PRINCIPLE OF THE METHOD (molecular biological basis)

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

Method principle polymerase chain reaction (PCR)(Polymerase chain reaction (PCR)) was developed by Cary Mullis (Cetus, USA) in 1983. and is now widely used for scientific research, and for diagnostics in practical health care and the service of the State Sanitary and Epidemiological Supervision (genotyping, diagnosis of infectious diseases).

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

Natural DNA replication involves several steps:

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

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

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

This process can be used to make copies short segments of DNA specific to specific microorganisms, those. to carry out a targeted search for such specific areas, which is the goal of gene diagnostics to identify pathogens of infectious diseases.

Discovery of thermostable DNA polymerase (Taq polymerase) from thermophilic bacteria Thermis aquaticus, the optimum of which is in the region of 70-72°C, made it possible to make the process of DNA replication cyclic and use it for work in vitro. The creation of programmable thermostats (amplifiers), which, according to a given program, carry out cyclic temperature changes, created the prerequisites for the widespread introduction of the PCR method into the practice of laboratory clinical diagnostics. With repeated repetition of synthesis cycles, an exponential increase in the number of copies of a specific DNA fragment occurs, which makes it possible to obtain a sufficient number of DNA copies from a small amount of the analyzed material, which may contain single cells of microorganisms, for their identification by electrophoresis.

Complementary completion of the chain does not begin at any point in the DNA sequence, but only in certain starting blocks - short double-stranded sections. By attaching such blocks to specific regions of DNA, it is possible to direct the process of synthesis of a new strand only in this region, and not along the entire length of the DNA strand. To create starting blocks in given DNA regions, two oligonucleotide primers (20 nucleotide pairs) are used, called primers. The primers are complementary to the DNA sequences on the left and right boundaries of a specific fragment and are oriented in such a way that the completion of a new DNA strand occurs only between them.

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

The following components are required for amplification:

A mixture of deoxynucleotide triphosphates (dNTPs)(a mixture of four dNTPs, which are the material for the synthesis of new complementary DNA strands)

Enzyme Taq polymerase(a thermostable DNA polymerase that catalyzes the lengthening of primer chains by sequentially adding nucleotide bases to a growing chain of synthesized DNA).

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

To obtain a sufficient number of copies of the desired characteristic DNA fragment, amplification includes several (20-40) cycles.

Each amplification cycle includes 3 stages proceeding in different temperature conditions Step 1: DNA Denaturation(double helix unwinding). It flows at 93-95°C for 30-40 seconds. Stage 2: Attaching primers (annealing). Primer attachment occurs complementary to the corresponding sequences on opposite DNA strands at the boundaries of a specific site. Each pair of primers has its own annealing temperature, the values ​​of which are in the range of 50-65°C. Annealing time -20-60 sec. Stage 3: Building DNA chains. Complementary completion of DNA chains occurs from the 5'-end to the 3'-end of the chain in opposite directions, starting from the sites of primer attachment. The material for the synthesis of new DNA chains are deoxyribonucleotide triphosphates (dNTPs) added to the solution. The synthesis process is catalyzed by the enzyme thermostable DNA polymerase (Taq polymerase) and takes place at a temperature of 70-72°C. Synthesis time - 20-40 sec.

The new DNA strands formed in the first amplification cycle serve as templates for the second amplification cycle, in which the desired specific DNA fragment (amplicon) is formed. (see fig. 2). In subsequent amplification cycles, amplicons serve as a template for the synthesis of new chains. Thus, the accumulation of amplicons in solution occurs according to the formula 2n, where n is the number of amplification cycles. Therefore, even if only one double-stranded DNA molecule was initially present in the initial solution, about 108 amplicon molecules accumulate in the solution after 30–40 cycles. This amount is sufficient for reliable visual detection of this fragment by agarose gel electrophoresis. The amplification process is carried out in a special programmable thermostat (amplifier), which, according to a given program, automatically changes temperatures according to the number of amplification cycles.

STAGES OF PCR - ANALYSIS

The PCR method, as a tool for laboratory diagnosis of infectious diseases, is based on the detection of a small DNA fragment of the pathogen (several hundred base pairs), specific only for this microorganism, using a polymerase chain reaction to accumulate the desired fragment.
The analysis technique using the PCR method includes three stages:

1. Isolation of DNA (RNA) from a clinical specimen

2. Amplification of specific DNA fragments
3. Detection of amplification products

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

Amplification of specific DNA fragments
At this stage, short specific DNA fragments are accumulated in the amount necessary for their further detection. Most methods for determining specific fragments of the genome use the so-called. “a classic version of directed PCR. To increase the specificity and sensitivity of the analysis, some methods use the “nested” PCR method, which uses 2 pairs of primers (“external” - for the 1st stage, and “internal” - for the 2nd stage).

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

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

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

- Direct detection of the presence of pathogens

Many traditional diagnostic methods, such as enzyme-linked immunosorbent assay, detect marker proteins that are waste products of infectious agents, which provides only indirect evidence of the presence of an infection. Identification of a specific DNA region of the pathogen by PCR gives a direct indication of the presence of the infectious agent.

- High specificity
The high specificity of the PCR method is due to the fact that a unique DNA fragment characteristic only for this pathogen is detected in the test material. Specificity is determined by the nucleotide sequence of the primers, which excludes
the possibility of obtaining false results, in contrast to the enzyme immunoassay method, where errors are not uncommon due to cross-reacting antigens.

- High sensitivity
The PCR method allows you to detect even single cells of bacteria or viruses. PCR diagnostics detects the presence of pathogens of infectious diseases in cases where other methods (immunological, bacteriological,
microscopic) is impossible. The sensitivity of PCR analysis is 10-1000 cells per sample (the sensitivity of immunological and microscopic tests is 103-105 cells).

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

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

It should be noted that the PCR method can detect pathogens not only in the clinical material obtained from the patient, but also in the material obtained from the objects. external environment(water, soil, etc.)

APPLICATION OF THE PCR METHOD IN PRACTICAL HEALTH CARE

The use of the PCR method for diagnosing infectious diseases of both bacterial and viral nature is of great importance for solving many problems of microbiology and epidemiology. The use of this method also contributes to the development fundamental research in the field of chronic and understudied infectious diseases.

The most effective and economically justified use of the method in:

urogynecological practice- to detect chlamydia, ureaplasmosis, gonorrhea, herpes, gardnerellosis, mycoplasma infection;

in pulmonology- for differential diagnosis viral and bacterial pneumonia, tuberculosis;

in gastroenterology- to detect helicobacteriosis;

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

in hematology- to detect cytomegalovirus infection, oncoviruses.

At the end of the article, see
Polymerase chain reaction (PCR, PCR) invented in 1983 by Carey Mullis (American scientist). Subsequently, he received the Nobel Prize for this invention. Currently, PCR diagnostics is one of the most accurate and sensitive methods for diagnosing infectious diseases.
Polymerase chain reaction (PCR)- an experimental method of molecular biology, a method of significantly increasing small concentrations of certain fragments of nucleic acid (DNA) in a biological material (sample).
The PCR method is based on the repeated doubling of a certain section of DNA with the help of enzymes under artificial conditions (in vitro). As a result, sufficient amounts of DNA are produced for visual detection. In this case, only the area that satisfies the specified conditions is copied, and only if it is present in the sample under study.
In addition to simply increasing the number of DNA copies (this process is called amplification), PCR allows many other manipulations with genetic material (introduction of mutations, splicing of DNA fragments), and is widely used in biological and medical practice, for example, to diagnose diseases (hereditary, infectious) , to establish paternity, to clone genes, introduce mutations, isolate new genes.

Specificity and application

Conducting PCR

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

• DNA template containing the section of DNA to be amplified;
• two primers complementary to the ends of the desired fragment;
• thermostable DNA polymerase;
• deoxynucleotide triphosphates (A, G, C, T);
• Mg2+ ions necessary for polymerase operation;
• buffer solution.

PCR is carried out in an amplifier - a device that provides periodic cooling and heating of test tubes, usually with an accuracy of at least 0.1 ° C. To avoid evaporation of the reaction mixture, a high-boiling oil, such as vaseline, is added to the test tube. The addition of specific enzymes can increase the yield of the PCR reaction.
Reaction progress

Typically, when conducting PCR, 20 - 35 cycles are performed, each of which consists of three stages. The double-stranded DNA template is heated to 94 - 96°C (or 98°C if a particularly thermostable polymerase is used) for 0.5 - 2 minutes to allow the DNA strands to separate. This stage is called denaturation - the hydrogen bonds between the two chains are destroyed. Sometimes, before the first cycle, the reaction mixture is preheated for 2–5 minutes to completely denature the template and primers.
When the strands are separated, the temperature is lowered to allow the primers to bind to the single stranded template. This stage is called annealing. The annealing temperature depends on the primers and is usually chosen 4 - 5°C below their melting point. Stage time - 0.5 - 2 minutes.

DNA polymerase replicates the template strand using the primer as a primer. This is the elongation stage. The elongation temperature depends on the polymerase. Frequently used polymerases are most active at 72°C. The elongation time depends both on the type of DNA polymerase and on the length of the fragment being amplified. Typically, the elongation time is taken to be one minute for every thousand base pairs. After the end of all cycles, an additional stage of final elongation is often carried out in order to complete all single-stranded fragments. This stage lasts 10 - 15 minutes.
Preparation of material for research and its transportation to the laboratory

For a successful analysis, it is important to correctly collect the material from the patient and properly prepare it. It is known that in laboratory diagnostics the majority of errors (up to 70%) are made at the stage of sample preparation. To take blood in the INVITRO laboratory, vacuum systems are currently used, which, on the one hand, minimally injure the patient, and, on the other hand, allow the material to be taken in such a way that it does not come into contact with either the personnel or the environment. This avoids contamination (contamination) of the material and ensures the objectivity of the PCR analysis.

DNA - deoxyribonucleic acid - a biological polymer, one of two types of nucleic acids that provide storage, transmission from generation to generation and implementation of the genetic program for the development and functioning of living organisms. The main role of DNA in cells is the long-term storage of information about the structure of RNA and proteins.

RNA-ribonucleic acid is a biological polymer, similar in its chemical structure to DNA. The RNA molecule is built from the same monomer units - nucleotides as DNA. In nature, RNA usually exists as a single strand. In some viruses, RNA is the carrier of genetic information. In the cell, it plays an important role in the transfer of information from DNA to protein. RNA is synthesized on a DNA template. This process is called transcription. There are sections in DNA that contain information responsible for the synthesis of three types of RNA, which differ in their functions: messenger or messenger RNA (mRNA), ribosomal (rRNA) and transport (tRNA). All three types of RNA are involved in protein synthesis in one way or another. However, information on protein synthesis is contained only in mRNA.

Nucleotides are the basic repeating unit in nucleic acid molecules, the product of a chemical compound of a nitrogenous base, a five-carbon sugar (pentose) and one or more phosphate groups. Nucleotides present in nucleic acids contain one phosphate group. They are named according to the nitrogenous base they contain - adenine (A) containing adenine, guanine (G) - guanine, cytosine (C) - cytosine, thymine (T) - thymine, uracil (U) - uracil. DNA consists of 4 types of nucleotides - A, T, G, C, RNA also has 4 types - A, U, G, C. The sugar in the composition of all DNA nucleotides is deoxyribose, RNA is ribose. During the formation of nucleic acids, nucleotides, by binding, form a sugar-phosphate backbone of the molecule, on one side of which there are bases.

A primer is a short DNA used to replicate the template strand. Each of the primers is complementary to one of the chains of the double-stranded template, framing the beginning and end of the amplified region.

Literature

1. Glick B., Pasternak J. Molecular biotechnology. Principles and application. Per. from English. - M.: Mir, 2002. - 589 p., illustration. ISBN 5-03-003328-9
2. Shchelkunov S.N. Genetic engineering - Novosibirsk: Sib. univ. publishing house, 2004. - 496 p.; ill. ISBN 5-94087-098-8
3. Patrushev L.I. Artificial genetic systems - M .: Nauka, 2005 - In 2 volumes - ISBN 5-02-033278-X

IMPORTANT!

The information in this section should not be used for self-diagnosis or self-treatment. In case of pain or other exacerbation of the disease, only the attending physician should prescribe diagnostic tests. For diagnosis and proper treatment, you should contact your doctor.

Polymerase chain reaction (PCR)

The essence of the PCR method. DNA polymerase

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

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

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

Conducting PCR

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

For the polymerase chain reaction to take place, a number of conditions must be met. For PCR, in the simplest case, the following components are required:

A DNA template containing the section of DNA to be amplified.

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

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

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

Mg 2+ ions necessary for polymerase to work.

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

To avoid evaporation of the reaction mixture, a high-boiling oil, such as vaseline, is added to the test tube. If a device with a heated lid is used, this is not required.

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

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

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

2. Annealing - binding of primers to template DNA. Once the strands have separated, the temperature is slowly lowered to allow the primers to bind to the single stranded template. The annealing temperature depends on the primer composition and is usually chosen at 50–65°C. Stage time - 20 - 60 seconds. Incorrect choice of annealing temperature leads either to poor binding of primers to the template (at elevated temperature), or to binding in the wrong place and the appearance of non-specific products (at low temperature).

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

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

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

To understand exactly how the amplification of a certain DNA segment occurs during PCR, it is necessary to clearly understand the position of all primers and their complementary sequences in the amplifiable chains in each round. In the first round, each of the newly synthesized chains is much longer than the distance from the 3"-hydroxyl group of its primer to the terminal nucleotide of the sequence complementary to the second primer. Such chains are called "long templates", it is on them that further synthesis will take place.

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

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

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

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

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

Basic principles of primer selection.

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

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

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

SEI HPE "Krasnoyarsk State Medical Academy

named after Yasenetsky Federal Agency for Health and Social Development »

Department of Medical Genetics and Clinical Neurophysiology, IPO

MAIN PRINCIPLES OF THE METHOD

POLYMERASE CHAIN ​​REACTION

Methodical manual for students of 3-4 courses

in the specialties of general medicine (060101) and

Krasnoyarsk - 2007

Shnaider, N. A., Butyanov, R. A. Basic principles of the polymerase chain reaction method. Methodological manual for extracurricular work of students of 3-4 courses in the specialties of general medicine (060101) and pediatrics (060103). - Krasnoyarsk: Publishing house of GOU VPO KrasGMA, 2007. - 42p.

The methodological manual fully complies with the requirements of the State Standard (2000) and reflects the main aspects modern method diagnosis of human hereditary diseases - the method of polymerase chain reaction, the educational material is adapted to educational technologies, taking into account the specifics of training in 3-4 courses of medical and pediatric faculties.

Reviewers: Head of the Department of Medical Genetics, State Educational Institution of Higher Professional Education

"Novosibirsk State Medical University of the Federal Agency for Health and Social Development", Doctor of Medical Sciences, Professor;

DNA replication

The object of study of this method is Deoxyribonucleic acid (DNA). DNA is a universal carrier of genetic information in all organisms existing on Earth (with the exception of RNA-containing microorganisms). DNA is a double strand twisted into a helix. Each strand consists of nucleotides connected in series. DNA strands have the opposite direction: the 5'-end of one strand corresponds to the 3'-end of the second strand. The unique property of DNA is its ability to duplicate itself. This process is called replication. Replication of the DNA molecule occurs during the synthetic period of interphase. Each of the two chains of the "parent" molecule serves as a template for the "daughter". After replication, the newly synthesized DNA molecule contains one "maternal" strand, and the second - a "daughter", newly synthesized (semi-conservative method). For template synthesis of a new DNA molecule, the old molecule must be despiralized and stretched. Replication begins at several locations in the DNA molecule. The section of a DNA molecule from the start of one replication to the start of another is called replicon.

The start of replication is activated primers(seeds), consisting of 100-200 base pairs. The DNA helicase enzyme unwinds and divides the parent DNA helix into two strands, on which, according to the principle of complementarity, with the participation of the DNA polymerase enzyme, “daughter” DNA strands are assembled. In order for the enzyme to start its work, the presence of a starting block is required - a small initial double-stranded fragment. The start block is formed when the primer interacts with the complementary region of the corresponding strand of the parent DNA. In each replicon, DNA polymerase can move along the "mother" strand in only one direction (5`=>3`).

On the leading strand, as the replicon unwinds, a “daughter” strand gradually grows continuously. On the lagging strand, the daughter strand also synthesizes in the (5`=>3`) direction, but in separate fragments as the replicon unwinds.

Thus, the attachment of complementary nucleotides of the "daughter" strands goes in opposite directions (antiparallel). Replication in all replicons occurs simultaneously. Fragments and parts of "daughter" strands synthesized in different replicons are ligated into a single strand by an enzyme ligase. Replication is characterized by semiconservative, antiparallelism and discontinuity. The entire genome of a cell is replicated once per time period corresponding to one mitotic cycle. As a result of the replication process, two DNA molecules are formed from one DNA molecule, in which one strand is from the parent DNA molecule, and the second, the daughter, is newly synthesized (Fig. 1).

Rice. one. Diagram of DNA molecule replication.

Thus, the DNA replication cycle includes three main stages:

1. unwinding of the DNA helix and divergence of strands (denaturation);

2. attachment of primers;

3. completion of the chain of the child thread.

Principle of the PCR method

It was DNA replication that formed the basis of PCR. In PCR, the processes listed above are carried out in a test tube in a cyclic mode. The transition from one stage of the reaction to another is achieved by changing the temperature of the incubation mixture. When the solution is heated to 93-95°C, DNA denaturation occurs. To proceed to the next step - the addition or "annealing" of primers - the incubation mixture is cooled to 50-65°C. Next, the mixture is heated to 70-72°C - the optimum operation of taq-DNA polymerase - at this stage, a new DNA strand is completed. Then the cycle repeats again. In other words PCR method is a multiple increase in the number of copies (amplification) a specific section of DNA catalyzed by the enzyme DNA polymerase.

The extension of the daughter DNA strands must occur simultaneously on both strands of the maternal DNA, so replication of the second strand also requires its own primer. Thus, two primers are introduced into the reaction mixture: one for the "+"-chain, the second for the "-"-chain. By attaching themselves to opposite strands of the DNA molecule, the primers limit themselves to that part of it, which will be subsequently repeatedly doubled or amplified. The length of such a fragment, which is called an amplicon, is usually several hundred nucleotides.

PCR steps

Each amplification cycle includes 3 stages occurring at different temperature conditions (Fig. 2).

· Stage 1: DNA denaturation . It flows at 93-95° for 30-40 seconds.

· Stage 2: primer annealing . Primer attachment occurs complementary to the corresponding sequences on opposite DNA strands at the boundaries of a specific site. Each pair of primers has its own annealing temperature, the values ​​of which are in the range of 50-65°C. Annealing time 20-60 sec.

· Stage 3: extension of DNA chains. Complementary extension of DNA chains occurs from the 5" end to the 3" end of the chain in opposite directions, starting from the primer attachment sites. The material for the synthesis of new DNA strands are deoxyribonucleoside triphosphates added to the solution. The synthesis process is catalyzed by the enzyme taq-polymerase and takes place at a temperature of 70-72°C. Synthesis time - 20-40 sec.

The new DNA strands formed in the first amplification cycle serve as templates for the second amplification cycle, in which a specific amplicon DNA fragment is formed (Fig. 3). In subsequent amplification cycles, amplicons serve as a template for the synthesis of new chains.

Thus, amplicons accumulate in the solution according to the formula 2", where n is the number of amplification cycles. Therefore, even if only one double-stranded DNA molecule was initially in the initial solution, about 108 amplicon molecules accumulate in the solution in 30-40 cycles. This the amount is sufficient for reliable visual detection of this fragment by agarose gel electrophoresis.

The amplification process is carried out in a special programmable thermostat ( cycler), which, according to a given program, automatically changes temperatures according to the number of amplification cycles.

The following components are required for amplification:

· DNA template(DNA or its part containing the desired specific fragment);

· Primers(synthetic oligonucleotides (20-30 nucleotide pairs) complementary to DNA sequences at the boundaries of the specific fragment being determined). The choice of a specific fragment and the selection of primers play a major role in the specificity of the amplification, which affects the quality of the analysis.

· A mixture of deoxynucleotide triphosphates (dNTPs)(a mixture of four dNTPs, which are the material for the synthesis of new complementary DNA strands in equivalent concentrations of 200-500 microns)

· EnzymeTaq-polymerase(thermostable DNA polymerase catalyzing the lengthening of primer chains by sequential addition of nucleotide bases to the growing chain of synthesized DNA, 2-3 mm).

· buffer solution(reaction medium containing Mg2+ ions necessary to maintain enzyme activity, PH 6.8-7.8).

To determine specific regions of the genome of RNA viruses, a DNA copy is first obtained from an RNA template using a reverse transcription (RT) reaction catalyzed by the enzyme reverse transcriptase (reverse transcriptase).

Rice. 2. Amplification (1st cycle).

Rice. 3. Amplification (2nd cycle).

Main Applications of PCR

clinical medicine:

o diagnosis of infections,

o detection of mutations, including the diagnosis of hereditary diseases,

o genotyping, including HLA genotyping,

o cellular technologies

ecology (as a way to monitor the state and quality of objects environment and food)

definition of transgenic organisms (GMOs)

Personal identification, paternity, forensics

general and particular biology,

Basic principles

organization of diagnostic laboratories

Work in the PCR laboratory is carried out in accordance with the "Rules for the design, safety, industrial sanitation, anti-epidemic regime and personal hygiene when working in laboratories (departments, departments) of sanitary and epidemiological institutions of the healthcare system.

Contamination of DNA samples

Carrying out PCR diagnostics is associated with a problem caused by the high sensitivity of the method - the possibility contamination. If trace amounts of positive DNA enter the reaction tube (specific DNA amplification products - amplicons; DNA standard used as a positive control; positive DNA of a clinical sample) leads to amplification of a specific DNA fragment during PCR and, as a result, to the appearance of false positive results.

In the course of work, you may meet two types of contamination:

1. cross contamination from sample to sample (during the processing of clinical samples or when digging out the reaction mixture), leading to the appearance of sporadic false positive results;

2. amplification product contamination(amplicons) having highest value, because during the PCR process, amplicons accumulate in huge quantities and are ideal products for reamplification.

Trace amplicon contamination of dishes, automatic pipettes and laboratory equipment, the surface of laboratory tables, or even the surface of the skin of laboratory workers leads to systematic false positive results. Determining the source of contamination can be very difficult and requires a significant investment of time and money. The experience accumulated to date in the work of laboratories using the PCR method for diagnostics allows us to formulate the basic requirements for the organization of such laboratories and the conduct of the analyzes themselves. Compliance with these requirements eliminates the possibility of contamination and obtaining false positive results.

Stages of PCR analysis

Geographically separated, placing them in separate rooms (Fig. 4.5):

· Pre-PCR room, where processing of clinical samples, DNA extraction, preparation of the reaction mixture for PCR and PCR is performed (if conditions are available, the last two steps are also recommended to be carried out in an additional separate room). In these rooms it is forbidden to carry out all other types of work with the studied agents, the PCR diagnostics of which are carried out in this laboratory.

· Post-PCR room, where the detection of amplification products is carried out. Other detection methods may be used in this room. It is desirable to locate the room for detection of amplification products as far as possible from the pre-PCR rooms.

Working rooms are equipped with ultraviolet lamps with a maximum radiation in the region of 260 nm (type DB-60) at the rate of 2.5 W per 1 m3. The lamps are located so that the surfaces of working tables, equipment and materials with which the operator comes into contact during the PCR analysis are exposed to direct radiation. Irradiation is carried out within 1 hour before the start of work and within 1 hour after the end of work.

Laboratory doctors work in special laboratory clothes, which are changed when moving from one room to another, and in disposable gloves. Processing of clothes from different rooms is carried out separately. Different employees work at different stages of the PCR analysis.

For work, separate sets of dispensers, plastic and glassware, laboratory equipment, gowns and gloves are used, designed for various stages of analysis and cannot be transferred from one room to another. Equipment, materials and inventory in each room are labeled accordingly.

All stages of work are carried out only with the use of disposable consumables: tips for automatic pipettes, test tubes, gloves, etc. Be sure to change the tips when moving from sample to sample. It is necessary to use tips with an aerosol barrier filter to prevent microdroplets of the solution from entering the pipette. Used test tubes and tips are discarded in special containers or containers containing a disinfectant solution. Clinical samples are stored separately from reagents.

For processing and cleaning the workplace, each room has cotton-gauze swabs (napkins), tweezers, disinfectant and inactivating solutions.

In the PCR diagnostic laboratory, work related to the production (cloning) and isolation of recombinant plasmids containing DNA sequences or gene fragments of pathogens that are diagnosed in this laboratory is excluded.

Collection of clinical material

The studied material for PCR can be scrapings of epithelial cells, blood, plasma, serum, pleural and cerebrospinal fluid, urine, sputum, mucus and other biological secretions, biopsy specimens.

The sampling of the material is carried out in the conditions of the treatment room of the corresponding profile. After sampling, the samples should be taken to the PCR diagnostic laboratory as soon as possible.

Sampling must be carried out using sterile, preferably disposable, instruments only in disposable sterile plastic tubes or glass tubes, pre-treated for an hour with a chromium mixture, thoroughly washed with distilled water and calcined in an oven at a temperature of 150 ° C for 1 hour.

Detection zone (another floor or another building).

Rice. 4. PCR laboratory device with detection by electrophoresis.

Detection zone (different floor or building)

Rice. five. PCR laboratory device with fluorescent detection (quantitative analysis).

Rice. 6. DNA extraction room. Shown is a tabletop box with a bactericidal lamp.

Rice. 7. amplification room.

Rice. 8. Detection room.

Rice. nine. Blood samples for DNA diagnostics of hereditary diseases.

Storage and transportation of samples

For the diagnosis of hereditary diseases, blood samples are stored on special paper forms or in epindorfs (plastic test tubes) in a frozen state for a long time (Fig. 9).

For the diagnosis of infectious diseases, samples are kept at room temperature for no more than 2 hours. If longer storage is required, the samples can be placed in a refrigerator at a temperature of 2-8°C for a period not exceeding 24 hours. Longer storage (up to 2 weeks) is acceptable when frozen in a freezer at a temperature of minus 20°C. Repeated freezing-thawing of samples is not allowed.

If the PCR diagnostic laboratory and the procedure room for sampling are geographically separated, then samples should be transported in thermoses or thermal containers in compliance with the rules for storing samples and the rules for transporting infectious materials.

Extraction of DNA from samples

The method of solid-phase sorption, which consists in adding a lysing agent containing a solution of guanidine, sorption of DNA on a sorbent, repeated washing and resorption of DNA with a buffer solution, has become widespread. In the case of serum, plasma or whole blood processing, the phenolic extraction method is usually used. The method involves deproteinization with phenol/chloroform followed by precipitation of DNA (or RNA) with ethanol or isopropanol. Processing is carried out in microcentrifuge test tubes of the Eppendor P type with a volume of 1.5 ml. The processing time is 1.5-2 hours (Fig. 10).

Rice. 10. Isolation of DNA.

Conducting PCR

A certain amount of the sample from the processed clinical sample is transferred to a special Eppendorf type microcentrifuge tube with a volume of 0.2 or 0.5 ml. An amplification mixture consisting of water, PCR buffer, dNTP solution, primer solution and solution is added to the same tube. Taq polymerase (added to the mixture last) Typically, the volume of the reaction mixture is 25 µl Then one drop of mineral oil is added to each tube to prevent evaporation of the reaction mixture during the amplification The tubes are transferred to a programmable thermostat (amplifier), where amplification is carried out in automatic mode according to a given program (Fig. 11).

Rice. eleven. Amplifier " Thermocycler ».

The reaction time, depending on the given program, is 2-3 hours. In parallel with the experimental samples, control samples are placed: the positive control includes all the components of the reaction, but instead of the material of the clinical sample, a control DNA preparation of the gene under study is introduced. The negative control includes all components of the reaction, but instead of the clinical material or DNA preparation, an appropriate amount of deionized water or an extract that does not contain the studied DNA is added. A negative control is necessary to check the components of the reaction for the absence of DNA in them due to contamination and to exclude false positive results.

Registration of results

The amplified specific DNA fragment is detected by agarose gel electrophoresis in the presence of ethidium bromide. Ethidium bromide forms a stable interstitial compound with DNA fragments, which appears as luminous bands when the gel is irradiated with UV radiation with a wavelength of 290-330 nm. Depending on the size of the resulting PCR amplicons, a gel containing 1.5% to 2.5% agarose is used. To prepare an agarose gel, a mixture of agarose, buffer, and water is melted in a microwave oven or in a water bath, and a solution of ethidium bromide is added. Cooled to 50-60°C, the mixture is poured into the mold with a layer of 4-6 mm thick, and using special combs, pockets are made in the gel for applying the sample. The combs are set so that between the bottom of the wells and the base of the gel remains a layer of agarose 0.5-1 mm. After the gel has hardened, an amplificate is applied to the pockets in an amount of 5-15 µl. It is recommended to carry out electrophoresis of a mixture of DNA fragment length markers in parallel with control and experimental samples. Typically, such a mixture contains ten DNA fragments 100, 200, 300, etc. long base pairs.

Setting up such a sample allows you to verify the length of the amplicons in the control and experimental samples. The gel with the applied sample is transferred to an electrophoresis chamber filled with a buffer, the chamber is connected to a power source and the electrophoretic separation of the amplification products is carried out for 30-45 minutes at an electric field strength of 10-15 V/cm. In this case, the front of the dye, which is part of the reaction mixture, must pass at least 3 cm.

After the end of electrophoresis, the gel is transferred to the transilluminator glass and viewed in ultraviolet light. For documentation, the gel is photographed on Mikrat 300 film or recorded using a video system connected to a computer.

The control samples are evaluated first. In the electrophoretic lane corresponding to the positive control, an orange luminous band should be present. Its electrophoretic mobility should correspond to the length of the amplicon specified in the instructions.

In the electrophoretic track corresponding to the negative control, such a band should be absent. The presence of such a band in the negative control indicates contamination - contamination of the reagents used with the studied DNA or amplicon. The test samples are evaluated by the presence in the respective lane of a band that is located at the same level as the band in the positive control sample. The intensity of the band glow corresponds to the amount of DNA under study in the sample, which allows for a semi-quantitative assessment of PCR. Usually positive results are evaluated on a four-point scale. If the glow of the band in the experimental sample is very weak, then such a sample should be rearranged (Fig. 12).

Rice. 12. Electrophoresis in agarose gel.

PCR applications fordiagnostics of point mutations and gene polymorphisms

One of the leading areas of application of PCR in practical healthcare is the diagnosis of point mutations and gene polymorphisms. . There are direct and indirect methods of DNA diagnostics. In those situations where a gene is known, the damage of which leads to the development of a hereditary disease, this damage can be detected by molecular genetic methods. Such methods are called direct. Using direct methods, disturbances in the primary nucleotide sequence of DNA (mutations and their types) are detected. Direct methods are characterized by accuracy reaching almost 100%.

However, in practice, these methods can be applied under certain conditions.:

with a known cytogenetic localization of the gene responsible for the development of a hereditary disease;

The disease gene must be cloned and its nucleotide sequence known.

The goal of direct DNA diagnostics is to identify mutant alleles.

Thus, in those situations where it is known what kind of DNA damage leads to a hereditary disease, the DNA fragment containing the damage is examined directly, i.e., the direct method of DNA diagnostics is used.

However, to date, the genes of many diseases have not been mapped, their exon-intron organization is unknown, and many hereditary diseases are characterized by pronounced genetic heterogeneity, which does not allow full use of direct DNA diagnostic methods. Therefore, in cases where the localization of damage is not known, a different approach is used, associated with the study of the vicinity of the gene responsible for the gene disease, in combination with family analysis, that is, indirect methods of molecular genetic diagnosis of hereditary diseases are used.

Various methods can be used to detect point mutations and small deletions, but all of them are based on the use of the PCR method. This reaction allows you to repeatedly multiply the nucleotide sequence of DNA, and then search for mutations. Methods for searching for DNA fragments carrying mutations are based on comparative analysis mutant and normal DNA nucleotide sequences.

Analysis of PCR products

in the process of direct DNA diagnostics

It involves the study of specific features of the amplified region of the gene. Thus, in diseases caused by the expansion of trinucleotide repeats, amplification products differ in their length (reflecting a different number of triplets in the studied gene region) and, as a result, in their speed of movement in the gel. Due to this, a clear electrophoretic separation of normal and mutant alleles and an accurate determination of the pathologically elongated fragment, i.e. DNA diagnosis of the disease (Fig. 13), is achieved.

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Rice. fourteen. Diagnosis of a deletion GAG in the gene DYT 1 in patients with dopa-independent dystonia (polyacrylamide gel electrophoresis). Tracks 2,3,6 - sick; lanes 1,4,5 - control. The thin arrow indicates the normal allele, the bold arrow indicates the mutant shorter allele (deletion of three nucleotides).

If the DNA region under study is entirely included in an extended deletion, then PCR amplification of DNA from this deleted allele will not be carried out due to the lack of places for primer hybridization. In this case, a homozygous deletion will be diagnosed based on total absence PCR reaction product (DNA synthesis is impossible from both copies of the gene). With a heterozygous deletion, it is possible to identify a PCR product synthesized from a normal (safe) allele, however, for reliable diagnosis of such a mutation, it is necessary to use more sophisticated DNA visualization methods that allow one to estimate the dose of the final PCR product.

To detect point mutations (most often nucleotide substitutions) at certain sites, the PCR method is used in combination with other methods of molecular genetic analysis. If the location and nature of the proposed point mutation are precisely known, then for the purposeful detection of such a mutation, restriction endonucleases (restrictases) are special cellular enzymes isolated from various strains of bacteria.

These enzymes recognize specific nucleotide sequences ranging from four to ten nucleotides in length. Then, restriction (lat. (cutting)) of these sequences is carried out as part of a double-stranded DNA molecule. Each restriction enzyme recognizes and cuts in a fixed place a strictly defined, specific nucleotide sequence - restriction site (recognition site).

In cases where a point mutation changes the natural site of recognition for a particular restriction enzyme, that enzyme will not be able to cleave the mutant PCR-amplified fragment. In some cases, the mutation leads to the appearance of a new recognition site for a particular restriction enzyme, which is absent in the norm.

In both situations, the mutant and normal PCR products treated with the selected restriction enzyme will give restriction fragments of different lengths, which can be easily detected by electrophoresis (Fig. 15).

Thus, if it is necessary to quickly detect any particular point mutation, the task is reduced to searching for the corresponding restriction enzyme, the recognition site of which is localized at the site of the disturbed nucleotide sequence. Treatment of PCR products with this restriction enzyme will allow easy differentiation of normal and mutant alleles. Restriction analysis greatly simplifies the detection of known point mutations and is currently widely used for direct DNA diagnosis of hereditary diseases.

final stage molecular genetic analysis of mutations is the determination of the nucleotide sequence of the studied DNA fragment (sequencing), which is compared with the norm and the final genetic diagnosis is formulated. Thanks to the advances in molecular genetics, DNA diagnostic methods have now been developed for more than 400 hereditary diseases.

Rice. 15. Detection of a point mutation using restriction analysis: A - amplifiable region of the gene containing a restriction siteAGCTfor restriction endonucleaseAlu I. MutationG→ Achanges this nucleotide sequence, resulting in restriction enzymeAluiblocked; B - electropherogram of restriction products: lane 1 - homozygosity for the normal allele; lane 2, homozygosity for the mutation; lane 3 - heterozygous state (normal allele + mutation).

Diagnosis of hereditary diseases based on direct examination of mutant alleles in patients, their family members or presumed heterozygous carriers of pathological mutations is suitable for pre-symptomatic and prenatal diagnosis, which can be applied to the most early stages fetal development, before the appearance of any clinical or biochemical symptoms of the disease.

Regardless of the method of mutation detection, accurate molecular characterization of each mutation can only be obtained by direct sequencing. To automate this process, in recent years, special devices have been widely used - sequencers, which make it possible to significantly speed up the process of reading DNA information.

The way for a wider application of molecular biological research in clinical diagnostic laboratories is opened by accelerating the analytical process by performing all procedures in one continuum, without sample transfer, creating conditions to prevent contamination during parallel testing of a number of analytes and with objective registration of results in each cycle.

Main modifications of the PCR method

Used to quickly scan and search for known gene mutations.

Multiplex (multiprimer) PCR

This method is based on the simultaneous amplification of several exons of the studied gene in one reaction. This allows economical rapid screening of the most common mutations. For example, to quickly diagnose the carriage of deletions in the dystrophin gene in patients with progressive Duchenne/Becker muscular dystrophy, simultaneous amplification of the set of the most frequently mutating exons of this gene is performed. Since these diseases are inherited in an X-linked recessive type and are associated with damage to the only X chromosome in boys, in the case of an extended deletion, electrophoresis of the reaction products will reveal the absence of one or more DNA fragments (exons), which can serve as a molecular confirmation of the diagnosis. In addition, by selecting specific gene regions for PCR amplification, a fairly accurate assessment of the total length of the deletion and gene break points (up to the exon) is possible.

The combined use of several multiplex reactions makes it possible to diagnose up to 98% of all deletions that occur in patients with progressive Duchenne/Becker muscular dystrophy. This is approximately 60% of total number known mutations in the dystrophin gene and indicates a very high efficiency of this screening method for DNA diagnosis of dystrophinopathy (Fig. 16).

Rice. 16. Direct DNA diagnosis of Duchenne muscular dystrophy using multiplex PCR (agarose gel electrophoresis). In each of the examined individuals, four exons of the dystrophin gene were simultaneously amplified (exons 17, 19, 44, and 45; arrows indicate the corresponding amplification products). Lane 1 - control, lanes 2-5 - patients with Duchenne muscular dystrophy with various deletions of the dystrophin gene (lanes 2 and 5 - deletion of exon 45, lane 3 - deletion of exon 44, lane 4 - deletion of exon 17 and 19).

Allele-specific amplification

The method is based on the use of two independent pairs of primers for a specific region of the gene: one primer in both pairs is common, and the second primer in each pair has a different structure and is complementary to either normal or mutant DNA sequences. As a result of such a reaction in solution, two types of PCR products can be simultaneously synthesized - normal and mutant. Moreover, the design of the primers used makes it possible to clearly differentiate normal and mutant amplification products by their molecular size. This method is very clear and allows you to verify both homo- and heterozygous carriage of the mutant allele.

Method for site-directed modification of amplified DNA

The method is based on the use in PCR of the so-called mismatch primer (not fully complementary to the template), which differs from the template DNA sequence by one nucleotide. As a result of the inclusion of the specified primer in the composition of the mutant PCR product, an artificially created restriction site for one of the restriction endonucleases is formed in it, which allows direct DNA diagnosis of a certain known mutation using restriction analysis. The creation of such an artificial restriction site may be necessary if the search did not reveal the existence of a known and accessible enzyme, the “natural” restriction site of which is affected as a result of the appearance of the studied mutation in the DNA molecule.

Reverse transcriptase PCR method (RT- PCR)

This method is used in cases where it is more convenient to use not genomic DNA as an object of study, but a more compact and informationally "saturated" cDNA obtained after appropriate processing of tissue samples, for example, biopsy material or cell lines of lymphocytes, fibroblasts, etc. Important the condition here is the expression (at least minimal) of the desired gene in the tissue under study.

At the first stage, reverse transcription of mRNA is carried out, and the resulting cDNA molecules serve as a template for PCR. Subsequently, the critical cDNA region amplified in sufficient quantity is subjected to sequencing and other mutation screening methods, direct electrophoretic study (detection of deletions, insertions, etc.) or integration into an expression system in order to obtain a protein product and its direct analysis.

This method is especially effective for the detection of mutations leading to the synthesis of a "truncated" protein (nonsense mutations, splicing mutations, large deletions) - the so-called PTT analysis (Protein Truncation Test). PTT analysis is commonly used when examining extended multi-exon genes, such as the gene for Duchenne/Becker muscular dystrophy, ataxia-telangiectasia, or neurofibromatosis type 1.

real time PCR(Real-Time PCR)

Every year, in practical healthcare, real-time PCR is becoming an increasingly popular diagnostic method. Its fundamental feature is the monitoring and quantitative analysis of the accumulation of polymerase chain reaction products and automatic registration and interpretation of the results. This method does not require an electrophoresis step, which reduces the requirements for a PCR laboratory. Thanks to savings in production space, a decrease in the number of personnel and the demand for DNA/RNA quantification, this method has been successfully used in recent years in the largest sanitary epidemic, diagnostic and research centers in the developed countries of the world, replacing PCR in its current ("classic") format.

Real-time PCR uses fluorescently labeled oligonucleotide probes to detect DNA during amplification. Real-time PCR allows a complete analysis of a sample within 20-60 minutes and is theoretically capable of detecting even a single DNA or RNA molecule in a sample.

Rice. 17. PCR in real time.

Real-time PCR uses the TaqMan system to control PCR kinetics directly during amplification using resonant fluorescence quenching. For detection, a probe carrying a fluorophore and a quencher complementary to the middle part of the amplified fragment is used. When the fluorophore and quencher are bound to the oligonucleotide probe, only a small amount of fluorescent emission is observed. During the amplification process, due to the 5'-exonuclease activity of Taq polymerase, the fluorescent label passes into solution, being released from the vicinity of the quencher, and generates a fluorescent signal that increases in real time in proportion to the accumulation of the amplificate (Fig. 17).

Main advantages of PCR-Real-Time over PCR with gel electrophoresis:

The whole method takes place in one test tube;

· The method takes 1 hour;

Enough 1-2 working rooms;

Along with a qualitative assessment of the result, it becomes possible to quantify it (for example, when prescribing antiviral therapy for AIDS or viral hepatitis, it is necessary to know the viral load, i.e. the amount of virus per 1 unit, which provides real-time PCR);

· Dramatically reduces the risk of contamination.

Conclusion

The PCR method is one of the most common methods of molecular biological research. This method should be used meaningfully by clinicians, and a doctor who decides to use PCR in his work must have certain knowledge about the features and capabilities of this method. Secondly, there must be close feedback between the clinician and the PCR laboratory, which is necessary for the analysis of complex cases and the development of the correct diagnostic strategy. Thirdly, PCR analysis is not a panacea in the diagnosis (primarily of infectious diseases) and does not replace existing research methods, but only complements them. And most importantly, PCR cannot replace the intuition and analytical thinking that a doctor who expects success should have.

P . S . Molecular-biological researches - change of reference points of diagnostics and treatment. The use of molecular biological methods is associated with the prospect of a radical change in emphasis in laboratory diagnostics. We can talk not just about timely information, but about its advance receipt. If now laboratory studies in most cases are carried out already with an advanced disease and treatment initiated, then molecular biological laboratory information is expected to make it possible to identify a person's inclination to certain types of pathology and the degree of sensitivity to certain drugs, which will allow substantiating predictive, preventive and personalized character of the medicine of the future.

CHANGE OF DIAGNOSIS AND TREATMENT FOCUSES

HEREDITARY DISEASES

Today In the future

Diagnosis Genetic passport

8. How many working rooms are required for a PCR laboratory with fluorescence detection (quantitative analysis, Real-Time PCR)?

9. What is detection?

10. What methods of DNA diagnostics are distinguished?

11. Which enzyme works on the basis of PCR?

12. Why does the detection zone need to be separated from other work zones?

13. What is a restriction site?

14. What is the difference between the direct method of DNA diagnostics and the indirect one?

15. What is sequencing?

16. What is multiplex PCR?

17. What types of mutations are determined by PCR?

18. What is contamination?

19. What is the essence of the allele-specific amplification method?

20. Storage conditions for PCR material?

21. What device is used for amplification?

22. What is the method of reverse transcriptase PCR (RT-PCR)?

23. What is the material for PCR diagnostics?

24. List the types of contamination?

Tests for self-study

1. Restriction endonucleases:

a) enzymes that “break” DNA in strictly specific places;

b) enzymes that sew breaks in the DNA molecule;

c) enzymes that provide compounds that carry out DNA repair.

2. Gene amplification:

3. Which of the methods of molecular genetics is used to diagnose diseases caused by a mutant gene of a known sequence?

a) the use of a specific restrictase;

b) direct detection using specific molecular probes;

c) family analysis of the distribution of normal restriction fragment length polymorphism.

4. DNA sequencing:

a) identification of the DNA base sequence;

b) repeated repetition of any DNA segment;

c) isolation of a DNA fragment containing the studied gene.

5. DNA samples can be obtained using :

b) chorionic villi;

c) amniotic fluid;

d) amniotic fluid cells;

e) biopsies of skin, muscles, liver,

e) everything is correct, except for point "c",

g) everything is correct, except for point "d",

h) All of the above are correct.

6. Which mutations are diagnosed by PCR?

a) genomic;

b) chromosomal;

c) gene (point).

7. Primer is:

a) a complementary section of DNA;

b) a synthetic oligonucleotide labeled (radioactively or fluorescently) sequence complementary to a mutant or normal gene;

c) an oligonucleotide acting as a "seed" and initiating the synthesis of a polynucleotide chain on a DNA or RNA template.

8. Who developed the principle of the PCR method?

b) K. Mullis

9. Is the PCR method used to diagnose the expansion of trinucleotide repeats (dynamic type of mutations)?

10. In what areas is PCR used?

a) clinical medicine;

b) definition of transgenic organisms (GMOs)

c) identification of the person, establishment of paternity, criminalistics

d) all of the above

d) none of the above.

Sample answers: 1 - a; 2 - b; 3 - b; 4 - a; 5 - e; 6 - in; 7 - in; 8 - b; 9 – a, 10 – d.

Main

1. Bochkov genetics. Moscow. GEOTAR, 2002.

Additional

1., Bakharev and the treatment of congenital and hereditary diseases in children. - Moscow, 2004.

2. DNA diagnostics and medical genetic counseling. - Moscow, 2004.

3. Ginter genetics. - Moscow, 2003.

4. Gorbunov fundamentals of medical genetics. - St. Petersburg: Intermedica, 1999.

5. J. McGee. Molecular clinical diagnostics. – World, 1999.

6. Menshikov - biological research in clinical laboratory diagnostics: the possibilities of the problem (lectures). Clinical laboratory diagnostics, № 3, 2006.

7. Kornienko of the work of the PCR laboratory during the in-line analysis of biological material. Clinical laboratory diagnostics, No. 10, 2006.

8. Organization of the work of the PCR laboratory. Methodical instructions. MU 1.3.1794-03. Chief Sanitary Doctor of the Russian Federation, 2003.

9. Erlich H. A. PCR technology. – Percin-Elmer Cetus, 1993.

10. Heid C. A., Stevens J. Real time quantitative PCR. Genome Res. - No. 6, 1996.

MAIN PRINCIPLES OF THE METHOD

POLYMERASE CHAIN ​​REACTION

Methodological manual for extracurricular work of students of 3-4 courses in the specialties of general medicine (060101) and pediatrics (060103).

SEI HPE "Krasnoyarsk State Medical Academy of the Federal Agency for Health and Social Development"

Russia, Krasnoyarsk,

The polymerase chain reaction has been known for 30 years. It is widely used in many fields, from archeology to genetics.

It is the PCR method that helps to establish paternity, but it is most often used to detect various infectious diseases in the human body.

How is PCR analysis carried out, and what is it? We will try to answer these questions in detail.

PCR analysis - what is it?

Polymerase chain reaction (PCR) is a high-precision method of molecular genetic diagnostics, which makes it possible to detect various infectious and hereditary diseases in humans, both in the acute and chronic stages, and long before the disease can manifest itself.

The PCR method is absolutely specific and, performed correctly, cannot give a false positive result. That is, if there is no infection, then the analysis will never show that it is. Therefore, now very often, in order to confirm the diagnosis, an additional PCR analysis is taken to determine the pathogen and its nature.

The polymerase chain reaction (PCR) was developed in 1983 by Cary Mullis (USA), for which he was awarded the Nobel Prize in Chemistry in 1993.

What is the advantage of this method?

Diagnosis by this method allows you to find the pathogen directly in the gene contained in the studied materials. This is the most accurate analysis for sexual infections, latent infections, various sexually transmitted diseases.

Differences between PCR diagnostics and other methods laboratory research are as follows:

• the method is aimed at identifying the pathogen itself;
• diagnostics by PCR is versatile: to detect several pathogens;
• diseases, only one biological sample of the patient is sufficient;
• the method is highly sensitive and is not accompanied by other cross-reactions.

In addition, the advantage of PCR diagnostics is that any biological material of the patient is suitable for analysis: blood, secretions from the genital organs, urine, semen.

What infections can be detected by a PCR smear?

A large number of infectious agents can be present in the body, including “hidden” ones that do not manifest themselves for a long time.

PCR smear analysis makes it possible to detect such infections:

• ureplasmosis of the genital organs;
• candidiasis ();
• herpes;
• the presence of cancer cells;
• assess the hormonal state;

The studied material for PCR is usually sputum, saliva, urine, blood. Before carrying out the analysis, it is necessary to carefully prepare for it, having received a preliminary consultation with a doctor.

Blood for PCR is usually donated on an empty stomach. Good results are shown by analysis when the material for research is taken from the cervical canal or urethra. In this case, it is best to carry out PCR diagnostics no later than one day after intercourse.

Varieties of PCR

PCR is used in many areas for analysis and in scientific experiments. There are different analysis methods:

1. reverse transcription PCR(Reverse Transcription PCR, RT-PCR (English)) - used to amplify, isolate or identify a known sequence from an RNA library.
2. Inverted PCR(Inverse PCR (English)) - is used if only a small area within the desired sequence is known. This method is especially useful when it is necessary to determine neighboring sequences after DNA has been inserted into the genome.
3. Nested PCR is used to reduce the number of side products of a reaction. Use two pairs of primers and carry out two consecutive reactions.
4. Asymmetric PCR(English Asymmetric PCR) - is carried out when it is necessary to amplify mainly one of the chains of the original DNA. Used in some sequencing and hybridization analysis techniques.
5. Quantitative PCR(Quantitative PCR, Q-PCR (English)) or real-time PCR - used to directly observe the measurement of the amount of a particular PCR product in each reaction cycle.
6. Stepped PCR (Touchdown PCR (English)) - using this approach, the influence of non-specific binding of primers is reduced.
7. Group-specific PCR(English group-specific PCR) - PCR for related sequences within one or between different species, using conservative primers for these sequences.

If the nucleotide sequence of the template is partially known or not known at all, degenerate primers can be used, the sequence of which contains degenerate positions in which any bases can be located. For example, the primer sequence could be: …ATH… where H is A, T, or C.

What biological materials are being studied?

Various biological media and human fluids can serve as a material for PCR research, in which foreign DNA of a bacterium or DNA or RNA of a virus can be detected:

1. Urine. It can be used for infectious lesions of the genitourinary tract in men and urinary organs in women (in men, the use of urine as a material replaces epithelial scraping).
2. Phlegm. It is used for the diagnosis of tuberculosis and less often for the diagnosis of respiratory forms of chlamydia and mycoplasmosis. Sputum in the amount of 15-20 ml is collected in a sterile (disposable) vial.
3. biological fluids. Prostate juice, pleural, cerebrospinal, amniotic, articular fluid, bronchoalveolar lavage, saliva are taken according to indications.
4. Epithelial scrapings from mucous membranes. Usually used to diagnose sexually transmitted diseases (STDs), such as gonorrhea, chlamydia, mycoplasmosis, ureaplasmosis, trichomoniasis, gardnerellosis, herpetic and other infections that affect the mucous membranes.
5. Biopsies. The most commonly used biopsy specimens of the stomach and duodenum to detect Helicobacter pylori infection.
6. Blood, plasma, serum. Used for PCR analysis of hepatitis B, C, D, G viruses, herpes, CMV, HIV, human genes.

How to prepare for the analysis?

The reliability of the PCR result directly depends on the correctness of the delivery of the material for examination. The material must not be contaminated, otherwise the result of the study will not be objective. The most important recommendations before taking a PCR test include the following requirements:

1. Urine is given in the morning in a sterile container.
2. A blood test for infections must be taken on an empty stomach in the morning.
3. You should not be sexually active the day before the test.

The result of the analysis will be ready in 1.5-2 days after the procedure in question. There are situations when the result can be prepared on the same day.

Deciphering the analysis of the PRP

The process of interpreting the presented study is notable for its simplicity. The results of PCR analysis can be obtained 1.5-2 days after the delivery of the material. In some cases, the result is ready on the first day, and this is what they can mean:

• Negative result shows that the material being diagnosed does not contain the desired infectious agent.
• PCR positive indicates that DNA or RNA of the pathogen is present in the human body.

In some cases, quantitative determination of microorganisms is carried out. This is especially true in diseases caused by opportunistic pathogens. Since these bacteria show their negative effects only when they are in excess.

Also, quantitative PCR analysis is important for the choice of therapeutic tactics and for the purpose of monitoring the treatment of such viral infections like HIV and hepatitis viruses.

How accurate is PCR in diagnosing infections?

The PCR method is characterized by high accuracy, specificity and sensitivity. It means that this analysis capable of:

• accurately determine the presence or absence of infection;
• specify exactly what kind of infection it is (specificity);
• detect infection even at a very low content of microbial DNA in biological material,
• which has been tested (sensitivity).

PCR analysis: price and terms

The price of a specific analysis will depend on which infection you will be tested for. Approximate prices and terms:

1. STI: 300-500 rubles, terms - 1 day;
2. Epstein-Barr virus, human papillomavirus, herpes, cytomegalovirus: 300-500 rubles, terms - 1 day;
3. Hepatitis A, B, C, D, G: qualitative analysis 650 rubles, quantitative analysis 2000 rubles. Terms - up to 5 days;
4. Antibodies to the hepatitis C virus, total (Anti-HCV) - 420 rubles;
5. Antibodies to the hepatitis C virus, IgM (Anti-HCV IgM) - 420 rubles;
6. Helicobacter pylori ( Helicobacter pylori): 300-400 rubles, terms - 1 day;
7. HIV (antibodies and antigens) - 380 rubles;
8. HIV RNA, qualitatively - 3,500 rubles;
9. HIV RNA, quantitatively - 11,000 rubles.

To save money, you can choose a fixed package of analyzes. This service is provided by most clinics where you can take an analysis using the PRC method (in vitro, onclinic, etc.).