PCR Mapping of LEU2 in Saccharomyces cerevisiae to Identify Deleterious Mutations

The LEU2 gene, a 1095 bp sequence, encodes 3-isopropylmalate dehydrogenase, an enzyme which plays a critical role in the leucine biosynthetic pathway.1 In the context of a three-part undergraduate genetics lab, Saccharomyces cerevisiae, baker’s yeast, was used as the model system on which the students performed genetic recombination because of the model’s high efficiency and simplicity. The yeast strain (EAY 235), which contained a deleterious mutation, was given to the students. The students were tasked with integrating the LEU2 gene as a linear fragment into the yeast genome through homologous recombination, and the results were scored for 235 LDO transformants. To determine the success of the recombination experiment, students amplified the yeast genome surrounding the LEU2 gene locus and performed PCR with known forward and reverse primers.2 The aim of our research is to determine the location of the ~ 450 bp deletion mutation in the LEU2 gene via the method of polymerase chain reaction (PCR) mapping. This procedure is ubiquitous for anyone attempting to define the location of an insertion or deletion within a known gene sequence and can be used to map a gene from the 5’ and 3’ ends. Using the known sequence of LEU2, primers were designed every hundred bases. Based on the primer data, the PCR fragments containing the deletion will be sent off for sequencing to confirm the location of the deleterious mutation. This research will then be used to develop the curriculum for future genetics labs.