Application of double-stranded cytosine deaminase in single-base resolution mapping of 5hmC modification in DNA

Abstract

The application discloses application of double-stranded cytosine deaminase in 5hmC modification single base resolution positioning analysis in DNA and relates to the technical field of gene sequencing detection. The application utilizes the double-stranded cytosine deaminase to realize the 5hmC modification single base resolution positioning analysis in DNA, does not need to perform denaturation treatment on the substrate DNA, avoids false positive signals caused by incomplete DNA denaturation or renaturation in the reaction process, eliminates the interference of 5fC and 5caC in the DNA, and significantly improves the accuracy and stability of the analysis result; the operation is simple, the cost is low, the two-step enzymatic reaction conditions are compatible, no bisulfite treatment and other purification treatment are needed, and after the deamination treatment, PCR amplification reaction can be directly performed; the deamination efficiency of the cytosine and 5-methylcytosine with different motifs is high (>99.9%), and the deamination efficiency of 5hmC in different motifs is low (<1%), which is beneficial to the positioning analysis of 5hmC.

Description

Technical Field

[0001] This invention relates to the field of gene sequencing and detection technology, and in particular to the application of double-stranded cytosine deaminase in the single-base resolution localization analysis of 5hmC-modified DNA. Background Technology

[0002] 5-Hydroxymethylcytosine (5hmC) is an important DNA hydroxymethylation modification, first discovered in the mammalian genome in 2009. It is widely found in eukaryotes and prokaryotes and is known as the "sixth base" of DNA. 5-Hydroxymethylcytosine plays a crucial role in a wide range of biological processes, from gene regulation to normal development. In the mammalian genome, the decadecyltransposase (TET) protein oxidizes the methyl group at the 5th carbon atom of 5-methylcytosine (5mC) to a hydroxymethyl group, forming 5hmC. 5hmC accounts for approximately 0.1-0.3% of cytosine in various tissues, with the vast majority located at CpG sites. Existing research indicates that 5hmC participates in the regulation of complex biological processes such as chromosome remodeling, gene expression regulation, cell differentiation, and disease occurrence and development. Because 5hmC levels are highly dynamic during development, differentiation, and cancer, genome-wide detection of 5hmC is necessary, even across different tissues, to improve our understanding of 5hmC in gene expression regulation, biological processes, and pathology. Therefore, it is important to develop a rapid, stable, and highly accurate whole-genome single-base resolution 5hmC detection method.

[0003] Currently, there are two main types of methods for single-base resolution sequencing of 5-hydroxymethylcytosine: one is the oxidized bisulfite sequencing method and the TET protein-assisted bisulfite sequencing method, which are based on bisulfite sequencing methods; the other is the sequencing method mediated by bioactive enzymes.

[0004] Bisulfite sequencing and TET (protein-assisted bisulfite sequencing) are widely used single-base resolution sequencing methods for 5hmC in DNA. This method first divides the sample into two parts. One part is treated directly with bisulfite; unmodified cytosine (C), 5fC, and 5caC are deaminated and read as thymine (T) during sequencing, while 5mC and 5hmC are not deaminated and are read as C. The other part is treated with potassium perruthenate to oxidize 5hmC, converting it to 5fC. Bisulfite is then used to deaminate C, 5fC (partially formed from the oxidation of 5hmC), and 5caC, making them read as T during sequencing, while 5mC remains unaminated and is read as C. The positioning information of 5hmC is obtained by utilizing the strategy of reading 5hmC as C in bisulfite sequencing and as T in bisulfite sequencing. TET protein-assisted bisulfite sequencing first uses glycosyltransferase to convert 5hmC in the sample into 5-glucosylcytosine (5gmC), then uses TET protein to convert 5mC into 5caC. Finally, bisulfite treatment deamination of C, 5fC, and 5caC (partially generated from the oxidation of 5mC) causes them to be read as T during sequencing, while 5gmC (the glycosylated product of 5hmC) remains undeamination and is read as C during sequencing. This strategy of reading 5hmC as C in TET protein-assisted bisulfite sequencing, while reading C, 5fC, and 5caC as T, obtains the location information of 5hmC in the sample. However, this method requires harsh chemical deamination conditions, resulting in up to 99.9% DNA degradation.

[0005] There are currently two sequencing methods mediated by bioactive enzymes. One method involves sequencing using an enzyme-linked chemical reagent, pyridineborane. This method requires splitting the sample DNA into two parts. One part uses TET protein to convert 5mC and 5hmC into 5caC. 5caC then reacts with pyridineborane to generate dihydrouracil (DHU), which is read as T during sequencing. Only C is read as C during sequencing. The other part uses β-glycosyltransferase to convert 5hmC into 5gmC. After TET protein treatment, 5mC can be converted into 5caC. 5caC then reacts with pyridineborane to generate dihydrouracil (DHU), which is read as T during sequencing. Both C and the protected 5hmC are read as C during sequencing. The strategy of reading 5hmC as T in methods without β-glycosyltransferase and as C in methods with β-glycosyltransferase allows for the acquisition of 5hmC loci information in the sample. Another method is the deaminase-mediated enzymatic sequencing approach. First, β-glycosyltransferase converts 5hmC into a product that cannot be deaminated by the deaminase APOBEC3A (human apolipoprotein B mRNA editing enzyme catalytic subunit 3A, A3A). Then, A3A deaminates C and 5mC to uracil (U) and T. In subsequent sequencing, C and 5mC are read as T, while 5hmC is read as C. However, most current enzymatic methods for determining DNA hydroxymethylation require DNA denaturation. Severe denaturation conditions can damage DNA, leading to false positives during annealing and failing to exclude interference from other large group modifications (such as 5fC and 5caC). Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a double-stranded cytosine deaminase. This double-stranded cytosine deaminase can deaminate C (ordinary cytosine), 5mC (5-methylcytosine), 5fC (5-formylcytosine or 5-aldehyde cytosine), and 5caC (5-carboxycytosine) in the intact genome, but cannot deaminate glycosylated protected 5hmC (5-hydroxymethylcytosine). Utilizing this double-stranded cytosine deaminase, this invention also provides a single-base resolution localization analysis method for 5-hydroxymethylcytosine modifications in DNA. This method can be widely applied in the field of DNA modification sequencing, featuring high sensitivity, high selectivity, high stability, simple operation, and no need for DNA substrate denaturation. Specifically, it is achieved through the following techniques.

[0007] In a first aspect, the present invention provides the application of double-stranded cytosine deaminase in single-base resolution localization analysis of 5hmC-modified DNA, wherein the amino acid sequence of the double-stranded cytosine deaminase is shown in SEQ ID NO.1.

[0008] Furthermore, the cytosine is deaminated using the double-stranded cytosine deaminase, wherein the cytosine is C, 5mC, 5fC, and 5caC.

[0009] A second aspect of the invention is the application of a double-stranded cytosine deaminase in cytosine deamination, wherein the cytosine is C, 5mC, 5fC, and 5caC.

[0010] A third aspect of the present invention provides a method for single-base resolution localization analysis of 5-hydroxymethylcytosine-modified DNA, comprising the following steps:

[0011] The 5hmC in the DNA to be tested was glycosylated for protection to obtain the first DNA fragment.

[0012] The first DNA fragment was deaminated using double-stranded cytosine deaminase to obtain the second DNA fragment;

[0013] The second DNA fragment was amplified by PCR and sequenced; the sequencing results were compared with the original sequence of the DNA to be tested, and the sites that were still read as C in the sequencing results were the 5hmC sites.

[0014] The amino acid sequence of the double-stranded cytosine deaminase is shown in SEQ ID NO.1.

[0015] The method for single-base localization analysis of 5mC modified single bases in DNA for single-base extension and linkage provided by the present invention can be used for purposes other than disease diagnosis or treatment.

[0016] Furthermore, the glycosylation reaction buffer used for glycosylation protection of 5hmC in the DNA to be tested, based on the final concentration, was formulated as follows: 50 nM potassium acetate, 10 mM magnesium acetate, 1 mM dithiothreitol, 10 U of β-glycosylation enzyme and 2 μM UDP-glucose, with a pH of 7.9.

[0017] Preferably, the reaction conditions for glycosylation protection are 37°C for 2-4 hours.

[0018] Further, based on the final concentration, the deamination reaction buffer for deamination of the first DNA fragment is formulated as follows: 100 mM 2-morpholinoethanesulfonic acid or tris(hydroxymethyl)aminomethane hydrochloride, 1 mM dithiothreitol, pH 5.5-9.0.

[0019] Preferably, the deamination treatment is carried out at 4-50°C for 30 min.

[0020] Furthermore, after the deamination process, the cytosine deaminase in the reaction system is inactivated and treated with proteinase K.

[0021] Preferably, the inactivation treatment conditions are 95°C for 5 min.

[0022] Preferably, the treatment with proteinase K is incubation at 55°C for 10 min.

[0023] Optionally, β-glycosylation enzymes can be used to protect the DNA to be tested by glycosylation.

[0024] After PCR amplification and sequencing of the second DNA fragment, the C, 5mC, 5fC, and 5caC sites in the DNA to be tested treated with SsdAcat are read as T in the sequencing, and the 5hmC site is read as C in the sequencing. The sequencing results are compared with the original sequence of the DNA to be tested. The site that is still read as C in the sequencing results is the 5hmC site, and the single-base resolution localization analysis of 5-hydroxymethylcytosine modification can be completed.

[0025] The principle of the analytical method of the present invention is as follows: First, 5hmC in double-stranded DNA is glycosylated and protected with β-glycosylation enzyme to form 5-glucosylcytosine (5gmC); then, double-stranded cytosine deaminase SsdAcat is used for deamination. SsdAcat deaminizes C, 5mC, 5fC, and 5caC of different motifs, becoming U, T, 5fU, and 5caU, respectively; finally, PCR amplification is performed, and the result is read as T in sequencing. 5gmC is resistant to the deamination effect of SsdAcat, and therefore, after PCR amplification, it is read as C in sequencing. This achieves the purpose of localizing and analyzing 5hmC in DNA at all sites.

[0026] In a fourth aspect, the present invention provides a double-stranded cytosine deaminase having the amino acid sequence shown in SEQ ID NO.1.

[0027] Furthermore, the coding gene sequence is shown in SEQ ID NO.2.

[0028] The double-stranded cytosine deaminase (SsdAcat) provided by this invention can deaminate C, 5mC, 5fC, and 5caC in double-stranded DNA, but not the glycosylated 5hmC. The entire process is mild and continuous, requiring no DNA denaturation. After processing the 5hmC-glycosylated DNA using the double-stranded cytosine deaminase, direct sequencing can obtain the single-base-resolved localization information of 5hmC.

[0029] Compared with the prior art, the advantages of the present invention are:

[0030] 1. This invention utilizes a special double-stranded cytosine deaminase to achieve single-base resolution localization analysis of 5-hydroxymethylcytosine modification in DNA. It eliminates the need for harsh denaturation treatment of the substrate DNA, is applicable to various difficult-to-denature secondary structures, avoids false positive signals caused by incomplete DNA denaturation or annealing during the reaction, and eliminates interference from 5fC and 5caC in DNA, significantly improving the accuracy and stability of the analysis results.

[0031] 2. The analytical method provided by this invention is simple to operate, the two-step enzymatic reaction conditions are compatible, no bisulfite treatment or other purification treatment is required, and PCR amplification reaction can be performed directly after deamination treatment.

[0032] 3. The double-stranded cytosine deaminase provided by this invention has a high deamination efficiency (>99.9%) for cytosine and 5-methylcytosine with different motifs, but a low deamination efficiency (<1%) for 5hmC with different motifs, which is beneficial for the localization analysis of 5hmC.

[0033] 4. No expensive third-generation sequencing platform is required; only second-generation sequencing platform technology is needed to complete single-base resolution localization analysis of 5 hmC.

[0034] 5. The method of the present invention can be widely applied to the study of 5hmC modification sites from different biological sources, and can help to conduct more in-depth research on the biological functions of 5hmC. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the deamination structures of cytosine (C), 5-methylcytosine (5mC), 5-aldehyde cytosine (5fC), and 5-carboxycytosine (5caC) in SsdAcat of the present invention.

[0036] Figure 2 This is a schematic diagram illustrating the deamination localization of 5-hydroxymethylcytosine (5caC) by SsdAcat in this invention.

[0037] Figures 3-6 In a specific embodiment of the present invention, the LC-MS / MS detection effects of artificially synthesized double-stranded DNA containing cytosine (C), 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) before and after SsdAcat deamination treatment are shown.

[0038] Figures 7-8 The figure above shows a comparison of sequencing results of artificially synthesized double-stranded DNA strands (i.e., DNA-C, DNA-5mC, and DNA-5hmC) containing cytosine (C), 5-methylcytosine (5mC), and 5-hydroxymethylcytosine (5hmC), respectively, after glycosylation protection and SsdAcat deamination, as described in a specific embodiment of the present invention.

[0039] Figure 9 and 10 These are comparative sequencing results of artificially synthesized double-stranded DNA strands (i.e., DNA-5fC and DNA-5caC) containing 5-aldehyde cytosine (5fC) and 5-carboxy cytosine (5caC), respectively, after glycosylation protection and SsdAcat deamination, respectively, in specific embodiments of the present invention. Detailed Implementation

[0040] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] Example 1: Repressed expression and activated purification of double-stranded deaminase

[0042] The truncated SsdAcat (i.e., double-stranded cytosine deaminase) used in this invention is the catalytic core region of the full-length SsdA protein, containing the sequence from amino acid 260 to amino acid 410 of the full-length SsdA protein.

[0043] SsdA protein is an intercellular deaminase derived from the bacterial type VI secretion system (T6SS) that acts as an interbacterial antagonist. SsdA exhibits strong double-strand deamination activity, effectively deaminating C molecules from different DNA sequence backgrounds to T molecules. Therefore, further characterizing the double-strand deamination activity of SsdA, as well as its differential deamination ability on cytosine and cytosine with different modifications, could replace A3A (deaminase APOBEC3A) as a new deaminase tool. If 5hmC is further protected by glycosylation, the localization analysis of 5hmC molecules with different motifs can be performed directly without requiring drastic denaturation of the DNA.

[0044] The amino acid sequence of the inhibitory protein SsdAI was referenced from the Universal Protein database (UniProtKB accession: A0 A3M2XVC5 and UniProtKB accession: A0A3M2XVH3).

[0045] Because SsdAcat exhibits double-strand deaminase toxicity to host cells, its repressor protein SsdAI needs to be co-expressed. Therefore, the coding sequences for the double-stranded cytosine deaminase SsdAcat and its repressor protein SsdAI were inserted into the pETDuet-1 plasmid vector, respectively, to achieve co-expression of these two proteins. SsdAcat has a six-His tag at its N-terminus for subsequent affinity chromatography purification; SsdAI is tagless for easy removal during denaturation. The recombinant plasmid vector was then transformed into host *E. coli* BL21(DE3) cells.

[0046] After protein expression, affinity chromatography was performed using Ni-NTA agarose beads. Subsequently, the inhibitory complex was denatured with 8 M urea to remove the SsdAI inhibitory protein. Then, the urea concentration was gradually reduced to allow SsdAcat to renature.

[0047] Finally, the protein was eluted with high-concentration imidazole buffer to obtain the activated SsdAcat protein.

[0048] The amino acid sequence of the SsdAcat protein is as follows:

[0049] KVSNIAESEAALGRASQARADLPQSKELKVKTVSSNDKKTLSGWGNKKPEGYERISAEQVKAKSEEIGHEVKSHPYDRDYKGQYFSSHAEKQMSIASPNHPLGVSKPMCTDCQGYFSQLAKYSKVEQTVADPKAIRIFKTDGSVETIMRSE, as shown in SEQ ID NO.1.

[0050] The gene sequence encoding the SsdAcat protein is as follows:

[0051] aaagttagcaatattgccgaaagcgaagcagcactgggtcgtgcaagtcaggcacgtgcagatttaccgcaaagcaaagaactgaaagttaagaccgttagcagcaatgataaga agaccctgagcggttggggtaataaaaaaccggaaggttatgaacgcatcagcgcagaacaggttaaagccaaaagcgaagaaattggccatgaagtgaaaagccatccgtatga ccgtgattataaaggccagtattttagcagccacgcagaaaaacagatgagcattgcaagcccgaatcatccgctgggtgttagcaaaccgatgtgtaccgattgtcagggttat tttagccagctggcaaaatatagcaaagtggagcagaccgttgccgatccgaaagcaattcgtatttttaaaaccgacggcagcgtggaaaccattatgcgtagcgaataa, as in SEQ Shown as ID NO.2.

[0052] Example 2: Properties analysis of double-stranded DNA cytosine deaminase

[0053] The specific steps are as follows.

[0054] 1. Prepare the reaction working solution: Dissolve 1 mM dithiothreitol (DTT), 100 mM 2-morpholinoethanesulfonic acid (MES) or tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) in deionized water and adjust the pH to 5.0-9.0.

[0055] 2. Commercially synthesized double-stranded DNA containing C, 5mC, 5hmC, and 5gmC, respectively named L-DNA-C, L-DNA-5mC, L-DNA-5hmC, and L-DNA-5gmC. The specific sequence of L-DNA-C is shown in Table 1 and SEQ ID NO.3. The specific sequences of L-DNA-5mC, L-DNA-5hmC, and L-DNA-5gmC are shown in Table 1, that is, replacing all cytosine (C) in the sequence of SEQ ID NO.3 with 5mC, 5hmC, or 5gmC.

[0056] Table 1

[0057]

[0058] 3. Take 20 ng of the double-stranded DNA sample obtained in step 2, add a certain amount of SsdAcat (final concentration approximately 800 nM), 2 μL of the reaction working solution prepared in step 1 (pH set to 5.0-9.0), and add deionized water to a total reaction volume of 20 μL. React for 30 min at a set temperature of 4-50℃. After the reaction, incubate the sample at 95℃ for 5 min and digest with proteinase K.

[0059] 4. Subsequently, the deamination-treated DNA strands from step 3 were subjected to enzymatic digestion. The enzymes used in the digestion reaction were 1 U DNase I, 100 U nuclease S1, 30 U alkaline phosphatase, and 1 U phosphodiesterase. The digestion reaction was carried out in Tris-HCl buffer at pH 7.0 at a temperature of 37°C for 5 hours.

[0060] The enzymatic digestion products were analyzed and detected using LC-MS / MS.

[0061] See results Figures 3-6 Mass spectra of DNA double strands containing C, 5mC, 5hmC, or 5gmC before and after treatment with SsdAcat showed that the mass spectrometric signals of C, 5mC, and 5hmC decreased below the detection limit after treatment. This indicates that SsdAcat can effectively remove the amino groups of C, 5mC, and 5hmC; however, the mass spectrometric signal intensity of 5gmC did not decrease significantly after treatment, indicating that SsdAcat does not have the ability to remove the amino group of glycosylated 5hmC (5gmC).

[0062] As shown in Table 2 below, SsdAcat exhibits very stable activity and can maintain strong deamination ability under various conditions.

[0063] Table 2

[0064]

[0065] The above data demonstrate that SsdAcat can replace A3A as a novel potent deaminase targeting 5hmC in cytosine.

[0066] Example 3: Analysis of Glycosylation Protection Efficiency

[0067] The glycosylation protection efficiency was determined using the synthesized 5hmC strand (L-DNA-5hmC). 20 ng of double-stranded DNA was used. The reaction mixture included β-glycosylation enzyme (10 U), 2 μL reaction buffer (50 nM potassium acetate, 10 mM magnesium acetate, 1 mM dithiothreitol, pH 7.9), and UDP-glucose (2 μM). Deionized water was added to a final volume of 20 μL, and the mixture was incubated at 37°C for 2 hours. After the reaction, the sample was incubated at 95°C for 5 minutes.

[0068] The glycosylated DNA strands were then subjected to enzymatic digestion using 1 U DNase I, 100 U nuclease S1, 30 U alkaline phosphatase, and 1 U phosphodiesterase. The digestion reaction was carried out in Tris-HCl buffer at pH 7.0 at 37°C for 5 hours. The digested products were analyzed using LC-MS / MS.

[0069] See results Figures 7-8 After protection ended, 99.7% of the 5hmC signal disappeared, and the 5gmC signal was generated. This indicates that β-glycosylation enzymes can effectively glycosylate 5hmC, ensuring the accuracy of subsequent 5hmC localization.

[0070] Example 4: Analysis of Standard Synthetic DNA Strains

[0071] Commercially synthesized DNA strands containing C, 5mC, 5hmC, 5fC, and 5caC were used. 10 ng of each DNA strand was added, along with 10 U of β-glycosylation enzyme, 1 μL of reaction buffer (50 nM potassium acetate, 10 mM magnesium acetate, 1 mM dithiothreitol, pH 7.9), and 2 μM UDP-glucose. Deionized water was added to a final volume of 10 μL, and the mixture was incubated at 37°C for 2 hours. Subsequently, 80 nM SsdAcat was added to the reaction product, along with 2 μL of reaction buffer (MES, 100 mM 2-morpholinoethanesulfonic acid, 1 mM dithiothreitol, pH 6.5). Deionized water was added to a final volume of 20 μL, and the mixture was incubated at 37°C for 30 min. Finally, the mixture was incubated at 95°C for 5 min before digestion with proteinase K.

[0072] Take the DNA (10 ng) after the above reaction and perform polymerase chain amplification. Reaction system: 5 μL of 10× amplification buffer, 2 μL each of 10 μmol / L forward and reverse primers, 10 ng of template DNA, 10 U of EpiMark® Hot Start Taq DNA polymerase, and add deionized water to a final volume of 50 μL.

[0073] Based on the TIM value of the substrate, select the appropriate annealing temperature for the amplification reaction; amplification cycle time program: (1) denaturation at 95℃ for 5 min; (2) denaturation at 95℃ for 30 sec; annealing at 50-72℃ for 30 sec (annealing temperature adjusted according to primer length); extension at 72℃ for 30 sec; repeat this step 25 times; (3) extension at 72℃ for 10 min, and store at 4℃. The sample was sequenced using Sanger sequencing.

[0074] The sequences of the DNA strands used and the primer sequences are shown in Table 3 below. In Table 3, the DNA-5mC sequence is obtained by replacing the C at positions 98, 105, 106, 112, and 121 of the DNA-C sequence (as shown in SEQ ID NO.4) with 5mC; the DNA-5hmC sequence is obtained by replacing the C at positions 98, 105, 106, 112, and 121 of the DNA-C sequence with 5hmC; the DNA-5fC sequence is obtained by replacing the C at position 59 of the sequence shown in SEQ ID NO.5 with 5fC; and the DNA-5caC sequence is obtained by replacing the C at position 59 of the sequence shown in SEQ ID NO.6 with 5caC.

[0075] Table 3

[0076]

[0077] See results Figure 9 and 10 SsdAcat can completely deaminate C, 5mC, 5fC, and 5caC in different motifs of double-stranded DNA, reading them as T during sequencing; however, it cannot deaminate 5gmC in different motifs after glycosylation protection in double-stranded DNA, still reading it as C during sequencing. This indicates that SsdAcat can perform localization analysis of 5hmC in different motifs.

[0078] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described 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 modifications all fall within the protection scope of the present invention.

Claims

1. The application of a double-stranded cytosine deaminase in the single-base resolution localization analysis of 5hmC-modified DNA, characterized in that, The amino acid sequence of the double-stranded cytosine deaminase is shown in SEQ ID NO.

1.

2. The application of the double-stranded cytosine deaminase according to claim 1 in the single-base resolution localization analysis of 5hmC-modified DNA, characterized in that, The cytosine is deaminated using the double-stranded cytosine deaminase, wherein the cytosine is C, 5mC, 5fC, and 5caC.

3. The application of a double-stranded cytosine deaminase in cytosine deamination, characterized in that, The cytosine is C, 5mC, 5fC, and 5caC.

4. A method for single-base resolution localization analysis of 5-hydroxymethylcytosine-modified DNA, characterized in that, Includes the following steps: The 5hmC in the DNA to be tested was glycosylated for protection to obtain the first DNA fragment. The first DNA fragment was deaminated using double-stranded cytosine deaminase to obtain the second DNA fragment; The second DNA fragment was amplified by PCR and sequenced. The sequencing results are compared with the original sequence. The sites that are still read as C in the sequencing results are the 5hmC sites. The amino acid sequence of the double-stranded cytosine deaminase is shown in SEQ ID NO.

1.

5. The method for single-base resolution localization analysis of 5-hydroxymethylcytosine-modified DNA according to claim 4, characterized in that, The glycosylation reaction buffer used for glycosylation protection of 5hmC in the DNA to be tested, based on the final concentration, was formulated as follows: 50 nM potassium acetate, 10 mM magnesium acetate, 1 mM dithiothreitol, 10 U of β-glycosylation enzyme and 2 μM UDP-glucose, with a pH of 7.

9. Preferably, the reaction conditions for glycosylation protection are 37°C for 2-4 hours.

6. The method for single-base resolution localization analysis of 5-hydroxymethylcytosine modification in DNA according to claim 4, characterized in that, The deamination reaction buffer for deamination of the first DNA fragment, based on the final concentration, is formulated as follows: 100 mM 2-morpholinoethanesulfonic acid or tris(hydroxymethyl)aminomethane hydrochloride, 1 mM dithiothreitol, pH 5.5-9.

0. Preferably, the deamination treatment is carried out at 4-50°C for 30 min.

7. The method for single-base resolution localization analysis of 5-hydroxymethylcytosine modification in DNA according to claim 4, characterized in that, After the deamination process was completed, the cytosine deaminase in the reaction system was inactivated and treated with proteinase K. Preferably, the inactivation treatment conditions are: inactivation at 95°C for 5 min; Preferably, the treatment with proteinase K is incubation at 55°C for 10 min.

8. A double-stranded cytosine deaminase, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

1.

9. The double-stranded cytosine deaminase according to claim 8, characterized in that, The encoding gene sequence is shown in SEQ ID NO.2.

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