46 results about "Single-stranded DNA binding" 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.

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.

The invention is a method and a kit for preparing single-stranded DNA from double-stranded DNA and the purification of single-stranded DNA derived from double-stranded DNA. A single-stranded-DNA binding substance is used in combination with a double-strand-specific endonuclease for the removal of undesired double-stranded DNA from a single-stranded DNA preparation and for other related purposes.

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 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 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 signal amplifying method of leaky surface acoustic wave-binary peptide nucleic acid biosensor comprising adding RecA protein-complementary single strand DNA probe in hybridization reaction of a binary peptide nucleic acid probe and nucleic acid specimen to react together; the RecA protein-complementary single strand DNA probe can be acquired by: designing and synthesizing two complementary single strand DNAs according to a target DNA sequence so that at least one single strand DNA sequence is adjacent to the binary peptide nucleic acid probe; then co-incubating the RecA protein and two single strand DNAs under existence of ATPrS so that the RecA protein and two single strand DNAs are combined to acquire; the invention uses biological property that the RecA protein-complementary single strand DNA probe can form multi-strand body with bis-PNA / bi-stran DNA compound to improve quality load on surface of the sensor, which effectively improves detection agility of the sensor. The method is simple in operation with short hybridization reaction time and low cost, which is suitable for genetic rapid detection of micro clinical pathogenic microorganism.

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 method is used for separating nucleic acids and other similar constructs. It involves selective introduction, enhancement, or stabilization of affinity handles such as single-strandedness in the undesired (or desired) nucleic acids as compared to the usual structure (e.g., double-strandedness) of the desired (or undesired) nucleic acids. The undesired (or desired) nucleic acids are separated from the desired (or undesired) nucleic acids due to capture by methods including but not limited to immobilized metal affinity chromatography, immobilized single-stranded DNA binding (SSB) protein, and immobilized oligonucleotides. The invention is useful to: remove contaminating genomic DNA from plasmid DNA; remove genomic DNA from plasmids, BACs, and similar constructs; selectively separate oligonucleotides and similar DNA fragments from their partner strands; purification of aptamers, (deoxy)-ribozymes and other highly structured nucleic acids; Separation of restriction fragments without using agarose gels; manufacture recombinant Taq polymerase or similar products that are sensitive to host genomic DNA contamination; and other applications

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.

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.

Provided is an adjuvant composition comprising a nucleic acid comprising a double-stranded RNA bound to a single-stranded DNA, the double-stranded RNA consisting of a nucleotide sequence of SEQ ID NO: 1 and its complementary sequence, the single-stranded DNA consisting of a nucleotide sequence of SEQ ID NO: 2. Also provided is a vaccine composition comprising the adjuvant composition and an antigen or an antigen component. The nucleic acid contained in the adjuvant composition is a chemically synthesizable nucleic acid having potent adjuvant activity and high safety.

The present invention relates to a method and reagents for determining extension products in reactions involving nucleic acid chain extension, e.g., in cyclic minisequencing, and to the use of this method when identifying specific point mutations and genetic variations. Three standard unmodified primers are needed and a non-specific fluorescent reagent (e.g. a ssDNA-binding dye) can be used as one of the fluorophores in the FRET assay used. The invention is also related to vertical light beam fluorometry.

The invention discloses a combination of a primer and a probe, a kit and method for detecting transgenic maize TC1507. The primer set consists of two specific primers, the nucleotide sequence of whichwas shown in SEQ ID NO: 1-2 and the probe sequence in SEQ ID NO: 3. The kit comprises a testing primer and probe solution, Buffer A, Buffer B and a positive control. The specific primers and probes were used to detect the recombinant enzyme, Single stranded DNA binding protein (SSB), strand substitution DNA polymerase (SSR-DNA polymerase) and exonuclease are used to amplify DNA template at 37-42DEG C. The fluorescence signal of probe was collected to determine whether the sample contained transgenic maize TC1507 or not. The invention does not need special instrument, has the characteristicsof fast and high efficiency, simple and convenient operation, high sensitivity, strong specificity and the like, and is suitable for on-site detection.

The invention relates to a detection primer group, kits and a detection method for porcine reproductive and respiratory syndrome virus fluorescent EMA. The detection primer group of the porcine reproductive and respiratory syndrome virus fluorescent EMA contains an upstream primer, a downstream primer and a probe for detecting the genome Nsp2 gene sequence of the porcine reproductive and respiratory syndrome virus; the EMA detection kits comprise a kit A and a kit B. The detection method comprises the following steps: carrying out normal temperature nucleic acid amplification technique by using the detection kits; carrying out multiple enzymatic reactions by using M-MLV reverse transcriptase, an Rnase inhibitor, DNA helicase, single-stranded DNA binding protein, DNA polymerase and the like; under the constant condition of 37 to 45 DEG C, carrying out reverse transcription on the to-be-tested RNA to obtain cDNA within 10 to 30 minutes; meanwhile, amplifying the cDNA for millions of times; realizing rapid detection of the to-be-tested RNA by matching a fluorescence detection technology. The detection method for the porcine reproductive and respiratory syndrome virus fluorescent EMA,disclosed by the invention has the advantages of simple operation, short time, low requirement on instrument and suitability for rapid diagnosis of porcine communicable diseases.