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

Mycology Reference Laboratory, Health Protection Agency, South-West Regional Laboratory, Kingsdown, Bristol, UK.
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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Available from: Christopher J Linton, Jul 31, 2014
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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 Experiments based on these methods were successively carried out: they did not improve PCR efficiency (unpublished observations).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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    ABSTRACT: Background Saccharomyces cerevisiae is extensively used in bio-industries. However, its genetic engineering to introduce new metabolism pathways can cause unexpected phenotypic alterations. For example, humanisation of the glycosylation pathways is a high priority pharmaceutical industry goal for production of therapeutic glycoproteins in yeast. Genomic modifications can lead to several described physiological changes: biomass yields decrease, temperature sensitivity or cell wall structure modifications. We have observed that deletion of several N-mannosyltransferases in Saccharomyces cerevisiae, results in strains that can no longer be analyzed by classical PCR on yeast colonies. Findings In order to validate our glyco-engineered Saccharomyces cerevisiae strains, we developed a new protocol to carry out PCR directly on genetically modified yeast colonies. A liquid culture phase, combined with the use of a Hot Start DNA polymerase, allows a 3-fold improvement of PCR efficiency. The results obtained are repeatable and independent of the targeted sequence; as such the protocol is well adapted for intensive screening applications. Conclusions The developed protocol enables by-passing of many of the difficulties associated with PCR caused by phenotypic modifications brought about by humanisation of the glycosylation in yeast and allows rapid validation of glyco-engineered Saccharomyces cerevisiae cells. It has the potential to be extended to other yeast strains presenting cell wall structure modifications.
    BMC Research Notes 05/2013; 6(1):201. DOI:10.1186/1756-0500-6-201
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    • "Although great effort has been made to understand the biological and molecular basis of brown rust infection, there is a lack of standardized and specific protocols for routine molecular biology research of this organism, although these are commonly available for other fungal research. Current methods of DNA extraction from P. triticina and other fungal pathogens are either time-consuming and or are based on expensive technologies (muller et al. 1998; fAggi et al. 2005; BormAn et al. 2006; Cheng and JiAng 2006). They include the use of SDS/CTAB/proteinase K (Wilson 1990), SDS lysis (syn and sWArup 2000), lysozyme/SDS (flAmm et al. 1984), high-speed cell disruption (muller et al. 1998) and beadvortexing/SDS lysis (sAmBrooK and Russel 2001). "
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    • "and M. oryzae), nine oomycetes (Phytophthora europaea, P. cactorum , P. quercina, P. citricola, P. citrophthora, P. cambivora, P. cactorum, P. botryose and Pythium sp.), five uncharacterized lichens, and four plant species (Arabidopsis thaliana, Oryza sativa, Solanum lycopersicum and Brassica oleracea). Although several simplified methods for fungal DNA extraction have been developed, they provide genomic DNA applicable only for PCR (Borman et al., 2006; Cenis, 1992; Liu et al., 2000; Thomson and Henry, 1995), use phenol and chloroform (Cassago et al., 2002; Guo et al., 2005), or require expensive instruments and supplies (Loeffler et al., 2002; Muller et al., 1998). The QS method provides not only efficiency in time, space, and cost but also high quality and quantity of DNA with organic solvent-free procedures. "
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