Fungal DNA Isolation

Saghai-Maroof MA, Soliman KM, Jorgensen RA, & Allard RW (1984) PNAS 81:8014-8018

DNA successfully isolated from fungal species of Cochliobolus, Aternaria, and Fusarium. The key elements in this prep are (1) the use of young lyophilized mycelial mats....young mats (4 days growth for C. carbonum)...yield less contaminating carbohydrates and other misc. junk (2) lots of proteinase K in the extraction buffer to kill DNases (final =0.3mg/ml).

Protocol

1.                                    place 0.2- 0.5 g (dry wt) lyophilized pad in a 5o ml disposable centrifuge tube, break up the pad wtih a spatula or glass rod, add c. 5 ml 3mm glass beads and powder the pad by brief shaking.

2.                                    add 10 ml (for a 0.5 g pad) of CTAB extraction buffer(see recipe, below), gently mix to wet all the powdered pad

3.                                    place in 65 C water bath for 30 min

4.                                    cool, add equal vol. ChCl3:IAA (24:1)

5.                                    mix, centrifuge in table top fuge 10 min at full speed

6.                                    transfer aqueous supernatant to a new tube

7.                                    add an equal volume of isopropanol

8.                                    High mw DNA should spool out upon mixing...spool out the DNA with a glass rod or hook, pour out the remaining supernatant

9.                                    rinse the spooled DNA with 70% ethanol

10.                                air dry, add 1- 5 ml TE containing 20 ug/ ml Rnase A. To resuspend the samples, place in 65 C bath, or allow pellets to resuspend overnight at 4 C

Notes: if the spooled DNA is discolored or has contaminating mycelial debris, phenol/chlorofrom extract and precipitate w/ ethanol.

This protocol can be scaled down using 0.1g pad in a 2ml eppendorf tube

For Southerns we routinely cut 50- 75 ul (2-4 ug) of a standard DNA prep in 200 ul rxn volumes, ETOH ppt, and resuspend in 30 ul.

Even after digestion the resuspended DNA can be very viscous at room temp; To load a Southern we keep the samples in a 50- 60 C heat block while loading to keep the samples at a lower viscosity.

CTAB extraction buffer: O.1M Tris, pH 7.5, 1% CTAB (mixed hexadecyl trimethyl ammonium bromide), 0.7M NaCl, 10mM EDTA, 1% 2-mercaptoethanol. Add proteinase K to a final concentration of 0.3 mg/ml prior to use. Less prot K may be acceptable for different fungi, and in fact we haven't determined if we can use less...this conc. was calibrated for a different C. carbonum DNA extraction buffer.

John Pitkin, MSU-DOE Plant Research Laboratory, Michigan State University, E. Lansing, MI 48824, (517)353-4886, JPITKIN@MSU.EDU

A novel method of growing fungi for DNA extraction

Preparation of fungi for DNA extraction typically involves growing cultures in liquid culture in Erlenmeyer flasks, Roux bottles or even microfuge tubes (Cenis 1992 Nucl. Acids Res. 20:2380). Growing fungal cultures in liquid may require formulating new media or determining aeration requirements, and there are no rapid means of confirming the identification of the resulting mycelium. Fungi are grown routinely on agar media for identification, but agar complicates DNA extraction.

Solutions of 'reverse agar' (BASF pluronic polyol F-127), a block polymer of polyoxypropylene and polyoxyethylene, are solid at normal room temperatures, but liquid at 4C. When the compound is used as a replacement for agar in solid media, its unusual properties allow the separation of mycelium and medium by simply placing a mature culture in a refrigerator. The compound has been employed for isolation of heat sensitive antagonistic microorganisms (Gardner and Jones 1984, J. Gen. Microbiol. 130:731-733; Olson and Lange 1989 Opera Bot. 100:197-199), isolation of enzymes associated with basidiome formation in Coprinus (Choi and Ross 1988 Exp. Mycol. 12:80-83), and for isolating mycelium from Neurospora race tubes (Munkres 1990, Fungal Genet. Newsl. 37:26). In this note, the possibility of extracting DNA suitable for PCR amplification from mycelium grown on reverse agar media is documented.

'Reverse Malt Agar' (RMA) medium was prepared containing 30% BASF pluronic polyol F-127 substituted for agar in the Malt Extract Agar medium of Pitt (1979 The Genus Penicillium, Academic Press). The liquid was poured over the F-127 granules and the resulting suspension left in the refrigerator overnight until dissolved. The solution was then autoclaved, resulting in a thick, sludge like substance, which was again refrigerated, until a homogenous, liquid solution resulted. The liquid was then poured into petri dishes, where it solidified as it warmed to room temperature. The resulting medium is not a true solid, but rather a dense gel.

Cultures of Penicillium spinulosum Thom. (DAOM 216698), Aspergillus japonicus Saito var. japonicus (DAOM 216695), Gliocladium roseum Bainier (Doyle SB-03a, not saved) and Trichoderma harzianum Rifai (DAOM 216501) were grown on RMA for 7 days at 25C in 6 cm petri dishes. Growth rates were slightly slower than on the same medium made with 2% agar, but the resulting colonies produced microscopically typical sporulating structures. For DNA extraction, the petri dishes were placed in the refrigerator for approximately 1 hour until the medium had liquified. Subsequent handling was done on ice. Mycelium was lifted from the medium using an autoclaved pipette tip, placed on the inverted, slanted lid, and allowed to drain for 30-60 seconds. The mycelium was then cut into smaller pieces using a sterile scalpel blade, and transferred into autoclaved 1.5 ml microfuge tubes. The tubes were then spun in a cold microfuge for 5-10 minutes and the excess medium removed. The mycelium was washed twice with 750 uL cold, autoclaved distilled water followed by cold centrifugation, and then used directly for DNA extraction. The DNA miniprep method of Edwards et al. (1991 Nucl. Acids Res. 19:1349), modified by the addition of a cold 70% ethanol wash of the final pellet, was used. The resulting DNA was treated with RNAase A for 1 hour at 37C. PCR amplification of the ITS1-ITS4 region of the ribosomal DNA was performed using the primers and conditions given by White et al. (pp. 282-287 In: PCR Protocols, Innis et al. eds Academic Press). The resulting products were digested for 2 hrs at 37C using HinfI in the buffer supplied with the enzyme.

The DNA yields obtained from mycelium grown on RMA were similar to those obtained from mycelium grown in liquid culture. Washing away excess reverse agar with cold water significantly improved yields, but DNA also was isolated from unwashed mycelium. The resulting DNA performed normally in the ITS PCR amplification and subsequent restriction digests (Figure 1).

Figure 1. Miniprep DNA (b-d), ITS 1-4 amplification (e-g) and HinfI restriction digests (h-j) from fungi grown on malt extract reverse agar. b, e, h Penicillium spinulosum. c, f, i Aspergillus japonicus var. japonicus. d, g, j Trichoderma harzianum. Lane a is the marker. Use of reverse agar for cultivation of fungi for DNA extraction may be convenient for certain studies. For fungi that do not produce characteristic structures in liquid broth, reverse agar provides a means of ensuring the correct identity of the mycelium before DNA extraction proceeds. Certain population genetics studies, for example, require the manipulation of a large number of cultures that must be cloned (eg. single-spore isolations) before genetic analysis can proceed. The use of reverse agar could eliminate one round of culture transfers, resulting in significant labour savings. Reverse agar solutions can be stored for some time at 4C and plates poured when required. Experience has shown that poured plates do not keep indefinitely at room temperature. After 4-6 weeks, the medium no longer liquifies, presumably because of higher polymer concentrations resulting from water evaporation. Also, because the polymer itself is slightly inhibitory to fungi, it does not seem to be suitable for weak nutrient media. Our trials with the Fusarium medium SNA (Nirenberg 1981 Can. J. Bot. 59:1599-1609), for example, resulted in sparse growth that could not be harvested following liquification of the medium. Acknowledgements: I am grateful to Dr. John Speakman (BASF, Limburghof, Germany) and BASF Performance Chemicals, Parsippany, NJ for providing samples of pluronic polyol F-127.

Note from FGSC: Dr. K.D. Munkres donated a large quantity of pluronic F-127 to the stock center. We will gladly make samples (100-200 g) available available at no cost to interested researchers.

A quick RNA mini-prep for Neurospora mycelial cultures

Most RNA isolation techniques currently in use have been developed for the processing of large quantities of material. These typically involve multiple phenol extractions (Reinert et al. 1981 Mol. Cell Biol. 1:829-836) or guanadinium isothio-cyanate/cesium chloride gradients (Chirgwin et al. 1979 Biochem 18:5294-5299) and can be both expensive and time consuming. Often, however, needs arise where quantitatively smaller amounts of RNA are needed from many different samples, for example, during time series analyses or when screening transfor-mants for expression of a transformed gene. Under such circumstances, existing tech-niques are overly time consuming and yield more RNA than is necessary. The availability of a rapid RNA mini-prep is thus desirable. Such a system has been developed for isola-ting plant RNA (Nagy et al. 1988 Plant Molecular Biology Manual, B4; ed. Gelvin and Schilperoort, Klewer Academic Publishing, pp. 1-29), and we have adapted this procedure for use with Neurospora and, potentially, other filamentous fungi. Below, we describe the use of this procedure with 50 ml mycelial cultures, although we have used in with equal success with 5 ml cultures without scaling down the amounts of any reagents.

The method involves the use of a triphenylmethane dye, aurintricarboxylic (ATA), to protect the RNA. ATA binds irreversibly to RNA and is a potent inhibitor of most nucleic acid binding enzymes (Hallick et al. 1977 Nucl. Acids Res. 4:3055-3064). Thus, RNA made with procedure cannot be used for in vitro transcription or translation or reverse transcription but works fine for RNA/DNA or RNA/RNA hybridizations.

To minimize RNase contamination, all glassware is baked at 182°C for at minimum of four hours. Work with gloved hands. The procedure is as follows:

1. Conidia from slants (grown in 16 x 150 mm test tubes containing 8 ml of solid medium) are resuspended in 50 ml of Horowitz complete medium (Horowitz 1947 J. Biol. Chem. 171:255-262) and the cultures grown overnight with shaking at 30°C. A 50 ml culture typically yields enough RNA for 200 gel lanes (see below), and, as noted, smaller culture volumes may be used.

2. Flat mycelial pads are easier to grind than mycelial balls. Therefore, filter cultures using a Buchner funnel onto Whatman #44 filter paper. Wrap flat mycelial pads in aluminum foil and freeze in dry ice. Do not freeze in EtOH/dry ice bath because alcohol might seep through foil. Pads can be stored at -70°C for at least three weeks.

3. Wash a mortar and pestle thoroughly with warm water and Alconox (Fisher Scientific); cool by filling with liquid N2. Remove frozen, flat mycelia from foil and add it to the liquid N2 in mortar. Add ~0.5 g of baked sand and grind mycelial pad to a fine powder. Add more N2 as needed. Mortar and pestle should be washed after every sample.

4. Working quickly before powder can thaw, pour or spoon ground mycelia into 15 ml round bottom Sarstedt tube (Sarstedt tubes #60.540, Princeton, NJ) containing 8 ml of E buffer at room temperature. [E buffer: 50 mM Tris-Cl pH 8.0, 300 mM NaCl, 5 mM EDTA, pH 8.0, 2% SDS; autoclave and add 1 mM ATA and 14 mM ß-mercaptoethanol. ATA=aurintricarboxylic acid, ammonium salt (Sigma #A0885, St. Louis, MO)]

5. Thaw the powder in E buffer in 42°C water bath, occasionally shaking, to get SDS into solution. This should take about 5 minutes.

6. Add 1.1 ml of 3M KCl, invert to mix, keep on ice for 10 min. Solution should form semi-solid, flocculent mass as K-SDS precipitate forms.

7. Spin at 3000g, 4°C in a fixed angle rotor. Make sure caps are screwed on tightly to prevent tubes from collapsing.

8. Pass supernatant through 50 micron Miracloth (Calbiochem #475855, La Jolla, CA) in a funnel into fresh Sarstedt tube.

9. Measure volume of average-sized sample. Add 0.5 vol. 8 M LiCl, mix and stand at 4°C overnight.

10. Spin at 12000g, 4°C, for 15 min in a fixed angle rotor. Thoroughly resuspend pellet in 4 ml sterile gd (glass distilled) H2O with pasteur pipette.

11. Extract twice with phenol/chloroform/isoamyl alcohol (25:24:1), spinning 12000g 10 min at 4°C in a fixed angle rotor. Save aqueous (upper) phase; add gd H2O if volume is less than 2 ml.

12. Add 0.1 vol 3M NaOAc pH 6.0, mix and add 2.5 vol EtOH, mix. Place at -20°C overnight or -70°C for 15 min.

13. Spin 12000g, 10 min, 4°C. Wash pellet with 70% EtOH and drain. Pellet should be a light pink or white.

14. Resuspend in 0.4 ml sterile gd H2O in a microfuge tube. Precipitate with NaOAc and EtOH as in step 12.

15. Spin 10-20 min in microfuge. Wash twice with 70% EtOH and dry pellet. Resuspend in 200 µl of sterile RNase-free gd H2O. Store at -70°C for up to three months. Spectophotometric quantification may be done at this point.

16. Load 1 µl onto a formaldehyde gel. Electrophorese overnight at 20 volts. Blot onto nitrocellulose. Probe with DNA fragment of choice. Expose to film.

Yields are typically on the order of 1-2 mg total RNA from an overnight 50 ml culture arising from an average slant. The number of samples able to be processed using this procedure is limited by the number of spaces in a centrifuge rotor. We have done as many as 24 samples in one day, and doing several times this many would be possible. We have observed on ethidium bromide stained gels that the fluorescence from the RNA deriving from this miniprep is brighter than that seen when the corresponding amount of standard-prep RNA is used. This may be due to enhanced fluorescence of RNA in the presence of ATA. However, autoradiography of the blots does not show any RNA degradation products (Figure 1). This procedure would probably work fine with other methods of tissue disruption. ATA inhibits many nucleic acid binding proteins, possibly by competing for binding sites (Blumenthal and Landers 1973. BBRC 55:680-688). Therefore, the most critical factor is getting the RNA in contact with ATA before nucleases can bind to the nucleic acid and degrade it. Supported by federal grants to J.J.L. and J.C.D.

Fig. 1. ATA mini-prep RNA probed with ccg-1 DNA fragment. Total RNA from a series of transformants into bd A and frq7 A was examined for the presence of the ccg-1 gene transcript (Loros et al. 1989 Science 243:385-388). Each lane contains 10 µg of RNA (1/200 of the preparation). While the fluorescence staining of the RNA extended from the 26S to below the 17S RNA bands (not shown), the hybridization revealed the presence of only a single undegraded transcript in each lane containing transformant RNA and no hybridization to the monkey cos cell RNA control.