Restriction Enzymes for Cloning

For cloning of DNA, often we need to cut the DNA at specific sites, which are recognized and cleaved by specific - enzymes (endonucleases, which cleave DNA at internal sites), described as restriction enzymes. Also by locating the positions of cleavage sites of a number of restriction enzymes, restriction maps can be prepared. These restriction enzymes recognize short sequences of double stranded DNA as targets for cleavage. Different enzymes recognize different, but specific sequences, each ranging in length from 4 to 8 base pairs.

Based on these attributes, restriction enzymes have been grouped into two classes: (i) types II restriction enzyme systems (e.g. EcoRI) which have separate enzymes for restriction and modification and (ii) type I (EcoK and ECoB) and type III (EcoPl, EcoP15) enzyme systems, in which same enzyme possesses both activities (bifunctional), although the restriction and modification sites differ in positions.

Of the above two classes of restriction enzymes, type II enzymes are most important for cloning purposes. The target sites for these enzymes are 4-8 bp with a symmetry (palindromes).Most type II restriction enzymes cleave DNA at unmethylated target sites. Some enzymes introduce staggered cuts, others generate blunt ends. Enzymes with 4 bp target sites are used when frequent cuts are desired and those with 8 bp (eg. Not) are used when rare cuts are desired to get long DNA segments.

Otherwise majority of enzymes used have 6 bp target sites. Some of them can cleave both methylated as well as unmethylated targets, but majority of them cleave only unmethylated targets.