Key Lab of Advanced Transducers and Intelligent Control System of Ministry of Education , Taiyuan University of Technology , 79 Yingze Street , Taiyuan 030024 , P. R. China.
Department of Biomedical Engineering , University of Michigan , 1101 Beal Avenue , Ann Arbor , Michigan 48109 , United States.
ACS Sens. 2018 Sep 28;3(9):1750-1755. doi: 10.1021/acssensors.8b00481. Epub 2018 Sep 5.
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 cavity Q-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.
我们使用基于 Fabry-Pérot 微腔的光流体激光进行 DNA 高分辨率熔解（HRM）分析。与基于荧光的 HRM 相比，基于激光的 HRM 具有更高的发射强度，从而实现更好的信噪比，以及更陡峭的跃迁，从而实现更好的温度分辨率。此外，可以通过优化激光条件（例如外部泵浦和腔 Q 因子）来降低熔点。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，我们以三个长 DNA 序列作为模型系统进行了实验，其中一个长 99 个碱基，另外两个长 130 个碱基，但 GC 含量不同。我们表明，基于激光的 HRM 能够区分目标和单碱基错配 DNA，只要长度达到 130 个碱基，且 GC 含量接近 50%。首先实验研究了激光阈值随温度的变化关系，通过优化外部泵浦，与基于荧光的 HRM 相比，长至 130 个碱基的 DNA 的熔点降低了 10°C 以上。最后，我们展示了一种基于激光的 HRM 的替代快速 DNA 筛选方法，而基于荧光的 HRM 则不存在这种方法，即在固定温度下扫描激光激发，以区分目标和碱基错配 DNA 序列。结果表明，GC 含量接近 50%的 130 个碱基长的 DNA ，其激光阈值的差异可达 20%，激光输出斜率的差异可达 40%，尽管其熔点差异仅为 0.5°C，这表明激光激发扫描方法也适用于 GC 含量较高的长 DNA 序列。
