CN106755348A - The detection method of microRNA detection probes group and microRNA - Google Patents
The detection method of microRNA detection probes group and microRNA Download PDFInfo
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Abstract
The present invention relates to field of molecular detection, more particularly to microRNA detection probes group and microRNA detection method.The invention provides the detection method for microRNA detection probes group and microRNA, the probe groups are reasonable in design, and replacing reaction using chain causes that fluorescence signal obtains Cascaded amplification, it is achieved thereby that the detection to intracellular miRNA.The method is simple to operate without strict operating condition, particularly has extraordinary advantage in the cell or tissue of miRNA molecule low expression.Solve the great difficulty lost along with object chain in signaling molecule output in there is detection architecture in detection technique before.Experiment shows that the probe provided using the present invention is detected to miRNA, fluorescence signal can be made to reach highest in 1h or so, and at utmost avoid the generation of fluorescence signal leakage phenomenon.
Description
Technical field
The present invention relates to field of molecular detection, more particularly to microRNA detection probes group and microRNA detection
Method.
Background technology
MicroRNA (miRNA) is raw, the non-coding single strand RNA molecule of about 19-24 nucleotides of length in a class.
The expression of open gene and then modulin after can be good at recognizing and matching due to miRNA, thus it with animal body in
Bioprocess is closely bound up.At present there are some researches show：The abnormal behaviour of miRNA can cause the appearance of disease, including painstaking effort
Pipe disease, neurodevelopment is not normal, or even cancer [Nature, 2012,482,347.].The overexpression of such as miRNA-32 is prostatitis
The basic important symbol thing [Oncogene 2012,31,4460.] of gland cancer, miRNA-122 is also fallen ill as HCC
A base mark thing [J.Am.Chem.Soc.2010,132,7976.].So in the diagnosis of cancer and therapeutic process
The concentration of miRNA is all an important Biological indicators.
At present, general miRNA detection methods have qRT-PCR, Northern blot, oligonucleotide microarray
[Chem.Rev., 2013,113,6207.], wherein Northern Blotting are most classical miRNA detection means, but
Itself detection sensitivity is relatively low, and needs substantial amounts of sample.And microarray has the advantage that high flux is detected, but detect
The time of needs is very long, and precision is not high.The characteristics of qRT-PCR detection means has high accuracy, high sensitivity, however it is necessary that
Prolonged amplification step and there are certain skill set requirements to operating personnel, this is one and very big asks in real-time detection
Topic.But a maximum problem of the above method is all of experiment is all based on cell cracking treatment, it is impossible to reflection in real time
Go out the mechanism of action of tumor tissues and the real miRNA of inside tumor cells.
Therefore in order to be able to more understand the miRNA real informations that controlling gene is expressed in the cell and exploration in depth
The dynamic process of miRNA reactions in the cell, realizes that the detection of intracellular miRNA is then particularly important.Nowadays it is internal
MiRNA detection means is broadly divided into hybridization in situ technique and dynamic imaging techniques.Molecular beacon is based in dynamic imaging techniques
Imaging is a very important means.It is by special rotaring dyeing technology that the molecular beacon importing of fluorescence labeling is intracellular, point
The transformation of recurring structure can cause the appearance of fluorescence signal after sub- beacon and the pairing of target miRNA molecule, finally realize miRNA's
Detection.[Biomaterials, 2012,33,6430.] but there are problems that in these technologies one it is obvious be exactly each letter
The output of number molecule is all along with the consumption of target molecule.And the miRNA molecule expression in tumour cell or tumor tissues is non-
Normal is limited, so above-mentioned technology has very big limitation in the detection for realizing miRNA molecule in tumor tissues living cells.
Therefore, in order to overcome the difficulty of intracellular miRNA molecule detection in the prior art, present inventor has performed deeply grinding
Study carefully, it is found that replacing reaction using the DNA of cascade realizes that microRNA detections have simple operations in tumour cell or tissue, can
To realize the features such as low concentration is detected, but, how to avoid concatenated dna chain from replacing letting out for the fluorescence signal of generation in course of reaction
Dew phenomenon, is still this area problem demanding prompt solution.
The content of the invention
In view of this, the technical problem to be solved in the present invention is to provide microRNA detection probes group and microRNA
Detection method, the probe that the present invention is provided sets reasonable, can be prevented effectively from the leakage of fluorescence signal, improves the special of detection
Property.
The probe groups of the detection microRNA that the present invention is provided, are made up of, respectively 4 DNA probes：Substrate chain, signal
Chain, leading fuel chain and protection fuel chain；
The substrate chain includes the complementary series of the quenching group, quasi- cohesive end and microRNA to be measured that are linked in sequence；
The signal chains are complementary with substrate chain, and described signal chains one end is modified with fluorophor；
The protection fuel chain is complementary with microRNA to be measured；
The leading fuel chain includes the consistent DNA sequence dna of the microRNA sequences to be measured being linked in sequence and complementary quasi- viscosity
End.
In probe groups of the invention, quenching group is located at 3 ' ends of substrate chain, then fluorophor is located at 5 ' ends of signal chains；
Quenching group is located at 5 ' ends of substrate chain, then fluorophor is located at 3 ' ends of signal chains.In the embodiment of the present invention, quenching group position
In 5 ' ends of substrate chain, and fluorophor is located at 3 ' ends of signal chains.
Sequences Design of the probe groups that the present invention is provided according to microRNA to be measured.Wherein substrate chain is mutual with signal chain part
Mend, protect fuel chain and leading fuel chain part complementary.The original that the probe groups provided with the present invention are detected to miRNA to be measured
Reason such as Fig. 1：
First, substrate chain and signal chains are hybridized, substrate chain is formed double-strand A with signal chains.After forming double-strand, signal chains
The fluorophor of end can very be close to the quenching group of substrate chain end, and fluorophor is quenched, and does not have fluorescence signal
Output.Then, by leading fuel chain and the chain hybridization of protection fuel, double-strand B is formed.Then, by double-strand A and double-strand B in transfection
In the presence of agent, it is transfected into animal body (or human body), tissue or cell.When double-strand A and double-strand B enters organism (or people
Body), in tissue or cell, in the presence of salt ion, there is chain and replace reaction, reactions steps are：
First, miRNA to be measured is acted on the exposed one end on double-strand A first, and then chain replacement reaction occurs, wherein, treat
Survey miRNA and form intermediate 1 with substrate chain；And signal chains are then released, fluorophor is separated with quenching group, sends fluorescence letter
Number；
2nd, with double-strand B there is chain replacement reaction in intermediate 1, wherein, miRNA forms intermediate 2 with protection fuel chain；And
Leading fuel chain and substrate chain combination form double-strand C, no longer light；
3rd, with double-strand A there is chain replacement reaction in intermediate 2, wherein, miRNA forms intermediate 1 with substrate chain combination, and
Protection fuel chain is then combined with signal chains, forms the signal chains of double-strand, and fluorophor is separated with quenching group, sends fluorescence.
In above process, DNA is replaced reaction and is caused, signal chains are released as single-stranded or and are chained with protection fuel
Conjunction forms double-strand signal chains, so that fluorophor is able to send fluorescence.And because miRNA forms centre with protection fuel chain
After body 2, remain able to for triggering the reaction with double-strand A, so that the DNA of cascade replaces reaction constantly occurring, realize
Fluorophor is constantly released, and then realizes fluorescence signal and be constantly exaggerated.The present invention takes full advantage of the DNA of cascade
Ability of the reaction in signal amplification is replaced, the detection of intracellular miRNA molecule is finally realized.
The method that the present invention is provided no longer needs to separate miRNA from sample, but is transfected into animal by by probe
In body (or human body), tissue or cell, by the detection to fluorescence signal, realize to internal, or intracellular miRNA molecule
Detection such that it is able to more understand the real information of miRNA controlling gene expression in the cell in depth and explore miRNA thin
The dynamic process of the reaction of intracellular.
Just there is chain in vitro in order to avoid probe and replace reaction, the present invention is to 4 length of probe, quasi- cohesive end
The position of length, the length of complementary series, fluorophor or quenching group and species are studied.
In the present invention, the complementary series of the quenching group, quasi- cohesive end and microRNA to be measured of the connection of substrate chain sequence；
Signal chains are complementary with substrate chain, and signal chains one end is modified with fluorophor.
In the present invention, the length of quasi- cohesive end is 4bp~5bp on substrate chain.Preferably, quasi- cohesive end on substrate chain
Length be 5bp.In the present invention, one end 5bp~6bp shorter than substrate chain of the unmodified fluorophor of signal chains.Preferably, signal
One end 6bp shorter than substrate chain of the unmodified fluorophor of chain.In the present invention, quenching group is BHQ-1, Dabcyl or BHQ-2；Institute
Fluorophor is stated for FAM or Cy3, Cy-5.
It is the complementary series of microRNA to be measured than the part that signal chains grow on substrate chain.Form exposed with complementary series
End, is conducive to chain to replace the generation of reaction, and experiment shows, the exposed end of 6bp is more beneficial for generation chain and replaces reaction, produces glimmering
The time of light is short and fluorescent value is higher.
In certain embodiments, 5 ' ends of substrate chain to 3 ' ends are sequentially connected quenching group, quasi- cohesive end and to be measured
The complementary series of microRNA.Wherein, quenching group is BHQ-2；The length of quasi- cohesive end be 5bp, it is described with it is to be measured
The complementary series of microRNA is the DNA sequence dna with microRNA total lengths complete complementary to be measured.The sequence of the quasi- cohesive end
Substrate chain is not set to produce secondary structure, such as hairpin structure.
In certain embodiments, signal chains 3 ' end to 5 ' end be sequentially connected fluorophor, complementary quasi- cohesive end and with treat
Survey the consistent DNA sequence dna of miRNA sequence.Wherein, fluorophor is Cy-5；The length of complementary quasi- cohesive end is 5bp, complementary accurate
Quasi- cohesive end complete complementary on cohesive end and substrate chain；On the DNA sequence dna consistent with miRNA sequence to be measured and substrate chain
Respective segments partial complementarity, the fragment compared with miRNA 5 ' end lack 6bp.
After the double-strand A that miRNA to be measured and signal chains and substrate chain are formed occurs chain replaces reaction, by miRNA and substrate
In the intermediate 1 that chain is formed, quasi- cohesive end is exposed on substrate chain, forms exposed end.It is middle in the embodiment of the present invention
The length of the exposed end in body 1 is 5bp.
In the present invention, the protection fuel chain is complementary with microRNA to be measured；The leading fuel chain including order connect
The microRNA sequences to be measured that connect consistent DNA sequence dna and complementary quasi- cohesive end.
In the present invention, the length for protecting fuel chain is 14bp~15bp.Preferably, the length of protection fuel chain is 14bp.
Preferably, sequence length complementary with microRNA to be measured on protection fuel chain is 13bp, in 5 ' end additions one of the sequence
Base, to promote chain to replace the generation of reaction.
In the present invention, complementary quasi- cohesive end on fuel chain is dominated complementary with quasi- cohesive end on the substrate chain；
The DNA sequence dna length consistent with microRNA sequences to be measured is 17bp~19bp on the leading fuel chain.
Preferably, DNA sequence dna length consistent with microRNA sequences to be measured on the leading fuel chain is 18bp.
In the present invention, fuel chain is dominated accurate into the complementation on the 3 ' ends for after double-strand B, dominating fuel chain with protection fuel chain
Cohesive end is exposed to be come, and the quasi- cohesive end on the end and substrate chain is complementary；5 ' the exposed 4bp in end of leading fuel chain.
During the course of the reaction, the exposed quasi- cohesive end of substrate chain is exposed with leading fuel chain in double-strand B in intermediate 1
Complementary quasi- cohesive end combine so that guiding chain replaces the generation of reaction, substrate chain and leading fuel chain combination form double-strand C
No longer light.And miRNA and protection fuel chain are then combined, intermediate 2 is formed.In intermediate 2,5 ' the exposed 9bp in end of miRNA,
The exposed part can be exposed with substrate chain in double-strand A partial complementarity so that guiding chain replace reaction proceed.
The present invention enables intracellular miRNA to be measured to trigger the DNA of cascade by reasonable design dna molecular probe
The generation of reaction, and then effective signal chains of the release with fluorophor modification are replaced, under conditions of limited object chain,
The presence of two double-strand B of fuel chain composition can to greatest extent amplify the signal chains with fluorophor modification for being discharged
Number, and then finally realized in the cell or in tumor tissues by laser confocal microscope or small animal imaging instrument
The detection of miRNA.And by the design of reasonable probe, effectively prevent the leakage of fluorescence signal.And the design of probe is cleverly
Avoid chain and replace the reverse generation of reaction, so that chain replaces reaction once occurring, just no longer reversible, the signal being released
Chain will not again with substrate chain combination.
The probe that the present invention is provided replaces the Cascaded amplification that fluorescence signal is realized in reaction by chain, because chain replaces reaction
Occurrence condition is simultaneously uncomplicated, widely occurs in cell, tissue or organism.As long as design of primers is rationally, chain replaces reaction
Can occur.Therefore, the form design probe of probe groups is provided according to the present invention, all can be real to not homotactic microRNA
Now detect.The probe groups can be widely used in the detection of various microRNA.But it is the length of quasi- cohesive end in probe, naked
Reveal the generation that the length at end, the length of complementary series may influence fluorescence signal.
The DNA replacement reaction that the probe groups that the present invention is provided are based on cascade is realized in intracellular or tumor tissues
MiRNA detects particularly there is extraordinary advantage in the cell or tissue of miRNA molecule low expression.Solve at it
There is the great difficulty lost along with object chain in signaling molecule output in detection architecture in preceding detection technique.The model of detection
Enclose and be also applied for other kinds of non-coding RNA molecule.The same technology is also the one kind in cell and other imaging of tissue
Important means.
The probe groups that the present invention is provided can be used in the detection of microRNA in living animal body, it can also be used to culture it is thin
The detection of the microRNA of intracellular, it is also possible to the detection with microRNA in vitro tissue.
The probe provided by the present invention, according to the situation that fluorescence after transfection is produced, can realize to the exhausted of microRNA
Quantitative determination or relative quantification are detected, so as to judge the expression of microRNA in product to be tested.Produced according to fluorescence after transfection
Raw position, can also realize the detection to microRNA distribution situations.And can realize expressing miRNA by repeated detection
The detection of the situation that changes with time.
Application of the probe groups that the present invention is provided in the product of intracellular detection of microRNA is prepared.
Because result of study shows in the prior art, the unconventionality expression of various miRNA is related to cancer, so the application is carried
The probe groups of confession can realize the imaging to tumor tissues, and can be according to imaging contexts, the tumorigenic degree of position judgment
And position.
Application of the probe groups that the present invention is provided in the product for preparing tumor tissues imaging.
Present invention also offers a kind of detection kit of microRNA, including the probe groups that the present invention is provided.
In the microRNA detection kits that the present invention is provided, also including transfection reagent.
The transfection reagent is Lipofectamine 2000.
In the microRNA detection kits that the present invention is provided, also including hybridization buffer.
The hybridization buffer is that concentration is the Tris-HCl buffer solutions of 10mmol/L.
Tris-HCl includes 0.3mol/L NaCl and 5mmol/L MgCl2, pH value is 7.5.
In the microRNA detection kits that the present invention is provided, substrate chain, signal chains, leading fuel chain and protection fuel chain
Mol ratio be (0.8~1.2):(0.8~1.2):(2~4):(2~4).
Present invention also offers a kind of method for detecting intracellular microRNA, including：
Step 1：In the probe groups that the present invention is provided, the substrate chain hybridizes to form double-strand A with the signal chains；It is described
Leading fuel chain and protection fuel chain hybridize to form double-strand B；
Step 2：After the double-strand A and double-strand B is transfected into cell to be measured, tissue or animal body, imaging.
In the method provided by the present invention, substrate chain is 1: 1 with the mol ratio of signal chains hybridization.
The buffer solution of hybridization is that concentration is the Tris-HCl buffer solutions of 10mmol/L.
Tris-HCl includes 0.3mol/L NaCl and 5mmol/L MgCl2, pH value is 7.5.
The condition that substrate chain hybridizes with signal chains is：90 DEG C of holding 15min, are then cooled to room temperature, keep at room temperature not
Less than 2h.Preferably, room temperature is 18 DEG C~30 DEG C.Retention time is 2h.Double-strand A after hybridization be stored in 4 DEG C it is stand-by.
In the method provided by the present invention, the mol ratio for dominating fuel chain and the chain hybridization of protection fuel is 1: 1.
Leading fuel chain and the condition of protection fuel chain hybridization are：90 DEG C of holding 15min, are then cooled to room temperature, room temperature
It is lower to keep being no less than 2h.Preferably, room temperature is 18 DEG C~30 DEG C.Retention time is 2h.Double-strand A after hybridization is stored in 4 DEG C and treats
With.
The concentration that hybridization obtains double-strand A is 10 μm of ol/L；The concentration that hybridization obtains double-strand B is 20 μm of ol/L.
The double-strand solution A and double-strand B solution that obtain will be hybridized with volume ratio 1:2 mixing after, add transfection reagent, then with
After hybridization buffer constant volume, 20min is combined, Transfection solution is obtained.Transfection solution is used to transfect testing sample.Wherein, described turn
Transfection reagent is Lipofectamine 2000.The consumption of transfection reagent is the 1/5 of double-strand B solution volume.Being settled to final volume is
2 times of double-strand B solution volume.
In final obtained Transfection solution, the concentration of double-strand A is 2.5 μm of ol/L；The concentration of double-strand B is 10 μm of ol/L.
After transfection, interval 0.5h~24h detection fluorescence signals.
For testing in vitro experiment, 0.5h~2h fluorescence signal intensities are that can reach peak value after transfection.And turn for injection
The animal body of dye, fluorescence signal needs the time of 24h or so, and enrichment reaches peak value in tumor tissues.
After transfected animal body, on the one hand probe groups are enriched in tumor tissues, are on the one hand discharged through kidney and liver metabolism
In vitro.Mainly excreted through kidney.
A kind of probe groups of detection miRNA-21 are supplied in the embodiment of the present invention, by SEQ ID NO：Core shown in 1~4
4 DNA probes composition of nucleotide sequence；
Wherein, SEQ ID NO:5 ' terminal modified quenching groups of the probe of nucleotide sequence shown in 1；
SEQ ID NO:3 ' terminal modified fluorophors of the probe of nucleotide sequence shown in 2.
Wherein, SEQ ID NO:The probe of nucleotide sequence shown in 1 is substrate chain；SEQ ID NO:Nucleotides sequence shown in 2
The probe of row is signal chains；SEQ ID NO:The probe of nucleotide sequence shown in 3 is main pilot material chain；SEQ ID NO:Shown in 4
The probe of nucleotide sequence is protection fuel chain.
The embodiment of the present invention proves, the probe combinations that the present invention is provided for other designs, can compared with
Fluorescence signal is set to reach peak value in short time, and fluorescence intensity is stronger, fluorescence signal leakage phenomenon is lowered.
The overexpression of miRNA-21 is closely related with tumour, such as breast cancer, cervical carcinoma.
By SEQ ID NO：Nucleotide sequence shown in 1~4 4 DNA probes composition probe groups prepare tumor tissues into
The application of the product of picture.The tumour is breast cancer.Mouse breast cancer model is built by MCF-7 cell lines.
MiRNA-21 detection kits are included by SEQ ID NO：4 DNA probe groups of nucleotide sequence shown in 1~4
Into probe groups, transfection reagent and hybridization buffer.
The transfection reagent is that the transfection reagent is Lipofectamine 2000.
The hybridization buffer is that concentration is the Tris-HCl buffer solutions of 10mmol/L.
In miRNA-21 detection kits, the mol ratio of substrate chain, signal chains, leading fuel chain and protection fuel chain is
(0.8~1.2):(0.8~1.2):(2~4):(2~4).
The embodiment of the present invention additionally provides a kind of method of tumor tissues imaging, including：
Step a：By SEQ ID NO:The probe of nucleotide sequence shown in 1~2 hybridizes to form double-strand A；By SEQ ID NO:3
The probe of nucleotide sequence shown in~4 hybridizes to form double-strand B；
Step b：After the double-strand A and double-strand B is transfected into test serum or animal body, imaging.
In the embodiment, SEQ ID NO:Substrate chain shown in 1 and SEQ ID NO:Signal chains shown in 2 hybridization mol ratio be
1∶1。
The buffer solution of hybridization is that concentration is the Tris-HCl buffer solutions of 10mmol/L.
Tris-HCl includes 0.3mol/L NaCl and 5mmol/L MgCl2, pH value is 7.5.
SEQ ID NO:Substrate chain shown in 1 and SEQ ID NO:Signal chains shown in 2 hybridization condition be：90 DEG C of holdings
15min, is then cooled to room temperature, keeps being no less than 2h at room temperature.Preferably, room temperature is 18 DEG C~30 DEG C.Retention time is 2h.
Double-strand A after hybridization be stored in 4 DEG C it is stand-by.
In the method provided by the present invention, SEQ ID NO:Fuel chain and SEQ ID NO are dominated shown in 3:Protection fuel chain shown in 4
The mol ratio of hybridization is 1: 1.
SEQ ID NO:Fuel chain and SEQ ID NO are dominated shown in 3:The conditions of the hybridization of protection fuel chains shown in 4 are：90℃
15min is kept, room temperature is then cooled to, keeps being no less than 2h at room temperature.Preferably, room temperature is 18 DEG C~30 DEG C.Retention time
It is 2h.Double-strand A after hybridization be stored in 4 DEG C it is stand-by.
The concentration that hybridization obtains double-strand A is 10 μm of ol/L；The concentration that hybridization obtains double-strand B is 20 μm of ol/L.
The double-strand solution A and double-strand B solution that obtain will be hybridized with volume ratio 1:2 mixing after, add transfection reagent, then with
After hybridization buffer constant volume, 20min is combined, Transfection solution is obtained.Transfection solution is used to transfect testing sample.Wherein, described turn
Transfection reagent is Lipofectamine 2000.
The consumption of the transfection reagent is related to the molal quantity of double-strand A or the molal quantity of double-strand B.
For mouse, when constant volume (total solution volume) is 300 μ L, for 75 μ L, (concentration is 10 to the volume of double-strand A
μm ol/L), then the consumption of transfection reagent is 30 μ L.
In final obtained Transfection solution, the concentration of double-strand A is 2.5 μm of ol/L；The concentration of double-strand B is 10 μm of ol/L.
The transfection is specially Transfection solution by mouse tail vein injection to Mice Body.After transfection, interval 24h inspections
Survey fluorescence signal.
The invention provides the detection method for microRNA detection probes group and microRNA, probe groups design is closed
Reason, replaces reaction and causes that fluorescence signal obtains Cascaded amplification, it is achieved thereby that the detection to intracellular miRNA using chain.The party
Method is simple to operate without strict operating condition, particularly has very in the cell or tissue of miRNA molecule low expression
Good advantage.Solve in there is detection architecture in detection technique before and lost along with object chain in signaling molecule output
Great difficulty.Experiment shows that the probe provided using the present invention is detected to miRNA, and fluorescence signal can be made left in 1h
The right side reaches highest, and at utmost avoids the generation of fluorescence signal leakage phenomenon.
Brief description of the drawings
Fig. 1 shows that the present invention provides the detection method principle of probe groups；
Fig. 2 shows the experimental result of embodiment 1；
Fig. 3 shows the experimental result of embodiment 2；
Fig. 4 shows the mouse tissue imaging organs of miRNA-21 examinations of the DNA molecular probe in MCF-7 tumor models
Figure, wherein, double-strand A concentration is 2.5 μM, and double-strand B concentration is 10 μM；
Fig. 5 shows the mouse tissue imaging organs of miRNA-21 examinations of the DNA molecular probe in MCF-7 tumor models
Figure；Double-strand A concentration is 2.5 μM, and double-strand B concentration is 0 μM；
Fig. 6 shows the mouse tissue imaging organs of miRNA-21 examinations of the DNA molecular probe in MCF-7 tumor models
Figure；Double-strand A concentration is 0 μM, and double-strand B concentration is 0 μM；
Fig. 7 shows in miRNA-21 examinations complex chart 2, Fig. 3 and Fig. 4 of the DNA molecular probe in MCF-7 tumor models
Mouse tumor imaging of tissue figure.
Specific embodiment
The invention provides the detection method of microRNA detection probes group and microRNA, those skilled in the art can be with
Present disclosure is used for reference, technological parameter realization is suitably modified.In particular, all similar replacements and change are to ability
It is it will be apparent that they are considered as being included in the present invention for field technique personnel.The method of the present invention and application have been led to
Cross preferred embodiment to be described, related personnel can be not substantially being departed from present invention, spirit and scope to this paper's
Methods and applications are modified or suitably change is realized and apply the technology of the present invention with combining.
The complete complementary refers to that the base number on two chains is identical, wherein a chain is held and another by 3 ' ends to 5 '
Chain can be all complementary by 5 ' ends to 3 ' ends.
The partial complementarity refers to remove the base that end has more, remainder complete complementary.
The sequence refers to unanimously that in addition to U is replaced with into T, the sequence of RNA is consistent with the sequence of DNA.
It is that DNA replacement reaction (strand displacement reaction) refers to two DNAs that chain replaces reaction
By completely or partially matching the process that the one or more of DNAs that will have previously hybridized are replaced.Replaced in chain and reacted
During, the single stranded DNA region on single stranded DNA part and intrusion chain (invading DNA) on compound (complex) leads to
Base pairing is crossed first to be combined together, then via branch migration (branch migration) forward random walk so as to will be multiple
Previously the DNA of hybridization is replaced on compound.
The examination material that the present invention is used is all common commercially available product, can all be bought in market.
Used in this experiment to the cell of MCF-7 buy in Shanghai Chinese Academy of Sciences cell bank.
All of initial feed, DNA is from raw work (Shanghai) bioengineering limited company or the purchase of Adamas companies
Buy, reagent is not unless explanation is all purified again.All of chemical reagent is all to analyze pure or higher purity, and what is used turns
Transfection reagent is Lipofectamine 2000 (Thermo Fisher), and hyclone is purchased from ExCell companies.
Ultraviolet-uisible spectrophotometer：Cary 300.
Sepectrophotofluorometer：F-7000(Hitachi High-Techonologies Corporation Japan).
Fluorescence microscope：IX71(Olymapus Japan).
Laser confocal fluorescence microscope：Leica TCS SP5.
With reference to embodiment, the present invention is expanded on further：
Embodiment 1
1st, fluorescence signal detection
Experiment is divided into 6 groups, wherein：
Experimental group 1~3 is tested using 4 probes,
And only contain 3 probes (lacking protection fuel chain) in control group 1~3.
Experimental probes design such as table 1：
In each group probe, it is FAM that the fluorophor in signal chains is located at 3 ' ends；
It is BHQ1 that quencher on substrate chain is located at 5 ' ends.
The each group probe sequence of table 1
| Substrate chain | Signal chains | Leading fuel chain | Protection fuel chain | |
| Experimental group 1 | SEQ ID NO:1 | SEQ ID NO:2 | SEQ ID NO:3 | SEQ ID NO:4 |
| Control group 1 | SEQ ID NO:1 | SEQ ID NO:2 | SEQ ID NO:3 | -- |
| Experimental group 2 | SEQ ID NO:5 | SEQ ID NO:6 | SEQ ID NO:7 | SEQ ID NO:8 |
| Control group 2 | SEQ ID NO:5 | SEQ ID NO:6 | SEQ ID NO:7 | -- |
| Experimental group 3 | SEQ ID NO:9 | SEQ ID NO:10 | SEQ ID NO:11 | SEQ ID NO:12 |
| Control group 3 | SEQ ID NO:9 | SEQ ID NO:10 | SEQ ID NO:11 | -- |
2nd, the preparation of DNA probe：
1), each probe is dissolved in 10mM Tris-HCl (pH 7.5,0.3M NaCl, 5mM MgCl respectively2) in buffer solution,
Single-stranded concentration is demarcated using 260nm length ultraviolet absorption values in solution；
2), substrate chain mixes with signal chains equimolar concentration, is kept for 15 minutes at 90 DEG C, and room is then gradually cooled to again
Temperature, keeps room temperature to be annealed, and annealing process was more than 2 hours.Double-strand A is obtained, in 4 DEG C of preservations, concentration is 100nM；
3), dominate fuel chain and protection fuel chain equimolar concentration mixes, holding 15 minutes at 90 DEG C, then again gradually
Room temperature is cooled to, keeps room temperature to be annealed, annealing process was more than 2 hours.Double-strand B is obtained, in 4 DEG C of preservations, concentration is
400nM。
4), with 10mM Tris-HCl (pH 7.5,0.3M NaCl, 5mM MgCl2) solution for dominating fuel chain is prepared, it is dense
Degree is 20 μM stand-by.
3rd, kinetics test
Experimental group：It is 20 μM of double-strands fuel chain (double-strand B), dissolving by the double-strand A that 4 microlitres of concentration is 10 μM, 8 microlitres of concentration
In Tris-HCl (pH 7.5,0.3M NaCl, 5mM MgCl2) buffer solution is to 400 microlitres of cumulative volume.Mixed liquor hybridizes chain concentration
It is 100nM, fuel chain concentration is 400nM.
Control group：By the double-strand A that 4 microlitres of concentration is 10 μM, 8 microlitres of concentration are 20 μM of single-stranded leading fuel chains, dissolving
In Tris-HCl (pH 7.5,0.3M NaCl, 5mM MgCl2) buffer solution is to 400 microlitres of cumulative volume.Mixed liquor hybridizes chain concentration
It is 100nM, fuel chain concentration is 400nM.
37 DEG C, in course of reaction, scan solution fluorescence signal with Fluorescence spectrophotometer at regular intervals.Choose FAM
The fluorescence intensity level of launch wavelength of the absworption peak at 518nm be ordinate, with the time as abscissa, draw each group fluorescence strong
Degree changes over time curve.Result such as Fig. 2, illustrates that the combination of 4 probes is more beneficial for reducing the generation of fluorescence leak case.
Embodiment 2
1st, the detection of in-vitro simulated miRNA-21.
Used as testing sample, concentration is the titer of configuration miRNA-21 (single-stranded with miRNA-21 sequence identicals DNA)
10μmol/L。
Experiment is divided into 6 groups, wherein：
Experimental group 1~3 is tested using 4 probes,
And only contain 3 probes (lacking protection fuel chain) in control group 1~3.
Experimental probes design such as table 1 in embodiment 1：
In each group probe, it is FAM that the fluorophor in signal chains is located at 3 ' ends；
It is BHQ1 that quencher on substrate chain is located at 5 ' ends.
2nd, the preparation of DNA probe：
1), each probe is dissolved in 10mM Tris-HCl (pH 7.5,0.3M NaCl, 5mM MgCl respectively2) in buffer solution,
Single-stranded concentration is demarcated using 260nm length ultraviolet absorption values in solution；
2), substrate chain mixes with signal chains equimolar concentration, is kept for 15 minutes at 90 DEG C, and room is then gradually cooled to again
Temperature, keeps room temperature to be annealed, and annealing process was more than 2 hours.Double-strand A is obtained, in 4 DEG C of preservations, concentration is 100nM；
3), dominate fuel chain and protection fuel chain equimolar concentration mixes, holding 15 minutes at 90 DEG C, then again gradually
Room temperature is cooled to, keeps room temperature to be annealed, annealing process was more than 2 hours.Double-strand B is obtained, in 4 DEG C of preservations, concentration is
400nM。
4), with 10mM Tris-HCl (pH 7.5,0.3M NaCl, 5mM MgCl2) solution for dominating fuel chain is prepared, it is dense
Degree is 20 μM stand-by.
3rd, kinetics test
Experimental group：By the double-strand A that 4 microlitres of miRNA-21 titers, 4 microlitres of concentration are 10 μM, 8 microlitres of concentration are 20 μM double
Chain fuel chain (double-strand B), is dissolved in Tris-HCl (pH 7.5,0.3M NaCl, 5mM MgCl2) buffer solution is micro- to cumulative volume 400
Rise.Mixed liquor hybridization chain concentration is 100nM, and fuel chain concentration is 400nM.
Control group：Just 4 microlitres miRNA-21 titers, 4 microlitres of concentration are 10 μM of double-strand A, and 8 microlitres of concentration are 20 μM
Single-stranded leading fuel chain, is dissolved in Tris-HCl (pH 7.5,0.3M NaCl, 5mM MgCl2) buffer solution is to cumulative volume 400
Microlitre.Mixed liquor hybridization chain concentration is 100nM, and fuel chain concentration is 400nM.
In course of reaction, solution fluorescence signal is scanned with Fluorescence spectrophotometer at regular intervals.Choose the absorption of FAM
The fluorescence intensity level of launch wavelength of the peak at 518nm is ordinate, with the time as abscissa, draws each group fluorescence intensity at any time
Between change curve.Result such as Fig. 3, as a result shows, SEQ ID NO:1~4 probe combinations can be obtained within the shorter time
Fluorescent value higher.
Embodiment 3
1st, the foundation of tumor model：
By MCF-7 (5 × 106Cells/50 μ L) (mouse is dams, about 4 weeks sizes, is weighed about to be transplanted to mouse left side oxter
20 grams).About 2 to 3 time-of-weeks of transplanting can be tested.MiRNA-21 in model mice body is detected,
Substrate chain-ordering (being from left to right 5' -3') is：
BHQ-2-CACAATCAACATCAGTCTGATAAGCTA(SEQ ID NO：1)；
The sequence (being from left to right 5' -3') of signal chains is：
ATCAGACTGATGTTGATTGTG-Cy5(SEQ ID NO：2)；
The sequence (being from left to right 5' -3') of leading fuel chain is：
TTATCAGACTGATGTTGATTGTG(SEQ ID NO：3)；
Protect fuel chain sequence (being from left to right 5' -3') be：
ATCAACATCAGTCT(SEQ ID NO：4).
2nd, the preparation of DNA probe：
1), each probe is dissolved in 10mM Tris-HCl (pH 7.5,0.3M NaCl, 5mM MgCl respectively2) in buffer solution,
Single-stranded concentration is demarcated using 260nm length ultraviolet absorption values in solution；
2), substrate chain mixes with signal chains equimolar concentration, is kept for 15 minutes at 90 DEG C, and room is then gradually cooled to again
Temperature, keeps room temperature to be annealed, and annealing process was more than 2 hours.Double-strand A is obtained, in 4 DEG C of preservations；
3), dominate fuel chain and protection fuel chain equimolar concentration mixes, holding 15 minutes at 90 DEG C, then again gradually
Room temperature is cooled to, keeps room temperature to be annealed, annealing process was more than 2 hours.Double-strand B is obtained, in 4 DEG C of preservations；
Gained double-strand A concentration is 10 μM, and double-strand fuel chain (double-strand B) DNA concentration is 20 μM.
3rd, using commercial transfection agent by DNA probe by mouse tail vein injection in Mice Body：
1), the preparation of transfection cocktail：
Experimental group：By 75 microlitres of concentration is 10 μM double-strand A, 150 microlitres of concentration be 20 μM of double-strands fuel chain (double-strand B) and
30 microlitres of commercial transfection agent Lipofectamine 2000 (Thermo Fisher) are dissolved in Tris-HCl (pH 7.5,0.3M
NaCl,5mM MgCl2) in buffer solution, Tris-HCl buffer solutions to 300 microlitres of cumulative volume are finally added, it is combined 20 minutes, treat
With.Mixed liquor hybridization chain concentration is 2.5 μM, and fuel chain concentration is 10 μM.
Control group：By 75 microlitres of double-strand A, and 30 microlitres of (Thermo of commercial transfection agent Lipofectamine 2000
Fisher Tris-HCl (pH 7.5,0.3M NaCl, 5mM MgCl) are dissolved in2) in buffer solution, finally add Tris-HCl and delay
Fliud flushing is combined 20 minutes to 300 microlitres of cumulative volume, stand-by.Mixed liquor hybridization chain concentration is 2.5 μM, and fuel chain concentration is 10 μM.
2), the DNA probe that will be combined is by mouse tail vein injection to Mice Body.
3), mouse is normally raised 24 hours.
4th, tumor tissues imaging：
By euthanasia mouse, mouse is dissected to mouse organs and tumor tissues imaging, the scanned region of selection is clapped
Take the photograph tumor tissues image.Result such as Fig. 4.
Comparative example 1
1st, the foundation of tumor model：
By MCF-7 (5 × 106Cells/50 μ L) (mouse is dams, about 4 weeks sizes, is weighed about to be transplanted to mouse left side oxter
20 grams).About 2 to 3 time-of-weeks of transplanting can be tested.MiRNA-21 in model mice body is detected,
Substrate chain-ordering (being from left to right 5' -3') is
BHQ-2-CACAATCAACATCAGTCTGATAAGCTA(SEQ ID NO：1),
The sequence (being from left to right 5' -3') of signal chains is
ATCAGACTGATGTTGATTGTG-Cy5(SEQ ID NO：2),
2nd, the preparation of DNA probe：
1), each probe is dissolved in 10mM Tris-HCl (pH 7.5,0.3M NaCl, 5mM MgCl respectively2) in buffer solution,
Single-stranded concentration is demarcated using 260nm length ultraviolet absorption values in solution；
2), substrate chain mixes with signal chains equimolar concentration, is kept for 15 minutes at 90 DEG C, and room is then gradually cooled to again
Temperature, keeps room temperature to be annealed, and annealing process was more than 2 hours.Double-strand A is obtained, in 4 DEG C of preservations；Gained double-strand A concentration is 10 μ
M。
3rd, using commercial transfection agent by DNA probe by mouse tail vein injection in Mice Body：
1), the preparation of transfection cocktail：By 75 microlitres of double-strand A, and 30 microlitres of commercial transfection agent Lipofectamine
2000 (Thermo Fisher) are dissolved in Tris-HCl (pH 7.5,0.3M NaCl, 5mM MgCl2) in buffer solution, finally mend
Plus Tris-HCl buffer solutions are to 300 microlitres of cumulative volume, it is combined 20 minutes, it is stand-by.Mixed liquor hybridization chain concentration is 2.5 μM.
2), the DNA probe that will be combined is by mouse tail vein injection to Mice Body.
3), mouse is normally raised 24 hours.
4th, tumor tissues imaging：
By euthanasia mouse, mouse is dissected to mouse organs and tumor tissues imaging, the scanned region of selection is clapped
Take the photograph tumor tissues image.Result such as Fig. 5.
Comparative example 2
1st, the foundation of tumor model：
By MCF-7 (5 × 106Cells/50 μ L) (mouse is dams, about 4 weeks sizes, is weighed about to be transplanted to mouse left side oxter
20 grams).About 2 to 3 time-of-weeks of transplanting can be tested.MiRNA-21 in model mice body is detected,
2nd, using commercial transfection agent by mouse tail vein injection to Mice Body：
1), the preparation of transfection cocktail：By 30 microlitres of (Thermo of commercial transfection agent Lipofectamine 2000
Fisher Tris-HCl (pH 7.5,0.3M NaCl, 5mM MgCl) are dissolved in2) in buffer solution, finally add Tris-HCl and delay
Fliud flushing is combined 20 minutes to 300 microlitres of cumulative volume, stand-by.
2), by transfection cocktail by mouse tail vein injection to Mice Body.
3), mouse is normally raised 24 hours.
3rd, tumor tissues imaging：
By euthanasia mouse, mouse is dissected to mouse organs and tumor tissues imaging, the scanned region of selection is clapped
Take the photograph tumor tissues image.Result such as Fig. 6.
The above is only the preferred embodiment of the present invention, it is noted that come for those skilled in the art
Say, under the premise without departing from the principles of the invention, can also make some improvements and modifications, these improvements and modifications also should be regarded as
Protection scope of the present invention.
SEQUENCE LISTING
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<120>The detection method of microRNA detection probes group and microRNA
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Claims (13)
1. a kind of probe groups of detection microRNA, are made up of, respectively 4 DNA probes：Substrate chain, signal chains, leading fuel
Chain and protection fuel chain；
The complementary series of the quenching group, quasi- cohesive end and microRNA to be measured including being linked in sequence of the substrate chain；
The signal chains are complementary with substrate chain, and described signal chains one end is modified with fluorophor；
The protection fuel chain is complementary with microRNA to be measured；
The DNA sequence dna consistent including the microRNA sequences to be measured being linked in sequence of the leading fuel chain and complementary quasi- viscosity end
End.
2. probe groups according to claim 1, it is characterised in that the length of quasi- cohesive end is 4bp on the substrate chain
~5bp.
3. probe groups according to claim 1, it is characterised in that compare base in one end of the unmodified fluorophor of signal chains
Short 5bp~the 6bp of bottom chain.
4. probe groups according to claim 1, it is characterised in that the length of the protection fuel chain is 14bp~15bp.
5. probe groups according to claim 1, it is characterised in that complementary quasi- cohesive end on the leading fuel chain with
Quasi- cohesive end on the substrate chain is complementary；The DNA sequence dna consistent with microRNA sequences to be measured on the leading fuel chain
Length is 17bp~19bp.
6. probe groups according to claim 1, it is characterised in that the quenching group is BHQ-1 or BHQ-2；It is described glimmering
Light group is FAM or Cy-5.
7. application of the probe groups described in any one of claim 1~6 in the product of intracellular detection of microRNA is prepared.
8. application of the probe groups described in any one of claim 1~6 in the product for preparing tumor tissues imaging.
9. a kind of detection kit of microRNA, it is characterised in that including the probe groups described in any one of claim 1~6.
10. a kind of method for detecting intracellular microRNA, it is characterised in that including：
Step 1：By in the probe groups described in any one of claim 1~6, the substrate chain hybridizes to form double with the signal chains
Chain A；The leading fuel chain and protection fuel chain hybridize to form double-strand B；
Step 2：After the double-strand A and double-strand B is transfected into cell to be measured, tissue or animal body, imaging.
11. a kind of probe groups of detection miRNA-21, it is characterised in that by SEQ ID NO：4 of nucleotide sequence shown in 1~4
Bar DNA probe is constituted；
Wherein, SEQ ID NO:5 ' terminal modified quenching groups of the probe of nucleotide sequence shown in 1；
SEQ ID NO:3 ' terminal modified fluorophors of the probe of nucleotide sequence shown in 2.
Probe groups described in 12. claims 11 are preparing the application of the product that tumor tissues are imaged.
A kind of 13. methods of tumor tissues imaging, it is characterised in that including：
Step a：By SEQ ID NO:The probe of nucleotide sequence shown in 1~2 hybridizes to form double-strand A；By SEQ ID NO:3~4
The probe of shown nucleotide sequence hybridizes to form double-strand B；
Step b：After the double-strand A and double-strand B is transfected into test serum or animal body, imaging.
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|---|---|---|---|
| CN201611092882.4A CN106755348B (en) | 2016-12-01 | 2016-12-01 | MicroRNA detection probe set and microRNA detection method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109055524A (en) * | 2018-07-24 | 2018-12-21 | 中山大学 | The probe and method of a variety of microRNA are detected simultaneously under a kind of constant temperature based on DNA assembling |
| CN110564657A (en) * | 2019-08-27 | 2019-12-13 | 北京理工大学 | Microbial sensor for early detection of breast cancer and detection system thereof |
| CN110628888A (en) * | 2019-06-12 | 2019-12-31 | 长沙理工大学 | Nucleic acid probe assembled by triggering miRNA-21 and cell fluorescence imaging |
| CN113637470A (en) * | 2021-08-24 | 2021-11-12 | 青岛科技大学 | Ratio type fluorescent probe and preparation method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103789435A (en) * | 2014-01-29 | 2014-05-14 | 西安交通大学 | Cascading isothermal amplification based miRNA fluorescence detection kit and miRNA fluorescence detection method |
| CN104962555A (en) * | 2015-06-10 | 2015-10-07 | 中国科学技术大学 | Method for detection of intracellular non-coding RNA by cascaded DNA chain replacement reaction |
| CN105950755A (en) * | 2016-06-17 | 2016-09-21 | 山东大学 | Method for detecting microRNA based on split-type recognition mode and cascade signal amplification strategy |
| CN106011235A (en) * | 2016-05-16 | 2016-10-12 | 南京大学 | Membrane protein analysis method based on DNA molecule cascade signal amplification |
-
2016
- 2016-12-01 CN CN201611092882.4A patent/CN106755348B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103789435A (en) * | 2014-01-29 | 2014-05-14 | 西安交通大学 | Cascading isothermal amplification based miRNA fluorescence detection kit and miRNA fluorescence detection method |
| CN104962555A (en) * | 2015-06-10 | 2015-10-07 | 中国科学技术大学 | Method for detection of intracellular non-coding RNA by cascaded DNA chain replacement reaction |
| CN106011235A (en) * | 2016-05-16 | 2016-10-12 | 南京大学 | Membrane protein analysis method based on DNA molecule cascade signal amplification |
| CN105950755A (en) * | 2016-06-17 | 2016-09-21 | 山东大学 | Method for detecting microRNA based on split-type recognition mode and cascade signal amplification strategy |
Non-Patent Citations (4)
| Title |
|---|
| JIE ZHU ET AL: "Accurate Quantification of microRNA via Single Strand Displacement Reaction on DNA Origami Motif", 《PLOS ONE》 * |
| XIAOJING WANG ET AL: "Robust Fuel Catalyzed DNA Molecular Machine for in Vivo MicroRNA Detection", 《ADV. BIOSYS》 * |
| 丁伟: "金属离子驱动的DNA分子折叠及其协助下的链替换反应", 《中国博士学位论文全文数据库 基础科学辑》 * |
| 杨斌: "基于DNA链置换反应的新型核酸适配体荧光探针的构建及应用研究", 《中国博士学位论文全文数据库 工程科技I辑》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109055524A (en) * | 2018-07-24 | 2018-12-21 | 中山大学 | The probe and method of a variety of microRNA are detected simultaneously under a kind of constant temperature based on DNA assembling |
| CN109055524B (en) * | 2018-07-24 | 2022-03-15 | 中山大学 | Probe and method for simultaneously detecting multiple microRNAs under constant temperature condition based on DNA assembly |
| CN110628888A (en) * | 2019-06-12 | 2019-12-31 | 长沙理工大学 | Nucleic acid probe assembled by triggering miRNA-21 and cell fluorescence imaging |
| CN110628888B (en) * | 2019-06-12 | 2022-05-31 | 长沙理工大学 | Nucleic acid probe assembled by triggering miRNA-21 and cell fluorescence imaging |
| CN110564657A (en) * | 2019-08-27 | 2019-12-13 | 北京理工大学 | Microbial sensor for early detection of breast cancer and detection system thereof |
| CN113637470A (en) * | 2021-08-24 | 2021-11-12 | 青岛科技大学 | Ratio type fluorescent probe and preparation method and application thereof |
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