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Chapter 61 : Identifying Microorganisms Involved in Specific In Situ Functions: Experimental Design Considerations for rRNA Gene-Based Population Studies and Sequence-Selective PCR Assays
Category: Environmental Microbiology
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This chapter examines experimental design considerations for a population-based approach for identifying microorganisms involved in specific in situ functions. Although this chapter focuses on a particular population-based approach, many of the experimental design considerations discussed here apply to a wide range of rRNA gene-based population studies and sequence selective PCR assays. This chapter examines an experimental approach that uses the population-based strategy. The approach has the following three phases: (i) identifying populations of rRNA genes whose abundances correlate with the functional parameter, (ii) validating the rRNA gene correlates identified in phase I by using an independent quantitative assay, and (iii) isolating the microorganisms identified by the rRNA gene correlates and reintroducing them into the environment to assess their functions in situ. This approach was recently used to identify microorganisms that suppress the population development of the plant parasitic nematode Heterodera schachtii in southern California soil. Functional gradients are created by manipulating the microbial community with methods such as differential heat treatments, targeted antimicrobial agents, and nutritional amendments. Microbial community composition is examined by rRNA gene analysis. Nucleotide sequence analysis of rRNA gene clone libraries can be used to generate detailed depictions of microbial community composition. PCR assays can be validated by using them to amplify DNAs extracted from different environmental samples and then cloning and sequencing several randomly selected clones from each sample. The assays can be considered selective if they exclusively amplify the target sequence.
Experimental strategy for OFRG.
rRNA operon and positions of PCR primers.
Effect of annealing temperature on a PCR assay.
Effect of template concentration on mismatch discrimination of a PCR assay.