The acronym PCR stands for “polymerase chain reaction”. This technique designed by Kary Mullis while driving on a long journey, thought that if, inside our cells, the different components were able to replicate our DNAIf he were able to recreate those conditions in his lab, he would still be able to obtain many copies of the same piece of DNA that he introduced.
And so it was, when he tested in his lab he was able to amplify his test sequence and this discovery won him the prize. Nobel Prize in Chemistry in 1993. Over time, the technique has been optimised and different variants have appeared. In the case of the coronavirus test, the technique used is a RT-qPCR.
Components of PCR
- Mould DNAis the DNA containing the sequence we want to amplify.
- Primersare small sequences complementary to a region of template DNA where the polymerase will bind. They are necessary because the polymerase cannot start adding nucleotides from a single-stranded sequence.
- Mixture of dNTPsthe nucleotides that will be added to the chain by the polymerase.
- Taq DNA polymeraseis the enzyme used for this reaction, because it is able to function at elevated temperatures. This polymerase was isolated from a bacterium called Thermus aquaticus which is able to live near hot springs and was first discovered in Yellowstone Park. This polymerase has an optimum temperature of 72°C.
- Magnesiumwhich is necessary for the polymerase to function optimally.
PCR steps:
- The first step consists of the denaturation of DNAThis can be achieved by raising the temperature of the solution to a temperature of around 96ºC.
- The second step consists of the annealing. This step is necessary for the primers to bind to their homologous regions so that the polymerase can then act. The temperature is usually between 55-65°C depending on the primers.
- And the third step consists of chain elongation. It occurs at the optimum temperature of the polymerase on the 72°C thus obtaining two chains identical to the initial mould chain.
So each time each of these three steps are completed we have performed what is called a PCR cycle. To start the next cycle, we would go back to the first step by increasing the temperature of the solution again. Therefore, each time we perform a cycle, we double the initial amount. So after a few 30 cycles we obtain an approximate quantity of 230 replicated moleculeswhich amounts to more than a billion copies!
RT-qPCR, coronavirus analysis
Because the genetic material of coronaviruses is RNAThe first step is to convert all the RNA found in our samples to DNA, using in the first cycle a retrotranscriptasewhich is a polymerase that is capable of transcribing a molecule DNA molecule from an RNA molecule. The qPCR o Real-time PCR is a variation in which the primers are attached to molecules capable of producing a fluorescent signal, which is found in the 5′ end end of the primers, which when the polymerase passes over it and adds a new nucleotide this molecule is released into solution.
This whole process takes place in a machine in which the samples are coupled and which at the end of each cycle records the increases, or not, of fluorescence in the sample. In the case of a sample with coronavirus, the fluorescence of the sample would have increased exponentially. If, on the other hand, it is negative, no fluorescence would be observed.
I hope you have learned more about this reaction, and if you want to learn how vaccines are made click here.