DNA Melting Analysis with Optofluidic Lasers Based on Fabry-Perot Microcavity
DNA Melting Analysis with Optofluidic Lasers Based on Fabry-Perot Microcavity
复制标题
基于法布里-珀罗微腔的光流控激光器 DNA 熔解分析
DOI：
10.1021/acssensors.8b00481
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发表时间：
2018
期刊：
影响因子：
8.9
通讯作者：
Fan Xudong
中科院分区：
文献类型：
--
作者：
Hou Mengdi;Liang Xiyue;Zhang Tingting;Qiu Chengyu;Chen Jingdong;Liu Shaoding;Wang Wenjie;Fan Xudong
我们使用基于法布里-珀罗微腔的光流体激光器进行DNA高分辨率熔解（HRM）分析。与荧光HRM相比，激光HRM具有发射强度高、信噪比好、跃迁陡、温度分辨率高等优点。此外，通过优化激光条件，如外泵浦和腔Q因子，可以降低熔化温度。在这项工作中，我们首先从理论上分析了基于激光的HRM。然后以三种不同GC含量的长DNA序列为模型系统进行实验，一种是99个碱基，另两种是130个碱基。我们表明，基于激光的HRM是能够区分目标和单碱基错配的DNA，只要130个碱基和近50%的GC含量。首先实验研究了每个DNA样品的激光阈值对温度的依赖性，通过优化外部泵浦，与基于荧光的HRM相比，对于长达130个碱基的长DNA序列，熔化温度降低了10 °C以上。最后，我们展示了一种替代方法，使用基于激光的HRM的快速DNA筛选，不存在基于荧光的HRM，其中激光激发在固定温度下扫描，以区分目标和碱基错配的DNA序列。结果表明，具有近50%GC含量的130个碱基长的DNA在靶序列和单碱基错配序列之间可以具有多达20%的激光阈值差异和40%的激光输出斜率差异，尽管它们的解链温度仅相差0.5 °C，表明激光激发扫描法也适用于GC含量较高的长DNA序列。
We conduct DNA high-resolution melting (HRM) analysis using optofluidic lasers based on a Fabry-Pérot microcavity. Compared to the fluorescence-based HRM, the laser-based HRM has advantages of higher emission intensity for better signal-to-noise ratio and sharper transition for better temperature resolution. In addition, the melting temperature can be lowered by optimizing the laser conditions such as external pump and cavityQ-factor. In this work, we first theoretically analyze the laser-based HRM. Then experiments are performed on three long DNA sequences as model systems, one being 99 bases and the other two being 130 bases long but with different GC contents. We show that the laser-based HRM is able to distinguish the target and the single-base mismatched DNA as long as 130 bases and with nearly 50% GC content. The dependence of laser threshold on the temperature for each DNA sample is first experimentally investigated and by optimizing the external pump, the melting temperature is reduced by more than 10 °C, compared to the fluorescence-based HRM for long DNA sequences up to 130 bases. Finally, we demonstrate an alternative method of using the laser-based HRM for rapid DNA screening that does not exist for the fluorescence-based HRM, in which laser excitation is scanned at a fixed temperature to distinguish the target and the base-mismatched DNA sequences. It is shown that the 130-bases-long DNA with nearly 50% GC content can have as much as 20% difference in the laser threshold and 40% difference in the laser output slope between the target and the single-base mismatched sequences, despite only 0.5 °C difference in their melting temperature, indicating that the laser-excitation-scanning method can also be suitable for long DNA sequences with higher GC content.