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≅2>3≅4≅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.

Strain-Dependent Variation in 18S Ribosomal DNA Copy Numbers in Aspergillus fumigatus

“…The hyphae were recovered after 24 h by filtering through an 18.5-cm, 0.45-mm-pore-size Whatman disk (Whatman, Florham Park, NJ) and washed with sterile saline. DNA isolation consisted of methods reported elsewhere (22,41), with slight modifications. After the saline wash, approximately 200 mg of wet hyphae were briefly dried by blotting between Whatman paper (Whatman) and then placed into a sterile mortar and frozen for 10 min at Ϫ70°C.…”

Two novel species of Calonectria isolated from soil in a natural forest in China

“…Total genomic DNA was extracted following the protocols "Extraction method 5: grinding and CTAB" described by Van Burik et al (1998). The extracted DNA was dissolved in 30 µL TE buffer (1 M Tris-HCl and 0.5 M EDTA, pH 8.0), and a Nano-Drop 2000 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to quantify the concentration.…”

Decreased expression of 14-3-3 inParacoccidioides brasiliensisconfirms its involvement in fungal pathogenesis

“…73 To confirm the presence of the hygromycin B resistance cassette, PCR (polymerase chain reaction) amplification was performed to detect the HPH 1000-bp Figure 8. Silencing of Pb14-3-3 decreases the virulence of P. brasiliensis in a Galleria mellonella infection model.…”