The absorption of ultraviolet light by DNA in the near UV (250-300 nm) is due to
ID: 89728 • Letter: T
Question
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?
Explanation / Answer
Answer:
a) DNA Concentration(C)=0.0-2 mg/ml=20 g/ml,
Path length(l)=width of the cuvette which is used to hold the solution=1cm
extinction coefficient for double-stranded DNA (e)= 0.020 (g/ml)1 cm1
So the abosorbance= C*e*l=20*0.020*1=0.4
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b) DNA in solution is heated above its melting temperature (more than 80 °C) in this case 90 °C, the double-stranded DNA unwinds to form single-stranded DNA. The bases become unstacked and can thus absorb more light. In their native state, the bases of DNA absorb light in the 260-nm wavelength region. When the bases become unstacked, the wavelength of maximum absorbance does not change, but the amount absorbed increases by 37%
So now the increased absorbance =0.4+(0.4*0.37)
=0.4+0.148=0.548
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