**3. Nucleic acid and Restriction Enzyme Analysis (REA)**

Restriction analysis is an easy-to-perform, inexpensive, and relatively fast method for the study of point mutations and identification of methylated regions in DNA. It can also be used for restriction profiling of micro- and macroorganisms and can serve as a basis for phylogenetic analysis.

In the case of fragmented nucleic acids, e.g., viruses with a segmented genome (Rotavirus), direct fingerprinting is applied, in which individual nucleic acid segments – due to different mobility in an electric field – are distributed at a different distance in an agarose or polyacry‐ lamide gel [24].

Direct electrophoresis, however, does not work in the case of non-segmented nucleic acids. That is why restriction enzymes (restrictases) are used. Restriction enzymes cut the nucleic acid molecule at a specific nucleotide sequence that they recognize. The method is both applicable to total homogeneous (from a single species) DNA and to PCR amplification products. The requirements for the quality of the nucleic acid sample are laid out above (see nucleic acid extraction methods). Additionally, it is recommended to purify the PCR amplifi‐ cation product in a gel (most kits recommend 2% gel, although 1.5% gels give better purifica‐ tion) or directly by a column, before restriction. Thus, if there is not enough DNA product in the reaction mixture, more DNA template sample can be added instead of water. It is essential to keep the enzyme/buffer/reaction volume ratio specified in the instructions provided by the enzyme manufacturer.
