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.
Figure 1-PCR results from SoilMaster™ DNA extract using one set of primers. In this reaction, the positive control was used as template in all wells. The positive control was run in varying dilutions as indicated by the amounts above each well, in either the soil DNA extract or in TE buffer.
Figure 2-PCR results from DNA extracted from soil using the PowerSoil® kit. DNA was successfully amplified with all four primer sets. Large bands and wells with multiple bands indicate the diversity of organisms picked up by each primer set.
Figure 3-The dominant basidiomycetes observed identified from genomic DNA extracted from a soil sample from the New York Botanical Garden. Eleven distinct organisms comprise 83% of the soil microbial community.
Figure 4-Sequencing gel containing PCR products from the SoilMaster™ kit and the PowerSoil® kit (in duplicate). Each band is a different sized piece of the rRNA gene region that was amplified by the primer set. Each band represents a unique organism. The blue arrow indicates a band that is the same for both kits. The red arrows indicate differences in the banding pattern between the two kits.
Soil microbial diversity is critically important to ecosystem functioning, but it can be very challenging to study. Microbial genomic DNA was extracted from forest soil samples using two chemical kits. From these extracts, the rRNA gene regions of prokaryotes and three types of fungi were amplified using four sets of primers. PCR was completely inhibited by the extract of one kit. Even after a clean-up procedure was applied, the remaining inhibitors still hindered PCR in several reactions. A second extraction kit produced a relatively clean extract, from which DNA was amplified without clean-up. These kits also varied dramatically in their ability to extract DNA from different organisms in the soil. The many unique sequences from a single PCR product were cloned in a DNA library. This library revealed that eleven unique organisms comprise the majority of the microbial community in the one soil sample. This technique circumvented the problems associated with using Sanger sequencing to read a diversity sequences in one PCR product.
This work was supported by the Department of Biological Sciences at Fordham University. Special thanks must be extended to Dr. Rubin for his guidance and uncanny knack for problem solving. Thank you to Alex Bulanov and Xie Xie for their remarkable patience and tireless efforts to help with this project. Thank you to Jessica Arcate Schuler at the New York Botanical Garden for the last-minute soil sample. An extra special thank you to the students of Techniques in Cell and Molecular Biology, past and present, for their advice and support.
|This document was last modified 05/19/2011.|
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