title: SYBR safeTMefficiently replaces ethidium bromide in Aspergillus fumigatus gene disruption.
abstract: Invasive aspergillosis is a disease responsible for high mortality rates, caused mainly by Aspergillus fumigatus. The available drugs are limited and this disease continues to occur at an unacceptable frequency. Gene disruption is essential in the search for new drug targets. An efficient protocol for A. fumigatus gene disruption was described but it requires ethidium bromide, a genotoxic agent, for DNA staining. Therefore, the present study tested SYBR safeTM, a non-genotoxic DNA stain, in A. fumigatus gene disruption protocol. The chosen gene was cipC, which has already been disrupted successfully in our laboratory. A deletion cassette was constructed in Saccharomyces cerevisiae and used in A. fumigatus transformation. There was no statistical difference between the tested DNA stains. The success rate of S. cerevisiae transformation was 63.3% for ethidium bromide and 70% for SYBR safeTM. For A. fumigatus gene disruption, the success rate for ethidium bromide was 100 and 97% for SYBR safeTM. In conclusion, SYBR safeTMefficiently replaced ethidium bromide, making this dye a safe and efficient alternative for DNA staining in A. fumigatus gene disruption.
authors.affiliation: Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil.
authors.forename: H M S
authors.initials: HM
authors.lastname: Canela
authors.affiliation: Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil.
authors.forename: L A
authors.initials: LA
authors.lastname: Takami
authors.affiliation: Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil mesfe@fcfrp.usp.br.
authors.forename: M E S
authors.initials: ME
authors.lastname: Ferreira
mesh_terms: D001232:Aspergillus fumigatus / Q000187:drug effects* / Q000235:genetics*; D004249:DNA Damage / Q000187:drug effects*; D004996:Ethidium / Q000633:toxicity*; D005456:Fluorescent Dyes / Q000633:toxicity*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics
chemical_list: D005456:Fluorescent Dyes; D004996:Ethidium