Cellophane based mini-prep method for DNA extraction from the filamentous fungus Trichoderma reesei

Abstract

Background: Methods for the extraction of DNA from filamentous fungi are frequently laborious and time consuming because most of the available protocols include maceration in liquid nitrogen after the mycelium has been grown in a liquid culture. This paper describes a new method to replace those steps, which involves the growth of the mycelium on cellophane disks overlaid on solid medium and the use of glass beads for cell wall disruption.

Results: Extractions carried out by this method provided approximately 2 microg of total DNA per cellophane disk for the filamentous fungus Trichoderma reesei. To assess the DNA's quality, we made a PCR (Polymerase Chain Reaction) amplification of a gene introduced by a transformation in this fungus's genome (hph gene), with successful results. We also confirmed the quality of the DNA by the use of Southern blotting to analyze the presence of the same gene, which was easily detected, resulting in a sharply defined and strong band.

Conclusions: The use of this method enabled us to obtain pure DNA from Trichoderma reesei, dispensing with the laborious and time-consuming steps involved in most protocols. The DNA obtained was found to be suitable for PCR and Southern blot analyses. Another advantage of this method is the fact that several samples can be processed simultaneously, growing the fungus on multiple well cell culture plates. In addition, the absence of maceration also reduces sample handling, minimizing the risks of contamination, a particularly important factor in work involving PCR.

Figure 1

DNA quality analysis. A – Agarose gel electrophoresis of DNA sample purified by the cellophane disk miniprep method. Lane 1 (1 Kb ladder), lane 2–3, genomic DNA from Trichoderma reesei transformants 1 and 2, respectively. B – Amplified hph fragment from DNAs prepared by the cellophane-base method. Agarose gel electrophoresis of PCR samples. Lane 1 (1 Kb ladder), lane 2–3, 750 pb PCR products from transformant 1 and 2, respectively. C – Detection of hph gene in DNA prepared from transformants of Trichoderma reesei. The DNA obtained by the cellophane method was BamHI and EcoRI restricted and subjected to Southern Blot, using an α-³²PdATP labeled fragment of the hph gene as a probe. Lane 1 and 4 (1 Kb DNA ladder, probed against itself), lane 2 and 5, restricted DNA from hph Trichoderma reesei transformant 1 and 2, respectively; 3 and 6, DNA from untransformed Trichoderma reesei (control).