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Restriction Enzymes Explained


Structure of the homodimeric restriction enzyme EcoRI (cyan and green cartoon diagram) bound to double stranded DNA (brown tubes).
Boghog2/Public Domain via Wikimedia Commons

Restriction endonucleases are a class of enzyme that cut DNA molecules. Each enzyme recognizes a unique sequence of nucleotides in the DNA strand, usually about 4-6 base-pairs long. The sequences are palindromic in that the complimentary DNA strand has the same sequence only in the reverse direction, so both strands of DNA are cut at the same location.

Restriction enzymes are found in many different strains of bacteria where their biological role is to participate in cell defense. These enzymes “restrict” foreign (e.g. viral) DNA that enters the cell, by destroying it. The host cell has a restriction-modification system that methylates its own DNA at sites specific for its respective restriction enzymes, thereby protecting it from cleavage. Over 800 known enzymes have been discovered that recognize over 100 different nucleotide sequences.

Restriction enzymes are used in biotechnology to cut DNA into smaller strands in order to study fragment length differences among individuals (Restriction Fragment Length Polymophism – RFLP) or for gene cloning. RFLP techniques have been used to determine that individuals or groups of individuals have distinctive differences in gene sequences and restriction cleavage patterns in certain areas of the genome. Knowledge of these unique areas is the basis for DNA fingerprinting. Each of these methods depends on the use of agarose gel electrophoresis for separation of the DNA fragments.

There are three different types of restriction enzymes. Type I cuts DNA at random locations as far as 1000 or more base-pairs from the recognition site. Type III cuts at approximately 25 base-pairs from the site. Types I and III require ATP and may be large enzymes with multiple subunits. Type II enzymes, which are predominantly used in biotechnology, cut DNA within the recognized sequence without the need for ATP, and are smaller and simpler. Type II restrcition enzymes are named according to the bacterial species from which they are isolated. For example, the enzyme EcoRI was isolated from E. coli.

Type II restriction enzymes can generate two different types of cuts depending on whether they cut both strands at the centre of the recognition sequence, or each strand closer to one end of the recognition sequence. The former cut will generate "blunt ends" with no nucleotide overhangs. The latter, generates "sticky" or "cohesive" ends, because each resulting fragment of DNA has an overhang that compliments the other fragments. Both are useful in molecular genetics for making recombinant DNA and proteins.

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