A practical method for the preparation of total DNA from filamentous fungi

Borges, M. I.; Azevedo, M. O.; Bonatelli, Renato; Felipe, M. S. S.; Filho, Seixas

doi:10.4148/1941-4765.1466

Cited by 45 publications

(39 citation statements)

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“…Afterward, the mixture was incubated at 37°C under agitation in a thermoshaker operated at 35 turns min Ϫ1 for 3 h. DNA was obtained after two phenol-chloroform extractions and ethanol precipitation. Finally, DNA quantification was performed by comparison with DNA standards electrophoresed in agarose gels (2).…”

Section: Methodsmentioning

confidence: 99%

Randomly Amplified Polymorphic DNA as a Valuable Tool for Epidemiological Studies ofParacoccidioides brasiliensis

et al. 2003

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Randomly amplified polymorphic DNA (RAPD) has been successfully used to detect genetic variations among isolates of Paracoccidioides brasiliensis. However, the usefulness of this technique for assessing important parasitic properties is still unconfirmed. In the present work we further investigated the applicability of RAPD in revealing important intrinsic and extrinsic features of this fungus associated with geographical origin, time of isolation, source of clinical specimen, clinical forms of human disease and also in vitro and in vivo susceptibility to antimicrobial and antifungal drugs. The RAPD patterns allowed us to distinguish all of the analyzed strains, which included 26 clinical isolates, 2 animal isolates, and 1 environmental isolate of P. brasiliensis obtained from different geographic regions, confirming the strong discriminating power of this technique. A phenetic tree, build from the RAPD data, showed that although the two nonclinical Brazilian strains were set together the majority of the clinical Brazilian strains were randomly distributed through different sub-branches of a major cluster without any correlation to any of the parameters analyzed. A second major cluster, however, has grouped isolates from Mato Grosso and Roraima (Brazil) that not only were susceptible in vitro to trimethoprim-sulfamethoxazole but also produced a good in vivo response. These results open new vistas for epidemiological and clinical studies of P. brasiliensis.Paracoccidioides brasiliensis is a dimorphic fungus alternating between the mycelial form observed in room temperatures and the yeast form found in infected tissues or in culture at 35 to 37°C. The human paracoccidioidomycosis caused by this fungus is characterized by a range of clinical manifestations from benign or asymptomatic forms to severe and disseminated disease that is often fatal. The exact causes of the clinical pleomorphism are not understood, but it is reasonable to presume that factors associated with both the fungus and the patient are involved. In relation to the fungus, although wide biological and biochemical diversity has been observed, no clear correlation with pathogenesis has been demonstrated (3,9,12,13,17,20,23). Until now very little has been known about the genome sequences of P. brasiliensis; hence, the use of molecular approaches for isolate typing has been difficult.The development of the randomly amplified polymorphic DNA (RAPD) technique represented a landmark in the molecular characterization of diverse organisms, especially for microbial strain identifications when very few genomic sequences are available. In relation to other molecular techniques, RAPD analysis offers the advantage of requiring small amounts of DNA, simpler procedures, and the use of arbitrary primers (26). Due to these characteristics, RAPD has been widely used for human diseases to detect genomic variations among isolates of fungi such as Histoplasma capsulatum (27), Cryptococcus neoformans (19), Aspergillus fumigatus (1), and P. brasiliensis (3,9,12,13,23). ...

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Section: Methodsmentioning

confidence: 99%

Randomly Amplified Polymorphic DNA as a Valuable Tool for Epidemiological Studies ofParacoccidioides brasiliensis

et al. 2003

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“…Basidiocarps were ground in liquid nitrogen and total RNA was extracted using a hot phenol method (Dudler and Hertig, 1992). Genomic DNA was isolated from dry basidiocarps using a method adapted for filamentous fungi (Borges et al, 1999). Plasmid DNA was purified from Escherichia coli using the GFX Micro plasmid prep (Amersham Pharmacia Biotech, Amersham, UK).…”

Section: Nucleic Acid Isolationmentioning

confidence: 99%

Hydrophobins DGH1, DGH2, and DGH3 in the Lichen-Forming Basidiomycete Dictyonema glabratum

Trembley

Ringli

Honegger

2002

Fungal Genetics and Biology

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“…H. grisea var. thermoidea total DNA was prepared according to Borges et al [14]. Southern blot analysis was carried out using standard techniques [13] with 5^10 Wg DNA from the wild-type strain and transformants.…”

Section: Nucleic Acid Manipulationsmentioning

confidence: 99%

Genetic transformation of germinated conidia of the thermophilic fungus Humicola grisea var. thermoidea to hygromycin B resistance

Dantas‐Barbosa

1998

FEMS Microbiology Letters

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Germinated conidia of the thermophilic fungus Humicola grisea var. thermoidea were transformed to hygromycin B resistance using the plasmid pAN7.1. Transformation was achieved using lithium acetate treatment or electroporation. The efficiency of transformation was up to 32 and 25 transformants per Wg of plasmid DNA with the two methods, respectively. Transformants obtained by the lithium acetate method were more stable and showed a high copy number of the hph gene integrated into their genome. The other transformants, from the electroporation procedure, were stable, but unable to grow in the presence of high levels of hygromycin, and detection of the hph gene was only possible by polymerase chain reaction analysis. z

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A practical method for the preparation of total DNA from filamentous fungi

Cited by 45 publications

References 0 publications

Randomly Amplified Polymorphic DNA as a Valuable Tool for Epidemiological Studies ofParacoccidioides brasiliensis

Randomly Amplified Polymorphic DNA as a Valuable Tool for Epidemiological Studies ofParacoccidioides brasiliensis

Hydrophobins DGH1, DGH2, and DGH3 in the Lichen-Forming Basidiomycete Dictyonema glabratum

Genetic transformation of germinated conidia of the thermophilic fungus Humicola grisea var. thermoidea to hygromycin B resistance

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