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PCR annealing is association of PCR primers with exposed complementary DNA regions by base pairing. It supplies the paired starting ends required for PCR extension. Cooling can permit this association without itself joining new nucleotides onto the PCR primers. The two templates can also pair with one another again, so exposure after PCR denaturation is an opportunity for binding, not a guarantee of productive copying.^[ref]

In the example below, $5^{\prime }\text{-}\mathrm{AGTC}\text{-}{3}^{\prime }$ pairs with the aligned template segment $3^{\prime }\text{-}\mathrm{TCAG}\text{-}{5}^{\prime }$. The opposite end labels show antiparallel nucleic acid strands. Pairing the right letters in the wrong directional display does not supply a correctly depicted starting end. These four-nucleotide strands are a teaching example, not a laboratory PCR primer recommendation.

The binding temperature balances enough productive pairing against unwanted binding. Its appropriate value depends on the PCR primers and reaction conditions; increasing it can improve PCR specificity but can also prevent useful binding. A predicted binding setting therefore needs experimental assessment rather than being inferred from the label PCR.^[ref]

PCR denaturation is the high-temperature stage that separates a DNA duplex into single-stranded templates for the next copying step. It disrupts the pairing and stacking arrangement of nucleobases; it does not require cutting the phosphodiester bonds along either strand. The distinction matters: breaking the backbone would damage the ordered nucleotide sequence that is meant to serve as a template.

For an original eight-base pair duplex, complete PCR denaturation exposes two eight-nucleotide strands. It leaves sixteen nucleotides altogether: separation has made the templates accessible without yet making more DNA. PCR extension, rather than heating alone, adds the new material. Already copied duplexes also need to separate at the beginning of subsequent cycles.

Separation is a condition-dependent process. Template composition and the buffer solution affect the required temperature and duration; heating longer is not automatically better because prolonged heating can reduce DNA polymerase activity. Incomplete separation leaves fewer accessible templates even when the PCR primers themselves are well chosen.^[ref]

PCR extension is template-directed construction of DNA from an annealed PCR primer by a DNA polymerase. Incoming deoxynucleoside triphosphates supply the new nucleotides. Each addition lengthens the growing strand at its $3^{\prime }$ end, so the product grows $5^{\prime }\to 3^{\prime }$ while the template is read $3^{\prime }\to 5^{\prime }$. The direction refers to the nucleic acid backbone, not to left or right on a page.^[ref]

For a complete worked copying step, start with template $3^{\prime }\text{-}\mathrm{TCAGGGAC}\text{-}{5}^{\prime }$ and paired PCR primer $5^{\prime }\text{-}\mathrm{AGTC}\text{-}{3}^{\prime }$. The next four template letters are GGAC, encountered in the template's $3^{\prime }$-to-$5^{\prime }$ direction. The base pairing rule therefore selects CCTG in order, givingThe PCR primer supplies four nucleotides, and PCR extension adds four more; this copying event consumes four deoxynucleoside triphosphates. On the oppositely oriented template $5^{\prime }\text{-}\mathrm{AGTCCCTG}\text{-}{3}^{\prime }$, the other PCR primer is $5^{\prime }\text{-}\mathrm{CAGG}\text{-}{3}^{\prime }$. It pairs at the right and extends leftward. Reading that template from right to left encounters CTGA after the paired region, so addition of GACT gives $5^{\prime }\text{-}\mathrm{CAGGGACT}\text{-}{3}^{\prime }$. Both finished products are the reverse complements of their respective templates.

The direction argument is short: the PCR primer and its template are antiparallel nucleic acid strands; extension takes place at the PCR primer's $3^{\prime }$ end; hence forward progress along the new strand is backward progress along the template. Reflecting a drawing reverses its arrows on the page without changing either chemical direction. This is why a leftward arrow can represent $5^{\prime }\to 3^{\prime }$ synthesis.

The time needed depends on how much DNA remains to be copied and the chosen DNA polymerase, while temperature must support that enzyme's activity.^[ref] Separating PCR annealing and PCR extension into different holds is convenient, but some compatible conditions allow a combined hold. The three chemical jobs therefore need not always require three distinct temperatures.^[ref]

A PCR plateau is the late phase in which further cycles add little product. Depletion of substrates and accumulation of reaction by-products can reduce PCR efficiency. Consequently, similar final amplicon amounts need not mean similar initial template amounts. In the stipulated green model, $0<N_n<K$ implies $N_n<N_{n+1}<K$, and growth approaches the imposed ceiling. That illustrates loss of starting-amount information without claiming to identify the limiting chemical process in an actual tube.^[ref]