Molecular genetic method of investigation

To study and identify variants in the structure of DNA, a molecular genetic method is used. For each DNA region that is examined, the region is a chromosome, a gene or an allele, the methods are different. At the heart of every molecular-genetic method contains some manipulation of RNA and DNA. All these methods are extremely complex, without laboratory conditions can not be conducted, and the staff must be highly qualified. This work is carried out in several stages.

Stages

First, samples of RNA or DNA need to be obtained. Here, the molecular genetic method can be applied to almost any material: a drop of blood, leukocytes, a culture of fibroblasts, a mucous membrane (scraping), even hair bulbs, - DNA can be obtained from any sample. It is suitable for applying any molecular genetic method and their various variants, and already allocated DNA is stored for a long time in the freezing. The second stage is devoted to the accumulation of necessary fragments (amplification) of DNA, to provide it helps the chain polymerase reaction in vitro (in vitro, without the participation of a living organism). As a result, the selected DNA fragment multiplies with this chain reaction, and the amount of DNA increases literally a million times.

The third stage of molecular genetic methods of investigation is the restriction of multiplied DNA (this is fragmentation, tearing or cutting). Restriction is carried out using polyacrylamide or agarose gel electrophoresis. This molecular genetic method of studying DNA allows each fragment to occupy a certain position in the gel. After that, the gel is treated with ethidium bromide, which is able to bind in DNA, ultraviolet irradiation is carried out, after which it is possible to observe the luminescence regions. Molecular genetic methods of diagnosis are diverse and numerous, however, the first two stages are typical for all. But in order to identify fragments of DNA, the gel can be stained with many other existing methods.

Varieties

The most direct and widespread methods for detecting mycobacteria include the above-described molecular genetic method of DNA research. The essence of it is to identify in the diagnostic material specific fragments of the chain of DNA pathogens. Molecular genetic methods of diagnosis do not yet have a more effective way of recognizing such a disease 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, there will be amplification, and this will allow visualizing the results. The sensitivity level here is very high - over ninety-five percent, which is the main advantage of this method.

The rest of the molecular genetic methods of research are twice as effective as duplicate copying, since in this case the editorial sample shows a specific oligonucleotide sequence increased by a hundred and six times. Even the cultural diagnosis of tuberculosis of respiratory organs is much lower in its sensitivity. That is why modern medicine is based on molecular genetic methods for diagnosing tuberculosis. And the described method is especially effective at meeting with pathogens of high antigenic variability, which is much more difficult to determine by another method - special nutrient media and long time of cultivation are required. Biochemical and molecular genetic methods give completely different effects.

Diagnosis of tuberculosis

They construct PCR diagnostics of tuberculosis most often using those DNA sequences that are specific for all four types of this disease. To achieve this goal, primers that detect sequences of IS elements (IS-986, IS-6110) are most often used, since these migrating elements characterize only kinds of mycobacteria of tuberculosis and are always present in several copies in the genome. Also, DNA can be isolated from pure cultures and clinical (sputum of patients) by any other acceptable method. For example, there is the Boom method, which uses a lysis buffer based on guanidine, silica and thiocyanate as a carrier of DNA. The number of patients with scant bacterial excretion increases year by year, and therefore a completely different level of organization has established itself in clinical practice: the molecular genetic method of studying DNA plays a major role in diagnosis.

However, we must admit that it is not without flaws either. The use of the PCR method often brings a huge amount of false positive results, and the fault here is not only technical errors, but also the peculiarities of the method itself. Among other things, using this method of diagnosis to determine the degree of viability of mycobacteria that are identified, it is simply impossible. But this drawback is not the most important one. Molecular genetic methods of PCR diagnostics entail the danger of contamination of mycobacterial DNA. Certification requirements for this reason for PCR laboratories are extremely rigid, they require the presence of three isolated rooms. PCR technology is modern and very complex, its use requires adequate equipment and highly trained personnel.

Bacterioscopy

When establishing the diagnosis, the results of the PCR study must necessarily be compared with the rest of the data: clinical examination, radiography, smear microscopy, sowing and even the response to a specific treatment are very important here. In this series of studies, PCR is only one of the components. Detect the pathogen at the very beginning of the diagnosis can be the most simple and rapid methods - bacteriological.

Here we use a light microscope (Tsil-Nielsen color) and a luminescent (fluorochrome coloration). The advantage of bacterioscopy is the speed with which results are obtained. A disadvantage of it is considered to be the limited possibilities due to low sensitivity. However, this method is given to WHO recommendation as the most economical and basic for identifying patients with tuberculosis. Detection of mycobacteria by a bacteriological method has the significance of a prognosis, and bacterial release is quantified. Molecular genetic methods of studying tuberculosis are much more confident with this.

Cultural research

The best detection of mycobacteria is recognized by culture studies. The sowing of pathological material is carried out in the egg media: Mordovsky, Finn II, Levenshtein-Jensen and the like. An indicative measure of the development of resistance of mycobacteria to drugs and indirect evidence of efficacy is the amount of mycobacteria or their colonies in vitro if a culture method of examination is used. To increase the percentage of allocation of mycobacteria, the pathological material is sown for several media.

Satisfying the numerous cultural needs, the causative agent is also provided with liquid media. At the same time, automated systems of accounting for growth of VANTES type are used. Crops should be incubated up to seven to eight weeks. By this time, sowing with a lack of growth can be considered negative. The most effective way to identify mycobacteria tuberculosis is biological tests: they infect the diagnostic material of guinea pigs, which are extremely sensitive to tuberculosis.

A few figures

The most interesting area of research, which was discovered through PCR diagnostics, was the study of M. tuberculosis, a latent infection. The current concept of tuberculosis infection suggests that of a hundred people who have been in contact with M. tuberculosis, ninety can be infected, but only 10 of them have active disease. The rest have antituberculous immunity, and therefore in ninety percent of cases the infection will remain latent. It was the molecular genetic method that helped to detect this pattern.

Genetics argue that fifty-five percent of those with negative pathological cultures and eighty percent of those infected with M. tuberculosis, but with a non-radiographic disease, have received positive PCR responses. It was the genetic method of diagnosis that helped to identify patients from risk groups using PCR studies, and the results of their analyzes (microscopy and crops) were negative, and the subclinical infection of M. tuberculosis was present.

Modern research

Bacteriological laboratories of the Russian Federation use an accelerated method of absolute concentrations: the nitrate reductase activity of mycobacteria is tested by means of the Griss reagent. Anti-tuberculosis centers use a method that allows to determine drug resistance. This is a culture in liquid media, where a radiometric and fluorescent system for recording the growth of mycobacteria is automated. Such an analysis is done quickly - up to two weeks.

At present, new methods are being developed: the drug resistance of mycobacteria is assessed at the genotype level. The 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 genes kasA, inhA, katG are resistant to isoniazid, rpoB to rifampicin, 16Sp RNA and rpsL to streptomycin, emb1 to ethambutol, gyrA to fluoroquinolone, and so on.

Mutations

In modern diagnostics, the molecular genetic level of the DNA method has significantly increased and allowed to carry out large-scale studies of mutations in their entire spectrum. Now we know that mutations in 516, 526 and 531 codons of the rpoB gene are most common, and resistance to various drugs has 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 reliable diagnostics methods for detecting monogenic diseases. They are based on DNA research in the exact region of a particular gene. This is usually a complex, time consuming and expensive process, but the data provided by 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 the organism, that it is in any nuclear cell, however, and this makes it possible to take absolutely any cells of the body for analysis, at any stages of ontogeny. A damaged gene can be detected before the appearance of the first symptoms, before the unfolded clinic of the disease, and also in healthy heterozygous people, but having a mutation in the gene. Molecular genetic methods for diagnosis of hereditary diseases can be identified by a direct DNA-diagnostic approach, and also by analyzing the segregation of the disease in the family with marker loci of DNA (polymorphic sites) that are closely linked to the damaged gene (ie, an indirect DNA-diagnostic approach). Direct or indirect - any DNA diagnosis is based on methods that identify a strictly defined area of human DNA.

Direct methods

Direct methods of DNA diagnosis are used in cases when the gene-culprit of the hereditary disease is known, and the types of its mutations are also known. For example, direct methods are appropriate for a variety of diseases. This is Huntington's chorea (expansion of CTG repeats), phenylketonuria (R408W), cystic fibrosis (delF508, major mutation) and the like. The main advantage of the direct method is a hundred percent accuracy of diagnosis, and there is no need to do a DNA analysis of the rest of the family. If a mutation in the corresponding gene is found, it allows you to accurately 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 relatives and parents of the deceased from the disease. This is especially true for diseases of autosomal recessive. Disadvantages of direct methods are also available. To apply them, you need to know exactly the localization of the pathological gene, the exon-intron structure and the spectrum of its mutations. Not all monogenic diseases have received such information today. The informativeness of direct methods can not 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 other cases: if the damaged gene is not identified, but is only localized on the chromosome, or if direct diagnosis has not yielded results (this happens if the gene has a complex molecular organization or a considerable length, if there is a lot in it Pathological mutations). Indirect methods are used to analyze the segregation of polymorphic markers in the family of alleles. Markers are in the same chromosomal region or are closely linked to the locus of the disease and represent deletions or insertions, point replacements, repetitions, 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 informativeness, 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 evaluation is determined to a large extent by the frequency of recombination between the polymorphic marker and the damage. Indirect methods of diagnosis also provide for the mandatory preliminary stage of the study of the allele frequency of the analyzed populations among the carriers of mutations and patients, plus the need to determine the probability of disequilibrium and recombination of the adhesion of markers and mutant alleles of the gene.

Other methods

Short segments of RNA or DNA, as well as a single gene can not be visualized by microscopic examination, therefore, in order to identify mutations, methods of molecular genetic diagnostics are needed. The existing "Human Genome Project", like other achievements in molecular genetics, has in many ways expanded the possibility of diagnosing hereditary diseases - both pre- and postnatal. These methods can provide early detection and make a prediction of poly- and monogenic diseases, in which the debut occurs in adulthood. Unfortunately, in terms of technical capabilities, molecular genetic studies sometimes go beyond the ethical framework that is established with respect to heredity, especially when diagnosis is carried out in adolescence and childhood.

Structural and quantitative anomalies of chromosomes are the most common causes of both oncological diseases and many developmental anomalies. Chromosomal aberrations should 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 methods of molecular genetic analysis can do more, because they use fluorescent labels based on cloning technologies, which can detect with their high sensitivity fine chromosomal changes that can not be detected by classical cytogenetic research. These techniques are increasingly expanding our diagnostic capabilities, when children with developmental defects, with mental retardation, and many other hereditary diseases are examined.

conclusions

Very important for humanity were knowledge of the structure and functions of genes, the types of their variability, the ability to recognize hereditary diseases, which occurred in connection with the development of molecular genetics. Its methods are aimed at studying the DNA molecule - and when it is normal, and if it is damaged. The preparation of nucleotide sequences of deoxyribonucleic acid (DNA) proceeds step by step from the preparation of the samples to the identification of individual fragments. Isolation of genomic DNA from cells, restriction (tearing), amplification (cloning), electrophoresis of fragments (separation of them by electric charge and molecular weight by means of agarose gel). Identification of certain fragments located on its surface by a discrete strip.

Then special filters enter into the matter, with the help of which the hybridization of each fragment with cloned DNA fragments or with synthetic radioactive probes that are control, through which each fragment under study will be equalized. If the position or its length is changed relative to the probe, if a new fragment appeared or disappeared, all this indicates that the studied gene underwent a rearrangement in the nucleotide sequence. There are eight basic methods of molecular genetic research: sequencing (DNA sequence determination), polymerase chain reaction (increasing the number of sequences), obtaining primers of known genes, cloning DNA, obtaining recombinant molecules, obtaining proteins through recombinant molecules, creating a complete set (collections, Library) of cloned fragments, which were obtained by restriction.