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A PCR thermal cycle separates three jobs. During PCR denaturation, heating separates the paired DNA strands without intentionally cutting their phosphodiester bonds. During PCR annealing, cooling permits base pairing between PCR primers and templates. During PCR extension, the chosen interval supports DNA polymerase synthesis from the paired $3^{\prime }$ ends. Each new nucleotide is supplied as a deoxynucleoside triphosphate; its incorporation lengthens the strand. The DNA polymerase is a catalyst, while these nucleotides are consumed substrates.^[ref]

Taq polymerase withstands repeated high-temperature strand separation better than a heat-sensitive DNA polymerase. Its extension activity still depends on temperature, buffer solution and magnesium ions. Heat resistance does not mean constant activity at every stage: the separation temperature serves the DNA, whereas the extension temperature serves the copying enzyme. As one institutional example, Addgene's Basic PCR Program specifies $94^{\circ }\mathrm{C}$ separation, $55^{\circ }\mathrm{C}$ primer binding and $72^{\circ }\mathrm{C}$ extension. These are example settings, not universal properties of all PCR primers or DNA polymerases.^[ref]

The polymerase chain reaction amplifies a selected DNA region using repeated copying steps. Amplified material can be examined by DNA sequencing; appropriate template controls help distinguish a new rearrangement from material already present in the starting preparation.

The essential trick is to choose two PCR primers on opposite DNA strands with their extendible $3^{\prime }$ ends facing into the same interval. A DNA polymerase extends each PCR primer $5^{\prime }\to 3^{\prime }$; the resulting DNA can itself become a template. The amplicon is therefore selected by two opposed PCR primers, rather than by the DNA polymerase recognizing a named gene. This makes PCR amplification useful for inspecting one interval within a much larger genome. Here PCR means polymerase chain reaction; the statistical abbreviation PCR means principal component regression.^[ref]

For a deliberately tiny nucleotide sequence example, display the upper strand as $5^{\prime }\text{-}\mathrm{ACGT}\text{-}\mathrm{GGAATT}\text{-}\mathrm{CAGC}\text{-}{3}^{\prime }$. The left PCR primer is $5^{\prime }\text{-}\mathrm{ACGT}\text{-}{3}^{\prime }$ and binds the lower strand. The right PCR primer, written in its own $5^{\prime }$-to-$3^{\prime }$ direction, is $5^{\prime }\text{-}\mathrm{GCTG}\text{-}{3}^{\prime }$: the reverse complement of CAGC. It binds the upper strand and extends leftward in the drawing. With flanks outside this interval, the bounded amplicon contains $4+6+4=14$ base pairs, including both PCR primer sequences. These four-nucleotide PCR primers illustrate direction and counting only; they are not a proposed experimental design.