28. Biotechnology & Genomics

Restriction Enzymes

Phosphodiester Bond DiagramDNA can be cut by restriction endonucleases (RE). Endonucleases are enzymes that can hydrolyze the nucleic acid polymer by breaking the phosphodiester bond between the phosphate and the pentose on the nucleic acid backbone. This is a very strong covalent bond while the weaker hydrogen bonds maintain their interactions and double strandedness.

As the name implies, restriction endonucleases (or restriction enzymes) are “restricted” in their ability to cut or digest DNA. The restriction that is useful to biologists is usually palindromic DNA sequences. Palindromic sequences are the same sequence forwards and backwards. Some examples of palindromes: RACE CAR, CIVIC, A MAN A PLAN A CANAL PANAMA. With respect to DNA, there are 2 strands that run antiparallelel to each other. Therefore, the reverse complement of one strand is identical to the other. Molecular biologists also tend to use these special molecular scissors that recognize palindromes of 6 or 8. By using 6-cutters or 8-cutters, the sequences occur throughout large stretches rarely, but often enough to be of utility.
HindIII Restriction site and sticky ends vector

EcoRI restriction enzyme recognition site

EcoRI generates sticky of cohesive ends

SmaI restriction enzyme recognition site

SmaI generates blunt ends


Restriction enzymes hydrolyze covalent phosphodiester bonds of the DNA to leave either “sticky/cohesive” ends or “blunt” ends. This distinction in cutting is important because an EcoRI sticky end can be used to match up a piece of DNA cut with the same enzyme in order to glue or ligate them back together. While endonucleases cut DNA, ligases join them back together. DNA digested with EcoRI can be ligated back together with another piece of DNA digested with EcoRI, but not to a piece digested with SmaI. Another blunt cutter is EcoRV with a recognition sequence of GAT | ATC.