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The page below is a sample from the LabCE course Fundamentals of Molecular Diagnostics. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Factors Affecting Hybridization

Because hybridization involves nucleotide bases and the separation and joining or reannealing of strands, several environmental factors can influence this process. Examples of environmental factors that may affect hybridization include:
  • Temperature: If the temperature is too high, the strands melt. If it is too low, they might be forced together.
  • The pH: A pH that is too alkaline will cause the strands to separate; too acidic and they are forced together.
  • The guanine to cytosine ratio (G:C ratio): Since this bond is stronger than the other nucleotide bonds, if the G:C ratio in the desired target strand is high, the separation process may take longer or require higher energy (temperature).
  • Salt concentration in the hybridization buffer
The term stringency is often used to collectively label the conditions under which the target is exposed to the probe during hybridization. In most cases, conditions that exhibit high stringency are more demanding of probe to target complementarity and length. Low stringency conditions are much more forgiving, allowing for a less-perfect match between probe and target.
  • If conditions of stringency are too high → Probe cannot bind to the target
  • If conditions of stringency are too low → Probe binds to unrelated targets