Ultra-rapid preparation of total genomic DNA from isolates of yeast and mould using Whatman FTA filter paper technology - A reusable DNA archiving system

University of Bristol, Bristol, England, United Kingdom
Medical Mycology (Impact Factor: 2.34). 09/2006; 44(5):389-98. DOI: 10.1080/13693780600564613
Source: PubMed


Conventional methods for purifying PCR-grade fungal genomic DNA typically require cell disruption (either physical or enzymatic) coupled with laborious organic extraction and precipitation stages, or expensive column-based technologies. Here we present an easy and extremely rapid method of preparing yeast and mould genomic DNAs from living cultures using Whatman FTA filter matrix technology. Aqueous suspensions of yeast cells or hyphal fragments and conidia (in the case of moulds) are applied directly (or after freeze-thawing) to dry FTA filters. Inoculated filters are then subjected to brief microwave treatment, to dry the filters and inactivate the organisms. Filter punches are removed, washed rapidly, dried and placed directly into PCR reactions. We show that this procedure inactivated all of the 38 yeast and 75 mould species tested, and generated PCR-grade DNA preparations in around 15 minutes. A total of 218 out of 226 fungal isolates tested liberated amplifiable DNA after application to FTA filters. Detection limits with yeast cultures were approximately 10 colony-forming units per punch. Moreover, we demonstrate that filter punches can be recovered after PCR, washed and used in fresh PCR reactions without detectable cross-contamination. Whatman FTA technology thus represents a cheap, ultra-rapid method of fungal genomic DNA preparation, and also potentially represents a powerful fungal DNA archiving and storage system.

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    • "Breaking this organelle is a prerequisite for DNA amplification by PCR on yeast colonies. Different techniques, applied to DNA or protein extraction, have been described for several decades: lysis by chemicals such as SDS (Sodium Dodecyl Sulfate) [36,37], cell wall disruption by thermal choc [38-40], sonication [41,42] and microwaves [43,44]. Experiments based on these methods were successively carried out: they did not improve PCR efficiency (unpublished observations). "
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