How PCR Works

Each cycle of PCR has three stages, controlled by the thermal cycler.1. Denaturation (about 95°C): the mixture is heated so the hydrogen bonds between complementary bases break. The double-stranded DNA separates into two single strands.2. Annealing (about 55°C): the mixture is cooled so the primers can bind by complementary base pairing to the ends of the target sequence on each single strand. The primers give Taq polymerase a starting point.3. Extension (about 72°C): this is the optimum temperature for Taq polymerase. The enzyme adds free nucleotides to the end of each primer, following complementary base pairing, and joins them to build a new complementary strand.At the end of one cycle, each original double-stranded DNA molecule has become two. The cycle is then repeated, typically 25 to 35 times. Each cycle takes only a few minutes.Because the amount of DNA doubles every cycle, the number of copies after n cycles is:number of copies = starting number × 2ⁿWorked example: a crime scene sample contains 5 molecules of the target DNA. How many copies are there after 10 cycles?
5 × 2¹⁰ = 5 × 1024 = 5120 copies.In practice the increase slows down after many cycles because the primers and nucleotides get used up.