( ng unique sequences from mix...)DGGE Analysis for Non-Invasive Studies of Primate Diet

ng unique sequences from mixed template PCR amplifications. Analyses of eighteen plant species consumed by captive animals showed that DGGE permitted resolution of 15 of the 18 species, or over 80% (Figure 3). The three unresolved species possessed identical sequences (unpublished) in the melting domain under examination, and thus differentiation could not be expected.

Additional results, from samples obtained from captive animals whose diets were known, indicate that, on average, only about 30% of all plants ingested by the monkeys were detected by PCR/DGGE. Further experiments on constructed plant mixtures confirmed that a moderate amount of amplification bias occurs in PCR amplifications of mixed templates (Figure 4). Experiments to assess the preferential amplification observed in both the control fecal samples and the constructed plant mixtures suggest multiple factors at play, including template concentration and differential primer fidelity to various templates.

These data suggest that the PCR/DGGE approach tested in this investigation should be useful in situations where a universal detection system is not required, or where nothing is known about the diet of an animal. Further optimization of the technique should permit greater fragment resolution and enhanced sensitivity. Yet, any future investigations utilizing these methods should carefully consider the effects of PCR bias (Reysenbach et al. 1992, Ferris et al. 1996, Suzuki and Giovannoni 1996).

References
1. Ferris, M. J., Muyzer, G. and Ward, D. M., Applied and Environmental Microbiology, 62, 340346 (1996).

2. Reysenbach, A., et al. Applied and
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