( . Mater...)DGGE Analysis for Non-Invasive Studies of Primate Diet

Materials and Methods
Genomic DNA was extracted from fecal samples and reference plants, and the chloroplast rbcL gene amplified via the PCR with one GC-clamped primer. PCR mixtures contained 1x PC2 PCR buffer (Ab Peptides), 0.1 units/l Klentaq DNA polymerase (Ab Peptides), MgCl₂ (5.0 mM), 0.25 M each primer, and 1.5% dimethyl sulfoxide. Thermal cycling conditions were: an initial denaturation at 94 C for four minutes, 3 cycles of 94 C, 1 minute; 62 C, 1 minute; 80 C, 45 seconds; 4 cycles of 94 C, 1 minute; 64 C, 1 minute; 80 C, 45 seconds; 32 cycles of 94 C, 1 minute; 68 C, 1 minute; 80 C, 45 seconds; and a 5 minutes final extension at 80 C. PCR products were subsequently subjected to DGGE, using the DCode universal mutation detection system from Bio-Rad, to resolve the various plant sequences produced by PCR amplification.

To determine the melting temperature of PCR products, they were first analyzed on a perpendicular denaturing gradient gel poured with Bio-Rads Model 475 Gradient Former. The gel was 6.5% 37.5:1 acrylamide/bis-acrylamide, and contained a gradient of 1100% denaturant. Approximately 100 l of PCR product were loaded on a gel, and electrophoresis performed at 130 V and 60 C for 3 hours (Figure 2).

Optimal resolution of fragments on subsequent parallel gels was obtained with gradients ranging from 3040% denaturant, with electrophoresis at 130 volts and 60 C for approximately 4 hours (Figure 3). Gels were stained with ethidium bromide, and visualized by UV-fluorescence.

Results and Discussion
Our results indicate that DGGE is an effective technique for resolvi
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