A method for detecting 5-hydroxymethylcytosine in the whole genome and its application

Abstract

The present invention discloses a method for detecting 5-hydroxymethylcytosine in the whole genome and its application, which relates to the technical field of biochemical detection. The method comprises the following steps: 1: Blocking the 3'-OH end of the DNA to be detected; 2: Preparation of glycosylated 5hmC DNA; 3: TdT-mediated DNA amplification; 4: Fluorescence measurement. The present invention constructs a homogeneous, template-independent signal amplification method for detecting 5-hydroxymethylcytosine in the whole genome. This method is based on specific enzyme-catalyzed reactions and multiple signal amplification strategies, and has the characteristics of high sensitivity, good specificity, no need for separation steps, sequence-independent amplification, and the ability to detect 5hmC at any position in the whole genome. Moreover, the present invention can achieve accurate and sensitive detection of actual clinical samples.

Description

Technical Field

[0001] The present invention relates to the field of biochemical detection technology, and more particularly to a method for detecting genome-wide 5-hydroxymethylcytosine and its application. Background Art

[0002] Epigenetic modification refers to the process of regulating gene expression through certain mechanisms without changing the DNA sequence. It is also one of the important factors affecting the occurrence and development of many diseases. Common types of epigenetic modification include DNA methylation, histone modification, non-coding RNA modification, RNA modification, and chromatin remodeling. Among them, DNA methylation is one of the most intensively studied epigenetic regulatory mechanisms. Imbalanced DNA methylation is associated with the occurrence of many diseases, such as breast cancer, lung cancer, colorectal cancer, and other cancers.

[0003] DNA methylation was once considered a stable epigenetic mark. However, research over the past decade has revealed that this modification is not static but rather dynamically changing. A proper balance between DNA methylation and demethylation maintains DNA methylation levels in healthy cells, but this stable state is severely disrupted in cancer cells. Methylated cytosine is oxidized to a series of intermediate states, including 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC), catalyzed by 10-11 translocation dioxygenase (TET). This is a dynamic demethylation process. 5-Acylcytidine and 5-carboxycytidine are further recognized by thymidine DNA glycosylase (TDG), which then undergoes base excision repair (BER) in collaboration with multiple other enzymes, converting them to unmodified cytosine, completing the demethylation process.

[0004] In 2009, 5hmC was discovered as another relatively abundant cytosine modification in embryonic stem cells (ESCs) and Purkinje neurons. Therefore, 5hmC is not only an intermediate in the demethylation process but is also widely present in mammalian tissues and cells. It is an epigenetic modification that plays an important role in individual development and has been dubbed the "sixth base." Genome-wide analyses of 5hmC further suggest its potential regulatory role in ESC regulation, myelogenesis, zygote development, and neural development. 5hmC levels are tissue-specific, with studies demonstrating that the highest levels are found in brain tissue. 5hmC is widely distributed in mammalian tissues and cells, but its levels are extremely low compared to 5mC (5-methylcytosine). 5hmC levels vary significantly between tissues, with the highest levels in the cerebral cortex, suggesting that 5hmC has the potential to serve as a better tissue-specific biomarker for tissue classification. There is ample evidence that decreased global 5hmC levels are a hallmark of malignant tumors. The researchers found that the overall levels of 5hmC were decreased in many tumor samples, including lung cancer, breast cancer, liver cancer, colorectal cancer, gastric cancer, pancreatic cancer, prostate cancer, skin cancer, glioma and hematopoietic system tumors, indicating that 5hmC can serve as an informative biomarker for many human cancers and other complex diseases.

[0005] Traditional bisulfite sequencing is the "gold standard" for detecting methylation at single-base resolution, but it cannot distinguish between 5mC and 5hmC. Bisulfite treatment severely degrades DNA, disrupting its integrity and compromising data readout. Early methods for studying 5hmC included thin-layer chromatography (TLC), liquid chromatography-mass spectrometry (LC-MS), high-performance liquid chromatography (HPLC), and immunoassays. However, these methods suffer from drawbacks such as difficulty in accurate quantification, low sensitivity, cumbersome procedures, the use of radiolabeled nucleotides, limited detection of sequence-specific 5-hydroxymethylcytosine, and difficulty accurately distinguishing 5hmC from 5mC. Recently, researchers have developed a number of novel methods for detecting 5-hydroxymethylcytosine. Chen et al. designed a method for spectroscopic quantification of 5-hydroxymethylcytosine in genomic DNA using a boronate-functionalized nanosphere fluorescent probe. However, this method lacks a signal amplification step and exhibits low sensitivity. Wang et al. constructed a glycosylation-mediated label-free fluorescent biosensor for zero-background detection of site-specific 5-hydroxymethylcytosine in cancer cells, but the scheme involved magnetic separation, which was cumbersome and could only detect 5-hydroxymethylcytosine at specific sites.

[0006] Therefore, developing a highly sensitive and genome-wide 5hmC quantification method is an urgent problem that needs to be solved by those skilled in the art in clinical research and common laboratory experiments. Summary of the Invention

[0007] In view of this, the present invention provides a genome-wide 5-hydroxymethylcytosine detection method and application.

[0008] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions.

[0009] A method for detecting 5-hydroxymethylcytosine in the whole genome comprises the following steps:

[0010] 1) Block the 3'-OH end of the DNA to be tested:

[0011] Terminal transferase (TdT) and adenine triphosphate dideoxynucleotide (ddATP) are added to the DNA to be tested. Under the action of terminal transferase (TdT), adenine triphosphate dideoxynucleotide (ddATP) is modified to the 3'-OH end of the double-stranded DNA to be tested;

[0012] This step can block the original 3'-OH end in the DNA, making it impossible to amplify, ensuring the specificity of the subsequent amplification steps and avoiding the occurrence of false positives;

[0013] Shrimp alkaline phosphatase (rSAP) was added to hydrolyze excess ddATP to obtain the blocked DNA to be tested;

[0014] rSAP can prevent other probes and newly generated 3'-OH termini from being blocked by unincorporated ddATP.

[0015] 2) Preparation of glycosylated 5hmC DNA:

[0016] T4 β-glucosyltransferase (T4 β-GT) and uridine diphosphate glucose (UDP-Glc) are added to the blocked DNA to be tested. Under the catalysis of T4 β-GT, the glucose group in uridine diphosphate glucose (UDP-Glc) is transferred to the hydroxymethyl group of 5hmC in the DNA to be tested for glycosylation. 5hmC is covalently modified with the glucose group to form 5ghmC, obtaining glycosylated 5hmC DNA.

[0017] Neither C (cytosine) nor 5mC can undergo glycosylation. Therefore, only 5hmC in genomic DNA can be covalently modified with glucose to form 5ghmC.

[0018] 3) TdT-mediated DNA amplification:

[0019] DNA modification-dependent endonuclease (AbaSI) is added to the glycosylated 5hmC DNA. AbaSI recognizes and selectively cuts the glycosylated 5hmC DNA double strands containing 5ghmC, generating double-stranded DNA breaks at 11-13 nt and 9-10 nt away from 5ghmC, respectively, forming 3'-OH protruding sticky ends with 2-3 bases.

[0020] Only when 5hmC is glycosylated can the target 5hmC be cleaved by AbaSI. However, C and 5mC cannot be glycosylated, so they cannot be cleaved by AbaSI to expose new 3'-hydroxyl ends.

[0021] TdT enzyme and deoxyadenosine triphosphate (dATP) are added. Under the catalysis of TdT enzyme, dATP polymerizes and extends at the 3'-OH protruding sticky end of glycosylated 5hmC DNA to generate poly-A chains, obtaining the DNA to be detected by fluorescence.

[0022] 4) Fluorescence measurement:

[0023] A signal probe containing an AP site and an APE1 enzyme are added to the DNA to be fluorescently detected. The signal probe and the poly-A chain in the DNA to be fluorescently detected are complementary and paired to form a double strand. The AP site in the signal probe is hydrolyzed by APE1, the fluorescent group is released, and fluorescence is restored.

[0024] The cut fluorescent probe falls off the double strand, allowing the poly-A chain to cyclically bind to the free signal probe. APE1 cyclically cuts and releases the fluorescent group, which significantly enhances the signal.

[0025] Furthermore, step 1) is specifically as follows:

[0026] The 3'-OH end of double-stranded DNA was modified with ddATP to prevent subsequent nonspecific amplification. 1 μM of the DNA sample to be tested was added with 8 U of terminal transferase, 200 μM ddATP, 1 × TdT reaction buffer, and 1 × CoCl2. The reaction was incubated at 37°C for 4 hours and terminated at 75°C for 20 minutes.

[0027] To remove incompletely reacted ddATP, 0.3 U of rSAP and 1 × rCutsmart reaction buffer were added to the product. The reaction was carried out at 37°C for 30 minutes and terminated at 65°C for 5 minutes.

[0028] Furthermore, step 2) is specifically as follows:

[0029] To construct glycosylated DNA (5ghmC-DNA), the 3'-OH-blocked double-stranded DNA (dsDNA) product was incubated in a solution containing 4U T4 β-GT, 40 μM UDP-glucose, and 1 × NEBuffer 4 (20 μL) at 37°C for 2 h and terminated at 65°C for 20 min.

[0030] Furthermore, step 3) is specifically as follows:

[0031] Glycosylated products were reacted in a mixture containing 4 U of AbaSI, 1 × rCutsmart reaction buffer, and 1 × DTT at 37°C for 2 hours. The reaction was terminated at 65°C for 20 minutes to generate new 3'-OH termini. During amplification, 8 U of TdT, 1 mM dATP, and 1 × TdT reaction buffer were added. The mixture was reacted at 37°C for 1 hour and inactivated at 75°C for 20 minutes.

[0032] Furthermore, step 4) is specifically as follows:

[0033] A mixture containing 125 nM signaling probe, 3 U APE1 enzyme, and 1 × NEBuffer 4 was added to the amplified product. The reaction was incubated at 37°C for 1 hour and terminated at 65°C for 20 minutes. ROX fluorescence was monitored using a Hitachi F-4600 fluorescence spectrometer with an excitation / emission slit of 5.0 nm and an excitation wavelength of 577 nm. The emission wavelength ranged from 590 to 700 nm, and data were analyzed using fluorescence intensity at 605 nm.

[0034] Furthermore, in step 4), the sequence of the signal probe is: TTTTTTTT ROX TTTTTXTTTTT BHQ-2 T, as shown in SEQ ID NO.7.

[0035] This signaling probe is a 19-nt poly-T sequence with an apurinic / apyrimidinic site (AP site) modified at the 13th nucleotide. Flanking the AP site are the fluorophore ROX (9th nucleotide) and the quencher BHQ-2 (19th nucleotide), respectively. The distance between the fluorophore and quencher is approximately 3.4 nm (<10 nm), enabling effective quenching and preventing high background signal. The signaling probe can pair with any stretch of the poly-A sequence generated by TdT amplification to form a DNA duplex. Apurinic / apyrimidinic endonuclease 1 (APE1) hydrolyzes the phosphodiester bond 5' of the apurinic / apyrimidinic site in the DNA duplex, generating single-stranded DNA breaks, separating the signaling probe into 12-nt and 6-nt fragments. The fluorophore and quencher then separate, restoring fluorescence. The signaling probe can also detach from the duplex DNA, allowing a new signaling probe to pair with the amplified poly-A sequence, initiating a new round of fluorescence recovery. This cycle achieves signal amplification.

[0036] This signal probe has two outstanding features: (1) The poly-T signal probe sequence is different from the traditional signal probe in that it has requirements for the sequence produced by amplification. A specific template is required to amplify the complementary sequence of the signal probe. This signal probe can pair with any segment of the amplified poly-A sequence to form a DNA double strand. The design is simple and ingenious, which can improve the reaction efficiency. (2) The traditional endonuclease recognition site is changed to an AP site. This design can not only match the poly-A sequence produced by TdT amplification in the previous step, but also makes the design of the enzyme cutting position more flexible. The ingenious design of the signal probe makes it possible for the entire system to not rely on any template, and there is no need to consider the impact of different test sequences on the detection.

[0037] Furthermore, the method further includes the following steps:

[0038] Extraction of DNA to be tested:

[0039] The tissue sample to be tested was taken and ground into powder in liquid nitrogen. The processed tissue sample was processed using the QIAamp DNA Mini Kit to extract the whole genomic DNA from the tissue sample. The long-chain DNA was fragmented using dsDNA fragmentase to obtain the DNA to be tested.

[0040] Furthermore, the method further includes the following steps:

[0041] Polyacrylamide gel electrophoresis analysis:

[0042] The reaction product of step 4) was loaded onto a 14% polyacrylamide gel electrophoresis (PAGE) using 1× Gel Red as a fluorescent indicator and placed in 1× TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.0). After electrophoresis at a constant voltage of 120 V for 45 minutes, the gel image was visualized using an imaging system.

[0043] Application of a genome-wide 5-hydroxymethylcytosine detection method in genome-wide 5hmC quantitative detection.

[0044] It can be seen from the above technical solution that compared with the prior art, the beneficial effects of the present invention are:

[0045] The present invention has developed a homogeneous, template-independent signal amplification method for genome-wide 5-hydroxymethylcytosine detection. Based on a specific enzyme-catalyzed reaction and a multiplexed signal amplification strategy, this method boasts high sensitivity and specificity, requires no separation steps, and exhibits sequence-independent amplification, enabling detection of 5hmC at any position in the entire genome. Furthermore, the present invention enables accurate and sensitive detection in actual clinical samples.

[0046] The amplification step in this method can produce poly-A chains. After the fluorescent probe binds to them, it is cleaved by the APE1 enzyme and falls off the poly-A chains. The free signal probe can continue to bind to the poly-A chains, and this cycle can amplify the signal. Therefore, the detection sensitivity is high and the detection limit is as low as 6.34 × 10 −17 M.

[0047] This method has strong specificity and can avoid interference from 5-methylcytosine, which has a similar structure to 5-hydroxymethylcytosine and is more abundant in the genome. It can detect 5hmC as low as 0.099% in mixed samples.

[0048] It can be applied to the detection of actual samples and can distinguish different types of tumor cells. The detection limit for U-118MG cells is as low as 2.33 × 10 −5 ng / μL. This method can distinguish different cancerous tissues in the human body, as well as between cancerous and normal tissues. Whole-genomic DNA extracted from actual samples eliminates the need for other potentially DNA-damaging processing steps. The 5hmC distribution levels in whole-genomic DNA from different cancer cells measured by this method are very similar to those obtained using commercial kits. Furthermore, this method is significantly more affordable than enzyme-linked immunosorbent assays and sequencing methods, costing only RMB 7.7 per sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0050] Figure 1 The accompanying drawings are schematic diagrams of the present invention;

[0051] Figure 2 The accompanying figure shows the feasibility of the gel electrophoresis verification scheme of the present invention; Figure 2 A in the middle is the gel electrophoresis analysis of 5hmC-modified dsDNA; Figure 2 Middle B is the gel electrophoresis analysis of cytosine (5C), 5-methylcytosine (5mC), and 5-hydroxymethylcytosine (5hmC);

[0052] Figure 3 The accompanying drawing is a fluorescence emission spectrum for verifying the feasibility of the present invention;

[0053] Figure 4 The accompanying drawings are fluorescence emission spectra of the target of different concentrations according to the present invention;

[0054] Figure 5 The accompanying drawing shows the corresponding relationship between the concentration and fluorescence intensity of the present invention; Figure 5 A in the middle is the relationship between different concentrations and fluorescence intensity; Figure 5 Middle B shows a linear relationship between fluorescence intensity and the logarithm of 5hmC-DNA concentration in the range of 0.5 fM-500 pM;

[0055] Figure 6 The accompanying drawing is a histogram of specific detection in the present invention;

[0056] Figure 7 The accompanying drawing is a radar chart for verifying the anti-interference capability of the present invention;

[0057] Figure 8 The accompanying figure shows the fluorescence intensity of nine biosensors independently prepared by the present invention when detecting targets of the same concentration;

[0058] Figure 9 The accompanying figure shows the fluorescence intensity and linear relationship generated by different cell genomic DNAs of the present invention; Figure 9 Center A is the fluorescence intensity generated by the same mass of genomic DNA from U-118 MG cells, SH-SY5Y cells, HeLa cells, HepG2 cells, and A549 cells; Figure 9 Middle B shows the linear relationship between fluorescence intensity and the logarithm of U-118 MG cell concentration in the range of 0.016 ng / μL to 4 ng / μL;

[0059] Figure 10 The accompanying figure shows the analysis of 5hmC content in genomic DNA of different cancer cells by the present invention and ELISA kit;

[0060] Figure 11 The accompanying figure is a histogram comparing the fluorescence intensities of three groups of cancer tissues and three groups of normal tissues of the present invention. DETAILED DESCRIPTION

[0061] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0062] Example 1

[0063] 1. Block the 3'-OH end of the DNA to be tested

[0064] First, the 3'-OH termini of double-stranded DNA were modified with ddATP to prevent subsequent nonspecific amplification. A 1 μM sample of double-stranded DNA was added with 8 U of terminal transferase, 200 μM ddATP, 1× TdT reaction buffer, and 1× CoCl2. The reaction was incubated at 37°C for 4 hours and terminated at 75°C for 20 minutes. To remove incompletely reacted ddATP, 0.3 U of rSAP and 1× rCutsmart reaction buffer were added to the product. The reaction was carried out at 37°C for 30 minutes and terminated at 65°C for 5 minutes.

[0065] 2. Preparation of Glycosylated 5hmC DNA Products

[0066] To construct glycosylated DNA (5ghmC-DNA), the 3'-OH-blocked double-stranded DNA (dsDNA) product was incubated in a solution containing 4U T4 β-GT, 40 μM UDP-glucose, and 1 × NEBuffer 4 (20 μL) at 37°C for 2 h and terminated at 65°C for 20 min.

[0067] 3. TdT-mediated DNA amplification

[0068] Glycosylated products were reacted in a mixture containing 4 U of AbaSI, 1 × rCutsmart reaction buffer, and 1 × DTT at 37°C for 2 hours. The reaction was terminated at 65°C for 20 minutes to generate new 3'-OH termini. During amplification, 8 U of TdT, 1 mM dATP, and 1 × TdT reaction buffer were added. The mixture was reacted at 37°C for 1 hour and inactivated at 75°C for 20 minutes.

[0069] 4. Fluorescence Measurement

[0070] A mixture containing 125 nM signaling probe, 3 U APE1 enzyme, and 1 × NEBuffer 4 was added to the amplified product. The reaction was incubated at 37°C for 1 hour and terminated at 65°C for 20 minutes. ROX fluorescence was monitored using a Hitachi F-4600 fluorescence spectrometer with an excitation / emission slit of 5.0 nm and an excitation wavelength of 577 nm. The emission wavelength ranged from 590 to 700 nm, and data were analyzed using fluorescence intensity at 605 nm.

[0071] Table 1 5hmC, 5mC, 5C and signal probe sequences

[0072]

[0073] Note: In DNA, bases with an italicized "hm" superscript denote 5-hydroxymethylcytosine (5hmC), and bases with an italicized "m" superscript denote 5-methylcytosine (5mC). In signaling probes, an italicized "X" denotes a base modified with an AP site, an italicized "ROX" denotes a base modified with a ROX fluorophore, and an italicized "BHQ-2" denotes a base modified with a BHQ-2 quencher. 5hmC sequence A and 5hmC sequence a are complementary; 5mC sequence A and 5mC sequence a are complementary; and 5C sequence A and 5C sequence a are complementary.

[0074] 5. Polyacrylamide Gel Electrophoresis Analysis

[0075] The reaction products were loaded onto 14% polyacrylamide gel electrophoresis (PAGE) using 1× Gel Red as a fluorescent indicator and placed in 1× TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.0). After electrophoresis at a constant voltage of 120 V for 45 minutes, the gel image was visualized using an imaging system.

[0076] Test Experiment

[0077] Preparation of double-stranded DNA substrate for experiment:

[0078] Double-stranded DNA (dsDNA) substrates were generated by hybridizing 10 μM target oligonucleotide with 10 μM complementary oligonucleotide in 1× annealing buffer (50 mM NaCl, 10 mM Tris-HCl, pH 8.0) at 95°C for 5 min and then gradually cooling to room temperature. The resulting dsDNA substrates were stored at 4°C until use.

[0079] The double-stranded DNA substrates used in the experiment were substrates containing different concentrations of cytosine (5C), 5-methylcytosine (5mC), and 5-hydroxymethylcytosine (5hmC).

[0080] 1. Feasibility Verification

[0081] In order to test the feasibility of the experiment, we first used 5hmC-modified dsDNA as a model and analyzed the products by polyacrylamide gel electrophoresis (PAGE). Figure 2Figure 5 (A). 5hmC-DNA is first glycosylated to generate 5ghmC-DNA. In the presence of AbaSI, two DNA fragments (i.e., AbaSI digestion products) are observed, indicating that AbaSI cleaves 5ghmC-DNA (lane 2). However, in the absence of AbaSI, only one fragment with a larger base number, representing the intact 5hmC-DNA, is observed (lane 1). In the presence of AbaSI, TdT, and dATP, a single amplified band is observed, indicating that 5ghmC cleavage by AbaSI generates a new 3'-OH group, which is then amplified by the action of terminal transferase and dATP (lane 3).

[0082] Next, the substrates of cytosine (5C), 5-methylcytosine (5mC), and 5-hydroxymethylcytosine (5hmC) were verified. Figure 2 As shown in Figure B, when dsDNA, T4 BGT, AbaSI, and TdT are present simultaneously, neither normal cytosine (lane 1) nor methylcytosine (lane 2) produces amplified bands. However, only the third lane, containing the 5hmC site, produces amplified bands. This indicates that glycosylation and DNA amplification reactions occur only in the presence of 5hmC, confirming the feasibility of the proposed method.

[0083] In order to verify the feasibility of this method for fluorescence experiments, the present invention measured the fluorescence intensity of 5hmC, 5C and blank control. Figure 3 As shown in the figure, the background signal and the signal of cytosine DNA are almost negligible, and only when 5hmC is present will a significant fluorescence signal be generated, indicating that fluorescence detection can be performed.

[0084] 2. Sensitivity Analysis

[0085] Under the optimized reaction conditions, the present invention used the constructed biosensor to measure the fluorescence intensity of a series of different concentrations of 5hmC-DNA to evaluate the detection sensitivity of the method. Figure 4 As shown in Figure 2, when there is no 5hmC, the fluorescence signal is extremely low. As the concentration of 5hmC gradually increases, the measured fluorescence intensity also increases. Figure 5 As shown, the logarithm of 5hmC-DNA concentration (C) is taken as the horizontal axis and the fluorescence intensity (F) is taken as the vertical axis. In the range of 0.5 fM-500 pM, the fluorescence intensity (F) and the logarithm of 5hmC-DNA concentration (C) show a good linear relationship, and the linear equation is F = 13.23 log 10 C + 288.46 (R 2 = 0.9955). By calculating the average signal of the blank group and adding 3 times the standard deviation, the detection limit was calculated to be 6.34 × 10 −17M. Compared with surface enhanced Raman spectroscopy (0.05 nM), nanosphere fluorescence (0.167 nM), electrochemiluminescence sensor (1.2× 10 -5 Compared with 10 nM, the sensitivity of this method was improved by 6, 7, and 3 orders of magnitude, respectively.

[0086] 3. Specificity Analysis

[0087] Since the distribution of 5hmC in the genome is random and the structure of 5mC is very similar to that of 5hmC, it often interferes with the detection of 5hmC. Therefore, in order to verify the specificity of the biosensor for 5hmC, the present invention uses unmethylated DNA (C-DNA), 5mC-DNA and 5hmC-DNA oligonucleotides to pair up in pairs to form nine DNA double strands to study the specificity of the biosensor. Figure 6 As shown, as long as one of the double-stranded chains contains 5hmC, a higher fluorescence signal can be generated, while the double-stranded chain containing only 5C and 5mC produces an extremely low fluorescence signal. These results clearly indicate that the biosensor has high selectivity for 5hmC-DNA.

[0088] The purpose of the present invention is to detect 5hmC in the whole genome, and the content of 5C and 5mC in genomic DNA is much higher than 5hmC. It is necessary to ensure that the detection of 5hmC is not interfered with by 5C and 5mC. In order to evaluate the anti-interference ability of this method, the present invention respectively incorporates 0, 10 nM, 25 nM, 50 nM, 100 nM, 250 nM, 2.5 μM and 25 μM of C-dsDNA and 5mC-DNA into the target 5hmC-DNA of the same concentration for experiments, so that the content of 5hmC is 100%, 71.43%, 50%, 33.33%, 20%, 9.09%, 0.99% and 0.099% respectively. Figure 7 As shown in the figure, adding different amounts of interfering substances to the target has no significant effect on the fluorescence intensity. Even when the 5hmC-DNA content in the mixture is as low as 0.099%, the fluorescence signal intensity is not significantly affected. This shows that this method has strong anti-interference ability.

[0089] 4. Reproducibility Analysis

[0090] In order to evaluate the reproducibility of the biosensor, the fluorescence signal intensity of 9 groups of 5hmC-DNA samples with the same concentration was measured. Figure 8 As shown, there was no significant difference in the fluorescence signal intensity of the nine samples, and the relative standard deviation (RSD) was 1.32%, indicating that the biosensor had good reproducibility.

[0091] 5. Cell Experiment

[0092] To verify that the proposed biosensor can be used for genome-wide 5hmC detection, the present invention measured the 5hmC content in five cancer cell lines (i.e., U118 cells, SH-SY5Y cells, HeLa cells, HepG2 cells, and A549 cells).

[0093] Cell culture and genomic DNA extraction

[0094] SH-SY5Y cells, U-118 MG cells, A549 cells, MCF-7 cells, HeLa cells, and HepG2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a cell culture incubator at 37°C and 5% CO2.

[0095] The genomic DNA was extracted from the cells using the QIAamp DNA Mini Kit, and then the long-chain DNA was fragmented using dsDNA fragmentase, and then detected using the detection method of the present invention.

[0096] Figure 9 Figure A shows that the 5hmC level in neural cancer cell lines (i.e., U118 cells and SH-SY5Y cells) is higher than that in other tumor cell lines (i.e., HeLa cells, HepG2 cells, and A549 cells), which is consistent with the research results reported in other literature. The present invention further used U-118 MG cells as a research model to study the relationship between genomic DNA concentration and fluorescence intensity. Figure 9 As shown in B, the fluorescence intensity increases with the increase of genomic DNA concentration (C) and is linearly correlated with the logarithm of the genomic DNA concentration in the range of 0.016 ng / μL to 4 ng / μL. The regression equation is F = 99.87 + 5.55 log 10 C (R 2 =0.9902). The detection limit was 2.33 × 10 −5 ng / μL.

[0097] Subsequently, the present invention used a commercial 5-hydroxymethylcytosine kit to detect 5hmC in whole-genome DNA extracted from U118 cells, SH-SY5Y cells, and HeLa cells, and compared the results obtained by this protocol with those obtained by the commercial kit. Figure 10 The results of the present invention are similar to those of the ELISA kit. The above results indicate that this method can be used for genome-wide analysis of 5hmC.

[0098] 6. Detection of 5-Hydroxymethylcytosine in Human Tissues

[0099] To further evaluate the ability of this method in analyzing clinical samples, the designed method was used to detect genomic DNA from lung cancer tissues and normal tissues from different patients.

[0100] An appropriate amount of tissue sample was weighed and ground into a powder in liquid nitrogen. The processed tissue sample was processed using the QIAamp DNA Mini Kit to extract whole-genomic DNA from the tissue sample. The long-chain DNA was then fragmented using dsDNA fragmentase and detected using the detection method of the present invention.

[0101] like Figure 11 As shown, the fluorescence intensities measured in the three groups of normal lung tissues were higher than those measured in the three groups of lung cancer tissues, proving that this method can distinguish between cancer tissues and normal tissues from the human body, and this method can be applied to the detection of actual clinical samples.

[0102] In summary, the present invention has constructed a homogeneous, template-independent signal amplification method for the detection of 5-hydroxymethylcytosine in the whole genome. This method is based on specific enzyme-catalyzed reactions and multiple signal amplification strategies, and has the characteristics of high sensitivity, good specificity, no need for separation steps, and no sequence dependence in amplification, and can detect 5hmC at any position in the whole genome. In addition, the present invention can achieve accurate and sensitive detection of actual clinical samples. The method is carried out in a homogeneous isothermal environment, without the need for amplification templates. Except for a signal probe, it does not require any carefully designed probes required for signal amplification and complex temperature change procedures, nor does it require other complex modifications or separation steps. This method can well distinguish the target 5hmC from 5mC and 5C, has strong specificity, and can avoid interference from 5-methylcytosine and cytosine in the genome that have similar structures to 5-hydroxymethylcytosine and are more abundant. The method has high detection sensitivity and a detection limit as low as 6.34 × 10 −17 The detection limit of M. in U-118 MG cells was 2.33 × 10 −5 ng / μL. This method also demonstrates good reproducibility and accuracy, consistent with results obtained from commercial kits. This method can distinguish between human cancer and normal tissue samples and is expected to be promoted for clinical application.

[0103] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for detecting 5-hydroxymethylcytosine in the whole genome for non-diagnostic purposes, characterized in that: The following steps are involved: 1) Block the 3'-OH end of the DNA to be tested: Terminal transferase TdT and adenine triphosphate dideoxynucleotide ddATP are added to the DNA to be tested. Under the action of TdT, ddATP is modified to the 3'-OH end of the double-stranded DNA to be tested. Shrimp alkaline phosphatase rSAP was added to hydrolyze excess ddATP to obtain the blocked DNA to be tested; 2) Preparation of glycosylated 5hmC DNA: T4β-glucosyltransferase T4β-GT and uridine diphosphate glucose UDP-Glc are added to the blocked test DNA. Under the catalysis of T4β-GT, the glucose group in UDP-Glc is transferred to the hydroxymethyl group of 5hmC in the test DNA to perform a glycosylation reaction. 5hmC is covalently modified with the glucose group to form 5ghmC, thereby obtaining glycosylated 5hmC DNA. 3) TdT-mediated DNA amplification: Adding DNA modification-dependent endonuclease AbaSI to the glycosylated 5hmC DNA, AbaSI recognizes and selectively cuts the glycosylated 5hmC DNA double strand containing 5ghmC, generating double-stranded DNA breaks at 11-13 nt and 9-10 nt away from 5ghmC, respectively, to form 3'-OH protruding sticky ends with 2-3 bases; TdT and deoxyadenosine triphosphate (dATP) are added. Under the catalysis of TdT, dATP polymerizes and extends at the 3'-OH protruding sticky end of the glycosylated 5hmC DNA to generate poly-A chains, obtaining the DNA to be detected by fluorescence. 4) Fluorescence measurement: A signal probe containing an AP site and an apurinic / apyrimidinic endonuclease APE1 are added to the DNA to be fluorescently detected. The signal probe and the poly-A chain in the DNA to be fluorescently detected are complementary and paired to form a double strand. The AP site in the signal probe is hydrolyzed by APE1, the fluorescent group is released, and the fluorescence is restored.

2. The detection method according to claim 1, wherein Step 1) is specifically as follows: Take 1 μM of the DNA sample to be tested, add 8 U TdT, 200 μM ddATP, 1× TdT reaction buffer, and 1× CoCl2, incubate at 37°C for 4 hours, and terminate the reaction at 75°C for 20 minutes; To remove incompletely reacted ddATP, 0.3 U rSAP and 1× rCutsmart reaction buffer were added to the product; the reaction was carried out at 37°C for 30 minutes and terminated at 65°C for 5 minutes.

3. The detection method according to claim 1, wherein Step 2) is specifically as follows: The blocked DNA to be tested was incubated in a solution containing 4 U T4β-GT, 40 μM UDP-glucose and 1×NEBuffer 4 (20 μL) at 37° C. for 2 h and terminated at 65° C. for 20 minutes.

4. The detection method according to claim 1, wherein Step 3) is specifically as follows: The glycosylated 5hmC DNA was reacted in a mixed solution containing 4U BaSI, 1× rCutsmart reaction buffer and 1× DTT at 37°C for 2 hours, and the reaction was terminated at 65°C for 20 minutes to generate new 3'-OH ends; during amplification, 8U TdT, 1mM dATP, and 1× TdT reaction buffer were added; the mixture was reacted at 37°C for 1 hour and inactivated at 75°C for 20 minutes.

5. The detection method according to claim 1, wherein Step 4) is specifically as follows: A mixed solution containing 125 nM signal probe, 3U APE1 enzyme, and 1× NEBuffer 4 was added to the DNA to be fluorescently detected, and the reaction was incubated at 37° C. for 1 hour and terminated at 65° C. for 20 minutes. The ROX fluorescence signal was monitored using a Hitachi F-4600 fluorescence spectrometer with an excitation / emission slit of 5.0 nm and an excitation wavelength of 577 nm. The emission wavelength was 590-700 nm, and the fluorescence intensity at 605 nm was used for data analysis.

6. The detection method according to claim 1, characterized in that Step 4) The sequence of the signal probe is: TTTTTTTT ROX TTTTTXTTTTT BHQ-2 T, as shown in SEQ ID NO.

7.

7. The detection method according to claim 1, characterized in that The following steps are also included: Extraction of DNA to be tested: Grind the tissue sample to be tested into powder in liquid nitrogen, and use the QIAamp DNA Mini Kit to extract the whole-genome DNA from the tissue sample. Use dsDNA fragmentase to fragment the long-chain DNA to obtain the DNA to be tested.

8. The detection method according to claim 1, wherein The following steps are also included: Polyacrylamide gel electrophoresis analysis: The reaction product of step 4) was added to a 14% polyacrylamide gel electrophoresis (PAGE) using 1× Gel Red as a fluorescent indicator and placed in 1× TBE buffer. After electrophoresis at a constant voltage of 120 V for 45 minutes, the gel image was visualized using an imaging system. The 1×TBE buffer comprises 89 mM Tris, 89 mM boric acid, 2 mM EDTA, and a pH of 8.

0.

9. Use of the detection method of claim 1 in the detection of whole-genome 5hmC quantification for non-diagnostic purposes.

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