PCR annealing

PCR annealing is association of PCR primers with exposed complementary DNA regions by base pairing. It supplies the paired starting ends required for PCR extension. Cooling can permit this association without itself joining new nucleotides onto the PCR primers. The two templates can also pair with one another again, so exposure after PCR denaturation is an opportunity for binding, not a guarantee of productive copying.^[ref]

In the example below, $5^{\prime }\text{-}\mathrm{AGTC}\text{-}{3}^{\prime }$ pairs with the aligned template segment $3^{\prime }\text{-}\mathrm{TCAG}\text{-}{5}^{\prime }$. The opposite end labels show antiparallel nucleic acid strands. Pairing the right letters in the wrong directional display does not supply a correctly depicted starting end. These four-nucleotide strands are a teaching example, not a laboratory PCR primer recommendation.

The binding temperature balances enough productive pairing against unwanted binding. Its appropriate value depends on the PCR primers and reaction conditions; increasing it can improve PCR specificity but can also prevent useful binding. A predicted binding setting therefore needs experimental assessment rather than being inferred from the label PCR.^[ref]