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The Experts below are selected from a list of 63753 Experts worldwide ranked by ideXlab platform

Erik V. Van Der Eycken - One of the best experts on this subject based on the ideXlab platform.

Yi He - One of the best experts on this subject based on the ideXlab platform.

Stephen J Elledge - One of the best experts on this subject based on the ideXlab platform.

  • magic an in vivo genetic method for the Rapid Construction of recombinant dna molecules
    Nature Genetics, 2005
    Co-Authors: Stephen J Elledge
    Abstract:

    We describe a highly engineered in vivo cloning method, mating-assisted genetically integrated cloning (MAGIC), that facilitates the Rapid Construction of recombinant DNA molecules. MAGIC uses bacterial mating, in vivo site-specific endonuclease cleavage and homologous recombination to catalyze the transfer of a DNA fragment between a donor vector in one bacterial strain and a recipient plasmid in a separate bacterial strain. Recombination events are genetically selected and result in placement of the gene of interest under the control of new regulatory elements with high efficiency. MAGIC eliminates the need for restriction enzymes, DNA ligases, preparation of DNA and all in vitro manipulations required for subcloning and allows the Rapid Construction of multiple constructs with minimal effort. We show that MAGIC can generate constructs for expression in multiple organisms. As this new method requires only the simple mixing of bacterial strains, it represents a substantial advance in high-throughput recombinant DNA production that will save time, effort and expense in functional genomics studies.

  • 32 Rapid Construction of recombinant dna by the univector plasmid fusion system
    Methods in Enzymology, 2000
    Co-Authors: Qinghua Liu, Dou Liu, Stephen J Elledge
    Abstract:

    Publisher Summary The functional analysis of a single gene often requires many cloning events of the same gene into various vectors for different purposes. Each of these manipulations consumes significant amounts of time and energy for many reasons. First, each gene must be individually tailored for each vector. This is not only because the sequence of every gene is different but also because the majority of existing vectors have been developed independently by different scientists and thus contain different sequences and restriction sites for the insertions of genes. Second, the conventional cut and paste cloning strategy is time-consuming and requires many in vitro manipulations including restriction endonuclease digestion, agrose gel electrophoresis, deoxyribonucleic acid (DNA) fragment isolation, and ligation. Finally, a rational cloning strategy must be designed prior to each cloning event by identifying a compatible vector and suitable restriction enzymes. This normally requires a detailed knowledge of the gene sequence and recipient vector. The advent of the polymerase chain reaction (PCR) and site-directed mutagenesis has greatly facilitated the alteration of gene sequences and the creation of compatible restriction sites for cloning purpose. The high error rate of thermostable polymerases, however, requires each PCR-derived DNA fragment to be verified by DNA sequencing, which is another time-consuming process. To facilitate the Rapid, efficient, and uniform Construction of recombinant DNA molecules, a novel cloning strategy, the univector plasmid-fusion system (UPS), has been developed based on the Cre– loxP site-specific recombination system of bacteriophage P1.

  • the univector plasmid fusion system a method for Rapid Construction of recombinant dna without restriction enzymes
    Current Biology, 1998
    Co-Authors: Mamie Z Li, Deborah Leibham, David Cortez, Stephen J Elledge
    Abstract:

    Abstract Background: Modern biological research is highly dependent upon recombinant DNA technology. Conventional cloning methods are time-consuming and lack uniformity. Thus, biological research is in great need of new techniques to Rapidly, systematically and uniformly manipulate the large sets of genes currently available from genome projects. Results: We describe a series of new cloning methods that facilitate the Rapid and systematic Construction of recombinant DNA molecules. The central cloning method is named the univector plasmid-fusion system (UPS). The UPS uses Cre– lox site-specific recombination to catalyze plasmid fusion between the univector – a plasmid containing the gene of interest – and host vectors containing regulatory information. Fusion events are genetically selected and place the gene under the control of new regulatory elements. A second UPS-related method allows for the precise transfer of coding sequences only from the univector into a host vector. The UPS eliminates the need for restriction enzymes, DNA ligases and many in vitro manipulations required for subcloning, and allows for the Rapid Construction of multiple constructs for expression in multiple organisms. We demonstrate that UPS can also be used to transfer whole libraries into new vectors. Additional adaptations are described, including directional PCR cloning and the generation of 3′ end gene fusions using homologous recombination in Escherichia coli . Conclusions: Together, these recombination-based cloning methods constitute a new comprehensive approach for the Rapid and efficient generation of recombinant DNA that can be used for parallel processing of large gene sets, a feature that will facilitate future genomic analysis.

Danjun Wu - One of the best experts on this subject based on the ideXlab platform.

Luc Van Meervelt - One of the best experts on this subject based on the ideXlab platform.