A simple method of genomic DNA extraction suitable for ... - CiteSeerX

Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

A simple method of genomic DNA extraction suitable for analysis of bulk fungal strains Y.J. Zhang1,2, S. Zhang1, X.Z. Liu2, H.A. Wen2 and M. Wang3 1 School of Life Sciences, Shanxi University, Taiyuan, China 2 Key Laboratory of Systematic Mycology and Lichenology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China 3 School of Plant Sciences & Technology, Agriculture and Animal Husbandry College of Tibet, Nyingchi, China

Keywords DNA extraction, fungi, internal transcribed spacer (ITS) region, Ophiocordyceps sinensis, PCR amplification, thermolysis. Correspondence Xing Zhong Liu, Institute of Microbiology, Chinese Academy of Sciences, No.3, 1st West Beichen Road, Chaoyang District, Beijing 100101, China. E-mail: [email protected] (X.Z. Liu)

2010 ⁄ 0284: received 17 February 2010, revised and accepted 30 April 2010 doi:10.1111/j.1472-765X.2010.02867.x

Abstract Aims: A simple and rapid method (designated thermolysis) for extracting genomic DNA from bulk fungal strains was described. Methods and Results: In the thermolysis method, a few mycelia or yeast cells were first rinsed with pure water to remove potential PCR inhibitors and then incubated in a lysis buffer at 85C to break down cell walls and membranes. This method was used to extract genomic DNA from large numbers of fungal strains (more than 92 species, 35 genera of three phyla) isolated from different sections of natural Ophiocordyceps sinensis specimens. Regions of interest from high as well as single-copy number genes were successfully amplified from the extracted DNA samples. The DNA samples obtained by this method can be stored at )20C for over 1 year. Conclusions: The method was effective, easy and fast and allowed batch DNA extraction from multiple fungal isolates. Significance and Impact of Study: Use of the thermolysis method will allow researchers to obtain DNA from fungi quickly for use in molecular assays. This method requires only minute quantities of starting material and is suitable for diverse fungal species.

Introduction As a very large group of eukaryotic organisms, the fungi include an enormous diversity of taxa with varied ecologies, life cycle strategies and morphologies ranging from single-celled aquatic chytrids to large mushrooms. Fungi perform an essential role in the decomposition of organic matter and are relevant to many human activities such as antibiotic, toxin and food production. Although the classification and identification of fungi is traditionally based on morphological characteristics, recently some gene sequences (e.g., parts of the nuclear ribosomal DNA locus) have been extensively used as essential phylogenetic and taxonomic tools (Hibbett et al. 2007). Gene sequencing typically requires PCR, and the initial and essential step for the amplification of a target gene is the extraction of genomic DNA. Extraction of fungal genomic DNA has generally involved two major 114

steps: the breaking of cell walls, and the extraction and purification of genomic DNA. The genomic DNA is usually extracted with CTAB (cetyl trimethyl ammonium bromide) extraction buffer (Doyle and Doyle 1987) and then purified through phenol ⁄ chloroform extraction and isopropanol or ethanol precipitation (Ashktorab and Cohen 1992). Various methods have been used to break down cell walls. In the most commonly used method, mycelia are ground using liquid nitrogen or glass rods (Lee et al. 1988; Wu et al. 2001). In addition, researchers have also used dry ice (Griffin et al. 2002), glass or magnetic beads (Faggi et al. 2005), enzyme digestion (Li et al. 2002), benzyl chloride (Xue et al. 2006), microwave exposure (Goodwin and Lee 1993) and combinations of different methods (Zhang et al. 2008a). Although these techniques generally provide DNA of satisfactory quantity and quality, most of these techniques are tedious and time consuming and involve the use of hazardous chemicals.

ª 2010 The Authors Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010) 114–118

Y.J. Zhang et al.

In recent years, studies on fungal communities and fungal diversity increased rapidly (Dighton et al. 2005). These studies often require the identification of a large number of fungal species and strains, and such identification currently requires extraction of genomic DNA followed by PCR amplification. Because the established techniques for extracting genomic DNA from fungi are time consuming, they are not suitable for use with a large number of samples, and a simpler and faster method is needed. The current article describes a simple ‘thermolysis method’ for extracting fungal genomic DNA. The efficiency of this method was examined by amplifying nrDNA ITS regions of a large number of strains recovered from natural Ophiocordyceps sinensis specimens. The storage stability and target gene applicability of the genomic DNA extracted with this method was also investigated. Materials and methods

Fungal DNA isolation by thermolysis

was autoclaved at 121C for 20 min and then stored at 4C. DNA extraction by the CTAB method As a comparison to the thermolysis method, genomic DNA was extracted with the established CTAB method (Lee et al. 1988; Wu et al. 2001). Briefly, cell walls of fungal mycelia were broken down by grinding with glass rods or in the presence of liquid nitrogen. The CTAB extraction buffer was then added, and after incubation at 65C, purification with phenol:chloroform:isoamyl alcohol (25 : 24 : 1) and precipitation with isopropanol were conducted. Finally, the DNA was dissolved in 50 ll of pure water. The quality of DNA samples extracted by the CTAB method and the thermolysis method was compared by amplifying the full-length nrDNA ITS region of H. sinensis. The time required for extracting DNA with both methods was recorded and compared using ten randomly selected fungal strains.

Isolation and storage of fungal strains Natural O. sinensis specimens (a type of traditional Chinese medicine) were collected from Tibet and Sichuan in China during 2003 and 2005. One strain of Hirsutella sinensis (the anamorph of O. sinensis) (Liu et al. 1989) and another 572 fungal strains (more than 92 species, 35 genera of three phyla) isolated from various sections (stromata, sclerotia and external mycelial vela) of natural O. sinensis specimens (Wang et al. 2006) were used in this study. All strains were stored in 15% glycerol in an ultralow temperature refrigerator ()80C). DNA extraction by the thermolysis method All the fungal strains were removed from storage and placed on PDA plates at 25C with the exception of H. sinensis (at 20C). Once a strain had formed a colony, a sterile toothpick was used to transfer a small amount of mycelia or yeast cells from the colony into 100 ll of pure water in a 1Æ5-ml microcentrifuge tube. The mixture was vortexed thoroughly and then centrifuged at 8000– 10 000 g for 1 min. After carefully discarding the supernatant using a pipette tip, 100 ll of lysis solution was added to the microcentrifuge tube. The mixture was finally incubated at 85C in a water bath for 20–30 min. The crude extract contained genomic DNA and was stored at )20C until use. The ingredients of the lysis solution, which referred to the breaking buffer in the manual of the EasySelectTM Pichia Expression Kit (Invitrogen, USA), contained 50 mmol l)1 sodium phosphate at pH 7Æ4, 1 mmol l)1 EDTA and 5% glycerol. Before use, the lysis solution

Amplification of nrDNA ITS regions Each PCR contained 2 ll of 10 · PCR buffer, 1Æ2 ll of dNTP mixture (2Æ5 mmol l)1 each), 0Æ8 ll of deioned formamide, 0Æ4 ll of MgCl2 (25 mmol l)1), 0Æ8 ll of each primer (10 lmol l)1), 0Æ2 ll of Taq DNA polymerase (5 U ll)1) and 1 ll of genomic DNA in a total volume of 20 ll. The primer pairs for amplifying ITS1 regions were ITS1 (5¢-TCCGTAGGTGAACCTGCGG-3¢) and ITS2 (5¢-GCTGCGTTCTTCATCGATGC-3¢); those for fulllength ITS regions were ITS1 and ITS4 (5¢-TCCTCCGCTTATTGATATGC-3¢). A PCR consisted of an initial denaturing step of 5 min at 94C followed by 35–40 cycles (50 s at 94C, 50 s at 54C and 50 s at 72C) finished by a final extension step at 72C for 10 min. PCR products were resolved by electrophoresis through 1Æ2% agarose gels in TAE (2 mmol l)1 EDTA, 80 mmol l)1 Tris-acetate, pH 8Æ0) and were visualized by staining with GoldView (SBS Genetech, China). Applicability and stability of the genomic DNA extracted with the thermolysis method The genomic DNA of H. sinensis extracted with the thermolysis method was used to amplify single-copy csp1 (primers CSP1-ZF ⁄ TSPa2) and MAT1-2-1 (primers MAT12F1 ⁄ MAT1-2R2) genes as reported previously (Zhang et al. 2008b, 2009). With ten randomly selected fungal strains, the stability of the DNA extracted with the thermolysis method was tested by amplifying full-length ITS regions with primers ITS1 ⁄ ITS4 after the DNA was stored for 1 year at )20C.


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L1 L2 M (a)

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M 2000

M (d)

(c)

1000 750 500

Figure 1 Electropherograms of PCR products amplified with DNA samples extracted with the thermolysis method or the established method. (a) Comparison of DNA samples extracted with the established CTAB method (L1) and the thermolysis method (L2) by amplifying the full-length nrDNA ITS regions (c. 550 bp) of Hirsutella sinensis. Markers (M) are shown in bps. (b) Partial results for the amplification of the nrDNA ITS1 regions (c. 250 bp) with the DNA isolated with the thermolysis method. (c) Amplification of the single-copy MAT1-2-1 (left, c. 1000 bp) and csp1 (right, c. 800 bp) genes of H. sinensis. (d) Amplification of the full-length nrDNA ITS regions with the DNA stored for over 1 year at )20C.

250 100

M (b)

Results The thermolysis method described in this study was compared with the traditional CTAB method that uses glass rods or liquid nitrogen to break down fungal cell walls. Good PCR products were obtained with the H. sinensis DNA samples extracted by those two methods (Fig. 1a), indicating that the quality of genomic DNA extracted by both methods was comparable. To determine whether the thermolysis method could be used to extract genomic DNA from many different genera, the new method was applied to various fungal strains. With this method, we extracted the genomic DNA of the 572 fungal strains (including ascomycetes, basidiomycetes and zygomycetes) that were isolated from natural O. sinensis specimens. It is not necessary to detect the genomic DNA by electrophoresis because no visible band of the genomic DNA is expected on agarose gels. They were used directly for PCR amplification of nrDNA ITS1 regions with primers ITS1 and ITS2. Without any dilution of genomic DNA solutions, good amplification was obtained for all strains within the genera of Mortierella, Trichocladium, Epicoccum, Mucor, Trichoderma, Tricladium, Gliocladium, Tolypocladium, Acremonium, Beauveria and yeasts; and for most strains within the genera of Penicillium, Paecilomyces, Gymnoascus, Fusarium, Cladosporium and Neonectria (Fig. 1b). Initial amplification, however, produced no band or only a weak band for most strains within the genera of Cylindrocarpon, Aspergillus, Chrysosporium, Zalerion, Tumularia, Cadophora, Fimetariella, Leohumicola and Stilbella. In this instance, using a 1 ⁄ 25th-1 ⁄ 100th dilution of the genomic DNA as a template corrected the problem (data not shown). The failure of the initial 116

amplification could have been caused by the presence of PCR inhibitors in the original DNA solution. Successful amplification was achieved with 85% of the 572 strains using an undiluted DNA extract, but this was increased to 100% when those that failed were diluted (data not shown). All the amplicons were used successfully for SSCP (single-strand conformation polymorphism) analyses, and full-length ITS regions were then amplified and sequenced for strains showing different SSCP patterns (Zhang et al. unpublished). The genomic DNA extracted with this simple method is of high amplification efficiency not only for high-copy genes (e.g., nrDNA ITS) but also for low- or single-copy genes, such as the single-copy csp1 (one cuticle-degrading serine protease gene) and MAT1-2-1 (one mating-type gene) genes of H. sinensis (Fig. 1c) (15, 16). The genomic DNA extracted with the traditional CTAB method can be preserved for several years at )20C (Porebski et al. 1997). We re-examined the DNA that had been extracted with the thermolysis method and then stored at )20C for 1 year. Good results were obtained with PCR amplification (Fig. 1d), indicating that the DNA extracted with the thermolysis method is stable for at least 1 year. Discussion A simple and rapid method for extracting fungal genomic DNA was proposed in this study. Compared to traditional methods, the thermolysis method has significant advantages. First, the thermolysis method does not involve the mechanical breaking of fungal cell walls or DNA purification using phenol ⁄ chloroform, and the time


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Table 1 Times required for three methods of extracting fungal genomic DNA Duration for different stages (min) Methods

No. of samples treated

Break ⁄ wash

Incubation

Purification

Precipitation

Total

CTAB (liquid nitrogen)

One Ten One Ten One Ten

5 50 3–5 30–50 2 20

30–60 30–60 30–60 30–60 20–30 20–30

35 50–60 35 50–60 – –

25 30 25 30 – –

95–125 165–200 93–125 145–200 22–32 40–50

CTAB (glass rods) Thermolysis

The thermolysis method was compared with established CTAB methods based on liquid nitrogen or glass rods. The process for extracting genomic DNA was divided into four stages: breaking of cell walls (for the CTAB method using liquid nitrogen or glass rods) or washing of fungal biomass (for the thermolysis method), incubation to release DNA into buffer solutions, purification using phenol ⁄ chloroform and precipitation using isopropanol or ethanol. The time for each stage is estimated according to our experience. Dash (–) means the lack of a stage.

required for DNA extraction is therefore reduced greatly (Table 1); with this method, our laboratory can process up to 100 fungal samples in a day. Second, the thermolysis method does not require liquid nitrogen or an ultralow temperature centrifuge, which may be unavailable to some laboratories; the method only requires a standard centrifuge and a water bath. It follows that this method is practicable for a general microbial laboratory and is easily learned by normal investigators. Third, the thermolysis method does not use toxic chemicals (e.g., phenol or chloroform) and so, it is safe for operators and does not require disposal of harmful wastes. Fourth, the thermolysis method requires only a small quantity of fungal biomass (as little as 0Æ01 g) and so, strains do not have to be cultivated for a long time, which is especially valuable for slow-growing fungi, such as H. sinensis, and for the screening of a large number of transformants in a short time during a study of fungal genetics. Finally, contaminant DNA can cause significant problems with PCR (Kwok and Higuchi 1989), but the thermolysis method reduces the chance of contamination because it omits many of the surface contacts (e.g., contact between DNA and mortar, pestle, spatula and other equipment) that occur with the traditional methods. The thermolysis method also has limitations. Because the method includes no purification procedures, PCR may be inhibited by certain chemicals released from the cells of some species ⁄ strains. These PCR inhibitors may vary with fungal species, media used for cultivation and growth status (Min et al. 1995; Paterson 2007, 2008). Additionally, this method is not recommended for extracting DNA from fungal tissues (e.g., stromata and sclerotia of O. sinensis) although weak amplification can be occasionally obtained if the genomic DNA is diluted (data not shown). Cell walls of those fungal tissues are more difficult to break with this method than those of mycelia, and their DNA solutions are usually accompanied by pigments and other chemical inhibitors of PCR amplification.

Another fast and efficient method for DNA extraction is based on the use of microwave radiation (Tendulkar et al. 2003). Although the quality and quantity of DNA obtained with the microwave radiation method was sufficient for PCR analysis and dot blot hybridization, that method has only been used for Magnaporthe grisea; whether the method can be applied to other fungal taxa without protocol variations is unknown. In contrast, the thermolysis method described here has been successfully applied to fungal species belonging to various taxa. Acknowledgements This study was supported by grants from the National Plans of Sciences and Technology (Grants No. 2007BAI32B04), the National Outstanding Youth Foundation (30625001), the National 863 Plan of China (2007AA021506) and the cooperative project between Guangdong Province and Chinese Academy of Sciences (2009B091300015). The Authors are grateful to Ms Manhong Sun, Dr Bingda Sun and Dr Guozhu Zhao for their original work in isolating and ⁄ or characterizing fungal strains. We acknowledge Mr Wenfeng Gong for his work in cultivating and storing the fungal strains. The authors also thank Prof Bruce Jaffee (the University of California at Davis) for serving as presubmission reviewers and for his valuable comments and suggestions. References Ashktorab, H. and Cohen, R.J. (1992) Facile isolation of genomic DNA from filamentous fungi. BioTechniques 13, 198– 200. Dighton, J., White, J.F. and Oudemans, P. (2005) The Fungal Community: Its Organization and Role in the Ecosystem. Boca Raton, FL: CRC Press.


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