A Review on Restriction Enzymes

The enzymes involved in cleaving the specific regions in the genome are known as restriction enzymes. They are also known as molecular scissors. They are used widely in molecular biology. A restriction site is a specific region in the DNA consisting of bases cleaved by these enzymes. The process in which the restriction enzyme cleaves at a particular site is known as restriction digestion. The products in the form of the nucleic acid fragments are known as the restriction digests. They are known to play a crucial role in mapping, sequencing, and cloning. Most of the restriction enzymes belong to the class of endonucleases. It is an enzyme cleaving or hydrolyzing the phosphodiester bond within a DNA. The knife and fork model of DNA replication represents a triangular structure. This triangular structure is a sign of an endonuclease enzyme. The DNA molecule consists of internal phosphodiester bonds which get cleaved through restriction endonucleases.

In somatic tissue or cells, these enzymes are capable of hydrolyzing DNA by introducing double-strand breaks. Naturally occurring restriction endonucleases are known as the cell protectors. They protect the cells from viral infections. For research and analysis purpose, the extraction source of the enzymes involves microorganism or chemical synthesis.

Properties of restriction endonucleases:

A restriction enzyme recognizes a restriction site at 3’ carbon and phosphate group of a phosphodiester bond. The resultant fragments have 5’ phosphate group and 3’ hydroxyl group. Bacteria and green algae chlorella have naturally occurring restriction enzymes. Bacteria undergo a process of methylation and modify the restriction sites. Hence, the bacterial cells get protected from the action of restriction enzymes. Arbor, Nathans, and Smith received a Nobel prize in discovering the restriction enzymes. Many restrictions enzymes select sites having an axis of symmetry. The number of cuts made by restriction enzymes gets determined by the frequency of occurrence of the restriction sites. The restriction sites occur in a randomly distributed base pair. The formula (1/4)ⁿ indicates the probability of the occurrence of the restriction site with a 50% GC content. (Where n indicates the no. of nucleotide pairs in the recognition sequence).

Image: Restriction digestion (Treatment of the DNA sample with restriction endonucleases leads to the synthesis of fragments of different sizes.

Nomenclature:

The nomenclature of the restriction enzymes involves the organisms from which they were isolated. The first letter belongs to the genus and the second and third letter from the species. It involves an italic or underlined font followed by Roman numerals. For example, EcoRI is an example of a restriction enzyme obtained from E. coli strain RY13. Thus, ‘Eco’ belongs to E. coli, and RI belongs to the strain RY13. There are four classes of the restriction enzymes based on the recognition sequences.

Enzymes with no. of recognition sequences in base pairs	Property
4	Symmetrical sequences
6	Symmetrical sequences
8	Symmetrical sequences
spacer sequence	Non-specific sequences

Table: Enzymes and their properties 

Constructing a restriction map:

A restriction map is a kind of a physical map. It consists of a piece of DNA with restriction sites specific for endonucleases. The number of bases decides the sites of separation. Restriction enzymes create two types of ends such as sticky or blunt ends. A staggered cut in the restriction site with symmetrical nucleotide sequence generates sticky ends. There are two types of ends such as 5’ or 3’ overhanging ends. When a restriction enzyme cuts at a symmetrical nucleotide sequence of a restriction site between the two base pairs, the blunt ends get created. When the DNA gets digested with the restriction enzymes, and the resultant fragments get separated on a gel, the banding patterns are visible after staining. They are known as restriction fragment length polymorphisms (RFLPs). Sometimes the restriction sites vary in different individuals. A restriction enzyme cleavage site present in an individual may be absent in another individual. RFLPs help in mapping the genes or polymorphic sites. However, the non-polymorphic sites get missed. Restriction mapping solves this problem. A single restriction enzyme or combination of restriction enzymes cleave the DNA efficiently. Thus we obtain the fragments of different sizes measured in kilobases. The electrophoretic gel separates the fragments based on their sizes. Autoradiography helps to visualize the bands and cut the desired portion of the gel to take the DNA fragment.

Cloning Vectors:

A cloning procedure primarily uses a piece or a fragment of DNA obtained from the restriction digestion. The fragment of DNA gets inserted into the vector. The technique involves two main steps. First, the plasmid gets cleaved at a unique restriction site with an appropriate restriction enzyme. The second step involves the insertion of a piece of DNA cut with the same enzyme. The procedure further involves mixing of the cloning vector and a DNA fragment. It results into base pairing and annealing the two single-stranded ends. An enzyme known as DNA ligase acts as a sealing agent. It seals the gaps formed due to a phosphodiester bond.

Applications of restriction enzymes:

1.     DNA typing:

DNA fingerprinting or DNA typing determines the paternity and maternity of an individual. It helps in crime investigation. The establishment of variability in the ethnic groups involves DNA typing. Endangered species determination and genetic variability studies involve DNA typing techniques. Forensic medicine largely involves DNA fingerprinting techniques. Initially, the DNA obtained from the suspect individual gets a treatment with the restriction enzymes. The restriction digests obtained from the procedure include DNA fragments of different sizes. The fragments get separated as per their sizes in an electrophoretic apparatus. Using a Southern blot and a probe hybridization technique, visualization of the DNA bands of the suspect involves autoradiography.

It is not possible to separate the DNA fragments without the restriction enzymes. Hence, obtaining the information of the suspect invites difficulties. In the detection of paternity or maternity cases, the DNA obtained from the child and his alleged parents have to undergo restriction enzyme treatment. Autoradiogram shows a banding pattern of each sample as a DNA fingerprint.

2.     DNA molecular testing:

Genetic testing such as disease gene detection requires restriction enzyme treatment. The requirement of the restriction enzymes using PCR determines the presence of microsatellites. RFLP analysis and restriction mapping utilize the restriction enzymes. Detection of genes associated with diseases such as sickle cell anemia, phenylketonuria (PKU), and others become easy to detect.

3.     Gene Cloning:

The isolated DNA gets cleaved through RE treatment. The fragments obtained through RE treatment mix with the cloning vector and get integrated into the restriction sites in the cloning vectors. The cloning vector gets transformed into the host and allowed to replicate. The process is known as molecular cloning. Identical copies of DNA of interest obtained from the above process are known as clones. Expression of genes, synthesis of protein products, gene therapy, and many other important applications require gene cloning.

4.     Preparing recombinant vectors:

Restriction enzyme treatment targets multiple cloning sites or the restriction sites in a plasmid or any other vector. The fragment of interest gets inserted into the multiple cloning sites or a polylinker and gets transformed into the host. The host (bacterial cells) plated on a suitable medium show the growth of the recombinant colonies.

5.     Industrial applications:

It is possible to grow plant hybrids showing resistance to herbicides, pesticides, and drought tolerant varieties. Restriction digests obtained through restriction enzyme treatment get inserted into a vector. Then the DNA gets microinjected into the animal’s pronucleus to obtain transgenic animals. Hence, it is possible to produce a recombinant protein product using a transgenic animal. Example, the gene of interest expressed in a mammary tissue helps protein secretion in the milk.

6.     Genomic and cDNA libraries:

 The recombinant DNA libraries consist of a collection of clones with the desired DNA sequence. The fragments subjected to restriction enzymes get cloned into a vector to obtain a collection of clones known as genomic or cDNA libraries. 

References:
[1] Essential Genetics: A Genomics Perspective, Hartl, Elizabeth W. Jones
[2] Molecular Biology, David P. Clark, Nanette J. Pazdernik
[3] Genetic Engineering, Verma P.S. & Agarwal V.K.
[4] Molecular Biology and Genomics, Cornel Mulhardt

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