Room for Improvement
Initially, PCR was slow, expensive, and required further analysis by gel electrophoresis. Even after automated thermal cyclers (PerkinElmer, Life Technologies) and thermally stable polymerases (Roche) were developed in the late 1980s, PCR protocols typically required four hours for amplification.
The feasibility of rapid cycle PCR (30 cycles in 15–30 min) was shown in the early 1990s and commercialized as the RapidCycler (Idaho Technology) with eventual adoption into the real-time capillary LightCycler (Roche) and SmartCycler (Cepheid) platforms. Amplification speed was limited by sample geometry and heat transfer, not reaction chemistry.
Nevertheless, microwell plates became the dominate format, reflecting user preference for convenience and automation over speed and temperature control. A trend toward faster amplification was recently rebranded as Fast PCR, often packaged with presumed enabling reagent modifications.
However, amplification speed remains instrument limited, not reagent or reaction limited.
Similar to the order of magnitude change in speed, the cost of PCR has diminished at least an order of magnitude. After the original Taq polymerase patents were deemed unenforceable, reagent manufacturers turned their focus to improved enzymes and hot-start methods to add value.
Physical separation of critical components with wax, inactivation of the polymerase with an antibody, heat-labile enzymes, primers, and dideoxynucleotides have all been commercialized to improve specificity. Although not necessary for the majority of applications, such improvements may compensate for poor reaction design and execution.
Twenty years ago, reaction volumes were typically 100 µL. Today, 10 µL is commonly used and microfluidic systems run a thousand times smaller yet at 1–10 nL per reaction.
Digital PCR can be performed to take advantage of limited template copies per reaction, or preamplification can expand the sensitivity limits inherent to small volumes. Even picoliter reactions are now routinely formed as emulsions or controlled droplets within oil, enabling massively parallel sequencing by expansion of individual templates or production of target libraries.
To the extent that reagent cost is proportional to reagent volume, the expense of individual reactions should be vanishingly small. However, in the case of digital PCR, many reactions are necessary for quantification, and droplets can only be analyzed individually with expensive equipment. Furthermore, if each reaction requires specific oligonucleotides, the initial synthesis costs remain the same and can be oppressive when many targets are analyzed.