We recommend procuring an oligonucleotide batch large enough to conduct an entire project. This should help to avoid any DGGE profile variations due to performance differences between repeat syntheses of GC-clamp oligonucleotide primers. Surveys of a range of environments such as soil, oceans, dental flora, the human gastrointestinal tract, and skin have revealed a bacterial diversity much higher than previously speculated (Janssen, 2006; Ley et al., 2006; Azam & Malfatti, 2007; Fierer et al., 2010;Kolenbrander et al., 2010). Early studies on the diversity of bacterial DNA from forest soil indicated click here a large discrepancy between

culture-based and culture-independent diversity (Torsvik RNA Synthesis inhibitor et al., 1990). These discoveries lead to a paradigm stating that the majority of bacteria cannot be cultured (Rappe & Giovannoni, 2003). Thus, bacterial communities are now characterized by a variety of culture-independent approaches, mostly consisting of

information derived from 16S rRNA gene sequences. Using 16S rRNA gene clone libraries to identify individual bacteria in mixed populations has been a popular tool (Beja et al., 2002; Elshahed et al., 2008). The increasing availability of high-throughput sequencing, particularly pyrosequencing, is driving migration to these more comprehensive approaches and revealing even higher bacterial diversity (Dowd et al., 2008). Because of the expense and time-consuming nature

of these inclusive techniques, the need remains for less intensive methods of interrogating the microbial biodiversity present in specific samples. Alternative techniques for characterizing microbial communities include terminal-restriction fragment length polymorphism, automated rRNA intergenic spacer analysis, denaturing gradient gel electrophoresis (DGGE), and temperature gradient gel electrophoresis (Fromin et al., 2002; Marzorati et al., PDK4 2008; Kovacs et al., 2010). These techniques have often been referred to as fingerprinting methods and provide a ‘snapshot’ of the overall structure and diversity in microbial populations (Nakatsu, 2007). They have proven to be particularly useful in comparative studies, such as detecting changes over time and effects of the addition or subtraction of substances on shifts in microbial community composition (Muyzer & Smalla, 1998; Fromin et al., 2002). The use of DGGE has proven to be one of the most popular methods for determination of microbial diversity (Muyzer & Smalla, 1998; Fromin et al., 2002; Yu & Morrison, 2004; Brons & van Elsas, 2008). DGGE, as used in molecular microbial ecology, is based on a series of discoveries and modifications since 1983. DNA duplex fragments of similar size migrate through an acrylamide matrix with constant mobility, but dissociation of the two strands leads to a considerable decrease in mobility through the gel.