81 results about "Concatemer" patented technology

This invention presents a new cDNA amplification method. RNA is first converted to cDNA. The synthesis of cDNA can include a promoter tagged oligonucleotide, and then this cDNA is ligated to form circles (or possibly concatemers). This is then amplified using a Phi29 DNA polymerase based rolling circle and strand displacement amplification. The invention allows for RNA promoter sequences to be attached to the cDNA to facilitate additional amplification through the generation of RNA from the amplified cDNA. The resulting product can then be used to make materials for gene expression studies or other RNA analysis procedures.

The present invention concerns concatemer and/or stabilized RNA constructs capable of forming dsRNA, optionally comprising a sequence capable of protecting the dsRNA against RNA processing in a host cell. The invention also relates to methods of producing these constructs and to methods for using these constructs. The constructs according to the present invention are particularly useful in plant pest control.

A method is disclosed for obtaining the 5′ends of transcribed regions from a plurality of nucleic acid fragments obtained from biological materials or synthetic pools. DNA fragments encoding the 5′ends are enriched for their individual analysis or for the analysis of concatemers thereof. The sequence information derived from 5′ ends can be used for characterization and cloning of the transcriptome.

Embodiments may include a method of determining a nucleic acid sequence. The method may include receiving a plurality of DNA fragments. The method may also include concatemerizing a first set of the DNA fragments to obtain a concatemer. The method may include performing single-molecule sequencing of the concatemer to obtain a first sequence of the concatemer. In some embodiments, single-molecule sequencing may be performed using a nanopore, and the method may include passing the concatemer through a nanopore. A first electrical signal may then be detected as the concatemer passes through the nanopore. The first electrical signal may correspond to a first sequence of the concatemer. In addition, the method may include analyzing the first electrical signal to determine the first sequence. Subsequences of the first sequence may be aligned to identify sequences corresponding to each of the first set of the DNA fragments.

The invention discloses a highly sensitive paper-based photoelectrochemical sensor for detecting microRNA. A paper chip is prepared by means of a wax printing technology, gold nano-particles are grownin situ in a hydrophilic working area to achieve functionalization of the paper chip, a cuprous oxide/bismuth sulfide/bismuth vanadate tertiary sensitizer is modified subsequently, and microRNA is captured through a fixed hairpin DNA chain; by means of identification of specificity and digestion effect of duplex-specific nuclease, signal amplification on microRNA is achieved, and then by means ofa multibranched hybrid chain, platinum nanoparticles are embedded into a main part of the chain, branch parts form a chlorhematin/G-tetrad structure, and a DNA concatemer with catalase bological characteristics is formed; signal amplification is further achieved, thus preparation of the photoelectrochemical sensor is completed, and highly sensitive and accurate detection on microRNA is achieved.

Methods and compositions are provided for introducing an expression cassette into a cell. In the subject methods, a population of at least two distinct adeno-associated viral particles is provided, where each distinct type of viral particle in the population comprises a different portion of the expression cassette to be introduced into the cell. The target cell is contacted with population of adeno-associated viral vectors under conditions sufficient to produce a hetero-concatemer in the cell, where the hetero-concatemer includes a functional expression cassette having an intron that includes an ITR sequence. Also provided by the subject invention are vector preparations for practicing the subject methods as well as kits for use in producing the vectors employed in the subject methods. The subject methods find use in a variety of different gene transfer applications, including both in vivo and in vitro gene transfer applications, and are particularly suited for use in the transfer of long genes.

The present invention relates to the DNA sequence for constituting T carrier, carrier containing the said sequence and the method of T carrier conversion. The DNA sequence GGTGACCANNNNT/NNNNNTGG is recognition sequence concatemer of limitative endoenzyme HphI and XcmI. The said two sequences are inserted into carrier without XcmI recognition site in reverse repeated mode to obtain one pre-I carrier. In the presence of competitive exoenzyme substrate in the concentration of 10 moles higher than pre-T carrier, the pre-T carrier is overdigested by endoenzyme XcmI to obtain mature T carrier. Cloning test shows that the T carrier has high cloning efficiency 95% or higher. The said method is simple and has universal application value.

The present invention relates to evolution of a cell or a composition of cells having a desired property or functionally. The principle behind the evolution of cells according to the invention is to produce a great diversity of genes in each cell subjected to evolution and a great diversity of genes among the cells in a composition according to the invention and to exchange the genes between the cells from time to time. In preferred embodiments the genes are arranged in expression cassettes in concatemers in the cells, as well as in artificial chromosomes. Said methods comprising the steps of a) obtaining a composition of cells, at least one cell of said composition comprising a1) at least two expressible nucleotide sequences, at least one of said sequences being incorporated into an artificial chromosome in the cell and/or a2) at least two expression cassettes of the following formula: [rs2-SP-PR-X-TR-SP-rs11 wherein rs1 and rs2 together denotes a restriction site, SP individually denotes a spacer, PR denotes a promoter, capable of functionning in the first cell, X denotes an expressible nucleotide sequence, TR denotes a terminator, and/or a3) at least two expressible nucleotide sequences, said sequences being heterologous to the cell, determining at least one screening functionality. The genes are preferably co-ordinately controllable for increasing diversity. The desired property of functionality is preferably a compound, or a series of compounds synergistically acting to each other.

The externally amplifying process of specific ring form or concatemer nucleic acid includes the pre-denaturation of ring type or concatemer nucleic acid containing target sequence as the template, one pair of 5'-phosphorylated oligonucleotide primers, four kinds of triphosphodeoxyribonucleotides and thermonucleic acid synthetase in proper buffing system at 90-99 deg.c for 1-30; and subsequent 1-40 thermal circulation treatment. The said thermal circulation includes three steps of: denaturation via maintaining at 90-99 deg.c for 1 sec to 5 min; renaturation and connection at 30-85 deg.c for 10 sec to 15 min; and extension at 65-85 deg.c for 1-30 min. The present invention can produce and amplify ring form or serial DNA externally and optionally.

In some aspects, the present disclosure provides methods for enriching amplicons, or amplification products, comprising a concatemer of at least two or more copies of a target polynucleotide. In some embodiments, a method comprises sequencing the amplicons comprising at least two or more copies of a target polynucleotide. In some embodiments, the target polynucleotides comprise sequences resulting from chromosome rearrangement, including but not limited to point mutations, single nucleotide polymorphisms, insertions, deletions, and translocations including fusion genes. In some aspects, the present disclosure provides compositions and reaction mixtures useful in the described methods.

In some aspects, the present disclosure provides methods for enriching amplicons, or amplification products, comprising a concatemer of at least two or more copies of a target polynucleotide. In some embodiments, a method comprises sequencing the amplicons comprising at least two or more copies of a target polynucleotide. In some embodiments, the target polynucleotides comprise sequences resulting from chromosome rearrangement, including but not limited to point mutations, single nucleotide polymorphisms, insertions, deletions, and translocations including fusion genes. In some aspects, the present disclosure provides compositions and reaction mixtures useful in the described methods.

A simple and robust method for genetic analysis of complex microbial communities involves the steps of PCR amplification of V1 region of rrs genes in the community DNA sample using two universal primers, followed by cleavage by BsgI and removal of dual-biotinylated primers using streptavidin-coated magnetic beads to purify RSTs, and concatemerization of the RSTs and size selection of resultant concatemers by agarose gel electrophoresis. The isolated concatamers are then cloned and sequenced and subjected to sequence analyses to enable identification of the members of the microbial community.

The invention discloses an artificially constructed araneus ventricosus dragline silk protein gene and a construction method thereof. The sequence of the araneus ventricosus dragline silk protein gene is shown as SEQIDNO.6. According to part of cDNA sequence of araneus ventricosus dragline silk protein, first, most-representative core repeat 108BP is selected, and artificially synthesized, both ends of the core repeat sequence are additionally added restriction enzyme cutting sites, then the core repeat sequence is connected with a pSIMPLE-19EcoRV / BAP vector, and transformed into Escherichia coli to obtain monomer recombinant plasmid, by an isocaudarner recursive directional ligation method, gene monomers are polymerized into a 16-time concatemer, 1836bp araneus pseudoventricosus dragline silk protein gene can be successfully obtained, the 1836bp araneus pseudoventricosus dragline silk protein gene can be successfully expressed for production of the araneus ventricosus dragline silk protein, and the whole process is simple and efficient, saves the cost, improves the work efficiency, and has good application prospects.

The present disclosure provides compositions and methods that employ the compositions for conducting pairwise sequencing and for generating concatemer template molecules for pairwise sequencing. The concatemers can be generated using a rolling circle amplification reaction which is conducted either on-support, or conducted in-solution and then distributed onto a support. The rolling circle amplification reaction generates concatemers containing tandem copies of a sequence of interest and at least one universal adaptor sequence. An increase in the number of tandem copies in a given concatemer increases the number of sites along the concatemer for hybridizing to multiple sequencing primers which serve as multiple initiation sites for polymerase-catalyzed sequencing reactions. When the sequencing reaction employs detectably labeled nucleotides and/or detectably labeled multivalent molecules (e.g., having nucleotide units), the signals emitted by the nucleotides or nucleotide units that participate in the parallel sequencing reactions along the concatemer yields an increased signal intensity for each concatemer.

The invention relates to the technical field of targeting transport of cell penetrating peptides, in particular to a method for transducing a buffalo embryo with PEP-1 peptide concatemer mediated green fluorescent protein. The method comprises steps as follows: design and optimization of PEP-1-PEP-1-EGFP, construction of a prokaryotic expression vector pCZN1-PEP-1-PEP-1-EGFP, prokaryotic expression of the protein, protein transfection and the like. By means of genetic engineering, the PEP-1 concatemer mediated expression vector plasmid pCZN1-PEP-1-PEP-1-EGFP is constructed, the optimal condition for transducing buffalo embryo cells with the PEP-1 peptide concatemer mediated green fluorescent protein is explored, the green fluorescent protein can be transduced into the buffalo embryo accurately and efficiently, a research foundation is laid for study of target transduction of nutrient substances and target effects of drugs, and the mediation effect of PEP-1 concatemers can be exerted efficiently.

Disclosed are methods for detecting spatial proximity relationships between nucleic acid sequences in a cell. The methods include: providing a sample of one or more cells comprising nucleic acids; fragmenting the nucleic acids present in the cells, wherein the fragmented nucleic acids have ends capable of joining to other fragmented nucleic acids; joining ends of fragmented nucleic acids to other ends fragmented nucleic acid to create at least one nucleic acid concatemer having at least one junction between the joined fragmented nucleic acids, and wherein the at least one nucleic acid concatemer encodes the information about the proximity of the DNA sequences in the cell; and determining the sequence at least one junction of the at least one nucleic acid concatemer, thereby detecting spatial proximity relationships between nucleic acid sequences in a cell.

The invention discloses a method for preparing an active small peptide by establishing an oligopeptide concatemer yeast expression plasmid, belongs to the technical field of biologic engineering. The method comprises the following steps of: amplifying by using an SOE-PCR (Splicing by Overlap Extension-Polymerase Chain Reaction) method so as to obtain a full-length gene of the oligopeptide; directionally and serially connecting the genes of a plurality of oligopeptides; establishing the obtained serial connected genes into an expression carrier of saccharomycete so as to convert the saccharomycete; and performing inducible expression on and the serial connected polypeptide and cutting into the target oligopeptide. The method has the characteristics that the various small molecule oligopeptides are effectively expressed by using saccharomycete, and the oligopeptides have important application values; and in addition, by utilizing the efficient expression of the serially connected oligopeptide plasmid, a product is very low in development cost.

The invention provides a detection module and method. The detection module and method are established on the basis of an EXPAR (exponential amplification reaction) tandem digestion type HCR (hybridization chain reaction). When a to-be-detected object contains a target substance, a large quantity of digestion single strands are formed by amplification with the established module and method for an EXPAR, detection signals of the target substance are amplified, then the formed digestion single strands are used for digesting a modified digestion type HCR product, and the detection signals of the target substance are converted. In one specific embodiment, supermolecular double-strand concatemers are modified and upgraded by modifying hairpin structures used in the HCR, so that not only can DNAdouble strands be self-assembled by nucleic acid hairpins, but also the nucleic acid hairpins can be digested efficiently by nucleic acid single strands. Dual induction of signals is realized. In another embodiment, when the designed and modified digestion type hairpin sequences and digestion sequences are applied to practical detection, detection sensitivity is high, and 1nM X substrate can be detected at lowest.

A method for analysing a target polynucleotide having distinct units of nucleic acid sequence comprising: (i) forming a first polynucleotide which is a concatemer having multiple repeating target polynucleotide sequences; (ii) forming on the first polynucleotide a second polynucleotide hybridised to a portion of one or more of the target polynucleotides, such that the portion hybridised, or the portion not hybridised, corresponds to a sequence unit on the target, and determining the sequence unit on the target.