Which of the following is (are) factor(s) that determine melting temperature (Tm) of linear DNA in solution? Select all correct answers.

The length of the DNA strands.

The absorption of 260 nm UV light.

The ion concentration of the solution.

The base composition (frequency of occurrence of A, C, G, or T).

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Casey DurganLv2

13 Aug 2019

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6. (10 points) 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.

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 Ã %(G + C)] â (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.

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 T_m.

A. Do you think T_m 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 T_m?

B. Under standard conditions, the expected melting temperature in degrees Celsius can be calculated from the equation T_m = 59.9 + [0.41 Ã %(G + C)] â (675/length of duplex). Does the T_m increase or decrease if there are more G + C (and thus fewer A + T) base pairs? Does the T_m increase or decrease as the length of DNA increases? Why?

C. Calculate the predicted T_m for a stretch of double helix that is 100 nucleotides long and contains 50% G + C content.

The absorption of ultraviolet light by DNA in the near UV (250-300 nm) is due to electronic transitions within the bases. Absorption at 260 nM is approximately twice as intense as absorption at 280 nm and increases by about 10% when the secondary structure present within the DNA is disrupted. Our dsDNA molecule has 25 base pairs. a) If the concentration of the DNA in a solution is 0.02 mg/mL, calculate its absorbance at 260 nm. Assume a path length of 1 cm. b) What will the absorbance be at 260 nM at a temperature of 90C, when all the secondary structure has been disrupted? c) To the same DNA solution, we add an equimolar amount of a DNA-binding protein. The protein has a molecular weight of 200,000 and contains 5 tryptophan residues and 25 tyrosines. What would the absorption of the complex be at 280 nm?

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		The length of the DNA strands.
		The absorption of 260 nm UV light.
		The ion concentration of the solution.
		The base composition (frequency of occurrence of A, C, G, or T).