Using molecular tools to assess microbial diversity

Abstract

Although the chemical balance of the biosphere depends on microbes, little is known about the makeup of the microbial ecosystems responsible for maintaining this balance. This project aims to assess the effects of different plant communities and nutrient availability on the microbial diversity in soil samples from a local prairie restoration project. This will be accomplished using the molecular technique, automated ribosomal intergenic spacer analysis (ARISA). It is a PCR-based method involving the amplification and anlysis of an intergenic region of bacterial genomes. This genomic region displays significant heterogeneity in length among bacterial species and can be used to distinguish bacterial strains. DNA has been isolated from over 360 soil samples from various test plots. Optimal PCR conditions were determined and the intergenic region of each sample was amplified. The specificity of the annealing temperature, the number of PCR cycles and the use of a hot start with the Taq polymerase enzyme were optimized to obtain specific PCR products. Successful DNA isolation and PCR were verified using agarose gel electrophoresis. Each PCR product will be subjected to ARISA and the date used to estimate the microbial diversity in the soil from each test plot.