Nucleic Acid Hybridization

Nucleic acid hybridization involves bonding together short complementary nucleic acid strands (probes) to a target sequence. The probe is generally labeled with a radioisotope or fluorescent molecule, and the target sequence is typically bound to a nylon membrane or other solid surface. A positive hybridization signal is obtained when complementary base pairing occurs between the probe and the target sequence. After any unbound probe is removed, a positive signal is visualized by exposing the hybridized sample to X-ray film, in the case of radiolabeled probes, or by use of fluorescence microscopy in the case of fluorescent probes. The type of probe used and the way the probe is labeled determine the applications of nucleic acid hybridization techniques. Table 4.1 provides examples of probes that are used commonly to address specific questions about the presence and location of selected organisms in a soil community. Further details on this approach can be found in Amann et al. (1995). The GenBank (http://www.ncbi.nlm.nih.gov/) sequence database and the Ribosomal Database Project II (http://rdp.cme.msu.edu/html/) have tools to assist users to design hybridization probes for use in molecular soil ecology studies. Information about probes that have already been designed for specific purposes can be found at the ProbeBase Web site (http://www.microbial-ecology.net/probebase/).

Techniques based upon nucleic acid colony hybridization (colony blotting) have particular value in rapidly screening bacterial isolates for their identity, such as identifying specific rhizobia strains occupying root nodules or screening libraries containing DNA clones obtained from a soil community. Nucleic acid hybridization probes can also be used to detect specific phylogenetic groups of bacteria in appropriately prepared soil samples. In the latter application, a nucleic acid probe is fluorescently labeled and hybridized to target sequences contained within micro-bial cells in situ using the FISH technique. These protocols have been described extensively in reviews by Amann et al. (1995) and Amann and Ludwig (2000).

TABLE 4.1 Hybridization Probes Used in the Analysis of Soil Microbial Communities

Probe name

Target gene

Community target

Reference

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