Restriction enzymes are bacterial enzymes that have the ability to cut double-stranded DNA at specific sites. These are also known as restriction endonucleases.
They occur naturally in bacteria as a weapon to fight against the invading viruses. Restrictions sites in the viral genome are cleaved by the restriction enzymes.
These are present in bacterium, fragmenting and destroying the DNA of invading virus before gets incorporated into the host’s genome and destroy it.
Restriction enzymes (also known as restriction endonucleases) are important in the construction of recombinant DNA molecules, hence play a very important role in genomics.
This course covers introduction to restriction enzymes (or restriction endonucleases), types of endonucleases, naming of restriction endonucleases, and restriction-modification system.
Introduction to Restriction Enzymes
Restriction enzymes were first discovered by Arber and Dussoix in 1962. In 1970, Smith, Wilcox and Kelly characterized and purified restriction enzymes. They also discovered recognition and cleavage site of a restriction enzyme, Hind II.
Restriction endonuclease can recognize a DNA molecule at a specific sequence, bind to it, and finally cleave the sugar-phosphate backbone of DNA strands at or near the recognition site (Figure 1).
In the cleaving process, the enzyme hydrolyzes the phosphodiester backbone on each strand by a process called nicking.
The mechanism of restriction occurs when cell produces restriction endonuclease. This endonuclease degrades phage DNA before it replicates and synthesizes new phage particles.
Applications of Restriction Enzymes
The ability of restriction endonucleases to cleave DNA at specific recognition sites makes these enzymes an essential tool in molecular biology.
Restriction endonucleases have a wide range of applications, such as gene cloning, DNA sequencing (Figure 2), and southern hybridization analysis.
Molecular cloning allows creation of multiple copies of genes, expression of genes, and study of specific genes.
Restriction enzymes are used to generate DNA fragments with specific complementary end sequences. These sequences are joined together with a DNA ligase, prior to transformation (Figure 3).
DNA or restriction mapping uses restriction endonucleases to obtain structural information of the DNA fragment or genome. Hence, restriction enzymes have proved to be invaluable tool for mapping.
A large DNA molecule can be sequenced by digesting it with restriction enzymes and processing the resulting fragments through a DNA sequencer.
There are two main types of DNA sequencing.
- The older and classical chain termination method, also called Sanger method.
- Newer methods, such as, High-Throughput Sequencing (HTS) techniques (Figure 4) and Next-Generation Sequencing (NGS). These methods process a large number of DNA molecules quickly.
Restriction Fragment Length Polymorphism (RFLP)
RFLP involves the digestion of a DNA sample using restriction enzymes. The fragments are separated based on length by gel electrophoresis and transferred onto a membrane.
These fragments are then bound to a radioactive or fluorescent labeled probe that targets specific sequences.
These sequences are bracketed by restriction enzyme sites. RFLP occurs when the resulting fragment lengths vary between individuals (Figure 5).