Comparison of six extraction techniques for isolation of DNA from filamentous fungi

Abstract

Filamentous fungi have a sturdy cell wall which is resistant to the usual DNA extraction procedures. We determined the DNA extraction procedure with the greatest yield of high quality fungal DNA and the least predilection for cross-contamination of equipment between specimens. Each of six extraction methods was performed using Aspergillus fumigatus hyphae. The six methods were: (1) glass bead pulverization with vortexing; (2) grinding with mortar and pestle followed by glass bead pulverization; (3) glass bead pulverization using 1% hydroxyacetyl trimethyl ammonium bromide (CTAB) buffer in a water bath sonicator; (4) water bath sonication in CTAB buffer; (5) grinding followed by incubation with CTAB; and (6) lyticase enzymatic cell lysis. Genomic DNA yields were measured by spectrophotometry and by visual reading of 2% agarose gels, with shearing assessed by the migration of the DNA on the gel. Genomic fungal DNA yields were highest for Method 1, followed by Methods 5 approximately = to 2 >3 approximately = to 4 approximately = to 6. Methods 2 and 5, both of which involved grinding with mortar and pestle, led to shearing of the genomic DNA in one of two trials each. We conclude that the use of glass beads with extended vortexing is optimal for extraction of microgramme amounts of DNA from filamentous fungal cultures.

Identification and Pathogenicity of Lasiodiplodia Species from Eucalyptus urophylla × grandis, Polyscias balfouriana and Bougainvillea spectabilis in Southern China

“…DNA was extracted using ‘method 5’ described by Van Burik et al. (). The resulting DNA was checked for purity and concentration using a NanoDrop 2000 Spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA).…”

Ecological study of Paracoccidioides brasiliensis in soil: growth ability, conidia production and molecular detection

“…The DNA was extracted from the yeast phase of P. brasiliensis (positive control) according to Van Burik et al [19]. The DNA extraction from soil seeded with P. brasiliensis (positive control) and from environmental samples was carried out by using Fast DNA ® Spin Kit for Soil (Qbio gene INC), with minor modifications according to Theodoro et al[20].…”

Evaluation of a polymerase chain reaction reverse hybridization line probe assay for the detection and identification of medically important fungi in bronchoalveolar lavage fluids

“…The fungal biomass was suspended in 400 ml of extraction buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA) [29]. The fungal biomass was suspended in 400 ml of extraction buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA) [29].…”