January 1, 1970 (Vol. , No. )

Scott D. Rose, Ph.D.

A brief history and review of what PCR can do.

The concept of amplifying small amounts of DNA using two oligonucleotide primers and a DNA polymerase, known as the polymerase chain reaction (PCR), was introduced to the broader scientific community in 1986. The technique was adopted quickly and became routine in just about every molecular biology lab. I remember watching a fellow graduate student set up several water baths in a row, grab three timers, and perform the cycling manually, the equivalent of a poor man’s thermal cycler. While the nature of the technique—DNA amplification—hasn’t changed, it is now an integral part of cutting-edge technologies such as nucleic acid quantification, molecular diagnostics, next-generation sequencing, and synthetic biology.

Until recently, one quantitative form of PCR, known as qPCR, was heralded as the gold standard of nucleic acid detection. A technology that was first described in 1999 by Vogelstein and Kinzler has recently been gaining rapid traction and is known as digital PCR (dPCR). dPCR is based on being able to set up a series of individual amplification events in partitioned reaction chambers (nano-wells or emulsion droplets), and determining the number of positive signals arising from the total population of reactions. The sample concentration can be determined by a Poisson distribution. The final readout is a simple binary form of signal versus no signal, and eliminates the need for a standard curve.

Rapid pathogen detection in blood samples and nasopharyngeal swabs via PCR is another application that is seeing wider usage. Invasive candidiasis (IC) is a fungal infection that can be acquired in a hospital setting and has a high mortality rate. Detection, and importantly strain identification of pathogens such as Candida, can be performed by multiplex PCR in hours as opposed to the gold standard of culture and histology which requires 24–48 hours for detection. The CDC list 20 different PCR-based tests approved for influenza detection and in some cases subtype differentiation. Rapid identification of new emerging strains will be key to fighting a potential pandemic. Sample prep and massively parallel high-throughput sequencing will enable strain identification in a matter of days.

The field of synthetic biology has recently exploded in part due to the availability of de novo synthesized sequences using oligonucleotides and PCR-based assembly approaches. Instead of having to obtain source DNA from an exotic or hard-to-grow/culture organism and then having to make cumbersome mutational changes to generate the desired sequence, you can order a sequence exactly as you want, from any number of companies, and have it available in a week or two. The molecular biologist tool box now contains functional DNA elements that can be stitched together in any combination using assembly techniques such as the Gibson Assembly™ Method. The utility of this approach was elegantly outlined in a recent paper by Dormitzer et al., in Science Translational Medicine. By utilizing the ability to rapidly synthesize de novo the H7N9 genes to a strain of influenza, they were able to generate MDCK cells producing viral particles in the span of just over two weeks, not the typical six-month time period. This proof of concept is an excellent example of how we can respond to pandemics with vaccines in radically shorter time periods, all made possible by PCR.

PCR is even being used in directed protein evolution. Emulsion PCR can be used to evolve other DNA polymerases with specialized functions by applying selective pressures to mutagenic libraries of a DNA polymerase. This evolutionary approach can be expanded to other enzymes that can be linked in some way to PCR. Ironically, this self-evolution will enable PCR to remain a major player in the molecular biology world for many years to come.

PCR is an integral part of technologies such as nucleic acid quantification, molecular diagnostics, next-generation sequencing, and synthetic biology.

Scott D. Rose, Ph.D. ([email protected]), is director of product development at Integrated DNA Technologies.

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