Selective enzymatic cleavage and labeling for sensitive capillary electrophoresis laser-induced fluorescence analysis of oxidized DNA bases
文献类型：期刊论文
| 作者 | Li, Cuiping; Wang, Hailin |
| 刊名 | JOURNAL OF CHROMATOGRAPHY A
 |
| 出版日期 | 2015-08-07 |
| 卷号 | 1406页码:324-330 |
| 关键词 | Oxidized DNA bases Formamidopyrimidine glycosylase (FPG) Capillary electrophoresis laser-induced fluorescence (CE-LIF) Quantum dot (QD) |
| 英文摘要 | Oxidatively generated DNA damage is considered to be a significant contributing factor to cancer, aging, and age-related human diseases. It is important to detect oxidatively generated DNA damage to understand and clinically diagnosis diseases caused by oxidative damage. In this study, using selective enzymatic cleavage and quantum dot (QD) labeling, we developed a novel capillary electrophoresislaser induced fluorescence method for the sensitive detection of oxidized DNA bases. First, oxidized DNA bases are recognized and removed by one DNA base excision repair glycosylase, leaving apurinic and apyrimidinic sites (AP sites) at the oxidized positions. The AP sites are further excised by the AP nicking activity of the chosen glycosylase, generating a nucleotide gap with 5'- and 3'- phosphate groups. After dephosphorylation with one alkaline phosphatase, a biotinylated ddNTP is introduced into the nucleotide space within the DNA strand by DNA polymerase I. The biotin-tagged DNA is further labeled with a QD-streptavidin conjugate via non-covalent interactions. The DNA-bound QD is well-separated from excess DNA-unbound QD by highly efficient capillary electrophoresis and is sensitively detected by online coupled laser-induced fluorescence analysis. Using this method, we can assess the trace levels of oxidized DNA bases induced by the Fenton reaction and UV irradiation. Interestingly, the use of the formamidopyrimidine glycosylase (FPG) protein and endonuclease VIII enables the detection of oxidized purine and pyrimidine bases, respectively. Using the synthesized standard DNA, the approach has low limits of detection of 1.1 x 10(-19) mol in mass and 2.9 pM in concentration. (C) 2015 Elsevier B.V. All rights reserved. |
| 研究领域[WOS] | Biochemical Research Methods ; Chemistry, Analytical |
| WOS记录号 | WOS:000358094200038 |
| 公开日期 | 2016-03-15 |
| 源URL | [http://ir.rcees.ac.cn/handle/311016/32658]  |
| 专题 | 生态环境研究中心_环境化学与生态毒理学国家重点实验室 |
| 推荐引用方式 GB/T 7714 | Li, Cuiping,Wang, Hailin. Selective enzymatic cleavage and labeling for sensitive capillary electrophoresis laser-induced fluorescence analysis of oxidized DNA bases[J]. JOURNAL OF CHROMATOGRAPHY A,2015,1406:324-330. |
| APA | Li, Cuiping,&Wang, Hailin.(2015).Selective enzymatic cleavage and labeling for sensitive capillary electrophoresis laser-induced fluorescence analysis of oxidized DNA bases.JOURNAL OF CHROMATOGRAPHY A,1406,324-330. |
| MLA | Li, Cuiping,et al."Selective enzymatic cleavage and labeling for sensitive capillary electrophoresis laser-induced fluorescence analysis of oxidized DNA bases".JOURNAL OF CHROMATOGRAPHY A 1406(2015):324-330. |
入库方式： OAI收割
来源：生态环境研究中心
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