PCR Turns 40! From Lab Curiosity to Revolutionary Tool - A Look Back and Ahead

In 1983, Kary Mullis, a researcher at Cetus Corporation, came up with an elegant solution that revolutionized biological and genetic research forever. He invented PCR - a process for the amplification of specific DNA sequences, that mimics the natural DNA replication cycle, but in a controlled and accelerated way.
PCR relies on three key components: a DNA template, two short DNA primers that flank the target sequence, and a heat-stable DNA polymerase enzyme. By repeatedly heating and cooling the mixture, the primers bind to the template, the polymerase extends the new DNA strand, and the cycle starts anew, doubling the amount of DNA each time. In just a few minutes, PCR can amplify millions or billions of copies of a specific DNA segment.
Originally, PCR was developed for the detection of mutations in the HBB gene that causes sickle cell anemia. Mullis noticed that the Sanger sequencing method yielded only weak signals and identified the reason: insufficient DNA concentration! Adding a denaturation step to split each dsDNA molecule into two ssDNAs, and a reverse primer to define the total amplicon length, resulted in the amplification of the DNA copies by two (1,2,3). For the PCR breakthrough, Mullis was awarded the Noble Prize in 1993.

During the early “dark ages of PCR”, the method was quite laborious. The polymerase was destroyed in each denaturation step and had to be added anew for each PCR cycle. The problem was later solved with the heat stable Taq polymerase from the bacteria Thermophilus aquaticus. Did you know that the home of the Taq DNA polymerase is the Yellow Stone National Park? In 1976, Thomas Brock isolated the extremophilic bacterium out of a hot spring.
The first PCR experiments were done manually, using simple tap water baths or block heaters for heating and cooling. The entire process could take several hours, and samples needed to be secured by mineral oil. However, even this "primitive" PCR was a game-changer, as it enabled researchers to amplify DNA from various sources, and to analyze them in unprecedented detail.
In 1988, the first commercial automated PCR cycler came to market. PCR thermal cyclers quickly became a standard in laboratories around the world and helped make PCR technology even more accessible.

In the following years, PCR was continuously developed and refined to achieve greater efficiency and sensitivity. The invention of multiplex PCR was an important milestone in the development of PCR diagnostic approaches. From then on multiple targets determined by different primer pairs could be amplified simultaneously in one PCR tube within a single PCR run - instead of performing many separate reactions. Today, the preparation of libraries of large gene panels for Next Generation Sequencing (NGS) relies on multiplex PCR.
After the invention of a method for monitoring PCR kinetics in real time, PCR techniques became fully quantitative. This led to the development of Real-Time PCR (or qPCR), which has the ability to detect PCR products in real time and provides rapid and accurate measurements of nucleic acid concentration. Thermal cyclers were designed for qPCR harboring optical systems for monitoring fluorescence during the reaction, like the ABI LS-50 or the Lightcycler from Roche. The incorporation of reverse transcription (RT) before thermal cycling allowed the use of PCR in RNA studies (RT-PCR or qRT-PCR).
In 1997, the first gradient cycler revolutionized the market: the Mastercycler Gradient. The gradient technology developed by Eppendorf for PCR optimization aimed to address the difficulties with manual optimization of the annealing temperature, usually carried out by multiple PCR runs with varying conditions. Imagine, ramp rates of 2 °C/s were super-fast then!

2D-Gradient technology: Two gradients in the same run allow to optimize the annealing and the denaturation temperature in parallel. Dimension 1 is a horizontal gradient from left to right across the block, giving 12 different temperatures. Dimension 2 is vertically from bottom to top, giving 8 temperatures across the thermal block. Both gradients can becombined in one PCR program, thus allowing concurrent optimization of the denaturation step and the annealing step revealing the best fitting temperatures regarding yield and specificity.

PCR has spawned a vast industry of PCR machines, reagents, and assays, and has inspired countless innovations and discoveries. In the 21st century, PCR has continued to evolve and expand, as new variants and technologies have emerged to address its limitations and challenges, expanding its applications, and enhancing its speed, sensitivity, and specificity.
Real-time PCR has revolutionized the 21st century due to its tremendous applications in quantitative genotyping, detecting genetic variations within and between organisms, and early diagnosis of diseases. The combination of real-time PCR with other molecular techniques has made it possible to monitor therapeutic interventions and individual responses to drugs, among other things. Also, qPCR has replaced the time-consuming and tedious slot blot technique for forensic investigations (5).
One of the most promising methods is loop-mediated isothermal amplification (LAMP) , which allows amplification at constant temperatures without a thermal cycler. LAMP has huge potential in applications in point-of-care diagnostics like pathogen detection from minimally processed sample such as the recently developed SARS-CoV-2 test that can be performed at home using only a smartphone and no laboratory equipment (6).

Digital droplet PCR (ddPCR) tooks PCR to the next level. PCR is performed in tiny water droplets toachieve higher precision and sensitivity making it possible to detect and quantify even the smallestamounts of nucleic acids.
Several breakthrough technologies rely on PCR, such as NGS, nanopore sequencing, and syntheticbiology. CRISPR is a revolutionary gene editing tool that relies on a bacterial defense system to cut andpaste DNA with unprecedented precision and ease. CRISPR uses PCR as a crucial step in its workflow, toamplify and prepare the DNA fragments for editing.
Ultimately, PCR is a ubiquitous and versatile tool that is used in almost every field of biology andmedicine, from forensics to food safety, from cancer diagnosis to infectious disease surveillance.

The future of PCR is bright and exciting, as it continues to push the boundaries of science and technology.The 40th anniversary of PCR is a remarkable occasion to celebrate the ingenuity, perseverance, and impact of Kary Mullis and his colleagues, and to acknowledge the transformative power of a simple yet elegant idea. Since its inception, PCR has become a cornerstone of modern biology and medicine, and its journey is far from over. PCR has shown us that sometimes the biggest breakthroughs can come from thesmallest things, and that science is a never-ending adventure full of surprises and opportunities. With its products, Eppendorf has contributed to the development and popularization of PCR and will continue to play an important role in the further development of PCR technology in the future. So, here's to PCR, and to the next 40 years of innovation and discovery!

Milestones in the history of PCR:

1952: Rosalind Franklin produces photographs of DNA fibers using X-ray diffraction
1953: Watson and Crick discover the double helix structure of DNA
1976: Taq Polymerase is isolated from Thermus aquaticus
1983: Kary Mullis develops the PCR technique
1985: The first paper describing the PCR technique is published in the journal Science by Saiki et al.
1986: PCR is used for the first time in a criminal case, to identify a suspect in a murder case
1987: The first commercial PCR machine is introduced, allowing researchers to automate the process
1988: PCR is used to amplify and sequence ancient DNA from a 40,000-year-old Neanderthal specimen
1989: DNA from single sperm cells is amplified
1991: First DNA polymerase with proof-reading activity isolated from Thermococcus litoralis is published
1993: First PCR chip for miniaturizing PCR is developed
1993: Kary Mullis is awarded for the Nobel Prize in Chemistry for the invention of PCR
1994: PCR is used to amplify and detect the first genetically modified organism (GMO), a tomato
1996: Real-time PCR is developed, allowing researchers to monitor the amplification process in real-time
1996: The first cloned mammal was produced - the sheep Dolly
2005: Next Generation Sequencing comes into market, using PCR for the generation of NGS libraries
2007: The first complete human genome is sequenced
2020: PCR is used extensively in the COVID-19 pandemic, for the detection of SARS-CoV-2
2023: PCR celebrates its 40th anniversary!

Literature

1. Mullis K, Faloona F, Scharf S, Saiki R, Horn G, Erlich H. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb. Symp. Quant. Biol. 51(Pt. 1), 263–273 (1986).
2. Saiki RK, Scharf S, Faloona F. et al. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle-cell anemia. Science 230(4732), 1350–1354 (1985)
3. Zhu H, Zhang H, Xu Y, Laššáková S, Korabečná M, Neužil P. PCR past, present and future. Biotechniques. 2020 Oct;69(4):317-325. doi: 10.2144/btn-2020-0057. Epub 2020 Aug 20.
4. https://en.wikipedia.org/wiki/Thermal_cycler
5. Deepak S, Kottapalli K, Rakwal R, Oros G, Rangappa K, Iwahashi H, Masuo Y, Agrawal G. Real-Time PCR: Revolutionizing Detection and Expression Analysis of Genes. Curr Genomics. 2007 Jun;8(4):234-51. doi: 10.2174/138920207781386960. PMID: 18645596; PMCID: PMC2430684.
6. Heithoff, D.M. et al. (2022). Assessment of a Smartphone-Based Loop-Mediated Isothermal Amplification Assay for Detection of SARS-CoV-2 and Influenza Viruses. JAMA Network Open, 5 (1).

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