Statistical assessment of DNA extraction methodology for culture-independent analysis of microbial community...

Cost-effectiveness, quality, time-effectiveness and ease of the methodology are the most crucial factors in isolating quality DNA from wide variety of samples. Thus, research efforts focusing on the development of an efficient DNA extraction protocol is the need of the hour. The present study therefore, focuses on development of an efficient, rapid and free of inhibitory substances based methodology for extracting metagenomic DNA from diverse environmental samples viz. anaerobic biogas digesta, ruminant stomach, human feces, soil, and microbial starter cultures used for preparation of fermented food. PCR-DGGE based analysis and quality metagenomic library preparation, using DNA extraction methodology, validates the developed protocol. The developed protocol is cost effective, capable of isolating DNA from small sample size (100-1000 µl), time efficient (1.5-2.0 h protocol) and results in significantly higher DNA yield (4-8 times increased yield) when compared to previously available DNA extraction method and a commercial DNA extraction kit. The DNA extracted from the samples using different protocols was evaluated based on its ability to identify diverse microbial species using PCR-DGGE profiles targeting variable region within the 16S rRNA gene. The results of microbial community analysis revealed comparability of the developed protocol to commercial kits, in effectively identifying dominant representatives of the microbial community in different samples. Using the DNA extracted from the presented methodology, metagenomic libraries were prepared, which were found suitable for sequencing on Illumina platform.