Abstract
BackgroundMethods 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.ResultsExtractions carried out by this method provided approximately 2 μg 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.ConclusionsThe 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.
Highlights
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 is usually because most of the available protocols include the growth of mycelium in a liquid culture, followed by maceration in liquid nitrogen
The method involves the growth of spores on small cellophane disks placed on a solid medium, ressuspension of the grown pellicle in an adequate buffer, cell disruption using glass beads, and subsequent DNA purification either by classical phenol-chloroform extraction followed by DNA precipitation or by a simple and fast direct precipitation
Summary
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. The methods applied to filamentous fungi, such as Trichoderma reesei, are often laborious and time consuming This is usually because most of the available protocols include the growth of mycelium in a liquid culture, followed by maceration in liquid nitrogen. The method involves the growth of spores on small cellophane disks placed on a solid medium, ressuspension of the grown pellicle in an adequate buffer, cell disruption using glass beads, and subsequent DNA purification either by classical phenol-chloroform extraction followed by DNA precipitation or by a simple and fast direct precipitation
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