47 results about "Single-strand DNA-binding protein" patented technology

The present invention relates, e.g., to in vitro method, using isolated protein reagents, for joining two double stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence identity, comprising contacting the two DNA molecules in a reaction mixture with (a) a non-processive 5′ exonculease; (b) a single stranded DNA binding protein (SSB) which accelerates nucleic acid annealing; (c) a non strand-displacing DNA polymerase; and (d) a ligase, under conditions effective to join the two DNA molecules to form an intact double stranded DNA molecule, in which a single copy of the region of sequence identity is retained. The method allows the joining of a number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes.

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 present invention relates, e.g., to in vitro method, using isolated protein reagents, for joining two double stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence identity, comprising contacting the two DNA molecules in a reaction mixture with (a) a non-processive 5′ exonculease; (b) a single stranded DNA binding protein (SSB) which accelerates nucleic acid annealing; (c) a non strand-displacing DNA polymerase; and (d) a ligase, under conditions effective to join the two DNA molecules to form an intact double stranded DNA molecule, in which a single copy of the region of sequence identity is retained. The method allows the joining of a number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes.

A method for cloning a nucleic acid fragment into a vector by flanking the fragment with first and second adapter sequences, and contacting the fragment with the vector having sequences homologous to the first and second adapter sequences under conditions such that the nucleic acid fragment is incorporated into the vector by homologous recombination in vivo in a host cell. Additionally, a method for selecting for a successful transformation of a vector by a nucleic acid insert. Also, systems for cloning a nucleic acid fragment into a vector without at least one of a restriction enzyme, a ligase, a gyrase, a single stranded DNA binding protein, or other DNA modifying enzymes. Further, a kit for cloning a nucleic acid fragment into a vector.

A method of amplifying a template DNA molecule in an isothermal reaction that can reduce background noise is provided. A method of amplifying a template DNA molecule using a strand-displacing DNA polymerase capable of carrying out isothermal amplification includes a step of conducting an amplification reaction with the addition of a single-strand DNA binding protein (SSB) from an extreme thermophile.

The invention discloses a recombinase-mediated nucleic acid isothermal amplification reaction detecting method. The nucleic acid isothermal amplification reaction detecting method comprises the following step that 1, an amplification reaction system is prepared. The amplification reaction system mainly comprises a, a nucleic acid sample to be detected; b, a pair of oligonucleotides primer 1 and primer 2, wherein the primer 1 and the primer 2 are cross-fertilized with the nucleic acid sample; c, a fluorescent probe, wherein the fluorescent probe is one or multiple of a molecular beacon, a fluorescent resonator, a scorpion probe and a molecular torch, and the concentration of the fluorescent probe ranges from 50-900 nM / L; d, recombinase; e, DNA polymerase; f, single-stranded DNA binding protein; g, amplification reaction buffer. The invention further provides a detection kit based on the nucleic acid isothermal amplification reaction detecting method. According to the nucleic acid isothermal amplification reaction detecting method and the detection kit based on the nucleic acid isothermal amplification reaction detecting method, the complexity of the system is reduced, reagent cost is reduced, sequence synthesis of the probe can be achieved easily, the probe can be designed easily, and actual application and popularization are facilitated.

In some embodiments, the present teachings provide a method for detecting a nucleotide of interest comprising; forming an amplification reaction mixture comprising a mismatched primer, a target polynucleotide, a strand-displacing polymerase lacking 3′ to 5′ exonuclease activity, a recombinase, and a single-stranded DNA binding protein; hybridizing a mismatched primer to the target polynucleotide to form a primer-target complex; and, detecting the nucleotide of interest by the absence of a primer extension product. In some embodiments, control reactions are performed in which a control polynucleotide is exponentially amplified. Additional methods, as well as reaction mixtures and kits, are also provided.

The invention relates to a primer pairs for rapid detection of toxoplasmosis nucleic acid, a kit and a detection method. The primer pairs for rapid detection of toxoplasmosis nucleic acid comprises aforward primer, a reverse primer and a probe for detecting genome B1 gene sequence of toxoplasmosis. The kit comprises the following contents: a lysate, a diluent, a lyophozyme tube containing the primers, a positive quality control and a negative quality control. The detection includes the process of carrying out a normal-temperature nucleic acid amplification technology by using the kit. Throughmultiple enzymatic reactions of DNA helicase, single-stranded DNA binding protein, DNA polymerase and the like, target DNA can be amplified by millions of times under the condition of constant temperature 37-45 DEG C within 10-30 min; and with the cooperation of the fluorescence detection technique, rapid detection of DNA to be tested can be realized. The detection method of the invention has advantages of simple operation, short time and low requirement on equipment, and is very suitable for rapid diagnosis of pet infectious diseases.

The invention relates to a colorimetric detection probe for detecting terramycin in food. A preparing method of the probe includes (1) bonding an aminated aptamer Apt and aminated magnetic beads MB under a function of a coupling agent to form a capturing probe; (2) modifying the surface of a synthesized metal-organic framework MOF-Pt composite material with single stranded DNA binding protein SSB to form a signal probe; and (3) preparing the capturing probe obtained in the step (1) and the signal probe obtained in the step (2) into a composite probe through affinity between the single stranded DNA binding protein and the aptamer Apt. A terramycin detecting method utilizing the probe is also disclosed. The detecting method does not depend on large-scale instruments and has a low detection limit. In addition, the colorimetric detection probe shows good specificity and selectivity. The detecting method is simple and rapid to operate, and can achieve on-site terramycin residue detection by virtue of a handheld chromometer, and an experiment result is visual by naked eyes.

The present invention relates to a colorimetric detection probe for detecting kanamycin. The preparation method comprises: (1) conjugating an aminated aptamer Apt and aminated magnetic beads MB under the action of a coupling agent to form a capture probe; (2) modifying the surface of a synthesized metal organic framework MOF-Pt composite material by using single-stranded DNA binding protein SSB to form a signal probe; and (3) forming a composite probe from the capture probe obtained in the step (1) and the signal probe obtained in the step (2) through the affinity between the single stranded DNA binding protein and the aptamer Apt. The invention further provides a kanamycin detection method of the probe. According to the present invention, the detection method does not require the large-size instrument equipment and has the low detection limit, the colorimetric detection probe shows good specificity and good selectivity, the operation of the method is simple and quick, the on-site detection of the kanamycin residue can be achieved by using the handheld type colorimeter, and the experimental results are visible to the naked eye.

The invention establishes a technology that allows non-specific amplification to be inhibited during nucleic acid amplification in an isothermal amplification reaction, such that the amplification efficiency is increased. The invention is a extreme thermophile single-stranded DNA binding mutant protein, having an amino acid sequence that expresses a function that can contribute to increasing an amplification efficiency of a template nucleic acid in an isothermal amplification reaction system that uses a strand displacement polymerase, and having in its amino acid sequence a mutation site where a mutation involving at least one of deletion, substitution, addition, and insertion of one or more amino acids in amino acid sequence of extreme thermophile single-stranded DNA binding protein has occurred, and a method of use thereof.

The invention discloses a reagent kit for quickly detecting nucleic acid of canine parvovirus (CPV). The reagent kit comprises lysate, diluent, lyophozyme tubes with primers, detection liquid and test strips. The reagent kit has the advantages that normal-temperature nucleic acid amplification technologies are adopted, different types of enzymatic reaction can be carried out by the aid of DNA (deoxyribonucleic acid) helicase, single-stranded DNA binding proteins, DNA polymerase and the like, and accordingly to-be-detected DNA can be amplified by several millions of times under the condition of the constant temperatures of 37-45 DEG C within 10-30 minutes; the reagent kit is matched with immune colloidal gold chromatographic detection technologies, and accordingly the to-be-detected DNA can be quickly verified; the reagent kit is easy to operate, short in time and quite suitable for quickly diagnosing infectious diseases of pets and has low requirements on instruments.

The invention relates to primer pairs for fluorescence EMA detection of canine distemper virus (CDV), a kit and a detection method. The primer pairs for fluorescence EMA detection of canine distempervirus (CDV) contain a forward primer, a reverse primer and a probe. The kit comprises the following contents: a redissolved solution, a lyophozyme tube containing primers, a positive quality control and a negative quality control. The detection includes the process of carrying out a normal-temperature nucleic acid amplification technology by using the kit. Through multiple enzymatic reactions of M-MLV reverse transcriptase, a Rnase inhibitor, DNA helicase, single-stranded DNA binding protein, DNA polymerase and the like, RNA to be tested can undergo reverse transcription into cDNA under the condition of constant temperature 37-45 DEG C within 10-30 min, and the cDNA is amplified by millions of times; and and with the cooperation of the fluorescence detection technique, rapid detection of RNA to be tested can be realized. The detection method of the invention has advantages of simple operation, short time and low requirement on equipment, and is suitable for rapid diagnosis of pet infectious diseases.

The invention specifically relates to a method for flexibly detecting DNA through exonuclease III assisted multiple circulation amplification. According to the technical scheme, through combination ofsingle-stranded DNA conjugated protein (SSB) with a barrette probe, the non-specific digestion of exonuclease III (Exo III) is effectively avoided, so that a background signal nearly zero is produced. The existence of a target DNA causes exonuclease III (Exo III) assisted multiple circulation amplification, the sensitivity is high, the detection limit is 3mol / litre, excellent selectivity is achieved, single alkali base mismatch can be recognized, and the method has tremendous potential in the respects of disease diagnosis and biomedical science research.

The invention discloses an RPA (recombinase polymerase amplification) detection primer and probe assembly for genetically modified maize MON810, a kit and a detection method. A primer group comprisestwo specific primers, nucleotide sequences of the two specific primers are represented as SEQ ID No:1-2, and a probe sequence is represented as SEQ ID No:3. The detection kit comprises detection primer and probe solutions, Buffer A, Buffer B and a positive control. The detection method comprises steps as follows: the specific primers and probes are adopted to perform amplification on a sample DNAtemplate at 37-42 DEG C under the actions of recombinase, single-strand binding protein (SSB), strand replacement DNA polymerase and exonuclease, and whether the sample contains the genetically modified maize MON810 component or not is judged with a probe fluorescent signal collecting method. The detection method does not need special instruments, has the characteristics of being rapid, efficient,simple and convenient to operate, high in sensitivity, high in specificity and the like and is suitable for in-situ detection.

The invention relates to an isothermal PCR nucleic acid amplification method based on dI modified primer and endonucleaseV. The amplified target nucleic acid primer is completely matched with the template; 1 to 5 dI nucleic acids are on the 10 to 30 site at the 5' end; 10 to 30 nucleic acids are at the downstream of the dI nucleic acids. The reaction components of isothermal PCR comprise dI modified forward primer and reverse primer, target nucleic acid template, four kinds of deoxyribonucleoside triphosphates dATP, dTTP, dCTP and dGTP, DNA polymerase, endonucleaseV, single DNA chain binding protein and helicase. After the initial thermal denaturation and annealing hybridization, the mixture of isothermal PCR carries out the PCR reaction. During the amplification process, the circulation and regeneration of the primer-template compound is realized by endonucleaseV, which induces the DNA synthesis reaction; the single DNA chain binding protein and helicase can improve the efficiency of isothermal PCR. The amplification of the target nucleic acid of the invention does not rely on the thermal cycler; the invention has easy and convenient operation, which can be used for a large scale and high yield amplification of the target nucleic acid.

The invention relates to a feline coronavirus fluorescence EMA detection primer group, a kit and a detection method. The feline coronavirus fluorescence EMA detection primer group comprises an upstream primer, a downstream primer and a probe, the kit comprises a reconstitution fluid, a lyophilized enzyme tube containing primers, a positive quality control material and a negative quality control material. The detection method comprises the steps that the kit is used for performing a normal-temperature nucleic acid amplification technology, through the multiple enzymatic reactions of M-MLV reverse transcriptase, Rnase inhibitor, DNA helicase, single stranded DNA binding protein, DNA polymerase and the like, to-be-detected DNA is reversely transcribed to cDNA within 10-30 minutes under the constant temperature of 37-45 DEG C, the cDNA is amplified by millions of times, and rapid verification of the to-be-detected DNA can be achieved by cooperating with a fluorescence detection technology.The feline coronavirus fluorescence EMA detection primer group, the kit and the detection method have the advantages that the operation is simple, the time is short, the requirements for an instrument are low, and the feline coronavirus fluorescence EMA detection primer group, the kit and the detection method are suitable for the rapid diagnosis for pet infectious diseases.

The invention relates to a preparation method?of a hot start DNA polymerase,?comprising the following steps:?obtaining a?single stranded DNA binding protein, a Taq DNA polymerasemonoclonal antibody?and?a Taq DNA polymerase; and mixing the?single stranded DNA binding protein, the Taq DNA polymerase?monoclonal antibody?and the Taq DNA polymerase in accordance with?aproportion. The contents of the?single stranded DNA binding protein, the Taq DNA polymerasemonoclonal antibody?and the Taq DNA polymerase are all 20-40%?respectively. The invention also relates to the hot start DNA polymerase prepared by the above method and an application thereof. The hot start DNA polymerase provided by the invention can also be used to solve problems which appear easily in hot start PCR technology such as DNA damage, product contamination?and bad effect of hot start caused by incomplete sealing.

The invention provides an expression vector containing a promoter and polynucleotide sequences for encoding a fusion protein. The invention also provides a fusion protein containing a single-stranded DNA binding protein and a target protein which is directly or indirectly infused with the end C or the end N of the single-stranded DNA binding protein, or polypeptide, and the fusion protein bonded with single-stranded DNA. The invention also provides a method for purifying proteins, which efficiently purifies the fusion protein by affinity chromatography bound with the single-stranded DNA.

The invention discloses an RPA detection primer and probe combination, a kit and a detection method of transgenic rape Oxy-235. A primer set consists of two specific primers, the nucleotide sequence of the primer set is shown in SEQ ID No:1-2, and the probe sequence is shown in SEQ ID No.3. The detection kit comprises a detection primer, a probe solution, Buffer A, Buffer B and a positive control.The detection method adopts a specific primer and a probe to amplify a sample DNA template at 37-42 DEG C under the action of recombinase, single-stranded DNA binding protein (SSB), strand displacement DNA polymerase and nucleic acid exonuclease, and judges whether the sample contains a transgenic rape Oxy-235 component through a probe fluorescence signal collection method. The method provided bythe invention does not need special instruments, has the characteristics of high speed and efficiency, simple and convenient operation, high sensitivity, strong specificity and the like, and is suitable for on-site detection.

The invention discloses a primer, a probe, a kit and a method for detecting transgenic soybean MON89788. A primer set consists of two specific primers, the nucleotide sequence of the primer set is shown in SEQ ID No:1-2, and the probe sequence is shown in SEQ ID No.3. The detection kit comprises a detection primer, a probe solution, Buffer A, Buffer B and a positive control. The detection method adopts a specific primer and a probe to amplify a sample DNA template at 37-42 DEG C under the action of recombinase, single-stranded DNA binding protein (SSB), strand displacement DNA polymerase and nucleic acid exonuclease, and judges whether the sample contains a transgenic soybean MON89788 component through a probe fluorescence signal collection method. The method provided by the invention doesnot need special instruments, has the characteristics of high speed and efficiency, simple and convenient operation, high sensitivity, strong specificity and the like, and is suitable for on-site detection.

A Herpes simplex virus (HSV) recombinase comprises a purified or isolated alkaline nuclease and a single stranded DNA binding protein. In HSV-1, the alkaline nuclease is the UL12 protein and the single stranded DNA binding protein is the ICP8 protein. The HSV recombinase can be purified from an in vitro expression system or can be expressed in an appropriate vector or vectors wherein the DNAs encoding the polypeptides are operatively linked to expression control sequences. Methods of use of the HSV recombinase include cloning, treating cells and organisms, and producing transgenic animals. The HSV recombinase can be in the form of a kit useful for cloning.

The invention relates to an analysis method for the geometrical structure characteristics of DNA binding protein interface, belonging to the technical field of biological computing. The present invention first derives proteins-DNA binding site information from proteins-DNA complex dataset; and then based on the protein-DNA Binding Site Information, residue Morphological Index and Spatial Environment of proteins-DNA Binding Sites are determined; finally, the morphological index and spatial environment of the identified protein-DNA binding sites are used as feature vectors to classify the obtained feature vectors by using classification model. As the binding specificity of double-stranded DNA binding protein (DSBs) and single-stranded DNA binding proteins (SSBs) is analyzed from the angles of the morphological structure of the residues and the characteristics of the space environment, the invention is conducive to the in-depth understanding of the proteins, and the binding specificity ofthe double-stranded DNA binding proteins and the single-stranded DNA binding proteins is analyzed. Based on the DNA binding mechanism, the present invention can also be applied to proteins-RNA, protein-Protein and other fields.

The invention discloses a novel recombination system in pseudomonas. The recombination system is established according to lambda Red-like operons (Orf38, Orf37 and Orf36) from pseudomonas aeruginosa phage Ab31 and RecET-like operon (recTEPsy) from pseudomonas syringae pv.syringae B728a prophage. According to the recombination system, apart from two genes of host specific exonuclease and single-stranded DNA annealing protein, a third gene is exonuclease recBCD profilin, and a third gene or a fourth gene also can be single-stranded DNA binding protein SSB from bacterial chromosome, bacterial plasmid or other sources. Function test results show that a hybrid system composed of the lambda Red-like operons and the RecET-like operon and Red gamma / Plu gamma can operate in pseudomonas and the SSB protein can remarkably enhance the recombination efficiency. Those combination systems can be utilized for efficient and succinct plasmid modification and point mutation and knock-in and knock-out genome modification, and have a wide application prospect.

Methods and compositions for cloning a donor DNA molecule into an acceptor vector at a predetermined location are described. The methods are based on homologous recombination mediated by in vitro treatment of the donor DNA and the acceptor vector with an enzyme cocktail containing an exonuclease and a single-stranded DNA binding protein.

The invention discloses a transgenic soybean GST40-3-2 test primer, probe, kit and method. A primer group comprises 2 specific primers, of which nucleotide sequences are as shown in SEQ ID No.1 and SEQ ID No.2. A probe sequence is as shown in SEQ ID No.3. The test kit comprises a test primer and a probe solution, a Buffer A, a Buffer B and a positive control. The test method is characterized in that the specific primers and the probe are used for amplification of a sample DNA template at a temperature of 37-42 DEG C under the action of a recombinase, single-stranded DNA-binding protein (SSB),a strand displacement DNA polymerase and an exonuclease; and fluorescence signals of the probe are collected to judge whether a sample contains the transgenic soybean GST40-3-2 content or not. The transgenic soybean GST40-3-2 test primer, probe, kit and method provided by the invention has the characteristics of being rapid, high in efficiency, convenient to operate, high in sensitivity, high in specificity and the like; no special instruments are required; and the applicability for field tests is achieved.

A Herpes simplex virus (HSV) recombinase comprises a purified or isolated alkaline nuclease and a single stranded DNA binding protein. In HSV-1, the alkaline nuclease is the UL12 protein and the single stranded DNA binding protein is the ICP8 protein. The HSV recombinase can be purified from an in vitro expression system or can be expressed in an appropriate vector or vectors wherein the DNAs encoding the polypeptides are operatively linked to expression control sequences. Methods of use of the HSV recombinase include cloning, treating cells and organisms, and producing transgenic animals. The HSV recombinase can be in the form of a kit useful for cloning.

A method is disclosed in which circular DNA molecules are amplified preferentially in a mixture of circular DNA molecules and linear DNA molecules by the inclusion of single strand DNA binding protein.