Restriction Enzyme: Definition, Types, Applications, Examples, Diagram

Restriction Enzyme Definition:

Restriction enzymes or restriction endonucleases, are enzymes produced by certain bacteria that can cleave DNA molecules at or near specific base sequences.

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This Story also Contains

1. Restriction Enzyme Definition:
2. What are Restriction Enzymes?
3. Types of Restriction Enzymes
4. Mechanism of Action
5. Specificity and Recognition Sites
6. Applications in Molecular Biology
7. Laboratory Techniques Involving Restriction Enzymes
8. Advances and Innovations
9. Practical Considerations in Using Restriction Enzymes
10. Recommended Video on Restriction Endonuclease

What are Restriction Enzymes?

Restriction enzymes, or restriction endonucleases, are crucial in molecular biology because of their DNA restriction function, or the cutting of DNA at specific recognition sites. They were isolated in the 1960s and signalled more change in genetic engineering since they provided the exact method for manipulating DNA molecules. These enzymes are widely used in different molecular approaches, for instance in cloning, gene modification, and even DNA profiling, among others.

Restriction enzymes are enzymes produced by bacteria and archaea that recognise and then cleave DNA. They also serve to cut the DNA molecule into fragments, where every fragment has an end or terminal boundary. Restriction enzymes have no doubt become one of the most crucial instruments in the context of molecular biology, especially regarding the manipulation and analysis of DNA. It lets researchers map the genomic structure, analyse the sequences of genes, and construct new DNA molecules or modified organisms.

The restriction enzyme is a protein produced by bacteria that cleaves DNA at specific locations. This location is known as the restriction site. They were first identified in the middle of the 1960s, basically through research on the mechanisms by which bacteria are against viruses. Werner Arber, Daniel Nathans, and Hamilton O. Smith shared the Nobel Prize for Physiology or Medicine for their discoveries concerning the restriction of bacterial viruses, which provided a new tool for analysing genetic material.

In general, restriction enzymes occur in bacteria and in some archaea, where they are part of the restriction-modification system. While restriction enzymes cut any foreign DNA that might invade the host cell, methyltransferases ‘protect’ host DNA from cutting itself by wrapping it around. This system has proved relevant in preventing viral infections and preventing bacteria from swapping genes.

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Types of Restriction Enzymes

Table: Comparison of Different Types of Restriction Enzymes or restriction endonucleases,

Resistant enzymes based on recognition sites, cleavage recognition sites, and mechanisms of action are distinguished.

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Type I

Recognition Sites: Some restriction enzymes are of Type I that cause DNA cleavage at a random position away from the site of recognition but recognise a specific sequence.

Cleavage Pattern: It must be noted that they generate fragments of variable size because they cleave at non-specific sites.

Mechanism of Action: The newly discovered type I restriction enzymes are multi-subunit enzymes whose subunits include the recognition, cleavage, and modification subunits. They need ATP for the cleavage of DNA and possess the characteristics of both endonuclease and methyl transferase enzymes.

Type II

Recognition Sites: The Type II restriction enzymes act on DNA at or near the specific sequence at which the enzyme can recognize the DNA molecules.

Cleavage Pattern: They reproduce DNA segments and provide specialised terminal characteristics to the ends of the segments as blunt ends or sticky ends, according to the enzyme type.

Mechanism of Action: Type II restriction enzymes exist as homodimers or homotetramers and cleave DNA at specific sites, and no energy in the form of ATP is utilised. They are generally used in molecular biology for genetic engineering.

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Type III

Recognition Sites: Type III restriction enzymes are specific in recognising DNA sequences, and there is DNA cleavage at a certain distance from the recognition site.

Cleavage Pattern: They produce DNA fragments with definite cut sites, like Type II enzymes.

Mechanism of Action: Type III restriction enzymes act as multicomponent molecules that consist of subunits that are designed for the recognition of the target DNA, the cutting of the DNA bonds, and the modification of DNA.

Type IV

Recognition Sites: Type IV restriction enzymes are those that cut DNA at specific base sequences on the DNA but do not use any ATP.

Cleavage Pattern: They cleave DNA non-specifically and produce DNA fragments with different lengths from those of the restriction endonucleases.

Mechanism of Action: Type IV restriction enzymes are endonucleases that make an incision on various DNA sequences and do not require ATP. They are considerably rare as compared to Type I, II, and III enzymes.

Mechanism of Action

The mechanisms of action include the recognition of specific DNA sequences and cleavage patterns:

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Recognition of Specific DNA Sequences

Restriction enzymes bind to a particular sequence on the DNA molecules and they are usually palindromic, This involves complementary base pairing between the enzyme and the DNA. The recognition sequence is different for all the enzymes and generally, it lies for four to eight nucleotide pairs.

Cleavage Patterns (Blunt Ends and Sticky Ends)

Once the particular sequence of the DNA has been identified by the restriction enzymes, they cut the DNA at specific sites separated by the particular base sequence, which causes either the formation of blunt ends or sticky ends. Cut ligation description Blunt ends: ends that are created when both of the DNA’s strands have been cleaved and a finish with a 90° angle is present, while sticky ends: are generated when the DNA is cleaved asymmetrically, leading to the presence of overhanging single strands of DNA. There are two types of cleavage patterns, depending on the enzymes to be used and the recognition sequence

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Specificity and Recognition Sites

Explanation Of Recognition Sequences

Recognition sequences are definable DNA sequences to which the restriction enzymes adhere and scission. These sequences are generally palindromic, as are the sequences on two complementary strands of DNA. For example, the recognition sequence for the restriction enzyme EcoRI is 5'-GAATTC-3', which reads the same on both strands: The reverse complement sequence is the ‘5’ to ‘3’ orientation, stated as ‘5’-GAATTC-‘3’.

Palindromic Sequences

Palindromes have a sequence; on one aspect, it is exactly the reverse of the other aspect of the other strand. This symmetry enables the restriction enzymes to efficiently bind to the DNA at precise sites to cleave the DNA and give predictable cleavage patterns.

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Diagram: Example of a Palindromic Sequence Recognized by a Restriction Enzyme

Applications in Molecular Biology

Genetic engineering

Under this category, restriction enzymes are very vital in genetic engineering since they can be used to alter DNA sequences. They are employed to cut the DNA at a particular site of recognition to enable the addition, removal, or alteration of DNA segments. This ensures the creation of recombinant DNA molecules with desired traits for various applications.

Cloning

To clone genes, restriction enzymes are applied to the vector DNA, and the DNA insert has to be cut at particular sites (40). Because they have sticky ends, the vector and insert DNA fragments can recombine by base pairing, resulting in the formation of recombinant DNA molecules. These recombinant molecules are then taken into host cells for replication as well as expression of the inserted gene.

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Recombinant DNA Technology

Recombinant DNA is the result of the formation of new DNA sequences from DNA fragments derived from different sources. In this process, restriction enzymes are crucial because they cut the DNA at predetermined sites and help in linking together new pieces of DNA with desirable properties.

DNA fingerprinting

In DNA fingerprinting, restriction enzymes can cut the genomic DNA randomly into smaller pieces of DNA fragments of different sizes. The obtained DNA fragments are run on a gel for electrophoresis, which generates specific band patterns that are referred to as fingerprints. This technique is used routinely, especially in the fields of forensics, DNA paternity testing, and DNA profiling.

CRISPR Technology

CRISPR also called cluster regularly Inter-spaced palindromic repetitive sequences is a remarkable modification that employs the capability of restriction enzymes for molecular sickleization. CRISPR-associated (Cas) proteins cut target DNA sequences that base pair with complementary RNA identities of the CRISPR organisation. This is a precise gene editing tool that has changed how molecular biology is done, especially in terms of gene editing.

Laboratory Techniques Involving Restriction Enzymes

Different techniques that involve restriction enzymes are:

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Gel Electrophoresis

The specific technique that is applied in the separation of DNA fragments is known as Gel electrophoresis. Restriction enzymes digest DNA in the presence of oxygen and thereafter the DNA fragments are applied on an agarose gel and submerged in an electric field. The DNA fragments that are smaller move in the gel much faster and can therefore be observed and quantified for fragments of choice.

DNA Ligation

A ligase catalyses the joining of DNA which is referred to as DNA ligation, where fragments of DNA are ligated to form recombinant DNA molecules. This is commonly done by using restriction enzymes that digest the DNA and DNA ligase that join compatible sticky ends. This process is very important in cloning and recombinant DNA technology.

PCR (Polymerase Chain Reaction)

PCR is a biochemical method of DNA replication that is applied in the amplification of targeted nucleic acid sequences. The desired DNA fragments of interest can be selectively amplified through the process of PCR after DNA digestion using restriction enzymes. This application is commonly employed in molecular biology, genetics, and diagnostics.

Restriction Fragment Length Polymorphism (RFLP)

RFLP analysis is a molecular method applicable to study polymorphism of nucleotide sequences in the genome. In digesting DNA with restriction enzymes the DNA samples get separated based on the size of the fragment by gel electrophoresis. In genetic mapping, RFLP analysis is broadly applied in forensic science and disease pathway diagnosis.

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Diagram: Gel Electrophoresis Process

Advances and Innovations

Engineered Restriction Enzymes

Some restriction enzymes are naturally occurring, while others have been engineered to have altered specificities or even to possess different desirable attributes. These engineered enzymes add to the available repertoire of molecular/synthetic biology to seek tighter control over DNA.

Use in Synthetic Biology

In synthetic biology, restriction enzymes are indispensable since they are applied to build special DNA sequences for designing synthetic gene circuits, genetic circuits, and biosensors. The creation of synthetic DNA has various uses in biotechnology, medicine, and even environmental studies.

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Development Of New Restriction Enzymes With Altered Specificity

Currently, the researchers are in the process of activity isolation and optimisation of new restriction enzymes with distinct specificities for selective DNA sequence recognition. These enzymes increase the potential of DNA manipulation techniques and facilitate the creation of new DNA constructions with desired functions.

Practical Considerations in Using Restriction Enzymes

Buffer Conditions

Some of the factors that are impacted while selecting the buffer conditions include the pH of the solution and the concentration of salts. The best buffer conditions should be chosen according to the needs of the enzyme as well as the specifics of the experiment.

Temperature Requirements

Restriction enzymes work at particular temperatures and can easily be destroyed via excessive heat. Fluctuations in temperature can impact enzymes and how effectively food is broken down. As is clear with the temperature, there are standard structures that must be followed to achieve efficient digestion of DNA.

Inhibitors and Activators

Some chemicals could influence restriction enzymes’ action; they are inhibitors or activators in this view. The effects of each of the inhibitors or activators should be closely looked at to minimise variability when using the method in DNA digestion experiments.

Troubleshooting Common Issues In Restriction Enzyme Digestion

Problems related to restriction enzymes are incomplete digestion, nonspecific digestion, and star activity. Corrective action plans could be the fine-tuning of reaction conditions, changing the enzyme concentration, or employing enzymes with other specificities.

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