CN102703594A - Method for detecting miRNA (micro ribonucleic acid) based on graphene/nucleic acid dye platform - Google Patents
Method for detecting miRNA (micro ribonucleic acid) based on graphene/nucleic acid dye platform Download PDFInfo
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Abstract
The invention discloses a method for detecting a miRNA (micro ribonucleic acid) based on a graphene oxide/nucleic acid dye detection platform and a nucleic acid constant-temperature amplification technology. The method comprises the following steps of: first, designing a specific DNA (deoxyribonucleic acid) hairpin probe and an amplification primer according to the sequence of a target miRNA, mixing the DNA hairpin probe, the primer, the target miRNA, DMSO (dimethylsulfoxide), an enzyme with a strand displacement amplification property and necessary reactants to carry out a constant-temperature amplification reaction, and then, adding the graphene oxide/nucleic acid dye detection platform to incubate, and carrying out fluorescence detection. When the target miRNA exists, a detection system has an obvious fluorescent signal enhancement phenomenon, and in a condition that the target miRNA does not exist, a fluorescent signal of the detection system is quite weak. By using the method, the purpose of highly sensitively, specifically, simply and quickly detecting a target molecule by using a non-modifying probe is realized, and the method is quite suitable to popularize in the application of actual detection.
Description
Technical field
The invention belongs to nucleic acid fluorescent detection technique field, particularly a kind of method that detects microRNA based on graphene oxide/nucleic acid dye detection platform bind nucleic acid isothermal amplification technology.
Background technology
MicroRNA (miRNA) is one type of endogenic non-coding RNA with adjusting function in eukaryote, finding, and its size is about 20 ~ 25 Nucleotide.Sophisticated miRNA is processed and produces through the shearing of a series of nucleicacidases by long primary transcript; Be assembled into RNA inductive silencing complex subsequently; Discern said target mrna through base complementrity paired mode, and instruct silencing complex degraded said target mrna perhaps to check the translation of said target mrna according to the difference of complementary degree.In in the past 10 years, the correlative study of miRNA has obtained significant progress.It is reported in mammiferous gene, have the gene of the coded protein about 30% to receive the regulation and control of miRNA approximately.Nearest research has shown that miRNA participates in various adjusting approach, comprises growth, virus defense, hematopoiesis, organ formation, cell proliferation and apoptosis, metabolism of fat or the like.For example, miR-181 control mammalian blood cytodifferentiation is the B cell; MiR-375 regulates mammalian islet cell and grows and insulin secretion; MiR-143 works in the adipocyte differentiation; MiR-196 has participated in the Mammals four limbs and has formed; MiR-1 is relevant with heart development; The expression of miR-21 just even with human cancer is closely related.Therefore, develop a kind of sensitivity, special, economy, the method for detection miRNA fast and safely are the focuses of research always.
At present, conventional miRNA detection means mainly contains Northern hybridization (Northern blotting) method, but it is consuming time, and sensitivity is not high.Other is based on capillary electrophoresis, and the nanoparticle signal amplifies, though the method for the technology of surface plasma resonance has higher detection sensitivity, necessary operations is loaded down with trivial details, and needs expensive instrument support.
Summary of the invention
The shortcoming that the objective of the invention is to overcome prior art is with not enough, and the method for a kind of highly sensitive that combines with the nucleic acid constant-temperature amplification technology based on graphene oxide/nucleic acid dye detection platform, detection miRNA accurate, quick, easy and simple to handle is provided.
The object of the invention is realized through following technical proposals: a kind of method based on graphene oxide/nucleic acid dye detection platform and nucleic acid constant-temperature amplification technology for detection miRNA specifically may further comprise the steps:
(1) design dna hair fastener probe: carry out design dna hair fastener probe according to the sequence of target miRNA to be detected, 5 ' end of DNA hair fastener probe with (or) 3 ' terminal modified zygote arranged;
(2) be designed for the primer of strand displacement isothermal amplification reactions: the sequence according to the DNA hair fastener probe of design in the step (1) designs;
(3) strand displacement isothermal amplification reactions: comprise in the reaction system DNA hair fastener probe and the primer of target miRNA to be measured, step (1) and (2) designing and preparing, nucleic acid amplification enzyme, 4 kinds of deoxyribonucleotides (dNTPs) with strand displacement character with and reaction buffer, accomplish the strand displacement isothermal amplification reactions;
(4) add graphene oxide/nucleic acid dye detection platform: in the strand displacement constant-temperature amplification product of step (3) gained, add with the nucleic acid dye and the graphene oxide solution that detect the damping fluid dilution and hatch;
(5) fluoroscopic examination: the detection thing that obtains step (4) is put into the power that fluorescence analyser is measured fluorescent signal, according to the fluorescent signal value and the typical curve that record, can detection by quantitative goes out the hit amount of miRNA of system.
Because the present invention detects the method for miRNA and has avoided some chemically modified steps, can accomplish the detection of sample fast.
DNA hair fastener probe described in the step (1) is for self forming specific double-stranded stem structure and the dna sequence dna composition that can carry out the single-stranded loop bilge construction of conversion according to target miRNA sequence to be detected, i.e. the strand ring portion sequence of DNA hair fastener probe and target miRNA sequence complementation fully to be detected; Specific double-stranded stem scantling length is preferably 5 '-CACATCGTCC-3 ' between 10～14bp; When there is target miRNA in detection architecture; The single-stranded loop bilge construction of DNA hair fastener probe combines with target miRNA through the base complementrity principle; The double-stranded stem structure that makes DNA hair fastener probe is owing to the rigidity effect is unwind, structure changes, thereby reaches the purpose of DNA hair fastener probe identification target miRNA;
Zygote described in the step (1) is not for influencing the sequence of DNA hair fastener probe specificity identification target miRNA, and being preferably one section gathers thymus pyrimidine (poly-thymine) or gather VITAMIN B4 (poly-adenine phosphate); For guaranteeing the stability of DNA hair fastener probe loop-stem structure, the length of zygote is 2～7nt;
The position of the zygote described in the step (1) can be held at the 5 ' end or 3 ' of DNA hair fastener probe, is preferably at the two ends of DNA hair fastener probe to exist simultaneously; The existence of zygote can improve the ratio of strand Nucleotide in the DNA hair fastener probe; Make the ratio of unpaired Nucleotide reach the over half of DNA hair fastener probe nucleotide quantity; Thereby improve the adsorption efficiency of graphene oxide to DNA hair fastener probe; Cancellation improves SNR because nucleic acid dye is inserted into the background signal that DNA hair fastener probe stem produces;
Primer described in the step (2) is the double-stranded stem structure sequence design according to DNA hair fastener probe, and under the condition that is not having target miRNA to exist, can not combine with DNA hair fastener probe;
The length of the primer described in the step (2) is 5～10nt, is preferably 8nt;
When the primer described in the step (2) was 5 '-CACATCGTCC-3 ' when corresponding specific double-stranded stem structure, its sequence was 5 '-CACATCGT-3 ' or 5 '-CATCGTCC-3 ';
The nucleic acid amplification enzyme with strand displacement character described in the step (3) is preferably Klenow fragment exo-polysaccharase;
The commercial damping fluid that reaction buffer described in the step (3) matches for its employed nucleic acid amplification enzyme with strand displacement character; Add DMSO 99.8MIN. (DMSO) in addition; The employed volume percent of DMSO is preferably 1% to 8% of reaction system volume, is preferably 3%;
The condition optimization of the isothermal amplification reactions described in the step (3) is under the optimal reactive temperature (37 ℃) of nucleic acid amplification enzyme, reacts 0.5～1.5 hour, is preferably 1 hour;
The composition of the detection damping fluid described in the step (4) is: Tris-HCl 10mM, MgCl
25mM, pH=8.2;
Nucleic acid dye described in the step (4) is can the double-stranded optical dye of specific insertion dsDNA; And it has only after being inserted into the dsDNA double-spiral structure; The fluorescence efficiency of self just can significantly strengthen; Like EB, Golden View, Gel Red or SYBR Green I etc., be preferably SYBR Green I;
The diameter of the graphene oxide described in the step (4) is 500nm～5 μ m, is preferably 1 μ m;
The final concentration of the nucleic acid dye described in the step (4) is 1～5 μ M, preferred 2 μ M;
The final concentration of the graphene oxide described in the step (4) is 6～14 μ g/mL, is preferably 8 μ g/mL;
The condition of hatching described in the step (4) is under the room temperature 5～20 minutes, is preferably 10 minutes;
Fluorescence analyser described in the step (5) is for measuring the instrument of fluorescence intensity, and like XRF, being preferably can automated operation, carries out the ELIASA that various article are analyzed simultaneously; The fluorescence analyser excitation wavelength is 488nm;
Described method based on graphene oxide/nucleic acid dye detection platform and nucleic acid constant-temperature amplification technology for detection miRNA, during for the miR-21 that is closely related with human cancer, concrete steps are following at miRNA:
(1) design dna hair fastener probe: 5 '-TTCACATCGTCCTCAACATCAGTCTGATAAGCTAGGACGATGTGTT-3 ', the TT that 5 ' end and 3 ' end exist is the zygote sequence;
(2) be designed for the primer of strand displacement isothermal amplification reactions: 5 '-CACATCGT-3 ';
(3) strand displacement isothermal amplification reactions: it is 3% DMSO and 0.4～60fmol (fmol=10 that 20 μ L reaction systems comprise 200nM DNA hair fastener probe, 400nM primer, 2U Klenow fragment exo-polysaccharase, Klenow fragment exo-polymerase buffer, dNTPs (every kind of 0.1mM), volume ratio
-15Mol) target miR-21 reacted 60 minutes down at 37 ℃;
(4) add graphene oxide/nucleic acid dye detection platform: add 60 μ L in the product after amplification and detect mixed solution (pH=8.2), hatched altogether 10 minutes under 25 ℃;
The concrete composition of described detection mixed solution is: Tris-HCl 10mM, MgCl
2The graphene oxide of the diameter 1 μ m of 5mM, 13.3 μ g/mL and nucleic acid dye SYBR Green I 2.7 μ M;
(5) fluoroscopic examination: the detection thing that will hatch altogether joins in 96 orifice plates, puts into ELIASA (Switzerland TECAN infiniteM200) and detects, and fluorescence exciting wavelength is set at 488nm, and the fluorescent emission wavelength of SYBR Green I is at 528nm.
Ultimate principle of the present invention is as shown in Figure 1: the present invention is at first to detecting the specific DNA hair fastener of target miRNA sequences Design probe; Graphene oxide can be effectively produces electrostatic adsorption (only adsorbing single-chain nucleic acid) and has high-efficiency fluorescence cancellation effect with the DNA of specific conformation.When experimental system did not contain target miRNA, isothermal amplification reactions can not be activated, and therefore can not produce dsDNA.After adding graphene oxide/nucleic acid dye detection platform, nucleic acid dye only can be inserted into excessive existence, be arranged in the duplex structure of DNA hair fastener probe stem.And the DNA hair fastener probe that contains big single-chain nucleic acid ratio can be by the stable surface that is adsorbed on graphene oxide, so be inserted into the efficient cancellation of fluorescence meeting quilt that the nucleic acid dye in the DNA hair fastener probe sends, the fluorescent signal of detection architecture maintains lower level.When having target miRNA in the detection architecture, DNA hair fastener probe can be hybridized with target miRNA mutually, even if with producing conformational change.At this moment, DNA hair fastener probe just can expose and primer bonded site, and primer can be hybridized on its certain location.Under the effect of the nucleic acid amplification enzyme with strand displacement character, 3 ' end of primer begins to duplicate.New synthetic nucleic acid chains overtime can be with hybridizing under the displacement of the target miRNA on the DNA hair fastener probe.The target miRNA that replaces can be hybridized with other DNA hair fastener probe again, triggers new amplified reaction.Through such cyclic amplification effect, generate the dna fragmentation (dsDNA) of a large amount of duplex structures in the detection architecture.The double-stranded dsDNA that forms can not attracted to the surface of graphene oxide, and being inserted into fluorescence that the nucleic acid dye in the structure of dsDNA sends can oxidized Graphene cancellation, thereby can measure very strong fluorescent signal.Therefore, how much target miRNA amount can judge through the power of fluorescent value.
The present invention has following advantage and effect with respect to prior art:
(1) the present invention utilizes excellent physical properties and the chemical property of this nano material of graphene oxide, and the graphene oxide of structure/nucleic acid dye detection platform has higher sensitivity, and need not pass through any chemically modified, and use is very convenient.
(2) since the present invention with simple oxidation Graphene/nucleic acid dye detection platform with combine based on the constant-temperature amplification of strand displacement efficiently, so the sensitivity of measurement is very high and quick.
(3) because the present invention uses is that the hair fastener probe with single base difference recognition capability removes to catch target miRNA, guaranteed the high specific of whole testing process.
(4) amplification procedure of the present invention carries out under constant temperature, does not need complicated temperature control instrument; And proofing unit is also very general, is fit to the on-the-spot needs that detect.
(5) the present invention is simple to operate, realizes that easily expense is low, is expected to realize robotization.
Description of drawings
Fig. 1 is based on the schematic diagram that graphene oxide/nucleic acid dye detection platform bind nucleic acid isothermal amplification technology detects miRNA.
Fig. 2 is the fluorescence spectrum figure of graphene oxide/SYBR Green I detection platform feasibility checking.
Fig. 3 is the non-sex change PAGE of strand displacement isothermal amplification reactions product electrophoresis check figure.
Fig. 4 is target miRNA (miR-21) the detected result figure to the difference amount.
Fig. 5 is target miRNA (miR-21) canonical plotting of the present invention to the difference amount.
Fig. 6 is the figure as a result that the present invention is directed to target miRNA (miR-21) detection specificity.
Embodiment
Below in conjunction with embodiment and accompanying drawing the present invention is described in further detail, but embodiment of the present invention is not limited thereto.
The used nucleotide sequence of each embodiment is as shown in table 1:
Used sequence among table 1 embodiment
The structure of embodiment 1 graphene oxide/SYBR Green I detection platform
(1) design a DNA hair fastener probe to miR-21 for target molecule, the zygote sequence of DNA hair fastener probe is that 5 ' end and 3 ' is held simultaneous TT;
(2) get the graphene oxide (pioneer's nano material company) of 1mL diameter 1 μ m, clean 2 times with distilled water under the room temperature condition, centrifugal for use;
The DNA hair fastener probe that (3) will design, the miR-21 target molecule that cleans graphene oxide, SYBR Green I and different concns for use and detection damping fluid join in 96 orifice plates, carry out hybridization; Reacting concrete parameter is: in damping fluid (pH 8.2) reaction system of 50 μ L, include the DNA hair fastener probe of 50nM, the graphene oxide of 8 μ g/mL, SYBR Green I nucleic acid dye (invitrogen company), Tris-HCl 10mM and the MgCl of 2 μ M
25mM was hybridized 30 minutes down for 37 ℃; The miR-21 concentration of target molecules is respectively 0nM, 2nM, 10nM, 50nM, 250nM in 5 samples; The ELIASA fluorescence exciting wavelength is 488nm.
The result is as shown in Figure 2: (b line) all do not have only very low background signal with the fluorescence spectrum (a line) of independent measurement water when having the miR-21 target molecule to exist; But when the concentration of miR-21 target molecule in the detection architecture increases gradually (c.2nM; D.10nM; E.50nM, f.250nM), the fluorescence intensity at system 528nm place also strengthens gradually.Therefore, the feasibility of graphene oxide of the present invention/SYBR Green I detection platform obtain the checking.
Embodiment 2 strand displacement isothermal amplification reactions
(1) preparation strand displacement isothermal amplification reactions system: it is 3% DMSO and the target miR-21 of 50fmol that 20 μ L reaction systems comprise 200nM DNA hair fastener probe, 400nM primer, 2U Klenow fragment exo-polysaccharase (MBI company), Klenow fragment exo-polymerase buffer, dNTPs (every kind of 0.1mM), volume ratio;
(2) reaction system of step (1) was reacted 30～60 minutes down at 37 ℃;
The result is as shown in Figure 3 for the strand displacement isothermal amplification reactions: 1.DNA hair fastener probe; 2.DNA hair fastener probe and primer; 3.DNA hair fastener probe, primer and strand displacement constant-temperature amplification enzyme; 4.DNA hair fastener probe, target miR-21 and primer, 5.DNA hair fastener probe, target miR-21, primer and strand displacement constant-temperature amplification enzyme (37 ℃ were reacted 30 minutes) are 6. with 5 (37 ℃ were reacted 60 minutes); 1,2 explanation DNA hair fastener probes are not having to keep stable in the presence of the target miR-21, all can not accomplish amplified reaction when 3,4 explanation amplification systems lack target miR-21 or amplification enzyme, and 5,6 can normally carry out the strand displacement isothermal amplification reactions obtains new band.The result show strand displacement isothermal amplification reactions of the present invention feasibility obtain the checking.
(1) according to the sequences Design DNA hair fastener probe and the primer that detect the miR-21 target molecule;
(2) with miR-21 target molecule, the DMSO of DNA hair fastener probe, primer, different amounts, have the enzyme of strand displacement amplification character and necessary reactant (the Klenow fragment of MBI company exo-polysaccharase and the standard buffer solution of being joined thereof, dNTPs) mixes, carry out the strand displacement isothermal amplification reactions; Reacting concrete parameter is: hair fastener probe, 400nM primer, 2U Klenow fragment exo-polysaccharase, dNTPs (every kind of 0.1mM), volume ratio that 20 μ L reaction systems comprise 200nM are that 3% DMSO, the amount of miR-21 target molecule are respectively 0.4fmol, 0.8fmol, 4fmol, 20fmol, 40fmol, 60fmol, 100fmol, react 60 minutes down at 37 ℃;
(3) add the detection mixed solution (pH=8.2) of 60 μ L graphene oxides and SYBR Green I in the product after amplification, hatched altogether 10 minutes under 25 ℃; The concrete composition of mixed solution is: Tris-HCl 10mM, MgCl
2The graphene oxide of the diameter 1 μ m of 5mM, 13.3 μ g/mL (pioneer's nano material company) and nucleic acid dye SYBR Green I (invitrogen company) 2.7 μ M;
(4) the detection thing that will hatch altogether joins in 96 orifice plates, puts into ELIASA (Switzerland TECAN infiniteM200) and detects, and fluorescence exciting wavelength is set at 488nm, and the fluorescent emission wavelength of SYBR Green I is at 528nm.
Result such as Fig. 4 and shown in Figure 5, the fluorescence intensity that records at the maximum emission wavelength 528nm place of SYBR Green I.Can find when the amount of target molecule miR-21 equals 0.4fmol by Fig. 4; Therefore fluorescent signal can judge that the present invention detects target molecule and has extraordinary sensitivity still greater than threshold value 119 (threshold value is that the mean fluorecence signal value of 3 groups of negative control samples adds its 3 times of standard deviations); Fig. 5 shows that the present invention detects target miRNA the better linearity relation is arranged in 0～60fmol scope.
The specific checking of embodiment 4 detection methods of the present invention
Get miR-21, miR-210, miR-214 and the miR-49 of 10fmol respectively, as shown in Figure 6 according to the 528nm place fluorescence intensity that embodiment 2 said methods record.Can know by Fig. 6; When having the different miRNA of same amount respectively in the sample; Have only to exist purpose miRNA target molecule just can obtain higher fluorescent signal, can judge that thus the present invention is that the method for the detection miRNA of representative has excellent specificity to detect miR-21.
The foregoing description is a preferred implementation of the present invention; But embodiment of the present invention is not restricted to the described embodiments; Other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; All should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1. method based on graphene oxide/nucleic acid dye detection platform and nucleic acid constant-temperature amplification technology for detection miRNA is characterized in that may further comprise the steps:
(1) design dna hair fastener probe: carry out design dna hair fastener probe according to the sequence of target miRNA to be detected, 5 ' end of DNA hair fastener probe and/or 3 ' terminal modifiedly has a zygote;
(2) be designed for the primer of strand displacement isothermal amplification reactions: the DNA hair fastener probe sequence according to design in the step (1) designs;
(3) strand displacement isothermal amplification reactions: comprise in the reaction system DNA hair fastener probe and the primer of target miRNA to be measured, step (1) and (2) designing and preparing, nucleic acid amplification enzyme, 4 kinds of deoxyribonucleotides with strand displacement character with and reaction buffer, accomplish the strand displacement isothermal amplification reactions;
(4) add graphene oxide/nucleic acid dye detection platform: in the strand displacement constant-temperature amplification product of step (3) gained, add with the nucleic acid dye and the graphene oxide solution that detect the damping fluid dilution and hatch;
(5) fluoroscopic examination: the detection thing that obtains step (4) is put into the power that fluorescence analyser is measured fluorescent signal, according to the fluorescent signal value and the typical curve that record, can detection by quantitative goes out the hit amount of miRNA of system.
2. the method based on graphene oxide/nucleic acid dye detection platform bind nucleic acid isothermal amplification technology detection miRNA according to claim 1 is characterized in that:
DNA hair fastener probe described in the step (1) is for self forming specific double-stranded stem structure and forming with the dna sequence dna of the complete complementary single-stranded loop of target miRNA sequence bilge construction to be detected;
Zygote described in the step (1) is not for influencing the sequence of DNA hair fastener probe specificity identification target miRNA, and its position is to exist simultaneously at 5 ' end of DNA hair fastener probe and 3 ' end;
Primer described in the step (2) is according to the double-stranded stem structure sequence design of DNA hair fastener probe.
3. the method based on graphene oxide/nucleic acid dye detection platform bind nucleic acid isothermal amplification technology detection miRNA according to claim 2 is characterized in that:
Described double-stranded stem scantling length is 10～14bp;
Described zygote is gathering thymus pyrimidine or gathering VITAMIN B4 of length 2～7nt;
The length of described primer is 5～10nt.
4. the method based on graphene oxide/nucleic acid dye detection platform bind nucleic acid isothermal amplification technology detection miRNA according to claim 3 is characterized in that:
Described double-stranded stem structure is 5 '-CACATCGTCC-3 ';
Described primer is 5 '-CACATCGT-3 ' or 5 '-CATCGTCC-3 '.
5. the method based on graphene oxide/nucleic acid dye detection platform bind nucleic acid isothermal amplification technology detection miRNA according to claim 1 is characterized in that:
The nucleic acid amplification enzyme with strand displacement character described in the step (3) is a Klenow fragment exo-polysaccharase;
Reaction buffer described in the step (3) is the commercial damping fluid that Klenow fragment exo-polysaccharase matches, and adds the DMSO 99.8MIN. of reaction system volume 1%～8% in addition;
The condition of the isothermal amplification reactions described in the step (3) is under 37 ℃, reacts 0.5～1.5 hour.
6. the method based on graphene oxide/nucleic acid dye detection platform bind nucleic acid isothermal amplification technology detection miRNA according to claim 5 is characterized in that:
The adding volume of described DMSO 99.8MIN. is 3% of a reaction system volume;
The condition of described isothermal amplification reactions is under 37 ℃, reacts 1 hour.
7. the method based on graphene oxide/nucleic acid dye detection platform bind nucleic acid isothermal amplification technology detection miRNA according to claim 1 is characterized in that:
The composition of the detection damping fluid described in the step (4) is: Tris-HCl 10mM, MgCl
25mM, pH=8.2;
Nucleic acid dye described in the step (4) is can the double-stranded optical dye of specific insertion dsDNA: EB, Golden View, Gel Red or SYBR Green I;
The diameter of the graphene oxide described in the step (4) is 500nm～5 μ m;
The final concentration of the nucleic acid dye described in the step (4) is 1～5 μ M;
The final concentration of the graphene oxide described in the step (4) is 6～14 μ g/mL;
Incubation conditions described in the step (4) is under the room temperature 5～20 minutes.
8. the method based on graphene oxide/nucleic acid dye detection platform bind nucleic acid isothermal amplification technology detection miRNA according to claim 7 is characterized in that:
Described nucleic acid dye is SYBR Green I;
The diameter of described graphene oxide is 1 μ m;
The final concentration of described nucleic acid dye is 2 μ M;
The final concentration of described graphene oxide is 8 μ g/mL;
Described condition of hatching is room temperature 10 minutes.
9. the method based on graphene oxide/nucleic acid dye detection platform bind nucleic acid isothermal amplification technology detection miRNA according to claim 1 is characterized in that:
Fluorescence detector described in the step (5) is an ELIASA;
The excitation wavelength of the fluorescence detector described in the step (5) is 488nm.
10. according to claim 1ly detect the method for miRNA based on graphene oxide/nucleic acid dye detection platform bind nucleic acid isothermal amplification technology, it is characterized in that: described target miRNA is during for the miR-21 that is closely related with human cancer, and concrete steps are following:
(1) design dna hair fastener probe: 5 '-TTCACATCGTCCTCAACATCAGTCTGATAAGCTAGGACGATGTGTT-3 ', the TT that 5 ' end and 3 ' end exist is the zygote sequence;
(2) be designed for the primer of strand displacement isothermal amplification reactions: 5 '-CACATCGT-3 ';
(3) strand displacement isothermal amplification reactions: it is 3% DMSO and the target miR-21 of 0.4～60fmol that 20 μ L reaction systems comprise 200nM DNA hair fastener probe, 400nM primer, 2U Klenow fragment exo-polysaccharase, Klenow fragment exo-polymerase buffer, 0.4mM dNTPs, volume ratio, 37 ℃ of reactions 60 minutes down;
(4) add graphene oxide/nucleic acid dye detection platform: add the detection mixed solution of 60 μ LpH=8.2 in the product after amplification, hatched altogether 10 minutes under 25 ℃;
The concrete composition of described detection mixed solution is: Tris-HCl 10mM, MgCl
2The graphene oxide of the diameter 1 μ m of 5mM, 13.3 μ g/mL and nucleic acid dye SYBR Green I 2.7 μ M;
(5) fluoroscopic examination: the detection thing that will hatch altogether joins in 96 orifice plates, puts into ELIASA and detects, and fluorescence exciting wavelength is set at 488nm, and the fluorescent emission wavelength of SYBR Green I is at 528nm.
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