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

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 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.

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 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.

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.

A method of detecting a target nucleic acid sequence in a test solution comprising: A. providing a test solution comprising: a. RPA single-stranded DNA-binding protein (RPA-SSB) molecule; b. cleavage

It has been established that one or more large double stranded DNA fragments (each 2,000 to 40,000 base pairs in size) can be captured and isolated from genomic DNA fragments using sequence specific PNA hybridization probes. Compositions and methods for enrichment of a multiplicity of long DNA sequences selected from the genome of any eukaryote are provided. Capture is performed using multiple PNA molecules with gamma-modified chiral backbones, comprising a mixture of neutral and positive chemical groups. Two or more PNA probes with covalently bound haptens, preferably biotin, target each DNA domain of interest for capture, isolation, and subsequent sequencing analysis of the multiplicity of enriched targets, including DNA methylation sequencing. The methods include enhancement of probe-DNA binding specificity through single strand binding proteins (SSB).

Methods, kits and compositions, in some embodiments, may include a thermostable DNA guided Argonaute protein for example TtAgo, a thermostable single-stranded DNA binding protein (SSB) for example, extreme thermostable single-stranded DNA binding protein (ET SSB), and, optionally, a strand-displacing polymerase. A SSB may allow (a) Argonaute / guide DNA complexes to substantially enhance cleavage efficiency of single- and double-stranded DNA substrates; (b) the use of longer guide DNAs (e.g., guide DNAs that are at least 24 nucleotides in length) and / or (c) increases in the sequence specificity of Argonaute-mediated binding and cleavage reactions.

The invention discloses a recombinase combination suitable for bacteroides, application and a homologous recombination method, and belongs to the technical field of microbial genetic engineering. The invention discloses a method for constructing and screening a combination of a single-chain annealing protein and a single-chain binding protein for the first time. A single-chain annealing protein and a single-chain binding protein are jointly constructed in an escherichia coli-bacteroides fragilis shuttle expression vector, and co-expression of the escherichia coli-bacteroides fragilis shuttle expression vector in bacteroides fragilis is achieved, so that the homologous recombination capacity of bacteroides fragilis is remarkably improved. According to the method disclosed by the invention, the homologous recombination efficiency of genome editing of bacteroides fragilis is greatly improved, the absolute editing efficiency of the genome is improved to 0.052% from the original level which is almost undetectable, and the research and application progress of genome engineering of the strain is remarkably promoted.

Engineered enzymes for amplification of nucleic acid sequences which function at constant temperatures thereby eliminating heating and cooling cycles associated with traditional polymerase chain reaction (PCR) are disclosed. Specifically, novel, genetically engineered PcrA helicase enzymes are utilized in conjunction with amplification components to provide a method for amplification of nucleic acid sequence by incubating at a substantially isothermal temperature i) a composition comprising a target sequence, ii) primers and iii) said engineered PcrA helicase, a single stranded binding protein (SSB), a polymerase, and / or a thermostable pyrophosphatase (PPase) buffer, or combinations thereof.

The invention provides a method for efficiently evolving the plant pathogenic fungus resisting activity of bacillus subtilis. The method comprises the following steps: connecting an APOBEC-1 gene for coding cytosine deaminase and an ssbA gene for coding DNA single-stranded binding protein of the bacillus subtilis to a pMA5 expression vector to obtain a base editor rBE; the method comprises the following steps: converting a base editor rBE into bacillus subtilis 168 to obtain a recombinant strain BS168-ASP; the recombinant strain BS168-ASP is subjected to continuous subculture for five times in an LB culture medium containing 20-50 mg / mL of chitin; fermenting the last subculture bacterial liquid, preparing supernate, screening the bacteriostatic activity, and screening a strain with high antagonistic activity for a single colony of a sample with the highest bacteriostatic activity improvement through a plate confrontation method. Therefore, bacillus subtilis gene mutation can be efficiently promoted, and strains with obviously improved plant pathogenic fungus inhibition activity can be screened out.

The invention discloses an application of a hybridization probe in binding with a target in a sample containing a single-stranded DNA binding protein, the hybridization probe at least comprises a segment of nucleic acid sequence, and the nucleic acid sequence comprises at least one of the following items: modification of a basic group, modification of ribose and modification of phosphate. In the application of the invention, the hybridization probe can efficiently hybridize with the target in the presence of the single-stranded DNA binding protein.

The present invention provides genetically modified microorganisms with reduced transmissibility, wherein the microorganism has been genetically modified to express a single stranded binding protein (SSB) regulated by an arabinose responsive promoter, wherein the microorganism has a reduced growth capacity in the absence of arabinose.

This invention relates to the field of molecular biology detection technology, specifically, to the application of a fluorescent fusion protein in the detection of DNA damage repair. The application includes: incubating the fluorescent fusion protein with a test sample and then performing fluorescence imaging to obtain the damage repair status of the DNA to be tested in the test sample; wherein the test sample contains the DNA to be tested; and the fluorescent fusion protein is formed by the fusion of a single-stranded DNA-binding protein and a fluorescent protein. This application not only enables in-situ detection of any DNA damage that exposes single-stranded DNA, but also provides accurate and quantifiable results. Furthermore, this invention is not limited to any species and can provide efficient visualization and quantitative analysis for DNA damage detection in various organisms.

The present invention features novel, diverse, hybrid and engineered recombinase enzymes, and the utility of such proteins with associated recombination factors for carrying out DNA amplification assays. The present invention also features different recombinase ‘systems’ having distinct biochemical activities in DNA amplification assays, and differing requirements for loading factors, single-stranded DNA binding proteins (SSBs), and the quantity of crowding agent employed.

The invention relates to the technical field of molecular biology, in particular to a single-stranded DNA molecular weight marker and a preparation method thereof. The method comprises the following steps: 1) mixing a sample containing single-stranded DNA with single-stranded DNA binding protein, and incubating for a period of time to obtain a conjugate containing the single-stranded DNA and the single-stranded DNA binding protein; (2) separating and purifying the conjugate obtained in the step (1), and removing impurities which are not combined with the single-chain binding protein to obtain a high-purity conjugate of the single-chain DNA and the single-chain DNA binding protein; and 3) purifying the high-purity conjugate of the single-stranded DNA and the single-stranded DNA binding protein obtained in the step 2) to at least remove the single-stranded DNA binding protein to obtain a target product, and (4) repeating the steps (1)-(3) for m times (m is greater than or equal to 1) to obtain target products of different single-stranded DNA, and mixing the target products of the different single-stranded DNA, so that the mixed mixture at least contains two kinds of single-stranded DNA with different lengths.