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The Optimization of Pulsed Field Gel Electrophoresis for Use with Profiling the Freshwater Viral Community
Glasner, Dustin Robert
Glasner, Dustin Robert
Abstract
Pulsed field gel electrophoresis (PFGE) has proven to be a useful tool for fingerprinting viral communities in environmental samples. PFGE has the ability to separate larger DNA segments, and it provides sharper resolution and better band separation than standard gel electrophoresis. Since virus genomes are essentially long segments of DNA, the ability to separate larger molecules is vital; as such, PFGE can provide a proxy measure of viral richness through genome size distribution. Despite its documented usefulness, however, PFGE has not been shown to work flawlessly with all samples -- especially those from freshwater environments. For samples taken from Lake Matoaka at the College of William & Mary, PFGE has produced non-distinct smearing and unclear banding patterns, limiting its use as a fingerprinting tool. Experiments were run with single phage isolates (species T4, λ, and CrimD) to determine the viral load at which PFGE ceases to produce clear banding. Pulsed field runs with these phage dilutions showed that a minimum of 10^7 viruses must be loaded into a given well of the gel in order to produce a distinguishable band. Artificial phage assemblages were also created using mixtures of T4 and λ. When run on a gel, these mixtures demonstrated that, so long as viruses are loaded at the threshold of detection, distinct bands will be visible, even in the presence of another virus. However, additional problems may arise in interpreting banding patterns due to DNA topology. The work carried out here more clearly illustrates the limitations of PFGE in fingerprinting aquatic viral assemblages, though further work must be done to gain even deeper insight into the method's usefulness.
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Thesis is part of Honors ETD pilot project, 2008-2013. Migrated from Dspace in 2016.
Date
2010-05-17
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Keywords
Pulsed Field Gel Electrophoresis, Environmental Virology, Freshwater Ecosystems, Lake Matoaka
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Department
Biology