Split-Marker-Mediated Transformation and Targeted Gene Disruption in Filamentous Fungi

Abstract

This article describes a split-marker approach, using two DNA fragments overlapping within the dominant selectable marker in fungi. This approach has been shown to increase homologous integration and thereby, facilitate targeted gene disruption in many fungi. Because the selectable marker gene is truncated in different fragments, the gene is not functional until homologous recombination takes place between two overlapping fragments. The truncated marker gene fragments flanking by homologous sequences of the target gene can be produced by two-round PCR without the need for cloning. The described method allows a faster and more efficient way of generating disruption strains and shall facilitate functional genomic analysis in filamentous fungi.