Gene Therapy: Lab Report

Introduction The purpose of this lab was to demonstrate the use of gene therapy on diseases that are caused by a single gene defect. This procedure was demonstrated on two different strains of baker’s yeast, EAY 235 and EAY 431, which both contained mutations in the LEU2 and TRP1 genes. Neither of these strains will grow without a proper medium that would supply both of these essential amino acids. The EAY 431 strain of yeast also contained a Rad 52 deletion, which caused EAY 431 to be a deficient, recombinant strain. The LEU2 gene is a linear fragment that does not contain an Autonomous Replication Sequence, so it could not replicate on its own and needed to be integrated by homologous recombination. The TRP1 gene was a circular plasmid that contained an ARS, which allowed for it to act as an extra chromosome in the gene. The objective was to insert a “wild gene” and replace the defective genes and then grow them on a medium that does not contain TRP1 or LEU2 to prove that the genes had been cured. To help determine if recombination took place in the LEU2 gene, and to compliment negative data from the 431 LEU2 drop out medium, the “cured” LEU2 gene was compared to the “diseased” LEU2 gene. The expectation was that the “cured” LEU2 gene would be a different size from that of the “diseased,” which would be proven through a PCR run of the two DNA strands after they were replicated under the same in vitro conditions. The purpose of the PCR was to show what kind of mutation occurred in the mutant to cause it to lose its LUE2 function.

Methods
Yeast Transformation Procedure Both hands and bench tops were sterilized by 10% ethyl alcohol and were continually wiped down at various times throughout the lab. Gloves were also worn for the duration of the lab to help prevent contamination. The first step was to obtain both strains of yeast, EAY 235 and EAY 431, with the fat end of a sterile tooth pick from an augur plate and place them into two separate Eppendorf