CN103014148B - Isothermal detection method of RNA (Ribonucleic Acid) - Google Patents
Isothermal detection method of RNA (Ribonucleic Acid) Download PDFInfo
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- CN103014148B CN103014148B CN201210419777.2A CN201210419777A CN103014148B CN 103014148 B CN103014148 B CN 103014148B CN 201210419777 A CN201210419777 A CN 201210419777A CN 103014148 B CN103014148 B CN 103014148B
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Abstract
The invention provides a qualitative and quantitive isothermal detection method of RNA (Ribonucleic Acid). The method comprises the steps of after cutting RNA at fixed points by dnazyme (DNAzyme), amplifying RNA through a strand displacement isothermal amplification technique (SDA); and carrying out qualitative or quantitive detection by inspecting a reporter group G-tetramer released from an SDA product. The method can measure trace RNA (comprising mRNA and miRNA) quickly, simply, conveniently, highly sensitively and specifically, and meanwhile, the risk of pollution is reduced.
Description
Technical field
The invention belongs to technical field of molecular biology, relate to RNA Amplification Analysis and detect, more particularly, the present invention relates to a kind of strand displacement isothermal duplication RNA that utilizes, discharge the detection method of reporter.
Background technology
Nucleic acid molecule amplification in vitro is the important means of biotechnology research, not only can increase and separated goal gene, at aspects such as clinical diagnosis, detection in Gene Mutation, forensic identifications, also has important use.Polymerase chain reaction (polymerase chain reaction, PCR) be the most widely used nucleic acid amplification technologies, its needs thermally denature is repeatedly to untie DNA double chain, in application, depend on high-quality thermal cycler and realize sex change, annealing, three steps of extension, cause temperature to control trouble.Difference along with scientific development and research purpose, since early 1990s, a lot of laboratory attempt development, without the DNA isothermal amplification technique of thermally denature, comprise the isothermal duplication (LAMP) of strand displacement amplification (SDA), rolling circle amplification (RCA), multiple displacement amplification (NASBA), ring mediation etc.And strand displacement amplification reaction has high sensitive, high specific and the characteristic such as easy and simple to handle, be employed gradually.
1992, the Walker in U.S. Becton Dickinson research centre etc. has delivered the research report about strand displacement amplification (Stra nd Displacement Am plification SDA), indicates the birth of a kind of new DNA cloning technology SDA.SDA is a kind of external isothermal amplification technique method of DNA based on enzymatic reaction.On the band of target DNA two ends, by the restriction endonuclease recognition sequence of chemically modified, endonuclease is made a breach double-stranded DNA at its recognition site, and archaeal dna polymerase then prolongs 3 ' of secondary breach and holds and replace next DNA chain.Being replaced the DNA single chain getting off can be combined with primer and be extended into two strands by archaeal dna polymerase.This process is constantly carried out repeatedly, and target sequence is efficiently increased.The method is simple to operate, has the feature that high sensitive, high specific and cost are low.
Enzyme is the biomacromolecule with catalysis, has Substratspezifitaet and the very high catalytic efficiency of height.Past people think that enzyme can only be comprised of protein always, but the rRNA of the discovery protozoon thermophilass such as T.R.Cech in 1981 has self splicing function, have disclosed nucleic acid and have also had the ability that catalysis biological reacts, and have proposed the concept of ribozyme.1994, Breaker utilized in-vitro screening scientific discovery, and some single strand dnas have enzymic activity equally, and these DNA moleculars with catalysis are called DNAzyme (Deoxyribozyme, DNAzyme).
1997, SANTORO etc. utilized in-vitro screening technology (SELEX) from 10
14in individual random dna library, filter out two efficient, general DNAzyme: 10-23 type DNAzyme.The function class of this enzyme is similar to a kind of sequence-specific restriction endonuclease ，Qi active centre district for " 10-23 structure (10-23motif) ", 15 deoxyribonucleotides, consists of.The two ends in active centre are respectively the arm of a long 7-9nt, combination for substrate RNA, by Waston-Crick base pairing, be combined pairing with substrate sequence-specific, its sequence can change according to the difference of substrate RNA, and cleavage site is the phosphodiester bond between unpaired purine and the pyrimidine of pairing on RNA molecule.The nicking activity of 10-23 structure DNAzyme wherein purine can not form base pairing, and pyrimidine needs to form base pairing with DNAzyme.Compare with ribozyme, 10-23DNAzyme is in the situation that temperature and ionic strength etc. are identical, and its stability is about 10 of RNA
9doubly, not only target RNA is had to special shear active, and have that relative molecular mass is little, chemical property is relatively stable, be not subject to nuclease degradation, be easy to the advantages such as synthetic and purifying.
Found subsequently various DNAzyme, they have different catalytically active, as Autophosphorylation, RNA hydrolytic activity, peroxidase activity etc.The concept of G-tetra-serobilas (G-qudruplex) was put forward by scholars such as Gellert in 1962 the earliest.Within 1991, Zalller seminar has reported at K
+under the condition existing, the telomeric dna that is folded into G-tetra-chain body structures can not become the effect substrate of Telomerase, and can suppress the extension of Telomerase to telomere.Dipankar Sen finds to be again folded into the tetrameric single stranded DNA zyme that is rich in guanine of G-subsequently.After this DNAzyme is combined with ferrous porphyrin (hemin) or ferrous porphyrin analogue, there is the Peroxidase activity of being similar to, can catalysis H
2o
2by chromogenic substrate (ABTS
2-) etc. be oxidized to coloring matter, therefore can directly by colour-change, detect nucleic acid.
Real-time fluorescence quantitative PCR is to have added fluorescence dye or fluorescent probe on conventional PCR basis, conventional fluorescence dye for example SYBR Green I can specificity mix DNA double chain, send fluorescent signal, and the dye molecule not mixing in two strands does not send fluorescent signal, thereby guarantee that the increase of fluorescent signal and PCR product increase Complete Synchronization.And a large amount of single stranded DNA of the final generation of strand displacement isothermal amplification technique, the chemical structure of triphenylmethane dye is easy to vibration and unstable, and the fluorescence sending during Individual existence is very weak.When tritane is when the tetramer is combined, because the tetramer can be stablized the structure of tritane, so fluorescent signal strengthened greatly, so tritane has the potentiality that are developed as G-tetramer fluorescent probe.
How further to expand existing isothermal amplification technique and the range of application of DNAzyme in life science and medical science, how further to improve or develop new simple and quick, not only can be qualitative but also can real-time quantitative, and be applicable to the detection method of mRNA and miroRNA simultaneously, remain the key subjects that detect RNA.
Summary of the invention
The invention provides the detection of a kind of RNA and the novel method of RNA isothermal duplication, the method does not need the variation of temperature, can be rapid, easy and highly sensitive single-minded ground can to RNA, carry out the detection method of quantitative and qualitative analysis, be particularly useful for mRNA and miRNA.
The technical scheme that realizes above object is as follows:
An isothermal detection methods of RNA, comprises the following steps:
A) DNAzyme based on shearing RNA, realizes the fixed point of RNA is sheared;
At polynueleotide kinase, under archaeal dna polymerase and restriction enzyme effect, there are three strand displacement isothermal duplication circulations in the RNA b) being sheared;
Wherein, through first strand displacement isothermal duplication circulation, discharge a series of DNA oligonucleotide small segment trigger; Then the oligonucleotide probe that can produce the tetrameric complementation of G-containing in testing sample is combined and second strand displacement isothermal duplication circulation occurs is discharged the G-tetramer; This G-tetramer with there is the DNAzyme that shears RNA function and be combined and the 3rd strand displacement isothermal duplication circulation occurs discharge again small segment trigger, cause again thus second strand displacement isothermal duplication, discharge the G-tetramer, this G-tetramer causes again the 3rd strand displacement isothermal duplication, then second and the 3 two moves in circles and carries out, thus the lower a large amount of G-tetramer of final displacement;
C) according to the tetrameric character of G-discharging, can be used as qualitative reporter group or quantitative reporter group, RNA is carried out to qualitative or real-time quantitative detection.
Particularly, the inventive method comprises three steps.First, adopt the DNAzyme that shears RNA to shear object RNA.Then, the RNA being sheared is at polynueleotide kinase, under archaeal dna polymerase and restriction enzyme effect, cause three strand displacement isothermal duplication circulations: the RNA being sheared of take is primer, the DNAzyme that shears RNA of take is template, there is first strand displacement isothermal duplication (SDA), the a series of DNA oligonucleotide small segment trigger of final generation, simultaneously, in testing sample, also contain one and can produce the oligonucleotide probe of the tetrameric complementation of G-, this probe can be combined with DNA oligonucleotide small segment, trigger thus second SDA amplification, discharge a large amount of G-tetramers, this G-tetramer includes one section of nucleotide sequence of a bit of non-G of being rich in, this segment nucleotide sequence can be complementary with the sequence at partially catalyzed center of DNAzyme of shearing RNA, thereby trigger the 3rd SDA amplified reaction, can discharge small segment trigger again, cause again thus second strand displacement isothermal duplication, discharge the G-tetramer, this G-tetramer causes again the 3rd strand displacement isothermal duplication, then second and the 3 two moves in circles and carries out, thereby the lower a large amount of G-tetramer of final displacement.The amplification that is primer by RNA thus change into take that DNA oligonucleotide small segment is primer amplification, avoided the easily shortcoming of degraded of RNA, realize efficient amplification efficiency simultaneously.After three SDA cyclic amplifications, produce a large amount of G-tetramers.Finally, according to the G-tetramer not, there is different character, the present invention has designed two kinds of different probe sequences, finally produce respectively two kinds of different G-tetramers, be respectively to there is the DNAzyme of peroxidase activity and can be combined with tritane to produce the D NAzyme of fluorescence, carry out qualitative or real-time quantitative and detect.
After three SDA cyclic amplifications, produce a large amount of G-tetramers.The present invention, by the different probe sequence of design, can produce the different G-tetramers, is respectively to have the DNAzyme of peroxidase activity and can be combined with tritane to produce the DNAzyme of fluorescence.
In method of the present invention, the pairing of terminal bases complete complementary after the DNAzyme of described shearing RNA and RNA are sheared, comprise catalytic activity loop section and with brachium conjunctivum two portions of RNA complementary pairing.
The probe relating in the inventive method by 3 ' to 5 ' comprise with the sequence of small segment trigger complementation, restriction endonuclease recognition sequence, with the sequence of G-tetramer complementation and the sequence identical with the partially catalyzed center of DNAzyme of shearing RNA.
The G-tetramer relating in the inventive method refers to and is rich in the four chain helical dna structures that interaction of hydrogen bond that the DNA fragmentation G base of guanylic acid (dG) matches by Hoogsteen forms.
The G-tetramer of the qualitative reporter group of conduct relating in the inventive method is the DNAzyme with peroxidase activity, and the result of its acquisition is macroscopic a kind of reporter group; As the G-tetramer of quantitative reporter group, being can be combined with a kind of dyestuff to produce the DNAzyme of fluorescence, is a kind of reporter group that can obtain quantitative result.
Preferably, above-mentioned dyestuff is a kind of triphenylmethane organic substance, for example malachite green preferably.
In SDA reaction system of the present invention, DNAzyme and the concentration and probe concentration of shearing RNA have certain demand in 0.1 μ M-0.5 μ M, sequences Design.The DNAzyme that shears RNA comprise catalytic activity loop section and with brachium conjunctivum two portions of RNA complementary pairing, according to RNA design brachium conjunctivum to be detected, by Waston-Crick base pairing, be combined pairing with substrate sequence-specific, the catalytic base ring of shearing the DNAzyme of RNA is fixed on RNA substrate molecule, the cleavage reaction of catalysis RNA privileged site, its sequence can change according to the difference of substrate RNA.It is the terminal bases complementary pairing after must being sheared with RNA that the DNAzyme of shearing RNA will satisfy condition.
DNAzyme involved in the present invention is the DNA sequence dna with catalysis.The DNAzyme finding at present falls into 5 types: the DNAzyme of cutting RNA, the DNAzyme of cutting DNA, has the DNAzyme of kinase activity, has the DNAzyme of ligase enzyme activity, has the DNAzyme of peroxidase activity with ligand binding.
Polynueleotide kinase described in the inventive method is to have hydrolysis 2 ', the nucleoside monophosphate kinase of 3 '-ring-type phosphate group, archaeal dna polymerase be normal temperature there is strand displacement ability and without the polysaccharase of exonuclease activity, restriction enzyme is to identify double-stranded specific site, and shears the restriction endonuclease of strand specific site.Preferably, polynueleotide kinase is T4 polynueleotide kinase (PNK); Archaeal dna polymerase is Klenow exo-DNA polysaccharase or Bsm archaeal dna polymerase; Restriction enzyme is Nb.Bpu10I.
The DNAzyme with peroxidase activity of the present invention can with chlorine iron protoheme (Hemin) in conjunction with, can catalysis H
2o
2oxidation ABTS
2-, make reaction solution from the colourless green that becomes.The colour-change that now can directly detect by an unaided eye, thus reach the object of qualitative detection RNA.
In the present invention, use and be combined the DNAzyme with peroxidase activity with Hemin while detecting, after finishing, SDA to regulate pH to 7.6,94 ℃ of heating 1min, this step is conducive to avoid the probe sealing G-tetramer, adds thermal distortion and makes probe discharge the G-tetramer being closed.This step increases sensitivity.Then add Hemin and ABTS
2-, room temperature is placed 5min, then adds H
2o
2observe colour-change.This step is fully combined the G-tetramer with Hemin, make the signal of negative control lower, thereby make detected result sensitiveer.
For improving sensitivity, in the color reaction of qualitative experiment of the present invention, preferred pH is 7.6.
Detection by quantitative of the present invention refers to and in reaction system, adds certain density triphenylmethane dye, the DNAzyme that utilizes another probe to produce through SDA amplification can produce in conjunction with triphenylmethane dye the character of fluorescence, make the carrying out along with amplified reaction, signal is progressively accumulated and is realized the whole amplification process of Real-Time Monitoring, finally by typical curve, unknown RNA is carried out to quantitative analysis.The present invention realizes the object of detection by quantitative RNA by detecting single stranded DNA, the false positive signal of having avoided the existence due to primer dimer to cause.Triphenylmethane dye and the exploitation of the G-tetramer are applied in real-time fluorescence detection as reporter group, there is stronger novelty, and reduced the cost of real-time detection, easy to operate, sensitive height.
For further strengthening sensitivity, the preferred triphenylmethane dye of the present invention are malachite green (Malachite green, MG).
The RNA that method of the present invention is applicable to detect is the RNA that contains AU or GU, and AU and the frequency of occurrences of GU in RNA be that approximately every 8 bases just there will be once, and the scope that therefore detects RNA is wider.RNA can synthesize, in-vitro transcription or from sample, extract.Sample can be any source, comprise virus, cell extraction, environmental sample, from sample, microbiological specimens, tissue or the humoral sample etc. of commercial run.
In addition, owing to shearing the characteristic of the DNAzyme of RNA, detect different RNA and will design corresponding DNAzyme, the present invention has very high specificity.
To be reaction solution can carry out of the present invention one large feature under 37 ℃ that optimize or 39 ℃ of constant temperatures.Owing to adopting strand displacement isothermal duplication RNA to carry out under constant temperature, thus temperature needn't periodically be changed up and down, thus can adopt easy device to realize the detection of the quantitative and qualitative analysis of RNA.
In addition, detection by quantitative of the present invention can adopt single stage method to implement in a reaction vessel, makes to operate further easy, and fast has reduced contaminated danger simultaneously.
Accompanying drawing explanation
Fig. 1 is the RNA schematic diagram that strand displacement amplification is sheared;
As shown in the figure, be first that the DNAzyme that shears RNA shears RNA at specific site, be then to carry out strand displacement isothermal duplication RNA to produce a series of DNA oligonucleotide small segment trigger, cause a series of SDA amplification, finally produce a large amount of reporter groups.Character by reporter group reaches the object that detects RNA.
Fig. 2 is the polyreaction figure after RNA is sheared;
Wherein A represents that 10-23DNAzyme (Gerald, 1997) shears RNA and produces 2 ', 3 ' ring-type phosphate group, but is unworthy of (A-A) to the appearance without extension due to terminal bases; B represents that extension appears in the DNAzyme (shearing end pairing A-T) of improvement.
Fig. 3 is that the RNA of present method detects schematic diagram.
In Fig. 4 A represent malachite green in conjunction with the G-tetramer schematic diagram as reporter; B represents that real-time quantitative detects the Flag-RNA result schematic diagram of gradient dilution; C represents the canonical plotting of real-time quantitative detected result.
Fig. 5 is single-minded detection by quantitative Let-7a result figure.
Embodiment
With specific embodiment, technical scheme of the present invention is described below; but be not the restriction to technical scheme of the present invention; those skilled in the art should understand that; still can modify or be equal to replacement invention; and not departing from any modification or partial replacement of the spirit and scope of the present invention, it all should be encompassed among protection scope of the present invention.
Embodiment 1: the strand displacement amplification experiment that the RNA of take is primer
(1) adopt the DNAzyme that shears RNA to shear the extension after RNA
First use in this experiment a kind of DNAzyme, in reaction solution 10 μ L systems, comprise 50nm Flag-RNA, 2 μ M DNAzyme, 10mM Tris-HCl (pH8.5), 100mM KCl, 10mM MgCl
2, and 0.1mg/ml BSA, 0.5unit/ μ l PNK and 0.4unit/ μ l Bsm archaeal dna polymerase, react after 1h at 37 ℃, with the polyacrylamide gel (PAGE) of sex change, analyzes, and without extending band, occurs.At this, adopt the DNAzyme of improvement, i.e. the terminal bases of RNA after being sheared and DNAzyme (fCat) complementary pairing of improvement.Through above same reaction conditions, there is extending band.Wherein the effect of PNK is 2 ', 3 ' the ring-type phosphate group of shearing end of hydrolysis RNA, and can be used as primer after RNA is sheared carries out extension.As shown in Figure 2.
In reaction solution, add certain density Flag-RNA, 0.5 μ M fCat, 0.25unit/ μ l PNK, 10mM Tris-HCl (pH8.5), 100mM KCl, 10mMMgCl
2, and 0.1mg/ml BSA, add ddH
2o adds to 10 μ L.
1 negative control pipe, does not only add Flag-RNA, and all the other are identical.All be placed under 37 ° of C and react 2 hours 30 minutes.
Flag-RNA(SEQ?ID?NO1)：
5’-GGAGGACGAAAUGGACUACAAGGACGAUGACGAUAAGCAGCUGCGUAACUCUAAAAA-3’
10-23(SEQ?ID?NO2)：
5’-CTT?ATC?GTC?A?GGC?TAG?CTA?CAA?CGA?C?GTC?CTT?GTAGTC?CAT-3’
fCat(SEQ?ID?NO3)：
5’-CAACAGCGACCCTCAGCCTTATCGTCAGGCTAGCTACAACGTCGTCCTTGTAGTCCAT-3’
(2) RNA being sheared prepared by (1) of take is primer, carries out strand displacement amplification reaction
Because the different G-tetramers has different character, so when quantitative and qualitative detection, the present invention adopts the probe that produces different G-tetramer sequences.The probe Ta of qualitative detection comprises by 3 ' to 5 ' and take the complementary sequence of the first paragraph oligonucleotide sequence (trigger) of RNA under primer amplification is sheared, restriction endonuclease recognition sequence, and has the sequence of PW-17 complementary pairing of peroxidase activity and the sequence identical with the partially catalyzed center of fCat.As shown in Figure 3.
Ta(SEQ?ID?NO4)：
5’-CTTATCGTCAAGCTAACTATTTCCCAACCCGCCCTACCCTCAGCCAACAGCGACCCTCA-3’
In (1) reaction solution, add 0.25 μ M probe Ta (qualitative probe), dNTPs (250 μ m), Nb.Bpu10I (0.125UmL/ μ l), Klenow exo-polymerase (0.125U m L/1), adds dd H
2o adds to 20 μ L, is placed at 37 ℃ and reacts 1h.
(3) qualitative detection of amplified production
1 of the negative control pipe of a qualitative detection, only contains ddH
2o.
Reaction tubes is at 94 ℃ of sex change 1min, and negative control pipe and reaction tubes all add 1.7 μ lTris-HCl (pH7.3), hemin (1.8 μ M), ABTS
2-(2.1mM), room temperature is placed 5min, H
2o
2(2.1mM).By digital camera record colour developing result.Wherein 94 ℃ of heating 1min, are conducive to avoid the probe sealing G-tetramer, to increase sensitivity.Then add Hemin and ABTS
2-, room temperature is placed 5min, and the G-tetramer is fully combined with Hemin, then add H
2o
2observe colour-change.Be conducive to make the signal of 2 negative control pipes lower, thereby make detected result sensitiveer.
The Chinese and English full name of the colouring reagents wherein occurring is as shown in table 1 below.
Table 1 colouring reagents Chinese and English full name
(4) qualitative detection result
Under the Flag-RNA of gradient dilution concentration, concentration from high to low presents the colour-change trend weakening gradually.When concentration is 1fmol, still there is macroscopic difference with blank pipe.Illustrate that the inventive method has higher sensitivity.
Embodiment 2: malachite green is developed as quantitative reporter experiment in conjunction with the Hum21 DNAzyme with G-tetramer structure of fluorescence (can be combined with dyestuff produce)
Quantitative probe Tb of the present invention is by 3 ' to the 5 ' sequence comprising with trgger complementation, and restriction endonuclease recognition sequence and can be combined with malachite green the sequence of the hum21 complementary pairing that produces fluorescence and the sequence identical with the partially catalyzed center of fCat.When primer is combined with Tb, occur to connect displacement amplification, thereby produce a large amount of Hum21, utilize this G-tetramer to be combined the character of generation fluorescence with malachite green, along with the accumulation of signal reaches real-time detection.Therefore, illustrate that malachite green can develop as quantitative reporter in conjunction with Hum21.
1 negative control pipe: the negative control pipe that does not add the malachite green of trigger
Do respectively two repetitions, be placed in real-time fluorescence quantitative PCR instrument (4 passages, excite at 620-650nm, are transmitted in 675-690nm), response procedures is: 39 ℃, 70 circulations, read number one time every 1min.The results are shown in accompanying drawing 4.
For detection by quantitative RNA, the present invention adopts single stage method to implement in a reaction vessel, makes to operate further easy, and fast has reduced contaminated danger simultaneously.In single stage method reaction, first the DNAzyme (fCat) of improvement shears RNA, with the effect of PNK, is 2 ', 3 ' ring-type phosphate group of the shearing end of hydrolysis RNA, and then cloning RNA occurs even to replace.By quantitative reporter, reached the object that detects RNA.
1 negative control pipe: just do not add Flag-RNA, all the other are identical.
Do respectively two repetitions, be placed in real-time fluorescence quantitative PCR instrument (Chanel4, Excitation600-640nm, Emission666-740nm.; PikoREAL, Thermo, USA) in.Response procedures is: 39 ℃, 120 circulations, read number one time every 1min.The results are shown in, detected result shows that this method has good discrimination to the Flag-RNA of gradient dilution, and becomes good linear relationship, R
2=0.969.
The RNA (sequence is as shown in table 2) of chemosynthesis Let-7 family, take Let-7 as example, and design can be sheared the DNAzyme (Let-7a-Cat) of the improvement of Let-7, and qualitative checking method is with embodiment 1, and quantitative detecting method is with embodiment 3.Detected result shows that this method can well detect Let-7a analogue, even if analogue and Let-7a exist very fine distinction, even the difference of one to two base all can differentiate by this method.Result as shown in Figure 5.
The RNA of table 2Let-7 family
Get single stranded DNA, the full RNA in double-stranded DNA and HepG2 cell, detection method is with embodiment 1.
2 positive control pipes: containing Flag-RNA, containing the full RNA in Flag-RNA and HepG2 cell.
Result shows single stranded DNA, and the full RNA in double-stranded DNA and HepG2 cell all shows colourless, only, in the situation that Flag-RNA exists, just has the generation of color reaction.Illustrate that the method has very high specificity.
Claims (9)
1. an isothermal detection methods of RNA, comprises following step:
A) DNAzyme based on shearing RNA, realizes the fixed point of RNA is sheared;
At polynueleotide kinase, under archaeal dna polymerase and restriction enzyme effect, there are three strand displacement isothermal duplication circulations in the RNA b) being sheared;
Wherein, through first strand displacement isothermal duplication circulation, discharge a series of DNA oligonucleotide small segment trigger; Then the oligonucleotide probe that can produce the tetrameric complementation of G-containing in testing sample is combined and second strand displacement isothermal duplication circulation occurs is discharged the G-tetramer; This G-tetramer with there is the DNAzyme that shears RNA function and be combined and the 3rd strand displacement isothermal duplication circulation occurs discharge again small segment trigger, cause again thus second strand displacement isothermal duplication, discharge the G-tetramer, this G-tetramer causes again the 3rd strand displacement isothermal duplication, then second and the 3 two moves in circles and carries out, thus the lower a large amount of G-tetramer of final displacement;
C) according to the tetrameric character of G-discharging, can be used as qualitative reporter group or quantitative reporter group, RNA is carried out to qualitative or real-time quantitative detection;
Wherein, described described RNA comprises mRNA and miRNA, and is the RNA that contains AU or GU, and AU and the frequency of occurrences of GU in RNA are that approximately every 8 bases just there will be once.
2. according to the process of claim 1 wherein that the DNAzyme of the described shearing RNA terminal bases complete complementary after being sheared with RNA matches, comprise catalytic activity loop section and with brachium conjunctivum two portions of RNA complementary pairing.
3. according to the method for claim 1, wherein said polynueleotide kinase is to have hydrolysis 2 ', the nucleoside monophosphate kinase of 3 '-ring-type phosphate group, archaeal dna polymerase be normal temperature there is strand displacement ability and without the polysaccharase of exonuclease activity, restriction enzyme is to identify double-stranded specific site, and shears the restriction endonuclease of strand specific site.
4. according to the method for claim 3, wherein said polynueleotide kinase can be T4 polynueleotide kinase (PNK), and archaeal dna polymerase can be Bsm archaeal dna polymerase, and restriction enzyme can be Nb.Bpu10I.
According to the process of claim 1 wherein described probe by 3 ' to 5 ' comprise with the sequence of small segment trigger complementation, restriction endonuclease recognition sequence, with the sequence of G-tetramer complementation and the sequence identical with the partially catalyzed center of DNAzyme of shearing RNA.
6. according to the process of claim 1 wherein that the described G-tetramer refers to, be rich in the four chain helical dna structures that interaction of hydrogen bond that the DNA fragmentation G base of guanylic acid (dG) matches by Hoogsteen forms.
7. according to the process of claim 1 wherein that the G-tetramer of the described qualitative reporter group of conduct is the DNAzyme with peroxidase activity, the result of its acquisition is macroscopic a kind of reporter group.
8. according to the process of claim 1 wherein that the G-tetramer of described quantitative reporter group is can be combined with a kind of dyestuff to produce the DNAzyme of fluorescence, be a kind of reporter group that can obtain quantitative result.
9. method according to Claim 8, wherein said dyestuff is a kind of triphenylmethane organic substance-malachite green.
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