258 results about "Restriction site" patented technology

The disclosure provides methods of capturing target nucleic acids (e.g., gene or gene fragments) onto a solid support for further analysis. The disclosed methods utilize a capture probe that selectively circularizes only the target nucleic acid. Following the circularization of the target, the linear, non-target, nucleic acids are removed from the sample. Next, the circularized target is linearized and bound to a solid support. To allow for linearization, the capture probe may include a cleavage site that can be a noncanonical nucleotide(s) (e.g., uracil in DNA) and/or a rare-cutter site (e.g., the Not I restriction site). In some embodiments, the target nucleic acid is captured onto a support without an intermediate amplification step.

The present invention concerns a family of nucleic acids, polypeptides and cloning vectors which direct expression of fusion proteins that can mimic aggregated IgG (AIG) and immune complex function with respect to their interactions with FcγR and which allow for the inclusion and targeting of a second protein domain to cells expressing FcγR. This was accomplished by expressing multiple linear copies of the hinge and CH2 domains (HCH2) of human IgG₁ fused to the framework region of human IgG₁. Convenient restriction sites allow for the facile introduction of additional amino-terminal domains. Methods for treating patients using fusion proteins are also disclosed. The HCH2 polymers described here represent a new strategy in the design of recombinant proteins for the therapeutic targeting of FcγR in autoimmune disorders.

The invention relates to enhancer Hr3 adopting the nucleotide sequence shown in the SEQIDNO: 1. The enhancer Hr3 can be used for preparing and recombining heterologous protein by bombyx mori. The invention further relates to a recombination carrier containing the enhancer Hr3, and the recombination carrier is prepared in such a manner that the enhancer Hr3 is connected with a Nco1 restriction site at the upstream of a promoter of a carrier containing no enhancer through the Nco1 restriction site of the enhancer Hr3. In the invention, functional elements such as the first grade enhancer, activating transcription factor IE1, untranslated region sequences (5' UTR and 3' UTR) and the like which are expressed by intensifier are identified in the cellular level, and an efficient and stable sericin I expression system is built by integrating optimum elements on the basis, so that efficient expression recombination heterologous protein can be made of middle silk gland of transgenic bombyx mori.

The present invention relates to an altered-type human ALT gene in which the codons for the five amino acids in the human ALT (alanine aminotransferase) gene are replaced, i. e. the fourth amino acid codon from the initiation codon for methionine (Met) is replaced by a codon for serine (Ser), the fifth by a codon for threonine (Thr), the seventh by a codon for aspartic acid (Asp), the 39th by a codon for glycine (Gly) and the 222nd by a codon for alanine (Ala), and concurrently, restriction sites are added at the upstream and downstream of said gene.The active human ALT having properties similar to those of the native enzyme can be effectively produced by culturing E. coli transformed with a recombinant plasmid in which the altered-type human ALT gene of the present invention is ligated to a vector.

The invention discloses a method for preparing rebaudioside M according to a saccharomyces cerevisiae enzymatic method. The method for preparing rebaudioside M comprises the following steps: utilizing recombinant saccharomyces cerevisiae containing UDP-glucosyltransferase or UDP-glucosyltransferase prepared from the recombinant saccharomyces cerevisiae to catalyze rebaudioside A or rebaudioside D in the presence of a glucosyl group donor, so as to generate rebaudioside M. The recombinant saccharomyces cerevisiae is obtained by introducing a strong promoter into a plasmid to obtain a vector plasmid, inserting a UDP-glucosyltransferase gene into the vector plasmid through a restriction site to obtain an expression vector under the control the strong promoter, and carrying out saccharomyces cerevisiae transformation. The method for preparing rebaudioside M has the advantages that the high-safety recombinant saccharomyces cerevisiae is utilized for catalytic production; the produced UDP-glucosyltransferase is higher in expression level and activity; the produced rebaudioside M can be directly utilized as a food additive, and is short in production period, higher in yield, and relatively low in cost.

A method for detection of nucleic acid targets using as reagents: (1) a stem loop probe having a long strand for hybridizing to a target, a short strand, usually with a free 3'-end for extension, hybridized to a portion of the long strand and a linker forming a loop and joining the short and long strands; and (2) a hybridizing reagent that hybridizes to the short strand when released by the target. The target is detected by extension of one or both the probe or hybridizing reagent along each other. A circular hybridizing reagent can be employed with DNA polymerase for concatenated extension of the probe 3'-end or extension of the 3'-end of the probe along a hybridizing reagent having a promoter sequence for forming transcripts of at least a portion of the long strand or the hybridizing reagent. A non-cleavable restriction site consensus sequence in the linker, where the hybridizing reagent is extended with dNTPs and DNA polymerase and the extended sequence is cleaved with a restriction enzyme, so that chains may be repetitively produced and cleaved. The presence of the target nucleic acid is established by detecting the concatenated chain, the RNA transcripts or the repetitively produced chains.

Methods are provided for evaluating the fidelity of a given set of polymerase chain reaction conditions. In the subject methods, a template polydeoxyribonucleotide is amplified under the to be evaluated polymerase chain reaction conditions, where the template polydeoxyribonucleotide includes a pseudo restriction endonuclease restriction site. The resultant amplified product population is then contacted with the corresponding restriction endonuclease and resultant cleavage products, if any, are detected. The fidelity of the polymerase chain reaction conditions is then derived from the detected cleavage products (or absence thereof). Also provided are kits for use in practicing the subject methods. The subject methods are suited for determining the fidelity of a given polymerase under PCR conditions, and are particularly suited for determining the fidelity of a thermostable polymerase under PCR conditions.

The invention discloses a single nucleotide polymorphism (SNP) site identification method based on digestion library-establishing and sequencing and bayesian statistics. The method is used for processing RAD (restriction site associated deoxyribonucleic acid) sequencing data, searching candidate SNP on an RAD sequencing fragment, and identifying the SNP reliability by employing a bioinformatics analysis method based on bayesian statistics. The method can be used for model and non-model organisms to eliminate the limitation that lots of species are lack of reference sequences and reduce the sequencing cost, and can be used for solving the bottleneck that a reliable statistical method is absent in the process of performing SNP identification by utilizing the RAD data at present, so that the obtained SNP site accuracy is greatly improved.

The invention relates to methods of making modular chimeric protein expression products and compositions utilized in the methods. In particular, the invention relates to sequential, directional cloning of polynucleotides encoding polypeptide modules. Each clonable element or module contains an open reading frame of interest flanked by predetermined restriction sites. The methods include using modules and vectors containing these modules as starting materials for recombinant DNA techniques. One advantage of the invention is that it allows for many variations of fusion proteins to be made quickly and easily without needing to design and evaluate each subsequent cloning step.

Proviral plasmids contain a modular gene expression cassette with one or a combination of (i) a wildtype 5′ AAV2 ITR sequence flanked by unique restriction sites that permit ready removal or replacement of said ITR; (ii) a promoter flanked by unique restriction sites that permit ready removal or replacement of the entire promoter sequence; (iii) a polylinker sequence that permits insertion of a gene coding sequence without modification thereof, wherein the gene is operatively linked to, and under the regulatory control of, the aforementioned promoter; (iv) a bovine growth hormone polyadenylation sequence flanked by unique restriction sites that permit ready removal or replacement of said polyA sequence; and (v) a wildtype 3′ AAV2 ITR sequence flanked by unique restriction sites that permit ready removal or replacement of the 3′ ITR. These plasmids enable rapid manipulation of the components of the cassette, e.g., rapid mutation and/or replacement of any component, and thereby increase the efficiency of recombinant viral vector, e.g., rAAV, production.

The invention belongs to the proteomics technology field, in particular relates to an efficient enzymolysis method of alternating current accessory protein. Protein and proteolytic enzymes such, as trypsin, are solubilized in buffer solution in a certain proportion by the method, and the solution is ensured to contact with an electrode applied by alternating voltage; the charged protein, enzyme and inorganic ions, etc. change the direction of motion in the electric field immediately, and the motions, such as vibration, collision, rotation and friction, etc., of the molecules such as protein and enzyme, etc., are caused. Simultaneously, shakings also occurs to the charged group in the peptide chain of the protein molecules under the exertion of the alternating electric field, and more restriction sites are exposed. The motions improve the energy level and interaction frequency of the protein and enzyme molecules, in order to increase the protein enzymolysis efficiency greatly. By adopting the alternating current accessory protein enzymolysis, the enzymolysis time is greatly shortened from twelve hours of traditional solution enzymolysis to five to ten minutes, and the enzymolysis efficiency is improved remarkably.

The invention provides a method for preparing a chicken genetic engineering immunopotentiator by an akirin2 gene sequence, relating to a method for preparing a chicken genetic engineering immunopotentiator. The invention solves the problem that the existing genetic engineering immunoenhancement nucleic acid vaccines can not extensively improve the immunity effects of vaccines and bacterins. The method comprises the following steps of: acquiring a chicken akirin2 gene coded sequence; acquiring a recombinant pMD18-T vector containing the chicken akirin2 gene; designing a primer and enabling the 5'- terminals to have HindIII and EcoRI restriction sites, subcloning the chicken akirin2 gene in the recombinant pMD18-T vector into an expression vector to construct a recombinant expression vector; and diluting the recombinant expression vector by a sterile PBS (phosphate buffer solution). The chicken genetic engineering immunopotentiator provided by the invention can improve the expression levels of multiple immunity related factors such as IL-1, IL-6, toll-like receptors and the like, and has the characteristics of wide applicable range and the like for various vaccines and bacterins.

The invention provides recombinant pichiapastoris for producing FAD-dependent glucose dehydrogenase as well as a construction method and application thereof and belongs to the technical field of genetic engineering. The invention provides a recombinant plasmid pMD-GDH containing FAD-dependent glucose dehydrogenase genes. After the FAD-dependent glucose dehydrogenase genes are subjected to codon preference adjustment, EcoRI restriction sites and NotI restriction sites are added at two ends of the sequence, and six histidine tags are added at the 3' end. The recombinant pichiapastoris takes Pichia pastoris as an expression host and comprises the recombinant plasmid. The invention further provides application of the recombinant pichiapastoris for producing FAD-dependent glucose dehydrogenase in fermentation production of the FAD-dependent glucose dehydrogenase. The embodiments show that when the recombinant pichiapastoris is subjected to induction culture for 136 hours during culture in a 10L of fermentation tank, the enzyme activity reaches 257600U/L and is 5.3 times that of the enzyme activity reported in the prior art.

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.

Methods and compositions are provided for generating a single-stranded extension on a polynucleotide molecule, the single-stranded extension having a desired length and sequence composition. Methods for forming single-stranded extensions include: the use of a cassette containing at least one nicking site and at least one restriction site at a predetermined distance from each other and in a predetermined orientation; or primer-dependent amplification which introduces into a polynucleotide molecule, a modified nucleotide which is excised to create a nick using a nicking agent. The methods and compositions provided can be used to manipulate a DNA sequence including introducing site specific mutations into a polynucleotide molecule and for cloning any polynucleotide molecule or set of joined polynucleotide molecules in a recipient molecule such as a vector of choice.

Disclosed is a computer-implemented method of determining at least one optimal alignment of at least part of a first map to at least part of a second map or a plurality of second maps, wherein the maps are physical genome maps and/or restriction maps. The method comprises: receiving first map data indicative of a first ordered list of distances between features of the first map, receiving second map data indicative of a second ordered list of distances between features of the second map or second maps; generating, from the second map data, seed data indicative of a plurality of seeds, each seed comprising at least one of the distances in the second ordered list, wherein the features are restriction sites and distances are fragment sizes. The said method further comprises generating a plurality of candidate alignments from the seed data by searching at least part of the first ordered list to find at least approximate matches for respective seeds, and extending the approximate matches by dynamic programming; determining respective alignment scores for respective candidate alignments; and selecting one or more of the candidate alignments as an optimal alignment or optimal alignments, based on the alignment scores.