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

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Steps of PCR - Annealing (Hybridization)

Annealing, or hybridization, is the spontaneous pairing of complementary DNA or RNA sequences by hydrogen bonding to create a double stranded molecule.
The reaction mixture is cooled to a temperature of 50-60°C for 20-40 seconds. The reaction is cooled quickly to allow annealing of the primers to the complementary sequences on the target. Annealing to the primers, as opposed to the original DNA strand, is ensured by the small size of the primers and their vast quantities in the original mixture.
Because annealing involves nucleotide bases and the separation and joining or reannealing of strands, several environmental factors can influence this process:
  • 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): G:C are bound by triple hydrogen bonds whereas adenine:thymine (A:T) are bound by double hydrogen bonds. It therefore takes more energy and may take longer to break G:C bonds than A:T bonds.