Key Laboratory of Biomedical Information Engineering of Education Ministry, School of Life Science and Technology, Xi'an Jiaotong University , Xianning West Road, Xi'an, Shaanxi 710049, P. R. China.
Division of Physical Biology, and Bioimaging Center, Shanghai Synchrotron Radiation Facility, CAS Key Laboraotory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences , Yuquan Road, Shanghai 201800, P. R. China.
Anal Chem. 2015 Sep 1;87(17):8718-23. doi: 10.1021/acs.analchem.5b01545. Epub 2015 Aug 13.
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 representative 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.
序列错配可能会诱导非特异性延伸反应，导致 SNP 诊断出现假阳性结果。在此，我们系统地研究了各种 3'-末端错配对代表性 DNA 聚合酶催化的等温扩增的影响。尽管它们的效率因错配类型和 DNA 聚合酶种类而异，但所有 12 种单 3'-末端错配均诱导了延伸反应。通常，只有几种错配（引物-模板、C-C、G-A、A-G 和 A-A）对扩增反应有明显的抑制作用，而其他错配则引发的扩增信号与 Watson-Crick 碱基对一样高。我们深入探讨了相关机制，并总结了用于特定 SNP 分析的引物设计指南。此外，我们发现添加适当的金纳米粒子（AuNPs）可以显著抑制错配延伸并增强扩增特异性。我们还通过 AuNPs 改进的等温扩增实现了对人血液基因组 DNA 的高精度 SNP 分析，并通过测序（SNP 分析的金标准方法）验证了结果。总的来说，这项工作深入了解了错配行为，并实现了 SNP 的精确诊断，在分子诊断和个性化医疗方面具有很大的应用潜力。
