SPION-mediated soil DNA extraction and comparative analysis with conventional and commercial kit-based protocol

Tanima Paul,Semanti Basu,Keka Sarkar

doi:10.1007/s13205-014-0232-y

Tanima Paul, Semanti Basu + Show 1 more

Open Access

https://doi.org/10.1007/s13205-014-0232-y

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Journal: 3 Biotech	Publication Date: Jun 24, 2014
Citations: 25	License type: CC BY 4.0

Affiliation: University of Kalyani

Abstract

Direct isolation of soil DNA comes as an emerging technology to understand the microbial diversity of a particular environment circumventing the dependency on culturable methods. Soil DNA isolation is tough due to the presence of various organic components present in soil which interfere in extraction procedure. Here, we report a novel direct soil DNA extraction protocol utilizing bare superparamagnetic iron oxide nanoparticles and its comparison with conventional and commercial kit-based soil DNA extraction methods. The quality, quantity and feasibility of the recovered DNA from all the three methods towards various molecular techniques were checked. Our magnetic nanoparticle-based soil DNA extraction successfully yields pure DNA without any RNA or protein contamination as revealed by the nanodrop spectrophotometer and agarose gel electrophoretic study. Different methods of soil DNA extraction were evaluated on the basis of PCR, denaturing gradient gel electrophoresis and real-time PCR. Soil DNA extracted using conventional method fails to carry out critical molecular biology techniques where as magnetic nanoparticle-based soil DNA extraction gave good results which is comparable to commercial kit. This comparative study suggests that protocol described in this report is novel, less time consuming, cost effective with fewer handling steps and yields high quantity, good quality DNA from soil.

Highlights

Soil is an immense reservoir of microbial diversity and it is estimated to have 109 cells per gram of soil (Whitman et al 1998)
Different methods of soil DNA extraction were evaluated on the basis of PCR, denaturing gradient gel electrophoresis and real-time PCR
Soil DNA extracted using conventional method fails to carry out critical molecular biology techniques where as magnetic nanoparticle-based soil DNA extraction gave good results which is comparable to commercial kit

Summary

Introduction

Soil is an immense reservoir of microbial diversity and it is estimated to have 109 cells per gram of soil (Whitman et al 1998). To overcome the limitation of culture-dependent method emphasis is being made to encourage the development of culture-independent approaches (Head et al 1998; Muyzer et al 1993) to provide an overview of species richness in soil. This information of microbial diversity can be utilized for the study of community physiology, novel approaches in bioremediation and recycling, and discovering new biotechnology applications. The methods of direct DNA extraction from soil made dramatic improvements in analysis of soil microbial communities. Soil DNA extraction is difficult due to the presence of humic acids which are coextracted during DNA isolation and leads to inhibition of Taq DNA polymerase during PCR (Smalla et al 1993), interfere with enzymatic restriction digestion (Porteous and Armstrong 1991), reduce transformation efficiency (Tebbe and Vahjen 1993) and DNA hybridization specificity (Steffan et al 1988)

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Conclusion