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Extension is achieved by using the loosened nucleotides of each base to grow the complementary DNA strand. The end result is two double-stranded products of DNA.

The temperature that is used during the extension phase is dependent on the DNA polymerase that is used. Generally at this stage, the reaction mixture is heated to a temperature intermediate between the denaturation and annealing temperatures. The optimal temperature for Taq polymerase is 72°C. The polymerase extends the primers in the 5' to 3' direction.

The extension time depends upon the length of the target DNA sequence but a general rule of thumb is one thousand bases per minute at optimal temperature. Under ideal conditions, the amount of target DNA should double in the extension step.