PCR material. polymerase chain reaction

Modern high-tech methods of laboratory research make it possible to identify diseases at an early stage. Infectious diseases develop and evolve, they become more and more, and it is more and more difficult to identify them. Diagnosis by PCR (polymerase chain reaction) is one of the latest and most accurate. For its development, the scientist Kary Mullis was awarded the Nobel Prize.

polymerase chain reaction- a method of molecular genetic diagnostics that allows you to find different types of infectious and hereditary pathologies in people, both in acute and chronic forms, and long before the pathology begins to manifest itself. Most doctors are faced with this analysis every day and no longer imagine how it is possible to make the correct diagnosis and stage of the disease without it.

The essence of PCR diagnostics

PCR analysis uses the principles of molecular biology. During its implementation, special enzymes are used that repeatedly copy fragments of RNA and DNA, as well as microorganisms that cause diseases and are found in human biological materials. Copying occurs in several stages, so a chain reaction is formed. After that, the laboratory specialists check the received data with a common database and identify the causative agents of the disease, as well as their concentration. Diagnostics is carried out in a special device that cools and heats a test tube with biomaterial and is called an amplifier. Compliance with the temperature regime affects the veracity of the analysis results.

Fragments of DNA of microorganisms can be contained in the following biological materials:

• biological fluids;
• blood and its derivatives;
• scraping of epithelial cells or smear;
• urine;
• sputum;
• mucus and other biological secretions;
• biopaths of the mucous membrane of the gastrointestinal tract.

Where is PCR diagnostics used?

The possibilities of diagnosing infectious diseases using the polymerase chain reaction are great; it can be used to identify a wide variety of viruses that cause infections such as:

This is not a complete list of diseases that are detected by PCR analysis. Most of these pathologies are almost invisible at an early stage, but the consequences of their impact on the body are extremely negative and often dangerous for human health and life.

Pregnant women are prescribed a PCR test to detect sexually transmitted infections, which significantly increase the risk of cervical cancer, adversely affecting the course of pregnancy and the health of the child. Therefore, the diagnosis of STDs is extremely important in order to prevent infection of the fetus with pathogenic microorganisms.

Based on all of the above, we can conclude that laboratory diagnostics using the PCR method helps the doctor to establish an accurate diagnosis and prescribe effective therapy in each individual case.

It is interesting! In addition to medicine, the PCR method is also used in other areas, for example, in forensics, when it is necessary to identify who owns the biomaterial found at the crime scene, and sometimes PCR is used to determine paternity, to conduct experiments with DNA mutation and other experiments.

Pros and cons of the method

The advantages of PCR diagnostics are:

• the high sensitivity of the test makes it possible to determine even single agents of the pathogen, which makes it possible to detect pathology at an early stage, with a chronic and latent form;
• the versatility of the method allows the use of almost any biomaterial for research from saliva to skin cells;
• large coverage - when examining one sample, it is possible to determine the presence of several different pathogens;
• accuracy - the high level of this survey method allows you not to give false indicators;
• speed - the result is ready in an average of five hours, that is, the patient can already pick up the conclusion the next day after the delivery of the biomaterial;
• relatively low price - this diagnostic method does not differ in cost from other laboratory blood tests;
• using this method, it is possible to make preclinical and retrospective diagnostics. The first one detects pathogenic microorganisms even before the manifestation of the disease, and the second one is carried out after the pathology has been transferred, which makes it possible to prevent the pathology from developing and cure it in time, as well as to determine the latent form of the disease that occurs without obvious symptoms.

But perfect diagnostic methods do not exist, therefore, PCR has disadvantages:

• high requirements for compliance with the technological process;
• high requirements for the professionalism of laboratory assistants.

Advice! It is better to conduct such an analysis only in the best and most professional laboratories, where strict quality control systems have been introduced.

For the reliability of the results, it is necessary to follow the rules for preliminary preparation for analysis:

• when donating saliva, you should not eat and drink medicines four hours before the analysis, before the procedure, rinse your mouth with boiled water, then carry out a small skin massage in order to secrete a secret from the gland;
• urine is usually collected at home. Before this, it is necessary to wash the genitals and collect 50 ml of the first morning urine in a special container; it is not advisable to collect the material during menstruation;
• before donating sperm, a man needs to not have sex for three days, do not go to the sauna, do not swim in a hot bath, do not take alcoholic drinks and spicy food. Immediately before the analysis, it is forbidden to urinate for three hours;
• for the delivery of a urogenital smear, the absence of sexual intercourse for three days is recommended. Antibacterial drugs are prohibited two weeks before the analysis. For a week, you need to stop using intimate gels, ointments, vaginal suppositories, and do not douche. Three hours before taking the biomaterial, it is necessary to refrain from going to the toilet.
• blood is given in the morning on an empty stomach.

Material for diagnostics should be collected under conditions that exclude its contamination, as this can significantly affect the veracity of the PCR result.

Deciphering the analysis

The analysis can show a positive or negative result. Negative means that there are no suspected infections in the body and the person is healthy. Positive says that the patient is ill and needs treatment. Any indicators should be deciphered and voiced by the doctor. You should not be upset and afraid of bad results, therapy is prescribed on the basis of them, the main thing is not to delay the process and not self-medicate.

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I am a general practitioner and general practitioner. My competence includes issues of early diagnosis of patients and treatment of many diseases of the gastrointestinal tract, lungs and respiratory tract, liver, kidneys, cardiovascular and genitourinary systems, skin diseases, metabolic disorders, etc. 15 years of experience as a general practitioner in polyclinics Moscow, 5 of which worked in one hospital in St. Petersburg .. I will be happy to answer questions from readers of my blog.

In modern medicine, more importance is given to high-precision laboratory research methods based on the Polymerase Chain Reaction. Thanks to the use of PCR technologies, it became possible to analyze at the molecular genetic level and identify acute and chronic forms of hereditary and infectious diseases in a patient long before the onset of clinical symptoms.

What is PCR - diagnostics

This method was developed by American biochemist and Nobel laureate Carrie Mullis in 1984.

Many qualified specialists are faced with a PCR study every day and cannot, without its results, accurately diagnose most active forms of pathologies in cases where immunological and microbiological methods do not work. It often happens that different viruses can cause the same clinical symptoms, PCR analysis will allow you to determine the pathogen at its lowest concentration in the biomaterial and to identify even single cells of viruses or bacilli.

About PCR diagnostics on video

The basis of PCR diagnostics is the in special laboratory conditions of repeated amplification (multiplication) of certain sections of DNA (deoxyribonucleic acid) - the human genetic material.

The entire technological process of copying consists of several stages:

1. Denaturation – preparation of the sample, by increasing the temperature of the biomaterial (up to 95 °C), 2-stranded DNA is split into two separate strands.
2. Annealing - the studied biomaterial is cooled and nitrogen primers (reagents) are added to it, which have the ability to specifically recognize sequences in the DNA molecule that are characteristic only of a pathogenic agent and combine with them.
3. elongations - the polymerase reaction itself, a unique molecular genetic site is completed, in each of the connections with the primer a new, structurally complementing the daughter DNA chain is formed.

The whole cycle is repeated 20-30 times. Ultimately, the number of complementary DNA strands is formed, which is sufficient for visual analysis and comparison of the results with the available data on the cellular structure of various pathogens. The virus is determined, the nature of its appearance, the strength of its effect on the body and the number of available bacilli are established. This information is of great value to the attending physician when prescribing effective methods of therapy and selecting drugs.

PCR diagnostic methods differ from other laboratory methods in the following:

• direct determination of the presence of pathogens;
• high sensitivity, specificity and versatility of the virus detection procedure;
• speed of analysis;
• the ability to diagnose asymptomatic pathologies.

The results of the study can be photographed or entered into information carriers for the possibility of their evaluation by independent experts.

How to prepare for the PCR test?

Various biomaterials are used for research:

• blood;
• slime;
• saliva
• urine;
• sputum;
• scrapings of the epithelium;
• prostate juice;
• scrapings of mucous membranes;
• amniotic fluid;
• placental tissue;
• cerebrospinal, articular or pleural fluid;
• secretion of the genital organs.

Modern laboratory equipment and the professionalism of the laboratory assistant guarantee the patient who undergoes PCR analysis to obtain a reliable result. But the accuracy of the study depends on the correct preparation for the test, and on compliance with all recommendations for the selection of biomaterial.

It is not difficult to properly prepare for the analysis, it is important to follow all the existing rules:

1. The day before the study, do not have sexual intercourse.
2. Cancel the gym.
3. Do not visit a bath or sauna before the examination.
4. You should have dinner the day before no later than 20 hours, do not get involved in spicy and fatty foods, do not take alcohol.
5. Venous blood should be taken in the morning, do not eat, drink or smoke before the procedure.

How women and men take PCR tests - features of the procedure

The main general requirement for the selection of biomaterial is to obtain the maximum concentration of microorganisms in the sample and the absence of undesirable impurities - mucus, blood or pus.

During examinations for infections that are transmitted through sexual contact (ureaplasmosis, gardnerellosis, chlamydia, mycoplasmosis, trichomoniasis), secretions from the genitals are taken:

• in men, a swab or scraping is taken from the urinary canal (urethra);
• in women - a smear or scraping from the vagina, cervical canal.

When taking material from the urogenital tract, it is important to avoid the ingress of impurities. For this purpose, scrapings are taken from men no earlier than 2 hours after the last urination, from women - taking into account the days of the menstrual cycle. Excess mucus or pus is removed with sterile cotton swabs, the biomaterial is taken using special plastic probes - this reduces the likelihood of blood entering the sample.

The procedure for taking a urogenital scraping is quite painful for men. , which is why the first portion of urine after a night delay, which contains the largest amount of epithelium, is often used for analysis. Urine is collected in a sterile container with a tight-fitting lid and delivered to the laboratory no later than two hours after collection. In the laboratory, for further work, a cellular urine sediment is obtained by centrifugation.

What infections are included in the PCR-12 complex?

PCR diagnostics is actively used by experienced specialists. The most popular is the diagnosis of viruses and infections.

Currently, polymerase chain reaction techniques expand the possibilities of conducting research - the introduction of genotyping and splicing of DNA fragments of tissues are widely used in various areas of modern medicine:

• gynecology;
• urology;
• pulmonology;
• gastroenterology;
• hematology;
• oncology.

Where can I get cheap tests at the URFO?

Modern methods of PCR diagnostics are constantly developing. The technique itself is being improved, new types of PCR and new test systems used for the chain reaction are emerging. Thanks to these innovations, the cost of these tests becomes more affordable for patients.

PCR smear in the diagnosis of infectious diseases has the following advantages:

• Direct detection of pathogens of infectious diseases.

Many traditional methods of laboratory diagnostics involve the identification of pathogens by various indirect signs. For example, ELISA diagnostics is based on the detection of proteins in the patient's blood, which are decay products of infectious pathogens, on the basis of which the doctor can make a particular diagnosis. The PCR analysis method gives a direct indication of the presence in the material taken from the patient of a specific region of the DNA of the causative agent of the disease.

• High specificity of PCR diagnostics.

During the analysis, a specific DNA fragment is isolated in the test material, i.e., inherent only in a specific pathogen - only a certain bacterium or virus. This section of DNA is unique and is not characteristic of any infection on earth.

• High sensitivity PCR.

Detection of infection is possible even if the material taken from the patient contains only one cell of a bacterium or virus. Compared with other immunological and microbiological diagnostic methods: the sensitivity of PCR analysis is 10-100 cells per sample, other methods - 103-105 cells.

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• Versatility of PCR analysis.

For PCR research, almost any materials can be used, including those that are not available for research by other methods: mucus, urine, blood, serum, sputum, ejaculate, scraping of epithelial cells - since the infection can be contained in any biological secretions and tissues.

• High speed of obtaining the result of PCR analysis.

A single method for processing the material taken for analysis from a patient, detection of reaction products, automated PCR amplification make it possible to carry out a complete PCR diagnosis in 4-5 hours. At the same time, much more time is spent on cultural research methods - from several days to several weeks, since it is necessary to isolate and then grow the pathogen in cell culture.

• The ability to diagnose any type of infection.

The high sensitivity of the PCR method makes it possible to diagnose an infection not only at the acute stage of the disease, but also chronic infections and even the presence of single bacteria or viruses.

A smear by PCR allows you to identify infections that cannot be detected in a smear for flora: chlamydia, ureaplasmosis, mycoplasmosis, genital herpes.

No matter how significant the research method has, PCR diagnostics also has some limitations. The disadvantages of PCR diagnostics include:

• Possibility of obtaining a false positive result

PCR analysis can show a positive result even if the infection is already dead, "killed" by antibiotics, but its dead cells are still contained in the patient's tissues. How is this possible? Very simple. For example, the infection lives in epithelial cells (on the mucous membrane of the genitals or eyes). And she was cured. But it takes time to “renew” epithelial cells. If the material is taken by a doctor before the period of complete cell renewal, the material may contain dead cells of the infection. Cells obviously contain genetic material - DNA or RNA of the pathogen, which is "looking for" PCR. PCR does not distinguish dead cells from living ones: it looks for DNA, and "clones" them in large quantities. The result of this analysis is positive. In fact, it is a false positive.

The inability of PCR to "distinguish" a dead infection from a live one imposes certain requirements when using PCR and for monitoring the effectiveness of treatment. The main rule, of course, is to wait until the dead remnants of the infection are completely eliminated from the body, which in the average person occurs within 4-8 weeks. After this period, after taking the last antibiotic tablet, the PCR method can and should be used to monitor the effectiveness of the treatment.

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At earlier periods, the control of cure can only be carried out using the cultural method, or inoculation: only viable multiplying microorganisms can serve as a sign of incurability.

• It is undesirable to use PCR analysis for the diagnosis of gardnerellosis, since gardnerella, the causative agents of this disease, normally live in the vagina in a small amount. The DNA of these bacteria, when using PCR, will be found over and over again. Gardnerella should not be in the smear, and bacterioscopy in this case is a sufficient method for diagnosing gardnerellosis and monitoring treatment.
• Variability of microorganisms

Any microbe has the ability to change, and at the DNA level. The most striking example in this area is the influenza virus. Having been ill once, a person develops antibodies to the virus. Purely theoretically, if we have had the flu at least once, then, like chickenpox, we should not get sick a second time. But we get sick almost every year. What is the reason? The virus "mutates", slightly "changing" its genome, and our immune system, our antibodies no longer "recognize" the old guest in a new guise. Sadly, you have to get sick with the flu again ...

When designing an amplifier (test PCR system), a DNA fragment specific to a given microorganism is used, which is the least susceptible to changes. It is "selected" from the so-called highly conserved region of DNA. But the variability of microorganisms can lead to the fact that some genotypes or strains of the pathogen under study can acquire mutations in the amplified (cloned) region of the genome, and thus become elusive by this test system.

Thus, different test systems are one of the reasons why analyzes made in different laboratories and in different clinics by the “same” PCR method can show diametrically opposite results. In order to avoid mutation errors as much as possible, standards have now been developed that govern the scope of testing (including testing for cross-reactions, as well as testing of known strains of a detectable pathogen) that a test system must pass before it enters the market and is used for diagnosing your health. That is why good clinics and laboratories use the latest generation of test kits. And our medical center "Euromedprestige" is one of the few who can offer their clients high-quality diagnostics.

Polymerase chain reaction (PCR for short) is a modern and ultra-precise diagnostic method at the genetic and molecular level. PCR diagnostics makes it possible to determine whether a person has an infection or a hereditary disease.

The essence of the method lies in the fact that the DNA or RNA of the material taken for the study is subjected to multiple multiplication (amplification), which is possible in the presence of enzymes. Due to this, a large mass of DNA or RNA is obtained, according to which a visual assessment is made. Specialists have a database of information about the structure of all pathogens and, in accordance with it, an assessment is made of which microorganism was obtained.

The test material (saliva, blood, sputum, etc.) is placed inside a special device (amplifier) ​​and certain enzymes are added. They combine with the studied DNA or RNA and cause the synthesis of their copies. Through PCR, it is possible to determine the type of pathogen, its quantity.

Interesting! The PCR method was invented in 1983 by the American scientist Carrie Mullis. For this discovery, he was awarded the Nobel Prize in Chemistry.

Test tubes with test material

Pros and cons

The PCR technique has been used in the field of medicine recently and has taken an important place in the diagnostic process.

Advantages of the method:

1. Direct identification of the pathogen. Some methods, for example, enzyme-linked immunosorbent assay, make it possible to identify proteins secreted by microbes. This is an indirect definition of the presence of pathogens. And by means of PCR, a piece of DNA belonging to a certain infection is detected with high accuracy.
2. The specificity of the technique. Since PCR detects DNA belonging to the pathogen, it is almost impossible to get a false conclusion (with the exception of an unknown pathogen). And with the immunological method, an error can occur due to cross-reaction between antigens.
3. Great sensitivity. The method detects the presence of even a very small amount of bacteria or viruses in the body. 10 disease-causing cells per sample are enough. For other methods, the minimum number of cells must be at least 105.
4. The versatility of the technique. The research process for different microorganisms is similar due to the similarity of all DNA, RNA. Based on this, it is possible to identify several pathogens in one sample.
5. Quick conclusion. From the moment of sampling to obtaining the result, no more than 4-5 hours pass. With PCR, no culture is needed, so the method is quite fast.
6. The effectiveness of diagnosis in latent types of infections. The method reveals those microorganisms that are difficult or not at all amenable to cultivation. Such pathogens are found in latent forms of the disease.
7. Wide scope of use. PCR can be used not only to diagnose human diseases, but also to determine microorganisms in soil, food, water, etc.

But you can also face the disadvantages of the PCR method:

1. Microbes can change. Pathogens can change and mutate. Therefore, they may not be detectable by PCR.
2. The possibility of multiplying the DNA of not only a living, but also a dead pathogen. To avoid such a situation, PCR can be used only 1-2 months after recovery from a previous illness.

Varieties of PCR

The research method is used for various purposes, so the PCR technique has varieties:

1. With reverse transcription - used for repeated doubling and subsequent determination of which RNA of which microorganism was isolated. To do this, use a library of data on the structures of various pathogens.
2. Inverted - used when there is a small area within a particular sequence.
3. Nested - used to reduce the amount of unwanted reaction products. For these purposes, two consecutive studies are carried out.
4. Asymmetric - used if you need to multiply to a greater extent one of all the available DNA chains.
5. Quantitative (real-time) - necessary for a direct study of how the amount of the product obtained changes in each new cycle of the PCR reaction.
6. Digital - the most modern and accurate.
7. Group-specific - this is a study that is carried out in order to study family ties.
8. Stepwise - reduces the effect of non-specific interaction of primers.

Filling tubes with blood samples

Deciphering the results

In the conclusion of the study, an unequivocal answer is given about the presence of a pathogen, which happens:

• negative, this means that there is no infection;
• positive - it is.

Identification of infection can occur even in the absence of symptoms of the disease in a person. This is the initial stage of infection. Sometimes you can get a false positive answer (on the verge of a negative and a positive result). In this case, the analysis must be repeated. Particular attention should be paid to ensuring that the material was collected in full compliance with all requirements.

Deadlines for PCR analysis

In practice, the result of the study can be obtained in 2 days. Sometimes it can be done faster - on the day of delivery.

Price for the procedure

The cost of PCR is determined by which infection will be tested. It also depends on the testing methodology. The average price of the analysis is in the range from 200 to 850 rubles. The price also includes a fee for the collection of material - about 400 rubles.

Approximate prices for PCR testing.

Often, medical institutions offer to analyze several infections at the same time in order to identify the right one. Such an examination will cost more, but it will eliminate the need for additional studies, which will save the patient's time.

Video "Polymerase chain reaction"

The video tells about the essence of the PCR method and its advantages. Filmed by the SiberianMedicalLaboratory channel.

Polymerase chain reaction (PCR)

The essence of the PCR method. DNA polymerase

Polymerase chain reaction is an experimental method of molecular biology that allows to achieve a significant increase in small concentrations of certain nucleic acid fragments in biological material. This process of increasing the number of copies of DNA is called amplification. DNA copying during PCR is carried out by a special enzyme - polymerase. DNA polymerase (Fig. 3) is an enzyme involved in the replication (amplification of DNA in living organisms) of DNA. Enzymes of this class catalyze the polymerization of deoxyribonucleotides along the DNA nucleotide chain, which the enzyme "reads" and uses as a template. The type of a new nucleotide is determined by the principle of complementarity with the template from which the reading is performed.

DNA polymerase adds free nucleotides to the 3 "end of the assembled chain. This leads to an elongation of the chain in the 5"-3 direction. None of the known DNA polymerases is able to create a chain "from scratch": they are only able to add nucleotides to already existing 3"-hydroxyl group. For this reason, DNA polymerase needs primer- a short sequence of nucleotides (usually 20-25), complementary to the end sections of the gene under study - to which she could add the first nucleotide. Primers are always composed of DNA and RNA bases, with the first two bases always being RNA bases. Primers are synthesized by another enzyme - primase. Another enzyme is helicase- necessary for the unwinding of the DNA double helix with the formation of a single-stranded structure, which ensures the replication of both strands in accordance with the semi-conservative model of DNA replication.

Some DNA polymerases also have the ability to correct errors in the newly assembled DNA strand. If an incorrect pair of nucleotides is detected, DNA polymerase rolls back one step, removes the wrong nucleotide from the chain, then inserts the correct one in its place, after which replication continues as usual.

Conducting PCR

Polymerase chain reaction (PCR) is a DNA amplification method that can isolate and multiply a certain 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.

For the polymerase chain reaction to take place, a number of conditions must be met. For PCR, in the simplest case, the following components are required:

A DNA template containing the section of DNA to be amplified.

Two primers complementary to the ends of the desired fragment. (A pair of artificially synthesized oligonucleotides, usually 15 to 30 bp in size, identical to the corresponding regions of the target DNA. They play a key role in the formation of amplification reaction products. Properly selected primers ensure the specificity and sensitivity of the test system.)

Thermostable DNA polymerase. The polymerase used in PCR must remain active at high temperature for a long time, therefore, enzymes isolated from thermophiles - Thermus aquaticus (Taq polymerase) and others are used.

Deoxynucleotide triphosphates (dATP, dGTP, dCTP, dTTP).

Mg 2+ ions necessary for polymerase to work.

A buffer solution that provides the necessary reaction conditions - pH, ionic strength of the solution. Contains salts, serum albumin.

To avoid evaporation of the reaction mixture, a high-boiling oil, such as vaseline, is added to the test tube. If a device with a heated lid is used, this is not required.

The addition of pyrophosphatase can increase the yield of the PCR reaction. This enzyme catalyzes the hydrolysis of pyrophosphate, a by-product of the addition of nucleotide triphosphates to the growing DNA strand, to orthophosphate. Pyrophosphate can inhibit the PCR reaction.

To multiply the number of copies of the original DNA, a cyclic reaction is needed. As a rule, each of the sequentially repeated PCR cycles consists of three stages:

1. Denaturation, or "melting" of DNA. The double-stranded DNA template is heated to 94 - 96°C (or 98°C if a particularly thermostable polymerase is used) for 0.5 - 2 minutes to allow the DNA strands to separate. This step is called denaturation because the hydrogen bonds between the two strands of DNA are broken. Sometimes, before the first cycle (before adding the polymerase), the reaction mixture is preheated for 2–5 minutes to completely denature the template and primers. This approach is called hot start, it allows to reduce the amount of non-specific reaction products.

2. Annealing - binding of primers to template DNA. Once the strands have separated, the temperature is slowly lowered to allow the primers to bind to the single stranded template. The annealing temperature depends on the primer composition and is usually chosen at 50–65°C. Stage time - 20 - 60 seconds. An incorrect choice of annealing temperature leads either to poor binding of primers to the template (at elevated temperature) or to binding in the wrong place and the appearance of non-specific products (at low temperature).

3. Synthesis (chain elongation). DNA polymerase replicates the template strand using the primer as a "seed". The polymerase starts the synthesis of the second strand from the 3" end of the primer, which has bound to the template and moves along the template. The elongation temperature depends on the polymerase. The commonly used Taq and Pfu polymerases are most active at 72°C. the length of the fragment to be amplified Typically, the elongation time is taken to be one minute for every thousand base pairs After all cycles have been completed, an additional step is often performed final elongation to complete all single-stranded fragments. This stage lasts 7 - 10 minutes.

Subsequently, the stages of denaturation, annealing, and elongation are repeated many times (30 or more times). At each cycle, the number of synthesized copies of a DNA fragment doubles.

All reactions are carried out in test tubes immersed in a thermostat. Changing the temperature regime and its maintenance is carried out automatically.

To understand exactly how the amplification of a certain DNA segment occurs during PCR, it is necessary to clearly understand the position of all primers and their complementary sequences in the amplifiable chains in each round. In the first round, each of the newly synthesized chains is much longer than the distance from the 3"-hydroxyl group of its primer to the terminal nucleotide of the sequence complementary to the second primer. Such chains are called "long templates", it is on them that further synthesis will take place.

In the second round, double-stranded DNA, consisting of similar and newly synthesized (long template) strands, is again denatured and then annealed with primers. During synthesis in this round, "long templates" are again synthesized, as well as a number of strands with a primer at one end and with a sequence complementary to the second primer at the other ("short templates"). During the third round, all heteroduplexes formed earlier are simultaneously denatured and annealed with primers, and then replicated. In subsequent rounds, there are more and more "short matrices", and by the 30th round their number is already 10 6 times greater than the number of initial chains or "long matrices".

The amount of specific reaction product (limited by primers) theoretically increases proportionally to 2 n , where n is the number of reaction cycles. In fact, the efficiency of each cycle can be less than 100%, so in reality:

where P is the amount of product, E is the average efficiency of the cycle.

The number of "long" DNA copies also grows, but linearly, so a specific fragment dominates in the reaction products. The growth of the required product is exponentially limited by the amount of reagents, the presence of inhibitors, and the formation of by-products.

PCR is a highly sensitive method, therefore, if there is even an insignificant amount of DNA in the test sample that accidentally got from one reaction mixture to another, false positive results can be obtained. This makes it necessary to carefully control all solutions and utensils used for PCR.

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:

1. Primers must be specific. Particular attention is paid to the 3 "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 undesirable processes will most likely 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, because it is easy to confuse a specific amplification product with synthesized foreign DNA.Some primers and dNTPs are consumed for the synthesis of nonspecific DNA, which leads to to a significant loss of sensation.

2. 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.