FIG. 3.

Methods to create new alleles. (A) Gene disruption by recombination with a plasmid containing a leu2 copy deleted at both the 5′ and 3′ ends. This results in a duplication, where both copies are mutated. This duplication can be obtained by selecting with the URA3 marker. (B) The pop-in/pop-out method. This two-step method requires first the integration of a plasmid with a mutated copy of leu2 (pop-in, selected for with the URA3 marker) and then the excision of the plasmid (pop-out, selected for as loss of URA3) leaves only one copy of leu2, which can be the original one or the mutated one (the case shown here). (C) One-step gene replacement. Some of the Ura⁺ transformants have also integrated the mutation in leu2 and become Leu⁻. (D) One-step method of gene knockout. Most of the central part of the gene is replaced by the selectable URA3 marker.