The univector plasmid-fusion system, a method for rapid construction of recombinant DNA without restriction enzymes.

摘要

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. A series of new cloning methods that facilitate the rapid and systematic construction of recombinant DNA molecules are described. The central cloning method is named the univector plasmid-fusion system (UPS). The UPS uses Cre-lox site-specific recombination to catalyse 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. It was demonstrated 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. 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.