35 results about "Single-Stranded DNA Binding Proteins" patented technology

Methods and a kit are provided for selectively and exponentially amplifying nucleic acids and include the use of a single strand helicase preparation or a thermostable helicase in the absence of a single strand binding protein and a DNA polymerase such that the amplification can be performed isothermally.

Gene targeting allows the deletion (knock out), the repair (rescuing) and the modification (gene mutation) of a selected gene and the functional analysis of any gene of interest. Targeting of nuclear genes has been a very inefficient process in most eukaryotes including plants and animals due to the dominance of illegitimate integration of the applied DNA into non-homologous regions of the genome. The present invention provides a method for increasing the ratio of homologous to non-homologous recombination of a polynucleotide into a host cell's DNA by suppressing non-homologous recombination. Surprisingly, the number of non-homologous recombination events can be reduced if the polynucleotide is applied as a purified single-stranded DNA, preferably coated with a single strand binding protein.

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 a normal-temperature and constant-temperature nucleic acid amplification method using a fluorescence probe. The normal-temperature and constant-temperature nucleic acid amplification method includes: under constant temperature, single-strand binding protein partially opens the double-strand parental strand into a single strand; recombinant enzyme is combined with primer to form complex, the complex is combined to the parental strand under the action of auxiliary protein, and the fluorescence probe is combined with a complementation area; DNA polymerase is combined to the 3' tail end of the primer to perform sub-strand extension; exonuclease identifies the tetrahydrofuran locus on the fluorescence probe under a double-strand state, fluorescent groups and quenching groups are separated after enzyme digestion to release fluorescence; after the fluorescence probe is cut off, the 3'-OH end originally sealed by probe 3' tail end modification is exposed, and DNA polymerase can continuously extend to form a sub-strand; qualitative and semi-quantitative detection can be performed on a to-be-detected sample by detecting the shape of an amplification curve and the strength of a fluorescence signal. The nucleic acid amplification method has the advantages that normal-temperature and constant-temperature detection can be performed, and qualitative and semi-quantitative detection can be performed on nucleic acid to-be-detected objects.

Disclosed are compositions preferably for use in nucleic acid synthesis that include one or more anti-reverse transcriptase (RT) antibodies and/or one or more anti-DNA polymerase (DNAP) antibodies and/or single strand binding proteins (SSBs). Some of the disclosed compositions include one or more anti-DNAP antibodies and/or one or more anti-RT antibodies and one or more SSBs. Other disclosed compositions include two or more SSBs. The disclosed nucleic acid synthesis compositions also can include one or more DNAPs, one or more RTs, one or more nucleotides, one or more primers, and/or one or more templates. Also disclosed are methods for using such compositions in nucleic acid synthesis, amplification and sequencing, where various combinations of anti-RT antibodies, anti-DNAP antibodies and/or SSBs can improve the yield and/or homogeneity of primer extension products.

The invention discloses a helicase-dependent isothermal DNA amplification-based method for detecting microRNA. The method comprises the following steps: (1) extracting total RNA in a sample; (2) adding a single-strand DNA probe and excessive exonuclease I into the extracted total RNA, incubating in a reacting buffer solution to realize specific combination of target microRNA and the single-strand DNA probe, and removing residual single-strand DNA probe by utilizing the excessive exonuclease I; and (3) adding upstream primer, downstream primer, single-strand binding protein and helicase to the reaction system, amplifying the target microRNA, and detecting expression of the target microRNA through a fluorescent signal. According to the method, the background is reduced by digesting the exonuclease I, and the signal is amplified by means of exonuclease assisted isothermal amplification (HDA) reaction, so that the microRNA can be quickly and sensitively detected.

The invention provides an isothermal nucleic acid amplification reaction reagent. The isothermal nucleic acid amplification reaction reagent is characterized by comprising components as follows: 100-800 mM of a Tris-HCl buffer solution, 10-150 mM of sodium chloride, 10-150 mM of potassium chloride, 10-50 mM of magnesium chloride, 5-15 mM of dithiothreitol, 5%-20% of polyvinylpyrrolidone, 10-20 mM of ATP (adenosine triphosphate), 1-5 mM of dNPTs, 10-50 mM of phosphoenolpyruvate, 500-1,500 ng/mu l of pyruvate kinase, 10-500 ng/mu l of BSA (bovine serum albumin), 25-200 pmol of each primer in a primer group, 50-200 ng/mu l of T4 bacteriophage DNA helicase gp41 protein, 100-500 ng/mu l of streptomyces coelicolor recA protein, 200-1,000 ng/mu l of single-strand binding protein and 50-200 ng/mu l of escherichia coli DNA polymerase I. The invention further provides an isothermal nucleic acid amplification method. According to the isothermal nucleic acid amplification reaction reagent and the isothermal nucleic acid amplification method, nucleic acid amplification under the isothermal condition at the lower temperature is realized, and a traditional nucleic acid amplification reaction process is greatly simplified.

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 a RPA detection method for detecting 3 type human adenovirus, a special primer, a probe and an application thereof in detecting 3 type human adenovirus. The detection method, the special primer and the probe thereof are designed on the basis of 3 type human adenovirus hexon gene conserved sequence, with oligonucleotide sequences shown as SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4. According to the invention, a novel isothermal amplification technology RPA is firstly applied to the detection of 3 type human adenovirus. According to the method, an enzyme reaction process ofin vivo DNA replication is simulated; amplification of DNA template relies on a specific combination of enzyme and protein (recombinase, single-stranded binding protein and DNA polymerase); amplification of specific nucleotide sequence is realized under a constant temperature at 37 DEG C; amplified products can be visually identified through a lateral chromatography test strip. The detection method established by the invention has the advantages of high sensitivity, limit of detection reaching up to 101 copies/reaction, high specificity, no cross reaction with other pathogens, high detection speed, only 20min reaction time, simple and convenient operation, and suitability for onsite detection.

The invention discloses an echinococcus shiquicus detection kit based on a RPA technology and application of the echinococcus shiquicus detection kit. A set of primer and probe combination capable ofbeing used for RPA detection is designed and screened according to the gene sequence of mitochondria of echinococcus shiquicus, and an echinococcus shiquicus specificity real-time fluorescent RPA detection method is established. According to the method, recombinant enzymes and single-stranded binding proteins are adopted to cooperate at the normal temperature to achieve specific binding of a primer and a template, and under the effect of DNA polymerases, the primer is extended to generate new DNA strands. The echinococcus shiquicus specificity fluorescent RPA detection method is easy, convenient and quick to operate, high in specificity, and suitable for site quick detection.

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).

The invention provides a RPA detection method for detecting 21 type human adenovirus, a special primer, a probe and an application thereof in detecting 21 type human adenovirus. The detection method,the special primer and the probe thereof are designed on the basis of 21 type human adenovirus hexon gene conserved sequence, with oligonucleotide sequences shown as SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4. According to the invention, a novel isothermal amplification technology RPA is firstly applied to the detection of 21 type human adenovirus. According to the method, an enzyme reaction processof in vivo DNA replication is simulated; amplification of DNA template relies on a specific combination of enzyme and protein (recombinase, single-stranded binding protein and DNA polymerase); amplification of specific nucleotide sequence is realized under a constant temperature at 37 DEG C; amplified products can be visually identified through a lateral chromatography test strip. The detection method established by the invention has the advantages of high sensitivity, limit of detection reaching up to 102 copies/reaction, high specificity, no cross reaction with other pathogens, high detection speed, only 20min reaction time, simple and convenient operation, and suitability for onsite detection.

To achieve the above object, the present invention discloses a fusion Taq DNA polymerase, which is formed by fusing a DNA single-strand binding protein Sac7d or a DNA single-strand binding protein sso7d with a wild-type Taq DNA polymerase, respectively. The Taq DNA polymerase provided by the invention enhances the affinity with the template chain in the amplification process so as to improve the amplification efficiency, realizes the amplification of difficult templates, has stable enzyme activity and can be directly amplified by using blood as a template.

The application relates to a constant temperature nucleic acid amplification reaction reagent. The constant temperature nucleic acid amplification reaction reagent is prepared from helicase, formamide, spermidine, magnesium chloride, glacial acetic acid, UvsX protein, UvsY protein, single strand binding protein, bovine serum albumin, dNTPs, Tris alkali and constant temperature DNA polymerase.

Compositions and methods are provided for loop mediated isothermal amplification in which single stranded binding proteins are shown to protect primers from non-specific extension and to stimulate the rate of threshold amplification.

The invention provides an RPA (recombinase polymerase amplification) method, an PRA special primer and an RPA probe for detecting universal human adenovirus, and application of the PRA special primerand RPA probe in detection of the universal human adenovirus. The detection method, the special primer and the probe are designed based on a common conserved sequence of type 3, type 7, type 11, type21 and type 55 human adenovirus hexon genes, and have the oligonucleotide sequences shown as SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5. According to the method, the novel isothermal amplification technology is applied to detection of the universal human adenovirus for the first time; the enzymatic reaction process of in-vivo DNA replication is simulated, a specific enzyme and protein combination(recombinase, single-stranded binding protein and DNA polymerase) is used for DNA template amplification, amplification of a specific nucleotide sequence can be realized at a constant temperature of37 DEG C, and amplification products can be visually judged through a lateral chromatographic test strip. The detection method has high sensitivity, and the detection limit can reach 101 copies/reaction; the specificity is high, and no cross-reaction with other pathogens exists; the detection speed is high, and the method is simple and convenient to operate.

The invention provides a method for detecting DNA of schistosoma through a fluorescent probe. An amplification system and the DNA of the schistosoma are mixed, isothermal amplification is carried out to obtain an amplified product, and the amplified product is subjected to real-time detection through a fluorescent probe method. The amplification system comprises the components of a Tris buffer solution, potassium acetate, magnesium acetate, dithiothreitol, polyethylene glycol, ATP, dNTPs, phosphocreatine single-strand binding protein, recombinase, UvsY protein, exonuclease, the fluorescent probe, DNA polymerase and a schistosoma DNA specific primer. The temperature of isothermal amplification is 35-45 DEG C. The time of isothermal amplification is 5-20 min. The method does not rely on a thermal cycler, operation is easy, the DNA of the schistosoma can be detected at the low temperature (35-45 DEG C), the time used for amplification is short (5-20 min), and the method is suitable for primary and on-site detection.

The invention discloses a constant-temperature detection method and kit for instantly detecting listeria monocytogenes. The constant-temperature detection kit does not depend on high-energy molecules such as ATP and phosphocreatine. Compared with the existing constant-temperature amplification system, the constant-temperature detection kit is relatively simple in components, relatively low in cost and relatively convenient to use. Key components comprise dNTPs, polymerase Bsu, single-strand binding protein and recombinase (E.coli Rect). The constant-temperature detection kit can be matched with a fluorescently-labeled probe based on a constant-temperature amplification reaction reagent to carry out instant fluorescence detection. The constant-temperature amplification system is applied to amplification of complex templates; amplification is rapid and stable; the amplification reaction can be completed in 30 minutes; amplification is high in sensitivity and accuracy; the possibility of mismatching is low; the amplification effect is excellent.

The invention relates to a preparation method, expression gene, recombinant expression vector and application of gp32 single-strand binding protein. The expression gene of the gp32 single-strand binding protein comprises a nucleotide sequence shown in SEQ ID No.1. The expression gene of the gp32 single-strand binding protein can achieve a large amount of soluble expression in escherichia coli, andthe purity of the expressed gp32 single-strand binding protein is greater than 95%.

The invention discloses an animal chlamydia detecting method based on an RPA (Recombinant Polymerase Amplification) technology. According to the method, recombinase and single-strand binding protein cooperate with each other at normal temperature to realize specific binding of a primer and a template, and a new DNA chain is generated by extending the primer under the action of DNA polymerase. Theanimal chlamydia detecting method is characterized in that a set of primer and probe combination used for RPA detection is designed and screened according to a chlamydia gene sequence envB, and an animal chlamydia specificity fluorescence RPA detecting method is established. The chlamydia specificity fluorescence RPA detecting method disclosed by the invention is easy, convenient and quick to operate and high in specificity, and is suitable for on-site quick detecting.

The invention provides a method for detecting blood fluke DNA. The method comprises the steps that an amplification system and the blood fluke DNA are mixed, isothermal amplification is conducted for 5-40 min at the temperature of 35 DEG C to 45 DEG C to obtain an amplification product, and agarose gel electrophoresis is conducted on the amplification product to detect the blood fluke DNA, wherein the amplification system is prepared from a Tris buffer solution, potassium acetate, magnesium acetate, dithiothreitol, polyethylene glycol, ATP, dNTPs, phosphocreatine, single-strand binding protein, recombinase, UvsY protein, DNA polymerase and specific primers of the blood fluke DNA. The method does not depend on a thermal cycler and is easy to operate, the blood fluke DNA can be detected at the low temperature (from 35 DEG C to 45 DEG C), the amplification time is short (5-40 min), and therefore the method is suitable for basic and site detection.

The invention provides an RPA detection method, special primers and a probe for detecting Rickettsia rickettsii, and uses of the RPA detection method in detection of Rickettsia rickettsii. According to the present invention, the special primers and the probe are designed based on the conserved sequence of a Rickettsia rickettsii gene, and have the oligonucleotide sequences represented by SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4; the novel constant temperature amplification technology RPA is firstly applied in the detection of Rickettsia rickettsii, wherein the enzymatic reaction process of DNAreplication in vivo is simulated, the DNA template is amplified by using the specific enzyme and protein combination including recombinase, single-stranded binding protein and DNA polymerase so as toamplify the specific nucleic acid sequence at the constant temperature of 37 DEG C, and the amplification product can be subjected to visualized discrimination through the lateral chromatography nucleic acid detection test paper strip; and the established detection method has the sensitivity of 6 copies/[mu]L, has high specificity, has low requirement on hardware equipment, can complete the detection within 30 min, does not require the complicated sample treatment, is suitable for on-site detection, and is suitable for popularization and application.

The invention provides a recombinase polymerase amplification (RPA) detection method, RPA special primers and a RPA probe for detecting streptococcus suis serotype 2 as well as application of the RPAmethod, the RPA special primers and RPA probe in detection of the streptococcus suis serotype 2. The detection method, the special primers and the probe are designed based on streptococcus suis cps2Jconserved sequence design, and have oligonucleotide sequences shown in SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4. An RPA technology is firstly applied to the detection of the streptococcus suis serotype 2; the method simulates an enzymatic reaction process of DNA replication in vivo, depends on a specific enzyme and protein combination including recombinase, single-stranded binding protein and DNA polymerase to amplify a DNA template, and realizes the amplification of the specific nucleic acid sequence at a constant temperature of 37 DEG C; the amplified product is visually distinguished bymeans of a lateral chromatographic test strip. The detection method established by the invention is high in sensitivity, and has a detection limit reaching up to 102 copies/reaction; the special primers are strong in specificity, and has no cross reaction with other pathogens; the requirement for hardware equipment is low, and the reaction time is only 15min; a sample does not need to be subjectedto complicated treatment.

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).

The invention relates to the molecular biological detection field and discloses an amplification system and a method for detecting RNA of Japanese encephalitis virus. The amplification system contains the following component: a Tris buffer solution, magnesium acetate, potassium chloride, dithiothreitol, polyethylene glycol, ATP, dNTPs, creatine phosphate, recombinase, single stranded binding protein, UvsY protein, exonuclease, a fluorescent probe, DNA polymerase, reverse transcriptase, an RNA enzyme inhibitor and a Japanese encephalitis virus specific primer. The amplification system and RNA of the Japanese encephalitis virus are mixed and are subjected to isothermal amplification, and an amplification product is detected in real time by virtue of a fluorescence probe method. The method provided by the invention has the beneficial effects that the operation is simple, an instrument is miniaturized and convenient to carry, the amplification time is short, and the method is high in specificity and sensitivity and suitable for basic level and field detection.