54 results about "Gene splicing" patented technology

The invention discloses a lyase of self-cleaving escherichia coli, and applications thereof. A brandnew lyase ClyN is constructed by adopting gene splicing means, the lyase can result in the self-cleaving of the host escherichia coli after being expressed in the escherichia coli, to release intracellularly expressed ATP and protein. Recombinant ClyN expression plasmid can be duplicated in the escherichia coli BL21(DE3), and express for 15 min under the induction of IPTG to observe the obvious phenomenon of inducing the escherichia coli to self-cleave, and the induced cleaving efficiency achieves 99.8%. When the host bacteria express heterologous enhanced green fluorescent protein (EGFP), EGFP can be released in solution after escherichia coli is cleaved, and the release efficiency is 89% of direct ultrasonic breaking efficiency. Therefore, the ClyN can be used for extracting and release of heterologous protein after expression in the escherichia coli, thus having wide application prospects.

The invention relates to a repeated module gene-splicing method, which includes the following steps: (1) two PCR primers are utilized to amplify target gene; (2) the PCR primers and plasmid pET-LIC are cut by double restriction enzymes, segments are connected with carrier, and after competent cells are transformed, plasmid pET-TS is obtained; (3) the plasmid pET-TS is cut by double restriction enzymes, and plasmid segments are recovered, and are then connected with the target gene segments to transform, so that plasmid pET-TS2 is obtained; (4) the plasmid pET-TS2 is cut by double restriction enzymes, and plasmid segments are recovered, and are then connected with the target gene segments to transform, so that plasmid pET-TS3 is obtained; (5) the plasmid and target gene PCR product are cutby double restriction enzymes, and the same method is utilized to continuously insert repeated segments. The invention repetitively splices a plurality of repeated module genes, moreover, no restriction enzyme cutting site residues are left between the modules, thereby the seamless connection between the repeated modules is truly realized, and moreover, splicing is directional.

The invention discloses a one-step seamless, non-homologous and multi-fragment gene splicing method. The steps of DNA fragment phosphorylation, DNA fragment bridging and exponential type amplification of a ligation product are finished in one pipe through thermal cycling similar to PCR. The method has the following advantages: (1) ligation reaction is finished by one step, so that high cost caused by using a phosphorylation primer is avoided; (2) the concentration of DNA, the concentration of a bridging primer and components and a proportion of a buffer solution are optimized, and the ligation reaction with high reliability and good repeatability is reached; (3) DNA fragments are seamlessly ligated with one another without introduction of excess nucleotide; (4) non-DNA sequence dependency is realized; (5) the multi-fragment gene splicing ability is efficient; (6) the ligation product is amplified while ligation, so that the concentration of a product is greatly increased, and efficient conversion and subsequent experiment needs are guaranteed; and (7) the cost is low compared with that of the conventional restriction enzyme ligation method.

Disclosed are systems and methods for simultaneous detection of DNA and RNA genetic alterations comprising gene splicing variants, mutations, indel, copy number changes, fusion and combination thereof, in a biofluid sample from the patient without physically separating RNA from DNA. The systems and methods are similarly applicable to the simultaneous detection of DNA and RNA genetic alterations in solid tissues comprising gene splicing variants, mutations, indel, copy number changes, fusion and combination thereof. The present method utilized a barcoding method for analysis. The streamlined methods improve the simplicity, quantification accuracy and detection sensitivity and specificity of non-invasive detections of biomarkers.