PCR analysis - what is it? This is an effective method of molecular biology, which is used in combination with already traditional immunological, morphological and biochemical studies. Infectious diseases evolve and develop, just like humanity itself. Every year there are more and more of them, and it is more and more difficult to diagnose them. The factors that provoke the appearance of diseases and influence their development also gradually adapt to the surrounding external conditions, changing along with them. This was the reason that new methods and technologies began to appear in medicine, which help to obtain more accurate results in the diagnosis of a particular disease.
This method of laboratory diagnosis of infectious diseases is based on detecting the causative agent of the infection that the doctor suspects in the research material. The polymerase chain reaction will identify them by identifying the appropriate genetic material (RNA or DNA) in the samples taken from the patient.
PCR was invented by the American scientist Cary Mullis in 1983.
Most infections, if detected in the early stages of development, respond well to treatment. That is why the PCR method is so effective, because it is able to detect even viruses or bacteria, whose cells are single in the material. Moreover, such a diagnosis determines the virus, establishes the nature of its appearance, the force with which it affects the body, even the number of microbes in the patient's body. All the information obtained by the polymerase chain reaction method, the doctor will be able to apply in order to select the appropriate drugs and prescribe the appropriate treatment.
PCR study
By the very principle of operation, everything happens quite simply. The biological material taken from the patient is placed in a special reactor. Then there are added such specific enzymes that can bind to the DNA of the microbe existing in the material and synthesize its copy.
Copying is based on the principle of a chain reaction. That is, the whole process will require several stages. First, 1 DNA molecule forms 2 new molecules, then 4 new ones will turn out of them, and so on up to hundreds and thousands of copies. After that, the analysis and its decoding will be carried out.
Microbial DNA fragments can be contained in various biological material:
- in biological fluids (saliva, articular, amniotic, pleural, cerebrospinal fluid, prostate juice);
- in blood and its serum, in plasma;
- in scraping of epithelial cells (scraping from the cervical canal, from the urethra - for both women and men);
- in the urine (the analysis will require morning urine, namely its first portion);
- in sputum;
- in mucus and other biological secretions;
- in biopaths of the stomach and duodenum.
What diseases can be detected by PCR?
Doctors consider this type of diagnosis one of the most accurate. This study allows you to detect almost all viral diseases that are currently known to medicine. A very important aspect is the fact that among them there are infections that live in the human body for many years, while not manifesting themselves in any way. They simply grow in anticipation of when it will be most comfortable for them to manifest themselves (decreased immunity, depletion of the body, etc.). The detection of such latent infections is especially important in the urological and gynecological areas.
Here are some diseases that are often analyzed by polymerase chain reaction:
- hepatitis B and C;
- many diseases that are sexually transmitted (diagnosis of chlamydia, ureaplasmosis, mycoplasmosis, genital candidiasis, bacterial vaginosis, trichomoniasis, infectious mononucleosis, human papillomavirus infection (HPV), AIDS, etc.);
- herpes infection (including genital herpes);
- tuberculosis and helicobacteriosis.
The PCR method gives good results because most of these diseases are characterized by the fact that their symptoms (especially at an early stage) are not very noticeable. But the consequences that they have on the body are very negative and often very dangerous for health and even life.
For example, sexually transmitted diseases can significantly increase a woman's chance of cervical cancer, adversely affect pregnancy, or cause infertility. In addition, the health of her unborn baby depends on the health of the mother. Therefore, it is very important, at the slightest suspicion of latent infections, to donate blood for diagnosis in time to prevent infection of the fetus through the penetration of pathogens. For men, the consequences are no less negative: a decrease in sperm motility and viability, the development of male infertility and prostatitis, damage to the urethra, and so on.
Such an analysis is also very relevant for the timely detection of viral hepatitis, tuberculosis, and various intestinal infections. When immediate treatment is required, it is very important to understand which pathogen has struck the body and what needs to be fought. Blood or any other biological material of the patient will help to carry out a complete and correct diagnosis and prescribe a treatment that will help restore body functions as soon as possible and promote recovery.
Herpes is also extremely persistent and dangerous. From an inactive mode, it can easily go into a progressive one, affecting the central nervous system, affecting the emergence and development of such terrible diseases as meningitis and encephalitis.
Deciphering the analysis
After conducting the study, you can get either a positive or a negative result. It is the positive results that will indicate that this or that infection is present in your body. A negative result is interpreted in such a way that there is no suspected infection in the patient's body. That is, nothing was found in the biological material that was submitted for research.
Any indicators should be deciphered and voiced to you by a doctor. Do not be upset and do not be afraid of bad results, because the reaction revealed the disease, which means that after additional examinations, the doctor will be able to prescribe a full-fledged treatment. The main thing is not to self-medicate and not delay the process.
Preparing for analysis: what you need to know?
Different biological materials will be required to detect different diseases. Before doing PCR, be sure to consult with the appropriate specialist and carefully prepare for the upcoming procedure.
To do this, you will need to strictly follow all the recommendations and instructions of your doctor. Do not forget that blood is usually taken on an empty stomach. To take a smear from the vagina or urethra, you need to refrain from sexual intercourse for 1-2 days, carry out all the necessary hygiene of the genital organs in the evening, and in the morning just rinse with warm water. It is also important to stop taking medications for about a week, unless they are discussed with your doctor. And within 2-3 hours before taking the test, do not urinate. For women, it is worth considering that the optimal time for the study is a few days before or immediately after menstruation.
PCR method: main advantages and disadvantages
This type of diagnosis has many advantages.
Firstly, any pathogens of infectious diseases are determined by direct indication to them, unlike other traditional laboratory studies.
Secondly, this analysis is simply universal, because almost any biological materials are available for its implementation, which are often not accepted by any other research method.
It is very important that when this diagnosis is carried out, then in the material under study, a DNA fragment is isolated, which will be inherent only to a specific pathogen, that is, a purely specific virus or bacterium. This indicates how specific this reaction is.
Another argument in favor of this diagnostic will be the high speed of its execution. If any culture studies require not days, but even weeks, necessary for the isolation and cultivation of the pathogen in cell culture, then this method will provide results within 4-5 hours.
PCR makes it possible to detect not only the infection that is already at the peak of the disease, but also chronic diseases, any single viruses or bacteria. Such a diagnosis is possible due to the very high sensitivity of the method. This should also include the fact that the infection will be detected even if only one cell of a particular virus or bacterium is present in the biological material that was submitted for analysis.
Reaction to false negative results is very rare.
However, the method also has its drawbacks. One of them is the possibility of obtaining a false positive result. This is often due to the fact that the infection has already been killed, but its epithelial cells have not yet been updated, so the reaction will still see its dead remnants and clone it. Therefore, it is worth either waiting for the complete removal of dead cells of the infection from the body (from a month to two after the treatment), or using other methods at an early stage: seeding or culture. Later it will be possible to carry out control using PCR.
Another weakness of the analysis is the variability of all microorganisms. This means that some genotypes of pathogens that have already mutated in the body will be elusive for the test system and no reaction will occur. For this, various developments are being made to improve this method.
Federal Agency for Education
State educational institution
Higher professional education
"Karelian State Pedagogical Academy"
Coursework on the topic:
Polymerase chain reaction (PCR) and its application
Completed by: student Koryagina Valeria Alexandrovna
Checked by: Karpikova Natalya Mikhailovna
Petrozavodsk 2013
Introduction
Chapter 1 Literature Review
1.5.4 Plateau effect
1.5.6 Amplification
Conclusion
Introduction
The last twenty years have been marked by the widespread introduction of molecular genetic methods into the biological, medical, and agricultural sciences.
By the early 1970s, it seemed that molecular biology had reached a certain degree of perfection. During this period, microorganisms were the main object of molecular genetic research. The transition to eukaryotes presented researchers with completely new problems that could not be solved using the methods of genetic analysis that existed at that time. A breakthrough in the development of molecular genetics became possible due to the emergence of a new experimental tool - restriction endonucleases. In subsequent years, the number of direct DNA analysis methods based on qualitatively different approaches began to increase rapidly.
In many cases, modern technologies have made it possible to start studying the fine structural and functional organization of the nuclear and extranuclear genomes of various organisms at a deeper level. This was of particular importance for the development of new methods for the diagnosis and treatment of various diseases. No less important was the possibility of using the achievements of molecular genetics in population biology and breeding to identify and analyze the genetic variability of populations, varieties and strains, identify and certify economically valuable individuals, create genetically modified organisms, and to solve other issues.
Each method has its own advantages and disadvantages. There is no universal method that could solve all the problems that arise. Therefore, the choice of a specific method for the ongoing research is one of the most important stages of any scientific work.
Chapter 1 Literature Review
1.1 History of the discovery of the polymerase chain reaction (PCR)
In 1983 K.B. Mullis et al. published and patented the polymerase chain reaction (PCR) method, which was destined to have a profound impact on all areas of research and application of nucleic acids. The significance of this method for molecular biology and genetics turned out to be so great and obvious that seven years later the author was awarded the Nobel Prize in Chemistry.
At the beginning of the use of the method, after each heating-cooling cycle, DNA polymerase had to be added to the reaction mixture, since it was inactivated at the high temperature necessary to separate the DNA helix chains. The reaction procedure was relatively inefficient, requiring a lot of time and enzyme. In 1986, the polymerase chain reaction method was significantly improved. It has been proposed to use DNA polymerases from thermophilic bacteria. These enzymes proved to be thermostable and were able to withstand many reaction cycles. Their use made it possible to simplify and automate PCR. One of the first thermostable DNA polymerases was isolated from bacteria Thermus aquaticusand named Taq-polymerase.
The possibility of amplifying any DNA segment whose nucleotide sequence is known, and obtaining it after PCR in a homogeneous form and preparative amount, makes PCR an alternative method for molecular cloning of short DNA fragments. In this case, there is no need to apply complex methodological techniques that are used in genetic engineering in conventional cloning. The development of the PCR method has greatly expanded the methodological possibilities of molecular genetics, and, in particular, genetic engineering, so much so that it has radically changed and strengthened the scientific potential of many of its areas.
1.2 Varieties of polymerase chain reaction (PCR)
· Nested PCR- used to reduce the number of by-products of the reaction. Use two pairs of primers and carry out two consecutive reactions. The second pair of primers amplifies the DNA region within the product of the first reaction.
· Inverted PCR- is used when only a small area within the desired sequence is known. This method is especially useful when it is necessary to determine neighboring sequences after DNA has been inserted into the genome. For the implementation of inverted PCR, a series of DNA cuts is carried out with restriction enzymes<#"justify">polymerase chain reaction primer
· Group-specific PCR- PCR for relatives<#"center">1.3 Polymerase chain reaction
Discovered in the mid-1980s, the polymerase chain reaction (PCR) can increase the number of copies of an original sample millions of times within a few hours. During each cycle of the reaction, two copies are formed from the original molecule. Each of the synthesized DNA copies can serve as a template for the synthesis of new DNA copies in the next cycle. Thus, repeated repetition of cycles leads to an increase in the number of copies exponentially. It follows from the calculations that even if there are 30 cycles, the number of copies of the original molecule will be more than 1 billion. Even if we take into account that not all amplicons are duplicated during each cycle, the total number of copies, despite this, is quite a large figure.
Each cycle of the polymerase chain reaction (PCR) consists of the following steps:
· Denaturation - An increase in temperature causes a double-stranded DNA molecule to unwind and split into two single-stranded ones;
· Annealing - Lowering the temperature allows primers to attach to complementary regions of the DNA molecule;
· Elongation - The enzyme DNA polymerase completes the complementary strand.
For amplification of the selected fragment, two oligonucleotide primers (seeds) flanking a certain DNA region are used. Primers oriented 3 - ends towards each other and in the direction of the sequence that needs to be amplified. DNA polymerase carries out the synthesis (completion) of mutually complementary DNA chains, starting with primers. During DNA synthesis, primers are physically inserted into the chain of newly synthesized DNA molecules. Each strand of the DNA molecule formed using one of the primers can serve as a template for the synthesis of a complementary DNA strand using the other primer.
1.4 Conducting a polymerase chain reaction (PCR)
The polymerase chain reaction is carried out in special thin-walled polypropylene test tubes, compatible in size with the used thermal cycler (amplifier) - a device that controls the temperature and time characteristics of the stages of the polymerase chain reaction (PCR).
1.5 Principle of the polymerase chain reaction method
Polymerase chain reaction (PCR) is an in vitro DNA amplification method that can isolate and multiply a specific DNA sequence billions of times within a few hours. The ability to obtain a huge number of copies of one strictly defined region of the genome greatly simplifies the study of an existing DNA sample.
To carry out a polymerase chain reaction, a number of conditions must be met:
1.5.1 Presence of a number of components in the reaction mixture
The main components of the reaction (PCR) mixture are: Tris-HCl, KCl, MgCl 2, a mixture of nucleotide triphosphates (ATP, GTP, CTP, TTP), primers (oligonucleotides), analyzed DNA preparation, thermostable DNA polymerase. Each of the components of the reaction mixture is directly involved in the polymerase chain reaction (PCR), and the concentration of reagents directly affects the course of amplification.
· Tris-HCl - determines the pH of the reaction mixture, creates a buffer capacity. The activity of DNA polymerase depends on the pH of the medium, so the value of the pH directly affects the course of the polymerase chain reaction. Usually the pH value is in the range of 8 - 9.5. The high pH is due to the fact that as the temperature rises, the pH of the Tril-HCl buffer drops.
· KCl - the concentration of potassium chloride up to 50 mm affects the course of the processes of denaturation and annealing, the concentration above 50 mm inhibits DNA polymerase.
· MgCl 2- because DNA polymerase is Mg 2+- dependent enzyme, then the concentration of magnesium ions affects the activity of the enzyme (Mg 2+forms complexes with NTP - it is these complexes that are the substrate for polymerase). A high concentration leads to an increase in nonspecific amplification, and a low one leads to inhibition of the reaction, the optimum (for various polymerases) is in the region of 0.5 - 5 mM. In addition, the concentration of magnesium salts affects the course of denaturation and annealing processes - an increase in the concentration of Mg 2+causes an increase in the melting temperature of DNA (i.e., the temperature at which 50% of double-stranded DNA strands are broken into single-stranded strands).
· NTP - nucleotide triphosphates are direct monomers of nucleic acids. To prevent chain termination, an equal ratio of all four nucleotide triphosphates is recommended. The low concentration of these components in the reaction mixture increases the probability of errors in the construction of the complementary DNA strand.
· Primers - The most optimal is the use of primers with a melting point difference of no more than 2 - 4 o C. Sometimes during long-term storage at a temperature of 4 o With, or after a large number of freezing - thawing, the primers form secondary structures - dimers, reducing the efficiency of the PCR. The elimination of this problem is reduced to incubation in a water bath (T=95 o C) for 3 minutes and subsequent rapid cooling to 0o WITH.
· DNA preparations - the quantity and quality of the DNA preparation (matrix) directly affects the course and parameters of the polymerase chain reaction. Excess DNA sample inhibits the polymerase chain reaction (PCR). Impurities of various substances in the DNA preparation can also reduce the efficiency of the polymerase chain reaction (PCR): sodium acetate, sodium chloride, isopropanol, ethanol, heparin, phenol, urea, hemoglobin, etc.
· DNA polymerase - when using a small amount of DNA polymerase, a decrease in the synthesis of the final product is observed in direct proportion to the size of the fragments. An excess of polymerase by 2 - 4 times leads to the appearance of diffuse spectra, and by 4 - 16 times - low molecular weight nonspecific spectra. The range of concentrations used is 0.5 - 1.5 units of activity in terms of 25 µl of the PCR mixture.
In addition to the main components of the PCR mixture, a number of additional substances are used that improve the qualitative and quantitative indicators of PCR: acetamide (5%) - an increase in the solubility of the main components; betaine (sodium salt) - stabilization of DNA polymerase, lowering the melting point of DNA, equalizing the melting point; bovine albumin (10-100 μg / ml) - stabilization of DNA polymerase; dimethyl sulfoxide (1-10%) - increasing the solubility of the main components; formamide (2-10%) - an increase in the specificity of annealing; glycerol (15-20%) - an increase in the thermal stability of the enzyme, a decrease in the temperature of denaturation of a DNA sample; ammonium sulfate - lowering the temperature of denaturation and annealing.
1.5.2 Cycle and temperature
The general view of the polymerase chain reaction (PCR) program is as follows:
stage. Prolonged primary denaturation of the DNA preparation.1 cycle
stage. Rapid denaturation of the DNA preparation. Primer annealing. Elongation.30 - 45 cycles.
stage. Prolonged elongation. Cooling of the reaction mixture. 1 cycle.
Each element of the stage - denaturation, annealing, elongation - has individual temperature and time characteristics. The parameters of temperature and flow time of each element are selected empirically, in accordance with the qualitative and quantitative indicators of the amplification products.
Denaturation. During this element of the polymerase chain reaction, a double-stranded DNA molecule is split into two single-stranded ones. Temperature parameters of denaturation are in the range of 90 - 95 o C, but in the case of a DNA sample with a high content of guanine and cytosine, the temperature should be increased to 98 o C. The temperature of denaturation should be sufficient to completely denature - cleave the DNA strands and avoid "sudden cooling" or rapid annealing, however, thermostable DNA polymerase is less stable at high temperatures. Thus, the selection of optimal denaturation temperature parameters for the primer/sample ratio (DNA preparation) is an important condition for amplification. If the denaturation temperature in the first step is above 95 o C, it is recommended to add DNA polymerase to the reaction mixture after primary denaturation. The duration of this element of the stage during the polymerase chain reaction (PCR) should be sufficient for complete DNA denaturation, but at the same time not significantly affect the activity of DNA polymerase at a given temperature.
Annealing. Annealing temperature (T a ) is one of the most important parameters of the polymerase chain reaction. The annealing temperature for each specific primer is selected individually. It depends on the length and nucleotide composition of the primer. Usually it is lower by 2 - 4 o From the melting point value (T m ) primer. If the annealing temperature of the system is below the optimum, then the number of nonspecific amplified fragments increases and, conversely, a higher temperature reduces the number of amplified products. In this case, the concentration of specific amplicons can sharply decrease, up to inhibition of the polymerase chain reaction (PCR). Increasing the annealing time also leads to an increase in the number of nonspecific amplicons.
Elongation. Typically, each type of thermostable DNA polymerase has an individual temperature optimum of activity. The rate of synthesis of a complementary DNA strand by an enzyme is also a value specific to each polymerase (on average, it is 30–60 nucleotides per second, or 1–2 thousand bases per minute), so the elongation time is selected depending on the type of DNA polymerase and the length of the amplified region.
1.5.3 Basic principles of primer selection
When creating a PCR test system, one of the main tasks is the correct selection of primers that must meet a number of criteria:
Primers must be specific. Particular attention is paid to 3 - the ends of the primers, since it is from them that Taq polymerase begins to complete the complementary DNA chain. If their specificity is insufficient, then it is likely that undesirable processes will occur in the test tube with the reaction mixture, namely, the synthesis of nonspecific DNA (short or long fragments). It is visible on electrophoresis in the form of heavy or light additional bands. This makes it difficult to evaluate the results of the reaction, since it is easy to confuse a specific amplification product with synthesized foreign DNA. Part of the primers and dNTPs is consumed for the synthesis of nonspecific DNA, which leads to a significant loss of sensitivity.
Primers should not form dimers and loops, i.e. no stable double strands should be formed by annealing the primers to themselves or to each other.
1.5.4 Plateau effect
It should be noted that the process of accumulation of specific amplification products exponentially lasts only a limited time, and then its efficiency drops critically. This is due to the so-called "plateau" effect.
term effect plateau used to describe the process of accumulation of PCR products in the last cycles of amplification.
Depending on the conditions and the number of cycles of the amplification reaction, at the time the effect is achieved plateau the utilization of substrates (dNTPs and primers), the stability of reactants (dNTPs and enzyme), the number of inhibitors, including pyrophosphates and DNA duplexes, competition for reactants with non-specific products or primer-dimers, the concentration of a specific product, and incomplete denaturation at high concentrations of amplification products are affected.
The lower the initial concentration of the target DNA, the higher the risk of the reaction plateau". This point can occur before the number of specific amplification products is sufficient to be analyzed. Only well-optimized test systems can avoid this.
1.5.5 Sample preparation of biological material
Different techniques are used for DNA extraction, depending on the tasks. Their essence lies in the extraction (extraction) of DNA from a biological product and the removal or neutralization of foreign impurities to obtain a DNA preparation with a purity suitable for PCR.
The method of obtaining a pure DNA preparation, described by Marmur, is considered standard and has already become classical. It includes enzymatic proteolysis followed by deproteinization and DNA reprecipitation with alcohol. This method makes it possible to obtain a pure DNA preparation. However, it is quite laborious and involves working with such aggressive and pungent substances as phenol and chloroform.
One of the currently popular methods is the DNA extraction method proposed by Boom et al. This method is based on the use of a strong chaotropic agent, guanidine thiocyanate (GuSCN), for cell lysis, and subsequent DNA sorption on a carrier (glass beads, diatomaceous earth, glass milk, etc.). After washings, DNA remains in the sample adsorbed on the carrier, from which it can be easily removed using an elution buffer. The method is convenient, technologically advanced and suitable for sample preparation for amplification. However, DNA losses are possible due to irreversible sorption on the carrier, as well as during numerous washes. This is especially important when working with small amounts of DNA in the sample. Moreover, even trace amounts of GuSCN can inhibit PCR. Therefore, when using this method, the correct choice of the sorbent and careful observance of technological nuances are very important.
Another group of sample preparation methods is based on the use of Chilex-type ion exchangers, which, unlike glass, do not adsorb DNA, but vice versa, impurities that interfere with the reaction. As a rule, this technology includes two stages: sample boiling and adsorption of impurities on an ion exchanger. The method is extremely attractive due to its simplicity of execution. In most cases, it is suitable for working with clinical material. Unfortunately, sometimes there are samples with impurities that cannot be removed using ion exchangers. In addition, some microorganisms cannot be destroyed by simple boiling. In these cases, it is necessary to introduce additional stages of sample processing.
Thus, the choice of the sample preparation method should be treated with an understanding of the purposes of the intended analyses.
1.5.6 Amplification
To carry out the amplification reaction, it is necessary to prepare the reaction mixture and add the analyzed DNA sample to it. In this case, it is important to take into account some features of primer annealing. The fact is that, as a rule, in the analyzed biological sample there are various DNA molecules, to which the primers used in the reaction have partial, and in some cases significant, homology. In addition, primers can anneal to each other to form primer-dimers. Both lead to a significant consumption of primers for the synthesis of side (nonspecific) reaction products and, as a result, significantly reduce the sensitivity of the system. This makes it difficult or impossible to read the results of the reaction during electrophoresis.
1.6 Composition of the standard PCR reaction mixture
x PCR buffer (100 mM Tris-HCl solution, pH 9.0, 500 mM KCl solution, 25 mM MgCl2 solution ) …….2.5 µl
Water (MilliQ) ……………………………………………………….18.8 µl
A mixture of nucleotide triphosphates (dNTPs)
mM solution of each………………………………………….……….0.5 µl
Primer 1 (10 mM solution) ………………………………………….….1 µl
Primer 2 (10 mM solution) ………………………………………….….1 µl
DNA polymerase (5 units / µl) ……………………………………………0.2 µl
DNA sample (20 ng/µl) …………………………………………..1 µl
1.7 Evaluation of reaction results
In order to correctly assess the results of PCR, it is important to understand that this method is not quantitative. Theoretically, amplification products of single target DNA molecules can be detected by electrophoresis already after 30-35 cycles. However, in practice this is done only in cases where the reaction takes place under conditions close to ideal, which is not often encountered in life. The degree of purity of the DNA preparation has a particularly great influence on the efficiency of amplification; the presence of certain inhibitors in the reaction mixture, which in some cases can be extremely difficult to get rid of. Sometimes, due to their presence, it is not possible to amplify even tens of thousands of target DNA molecules. Thus, there is often no direct relationship between the initial amount of target DNA and the final amount of amplification products.
Chapter 2: Applications of the Polymerase Chain Reaction
PCR is used in many areas for analysis and in scientific experiments.
Criminalistics
PCR is used to compare so-called "genetic fingerprints". We need a sample of genetic material from the crime scene - blood, saliva, semen, hair, etc. It is compared with the suspect's genetic material. A very small amount of DNA is enough, theoretically - one copy. The DNA is cut into fragments, then amplified by PCR. The fragments are separated by DNA electrophoresis. The resulting picture of the location of the DNA bands is called the genetic fingerprint.
Establishing paternity
Results of electrophoresis of DNA fragments amplified by PCR. Father. Child. Mother. The child inherited some features of the genetic imprint of both parents, which gave a new, unique imprint.
Although "genetic fingerprints" are unique, family ties can still be established by making several such fingerprints. The same method can be applied, with slight modifications, to establish evolutionary relationships among organisms.
Medical diagnostics
PCR makes it possible to significantly speed up and facilitate the diagnosis of hereditary and viral diseases. The gene of interest is amplified by PCR using appropriate primers and then sequenced to determine mutations. Viral infections can be detected immediately after infection, weeks or months before symptoms of the disease appear.
Personalized medicine
Sometimes drugs are toxic or allergenic for some patients. The reasons for this are partly in individual differences in the susceptibility and metabolism of drugs and their derivatives. These differences are determined at the genetic level. For example, in one patient, a certain cytochrome may be more active, in another - less. In order to determine what kind of cytochrome a given patient has, it is proposed to perform a PCR analysis before using the drug. This analysis is called preliminary genotyping.
Gene cloning
Gene cloning is the process of isolating genes and, as a result of genetic engineering manipulations, obtaining a large amount of the product of a given gene. PCR is used to amplify a gene, which is then inserted into a vector, a piece of DNA that carries the foreign gene into the same organism or another organism that is easy to grow. As vectors, for example, plasmids or viral DNA are used. The insertion of genes into a foreign organism is usually used to obtain a product of this gene - RNA or, most often, a protein. In this way, many proteins are obtained in industrial quantities for use in agriculture, medicine, etc.
DNA sequencing
In the method of sequencing using labeled with a fluorescent label or a radioactive isotope of dideoxynucleotides, PCR is an integral part, since it is during polymerization that derivatives of nucleotides labeled with a fluorescent or radioactive label are inserted into the DNA chain. This stops the reaction, allowing the positions of specific nucleotides to be determined after separation of the synthesized strands in the gel.
Mutagenesis
Currently, PCR has become the main method of mutagenesis. The use of PCR made it possible to simplify and speed up the mutagenesis procedure, as well as to make it more reliable and reproducible.
The PCR method made it possible to analyze the presence of human papillomavirus sequences in biopsy sections of human cervical neoplasms embedded in paraffin 40 years before this study. Moreover, with the help of PCR, it was possible to amplify and clone fragments of mitochondrial DNA from the fossil remains of the human brain of the age of 7 thousand years!
On lysates of individual human spermatozoa, the possibility of simultaneously analyzing two loci located on different nonhomologous chromosomes was demonstrated. This approach provides a unique opportunity for fine genetic analysis and the study of chromosomal recombination, DNA polymorphism, etc. The method of analyzing individual spermatozoa immediately found practical application in forensic medicine, since HLA typing of haploid cells makes it possible to determine paternity or identify a criminal (the HLA complex is a set of genes of the human major histocompatibility complex; the loci of the HLA complex are the most polymorphic of all known in higher vertebrates: within a species, at each locus, there is an unusually large number of different alleles - alternative forms of the same gene).
Using PCR, it is possible to identify the correctness of the integration of foreign genetic structures in a predetermined region of the genome of the studied cells. The total cellular DNA is annealed with two oligonucleotide primers, one of which is complementary to the host DNA region near the insertion point, and the other to the sequence of the integrated fragment in the antiparallel DNA strand. Polymerase chain reaction in the case of an unchanged structure of chromosomal DNA at the proposed insertion site leads to the formation of single-stranded DNA fragments of an indefinite size, and in the case of a planned insertion, double-stranded DNA fragments of a known size, determined by the distance between the annealing sites of two primers. Moreover, the degree of amplification of the analyzed region of the genome in the first case will be linearly dependent on the number of cycles, and in the second - exponentially. The exponential accumulation of an amplified fragment of a predetermined size during PCR makes it possible to visually observe it after electrophoretic fractionation of a DNA preparation and make an unambiguous conclusion about the insertion of a foreign sequence into a given region of chromosomal DNA.
Conclusion
The PCR method is currently the most widely used as a method for diagnosing various infectious diseases. PCR allows you to identify the etiology of the infection, even if the sample taken for analysis contains only a few DNA molecules of the pathogen. PCR is widely used in the early diagnosis of HIV infections, viral hepatitis, etc. To date, there is almost no infectious agent that cannot be detected using PCR.
List of used literature
1.Padutov V.E., Baranov O.Yu., Voropaev E.V. Methods of molecular - genetic analysis. - Minsk: Unipol, 2007. - 176 p.
2.PCR "in real time" / Rebrikov D.V., Samatov G.A., Trofimov D.Yu. and etc.; ed. b. n. D.V. Rebrikov; foreword L.A. Osterman and acad. RAS and RAAS E.D. Sverdlov; 2nd ed., rev. and additional - M.: BINOM. Knowledge Laboratory, 2009. - 223 p.
.Patrushev L.I. Artificial genetic systems. - M.: Nauka, 2005. - In 2 tons
.B. Glick, J. Pasternak Molecular biotechnology. Principles and application 589 pages, 2002
5.Shchelkunov S.N.
Edited by A.A. Vorbyeva
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SEI HPE "Krasnoyarsk State Medical Academy
named after Yasenetsky Federal Agency for Health and Social Development »
Department of Medical Genetics and Clinical Neurophysiology, IPO
MAIN PRINCIPLES OF THE METHOD
POLYMERASE CHAIN REACTION
Methodical manual for students of 3-4 courses
in the specialties of general medicine (060101) and
Krasnoyarsk - 2007
Shnaider, N. A., Butyanov, R. A. Basic principles of the polymerase chain reaction method. Methodological manual for extracurricular work of students of 3-4 courses in the specialties of general medicine (060101) and pediatrics (060103). - Krasnoyarsk: Publishing house of GOU VPO KrasGMA, 2007. - 42p.
The methodological manual fully complies with the requirements of the State Standard (2000) and reflects the main aspects of the modern method for diagnosing hereditary human diseases - the polymerase chain reaction method, the educational material is adapted to educational technologies, taking into account the specifics of training in 3-4 courses of medical and pediatric faculties.
Reviewers: Head of the Department of Medical Genetics, State Educational Institution of Higher Professional Education
"Novosibirsk State Medical University of the Federal Agency for Health and Social Development", Doctor of Medical Sciences, Professor;
DNA replication
The object of study of this method is Deoxyribonucleic acid (DNA). DNA is a universal carrier of genetic information in all organisms existing on Earth (with the exception of RNA-containing microorganisms). DNA is a double strand twisted into a helix. Each strand consists of nucleotides connected in series. DNA strands have the opposite direction: the 5'-end of one strand corresponds to the 3'-end of the second strand. The unique property of DNA is its ability to duplicate itself. This process is called replication. Replication of the DNA molecule occurs during the synthetic period of interphase. Each of the two chains of the "parent" molecule serves as a template for the "daughter". After replication, the newly synthesized DNA molecule contains one "maternal" strand, and the second - a "daughter", newly synthesized (semi-conservative method). For template synthesis of a new DNA molecule, it is necessary that the old molecule be despiralized and stretched. Replication begins at several locations in the DNA molecule. The section of a DNA molecule from the start of one replication to the start of another is called replicon.
The start of replication is activated primers(seeds), consisting of 100-200 base pairs. The DNA helicase enzyme unwinds and divides the parent DNA helix into two strands, on which, according to the principle of complementarity, with the participation of the DNA polymerase enzyme, “daughter” DNA strands are assembled. In order for the enzyme to start its work, the presence of a starting block is required - a small initial double-stranded fragment. The starting block is formed when the primer interacts with the complementary region of the corresponding strand of the parent DNA. In each replicon, DNA polymerase can move along the "mother" strand in only one direction (5`=>3`).
On the leading strand, as the replicon unwinds, a “daughter” strand gradually grows continuously. On the lagging strand, the daughter strand also synthesizes in the direction (5`=>3`), but in separate fragments as the replicon unwinds.
Thus, the attachment of complementary nucleotides of the "daughter" strands goes in opposite directions (antiparallel). Replication in all replicons occurs simultaneously. Fragments and parts of "daughter" strands synthesized in different replicons are ligated into a single strand by an enzyme ligase. Replication is characterized by semi-conservation, anti-parallelism and discontinuity. The entire genome of a cell is replicated once per time period corresponding to one mitotic cycle. As a result of the replication process, two DNA molecules are formed from one DNA molecule, in which one strand is from the parent DNA molecule, and the second, the daughter, is newly synthesized (Fig. 1).
Rice. one. Diagram of DNA molecule replication.
Thus, the DNA replication cycle includes three main stages:
1. unwinding of the DNA helix and divergence of strands (denaturation);
2. attachment of primers;
3. completion of the chain of the child thread.
Principle of the PCR method
It was DNA replication that formed the basis of PCR. In PCR, the processes listed above are carried out in a test tube in a cyclic mode. The transition from one stage of the reaction to another is achieved by changing the temperature of the incubation mixture. When the solution is heated to 93-95°C, DNA denaturation occurs. To proceed to the next step - the addition or "annealing" of primers - the incubation mixture is cooled to 50-65°C. Next, the mixture is heated to 70-72°C - the optimum operation of taq-DNA polymerase - at this stage, a new DNA strand is completed. Then the cycle repeats again. In other words PCR method is a multiple increase in the number of copies (amplification) a specific section of DNA catalyzed by the enzyme DNA polymerase.
The extension of the daughter DNA strands must occur simultaneously on both strands of the maternal DNA, so replication of the second strand also requires its own primer. Thus, two primers are introduced into the reaction mixture: one for the "+"-chain, the second for the "-"-chain. Having joined the opposite strands of the DNA molecule, the primers limit themselves to that part of it, which will be subsequently repeatedly doubled or amplified. The length of such a fragment, which is called an amplicon, is usually several hundred nucleotides.
PCR steps
Each amplification cycle includes 3 stages occurring at different temperature conditions (Fig. 2).
· Stage 1: DNA denaturation . It flows at 93-95° for 30-40 seconds.
· Stage 2: primer annealing . Primer attachment occurs complementary to the corresponding sequences on opposite DNA strands at the boundaries of a specific region. Each pair of primers has its own annealing temperature, the values of which are in the range of 50-65°C. Annealing time 20-60 sec.
· Stage 3: extension of DNA chains. Complementary extension of DNA chains occurs from the 5" end to the 3" end of the chain in opposite directions, starting from the primer attachment sites. The material for the synthesis of new DNA strands are deoxyribonucleoside triphosphates added to the solution. The synthesis process is catalyzed by the enzyme taq-polymerase and takes place at a temperature of 70-72°C. Synthesis time - 20-40 sec.
The new DNA strands formed in the first amplification cycle serve as templates for the second amplification cycle, in which a specific amplicon DNA fragment is formed (Fig. 3). In subsequent amplification cycles, amplicons serve as a template for the synthesis of new chains.
Thus, amplicons accumulate in the solution according to the formula 2", where n is the number of amplification cycles. Therefore, even if only one double-stranded DNA molecule was initially in the initial solution, about 108 amplicon molecules accumulate in the solution in 30-40 cycles. This the amount is sufficient for reliable visual detection of this fragment by agarose gel electrophoresis.
The amplification process is carried out in a special programmable thermostat ( cycler), which, according to a given program, automatically changes temperatures according to the number of amplification cycles.
The following components are required for amplification:
· DNA template(DNA or its part containing the desired specific fragment);
· Primers(synthetic oligonucleotides (20-30 nucleotide pairs) complementary to DNA sequences at the boundaries of the specific fragment being determined). The choice of a specific fragment and the selection of primers play a major role in the specificity of the amplification, which affects the quality of the analysis.
· A mixture of deoxynucleotide triphosphates (dNTPs)(a mixture of four dNTPs, which are the material for the synthesis of new complementary DNA strands in equivalent concentrations of 200-500 microns)
· EnzymeTaq-polymerase(thermostable DNA polymerase, catalyzing the lengthening of primer chains by sequential addition of nucleotide bases to the growing chain of synthesized DNA, 2-3 mm).
· buffer solution(reaction medium containing Mg2+ ions necessary to maintain enzyme activity, PH 6.8-7.8).
To determine specific regions of the genome of RNA viruses, a DNA copy is first obtained from an RNA template using a reverse transcription (RT) reaction catalyzed by the enzyme reverse transcriptase (reverse transcriptase).
Rice. 2. Amplification (1st cycle).
Rice. 3. Amplification (2nd cycle).
Main Applications of PCR
clinical medicine:
o diagnosis of infections,
o detection of mutations, including the diagnosis of hereditary diseases,
o genotyping, including HLA genotyping,
o cellular technologies
ecology (as a way to monitor the state and quality of environmental objects and food)
definition of transgenic organisms (GMOs)
Personal identification, paternity, forensics
general and particular biology,
Basic principles
organization of diagnostic laboratories
Work in the PCR laboratory is carried out in accordance with the "Rules for the design, safety, industrial sanitation, anti-epidemic regime and personal hygiene when working in laboratories (departments, departments) of sanitary and epidemiological institutions of the healthcare system.
Contamination of DNA samples
Carrying out PCR diagnostics is associated with a problem caused by the high sensitivity of the method - the possibility contamination. If trace amounts of positive DNA enter the reaction tube (specific DNA amplification products - amplicons; DNA standard used as a positive control; positive DNA of a clinical sample) leads to amplification of a specific DNA fragment during PCR and, as a result, to the appearance of false positive results.
In the course of work, you may meet two types of contamination:
1. cross contamination from sample to sample (during the processing of clinical samples or when digging out the reaction mixture), leading to the appearance of sporadic false positive results;
2. amplification product contamination(amplicons), which is of the greatest importance, because during the PCR process, amplicons accumulate in huge quantities and are ideal products for reamplification.
Trace amplicon contamination of dishes, automatic pipettes and laboratory equipment, the surface of laboratory tables, or even the surface of the skin of laboratory workers leads to systematic false positive results. Determining the source of contamination can be very difficult and requires a significant investment of time and money. The experience accumulated to date in the work of laboratories using the PCR method for diagnostics allows us to formulate the basic requirements for the organization of such laboratories and the conduct of the analyzes themselves. Compliance with these requirements eliminates the possibility of contamination and obtaining false positive results.
Stages of PCR analysis
Geographically separated, placing them in separate rooms (Fig. 4.5):
· Pre-PCR room, where processing of clinical samples, DNA extraction, preparation of the reaction mixture for PCR and PCR is performed (if conditions are available, the last two steps are also recommended to be carried out in an additional separate room). In these rooms it is forbidden to carry out all other types of work with the studied agents, the PCR diagnostics of which are carried out in this laboratory.
· Post-PCR room, where the detection of amplification products is carried out. Other detection methods may be used in this room. It is desirable to locate the room for detection of amplification products as far as possible from the pre-PCR rooms.
Working rooms are equipped with ultraviolet lamps with a maximum radiation in the region of 260 nm (type DB-60) at the rate of 2.5 W per 1 m3. The lamps are located so that the surfaces of working tables, equipment and materials with which the operator comes into contact during the PCR analysis are exposed to direct radiation. Irradiation is carried out within 1 hour before the start of work and within 1 hour after the end of work.
Laboratory doctors work in special laboratory clothes, which are changed when moving from one room to another, and in disposable gloves. Processing of clothes from different rooms is carried out separately. Different employees work at different stages of the PCR analysis.
For work, separate sets of dispensers, plastic and glassware, laboratory equipment, gowns and gloves are used, designed for various stages of analysis and cannot be transferred from one room to another. Equipment, materials and inventory in each room are labeled accordingly.
All stages of work are carried out only with the use of disposable consumables: tips for automatic pipettes, test tubes, gloves, etc. Be sure to change the tips when moving from sample to sample. It is necessary to use tips with an aerosol barrier filter to prevent microdroplets of the solution from entering the pipette. Used test tubes and tips are discarded in special containers or containers containing a disinfectant solution. Clinical samples are stored separately from reagents.
For processing and cleaning the workplace, each room has cotton-gauze swabs (napkins), tweezers, disinfectant and inactivating solutions.
In the PCR diagnostic laboratory, work related to the production (cloning) and isolation of recombinant plasmids containing DNA sequences or gene fragments of pathogens that are diagnosed in this laboratory is excluded.
Collection of clinical material
The studied material for PCR can be scrapings of epithelial cells, blood, plasma, serum, pleural and cerebrospinal fluid, urine, sputum, mucus and other biological secretions, biopsy specimens.
The sampling of the material is carried out in the conditions of the treatment room of the corresponding profile. After sampling, the samples should be taken to the PCR diagnostic laboratory as soon as possible.
Sampling must be carried out using sterile, preferably disposable, instruments only in disposable sterile plastic tubes or glass tubes, pre-treated for an hour with a chromium mixture, thoroughly washed with distilled water and calcined in an oven at a temperature of 150 ° C for 1 hour.
Detection zone (another floor or another building).
Rice. 4. PCR laboratory device with detection by electrophoresis.
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Detection zone (different floor or building)
Rice. 5. PCR laboratory device with fluorescent detection (quantitative analysis).
Rice. 6. DNA extraction room. Shown is a tabletop box with a bactericidal lamp.
Rice. 7. amplification room.
Rice. eight. Detection room.
Rice. nine. Blood samples for DNA diagnostics of hereditary diseases.
Storage and transportation of samples
For the diagnosis of hereditary diseases, blood samples are stored on special paper forms or in epindorfs (plastic test tubes) in a frozen state for a long time (Fig. 9).
For the diagnosis of infectious diseases, samples are kept at room temperature for no more than 2 hours. If longer storage is required, the samples can be placed in a refrigerator at a temperature of 2-8°C for a period not exceeding 24 hours. Longer storage (up to 2 weeks) is acceptable when frozen in a freezer at a temperature of minus 20°C. Repeated freezing-thawing of samples is not allowed.
If the PCR diagnostic laboratory and the procedure room for sampling are geographically separated, then samples should be transported in thermoses or thermal containers in compliance with the rules for storing samples and the rules for transporting infectious materials.
Extraction of DNA from samples
The method of solid-phase sorption, which consists in adding a lysing agent containing a solution of guanidine, sorption of DNA on a sorbent, repeated washing and resorption of DNA with a buffer solution, has become widespread. In the case of serum, plasma or whole blood processing, the phenolic extraction method is usually used. The method involves deproteinization with phenol/chloroform followed by precipitation of DNA (or RNA) with ethanol or isopropanol. Processing is carried out in microcentrifuge test tubes of the Eppendor P type with a volume of 1.5 ml. The processing time is 1.5-2 hours (Fig. 10).
Rice. ten. Isolation of DNA.
Conducting PCR
A certain amount of the sample from the processed clinical sample is transferred to a special Eppendorf type microcentrifuge tube with a volume of 0.2 or 0.5 ml. An amplification mixture consisting of water, PCR buffer, dNTP solution, primer solution and solution is added to the same tube. Taq polymerase (added to the mixture last) Typically, the volume of the reaction mixture is 25 µl Then one drop of mineral oil is added to each tube to prevent evaporation of the reaction mixture during the amplification The tubes are transferred to a programmable thermostat (amplifier), where amplification is carried out in automatic mode according to a given program (Fig. 11).
Rice. eleven. Amplifier " Thermocycler ».
The reaction time, depending on the given program, is 2-3 hours. In parallel with the experimental samples, control samples are placed: the positive control includes all the components of the reaction, but instead of the material of the clinical sample, a control DNA preparation of the gene under study is introduced. The negative control includes all components of the reaction, but instead of the clinical material or DNA preparation, an appropriate amount of deionized water or an extract that does not contain the studied DNA is added. A negative control is necessary to check the components of the reaction for the absence of DNA in them due to contamination and to exclude false positive results.
Registration of results
The amplified specific DNA fragment is detected by agarose gel electrophoresis in the presence of ethidium bromide. Ethidium bromide forms a stable interstitial compound with DNA fragments, which appears as luminous bands when the gel is irradiated with UV radiation with a wavelength of 290-330 nm. Depending on the size of the resulting PCR amplicons, a gel containing 1.5% to 2.5% agarose is used. To prepare an agarose gel, a mixture of agarose, buffer, and water is melted in a microwave oven or in a water bath, and a solution of ethidium bromide is added. Cooled to 50-60°C, the mixture is poured into the mold with a layer of 4-6 mm thick, and using special combs, pockets are made in the gel for applying the sample. The combs are set so that between the bottom of the wells and the base of the gel remains a layer of agarose 0.5-1 mm. After the gel has hardened, an amplificate is applied to the pockets in an amount of 5-15 µl. It is recommended to carry out electrophoresis of a mixture of DNA fragment length markers in parallel with control and experimental samples. Typically, such a mixture contains ten DNA fragments 100, 200, 300, etc. long base pairs.
Setting up such a sample allows you to verify the length of the amplicons in the control and experimental samples. The gel with the applied sample is transferred to an electrophoresis chamber filled with a buffer, the chamber is connected to a power source and the electrophoretic separation of the amplification products is carried out for 30-45 minutes at an electric field strength of 10-15 V/cm. In this case, the front of the dye, which is part of the reaction mixture, must pass at least 3 cm.
After the end of electrophoresis, the gel is transferred to the transilluminator glass and viewed in ultraviolet light. For documentation, the gel is photographed on Mikrat 300 film or recorded using a video system connected to a computer.
The control samples are evaluated first. In the electrophoretic lane corresponding to the positive control, an orange luminous band should be present. Its electrophoretic mobility should correspond to the length of the amplicon specified in the instructions.
In the electrophoretic track corresponding to the negative control, such a band should be absent. The presence of such a band in the negative control indicates contamination - contamination of the reagents used with the studied DNA or amplicon. Test samples are evaluated by the presence of a band in the corresponding lane that is located at the same level as the band in the positive control sample. The intensity of the band glow corresponds to the amount of DNA under study in the sample, which allows for a semi-quantitative assessment of PCR. Usually positive results are evaluated on a four-point scale. If the glow of the band in the experimental sample is very weak, then such a sample should be rearranged (Fig. 12).
Rice. 12. Electrophoresis in agarose gel.
PCR applications fordiagnostics of point mutations and gene polymorphisms
One of the leading areas of application of PCR in practical healthcare is the diagnosis of point mutations and gene polymorphisms. . There are direct and indirect methods of DNA diagnostics. In those situations where a gene is known, the damage of which leads to the development of a hereditary disease, this damage can be detected by molecular genetic methods. Such methods are called direct. Using direct methods, disturbances in the primary nucleotide sequence of DNA (mutations and their types) are detected. Direct methods are characterized by accuracy reaching almost 100%.
However, in practice, these methods can be applied under certain conditions.:
with a known cytogenetic localization of the gene responsible for the development of a hereditary disease;
The disease gene must be cloned and its nucleotide sequence known.
The goal of direct DNA diagnostics is to identify mutant alleles.
Thus, in those situations where it is known what kind of DNA damage leads to a hereditary disease, the DNA fragment containing the damage is examined directly, i.e., the direct method of DNA diagnostics is used.
However, to date, the genes of many diseases have not been mapped, their exon-intron organization is unknown, and many hereditary diseases are characterized by pronounced genetic heterogeneity, which does not allow full use of direct DNA diagnostic methods. Therefore, in cases where the localization of damage is not known, a different approach is used, associated with the study of the vicinity of the gene responsible for the gene disease, in combination with family analysis, that is, indirect methods of molecular genetic diagnosis of hereditary diseases are used.
Various methods can be used to detect point mutations and small deletions, but all of them are based on the use of the PCR method. This reaction allows you to repeatedly multiply the nucleotide sequence of DNA, and then search for mutations. Methods for searching for DNA fragments carrying mutations are based on a comparative analysis of mutant and normal DNA nucleotide sequences.
Analysis of PCR products
in the process of direct DNA diagnostics
It involves the study of specific features of the amplified region of the gene. Thus, in diseases caused by the expansion of trinucleotide repeats, amplification products differ in their length (reflecting a different number of triplets in the studied gene region) and, as a result, in their speed of movement in the gel. Due to this, a clear electrophoretic separation of normal and mutant alleles and an accurate determination of the pathologically elongated fragment, i.e. DNA diagnosis of the disease, is achieved (Fig. 13).
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Rice. fourteen. Diagnosis of a deletion GAG in the gene DYT 1 in patients with dopa-independent dystonia (polyacrylamide gel electrophoresis). Tracks 2,3,6 - sick; lanes 1,4,5 - control. The thin arrow indicates the normal allele, the bold arrow indicates the mutant shorter allele (deletion of three nucleotides).
If the DNA region under study is entirely included in an extended deletion, then PCR amplification of DNA from this deleted allele will not be carried out due to the lack of places for primer hybridization. In this case, a homozygous deletion will be diagnosed based on the complete absence of the PCR product of the reaction (DNA synthesis is impossible from both copies of the gene). With a heterozygous deletion, it is possible to identify a PCR product synthesized from a normal (safe) allele, however, for reliable diagnosis of such a mutation, it is necessary to use more sophisticated DNA visualization methods that allow one to estimate the dose of the final PCR product.
To detect point mutations (most often nucleotide substitutions) at certain sites, the PCR method is used in combination with other methods of molecular genetic analysis. If the site of localization and the nature of the proposed point mutation are precisely known, then for the purposeful detection of such a mutation, restriction endonucleases (restrictases) are special cellular enzymes isolated from various strains of bacteria.
These enzymes recognize specific nucleotide sequences ranging from four to ten nucleotides in length. Then, restriction (lat. (cutting)) of these sequences is carried out as part of a double-stranded DNA molecule. Each restriction enzyme recognizes and cuts in a fixed place a strictly defined, specific nucleotide sequence - restriction site (recognition site).
In cases where a point mutation changes the natural recognition site for a particular restriction enzyme, that enzyme will not be able to cleave the mutant PCR-amplified fragment. In some cases, the mutation leads to the appearance of a new recognition site for a particular restriction enzyme, which is absent in the norm.
In both situations, the mutant and normal PCR products treated with the selected restriction enzyme will give restriction fragments of different lengths, which can be easily detected by electrophoresis (Fig. 15).
Thus, if it is necessary to quickly detect any particular point mutation, the task is reduced to searching for the corresponding restriction enzyme, the recognition site of which is localized at the site of the disturbed nucleotide sequence. Treatment of PCR products with such a restriction enzyme will allow easy differentiation of normal and mutant alleles. Restriction analysis greatly simplifies the detection of known point mutations and is currently widely used for direct DNA diagnosis of hereditary diseases.
final stage molecular genetic analysis of mutations is the determination of the nucleotide sequence of the studied DNA fragment (sequencing), which is compared with the norm and the final genetic diagnosis is formulated. Thanks to the advances in molecular genetics, DNA diagnostic methods have now been developed for more than 400 hereditary diseases.
Rice. fifteen. Detection of a point mutation using restriction analysis: A - amplifiable region of the gene containing a restriction siteAGCTfor restriction endonucleaseAlu I. MutationG→ Achanges the given nucleotide sequence, resulting in restriction enzymeAluiblocked; B - electropherogram of restriction products: lane 1 - homozygosity for the normal allele; lane 2, homozygosity for the mutation; lane 3 - heterozygous state (normal allele + mutation).
Diagnosis of hereditary diseases based on direct examination of mutant alleles in patients, their family members or presumed heterozygous carriers of pathological mutations is suitable for pre-symptomatic and prenatal diagnosis, which can be applied at the earliest stages of fetal development, before the appearance of any clinical or biochemical symptoms. illness.
Regardless of the method of mutation detection, accurate molecular characterization of each mutation can only be obtained by direct sequencing. In recent years, to automate this process, special devices have been widely used - sequencers, which make it possible to significantly speed up the process of reading DNA information.
The way for a wider application of molecular biological research in clinical diagnostic laboratories is opened by accelerating the analytical process by performing all procedures in one continuum, without sample transfer, creating conditions to prevent contamination during parallel testing of a number of analytes and with objective registration of results in each cycle.
Main modifications of the PCR method
Used to quickly scan and search for known gene mutations.
Multiplex (multiprimer) PCR
This method is based on the simultaneous amplification of several exons of the studied gene in one reaction. This allows economical rapid screening of the most common mutations. For example, to quickly diagnose the carriage of deletions in the dystrophin gene in patients with progressive Duchenne/Becker muscular dystrophy, simultaneous amplification of the set of the most frequently mutating exons of this gene is performed. Since these diseases are inherited in an X-linked recessive type and are associated with damage to the only X chromosome in boys, in the case of an extended deletion, electrophoresis of the reaction products will reveal the absence of one or more DNA fragments (exons), which can serve as a molecular confirmation of the diagnosis. In addition, by selecting specific gene regions for PCR amplification, a fairly accurate assessment of the total length of the deletion and gene break points (up to the exon) is possible.
The combined use of several multiplex reactions makes it possible to diagnose up to 98% of all deletions that occur in patients with progressive Duchenne/Becker muscular dystrophy. This is approximately 60% of the total number of known mutations in the dystrophin gene and indicates a very high efficiency of this screening method for DNA diagnosis of dystrophinopathy (Fig. 16).
Rice. 16. Direct DNA diagnosis of Duchenne muscular dystrophy using multiplex PCR (agarose gel electrophoresis). In each of the examined individuals, four exons of the dystrophin gene were simultaneously amplified (exons 17, 19, 44, and 45; arrows indicate the corresponding amplification products). Lane 1 - control, lanes 2-5 - patients with Duchenne muscular dystrophy with various deletions of the dystrophin gene (lanes 2 and 5 - deletion of exon 45, lane 3 - deletion of exon 44, lane 4 - deletion of exon 17 and 19).
Allele-specific amplification
The method is based on the use of two independent pairs of primers for a specific region of the gene: one primer in both pairs is common, and the second primer in each pair has a different structure and is complementary to either normal or mutant DNA sequences. As a result of such a reaction in solution, two types of PCR products can be simultaneously synthesized - normal and mutant. Moreover, the design of the primers used makes it possible to clearly differentiate normal and mutant amplification products by their molecular size. This method is very clear and allows you to verify both homo- and heterozygous carriage of the mutant allele.
Method for site-directed modification of amplified DNA
The method is based on the use in PCR of the so-called mismatch primer (not fully complementary to the template), which differs from the template DNA sequence by one nucleotide. As a result of the inclusion of the specified primer in the composition of the mutant PCR product, an artificially created restriction site for one of the restriction endonucleases is formed in it, which allows direct DNA diagnosis of a certain known mutation using restriction analysis. The creation of such an artificial restriction site may be necessary if the search did not reveal the existence of a known and accessible enzyme, the “natural” restriction site of which is affected as a result of the appearance of the studied mutation in the DNA molecule.
Reverse transcriptase PCR method (RT- PCR)
This method is used in cases where it is more convenient to use not genomic DNA as an object of study, but a more compact and informationally "saturated" cDNA obtained after appropriate processing of tissue samples, for example, biopsy material or cell lines of lymphocytes, fibroblasts, etc. Important the condition here is the expression (at least minimal) of the desired gene in the tissue under study.
At the first stage, reverse transcription of mRNA is carried out, and the resulting cDNA molecules serve as a template for PCR. Subsequently, the critical cDNA region amplified in sufficient quantity is subjected to sequencing and other mutation screening methods, direct electrophoretic study (detection of deletions, insertions, etc.) or integration into an expression system in order to obtain a protein product and its direct analysis.
This method is especially effective for the detection of mutations leading to the synthesis of a "truncated" protein (nonsense mutations, splicing mutations, large deletions) - the so-called PTT analysis (Protein Truncation Test). PTT analysis is commonly used when examining extended multi-exon genes, such as the gene for Duchenne/Becker muscular dystrophy, ataxia-telangiectasia, or type 1 neurofibromatosis.
real time PCR(Real-Time PCR)
Every year, in practical healthcare, real-time PCR is becoming an increasingly popular diagnostic method. Its fundamental feature is the monitoring and quantitative analysis of the accumulation of polymerase chain reaction products and automatic registration and interpretation of the results. This method does not require an electrophoresis step, which reduces the requirements for a PCR laboratory. Thanks to savings in production space, a decrease in the number of personnel and the demand for DNA/RNA quantification, this method has been successfully used in recent years in the largest sanitary epidemic, diagnostic and research centers in the developed countries of the world, replacing PCR in its current ("classic") format.
Real-time PCR uses fluorescently labeled oligonucleotide probes to detect DNA during amplification. Real-time PCR allows a complete analysis of a sample within 20-60 minutes and is theoretically capable of detecting even a single DNA or RNA molecule in a sample.
Rice. 17. PCR in real time.
Real-time PCR uses the TaqMan system to control PCR kinetics directly during amplification using resonant fluorescence quenching. For detection, a probe carrying a fluorophore and a quencher complementary to the middle part of the amplified fragment is used. When the fluorophore and quencher are bound to the oligonucleotide probe, only a small amount of fluorescent emission is observed. During the amplification process, due to the 5'-exonuclease activity of Taq polymerase, the fluorescent label passes into solution, being released from the vicinity of the quencher, and generates a fluorescent signal that increases in real time in proportion to the accumulation of the amplificate (Fig. 17).
Main advantages of PCR-Real-Time over PCR with gel electrophoresis:
The whole method takes place in one test tube;
· The method takes 1 hour;
Enough 1-2 working rooms;
Along with a qualitative assessment of the result, it becomes possible to quantify it (for example, when prescribing antiviral therapy for AIDS or viral hepatitis, it is necessary to know the viral load, i.e. the amount of virus per 1 unit, which provides real-time PCR);
· Dramatically reduces the risk of contamination.
Conclusion
The PCR method is one of the most common methods of molecular biological research. This method should be used meaningfully by clinicians, and a doctor who decides to use PCR in his work must have certain knowledge about the features and capabilities of this method. Secondly, there must be close feedback between the clinician and the PCR laboratory, which is necessary for the analysis of complex cases and the development of the correct diagnostic strategy. Thirdly, PCR analysis is not a panacea in the diagnosis (primarily of infectious diseases) and does not replace existing research methods, but only complements them. And most importantly, PCR cannot replace the intuition and analytical thinking that a doctor who expects success should have.
P . S . Molecular-biological researches - change of reference points of diagnostics and treatment. The use of molecular biological methods is associated with the prospect of a radical change in emphasis in laboratory diagnostics. We can talk not just about timely information, but about its advance receipt. If now laboratory studies in most cases are carried out already with an advanced disease and treatment initiated, then molecular biological laboratory information is expected to make it possible to identify a person's inclination to certain types of pathology and the degree of sensitivity to certain drugs, which will allow substantiating predictive, preventive and personalized character of the medicine of the future.
CHANGE OF DIAGNOSIS AND TREATMENT FOCUSES
HEREDITARY DISEASES
Today In the future
Diagnosis Genetic passport
8. How many working rooms are required for a PCR laboratory with fluorescence detection (quantitative analysis, Real-Time PCR)?
9. What is detection?
10. What methods of DNA diagnostics are distinguished?
11. Which enzyme works on the basis of PCR?
12. Why does the detection zone need to be separated from other work zones?
13. What is a restriction site?
14. What is the difference between the direct method of DNA diagnostics and the indirect one?
15. What is sequencing?
16. What is multiplex PCR?
17. What types of mutations are determined by PCR?
18. What is contamination?
19. What is the essence of the allele-specific amplification method?
20. Storage conditions for PCR material?
21. What device is used for amplification?
22. What is the method of reverse transcriptase PCR (RT-PCR)?
23. What is the material for PCR diagnostics?
24. List the types of contamination?
Tests for self-study
1. Restriction endonucleases:
a) enzymes that “break” DNA in strictly specific places;
b) enzymes that sew breaks in the DNA molecule;
c) enzymes that provide compounds that carry out DNA repair.
2. Gene amplification:
3. Which of the methods of molecular genetics is used to diagnose diseases caused by a mutant gene of a known sequence?
a) the use of a specific restrictase;
b) direct detection using specific molecular probes;
c) family analysis of the distribution of normal restriction fragment length polymorphism.
4. DNA sequencing:
a) identification of the DNA base sequence;
b) repeated repetition of any DNA segment;
c) isolation of a DNA fragment containing the studied gene.
5. DNA samples can be obtained using :
b) chorionic villi;
c) amniotic fluid;
d) amniotic fluid cells;
e) biopsies of skin, muscles, liver,
e) everything is correct, except for point "c",
g) everything is correct, except for point "d",
h) All of the above are correct.
6. Which mutations are diagnosed by PCR?
a) genomic;
b) chromosomal;
c) gene (point).
7. Primer is:
a) a complementary section of DNA;
b) a synthetic oligonucleotide labeled (radioactively or fluorescently) sequence complementary to a mutant or normal gene;
c) an oligonucleotide acting as a "seed" and initiating the synthesis of a polynucleotide chain on a DNA or RNA template.
8. Who developed the principle of the PCR method?
b) K. Mullis
9. Is the PCR method used to diagnose the expansion of trinucleotide repeats (dynamic type of mutations)?
10. In what areas is PCR used?
a) clinical medicine;
b) definition of transgenic organisms (GMOs)
c) identification of the person, establishment of paternity, criminalistics
d) all of the above
d) none of the above.
Sample answers: 1 - a; 2 - b; 3 - b; 4 - a; 5 - e; 6 - in; 7 - in; 8 - b; 9 – a, 10 – d.
Main
1. Bochkov genetics. Moscow. GEOTAR, 2002.
Additional
1., Bakharev and the treatment of congenital and hereditary diseases in children. - Moscow, 2004.
2. DNA diagnostics and medical genetic counseling. - Moscow, 2004.
3. Ginter genetics. - Moscow, 2003.
4. Gorbunov fundamentals of medical genetics. - St. Petersburg: Intermedica, 1999.
5. Herrington S., J. McGee. Molecular clinical diagnostics. – World, 1999.
6. Menshikov - biological research in clinical laboratory diagnostics: the possibilities of the problem (lectures). Clinical laboratory diagnostics, No. 3, 2006.
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MAIN PRINCIPLES OF THE METHOD
POLYMERASE CHAIN REACTION
Methodological manual for extracurricular work of students of 3-4 courses in the specialties of general medicine (060101) and pediatrics (060103).
SEI HPE "Krasnoyarsk State Medical Academy of the Federal Agency for Health and Social Development"
Russia, Krasnoyarsk,
For adequate and effective treatment of many infectious diseases, it is necessary to establish an accurate diagnosis in a timely manner. In solving this problem today, high-tech diagnostic methods based on molecular biology methods are involved. At the moment, the polymerase chain reaction (PCR) is already widely used in practical medicine as the most reliable laboratory diagnostic tool.
What explains the popularity of PCR at the present time?
Firstly, this method is used to identify pathogens of various infectious diseases with high accuracy.Secondly, to monitor the effectiveness of the treatment.
In various manuals, prospectuses, articles, as well as explanations of medical specialists, we often encounter the use of incomprehensible terms and words. It is really difficult to talk about high-tech products of science in ordinary words.
What is the essence and mechanics of PCR diagnostics?
Every living organism has its own unique genes. Genes are located in the DNA molecule, which in fact is the "calling card" of each specific organism. DNA (genetic material) is a very long molecule that is made up of building blocks called nucleotides. For each pathogen of infectious diseases, they are located strictly specific, that is, in a certain sequence and combination. When it is necessary to understand whether a person has a particular pathogen, biological material (blood, urine, saliva, smear) is taken, which contains DNA or DNA fragments of a microbe. But the amount of the genetic material of the pathogen is very small, and it is impossible to say which microorganism it belongs to. To solve this problem, PCR serves. The essence of the polymerase chain reaction is that a small amount of material for research containing DNA is taken, and during the PCR process, the amount of genetic material belonging to a particular pathogen increases and, thus, it can be identified.PCR diagnostics is a genetic study of a biomaterial.
The idea of the PCR method belongs to the American scientist K. Mullins, which he proposed in 1983. However, it received wide clinical use only in the middle of the 90s of the XX century. Let's deal with the terminology, what is it - DNA, etc. Each cell of any living being (animal, plant, human, bacteria, virus) has chromosomes. Chromosomes are the custodians of genetic information that contain the entire sequence of genes of each particular living being.
Each chromosome is made up of two strands of DNA that are twisted into a helix relative to each other. DNA is chemically deoxyribonucleic acid, which consists of structural components - nucleotides. There are 5 types of nucleotides - thymine (T), adenosine (A), guanine (G), cytosine (C) and uracil (U). Nucleotides are arranged one after another in a strict individual sequence, forming genes. One gene may consist of 20-200 such nucleotides. For example, the gene encoding insulin production is 60 base pairs long.
Nucleotides have the property of complementarity. This means that opposite adenine (A) in one strand of DNA there is always thymine (T) in the other strand, and opposite guanine (G) - cytosine (C). Schematically looks like this:
G - C
T - A
A - T
This property of complementarity is key for PCR.
In addition to DNA, RNA has the same structure - ribonucleic acid, which differs from DNA in that it uses uracil instead of thymine. RNA - is the keeper of genetic information in some viruses, which are called retroviruses (for example, HIV).
DNA and RNA molecules can "multiply" (this property is used for PCR). This happens as follows: two strands of DNA or RNA move away from each other to the sides, a special enzyme sits on each thread, which synthesizes a new chain. Synthesis proceeds according to the principle of complementarity, that is, if nucleotide A is in the original DNA chain, then T will be in the newly synthesized one, if G - then C, etc. This special "builder" enzyme needs a "seed" - a sequence of 5-15 nucleotides - to start synthesis. This "seed" is defined for each gene (chlamydia gene, mycoplasma, viruses) experimentally.
So, each PCR cycle consists of three stages. In the first stage, the so-called unwinding of DNA occurs - that is, the separation of the two strands of DNA connected to each other. In the second, the “seed” is attached to a section of the DNA strand. And, finally, the elongation of these DNA strands, which is produced by the "builder" enzyme. Currently, this entire complex process takes place in one test tube and consists of repeated cycles of reproduction of the DNA being determined in order to obtain a large number of copies that can then be detected by conventional methods. That is, from one strand of DNA, we get hundreds or thousands.
Stages of a PCR study
Collection of biological material for research
Various biological material serves as a sample: blood and its components, urine, saliva, secretions of mucous membranes, cerebrospinal fluid, discharge from wound surfaces, the contents of body cavities. All biosamples are collected with disposable instruments, and the collected material is placed in sterile plastic tubes or placed on culture media, followed by transportation to the laboratory.The necessary reagents are added to the taken samples and placed in a programmable thermostat - a thermal cycler (amplifier). In the cycler, the PCR cycle is repeated 30-50 times, consisting of three stages (denaturation, annealing and elongation). What does this mean? Let's consider in more detail.
Stages of immediate PCR reaction, copying of genetic material
IPCR stage - Preparation of genetic material for copying.Occurs at a temperature of 95 ° C, while the DNA strands are disconnected, and “seeds” can sit on them.
"Seeds" are manufactured industrially by various research and production associations, and laboratories buy ready-made ones. At the same time, the “seed” for detecting, for example, chlamydia, works only for chlamydia, etc. Thus, if a biomaterial is tested for the presence of a chlamydial infection, then a “seed” for chlamydia is placed in the reaction mixture; if testing the biomaterial for the Epstein-Barr virus, then the "seed" for the Epstein-Barr virus.
IIstage - Combining the genetic material of the infectious agent and the "seed".
If there is DNA of the virus or bacterium to be determined, the "seed" sits on this DNA. This primer addition process is the second step of the PCR. This stage takes place at a temperature of 75°C.
IIIstage - Copying the genetic material of the infectious agent.
This is the process of the actual elongation or reproduction of genetic material, which occurs at 72°C. An enzyme-builder approaches the "seeds" and synthesizes a new strand of DNA. With the end of the synthesis of a new DNA strand, the PCR cycle also ends. That is, in one PCR cycle, the amount of genetic material doubles. For example, in the initial sample there were 100 DNA molecules of a virus; after the first PCR cycle, the sample will already contain 200 DNA molecules of the tested virus. One cycle lasts 2-3 minutes.
In order to generate enough genetic material for identification, 30-50 PCR cycles are usually performed, which takes 2-3 hours.
Stage of identification of the propagated genetic material
The PCR itself ends here and then comes the equally significant stage of identification. For identification, electrophoresis or labeled "seeds" are used. When using electrophoresis, the resulting DNA strands are separated by size, and the presence of DNA fragments of different lengths indicates a positive result of the analysis (that is, the presence of a particular virus, bacterium, etc.). When labeled "seeds" are used, a chromogen (dye) is added to the final product of the reaction, as a result of which the enzymatic reaction is accompanied by the formation of a color. The development of a color indicates directly that a virus or other detectable agent is present in the original sample. To date, using labeled "seeds", as well as appropriate software, it is possible to immediately "read" the PCR results. This is the so-called real-time PCR.
Why is PCR diagnostics so valuable?
One of the significant advantages of the PCR method is its high sensitivity - from 95 to 100%. However, these advantages must be based on the indispensable observance of the following conditions:
- correct sampling, transportation of biological material;
- availability of sterile, disposable instruments, special laboratories and trained personnel;
- strict adherence to the methodology and sterility during the analysis
The capabilities inherent in PCR analysis allow you to achieve unsurpassed analytical specificity. This means identifying exactly the microorganism that was searched for, and not a similar or closely related one.
The diagnostic sensitivity and specificity of the PCR method often exceeds those of the culture method, which is called the "gold standard" for the detection of infectious diseases. Considering the duration of culture growth (from several days to several weeks), the advantage of the PCR method becomes obvious.
PCR in the diagnosis of infections
The advantages of the PCR method (sensitivity and specificity) determine a wide range of applications in modern medicine.
The main areas of application of PCR diagnostics:
- diagnosis of acute and chronic infectious diseases of various localization
- monitoring the effectiveness of the therapy
- clarification of the type of pathogen
The use of PCR diagnostics is carried out in conjunction with other research methods (ELISA, PIF, RIF, etc.). Their combination and expediency is determined by the attending physician.
Infectious agents detected by PCR
Viruses:
- HIV-1 and HIV-2 retroviruses
- herpetiform viruses
- herpes simplex virus types 1 and 2
The highly informative PCR method (polymerase chain reaction) enables early detection of various genetic and infectious diseases that occur acutely or chronically. Moreover, they can be identified even at the stage when they do not show any symptoms. Most often, PCR analysis is used to detect sexually transmitted infections (STDs, STIs).
PCR analysis refers to a molecular diagnostic method, which is based on a multiple increase in small concentrations of certain fragments of the nucleic acid (DNA) of the pathogen in any biological material (smear from the cervix, vagina, urethra, blood, saliva, sputum, etc.) and comparison its DNA or RNA with a database of known species of infectious agents.
The technique was developed by the American Carrie Mullis in the 80s of the last century. In 1993, the scientist won the Nobel Prize in Chemistry. Today, the PCR study is considered a kind of "gold standard" for diagnosing the vast majority of infections. PCR analysis is widely used in medical practice to clarify the nature of the disease and make an accurate diagnosis. Very often there are cases when all known immunological, virological and bacteriological methods do not work. Then PCR becomes the only way to identify the active stage of the disease.
Advantages of PCR diagnostics over other methods
The PCR technique has found wide application in modern medicine due to a number of indisputable advantages. Let's talk about them in more detail.
Ability to directly detect the presence of a pathogen
Many of the traditional diagnostic methods are based on the determination of markers - proteins that are the waste products of the pathogen. Such a diagnostic principle can only provide indirect confirmation of the pathology. The PCR method provides a direct identification of the pathogen, since it identifies specific sections of the DNA of pathogenic organisms.
High specificity
The PCR technique is highly specific due to the fact that it allows to detect DNA fragments that are characteristic only for a particular infectious agent. When using immunological methods, it remains possible to get a false result (mistakes in diagnosis are associated with cross-reacting antigens). As for PCR, errors are excluded here, since the specificity in this case is determined by the nucleotide sequence of the primers.
High sensitivity
Through this method, even single pathogens are determined. Infectious agents are detected in the human body even when other diagnostic methods do not clarify the situation (we are talking about various immunological microscopic and bacteriological research methods).
We present data for comparison. The sensitivity of microscopic and immunological methods is 103-105 cells, and the sensitivity of PCR is 10-100 cells per sample.
Universality of the method
The PCR technique is based on the study of the DNA of pathogenic organisms. During the study, fragments of RNA or DNA are determined that are specific to specific infectious agents. Since all nucleic acids have a similar chemical composition, standardized procedures can be used in laboratory analysis. This means that the study of one sample makes it possible to identify several pathogens at once.
Fast results
This technique does not require the cultivation of cultures of pathogens, which takes quite a lot of time. Thanks to the use of a unified technology for processing the material and identifying reaction products, as well as an automated amplification process, the entire research procedure takes only a few hours.
Ability to detect latent infections
The PCR technique makes it possible to effectively perform preclinical diagnostics (detection of pathogens before the onset of symptoms) and retrospective diagnostics (determination of pathogens after an illness). So, preclinical diagnostics is of great importance when examining a patient in the incubation period of a possible disease - after the alleged infection before the first signs appear.
One of the important advantages of PCR is the possibility of using biological residues or archival materials for analysis. This makes it possible to identify paternity and identity.
To date, PCR diagnostic methods continue to develop. The technology of analysis itself is being improved, and new types of PCR are also emerging. Innovative test systems for this reaction are being introduced into medical practice. Thanks to this rapid development of science, the cost of the procedure is decreasing, and now the PCR examination can be applied to many categories of patients.
This diagnostic method is based on multiple doubling of a given section of RNA or DNA. This process is carried out in the laboratory using special enzymes. As a result, as many sections of DNA (RNA) are formed as needed for visual examination. During the procedure, only the area corresponding to the specified conditions is copied (if it is present in the studied sample).
Biological material that needs to be examined for the presence of DNA or RNA of pathogens is placed in an amplifier. (Depending on the specific situation, blood, urine, saliva, discharged from the genitals are taken for analysis). Special enzymes are then added to the samples. They bind to the RNA or DNA of pathogenic microbes, and the synthesis of copies begins. Copying is a multi-stage process that proceeds like a chain reaction. As a result, hundreds or even thousands of copies may appear.
At the next stage of diagnostics, the results are analyzed and compared with the database of infectious agents.
The PCR technique not only makes it possible to determine the type of pathogenic organism, but also allows us to draw conclusions about the number of infectious agents in the human body.
Today, the use of such technologies opens up wide opportunities in the study of mutations, splicing of DNA sections. In modern medicine, the method began to be used to determine paternity, identify new genes, and much more.
Due to its versatility, the PCR method has found wide application in urology, gynecology, pulmonology, oncology, hematology, phthisiology, infectious disease practice and other areas of medicine.
Material for PCR analysis
For PCR diagnostics, various biological media and fluids taken from the human body are used: sputum, mucus, saliva, urine, blood, scraping of epithelial cells, placental tissues, pleural fluid, amniotic fluid, prostate juice, etc.
When diagnosing STDs (STIs), discharges from the male and female genital organs are analyzed. To do this, perform a smear or scraping from the urethra or cervix. Urine is also used for research.
To detect infections (herpes, mononucleosis, CMVI, toxoplasmosis, HIV, hepatitis B and C), blood is taken for analysis. If there is a suspicion of damage to the nervous system, cerebrospinal fluid is taken.
For pulmonological studies, pleural fluid and sputum are used.
To detect intrauterine infections, placental tissues and amniotic fluid are analyzed.
PCR for STIs and other infections
What infections can be detected by PCR analysis
HIV infection (human immunodeficiency virus HIV-1 can be detected).
Viral hepatitis A, B, C, G (RNA-HAV, DNA-HBV, RNA-HCV, RNA-HGV).
STIs (sexually transmitted infections) - ureaplasmosis, gardnerellosis, chlamydia, mycoplasmosis, trichomoniasis.
Infectious mononucleosis (DNA of the Epstein-Barr virus - EBV).
Cytomegalovirus infection (DNA-CMV).
Herpetic infection (DNA - herpes simplex virus HSV types 1 and 2).
Tuberculosis (mycobacterium tuberculosis).
Oncogenic viruses - papillomavirus infection (human papillomavirus (including its oncogenic species 16, 18, 31, 33, 45, 51, 52, 56, 58, and 59).
Borreliosis, tick-borne encephalitis.
Listeriosis.
Candidiasis (fungi of the genus Candida).
Helicobacter pylori infection
other.
Preparation and submission of PCR analysis
Patients who submitted material for PCR diagnostics expect to receive a quick and accurate result. At the same time, one important point should be taken into account - the reliability of diagnostics depends not only on the professionalism of specialists and the capabilities of the medical laboratory. In order for the study to be informative, the patient himself must make an effort. So, it is necessary to strictly adhere to all the recommendations of the doctor and carefully follow the rules for preparing for the collection of material. It is very important to prevent contamination of biological samples, otherwise the results of the examination will be distorted.
Preparation for PCR analysis
Preparation for the procedure is not associated with special difficulties. Just remember a few rules:
Blood for PCR analysis is taken on an empty stomach. Blood is usually taken from a vein in the morning with a sterile needle into a special container.
During the day before the sampling of the material, sexual abstinence is shown when taking a PCR smear for STIs.
For urine analysis for PCR, the first portion is taken, the container must be sterile.
How to take a PCR analysis in men and women
PCR analysis is taken from men and women in the vaccination room, this is blood from a vein, saliva, swabs from the pharynx, tonsils, swabs from the nasopharynx, etc. The collection method is no different.
PCR for STIs is taken at the antenatal clinic during a gynecological examination in women, these are smears from the vagina, cervix, and urethra. In men, during a visit to an andrologist or venereologist, this is a smear from the urethra.
Schemes for sampling material for PCR
How long does PCR take
Patients do not have to wait long for the results of the PCR examination. The entire analysis procedure usually takes from several hours (real-time PCR) to 2-10 days. As a rule, the patient receives the results of the analysis in 2-5 days, up to a maximum of 10 days, it depends on the type of analysis. The longest time is PCR blood diagnostics for HIV and hepatitis, and the shortest is smears and scrapings - 2-3 days.
A negative result indicates that at the moment no traces of infectious agents are detected in the biological material. That is, the infection for which the examination was carried out is absent.
A positive result indicates the detection of traces of the pathogen in biological samples. This means that at this time there is an infection in the human body.
There may be cases when PCR gives a positive result, but an active infectious process is not observed. This is a phenomenon that is called "healthy carriage." Treatment of such a patient is not required, but they must be under constant dynamic supervision. Such situations are typical for viral infections: Epstein-Barr virus (EBV), genital herpes, cytomegalovirus infection (CMVI), human papillomavirus (HPV), in which samples are taken for research from local foci (scraping of the urethra, cervical canal, saliva). At the same time, we must not forget that a healthy carrier can transmit the infection to other people. In addition, the activation of the infectious process is not excluded. In cases where PCR gives a positive result in a blood test, this can no longer be considered a carrier. Such patients need specific treatment for the disease caused by the detected pathogen.
Quantitative indicators do not have general gradations. They are evaluated by the doctor individually for each specific infection. A quantitative result makes it possible to determine how active the infection is and to identify the stage of the process.
Reliability of the PCR method
To evaluate the effectiveness of the methodology, there are three criteria:
- Accuracy- detection of infection (or its absence) with a high degree of probability.
- Specificity– the accuracy of determining a specific pathogen.
- Sensitivity– the possibility of identifying the pathogen even with a small amount of genetic material in biological samples.
When using the PCR method, obtaining false positive results is almost impossible (that is, if the pathogen is absent, then there will be no positive sample).
A false negative result is possible, but this happens quite rarely. Similar situations arise if the infection is inactive at the time of the study. For example, a chronic infection without activity or a latent infection.
PCR analysis: video