Most of the techniques described in this chapter (blotting, cloning, PCR, etc.) are dependent on hybridization (annealing) between different populations of nucleic acids. Length of the strands, temperature, and percentage of GC nucleotides weigh considerably on hybridization. Two other components commonly used in hybridization protocols are monovalent ions and formamide. A formula that takes monovalent Na⁺ ions ((M[Na⁺]) and formamide concentrations into consideration to compute a Tₘ (temperature of melting) is as follows:

Tₘ=81.5+16.6(log M[Na+])+0.41(%GC)−0.72(%formamide)


For the following concentrations of Na⁺ and formamide, calculate the Tₘ. Assume 45% GC content.
    [Na⁺]     % Formamide
   0.825             20
   0.825             40
   0.165             20
   0.165             40

Key Concepts

Hybridization

Hybridization refers to the process where two complementary strands of nucleic acids (DNA or RNA) bind together to form a double-stranded molecule. This process is crucial in various molecular biology techniques, as it allows for the specific pairing of nucleotides based on complementary base pairing rules. Factors such as temperature, strand length, and GC content significantly influence the stability and efficiency of hybridization.

Melting Temperature (Tₘ)

The melting temperature (Tₘ) is the temperature at which half of the DNA strands are in the double-helix state and half are in the 'melted' single-strand state. Tₘ is an important parameter in molecular biology, as it indicates the stability of the DNA duplex. It is influenced by factors such as the concentration of monovalent ions, the percentage of GC content, and the presence of denaturing agents like formamide.

Role of Monovalent Ions and Formamide

Monovalent ions, such as sodium (Na⁺), stabilize the DNA duplex by shielding the negative charges on the phosphate backbone, which enhances hybridization. Formamide, on the other hand, is a denaturing agent that lowers the Tₘ by disrupting hydrogen bonding between base pairs. Both components are critical in hybridization protocols, as they help control the conditions under which nucleic acid interactions occur, thereby affecting the overall efficiency of techniques like PCR and blotting.