Molecular genetic methods

DNA technology methods are used to determine the localization in a particular chromosome of a mutant gene responsible for the origin of certain forms of hereditary pathology. Since the gene is a region of DNA, and the mutation of the genes is the damage to the primary structure of DNA( by mutation they mean all the changes in the DNA sequence, regardless of their localization and influence on the viability of the individual), then, by probing the metaphase chromosomes of a patient with a hereditary disease,localization of pathological gene. Methods of molecular genetics create opportunities for diagnosing diseases at the level of the altered DNA structure, they allow to find out the localization of hereditary disorders. Molecular genetic methods can reveal mutations associated with the replacement of even a single base.

The most important stage of gene identification is its isolation. DNA can be isolated from any type of tissue and cell containing nuclei. Stages of DNA isolation include: rapid cell lysis, removal of cell organelles and membranes by centrifugation, enzymatic degradation of proteins and their extraction from the solution by phenol and chloroform, concentration of DNA molecules by precipitation in ethanol.

In genetic laboratories, DNA is most often isolated from blood leukocytes, for which the patient takes 5-20 ml of venous blood in a sterile tube with an anticoagulant solution( heparin).Leukocytes are then separated and processed according to the steps outlined above.

The next step in preparing the material for the study is "cutting" the DNA into fragments in areas with a strictly specific base sequence, which is carried out with the help of bacterial enzymes - restriction endonucleases( restriction enzymes).Restrictases recognize specific sequences of 4-6, less often 8-12 nucleotides in a double-stranded DNA molecule and divide it into fragments at the sites of localization of these sequences, called restriction sites. The number of restriction DNA fragments produced is determined by the frequency of restriction sites, and the size of the fragments is determined by the distribution of these sites along the length of the original DNA molecule. The more often the restriction sites are located, the shorter the DNA fragments after restriction. Currently, more than 500 different types of restriction enzymes of bacterial origin are known, and each of these enzymes recognizes its specific sequence of nucleotides. In the future, restriction sites can be used as genetic markers for DNA.The DNA fragments formed as a result of restriction can be ordered along the length by electrophoresis in an agarose or polyacrylamide gel, and thus their molecular weight can be determined. Usually, a specific staining( more often ethidium bromide) is used to detect DNA in the gel and the gel is viewed in the transmitted light of the ultraviolet region of the spectrum. Locations of DNA localization have a red color. However, in humans, when processing DNA with several restriction enzymes, so many fragments of different lengths are formed that they can not be separated by electrophoresis, that is, it is not possible to visually identify individual DNA fragments on the electrophore gram( get an even color throughout the length of the gel).Therefore, a hybridization method with labeled DNA probes is used to identify the desired DNA fragments in such a gel.

Any single-stranded segment of DNA or RNA is able to bind( hybridize) to its complementary chain, and guanine is always associated with cytosine, adenine with thymine. This is the formation of a double-stranded molecule. If a single-stranded copy of the cloned gene is labeled with a radioactive label, a probe will be obtained. The probe is able to find a complementary segment of DNA, which is then easily identified by radioautography. A radioactive probe added to a drug of stretched chromosomes allows the gene to be localized on a certain chromosome: certain DNA samples can be identified with a DNA probe in Southern blotting. Hybridization occurs if the test portion of the DNA contains a normal gene. In the case where an abnormal sequence of nucleotides is present, that is, the corresponding chromosome structures contain a mutant gene, hybridization will not occur, which allows to determine the localization of the pathological gene.

To obtain DNA probes, the gene cloning method is used. The essence of the method is that a DNA fragment corresponding to a gene or a gene site is inserted into the cloning particle, usually a bacterial plasmid( annular extrachromosomal DNA present in bacterial cells and carrying antibiotic resistance genes), and then bacteria,having a plasmid with a built-in human

gene, multiply. Thanks to the synthesis processes in the plasmid, it is possible to obtain billions of copies of the human gene or its site.

Further copies of DNA labeled with a radioactive label or fluorochromes are used as probes to search for complementary sequences among the pool of DNA molecules studied.

Currently, there are many varieties of methods using DNA probes for the diagnosis of gene mutations.