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

Importance of the topic

Nucleic acid thermal melting experiments are essential for characterizing DNA and RNA stability, base composition, and hybridization properties. The melting temperature (Tm) provides insights into GC content and molecular interactions, making it a critical parameter in drug discovery, quality control, and biopharmaceutical manufacturing processes.

Objectives and overview of the study

This application note demonstrates a rapid and reproducible method for determining the thermal melt temperature of double-stranded herring sperm DNA using UV-Vis spectroscopy. The study evaluates the impact of increasing temperature ramp rates on measured Tm values, aiming to reduce analysis time without compromising accuracy.

Methodology and instrumentation

A phosphate-buffered saline solution containing ~15 µg/mL herring sperm DNA was measured in Agilent quartz semimicro cells with in-cuvette temperature probes. The Agilent Cary 3500 Multizone UV-Vis spectrophotometer, controlled by Cary UV Workstation software, monitored absorbance at 260 nm over a temperature range from 25 °C to 100 °C and back at six different ramp rates (1, 5, 10, 20, 30, and 40 °C/min). Signal averaging and data intervals were set to ensure optimal data quality, and mineral oil was used to prevent evaporation.

Instrumentation used

• Agilent Cary 3500 Multizone UV-Vis spectrophotometer
• Agilent quartz semimicro cells (10 mm pathlength) with integrated temperature probes
• Agilent Cary UV Workstation software
• 0.01 M phosphate buffered saline (pH 7.4)

Main results and discussion

Measured Tm values remained within ±0.2 °C across all six temperature ramp rates, with an average Tm of 86.9 °C and a standard deviation of 0.2 °C (n=6). Increasing the ramp rate from the conventional 0.5 °C/min to up to 40 °C/min did not adversely affect the accuracy of the melting temperature. At a ramp rate of 30 °C/min, a complete melt experiment was completed in approximately 10 minutes, compared to 2.5 hours at slower ramp rates.

Benefits and practical applications of the method

• Significantly reduced analysis time enhances laboratory throughput.
• High reproducibility supports reliable quality control checks.
• Simultaneous measurement of multiple samples increases productivity.
• Flexibility to adapt ramp rates for a variety of nucleic acid melting studies.

Future trends and potential applications

Advances in spectrophotometric hardware and software integration point to further reductions in analysis time and enhanced temperature accuracy. Future developments may include automated high-throughput workflows, integration with regulatory-compliant data management systems, and application to complex nucleic acid structures and real-time melting analyses.

Conclusion

The Agilent Cary 3500 UV-Vis spectrophotometer paired with optimized ramp rates offers a fast, accurate, and reproducible method for determining nucleic acid melting temperatures. Laboratories can reduce experimental durations from hours to minutes without sacrificing data quality, supporting efficient biopharmaceutical research and quality control operations.

Reference

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