DNA methylation label-free fluorescence detection method, reagent composition and application

Abstract

The invention belongs to the technical field of gene detection, and particularly relates to a DNA methylation label-free fluorescence detection method, a reagent composition and application. The method comprises the following steps: cracking target DNA (Deoxyribose Nucleic Acid) by adopting GLaI endonuclease, and carrying out GLaI endonuclease reaction to obtain a digestion product; carrying out a double-cascade chain displacement amplification reaction on the obtained digestion product to obtain a double-cascade chain displacement amplification reaction product; carrying out CRISPR / Cas12a reaction on the obtained double-cascade chain displacement amplification reaction product, so as to obtain a CRISPR / Cas12a reaction product; in the CRISPR / Cas12a reaction, a G triplex is used as a signal source; and adding a fluorescent reaction reagent into the CRISPR / Cas12a reaction product, and measuring the fluorescence intensity of the CRISPR / Cas12a reaction product to obtain a methylation detection result of the target DNA. The detection method has the characteristics of low cost, universality, high sensitivity, high accuracy, good selectivity and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection, and specifically relates to a label-free fluorescence detection method for DNA methylation, a reagent composition and an application thereof. Background Art

[0002] DNA methylation is a highly conserved epigenetic modification mechanism, catalyzed by DNA methyltransferases, which covalently attach methyl groups to specific DNA bases using S-adenosylmethionine as the methyl donor. In mammals, methylation primarily occurs through the covalent attachment of a methyl group to the fifth carbon atom of cytosine, forming 5-methylcytosine, which plays a central role in genome stability and transcriptional regulation. This dynamic and reversible modification transcends the irreversible limitations of traditional gene mutations, making it a novel molecular target for disease treatment.

[0003] Currently, a variety of molecular assays have been developed for analyzing DNA methylation, including methylation-sensitive restriction endonucleases (MSREs), polymerase chain reaction (PCR), bisulfite sequencing, and mass spectrometry. These methods are primarily based on bisulfite conversion or MSRE digestion. Bisulfite conversion is considered the gold standard for measuring DNA methylation. It converts unmethylated cytosine to uracil using bisulfite. However, this process requires acidic and high-temperature conditions, which can lead to DNA degradation and limit its applicability to trace samples. Techniques based on the MSRE principle offer a bisulfite-independent approach for measuring DNA methylation. MSREs are a class of methylation-sensitive restriction endonucleases that effectively distinguish methylated from unmethylated sequences. Furthermore, the reaction is performed under mild conditions, avoiding the risk of chemical damage. However, the content of methylated DNA in cancer cells is very low, even as low as 0.1%, while the detection limit of existing MSRE-PCR techniques is only 0.5%-1.0%. Therefore, the sensitivity and accuracy of DNA methylation detection based on MSRE digestion need to be further improved.

[0004] In the prior art, patent CN118028438A discloses a method for rapid detection of DNA methylation at specific sites triggered by methylation-dependent enzyme cleavage, comprising: 1) GlaI specifically recognizes and cuts the 5'-GmCGmC-3' methylation site of DNA and produces DNA fragments with free 3'-OH ends to initiate EXPAR; 2) EXPAR amplifies a large amount of single-stranded DNA in a short period of time; 3) the accumulated single-stranded DNA activates the trans-cleavage activity of CRISPR / Cas12a, generating a fluorescent signal with a thousand-fold efficiency amplification.

[0005] The fluorescent probes of the CRISPR / Cas system can achieve signal amplification through the trans-cleavage characteristics of the Cas protein (cas12a and cas13a), making the detection sensitive and able to detect extremely low concentrations of target molecules. The crRNA-guided targeted recognition mechanism ensures high specificity for specific sequences. The fluorescent probes of the CRISPR / Cas system are usually composed of fluorescent groups-ssDNA-quenching groups. There are still some challenges, such as the difficulty of short chain synthesis and purification, the photobleaching of fluorescent dyes (such as FAM) under continuous excitation, and the high cost of fluorescent probes. Therefore, it is necessary to develop a highly sensitive, highly accurate, simple and easy to use, and low-cost DNA methylation detection method. Summary of the Invention

[0006] In view of this, in order to solve the problems of existing DNA methylation detection methods, such as easy DNA degradation and detection failure, and insufficient sensitivity and accuracy in detecting low-concentration methylated DNA, the present invention provides a label-free fluorescent detection method for DNA methylation based on GLaI enzyme-assisted double-cascade strand displacement amplification coupled with CRISPR / Cas12a system, a reagent composition and application. This method is mainly based on nuclease-assisted constant-temperature signal amplification and G-triplex label-free fluorescent biosensor to achieve DNA methylation detection. It has the advantages of being universal, mild, highly sensitive, and selective, and has good application prospects.

[0007] One of the objectives of the present invention is to provide a reagent composition for detecting DNA methylation.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] A reagent composition for detecting DNA methylation, the reagent composition comprising a GLaI endonuclease cleavage reaction reagent, a double-cascade strand displacement amplification reaction reagent, a CRISPR / Cas12a reaction reagent, and a fluorescence reaction reagent;

[0010] The GLaI endonuclease cleavage reaction reagent includes GLaI endonuclease;

[0011] The CRISPR / Cas12a reaction reagent includes a guanine-rich sequence, and the guanine-rich sequence includes any one or more of Oxy3.5, Oxy3, Cat G4, G31, 22AG, 16AG, Telo 24, Telo 18, C-MYC22, and C-MYC15; the nucleotide sequences of Oxy 3.5, Oxy 3, Cat G4, G31, 22AG, 16AG, Telo 24, Telo 18, Cmyc-22, and C-MYC15 are shown in SEQ ID NO: 1 to SEQ ID NO: 10, respectively;

[0012] The fluorescent reaction reagent includes Thioflavin T.

[0013] As a specific functional nucleic acid, a guanine (G)-rich sequence specifically binds to Thioflavin T to form a G-triplex / Thioflavin T complex, which enhances the fluorescence of Thioflavin T. The fluorescence of this complex is approximately 20 times higher than that of Thioflavin T alone, significantly improving the fluorescence properties of Thioflavin T itself. The principle is simple, and G-quadruplex / G-triplex and Thioflavin T have been widely used as fluorescent indicators. G-triplet is less stable than G-quadruplex. The present invention uses G-triplex / Thioflavin T to replace the fluorescent probe of the CRISPR / Cas system, which can effectively improve the sensitivity and stability of the detection method and reduce its cost.

[0014] Furthermore, the GLaI endonuclease cleavage reaction reagent includes GLaI endonuclease, SE restriction endonuclease buffer and enzyme-free water;

[0015] and / or the double-cascade strand displacement amplification reaction reagents include NEB restriction endonuclease buffer, NEB buffer 2, template 1, template 2, Klenow Fragment polymerase, Nb.BbvCI endonuclease, dNTPs, and enzyme-free water, the nucleotide sequence of template 1 is shown in SEQ ID NO: 12, and the nucleotide sequence of template 2 is shown in SEQ ID NO: 13;

[0016] And / or the CRISPR / Cas12a reaction reagent includes Cas12a nuclease, crRNA, the guanine-rich sequence, NEB buffer and enzyme-free water, and the nucleotide sequence of the crRNA is shown in SEQ ID NO: 14;

[0017] And / or the fluorescent reaction reagent includes Thioflavin T, Tris-HCl buffer and enzyme-free water.

[0018] Furthermore, the guanine-rich sequence is G31.

[0019] A second object of the present invention is to provide a label-free fluorescent biosensor for detecting DNA methylation.

[0020] To achieve the above object, the present invention adopts the following technical solutions:

[0021] A label-free fluorescent biosensor for detecting DNA methylation contains the aforementioned reagent composition.

[0022] A third object of the present invention is to provide a label-free fluorescence detection method for DNA methylation.

[0023] To achieve the above object, the present invention adopts the following technical solutions:

[0024] A label-free fluorescent detection method for DNA methylation, using the aforementioned reagent composition and / or label-free fluorescent biosensor for detection, wherein the method is not intended for disease treatment or diagnosis; the method comprises the following steps:

[0025] S1: cleavage of target DNA using GLaI endonuclease, and obtaining digestion products through GLaI endonuclease reaction;

[0026] S2: The digestion product obtained in S1 is subjected to a double-cascade strand displacement amplification reaction to obtain a double-cascade strand displacement amplification reaction product;

[0027] S3: The double-cascade strand displacement amplification reaction product obtained in S2 is subjected to a CRISPR / Cas12a reaction to obtain a CRISPR / Cas12a reaction product; in the CRISPR / Cas12a reaction, a G triplex is used as a signal source;

[0028] S4: Add a fluorescent reaction reagent to the CRISPR / Cas12a reaction product obtained in S3, measure its fluorescence intensity, and obtain the methylation detection result of the target DNA.

[0029] The detection principle of the present invention is as follows:

[0030] The GLaI enzyme can distinguish between methylated and unmethylated DNA double strands. Only when the recognition site is methylated will the target DNA be cut, triggering double-cascade strand displacement amplification. The high amplification capacity of double-cascade strand displacement amplification can produce a large amount of ssDNA, which can then be recognized by Cas12a / crRNA and activate the trans-cleavage function of Cas12a / crRNA. Thioflavin T specifically binds to the G-triplex to form a G-triplex / thioflavin T complex, which efficiently enhances the fluorescence reaction of Thioflavin T. Under the action of Cas12a / crRNA, the structure of the G-triplex is destroyed, and DNA methylation is detected by changes in fluorescence values.

[0031] Furthermore, the GLaI endonuclease reaction system includes 1.6 μL of target DNA, 0.04-0.5 U / μL of GLaI endonuclease, 0.32 μL of SE restriction endonuclease buffer, and enzyme-free water to 3.2 μL;

[0032] And / or the double-cascade strand displacement amplification reaction system includes 0.64 μL of NEB restriction endonuclease buffer, 20.64 μL of NEB buffer, 11-40 nM of template, 21-40 nM of template, 3.2 μL of digestion product, 75-200 U / mL of Klenow Fragment polymerase, 75-300 U / mL of Nb.BbvCI endonuclease, 0.125-1.5 mM of dNTP, and enzyme-free water to 6.4 μL;

[0033] And / or the CRISPR / Cas12a reaction system includes 2 μL of NEB buffer, 2 μL of double-cascade strand displacement amplification product, 40-140 nM of Cas12a, 40-140 nM of crRNA, 100-600 nM of guanine-rich sequence, and enzyme-free water to 20 μL;

[0034] And / or the fluorescence reaction system includes 20 μL of CRISPR / Cas12a reaction product, 1-6 μM Thioflavin T, and Tris-HCl buffer supplemented to 100 μL.

[0035] Preferably, the GLaI endonuclease reaction system includes 1.6 μL of target DNA, 0.1 U / μL of GLaI endonuclease, 0.32 μL of SE restriction endonuclease buffer, and enzyme-free water to make up to 3.2 μL.

[0036] Preferably, the double-cascade strand displacement amplification reaction system includes 0.64 μL of NEB restriction endonuclease buffer, 20.64 μL of NEB buffer, 110 nM of template, 210 nM of template, 3.2 μL of digestion product, 125 U / mL of Klenow Fragment polymerase, 125 U / mL of Nb.BbvCI nuclease, 0.25 mM of dNTP, and enzyme-free water to 6.4 μL.

[0037] Preferably, the CRISPR / Cas12a reaction system includes 2 μL of NEB buffer, 2 μL of double-cascade strand displacement amplification product, 100 nM of Cas12a, 100 nM of crRNA, 1500 nM of G3, and enzyme-free water to 20 μL.

[0038] Preferably, the fluorescence reaction system includes 20 μL of CRISPR / Cas12a reaction product, 5 μM Thioflavin T, and Tris-HCl buffer supplemented to 100 μL.

[0039] Furthermore, the GLaI endonuclease cleavage reaction conditions are: 25-40°C for 30-120 min, then heated to 60-80°C for 5-15 min;

[0040] And / or the double cascade strand displacement amplification reaction conditions are: 30-45° C. for 10-150 min;

[0041] And / or the CRISPR / Cas12a reaction conditions are: 30-45° C. for 10-120 min;

[0042] And / or the fluorescence reaction conditions are: 20-30° C. reaction time 1-3 min.

[0043] Preferably, the GLaI endonuclease cleavage reaction conditions are: reaction at 30°C for 60 minutes, then heating to 70°C for 10 minutes.

[0044] Preferably, the double-cascade strand displacement amplification reaction conditions are: 37° C. reaction for 60 min.

[0045] Preferably, the CRISPR / Cas12a reaction conditions are: 37° C. reaction for 60 min.

[0046] Preferably, the fluorescence reaction condition is: 25° C. reaction for 1 min.

[0047] Furthermore, the DNA to be tested is genomic DNA of cells or tissues suspected of being tumors; the tumors include colorectal cancer, breast cancer and / or gastric cancer.

[0048] Furthermore, the tumor is colorectal cancer, its DNA target gene is Septin 9-C, and its methylated DNA target gene is Septin 9-mC; the nucleotide sequence of Septin 9-C is shown in SEQ ID NO:11, and the nucleotide sequence of Septin 9-mC is shown in SEQ ID NO:19.

[0049] A fourth object of the present invention is to provide a use of the aforementioned reagent composition and / or the aforementioned sensor in preparing a cancer detection kit.

[0050] To achieve the above object, the present invention adopts the following technical solutions:

[0051] The use of the aforementioned reagent composition and / or the aforementioned label-free fluorescent biosensor in the preparation of a cancer detection kit is characterized in that whether cancer occurs is determined by detecting whether methylation occurs in genomic DNA; the cancer includes colorectal cancer, breast cancer and / or gastric cancer.

[0052] Preferably, the cancer is colorectal cancer.

[0053] The beneficial effects of the present invention are:

[0054] 1. The present invention provides a label-free fluorescence detection method for DNA methylation based on GLaI enzyme-assisted double cascade strand displacement amplification coupled with CRISPR / Cas12a system. Compared with traditional methylation-specific PCR, double cascade strand displacement amplification based on nuclease endonuclease GLaI has mild conditions and fast detection speed. It shows good performance in DNA methylation detection and can accurately detect low concentration methylation levels, and has good application prospects.

[0055] 2. The method of the present invention utilizes the specificity of GLaI to effectively distinguish methylated and unmethylated DNA. At the same time, the high amplification ability of the double-cascade strand displacement amplification can produce a large amount of ssDNA containing guanine-rich sequences, and the obtained ssDNA can then be recognized by Cas12a; and the high specificity and self-amplification ability of CRISPR / Cas12a further improve the sensing performance, thereby making the detection method of the present invention have higher sensitivity and selectivity.

[0056] 3. The present invention uses the screened G triplex (G31) as a signal source to replace the fluorescently labeled probe traditionally used in the CRISPR / Cas12a system to construct a label-free fluorescent sensor for detecting DNA methylation, thereby making the detection method of the present invention have the characteristics of high specificity and low cost.

[0057] 4. The label-free fluorescent detection method for DNA methylation provided by this invention is versatile and can detect different target substances by varying the amplified template sequence. The detection method provided by this invention can be further used to establish a universal platform for detecting multiple methylation sites and even nucleic acids, showing great application prospects.

[0058] 5. Experiments have shown that the label-free fluorescence detection method for DNA methylation of the present invention has a low detection limit and a wide detection range for the methylated DNA target gene (Septin 9-mC) in colorectal cancer, with a detection limit as low as 165.13 fM and a linear detection range of 0.2 pM to 200 nM. The method also has excellent accuracy and selectivity, capable of distinguishing methylation levels as low as 0.05%, and is unaffected by interference from Septin 9-C and other random DNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is a schematic diagram of the DNA methylation detection strategy based on GLaI enzyme-assisted double cascade strand displacement amplification coupled with CRISPR / Cas12a system, wherein: Figure 1 -A is a schematic diagram of the Septin 9-mC methylation detection process; Figure 1 -B is a schematic diagram of the Septin9-C methylation detection process;

[0060] Figure 2 -A is the fluorescence response diagram of different samples; Figure 2 -B is the LFA detection image of different targets Blank, Septin 9-C, and Septin 9-mC; Figure 2 -C is the fluorescence response diagram of ThT under different guanine-rich sequences;

[0061] Figure 3 PAGE images of double-cascade strand displacement amplification reaction, lane 1: marker; lane 2: Septin 9-C / GLaI; lane 3: Septin 9-mC / GLaI; lane 4: template 1; lane 5: template 2; lane 6: GLaI / template 1 / template 2 / Klenow Fragment enzyme / Nb.BbvCI enzyme / dNTP; lane 7: Septin 9-C / GLaI / template 1 / template 2 / Klenow Fragment enzyme / Nb.BbvCI enzyme / dNTP; lane 8: Septin 9-mC / GLaI / template 1 / template 2 / Klenow Fragment enzyme / Nb.BbvCI enzyme / dNTP;

[0062] Figure 4 This is a graph showing the results of optimizing the GLaI endonuclease reaction conditions and the double-cascade strand displacement amplification reaction conditions. Figure 4 -A is a graph showing the fluorescence response of the GLaI enzyme at different concentrations; Figure 4 -B is a graph showing the fluorescence response of templates (template 1 / template 2) at different concentrations for double-cascade strand displacement amplification; Figure 4 -C is a graph showing the fluorescence response of Klenow Fragment enzyme at different concentrations; Figure 4 -D is a graph showing the fluorescence response of the Nb.BbvCI enzyme at different concentrations;

[0063] Figure 5 This is a result diagram for optimizing the double-cascade strand displacement amplification reaction conditions and CRISPR / Cas12a reaction conditions, where: Figure 5 -A is a graph showing the fluorescence response of dNTP at different concentrations; Figure 5 -B is a graph of fluorescence response at different double-cascade strand displacement amplification reaction times; Figure 5 -C is a graph showing the fluorescence response of cas12a / crRNA at different concentrations; Figure 5 -D is a graph showing the fluorescence response of G31 at different concentrations;

[0064] Figure 6 -A is a graph showing the fluorescence response of CRISPR / cas12a at different reaction times; Figure 6 -B is a graph of the fluorescence response of ThT at different concentrations; Figure 6 -C is the fluorescence response graph of Septin 9-mC at different concentrations from 0.2 pM to 200 nM; Figure 6 -D is the linear relationship between fluorescence intensity (ΔF) and the logarithm of Septin 9-mC concentration in the range of 0.2 pM to 200 nM; Figure 6 -E is the LFA detection image of Septin 9-mC at different concentrations from 0.2 pM to 200 nM;

[0065] Figure 7 is a linear fitting curve plot between the input methylation level and the measured methylation level;

[0066] Figure 8 Fluorescence response diagrams of different targets: Septin 9-C, Septin 9-mC, and random DNA;

[0067] Figure 9 LFA detection images of different targets: Septin 9-C, Septin 9-mC, and random DNA. DETAILED DESCRIPTION

[0068] The technical solutions of the present invention will be further described in detail below with reference to specific embodiments. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Therefore, all other embodiments derived by those skilled in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0069] The present invention mainly develops a label-free fluorescence detection method for DNA methylation based on GLaI enzyme-assisted double cascade strand displacement amplification coupled with CRISPR / Cas12a system. The method is mainly based on nuclease-assisted constant temperature signal amplification and G-triplex label-free fluorescence biosensor to achieve DNA methylation detection. The detection principle is as follows Figure 1As shown: The GLaI enzyme can distinguish between methylated and unmethylated DNA double strands. Only when the recognition site is methylated will the target DNA be cut, triggering double-cascade strand displacement amplification. The high amplification capacity of double-cascade strand displacement amplification can produce a large amount of thymine-rich ssDNA, which can then be recognized by Cas12a / crRNA and activate the trans-cleavage function of Cas12a / crRNA. Thioflavin T specifically binds to the G-triplex to form a G-triplex / thioflavin T complex, which efficiently enhances the Thioflavin T fluorescence reaction. Under the action of Cas12a / crRNA, the structure of the G-triplex is destroyed, and DNA methylation is detected by changes in fluorescence values.

[0070] The present invention provides a label-free fluorescence detection method for DNA methylation based on GLaI enzyme-assisted double-cascade strand displacement amplification coupled with CRISPR / Cas12a system, which specifically comprises the following steps:

[0071] (1) GLaI endonuclease reaction: 1.6 μL of target DNA, 0.32 μL of SE restriction endonuclease buffer, 0.04-5 U / μL of GLaI, and enzyme-free water were mixed to a total volume of 3.2 μL. The mixture was reacted at 30°C for 60 minutes, and then heated to 70°C for 15 minutes to terminate the digestion and obtain the digestion product.

[0072] (2) Double cascade strand displacement amplification reaction: add 0.64 μL NEB restriction endonuclease buffer, 0.64 μL NEB buffer 2, 1-40 nM template 1, 1-40 nM template 2, 75-200 U / mL Klenow Fragment polymerase, 75-300 nM Nb.BbvCI nuclease, 0.125-1.5 mM dNTP and enzyme-free water to the digestion product obtained in step (1) to a total volume of 6.4 μL, and react at 37°C for 10-150 minutes to obtain a reaction product;

[0073] (3) CRISPR / Cas12a reaction: 2 μL of the reaction product from step (2), 2 μL of NEB buffer, 40-140 nM cas12a, 40-140 nM crRNA, and 100-600 nM guanine-rich sequence were added to a total volume of 20 μL and reacted at 37°C for 10-120 minutes to obtain a reaction product; for lateral flow detection, the guanine-rich sequence was replaced by 500 nM lateral flow probe Bio-DNA-FAM. The other reaction steps were the same as above. The test strip was then inserted into the reaction solution for 2 minutes, and the results were finally photographed with a camera.

[0074] (4) Fluorescence reaction: 1-6 μM Thioflavin T and Tris-HCl buffer were mixed to a total volume of 80 μL, and added to the reaction product of step (3), and reacted at 25° C. for 1 minute.

[0075] In specific implementation, in step (4), the fluorescence intensity is measured using a multifunctional microplate reader (BioTek, SYNERGYHTX) under 442 nm excitation and 487 nm emission to obtain the detection result of the target DNA.

[0076] Unless otherwise specified, the materials, reagents or instruments involved in the present invention can be obtained through conventional commercial channels. For example, in the embodiments of the present invention, S-adenosylmethionine, buffer, CpG methyltransferase (M.SssI), Klenow Fragment enzyme, Nb.BbvCI enzyme and Cas12a enzyme were all purchased from New England BioLabs in Beijing, China. GLaI enzyme was purchased from Western Enzyme Technology (Beijing) Co., Ltd. HybriDetect test paper (MGHD 1) was provided by Milenia Biotec GmbH. Enzyme-free water (DEPC water) was purchased from Shanghai McLean Biochemical Technology Co., Ltd. Thioflavin T was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. All nucleic acid sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd. PAGE-related reagents (Acryl / Bis 30% Solution (29:1), nucleic acid dyes, and TBE buffer were all from Sangon Biotech (Shanghai) Co., Ltd. dNTP, loading buffer and DNA molecular weight standard Marker were all purchased from Baori Medical Biotechnology (Beijing) Co., Ltd.

[0077] Example 1

[0078] The present invention takes colorectal cancer as an example. The specific site (site 773737474) of 5'-GmCGmC-3' / 3'-mCGmCG-5' in Septin 9 gene is considered to be a recognized site for methylation detection of colon cancer, and is detected by the following method.

[0079] 1. Sequence Design and Optimization

[0080] All nucleic acid sequences were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0081] Table 1. DNA sequence information table involved in this embodiment

[0082]

[0083]

[0084] Note: Septin 9-C, Random 1, Random 2, Random 3, and Random 4 are all double-stranded DNA.

[0085] Template 1 and template 2 were modified with 3'-NH2, where "-NH2" refers to amino group modification; the specific sequence of template 1 is: AAAAAAAAAAAAAAAAAAAAAAAACCTCAGCAACCCCTCAGCGCGGAGGGCAGCG GCGAG-NH2; the specific sequence of template 2 is as follows: AAAAAAAAAAAAAAAAAAAAAAAACCTCAG CAACCCCTCAGCGCGACCCGCTGCCCACCAGCCA-NH2.

[0086] The 22nd and 24th cytosine nucleotides of Septin 9-mC are mC, and "mC" indicates C5 methylation of cytosine; its specific sequence is: TGGCTGGTGGGCAGCGGGTCGmCGmCGGAGGGCAGCGGCGAG.

[0087] 2. Preparation of Methylated DNA Target Genes

[0088] 2 μL of S-adenosylmethionine (32 mM), 5 μL of NEB biological buffer 2, 2 μL of M.SSSI methyltransferase (4 U / μL), 100 nM DNA target gene (Septin 9-C) and water were mixed into a 50 μL reaction solution, and then incubated at 37°C for 5 hours; 2 μL of M.SSSI methyltransferase (4 U / μL) and 1 μL of S-adenosylmethionine (32 mM) were added, and the incubation continued at 37°C for 12 hours, and then the reaction was terminated after reacting at 65°C for 20 minutes to obtain the methylated DNA target gene (Septin9-mC).

[0089] 3. GLaI endonuclease cleaves target DNA

[0090] Digest target DNA (Septin 9-C and Septin 9-mC) with GlaI in a total reaction volume of 3.2 μL. Mix 1.6 μL of target DNA, 0.32 μL of SE restriction enzyme buffer, 0.04-5 U / μL of GlaI, and enzyme-free water. Incubate at 30°C for 60 minutes, then heat to 70°C and incubate for 15 minutes to terminate the digestion. This yields the GlaI digestion product.

[0091] 4. Double-cascade strand displacement amplification reaction and ssDNA generation

[0092] Mix 3.2 μL of GLaI digestion product, 0.64 μL of NEB restriction endonuclease buffer, 0.64 μL of NEB buffer 2, 1-40 nM template 1, 1-40 nM template 2, 75-200 U / mL Klenow Fragment polymerase, 75-300 nM Nb.BbvCI endonuclease, 0.125-1.5 mM dNTP, and enzyme-free water to a total volume of 6.4 μL. React at 37° C. for 10-150 minutes to obtain a double cascade strand displacement amplification reaction product.

[0093] 5. CRISPR / Cas12a reaction

[0094] Then, 2 μL of the double-cascade strand displacement amplification reaction product, 2 μL of NEB buffer, 40–140 nM Cas12a, 40–140 nM crRNA, and 100–600 nM of a guanine-rich sequence were mixed in a total volume of 20 μL. The reaction was incubated at 37°C for 10–120 minutes to generate the CRISPR / Cas12a reaction product. For lateral flow detection, the guanine-rich sequence was replaced with 500 nM of the lateral flow probe Bio-DNA-FAM. The remaining reaction steps were identical to those described above. The test strip was then immersed in the reaction solution for 2 minutes, and the results were captured using a camera.

[0095] 6. Fluorescence reaction

[0096] Then, 1-6 μM Thioflavin T and Tris-HCl buffer were mixed to a total volume of 80 μL and added to the CRISPR / Cas12a reaction product. The reaction was allowed to react at 25°C for 1 minute. The fluorescence intensity was measured using a multifunctional microplate reader (BioTek, SYNERGYHTX) at 442 nm excitation and 487 nm emission to obtain the detection results of the target DNA.

[0097] 7. Electrophoresis Analysis

[0098] The reaction principle of this method was verified using a non-denaturing 10% polyacrylamide gel. A 40bp double-stranded DNA target gene was used as the target for electrophoresis analysis. The reacted sample was mixed with 6X loading buffer at room temperature for 2 minutes and then transferred to the gel well. Electrophoresis was performed in 1× TBE buffer at a voltage of 120V for 40 minutes. Finally, the electrophoresis gel was reacted in a nucleic acid dye for 30 minutes and analyzed with Gel Doc. TM Images were acquired using EZ (Bio-Rad).

[0099] Example 2. Optimization of detection conditions

[0100] 1. Label-free fluorescence detection of DNA methylation

[0101] like Figure 2 As shown in Figure 1-A, when only the methylated target (Septin 9-mC) is present, the fluorescence signal changes significantly after GLaI enzyme treatment, double cascade strand displacement amplification reaction, and CRISPR / Cas reaction. However, when Blank and non-methylated target (Septin9-C) are present, the fluorescence signal does not change significantly. The lateral flow results are shown in Figure 1-A. Figure 2 As shown in Figure 2B, double bands of Control and Test are present only when the methylated target (Septin9-mC) is present. These results demonstrate that the label-free fluorescence detection method for DNA methylation based on GLaI enzyme-assisted double cascade strand displacement amplification coupled with the CRISPR / Cas12a system is feasible.

[0102] 2. Guanine-rich sequence optimization

[0103] Different guanine-rich sequences have different abilities and stabilities to enhance the fluorescence of Thioflavin T. Optimizing the guanine-rich sequences is aimed at improving the performance of the detection method and screening out guanine-rich sequences suitable for CRISPR / Cas12a trans-cleavage. Figure 2 As shown in Figure 3-C, the fluorescence response is sequence-dependent, with G31 having the highest fluorescence response and being fully hydrolyzed by CRISPR / Cas12a. Therefore, G31 was selected as the guanine-rich sequence for subsequent experiments, and its complementary sequence was the corresponding cytosine-rich sequence in the double-cascade strand displacement amplification reaction template.

[0104] 3. Optimization of GLaI endonuclease reaction conditions

[0105] The concentration of GLaI enzyme was optimized to fully degrade methylated Septin 9-mC and detect it quickly. First, the concentration of GLaI enzyme was optimized, such as Figure 4 As shown in Figure 2A, the fluorescence response was concentration-dependent and reached a peak at 0.1 U / μL. Therefore, 0.1 U / μL was selected as the optimal condition. At the same time, the cleavage effect of the GLaI enzyme was verified by 10% polyacrylamide gel electrophoresis. Figure 3 As shown, lanes 2 and 3 are Septin 9-C and Septin 9-mC, respectively, and only lane 3 has a band at 30-40 bp. This indicates that methylated Septin 9-mC is effectively digested, while unmethylated Septin 9-C is not digested.

[0106] 4. Optimization of Double-Cascade Strand Displacement Amplification Reaction Conditions

[0107] First, the template concentration of the double-cascade strand displacement amplification was optimized, such as Figure 4As shown in Figure 2-B, the fluorescence response is template concentration dependent and reaches saturation at 10 nM. Therefore, 10 nM was selected as the concentration of template 1 / template 2. Next, the concentration of KlenowFragment polymerase was optimized, as shown in Figure 2-B. Figure 4 As shown in Figure 3-C, as the enzyme concentration increases, the fluorescence response increases and reaches saturation at 125 U / mL. Therefore, 125 U / mL was selected as the concentration of Klenow Fragment polymerase. Then, the concentration of Nb.BbvCI enzyme was optimized, as shown in Figure 3-C. Figure 4 As shown in Figure 2-D, the fluorescence response showed enzyme concentration dependence, reaching a peak at 125 U / mL and then decreasing. Therefore, 125 U / mL was selected as the optimal condition. Finally, the dNTP concentration and reaction time were optimized. Figure 5 As shown in -A, 0.25mM dNTP has the best fluorescence response. Figure 5 As shown in -B, the fluorescence response increases with time and reaches a plateau at 60 min, which is selected as the optimal condition. The amplification effect of the double cascade strand displacement amplification reaction was verified by 10% polypropylene gel electrophoresis, as shown in Figure 3 As shown, channels 6, 7, and 8 are the amplification products of the blank control, Septin 9-C, and Septin 9-mC, respectively, and amplification products are only present in channel 8. This indicates that only methylated Septin 9-mC can trigger the double-cascade strand displacement amplification reaction to produce ssDNA.

[0108] 5. CRISPR / Cas12a reaction condition optimization

[0109] First, the concentrations of cas12a enzyme and crRNA were optimized. Figure 5 As shown in Figure 2-C, the fluorescence response is concentration-dependent and reaches saturation at 100 nM. Therefore, 100 nM cas12a / crRNA was selected as the optimal condition. Next, the concentration of G31 was optimized to obtain better detection performance. Figure 5 As shown in Figure 3-D, the fluorescence response increases with increasing concentration, reaches a peak at 500nM, and finally decreases. Therefore, 500nM is selected as the optimal condition. Finally, the CRISPR / Cas12a reaction time is optimized for rapid detection. Figure 6 As shown in Figure 2A, the fluorescence response increases with time, and combined with ΔF and ΔF / F0, it reaches a peak at 60 minutes and then decreases. Therefore, 60 minutes is selected as the optimal condition.

[0110] 6. Fluorescence Reaction Optimization

[0111] The concentration of Thioflavin T (ThT) was optimized to reduce the background signal. Figure 6As shown in Figure 2-B, the signal-to-background ratio reached its peak at a concentration of 5 μM Thioflavin T. Therefore, 5 μM was selected as the optimal condition.

[0112] 7. Fluorescent Biosensor Sensitivity Analysis

[0113] In order to study the performance of this method, the concentration range of Septin 9-mC detection was measured under the optimal conditions. Figure 6 As shown in Figure 2-C, the fluorescence response gradually increases with the increase of Septin 9-mC concentration from 0.2pM to 200nM. There is a good linear relationship between the fluorescence intensity (F) and the logarithm of Septin 9-mC concentration from 0.2pM to 200nM. Figure 6 -D. Fluorescence intensity (ΔF) is the fluorescence intensity of the experimental group minus the fluorescence value of the blank group. The linear fitting equation is ΔF = 3890.40LogC + 15712.00 (R 2 =0.99), C is the concentration of Septin 9-mC (nM). The limit of detection (LOD) was calculated to be 165.13 fM. The lateral flow results are shown in Figure 2. Figure 6 As shown in Figure 1-E, from 0.2 pM to 200 nM, as the concentration of Septin 9-mC increases, the control band gradually becomes lighter and the test band gradually becomes darker. Compared with the existing DNA methylation detection methods, the method of the present invention has a lower detection limit and a wider detection range.

[0114] 8. Analysis of Fluorescent Biosensor Accuracy

[0115] Epigenomic studies have shown that 5-methylcytosine accounts for 2-7% of cytosine in the mammalian genome. Therefore, in the presence of a large number of unmethylated samples, the detection method must be able to accurately detect methylated samples. The total concentration of the unmethylated target (Septin 9-C) and methylated target (Septin 9-mC) combination is 20nM, and different concentrations of Septin 9-mC are added to the combination. Figure 7 As shown in Figure 2, as the methylation level increases, the fluorescence signal gradually increases from 0.05% to 100%, and there is a good linear relationship between the input methylation level and the measured methylation level. The linear fitting equation is y = 0.91x + 0.017 (R 2 =0.999), y is the measured methylation level, and x is the input methylation level. A methylation level of 0.05% (10 pM) can be detected, indicating that the detection strategy adopted by the present invention has high accuracy.

[0116] 9. Fluorescent Biosensor Selectivity Analysis

[0117] Selectivity is the key to the detection method. Therefore, first, random DNA such as Septin 9-C was used as an interfering substance to study the selectivity of the method of the present invention for site-specific methylation detection. Figure 8 It can be seen that only when Septin 9-mC (20nM) is present, the fluorescence reaction is strong. However, when Septin 9-mC (20nM) is present and the concentration is five times that of random DNA (100nM), the fluorescence reaction is weak. Figure 9 As shown, only the control and test bands appeared when Septin 9-mC (20 nM) was present. However, when the same concentration of Septin 9-mC (20 nM) was used with five times the concentration of random DNA (100 nM), only the control band appeared. The detection method of the present invention has good selectivity.

[0118] In summary, the present invention provides a method for detecting DNA methylation by a label-free fluorescent biosensor based on nuclease-assisted constant temperature signal amplification and G-triplex. The present invention adopts the screened G-triplex (G31) as a signal source to replace the traditional fluorescent labeled probe in the CRISPR / cas system, so that the detection method of the present invention has the characteristics of high specificity and low cost. Compared with traditional methylation-specific PCR, the double cascade chain displacement amplification based on GLaI and has mild reaction conditions and fast detection speed. The high amplification capacity of the double cascade chain displacement amplification can produce a large amount of ssDNA, which can then be recognized by Cas12a / crRNA and activate the trans-cleavage function of Cas12a / crRNA, and destroy the structure of the G-triplex / thioflavin T complex, so that the fluorescence signal changes significantly, thereby improving the specificity and sensitivity of the detection method. The linear range of its detection is 0.2pM~200nM, the fluorescence detection limit is 165.13fM, and methylation levels as low as 0.05% can be distinguished. The programmability of the double-cascade strand displacement amplification template enables the label-free fluorescence detection method of DNA methylation based on GLaI enzyme-assisted double-cascade strand displacement amplification coupled with the CRISPR / Cas12a system to be further used to establish a universal platform for multiple methylation sites and even nucleic acid detection, which has great application prospects.

Claims

1. A reagent composition for detecting DNA methylation, characterized in that: The reagent composition includes a GLaI endonuclease cleavage reaction reagent, a double-cascade strand displacement amplification reaction reagent, a CRISPR / Cas12a reaction reagent and a fluorescence reaction reagent; The GLaI endonuclease cleavage reaction reagent includes GLaI endonuclease; The CRISPR / Cas12a reaction reagent includes a guanine-rich sequence, and the guanine-rich sequence includes any one or more of Oxy 3.5, Oxy3, Cat G4, G31, 22AG, 16AG, Telo 24, Telo 18, C-MYC22, and C-MYC15; the nucleotide sequences of Oxy 3.5, Oxy 3, Cat G4, G31, 22AG, 16AG, Telo 24, Telo 18, Cmyc-22, and C-MYC15 are shown in SEQ ID NO: 1 to SEQ ID NO: 10, respectively; The fluorescent reaction reagent includes Thioflavin T.

2. The reagent composition according to claim 1, characterized in that The GLaI endonuclease cleavage reaction reagent includes GLaI endonuclease, SE restriction endonuclease buffer and enzyme-free water; and / or the double-cascade strand displacement amplification reaction reagents include NEB restriction endonuclease buffer, NEB buffer 2, template 1, template 2, Klenow Fragment polymerase, Nb.BbvCI endonuclease, dNTPs, and enzyme-free water, the nucleotide sequence of template 1 is shown in SEQ ID NO: 12, and the nucleotide sequence of template 2 is shown in SEQ ID NO: 13; And / or the CRISPR / Cas12a reaction reagent includes Cas12a nuclease, crRNA, the guanine-rich sequence, NEB buffer and enzyme-free water, and the nucleotide sequence of the crRNA is shown in SEQ ID NO: 14; And / or the fluorescent reaction reagent includes Thioflavin T, Tris-HCl buffer and enzyme-free water.

3. The reagent composition according to claim 1 or 2, characterized in that The guanine-rich sequence is G31.

4. A label-free fluorescent biosensor for detecting DNA methylation, characterized in that A reagent composition comprising the reagent composition according to any one of claims 1 to 3.

5. A label-free fluorescence detection method for DNA methylation, characterized in that: Detection is performed using the reagent composition according to any one of claims 1 to 3 and / or the label-free fluorescent biosensor according to claim 4, wherein the method is not intended for disease treatment or diagnosis; the method comprises the following steps: S1: cleavage of target DNA using GLaI endonuclease, and obtaining digestion products through GLaI endonuclease reaction; S2: The digestion product obtained in S1 is subjected to a double-cascade strand displacement amplification reaction to obtain a double-cascade strand displacement amplification reaction product; S3: The double-cascade strand displacement amplification reaction product obtained in S2 is subjected to a CRISPR / Cas12a reaction to obtain a CRISPR / Cas12a reaction product; in the CRISPR / Cas12a reaction, a G triplex is used as a signal source; S4: Add a fluorescent reaction reagent to the CRISPR / Cas12a reaction product obtained in S3, measure its fluorescence intensity, and obtain the methylation detection result of the target DNA.

6. The method according to claim 5, characterized in that The GLaI endonuclease reaction system includes 1.6 μL of target DNA, 0.04-0.5 U / μL of GLaI endonuclease, 0.32 μL of SE restriction endonuclease buffer, and enzyme-free water to 3.2 μL; And / or the double-cascade strand displacement amplification reaction system includes 0.64 μL of NEB restriction endonuclease buffer, 20.64 μL of NEB buffer, 11-40 nM of template, 21-40 nM of template, 3.2 μL of digestion product, 75-200 U / mL of Klenow Fragment polymerase, 75-300 U / mL of Nb.BbvCI endonuclease, 0.125-1.5 mM of dNTP, and enzyme-free water to 6.4 μL; And / or the CRISPR / Cas12a reaction system includes 2 μL of NEB buffer, 2 μL of double-cascade strand displacement amplification product, 40-140 nM of Cas12a, 40-140 nM of crRNA, 100-600 nM of guanine-rich sequence, and enzyme-free water to 20 μL; And / or the fluorescence reaction system includes 20 μL of CRISPR / Cas12a reaction product, 1-6 μM Thioflavin T, and Tris-HCl buffer supplemented to 100 μL.

7. The method according to claim 5, characterized in that The GLaI endonuclease cleavage reaction conditions are: react at 25-40°C for 30-120 minutes, then heat to 60-80°C for 5-15 minutes; And / or the double cascade strand displacement amplification reaction conditions are: 30-45° C. for 10-150 min; And / or the CRISPR / Cas12a reaction conditions are: 30-45° C. for 10-120 min; And / or the fluorescence reaction conditions are: 20-30° C. reaction time 1-3 min.

8. The method according to claim 5, characterized in that The DNA to be tested is genomic DNA from cells or tissues suspected of being tumors; the tumors include colorectal cancer, breast cancer and / or gastric cancer.

9. The method according to claim 8, characterized in that The tumor is colorectal cancer, its DNA target gene is Septin 9-C, and its methylated DNA target gene is Septin 9-mC; the nucleotide sequence of Septin 9-C is shown in SEQ ID NO: 11, and the nucleotide sequence of Septin 9-mC is shown in SEQ ID NO:

19.

10. Use of the reagent composition according to any one of claims 1 to 3 and / or the label-free fluorescent biosensor according to claim 4 in preparing a cancer detection kit, characterized in that: By detecting whether methylation occurs in genomic DNA, it is determined whether cancer occurs; the cancer includes colorectal cancer, breast cancer and / or gastric cancer.

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