Introduction
Microorganisms play a fundamental role in the transformation of energy and matter. For this reason, knowledge of microbial diversity is essential to understanding how ecosystems function (Torsvik et al. 1996, van der Heijden et al. 2008). However, genetic approaches for studying soil microbial communities come with a unique set of challenges. In this study two different chemical kits were used to extract genomic DNA from forest soil samples. A methodology for amplifying short DNA sequences from three phyla of fungi and prokaryotes was developed. In order to overcome the challenges associated with sequencing, a DNA library was made to isolate and identify the diversity of fungi amplified by one primer set. Finally, the relative extraction efficiencies of the two different soil DNA extraction kits were examined.
Materials and Methods
Soil DNA was extracted by using either the SoilMaster™ DNA extraction kit by EPICENTRE® Biotechnologies or the PowerSoil® DNA Isolation Kit by Mo Bio Laboratories. The SoilMaster™ DNA extract was also purified using the PowerClean® Soil DNA Clean-Up Kit by Mo Bio Laboratories. Short segments of the ribosomal RNA gene region were PCR amplified using four primer sets to target three phyla of fungi and prokaryotes. The PCR product that produced the strongest band was purified, and the individual amplicons were separated by cloning. One hundred randomly-selected clones were sequenced and compared to the Nucleotide Collection database of GenBank for identification. Radiolabeled PCR products of the two extraction kits, both applied to the same soil sample and amplified with the same set of primers, were run side-by-side on a sequencing gel that separates DNA segments that differ in size by one base. The results were visualized on photographic film to determine the relative extraction efficiencies of the two kits.
Results
PCR could not successfully amplify DNA in the SoilMaster™ DNA extract, nor could it amplify positive control DNA that was diluted in the extract (Fig.1). Even after applying the PowerClean® Soil DNA Clean-Up procedure to the SoilMaster™ DNA extract, only two primer sets could successfully amplify the soil DNA. The PowerSoil® DNA Isolation Kit produced an extract that could be amplified by all four primer sets (Fig.2). Due to the diversity of organisms present in the soil extract, Sanger sequencing could not produce a readable sequence for a single PCR product. Only when the individual amplicons of the PCR product were cloned to form a DNA library and the clones were sequenced independently could Sanger sequencing give a picture of the richness and abundance of soil microorganisms (Fig.3). A total of eleven organisms comprised 83% of the Basidiomycota community present in that soil sample. When the two soil DNA extracts were compared side-by-side on a sequencing gel, it was clear from the banding pattern that the two kits differ in their ability to extract DNA from certain microorganisms (Fig.4).
Discussion
This study demonstrated that soil DNA extraction kits, particularly the SoilMaster™ kit, may leave behind contaminants that inhibit PCR. These inhibitors were so potent that they effectively inhibited PCR at concentrations as low as 1%. Even after applying a clean-up methodology to the SoilMaster™ extract, PCR was still inhibited at higher concentrations of template. When soil DNA is successfully amplified, it is difficult to sequence because it represents many different organisms. When soil PCR products are sequenced by the Sanger method, multiple bases are detected at every step, which prevents the identification of one readable sequence. To overcome this problem, the individual pieces need to be separated from one another by ligation and transformation into cells, creating a DNA library. However, this picture of richness and abundance could be influenced by the kit that was used to extract the DNA. The side-by-side comparison of the two extraction kits on the sequencing gel revealed profound differences in the extraction efficiencies of the two kits. If this variability affects all chemical extraction kits, chemical extraction alone could introduce bias into research by selecting for a particular set of organisms.
Literature Cited
Torsvik, V., R. Sorheim, and J. Goksoyr. 1996. Total bacterial diversity in soil and sediment communitites - a review. Journal of Industrial Microbiology 17:170-178.
van der Heijden, M. G. A., Richard D. Bardgett, and Nico M. van Straalen. 2008. The unseen majority: soil microbes as drivers of plant diversity and productivity in terrestrial ecosystems. Ecology Letters 11:296-310.
