Simple identification of mutant clones.

Screening or selection of mutants is an essential step in genetics. Mutants are invaluable tools for the elucidation of biochemical pathways, for defining the roles of genetic units in a particular pathway and for understanding the relationship between enzyme structure and function (3). For mutant searches, there are two approaches: (i) positive selection for mutants that can grow under selective conditions or (ii) negative screening for mutants incapable of growth under specified conditions, such as auxotrophic, temperature-sensitive and UV-sensitive mutants (2,3). Positive selection is relatively easy and cost-effective, since high cell densities can be plated and the outgrowing mutants can be identified easily. In contrast, negative screening is quite time-consuming and laborious for several reasons. First, it requires a replica plating procedure, which enables the transfer of colonies from a master plate containing hundreds of colonies to plates for growth under both permissive and a nonpermissive condition (2,3). Second, usually only 200–300 colonies can be plated for effective comparisons of replicas grown under permissive and nonpermissive conditions. The identification of mutants is usually accomplished by either overlaying or placing side-by-side plates incubated under selective vs. nonselective conditions. The mutant clones are identified by their absence on the replica grown under selective conditions. When plates are crowded or the colonies are small, the procedure can be tedious and errorprone. The simple procedure described here takes advantage of the power of photo-imaging systems that are routinely used for capturing gel electrophoresis patterns of DNA, RNA and proteins. By obtaining high-resolution images that can be printed either on transparencies or paper, identification of mutants can be rapidly and accurately achieved. The use of this procedure for identifying temperature-sensitive mutants in yeast is described here. Wild type SacBenchmarks