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

Area of Science:

• Biophysics
• Molecular Biology
• Optics

Background:

• High-resolution melting (HRM) analysis is crucial for DNA analysis.
• Current fluorescence-based HRM has limitations in signal-to-noise ratio and temperature resolution.
• Optofluidic lasers offer potential for improved HRM performance.

Purpose of the Study:

• To investigate the application of optofluidic lasers for DNA high-resolution melting (HRM) analysis.
• To compare laser-based HRM with traditional fluorescence-based HRM.
• To explore novel applications of laser-based HRM for DNA screening.

Main Methods:

• Theoretical analysis of laser-based HRM.
• Experimental HRM analysis of long DNA sequences (99 and 130 bases) using a Fabry-Pérot microcavity laser.
• Optimization of laser conditions (external pump, cavity Q-factor) to lower melting temperature.
• Development and testing of a laser-excitation-scanning method for rapid DNA screening.

Main Results:

• Laser-based HRM demonstrated higher emission intensity and sharper transitions than fluorescence-based HRM.
• Successfully distinguished single-base mismatched DNA sequences up to 130 bases with nearly 50% GC content.
• Reduced melting temperature by over 10 °C by optimizing laser parameters.
• The laser-excitation-scanning method showed significant differences in laser threshold and output slope for mismatched DNA, even with minimal melting temperature differences.

Conclusions:

• Optofluidic laser-based HRM provides enhanced performance for DNA analysis, including improved resolution and signal.
• Laser-based HRM is effective for detecting single-base mismatches in long, high-GC DNA sequences.
• The novel laser-excitation-scanning method offers a rapid and sensitive approach for DNA screening, particularly for challenging sequences.