Advances in PCR would not be possible without the evolution of DNA polymerases
The story of modern PCR begins in 1976 with the isolation of Taq DNA polymerase from the thermophilic bacterium Thermus aquaticus. Its isolation meant that molecular biologists now had a thermostable enzyme that was capable of repeat PCR cycling without the need to add fresh DNA polymerase after each cycle. For those of us who can remember that far back, resetting the PCR reaction as many as 40 times over a four or five hour period was not much fun and did not feel like a great use of time, so the Taq enzyme made our lives better in so many ways!
However, the impact that the Taq enzyme would have on molecular biology wouldn't become clear until 1988 when Kary Mullis and the Cetus Corporation commercialized the enzyme for widespread use. Taq DNA polymerase was an instant success, even winning Science magazines 'molecule of the year' in 1989. In combination with the release of the first PCR cyclers, we could now finally say goodbye to those laborious afternoons in front of the water bath, and instead amplify our DNA using automated equipment.
Although these developments represented significant progress, Taq DNA polymerase wasn't perfect. It was unstable at high temperatures and error prone, and had difficulty amplifying DNA rich in GC content or with strong secondary structure. These factors played a role in the stunted development of PCR early on, particularly in applications that required high specificity and reliability. It quickly became apparent that the development of DNA polymerases was intrinsically linked to the effective leverage of PCR, and that to increase the power of PCR and open up a wider range of applications there was a need to engineer more advanced DNA polymerases.
Hot starting polymerase development
The late 1980s saw a number of techniques spring up using "hot start" to overcome Taq DNA polymerase's inefficiency and low specificity at high temperatures. These hot-start techniques revolved around heating PCR reactions to 95°C and then letting them cool to 60–70°C , before adding your polymerase. Although effective, hot-start techniques were time consuming and often caused sample cross contamination. PCR needed a long term solution.
Pfu & PCR
1991 saw the isolation and development of Pfu polymerases, derived from the hyperthermophilic archaeon Pyrococcus furiosus. Pfu DNA polymerase, unlike Taq DNA polymerase, has built in 3' to 5' exonuclease proofreading activity, meaning that it could correct nucleotide-incorporation errors, dramatically lower error rate, and offer increased specificity. The development and use of both Pfu and Taq polymerases continued for some time, with PCR playing a major role in a number of ground breaking studies, such as the sequencing of Haemophilus influenzae by Venter and colleagues in 1995 and the development and release of real-time PCR instruments in 1996. However, while useful for many basic applications, these polymerases could not provide the required accuracy, reliability, and read length required to push PCR into new and exciting areas.
The introduction of fusion DNA polymerases in 2003 was probably the first step in the development of truly next-generation polymerases and high-fidelity PCR. These specially engineered DNA polymerases could overcome or reduce many of the problems still limiting PCR development. Created by fusing the core components of an Archaebacterial polymerase with a thermostable DNA-binding domain, the first Phusion High Fidelity DNA polymerases possessed strong proofreading ability and were incredibly stable at high reaction temperatures. Additionally, by using a specialized DNA-binding domain, the affinity of the polymerase for double-stranded DNA was increased exponentially.
The development of these next-generation polymerases alongside the development of both real-time and digital PCR technologies has guaranteed that PCR will play an important part in the future of life science research and human healthcare. Best of all, researchers can now be much more confident that the results of their PCR analyses are truly reflective of the DNA sequences they are trying to amplify, rather than mistakes caused by a sloppy polymerase.
Check out this informative timeline(1950–2010) to learn about the evolution of PCR
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