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Analysis of Bacterial Assemblage Genetic Diversity in Environmental Samples Using the DCode System

, NJ), 5 M bovine serum albumin, 200 M of each primer, 4 mm MgCl2, 1 l (60100 ng) template DNA, and 10 units of Stoffel Fragment Taq polymerase (Perkin Elmer).

Site-specific genetic profiles were produced by analyzing PCR products with DGGE in the manner described by Muyzer et al.1 Six percent acrylamide stock solutions were prepared containing 0 and 100% denaturant. The 0% denaturant stock was prepared by adding 15 ml of 40% acrylamide (Fisher Scientific) plus 2 ml of 50x Tris-Acetic acid-EDTA (TAE) buffer to 83 ml of distilled water. The 100% denaturant stock solution, defined as 7 M urea and 40% formamide, was prepared by mixing 15 ml of 40% acrylamide (Fisher Scientific) with 2 ml of 50x TAE buffer with 40 ml of molecular biology grade deionized formamide (Sigma Chemical) and 42 g of molecular biology grade urea (Sigma Chemical, St. Louis, MO). Stock solutions were degassed under vacuum for 510 minutes and filtered through 0.45 m filters. Parallel-style gels were cast using the Model 475 Gradient Former (Bio-Rad, Hercules, CA). Gels were run in a DCode unit in 1x TAE at 60 C for 912 h at 170 V (Bio-Rad Model 200/2.0 Power Supply). Gels were subsequently stained with a 1:10,000 dilution of Syber Green I (Molecular Probes, Eugene, OR). Gels were photographed in a dark room with a Nikon 35 mm camera and Ilford 400 black and white film and the film negatives were digitized with a Hamamatsu CCD camera (Hamamatsu, Japan). Image analysis of DGGE gels was performed using quantitative analysis software and lane intensity profiles measured by optical density highlighted the position of individual bands. Numerical representations of the profiles were exported to Microsoft Excel spreadsheet s
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