Date Published: February 13, 2016
Publisher: Springer Berlin Heidelberg
Author(s): Avinash Narayan, Kunal Jain, Amita R. Shah, Datta Madamwar.
The present study describes the rapid and efficient indirect lysis method for environmental DNA extraction from athalassohaline soil by newly formulated cell extraction buffer. The available methods are mostly based on direct lysis which leads to DNA shearing and co-extraction of extra cellular DNA that influences the community and functional analysis. Moreover, during extraction of DNA by direct lysis from athalassohaline soil, it was observed that, upon addition of poly ethylene glycol (PEG), isopropanol or absolute ethanol for precipitation of DNA, salt precipitates out and affecting DNA yield significantly. Therefore, indirect lysis method was optimized for extraction of environmental DNA from such soil containing high salts and low microbial biomass (CFU 4.3 × 104 per gram soil) using newly formulated cell extraction buffer in combination with low and high speed centrifugation. The cell extraction buffer composition and its concentration were optimized and PEG 8000 (1 %; w/v) and 1 M NaCl gave maximum cell mass for DNA extraction. The cell extraction efficiency was assessed with acridine orange staining of soil samples before and after cell extraction. The efficiency, reproducibility and purity of extracted DNA by newly developed procedure were compared with previously recognized methods and kits having different protocols including indirect lysis. The extracted environmental DNA showed better yield (5.6 ± 0.7 μg g−1) along with high purity ratios. The purity of DNA was validated by assessing its usability in various molecular techniques like restriction enzyme digestion, amplification of 16S rRNA gene using PCR and UV–Visible spectroscopy analysis.
The molecular analysis of community DNA is the ultimate route to study the diversity of microbial wealth and genetic variation in natural conditions, to recover novel genes for understanding their metabolic functions, to track metabolic pathways and genetic adaptations for surviving under various environmental conditions (Kakirde et al. 2010; Delmont et al. 2012; Qu et al. 2009; Cary et al. 2010; Sharma et al. 2014). Subsequently, extraction of highly pure and unbiased environmental DNA is very fundamental and significant process. It requires basic understanding of physicochemical properties of soil (viz. organic content, presence of metal ions, salts, etc.) that always hinders the effectiveness of various treatment procedures and chemicals used during DNA extraction, which inturn affects the quality and quantity of extracted environmental DNA (Lombard et al. 2011; Young et al. 2014). Moreover, every environmental sample has its own set of physicochemical composition and biomass abundance. Therefore, every type of soil needs protocol optimization for environmental DNA extraction.
The presented protocol was highly efficient for metagenomics DNA extraction athalasohaline soil. To the best of our knowledge, the study first time demonstrated the use of PEG 8000 in combination of 1 M NaCl at pH 9.2 for the extraction of microbial cell biomass from the soil. The purified environmental DNA was highly compatible for further molecular analysis like PCR amplification, restriction enzyme digestion and community analysis by next generation sequencing technology.