To study and identify options in the structure of DNA, the molecular genetic method is used. For each DNA region that is being studied, the region is a chromosome, gene or allele, the methods are different. At the heart of each molecular genetic method contains certain manipulations of RNA and DNA. All these methods are very complex, cannot be carried out without laboratory conditions, and the staff must be highly qualified. This work is carried out in several stages.
Stages
RNA or DNA samples must first be obtained. Here, the molecular genetic method can be applied to almost any material: a drop of blood, white blood cells, fibroblast culture, mucous membrane (scraping), even hair follicles - DNA can be obtained from any sample. It is suitable for applying any molecular genetic method and their various variants, and already isolated DNA is stored in a freeze for a long time. The second stage is devoted to the accumulation of the necessary DNA fragments (amplification), but the in vitro polymerase chain reaction (in vitro, without the participation of a living organism) helps to ensure it. As a result, the selected DNA fragment is propagated using this chain reaction, and the amount of DNA increases literally a million times.
The third stage of molecular genetic research methods involves restriction of the propagated DNA (this is fragmentation, tearing, or cutting). Restriction is carried out using polyacrylamide or agarose gel electrophoresis. This molecular genetic method for studying DNA allows each fragment to occupy a specific position in the gel. After that, the gel is treated with ethidium bromide, which can bind to DNA, is irradiated with ultraviolet light, after which it is possible to observe the glow areas. Molecular genetic diagnostic methods are diverse and numerous, however, the first two stages are characteristic of all. But in order to identify DNA fragments, the gel can be stained with many other existing methods.
Varieties
The most direct and common methods for detecting mycobacteria include the above molecular genetic method for studying DNA. Its essence is to identify specific fragments of a chain of DNA pathogens in the diagnostic material. Molecular genetic diagnostic methods do not yet have a more effective way to recognize a disease such as tuberculosis. Using polymerase chain reaction (PCR), you can be sure that the original DNA will increase the number of copies in a million times, that is, amplification will occur, and this will allow you to visualize the results. The level of sensitivity here is very high - more than ninety-five percent, which is the main advantage of this method.
Other molecular genetic methods of research in efficiency are inferior to multiple copying literally by half, because in this case the editorial sample shows a specific oligonucleotide sequence that has increased one hundred and six times. Even the cultural diagnosis of respiratory tuberculosis is much lower in sensitivity. That is why modern medicine relies on molecular genetic methods for the diagnosis of tuberculosis. And the described method is especially effective when meeting with pathogens of high antigenic variability, which is much more difficult to determine in another way - it requires special nutrient media and a long cultivation time. Biochemical and molecular genetic methods give completely different results.
Diagnosis of tuberculosis
PCR diagnostics of tuberculosis is built most often using those DNA sequences that are specific for all four types of this disease. To achieve this goal, primers are most often used that identify sequences of IS elements (IS-986, IS-6110), since these migrating elements characterize purely types of mycobacterium tuberculosis and are always present in several copies in the genome. DNA can also be isolated from pure cultures and clinical (sputum patients) by any other suitable method. For example, there is the Boom method, which uses a lysing buffer based on guanidine, silicon dioxide and thiocyanate as a DNA carrier. The number of patients characterized by scanty bacterial excretion is increasing every year, and therefore a completely different level of organization has been established in clinical practice: the molecular genetic method for studying DNA already plays a major role in diagnosis.
However, we must admit that he is not without flaws. The use of the PCR method often brings a huge amount of false positive results, and the fault is not only technical errors, but also the features of the method itself. Among other things, using this diagnostic method to determine the degree of viability of the mycobacteria that are identified is simply impossible. But this drawback is not the most important. Molecular genetic methods of PCR diagnostics entail the risk of mycobacterial DNA contamination. For this reason, certification requirements for PCR laboratories are developed exclusively rigorously; they require three isolated rooms. PCR technology is modern and very complex, its use requires appropriate equipment and highly trained personnel.
Bacterioscopy
When establishing a diagnosis, the results of PCR studies must necessarily be compared with other data: clinical examination, radiography, smear microscopy, culture and even the response to a specific treatment are very important here. In this series of studies, PCR is only one component. The pathogen can be detected at the very beginning of diagnosis by the simplest and fastest methods - bacteriological.
A light microscope (Ziehl-Nielsen stain) and a luminescent (fluorochrome stain) are used here. The advantage of bacterioscopy is the speed of obtaining results. A limitation of its capabilities due to its low sensitivity is rightly considered a disadvantage of it. However, it was to this method that the WHO recommendation was given as the most economical and basic for the detection of tuberculosis patients. The detection of mycobacteria by the bacteriological method has a forecast value, and bacterial excretion is quantified. The molecular genetic methods for studying tuberculosis are much more confident in this.
Cultural studies
The best detection of mycobacteria is recognized by cultural studies. Sowing pathological material is carried out in egg environments: Mordovian, Finn II, Levenshtein-Jensen and the like. A tentative indicator of the development of resistance of mycobacteria to drugs and indirect evidence of effectiveness is the number of mycobacteria or their colonies in vitro, if the cultural method of research is used. To increase the percentage of mycobacteria, sowing pathological material is carried out on several media.
Satisfying numerous cultural needs, the causative agent is also provided with liquid media. At the same time, automated growth accounting systems such as VANTES are also used. Crops should be carried out in incubation for up to seven to eight weeks. By this time, sowing with a lack of growth can be considered negative. Biological tests are considered the most effective way to detect mycobacterium tuberculosis : they infect with guinea pigs diagnostic material, which are extremely sensitive to tuberculosis.
Few numbers
An interesting area of research that was discovered through PCR diagnostics was the study of M. tuberculosis, a latent infection. The modern concept of tuberculosis infection suggests that out of a hundred people who were in contact with M. tuberculosis, ninety may well become infected, but only ten of them have active disease. The rest have anti-tuberculosis immunity, and therefore, in ninety percent of cases, the infection will remain latent. It was the molecular genetic method that helped to discover such a pattern.
Genetics say that fifty-five percent of people whose pathological material was negative and eighty percent of people infected with M. tuberculosis but who had a disease without any radiographic manifestations received positive PCR responses. It was the genetic diagnostic method that helped identify patients at risk using PCR studies, and the results of their analyzes (microscopy and culture) were negative, and a subclinical infection of M. tuberculosis was present.
Modern research
Bacteriological laboratories of the Russian Federation also use the accelerated method of absolute concentrations: the nitrate reductase activity of mycobacteria is tested using the Griss reagent. Tuberculosis centers use a method that can determine drug resistance. This is sowing in liquid media, where the radiometric and fluorescence system for recording the growth of mycobacteria is automated. Such an analysis is done quickly - up to two weeks.
Currently, new methods are being developed: drug resistance of mycobacteria is evaluated at the genotype level. A study of the molecular mechanisms of resistance shows the presence of genes in mycobacteria. These genes are associated with resistance to certain drugs. For example, the kasA, inhA, and katG genes are resistant to isoniazid, the rpoB gene is resistant to rifampicin, the 16Sp RNA and rpsL genes are resistant to streptomycin, emb1 is to ethambutol, gyrA is to fluoroquinolone, and so on.
Mutations
In modern diagnostics, the molecular genetic level of the method for studying DNA has significantly increased and has allowed conducting large-scale studies of mutations in their entire spectrum. Now we know that the most common mutations are in 516, 526 and 531 codons of the rpoB gene, and resistance to various drugs has also been identified. There is a whole range of methods for typing mycobacteria using not only traditional methods - biochemical, biological and cultural, but modern molecular genetic methods are also widely used. Already there are adequate and providing correct diagnosis methods for the detection of monogenic diseases. They are based on DNA studies in the exact region of a particular gene. This, as a rule, is a complex, time-consuming and expensive process, but the data provided by the methods of molecular genetic analysis are much more accurate and informative than the data of all other analyzes.
It has long been known that DNA does not change over the entire life of an organism, that it is the same in any nuclear cell, and this makes it possible to take absolutely any cells of the body for analysis at any stages of ontogenesis. The damaged gene can be detected before the onset of the first symptoms, before the expanded clinic of the disease, as well as in healthy heterozygous people, but with a mutation in the gene. Molecular genetic methods for the diagnosis of hereditary diseases make it possible to identify it (by the direct approach of DNA diagnostics), as well as analyze the segregation of the disease in the family with marker DNA loci (polymorphic regions) that are closely linked to the damaged gene (i.e., an indirect approach to DNA diagnostics). Direct or indirect - any DNA diagnosis is based on methods that identify a strictly defined area of human DNA.
Direct methods
Direct DNA diagnostic methods are used in cases where the culprit of the hereditary disease is known, and the types of mutations are known. For example, direct methods are advisable for a number of diseases. These are Huntington's chorea (extension of CTG repeats), phenylketonuria (R408W), cystic fibrosis (delF508, major mutation) and the like. The main advantage of the direct method is the absolute accuracy of diagnosis, and there is no need to do DNA analysis of the rest of the family. If a mutation is found in the corresponding gene, this allows you to exactly confirm the diagnosis of heredity, determine the genotype for the rest of the burdened family.
Another advantage of direct diagnosis is the identification of heterozygous carriage of bad mutations in the relatives and parents of the deceased from the disease. This is especially true for autosomal recessive diseases. Direct methods also have disadvantages. To apply them, you need to know exactly the location of the pathological gene, its exon-intron structure and the spectrum of its mutations. Not all monogenic diseases today received similar information. The informational content of direct methods cannot be considered complete, since the same gene can have a large number of pathological mutations, which determines the development of hereditary diseases.
Indirect methods
Indirect methods in DNA diagnostics are used in completely different cases: if the damaged gene is not identified, but only localized on the chromosome, or if direct diagnosis does not give a result (this happens if the gene has a complex molecular organization or a considerable length, if it has a lot pathological mutations). Indirect methods are used to analyze the segregation of polymorphic markers in the allele family. Markers are located in the same chromosomal region or are closely linked to the locus of the disease and represent deletions or insertions, point replacements, repeats, and their polymorphism is due to different numbers in the block of elements.
The most convenient for indirect diagnosis are microsatellite and minisatellite polymorphic markers, which are widely distributed in the human genome. Their value is expressed in high information content if the genetic distance between the damage in the gene and the marker is not too large. In the latter case, the accuracy of the assessment is determined to a large extent by the frequency of recombination between the polymorphic marker and damage. Indirect diagnostic methods also include an obligatory preliminary stage of studying the frequency of alleles of the analyzed populations among carriers of mutations and patients, plus the need to determine the likelihood of disequilibrium and recombination of linkage of markers and mutant gene alleles.
Other methods
Short segments of RNA or DNA, as well as a single gene cannot be visualized by microscopic examination , therefore, methods of molecular genetic diagnostics are necessary to identify mutations. The existing “Human Genome Project”, like other advances in molecular genetics, has greatly expanded the ability to diagnose hereditary diseases - both pre- and postnatal. These methods can provide early detection and prognosis of poly- and monogenic diseases in which debut occurs in adulthood. Unfortunately, in terms of technical capabilities, molecular genetic studies sometimes go beyond the ethical framework that is established regarding heredity, especially when the diagnosis is carried out in adolescence and childhood.
Structural and quantitative anomalies of chromosomes are the most common causes of cancer and many malformations. Chromosomal aberrations must be identified, which is important for family counseling - to assess the prognosis along with the reproductive risk in future pregnancies. Chromosomal analysis is the "gold standard" of genetic diagnosis, but it also has limited capabilities. Only molecular genetic analysis methods can do more, because fluorescent labels based on cloning technologies are used there, with their high sensitivity they can detect subtle chromosomal changes that cannot be detected by classical cytogenetic studies. These techniques are increasingly expanding our diagnostic capabilities when examining children with developmental defects, mental retardation, and many other hereditary diseases.
conclusions
Very important for humanity were knowledge of the structure and functions of genes, their types of variability, the ability to recognize hereditary diseases, which happened in connection with the development of molecular genetics. Its methods are aimed at studying the DNA molecule - both when it is normal and when it is damaged. () . , (), (), ( ). , .
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