hydrazide magnetic beads and oxidized DNA fragments comprises:

[0020] Transfer 100 μl of the well-mixed 50% hydrazide magnetic bead suspension solution to a 1.5 ml spin column, wash with DEPC water and centrifuge at 6000 rpm for 5 minutes to remove the supernatant to obtain the pretreated hydrazine hydrate resin;

[0021] 600 μl of the oxidized DNA fragment sample and 60 μl of 100 mmol aniline were added to the pretreated hydrazine hydrate resin in sequence, and the coupling reaction was carried out at room temperature with gentle shaking for 3 hours. After the reaction was completed, the supernatant was removed by centrifugation at 6000 rpm for 5 minutes;

[0022] Subsequently, 1 ml of 1.5 M sodium chloride solution was used for washing by vigorous shaking for 1 minute, and the supernatant was removed by centrifugation at 6000 rpm for 5 minutes. This step was repeated 3 times, and the same washing steps were continued using 1 ml of DEPC water and 1 ml of freshly prepared 25 mmol ammonium bicarbonate buffer. Each solution was required to be washed 3 times. Finally, the washed resin was resuspended in 200 μl of freshly prepared 25 mmol ammonium bicarbonate buffer to obtain the sample solution to be identified.

[0023] As a preferred embodiment of the solid phase chemical enzyme method for enriching and identifying glycosylated DNA and its sites of the present invention, the method for obtaining the analysis solution I comprises:

[0024] Replace the DEPC water used in the PNGase F reaction system with 18 O-labeled water, replaced 18 O-labeled water buffer;

[0025] The sample solution to be identified and the replacement 18 The buffer solution of O-labeled water was divided into two samples, to which 1 μl Endo H and 1 μl PNGase F were added respectively, and the samples were incubated with gentle shaking overnight at 37°C, and washed twice with 200 μl 25 mmol ammonium bicarbonate solution. The eluate was collected to obtain analysis solution I for NGS sequencing analysis and secondary mass spectrometry analysis.

[0026] As a preferred embodiment of the solid phase chemoenzymatic method for enriching and identifying glycosylated DNA and its sites of the present invention, the method for obtaining the analytical solution II includes:

[0027] The DEPC water used in the O-glycosidase reaction system is replaced with heavy water to obtain a buffer labeled with heavy water;

[0028] Add 20 μl of 40,000 U / μl O-glycosidase to analysis solution I, incubate at 37°C for 1-4 hours for enzyme digestion and wash twice with 200 μl of 25 mmol ammonium bicarbonate solution, collect the eluate to obtain analysis solution II for secondary mass spectrometry analysis and NGS sequencing analysis.

[0029] As a preferred embodiment of the solid phase chemoenzymatic method for enriching and identifying glycosylated DNA and its sites of the present invention, wherein: the analysis solution I contains N-glycosylated DNA fragments with N-acetylglucosamine residues and glycosylation sites with 18 OH-labeled N-linked glycosylated DNA fragments.

[0030] As a preferred embodiment of the solid phase chemoenzymatic method for identifying glycosylated DNA and its sites of the present invention, wherein: the analysis solution II contains 2 D labeled O-glycosylated DNA fragments.

[0031] Beneficial effects of the present invention:

[0032] This application establishes a novel, highly specific solid-phase chemical enzyme method for enriching and identifying glycosylated DNA and its sites, providing important technical support for research in related fields. This method can be simultaneously applied to multiple types of biological samples, including paraffin-embedded tissues, fresh tissues, and blood / cell samples, and has wide applicability.

[0033] This method can obtain complete DNA glycosylation site information and provide an important technical means for studying DNA epigenetic modifications through multi-step analysis of DNA glycosylation sites and sequences. It has important application value in the fields of tumor marker screening, disease diagnosis and drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0035] Figure 1Schematic diagram of the solid phase chemical enzyme method for enriching and identifying glycosylated DNA and its sites in Example 1 of the present invention. DETAILED DESCRIPTION

[0036] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0037] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0038] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0039] Unless otherwise specified, the raw materials used in the present invention are all commercially available in the art, and DEPC water is required to be used in the preparation of all solutions used in the entire analysis process.

[0040] Example 1

[0041] Reference Figure 1 This embodiment provides a solid phase chemical enzyme method for enriching and identifying glycosylated DNA and its sites. Figure 1 The green double helix fragment in the middle represents the DNA sequence fragment, the yellow dots represent galactose, the yellow squares represent N-acetylgalactosamine, the green dots represent mannose, the blue squares represent N-acetylglucosamine, the gray-black spheres represent super-linked hydrazide magnetic beads, the red and blue short lines represent the modification sites of N-sugar and O-sugar, respectively, and the arrows indicate the direction of the experiment. Specifically:

[0042] 1) Extraction of DNA to be analyzed from paraffin-embedded tissue sections:

[0043] Take 3 slices with a thickness of about 4 μm and a tumor tissue area of ​​about 2 cm 2 For the slice samples, use a sterile blade to scrape the tumor cell area and place it in a 1.5ml centrifuge tube. The total thickness of the slice must be controlled below 100μm to ensure the efficiency of DNA extraction.

[0044] Dewax the sections with mineral oil. When the total thickness of the sections is less than or equal to 5 μm, add 300 μl of mineral oil. When the total thickness of the sections is greater than 50 μm, add 500 μl of mineral oil. Incubate in an 80°C metal bath for 1 minute and vortex to mix. Then add 200 μl of lysis buffer. Centrifuge at 10,000 × g for 15 seconds to separate the layers. Add 20 μl of proteinase K to the lower blue aqueous phase and incubate at 56°C for 1 hour to fully dissolve the tissue. Then incubate at 80°C for 2 hours. After the sample is cooled to room temperature, add 10 μl of RNase A for 5 minutes, then add 220 μl of BL buffer and 240 μl of 100% ethanol for nucleic acid binding (this step removes possible residual RNA in the sample) to obtain a crude DNA solution.

[0045] 2) DNA fragmentation;

[0046] ① The crude DNA solution is purified by adsorption column, washed repeatedly with a dedicated washing solution, and finally eluted with 50 μl of DEPC water to obtain a purified DNA solution for subsequent fragmentation treatment;

[0047] ② The extracted DNA was fragmented using a non-contact ultrasonic disruptor. A total of 2000ng of DNA sample was taken, fixed to 40μl (the insufficient part was supplemented with 1×TE) and placed in a low-adsorption 0.2ml disruption tube. The ultrasonic disruption parameters were set to ON / OFF: 30 seconds / 30 seconds. After every 5 cycles, the reaction tube was taken out, vortexed and briefly centrifuged. A total of 13 cycles were performed to fragment the complete genomic DNA into 100-300bp;

[0048] ③ The fragmented DNA was purified using the magnetic bead method: 1.8 times the volume of DNA Clean Beads was added, incubated at room temperature for 5 minutes, and then separated on a magnetic rack. After discarding the supernatant, it was washed twice with 80% ethanol. After drying, 45 μl of DEPC water was added for elution, and finally 40 μl of purified DNA fragments were obtained.

[0049] 3) Oxidation treatment of fragmented DNA

[0050] Dissolve 2 μg of the spin-dried DNA fragment in 200 μl of 100 mmol sodium acetate solution, add 1 / 10 of the final volume of 200 mmol sodium periodate (final concentration is 20 mmol NaIO4), and gently shake for 2 hours at room temperature in the dark for oxidation reaction. After the reaction is completed, dilute the sample to 600 μl with 1× PBS or other appropriate solution to obtain glycosylated DNA fragments.

[0051] 3) Coupling reaction of glycosylated DNA fragments

[0052] Transfer 100 μl of the well-mixed 50% hydrazide magnetic bead suspension solution (hydrazide resin) to a 1.5 ml snap-cap spin-column, wash with DEPC water and centrifuge at 6000 rpm for 5 minutes to remove the supernatant to obtain the pre-treated hydrazide hydrate resin;

[0053] The oxidized DNA fragment sample (600 μl) and 60 μl of 100 mmol aniline were sequentially added to the pretreated hydrazine hydrate resin, and the coupling reaction was carried out by gentle shaking at room temperature for 3 hours. After the reaction was completed, the supernatant was removed by centrifugation at 6000 rpm for 5 minutes (the uncoupled DNA supernatant was saved), and then 1 ml of 1.5 M sodium chloride solution was vigorously shaken for 1 minute for washing, and the supernatant was removed by centrifugation at 6000 rpm for 5 minutes. This step was repeated 3 times, and 1 ml of DEPC water and 1 ml of freshly prepared 25 mmol ammonium bicarbonate (NH4HCO3) buffer were used to perform the same washing steps. Each solution needed to be washed 3 times. Finally, the washed resin was resuspended in 200 μl of freshly prepared 25 mmol ammonium bicarbonate buffer to obtain a sample solution to be identified.

[0054] 4) Identify the glycosylated DNA species in the sample solution to be identified

[0055] The DEPC water used in the PNGase F reaction system was replaced with 18 O labeled water, i.e. use 200 μl 18 O-labeled water was used to prepare 25 mmol ammonium bicarbonate buffer to obtain the replacement 18 O-labeled water buffer;

[0056] The sample solution to be identified obtained in step 3) and the replacement 18 After the buffer of O-labeled water is divided into two samples, add 1 μl of endoglycosidase H (Endo H) and 1 μl of proteinase F (do not vortex, the enzyme needs to be dissolved in 18 O-labeled water, the enzyme stock solution needs to be freeze-dried and re-dissolved in 18 O-labeled water), incubated at 37°C with gentle shaking overnight, and washed twice with 200 μl of 25 mmol ammonium bicarbonate solution, and the eluate was collected to obtain analysis solution I (which contained N-glycosylated DNA fragments with N-acetylglucosamine residues and 18 OH-labeled N-linked glycosylated DNA fragments) for NGS sequencing analysis and secondary mass spectrometry analysis.

[0057] The DEPC water used in the O-glycosidase reaction system was replaced with heavy water, i.e., 200 μl of 25 mmol ammonium bicarbonate solution was prepared using heavy water (D2O);

[0058] Add 20 μl of O-glycosidase (40000 U / μl) (prepared with heavy water) to the analysis solution I, incubate for 2 hours at 37°C for enzyme digestion, and wash twice with 200 μl of 25 mmol ammonium bicarbonate solution, collect the eluate, and obtain the analysis solution II (obtain the glycosylation site band 2 D-labeled O-glycosylated DNA fragments) for secondary mass spectrometry analysis and NGS sequencing analysis.

[0059] The analytical solution I and analytical solution II were placed in a vacuum centrifugal concentrator (SpeedVac) to dry, and finally resuspended with an appropriate amount of HPLC, wherein the N-glycosylated fragments with N-acetylglucosamine residues were used for mass spectrometry analysis to identify the glycosylation sites; 18 OH labeled N-glycosylated DNA fragments and bands 2 D (deuterated water) labeled O-glycosylated DNA fragments were used for both NGS sequencing analysis and secondary mass spectrometry analysis.

[0060] Detection and analysis

[0061] MCF-7 tumor cell line was used as a sample for identification. The sample that was not treated by the method of Example 1 (DIGMA) was recorded as MCF-7-A, and the sample that was treated by the method of Example 1 was recorded as MCF-7-B:

[0062] ① Prepare a reaction system with a total volume of 50 μl: 40 μl of DNA solution (2000 ng), 5 μl of 10X DNA degrading enzyme reaction solution, 2 μl of DNA degrading enzyme (2U / μl), and 3 μl of DEPC water.

[0063] ② Reaction incubation: Incubate in 37℃ water bath or at room temperature for 15 minutes.

[0064] ③Terminate the reaction: add 5 μl of 0.05 M EDTA (final concentration 5 mmol) and heat at 75°C for 10 minutes to inactivate the DNA degrading enzyme in the reaction system.

[0065] ④ Place the reaction tube on ice to cool and use for subsequent reactions.

[0066] ⑤ Use the Qubit4 instrument to detect the DNA concentration. This method can distinguish the DNA and RNA concentrations in the sample through fluorescent markers, which helps to obtain more reliable experimental results. The results are shown in Table 1.

[0067] Table 1

[0068]

[0069] The above results show that the DIGMA method can effectively enrich and detect glycosylated DNA fragments in samples. This conclusion has been fully verified by control experiments: a higher concentration of glycosylated DNA fragments was detected in the untreated MCF-7-A sample, while the concentration of the target fragment was significantly reduced to near the detection limit in the MCF-7-B sample treated with DNA degrading enzyme. The significant difference in the detection results of the two groups of samples reflects the specificity of the DIGMA method. At the same time, the accurate distinction between DNA and RNA by the Qubit4 fluorescence detection system further ensures the reliability of the experimental results. These findings provide important experimental basis for the application of the DIGMA method in the field of glycosylated DNA research.

[0070] Subsequently, the glycosylated DNA fragments obtained from the MCF-7-A sample were subjected to NGS sequencing and compared with the NGS sequencing results of the glycosylated DNA fragments enriched in the corresponding normal cell MCF-10A sample. Table 2 shows some glycosylated DNA fragments with significantly increased abundance screened out based on the data.

[0071] Table 2

[0072]

[0073]

[0074]

[0075] It can be seen that the method of the present invention can realize the identification of the obtained glycosylated DNA fragments and the identification of the sites at the same time, that is, identify the base at which the glycosylation occurs.

[0076] Example 2

[0077] The difference between this embodiment and embodiment 1 is that the type of biological sample is adjusted to blood or cell sample, and the DNA extraction process is adjusted to:

[0078] Take 200μl sample (if the volume is insufficient, use ddH2O to make up) and place it in a 1.5ml centrifuge tube. Add 20μl proteinase K (to remove residual protein), 4μl RNase A and 200μl Buffer AL in sequence. Vortex for 15 seconds and incubate at 56℃ for 10 minutes. After cooling, add 200μl anhydrous ethanol and vortex to mix. Regardless of the sample extraction method used, the obtained DNA solution must be purified by adsorption column, repeatedly washed with a dedicated washing solution, and finally eluted with 50μl DEPC water to obtain a purified DNA solution.

[0079] The remaining steps and processes are all referred to Example 1 to perform glycosylated DNA identification of the biological sample.

[0080] In summary, this application has established a novel, highly specific solid-phase chemical enzyme method for enriching and identifying glycosylated DNA and its sites, providing important technical support for research in related fields. This method can be simultaneously applied to various types of biological samples, including paraffin-embedded tissues, fresh tissues, and blood / cell samples, and has wide applicability.

[0081] This method can obtain complete DNA glycosylation site information and provide an important technical means for studying DNA epigenetic modifications through multi-step analysis of DNA glycosylation sites and sequences. It has important application value in the fields of tumor marker screening, disease diagnosis and drug development.

[0082] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A solid phase chemoenzymatic method for enriching and identifying glycosylated DNA and its sites, characterized in that: include, Extract DNA from the sample to be identified, and perform fragmentation to obtain DNA fragments; The DNA fragments are first oxidized, and then the super-linked hydrazide magnetic beads are used to couple with the oxidized DNA fragments to obtain a sample solution to be identified; Add endoglycosidase H (Endo H) and proteinase F (PNGase F) to the sample solution to be identified, and replace the DEPC water in the reaction system with 18 O-labeled water to obtain analytical solution I, in which the N-glycosylated DNA fragments and their glycosylation sites were analyzed by secondary mass spectrometry and NGS sequencing; O-glycosidase was then added to the analytical solution I, and the DEPC water in the reaction system was replaced with heavy water to obtain analytical solution II, and the O-glycosylated DNA fragments and their glycosylation sites therein were analyzed by secondary mass spectrometry analysis and NGS sequencing.

2. The solid phase chemical enzyme method for enriching and identifying glycosylated DNA and its sites according to claim 1, characterized in that: The length of the DNA fragment is 100 to 300 bp.

3. The solid phase chemical enzyme method for enriching and identifying glycosylated DNA and its sites according to claim 1, characterized in that: The fragmentation method comprises: Take a total of 2 μg of DNA sample, dilute to 40 μl and put it into a low-adsorption 0.2 ml crushing tube. The ultrasonic crushing parameters are set to ON / OFF: 30 seconds / 30 seconds. After every 5 cycles, the reaction tube needs to be taken out, vortexed and briefly centrifuged. A total of 13 cycles are performed to fragment the complete genomic DNA into 100-300 bp.

4. The solid phase chemoenzymatic method for enriching and identifying glycosylated DNA and its sites according to claim 1, characterized in that: The oxidizing agent for the oxidation treatment is sodium periodate.

5. The solid phase chemical enzyme method for enriching and identifying glycosylated DNA and its sites according to claim 4, characterized in that: The oxidation treatment method comprises: Dissolve the 2 μg DNA fragment after spin drying in 200 μl of 100 mmol sodium acetate solution, add 1 / 10 of the final volume of 200 mmol sodium periodate, and gently shake for 2 hours at room temperature in the dark for oxidation reaction. After the reaction is completed, dilute the sample to 600 μl with 1×PBS or other appropriate solution to obtain the oxidized DNA fragment.

6. The solid phase chemical enzyme method for enriching and identifying glycosylated DNA and its sites according to claim 1, characterized in that: The method of using super-linked hydrazide magnetic beads to carry out coupling reaction with oxidized DNA fragments comprises: Transfer 100 μl of the well-mixed 50% hydrazide magnetic bead suspension solution to a 1.5 ml spin column, wash with DEPC water and centrifuge at 6000 rpm for 5 minutes to remove the supernatant to obtain the pretreated hydrazine hydrate resin; 600 μl of the oxidized DNA fragment sample and 60 μl of 100 mmol aniline were added to the pretreated hydrazine hydrate resin in sequence, and the coupling reaction was carried out at room temperature with gentle shaking for 3 hours. After the reaction was completed, the supernatant was removed by centrifugation at 6000 rpm for 5 minutes; Subsequently, 1 ml of 1.5 M sodium chloride solution was used for washing by vigorous shaking for 1 minute, and the supernatant was removed by centrifugation at 6000 rpm for 5 minutes. This step was repeated 3 times, and the same washing steps were continued using 1 ml of DEPC water and 1 ml of freshly prepared 25 mmol ammonium bicarbonate buffer. Each solution was required to be washed 3 times. Finally, the washed resin was resuspended in 200 μl of freshly prepared 25 mmol ammonium bicarbonate buffer to obtain the sample solution to be identified.

7. The solid phase chemoenzymatic method for enriching and identifying glycosylated DNA and its sites according to claim 1, characterized in that: The method for obtaining the analysis liquid I comprises: The DEPC water used in the PNGase F reaction system was replaced with 18 O-labeled water, replaced 18 O-labeled water buffer; The sample solution to be identified and the replacement 18 The buffer solution of O-labeled water was divided into two samples, 1 μl of Endo H and 1 μl of PNGase F were added to each sample, and the samples were incubated with gentle shaking overnight at 37°C, and washed twice with 200 μl of 25 mmol ammonium bicarbonate solution. The eluate was collected to obtain analysis solution I for NGS sequencing analysis and secondary mass spectrometry analysis.

8. The solid phase chemoenzymatic method for enriching and identifying glycosylated DNA and its sites according to claim 1, characterized in that: The method for obtaining the analysis liquid II comprises: The DEPC water used in the O-glycosidase reaction system is replaced with heavy water to obtain a buffer solution labeled with heavy water; Add 20 μl of 40,000 U / μl O-glycosidase to analysis solution I, incubate at 37°C for 1-4 hours for enzyme digestion, and wash twice with 200 μl of 25 mmol ammonium bicarbonate solution. Collect the eluate to obtain analysis solution II for secondary mass spectrometry analysis and NGS sequencing analysis.

9. The solid phase chemoenzymatic method for enriching and identifying glycosylated DNA and its sites according to claim 7, characterized in that: The analysis solution I contains N-glycosylated DNA fragments with N-acetylglucosamine residues and glycosylation sites having 18 OH-labeled N-linked glycosylated DNA fragments.

10. The solid phase chemical enzyme method for enriching and identifying glycosylated DNA and its sites according to claim 8, characterized in that: The analysis liquid II contains 2 D labeled O-glycosylated DNA fragments.

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