CN107760762B - Fluorescent chemical sensor for detecting DNA adenine methyltransferase and detection method thereof - Google Patents
Fluorescent chemical sensor for detecting DNA adenine methyltransferase and detection method thereof Download PDFInfo
- Publication number
- CN107760762B CN107760762B CN201710945583.9A CN201710945583A CN107760762B CN 107760762 B CN107760762 B CN 107760762B CN 201710945583 A CN201710945583 A CN 201710945583A CN 107760762 B CN107760762 B CN 107760762B
- Authority
- CN
- China
- Prior art keywords
- probe
- dam
- dam methyltransferase
- methyltransferase
- detection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6825—Nucleic acid detection involving sensors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/682—Signal amplification
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a fluorescence chemical sensor for detecting Dam methyltransferase, which comprises: a Dam methyltransferase detection probe and a helper probe; the Dam methyltransferase detection probe is a hairpin DNA probe with a stem-loop structure, two stem structures are complementary to form double-stranded DNA, and the double-stranded DNA has a recognition site of Dam methyltransferase; the 3' terminal of the hairpin DNA probe is modified by amino; the recognition site of Dam methyltransferase is palindromic sequence of 5 '-G-A-T-C-3'; the auxiliary probe is a T-rich single-stranded DNA sequence with an apurinic pyrimidine site (AP site) close to the 3' end. The preparation and detection method of the fluorescence chemical sensor is simple, greatly improves the detection sensitivity, effectively prevents the occurrence of nonspecific reaction, and can realize the accurate detection of Dam methyltransferase in complex biological samples.
Description
Technical Field
The invention belongs to the technical field of biological analysis, and particularly relates to a fluorescence chemical sensor for detecting DNA (deoxyribonucleic acid) adenine methyltransferase based on hyper-branched amplification zero background and a detection method thereof.
Background
DNA methylation is the most prominent form of epigenetic gene regulation and plays a key role in cell proliferation, gene transcription and senescence. Targeting adenine/cytosine residues in recognition sequences by covalent addition of methyl groups catalyzed by DNA methyltransferases with S-adenosyl-L-methionine (SAM) as methyl donor. DNA methylation typically occurs at the C-5/N-4 position of cytosine and at the N-6 position of adenine. Hypermethylation and lack of methylation have been found in various types of cancer, such as breast, ovarian and lung cancer. Whereas abnormalities in DNA methylation are associated with abnormal expression and activity of DNA methyltransferases. Therefore, DNA methyltransferases have also become targets in clinical diagnostics and drug screening. Therefore, the development of ultrasensitive detection of DNA methyltransferase is not only helpful for the deep research of basic biochemistry, but also has great significance for the development of new therapeutic methods and strategies for human diseases.
To date, traditional detection methods for DNA methyltransferases include radiolabelling, High Performance Liquid Chromatography (HPLC), immuno-based analytical methods, and gel electrophoresis. These methods use radioactively labeled substrates, specific antibodies, expensive instruments, which not only increase the cost of the experiment but also increase the complexity of the experiment, are time consuming and laborious, and have low sensitivity.
In order to overcome these problems, in recent years, related researchers have introduced colorimetric methods, fluorometric methods and bioluminescence measurement methods, which have the advantages of intuition, safety, simplicity and high sensitivity. However, they generally require cumbersome nanoparticle preparation, rely on external labeling with fluorophores and quenchers, are long in analysis time, involve complicated designs and are costly. The electrochemical method has high response speed and low design cost, but the application of the electrochemical method is limited by the complicated electrode surface modification. The method of introducing nucleic acid amplification greatly improves the detection sensitivity, however, the existing nucleic acid amplification usually requires a DNA template, a specific recognition sequence of endonuclease and a fluorescent label of nucleic acid substrate, which increases the difficulty of scheme design and the complexity of experimental operation. Therefore, there is an urgent need to develop a method for sensitively detecting DNA methyltransferases in complex biological samples, which is simple to operate, does not need fluorescent labels, and has zero background signals.
Disclosure of Invention
Aiming at the defects of the prior art, the inventor provides a fluorescent chemical sensor for detecting DNA adenine methyltransferase (Dam methyltransferase) through long-term technical and practical exploration, the sensor can realize the characteristic of catalyzing deoxyadenosine triphosphate (dATPs) to be repeatedly added into 3' -hydroxyl terminal (3' -OH) of a DNA molecule based on terminal transferase (TDT) without any DNA template, can specifically identify complete Apurinic Pyrimidine (AP) sites in hydrolyzed double-stranded DNA by combining endonuclease IV so as to expose 3' -hydroxyl of the DNA molecule to realize a detection strategy of hyperbranched amplification, can realize the ultra-sensitive detection of the DNA adenine methyltransferase without fluorescent marks, and has the advantages of simple and rapid operation and accurate and reliable test result.
Specifically, the invention relates to the following technical scheme:
in a first aspect of the present invention, there is provided a fluorescent chemosensor for detecting Dam methyltransferase, the fluorescent chemosensor comprising: a Dam methyltransferase detection probe and a helper probe;
wherein the Dam methyltransferase detection probe is a hairpin DNA probe with a stem-loop structure, two stem structures are complementary to form double-stranded DNA, and the double-stranded DNA has a recognition site of Dam methyltransferase; the hairpin DNA probe is modified at the 3' end with an amino group, thereby preventing TdT (terminal transferase) activation of non-specific amplification;
specifically, the recognition site of Dam methyltransferase is a palindromic sequence of 5 '-G-A-T-C-3';
the auxiliary probe is a T-rich single-stranded DNA sequence with an apurinic pyrimidine site (AP site) near the 3 'end, and the 3' end of the auxiliary probe is modified by an amino group so as to prevent TdT (terminal transferase) from activating non-specific amplification;
the fluorescence chemical sensor also comprises methylation dependent endonuclease DpnI, terminal transferase (TdT), endonuclease IV and deoxyadenosine triphosphate (dATPs), wherein the methylation dependent endonuclease DpnI can recognize and cut a Dam methyltransferase detection probe after methylation of Dam methyltransferase, so that the detection probe is decomposed into three single-stranded DNA fragments, two single-stranded DNA fragments comprise a free 3'-OH end, deoxyadenosine triphosphate is added into the single-stranded DNA fragments with the 3' -OH end under the action of the terminal transferase (TdT) to obtain DNA fragments with an A-rich sequence, the DNA fragments with the A-rich sequence are hybridized and complemented with the auxiliary probe to form stable double-stranded DNA, and an apurinic pyrimidine site (AP site) in the double-stranded DNA is catalyzed by the endonuclease IV (endo IV), resulting in cleavage of the helper probe and the generation of a free 3' -OH terminus. The new DNA fragment with a free 3' -OH terminus initiates a new terminal transferase (TdT) -mediated extension reaction to form a longer a-rich sequence. Notably, the excess of the auxiliary probe hybridizes to the a-rich sequence, initiates a new cycle of cleavage extension, induces hyper-branched amplification, and generates a large number of DNA fragments; the obtained DNA fragments with different numbers generate different fluorescence values through a fluorescence indicator, so that the Dam methyltransferase activity is measured.
Preferably, the length of the Dam methyltransferase detecting probe is 37nt, and the base sequence of the Dam methyltransferase detecting probe is：5'-GAAGGA TCT TCT CGA CTT GCT GAAGAT CCT TCT TAA T-NH2-3', wherein the base sequence GATC, underlined, is the recognition site for Dam methyltransferase, which is amino-modified at the 3' end of the Dam methyltransferase detection probe.
Preferably, the length of the auxiliary probe is 26nt, and the base sequence of the auxiliary probe is: 5' -TTT TTT TTTTTT TTT TTT TTX TTT TT-NH2-3', wherein X represents an apurinic pyrimidine site, and the 3' end of the helper probe is modified with an amino group;
preferably, the fluorescence chemical sensor further comprises a fluorescence indicator, wherein the fluorescence indicator is SYBR Gold;
preferably, the fluorescence chemosensing further comprises a Dam methyltransferase reaction buffer comprising: 50 mmoles per liter Tris-HCl buffer, 10 mmoles per liter EDTA, 5 mmoles per liter 2-mercaptoethanol, pH 7.5;
preferably, the fluorescence chemosensing further comprises a terminal transferase buffer, the terminal transferase buffer comprising: 50 mmoles per liter of potassium acetate, 20 mmoles per liter of Tris-Ac, 10 mmoles per liter of magnesium acetate, pH 7.9;
the invention also discloses a method for detecting Dam methyltransferase by using the fluorescence chemical sensor, which specifically comprises the following steps:
1) adding a sample to be tested into the reaction solution I for incubation reaction, and then performing high-temperature inactivation treatment;
2) adding a reaction solution II into the solution subjected to high-temperature inactivation treatment in the step 1) for polymerization reaction;
3) performing fluorescence chemical detection on the solution reacted in the step 2) to realize quantitative analysis on Dam methyltransferase in the sample to be detected.
Wherein, the reaction solution I in the step 1) comprises a Dam methyltransferase detection probe, S-adenosylmethionine, restriction endonuclease Dpn I and Dam methyltransferase reaction buffer solution;
the incubation reaction conditions in the step 1) are as follows: incubating for 1.5-3 h (preferably 2h) at 37 ℃; the high-temperature inactivation treatment temperature is 80 ℃, and the treatment time is 10-30 min (preferably 20 min);
the reaction solution II in the step 2) comprises an auxiliary probe, deoxyadenosine triphosphate, terminal transferase (TdT), endonuclease IV, cobalt dichloride, a terminal transferase buffer solution and SYBR Gold;
the reaction conditions in the step 2) are as follows: the reaction time is 60-200 min (preferably 100min) at 37 ℃;
and 3) carrying out real-time quantitative detection on the fluorescence intensity by adopting a real-time quantitative PCR instrument.
The invention also discloses application of the fluorescence chemical sensor and/or the detection method in quantitative detection of Dam methyltransferase and/or screening Dam methyltransferase inhibitor/activator.
The principle of the fluorescence chemical sensor detection method provided by the invention is as follows: the invention relates to a fluorescence method for detecting DNA methyltransferase based on hyper-branched amplification zero background.
The inventors designed a hairpin DNA probe with a 5'-G-A-T-C-3' palindromic sequence as a substrate. To prevent TdT (terminal transferase) -activated non-specific amplification, the 3' ends of the hairpin and helper probes were modified with amino groups. After treatment with Dam methyltransferase, the 5'-G-A-T-C-3' sequence in the hairpin DNA probe stem is methylated to give 5 '-G-mA-T-C-3'. The methylated hairpin DNA probe is then cleaved by DNA methylation dependent endonuclease DpnI, releasing three single stranded DNA fragments, two of which contain a free 3' -OH terminus.
Multiple dATPs (deoxyadenosine triphosphate) were added sequentially to the free 3' -OH end of single-stranded DNA in the presence of TdT (terminal transferase) to obtain an A-rich sequence. A T-rich sequence helper probe (3' end modified with an amino group) with an AP site hybridizes to the resulting A-rich sequence to form a stable double-stranded DNA. The AP site in double-stranded DNA is catalyzed by Endo IV (endonuclease IV), resulting in cleavage of the helper probe and the generation of a free 3' -OH terminus. The new DNA fragment with a free 3' -OH terminus can initiate a new TdT (terminal transferase) -mediated extension reaction to form a longer a-rich sequence. Notably, the excess of helper probes hybridizes to the a-rich sequence, initiating a new cycle of cleavage extension, inducing hyper-branched amplification, and generating a large number of DNA fragments. The resulting different numbers of DNA fragments can be used as indicators to generate different fluorescence values by SYBR Gold. In the absence of Dam methyltransferase, neither TdT (terminal transferase) -mediated extension nor Endo iv (endonuclease iv) -mediated cleavage of the helper probe could be initiated and no significant fluorescence was observed.
The invention has the following beneficial effects:
(1) existing methods introduce a variety of nucleic acid amplification methods to improve sensitivity, but these methods often require specific endonuclease recognition sequences, increasing the complexity of DNA probe design. The invention utilizes the characteristic that terminal transferase (TDT) can catalyze deoxyadenosine triphosphate to be repeatedly added into the 3 '-hydroxyl terminal (3' -OH) of a DNA molecule without any DNA template, thereby greatly simplifying the design of a DNA probe;
(2) the invention utilizes endonuclease IV (Endo IV) to specifically identify complete depurination pyrimidine (AP) sites in the hydrolyzed double-stranded DNA, thereby exposing the 3' -hydroxyl of the DNA molecule, continuously amplifying under the catalysis of TDT, realizing hyperbranched amplification and greatly improving the detection sensitivity;
(3) the invention effectively prevents TdT from activating non-specific amplification through the 3 'ends of the hairpin probe and the auxiliary probe modified by amino, the high-accuracy identification of TdT leads to the amplification only at the free 3' -OH end, and Endo IV can only hydrolyze complete AP locus, thus realizing zero background signal and greatly improving the specificity of the detection method;
(4) the invention takes SYBR Gold as an indicator, does not need fluorescent labeling, has simple operation and reduces the experiment cost.
In conclusion, the preparation and detection method of the fluorescence chemical sensor is simple, large-scale expensive instruments and equipment are not needed, and meanwhile, because the reaction conditions are carefully optimized, the detection sensitivity is greatly improved and the occurrence of non-specific reaction is effectively prevented in the detection process, so that the accurate detection of Dam methyltransferase in a complex biological sample can be realized. Tests prove that compared with a fluorescence measurement method based on exonuclease mediated target circulation, a fluorescence measurement method based on endonuclease assisted signal amplification and a fluorescence measurement method based on transcription mediated double-strand specific nuclease assisted circulation signal amplification, the sensitivity of the detection method is improved by 1 order of magnitude, and compared with a fluorescence measurement method based on hairpin-shaped DNase signal amplification and a colorimetric method based on DNase mediated signal amplification, the sensitivity of the detection method is improved by 2 orders of magnitude.
Drawings
FIG. 1 is a schematic diagram of the mechanism of the fluorescence chemical sensor of the present invention for Dam methyltransferase detection;
FIG. 2(A) a schematic representation of methylation of hairpin substrates by Dam and subsequent DpnI cleavage by PAGE (polyacrylamide gel electrophoresis) wherein lane M is DNA marker (molecular mass reference); lane 1 is hairpin probe (0.5 micromole per liter) + Dam methyltransferase (20 units per ml) + restriction endonuclease DpnI (50 units per ml); lane 2 hairpin substrate (0.5 micromoles per liter) + DpnI (50 units per ml); lane 3, hairpin probe (0.5 micromoles per liter) + Dam methyltransferase (20 units per ml); FIG. 2(B) is a PAGE analysis of the hyperbranched amplification product, wherein the lane M is DNAmarker (molecular mass reference); lane 1 is a hyperbranched amplification product in the presence of Dam methyltransferase (20 units per ml) + restriction endonuclease DpnI (50 units per ml); lane 2 is the product of the hyperbranched amplification in the presence of the restriction endonuclease DpnI (50 units per ml), and figure 2(C) is a real-time fluorescence monitoring of the hyperbranched amplification in the presence of Dam methyltransferase (20 units per ml) + the restriction endonuclease DpnI (50 units per ml) (i.e., experimental group) and in the absence of Dam methyltransferase with the addition of only 50 units per ml of the restriction endonuclease DpnI (i.e., control group).
FIG. 3(A) shows F-F corresponding to various concentrations of auxiliary probes0A value; FIG. 3(B) is a graph showing F-F corresponding to Endo IV of different dosages when TdT is fixed to 8 units0A value; FIG. 3(C) is a graph showing F-F corresponding to TdT at different dosages when the dose of Endo IV is fixed to 4 units0A value; FIG. 3(D) shows F-F for different concentrations of dATP0A value; wherein F and F0Fluorescence intensity in the presence and absence of Dam methyltransferase, respectively; error bars indicate the standard deviation of triplicate experiments.
FIG. 4(A) is a real-time fluorescence curve obtained from different concentrations of Dam-initiated amplification; FIG. 4(B) is a linear correlation between the fluorescence intensity of Dam methyltransferase in the range of 0.005 to 40 units per milliliter and the logarithm of its concentration, and the fluorescence intensity in FIG. 4(B) is obtained at 100 minutes; error bars indicate the standard deviation of triplicate experiments.
FIG. 5 is a graph showing the selectivity of the detection method of the present invention for Dam methyltransferase, and FIG. 5(A) is a real-time fluorescence curve in response to Dam methyltransferase, M.Sss I methyltransferase, Bovine Serum Albumin (BSA) and a buffer-only control; fig. 5(B) is a measurement value of fluorescence intensity in response to Dam methyltransferases, m.ss I methyltransferases, BSA, and a buffer-only control group, the fluorescence intensity in fig. 5(B) being obtained at 100 minutes, wherein the concentrations of Dam methyltransferases, m.ss I methyltransferases, Bovine Serum Albumin (BSA) of each group were 20 units per ml; error bars indicate the standard deviation of triplicate experiments.
FIG. 6 shows the detection of Dam methyltransferase in reaction buffer and LB medium; diluting LB with reaction buffer solution in a ratio of 1: 10; error bars indicate the standard deviation of triplicate experiments.
FIG. 7(A) is a graph of 5-fluorouracil effect on TdT and Endo IV activity analyzed by polyacrylamide gel electrophoresis, in which lane M is DNA marker (molecular mass reference); lane 1 is primers (67 nmol per liter); lane 2 is primer (67 nmol per liter) + helper probe (0.13 micromol per liter) + TdT (8 units); lane 3 is primer (67 nmol per liter) + helper probe (0.13 micromol per liter) + TdT (8 units) + 5-fluorouracil (10 micromol per liter); lane 4 is primer (67 nmol per liter) + helper probe (0.13 micromol per liter) + TdT (8 units) + Endo IV (4 units); lane 5 is primer (67 nmol per liter) + auxiliary probe (0.13 micromol per liter) + TdT (8 units) + Endo IV (4 units) + 5-fluorouracil (10 micromol per liter), and figure 7(B) is fluorescence intensity measurements in the presence and absence of 10 micromol per liter 5-fluorouracil.
FIG. 8 is a graph of the inhibition of Dam methyltransferase activity by different concentrations of 5-fluorouracil.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular is intended to include the plural unless the context clearly dictates otherwise, and it should be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of features, steps, operations, devices, components, and/or combinations thereof.
As introduced in the background art, the prior art has various problems of low sensitivity, complicated detection method, expensive required instruments and the like for the determination of the methyltransferase;
in view of the above, in an exemplary embodiment of the present invention, there is provided a fluorescent chemical sensor for detecting Dam methyltransferase, the fluorescent chemical sensor including: a Dam methyltransferase detection probe and a helper probe;
wherein the Dam methyltransferase detection probe is a hairpin DNA probe with a stem-loop structure, two stem structures are complementary to form double-stranded DNA, and the double-stranded DNA has a recognition site of Dam methyltransferase; the hairpin DNA probe is modified at the 3' end with an amino group, thereby preventing TdT (terminal transferase) activation of non-specific amplification;
the recognition site of Dam methyltransferase is palindromic sequence of 5 '-G-A-T-C-3';
the auxiliary probe is a T-rich single-stranded DNA sequence with an apurinic pyrimidine site (AP site) close to the 3' end; the 3' end of the auxiliary probe is modified by amino so as to prevent TdT (terminal transferase) from activating non-specific amplification;
the fluorescent chemical sensor further comprises a methylation dependent endonuclease, DpnI, a terminal transferase (TdT), an endonuclease IV, and deoxyadenosine triphosphate (dATPs);
in another embodiment of the present invention, the Dam methyltransferase detection probe has a length of 37nt, and the Dam methyltransferase detection probe has a base sequence of: 5' -GAA GGA TCT TCT CGA CTT GCT GAAGATCCTTCT TAAT-NH2-3', wherein the base sequence GATC, underlined, is the recognition site for Dam methyltransferase, which is amino-modified at the 3' end of the Dam methyltransferase detection probe;
in another embodiment of the present invention, the length of the auxiliary probe is 26nt, and the base sequence of the auxiliary probe is: the base sequence of the auxiliary probe is as follows: 5' -TTT TTT TTT TTT TTT TTT TTX TTT TT-NH2-3', wherein X represents an apurinic pyrimidine site, and the 3' end of the helper probe is amino-modified;
in yet another embodiment of the present invention, the fluorescence chemical sensor further comprises a fluorescence indicator, wherein the fluorescence indicator is SYBR Gold;
in yet another embodiment of the present invention, the fluorescence chemosensing further comprises a Dam methyltransferase reaction buffer comprising: 50 mmoles per liter Tris-HCl buffer, 10 mmoles per liter EDTA, 5 mmoles per liter 2-mercaptoethanol, pH 7.5;
in yet another embodiment of the present invention, the fluorescence chemosensing further comprises a terminal transferase buffer comprising: 50 mmoles per liter of potassium acetate, 20 mmoles per liter of Tris-Ac, 10 mmoles per liter of magnesium acetate, pH 7.9;
in another embodiment of the present invention, a method for using the fluorescence chemical sensor for Dam methyltransferase detection is disclosed, which comprises:
1) adding a sample to be tested into the reaction solution I for incubation reaction, and then performing high-temperature inactivation treatment;
2) adding a reaction solution II into the solution subjected to high-temperature inactivation treatment in the step 1) for polymerization reaction;
3) performing fluorescence chemical detection on the solution reacted in the step 2) to realize quantitative analysis on Dam methyltransferase in the sample to be detected.
Wherein, the reaction solution I in the step 1) comprises a Dam methyltransferase detection probe, S-adenosylmethionine, restriction endonuclease Dpn I and Dam methyltransferase reaction buffer solution;
the incubation reaction conditions in the step 1) are as follows: incubating for 1.5-3 h (preferably 2h) at 37 ℃; the high-temperature inactivation treatment temperature is 80 ℃, and the treatment time is 10-30 min (preferably 20 min);
the reaction solution II in the step 2) comprises an auxiliary probe, deoxyadenosine triphosphate, terminal transferase (TdT), endonuclease IV, cobalt dichloride, a terminal transferase buffer solution and SYBR Gold;
the reaction conditions in the step 2) are as follows: the reaction time is 60-200 min (preferably 100min) at 37 ℃;
and 3) carrying out real-time quantitative detection on the fluorescence intensity by adopting a real-time quantitative PCR instrument.
In yet another embodiment of the present invention, the use of the above-described fluorescence chemical sensor and/or detection method for quantitative detection of Dam methyltransferases and/or screening Dam methyltransferase inhibitors/activators is disclosed.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto.
Examples
Procedure of the Experimental method
Detection of Dam methyltransferase: methylation and cleavage of hairpin probes were carried out in 200. mu.l reaction mixtures containing 0.5. mu. mol per liter of hairpin probe, 160. mu. mol per liter of SAM (S-adenosylmethionine), 10 units of restriction endonuclease Dpn I, 1 XDNAdam buffer (50 mmol per liter Tris-HCl buffer, 10 mmol per liter EDTA, 5 mmol per liter 2-mercaptoethanol, pH7.5) and varying amounts of Dam methyltransferase. The mixture was incubated at 37 ℃ for 2 hours and then inactivated at 80 ℃ for 20 minutes. Hyperbranched amplification was carried out in 30. mu.l of a buffer containing 1 Xterminal transferase (50 mmole per liter of potassium acetate, 20 mmole per liter of Tris-Ac, 10 mmole per liter of magnesium acetate, pH7.9), 0.25 mmole per liter of cobalt dichloride, 1 XSYBR Gold, 0.13. mu.l per liter of the helper probe reaction solution, 1 mmole per liter of dATPs (deoxyadenosine triphosphate), 4 units of Endo IV (endonuclease IV), 8 units of TDT (terminal transferase) and 4. mu.l of the methylation product. The polymerization was carried out on a Bio-Rad CFX 96 real-time quantitative PCR instrument (Bio-Rad, USA) at 37 ℃ for 100 minutes, and the fluorescence intensity was monitored at 30-second intervals.
2. Gel electrophoresis analysis: 12% native polyacrylamide gel electrophoresis (PAGE) in 1 XTBE buffer (9 mmol per liter Tris-HCl, 9 mmol per liter boric acid, 0.2 mmol per liter EDTA, pH7.9) at 110V constant voltage for 50 min at room temperature using 1 XSSYBR Gold as the fluorescence indicator.
Selectivity of Dam methyltransferase assay: to investigate the selectivity of the proposed method, we used m.sssi methyltransferase and bovine serum albumin as interfering enzymes. Experiments were performed using the above method with 20 units per ml of interfering enzyme.
4. Detection of Dam methyltransferase in a sample of bacteriolytic yeast medium: a total volume of 200. mu.l of a sample mixture containing 10% bacteriolytic yeast medium (LB) was added with various concentrations of Dam methyltransferase, 0.5. mu. mol per liter of hairpin probe, 160. mu. mol per liter of SAM (S-adenosylmethionine), 10 units of restriction endonuclease Dpn I, 1 XDam reaction buffer (50. mu. mol per liter Tris-HCl buffer, 10. mu. mol per liter EDTA, 5. mu. mol per liter 2-mercaptoethanol, pH7.5), incubated at 37 ℃ for 2 hours, then inactivated at 80 ℃ for 20 minutes. Dam methyltransferase activity was determined as described above.
5. Inhibitor analysis: to investigate the effect of 5-fluorouracil on Dam methyltransferase activity, various concentrations of 5-fluorouracil, 0.5 micromoles per liter of hairpin probe and 1 XDam reaction buffer (50 mmoles per liter Tris-HCl buffer, 10 mmoles per liter EDTA, 5 mmoles per liter 2-mercaptoethanol, pH7.5) were incubated at 37 ℃ for 15 minutes. Then, 20 units per ml Dam methyltransferase, 50 units per ml restriction endonuclease Dpn I and 160. mu. mol per liter SAM (S-adenosylmethionine) were added to the solution, and reacted at 37 ℃ for 2 hours and at 80 ℃ for 20 minutes. Dam methyltransferase activity was determined using the procedure analysis described above. The Relative Activity (RA) of Dam methyltransferase was calculated by equation 1:
wherein F0Is the fluorescence intensity in the absence of Dam methyltransferase, Ft is the fluorescence intensity in the presence of 20 units per ml Dam methyltransferase, FiIs the fluorescence intensity in the presence of 20 units per ml Dam methyltransferase and 5-fluorouracil.
Analysis and discussion of results
1. Experimental verification of the principles of the invention
To verify the feasibility of this protocol, we investigated the methylation process of Dam methyltransferase using native polyacrylamide gel electrophoresis (FIG. 2). In the absence of Dam methyltransferase or DpnI, only a 20bp band was observed ( lanes 2 and 3 in FIG. 2A), indicating that the hairpin was not cleaved. When Dam methyltransferase and DpnI were both present, a new 20nt band appeared (FIG. 2A, lane 1), indicating that methylation and cleavage processes occurred. To validate TdT-activated Endo IV-assisted hyperbranched amplification, we used a non-denaturing polyacrylamide gel to detect the amplification products. In the presence of 20 units per ml Dam methyltransferase and 50 units per ml DpnI, a distinct amplified product band was observed (fig. 2B, lane 1), indicating that a large number of DNA fragments were generated due to hyperbranched amplification. In contrast, no amplification product band was observed in the control group without Dam methyltransferase (FIG. 2B, lane 2). To further confirm TdT-activated Endo IV-assisted hyperbranched amplification, we also performed real-time fluorescence detection (fig. 2C). The fluorescence intensity increased linearly with time in the presence of Dam methyltransferase and Dpn I (FIG. 2C). In the control group without Dam methyltransferase, zero background signal was observed (fig. 2C). The achievement of a zero background signal is likely due to the modification of the 3' end of the hairpin probe and the ability of the helper probe with an amino modification to effectively prevent non-specific amplification of TdT activation in the absence of Dam methyltransferase.
2. Optimizing the experimental conditions
To obtain the best results, we optimized the concentrations of the helper probe and dATP, as well as the amounts of Endo IV and TdT. As shown in FIG. 3A, as the concentration of the auxiliary probe increases, F-F0(F and F)0Fluorescence intensity in the presence and absence of Dam, respectively) increased and a plateau was reached at an auxiliary probe concentration of 0.13 micromole per liter. Thus, the concentration of the helper probe used in subsequent experiments was 0.13 micromoles per liter. The generation of the fluorescent signal is dependent on TdT-mediated DNA strand extension and Endo IV-mediated cleavage of the helper probe, and therefore the amounts of Endo IV and TdT also need to be optimized. We tested the effect of different amounts of Endo IV on the fluorescence signal with TdT fixed at 8 units. As shown in FIG. 3B, F-F increases with Endo IV0(F and F)0Fluorescence intensity in the presence and absence of Dam, respectively) and reached a maximum at a dose of 4 units. We then investigated the effect of TdT on the fluorescence signal, when the amount of Endo IV was fixed at 4 units. As shown in FIG. 3C, TdT is from 4 units to 8 units within the interval F-F0(F and F)0Fluorescence intensity in the presence and absence of Dam, respectively) increased with increasing amounts of TDT, F-F exceeding 8 units0The value of (c) decreases. Thus, 4 units of Endo IV and 8 units of TdT were used in subsequent experiments.
We further investigated the effect of dATP concentration on the fluorescence signal. FIG. 3D shows that F-F increases with dATP concentration0(F and F)0Fluorescence intensity in the presence and absence of Dam, respectively) increased to a maximum at a concentration of 1 millimole per liter. Therefore, 1 mmol per liter of dATP was used in subsequent experiments.
3. Sensitivity detection
Under the best experimental conditions, we monitored the fluorescence signals generated by different concentrations of Dam methyltransferase in real time. As shown in fig. 4A, the fluorescence signal increases linearly in a time-dependent and concentration-dependent manner. The higher the concentration of Dam methyltransferase, the more DNA substrate methylated and the more free 3' -OH end is produced by subsequent cleavage with DpnI, and therefore the higher the fluorescence signal. In addition, there was a good linear correlation between fluorescence intensity and the logarithm of Dam methyltransferase concentration in the range of 4 orders of magnitude from 0.005 to 40 units per milliliter (fig. 4B). The regression equation was F2889.8 +1164.4log10C with a correlation coefficient of 0.991, where F and C represent fluorescence intensity and Dam methyltransferase concentration (in ml), respectively. By calculating the average response value of the blank plus three times the standard deviation into the linear equation, a detection limit of 0.003 units per milliliter can be obtained. Notably, the sensitivity of this method was improved by 1 order of magnitude compared to the exonuclease mediated target cycle fluorometry (0.01 units per milliliter), endonuclease assisted signal amplification based fluorometry (0.06 units per milliliter), and transcription mediated double strand specific nuclease assisted cycle signal amplification based fluorometry (0.015 units per milliliter), and 2 orders of magnitude compared to hairpin dnase signal amplification based fluorometry (0.4 units per milliliter) and dnase mediated signal amplification based colorimetric methods (0.25 units per milliliter). Importantly, the method proposed by the invention is very simple, does not need to design a specific recognition sequence of any endonuclease, and shows excellent specificity, namely the background signal is almost zero when no target Dam methyltransferase exists. The improvement in sensitivity can be attributed to three factors: (1) TdT activates the enhancement of the fluorescence signal induced by Endo IV assisted hyperbranched amplification; (2) the high-accuracy identification of TdT leads to that amplification can only occur at the free 3' -OH end, and Endo IV can only hydrolyze the complete AP locus, thus realizing zero background signal; (3) the 3' ends of the hairpin probe and the helper probe are effectively prevented from activating non-specific amplification by amino modification.
4. Selective detection
To investigate the selectivity of the assay method of the invention, we used m.sssi methyltransferase and BSA as interfering enzymes. SssI methyltransferases can specifically methylate cytosine residues in the 5'-C-G-3' recognition sequence of double stranded DNA, whereas BSA is an unrelated protein. As shown in fig. 5A, the fluorescence signal increased in a time-dependent manner in the presence of 20 units per ml Dam methyltransferase, whereas no fluorescence signal was observed in the presence of 20 units per ml m.ss I methyltransferase, 20 units per ml BSA, and the control. In addition, the fluorescence intensity in response to Dam methyltransferase was much higher than that in response to m.ss I methyltransferase, BSA and control (fig. 5B). This can be explained by the following facts: the specific recognition sequence of 5'-G-A-T-C-3' can only be methylated by Dam methyltransferase instead of M.Sss I and BSA. These results clearly demonstrate the excellent selectivity of the proposed method for Dam methyltransferases.
5. Detection of Dam methyltransferase in lytic Yeast Medium
To further validate the feasibility of this approach in the analysis of real samples, we tested Dam methyltransferases activity in complex samples containing 10% Lysozyme (LB) medium. As shown in FIG. 6, the fluorescence intensity of Dam methyltransferase in the diluted sample of the lysed yeast medium was consistent with its response in the reaction buffer, and the recovery rates of Dam methyltransferase were 99.8% + -4.62% and 103.8% + -4.18% at 0.5 units per ml and 20 units per ml, respectively. These results indicate that the proposed method has great potential for further applications in complex biological samples.
6. Inhibitor assay
Dam methyltransferases play an important role in the virulence of an increasing number of bacterial pathogens and have been the target of antimicrobial drug development. To demonstrate the feasibility of the proposed inhibition assay, we used 5-fluorouracil as a model inhibitor. Previous studies have shown that concentrations of 5-fluorouracil of less than 10 micromoles per liter have no effect on the activity of DpnI. The influence of 5-fluorouracil on the activity of TDT and Endo IV was studied by gel electrophoresis analysis and real-time fluorescence detection. As shown in FIG. 7A, in the TDT-only system, two bands reflecting TdT-mediated amplification products were observed with 10. mu. moles per liter of 5-fluorouracil (FIG. 7A, lane 2) and without 5-fluorouracil (FIG. 7A, lane 3), indicating that 10. mu. moles per liter of 5-fluorouracil had no effect on TdT activity. Furthermore, in the system with TDT and Endo IV present, with 10 micromoles per liter of 5-fluorouracil (fig. 7A, lane 4) and without 5-fluorouracil (fig. 7A, lane 5), no significant difference was observed in the two bands reflecting TDT-activated Endo IV-assisted hyperbranched amplification product, indicating that 10 micromoles per liter of 5-fluorouracil had no effect on the activity of Endo IV. In addition, the fluorescence intensity obtained in the presence of 10. mu. mol/l of 5-fluorouracil (FIG. 7B) was not significantly different from that obtained in the absence of 5-fluorouracil (FIG. 7B), indicating that 10. mu. mol/l of 5-fluorouracil had no significant effect on the activity of TDT and Endo IV, regardless of the presence of TdT alone or TDT and Endo IV together. These results indicate that 5-fluorouracil does not affect the enzymatic activity of DpnI, TdT and Endo IV.
FIG. 8 shows the effect of 5-fluorouracil on Dam methyltransferase activity. The relative activity of Dam methyltransferase gradually decreased with increasing concentration of 5-fluorouracil. IC (integrated circuit)50Values are the concentration of inhibitor required to reduce enzyme activity by 50%. For 5-Fluorouracil, IC was calculated50IC obtained with hairpin probe-based primer-generated Rolling circle amplification (PG-RCA) induced chemiluminescence assay (1.42. + -. 0.07. mu.M) at a value of 1.29 micromol per liter50The values are consistent. These results indicate that the proposed method can be used to screen Dam methyltransferase inhibitors.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
SEQUENCE LISTING
<110> university of Shandong Master
<120> fluorescent chemical sensor for detecting DNA adenine methyltransferase and detection method thereof
<130>
<160>2
<170>PatentIn version 3.3
<210>1
<211>37
<212>DNA
<213> Artificial Synthesis
<400>1
gaaggatctt ctcgacttgc tgaagatcct tcttaat 37
<210>2
<211>26
<212>DNA
<213> Artificial Synthesis
<400>2
tttttttttt tttttttttt xttttt 26
Claims (9)
1. A fluorescent chemical sensor for detecting Dam methyltransferase, comprising: a reaction solution I and a reaction solution II; the reaction solution I comprises a Dam methyltransferase detection probe, S-adenosylmethionine, restriction endonuclease Dpn I and Dam methyltransferase reaction buffer solution; the reaction solution II comprises an auxiliary probe, deoxyadenosine triphosphate, terminal transferase (TdT), endonuclease IV, cobalt dichloride, a terminal transferase buffer solution and SYBR Gold;
the Dam methyltransferase detection probe is a hairpin DNA probe with a stem-loop structure, two stem structures are complementary to form double-stranded DNA, and the double-stranded DNA has a recognition site of Dam methyltransferase; the length of the Dam methyltransferase detection probe is 37nt, and the base sequence of the Dam methyltransferase detection probe is as follows: 5' -GAA GGA TCT TCT CGA CTT GCTGAA GAT CCT TCT TAA T-NH2-3'; the 3' end of the Dam methyltransferase detection probe is modified by amino;
the recognition site of Dam methyltransferase is palindromic sequence of 5 '-G-A-T-C-3';
the auxiliary probe is a T-rich single-stranded DNA sequence with an apurinic pyrimidine site (AP site) near the 3 'end, the length of the auxiliary probe is 26nt, and the base sequence of the auxiliary probe is 5' -TTT TTT TTTTTTTTTTTTX TTTTT-NH2-3', wherein X represents an apurinic pyrimidine site, and the 3' end of the helper probe is amino-modified.
2. The fluorochemical sensor of claim 1, wherein said Dam methyltransferase reaction buffer comprises: 50 mmoles per liter Tris-HCl buffer, 10 mmoles per liter EDTA, 5 mmoles per liter 2-mercaptoethanol, pH 7.5.
3. The fluorochemical sensor of claim 1, wherein said terminal transferase buffer comprises: 50 mmoles per liter of potassium acetate, 20 mmoles per liter of Tris-Ac, 10 mmoles per liter of magnesium acetate, pH 7.9.
4. A method for Dam methyltransferase detection with the aim of non-disease diagnostic therapy using a fluorogenic chemical sensor according to any of claims 1-3, characterized by the steps comprising:
1) adding a sample to be tested into the reaction solution I for incubation reaction, and then performing high-temperature inactivation treatment;
2) adding a reaction solution II into the solution subjected to high-temperature inactivation treatment in the step 1) for polymerization reaction;
3) performing fluorescence chemical detection on the solution reacted in the step 2) to realize quantitative analysis on Dam methyltransferase in the sample to be detected.
5. The detection method according to claim 4, wherein the incubation reaction conditions in step 1) are as follows: incubating for 1.5-3 h at 37 ℃; the high-temperature inactivation treatment temperature is 80 ℃, and the treatment time is 10-30 min.
6. The assay of claim 5, wherein the incubation time in step 1) is 2 h.
7. The detection method according to claim 5, wherein the time for the high-temperature inactivation treatment in step 1) is 20 min.
8. Use of the fluorescence chemical sensor according to any one of claims 1 to 3 for the quantitative detection of Dam methyltransferases and/or for the screening of Dam methyltransferase inhibitors or activators for purposes other than disease diagnosis and treatment.
9. Use of the assay of any one of claims 5 to 7 for the quantitative detection of Dam methyltransferases and/or for screening Dam methyltransferase inhibitors or activators for purposes other than disease diagnosis and treatment.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710945583.9A CN107760762B (en) | 2017-09-30 | 2017-09-30 | Fluorescent chemical sensor for detecting DNA adenine methyltransferase and detection method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710945583.9A CN107760762B (en) | 2017-09-30 | 2017-09-30 | Fluorescent chemical sensor for detecting DNA adenine methyltransferase and detection method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107760762A CN107760762A (en) | 2018-03-06 |
| CN107760762B true CN107760762B (en) | 2020-05-08 |
Family
ID=61267994
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710945583.9A Active CN107760762B (en) | 2017-09-30 | 2017-09-30 | Fluorescent chemical sensor for detecting DNA adenine methyltransferase and detection method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107760762B (en) |
Families Citing this family (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109251960B (en) * | 2018-03-27 | 2022-04-26 | 兰州大学 | Method for detecting Dam methyltransferase activity based on base excision repair induction |
| CN109270144B (en) * | 2018-10-31 | 2020-09-29 | 山东师范大学 | Method for detecting 5-hydroxymethylcytosine based on non-labeled and non-immobilized electrochemical magnetic biosensor |
| CN109406477A (en) * | 2018-12-29 | 2019-03-01 | 济南大学 | Active biological sensor of a kind of detection dnmt rna and preparation method thereof |
| CN109613096B (en) * | 2019-01-21 | 2021-09-10 | 宁波大学 | DNA-related enzyme electrochemical biosensor constructed based on DNA-copper nanoclusters and application thereof |
| CN109750088B (en) * | 2019-03-22 | 2022-06-21 | 重庆医科大学 | TdT-RCA-based sensor and application thereof in DNA methyltransferase detection |
| CN110144384B (en) * | 2019-06-03 | 2023-03-24 | 山东师范大学 | Fluorescent chemical sensor for detecting oxidative damage in telomere as well as detection method and application thereof |
| CN110609020B (en) * | 2019-08-15 | 2021-10-01 | 济南大学 | Biosensor for detecting ATP based on palindromic molecular beacon and preparation method and application thereof |
| CN110646487B (en) * | 2019-10-14 | 2022-06-14 | 宁波大学 | Electrochemical sensor for detecting hydrogen peroxide and terminal transferase and application |
| CN111088324B (en) * | 2019-12-30 | 2023-02-24 | 山东师范大学 | Single quantum dot nano sensor for detecting methyltransferase and detection method and application thereof |
| CN112094889A (en) * | 2020-08-31 | 2020-12-18 | 山东师范大学 | Chemiluminescence sensor and application in detection of bacterial or human methyltransferase |
| CN112322702A (en) * | 2020-10-14 | 2021-02-05 | 山东师范大学 | Biosensor for simultaneously detecting multiple DNA (deoxyribonucleic acid) methyltransferases as well as detection method and application thereof |
| CN112226484B (en) * | 2020-10-27 | 2022-08-26 | 山东师范大学 | Fluorescent biosensor of beta-glucose transferase, detection method and application |
| CN112852922B (en) * | 2021-01-14 | 2022-12-27 | 山东师范大学 | Fluorescent biosensor for detecting DNA methylation, detection method and application |
| CN113652473B (en) * | 2021-06-30 | 2023-05-05 | 山东师范大学 | Fluorescent chemical sensor for detecting DNA damage site by single molecule, method and application |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103589777A (en) * | 2012-08-14 | 2014-02-19 | 中国科学院深圳先进技术研究院 | DNA methylation detection probe, and detection method and detection kit thereof |
| CN102816855B (en) * | 2012-09-03 | 2014-04-16 | 华南师范大学 | Method for detecting food-borne pathogenic bacteria by using nucleic acid test strip based on hyper-branched rolling cycle amplification and kit |
| CN103710453A (en) * | 2013-12-30 | 2014-04-09 | 深圳先进技术研究院 | Chemiluminescence reaction-based methylase detection probe, detection kit and detection method |
| CN104212792A (en) * | 2014-04-22 | 2014-12-17 | 上海大学 | Nicking endonuclease-based netted rolling cycle amplification system and use thereof |
| CN103993083B (en) * | 2014-05-22 | 2016-01-06 | 东南大学 | The unmarked fluoroscopic examination DNA methylation of restriction enzyme and exonucleaseⅲ and the detection method of methyl transferase activity |
| CN105112540B (en) * | 2015-09-17 | 2019-03-05 | 山东大学 | A kind of active method of detection dnmt rna based on strand displacement amplification and DNAzyme amplification |
| CN105506078B (en) * | 2015-12-18 | 2019-07-09 | 山东大学 | One kind is for being measured in parallel uracil-DNA glycosylase and the active method and its application of restriction endonuclease IV and kit |
| CN106995840B (en) * | 2017-03-20 | 2020-05-05 | 山东师范大学 | Method for detecting activity of thymine DNA glycosylase based on double-signal amplification strategy mediated by cyclic enzyme repair |
-
2017
- 2017-09-30 CN CN201710945583.9A patent/CN107760762B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN107760762A (en) | 2018-03-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107760762B (en) | Fluorescent chemical sensor for detecting DNA adenine methyltransferase and detection method thereof | |
| Huang et al. | Sensitive fluorescent detection of DNA methyltransferase using nicking endonuclease-mediated multiple primers-like rolling circle amplification | |
| HUP0101385A2 (en) | Method for detection of target nucleic acids using for | |
| CN109750088B (en) | TdT-RCA-based sensor and application thereof in DNA methyltransferase detection | |
| CN107937482B (en) | Kit for detecting polynucleotide kinase and detection method thereof | |
| CN105112540B (en) | A kind of active method of detection dnmt rna based on strand displacement amplification and DNAzyme amplification | |
| Du et al. | Terminal deoxynucleotidyl transferase-activated nicking enzyme amplification reaction for specific and sensitive detection of DNA methyltransferase and polynucleotide kinase | |
| CN109161579B (en) | Ligase-based isothermal amplification method and application thereof in polynucleotide kinase detection | |
| CN112326637B (en) | Chemiluminescence biosensor for detecting 5-hydroxymethylcytosine and detection method and application thereof | |
| Hu et al. | A DNA structure-mediated fluorescent biosensor for apurinic/apyrimidinic endonuclease 1 activity detection with ultra-high sensitivity and selectivity | |
| CN105136882B (en) | The detection method of Electrochemical Detection dnmt rna activity based on DNA simulation enzyme induction aniline polymerizations | |
| CN107151694A (en) | The Cascaded amplification strategy of ring mediation is used for highly sensitive detection dnmt rna activity | |
| Xi et al. | A highly sensitive strategy for base excision repair enzyme activity detection based on graphene oxide mediated fluorescence quenching and hybridization chain reaction | |
| Wang et al. | Target-mediated hyperbranched amplification for sensitive detection of human alkyladenine DNA glycosylase from HeLa cells | |
| CN113088557A (en) | Fluorescent chemical sensor for simultaneously detecting multiple DNA glycosylases and detection method and application thereof | |
| Sun et al. | Hybridization-initiated exonuclease resistance strategy for simultaneous detection of multiple microRNAs | |
| CN109251960A (en) | Detection Dam methyl transferase activity method based on base excision repair induction | |
| CN109988822B (en) | Sensor and method for detecting hAAG through controllable autocatalysis cleavage mediated fluorescence recovery | |
| Ma et al. | A sensitive strategy for the fluorescence detection of DNA methyltransferase activity based on the graphene oxide platform and T7 exonuclease-assisted cyclic signal amplification | |
| Zhang et al. | Base excision repair mediated cascading triple-signal amplification for the sensitive detection of human alkyladenine DNA glycosylase | |
| CN110923314A (en) | Primer group for detecting SNP locus rs9263726, crRNA sequence and application thereof | |
| CN107083437B (en) | Method for simultaneously detecting multiple DNA glycosylases by using inherent fluorescent nucleotide ultrasensitiveness | |
| Cheng et al. | Rolling circle transcription and CRISPR/Cas12a-assisted versatile bicyclic cascade amplification assay for sensitive uracil-DNA glycosylase detection | |
| CN112094889A (en) | Chemiluminescence sensor and application in detection of bacterial or human methyltransferase | |
| CN114250272B (en) | Fluorescent biosensor based on CRISPR and application of fluorescent biosensor in DNA glycosylase detection |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |