Ultra-rapid preparation of total genomic DNA from isolates of yeast and mould using Whatman FTA filter paper technology - a reusable DNA archiving system.
ABSTRACT 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.
- SourceAvailable from: nih.govNucleic Acids Research 06/1992; 20(9):2380. · 8.28 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Invasive pulmonary aspergillosis (IPA) is an important cause of mortality and morbidity in the immunocompromised host. However, the diagnosis of this condition may be difficult, and it is sometimes missed because of the lack of sensitivity of available tests. Therefore, we used polymerase chain reaction (PCR)-based amplification of fragments of genes-encoding alkaline proteases from Aspergillus fumigatus and A. flavus to detect these organisms in bronchoalveolar lavage fluid specimens. The predicted size of the product (747 base pairs) after amplification of A. fumigatus was larger than that for A. flavus (690 base pairs). The reaction was highly sensitive (after amplification of 500 fg of A. fumigatus DNA, product could be detected by Southern analysis), and it was specific for A. fumigatus and A. flavus. Bronchoalveolar lavage fluid from four immunosuppressed patients with proved or probable IPA was positive by this assay (sensitivity, 100%); in addition, the sample from one patient with possible IPA was PCR-positive. Only one specimen from 18 immunosuppressed patients with no evidence of IPA was PCR-positive (specificity, 94.4%). Five of 28 bronchoalveolar lavage samples from nonimmunosuppressed patients were PCR-positive, probably representing colonization of the respiratory tract. PCR-based detection may prove useful in the diagnosis of IPA.The American review of respiratory disease 12/1993; 148(5):1313-7. · 10.19 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The aim of this study was to establish and validate a method to permit microsatellite analysis of DNA profiles obtained from frozen-thawed stallion sperm cells. This would provide reliable and accurate verification of the identification of a semen donor. Ejaculates from 5 pony stallions were collected, processed and frozen in 0.5 ml plastic straws. Aliquots of 100 microl of the frozen-thawed semen thus obtained were either placed directly, or diluted (1:10; 1:100; and 1:1000) and placed on slides of FTA paper. Similarly, blood samples obtained from each of the stallions were placed onto slides of FTA paper. A punch was removed from each sample after drying Each sample was mixed with FTA purification reagent, Dithiothreitol and Proteinase K before incubation and processing. All samples were processed with a set of 13 microsatellite markers. Further analysis permitted a comparison of the DNA profiles of the frozen-thawed semen and the blood samples. A full profile of markers was obtained from the 1:10 and 1:100 dilutions of the frozen-thawed semen samples as well as from the blood samples. The DNA profiles from the frozen-thawed semen and blood samples obtained from the stallions matched in all cases.Journal of the South African Veterinary Association 01/2003; 73(4):222-3.