Isolation of high molecular weight DNA and double-stranded RNAs from fungi
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Abstract
An efficient method for the extraction of DNA and RNA from fungi is described. Urediosporelings and sporidia of two basidiomycete species and mycellia from several species of Ascomycetes and Oomycetes were homogenized in a lysis buffer containing sodium dodecyl sulfate followed by cetyltrimethylammonium bromide extraction of carbohydrates in 1.4 M NaCl, leaving nucleic acids in the supernatant. After chloroform – isoamyl alcohol extraction of proteins, nucleic acids were precipitated with ethanol. Total nucleic acids prepared in this way contained nuclear, ribosomal, and mitochondrial DNA as well as double-stranded and single-stranded RNA. DNA was eluted from agarose gels and digested with endonucleases, labelled by nick translation, and used for hybridization without nonspecific background signal. A method is also described for RNase digestion of single-stranded and double-stranded RNA in agarose gels. Key words: DNA extraction, double-stranded RNA, cetyltrimethylammonium bromide, sodium dodecyl sulfate.
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