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Chapter 12 : Molecular Approaches for the Measurement of Density, Diversity, and Phylogeny

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Abstract:

This chapter considers the use of molecular methods for direct measures of abundance, diversity and phylogeny of environmental populations of microorganisms. These molecular methods are mainly based on direct nucleic acid sequence recovery, genomic DNA hybridization, and nucleic acid fingerprinting. There are three basic formats now used to recover DNA sequence information isolated from either pure culture or environmental samples: DNA probe hybridization, restriction enzyme digestion, and chain termination sequencing of cloned (or PCR-amplified) DNA templates. Restriction fragment length polymorphism (RFLP) analysis has been used to characterize extracted total DNA or specific PCR-amplified DNA. Sequence diversity is evaluated by digesting the native or amplfied DNA with a restriction endonuclease(s) followed by size fractionation by electrophoresis on an agarose or a polyacrylamide gel. Genomic DNA hybridization is mainly used in bacterial systematics to determine the degree of genetic similarity between genomic DNA sequences and thus provides limited information of specific sequence content. Molecular fingerprinting methods can be used for rapid surveys using genes that provide for either phylogenetic or functional assessment of populations present in an environmental sample. Terminal RFLP (T-RFLP) of PCR-amplified DNAs is a refined fingerprinting technique based on RFLP. The general steps include PCR amplification of a conserved target sequence (most commonly a region of the small-subunit (SSU) rRNA gene) followed by restriction enzyme digestion and gel fractionation of resulting fragments.

Citation: Liu W, Stahl D. 2007. Molecular Approaches for the Measurement of Density, Diversity, and Phylogeny, p 139-156. In Hurst C, Crawford R, Garland J, Lipson D, Mills A, Stetzenbach L (ed), Manual of Environmental Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815882.ch12

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Denaturing Gradient Gel Electrophoresis
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FIGURE 1

Partial listing of group-specific probes in relationship to prokaryote phylogeny. The phylogeny was adapted by Stahl ( ) using the maximum likelihood analysis of Olsen, Woese, and Overbeek ( ) to provide the relative branching order of the major prokaryotic lineages. The probes for the indicated target groups have been described: archaeal domain ( ), bacterial domain ( ), archaeal kingdoms ( ), gram-positive organisms (S. Toze and D. Stahl, unpublished data), low-G+C gram-positive organisms ( ), high-G+C gram-positive organisms ( ), and ( ), proteobacterial subdivisions ( ), sulfate-reducing bacteria ( ), methylotrophic bacteria ( ), and nitrifying bacteria ( ). The reader is referred to these references for more complete descriptions of probe design and characterization.

Citation: Liu W, Stahl D. 2007. Molecular Approaches for the Measurement of Density, Diversity, and Phylogeny, p 139-156. In Hurst C, Crawford R, Garland J, Lipson D, Mills A, Stetzenbach L (ed), Manual of Environmental Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815882.ch12
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Image of FIGURE 2
FIGURE 2

Typical curves obtained by reassociation of nucleic acids. The method can be used to estimate genome size or number by comparison to a reference genome represented by the left curve. The left and middle curves display pseudo-first-order kinetics of unique sequence DNA of different complexities, while the right curve displays pseudo-second-order kinetics.

Citation: Liu W, Stahl D. 2007. Molecular Approaches for the Measurement of Density, Diversity, and Phylogeny, p 139-156. In Hurst C, Crawford R, Garland J, Lipson D, Mills A, Stetzenbach L (ed), Manual of Environmental Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815882.ch12
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Tables

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TABLE 1

Molecular fingerprinting methods for microbial community identification, monitoring, and tracking

Citation: Liu W, Stahl D. 2007. Molecular Approaches for the Measurement of Density, Diversity, and Phylogeny, p 139-156. In Hurst C, Crawford R, Garland J, Lipson D, Mills A, Stetzenbach L (ed), Manual of Environmental Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815882.ch12

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