WO2012009828A1

WO2012009828A1 - Chemical modification method and detection method of 5-hydroxymethylcytosine in dna molecules

Info

Publication number: WO2012009828A1
Application number: PCT/CN2010/001107
Authority: WO
Inventors: 邓大君; 陆哲明; 周静
Original assignee: 北京市肿瘤防治研究所
Priority date: 2010-07-22
Filing date: 2010-07-22
Publication date: 2012-01-26

Abstract

The invention provides a method for chemically modifying 5-hydroxymethylcytosine in DNA molecules by sodium bisulfite, and a method for detecting the presence of 5-hydroxymethylcytosine or the locus of 5-hydroxymethylcytosine existing in DNA molecules. The invention also provides a use of sodium bisulfite for modifying or detecting 5-hydroxymethylcytosine existing in DNA molecules.

Description

Chemical modification method and detection method of 5-hydroxymethylcytosine in DNA molecule

The present invention belongs to the field of epigenetics research, and in particular to a method for chemically modifying 5-hydroxymethylcytosine in a DNA molecule, and detecting 5-hydroxymethylcytosine or 5-hydroxymethyl group present in a DNA molecule. A method of cytosine site and the use of sodium bisulfite for modifying 5-hydroxymethylcytosine present in a DNA molecule. Background technique

DNA cytosine methylation, histone modification, non-coding R A is an important epigenetic mechanism that regulates gene expression and plays an important role in normal cell differentiation, embryonic development, and disease development. Among the three epigenetic mechanisms, the 5-methylation modification of DNA cytosine was the most stable. In adult vertebrate cell DNA, methylation of this DNA cytosine occurs only at the CpG site. There are many regions in the genomic DNA in which CpG sites are highly dense, and one of the sequences longer than 250 bp is called a CpG island. The state of methylation of the CpG island located around the initiation point (TSS) of the gene plays a crucial role in determining the transcriptionality of the gene: the region near the TSS site is reproducible when it is unmethylated, Those who develop methylation are inactivated because they cannot be transcribed. There is a close relationship between CpG island methylation and the occurrence and development of abnormal cell differentiation diseases such as tumors. This methylation information can be stably maintained for a long time in various tissue samples and can be sensitively detected. These not only make CpG island methylation analysis an important means of gene expression regulation research, but also have important application value in the occurrence and development of tumors and other diseases and new drug development.

5-methylcytosine can be further oxidized to 5-hydroxymethylcytosine by enzymatic catalysis such as TET1. Using chromatography or mass spectrometry separation techniques, 5-hydroxymethylcytosine has been found in embryonic stem cells and certain brain stem tissue cells. However, chromatographic or mass spectrometry separation techniques can only be used to determine the presence of 5-hydroxymethylcytosine in the DNA of the assay sample, and the specific location of 5-hydroxymethylcytosine in the DNA sequence cannot be determined. Since the various DNA-sequence-specific 5-methylcytosine assays currently used do not distinguish 5-hydroxymethylcytosine from 5-methylcytosine, the location of 5-hydroxymethylcytosine Research on biological functions and their relationship with disease development is difficult to carry out. Abnormal ectopic fusion of the TET1 enzyme gene with the MLL gene has been reported to be a common phenomenon in leukemia cells, suggesting that abnormalities in 5-hydroxymethylcytosine may play an important role in the development of the disease. The invention of a technique suitable for DNA sequence-specific 5-hydroxymethylcytosine analysis is expected by the international epigenetic basis and applied research field. The currently used 5-hydroxymethylcytosine assay is a chromatographic total analysis method that analyzes DNA after digestion into nucleosides or single nucleotides. It does not provide information on which sites of cytosine in the DNA are hydroxyl. Methylation information. The currently used DNA sequence-specific 5-methylcytosine analysis technique is not suitable for the analysis of 5-hydroxymethylcytosine. For example, the existing sodium bisulfite chemical modification method cannot convert 5-hydroxymethylcytosine into uracil, and cannot distinguish it from 5-methylcytosine; 5-methylcytosine monoclonal antibody, N-methylation-sensitive enzymes and methylated DNA-binding proteins do not recognize 5-hydroxymethylcytosine and cannot be distinguished from unmethylated cytosine. That is, chromatin co-precipitation using a 5-hydroxymethylcytosine-specific antibody did not identify the specific position of 5-hydroxymethylcytosine in the precipitated DNA sequence.

Therefore, there is a need for a method for conveniently detecting the presence or absence of 5-hydroxymethylcytosine in a DNA molecule, and a method for detecting a 5-hydroxymethylcytosolic site in a DNA molecule. Disclosure of invention

The object of the present invention is to provide a method for chemically modifying 5-hydroxymethylcytosine in a DNA molecule, and a method for detecting a 5-hydroxymethylcytosine or a 5-hydroxymethylcytosine site present in a DNA molecule The use of sodium bisulfate for modifying 5-hydroxymethylcytosine present in DNA molecules.

The present invention is based on the fact that the applicant has found that the 5-hydroxymethylcytosine in the DNA has been modified in the existing chemical modification method of sodium hydrogen sulfite, and the 5-hydroxymethylcytosine is converted after modification. The transformation product is used as a PCR amplification template. In conventional PCR, if dTTP is used in the PCR system, it is amplified to thymine T or if dUTP is used in the PCR system, it is amplified into uracil U.

Therefore, the present invention proposes the following technical solutions:

The invention provides a chemical modification method of 5-hydroxymethylcytosine in a DNA molecule, the method comprising the following steps: treating the DNA with sodium bisulfite chemical modification method to make 5-hydroxyl group present in the DNA The cytosine is transformed and the transformed product is used as a template for PCR amplification and amplified into thymine T or uracil U in conventional PCR.

The method for chemically modifying 5-hydroxymethylcytosine in the DNA molecule of the present invention, wherein the treatment of the DNA by the chemical modification method of sodium hydrogen sulfite is one of the following methods:

A1. Aqueous sodium hydroxide solution was added to the DNA sample solution, and a water bath at 95 ° C for 5 minutes was added to a final concentration of 5 mol/L of sodium hydrogen sulfite, followed by 95. C, 5 minutes, 60 ° C, 25 minutes, 95 ° C, 5 minutes, 60 ° (:, 85 minutes, 95 ° C, 5 minutes, 60 ° C, 175 minutes, heat cycle DNA treatment, plus final concentration 0.3 Mol/L sodium hydroxide terminates the sulfonation reaction and desulfurization, purifies the modified DNA, precipitates with ethanol, and dissolves;

A2. Add a final concentration of 9mol/L sodium bisulfite to the DNA sample solution, and hydrate the modified DNA for 1 hour at 70 °C to precipitate and dissolve the ethanol; or

A3. Use EpiTect Bisulfite kit: Qiagen GmbH, D40724 Hilden, Cat. No. 59104; or

A4. Using the Methylation-God kit: ZYMO Research China, Cat. No. D5005 or the present invention provides a method for detecting a 5-hydroxymethylcytosine site present in a DNA molecule, the method comprising the steps of:

A. A piece of DNA is treated with sodium bisulfite chemical modification method to convert 5-hydroxymethylcytosine present in the DNA, and the transformation product can be used as a template for PCR amplification and amplified in conventional PCR. Thymine T or uracil U;

B. Another identical DNA is treated by sodium bisulfite chemical modification method, so that the 5-hydroxymethylcytosine present in the DNA is not transformed, and is amplified into cytosine C in conventional PCR;

C. using the two treated DNAs as a template, and respectively amplifying the DNA molecules with sequence-specific primers;

D. Sequencing the amplified two DNAs separately, comparing the C/T or C/U differential sites in the DNA after sequencing, the C/T or CU differential sites are the 5-hydroxyl groups present in the DNA molecule. Methylcytosine site.

The method for detecting a 5-hydroxymethylcytosine site present in a DNA molecule according to the present invention is characterized in that: Step D is to digest the amplified two DNAs by restriction endonuclease, if step A is expanded The amplified DNA product is not digested, and the DNA product amplified in step B is digested or if the DNA product amplified in step A is cleaved, and the DNA product amplified in step B is not digested. Then, a 5-hydroxymethylcytosine site is present on the restriction endonuclease recognition site in the DNA molecule.

The method for detecting a 5-hydroxymethylcytosine site present in a DNA molecule according to the present invention, wherein the treatment of the DNA by the sodium hydrogen sulfite chemical modification method in the step A is one of the following methods :

A1. Aqueous sodium hydroxide solution was added to the DNA sample solution, and a water bath at 95 ° C for 5 minutes was added to a final concentration of 5 mol/L of sodium hydrogen sulfite, followed by 95. C, 5 minutes, 60. C, 25 minutes, 95. C, 5 minutes, 60° (, 85 minutes, 95° (:, 5 minutes, 60. (, 175 minutes, heat cycle treatment of DNA, addition of final concentration of 0.3mol/L sodium hydroxide to terminate the sulfonation reaction and desulfurization, purification Modified DNA, ethanol precipitation, dissolution; or

A3. Using EpiTect Bisulfite Kit: Qiagen GmbH, D40724 Hilden, Cat. No.

59104; or

A4. Use Methylation-God kit: ZYMO Research China, Cat. No. D5005 or D5006.

A method for detecting a 5-hydroxymethylcytosine site present in DNA according to the present invention, characterized N2010/001107

-4- lie in: Step B: The sodium bisulphite chemical modification method is used to treat the DNA: adding 0.1 mol/L sodium hydroxide aqueous solution to the DNA sample solution, 95 ° C water bath for 5 minutes, adding the final concentration 5 mol/L of sodium hydrogen sulfite, 55 ° C water bath for 16 hours, adding a final concentration of 0.3 mol / L sodium hydroxide to terminate the sulfonation reaction and desulfurization, purification of the modified DNA, ethanol precipitation, dissolution.

The present invention provides a method for detecting the presence of 5-hydroxymethylcytosine in a DNA molecule, the method comprising the steps of:

A. A piece of DNA is treated with sodium bisulfite chemical modification method to convert hydroxymethylcytosine present in the DNA, and the transformed product is used as a template for PCR amplification and amplified into thymine T in conventional PCR. Or uracil U;

C. using the two treated DNAs as a template, and respectively amplifying the DNA molecules with sequence-specific primers to obtain a PCR product;

D. The denaturing temperature of the DNA molecule was predicted by denaturing high performance liquid chromatography workstation software, and the PCR product was subjected to mutation mode analysis by denaturing high performance liquid chromatography under the temperature condition;

E. After PCR amplification of the DNA treated in step A, the PCR product appears as a single-stranded DNA chromatographic peak, and the DNA processed in step B is PCR-amplified and the PCR product appears as a double-stranded DNA peak, and the DNA molecule is determined. There is 5-hydroxymethylcytosine.

The method for detecting 5-hydroxymethylcytosine present in a DNA molecule according to the present invention, wherein the treatment of the DNA by sodium bisulfite chemical modification as described in the step A is as follows:

A1. Aqueous sodium hydroxide solution was added to the DNA sample solution, and a water bath at 95 ° C for 5 minutes was added to a final concentration of 5 mol/L of sodium hydrogen sulfite, followed by 95. C, 5 minutes, 60 ° C, 25 minutes, 95. C, 5 minutes, 60° ( , 85 minutes, 95 ° C, 5 minutes, 60 ° C, 175 minutes, heat cycle DNA treatment, add final concentration 0.3mol / L sodium hydroxide to terminate the sulfonation reaction and desulfurization, purification modification After DNA, ethanol precipitation, dissolution; or

A2. Add a final concentration of 9 mol/L sodium bisulfite to the DNA sample solution, and heat the modified DNA for 1 hour at 70 ° C to precipitate and dissolve the ethanol; or

A3. Using EpiTect Bisulfite Kit: Qiagen GmbH, D40724 Hilden, Cat. No.

59104; or

A4. Use Methylation-God kit: ZYMO Research China, Cat. No. D5005 or D5006.

The method for detecting 5-hydroxymethylcytosine present in DNA according to the present invention is characterized in that: in step B, the DNA is treated by sodium bisulfite chemical modification method: 0.1 mol is added to the DNA sample solution /L aqueous sodium hydroxide solution, 95 ° C water bath for 5 minutes, adding a final concentration of 5 mol / L The sodium hydrogen sulfite was subjected to a water bath at 55 ° C for 16 hours, and the sulfonation reaction and desulfurization were terminated by adding a final concentration of 0.3 mol/L sodium hydroxide, and the modified DNA was purified, precipitated by ethanol, and dissolved.

The invention also provides the use of sodium bisulfite for modifying or detecting the presence of 5-hydroxymethylcytosine in a DNA molecule.

The use according to the present invention is characterized in that: the sodium hydrogen sulfite modification converts 5-hydroxymethylcytosine present in the DNA molecule, and the transformed product is used as a template for PCR amplification and is amplified in conventional PCR. Thymine T or uracil U.

The use according to the invention is characterized in that: the sodium hydrogen sulfite is an EpiTect Bisulfite kit containing sodium bisulfite: Qiagen GmbH, D40724 Hilden, Cat. No. 59104 or Methylation-God kit: ZYMO Research China, Cat. No. D5005 or D5006.

The method of the present invention can conveniently detect the presence of 5-hydroxymethylcytosine or its site in the DNA sequence of interest, providing a powerful tool for epigenetic research.

The invention will now be described in detail with reference to the drawings and specific embodiments. It is to be understood that the examples are merely illustrative of the invention and should not be construed as limiting the invention.

Further, the experimental methods, experimental reagents, experimental materials and the like used in the present invention are all conventional experimental methods, experimental reagents, and experimental materials well known to those skilled in the art unless otherwise specified. BRIEF DESCRIPTION OF THE DRAWINGS

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic illustration of the invention.

Figure 2 is a HPLC analysis of the presence of various single nucleotides in synthetic H-DNA, M-DNA and C-DNA molecules. Column: Supelcosil C-128; Mobile phase: 0.1% ammonium formate + 0~100% methanol linear gradient; UV detector 260 nm.

Figure 3A and Figure 3B are DHPLC chromatograms of six PCR amplification products of three DNA templates modified by Method A and Method B.

Figure 4 shows the Ncol fragment length polymorphism of the six PCR products. The best way to implement the invention

Referring to Figure 1, there is shown a schematic diagram of the principles of the present invention:

Using two kinds of sodium bisulfite chemical modification techniques (referred to as method A and method B, respectively), in the method A, sodium bisulphite chemical modification method is used to convert 5-hydroxymethylcytosine H in DNA, and the conversion product is S. In Method B, the sodium bisulfite chemical modification method does not modify the 5-hydroxymethylcytosine H in the DNA to be S, but Η'; in the subsequent PCR amplification reaction, S is amplified to thymidine (eg Substituting dUTP for dTTP in the PCR system is uracil U), and H' without modification to S is amplified. It is cytosine C (if 5-C cytosine M is replaced by 5C-dCTP instead of dCTP in PCR system); 5-methylcytosine will not be modified by these two methods, and will be modified in subsequent PCR amplification reactions. Amplification to cytosine; cytosine without 5-methylation or 5-hydroxymethylation modification is modified by these two methods to form uracil, which is amplified into thymidine in subsequent PCR amplification reactions. The sequence C/T or C/U differential site in the PCR products of these two chemically modified templates is the 5-hydroxymethylcytosine presence site.

Obviously, how to modify DNA using sodium bisulfite chemical modification is a well-known technique in the art, and the present invention has only found a new application of the chemical modification method of sodium hydrogen sulfite. Example 1

1. Directly use dCTP-containing dNTPs and amplification primer pair 1 (forward primer: SEQ ID No.: l and reverse primer: SEQ ID No.: 2), and all C sites are unmethylated by PCR. Representative and non-hydroxymethylated representative DNA molecules (C-DNA) (SEQ ID No.: 3);

Replace dCTP in the PCR amplification system with hm-dCTP, and synthesize all C sites by amplification primer pair 1 (forward primer: SEQ ID No.: 1 and reverse primer: SEQ ID No.: 2). a DNA molecule (H-DNA) of a representative hydroxymethylcytosine that is replaced by H;

Replacing dCTP in the PCR amplification system with 5m-dCTP, and synthesizing all C sites by amplification primer pair 1 (forward primer: SEQ ID No.: 1 and reverse primer: SEQ ID No.: 2) Representative DNA molecules (M-DNA) each replaced by M;

2. Remove free dNTPs; take 2 μg of purified H-DNA, M-DNA and C-DNA samples, digest and digest into single nucleotides, and isolate them by HPLC: H-DNA, M-DNA and C-DNA Only hm-dCMP, 5m-dCMP and dCMP were detected in the digested products; dTMP, dGMP and dAMP were detected in all three DNA digestion products, see Figure 2;

3. Take 2 μg of each of the above purified DNAs and treat the three DNA samples separately using the following A method:

A 0.1 mol/L sodium hydroxide aqueous solution was added to the DNA sample solution, and a water bath at 95 ° C for 5 minutes was added to a final concentration of 5 mol/L sodium hydrogen sulfite, 95. (:, 5 min, 60 ° C, 25 min, 95. (:, 5 min, 60. (, 85 min, 95. (:, 5 min, 60 ° C, 175 min, heat cycle DNA treatment, purification modification) After the DNA, the final concentration of 0.3 mol / L sodium hydroxide was terminated by sulfonation and desulfurization, and the modified DNA was purified by Promega kit (Cat. No. A7280) and precipitated with ethanol;

4. Take 2 micrograms of each of the above purified DNAs, and use the following B method to treat 3 DNA samples separately:

Add 0.1 mol/L sodium hydroxide aqueous solution to the DNA sample solution, water bath at 95 ° C for 5 minutes, add 5 mol / L sodium bisulfite at a final concentration, and bath at 55 ° C for 16 hours to promote C deamination and add Termination of sulfonation and desulfurization with a concentration of 0.3 mol/L sodium hydroxide, with Promega The modified DNA (Cat. No. A7280) was purified by ethanol precipitation, dissolved in deionized water or TE buffer after drying;

5. Using primer pair 2 (forward primer: SEQ ID No.: 4 and reverse primer: SEQ ID No.: 5), respectively, three kinds of DNA templates modified by the above method A and method B were amplified, respectively, to obtain C. - PCR2A, C-PCR2B, M-PCR2A. M-PCR2B, H-PCR2A. H-PCR2B six products, identified by agarose gel electrophoresis;

6. Determinate the partial denaturation temperature (53.5 °C) of the PCR product of the DNA fragment to be tested by denaturing high performance liquid chromatography (DHPLC) workstation software (WaveMaker or Navigator), and use DHPLC (DNASep® analytical column) to perform the step at this temperature. Five or six PCR products were subjected to mutation pattern analysis. The results are as follows: The two amplification products of methylolated DNA are single-stranded DNA and double-stranded DNA peaks, while the two amplified products of methylated DNA exhibit double-stranded DNA peaks, unmethylated. Both amplification products of DNA showed single-stranded DNA peaks (Fig. 3A and Fig. 3B):

[1] In the C-PCR2A and C-PCR2B products, a single-stranded DNA peak having a low denaturation temperature due to the occurrence of non-methylated C"T substitution was detected only at 4.2 minutes;

[2] In the M-PCR2A and M-PCR2B products, double-stranded DNA peaks with high denaturation temperature were detected only in 5.5 minutes due to methylation of C;

[3] In the H-PCR2A product, a single-stranded DNA peak with a low denaturation temperature due to CT substitution of methylolation was detected only at 4.2 minutes; whereas in the H-PCR2B product, only The DNA peak of the double-stranded DNA with higher denaturation temperature was detected in 5.5 minutes due to no substitution of methylolated C;

7. Judgment of results: A single-stranded DNA peak is shown in Method A, and DNA methylation is present in the product of Method B as a double-stranded DNA peak.

Replace the method A in step 3 with one of the following methods, and the result is the same as step 7.

A3. Using EpiTect Bisulfite Kit: Qiagen GmbH, D40724 Hilden, Cat. No.

59104; or

A4. Using Methylation-God Kit: ZYMO Research China, Cat No. D5005 or D5006o

Example 2

1-5. Same as Embodiment 1 Step 1-5

6. Six freshly amplified PCR2 products were ligated to AT clones for sequencing;

7. All H sites in the PCR amplification product (H-PCR2A) of the H-DNA template modified by Method A were replaced by T, and the PCR amplification product of the H-DNA template modified by Method B (H- Most of the H sites in PCR2B) are not replaced by T and still be expressed as C; and 2 amplifications of methylated DNA All M sites in the product are expressed as C, and all C sites in the two amplification products of unmethylated DNA exhibit T;

8. Judgment of results: T is expressed in Method A, and the site represented as C in Method B is the site where 5-hydroxymethylation occurs.

Replace the method A in step 3 with one of the following methods, and the result is the same as step 8.

A2. Add a final concentration of 9 mol/L sodium bisulfite, 70 to the DNA sample solution. C water bath

1 hour, purification of the modified DNA, ethanol precipitation, dissolution; or

A3. Using EpiTect Bisulfite Kit: Qiagen GmbH, D40724 Hilden, Cat. No.

59104; or

A4. Use the Methylation-God kit: ZYMO Research China, Cat. No. D5005 or D5006.

Example 3

1-5. Same as Embodiment 1 Step 1-5

6. Ncol digestion analysis of the presence of cytosine positions (including C/H/M) at positions 141 and 142 in the PCR amplification product (Fig. 4);

7. Results: All C-PCR2A and C-PCR2B molecules have no cleavage point (5'-C||CTAGG-3'), can not be digested, and are still 393 bp intact DNA molecules;

Both M-PCR2A and M-PCR2B molecules retained the cleavage point of the enzyme, and were completely digested into two fragments of 251 bp and 142 bp by the enzyme; all H-PCR2A did not have the enzyme cleavage point and could not be digested; part of H-PCR2B retained The enzyme cleavage site is digested by the enzyme;

8. Judgment of results: The amplification products of the two modification methods were completely digested, and the cytosine of the two cleavage sites was methylated; the amplification products of the two modification methods were not digested by the enzyme. The cytosine is not methylated; the amplification product of the modification method A is not digested, and the amplification product of the modification method B is digested by the enzyme digestor or the amplification product of the modification method A, and the modification method B is expanded. If the product is not cleaved, the cytosine has been methylolated.

A2. Add a final concentration of 9 mol/L sodium bisulfite to the DNA sample solution, 70. C water bath

A3. Using EpiTect Bisulfite Kit: Qiagen GmbH, D40724 Hilden, Cat. No.

59104; or

A4. Use the Methylation-God kit: ZYMO Research China, Cat. No. D5005 or D5006.

Claims

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A method for chemically modifying 5-hydroxymethylcytosine in a DNA molecule, the method comprising the steps of: treating the DNA with a sodium bisulfite chemical modification method to cause a 5-hydroxymethyl group present in the DNA The pyrimidine is transformed and the transformed product is used as a PCR amplification template and amplified into thymine T or uracil U in PCR.

The method for chemically modifying 5-hydroxymethylcytosine in a DNA molecule according to claim 1, wherein the treatment of the DNA by a chemical modification method of sodium hydrogen sulfite is one of the following methods:

A1. Aqueous sodium hydroxide solution was added to the DNA sample solution, and a water bath at 95 ° C for 5 minutes was added to a final concentration of 5 mol/L of sodium hydrogen sulfite, followed by 95 ° C, 5 minutes, 60 ° C. C, 25 minutes, 95 ° C, 5 points, 60. C, 85 minutes, 95° (:, 5 minutes, 60. (, 175 minutes, heat cycle DNA treatment, final concentration of 0.3mol/L sodium hydroxide to terminate the sulfonation reaction and desulfurization, purification of modified DNA, ethanol precipitation , dissolved; or

A3. Using EpiTect Bisulfite Kit: Qiagen GmbH, D40724 Hilden, Cat. No.

59104; or

A4. Use Methylation-God kit: ZYMO Research China, Cat. No. D5005 or D5006.

3. A method of detecting a 5-hydroxymethylcytosine site present in a DNA molecule, the method comprising the steps of:

A. A piece of DNA is treated with sodium bisulfite chemical modification method to convert the hydroxymethylcytosine present in the DNA, and the transformed product is used as a PCR amplification template to be amplified into thymine T in PCR. Uracil U;

B. The same DNA is treated with sodium bisulfite chemical modification method, so that the 5-hydroxymethylcytosine present in the DNA is not transformed, and is amplified into cytosine C in PCR;

C. using the two treated DNAs as a template, and sequentially amplifying the DNA molecules with sequence-specific primers;

D. Sequencing the amplified two DNAs separately, comparing the C/T or CU difference sites in the DNA after sequencing, the C/T or CU difference site is the 5-hydroxymethyl group present in the DNA molecule. Cytosine site.

4. The method for detecting a 5-hydroxymethylcytosine site present in a DNA molecule according to claim 3, wherein: step D is digesting the amplified two DNAs with a restriction enzyme, If the DNA product amplified in step A is not digested, and the DNA product amplified in step B is digested or the DNA product amplified in step A is cleaved, and step B is amplified. The DNA product is not digested, and a 5-hydroxymethylcytosine site is present on the restriction endonuclease recognition site in the DNA molecule.

The method for detecting a 5-hydroxymethylcytosine site present in a DNA molecule according to claim 3 or 4, wherein the step of treating the DNA with sodium hydrogen sulfite chemical modification method in step A is as follows The methods listed:

A1. Add a sodium hydroxide aqueous solution to the DNA sample solution, water bath at 95 ° C for 5 minutes, add 5 mol / L of sodium bisulfite, then 95 ° C, 5 minutes, 60 ° C, 25 minutes, 95 ° ( , 5 minutes, 60 ° C, 85 minutes, 95 ° (, 5 minutes, 60. (, 175 minutes, heat cycle treatment of DNA, adding a final concentration of 0.3 mol / L sodium hydroxide to terminate the sulfonation reaction and desulfurization, after purification and modification DNA, ethanol precipitation, dissolution; or

A3. Using EpiTect Bisulfite Kit: Qiagen GmbH, D40724 Hilden, Cat. No.

59104; or

A4. Use Methylation-God Kit: ZYMO Research China, Cat. No. D5005 or

6. The method for detecting a 5-hydroxymethylcytosine site present in DNA according to claim 3 or 4, wherein: the step B is treated with sodium bisulfite chemical modification method to: An aqueous sodium hydroxide solution was added to the DNA sample solution, and a water bath at 95 ° C for 5 minutes was added to a final concentration of 5 mol/L of sodium hydrogen sulfite, 55. After 16 hours of C water bath, the sulfonation reaction and desulfurization were terminated by adding a final concentration of 0.3 mol/L sodium hydroxide, and the modified DNA was purified, precipitated by ethanol, and dissolved.

7. A method for detecting the presence of 5-hydroxymethylcytosine in a DNA molecule, the method comprising the steps of:

A. A piece of DNA is treated with sodium bisulfite chemical modification method to convert the hydroxymethylcytosine present in the DNA, and the transformed product is used as a template for PCR amplification and amplified into thymine T in PCR. Uracil U;

D. Using denatured high performance liquid chromatography workstation software to predict the DNA molecular denaturation temperature, and then use the denaturing high performance liquid chromatography to analyze the mutation pattern of the PCR product;

E. After PCR amplification of the DNA treated in step A, the PCR product appears as a single-stranded DNA peak, and the PCR-amplified DNA of the DNA treated in step B is expressed by PCR. As a double-stranded DNA chromatographic peak, it was confirmed that 5-hydroxymethylcytosine was present in the DNA molecule.

8. The method for detecting 5-hydroxymethylcytosine present in a DNA molecule according to claim 7, wherein the step of treating the DNA with sodium hydrogen sulfite chemical modification in step A is one of the following methods : .

A1. Add sodium hydroxide solution to the DNA sample solution, water bath at 95 ° C for 5 minutes, add 5 mol / L sodium bisulfite, then 95 ° C, 5 min, 60 ° C, 25 min, 95 ° C , 5 points, 60. C, 85 minutes, 95 ° C, 5 minutes, 60 ° C 175 minutes, heat cycle DNA treatment, add a final concentration of 0.3 mol / L sodium hydroxide to terminate the sulfonation reaction and desulfurization, purification of the modified DNA, ethanol precipitation, dissolution ; or

A2. Add a final concentration of 9 mol/L sodium bisulfite to the DNA sample solution, 70 ° C water bath

A3. Using EpiTect Bisulfite Kit: Qiagen GmbH, D40724 Hilden, Cat. No.

59104; or

A4. Use Methylation-God kit: ZYMO Research China, Cat. No. D5005 or D5006.

The method for detecting 5-hydroxymethylcytosine present in DNA according to claim 7 or 8, wherein the DNA is treated with sodium hydrogen sulfite chemical modification method in step B: Add sodium hydroxide aqueous solution to the sample solution, water bath at 95 ° C for 5 minutes, add 5 mol / L sodium hydrogen sulfite, the water bath at 55 ° C for 16 hours, and add the final concentration of 0.3 mol / L sodium hydroxide to terminate the sulfonation reaction. And desulfurization, purification of the modified DNA, ethanol precipitation, and dissolution.

10. Sodium bisulfite is used to modify or detect the use of 5-hydroxymethylcytosine present in DNA molecules.

The use according to claim 8, wherein: the sodium hydrogen sulfite modification converts 5-hydroxymethylated cytosine present in the DNA molecule, and the transformed product is used as a PCR amplification template in PCR. Amplified to thymine T or uracil U.

The use according to claim 10 or 11, wherein the sodium hydrogen sulfite is an EpiTect Bisulfite kit containing sodium hydrogen sulfite: Qiagen GmbH, D40724 Hilden, Cat. No. 59104 or Methylation- God kit: ZYMO Research China, Cat. No. D5005 or D5006.