CN104630363A - Method for detecting activity of uracil-DNA glycosylase (UDG) based on fluorescence amplification strategy of label-free non-enzyme DNA machines - Google Patents
Method for detecting activity of uracil-DNA glycosylase (UDG) based on fluorescence amplification strategy of label-free non-enzyme DNA machines Download PDFInfo
- Publication number
- CN104630363A CN104630363A CN201510061929.XA CN201510061929A CN104630363A CN 104630363 A CN104630363 A CN 104630363A CN 201510061929 A CN201510061929 A CN 201510061929A CN 104630363 A CN104630363 A CN 104630363A
- Authority
- CN
- China
- Prior art keywords
- dna
- chain
- sequence
- udg
- probe
- 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.)
- Pending
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
- C12Q1/682—Signal amplification
-
- 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
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (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)
Abstract
The invention discloses a method for detecting the activity of uracil-DNA glycosylase (UDG) based on a fluorescence amplification strategy of label-free non-enzyme DNA machines, and the method comprises the following steps: (1) the preparation of a probe for the recognition and signal transduction of the UDG: firstly, designing an uracil base and initiation sequence containing double-stranded DNA probe P1-P2, wherein the P1 chain is an inhibition chain and the nucleotide sequence thereof is show in SEQ ID NO.1 in a sequence table; the P2 chain is a uracil-DNA sequence and initiation sequence containing chimeric conjugated chain and the nucleotide sequence thereof is show in SEQ ID NO.2 in the sequence table; and the P1 chain and the P2 chain are partially complemented so as to form the double-stranded DNA probe P1-P2; (2) the construction of a label-free non-enzyme DNA machine: according to an initiation sequence of the P2 chain, designing hairpin probes H1 and H2 which are partially complemented and used for constructing the label-free non-enzyme DNA machine, and grafting a G-quadruplet sequence to the tail end of the hairpin probe H2; and (3) the activity detection of UDG. The method disclosed by the invention successfully realizes background diminishing and signal amplification, and the LOD (limit of detection) is 0.00044 U/mL.
Description
Technical field
The present invention relates to a kind of based on exempting from the method for mark without enzyme dna machine Fluorescence amplification strategy detection uracil-DNA glycosylase activity, belonging to protein urine and quantitative detection field.
Background technology
Uracil-DNA glycosylase (UDG) is a kind of DNA repair enzyme, and it plays keying action in maintenance cellular genome integrity.It is connected to the N-glycosidic link of uridylic and ribodesose by catalytic hydrolysis, removes uridylic damage in DNA.The response that abnormal UDG is active to be damaged T suppression cell uridylic is also directly related with various disease, comprises human immune deficiency disease, lymphoma and facial telangiectasis of dwarfs syndrome.Result of study shows for these diseases, and UDG activity has become a kind of promising biomarker, and is a kind of potential candidate's instrument in UDG functional study and clinical diagnosis to the detection method of UDG activity.
The common method detecting UDG activity is gel electrophoresis, electrochemical process and colorimetry.Except these methods, based on the UDG activity test method of fluorescence, due to it, there is the advantage such as safe, simple and sensitive and cause the extensive concern of investigator.Designed many fluorescent methods for UDG Activity determination at present, they carry out identification and the signal output of target compound with the DNA probe containing uridylic, achieve easy detection UDG active.But the sensitivity for the fluorescent method detecting UDG activity reported is not entirely satisfactory, therefore method for amplifying signal starts to be introduced in UDG active fluoro detection method.It is active for fluoroscopic examination UDG that Yu seminar proposes a kind of DNA enzymatic amplification method based on autocatalysis, and improve the sensitivity of detection, the detection of the method is limited to 0.002U/mL.In addition, Lu and colleague thereof are demonstrated and a kind ofly amplify the detection by quantitative of fluorescence strategy for UDG activity, the inactivation of the DNA enzymatic of being correlated with by UDG-or activation, produce low detection and are limited to 0.0034U/mL.
Although the Fluorescence amplification method of above-mentioned detection UDG activity has achieved good performance, these methods have relied on the fluorescent probe of double-tagging, or/and proteolytic enzyme.For the dual labelled fluorogeilic probe that covalent labeling has fluorophore and quencher group, quencher group may not the fluorescence of quench donor completely, causes high background signal and limit the further raising of detection sensitivity.In addition, proteolytic enzyme is responsive and easy in inactivation to reaction conditions, this can undesired signal amplifying power and cause the problem of detected result poor reproducibility.
DNA machine is supramolecule nucleic acid construct, and when there is suitable primer, it can show the function of similar machine on a molecular scale.DNA machine has been used as switch, ambulacral organ or amplification sensor.Especially, because DNA machine only just can carry out effectively amplifying from advocating peace under simple operations, so they have been used to amplification detection nucleic acid, small molecules and metal ion.But not yet there is at present the Smart fluorescent based on DNA machine to amplify strategy report for the open source literature of UDG Activity determination, therefore, develop a kind of new fluorescence strategy and be used for exempting from marking, be current problem demanding prompt solution without enzyme and Sensitive Detection UDG activity.
Summary of the invention
For above-mentioned the deficiencies in the prior art, the object of this invention is to provide a kind of based on exempting from the method for mark without enzyme dna machine Fluorescence amplification strategy detection uracil-DNA glycosylase activity.The present invention adopts double-stranded DNA (dsDNA) the probe identification UDG target compound the adjoint release causing chain that comprise uridylic base and cause sequence, and this initiation chain can activate exempts from mark without enzyme dna machine, produces the fluorescent signal amplified.Due to dsDNA probe and specific DNA machine design, detection method of the present invention successfully achieves background and reduces and signal amplification, creates low detectability (0.00044U/mL).
Another object of the present invention is to provide a kind of method of the UDG activity detected in cervical cancer (Hela) cell pyrolysis liquid.
For achieving the above object, the present invention adopts following technical proposals:
Marking based on exempting from the method detecting uracil-DNA glycosylase activity without enzyme dna machine Fluorescence amplification strategy, comprising the following steps:
(1) for the preparation of the identification of UDG and the probe of signal transduction:
First design one containing uridylic base and double-stranded DNA (dsDNA) the probe P1-P2 causing sequence, wherein P1 chain is for suppressing chain, and its nucleotide sequence is as shown in SEQ ID NO.1 in sequence table; P2 chain is that its nucleotide sequence is as shown in SEQ ID NO.2 in sequence table containing ura DNA sequence and the chimeric conjugated chain causing sequence; P1 chain and the complementation of P2 chain portion form double-stranded DNA (dsDNA) probe P1-P2;
P1：5’-GAAATTCTTAAGTAGTCAG-3’；
P2:5 '-CGACATCTAACCTAGCTGACTACUUAAGAAUUTC-3 '; (wherein, italicized item is for causing sequence)
(2) exempt to mark the structure without the DNA machine operation part of enzyme:
According to the initiation sequence of P2 chain, hairpin probe H1 and H2 of design two partial complementarity exempts to mark the operation part without the DNA machine of enzyme for building, and the nucleotide sequence of hairpin probe H1 is as shown in SEQ ID NO.3 in sequence table; The nucleotide sequence of hairpin probe H2 is as shown in SEQ ID NO.4 in sequence table; The grafting of G-tetrad (G4) sequence is at the end of hairpin probe H2;
H1:5’-AGTCAGCTAGGTTAGATGTCGCTGACTGGGTAGGGCCGACATCTAACCTAG-3’,
H2:5’-ATGTCGGCCCTACCCAGTCAGCGACATCTAACCTAGCTGACTGGGTAGGGCGGGTTGGG-3’；
(3) Activity determination of UDG:
Double-stranded DNA (dsDNA) probe P1-P2 step (1) prepared and UDG to be detected joins in reaction buffer, the final volume obtaining system is 50 μ L, the concentration of system double center chain DNA (dsDNA) probe P1-P2 is 50-500nM, be preferably 200nM, system hatches 30-180min at 37 DEG C, make base remove reaction to occur, then by the hairpin probe H1 in step (2), grafting has the hairpin probe H2 of G-tetrad sequence to join in the solution after hatching, the final volume obtaining system is 100 μ L, in system, the concentration of hairpin probe H1 is 100-600nM, the concentration of hairpin probe H2 is 400-900nM, at 37 DEG C, DNA machine carries out the iodine of 20-120min, N-methyl porphyrin IX (NMM) of 0.5-20 μ L 200 μMs is added again in reaction system, and 10-120min is hatched at 37 DEG C, be preferably 30min, obtain reaction product, fluorometric assay is carried out to reaction product, builds the linearity curve between fluorescence intensity and UDG concentration, to a certain sample, by the fluorescence intensity of working sample, substitute into linearity curve, calculate UDG concentration.
In step (3), consisting of of described reaction buffer: 20mM Tris-HCl, 1.0mM EDTA, 1.0mM dithiothreitol (DTT) (DTT), pH 8.0.
In step (3), carrying out fluorimetric method to reaction product is: joined by reaction product solution in the quartzy fluorescence pond of 100 μ L, insert in spectrophotofluorometer and carry out fluorometric assay, optimum configurations is: exciting voltage is 700V; Excitation wavelength is 399nm; Emission wavelength is 618nm; Slit width is excited to be 10nm; Transmitting slit width is 10nm.
Of the present inventionly based on exempting from the principle marked without the Fluorescence amplification strategy detection UDG activity of enzyme dna machine be:
As shown in Figure 1, for obtaining gratifying result, the nucleic acid chains used here is by appropriate design for concrete principle.Wherein, P1 is a suppression chain.In addition, P2 is containing ura DNA sequence (black) and the chimeric conjugated chain causing sequence (redness).This single stranded DNA (ssDNA) P2 comprising uridylic base and initiation sequence with suppression chain P1 partial complementarity and then can form P1-P2dsDNA probe.P1-P2dsDNA probe demonstrates relatively high melting temperature(Tm), makes this probe become a kind of stable hybridization and close to cause sequence.According to initiation sequence, hairpin probe H1 and H2 that the present invention devises two partial complementarity exempts to mark the DNA machine without enzyme for building.For avoiding complicated chemical labeling, G-tetrad (G4) sequence is grafted the end (region 4 and region 5) at H2.When H2 exists with hairpin structure, region 4 is hidden in the neck of H2.Therefore, now G4 configuration can not be formed.Under UDG effect, uridylic base is removed by from the deoxidation phosphate backbones of P2, produces without base (AP) site, causes P1-P2dsDNA probe to demonstrate lower melting temperature(Tm), and then cause P2 ' chain to discharge from P1-P2dsDNA probe.P2 ' the chain of release can cause the signal amplification process of DNA machine.Especially, the region 1 of causing in chain P2 ' is exposed, with foothold 1 come-at-able in hair clip H1
*hybridization, and then cause chain transition process and expose 3,4 of hair clip H1,1 region.The complete hybrid H1-P2 ' generated opens H1 and also exposes the foothold made new advances, region 3, it can with 3 of hair clip H2
*region nucleation.As a result, moved by chain, 3,2 of hair clip H2, Isosorbide-5-Nitrae, 5 regions be completely can and.Because H1-H2 mixture is more stable than H1-P2 ' hybrid, so the accessible areas 3,2,1 of hair clip H2 can replace P2 ', form H1-H2 mixture and discharge 4,5 regions of H2.Discharge in H2 4 and 5 regions can be folded into G4 configuration under univalent cation exists.And then, the P2 ' of release can cause next reaction cycle and the H1-H2 mixture produced continuously containing G4 structure.Finally, NMM G4 structure to obvious structure selectivity is added in reactive system, and the G4-NMM mixture of formation can produce the fluorescent signal exempting to mark.In this way, utilize and exempt to mark the amplification process without enzyme dna machine, an initiation chain of release can produce the fluorescent signal of enhancing for Sensitive Detection UDG activity.
Beneficial effect of the present invention:
(1), in detection method design of the present invention, carry out the identification of UDG and the release of adjoint initiation chain with a dsDNA probe, the initiation chain of release can cause the signal amplification of DNA machine.The designed dsDNA probe comprising uridylic base and initiation sequence has high hybridization efficiency, and to the good inhibition that initiation sequence demonstrates, causes low background signal.
(2) the present invention adopts the design of exempting to mark not need fluorophore and quencher group to be covalently bound on DNA, reduces the impact of mark on background.
(3) the present invention adopts the signal amplifying power of DNA machine to further increase the sensitivity of detection, detection method of the present invention can detect, the UDG being low to moderate 0.00044U/mL is active, is significantly less than the Fluorescence amplification strategy of the detection UDG activity reported in prior art.
(4) design without enzyme makes the signal amplifying power of DNA machine not rely on easily damaged enzyme, and bring forth good fruit circulation ratio.
(5) detection method of the present invention can also be used for the UDG activity in analyzing and testing Hela cell pyrolysis liquid, and the impact of cellular component on this strategy is low.
Accompanying drawing explanation
Fig. 1 is the schematic diagram based on exempting from the Fluorescence amplification strategy detection UDG activity marked without enzyme dna machine of the present invention, and in figure, U and A represents uridylic and adenyl-deoxyribonucleotide respectively, and numeral and the corresponding unmarked numeral of mark * are complementary;
Fig. 2 be system when 60%Hela cell pyrolysis liquid presence or absence fluorescence emission spectrum, and UGI is to the inhibition of UDG activity in Hela cell pyrolysis liquid;
Fig. 3 is the relative intensity of fluorescence of system when different concns UGI exists;
Fig. 4 is the fluorescence emission spectrogram of different system, in figure, and (a) H2, (b) H1+H2, (c) P1-P2+H1+H2, (d) P1-P2+UDG+H1+H2;
Fig. 5 is that Polyacrylamide Gel Electrophoresis dsDNA probe P1-P2 dissociates figure under UDG effect, and band 1 is containing 5.0 μMs of P1; Band 2 is containing 1.0 μMs of P2; Band 3 is containing 1.0 μMs of P1-P2; Band 4 is containing 1.0 μMs of P1-P2 and 10U/mLUDG;
Fig. 6 is the fluorescence response of system to different concns UDG;
Fig. 7 is the calibration curve of Δ F relative to UDG concentration;
Fig. 8 is reactive system to the relative intensity of fluorescence of UDG (1.0U/mL) or 10 times of excessive hOGG1, Exo I, Exo III (10U/mL).
Embodiment
The present invention is further illustrated in conjunction with the embodiments, should be noted that following explanation is only to explain the present invention, not limiting its content.
Experiment reagent and instrument:
Experiment reagent used in the embodiment of the present invention and instrument as follows:
1. experiment reagent: DNA oligonucleotide is by (China, the Shanghai) synthesis of raw work and purifying.Uracil-DNA glycosylase (UDG), uracil glycosylase enzyme inhibitors (UGI) and 8-oxoguanine DNA glycosylase (hOGG1) obtain from New England's biology laboratory (China, Beijing).Excision enzyme I (Exo I) and exonucleaseⅢ (Exo III) are purchased from Thermo Fisher Scientific Inc. (China).N-methyl porphyrin IX (NMM) is purchased from front line science company limited (U.S., the Utah State, sieve root).The storage liquid of NMM prepares in dimethyl sulfoxide (DMSO) (DMSO) and lucifuge storage at-20 DEG C.Other pharmaceutical chemicalss all are all analytical pure and directly use.Time prepared by solution, ultrapure water used is all from Millipore Milli-Q water purification system (>18.25M Ω).
2. laboratory apparatus: fluorescence measurement uses Hitachi F-7000 fluorescence spectrophotometer to carry out measuring (Japan, Hitachi, Ltd).Excitation wavelength is 399nm, and the emission wavelength ranges of report is 570-700nm.Be used to evaluate institute in the fluorescence intensity at 618nm place and propose tactful performance.The slit width excited and launch is 10nm, and Photomultiplier tube voltage is 700V.
Embodiment 1: pure UDG Activity determination
First design one containing uridylic base and double-stranded DNA (dsDNA) the probe P1-P2 causing sequence, wherein P1 chain is for suppressing chain, and its nucleotide sequence is as shown in SEQ ID NO.1 in sequence table; P2 chain is that its nucleotide sequence is as shown in SEQ ID NO.2 in sequence table containing ura DNA sequence and the chimeric conjugated chain causing sequence; P1 chain and the complementation of P2 chain portion form double-stranded DNA (dsDNA) probe P1-P2;
P1：5’-GAAATTCTTAAGTAGTCAG-3’；
P2:5 '-CGACATCTAACCTAGCTGACTACUUAAGAAUUTC-3 '; (wherein, italicized item is for causing sequence)
According to the initiation sequence of P2 chain, hairpin probe H1 and H2 of design two partial complementarity exempts to mark the DNA machine without enzyme for building, and the grafting of G-tetrad (G4) sequence is at the end of hairpin probe H2;
H1:5’-AGTCAGCTAGGTTAGATGTCGCTGACTGGGTAGGGCCGACATCTAACCTAG-3’,
H2:5’-ATGTCGGCCCTACCCAGTCAGCGACATCTAACCTAGCTGACTGGGTAGGGCGGGTTGGG-3’；
Double-stranded DNA (dsDNA) probe P1-P2 step (1) prepared and the UDG of different concns joins in reaction buffer, consisting of of reaction buffer: 20mM Tris-HCl, 1.0mM EDTA, 1.0mM DTT, pH 8.0, the final volume obtaining system is 50 μ L, the concentration of system double center chain DNA (dsDNA) probe P1-P2 is 200nM, system hatches 120min at 37 DEG C, make base remove reaction to occur, then the hairpin probe H2 of G-tetrad sequence hairpin probe H1 and grafting is had to join in the solution after hatching, the final volume obtaining system is 100 μ L, at 37 DEG C, DNA machine carries out the iodine of 60min, N-methyl porphyrin IX (NMM) is added again in reaction system, and 30min is hatched at 37 DEG C, obtain reaction product, reaction product solution is joined in the quartzy fluorescence pond of 100 μ L, insert in spectrophotofluorometer and carry out fluorometric assay, optimum configurations is: exciting voltage is 700V, excitation wavelength is 399nm, emission wavelength is 618nm, slit width is excited to be 10nm, transmitting slit width is 10nm, and build the linearity curve between fluorescence intensity and UDG concentration, linearity curve is Δ F=16.542+12948C
uDG, R
2=0.99896, linearity range is 0-0.025U/mL.
By pure UDG sample to be detected, by the fluorescence intensity of working sample, substitute into linearity curve, calculate UDG concentration.
Embodiment 2: the UDG Activity determination in cervical cancer (Hela) cell pyrolysis liquid
Adopt detection method of the present invention to carry out analyzing and testing to the UDG activity in Hela cell pyrolysis liquid, concrete steps are as follows:
Hela cell sample is pressed into pellet by centrifugation (5min, 3000rpm, 4 DEG C), and is again dispersed in by ultrasonoscope on ice in lysis buffer (10mM Tris-Hcl, pH 7.0).Mixing solutions carries out centrifugation 30min to remove insoluble material at 4 DEG C with the speed of 12,000rpm subsequently.Collect supernatant liquor also by the membrane filtration of 0.45 μm, prepare rough Hela cell pyrolysis liquid.
Rough Hela cell pyrolysis liquid directly can carry out UDG Activity determination, without the need to further process.Detection method is identical with UDG activity test method pure in embodiment 1.
For verifying that detection method of the present invention is detecting the suitability in UDG activity, we have also carried out analyzing and testing by method of the present invention to the UDG activity in Hela cell pyrolysis liquid.As shown in Figure 2, cracking buffered soln only can cause low-down fluorescent signal to result.By comparison, when analyzing and testing 60%Hela cell pyrolysis liquid, due to the existence of UDG activity, the fluorescent signal of system obviously strengthens.In order to confirm Fluorescence Increasing be caused by UDG instead of caused by other component any in lysate, UGI is also added in cell pyrolysis liquid, and result does not demonstrate obvious Fluorescence Increasing.These results show that detection method of the present invention has tolerance to cellular constituent, and will become a kind of candidate's instrument detecting UDG activity in UDG functional study and clinical diagnosis.
The activity of embodiment 3:UDG suppresses to detect
For the activity detecting UDG suppresses, the UDG of dsDNA probe P1-P2 and the 1U/mL of preparation and the UGI mixing of different concns, and certain hour is hatched at 37 DEG C.Then, hair clip H1 and H2 is added in mixture, and the final volume obtaining system is 100 μ L.At 37 DEG C, DNA machine carries out the iodine of certain hour.Finally, NMM is added in product, and hatches 30min at 37 DEG C.All experiments all in triplicate.
UGI is selected as a kind of model inhibitor.UGI can be formed closely and the irreversible mixture of physiology with the mol ratio of 1:1 with UDG, and as shown in Figure 3, when there is UGI, the relative intensity of fluorescence of system reduces with the increase of UGI concentration.Result shows that this strategy can be used for detecting the activity suppression of UDG.
Embodiment 4: the feasibility study of detection method of the present invention
(1) activity of UDG and active suppression thereof is detected:
For obtaining double-stranded DNA (dsDNA) probe (P1-P2) and hairpin structure, the mixture of P1 and P2 and H1, H2 are respectively at the hybridization buffer (composition of hybridization buffer: 10mM Tris, 5mM KCl, 100mM NaCl, pH 7.5) at 95 DEG C heat denatured 10min, be then slowly down to room temperature.Active for detecting UDG, by the dsDNA probe P1-P2 of preparation and different concns (0.0025U/mL, 0.0050U/mL, 0.0075U/mL, 0.010U/mL, 0.015U/mL, 0.020U/mL, 0.025U/mL, 0.050U/mL, 0.20U/mL, 0.50U/mL, 1.0U/mL) UDG join (20mM Tris-HCl in reaction buffer, 1.0mM EDTA, 1.0mM DTT, pH 8.0), the final volume obtaining system is 50 μ L.Mixture hatches 150min at 37 DEG C, makes base remove reaction and occurs.Then, hair clip H1 and H2 is added in above-mentioned solution, and the final volume obtaining system is 100 μ L.At 37 DEG C, DNA machine carries out the iodine of 60min.Finally, NMM is added in product, and hatches 30min at 37 DEG C.Controlled trial is carried out under the same conditions, does not just add UDG.
(2) the dissociating of gel electrophoresis experimental study dsDNA probe:
P1-P2 and UDG of 0.5 μM-5.5 μMs hatches certain hour at 37 DEG C, obtains the product of UDG-process.P1, P2 and dsDNA probe is also analyzed.Sample mixes with sample-loading buffer and is loaded in the polyacrylamide gel of 15%.With 1 × TBE (89mM Tris, 89mM Boric Acid, 2.0mM EDTA, pH 8.3), as electrophoresis running buffer, electrophoresis runs 2h under normal electric current 30mA.Gel dyes 5min in ethidium bromide, and the 5min that decolours in distilled water, be then placed in ultraviolet imagery system (U.S., Bio-RAD R&D units company limited) and take pictures.
The feasibility of fluorescence emission spectrum to UDG activity test method of the present invention is adopted to study, result as shown in Figure 4, the system only containing H2 shows low-down fluorescence intensity, and (in Fig. 4, curve a), shows when Sequence is hidden in the neck of H2, is difficult to form G4 structure.System containing H1 and H2 demonstrates the fluorescence intensity (in Fig. 4 curve b) strengthened a little, and when showing to there is not target compound, the interaction of H1 and H2 is weak.When P1-P2 is added in H1, H2 system, fluorescence intensity display further strengthens (in Fig. 4 curve c), and this shows that the interaction of H1 and H2 is promoted and production background signal by P1-P2.But when in the system that UDG joins containing P1-P2, H1 and H2, fluorescence intensity has obvious enhancing (in Fig. 4 curve d).This shows that UDG can cause the self-assembly of H1 and H2.
By removing the uridylic base also adjoint release causing chain P2 ' in dsDNA probe P1-P2.Polyacrylamide gel electrophoresis (PAGE) is also used to verify dissociating of dsDNA probe.After UDG process, dsDNA probe is dissociated into the band of separation, as shown in Figure 5.Therefore, these results show that the detection method of the present invention proposed can be used for the detection of UDG activity.
Embodiment 5: the research of the analytical performance of detection method
(1) detection method of the present invention is to the fluorescence response of different concns UDG:
As shown in Figure 6, fluorescence intensity strengthens along with the increase of UDG concentration from 0 to 1.0U/mL, shows that the height of formation of G4-NMM fluorescent composition depends on the concentration of UDG.
Detect the calibration curve of UDG activity as shown in Figure 7.As shown in the illustration in Fig. 7, net signal Δ F and 0.0025 demonstrates linear dependence, R to the UDG activity within the scope of 0.020U/mL
2value is 0.999.According to 3 δ rules, the detection of UDG is limited to 0.00044U/mL, the Fluorescence amplification strategy of this detection UDG activity reported lower than great majority.The highly sensitive of detection method is amplified owing to the signal of designed DNA machine, and the background of the dsDNA probe of uniqueness reduces.Therefore, detection method of the present invention has the effective tool that very large potentiality become a kind of UDG active level.
(2) selectivity of detection method
For evaluating the selectivity of detection method of the present invention further, we have studied it and hOGG1 is comprised to UDG and other nuclease, the fluorescence response of Exo I, Exo III.As shown in Figure 8, only have UDG can cause obvious relative intensity of fluorescence, and hOGG1, Exo I or Exo III only provide the low relative intensity suitable with blank solution.This result shows the good selectivity that detection method of the present invention demonstrates active UDG.
(3) precision of detection method and circulation ratio
Be respectively 0.0025U/mL according to UDG concentration, the experimental result of the same batch sample of 0.010U/mL and 0.020U/mL, relative standard deviation (RSD) is respectively 4.36%, 3.74% and 3.67%.Under same experimental conditions, detect respectively the sample of each concentration above-mentioned in three days, the RSD of these three the different batches samples obtained is respectively 5.00%, 4.19% and 3.96%.These results show that detection method of the present invention has acceptable precision and circulation ratio.
Sum up:
It is active for Sensitive Detection UDG that the present invention has developed a kind of novel fluorescence amplification strategy based on exempting to mark without enzyme dna machine.This strategy carries out the identification of UDG with a dsDNA probe and with the release causing chain, the initiation chain of release can cause the signal amplification of DNA machine.The designed dsDNA probe comprising uridylic base and initiation sequence has high hybridization efficiency, and to the good inhibition that initiation sequence demonstrates, causes low background signal.In addition, the design of exempting to mark does not need fluorophore and quencher group to be covalently bound on DNA, reduces the impact of mark on background.In addition, the signal amplifying power of the DNA machine of design is also used to improve detection sensitivity further.It is active that this strategy can detect the UDG being low to moderate 0.00044U/mL, lower than the Fluorescence amplification strategy of the detection UDG activity of great majority report.In addition, the design without enzyme makes the signal amplifying power of DNA machine not rely on easily damaged enzyme, and bring forth good fruit circulation ratio.Finally, the UDG that this strategy is also used in analyzing and testing Hela cell pyrolysis liquid is active, and the impact of cellular component on this strategy is low.Result shows that detection method of the present invention provides quantitatively sensitive for UDG activity in UDG functional study and clinical diagnosis of a kind of potential instrument.
Claims (7)
1. one kind for the identification of UDG and the probe of signal transduction, it is characterized in that, described probe is containing uridylic base and the double-stranded DNA causing sequence, comprises P1 chain and the P2 chain of partial sequence complementarity, wherein P1 chain is for suppressing chain, and its nucleotide sequence is as shown in SEQ ID NO.1 in sequence table; P2 chain is that its nucleotide sequence is as shown in SEQ ID NO.2 in sequence table containing ura DNA sequence and the chimeric conjugated chain causing sequence.
2. probe according to claim 1 is detecting the application in uracil-DNA glycosylase activity.
3. marking based on exempting from the method detecting uracil-DNA glycosylase activity without enzyme dna machine Fluorescence amplification strategy, it is characterized in that, comprise the following steps:
(1) design is containing uridylic base and the double chain DNA probe P1-P2 causing sequence, and for identification and the signal transduction of UDG, wherein P1 chain is for suppressing chain, and its nucleotide sequence is as shown in SEQ ID NO.1 in sequence table; P2 chain is that its nucleotide sequence is as shown in SEQ ID NO.2 in sequence table containing ura DNA sequence and the chimeric conjugated chain causing sequence; P1 chain and the complementation of P2 chain portion form double chain DNA probe P1-P2;
(2) according to the initiation sequence of P2 chain, hairpin probe H1 and H2 of design two partial complementarity, exempt to mark the operation part without the DNA machine of enzyme for building, the nucleotide sequence of hairpin probe H1 is as shown in SEQ ID NO.3 in sequence table; The nucleotide sequence of hairpin probe H2 is as shown in SEQ ID NO.4 in sequence table; The grafting of G-tetrad sequence is at the end of hairpin probe H2;
(3) double chain DNA probe P1-P2 step (1) prepared and UDG to be detected joins in reaction buffer, the final volume obtaining system is 50 μ L, the concentration of system double center chain DNA probe P1-P2 is 50-500nM, system hatches 30-180min at 37 DEG C, there is base and remove reaction, then by the hairpin probe H1 in step (2), grafting has the hairpin probe H2 of G-tetrad sequence to join in the solution after hatching, the final volume obtaining system is 100 μ L, in system, the concentration of hairpin probe H1 is 100-600nM, the concentration of hairpin probe H2 is 400-900nM, at 37 DEG C, DNA machine carries out the iodine of 20-120min, the N-methyl porphyrin IX of 0.5-20 μ L 200 μMs is added again in reaction system, and 10-120min is hatched at 37 DEG C, obtain reaction product, fluorometric assay is carried out to reaction product, builds the linearity curve between fluorescence intensity and UDG concentration, to a certain sample, by the fluorescence intensity of working sample, substitute into linearity curve, calculate UDG concentration.
4. mark based on exempting from the method detecting uracil-DNA glycosylase activity without enzyme dna machine Fluorescence amplification strategy as claimed in claim 3, it is characterized in that, in step (3), consisting of of described reaction buffer: 20mM Tris-HCl, 1.0mM EDTA, 1.0mM DTT, pH 8.0.
5. mark based on exempting from the method detecting uracil-DNA glycosylase activity without enzyme dna machine Fluorescence amplification strategy as claimed in claim 3, it is characterized in that, in step (3), carrying out fluorimetric method to reaction product is: joined by reaction product solution in the quartzy fluorescence pond of 100 μ L, insert in spectrophotofluorometer and carry out fluorometric assay, optimum configurations is: exciting voltage is 700V; Excitation wavelength is 399nm; Emission wavelength is 618nm; Slit width is excited to be 10nm; Transmitting slit width is 10nm.
6. mark based on exempting from the method detecting uracil-DNA glycosylase activity without enzyme dna machine Fluorescence amplification strategy as claimed in claim 3, it is characterized in that, in step (3), final volume is in the system of 50 μ L, and the concentration of double chain DNA probe P1-P2 is 200nM.
7. marking based on exempting from the method detecting uracil-DNA glycosylase activity without enzyme dna machine Fluorescence amplification strategy as claimed in claim 3, it is characterized in that, in step (3), after adding N-methyl porphyrin IX, at 37 DEG C, hatch 30min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510061929.XA CN104630363A (en) | 2015-02-05 | 2015-02-05 | Method for detecting activity of uracil-DNA glycosylase (UDG) based on fluorescence amplification strategy of label-free non-enzyme DNA machines |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510061929.XA CN104630363A (en) | 2015-02-05 | 2015-02-05 | Method for detecting activity of uracil-DNA glycosylase (UDG) based on fluorescence amplification strategy of label-free non-enzyme DNA machines |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104630363A true CN104630363A (en) | 2015-05-20 |
Family
ID=53209607
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510061929.XA Pending CN104630363A (en) | 2015-02-05 | 2015-02-05 | Method for detecting activity of uracil-DNA glycosylase (UDG) based on fluorescence amplification strategy of label-free non-enzyme DNA machines |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104630363A (en) |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105132522A (en) * | 2015-09-08 | 2015-12-09 | 山东大学 | High-sensitivity uracil DNA glycosylase (UDG) detection using DNA three-direction section activated hybridization chain reaction |
| CN106244703A (en) * | 2016-08-26 | 2016-12-21 | 山东大学 | Strand replacement reaction based on sticky end mediation combines the method for polymerization nicking isothermal amplification technique detection UDG activity |
| CN107119031A (en) * | 2017-06-21 | 2017-09-01 | 南京大学 | A kind of new thermophilic serobila DNA enzymatics of quaternary G tetra- |
| CN107727621A (en) * | 2017-09-29 | 2018-02-23 | 济南大学 | A kind of label-free DNA System of Logics detection miRNA method |
| CN108088826A (en) * | 2017-12-14 | 2018-05-29 | 济南大学 | A kind of detection uracil-DNA glycosylase（UDG）Biological sensor |
| CN109406467A (en) * | 2018-10-16 | 2019-03-01 | 商丘师范学院 | Division aptamer sensor and its application for ATP detection |
| CN109504734A (en) * | 2018-11-13 | 2019-03-22 | 西安交通大学 | A kind of DNA motor and its preparation method and application accelerated in situ in living cells |
| CN109884304A (en) * | 2019-03-27 | 2019-06-14 | 四川大学 | A kind of the CHA iodine system and hypersensitive visible detection method of HCV Core Protein |
| CN109957608A (en) * | 2019-03-05 | 2019-07-02 | 广东省生态环境技术研究所 | A kind of method and kit of label-free fluorescence detection gene |
| CN110734961A (en) * | 2019-11-29 | 2020-01-31 | 福州大学 | enzyme-free biosensors for detecting uracil-DNA glycosylase activity |
| CN111879738A (en) * | 2020-06-15 | 2020-11-03 | 山东师范大学 | Non-labeled aptamer probe system and detection method and application thereof |
| CN112662741A (en) * | 2021-01-16 | 2021-04-16 | 湘潭大学 | High-universality nucleic acid detection method and kit |
| CN112760363A (en) * | 2020-12-31 | 2021-05-07 | 诸暨加向生物科技有限公司 | Method for detecting uracil-DNA glycosylase activity by fluorescent probe |
| CN113512579A (en) * | 2021-07-29 | 2021-10-19 | 山东大学 | Fluorescent biosensor for detecting uracil DNA glycosylase and detection method and application thereof |
| CN113720819A (en) * | 2021-08-30 | 2021-11-30 | 安徽师范大学 | Aptamer DNA-fluorescent probe sensor, preparation method thereof and method for quantitatively detecting GTX1/4 by using aptamer DNA-fluorescent probe sensor |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0624643A2 (en) * | 1993-05-11 | 1994-11-17 | Becton, Dickinson and Company | Decontamination of nucleic acid amplification reactions |
| CN101008030A (en) * | 2007-01-25 | 2007-08-01 | 上海交通大学 | Method for detecting ura DNA glycosidase activity adopting molecular beacon as substrate |
-
2015
- 2015-02-05 CN CN201510061929.XA patent/CN104630363A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0624643A2 (en) * | 1993-05-11 | 1994-11-17 | Becton, Dickinson and Company | Decontamination of nucleic acid amplification reactions |
| CN101008030A (en) * | 2007-01-25 | 2007-08-01 | 上海交通大学 | Method for detecting ura DNA glycosidase activity adopting molecular beacon as substrate |
Non-Patent Citations (2)
| Title |
|---|
| DAN HU等: "A Label-Free, Quadruplex-Based Functional Molecular Beacon (LFG4-MB) for Fluorescence Turn-On Detection of DNA and Nuclease", 《CHEMISTRY EUROPEAN JOURNAL》 * |
| YUSHU WU等: "A DNA machine-based fluorescence amplification strategy for sensitive detection of uracil-DNA glycosylase activity", 《BIOSENSORS AND BIOELECTRONICS》 * |
Cited By (24)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105132522B (en) * | 2015-09-08 | 2018-07-03 | 山东大学 | The hybridization chain reaction of DNA three-dimensionals section activation is used for the highly sensitive detection of uracil dna glycosylase |
| CN105132522A (en) * | 2015-09-08 | 2015-12-09 | 山东大学 | High-sensitivity uracil DNA glycosylase (UDG) detection using DNA three-direction section activated hybridization chain reaction |
| CN106244703B (en) * | 2016-08-26 | 2019-12-13 | 山东大学 | method for detecting UDG activity based on sticky end-mediated strand displacement reaction combined with polymerization nicking isothermal amplification technology |
| CN106244703A (en) * | 2016-08-26 | 2016-12-21 | 山东大学 | Strand replacement reaction based on sticky end mediation combines the method for polymerization nicking isothermal amplification technique detection UDG activity |
| CN107119031A (en) * | 2017-06-21 | 2017-09-01 | 南京大学 | A kind of new thermophilic serobila DNA enzymatics of quaternary G tetra- |
| CN107119031B (en) * | 2017-06-21 | 2022-12-27 | 南京大学 | Novel thermophilic quaternary G quadruplex DNA enzyme |
| CN107727621A (en) * | 2017-09-29 | 2018-02-23 | 济南大学 | A kind of label-free DNA System of Logics detection miRNA method |
| CN108088826A (en) * | 2017-12-14 | 2018-05-29 | 济南大学 | A kind of detection uracil-DNA glycosylase（UDG）Biological sensor |
| CN108088826B (en) * | 2017-12-14 | 2020-08-04 | 济南大学 | Fluorescent biosensor for detecting uracil-DNA glycosylase (UDG) |
| CN109406467A (en) * | 2018-10-16 | 2019-03-01 | 商丘师范学院 | Division aptamer sensor and its application for ATP detection |
| CN109406467B (en) * | 2018-10-16 | 2021-01-29 | 商丘师范学院 | Split aptamer sensor for ATP detection and application thereof |
| CN109504734A (en) * | 2018-11-13 | 2019-03-22 | 西安交通大学 | A kind of DNA motor and its preparation method and application accelerated in situ in living cells |
| CN109957608A (en) * | 2019-03-05 | 2019-07-02 | 广东省生态环境技术研究所 | A kind of method and kit of label-free fluorescence detection gene |
| CN109884304A (en) * | 2019-03-27 | 2019-06-14 | 四川大学 | A kind of the CHA iodine system and hypersensitive visible detection method of HCV Core Protein |
| CN110734961B (en) * | 2019-11-29 | 2021-10-29 | 福州大学 | Enzyme-free biosensor for detecting activity of uracil-DNA glycosylase |
| CN110734961A (en) * | 2019-11-29 | 2020-01-31 | 福州大学 | enzyme-free biosensors for detecting uracil-DNA glycosylase activity |
| CN111879738A (en) * | 2020-06-15 | 2020-11-03 | 山东师范大学 | Non-labeled aptamer probe system and detection method and application thereof |
| CN111879738B (en) * | 2020-06-15 | 2023-03-14 | 山东师范大学 | Non-labeled aptamer probe system and detection method and application thereof |
| CN112760363A (en) * | 2020-12-31 | 2021-05-07 | 诸暨加向生物科技有限公司 | Method for detecting uracil-DNA glycosylase activity by fluorescent probe |
| CN112662741A (en) * | 2021-01-16 | 2021-04-16 | 湘潭大学 | High-universality nucleic acid detection method and kit |
| CN113512579A (en) * | 2021-07-29 | 2021-10-19 | 山东大学 | Fluorescent biosensor for detecting uracil DNA glycosylase and detection method and application thereof |
| CN113512579B (en) * | 2021-07-29 | 2024-01-16 | 山东大学 | Fluorescent biosensor for detecting uracil DNA glycosylase, and detection method and application thereof |
| CN113720819A (en) * | 2021-08-30 | 2021-11-30 | 安徽师范大学 | Aptamer DNA-fluorescent probe sensor, preparation method thereof and method for quantitatively detecting GTX1/4 by using aptamer DNA-fluorescent probe sensor |
| CN113720819B (en) * | 2021-08-30 | 2023-10-17 | 安徽师范大学 | Aptamer DNA-fluorescent probe sensor, preparation method thereof and method for quantitatively detecting GTX1/4 by using aptamer DNA-fluorescent probe sensor |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104630363A (en) | Method for detecting activity of uracil-DNA glycosylase (UDG) based on fluorescence amplification strategy of label-free non-enzyme DNA machines | |
| CN107723338B (en) | Fluorescent chemical sensor for simultaneously detecting multiple DNA glycosylases at single-molecule level and detection method and application thereof | |
| CN105132524A (en) | Application of dual application reaction of Exo (exonuclease) III-assisted cycle and DNAzyme cycle to Hg<2+> detection | |
| CN109161579B (en) | Ligase-based isothermal amplification method and application thereof in polynucleotide kinase detection | |
| CN105506078A (en) | Method for parallel determination of activity of uracil-DNA glycosylase and endonuclease IV, application thereof and reagent kit | |
| Sha et al. | A label-free and enzyme-free ultra-sensitive transcription factors biosensor using DNA-templated copper nanoparticles as fluorescent indicator and hairpin DNA cascade reaction as signal amplifier | |
| Chi et al. | CRISPR-Cas14a-integrated strand displacement amplification for rapid and isothermal detection of cholangiocarcinoma associated circulating microRNAs | |
| CN103589777A (en) | DNA methylation detection probe, and detection method and detection kit thereof | |
| CN109439735A (en) | A kind of fluorescent detection probe, kit and the application of abasic endonuclease 1 | |
| Sohail et al. | Molecular reporters for CRISPR/Cas: From design principles to engineering for bioanalytical and diagnostic applications | |
| Zhang et al. | A rapid label-and enzyme-free G-quadruplex-based fluorescence strategy for highly-sensitive detection of HIV DNA | |
| CN109406477A (en) | Active biological sensor of a kind of detection dnmt rna and preparation method thereof | |
| CN103952497B (en) | A kind of hepatitis B virus detecting method based on DNAzyme probes | |
| Liu et al. | A label-free electrochemical sensor for the detection of two kinds of targets based on CRISPR/Cas12a system | |
| Tian et al. | A universal CRISPR/Cas12a nucleic acid sensing platform based on proximity extension and transcription-unleashed self-supply crRNA | |
| Zhang et al. | A highly sensitive and versatile fluorescent biosensor for pathogen nucleic acid detection based on toehold-mediated strand displacement initiated primer exchange reaction | |
| Xu et al. | Isothermal cycling and cascade signal amplification strategy for ultrasensitive colorimetric detection of nucleic acids | |
| Iwe et al. | A dual-cycling fluorescence scheme for ultrasensitive DNA detection through signal amplification and target regeneration | |
| Zhao et al. | G-quadruplex molecular beacon: A versatile CRISPR/Cas12a reporter for rapid and label-free biosensing | |
| Zhang et al. | Accurate identification of exosomes based on proximity-induced autonomous assembly of DNAzyme wires | |
| CN107083437B (en) | Method for simultaneously detecting multiple DNA glycosylases by using inherent fluorescent nucleotide ultrasensitiveness | |
| CN102676656A (en) | Structure-switching aptamer based highly-sensitive unmarked fluorescent detection method | |
| Hu et al. | Discrimination between 5-hydroxymethylcytosine and 5-methylcytosine in DNA by selective chemical labeling | |
| CN116083575A (en) | Kit for detecting cervical cancer and application thereof | |
| Han et al. | A novel fluorescent biosensor for sensitive detection of pathogenic bacteria based on catalytic hairpin assembly and λ-Exonuclease cleavage reaction |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150520 |