DNA Melting Analysis with Optofluidic Lasers Based on Fabry-Pérot Microcavity.

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.