阅读说明：本技术 一种基于G-四链体的鉴别BstUI甲基化敏感位点的方法 (Method for identifying BstUI methylation sensitive site based on G-quadruplex ) 是由 邓惠敏 范子彦 刘珊珊 杨飞 王颖 边照阳 唐纲岭 刘洋 于 2019-09-30 设计创作，主要内容包括：本发明涉及一种基于G-四链体的鉴别BstUI甲基化敏感位点的方法,属于生化分析领域。首先,以含G-四链体序列、经不同甲基化修饰的Hairpin DNA为底物,加入BstUI,于60℃反应,含BstUI甲基化敏感位点的Hairpin DNA底物不能被识别,从而不能被剪切,而不含BstUI甲基化敏感位点的Hairpin DNA底物能被剪切,释放出G-四链体片段；最后,向体系中加入N-甲基卟啉二丙酸IX,与G-四链体结合产生强荧光信号,可根据荧光信号的强弱鉴别BstUI的甲基化敏感位点。此外,也可通过凝胶电泳对反应后的DNA进行表征以判断甲基化敏感位点。本方法提供了两种灵活的鉴别方式,并鉴别了新的BstUI的甲基化敏感位点。(The invention relates to a method for identifying BstUI methylation sensitive sites based on a G-quadruplex, belonging to the field of biochemical analysis. Firstly, taking the Hairpin DNA containing the G-quadruplex sequence and modified by different methylation as a substrate, adding BstUI, reacting at 60 ℃, wherein the Hairpin DNA substrate containing BstUI methylation sensitive sites cannot be identified and cannot be sheared, and the Hairpin DNA substrate without BstUI methylation sensitive sites can be sheared, so that G-quadruplex fragments are released; and finally, adding N-methylporphyrindipropionic acid IX into the system, combining with a G-quadruplex to generate a strong fluorescent signal, and identifying the methylation sensitive site of BstUI according to the strength of the fluorescent signal. In addition, the DNA after reaction can be characterized by gel electrophoresis to judge methylation sensitive sites. The method provides two flexible identification modes and identifies the methylation sensitive sites of the novel BstUI.)

1. A method for identifying BstUI methylation sensitive sites based on G-quadruplexes, which is characterized by comprising the following steps: taking a G-quadruplex sequence-containing Hairpin DNA modified by different methylation as a substrate, adding a methylation-sensitive restriction enzyme BstUI, carrying out a shearing reaction at 60 ℃, and identifying BstUI methylation-sensitive sites by polyacrylamide gel electrophoresis (PAGE) or fluorescence measurement after the shearing reaction.

2. The G-quadruplex based method for identifying BstUI methylation sensitive sites according to claim 1, wherein: the method comprises the following steps:

(1) dissolving substrate DNA containing a G-quadruplex sequence and modified by different methylation in TE buffer solution to prepare 100 mu M substrate DNA, heating to 90 ℃ in a water bath, keeping for 5min, and slowly cooling to room temperature to obtain 100 mu M Hairpin DNA substrate;

(2) mu.L of 100. mu.M Hairpin DNA substrate was added to 100. mu.L of 1 XCutSmart buffer, 3. mu.L of 1000U/mL BstUI was added, and BstUI catalyzed cleavage reaction was performed in a water bath at 60 ℃ for 2 h;

(3) after the shearing reaction is finished, BstUI methylation sensitive sites can be identified by two modes of polyacrylamide gel electrophoresis (PAGE) and fluorescence determination, namely 15% PAGE is used for representing the condition that substrate DNA subjected to different methylation modifications is sheared; alternatively, 2. mu.L of 100. mu. M N-methylporphyrindipropionic acid IX (NMM) was added to the system, vortexed for 1min, and the fluorescence intensity was measured at an excitation wavelength of 399 nm.

3. The G-quadruplex based method for identifying BstUI methylation sensitive sites according to claim 2, wherein: the TE buffer component is 10mM Tris-HCl,1mM EDTA, pH 8.0.

4. The G-quadruplex based method for identifying BstUI methylation sensitive sites according to claim 2, wherein: the CutSmart buffer composition was 50mM Potashium Acetate,20mM Tris-Acetate,10mM Mgnesium Acetate, 100. mu.g/ml BSA, pH 7.9.

5. The G-quadruplex based method for identifying BstUI methylation sensitive sites according to claim 2, wherein: the N-methylporphyrin dipropionic acid IX (NMM) is dissolved in DMSO.

Technical Field

The invention relates to a method for identifying BstUI methylation sensitive sites based on a G-quadruplex, belonging to the field of biochemical analysis.

Background

Restriction endonucleases, also called restriction enzymes, are enzymes that recognize a specific deoxyribonucleotide sequence and cleave the phosphodiester bond between two deoxyribonucleotides at a specific position in each strand. The restriction is actually a protective mechanism for degrading exogenous DNA and maintaining the genetic stability of a host by using restriction enzymes. Methylation is a common modification that protects adenine A and cytosine C from methylation. The purpose of identifying self genetic material and foreign genetic material is achieved through methylation. Most restriction enzymes are sensitive to DNA methylation, and when the restriction enzyme target sequence overlaps with the methylation site, the restriction enzyme reaction is affected. Thus, for efficient cleavage of DNA, both DNA methylation and the sensitivity of restriction enzymes to this type of methylation must be considered.

BstuI is a commercial restriction enzyme and is widely used in DNA methylation related research. The current methylation sensitive information of BstUI is vaguely reported in the literature, and only indicates that the BstUI is a CpG methylation sensitive restriction enzyme, but the specific methylation sensitive site is not clear. REBASE is a very useful resource pool, and can inquire the comprehensive information and updated information of restriction enzyme, including the specificity, sensitivity and commercial source of restriction enzyme. The identification sequence of BstUI and methylation sensitivity related information provided in REBASE are shown in FIG. 1.

The invention aims to establish a method for identifying BstUI methylation sensitive sites based on a G-quadruplex, which realizes the identification of the BstUI methylation sensitive sites by reasonably designing a substrate DNA sequence and taking polyacrylamide gel electrophoresis or strong fluorescence generated by the combination of N-methylporphyrin dipropionic acid IX (NMM) and the G-quadruplex as a signal source, thereby supplementing the existing data.

Disclosure of Invention

The invention aims to provide a method for identifying BstUI methylation sensitive sites based on a G-quadruplex. Firstly, taking Hairpin DNA containing a G-quadruplex sequence and modified by different methylation as a substrate, adding BstUI (restriction enzyme) which is sensitive to methylation, and carrying out a shearing reaction at 60 ℃, wherein the Hairpin DNA substrate containing BstUI methylation sensitive sites cannot be recognized by BstUI and cannot be sheared, and the Hairpin DNA substrate without BstUI methylation sensitive sites can be sheared, so that a G-quadruplex fragment can be released; finally, N-methylporphyrindipropionic acid IX (NMM) is added into the system, and after the N-methylporphyrindipropionic acid IX (NMM) is combined with the G-quadruplex, a strong fluorescence signal is generated, and the methylation sensitive site of BstUI can be identified according to the strength of the fluorescence signal. In addition, the DNA after the shearing reaction can be characterized by polyacrylamide gel electrophoresis to judge methylation sensitive sites. The method provides two flexible identification modes and identifies the methylation sensitive sites of the novel BstUI.

The purpose of the invention is realized by the following technical scheme:

a method for identifying BstUI methylation sensitive sites based on G-quadruplexes, comprising the following steps:

(1) substrate DNA containing the G-quadruplex sequence (modified by different methylation) is dissolved in TE buffer (10mM Tris-HCl,1mM EDTA, pH 8.0) to prepare 100 mu M substrate DNA, the substrate DNA is heated to 90 ℃ in a water bath, kept for 5min and slowly cooled to room temperature to obtain 100 mu M Hairpin DNA substrate.

(2) mu.L of 100. mu.M Hairpin DNA substrate was added to 100. mu.L of 1 XCutSmart buffer (50mM Potashium Acetate,20mM Tris-Acetate,10mM Magnesium Acetate, 100. mu.g/mL BSA, pH7.9) and 3. mu.L of 1000U/mL Bstui was added to carry out BstUI catalyzed cleavage reaction in a water bath at 60 ℃ for 2 h.

(3) After the shearing reaction is finished, BstUI methylation sensitive sites can be identified by two modes of polyacrylamide gel electrophoresis (PAGE) and fluorescence determination, namely 15% PAGE is used for representing the condition that substrate DNA subjected to different methylation modifications is sheared; alternatively, 2. mu.L of 100. mu. M N-methylporphyrindipropionic acid IX (NMM) was added to the system, vortexed for 1min, and the fluorescence intensity was measured at an excitation wavelength of 399 nm.

The invention has the advantages of

The invention establishes a method for identifying BstUI methylation sensitive sites based on a G-quadruplex. The method is simple to operate, provides two flexible identification modes, identifies a new methylation sensitive site of BstUI, supplements the existing methylation sensitive information of BstUI, and provides reference for methylation research related to the use of BstUI.

Drawings

FIG. 1 recognition sequence and methylation sensitive sequence of BstUI in REBASE library;

FIG. 2 is a schematic diagram of the detection principle;

FIG. 3 shows the results of fluorescence analysis for feasibility verification;

FIG. 4 shows the results of linear regression analysis of Dnmt1 activity assay.

Detailed Description

The invention is further described with reference to examples, but not limited thereto.

Example 1:

(1) reagent and apparatus

Substrate DNAs (different in methylation degree) purified by HPLC are purchased from Biotechnology engineering Co., Ltd (Shanghai), dissolved in TE buffer solution, and subjected to annealing treatment at 90 ℃ to obtain 100 mu M of Hairpin DNA substrate stock solution; 10000U/mL of Bstui, 10 × CutSmart buffer were purchased from New England Biolabs (Epstein, England); N-Methylporphyrin dipropionic acid IX was purchased from Frontier Scientific (USA).

Gel electrophoresis separation was performed using a general Mini vertical electrophoresis apparatus (Biotechnology, Ltd., Shanghai).

Fluorescence analysis was carried out using a HORIBA Fluoromax-4 fluorometer (Japan), with an excitation wavelength of 399nm and a slit width of 5 nm.

(2) BstUI methylation sensitive site identification

The method adopts 4 substrate DNAs with different methylation degrees, and the sequences of the substrate DNAs are shown in Table 1. Wherein the wavy line is a sequence capable of forming a G-quadruplex, the underlined part is a recognition sequence of BstuI,/i 5 MedC/indicates a methylation modification at the carbon atom at position 5 of cytosine C. The substrate DNA is annealed at 90 ℃ to obtain the Hairpin DNA, on one hand, the G-quadruplex is blocked and protected, and on the other hand, a double-stranded DNA region required by Bstui recognition is formed.

TABLE 1 substrate DNA of different methylation degrees

The detection principle of the method is shown in FIG. 2, firstly, Hairpin DNA with different methylation degrees is used as a substrate, dotted circles represent fragments capable of forming a G-quadruplex, and thick solid lines represent the identification region of BstUI. When BstUI is added, the substrate DNA can be recognized by BstUI, and is cut, so that a DNA fragment capable of forming a G-quadruplex is released, and the fragment can be combined with NMM, and strong fluorescence far stronger than the self-fluorescence intensity of the NMM is generated. On the contrary, when the substrate DNA cannot be recognized by BstUI (BstUI block), the substrate DNA cannot be cut and a G-quadruplex-NMM conjugate cannot be formed, and thus the fluorescence is weak, so that the methylation sensitive site of BstUI can be identified based on the fluorescence intensity.

Based on the above principle, the following experiments were performed:

mu.L of 100. mu.M Hairpin DNA substrate was added to 1 XCutSmart buffer (50mM Potashium Acetate,20mM Tris-Acetate,10mM Magnesium Acetate, 100. mu.g/mL BSA, pH7.9), 3. mu.L of 10000U/mL Bstui was added, diluted with ultrapure water to a final volume of 100. mu.L, and Bstui-catalyzed cleavage reaction was carried out in a 60 ℃ water bath for 2 h. After the cleavage reaction was completed, the sample was separated on a 15% polyacrylamide gel (PAGE), and cleavage of the substrate DNA was observed. Alternatively, 10. mu.L of the DNA sample after completion of the cleavage reaction was added with 0.2. mu.L of 100. mu. M N-methylporphyrindipropionic acid IX (NMM in DMSO), diluted to 200. mu.L with TE buffer, vortexed for 1min, and subjected to fluorescence measurement.

The results of the characterization of the 15% PAGE are shown in FIG. 3, for four substrate DNAs, each substrate is in duplicate, where 1 and 2 are S-A, 3 and 4 are S-B, 5 and 6 are S-C, and 7 and 8 are S-D. As can be seen, neither S-A nor S-C is cleaved, and its methylation site prevents recognition by BstUI, and both S-B and S-D are cleaved, indicating that BstUI recognizes it. Thus, it was found that, in the BstUI recognition sequence 5 '-CGCG-3', the recognition of BstUI was blocked once the first cytosine C was methylated (S-A, S-C), and that when the first cytosine C was not methylated, the recognition of BstUI was not affected by whether the second cytosine C was methylated (S-B, S-D).

The results of the fluorescence measurements are shown in FIG. 4, where the fluorescence is weak in the presence of NMM alone; when the substrate DNA can be recognized by BstUI, the G-quadruplex fragment can be released by shearing, and thus the enhanced fluorescence intensity can be formed by combining with NMM; when the substrate DNA cannot be recognized by BstUI, the G-quadruplex fragment cannot be released, and thus the fluorescence intensity is weak.

The above results demonstrate the feasibility of the process.