Extraction and PCR amplification of DNA from minute ectoparasitic fungi

Laboulbeniales is a large order of ascomycete fungi characterized by microscopic size, determinate growth patterns, thalloid morphology, and obligately parasitic lifestyle that complicate the application of molecular phylogenetic methods. Here, we describe improved techniques for the isolation of genomic DNA from Laboulbeniales, and use of the resulting extracts for PCR amplification of 18S rDNA and 28S rDNA. The important modification we report is the extraction and retrieval of DNA on dry ice; the dramatic improvements in consistency and yield indicate that these protocols could be applied successfully to a wide range of small, melanized organisms that cannot be grown in culture.