Fast Determination of Thermal Melt Temperature of Double-Stranded Nucleic Acids by UV-Vis Spectroscopy

Importance of the Topic

In biopharmaceutical research and quality control, reliable determination of DNA or RNA duplex stability is essential for genetic analysis, drug discovery, and manufacturing of nucleic acid therapeutics.

Objectives and Study Overview

This study evaluates the effect of rapid temperature ramp rates on the melting temperature (Tm) of herring sperm DNA using an Agilent Cary 3500 UV-Vis spectrophotometer. Ramp rates from 1 to 40 °C/min were compared to assess reproducibility and throughput improvements.

Methodology

Samples of herring sperm DNA (~15 µg/mL) in phosphate buffered saline were measured for absorbance at 260 nm during controlled temperature increases. Mineral oil was applied to minimize evaporation, and measurements were recorded every 1 °C with 0.1 s signal averaging. Six ramp rates (1, 5, 10, 20, 30, 40 °C/min) were tested in triplicate across eight cuvette positions.

Used Instrumentation

The key instrument was the Agilent Cary 3500 Multizone UV-Vis spectrophotometer equipped with Peltier-cooled temperature control (0–110 °C) and integrated in-cuvette probes. Quartz semimicro cuvettes (10 mm pathlength) and the Cary UV Workstation software with Savitzky–Golay smoothing and derivative functions were employed.

Key Results and Discussion

The Tm values determined at all ramp rates were within ±0.2 °C of each other, averaging 86.9 °C. Fast ramp rates reduced experiment time from ~2.5 hours to approximately 10 minutes at 30 °C/min without compromising accuracy. The built-in smoothing and derivative analysis streamlined data processing and peak identification.

Benefits and Practical Applications

• Significant reduction in analysis time and increased sample throughput.
• High reproducibility supports routine QC checks in nucleic acid manufacturing.
• Enhanced accuracy at elevated ramp rates benefits drug discovery and research workflows.

Future Trends and Opportunities

Advances in multi-well UV-Vis automation and integration with compliance software (e.g., Agilent OpenLab) will further accelerate thermal analysis. Real-time data analytics and expanded protocols for RNA structures, G-quadruplexes, and complex nucleic acid assemblies are expected.

Conclusion

Agilent Cary 3500 UV-Vis spectroscopy delivers fast, accurate, and reproducible nucleic acid melting temperature measurements, enabling significant productivity gains in research and QC environments.

Reference

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