A simple method for a mini-preparation of fungal DNA

Abstract

A simple method was established to prepare DNA from fungal mycelia cultured on an agar plate. The fungi tested successfully with this method contained Zygomycetes, Ascomycetes, Basidiomycetes, and Oomycetes. This method did not require any time-consuming steps to crush or digest mycelia or fractionation in a phenol-chloroform mixture. The DNA was easily extracted by immersing and dispersing the mycelial plugs in a specific buffer (200 mM Tris-HCl, 50 mM ethylenediaminetetraacetic acid, 200 mM NaCl, 1% n-lauroylsarcosine, pH 8.0), then concentrated by ethanol precipitation. The total time to complete the whole procedure was less than 1 h. The quality and quantity were sufficient for polymerase chain reaction amplification and Southern blot analysis.Extraction of genomic DNA is an essential step for molecular analyses of fungi. The standard method to prepare fungal DNA consists of lyophilization of mycelia, disruption of cell walls by grinding, extraction of DNA in buffers containing sodium dodecyl sulfate, removal of proteins with a mixture of phenol and chloroform, and precipitation of DNA with 2-propanol ( Kawabe et al. 2004). This method is suitable to obtain a large amount of pure DNA, but is time consuming, labor intensive, and pollutes water with phenol and chloroform. Therefore, a rapid and simple method for DNA preparation on a small scale has been needed for polymerase chain reaction (PCR)-based identification and determination of genotypes such as mating types or drug resistance, and for screening transformants to obtain isolates with a targeted gene modification. Generally, a fungus isolated from the field or a mixture of transformants is grown in liquid culture before DNA extraction. If DNA can be successfully extracted directly from thalli on agar media, the total time for the identification or screening will be markedly decreased. Several methods for rapid DNA extraction have been proposed (they were not fully suitable for this purpose.In this report, a simple and reliable method was developed for the mini preparation of fungal DNA from thalli on agar media, based on the method of Liu et al. (2000). This method has several advantages:1. Preculture in liquid media is not necessary. 2. The duration of culture is more flexible and can be chosen before the preparation. 3. Potentially harmful organic solvents, such as phenol and chloroform, which are troublesome to discard, are excluded as exhaustively as possible. 4. DNA is less damaged by mechanical forces because freezing and homogenizing the thalli are not needed. 5. The minimized number of preparation steps and the lyophilized powder-free procedure reduce the risk of crosscontamination. 6. Total time of the procedure is less than 1 h.Magnaporthe grisea was cultured on Misato-Hara agar medium (0.2% yeast extract, 1% soluble starch, 1.5% agar) at 26°C. Fusarium oxysporum was cultured on potatosucrose agar medium [20% (w/v) potato extract, 0.5% sucrose, 1.5% agar] at 26°C. A small piece of mycelia (7-15 mm on a side) with agar medium (100-300 mg total m...

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