Geomicrobiological Properties and Processes of Travertine by Akihiro Kano & Tomoyo Okumura & Chizuru Takashima & Fumito Shiraishi

Author:Akihiro Kano & Tomoyo Okumura & Chizuru Takashima & Fumito Shiraishi
Language: eng
Format: epub
ISBN: 9789811313370
Publisher: Springer Singapore

4.5.2 PCR-DGGE

Denaturing gradient gel electrophoresis (DGGE) is an effective method to separate DNA fragments according to their mobilities under increasingly denaturing conditions. Separation using electrophoresis is normally performed on gel material containing a denaturing agent, where microbial taxa appear in separated bands. Advantage of this method is that the abundance of each microbe can be visually seen in the thickness of the band.

This method is usually applied to PCR products prepared by the following procedures. The eubacterial 16S rDNA fragments of each sample are amplified with the primer set of 357F-GC and 518R (Muyzer et al. 1993), which includes the V3 region of the 16S rRNA genes (corresponding to positions 341–534 in E. coli). The total volume of the reaction mixture is 25 μL, containing of 10 pmol of each primer, 10 mM of each dNTP mixture, Ex Taq buffer (20 mmol/L Mg2+), 2 U of Taq polymerase (Ex Taq; TaKaRa, Otsu, Japan), and 1 μL of extracted DNA. The thermocycling program consists of the following steps: initial denaturation at 94 °C for 6 min and 98 °C for 30 s, followed by touchdown primer annealing from 65 to 55 °C (1 °C reduction in annealing temperature every second cycle for the first 22 cycles, to touchdown at 54 °C), extension of the 22 cycles at 72 °C for 3 min, and a final extension at 72 °C for 5 min.

The DGGE analysis of PCR products is performed using the D-Code System (Bio-Rad Laboratories, Hercules, CA, USA) with 8% polyacrylamide gel (16 × 16 cm) and a denaturing gradient ranging from 30% to 70% in 1× TAE buffer (40 mmol/L Tris, 20 mmol/L acetic acid, 1 mmol/ L EDTA; pH 8.0). Electrophoresis is performed for 5 h at 60 °C with 130 V. The loaded samples are a mixture of 10 μL PCR product and 2 × DGGE loading buffer (Nippon Gene, Tokyo, Japan). After electrophoresis, the gel is stained in an ethidium bromide solution (0.5 μg/mL). Identified bands are excised with a sterilized knife and washed three times with sterilized water. The excised bands are amplified with the same primer set, and the same DGGE procedure is performed to confirm the formation of single bands in the proper positions. The confirmed amplicons are purified and sequenced by the procedure described in the previous section.

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