J Gen Plant Pathol (2006) 72:348–350 DOI 10.1007/s10327-006-0300-1
© The Phytopathological Society of Japan and Springer 2006
TECHNIQUE Ken-ichiro Saitoh · Kana Togashi · Tsutomu Arie Tohru Teraoka
A simple method for a mini-preparation of fungal DNA
Received: January 27, 2006 / Accepted: March 31, 2006
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. Key words Fungal DNA extraction · Zygomycetes · Ascomycetes · Basidiomycetes · Oomycetes
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
K. Saitoh · K. Togashi · T. Arie · T. Teraoka Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, Japan T. Teraoka (*) Laboratory of Plant Pathology, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan Tel./Fax +81-42-367-5692 e-mail:
[email protected]
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 (Cenis 1992; Ferreira and Glass 1996; Griffin et al. 2002; Xu and Hamer 1995), but 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 mass) was excised with a toothpick or an inoculation needle from a 4- to 10-day-old culture plate (Fig. 1A) and transferred to a microtube with 500 µl of the lysis buffer (200 mM TrisHCl, 50 mM ethylenediaminetetraacetic acid, 200 mM NaCl, 1% n-lauroylsarcosine sodium salt, pH 8.0). The mycelia were dispersed in the buffer with the toothpick and incubated for 10 min at room temperature. The mixture was
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then centrifuged at 18 000 g (15 000 rpm in a T15AP21 rotor, Hitachi Koki, Tokyo, Japan) for 5 min at 4°C, and the supernatant (300 µl) was transferred to a fresh microtube. After mixing with 750 µl of ethanol by inverting the tube, the DNA was precipitated by centrifugation at 18 000 g for 2 min at 4°C. After a wash with 70% ethanol, the DNA pellet was air-dried and dissolved in 50 µl of TE buffer (pH 8.0). To check the quality and quantity of the DNA prepared by this method, 5 µl of the DNA solution was electrophoresed on 0.8% agarose gel and stained with ethidium bromide (Fig. 1B). About 50–200 ng of DNA were obtained from M. grisea isolate P2 and F. oxysporum f. sp. lycopersici isolate 880621a-1 by this method. In these DNA samples, RNAs were probably degraded; they were hardly detected at more than 500 bp (Fig. 1B). DNA prepared by this method was used as a template for PCR to amplify the internal transcribed spacer (ITS) region. PCR primers were ITS1 (5′-TCCGTAGGTGAA CCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGAT
ATGC-3′) (White et al. 1990). Isolates of fungal species within the Zygomycota, Ascomycota, Basidiomycota, or Oomycota (Chromista) used in this experiment are listed in Table 1. All isolates were cultured on potato-sucrose agar medium at 26°C, except for Phytophthora infestans grown on rye agar medium [6.7% (w/v) rye extract, 2% sucrose, 0.35% agar] at 20°C. The PCR mixture (20 µl) contained 0.5 U Taq DNA polymerase (Sigma Aldrich, St. Louis, MO, USA), 1 × PCR buffer (Sigma Aldrich), 2.5 mM MgCl2, 0.2 mM (each) dNTPs, 0.4 µM (each) primers, and 1 µl of the prepared DNA. Thermal conditions were as follows: denaturing at 94°C for 2 min; 30 cycles of 94°C for 30 s, 57°C for 30 s, 72°C for 1 min; and a final extension at 72°C for 7 min. Fragments of the ITS region were successfully amplified from all tested isolates as expected (Fig. 2). The quality of the DNA obtained by this method was sufficient for digestion by restriction endonucleases, and the extraction protocol can be easily scaled up. The DNA preparations in this case were used in Southern blot analyses (Fig. 3).
Table 1. Fungal species and isolates used for DNA preparation Species
B (kbp)
A
λ Mg Fo
23.1 9.4 4.4 2.0 -
0.6 Fig. 1A, B. Mini preparation method. A Excision of a mycelial block on agar medium to start the mini preparation and B agarose gel (0.8%) electophoresis of the DNA prepared by this method from Magnaporthe grisea (Mg) and Fusarium oxysporum f. sp. lycopersici (Fo). Lambda DNA digested (50 ng) with HindIII was used as a DNA molecular marker (l)
1
2
3
4 5
6
Zygomycetes Mucor sp. Ascomycetes and mitosporic Ascomycetes Magnaporthe griesa Aspergillus oryzae Penicillium sp. Botryotinia fuckeliana Sclerotinia sclerotiorum Fusarium oxysporum f. sp. lycopersici Gibberella fujikuroi Trichoderma sp. Cladosporium sp. Alternaria sp. Ulocladium sp. Basidiomycetes Athelia rolfsii Rhizoctonia solani Oomycetes (Chromista) Phytophthora infestans Phytophthora nicotianae
Isolate
Reference
0016-2-2
Okabe et al. (2005)
P2 RIB40 0044-1-2 031024d 910624a 880621a-1
Saitoh et al. (2003) Machida et al. (2005) Okabe et al. (2005) Takahashi et al. (2005) Takahashi et al. (2005) Kawabe et al. (2004)
FGSC 7611 0039-1-6 0043-1-3 0043-2-5 0036-1-5
Arie et al. (1999) Okabe et al. (2005) Okabe et al. (2005) Okabe et al. (2005) Okabe et al. (2005)
010829a-1 TKF-44
Takahashi et al. (2005) Shigemoto et al. (1992)
TKF-411 TPS1234
Takahashi et al. (2005) Umezawa et al. (2004)
7 8 99 10 11 12 13 14 15 16 M - 1000 bp - 500 bp - 100 bp
Fig. 2. Polymerase chain reaction (PCR) amplification using DNA from mini preparation. The prepared DNA from Mucor sp. (lane 1), Magnaporthe grisea (lane 2), Aspergillus oryzae (lane 3), Penicillium sp. (lane 4), Botryotinia fuckeliana (lane 5), Sclerotinia sclerotiorum (lane 6), Fusarium oxysporum f. sp. lycopersici (lane 7), Gibberella fujikuroi (lane 8), Trichoderma sp. (lane 9), Cladosporium sp. (lane 10), Alternaria sp. (lane 11), Ulocladium sp. (lane 12), Athelia rolfsii
(lane 13), Rhizoctonia solani (lane 14), Phytophthora infestans (lane 15), and Phytophthora nicotianae (lane 16) were used as templates. The PCR mixture and reaction conditions are described in the text. A sample (1 µl) of the reaction mixture (20 µl) was separated by electrophoresis using a 1.5% agarose gel. Primers used for the internal transcribed spacer region are described in the text. Lane M is a 100-bp DNA ladder size marker (New England Biolabs, Ipswich, MA, USA)
350
A
1 kb
Mg-NCS-1
P
B
(kbp)
P
E
P
E
9.4 6.6 4.4 2.0 Fig. 3A, B. Southern blot analysis using the genomic DNA from the mini preparation. A Mg-NCS-1 probe labeled with DIG was prepared with a standard method using DIG DNA labeling and detection kit (Roche Diagnostics, Manheim, Germany). A Genomic structure around Mg-NCS-1 (Saitoh et al. 2003) in Magnapothe grisea isolate P2 is presented with recognition sites for PstI (P) and EcoRV (E). The dotted line indicates the fragment used to prepare the probe. B The genomic DNA (10 µl) prepared from M. grisea was digested with PstI (P) or EcoRV (E), separated on 0.8% agarose gel, and transferred to a nylon membrane (positively charged, Roche Diagnostics). DNA fragments that hybridized with the probe were detected with 4nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate (Roche Diagnostics). These procedures were carried out as described in the manufacturer’s protocol
This method was successfully used with mycelia cultured on diverse media such as oatmeal agar, V-8 agar, and potato-dextrose agar. Although the quality of the DNA after long-term storage has not yet been checked, the DNA was stable enough for use as a template for PCR after storage for 3 weeks at 4°C. Acknowledgments We thank Mr. Ryo Ishikawa (Sumitomo Chemical Co., Ltd., Hyogo, Japan), Ms. Junko Umezawa Sekihara (Toyama Agricultural Research Center, Toyama, Japan), and Dr. Michio Takeuchi (Tokyo University of Agriculture and Technology, Tokyo, Japan) for providing the isolates. This study was supported in part by a Grant-in-Aid for Scientific Research (A) from the Ministry of Education, Science, Sports, and Culture, Japan (no. 16208004).
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