Applications of Nucleic Acid Hybridization in Microbial Ecology

Abstract

Nucleic acid hybridization techniques offer a new approach to answer old, intractable questions in microbial ecology as well as new questions. These include characterization of the predominant, yet unculturable populations in nature, the role of the environment in gene expression, and the extent of gene exchange among communities in nature. The essence of this methodology is the denaturing and annealing of complementary strands of nucleic acid molecules. The specificity of this hybridization reaction can be controlled such that only identical, or nearly identical, sequences in a complex mixture of nucleic acids extracted from a population or community can anneal. Labeled DNA or RNA sequences (probes) are introduced into hybridization reactions to identify and quantitate a particular organism containing the complementary target sequence. Methods for the recovery and purification of DNA from soils and sediments are given, as well as important considerations for the selection of probe and target sequences, and for the methods of detection and quantitation. Some advantages of this methodology include the abilities to: (1) detect populations without prior culturing of organisms, (2) detect specific organisms without the need for selectable markers, (3) detect multiple populations in the same analysis, and (4) detect genetic rearrangements or gene transfer in natural communities.

Hidden diversity among aquatic heterotrophic flagellates: ecological potentials of zoosporic fungi

“…In addition to providing linkage of cell identity to estimates of the abundance and biomass which is highly important for carbon flows and the related biogeochemical cycles in aquatic systems, the application of molecular technology to pelagic HF allows the detection of: (i) populations without prior culturing the organisms, (ii) specific organism without the need of selected markers, (iii) multiple populations in the same analysis and (iv) genetic rearrangement and genetic transfer in natural communities, (v) and the provision of proxies of population/community activity via RNA signals (Holben & Tiedje, 1988). Because HF are not a monophyletic group of organisms in contrast to ciliates (Patterson, 1993), the use of a single probe for their identification is not possible.…”

Interplay of Physical, Chemical and Biological Components in Estuarine Ecosystem with Special Reference to Sundarbans, India

“…Only a minor fraction (0. 1-10%) of the bacteria can be cultivated using standard techniques; (4) The biggest drawback in exploring bacterial biodiversity is that phenotypic methods can be applied only on bacteria which can be cultured and (4) it offers a very limited insight into the spatial distribution of the microorganisms (Pace et al, 1986;Holben & Tiedje, 1988;Ward et al, 1992;Amann et al, 1995). Microbial ecologists are turning increasingly to culture-independent methods of community analysis because of the inherent limitations of culture-based methods.…”

Characterization of Microbial Communities in an Anaerobic Fluidized Bioreactor Treating TNT Using Molecular Technologies

“…Nucleic acid hybridization techniques that use oligonucleotide probes to target regions of the 16S and 23S ribosomal ribonucleic acid (rRNA) can be used to identify microbial populations in the environment (Stahl et al 1988). One of the biggest advantages of this technology is the ability to detect and identify microbial populations without having to culture them (Holben and Tiedje 1988). The hybridization assay used in this study involves extraction of RNA from bioreactor samples, denaturation of the RNA, and immobilization of RNA on nylon membranes.…”