What Are Restriction Enzymes?
Restriction endonucleases are a class of enzyme that cut DNA molecules. Each enzyme recognizes unique sequences of nucleotides in a DNA strand—usually about 4 to 6 base-pairs long. The sequences are palindromic in that the complementary DNA strand has the same sequence in the reverse direction. In other words, both strands of DNA are cut at the same location.
Where These Enzymes Are Found
Restriction enzymes are found in many different strains of bacteria where their biological role is to participate in cell defense. These enzymes restrict foreign (viral) DNA that enters the cells by destroying them. The host cells have a restriction-modification system that methylates their own DNA at sites specific for their respective restriction enzymes, thereby protecting them from cleavage. More than 800 known enzymes have been discovered that recognize more than 100 different nucleotide sequences.
Types of Restriction Enzymes
There are five different types of restriction enzymes. Type I cuts DNA at random locations as far as 1,000 or more base-pairs from the recognition site. Type III cuts at approximately 25 base-pairs from the site. Both of these types require ATP and may be large enzymes with multiple sub-units. 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 restriction enzymes are named according to the bacterial species from which they are isolated. For example, the enzyme EcoRI was isolated from E. coli. Most of the public is familiar with E. coli outbreaks in food.
Type II restriction enzymes can generate two different types of cuts depending on whether they cut both strands at the center 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. This form of DNA stands out because it is produced by the ligation (bonding together) of two or more different strands that weren't originally linked together.
Type IV enzymes recognize methylated DNA, and Type V enzymes use RNAs to cut sequences on invading organisms that are not palindromic.
Use in Biotechnology
Restriction enzymes are used in biotechnology to cut DNA into smaller strands in order to study fragment length differences among individuals. This is referred to as Restriction Fragment Length Polymorphism (RFLP). They're also used 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. TBE buffer, which is made up of Tris base, boric acid, and EDTA, is commonly used for agarose gel electrophoresis to examine DNA products.
Use in Cloning
Cloning often requires inserting a gene into a plasmid, which is a type of a piece of DNA. Restriction enzymes can assist with the process because of the single-stranded overhangs they leave when they make cuts. DNA ligase, a separate enzyme, can join together two DNA molecules with matching ends.
So, by using restriction enzymes with DNA ligase enzymes, pieces of DNA from different sources can be used to create a single DNA molecule.