Molecular phylogenetic analysis of uncultivated organisms

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Most of what we know about the microbial world was (and is being) learned by examination of cultivated organisms. But most microbial species are not readily cultivatable; the often-quoted number is "less than 1%", and the real number is probably many, many orders of magnitude less than 1%. This means inevitably that you end up examining unrepresentative organisms if you rely on cultivation. Inferences from these species are unlikely to be realistic; imagine how poorly we would understand plants if we could only study a handful of weeds.

Molecular phylogenetic analysis can solve this problem, because cultivation is not required. Molecular phylogenetic analysis can be performed on isolated (cultivated or not) material, or even of microbial populations. Molecular phylogenetic analysis of uncultivated organisms is similar to analysis of cultivated species, but starts with DNA extracted directly from a purified sample or the environment, rather than from a pure culture:

  1. Extract DNA directly from purified sample or an environmental sample
  2. Amplify rRNA genes by PCR using universal primers
  3. Clone, sequence, & tree the amplified genes
  4. Use these sequences to design specific hybridization probes to test the occurence, abundance, morphology, etc, of the specie in the population

the Search

Some uses of molecular phylogenetic analysis in microbial ecology:

  • Identify the predominant microbial groups in a population
  • Count sequences from various phylogenetic group to assess their abundance in the ecosystem
  • Assess enrichments aimed at cultivating organisms previously identified by rRNA sequence.
  • Use fluorescently-labeled rRNA sequences (oligonucleotides) as probes for the identification orenumeration of specific organisms or groups in environmental samples.
  • Identification of unculturable organisms. e.g. the identification of uncultivatable pathogens in tissue samples.