Restriction enzymes are bacterial enzymes used to cut DNA in a sequence-specific manner. To cleave DNA, they bind to specific palindromic sequences called restriction sites. Such palindromic DNA sequences or inverted repeats are commonly found in regions of functional significance, such as the origin of replication, gene operator sites, and regions containing transcription termination signals.

The host bacteria protect their own genomic DNA from these enzymes by methylating these sites. Some bacteria have enzymes that have both abilities to cut the DNA and methylate it with the same sequence specificity. EcoRI acts as a restriction enzyme when it is a dimer of identical subunits. As a monomer, it acts as a methylase. Other bacteria have two different enzymes to carry out each function. This strategy of restriction and modification prevents bacterial viruses from attacking the bacterial genome.

Because different bacterial species produce different restriction enzymes, each enzyme has a unique restriction site and is named after the bacterium of origin. For instance, EcoRI is isolated from the E.coli strain RY13.

When DNA is digested with a particular restriction enzyme, all the fragments produced have the same sequence at their 5'and 3'ends. Thus, when a plasmid DNA and an insert are cut with the same restriction enzyme, they have complementary ends that can be easily ligated. The fragments are usually run on an agarose gel to confirm that the length of digested DNA matches the expected length of the fragment.