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 Hyperchromic 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 riches in G and C needs more energy to be broken than one that is riches in A and T, meaning higher melting temperature(Tm). Above the Tm, DNA denaturaizes, below it, DNA anneals. Annealing is the reverse of denaturation.
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.