An efficient and economical method for extraction of DNA amenable to biotechnological manipulations, from diverse soils and sediments

Abstract

Aims: An attempt was made to optimize a new protocol for isolation of pure metagenomic DNA from soil samples. Methods and Results: Various chemicals (FeCl 3 , MgCl 2 , CaCl 2 and activated charcoal) were tested for their efficacy in isolation of metagenomic DNA from different soil and compost samples. Among these trials, charcoal and MgCl 2 when used in combination yielded highly pure DNA free from humic acids and other contaminants. The DNA extracted with the optimized protocol was readily digested, amplified and cloned. Moreover, compared with a wellestablished commercial DNA isolation kit (UltraClean TM Soil DNA Isolation Kit), our method for DNA isolation was found to be economical. This demonstrated that the method developed can be applied to a wide variety of soil samples and allows handling of multiple samples at a given time. Conclusions: The optimized protocol developed has successfully yielded pure metagenomic DNA amenable to biotechnological manipulations. Significance and Impact of the Study: A user-friendly and economical protocol for isolation of DNA from soil and compost samples has been developed.

A Rapid and Economical Method for Efficient DNA Extraction from Diverse Soils Suitable for Metagenomic Applications

“…An assessment of the extracted DNA purity demonstrates that the method M6 provides more pure DNA with an average absorbance ratio (A 260/280 ) of 1.76 ± 0.05, as compared to absorbance ratios lower than 1.5 for the other methods. The A 340 absorbance value was taken as an indication of humic acid contamination rather than A 230 because it has less overlap with the absorbance of DNA which is measured at A 260 and the associated use of A 260 /A 230 as a typical ratio indicator of DNA purity [ 22 ]. The average A 340 value for method M6 is low (0.047 ± 0.03) as compared to the other methods ( Table 4 , S4 and S5 Tables) which is consistent with the recent report of Sharma et al (2014) [ 22 ] that demonstrated effective use of PAC for the reduction in humic substances.…”

“…PAC is very porous with a vast surface area which allows the absorption of humic substances, lignin sulphonate, tannic acid, heavy metals and non-degradable coloured substances [ 33 ]. PAC has previously been used as a purifying agent in other reports of soil DNA extraction [ 11 , 22 ]. In the present study, 1% PAC was added directly to the extraction buffer in order to absorb contaminating components of the metagenomic DNA that would otherwise inhibit subsequent DNA manipulation.…”

“…However, a comparable glass powder based DNA extraction is not yet reported for metagenomic DNA from soils. Purification is accomplished with powdered activated charcoal (PAC), because it absorbs humic substances and allows the release of pure DNA [ 22 , 11 ]. Hence, PAC was included in the extraction buffer of this study with the goal of eliminating the need for subsequent purification steps.…”

Rapid and economical protocols for genomic and metagenomic DNA extraction from oak (Quercus brantii Lindl.)

“…If the ratio is appreciably lower than expected, it may indicate the presence of polyphenolics, salts, and humic acid contaminants which absorb at 230 nm whereas 260/280 nm ratio of~1.8 is generally accepted as pure for DNA. If the ratio is appreciably lower in either case, it may indicate the presence of protein, phenol, or other contaminants that absorb strongly at or near 280 nm (Sharma et al 2014). The means followed by different lowercase letters within each row are significantly different (p < 0.01) a The statistical analysis was separately performed between different methods for each sample All the extracted DNAs were used for further molecular analysis through qualitative and quantitative polymerase chain reaction (PCR and Q-PCR).…”

High-Throughput Isolation of Nucleic Acids from Soil

“…These substances are the major obstacle to the following PCR amplification steps. Inhibitors can co-precipitate with DNA, adversely affecting the quality and quantity of the extract [11,12]. Among inhibitory substances, humic compounds are the most common, followed by heavy metals and aromatic compounds.…”