Method for making recombinant yeast artificial chromosomes by minimizing diploid doubling during mating

A method of producing a desired recombinant YAC, comprising the steps of

• (a) obtaining a first haploid yeast cell comprising a first input YAC,
• (b) obtaining a second haploid yeast cell comprising a second input YAC,
• (c) obtaining a third haploid yeast cell comprising a linking YAC having a first homology region with the first input YAC and a second homology region with the second input YAC,
• (d) obtaining an intermediate haploid yeast cell comprising an intermediate recombinant YAC by meiotic homologous recombination between the linking YAC and the first input YAC, and
• (e) obtaining a final yeast cell comprising the desired recombinant YAC by meiotic homologous recombination between the intermediate YAC and the second input YAC, wherein steps (a), (b), and (c) are performed in any order wherein the steps (d) and/or (e) comprise the steps of
• (1) mating the first haploid yeast cell to the third haploid yeast cell in the case of step (d) to obtain a first diploid yeast cell and mating the intermediate haploid yeast cell to the second haploid yeast cell in the case of step (e) to obtain a second diploid yeast cell, said YACs being maintained without rearrangements prior to meiotic recombination by limiting mitotic doubling of the diploid to less than or equal to 8 doublings.