Every modern person, even those not knowing about it, has probably met with the polymerase chain reaction method (PCR) because it is used in a lot of analyzes such as infections analyzes — HIV or hepatitis. As a result of PCR, many sequences can be obtained from a small number of tissue samples. This is useful because PCR can work even with the smallest of samples — a drop of saliva or blood. In this topic, we will analyze what PCR is, what stages this reaction has, and how to use this method in practice.
History
The idea of amplification — cloning of DNA section to increase the total amount of the sample — appeared in the early 70s of the last century. A decade later, Kary Mullis, an American biochemist, managed to achieve multiple consecutive DNA duplications using the enzyme DNA-polymerase. The creation of the protocol was incredibly difficult since Mullis had many tasks: on the one hand, to conduct PCR without errors, so that the child nucleotides matched the parent with 100% accuracy; on the other hand, to provide the necessary speed, and on the third, to find a suitable thermostable enzyme (high temperature is a necessary part of PCR, which allows breaking double-stranded DNA into separate chains). After much research, it was decided to use a thermophilic DNA polymerase named Taq from the bacteria Thermus aquaticus. This bacterium lives in hot springs, so all its enzymes can work effectively even at very high temperatures — above 55°C.
Steps of PCR
PCR is a method of increasing the concentration of DNA fragments in a sample by repeatedly cloning the area under study.
PCR analysis is carried out in three stages:
1) DNA isolation;
2) DNA denaturation;
2.1) Amplification of DNA fragments — it includes renaturation (attachment of special sequences to DNA from a sample) and elongation (synthesis of new DNA strands). This PCR step is repeated many times in the same pattern, which ultimately leads to an increase in the amount of DNA.
3) Detection of DNA amplification products.
Isolation of DNA occurs after the sampling of the material. For DNA to be analyzed, it is isolated from the sample. A reagent is added to the sample that dissolves lipids, peptides, proteins, carbohydrates, and other organic substances that interfere with the "purity" of the reaction. DNA enzymes are very sensitive and fragile substances that break down extremely quickly, especially in the aggressive conditions of a "shaken" cell. Therefore, for the correct conduct of PCR, it is very important to remove absolutely all molecules, ions, and substances.
After isolation double helix is split into separate strands. This process takes place at a temperature of 94-96°C and is called denaturation. At this temperature, the hydrogen bonds between DNA bases melt, and single strands are formed. After that, the process of PCR itself begins.
The next step in PCR is DNA amplification. This phase requires:
1) a sample — isolated and purified DNA;
2) a DNA-polymerase — thermophilic Taq-polymerase is most often used in laboratory practice and has some shortcomings (in particular, it cannot "correct" errors), so at some point, another DNA-polymerase isolated from the archaea Pyrococcus furiosus — Pfu and Pwo — began to be used together with it. Archaeal polymerases can correct errors made during copying, and their use significantly reduces the number of mutations in DNA, although their speed is lower than that of Taq. Now in laboratory practice mixtures of Taq and Pfu are used — this allows the reaction to be carried out quickly and accurately;
3) a DNA primer or DNA template — DNA molecules on which "cloning" will subsequently occur. The primer floats in solution and at some point binds complementarily to the corresponding site on the thread under study, forming a new double helix;
4) free nucleotides that are used for synthesis.
This stage goes through the same cycle many times with determined stages, that run at a certain temperature. By changing the temperature, the scientist creates the conditions for the next stage of the cycle.
The first stage starts the process of primer binding to sample DNA and is called renaturation. It takes place at a temperature of approximately 68°C. A complex of single-stranded DNA with a primer is formed, and it will be completed further.
The second stage is elongation — the completion of the DNA segment by DNA polymerase. It occurs at 72°C. DNA polymerase recognizes the "start" site and, starting from the primer, completes the second strand complementarily using free nucleotides in solution. After elongation, DNA is formed twice as much as it was in the original sample.
Then the cycle repeats — denaturation, renaturation, and elongation take place, and so on until a sufficient amount of DNA is acquired. This is how any PCR works — from paternity testing to identifying the pathogen: the process of DNA replication is the basis. From one single piece of DNA after the first cycle, you already have 2 fragments, after the second cycle — 4, after the fourth — 16, and so on.
During the detection of amplification products, the resulting mixture of amplification products is separated. Special solutions are added to the mixture, which endows DNA fragments with the ability to fluoresce — it reflects orange-red luminous stripes. If the primer found the desired section of DNA, then during the detection it will be possible to see many fragments. If there was no such section in the sample, then only the primer and the original DNA will remain in the solution.
qPCR
The standard PCR protocol allows only a qualitative determination of the presence of the desired DNA sequence in the sample. For a quantitative study, there is a special PCR method — real-time PCR, or qPCR. qPCR is often used to detect the quantity of RNA in a sample. On its basis, scientists "make" DNA using the process of reverse transcription.
Then the method uses the general principles of PCR. The main difference is that the amount of amplified DNA is measured in real-time after each amplification cycle. For quantitative determination, two methods are used — fluorescent dyes that are embedded in double-stranded DNA molecules, and modified nucleotides, the so-called DNA probes, which fluoresce after they bind to DNA. The more intense the fluorescence, the more amplification products are in the sample. In this case, the probes are more specific, since they are associated only with the desired area.
PCR in use
PCR analysis is used in many laboratory studies as a preparation step. For example, PCR is used as a preparation step to establish paternity. Kinship can only be established by the similarity of genetic information (this is done by using genome sequencing), but in order for it to be sufficient for determination in the sample, PCR is performed — this allows for the accumulation of many copies of a particular section of DNA.
PCR allows for diagnosing the presence of pathogens (viruses, bacteria) that grow in the laboratory for a long time without resorting to time-consuming microbiological methods. That's why PCR is used to determine a huge number of other infections: STIs, including HIV, hepatitis, and so on. In such cases, scientists can use primers specific for specific infections — if there is no complementary site in the sample, then binding and further accumulation of DNA will not occur.
Real-time PCR is also used to determine covid infection: a special polymerase is added to the sample so that the virus RNA is converted into DNA, and then PCR is performed with a specific primer. If a virus is present in the sample, these fragments are attached to the specified regions of the viral DNA and amplification occurs. Then the fluorescence of the sample is evaluated — if it is more than a certain level, then the virus is considered detected. Such an analysis also allows you to assess the level of viral load: the earlier the threshold value was reached, the higher it is.
In addition, PCR is used as a method for research purposes. This is often needed by molecular biologists, immunochemists, and other scientists working with DNA and the processes associated with it.
Conclusion
PCR is a method in which DNA from a sample is replicated many times. To identify a particular site, primers specific to that site are used. Some PCR steps take place at very high temperatures, so a thermostable DNA polymerase is used. One PCR cycle proceeds in three stages: at the stage of denaturation, DNA breaks up into separate strands, at the stage of renaturation a primer is attached to the strand, and at the elongation stage the polymerase builds a complementary strand, starting from the primer. Sometimes scientists use a fluorescent dye, as it can help quantify the pathogen.