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"Samples are divided into analytical replicates 1 and 2 for each method.

"Samples are divided into analytical replicates 1 and 2 for each method.

among many other applications. This technique need not be restricted to studying the 16S rRNA gene. T-RFLP can be used as a quick screen for any gene for which specific primers can be devised to look at differences between communities in environmental samples, such as nifH to compare populations of nitrogen-fixing bacteria or amoA to study ammonia-oxidizing bacterial populations in soil (Thies, in press). The main drawback of the use of this approach is the inability to characterize TRFs further or obtain sequence information, as the sample is lost shortly after it is sized. However, once profiles are compared, the original PCR products from samples of interest can be used for cloning and sequencing experiments as described above.

T-RFLP often yields a higher number of OTUs for use in comparative analyses than DGGE (Table 4.4). However, all of these techniques yield numbers of OTUs that do not come close to the estimates of extant diversity in soil populations as estimated by DNA:DNA reassociation kinetics (discussed previously). Hence, we are still viewing the tip of the iceberg as far as characterizing soil microbial diversity with these higher throughput DNA fingerprinting techniques.

A new approach, which combines gel separation of PCR amplicons of the ITS regions by size in a nondenaturing gel and then by melting characteristics in a second, denaturing gel has been developed (2D-PAGE; Jones and Thies, in press). This approach yields an order of magnitude greater number of OTUs than DGGE alone and three times the number of OTUs obtained by use of T-RFLP or automated ribosomal intergenic spacer analysis (ARISA) (Table 4.4). The disadvantage of this technique is that it is more laborious, therefore it does not lend itself to high sample throughput. Yet, its improved ability to discriminate between soil communities and retrieve sequence information make it a powerful technique for elucidating key differences in community structure between studied samples.

Several additional PCR fingerprinting techniques target the ribosomal gene sequences. Ribotyping makes use of differences in the chromosomal positions or structure of rRNA genes to identify or group isolates of a particular genus or species. Ribotyping has been shown to be reproducible and hence has gained popularity for isolate fingerprinting and has found use in bacterial source tracking and other studies in which the similarity of isolates obtained from different samples needs to be compared. The most frequently used ribotyping method is to identify RFLPs of rRNA genes by probing a Southern transfer of genomic DNA that has been hydrolyzed with an endonuclease. In ARDRA, rRNA gene sequences are amplified. In ARISA, the ITS region is amplified. PCR amplicons resulting from use of both methods are hydrolyzed subsequently with restriction enzymes and the resulting variations in restriction fragment sizes are analyzed on a gel. Bands in the gel are again termed OTUs and similarities and differences between the fingerprints from different samples are analyzed using multivariate techniques. Use of ARISA may yield more OTUs from a given sample, but as the number of bands on the gel increases, so does the difficulty one has in resolving individual bands in the analysis.

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