Polymerase chain reaction (PCR)

The essence of the PCR method. DNA polymerase

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

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

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

Carrying out PCR

Polymerase chain reaction (PCR) is a DNA amplification method that can be used to 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. To carry out PCR in the simplest case, the following components are required:

A DNA template containing the portion of DNA that needs to be amplified.

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

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

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

Mg 2+ ions necessary for the polymerase to function.

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

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

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

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

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

2. Annealing - binding of primers to template DNA. Once the chains have separated, the temperature is slowly lowered to allow the primers to contact the single-stranded template. The annealing temperature depends on the composition of the primers and is usually selected at 50-65°C. Stage time is 20 - 60 seconds. An incorrect choice of annealing temperature leads either to poor binding of primers to the template (at too high a temperature) or to binding in the wrong place and the appearance of nonspecific products (at too low a temperature).

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

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

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

To understand exactly how a specific DNA segment is amplified during PCR, you need to clearly imagine the position of all primers and their complementary sequences in the amplified strands in each round. In the first round, each of the newly synthesized chains has a much greater length than the distance from the 3"-hydroxyl group of its primer to the terminal nucleotide of the sequence complementary to the second primer. Such chains are called “long templates”; it is on them that further synthesis will take place.

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

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

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

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

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

Basic principles of primer selection.

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

1. Primers must be specific. Particular attention is paid to the 3" ends of the primers, since it is from them that Taq polymerase begins to complete the complementary DNA chain. If their specificity is insufficient, then 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 interferes with the assessment of the reaction results, since it is easy to confuse the specific amplification product with synthesized foreign DNA. Some of the primers and dNTPs are spent on the synthesis of non-specific DNA, which leads to to significant loss of sensitivity.

2. Primers should not form dimers and loops, i.e. stable double strands should not be formed as a result of primers annealing to themselves or to each other.

Which allows you to detect small amounts of, or rather, certain fragments of it in biological material, and multiply them many times. They are then identified visually by gel electrophoresis. The reaction was developed in 1983 by K. Mullis and is included in the list of outstanding discoveries of recent years.

What are the mechanisms of PCR

The whole technique is based on the ability of nucleic acids to replicate independently, which in this case is carried out artificially in a laboratory. DNA reproduction can begin not in any region of the molecule, but only in areas with a certain nucleotide sequence - starting fragments. In order for the polymerase chain reaction to begin, primers (or DNA probes) are needed. These are short fragments of a DNA chain with a given nucleotide sequence. They are complementary (i.e. corresponding) to the starting sites

Of course, in order to create primers, scientists must study the nucleotide sequence of the one involved in the technique. It is these DNA probes that provide the specificity of the reaction and its initiation. will not work if the sample does not contain at least one molecule of the desired DNA. In general, the above primers, a set of nucleotides, and a heat-stable DNA polymerase are required to carry out the reaction. The latter is an enzyme that catalyzes the reaction of synthesis of new nucleic acid molecules based on the sample. All these substances, including the biological material in which DNA needs to be detected, are combined into a reaction mixture (solution). It is placed in a special thermostat, which performs very rapid heating and cooling within a given time - a cycle. Usually there are 30-50 of them.

How does this reaction happen?

Its essence is that during one cycle, primers are attached to the desired sections of DNA, after which it is doubled under the action of an enzyme. Based on the resulting DNA strands, more and more identical fragments of the molecule are synthesized in subsequent cycles.

The polymerase chain reaction proceeds sequentially; the following stages are distinguished. The first is characterized by doubling the amount of product during each heating and cooling cycle. In the second stage, the reaction slows down as the enzyme is damaged and also loses activity. In addition, reserves of nucleotides and primers are depleted. At the last stage - plateau - products no longer accumulate because the reagents have run out.

Where is it used?

Undoubtedly, the polymerase chain reaction is widely used in medicine and science. It is used in general and special biology, veterinary medicine, pharmacy and even ecology. Moreover, in the latter they do this to monitor the quality of food and environmental objects. Polymerase chain reaction is actively used in forensic practice to confirm paternity and identify a person’s identity. In forensic medicine, as well as in paleontology, this technique is often the only way out, since usually an extremely small amount of DNA is available for research. Of course, the method has found very wide application in practical medicine. It is necessary in such areas as genetics, infectious diseases and oncological diseases.

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

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

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

Carrying out PCR

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

Reaction components

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

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

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

Primers

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

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

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

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

Amplifier

Rice. 1: Cycler for PCR

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

Progress of the reaction

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

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

Denaturation

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

Annealing

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

Elongation

Types of PCR

“Nested” PCR (Nested PCR) is used to reduce the number of reaction byproducts. Two pairs of primers are used and two sequential reactions are carried out. The second pair of primers amplifies a region of DNA within the product of the first reaction.
“Inverted” PCR (Inverse PCR) - is used if only a small region within the desired sequence is known. This method is particularly useful when it comes to determining neighboring sequences after DNA has been inserted into the genome. To carry out inverted PCR, a series of DNA cuts with restriction enzymes are carried out, followed by joining of fragments (ligation). As a result, known fragments end up at both ends of the unknown region, after which PCR can be carried out as usual.
Reverse Transcription PCR (RT-PCR) is used to amplify, isolate, or identify a known sequence from an RNA library. Before conventional PCR, a single-stranded DNA molecule is synthesized on an mRNA template using reversease 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 Asymmetric PCR) - is carried out when it is necessary to amplify predominantly one of the source DNA strands. 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.
Quantitative PCR (Q-PCR) is used to quickly measure the amount of specific DNA, cDNA or RNA in a sample.
Quantitative real-time PCR - this method uses fluorescently labeled reagents to accurately measure the amount of reaction product as it accumulates.
Touchdown (Stepdown) PCR (Touchdown PCR) - using this method, the effect of nonspecific binding of primers on the formation of the product is reduced. The first cycles are carried out at a temperature above the annealing temperature, then the temperature is reduced every few cycles. At a certain temperature, the system will pass through the band of optimal primer specificity for DNA.
Molecular colony method (PCR in gel, English. Polony - 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 Rapid amplification of cDNA ends, RACE-PCR )
Long fragment PCR Long-range PCR) - a modification of PCR for the amplification of extended sections of DNA (10 thousand bases or more). Two polymerases are used, one of which is Taq polymerase with high processivity (that is, capable of synthesizing a long chain of DNA in one pass), and the second is DNA polymerase with 3"-5" endonuclease activity. The second polymerase is necessary in order to correct the errors introduced by the first.
RAPD PCR Random Amplification of Polymorphic DNA PCR , 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 one small primer (20 - 25 bp). This primer will be partially complementary to random sections of the DNA of the organisms being studied. By selecting the conditions (primer length, its composition, temperature, etc.), it is possible to achieve a satisfactory difference in the PCR pattern for two organisms.

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

Application of PCR

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

Forensics

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

Establishing paternity

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

Although genetic fingerprints are unique (except in the case of identical twins), family relationships can still be established by making several fingerprints (Figure 3). The same method can be applied, slightly modified, to establish evolutionary relatedness among organisms.

Medical diagnostics

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

Personalized medicine

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

Gene cloning

Gene cloning (not to be confused with cloning of organisms) is the process of isolating genes and, as a result of genetic engineering manipulations, obtaining a large amount of the product of a given gene. PCR is used to amplify a gene, which is then inserted into vector- a DNA fragment that transfers a foreign gene into the same or another organism convenient for cultivation. For example, plasmids or viral DNA are used as vectors. The insertion of genes into a foreign organism is usually used to produce the product of that gene - RNA or, most often, a protein. In this way, many proteins are obtained in industrial quantities for use in agriculture, medicine, etc.

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

DNA sequencing

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

Mutagenesis

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

1. Polymerase chain reaction (PCR)

2. The principle of the polymerase chain reaction method

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

2.2 Cyclic temperature regime

2.3 Basic principles for selecting primers

2.4 Plateau effect

3. Stages of PCR

3.2 Amplification

3.4.1 Positive controls

3.4.2 Internal controls

4.1 Qualitative analysis

4.1.2 Detection of RNA molecules

3.1 Preparation of a biological sample

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

The method for obtaining a pure DNA preparation described by Marmur is considered standard and has already become classical. It includes enzymatic proteolysis followed by deproteinization and reprecipitation of DNA with alcohol. This method allows you to obtain a pure DNA preparation. However, it is quite labor-intensive and involves working with such aggressive and strong-smelling substances as phenol and chloroform.

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

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

Thus, the choice of sample preparation method should be taken into account with an understanding of the purposes of the intended analysis.

3.2 Amplification

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

3.3 Evaluation of reaction results

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

3.3.1 Horizontal electrophoresis method

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

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

3.3.2 Vertical electrophoresis method

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

3.4 Monitoring the progress of the amplification reaction

3.4.1 Positive controls

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

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

3.4.2 Internal controls

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

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

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

4. Methods based on polymerase chain reaction

4.1 Qualitative analysis

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

4.1.1 Method of performing PCR using a “hot start”

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

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

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

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

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

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

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

4.1.2 Detection of RNA molecules

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

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

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

5. Organization of the technological process of performing PCR

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

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

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

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

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

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

Conclusion

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

Not long ago, a reliable, highly sensitive and rapid method for diagnosing various human infectious diseases was developed. This method is called “PCR analysis”. What it is, what its essence is, what microorganisms it can identify and how to take it correctly, we will tell you in our article.

History of discovery

PCR methods are also used in the diagnosis of cancer.

Advantages of the method

PCR diagnostics has a number of advantages:

High sensitivity. Even if only a few microorganism DNA molecules are present, PCR analysis determines the presence of infection. The method will help with chronic and latent diseases. Often in such cases the microorganism is not otherwise culturable.
Any material is suitable for research, for example saliva, blood, genital secretions, hair, epithelial cells. The most common is the PCR test of blood and urogenital smear.
No long-term cultivation of crops is required. The automated diagnostic process allows you to obtain research results after 4-5 hours.
The method is almost one hundred percent reliable. Only isolated cases of false negative results have been recorded.
The ability to identify several types of pathogens from one sample of material. This not only speeds up the process of diagnosing the disease, but also significantly reduces material costs. Often, the doctor prescribes a complex PCR test. The cost of an examination consisting of identifying six pathogens is about 1,500 rubles.

In order for the results to be reliable when conducting a PCR study, you need to take the test, following the recommendations for preliminary preparation for diagnosis:

Before donating saliva, you should refrain from eating and taking medications 4 hours before collecting the material. Immediately before the procedure, rinse your mouth with boiled water.
The above rules should also be followed when taking a sample from the inner surface of the cheek. After rinsing, it is recommended to perform a light massage of the skin to release the secretion of the gland.
Urine is usually collected at home. To do this, you need to thoroughly clean the genitals. 50-60 ml of urine should be collected in a sterile plastic container. To ensure the purity of the material, it is recommended for women to insert a tampon into the vagina, and for men to pull back the skin fold as much as possible. You cannot donate material during your menstrual period.
To donate sperm, you must abstain from sexual intercourse for 3 days before collecting the material. Doctors also advise avoiding visiting the sauna and taking a hot bath, drinking alcohol and spicy foods. You should refrain from urinating 3 hours before the test.
For example, if a PCR test for chlamydia is carried out, both women and men are recommended to have sexual rest for 3 days. 2 weeks before the test you should not take antibacterial drugs. For a week, you need to stop using intimate gels, ointments, vaginal suppositories, and douching. 3 hours before the test you should refrain from urinating. During menstruation, material is not collected; only 3 days after bleeding has stopped, a urogenital smear can be taken.

PCR during pregnancy

While waiting for a baby, many sexually transmitted infectious diseases are extremely dangerous for the normal development of the fetus. STDs can cause intrauterine growth retardation, miscarriage or premature birth, and congenital defects of the child. Therefore, it is extremely important to undergo PCR testing in the early stages of pregnancy. The test must be taken upon registration - up to 12 weeks.

The material is collected from the cervical canal using a special brush. The procedure is painless and does not pose a danger to the baby. Typically, during pregnancy, an analysis is carried out for chlamydia using the PCR method, as well as for ureaplasmosis, mycoplasmosis, cytomegalovirus, herpes, and papillomavirus. This set of examinations is called PCR-6.

PCR for HIV diagnosis

Due to the fact that the method is very sensitive to changes in the body and diagnostic conditions, many factors can affect the result. Therefore, PCR analysis for HIV infection is not a reliable method; its effectiveness is 96-98%. In the remaining 2-4% of cases, the test gives false positive results.

But in some situations, you cannot do without PCR diagnostics of HIV. It is usually performed on people with a false negative ELISA result. Such indicators indicate that a person has not yet developed antibodies to the virus and they cannot be detected without a multiple increase in the number. This is exactly what can be achieved by performing a blood test using the PCR method.

Such diagnostics is also necessary for children in the first year of life born from an HIV-positive mother. The method is the only way to reliably determine the status of a child.

PCR for diagnosing hepatitis

The polymerase chain reaction method allows you to detect the DNA of the hepatitis A, B, C virus long before the formation of antibodies to the infection or the appearance of symptoms of the disease. The PCR test for hepatitis C is especially effective, since in 85% of cases this disease is asymptomatic and without timely treatment becomes chronic.

Timely detection of the pathogen will help to avoid complications and long-term treatment.

Comprehensive PCR examination

Comprehensive PCR analysis: examination using the polymesic chain reaction method, which includes the simultaneous determination of several types of infections: mycoplasma genitalium, mycoplasma hominis, Gardnerella vaginalis, candida, trichomonas, cytomegalovirus, herpes types 1 and 2, gonorrhea, papillomavirus. The price of such diagnostics ranges from 2000 to 3500 rubles. depending on the clinic, the materials and equipment used, as well as the type of analysis: qualitative or quantitative. The doctor will decide which one is necessary in your case. In some cases, it is enough to simply determine the presence of the pathogen; in others, for example, with HIV infection, a quantitative titer plays an important role. When diagnosing all of the above pathogens, the examination is called “PCR-12 analysis.”

Decoding the analysis results

Deciphering the PCR analysis is not difficult. There are only 2 indicator scales - “positive result” and “negative result”. If a pathogen is detected, doctors can confirm the presence of the disease with 99% confidence and begin treating the patient. With the quantitative method of determining infection, the numerical indicator of the detected bacteria will be indicated in the corresponding column. Only a doctor can determine the extent of the disease and prescribe the necessary treatment.

In some cases, for example, when determining HIV infection using the PCR method, if the result is negative, it becomes necessary to conduct additional examinations to confirm the obtained indicators.

Where can I get tested?

Where to take a PCR test: in a public clinic or in a private laboratory? Unfortunately, in municipal medical institutions, equipment and methods are often outdated. Therefore, it is better to give preference to private laboratories with modern equipment and highly qualified personnel. In addition, in a private clinic you will get results much faster.

In Moscow, many private laboratories offer PCR testing for various infections. For example, in such clinics as “Vita”, “Complex Clinic”, “Happy Family”, “Uro-Pro”, PCR analysis is carried out. The price of the examination is from 200 rubles. for identifying one pathogen.

It can be concluded that the diagnosis of infectious diseases using the PCR method in most cases is a quick and reliable way to detect the pathogen in the body in the early stages of infection. But still, in certain cases it is worth choosing other diagnostic methods. Only a specialist can determine the need for such a study. Deciphering the PCR analysis also requires a professional approach. Follow your doctor's recommendations and do not take unnecessary tests yourself.