Molecular Approach for the Diagnosis of Genetic Disorders
Molecular diagnosis and gene therapy are two important branches of molecular medicine that use techniques and methods of molecular biology to diagnose and treat human genetic diseases. Molecular diagnosis refers to the determination of the type, degree, and prognosis of a disease, as well as an individual's response and sensitivity to drugs, by detecting changes in genes or proteins. Gene therapy refers to the introduction of normal or modified genes into cells or tissues to correct or compensate for gene defects or dysfunction so as to achieve the purpose of treating or preventing diseases. The main methods and technologies of molecular diagnosis and gene therapy include nucleic acid isolation, restriction fragment length polymorphism (RFLP), hybridization technology, gene chips, single nucleotide polymorphism (SNP), fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), microsatellite instability (MSI), transcriptomics, proteomics, epigenetics, gene transfer, gene targeting, vector design, transduction efficiency, immune response, etc. The application fields and prospects of molecular diagnosis and gene therapy are very broad. They can be used to diagnose and treat various genetic diseases, such as blood system diseases, liver diseases, cardiovascular diseases, nervous system diseases, rheumatoid arthritis, etc.; they can also be used to diagnose and treat various non-genetic diseases, such as Infectious diseases, tumors, etc.; and they can also be used in personalized medicine, drug screening, organ transplantation, stem cell therapy, etc. With the advancement of science and technology and the needs of society, molecular diagnosis and gene therapy will play a greater role in the future.
Nucleic Acid Isolation Methods
Nucleic acid isolation is the process of extracting DNA or RNA from biological samples, which is the first and most critical step for molecular diagnosis and gene therapy. There are many methods for nucleic acid isolation that can be classified according to different principles, methods, and steps. Generally speaking, nucleic acid isolation methods can be divided into three major categories: organic solvent methods, inorganic salt methods, and solid phase methods. The organic solvent method is the method of using organic solvents (such as phenol and chloroform) to mix with water to form a two-phase system and separating nucleic acids from other impurities by the different hydrophilicity and hydrophobicity of different phases for nucleic acids. This method can effectively remove contaminants such as proteins, polysaccharides, etc. and obtain high-purity nucleic acids. The inorganic salt method involves using inorganic salts (such as NaCl and NH4Ac) to mix with water to form a high-concentration salt solution and precipitating nucleic acids from other impurities by changing the ionic strength and pH value of the solution. This method is simple, fast, non-toxic, and harmless. The solid phase method uses solid phase carriers (such as cellulose, silica gel, and magnetic beads) to mix with the water phase to form a solid-liquid system, adsorbing or eluting nucleic acids from other impurities by the different adsorption capacities of different carriers for nucleic acids. This method is convenient, efficient, and stable.
Different types of nucleic acid isolation methods have their own advantages, disadvantages, and application scopes. They need to be selected and optimized according to factors such as the source, quantity, quality, target nucleic acid type, and purpose of the sample.
Restriction Fragment Length Polymorphism
Restriction fragment length polymorphism (RFLP) is a concept, principle, and method that use the differences (or variations) in DNA sequences at sites recognized by restriction enzymes to diagnose and treat human genetic diseases. RFLP is based on the fact that a single nucleotide difference between two individuals could result in either the presence or absence of a restriction site, which can be detected by the presence of fragments of different lengths after digestion of the DNA samples with specific restriction enzymes. RFLP can be used as a genetic marker, which can be used to follow the inheritance of DNA through families or populations or to locate genes within a sequence. RFLP has some advantages, disadvantages, and application scopes. It is simple, fast, non-toxic, and harmless, and can effectively remove contaminants such as proteins, polysaccharides, etc. However, it also has some disadvantages, such as low resolution, low polymorphism rate, large amount of DNA required, etc. It can be used for the diagnosis and treatment of various genetic diseases, such as blood system diseases, liver diseases, cardiovascular diseases, neurological diseases, and rheumatoid arthritis. It can also be used for the diagnosis and treatment of various non-genetic diseases, such as infectious diseases, tumors, etc.
Fig.1 How restriction fragment length polymorphism (RFLP) works
Hybridization-Based Methods
Hybridization-based methods are a group of techniques that use the specific binding of DNA or RNA molecules to identify and quantify target nucleic acid sequences in a complex sample. Hybridization-based methods rely on the use of probes that are labeled with a detectable signal, such as fluorescence, radioactivity or chemiluminescence, and that can anneal to the complementary sequences of the target under controlled conditions. Hybridization-based methods can be used to measure the expression level, mutation status or copy number of genes or transcripts in a sample. Hybridization-based methods have some advantages and disadvantages and application scopes. They are powerful, flexible and diverse, and can detect both DNA and RNA sequences with high sensitivity and specificity. However, they also have some limitations, such as requiring high-quality probes and samples, being influenced by temperature and salt concentration, being susceptible to cross-hybridization and background noise, etc. They can be applied to various fields of molecular diagnosis and gene therapy, such as blood system diseases, liver diseases, cardiovascular diseases, neurological diseases, and rheumatoid arthritis. They can also be applied to various fields of non-genetic diseases, such as infectious diseases and tumors.
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