A self-assembled deoxyribonucleic acid concatemer for sensitive detection of single nucleotide polymorphism |
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Authors: | Wei Wu Junhua Chen Zhiyuan Fang Chenchen GeZhicheng Xiang Chuanyan OuyangPuchang Lie Zhuo XiaoLuxin Yu Lin WangLingwen Zeng |
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Affiliation: | Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China |
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Abstract: | Polymerase-free and label-free strategies for DNA detection have shown excellent sensitivity and specificity in various biological samples. Herein, we propose a method for single nucleotide polymorphism (SNP) detection by using self-assembled DNA concatemers. Capture probes, bound to magnetic beads, can joint mediator probes by T4 DNA ligase in the presence of target DNA that is complementary to the capture probe and mediator probe. The mediator probes trigger self-assembly of two auxiliary probes on magnetic beads to form DNA concatemers. Separated by a magnetic rack, the double-stranded concatemers on beads can recruit a great amount of SYBR Green I and eventually result in amplified fluorescent signals. In comparison with reported methods for SNP detection, the concatemer-based approach has significant advantages of low background, simplicity, and ultrasensitivity, making it as a convenient platform for clinical applications. As a proof of concept, BRAFT1799A oncogene mutation, a SNP involved in diverse human cancers, was used as a model target. The developed approach using a fluorescent intercalator can detect as low as 0.1 fM target BRAFT1799A DNA, which is better than those previously published methods for SNP detection. This method is robust and can be used directly to measure the BRAFT1799A DNA in complex human serum with excellent recovery (94–103%). It is expected that this assay principle can be directed toward other SNP genes by simply changing the mediator probe and auxiliary probes. |
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Keywords: | MB, magnetic bead SNP, single nucleotide polymorphism PBS, phosphate buffer saline dsDNA, double-stranded DNA T1, target DNA CP, capture probe MP, mediator probe AP, auxiliary probe WT1, wildtype DNA bsDNA, biotin-modified single-stranded DNA SA, streptavidin |
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