738 results about "Insertion" patented technology

An inactive CRISPR/Cas system-based fusion protein and its applications in gene editing are disclosed. More particularly, chimeric fusion proteins including an inCas fused to a DNA modifying enzyme and methods of using the chimeric fusion proteins in gene editing are disclosed. The methods can be used to induce double-strand breaks and single-strand nicks in target DNAs, to generate gene disruptions, deletions, point mutations, gene replacements, insertions, inversions and other modifications of a genomic DNA within cells and organisms.

The present invention provides a method for identifying one or more low abundance sequences differing by one or more single-base changes, insertions, or deletions, from a high abundance sequence in a plurality of target nucleotide sequences. The high abundance wild-type sequence is selectively removed using high fidelity polymerase chain reaction analog conversion, facilitated by optimal buffer conditions, to create a restriction endonuclease site in the high abundance wild-type gene, but not in the low abundance mutant gene. This allows for digestion of the high abundance DNA. Subsequently the low abundant mutant DNA is amplified and detected by the ligase detection reaction assay. The present invention also relates to a kit for carrying out this procedure.

An improved method of amplifying nucleic acids comprising the use of four discrete temperature steps in a thermocyclic amplification reaction, as well as, a method of detecting large nucleic acid insertions or deletions such as those that occur from gene duplication or deletion.

Provided are compositions comprising recombinant polymerases that include amino acid substitutions, insertions, deletions, and/or exogenous features that confer modified properties upon the polymerase for sequencing RNA or RNA/DNA templates. Polymerases that topologically encircle the template nucleic acid are provided. Also provided are methods of using such polymerases to make a DNA or to sequence a template comprising RNA.

The invention provides a functional molecular marker for rice anti-blast gene Pi9 and application thereof, belonging to the field of crop molecular genetic breeding science. The invention finds out that the rice anti-blast gene Pi9 has a 10bp insertion/deletion site positioned between the gene promoter -516 and -517, wherein the nucleotide sequence is disclosed as SEQ ID NO.1; and on such basis, the invention develops a method of the functional molecular marker for gene Pi9. By detecting the functional molecular marker, the invention can accurately detect whether genomes of different rice species contain the Pi9 functional gene and detect the homozygotic state, and can be used for screening rice hybrid transformed descendant plants to enhance the breeding efficiency of the rice anti-blast material, thereby effectively controlling the size of the breeding population, obviously saving the breeding and screening cost and obtaining the anti-blast rice species containing the Pi9 functional gene.

The invention provides an STAG2 gene mutant sequence and a detection method thereof as well as use of STAG2 gene mutation in detecting bladder cancer and belongs to the technical field of the molecular biology gene detection. The detection method comprises the following steps: (1) extracting a tumor tissue DNA and a peripheral blood DNA from a tumor tissue and the peripheral blood of a bladder cancer patient; (2) performing whole genome sequencing and whole exon sequencing on the two DNAs extracted in the step (1), respectively; (3) performing whole exome sequence alignment and somatic mutation detection on the sequencing results of the step (2), thereby obtaining a mutant gene; (4) verifying the somatic replacement, insertion and deletion of the mutant gene obtained in the step (3) by use of Sanger sequencing; and (5) identifying the mutant gene as the remarkable mutant gene. The detection method has the advantage that the mutation of the STAG2 is one of unfavorable indexes of the bladder cancer and also is capable of serving as a new acting target for treating the bladder cancer.

The invention belongs to the field of crop biotechnology and in particular relates to a molecular marker of a rice-blast-resistant gene Pi2 and application of the molecular marker. The upstream and downstream molecular markers are based on specific insertion/deletion loci polymorphism of nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2. The two molecular markers are co-dominant molecular markers developed for close linkage regions on two sides of the functional gene Pi2, and have the advantages of being high in accuracy and capable of being widely applied to different breeding parents. By detecting the two molecular markers, whether a detected rice material has the rice-blast-resistant gene Pi2 or not can be accurately judged. By virtue of a molecular marker combination, whether a hybrid transferred progeny plant genome of a parent of the functional gene Pi2 and other parents has the functional gene Pi2 or not and the homozygous state of the functional gene Pi2 can be accurately detected, and the breeding efficiency of a rice-blast-resistant rice variety with the functional gene Pi2 is improved.

The invention belongs to the technical field of biological detection, and in particular relates to a fluorescently-labeled insertion/deletion (InDel) genetic polymorphism locus composite amplification system and application thereof. The application comprises the following steps of: selecting 35 InDel loci, designing polymerase chain reaction (PCR) amplification primers of the 35 InDel loci, matching fluorescein labels of the PCR primers, compositely amplifying and detecting the 35 InDel loci, and the like. The system can analyze 35 InDel genetic polymorphism loci by the composite amplification. The 35 InDel loci are labeled by FAM, HEX, TAMRA and ROX fluorescein with four different colors respectively. The composite amplification system based on the invention can be made into a kit, serves as an InDel locus fluorescence composite amplification kit with Chinese characteristics, and is applied to the fields, such as triplet paternity test, doublet paternity test, grandparent and grandchild test, sibling test, individual recognition, disease diagnosis, anthropology and the like.

The invention belongs to the technical field of plant biology, and relates to a transgenic soybean event B4J8049 exogenous inserted fragment flanking sequence and applications thereof. The provided anti-disease transgenic soybean event B4J8049 is identified as unit point insertion through the Southern hybrid, and the right boundary flanking sequence of the exogenous fragment of the insertion site is represented by SEQ-1. The specific primers, which are designed according to the flanking sequence, are represented by the SEQ-5 and SEQ-6, and a specific PCR detection method of the anti-disease transgenic soybean event B4J8049 is established. The provided detection primer and detection method are suitable for carrying out specific detection on the anti-disease transgenic soybean event B4J8049, which comprises parent, derivation species, and products (plants, tissue, seed, and product).

The invention discloses a genetic engineering strain for producing phenazine-1-carboxylic acid, which is produced by taking pseudomonas chlororaphis as a culture, as well as application of the genetic engineering strain; the genetic engineering strain is obtained by knocking out a phzH gene in pseudomonas chlororaphis genome. The genetic engineering strain for producing phenazine-1-carboxylic acid is finally prepared by deleting the phzH gene in the genome of the strain by virtue of insertion mutation or marker-less deletion from pseudomonas chlororaphis HT66 CCTCC NO: M2013467 which can naturally secrete phenazine-1-formamide as well as derivatives of the pseudomonas chlororaphis so as to convert a secondary metabolite from the phenazine-1-formamide into the phenazine-1-carboxylic acid. The invention also discloses a preparation method and a detection method of the fungicide.

The invention relates to a CRISPR/Cas12a gene editing system and application thereof. The invention comprehensively utilizes biochemical, molecular biology, cell biology and other methods to screen and identify novel LiCas12a protein with endonuclease activity from a plurality of different bacteria. Based on LiCas12a, a CRISPR/LiCas12a gene editing system with high editing efficiency and high specificity is established in eukaryotic cells and prokaryotic cells separately, and genetic manipulation such as knockout, insertion and point mutation of genes is achieved; a molecular mechanism that the mutation of a TERT gene promoter promotes the cell proliferation of bladder cancer and liver cancer is disclosed. The genetic editing system based on CRISPR-LiCas12a provides a more accurate and efficient gene editing method for research fields such as disease pathogenesis and metabolic engineering transformation.

The present invention relates to methods and apparatus for rapid assessment of genomic sequences using the difference set model. The invention provides methods to determine the presence and identity of similarities and differences in genomic sequences. In particular, the invention provides methods and apparatus to assess homology, the presence and identity of insertion and deletion segments and the presence and identity of single nucleotide polymorphisms in genomic sequences.

The invention discloses application of a gene FoPDCD5 (Program Cell Death Protein 5) to regulation of pathogenicity of fusarium oxysporum, and belongs to the field of plant genetic engineering. According to the application of the gene FoPDCD5 to regulation of the pathogenicity of the fusarium oxysporum, the gene is knocked out from the fusarium oxysporum by a homologous recombination method, and aknockout mutant [delta]Fopdcd5 is obtained; by constructing a gene complement vector, the gene complement vector is introduced into a [delta]Fopdcd5 protoplast; and by using a method of random insertion, the gene is completed into the knockout mutant, a complement mutant [delta]Fopdcd5-com is obtained. A pathogenicity test shows that pathogenicity of the knockout mutant [delta]Fopdcd5 is significantly reduced; and pathogenicity of the complement mutant [delta]Fopdcd5-com is restored to a wild-type level. The application of the gene FoPDCD5 to regulation of the pathogenicity of the fusarium oxysporum proves that the FoPDCD5 is necessary for production of conidia of the fusarium oxysporum and response to oxidative stress and pathogenicity. The application is helpful to elucidate a pathopoiesia molecular mechanism of the fusarium oxysporum thoroughly, and target genes are provided for development of effective fungicides.

The invention discloses a method for enriching a gene 56 target region based on multiple probes. The method comprises the following steps: designing a capture probe library, and enriching a target gene sequence by adopting a probe capture mode in a DNA level so as to detect all variation types of target genes such as mutation, deficiency, insertion, fusion and copy number amplification. By adopting the method disclosed by the invention, the defects that only the mutation and deficiency of the target genes can be detected and the fusion cannot be detected when the target gene sequence is enriched by virtue of a PCR method in a DNA level at present can be overcome.

The invention relates to an allele double knockout targeting vector system and a construction method thereof. The system of the invention consists of two complementary vectors, namely pGT-V1 and pGT-V2, wherein each vector contains two in phase LoxP elements which contain a positive selection marker gene Neo/GFP and Hyg/RFP respectively; and the outside of each vector contains a negative selection marker gene TK. Meanwhile, two 8-basic group multiple cloning sites are designed and arranged between the two LoxP elements and the negative selection marker for the insertion of the homology arm. By adopting the constructed targeting vector system of the invention, two complementary targeting vectors are cotransfected into the recipient cell; through the selection of drug and fluorescent double-selection marker, the genetic modification or knockout of the two alleles of the target gene can be realized once; and the interaction of the transfected Cre enzyme and the LoxP elements can be utilized to remove the selection marker gene integrated with the genome, the time of obtaining the homozygous knockout target cell can be shortened, the safety of the transgenic animal can be increased and a valuable technology platform is provided to develop the animal transgenic researches.

Macrolide resistance associated with macrolide efflux (mef) in Streptococcus pneumoniae has been defined with respect to the genetic structure and dissemination of a novel mefE-containing chromosomal insertion element. The mefE gene is found on the 5′-end of a 5.5 kb or 5.4 kb insertion designated mega (macrolide efflux genetic assembly) found in at least four distinct sites of the pneumococcal genome. The element is transformable and confers macrolide resistance to susceptible S. pneumoniae. The first two open reading frames (ORFs) of the element form an operon composed of mefE and a predicted ATP-binding cassette homologous to msrA. Convergent to this efflux operon are three ORFs with homology to stress response genes of Tn5252. Mega is related to mefA-containing element Tn1207.1. Macrolide resistance due to mega has been rapidly increased by clonal expansion of bacteria containing it and horizontally by transformation of previously sensitive bacteria.