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Statistical assessment of DNA extraction methodology for culture-independent analysis of microbial community associated with diverse environmental samples

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Abstract

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.

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Funding

The research is funded by Department of Science and Technology, Government of India for Indo-Russian collaborative project “Elucidating the linkage between key limiting processes and microorganisms during anaerobic degradation of lignocellulosic waste” INT/RUS/RFBR/P-175.

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Authors and Affiliations

  1. Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Solan, 173234, India

    Rishi Mahajan, Sampan Attri, Kavita Sharma, Niharika Singh, Deepika Sharma & Gunjan Goel

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  1. Rishi Mahajan
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  2. Sampan Attri
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  3. Kavita Sharma
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  4. Niharika Singh
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  5. Deepika Sharma
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  6. Gunjan Goel
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Contributions

RM and GG designed the research, assessed and interpreted the results and prepared the manuscript. RM, SA, NS, KS, DS analyzed samples using the developed protocols. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Gunjan Goel.

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Conflict of interest

The authors have no conflict of interest to declare.

Ethical approval

All experimental protocols used in the study were carried out in accordance with the guidelines and regulations of Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, India. The stool sample collection, storage and analysis were approved by Jaypee University institutional ethical and biosafety committee (IEC No. 22/2015).

Informed consent

Informed consent was obtained from the parents of all the infants, for using their stool samples.

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Mahajan, R., Attri, S., Sharma, K. et al. Statistical assessment of DNA extraction methodology for culture-independent analysis of microbial community associated with diverse environmental samples. Mol Biol Rep 45, 297–308 (2018). https://doi.org/10.1007/s11033-018-4162-3

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  • DOI: https://doi.org/10.1007/s11033-018-4162-3

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