Separating the two Strands of a DNA double Helix

While the ratios of G to C and A to T in an organism's DNA are fixed, the GC content (percentage of G +C) can vary considerably from one DNA to another.When a DNA solution is heated enough, the non-covalent forces that hold the two strands together weaken and finally break.When this happens, the two strands come apart in a process known as DNA denaturation, or DNA melting.The temperature at which the DNA strands are half denatured is called the melting temperature, or 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 hyperchromic shift.
The GC content of DNA has a significant effect on its Tm.The higher a DNA's GC content, the higher its Tm.Why should this be?One of the forces holding the two strands of DNA together is hydrogen bonding.Also G-C pairs form three hydrogen bonds, whereas A-T pairs have only two.It stands to reason, then that two strands of DNA rich in G and C will hold to each other more tightly than those of AT-rich DNA.
Heating is not the only way to denature DNA.Organic solvents such as dimethyl sulfoxide and formamide, or high pH, disrupt the hydrogen bonding between DNA strands and promote denaturation.Lowering the salt concentration of the DNA solution also aids denaturation by removing the ions that shield the negative charges on the two strands from one another.At low ionic strength, the mutually repulsive forces of the negative charges are strong enough to denature the DNA ar a relatively low temperature.