Application of green monkey-derived and dog-derived deaminase in resolution sequencing analysis of 5hmC modified single base in DNA
By using deaminase from green monkey and dog sources to perform one-step enzyme treatment on DNA, the problem of DNA degradation and cumbersome operation caused by bisulfite treatment in the existing 5hmC sequencing methods was solved, and high sensitivity and high specificity 5hmC single-base resolution sequencing was achieved.
Patent Information
- Application Number
- CN202510030553.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-08
- Publication Date
- 2025-04-15
AI Technical Summary
The existing 5hmC single-base resolution sequencing method is limited by the severe conditions of bisulfite treatment, which leads to DNA degradation, and is cumbersome to operate and has a high false positive rate.
The DNA was treated with one-step enzyme with deaminase from green monkey and dog sources. The C and 5mC of TC/CC and GC/AC sites were deaminated by gmA3A and dogA3A, without deaminating 5hmC, thereby achieving single-base resolution sequencing of 5hmC.
It realizes one-step enzyme treatment with high sensitivity and high specificity without bisulfite treatment, simplifies the operation process, reduces the false positive rate, and improves the efficiency of 5hmC positioning analysis.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of genetic engineering, and in particular to application of green monkey-derived and dog-derived deaminases in single-base resolution sequencing analysis of 5hmC modification in DNA. Background Art
[0002] Epigenetic modification of cytosine base (C) plays a key role in the regulation of gene expression in mammals. 5-Methylcytosine (5mC) is the first discovered and most studied modified base, playing a key role in basic biological processes such as gene expression regulation and normal developmental progression. 5-Hydroxymethylcytosine (5hmC) is generated by oxidation of 5mC by ten-eleven translocation (TET). In addition to being an intermediate for DNA demethylation, 5hmC also participates in the regulation of important biological processes as a stable epigenetic modification in the mammalian genome.
[0003] There are currently a variety of methods for mapping 5hmC in DNA. Antibody-assisted hydroxymethylated DNA immunoprecipitation (hMeDIP) and affinity-enrichment-based purification and sequencing have been used to map the whole genome of 5hmC. However, the resolution of these methods is limited by the size of genomic DNA fragments, and the precise location of 5hmC cannot be determined. Subsequently, two methods involving bisulfite were developed, namely oxidative bisulfite sequencing (oxBS-seq) and TET-assisted bisulfite sequencing (TAB-seq), which can achieve single-base resolution analysis of 5hmC in mammalian genomes; however, the reaction conditions of bisulfite treatment are severe and will cause a large amount of DNA degradation. In addition, the method of combining single-molecule real-time (SMRT) sequencing with selective chemical labeling and nanopore sequencing technology has achieved single-base resolution analysis of 5hmC in mammalian genomes. This method still has a high false positive rate when mapping 5hmC.
[0004] Another method for single-base resolution sequencing of 5hmC is to use a sequencing method mediated by a biologically active enzyme. APOBEC3A protein (lipoprotein B mRNA editing catalytic polypeptide-like 3A, A3A) is present in most placental mammals, which can deaminize cytosine (C) in DNA to uracil (U). Human A3A (hA3A) protein can effectively deaminize cytosine C, 5mC and 5hmC in DNA, but has no deamination activity on glycosylated 5hmC (5gmC). The mechanism of action of this deaminase-mediated sequencing method is: 5hmC is converted into 5gmC using β-glycosyltransferase. After treatment with the deaminase hA3A, cytosine C and 5mC can be converted into uracil (U) or thymine (T). The site read as C in the sequencing result is the measured 5hmC site. However, this method requires glycosylation protection of 5hmC, making the operation more cumbersome.
[0005] Previous studies have shown that mammalian homologous A3A proteins exhibit different degrees of deamination activity for C and 5mC. Except for hA3A, the deamination properties of other mammalian homologous A3A proteins for 5mC and 5hmC have not been fully explored. If it is possible to screen out proteins with good deamination ability for C and 5mC but not for 5hmC from mammalian homologous A3A proteins, it is possible to perform localization analysis of 5hmC at all sites without converting 5hmC to 5gmC. Summary of the invention
[0006] The present invention provides the application of green monkey-derived and dog-derived deaminases in single-base resolution sequencing analysis of 5hmC modification in DNA, that is, provides a single-base resolution 5hmC localization sequencing method that can be applied to the field of DNA modification sequencing, does not require bisulfite treatment, only requires one-step enzyme treatment, has high sensitivity, high specificity, and is easy to operate. It is specifically achieved through the following technologies.
[0007] In a first aspect of the present invention, a method for applying a green monkey-derived deaminase and a dog-derived deaminase in single-base resolution sequencing analysis of 5hmC modification in DNA is provided, wherein a DNA fragment to be sequenced is denatured to form a single-stranded DNA, and the single-stranded DNA is deaminated using the green monkey-derived deaminase or the dog-derived deaminase, respectively, to inactivate the green monkey-derived deaminase (hereinafter referred to as gmA3A) or the dog-derived deaminase (hereinafter referred to as dogA3A), and the treated samples are amplified and sequenced, and the site read as C in the sequencing result is the 5hmC site in the DNA fragment to be sequenced.
[0008] After extensive research, the present invention found that green monkey deaminase (gmA3A) can completely deaminate the base C at the TC and CC sites in the DNA sequence, and can also completely deaminate the 5mC at the TC and CC sites in DNA-5mC, but will not deaminate the 5hmC at the TC and CC sites in DNA-5hmC. Therefore, the green monkey deaminase (gmA3A) is used to deaminate the DNA fragment to be sequenced, and all bases C and 5mC are deaminated, while 5hmC is not deaminated and is retained, so that in the final sequencing result, 5hmC is still read as C, while the deaminated bases C and 5mC are read as T.
[0009] After extensive research, the inventors found that dog-derived deaminase (dogA3A) can completely deaminate the base C at the GC and AC sites in the DNA sequence, and can also completely deaminate the 5mC at the GC and AC sites in DNA-5mC, but will not deaminate the 5hmC at the GC and AC sites in DNA-5hmC. Therefore, the green monkey-derived deaminase (gmA3A) is used to deaminize the DNA fragment to be sequenced, and all bases C and 5mC are deaminated, while 5hmC is not deaminated and is retained, so that in the final sequencing result, 5hmC is still read as C, while the deaminated bases C and 5mC are read as T. The deamination process of gmA3A and dogA3A is as follows Figure 1 and Figure 2 shown.
[0010] Therefore, based on the above findings, the sites read as C in the sequencing results after deamination treatment are the 5hmC modification sites in the DNA fragment to be tested, thereby achieving single-base resolution sequencing analysis of 5-hydroxymethylcytosine.
[0011] Furthermore, the green monkey-derived deaminase or dog-derived deaminase is digested and inactivated using a protease.
[0012] The second aspect of the present invention provides a single-base resolution sequencing analysis method for 5hmC modification in DNA, wherein the DNA fragment to be sequenced is denatured to form single-stranded DNA, the single-stranded DNA is deaminated using the green monkey-derived deaminase or the dog-derived deaminase to inactivate the green monkey-derived deaminase or the dog-derived deaminase, the treated sample is amplified and sequenced, and the site read as C in the sequencing result is the 5hmC site in the DNA fragment to be sequenced.
[0013] Furthermore, the method for denaturing the DNA fragment to be sequenced is: adding DMSO to the solution containing the DNA fragment to be sequenced, incubating at 95° C. for 10-20 min, and immediately transferring to an ice water bath for 2-5 min to denature the DNA fragment to be sequenced into single-stranded DNA.
[0014] Furthermore, the DNA fragment to be sequenced is subjected to deamination treatment using a deamination reaction buffer, wherein the deamination reaction buffer contains the green monkey-derived deaminase or the dog-derived deaminase.
[0015] Optionally, the deamination reaction buffer further contains 20 mM 2-morpholineethanesulfonic acid and has a pH value of 6.5.
[0016] Furthermore, in the deamination reaction buffer, the concentrations of the green monkey-derived deaminase and the dog-derived deaminase are 10-20 μM, respectively.
[0017] Optionally, the temperature of the deamination treatment is 37° C., and the treatment time is 2-4 h.
[0018] Furthermore, the green monkey-derived deaminase or dog-derived deaminase is digested and inactivated using a protease.
[0019] Optionally, inactivate by digestion using proteinase K.
[0020] The application method of the green monkey-derived deaminase and the dog-derived deaminase in single-base resolution sequencing analysis of 5hmC modification in DNA, and the single-base resolution sequencing analysis method of 5hmC modification in DNA provided by the present invention can be used for purposes other than disease diagnosis or treatment.
[0021] The third aspect of the present invention provides a product for single-base resolution sequencing analysis of 5hmC modification in DNA, wherein the product contains green monkey-derived deaminase or dog-derived deaminase.
[0022] Furthermore, the product is a detection kit.
[0023] Compared with the prior art, the present invention is beneficial in that:
[0024] 1. The present invention provides a method for applying green monkey-derived deaminase and dog-derived deaminase in single-base resolution sequencing analysis of 5hmC modification in DNA. The method is easy to operate, does not require bisulfite treatment and subsequent purification, and does not require cumbersome sample pretreatment. After one-step deamination treatment, PCR amplification reaction can be directly performed.
[0025] 2. The present invention does not require glycosylation treatment or chemical oxidation, which greatly simplifies the experimental steps.
[0026] 3. The deamination reaction involved in the present invention has a high deamination efficiency and is conducive to positioning analysis. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 Schematic diagram of the single-base resolution positioning analysis method for 5-hydroxymethylcytosine in DNA using the combination of gmA3A and dogA3A.
[0028] Figure 2 Schematic diagram of the deamination effect of gmA3A and dogA3A on cytosine and its oxidized derivatives in the present invention.
[0029] Figure 3 The mass spectra of TC-C and CC-C, TC-5mC and CC-5mC, and TC-5hmC and CC-5hmC before and after treatment with gmA3A. After treatment, the mass spectrometry signals of C and 5mC were below the detection limit, so it can be considered that gmA3A can almost completely deaminate C and 5mC at the TC and CC sites. The mass spectrometry signal intensity of 5hmC did not decrease significantly, so it can be considered that gmA3A can hardly deaminate 5hmC at the TC and CC sites.
[0030] Figure 4 The mass spectra of GC-C and AC-C, GC-5mC and AC-5mC, and GC-5hmC and AC-5hmC before and after treatment with dogA3A. After treatment, the mass spectrometry signals of C and 5mC were below the detection limit, so it can be considered that dogA3A can almost completely deaminate C and 5mC at the GC and AC sites. The mass spectrometry signal intensity of 5hmC did not decrease significantly, so it can be considered that dogA3A can hardly deaminate 5hmC at the GC and AC sites.
[0031] Figure 5 This is a comparison chart of the sequencing results of the artificially synthesized DNA chains containing C, 5mC and 5hmC (DNA-C, DNA-5mC and DNA-5hmC) before and after deamination treatment by gmA3A.
[0032] Figure 6 This is a comparison chart of the sequencing results of artificially synthesized DNA chains containing C, 5mC and 5hmC (DNA-C, DNA-5mC and DNA-5hmC) before and after deamination treatment by dogA3A. DETAILED DESCRIPTION
[0033] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0034] Example 1: Expression and purification of green monkey deaminase (gmA3A) and dog deaminase (dogA3A)
[0035] The amino acid sequence of the green monkey deaminase (gmA3A) is referenced from the NCBI database (GenBank: ADO85881.1), as shown in SEQ ID NO. 1. The gene sequence encoding the gmA3A protein is shown in SEQ ID NO. 2.
[0036] The amino acid sequence of dog-derived deaminase (dogA3A) is referenced to the NCBI database (NCBI Reference Sequence: NP_001333061.1), as shown in SEQ ID NO. 3. The gene sequence encoding dogA3A protein is shown in SEQ ID NO. 4.
[0037] The expression and purification procedures of these two homologous mammalian deaminases are as follows:
[0038] (1) The gene sequence DNA encoding gmA3A and dogA3A (described in SEQ ID NO. 2 and SEQ ID NO. 4) were cloned into pET-41a(+) plasmid respectively to construct a recombinant plasmid capable of expressing proteins.
[0039] (2) The recombinant plasmid was transformed into Escherichia coli BL21(DE3) pLysS strain.
[0040] (3) The transformed E. coli cells were grown in LB medium at 37°C and 180 rpm. The LB medium formula is: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L; kanamycin and chloramphenicol 10 μg / mL were also added to the medium at a final concentration of 30 μg / mL to inhibit the growth of foreign bacteria and prevent the transformed bacteria from losing the recombinant plasmid.
[0041] (4) When the OD of the E. coli cell suspension is 600 When the value reaches 0.6-0.8, IPTG (isopropyl-β-D-thiogalactoside) with a final concentration of 1 mM is added to the culture medium, and the culture temperature is lowered to 25°C, and the culture is shaken at 180 rpm for 20 h to fully induce Escherichia coli to synthesize the target protein.
[0042] (5) After the culture is completed, the E. coli cells are collected in a 50 mL centrifuge tube after centrifugation at 8000 rpm for 3 min. The precipitated bacteria are washed twice with 30 mL of PBS solution, and the precipitate is collected by centrifugation each time.
[0043] (6) The precipitated E. coli cells were resuspended in 30 mL of PBS solution and then disrupted using an ultrasonic disruptor. The disrupted bacterial solution was centrifuged at 8000 rpm for 20 min to obtain the supernatant containing the target protein.
[0044] (7) After incubating the supernatant with glutathione agarose beads (purchased from Bio-Technology Co., Ltd.) for 2 h, the supernatant was removed by centrifugation at 500 g and the agarose beads were retained;
[0045] (8) Resuspend the agarose beads in the reaction buffer solution of HRV 3C protease, add 10 U HRV 3C protease to remove the GST tag and release the deaminase protein from the agarose beads. The released protein is collected by centrifugation using a 10 kDa concentrating spin column (purchased from Millipore). The concentrated protein is equilibrated with a protein storage solution containing 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.01 mM EDTA, 0.5 mM dithiothreitol and 0.01% Tween-20. Add 25% glycerol to the concentrated protein and store it in aliquots at -80°C until use.
[0046] Example 2: Analysis of the deamination properties of gmA3A and dogA3A
[0047] The deamination characteristics of gmA3A and dogA3A prepared in Example 1 were tested and analyzed. The specific steps are as follows:
[0048] (1) Commercially synthesize the oligonucleotide chains listed in Table 1 below;
[0049] Table 1
[0050]
[0051] In Table 1 above, the sequence of TC-5mC is that the base C at the 9th position in the TC-C sequence is replaced by 5mC, the sequence of CC-5mC is that the base C at the 9th position in the CC-C sequence is replaced by 5mC, the sequence of GC-5mC is that the base C at the 9th position in the GC-C sequence is replaced by 5mC, and the sequence of AC-5mC is that the base C at the 9th position in the AC-C sequence is replaced by 5mC. The sequence of TC-5hmC is that the base C at the 9th position in the TC-C sequence is replaced by 5hmC, the sequence of CC-5hmC is that the base C at the 9th position in the CC-C sequence is replaced by 5hmC, the sequence of GC-5hmC is that the base C at the 9th position in the GC-C sequence is replaced by 5hmC, and the sequence of AC-5hmC is that the base C at the 9th position in the AC-C sequence is replaced by 5hmC.
[0052] (2) 60 ng of each of the three different DNA combinations (TC-C and CC-C; TC-5mC and CC-5mC; TC-5hmC and CC-5hmC) were deaminated using green monkey deaminase (gmA3A) at a final concentration of 10 µM.
[0053] 60 ng of each of the other three different DNA combinations (GC-C and AC-C; GC-5mC and AC-5mC; GC-5hmC and AC-5hmC) were deaminated using dog-derived deaminase (dogA3A) at a final concentration of 10 µM. The deamination reaction solution was 20 mM MES (pH 6.5), 0.1% Triton X-100, and the reaction system was 20 μL. The reaction was then carried out at 37°C for 2 h.
[0054] (3) Add 1.5 μL of proteinase K solution to the deaminated solution, incubate at 55°C for 30 min, and then digest and inactivate the solution at 95°C for 30 min.
[0055] (4) The deaminated DNA strand in step (3) is then subjected to enzymatic digestion.
[0056] Based on the final concentration of the enzymatic system, the enzymes used in the enzymatic reaction were 2.5 U of DNase I, 90 U of nuclease S1, 7.5 U of alkaline phosphatase, and 0.125 U of phosphodiesterase.
[0057] The enzymatic hydrolysis reaction was carried out in a neutral buffer at a temperature of 37°C and a reaction time of 6 h.
[0058] Optionally, the neutral buffer contains 500 mM Tris–HCl, 10 mM MgCl2, 100 mM NaCl, 10 mM ZnSO4, pH 7.0 based on the final concentration of the enzymatic system.
[0059] (5) The enzymatic products were analyzed using LC-MS / MS.
[0060] The results are as follows Figure 3 , 4 As shown, the mass spectrometry signal intensity of C on TC-C and CC-C, and 5mC on TC-5mC and CC-5mC decreased to below the detection limit after treatment with gmA3A; the mass spectrometry signal intensity of 5hmC on TC-5hmC and CC-5hmC did not change significantly after treatment with gmA3A.
[0061] Similarly, the mass spectrometry signal intensity of C on GC-C and AC-C, and 5mC on GC-5mC and AC-5mC decreased to below the detection limit after treatment with dogA3A; the mass spectrometry signal intensity of 5hmC on GC-5hmC and AC-5hmC did not change significantly after treatment with dogA3A.
[0062] The above results show that: (1) gmA3A can completely deaminate C on TC-C and CC-C, and 5mC on TC-5mC and CC-5mC, but cannot deaminate 5hmC on TC-5hmC and CC-5hmC; (2) dogA3A can completely deaminate C on GC-C and AC-C, and 5mC on GC-5mC and AC-5mC, but cannot deaminate 5hmC on GC-5hmC and AC-5hmC. The deamination process of gmA3A and dogA3A is shown in Figure 2. Figure 1 and Figure 2 shown.
[0063] All cytosine sites in DNA can be divided into four types: GC, AC, TC and CC. Therefore, gmA3A can be used to detect 5hmC at TC and CC sites, and dogA3A can be used to detect 5hmC at GC and AC sites.
[0064] Example 3: Analysis of standard DNA by gmA3A and dogA3A
[0065] The standard DNA was analyzed using gmA3A and dogA3A. The specific steps are as follows:
[0066] (1) Double-stranded DNA containing C (DNA-C), 5mC (DNA-5mC) and 5hmC (DNA-5hmC) were prepared respectively. The DNA sequences are shown in Table 2.
[0067] Table 2
[0068]
[0069] In Table 2 above, the sequence of DNA-5mC is obtained by replacing the base C at positions 97, 104, 105, 111 and 120 of DNA-C with 5mC; and the sequence of DNA-5hmC is obtained by replacing the base C at positions 97, 104, 105, 111 and 120 of DNA-C with 5hmC.
[0070] (2) Take 60 ng of each of the three DNAs above, add 2 μL of DMSO, and add deionized water to make the system 10 μL. Incubate at 95°C for 10-20 min, then immediately transfer to an ice water bath for 2-5 min to quench the double-stranded DNA into single-stranded DNA.
[0071] (3) Add MES (pH 6.5) with a final concentration of 20 mM and 10-20 μM gmA3A or dogA3A to the system denatured into single-stranded DNA, and react at 37°C for 2 h.
[0072] (4) Add 1.5 μL of proteinase K solution to the deaminated solution, incubate at 55°C for 30 min, and then inactivate at 95°C for 30 min.
[0073] (5) Take the DNA after the above reaction and perform polymerase chain amplification reaction. The reaction system: 5 μL of 10× amplification buffer, 0.4 μM forward primer (5'-AGTGATGTTGAGTTTGATGTTGTGT-3', as shown in SEQ ID NO.10), 0.4 μM reverse primer (5'-CCAACATTCCACTAACAATTACTCTCT-3', as shown in SEQ ID NO.11), 1.25 U of EpiMark Hot Start Taq DNA polymerase, 0.2 mM dNTPs, 1 μL of the solution treated in step (4), and add deionized water to make the system 50 μL.
[0074] The PCR program was as follows: (a) denaturation at 95°C for 5 min; (b) denaturation at 95°C for 30 sec; (c) annealing at 55°C for 30 sec; (d) extension at 68°C for 30 sec; steps (b) to (d) were repeated 25 times; and extension at 68°C for 5 min. The PCR products were then sequenced by Sanger sequencing.
[0075] Results Figure 5 and 6 gmA3A can completely deaminate C at the TC and CC sites in DNA-C, and can also completely deaminate 5mC at the TC and CC sites in DNA-5mC, and they are read as T during the sequencing process, but it cannot deaminate 5hmC at the TC and CC sites in DNA-5hmC, so they are still read as C during the sequencing process.
[0076] dogA3A can completely deaminate C at the GC and AC sites in DNA-C, and can also completely deaminate 5mC at the GC and AC sites in DNA-5mC, which are read as T during the sequencing process, but it cannot deaminate 5hmC at the GC and AC sites in DNA-5hmC, which are still read as C during the sequencing process.
[0077] The above results indicate that gmA3A can be used for positioning analysis of 5hmC at TC and CC sites, while dogA3A can be used for positioning analysis of 5hmC at GC and AC sites.
[0078] The above specific embodiments describe the implementation of the present invention in detail, but 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, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.
Claims
1. An application of a green monkey-derived deaminase and a dog-derived deaminase in single-base resolution sequencing analysis of 5hmC modification in DNA, characterized in that: The DNA fragment to be sequenced is denatured to form a single-stranded DNA, and the single-stranded DNA is deaminated using the green monkey deaminase or the dog deaminase to inactivate the green monkey deaminase or the dog deaminase, and the treated samples are amplified and sequenced, and the site read as C in the sequencing result is the 5hmC site in the DNA fragment to be sequenced.
2. The use of the green monkey-derived deaminase and the dog-derived deaminase in single-base resolution sequencing analysis of 5hmC modification in DNA according to claim 1, characterized in that: The green monkey-derived deaminase or dog-derived deaminase is digested and inactivated by using a protease.
3. A single-base resolution sequencing analysis method for 5hmC modification in DNA, characterized in that: The DNA fragment to be sequenced is denatured to form a single-stranded DNA, the single-stranded DNA is deaminated with the green monkey-derived deaminase or the dog-derived deaminase to inactivate the green monkey-derived deaminase or the dog-derived deaminase, the treated sample is amplified and sequenced, and the site read as C in the sequencing result is the 5hmC site in the DNA fragment to be sequenced.
4. The single-base resolution sequencing analysis method for 5hmC modification in DNA according to claim 3, characterized in that: The method for denaturing the DNA fragment to be sequenced is: adding DMSO to the solution containing the DNA fragment to be sequenced, incubating at 95° C. for 10-20 min, and immediately transferring to an ice water bath for 2-5 min to denature the DNA fragment to be sequenced to form single-stranded DNA.
5. The single-base resolution sequencing analysis method for 5hmC modification in DNA according to claim 3, characterized in that: Performing deamination treatment on the DNA fragment to be sequenced using a deamination reaction buffer, wherein the deamination reaction buffer contains the green monkey-derived deaminase or the dog-derived deaminase; Preferably, it also contains 20 mM 2-morpholineethanesulfonic acid and has a pH value of 6.
5.
6. The single-base resolution sequencing analysis method for 5hmC modification in DNA according to claim 5, characterized in that: In the deamination reaction buffer, the concentrations of the green monkey-derived deaminase and the dog-derived deaminase are 10-20 μM, respectively.
7. The single-base resolution sequencing analysis method for 5hmC modification in DNA according to claim 6, characterized in that: The temperature of the deamination treatment is 37°C and the treatment time is 2-4 h.
8. The single-base resolution sequencing analysis method for 5hmC modification in DNA according to claim 3, characterized in that: The green monkey-derived deaminase or dog-derived deaminase is digested and inactivated by using a protease.
9. A product for single-base resolution sequencing analysis of 5hmC modification in DNA, characterized in that: The product contains green monkey-derived deaminase or dog-derived deaminase.
10. The product for single-base resolution sequencing analysis of 5hmC modification in DNA according to claim 9, characterized in that: The product is a detection kit.