The extraction of fungal DNA from multiple large soil samples

Abstract

A commercial electric paint shaker was utilized to enable the simultaneous extraction of DNA from multiple large soil samples. A bead homogenization procedure was used to extract DNA from soil samples added to tubes. Soils infested with chlamydospores of the phytopathogenic fungus Cylindrocarpon destructans were used to evaluate the procedure. Glass beads and zirconium-oxide beads of various diameters were compared for their ability to extract DNA and reduce fungal colony-forming units (CFU) on agar media after shaking treatments. Total extracted DNA was measured using fluorometry, and DNA quality was assessed using gel electrophoresis. Twenty minutes of shaking with 1-mm-diameter zirconium-oxide beads provided the highest recovery of DNA from soil and resulted in 98% reduction of colony forming units, compared with nonshaken soil. Components of the extraction solution (calcium chloride, proteinase K, sodium dodecyl sulfate) were evaluated for their effects on DNA yield and on amplification by polymerase chain reaction. Minimum amounts of these materials provided maximum yield, but their impacts on amplification by polymerase chain reaction were dependent upon the primer set used. Amplification of extracts, using the optimized extraction procedure, successfully detected C. destructans in naturally infested soil. Total DNA yields resulting from this procedure were at least as high as those obtained with commercial extraction kits

Keywords:

• calcium chloride
• fluorometry
• soilborne plant pathogens
• zirconium-oxide beads