CN114591440B

CN114591440B - Recombinant TET enzyme MBD4-NgTET1 and application thereof in improving 5caC (cubic-alternating current) ratio in TET enzyme oxidation product

Info

Publication number: CN114591440B
Application number: CN202111541359.6A
Authority: CN
Inventors: 侯策; 韦磊; 江翱; 陈晶晶; 黄开瑜; 滕以刚; 曹振; 宋东亮
Original assignee: Yisheng Biotechnology Shanghai Co ltd
Current assignee: Yisheng Biotechnology Shanghai Co ltd
Priority date: 2021-10-18
Filing date: 2021-10-18
Publication date: 2023-06-20
Anticipated expiration: 2041-10-18
Also published as: CN114591441A; CN114591439B; CN113637053A; CN114591441B; CN114195902A; CN114591440A; CN114591439A; CN114195902B; CN113637053B

Abstract

The invention provides a recombinant protein domain for enhancing TET enzyme activity, which is a DNA Methylation Binding Domain (MBD), and the amino acid sequences of MBDs 1-4 are shown in SEQ ID 1-4. Recombinant proteins MBD-TET formed by fusion of MBD with a TET enzyme can significantly enhance the oxidative activity of TET enzymes including NgTET1, mTET1CD, mTET2CDT, hTET1CD and hTET2CDT on 5 mC. In addition, the invention also provides a simple, convenient and rapid high-flux DNA methylation detection flow, and improves the sensitivity and accuracy of DNA methylation detection technology based on TET enzyme oxidation reaction.

Description

Recombinant TET enzyme MBD4-NgTET1 and application thereof in improving 5caC (cubic-alternating current) ratio in TET enzyme oxidation product

The present application is a divisional application of patent application with application number 202111207210.4 entitled "recombinant protein domain and its coding DNA, enhanced TET enzyme and whole genome DNA methylation detection method" filed 10/18/2021.

Technical Field

The invention relates to a recombinant protein structural domain, encoding DNA thereof, enhanced TET enzyme and a whole genome DNA methylation detection method, belonging to the technical field of biology.

Background

DNA cytosine methylation (5 mC) is the most common form of base modification in DNA, accounting for about 1% -8% of all cytosines, and is called the "fifth base". The DNA methylation has obvious correlation with the chromatin state and the gene transcription activity level, and is an effective basis for predicting the gene expression level. Thus, DNA methylation level detection is an effective means of clinical disease diagnosis. The existing DNA methylation detection technology mainly relies on a reverse screening bisulfite conversion method, and the principle is that unmethylated cytosine is converted into uracil by utilizing bisulfite and then is converted into thymine by PCR amplification. The method has the defects of large DNA damage, high background noise, low accuracy and the like. In recent years, methylation detection technology by an enzyme conversion method has the advantages of small DNA damage, low background noise, high accuracy, good data quality and the like, and becomes an important concern in the field of DNA methylation detection.

DNA methylol transferase TET is a ubiquitous alpha-ketoglutarate (alpha-KG) and Fe2+ -dependent dioxygenase enzyme in eukaryotes, and has a high degree of conservation during bioaugmentation. TET enzyme is a key protein in DNA demethylation, and can convert 5mC into 5caC by three-step oxidation (5 mC-5hmC-5fC-5 caC). The current DNA cytosine methylation detection technology by an enzyme conversion method is dependent on the ability of TET enzyme to catalyze methylated cytosine, and TET protein is a core protein of the DNA methylation detection technology by the enzyme conversion method, so that the method has great engineering and application values. The obtained recombinant TET enzyme mutant with high activity has important value for the development of in-vitro DNA methylation technology and the application in the field of disease diagnosis.

The invention provides a recombinant protein domain for enhancing TET enzyme activity, which is a DNA Methylation Binding Domain (MBD), and the amino acid sequences of MBDs 1-4 are shown in SEQ ID 1-4. Recombinant proteins MBD-TET formed by fusion of MBD with a TET enzyme can significantly enhance the oxidative activity of TET enzymes including NgTET1, mTET1CD, mTET2CDT, hTET1CD, and hTET2 CDT. In addition, the invention also provides a reaction buffer suitable for MBD-TET and a simple and rapid high-throughput DNA methylation detection flow, which can effectively enhance the oxidative activity of MBD-TET to 5mC and improve the sensitivity and accuracy of DNA methylation detection technology based on TET enzyme oxidation reaction.

Disclosure of Invention

It is a first object of the present invention to provide a recombinant protein domain that enhances TET enzyme activity.

The recombinant protein domain is a DNA Methylation Binding Domain (MBD), and the amino acid sequences of the MBDs 1-4 are shown as SEQ ID 1-4, wherein the MBD1 has the optimal effect.

And the coding DNA of the MBD, the nucleotide sequence of which is shown in any one of SEQ ID No. 10-13.

The second object of the present invention is to provide an enhanced TET enzyme, wherein the amino acid sequence of hTET2CDT of NgTET1, mTET1CD, mTET2CDT, hTET1CD or hTET2CDT is shown in SEQ ID 5-9, respectively, and the NgTET1 is optimal, wherein the amino acid sequence of the above recombinant protein domains MBD1, MBD2, MBD3 or MBD4 is linked to the amino terminus of NgTET1, mTET1CD, mTET2CDT, hTET1CD, or hTET2CDT through a GGGS linker peptide.

Still another object of the present invention is to provide a method for detecting methylation of whole genome DNA, characterized by comprising the steps of:

(1) Oxidizing the template DNA using the enhanced TET enzyme described above;

(2) Treating with a reducing agent, and neutralizing with alkali;

(3) Recovering DNA;

(4) Constructing a DNA library.

Preferably, the oxidation reaction described in step (1) is followed by the addition of a TET enzyme reaction buffer which increases the 5caC product duty cycle of the TET enzyme oxidation 5mC reaction.

Preferably, the TET enzyme reaction buffer contains: 1-100 mM 3- (N-morpholino) propanesulfonic acid sodium salt, 1-100 mM bis (2-hydroxyethyl) amino-tris (hydroxymethyl) methane, 1-100 mM hydroxyethyl piperazine ethanethiol sulfonic acid, 1-300 mM sodium chloride, 0.1-10 mM ascorbic acid, 0.1-10 mM citric acid, 0.1-20 mM alpha-ketoglutaric acid, 0.1-20 mM 1, 3-acetonedicarboxylic acid, 0.1-20 mM adenosine triphosphate, 0.1-10 mM tetrafluoro-p-benzoquinone, 0.1-10 mM tetrachloro-p-benzoquinone, 0.1-10 mM tetrabromo-p-benzoquinone, 0.1-10 mM tetraiodobenzoquinone, 0.01-2 mM tris (2-carboxyethyl) phosphine, 0.01-2 mM dithiothreitol.

Preferably, the TET enzyme reaction buffer further contains an iron salt compound.

Preferably, the ferric salt compound is used at a concentration of 0.1 to 50 mM, with 1 to 10 mM being most preferred.

Preferably, the iron salt compound is ferrous sulfate, ferrous ethylenediamine sulfate, ferrous ammonium sulfate, ferrous citrate, ferrous chloride, 2, 6-bis (1, 1-bis (2-pyridine) ethyl) pyridine-ferric oxide complex ([ Fe) ^IV (O)(Py ₅ Me ² H)] ²⁺ ) One or more of ferrous sulfate and [ Fe ] ^IV (O)(Py ₅ Me ² H)] ²⁺ Optimally.

Preferably, the TET enzyme reaction buffer solution also comprises 0.001-1% concentration hydrogen peroxide and 0.001-1% concentration potassium permanganate.

Preferably, the reaction temperature of the oxidation reaction described in step (1) is 10-40 ℃, with 30-40 ℃ being optimal.

Preferably, the oxidation reaction in step (1) is carried out for a reaction time of 0.5 to 2. 2 h.

Preferably, the reducing agent in step (2) comprises one or more of lithium aluminum hydride, lithium boron hydride, sodium borohydride acetate, sodium cyanoborohydride, diisobutylaluminum hydride, lithium triethylborohydride, ammonia borane, pyridine borane, 2-methylpyridine borane, sodium borohydride, ammonia borane lithium, pyrrolidone lithium borohydride, borane ethylenediamine, borane dimethylamine, borane morpholine complex, borane triphenylphosphine, borane diphenylphosphine, borane triethylamine, 4-methylmorpholine borane, borane trimethylamine complex, 5-ethyl-2-methylpyridine borane, N-diisopropylethylamine borane, borane tetrahydrofuran complex, borane dimethyl sulfide complex. Of these, lithium aluminum hydride, ammonia borane, pyridine borane, 2-methyl pyridine borane, ammonia borane lithium, tert-butylamine borane, borane morpholine complexes and borane tetrahydrofuran complexes are most preferred.

Preferably, the treatment concentration of the reducing agent in step (2) is in the range of 100-2000 mM, with 200-500 mM being most preferred.

Preferably, the treatment temperature of the reducing agent in step (2) is 20-50 ℃, with 30-40 ℃ being the most preferred.

Preferably, the treatment time of the reducing agent in step (2) is 2-24 h, with 4-12 h being most preferred.

Preferably, the alkali neutralization in the step (2) is performed by sodium hydroxide or potassium hydroxide, and the final concentration in the reaction system is 0.5-1.5. 1.5M.

Preferably, the DNA recovery method in step (3) refers to magnetic bead recovery, column recovery or extraction precipitation recovery, wherein the magnetic bead recovery is optimal.

The DNA library construction method in the step (4) adopts a conventional double-stranded DNA library construction method or a single-stranded DNA library construction method.

The invention provides a recombinant protein domain for enhancing TET enzyme activity, which is a DNA Methylation Binding Domain (MBD), and the amino acid sequences of MBDs 1-4 are shown in SEQ ID 1-4. The recombinant protein MBD-TET formed by fusion of MBD and a TET enzyme active structural region can significantly enhance the oxidation activity of TET enzymes including NgTET1, mTET1CD, mTET2CDT, hTET1CD and hTET2 CDT. In addition, the invention also provides a reaction buffer suitable for MBD-TET and a simple and rapid high-throughput DNA methylation detection flow, can effectively enhance the oxidation activity of MBD-TET to 5mC, improves the sensitivity and accuracy of DNA methylation detection technology based on TET enzyme oxidation reaction, and can be applied to disease diagnosis, in particular to the fields of disease diagnosis and tumor early screening.

Drawings

FIG. 1 is a graph comparing conventional TAPS and optimized TAPS conversion.

FIG. 2 effect of MBD domain on TET enzyme activity.

FIG. 3 effect of MBD fusion TET enzyme on m5C oxidation product ratio.

FIG. 4 is a schematic diagram of a conventional TAPS and optimized TAPS flow.

FIG. 5 comparison of conventional TAPS and optimized TAPS library yields.

FIG. 6 comparison of TET enzyme treatment time versus TAPS conversion.

FIG. 7 comparison of reductant species versus TAPS conversion.

FIG. 8 comparison of reductant treatment time versus TAPS conversion.

FIG. 9 comparison of the effect of MBD-NgTET1 and optimization procedure on TAPS detection on DNA methylation.

Detailed Description

The following describes the embodiments of the present invention further with reference to the drawings. In the sequence table, SEQ ID No.1 is the amino acid sequence of MBD1, SEQ ID No.2 is the amino acid sequence of MBD2, SEQ ID No.3 is the amino acid sequence of MBD3, SEQ ID No.4 is the amino acid sequence of MBD4, SEQ ID No.5 is the amino acid sequence of NgTET1, SEQ ID No.6 is the amino acid sequence of mTET1CD, SEQ ID No.7 is the amino acid sequence of mTET2CDT, SEQ ID No.8 is the amino acid sequence of hTET1CD, SEQ ID No.9 is the amino acid sequence of hTET2CDT, SEQ ID No.10 is the nucleotide sequence of MBD1, SEQ ID No.11 is the nucleotide sequence of MBD2, SEQ ID No.12 is the nucleotide sequence of MBD3, and SEQ ID No.13 is the nucleotide sequence of MBD 4.

The examples provided are merely illustrative of the methods of the present invention and are not intended to limit the remainder of the disclosure in any way whatsoever.

Example 1: measurement of specific Activity of MBD-TET recombinant proteases.

Enzyme specific activities of the respective MBD-TET recombinant proteins were determined according to the procedure of the specification using an Epigenase 5 mC-hydroxylase TET activity/inhibition assay kit (fluorescence method).

The schematic diagram of the enzyme activity effect after transformation is shown in fig. 1, and the result is shown in fig. 2, and the activity of each TET enzyme can be obviously enhanced by the MBD-fused TET enzyme.

Example 2: measurement of the oxidative Capacity of each recombinant protease MBD-TET on 5mC oligo.

In this example, the ratio of MBD-TET enzyme to 5mC oxidation product was determined as follows:

TABLE 1

Component (A)	Dosage of
		5mC oligo	1-100 ng
10 XTET enzyme reaction buffer	3 μL
		10. Mu M of each TET enzyme	2-10 μL
Supplement ddH ₂ O to	30 μL

The 10 XTE enzyme reaction buffer comprises 1-100 mM 3- (N-morpholinyl) propanesulfonic acid sodium salt, 1-100 mM bis (2-hydroxyethyl) amino-tris (hydroxymethyl) methane, 1-100 mM hydroxyethyl piperazine ethanesulfonic acid, 1-300 mM sodium chloride, 0.1-10 mM ascorbic acid, 0.1-10 mM citric acid, 0.1-20 mM alpha-ketoglutarate, 0.1-20 mM 1, 3-acetone dicarboxylic acid, 0.1-20 mM adenosine triphosphate, 0.1-10 mM tetrafluoro-p-benzoquinone, 0.1-10 mM tetrachloro-p-benzoquinone, 0.1-10 mM tetrabromo-benzoquinone, 0.1-10 mM tetraiodobenzoquinone, 0.01-2 mM tris (2-carboxyethyl) phosphine, 0.01-2 mM dithiothreitol, 0.1-50 mM subunitFerric salt (ferrous sulfate, ethylenediamine ferrous sulfate, ferrous ammonium sulfate, ferrous citrate, ferrous chloride, 2, 6-bis (1, 1-bis (2-pyridine) ethyl) pyridine-ferric oxide complex ([ Fe) ^IV (O)(Py ₅ Me ² H)] ²⁺ ) Hydrogen peroxide or potassium permanganate, etc. in an amount of 0.001% -1%.

The reaction is carried out at 37 ℃ for 0.5-2 h.

After the reaction is finished, 1 ul is added to stop the reaction solution, and the reaction is carried out for 3-10 min at 50 ℃. The DNA was recovered using either the magnetic bead method or QIAquick Nucleotide Removal Kit (Qiagen) for LC-MS/MS analysis. The 5mC, 5hmC, 5fC and 5caC content ratio analysis flow is Hideharu Hashimotoet al（Nature，2013）。

TABLE 2

The experimental results are shown in table 2 and fig. 3, the MBD-fused TET enzyme was able to significantly enhance the activity of each TET enzyme, with the final oxidation product 5caC significantly higher than the non-MBD-fused TET enzyme. Wherein the maximum ratio of the final oxidation product of MBD1-NgTET1 can reach 90.4%, which is obviously higher than that of other MBD functional domains.

Example 3: MBD1-NgTET 1-based simple high throughput sequencing methods.

In example 2, we verified that the final product 5caC produced by NgTET1 during oxidation of 5mC was the most abundant, and that the MBD 1-fused NgTET1 was able to significantly increase the 5caC in the oxidized product, indicating that MBD1-NgTET1 proteins have application in the high throughput DNA methylation detection technology TAPS based on TET proteins (Liu Y, siejka-Zieli ń ska P, et al.Nature Biotechnology2019). However, the existing TAPS technology has complicated flow operation, long time consumption and large loss, and is difficult to realize large-scale industrialization. According to the optimized TAPS flow and processing conditions of the MBD-NgTET1 and the corresponding TET enzyme reaction buffer, the operation of TAPS is greatly simplified, the processing time of TAPS is shortened, and the sensitivity and accuracy of detecting DNA methylation by TAPS are improved. The specific implementation mode is as follows:

(1) MBD1-NgTET1 oxidizes template DNA:

TABLE 3 Table 3

Component (A)	Dosage of
		Control DNA CpG methylated pUC19	1-100 ng
10 XTET enzyme reaction buffer	3 μL
		10. Mu M of each TET enzyme	2-10 μL
Supplement ddH ₂ O to	30 μL

Treatment is carried out at 37 ℃ for 0.5-2 h.

(2) Reducing agent treatment, alkali neutralization:

adding 6-12 mu L sodium acetate and 100-4000 mM reducer (including aluminum lithium hydride, boron lithium hydride, sodium acetate boron hydride, sodium cyanoborohydride, diisobutyl aluminum hydride, triethyl boron hydride, ammonia boron hydride, pyridine boron hydride, 2-methyl pyridine boron hydride, sodium boron hydride, ammonia boron hydride, pyrrolidine boron hydride, tert-butyl amine boron hydride, boron ethane diamine, boron methane dimethylamine, boron morpholine complex, boron triphenyl phosphine, boron diphenyl phosphine, boron triethylamine, 4-methyl morpholine boron hydride, boron trimethylamine complex, 5-ethyl-2-methyl pyridine boron hydride, N-diisopropyl ethylamine boron hydride, boron tetrahydrofuran complex and boron dimethyl sulfide complex). The reaction is carried out at room temperature for 4-12 and h. After the reaction is finished, 5-30 ul of 3M NaOH is added and mixed uniformly.

(3) DNA recovery:

recovering by a magnetic bead method: adding 1.6 XDNA Hieff NGS cube DNA Selection Beads, and incubating for 5-10 min at room temperature. The PCR tube was placed on a magnetic rack. After the solution was clear, the supernatant was aspirated. After washing the beads twice with 80% ethanol, the supernatant was blotted. After standing at room temperature for 3-5 min, 21 ul of ddH2O was added for elution.

And (3) recovering by a column method: DNA was recovered using the DNA Clean & Concentrator-5 of Zymo Research according to the instructions.

Extraction and precipitation method: adding an equal volume of DNA extraction reagent, mixing uniformly, centrifuging for 10 min at 12000 and x g, taking the supernatant, and adding 1/10 volume of 3M sodium acetate and equal volume of isopropanol. After 2 h treatment at-80 ℃,12000x g was centrifuged at 4 ℃ for 20 min. The precipitate was washed once with 75% pre-chilled absolute ethanol, dried at room temperature and dissolved in 21 ul ddH2O.

(4) Constructing a DNA library:

and constructing a DNA library by using Hieff NGS Fast Tagment DNA Library Prep Kit for Illumina of the next holy organism.

The comparison of the conventional TAPS and the optimized TAPS flow is shown in FIG. 4. The results are shown in fig. 5-9, and the optimized TAPS significantly improved library yield (fig. 5). MBD 1-fused NgTET1 significantly enhanced the conversion of the TAPS procedure and reached essentially the highest value (98% or more) after 1h of treatment (fig. 6). The conversion of TAPS by the different reducing agents was different (FIG. 7), with the highest conversion (over 98%) achieved by treatment with lithium aluminum hydride, ammonia borane, pyridine borane, 2-methylpyridine borane, ammonia borane lithium, tert-butylamine borane, borane morpholine complex, and borane tetrahydrofuran complex. The treatment time of the reducing agent reaches the highest value (more than 98.5 percent) approximately at 4 to 8 hours. Therefore, we examined the effect of MBD-NgTET1, optimized TET enzyme reaction buffer, optimized reducing agent, and optimized TAPS procedure on DNA methylation detection, and found that MBD-NgTET1, optimized TET enzyme reaction buffer, optimized reducing agent all significantly improved the efficiency and accuracy of TAPS to detect DNA methylation (fig. 9). The optimization procedure did not reduce the efficiency and accuracy of the TAPS detection of DNA methylation (FIG. 9). The optimized TAPS has better accuracy, higher efficiency and stronger sensitivity.

In summary, the invention provides a recombinant protein domain for enhancing the activity of TET enzyme, which is a DNA Methylation Binding Domain (MBD), and the amino acid sequences of MBDs 1-4 are shown in SEQ ID 1-4. Recombinant proteins MBD-TET formed by fusion of MBD with a TET enzyme can significantly enhance the oxidative activity of TET enzymes including NgTET1, mTET1CD, mTET2CDT, hTET1CD, and hTET2 CDT. In addition, the invention also provides a reaction buffer suitable for MBD-TET and a simple and rapid high-throughput DNA methylation detection flow, can effectively enhance the oxidation activity of MBD-TET to 5mC, improves the sensitivity and accuracy of DNA methylation detection technology based on TET enzyme oxidation reaction, and can be applied to disease diagnosis, in particular to the fields of disease diagnosis and tumor early screening.

Sequence listing

<110> next san Jose Biotech (Shanghai) stock Co., ltd

<120> recombinant TET enzyme MBD4-NgTET1 and its use for increasing the 5caC occupancy in the oxidation products of TET enzymes

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 68

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 1

Ala Glu Asp Trp Leu Asp Cys Pro Ala Leu Gly Pro Gly Trp Lys Arg

1 5 10 15

Arg Glu Val Phe Arg Lys Ser Gly Ala Thr Cys Gly Arg Ser Asp Thr

20 25 30

Tyr Tyr Gln Ser Pro Thr Gly Asp Arg Ile Arg Ser Lys Val Glu Leu

35 40 45

Thr Arg Tyr Leu Gly Pro Ala Cys Asp Leu Thr Leu Phe Asp Phe Lys

50 55 60

Gln Gly Ile Leu

65

<210> 2

<211> 69

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 2

Glu Ser Gly Lys Arg Met Asp Cys Pro Ala Leu Pro Pro Gly Trp Lys

1 5 10 15

Lys Glu Glu Val Ile Arg Lys Ser Gly Leu Ser Ala Gly Lys Ser Asp

20 25 30

Val Tyr Tyr Phe Ser Pro Ser Gly Lys Lys Phe Arg Ser Lys Pro Gln

35 40 45

Leu Ala Arg Tyr Leu Gly Asn Thr Val Asp Leu Ser Ser Phe Asp Phe

50 55 60

Arg Thr Gly Lys Met

65

<210> 3

<211> 71

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 3

Glu Arg Lys Arg Trp Glu Cys Pro Ala Leu Pro Gln Gly Trp Glu Arg

1 5 10 15

Glu Glu Val Pro Arg Arg Ser Gly Leu Ser Ala Gly His Arg Asp Val

20 25 30

Phe Tyr Tyr Ser Pro Ser Gly Lys Lys Phe Arg Ser Lys Pro Gln Leu

35 40 45

Ala Arg Tyr Leu Gly Gly Ser Met Asp Leu Ser Thr Phe Asp Phe Arg

50 55 60

Thr Gly Lys Met Leu Met Ser

65 70

<210> 4

<211> 73

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 4

Ala Thr Ala Gly Thr Glu Cys Arg Lys Ser Val Pro Cys Gly Trp Glu

1 5 10 15

Arg Val Val Lys Gln Arg Leu Phe Gly Lys Thr Ala Gly Arg Phe Asp

20 25 30

Val Tyr Phe Ile Ser Pro Gln Gly Leu Lys Phe Arg Ser Lys Ser Ser

35 40 45

Leu Ala Asn Tyr Leu His Lys Asn Gly Glu Thr Ser Leu Lys Pro Glu

50 55 60

Asp Phe Asp Phe Thr Val Leu Ser Lys

65 70

<210> 5

<211> 320

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 5

Thr Thr Phe Lys Gln Gln Thr Ile Lys Glu Lys Glu Thr Lys Arg Lys

1 5 10 15

Tyr Cys Ile Lys Gly Thr Thr Ala Asn Leu Thr Gln Thr His Pro Asn

20 25 30

Gly Pro Val Cys Val Asn Arg Gly Glu Glu Val Ala Asn Thr Thr Thr

35 40 45

Leu Leu Asp Ser Gly Gly Gly Ile Asn Lys Lys Ser Leu Leu Gln Asn

50 55 60

Leu Leu Ser Lys Cys Lys Thr Thr Phe Gln Gln Ser Phe Thr Asn Ala

65 70 75 80

Asn Ile Thr Leu Lys Asp Glu Lys Trp Leu Lys Asn Val Arg Thr Ala

85 90 95

Tyr Phe Val Cys Asp His Asp Gly Ser Val Glu Leu Ala Tyr Leu Pro

100 105 110

Asn Val Leu Pro Lys Glu Leu Val Glu Glu Phe Thr Glu Lys Phe Glu

115 120 125

Ser Ile Gln Thr Gly Arg Lys Lys Asp Thr Gly Tyr Ser Gly Ile Leu

130 135 140

Asp Asn Ser Met Pro Phe Asn Tyr Val Thr Ala Asp Leu Ser Gln Glu

145 150 155 160

Leu Gly Gln Tyr Leu Ser Glu Ile Val Asn Pro Gln Ile Asn Tyr Tyr

165 170 175

Ile Ser Lys Leu Leu Thr Cys Val Ser Ser Arg Thr Ile Asn Tyr Leu

180 185 190

Val Ser Leu Asn Asp Ser Tyr Tyr Ala Leu Asn Asn Cys Leu Tyr Pro

195 200 205

Ser Thr Ala Phe Asn Ser Leu Lys Pro Ser Asn Asp Gly His Arg Ile

210 215 220

Arg Lys Pro His Lys Asp Asn Leu Asp Ile Thr Pro Ser Ser Leu Phe

225 230 235 240

Tyr Phe Gly Asn Phe Gln Asn Thr Glu Gly Tyr Leu Glu Leu Thr Asp

245 250 255

Lys Asn Cys Lys Val Phe Val Gln Pro Gly Asp Val Leu Phe Phe Lys

260 265 270

Gly Asn Glu Tyr Lys His Val Val Ala Asn Ile Thr Ser Gly Trp Arg

275 280 285

Ile Gly Leu Val Tyr Phe Ala His Lys Gly Ser Lys Thr Lys Pro Tyr

290 295 300

Tyr Glu Asp Thr Gln Lys Asn Ser Leu Lys Ile His Lys Glu Thr Lys

305 310 315 320

<210> 6

<211> 674

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 6

Gln Glu Ala Ala Pro Cys Asp Cys Asp Gly Gly Thr Gln Lys Glu Lys

1 5 10 15

Gly Pro Tyr Tyr Thr His Leu Gly Ala Gly Pro Ser Val Ala Ala Val

20 25 30

Arg Glu Leu Met Glu Thr Arg Phe Gly Gln Lys Gly Lys Ala Ile Arg

35 40 45

Ile Glu Lys Ile Val Phe Thr Gly Lys Glu Gly Lys Ser Ser Gln Gly

50 55 60

Cys Pro Val Ala Lys Trp Val Ile Arg Arg Ser Gly Pro Glu Glu Lys

65 70 75 80

Leu Ile Cys Leu Val Arg Glu Arg Val Asp His His Cys Ser Thr Ala

85 90 95

Val Ile Val Val Leu Ile Leu Leu Trp Glu Gly Ile Pro Arg Leu Met

100 105 110

Ala Asp Arg Leu Tyr Lys Glu Leu Thr Glu Asn Leu Arg Ser Tyr Ser

115 120 125

Gly His Pro Thr Asp Arg Arg Cys Thr Leu Asn Lys Lys Arg Thr Cys

130 135 140

Thr Cys Gln Gly Ile Asp Pro Lys Thr Cys Gly Ala Ser Phe Ser Phe

145 150 155 160

Gly Cys Ser Trp Ser Met Tyr Phe Asn Gly Cys Lys Phe Gly Arg Ser

165 170 175

Glu Asn Pro Arg Lys Phe Arg Leu Ala Pro Asn Tyr Pro Leu His Asn

180 185 190

Tyr Tyr Lys Arg Ile Thr Gly Met Ser Ser Glu Gly Ser Asp Val Lys

195 200 205

Thr Gly Trp Ile Ile Pro Asp Arg Lys Thr Leu Ile Ser Arg Glu Glu

210 215 220

Lys Gln Leu Glu Lys Asn Leu Gln Glu Leu Ala Thr Val Leu Ala Pro

225 230 235 240

Leu Tyr Lys Gln Met Ala Pro Val Ala Tyr Gln Asn Gln Val Glu Tyr

245 250 255

Glu Glu Val Ala Gly Asp Cys Arg Leu Gly Asn Glu Glu Gly Arg Pro

260 265 270

Phe Ser Gly Val Thr Cys Cys Met Asp Phe Cys Ala His Ser His Lys

275 280 285

Asp Ile His Asn Met His Asn Gly Ser Thr Val Val Cys Thr Leu Ile

290 295 300

Arg Ala Asp Gly Arg Asp Thr Asn Cys Pro Glu Asp Glu Gln Leu His

305 310 315 320

Val Leu Pro Leu Tyr Arg Leu Ala Asp Thr Asp Glu Phe Gly Ser Val

325 330 335

Glu Gly Met Lys Ala Lys Ile Lys Ser Gly Ala Ile Gln Val Asn Gly

340 345 350

Pro Thr Arg Lys Arg Arg Leu Arg Phe Thr Glu Pro Val Pro Arg Cys

355 360 365

Gly Lys Arg Ala Lys Met Lys Gln Asn His Asn Lys Ser Gly Ser His

370 375 380

Asn Thr Lys Ser Phe Ser Ser Ala Ser Ser Thr Ser His Leu Val Lys

385 390 395 400

Asp Glu Ser Thr Asp Phe Cys Pro Leu Gln Ala Ser Ser Ala Glu Thr

405 410 415

Ser Thr Cys Thr Tyr Ser Lys Thr Ala Ser Gly Gly Phe Ala Glu Thr

420 425 430

Ser Ser Ile Leu His Cys Thr Met Pro Ser Gly Ala His Ser Gly Ala

435 440 445

Asn Ala Ala Ala Gly Glu Cys Thr Gly Thr Val Gln Pro Ala Glu Val

450 455 460

Ala Ala His Pro His Gln Ser Leu Pro Thr Ala Asp Ser Pro Val His

465 470 475 480

Ala Glu Pro Leu Thr Ser Pro Ser Glu Gln Leu Thr Ser Asn Gln Ser

485 490 495

Asn Gln Gln Leu Pro Leu Leu Ser Asn Ser Gln Lys Leu Ala Ser Cys

500 505 510

Gln Val Glu Asp Glu Arg His Pro Glu Ala Asp Glu Pro Gln His Pro

515 520 525

Glu Asp Asp Asn Leu Pro Gln Leu Asp Glu Phe Trp Ser Asp Ser Glu

530 535 540

Glu Ile Tyr Ala Asp Pro Ser Phe Gly Gly Val Ala Ile Ala Pro Ile

545 550 555 560

His Gly Ser Val Leu Ile Glu Cys Ala Arg Lys Glu Leu His Ala Thr

565 570 575

Thr Ser Leu Arg Ser Pro Lys Arg Gly Val Pro Phe Arg Val Ser Leu

580 585 590

Val Phe Tyr Gln His Lys Ser Leu Asn Lys Pro Asn His Gly Phe Asp

595 600 605

Ile Asn Lys Ile Lys Cys Lys Cys Lys Lys Val Thr Lys Lys Lys Pro

610 615 620

Ala Asp Arg Glu Cys Pro Asp Val Ser Pro Glu Ala Asn Leu Ser His

625 630 635 640

Gln Ile Pro Ser Arg Val Ala Ser Thr Leu Thr Arg Asp Asn Val Val

645 650 655

Thr Val Ser Pro Tyr Ser Leu Thr His Val Ala Gly Pro Tyr Asn Arg

660 665 670

Trp Val

<210> 7

<211> 446

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 7

Gln Ser Gln Asn Gly Lys Cys Glu Gly Cys Asn Pro Asp Lys Asp Glu

1 5 10 15

Ala Pro Tyr Tyr Thr His Leu Gly Ala Gly Pro Asp Val Ala Ala Ile

20 25 30

Arg Thr Leu Met Glu Glu Arg Tyr Gly Glu Lys Gly Lys Ala Ile Arg

35 40 45

Ile Glu Lys Val Ile Tyr Thr Gly Lys Glu Gly Lys Ser Ser Gln Gly

50 55 60

Cys Pro Ile Ala Lys Trp Val Tyr Arg Arg Ser Ser Glu Glu Glu Lys

65 70 75 80

Leu Leu Cys Leu Val Arg Val Arg Pro Asn His Thr Cys Glu Thr Ala

85 90 95

Val Met Val Ile Ala Ile Met Leu Trp Asp Gly Ile Pro Lys Leu Leu

100 105 110

Ala Ser Glu Leu Tyr Ser Glu Leu Thr Asp Ile Leu Gly Lys Cys Gly

115 120 125

Ile Cys Thr Asn Arg Arg Cys Ser Gln Asn Glu Thr Arg Asn Cys Cys

130 135 140

Cys Gln Gly Glu Asn Pro Glu Thr Cys Gly Ala Ser Phe Ser Phe Gly

145 150 155 160

Cys Ser Trp Ser Met Tyr Tyr Asn Gly Cys Lys Phe Ala Arg Ser Lys

165 170 175

Lys Pro Arg Lys Phe Arg Leu His Gly Ala Glu Pro Lys Glu Glu Glu

180 185 190

Arg Leu Gly Ser His Leu Gln Asn Leu Ala Thr Val Ile Ala Pro Ile

195 200 205

Tyr Lys Lys Leu Ala Pro Asp Ala Tyr Asn Asn Gln Val Glu Phe Glu

210 215 220

His Gln Ala Pro Asp Cys Cys Leu Gly Leu Lys Glu Gly Arg Pro Phe

225 230 235 240

Ser Gly Val Thr Ala Cys Leu Asp Phe Ser Ala His Ser His Arg Asp

245 250 255

Gln Gln Asn Met Pro Asn Gly Ser Thr Val Val Val Thr Leu Asn Arg

260 265 270

Glu Asp Asn Arg Glu Val Gly Ala Lys Pro Glu Asp Glu Gln Phe His

275 280 285

Val Leu Pro Met Tyr Ile Ile Ala Pro Glu Asp Glu Phe Gly Ser Thr

290 295 300

Glu Gly Gln Glu Lys Lys Ile Arg Met Gly Ser Ile Glu Val Leu Gln

305 310 315 320

Ser Phe Arg Arg Arg Arg Val Ile Arg Ile Gly Glu Leu Pro Lys Ser

325 330 335

Cys Lys Lys Gly Gly Gly Gly Ser Val Ser Gly Gln Asp Ala Ala Ala

340 345 350

Val Gln Glu Ile Glu Tyr Trp Ser Asp Ser Glu His Asn Phe Gln Asp

355 360 365

Pro Cys Ile Gly Gly Val Ala Ile Ala Pro Thr His Gly Ser Ile Leu

370 375 380

Ile Glu Cys Ala Lys Cys Glu Val His Ala Thr Thr Lys Val Asn Asp

385 390 395 400

Pro Asp Arg Asn His Pro Thr Arg Ile Ser Leu Val Leu Tyr Arg His

405 410 415

Lys Asn Leu Phe Leu Pro Lys His Cys Leu Ala Leu Trp Glu Ala Lys

420 425 430

Met Ala Glu Lys Ala Arg Lys Glu Glu Glu Cys Gly Lys Asn

435 440 445

<210> 8

<211> 720

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 8

Ser Glu Leu Pro Thr Cys Ser Cys Leu Asp Arg Val Ile Gln Lys Asp

1 5 10 15

Lys Gly Pro Tyr Tyr Thr His Leu Gly Ala Gly Pro Ser Val Ala Ala

20 25 30

Val Arg Glu Ile Met Glu Asn Arg Tyr Gly Gln Lys Gly Asn Ala Ile

35 40 45

Arg Ile Glu Ile Val Val Tyr Thr Gly Lys Glu Gly Lys Ser Ser His

50 55 60

Gly Cys Pro Ile Ala Lys Trp Val Leu Arg Arg Ser Ser Asp Glu Glu

65 70 75 80

Lys Val Leu Cys Leu Val Arg Gln Arg Thr Gly His His Cys Pro Thr

85 90 95

Ala Val Met Val Val Leu Ile Met Val Trp Asp Gly Ile Pro Leu Pro

100 105 110

Met Ala Asp Arg Leu Tyr Thr Glu Leu Thr Glu Asn Leu Lys Ser Tyr

115 120 125

Asn Gly His Pro Thr Asp Arg Arg Cys Thr Leu Asn Glu Asn Arg Thr

130 135 140

Cys Thr Cys Gln Gly Ile Asp Pro Glu Thr Cys Gly Ala Ser Phe Ser

145 150 155 160

Phe Gly Cys Ser Trp Ser Met Tyr Phe Asn Gly Cys Lys Phe Gly Arg

165 170 175

Ser Pro Ser Pro Arg Arg Phe Arg Ile Asp Pro Ser Ser Pro Leu His

180 185 190

Glu Lys Asn Leu Glu Asp Asn Leu Gln Ser Leu Ala Thr Arg Leu Ala

195 200 205

Pro Ile Tyr Lys Gln Tyr Ala Pro Val Ala Tyr Gln Asn Gln Val Glu

210 215 220

Tyr Glu Asn Val Ala Arg Glu Cys Arg Leu Gly Ser Lys Glu Gly Arg

225 230 235 240

Pro Phe Ser Gly Val Thr Ala Cys Leu Asp Phe Cys Ala His Pro His

245 250 255

Arg Asp Ile His Asn Met Asn Asn Gly Ser Thr Val Val Cys Thr Leu

260 265 270

Thr Arg Glu Asp Asn Arg Ser Leu Gly Val Ile Pro Gln Asp Glu Gln

275 280 285

Leu His Val Leu Pro Leu Tyr Lys Leu Ser Asp Thr Asp Glu Phe Gly

290 295 300

Ser Lys Glu Gly Met Glu Ala Lys Ile Lys Ser Gly Ala Ile Glu Val

305 310 315 320

Leu Ala Pro Arg Arg Lys Lys Arg Thr Cys Phe Thr Gln Pro Val Pro

325 330 335

Arg Ser Gly Lys Lys Arg Ala Ala Met Met Thr Glu Val Leu Ala His

340 345 350

Lys Ile Arg Ala Val Glu Lys Lys Pro Ile Pro Arg Ile Lys Arg Lys

355 360 365

Asn Asn Ser Thr Thr Thr Asn Asn Ser Lys Pro Ser Ser Leu Pro Thr

370 375 380

Leu Gly Ser Asn Thr Glu Thr Val Gln Pro Glu Val Lys Ser Glu Thr

385 390 395 400

Glu Pro His Phe Ile Leu Lys Ser Ser Asp Asn Thr Lys Thr Tyr Ser

405 410 415

Leu Met Pro Ser Ala Pro His Pro Val Lys Glu Ala Ser Pro Gly Phe

420 425 430

Ser Trp Ser Pro Lys Thr Ala Ser Ala Thr Pro Ala Pro Leu Lys Asn

435 440 445

Asp Ala Thr Ala Ser Cys Gly Phe Ser Glu Arg Ser Ser Thr Pro His

450 455 460

Cys Thr Met Pro Ser Gly Arg Leu Ser Gly Ala Asn Ala Ala Ala Ala

465 470 475 480

Asp Gly Pro Gly Ile Ser Gln Leu Gly Glu Val Ala Pro Leu Pro Thr

485 490 495

Leu Ser Ala Pro Val Met Glu Pro Leu Ile Asn Ser Glu Pro Ser Thr

500 505 510

Gly Val Thr Glu Pro Leu Thr Pro His Gln Pro Asn His Gln Pro Ser

515 520 525

Phe Leu Thr Ser Pro Gln Asp Leu Ala Ser Ser Pro Met Glu Glu Asp

530 535 540

Glu Gln His Ser Glu Ala Asp Glu Pro Pro Ser Asp Glu Pro Leu Ser

545 550 555 560

Asp Asp Pro Leu Ser Pro Ala Glu Glu Lys Leu Pro His Ile Asp Glu

565 570 575

Tyr Trp Ser Asp Ser Glu His Ile Phe Leu Asp Ala Asn Ile Gly Gly

580 585 590

Val Ala Ile Ala Pro Ala His Gly Ser Val Leu Ile Glu Cys Ala Arg

595 600 605

Arg Glu Leu His Ala Thr Thr Pro Val Glu His Pro Asn Arg Asn His

610 615 620

Pro Thr Arg Leu Ser Leu Val Phe Tyr Gln His Lys Asn Leu Asn Lys

625 630 635 640

Pro Gln His Gly Phe Glu Leu Asn Lys Ile Lys Phe Glu Ala Lys Glu

645 650 655

Ala Lys Asn Lys Lys Met Lys Ala Ser Glu Gln Lys Asp Gln Ala Ala

660 665 670

Asn Glu Gly Pro Glu Gln Ser Ser Glu Val Asn Glu Leu Asn Gln Ile

675 680 685

Pro Ser His Lys Ala Leu Thr Leu Thr His Asp Asn Val Val Thr Val

690 695 700

Ser Pro Tyr Ala Leu Thr His Val Ala Gly Pro Tyr Asn His Trp Val

705 710 715 720

<210> 9

<211> 450

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 9

Asp Phe Pro Ser Cys Arg Cys Val Glu Gln Ile Ile Glu Lys Asp Glu

1 5 10 15

Gly Pro Phe Tyr Thr His Leu Gly Ala Gly Pro Asn Val Ala Ala Ile

20 25 30

Arg Glu Ile Met Glu Glu Arg Phe Gly Gln Lys Gly Lys Ala Ile Arg

35 40 45

Ile Glu Arg Val Ile Tyr Thr Gly Lys Glu Gly Lys Ser Ser Gln Gly

50 55 60

Cys Pro Ile Ala Lys Trp Val Val Arg Arg Ser Ser Ser Glu Glu Lys

65 70 75 80

Leu Leu Cys Leu Val Arg Glu Arg Ala Gly His Thr Cys Glu Ala Ala

85 90 95

Val Ile Val Ile Leu Ile Leu Val Trp Glu Gly Ile Pro Leu Ser Leu

100 105 110

Ala Asp Lys Leu Tyr Ser Glu Leu Thr Glu Thr Leu Arg Lys Tyr Gly

115 120 125

Thr Leu Thr Asn Arg Arg Cys Ala Leu Asn Glu Glu Arg Thr Cys Ala

130 135 140

Cys Gln Gly Leu Asp Pro Glu Thr Cys Gly Ala Ser Phe Ser Phe Gly

145 150 155 160

Cys Ser Trp Ser Met Tyr Tyr Asn Gly Cys Lys Phe Ala Arg Ser Lys

165 170 175

Ile Pro Arg Lys Phe Lys Leu Leu Gly Asp Asp Pro Lys Glu Glu Glu

180 185 190

Lys Leu Glu Ser His Leu Gln Asn Leu Ser Thr Leu Met Ala Pro Thr

195 200 205

Tyr Lys Lys Leu Ala Pro Asp Ala Tyr Asn Asn Gln Ile Glu Tyr Glu

210 215 220

His Arg Ala Pro Glu Cys Arg Leu Gly Leu Lys Glu Gly Arg Pro Phe

225 230 235 240

Ser Gly Val Thr Ala Cys Leu Asp Phe Cys Ala His Ala His Arg Asp

245 250 255

Leu His Asn Met Gln Asn Gly Ser Thr Leu Val Cys Thr Leu Thr Arg

260 265 270

Glu Asp Asn Arg Glu Phe Gly Gly Lys Pro Glu Asp Glu Gln Leu His

275 280 285

Val Leu Pro Leu Tyr Lys Val Ser Asp Val Asp Glu Phe Gly Ser Val

290 295 300

Glu Ala Gln Glu Glu Lys Lys Arg Ser Gly Ala Ile Gln Val Leu Ser

305 310 315 320

Ser Phe Arg Arg Lys Val Arg Met Leu Ala Glu Pro Val Lys Thr Cys

325 330 335

Arg Gln Arg Lys Leu Glu Ala Lys Lys Ala Ala Ala Glu Lys Leu Ser

340 345 350

Gly Gly Gly Gly Ser Asp Glu Val Trp Ser Asp Ser Glu Gln Ser Phe

355 360 365

Leu Asp Pro Asp Ile Gly Gly Val Ala Val Ala Pro Thr His Gly Ser

370 375 380

Ile Leu Ile Glu Cys Ala Lys Arg Glu Leu His Ala Thr Thr Pro Leu

385 390 395 400

Lys Asn Pro Asn Arg Asn His Pro Thr Arg Ile Ser Leu Val Phe Tyr

405 410 415

Gln His Lys Ser Met Asn Glu Pro Lys His Gly Leu Ala Leu Trp Glu

420 425 430

Ala Lys Met Ala Glu Lys Ala Arg Glu Lys Glu Glu Glu Cys Glu Lys

435 440 445

Tyr Gly

450

<210> 10

<211> 204

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

gctgaagatt ggctggattg tccagcactg ggtccaggtt ggaaacgtcg cgaagtgttc 60

cgtaaatctg gtgcgacttg cggtcgttct gacacctatt accagagccc gactggcgat 120

cgcattcgtt ccaaagttga actgacccgt tacctgggcc cggcctgtga tctgactctg 180

ttcgatttca aacagggtat cctg 204

<210> 11

<211> 207

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

gaatctggta aacgtatgga ttgtccagca ctgccgcctg gttggaaaaa agaagaagtt 60

atccgtaaat ctggcctgtc tgcaggcaaa tccgacgtat actacttcag cccgtctggc 120

aaaaaattcc gctctaaacc acagctggcg cgttacctgg gtaacaccgt tgatctgtcc 180

tctttcgatt tccgtaccgg caaaatg 207

<210> 12

<211> 213

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

gaacgtaaac gttgggaatg tcctgctctg ccacagggtt gggaacgtga agaagttccg 60

cgtcgttctg gtctgtctgc tggccatcgt gatgtgttct actattctcc gtccggtaaa 120

aaattccgta gcaaaccgca gctggcacgt tacctgggcg gttctatgga tctgtctact 180

tttgatttcc gtactggcaa aatgctgatg tct 213

<210> 13

<211> 219

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

gctaccgctg gtaccgaatg tcgtaaatct gttccttgtg gttgggaacg tgtggtgaaa 60

cagcgtctgt tcggtaaaac tgcaggccgt ttcgatgttt attttatctc tccgcagggc 120

ctgaaattcc gcagcaaaag ctccctggcg aactacctgc acaagaacgg tgaaacttcc 180

ctgaaacctg aagacttcga cttcaccgtg ctgtccaaa 219

Claims

1. A recombinant TET enzyme MBD4-NgTET1, characterized by: the recombinant TET enzyme MBD4-NgTET1 is a recombinant protein domain MBD4 with a sequence shown as SEQ ID No.4, and is connected with an NgTET1 protein with a sequence shown as SEQ ID No.5 through GGGS connecting peptide.

2. Use of recombinant tetase MBD4-NgTET1 according to claim 1 for the preparation of a DNA methylation detection reagent based on a TET enzyme oxidation reaction, wherein said recombinant tetase MBD4-NgTET1 catalyzes 5mC, resulting in oxidation products 5hmC, 5fC and 5caC, said recombinant tetase MBD4-NgTET1 increasing the 5caC ratio in the TET enzyme oxidation product compared to the NgTET1 protein shown in SEQ ID No. 5.

3. Use according to claim 2, characterized in that it comprises the steps of:

(1) Performing an oxidation reaction on the template DNA using the recombinant tetase MBD4-NgTET1 of claim 1;

(2) Treating with a reducing agent, and neutralizing with alkali;

(3) DNA recovery, wherein the recovered products form a whole genome DNA methylation detection library;

wherein in step (1) a TET enzyme reaction buffer is added, said TET enzyme reaction buffer comprising: 1-100 mM 3- (N-morpholino) propanesulfonic acid sodium salt, 1-100 mM bis (2-hydroxyethyl) amino-tris (hydroxymethyl) methane, 1-100 mM hydroxyethyl piperazine ethanethiol sulfonic acid, 1-300 mM sodium chloride, 0.1-10 mM ascorbic acid, 0.1-10 mM citric acid, 0.1-20 mM alpha-ketoglutaric acid, 0.1-20 mM 1, 3-acetonedicarboxylic acid, 0.1-20 mM adenosine triphosphate, 0.1-10 mM tetrafluoro-p-benzoquinone, 0.1-10 mM tetrachloro-p-benzoquinone, 0.1-10 mM tetrabromo-p-benzoquinone, 0.1-10 mM tetraiodobenzoquinone, 0.01-2 mM tris (2-carboxyethyl) phosphine, 0.01-2 mM dithiothreitol.

4. A use according to claim 3, characterized in that: the TET enzyme reaction buffer also contains 0.1-50 mM ferric salt compound.

5. A use according to claim 3, characterized in that: the reaction temperature of the oxidation reaction in the step (1) is 10-40 ℃.

6. A use according to claim 3, characterized in that: the reaction time of the oxidation reaction described in step (1) is 0.5 to 2 h.

7. A use according to claim 3, characterized in that: the reducing agent in step (2) comprises one or more of lithium aluminum hydride, lithium boron hydride, sodium borohydride acetate, sodium cyanoborohydride, diisobutyl aluminum hydride, lithium triethylborohydride, ammonia borane, pyridine borane, 2-methylpyridine borane, sodium borohydride, ammonia borane lithium, pyrrolidine borohydride, borane ethylenediamine, borane dimethylamine, borane morpholine complex, borane triphenylphosphine, borane diphenylphosphine, borane triethylamine, 4-methylmorpholine borane, borane trimethylamine complex, 5-ethyl-2-methylpyridine borane, N-diisopropylethylamine borane, borane tetrahydrofuran complex, borane dimethyl sulfide complex.

8. A use according to claim 3, characterized in that: the treatment concentration of the reducing agent in the step (2) is 100-2000 mM.

9. A use according to claim 3, characterized in that: the treatment temperature of the reducing agent in the step (2) is 20-50 ℃.

10. A use according to claim 3, characterized in that: the treatment time of the reducing agent in the step (2) is 2-24 h.

11. A use according to claim 3, characterized in that: the alkali neutralization in the step (2) uses sodium hydroxide or potassium hydroxide, and the final concentration in the reaction system is 0.5-1.5. 1.5M.

12. A use according to claim 3, characterized in that: the DNA recovery mode in the step (3) refers to magnetic bead recovery, column recovery or extraction precipitation recovery.