Hybridization-Based Methods for the Diagnosis of Genetic Disorders

Hybridization techniques are important methods in molecular biology that can be used to detect specific sequences in complex mixtures of DNA or RNA molecules. The principle of hybridization techniques is to use two complementary single-stranded DNA or RNA molecules to form a double-stranded molecule by hydrogen bonding. Hybridization techniques can be classified in different ways, one of which is based on the type of solid support, such as membrane-based methods and array-based methods. Membrane-based methods involve transferring and immobilizing DNA or RNA to nitrocellulose or nylon membranes, and then hybridizing with labeled probes. Array-based methods involve immobilizing DNA or RNA on glass or plastic chips, and then hybridizing with labeled probes. Hybridization techniques have wide applications in gene detection, expression analysis, protein identification, genomics, transcriptomics, proteomics and other fields.

Fig.1 How DNA hybridization works

Membrane-Based Methods

Membrane-based methods are techniques that use the principle of complementary base pairing to hybridize labeled probes with target DNA fixed on a membrane. This technique can be divided into different types depending on the type of nucleic acid transferred to the membrane, such as Southern hybridization (transfer DNA), Northern hybridization (transfer RNA), and Dot hybridization (transfer dot-like nucleic acids). The basic steps and principles of these techniques are similar. First of all, the cells or tissues containing the target DNA are lysed, and the DNA is extracted and digested with restriction enzymes. Second, the digested DNA is separated by electrophoresis on an agarose gel, forming bands at different positions according to the size of the DNA fragments. Third, the agarose gel and nylon membrane or nitrocellulose membrane are closely contacted, and the DNA is transferred from the gel to the membrane by capillary action or vacuum suction and fixed on the membrane by ultraviolet light or heat treatment. Fourth, the membrane is soaked in an alkaline solution to make the DNA fixed to the membrane single-stranded. Finally, the probe labeled with radioactive or non-radioactive substances is hybridized with the membrane so that the probe binds with the complementary target DNA, and then the unbound probe is washed away, and the position and intensity of the bound probe are detected by an X-ray film or fluorescence instrument, thus determining the presence and quantity of the target DNA.

Table 1. Comparison of different types of membrane-based methods

Membrane-based methods have a wide range of applications in human genetic disease diagnosis and gene therapy. For example, in diagnosis, Southern hybridization can be used to detect chromosomal abnormalities, gene deletions, insertions, duplications, inversions, and other structural variations, such as for diagnosing β-thalassemia, hemophilia, and hereditary breast cancer; Northern hybridization can be used to detect gene expression levels, splicing isoforms, non-coding RNAs, and other functional variations, such as for diagnosing muscular dystrophy, neurodegenerative diseases, cancer, and others. Dot hybridization can be used to detect the presence of specific sequences in trace nucleic acid samples, such as for diagnosing hereditary metabolic diseases and infectious diseases. In gene therapy, Southern hybridization can be used to detect the integration of exogenous genes in recipient cells, such as for evaluating the safety and efficacy of gene therapy vectors. Northern hybridization can be used to detect the expression level of exogenous genes in recipient cells, such as for evaluating the stability and persistence of gene therapy vectors. Dot hybridization can be used to detect the copy number of exogenous genes in recipient cells, such as for evaluating the dose and distribution of gene therapy vectors. In addition, membrane-based methods also have some other applications. For example, in biotechnology, Southern hybridization can be used to detect the presence and copy number of exogenous genes in transgenic plants or animals; Northern hybridization can be used to detect the expression level of exogenous genes in transgenic plants or animals; Dot hybridization can be used to detect nucleic acid samples immobilized on biochips.

Membrane-based methods have some advantages over other nucleic acid detection techniques. For example, in terms of sensitivity, membrane-based methods can reach picomolar levels or even femtomolar levels; in terms of specificity, membrane-based methods can adjust the affinity between probe and target DNA by adjusting temperature, salinity, and other conditions; in terms of stability, membrane-based methods use nylon membrane or nitrocellulose membrane as carriers, which have high resistance to temperature, acid-base, and biodegradation; and in terms of cost, membrane-based methods use simple and cheap instruments and reagents. Of course, membrane-based methods also have some limitations. For example, in terms of time, membrane-based methods take a long time to complete the whole process (usually takes several hours or even days); in terms of operation, membrane-based methods require more steps and are prone to human errors (such as incomplete transfer and contamination); in terms of resolution, membrane-based methods are affected by factors such as electrophoresis separation effect and probe design.

Array-Based Methods

Array-based methods are techniques that use the principle of complementary base pairing to hybridize labeled probes with target DNA or RNA fixed on a solid surface, such as a glass slide or a microarray chip. This technique can be divided into different types depending on the type and number of probes and targets, such as DNA array, RNA array, and protein array. The basic steps and principles of these techniques are similar: first of all, the target DNA or RNA is extracted from the sample and labeled with fluorescent or chemiluminescent molecules; then, the target DNA or RNA is hybridized with the probes that are immobilized on the surface in a specific pattern; and finally, the hybridization signal is detected by a scanner or a reader, thus determining the presence and quantity of the target DNA or RNA.

Table 2. Comparison of different types of array-based methods

Array-based methods have a wide range of applications in human genetic disease diagnosis and gene therapy. For example, in diagnosis, DNA arrays can be used to detect single nucleotide polymorphisms (SNPs), copy number variations (CNVs), gene expression profiles, and so on, such as for diagnosing cystic fibrosis, Down syndrome, and cancer; RNA arrays can be used to detect gene expression levels, alternative splicing events, and non-coding RNAs, such as for diagnosing muscular dystrophy, neurodegenerative diseases, and cancer; protein arrays can be used to detect protein expression levels, protein interactions, protein modifications, and others, such as for diagnosing Alzheimer's disease, diabetes, and cancer. In gene therapy, DNA arrays can be used to detect genome integration sites and gene regulation networks, such as for evaluating the safety and efficacy of gene therapy vectors; RNA arrays can be used to detect gene expression levels and gene silencing effects, such as for evaluating the stability and persistence of gene therapy vectors; and protein arrays can be used to detect protein expression levels, protein-protein interactions, and protein modifications, such as for evaluating the function and activity of gene therapy products.

Array-based methods also have some advantages over other nucleic acid detection techniques. For example, in terms of throughput, array-based methods can analyze thousands or millions of probes and targets simultaneously in a single experiment; in terms of sensitivity, array-based methods can detect low-abundance targets with a high signal-to-noise ratio; in terms of specificity, array-based methods can discriminate subtle sequence differences with high accuracy; in terms of scalability, array-based methods can be easily adapted to different formats and sizes according to different needs. Of course, array-based methods also have some limitations. For example, in terms of cost, array-based methods require expensive equipment and reagents; in terms of complexity, array-based methods require sophisticated data analysis and interpretation; in terms of variability, array-based methods are affected by factors such as probe design, hybridization conditions, and signal detection.

Conclusion

Based on the above, the principles, types, features, applications, and challenges of hybridization-based methods for the diagnosis and gene therapy of human genetic disorders have been introduced. It has been shown that hybridization-based methods are powerful tools for the detection and characterization of genomic variations in human genetic disorders, which can provide valuable information for clinical and research purposes. However, the limitations and difficulties of these methods, such as low resolution, high cost, complexity, and data analysis challenges have also been pointed out. The differences in the advantages and disadvantages of membrane-based and array-based methods have been discussed, and their applications to various aspects of human genetic disorders, such as diagnosis, prognosis, classification, and gene therapy have been illustrated. Some future directions and recommendations for the improvement and integration of hybridization-based methods with other technologies have also been suggested. It is believed that hybridization-based methods have a significant impact on the understanding and treatment of human genetic disorders and will continue to contribute to the advancement of molecular medicine.

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