CN107760762A - A kind of fluorescence chemical sensor and its detection method of detection DNA adenine methyltransferases - Google Patents
A kind of fluorescence chemical sensor and its detection method of detection DNA adenine methyltransferases Download PDFInfo
- Publication number
- CN107760762A CN107760762A CN201710945583.9A CN201710945583A CN107760762A CN 107760762 A CN107760762 A CN 107760762A CN 201710945583 A CN201710945583 A CN 201710945583A CN 107760762 A CN107760762 A CN 107760762A
- Authority
- CN
- China
- Prior art keywords
- dam
- transmethylases
- detection
- dna
- fluorescence chemical
- 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.)
- Granted
Links
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
Abstract
The invention discloses a kind of fluorescence chemical sensor of detection Dam transmethylases to include：Dam transmethylases detection probe and assist probes；The Dam transmethylases detection probe is the hair clip DNA probe of a tool loop-stem structure, and two stem structures are complementarily shaped to double-stranded DNA, and the double-stranded DNA has the recognition site of Dam transmethylases；The hair clip DNA probe 3' ends use amido modified；The recognition site of the Dam transmethylases is 5'G A T C 3' palindromic sequence；The assist probes are the rich T single-stranded DNA sequences that a nearly 3' end has AP site (AP site).Prepared by fluorescence chemical sensor of the present invention and detection method is simple, greatly improves detection sensitivity while effectively prevents the generation of nonspecific reaction, may be implemented in the accurate detection to Dam transmethylases in complex biological sample.
Description
Technical field
The invention belongs to bioassay technique field, and in particular to based on one kind based on oversubscription branched amplification zero background inspection
Survey the fluorescence chemical sensor and its detection method of DNA adenine methyltransferases.
Background technology
DNA methylation is the most projecting form of epigenetics gene regulation, is bred in cell, genetic transcription and aging
In play a crucial role.Using SAMe (SAM) as methyl donor, by DNA methyl transferase catalytics, identifying
The covalent addition methyl group of adenine/cytosine residues is targetted in sequence.C-5/ in cytimidine generally occurs for DNA methylation
N-4 positions and the N-6 positions of adenine.Excessive first is found that in polytype cancer such as breast cancer, oophoroma and lung cancer
Base and shortage methylate phenomenon.And the exception of DNA methylation is related to the unconventionality expression and activity of dnmt rna.Cause
This, DNA transmethylases also have become the target spot in clinical diagnosis and drug screening.Also therefore, therefore, carry out to DNA
The super sensitivity detection of methylated transferase not only has very great help to the further investigation of basic biochemistry, while to developing people
The new treatment method and strategy of class disease are significant.
So far, radioactive label method, efficient liquid phase are included for the traditional detection method of DNA methylation transferase
Chromatogram (HPLC), based on immune analysis method and gel electrophoresis.Radiolabeled substrate is used in these methods, specifically
Property antibody, expensive instrument this not only adds experimental cost and adds the complexity of experiment, wasted time and energy, sensitive
Spend low.
In order to overcome these problems, in recent years, related researcher has been introduced into colorimetric method, fluorimetry and life
Thing luminescent assays, these methods have the advantages of directly perceived, safe, simple, high sensitivity.However, they generally require it is cumbersome
Nano particle prepare, carry out external label dependent on fluorogen and quencher, analysis time length, be related to complicated set
Count and costly.Electrochemical method fast response time, design cost is relatively low, but complicated electrode face finish limits it
Application.The method for introducing nucleic acid amplification substantially increases the sensitivity of detection, but existing nucleic acid amplification usually requires
DNA profiling, the specific identification sequence of endonuclease and the fluorescence labeling of nucleic acid primer, this all increases conceptual design
The complexity of difficulty and experimental implementation.Therefore, there is an urgent need to develop it is a kind of it is simple to operate, without fluorescence labeling, zero background
The method for being used for dnmt rna in Sensitive Detection complex biological sample of signal.
The content of the invention
For above-mentioned the deficiencies in the prior art, inventor is through long-term technology and practical exploration, there is provided one kind detection DNA
The fluorescence chemical sensor of adenine methyltransferase (Dam transmethylases), the sensor are based on terminal enzyme (DNA) (TDT)
Do not need any DNA profiling that catalysis deoxyadenosine triphosphate (dATPs) can be achieved to repeat to add the 3'- hydroxyls end of DNA molecular
The characteristics of holding (3'-OH), being capable of complete depurination pyrimidine in specific recognition hydrolysis double-stranded DNA with reference to endonuclease IV
(AP) site, can be real without fluorescence labeling so as to expose the inspection policies that the 3'- hydroxyls of DNA molecular realize hyperbranched amplification
Easy to operate, quick now to the super sensitivity detection of DNA adenine methyltransferases, result of the test is accurate, reliable.
Specifically, the present invention relates to following technical scheme：
The first aspect of the invention, there is provided a kind of fluorescence chemical sensor of detection Dam transmethylases, it is described
Fluorescence chemical sensor includes：Dam transmethylases detection probe and assist probes；
Wherein, the Dam transmethylases detection probe be one tool loop-stem structure hair clip DNA probe, two stem knots
Structure is complementarily shaped to double-stranded DNA, and the double-stranded DNA has the recognition site of Dam transmethylases；The hair clip DNA probe 3' ends
End use is amido modified, so as to prevent TdT (terminal enzyme (DNA)) from activating non-specific amplification；
Specifically, the recognition site of the Dam transmethylases is 5'-G-A-T-C-3' palindromic sequence；
The assist probes are the rich T single stranded DNAs sequence that a nearly 3' end has AP site (AP site)
Row, the assist probes 3' ends are using amido modified, so as to prevent TdT (terminal enzyme (DNA)) from activating non-specific amplification；
The fluorescence chemical sensor also includes relying on restriction endonuclease DpnI, terminal enzyme (DNA) (TdT), the nucleic acid to methylate
Restriction endonuclease IV and deoxyadenosine triphosphate (dATPs), the restriction endonuclease DpnI to methylate that relies on can be identified and be sheared Dam
Transmethylase methylate after Dam transmethylase detection probes so that the detection probe be decomposed into three it is single-stranded
DNA fragmentation, two of which Single-stranded DNA fragments contain free 3'-OH ends, under terminal enzyme (DNA) (TdT) effect, three phosphorus
Sour desoxyadenossine is added in the Single-stranded DNA fragments with 3'-OH ends and obtains the DNA fragmentation with rich A sequences, the tool
There are the DNA fragmentations of rich A sequences and assist probes hybridization to be complementarily shaped to stable double-stranded DNA, it is de- in the double-stranded DNA
Purine pyrimidine site (AP site) is catalyzed by endonuclease IV (Endo IV), is caused the fracture of assist probes and is produced free
3'-OH ends.Novel DNA fragments with free 3'-OH ends trigger the extension of new terminal enzyme (DNA) (TdT) mediation anti-
Should be to form longer rich A sequences.It is worth noting that, excessive assist probes hybridize with rich A sequences, trigger cutting extension
New circulation, induce hyperbranched amplification, produce a large amount of DNA fragmentations；The DNA fragmentation of obtained varying number is indicated by fluorescence
Agent produces different fluorescent values, so as to realize the measure to Dam methyl transferase activities.
Preferably, the Dam transmethylases detection probe length is 37nt, the Dam transmethylases detection probe
Base sequence be：5'-GAAGGA TCT TCT CGA CTT GCT GAA GAT CCT TCT TAA T-NH2- 3', wherein
The base sequence GATC that underscore the marks i.e. recognition site of Dam transmethylases, the Dam transmethylases detection probe
3' ends carry out amido modified.
Preferably, the assist probes length is 26nt, and the base sequence of the assist probes is：5'-TTT TTT TTT
TTT TTT TTT TTX TTT TT-NH2- 3', wherein X represent AP site, and the assist probes 3' ends carry out ammonia
Base is modified；
Preferably, the fluorescence chemical sensing also includes fluorescence indicator, and the fluorescence indicator is SYBR Gold；
Preferably, the fluorescence chemical sensing also includes Dam transmethylase reaction buffers, the Dam methyl transfer
Enzyme reaction buffer solution includes：50 mMs of every liter of Tris-HCl buffer solutions, 10 mMs of every liter of EDTA, 5 mMs of every liter of 2- mercaptos
Base ethanol, pH 7.5；
Preferably, the fluorescence chemical sensing also includes terminal enzyme (DNA) buffer solution, the terminal enzyme (DNA) buffer solution bag
Include：50 mMs of every liter of potassium acetates, 20 mMs of every liter of Tris-Ac, 10 mMs of every liter of magnesium acetates, pH 7.9；
The invention also discloses the method that the fluorescence chemical sensor is used for the detection of Dam transmethylases, specific bag
Include：
1) testing sample is added in reaction solution I and carries out incubation reaction, then carry out high-temperature inactivation processing；
2) add reaction solution II into the solution after the processing of step 1) high-temperature inactivation and carry out polymerisation；
3) fluorescence chemical detection is carried out to the reacted solution of step 2), realized to Dam transmethylases in testing sample
Quantitative analysis.
Wherein, step 1) the reaction solution I includes Dam transmethylase detection probes, S- adenosine METs,
Restriction endonuclease Dpn I and Dam transmethylase reaction buffers；
Incubation reaction condition is in step 1)：37 DEG C, incubation time is 1.5~3h (being preferably 2h)；High-temperature inactivation processing
Temperature is 80 DEG C, and processing time is 10~30min (being preferably 20min)；
Reaction solution II described in step 2) includes assist probes, deoxyadenosine triphosphate, terminal enzyme (DNA) (TdT), core
Sour restriction endonuclease IV, cobaltous dichloride and terminal enzyme (DNA) buffer solution and SYBR Gold；
Reaction condition is in step 2)：37 DEG C, the reaction time is 60~200min (being preferably 100min)；
Real-time PCR Real_time quantitative detection fluorescence intensity is used in step 3).
The invention also discloses above-mentioned fluorescence chemical sensor and/or detection method in quantitative detection Dam transmethylases
And/or the application in screening Dam methyltransferase inhibitors/activator.
The principle of fluorescence chemical sensor detection method of the present invention is：The present invention is based on the branched amplification zero of oversubscription
The fluorescent method of background detection dnmt rna.
Inventor devises a kind of hair clip DNA probe with 5'-G-A-T-C-3' palindromic sequences as substrate.In order to
The non-specific amplification for preventing TdT (terminal enzyme (DNA)) from activating, has used hairpin probe amido modified and the 3' of assist probes ends
End.After being handled with Dam transmethylases, the 5'-G-A-T-C-3' sequences in hair clip DNA probe stem are methylated, and obtain 5'-
G-mA-T-C-3'.The hair clip DNA probe to methylate is then sheared by the endonuclease DpnI of DNA methylation dependence,
Three Single-stranded DNA fragments are discharged, two of which contains free 3'-OH ends.
In the presence of TdT (terminal enzyme (DNA)), multiple dATPs (deoxyadenosine triphosphate) are sequentially added to single-stranded
3'-OH ends free DNA are to obtain rich A sequences.Rich T-sequence assist probes with AP site (are repaiied 3' ends by amino
Decorations) hybridize with gained richness A sequences to form stable double-stranded DNA.AP site in double-stranded DNA is by Endo IV (endonucleases
Enzyme IV) catalysis, cause the fracture of assist probes and produce free 3'-OH ends.New DNA pieces with free 3'-OH ends
Section can trigger the extension that new TdT (terminal enzyme (DNA)) is mediated to form longer rich A sequences.It is worth noting that,
Excessive assist probes hybridize with rich A sequences, trigger the new circulation of cutting extension, induce hyperbranched amplification, produce a large amount of DNA
Fragment.The DNA fragmentation of obtained varying number can be used as indicator by SYBR Gold and produce different fluorescent values.And
In the case of not having Dam transmethylases, the extension of TdT (terminal enzyme (DNA)) mediations and Endo IV (endonuclease IV) are situated between
The cutting for the assist probes led can not all start, and would not observe that obvious fluorescence.
Beneficial effects of the present invention are as follows：
(1) existing method introduces multiple nucleic acids amplification method to improve sensitivity, but these methods usually require
Specific endonuclease enzyme recognition sequence, add the complexity of DNA probe design.And present invention utilizes terminal enzyme (DNA)
(TDT) do not need any DNA profiling that catalysis deoxyadenosine triphosphate can be achieved to repeat to add the 3'-hydroxyl terminal of DNA molecular
The characteristics of (3'-OH), it greatly simplify the design of DNA probe；
(2) present invention can completely be taken off using endonuclease IV (Endo IV) in specific recognition hydrolysis double-stranded DNA
Purine pyrimidine (AP) site, so as to the 3'- hydroxyls of exposure DNA molecular, continue to expand under TDT catalysis, realize hyperbranched expansion
Increase, substantially increase detection sensitivity；
(3) present invention effectively prevents TdT from activating non-spy by the 3' ends of amido modified hairpin probe and assist probes
Specific amplification, TdT high precision identification result in and could only be expanded in free 3'-OH ends, and Endo IV can only
Complete AP site is hydrolyzed, realizes zero background signal, so as to which the specificity of detection method be greatly improved；
(4) present invention is simple to operate without fluorescence labeling using SYBR Gold as indicator, reduces experimental cost.
To sum up, prepared by fluorescence chemical sensor of the present invention and detection method is simple, without Large expensive instrument and equipment, together
When because we have also carried out careful optimization to each reaction condition in the present invention, therefore in detection process, be greatly improved
Detection sensitivity effectively prevents the generation of nonspecific reaction simultaneously, may be implemented in complex biological sample and Dam methyl is turned
Move the accurate detection of enzyme.Experiment proves that mediate the fluorimetry of target circulation, based on endonuclease with exonuclease
The fluorimetry of enzyme auxiliary signal amplification and double-stranded specific nuclease auxiliary circulation amplification of signal based on transcriptive intermediate
Fluorimetry is compared, and the sensitivity of detection method of the present invention improves 1 order of magnitude, with being believed based on hair clip shape DNA enzymatic
Number amplification fluorimetry with based on DNA enzymatic mediation amplification of signal colorimetric method compare, detection method of the present invention
Sensitivity improves 2 orders of magnitude.
Brief description of the drawings
Fig. 1 is the mechanism schematic diagram that fluorescence chemical sensor of the present invention is used for the detection of Dam transmethylases；
Fig. 2 (A) by PAGE (polyacrylamide gel electrophoresis) analyze Dam to hair clip substrate methylate and it is subsequent
DpnI cutting schematic diagrams, wherein swimming lane M are DNA marker (molecular mass reference)；Swimming lane 1 is hairpin probe (0.5 micromole
Every liter)+Dam transmethylases (20 units per ml)+restriction endonuclease DpnI (50 units per ml)；Swimming lane 2 is
Hair clip substrate (0.5 every liter of micromole)+DpnI (50 units per ml)；Swimming lane 3, hairpin probe (0.5 every liter of micromole)+Dam
Transmethylase (20 units per ml)；Fig. 2 (B) is that the PAGE of hyperbranched amplified production is analyzed, wherein, swimming lane M is DNA
Marker (molecular mass reference)；Swimming lane 1 is in Dam transmethylases (20 units per ml)+restriction endonuclease
Hyperbranched amplified production in the presence of DpnI (50 units per ml)；Swimming lane 2 is that (50 is single in restriction endonuclease DpnI
Every milliliter of position) in the presence of hyperbranched amplified production, Fig. 2 (C) is in Dam transmethylases (20 units per ml)+restricted
Endonuclease DpnI (50 units per ml) while exist and (i.e. experimental group) and only add 50 in the absence of Dam transmethylases
In the case of units per ml restriction endonuclease DpnI (i.e. control group), real-time fluorescence monitors hyperbranched amplification situation.
Fig. 3 (A) is F-F corresponding to the assist probes of various concentrations0Value；Fig. 3 (B) is that TdT is fixed as difference during 8 unit
F-F corresponding to the Endo IV of dosage0Value；The TdT that Fig. 3 (C) is fixed as different amounts during 4 unit for Endo IV dosages is corresponding
F-F0Value；Fig. 3 (D) is F-F corresponding to the dATP of various concentrations0Value；Wherein F and F0It is to exist and in the absence of Dam methyl respectively
Fluorescence intensity during transferase；What error bars represented is the standard deviation for repeating experiment three times.
Fig. 4 (A) is the real-time fluorescence curves obtained by the Dam of the various concentrations amplifications triggered；Fig. 4 (B) is Dam methyl
Shift the linear correlation between the logarithm of fluorescence intensity and its concentration of the enzyme concentration in the range of 0.005~40 units per ml
Property, fluorescence intensity in Fig. 4 (B) is obtained at 100 minutes；What error bars represented is the standard deviation for repeating experiment three times.
Fig. 5 is selective determination of the detection method to Dam transmethylases, and Fig. 5 (A) is in response to Dam methyl
Transferase, M.Sss I transmethylases, the real-time fluorescence of bovine serum albumin(BSA) (BSA) and the only control group of buffer solution are bent
Line；Fig. 5 (B) is the M.Sss I transmethylases in response to Dam transmethylases, the control group of BSA and only buffer solution it is glimmering
The measured value of luminous intensity, the fluorescence intensity in Fig. 5 (B) obtained at 100 minutes, wherein the Dam transmethylases of each group,
M.Sss I transmethylases, the concentration of bovine serum albumin(BSA) (BSA) is 20 units per mls；What error bars represented is three times
Repeat the standard deviation of experiment.
Fig. 6 is the Dam methylated transferases in detection reaction buffer and LB culture mediums；With reaction buffer with 1:10
Dilution proportion LB；What error bars represented is the standard deviation for repeating experiment three times.
Fig. 7 (A) be with Polyacrylamide Gel Electrophoresis 5 FU 5 fluorouracil to TdT and Endo IV activity influence,
Wherein, swimming lane M is DNA marker (molecular mass reference)；Swimming lane 1 is primer (67 every liter of nanomole)；Swimming lane 2 is primer
(67 every liter of nanomole)+assist probes (0.13 every liter of micromole)+TdT (8 unit)；Swimming lane 3 is primer (67 every liter of nanomole)
+ assist probes (0.13 every liter of micromole)+TdT (8 unit)+5 FU 5 fluorouracil (10 every liter of micromole)；Swimming lane 4 is primer (67
Every liter of nanomole)+assist probes (0.13 every liter of micromole)+TdT (8 unit)+Endo IV (4 unit)；Swimming lane 5 is primer
(67 every liter of nanomole)+assist probes (0.13 every liter of micromole)+TdT (8 unit)+Endo IV (4 unit)+5 FU 5 fluorouracil
(10 every liter of micromole), Fig. 7 (B) are the feelings in the presence of every liter of 5 FU 5 fluorouracil of 10 micromole and in the absence of 5 FU 5 fluorouracil
Fluorescence intensity measurements under condition.
Fig. 8 is inhibitory action of the various concentrations 5 FU 5 fluorouracil to Dam methyl transferase activities.
Embodiment
It is noted that described further below is all exemplary, it is intended to provides further instruction to the application.Unless
Otherwise indicated, all technologies used herein and scientific terminology have and the application person of an ordinary skill in the technical field
The identical meanings being generally understood that.
It should be noted that term used herein above is merely to describe embodiment, and be not intended to restricted root
According to the illustrative embodiments of the application.As used herein, unless the context clearly indicates otherwise, otherwise odd number shape
Formula is also intended to include plural form, additionally, it should be understood that, when in this manual use term "comprising" and/or
During " comprising ", it indicates existing characteristics, step, operation, device, component and/or combinations thereof.
As background technology is introduced, prior art has that sensitivity is relatively low, detection side to methylated transferase measure
The various problems such as method is cumbersome, required expensive equipment；
In view of this, in an exemplary embodiment of the invention, there is provided a kind of fluorescence of detection Dam transmethylases
Chemical sensor, the fluorescence chemical sensor include：Dam transmethylases detection probe and assist probes；
Wherein, the Dam transmethylases detection probe be one tool loop-stem structure hair clip DNA probe, two stem knots
Structure is complementarily shaped to double-stranded DNA, and the double-stranded DNA has the recognition site of Dam transmethylases；The hair clip DNA probe 3' ends
End use is amido modified, so as to prevent TdT (terminal enzyme (DNA)) from activating non-specific amplification；
The recognition site of the Dam transmethylases is 5'-G-A-T-C-3' palindromic sequence；
The assist probes are the rich T single stranded DNAs sequence that a nearly 3' end has AP site (AP site)
Row；The assist probes 3' ends are using amido modified, so as to prevent TdT (terminal enzyme (DNA)) from activating non-specific amplification；
The fluorescence chemical sensor also includes relying on restriction endonuclease DpnI, terminal enzyme (DNA) (TdT), the nucleic acid to methylate
Restriction endonuclease IV and deoxyadenosine triphosphate (dATPs)；
In the still another embodiment of the present invention, the Dam transmethylases detection probe length is 37 nt, described
The base sequence of Dam transmethylase detection probes is：5'-GAA GGA TCT TCT CGA CTT GCT GAA GAT CCT
TCT TAAT-NH2The base sequence GATC that -3', wherein underscore the are marked i.e. recognition site of Dam transmethylases, it is described
Dam transmethylase detection probe 3' ends carry out amido modified；
In the still another embodiment of the present invention, the assist probes length is 26nt, the base of the assist probes
Sequence is：The base sequence of the assist probes is：5'-TTT TTT TTT TTT TTT TTT TTX TTT TT-NH2- 3',
Wherein X represents AP site, and the assist probes 3' ends carry out amido modified；
In the still another embodiment of the present invention, the fluorescence chemical sensing also includes fluorescence indicator, the fluorescence
Indicator is SYBR Gold；
In the still another embodiment of the present invention, it is slow that the fluorescence chemical sensing also includes the reaction of Dam transmethylases
Fliud flushing, the Dam transmethylases reaction buffer include：50 mMs of every liter of Tris-HCl buffer solutions, 10 mMs every liter
EDTA, 5 mMs of every liter of 2 mercapto ethanols, pH 7.5；
In the still another embodiment of the present invention, the fluorescence chemical sensing also includes terminal enzyme (DNA) buffer solution, institute
Stating terminal enzyme (DNA) buffer solution includes：50 mMs of every liter of potassium acetates, 20 mMs of every liter of Tris-Ac, 10 mMs of every liter of vinegar
Sour magnesium, pH 7.9；
In the still another embodiment of the present invention, disclose the fluorescence chemical sensor and be used for Dam transmethylases
The method of detection, including：
1) testing sample is added in reaction solution I and carries out incubation reaction, then carry out high-temperature inactivation processing；
2) add reaction solution II into the solution after the processing of step 1) high-temperature inactivation and carry out polymerisation；
3) fluorescence chemical detection is carried out to the reacted solution of step 2), realized to Dam transmethylases in testing sample
Quantitative analysis.
Wherein, step 1) the reaction solution I includes Dam transmethylase detection probes, S- adenosine METs,
Restriction endonuclease Dpn I and Dam transmethylase reaction buffers；
Incubation reaction condition is in step 1)：37 DEG C, incubation time is 1.5~3h (being preferably 2h)；High-temperature inactivation processing
Temperature is 80 DEG C, and processing time is 10~30min (being preferably 20min)；
Reaction solution II described in step 2) includes assist probes, deoxyadenosine triphosphate, terminal enzyme (DNA) (TdT), core
Sour restriction endonuclease IV, cobaltous dichloride and terminal enzyme (DNA) buffer solution and SYBR Gold；
Reaction condition is in step 2)：37 DEG C, the reaction time is 60~200min (being preferably 100min)；
Real-time PCR Real_time quantitative detection fluorescence intensity is used in step 3).
In the still another embodiment of the present invention, above-mentioned fluorescence chemical sensor and/or detection method are disclosed fixed
Application in amount detection Dam transmethylases and/or screening Dam methyltransferase inhibitors/activator.
Explanation is further explained to the present invention by the following examples, but is not construed as limiting the invention.
Embodiment
Experimental method step
The detection of 1.Dam transmethylases：Containing every liter of hairpin probe of 0.5 micromole, 160 every liter of micromole SAM
(S- adenosines MET), 10 units of restriction endonuclease Dpn I, (50 mMs every liter of 1 × DNADam buffer solutions
Tris-HCl buffer solutions, 10 mMs of every liter of EDTA, 5 mMs of every liter of 2 mercapto ethanols, pH 7.5) and varying number Dam
Methylating and cutting for hairpin probe is carried out in 200 microlitres of reactant mixtures of transmethylase.Mixture is incubated at 37 DEG C
2 hours, then inactivated 20 minutes at 80 DEG C.Contain 1 × terminal enzyme (DNA) buffer solution (50 mMs of every liter of acetic acid at 30 microlitres
Potassium, 20 mMs of every liter of Tris-Ac, 10 mMs of every liter of magnesium acetates, pH 7.9), 0.25 mM of every liter of cobaltous dichloride, 1
× SYBR Gold, hyperbranched amplification, 1 mM of every liter of dATPs are carried out in 0.13 every liter of micromole assist probes reaction solution
(deoxyadenosine triphosphate), 4 unit Endo IV (endonuclease IV), 8 unit TDT (terminal enzyme (DNA)) and 4 microlitres methylate
Product.Polymerisation is carried out 100 minutes on the real-time PCRs of Bio-Rad CFX 96 (Bio-Rad, USA) in 37 DEG C,
And fluorescence intensity was monitored with the interval of 30 seconds.
2. gel electrophoresis analysis：1 × tbe buffer liquid (9 mMs of every liter of Tris-HCl, 9 mMs of every liter of boric acid,
0.2 mM of every liter of EDTA, pH7.9) in, using 1 × SYBR Gold as fluorescence indicator, the room temperature under 110V constant voltages
Carry out 12% native polyacrylamide gel electrophoresis (PAGE) 50 minutes.
The selectivity of 3.Dam transmethylases detection：For the selectivity of method researched and proposed, we use
M.SssI transmethylases and bovine serum albumin are as interferases.Carried out with 20 units per ml interferases using the above method
Experiment.
4. Dam transmethylases are detected in bacteriolyze Yeast Cultivation based specimen：Cumulative volume is 200 microlitres and contains 10% bacteriolyze
The sample mixture of yeast culture medium (LB), adds the Dam transmethylases of various concentration, and 0.5 every liter of micromole hair clip is visited
Pin, 160 every liter of micromole SAM (S- adenosines MET), 10 units of restriction endonuclease Dpn I, 1 × Dam reaction are slow
Fliud flushing (50 mMs of every liter of Tris-HCl buffer solutions, 10 mMs of every liter of EDTA, 5 mMs of every liter of 2 mercapto ethanols, pH
7.5), it is incubated at 37 DEG C 2 hours, is then inactivated 20 minutes at 80 DEG C.Dam transmethylases are determined with method as described above
Activity.
5. inhibitor is analyzed：In order to study influence of the 5 FU 5 fluorouracil to Dam methyl transferase activities, various concentration
5 FU 5 fluorouracil, 0.5 every liter of micromole hairpin probe and 1 × Dam reaction buffers (50 mMs of every liter of Tris-HCl bufferings
Liquid, 10 mMs of every liter of EDTA, 5 mMs of every liter of 2 mercapto ethanols, pH 7.5), it is incubated 15 minutes at 37 DEG C.Then exist
20 units per ml Dam transmethylases are added in solution, 50 units per ml restriction endonuclease Dpn I and 160 are micro-
Mole every liter of SAM (S- adenosines MET), inactivated 20 minutes at reacting 2 hours, 80 DEG C at 37 DEG C.Use said procedure point
The activity of analysis measure Dam transmethylases.Relatively movable (RA) of Dam transmethylases is calculated by formula 1：
Wherein F0It is the fluorescence intensity in the case of no Dam transmethylases, Ft is 20 units per mls be present
Fluorescence intensity during Dam transmethylases, FiIt is when 20 units per ml Dam transmethylases and 5 FU 5 fluorouracil be present
Fluorescence intensity.
Interpretation of result is with discussing
1. the experimental verification of the principle of the invention
In order to verify the feasibility of the technical program, we study Dam using native polyacrylamide gel electrophoresis
The methylation procedure (Fig. 2) of transmethylase.In the case of no Dam transmethylases or DpnI, one is only observed
20bp band (swimming lane 2 and 3 in Fig. 2A), shows that hair clip is not sheared.When Dam transmethylases and DpnI all in the presence of, go out
Existing one new 20nt band (Fig. 2A, swimming lane 1), shows to be methylated and shear history.In order to verify TdT activation
Endo IV aid in hyperbranched amplification, and we detect amplified production using non-denaturing polyacrylamide gel.It is every in 20 units
In the presence of milliliter Dam transmethylases and 50 units per ml DpnI, it was observed that obvious amplified production band (Fig. 2 B,
Swimming lane 1), show because hyperbranched amplification generates a large amount of DNA fragmentations.By contrast, in the control of no Dam transmethylases
Amplified production band (Fig. 2 B, swimming lane 2) is not observed in group.In order to further confirm that the Endo IV auxiliary of TdT activation is super
Branched amplification, we have also carried out real-time fluorescence detection (Fig. 2 C).In the presence of Dam transmethylases and Dpn I, fluorescence
Intensity linearly increases (Fig. 2 C) with the time.In the control group of no Dam transmethylases, it was observed that zero background signal (figure
2C).The realization of zero background signal may be attributed to the modification of hairpin probe 3' ends and have amido modified assist probes can
The non-specific amplification that TdT is activated in the case of effectively to prevent no Dam transmethylases.
2. Optimal Experimental condition
In order to obtain optimum efficiency, we optimize assist probes and dATP concentration, and Endo IV and TdT use
Amount.As shown in Figure 3A, with the increase of assist probes concentration, F-F0(F and F0Respectively Dam exist and in the absence of when fluorescence
Intensity) value increase, assist probes concentration reaches platform when being every liter of 0.13 micromole.Therefore, used in subsequent experiment
Assist probes concentration be 0.13 every liter of micromole.Fluorescence signal generation dependent on TdT mediation DNA extension and
The cutting to assist probes of Endo IV mediations, therefore Endo IV and TdT dosage are also required to optimize.We have tested TdT
Dosage is fixed as influence of the Endo IV different during 8 unit dosage to fluorescence signal.As shown in Figure 3 B, with Endo IV
Increase, F-F0(F and F0Respectively Dam exist and in the absence of when fluorescence intensity) value increase, and when dosage is 4 unit
Reach maximum.Then we have studied influences of the TdT to fluorescence signal, now Endo IV dosages are fixed as 4 units.Such as figure
Shown in 3C, TdT is from 4 units to F-F in the section of 8 units0(F and F0Respectively Dam exist and in the absence of when fluorescence intensity)
Value increase with the increase of TDT dosages, F-F during more than 8 unit0Value reduce.Therefore, 4 unit Endo IV and 8 are single
Position TdT is used for subsequent experimental.
We further study dATP influence of the concentration to fluorescence signal.Fig. 3 D are shown, with the increasing of dATP concentration
Add, F-F0(F and F0Respectively Dam exist and in the absence of when fluorescence intensity) value increase, concentration be 1 mM every liter when
Reach maximum.Therefore, 1 mM of every liter of dATP is used in subsequent experiment.
3. sensitivity technique
Under optimum experimental condition, we monitor fluorescence signal caused by the Dam transmethylases of various concentrations in real time.
As shown in Figure 4 A, fluorescence signal is linearly increasing with time dependence and concentration dependant manner.The concentration of Dam transmethylases
Higher, the DNA substrates being methylated are more, then cut by DpnI and caused free 3'-OH ends are more, therefore fluorescence
Signal is higher.In addition, in the range of 4 orders of magnitude of 0.005 to 40 units per ml, fluorescence intensity and the transfer of Dam methyl
Good linear dependence (Fig. 4 B) between the logarithm of enzyme concentration be present.Regression equation is F=2889.8+1164.4log10C,
Coefficient correlation is that 0.991, wherein F and C represent fluorescence intensity and Dam transmethylases concentration (units per ml) respectively.It is logical
Cross and calculate the average response value of blank and add three times standard deviation to substitute into linear equation, can be detected that to be limited to 0.003 unit every
Milliliter.It is worth noting that, mediate the fluorimetry (0.01 units per ml) of target circulation with exonuclease, be based on
The fluoremetry (0.06 units per ml) of endonuclease auxiliary signal amplification and the double-stranded specific core based on transcriptive intermediate
The fluorimetry (0.015 units per ml) of sour enzyme auxiliary circulation amplification of signal is compared, and the sensitivity of this method improves 1
The individual order of magnitude, mediated with the fluorimetry (0.4 units per ml) based on hair clip shape DNA enzymatic amplification of signal and based on DNA enzymatic
The colorimetric method (0.25 units per ml) of amplification of signal is compared, and the sensitivity of this method improves 2 orders of magnitude.Important
It is that the method that this invention proposes is very simple, it is not necessary to designs the unique identification sequence of any endonuclease, and table
Revealing excellent specificity, background signal is almost nil i.e. in the presence of no object Dam transmethylases.Sensitivity carries
Height is attributable to following three factors：(1) TdT activates the hyperbranched amplification induced fluorescence signal enhancing of Endo IV auxiliary；(2)
TdT high precision identification be result in and could only be expanded in free 3'-OH ends, and Endo IV can only be hydrolyzed completely
AP site, realize zero background signal；(3) effectively prevented by the 3' ends of amido modified hairpin probe and assist probes
TdT activates non-specific amplification.
4. selective enumeration method
In order to study the selectivity of detection method, we use M.SssI transmethylases and BSA as interference
Enzyme.M.SssI transmethylases can in the 5'-C-G-3' recognition sequences of double-stranded DNA specific methylation cytosine residues,
And BSA is a kind of incoherent protein.As shown in Figure 5A, there are 20 units per ml Dam transmethylases in fluorescence signal
When increase in a manner of time dependence, and 20 units per ml M.Sss I transmethylases, 20 units per ml BSA and
Fluorescence signal is not observed in the presence of control group.In addition, it is far above in response to the fluorescence intensity of Dam transmethylases
In response to M.Sss I transmethylases, the fluorescence intensity (Fig. 5 B) of BSA and control group.This can be with by the fact that to solve
Release：5'-G-A-T-C-3' unique identification sequence can only be carried out by Dam transmethylases rather than M.Sss I and BSA
Methylate.These results clearly demonstrate that the method for proposition has fabulous selectivity to Dam transmethylases.
5. Dam transmethylases are detected in bacteriolyze yeast culture medium
In order to further verify feasibility of this method in actual sample analysis, we have detected Dam transmethylases
Activity in the complex sample containing 10% bacteriolyze yeast (LB) culture medium.As shown in fig. 6, Dam transmethylases are diluting
Bacteriolyze Yeast Cultivation based specimen in response fluorescence intensity it is consistent with its response in reaction buffer, 0.5 unit is every
The rate of recovery of milliliter and 20 units per ml Dam transmethylases is respectively 99.8% ± 4.62% and 103.8% ±
4.18%.These results indicate that the method proposed has the great potential further applied in complex biological sample.
6. inhibitor is analyzed
Dam transmethylases play an important role in the toxicity of increasing bacterial pathogens, it has also become antibacterials
The target of development.In order to prove the feasibility of proposed Inhibition test method, we suppress by model of 5 FU 5 fluorouracil
Agent.Existing research shows that the 5 FU 5 fluorouracil less than every liter of concentration of 10 micromole does not influence on DpnI activity.We
The method detected using gel electrophoresis analysis and real-time fluorescence, influence of the research 5- fluorouracils to TDT and Endo IV activity.
As shown in Figure 7 A, only exist in TDT system containing every liter of 5 FU 5 fluorouracil of 10 micromole (Fig. 7 A, swimming lane 2) and without 5-
During fluorouracil (Fig. 7 A, swimming lane 3), it was observed that two swimming bands of reflection TdT mediated amplification products do not have significant difference, show
10 every liter of micromole 5 FU 5 fluorouracils do not influence on TdT activity.In addition, in TDT and Endo IV existing system
In, during containing every liter of 5 FU 5 fluorouracil of 10 micromole (Fig. 7 A, swimming lane 4) and without 5 FU 5 fluorouracil (Fig. 7 A, swimming lane 5), observation
Two swimming bands for aiding in hyperbranched amplified production to reflection TdT activation Endo IV do not have notable difference, show that 10 micromoles are every
Rise 5 FU 5 fluorouracil does not influence on Endo IV activity.In addition, obtained in the presence of every liter of 5 FU 5 fluorouracil of 10 micromole
Fluorescence intensity (Fig. 7 B) and the fluorescence intensity no significant difference obtained during 5 FU 5 fluorouracil (Fig. 7 B) is not present, show no matter
When only existing TdT or TDT and Endo IV simultaneously be present, activity of 10 every liter of the micromole 5 FU 5 fluorouracils to TDT and Endo IV
Have not significant impact.These results show that 5 FU 5 fluorouracil does not influence DpnI, TdT and Endo IV enzymatic activity.
Fig. 8 shows influence of the 5 FU 5 fluorouracil to Dam methyl transferase activities.With the increase of 5 FU 5 fluorouracil concentration,
Dam transmethylase relative activities gradually reduce.IC50Value is that enzymatic activity is reduced to the concentration of the inhibitor needed for 50%.For
5 FU 5 fluorouracil, calculate to obtain IC50It is worth for 1.29 every liter of micromoles, with the primer generation rolling circle amplification (PG- based on hairpin probe
RCA) the IC of (1.42 ± 0.07 μM) acquisitions of the chemiluminescence assay of induction50Value is consistent.These results indicate that proposed
Method can be used for screening Dam methyltransferase inhibitors.
The preferred embodiment of the application is the foregoing is only, is not limited to the application, for the skill of this area
For art personnel, the application can have various modifications and variations.It is all within spirit herein and principle, made it is any
Modification, equivalent substitution, improvement etc., should be included within the protection domain of the application.
SEQUENCE LISTING
<110>Shandong Normal University
<120>A kind of fluorescence chemical sensor and its detection method of detection DNA adenine methyltransferases
<130>
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 37
<212> DNA
<213>It is artificial synthesized
<400> 1
gaaggatctt ctcgacttgc tgaagatcct tcttaat 37
<210> 2
<211> 26
<212> DNA
<213>It is artificial synthesized
<400> 2
tttttttttt tttttttttt xttttt 26
Claims (10)
- A kind of 1. fluorescence chemical sensor of detection Dam transmethylases, it is characterised in that the fluorescence chemical sensor bag Include：Dam transmethylases detection probe and assist probes；Wherein, the Dam transmethylases detection probe is the hair clip DNA probe of a tool loop-stem structure, and two stem structures are mutual Benefit forms double-stranded DNA, and the double-stranded DNA has the recognition site of Dam transmethylases；The hair clip DNA probe 3' ends make With amido modified；The recognition site of the Dam transmethylases is 5'-G-A-T-C-3' palindromic sequence；The assist probes have the rich T single-stranded DNA sequences of AP site, the assist probes for a nearly 3' end 3' ends use amido modified；The fluorescence chemical sensor also include rely on methylate restriction endonuclease DpnI, terminal enzyme (DNA), endonuclease IV and Deoxyadenosine triphosphate.
- 2. a kind of fluorescence chemical sensor as claimed in claim 1, it is characterised in that the Dam transmethylases detection is visited Pin length is 37nt, and the base sequence of the Dam transmethylases detection probe is：5'-GAA GGA TCT TCT CGA CTT GCT GAA GAT CCT TCT TAA T-NH2-3'；The Dam transmethylases detection probe 3' ends carry out amido modified.
- A kind of 3. fluorescence chemical sensor as claimed in claim 1, it is characterised in that the assist probes length is 26nt, The base sequence of the assist probes is 5'-TTT TTT TTT TTT TTT TTT TTX TTT TT-NH2- 3', wherein X generations Table AP site, the assist probes 3' ends carry out amido modified.
- 4. a kind of fluorescence chemical sensor as claimed in claim 1, it is characterised in that the fluorescence chemical sensing also includes glimmering Light indicator, it is preferred that the fluorescence indicator is SYBR Gold.
- 5. a kind of fluorescence chemical sensor as claimed in claim 1, it is characterised in that the fluorescence chemical sensing also includes Dam transmethylase reaction buffers, the Dam transmethylases reaction buffer include：50 mMs of every liter of Tris-HCl Buffer solution, 10 mMs of every liter of EDTA, 5 mMs of every liter of 2 mercapto ethanols, pH 7.5.
- 6. a kind of fluorescence chemical sensor as claimed in claim 1, it is characterised in that the fluorescence chemical sensing also includes end End transfer enzyme buffer liquid, the terminal enzyme (DNA) buffer solution include：50 mMs of every liter of potassium acetates, 20 mMs of every liter of Tris- Ac, 10 mMs of every liter of magnesium acetates, pH 7.9.
- 7. any one of the claim 1-5 fluorescence chemical sensors are used for the method for Dam transmethylases detection, its feature exists In step includes：1) testing sample is added in reaction solution I and carries out incubation reaction, then carry out high-temperature inactivation processing；2) add reaction solution II into the solution after the processing of step 1) high-temperature inactivation and carry out polymerisation；3) fluorescence chemical detection is carried out to the reacted solution of step 2), realizes and Dam transmethylases in testing sample are determined Amount analysis.
- 8. a kind of detection method as claimed in claim 7, it is characterised in that step 1) the reaction solution I includes Dam first Based transferase detection probe, S- adenosine METs, restriction endonuclease Dpn I and Dam transmethylases reaction buffering Liquid；Incubation reaction condition is in step 1)：37 DEG C, incubation time is 1.5~3h (being preferably 2h)；High-temperature inactivation treatment temperature For 80 DEG C, processing time is 10~30min (being preferably 20min).
- 9. a kind of detection method as claimed in claim 7, it is characterised in that reaction solution II described in step 2) includes auxiliary Probe, deoxyadenosine triphosphate, terminal enzyme (DNA), endonuclease IV, cobaltous dichloride and terminal enzyme (DNA) buffer solution and SYBR Gold。
- 10. any one of any one of the claim 1-6 fluorescence chemical sensors and/or claim 7-9 detection method In application of the quantitative detection in quantitative detection Dam transmethylases and/or screening Dam methyltransferase inhibitors/activator.
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 true CN107760762A (en) | 2018-03-06 |
| CN107760762B 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) |
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109251960A (en) * | 2018-03-27 | 2019-01-22 | 兰州大学 | Detection Dam methyl transferase activity method based on base excision repair induction |
| CN109270144A (en) * | 2018-10-31 | 2019-01-25 | 山东师范大学 | A method of the electrochemistry magnetic biosensor based on non-marked, on-fixed detects 5-hydroxymethyl cytosine |
| 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 |
| CN109613096A (en) * | 2019-01-21 | 2019-04-12 | 宁波大学 | DNA relevant enzyme electrochemica biological sensor and its application based on the building of DNA- copper nano-cluster |
| CN109750088A (en) * | 2019-03-22 | 2019-05-14 | 重庆医科大学 | Sensor based on TdT-RCA and its application in dnmt rna detection |
| CN110144384A (en) * | 2019-06-03 | 2019-08-20 | 山东师范大学 | The fluorescence chemical sensor and its detection method of a kind of test side intragranular oxidative damage and application |
| CN110609020A (en) * | 2019-08-15 | 2019-12-24 | 济南大学 | Biosensor for detecting ATP based on palindromic molecular beacon and preparation method and application thereof |
| CN110646487A (en) * | 2019-10-14 | 2020-01-03 | 宁波大学 | Electrochemical method based on G4 stacked self-assembly and application of electrochemical method in hydrogen peroxide and terminal transferase detection |
| CN111088324A (en) * | 2019-12-30 | 2020-05-01 | 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 |
| CN112226484A (en) * | 2020-10-27 | 2021-01-15 | 山东师范大学 | Fluorescent biosensor of beta-glucose transferase, detection method and application |
| 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 |
| CN112852922A (en) * | 2021-01-14 | 2021-05-28 | 山东师范大学 | Fluorescent biosensor for detecting DNA methylation, detection method and application |
| CN113652473A (en) * | 2021-06-30 | 2021-11-16 | 山东师范大学 | Fluorescent chemical sensor for single-molecule detection of DNA damage sites, method and application |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102816855A (en) * | 2012-09-03 | 2012-12-12 | 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 |
| 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 |
| 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 |
| CN103993083A (en) * | 2014-05-22 | 2014-08-20 | 东南大学 | Method for detecting activity of DNA methylase and DNA methyltranseferase by unlabeled fluorescent detection based on restriction endonuclease and exonuclease III |
| CN104212792A (en) * | 2014-04-22 | 2014-12-17 | 上海大学 | Nicking endonuclease-based netted rolling cycle amplification system and use thereof |
| CN105112540A (en) * | 2015-09-17 | 2015-12-02 | 山东大学 | Method for detecting DAN methyltransferase activity based on strand displacement amplification and DNAzyme amplification |
| CN105506078A (en) * | 2015-12-18 | 2016-04-20 | 山东大学 | Method for parallel determination of activity of uracil-DNA glycosylase and endonuclease IV, application thereof and reagent kit |
| CN106995840A (en) * | 2017-03-20 | 2017-08-01 | 山东师范大学 | A kind of method of the dual signal amplification strategy detection thymidine DNA glycosylase activity mediated based on cyclophorase reparation |
-
2017
- 2017-09-30 CN CN201710945583.9A patent/CN107760762B/en active Active
Patent Citations (9)
| 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 |
| CN102816855A (en) * | 2012-09-03 | 2012-12-12 | 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 |
| CN103993083A (en) * | 2014-05-22 | 2014-08-20 | 东南大学 | Method for detecting activity of DNA methylase and DNA methyltranseferase by unlabeled fluorescent detection based on restriction endonuclease and exonuclease III |
| 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 |
| CN105112540A (en) * | 2015-09-17 | 2015-12-02 | 山东大学 | Method for detecting DAN methyltransferase activity based on strand displacement amplification and DNAzyme amplification |
| CN105506078A (en) * | 2015-12-18 | 2016-04-20 | 山东大学 | Method for parallel determination of activity of uracil-DNA glycosylase and endonuclease IV, application thereof and reagent kit |
| CN106995840A (en) * | 2017-03-20 | 2017-08-01 | 山东师范大学 | A kind of method of the dual signal amplification strategy detection thymidine DNA glycosylase activity mediated based on cyclophorase reparation |
Non-Patent Citations (6)
| Title |
|---|
| CHANGBEI MA等: "Label-free monitoring of DNA methyltransferase activity based on terminal deoxynucleotidyl transferase using a thioflavin T probe", 《MOLECULAR AND CELLULAR PROBES》 * |
| LI-JUAN WANG: "Excision Repair-Initiated Enzyme-Assisted Bicyclic Cascade Signal Amplification for Ultrasensitive Detection of Uracil-DNA Glycosylase", 《ANAL. CHEM》 * |
| XI-WEN XING等: "Sensitive Detection of DNA Methyltransferase Activity Based on", 《ANAL. CHEM.》 * |
| YONGXI ZHAO等: "Isothermal Amplification of Nucleic Acids", 《CHEM. REV.》 * |
| 汪谦: "《现代医学实验方法》", 31 August 1997, 人民卫生出版社 * |
| 金晶等: "《分子诊断学实验指导》", 31 August 2015, 中国医药科技出版社 * |
Cited By (22)
| 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 |
| CN109251960A (en) * | 2018-03-27 | 2019-01-22 | 兰州大学 | Detection Dam methyl transferase activity method based on base excision repair induction |
| CN109270144A (en) * | 2018-10-31 | 2019-01-25 | 山东师范大学 | A method of the electrochemistry magnetic biosensor based on non-marked, on-fixed detects 5-hydroxymethyl cytosine |
| 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 |
| CN109613096A (en) * | 2019-01-21 | 2019-04-12 | 宁波大学 | DNA relevant enzyme electrochemica biological sensor and its application based on the building of DNA- copper nano-cluster |
| CN109750088A (en) * | 2019-03-22 | 2019-05-14 | 重庆医科大学 | Sensor based on TdT-RCA and its application in dnmt rna detection |
| CN110144384A (en) * | 2019-06-03 | 2019-08-20 | 山东师范大学 | The fluorescence chemical sensor and its detection method of a kind of test side intragranular oxidative damage and application |
| 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 |
| CN110609020A (en) * | 2019-08-15 | 2019-12-24 | 济南大学 | Biosensor for detecting ATP based on palindromic molecular beacon and preparation method and application thereof |
| CN110646487A (en) * | 2019-10-14 | 2020-01-03 | 宁波大学 | Electrochemical method based on G4 stacked self-assembly and application of electrochemical method in hydrogen peroxide and terminal transferase detection |
| CN111088324A (en) * | 2019-12-30 | 2020-05-01 | 山东师范大学 | Single quantum dot nano sensor for detecting methyltransferase and detection method and application thereof |
| 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 |
| CN112226484A (en) * | 2020-10-27 | 2021-01-15 | 山东师范大学 | Fluorescent biosensor of beta-glucose transferase, detection method and application |
| CN112226484B (en) * | 2020-10-27 | 2022-08-26 | 山东师范大学 | Fluorescent biosensor of beta-glucose transferase, detection method and application |
| CN112852922A (en) * | 2021-01-14 | 2021-05-28 | 山东师范大学 | Fluorescent biosensor for detecting DNA methylation, 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 |
| CN113652473A (en) * | 2021-06-30 | 2021-11-16 | 山东师范大学 | Fluorescent chemical sensor for single-molecule detection of DNA damage sites, method and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107760762B (en) | 2020-05-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107760762A (en) | A kind of fluorescence chemical sensor and its detection method of detection DNA adenine methyltransferases | |
| Huang et al. | Sensitive fluorescent detection of DNA methyltransferase using nicking endonuclease-mediated multiple primers-like rolling circle amplification | |
| CN104372076B (en) | Notch and extension amplified reaction for nucleic acid exponential amplification | |
| JP3437816B2 (en) | Methods for measuring nucleic acids | |
| HUP0101385A2 (en) | Method for detection of target nucleic acids using for | |
| CN105112540B (en) | A kind of active method of detection dnmt rna based on strand displacement amplification and DNAzyme amplification | |
| CN103614489B (en) | Constant-temperature amplification detection kit for dengue viruses and detection method | |
| JP2010533494A5 (en) | ||
| JP6126381B2 (en) | Target nucleic acid detection method and kit | |
| CN106995840A (en) | A kind of method of the dual signal amplification strategy detection thymidine DNA glycosylase activity mediated based on cyclophorase reparation | |
| CN109750088B (en) | TdT-RCA-based sensor and application thereof in DNA methyltransferase detection | |
| Du et al. | Terminal deoxynucleotidyl transferase-activated nicking enzyme amplification reaction for specific and sensitive detection of DNA methyltransferase and polynucleotide kinase | |
| CN107937482A (en) | A kind of kit and its detection method for detecting polynucleotide kinase | |
| Que et al. | Terminal deoxynucleotidyl transferase and rolling circle amplification induced G-triplex formation: A label-free fluorescent strategy for DNA methyltransferase activity assay | |
| Cheng et al. | Label-free and sensitive detection of T4 polynucleotide kinase activity via coupling DNA strand displacement reaction with enzymatic-aided amplification | |
| Wang et al. | Cytosine-5 methylation-directed construction of a Au nanoparticle-based nanosensor for simultaneous detection of multiple DNA methyltransferases at the single-molecule level | |
| CN107151694A (en) | The Cascaded amplification strategy of ring mediation is used for highly sensitive detection dnmt rna activity | |
| Huang et al. | Detection and quantification of RNA 2′-O-methylation and pseudouridylation | |
| CN109251960A (en) | Detection Dam methyl transferase activity method based on base excision repair induction | |
| CN106460071B (en) | Compositions and methods for enhancing and/or predicting DNA amplification | |
| Ma et al. | Label-free monitoring of DNA methyltransferase activity based on terminal deoxynucleotidyl transferase using a thioflavin T probe | |
| WO2013154138A1 (en) | Method for detecting target nucleic acid using molecular beacon-type probe | |
| GB2401175A (en) | Detection of SARS virus by PCR | |
| Hu et al. | Discrimination between 5-hydroxymethylcytosine and 5-methylcytosine in DNA by selective chemical labeling | |
| CN104726548A (en) | DNA, RNA or protein detection probe, detection method and kit based on hybridization chain reaction |
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 |