Factors which affect the success of nucleic acid hybridization include:
Probe length: Longer probes generally provide more specificity; the probe should also be single stranded.
Sequence: The sequence of the probe should be complementary to the target sequence and not have regions prone to self-annealing.
Composition: The nucleotide composition of the probe (particularly GC content) affects the stability and specificity of hybridization. GC-rich probes typically exhibit stronger binding compared to AT-rich probes
Concentration of the target strand and probe: Higher concentrations of target and probe molecules generally lead to increased hybridization efficiency.
Hybridization buffer composition: The composition of the hybridization buffer influences the stability and specificity of probe-target interactions. Factors such as salt concentration, pH, and presence of denaturing agents (e.g., formamide) affect the stringency of hybridization, controlling the balance between specificity and sensitivity.
Temperature of hybridization: If the temperature is too high, the DNA strands will separate. In contrast, if it's too low, they might stick together.
Assay format: The nature of a hybridization assay, whether it's solution-based or involves one immobilized strand, significantly affects the time required for hybridization to occur. For instance, in the polymerase chain reaction (PCR), hybridization happens rapidly, typically within a minute. On the other hand, in techniques like Southern blotting, hybridization can take up to 16 hours or more due to the solid-phase setup.