A preliminary application of PCR-DGGE to study microbial diversity in soil
PCR-DGGE技术在农田土壤微生物多样性研究中的应用

Few traditional methods are applicable in study of the microbial community in the field of microbial ecology in recent years, since most of them rely on the microscope and cultivation of bacteria on laboratory media, and it had been proved that only about 1% of total number of actively respiring bacterial cells can be cultured on laboratory media. In addition, less than 1% bacterial species have been isolated and characterized from soil samples. As a new DNA fingerprinting technique, denaturing gradient gel electrophoresis (DGGE) can be used to analyze the microbial diversity in different environmental samples. Application of DGGE in this study had provided new ideas for study of the microbial community in soil. Seven soils in different farmland were sampled and their genomic DNA of microbial community were extracted directly in chemical method, which is based on lysis with a high-salt extraction buffer (1^5mol/L NaCl) and extend heating (2 to 3 h) of the soil suspension in the presence of sodium dodecyl sulfate (SDS), CTAB and proteinase K. For seven soils with different texture in this study, different DNA yields were obtained. After purification of the genomic DNA by DNA gel Recovery kit, the 16S rRNA genes (V3 region) were amplified by using the specific primers (F 357GC and R 518). The result of agarose gel (1.7%) electrophoresis showed that the PCR products were about 230 bp. These amplified DNA fragments were then separated by paralleled DGGE with the denaturant (urea and formamide) from 30% to 50%. The profile of DGGE showed that different soils had the different bands' patterns. DGGE could clearly separate the amplified fragments of 16S rRNA genes (V3 region) of different microbial community in soils, which make it possible to identify and define these 16S rDNA fragments by sequencing. For all DGGE profiles, there exist some common bands in all soil samples, which indicated that some kinds of microorganisms exist in each soil. On the other hand, the specific bands in some soils showed that different soils had the their own specific microorganisms. Compared to the traditional methods, DGGE could give more comprehensive information about the microbial community in soils. Our results showed that DGGE is a new and effective technique in analyzing microbial diversity.