This year marks an important birthday for molecular biologists: the polymerase chain reaction (PCR) turns 30! This is my eulogy to celebrate life so far for the technique that has rocked the world.
PCR has become such an integral part of the bioscientist’s toolbox that new generations of researchers probably cannot imagine life without it. It truly has transformed molecular research and diagnostics.
The invention and initial development of PCR
Kary Mullis is credited with inventing PCR. In 1983, he was agonizing over the technical problem of how to replicate a piece of DNA to detect point mutations within. An elegant solution came to him as he was driving his car.
He realized he could amplify a stretch of target DNA by using opposing oligonucleotide primers to replicate both complementary strands of DNA in unison. Through cycles of denaturation, annealing and polymerization he could logarithmically amplify a single copy of DNA. Thus, the polymerase chain reaction (PCR) was conceived.
As with most landmark inventions that drive human progress, Mullis’ invention had to combine with other simultaneous and subsequent inventions to reach its full potential.
Without a way to efficiently synthesize oligonucleotides, PCR would have been stuck in the starting blocks. Luckily, Dr. Ronald Cook, founder of Biosearch Inc. (now Biosearch Technologies) had been working on that problem. He supplied Mullis with one of the first SAM I DNA synthesizers that enabled him to automatically generate the oligonucleotides he needed for his experiments (see bottom of this page for more).
This got PCR off and running, but it was still not practical. Early PCR experiments were cumbersome because researchers had to manually perform thermal cycling, and replenish the heat-sensitive DNA polymerase after every cycle. A standard PCR experiment uses >30 cycles, so that is a lot of waiting around and pipetting for each experiment.
Two main things changed to make PCR practical. One of these was the harnessing of the heat-tolerant Taq DNA polymerase, which was purified from a thermophilic bacterium Thermus aquaticus discovered in Yellowstone National Park in the 1960‘s. The other was the development and commercialization of the DNA thermal cycler by Cetus (Mullis’ employers at that time), in partnership with Perkin Elmer.
Through the combination of Mullis’ breakthrough invention with SAM-1, Taq and the DNA thermal cycler, PCR became the powerful, convenient and accessible technique that we know so well. Kary Mullis later shared the 1993 Nobel Prize in Chemistry for his invention.
New ideas, building on the initial concept of PCR
But the basic technique of PCR, powerful as it was, proved to be just the beginning. As researchers embraced the technique, they combined PCR with other ideas, inventions and techniques, creating a seemingly endless stream of new techniques and applications for research and diagnostics. Here are just some of the advances we can thank PCR for:
- Instead of spending years trying to clone a gene, today’s researchers can clone, sequence and identify a new gene in one week.
- PCR can be combined with reverse transcription (RT-PCR) to routinely detect, quantify and clone messenger RNA.
- Real-time PCR or quantitative PCR (qPCR) offers an extremely sensitive assay to quantify DNA across an impressive dynamic range, by recording the kinetics of the amplification in “real-time” as the PCR reaction proceeds.
- Using multiplexed qPCR, researchers can easily monitor changes in expression of a set of genes in response to various treatments, conditions or diseases.
- Techniques such as site-directed mutagenesis employ PCR to specifically introduce a mutation to study its effect on the gene’s function. Thanks to PCR, these assays have become faster and easier allowing even the smallest of labs the opportunity to perform these techniques.
- In medicine, PCR is used to screen for hereditary diseases. Prior to conception, at-risk individuals can be counseled on the likelihood that their children may inherit a genetic deficiency. Identification of a hereditary disease in an individual can lead to earlier diagnosis and treatment, or predict the severity of the disease. Over 3,500 genetic conditions with available diagnostic tests are included in the National Institutes of Health Genetic Testing Registry. As new genetic variations are discovered and quick diagnostic tests are developed, the analysis of a person’s genome may lead to individualized medicine.
- PCR has dramatically changed the study of emerging infectious disease. Incredibly, it took researchers just a year to identify the virus responsible for the severe acute respiratory syndrome (SARS) epidemic in 2002-2003. Using PCR, researchers were able to classify the organism as a Coronavirus and then take field samples to determine the origin of the outbreak. Without PCR this could have taken decades.
- PCR has also changed forensic science. Since PCR can amplify small amounts of DNA, paternity testing can now be performed on a single swab taken from the mouth. Likewise, the ability to amplify minute traces of genetic material can lead to a conviction or exonerate individuals indicted of a crime. ~300 prisoners have been released from prisons in the United States based on DNA tests that were not available at the time of their conviction.
- PCR has spurred the creation of a new scientific discipline, paleobiology in which comparison of DNA from fossils to current relatives is helping discover the evolutionary history of life. In fact, molecular methods like PCR have fundamentally revised the phylogenic relationships between entire domains of life. Scientists continue to use PCR to catalog and understand the multitude of organisms on Earth.
- As globalization provides for a more rapid spread of infectious disease and drug resistant strains, PCR will be crucial in detecting the causative agents through diagnostic tests.
If you want to learn more about PCR, check out the webinar that was hosted by The Scientist on Wednesday September 11, 2013. In honor of PCR’s 30th birthday, Dr. Mullis and other experts in the field discuss the history and future of PCR.