DNA cloning using in vitro site-specific recombination

As a result of numerous genome sequencing projects, large numbers of candid ate open reading frames are being identified, many of which have no known f unction. Analysis of these genes typically involves the transfer of DNA seg ments into a variety of vector backgrounds for protein expression and funct ional analysis. We describe a method called recombinational cloning that us es in vitro site-specific recombination to accomplish the directional cloni ng of PCR products and the subsequent automatic subcloning of the DNA segme nt into new vector backbones at high efficiency. Numerous DNA segments can be transferred in parallel into many different vector backgrounds, providin g an approach to high-throughput, in-depth functional analysis of genes and rapid optimization of protein expression. The resulting subclones maintain orientation and reading frame register, allowing amino- and carboxy-termin al translation fusions to be generated. In this paper, we outline the conce pts of this approach and provide several examples that highlight some of it s potential.