Method for increased gene targeting.

<p>Cells are grown in the presence of hydroxyurea to induce cell cycle arrest in S-phase with high HR activity (a). <i>Y</i>. <i>lipolytica</i> YB-392 cells untreated or arrested at the large-budded stage are shown. HU-arrested cells are transformed with an antibiotic resistance cassette bearing the marker flanked by short regions of homology to the promoter and terminator of the target gene (b). Homologous recombination between the cassette and genomic DNA leads to replacement of the target gene with the marker (c). Antibiotic-resistant colonies are screened by PCR to distinguish between random and targeted integration using primer sets specific to each integration outcome (d).