1528 results about "Molecular Biology Technique" patented technology

A method of designing oligonucleotides useful in molecular biology techniques as PCR primers or in other techniques as identification and / or quantification probes is disclosed. The method permits designing specific oligonucleotides for the identification of a determined sequence in a metagenomic sample. The method includes selecting or constructing a database of reference sequences, selecting a subset of sequences belonging to target organisms, selecting candidate oligonucleotides from such sequences, depurating these candidate oligonucleotides according to hybridization specificity and thermodynamic stability criteria, obtaining a list of designed oligonucleotides that fulfill the hybridization specificity and thermodynamic stability criteria, producing materially by chemical synthesis the designed oligonucleotides, and selecting the oligonucleotides which comply with the requirements of the desired process.

The present invention provides methods for constructing peptide construct sets and methods of use of these peptide construct sets in assay systems for peptide analysis, and in particular for use in high throughput peptide analysis. The methods allow for analysis of large sets of peptide constructs in a cost-effective manner, employing molecular biological techniques that are both robust and easily parallelized. Thus, the methods allow for the construction of peptide construct sets encompassing, e.g., the human proteome.

The invention belongs to the technical field of molecular biology and relates to application of an exosome derived from human mesenchymal stem cells to preparation of medicines for antagonizing scars or tissue and organ fibrosis. According to the application, the high-purity and high-bioactivity exosome is obtained by subjecting healthy human umbilical cords to primary in-vitro culture, collecting liquid supernatant after amplification, performing low-temperature ultracentrifugation, purifying and the like; the exosome is used individually or added into medicines, health care products and cosmetics according to a proportion to be developed into a product with a bioactivity function, thereby being capable of regulating organism cell signal transduction effectively, inhibiting fibrocyte differentiation and then antagonizing the tissue and organ fibrosis and scar forming. Through experimental verification, the exosome can play a remarkable role in repairing promotion in hypertrophic scar after skin injury, particularly pathological fibrosis proliferation such as scar contracture of the joint parts, hepatic fibrosis and cardiac fibrosis, and is high in safety, little in toxicity and convenient to storage and transport, thereby being wide in application prospect.

The invention provides a gene knockout vector and a zebra fish glioma model thereof. The gene knockout vector is obtained by connecting a target sequence to plasmids capable of expressing CRISPR-Cas9gene editing system related enzymes, and the target gene is a p53, Rb1 or Nf1 gene. The CRISPR / CAS9 technology is utilized for performing targeted knockout of rb1, nf1 and tp53 genes, an established transgene-induced malignant glioma zebra fish model can observe occurrence of malignant gliomas, tumor-induced angiogenesis and glioma stem cell generating processes through real-time fluorescence, andthe difference of pathogenesis of gliomas under different genetic background conditions is studied through the molecular biotechnology.

The present invention discloses a tobacco cultivation method. Said method is characterized by that it adopts a molecular biological technique to identify key gene capable of promoting axillary bud germination and growth after the tip is pruned, then uses nocuity inducible promoter to drive antisense RNA and make it promptly express after the tip is pruned so as to inhibit the expression of said key gene capable of promoting axillary bud germination and growth and make the axillary bud can not be germinated or grown.

The invention relates to the microbiology and molecular biology technical field, in particular to a method for extracting pig manure sample total DNA. The method mainly comprises manure sample pretreatment and sample total DNA extraction. Moreover, the method comprises the following steps that: collected fresh manure sample or frozen manure sample is taken as a material; the pretreatment of the manure sample is carried out by means of a PBS buffer solution, a paraformaldehyde solution and anhydrous alcohol so as to obtain manure sample bacteria suspension; and a CTAB solution and beta-mercaptoethanol, etc. are used to carry out cell fragmentation, thereby releasing DNA. The method has the characteristics of simple operation, quickness, high efficiency and ideal repeatability and economical efficiency, etc.; moreover, the extracted DNA has high concentration, purity and yield, and can be directly used in subsequent molecular biology experimental researches such as PCR amplification without purification.

Disclosed is a method using an alkane response element (ARE) from, e.g., Acinetobacter spp. to (i) identify and clone hydrocarbon biosynthesis genes, (ii) identify and clone hydrocarbon transporter genes (iii) identify and clone hydrocarbon response genes. Screening cells were developed that expressed a transcriptional activator, e.g., alkR, and included a reporter gene, e.g., GFP operatively linked to an ARE promoter, e.g., the alkM promoter. The cells were transformed with libraries from organisms capable of hydrocarbon biosynthesis. Transformed cells that expressed the reporter gene harbored library-derived genes involved in one or more of the above-mentioned processes; and these genes were isolated from the cells using standard molecular biology techniques. Additional systems were designed wherein screening cells also expressed a gene identified in the original screen, e.g., an additional hydrocarbon pathway gene, e.g., an enhancer.

The invention discloses an SSR (Simple Sequence Repeat) core primer group developed based on whole genome sequence of foxtail millet and an application of the SSR core primer group, and belongs to the technical field of molecular biology. The core primer group comprises 30 pairs of primers, wherein nucleotide sequences are represented by SEQ ID NO. 1-60. The primer has advantages of clear electrophoretic band and rich polymorphism, and is uniformly distributed and stable in amplification. The invention also provides the application of the SSR core primer group in identifying the genetic diversity and variety of the foxtail millet. The primer group can be used for precisely and quickly identifying the variety of the foxtail millet and precisely reflecting a genetic relationship among the varieties of the foxtail millet.

The invention belongs to the fields of molecular biotechnology and experimental methods and specifically relates to a fluorescence quantitative PCR reaction solution. The solution is characterized by comprising a PCR reaction reinforcing agent which is one or more selected from the group consisting of dimethyl sulfoxide, glycerin, bovine serum albumin, methanamide, polyethylene glycol 6000, spermidine, ammonium sulfate and betaine, e.g., dimethyl sulphoxide, bovine serum albumin and betaine. The fluorescence quantitative PCR reaction solution further comprises heatproof DNA polymerase having 5'-3' polymerase activity and 5'-3' exonuclease activity and preferably comprises heatproof DNA polymerase and hot-start heatproof DNA polymerase, both of which having 3'-5' exonuclease activity. The invention also relates to a kit containing the reaction solution and a method of applying the reaction solution in fluorescence quantitative PCR.

The present invention relates to a bovine VDJ cassette (BF1H1) that provides the novel ability to develop chimeric immunoglobulin molecule capable of incorporating both linear T cell epitope(s) (CDR1H and CDR2H) as well as conformational B cell epitope(s) (exceptionally long CDR3H). Further, multiple epitopes can be incorporated for development of multivalent vaccine by replacing at least a portion of an immunoglobulin molecule with the desired epitope such that functional ability of both epitope(s) and parent VDJ rearrangement is retained. The antigenized immunoglobulin incorporating both T and B epitopes of interest is especially useful for development of oral vaccines for use in humans apart from other species including cattle. The long CDR3H in BF1H1 VDJ rearrangement originates from long germline D-genes. The novel bovine germline D-genes provide unique molecular genetic marker for sustaining the D-gene pool in cattle essential for immunocompetence via selective breeding. D-gene specific DNA probe permits typing and selection of breeding cattle stock for maximum gemline D gene pool for better health and disease prevention. The bovine D-genes are unique to cattle and, therefore, provide sensitive and specific forensic analytical tool using molecular biology techniques to determine tissues suspected of bovine origin.

The invention discloses an omega-aminotransferase mutant gene and an application thereof, and belongs to the field of the molecular biological technology. The nucleotide sequence of the omega-aminotransferase mutant gene is shown as SEQ ID NO.1. The omega-aminotransferase amino acid sequence embodied by the code of the omega-aminotransferase mutant gene is shown as SEQ ID NO.2. The omega-aminotransferase mutant gene is firstly provided, and the omega- aminotransferase corresponding to the code of the omega-aminotransferase mutant gene has a high enzyme amount, so that the biological catalysis and conversion efficiency is improved in industrial production. The invention further provides the application of the omega-aminotransferase embodied by the code of the omega-aminotransferase mutant gene in preparation of trifluoro-propylamine. The trifluoro-propylamine is added to a buffer solution to serve as the substrate for the reaction, and then products of trifluoromethyl amine compounds are collected from the reaction liquid. The method for preparing the trifluoro-propylamine has the obvious advantages that the reaction conditions are mild, no pollution is caused, and the process is simple.

The invention relates to lipase mutants with enhanced thermal stability, which belongs to the technical field of enzyme gene engineering. Rhizopus chinensis CCTCC No.M201021 lipase used as the parent is processed by molecular biotechnology to obtain the lipase mutants with enhanced thermal stability. In the amino acid sequences of the mutants, the related amino acid mutation(s) is(are) one or a plurality of Met101Thr, Glu107Gly, Ala129Ser, Ser151Asn, Cys160Leu, Lys161Arg, Pro168Leu, Pro168His, Leu180His, Asp182Tyr, Thr183Ala, Thr218Ser, Lys219Asp, Ala230Phe, Ser234Phe, Val261Gly, His317Pro, Val329Ala, Glu363Arg, Asn366Asp and Ser373Cys. The mutants are expressed by half-life period t50 at 65 DEG C. The thermal stability of the mutants is enhanced as compared with the parent Rhizopus chinensis lipase. The invention also discloses a DNA sequence, an expression carrier and a host cell for coding the lipase mutants.

The invention relates to a quick, sensitive and high-throughput intestines source pathogenic bacterium detection method. The detection method provided by the invention integrates two powerful molecular biological techniques: polymerase chain reaction PCR and a micro-array, and directly fixes a probe of PCR hybridization in a hybridization cabin of the micro-array on a same chip with a PCR reaction chamber. The detection method comprises the following steps of enriching bacteria; extracting a DNA solution; carrying out PCR amplification; hybridizing; cleaning; and judging the result. The method provided by the invention can quickly detect genes of vibrio parahaemolyticus, Shigella, staphylococcus aureus, listeria monocytogenes and salmonella in high throughput, and the detection efficiency of front-line inspection and quarantine personnel of import and export ports can be greatly improved, thereby not only reducing the workload, but also solving the undetected positive result problem probably caused by conventional detection method to the maximum extent. Therefore, food safety incidents are prevented to the maximum extent.

An Escherichia coli recombinant strain producing shikimic acid, and a construction method and application thereof belong to the technical field of microbial gene engineering. The invention firstly utilizes a molecular biology technique to delete shikimic acid kinase I gene (aroK) and shikimic acid kinase II gene (aroL) of Escherichia coli CICIMB0013, and a gene (ptsG) of a key protein EIIBC<Glc> and a quinin acid / shikimic acid dehydrogenase gene (ydiB) of a glucose phosphotransferase system to obtain an Escherichia coli mutant strain CICIMB0013.SA4 (delta aroK, delta aroL, delta ptsG, delta ydiB). The invention also constructs a recombinant expression plasmid pTHGAA containing key genes comprising aroG*,ppsA and tktA in a metabolic pathway of shikimic acid; and the recombinant expression plasmid pTHGAA is transferred into the recombinant strain CICIMB0013.SA4 to obtain a recombinant Escherichia coli B0013 (SA4 / pTHGAA) capable of producing shikimic acid efficiently. The Escherichia coli recombinant strain provided by the invention can realize efficient accumulation of shikimic acid in a fermentation process.

The invention belongs to the technical field of molecular biology and provides primers, a probe composition and a kit for rapid identification of nine animal origin ingredients in food or feed, a detection method for identification of the nine animal origin ingredients in the food or feed and application of the primers, the probe composition, the kit and the detection method. Origin ingredients of the food or feed containing multiple species can be identified rapidly by the primers, the probe composition and the real-time fluorescent PCR (polymerase chain reaction) joint detection kit. The detection method includes: designing a universal primer by taking 16SrDNA as a target gene, and designing specific probes of nine species by designing to construct internal amplification control and designing the specific probes aiming at internal control sequences; performing PCR amplification by three PCR reaction systems; interpreting the origin ingredients of the nine species directly through corresponding fluorescent probe signals and Ct values. The detection method is low in cost, time saving and high in efficiency and can achieve identification of multiple species simultaneously.

The present invention is generally directed to novel functionalized biomolecules and methods for generating such biomolecules. Biomolecules may generally include nucleic acids, peptides, multicomponent molecular complexes and / or any other molecular products that may be produced by living organisms. The present invention is further directed to cells and / or organisms manipulated to produce such functionalized biomolecules. The cells contemplated by the present invention include both prokaryotic as well as eukaryotic cells. The functionalized biomolecules are produced via materials introduced into the cell using standard molecular biology techniques or are incorporated within the genomic nucleic acid of a cell by standard recombination techniques. Further contemplated is the use of such cells for sequestration of target molecules within the cells.

The invention relates to a bacillus licheniformis engineering bacterium for efficiently synthetizing poly-gamma-glutamic acid, and belongs to the field of microbial metabolism engineering. A conventional molecular biological technique is used; on the basis of bacillus licheniformis WX-02 stored in a laboratory, a double-ingredient regulation and control factor degU is expressed in an enhanced way through external sources; the bacillus licheniformis engineering bacterium WX-02 / pHY-degU capable of efficiently synthetizing the poly-gamma-glutamic acid under the condition of no addition of an external source of glutamic acid is obtained. Liquid fermentation proves that the concentration of the poly-gamma-glutamic acid at a fermentation end point reaches 32.78 g / L; compared with that of an original bacterial strain of bacillus licheniformis WX 02, the concentration is improved by 47.59 percent; the synthesis capability of the poly-gamma-glutamic acid is obviously enhanced. The bacterial strain is preserved in CCTCC (China Center For Type Culture Collection) on May 27, 2016, and the preservation number is CCTCC NO:M2016294.

The invention discloses a method for extracting virus DNA contained in a biological sample by utilizing a micro-nucleic acid releasing agent, belongs to the technical field of molecular biology, and particularly relates to a reagent for extracting virus DNA by utilizing the micro-nucleic acid releasing reagent and a using method thereof. The micro-nucleic acid releasing agent cracks the virus DNA contained in the biological sample (a serum or a plasma) and can more effectively ensure the cracking and release efficiency through the assistance of a release promoting agent; the micro-nucleic acid releasing agent completely releases nucleic acid and also has the function of closing various factor substances, namely protein, drugs, hemolyzed blood and the like which are contained in a sample and have interference effects on PCR amplification, so that false negative in a detection process is prevented. The method disclosed by the invention is simple, convenient, flexible, fast and accurate in sample extraction, prevents the pollution and nucleic acid loss which are caused by repeated centrifugation and elution uncapping, and realizes the 'one-step' and 'one-room' operation of PCR extraction.

The invention relates to a method for studying the structural diversity of a daqu bacterial community, i.e. the denaturing gradient gel electrophoresis (DGGE) technology, which belongs to the technical field of microbial ecology and mainly comprises the following steps that: 1), daqu genomic deoxyribonucleic acid (DNA) is directly extracted; 2), bacterial universal primers are selected, and specific DNA fragments in the bacterial ribosome DNA are amplified; 3), polymerase chain reaction (PCR) products are separated through DGGE; 4), corresponding strips of microbes in the DGGE fingerprint map are recovered in a gel cutting way; 5), PCR is carried out again, products are connected to a T carrier, the blue and white spot screening is carried out, and positive clone verification is carried out; and 6), the species information of the corresponding microbes of the DGGE strips is obtained through the sequence test. The method does not rely on the molecular biology technology of the traditional culture method, has the characteristics of sensitivity, convenience and accuracy and solves the difficult problems to study some microbes incapable of being cultured or difficult to culture, and the method provides the technical basis for the verification of the daqu bacterial community structure and the discovery of new wine brewing microbes or functional microbes.

The invention belongs to the molecular biology technology field, and concretely relates to a method for sequencing and development of Asplenium nidus L. EST-SSR primers based on transcriptome. The method comprises the following steps: firstly, a transcriptome library is constructed; secondly, transcriptome data is obtained, namely, sequencing data is subjected to splicing by utilization of a Trinity software and sequences are spliced into a complete transcriptome; thirdly, SSR site search is carried out, namely, combined with a Perl programming language method, a lot of sequence information is subjected to batch processing, and SSR site search is carried out; fourthly, batch design of EST-SSR primers is carried out. Different fiddlehead materials are selected to verify the designed SSR primers, if strips are detected, successful SSR primers are obtained. Through the method, 4063 pairs of SSR primers are designed, and a new method and thought are provided for development of Asplenium nidus L. EST-SSR primers and further for achieving molecule label auxiliary selection breeding.

The invention belongs to the technical field of molecular biology, and particularly relates to a kit for extracting genome DNA (Deoxyribose Nucleic Acid) from plant leaves based on a paramagnetic particle method and an extracting method thereof. The kit for extracting genome DNA from plant leaves based on the paramagnetic particle method comprises a pretreatment solution, a rapid cracking liquid, a DNA binding liquid, a magnetic bead suspension and an eluent, wherein cell walls and cell membranes can be effectively cracked through the pretreatment solution; and the interference of impurities such as polysaccharides, polyphenol, tannin and the like on genome DAN extraction can be effectively eliminated through the pretreatment solution. In the rapid cracking liquid, guanidine hydrochloride and Tween 20 are taken as main components, so that plant cells can be fully cracked at the normal temperature within 2 minutes, only two minutes are required in a subsequent centrifuging step, and the operation time is greatly saved. The guanidine hydrochloride is a strong denaturant, has a good cell cracking effect. Moreover, organic solvents such as chloroform and the like are not required to be added, so that damages to operating personnel are avoided, and safety and reliability are achieved.

The invention discloses primers, a kit and a detection method for detecting the genders of cotuenix coturnix, and belongs to the technical field of molecular biology. According to the invention, specific primers are designed by using length difference of CHD1 (Chromodomain-Helicase-DNA-binding protein 1) genes of the cotuenix coturnix on Z chromosome and W chromosome; genome DNAs of the cotuenix coturnix are taken as templates for performing PCR (Polymerase Chain Reaction) amplification and dissolution curve analysis; the karyotype of female cotuenix coturnix is ZW type, a PCR product contains two fragments with different sizes, and the sizes of strips are 470bp and 638bp respectively, so that the dissolution curve has two peaks; the karyotype of male cotuenix coturnix is ZZ type, a PCR product only contains one fragment, and the size of the strip is 638bp, so that the dissolution curve has one peak; the male cotuenix coturnix and the female cotuenix coturnix can be clearly distinguished by using the difference of the dissolution curve. The detection method for the genders of the cotuenix coturnix, disclosed by the invention, has the characteristics of quickness, accuracy, economy and practicality, and is suitable for high-throughput operation.

The invention discloses recombinant pichia pastoris for heterogenous high level secretory expression of a rhizomucor miehei lipase. The recombinant pichia pastoris is obtained by introducing a self leader peptide gene for encoding the rhizomucor miehei lipase by employing a molecular biology technology, then optimizing a copy number of an objective gene, and constructing a vector which can be expressed in pasteur pichia pastoris. Researches show that: the introduction of the self leader peptide greatly increases the secretion amount of the objective lipase, a pichia pastoris recombinant of a lipase gene (pro-rml) containing two copies has highest enzyme activity, and the lipase Pro-RML has higher activity of hydrolysis of tri-acylglycerol.

The invention relates to miRNAs (micro ribonucleic acids) used for detecting prostatic cancer and a kit, and belongs to the technical field of molecular biology, wherein the miRNAs comprise carcinogenic miRNAs and cancer-inhibiting miRNAs; expression quantity of the carcinogenic miRNAs in real-time PCR (polymerase chain reaction) method detection, and expression quantity of the cancer-inhibiting miRNAs in the real-time PCR method detection are lowered. According to different degrees of the prostatic cancer, different carcinogenic miRNAs and cancer-inhibiting miRNAs are adopted to respectively carry out diagnosis in a targeted manner. A technical scheme provided by the invention is adopted, so that diagnosis and prognosis prediction results of the prostatic cancer can be quickly, simply, efficiently and stably obtained.

The invention belongs to the technical field of molecular biology and discloses an extracting method of polysaccharide and polyphenol plant genomes. The method includes: grinding plant materials, then adding a nucleic acid separation buffer solution and 2-mercaptoethanol, well mixing, and placing into water bath; centrifuging in a centrifuge, and then discarding supernate; adding a 1x PBS solution, well mixing on a grinding instrument in an oscillation manner, and discarding supernate after centrifuging; adding preheated 3x CTAB lysate into sediment, well mixing, and performing splitting decomposition under water bath; adding equal-volume phenol / chloroform / isoamyl alcohol mixed liquor, well mixing and centrifuging; taking supernate, adding equal-volume chloroform / isoamyl alcohol mixed liquor, well mixing and centrifuging; taking supernate, adding NaCl and icy isopropanol, well mixing, and precipitating for 1-3 hours; taking out and centrifuging, then washing sediment with ethanol, and adding TE for dissolving after air drying so as to complete the extracting process. The extracting method has the advantages that polysaccharides and polyphenols can be removed effectively, the influence of the polysaccharides and polyphenols on nucleic acid extraction is reduced, and DNA quality and concentration are increased.

The invention relates to a cloning vector and preparation and application thereof and discloses a cloning vector pUC57-ccdB which is a modified vector with ccdB genes inserted at multiple cloning sites of a pUC57 vector, wherein the ccdB genes have blunt-end restriction enzyme digested recognition sites. By means of the lethal effect of CcdB protein on escherichia coli not containing F plasmids, the ccdB genes having the restriction enzyme Sma I digested sites are inserted on the pUC57 plasmids through the molecular biological technology to obtain the vector pUC57-ccdB. Blunt ends are generated through Sma I digestion and are connected with the genes needing to be cloned so that insertion of the genes needing to be cloned can be achieved. Meanwhile, bacterial colonies with empty vectors are avoided. The cloning vector plays an important role in the fields of molecular biology and genetic engineering.

The recombination, expression and application of Chinese prawn's antibacterial peptide gene are disclosed. Its recombination expression method includes configuring recombinant carrier (pET series, pGEX-4T series, pPIC series, pAO815, and baculovirus expression carrier), transferring the carrier to colibacillus and yeast and transfecting insect cells, screening engineered bacterial strain or cell, and inducing expression. The expressed product can be used to prepare feed additive, antistaling agent, cosmetics and medical products.

The invention discloses a lipase mutant with increased optimum temperature, which belongs to the technical field of enzymatic genetic engineering. The lipase mutant with increased optimum temperature disclosed by the invention is obtained through taking rhizopus chinensis China center for type culture collection (CCTCC) M201021 lipase as a parent and utilizing a molecular biological technique, and the mutant amino acid of the mutant is Asp310Val. The optimum temperature of the lipase mutant is increased compare with the optimum temperature of the parent of rhizopus chinensis lipase and has an important industrial application value.

Extracts obtained from the genus Hippophae or other plant sources or compositions produced by chemical or molecular biological techniques each having in common certain moieties in amounts effective when combined with reproductive cells to reduce the loss of function.

The invention discloses a nasopharyngeal carcinoma distant metastasis predicting system based on a deep learning algorithm. The predicting system comprises the following modules of: an image acquisition module, an image preprocessing module, a patient information collecting module, a distant metastasis predicting module based on the deep learning algorithm, and a predicting information output module. The system of the invention realizes risk predicting to nasopharyngeal carcinoma distant metastasis of a nasopharyngeal carcinoma patient. Compared with an analysis-diagnosis-treatment mode in which traditional molecular biological technology and gene sequencing and the like are used as main means, the system has advantages of improving efficiency in acquiring a diagnosis result and realizingpredictive judgment which cannot be independently performed by a clinical doctor.