PCR reaction what. See what "PCR" is in other dictionaries

Date of: 03.03.2020

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. Especially great importance this has when working with small amounts of DNA in a 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, sorb not 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 the apparatus for horizontal gel electrophoresis and the source is connected constant voltage. 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 the 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 introduced into 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.

Exist various options"hot start" implementations:

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 monoclonal antibodies irreversibly denature and release active centers Taq polymerases.

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, in their life cycles there is no intermediate phase of transformation into DNA. 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.

Federal Agency for Education

State educational institution

Higher professional education

"Karelian State Pedagogical Academy"

Coursework on the topic:

Polymerase chain reaction (PCR) and its application

Completed by: student Koryagina Valeria Alexandrovna

Checked by: Karpikova Natalya Mikhailovna

Petrozavodsk 2013

Introduction

Chapter 1 Literature Review

1.5.4 Plateau effect

1.5.6 Amplification

Conclusion

Introduction

The last twenty years have been marked by the widespread introduction of molecular genetic methods into the biological, medical, and agricultural sciences.

By the early 1970s, it seemed that molecular biology had reached a certain degree of perfection. During this period, microorganisms were the main object of molecular genetic research. The transition to eukaryotes presented researchers with completely new problems that could not be solved using the methods of genetic analysis that existed at that time. A breakthrough in the development of molecular genetics became possible due to the emergence of a new experimental tool - restriction endonucleases. In subsequent years, the number of direct DNA analysis methods based on qualitatively different approaches began to increase rapidly.

Modern technologies in many cases, they allowed at a deeper level to begin studying the fine structural and functional organization of the nuclear and extranuclear genomes of various organisms. This was of particular importance for the development of new methods of diagnosis and treatment. various diseases. No less important was the possibility of using the achievements of molecular genetics in population biology and breeding to identify and analyze the genetic variability of populations, varieties and strains, identify and certify economically valuable individuals, create genetically modified organisms, and to solve other issues.

Each method has its own advantages and disadvantages. There is no universal method that could solve all the problems that arise. Therefore, the choice of a specific method for the study is one of the most important stages of any scientific work.

Chapter 1 Literature Review

1.1 History of the discovery of the polymerase chain reaction (PCR)

In 1983 K.B. Mullis et al. published and patented the polymerase chain reaction (PCR) method, which was destined to have a profound impact on all areas of research and application of nucleic acids. The significance of this method for molecular biology and genetics turned out to be so great and obvious that seven years later the author was awarded the Nobel Prize in Chemistry.

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

The possibility of amplifying any DNA segment, the nucleotide sequence of which is known, and obtaining it after the completion of PCR in a homogeneous form and in a preparative amount make PCR alternative method molecular cloning of short DNA fragments. This eliminates the need for complex methodological techniques, which are used in genetic engineering in conventional cloning. The development of the PCR method has greatly expanded the methodological possibilities of molecular genetics, and, in particular, genetic engineering, so much so that it has radically changed and strengthened the scientific potential of many of its areas.

1.2 Varieties of polymerase chain reaction (PCR)

· Nested PCR- used to reduce the number of by-products of the reaction. Use two pairs of primers and carry out two consecutive reactions. The second pair of primers amplifies the DNA region within the product of the first reaction.

· Inverted PCR- is used when only a small area within the desired sequence is known. This method is especially useful when it is necessary to determine neighboring sequences after DNA has been inserted into the genome. For the implementation of inverted PCR, a series of DNA cuts is carried out with restriction enzymes<#"justify">polymerase chain reaction primer

· Group-specific PCR- PCR for relatives<#"center">1.3 Polymerase chain reaction

Discovered in the mid-1980s, the polymerase chain reaction (PCR) can increase the number of copies of an original sample millions of times within a few hours. During each cycle of the reaction, two copies are formed from the original molecule. Each of the synthesized DNA copies can serve as a template for the synthesis of new DNA copies in the next cycle. Thus, repeated repetition of cycles leads to an increase in the number of copies exponentially. It follows from the calculations that even if there are 30 cycles, the number of copies of the original molecule will be more than 1 billion. Even if we take into account that not all amplicons are duplicated during each cycle, the total number of copies, despite this, is quite a large figure.

Each cycle of the polymerase chain reaction (PCR) consists of the following steps:

· Denaturation - An increase in temperature causes a double-stranded DNA molecule to unwind and split into two single-stranded ones;

· Annealing - Lowering the temperature allows primers to attach to complementary regions of the DNA molecule;

· Elongation - The enzyme DNA polymerase completes the complementary strand.

For amplification of the selected fragment, two oligonucleotide primers (primers) flanking a certain DNA region are used. Primers oriented 3 - ends towards each other and in the direction of the sequence that needs to be amplified. DNA polymerase carries out the synthesis (completion) of mutually complementary DNA chains, starting with primers. During DNA synthesis, primers are physically inserted into the chain of newly synthesized DNA molecules. Each strand of the DNA molecule formed using one of the primers can serve as a template for the synthesis of a complementary DNA strand using the other primer.

1.4 Conducting a polymerase chain reaction (PCR)

The polymerase chain reaction is carried out in special thin-walled polypropylene test tubes, compatible in size with the used thermal cycler (amplifier) - a device that controls the temperature and time characteristics of the stages of the polymerase chain reaction (PCR).

1.5 Principle of the polymerase chain reaction method

Polymerase chain reaction (PCR) is an in vitro DNA amplification method that can isolate and multiply a specific DNA sequence billions of times within a few hours. The ability to obtain a huge number of copies of one strictly defined region of the genome greatly simplifies the study of an existing DNA sample.

To carry out a polymerase chain reaction, a number of conditions must be met:

1.5.1 Presence of a number of components in the reaction mixture

The main components of the reaction (PCR) mixture are: Tris-HCl, KCl, MgCl 2, a mixture of nucleotide triphosphates (ATP, GTP, CTP, TTP), primers (oligonucleotides), analyzed DNA preparation, thermostable DNA polymerase. Each of the components of the reaction mixture is directly involved in the polymerase chain reaction (PCR), and the concentration of reagents directly affects the course of amplification.

· Tris-HCl - determines the pH of the reaction mixture, creates a buffer capacity. The activity of DNA polymerase depends on the pH of the medium, so the value of the pH directly affects the course of the polymerase chain reaction. Usually the pH value is in the range of 8 - 9.5. The high pH is due to the fact that as the temperature rises, the pH of the Tril-HCl buffer drops.

· KCl - the concentration of potassium chloride up to 50 mm affects the course of the processes of denaturation and annealing, the concentration above 50 mm inhibits DNA polymerase.

· MgCl 2- because DNA polymerase is Mg 2+- dependent enzyme, then the concentration of magnesium ions affects the activity of the enzyme (Mg 2+forms complexes with NTP - it is these complexes that are the substrate for polymerase). A high concentration leads to an increase in nonspecific amplification, and a low one leads to inhibition of the reaction, the optimum (for various polymerases) is in the region of 0.5 - 5 mM. In addition, the concentration of magnesium salts affects the course of denaturation and annealing processes - an increase in the concentration of Mg 2+causes an increase in the melting temperature of DNA (i.e., the temperature at which 50% of double-stranded DNA strands are broken into single-stranded strands).

· NTP - nucleotide triphosphates are direct monomers of nucleic acids. To prevent chain termination, an equal ratio of all four nucleotide triphosphates is recommended. The low concentration of these components in the reaction mixture increases the probability of errors in the construction of the complementary DNA strand.

· Primers - The most optimal is the use of primers with a melting point difference of no more than 2 - 4 o C. Sometimes during long-term storage at a temperature of 4 o With, or after a large number of freezing - thawing, the primers form secondary structures - dimers, reducing the efficiency of the PCR. The elimination of this problem is reduced to incubation in a water bath (T=95 o C) for 3 minutes and subsequent rapid cooling to 0o FROM.

· DNA preparations - the quantity and quality of the DNA preparation (matrix) directly affects the course and parameters of the polymerase chain reaction. Excess DNA sample inhibits the polymerase chain reaction (PCR). impurities various substances, which are in the DNA preparation, can also reduce the efficiency of the polymerase chain reaction (PCR): sodium acetate, sodium chloride, isopropanol, ethanol, heparin, phenol, urea, hemoglobin, etc.

· DNA polymerase - when using a small amount of DNA polymerase, a decrease in the synthesis of the final product is observed in direct proportion to the size of the fragments. An excess of polymerase by 2 - 4 times leads to the appearance of diffuse spectra, and by 4 - 16 times - low molecular weight nonspecific spectra. The range of concentrations used is 0.5 - 1.5 units of activity in terms of 25 µl of the PCR mixture.

In addition to the main components of the PCR mixture, a number of additional substances are used that improve the qualitative and quantitative indicators of PCR: acetamide (5%) - an increase in the solubility of the main components; betaine (sodium salt) - stabilization of DNA polymerase, lowering the melting point of DNA, equalizing the melting point; bovine albumin (10-100 μg / ml) - stabilization of DNA polymerase; dimethyl sulfoxide (1-10%) - increasing the solubility of the main components; formamide (2-10%) - an increase in the specificity of annealing; glycerol (15-20%) - an increase in the thermal stability of the enzyme, a decrease in the temperature of denaturation of a DNA sample; ammonium sulfate - lowering the temperature of denaturation and annealing.

1.5.2 Cycle and temperature

General form polymerase chain reaction (PCR) programs are as follows:

stage. Prolonged primary denaturation of the DNA preparation.1 cycle

stage. Rapid denaturation of the DNA preparation. Primer annealing. Elongation.30 - 45 cycles.

stage. Prolonged elongation. Cooling of the reaction mixture. 1 cycle.

Each element of the stage - denaturation, annealing, elongation - has individual temperature and time characteristics. The parameters of temperature and flow time of each element are selected empirically, in accordance with the qualitative and quantitative indicators of the amplification products.

Denaturation. During this element of the polymerase chain reaction, a double-stranded DNA molecule is split into two single-stranded ones. Temperature parameters of denaturation are in the range of 90 - 95 o C, but in the case of a DNA sample with a high content of guanine and cytosine, the temperature should be increased to 98 o C. The temperature of denaturation should be sufficient to completely denature - cleave the DNA strands and avoid "sudden cooling" or rapid annealing, however, thermostable DNA polymerase is less stable at high temperatures. Thus, the selection of optimal denaturation temperature parameters for the primer/sample ratio (DNA preparation) is an important condition for amplification. If the denaturation temperature in the first step is above 95 o C, it is recommended to add DNA polymerase to the reaction mixture after primary denaturation. The duration of this element of the stage during the polymerase chain reaction (PCR) should be sufficient for complete DNA denaturation, but at the same time not significantly affect the activity of DNA polymerase at a given temperature.

Annealing. Annealing temperature (T but ) is one of the most important parameters of the polymerase chain reaction. The annealing temperature for each specific primer is selected individually. It depends on the length and nucleotide composition of the primer. Usually it is lower by 2 - 4 o From the melting point value (T m ) primer. If the annealing temperature of the system is below the optimum, then the number of nonspecific amplified fragments increases and, conversely, more heat reduces the amount of amplified products. In this case, the concentration of specific amplicons can sharply decrease, up to inhibition of the polymerase chain reaction (PCR). Increasing the annealing time also leads to an increase in the number of nonspecific amplicons.

Elongation. Typically, each type of thermostable DNA polymerase has an individual temperature optimum of activity. The rate of synthesis of a complementary DNA strand by an enzyme is also a value specific to each polymerase (on average, it is 30–60 nucleotides per second, or 1–2 thousand bases per minute), so the elongation time is selected depending on the type of DNA polymerase and the length of the amplified region.

1.5.3 Basic principles of primer selection

When creating a PCR test system, one of the main tasks is the correct selection of primers that must meet a number of criteria:

Primers must be specific. Particular attention is paid to 3 - the ends of the primers, since it is from them that Taq polymerase begins to complete the complementary DNA chain. If their specificity is insufficient, then it is likely that undesirable processes will occur in the test tube with the reaction mixture, namely, the synthesis of nonspecific DNA (short or long fragments). It is visible on electrophoresis in the form of heavy or light additional bands. This makes it difficult to evaluate the results of the reaction, since it is easy to confuse a specific amplification product with synthesized foreign DNA. Part of the primers and dNTPs is consumed for the synthesis of nonspecific DNA, which leads to a significant loss of sensitivity.

Primers should not form dimers and loops, i.e. no stable double strands should be formed by annealing the primers to themselves or to each other.

1.5.4 Plateau effect

It should be noted that the process of accumulation of specific amplification products exponentially lasts only a limited time, and then its efficiency drops critically. This is due to the so-called "plateau" effect.

term effect plateau used to describe the process of accumulation of PCR products in the last cycles of amplification.

Depending on the conditions and the number of cycles of the amplification reaction, at the time the effect is achieved plateau the utilization of substrates (dNTPs and primers), the stability of reactants (dNTPs and enzyme), the amount of inhibitors, including pyrophosphates and DNA duplexes, competition for reactants by non-specific products or primer-dimers, the concentration of a specific product, and incomplete denaturation at high concentration amplification products.

The lower the initial concentration of the target DNA, the higher the risk of the reaction plateau". This point can occur before the number of specific amplification products is sufficient to be analyzed. Only well-optimized test systems can avoid this.

1.5.5 Sample preparation of biological material

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.

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

1.5.6 Amplification

1.6 Composition of the standard PCR reaction mixture

x PCR buffer (100 mM Tris-HCl solution, pH 9.0, 500 mM KCl solution, 25 mM MgCl2 solution ) …….2.5 µl

Water (MilliQ) ……………………………………………………….18.8 µl

A mixture of nucleotide triphosphates (dNTPs)

mM solution of each………………………………………….……….0.5 µl

Primer 1 (10 mM solution) ………………………………………….….1 µl

Primer 2 (10 mM solution) ………………………………………….….1 µl

DNA polymerase (5 units / µl) ……………………………………………0.2 µl

DNA sample (20 ng/µl) …………………………………………..1 µl

1.7 Evaluation of reaction results

Chapter 2: Applications of the Polymerase Chain Reaction

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

Criminalistics

PCR is used to compare so-called "genetic fingerprints". We need a sample of genetic material from the crime scene - blood, saliva, semen, hair, etc. It is compared with the suspect's genetic material. A very small amount of DNA is enough, theoretically - one copy. The DNA is cut into fragments, then amplified by PCR. The fragments are separated by DNA electrophoresis. The resulting picture of the location of the DNA bands is called the genetic fingerprint.

Establishing paternity

Results of electrophoresis of DNA fragments amplified by PCR. Father. Child. Mother. The child inherited some features of the genetic imprint of both parents, which gave a new, unique imprint.

Although "genetic fingerprints" are unique, family ties can still be established by making several such fingerprints. The same method can be applied, with slight modifications, to establish evolutionary relationships among organisms.

Medical diagnostics

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

Personalized medicine

Sometimes drugs are toxic or allergenic for some patients. The reasons for this are partly individual differences in the susceptibility and metabolism of drugs and their derivatives. These differences are determined at the genetic level. For example, in one patient, a certain cytochrome may be more active, in another - less. In order to determine what kind of cytochrome a given patient has, it is proposed to perform a PCR analysis before using the drug. This analysis is called preliminary genotyping.

Gene cloning

Gene cloning is the process of isolating genes and, as a result of genetic engineering manipulations, obtaining a large amount of the product of a given gene. PCR is used to amplify a gene, which is then inserted into a vector, a piece of DNA that carries the foreign gene into the same organism or another organism that is easy to grow. As vectors, for example, plasmids or viral DNA are used. insertion of genes into foreign organism usually used to obtain the product of this gene - RNA or, most often, protein. In this way, many proteins are obtained in industrial quantities for use in agriculture, medicine, etc.

DNA sequencing

In the method of sequencing using labeled with a fluorescent label or a radioactive isotope of dideoxynucleotides, PCR is an integral part, since it is during polymerization that derivatives of nucleotides labeled with a fluorescent or radioactive label are inserted into the DNA chain. This stops the reaction, allowing the positions of specific nucleotides to be determined after separation of the synthesized strands in the gel.

Mutagenesis

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

The PCR method made it possible to analyze the presence of human papillomavirus sequences in biopsy sections of human cervical neoplasms embedded in paraffin 40 years before this study. Moreover, with the help of PCR, it was possible to amplify and clone fragments of mitochondrial DNA from the fossil remains of the human brain of the age of 7 thousand years!

The ability to simultaneously analyze two loci located on different nonhomologous chromosomes was demonstrated on lysates of individual human spermatozoa. This approach provides a unique opportunity for fine genetic analysis and the study of chromosomal recombination, DNA polymorphism, etc. The method of analyzing individual spermatozoa immediately found practical use in forensic medicine, since HLA typing of haploid cells allows determining paternity or identifying a criminal (the HLA complex is a set of human major histocompatibility complex genes; the loci of the HLA complex are the most polymorphic of all known in higher vertebrates: within a species, at each locus there is an unusual a large number of different alleles - alternative forms of the same gene).

Using PCR, it is possible to identify the correctness of the integration of foreign genetic structures in a predetermined region of the genome of the studied cells. The total cellular DNA is annealed with two oligonucleotide primers, one of which is complementary to the host DNA region near the insertion point, and the other to the sequence of the integrated fragment in the antiparallel DNA strand. Polymerase chain reaction in the case of an unchanged structure of chromosomal DNA at the proposed insertion site leads to the formation of single-stranded DNA fragments of an indefinite size, and in the case of a planned insertion, double-stranded DNA fragments of a known size, determined by the distance between the annealing sites of two primers. Moreover, the degree of amplification of the analyzed region of the genome in the first case will be linearly dependent on the number of cycles, and in the second - exponentially. The exponential accumulation of an amplified fragment of a predetermined size during PCR makes it possible to visually observe it after electrophoretic fractionation of a DNA preparation and make an unambiguous conclusion about the insertion of a foreign sequence into a given region of chromosomal DNA.

Conclusion

List of used literature

1.Padutov V.E., Baranov O.Yu., Voropaev E.V. Methods of molecular - genetic analysis. - Minsk: Unipol, 2007. - 176 p.

2.PCR "in real time" / Rebrikov D.V., Samatov G.A., Trofimov D.Yu. and etc.; ed. b. n. D.V. Rebrikov; foreword L.A. Osterman and acad. RAS and RAAS E.D. Sverdlov; 2nd ed., rev. and additional - M.: BINOM. Knowledge Laboratory, 2009. - 223 p.

.Patrushev L.I. Artificial genetic systems. - M.: Nauka, 2005. - In 2 tons

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Edited by A.A. Vorbyeva "Polymerase chain reaction and its application for diagnostics in dermatovenereology"; Medical News Agency - 72 pages

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Content

Those who are interested in new diagnostic methods should find out what the PCR method is. Modern technical capabilities in the field of laboratory research provide the ability to detect many diseases in the initial stages. Polymerase chain reaction (PCR) is currently considered the most accurate and new method.

PCR analysis

PCR analysis - what is it? This method uses the principles of molecular biology. To study the material, special enzymes are used that repeatedly and quickly copy DNA, RNA fragments of pathogens. Exists different types PCR analysis depending on the material being studied (blood, urine, feces, etc.). After processing, laboratory staff compare the result with the database, identify the concentration, type of pathogen.

The PCR analysis is placed in a special amplifier (device) that heats and cools the test tubes with the biomaterial. Temperature changes are needed for fragment replication. The accuracy of the result will depend on the accuracy of the temperature regime. The polymerase chain reaction method helps to identify:

Infectious mononucleosis;
cytomegalovirus infection;
viral hepatitis G, C, B, A;
sexually transmitted infections / diseases (STIs / STDs): gardnerellosis, trichomoniasis, ureaplasmosis;
herpetic infection;
oncogenic viruses;
listeriosis;
helicobacter infection;
tick-borne encephalitis, borreliosis;
tuberculosis;
candidiasis.

Blood

At the moment, due to the novelty of the technology, the PCR blood test still has a high price. For the preparation of biomaterial it is not necessary to comply with certain requirements. Even caused physical activity, stress, change in diet, changes in the composition do not affect the result of the study. A PCR blood test can only spoil the intake of antibacterial agents, therefore, before taking it, it is necessary to pause between treatment and the test.

PCR blood test is the most common option for diagnosing chronic, acute infectious pathologies with a viral or atypical manifestation. Serological research methods have a certain difficulty in carrying out - the presence of a pathogen is determined by the presence of antibodies in the human body. The result could be false negative if the patient's condition did not give time for their development.

smear

In the field of gynecology, PCR smear analysis is used to study the presence of infectious microorganisms. Working with the material is carried out according to the same principle as with blood: a multiple increase in DNA fragments of the pathogen in order to easily identify it. It also helps to discover hidden infections at a woman. Various biological fluids can be taken for analysis: saliva, sputum, urine, blood. In gynecology, for the accuracy of the determination, a smear from the vaginal mucosa from the cervical canal is more often used.

There are certain indications for PCR. Often it needs to be done to identify an antibiotic-resistant type of pathogen. In women, the main indications for diagnosis by this method are:

a pregnancy that is difficult;
acute phase of STIs;
if there is a suspicion of the transfer of STIs to chronic stage;
search for causes of infertility.

Cala

To detect infection, a fecal PCR test may be prescribed by the doctor. In order to obtain the most reliable results after the test, it is necessary to adhere to the following rules before taking the biomaterial:

stop taking laxatives for a few days: oils, suppositories;
exclude medicines that give a specific color to the stool, for example, with iron content.

Urine

If necessary, the doctor may take urine for testing. High accuracy opens the possibility to work with any biological fluid from which the DNA of the virus can be extracted. To pass a PCR urine test, you must adhere to the following restrictions before taking the material:

at least 1 day before the procedure, stop sexual intercourse;
3 weeks before the delivery, any antibacterial treatment should be completed, because the medicines will blur the picture;
you need to take the test on an empty stomach (liquid is also prohibited);
you need to take the first morning portion of the material.

PCR test results

From the above, it is clear what PCR analysis is and the clear advantages of this research method are visible. Another plus of this diagnostic procedure is the ease of deciphering the results. Considering how much PCR analysis is done (the process itself takes about 5 hours, but the laboratory issues data in 1-2 days), this diagnostic method becomes the best option to identify a variety of infections. Based on the results, your doctor may tell you that the test:

Negative - the studied material did not contain the desired pathogen.
Positive - RNA, DNA of the pathogen were found.

Sometimes quantitative determination of microorganisms is carried out. This is necessary for diseases that cause opportunistic pathogens. The peculiarity of these viruses is that they appear only in excess and it is extremely problematic to find them with conventional studies. This factor is important for choosing therapeutic tactics to effectively treat viral infections, such as hepatitis, HIV.

For 12 infections

To fully understand what is PCR diagnostics infections and how effective it is, you need to know that it is capable of isolating up to 12 pathogens. The text is carried out only in laboratory conditions. For research, special enzymes are used, which increase many times the amount of RNA, DNA fragments of the virus. PCR analysis for 12 infections can reveal:

mycobacterium tuberculosis;
cytomegalovirus;
hepatitis C, G, B, A;
herpes 1, 2 types;
Epstein-Barr virus (infectious mononucleosis);
infections that are sexually transmitted by, for example, chlamydia;
listeriosis;
candida infection;
helicobacter pylori;
borreliosis, tick-borne encephalitis.

For hepatitis C

This diagnostic method helps to determine the presence of the virus in the blood. This gives doctors the opportunity to talk about its presence or absence. There are two types of PCR analysis for hepatitis C: qualitative and quantitative. The first option indicates only its presence and can be worded "detected" / "not detected". This type of test has a sensitivity of 10-500 IU/ml. This suggests that with a low content of the pathogen in the body, the analysis will be “not detected”.

Quantitative analysis is more accurate and will show the concentration of infection in the blood. This indicator is designated as "viral load", measured in the amount of viral RNA per specific volume of blood. Decryption in different laboratories may vary. In addition to measuring in IU / ml, "copy" units are used. You can recalculate copies per IU using the formula: 1 IU = 4 copies. If in the transcript the value of the presence of the virus exceeds 800,000 IU / ml (or 800 * 103), this indicates a high content of the pathogen.

For tuberculosis

The test should be done in the morning. This is important in order to prevent all the sputum that formed during the night from leaving the stomach. PCR analysis for tuberculosis is as important as ELISA, Mantoux, tomography. The test helps to highlight the presence of mycobacteria, the state of urine, total immunoglobulin, ESR, determine the state of the lungs at the moment. For the accuracy of obtaining results in the analysis of PCR, it is necessary to carry out it in compliance with the following rules:

Sowing is carried out 3 times, but complete aspiration of the contents of the stomach should be carried out only in a hospital.
Detects mycobacteria by culture of existing masses in the stomach in less than 50% of diagnoses. Even when optimal conditions are obtained, ultrasound is recommended instead.
Even with a negative result, the likelihood of developing tuberculosis with a change in ESR, immunoglobulin or other indicators cannot be completely excluded.
PCR culture is less susceptible to pathological conditions if it is obtained as part of a bronchoscopic examination, which excludes suspicion of TB in a child.

For HIV

For many people, this diagnosis is considered a death sentence. For this reason, after frequent sexual intercourse, a person becomes more attentive to the signals that his body gives (and sometimes comes up with them). The most reliable option to get confirmation or refutation this disease– PCR analysis for HIV. The test can be used to identify the following possible health problems:

Denial/confirmation of the presence of HIV during the period of the seronegative horse.
Determination of the genotype of HIV-1, HIV-2.
Clarification of the description of the pathological process with a doubtful result of the immunoblot.
Infection after blood transfusion.
Determining HIV status in children born to carrier mothers.
Helps to establish monitoring of the viral load of the body.

For HPV

The papilloma virus can be detected in any person, long time it may be in a dormant state. Development provokes a weakening of the immune system, stress or emotional outbursts. PCR analysis for HPV helps to determine the concentration of the virus in the blood. For this reason, it is recommended to carry out a quantitative determination rather than a qualitative one. These data will help predict the likelihood of developing a malignant nature of the infection.

The technique for diagnosing the presence of HPV is based on the main property of PCR to isolate virus DNA from the material. Due to the high sensitivity of the test, even a small amount of bacteria will be detected. Quantitative research gives doctors the opportunity to determine the degree of danger of the disease, to make a prognosis for the future. This diagnosis is mandatory for all men and women who have found themselves with warts. Quantitative PCR analysis will help determine what caused the development of HPV: a temporary decrease in immunity or a chronic disease.

For herpes

This type of diagnostics in microbiology helps to carry out PCR analysis for herpes with high accuracy. Copying of DNA fragments of the virus will occur only if the desired gene is present in the material. In this case, the test based on the results of the conduct may indicate the presence or absence of the pathogen. It will be possible to detect it even at low concentration in the blood.

Another plus of the PCR analysis is that it can detect a herpes virus infection immediately after infection, before the appearance clinical symptoms. It is possible to determine the type of herpes (1 or 2), no specific preparation is required to pass the analysis, but doctors recommend giving up before taking blood:

fried;
acute;
alcohol;
fatty.

During pregnancy

When carrying a child, it is very important to conduct this study in order to take into account the condition of the woman. PCR analysis during pregnancy is one of the most effective methods determining the presence various diseases. It is necessary to conduct a test not only to identify pathologies, but also to determine the likelihood of infection of the child in utero. Only thanks to PCR diagnostics it became possible to identify the degree of progression, the development of many infections inside the mother's womb.

Delivery of PCR analyzes

If you are interested in how the PCR analysis is taken, then each individual case should be considered, taking into account the type of biomaterial. Scraping, smear or blood sampling has its own characteristics, for example:

plasma is donated in the morning;
urine is taken only the first in the morning, under laboratory conditions in a sterile container;
a smear or scraping will be indicative only after abstinence from sexual intercourse for at least 3 days;
you can not take a smear during menstruation and 2 days after it.

Where to get tested for PCR

This type of research refers to modern and high-tech diagnostic methods. PCR tests should be taken in laboratories that have everything the necessary complex for complete results. Qualified and trained personnel play an equally important role. Give preference to large, serious, well-known laboratories. This will help not only get the results quickly, but also ensure their reliability.

Price

Another question that is often of interest to patients is: how much does a PCR test cost? Due to the novelty of the method, the need to purchase expensive equipment, the price of the test is relatively high. The cost of PCR is affected by the type of infection for which a person will be tested. Estimated price and terms of the tests are as follows:

STIs will be checked in 1 day, the price is 400-500 rubles.
Herpes, HPV, Epstein-Barr virus, cytomeglovirus are detected per day, the price is 300-500 rubles.
An analysis for hepatitis is carried out in 5 days, the price for a qualitative option is 500 rubles, for a quantitative one - 2000 rubles.
Helicobacter pylori is detected per day, the price is 400 rubles.
Antigens, HIV antibodies, price - from 380 rubles.
Qualitative analysis of HIV RNA, price - from 3,500 rubles.
Quantitative analysis of HIV RNA, price - from 11,000 rubles.

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Attention! The information provided in the article is for informational purposes only. The materials of the article do not call for self-treatment. Only qualified doctor can make a diagnosis and make recommendations for treatment based on individual characteristics specific patient.

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Bacterial genetics. Information for the second lesson.

polymerase chain reaction

Polymerase chain reaction is a method that allows for a multiple increase (amplification) of the number of certain DNA molecules in the analyzed sample (including biological material or pure culture).

The main advantages of PCR as a diagnostic method in microbiology are its very high sensitivity, which allows the detection of extremely low concentrations of pathogens in samples, as well as adjustable specificity, which makes it possible to detect or identify pathogens at the generic, species, or subspecies level. The main disadvantage of PCR stems from its extremely high sensitivity - it is very easy for images to contaminate DNA from a positive control, another sample, or a PCR product, leading to a false positive reaction. This imposes severe restrictions on the conditions under which PCR is mixed and processed with finished PCR products.

Conducting PCR. A reaction mixture is prepared containing the following components:

isolated DNA from the test sample,

buffer solution,

Mg2+ ions (required for the enzyme to work),

Two primers are single-stranded short DNA molecules (most often 18 to 24 nucleotides in length) complementary to the ends of different strands of the DNA sequence to be detected.

A mixture of deoxynucleotide triphosphates.

Heat-resistant DNA polymerase (most commonly used is Taq polymerase, a polymerase isolated from Thermus aquaticus).

Then this reaction mixture is placed in the cycler, which is actually a programmable thermostat. In the cycler, 30-40 cycles of temperature changes are carried out. Each of these cycles consists of three stages (see Fig. 1):

Denaturation (temperature 94 ° C) - hydrogen chains are broken, and DNA chains diverge.

Primer annealing (temperature is usually in the region of 50-60 ° C) - primers are attached to the ends of the DNA chains. In general, when the temperature is lowered, the reunification of the original DNA strands from the sample under study (renaturation) is energetically more favorable, however, the concentration of primers in the reaction mixture is many orders of magnitude higher than the concentration of DNA from the sample (at least in the initial PCR cycles), so the primer annealing reaction proceeds faster than renaturation. DNA. The annealing temperature is selected depending on the melting (denaturation) temperatures of the primers.

Elongation (temperature is usually 72 ° C) - DNA polymerase completes the primers along the template of long DNA chains. The temperature corresponds to the optimum operating temperature for the DNA polymerase used.

Detection of results differs in different PCR formulations and is described in the "PCR Varieties" section.

Dynamics of PCR

In early PCR cycles, the number of double-stranded DNA molecules, whose size is determined by the distance between primer sites, doubles with each cycle. A small number of longer DNA molecules are also formed, which can be neglected (see Figure 2).

Thus, in the early cycles, the amount of the PCR product is described by the formula m*2 n , where m is the initial amount of the desired DNA in the sample, n is the number of cycles. Then the reaction reaches a plateau. This is due to the accumulation of the reaction product, a decrease in the concentration of primers and deoxynucleotide triphosphates, and also due to an increase in the concentration of pyrophosphate (see Fig. 3).

Varieties of PCR

Conventional PCR

In this version of the PCR setting, the reaction goes on for a preselected number of cycles (30-40), after which it is analyzed whether the accumulation of double-stranded DNA molecules in the reaction mixture has occurred.

This variant of PCR, when used as a diagnostic method, is a qualitative method. A positive reaction indicates the presence of at least trace amounts of the desired DNA molecules in the sample. A negative reaction indicates their absence. A quantitative assessment of the content of the initial DNA molecules in the sample is impossible due to the reaction reaching a plateau.

The main method for detecting the presence of the product is electrophoresis in agarose or polyacrylamide gel. The PCR products are separated in the gel under the action of an electric field according to their molecular weight. An intercalating dye is added to the gel (fluorescent in the state associated with double-stranded DNA - most often ethidium bromide). Thus, when exposed to ultraviolet light, it will be possible to see the presence or absence of a strip corresponding to DNA of the required molecular weight. When conducting PCR for diagnostic purposes, positive and negative reaction controls are always placed, with which the samples are compared (see Fig. 4).

real time PCR

In this version of the PCR setup, the amount of the PCR product in the reaction mixture is constantly recorded during the course of the reaction. This allows you to build a reaction curve (see Fig. 3) and, based on it, calculate the number of desired DNA molecules in the samples.

One of the types of real-time PCR is using an intercalating dye that is added directly to the reaction mixture (SYBRGreen is most often used). Another type is using one of the types of fluorescent probes that bind to a site inside the PCR product, which makes it possible to increase the specificity of detection (see Fig. 5). Fluorescence detection occurs directly in the device during the reaction.

In addition to the possibility of quantitative detection, there are other advantages of real-time PCR compared to conventional PCR. This PCR variant is simpler, faster, and does not require opening tubes with PCR products, which reduces the possibility of contaminating other samples. The main disadvantage is the higher cost of an amplifier with a built-in fluorescence detection capability compared to a conventional one.

Digital quantitative PCR

A new, expensive and still not widely used version of PCR, which allows more accurate determination of the amount of DNA in a sample. In this version, the reaction mixture containing a fluorescent dye is divided into a huge number of microscopic volumes (for example, droplets in an emulsion). After the PCR, it is analyzed in which proportion of the droplets the reaction turned out to be positive and, accordingly, fluorescence is observed. This proportion will be proportional to the number of DNA molecules of interest in the sample.

reverse transcription PCR

In this case, before one or another PCR variant, a reverse transcription reaction (RNA to DNA) is performed using the reverse enzyme. Thus, this method allows qualitative or quantitative detection of RNA molecules. This can be used to detect RNA-containing viruses or determine the level of transcription (the amount of mRNA) of a particular gene.

Picture 1. PCR steps. Primers are marked in red.

Figure 2. Accumulation of primer-limited double-stranded DNA molecules during PCR.

Figure 3 The dynamics of the PCR reaction at different initial concentrations of the desired DNA molecules in the sample. (a) - the highest concentration (b) - intermediate concentration (c) - the lowest concentration

Figure 4 Agarose electrophoresis of PCR products. K+ - positive control (obviously the required DNA is present). 1-7 - test samples (of which 1-2 are positive, 3-7 are negative). K- -negative control (definitely missing the desired DNA). In many cases, in addition to the target product, lighter nonspecific reaction products (primer-dimers) are visible.

Figure 5 Detection methods using real-time PCR. (a) - intercalating dye - fluoresces when bound to double-stranded DNA (b) - Taqman probe - fluorescence occurs when the probe is cleaved by DNA polymerase with 5'-3' endonuclease activity due to the separation of the fluorophore and quencher. (c) MolecularBeacon probe - fluorescence occurs when the probe hybridizes with the target fragment due to the spatial separation of the fluorophore and quencher (d) - LightCycler probes - acceptor fluorescence occurs when the probes (containing an acceptor and donor) hybridize with the target fragment due to the resonant fluorescence energy transfer (FRET).

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. IN currently 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, mycoplasmas, 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 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 analysis (that is, the presence of a particular virus, bacteria, etc.). When labeled "seeds" are used, a chromogen (dye) is added to the final product of the reaction, as a result of which the enzymatic reaction is accompanied by the formation of a color. The development of a color indicates directly that a virus or other detectable agent is present in the original sample.

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

Why is PCR diagnostics so valuable?

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

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

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

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

diagnosis of acute and chronic infectious diseases different localization
monitoring the effectiveness of the therapy
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:

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