DNA diagnostics and gene therapy

Molecular genetic method: detection of mutations

To confirm or exclude the carriage of a specific mutation, a strict sequence of laboratory procedures is applied. The method makes it possible to differentiate normal and altered regions of the genome with high accuracy.

Main stages of diagnostics:

1. Amplification. The target gene region suspected of containing a mutation is replicated many times. Polymerase chain reaction (PCR) is used for this, allowing a sufficient amount of material to be obtained for analysis.
2. Fixation. The DNA samples obtained during PCR are applied and fixed onto special narrow strips of nitrocellulose.
3. Hybridization. The fixed material is treated with specific probes—labeled oligonucleotides. These probes carry either a strictly normal or a mutant nucleotide sequence.
4. Visualization. The evaluation of hybridization results is carried out using the autoradiographic method. It makes it possible to determine whether the patient's DNA has bound to the normal probe or the mutation probe, which directly indicates the genetic status of the examined individual.

Fundamentals of gene therapy

Gene therapy opens up possibilities for treating both hereditary pathologies and non-hereditary (e.g., infectious) diseases. The essence of the method lies in introducing therapeutic genes into the patient's cells, which are capable of either eliminating existing genetic defects or endowing the cell with completely new functions.

Historical precedent The starting point in the clinical application of the method was 1990. The patient was a four-year-old girl suffering from severe inherited immunodeficiency caused by a mutation in the adenosine deaminase (ADA) gene. The treatment regimen (ex vivo):

• The patient's own lymphocytes were harvested.
• In laboratory conditions (in vitro), a normal, functioning copy of the ADA gene was introduced into these cells using a retroviral DNA vector as a delivery vehicle.
• Modified lymphocytes with a corrected genetic apparatus were transplanted back into the girl's body.

Today, the scope of gene therapy has expanded significantly. Methods are actively being developed and implemented to treat oncological diseases, hereditary syndromes, and severe viral pathologies, including HIV infection.

The Human Genome Project and the nature of hereditary diseases

The international Human Genome Project, launched in 1990, became the foundation for the modern development of molecular medicine. Its global goal was to accurately determine the nucleotide sequence in all human DNA molecules and map (establish the localization of) all genes. To solve this large-scale task, cutting-edge second- and third-generation sequencing technologies are used.

Etiology and perspectives According to statistics, out of approximately 10,000 known human diseases, about 3,000 are classified as hereditary. Their etiology is based on direct damage to the hereditary apparatus of the cell. It is important to understand that such damage does not always imply the classical transmission of a defect from generation to generation. Complete sequencing of genes involved in the pathogenesis of various diseases is the main prospect of modern biochemistry and genetics. This will radically change approaches to early diagnosis and bring patient treatment to a qualitatively new level.

Briefly: answers to topical questions

Class and exam questions

What else to study on this topic

• Features of polymerase chain reaction (PCR) at the amplification stage
• Mechanisms of hybridization of labeled oligonucleotide probes with target DNA
• Principles of in vitro retroviral DNA vector operation
• Second- and third-generation sequencing technologies
• Pathogenesis of hereditary immunodeficiency (ADA gene mutation)

Other topics in the section "Template Biosyntheses"

Prepared by WhiteCoat Pro faculty in accordance with the medical school curriculum. For educational purposes only.