Foundations of Molecular Biotechnology

Restriction endonucleases (or restriction enzymes) are proteins that act as biological scissors. Originally discovered in bacteria as a defense mechanism against viral DNA, these enzymes recognize specific sequences called recognition sites. Most of these sites are palindromic, meaning the sequence reads the same from $5'$ to $3'$ on both strands. For example, the enzyme EcoRI looks for the sequence $5'-GAATTC-3'$. When the enzyme finds this sequence, it breaks the phosphodiester bonds of the DNA backbone at specific points. This precision allows scientists to isolate specific genes from a massive genome with surgical accuracy.

Not all cuts are created equal. Restriction enzymes produce two types of ends: sticky ends and blunt ends. Sticky ends occur when the enzyme makes a staggered cut, leaving short, single-stranded overhangs. These overhangs are 'sticky' because they can readily form hydrogen bonds with complementary sequences. In contrast, blunt ends occur when the enzyme cuts straight through both strands at the same position, leaving no overhangs. While blunt ends are more 'universal' (any blunt end can join another), sticky ends are far more efficient for cloning because the overhangs act like molecular Velcro, holding the DNA pieces together long enough for the next step.

Once restriction enzymes have cut the DNA, DNA ligase is required to join the fragments permanently. While sticky ends might 'stick' together via hydrogen bonds, these bonds are weak and temporary. DNA ligase acts as the 'glue' by catalyzing the formation of a covalent phosphodiester bond between the $3'-OH$ group of one nucleotide and the $5'-phosphate$ group of another. This process, called ligation, requires energy in the form of ATP. By using the same restriction enzyme on a bacterial plasmid and a human gene, then adding ligase, scientists create recombinant DNA—a single molecule containing DNA from two different species.