Received the Nobel Prize.

At the beginning of the use of the method, after each heating-cooling cycle, DNA polymerase had to be added to the reaction mixture, since it was inactivated at the high temperature necessary to separate the strands of the DNA helix. The reaction procedure was relatively inefficient, requiring a lot of time and enzyme. In 1986, the polymerase chain reaction method was significantly improved. It has been proposed to use DNA polymerases from thermophilic bacteria. These enzymes proved to be thermostable and were able to withstand many reaction cycles. Their use made it possible to simplify and automate PCR. One of the first thermostable DNA polymerases was isolated from bacteria Thermus 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 lacks 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. Currently using mixtures Taq and pfu to achieve both high polymerization speed and high copy accuracy.

At the time of the invention of the method, Cary Mullis worked as a synthetic chemist (he synthesized oligonucleotides, which were then used to detect point mutations by hybridization with genomic DNA) at Cetus Corporation, which patented the PCR method. In 1992, Cetus sold the rights to the method and the patent to use Taq polymerase company Hoffman-La Roche for $300 million. However, it turned out that Taq-polymerase was characterized by Soviet biochemists A. Kaledin, A. Slyusarenko and S. Gorodetsky in 1980, and also 4 years before this Soviet publication, that is, in 1976, by American biochemists Alice Chien, David B. Edgar and John M. Trela. In this regard, the company Promega (Promega) tried in court to force Roche to give up exclusive rights to this enzyme. The American patent for the PCR method expired in March 2005.

Conducting PCR

The method is based on multiple selective copying of a certain DNA region with the help of enzymes under artificial conditions ( in vitro). In this case, only the area that satisfies the specified conditions is copied, and only if it is present in the sample under study. In contrast to DNA amplification in living organisms (replication), relatively short sections of DNA are amplified using PCR. In a conventional PCR process, the length of the replicated DNA regions is no more than 3000 base pairs (3 kbp). With the help of a mixture of different polymerases, with the use of additives and under certain conditions, the length of the PCR fragment can reach 20-40 thousand base pairs. This is still much less than the length of the chromosomal DNA of a eukaryotic cell. For example, the human genome is approximately 3 billion base pairs long.

Reaction components

For PCR, in the simplest case, the following components are required:

• DNA template, which contains the section of DNA that needs to be amplified.
• Two primers, complementary to opposite ends of different strands of the desired DNA fragment.
• thermostable DNA polymerase is an enzyme that catalyzes the polymerization of DNA. The polymerase for use in PCR must remain active at high temperature for a long time, therefore, enzymes isolated from thermophiles are used - Thermus aquaticus(Taq polymerase), Pyrococcus furiosus(Pfu polymerase), Pyrococcus woesei(Pwo-polymerase) and others.
• Deoxyribonucleoside triphosphates(dATP, dGTP, dCTP, dTTP).
• Mg 2+ ions necessary for polymerase to work.
• 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, a high-boiling oil, such as vaseline, is added to the test tube. If a heated lid cycler 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.

Primers

The specificity of PCR is based on the formation of complementary complexes between template and primers, short synthetic oligonucleotides 18-30 bases long. Each of the primers is complementary to one of the chains 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 in the synthesis of the complementary strand of the template (see).

The most important characteristic of primers is the melting point (Tm) of the primer-matrix complex.

T m is the temperature at which half of the DNA templates forms a complex with the oligonucleotide primer. The average formula for calculating T m for a short oligonucleotide (and for long DNA fragments), taking into account the concentration of K + ions and DMSO:

where L is the number of nucleotides in the primer, K + is the molar concentration of potassium ions, G+C is the sum of all guanines and cytosines.

If the length and nucleotide composition of the primer or the annealing temperature are chosen incorrectly, 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 drops at temperatures above 80 °C.

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

amplifier

Rice. one: PCR cycler

PCR is carried out in an amplifier - 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 possibility of "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. Instruments are also available with an automatic lid and microplate compartment, allowing them to be integrated into automated systems.

Reaction progress

Photograph of a gel containing marker DNA (first and last slots) and PCR products

Usually, during PCR, 20-35 cycles are performed, each of which consists of three stages (Fig. 2).

Denaturation

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 min to allow the DNA strands to separate. This stage 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 2–3 min to completely denature the template and primers. Such an approach is called hot start, it allows to reduce the amount of non-specific reaction products.

Annealing

When the strands are 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 chosen equal to the melting temperature of the primers. 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). The time of the annealing stage is 30 sec, at the same time, during this time the polymerase already has time to synthesize several hundred nucleotides. Therefore, it is recommended to select primers with a melting point above 60 °C and carry out annealing and elongation at the same time, at 60-72 °C.

Elongation

DNA polymerase replicates the template strand using the primer as a primer. This is the stage elongation. The polymerase starts the synthesis of the second strand from the 3" end of the primer that has bound to the template and moves along the template, synthesizing a new strand in the direction from the 5" to the 3" end. 72 ° C. The elongation time depends on both the type of DNA polymerase and the length of the amplified fragment.Typically, the elongation time is taken to be one minute for every thousand base pairs.After all the cycles, an additional step is often carried out final elongation to complete all single-stranded fragments. This stage lasts 7-10 minutes.

Rice. 2: Schematic representation of the first PCR cycle. (1) Denaturation at 94-96°C. (2) Annealing at 68°C (for example). (3) Elongation at 72°C (P=polymerase). (4) The first cycle is finished. The two resulting DNA strands serve as a template for the next cycle, so the amount of template DNA doubles during each cycle.

The amount of the specific reaction product (limited by primers) theoretically increases in proportion to 2n - 2n, where n is the number of reaction cycles. In fact, the efficiency of each cycle can be less than 100%, therefore, in reality, P ~ (1 + E) n, 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. In the last cycles of the reaction, growth slows down, this is called the "plateau effect".

Varieties of PCR

• Nested PCR(Nested PCR (eng.) ) - used to reduce the number of by-products of the reaction. Use two pairs of primers and carry out two consecutive reactions. The second pair of primers amplifies the DNA region within the product of the first reaction.
• Inverted PCR(Inverse PCR (English) ) - is used if only a small area within the desired sequence is known. This method is especially useful when it is necessary to determine neighboring sequences after DNA has been inserted into the genome. For the implementation of inverted PCR, a series of cuts of DNA with restriction enzymes is carried out, followed by the connection of fragments (ligation). As a result, known fragments are at both ends of the unknown region, after which PCR can be carried out as usual.
• reverse transcription PCR(Reverse Transcription PCR, RT-PCR (English) ) - used to amplify, isolate or identify a known sequence from an RNA library. Before conventional PCR, a single-stranded DNA molecule is synthesized on the mRNA template using reversetase and a single-stranded cDNA is obtained, which is used as a template for PCR. This method often determines where and when these genes are expressed.
• Asymmetric PCR(English) Asymmetric PCR) - is carried out when it is necessary to amplify mainly one of the chains of the original DNA. Used in some sequencing and hybridization analysis techniques. PCR is carried out as usual, except that one of the primers is taken in large excess. Modifications of this method is English. L inear- A after- T he- E xponential-PCR (LATE-PCR), which uses primers with different concentrations, and the low concentration primer is selected with a higher (melting point) than the high concentration primer. PCR is carried out at a high annealing temperature, thereby maintaining the efficiency of the reaction throughout all cycles.
• Quantitative PCR(Quantitative PCR, Q-PCR) or real time PCR- used to directly monitor the measurement of the amount of a specific PCR product in each reaction cycle. This method uses fluorescently labeled primers or DNA probes to accurately measure the amount of the reaction product as it accumulates; or a fluorescent intercalating dye is used Sybr Green I that binds to double-stranded DNA. Sybr Green I provides a simple and cost-effective option for real-time PCR detection and quantification of PCR products without the need for specific fluorescent probes or primers. During amplification, the dye SYBR Green I integrates into the minor groove of the DNA of PCR products and emits a stronger fluorescent signal than unbound dye when irradiated with a blue laser. SYBR Green I compatible with all currently known real-time PCR instruments. Maximum absorption for SYBR Green I is at a wavelength of 494 nm. In addition to the main one, there are two small additional absorption maxima in the dye spectrum - at 290 nm and 380 nm. Maximum emission for SYBR Green I is at a wavelength of 521 nm (green).
• Step PCR(Touchdown PCR (English) ) - using this approach, the influence of non-specific binding of primers is reduced. The first cycles are carried out at a temperature above the optimum annealing temperature, then every few cycles the annealing temperature is gradually reduced to the optimum. This is to ensure that the primer hybridizes to the complementary strand throughout its length; whereas at the optimum annealing temperature, the primer partially hybridizes to the complementary strand. Partial hybridization of the primer on genomic DNA leads to non-specific amplification if there are enough binding sites for the primer. In most cases, the first ten PCR cycles can be carried out at an annealing temperature of 72-75°C, and then immediately lowered to the optimum, for example, to 60-65°C.
• Molecular colony method(PCR in gel) Colony-PCR Colony) - acrylamide gel is polymerized with all PCR components on the surface and PCR is carried out. At points containing the analyzed DNA, amplification occurs with the formation of molecular colonies.
• PCR with rapid amplification of cDNA ends(English) Rapid amplification of cDNA ends, RACE-PCR ).
• PCR of long fragments(English) Long range PCR) - modification of PCR for amplification of extended DNA segments (10 thousand or more bases). A mixture of two polymerases is used, one of which is a Taq polymerase with high processivity (that is, capable of synthesizing a long DNA chain in one pass), and the second is a DNA polymerase with 3 "-5" exonuclease activity, usually Pfu polymerase. The second polymerase is needed to correct the errors introduced by the first, since Taq polymerase stops DNA synthesis if a non-complementary nucleotide has been added. This non-complementary nucleotide is removed by Pfu polymerase. The mixture of polymerases is taken in a ratio of 50:1 or even less than 100:1, where Taq polymerase is taken 25-100 times more in relation to Pfu polymerase.
• RAPD(English) Random Amplification of Polymorphic DNA ), PCR with random amplification of polymorphic DNA - is used when it is necessary to distinguish between organisms that are close in genetic sequence, for example, different varieties of cultivated plants, dog breeds or closely related microorganisms. This method usually uses a single small primer (about 10 bp). This primer will be partially complementary to random DNA regions of the organisms under study. By selecting the conditions (primer length, primer composition, temperature, etc.), it is possible to achieve a satisfactory difference in the PCR pattern for two organisms.
• Group-specific PCR(English) group-specific PCR) - PCR for related sequences within the same or between different species using conservative primers to these sequences. For example, the selection of universal primers for ribosomal 18S and 26S genes for amplification of a species-specific intergenic spacer: gene sequence 18S and 26S is conservative between species, so PCR between these genes will take place for all studied species. The opposite of this method is - unique PCR(English) unique PCR), in which the task is to select primers to amplify only a specific sequence among related sequences.
• PCR using hot start(English) Hot start PCR) - modification of PCR using DNA polymerase, in which polymerase activity is blocked at room temperature by antibodies or small molecules mimicking antibodies like Affibody, that is, at the time of the reaction before the first denaturation in PCR. Typically, the first denaturation is carried out at 95°C for 10 minutes.
• Virtual PCR(eng. in silico PCR, digital PCR, electronic PCR, e-PCR) - a mathematical method for computer analysis of a theoretical polymerase chain reaction using a list of primer sequences (or DNA probes) to predict potential DNA amplification of the studied genome, chromosome, circular DNA or any other piece of DNA.

If the nucleotide sequence of the template is partially known or not known at all, one can use degenerate primers, the sequence of which contains degenerate positions, which can contain any bases. For example, the primer sequence might be: …ATH…, where N - A, T or C.

Application of PCR

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

Criminalistics

PCR is used to compare so-called "genetic fingerprints". A sample of genetic material from the crime scene is needed - blood, saliva, semen, hair, etc. It is compared with the genetic material of the suspect. A very small amount of DNA is enough, theoretically - one copy. The DNA is cut into fragments, then amplified by PCR. The fragments are separated by DNA electrophoresis. The resulting picture of the 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, which gave a new, unique imprint.

Although "genetic fingerprints" are unique (except in the case of identical twins), family ties can still be established by making several such fingerprints (Fig. 3). The same method can be applied, with slight modifications, to establish evolutionary relationships among organisms.

Medical diagnostics

PCR makes it possible to significantly speed up and facilitate the diagnosis of hereditary and viral diseases. The desired gene is amplified by PCR using appropriate primers and then sequenced to determine mutations. Viral infections can be detected immediately after infection, weeks or months before symptoms of the disease appear.

Personalized medicine

Sometimes drugs are toxic or allergenic for some patients. The reasons for this are partly in individual differences in the susceptibility and metabolism of drugs and their derivatives. These differences are determined at the genetic level. For example, in one patient, a certain cytochrome (a liver protein responsible for the metabolism of foreign substances) may be more active, in another - less. In order to determine what kind of cytochrome a given patient has, it is proposed to perform a PCR analysis before using the drug. This analysis is called preliminary genotyping. 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 the gene, which is then inserted into vector- a DNA fragment that transfers a foreign gene into the same or another organism convenient for growing. As vectors, for example, plasmids or viral DNA are used. The insertion of genes into a foreign organism is usually used to obtain a product of this gene - RNA or, most often, a protein. In this way, many proteins are obtained in industrial quantities for use in agriculture, medicine, etc.

Rice. 4: Gene cloning using a plasmid.
(1) Chromosomal DNA of organism A. (2) PCR. (3) Multiple 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 copy number of the gene of organism A in organism B.

DNA sequencing

In the sequencing method using dideoxynucleotides labeled with a fluorescent label or a 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. The addition of a dideoxynucleotide to the synthesized strand terminates the synthesis, allowing the position of specific nucleotides to be determined after separation in the gel.

Mutagenesis

Currently, PCR has become the main method for conducting mutagenesis (introducing changes in the nucleotide sequence of DNA). The use of PCR made it possible to simplify and speed up the mutagenesis procedure, as well as to make it more reliable and reproducible.

For adequate and effective treatment of many infectious diseases, it is necessary to establish an accurate diagnosis in a timely manner. In solving this problem today, high-tech diagnostic methods based on molecular biology methods are involved. At the moment, the polymerase chain reaction (PCR) is already widely used in practical medicine as the most reliable laboratory diagnostic tool.

What explains the popularity of PCR at the present time?

Firstly, this method is used to identify pathogens of various infectious diseases with high accuracy.

Secondly, to monitor the effectiveness of the treatment.

In various manuals, prospectuses, articles, as well as explanations of medical specialists, we often encounter the use of incomprehensible terms and words. It is really difficult to talk about high-tech products of science in ordinary words.

What is the essence and mechanics of PCR diagnostics?

Every living organism has its own unique genes. Genes are located in the DNA molecule, which in fact is the "calling card" of each specific organism. DNA (genetic material) is a very long molecule that is made up of building blocks called nucleotides. For each pathogen of infectious diseases, they are located strictly specific, that is, in a certain sequence and combination. When it is necessary to understand whether a person has a particular pathogen, biological material (blood, urine, saliva, smear) is taken, which contains DNA or DNA fragments of a microbe. But the amount of the genetic material of the pathogen is very small, and it is impossible to say which microorganism it belongs to. To solve this problem, PCR serves. The essence of the polymerase chain reaction is that a small amount of material for research containing DNA is taken, and during the PCR process, the amount of genetic material belonging to a particular pathogen increases and, thus, it can be identified.

PCR diagnostics is a genetic study of a biomaterial.

The idea of ​​the PCR method belongs to the American scientist K. Mullins, which he proposed in 1983. However, it received wide clinical use only in the middle of the 90s of the XX century.

Let's deal with the terminology, what is it - DNA, etc. Each cell of any living being (animal, plant, human, bacteria, virus) has chromosomes. Chromosomes are the custodians of genetic information that contain the entire sequence of genes of each particular living being.

Each chromosome is made up of two strands of DNA that are twisted into a helix relative to each other. DNA is chemically deoxyribonucleic acid, which consists of structural components - nucleotides. There are 5 types of nucleotides - thymine (T), adenosine (A), guanine (G), cytosine (C) and uracil (U). Nucleotides are arranged one after another in a strict individual sequence, forming genes. One gene may consist of 20-200 such nucleotides. For example, the gene encoding insulin production is 60 base pairs long.

Nucleotides have the property of complementarity. This means that opposite adenine (A) in one strand of DNA there is always thymine (T) in the other strand, and opposite guanine (G) - cytosine (C). Schematically looks like this:
G - C
T - A
A - T
This property of complementarity is key for PCR.

In addition to DNA, RNA has the same structure - ribonucleic acid, which differs from DNA in that it uses uracil instead of thymine. RNA - is the keeper of genetic information in some viruses, which are called retroviruses (for example, HIV).

DNA and RNA molecules can "multiply" (this property is used for PCR). This happens as follows: two strands of DNA or RNA move away from each other to the sides, a special enzyme sits on each thread, which synthesizes a new chain. Synthesis proceeds according to the principle of complementarity, that is, if nucleotide A is in the original DNA chain, then T will be in the newly synthesized one, if G - then C, etc. This special "builder" enzyme needs a "seed" - a sequence of 5-15 nucleotides - to start synthesis. This "seed" is defined for each gene (chlamydia gene, mycoplasma, viruses) experimentally.

So, each PCR cycle consists of three stages. In the first stage, the so-called unwinding of DNA occurs - that is, the separation of the two strands of DNA connected to each other. In the second, the “seed” is attached to a section of the DNA strand. And, finally, the elongation of these DNA strands, which is produced by the "builder" enzyme. Currently, this entire complex process takes place in one test tube and consists of repeated cycles of reproduction of the DNA being determined in order to obtain a large number of copies that can then be detected by conventional methods. That is, from one strand of DNA, we get hundreds or thousands.

Stages of a PCR study

Collection of biological material for research

Various biological material serves as a sample: blood and its components, urine, saliva, secretions of mucous membranes, cerebrospinal fluid, discharge from wound surfaces, the contents of body cavities. All biosamples are collected with disposable instruments, and the collected material is placed in sterile plastic tubes or placed on culture media, followed by transportation to the laboratory.

The necessary reagents are added to the taken samples and placed in a programmable thermostat - a thermal cycler (amplifier). In the cycler, the PCR cycle is repeated 30-50 times, consisting of three stages (denaturation, annealing and elongation). What does this mean? Let's consider in more detail.

Stages of immediate PCR reaction, copying of genetic material

IPCR stage - Preparation of genetic material for copying.
Occurs at a temperature of 95 ° C, while the DNA strands are disconnected, and “seeds” can sit on them.

"Seeds" are manufactured industrially by various research and production associations, and laboratories buy ready-made ones. At the same time, the “seed” for detecting, for example, chlamydia, works only for chlamydia, etc. Thus, if a biomaterial is tested for the presence of a chlamydial infection, then a “seed” for chlamydia is placed in the reaction mixture; if testing the biomaterial for the Epstein-Barr virus, then the "seed" for the Epstein-Barr virus.

IIstage - Combining the genetic material of the infectious agent and the "seed".
If there is DNA of the virus or bacterium to be determined, the "seed" sits on this DNA. This primer addition process is the second step of the PCR. This stage takes place at a temperature of 75°C.

IIIstage - Copying the genetic material of the infectious agent.
This is the process of the actual elongation or reproduction of genetic material, which occurs at 72°C. An enzyme-builder approaches the “seeds” and synthesizes a new strand of DNA. With the end of the synthesis of a new DNA strand, the PCR cycle also ends. That is, in one PCR cycle, the amount of genetic material doubles. For example, in the initial sample there were 100 DNA molecules of a virus, after the first PCR cycle there will already be 200 DNA molecules of the tested virus in the sample. One cycle lasts 2-3 minutes.

In order to generate enough genetic material for identification, 30-50 PCR cycles are usually performed, which takes 2-3 hours.

Stage of identification of the propagated genetic material

The PCR itself ends here and then comes the equally significant stage of identification. For identification, electrophoresis or labeled "seeds" are used. When using electrophoresis, the resulting DNA strands are separated by size, and the presence of DNA fragments of different lengths indicates a positive result of the analysis (that is, the presence of a particular virus, bacterium, etc.). When using labeled "seeds", a chromogen (dye) is added to the final product of the reaction, as a result of which the enzymatic reaction is accompanied by the formation of a color. The development of a color indicates directly that a virus or other detectable agent is present in the original sample.

Today, using labeled "seeds", as well as appropriate software, it is possible to immediately "read" the PCR results. This is the so-called real-time PCR.

Why is PCR diagnostics so valuable?

One of the significant advantages of the PCR method is its high sensitivity - from 95 to 100%. However, these advantages must be based on the indispensable observance of the following conditions:

1. correct sampling, transportation of biological material;
2. availability of sterile, disposable instruments, special laboratories and trained personnel;
3. strict adherence to the methodology and sterility during the analysis

Sensitivity varies for different microbes detected. So, for example, the sensitivity of the PCR method for detecting the hepatitis C virus is 97-98%, the sensitivity for detecting ureaplasma is 99-100%.

The capabilities inherent in PCR analysis allow you to achieve unsurpassed analytical specificity. This means identifying exactly the microorganism that was searched for, and not a similar or closely related one.
The diagnostic sensitivity and specificity of the PCR method often exceeds those of the culture method, which is called the "gold standard" for the detection of infectious diseases. Considering the duration of culture growth (from several days to several weeks), the advantage of the PCR method becomes obvious.

PCR in the diagnosis of infections
The advantages of the PCR method (sensitivity and specificity) determine a wide range of applications in modern medicine.
The main areas of application of PCR diagnostics:

1. diagnosis of acute and chronic infectious diseases of various localization
2. monitoring the effectiveness of the therapy
3. clarification of the type of pathogen

PCR is used in obstetrics, gynecology, neonatology, pediatrics, urology, venereology, nephrology, clinic of infectious diseases, ophthalmology, neurology, phthisiopulmonology, etc.

The use of PCR diagnostics is carried out in conjunction with other research methods (ELISA, PIF, RIF, etc.). Their combination and expediency is determined by the attending physician.

Infectious agents detected by PCR

Viruses:

1. HIV-1 and HIV-2 retroviruses
2. herpetiform viruses
3. herpes simplex virus types 1 and 2

Polymerase chain reaction (PCR)- experimental method of molecular biology, which is a specific amplification of nucleic acids induced by synthetic oligonucleotide primers in vitro.

The idea of ​​developing the PCR method belongs to the American researcher Kary Mullis, who in 1983 created a method that made it possible to amplify DNA during cyclic doublings using the DNA polymerase enzyme under artificial conditions. A few years after the publication of this idea, in 1993, K. Mullis received the Nobel Prize for it.

At the beginning of using the method, after each heating-cooling cycle, it was necessary to add DNA polymerase to the reaction mixture, since it was quickly inactivated at high temperature. The procedure was very inefficient, requiring a lot of time and enzyme. In 1986, it was significantly modified by using DNA polymerase from thermophilic bacteria. These enzymes are able to withstand many reaction cycles, which allows you to automate the PCR. One of the most commonly used thermostable DNA polymerases has been isolated from bacteria. Thermus aquaticus and named Taq-DNA polymerase.

The essence of the method. The method is based on multiple selective copying of a certain DNA region using the enzyme Taq-DNA polymerase. The polymerase chain reaction makes it possible to obtain amplificates up to several thousand base pairs in length. To increase the length of the PCR product to 20-40 thousand base pairs, a mixture of various polymerases is used, but it is still much less than the length of the chromosomal DNA of a eukaryotic cell.

The reaction is carried out in a programmable thermostat (amplifier) ​​- a device that can carry out fast enough

cooling and heating of test tubes (usually with an accuracy of at least 0.1 °C). Amplifiers allow you to set complex programs, including those with the possibility of a "hot start" and subsequent storage. For real-time PCR, devices equipped with a fluorescent detector are produced. Instruments are also available with an automatic lid and microplate compartment, allowing them to be integrated into automated systems.

Usually, during PCR, 20-45 cycles are performed, each of which consists of three stages: denaturation, primer annealing, elongation (Fig. 6.1 and 6.2). On fig. 6.1 shows the dynamics of temperature changes in the test tube during the PCR cycle.

Rice. 6.1. Graph of temperature change in a test tube during one cycle of polymerase chain reaction

DNA template denaturation is carried out by heating the reaction mixture to 94-96 ° C for 5-90 s so that the DNA chains disperse. It should be noted that before the first cycle, the reaction mixture is preheated for 2–5 min to completely denature the initial matrix, which makes it possible to reduce the amount of nonspecific reaction products.

Rice. 6.2. Scheme of the first cycle of the polymerase chain reaction

Primer annealing stage. With a gradual decrease in temperature, the primers bind complementarily to the template. The annealing temperature depends on the composition of the primers and is usually 4-5°C lower than the calculated melting temperature. The duration of the stage is 5-60 s.

During the next stage - elongation- there is a synthesis of the daughter strand of DNA on the maternal matrix. The elongation temperature depends on the polymerase. The commonly used Taq and Pfu DNA polymerases are most active at 72°C. The elongation time, mainly dependent on the length of the PCR product, is typically 1 minute per 1000 base pairs.

PRINCIPLE OF THE METHOD (molecular biological basis)

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

Method principle polymerase chain reaction (PCR)(Polymerase chain reaction (PCR)) was developed by Cary 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 Supervision service (genotyping, diagnosis of infectious diseases).

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

Natural DNA replication involves several steps:

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

2) Formation of short double-stranded DNA segments(seeds required to initiate DNA synthesis);

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

This process can be used to obtain copies short segments of DNA specific to specific microorganisms, those. to 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, according to a given program, carry out a cyclic change in temperature, created the prerequisites for the widespread introduction of the PCR method into the practice of laboratory clinical diagnostics. With repeated repetition 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 the analyzed material, which may contain single cells of microorganisms, for their identification by electrophoresis.

Complementary completion of the chain 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 regions of DNA, it is possible to direct the process of synthesis of a new strand only in this region, and not along the entire length of the DNA strand. To create starting blocks in given DNA regions, two oligonucleotide primers (20 nucleotide pairs) are used, called primers. The primers are complementary to the 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 strand occurs only between them.

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

The following components are required for amplification:

A 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 lengthening of primer chains by sequentially adding nucleotide bases to a growing chain of synthesized DNA).

buffer solution(reaction medium containing Mg2+ ions necessary to maintain enzyme activity)
To determine specific regions of the genome of RNA-containing viruses, a DNA copy is first obtained from an RNA template using a reverse transcription (RT) reaction catalyzed by the enzyme reverse transcriptase (reverse transcriptase).

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 proceeding in different temperature conditions Step 1: DNA Denaturation(double helix unwinding). It flows at 93-95°C for 30-40 seconds. Stage 2: Attaching primers (annealing). Primer attachment occurs complementary to the corresponding sequences on opposite DNA strands at the boundaries of a specific site. 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: Building DNA chains. Complementary completion of DNA chains occurs from the 5'-end to the 3'-end of the chain in opposite directions, starting from the sites of primer attachment. The material for the synthesis of new DNA chains are 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. Synthesis time - 20-40 sec.

The new DNA strands formed in the first amplification cycle serve as templates for the second amplification cycle, in which 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, the accumulation of amplicons in solution occurs according to the formula 2n, where n is the number of amplification cycles. Therefore, even if only one double-stranded DNA molecule was initially present in the initial solution, about 108 amplicon molecules accumulate in the solution after 30–40 cycles. 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 this microorganism, using a polymerase chain reaction to accumulate the desired fragment.
The analysis technique 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

Isolation of DNA (RNA)
At this stage of the analysis, the clinical sample is subjected to special processing, which results in the lysis of cellular material, the removal of protein and polysaccharide fractions, and the preparation of a DNA or RNA solution free of
inhibitors and ready for further amplification.
The choice of DNA (RNA) extraction technique is mainly determined by the nature of the processed clinical material.

Amplification of specific DNA fragments
At this stage, short specific DNA fragments are accumulated in the amount necessary for their further detection. Most methods for determining specific fragments of the genome use the so-called. “a classic version of guided 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 the 1st stage, and “internal” - for the 2nd stage).

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

As an alternative to the electrophoretic method of detection, which has some disadvantages: subjectivity in reading the results, restrictions on the determination of DNA of various microorganisms in one reaction, can be proposed. hybridization detection schemes. In these schemes, the DNA fragment resulting from the amplification hybridizes (forms 2-strand complexes - "hybrids") with a specific oligonucleotide probe. Registration of such complexes can be carried out colorimetrically or fluorimetrically. SPC "Litekh" created detection kits based on hybridization with fluorimetric registration of results

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

- Direct detection of the presence of pathogens

Many traditional diagnostic methods, such as enzyme-linked immunosorbent assay, detect marker proteins that are waste products of infectious agents, which provides only indirect evidence of the presence of an infection. Identification of a specific DNA region of the pathogen 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 this pathogen is detected in the test material. Specificity is determined by the nucleotide sequence of the primers, which excludes
the possibility of obtaining false results, in contrast to the enzyme immunoassay method, where errors are not uncommon due to cross-reacting antigens.

- 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) is impossible. The sensitivity of PCR analysis is 10-1000 cells per sample (the sensitivity of immunological and microscopic tests is 103-105 cells).

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

- High speed of obtaining the result of the analysis
PCR analysis does not require the isolation and cultivation of the pathogen culture, which takes a lot of time. A unified method for biomaterial processing and detection of reaction products, and automation of the amplification process make it possible to carry out a complete analysis in 4-4.5 hours.

It should be noted that the PCR method can detect pathogens not only in the clinical material obtained from the patient, but also in the 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 great importance for solving many problems of microbiology and epidemiology. The use of this method also contributes to the development of fundamental research in the field of chronic and little-studied infectious diseases.

The most effective and economically justified use of the method 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 detect helicobacteriosis;

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

in hematology- to detect cytomegalovirus infection, oncoviruses.

1. Polymerase chain reaction (PCR)

2. Principle of polymerase chain reaction method

2.1 Presence of a number of components in the reaction mixture

2.2 Temperature cycling

2.3 Basic principles of primer selection

2.4 Plateau effect

3. Stages of PCR setting

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 Sample preparation of biological material

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

The method of obtaining a pure DNA preparation, described by Marmur, is considered standard and has already become classical. It includes enzymatic proteolysis followed by deproteinization and DNA reprecipitation with alcohol. This method makes it possible to obtain a pure DNA preparation. However, it is quite laborious and involves working with such aggressive and pungent substances as phenol and chloroform.

One of the currently popular methods is the DNA extraction method proposed by Boom et al. This method is based on the use of a strong chaotropic agent, guanidine thiocyanate (GuSCN), for cell lysis, and subsequent DNA sorption on a carrier (glass beads, diatomaceous earth, glass milk, etc.). After washings, DNA remains in the sample adsorbed on the carrier, from which it can be easily removed using an elution buffer. The method is convenient, technologically advanced and suitable for sample preparation for amplification. However, DNA losses are possible due to irreversible sorption on the carrier, as well as during numerous washes. This is especially important when working with small amounts of DNA in the sample. Moreover, even trace amounts of GuSCN can inhibit PCR. Therefore, when using this method, the correct choice of the sorbent and careful observance of technological nuances are very important.

Another group of sample preparation methods is based on the use of Chilex-type ion exchangers, which, unlike glass, do not adsorb DNA, but vice versa, impurities that interfere with the reaction. As a rule, this technology includes two stages: sample boiling and adsorption of impurities on an ion exchanger. The method is extremely attractive due to its simplicity of execution. In most cases, it is suitable for working with clinical material. Unfortunately, sometimes there are samples with impurities that cannot be removed using ion exchangers. In addition, some microorganisms cannot be destroyed by simple boiling. In these cases, it is necessary to introduce additional stages of sample processing.

Thus, the choice of the sample preparation method should be treated with an understanding of the purposes of the intended analyses.

3.2 Amplification

To carry out the amplification reaction, it is necessary to prepare the reaction mixture and add the analyzed DNA sample to it. In this case, it is important to take into account some features of primer annealing. The fact is that, as a rule, in the analyzed biological sample there are various DNA molecules, to which the primers used in the reaction have partial, and in some cases significant, homology. In addition, primers can anneal to each other to form primer-dimers. Both lead to a significant consumption of primers for the synthesis of side (nonspecific) reaction products and, as a result, significantly reduce the sensitivity of the system. This makes it difficult or impossible to read the results of the reaction during electrophoresis.

3.3 Evaluation of reaction results

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

3.3.1 Horizontal electrophoresis method

Various methods are used to visualize the results of amplification. The most common today is the method of electrophoresis, based on the separation of DNA molecules by size. To do this, a plate of agarose gel is prepared, which is agarose frozen 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. The frozen agarose forms a spatial lattice. When pouring with the help of 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. At the same time, shorter DNA molecules move faster than long ones. The speed of DNA movement in the gel is affected by the concentration of agarose, electric field strength, temperature, the composition of the electrophoresis buffer, and, to a lesser extent, the GC composition of DNA. All molecules of the same size move at the same speed. The dye is embedded (intercalates) in planar groups into DNA molecules. After the end of electrophoresis, which lasts from 10 minutes to 1 hour, the gel is placed on the filter of a transilluminator emitting light in the ultraviolet range (254 - 310 nm). The UV energy absorbed by the 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 bands of amplification products can be different. 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 lies in the fact 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 a higher resolution than agarose electrophoresis and makes it possible to distinguish DNA molecules of different sizes with an accuracy of one nucleotide. Preparation of polyacrylamide gel is somewhat more complicated than agarose. In addition, acrylamide is a toxic substance. Since the need to determine the size of the amplification product with an accuracy of 1 nucleotide rarely arises, the horizontal electrophoresis method is used in routine work.

3.4 Monitoring the progress of the amplification reaction

3.4.1 Positive controls

As a "positive control" use the DNA preparation of the desired microorganism. Non-specific amplicons differ in size from the amplicons generated by amplification with a control DNA preparation. The size of non-specific products can be either larger or smaller than the positive control. In the worst case, these dimensions may coincide and are read in electrophoresis as positive.

To control the specificity of the resulting amplification product, hybridization probes (DNA regions located within the amplifiable sequence) labeled with enzyme labels or radioactive isotopes and interacting with DNA in accordance with the same principles as primers can be used. This greatly complicates and lengthens the analysis, and its cost increases significantly.

3.4.2 Internal controls

It is necessary to control the progress of amplification in each tube with the reaction mixture. For this purpose, an additional, so-called "internal control" is used. It is any preparation of DNA that is not similar to the DNA of the desired microorganism. If the internal control is added to the reaction mixture, then 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 generated from the amplification of the target DNA of the microorganism. As a result, if internal control DNA is introduced into the reaction mixture together 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 much longer (heavier) than the amplicon of the microorganism. The presence of heavy amplicons in the reaction mixture will indicate the normal course of the amplification reaction and the absence of inhibitors. If the amplicons of the required size were not formed, but the internal control amplicons were not formed either, it can be concluded that the analyzed sample contains undesirable impurities that should be eliminated, but not the absence of the desired DNA.

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

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

4. Methods based on the polymerase chain reaction

4.1 Qualitative analysis

The classical method of setting up 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 How to set up PCR using “hot start”

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

The fact is that, depending on the HC composition and size, primers have a certain melting temperature (Tm). If the temperature of the system exceeds Tm, the primer is unable to adhere to the DNA strand and denatures. Under optimal conditions, i.e. 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":

Introducing Taq polymerase into 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 (primers in the lower part, Taq polymerase and target DNA in the upper part), which are mixed when the paraffin is melted (~65-75 0 С).

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 irreversibly denature and release the active sites of Taq polymerase.

In all these cases, even if nonspecific annealing occurred before the onset of thermal cycling, elongation does not occur, and primer-DNA complexes are denatured upon heating, so no nonspecific products are formed. Subsequently, the temperature in the tube does not fall below the melting point, 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. At the same time, there is no intermediate phase of transformation into DNA in their life cycles. To detect RNA, it must first be converted into the form of DNA. For 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 a temperature not exceeding 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 disadvantage 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 the reverse transcription reaction, the reaction mixture, as well as in PCR, must contain primers as a seed and a mixture of 4 dNTPs as a building material.

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

5. Organization of the technological process of setting PCR

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

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

There are several ways to deal with 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 tube will contain uracil. If HC is added to the reaction mixture before amplification, then the amplicons that enter the reaction mixture will be destroyed, while 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 deal with the results of contamination is a significant reduction in the number of reaction cycles (up to 25-30 cycles). But even with this approach, the risk of obtaining false positive results is high, because in this case, in the absence of inhibitors, it is easy to obtain an amplification product due to contamination.

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

Conclusion

The PCR method is currently the most widely used as a method for diagnosing various infectious diseases. PCR allows you to identify the etiology of the infection, even if the sample taken for analysis contains only a few DNA molecules of the pathogen. PCR is widely used in the early diagnosis of HIV infections, viral hepatitis, etc. To date, there is almost no infectious agent that cannot be detected using PCR.