In the ’80s, researchers explored a variety of signal or probe amplification systems for research and diagnostic applications. In contrast, PCR, with its sequential cycles of targeted oligonucleotide primed synthesis, is a target-amplification system, capable of amplifying unknown genomic regions between the primer sites.
While sequence information is necessary for primer design, some mismatches between primer and template, primarily those at the 5´ end of the primer, could be tolerated. As a result, primers based on a sequence from one species could be used to amplify the homologous gene from related species, or those from one gene in a multigene family could amplify related genes.
In addition, valuable sequence information, such as restriction sites to facilitate cloning or, more recently, adaptor and sample barcode sequences for next-generation sequencing (NGS), could be introduced at the ends of the amplicon via tolerated mismatches at the 5´ end of the primer. Labels could also be introduced into the amplicon using primers labeled at the 5´ end, facilitating both gel electrophoresis and probe hybridization analyses.
PCR could also be used to confer on any DNA fragment the capacity to be replicated by simply ligating primer sites onto the ends of the targeted fragment(s), a property currently used in the preparation of shotgun libraries for NGS.
PCR is essentially agnostic with respect to analytic methods: gel electrophoresis, probe hybridization, Tm melting analysis, sequencing, and a variety of other techniques have all been successfully applied to the analysis of amplicons.
PCR’s ability to generate high concentrations of a specific labeled DNA target created a paradigm shift in oligonucleotide probe hybridization analysis. Previously, target DNA had been immobilized on a substrate, denatured, and hybridized under stringent conditions (so that a single mismatched base-pair would destablilize the target-probe duplex) to a vast molar excess of labeled probe (the “dot blot”).
With PCR, a panel of sequence-specific oligonucleotide probes could be immobilized and then hybridized to a labeled amplicon (“reverse dot blot”). This basic principle became the basis of the linear array, probe-coupled beads, and microarray analysis, in which oligonucleotide probes are immobilized (by synthesis or deposition) on a membrane, bead, or chip.