Optimization of Soil DNA Extraction Protocol Using Na2EDTA, SDS, Heating, Vortexing and CaCl2 and Its Validation for Metagenomic Studies

Main Article Content

Malik Ahmed Pasha
Sachin A. More
P. U. Krishnaraj


Aim: Several methods described previously for isolation and purification of soil DNA. Most of these protocols use combination of techniques or methods but the role and contribution of each individual method or component used is not clearly discussed. This study aims at analysing the effect of individual components used in extraction of DNA from soil and finally to optimize soil DNA isolation protocol and its validation by using 16SrDNA sequence analysis.

Methods and Results: The soil was washed with anionic buffers before lysis step to reduce humic substances and release microbial cells from soil matrix, then the cells were lysed using combination of SDS, heating and vortexing and finally humic substances were removed using chemical flocculation. Pre-lysis washing of soil with 100 mmol l-1 Na2EDTA proved good for releasing microbial cells from soil matrix. Heating the soil sample at 75°C yielded good quantity (15.73 µg g-1 soil) DNA followed by 2% SDS (10.28 µg g-1 soil) and vortexing at 1400 rpm (8.94 µg g-1 soil). Combination of heating, SDS and vortexing yielded 25 µg DNA per gram of soil. Different concentrations of chemical flocculants like AlNH4(SO4)2, FeCl3, CaCl2 and MgCl2 were used to reduce humic substances. Flocculation with 100 mmol l-1 CaCl2 removed 5.2 mg humic substances without significant loss of DNA. 16S rDNA sequence analysis of DNA extracted from soil reveals presence of all the common soil bacterial species indicating the protocol is unbiased.

Conclusion: Combination of chemical (SDS) and physical (heating and vortexing) methods yield good DNA whereas addition of enzyme (lysozyme) did not show significant effect on cell lysis. The digestion of isolated DNA with restriction enzyme and amplification of 16S rDNA using Taq DNA polymerase indicates the isolated DNA is pure enough for metagnomic analysis. 16Sr DNA sequencing of soil DNA indicates that this protocol can extract good quality and quantity DNA from range of bacteria present in soil varying in their cell wall composition. The optimised protocol is unbiased, very simple, does not need special equipments and many samples can be processed simultaneously.

Metagenomics, soil DNA, humic acid, PCR, protocol, sequencing

Article Details

How to Cite
Pasha, M. A., More, S. A., & Krishnaraj, P. U. (2020). Optimization of Soil DNA Extraction Protocol Using Na2EDTA, SDS, Heating, Vortexing and CaCl2 and Its Validation for Metagenomic Studies. International Journal of Environment and Climate Change, 10(6), 1-13. https://doi.org/10.9734/ijecc/2020/v10i630201
Original Research Article


Bates ST, Berg-Lyons D, Caporaso JG, Walters WA, Knight R, Fierer N. Examining the global distribution of dominant archaeal populations in soil. ISME J. 2011;5:908–917.

Maron PA, Mougel C, Ranjard L. Soil microbial diversity: Methodological strategy, spatial overview and functional interest. C R Biol. 2011;334:403-411.

Torsvik V, Ovreas L. Microbial diversity and function in soil: From genes to ecosystems. Curr Opin Microbiol. 2002;5:240–245

Krishnaraj PU, Pasha MA. Metagenomics concepts, tools and applications. In Environment Science and Engineering. Instrumentation, Modelling and Analysis. Studium Press, USA. 2016;7: 272-307.

Holben WE, Jansson JK, Chelm BK, Tiedje JM. DNA probe method for the detection of specific microorganisms in the soil bacterial community. Appl Environ Microbiol. 1988;54:703–711.

Ogram A, Sayler GS, Barkay T. The extraction and purification of microbial DNA from sediments. J Microbiol Methods. 1987;7:57-66.

Zhou J, Bruns MA, Tiedje JM. DNA recovery from soils of diverse composition. Appl Environ Microbiol. 1996;62:316-322.

Kuske CR, Banton KL, Adorada DL, Stark PC, Hill KK, Jackson PJ. Small-scale DNA sample preparation method for field PCR detection of microbial cells and spores in soil. Appl Environ Microbiol. 1998;64:2463-72.

Tebbe CC, Vahjen W. Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast. Appl Environ Microbiol. 1993;59:2657-2665.

Malik M, Kain J, Pettigrew C, Ogram A. Purification and molecular analysis of microbial DNA from compost. J Microbiol Methods. 1994;20:183-196.

De Grange V, Bardin R. Detection and counting of Nitrobacter populations in soil by PCR. Appl Environ Microbiol. 1995;61:2093–2098

Leff LG, Dana JR, McArthur JV, Shimkets LJ. Comparison of methods of DNA extraction from stream sediments. Appl Environ Microbiol. 1995;61:1141-1143.

Herrick JB, Miller DN, Madsen EL Ghiorse WC. Extraction, purification and amplification of microbial DNA from sediments and soils, In Burke JF. editors. PCR: Essential techniques. John Wiley & Sons; 1996.

Braid MD, Daniels LM, Kitts CL. Removal of PCR inhibitors from soil DNA by chemical flocculation. J. Microbiol Methods. 2003;52:389–93.

Arbeli Z, Fuentes CL. Improved purification and PCR amplification of DNA from environmental samples. FEMS Microbiol Lett. 2007;272:269-275.

Olson ND, Morrow JB. DNA extract characterization process for microbial detection methods development and validation. BMC Res Notes. 2012;5:668.
DOI: 10.1186/1756-0500-5-668

Nakatsu CH, Torsvik V, Ovreas L. Soil community analysis using DGGE of 16S rDNA polymerase chain reaction products. Soil Sci Soc Am J. 2000;64:1382-1388.

Pasha MA, More SA, Krishnaraj PU. In silico and denaturing gradient gel electrophoresis-based evaluation of 16S rDNA primers for metagenomic studies. Res. J. Biotech. 2019;14(2):54-60.

Meyer F, Paarmann D, Souza M, Olson R, Glass EM, Kubal M, Paczian T, Rodriguez A, Stevens R, Wilke A, Wilkening J Edwards RA. The metagenomics RAST server-a public resource for the automatic phylogenetic and functional analysis of metagenomes. BMC Bioinformatics. 2008;9:386.

Chao A, Shen TJ. Program SPADE (Species prediction and diversity estimation). Program and User’s Guide; 2010.

Frostegard A, Courtois S, Ramisse V, Clerc S, Bernillon D, et al. Quantification of bias related to the extraction of DNA directly from soils. Appl Environ Microbiol. 1999;65:5409–5420.

Schneegurt MA, Dore S, Kulpa CF. Direct extraction of DNA from soils for studies in microbial ecology. Curr Issues Mol Biol. 2003;5:1-8.

Van Es FB, Laanbroek HJ, Veldkamp H. Microbial ecology: An overview, In Codd GA. editors. Aspects of microbial metabolism and ecology. New York: Academic Press, Inc; 1984.

Ling TY, Achberger EC, Drapcho CM, Bengtson RL. Quantifying adsorption of an indicator bacteria in soil-water system. Transactions of the American Society of Agricultural Engineers. 2002;45:669-674.

Huang PM. Soil mineral–organic matter–microorganism interactions: fundamentals and impacts. Adv Agron. 2004;82:391-472.

He J, Xu Z, Hughes J. Pre-lysis washing improves DNA extraction from a forest soil. Soil Biol Biochem. 2005;37:2337–2341.

Moore E, Arnscheidt A, Kruger A, Strompl C, Mau M. Simplified protocols for the preparation of genomic DNA from bacterial cultures. Molecular Microbial Ecology Manual, Second Edition. 2004;101:3-18.

Pietramellara G, Ascher J, Borgogni F, Ceccherini MT, Guerri G, Nannipieri P. Extracellular DNA in soil and sediment: fate and ecological relevance. Biol Fert Soils. 2009;45:219-235.

Portillo MC, Leff JW, Lauber CL, Fierer N. Cell size distributions of soil bacterial and archaeal taxa. Appl Environ Microbiol. 2013;79:7610-7617.

Delmont TO, Robe P, Clark I, Simonet P, Vogel TM. Metagenomic comparison of direct and indirect soil DNA extraction approaches. J Microbiol Methods. 2011;86:397–400.

Packard MM, Wheeler EK, Alocilja EC, Shusteff M. Performance evaluation of fast microfluidic thermal lysis of bacteria for diagnostic sample preparation. Diagnostics. 2013;3:105-116.

Mahalanabis M, AlMuayad H, Kulinski MD, Altman D, Klapperich CM. Cell lysis and DNA extraction of gram-positive and gram-negative bacteria from whole blood in a disposable microfluidic chip. Lab Chip. 2009;9:2811-2817.

Yeates C, Gillings MR, Davison AD, Altavilla N, Veal DA. Methods for microbial DNA extraction from soil for PCR amplification Biol Proc. 1998;1:40-47.

Li Y, Tan W, Koopal LK, Wang M, Liu F, Norde W. Influence of soil humic and fulvic acid on the activity and stability of lysozyme and urease. Environ Sci Technol. 2013;47(10):5050-5056.

Martzy R, Bica-Schröder K, Pálvölgyi ÁM. et al. Simple lysis of bacterial cells for DNA-based diagnostics using hydrophilic ionic liquids. Sci Rep. 2019;9:13994.

Bollet C, Gevaudan MJ, de Lamballerie X, Zandotti C, de Micco P. A simple method for the isolation of chromosomal DNA from Gram positive or acid-fast bacteria. Nucleic Acids Res. 1991;19(8):1955.

Yuan S, Cohen DB, Ravel J, Abdo Z, Forney LJ. Evaluation of Methods for the Extraction and Purification of DNA from the Human Microbiome. PLoS ONE. 2012;7(3): e33865.

Shahriar M, Haque R, Kabir S, Dewan I, Bhuyian MA. Effect of Proteinase-K on Genomic DNA Extraction from Gram-positive Strains. J Pharm Sci. 2011;4(1):53-57.

Navarre WW, Schneewind O. Surface proteins of gram-positive bacteria and mechanisms of their targeting to the cell wall envelope. Microbiol Mol Biol Rev. 1999;63:174-229.

Lakay FM, Botha A, Prior BA. Comparative analysis of environmental DNA extraction and purification methods from different humic acid-rich soils. J Appl Microbiol. 2007;102(1):265-73.

Tsai Y, Olson BH. Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction. Appl Environ Microbiol. 1992;58:2292-2295.

Sagova-Mareckova M, Cermak L, Novotna J, Plhackova K, Forstova J, Kopecky J. Innovative methods for soil DNA purification tested in soils with widely differing characteristics. Appl Environ Microbiol. 2008;74:2902-2907.

Schulze HJ. Schwefelarsen im wasseroger Losung Prakt. Chem. 1882;25:431-452.

Hardy WB. A preliminary investigation of the conditions which determine the stability of irreversible hydrosols. Proc Roy Soc Lon. 1900;66:110–125.

Acosta-Martinez V, Dowd S, Sun Y, Allen V. Tag-encoded pyrosequencing analysis of bacterial diversity in a single soil type as affected by management and land use. Soil Biol Biochem. 2008; 40:2762–2770

Fierer N, Breitbart M, Nulton J, Salamon P, Lozupone C, Jones R, Robeson M, Edwards RA, Felts B, Rayhawk S. et al. Metagenomic and small-subunit rRNA analyses reveal the genetic diversity of bacteria, archaea, fungi, and viruses in soil. Appl Environ Microbiol. 2007;73:7059–7066

Acosta-Martinez V, Dowd SE, Bell CW, Lascano R, Booker JD, et al. Microbial community composition as affected by dryland cropping systems and tillage in a semiarid sandy soil. Diversity. 2010;2:910–931.

Janssen PH. Identifying the dominant soil bacterial taxa in libraries of 16S rRNA and 16S rRNA genes. Appl Environ Microbiol. 2006;72:1719–1728.

Dedysh SN, Kulichevskaya IS, Huber KJ, Overmann J. Defining the taxonomic status of described subdivision 3 Acidobacteria: The proposal of Bryobacteraceae fam. Nov. Int J Syst Evol Microbiol. 2016;67: 498-501.

Ward NL, Challacombe JF, Janssen PH, Henrissat B, Coutinho PM, Wu M, Xie G, Haft DH, Sait M, et al. Three genomes from the phylum Acidobacteria provide insight into the lifestyles of these microorganisms in soils. Appl Environ Microbiol. 2009;75:2046-2056.

Lori M, Symnaczik S, Mäder P, De Deyn G, Gattinger A. Organic farming enhances soil microbial abundance and activity: A meta-analysis and meta-regression. PLoS ONE. 2017;12(7): e0180442.

Aparna K, Pasha MA, Rao DLN, Krishnaraj PU. Organic amendments as ecosystem engineers: Microbial, biochemicaland genomic evidence of soil health improvement in a tropical aridzone field site. Ecol Engg. 2014;71:268-277