Fragmentation of large sequences for allele transfer. (A) When the mutation is close to the 3′ end of a large gene (>2.5 kb), a new adaptamer Aint is needed to amplify the 3′ portion of the ORF. After amplification using adaptamerA int and adaptamer B, the fragment is fused to K. lactis URA3 fragments and cotransformed into yeast as described in Figs. 2 and 3. Integration results in a full-length ORF only in the left copy following recombination that fuses the promoter (purple box labeled Pro) and the endogenous, nonamplified region of the ORF (shaded box) with the duplicated 3′-amplified fragment (open box). The fragment on the right is truncated upstream of the sequence homologous to adaptamer Aint. (B) When the mutation is close to the 5′ end in long essential genes, allele transfer requires two new adaptamers. Adaptamer Apro and adaptamer Bint are used to amplify the 5′ portion of the ORF including the promoter. After fusion to K. lactis URA3 and cotransformation (Figs. 2 and 3), integration results in the generation of the full-length ORF with its promoter in the right repeat. The left copy contains a 3′ truncation downstream of the sequence homologous to adaptamer Bint.
