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Photoactivated Ethidium Monoazide Directly Cleaves Bacterial DNA and Is Applied to PCR for Discrimination of Live and Dead Bacteria
Author(s) -
Soejima Takashi,
Iida Kenichiro,
Qin Tian,
Taniai Hiroaki,
Seki Masanori,
Takade Akemi,
Yoshida Shinichi
Publication year - 2007
Publication title -
microbiology and immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.664
H-Index - 70
eISSN - 1348-0421
pISSN - 0385-5600
DOI - 10.1111/j.1348-0421.2007.tb03966.x
Subject(s) - biology , dna , dna supercoil , bacteria , agarose gel electrophoresis , microbiology and biotechnology , dna gyrase , agarose , topoisomerase iv , topoisomerase , escherichia coli , biochemistry , dna replication , genetics , gene
Ethidium monoazide (EMA) is a DNA intercalating agent and a eukaryotic topoisomerase II poison. We found that EMA treatment and subsequent visible light irradiation (photoactivation or photolysis) shows a bactericidal effect, hence the mechanism was analyzed. When bacterial cells were treated with more than 10 μg/ml of EMA for 1 hr plus photoactivation for 20 min, cleavage of bacterial DNA was confirmed by agarose gel electrophoresis and electron microscopic studies. The cleavage of chromosomal DNA was seen when it was treated in vitro with EMA and photolysis, which showed that the cleavage directly took place without the assistance of DNA gyrase/topoisomerase IV and the DNA repair enzymes of bacteria. It was also verified, by using negatively supercoiled pBR322 DNA, that medium/high concentrations of EMA (1 to 100 μg/ml) led to breaks of double‐stranded DNA and that low concentrations of EMA (10 to 100 ng/ml) generated a single‐stranded break. EMA is known to easily penetrate dead but not live bacteria. After treatment of 10 μg/ml of EMA for 30 min and photoactivation for 5 min, EMA cleaved the DNA of dead but not live Klebsiella oxytoca . When the cleaved DNA was used for templates in PCR targeting 16S rDNA, PCR product from the dead bacteria was completely suppressed. We demonstrated that EMA and photolysis directly cleaved bacterial DNA and are effective tools for discriminating live from dead bacteria by PCR.