Molecular genetic methods for diagnosing hereditary diseases

DNA technology methods are used to determine the localization of a mutant gene in a particular chromosome, responsible for the origin of certain forms of hereditary pathology. Since a gene is a section of DNA, and a gene mutation is damage to the primary structure of DNA (mutation is understood as all changes in the DNA sequence, regardless of their localization and impact on the viability of an individual), then, by probing preparations of metaphase chromosomes of a patient with a hereditary disease, it is possible to establish the localization of the pathological gene. Molecular genetic methods create opportunities for diagnosing diseases at the level of altered DNA structure, they allow us to determine the localization of hereditary disorders. Molecular genetic methods can identify mutations associated with the replacement of even one single base.

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

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

The next stage of preparing the material for research is “cutting” the DNA into fragments in areas with a strictly specific base sequence, which is carried out using bacterial enzymes - restriction endonucleases (restriction enzymes). Restriction enzymes recognize specific sequences of 4-6, less often 8-12 nucleotides in a double-stranded DNA molecule and divide it into fragments at the locations of these sequences, called restriction sites. The number of resulting restriction fragments of DNA is determined by the frequency of occurrence of restriction sites, and the size of the fragments is determined by the nature of 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 own specific nucleotide sequence. In the future, restriction sites can be used as genetic markers of DNA. The DNA fragments formed as a result of restriction can be ordered by length by electrophoresis in an agarose or polyacrylamide gel, and thus their molecular weight can be determined. Typically, DNA in a gel is identified by specific staining (usually ethidium bromide) and viewing the gel in transmitted ultraviolet light. The sites of DNA localization are colored red. However, in humans, when DNA is processed by several restriction enzymes, so many fragments of different lengths are formed that they cannot be separated by electrophoresis, i.e. it is not possible to visually identify individual DNA fragments on the electrophoresis (uniform staining is obtained along the entire length of the gel). Therefore, to identify the desired DNA fragments in such a gel, the hybridization method with labeled DNA probes is used.

Any single-stranded segment of DNA or RNA is capable of binding (hybridizing) with a complementary strand, with guanine always binding to cytosine, and adenine to thymine. This is how a double-stranded molecule is formed. If a single-stranded copy of a cloned gene is labeled with a radioactive label, a probe is obtained. The probe is capable of finding a complementary segment of DNA, which is then easily identified using autoradiography. A radioactive probe added to a preparation of stretched chromosomes allows the gene to be localized on a specific chromosome: using a DNA probe, specific areas can be identified during Southern blotting. Hybridization occurs if the DNA section being tested contains a normal gene. In the case where an abnormal nucleotide sequence is present, i.e. the corresponding chromosome structures contain a mutant gene, hybridization will not occur, which allows the localization of the pathological gene to be determined.

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 section of a gene is inserted into a cloning particle, usually a bacterial plasmid (annular extrachromosomal DNA present in bacterial cells and carrying antibiotic resistance genes), and then the bacteria with the plasmid with the inserted human gene are multiplied. Thanks to the synthesis processes in the plasmid, billions of copies of the human gene or its section can be obtained.

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

Currently, there are many different types of methods using DNA probes to diagnose gene mutations.

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