The S. cerevisiae strains that we are using this semester were constructed by the Saccharomyces Gene Deletion Project. In the project, yeast investigators systematically replaced every ORF in the yeast genome with a bacterial kanamycin resistance (KANR) gene. In this lab, you will use the polymerase chain reaction (PCR) to identify the disrupted MET genes in your deletion strains. Thermostable DNA polymerases (above) play a key role in PCR.
At the end of this lab, students should be able to:
- describe the reactions that are occuring at the different temperatures used in PCR cycles.
- design oligonucleotide primers to amplify sequences with PCR.
- explain how changes to the annealing temperature and extension time affect the production of PCR products.
- design and carry out a PCR strategy that distinguishes three met deletion strains.
In a previous lab, you used different culture media to distinguish between different S. cerevisiae met mutants. Your results may have allowed you to tentatively identify your strains. In this lab, you will use the polymerase chain reaction (PCR) to more conclusively identify the mutant strains. This chapter begins with an overview of the PCR and the Saccharomyces Gene Deletion Project. You will use this knowledge to design and carry out a strategy for identifyingmet deletion strains by yeast colony PCR.