Nonspecific binding primer

PCR is a cycle reaction and is carried out in a thermocycler. Each cycle consists of three steps. In the first step, denaturation, the initial DNA and primers are healed at 95 C and denatured into single strands. In the second step, called annealing, the primers hybridize to their opposite sequence at both DNA strands. The annealing temperature is usually 3-5 °C below the melting temperature of the primer. If the selected annealing tanperatnre chosen is too low the primers may also bind at positions which are not 100% complanentary and thus lead to nonspecific products. If the temperature is too high the primers do not bind or bind incompletely and no product, or only a small amount, is formed. 

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