Effect of chromosomal locus, GC content and length of homology on PCR-mediated targeted gene replacement in Saccharomyces

Targeted gene replacement (TGR) using fragments generated by PCR is a widely-used technique for deleting genes in Saccharomyces cerevisiae. We found that the efficiency of this procedure, defined as the fraction of transformants that delete the targeted gene, varied by >10-fold depending on the sequence being targeted. We examined the effect of chromosomal position, length of homology and GC content on TGR efficiency. When URA3 was positioned at five different chromosomal locations, the efficiency of replacing this gene with LEU2 remained the same. Similarly, varying the length of homology from 35 to 60 bp had only a small effect on the efficiency of targeting (<50%), though an increase in the length of homology to 200 bp on one end of the disruption fragment did increase TGR efficiency. Strikingly, as GC content in the target sequence increased, the efficiency of targeting also increased. When TGR efficiency was high, the frequency of untargeted integration events was low. These results suggest two strategies for designing TGR primers: (i) use 40 bp targeting sequences containing 40–50% GC, and (ii) if necessary, increase TGR efficiency by extending the length of homology on one end of the disruption fragment.