Polymerase chain reaction, its essence and areas of application. Polymerase chain reaction (PCR) and its application Polymerase chain reaction PCR method

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.

Often used as a rapid method for the indication and identification of viruses.

This method was first developed by K. Mullis (USA) in 1983. Due to its high sensitivity, specificity and ease of implementation, it is widely used in genetics, forensic medicine, diagnostics and other fields.

The essence of the method is amplification, i.e. increasing the number of copies of strictly defined fragments of a DNA molecule in vitro. This method uses a matrix mechanism and the principle of complementarity. Two single polynucleotide chains (nucleic acids) are capable of being linked by hydrogen bonds into one double-stranded chain if the nucleotide sequences of one exactly match the nucleotide sequence of the other so that their nitrogenous bases can form adenine-thymine and guanine-cytosine pairs.

PCR is based on DNA amplification using a thermostable DNA polymerase, which synthesizes mutually complementary DNA strands, starting with two primers. A primer is a DNA fragment consisting of 20-30 nucleotides. These primers are complementary to the opposite DNA strands. During DNA synthesis, primers are inserted into the chain of newly synthesized DNA molecules.

Usually PCR is performed in 25-40 cycles. Each cycle includes three stages: the first is denaturation at 92-95 °C. In this case, the two DNA strands diverge; the second is annealing, or adding primers at 50-65 ° C; the third is elongation, or polymerization at 68-72 ° C, while DNA polymerase carries out complementary completion of the DNA template chains using four types of nucleotides. As a result of one cycle, the desired genetic material is doubled. The DNA chains formed in the first cycle serve as templates for the second cycle, etc. After the first cycle, only the fragment between the two primers is amplified. Thus, the number of copies of the amplified region is doubled, which makes it possible to synthesize millions (2 n) DNA fragments in 25-40 cycles - an amount sufficient for their indication by various methods (the method of hybridization probes containing a certain label, electrophoresis, etc.) . More often, agarose gel electrophoresis with ethidium bromide staining is used for this purpose.

In PCR from DNA sections of a pathogen, primers are used that have a unique sequence of nucleotides characteristic only of a particular pathogen.

The procedure for performing PCR boils down to the following: a DNA matrix is isolated from the material being studied; in a test tube, the isolated DNA is combined with an amplification mixture, which includes DNA polymerase, all 4 types of nucleotides, 2 types of primers, MgCl, buffer, deionized water and mineral oil. Then the tubes are placed in a cycler, and amplification is carried out automatically according to a given program corresponding to the type of pathogen. The results are recorded more often by electrophoresis in a 1-2% agarose gel in the presence of ethidium bromide, which combines with DNA fragments and is revealed in the form of luminous bands when the gel is irradiated with UV rays on a transilluminator. All PCR procedures take 1-2 business days.

In order to increase the specificity and sensitivity of PCR, various options are used: nested PCR; PCR with a “hot start” using a paraffin layer or blocking the active centers of the polymerase with monoclonal antibodies. In addition, some companies produce lyophilized reagent kits for DNA amplification, which speed up the PCR process and reduce the possibility of false-positive results.

A new technology, Real-Time PCR, is currently being introduced. Its fundamental feature is monitoring and quantitative analysis of the accumulation of polymerase chain reaction products and automatic registration and interpretation of the results obtained. This method does not require an electrophoresis step, which reduces laboratory requirements for PCR. Real-time PCR uses fluorescently labeled oligonucleotide probes to detect DNA as it is amplified. Real-time PCR allows for complete analysis of a sample within 20-60 minutes and is theoretically a way to detect even one DNA or RNA molecule in a sample.

The product detection system in real-time polymerase chain reaction (PCR monitoring) allows you to monitor the accumulation of amplified DNA cycle by cycle. The system also includes an oligonucleotide probe that is capable of attaching (hybridizing) to the internal segment of the target DNA. The probe is labeled at the 5′ end with a fluorescent reporter dye, and at the 3′ end with a blocker dye (quencher dye). As the PCR product accumulates, the probe hybridizes to it, but no luminescence occurs due to the proximity between the reporter and the blocker. As a result of copying the sequence, the polymerase reaches the 5′ end of the probe. The 5'-3' exonuclease activity of the polymerase detaches the fluorescent tag from the 3' end of the probe, thereby freeing the fluorescent reporter from its connection with the signal blocker, which leads to an increase in fluorescence. The level of fluorescence is thus proportional to the amount of the specific reaction product. It is important that PCR results are recorded by the presence of fluorescence in closed tubes and, thus, another of the main problems of this method is solved - the problem of contamination with amplicons.

Advantages of PCR: speed of analysis; high sensitivity and specificity; minimum amount of test material; simplicity of execution and the possibility of full automation.

Due to the fact that the sensitivity of PCR can reach up to the detection of one copy of the DNA template, there is a high degree of danger of obtaining false positive results. Therefore, when performing PCR testing, a genetic diagnostic laboratory must strictly comply with special requirements for layout and operating mode.

PCR is one of the complementary methods existing in virological diagnostics. This reaction is very important for the diagnosis of viral infections when viral antigens or virus-specific antibodies cannot be detected and when the presence of viral nucleic acid may be the only evidence of infection, especially in latent and mixed infections.

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Received the Nobel Prize.

At the beginning 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 required to separate the strands of the DNA helix. The reaction procedure was relatively inefficient and required 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 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, Kary Mullis worked as a synthetic chemist (he synthesized oligonucleotides, which were then used to detect point mutations by hybridization with genomic DNA) at 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 Hofmann-La Roche for $300 million. However, it turned out that Taq-polymerase was characterized by Soviet biochemists A. Kaledin, A. Slyusarenko and S. Gorodetsky in 1980, and also 4 years before this Soviet publication, that is, in 1976, by American biochemists Alice Chien, David B. Edgar and John M. Trela. In this regard, the Promega company tried to force Roche to give up exclusive rights to this enzyme in court. 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.
Deoxyribonucleoside triphosphates(dATP, dGTP, dCTP, dTTP).
Mg 2+ ions necessary for the operation of the polymerase.
Buffer solution, providing the necessary reaction conditions - pH, ionic strength of the solution. Contains salts, bovine serum albumin.

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

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

Primers

The specificity of PCR is based on the formation of complementary complexes between the template and primers, short synthetic oligonucleotides 18-30 bases long. Each of the primers 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.

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

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

If the length and nucleotide composition of the primer or 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 (first and last slots) and PCR products

Typically, PCR carries out 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 min 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 the polymerase), the reaction mixture is preheated for 2-3 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.

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

Elongation

DNA polymerase replicates the template strand using a primer as a primer. This is the stage elongation. The polymerase begins synthesizing the second strand from the 3" end of the primer that has bound to the template, and moves along the template, synthesizing a new strand in the direction from the 5" to 3" end. The elongation temperature depends on the polymerase. The commonly used polymerases Taq and Pfu are most active at 72 °C. The elongation time depends on both the type of DNA polymerase and the length of the amplified fragment. Typically, the elongation time is set to one minute per thousand base pairs. After all cycles are completed, an additional step is often performed final elongation, to complete all single-stranded fragments. This stage lasts 7-10 minutes.

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

The amount of specific reaction product (limited by primers) theoretically increases in proportion to 2n - 2n, where n is the number of reaction cycles. In fact, the efficiency of each cycle may be less than 100%, so in reality P ~ (1+E) n, where P is the amount of product, E is the average efficiency of the cycle.

The number of “long” DNA copies also 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. During the last cycles of the reaction, growth slows down, this is called the “plateau effect.”

Types of PCR

Nested PCR(Nested PCR (English)) - 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 (English)) - 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(Reverse Transcription PCR, RT-PCR (English)) - used to amplify, isolate or identify a known sequence from an RNA library. Before conventional PCR, a single-stranded DNA molecule is synthesized on 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(English) 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. Modifications of this method are English. L inear- A fter- T he- E xponential-PCR (LATE-PCR), in which primers with different concentrations are used, and the low concentration primer is selected to have a higher (melting point) than the high concentration primer. PCR is carried out at a high annealing temperature, thereby maintaining the efficiency of the reaction throughout all cycles.
Quantitative PCR(Quantitative PCR, Q-PCR (English)) or Real-time PCR- used to directly monitor the measurement of the amount of a specific PCR product in each reaction cycle. This method uses fluorescently labeled primers or DNA probes to accurately measure the amount of reaction product as it accumulates; or a fluorescent intercalating dye is used Sybr Green I, which binds to double-stranded DNA. Sybr Green I provides a simple and cost-effective option for detection and quantitation of PCR products during real-time PCR without the need for specific fluorescent probes or primers. During amplification, the dye SYBR Green I is embedded in the minor groove of the DNA of PCR products and emits a stronger fluorescent signal when irradiated with a blue laser compared to unbound dye. SYBR Green I compatible with all currently known devices for real-time PCR. Maximum absorption for SYBR Green I is located at a wavelength of 494 nm. In addition to the main one, the spectrum of the dye contains two small additional absorption maxima - at 290 nm and 380 nm. Maximum emission for SYBR Green I is located at a wavelength of 521 nm (green).
Stepwise PCR(Touchdown PCR (English)) - using this approach, the influence of nonspecific primer binding is reduced. The first cycles are carried out at a temperature above the optimal annealing temperature, then every few cycles the annealing temperature is gradually reduced to the optimal one. This is done so that the primer hybridizes with the complementary strand along its entire length; whereas at the optimal annealing temperature, the primer partially hybridizes with the complementary strand. Partial hybridization of the primer on genomic DNA leads to nonspecific amplification if there are many binding sites for the primer. In most cases, the first ten PCR cycles can be carried out at an annealing temperature of 72-75°C, and then immediately reduced to the optimal temperature, for example, to 60-65°C.
Molecular colony method(PCR in gel, English. Colony - PCR Colony) - acrylamide gel is polymerized with all PCR components on the surface and PCR is carried out. At points containing the analyzed DNA, amplification occurs with the formation of molecular colonies.
PCR with rapid amplification of cDNA ends(English) Rapid amplification of cDNA ends, RACE-PCR ).
Long fragment PCR(English) Long-range PCR) - a modification of PCR for the amplification of extended sections of DNA (10 thousand bases or more). A mixture of two polymerases is 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" exonuclease activity, usually Pfu polymerase. The second polymerase is necessary in order to correct errors introduced by the first, since Taq polymerase stops DNA synthesis if a non-complementary nucleotide has been added. This non-complementary nucleotide is removed by Pfu polymerase. The mixture of polymerases is taken in a ratio of 50:1 or even less than 100:1, where Taq polymerase is taken 25-100 times more in relation to Pfu polymerase.
RAPD(English) Random Amplification of Polymorphic DNA ), PCR with random amplification of polymorphic DNA - is used when it is necessary to distinguish between organisms that are close in genetic sequence, for example, different varieties of cultivated plants, dog breeds or closely related microorganisms. This method usually uses one small primer (about 10 bp). This primer will be partially complementary to random sections of the DNA of the organisms being studied. By selecting conditions (primer length, its composition, temperature, etc.), it is possible to achieve a satisfactory difference in the PCR pattern for two organisms.
Group-specific PCR(English) group-specific PCR) - PCR for related sequences within the same or between different species, using conserved primers for these sequences. For example, the selection of universal primers for ribosomal 18S And 26S genes for species-specific intergenic spacer amplification: gene sequence 18S And 26S is conserved between species, so PCR between these genes will work for all species tested. The opposite of this method is - unique PCR(English) unique PCR), in which the task is to select primers to amplify only a specific sequence among related sequences.
PCR using hot start(English) Hot-start PCR) - a modification of PCR using DNA polymerase, in which the polymerase activity is blocked at room temperature by antibodies or small molecules simulating antibodies such as Affibody, that is, at the time of setting up the reaction before the first denaturation in PCR. Typically, the first denaturation is carried out at 95 °C for 10 minutes.
Virtual PCR(eng. in silico PCR, digital PCR, electronic PCR, e-PCR) - a mathematical method of computer analysis of a theoretical polymerase chain reaction using a list of primer sequences (or DNA probes) to predict the potential DNA amplification of the genome, chromosome, circular DNA or any other piece of DNA.

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 N 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, which gave 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

Sometimes 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. The addition of a dideoxynucleotide to the synthesized chain terminates the synthesis, allowing the position of specific nucleotides to be determined after separation in the gel.

Mutagenesis

Currently, PCR has become the main method for carrying out mutagenesis (amending the nucleotide sequence of DNA). 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.

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

What is polymerase chain reaction

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

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

Actively used:

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

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

Who is it prescribed to?

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

Laboratory PCR test is used:

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

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

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

Why undergo such a procedure?

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

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

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

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

Detailed pathogen DNA analysis is used:

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

Types of diagnostics

Standard method

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

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

This detailed video with useful diagrams explains how PCR works:

Other methods

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

Indications for testing

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

Indications for testing:

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

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

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

It is also used to determine infections:

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

Contraindications for

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

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

Is the method safe?

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

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

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

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

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

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

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

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

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

How does the procedure work?

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

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

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

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

Decoding the results

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

Qualitative analysis

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

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

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

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

Quantitative Analysis

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

average cost

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

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

Approximate cost of PCR analysis in rubles:

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

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

Genetics of bacteria. Information for the second lesson.

Polymerase chain reaction

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

The main advantages of PCR as a diagnostic method in microbiology are very high sensitivity, allowing the detection of extremely low concentrations of pathogens in samples, as well as adjustable specificity, allowing the detection or identification of pathogens at the genus, species or subspecies level. The main disadvantage of PCR arises from its extremely high sensitivity—it is very easy for samples to be contaminated with DNA from a positive control, another sample, or a PCR product, resulting in a false-positive reaction. This imposes severe restrictions on the conditions under which PCR mixing and work with finished PCR products is carried out.

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

Isolated DNA from the test sample,

Buffer solution,

Mg2+ ions (necessary for the enzyme to function),

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 being detected.

A mixture of deoxynucleotide triphosphates.

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

This reaction mixture is then placed in a thermal cycler, which is essentially a programmable thermostat. The thermal cycler carries out 30-40 cycles of temperature changes. Each of these cycles consists of three stages (see Fig. 1):

Denaturation (temperature 94 o C) - hydrogen chains are broken and DNA strands diverge.

Annealing of primers (temperature is usually around 50-60 o C) - primers are attached to the ends of the DNA chains. In general, when the temperature decreases, it is energetically more favorable to reunite the original DNA chains from the test sample (renaturation), however, the concentration of primers in the reaction mixture is many orders of magnitude greater 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 o C) - DNA polymerase completes the primers along the template of long DNA chains. The temperature corresponds to the optimal operating temperature of the DNA polymerase used.

Detection of results differs in different versions of PCR and is described in the “Types of PCR” section.

PCR dynamics

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

Thus, in early cycles, the amount of 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 occurs 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 Figure 3).

Types of PCR

Conventional PCR

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

This option for performing 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. Quantitative assessment of the content of the original DNA molecules in the sample is impossible due to the reaction reaching a plateau.

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

Real-time PCR

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

One type of real-time PCR is using an intercalating dye, which 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 option is simpler, faster, and also does not require opening tubes with PCR products, which reduces the likelihood of contamination of other samples. The main disadvantage is the higher cost of a thermal cycler with built-in fluorescence detection capabilities compared to a conventional one.

Digital quantitative PCR

A new, expensive and still rarely used version of PCR that allows you to more accurately determine 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 PCR, it is analyzed in what fraction of droplets the reaction was positive and, accordingly, fluorescence is observed. This fraction will be proportional to the number of DNA molecules being sought in the sample.

Reverse transcription PCR

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

Picture 1. PCR stages. Primers are indicated in red.

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

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

Figure 4. Agarose electrophoresis of PCR products. K+ – positive control (the desired DNA is known to be present). 1-7 – test samples (of which 1-2 are positive, 3-7 are negative). K- – negative control (the desired DNA is obviously absent). 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 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 resonance transfer of fluorescence energy (FRET).