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Microbial Diversity in Ground and Surface Water Analyzed by Denaturing Gradient Gel Electrophoresis Using the DCode System

-Elmer Corp.), 10 l DNA solution. The temperature cycle for the PCR was 60 seconds of denaturation at 94 C, 60 seconds of annealing (see below), and 90 seconds of primer extension at 72 C. During an initial touchdown cycle, the annealing temperature was lowered from 65 C to 55 C in intervals of 1 C per cycle. The additional annealing cycles were done at 55 C. Ten PCR cycles were performed for the touchdown procedure and then 20 additional cycles at the actual annealing temperature of 55 C. This touchdown procedure reduces the formation of spurious by-products during the amplification process.4, 5 DGGE was performed with the DCode system. The polyacrylamide gels (7.5%) contained a denaturing gradient from 40 to 70% (100% denaturant: 7 M urea and 40% formamide) and were run in 0.5x TAE buffer (40 mM Tris base, pH 7.4, 20 mM sodium acetate, 1 mM EDTA). Gels were poured with a stacker on top (7.5% polyacrylamide, no denaturant). The optimum denaturant gradient was determined by performing perpendicular DGGE according to the instruction manual (Bio-Rad). Electrophoresis was performed at constant voltage (70 V) and temperature (57 C) for 16 hours. After electrophoresis, the gels were stained with silver nitrate,6 dried and photographed.


Results and Discussion

Isolated and PCR amplified DNA from surface water is shown in Figure 1. Separation of 16S rDNA amplified fragments by DGGE revealed the great diversity in ground and surface water samples. Complex banding patterns could be observed in samples with different hydrochemical conditions. These patterns were characteristic for each sample and showed the differences and common features in species compositi
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