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8. Beskrive hypokromisk og hyperkromisk effekt og middeltemperatur (Tm)
Devlin, s.43 - fig. 2.20; fig.2.22

The denaturizing of nucleic acids can be easily followed by monitoring their absorption of UV-light at 260 nm. There are 2 possibilities:

hypochromicity - absorption of less UV-light due to stacking. The total absorbance of stacked bases may reduce the UV absorption by as much as 40% compared to an unstacked state.

hyperchromicity -  absorption of more UV-light due to denaturizing of the DNA and unstacking of bases. When DNA is heated, its absorbance of UV-light at 260 nm increases with rising temperature, because more and more bases are unstacked. When the DNA is completely denaturized, the absorption is 100%.

Tm - at this temperature, half of the DNA is single-stranded (denaturized), and half of it is found in its double stranded helix form. This basically means that a half of the maximum optical absorbtion is reached.

The Tm gets higher, when the DNA has more cytosine (C) - guanine (G) content, because more heat is needed to break the 3 hydrogen bonds that link these 2 bases. The Tm is, as expected, lower if there are more A=T base pairs.

In general nucleosides absrob the most UV-light followed by nucleotides, single stranded DNA absorbes even less and double stranded DNA absorbs the least. The reason is, of course, that stacking is smalllest in nucleosids and biggest in double stranded DNA.

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