BIOTECHNOLOGY - Inshyna N.M. - 2009

CHAPTER 4. MEDICAL BIOTECHNOLOGY

Gene Diagnostics

It is known that The Human Genome contains 3.5 billion NUCLEOTIDES and more than 30,000 genes. Protein-coding nucleotide sequences account for only 1.1 - 1.4% of the total DNA length in the human genome. Mutations in structural genes lead to severe cellular, tissue, or organ dysfunctions. Most often, mutations alter The activity of a specific enzyme, resulting in the accumulation of a toxic substrate or a deficiency of a compound essential for normal Cell function.

To date, about 5,000 inherited Metabolic Disorders are known, yet molecular causes have been identified for only 500 of them. Tests determining genetic predisposition to Hereditary diseases, as well as Methods for their molecular Diagnosis, have been developed. Currently, newborns in Japan are screened for 11 Genetic Disorders, in the USA for 7, and in Ukraine for 2 (phenylketonuria and hypothyroidism).

Modern Methods for the molecular diagnosis of hereditary diseases must meet the following requirements:

- high efficiency, productivity, and low cost;

- high Specificity (positive reaction exclusively with the target molecule);

- high sensitivity (detection of extremely small quantities of target molecules).

The molecular diagnosis of hereditary diseases is based on immunological approaches or methods for detecting specific DNA sequences.

To detect Gene Mutations, nucleic acid Hybridization is used, which involves the pairing of two complementary fragments from different DNA molecules. Probes are utilized to identify specific DNA sequences. These probes can be:

- DNA or RNA molecules;

- long (more than 100 nucleotides) or short (less than 50);

- products of chemical synthesis or cloned genes.

Over 100 different DNA probes have been obtained and characterized, which

make it possible to detect pathogenic strains of various Bacteria, Viruses, and Protozoa. Probes have been developed for diagnosing human bacterial Infections caused by Legionellapneumophila (respiratory diseases), Salmonella typhi (food poisoning), and Camphylobacter hyointestinalis (gastritis).

The Use of molecular diagnostic methods allows for the detection of minimal concentrations of pathogenic microorganism DNA in patients' Blood. For instance, employing a DNA probe makes it possible to detect 1 mg of DNA from the malaria pathogen Plasmodium falciparum in a patient's blood. Thus, DNA probes can be used to identify virtually any pathogenic microorganism.

In addition to probes and target DNA, an essential component of DNA Diagnostic Systems is the method used to detect hybrid molecules. In 1983, Conner developed a diagnostic method for Sickle cell anemia based on nucleic acid hybridization. The molecular cause of sickle cell anemia is the substitution of valine for glutamic acid at the 6th position of the globin β-chain. The diagnosis utilizes two oligonucleotides 19 bp in length: one complementary to the normal allele of the β-globin gene (β^A), and the other to the mutant allele (β*). DNA from a healthy individual (β^Aβ^A) hybridizes only with the β^A probe, DNA from a sickle cell anemia patient (β^Sβ^S) hybridizes exclusively with the β^A probe, and DNA from a heterozygous individual for this gene (β^Aβ^S) hybridizes with both probes. This model system first demonstrated the feasibility of determining genotypes via DNA hybridization.

In forensic medicine, genetic fingerprinting is used to identify biological samples. Human minisatellite DNA, which does not encode Proteins and exhibits high Variability, is used as probes. Each person's DNA fingerprint is unique, consisting of a set of fragments of varying lengths corresponding to the minisatellite sequences of The Genome.

The application of DNA diagnostic methods enables the detection of hereditary diseases at the Embryonic Stage. There have been reports of gene diagnostics being used to ensure the birth of a healthy child in a family affected by Alzheimer's disease. The mother's egg was fertilized with the father's sperm in vitro. Fifteen zygotes were analyzed for the presence of mutations in the gene responsible for Alzheimer's disease. In most cases, a lethal gene combination was detected, and only one zygote appeared completely normal. This "healthy" zygote was implanted into a surrogate mother, resulting in the birth of a healthy child.

One of the Applications of gene diagnostics is identifying human genetic predisposition to various activities, particularly sports. The primary genetic marker is the angiotensin-converting enzyme gene, which is involved in Blood Pressure Regulation. This enzyme mediates the synthesis of angiotensin, a vasoconstrictor and growth factor that enhances structural Protein Synthesis in myocardial Cells and leads to cardiac Muscle hypertrophy. A person's physical activity depends on the presence of a specific gene variant in their genome. This gene is characterized by polymorphism (the presence or absence of a 287 bp fragment in the 16th intron).

In the future, gene diagnostics will make it possible to determine the full spectrum of disease-susceptibility genes for each individual, creating a personal "Genetic Passport."