Mismatch Extension of DNA Polymerases and High-Accuracy Single Nucleotide Polymorphism Diagnostics by Gold Nanoparticle-Improved Isothermal Amplification
Mismatch Extension of DNA Polymerases and High-Accuracy Single Nucleotide Polymorphism Diagnostics by Gold Nanoparticle-Improved Isothermal Amplification
复制标题
DNA 聚合酶的错配延伸和金纳米粒子改进等温扩增的高精度单核苷酸多态性诊断
DOI：
10.1021/acs.analchem.5b01545
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发表时间：
2015-09-01
影响因子：
7.4
通讯作者：
Zhao, Yongxi
中科院分区：
文献类型：
--
作者：
Chen, Feng;Zhao, Yue;Zhao, Yongxi
序列错配可能会引起非特异性延伸反应，从而导致 SNP 诊断出现错误结果。在此，我们。系统地研究了各种 3' 末端错配对重复 DNA 聚合酶催化的等温扩增的影响。尽管它们的效率取决于错配的类型和 DNA：聚合酶的种类，但所有 12 种单一 3' 末端错配都会诱导延伸反应。一般来说，只有几种错配（引物-模板、C-C、G-A、A-G 和 A-A）对扩增反应呈现可观察到的抑制作用，而其他错配触发的扩增信号与 Watson-Crick 对的信号一样高。深入讨论了相关机制，总结了特异性SNP分析的引物设计指南。此外，我们发现添加适当的金纳米颗粒（AuNPs）可以显着抑制错配延伸并增强扩增特异性。此外，通过 AuNP 改进的等温扩增，可以对人类血液基因组 DNA 进行高精度 SNP 分析，并通过测序（SNP 测定的金标准方法）验证其结果。总的来说，这项工作提供了对错配行为的机制洞察，并实现了准确的 SNP 诊断，在分子诊断和个性化医疗中具有巨大的应用潜力。
Sequence Mismatches may induce nonspecific extension reaction, causing false results for SNP diagnostics. Herein, we. systematically investigated the impact of various 3'-terminal mismatches on isothermal amplification catalyzed by reptesentative DNA polymerases. Despite their diverse efficiencies depending on types of mismatch and kinds, of DNA: polymerase, all 12 kinds of single 3'-terminal mismatches induced the extension reaction. Generally, only several mismatches (primer-template, C-C, G-A, A-G, and A-A) present an observable inhibitory effect on the amplification reaction, whereas other mismatches trigger amplified signals as high as those of Watson-Crick pairs. The related mechanism was deeply discussed, and a primer-design guideline for specific SNP analysis was summarized. Furthermore, we found that the addition of appropriate gold nanoparticles (AuNPs) can significantly inhibit mismatch extension and enhance the amplification specificity. Also the high-accuracy SNP analysis of human blood genomic DNA has been demonstrated by AuNPs-improved isothermal amplification, the result of which was verified by sequencing (the gold standard method for SNP assay). Collectively, this work provides mechanistic insight into mismatch behavior and achieves accurate SNP diagnostics, holding great potential for the application in molecular diagnostics and personalized medicine.