26 results about "Complementary DNA" patented technology

This invention relates to an IL5 / Bi2MoO6 / MXene photoactive composite material, a low-background photoelectrochemical aptamer sensor, its preparation method, and its applications. The sensor is constructed by sequentially modifying chitosan, glutaraldehyde, complementary DNA strands, and a gold nanoparticle-labeled aptamer. A fixed capture probe and APT-Au synergistically achieve dual signal quenching, constructing an extremely low detection baseline. When the target analyte MUC1 is present, its specific binding to the aptamer causes APT-Au to detach, dissolving the dual quenching and significantly restoring the photocurrent, thereby achieving quantitative detection. This invention solves the problems of low efficiency, heterojunction instability, and high baseline in single quenching modes of existing photoelectric materials, achieving highly sensitive, highly specific, rapid, and low-cost detection of the breast cancer biomarker MUC1 in human serum samples, providing a novel, industrially feasible technical solution for large-scale early screening of breast cancer.

The invention discloses a cell membrane in-situ drug membrane protein target screening method based on variable configuration DNA, and belongs to the technical field of biological medicine. Two pairs of functionalized nucleic acid structures are constructed, the first pair is S1S2 semi-complementary DNA double strands, the second pair is S3S4 DNA-RNA hybrid double strands, and the S1S2 structure is covalently coupled to a non-natural amino acid site-directed modified membrane protein through a click chemical reaction in a living cell to realize tagging of the membrane protein; when the candidate drug coupled in S3 interacts with the membrane protein, triggering an allosteric response system to release the tagged chain in S1; the released DNA tag is subjected to PCR amplification and sequencing, and accurate identification and analysis of a membrane protein target are realized according to a tag sequence. The invention provides a high-specificity and non-in-vitro drug membrane protein target screening technology. According to the technology, active compound discovery or accurate screening of drug membrane protein targets in a living cell in-situ environment can be realized.

Disclosed is a cDNA amplification method capable of comprehensively amplifying mRNA with a high efficiency and a low bias even from a very small amount of cell sample. The method of amplifying a complementary DNA strand of the present invention comprises steps of capturing a target RNA and synthesizing a complementary DNA strand using a reverse transcriptase having terminal transferase activity in a liquid-phase system or a solid-phase system such that bases such as CCC are added to an end of the complementary DNA strand, and thereafter carrying out homopolymer tailing by the TdT reaction in the presence of a chain-terminating nucleotide triphosphate. By combination with a template-switching reaction, the method enables amplification of the complementary DNA strand with a higher efficiency and a lower bias.

The invention discloses an oil immersion lens microscopic imaging multi-target simultaneous detection platform based on machine vision and application, and belongs to the field of biochemical analysis. The aptamer is adopted as a recognition element and matched with the corresponding complementary DNA chain and the combined DNA chain, the aptamer and the DNA chain can be replaced according to a target object, operation is easy and convenient, good applicability and universality are achieved, the size of an observation area is reduced to the dry size of a liquid drop area from the size of a conventional cover glass, visual field loss is greatly reduced, and the detection accuracy is improved. The visual imaging of a single microsphere can be realized, the signal sensing is direct and efficient, and the detection sensitivity is high; the multi-detection capability of the color and particle size combination of the microspheres is strong; the used recognition elements are all DNA sequences, so that the use cost of the recognition elements such as antibodies can be greatly reduced.

A cDNA amplification method is disclosed that can comprehensively amplify mRNA from even extremely small cell samples with high efficiency and low bias. The complementary DNA chain amplification method of the present invention comprises the steps of: capturing target RNA and synthesizing a complementary DNA chain in a liquid or solid system using a reverse transcriptase having terminal transferase activity; adding a base such as CCC to the end of the complementary DNA chain; and performing homopolymer tailing by a TdT reaction in the presence of a chain-stopping nucleotide triphosphate. By further combining a template switching reaction, the complementary DNA chain can be amplified with even higher efficiency and low bias.

The present disclosure relates to a method of determining the presence of at least one messenger RNA (mRNA) molecule in a composition, the method comprising: (a) reverse transcription of the at least one mRNA molecule to obtain a template comprising at least one first strand of complementary DNA (cDNA); (b) generating at least one double-stranded cDNA molecule from the template; (c) amplifying the at least one double-stranded cDNA molecule; (d) digesting the at least one amplified double-stranded cDNA molecule with at least one restriction enzyme to obtain a test cDNA fragment; (e) separating the test cDNA fragments so as to form a test cDNA fragment map; and (f) comparing the test cDNA fragment map to a control cDNA fragment map wherein the at least one mRNA molecule is present in the composition when the test cDNA fragment map comprises the control cDNA fragment map. Also disclosed are methods of quantifying and / or assessing the integrity of at least one mRNA molecule in a composition, processes of making a composition comprising at least one mRNA molecule and a primer pair for use in the methods of the invention.

The invention discloses a preparation method and application of reverse transcriptase derived from thermophilic bacteria, and relates to the technical field of bioengineering, the reverse transcriptase is an enzyme which is obtained by screening, is genetically engineered and has high heat resistance, and the reverse transcriptase has enzymatic activity equivalent to that of wild type reverse transcriptase. The reverse transcriptase disclosed by the invention can be used for synthesizing complementary DNA chains (cDNA) by taking RNA as a template. The recombinant plasmid is cloned to a pET28 expression vector, inducible expression is carried out in escherichia coli, and the reverse transcriptase is prepared through immobilized nickel ion affinity purification. The gene is used for reverse transcription reaction to synthesize cDNA, and RNA detection and cDNA coding gene cloning are carried out.

The invention discloses a glyphosate fluorescence detection method based on RNase H cycle amplified magnetic bead-aptamer-Cas9 / deoxyribozyme, belongs to the technical field of environmental monitoring and biological detection, and can realize high-sensitivity and rapid quantitative detection of glyphosate residues in soil, water bodies and agricultural products. According to the method, the glyphosate is combined with an aptamer to release complementary DNA (cDNA) combined with the aptamer, then signal cascade amplification is realized through RNase H-mediated cDNA cyclic utilization, the anti-interference advantage brought by magnetic bead separation is combined, and the known characteristics of Cas9 endonuclease and DNAzyme in the aspects of nucleic acid recognition and cutting are used as signal amplification and transduction means, so that the detection sensitivity of the glyphosate is improved, and the detection sensitivity of the glyphosate is improved. Therefore, trace detection of glyphosate is realized. According to the invention, sensitive detection of other targets can be realized by replacing nucleic acid aptamers capable of specifically recognizing other targets and correspondingly adjusting the sequence of cDNA to maintain base pairing.

Methods are provided for analyzing genomic DNA where the genomic DNA is subject to in situ fragmentation, in situ transcription, complementary DNA synthesis and barcoding complementary DNA.

The present invention relates to a composition for DNA sequence analysis and a method for DNA sequence analysis, the method comprising treating a sample with the composition. The composition of the present invention can attain efficient optical identification at a single-DNA molecule level by linking both an A / T-specific DNA-binder agent and an A / T-non-specific complementary DNA-binder agent to DNA, and thus can be helpfully used in studying chromosomal organization of genomes, protein immunolocalization, and the like.

The invention belongs to the technical field of biology, and particularly relates to a virus antigen detection method. The method comprises the following steps: modifying a complementary DNA chain of a virus antigen nucleic acid aptamer on the surface of an optical fiber, reacting a virus antigen in a sample with a fluorescently-labeled nucleic acid aptamer to change the conformation of the nucleic acid aptamer, and introducing a mixed solution containing the virus antigen and the fluorescently-labeled nucleic acid aptamer into the surface of a functionalized optical fiber sensing element, so as to detect the surface of the functionalized optical fiber sensing element. The fluorescence labeling nucleic acid aptamer with changed conformation is combined with complementary DNA (cDNA) on the surface of the optical fiber sensing element, the intensity of fluorescence received by the optical fiber sensing element is improved along with the increase of the combination amount, and the quantitative detection purpose is achieved by detecting the change of the intensity of the fluorescence received by the optical fiber sensing element. The method is high in accuracy, strong in specificity, small in required sample size and simple to operate, and can be used for carrying out specific detection on virus antigens.

The present application belongs to the technical field of environmental pollution detection, and relates to a porphyrin-based metal-organic framework nanoenzyme-mediated chemiluminescence optical fiber biosensor for ricin detection. The biosensor of the present application realizes the detection of ricin based on nanoenzyme catalysis, indirect competition nucleic acid aptamer affinity and optical fiber biosensing principle. The porphyrin-based metal-organic framework nanoenzyme probe labeled with nucleic acid aptamer is used to catalyze the luminol / hydrogen peroxide system to generate a luminescence signal. The higher the concentration of ricin is, the more nanoenzyme probe binding sites are occupied, which leads to the reduction of nanoenzyme probes combined with the optical fiber probe of the modified complementary DNA chain, and the generated luminescence signal is also reduced. Based on the high specificity of nucleic acid aptamer, the biosensor has good selectivity and anti-interference for ricin detection. The sensor has simple structure, high sensitivity and good selectivity, and can realize the rapid detection of ricin in medical and environmental samples.

Improved compositions and methods for using modified non-retroviral reverse transcriptase to perform 3′ extension of a nucleic acid, employ non-templated deoxynucleotide addition to a single-stranded nucleic acid and / or synthesis of complementary DNA using non-complementary nucleic acids as primer and template (RNA- or DNA-templated DNA polymerase activity.

This invention discloses an exosome enrichment and detection system for the diagnosis of peritoneal metastases in gastric cancer and its applications, primarily relating to the field of biomedical detection. It comprises a DNA nanoscaffold complex functionalized with multiple nucleic acid aptamers for specific capture of exosomes; a microfluidic chip, the solid surface of which is modified with the complex; an exosome release buffer containing complementary DNA strands for displacing the nucleic acid aptamers to release the exosomes; and an exosome content detection reagent containing a depurinyl / depyrimidine endonuclease 1 and fuel probes corresponding to various target miRNAs. The advantages of this invention are: it solves the problem of poor universality of traditional unit recognition in the highly heterogeneous gastric cancer, and can broadly identify multiple subtypes of tumor-derived exosomes.

The present invention relates to a method of quantifying the amount of at least one nucleic acid target, the method comprising: a) providing a prescribed number of cells wherein the prescribed number of cells is at least one cell; and b) lysing the at least one cell, thereby releasing RNA and DNA; and c) performing a reverse transcription reaction on the released RNA thereby producing complementary DNA (cDNA) and further performing a digital polymerase chain reaction (dPCR) on at least one reverse transcribed RNA target within the cDNA and / or performing a digital polymerase chain reaction (dPCR) on at least one DNA target within the released DNA wherein the dPCR is performed in a plate, and d) quantifying the amount of the at least one nucleic acid target.

The invention provides a beta amyloid protein oligomer detection electrode and a preparation method thereof and a dual-signal amplification sensing chip, the electrode comprises a first current collector, gold nanoparticles are attached to the surface of the first current collector, the gold nanoparticles are in bonding connection with complementary double-stranded DNA formed by complementation of cDNA-FAM and Apt-TAMRA, TAMRA is tetramethyl rhodamine, and the surface of the first current collector is in bonding connection with the complementary double-stranded DNA formed by complementation of cDNA-FAM and Apt-TAMRA. The Apt is an aptamer of a beta amyloid protein oligomer, the cDNA is complementary DNA of the Apt, and the FAM is carboxyl fluorescein. By combining fluorescence resonance energy transfer and electrochemical signals, efficient, sensitive and stable dual detection of AbetaO can be realized, the detection precision and the signal-to-noise ratio are improved, and the influence of single signal error is avoided.

Disclosed is a cDNA amplification method capable of comprehensively amplifying mRNA with high efficiency and low deviation from a very small amount of a cell sample. The complementary DNA chain amplification method according to the present invention comprises the steps of capturing a target RNA and synthesizing a complementary DNA chain in a liquid phase system or a solid phase system using a reverse transcriptase having terminal transferase activity, adding a base such as CCC to the terminal of the complementary DNA chain, and then performing homopolymer tailing by a TdT reaction in the presence of chain-terminated nucleotide triphosphate. By further combining the template conversion reactions, the complementary DNA strand can be amplified with higher efficiency and lower deviation.

The present invention is in the field of nucleic acid detection and quantification and relates to a method for detecting and quantifying target RNA sequences in a sample, the method comprising the steps of: a) reverse transcribing RNA present in a biological sample into complementary DNA (cDNA) using a plurality of oligonucleotides, wherein the oligonucleotides in the plurality of oligonucleotides comprise (i) a random nucleotide sequence (B) at the 3' end having a length of 3 to 12 nucleotides, wherein the plurality of oligonucleotides comprises at least 2 different random nucleotide sequences (B); and (ii) a unique sequence tag (X) at the 5' end; b) amplifying the tagged cDNA obtained in step (a) using a pair of primers, wherein the forward primer specifically anneals to a target sequence within the cDNA and wherein the reverse primer comprises a nucleic acid sequence that is at least partially identical in sequence to the nucleic acid sequence of the unique sequence tag (X); and c) detecting and quantifying the nucleic acids amplified in step (b). Further provided herein is a plurality of oligonucleotides and kits comprising the same.

In an example of a method, a primer functionalized, ribosomal RNA (rRNA) depleted sample is generated. The primer functionalized, rRNA depleted sample is combined with a composition to form a mixture, where the composition includes a reverse transcriptase, an RNA nickase, a DNA polymerase with strand displacement activity or 5'-3' exonuclease activity, and a plurality of deoxynucleotide trisphosphates. The mixture is introduced into a flow cell including a substrate, first and second primers immobilized on the substrate, and first and second transposome complexes immobilized on the substrate. A temperature protocol is initiated to form double-stranded complementary DNA (cDNA) from the primer functionalized, rRNA depleted sample and to form partially adapted cDNA fragments from the double-stranded cDNA.

This invention discloses a molecular switch-type nucleic acid framework fluorescent probe and its application in the detection of ATP molecules, a biomarker for depression, belonging to the field of biodetection technology. The fluorescent probe of this invention has a tetrahedral structure, comprising three complementary DNA single strands and four additional DNA single strands forming the tetrahedral structure. The three complementary DNA single strands include a Cy3 dye modified with a central backbone, a phosphate group modified at the 5' end, and a Cy5 dye modified at the 3' end. The fluorescent probe of this invention is activated by ATP, rapidly responding to ATP to emit a fluorescent signal, and is deactivated by an endonuclease, automatically returning to its initial state. This probe enables dynamic cyclic detection of ATP molecules and can directly perform rapid, highly sensitive, and highly selective quantitative analysis of the purine neurotransmitter ATP in complex biological samples such as plasma and urine, providing an effective means for early screening of neurological diseases.

Methods are described for enhancing complementary DNA (cDNA) synthesis from a ribonucleic acid (RNA) template during reverse transcription by an enzyme having template switching activity. The methods provide for enhancing the processivity of a reverse transcriptase.

The application discloses a ratio biosensor for marine biological toxin detection and preparation and application thereof, and the ratio biosensor comprises a specific detection biomagnetic element FA, a catalytic reaction solution and a SERS enhanced nanosubstrate; wherein, magnetic particles (Fe3O4-Apt) anchored by aptamers are combined with gold-labeled short complementary DNA (Au-cDNA) to generate the biomagnetic element (FA). In the detection, if the target exists, the Au-cDNA is dissociated, the separated Au cDNA is used as a catalyst to initiate a reduction reaction of 4-nitrothiophenol (4-NTP) in the presence of a hydrogen source, and a ratio change of SERS spectrum of 4-NTP is caused. The change is finally collected on the SERS enhanced nanosubstrate. The design of the ratio biosensor can effectively overcome unstable detection response caused by spectral fluctuation, and has self-calibration ability and high robustness.

The application belongs to the technical field of molecular biology, and particularly relates to a detection method and a detection kit for a poly-A tail of RNA. The first aspect provides a detection method for a poly-A tail of RNA, comprising the following steps: incorporating first dNTPs into RNA by reverse transcriptase to synthesize a complementary DNA chain; amplifying and sequencing the complementary DNA chain by using DNA polymerase and taking second dNTPs as a substrate; and analyzing the length of the poly-A tail according to the sequencing result. The application develops a method for replacing raw materials based on deoxyinosine triphosphate (dITP) of hypoxanthine, in which, after template conversion of RNA to form cDNA, the pairing mechanism of dITP and other bases is used to replace the poly-A chain with a random chain, so that a relatively complex sequence can be obtained, the stability of the sequencing signal is improved, and the length of the poly-A tail can be more accurately obtained based on the complex sequence.

Provided is a method for continuous detection of a miRNA by a metal-organic framework (MOF)-based nanozyme, including the following steps: step 1, conducting a duplex-specific nuclease (DSN)-assisted signal amplification strategy; and step 2, conducting the flow analysis on a target using a flow injection-batch method, and recording a differential pulse voltammetry (DPV) response of a biosensor in a batch mode; and step 3, in the flow mode, conducting competitive elution on a MOF / indium tin oxide (ITO) electrode with a complementary DNA (cDNA) as a mobile phase. In the present invention, a two-dimensional MOF-based nanozyme is simply synthesized by an ultrasonic method and then applied to a homogeneous electrochemical biosensor without a complicated electrode modification process, showing a continuous detection function, a high selectivity and a desirable sensitivity.

The application discloses an RNA response controlled-release DNA nanocarrier system, which comprises a DNA nanotube, a drug effective component and a nanodevice lock; the DNA nanotube comprises a tube wall and the nanodevice lock; the tube wall is unfolded in a rectangular shape; the nanodevice lock comprises a plurality of sealing chains, each of which is composed of two partially complementary DNA single strands, and the two DNA single strands are connected to two long sides of the tube wall; the sealing chains can undergo an isothermal chain displacement reaction with target RNA, open the nanodevice lock and unfold the tube wall; and the drug effective component is loaded in the inside of the DNA nanotube. The DNA nanotube is not easy to be enzymatically hydrolyzed, has high biocompatibility and is easy to be endocytosed by cells, and is used for wrapping the RNA drug with poor stability, so that the RNA drug is completely and sufficiently delivered to a target position; under the action of high expression of the RNA at the target position, the nanodevice lock on the DNA nanotube is rapidly opened, and the RNA drug is released.