Biological Basis and Principle of the Method
Specific enzymes known as restriction enzymes (or restriction endonucleases) play a key role in restriction analysis. Their mechanism of action involves cleaving the DNA molecule. The enzyme physically breaks the phosphodiester bonds within the nucleotide sequence, not randomly, but strictly at specific locations.
The segments of the molecule recognized by the enzyme are called restriction sites (recognition sites). They have a characteristic structure—they possess central symmetry, meaning they are palindromes. This implies that the nucleotide sequence reads identically in both directions from the axis of symmetry. The actual point of DNA cleavage may lie directly on the axis of symmetry or be slightly shifted relative to it.
To date, over 175 different restriction enzymes have been isolated, and more than 80 types of recognition sites are known. This diversity is extremely important because the genome of each taxon (e.g., a specific bacterial species) contains a genetically determined, strictly defined number of cleavage sites for a particular restriction enzyme. As a result, the cleavage pattern remains constant and unique for each microorganism.
Technique and Visualization of Results
The laboratory workflow of restriction analysis consists of several sequential steps, each requiring high precision:
- DNA digestion: At the first stage, previously isolated DNA from the test microbe is mixed with a chosen specific restriction enzyme. The enzyme locates its recognition sites and cuts the molecule.
- Fragment pool formation: The enzymatic reaction yields a specific set of DNA fragments, each of a strictly fixed size.
- Electrophoresis: To analyze the obtained fragments, they must be separated. Agarose gel electrophoresis is used for this purpose. The separation principle is based on the dependence of migration speed on mass: smaller DNA fragments move significantly faster through the gel and cover a greater distance, whereas larger fragments become trapped in the gel pores and move more slowly.
- Detection: To visualize the invisible DNA fragments, the gel is stained with a specific dye, ethidium bromide. The result is then photographed under ultraviolet (UV) light, where the fragments begin to fluoresce.
Clinical and Scientific Applications
The ultimate result of the study is the generation of a restriction map for a specific microbial species. This map is a visual representation of the distribution of DNA fragments of varying lengths.
In microbiology and medicine, restriction analysis is used for the following tasks:
- Microbial identification: The method allows for the highly accurate determination of whether a tested sample belongs to a specific bacterial species or genus.
- Assessment of genetic relatedness: By comparing restriction maps of different strains, researchers can establish how closely related they are (which is critical in epidemiological investigations).
- Mutation detection: The method effectively detects regions of the genome where mutational changes have occurred (as a mutation may create a new restriction site or abolish an old one).
Furthermore, restriction analysis is rarely used in isolation. It serves as an essential initial step for more complex molecular genetic studies, such as sequencing (direct determination of nucleotide sequence) and molecular hybridization.