Ultra-rapid preparation of total genomic DNA from isolates of yeast and mould using Whatman FTA filter paper technology - a reusable DNA archiving system.
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.
Borman
Andrew M
AM
Mycology Reference Laboratory, Health Protection Agency, South-West Regional Laboratory, Kingsdown, Bristol, UK. Andy.Borman@ubht.swest.nhs.uk
Linton
Christopher J
CJ
Miles
Sarah-Jane
SJ
Campbell
Colin K
CK
Johnson
Elizabeth M
EM
D004271:DNA, Fungal / Q000302:isolation & purification*; D005658:Fungi / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D050296:Microbial Viability; D008872:Microwaves; D009181:Mycoses / Q000175:diagnosis / Q000382:microbiology; D010209:Paper; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D004271:DNA, Fungal