The Denaturation of DNA

 At elevated temperatures, the 3D structure of both proteins and nucleic acids (or any strucuture held together by H bonding) is disrupted.

 denaturation - disordered 3D structure, disrupted due to broken H bonds
native - ordered 3D state, presumably as in biological systems.

Native DNA is double-stranded DNA

 Upon heating double-stranded DNA------------------> single stranded DNA

 Absorbance - ability of a substance to absorb light

Visible light 400-700 nm
DNA absorbs maximally at 260 nm (in the UV range)
Proteins absorb maximally at 280 nm
The ability of DNA to absorb light (260nm, UV) increases as denaturation progresses. Absorbance is primarily a property of the nitrogenous bases. When they are hydrogen bonded and in close proximity they cannot absorb light as well as when they are exposed. Absorbance goes up at 260nm even higher when the DNA is completely hydrolysed (broken down into single nulcleotides).

 If you measure the absorbance of a sample under increasing denaturing conditions (i.e. heat) you can generate a melting curve (Fig. 1).

For example, lets assume that for a particular sample of DNA:
Absorbance of double stranded DNA =1.00
Absorbance of single stranded DNA=1.37

Tm=melting temperature
or the temperature at which the rise in A260 is half complete

 Notice from the curve:
Absorbance remains constant up to temperatures well above those encountered by living cells in nature.

 When absorbance rises, the rise is steep over 6-8C.

 Curve plateaus as the 2 strands separate.

 If you hold DNA length constant and the concentration of DNA in the sample constant, the Tm is a pretty good indication of base content.

 RULE: Tm increases as [G+C] increases. The higher the GC content of the DNA the higher the Tm.

 GC base pairing involves 3 hydrogen bonds (vs two in AT pairing) Therefore it takes more heat to disrupt the pairing.

 pH changes can also be used to denature DNA

Hydrogen bonding is very sensitive to H+ concentration

 At pH greater than 11.3 (alkaline conditions) all hydrogen bonds are eliminated and DNA is completely denatured (in single stranded form).

 Acid conditions also denature DNA but... there is damage to phosphodiester linkages. Phosphodiester bonds are resistant to alkaline pH so use of alkaline pH is method of choice for deliberately denaturing DNA in the laboratory.

 Interestingly - repulsive forces also are at play in the DNA double helix Recall the negatively charged phosphate groups. Charges are neutralized by bound + ions such as Na+ and Mg++. *In dH2O, repulsions are so great, strand sepration or denaturation occurs.

Double stranded state is stable, but double stranded regions frequently open to become single-stranded bubbles.

 In AT rich areas, breathing is much more common.

 Denaturation is reversible