One-Step PCR Cloning System Based On FLP-Recombination

The goal of this proposal is to develop a "vector-free" system for cloning PCR products which is efficient and permits the direct transformation of bacterial cells with PCR products. The system will utilize the yeast FLP recombination system in vivo to clone PCR product into a recombinase target site located in a chromosomally integrated, rescuable plasmid vector. Subsequent generations of the target vector will include features which allow analysis and expression in eukaryotic systems (e. g. tissue culture cells, baculovirus expression vectors, yeast YAC vectors), facilitate large scale genome mapping when used in specialized prokaryotic cloning systems such as P1 and cosmid vectors, and allow high level expression and purification of the desired product. Application for cDNA library construction will also be explored. In light of the widespread use of PCR, and the relative inconsistency involved with cloning PCR products by conventional means, there is a broad general application for an efficient one-step PCR cloning system. The current "state of the art" systems all require enzymatic and purification steps following PCR. Prior to transformation, FLP-mediated PCR cloning will offer a decided advantage in that no in vitro manipulation is needed beyond the PCR reaction.