Best Practice for Nucleic Acid Thermal Stability Measurements Using the Cary 3500 UV-Vis Spectrophotometer - Thermal melt (Tm) analysis using rapid, precise temperature-dependent UV-Vis absorbance measurements
UV-Vis spectrophotometers have been used widely for nucleic acid quantification and quality control (QC) utilizing the fact that nucleic acids have a maximum absorbance at 260 nm (1). The concentration of nucleic acids can be easily estimated using the absorbance at 260 nm and the established absorption coefficient. Often a background correction is also performed, for example collecting a baseline using a solution containing everything but the nucleic acid or by measuring the absorbance at a wavelength that nucleic acids do not absorb. Double stranded nucleic acids are bound by hydrogen bonds between the base pairs. The temperature at which double stranded nucleic acids denature to become single stranded depends on the: – sequence and length of the nucleic acid – the pH and buffer conditions – and any mismatches in base pairs between the two strands As such, the melting temperature is very useful analytical tool and can be studied by monitoring the absorbance at 260 nm as temperature is increased or decreased. As the temperature is increased, the hydrogen bonds between the strands are broken and the double stranded nucleic acid separates into two separate strands. When the strands separate, the absorbance at 260 nm increases. The transition temperature is called “melting temperature” (Tm) (1).
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