The DNA polymerase reaction is positive. PCR analysis: what is it? How to take a PCR test correctly. PCR is the main method for detecting hidden infections

Polymerase chain reaction (PCR) is a method of biochemical technology in molecular biology, carried out to increase one or several copies of DNA fragments by several degrees, which makes it possible to create from several thousand to millions of copies of a specific DNA sequence.

Developed in 1983 by Cary Mullis, PCR is now a common and often indispensable technique used in medical and biological research laboratories for a variety of different applications. These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; diagnosis of hereditary diseases; detection of genetic fingerprints (used in forensic science and paternity testing), and detection and diagnosis of infectious diseases. In 1993, Mullis was awarded the Nobel Prize in Chemistry along with Michael Smith for their work on PCR.

The method is based on thermal cycling, consisting of repeated cycles of heating and cooling a reaction to denature and replicate DNA by enzymes. Primers, (short pieces of DNA) containing sequences complementary to the target site along with DNA polymerase (from which the method gets its name), are key components for triggering selective and repeated amplification. In the PCR process, the synthesized DNA itself is used as a template for replication, setting in motion a chain reaction in which the DNA template is amplified exponentially. PCR can be significantly modified to perform a wide range of genetic manipulations.

Almost all PCR applications use a thermostable DNA polymerase such as Taq polymerase, an enzyme originally isolated from bacteria Thermusaquaticus. This DNA polymerase enzymatically assembles a new strand of DNA from the building blocks of DNA - nucleotides, using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers) that are necessary to initiate DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternating heating and cooling of the PCR sample over a certain series of temperature steps. These thermal cycling steps are necessary to first physically separate the two strands of the DNA double helix at high temperature in a process called DNA denaturation. At lower temperatures, each strand will be used as a template in DNA synthesis by DNA polymerase in order to selectively amplify the target DNA region. Selectivity of PCR results using primers that are complementary to the target DNA region for amplification under certain thermal cycling conditions.

Principles of PCR diagnostics

PCR is used to amplify a specific section of a DNA strand (target DNA). Most PCR methods typically amplify DNA fragments up to ~10,000 base pairs (kb), although some methods can increase fragments up to 40 kb in size. The reaction produces a limited amount of the final amplified product, which is regulated by the available reagents in the reaction and feedback inhibition of the reaction products.

The basic PCR kit requires several components and reagents. These include:

• DNA template, containing the target DNA region that needs to be amplified.
• Two primers complementary to the 3" ends of each of the sense and antisense strands of the target DNA.
• Taq polymerase or other DNA polymerase operating at an optimal temperature of about 70 °C.
• Deoxynucleoside triphosphates(dNTPs; triphosphate groups containing nucleotides), the building blocks from which DNA polymerase synthesizes a new strand of DNA.
• Buffer solution, providing suitable chemical conditions for optimal DNA polymerase activity and stability.
• Divalent cations magnesium or manganese ions; Mg2+ is commonly used, but Mn2+ can also be used for PCR-mediated DNA mutagenesis, since higher concentrations of Mn2+ increase the error rate during DNA synthesis.
• Monovalent cations potassium ions.

PCR is usually carried out in a reaction volume of 10-200 µl in small reaction tubes (volume 0.2-0.5 ml) in a thermal cycler amplifier. The amplifier heats and cools the reaction tubes to achieve the temperatures required for each step of the reaction. Many modern thermal cyclers use the Peltier effect, which allows a block of PCR tubes to be heated and cooled simply by changing the direction of the electric current. Thin-walled reaction tubes promote favorable thermal conductivity to ensure rapid thermal equilibrium. Older cyclers that do not have a heated lid require a layer of oil on the surface of the reaction mixture or a bead of wax in a test tube.

Procedure

Typically, PCR consists of a series of 20-40 repeated temperature changes called cycles, with each cycle typically consisting of 2-3 discrete temperature steps, usually three. Cycling often begins and ends with one temperature step (called waiting) at high temperature (>90°C) for final product expansion or short-term storage. The temperatures used and the length of time they are applied in each cycle depend on many parameters. These include the enzyme used to synthesize the DNA, the concentration of divalent ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers.

• Initialization phase: This step consists of heating the reaction to a temperature of 94-96 °C (or 98 °C if highly thermostable polymerases are used) for 1-9 minutes. The step is required only for DNA polymerases that require activation by heat, the so-called hot start PCR.
• Denaturation stage: Is the first regular thermal cycling event and consists of heating the reaction to 94-98°C for 20-30 seconds. This causes cleavage of the DNA template with the destruction of hydrogen bonds between complementary bases and the formation of single-stranded DNA molecules.
• Annealing stage: The reaction temperature is reduced to 50-65°C within 20-40 seconds, which allows the primers to bind to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are formed only when the primer sequence more closely matches the sequence template. The polymerase binds to the primer-template hybrid and begins DNA synthesis.
• Expansion/elongation stage: The temperature at this stage depends on the DNA polymerase used; Taq polymerase has its optimal temperature of activity at 75-80°C; A temperature of 72°C is commonly used for this enzyme. At this stage, DNA polymerase synthesizes a new DNA strand complementary to the template DNA strand, adding dNTPs that are complementary to the template in a 5" to 3" direction, linking the 5"-phosphate group of the dNTP with the 3"-hydroxyl group at the end of the resulting (extending) ) DNA. The extension time depends on both the DNA polymerase used and the length of the DNA fragment that needs to be amplified. Typically, at its optimal temperature, DNA polymerase polymerizes one thousand bases per minute. Under optimal conditions, i.e. in the absence of limitations due to limiting substrates or reagents, at each expansion step, the amount of target DNA doubles, resulting in exponential (geometric) amplification of the DNA fragment.
• Final extension: This is a single step, sometimes performed at 70-74°C for 5-15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA has been fully elongated.
• Final expectation: This step at 4-15°C indefinitely can be used to maintain the reaction for a short time. To test whether PCR has synthesized the expected DNA fragment (also sometimes called an "amplimer" or "amplicon"), agarose gel electrophoresis is used to separate the PCR products by size. The size of PCR products is determined by comparison with a DNA ladder (molecular weight marker), which contains DNA fragments of known size, performed on a gel along with the PCR products.

Polymerase chain reaction steps

The PCR process can be divided into three stages:

1. Exponential amplification: During each cycle, the amount of product doubles (assuming 100% reaction efficiency). The reaction is very sensitive: only a small amount of DNA is required.
2. Leveling stage: The reaction slows down as DNA polymerase loses activity and consumption of reagents such as dNTPs and primers causes them to become limiting .
3. Plateau: Product no longer accumulates due to depletion of reagents and enzymes.

PCR optimization

In practice, PCR may fail for various reasons, particularly its sensitivity to contamination, which causes amplification of DNA by-products. In this regard, a number of techniques and procedures have been developed to optimize PCR conditions. Contamination of extraneous DNA is addressed by laboratory protocols and procedures that purify pre-PCR mixtures of potential DNA contaminants. This typically involves spatially separating PCR kits from areas for analysis or purification of PCR products, using disposable plastic utensils, and thoroughly cleaning the work surface between reaction steps. Primer design techniques play an important role in improving the recovery of PCR products and avoiding the formation of by-products, and the use of alternative buffer components or polymerase enzymes can aid in the amplification of long or otherwise problematic stretches of DNA. Addition of reagents such as formamide to buffer systems can increase the specificity and recovery of PCR. Computer simulation of theoretical PCR results (electronic PCR) can be performed to assist in primer design.

Application of PCR

Selective DNA extraction

PCR allows the isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA. This application of PCR complements many techniques, such as the generation of hybridization probes for Southern or Northern blotting and DNA cloning, which require large quantities of DNA representing a specific region of DNA. PCR provides these methods with a high content of pure DNA, allowing DNA samples to be analyzed even with small amounts of starting material.

Other applications of PCR include DNA sequencing to identify unknown PCR-amplified sequences, in which one of the amplification primers can be used in Sanger sequencing, DNA sequence extraction to accelerate recombinant DNA technologies involving the insertion of a DNA sequence into a plasmid or genetic material of another organism. Colonies of bacteria (E. coli) can be quickly screened by PCR to correct the vector DNA design. PCR can also be used for genetic fingerprinting; a technique used in forensic medicine to identify a person or organism by comparing experimental DNA using various PCR methods.

Some PCR fingerprinting methods have high discriminatory power and can be used to determine genetic relationships between individuals, such as parent-child or between siblings, and are used in paternity detection. This technique can also be used to determine evolutionary relationships between organisms.

DNA amplification and quantification

Because PCR increases the copy number of DNA regions that are targeted, PCR can be used to analyze very small quantities of a sample. This is often critical in forensic cases where only trace amounts of DNA are available as evidence. PCR can also be used to analyze ancient DNA that is tens of thousands of years old. These PCR methods have been successfully used on animals such as the forty-thousand-year-old mammoth, as well as human DNA, in applications ranging from the analysis of Egyptian mummies to the identification of the Russian Tsar.

Quantitative PCR methods estimate the amount of a given sequence present in a sample, a method often used to quantify the level of gene expression. Real-time PCR is an established tool for quantitative DNA analysis that measures the accumulation of product DNA after each PCR amplification cycle.

PCR in disease diagnosis

PCR allows early diagnosis of malignant diseases such as leukemia and lymphoma, which is currently highly developed in cancer research and is already used routinely. PCR can be performed directly on genomic DNA samples to detect translocation-specific malignant cells with a sensitivity that is at least 10,000 times higher than other methods.

PCR can also detect uncultured or slow-growing microorganisms such as mycobacteria, anaerobic bacteria, and viruses from tissue culture and animal models. The basis for PCR diagnostic applications in the field of microbiology is the identification of infectious agents and the differentiation of non-pathogenic strains from pathogenic ones due to specific genes.

Viral DNA can also be detected using PCR. Primers must be specific to target viral DNA sequences, and PCR can be used for diagnostic DNA tests or sequencing of the viral genome. The high sensitivity of PCR allows you to detect viruses soon after infection and even before the onset of the disease. This early detection of the virus could give doctors significant treatment options. The amount of virus (“viral load”) in a patient can also be quantified using PCR-based DNA testing.

Variations of Basic Polymerase Chain Reaction Methods

• Allele-specific PCR: A diagnostic or cloning method based on single nucleotide polymorphisms (SNPs) (differences of a single base in DNA). Requires prior knowledge of DNA sequence, including differences between alleles, and uses primers whose 3" ends span the SNP. PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer, so successful amplification with an SNP-specific primer signals about the presence of specific SNPs in the sequence.
• PCR assembly or polymerase cycling assembly (PCR): artificial synthesis of long DNA sequences by performing PCR on a reserve of long oligonucleotides with short overlapping segments. The oligonucleotides alternate between the sense and antisense strand directions, and the overlapping segments determine the order of the PCR fragments, thereby selectively producing the final long DNA product.
• Asymmetric PCR: Preferentially amplifies one DNA strand in a double-stranded DNA template. Used in sequencing and hybridization probing where amplification of only one of the two complementary strands is required. PCR is carried out as usual, but with a large excess of primers for the strand to be amplified. Due to the slow (arithmetic progression) amplification at the end of the reaction after using the limiting primer, additional PCR cycles are required. The latest modification of this process, known as "LATE-PCR" (linearity after exponential phase - PCR), uses a limiting primer with a higher melting temperature (Tm) than the excess primer to maintain reaction efficiency as the concentration of the limiting primer decreases midway through the reaction.
• Dial-out PCR: A highly parallel method to obtain precise DNA molecules for gene synthesis. The complex pool of DNA molecules is modified by unique flanking tags prior to massively parallel sequencing. Tag-directed primers then produce molecules with a given sequence using PCR.
• Helicase-dependent amplification: similar to traditional PCR but requires a constant temperature than cycling through denaturation and annealing/extension cycles. DNA helicase, an enzyme that unwinds DNA, is used instead of heat denaturation.
• Hot start PCR: A technique that reduces nonspecific amplification during the initial setup of PCR steps. Can be done manually by heating reaction components to denaturation temperature (e.g. 95 °C) before adding polymerase. Specialized enzyme systems have been developed that inhibit polymerase activity at room temperature, either by binding antibodies or in the presence of covalently bound inhibitors that dissociate only after a high temperature activation step. “Hot start/cold finish” PCR is achieved using new hybrid polymerases that are inactive at ambient temperature and immediately activated at elongation temperature.
• Intermicrosatellite sequence specific PCR (ISSR): PCR is a DNA fingerprinting method that increases the copy number of regions between simple repeat sequences to produce a unique fingerprint from the amplified length of the fragment.
• Inverted PCR widely used to identify sequence regions around genomic insertions. It involves a series of DNA cleavages and self-ligations that produce known sequences at either end of an unknown sequence.
• Ligation-mediated PCR: Uses small DNA linkers connected to the DNA of interest and multiple primers linked to the DNA linkers; used for DNA sequencing, genome walking, and DNA footprinting.
• Methylation-specific PCR(MSP): developed by Stephen Baylin and Jim Herman at the Johns Hopkins School of Medicine, used to detect methylation of CpG islands in genomic DNA. The DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is recognized by PCR primers as thymine. Two PCRs are then performed on the modified DNA using sets of identical primers, except at any CpG island within the primer sequence. At these points, one set of primers recognizes DNA with cytosines to increase the copy number of methylated DNA, and one set recognizes DNA with uracil or thymine to amplify unmethylated DNA. MSP using qPCR can also be performed to obtain quantitative rather than qualitative methylation information.
• Miniprimer - PCR: thermostable polymerases (S-Tbr) are used, which can expand from short primers (“smalligos”), with a number of 9 or 10 nucleotides. This method allows PCR to target regions bound by smaller primers and is used to amplify conserved DNA sequences such as the 16S (or eukaryotic 18S) rRNA gene.
• Multiplex ligation-dependent probe amplification (MLPA): allows the amplification of multiple targets with only one pair of primers, thus avoiding the resolution limitations of multiplex PCR.
• Multiplex PCR consists of multiple sets of primers in a single PCR mixture to produce amplicons of different sizes that are specific for different DNA sequences. By targeting several genes simultaneously, it is possible to obtain additional information during a single test, which would otherwise require several times more reagents and more time to perform. Annealing temperatures for each primer set must be optimized to work correctly within a single reaction and with amplicon sizes. That is, their base pair lengths must be different enough to produce distinct bands when visualized by gel electrophoresis.
• Nested PCR: Increases the specificity of DNA amplification by reducing background due to non-specific DNA amplification. Two sets of primers are used in two sequential PCRs. In the first reaction, one pair of primers is used to synthesize DNA products that, in addition to the intended target, may still consist of nonspecifically amplified DNA fragments. The products are then used in a second PCR with a set of primers whose binding sites are completely or partially different from the 3" ends of each of the primers used in the first reaction. Nested PCR is often more successful at specifically amplifying long DNA fragments than traditional PCR, but it requires more detailed knowledge of target sequences.
• PCR with overlapping extensions or splicing by overlapping extensions(SOE): A genetic engineering technique that is used to join two or more pieces of DNA that contain complementary sequences. Used to connect parts of DNA containing genes, regulatory sequences, or mutations; The technique allows you to create specific and long DNA structures.
• Quantitative PCR (qPCR): used to measure the amount of PCR product (usually in real time). Quantitatively measures initial amounts of DNA, cDNA or RNA. qPCR is widely used to determine the presence of a DNA sequence in a sample, and the number of its copies in the sample. Quantitative real-time PCR has a very high degree of accuracy. QRT-PCR (or QF-PCR) methods use fluorescent dyes such as Sybr Green, EvaGreen, or fluorophore-containing DNA probes such as TaqMan to measure the amount of amplified product in real time. Sometimes referred to by the abbreviation RT-PCR (real-time PCR) or RQ-PCR. QRT-PCR or RTQ-PCR are more appropriate abbreviations, since RT-PCR usually refers to reverse transcription PCR, often used in combination with qPCR.
• Reverse transcription PCR (RT-PCR): to increase the number of copies of DNA from RNA. Reverse transcriptase transcribes the RNA into cDNA, which is then amplified using PCR. RT-PCR is widely used in expression profiling to detect gene expression or to determine the sequence of an RNA transcript, including transcription start and stop sites. If the genomic DNA sequence of a gene is known, RT-PCR can be used to map the location of exons and introns in the gene. The 5" end of the gene (corresponding to the transcription start site) is usually determined by RACE-PCR (rapid amplification of cDNA ends).
• Solid phase PCR: covers several meanings, including "Polonium Amplification" (where colony PCRs are performed on a gel matrix, for example), "Bridge PCR" (primers are covalently linked to a solid support surface), traditional solid phase PCR (where "asymmetric PCR" is used in presence of primers bearing a solid support with a sequence corresponding to one of the aqueous primers), and enhanced solid phase PCR (where traditional solid phase PCR can be improved by the use of high Tm and nested solid support primers with the option of applying a thermal "step" to promote the formation of solid-supported primers).
• Thermal Asymmetric Alternating PCR (TAIL-PCR): used to isolate an unknown sequence following a known sequence. In a known sequence, TAIL-PCR uses a nested pair of primers with different annealing temperatures; a degenerate primer is used to amplify in a different direction from the unknown sequence.
• Touchdown PCR (step-by-step PCR): a variant of PCR aimed at reducing nonspecific background by gradually lowering the annealing temperature as PCR cycles progress. The annealing temperature on initial cycles is typically several degrees (3-5°C) above the Tm of the primers used, while on later cycles, the temperature is several degrees (3-5°C) below the Tm of the primers. Higher temperatures provide greater specificity for primer binding, and lower temperatures allow more efficient amplification from specific products generated during the initial cycles.
• PAN-AC: Uses isothermal conditions for amplification and can be used on living cells.
• A universal quick walk through the genome: for genome walking and genetic fingerprinting using more specific "two-way" PCRs than traditional "one-way" approaches (using only one gene-specific primer and one general primer - which can lead to artifactual "noise") due to the mechanism, including the formation of a lasso structure. Simplified derivatives of UFW are "Lane RAGE" (lasso-dependent nested PCR for rapid amplification of genomic DNA ends), "5"RACE Lane" and "3"RACE Lane".
• InsilicoPCR(digital PCR, virtual PCR, e-PCR, e-PCR) refers to computational tools used to calculate the results of a theoretical polymerase chain reaction using a given set of primers (probes) to amplify DNA sequences from a sequenced genome or transcriptome.

History of PCR

An article in the Journal of Molecular Biology in 1971 by Kleppe and his co-authors first described a method using an enzymatic assay to replicate a short DNA template with primers in vitro. However, this early manifestation of the basic principle of PCR did not receive much attention, and the invention of the polymerase chain reaction in 1983 is generally credited to Kary Mullis.

When Mullis developed PCR in 1983, he was working in Emeryville, California for Cetus Corporation, an early biotechnology company. There he was responsible for the synthesis of short DNA chains. Mullis wrote that he conceived the idea of ​​PCR while driving along the Pacific Coast Highway one night in his car. He was replaying in his mind a new way of analyzing changes (mutations) in DNA when he realized that he had instead invented a method for increasing the number of copies of any piece of DNA through repeated cycles of duplication driven by DNA polymerase. In Scientific American, Mullis summarized the procedure: “Starting with one molecule of DNA genetic material, PCR can generate 100 billion such molecules in a single day. This reaction is easy to perform. It requires no more than a test tube, a few simple reagents and a heat source." He was awarded the Nobel Prize in Chemistry in 1993 for his invention, seven years after he and his colleagues at Cetus first put his proposal into practice. However, some controversy remained about the intellectual and practical contributions of other scientists to Mullis's work, and whether he was the sole inventor of the PCR principle.

The PCR method relies on the use of a suitable DNA polymerase that can withstand the high temperatures >90°C (194°F) required to cleave the two DNA strands in the DNA double helix after each replication cycle. DNA polymerases originally used for in vitro experiments, foreshadowing PCR, were unable to withstand such high temperatures. Therefore, early DNA replication procedures were very inefficient and time-consuming, requiring large amounts of DNA polymerase and continuous processing throughout the process.

Discovery in 1976 of Taq polymerase, a DNA polymerase isolated from a thermophilic bacterium, Thermusaquaticus, which naturally lives in hot (50 to 80°C (122 to 176°F)) environments such as hot springs - paved the way for a dramatic improvement in the PCR method. DNA polymerase isolated from T.Aquaticus, is stable at high temperatures and remains active even after DNA denaturation, thereby eliminating the need to add new DNA polymerases after each cycle. This made it possible to automate the process of DNA amplification based on a thermocycler amplifier.

Patent wars

The proposed PCR method was patented by Cary Mullis and attributed to Cetus Corporation, where Mullis worked when he invented the technique in 1983. The enzyme Taq polymerase was also protected by patents. There have been several high-profile lawsuits related to the technique, including an unsuccessful lawsuit brought by DuPont. The pharmaceutical company Hoffmann-La Roche acquired the rights to the patents in 1992 and currently holds those that are still protected.

A similar patent battle over the Taq polymerase enzyme is still ongoing in some jurisdictions around the world between Roche and Promega. Legal arguments extended beyond the terms of the original PCR and Taq polymerase patents, which expired on March 28, 2005.

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 donor 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:

1. When examining for sexually transmitted infections, secretions from the genital organs, urine, and a smear from the urethra are collected.
2. 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.
3. In order to diagnose various types, cerebrospinal fluid is collected.
4. In pulmonology, samples for analysis are sputum and pleural fluid.
5. 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):

1. 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.
2. 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.
3. 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:

Received the Nobel Prize.

At the beginning of the method, after each heating-cooling cycle, it was necessary to add DNA polymerase 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 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.

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.

S.V. Pospelova, M.V. Kuznetsova

Polymerase chain reaction

S.V. Pospelov– Ph.D. honey. Sciences, Associate Professor of the Department of Microbiology, Virology and Immunology, M.V. Kuznetsova– Ph.D. biol. Sciences, employee of IEGM Ural Branch of the Russian Academy of Sciences

Pospelova, S.V.

Designed for independent work by students of all faculties: medical, pediatric, medical and preventive, dental and the Faculty of Higher Nursing Education (FVSO) of the Medical Academy.

Reviewer:

head Department of Biology, Ecology and Medical Genetics PGMA, Professor A.B. Vinogradov

Printed by decision of the central coordination
Methodological Council of State Educational Institution of Higher Professional Education PGMA
them. ak. E.A. Wagner Roszdrav

UDC 616-078.33

© Pospelova S.V., Kuznetsova M.V., 2007

© State Educational Institution of Higher Professional Education PGMA named after. ak. E.A. Wagner Roszdrav, 2007

Polymerase chain reaction in clinical
microbiological diagnostics

Modern medicine successfully uses the achievements of natural sciences and intensively applies new technologies for the diagnosis and treatment of diseases. Recently, new ones based on the use of molecular genetic technologies have been added to traditional microbiological and immunological methods for laboratory diagnosis of infectious diseases. The use of these methods not only for scientific purposes, but also in practical laboratory diagnostics became possible to a large extent thanks to the creation in the mid-80s of the process of artificial multiple copying of DNA and the further rapid development of this technology, currently known as polymerase chain reaction(PCR). In less than 15 years of its existence, PCR has made routine analysis of specific DNA sequences of many pathogenic microorganisms. Its versatility, high sensitivity and relative ease of execution have made the PCR method indispensable for solving various clinical diagnostic problems, such as direct detection and identification of pathogens, molecular typing and study of the properties of pathogenic microorganisms, analysis of mutations associated with genetic diseases in humans, and identification of human personality.

What is PCR?

Polymerase chain reaction(PCR) - an artificial process of repeated copying (amplification) specific DNA sequence, carried out in vitro(Fig. 1). DNA copying during PCR is carried out by a special enzyme - DNA polymerase as in the cells of living organisms. DNA polymerase, moving along a single DNA strand (template), synthesizes a DNA sequence complementary to it. It is important that DNA polymerase cannot start synthesizing a DNA strand “from scratch”; it needs a short “seed” strand of RNA or DNA to which it can begin to attach nucleotides. The basic principle of PCR is that the polymerization reaction (synthesis of a DNA polymer chain from monomeric nucleotide units) is initiated by specific primers(short fragments of “seed” DNA) in each of many repeating cycles. The specificity of PCR is determined by the ability of primers to “recognize” a strictly defined section of DNA and bind to it according to the molecular principle complementarity.

A typical PCR reaction uses a pair of primers that “limit” the region to be amplified on both sides by binding to opposite strands of the DNA template. 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 double-stranded DNA: before the start of the reaction, the target DNA is double-stranded; at a temperature of 94-95 0 C, the complementary DNA strands diverge and transform into a single-stranded state;

2) binding (annealing) primers: at a temperature optimal for the selected primers, they bind to the complementary region of the template DNA;

3)elongation, or chain elongation: DNA polymerase adds nucleotides to primers, synthesizing new DNA strands that become targets for primers in subsequent PCR cycles.

Changing the stages of each cycle is carried out by changing the temperature of the reaction mixture (see Fig. 1).

Rice. 1. Main stages of the PCR cycle

At first, the primers can only bind to a specific sequence of the original DNA, but in subsequent cycles they bind to copies of that sequence synthesized in previous cycles. In this case, the amount of the main PCR product (a copy of the DNA sequence limited by the primers) theoretically doubles in each cycle. If at the initial cycle there was only one DNA target in the material under study, after the first cycle there will already be two copies, after two cycles – 4 copies, the result of the third cycle will be 8 copies, and the thirty-fifth – already 68 billion copies (Fig. 2).

Rice. 2. Multiple copying process
Target DNA during sequential
changing cycles

The main method for analyzing reaction products, which is traditionally used in many laboratories to detect amplified DNA and determine its size, is the method gel electrophoresis followed by staining with a DNA-specific dye, such as ethidium bromide (Fig. 3).

Control - various DNA fragments with a known number of their constituent nucleotides. It is known that the distance between different fragments has a logarithmic dependence on their size and mass. Line 1 – PCR fragments approximately 1850 bases long were detected. Lines 2 and 4 are fragments about 800 bases long.

Rice. 3. Analysis of reaction products by method
gel electrophoresis

Line 3 – the required fragments have not been identified, the reaction result is negative. Line 5 - multiple lines were formed because the primers were complementary to several DNA fragments of different lengths: about 550, 800 and 1500 bases.

Improvement of PCR technology

Initially, conventional DNA polymerases were used to carry out PCR, which were subjected to temperature inactivation in each cycle at the stage of DNA denaturation. The polymerase had to be added to the reaction mixture many times, which was quite labor-intensive and did not allow the process to be automated.

The reaction uses thermostable DNA polymerases that can withstand high temperatures at all stages of the PCR cycle for several tens of cycles. The number of commercially available thermostable DNA polymerases, differing in some of their properties, is quite large. Most commonly used Taq polymerase originally isolated from a thermophilic microorganism Thermus aquaticus. Other polymerases are more commonly used for specific PCR applications. Modern commercial preparations of thermostable polymerases, as a rule, provide stable, reproducible activity, which allows the use of PCR technology in standard laboratory practice.

The technical design of changing the temperature of the reaction mixture has also developed rapidly recently. Initially, PCR was carried out using three water baths set at different temperatures: for DNA denaturation, primer annealing and polymerization. The test tubes were transferred from one water bath to another “in a circle”, due to which the temperature changed at different stages of the cycle. There were also variants of devices where water of different temperatures was alternately supplied to a water bath in which test tubes with a reaction mixture were located. Changing cycles in these cases took a long time, and the process was difficult to automate. To carry out PCR, devices are mainly used (thermocyclers), which change the temperature automatically based on a given program. In thermal cyclers, tubes with a reaction mixture are placed in a metal block, the temperature of which changes at a high speed, which reduces the duration of each PCR cycle.

Modern thermal cyclers are adapted to use special thin-walled plastic tubes for the reaction mixture, which speeds up the heat exchange between the device block and the reaction mixture and ultimately further reduces the reaction time.

Thus, standard PCR can be carried out in 1-3 hours. Many devices allow programming of special, sophisticated temperature profiles necessary for specific modifications of the PCR process.

In parallel with the improvement of PCR technology, methods for analyzing reaction products also developed. Method gel electrophoresis followed by staining with a DNA-specific dye, such as ethidium bromide, is traditionally used in many laboratories to detect amplified DNA and determine its size. Usage hybridization with internal DNA probes allows in some cases to significantly increase the sensitivity and specificity of detection of PCR products. By eliminating the need to prepare and perform electrophoretic separations, the ability to automate the analysis of large numbers of samples, and the use of a non-radioactive detection format, this method is becoming increasingly common. In some cases, the use of special fluorescent “markers” makes it possible to control the amplification or detection of final PCR products directly in the reaction tube.

Use of PCR
in medical microbiology

Among the many different areas of clinical diagnostics, medical microbiology occupies, perhaps, a leading position in the number and variety of applications using PCR technology. The introduction of this method into practice, along with serological diagnostics, has significantly expanded the capabilities of modern clinical microbiology, which is still based on methods for isolating and cultivating microorganisms on artificial nutrient media or in cell culture.

Opportunities and limitations of traditional
cultivation methods

The traditional culture diagnostic method for microbiological laboratories, as a rule, works well for identifying and studying properties such as sensitivity to antibiotics and virulence of easily cultivated microorganisms. However, some microorganisms (pneumococci, hemophilus, neisseria, mycoplasmas, obligate anaerobes, etc.) can be extremely sensitive to the conditions of collection of clinical material, transportation and cultivation, the presence of special growth factors, or are capable of reproduction in vitro only in cell culture (viruses, chlamydia, rickettsia).

The slow growth of microorganisms such as mycobacteria and fungi on artificial media is another natural limitation associated with the use of culture methods for the diagnosis of these microorganisms. In addition, working with live cultures of isolated pathogens, not only especially dangerous ones, but sometimes also opportunistic pathogens, can pose a threat to the health of laboratory personnel.

Among the causative agents of human diseases, unculturable species of bacteria are also known, for example Mycobacterium leprae, Treponema pallidum, and many types of viruses, including human papilloma and hepatitis C viruses, attempts to grow them in cell culture have so far remained unsuccessful. Finally, even with successful cultivation, there is a need for subsequent identification of the isolated microorganisms.

Traditional microbiological identification methods are based on the use of various phenotypic tests, such as the detection of specific enzymatic activity, the ability to metabolize sugars or support growth on media with selective additives. The difficulty of standardizing the conditions of such tests, as well as the natural phenotypic variability inherent in many microorganisms, can cause misidentification.

Using PCR for direct diagnosis
and identification of pathogens
infectious diseases

In cases where the use of cultural methods is problematic or associated with insufficient diagnostic efficiency, the possibility of replacing biological amplification (that is, growth on artificial media) with enzymatic duplication of nucleic acids in vitro with using PCR seems particularly attractive. There are various approaches to using PCR to diagnose infectious agents. The most common PCR option (specific PCR) involves the use of primers complementary specific sequence DNA characteristic of a strictly defined type of microorganism. For example, PCR amplification of a specific region of the gene encoding the major outer membrane protein (MOMP) Chlamydia trachomatis, in combination with non-radioactive hybridization to detect reaction products, it makes it possible to detect single copies of chlamydial DNA in the studied samples. At the same time, PCR is significantly superior in diagnostic efficiency to cultivation and methods of direct detection of chlamydial antigen (microimmunofluorescence and enzyme-linked immunosorbent assay), traditionally used to detect C. trachomatis.

It is also possible to use several pairs of species-specific primers in one reaction tube for simultaneous amplification of DNA from various pathogens. This modification is called multiplex PCR. (multiplex PCR). Multiplex PCR can be used to identify the etiological role of various microorganisms that cause a particular type of disease. For example, options for using multiple PCR for the simultaneous detection of two (S. trachomatis And N.gonorrhoeae for diseases of the urogenital tract) or even four pathogens (I. influenzae, S. pneumoniae, M. catarrhalis And A. otitidis with chronic purulent otitis).

An alternative approach to PCR diagnostics involves the use of universal primers, which allow amplification of gene fragments present in all microorganisms of a certain taxonomic group. The number of species that can be identified using this method can be limited both by small systematic groups (genus, family) and large taxa at the level of order, class, and phylum. In the latter case, the targets for PCR are most often ribosomal genes (16S and 23S rRNA), which have a similar structure in various prokaryotic microorganisms.

The use of primers complementary to conserved regions of these genes allows DNA amplification of most bacterial species. The resulting PCR fragments of ribosomal genes can then be analyzed using various laboratory methods to identify the bacteria to which they belong. The most accurate method of "molecular" identification is to determine the complete nucleotide sequence (sequencing) of the amplified DNA and compare it with the corresponding sequences of known species.

Despite the availability of automated systems that use the described identification principle, in practice, less labor-intensive and expensive methods are usually used, which nevertheless make it possible to reliably detect certain differences in the sequence of DNA fragments. The most common methods are based on the analysis of the location of DNA cleavage sites with restriction enzymes (RFLP method - restriction fragment length polymorphism), or by determining the electrophoretic mobility of DNA in single-stranded form (SSCP-method single-strand conformational polymorphism).

PCR using universal primers can be used both to identify microorganisms isolated in pure culture and to directly diagnose a wide range of pathogens directly in clinical samples. It should be noted, however, that the sensitivity of “broad-spectrum” PCR is generally lower compared to “species-specific” test systems. In addition, PCR with universal primers is not typically used for samples that may contain a large number of different microorganisms due to the difficulty of analyzing reaction products resulting from amplification of DNA from different species.

Molecular typing methods
PCR-based microorganisms

PCR is widely used not only for diagnosis and identification, but also for subspecies typing and analysis of genetic relatedness (clonality) of isolated strains of microorganisms, especially when conducting epidemiological studies. Compared to traditional phenotypic methods (bio-, phage- and serotyping), PCR-based genotyping is distinguished by its versatility, a deeper level of differentiation, the ability to use quantitative methods to assess the identity of strains, and high reproducibility. Many genotyping methods have been described that can be considered derivatives of PCR technology.

Despite the variety of PCR typing methods, what most have in common is the use of gel electrophoresis to separate DNA fragments of different lengths obtained from each individual strain. At the same time, a comparative analysis of individual electrophoretic profiles, carried out visually or using a computer, allows one to assess the degree of genetic relatedness of the studied strains.

Using PCR to detect drug
resistance in microorganisms

Recently, PCR is increasingly used to study various properties of pathogenic microorganisms, in particular to identify the resistance of certain types of pathogens to certain drugs. As a rule, the use of PCR to determine the sensitivity of microorganisms is advisable only in cases where traditional phenotypic methods are not applicable or are not effective enough. For example, the definition of sensitivity Mycobacterium tuberculosis to anti-tuberculosis drugs using culture methods usually takes from 4 to 8 weeks. In addition, the results of phenotypic tests in such cases may be distorted due to a decrease in the activity of antimicrobial drugs during long-term cultivation of microorganisms. Study of molecular mechanisms of drug resistance M. tuberculosis and some other pathogens has made it possible to develop PCR-based methods for the rapid identification of genetic markers of resistance.

For such an analysis, DNA or RNA of the pathogen isolated in pure culture is usually used. However, in some cases there is the possibility of direct PCR analysis for antibiotic resistance without prior cultivation of the pathogen. The studied sample of clinical material is used as a source of target DNA for PCR, and the copied PCR product is analyzed to identify mutations associated with antibiotic resistance. For example, a method has been developed that makes it possible to detect, using PCR, the resistance of the pathogen to rifampicin in patients suffering from tuberculous meningitis.

There are, however, natural limitations to the use of genetic methods for assessing drug resistance of microorganisms:

Data on specific genetic mechanisms of resistance may be lacking;

Resistance to certain drugs is often associated with different mechanisms and mutations in different genes that independently affect the phenotype.

For example, resistance of Gram-negative bacteria to aminoglycoside antibiotics can be caused by the production of various aminoglycoside-modifying enzymes or changes in cell wall permeability. In this case, the results of PCR analysis, which always characterize a strictly defined specific section of DNA, cannot serve as a basis for assessing the sensitivity of the microorganism as a whole.

In addition, the lack of international standards and recommendations for the use of PCR to determine sensitivity to antimicrobial drugs is an additional factor limiting the possibility of widespread use of this approach in practical diagnostics.

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:

1. 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.
2. 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.

   

3. No long-term cultivation of crops is required. The automated diagnostic process allows you to obtain research results after 4-5 hours.
4. The method is almost one hundred percent reliable. Only isolated cases of false negative results have been recorded.
5. 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:

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.