The Polymerase Chain Reaction (PCR)

    PCR begins with a mixture containing a dsDNA template, a pair of short ssDNA oligonucleotide primers, a pool of the four dNTPs, and a heat-resistant DNA polymerase, Taq Enzyme. The reaction is carried out in a computer-regulated heating block, a thermal cycler, which permits rapid, controlled heating & cooling. The primers are chosen so that they are base-complementary to opposite ends of either strand of a short stretch of DNA containing the gene region of interest: PCR thus requires some prior knowledge of the gene.

    The reaction is first heated to 95oC to melt (denature) the dsDNA into separate starnds. The reaction is then cooled to ~50oC, at which temperature the primers will find base-complementary regions in the ssDNA, to which they will stick (anneal). The reaction is finally heated to 72oC, at which temperature the Taq enzyme replicates the primed ssDNA (extension). At the end of one cycle, the region between the two primers has been copied once, producing two copies of the original gene region. [This is slightly oversimplified: see your text for details].

    Because a heat-resistant polymerase is used, the reaction can be repeated continuously without addition of more enzyme. Each cycle doubles the copy number of the amplified gene: ten cycles ideally produces 2   8   16   32   64   128   256   512  1,024 (210) copies. Thus, 30 cycles yields a (210x3) = 109-fold amplification. This produces a sufficient quantity of the gene region of interest for direct analysis, for example by DNA sequencing.


Figure & Text 2013 by Steven M. Carr