Application of glycosidase eROS1 in single base resolution quantitative analysis of 5mC modification in DNA

Abstract

The invention discloses an application of glycosidase eROS1 in single base resolution quantitative analysis of 5mC modification in DNA, and relates to the technical field of gene engineering. On the basis of artificially modified glycosidase eROS1, 5mC in double-stranded DNA can be specifically cut, a mononucleotide gap is generated at the 5mC site, 3 '-phosphoric acid-alpha, beta-unsaturated aldehyde and a 5'-phosphoric acid terminal are formed, and extension of Bst 3.0 DNA polymerase is stopped. Therefore, the to-be-detected DNA is subjected to glycosidase eROS1 treatment, the qPCR technology is utilized after extension, the difference of extension products can be amplified, and then the content of the specific site 5mC is directly detected. The method is high in sensitivity, good in specificity, simple to operate and high in efficiency, is carried out under mild reaction conditions, can effectively prevent degradation of DNA samples, and is very suitable for quantitative analysis of specific sites in biological and clinical samples.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly to the application of glycosidase eROS1 in the quantitative analysis of 5mC modification in DNA at single-base resolution. Background Art

[0002] DNA methylation, especially methylation at the fifth position of cytosine (5-methylcytosine, 5mC), plays a key role in a variety of biological processes, including gene expression regulation and disease pathogenesis. In mammals, active DNA demethylation is achieved through a stepwise oxidation process mediated by TET (ten-eleven translocation) proteins; among them, 5mC is sequentially converted to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). These intermediate molecules are then further processed through pathways such as base excision repair, deformylation, or decarboxylation, and finally restored to unmodified cytosine. The active demethylation mechanism in plants is significantly different from that in mammals. Plants do not rely on the oxidation of 5mC, but use bifunctional glycosidase / lyases, such as repressor of silencing 1 (ROS1), to directly excise the 5mC base from DNA, generating an abasic site (AP site). These AP sites are then further processed through β- or δ-elimination to form a nucleotide gap. Through the action of DNA repair-related enzymes, unmodified cytosine can be filled into these gaps, thus completing the demethylation process. However, the activity of wild-type ROS1 protein does not meet the requirements for 5mC quantitative detection.

[0003] In order to comprehensively understand the biological function of 5mC in the genome, it is necessary to accurately quantify 5mC at specific genomic loci. Bisulfite sequencing (BS-seq) has long been regarded as the gold standard for detecting 5mC at single-base resolution. This method uses bisulfite to convert cytosine to 5mC, resulting in a C to T conversion in the sequencing results, while 5mC remains unchanged and is read as C during sequencing. However, in the BS-seq method, due to the harsh reaction conditions required for bisulfite treatment, more than 99% of the DNA may be degraded; the C to 5mC conversion reduces sequence complexity, resulting in nucleotide imbalance in the input DNA. These all lead to limitations of BS-seq, affecting the accurate quantitative analysis of 5mC.

[0004] Recently, TET-assisted pyridine borane sequencing (TAPS) has been developed to detect 5mC at single-base resolution. This method utilizes TET proteins to oxidize 5mC to 5caC, which is then reduced by pyridine borane to form dihydrouracil (DHU). During subsequent PCR amplification, DHU pairs with adenine, resulting in the conversion of 5mC to T, which can be detected by sequencing. However, DHU may affect the amplification efficiency of DNA polymerase, thereby affecting the accuracy of 5mC quantitative detection. Moreover, this method cannot distinguish 5mC from 5fC / 5caC, further reducing the accuracy of 5mC quantitative detection.

[0005] Therefore, the existing 5mC quantitative detection methods all have their own limitations, such as being prone to base imbalance and being susceptible to interference from other types of cytosine. Summary of the Invention

[0006] In view of the above deficiencies of the prior art, the present invention provides an application of glycosidase eROS1 in the quantitative analysis of 5mC modification in DNA at single-base resolution, and specifically obtains a site-specific 5mC quantitative method with advantages such as single-base resolution, high sensitivity, high specificity, and simple operation. The specific implementation is as follows.

[0007] In the first aspect of the present invention, a method for quantitatively analyzing 5mC modification in DNA at single-base resolution is provided, including the following steps:

[0008] Design a DNA probe and a qPCR primer pair for the target site on the DNA to be tested; the DNA probe is composed of a first fragment and a second fragment connected from the 3' end. The first fragment is completely complementary to the 3' end of the site to be tested, and the second fragment is a sequence that is not complementary to the DNA to be tested; in the qPCR primer pair, the forward primer is the same as the sequence at the 5' end of the target site in the DNA to be tested, and the reverse primer is the same as the sequence of the second fragment.

[0009] Extend a mixed system containing the DNA standard and the DNA probe, and after gradient dilution of the extension product, perform qPCR reactions using the qPCR primer pair to obtain the corresponding C T value; use the gradient-diluted concentration as the abscissa and the C T value as the ordinate to plot and obtain a standard curve; the sequence of the DNA standard contains the same target site as the DNA sample to be tested.

[0010] Take equal amounts of the DNA to be tested as the experimental group and the blank group, and use glycosidase eROS1 to treat the experimental group; add the DNA probe to the experimental group and the blank group respectively, and extend; use the qPCR primer pair to perform qPCR quantitative analysis on the extension product to obtain the amplification cycle number C of the experimental group T and the amplification cycle number C of the blank group T0 ;

[0011] Obtain the corresponding concentration c according to the standard curve and the C T corresponding concentration c0; T0 ;

[0012] Calculate the 5mC modification level in the DNA to be tested; the calculation formula is:

[0013] 5mC modification level = ;

[0014] where c is the concentration of cytosine at the target site in the DNA to be tested, and c0 is the total concentration of cytosine and methylcytosine at the target site in the DNA to be tested;

[0015] The nucleotide sequence of the glycosidase eROS1 is shown in SEQ ID NO.1.

[0016] Furthermore, the nucleotide sequence encoding the glycosidase eROS1 is shown in SEQ ID NO.2.

[0017] Furthermore, the length of the first fragment is 20-30 bases, and / or the length of the second fragment is 20-30 bases.

[0018] Furthermore, the forward primer is the same as the 20-30 base sequence at the 5' end of the target site in the DNA to be tested.

[0019] Furthermore, the condition for treating the DNA to be tested with the glycosidase eROS1 is: reacting at 37°C for 6-8 h.

[0020] Furthermore, the reaction system formulation for treating the DNA to be tested with the glycosidase eROS1 is: 50 mM Tris-HCl pH 7.0, 50 mM DTT, 40 mM vitamin C, 15 μmol eROS1, 1.5-2 pmol DNA to be tested, and add water to make up to 20 μL.

[0021] Furthermore, the DNA polymerase is Bst 3.0 DNA polymerase.

[0022] In the above method, a glycosidase eROS1 was selected to process the methylcytosine at the target site of the DNA to be tested. The glycosidase eROS1 was obtained by engineering the wild-type ROS1 protein. As Figure 1 shown, the glycosidase eROS1 has enhanced glycosidase / lyase activity specific for 5mC, and can efficiently and specifically recognize and cleave 5mC in DNA, thereby generating a 1-nucleotide gap at the 5mC site. The glycosidase eROS1 cannot recognize cytosine C in DNA, and thus will not generate a nucleotide gap at the site of cytosine.

[0023] Based on the above characteristics of the glycosidase eROS1, after the DNA to be tested with the target site of 5mC is treated with the glycosidase eROS1, during the subsequent extension process, DNA polymerase terminates the extension at the nucleotide gap on the DNA to be tested, resulting in fewer extension products. The DNA to be tested without 5mC will not be affected by the glycosidase eROS1, and thus can be extended normally, and the extension products will not decrease, as Figure 2 shown.

[0024] Based on the above principle, in the later stage of the present invention, by detecting the difference in the content of the extension products of the DNA to be tested before and after being treated with eROS1 by real-time quantitative PCR (qPCR), the content of the target site of 5mC modification in the DNA to be tested can be quantitatively evaluated, and the content of 5mC in the DNA can be detected at single-base resolution.

[0025] When processing actual samples, first, DNA standards containing the target site with gradient dilution are used to obtain Ct T values of different gradient concentrations through extension and qPCR, and a standard curve is drawn with the Ct T values and the gradient concentrations; then the actual DNA samples to be tested are divided into an experimental group and a blank group. The experimental group is treated with the glycosidase eROS1, and the blank group is not treated with the glycosidase eROS1. DNA polymerase is used for extension respectively, and then qPCR detection is carried out; combined with the standard curve, the total amount of DNA with the target sites of C and 5mC is obtained corresponding to the blank group, and the amount of DNA with the target site of cytosine C is obtained corresponding to the experimental group, and finally the 5mC modification level of the target site of the DNA to be tested is calculated.

[0026] In the method for quantitatively analyzing 5mC modification in DNA with single-base resolution provided by the present invention, the DNA standards are obtained by pre-artificial synthesis.

[0027] For the blank group, generally, a blank buffer without the glycosidase eROS1 can be added.

[0028] The formulation of the blank buffer is generally: 50 mM Tris-HCl pH 7.0, 50 mM DTT, and 40 mM vitamin C.

[0029] In a second aspect of the present invention, there is provided an application of glycosidase eROS1 in the single-base resolution quantitative analysis of 5mC modification in DNA. The specific application method is to quantitatively analyze the 5mC modification in DNA at single-base resolution by using the method described in any one of the above.

[0030] In a third aspect of the present invention, there is provided a product for the single-base resolution quantitative analysis of 5mC modification in DNA. The product is used to quantitatively analyze the 5mC modification in DNA at single-base resolution by using the method described in any one of the above; the product includes the glycosidase eROS1 and DNA polymerase.

[0031] Further, the product is a kit.

[0032] Even further, the product further contains a blank reaction buffer.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] 1. The method of the present invention has high sensitivity, good specificity, simple operation, and high efficiency; it can not only be used for the accurate quantitative determination of 5mC at single-base resolution in biological samples with low abundance, but also provides a new method for the functional research of 5mC in organisms.

[0035] 2. The present invention can quickly evaluate the content of 5mC at different types of sites on the genome without complex optimization and quantification.

[0036] 3. The present invention does not require the use of complex, time-consuming, and expensive second-generation and third-generation sequencing technologies. The method is simple and easy to understand, the process is concise and easy to operate, the cost is low, the instrument requirements are low, and the entire detection experiment can be completed within one day, which is convenient for popularization and use.

[0037] 4. The present invention can be used to analyze the content change of 5mC at important sites in clinical samples, providing a powerful tool for further research on the role of 5mC in different diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is a schematic diagram of the single-base resolution quantitative detection of 5mC in DNA by specific enzymatic digestion combined with qPCR in the present invention.

[0039] Figure 2This is a schematic diagram of the principle for quantitatively detecting 5mC in DNA using qPCR in the present invention. In the figure, the blue curve and the red curve respectively represent the fluorescence amplification curves of the DNA with the target site modified by 5mC without eROS1 treatment and with eROS1 treatment.

[0040] Figure 3 This is the preparation of the standard curve in the present invention. (A) qPCR fluorescence amplification curves generated using dsDNA-C / G duplexes with different concentrations. (B) T The linear relationship between the C value and the content of dsDNA-C / G duplex.

[0041] Figures 4-6 Samples of mixed strands with different double-stranded DNA-5mC / G percentages (20%, 60% and 0) were respectively selected to simulate the actual complex background and quantitatively determine the 5mC modification level at specific sites. Figures 4-6 In this, the red and blue curves respectively represent the mixed strands with eROS1 treatment and without eROS1 treatment; the left figure is the real-time fluorescence amplification curve and the bar chart of the C value, and the right figure is the comparison between the actually measured 5mC percentage and the theoretical 5mC percentage. T Value bar chart, and the right figure is the comparison between the actually measured 5mC percentage and the theoretical 5mC percentage. Specific embodiments

[0042] The technical solutions of the present invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art without creative efforts based on the embodiments in the present invention belong to the scope of protection of the present invention.

[0043] Example 1: Design of artificially modified 5-methylcytosine glycosidase

[0044] The amino acid sequence of the wild-type 5-methylcytosine glycosidase ROS1 used in this example refers to the National Center for Biotechnology Information (NCBI Gene ID: 818224) in the United States, and a mutant protein eROS1 with good cleavage activity for 5mC was obtained through engineering modification of it.

[0045] Compared with wtROS1, eROS1 has two differences, namely, the 947th amino acid is changed from phenylalanine (Phe, abbreviated as F) to isoleucine (Ile, abbreviated as I), and the 948th amino acid is changed from aspartic acid (Asp, abbreviated as D) to arginine (Arg, abbreviated as R).

[0046] The coding sequence of eROS1 protein was inserted into the pET-41a(+) plasmid vector. The N-terminus of eROS1 protein carried a GST tag, and the C-terminus carried an 8×His tag. There was a cleavage site of human rhinovirus 3C protease (HRV 3C) between the GST tag and eROS1 protein; the protein was expressed in Escherichia coli TSsetta (DE3) cells. After protein expression, it was purified using glutathione agarose beads, and after digestion with HRV3C enzyme, the full-length eROS1 protein could be obtained.

[0047] The amino acid sequence of eROS1 protein is specifically as follows:

[0048] GAGAIVPVTPVKKPRPRPKVDLDDETDRVWKLLLENINSEGVDGSDEQKAKWWEEERNVFRGRADSFIARMHLVQGDRRFTPWKGSVVDSVVGVFLTQNVSDHLSSSAFMSLASQFPVGSSGNKKAFDWDCLRREAQARAGIREKTRSTMDTVDWKAIRAADVKEVAETIKSRGMNHKLAERIQGFLDRLVNDHGSIDLEWLRDVPPDKAKEYLLSIRGLGLKSVECVRLLTLHHLAFPVDTNVGRIAVRLGWVPLQPLPESLQLHLLEMYPMLESIQKYLWPRLCKLDQKTLYELHYQMITFGKVFCTKSKPNCNACPMKGECRHFASAFASARLALPSTEKGMGTPDKNPLPLHLPEPFQREQGSEVVQHSEPAKKVTCCEPIIEEPASPEPETAEVSIADIEEAFFEDPEEIPTIRLNMDAFTSNLKKIMEHNKELQDGNMSSALVALTAETASLPMPKLKNISQLRTEHRVYELPDEHPLLAQLEKREPDDPCSYLLAIWTPGETADSIQPSVSTCIFQANGMLCDEETCFSCNSIKETRSQIVRGTILIPCRTAMRGSFPLNGTYFQVNEVFADHASSLNPINVPRELIWELPRRTVYFGTSVPTIFKGLSTEKIQACFWKGYVCVRGFDRKTRGPKPLIARLHFPASKLKGQQANLA, as shown in SEQ ID NO.1.

[0049] The nucleotide sequence encoding eROS1 protein is as follows:

[0050]

[0051] Example 2: Drawing and obtaining a standard curve

[0052] (1)For the target site of a double-stranded DNA sample with a known sequence, design a DNA probe and a pair of qPCR primers (forward primer and reverse primer).

[0053] As Figure 1 shown, the DNA probe is composed of the ligation of a first fragment and a second fragment starting from the 3'-end. The first fragment is completely complementary to the 3'-end of the site to be measured, and the second fragment is a sequence that is not complementary to the DNA to be measured.

[0054] Optionally, the first fragment can be selected with any length of 20-30 bases, and / or, the second fragment can be selected with any length of 20-30 bases.

[0055] As Figure 1 shown, in the pair of qPCR primers, the forward primer has the same sequence as the 5'-end of the target site in the DNA to be measured, and the reverse primer has the same sequence as the second fragment.

[0056] Optionally, the forward primer has the same sequence as the 5'-end of the target site in the DNA to be measured with a length of 20-30 bases.

[0057] Specifically, the sequences of the DNA standard product and the DNA to be measured selected in this example are both dsDNA-C / G, and the DNA sequence with the target site modified to 5mC is dsDNA-5mC / G. The sequences of dsDNA-C / G, dsDNA-5mC / G, the DNA probe, and the pair of qPCR primers are shown in Table 1 below, and these sequences are all synthesized by a third-party company.

[0058] Table 1

[0059]

[0060] In Table 1, the sequence of dsDNA-5mC / G is that the 23rd base C starting from the 5'-end of the sequence shown in SEQ ID NO.3 is replaced with 5mC. The DNA probe sequence for extending dsDNA-C / G and dsDNA-5mC / G is shown in SEQ ID NO.4. The forward primer sequence for amplifying the extension product is shown in SEQ ID NO.5, and the reverse primer sequence for amplifying the extension product is shown in SEQ ID NO.6.

[0061] (2)Take 1.8 pmol of a double-stranded DNA sample (dsDNA-C / G) containing the target site, and add 10 pmol of the DNA probe. Add the reaction system shown in Table 2 below for reaction.

[0062] The reaction procedure is as follows: React at 95°C for 10 - 15 min to open the DNA double strand; then react at 50 - 55°C for 20 - 25 min to anneal the DNA template (dsDNA-C / G) with the DNA probe; react at 68 - 72°C for 20 - 25 min.

[0063] Table 2

[0064] (3)Gradient dilute the sample of 180 nM dsDNA-C / G, and the final concentrations are 1 fM, 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, and 1 nM respectively.

[0065] Perform qPCR reaction on a CFX Connect real-time system (Bio-Rad Laboratories, Hercules, 5mCSA) to obtain the C dsDNA-C / G values corresponding to different gradient concentrations (C T ). The qPCR system is shown in Table 3 below. The specific qPCR procedure is as follows: React at 95°C for 10 min; react at 95°C for 5 s, 61°C for 30 s, and 72°C for 45 s, for a total of 45 cycles.

[0066] Table 3

[0067] Using the C T values corresponding to different gradient concentrations as the ordinate and the logarithm (lg) values of the respective gradient concentrations of dsDNA-C / G as the abscissa, plot to generate a standard curve. As Figure 3 shown, the standard curve of the C T value and the dsDNA-C / G concentration is:

[0068] C T= -3.469 lgC dsDNA-C / G -20.59, R 2 = 0.9993 Equation Ⅰ.

[0069] It can be seen that the above equation shows a good linear relationship. Among them, the C T value of 1 fM dsDNA-C / G is 31.12, which is within the acceptable range for qPCR quantification.

[0070] Example 3: Verification of the 5mC modification level of the target site by quantifying the eROS1 protein in a complex background

[0071] The specific steps of the verification method are as follows:

[0072] (1)Prepare a mixed-strand sample for simulating a real biological sample

[0073] Synthesize double-stranded DNA containing the target site and modified with 5mC at the target site (i.e., dsDNA-5mC / G), and double-stranded DNA containing the target site without 5mC modification at the target site (i.e., dsDNA-C / G) according to the nucleotide sequence shown in Table 3 below. Mix dsDNA-5mC / G and dsDNA-C / G in different content ratios so that the proportion of dsDNA-5mC / G is 60%, 20%, and 0% to obtain mixed strand samples for simulating real biological samples.

[0074] Extract genomic DNA from 293T cells and use ultrasonic fragmentation to cut it into several gene fragments of 300 - 500 bp to obtain a gene fragment mixture.

[0075] (2) Add the mixed strand samples with the proportion of dsDNA-5mC / G being 60%, 20%, and 0 respectively to 7.5 μg of the gene fragment mixture to prepare the DNA samples to be tested.

[0076] (3) Divide the DNA samples to be tested into two equal parts. One part is not treated with eROS1 (blank group), and the other part is treated with eROS1 (experimental group).

[0077] The reaction system (20 μL) for treating the DNA samples to be tested with eROS1 is shown in Table 4 below. The reaction system reacts at 37 °C for 6 - 8 h.

[0078] Table 4 Reaction system for eROS1 treatment

[0079] (4) Add 1 nmol of DNA probe (shown in Table 1) to each of the two DNA samples to be tested. Use Bst 3.0 DNA polymerase to extend the two DNA samples to be tested to obtain the corresponding extended products.

[0080] (5) Perform qPCR on the two extended products respectively to obtain the corresponding C T and C T0 . Among them, C T represents the number of amplification cycles of the sample to be tested treated with eROS1, and C T0 represents the number of amplification cycles of the sample to be tested not treated with eROS1.

[0081] (6) According to C T and C T0, substituted into Formula Ⅰ obtained in Example 2, and the corresponding c and c0 are obtained, wherein c represents the concentration of cytosine C at the target site in the DNA sample to be tested (i.e., dsDNA-C / G), and c0 represents the total concentration of cytosine C and methylcytosine 5mC (i.e., dsDNA-C / G and dsDNA-5mC / G) in the DNA sample to be tested, and then the level of 5mC modification (5mC%) is calculated according to the following Formula Ⅱ.

[0082] 5mC%= ; Formula II.

[0083] When the content of dsDNA-5mC / G in the mixture was 20% (i.e., the content of dsDNA-5mC / G accounted for 20% of the total content of dsDNA-5mC / G and dsDNA-C / G), the C T The values ​​are 20.42 and 20.06 respectively. Figure 4 According to the linear equation (Formula I) obtained in Example 2, the content of dsDNA-C / G was calculated to be 1.51 pM, and the total content of dsDNA-C / G and dsDNA-5mC / G was 1.91 pM; furthermore, the level of 5mC modification at the specific site was deduced to be 20.9% ([(1.91 pM-1.51 pM) / 1.91 pM]×100%=20.9%), as shown in FIG. Figure 4 shown.

[0084] When the content of dsDNA-5mC / G in the mixture was 60% (i.e., the content of dsDNA-5mC / G accounted for 60% of the total content of dsDNA-5mC / G and dsDNA-C / G), the C T The values ​​are 20.76 and 19.33 respectively. Figure 5 According to the linear equation formula (Formula I) obtained in Example 2, the content of dsDNA-C / G was calculated to be 1.20 fM, and the total content of dsDNA-C / G and dsDNA-5mC / G was 3.09 fM; furthermore, the level of 5mC modification at the specific site was deduced to be 61.2% ([(3.09 pM-1.20 pM) / 3.09 pM]×100%=61.2%), as shown in FIG. Figure 5 shown.

[0085] When the mixture did not contain dsDNA-5mC / G (i.e., the content of dsDNA-5mC / G accounted for 0% of the total content of dsDNA-5mC / G and dsDNA-C / G), the C T The values ​​are 28.78 and 28.76 respectively. Figure 6As shown, according to the linear equation formula (Equation I) obtained in Example 2, the content of dsDNA-C / G was calculated to be 5.86 fM, and the total content of dsDNA-C / G and dsDNA-5mC / G was 5.94 fM; furthermore, the level of 5mC modification at a specific locus was deduced to be 1.3% ([(5.94 pM - 5.86 pM) / 5.94 pM]×100% = 1.3%), close to 0, as Figure 6 shown.

[0086] The above results indicate that the accuracy of the measurement is consistent with the actual value by using the glycosidase eROS1 provided in Example 1 of the present invention and the test method for the 5mC modification level of the corresponding DNA target site to be measured.

[0087] The above specific embodiments have described the implementation of the present invention in detail. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple variations all fall within the protection scope of the present invention.

Claims

1. A method for quantitatively analyzing 5mC modification in DNA with single-base resolution, characterized in that, Comprising the following steps: Designing a DNA probe and amplification primers for a target site on the DNA to be tested; the DNA probe is composed of a first fragment and a second fragment connected from the 3'-end, the first fragment is completely complementary and paired with the 3'-end of the site to be tested, and the second fragment is not complementary to the DNA to be tested; among the amplification primers, the forward primer has the same sequence as the sequence at the 5'-end of the target site in the DNA to be tested, and the reverse primer has the same sequence as the second fragment; Extend the system containing the DNA standard and the DNA probe, gradient-dilute the extension product, and use the amplification primers to obtain the corresponding C values through qPCR reactions respectively; use the gradient-diluted concentration as the abscissa and the C value as the ordinate to plot a standard curve; the DNA standard contains the same target site as the DNA sample to be tested; T value; use the gradient-diluted concentration as the abscissa and the C T value as the ordinate to plot a standard curve; the DNA standard contains the same target site as the DNA sample to be tested; Take an equal amount of the DNA to be tested as the experimental group and the blank group, and treat the experimental group with glycosidase eROS1; add the DNA probe to the experimental group and the blank group, and extend; use the amplification primer to perform qPCR quantification on the extension product to obtain the amplification cycle number C of the experimental group T and the amplification cycle number C of the blank group T0 , and obtain the concentration c corresponding to the C T according to the standard curve, and the concentration c0 corresponding to the C T0 ; Calculating the 5mC modification level in the DNA to be tested, and the calculation formula is: 5mC modification level = ; Wherein, c is the concentration of cytosine at the target site in the DNA to be tested, and c0 is the total concentration of cytosine and methylcytosine at the target site in the DNA to be tested; The nucleotide sequence of the glycosidase eROS1 is shown in SEQ ID NO.

1.

2. The method for quantitatively analyzing 5mC modification in DNA with single-base resolution according to claim 1, wherein The nucleotide sequence encoding the glycosidase eROS1 is shown in SEQ ID NO.

2.

3. The method for quantitatively analyzing 5mC modification in DNA with single-base resolution according to claim 1, characterized in that, The length of the first fragment is 20-30 bases, and / or the length of the second fragment is 20-30 bases.

4. The method for quantitatively analyzing 5mC modification in DNA with single-base resolution according to claim 1, wherein, The forward primer has the same sequence as the 20-30 base sequence at the 5'-end of the target site in the DNA to be tested.

5. The method for quantitatively analyzing 5mC modification in DNA with single-base resolution according to claim 1, wherein The conditions for treating the DNA to be tested with the glycosidase eROS1 are: reacting at 37°C for 6-8 h.

6. The method for quantitatively analyzing 5mC modification in DNA with single-base resolution according to claim 5, wherein The reaction system formulation when treating the DNA to be tested with the glycosidase eROS1 is: 50 mM Tris-HCl pH7.0, 50 mM DTT, 40 mM vitamin C, 15 μmol eROS1, 1.5-2 pmol DNA to be tested, and adding water to make up to 20 μL.

7. The method for quantitatively analyzing 5mC modification in DNA with single-base resolution according to claim 1, wherein The DNA polymerase is Bst 3.0 DNA polymerase.

8. Use of a glycosidase eROS1 in the quantitative analysis of 5mC modification in DNA at single-base resolution, characterized in that, Using the method according to any one of claims 1-7 for single-base resolution quantitative analysis of 5mC modification in DNA.

9. A product for single-base resolution quantitative analysis of 5mC modification in DNA, characterized in that, The product is used for single-base resolution quantitative analysis of 5mC modification in DNA by using the method according to any one of claims 1-7; the product includes the glycosidase eROS1 and DNA polymerase.

10. The product for single-base resolution quantitative analysis of 5mC modification in DNA according to claim 9, wherein, The product is a kit.

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