Mutation detection by electrocatalysis at DNA-modified electrodes
Mutation detection by electrocatalysis at DNA-modified electrodes
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DOI：
10.1038/80301
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发表时间：
2000-10-01
影响因子：
46.9
通讯作者：
Barton, JK
中科院分区：
文献类型：
--
作者：
Boon, EM;Ceres, DM;Barton, JK
突变和DNA碱基受损的检测对于遗传病的早期诊断非常重要。在这里，我们描述了一种基于通过DNA膜的电荷传输的电催化方法，用于检测完全杂交的双链中的单碱基错配和DNA碱基损伤。用预组装的DNA双链体修饰的金电极用于监测氧化还原活性DNA嵌入剂亚甲基蓝与PFe(CN)6(3-)的电催化信号。不匹配或损坏的DNA碱基的存在会大大减弱电催化信号。由于这项检测不是差异杂交的一种手段，所有单碱基错配，包括热力学上稳定的GT和GA错配，都可以在没有严格的杂交条件的情况下被检测到。此外，人类p53基因组中许多常见的DNA损伤和“热点”突变可以与完美双链区分开来。最后，我们演示了该技术在基于芯片的格式中的应用。该系统为检测DNA序列的完整性提供了一种灵敏的方法，也为单碱基错配检测提供了一种全新的方法。
Detection of mutations and damaged DNA bases is important for the early diagnosis of genetic disease. Here we describe an electrocatalytic method for the detection of single-base mismatches as well as DNA base lesions in fully hybridized duplexes, based on charge transport through DNA films. Gold electrodes modified with preassembled DNA duplexes are used to monitor the electrocatalytic signal of methylene blue, a redox-active DNA intercalator, coupled to pFe(CN)6](3-). The presence of mismatched or damaged DNA bases substantially diminishes the electrocatalytic signal. Because this assay is not a measure of differential hybridization, all single-base mismatches, including thermodynamically stable GT and GA mismatches, can be detected without stringent hybridization conditions. Furthermore, many common DNA lesions and "hot spot" mutations in the human p53 genome can be distinguished from perfect duplexes. Finally, we have demonstrated the application of this technology in a chip-based format. This system provides a sensitive method for probing the integrity of DNA sequences and a completely new approach to single-base mismatch detection.