Since the discovery of restriction enzymes, the field of molecular biology has rapidly advanced due to the unique ability of these proteins to cleave DNA in a specific manner. These simple enzymes have had a profound effect on research all over the world; oddly enough, we have bacteria to thank for this scientific gift.
Restriction Enzyme Properties and Types
Restriction enzymes, also called restriction endonucleases, bind to DNA and cleave the double strand, forming smaller pieces of DNA. There are three types of restriction enzymes; Type I restriction enzymes recognize a DNA sequence and cut the strand randomly more than one thousand base pairs away from the site. Type II restriction enzymes, the most useful for molecular biology laboratories, recognize and cut the DNA strand predictably at a specific sequence which is usually less than ten base pairs long. Type III restriction enzymes are similar to Type I, but these cut the DNA about thirty base pairs from the recognition sequence.
Bacterial species are the major source of commercial restriction enzymes. These enzymes serve to defend the bacterial cells from invasion by foreign DNA, such as nucleic acid sequences used by viruses to replicate themselves inside a host cell. Basically, the enzyme will chop DNA into much smaller pieces which pose little danger to the cell. The enzymes are named for the species and strain of bacteria that produces it. For instance, the first restriction enzyme extracted from Escherichia coli strain RY13 is called EcoRI, and the fifth enzyme extracted from the same species is called EcoRV.
The use of Type II restriction enzymes is nearly universal in laboratories across the world. DNA molecules are extremely long and difficult to manage properly, especially if a researcher is only interested in one or two genes. Restriction enzymes allow the scientist to reliably cut the DNA into much smaller portions. This ability to manipulate DNA has allowed for the advance of restriction mapping and molecular cloning.
In a laboratory setting, knowing exactly where certain restriction sites are on a DNA strand is extremely helpful and convenient. If the DNA sequence is known, restriction mapping can be done by computer, which can quickly map all possible restriction enzyme recognition sequences. If the DNA sequence is not known, a researcher can still create a general map by using different enzymes by themselves and in conjunction with other enzymes to cleave the molecule. Using deductive reasoning, the general restriction map can be created. Having a restriction map available is critical when cloning genes.
Molecular cloning is a laboratory technique in which a gene is cut from a target DNA molecule, usually extracted from an organism, by restriction enzymes. Next, the gene is inserted into a molecule called a vector, which are usually small pieces of circular DNA called plasmids which have been modified to carry several restriction enzyme target sequences. The vector is cleaved open by restriction enzymes, and then the gene is inserted into the circular DNA. An enzyme called DNA ligase can then reform the circle to include the target gene. Once the gene is 'cloned' in such a way, the vector can be inserted into a bacterial cell so that the gene can produce protein.
- Molecular Biology of the Gene, Fifth Edition; Watson et al.; 2004
- Biochemistry, Third Edition; Voet and Voet; 2004
- Image by Flickr.com, courtesy of Michael Knowles