Cloning-free PCR-based allele replacement methods

Efficient homologous recombination permits the directed introduction of specific mutations into the yeast genome. Here we describe a cloning-free, PCR-based allele replacement method that simplifies allele transfer between yeast strains. The desired allele from one strain is amplified by PCR, along with a selectable/counterselectable marker. After transformation, the resident allele in the target strain is replaced by creating a duplication of the new allele. Selection for direct repeat recombinants results in a single copy of the new allele in the target strain. Specifically, the desired allele is amplified by PCR with a pair of adaptamers, which are chimeric oligonucleotides that are used to amplify the allele and differentially tag its 5' and 3' ends. These tags allow the directed fusion to two different, but overlapping, regions of an appropriately tagged selectable/counterselectable marker after a second round of PCR amplification. Following cotransformation of the two fusion fragments into yeast, homologous recombination efficiently generates a duplication of the amplified allele flanking the intact selectable marker in the genome. After counterselection, only the desired allele is retained as a result of direct repeat recombination. A simple modification of this method allows the creation of de novo mutations in the genome.