Measuring microbial diversity typically involves sequencing individual 16S rDNA gene se­quences, to obtain species-level resolution. The power of the approach comes from the ability to amplify, clone, and analyze homologous regions of 16S rDNA from small amounts of sample DNA. Some studies focus on only a small portion of the 16S rDNA, while others survey the entire gene sequence. Sequencing the entire 16S rDNA enhances the accuracy of estimating the species variability of microbial communities, and is preferred when broad comparative determinations are preformed.

The question of whether two populations of microorganisms have different numbers of species and the level ofgenetic diversitycan be answered bycomparing the relationships and degrees of divergence among sequences. Depending on amplification and cloning conditions the technique can overrepresented some species while underrepresenting others. There is also a relatively small potential for error from sequence variation due to PCR replication errors. Variations of the 16S approach include denaturing gradient gel electrophoresis and in situ hybridization.

Bacterial populations, including soil communities, have been characterized using rRNA intergenic spacer analysis (RISA) which determines the variability in the length of the in­tergenic spacer between the small (16S) and large (23S) ribosomal subunits. The method has been automated (ARISA), and the sensitivity increased by the use of fluorescence — tagged oligonucleotide primers for PCR amplification and for subsequent electrophoresis in an automated capillary electrophoresis system. Fungal ARISA makes use of the length polymorphism of the nuclear ribosomal DNA (rDNA) region that contains the two in­ternal transcribed spacers (ITS) and the 5.8S rRNA gene. Comparative sequencing and quantification of rRNA genes using universal and phylogenetically specific primers has become an established method for detecting and characterizing subgroups of prokary­otic and eukaryotic microorganisms. For instance, constructed 16S and 18S rRNA gene libraries have been constructed to examine the effects of elevated CO2 levels on the composition of microbial communities associated with the rhizosphere of trembling aspen (88). However, a thorough analysis of complex microbial communities using rRNA gene-based libraries requires a huge sequencing effort. To overcome this sequenc­ing limitation, several rapid high-throughput DNA-based molecular methods for rRNA gene-based analysis of microbial communities have been developed. These methods, includ­ing terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE), provide a DNA fingerprint of the microbial community present in the sample (89-92). While T-RFLP and DDGE are sensitive methods for differentiating between microbial communities, these methodologies do not provide actual DNA sequence information unless the resolved bands are recovered and sequenced (93).