ALTERNATIVE METHODS OF PREPARING WHOLE-CELL DNA FROM FUNGI FOR DOT-BLOT, RESTRICTION ANALYSIS, AND COLONY FILTER HYBRIDIZATION

There is a large and increasing number of methods for preparing whole- cell DNA from fungi. Modifications have evolved for two reasons. This first is to simplify the protocol as much as possible to allow process ing of large sample numbers, in some cases for very specific uses, e.g ., dot-blots. The second is to increase the quality of the DNA. Most p reparations are contaminated with varying amounts of polysaccharides a nd unknown wall contaminants that can inhibit subsequent restriction o r ligation. The extent of contamination varies with the species, the i ndividual isolate, and at least in Neurospora, with the method or exte nt of growth. This paper offers three new methods. The first is a simp lified procedure for isolating denatured DNAs from filamentous fungi f or dot-blot analysis. The second is a rapid method for isolating DNAs from large numbers of small- to medium-scale cultures of filamentous f ungi. These preparations are sufficiently pure for a variety of enzyma tic reactions. The third is a nonenzymatic method for yeast colony fil ter hybridization that is simple, inexpensive, and efficient and resul ts in uniform signals for a variety of species. (C) 1995 Academic Pres s, Inc.