Parameters affecting the frequencies of transformation and co‐transfromation with synthetic oligonucleotides in yeast

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Abstract
Factors influencing the direct transformation of the yeast Saccharomyces cerevisiae with synthetic oligonucleotides were investigated by selecting for cyc1 transformants that contained at least partially fuctional iso‐1‐cytochrome c. Aproximately 3 × 104 transformanrs, constituting 0·1% of the cells, were obtained by using 1 mg of oligonucleotide in the reaction mixture. Carrier, such as heterogenous oligonucleotides, enhanced transformation frequencies. Transformation frequencies were dramatically reduced if the oligonucleotides had a large number of mismatches or had terminally located mismatches. Transformation with oligonucleotides, but not with linearized double‐strand plasmid, was efficient in a rad52− strain, ssuggesting that the pathway for transformation with oligonucleotides is different from that with linearized double‐strand plasmid. We describe a procedure of co‐transformation with two oligonucleotides, one correcting the cyc1 defect of the target allele in the host strain, and the other producing a desired amono acid alteration elsewhere in the iso‐1‐cytochrome c molecule; approximately 20% of the transformants obtained by co‐transformation contained these desired second alterations.