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

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.

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.

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 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.

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.