Research
| Title： | Sensitive detection of oxidative DNA damage in cyanobacterial cells using supercoiling-sensitive quantitative PCR |
|---|---|
| First author： | Chen, Zhilan; Tian, Yun; Zhu, Chenhong; Liu, Biyun; Zhang, Yongyuan; Lu, Zhiying; Zhou, Qiaohong; Wu, Zhenbin |
| Journal： | CHEMOSPHERE |
| Years： | 2018 |
| DOI： | 10.1016/j.chemosphere.2018.06.154 |
| Abstract： | Supercoiling-sensitive quantitative PCR (ss-qPCR) is a sensitive technique to detect DNA damage in cultured animal cells and cultured/clinical human cells in vitro. In this study, we investigated whether the ss-qPCR method can be applied as a sensitive means to detect oxidative DNA damage in unicellular organisms. We used the model cyanobacterium Synechococcus elongatus PCC 7942 as a test organism and H2O2 as an exogenetic oxidative toxicant. Results showed that a significant increase in the plasmid DNA damage of S. elongatus PCC 7942 was induced by H2O2 in a dose- and time-dependent manner. The sensitivity of ss-qPCR in detecting DNA damage of the cyanobacterium was higher than the cell inhibition method (up to 255 times) as calculated from the slopes of fitted curves in the tested sub-toxic concentration range of 1-5 mM H2O2. Ss-qPCR also detected repairable low-intensity DNA damage in the cyanobacterium when DNA repair inhibitors were used. The detection limit of modified ss-qPCR was one tenth of that of previous methods. We also observed that ss-qPCR can be used to detect genomic DNA conformation change of cyanobacterium exposed to H2O2. Thus, this method will provide a powerful technical support for investigating the mechanisms of cyanobacterial DNA damage by environmental factors, especially intracellular reactive oxygen species enhancement-related factors. (C) 2018 Published by Elsevier Ltd. |
