Rapid and efficient extraction of genomic DNA from differentphytopathogenic fungi using DNAzol reagent

Abstract

A modified procedure using the commercial DNAzol reagent was successfully applied to extract genomic DNA from 25 fungal species. The DNA yield varied from 306 to 1,927 microg g(-1) dry mycelia and the A(260)/A(280) ratio from 1.59 to 1.93. Compared with the method of J.L. Cenis (Nucleic Acids Res. 1992, 20: 2380) this procedure generated a higher DNA yield from 17 species and a higher A(260)/A(280) ratio from 23 species. But for four species, Cenis (1992) method was more suitable. No inhibitor of polymerase chain reaction was evident for the DNA extracted by the modified procedure, whereas some inhibitors remained in DNA of eight species extracted by the previous method.

Comparison of methods for isolating fungal DNA

“…The isolation using this commercially available compound is based on the presence of guanidine in the lysing solution, followed by chloroform extraction of DNA precipitated by ethanol. The results we obtained are in accordance with the results published by Guo et al (2005), i.e. better results were achieved in isolating DNA from the potentially aflatoxigenic mould of the genus Aspergillus than in isolating DNA with the use of the classic method according to Cenis (1992).…”

“…Each method for DNA isolation was simultaneously used for the various species of the genus Aspergillus, for reasons of the varying extractability of their DNA. The diverse results in extraction of DNA among the individual strains and species of moulds were pointed out by Guo et al (2005). In our study, four methods were used to evaluate DNA concentration: spectrophotometric detection, fluorometric detection using PicoGreen compound (Ahn et al 1996;Haque et al 2003;Nicklas & Buel 2003), determination of the maximum amplifiable dilution of DNA (Olexová et al 2004), and semi-quantitative determination of the amount of DNA by staining with ethidium bromide and evaluation on agarose gel.…”

“…The acquired amount of DNA varied according to the method used for the initial cell wall disruption and decreased in 1, 2 -cell wall disruption by Eppendorf micropestle; 3, 4 -cell wall disruption by ultrasound; 5, 6 -cell wall disruption by liquid nitrogen; N -negative control; M -DNA marker (155-970 pb) Very good results concerning DNA quality and quantity were provided by the isolation procedure using the commercially available compound DNAzol ® ES (Table 2). DNAzol ® ES was tested according to the publication by Guo et al (2005). Those authors have used DNAzol ® ES for isolating DNA from 25 species of moulds and compared the method with the DNA isolation method according to Cenis (1992).…”

Molecular characterization of the pathogenic plant fungus Rhizoctonia solani (Ceratobasidiaceae) isolated from Egypt based on protein and PCR-RAPD profiles

“…Cultures were filtered through microglass filters, and the mycelia were frozen in liquid nitrogen and ground to a fine powder in a mortar. DNA was extracted from 50 mg fresh mat according to Guo et al (2005).…”

An improved method of DNA extraction from plants for pathogen detection and genotyping by polymerase chain reaction

“…To date, many simplified DNA extraction methods have been developed for fungi and plant tissues, each having its own advantages and disadvantages (Cenis, 1992;Thomson and Henry, 1995;Liu et al, 2000;Cassago et al, 2002;Guo et al, 2005;Chi et al, 2009). In this study, a DNA extraction protocol was standardized, which is a modification of the methods reported by Kang et al (1998) and Wulff et al (2002).…”