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The temperature is rapidly shifted to an annealing temperature between 37 and 72 C (the best temperature is determined experimentally for a particular set of primers), and at this temperature, the single strands anneal, or form heteroduplexes, with the primers The primers are single-stranded, 20 26 bases in length and 40 60% GC content Two unique primers are needed for PCR ampli cation, with one annealing to each strand, near the 30 -end of the region of interest Following the annealing step, the temperature is rapidly changed to 70 72 C, where the DNA polymerase sequentially extends the primers one nucleotide at a time, moving toward the 50 -end of the region of interest The nucleotides that are incorporated into the new double strands are complementary in sequence to the analyte strands After this cycle, one copy of each analyte DNA molecule has been produced.

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Repeated thermal cycling through the denaturation, annealing and extension temperatures yields copies of the copies, and an exponential ampli cation of the DNA concentration In principle, the number of copies produced is equal to 2n 1, where n is the number of complete thermal cycles used Most PCR protocols specify over 25 cycles In practice, the ampli cation is somewhat less than predicted, due to incomplete extension through the second primer sites The usual size of the ampli ed region is 100 400 bases in length Figure 109 shows a schematic representation of PCR ampli cationRelated: .

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