- Introduction
Restriction enzymes, or endonucleases, have become one of the most important tools in molecular biology. They were discovered about 40 years ago when scientists investigated the host-specific restriction and modification of bacterial viruses (see 'Homeland defence'). Among the first restriction enzymes purified were EcoR I and EcoR II from Escherichia coli, and Hind II and Hind III from Haemophilus influenzae. These enzymes turned out to often function as dimers that specifically recognize and cleave DNA, generating discrete gene-sized fragments. Researchers quickly realized that these enzymes provided them with remarkable new ways to manipulate and investigate gene organization, function, and expression. Today, approximately 3,000 restriction enzymes have been discovered, recognizing over 230 different DNA sequences.
- DNA Analysis
One of the most used applications of restriction enzymes in the laboratory is in DNA analysis. By making use of the sequence specificity of these enzymes, scientists can map the structural organization of genes, DNA features, et-cetera. First, the DNA is incubated together with one or more restriction enzymes under conditions where the restriction enzyme works optimally. This varies from enzyme to enzyme, and includes factors like temperature (often 37 degrees Celsius), salt concentrations, pH, presence of divalent ions like magnesium, protein content, and time of incubation. After the digestion the resulting fragments are separated on size using an gel electrophoresis. As DNA is negatively charged, it will run towards the positive pole when exposed to an electrical field. While running, the agarose gel separates smaller fragments from larger ones as these run faster through the mazes of the gel, resulting in ladder patterns that can be visualized by DNA staining dyes like Ethydium Bromide. As a reference, scientists often take along a so-called marker, a pre-digested DNA mixture with fragments of known sizes (see first lane of example). The unique fragments patterns in combination with the known sequence specificity of the restriction enzymes makes it possible to exactly map pieces of DNA.
- Molecular Cloning
The discovery of restriction enzymes opened up possibilities to scientists unseen before, and started a completely new technique: molecular cloning. Now it suddenly became possible to cut two pieces of DNA and rejoin them to form a recombinant new DNA fragment. In the figure an example is given. Using the restriction enzyme Eco RI two different pieces of DNA are digested. After cleavage Eco RI produces a so-called sticky end, a short single stranded overhang. This is a common feature of many restriction enzymes, although there are also enzymes that produce a 'blunt' end. Because both pieces of DNA were cut using the same enzyme, the resulting sticky ends are complementary and can bind each other when both fragments are mixed. A different type of enzyme, DNA ligases, can then glue the parts together in a definitive way. By constructing chimeric genes, or genes with fragments inserted or deleted, the function of genes can be studied in far greater detail by relatively simple cutting and pasting. One popular implementation of this technique is to generate a fusion protein with another protein called GFP. This Green Fluorescent Protein allows direct visualization of the protein of interest inside a living cell, and helps to determine the protein's localization and function.
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Restriction enzymes play an important role in the bacteria where they are isolated from. They can be seen as the microbial equivalent of our immune system as they protect the bacterium against viral infections. Foreign DNA injected by a virus is rapidly digested by the restriction enzymes into small fragments that are no longer harmful.