An Improved Method for Soil DNA Extraction to Study the Microbial Assortment within Rhizospheric Region

The need for identification of soil microbial community mainly depends on direct extraction of DNA from soil, a multifaceted environment that is a major pool for microbial genetic diversity. The soil DNA extraction procedures usually suffer from two major problems, namely, inappropriate rupturing of cells and contamination with humic substances. In the present study, five protocols for single type of rhizospheric soil were investigated and their comparison indicated that the inclusion of 120？mM phosphate buffered saline (PBS) for washing and mannitol in the lysis buffer allowed the processing of soil sample in minimal time with no specific equipment requirement. Furthermore, DNA purity and yield were also improved, which allowed the exploitation of genetic potential of soil microbes within soil sample thereby facilitating the amplification of metagenomic DNA. The effectiveness of methods was analyzed using random amplification of polymorphic DNA. The banding patterns revealed that both the abundance and the composition of indigenous microbial community depend on the DNA recovery method. 1. Introduction The biodiversity of microbes within soil is significant for the maintenance of healthy soil because these microbes are involved in many vital functions like crucial cycles of C, N, P, formation of soil, toxin removal, and so on. Previously, studies on the development of microbial communities required the isolation of these microbes from soil sample by culture dependent techniques followed by a series test for phenotypic evaluation and their identification. However, the microbial diversity studies conducted in soil have been biased essentially due to the unculturability of many microbes. Specific media, which are used to culture microbes, are selective in nature and only subpopulations of microbes from environment sample that will grow mainly depend on the particular conditions. It is reported that only 1% of microbes can be cultured in the laboratory using traditional culture techniques [1]. To study the microbial community, microbiologists have adopted culture-independent techniques. These techniques employ molecular biology based methods, in which soil extracted nucleic acid is subjected to PCR amplification [2]. These methods provide a unique insight into richness, composition, and structure of microbial community, that is, species richness and species evenness. The results thus rely not only on DNA extraction procedures but also on the factors affecting PCR amplification. Moreover, these culture-independent methods should address the problems like