Analysis of restriction fragment length polymorphism

The wide use of various restriction endonucleases for the analysis of chromosomal DNA revealed a huge variability in the human genome. Even small changes in the coding and regulatory regions of structural genes can lead to the termination of the synthesis of a certain protein or to the loss of its function in the human body, which usually affects the phenotype of the patient. However, approximately 90% of the human genome consists of non-coding sequences that are more variable and contain many so-called neutral mutations, or polymorphisms, and do not have a phenotypic expression. Such polymorphic sites (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. Having determined the localization of the polymorphic locus, it is possible to establish with which genome a mutation is associated that caused the disease in the patient.

To isolate polymorphic regions of DNA, bacterial enzymes are used - restriction enzymes, 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 restriction enzyme.

Mutational variability in restriction sites can be detected by changing the length of the restricted DNA fragments, 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 the electrophoregrams, and if present, a smaller fragment will be present. The presence or absence of a restriction site in the identical loci of homologous chromosomes makes it possible to reliably label the mutant and normal gene and trace its transmission to the offspring. Thus, in the study of DNA of patients, in both chromosomes of which a restriction site is present in the polymorphic region, short fragments of DNA will be revealed on the electrophoregram. In patients homozygous for a mutation that modifies a polymorphic restriction site, fragments of greater length will be detected, and in heterozygous fragments, short and long fragments will be identified.