SYBR safeTM efficiently replaces ethidium bromide in Aspergillus fumigatus gene disruption.

H. M. S. Canela, L. A. Takami, M. Ferreira
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引用次数: 1

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 safeTM efficiently replaced ethidium bromide, making this dye a safe and efficient alternative for DNA staining in A. fumigatus gene disruption.
SYBR safeTM在烟曲霉基因破坏中有效替代溴化乙啶。
侵袭性曲霉病是一种死亡率很高的疾病,主要由烟曲霉引起。可获得的药物有限,这种疾病继续以不可接受的频率发生。在寻找新的药物靶点时,基因破坏是必不可少的。描述了一种有效的烟曲霉基因破坏方案,但它需要溴化乙啶(一种基因毒性剂)进行DNA染色。因此,本研究在烟曲霉基因破坏方案中测试了SYBR safeTM,一种无基因毒性的DNA染色剂。所选择的基因是cipC,我们的实验室已经成功地对其进行了破坏。在酿酒酵母中构建了一个缺失盒,并将其用于烟曲霉的转化。测试的DNA染色之间没有统计学差异。溴化乙啶的转化成功率为63.3%,SYBR safeTM的转化成功率为70%。对烟曲霉基因破坏,溴化乙啶的成功率为100%,SYBR safeTM的成功率为97%。综上所述,SYBR safeTM有效地取代了溴化乙啶,使其成为烟曲霉基因破坏DNA染色的安全有效的替代染料。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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