Detection method and application of whole genome 5-hydroxymethylcytosine

Abstract

The invention discloses a detection method and application of whole genome 5-hydroxymethylcytosine, and relates to the technical field of biochemical detection. Comprising the following steps: 1, closing a 3 '-OH end of DNA to be detected; 2, preparation of glycosylated 5hmC DNA (Deoxyribose Nucleic Acid); 3, carrying out TdT mediated DNA (Deoxyribonucleic Acid) amplification; and 4, fluorescence measurement. According to the invention, a homogeneous and non-template dependent type signal amplification method for detecting the 5-hydroxymethylcytosine in the whole genome is constructed. The method is based on a specific enzyme catalytic reaction and a multiple signal amplification strategy, and has the characteristics of high sensitivity, good specificity, no need of a separation step, no sequence dependence in amplification and capability of detecting 5hmC at any position in a whole genome. In addition, accurate and sensitive detection of actual clinical samples can be realized.

Description

Technical Field

[0001] The present invention relates to the field of biochemical detection technology, and more specifically to a method for detecting whole-genome 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 modifications 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. Imbalance in DNA methylation is related to 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, studies over the past decade have shown that this modification is not static, but dynamically changing. The proper balance between DNA methylation and demethylation maintains the DNA methylation level of 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), under the catalysis of 10-11 translocation dioxygenase (TET). This is a dynamic demethylation process. Among them, 5-acylcytidine and 5-carboxycytidine can be further recognized by thymidine DNA glycosylase (TDG), and then cooperate with other enzymes to perform base excision repair (BER) to convert them into unmodified cytosine and complete the demethylation process.

[0004] In 2009, 5hmC was found to be another relatively abundant form of cytosine modification in embryonic stem cells (ESCs) and Purkinje neurons. Therefore, 5hmC is not only an intermediate in the demethylation process, but also widely present in mammalian tissues and cells. It is an epigenetic modification that plays an important role in individual development and is called the "sixth base". Whole-genome analysis of 5hmC further showed that it has a potential regulatory role in ESC regulation, bone marrow formation, fertilized egg development, and neural development. The content of 5hmC is tissue-type specific, and studies have shown that the content is highest in brain tissue. 5hmC is widely distributed in mammalian tissues and cells, and its content is extremely low compared to 5mC (5-methylcytosine). The content of 5hmC varies significantly between different tissues, with the highest content in the cerebral cortex, indicating that 5hmC has the potential to serve as a better tissue-specific biomarker for tissue type classification. There is ample evidence that a reduction in the overall level of 5hmC is one of the characteristics of malignant tumors. The researchers found that the overall level of 5hmC was 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. After bisulfite treatment, DNA will be severely degraded, destroying the integrity of DNA and affecting data reading. The main methods for studying 5hmC in the early stage include thin layer chromatography (TLC), liquid chromatography-mass spectrometry (LC-MS), high performance liquid chromatography (HPLC), immunoassay, etc. However, these methods have the disadvantages of difficulty in accurate quantification, low sensitivity, cumbersome operation, involving the use of radiolabeled nucleotides, only being able to detect 5-hydroxymethylcytosine of a specific sequence, and difficulty in accurately distinguishing 5hmC from 5mC. Recently, researchers have developed many new methods for detecting 5-hydroxymethylcytosine. Chen et al. designed a method for spectroscopic quantification of 5-hydroxymethylcytosine in genomic DNA using a boric acid-functionalized nanosphere fluorescent probe, but this method has no signal amplification step and low detection 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, cumbersome steps, 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 to be solved by technicians in this field in clinical research and common laboratory experiments. Summary of the invention

[0007] In view of this, the present invention provides a method and application for detecting 5-hydroxymethylcytosine in the whole genome.

[0008] In order to achieve the above object, the present invention adopts the following technical solution.

[0009] A method for detecting 5-hydroxymethylcytosine in the whole genome comprises the following steps: 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 terminal transferase (TdT), adenine triphosphate dideoxynucleotide (ddATP) is modified to the 3'-OH end of the double-stranded DNA to be tested; 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; Shrimp alkaline phosphatase (rSAP) was added to hydrolyze the excess ddATP to obtain the blocked DNA to be tested; rSAP can prevent other probes and newly generated 3'-OH termini from being blocked by unincorporated ddATP.

[0010] 2) Preparation of glycosylated 5hmC DNA: T4 β-glucosyltransferase (T4 β-GT) and UDP-Glc are added to the blocked DNA to be tested. Under the catalysis of T4 β-GT, the glucose group in UDP-Glc is transferred to the hydroxymethyl group of 5hmC in the DNA to be tested for glycosylation reaction. 5hmC is covalently modified by the glucose group to form 5ghmC, and glycosylated 5hmC DNA is obtained. Neither C (cytosine) nor 5mC can undergo glycosylation. Therefore, only 5hmC in genomic DNA can be covalently modified by glucose to form 5ghmC.

[0011] 3) TdT-mediated DNA amplification: DNA modification-dependent endonuclease (AbaSI) was added to the glycosylated 5hmC DNA. AbaSI recognized and selectively cut 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. 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. 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 and obtain DNA to be detected by fluorescence.

[0012] 4) Fluorescence measurement: A signal probe containing an AP site and an APE1 enzyme are added to the DNA to be detected by fluorescence. The signal probe and the poly-A chain in the DNA to be detected by fluorescence are complementary 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. The cut fluorescent probe falls off the double-stranded chain, 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.

[0013] Furthermore, step 1) is specifically as follows: 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 terminal transferase, 200 μM ddATP, 1 × TdT reaction buffer, and 1 × CoCl2, incubated at 37°C for 4 hours, and terminated at 75°C for 20 minutes. To remove the 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.

[0014] Furthermore, step 2) is specifically as follows: 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.

[0015] Furthermore, step 3) is specifically as follows: The glycosylation product was reacted in a mixed solution containing 4 U AbaSI, 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, 8 U 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.

[0016] Furthermore, 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 amplified product, reacted 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.

[0017] Furthermore, in step 4), the sequence of the signal probe is: TTTTTTTT ROX TTTTTXTTTTT BHQ-2 T, as shown in SEQ ID NO.7.

[0018] This signal probe is a 19 nt poly-T sequence, the 13th nucleotide is modified with an apurinic pyrimidine site (AP site), and the two sides of the AP site are modified with a fluorescent group ROX (9th nucleotide) and a quenching group BHQ-2 (19th nucleotide), respectively. The distance between the fluorescent group and the quenching group is about 3.4 nm (<10 nm), which can achieve effective quenching and prevent high background signals. The signal probe can be paired with any sequence of the poly-A sequence generated by TdT amplification to form a DNA double strand. Apurinic pyrimidine endonuclease 1 (APE1) can hydrolyze and break the phosphodiester bond at the 5' end of the apurinic / apyrimidine site in the DNA double strand, resulting in a single-stranded DNA break, and the signal probe is divided into 12 nt and 6 nt parts. The fluorescent group and the quenching group are separated, and the fluorescence is restored. In addition, the signal probe can also fall off the double-stranded DNA, and the new signal probe can be paired with the amplified poly-A sequence to start a new round of fluorescence recovery. This process is repeated to achieve signal amplification.

[0019] 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 must be present to amplify the complementary sequence of the signal probe. This signal probe can pair with any segment of the poly-A sequence produced by amplification to form a DNA double strand. The design is simple and ingenious, and 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 make the design of the enzyme cutting position more flexible. The ingenious design of the signal probe enables the entire system to be independent of any template, and there is no need to consider the impact of different sequences to be tested on the detection.

[0020] Furthermore, the method further comprises the following steps: Extraction of DNA to be tested: Take the tissue sample to be tested, grind it into powder in liquid nitrogen, process the processed tissue sample with QIAamp DNA Mini Kit, extract the whole genome DNA in the tissue sample; use dsDNA fragmentase to fragment the long-chain DNA to obtain the DNA to be tested.

[0021] Furthermore, the method further comprises the following steps: Polyacrylamide gel electrophoresis analysis: The reaction product of step 4) was added to 14% polyacrylamide gel electrophoresis (PAGE) with 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 120 V constant voltage for 45 minutes, the gel image was visualized using an imaging system.

[0022] A genome-wide 5-hydroxymethylcytosine detection method and its application in genome-wide 5hmC quantitative detection.

[0023] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects of the present invention are: The present invention constructs a homogeneous, template-independent signal amplification method for detecting 5-hydroxymethylcytosine in the whole genome. This method is based on a specific enzyme catalytic reaction and a multiple signal amplification strategy, has the characteristics of high sensitivity, good specificity, no need for separation steps, amplification without sequence dependence, 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.

[0024] The amplification step in this method can produce poly-A chains. After the fluorescent probe binds to them, it will be cut by APE1 enzyme and fall off the poly-A chains. The free signal probe can continue to bind to the poly-A chains. This cycle can play a role in signal amplification. Therefore, the detection sensitivity is high and the detection limit is as low as 6.34 × 10 −17 M.

[0025] This method has high 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.

[0026] 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 −5ng / μL. It can distinguish different cancerous tissues in the human body and between cancerous tissues and normal tissues, and after extracting whole genome DNA from actual samples, no other processing steps that may damage DNA are required. The 5hmC distribution levels of whole genome DNA of different cancer cells measured by this method are very similar to those obtained using commercial kits, and this method has a lower detection price than ELISA and sequencing methods, and it only costs RMB 7.7 to detect one sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0028] Figure 1 The accompanying drawings are schematic diagrams of the present invention; Figure 2 The accompanying drawing is a gel electrophoresis verification scheme feasibility of the present invention; Figure 2 A in the middle is the gel electrophoresis analysis of 5hmC modified dsDNA; Figure 2 B is the gel electrophoresis analysis of cytosine (5C), 5-methylcytosine (5mC), and 5-hydroxymethylcytosine (5hmC); Figure 3 The accompanying drawing is a fluorescence emission spectrum for verifying the feasibility of the present invention; Figure 4 The accompanying drawings are fluorescence emission spectra of the target of different concentrations according to the present invention; Figure 5 The accompanying drawing shows the corresponding relationship between the concentration and the fluorescence intensity of the present invention; Figure 5 A in the middle is the relationship between different concentrations and fluorescence intensity; Figure 5 In the middle B, the fluorescence intensity is linearly related to the logarithm of the 5hmC-DNA concentration in the range of 0.5 fM-500 pM; Figure 6 The accompanying drawing is a bar graph of specific detection in the present invention; Figure 7 The accompanying drawing is a radar diagram for verifying the anti-interference capability of the present invention; Figure 8 The attached figure shows the fluorescence intensity of 9 biosensors independently prepared by the present invention to detect the same concentration of target; Fig. 9 The accompanying drawing shows the fluorescence intensity and linear relationship produced by different cell genomic DNAs of the present invention; Fig. 9A in the middle is the fluorescence intensity generated by the same mass of genomic DNA of U-118 MG cells, SH-SY5Y cells, HeLa cells, HepG2 cells and A549 cells; Fig. 9 Middle B is the linear relationship between fluorescence intensity and the logarithm of U-118 MG cell concentration in the range of 0.016ng / μL - 4ng / μL; Fig.10 The accompanying drawings show the analysis of the content of 5hmC in the genomic DNA of different cancer cells by the present invention and ELISA kit; Fig.11 The attached 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

[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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 creative work are within the scope of protection of the present invention.

[0030] Example 1 1. Block the 3'-OH end of the DNA to be tested First, ddATP was used to modify the 3'-OH end of double-stranded DNA to prevent subsequent nonspecific amplification. Take 1 μM double-stranded DNA sample, add 8 U terminal transferase, 200 μM ddATP, 1 × TdT reaction buffer, 1 × CoCl2, incubate at 37°C for 4 hours, and terminate the reaction at 75°C for 20 minutes. In order to remove the 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.

[0031] 2. Preparation of Glycosylated 5hmC DNA Products 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.

[0032] 3. TdT-mediated DNA amplification The glycosylation product was reacted in a mixed solution containing 4 U AbaSI, 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, 8 U 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.

[0033] 4. Fluorescence Measurement A mixed solution containing 125 nM signal probe, 3U APE1 enzyme and 1 × NEBuffer 4 was added to the amplified product, reacted 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.

[0034] Table 1 5hmC, 5mC, 5C and signal probe sequences

[0035]

[0036] Note: In DNA, bases with italic "hm" superscripts represent 5-hydroxymethylcytosine (5hmC), and bases with italic "m" superscripts represent 5-methylcytosine (5mC). In signal probes, italic "X" indicates that the base is modified with an AP site, italic "ROX" indicates that the base is modified with a ROX fluorescent group, and italic "BHQ-2" indicates that the base is modified with a BHQ-2 quenching group. 5hmC sequence A and 5hmC sequence a are base complementary sequences; 5mC sequence A and 5mC sequence a are base complementary sequences; 5C sequence A and 5C sequence a are base complementary sequences.

[0037] 5. Polyacrylamide Gel Electrophoresis Analysis The reaction products were loaded onto 14% polyacrylamide gel electrophoresis (PAGE) with 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 120 V constant voltage for 45 min, the gel image was visualized using an imaging system.

[0038] Test experiment Preparation of double-stranded DNA substrate for experiment: 10 μM target oligonucleotide and 10 μM complementary oligonucleotide were hybridized in 1 × annealing buffer (50 mM NaCl, 10 mM Tris-HCl, pH 8.0) at 95°C for 5 min, and then gradually cooled to room temperature to obtain double-stranded DNA (dsDNA) substrate. The obtained dsDNA substrate was stored at 4°C for future use.

[0039] The double-stranded DNA substrates used in the experiment were substrates of different concentrations containing cytosine (5C), 5-methylcytosine (5mC), and 5-hydroxymethylcytosine (5hmC).

[0040] 1. Feasibility verification 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 2 A in the middle). 5hmC-DNA is first glycosylated to generate 5ghmC-DNA. When AbaSI is present, two DNA fragments (i.e., AbaSI digestion products) can be observed, indicating that AbaSI can cut 5ghmC-DNA (the second lane). However, in the absence of AbaSI, there is only one fragment with a large number of bases, i.e., the complete 5hmC-DNA (the first lane). When AbaSI+TdT+dATP is present, an amplification band can be observed, indicating that 5ghmC generates a new 3'-OH under the cleavage of AbaSI and is amplified under the action of terminal transferase and dATP (the third lane).

[0041] Next, the substrates of cytosine (5C), 5-methylcytosine (5mC), and 5-hydroxymethylcytosine (5hmC) were verified. Figure 2 As shown in B, when dsDNA+T4 BGT+AbaSI+TdT co-exist, normal cytosine (first lane) and methylcytosine (second lane) do not produce amplification bands, while only the third lane containing the 5hmC site produces an amplification band. This indicates that a series of reactions such as glycosylation and DNA amplification will only occur when 5hmC is present, proving that the scheme is feasible.

[0042] 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, 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.

[0043] 2. Sensitivity analysis Under the optimized optimal reaction conditions, the present invention uses 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 1, 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 −17 M. 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 is improved by 6, 7, and 3 orders of magnitude, respectively.

[0044] 3. Specificity Analysis 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 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 a high selectivity for 5hmC-DNA.

[0045] 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, so it is necessary to ensure that the detection of 5hmC is not interfered 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 same concentration of target 5hmC-DNA 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.

[0046] 4. Reproducibility Analysis 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.

[0047] 5. Cell experiments In order to verify that the proposed biosensor can be used for 5hmC detection in the whole genome, 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).

[0048] Cell culture and genomic DNA extraction 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) containing 10% fetal bovine serum and 1% penicillin-streptomycin. The cells were cultured in a cell culture incubator at 37°C and 5% CO2.

[0049] The genomic DNA is extracted from the cells using QIAamp DNA Mini Kit, and then the long-chain DNA is fragmented using dsDNA fragmentase, and then detected using the detection method of the present invention.

[0050] Fig. 9Figure A shows that the 5hmC level of neural cancer cell lines (i.e., U118 cells and SH-SY5Y cells) is higher than that of 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 uses U-118 MG cells as a research model to study the relationship between genomic DNA concentration and fluorescence intensity. Fig. 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 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 is 2.33 × 10 −5 ng / μL.

[0051] 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 scheme with those obtained by the commercial kit. Fig.10 The results of the present invention are similar to those of the ELISA kit. The above results show that this method can be used for the analysis of 5hmC in the whole genome.

[0052] 6. Detection of 5-Hydroxymethylcytosine in human tissues 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.

[0053] An appropriate amount of tissue sample is weighed, ground into powder in liquid nitrogen, and treated with QIAamp DNA Mini Kit to extract whole genome DNA from the tissue sample. Then, dsDNA fragmentase is used to fragment the long-chain DNA, and the detection method of the present invention is used for detection.

[0054] like Fig.11 As shown, the fluorescence intensities measured in the three groups of normal lung tissues are higher than those measured in the three groups of lung cancer tissues, which proves that this method can distinguish cancer tissues and normal tissues from the human body, and this method can be applied to the detection of actual clinical samples.

[0055] In summary, 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, and sequence-independent 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. In addition to a signal probe, it does not require the careful design of any probes required for signal amplification and complex temperature change procedures, nor does it require other complex modifications or separation steps. The 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. And the method has good reproducibility and accuracy, which is consistent with the results obtained by commercial kits. This method can distinguish human cancer tissue samples from normal tissue samples and is expected to be promoted to clinical applications.

[0056] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be 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 will not be limited to the embodiments shown herein, but rather 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, 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 the excess ddATP to obtain the blocked DNA to be tested; 2: Preparation of glycosylated 5hmC DNA: T4 β-glucosyltransferase T4 β-GT and uridine diphosphate glucose UDP-Glc are added to the blocked DNA to be tested, and under the catalysis of T4 β-GT, the glucose group in UDP-Glc is transferred to the hydroxymethyl group of 5hmC in the DNA to be tested for glycosylation reaction, and 5hmC is covalently modified by the glucose group to form 5ghmC, thereby obtaining glycosylated 5hmC DNA; 3: TdT-mediated DNA amplification: A DNA modification-dependent endonuclease AbaSI is added to the glycosylated 5hmC DNA, and 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, 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 a poly-A chain, thereby obtaining the DNA to be detected by fluorescence. 4: Fluorescence measurement: A signal probe containing an AP site and an apurinic / apyrimidinic nuclease APE1 are added to the DNA to be fluorescently detected. The signal probe is complementary to the poly-A chain in the DNA to be fluorescently detected 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, characterized in that: Step 1 is as follows: Take 1 μM of the DNA sample to be tested, add 8 U TdT, 200 μM ddATP, 1 × TdT reaction buffer, 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, characterized in that: Step 2 is as follows: The blocked DNA to be tested was incubated in a solution containing 4 U 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 minutes.

4. The detection method according to claim 1, characterized in that: Step 3 is as follows: The glycosylated 5hmC DNA was reacted in a mixed solution containing 4 U AbaSI, 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, 8 UTdT, 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.

5. The detection method according to claim 1, characterized in that: Step 4 is as follows: A mixed solution containing 125 nM signal probe, 3U APE1 enzyme and 1 × NEBuffer 4 was added to the DNA to be detected by fluorescence, and the reaction was carried out at 37°C for 1 hour and 65°C for 20 minutes to terminate the reaction; the ROX fluorescence signal was monitored using a F-4600 fluorescence spectrometer from Hitachi, Japan, 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: The sequence of the signal probe in step 4 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: Take the tissue sample to be tested, grind it into powder in liquid nitrogen, process the processed tissue sample with QIAamp DNA Mini Kit, and extract the whole genome DNA in 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, characterized in that: The following steps are also included: Polyacrylamide gel electrophoresis analysis: The reaction product of step 4 was added to 14% polyacrylamide gel electrophoresis PAGE, with 1× Gel Red as the fluorescent indicator, and placed in 1× TBE buffer. After electrophoresis at 120 V constant voltage for 45 minutes, the gel image was visualized using an imaging system; The 1×TBE buffer contains 89 mM Tris, 89 mM boric acid, 2 mM EDTA, and pH 8.

0.

9. Use of the detection method according to claim 1 in the quantitative detection of 5hmC in the whole genome.

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