Optimized primers and other critical conditions for efficient fusion PCR to generate knockout vectors in filamentous fungi

Figure 1. Diagram of a split-marker gene replacement strategy using fusion-PCR (A) FPCR step 1 amplification of flanking regions and partial marker segments. Dotted tails indicate the overlap sequences on selected primers which allow fusion in step 2. (B) FPCR step 2 flanking regions are fused to the marker segments at the overlap sights, forming two constructs. The fusion products are then amplified. (C) Transformation. Three crossovers occur during the gene replacement. Each genomic flanking region crosses over with its complementary sequence in the FPCR constructs. The complementary portions of the marker segments crossover to make the completed selectable marker. Optimized primers and other critical conditions for efficient fusion PCR to generate knockout vectors in filamentous fungi.