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As with some of the greatest discoveries in science, from penicillin to microwave ovens and play-doh, PCR was discovered serendipitously. Thanks to the work of many scientists, including Watson and Crick, Kornberg, Khorana, Klenow, Kleppe (so many K’s…) and Sanger, all the main ingredients for PCR had been described by 1980. Like butter, flour, eggs, and sugar lined up on a kitchen table, the ingredients of PCR were waiting for someone to scream out “CAKE!” and open up the scientific community to a technique with a myriad of applications.
The father of PCR
Kary B. Mullis (another point for the Ks!) who worked for Cetus Corporation perfecting oligonucleotide synthesis received the Nobel Prize in chemistry in 1993 (along with Michael Smith) for his work on PCR and is accredited with its invention. Like many great inventions and discoveries that later prove immensely important, it took time for the scientific community to become interested PCR.
When Mullis presented an early PCR recipe and the thought process behind it at a conference, no one could conceive the final product. At his lowest point, Mullis’ career was nearly derailed as his defense of his idea led to an altercation at the conference and he was removed as head of the oligonucleotide synthesis lab. Science would reject his paper on PCR only to to name it and Taq polymerase ‘Molecule of the Year’ three years later.
Thanks to a collaboration with the Erlich lab, the PCR project was back in the oven and over the next few years it was perfected and various applications developed, including DNA fingerprinting (1986), gene amplification systems (1988), real-time PCR with ethidium bromide (1992) and genome sequencing (2001).
Conception of PCR
Mullis had been interested in chemistry since childhood when he and his friends enjoyed hours blowing things up. After hearing a seminar on DNA synthesis and gene cloning, he realized that chemistry could be used to make DNA and his excitement led him to successfully applying for a position at Cetus’ synthesizing oligonucleotides.
Next door to Mullis was the Henry Erlich lab who were trying to develop methods to detect point mutations in genetic material. The Erlich lab had designed a technique which became known as oligomer restriction and would become the founding idea leading to PCR. A labelled oligonucleotide probe hybridizes to the complementary target sequence and the dimer is then cleaved using a restriction enzyme. However, if the sequence did not match the probe perfectly the restriction enzyme could not recognize the sequence and cleave it. There were many problems with this technique, however, and in thinking about solutions Mullis conceived the concept of PCR in 1983.
Oligomer restriction could only be applied to DNA polymorphisms that alter an enzyme restriction for a known sequence of nucleic acids, meaning it couldn’t be used for just any mutation. Additionally, there needed to be a number of copies present in order for the cleaved product to be detected. Thus, in order to use oligomer restriction for more than plasmids, the target sequence needed to be amplified to increase binding to the target site of interest. The sequence would need to be very unique or the probe would bind to hundreds or even thousands of sites along the DNA.
Mullis eventually struck on an idea: instead of a single round of replication, multiple rounds could be used in the presence of large quantities of the four nucleoside triphosphates and primers and using a second oligonucleotide probe for the complementary strand of the target sequence. This would eliminate the need for restriction enzymes and, thus, any sequence could be used once a specific primer had been made for it. Since with each successive round of replication, the number of target sequence products would increase to eventually far outnumber any contaminating sequencing, a purer starting sample with the target sequence in abundance would not be needed. As target sequences could be made to be of a specific length, they could be detected on a gel as restriction fragments and so non-specific binding or binding to a sequence not of interest would not interfere with results.
First PCR runs
When Mullis first tried PCR, his hope was that rather than needing temperature cycles, the ingredients would take care of themselves. A few experiments with no apparent product showed him what he dreaded most: that the temperatures would have to be readjusted to cycle the reaction from single to double-stranded DNA temperatures and, since the polymerase used at the time was thermally unstable, fresh enzyme would need to be added for every one of the 30 round requires to create an almost pure product.
Painstakingly, the temperatures would have to controlled by hand. This meant heating the reaction up to 95°C, then allowing it to cool, adding DNA polymerase and heating the temperature back up 30 times over. Thus is was time-consuming, exhausting, and tedious work. Imagine troubleshooting the optimal temperature for a primer to bind? Rather than popping your plate into a PCR cycler and giving different annealing temperatures to the different rows, one would need to do every single sample by hand, moving from bath to bath every 30-60 seconds. At this time, the samples were not done on convenient 96-well plates, but instead in tubes so you can imagine that hours of work would go into running from waterbath to waterbath to a timer ringing constantly.
Invention of the thermocyclers
By 1985 and 1987, thermostable Taq polymerase and the first PCR machine, the PCR-1000 Thermal Cycler, became commercially available following a joint venture by Cetus and Perkin-Elmer. These contributed to reducing the cost and hours spent performing this technique and opened up numerous new application for its commercial use and use in research.
Many others could be cited as playing a part in the discovery of PCR as far back as the Father of Genetics, Gregor Mendel, or even further back. When we can now go for a coffee when performing PCR, it used to be excruciatingly slow and labor intensive technique. In 2-3 hours we can now synthesize an almost pure product of genetic material containing a billion copies of the target sequence with the most intense labor being carried out by a machine. How thankful we all can be that we weren’t the poor postgrad student doing PCR in the early days!