High-Molecular-Weight Genomic DNA Isolation from Doratomyces microsporus and Synthesis of a Genomic DNA Library

Mojca BenČina*, Melita JakopiČ, Jožefa Friedrich
National Institute of Chemistry, Department of Biotechnology, Hajdrihova 19, SI-1000 Ljubljana, Slovenia.
Tel.: +38614760334, Fax: +38614760300,
E-mail: mojca.bencina@ki.si

A protocol for isolation of genomic DNA from a filamentous fungus was optimised and a genomic library was constructed. Various methods for preparing genomic DNA from different fungi are known: simplified methods to allow processing of large sample numbers and methods to increase the quality of DNA, which is especially important for synthesis of a genomic DNA library. An isolation of high-molecular-weight DNA from a fungus is a challenging process since cell wall composition and cellular components differ with the fungal species. A procedure for isolation of high-molecular- weight DNA from the keratinolytic filamentous fungus Doratomyces microsporus, strain MZKI B399, is described and compared to the method used for filamentous bacteria and to the method including commercially available DNA extraction buffer. The isolation protocol that gave the desired quality of DNA was optimised in the following steps: disruption of cells by grinding in liquid nitrogen, followed by removal of polysaccharides and proteins by a phenol extraction buffer and finally precipitation of DNA with isopropanol. The obtained DNA was used to produce the bacteriophage genomic DNA library of the fungus.

Keywords: Keratinolytic fungus, Doratomyces microsporus, genomic DNA library, DNA isolation