Protocol

Isolation and Cloning of High-Molecular-Weight Metagenomic DNA from Soil Microorganisms

¹ Department of Biological Sciences, Auburn University, Auburn, AL 36849, USA
² Department of Bacteriology, University of Wisconsin, Madison, WI 53706, USA
³ Department of Biotechnology, Mahidol University, Bangkok 10400, Thailand
⁴ Department of Microbiology, University of Bergen, N-5020 Bergen, Norway
⁵ School of Environmental & Biological Sciences, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901, USA

⁶Corresponding author (lilesma{at}auburn.edu)

INTRODUCTION

The successful construction of large-insert community DNA (i.e., metagenomic) libraries from natural environments is dependent on several parameters, including effective cell lysis, DNA purity, and a high transformation efficiency. One problem associated with constructing metagenomic libraries from soil microbes is the co-isolation of contaminants, leading to the degradation of DNA as a result of nuclease activity. Because the isolation of intact genetic pathways from soil microbes is necessary to characterize their genetic and functional diversity, obtaining high-purity, high-molecular-weight (HMW) DNA for library construction is absolutely critical. This protocol describes the steps for the indirect extraction of bacterial DNA from soil, embedding the DNA in an agarose matrix, using a formamide and high-salt treatment to eliminate nucleases, size-selecting DNA by restriction digestion and pulsed-field gel electrophoresis (PFGE), and cloning the HMW DNA into a large-insert vector. The resulting metagenomic libraries contain high-purity, stable, HMW DNA that can be screened for various genetic loci (sequence-based) or phenotypic traits (function-based).