[32] Rapid construction of recombinant DNA by the univector plasmid-fusion system

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.