54 results about "Single-stranded binding protein" patented technology

The present invention discloses a recombinant glycosylase base editing system and its application. The recombinant glycosylase base editing system of the present invention is an APOBEC-nCas9-Ung1 or APOBEC(R33A)-nCas9-Rad51-Ung1 base editing system; the base editing system APOBEC-nCas9-Ung1 includes cytosine deaminase APOBEC, nuclease nCas9 and uracil DNA glycosylase Ung1; the base editing system APOBEC(R33A)-nCas9-Rad51-Ung1 includes cytosine deaminase APOBEC(R33A), nuclease nCas9, single-stranded DNA binding protein Rad51 and uracil DNA glycosylase Ung1. Experiments have shown that the recombinant glycosylase base editing system of the present invention can improve the editing efficiency of base C to base G in the genome sequence of an organism or a biological cell and the purity of the editing product.

The invention provides a plant cytosine single-base editor as well as a construction method and application thereof, and relates to the technical field of plant genetic engineering. The single-base editor provided by the invention is named as hyPopCBE, and has a structural formula shown in the description, the editor comprises hA3A / Y130F cytosine deaminase, an optimized human-derived Rab51 single-stranded DNA binding protein structural domain, a fusion protein of nCas9 nuclease and a uracil glycosylase inhibitor, a fusion protein expression unit of phage MS2 coat protein and the uracil glycosylase inhibitor, and a guide RNA transcription expression unit integrated with an MS2 binding sequence. Compared with an existing plant cytosine single-base editor, the single-base editor constructed by the method can remarkably improve the editing efficiency of plant alleles, and the stability is higher; when the herbicide-resistant poplar gene is applied to poplar, a new herbicide-resistant poplar germplasm can be successfully created, and a technical support is provided for research and molecular breeding of a poplar functional genome.

The invention provides a method for detecting the relative activity of a single-stranded DNA binding protein. The method comprises the following steps: incubating a to-be-detected sample and a fluorescent probe for detecting the single-stranded DNA binding protein, collecting a fluorescence signal, and judging the relative activity of the single-stranded DNA binding protein in the to-be-detected sample according to the intensity of the fluorescence signal, the fluorescent probe is of a single-stranded DNA structure, the length is 13-21 bp, and the fluorescent probe contains a reporter group and a quenching group; the reporter group and the quenching group are respectively positioned at the 5'end and the 3 'end of the fluorescent probe, or the reporter group and the quenching group are respectively positioned at the 3' end and the 5 'end of the fluorescent probe. The fluorescence intensity of the detection system after incubation of the to-be-detected sample and the fluorescent probe is detected so as to judge the relative activity of the single-stranded DNA protein in the to-be-detected sample, the purpose of detecting the relative activity of the single-stranded DNA binding protein can be achieved, the detection result is accurate, and the detection time is short.

The invention belongs to the field of gene engineering, and particularly relates to a novel recombination system in aeromonas and application of the novel recombination system. A novel recombination system in the aeromonas disclosed by the invention contains a homologous recombination system expression vector, the expression vector expresses an operon under the control of an inducible promoter, and the operon contains three genes of a coding host specific exonuclease RedAahy, a single-stranded DNA annealing protein RedBahy and a single-stranded DNA binding protein RedSahy; the operon is derived from an aeromonas hydrophila (Aeromonas hydrophila) plasmid pR148, and is characterized in that the operon is derived from the aeromonas hydrophila (Aeromonas hydrophila) plasmid pR148. By utilizing the recombination system, efficient, simple and convenient gene insertion and deletion of aeromonas are successfully realized, and the application prospect is wide.

The invention is entitled "Formation of Synthons". The present disclosure provides, among other things, a composition comprising: a 5'-3' exonuclease; a strand displacement polymerase; and optionally, a single-stranded DNA binding protein and / or a ligase. Also described are polynucleotide assembly methods for forming synthons and kits for performing the methods.

The invention belongs to the field of microbial genetic engineering, and particularly relates to an efficient homologous recombination method suitable for bifidobacteria and application of the efficient homologous recombination method. Specifically, the invention discloses a method for constructing and screening the combination of the single-chain annealing protein and the single-chain binding protein for the first time. According to the method, a single-chain annealing protein and a single-chain binding protein are jointly constructed in an escherichia coli-bifidobacterium shuttle expression vector, and co-expression of the single-chain annealing protein and the single-chain binding protein in bifidobacterium longum is realized, so that the homologous recombination capability of the bifidobacterium longum is remarkably improved. According to the method disclosed by the invention, the homologous recombination efficiency of genome editing of the bifidobacterium longum is greatly improved, the absolute genome editing efficiency of the bifidobacterium longum is improved to 0.00009792% from the original level which is almost undetectable, and the research and application progress of genome engineering of the strain is remarkably promoted, so that the method has a good practical application value.

This invention discloses direct amplification qPCR reagents, kits, and applications for whole blood samples. The amplification reagents include: a composite hot-start DNA polymerase system, a buffer system, a surfactant, an anti-contamination system, and an anti-inhibition enhancer system. The composite hot-start DNA polymerase system is obtained by mixing antibody-modified hot-start Taq enzyme and antibody-modified Taq1C2 mutant Taq enzyme in a predetermined ratio. The buffer system includes Tris-HCl, MgCl2, (NH4)2SO4, and KCl. The surfactants include Tween-20 and NP-40. The anti-contamination system includes UDG enzyme and dNTPs, wherein the dNTPs are composed of dATP, dGTP, dCTP, dTTP, and dUTP. The anti-inhibition enhancer system includes D-trehalose, L-carnitine, DMSO, BSA, and SSB single-chain binding protein.

The application discloses a buffer composition for inhibiting rolling circle amplification product-mediated microsphere aggregation and a detection method. The buffer composition comprises a zwitterionic compound, a single-stranded DNA binding protein and an inert hydrophilic polymer. Preferably, the buffer composition contains 50-200 mM betaine, 0.05-2 mu g / mL SSB protein and 1-5% (w / v) PEG. More preferably, the buffer composition contains 100 mM betaine, 0.5 mu g / mL SSB protein and 2% PEG 8000. A rolling circle amplification-based microsphere detection method is also provided. Through the synergistic effect of multiple components, the application systematically inhibits microsphere aggregation, and the proportion of single microspheres can reach 89.2%, and the CV can be reduced to 4.8%, thereby significantly improving the detection accuracy and repeatability.

The invention belongs to the technical field of food-borne pathogenic microorganism detection, and particularly relates to an ortho-position nucleic acid probe for detecting norovirus and application of the ortho-position nucleic acid probe. The invention provides an ortho-position nucleic acid probe for identifying norovirus, the ortho-position nucleic acid probe specifically comprises a polystyrene nanoprobe and a single-chain binding protein coupling probe, and the polystyrene nanoprobe is formed by covalent binding of polystyrene microspheres, lectin and hairpin DNA. The single-chain binding protein coupling probe is formed by combining a norovirus antibody and a nucleic acid molecule through a connecting molecule. The probe is simple in synthesis condition and does not need complex purification steps. According to the present invention, a combined reaction with an activation chain, a hybridization chain reaction reagent and a catalytic self-assembly reagent is performed, and the detection sensitivity is improved through a signal cascade amplification reaction, and can achieve the minimum of 0.15 pg mL <-1 >; and moreover, the gene has excellent specificity, and the delta F'difference between the gene and pathogens of staphylococcus aureus, escherichia coli, rotavirus and astrovirus is extremely remarkable.

The invention discloses a flushing reagent for high-throughput sequencing, a kit and a use method, and the flushing reagent comprises a Tris buffer solution, a soluble salt, a PCR enhancer and a single-chain binding protein. The washing reagent contains the single-chain binding protein, so that the amplification efficiency can be greatly improved, the small difference of the amplification efficiency among different DNCs is ensured, the amplified DNCs in the chip are bright and uniform, and the problem of non-uniform sequencing signals in the traditional bridge amplification technology is solved.

The invention discloses a construction method and application of a kluyveromyces marxianus mutant, and belongs to the field of synthetic biology and molecular biology. According to the method, single-stranded DNA binding protein and cytosine deaminase pmCDA1 are fused, a random base editing system is constructed, and whole-genome efficient C-T / G-A mutation is realized in kluyveromyces marxianus by taking MCM2-7 as a verification target. The system does not need a specific target or an exogenous DNA template, the mutation efficiency is high, the distribution is wide, the mutation rate is improved by 303.1 times compared with that of a control group through optimal combination, and the application potential of kluyveromyces marxianus in industrial production is expanded. The method is combined with an efficient screening system, mutant strains with the intracellular protein expression quantity remarkably improved are screened out, the constructed mutation system is not only suitable for kluyveromyces marxianus cell factory transformation, but also can be popularized to genome evolution of other yeasts or eukaryotic hosts, and a new strategy is provided for industrial microorganism breeding and protein engineering.

The invention relates to a method of characterising a target polynucleotide using a single-stranded binding protein (SSB). The SSB is either an SSB comprising a carboxy-terminal (C-terminal) region which does not have a net negative charge or a modified SSB comprising one or more modifications in its C-terminal region which decreases the net negative charge of the C-terminal region.

The present application discloses a yeast Rad protein hybrid and a use of yeast Rad protein in combination in nucleic acid amplification and a nucleic acid amplification kit. The present application utilizes yeast protein homologues to form a hybrid dimer, which has a conserved ATP binding domain and the ability to catalyze ATP hydrolysis, and can promote yeast Rad protein to displace single-stranded DNA binding protein bound to single-stranded DNA, thereby improving the strand displacement activity of yeast Rad protein, mediating the strand displacement activity between primers and nucleic acid templates, and improving the affinity of yeast Rad protein for low-abundance templates, thereby improving the specificity and sensitivity of nucleic acid isothermal amplification.

Methods and compositions for polynucleotide sequencing. The methods include incorporating a blocked, labeled nucleotide into a copy polynucleotide strand; detecting the identity of the blocked, labeled nucleotide; and removing the blocked, labelled nucleotide. The steps may be repeated. At least one of the steps is performed in the presence of a single-stranded binding protein.

A method for purifying single-stranded DNA is provided. The method can comprise: mixing a sample containing single-stranded DNA with a single-stranded DNA binding protein, and incubating for a first time period to obtain a first product, the first product containing a conjugate of the single-stranded DNA and the single-stranded DNA binding protein; purifying the first product by using affinity chromatography to obtain a second product; and purifying the second product to at least remove the single-stranded DNA binding protein to obtain a target product containing a purified single-stranded DNA. Also provided are a kit for purifying single-stranded DNA, the kit comprising a single-stranded DNA binding protein and a purification reagent; and a use of a single-stranded DNA binding protein in purifying single-stranded DNA.

The application provides a RT-qPCR full premix reaction reagent and application and product thereof, and belongs to the technical field of biochemical reagents.The RT-qPCR full premix reaction reagent provided by the application comprises PCR reaction additives, DNA polymerase, reverse transcriptase, heat-sensitive uracil DNA glycosylase, RNAase inhibitor, soluble cation and dNTPs; the PCR reaction additives are composed of 50-1000 mM trehalose, 0.5-10.0 %v / v DMSO, 50-500 mM betaine, 50-500 ng / μL SSB single-strand binding protein and a stabilizer; and the stabilizer is composed of lauryl alcohol polyoxyethylene ether, glycerol and BSA.The RT-qPCR full premix reaction reagent provided by the application is not only good in universality, high in specificity and sensitivity, but also has good stability and can be stably stored in a-20 DEG C refrigerator for one year.

This invention belongs to the field of gene editing technology and discloses an inosine-enriched adenine base editor off-target detection method (Ino-seq) and its applications. The method of this invention utilizes endonuclease V to specifically cleave inosine-containing DNA, combined with the stabilization of a thermostable single-stranded DNA-binding protein and the high affinity enrichment of streptavidin-biotin, to achieve direct capture of ABE enzymatic products. This method can simultaneously detect sgRNA-dependent and independent off-target effects, with systematic comparisons showing F1 scores of 0.892–0.931. Targeted deep sequencing validation showed an overall accuracy of 95.3%. Ino-seq can also detect endogenous genomic inosine, observing a variation range of >1,000-fold in 10 cell lines. This invention provides a crucial safety assessment tool for the clinical translation of ABE technology, supporting comprehensive off-target analysis in preclinical and clinical studies.

The application provides a single-chain binding protein mutant with increased thermal stability, a nucleotide sequence and an application, and aims to solve the problem of poor thermal stability of existing single-chain binding protein mutants. The application provides a single-chain binding protein mutant with increased thermal stability, and the amino acid sequence of the single-chain binding protein mutant is shown in SEQ ID NO. 1. The single-chain binding protein mutant has better thermal stability, and unlike the wild-type protein which needs to be stored at-20 DEG C, the single-chain binding protein mutant can be stored and transported at room temperature. In addition, under the condition of constant temperature amplification reaction at 60-65 DEG C, the mutant protein has better reaction performance compared with the wild-type.

The invention relates to the field of nucleic acid amplification, in particular to a method for isothermal amplification of nucleic acid molecules. The invention provides a method for isothermal amplification of nucleic acid molecules. The method comprises the following steps: mixing nucleic acid to be amplified with helicase to obtain single-stranded nucleic acid; mixing the single-stranded nucleic acid with the single-stranded binding protein and the primer enzyme to obtain oligonucleotide; under the condition that dNTP exists, oligonucleotide and DNA polymerase are mixed, and an extension product is obtained; repeating the steps for one or more times on the extension product to obtain the sequence of the nucleic acid to be amplified. The HPA disclosed by the invention is an isothermal amplification method which comprises the following steps of: unfastening double strands of a template by utilizing helicase, generating a primer by utilizing primer enzyme, and then amplifying by utilizing DNA (Deoxyribose Nucleic Acid) polymerase. According to the method, no additional primer needs to be added, non-specific amplification products such as primer dimers are prevented from being generated, high-temperature denaturation is also not needed, and the reaction can be completed at a constant temperature. Whole genome amplification can be carried out on an original template, and the DNA concentration of the original template is improved, so that downstream detection is facilitated.

The invention provides an RT-qPCR (real-time quantitative polymerase chain reaction) full premix reaction reagent as well as application and a product thereof, and belongs to the technical field of biochemical reagents. The RT-qPCR full premix reaction reagent provided by the invention comprises a PCR reaction additive, a DNA polymerase, a reverse transcriptase, a thermosensitive uracil DNA glycosidase, an RNA enzyme inhibitor, soluble cations and dNTPs (deoxyribonucleoside triphosphates). The PCR reaction additive is composed of 50 to 1000 mM of trehalose, 0.5 to 10.0% v / v of DMSO, 50 to 500 mM of betaine, 50 to 500 ng / [mu] L of SSB single-chain binding protein, and a stabilizing agent; the stabilizer is prepared from polyoxyethylene lauryl ether, glycerol and BSA (Bovine Serum Albumin). The RT-qPCR full premix reaction reagent provided by the invention is not only good in universality, high in specificity and sensitivity, but also relatively good in stability, and can be stably stored for one year in a refrigerator at-20 DEG C.

The invention provides a preparation method and application of a single-chain binding protein, and relates to the field of protein purification, the preparation method comprises the following steps: (1) affinity chromatography of the single-chain binding protein: using Ni-FF as a filler of a chromatographic column; (2) ion exchange chromatography: using DEAE Sepharose FF as a filler of a chromatographic column; (3) hydrophobic chromatography: using Phenyl-FF as a filler of a chromatographic column; (4) affinity chromatography: using Ni-FF as a filler of a chromatographic column; (5) ion exchange chromatography: using QHP as a filler of a chromatographic column; and (6) molecular sieve chromatography: using Sephadex G-25 as a filler of a chromatographic column. The method can effectively remove nuclease and nucleic acid residues of host cells, and is simple in purification process, low in cost and high in protein purity.

The invention discloses a real-time fluorescence isothermal amplification detection kit for detecting whether meat products are doped with chicken components and application. The real-time fluorescence isothermal amplification detection kit for detecting whether the meat product is doped with the chicken component or not is constructed based on Cas9-H840A and exo probes, sgRNA-guided Cas9-H840A can accurately recognize target genome DNA, and a target fragment is amplified under the action of Bsu DNA polymerase with strand displacement activity and single-stranded binding protein; the exo probe can specifically recognize a complementary sequence and cut a THF site with the help of exonuclease III to realize signal amplification. The real-time fluorescent isothermal amplification detection kit disclosed by the invention has extremely high specificity and sensitivity on chicken detection in a complex sample environment, can accurately and sensitively detect whether chicken is doped in meat products or not, and has good accuracy, practicability and universality on actual sample detection.

Methods and compositions for polynucleotide sequencing. The methods include incorporating a blocked, labeled nucleotide into a copy polynucleotide strand; detecting the identity of the blocked, labeled nucleotide; and removing the blocked, labelled nucleotide. The steps may be repeated. At least one of the steps is performed in the presence of a single-stranded binding protein.

The present invention discloses a method for editing the genome of Bacillus subtilis, which belongs to the field of genetic engineering. The present invention uses Bacillus subtilis as an expression host, integrates a single-stranded DNA binding protein and a DNA annealing protein into the Bacillus subtilis genome, and obtains a recombinant Bacillus subtilis, wherein the amino acid sequence of the single-stranded DNA binding protein is shown in SEQ ID NO.1, and the amino acid sequence of the DNA annealing protein is shown in SEQ ID NO.2. By integrating and expressing the single-stranded DNA binding protein and the DNA annealing protein in the Bacillus subtilis genome, the length of the homology arm of the Bacillus subtilis genome integration can be shortened to 35bp, providing a tool for achieving efficient and simple gene editing in Bacillus subtilis.

Provided are a fusion protein that improves gene editing efficiency and an application thereof. The fusion protein comprises a single-stranded DNA binding protein functional domain, nucleoside deaminase and nuclease. According to CBEs, when carrying our base conversion from C-G to T-A, nucleoside deaminase such as cytosine deaminase carries out deamination by using single-stranded DNA as a substrate, and by re-fusing the single-stranded DNA binding protein functional domain on the fusion protein of the nucleoside deaminase and nuclease, the chance of single-stranded DNA being exposed to the nucleoside deaminase is greatly increased, thereby significantly improving base editing efficiency. The present disclosure provides a breakthrough improvement of single-base gene editing technology and can greatly promote the application thereof in aspects such as gene editing, gene therapy, cell therapy, animal model making, and crop genetic breeding.

This invention belongs to the field of bioengineering and discloses a *Yersinia lipolytica* chassis strain with homologous recombination preference, its construction method, and its applications. It involves inserting endogenous genes Rad52, Rad50, Mus81, and Mms4, as well as the single-stranded DNA annealing protein (PapRecT) encoded by *Pseudomonas aeruginosa* bacteriophage and its corresponding host *P. aeruginosa*-derived single-stranded DNA binding protein (PaSSB), into the genome of the *Yersinia lipolytica* strain, followed by knocking out its endogenous Rad9, resulting in a *Yersinia lipolytica* chassis strain with homologous recombination preference. This invention's *Yersinia lipolytica* chassis strain achieves effective improvement in homologous recombination efficiency through multi-dimensional regulation of the homologous recombination process, constructing an excellent *Yersinia lipolytica* platform, which is beneficial for further genome editing and has significant implications for using *Yersinia lipolytica* as a chassis cell in basic or applied research.

The invention relates to a lateral flow immune nucleic acid detection sensor and a preparation method and application thereof.According to the sensor, fluorescent microspheres marked by nucleic acid aptamers serve as detection probes, a multiple signal amplification platform is combined, and the novel multiple signal amplification sensor for multiple fluorescent lateral flow immune nucleic acid detection is constructed. And high-sensitivity and high-specificity multiple quantitative amplification detection of the concentration of a target object is realized. Compared with traditional monoclonal antibody paired multiple immunochromatography detection, the fluorescent microspheres labeled by spore particle aggregation nucleic acid of bacillus and the fluorescent microspheres labeled by single-chain binding protein (RPA1) are used as multiple signal amplification sensors, so that the signal intensity on a detection line is remarkably enhanced, and the sensitivity of the detection method is greatly improved.

The present invention relates to the field of biotechnology, and particularly to a highly salt-tolerant single-stranded binding protein mutant and an application thereof. The amino acid sequence of the highly salt-tolerant mutant is shown in SEQ ID NO: 2 or SEQ ID NO: 3. Compared with the wild type, the multi-site mutation / deletion SSB mutant SSBV1 comprises V11I+A30C+I79F+T98S, while SSBV2 comprises V11I+A30C+I79F+T98S+144-148del (GNQF). The binding efficiencies of the mutants under different salt concentrations are tested. Compared with the wild-type Escherichia coli SSB, the binding efficiencies of SSBV1 and SSBV2 are significantly improved under different sodium chloride concentrations, different magnesium chloride concentrations, and different ammonium sulfate concentrations, thereby facilitating further expansion of the application scenarios of SSB.

The invention relates to a method of characterising a target polynucleotide using a single-stranded binding protein (SSB). The SSB is either an SSB comprising a carboxy-terminal (C-terminal) region which does not have a net negative charge or a modified SSB comprising one or more modifications in its C-terminal region which decreases the net negative charge of the C-terminal region.

The invention relates to the technical field of biology, in particular to a high-salt-tolerance single-chain binding protein mutant and application thereof.The amino acid sequence of the high-salt-tolerance mutant is shown as SEQ ID NO: 2 or SEQ ID NO: 3, compared with a wild type, a multi-site mutation / deletion SSB mutant SSBV1 contains V11I + A30C + I79F + T98S, SSBV2 contains V11I + A30C + I79F + T98S + 144-148del (GNQF), the binding efficiency of the SSBV1 and the SSBV2 under different salt concentrations is tested, and the high-salt-tolerance single-chain binding protein mutant can be obtained. Compared with wild escherichia coli SSB, the combination efficiency of the SSBV1 and the SSBV2 is remarkably improved under the conditions of different sodium chloride concentrations, different magnesium chloride concentrations and different ammonium sulfate concentrations, so that the application scene of the SSB is further expanded.