CN106868118A - A kind of molecular beacon based on two ends locking DNA enzymatic recognizes miRNA new methods - Google Patents
A kind of molecular beacon based on two ends locking DNA enzymatic recognizes miRNA new methods Download PDFInfo
- Publication number
- CN106868118A CN106868118A CN201710072705.8A CN201710072705A CN106868118A CN 106868118 A CN106868118 A CN 106868118A CN 201710072705 A CN201710072705 A CN 201710072705A CN 106868118 A CN106868118 A CN 106868118A
- Authority
- CN
- China
- Prior art keywords
- mirna
- dna
- solution
- incubated
- added
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
Abstract
Establish a kind of low background, unmarked, high sensitivity and recognize the Novel Chemiluminescence Method of miRNAs, close the rich G aptamers sequence closed at both ends on DNA probe chain in advance by blocking dna chain, prevent the formation of the G tetrad DNA enzymatics of intermolecular and intramolecular, the target miRNA of introducing, blocking dna chain is displaced by complementary pairing, activate richness G aptamers sequence and form the intramolecular G tetrad DNA enzymatic cataluminescences of high catalytic activity, realize specific recognition miRNA.
Description
Technical field
MiRNA new methods are recognized the present invention relates to a kind of molecular beacon based on two ends locking DNA enzymatic, belongs to bio-sensing
Technical field.
Background technology
MiRNAs plays key player in numerous diseases (particularly tumor disease) gene regulation, and its unconventionality expression is total
The generation of disease is accompanied by, diagnosis is carried out to disease by determining miRNAs expressions very promising will examine as a kind of
Cut off the hands section, hence set up simple, highly sensitive detection miRNAs analysis methods extremely important.
G- tetramers DNA enzymatic can be catalyzed luminol and H2O2Chemiluminescence is produced, with reference to nano material, aptamer group
The technological means such as dress, electrochemistry, establish numerous detection miRNAs analysis methods.But, all there is background signal in many methods
Problem high.The method of the highly sensitive detection miRNAs for having set up at present is based primarily upon the means such as signal amplification or mark,
Have the shortcomings that high cost, experimentation are complicated.Therefore, it is necessary to develop a kind of new low background, it is unmarked, without signal
The method that the high sensitivity of amplification procedure recognizes miRNAs.
The content of the invention
The present invention closes the rich G aptamers sequence two ends on DNA probe chain using blocking dna in advance, reduces background signal,
Target miRNA-21 displaces blocking dna by complementary pairing, and richness G aptamers sequences are closed in activation in advance, forms catalysis high and lives
The intramolecular G- tetramer DNA enzymatics of property.Add luminol and H2O2Afterwards, chemiluminescence signal is greatly enhanced.
Technical scheme is as follows：
(1) 500pM DNA probes and 1000pM blocking dnas are incubated a hour in HEPES bufferings.
(2) target miRN-21 is added to continue to be incubated three hours in the mixed solution described in (1)；
(3) 2.5nM hemins are added to continue to be incubated half an hour in the mixed solution described in (2)；
(4) the solution 100uL described in (3) is taken, 50uL 2 × 10 is added-3The luminol solution and 50uL 20mM H of M2O2
Solution, chemiluminescence signal is gathered with Chemiluminescence Apparatus, analyzes measurement result.
Invention effect
Compared with prior art, the invention has the advantages that：
(1) experimentation is simple, low cost, the technology such as amplify without mark and with signal；
(2) compared with being designed without the unmarked DNA enzymatic of amplification at present, the signal-to-background ratio of this method improves more than 4 times；
(3) sensitivity is high, can distinguish different miRNA, and the miRNA containing gene mutation can also be recognized very well；
(4) can realize recognizing different miRNAs by changing DNA probe partial sequence.
Specific embodiment
Embodiment 1
500pM DNA probes and 1000pM blocking dnas are incubated a hour in HEPES bufferings.Add in mixed solution
After entering different types of miRNA continuation 3 hours of incubation, 2.5nM hemins are added to be incubated half an hour again.Take incubation
Solution 100uL afterwards, adds 50uL 2 × 10-3The luminol solution and 50uL 20mM H of M2O2Solution, is adopted with Chemiluminescence Apparatus
Collection chemiluminescence signal, analyzes its measurement result.Result shows that only miRNA-21 produces stronger chemiluminescence signal, dense
MiRNA-let 7a, the miRNA-let 7c and miRNA-let 7j spent for 10 times of miRNA-21 increase without obvious signal
Plus or reduction phenomenon.The method can very well recognize target miRNA.
Embodiment 2
500pM DNA probes and 1000pM blocking dnas are incubated a hour in HEPES bufferings.Add in mixed solution
Enter miRNA-21, after a base mutation and two miRNA-21 of base mutation continue to be incubated 3 hours, add 2.5nM chlorine
Change ferroheme and be incubated half an hour again.The solution 100uL after being incubated is taken, 50uL 2 × 10 is added-3The luminol solution of M and
50uL 20mM H2O2Solution, chemiluminescence signal is gathered with Chemiluminescence Apparatus, analyzes its measurement result.Result shows that concentration is
The chemiluminescence signal that the miRNA-21 containing gene mutation of 10 times of miRNA-21 is produced substantially does not increase or decrease existing
As the method can be good at distinguishing the gene mutation of miRNA.
Claims (4)
1. a kind of molecular beacon based on two ends locking DNA enzymatic recognizes miRNA new methods, it is characterised in that including following four step
Suddenly：
(1) 500pM DNA probes and 1000pM blocking dnas are incubated a hour in HEPES bufferings.
(2) target miRNA-21 is added to continue to be incubated 3 hours in the mixed solution described in (1)；
(3) 2.5nM hemins are added to continue to be incubated half an hour in the mixed solution described in (2)；
(4) the solution 100uL described in (3) is taken, the luminol solution and 50uL20mM H of 2 μM of 50uL is added2O2Solution, with chemistry
Light-emitting appearance detects chemiluminescence signal, analyzes its measurement result.
2. the method as described in claim 1, it is characterised in that selected DNA probe sequence is 5 '-TCA ACA TCA GTC TGA
TAA GCT AAG GGT AGG GCG GGT TGG G-3’；Blocking dna sequence is 5 '-CCC AAC CCT AGC TTA TCA
GAC T-3’；MiRNA-21 sequences are 5 '-UAG CUU AUC AGA CUG AUG UUG A-3 '.
3. the method as described in claim 1, it is characterised in that HEPES (hydroxyethyl piperazine second thiosulfonic acid) buffers packet contains 25mM
N- (2- ethoxys) piperazine-N'- (2-ethanesulfonic acid) sodium salt, 200mM NaCl, 25mM KCl, 150mM NH4Cl, 1% diformazan is sub-
Sulfone (V/V0), 0.05%Triton X-100 (V/V0), pH 7.4.
4. the method as described in claim 1, it is characterised in that the pH of chemiluminescent solution is 7.4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710072705.8A CN106868118A (en) | 2017-02-10 | 2017-02-10 | A kind of molecular beacon based on two ends locking DNA enzymatic recognizes miRNA new methods |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710072705.8A CN106868118A (en) | 2017-02-10 | 2017-02-10 | A kind of molecular beacon based on two ends locking DNA enzymatic recognizes miRNA new methods |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106868118A true CN106868118A (en) | 2017-06-20 |
Family
ID=59166032
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710072705.8A Pending CN106868118A (en) | 2017-02-10 | 2017-02-10 | A kind of molecular beacon based on two ends locking DNA enzymatic recognizes miRNA new methods |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106868118A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110241180A (en) * | 2019-06-05 | 2019-09-17 | 成都理工大学 | A kind of chemiluminescence method for sensing based on dsDNA-SYBR Green I photocatalysis |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102618664A (en) * | 2012-05-03 | 2012-08-01 | 武汉大学 | MiRNA (Micro Ribonucleic Acid) detection probe and method for visually detecting miRNA |
-
2017
- 2017-02-10 CN CN201710072705.8A patent/CN106868118A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102618664A (en) * | 2012-05-03 | 2012-08-01 | 武汉大学 | MiRNA (Micro Ribonucleic Acid) detection probe and method for visually detecting miRNA |
Non-Patent Citations (1)
| Title |
|---|
| DI LI, ET AL: "Amplified Analysis of Low-Molecular-Weight Substrates or Proteins by the Self-Assembly of DNAzyme−Aptamer Conjugates", 《J. AM. CHEM. SOC.》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110241180A (en) * | 2019-06-05 | 2019-09-17 | 成都理工大学 | A kind of chemiluminescence method for sensing based on dsDNA-SYBR Green I photocatalysis |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Hao et al. | A highly sensitive ratiometric electrochemiluminescent biosensor for microRNA detection based on cyclic enzyme amplification and resonance energy transfer | |
| CN102154489B (en) | Singly labeled oligonucleotide fluorescent probe and method for detecting nuclease | |
| CN101845503B (en) | Kit for detecting drug resistant mycobacterium tuberculosis (MDR-TB) | |
| CN107151694B (en) | Loop-mediated cascade amplification strategy for high-sensitivity detection of DNA methyltransferase activity | |
| JP6243013B2 (en) | 5-Formylcytosine-specific chemical labeling method and use thereof | |
| CN102618651B (en) | Omega structure oligonucleotide primer for detecting short chain ribonucleic acid (RNA) and application thereof | |
| US20190040465A1 (en) | Simple Method for Detecting RNA Sequences | |
| Zhang et al. | Rational design of signal-on biosensors by using photoinduced electron transfer between Ag nanoclusters and split G-quadruplex halves–hemin complexes | |
| Ma et al. | Rapid, sensitive and highly specific label-free fluorescence biosensor for microRNA by branched rolling circle amplification | |
| Cheng et al. | Target-triggered triple isothermal cascade amplification strategy for ultrasensitive microRNA-21 detection at sub-attomole level | |
| Cai et al. | Label-free technology for the amplified detection of microRNA based on the allosteric hairpin DNA switch and hybridization chain reaction | |
| KR101844541B1 (en) | Novel Biomarkers for Predicting Susceptibility to PARP Inhibitor and Uses Thereof | |
| CN109097446A (en) | A kind of method and kit detecting miRNA | |
| Zhao et al. | Demethylase FTO activity analysis based on methyl sensitive enzyme MazF and hybridization chain reaction | |
| CN106868118A (en) | A kind of molecular beacon based on two ends locking DNA enzymatic recognizes miRNA new methods | |
| Huang et al. | A sensitive aptasensor based on rolling circle amplification and G-rich ssDNA/terbium (III) luminescence enhancement for ofloxacin detection in food | |
| CN102251023A (en) | Method, primer and TaqMan-MGB probe for detecting mutation of alcohol metabolism-related gene | |
| Nesterova et al. | Near-IR single fluorophore quenching system based on phthalocyanine (Pc) aggregation and its application for monitoring inhibitor/activator action on a therapeutic target: L1-EN | |
| Chen et al. | A triplex-forming linear probe for sequence-specific detection of duplex DNA with high sensitivity and affinity | |
| CN107083437B (en) | Method for simultaneously detecting multiple DNA glycosylases by using inherent fluorescent nucleotide ultrasensitiveness | |
| CN109913534A (en) | A method of two kinds of miRNA based on concatenated dna chain circular response and fluorescence resonance energy transfer effect are detected simultaneously | |
| CN108251528A (en) | Applications of the LINC01814 in gastric cancer diagnosis and treatment | |
| KR101438531B1 (en) | Single-stranded nucleic acid aptamers specifically binding to E.coli and method for detecting E.coli using the same | |
| Li et al. | Branched DNA switchable CRISPR-Cas12a system for sensing FEN1 activity | |
| CN104165999B (en) | Homogeneous chemistry chemiluminescence immunoassay method based on ortho position striking effect |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20170620 |
|
| WD01 | Invention patent application deemed withdrawn after publication |