A simple procedure for the isolation of fungal DNA for dot blot analysis

Abstract

A rapid method for the detection oftransforming sequences in a fungal strain would be advantageous when trying to determine if unselected sequences are present. Colony hybridization protocols for filamentous fungi have been developed (Stohl and Lambowitz, 1983. Anal. Biochem. 134:82-85; Paietta and Marzluf, 1984. Neurospora Newsletter 31:40) and a modified method thereof was described (McClung and Dunlap, 1988. Fungal Genetics Newsletter 35:26-27). In this report a simple method for the isolation of fungal DNA from a single transformed colony suitable for dot blot analysis is described. If the original transformant colony is not too small it is sufficient to extract the DNA of one half of that colony. That means that no subculturing is necessary and the results can be available within 24 h starting from the transformant colony. 1. Transfer one half of the transformant colony (or approximately 0.1 g of mycelium from a liquid culture) into a microfuge tube. 2. Add 1 ml oflysis buffer (50 mM EDTA, 0.2% SDS) and 0.1 g of Alumina (Type A5, Sigma). 3. Mix in a microfuge tube mixer (for this protocol an Eppendorf micro tube mixer was used) for 30 mm. 4. Centrifuge at 5000 rpm for 5 min. 5. Transfer the supematant (750 l) to a new microfuge tube. 6. Extract once with phenol, phenol/chloroform and chloroform each. 7. Add 2 M sodium acetate to a final concentration of 0.2 M. 8. Precipitate the DNA with an equal volume ofisopropanol. 9. Dry the pellet and redissolve in an appropriate volume ofTE buffer. With this DNA isolation procedure a contransformation experiment using Penici/lium nalgiovense ATCC 66742 as host organism was checked by dot blot analysis. The vector p3SR2 which carries the amdS gene (Hynes et al. 1983. Mol. Cell. Biol. 3:1430-1439) as a marker was used as a selectable plasmid. As a nonselectable plasmid, pELN5-lac was used. pELN5-lac carries the E. coli I3- galactosidase gene under the control ofthe promoter ofthe oliC31 gene (Ward and Tumer 1986. Mol. Gen. Genet. 205:331-338) and the terminator ofthe trpC gene (Mullaney et al. 1985. Mol. Gen. Genet. 199:37-45) from Aspergillus nidulans. One microgram of each ofthe two plasmids were cotransformed and positive transformants were selected for the presence of the amdS marker by growth on acetamide minimal medium. The DNA of one half ofthe resulting transformant colonies (diameter approximately 1 cm) was isolated as described above. Under these conditions 0.1-0.5 g ofDNA was isolated. The whole amount ofDNA was used for dot blot analysis. Hybridization was carried out using a digoxygenin labelled DNA.