31 results about "DNA ligase" patented technology

The invention belongs to the technical field of biology, and particularly relates to a T4 DNA ligase mutant with improved connection fidelity. The T4 DNA ligase mutant is obtained by mutating any one or more amino acid sites of lysine at the 365th site and lysine at the 367th site of wild type T4 DNA ligase. Compared with a wild type T4 DNA ligase, the T4 DNA ligase has higher fidelity, and the fidelity of the T4 DNA ligase is maximally improved to more than 10 times of that of the wild type T4 DNA ligase.

This invention discloses a dual-linker head, its introduction method, and its applications, relating to the field of biotechnology. The dual-linker head of this invention comprises a long nucleic acid chain and a short nucleic acid chain; the long nucleic acid chain includes a first guide sequence and a support sequence; the long nucleic acid chain is complementary to the short nucleic acid chain through the support sequence; the first guide sequence is 2-30 nt long; the first guide sequence is used to complementarily pair with a second guide sequence at the end of the target nucleic acid. This dual-linker head can freely carry various nucleic acid or nucleic acid aptamer functional modules and is compatible with different sample types, different sequencing platforms, and one-tube or step-by-step workflows. After the end of the target nucleic acid forms or exposes a second guide sequence complementary to the first guide sequence, the dual-linker head can undergo directional annealing with the target nucleic acid to construct a controllable anchoring complex, and further complete the adapter incorporation under the action of DNA ligase and / or DNA polymerase.

The application discloses a kind of for sweat glucose concentration monitoring method, it is related to sweat monitoring field, comprising the following steps: step 1, enzyme-fixed chain conjugate synthesis: glucose dehydrogenase is reacted with bilirubin oxidase respectively with bifunctional crosslinking agent and is purified, obtain enzyme-crosslinking agent intermediate;It is clicked with azide modified DNA fixed chain chemistry reaction again, and after purification, obtain GDH-fixed chain and BOx-fixed chain conjugate;Step 2, preparation of circular DNA template: after annealing hybridization of phosphorylated template chain and primer chain, it is connected into ring by T4 DNA ligase;Step 3, rolling circle amplification self-assembly package enzyme DNA hydrogel;The present application uses super-flexible DNA hydrogel as flexible electrode material, realizes the efficient fixation and continuous stable catalysis of GDH and BOx, and constructs flexible sweat-based EBFC self-powered sensor as flexible bioelectrode, to realize continuous non-invasive blood glucose monitoring.

The invention provides a construction method and application of bacillus amyloliquefaciens capable of enhancing yhcA expression, and the construction method comprises the following steps: taking bacillus amyloliquefaciens XH7 genome DNA as a template, carrying out PCR amplification on a transport protein coding gene yhcA, and connecting a promoter and a terminator to obtain a target gene segment; respectively carrying out double enzyme digestion on a free plasmid vector and a target gene segment by adopting the restriction enzyme to obtain a linear plasmid segment and an enzyme digestion gene segment; connecting the enzyme-digested gene fragment and the linear plasmid fragment through DNA ligase to obtain a free expression vector; and finally, transferring the free expression vector into a competent cell of bacillus amyloliquefaciens XH7, and adding a resistance selection marker to obtain a positive transformant. The research proves that the enhanced expression of the transporter gene yhcA is also a very effective method for improving the inosinic acid yield of bacillus amyloliquefaciens.

The invention provides a method for detecting Ggt in gene mutation. The invention relates to a lock type probe for detecting A mutation, which comprises a recognition sequence complementary with a target p53 R175H and a sequence recognized by CRISPR / Cas12a, and is modified with 8-oxoguanine. The invention also provides a Ggt based on the combination of the Fpg enzyme and CRISPR / Cas12a (clustered regularly interspaced short palindromic repeats / CRISPR / Cas12a). The universal type detection kit for A mutation comprises the lock-type probe, a fluorescence report probe, a primer, crRNA, a T4 DNA ligase, an Fpg enzyme, a phi29 DNA polymerase and a Cas12a protein. The invention also provides a Ggt; the invention discloses an A mutation detection method and application. According to the invention, the Ggt based on the combination of the Fpg enzyme and CRISPR / Cas12a is successfully constructed; according to the universal detection method for the A mutation, a 8-oxoG modified lock-type probe is used for recognizing a mutation site, and rolling circle amplification and CRISPR / Cas12a cascade signal amplification system are combined, so that the core bottlenecks of insufficient universality caused by restriction enzyme dependence and strict dependence of Cas protein on a target PAM site in the existing mutation detection technology are solved in a breakthrough manner, and the purpose of detecting all Ggt, Ggt, Ggt, Ggt, Ggt, Ggt, Ggt, Ggt, Ggt, Ggt and Ggt mutation is achieved. And broad-spectrum and accurate detection of point A mutation is realized.

The invention relates to a method for detecting a single-stranded break (SSB) site in double-stranded (ds) DNA. A dsDNA sample is contacted with an oligonucleotide probe comprising a first sequencing linker and a tag, and then contacted with a DNA ligase. And the DNA ligase is used for covalently linking the oligonucleotide probe to the dsDNA chain at the dsDNA single-chain breakage site. The dsDNA sample is then contacted with a transposase loaded with a second sequencing linker such that the transposase cleaves the dsDNA to produce a population of DNA fragments comprising the second sequencing linker. DNA strands comprising the first and second sequencing linkers are isolated from the population and the sequence of the isolated DNA strands is determined. These methods and kits for carrying out these methods are provided.

The invention relates to the technical field of biochemical analysis, in particular to a preparation method of a non-destructive exosome efficient drug loading system, an anti-tumor drug and application. The method comprises the steps of exosome separation and extraction and EVs-RCT synthesis, the exosome is used as a siRNA carrier, Primer-5 'cholesterol connects a Primer sequence to the surface of the exosome through hydrophilic and hydrophobic interaction, the Primer-5' cholesterol is hybridized with an annular template containing siRNA antisense and antisense complementary sequences, and a gap in annular DNA is chemically blocked by T4 DNA ligase; the closed-loop DNA generates RNA transcription products under the condition of T7 RNA polymerase, and a large number of hairpin RNA structures are generated. The high-density RNA structure on the efficient drug loading system not only can improve the drug loading capacity of siRNA, but also can improve the stability of the exosome in a complex environment, and has important significance in the aspects of tumor fluorescence imaging and antitumor drugs.

The present disclosure relates generally to the enzymatic conversion of methylated nucleic acids in order to differentiate methylated cytosine from unmethylated cytosine in DNA, and more particularly to improved methods and compositions for enzymatic methylation sequencing. In one aspect, various compositions and methods for improving the recovery of methylation signals are provided. The methods include one or more of a nick repair step, methylation signal restoration, use of a modified methylcytosine nucleic acid adapter, and use of a helicase, an ssDNA binding protein, an engineered DNA ligase, or a combination thereof.

The invention belongs to the technical field of biological detection, and particularly discloses a background-free DNA amplification method based on CRISPR-Cas12a pretreatment and application of the background-free DNA amplification method. The background-free DNA amplification method comprises the following steps: S1, carrying out precutting on interference oligonucleotide and a pre-primer by taking double-stranded target nucleic acid in a sample as a CRISPR-Cas12 primer; adding an ASFV dsDNA target into a PCR (Polymerase Chain Reaction) tube containing a buffer solution, a magnetic bead-coated DNA / RNA pre-primer and Cas12a-crRNA for incubation; s2, magnetic separation and amplification of the pre-primer: collecting a precipitate by separating magnetic beads with the help of a magnetic frame; transferring the precipitate into another PCR tube containing a circular padlock template, T4DNA ligase, phi29 DNA polymerase and an SERS label for incubation, and then taking out the solution for enzyme inactivation treatment; and S3, signal detection of an amplification product: adding a NaCl solution with the same volume into the solution subjected to enzyme inactivation treatment obtained in the step S2, and then carrying out Raman detection. The nucleic acid pretreatment scheme provided by the invention can be used as an ideal nucleic acid treatment platform for high-efficiency and high-specificity amplification of target nucleic acid.

The application belongs to the technical field of biological analysis detection, and specifically discloses an acute myocardial infarction biological detection system based on multiplex isothermal transcription amplification, application and method thereof, the system comprises a probe group, PBCV-1 DNA ligase, T7 RNA polymerase and thioflavin T, the probe group comprises probes P1, P2, double-stranded probe P2 / A1 and double-stranded probe A2 / A3, the double-stranded probe A2 / A3 is composed of probe A2 and probe A3, and the double-stranded probe P2 / A1 is composed of probe P2 and probe A1. The application innovatively combines target recognition based on PBCV-1 DNA ligase, double-cycle transcription signal amplification mediated by T7 RNA polymerase and split G-quadruplex / ThT fluorescence signal amplification system, and constructs a triple signal cycle amplification detection system, which has the characteristics of ultra-high sensitivity, excellent specificity and simple operation.

The present invention discloses a method and application for functionalization of engineered DNA crystals after crystallization. The present invention constructs a rigid triangular tensile structural unit, including three different DNA component chains, a total of seven chains, the side chains extending in three double helix directions, and the central chain with three repeating sequences is located in the center of the structural unit. Each double helix structure has two sticky ends with two bases at both ends, which interact with each other to form a very ordered 3D crystal network. T4 DNA ligase is used to connect the sticky ends to obtain stable DNA crystals. The crystal structure of the present invention is stable, the target central chain can be added with complex modifications, and it has universality. The crystal has a wide range of uses in medical disease detection.

The invention includes a mutant T4 DNA ligase or a biologically active fragment thereof, which has greater activity than wild type T4 DNA ligase. The mutant T4 DNA ligase, or the biologically active fragment, has one or more substitutions differing from the wild type, as described more fully in the Summary.

The invention provides a T4 DNA ligase variant which has increased T / A connection specificity, in the library construction process, the library yield and the library quality can be improved by using the T4 DNA ligase variant, and the A / T separation problem in offline data is solved to a certain extent.

The invention relates to the technical field of sequencing, in particular to a preparation method of a sequencing library and a reagent. The invention provides a preparation method of a sequencing library, which comprises the following steps of: additionally adding an additive or an additive combination capable of reducing a DNA secondary structure and / or reducing single-stranded DNA degradation in single-stranded cyclization reaction and / or rolling circle amplification reaction; an additive or an additive combination capable of reducing a DNA secondary structure, reducing single-stranded DNA degradation and / or reducing double-stranded DNA double-helix stability is additionally added in a double-stranded cyclization library rolling circle amplification reaction, so that the binding capacity of DNA ligase and / or strand displacement DNA polymerase and target nucleic acid is improved, and the situation that the rolling circle amplification efficiency of different circular DNAs is not uniform due to the secondary structure is reduced; the probability that the loose single-stranded DNA is easy to degrade is reduced, and the amplification uniformity of different GC regions and different sequence characteristic regions of the library is improved, so that the sequencing quality and the sequencing data yield are improved.

The invention relates to the technical field of gene editing, and discloses a gene editing efficiency detection vector based on double-color fluorescent protein as well as a construction method and application thereof. The construction method comprises the following steps: synthesizing a full-length DNA fragment containing a specific targeting sequence, an mCherry reporter gene and a C-terminal fused PEST degrading peptide sequence; performing double enzyme digestion and purification to obtain a target fragment; connecting the target fragment with the vector skeleton subjected to enzyme digestion linearization through T4 DNA ligase; the gene editing efficiency detection vector pLV3-CMV-RGFP-PEST-Neo is prepared by the following steps: carrying out gene editing efficiency detection on the gene editing efficiency vector pLV3-CMV-RGFP-PEST-Neo, transforming the gene editing efficiency vector pLV3-CMV-RGFP- The system containing the gene editing efficiency detection vector co-expresses red and green, during editing, an mCherry coding sequence is cut off, cells are converted from double positive (yellow) to single positive (green), cell lysis is not needed, the editing efficiency is quantified by calculating the change of the green / red fluorescence ratio, and the method is convenient and fast.

This invention discloses a highly active T4 DNA ligase mutant, its preparation method, and its applications. In this invention, through crystal structure and bioinformatics analysis combined with site-directed mutagenesis, saturation mutagenesis, and combinatorial mutagenesis, as well as rigorous experimental analysis, several T4 DNA ligase mutants with significantly improved performance were screened, including single mutants and combinatorial mutants. The improved performance includes: enzyme activity, thermal stability, and / or ligation efficiency.

B cells with an altered function. FIG. 1A Rearrangement at the Ig locus n - 250-1,000) VH1 VHP DH1-12 T FEu DNA-PKcs RAG1 / 2 VIDN Excised Ku70, Ku80 NHEJ recombination intervening DNA ARTEMIS XROC4 DNA Ligase N V DJ Cp C6 OB Cr1 0120 area Ce Cex 3' RR Eu Su Se At S regions: DNA breaks AD Class-switch DNA repair recombination IgM V D JCu Transcription Recombined CH locus V DJ Ce Ca 3' RR Cy2a Cu Eµ C8 Excised Cy2b intervening DNA IgE V DJCe Cy3 Cyl 20 26 20 47 61 19 J un 2 02 6 A B S T R A C T 1 9 J u n 2 0 2 6 2 0 2 6 2 0 4 7 6 1 F I G . 1 A R e a r r a n g e m e n t a t t h e I g l o c u s V H 1 V H n D H 1 - 1 2 7 F E u r D N A - P K c s R A G 1 / 2 V I D N N H E J V D J C p C 6 O B 0 1 2 0 O R D C e E u S u S e A D r e c o m b i n a t i o n I g M V D J C u R e c o m b i n e d V D J C e C a 3 ' R R C y 2 a C u E u r C 8 E x c i s e d C y 2 b I g E V D J C e C y 3 C y l

The invention includes a mutant T7 DNA ligase or a biologically active fragment thereof, which has greater activity than wild type T7 DNA ligase. The mutant T7 DNA ligase, or the biologically active fragment, has one or more substitutions differing from the wild type, as described more fully in the Summary. The preferred mutant T7 DNA ligase has at least one of the following mutations: E63K (SEQ ID NO:4), K73E (SEQ ID NO:6), K137E (SEQ ID NO:7), K174E (SEQ ID NO:9), E182K (SEQ ID NO:11), K210E (SEQ ID NO: 13), E243K (SEQ ID NO:15), D245R (SEQ ID NO: 17), E268K (SEQ ID NO:19), E272K (SEQ ID NO:21), E289K (SEQ ID NO:23), K295E (SEQ ID NO:25) and D336R (SEQ ID NO:27).

The invention provides a multifunctional nano-composite, which is formed by electronegative DNA nano-flower and cationic liposome through electrostatic self-assembly, the DNA nano-flower takes single-stranded DNA containing CpG motif as a template, and is synthesized through Phi29 DNA polymerase rolling circle amplification reaction after T4 DNA ligase cyclization; the cationic liposome is composed of a single component cationic lipid NGPE, and an NR1D1 agonist SR9011 is entrapped in the cationic liposome. The invention also provides a preparation method of the nanocomposite. The invention also provides an application of the nano-composite in preparation of drugs for radioimmunotherapy of triple negative breast cancer. According to the invention, the technical problems of insufficient immune activation, high tumor radiation resistance, low hydrophilic / hydrophobic drug co-delivery efficiency, tumor microenvironment immunosuppression and the like in the existing radiotherapy are solved, and the radioimmunotherapy curative effect of the triple negative breast cancer is improved.

The present invention relates to a method for detecting single-strand breaks (SSBs) in double-stranded (ds) DNA. A sample of dsDNA is brought into contact with an oligonucleotide probe containing a first sequencing adapter and tag, and then into contact with a DNA ligase. The DNA ligase covalently binds the oligonucleotide probe to the dsDNA strand at the single-strand break sites in the dsDNA. The dsDNA sample is then brought into contact with a transposase loaded with a second sequencing adapter, resulting in the cleavage of the dsDNA by the transposase and the generation of a population of DNA fragments containing the second sequencing adapter. DNA strands containing the first and second sequencing adapters are isolated from the population, and the sequences of the isolated DNA strands are determined. A method and a kit for carrying out the method are provided.

The present application relates to a product based on dumbbell-shaped probe and RCA for KRAS gene detection, the product comprises a dumbbell-shaped probe, a DNA ligase and RCA amplification reagents, the dumbbell-shaped probe comprises a stem and two loop parts, the ends of the two loop parts are connected through the stem, one of the loops of the dumbbell-shaped probe contains a G-quadruplex aptamer PW17 complementary sequence, and the other loop contains a G-quadruplex aptamer T30695 complementary sequence, the RCA amplification reagents comprise Phi29 DNA polymerase, dNTPs and Phi29 DNA polymerase reaction buffer, under the mediation of the DNA ligase and the detected gene, the stem of the dumbbell-shaped probe is opened, the nick ligation forms a closed loop structure, and serves as a template for RCA. By adopting the technical scheme, the present application provides a product based on dumbbell-shaped probe and RCA for KRAS gene detection, compared with the traditional method, when testing single nucleotide mutation of the gene, it is more convenient and more efficient.

The invention belongs to the technical field of biological analysis and detection, and particularly discloses an acute myocardial infarction biological detection system based on multiple isothermal transcription amplification and application and method thereof.The system comprises a probe set, PBCV-1 DNA ligase, T7 RNA polymerase and thioflavin T. The probe set comprises a probe P1, a probe P2, a double-stranded probe P2 / A1 and a double-stranded probe A2 / A3. The double-stranded probe A2 / A3 is formed by connecting a probe A2 and a probe A3, and the double-stranded probe P2 / A1 is formed by connecting a probe P2 and a probe A1. According to the application, target recognition based on PBCV-1 DNA ligase, T7 RNA polymerase mediated dual-cycle transcription signal amplification and a split G-quadruplex / ThT fluorescence signal amplification system are innovatively and organically combined to construct a triple signal cycle amplification detection system, and the triple signal cycle amplification detection system has the characteristics of ultrahigh sensitivity, excellent specificity, simplicity and convenience in operation and the like.

This invention discloses a PSA detection method based on a black phosphorus / bismuth vanadate composite material, comprising: S1, coating a microplate with a PSA monoclonal antibody, blocking it, adding the PSA sample to be tested and incubating it, then incubating it with a PSA polyclonal antibody, a thiolized oligonucleotide sequence 1, and a gold nanoparticle conjugate to form a sandwich immune complex; S2, adding a circular DNA template, DNA ligase, and other reagents, and incubating to complete RCA amplification; S3, adding a thiolized oligonucleotide sequence 2 and a conjugate with alkaline phosphatase, and adding a substrate for hydrolysis to generate ascorbic acid; S4, using a biosensor to perform photoelectrochemical testing under simulated sunlight and voltage, recording the oxidation current signal to quantify the PSA concentration. The PSA detection method disclosed in this invention, using a biosensor modified with this composite material and employing a signal amplification strategy, achieves highly sensitive and stable detection of PSA, which has significant clinical implications for the early diagnosis of prostate cancer.

The invention discloses a plant insect-resistant related protein as well as a coding gene and application thereof. According to the invention, the full length of the SlJAIB14 gene is amplified from tomato, and two restriction enzyme cutting sites of BamHI and Sac I are introduced to the 5'and 3 'ends of the gene through primer sequences. The newly synthesized gene is connected with a pYPX158 plant expression vector containing double 35S promoters through a T4 DNA (Deoxyribose Nucleic Acid) ligase, so that a recombinant plasmid pYPX-S1JAIB14 containing a target gene S1JAIB14 is obtained. According to the invention, plasmids are introduced into agrobacterium tumefaciens (GV3101) by using an electric shock method. The SlJAIB14 gene is transformed into tomato by using an agrobacterium-mediated method, and the resistance of the transgenic plant to cotton bollworm is verified through a feeding experiment.

This application discloses a magnetic capture probe, its preparation method, kit, and applications. The preparation method includes mixing amino magnetic beads with glutaraldehyde and incubating to obtain aldehyde magnetic beads; mixing aldehyde magnetic beads with primers and incubating to obtain primer-modified magnetic beads; mixing primer-modified magnetic beads with a template, followed by high-temperature denaturation and low-temperature annealing, and then adding T4 DNA ligase to ligate gaps to obtain circular template-modified magnetic beads; and performing rolling circle amplification on the circular template-modified magnetic beads to obtain polynucleotide aptamer-modified magnetic beads, i.e., the magnetic capture probe. This application utilizes the specific recognition of Acinetobacter baumannii by polynucleotide aptamers, combined with magnetic separation technology of magnetic beads, to form a magnetic DNA capture probe based on polynucleotide aptamers, which can effectively achieve the capture, separation, and enrichment of Acinetobacter baumannii.

A preparation method of a super high dose loaded compressed nucleic acid DNA drug delivery platform, according to the disease type, select CpG type, mix the 5' end phosphorylated DNA template containing CpG sequence with the first primer solution, add T4 ligase buffer, mix with ultrapure water to form a circularization system, heat annealing in a PCR instrument; add T4 DNA ligase solution, overnight reaction, after the reaction is completed, inactivate the ligase, obtain the circular DNA template; add phi29 DNA polymerase, 10x phi29 buffer, mixed solution of dATP, dTTP, dCTP, dGTP, Milli-Q to the circular DNA template to form a reaction solution, and the reaction solution is placed in a constant temperature mixing instrument for reaction; then, the second primer and the third primer are added, and constant temperature reaction is carried out to form a DNA drug delivery platform; after the reaction is completed, the polymerase is inactivated, and the reaction is terminated. The application also provides application of the above-mentioned DNA drug delivery platform in preparation of treatment of cancer or inflammatory diseases. The application can super-compress and efficiently load nucleic acid drugs.

The invention discloses a plant gene expression binary vector construction method based on T4DNA ligase, relates to the technical field of plant gene expression binary vectors, and aims at solving the problems that a traditional step-by-step enzyme digestion connection method is long in period and the reverse insertion rate reaches up to 40%-60%. The promoter-target gene-terminator directional assembly is realized by utilizing stepped enzyme cutting site design, combining with self-connection of a CIP alkaline phosphatase dephosphorylation inhibition vector, accurately controlling the fragment proportion and incubating for 16 hours through T4 ligase at 16 DEG C. The invention further discloses a preparation method of the promoter-target gene-terminator directional assembly of the promoter-target gene-terminator based on the CIP alkaline phosphatase dephosphorylation inhibition vector. According to the method, the positive cloning rate is increased to 70% or above, the construction period is shortened to 2-3 days, and the efficiency is increased by 40% or above compared with that of a traditional method. Modularized design is adopted in the elements, the promoter can be replaced into a tissue specific type, and resistance genes support flexible selection and meet screening requirements of different plants. The experiment only needs a conventional molecular biology reagent, is low in cost and easy to operate, and does not need an expensive seamless cloning kit.

Reaction condition compositions for detecting a genomic variation from a small sample amount from 5 nano grams (ng) to 1 microgram (ug) includes DNA ligase, DNA polymerase, at least one COP, a DNA polymerase buffer, NAD+, at least two primers, and deoxynucleotide triphosphates (dNTPs). Detection of the genomic variation utilizes COPs with increased ligation efficiency and RCA with fluorescence detection due to simultaneous ligation of COPs to CPs and replication of the genomic variation. The reaction condition composition eliminates the need to perform background reduction of un-hybridized or un-ligated COPs.

The invention designs a preparation method of an electrochemical sensor based on a multi-branched chain annular DNA signal probe. C0 and S1 form an annular template under the action of T4 DNA ligase, and S2 is added to form a multi-branched chain annular DNA structure. Thi / Au (at) PBA and S3 are incubated to synthesize a signal probe, and the signal probe and the multi-branched-chain annular DNA structure are incubated to form the multi-branched-chain annular DNA signal probe. In a magnetic separation system, a histamine aptamer chain Apt is connected to a magnetic bead, a multi-branched chain annular DNA signal probe is added to be connected with an aptamer, when histamine exists, histamine and the aptamer chain Apt are combined to enable the multi-branched chain annular DNA signal probe to fall off, and the multi-branched chain annular DNA signal probe is detected. Dropwise adding a multi-branched chain annular DNA signal probe in the supernate to the surface of an electrode modified by the gold-palladium core-shell nano material, connecting through a palladium-sulfur bond, and determining the signal change of Thi to realize quantitative detection of histamine. The multi-branched chain annular DNA signal probe and the nano material are introduced, so that the accuracy and the sensitivity of the sensor are improved.

The invention relates to a simple and novel EXPAR-assisted self-supplied crRNA-mediated CRISPR (clustered regularly interspaced short palindromic repeats) / Cas12a (CRISPR / Cas12a) fluorescent biosensor, which is used for ultra-sensitive detection of miRNA (micro Ribonucleic Acid) in breast cancer. The detection process of the sensor comprises three key steps: EXPAR amplification, connection identification and Cas12a trans-splitting decomposition. By combining EXPAR, a transcription process driven by connection recognition and a CRISPR / Cas12a signal amplification mechanism, the system realizes triple signal amplification, so that the detection sensitivity is remarkably improved, and miR-155 as low as 375.8 aM can be detected. A transcription process driven by connection recognition is introduced between EXPAR and CRISPR / Cas12a, so that background interference is effectively reduced, which is benefited from the accurate recognition capability of special DNA ligase on a mononucleotide specific sequence. In addition, due to the flexible design of the EXPAR template, the strategy can detect various different miRNA targets with high sensitivity. It is worth noting that the sensor shows good reliability in a complex sample and is successfully applied to detection of miR-155 in tumor cells.