Structural Biochemistry/Nucleic Acid/DNA/DNA Denaturation
When a DNA solution is heated enough, The double-stranded DNA unwinds, and the Hydrogen bonds that hold the two strands together weaken and finally break. The process of breaking a double-stranded DNA into single strands is known as DNA denaturation, or DNA melting. The temperature at which the DNA strands are half denatured, meaning half double-stranded, half single-stranded, is called the melting temperature(Tm). The amount of strand separation, or melting, is measured by the absorbance of the DNA solution at 260nm. Nucleic acids absorb light at this wavelength because of the electronic structure in their bases, but when two strands of DNA come together, the close proximity of the bases in the two strands quenches some of this absorbance. When the two strands separate, this quenching disappears and the absorbance rises 30%-40%.This is called Hyperchromicity. The Hypochromic effect is the effect of stacked bases in a double helix absorbing less ultra-violet light.
Applications of DNA denaturation
Sequence differences between two different DNA sequences can also be detected by using DNA denaturation. DNA is heated and denatured into single-stranded state, and the mixture is cooled to permit strands to rehybridize. Hybrid molecules are formed between similar sequences and any differences between those sequences will give a disruption of the base-pairing
What determines the Melting Temperature (Tm)?
While the ratio of G to C and A to T in an organism's DNA is fixed, the GC content (percentage of G +C) can vary considerably from one DNA to another. The percentage of GC content of DNA has a significant effect on its Tm. Because G-C pairs form three hydrogen bonds, while A-T pairs form only two, the higher the percentage of GC content, the higher its Tm. Thus, A double-stranded DNA rich in G and C needs more energy to be broken than one that is rich in A and T, meaning higher melting temperature(Tm). Above the Tm, DNA denatures, and below it, DNA anneals. Annealing is the reverse of denaturation.
One aspect of thermal denaturation is never discussed. The heat supplied to effect such denaturation has no preferred direction and is therefore a scalar quantity. However, unwinding a double helix involves unwinding and this has direction and is therefore a vector. The issue is this: how does a scalar change induce a vector change ?
Other methods to denature DNA
Heating is not the only way to denature DNA. Organic solvents such as dimethyl sulfoxide and formamide, or high pH, could break the hydrogen bonding between DNA strands too. Low salt concentration could also denature DNA double-strands by removing ions that stabilize the negative charges on the two strands from each other.
The central difficulty with denaturation of the double helix remains. How would two strands, typically consisting of many turns, and often many hundreds of turns, actually effect strand separation after the hydrogen bonds have been severed ?
A further, major difficulty lies in the fact that the application of heat to a suspension of nucleic acids amounts to the application of a scalar quantity because the heat applied in this way has no direction. However, unwinding the strands requires an angular force and this is a vector as it has a preferred direction. It has never been explained how a scalar quantity (heat) can effect a vector change (rotation) in a solution.
A solution to these problems is offered by the side-by-side models in which there is no net winding of strands around each other.