What is PCR analysis? This is an effective method of molecular biology, which is used in combination with traditional immunological, morphological and biochemical studies. Infectious diseases evolve and develop, just like humanity itself. Every year there are more and more of them, and it is more and more difficult to diagnose them. The factors that provoke the appearance of diseases and affect their development, also gradually adapt to the surrounding external conditions, changing along with them. This was the reason that new methods and technologies began to appear in medicine that help to obtain more accurate results in the diagnosis of a particular disease.
Such a technique for laboratory diagnosis of infectious diseases is based on detecting the causative agent of the infection, which the doctor suspects in the research material. The polymerase chain reaction will identify them by identifying the appropriate genetic material (RNA or DNA) in the samples taken from the patient.
PCR was invented by the American scientist Carey Mullis in 1983.
Most infections, if detected early, respond well to treatment. That is why the PCR method is so effective, because it is able to detect even viruses or bacteria, whose cells are single in the material. Moreover, such a diagnosis determines the virus, establishes the nature of its appearance, the force with which it affects the body, even the number of microbes in the patient's body. All the information obtained by the polymerase chain reaction method, the doctor will be able to use in order to select the appropriate drugs and prescribe the appropriate treatment.
PCR study
By the very principle of work, everything happens quite simply. The biological material taken from the patient is placed in a special reactor. Then there are added such specific enzymes that can bind to the DNA of the microbe existing in the material and synthesize its copy.
Copying is based on the principle of a chain reaction. That is, it will take several stages to complete the entire process. First, 1 DNA molecule forms 2 new molecules, then 4 new ones will turn out of them, and so on up to hundreds and thousands of copies. After that, the analysis and its decoding will be carried out.
Fragments of microbe DNA can be contained in various biological material:
- in biological fluids (saliva, joint, amniotic fluid, pleural, cerebrospinal fluid, prostate juice);
- in blood and its serum, in plasma;
- in scraping of epithelial cells (scraping from the cervical canal, from the urethra - for both women and men);
- in urine (analysis will require morning urine, namely its first portion);
- in sputum;
- in mucus and other biological secretions;
- in biopaths of the stomach and duodenum.
What diseases can be detected by PCR method?
Doctors consider this type of diagnosis to be one of the most accurate. This study allows you to detect almost all viral diseases that are currently known to medicine. A very important aspect is the fact that among them there are infections that live in the human body for many years, while not manifesting themselves in any way. They just grow in anticipation of when it will be most comfortable for them to manifest themselves (decreased immunity, depletion of the body, etc.). The identification of such latent infections is especially important in the urological and gynecological areas.
Here are some diseases that are often analyzed using the polymerase chain reaction method:
- hepatitis B and C;
- many diseases that are sexually transmitted (diagnosis of chlamydia, ureaplasmosis, mycoplasmosis, genital candidiasis, bacterial vaginosis, trichomoniasis, infectious mononucleosis, human papillomavirus infection (HPV), AIDS, etc.);
- herpes infection (including genital herpes);
- tuberculosis and helicobacteriosis.
The PCR method gives good results, because most of these diseases are characterized by the fact that their symptoms (especially at an early stage) are not very noticeable. But the consequences that they have on the body are very negative and often very dangerous to health and even life.
For example, sexually transmitted diseases can significantly increase the chance of cervical cancer in women, negatively affect the course of pregnancy, or cause infertility. In addition, the health of her unborn baby depends on the health of the mother. Therefore, at the slightest suspicion of latent infections, it is very important to donate blood for diagnosis in time in order to prevent infection of the fetus through the penetration of pathogens. For men, the consequences are no less negative: a decrease in the mobility and viability of spermatozoa, the development of male infertility and prostatitis, damage to the urethra, and so on.
Such an analysis is also very relevant for the timely detection of viral hepatitis, tuberculosis, and various intestinal infections. When immediate treatment is required, it is very important to understand what kind of pathogen hit the body and what to fight with. The patient's blood or any other biological material will help to carry out a complete and correct diagnosis and prescribe treatment that will help the speedy restoration of body functions and will contribute to recovery.
Herpes is also extremely resistant and dangerous. From an inactive mode, it can easily turn into a progressive one, affecting the central nervous system, affecting the onset and development of such terrible diseases as meningitis and encephalitis.
Analysis decoding
After doing the research, you can get either a positive or negative result. It is the positive results that will indicate that this or that infection is present in your body. A negative result is deciphered so that there is no suspected infection in the patient's body. That is, nothing was found in the biological material that was submitted for research.
Any indicators should be deciphered and voiced to you by a doctor. Do not be discouraged or intimidated by poor results, because the reaction revealed a disease, which means that after additional examinations, the doctor will be able to prescribe a full-fledged treatment. The main thing is not to self-medicate and not to delay the process.
Preparing for analysis: what you need to know?
Different biological materials are required to detect different diseases. Before doing PCR, be sure to consult with the appropriate specialist and carefully prepare for the upcoming procedure.
To do this, you will need to strictly follow all the recommendations and instructions of your doctor. Remember that blood is usually taken on an empty stomach. To take a smear from the vagina or urethra, you need to refrain from sexual intercourse for 1-2 days, carry out all the necessary hygiene of the genitals in the evening, and in the morning just rinse with warm water. It is also important to stop taking medications about a week in advance if they have not been discussed with your doctor. And for 2-3 hours before taking the test, do not urinate. For women, it is worth considering that the optimal time to conduct a study is a few days before or immediately after menstruation.
PCR method: main advantages and disadvantages
This type of diagnosis has many advantages.
Firstly, any causative agents of infectious diseases are determined by direct instruction, in contrast to other traditional laboratory tests.
Secondly, this analysis is simply universal, because almost any biological materials are available for it, which are often not acceptable to any other research method.
It is very important that when this diagnosis is carried out, a DNA fragment is isolated in the material under study, which will be inherent only in a specific pathogen, that is, a purely specific virus or bacteria. This indicates how specific this reaction is.
Another argument in favor of this diagnostics will be the high speed of its execution. If any culture studies require not days, but even weeks required to isolate and grow the pathogen on a cell culture, then this method will provide results within 4-5 hours.
PCR makes it possible to detect not only the infection that is already at the peak of the disease, but also chronic diseases, any single viruses or bacteria. Such a diagnosis is possible due to the very high sensitivity of the method. This should include the fact that the infection will be detected even if only one cell of a particular virus or bacteria is present in the biological material that was submitted for analysis.
False negative results are very rare.
True, the method also has its drawbacks. One of them is the possibility of getting a false positive result. This is often due to the fact that the infection has already been killed, but its epithelial cells have not yet been renewed, so the reaction will still see its dead remains and will clone it. Therefore, you should either wait until the dead cells of the infection are completely removed from the body (from a month to two after the treatment), or use other methods at an early stage: inoculation or culture. Later it will be possible to carry out control using PCR.
Another weakness of the analysis is considered to be the variability of all microorganisms. This means that some genotypes of pathogens that have already mutated in the body will be elusive for the test system and no reaction will occur. For this, various developments are being made to improve this method.
Federal Agency for Education
State educational institution
Higher professional education
"Karelian State Pedagogical Academy"
Course work on the topic:
Polymerase chain reaction (PCR) and its applications
Completed by: student Koryagina Valeria Aleksandrovna
Checked by: Karpikova Natalia Mikhailovna
Petrozavodsk 2013
Introduction
Chapter 1. Literary Review
1.5.4 The "Plateau" effect
1.5.6 Amplification
Conclusion
Introduction
The last twenty years have been marked by the widespread introduction of molecular genetic methods into biological, medical and agricultural sciences.
By the early 1970s, molecular biology seemed to have reached a certain stage of completion. During this period, the main object of molecular genetic research was microorganisms. The transition to eukaryotes posed completely new problems for researchers that could not be solved using the methods of genetic analysis that existed at that time. A breakthrough in the development of molecular genetics became possible thanks to the emergence of a new experimental tool - restriction endonucleases. In subsequent years, the number of direct DNA analysis methods based on qualitatively different approaches began to increase rapidly.
Modern technologies in many cases have made it possible to begin the study of the fine structural and functional organization of nuclear and extra-nuclear genomes of various organisms at a deeper level. This was of particular importance for the development of new methods for the diagnosis and treatment of various diseases. Equally important was the possibility of using the achievements of molecular genetics in population biology and in breeding for identifying and analyzing the genetic variability of populations, varieties and strains, identifying and certifying economically valuable individuals, creating genetically modified organisms, and for solving other issues.
Each method has its own advantages and disadvantages. There is no one-size-fits-all method that can solve all the problems that arise. Therefore, the choice of a specific method for the research being carried out is one of the most important stages of any scientific work.
Chapter 1. Literary Review
1.1 History of the discovery of the Polymerase chain reaction (PCR)
In 1983, K.B. Mullis et al. Published and patented the polymerase chain reaction (PCR) method, which was destined to have a profound impact on all areas of research and applications of nucleic acids. The significance of this method for molecular biology and genetics turned out to be so great and obvious that seven years later the author was awarded the Nobel Prize in Chemistry.
At the beginning of using 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 ineffective, requiring a lot of time and enzyme. In 1986, the polymerase chain reaction method was significantly improved. It was proposed to use DNA polymerases from thermophilic bacteria. These enzymes proved to be thermally stable and were able to withstand multiple reaction cycles. Their use made it possible to simplify and automate PCR. One of the first thermostable DNA polymerases was isolated from bacteria Thermus aquaticusand named Taq-polymerase.
The possibility of amplifying any DNA segment, the nucleotide sequence of which is known, and obtaining it after the completion of PCR in a homogeneous form and preparative amount make PCR an alternative method for molecular cloning of short DNA fragments. At the same time, there is no need to use complex methodological techniques that are used in genetic engineering in conventional cloning. The development of the PCR method has largely expanded the methodological capabilities of molecular genetics, and, in particular, genetic engineering, and so much that it has radically changed and strengthened the scientific potential of many of its areas.
1.2 Varieties of polymerase chain reaction (PCR)
· Nested PCR- used to reduce the number of by-products of the reaction. Two pairs of primers are used and two consecutive reactions are carried out. A second pair of primers amplifies a stretch of DNA within the product of the first reaction.
· Inverted PCR- is used if only a small area within the required sequence is known. This method is especially useful when it is necessary to determine adjacent sequences after inserting DNA into the genome. To carry out inverted PCR, a series of DNA cuts is performed with restriction endonucleases<#"justify">polymerase chain reaction primer
· Group-specific PCR- PCR for relatives<#"center">1.3 Polymerase chain reaction
Discovered in the mid-1980s, polymerase chain reaction (PCR) is capable of increasing the copy number of an original sample by millions of times over a period of several hours. During each reaction cycle, two copies are formed from the original molecule. Each of the synthesized DNA copies can serve as a template for the synthesis of new DNA copies in the next cycle. Thus, multiple repetition of cycles leads to an increase in the number of copies exponentially. It follows from the calculations that even with 30 cycles, the number of copies of the original molecule will be more than 1 billion. Even if we take into account that not all amplicons are duplicated during each cycle, the total number of copies, in spite of this, is quite a large figure.
Each cycle of the polymerase chain reaction (PCR) consists of the following steps:
· Denaturation - An increase in temperature causes unwinding and splitting of a double-stranded DNA molecule into two single-stranded ones;
· Annealing - Lowering the temperature allows the primers to attach to the complementary regions of the DNA molecule;
· Elongation - The enzyme DNA polymerase completes the complementary strand.
To amplify the selected fragment, two oligonucleotide primers (primers) are used, flanking a specific DNA region. Primer oriented 3 - ends towards each other and towards the sequence that needs to be amplified. DNA polymerase carries out the synthesis (extension) of mutually complementary DNA strands, starting with primers. During DNA synthesis, primers are physically incorporated into the chain of newly synthesized DNA molecules. Each strand of a DNA molecule formed with one of the primers can serve as a template for the synthesis of a complementary DNA strand using another primer.
1.4 Carrying out polymerase chain reaction (PCR)
The polymerase chain reaction is carried out in special thin-walled polypropylene tubes, compatible in size with the used thermocycler (amplifier) - a device that controls the temperature and time characteristics of the stages of the polymerase chain reaction (PCR).
1.5 Principle of the polymerase chain reaction method
Polymerase chain reaction (PCR) is an in vitro DNA amplification method, with which, within a few hours, a specific DNA sequence can be isolated and multiplied billions of times. The possibility of obtaining a huge number of copies of one strictly defined region of the genome greatly simplifies the study of an available DNA sample.
To carry out the polymerase chain reaction, a number of conditions must be met:
1.5.1 The presence of a number of components in the reaction mixture
The main components of the reaction (PCR) mixture are: Tris-HCl, KCl, MgCl 2, a mixture of nucleotide triphosphates (ATP, GTP, CTP, TTF), primers (oligonucleotides), a preparation of the analyzed DNA, thermostable DNA polymerase. Each of the components of the reaction mixture is directly involved in the polymerase chain reaction (PCR), and the concentration of reagents directly affects the course of amplification.
· Tris-HCl - determines the pH of the reaction mixture, creates a buffer capacity. The activity of DNA polymerase depends on the pH of the medium, therefore the pH value directly affects the course of the polymerase chain reaction. Typically the pH is between 8 and 9.5. A high pH value is taken due to the fact that as the temperature rises, the pH of the Tril-HCl buffer drops.
· KCl - concentration of potassium chloride up to 50 mM affects the course of denaturation and annealing processes, concentration over 50 mM inhibits DNA polymerase.
· MgCl 2- since DNA polymerase is Mg 2+- dependent enzyme, the concentration of magnesium ions affects the activity of the enzyme (Mg 2+forms complexes with NTF - these complexes are the substrate for polymerase). A high concentration leads to an increase in nonspecific amplification, and a low concentration leads to inhibition of the reaction, the optimum (for various polymerases) is in the range of 0.5 - 5 mM. In addition, the concentration of magnesium salts affects the course of denaturation and annealing processes - an increase in the concentration of Mg 2+causes an increase in the melting temperature of DNA (i.e., temperature, with a bark, 50% of double-stranded DNA strands are separated into single-stranded ones).
· NTF - nucleotide triphosphates are direct monomers of nucleic acids. An equal proportion of all four nucleotide triphosphates is recommended to prevent chain termination. A low concentration of these components in the reaction mixture increases the likelihood of errors in the construction of a complementary DNA strand.
· Primers - The most optimal is the use of primers with a melting point difference of no more than 2 - 4 o C. Sometimes during prolonged storage at a temperature of 4 o With, or after a large number of freezing - thawing, primers form secondary structures - dimers, reducing the efficiency of PCR. Elimination of this problem is reduced to incubation in a water bath (T = 95 o C) for 3 minutes and subsequent sharp cooling to 0o WITH.
· DNA preparations - the quantity and quality of the DNA preparation (matrix) directly affects the course and parameters of the polymerase chain reaction. An excess amount of DNA sample inhibits the polymerase chain reaction (PCR). Impurities of various substances in the DNA preparation can also reduce the efficiency of the polymerase chain reaction (PCR): sodium acetate, sodium chloride, isopropanol, ethanol, heparin, phenol, urea, hemoglobin, etc.
· DNA polymerase - when a small amount of DNA polymerase is used, a decrease in the synthesis of the final product is observed in direct proportion to the size of the fragments. An excess of polymerase by a factor of 2 - 4 leads to the appearance of diffuse spectra, and by a factor of 4 - 16 - of low molecular weight nonspecific spectra. The range of concentrations used is 0.5 - 1.5 activity units per 25 μl of PCR mixture.
In addition to the main components of the PCR mixture, a number of additional substances are used that improve the qualitative and quantitative parameters of PCR: acetamide (5%) - an increase in the solubility of the main components; betaine (sodium salt) - stabilization of DNA polymerase, lowering the melting point of DNA, leveling the melting point; bovine albumin (10-100 μg / ml) - stabilization of DNA polymerase; dimethyl sulfoxide (1-10%) - increasing the solubility of the main components; formamide (2-10%) - an increase in the specificity of annealing; glycerol (15-20%) - an increase in the thermal stability of the enzyme, a decrease in the denaturation temperature of the DNA sample; ammonium sulfate - lowering the denaturation and annealing temperature.
1.5.2 Cyclic and temperature conditions
The general view of the polymerase chain reaction (PCR) program is as follows:
stage. Long-term primary denaturation of the DNA preparation. 1 cycle
stage. Rapid denaturation of the DNA preparation. Annealing the primers. Elongation. 30 - 45 cycles.
stage. Long-term elongation. Cooling the reaction mixture. 1 cycle.
Each stage element - denaturation, annealing, elongation - has individual temperature and time characteristics. The parameters of temperature and time of flow of each element are selected empirically, in accordance with the qualitative and quantitative indicators of the amplification products.
Denaturation. During this element of the polymerase chain reaction, a double-stranded DNA molecule is split into two single-stranded ones. Temperature parameters of denaturation are in the range of 90 - 95 o C, but in the case of a DNA sample with a high content of guanine and cytosine, the temperature should be increased to 98 o C. The temperature of denaturation must be sufficient for complete denaturation - cleavage of DNA strands and avoiding "sudden cooling" or rapid annealing, however, thermostable DNA polymerase is less stable at high temperatures. Thus, the selection of the optimal temperature parameters of denaturation for the primer / sample ratio (DNA preparation) is an important condition for carrying out amplification. If the denaturation temperature in the first stage is above 95 o C, it is recommended to add DNA polymerase to the reaction mixture after primary denaturation. The duration of this stage element in the course of the polymerase chain reaction (PCR) should be sufficient for complete DNA denaturation, but at the same time not have a significant effect on the activity of DNA polymerase at a given temperature.
Annealing. Annealing temperature (T a ) is one of the most important parameters of the polymerase chain reaction. Annealing temperature for each specific primer is selected individually. It depends on the length and nucleotide composition of the primer. Usually it is lower by 2 - 4 o Melting point value (T m ) primer. If the annealing temperature of the system is lower than the optimal one, then the number of nonspecific amplified fragments increases and, conversely, a higher temperature decreases the amount of amplified products. In this case, the concentration of specific amplicons can sharply decrease, up to inhibition of the polymerase chain reaction (PCR). An increase in the annealing time also leads to an increase in the number of nonspecific amplicons.
Elongation. Usually, each type of thermostable DNA polymerase has an individual temperature optimum of activity. The rate of synthesis of a complementary DNA strand by the enzyme is also a value specific to each polymerase (on average, it is 30-60 nucleotides per second, or 1-2 thousand bases per minute), therefore the elongation time is selected depending on the type of DNA polymerase and the length of the amplified region.
1.5.3 Basic principles of primer selection
When creating a PCR test system, one of the main tasks is the correct selection of primers, which must meet a number of criteria:
Primers must be specific. Pay special attention to 3 - the ends of the primers, because it is from them that Taq polymerase begins to build up the complementary DNA strand. If their specificity is insufficient, then it is likely that undesirable processes will occur in the test tube with the reaction mixture, namely, the synthesis of nonspecific DNA (short or long fragments). It is visible on electrophoresis in the form of heavy or light additional bands. This interferes with the evaluation of the reaction results, since it is easy to confuse a specific amplification product with synthesized extraneous DNA. Some primers and dNTPs are consumed for the synthesis of nonspecific DNA, which leads to a significant loss of sensitivity.
Primers should not form dimers and loops, i.e. no stable double chains should be formed as a result of annealing the primers to themselves or to each other.
1.5.4 The "Plateau" effect
It should be noted that the process of accumulation of specific amplification products in a geometric progression lasts only for a limited time, and then its efficiency drops critically. This is due to the so-called "plateau" effect.
Effect term plateau are used to describe the accumulation of PCR products during the last amplification cycles.
Depending on the conditions and the number of cycles of the amplification reaction, at the time of achieving the effect plateau Utilization of substrates (dNTPs and primers), stability of reactants (dNTPs and enzymes), the number of inhibitors, including pyrophosphates and DNA duplexes, competition for reactants with nonspecific products or primer-dimers, the concentration of a specific product, and incomplete denaturation at a high concentration of amplification products are affected.
The lower the initial concentration of the target DNA, the higher the risk of the reaction to plateau. ”This point may come before the amount of specific amplification products is sufficient to be analyzed, and this can only be avoided by well-optimized test systems.
1.5.5 Preparation of a sample of biological material
For DNA extraction, various techniques are used, depending on the tasks. Their essence lies in the extraction (extraction) of DNA from a biological product and the removal or neutralization of impurities to obtain a DNA preparation with a purity suitable for PCR.
The method for obtaining a pure DNA preparation, described by Marmur, is considered standard and has already become classic. It includes enzymatic proteolysis followed by deproteinization and DNA reprecipitation with alcohol. This method allows you to obtain a pure DNA preparation. However, it is quite laborious and involves working with aggressive and pungent substances such as phenol and chloroform.
One of the currently popular methods is the DNA extraction method proposed by Boom et al. This method is based on the use of a strong chaotropic agent, guanidine thiocyanate (GuSCN), for cell lysis, and subsequent sorption of DNA on a carrier (glass beads, diatomaceous earth, glass "milk", etc.). After washing, DNA remains in the sample, adsorbed on the carrier, from which it can be easily removed using an elution buffer. The method is convenient, technological, and suitable for preparing a sample for amplification. However, DNA losses are possible due to irreversible sorption on the carrier, as well as in the process of numerous washes. This is especially important when working with small amounts of DNA in the sample. In addition, even trace amounts of GuSCN can inhibit PCR. Therefore, when using this method, the correct choice of sorbent and careful observance of technological nuances are very important.
Another group of sample preparation methods is based on the use of Chilex-type ion exchangers, which, unlike glass, do not adsorb DNA, but, on the contrary, impurities that interfere with the reaction. As a rule, this technology includes two stages: boiling the sample and sorption of impurities on the ion exchanger. The method is extremely attractive for its simplicity of execution. In most cases, it is suitable for working with clinical material. Unfortunately, sometimes there are samples with such impurities that cannot be removed using ion exchangers. In addition, some microorganisms cannot be destroyed by simple boiling. In these cases, it is necessary to introduce additional stages of sample processing.
Thus, the choice of the sample preparation method should be considered with an understanding of the objectives of the intended analyzes.
1.5.6 Amplification
To carry out the amplification reaction, it is necessary to prepare a reaction mixture and add the analyzed DNA sample to it. In this case, it is important to take into account some features of primer annealing. The fact is that, as a rule, the analyzed biological sample contains a variety of DNA molecules, to which the primers used in the reaction have partial, and in some cases significant, homology. In addition, primers can anneal to each other to form primer dimers. Both leads to a significant consumption of primers for the synthesis of by-product (nonspecific) reaction products and, as a consequence, significantly reduces the sensitivity of the system. This makes it difficult or impossible to read the results of the reaction during electrophoresis.
1.6 Composition of the standard PCR reaction mixture
x PCR buffer (100 mM Tris-HCl solution, pH 9.0, 500 mM KCl solution, 25 mM MgCl2 solution ) …… .2.5 μl
Water (MilliQ) …………………………………………………… .18.8 μl
Mixture of nucleotide triphosphates (dNTPs)
mm solution of each ………………………………………. ……… .0.5 μl
Primer 1 (10 mM solution) ………………………………………….… .1 μl
Primer 2 (10 mM solution) ………………………………………….… .1 μl
DNA polymerase (5 units / μl) ……………………………………… 0.2 μl
DNA sample (20 ng / μl) ………………………………………… ..1 μl
1.7 Evaluation of reaction results
For a correct assessment of PCR results, it is important to understand that this method is not quantitative. Theoretically, the products of amplification of single target DNA molecules can be detected by electrophoresis after 30-35 cycles. However, in practice, this is done only in cases where the reaction takes place under conditions close to ideal, which is not often encountered in life. The degree of purity of the DNA preparation has a particularly great influence on the amplification efficiency, i.e. the presence of certain inhibitors in the reaction mixture, which in some cases can be extremely difficult to get rid of. Sometimes, due to their presence, it is not possible to amplify even tens of thousands of target DNA molecules. Thus, there is often no direct relationship between the initial amount of target DNA and the final amount of amplification products.
Chapter 2: Applications of the Polymerase Chain Reaction
PCR is used in many fields for analysis 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. He is being compared to the suspect's genetic material. A very small amount of DNA is enough, theoretically - one copy. DNA is cleaved into fragments, then amplified by PCR. The fragments are separated by DNA electrophoresis. The resulting picture of the location of the DNA bands is called a genetic fingerprint.
Establishing paternity
Results of electrophoresis of DNA fragments amplified by PCR. Father. Child. Mother. The child inherited some of the characteristics of the genetic imprint of both parents, which gave a new, unique imprint.
Although genetic fingerprints are unique, family ties can still be established by making multiple such fingerprints. The same method can be applied, slightly modified, to establish evolutionary kinship among organisms.
Medical diagnostics
PCR makes it possible to significantly speed up and facilitate the diagnosis of hereditary and viral diseases. The desired gene is amplified by PCR using the appropriate primers and then sequenced to detect mutations. Viral infections can be detected immediately after infection, weeks or months before symptoms of the disease appear.
Personalized medicine
Sometimes medications are toxic or allergenic for some patients. The reasons for this are partly in individual differences in the susceptibility and metabolism of drugs and their derivatives. These differences are determined at the genetic level. For example, in one patient, a certain cytochrome may be more active, in another - less. In order to determine what kind of cytochrome a given patient possesses, it was proposed to carry out a PCR analysis before using the drug. This analysis is called pre-genotyping.
Gene cloning
Gene cloning is the process of isolating genes and, as a result of genetic engineering manipulations, obtaining a large amount of the product of a given gene. PCR is used to amplify a gene, which is then inserted into a vector - a piece of DNA that transfers a foreign gene into the same or another, convenient for growing, organism. As vectors are used, for example, plasmids or viral DNA. The insertion of genes into a foreign organism is usually used to obtain the product of this gene - RNA or, more often, a protein. Thus, many proteins are obtained in industrial quantities for use in agriculture, medicine, etc.
DNA sequencing
In the sequencing method using fluorescently labeled or radioactive isotope dideoxynucleotides, PCR is an integral part, since it is during polymerization that nucleotide derivatives labeled with a fluorescent or radioactive label are incorporated into the DNA strand. This stops the reaction, allowing the position of specific nucleotides to be determined after separation of the synthesized strands in the gel.
Mutagenesis
Currently, PCR has become the main method for carrying out mutagenesis. The use of PCR made it possible to simplify and speed up the procedure for carrying out mutagenesis, as well as to make it more reliable and reproducible.
The PCR method made it possible to analyze the presence of human papillomavirus sequences in biopsy sections of human cervical neoplasms embedded in paraffin 40 years before this study. Moreover, with the help of PCR, it was possible to amplify and clone fragments of mitochondrial DNA from the fossil remains of the human brain aged 7 thousand years!
The ability to simultaneously analyze two loci located on different non-homologous chromosomes has been demonstrated on lysates of individual human spermatozoa. This approach provides a unique opportunity for fine genetic analysis and the study of chromosomal recombination, DNA polymorphism, etc. The method of analysis of individual spermatozoa immediately found practical application in forensic medicine, since HLA typing of haploid cells allows determining paternity or identifying the culprit (the HLA complex is a set genes of the main human histocompatibility complex; loci of the HLA complex are the most polymorphic of all known in higher vertebrates: within a species in each locus there is an unusually large number of different alleles - alternative forms of the same gene).
Using PCR, it is possible to reveal the correctness of the integration of foreign genetic structures into a predetermined region of the genome of the cells under study. Total cellular DNA is annealed with two oligonucleotide primers, one of which is complementary to the host DNA region near the insertion point, and the other to the sequence of the integrated fragment in the antiparallel DNA strand. In the case of an unchanged chromosomal DNA structure at the proposed insertion site, the polymerase chain reaction leads to the formation of single-stranded DNA fragments of an undetermined size, and in the case of a planned insertion, double-stranded DNA fragments of a known size, determined by the distance between the annealing sites of the two primers. Moreover, the degree of amplification of the analyzed region of the genome in the first case will be linearly dependent on the number of cycles, and in the second - in exponential. The exponential accumulation of an amplified fragment of a previously known size during PCR makes it possible to visually observe it after electrophoretic fractionation of a DNA preparation and make an unambiguous conclusion about the insertion of a foreign sequence into a given region of chromosomal DNA.
Conclusion
The most widespread method of PCR is currently received as a method for diagnosing various infectious diseases. PCR allows you to identify the etiology of the infection, even if the sample taken for analysis contains only a few DNA molecules of the pathogen. PCR is widely used in the early diagnosis of HIV infections, viral hepatitis, etc. Today, there is almost no infectious agent that could not be detected using PCR.
List of used literature
1.Padutov V.E., Baranov O.Yu., Voropaev E.V. Molecular genetic analysis methods. - Minsk: Unipol, 2007 .-- 176 p.
2.PCR "in real time" / Rebrikov DV, Samatov GA, Trofimov D.Yu. and etc.; ed. d. b. n. D.V. Rebrikov; foreword L.A. Osterman and Acad. RAS and RAAS E.D. Sverdlov; 2nd ed., Rev. and add. - M .: BINOM. Knowledge Laboratory, 2009 .-- 223 p.
.Patrushev L.I. Artificial genetic systems. - M .: Nauka, 2005. - In 2 volumes
.B. Glick, J. Pasternak Molecular Biotechnology. Principles and Application 589 pp., 2002
5.Shchelkunov S.N.
Edited by A.A. Vorbieva
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GOU VPO "Krasnoyarsk State Medical Academy
named after -Yasenetsky Federal Agency for Healthcare and Social Development "
Department of Medical Genetics and Clinical Neurophysiology IPO
BASIC PRINCIPLES OF THE METHOD
POLYMERASE CHAIN REACTION
Methodological guide for 3-4 year students
in the specialties of general medicine (060101) and
Krasnoyarsk - 2007
Schneider, N. A., Butyanov, R. A. Basic principles of the polymerase chain reaction method. Methodological manual for extracurricular work of 3-4 year students in the specialties of general medicine (060101) and pediatrics (060103). - Krasnoyarsk: Publishing house of GOU VPO KrasGMA, 2007. - 42 p.
The methodological manual fully complies with the requirements of the State Standard (2000) and reflects the main aspects of the modern method for diagnosing hereditary human diseases - the polymerase chain reaction method, 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, GOU VPO
Novosibirsk State Medical University of the Federal Agency for Healthcare and Social Development, Doctor of Medical Sciences, Professor;
DNA replication
The object of research of this method is deoxyribonucleic acid (DNA). DNA is a universal carrier of genetic information for 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. A unique property of DNA is its ability to duplicate. This process is called replication... Replication of the DNA molecule occurs during the synthetic period of the interphase. Each of the two chains of the "parent" molecule serves as a matrix for the "daughter". After replication, the newly synthesized DNA molecule contains one “mother” chain, and the second “daughter”, newly synthesized (semi-conservative method). For the matrix synthesis of a new DNA molecule, it is necessary that the old molecule be despiralized and stretched. Replication begins at several locations in the DNA molecule. A section of a DNA molecule from the point of origin of one replication to the point of origin of another is called replicon.
Replication start is activated primers(seeds), consisting of 100-200 base pairs. The DNA helicase enzyme unwinds and divides the maternal 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, a starting block is required - a small initial double-stranded fragment. The starting block is formed when the primer interacts with the complementary region of the corresponding strand of the parental DNA. In each replicon, DNA polymerase can move along the "mother" strand in only one direction (5` => 3`).
On the leading thread, as the replicon unwinds, the "daughter" chain is gradually built up continuously. On the lagging thread, the daughter chain also synthesizes in the direction (5` => 3`), but in separate fragments as the replicon unwinds.
Thus, the addition of complementary nucleotides of the "daughter" strands is in opposite directions (antiparallel). Replication in all replicons occurs simultaneously. Fragments and parts of "daughter" threads, synthesized in different replicons, are stitched into a single thread by the enzyme ligase. Replication is characterized by semi-conservatism, anti-parallelism, 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, daughter, is newly synthesized (Fig. 1).
Rice. 1. DNA molecule replication scheme.
Thus, the DNA replication cycle includes three main stages:
1. unweaving of the DNA helix and strand separation (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 above processes 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 stage - attachment or "annealing" of primers - the incubation mixture is cooled to 50-65 ° C. Then the mixture is heated to 70-72 ° C - the optimum work of taq-DNA polymerase - at this stage, a new DNA strand is completed. Then the cycle is repeated again. In other words PCR method is a multiple increase in the number of copies (amplification) specific area of DNA catalyzed by the enzyme DNA - polymerase.
The extension of daughter DNA strands must occur simultaneously on both strands of the mother's DNA, therefore, a primer is also required for the replication of the second strand. Thus, two primers are introduced into the reaction mixture: one for the "+" - chain, the second for the "-" - chain. Having attached to the opposite strands of the DNA molecule, the primers restrict that part of it, which will be subsequently duplicated or amplified many times. The length of such a fragment, called an amplicon, is usually several hundred nucleotides.
PCR stages
Each amplification cycle includes 3 stages that take place at different temperature conditions (Fig. 2).
· Stage 1: DNA denaturation . It runs at 93-95 ° for 30-40 seconds.
· Stage 2: annealing primers . The attachment of primers is 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: DNA strand extension. Complementary DNA strand extension occurs from the 5 "end to the 3" end of the chain in opposite directions, starting from the primer attachment sites. Deoxyribonucleoside triphosphates added to the solution serve as the material for the synthesis of new DNA strands. The synthesis process is catalyzed by the enzyme taq-polymerase and takes place at a temperature of 70-72 ° C. The duration of the synthesis is 20-40 sec.
The new DNA strands formed in the first cycle of amplification serve as templates for the second cycle of amplification, in which a specific fragment of DNA amplicon is formed (Fig. 3). In subsequent rounds of amplification, the amplicons serve as a template for the synthesis of new strands.
Thus, there is an accumulation of amplicons in the solution according to the formula 2 ", where n is the number of amplification cycles. Therefore, even if initially there was only one double-stranded DNA molecule in the initial solution, about 108 amplicon molecules accumulate in the solution over 30-40 cycles. 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 ( amplifier), which, according to a given program, automatically changes temperatures according to the number of amplification cycles.
To carry out amplification, the following components are required:
· DNA matrix(DNA or a part thereof containing the desired specific fragment);
· Primers(synthetic oligonucleotides (20-30 nucleotide pairs), complementary to DNA sequences at the boundaries of the determined specific fragment). The selection of a specific fragment and the selection of primers play an important role in the specificity of the amplification, which affects the quality of the assay.
· Deoxynucleotide triphosphate (dNTP) mixture(a mixture of four dNTPs, which are material for the synthesis of new complementary DNA strands in equivalent concentrations of 200-500 μm)
· EnzymeTaq-polymerase(thermostable DNA polymerase, catalyzing the lengthening of primer chains by sequential attachment of nucleotide bases to the growing chain of the synthesized DNA, 2-3 mM).
· Buffer solution(a reaction medium containing Mg2 + ions necessary to maintain the activity of the enzyme, PH 6.8-7.8).
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).
Rice. 2. Amplification (1st cycle).
Rice. 3. Amplification (2nd cycle).
The main areas of application of PCR
Clinical medicine:
o diagnosis of infections,
o detection of mutations, including diagnostics of hereditary diseases,
o genotyping, including HLA genotyping,
o cell technologies
Ecology (as a way to monitor the state and quality of environmental objects and food products)
Determination of transgenic organisms (GMO)
Personal identification, paternity, forensics
General and private biology,
Basic principles
organization of diagnostic laboratories
Work in the PCR laboratory is carried out in accordance with the "Rules for the device, safety precautions, industrial sanitation, anti-epidemic regime and personal hygiene when working in laboratories (departments, departments) of sanitary and epidemiological institutions of the health care system."
Contamination of DNA samples
Carrying out PCR diagnostics is associated with a problem due to the high sensitivity of the method - the possibility contamination. The ingestion of trace amounts of positive DNA (specific products of DNA amplification - amplicons; DNA standard used as a positive control; positive DNA of a clinical sample) into a reaction tube leads to amplification of a specific DNA fragment during PCR and, as a consequence, to false positive results.
In the process of work, there may be two types of contamination:
1. cross contamination from sample to sample (during the processing of clinical samples or when dispensing a reaction mixture), leading to the appearance of sporadic false positive results;
2. contamination with amplification products(amplicons), which is of the greatest importance, since amplicons accumulate in huge quantities during the PCR process and are ideal products for re-amplification.
Trace amplicon contamination of glassware, automatic pipettes and laboratory equipment, the surface of laboratory tables, or even the surface of the skin of laboratory staff leads to systematic false positive results. Determining the source of contamination can be very difficult and time-consuming and costly. The experience gained to date in the work of laboratories using the PCR method for diagnostics makes it possible 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
They are territorially divided by placing them in separate rooms (Figure 4.5):
· Pre-PCR room, where the processing of clinical samples, DNA extraction, preparation of the reaction mixture for PCR and the setting of PCR are carried out (if conditions exist, the last two stages are also recommended to be carried out in an additional separate room). In these rooms, it is prohibited to carry out all other types of work with the investigated agents, the PCR diagnostics of which is 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 advisable to place the room for the detection of amplification products as far as possible from the pre-PCR rooms.
Workrooms are equipped with ultraviolet lamps with a maximum radiation in the 260 nm region (DB-60 type) at the rate of 2.5 W per 1 m3. The lamps are located so that the surfaces of the work 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.
Doctors-laboratory assistants work in special laboratory clothes, which are changed when moving from one room to another, and in disposable gloves. Garments from different premises are processed separately. Different employees work at different stages of the PCR analysis.
For work, use separate sets of dispensers, plastic and glassware, laboratory equipment, gowns and gloves designed for various stages of analysis and not transferred from one room to another. Equipment, materials and inventory in each room are marked accordingly.
All stages of work are carried out only using disposable consumables: tips for automatic pipettes, test tubes, gloves, etc. Be sure to change tips when passing from sample to sample. Use tips with an aerosol barrier filter to prevent micro-droplets of solution from entering the pipette. Used tubes and tips are disposed of in special containers or containers containing a disinfectant solution. Store clinical specimens separately from reagents.
For processing and cleaning the workplace, each room has cotton-gauze swabs (napkins), tweezers, disinfecting and inactivating solutions.
In a PCR diagnostic laboratory, it is excluded to carry out 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.
Collection of clinical material
The test material for PCR can be scraped epithelial cells, blood, plasma, serum, pleural and cerebrospinal fluid, urine, sputum, mucus and other biological secretions, biopsies.
The material is taken in a treatment room of the corresponding profile. After collection, samples should be delivered to the PCR diagnostic laboratory as soon as possible.
Samples should be taken using sterile, preferably disposable, instrumentation only into disposable sterile plastic test tubes or glass test tubes pretreated for an hour with a chromium mixture, thoroughly washed with distilled water and calcined in an oven at 150 ° C for 1 hour.
Detection area (another floor or another building).
Rice. 4. PCR laboratory device with electrophoresis detection.
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Detection area (another floor or another building)
Rice. 5. PCR laboratory device with fluorescence detection (quantitative analysis).
Rice. 6. DNA extraction room. Shown is a tabletop box with a germicidal lamp.
Rice. 7. Amplification room.
Rice. eight. Detection room.
Rice. nine. Blood samples for DNA diagnostics of hereditary diseases.
Storage and transportation of samples
To diagnose hereditary diseases, blood samples are stored on special paper forms or in epindorfs (plastic tubes) frozen 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 a longer storage is required, the samples can be placed in a refrigerator with a temperature of 2-8 ° C for a period not exceeding a day. Longer storage (up to 2 weeks) is permissible 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 the transportation of samples should be carried out in thermoses or thermal containers in compliance with the rules for storing samples and the rules for transporting infectious materials.
Isolation of DNA from samples
The method of solid-phase sorption has become widespread, which consists in adding a lysing agent containing a guanidine solution, sorption of DNA on a sorbent, and repeated washing and resorption of DNA with a buffer solution. In the case of serum, plasma or whole blood, the phenolic extraction method is usually used. The method involves deproteinization with phenol / chloroform followed by DNA (or RNA) precipitation with ethanol or isopropanol. Processing is carried out in 1.5 ml Eppendor P microcentrifuge tubes. The processing time is 1.5-2 hours (Fig. 10).
Rice. ten. Isolation of DNA.
Carrying out PCR
A certain amount of the sample from the processed clinical sample is transferred into a special microcentrifuge tube of the Eppendorf type 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 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 set program, is 2-3 hours. In parallel with the experimental samples, control samples are set: 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 the 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 DNA under investigation is added. Negative control is necessary to check the components of the reaction for the absence of DNA in them due to contamination and to exclude the accounting of 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 implantation compound with DNA fragments, which appears in the form of 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 with an agarose content of 1.5% to 2.5% 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. The mixture cooled to 50-60 ° C is poured into the mold with a layer 4-6 mm thick and, using special combs, pockets are made in the gel for applying the sample. The combs are set so that a 0.5-1 mm agarose layer remains between the bottom of the wells and the base of the gel. After the gel has solidified, an amplificate is applied to the pockets in an amount of 5-15 μl. It is recommended to conduct electrophoresis of a mixture of DNA fragment length markers in parallel with control and experimental samples. Typically, such a mixture contains ten DNA fragments of 100, 200, 300, etc. base pairs.
Setting up such a sample allows you to verify the length of the amplicons in control and experimental samples. The gel with the applied sample is transferred into an electrophoresis chamber filled with a buffer, the chamber is connected to a power source and electrophoretic separation of 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 included in the reaction mixture must pass at least 3 cm.
After the end of electrophoresis, the gel is transferred to the glass of the transilluminator and viewed in ultraviolet light. For documentation, the gel is photographed on a Micrat 300 film or recorded using a video system connected to a computer.
Control samples are evaluated first. The positive control electrophoretic lane should have an orange luminous band. Its electrophoretic mobility must correspond to the length of the amplicon specified in the instructions.
In the electrophoretic lane 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 used reagents with the analyzed DNA or amplicon. Test samples are evaluated for the presence of a strip in the corresponding lane, which is located at the same level as the strip in the positive control sample. The intensity of the luminescence of the band corresponds to the amount of analyzed DNA in the sample, which makes it possible to carry out a semi-quantitative assessment of PCR. Usually, positive results are assessed on a four-point scale. If the luminescence of the band in the experimental sample is very weak, then such a sample should be rearranged (Fig. 12).
Rice. 12. Agarose gel electrophoresis.
PCR applications fordiagnostics of point mutations and gene polymorphisms
One of the leading areas of application of PCR in practical health care is the diagnosis of point mutations and gene polymorphisms. . Direct and indirect methods of DNA diagnostics are distinguished. In situations where a gene is known, damage to 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, violations in the primary nucleotide sequence of DNA (mutations and their types) are detected. Direct methods are characterized by an accuracy of 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 purpose of direct DNA diagnostics is to identify mutant alleles.
Thus, in situations where it is known exactly which DNA damage leads to a hereditary disease, the DNA fragment containing the damage is directly examined, i.e., a 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 methods of DNA diagnostics. 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 they are all based on the use of the PCR method. This reaction allows you to multiply the DNA nucleotide sequence many times, and then search for mutations. Methods for searching for DNA fragments carrying mutations are based on a comparative analysis of mutant and normal DNA nucleotide sequences.
Analysis of PCR products
in the process of direct DNA diagnostics
Assumes the study of specific features of the amplified gene region. Thus, in diseases caused by the expansion of trinucleotide repeats, the amplification products differ in their length (reflecting a different number of triplets in the studied gene region) and, as a consequence, 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, ie, DNA diagnosis of the disease, is achieved (Fig. 13).
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Rice. fourteen. Deletion diagnosis GAG in the gene DYT 1 in patients with dopa-independent dystonia (polyacrylamide gel electrophoresis). Lanes 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 studied DNA region is entirely part of 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 the complete absence of the PCR reaction product (DNA synthesis is impossible from both copies of the gene). With a heterozygous deletion, it is possible to detect a PCR product synthesized from a normal (intact) allele; however, for a reliable diagnosis of such a mutation, it is necessary to use more sophisticated DNA imaging methods that allow estimating 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 site of localization and the nature of the putative point mutation are precisely known, then for the targeted detection of such a mutation, restriction endonucleases (restriction enzymes) - special cellular enzymes secreted from various strains of bacteria.
These enzymes recognize specific nucleotide sequences from four to ten nucleotides in length. Then carry out restriction (lat. (Cutting) of these sequences as part of a double-stranded DNA molecule. Each restriction enzyme recognizes and cuts in a fixed place a strictly defined, specific for itself nucleotide sequence - restriction site (recognition site).
In cases where a point mutation changes the natural recognition site for a particular restriction enzyme, this 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, mutant and normal PCR products treated with the selected restriction endonuclease will yield restriction fragments of different lengths, which can be easily detected by electrophoresis (Fig. 15).
Thus, if it is necessary to quickly detect any specific point mutation, the task is reduced to finding the corresponding restriction enzyme, the recognition site of which is located at the site of the disturbed nucleotide sequence. Processing of PCR products with such a restriction enzyme will make it easy to differentiate between normal and mutant alleles. Restriction analysis greatly simplifies the detection of known point mutations and is now widely used for direct DNA diagnostics of hereditary diseases.
The final stage molecular genetic analysis of mutations is the determination of the nucleotide sequence of the investigated DNA fragment (sequencing), which is compared with the norm and the final genetic diagnosis is formulated. Thanks to the successes of molecular genetics, methods of DNA diagnostics have now been developed for more than 400 hereditary diseases.
Rice. 15. Detection of point mutations using restriction analysis: A - amplifiable gene region containing a restriction siteAGCTfor restriction endonucleaseAlu I... MutationG→ Aalters this nucleotide sequence, resulting in restriction enzymeAluIblocked; B - electrophoregram of restriction products: lane 1 - homozygosity for the normal allele; lane 2, mutation homozygosity; lane 3 - heterozygous state (normal allele + mutation).
Diagnostics of hereditary diseases, based on the direct study of mutant alleles in patients, their family members or suspected heterozygous carriers of pathological mutations, is suitable for pre-symptomatic and prenatal diagnostics, which can be used at the earliest stages of fetal development, before any clinical or biochemical symptoms appear. illness.
Regardless of the method for detecting mutations, precise molecular characteristics 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 opens up the acceleration of the analytical process due to the performance of all procedures in one continuum, without sample transfer, the creation of conditions to prevent contamination during the parallel study of a number of analytes and with the objective registration of results in each cycle.
Basic modifications of the PCR method
Used to quickly scan for known gene mutations.
Multiplex (multi-primer) PCR
This method is based on the simultaneous amplification in one reaction of several exons of the gene under study. This allows for cost-effective 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, a set of the most frequently mutated exons of this gene is simultaneously amplified. Since these diseases are inherited according to the X-linked recessive type and are associated with damage to a single X-chromosome in boys, in the case of an extended deletion during electrophoresis of the reaction products, the absence of one or more DNA fragments (exons) will be revealed, which can serve as a molecular confirmation of the diagnosis. In addition, by selecting specific regions of the gene for PCR amplification, a fairly accurate assessment of the total length of the deletion and gene breakdown points (in the flesh 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 the total number of known mutations in the dystrophin gene and indicates a very high efficiency of this screening method for DNA diagnostics of dystrophinopathies (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 to 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 the normal or mutant DNA sequence. As a result of such a reaction, two types of PCR products, normal and mutant, can be simultaneously synthesized in solution. Moreover, the design of the primers used makes it possible to clearly differentiate between normal and mutant amplification products by their molecular size. This method is very illustrative 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 of a so-called mismatch primer in PCR (not completely complementary to the template), which differs from the template DNA sequence by one nucleotide. As a result of the inclusion of this 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 diagnostics of a certain known mutation using restriction analysis. The creation of such an artificial restriction site is sometimes necessary if the search did not reveal the existence of a known and available 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 (RT- PCR)
This method is used in cases when it is more convenient to use not genomic DNA as an object of research, but more compact and informationally “rich” 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 region of cDNA amplified in sufficient quantity is subjected to sequencing and other methods of mutational screening, direct electrophoretic study (detection of deletions, insertions, etc.) or insertion into the expression system in order to obtain a protein product and its direct analysis.
This method is especially effective for detecting mutations leading to the synthesis of a "truncated" protein (nonsense mutations, splicing mutations, large deletions) - the so-called PTT-analysis (Protein Truncation Test). The PTT assay is commonly used in the study of extended multi-exon genes, such as the Duchenne / Becker muscular dystrophy gene, ataxia-telangiectasia, or type 1 neurofibromatosis.
Real time PCR(Real-Time PCR)
Every year in practical healthcare, real-time PCR is becoming an increasingly popular diagnostic method. Its principal feature is monitoring and quantitative analysis of the accumulation of polymerase chain reaction products and automatic registration and interpretation of the results obtained. This method does not require an electrophoresis step, which makes it possible to reduce the requirements for a PCR laboratory. Due to the economy of production space, a decrease in the number of personnel and the demand for quantitative determination of DNA / RNA, this method has been successfully used in recent years in the largest sanitary-epidemic, diagnostic and research centers of the developed countries of the world, replacing PCR in its current ("classical") format.
Real-time PCR uses fluorescently labeled oligonucleotide probes to detect DNA during its amplification. Real-time PCR allows a complete analysis of a sample within 20-60 minutes and is theoretically capable of detecting even one DNA or RNA molecule in a sample.
Rice. 17. Real-time PCR.
Real-time PCR uses the TaqMan system, which monitors the kinetics of PCR directly during amplification using fluorescence resonance quenching. For detection, a probe is used that carries a fluorophore and a quencher complementary to the middle part of the amplified fragment. When the fluorophore and quencher are bound to the oligonucleotide probe, only negligible fluorescence emission is observed. During the amplification process, due to the 5'-exonuclease activity of Taq polymerase, the fluorescent label passes into the solution, being freed from the proximity to the quencher, and generates a fluorescent signal that amplifies in real time in proportion to the accumulation of the amplificate (Fig. 17).
The 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 work rooms;
· Along with a qualitative assessment of the result, it becomes possible to quantitatively assess (for example, when prescribing antiviral therapy for AIDS or viral hepatitis, it is necessary to know the viral load, that is, the amount of virus per unit, which provides real-time PCR);
· The risk of contamination is sharply reduced.
Conclusion
The PCR method is one of the most common methods of molecular biological research. This method should be used by clinicians meaningfully, and a doctor who decides to use PCR in his work should have some knowledge about the features and capabilities of this method. Second, there must be close feedback between the clinician and the PCR laboratory to analyze complex cases and develop the right 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 supplements them. And most importantly, PCR cannot replace the intuition and analytical thinking that a doctor who is counting on success should have.
P . S ... Molecular biological research - a change in diagnostic and treatment guidelines. The prospect of a radical change in emphasis in laboratory diagnostics is associated with the use of molecular biological methods. We can talk not just about timely information, but about getting it in advance. If now laboratory studies in most cases are carried out already with the developed disease and the treatment begun, then molecular biological laboratory information is expected to make it possible to reveal a person's tendency to certain types of pathology and the degree of sensitivity to certain drugs, which will justify predictive, prophylactic and personalized the nature of the medicine of the future.
CHANGE OF DIAGNOSTIC AND TREATMENT LINES
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. What enzyme is the basis of PCR?
12. Why should the detection area be removed from other working areas?
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 of material for PCR?
21. In what device does the amplification take place?
22. What is the reverse transcriptase PCR (RT-PCR) method?
23. What is the material for PCR diagnostics?
24. List the types of contamination?
Self-study tests
1. Endonuclease restriction enzymes:
a) enzymes that "break" DNA in strictly specific places;
b) enzymes that cross-link 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 restriction enzyme;
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) multiple repetition of a piece of DNA;
c) isolation of a DNA fragment containing the gene under study.
5. To obtain DNA samples, you can use :
b) chorionic villi;
c) amniotic fluid;
d) amniotic fluid cells;
e) biopsies of skin, muscles, liver,
f) everything is correct, except for point "c",
g) everything is correct, except for point "d",
h) all of the above is true.
6. To diagnose which mutations the PCR method is used:
a) genomic;
b) chromosomal;
c) gene (point).
7. A primer is:
a) complementary DNA region;
b) a synthetic oligonucleotide labeled (radioactive or fluorescent) sequence complementary to the mutant or normal gene;
c) an oligonucleotide that acts as a "seed" and initiates 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) determination of transgenic organisms (GMO)
c) personal identification, paternity, forensics
d) all of the above,
e) none of the above ..
Sample answers: 1 - a; 2 - b; 3 - b; 4 - a; 5 - e; 6 - c; 7 - c; 8 - b; 9 - a, 10 - d.
The main
1.Barrels genetics. Moscow. GEOTAR, 2002.
Additional
1., Bakharev and 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. - SPb .: Intermedica, 1999.
5. Herrington S., J. McGee. Molecular clinical diagnostics. - Peace, 1999.
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BASIC PRINCIPLES OF THE METHOD
POLYMERASE CHAIN REACTION
Methodological manual for extracurricular work of 3-4 year students in the specialties of general medicine (060101) and pediatrics (060103).
GOU VPO "Krasnoyarsk State Medical Academy of the Federal Agency for Healthcare and Social Development"
Russia, Krasnoyarsk,
For adequate and effective treatment of many infectious diseases, timely establishment of an accurate diagnosis is necessary. In solving this problem, high-tech diagnostic methods based on the methods of molecular biology are involved today. At the moment, polymerase chain reaction (PCR) is already widely used in practical medicine as the most reliable tool for laboratory diagnostics.
What explains the popularity of PCR at the present time?
Firstly, this method is used to identify the causative agents of various infectious diseases with high accuracy.Secondly, to monitor the effectiveness of the treatment.
In various manuals, brochures, articles, as well as explanations of medical specialists, we often come across the use of incomprehensible terms and words. It is really difficult to talk about high-tech products of science in ordinary words.
What is the essence and mechanics of PCR diagnostics?
Every living organism has its own unique genes. Genes are located in the DNA molecule, which, in fact, is the "calling card" of each particular organism. DNA (genetic material) is a very long molecule made up of building blocks called nucleotides. For each causative agent of infectious diseases, they are located strictly specifically, that is, in a certain sequence and combination. When it is necessary to understand whether a person has a particular pathogen, biological material (blood, urine, saliva, smear) is taken, which contains DNA or DNA fragments of the microbe. But the amount of genetic material of the pathogen is very small, and it is impossible to say which microorganism it belongs to. To solve this problem, PCR is used. The essence of the polymerase chain reaction is that a small amount of material for research containing DNA is taken, and during the PCR process there is an increase in the amount of genetic material belonging to a specific pathogen and, thus, it can be identified.PCR diagnostics is a genetic study of a biomaterial.
The idea of the PCR method belongs to the American scientist K. Mullins, which he proposed in 1983. However, it received wide clinical use only in the middle of the 90s of the XX century. Let's figure out the terminology, what is it - DNA, etc. Every cell of any living being (animal, plant, human, bacteria, virus) has chromosomes. Chromosomes are the custodians of genetic information, which contain the entire sequence of genes of each particular living being.
Each chromosome consists of two strands of DNA twisted into a helix relative to each other. DNA is chemically deoxyribonucleic acid, which consists of structural components - nucleotides. There are 5 types of nucleotides - thymine (T), adenosine (A), guanine (G), cytosine (C) and uracil (U). Nucleotides are located one after another in a strict individual sequence, forming genes. One gene can consist of 20-200 such nucleotides. For example, the gene for insulin production is 60 base pairs in length.
Nucleotides have the property of complementarity. This means that opposite to adenine (A) in one DNA strand there is necessarily thymine (T) in the other strand, and opposite to guanine (G) - cytosine (C). It looks like this schematically:
G - C
T - A
A - T
This property of complementarity is key for PCR.
In addition to DNA, RNA has the same structure - ribonucleic acid, which differs from DNA in that it uses uracil instead of thymine. RNA - is the custodian of genetic information in some viruses called retroviruses (for example, HIV).
DNA and RNA molecules can "multiply" (this property is used for PCR). It happens as follows: two strands of DNA or RNA move apart from each other, a special enzyme sits on each strand, which synthesizes a new strand. The synthesis proceeds according to the principle of complementarity, that is, if there is a nucleotide A in the original DNA strand, then in the newly synthesized there will be T, if G - then C, etc. This special enzyme - "builder" for the beginning of synthesis needs a "seed" - a sequence of 5-15 nucleotides. This "seed" is defined for each gene (chlamydia gene, mycoplasma, viruses) experimentally.
So, each PCR cycle consists of three stages. In the first stage, the so-called DNA unwinding takes place - that is, the separation of two linked DNA strands. In the second, the "seed" is attached to a section of the DNA strand. And, finally, the lengthening of these DNA strands, which is produced by the "builder" enzyme. Currently, this entire complex process takes place in one test tube and consists of repeated cycles of reproduction of the determined DNA in order to obtain a large number of copies, which can then be detected by conventional methods. That is, from one strand of DNA we get hundreds or thousands.
Stages of PCR research
Collection of biological material for research
As a sample, various biological materials are used: blood and its components, urine, saliva, mucous membrane discharge, cerebrospinal fluid, discharge from wound surfaces, and the contents of body cavities. All bioassays are collected with disposable instruments, and the collected material is enclosed in plastic sterile tubes or placed on culture media, followed by transportation to the laboratory.The required reagents are added to the collected samples and placed in a programmable thermostat - a thermal cycler (amplifier). In the amplifier, the PCR cycle is repeated 30-50 times, consisting of three stages (denaturation, annealing and elongation). What does this mean? Let's take a closer look.
Stages of direct PCR reaction, copying of genetic material
IPCR stage - Preparation of genetic material for copying.It occurs at a temperature of 95 ° C, while the DNA strands are separated, and "seeds" can sit on them.
"Seeds" are manufactured industrially by various research and production associations, and laboratories are bought ready-made. At the same time, the "seed" for detecting, for example, chlamydia, works only for chlamydia, etc. Thus, if a biomaterial is tested for the presence of chlamydial infection, then a "seed" for chlamydia is placed in the reaction mixture; if a biomaterial is tested for the Epstein-Barr virus, then it is also a "seed" for the Epstein-Barr virus.
IIstage - Combining the genetic material of the infectious agent and the "seed".
If there is DNA of the detected virus or bacteria, the "seed" sits on this DNA. This process of attaching the "primer" is the second stage of PCR. This stage takes place at a temperature of 75 ° C.
IIIstage - Copying the genetic material of the infectious agent.
This is the process of actually lengthening or reproduction of genetic material, which occurs at 72 ° C. The “builder” enzyme approaches the “seeds” and synthesizes a new DNA strand. With the end of the synthesis of a new DNA strand, the PCR cycle also ends. That is, in one PCR cycle, the amount of genetic material doubles. For example, in the original sample there were 100 DNA molecules of any virus; after the first PCR cycle, there will already be 200 DNA molecules of the tested virus in the sample. One cycle lasts 2-3 minutes.
To generate a sufficient amount of genetic material for identification, usually 30-50 PCR cycles are performed, which takes 2-3 hours.
Stage of identification of the propagated genetic material
The PCR itself ends at this point, and then there is an equally significant stage of identification. For identification, the method of electrophoresis or labeled "primers" is used. When using electrophoresis, the obtained DNA strands are separated by size, and the presence of DNA fragments of different lengths indicates a positive analysis result (that is, the presence of a particular virus, bacteria, etc.). When using labeled "primers", a chromogen (dye) is added to the final reaction product, as a result of which the enzymatic reaction is accompanied by the formation of a color. The development of color directly indicates that a virus or other detectable agent is present in the original sample. Today, using labeled "primers", as well as the appropriate software, it is possible to immediately "read" the results of PCR. This is the so-called real-time PCR.
Why is PCR diagnostics so valuable?
One of the significant advantages of the PCR method is its high sensitivity - from 95 to 100%. However, these advantages should be based on the indispensable observance of the following conditions:
- correct collection, transportation of biological material;
- availability of sterile, disposable instruments, special laboratories and trained personnel;
- strict adherence to the technique and sterility during the analysis
The capabilities inherent in PCR analysis allow you to achieve unsurpassed analytical specificity. This means identifying exactly the microorganism that you were looking for, and not a similar or closely related one.
The diagnostic sensitivity and specificity of the PCR method are often superior to those of the culture method, called the "gold standard" for detecting infectious diseases. Considering the duration of culture growth (from several days to several weeks), the advantage of the PCR method becomes obvious.
PCR in the diagnosis of infections
The advantages of the PCR method (sensitivity and specificity) determine a wide range of applications in modern medicine.
The main areas of application of PCR diagnostics:
- diagnostics of acute and chronic infectious diseases of various localization
- monitoring the effectiveness of the therapy
- clarification of the type of pathogen
The use of PCR diagnostics is carried out in conjunction with other research methods (ELISA, PIF, RIF, etc.). Their combination and appropriateness is determined by the attending physician.
Infectious agents detected by PCR
Viruses:
- retroviruses HIV-1 and HIV-2
- herpetiform viruses
- herpes simplex virus types 1 and 2
The highly informative PCR method (polymerase chain reaction) makes it possible for the early detection of various genetic and infectious diseases, acute or chronic. Moreover, they can be identified even at the stage when they do not manifest themselves with any symptoms. Most often, PCR analysis is used to detect sexually transmitted infections (STDs, STIs).
PCR analysis refers to a molecular diagnostic method, which is based on a multiple increase in small concentrations of certain fragments of nucleic acid (DNA) of the pathogen in any biological material (smear from the cervix, vagina, urethra, blood, saliva, sputum, etc.) and comparison its DNA or RNA with a database of known types of infectious agents.
The technique was developed by the American Carrie Mullis in the 80s of the last century. In 1993, the scientist was awarded the Nobel Prize in Chemistry. Today, PCR research is considered a kind of "gold standard" for diagnosing the vast majority of infections. PCR analysis is widely used in medical practice to clarify the nature of the disease and make an accurate diagnosis. Very often there are cases when all known immunological, virological and bacteriological methods do not work. Then PCR becomes the only way to identify the active stage of the disease.
Advantages of PCR diagnostics over other methods
The PCR technique has found wide application in modern medicine due to a number of indisputable advantages. Let's talk about them in more detail.
Possibility of direct detection of the presence of the pathogen
Many of the traditional diagnostic techniques are based on the identification of markers - proteins that are metabolic products of the causative agent of the disease. This principle of diagnosis can only provide an indirect confirmation of the pathology. The PCR method provides a direct determination of the pathogen, since it identifies specific regions of the DNA of pathogenic organisms.
High specificity
The PCR technique is highly specific due to the fact that it allows detecting DNA fragments that are characteristic only of a specific infectious agent. When using immunological methods, the probability of obtaining a false result remains (errors in diagnosis are associated with cross-reacting antigens). As for PCR, errors are excluded here, since the specificity in this case is set by the nucleotide sequence of the primers.
High sensitivity
Even single pathogens are detected by this method. The causative agents of infections are detected in the human body even when other diagnostic methods do not clarify the situation (we are talking about various immunological microscopic and bacteriological research methods).
Here are the data for comparison. The sensitivity of microscopic and immunological methods is 103-105 cells, and the sensitivity of PCR is 10-100 cells in a sample.
Method versatility
The PCR technique is based on the study of the DNA of pathogenic organisms. During the study, fragments of RNA or DNA are identified that are specific for specific infectious agents. Since all nucleic acids have a similar chemical composition, unified methods can be used in laboratory analysis. This means that the study of one sample makes it possible to identify several pathogens at once.
Fast results
This technique does not require the cultivation of pathogen cultures, which takes quite a long time. Thanks to the use of a unified material processing technology and the detection of reaction products, as well as an automated amplification process, the entire research procedure takes only a few hours.
Possibility of detecting infections in latent form
The PCR technique makes it possible to effectively perform preclinical diagnostics (identification of pathogens before symptoms appear) and retrospective diagnostics (determination of pathogens after a past illness). So, preclinical diagnostics is of great importance when examining a patient in the incubation period of a possible disease - after the alleged infection before the first signs appear.
One of the important advantages of PCR is the possibility of using it for the analysis of biological residues or archival materials. This makes it possible to identify paternity and identity.
Today, PCR diagnostic methods continue to develop. The analysis technology itself is being improved, and new types of PCR appear. Innovative test systems for this reaction are being introduced into medical practice. Thanks to such a rapid development of science, the cost of the procedure is decreasing, and now the PCR examination can be used for many categories of patients.
This diagnostic method is based on multiple doubling of a given section of RNA or DNA. This process is carried out in the laboratory using special enzymes. As a result, as many pieces of DNA (RNA) are formed as needed for visual examination. During the procedure, only the area corresponding to the specified conditions is copied (if it is present in the studied sample).
Biological material that needs to be examined for the presence of DNA or RNA pathogens is placed in an amplifier. (Depending on the specific situation, blood, urine, saliva, separated from the genitals are taken for analysis). Then special enzymes are added to the samples. They bind to the RNA or DNA of pathogenic microbes, and copy synthesis begins. Copying is a multi-stage process, proceeding according to the type of a chain reaction. As a result, hundreds or even thousands of copies may appear.
At the next stage of diagnostics, the results are analyzed and compared with a database of infectious agents.
The PCR technique not only makes it possible to determine the type of pathogenic organism, but also allows one to draw conclusions about the number of infectious agents in the human body.
Today, the use of such technologies opens up wide opportunities in the study of mutations, splicing of DNA sections. In modern medicine, the method began to be used to determine paternity, identify new genes, and much more.
Due to its versatility, the PCR method has found wide application in urology, gynecology, pulmonology, oncology, hematology, phthisiology, infectious disease practice and other fields of medicine.
Material for PCR analysis
For PCR diagnostics, various biological media and fluids taken from the human body are used: sputum, mucus, saliva, urine, blood, scraping of epithelial cells, placental tissue, pleural fluid, amniotic fluid, prostate juice, etc.
When diagnosing STDs (STIs), the discharge from the male and female genital organs is analyzed. For this, a smear or scraping is performed from the urethra or cervix. Also, urine is used for research.
To detect infections (herpes, mononucleosis, CMVI, toxoplasmosis, HIV, hepatitis B and C), blood is taken for analysis. If there is a suspicion of damage to the nervous system, cerebrospinal fluid is taken.
For pulmonary studies, pleural fluid and sputum are used.
To detect intrauterine infections, placental tissue and amniotic fluid are analyzed.
PCR for STIs and other infections
What infections can be detected using PCR analysis
HIV infection (HIV-1 human immunodeficiency virus can be detected).
Viral hepatitis A, B, C, G (RNA-HAV, DNA-HBV, RNA-HCV, RNA-HGV).
STIs (sexually transmitted infections) - ureaplasmosis, gardnerellosis, chlamydia, mycoplasmosis, trichomoniasis.
Infectious mononucleosis (DNA of the Epstein-Barr-EBV virus).
Cytomegalovirus infection (DNA-CMV).
Herpes infection (DNA-herpes simplex virus HSV types 1 and 2).
Tuberculosis (Mycobacterium tuberculosis).
Oncogenic viruses - human papillomavirus infection (human papillomavirus (including its oncogenic types 16, 18, 31, 33, 45, 51, 52, 56, 58, and 59).
Borreliosis, tick-borne encephalitis.
Listeriosis.
Candidiasis (fungi of the genus Candida).
Helicobacter pylori infection
other.
Preparation and delivery of PCR analysis
Patients who have submitted material for PCR diagnostics expect to receive a quick and accurate result. At the same time, one important point must be taken into account - the reliability of the diagnosis depends not only on the professionalism of the specialists and the capabilities of the medical laboratory. For the study to be informative, the patient himself must make efforts. So, it is necessary to strictly adhere to all the doctor's recommendations and carefully follow the rules for preparing for the collection of material. It is very important to avoid contamination of biological samples, otherwise the test results will be distorted.
Preparing for PCR Analysis
Preparation for the procedure is not associated with any particular difficulties. It is enough to remember a few rules:
Blood for PCR analysis is given on an empty stomach. Blood is taken from a vein, usually in the morning with a sterile needle, into a special container.
During the day before taking the material, sexual abstinence is shown when taking a PCR smear for STIs.
For the analysis of urine for PCR, the first portion is taken, the container must be sterile.
How to take a PCR test in men and women
PCR analysis is taken from men and women in the vaccination room, this is blood from a vein, saliva, swabs from the pharynx, tonsils, swabs from the nasopharynx, etc. The sampling method is no different.
PCR for STIs is taken in the antenatal clinic during a gynecological examination in women, these are smears from the vagina, cervix, urethra. In men, when visiting an andrologist or venereologist, this is a swab from the urethra.
Schemes of sampling material for PCR
How long does PCR take?
Patients do not have to wait long for the results of a PCR examination. The entire analysis procedure usually takes from several hours (real-time PCR) to 2-10 days. As a rule, the patient receives the results of the analysis on his hands in 2-5 days, up to a maximum of 10 days, it depends on the type of analysis. The longest time is PCR-diagnostics of blood for HIV and hepatitis, and least of all smears and scrapings - 2-3 days.
A negative result indicates that at the moment no traces of infectious agents are detected in the biological material. That is, the infection for which the examination was carried out is absent.
A positive result indicates the detection of traces of the pathogen in biological samples. This means that an infection is present in the human body at this time.
There are cases when PCR gives a positive result, but an active infectious process is not observed. This is a phenomenon called "healthy carriage". Treatment of such a patient is not required, but they must be under constant dynamic supervision. Such situations are typical for viral infections: Epstein-Barr virus (EBV), ginital herpes, cytomegalovirus infection (CMVI), human papillomavirus (HPV), in which samples are taken from local foci for research (scraping of the urethra, cervical canal, saliva). At the same time, we must not forget that a healthy carrier can transmit the infection to other people. In addition, activation of the infectious process is not excluded. In cases where PCR gives a positive result in the study of blood, this can no longer be considered a carrier. Such patients need specific treatment for the disease caused by the detected pathogen.
Quantitative indicators do not have general gradations. They are assessed by the doctor individually for each specific infection. The quantitative result makes it possible to determine how active the infection is and to identify the stage of the process.
Reliability of the PCR method
There are three criteria for assessing the effectiveness of a technique:
- Accuracy- detection of infection (or its absence) with a high degree of probability.
- Specificity- the accuracy of determining a specific pathogen.
- Sensitivity- the ability to identify the pathogen even with a small amount of genetic material in biological samples.
When using the PCR method, obtaining false positive results is almost impossible (that is, if the pathogen is absent, then there will be no positive sample).
A false negative result is possible, but it rarely happens. Similar situations arise if the infection is inactive at the time of the study. For example, a chronic infection with no activity or a latent infection.
PCR analysis: video