DNA Melting Temperature (Tm): Estimation Using UV Spectrophotometry

DNA Melting Temperature (Tm) Estimation Using UV Spectrophotometer

The melting temperature (Tm) of DNA is a key parameter that reflects the stability of the double helix. Using UV spectrophotometry, we can determine the temperature at which DNA strands separate into single strands.

Theory and Principle

DNA absorbs ultraviolet light strongly due to conjugated double bonds present in nitrogenous bases. The peak absorbance occurs at 260 nm.

In its double-stranded form, DNA shows lower absorbance due to tight base stacking. When temperature increases, hydrogen bonds break and strands separate, causing denaturation.

This leads to an increase in absorbance, known as the hyperchromic effect.

Melting temperature (Tm) is the point where 50% DNA is double-stranded and 50% is single-stranded.

Materials Required

Reagents

• Plasmid DNA
• Milli-Q water

Lab Supplies

• Eppendorf tubes
• Micropipettes

Equipment

• Water bath
• UV Spectrophotometer

Procedure

1. Take 700 µL DNA into 10 tubes
2. Heat at 10°C to 100°C (stepwise)
3. Cool immediately in ice
4. Measure absorbance at 260 nm

Data Analysis

Plot temperature vs absorbance to obtain the melting curve. The midpoint of this curve gives the Tm value.

Higher temperature → more strand separation → higher absorbance

Concept

DNA undergoes helix-to-coil transition:

• Helix = Double-stranded DNA
• Coil = Single-stranded DNA

Absorbance increase is directly related to base unstacking.

Tm occurs when fraction of paired bases (f) = 0.5

Conclusion

This method provides a simple and reliable way to determine DNA stability. The hyperchromic effect observed at 260 nm allows accurate estimation of melting temperature.

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