One of the words of 2021 is PCR. We’ve heard it a thousand times. In this article I will talk about what exactly this technique is and what it is used for. First say that PCR comes from Polymerase Chain Reaction.
By convention, scientists use the acronym for english words. In this way you have a standard with which to talk to other scientists. Just as if you are an ornithologist to address another ornithologist you have to use the scientific name of a bird, since regardless of the country, the scientific name derived from Latin is unique. Therefore, it is a good idea to use PCR and not the Spanish acronym RCP. And say DNA (DeoxyriboNucleic Acid) instead of ADN(ácido desoxirribonucleico).
What PCR means
PCR stands for polymerase chain reaction. Therefore, it is a chemical reaction since an enzyme (biological catalyst carrying out the reaction) and a substrate (the target nucleic acid to be studied) are used. Chained because it is performed multiple times chained (several cycles). And polymerase since it is the enzyme in charge of carrying it out. As the name suggests, the polymerase enzyme, polymerizes, binds nucleocids to a chain that is forming.
Its goal is to obtain a large number of copies of a particular DNA fragment, starting from a minimum; in theory it is sufficient to start from a single copy of that original fragment, or mold.
PCR is based on repeating in vitro (in laboratory) what the cell would do in vivo (inside the living). It is a recipe for cooking but instead of potatoes and grilos, the components are nitrogen bases, phosphorus, heat, magnesium chloride, barleys, etc. Several cycles occur in which the temperature is changed to denatur DNA. Denaturing DNA is for the two strands of DNA to separate, turning the double helix into two independent propellers.
DNA is a double helix of nucleic acid in which a nitrogenous base (which can be 4, Adenine, Timine, Cytosine and Guanine) faces another that will always be the same since the bond only allows it in one way. In this way Adenina faces a Timina, and an Citosine faces a Guanine It is like a spiral staircase in which each rung is formed by two tiles joined together.
One of the most important processes in a cell is replication. By replicaning from one cell we get two and the two are identical copies of each other. In the PCR there are successive cycles of denaturation, renaturalization and various replicative processes, so in the end we will obtain thousands of molecules from the DNA from which we start.
History and description of the method
The PCR method was described by Kary Mullis in 1985. It consists of looking at DNA replication in a cell and taking it to a process in the lab. We start from DNA and apply heat. As a result, the two strands separate. Polymerase DNA needs a first, a primer, a complementary RNA is produced. The cell needs the corresponding polydesoxynucleoticides, polymerase DNA and some Cl2Mg. Mullis occurs to you that by taking a strand, denaturing it by heat, the DNA separates. If at this time I have some first that had the complementary sequence of any of the DNA fragments and I add the DNA polymerase, the deoxytriphosphates and Cl2Mg, the DNA is copied. If I put another first to a dilution of 1 the same thing happens in that chain and the DNA is elongated.
Mullis had three buckets at different temperatures and some eppendorf where he introduces the standard DNA, where he adds the first or primers (about 20 nucleotides each). The dNTP deoxy of T, C, C and A, Cl2Mg. The tubes are inserted at 92 oC, the standard DNA is denatured, for 30-1 minutes and I pass it to another bath at about 50 oC for 1 minute and what happens is that the DNA is renatured. To force the DNA to be renaturaled I put with the first a high concentration of primers. Now the DNA polymerase Y is introduced. It is passed to another bath of about 70 oC for 30-1 minute.
It returns to the first bucket, DNA polymerase is lost but DNA is denatured after the minute is passed to the third bucket. What is achieved is to amplify the amount of DNA,from very few molecules to many. It’s a pre-step for other things.
Problems detected in the beginning in the PCR
From a technical point of view there were several problems:
- On the one hand, DNA polymerase had to be introduced into each cycle since when applying heat the enzyme, which is a protein, it was denatured (lost its properties). Therefore, it involved a significant expense of DNA polymerase. And foreign DNA contamination should also be avoided since each cycle increased that possibility.
- Another problem is that the person in charge of carrying out the reaction had to be concentrated during the 3 hours of the process.
- When sequencing DNA with PCR you need to know the sequence in advance. You don’t always want the PCR to do a sequence.
The Tag polymerase
The first problem was solved when a DNA polymerase was found that endured high temperatures without losing its properties. It was known that bacteria existed in places with high temperature that performed all the normal processes of a cell. The bacterium Thermophylus acuaticus,lives in extremely hotenvironments because it has enzymes that support them. It was one of those enzymes was the enzyme TAG polymerase that lasts until 92 oC they degrade.
To solve the second problem what was done was to try to machine this process. The thermocycler was invented. It is an instrument where eppendorf tubes of a certain size are inserted. It has a rapid heating and cooling system. The thermocycler is programmed to perform a series of cycles.
Many thermocyclers do not lower the temperature beyond the ambient one. There are others with a cooling mechanism to reach 4 oC.
All this achieved the automation of the PCR. However, TAG polymerase makes mistakes and had problems in certain lengths of DNA. Therefore, enzymes were sought that made fewer mistakes and endured longer DNAs. In addition, the TAG could not sequence more than 15,000 bp. Therefore, its use was brazen.
But what is a virus? What is your genetic material? A virus is a microscopic package of genetic material surrounded by a wrappedone. This genetic material can be either DNA or RNA
- DNA is a molecule formed by a double chain found in all organisms, such as animals, plants and viruses, and which contains the genetic material, by which all organisms are made.
- RNA is usually a single-strand molecule that copies, transcribes, and transmits parts of the genetic code to proteins so that they can synthesize and perform functions that keep organisms alive and developing. Different RNA variations are responsible for copying, transcribing, and transmitting. Some viruses, such as SARS-CoV-2 coronavirus,whichcauses COVID-19 disease, only contain RNA, meaning they rely on infiltration of healthy cells to multiply and survive.
To use the PCR technique it is necessary that we are working with DNA, it does not serve the RNA, because double chains have to be denatured. From one of the strings we can synthesize the other. Therefore we cannot work directly on the RNA of the sars-cov-2 virus.
Choosing the RNA solves a problem that has this microscopic package of genetic material called viruses, and is that conveniently for him is able to use the cellular machinery at his disposal to create more copies of himself.
Once inside the cell, the virus uses its own genetic code – RNA in the case of the COVID-19 virus to take control and reprogram the cell to make them a factor in creating new copies of viruses.
For a virus like COVID-19 LA to be detected early in the body using real-time RT-PCR, scientists must convert RNA into DNA. This is a process called “reverse transcription”. They do this because they can only copy or amplify DNA, which is a key part of the real-time RT-PCR process for detecting viruses. Scientists amplify a specific part of transscribed viral DNA hundreds of thousands of times. Amplification is important so that, instead of trying to detect a tiny amount of the virus among millions of strands of genetic information, scientists have a large enough number of target sections of viral DNA to accurately confirm that the virus is present.
How RT-PCR used against COVID-19 works
First, a sample is taken from an area where the patient may have traces of viruses such as the throat, nose or saliva. The sample obtained is purified to remove all material that is not necessary for the PCR test. The extract obtained is an amalgam of the person’s genetic material and, if present, genetic material from the virus.
The next step is done by the protein called reverse transcription, an enzyme that is capable of converting it into DNA from RNA. Additional small dna fragments are then added to complement certain parts of transcribed viral DNA. These fragments adhere to specific parts of viral DNA if the virus is present in the sample. These added genetic fragments allow you to create and amplify new DNA chains that allow you to subsequently detect the virus.
The process is automated by means of a device performed by RT-PCR, in which this material undergoes continuous cycles of raising the temperature (to denatur) and lowering the temperature (to naturalize) and each cycle results in new identid copies of specific parts of viral DNA.
Viral DNA meter
This combination is then introduced into an RT-PCR apparatus, where they undergo heat-cold cycles to cause certain chemical reactions that result in new identical copies of specific parts of viral DNA. These cycles are repeated over and over again to continue copying the specific parts of viral DNA.
In each of them the quantities are doubled: that is, from two copies, they are passed to four; from four, to eight, and so on. A common real-time RT-PCR system typically consists of 35 cycles, i.e. some 35 billion new copies of the viral DNA parts of each of the virus chains present in the sample will have been created at the end of the process.
As new copies of the invited parts of the invited viral DNA are produced, the markers are coupled to the DNA chains and emitted a fluorescence. The device that performs the RT-PCR measures and will present on the computer screen the result in real time.
The computer tracks the scale of the fluorescence of the sample that is measured after each cycle. When it exceeds a certain level, a detection threshold, the presence of the virus is confirmed and the patient is declared positive in sars-cov-2 viruses. The fewer cycles to obtain the highest fluorescence will be the viral load.
- Apuntes carrera de Biología.