Abstract
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.
Highlights
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
The results rely on DNA extraction procedures and on the factors affecting PCR amplification. These culture-independent methods should address the problems like incomplete rupturing of cells and presence of soil organic substances, namely, fulvic and humic acid, the presence of which inhibit the activity of DNA polymerase, and interfere with the hybridization protocols [3]
It has been demonstrated that an additional step of using phosphate buffer saline with inclusion of mannitol was useful to achieve these objectives
Summary
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. 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]. The results rely on DNA extraction procedures and on the factors affecting PCR amplification These culture-independent methods should address the problems like incomplete rupturing of cells and presence of soil organic substances, namely, fulvic and humic acid, the presence of which inhibit the activity of DNA polymerase, and interfere with the hybridization protocols [3]. Purification of silica and other biogel columns has been reported to minimize the humic acid contamination These procedures, make DNA isolation process expensive involving a large number of steps, Molecular Biology International which makes these procedures lengthy, time-consuming, and tedious. An improved method is required for soil DNA extraction that would allow efficient rupturing of microbial cells and simultaneously decrease the contamination of organic materials (humic acid) in an easy and costeffective manner
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