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Restriction fragment length polymorphism analysis
Last reviewed: 05.07.2025
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The widespread use of various restriction endonucleases for the analysis of chromosomal DNA has revealed the enormous variability of the human genome. Even small changes in the coding and regulatory regions of structural genes can lead to the cessation of the synthesis of a certain protein or to the loss of its function in the human body, which usually affects the patient's phenotype. However, approximately 90% of the human genome consists of non-coding sequences, which are more variable and contain many so-called neutral mutations, or polymorphisms, and have no phenotypic expression. Such polymorphic regions (loci) are used in the diagnosis of hereditary diseases as genetic markers. Polymorphic loci are present in all chromosomes and are linked to a specific region of the gene. By determining the localization of a polymorphic locus, it is possible to establish which gene is associated with the mutation that caused the disease in the patient.
To isolate polymorphic DNA regions, bacterial enzymes are used - restrictases, the product of which are restriction sites. Spontaneous mutations that occur in polymorphic sites make them resistant or, conversely, sensitive to the action of a specific restrictase.
Mutational variability in restriction sites can be detected by changing the length of restriction fragments of DNA by separating them using electrophoresis and subsequent hybridization with specific DNA probes. In the absence of restriction in a polymorphic site, one large fragment will be detected on electrophoregrams, and in its presence, a smaller fragment will be present. The presence or absence of a restriction site in identical loci of homologous chromosomes allows for fairly reliable labeling of the mutant and normal gene and tracking its transmission to offspring. Thus, when studying DNA of patients in both chromosomes of which a restriction site is present in a polymorphic region, short DNA fragments will be detected on the electrophoregram. In patients homozygous for a mutation that changes the polymorphic restriction site, longer fragments will be detected, and in heterozygous patients, short and long fragments.
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