What are Restriction Enzymes?
Restriction enzymes or to use their correct name, restriction endonucleases, are a type of enzyme which have the ability to "cut" molecules of DNA. They are often referred to as "genetic scissors".
The restriction enzyme recognises a unique sequence of nucleotides in the DNA strand, which is usually between four to six base-pairs in length. The complimentary DNA strand has the same sequence but in the reverse direction, thus ensuring both strands of DNA are cut at the same location.
Where are Restriction Enzymes found?
Restriction enzymes are found in many different strains of bacteria and their biological purpose is to participate and assist actively in cell defence. These enzymes prevent and "restrict" (hence their name) any foreign, i.e. viral DNA that may enter the cell, by destroying it. The host cell has an inbuilt restriction-modification system that methylates its own DNA at sites specific for its respective restriction enzymes, thereby protecting it from self-cleavage.
How are Restriction Enzymes used in Biotechnology?
In relation to biotechnology, restriction enzymes are used to cut DNA into smaller strands in order to study differences and similarities in fragment length amongst individuals such as in Restriction Fragment Length Polymorphism (RFLP) or in gene cloning. Such techniques have been used to determine that individuals or groups of individuals have distinctive differences in gene sequences and restriction cleavage patterns in specific areas of the genome. The basis for DNA fingerprinting is taken from this knowledge. Both RFLP and gene cloning processes are dependent on the use of agarose gel electrophoresis to allow complete and precise separation of the DNA fragments.
What types of Restriction Enzymes are there?
There are three different types of restriction enzymes simply named Type I, Type II and Type III.
Type I restriction enzymes cut DNA at random locations as remote as 1000 or more base-pairs from the recognition site.
Type III restriction enzymes cut at approximately 25 base-pairs from the recognition site.
Both Type I and Type III restriction enzymes require energy in the form of Adenosine Triphosphate (ATP) and may exist as larger enzymes consisting of multiple subunits.
However, Type II enzymes, such as those predominantly used in biotechnology, cut DNA within the recognised sequence without the need for ATP, and are smaller and less complex than Types I and III. Type II restriction enzymes are given specific names according to which bacterial species they are isolated from. By way of example, the Type II restriction enzyme isolated from E. Coli is named EcoR1.
Type II restriction enzymes are able to create two different types of cut, which is dependent 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, while the latter cut generates "sticky" or "cohesive" ends, due to the fact that each resulting fragment of DNA has an overhang that compliments the other fragments. Both types are widely used in biotechnology, particularly in the fields of molecular genetics and protein engineering processes.
Biotechnology exploits the ability of restriction enzymes to reliably and precisely cleave DNA at specific sequences, which has led to the widespread use of these genetic tools in many molecular genetics techniques. Restriction enzymes can be used to map DNA fragments or the entire genome, thus determining the specific order of the restriction enzyme sites in the genome. Restriction enzymes are also frequently used to verify the identity of a specific DNA fragment, based on the known restriction enzyme sites sequence that it contains.
An extremely important use of restriction enzymes has been in the generation of recombinant DNA molecules. These are DNA molecules which consist of consist of genes or DNA fragments from two different organisms. Typically, a small circular DNA molecule known as a plasmid, obtained from bacteria, is joined to another piece of DNA from another gene of interest.
Type II restriction enzymes are used at several points during this process. They are used to digest the DNA from the experimental organism, in order to prepare the DNA for cloning. Thereafter, a bacterial plasmid or bacterial virus is cleaved with an enzyme that yields compatible ends. These compatible ends could be blunt with no overhang, or have complementary overhanging sequences. DNA from the gene of interest is combined with DNA from the plasmid or virus, and both types of DNA are joined with an enzyme called DNA ligase.
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