Method for accurately distinguishing and sequencing 5-methylcytosine and 5-hydroxymethylcytosine in DNA

Abstract

The invention provides a method for accurately distinguishing and sequencing 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in DNA (Deoxyribose Nucleic Acid). The core of the method is that the CMD1 enzyme is used for specifically converting the 5mC into 5-glyceryl methyl cytosine (5g mC), so that the 5mC and 5hmC are distinguished. Specifically, after CMD1 enzyme treatment, 5mC and 5hmC are sequenced respectively in combination with deaminase treatment (CD-seq) or TET protein oxidation and hydrosulfite treatment (CT-seq). According to the method disclosed by the invention, the sequencing accuracy is improved, the 5mC and 5hmC which are originally difficult to distinguish can be accurately identified, and the research range of epigenetics is expanded. Meanwhile, the method is compatible with the prior art, and a new tool is provided for early diagnosis, precise treatment and biological science research of diseases. The implementation of the invention can promote the development of epigenetics and related fields.

Description

Technical Field

[0001] The present invention relates to the fields of molecular biology, epigenetics and sequencing technology, and in particular to a method for accurately distinguishing and sequencing 5-methylcytosine and 5-hydroxymethylcytosine in DNA. Background Art

[0002] In mammals, DNA methylation is one of the important mechanisms of epigenetic regulation, among which 5mC, as the main form of DNA methylation, plays a key role in biological processes such as gene expression regulation, X chromosome inactivation, genomic imprinting and transposon suppression. However, 5mC can be oxidized to 5hmC by the TET protein family, and in some cell types, 5hmC can account for up to 20% of the total 5mC. Traditional bisulfite sequencing (BS-seq) methods cannot distinguish between 5hmC and 5mC, which limits the in-depth understanding and functional analysis of these two DNA modifications.

[0003] Although some methods have been proposed to detect 5hmC and infer 5mC levels by subtracting 5hmC from whole-genome BS-seq data, these methods have the disadvantage of underestimating 5mC levels at sites with high 5hmC abundance. Therefore, it is particularly important to develop a method that can directly and accurately distinguish and sequence 5mC and 5hmC.

[0004] In order to solve the above problems, the applicant proposes a method for accurately distinguishing and sequencing 5-methylcytosine and 5-hydroxymethylcytosine in DNA. Summary of the invention

[0005] The object of the present invention is to provide a method for accurately distinguishing and sequencing 5-methylcytosine and 5-hydroxymethylcytosine in DNA, so as to solve the problems in the prior art.

[0006] To achieve the above object, the present invention provides the following technical solution: a method for accurately distinguishing and sequencing 5-methylcytosine and 5-hydroxymethylcytosine in DNA, comprising the following steps:

[0007] The CMD1 enzyme converts 5mC in DNA into 5-glycerylmethylcytosine;

[0008] Depending on the sequencing technology selected, do one of the following:

[0009] Treating DNA with a specific deaminase completely converts 5hmC into uracil, while 5gmC resists deaminase activity and remains unchanged. 5gmC is directly read as cytosine by sequencing analysis, thereby distinguishing and sequencing 5mC.

[0010] DNA is treated with TET proteins for oxidation, converting 5hmC to 5-carboxycytosine, which is then converted to U or T after bisulfite treatment, whereas 5gmC resists TET oxidation and bisulfite treatment and remains unchanged, being read as C by sequencing analysis, thereby distinguishing and sequencing 5mC.

[0011] Optionally, the specific deaminase is a deaminase that can efficiently convert 5hmC but not 5gmC.

[0012] Optionally, the TET protein is at least one of TET1, TET2 or TET3.

[0013] Optionally, it also includes combining TAB-seq or ACE-seq technology to obtain the precise distribution spectrum of 5mC and 5hmC in the genome.

[0014] Use of the method according to any one of claims 1 to 4 in the preparation of a reagent or kit for studying the role of DNA methylation and hydroxymethylation in biological processes or diseases.

[0015] Optionally, the biological process includes but is not limited to gene expression regulation, X chromosome inactivation, genomic imprinting and transposon suppression; the disease includes but is not limited to cancer and neurodegenerative diseases, such as Alzheimer's disease.

[0016] Beneficial effects: 1. Improve sequencing accuracy:

[0017] The present invention uses CMD1 enzyme to specifically convert 5mC into 5-glycerylmethylcytosine (5gmC), thereby achieving effective distinction between 5mC and 5hmC. This conversion enables accurate identification of 5mC and 5hmC, which were originally difficult to distinguish, in the subsequent sequencing process, thereby greatly improving the accuracy of sequencing.

[0018] Expanding research scope:

[0019] Traditional sequencing methods cannot effectively distinguish 5mC and 5hmC, which limits in-depth research on these two DNA modifications. The emergence of this invention provides researchers with a new tool that enables them to more deeply explore the role of 5mC and 5hmC in biological processes, thereby expanding the scope of epigenetic research.

[0020] 2. Promote disease diagnosis and treatment:

[0021] Abnormal modifications of 5mC and 5hmC are closely related to the occurrence and development of a variety of diseases. The method of the present invention can accurately sequence the distribution of these two modifications in the genome, providing the possibility for early diagnosis and precise treatment of diseases. For example, in the study of complex diseases such as cancer and neurodegenerative diseases, the method of the present invention will help reveal the epigenetic mechanism of disease occurrence and provide an important basis for the development of new drugs and the formulation of treatment strategies.

[0022] 3. Improve technical compatibility:

[0023] The method of the present invention has good compatibility with existing technologies such as TAB-seq or ACE-seq, and can be used in combination to obtain a more comprehensive 5mC and 5hmC distribution spectrum. This technical compatibility not only improves the flexibility of research, but also provides researchers with more diversified research methods.

[0024] 4. Promote the development of epigenetics:

[0025] The successful implementation of the present invention not only provides a new research tool for the field of epigenetics, but also injects new vitality into the development of this field. With the wide application of the method of the present invention, it is expected to promote the in-depth development of epigenetic research and make greater contributions to revealing the mysteries of life and preventing and treating diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of CT-seq and CD-seq principles established for the embodiment of the present invention DETAILED DESCRIPTION

[0027] The following describes the preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0028] Example 1

[0029] In order to solve the above technical problems, the present invention provides a method for accurately distinguishing and sequencing 5mC and 5hmC in DNA. The method is based on the specific enzymatic activity of CMD1 enzyme on 5mC, converting it into 5-glycerylmethylcytosine (5gmC), thereby achieving accurate distinction between 5mC and 5hmC. Further, by combining specific deaminase treatment or TET protein oxidation treatment and bisulfite sequencing technology, 5mC and 5hmC can be sequenced separately.

[0030] The technical solution of the present invention mainly includes the following two core parts: CMD1-Deaminase sequencing (CD-seq) and CMD1-TET bisulfite sequencing (CT-seq).

[0031] CMD1-Deaminase Sequencing (CD-seq)

[0032] (1) CMD1 enzyme treatment:

[0033] First, select DNA samples containing 5mC and 5hmC.

[0034] The DNA samples were then treated with CMD1, an enzyme that has specific activity for 5mC, converting it to 5-glycerylmethylcytosine (5gmC) without affecting 5hmC.

[0035] By controlling the treatment conditions of the CMD1 enzyme (such as enzyme concentration, treatment time, etc.), it can be ensured that 5mC is completely converted into 5gmC.

[0036] (2) Deaminase treatment:

[0037] The CMD1-treated DNA samples were then treated with a specific deaminase that efficiently converts 5hmC to other forms, such as uracil, without affecting 5gmC.

[0038] By choosing the appropriate deaminase and treatment conditions, one can ensure that 5hmC is fully converted while 5gmC remains unchanged.

[0039] (3) Sequencing analysis:

[0040] Finally, the DNA samples treated with deaminase were sequenced and analyzed. Since 5gmC will produce specific sequencing signals during the sequencing process, these signals can be identified to distinguish and sequence the original 5mC.

[0041] By comparing the sequencing results before and after treatment, the location and distribution of 5mC and 5hmC in the genome can be accurately determined.

[0042] CMD1-TET bisulfite sequencing (CT-seq)

[0043] (1) CMD1 enzyme treatment:

[0044] Similar to CD-seq, a DNA sample containing 5mC and 5hmC is first selected, and the DNA sample is treated with the CMD1 enzyme to convert 5mC into 5gmC.

[0045] (2) TET protein oxidation treatment:

[0046] Next, the DNA samples treated with CMD1 were oxidized using TET proteins, which are enzymes that can oxidize 5hmC to 5-formylcytosine (5fC) or 5-carboxylcytosine (5caC).

[0047] By controlling the treatment conditions of TET proteins, we can ensure that 5hmC is fully oxidized to a detectable modified form, while 5gmC resists TET oxidation and remains unchanged.

[0048] (3) Bisulfite treatment:

[0049] Then, the DNA samples treated with TET protein oxidation were treated with bisulfite. Bisulfite can convert unmodified cytosine and oxidized 5hmC (5fC and 5caC) into uracil, but has no effect on 5gmC.

[0050] By treating with bisulfite, 5gmC (representing the original 5mC) and oxidized 5hmC can be further distinguished.

[0051] (4) Sequencing analysis:

[0052] Finally, the bisulfite-treated DNA samples were sequenced and analyzed. Since 5gmC and oxidized 5hmC will produce different sequencing signals during the sequencing process, these signals can be identified to distinguish and sequence the original 5mC and 5hmC.

[0053] By comparing the sequencing results before and after treatment, the location and distribution of 5mC and 5hmC in the genome can be accurately determined.

[0054] Selection and optimization of CMD1 enzyme

[0055] When selecting a CMD1 enzyme, its enzymatic activity, stability, and specificity for 5mC need to be considered. The CMD1 enzyme can be modified by genetic engineering to improve its enzymatic activity and specificity.

[0056] The treatment conditions of CMD1 enzyme, such as enzyme concentration, treatment time, temperature, etc., were optimized to ensure that 5mC was completely converted into 5gmC while avoiding damage to the DNA sample.

[0057] Selection and optimization of deaminase

[0058] Deaminases with the ability to efficiently convert 5hmC were selected, and their treatment conditions were optimized to ensure that 5hmC was fully converted while 5gmC remained unchanged.

[0059] The conversion efficiency and specificity of deaminases can be verified by in vitro experiments to ensure their reliability in practical applications.

[0060] Selection and optimization of TET proteins

[0061] Select TET proteins with the ability to efficiently oxidize 5hmC and optimize their treatment conditions to ensure that 5hmC is fully oxidized to a detectable modified form.

[0062] The oxidation efficiency and specificity of TET proteins can be verified by in vitro experiments to ensure their reliability in practical applications.

[0063] Optimization of Bisulfite Treatment Conditions

[0064] Optimize the bisulfite treatment conditions, such as concentration, treatment time, temperature, etc., to ensure that unmodified cytosine and oxidized 5hmC are completely converted to uracil, while 5gmC remains unchanged.

[0065] The effect of bisulfite treatment can be verified by in vitro experiments to ensure its reliability in practical applications.

[0066] Selection and optimization of sequencing technology

[0067] Select sequencing technology with high sensitivity, high resolution, and high throughput, such as second-generation sequencing or third-generation sequencing technology, to ensure accurate and efficient sequencing analysis of the processed DNA samples.

[0068] Optimize the parameter settings of sequencing technology, such as sequencing depth, sequencing length, etc., to improve the accuracy and reliability of sequencing results.

[0069] Combining with other technologies

[0070] The technical solution of the present invention can be combined with existing technologies such as TAB-seq or ACE-seq to obtain a more comprehensive 5mC and 5hmC distribution spectrum. This combination can further improve the accuracy and reliability of sequencing and provide researchers with more diversified research methods.

[0071] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the attached claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention. Any figure mark in the claims should not be regarded as limiting the claims involved.

[0072] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for accurately distinguishing and sequencing 5-methylcytosine and 5-hydroxymethylcytosine in DNA, characterized in that: The following steps are involved: The CMD1 enzyme converts 5mC in DNA into 5-glycerylmethylcytosine; Depending on the sequencing technology selected, do one of the following: Treating DNA with a specific deaminase completely converts 5hmC into uracil, while 5gmC resists deaminase activity and remains unchanged. 5gmC is directly read as cytosine by sequencing analysis, thereby distinguishing and sequencing 5mC. DNA is treated with TET proteins for oxidation, converting 5hmC to 5-carboxycytosine, which is then converted to U or T after bisulfite treatment, whereas 5gmC resists TET oxidation and bisulfite treatment and remains unchanged, being read as C by sequencing analysis, thereby distinguishing and sequencing 5mC.

2. The method according to claim 1, characterized in that The specific deaminase is a deaminase that can efficiently convert 5hmC but does not convert 5gmC.

3. The method according to claim 1, characterized in that The TET protein is at least one of TET1, TET2 or TET3.

4. The method according to claim 1, characterized in that: It also includes combining TAB-seq or ACE-seq technology to obtain the precise distribution spectrum of 5mC and 5hmC in the genome.

5. Use of the method according to any one of claims 1 to 4 in the preparation of a reagent or kit for studying the role of DNA methylation and hydroxymethylation in biological processes or diseases.

6. The use according to claim 5, characterized in that: The biological processes include, but are not limited to, gene expression regulation, X chromosome inactivation, genomic imprinting, and transposon suppression; the diseases include, but are not limited to, cancer and neurodegenerative diseases such as Alzheimer's disease.

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