| Title | Development of Molecular Biological Methods to Analyze Bacterial Species Diversity in Freshwater and Soil Ecosystems |
|---|---|
| Author | Dong-Hun Lee *, Sung-Ae Noh, and Chi-Kyung Kim |
| Address | Department of Microbiology and Research Institute for Genetic Engineering, Chungbuk National University, Cheongju 361-763, Korea |
| Bibliography | Journal of Microbiology, 38(1),11-17, 2000, |
| DOI | |
| Key Words | PCR, SSCP, 16S rRNA, diversity analysis, phenol-degrading bacteria |
| Abstract | A new method was developed for the rapid analysis of diverse bacterial species in the natural envi-ronment. Our method is based on PCR-single-strands-conformation polymorphism (PCR-SSCP) and selective isolation technique of single-stranded DNA. Variable V3 fragments of 16S rDNA were amplified by PCR with bacterial 16S rDNA primers, where one of the primers was biotinylated at the 5'-end. The biotinylated strands of the PCR products were selectively isolated by using streptavidin paramagnetic particles and a magnetic stand, to prevent SSCP analysis producing heteroduplexes from heterogeneous DNA samples. The selected strands were separated by electrophoresis on a polyacrylamide gel, and detected by silver staining. Analysis of PCR products from 8 bacterial strains demonstrated their characteristic DNA band patterns. In addition, changes in the structure of the bacterial community and species diversity in the microcosm treated with phenol could be monitored. After 3 weeks of incubation, phenol and its intermediate, 2-hydroxy-muconic-semialdehyde, were degraded by indig-enous bacteria. These dominating bacterial populations were identified as strong bands on an SSCP gel. Therefore, this study provides useful tools for microbial community analysis of natural habitats. |
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