.

Procedure

1.                         If using petri plates, the 3-7 day culture is then diced up, using asterile scalpel, and small pieces (ca. 50-100mg) are placed in microfuge tubes containing sterile sand (ca. 100mg) and 500uL of extraction buffer. If using microfuge tubes, simply add the sand and extraction buffer:

o                         100mM Tris, pH8.0

o                         10mM EDTA

o                         2% SDS

o                         100ug/mL Proteinase K

o                         1% B-mercaptoethanol

(tubes can be stored in this extraction buffer at minus 20C for greater than a year)

2.                         Using a Kontes micro-homogenizer with sterilized tips (Fisher Scientific Cat. # K749540-0000) samples are ground into a slurry and incubated for 60 min. at 60C.

3.                         Salt concentration is adjusted to 1.4M with 5M NaCl, 1/10 vol. of 10% CTAB added and samples incubated a further 10 min. at 65C.

4.                         Add 1 vol. chloroform:isoamyl alcohol, gently emulsify by inversion, incubate  at 0C for 30 min. Spin 10 min. at 4C at rpm max. Transfer top phase to fresh 1.5mL microfuge tube, add 1/2 vol. 5M NH4OAc, mix gently, ice for 60 min.; spin at 4C at rpm max.

5.                         Transfer supernatant to fresh tube (add stock RNase 10mg/mL to a final concentration of 0.02ug/uL) add 0.55 vol. isopropanol to precipitate the DNA. Spin immediately 5-10 min. at rpm max. Aspirate off supernatant, wash DNA pellet twice with 70% ETOH, air dry pellet 20 min. and resuspend in 50uL  TE buffer. Incubate 4C overnight.

6.                         Quantify yield with the Hoefer DyNA Quant 200 fluorometer.

This procedure does not require phenol extraction. The B-mercaptoethanol can be omitted from the extraction buffer for safety, but yields will be slightly lower. The DNA is pure enough for restriction digests, PCR and genomic library construction.

Note

Modified from Moller, Bahnweg, Sandermann and Geiger, 1992, Nucleic Acids Res. 20: 6115-16.

A safe method of extracting DNA from Coccidioides immitis

Human-pathogenic fungi such as Coccidioides immitis and Histoplasma capsulatum must be handled in Biosafety level 3 containment facilities which make for a very awkward working environment (J.Y. Richmond and R.W. McKinney, eds. Biosafety in Microbiological and Biomedical Laboratories, 3rd ed. NIH, Washington). In this paper we describe a safe and convenient method of extracting DNA from such fungi in which the culture is killed by steaming, allowing removal from the containment facilities, as soon as possible. The method was first developed with the non-pathogen Neurospora crassa, has worked well for both C. immitis and H. capsulatum, and should be useful for extracting DNA from any pathogenic fungus.

Cultures of C. immitis are grown in the saprophytic phase at 37 C in 100 ml of 2X GYE medium (20 g glucose and 10 g yeast extract in 1 liter medium, autoclaved for 15 min.) in 500 ml plastic Nalgene flasks with plastic foam stoppers (Identi-plug, Fisher). When there has been sufficient growth, the whole container is transferred to an autoclave and steamed at 1 atm (100 C) for 15 min. This raises the temperature of the culture to about 95 C (as measured in trials with a thermocouple). The culture is allowed to cool, 1 ml is plated on 2X GYE or potato dextrose agar, and the rest frozen at -20 C. If there is no growth on the plate after 1 week, the culture is assumed to be dead and is removed from the containment room to proceed with the DNA extraction.

Cultures are thawed, filtered, frozen in liquid nitrogen, lyophilized, and ground with a mortar and pestle. About 10 mg of dry ground tissue is mixed with 400 ul lysis buffer (50 mM Tris, 100 uM NaCl, 5 mM EDTA, 1% sodium dodecyl sulfate), and incubated at 80 C for 10 min., cooled to 40 C and incubated with 10 ul of 10 mg/ml Proteinase K for 2-3h, and then incubated again at 80 C for 10 min. The DNA is then extracted with chloroform- phenol and precipitated with isopropanol, following Lee and Taylor (In: M. A. Innis et al, eds. PCR Protocols, pp. 282-287, Academic Press, San Diego). The pellet is resuspended in 100 ul TE, and 5 ul of this is electrophoresed in agarose to check the yield - usually there is an easily visible band of high molecular weight DNA (Figure 1). We have successfully amplified the DNA by PCR and sequenced the product directly.

The main innovation of this protocol is the use of steam to kill the culture. By doing this at an early stage, right in the culture flask, exposure to the fungus is minimized. In trials with Neurospora crassa, steaming at 1 atm gave a much higher yield of DNA than autoclaving under pressure. The addition of Proteinase K to the lysis buffer also increases yield (Figure 1), presumably by digesting away proteins which are bound to the DNA and denatured by the heat.

Acknowledgements: Norman Pace suggested the lysis buffer recipe and the use of Proteinase K. Financial support came from NIH-NIAID grant AI28545.

Figure 1. Genomic DNA extracted from 10 mg lyophilized C. immitis after killing with steam. a, b: strain 1; c, d: strain 2; a, c: without Proteinase K; b, d: with Proteinase K; L: 1 kb DNA size ladder (Gibco BRL). Gel was treated with RNAse after electrophoresis.

A simple, rapid procedure for the isolation of DNA for PCR from Gibberella fujikuroi (Fusarium section Liseola)

The polymerase chain reaction (PCR) is a method for amplifying specific segments of DNA defined by the small primers used to start the reaction. Using arbitrarily chosen 10-base primers, one can generate "random amplified polymorphic DNA" (RAPD) markers (Williams et al. 1991 Nucl. Acids Res. 18:6531-6535). These DNA fragments, separated by electrophoresis in an agarose gel, can be used as markers for studying genetic variation within and among fungal populations. A rapid and simple procedure for isolating fungal DNA from multiple isolates is needed if this technique is to be useful for diagnosis and screening of natural populations. We have developed a method for isolating DNA without grinding from Fusarium cultures grown on agar slants using a modification of the SDS miniprep method of Lee et al. (1988 Fungal Genetics Newsletter 35:23-24).

1. Place a cube (0.5 cc) of mycelia-covered agar from a 5 day-old slant in a 1.5 ml microcentrifuge tube.
2. Fill tube with liquid nitrogen and let it evaporate. Repeat. No grinding is necessary.
3. Immediately add 500 ul of 65 C lysis buffer (50 mM Tris pH 8, 50 mM EDTA, 3% SDS, 1% BME, and 0.1 mg/ml Proteinase K). Vortex tube and place in 65 C water bath for 1 hr. Vortex after 30 min and 60 min.
4. Extract with 500 ul phenol. Spin tube 5 min at 8000 rpm in microcentrifuge to separate phases. Remove 450 ul of the aqueous phase.
5. Extract with 450 ul buffered phenol. Spin. Remove 400 ul of the aqueous phase. 6. Extract with 400 ul chloroform:isoamyl alcohol::24:1. Spin. Remove 350 ul of the aqueous phase.
7. Add 50 ul 7.5 M ammonium acetate. Gently mix. Add 880 ul 95% ethanol. Invert to mix. Place in -20 C freezer 30 min to overnight.
8. Spin down DNA pellet for 20 min at 13,000 rpm. Rinse pellet in 70% ethanol. Dry pellet and resuspend in 20 ul TE (10 mM Tris, 1 mM EDTA, pH 8).

DNA was prepared from isolates grown on complete and minimal slants (Correll et al. 1987 Phytopathology 77:1640-1646). We obtained enough DNA from each isolate for 20 reactions. We used 1 ul of DNA for PCR with our primer, designated ECORI (5'- ATGAATTCGC-3'). To each reaction tube on ice was added 42 ul sterile, glass-distilled and deionized water; 5 ul 10X buffer from Promega (500 mM KCl, 100 mM Tris-HCl pH 9, 15 mM MgCl, 0.1% gelatin w/v, 1% Triton X-100), 1 ul of 50 uM primer, 1 ul of 1 mM dNTP's and 1 ul DNA solution. Tubes were boiled for 2.5 min. Tubes were returned to ice, then 1 unit of Taq polymerase (Promega) was added. Tubes were spun for 5 sec in a 4 C microfuge and then 100 ul mineral oil was added. Tubes were placed in a PTC-100 programmable thermal controller from M.J. Research with the following program: Step 1. 92 C 30 sec. Step 2. 35 C 1 min. Step 3. Slope 35 to 72 at 1 degree C every 8 sec. Step 4. 72 C 2 min. Step 5. cycle to Step 1. 45 times. Step 6. 72 C 7 min. and Step 7. end. 20 ul of each reaction mix was loaded into a 1.3% agarose gel with TBE buffer. After 2.0 hours at 56 V, the gel was stained with ethidium bromide and photographed. The resulting patterns. of amplified DNA fragments are listed in Table 1.

Table 1. RAPD marker patterns for Gibberella fujikuroi isolates using the ECORI primer

Strain    DNA fragments,  kb +/- SE

A2910     1.661 +/- .044, 0.597 +/- .015

A2911     1.698 +/- .098, 0.561 +/- .031

A3957     1.678 +/- .032, 0.639 +/- .015

X3974     1.661 +/- .044

Acknowledgements: Contribution 91-404-J, Kansas Agricultural Experiment Station, Kansas State University, Manhattan. Supported by a grant from the Kansas State Board of Agriculture (Corn Commission and Grain Sorghum Commission), by the Kansas Agricultural Experiment State Hatch Project 547, and by the Sorghum/Millet Collaborative Research Support Program (INTSORMIL) AID/DAN-1254-G-00-0021-00 from the Agency for International Development, Washington, DC.