PCR, what is it?

PCR (Polymerase Chain Reaction – polymerase chain reaction) is a technique that detects the presence of DNA fragments in a sample, part of the sequence of which is known. The trick is to make many copies of the DNA strand to make its presence detectable. This method is fast and very sensitive as the presence of a single DNA strand of interest can be detected. It is also very efficient as copies are made exponentially. Today, PCR is used in genome sequencing, disease diagnosis, paleogenetics, and forensic medicine. Its first description in a scientific journal dates back to 1987, and in 1993 its inventor Kary Mullis (1944-2019) was awarded the Nobel Prize in Chemistry.

PCR is a method that has become routine in laboratories due to the discovery of thermophilic bacteria.

PCR is based on a special enzyme called Taq polymerase. Interest in this molecule is due to its ability to copy DNA and at the same time withstand very high temperatures. And for good reason: it was first isolated in 1976 from a thermophilic bacterium, water termusliving in a hot spring in Yellowstone Park, USA. Three steps follow each other in PCR, with Taq polymerase interfering with the last one. The first step is the denaturation of the DNA molecule. This is done in order to open the double strands of DNA present in the sample. The recipe is simple: heat the sample to 95°C, and the two strands that make up the famous DNA double helix separate into single strands. Then it is necessary to recognize the individual strands of DNA that need to be reproduced; so we will mark them with a small piece of complementary DNA called a primer. This step, hybridization, requires the temperature of the sample to be reduced between 50°C and 60°C. The last step will be the copy of one strand of DNA itself. This is where Taq polymerase comes in. It will stick to the primer and restore the double-stranded DNA molecule. For this step, called polymerization, the sample must be heated again, this time to 72°C. At the end of this cycle, two double-stranded DNA molecules are produced.

This video from the Sorbonne is a very clear illustration of the principle of PCR.

Of course, two copies of the DNA are not enough to make the desired DNA visible. Therefore, several copy cycles are necessary. For example, in the case of a search for the Sras-CoV-2 virus, it is estimated that more than twenty cycles allow one to conclude whether viral sequences are present in the sample. Carrying out these multiple cycles with the “classic” enzyme means the need for intervention to replace it at the end of each cycle. Thanks to Taq polymerase, which can withstand the high temperature of the denaturation phase, human intervention is no longer required.

https://www.youtube.com/watch?v=NSokdA2IjrU

Original video footage from Sciences et Avenir showing the use of PCR in a specific case of horse meat found in beef lasagne.

PCR, history of disputed patents

In the 1990s, when the Nobel Prize in Chemistry was awarded to Carey Mullis, scientists and industrialists worked behind the scenes to prevent patents filed for PCR from being validated. It’s about making this revolutionary technology available to the public. Especially since those patents were sold for $300 million to Roche Pharmaceutical Lab, which has the funds to take legal action. In the US, Dupont de Nemours is suing the patents. Promega decides to sell the Taq polymerase enzyme without paying royalties to Roche. In Europe, fifteen days before the Nobel Prize was awarded, six companies filed an opposition to Roche’s patent application with the European Patent Office. Defenders of the free use of PCR have arguments: PCR was invented in 1968 by Nobel laureate Har Gobind Khorana and described in articles published in the 1970s. In addition, a weighty argument, Taq polymerase is not new: ten years earlier, a similar thermostable enzyme was discovered in the USSR. Eventually, the three patents will be filed and validated and generate about $2 billion in royalties for Roche Laboratories before they enter the public domain.

Limits of PCR

Today, PCR is present in all laboratories of molecular biology. Scientists have learned to measure its limits. Thus, in forensic medicine, contamination has sometimes led to false results. In Treiber’s case, they led to the suspicion of the forensic investigator, not the alleged killer. In paleogenetics, it is often difficult to amplify fossil DNA due to degradation or fragmentation, and spectacular claims of dinosaur or insect DNA amplification preserved in amber have been proven false. Closer to home, the first PCR tests to identify people who test positive for COVID have become the subject of controversy condemning false positives. Indeed, the further PCR is carried out, the more likely it is to detect traces of the virus. This is why the French Society for Microbiology (SFM) determines the number of cycles to declare a positive person. Because, like any biological test, PCR transmits information that then needs to be interpreted.

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