DNA Analysis

DNA Extraction from Environmental Soils

We begin DNA extractions by measuring out 0.25 grams of the soil sample. This is done using an analytical balance and Qiagen Power Soil Pro bead tubes. Several steps are taken to ensure no contamination during the weighing process. We disinfect the workspace and balance with ethanol and we use a Bunsen burner to cleanse the Scoopula in between samples. Once samples are weighed and placed into bead tubes, they are taken to the molecular lab for extraction. We use the Qiagen DNeasy PowerSoil Pro Kit, which uses chemical and mechanical homogenization to lyse samples. Samples then undergo inhibitor removal, and a DNA binding solution is added. Samples are then passed through a spin filter membrane in a two-step washing process. The DNA then undergoes elution, and after is ready for polymerase chain reaction (PCR) amplification.

DNA Extraction from Single Spores

DNA can also be extracted from individual spores by placing spores into microcentrifuge tubes along with approximately 2.0 µl Taq polymerase buffer. Spores are then crushed using either a pipette tip or a microfuge pestle to release the contents of the spore. This process is done under a stereomicroscope. Once the spore contents are released, we add 14 µl cold Taq polymerase and shake to mix the contents. The tube is then put into a water bath at 94C for four minutes in order to denature DNase. The DNA is then ready for PCR amplification.

PCR Amplification

Once DNA has been extracted using either method, samples can be stored in -20C freezers until ready for PCR. There are many different primers and reagents that can be used for PCR to help refine and select for different types of DNA. For AMF (arbuscular mycorrhizal fungi) selection, we generally use either Phusion or the Q5 PCR kit, and LROR and FLR2 primers. Most of the PCR process is done inside a clean hood to prevent any contamination. Primers, reagents, and DNA samples are thawed inside the hood and spun down in a mini centrifuge. We create a master-mix using either the Q5 kit or Phusion, ultrapure water, and primers. This master-mix is transferred into PCR tubes and 1µl of DNA is added and mixed thoroughly. PCR tubes are then spun down using the mini centrifuge and placed into the thermocycler. The thermocycler runs on a pre-programmed cycle which changes the temperature of the reactions. This allows for the denaturing and cloning of the targeted DNA sequence. After this process is finished, the PCR product goes through several more steps including cleanups using the Machery-Nagel NucleoMag kit, gel electrophoresis, barcoding, and qubit assays. These steps prepare samples for submission to the University of Kansas Genome Sequencing Core, where they are pooled and sequenced using Illumina Next-Gen Sequencers.

DNA Methods

In this section, which is being developed as time permits, methods used at INVAM to carefully select spores, extract DNA from individual spores, amplify a target gene region, and clone and sequence amplicons to individuate sequence variants are discussed. Only two genes have been sequenced at INVAM, with the majority pertaining to an rRNA gene.

  1. The full length beta-tubulin gene ( TUB2 ) was characterized by Msiska and Morton (2009a) and an approximate 1000 bp region was selectively amplified for phylogenetic analysis which contains three highly variable introns (Msiska and Morton 2009b). No further work was done with this gene after Msiska left the lab because fungi many clades contained more than one copy (paralogs) which would have required much greater sampling of transformant clones to sort these from orthologs that gave a truer picture of gene evolution. There are 103 sequences representing 47 species (all described) in the INVAM library (stored in a FileMaker Pro database module).
  2. A 750-800 bp region near the 5’ end of the 28S rRNA gene ( LSU ) which spans two highly variable regions, D1 and D2. A fairly extensive library had been developed before Krueger et al. (2012) developed a set of primers to amplify a region of the 18S rRNA gene (SSU), ITS, 5.8S, ITS2, and a similar length of the LSU. Because of that database, cost considerations, and our attention mostly on species-level differences, we continue to select the LSU fragment. Our analysis of its power of resolution relative to concatenation of other rRNA gene repeats, which shows only minor differences in tree topology at the genus and species levels. There are 328 sequences representing 78 species (14 of which are undescribed) in the INVAM library as of July, 2013. We are using these sequences internally to better define reference strains and the boundaries between species, especially when morphological or genetic polymorphisms are apparent.

References

  • Krüger M, C. Krüger, C. Walker, H. Stockinger, and A. Schüßler A. 2012. Phylogenetic reference data for systematics and phylotaxonomy of arbuscular mycorrhizal fungi from phylum to species level. New Phytologist 193:970-984.

  • Msiska, Z. and J.Morton. 2009. Isolation and sequence analyses of a beta-tubulin gene from arbuscular mycorrhizal fungi. Mycorrhiza 19:501-513.

  • Msiska, Z. and J. Morton. 2009. Phylogenetic analysis of the Glomeromycota by a partial beta-tubulin gene. Mycorrhiza 19:247-254.