home Consultations Polymerase chain reaction PCR is used for. Polymerase chain reaction, its essence and areas of application. How to prepare for the test

Polymerase chain reaction PCR is used for. Polymerase chain reaction, its essence and areas of application. How to prepare for the test

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Method polymerase chain reaction was discovered almost thirty years ago by an American scientist named Carrie Mullis. The technique is widely used in medicine as a diagnostic tool, and its essence is to copy a section of DNA using a special enzyme ( polymerases) artificially in vitro.

In what areas of medicine is this method used?

Why is DNA copying performed and how can it serve medicine?
This technique allows:

Isolate and clone genes.
Diagnose genetic and infectious diseases.
Determine paternity. A child inherits some of its genetic characteristics from its biological parents, but has its own unique genetic identity. The presence of some genes that are identical to the parental genes allows us to talk about establishing a relationship.

Polymerase chain reaction is also used in forensic practice.

At crime scenes, forensic scientists collect samples of genetic materials. These include: hair, saliva, blood. Subsequently, thanks to the polymerase reaction technique, the DNA can be amplified and the identity of the sample taken can be compared with the genetic material of the suspected person.

The polymerase chain reaction is effectively used in medicine:

In pulmonological practice - to differentiate bacterial and viral types of pneumonia, tuberculosis.
In gynecological and urological practice - to determine ureaplasmosis, chlamydia, mycoplasma infection, gardnerellosis, herpes, gonorrhea.
In gastroenterological practice.
In hematology - for the determination of oncoviruses and cytomegalovirus infection.
In the rapid diagnosis of infectious diseases such as viral hepatitis, diphtheria, salmonellosis.

Currently, this method is most common in the diagnosis of infectious diseases ( hepatitis of viral etiology, HIV, sexually transmitted diseases, tuberculosis, tick-borne encephalitis).

What happens during the reaction?

The reaction itself is chemically simple. The source of DNA for the reaction can be a drop of blood, hair, a piece of skin, etc. In theory, the reaction requires the necessary reagents, a test tube, a biological sample, and a heat source.

The polymerase reaction makes it possible to detect an infection, even if the sample containing biological material contains only one or several DNA molecules of the pathogen.

During the reaction, thanks to the enzyme DNA polymerase, doubling occurs ( replication) section of DNA. Deoxyribonucleic acid itself ( abbreviated as DNA) is important for us because it ensures the storage and transmission of genetic information to daughter cells. DNA has the shape of a helix, which consists of repeating blocks. These blocks make up nucleotides, which are the smallest unit of DNA. Nucleotides are formed from amino acids.

The process of replication of DNA sections occurs during repeated cycles. In each such cycle, not only the original DNA fragment is copied and doubled, but also those fragments that were already doubled in the previous amplification cycle. All this resembles the process of geometric progression.

Exists:

Natural amplification ( that is, the process of copying and multiplying DNA), which occurs in our body and is a deterministic, predetermined process.
Artificial amplification, which occurs due to the polymerase chain reaction. In this case, the copying process is controlled and allows even short sections of nucleic acid to be duplicated.

After each copying cycle is completed, the number of nucleic acid fragments increases exponentially. That is why the process itself is called a “chain reaction.”

After thirty to forty cycles, the number of fragments reaches several billion.

For amplification in vitro (in vitro) it is necessary that a specific foreign DNA fragment ( that is, the DNA is not of the patient, but of the pathogen). If the created solution does not contain a specific fragment, the chain reaction under the action of the polymerase will not proceed. This explains the fact of high specificity of PCR.

Stages of PCR diagnostics

1. DNA is isolated from the material under study.
2. DNA is added to a special solution of nucleotides.
3. The solution is heated to a temperature of 90 - 95 degrees Celsius so that the DNA protein coagulates.
4. Reduce temperature to 60 degrees.
5. As cycles of increasing and decreasing temperature are repeated, the number of nucleic acid sites increases.

6. By performing electrophoresis, the results are summed up and the doubling results are calculated.

What are the advantages of this diagnosis?

Versatility: Any nucleic acid sample is suitable for this method.
High specificity: the pathogen has unique sequences of DNA chains that are specific to it. Therefore, the results of the PCR will be reliable; it is impossible to confuse the gene of one pathogen with the gene of another pathogen.
Sensitivity to the presence of even a single pathogen molecule.

Small amount of material needed for research. Even a drop of blood will do. The ability to obtain results using a minimal sample volume is very important for pediatric, neonatological, neurological studies, as well as in the practice of forensic medicine.
The ability to determine indolent, chronic infection, and not just acute.
Many pathogenic cultures are very difficult to cultivate in vitro using other methods, but the polymerase reaction allows the culture to be propagated in the required quantity.

What disadvantages does this diagnosis have?

If the material intended for PCR contains the DNA of not only a living pathogen, but also a dead one, amplification of both DNA will occur. Accordingly, the treatment prescribed after diagnosis may not be entirely correct. After some time, it is better to monitor the effectiveness of the treatment.
Increased sensitivity to the presence of microorganisms can also be considered, in some way, a disadvantage. After all, the human body normally contains opportunistic microflora, that is, these are microorganisms that live in the intestines, stomach, and other internal organs. These microorganisms can cause harm to humans only under certain unfavorable conditions - failure to comply with hygiene requirements, contaminated drinking water, etc. The PCR technique amplifies the DNA of even these microorganisms, although they do not lead to pathology.
PCR of different test systems may show results that will differ from each other. There are many modifications of this technique: “ nested», « asymmetrical», « inverted», « quantitative» PCR and others.

Polymerase chain reaction (PCR) is a high-precision method in the field of diagnosing hereditary pathologies, infections, viral diseases at any stage (acute or chronic), as well as - at an early stage - before obvious manifestations of the disease by identifying pathogens based on their DNA, RNA , which are genetic material, in samples obtained from the patient. And today we will talk about the essence, diagnostic stages and principles of polymerase chain reaction (PCR) methods, as well as its cost.

What is polymerase chain reaction

The basis of the analysis is amplification (doubling) - the creation of many copies from a short section of DNA (deoxyribonucleic acid), which represents the human genetic complex. The study requires a very small amount of physiological substance (sputum, feces, epithelial scrapings, prostate juice, blood, sperm, amniotic fluid, mucus, placental tissue, urine, saliva, pleural fluid, cerebrospinal fluid). In this case, for example, even a single harmful microbe can be detected in the patient’s genitourinary tract.

The PCR (polymerase chain reaction) technique was developed by the American scientist K. Mullis, who received the Nobel Prize in 1993.

Actively used:

in the early diagnosis of infections, genetic;
in forensic medical examination when there is an extremely small amount of DNA available for examination;
in veterinary medicine, pharmaceuticals, biology, molecular genetics;
for identification of a person by DNA, confirmation of paternity;
in paleontology, anthropology, ecology (when monitoring the quality of products, environmental factors).

This video will tell you in detail what a polymerase chain reaction is:

Who is it prescribed to?

Polymerase chain reaction in the diagnosis of infectious diseases is one of the most reliable methods of particular accuracy and reliability. For example, the reliability of the PCR analysis for chlamydia and many other pathogens is close to 100% (absolute). Most often, the polymerase chain reaction procedure is prescribed to patients who have difficulty identifying a specific pathogen during diagnosis.

Laboratory PCR test is used:

to detect pathogens that cause infections of the urinary and genital organs that are difficult to identify using culture or immunological methods;
for re-diagnosis of HIV at the initial stage in case of a positive but questionable result of the initial test (for example, in newborns from AIDS-infected parents);
to identify cancer at an early stage (study of oncogene mutations) and individually adjust the treatment regimen for a particular patient;
for the purpose of early detection and potential treatment of hereditary pathologies.

Thus, future parents take a test to find out whether they are carriers of a genetic pathology; in children, PCR determines the likelihood of exposure to a disease transmitted by inheritance.

to detect fetal pathologies in the early stages of gestation (individual cells of the growing embryo are examined for the presence of possible mutations);
in patients before organ transplantation - for “tissue typing” (determining tissue compatibility);
to identify dangerous pathogenic organisms in donated blood;
in newborns - to identify hidden infections;
to evaluate the results of antiviral and antimicrobial treatment.

Why undergo such a procedure?

Since PCR is a highly effective diagnostic method, giving almost 100% results, the procedure is used:

to confirm or exclude the final diagnosis;
rapid assessment of the effectiveness of therapy.

In many cases, PCR is the only possible test for detecting a developing disease if other bacteriological, immunological and virological diagnostic methods are useless.

Viruses are detected using a PCR procedure immediately after infection and before signs of illness appear. Early detection of the virus allows prompt treatment.
The so-called “viral load” (or the number of viruses in the body) is also determined by DNA analysis using a quantitative method.
Specific pathogens (eg, Koch's tuberculosis bacillus) are difficult and take too long to culture. PCR testing allows rapid detection of minimal pathogens (live and dead) in samples convenient for testing.

Detailed pathogen DNA analysis is used:

to determine its sensitivity to specific types of antibiotics, which allows immediate treatment;
to control the spread of epidemics among domestic and wild animals;
to identify and track new infectious microbial species and pathogen subtypes that have fueled previous epidemics.

Types of diagnostics

Standard method

Polymerase chain reaction analysis is carried out on the basis of multiple amplification (doubling) of a specific fragment of DNA and RNA using special primer enzymes. As a result of the copying chain, a sufficient amount of material is obtained for research.

During the procedure, only the desired fragment (corresponding to the specified specific conditions) is copied and if it is actually present in the sample.

This detailed video with useful diagrams explains how PCR works:

Other methods

Real-time PCR. In this type of research, the process of identifying a given DNA fragment starts after each cycle, and not after completing the entire chain of 30 - 40 cycles. This type of research allows you to obtain information about the amount of a pathogen (virus or microbe) in the body, that is, carry out a quantitative analysis.
RT-PCR (reverse transcription mode). This test is used to look for single-stranded RNA to detect viruses whose genetic base is RNA (for example, hepatitis C virus, immunodeficiency virus). In this study, a special enzyme is used - reverse transcriptase and a specific primer, and single-stranded DNA is built on the basis of RNA. Then the second DNA strand is recovered from this strand and the standard procedure is performed.

Indications for testing

The PCR procedure is used in the clinic of infectious diseases, neonatology, obstetrics, pediatrics, urology, gynecology, venereology, neurology, nephrology, and ophthalmology.

Indications for testing:

determining the risk of developing genetic abnormalities in a child with the likelihood of hereditary pathologies;
diagnosing both parents when planning a pregnancy or the serious condition of the mother during an ongoing pregnancy;
difficulties with conception, identifying the causes of infertility;
suspicion of sexually transmitted infections in the acute stage and with symptoms of their transition to chronic;
detection of causes of inflammatory processes of unknown origin;
unprotected casual and regular sexual contacts;
determining the sensitivity of a pathogenic microorganism to specific antibiotics;
patients with suspected latent infection to detect pathogens before the development of obvious symptoms (preclinical diagnosis);
patients to confirm recovery after illness (retrospective diagnosis);:

Diagnostics is also used if it is necessary to accurately identify the following pathogens::

hepatitis viruses (A B C G), human immunodeficiency, cytomegalovirus;
Vibrio cholerae;
herpes simplex virus, herpetiform species;
retro - adeno - and rhinoviruses;
rubella, Epstein-Barr, varicella (Zoster) viruses;
parvo and picornoviruses;
bacterium Helicobacter pylori;
Legionella, pathogenic types of Escherichia coli;
Staphylococcus aureus;
pathogen;
clostridia, diphtheria and hemophilus influenzae;

It is also used to determine infections:

Infectious mononucleosis;
borreliosis, listeriosis, tick-borne encephalitis;
candidiasis caused by Candida fungi;
sexually transmitted infections – trichomoniasis, ureaplasmosis, treponema pallidum, gardnerellosis, gonorrhea, mycoplasmosis, chlamydia;
tuberculosis.

Contraindications for

Since the procedure is not carried out with the patient, without any impact on the body, but with biological material taken for research, there are no contraindications for PCR due to the absence of potential danger.

However, biomaterial is not collected from the cervical canal of the uterus after the colposcopy procedure. Submission of smears and scrapings for analysis is allowed only 4–6 days after the end of menstruation and the complete cessation of discharge.

Is the method safe?

No negative impact on the patient during an isolated study of his biomaterial in the laboratory is possible.

Preparation for the procedure (submission of biological substances for analysis)

Any biological fluid, tissue, or body secretions serve as a sample for PCR analysis, which detects the DNA of a foreign pathogen. The test substance is taken in the form of taking blood from a vein, scraping from the larynx, nasal cavity, urethra, pleural cavity, cervix.

Before the diagnostic procedure, the doctor explains to the patient what material will be collected:

When examining for sexually transmitted infections, secretions from the genital organs, urine, and a smear from the urethra are collected.
When analyzing for herpetic infections, cytomegalovirus, mononucleosis, urine and a throat swab are taken for analysis; for hepatitis, toxoplasmosis, blood from a vein is taken.
In order to diagnose various types, cerebrospinal fluid is collected.
In pulmonology, samples for analysis are sputum and pleural fluid.
When conducting a study of possible intrauterine infections during pregnancy, amniotic fluid and placental cells are used for analysis.

The reliability and accuracy of the analysis depends on the sterility of the conditions when taking the material. Since PCR testing is highly sensitive, any contamination of the test substance can distort the result.

Competent preparation for the delivery of biomaterial does not present any difficulties for patients. There are certain recommendations:

when analyzing for sexually transmitted infections:
- exclude intimate contacts 72 hours before submitting the material;
- stop using any vaginal products 3 days before;
- from the evening of the previous day, do not carry out hygiene of the area being examined;
- exclude urination 3–4 hours before taking a sample from the urethra;
stop taking antibiotics a month before testing for infections;
blood is donated in the morning before eating and drinking;
The first morning urine sample is collected in a sterile container after a thorough intimate toilet.

Read below about how diagnostics are carried out using the polymerase chain reaction method.

How does the procedure work?

When performing a PCR study, certain cycles are repeated over and over again in a reactor (amplifier or thermal cycler):

The first step is denaturation. Saliva, blood, biopsy material, gynecological samples, sputum, in which the presence of DNA (or RNA) of a pathogen is suspected, is placed in an amplifier, where the material is heated and the DNA is split into two separate chains.
The second step is annealing or slight cooling of the material and adding primers to it that can recognize the desired regions in the DNA molecule and bind to them.
The third step is elongation– occurs after 2 primers are attached to each of the DNA strands. During the process, the DNA fragment of the pathogen is completed, and its copy is formed.

These cycles are repeated like a “chain reaction,” each time leading to doubling of copies of a specific DNA fragment (for example, the segment where a specific virus is programmed). Within a few hours, many copies of the DNA fragment are formed, and their presence in the sample is detected. After this, the results are analyzed and compared with data from a database of various types of pathogens to determine the type of infection.

Read below about decoding the results and conclusion based on the PCR reaction.

Decoding the results

The final result of the study is issued 1 – 2 days after the submission of biological material. Often - already on the first day after the analysis.

Qualitative analysis

Negative the result means that no traces of infectious agents were found in the substance submitted for testing.
Positive the result means the detection of pathogenic viruses or bacteria in a biological sample with a very high degree of accuracy at the time of submission of the material.

If the result is positive, but no signs of increased infection are detected, this state of the body is called asymptomatic “healthy carriage.” Most often observed when taking biomaterial from a certain place (cervical canal, urethra, oral cavity) in viral diseases. In this case, treatment is not required, but constant medical supervision is required, since there is a possibility of:

spread of the virus from carriers and infection of healthy people;
activation of the process and transition of the disease to a chronic form.

However, if the blood test is positive, this indicates that the infection has struck the body, and this is no longer a carrier state, but a pathology that requires immediate specific therapy.

Quantitative Analysis

The quantitative result is determined by a specialist specifically for a specific type of infection. Based on it, it is possible to assess the degree of development and stage of the disease, which makes it possible to promptly prescribe the correct treatment.

average cost

Prices for polymerase chain reaction are determined by: the type of research, the difficulty of identifying the pathogen, the difficulty of collecting biological material, the type of analysis (qualitative or quantitative), and the price level in the laboratory.

On the other hand, when studying PCR, it is possible to identify several pathogens at once when collecting one type of material for analysis. This allows you to save on other laboratory tests.

Approximate cost of PCR analysis in rubles:

gonococcus, gardnerella, trichomonas vaginalis – from 180
chlamydia trachomatis – from 190
papillomavirus – from 380 to 500
biocenosis of the urogenital tract in women (quantitative and qualitative assessment of microflora) – from 800.

Even more useful information regarding PCR testing is contained in the video below:

PRINCIPLE OF THE METHOD (molecular biological basis)

Among the wide variety of hybridization methods for DNA analysis, the PCR method is most widely used in clinical laboratory diagnostics.

Principle of the method polymerase chain reaction (PCR)(Polymerase chain reaction (PCR)) was developed by Kary Mullis (Cetus, USA) in 1983. and is currently widely used both for scientific research and for diagnostics in practical healthcare and the State Sanitary and Epidemiological Surveillance Service (genotyping, diagnosis of infectious diseases).

The PCR method is based on a natural process - complementary completion of the DNA matrix, carried out using the enzyme DNA polymerase. This reaction is called DNA replication.

Natural DNA replication includes several stages:

1) Denaturation of DNA(unwinding of the double helix, divergence of DNA strands);

2) Formation of short double-stranded DNA sections(primers necessary to initiate DNA synthesis);

3) Synthesis of a new DNA strand(complementary completion of both threads)

This process can be used to obtain copies short sections of DNA specific for specific microorganisms, those. carry out a targeted search for such specific areas, which is the goal of gene diagnostics to identify pathogens of infectious diseases.

Discovery of thermostable DNA polymerase (Taq polymerase) from thermophilic bacteria Thermis aquaticus, the optimum of which is in the region of 70-72°C, made it possible to make the process of DNA replication cyclic and use it for work in vitro. The creation of programmable thermostats (amplifiers), which carry out cyclic temperature changes according to a given program, has created the prerequisites for the widespread introduction of the PCR method into the practice of laboratory clinical diagnostics. With multiple repetitions of synthesis cycles, an exponential increase in the number of copies of a specific DNA fragment occurs, which makes it possible to obtain a sufficient number of DNA copies from a small amount of analyzed material, which may contain single microorganism cells, to identify them by electrophoresis.

Complementary chain completion does not begin at any point in the DNA sequence, but only in certain starting blocks - short double-stranded sections. By attaching such blocks to specific sections of DNA, it is possible to direct the process of synthesis of a new chain only in this section, and not along the entire length of the DNA chain. To create starting blocks in given DNA sections, two oligonucleotide primers (20 nucleotide pairs), called primers. Primers are complementary to DNA sequences on the left and right boundaries of a specific fragment and are oriented in such a way that the completion of a new DNA chain occurs only between them.

Thus, PCR is a multiple increase in the copy number (amplification) of a specific DNA region catalyzed by the enzyme DNA polymerase.

To carry out amplification, the following components are required:

Mixture of deoxynucleotide triphosphates (dNTPs)(a mixture of four dNTPs, which are the material for the synthesis of new complementary DNA strands)

Enzyme Taq polymerase(a thermostable DNA polymerase that catalyzes the elongation of primer chains by sequentially adding nucleotide bases to the growing chain of synthesized DNA).

Buffer solution(reaction medium containing Mg2+ ions necessary to maintain enzyme activity)
To identify 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 revertase (reverse transcriptase).

To obtain a sufficient number of copies of the desired characteristic DNA fragment, amplification includes several (20-40) cycles.

Each amplification cycle includes 3 stages occurring in different temperature conditions

Stage 1: DNA denaturation(unbraiding of the double helix). Occurs at 93-95°C for 30-40 seconds.

Stage 2: Attaching primers (annealing). The joining of primers 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: Completion of DNA chains. Complementary addition of DNA strands 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 chains is deoxyribonucleotide triphosphates (dNTPs) added to the solution. The synthesis process is catalyzed by the enzyme thermostable DNA polymerase (Taq polymerase) and takes place at a temperature of 70-72°C. The synthesis time is 20-40 seconds.

The new DNA chains formed in the first amplification cycle serve as templates for the second amplification cycle, in which the desired specific DNA fragment (amplicon) is formed. (see Fig. 2). In subsequent amplification cycles, amplicons serve as a template for the synthesis of new chains. Thus, amplicons accumulate in solution according to the formula 2n, where n is the number of amplification cycles. Therefore, even if the initial solution initially contained only one double-stranded DNA molecule, then in 30-40 cycles about 108 amplicon molecules accumulate in the solution. This 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 (amplifier), which, according to a given program, automatically changes temperatures according to the number of amplification cycles.

STAGES OF PCR ANALYSIS

The PCR method, as a tool for laboratory diagnosis of infectious diseases, is based on the detection of a small DNA fragment of the pathogen (several hundred base pairs), specific only for a given microorganism, using a polymerase chain reaction to accumulate the desired fragment.
The analysis method using the PCR method includes three stages:

1. Isolation of DNA (RNA) from a clinical sample

2. Amplification of specific DNA fragments
3. Detection of amplification products

DNA (RNA) Isolation
At this stage of the analysis, the clinical sample is subjected to special treatment, as a result of which lysis of cellular material occurs, removal of protein and polysaccharide fractions, and obtaining a solution of DNA or RNA free from
inhibitors and ready for further amplification.
The choice of DNA (RNA) isolation technique is mainly determined by the nature of the clinical material being processed.

Amplification of specific DNA fragments
At this stage, short specific DNA fragments accumulate in the amount necessary for their further detection. Most methods for determining specific genome fragments use the so-called. “a classic version of targeted PCR. To increase the specificity and sensitivity of the analysis, some methods use the “nested” PCR method, which uses 2 pairs of primers (“external” - for stage 1, and “internal” - for stage 2).

Detection of amplification products
In most methods, at this stage, the mixture of amplification products obtained at the 2nd stage is separated by horizontal electrophoresis in an agarose gel. Before electrophoretic separation, a solution of ethidium bromide is added to the amplification mixture, which forms strong interstitial compounds with double-stranded DNA fragments. These compounds are capable of fluorescing under UV irradiation, which is recorded in the form of orange-red luminous bands after electrophoretic separation of the amplification mixture in an agarose gel.

As an alternative to the electrophoretic detection method, which has some disadvantages: subjectivity in reading the results, limitations on determining the DNA of various microorganisms in one reaction, it can be proposed hybridization detection schemes. In these schemes, the DNA fragment formed as a result of amplification hybridizes (forms 2-stranded complexes - “hybrids”) with a specific oligonucleotide probe. Registration of such complexes can be carried out colorimetrically or fluorimetrically. SPF "Litekh" has created detection kits based on hybridization with fluorimetric registration of results

ADVANTAGES OF THE PCR METHOD as a method for diagnosing infectious diseases:

- Direct determination of the presence of pathogens

Many traditional diagnostic methods, such as enzyme immunoassay, identify marker proteins that are waste products of infectious agents, which provides only indirect evidence of the presence of infection. Identification of a specific section of pathogen DNA by PCR gives a direct indication of the presence of the infectious agent.

- High specificity
The high specificity of the PCR method is due to the fact that a unique DNA fragment, characteristic only for a given pathogen, is detected in the material under study. Specificity is determined by the nucleotide sequence of the primers, which eliminates
the possibility of obtaining false results, in contrast to the enzyme immunoassay method, where errors due to cross-reacting antigens are common.

- High sensitivity
The PCR method allows you to detect even single cells of bacteria or viruses. PCR diagnostics detects the presence of pathogens of infectious diseases in cases where other methods (immunological, bacteriological,
microscopic) this cannot be done. The sensitivity of PCR analysis is 10-1000 cells per sample (the sensitivity of immunological and microscopic tests is 103-105 cells).

-University of the procedure for identifying various pathogens
The material for research using the PCR method is the DNA of the pathogen. The method is based on identifying a fragment of DNA or RNA that is specific to a particular organism. The similarity of the chemical composition of all nucleic acids allows the use of unified methods for conducting laboratory research. This makes it possible to diagnose several pathogens from one biosample. Various biological secretions (mucus, urine, sputum), scrapings of epithelial cells, blood, and serum can be used as test material.

- High speed of obtaining analysis results
PCR analysis does not require isolating and growing a culture of the pathogen, which takes a lot of time. A unified method for processing biomaterial and detecting reaction products, and automation of the amplification process make it possible to conduct a complete analysis in 4-4.5 hours.

It should be noted that the PCR method can detect pathogens not only in clinical material obtained from a patient, but also in material obtained from environmental objects (water, soil, etc.)

APPLICATION OF THE PCR METHOD IN PRACTICAL HEALTH CARE

The use of the PCR method for diagnosing infectious diseases of both bacterial and viral nature is of enormous importance for solving many problems of microbiology and epidemiology. The use of this method also contributes to the development of basic research in the field of studying chronic and poorly understood infectious diseases.

The most effective and economically feasible use of the method is in:

urogynecological practice- to detect chlamydia, ureaplasmosis, gonorrhea, herpes, gardnerellosis, mycoplasma infection;

in pulmonology- for differential diagnosis of viral and bacterial pneumonia, tuberculosis;

in gastroenterology- to identify helicobacteriosis;

in the infectious diseases clinic- as an express method for diagnosing salmonellosis, diphtheria, viral hepatitis B, C and G;

in hematology- to detect cytomegalovirus infection, oncoviruses.

1. Polymerase chain reaction (PCR)

2. The principle of the polymerase chain reaction method

2.1 The presence of a number of components in the reaction mixture

2.2 Cyclic temperature regime

2.3 Basic principles for selecting primers

2.4 Plateau effect

3. Stages of PCR

3.2 Amplification

3.4.1 Positive controls

3.4.2 Internal controls

4.1 Qualitative analysis

4.1.2 Detection of RNA molecules

3.1 Preparation of a biological sample

To isolate DNA, various techniques are used depending on the tasks at hand. Their essence lies in the extraction (extraction) of DNA from a biological preparation and the removal or neutralization of foreign impurities to obtain a DNA preparation with a purity suitable for PCR.

The method for obtaining a pure DNA preparation described by Marmur is considered standard and has already become classical. It includes enzymatic proteolysis followed by deproteinization and reprecipitation of DNA with alcohol. This method allows you to obtain a pure DNA preparation. However, it is quite labor-intensive and involves working with such aggressive and strong-smelling 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 sorption of DNA on a carrier (glass beads, diatomaceous earth, glass milk, etc.). After washing, DNA remains in the sample, sorbed on the carrier, from which it is easily removed using an elution buffer. The method is convenient, technologically advanced and suitable for preparing a sample for amplification. However, DNA loss is possible due to irreversible sorption on the carrier, as well as during numerous washings. This is especially important when working with small amounts of DNA in a sample. In addition, even trace amounts of GuSCN can inhibit PCR. Therefore, when using this method, the correct choice of sorbent and careful adherence to 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 sorb DNA, but rather, impurities that interfere with the reaction. As a rule, this technology includes two stages: boiling the sample and sorption 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 sample preparation method should be taken into account with an understanding of the purposes of the intended analysis.

3.2 Amplification

To carry out the amplification reaction, it is necessary to prepare a reaction mixture and add the analyzed DNA sample to it. It is important to take into account some features of primer annealing. The fact is that, as a rule, the analyzed biological sample contains 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, forming primer dimers. Both lead to a significant consumption of primers for the synthesis of by-products (non-specific) reaction products and, as a result, significantly reduce the sensitivity of the system. This makes it difficult or impossible to read the reaction results during electrophoresis.

3.3 Evaluation of reaction results

To correctly evaluate PCR results, it is important to understand that this method is not quantitative. Theoretically, amplification products of single target DNA molecules can be detected using electrophoresis after 30-35 cycles. However, in practice, this is only done in cases where the reaction takes place under conditions close to ideal, which is not often the case in life. The degree of purity of the DNA preparation has a particularly great influence on the efficiency of amplification, i.e. the presence in the reaction mixture of certain inhibitors, which in some cases can be extremely difficult to get rid of. Sometimes, due to their presence, even tens of thousands of target DNA molecules cannot be amplified. Thus, there is often no direct relationship between the initial amount of target DNA and the final amount of amplification products.

3.3.1 Horizontal electrophoresis method

Various methods are used to visualize amplification results. The most common method today is electrophoresis, based on the separation of DNA molecules by size. To do this, prepare a plate of agarose gel, which is agarose solidified after melting in an electrophoresis buffer at a concentration of 1.5-2.5% with the addition of a special DNA dye, for example, ethidium bromide. Solidified agarose forms a spatial lattice. When pouring using combs, special wells are formed in the gel, into which amplification products are subsequently added. The gel plate is placed in a horizontal gel electrophoresis apparatus and a constant voltage source is connected. Negatively charged DNA begins to move in the gel from minus to plus. In this case, shorter DNA molecules move faster than longer ones. The speed of DNA movement in the gel is affected by the concentration of agarose, the electric field strength, temperature, the composition of the electrophoresis buffer and, to a lesser extent, the GC composition of the DNA. All molecules of the same size move at the same speed. The dye is incorporated (intercalated) by planar groups into DNA molecules. After completion of electrophoresis, which lasts from 10 minutes to 1 hour, the gel is placed on a transilluminator filter that emits light in the ultraviolet range (254 - 310 nm). Ultraviolet energy absorbed by DNA at 260 nm is transferred to the dye, causing it to fluoresce in the orange-red region of the visible spectrum (590 nm).

The brightness of the amplification product bands may vary. However, this cannot be related to the initial amount of target DNA in the sample.

3.3.2 Vertical electrophoresis method

The method of vertical electrophoresis is fundamentally similar to horizontal electrophoresis. Their difference is that in this case, polyacrylamide gels are used instead of agarose. It is carried out in a special chamber for vertical electrophoresis. Polyacrylamide gel electrophoresis has greater resolution compared to agarose electrophoresis and allows one to distinguish DNA molecules of different sizes with an accuracy of one nucleotide. Preparation of polyacrylamide gel is somewhat more complicated than agarose gel. In addition, acrylamide is a toxic substance. Since the need to determine the size of an amplification product with an accuracy of 1 nucleotide rarely arises, the method of horizontal electrophoresis is used in routine work.

3.4 Monitoring the progress of the amplification reaction

3.4.1 Positive controls

A DNA preparation of the desired microorganism is used as a “positive control”. Nonspecific amplicons differ in size from amplicons formed as a result of amplification with a control DNA preparation. Nonspecific products may be either larger or smaller in size compared to the positive control. In the worst case, these sizes may coincide and are read as positive in electrophoresis.

To control the specificity of the resulting amplification product, you can use hybridization probes (DNA sections located within the amplified sequence), labeled with enzyme tags or radioactive isotopes and interacting with DNA in accordance with the same principles as primers. This significantly complicates and lengthens the analysis, and its cost increases significantly.

3.4.2 Internal controls

It is necessary to monitor the progress of amplification in each tube with the reaction mixture. For this purpose, additional, so-called “internal control” is used. It is any DNA preparation that is dissimilar to the DNA of the desired microorganism. If an internal control is added to the reaction mixture, it will become the same target for primer annealing as the chromosomal DNA of the desired infectious agent. The size of the internal control amplification product is selected so that it is 2 or more times larger than the amplicons formed from amplification of the desired microorganism DNA. As a result, if internal control DNA is added to the reaction mixture along with the test sample, then regardless of the presence of a microorganism in the biological sample, the internal control will cause the formation of specific amplicons, but significantly longer (heavier) than the amplicon of the microorganism. The presence of heavy amplicons in the reaction mixture will indicate the normal progress of the amplification reaction and the absence of inhibitors. If amplicons of the required size are not formed, but also internal control amplicons are not formed, we can conclude that there are undesirable impurities in the analyzed sample that should be eliminated, but not about the absence of the desired DNA.

Unfortunately, despite all the attractiveness of this approach, it has a significant flaw. If the desired DNA is present in the reaction mixture, then the efficiency of its amplification sharply decreases due to competition with internal control for primers. This is especially important at low DNA concentrations in the test sample, which can lead to false negative results.

However, provided that the problem of competition for primers is solved, this method of monitoring amplification efficiency will certainly be very useful.

4. Methods based on polymerase chain reaction

4.1 Qualitative analysis

The classical method of performing PCR, the principles of which were outlined above, has been developed in some modifications aimed at overcoming the limitations of PCR and increasing the efficiency of the reaction.

4.1.1 Method of performing PCR using a “hot start”

To reduce the risk of the formation of nonspecific amplification reaction products, an approach called “Hot-start” is used. Its essence is to prevent the start of the reaction until conditions in the test tube are achieved that ensure specific annealing of the primers.

The fact is that, depending on the GC composition and size, primers have a certain melting temperature (Tm). If the system temperature exceeds Tm, the primer is unable to adhere to the DNA strand and denatures. Subject to optimal conditions, i.e. With an annealing temperature close to the melting temperature, the primer forms a double-stranded molecule only if it is fully complementary and thus ensures the specificity of the reaction.

There are various options for implementing a “hot start”:

Adding Taq polymerase to the reaction mixture during the first cycle after heating the tube to the denaturation temperature.

Separation of the ingredients of the reaction mixture by a paraffin layer into layers (in the lower part - primers, in the upper part - Taq polymerase and DNA targets), which are mixed when the paraffin melts (~ 65-75 0 C).

Use of monoclonal antibodies to Taq polymerase. The enzyme bound by monoclonal antibodies becomes active only after the first denaturation stage, when the monoclonal antibodies are irreversibly denatured and release the active sites of Taq polymerase.

In all of the above cases, even if nonspecific annealing occurred before the start of temperature cycling, elongation does not occur, and upon heating, the primer-DNA complexes are denatured, so nonspecific products are not formed. Subsequently, the temperature in the test tube does not fall below the melting temperature, which ensures the formation of a specific amplification product.

4.1.2 Detection of RNA molecules

The possibility of using RNA as a target for PCR significantly expands the range of applications of this method. For example, the genomes of many viruses (hepatitis C, influenza virus, picornaviruses, etc.) are represented by RNA. Moreover, in their life cycles there is no intermediate phase of transformation into DNA. To detect RNA, it must first be converted into DNA form. To do this, reverse transcriptase is used, which is isolated from two different viruses: avian myeloblastosis virus and Moloney murine leukemia virus. The use of these enzymes is associated with some difficulties. First of all, they are thermolabile and therefore can be used at temperatures no higher than 42° C. Since at this temperature RNA molecules easily form secondary structures, the reaction efficiency decreases markedly and, according to various estimates, is approximately 5%. Attempts are being made to circumvent this drawback by using a thermostable polymerase obtained from the thermophilic microorganism Thermus Thermophilus, which exhibits transcriptase activity in the presence of Mn 2+ , as a reverse transcriptase. It is the only known enzyme capable of exhibiting both polymerase and transcriptase activity.

To carry out a reverse transcription reaction, the reaction mixture, just like in PCR, must contain primers as a primer and a mixture of 4 dNTPs as a building material.

After performing a reverse transcription reaction, the resulting cDNA molecules can serve as a target for PCR

5. Organization of the technological process of performing PCR

The potentially high sensitivity of the polymerase chain reaction makes particularly careful design of the PCR laboratory absolutely necessary. This is due to the most acute problem of the method - contamination.

Contamination is the entry of specific DNA molecules from the external environment into the reaction mixture that can serve as targets in the amplification reaction and give false-positive results.

There are several ways to combat this unpleasant phenomenon. One of them is the use of the enzyme N-uracil glycosylase (UG). This method is based on the ability of UG to cleave DNA molecules with embedded uracil. The amplification reaction is carried out using a dNTP mixture in which dTTP is replaced by uracil, and after thermal cycling, all amplicons formed in the test tube will contain uracil. If CG is added to the reaction mixture before amplification, then the amplicons entering the reaction mixture will be destroyed, while the native DNA will remain intact and will subsequently serve as a target for amplification.

Thus, this method only to some extent eliminates the source of contamination and does not guarantee against false positive results.

Another way to combat the results of contamination is to significantly reduce the number of reaction cycles (up to 25-30 cycles). But even with this approach, the risk of obtaining false-positive results is high, since in this case, in the absence of inhibitors, it is easy to obtain an amplification product due to contamination.

Thus, despite the benefits of preamplification measures aimed at inactivating DNA molecules that cause false-positive results, the most radical remedy is a well-thought-out laboratory organization.

Conclusion

The PCR method is currently most widely used as a method for diagnosing various infectious diseases. PCR allows you to identify the etiology of an 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. Today there is almost no infectious agent that cannot be detected using PCR.

However, at that time this idea remained unclaimed. The polymerase chain reaction was rediscovered in 1983 by Kary Mullis. His goal was to create a method that would allow DNA to be amplified through multiple successive duplications of the original DNA molecule using the enzyme DNA polymerase. 7 years after the publication of this idea, in 1993, Mullis received the Nobel Prize for it.

At the beginning of using the method, after each heating-cooling cycle, DNA polymerase had to be added to the reaction mixture, since it was quickly inactivated at the high temperature required to separate the strands of the DNA helix. The procedure was very inefficient and required a lot of time and enzyme. In 1986 it was significantly improved. It has been proposed to use DNA polymerases from thermophilic bacteria. These enzymes turned out 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 aquaticus and named Taq-polymerase. The disadvantage of this polymerase is that the probability of introducing an erroneous nucleotide is quite high, since this enzyme does not have error correction mechanisms (3"→5" exonuclease activity). Polymerases Pfu And Pwo, isolated from archaea, have such a mechanism; their use significantly reduces the number of mutations in DNA, but the speed of their work (processivity) is lower than that of Taq. Nowadays mixtures are used Taq And Pfu to achieve both high polymerization speed and high copying accuracy.

At the time of the invention of the method, Mullis worked for the Cetus Corporation, which patented the PCR method. In 1992, Cetus sold the rights to the method and the patent to use Taq-polymerase company Hoffmann-La Roche (en: Hoffmann-La Roche) for $300 million. However, it turned out that Taq-polymerase was characterized by Russian biochemist Alexei Kaledin in 1980, and Promega tried to force Roche to give up exclusive rights to this enzyme. The US patent for the PCR method expired in March 2005.

Carrying out PCR

The method is based on repeated selective copying of a certain section of DNA using enzymes under artificial conditions ( in vitro). In this case, only the section that satisfies the specified conditions is copied, and only if it is present in the sample under study. Unlike DNA amplification in living organisms (replication), relatively short sections of DNA are amplified using PCR. In a conventional PCR process, the length of the copied DNA sections is no more than 3000 base pairs (3 kbp). Using a mixture of various polymerases, using additives and under certain conditions, the length of a PCR fragment can reach 20-40 thousand nucleotide pairs. This is still significantly less than the length of the chromosomal DNA of a eukaryotic cell. For example, the human genome consists of approximately 3 billion base pairs.

Reaction components

To carry out PCR in the simplest case, the following components are required:

DNA matrix, containing the section of DNA that needs to be amplified.
Two primers, complementary to the opposite ends of different strands of the desired DNA fragment.
Thermally stable DNA polymerase- an enzyme that catalyzes the polymerization reaction of DNA. Polymerase for use in PCR must remain active at high temperatures for a long time, so enzymes isolated from thermophiles are used - Thermus aquaticus(Taq polymerase), Pyrococcus furiosus(Pfu polymerase), Pyrococcus woesei(Pwo polymerase) and others.
Deoxynucleoside triphosphates(dATP, dGTP, dCTP, dTTP).
Mg 2+ ions necessary for the operation of the polymerase.
Buffer solution, providing the necessary reaction conditions - pH, ionic strength of the solution. Contains salts, bovine serum albumin.

To avoid evaporation of the reaction mixture, add high-boiling oil, such as Vaseline, to the test tube. If you are using a thermal cycler with a heated lid, this is not required.

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

Primers

The specificity of PCR is based on the formation of complementary complexes between the template and primers, short synthetic oligonucleotides 18-30 bases long. Each primer is complementary to one of the strands of the double-stranded template and limits the beginning and end of the amplified region.

After hybridization of the template with the primer (annealing), the latter serves as a primer for DNA polymerase during the synthesis of the complementary template strand (see).

The most important characteristic of primers is the melting temperature (Tm) of the primer-matrix complex. T m is the temperature at which half of the DNA templates form a complex with the oligonucleotide primer. The melting temperature can be approximately determined by the formula , where n X is the number of nucleotides X in the primer. If the length and nucleotide composition of the primer or annealing temperature are incorrectly selected, the formation of partially complementary complexes with other regions of the template DNA is possible, which can lead to the appearance of nonspecific products. The upper limit of the melting temperature is limited by the optimum temperature of action of the polymerase, the activity of which decreases at temperatures above 80 °C.

When choosing primers, it is advisable to adhere to the following criteria:

Amplifier

Rice. 1: Cycler for PCR

PCR is carried out in a thermal cycler - a device that provides periodic cooling and heating of test tubes, usually with an accuracy of at least 0.1 °C. Modern cyclers allow you to set complex programs, including the ability to “hot start”, Touchdown PCR (see below) and subsequent storage of amplified molecules at 4 °C. For real-time PCR, devices equipped with a fluorescent detector are produced. There are also devices with an automatic lid and a compartment for microplates, which allows them to be integrated into automated systems.

Progress of the reaction

Photograph of a gel containing marker DNA (1) and PCR reaction products (2,3). The numbers show the length of DNA fragments in nucleotide pairs

Typically, PCR involves 20-35 cycles, each of which consists of three stages (Fig. 2).

Denaturation

The double-stranded DNA template is heated to 94-96°C (or to 98°C if a particularly thermostable polymerase is used) for 0.5-2 minutes to separate the DNA strands. This stage is called denaturation, since the hydrogen bonds between the two DNA strands are destroyed. Sometimes, before the first cycle (before adding polymerase), the reaction mixture is preheated for 2-5 minutes. for complete denaturation of the template and primers. This technique is called hot start, it allows you to reduce the amount of nonspecific reaction products.

Annealing

Once the strands have separated, the temperature is lowered to allow the primers to bind to the single-stranded template. This stage is called annealing. The annealing temperature depends on the composition of the primers and is usually selected 4-5°C below their melting temperature. Stage time - 0.5-2 minutes. An incorrect choice of annealing temperature leads either to poor binding of primers to the template (at too high a temperature) or to binding in the wrong place and the appearance of nonspecific products (at too low a temperature).

Elongation

Types of PCR

If the nucleotide sequence of the template is partially known or unknown at all, you can use degenerate primers, the sequence of which contains degenerate positions in which any bases can be located. For example, the primer sequence could be: ...ATH..., where H is A, T or C.

Application of PCR

PCR is used in many areas for testing and scientific experiments.

Forensics

PCR is used to compare so-called “genetic fingerprints.” A sample of genetic material from the crime scene is required - blood, saliva, semen, hair, etc. This is compared with the genetic material of the suspect. A very small amount of DNA is enough, theoretically one copy. The DNA is broken down into fragments and then amplified using PCR. The fragments are separated using DNA electrophoresis. The resulting picture of the arrangement of DNA bands is called genetic fingerprint(English) genetic fingerprint).

Establishing paternity

Rice. 3: Results of electrophoresis of DNA fragments amplified by PCR. (1) Father. (2) Child. (3) Mother. The child inherited some features of the genetic imprint of both parents, resulting in a new, unique imprint.

Although genetic fingerprints are unique (except in the case of identical twins), family relationships can still be established by making several fingerprints (Figure 3). The same method can be applied, slightly modified, to establish evolutionary relatedness 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 identify mutations. Viral infections can be detected immediately after infection, weeks or months before symptoms appear.

Personalized medicine

It is known that most drugs do not act on all patients for whom they are intended, but only on 30-70% of their number. In addition, many medications turn out to be toxic or allergenic for some patients. The reasons for this are partly due to 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 (a liver protein responsible for metabolizing foreign substances) may be more active, in another - less. In order to determine what type of cytochrome a given patient has, it is proposed to conduct a PCR analysis before using the medicine. This analysis is called preliminary genotyping. prospective genotyping).

Gene cloning

Gene cloning (not to be confused with cloning of organisms) 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 vector- a DNA fragment that transfers a foreign gene into the same or another organism convenient for cultivation. For example, plasmids or viral DNA are used as vectors. The insertion of genes into a foreign organism is usually used to produce the product of that gene - RNA or, most often, a protein. In this way, many proteins are obtained in industrial quantities for use in agriculture, medicine, etc.

Rice. 4: Gene cloning using a plasmid. .
(1) Chromosomal DNA of organism A. (2) PCR. (3) Many copies of the gene of organism A. (4) Insertion of the gene into a plasmid. (5) Plasmid with the gene of organism A. (6) Introduction of the plasmid into organism B. (7) Multiplication of the number of copies of the gene of organism A in organism B.

DNA sequencing

In the sequencing method using dideoxynucleotides labeled with a fluorescent label or radioactive isotope, 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 the synthesized chains are separated in the gel.

Mutagenesis

Currently, PCR has become the main method for carrying out mutagenesis. The use of PCR has made it possible to simplify and speed up the mutagenesis procedure, as well as make it more reliable and reproducible.