CN110184327B - Double-stranded DNA detection method based on stem-loop claw probe and biosensor - Google Patents
Double-stranded DNA detection method based on stem-loop claw probe and biosensor Download PDFInfo
- Publication number
- CN110184327B CN110184327B CN201910394231.8A CN201910394231A CN110184327B CN 110184327 B CN110184327 B CN 110184327B CN 201910394231 A CN201910394231 A CN 201910394231A CN 110184327 B CN110184327 B CN 110184327B
- Authority
- CN
- China
- Prior art keywords
- stranded dna
- double
- stem
- probe
- sequence
- 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.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/682—Signal amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6825—Nucleic acid detection involving sensors
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a double-stranded DNA detection method based on a stem-loop-claw probe, belonging to the technical field of biological detection. The detection method comprises the following steps: designing hairpin probes H1 and H2 and stem-loop claw probes SLCP1 and SLCP2 according to target single-stranded DNA and complementary single-stranded DNA respectively, adding H1, SLCP1 and SLCP2 into a solution containing the target double-stranded DNA during detection, uniformly mixing, heating until the target double-stranded DNA is denatured and melted, cooling to a temperature between the melting point temperature of the stem-loop claw probe and the melting point temperature of the double-stranded DNA, so that the stem-loop claw probe and the complementary single-stranded DNA are hybridized to form a hybrid compound, cooling again, identifying and combining the target single-stranded DNA with H1, then adding H2 into the detection solution to initiate chain hybridization reaction, and finally determining the content of the target double-stranded DNA according to the change of a detection signal. The invention promotes the release of the target single strand by the stem-loop claw probe to obviously improve the detection performance of the double-strand DNA.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a stem-loop-paw probe sequence-based specific double-stranded DNA detection method and application thereof to accurate analysis and detection of double-stranded DNA or double-stranded RNA in liquid biopsy.
Background
In the field of biomedical detection, by identifying free DNA released by cells in human peripheral blood, the conditions of cancer treatment and recurrence, drug resistance judgment, tiny residual lesion diagnosis and the like can be monitored. In the past decades, researchers around the world have developed various DNA detection biosensors having high specificity and sensitivity, however, most of the reported DNA detection biosensors are used to detect a selected single-stranded DNA sequence, and most of the currently developed DNA detection biosensors are not suitable because DNA exists substantially in a double-stranded form in nature (e.g., human peripheral blood).
Sensors for detection of double-stranded DNA are still less reported, and in particular biosensors capable of sequence-specific detection of double-stranded DNA are more rare. Currently, there are three main detection strategies for constructing sequence-specific double-stranded DNA detection sensors: 1. the ability of peptide nucleic acids to form specific triple-helical complexes with double-stranded DNA makes them ideal probes for the construction of sequence-specific double-stranded DNA biosensors. Dervan et al synthesized hairpin polyamide fluorophore conjugates capable of sequence-specific detection of short fragments of double-stranded DNA (typically 4 base pairs). 3. Zinc finger proteins, restriction enzymes and transcription factors can recognize and detect double-stranded DNA with sequence specificity.
However, the three strategies described above still face significant challenges when applied to the detection of double stranded DNA in liquid biopsy samples. First, peptide nucleic acids form triple-helical complexes with double strands, only T-A-T and C-G-C are formed, and there are special requirements for the sequence of the double strands, and fewer specific double-stranded DNAs are satisfied in an organism. Second, the method developed by Dervan et al is capable of sequence-specific detection of less sequences of double-stranded DNA. Third, there are fewer kinds of proteins or enzymes that can sequence-specifically recognize double-stranded DNA, and the number of double-stranded DNA that can be detected in an actual sample is small.
The Kelley group creatively proposed the use of DNA paw probes to detect double-stranded DNA from serum samples from cancer patients using electrochemical biosensors. In the annealing process after the target double-stranded DNA is denatured at high temperature, the DNA claw probe can be hybridized with the complementary single-stranded DNA to release the target single-stranded DNA, and the target single-stranded DNA is recognized by the probe on the electrode interface to output an electric signal to realize the specific detection of the double-stranded DNA sequence.
However, in the above-described double-stranded DNA detection method using the DNA paw probe, the double-stranded DNA in the serum sample needs to be extracted and amplified by PCR. Furthermore, the proposed DNA claw probe method described above requires balancing the hybridization stability of the DNA claw probe with the complementary single-stranded DNA probe: because the DNA paw probe can also be complementary with the recognition probe at the electrode interface, although the hybridization stability of the DNA paw probe and the recognition probe at the electrode interface can be reduced after the DNA paw probe is divided into two sequences, the hybridization capability of the DNA paw probe and the complementary single-stranded DNA is reduced, so that the number of released target single-stranded DNA is reduced, and the detection performance of the double-stranded DNA is finally influenced; therefore, the release of the target single-stranded DNA is facilitated by improving the hybridization ability of the DNA claw probe with the complementary single-stranded DNA, while the capture of the target single-stranded DNA by the electrode interface recognition probe is seriously interfered by an excessive amount of the DNA claw probe.
Disclosure of Invention
The invention aims to provide a double-stranded DNA detection method based on a stem-loop claw probe, so as to solve the defects in the current double-stranded DNA analysis. The target single-stranded DNA is released by hybridizing the two stem-loop claw probes with the complementary single-stranded DNA, and the HCR reaction is initiated by the target single-stranded DNA to amplify and output a fluorescent signal, so that the detection of the sequence-specific double-stranded DNA is realized.
In order to realize the purpose, the invention adopts the following technical scheme:
a double-stranded DNA detection method based on a stem-loop claw probe, the double-stranded DNA comprising a target single-stranded DNA and a complementary single-stranded DNA, the method comprising the steps of:
(1) Synthesizing a hairpin probe: designing chain hybridization reaction hairpin probes H1 and H2 according to the target single-stranded DNA sequence, wherein the H1 comprises a sequence complementary to the target single-stranded DNA, and the H2 comprises a sequence complementary to a partial sequence of the H1; a signal mark is modified on the H1 or the H2;
(2) Synthesizing a stem-loop paw probe: designing a stem-loop claw probe SLCP1 and an SLCP2 according to a complementary single-stranded DNA sequence, wherein the complementary single-stranded DNA comprises an A sequence and a B sequence, the SLCP1 comprises three parts of an SLCP1e, an SLCP1f and an SLCP1g, a partial sequence of the SLCP1f and a partial sequence of the SLCP1g are complemented into a double strand to serve as a stem part of a hairpin claw structure of the SLCP1, and the SLCP1e is complemented with the A sequence of the complementary single-stranded DNA; the SLCP2 comprises three parts, namely an SLCP2h part, an SLCP2i part and an SLCP2j part, wherein the partial sequence of the SLCP2h and the partial sequence of the SLCP2i are complemented into a double strand which is used as a stem part of a hairpin claw structure of the SLCP2, and the SLCP2j is complemented with a B sequence of a complementary single-stranded DNA; the melting points of the stem-loop claw probes SLCP1 and SLCP2 are greater than that of the double-stranded DNA;
(3) Detecting double-stranded DNA based on sequence specificity of the stem-loop claw probe: adding H1, SLCP1 and SLCP2 into a solution containing a target double-stranded DNA, uniformly mixing, heating until the target double-stranded DNA is denatured and melted, cooling to a temperature between the melting point temperature of the stem-loop paw probes SLCP1 and SLCP2 and the melting point temperature of the double-stranded DNA, so that the stem-loop paw probe and the complementary single-stranded DNA are hybridized to form a hybrid complex, cooling to room temperature, identifying and combining the target single-stranded DNA with the H1 in the process, adding H2 into the detection solution to initiate a chain hybridization reaction, and determining the content of the target double-stranded DNA in the solution according to the signal change of the detection solution.
The principle of the detection method of the invention is as follows:
adding H1, SLCP1 and SLCP2 into a sample containing the target double-stranded DNA, uniformly mixing, heating the solution to the temperature of denaturation and melting of the double-stranded DNA, and then cooling to the temperature between the melting point temperature of the stem-loop claw probes SLCP1 and SLCP2 and the melting point temperature of the double-stranded DNA, wherein the double-stranded DNA is melted into two single strands at the temperature, and the SLCP1 and SLCP2 form a stable stem-loop structure and can be hybridized with the complementary single-stranded DNA to form a more stable hybrid complex. Then the temperature is reduced to room temperature or below within 10 minutes, in the process, H1 can identify and bind the target DNA single strand and compete with the free complementary DNA single strand, and the introduction of the stem-loop paw probe effectively improves the identification and hybridization efficiency of the target single strand DNA and H1. And then adding H2 into the detection solution, opening H1 by the target DNA single strand, taking H1 and H2 as raw materials, initiating a chain type hybridization reaction, and finally determining the content of the target double strand DNA in the solution according to the signal change of the detection solution, thereby calculating the concentration of the target object in the sample to be detected.
Aiming at the design of the hairpin probe, a traditional sticky end chain substitution method can be used for initiating a chain type hybridization reaction, and a segment of sequence can be inserted into the ring part of the traditional hairpin structure H1 or H2 for identifying the target single-stranded DNA, so that the chain type hybridization reaction is initiated bidirectionally.
Preferably, in step (1), H1 comprises three parts, i.e., H1a, H1b and H1c, wherein the partial sequence of H1a is complementary to the partial sequence of H1c to form a double strand as the stem of the hairpin structure of H1, and H1b is complementary to the target single-stranded DNA; h2 comprises two parts, H2a 'and H2c', wherein the partial sequence of H2a 'and the partial sequence of H2c' are complementary to form a double strand serving as a stem of the hairpin structure of H2, H2a 'is complementary to H1a, and H2c' is complementary to H1 c.
According to the invention, through the innovative design of the SLCP1 and SLCP2 stem-loop paw probes, a more stable hybridization product formed by the probes and a complementary DNA single strand can be effectively improved, the release of a target DNA single strand is promoted, and the detection performance of the double-strand DNA is finally improved.
The design of the stem-loop claw probe, the DNA claw probe can be hybridized with complementary single-stranded DNA for releasing target single-stranded DNA, and can also be hybridized with the recognition probe, so that the claw probe is divided into two sections to reduce the hybridization capacity of the claw probe and the recognition probe. However, the hybridization capability of the claw probe and the complementary single-stranded DNA is weakened, so that a stem-loop structure is added on the basis of the DNA claw probe, and the hybridization capability of the claw probe and the complementary single-stranded DNA can be obviously improved by the hybridization sequence of the stem part, so that more target single-stranded DNA is released. Due to factors such as steric hindrance, the hybridization capacity of the stem-loop paw probe and the recognition probe is partially reduced (background is reduced), and the detection performance of the double-stranded DNA is finally improved.
And (3) promoting the release of the target single-stranded DNA with the aid of the stem-loop claw, hybridizing with a recognition sequence in H1 to open H1, opening H1 to expose H1a and H1c of the stem part to open H2 bidirectionally, opening H2 to expose H2a 'or H2c' of the stem part to open H1, and repeating the steps to initiate the HCR reaction bidirectionally. When the H1 or H2 modifies the fluorescent group and the quenching group, the two groups are close to each other and the fluorescence is quenched when the stem-loop structure is maintained, and after the HCR reaction is initiated, the two groups are far away from each other, so that the fluorescence intensity is obviously increased, and the amplification detection of the double-stranded DNA fluorescence signal is realized. The research of the invention shows that when no target double-stranded DNA exists, the hairpin probe and the stem-loop claw probe can keep self-stability in a hybridization solution and can not cause obvious change of a detection signal.
The double-stranded DNA detection can be achieved by designing the H1, H2, SLCP1 and SLCP2 sequences by substitution, deletion or addition of bases, as the case may be, by those skilled in the art.
Preferably, H1b has a length of 15 to 30nt, and the sequence lengths of H1a, H1c, H2a 'and H2c' are all 18 to 28nt. This length range is suitable in view of cost and stability.
More preferably, H1b is 18-24nt in length, and the sequences H1a, H1c, H2a ', H2c' are all 24nt in length, wherein the stem sequence of the hairpin structure is 18bp in length.
The stem and loop part of the stem loop paw probe sequence is any nucleic acid sequence, the length of the stem is 5-50bp, and the number of bases of the loop is 1-50nt.
Preferably, the stem length of the stem-loop paw probe is 18bp, and the number of bases of the loop is 20nt. The number of bases of the 5 '-end single-stranded DNA terminal SLCP1e of SLCP1 and the 3' -end single-stranded DNA terminal SLCP2j of SLCP2 was 10nt.
And amplifying a detection signal through a chain hybridization reaction, and determining the content of the target double-stranded DNA in the solution according to the signal change of the detection solution. The sample to be detected is double-stranded DNA or double-stranded RNA.
The detection method has no specific requirement on the buffer system of the hybridization solution, and generally adopts high-salt, neutral and slightly alkaline buffer conditions which are favorable for hybridization reaction. Preferably, the buffer system containing the target double-stranded DNA solution is SPSC buffer solution. The SPSC solution consists of: 50mM Na 2 HSO 4 /NaH 2 SO 4 ，1M NaCl，pH 7.5。
Preferably, the working concentration of H1, H2, SLCP1 and SLCP2 is between 1nM and 20. Mu.M.
Preferably, a fluorescent group FAM and a quencher group dabcyl are modified at both ends of H1 or H2, respectively, and the change of fluorescence intensity is directly related to the amount of the target double-stranded DNA, so that the target double-stranded DNA in the solution to be detected can be quantitatively analyzed through the fluorescence intensity, specifically, a standard curve is drawn, and then the quantitative analysis is performed according to the standard curve.
Another object of the present invention is to provide a biosensor for specifically detecting double-stranded DNA including a target single-stranded DNA and a complementary single-stranded DNA including an a sequence and a B sequence based on a stem-loop paw probe sequence, comprising:
hairpin probes H1 and H2, wherein H1 comprises three parts of H1a, H1b and H1c, a partial sequence of H1a and a partial sequence of H1c are complementary to form a double strand serving as a stem part of an H1 hairpin structure, and H1b is complementary to a target single-stranded DNA; h2 comprises two parts, namely H2a 'and H2c', wherein the partial sequence of H2a 'and the partial sequence of H2c' are complementary to form a double strand serving as a stem part of an H2 hairpin structure, H2a 'is complementary to H1a, and H2c' is complementary to H1 c; two ends of the H1 or H2 are respectively marked with a fluorescent group and a quenching group;
the stem-loop claw probes SLCP1 and SLCP2, wherein the SLCP1 comprises three parts of SLCP1e, SLCP1f and SLCP1g, a partial sequence of the SLCP1f and a partial sequence of the SLCP1g are complemented into a double strand as a stem part of a hairpin claw structure of the SLCP1, and the SLCP1e is complemented with an A sequence of the complementary single-stranded DNA; the SLCP2 comprises three parts of SLCP2h, SLCP2i and SLCP2j, wherein the partial sequence of the SLCP2h and the partial sequence of the SLCP2i are complemented into a double strand as a stem part of a hairpin claw structure of the SLCP2, and the SLCP2j is complemented with a B sequence of the complementary single-stranded DNA.
The stem-loop claw probe provided by the invention assists in releasing the target single-stranded DNA, and can be used for detecting by any DNA sensor already invented. Therefore, in addition to the above-mentioned fluorescence sensor, the double-stranded DNA detection technology based on the stem-loop-claw probe provided by the present invention can also be applied to other sensing fields, such as: SPR, microbalance, electrochemistry, fluorescence enhancement, color development, raman and other sensing fields.
Preferably, the kit further comprises an auxiliary strand for assisting the target DNA in opening the hairpin structure of H1, wherein the 3' end of H1 has a single-stranded sticky end H1x, and the auxiliary strand is complementary to partial sequences of H1x and H1 c.
The buffer system of the detection sensor adopts SPSC solution.
The hairpin probes (H1, H2) and stem-loop claw probes (SLCP 1, SLCP 2) may be DNA, RNA, peptide Nucleic Acid (PNA) or Locked Nucleic Acid (LNA).
Specifically, when the target double-stranded DNA is shown as SEQ ID NO.1, the designed H1 sequence is shown as SEQ ID NO.3, the H2 sequence is shown as SEQ ID NO.4, the SLCP1 sequence is shown as SEQ ID NO.6, and the SLCP2 sequence is shown as SEQ ID NO. 7.
When different DNA dynamic concentration ranges are detected, the stability of H1 is changed by using an auxiliary chain, and the sequence of the auxiliary chain is shown as SEQ ID NO. 5.
Based on the above design, 100nM of SLCP1, SLCP2 and H1 were added to 100. Mu.L of SPSC solution containing the target double-stranded DNA, the solution was heated to 90 ℃ for 5 minutes for denaturation and melting, then cooled to 80 ℃ for 20 minutes, and then cooled to 4 ℃ within 10 minutes. Then, 100nM H2 was added to the assay solution and the reaction was carried out for 1h. Under the above conditions, the best signal-to-noise ratio can be obtained.
The invention has the following beneficial effects:
(1) The research of the sequence specificity double-stranded DNA detection technology is still in the early stage, the double-stranded DNA detection method based on the stem-loop claw probe provided by the invention solves the defect of the current double-stranded DNA detection, the design of the stem-loop claw probe obviously improves the release of the target single-stranded DNA, and finally the double-stranded DNA sensitive detection is realized.
(2) The fluorescence sensor provided by the invention can be applied to the detection of double-stranded DNA in a diluted serum sample, and the double-stranded DNA detection sensor based on the technology has market commercialization prospect.
Drawings
FIG. 1 is a schematic diagram of a process for detecting double-stranded DNA based on a stem-loop claw probe.
FIG. 2 is a theoretical feasibility of using NUPACK software to simulate hybridization of a stem-loop paw probe and complementary single-stranded DNA at 80 ℃ to release target single-stranded DNA.
FIG. 3 is a diagram showing the verification of the hybridization of the stem-loop paw probe with the complementary single-stranded DNA by gel electrophoresis.
FIG. 4 shows fluorescence verification of target single-stranded DNA released by hybridization of the stem-loop paw probe with the complementary single-stranded DNA, and (A) fluorescence verification of target single-stranded DNA released by hybridization of the stem-loop paw probe with the complementary single-stranded DNA when H1 is not added during annealing of denatured double-stranded DNA. (B) When H1 is added in the annealing process of the denatured double-stranded DNA, the stem-loop paw probe hybridizes with the complementary single-stranded DNA to release the fluorescence verification of the target single-stranded DNA. Compared with a stem-loop grab probe, when H1 is not added in the annealing process, the single-stranded grab probe cannot hybridize with complementary single-stranded DNA to release target single-stranded DNA, and cannot detect the double-stranded DNA.
FIG. 5 shows the optimization of the detection conditions for double-stranded DNA. (A) And (3) investigating the influence of the concentration of the stem-loop paw probe on the fluorescence detection of the double-stranded DNA. (B) And (3) investigating the influence of the complementary base numbers of the stem-loop claw probe and the complementary single-stranded DNA on the signal-to-noise ratio of the fluorescence detection double-stranded DNA.
FIG. 6 shows fluorescence detection curves (A) and fluorescence intensity superposition (C) of double-stranded DNA, after curve fitting, concentrationThe conversion relation with the fluorescence signal is as follows: y =0.056+ 0.0078X 0.23 Where X is the double-stranded DNA concentration in pM and Y is the relative value of the measured fluorescence intensity (normalized according to the maximum fluorescence value). When an auxiliary chain is added into a detection system, the detection range of double-stranded DNA is regulated and controlled by imitating an allosteric effect, after activator is added, the detection sensitivity is obviously improved as can be seen from a double-stranded DNA fluorescence detection curve (B) and fluorescence intensity superposition (D), and after curve fitting, the conversion relation formula of the concentration and the fluorescence signal is as follows: y =0.086+ 0.026X 0.26 Where X is the double-stranded DNA concentration in pM and Y is the relative value of the measured fluorescence intensity (normalized according to the maximum fluorescence value).
FIG. 7 is a graph showing the ability to detect single-base mismatches in a double-stranded DNA detection region.
FIG. 8 is a gel electrophoresis to verify that double-stranded DNA of different lengths initiates chain hybridization reaction by stem-loop-claw probe assisted method.
FIG. 9 is a diagram showing detection of double-stranded DNAs of different lengths by the double-stranded DNA detection sensor of the present invention.
FIG. 10 shows fluorescence detection of double-stranded DNA in 10% serum using H1-a structure, (A) superposition of fluorescence intensity curves at different double-stranded DNA concentrations; (B) a detection curve for detecting double-stranded DNA in 10% serum; (C) Detection curve for direct detection of target single stranded DNA in 10% serum.
Detailed Description
The features and advantages of the invention will be further illustrated by the following examples. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way.
Example 1
Detection of double-stranded DNA based on sequence specificity of stem-loop claw probe
1. Designing a hairpin probe and a stem-loop claw probe: on the basis of a classical hybridization chain reaction sequence, a related hairpin probe is designed according to a target single-chain DNA in a detection double-chain, a stem-loop-claw probe is designed according to a complementary single-chain DNA in the detection double-chain, and a related nucleic acid sequence is synthesized and modified by the Competition Biotechnology engineering corporation (Shanghai).
Ultraviolet light is used for the concentration of hairpin probe and stem-loop paw probeThe amount was determined accurately in a spectrophotometer using SPSC buffer (50 mM Na) 2 HSO 4 /NaH 2 SO 4 1M NaCl, pH 7.5) all hairpin probes were diluted to the desired assay concentration.
DNA Probe sequences used in Table 1
Note: H1-dsDNA-18, H2, SLCP-18-9-1 and SLCP-18-9-2 were used to detect double stranded DNA-18 (T-ssDNA-18 and C-ssDNA-18, 18 base pairs); H1-dsDNA-21, H2, SLCP-21-10-1 and SLCP-21-10-2 were used to detect double stranded DNA-21 (T-ssDNA-21 and C-ssDNA-21, 21 base pairs); H1-dsDNA-24, H2, SLCP-24-10-1 and SLCP-24-10-2 were used to detect double stranded DNA-24 (T-ssDNA-24 and C-ssDNA-24, 24 base pairs); LCP-24-10-1 and LCP-24-10-2 are used for comparing the stem-loop paw probe, and have outstanding advantages. SLCP-24-8-1, SLCP-24-8-2, SLCP-24-10-1, SLCP-24-10-2, SLCP-24-12-1, SLCP-24-12-2 were used to optimize paw length. M1-M5, single base mismatch double stranded DNA sequence.
FIG. 1 shows the principle of sequence-specific double-stranded DNA detection: under a proper high temperature environment, double-stranded DNA is melted into two single strands, and SLCP1 and SLCP2 form a stable stem-loop structure, which can be hybridized with complementary single-stranded DNA to form a more stable hybridization complex. In the process of cooling, H1 can identify and combine a target DNA single chain to compete with a free complementary DNA single chain, and the introduction of the stem-loop paw probe effectively improves the identification hybridization efficiency of the target single-chain DNA and H1. After H1 of a target DNA single strand is opened, H1 and H2 are used as raw materials to initiate a chain type hybridization reaction in a two-way mode, fluorescence intensity is changed in the HCR reaction process, and the concentration of the target double strand DNA is determined according to the change of fluorescence signals in a detection solution. In the absence of double-stranded DNA, the stem-loop paw DNA cannot open H1 priming HCR.
FIG. 2 uses NUPACK software to simulate the melting point temperatures of double-stranded DNA and stem-loop paw probes, and the theoretical feasibility of hybridization between the stem-loop paw probe and complementary single-stranded DNA is judged according to the respective melting point temperatures. From the simulation result, the melting point temperature of the selected double-stranded DNA is about 71 ℃, the melting point temperature of the two stem-loop paw probes is about 85 ℃, when the temperature is set to 80 ℃, the double-stranded DNA exists in the form of two single-stranded DNAs, and the stem-loop paw probes form a stem-loop structure, so that the stem-loop paw probes can be hybridized with complementary DNA single strands to form a more stable hybridization product, and finally, the target single-stranded DNA is released to realize quantitative analysis of the target single-stranded DNA.
FIG. 3 verifies by gel electrophoresis that the loop-loop probe can hybridize with the complementary DNA single strand to form a more stable hybridization product, and band 6 can prove that the corresponding product is generated. In order to highlight the unique performance of the stem-loop claw probe in the detection of the double-stranded DNA, the traditional single-stranded DNA claw probe is introduced, and because the hybridization product of the single-stranded DNA claw probe and the complementary single-stranded DNA is similar to the double-stranded DNA, the hybridization condition of the single-stranded DNA claw probe and the complementary single-stranded DNA cannot be judged through the gel electrophoresis result.
FIG. 4 is a graph showing the further confirmation of the presence of a change in fluorescence signal that the stem-loop claw probe can hybridize to a single strand of complementary DNA to form a stable hybridization product. (A) When the double strand is denatured and no H1 is added in the process of hybridizing with the stem-loop paw, the fluorescence intensity is obviously changed only when the double strand DNA and the stem-loop paw probe exist simultaneously, which indicates that the stem-loop paw probe can hybridize with the complementary DNA single strand to form a stable hybridization product, and the released target single strand DNA triggers HCR to change the fluorescence intensity. And when the double-stranded DNA and the single-stranded DNA claw probe coexist, the fluorescence intensity is not obviously changed, which indicates that the single-stranded DNA claw probe cannot be hybridized with the complementary single-stranded DNA to release the target single-stranded DNA in the processes of high-temperature denaturation and cooling. (B) When H1 is added in the process of double-strand denaturation and stem-loop paw hybridization, H1 can compete with free complementary single-strand DNA to hybridize with target single-strand DNA, and the change of fluorescence intensity is obviously improved. When the stem-loop claw probe exists, the change of fluorescence intensity is more obvious, and the obvious advantages of the designed stem-loop claw probe in detection of double-stranded DNA are well illustrated. In addition, it can be seen from the figure that, in the absence of double-stranded DNA, the probability of initiating HCR by opening H1 by the stem-loop paw probe is slightly less than that by opening H1 by the single-stranded DNA paw probe, and the former has better detection signal-to-noise ratio, which is probably due to the existence of a certain steric hindrance in the process of recognizing H1 and initiating HCR by the stem-loop paw structure.
FIG. 5 is a diagram of the optimized detection conditions of the double-stranded DNA detection fluorescence sensor. (A) When the concentration of the stem-loop paw probe is different, the hybridization efficiency of the stem-loop paw probe with complementary single-stranded DNA and H1 is different, and as can be seen from the figure, when the concentration of the stem-loop paw probe is 100nM, a higher detection signal-to-noise ratio of the double-stranded DNA is obtained compared with 50nM and 200nM, so that the concentration of the stem-loop paw probe is fixed to 100nM. (B) The number of bases capable of hybridizing with complementary single-stranded DNA in each stem-loop claw probe not only affects the release of the target single-stranded DNA, but also affects the efficiency of opening H1 to initiate HCR, and the stability of the hybridization of the stem-loop claw probe and the complementary single-stranded DNA can be improved by increasing the base number of the stem, so that the number of bases capable of hybridizing with the complementary single-stranded DNA in the stem-loop claw probe can be properly reduced, background signals can be effectively reduced, and the detection sensitivity of the double-stranded DNA is improved. From the optimized results, it can be seen that when the number of bases capable of hybridizing with the complementary single-stranded DNA in the stem-loop paw probe is 10 bases, the best signal-to-noise ratio of the detection of the double-stranded DNA is obtained.
FIG. 6 designs 2 different H1 structures, which are H1 and H1/Activator, respectively, wherein the H1/Activator is an Activator added to the H1, and the Activator (see the sequence in Table 1) is added to assist the target single-stranded DNA in opening the H1, so that the detection sensitivity of the double-stranded DNA is improved. In the SPSC buffer solution, 100nM of H1, SLCP1, and SLCP2 were mixed with different concentrations of double-stranded DNA, the solution was heated to 90 degrees for 5 minutes to denature and melt, then cooled to 80 degrees for 20 minutes, and then cooled to 4 degrees within 10 minutes. Adding 100nM H2 into the detection solution, reacting for 1h, and measuring the fluorescence intensity corresponding to the target double-stranded DNA at each concentration by using a fluorescence spectrometer (excitation voltage 800V, excitation slit 5nm, emission slit 5nm, excitation wavelength 492nm, wavelength scanning range 500-600 nm). FIGS. 6 (A) and (C) are graphs in which the fluorescence detection curve of double-stranded DNA using H1 as a discrimination probe and the fluorescence intensity are superimposedAfter fitting, the conversion relation between the concentration and the fluorescence signal is as follows: y =0.056+0.0078 x 0.23 Where X is the concentration of double-stranded DNA in pM, Y is the relative value of the measured fluorescence intensity (normalized according to the maximum fluorescence value), and the limit of detection is about 50pM. After the activator is added, as can be seen from a double-stranded DNA fluorescence detection curve diagram 6 (B) and a fluorescence intensity superposition diagram 6 (D), the detection sensitivity is obviously improved, and after curve fitting, the conversion relation of the concentration and the fluorescence signal is as follows: y =0.086+ 0.026X 0.26 Wherein X is double-stranded DNA concentration, the unit pM, Y is the relative value of the measured fluorescence intensity (normalized according to the maximum fluorescence value), and the detection limit is improved to 0.5pM.
In the design of the double-stranded DNA detection sensor, a DNA mutation sequence can interfere the detection of a target double-stranded DNA, particularly, the influence of single base mutation is more obvious, in order to verify the single base mismatch capability of the designed double-stranded DNA detection, single base mismatch is introduced at different positions of the target double-stranded DNA, and as can be seen from figure 7, under the same target double-stranded DNA concentration, the fluorescence intensity signal change caused by the single base mismatch at different positions is obviously smaller than that of the fully complementary target single-stranded DNA. When the sequence where the single base mismatch is located can be hybridized with the stem-loop paw probe, the single base mismatch capability of the double-stranded DNA is superior to the mismatch capability when the single-stranded DNA is directly detected, because the mismatch at the position weakens the hybridization capability of the stem-loop paw probe and the complementary single-stranded DNA, the released target single-base mismatch single-stranded DNA is reduced, and finally the single base mismatch capability when the double-stranded DNA is detected is improved.
The length of the selected target double-stranded DNA is 24 base pairs, and the double-stranded DNA fluorescent sensor designed by the invention can also detect other double-stranded DNA with different lengths. FIGS. 8 and 9 respectively verify that the detection can be realized by the double-stranded DNA of 21 base pairs and 18 base pairs through gel electrophoresis and fluorescence detection, and only the target single-stranded DNA recognition sequence of the H1b part and the sequence of the part hybridized with the complementary single-stranded DNA in the stem-loop claw need to be replaced.
Example 2
Double-stranded DNA (deoxyribonucleic acid) detection in serum based on sequence specificity of stem-loop claw probe
When double-stranded DNA is detected in serum, proteins are denatured into solids due to the heating process, and autofluorescent proteins of serum itself interfere with the output of fluorescent signals. Therefore, the double-stranded DNA detection sensor designed by the present invention attempts double-stranded DNA detection in diluted serum. The detection process is essentially the same as the detection of double stranded DNA in SPSC solution, except that the denatured protein solids need to be removed by centrifugation prior to fluorescence intensity measurement.
FIG. 10 (A) shows the superposition of fluorescence curves of H1-a as a double-stranded DNA recognition structure in fluorescence detection at different double-stranded DNA concentrations in 10% serum. FIG. 10 (B) is a curve of detection of double-stranded DNA in 10% serum, with a detection range of 1pM to 1. Mu.M, and after curve fitting, the conversion of the double-stranded DNA concentration to fluorescence intensity is as follows: y =344.9+ 33.8X 0.31 . FIG. 10 (C) is the detection curve of the target single-stranded DNA in 10% serum, also with the detection range of 1pM to 1. Mu.M. As can be seen from the two detection curves, the designed double-stranded DNA detection fluorescent sensor obtains the detection sensitivity equivalent to the direct detection of single-stranded DNA.
Sequence listing
<110> Jiaxing school
<120> double-stranded DNA detection method based on stem-loop paw probe and biosensor
<160> 33
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tacccgtaat cttcataatc cgag 24
<210> 2
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ctcggattat gaagattacg ggta 24
<210> 3
<211> 78
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ttaacccacg ccgaatccta gactctcgga ttatgaagat tacgggtaca aagtagtcta 60
ggattcggcg tgccaatt 78
<210> 4
<211> 48
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
agtctaggat tcggcgtggg ttaacacgcc gaatcctaga ctactttg 48
<210> 5
<211> 15
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
aattggcacg ccgaa 15
<210> 6
<211> 66
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ataatccgag ggtttggctt tcttgttagg caaccaccca gggtgtcgta acaagaaagc 60
caaacc 66
<210> 7
<211> 66
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
ggtttggctt tcttgttagg caaccaccca gggtgtcgta acaagaaagc caaacctacc 60
cgtaat 66
<210> 8
<211> 64
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
aatccgaggg tttggctttc ttgttaggca accacccagg gtgtcgtaac aagaaagcca 60
aacc 64
<210> 9
<211> 64
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
ggtttggctt tcttgttagg caaccaccca gggtgtcgta acaagaaagc caaacctacc 60
cgta 64
<210> 10
<211> 68
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
tcataatccg agggtttggc tttcttgtta ggcaaccacc cagggtgtcg taacaagaaa 60
gccaaacc 68
<210> 11
<211> 68
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
ggtttggctt tcttgttagg caaccaccca gggtgtcgta acaagaaagc caaacctacc 60
cgtaatct 68
<210> 12
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
tacgccatca gctggaac 18
<210> 13
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gttccagctg atggcgta 18
<210> 14
<211> 72
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
ttaacccacg ccgaatccta gactgttcca gctgatggcg tacaaagtag tctaggattc 60
ggcgtgccaa tt 72
<210> 15
<211> 65
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
agctggaacg gtttggcttt cttgttaggc aaccacccag ggtgtcgtaa caagaaagcc 60
aaacc 65
<210> 16
<211> 65
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
ggtttggctt tcttgttagg caaccaccca gggtgtcgta acaagaaagc caaacctacg 60
ccatc 65
<210> 17
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
tacgccatca gctggaacgc c 21
<210> 18
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
ggcgttccag ctgatggcgt a 21
<210> 19
<211> 75
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
ttaacccacg ccgaatccta gactggcgtt ccagctgatg gcgtacaaag tagtctagga 60
ttcggcgtgc caatt 75
<210> 20
<211> 66
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
ctggaacgcc ggtttggctt tcttgttagg caaccaccca gggtgtcgta acaagaaagc 60
caaacc 66
<210> 21
<211> 66
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
ggtttggctt tcttgttagg caaccaccca gggtgtcgta acaagaaagc caaacctacg 60
ccatca 66
<210> 22
<211> 10
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
ataatccgag 10
<210> 23
<211> 10
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
tacccgtaat 10
<210> 24
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
cacccgtaat cttcataatc cgag 24
<210> 25
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
tacccgaaat cttcataatc cgag 24
<210> 26
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
tacccgtaat ctacataatc cgag 24
<210> 27
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
tacccgtaat ctatcataat ccgag 25
<210> 28
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
tacccgtaat ctcataatcc gag 23
<210> 29
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
ctcggattat gaagattacg ggtg 24
<210> 30
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
ctcggattat gaagatttcg ggta 24
<210> 31
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
ctcggattat gtagattacg ggta 24
<210> 32
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
ctcggattat gatagattac gggta 25
<210> 33
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
ctcggattat gagattacgg gta 23
Claims (7)
1. A biosensor for specifically detecting double-stranded DNA based on a stem-loop claw probe sequence, wherein the double-stranded DNA comprises a target single-stranded DNA and a complementary single-stranded DNA, and the complementary single-stranded DNA comprises an A sequence and a B sequence, the biosensor comprising:
hairpin probes H1 and H2, wherein H1 comprises three parts of H1a, H1b and H1c, a partial sequence of H1a and a partial sequence of H1c are complementary to form a double strand serving as a stem part of an H1 hairpin structure, and H1b is complementary to a target single-stranded DNA; h2 includes two portions, H2a 'and H2c', wherein the partial sequence of H2a 'and the partial sequence of H2c' are complementary to form a double strand as the stem of the hairpin of H2, H2a 'is complementary to H1a, and H2c' is complementary to H1 c; two ends of the H1 or H2 are respectively marked with a fluorescent group and a quenching group;
the stem-loop claw probes SLCP1 and SLCP2, wherein the SLCP1 comprises three parts of SLCP1e, SLCP1f and SLCP1g, a partial sequence of the SLCP1f and a partial sequence of the SLCP1g are complemented into a double strand as a stem part of a hairpin claw structure of the SLCP1, and the SLCP1e is complemented with an A sequence of the complementary single-stranded DNA; the SLCP2 comprises three parts, namely an SLCP2h part, an SLCP2i part and an SLCP2j part, wherein the partial sequence of the SLCP2h and the partial sequence of the SLCP2i are complemented into a double strand which is used as a stem part of a hairpin claw structure of the SLCP2, and the SLCP2j is complemented with a B sequence of a complementary single-stranded DNA;
the double-stranded DNA detection method using the biosensor comprises the following steps:
(1) Synthesizing a hairpin probe: designing hairpin probes H1 and H2 for chain hybridization reaction according to a target single-stranded DNA sequence, wherein the length of H1b is 15-30nt, and the length of the sequences of H1a, H1c, H2a 'and H2c' is 18-28nt;
(2) Synthesizing a stem-loop paw probe: designing stem-loop claw probes SLCP1 and SLCP2 according to a complementary single-stranded DNA sequence, wherein the melting points of the stem-loop claw probes SLCP1 and SLCP2 are greater than the melting point of the double-stranded DNA;
(3) Detecting double-stranded DNA based on sequence specificity of the stem-loop claw probe: adding H1, SLCP1 and SLCP2 into a solution containing target double-stranded DNA, uniformly mixing, heating until the target double-stranded DNA is denatured and melted, cooling to a temperature between the melting point temperature of the stem-loop paw probes SLCP1 and SLCP2 and the melting point temperature of the double-stranded DNA, so that the stem-loop paw probe and the complementary single-stranded DNA are hybridized to form a hybrid complex, cooling to room temperature, identifying and combining the target single-stranded DNA with the H1 in the process, then adding H2 into a detection solution, initiating a chain hybridization reaction, and determining the content of the target double-stranded DNA in the solution according to the signal change of the detection solution.
2. The biosensor for detecting double-stranded DNA based on the sequence specificity of the stem-loop claw probe as claimed in claim 1, wherein the length of H1b is 18-24nt, and the length of the sequences H1a, H1c, H2a 'and H2c' are all 24nt, wherein the length of the stem sequence of the hairpin structure is 18bp.
3. The biosensor for detecting double-stranded DNA based on the sequence specificity of the stem-loop claw probe as claimed in claim 1, wherein the stem length of the stem-loop claw probe is 18bp, the number of bases of the loop is 20nt, and the number of bases of SLCP1e and SLCP2j are 10nt when the target double-stranded DNA is 24 base pairs.
4. The biosensor for detecting double-stranded DNA based on the sequence specificity of the stem-loop claw probe as claimed in claim 1, wherein the buffer system containing the target double-stranded DNA solution is SPSC buffer solution.
5. The biosensor for sequence-specific detection of double-stranded DNA based on stem-loop paw probe as claimed in claim 1, wherein the working concentration of H1, H2, SLCP1, SLCP2 is 1nM-20 μ M.
6. The biosensor for sequence-specific detection of double-stranded DNA based on stem-loop-claw probe as claimed in claim 1, wherein said hairpin probe and stem-loop-claw probe are DNA probe, RNA probe, locked nucleic acid probe or peptide nucleic acid probe.
7. The biosensor for specifically detecting double-stranded DNA based on the stem-loop claw probe sequence as claimed in claim 1, wherein when the target double-stranded DNA is represented by SEQ ID No.1, the designed H1 sequence is represented by SEQ ID No.3, the designed H2 sequence is represented by SEQ ID No.4, the designed SLCP1 sequence is represented by SEQ ID No.6, and the designed SLCP2 sequence is represented by SEQ ID No. 7.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910394231.8A CN110184327B (en) | 2019-05-13 | 2019-05-13 | Double-stranded DNA detection method based on stem-loop claw probe and biosensor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910394231.8A CN110184327B (en) | 2019-05-13 | 2019-05-13 | Double-stranded DNA detection method based on stem-loop claw probe and biosensor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110184327A CN110184327A (en) | 2019-08-30 |
| CN110184327B true CN110184327B (en) | 2023-04-07 |
Family
ID=67716101
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910394231.8A Active CN110184327B (en) | 2019-05-13 | 2019-05-13 | Double-stranded DNA detection method based on stem-loop claw probe and biosensor |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110184327B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113495091B (en) * | 2021-05-28 | 2024-01-23 | 中国科学院苏州生物医学工程技术研究所 | Electrochemical detection method for trace micro ribonucleic acid |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154276A (en) * | 2011-02-28 | 2011-08-17 | 中国人民解放军第三军医大学第一附属医院 | End joining-type hairpin DNA probe |
| CN109321635A (en) * | 2018-09-19 | 2019-02-12 | 嘉兴学院 | It is a kind of based on more hybridize chain reaction nucleic acid detection method and application |
-
2019
- 2019-05-13 CN CN201910394231.8A patent/CN110184327B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154276A (en) * | 2011-02-28 | 2011-08-17 | 中国人民解放军第三军医大学第一附属医院 | End joining-type hairpin DNA probe |
| CN109321635A (en) * | 2018-09-19 | 2019-02-12 | 嘉兴学院 | It is a kind of based on more hybridize chain reaction nucleic acid detection method and application |
Non-Patent Citations (4)
| Title |
|---|
| DNA Clutch Probes for Circulating Tumor DNA Analysis;Jagotamoy Das等;《Journal of the American Chemical Society》;20160811;1-10 * |
| Hybridization of single-stranded DNA targets to immobilized complementary DNA probes: comparison of hairpin versus linear capture probes;P V Riccelli等;《Nucleic Acids Res.》;20010215;第29卷(第4期);996-1004 * |
| Single-step multivalent capture assay for nucleic acid detection with dual-affinity regulation using mutation inhibition and allosteric activation;Xing Lu等;《Chem. Sci.》;20190405;第10卷;5025-5030 * |
| Stem-loop clutch probes for sequence specific dsDNA analysis with improved single-mismatch selectivity;Xing Lu等;《Chem Commun (Camb).》;20190612;第55卷(第52期);7514-7517 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110184327A (en) | 2019-08-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR100702338B1 (en) | Detection of labeled primers and target nucleic acids for use in detection of target nucleic acids | |
| KR100245283B1 (en) | In situ hybridization method | |
| US20220154297A1 (en) | Compositions and methods for detection of candida species | |
| CN109971883B (en) | Primer-probe combination, kit, detection method and application for candida species classification detection | |
| WO2011001496A1 (en) | Sample analysis method and assay kit for use in the method | |
| EP0896630A1 (en) | Method for detecting a nucleic acid base sequence | |
| US9845495B2 (en) | Method and kit for detecting target nucleic acid | |
| CA2692633A1 (en) | Method for the simultaneous detection of multiple nucleic acid sequences in a sample | |
| JP7392048B2 (en) | Analysis method and kit | |
| KR102323375B1 (en) | Multiplex Probes | |
| Tian et al. | dsDNA/ssDNA-switchable isothermal colorimetric biosensor based on a universal primer and λ exonuclease | |
| CN110184327B (en) | Double-stranded DNA detection method based on stem-loop claw probe and biosensor | |
| JP5258760B2 (en) | Method for amplifying methylated or unmethylated nucleic acid | |
| CN114507706B (en) | Enzyme DNA repair cascade driving fluorophore coding/de-coding based biosensor and application thereof in telomerase detection | |
| Bi et al. | Ultrasensitive detection of mRNA extracted from cancerous cells achieved by DNA rotaxane-based cross-rolling circle amplification | |
| KR101569127B1 (en) | Probe for detecting polymorphism, method of detecting polymorphism, method of evaluating drug efficacy, disease prediction method and reagent kit for detecting polymorphism | |
| CN113462753B (en) | Click chemistry mediated single quantum dot nano sensor, method for detecting miRNAs and application | |
| KR101845043B1 (en) | Method of Solving Nucleic Acid Detection Problem Based on Threshold Cycle(Ct) of Polymerase Chain Reaction Using Peptide Nucleic Acid Probe | |
| US20230031670A1 (en) | Method and kit for detection of polynucleotide | |
| CN110004214B (en) | Method for detecting microRNA-21 by double DNA machine | |
| JP2014180278A (en) | Nucleic acid analyzing method and assay kit used thereby | |
| WO2005021743A1 (en) | Primers for nucleic acid amplification and method of examining colon cancer using the same | |
| JP2011004733A (en) | Microarray for detecting target nucleic acid in a plurality of specimens | |
| CN114144530A (en) | Multivalent nucleic acid nanostructures for nucleic acid detection and highly sensitive nucleic acid probes using the same | |
| JP7346533B2 (en) | Analysis method and kit |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |