Optimizing Direct Polymerase Chain Reaction (PCR) for Germinated Sporisorium ellisii teliospores, Mycelial Ball Cultures, and Fragments

Emily Nowicki,  Biology

Faculty Mentor:  Dr. Janet A. Morrison

              This summer my lab group continued our study of the grass Andropogon virginicus and the smut fungus Sporisorium ellisii, which infects populations of A. virginicus on the east coast.  On the west coast, this plant has eluded infection by S. ellisii.   The plant is classified as introduced in California and invasive in Hawaii.  In order to analyze the differences between A. virginicus populations, data must be recorded from each plant previously collected by Dr. Morrison.  This summer, I continued with this large scale project of processing each plant.  In addition, I collected data for an ongoing experiment in the greenhouse, studying the Evolution of Increased Competitive Ability hypothesis.

              One of the primary goals of our lab is to study the genetic differences of this fungus within a single plant, within a population of plants, and among populations of plants.  Currently, in order to obtain a genetic marker whose DNA can be sequenced, S. ellisii DNA must first be extracted from the teliospores of infected plants.  This process is very time consuming, requires a large sample of spores, and could potentially become costly when done on a large scale.  Designing and optimizing a new method (direct PCR) to use genetic markers without first extracting S. ellisii DNA is very desirable, and this effort has been my primary focus this summer.

              The protocol I have developed begins with spreading sterilized teliospores on plates of agar.  Germinated teliospores are then plucked from the plate after 24 hours, using an inverted microscope and a platinum tipped tool, and placed directly into PCR reaction tubes to amplify a specific sequence of DNA. The reaction products are run on agarose gels and bands of DNA are visualized with a computerized imaging system. Replicates of single spores, three spores, and six spores were run multiple times to test this procedure, with a 50 to 60 percent success rate.  Germinated teliospores also were transferred to new plates and incubated for seven to ten days and then transferred into flasks of nutrient broth.  These mycelial ball cultures were grown for a week, individually centrifuged, diluted and used in PCR.  Mycelial fragments were also centrifuged and diluted and used in PCR.  Both of these procedures yielded bands of DNA nearly 100 percent of the time.  This research will continue throughout the next academic year.  Ultimately, Dr. Morrison and I hope to publish a note about these new techniques of direct PCR on fungal spore germlings and mycelium.

Personal Statement

              This program has been a very positive and educational experience for me.  I learned new and extremely useful lab techniques, such as PCR and electrophoresis, and ways to troubleshoot the many problems a research scientist encounters.  I have also built stronger relationships with the Biology professors, as well as my colleagues in my department.  I feel that the many social opportunities, including the frisbee games and the bi-weekly lunches and tea hours really ameliorated this program.