( Derek Cody and Laura G. ...)Analysis of Bacterial Assemblage Genetic Diversity in Environmental Samples Using the DCode System

Derek Cody and Laura G. Leff, Department of Biological Sciences, Kent State University, Kent, Ohio

Introduction
Assessing bacterial genetic diversity in natural environments is problematic because of difficulties in culturing native bacteria and the large number of species that may be encountered. The morphological and physiological traits of most microbes are ambiguous and provide few clues as to their identity; nucleic acid sequence comparison is the most fundamental way to classify microorganisms. Molecular techniques like the polymerase chain reaction (PCR)* have enhanced the ability to examine ribosomal genes of bacteria. Denaturing gradient gel electrophoresis (DGGE) has proven to be a powerful tool for analyzing PCR-amplified bacterial 16S rDNA fragments. The major advantage of DGGE is that it allows direct determination of bacterial genetic diversity, making it superior to cloning and subsequent sequencing.

Materials and Methods
Sediment samples were collected in triplicate from three sites in Four Mile Creek on the U.S. Department of Energys Savannah River Site near Aiken, South Carolina that differed in the extent of anthropogenic disturbance. DNA was extracted from samples using the Tsai and Olson method.² Briefly, 10 grams of sediment were mixed with 120 mM sodium phosphate buffer, then centrifuged at 6,000 x g for 10 minutes. Pellets were resuspended and incubated for 1.5 hours in 10 ml of lysis solution (0.15 M NaCl, 0.1 M Na₂ EDTA) containing 15 g/ml of freshly added lysozyme (Sigma Chemical, St. Louis, MO) and heated at 37 C to aid in dissolution. Two mg/ml of proteinase K (Sigma Chemical, St. Louis
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