Thermal stability of quadruplex primers for highly versatile isothermal DNA amplification
Thermal stability of quadruplex primers for highly versatile isothermal DNA amplification
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DOI：
10.1016/j.bpc.2013.10.008
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发表时间：
2014-01-01
影响因子：
3.8
通讯作者：
Kankia, Besik
中科院分区：
文献类型：
--
作者：
Mathias, Jordan;Okyere, Robert;Kankia, Besik
四联引物扩增 (QPA) 允许核酸等温扩增，提高产量并简化检测。该测定基于 DNA 四链体 GGGTGGGTGGGTGGG (G3T)，它在特定阳离子存在下具有异常高的热稳定性。 QPA 采用截短的 G3T 序列作为引物，在聚合酶延伸时，其从结合位点自行解离，并允许下一轮引发，而无需扩增子热解折叠。扩增速率很大程度上取决于引物/引物结合位点 (PBS) 复合物的热稳定性，迄今为止 QPA 已被证明可以在较窄的温度范围内工作。为了扩展 QPA 的功能，在本研究中，我们研究了野生型 G3T 和包含 5' 端序列修饰或延伸的变体的折叠和热力学特性。圆二色性研究表明，用其他核苷酸取代胸苷或在 5' 端添加 GC 不会改变 G3T 的平行折叠。热解折叠实验表明，在环位置掺入的嘌呤碱基和5'端二核苷酸延伸显着破坏了四链体的稳定性，而环嘧啶几乎没有影响。总体而言，这些研究的结果表明，线性等温 QPA 可以在很宽的温度范围内进行，以适应嗜热和嗜温 DNA 聚合酶。 (C) 2013 Elsevier B.V. 保留所有权利。
Quadruplex priming amplification (QPA) allows isothermal amplification of nucleic acids with improved yield and simplified detection. This assay is based on a DNA quadruplex, GGGTGGGTGGGTGGG (G3T), which in the presence of specific cations possesses unusually high thermal stability. QPA employs truncated G3T sequences as primers, which upon polymerase elongation, self-dissociate from the binding site and allow the next round of priming without thermal unfolding of amplicons. The rate of amplification strongly depends on the thermal stability of the primer/primer binding site (PBS) complex and to date QPA has been demonstrated to work over a narrow temperature range. To expand the capabilities of QPA, in the present study, we studied the fold and thermodynamic properties of the wild-type G3T and variants containing sequence modifications or extensions at the 5'-end. Circular dichroism studies demonstrate that the substitution of thymidines by other nucleotides or GC addition at the 5'-end does not change the parallel fold of G3T. Thermal unfolding experiments revealed that purine bases incorporated at loop positions and 5'-end dinucleotide extension significantly destabilize the quadruplex, while loop pyrimidines have almost no effect. Overall, the results of these studies suggest that linear isothermal QPA can be performed over a wide temperature range to accommodate both thermophilic and mesophilic DNA polymerases. (C) 2013 Elsevier B.V. All rights reserved.