POLYMERASE CHAIN REACTION (PCR)
PCR is a technique for the “in vitro” synthesis of specific DNA sequences. It is a simple and very fast way of multiplying the DNA present in different biological samples, obtaining millions of copies of a certain DNA sequence in a short time this technique has managed to be widely used not only in the field of molecular genetics, but in many other sciences. The acronym PCR stands for Polymerase Chain Reaction, it is also known as Peoples choice Reaction.
It is possible to amplify any DNA sequence obtained from blood, urine, tissue fragments, and also microorganisms, animal or plant cells, even if they are thousands of years old.
The reaction is based on the natural function of a thermostable enzyme, called Taq DNA polymerase, extracted from the bacterium Thermus aquatics, an extremophile found in hydrothermal vents. This fact is extremely important, since the reaction takes place in a device called a thermocycler, which heats and cools the material contained in it in pre-established temperature cycles.
History of PCR :
The inventor of this interesting technique was Kary B Mullis for which he was awarded the Nobel Prize in Chemistry in 1993. He used PCR for the amplification of the human β-globin gene since then PCR has revolutionized all the fields that study and manipulate nucleic acids.
Mullis relied on DNA replication in eukaryotic organisms by DNA polymerase. These enzymes synthesize a complementary DNA strand in the 5′-> 3 ‘sense using a single-stranded template, but starting from a double-stranded region. To create this double chain region, the so-called primers are used. They are a pair of oligonucleotides synthesized so that they are complementary to each of the 3 ‘ends of the DNA fragment to be amplified.
After extracting and obtaining the DNA from the sample, the next step in any genetic analysis is the selection and amplification of the region of the genome that we want to study. For this, it is necessary to use primers or primers that delimit our region of interest and an enzyme polymerase that is in charge of replicating (making thousands of copies) that fragment of the genome.
What is PCR?
Polymerase chain reaction (PCR) is one of the most common techniques in a genetics laboratory and its purpose is to obtain many copies of a specific region of DNA. Studying a region of DNA can be interesting to know the function of a gene, analyze a genetic marker that allows us to identify a person or a gene in which we want to find a mutation that produces a certain disease.
Therefore, the ultimate goal of PCR is to produce enough DNA, from the region of interest, so that it can be analyzed or used in some other technique: for example, for sequencing, to visualize by gel electrophoresis, or to perform a restriction. enzymatic.
Definition of PCR :
The PCR (Polymerase Chain Reaction) technique allows multiplying a specific stretch of DNA thousands of times without the use of living organisms. In a few hours, with minimal amounts of genetic material, it is possible to obtain enough copies to detect and analyze the sequence that is the target of the study.
PCR technique is used in biological and medical research laboratories, to identify hereditary diseases, clone genes, detect pathogenic organisms, produce transgenic organisms, making paternity tests, among others.
Types of PCR
Nested PCR: This is a variant of basic PCR that uses two pairs of primers. In a first step, the amplification of a region of the genome is carried out, to later specify the region more using a second, more specific amplification. This PCR is used to amplify very specific fragments of the genome.
RT-PCR: This technique converts the RNA from a sample into DNA. To do this, it uses reverse transcriptase, an enzyme used by retroviruses. It is used for multiple purposes. For example, it can be used to find out if a gene is being expressed in a biological sample. It is also used to genotype different RNA viruses, such as SARS-Co-V or HIV.
Quantitative PCR: it is a PCR that allows the number of fragments that are produced to be measured in real-time. It is often used to analyze the expression of genes.
Multiplex PCR: in this type of PCR, simultaneous amplification of more than one DNA fragment is carried out. For this, several different primers are used in the same reaction.
In situ PCR: This PCR is performed on cells or tissues. It is used to be able to detect DNA sequences inside cells that are not detectable by other techniques.
Digital PCR: It is one of the last generations in DNA amplification techniques. It is based on the separation of each sample into multiple partitions (microdroplets), so that the amplification reaction occurs independently of each of them.
These are just some of the better-known types of PCR, although there are also many other variations of PCR, such as asymmetric PCR, or allele-specific PCR, that achieve different results from the DNA samples obtained.
Components of PCR
To perform genome amplification using PCR, there are several basic components, including:
- DNA sample.
- Polymerase enzyme (Taq polymerase).
- Stable medium for the reaction to occur.
- Nucleotide solution that the enzyme will use to make copies of the genome together with the cofactors that the enzyme needs.
- Buffer solution
- Divalent magnesium ions
- PCR Machine ( Thermal cycler )
There are several simple rules so that template DNA is not a problem in the reaction:
- DNA integrity: it cannot be fragmented into pieces smaller than what we want to amplify.
- Origin of the sample and extraction process: the sample must not contain chelating agents (EDTA) that reduce the concentration of Mg ions. in dissolution. Not should there be certain blood factors, phenol, detergents that would inhibit the activity of the polymerase?
- Sample quantity: if sufficient quantity is available for single-copy genomic DNA amplification, quantities of 100-500 ng are used. In the case of repeated areas, this amount can be reduced to 10-50 ng.
- The minimum ranges from 10-100 ng. and the maximum between 400-500 ng.
- For the choice of primers, there are a series of rules that can help us, although it should also be noted that there are computer programs that facilitate this task (DNAsis, Primer3, etc.).
- The G + C content should be approximately 50%. The maximum purine / pyrimidine ratio will be 60% / 40%.
- Areas with long single base sequences should be avoided.
- Do not select primers that have an important secondary structure at their 3 ‘end.
- It is recommended that the ends of the last bases be G or C.
- Complementarity between the pair of primers should be avoided. If this exists between the 3 ‘ends, the chance of primer dimers being created is increased.
- They should normally be 18-30 bp in size.
- The hybridization temperature of the primers must be similar in both and will vary depending on their sequence. It generally ranges between 45 and 65ºC.
- If the primer is less than 20 bp, the melting temperature (Tm) is calculated based on the following formula: Tm = 4 (G + C) + 2 (A + T) G, C, T, and A being the number of each of the bases that make up each of the oligos.
- The annealing temperature should be approximately 5 ° C lower than the calculated temperature.
Taq DNA polymerase Enzyme
- As in the case of DNA replication in the cells of an organism, PCR requires a DNA polymerase enzyme to produce new DNA strands by using existing strands as a template.
- However, this enzyme must possess special characteristics: that it withstands the increase in temperature well. The amplification process is carried out in cycles, in each of which it is necessary to increase the temperature (96ºC) to denature and separate the DNA strands, without denaturing the proteins.
- Furthermore, the synthesis process of new DNA strands must be carried out at a high temperature (around 72ºC) to prevent the strands from rejoining in the middle of the process.
- For these reasons, it is necessary to use an enzyme capable of withstanding and working at high temperatures.
- The DNA polymerase normally used in PCR is called Taq polymerase, which gets its name from the hyperthermophilic bacteria from which it was isolated (Thermus aquaticus present in hot spring).
- The natural habitat of this bacterium is the fumaroles and hydrothermal vents, for this reason, its DNA polymerase is very thermostable and its highest activity occurs near 70 ° C (the temperature at which the DNA polymerase of the human being would not work).
- The primers are short single-stranded DNA fragments (generally about 20 nucleotides in length) whose function is to limit the region of the genome that we want to amplify, since they serve as a starting point for DNA polymerase.
- These primers must be designed in such a way that their sequence is complementary to the previous region of which we want to amplify.
- This will allow the union by the complementarity of bases and will serve as a starting point for DNA polymerase, all this can be observed in the following figure.
Divalent magnesium ions :
- Positively charged ions are used as polymerase cofactors in PCR.
- These cations are essential for the function of DNA polymerase.
- Magnesium chloride is normally added to release magnesium upon dissociation (with a +2 charge).
Buffer solution :
- A buffer solution is a solution that is capable of regulating the pH.
- That is the acidity or basicity conditions of our PCR Reaction.
- This is very important because changes in the pH of the solution can alter the results of our PCR Reaction or prevent it from occurring.
Thermal cycler :
- A thermal cycler is a device that regulates the temperature in each cycle of the PCR .
- Thanks to him it is possible to carry out this technique is much less time since to carry it out.
- The temperature of the solution must be modified several times.
All right! We already know what basic elements we need to perform a PCR. Now, we only have to know the procedure to follow in this technique:
Steps of PCR :
The DNA amplification using the PCR technique is carried out all of it in a single 0.5 ml Eppendorf tube, which will contain: problem DNA, oligonucleotide primers or primers, free deoxynucleotide triphosphates, Taq polymerase, magnesium, and reaction buffer. Once we have analyzed the parts that make up the polymerase chain reaction,
The process takes place in three stages, which together are called a PCR cycle that is repeated a specific number of times:
1. Denaturation: in this step, we raise the temperature to 96ºC to favor the separation, or denaturation, of the DNA chains. This provides us with the single-chain strands that will serve as the template for the next step.
2. Primer Binding : The temperature decreases to 55-65ºC (depending on the specific banding temperature of each primer) to allow primers to bind to their complementary sequences in the DNA template strand.
3. Primer Extention : this is the final step of the cycle, in which the temperature of the reaction rises to around 72ºC to thus favor the activity of the Taq enzyme. This will proceed to extend the primers and thus synthesize new DNA chains.
This PCR cycle is repeated between 25-45 times in a complete PCR reaction, which allows us to obtain in each cycle an exponential number of copies of the region we want to study, until we obtain millions of copies of the region of interest.
Applications of PCR
- The applications of PCR are multiple, from archeology, forensic medicine, and clinical medicine.
- It allows the diagnosis of infectious diseases in a few hours compared to waiting for the cultures.
- This technique is also useful for the study of certain tumors.
- The amplification of specific DNA fragments by PCR quickly and efficiently produces many copies of DNA that allow a rapid diagnosis of tuberculosis, papillomavirus, cytomegalovirus, and chlamydia among other germs.
- Polymerase chain reaction Application has great importance in medicine, for the prevention, diagnosis, and control of human cancer.
- The analysis of karyotypes can be done by a methodology called Molecular Cytogenetics, whose execution protocols involve DNA analysis by Polymerase Chain Reaction, followed by electrophoresis and laser densitometry.
- Polymerase Chain Reaction is used in the analysis for the determination of a baby’s sex (fetal sexing) even before the sex is visible.
- The polymerase chain reaction is useful for the detection of various kinds of diseases.
- The Polymerase Chain Reaction can also be used to diagnose diseases caused by external agents, such as: Chikungunya (virus), Dengue (virus), Zika virus), Gonorrhea (bacteria), Chlamydia (bacteria), HPV (virus)
- Some genetic diseases that can be detected by PCR, among several others, are: Variant hemoglobinopathies, Severe Combined Immunodeficiency, Detection of the methylene hydroxireductase (MTHFR) mutation, Cystic fibrosis, Thrombo ﬁlia Detection of the G1691A mutation (FactorV).