A simple and efficient protocol for isolation of high molecular weight DNA from filamentous fungi, fruit bodies, and infected plant tissues

Abstract

Molecular approaches have become an important tool for mycologists studying fungal systematics, molecular evolution, population genetics or plant-fungus interactions.For this purpose the analysis of restriction fragment length polymorphisms (RFLPs), PCR-based techniques like amplification of polymorphic sequences by one random primer (RAPDs) or amplification of defined genomic regions are applied.Prerequisite for such studies is the rapid isolation of pure genomic DNA of high molecular weight not only allowing simultaneous processing of large numbers of samples but also providing reproducibility with regard to restriction, ligation, and PCR.Most common problems encountered in fulfilling these conditions are the rather high production of polysaccharides by some fungal cultures, and extraction of insufficient amounts of DNA from fruit bodies or from lignified and/or infected plant tissues.Few published protocols include steps for efficient removal of polysaccharides (Do and Adams 1991).Moreover, most procedures contain phenol-chloroform treatments (e.g.Garber and Yoder 1983, Blaiseau et al. 1992) and/or more than one precipitation (e.g.Rodriguez and Yoder 1991: four precipitations) -both latter steps are possible causes of shearing.The short protocol given below combines inactivation of proteins by SDS/Proteinase K with precipitation of acidic polysaccharides by hot CTAB in the presence of SDS and high salts (Kim et al. 1990) and only a single selective precipitation of DNA with isopropanol (Marmur 1961, Cryer et al. 1975).The method is appropriate for simultanous processing of many samples because all steps can be done in Eppendorf tubes, but it can also be scaled up easily for bulk preparations.A treatment with methanol containing 0.1% mercaptoethanol preceding DNA extraction was performed when basidiocarps and plant tissues contained large amounts of reactive compounds (resins, phenolics) interfering with solubilization of DNA.Methanol extraction was found to be superior over other solvents such as acetone (Schneiderbauer et al. 1991).In this way we isolated DNAs from a wide variety of fungi including Fusarium, Pseudocercosporella, Phytophthora, Setosphaeria, Armillaria, Heterobasidion, Boletus, Russula, Lactarius, Suillus, Cortinarius, and from conifer roots and needles (Picea abies, Pinus sylvestris).DNAs were subjected to Pulsed Field Gel Electrophoresis to verify their high molecular weight: the majority of DNA molecules was in the size range of 50-150 kbp and only insignificant amounts of DNA <50 kbp were present (Figure 1, lane 1).DNAs are suited as substrate for restriction,