Probing the Changes of Microbial Diversity with Land Use Changes on the Cumberland Plateau
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AbstractAlthough there has been a great deal of interest in cataloging and studying the biodiversity of life on the planet, bacterial biodiversity has largely been underestimated until recently because over 99% of all bacteria cannot be cultured. Hence, they could not be identified until scientists learned to identify and classify bacteria by sequencing bacterial 16S rDNA, the gene that encodes the 16S rRNA of the ribosome. Three protocols were adapted to break bacteria cells and release their DNA: Two freeze-thaw methods using either lysozyme or sodium dodecyl sulfate (SDS) did not yield repeatable results, whereas bead-beating produced the most consistent results. The efficiencies of bead-beating with FastPrep Instrument and the SPEX 8000 Mixer Mill to break soil bacterial cells were compared during the beating method; the extraction of bacterial DNA with the Mixer Mill was more effective at breaking soil bacterial cells. Universal and Pseudomonas spp. specific primers were used to amplify 16S rDNA from soil bacteria in polymerase chain reaction (PCR) after isolating DNA by bead beating. Comparison of DNA bands following gel electrophoresis demonstrated that universal bacterial DNA was amplified by PCR from all the different seasonal samples studied at the Cross Creek and Smith Tract, while Pseudomonas sp. DNA was amplified mostly in Smith Tract soil samples.
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