A 2-microm DNA-based marker recycling system for multiple gene disruption in the yeast Saccharomyces cerevisiae

A molecular FRT (Flp recombinase recognition target)-based cassette system for multiple gene disruption in the yeast Saccharomyces cerevisiae was developed. FRT DNA sequences were designed with different core mutations and subsequently cloned in direct orientation upstream and downstream of a marker gene to serve as template for the amplification of a set of different gene disruption cassettes. After each disruption, the marker can be easily eliminated from its integration site by in vivo site-specific recombination between the two identical, mutated FRT sequences flanking the marker, leaving behind one FRT sequence with a particular point mutation. Since recombination between two FRTs with a different core mutation is extremely rare, the possibility of chromosome rearrangements, due to site-specific recombination between residual FRTs, is very low. In strains containing 2-microm ([cir+]) the site-specific reaction is catalysed by the endogenous Flp gene product, whereas in strains without 2-microm ([cir0]), the FLP gene is carried on the cassette, together with the marker gene. This system can be applied for haploid and diploid [cir+] and [cir0] strains.