SYBR safe^TM efficiently replaces ethidium bromide in Aspergillus fumigatus gene disruption

Abstract

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 safe TM , 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 safe TM . For A. fumigatus gene disruption, the success rate for ethidium bromide was 100 and 97% for SYBR safe TM . In conclusion, SYBR safe TM efficiently replaced ethidium bromide, making this dye a safe and efficient alternative for DNA staining in A. fumigatus gene disruption.

Moving towards improved surveillance and earlier diagnosis of aquatic pathogens: From traditional methods to emerging technologies

“…Each PCR cycle provides a doubling of the targeted fragment resulting in over a billion copies (1.07x10 9 ) from 30 amplification cycles. DNA generating products can be visualised through gel electrophoresis where the size (in bp) can be confirmed against known size markers; a visualisation process that historically used the carcinogen ethidium bromide, but there are now alternatives, such as SYBR Safe 127 . Key to the success of PCR are the primers, which can either be designed specifically for a group or species of pathogens or nonspecific/degenerate when looking for more general groups of pathogens.…”

“…DNA generating products can be visualised through gel electrophoresis where the size (in bp) can be confirmed against known size markers; a visualisation process that historically used the carcinogen ethidium bromide, but there are now alternatives, such as SYBR Safe. 127 Key to the success of PCR are the primers, which can either be designed specifically for a group or species of pathogens or nonspecific/degenerate when looking for more general groups of pathogens. Sequencing of PCR products is particularly beneficial for disease diagnostics to identify pathogens to species and even strain level, mainly if general primers have been used.…”