ABSTRACT
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.