When double-stranded DNA is heated, the two strands separate into single strands in a process called melting or denaturation. The temperature at which half of the duplex DNA molecules are intact and half have melted is defined as the Tm

Question

A. Do you think Tm is a constant, or can it depend on other small molecules in the solution? Do you think high salt concentrations increase, decrease, or have no effect on Tm?
B. Under standard conditions, the expected melting temperature in degrees Celsius can be calculated from the equation Tm = 59.9 + [0.41 ×%(G + C)] – (675/length of duplex). Does the Tm increase or decrease if there are more G + C (and thus fewer A + T) base pairs? Does the Tm increase or decrease as the length of DNA increases? Why?
C. Calculate the predicted Tm for a stretch of double helix that is 100 nucleotides long and contains 50% G + C content.

Answer 1

A. Tm depends on the identity and concentration of other molecules in the solution. High salt concentrations are more effective at shielding the two negatively charged sugar–phosphate backbones in the double helix from each other, so the two strands repel each other less strongly. Thus, a high salt concentration stabilizes the duplex and increases the melting temperature.
B. The Tm increases as the proportion of G + C bases increases and as the length increases. The thermal energy required for melting depends on how many hydrogen bonds between the strands must be broken. Each G-C base pair contributes three hydrogen bonds, whereas an A-T base pair contributes only two.
C. Inserting values into the equation in part B gives Tm = 59.9 + (0.41 × 50) – (675/100) = 73.65°C, which is about twice the normal temperature of the human body and nearly too hot to touch.