Soil DNA Extraction Methods And Application Of These Methods To Analyze Ammonia Oxidizer From Wetland Soils

DNA-based molecular biology techniques have widely been used as a powerful tool to understand the microbial community. In this paper, two new methods to extract microbial genomic DNA from wetland soil were established, namely Calcium Chloride/SDS/Enzymatic method and glass bead/calcium chloride/SDS method. Calcium chloride rather than EDTA chelating agent was used to remove humic acids in the process of direct DNA extraction. The extracting time is less than 4 hours.In comparing with other two methods, this method is more efficient in removing humic acids from wetland soil, and the purity of extracted DNA is higher which can be applied to 16S rDNA PCR amplification. It provides an efficient technology to extract and purify microbial genome DNA from soil for microbial ecological studies. However, the method is limited for studying microbial functional genes, based on the above reasons, we have to improve the calcium chloride/SDS/enzymatic method.Based on the dispersal of soils with glass beads is used to release ammonia-oxidizing microbes that are strongly adherent on soil colloids or located within the inner microporosity of soil aggregates and AOB's adsorption capacity on a solid surface, one commercial DNA purification kit and two laboratory-devised methods, a calcium chloride/SDS/enzyme DNA extraction method and an improved glass bead/calcium/chloride/SDS method, were used to extract DNA directly from soil. The amoA gene and 16S rDNA were amplified to estimate the effectiveness of the different DNA extraction procedures.The glass beads/calcium chloride/SDS extraction method was used to extract the total DNA from wetland soils, amoA gene fragments of the ammonia oxidizing bacteria and the ammonia oxidizing archaea were PCR-amplified, then the products were cloned and sequenced for constructing the phylogenetic tree. The size of AOB's PCR products is 647 bp. Phylogenetic analysis show that 19 sequencese of AOB can be divided into three clusters that were related to Betaproteobacteria Nitrosospira. The AmoA gene sequences indicated that Betaproteobacteria Nitrosospira of AOB may be the main nitrifiers in these four different soils. The size of AOA's PCR products is 647 bp. Phylogenetic analysis show that 20 sequencese of AOA which belong to uncultured Crenarchaeota can be divided into six clusters, but can not determine the specific genus.It provides the basic data for better understanding the ammonia oxidation process of natural wetlands.