The term “Restriction enzyme” originated from the studies of Enterobacteria phage λ (lambda phage) in the laboratories of Werner Arber and Matthew Meselson. The ability of certain E. coli strains to inhibit the activity of lambda phage by the enzymatic cleavage of the phage DNA was studied and the enzyme responsible for this growth restriction was termed a restriction enzyme.1, 2, 3
Werner Arber, Daniel Nathans, and Hamilton O. Smith were awarded the Nobel Prize for Physiology or Medicine in 1978 for their discovery and characterization of restriction enzymes, which led to the development of recombinant DNA technology.
Restriction enzymes are also called "molecular scissors" as they cleave DNA at or near specific recognition sequences known as restriction sites. These enzymes make one incision on each of the two strands of DNA and are also called restriction endonucleases.4
Viruses infect the host cells by injecting their DNA into the cells. This viral DNA hijacks the host cell’s machinery for reproduction of viral progeny, resulting in the host cell’s death. To overcome the viral infection, many bacteria and archaea have evolved several mechanisms. A major protective mechanism involves the use of restriction enzymes to degrade the invading viral DNA by cleaving it at specific restriction sites. At the same time, the host cell protects its own DNA from being cleaved by employing other enzymes called methylases, which methylate adenine or cytosine bases within host recognition sequences. For each of the restriction enzyme, the host cell produces a corresponding methylase that methylates and protects the host DNA from degradation. These enzymes make up the restriction-modification (R-M) systems.
The restriction enzymes catalyze the hydrolysis of the bond between the 3’-oxygen atom and the phosphorus atom in the phosphodiester backbone of DNA. The enzymes require Mg2+ or other divalent ions for their activity.
Smith and Nathans suggested the naming guidelines for restriction endonucleases in 1973. According to these guidelines, the names of the enzymes begin with an italicized three-letter acronym. The first letter indicates the first letter of the bacterial genus from which the enzyme has been isolated and the next two letters are derived from the bacterial species. These may be followed by extra letters or numbers to indicate the serotype or strain. This is followed by a space and a Roman numeral to indicate the chronology of identification. For example, Hind III was the third of four enzymes isolated from Haemophilus influenza serotype d.6
Based on the composition, characteristics of the cleavage site, and the cofactor requirements, the restriction endonucleases are classified into four groups, Type I, II, III, and IV.
The site of cleavage is indicated by the red arrow in the table.
Depending on the substrate DNA and the reaction conditions, restriction enzymes show a wide variation of cleavage and possible star activity. In order to obtain the desired cleavage, it becomes important to control the following factors:
Isoschizomers are restriction enzymes with the same recognition sequence and cleavage sites. Example: Sph I (CGTAC/G) and Bbu I (CGTAC/G)
Neoschizomers are restriction enzymes with the same recognition sequence but cleave the DNA at a different site within that sequence. Example: Tai I (ACGT/) and Mae II (A/CGT)
Restriction digestion of double-stranded DNA produces two kinds of ends: Sticky ends and Blunt ends.
Blunt ends possess a 5’-phosphate group that promotes ligation. They are universally compatible with other blunt-ended DNA.
Blunt ends generated by EcoR V
Sticky ends are small stretches of single-stranded DNA capable of self-ligation or ligation with a complementary region from another DNA molecule. The sticky ends possess 3’- or 5’-overhangs of 1–4 nucleotides.
5’ Cohesive end generated by Bln I (Catalog No. 11558170001)
3’ Cohesive end generated by Kpn I
Our restriction enzyme collection has been optimized for digestion using five unique buffers. When digesting DNA using a single enzyme, use the buffer supplied with the enzyme. For double digestion of DNA, use the buffer in which both the enzymes show 100% activity. Alternatively, the optimal buffer can be determined from the chart of common double digestions. In some cases, sequential digestion is recommended due to buffer incompatibility (composition or temperature). For protocols on restriction digestion with single enzyme, two restriction enzymes and sequential DNA Digestion, refer to Restriction Enzyme Digest Protocol. The selection of the correct buffer is crucial for obtaining high activity of both enzymes and for avoiding star activity. The choice of five buffers allows the user to select compatible buffers for the desired digestion. Most digestions require 1–2 buffers, which becomes cost-effective.
The ability of restriction endonucleases to cleave DNA at specific recognition sites has enabled extensive use of these enzymes as essential tools in several molecular biology techniques. Some of the major applications are explained below: