Restriction analysis

Biological basis and principle of the method

Specific enzymes—restriction enzymes (or restriction endonucleases)—play a key role in performing restriction analysis. The mechanism of their action consists in cleaving the DNA molecule. The enzyme physically breaks the phosphodiester bonds in the nucleotide sequence, but it does so not randomly, but strictly at specific locations.

The regions 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 means that the nucleotide sequence is read in the exact same way in both directions from the axis of symmetry. At the same time, the actual point of DNA cleavage may be located 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. Such diversity is extremely important because the genome of each taxon (for example, a specific bacterial species) contains a genetically determined, strictly defined number of sites for cleavage by a specific restriction enzyme. Due to this, the cleavage pattern remains constant and unique for each microorganism.

Technique of the analysis and visualization of results

The laboratory process of restriction analysis consists of several consecutive steps, each of which requires high precision:

1. DNA treatment. At the first stage, pre-extracted DNA of the studied microbe is mixed with a specific chosen restriction enzyme. The enzyme finds its recognition sites and cuts the molecule.
2. Formation of the fragment pool. As a result of the enzymatic reaction, a specific set of DNA fragments is formed, each of which has a strictly fixed size.
3. Electrophoresis. To analyze the obtained fragments, they need to be separated. Agarose gel electrophoresis is used for this purpose. The separation principle is based on the dependence of movement speed on mass: smaller DNA fragments move significantly faster in the gel and manage to travel a greater distance, while larger fragments get stuck in the gel pores and move slower.
4. Detection. To visualize the DNA fragments invisible to the naked eye, the gel is stained with a special dye—ethidium bromide. After that, the result is photographed under ultraviolet (UV) light, where the fragments begin to glow.

Clinical and scientific application

The final result of the study is obtaining a restriction map of a specific microbial species. This map is a visual representation of the distribution of DNA fragments of different lengths.

In microbiology and medicine, restriction analysis is used to solve the following tasks:

• Microorganism identification: the method allows with high accuracy to determine whether the studied sample belongs to a certain bacterial species or genus.
• Assessment of genetic relatedness: by comparing restriction maps of different strains, it is possible to establish how close they are to each other (which is critically important in epidemiological investigations).
• Mutation search: the method allows effectively detecting regions of the genome where mutational changes have occurred (since a mutation can create a new restriction site or destroy an old one).

In addition, restriction analysis is rarely used in isolation. It serves as a crucial initial stage for more complex molecular genetic studies, such as sequencing (direct determination of the nucleotide sequence) and molecular hybridization.

Briefly: answers to topical questions

Class and exam questions

What else to study on this topic

• Mechanism of phosphodiester bond cleavage
• Types of restriction enzymes and their classification
• Properties and structure of agarose gel
• Principles of molecular hybridization
• Basics of the DNA sequencing method

Other topics in the "Microbial Genetics" section

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