A single-base resolution positioning analysis method and kit for 5-formylcytosine in DNA

Abstract

The present invention provides a single-base resolution positioning analysis method and kit for 5-aldehyde cytosine in DNA. The method comprises: subjecting DNA containing 5-aldehyde cytosine to a chemical derivatization labeling reaction with biotin hydroxylamine, followed by purification to obtain a labeled DNA double-strand; denaturing the labeled DNA double-strand to obtain a DNA single-strand; deaminating the single-stranded DNA using cytosine deaminase A3A protein; and then enzymatically digesting and removing the cytosine deaminase A3A protein to obtain a deaminated single-stranded DNA; subjecting the deaminated single-stranded DNA to a PCR reaction to obtain an amplified product; and sequencing the amplified product to obtain the site information of 5-aldehyde cytosine. This method does not rely on bisulfite treatment, has high selectivity, is simple to operate, and can directly obtain single-base resolution positioning information of 5-aldehyde cytosine in DNA.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a single-base resolution positioning analysis method and a kit for 5-formylcytosine in DNA. Background Art

[0002] 5-Methylcytosine (5mC), considered the fifth base in genomic DNA, can be further oxidized by TET (ten-eleven translocation) dioxygenases to form 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC and 5caC are recognized and excised from DNA by thymine DNA glycosylase (TDG), ultimately converting them to unmodified cytosine (C). Therefore, 5fC and 5caC clearly mark the activation of methylation in the mammalian genome. In 2011, 5fC and 5caC were first discovered in mouse embryonic stem cells (mESCs). However, the abundance of 5fC in mESCs (0.002% of all C) is much lower than that of 5hmC (0.39% of all C) and 5mC (3% of all C). Despite its very low abundance, 5fC represents an important epigenetic mark in mammals and plays a crucial role in gene regulation and the development of certain diseases. To further understand the biological functions of 5fC, it is necessary to accurately determine the distribution of 5fC across the genome.

[0003] Several methods are currently available for localizing and analyzing 5fC. One type involves non-single-base resolution methods, such as chemical labeling (O-(biotinylcarbazoylmethyl)hydroxylamine, ARP), which utilizes an aldehyde-amine condensation reaction between an aldehyde group and an amino group to specifically label and enrich 5fC. Enzymatic methods (β-glucosyltransferase) utilize sodium borohydride to specifically reduce 5fC to 5hmC, and then use β-glucosyltransferase to add an azide group to the resulting 5hmC, thereby enriching 5fC. Antibody-based methods (modification-specific antibodies) utilize antibodies to enrich 5fC. These methods all enrich DNA fragments containing 5fC modifications through reactions (chemical or enzymatic) and then sequence them, resulting in low resolution. Another type involves bisulfite sequencing, such as 5fC-assisted bisulfite sequencing (fCAB-seq) and reduced bisulfite sequencing (redBS-seq). In standard bisulfite sequencing (BS-seq), bisulfite treatment deaminates C, 5fC, and 5caC in DNA, which are read as thymine (T) during PCR amplification. However, 5mC and 5hmC are not deaminated, and the C remains unchanged during the subsequent PCR process. In fCAB sequencing, hydroxylamine first reacts with 5fC, and the resulting group acts as a protective group, preventing 5fC from being deaminated. Therefore, by comparing the data obtained from BS-seq (where 5fC is read as T) with the data obtained from fCAB-seq (where 5fC is read as C), 5fC can be located. The redBS-seq sequencing method is similar, using sodium borohydride to reduce 5fC to 5hmC, so it is not deaminated during subsequent bisulfite treatment and the C remains unchanged during sequencing. These two bisulfite-based sequencing methods require harsh chemical deamination conditions, which leads to the degradation of a large amount of DNA; and because the 5fC content is extremely low, subsequent sequencing requires very high costs; and site analysis requires comparing sequencing data obtained from two different experiments.

[0004] Therefore, in order to solve the above technical problems, it is necessary to develop a single-base resolution positioning analysis method for 5-formylcytosine in DNA. Summary of the Invention

[0005] In order to solve the technical problem, the present invention provides a single-base resolution positioning analysis method for 5-formylcytosine in DNA, which has strong selectivity, high sensitivity and easy operation, and does not require a bisulfite treatment process.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect of the present invention, a method for single-base resolution positioning analysis of 5-formylcytosine in DNA is provided, the method comprising:

[0008] DNA containing 5-formylcytosine is chemically derivatized and labeled with biotin hydroxylamine, and then purified to obtain labeled DNA double strands;

[0009] Denaturing the labeled DNA double strand to obtain a single strand of DNA, then deaminating the single strand of DNA using cytosine deaminase A3A protein, and then enzymatically digesting and removing the cytosine deaminase A3A protein to obtain deaminated single strand of DNA;

[0010] Performing a PCR reaction on the deaminated single-stranded DNA to obtain an amplified product;

[0011] The amplified product is sequenced to obtain the site information of 5-aldehyde cytosine.

[0012] Furthermore, the DNA containing 5-formylcytosine is subjected to a chemical derivatization labeling reaction with biotin hydroxylamine, comprising:

[0013] The DNA containing 5-formylcytosine, biotin hydroxylamine and 2-morpholinoethanesulfonic acid buffer were mixed and shaken.

[0014] Furthermore, the concentration of the 2-morpholineethanesulfonic acid buffer is 80-120 mM, pH 5.0; the concentration of the biotin hydroxylamine is 4-6 μM.

[0015] Furthermore, the deamination reaction conditions are as follows: DNA is reacted in a reaction system containing 10-30 mM 2-(N-morpholino)ethanesulfonic acid, 0.05-0.5% Triton X-100, pH 5.5-7.5, and 1-10 μM A3A protein at 30-45° C. for 0.5-4 hours.

[0016] Furthermore, the enzymatic digestion to remove the cytosine deaminase A3A protein comprises:

[0017] After the deamination reaction is completed, proteinase K is added and reacted at 52-58° C. for 25-35 minutes to digest and remove the A3A protein, and then the proteinase K is inactivated by high temperature treatment. The conditions of the high temperature inactivation treatment include: incubating in a 92-98° C. water bath for 5-20 minutes.

[0018] In a second aspect of the present invention, there is provided a use of a combination of biotin hydroxylamine and cytosine deaminase A3A protein in an analytical method for single-base resolution positioning of 5-formylcytosine in DNA.

[0019] In a third aspect of the present invention, a kit for locating 5-formylcytosine in DNA at single-base resolution is provided, the kit comprising biotin hydroxylamine and cytosine deaminase A3A protein.

[0020] Furthermore, the kit further comprises:

[0021] The reaction system for chemical derivatization labeling reaction using biotin hydroxylamine: biotin hydroxylamine and 2-morpholineethanesulfonic acid buffer;

[0022] The reaction system for deamination using cytosine deaminase A3A protein: 2-(N-morpholino)ethanesulfonic acid, TritonX-100.

[0023] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0024] The present invention provides a single-base resolution positioning analysis method for 5-formylcytosine in DNA. The principle is shown in Figure 1 A3A deaminase deaminates C, 5mC, 5hmC, and 5fC in DNA to U, thereby converting C to T. The amino group in biotin hydroxylamine specifically reacts with the aldehyde group of 5fC. The resulting group disrupts the A3A protein's deamination of the amino group on 5fC. Therefore, only labeled 5fC remains undeaminated. Therefore, we exploit the differential deamination of 5fC-biotin and C, 5mC, and 5hmC by A3A protein to achieve single-base localization analysis of 5fC modifications in DNA. This approach offers the following advantages:

[0025] (1) The present invention is simple to operate, does not require complicated sample processing, and can be directly used for PCR after deamination.

[0026] (2) The present invention does not involve bisulfite treatment.

[0027] (3) The A3A protein used in the present invention has a high deamination efficiency for C, 5mC, and 5hmC (C>99.9%, 5mC>99.5%, 5hmC>93%), while the deamination efficiency for 5fC-biotin is extremely low (<4%), thus enabling single-base resolution localization analysis of 5fC.

[0028] (4) The present invention does not require expensive third-generation sequencing technology, and second-generation sequencing can complete the single-base resolution positioning analysis of 5fC modification on the entire genome.

[0029] (5) The method of the present invention can be widely used to analyze 5fC sites in different samples, which is conducive to further studying the biological function of 5fC.

[0030] (6) The present invention does not involve bisulfite treatment and can achieve single-base resolution positioning analysis of 5fC. Compared with previous methods, it is simpler and is conducive to popularization and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following is a brief introduction to the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0032] Figure 1 The chemical formula for the reaction of 5fC with biotin hydroxylamine in the present invention is ( Figure 1 A) and schematic diagram of single-base resolution localization of 5fC using the deamination effect of A3A protein ( Figure 1 B).

[0033] Figure 2 is the result of Experimental Example 1 in the present invention; wherein Figure 2 A is the detection effect of LC-MS / MS before and after biotin-hydroxylamine labeling of 5fC; Figure 2 B is the enrichment result.

[0034] Figure 3 This is the LC-MS / MS detection effect of the artificially synthesized DNA chain containing 5mC, 5mC and 5fC-biotin before and after treatment with A3A protein.

[0035] Figure 4 This is a comparison chart of the Sanger sequencing results of the artificially synthesized DNA chains containing 5mC, 5hmC and 5fC-biotin before and after treatment with A3A protein. DETAILED DESCRIPTION

[0036] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than for limiting the present invention.

[0037] Throughout this specification, unless otherwise specified, the terms used herein should be understood as having the same meaning as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of any conflict, the present specification shall take precedence.

[0038] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0039] The technical solution provided by the embodiments of the present invention is to solve the above technical problems, and the overall idea is as follows:

[0040] According to a typical embodiment of the present invention, a single-base resolution positioning analysis method for 5-formylcytosine in DNA is provided, the method comprising:

[0041] S1, chemically derivatizing and labeling DNA containing 5-formylcytosine with biotin hydroxylamine, followed by purification to obtain labeled DNA double strands;

[0042] In the step S1,

[0043] The chemical derivatization labeling reaction comprises: mixing and shaking DNA containing 5-formylcytosine, biotin hydroxylamine and 2-morpholineethanesulfonic acid buffer.

[0044] Preferably, the concentration of the 2-morpholineethanesulfonic acid buffer is 80-120 mM, pH 5.0; the concentration of the biotin hydroxylamine is 4-6 μM.

[0045] In a specific embodiment, 5fC is specifically labeled using 100 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.0) and 5 μM biotin hydroxylamine at 37° C. with shaking for 4 hours.

[0046] The conditions of the chemical derivatization labeling reaction are conducive to ensuring the activity of biotin hydroxylamine and the efficiency of the chemical derivatization labeling reaction.

[0047] The biotin hydroxylamine of the present application can be purchased from Shenzhen Ruijit Biotechnology Co., Ltd., Changsha Yifu Biotechnology Co., Ltd., Shanghai Zhenzhun Biotechnology Co., Ltd., etc., CAS No.: 139585-03-8.

[0048] The biotin hydroxylamine of the present application can also be obtained by synthesis, and the specific method of synthesis is as follows:

[0049] ① The following reagents were added to a round-bottom flask: diethylene glycol (1.0 g, triphenylphosphine (7.4 g) and N-hydroxyphthalimide (4.6 g). After stirring for 15 minutes, diisopropyl azodicarboxylate was added dropwise under ice bath. The mixture was heated to 25°C and stirred for 12 hours. The solvent was removed, and the resulting solid was dissolved with ether and vigorously stirred at 4°C for 30 minutes. The resulting precipitate was collected by filtration, and the resulting precipitate was recrystallized from ether once and then dried in vacuo. The white solid (Compound 1) was collected and directly used for the next step of hydrazinolysis.

[0050] ② Compound 1 (1.0 g) was dissolved in ethanol, and then hydrazine hydrate (1.2 mL) was added. The reaction was stirred vigorously at 25°C for 6 hours, and then the solvent was evaporated under vacuum. The resulting product was filtered to remove the solids, and the remaining oily substance was purified by silica gel column chromatography to obtain a clean oily substance, Compound 2.

[0051] ③ Add biotin, O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroboric acid, N,N-dimethylformamide (DMF), and triethylamine to a flask. Stir the mixture at 25°C for 2 hours. Then, add compound 2 containing DMF to the reaction solution. After continuing the reaction at 25°C for 12 hours, remove the solvent under vacuum. Purify the resulting white solid by silica gel column chromatography to obtain biotin hydroxylamine.

[0052] S2, denaturing the labeled DNA double strand to obtain a single strand of DNA, then deaminating the single strand of DNA using cytosine deaminase A3A protein, and then enzymatically digesting and removing the cytosine deaminase A3A protein to obtain deaminated single strand of DNA;

[0053] In the step S2,

[0054] The deamination reaction conditions are as follows: DNA is reacted at 30-45°C for 0.5-4 hours in a reaction system containing 10-30 mM 2-(N-morpholino)ethanesulfonic acid, 0.05-0.5% Triton X-100, pH 5.5-7.5, and 1-10 μM A3A protein. This buffer composition is chosen to ensure deaminase activity and deamination reaction efficiency.

[0055] The enzymatic digestion to remove the cytosine deaminase A3A protein comprises:

[0056] After the deamination reaction is complete, proteinase K is added and reacted at 52-58°C for 25-35 minutes to digest and remove the A3A protein. The proteinase K is then inactivated by high-temperature inactivation. The high-temperature inactivation conditions include incubation in a 92-98°C water bath for 5-20 minutes. These reaction conditions facilitate the inactivation of the cytosine deaminase A3A protein, thereby facilitating the subsequent PCR amplification process.

[0057] The cytosine deaminase A3A protein is described in the literature “Emily K. Schutsky, Jamie E. DeNizio1, Peng Hu, Monica Yun Liu, Christopher S. Nabel, Emily B. Fabyanic, Young Hwang, Frederic D. Bushman, Hao Wu,*, and Rahul M. Kohli. Nondestructive, base-resolution sequencing of 5-hydroxymethylcytosine using a DNA deaminase. Nat Biotechnol. 36. 1083–1090.” The amino acid sequence is shown below:

[0058] MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN.

[0059] S3, performing a PCR reaction on the deaminated DNA sample to obtain an amplified product;

[0060] The PCR reaction requires the use of specific primers to amplify the carboxymethyl-protected and deaminated DNA. The amplified product is subjected to agarose gel electrophoresis, gel excision, purification, and Sanger sequencing. Corresponding primers need to be designed for different DNA sequences to be tested.

[0061] S4. Sequence the amplified product to obtain the site information of 5-aldehyde cytosine.

[0062] If PCR amplification yields a DNA fragment of a specific sequence, the amplified product can be directly subjected to Sanger sequencing; if PCR amplification forms a DNA library, the amplified product will be subjected to high-throughput sequencing.

[0063] The method provided by the present invention can enrich 5fC with an extremely low content in DNA because it contains biotin.

[0064] According to another typical embodiment of the present invention, there is provided an application of a combination of biotin hydroxylamine and cytosine deaminase A3A protein in an analytical method for single-base resolution positioning of 5-formylcytosine in DNA.

[0065] According to another typical embodiment of the present invention, a kit for locating 5-formylcytosine in DNA with single-base resolution is provided, the kit comprising: biotin hydroxylamine and cytosine deaminase A3A protein.

[0066] The kit further comprises:

[0067] Reaction components for chemical derivatization labeling using biotin-hydroxylamine: biotin-hydroxylamine and 2-morpholinoethanesulfonic acid buffer;

[0068] The reaction components for deamination using cytosine deaminase A3A protein are: 2-(N-morpholino)ethanesulfonic acid and Triton X-100.

[0069] The common terms involved in the present invention are:

[0070] fC-DNA: DNA containing 5-formylcytosine;

[0071] The effects of the present invention will be described in detail below with reference to the examples and experimental data. Unless otherwise specified, the technical means used in the examples, including nucleic acid extraction, enzymatic hydrolysis, and polymerase chain reaction, are conventional means well known to those skilled in the art.

[0072] Example 1: A single-base resolution positioning analysis method for 5-formylcytosine in DNA

[0073] 1. Synthesize DNA chains containing 5mC, 5hmC, and 5fC-biotin modifications (5mC-DNA, 5hmC-DNA, and 5fC-biotin-DNA), respectively, as shown in Table 1.

[0074] Table 1

[0075]

[0076] 2. Take 50 ng of each of the three DNA samples above, denature the double-stranded DNA into single strands, add A3A protein (final concentration 5 μM), MES reaction buffer, pH 6.5 (final concentration 100 mM), and add water to a 20 μL reaction volume. Incubate the reaction at 37°C for 4 hours. After the reaction, add 2 μL of proteinase K and incubate at 55°C for 30 minutes. Heat at 95°C for 10 minutes to inactivate proteinase K.

[0077] 3. Take a certain amount of DNA after the above reaction for PCR amplification. The reaction system (50 μL) contains a certain amount of deaminated DNA chain, 10 μL Hot Start Taq reaction buffer, 1 μL 10 mM dNTPs, 0.5 μL Hot Start Taq DNA polymerase, 2.5 μL forward primer (10 μM), 2.5 μL reverse primer (10 μM), and H O. PCR amplification protocol: (1) denaturation at 95°C for 30 seconds; (2) denaturation at 95°C for 20 seconds; (3) annealing at 58°C for 30 seconds; (4) extension at 72°C for 1 minute; steps (2)-(4) repeated 30 times; and finally extension at 72°C for 5 minutes. PCR products were then subjected to Sanger sequencing.

[0078] See the results Figure 4 The A3A protein completely deaminated the C, 5mC, and 5hmC in 5mC-DNA and 5hmC-DNA, and these were read as Ts during subsequent sequencing. The C in 5fC-biotin-DNA was completely deaminated and read as Ts during sequencing, but the 5fC in 5fC-biotin-DNA was not completely deaminated by the A3A protein and was still read as C during sequencing. This result indicates that chemical derivatization combined with A3A deaminase can be used to locate and analyze 5fC in DNA.

[0079] Experimental Example 1: Biotin-hydroxylamine labeling and enrichment of 5fC

[0080] 1. Incubate 5 μM biotin-hydroxylamine, 100 mM MES buffer (pH 5.0), and 200 ng of 5fC-DNA at 37°C and 1200 rpm for 4 hours. Then, purify the DNA in the reaction system by ethanol precipitation to remove excess biotin-hydroxylamine.

[0081] 2. Ethanol precipitation: Add half the volume of NH4Ac to the reaction mixture from the previous step, vortex to mix, then add 2μL of glycogen, vortex to mix, and add 4 volumes of anhydrous ethanol. After mixing, place the system in a -20°C refrigerator overnight or -70°C refrigerator to precipitate for at least 5 hours. Centrifuge at 13,000 rpm, 4°C for 30 minutes, remove the supernatant, and rinse twice with 1 mL of 80% ice-cold ethanol to remove salts. Then, open the lid and allow to stand at room temperature to remove any residual ethanol. Finally, reconstitute with a predetermined volume of water.

[0082] 3. Enrichment of DNA containing 5fC with biotin-hydroxylamine:

[0083] (1) Wash the streptavidin magnetic beads. Take two 10 μL aliquots of streptavidin magnetic beads and wash them three times with 100 μL 1x wash (bind) buffer.

[0084] (2) Loading: Add 50 μL of 2x bind buffer to 50 μL of DNA (0.1-10 ng of 5fC-biotin-DNA and 1 μg of ordinary double-stranded DNA) and rotate at room temperature for 20 minutes.

[0085] (3) Wash the DNA, discard the supernatant, and retain the magnetic beads. Then, wash three times with 1 mL of 1x bind buffer and twice with 1 mL of H2O. Redissolve the beads in 20 μL of H2O and perform qPCR to determine the enrichment factor. 2x bind buffer: 40 mM tris; 2 M NaCl; 2 mM EDTA; 0.04% tween.

[0086] See the results Figure 2 After reaction with biotin-hydroxylamine, 5fC was almost completely consumed, with a labeling efficiency of up to 95%, demonstrating that biotin-hydroxylamine can specifically and efficiently label 5fC in DNA. qPCR results showed that the enrichment of 5fC-DNA after biotin labeling and enrichment was as high as 4000-fold, demonstrating that our method can enrich extremely low-abundance 5fC in the genome.

[0087] Experimental Example 2: Analysis of Cytosine Deaminase Activity of A3A Protein

[0088] 1. Prepare reaction buffer: dissolve MES in deionized water to a final concentration of 200 mM and adjust its pH to 6.5.

[0089] 2. Synthesize DNA chains containing C, 5mC, 5hmC and 5fC-biotin respectively.

[0090] 3. Take 50 ng of each of the four DNA samples above, denature the double-stranded DNA into single strands, add A3A protein (final concentration 5 μM), MES reaction buffer, pH 6.5 (final concentration 100 mM), and add water to a 20 μL reaction volume. Incubate the reaction at 37°C for 4 hours. After the reaction, add 2 μL of proteinase K and incubate at 55°C for 30 minutes. Heat at 95°C for 10 minutes to inactivate proteinase K.

[0091] 4. The DNA chain treated with deamination in step 3 is enzymatically hydrolyzed, and the hydrolysis product is analyzed and detected by LC-MS / MS.

[0092] See the results Figure 3 Extracted ion chromatograms of DNA strands containing C, 5mC, 5hmC, and 5fC-biotin before and after treatment with A3A protein showed that the peaks for C, 5mC, and 5hmC almost completely disappeared after treatment, indicating that A3A protein efficiently removes the amino groups from C, 5mC, and 5hmC. However, the peak intensity for 5fC-biotin did not change significantly after treatment, indicating that A3A protein has no ability to deaminize 5fC-biotin. These experimental results demonstrate that the differential deamination ability of A3A protein enables the differentiation of 5fC from C, 5mC, and 5hmC in cytosine.

[0093] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements, but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0094] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0095] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A single-base resolution positioning analysis method for 5-formylcytosine in DNA, characterized in that: The method comprises: DNA containing 5-formylcytosine is chemically derivatized and labeled with biotin hydroxylamine, and then purified to obtain labeled DNA double strands; The labeled DNA double strand is denatured to obtain a single strand of DNA, and then the single strand of DNA is deaminated using cytosine deaminase A3A protein, followed by enzymatic digestion to remove the cytosine deaminase A3A protein, to obtain deaminated single strand of DNA; the amino acid sequence of the cytosine deaminase A3A protein is as follows: MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN; Performing a PCR reaction on the deaminated single-stranded DNA to obtain an amplified product; The amplified product is sequenced to obtain the site information of 5-aldehyde cytosine, wherein the method is not used for diagnosis or treatment of diseases.

2. A single-base resolution positioning analysis method for 5-formylcytosine in DNA according to claim 1, characterized in that: The chemical derivatization labeling reaction of the DNA containing 5-formylcytosine and biotin hydroxylamine comprises: The DNA containing 5-formylcytosine, biotin hydroxylamine and 2-morpholinoethanesulfonic acid buffer were mixed and shaken.

3. A single-base resolution positioning analysis method for 5-formylcytosine in DNA according to claim 2, characterized in that: The concentration of the 2-morpholinoethanesulfonic acid buffer is 80-120 mM, pH 5.0; the concentration of the biotin hydroxylamine is 4-6 μM.

4. The single-base resolution positioning analysis method for 5-formylcytosine in DNA according to claim 1, characterized in that: The conditions for the deamination reaction are as follows: DNA is reacted in a reaction system containing 10-30 mM 2-(N-morpholino)ethanesulfonic acid, 0.05-0.5% Triton X-100, pH 5.5-7.5, and 1-10 μM A3A protein at 30-45° C. for 0.5-4 hours.

5. The single-base resolution positioning analysis method for 5-formylcytosine in DNA according to claim 1, characterized in that: The enzymatic digestion to remove the cytosine deaminase A3A protein comprises: After the deamination reaction is completed, proteinase K is added and the mixture is heated at 52 to 58 o The mixture was reacted at 92-98°C for 25-35 minutes to digest and remove the A3A protein, and then the proteinase K was inactivated by high temperature treatment. The conditions of the high temperature inactivation treatment include: incubating in a 92-98°C water bath for 5-20 minutes.

6. Use of a combination of biotin hydroxylamine and the cytosine deaminase A3A protein according to claim 1 in the preparation of a kit for locating 5-formylcytosine in DNA at single base resolution.

7. A kit for locating 5-formylcytosine in DNA at single base resolution, characterized in that: The kit comprises: biotin hydroxylamine and the cytosine deaminase A3A protein according to claim 1.

8. A kit for locating 5-formylcytosine in DNA at single base resolution according to claim 7, characterized in that: The kit further comprises: Reaction components for chemical derivatization labeling using biotin-hydroxylamine: biotin-hydroxylamine and 2-morpholinoethanesulfonic acid buffer; Reaction components for deamination using cytosine deaminase A3A protein: 2-(N-morpholino)ethanesulfonic acid, Triton X-100.

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