Tools of Recombinant DNA Technology: Restriction Enzymes | Biology Class 12 - NEET PDF Download

Restriction Enzymes

“Restriction enzymes are the enzymes produced by certain bacteria that have the property of cleaving DNA molecule at or near specific base sequences.”

Introduction

The restriction enzyme is a protein produced by bacteria that cleaves the DNA at specific sites. This site is known as the restriction site.
The restriction enzymes protect the live bacteria from bacteriophages. They recognize and cleave at the restriction sites of the bacteriophage and destroy its DNA.
Restriction enzymes are important tools for genetic engineering. They can be isolated from the bacteria and used in the laboratories.
The restriction enzymes recognize short and specific nucleotide sequences in the DNA known as the recognition sequences. When the restriction enzyme recognizes a DNA sequence, it hydrolyzes the bond between adjacent nucleotide and cuts through the DNA molecule.
The bacteria prevents its own DNA sequences from degradation by the addition of the methyl group at the adenine or cytosine bases within the recognition sequence with the help of enzyme methylases.

Types of Restriction Enzymes

Type I

These restriction enzymes cut the DNA far from the recognition sequences. However, they do not produce discrete restriction fragments, hence, are of not much practical value.

These are complex, multi-subunit restriction and modification enzymes. They were initially thought to be rare, but through genomic analysis, they are found to be common and are of considerable biochemical interest.

Type II

These enzymes cut at specific positions closer to or within the restriction sites. Discrete restriction fragments and gel banding patterns are observed. They are exclusively used for DNA analysis and gene cloning in the laboratories. These are a family of unrelated proteins. They are named after the bacterial species from which they are isolated. For eg., EcoRI is isolated from bacterial species E.coli. The restriction enzymes generate two different types of cuts. Blunt ends are produced when they cut the DNA at the centre of the recognition sequence, and sticky ends produce an overhang.

Type III

These are multi-functional proteins with two subunits- Res and Mod. It is a modification methyltransferase. The DNA sequence specific for the system is recognized by the Mod subunit.

Applications of Restriction Enzymes

They are used in RFLP techniques to cut the DNA into smaller fragments to study the fragment length differences among the individuals.

In Gene Cloning

During cloning, a gene is inserted into a plasmid. Restriction enzymes cut the plasmid producing single-stranded overhangs. The two DNA molecules are ligated with the help of DNA ligase to form a single DNA molecule.

Nomenclature of Enzyme: The first letter used for the enzyme is the first letter of the bacterium genus name (in Italics) then comes the first two letter of it's species (In Italics), next is the strain of the organism, last is Roman numerical signifying the order in which the enzymes were isolated from that strain of bacteria.

Examples of Restriction Enzymes

Restriction Enzyme

Recognition Sequences of Some Restriction Endonucleases

Name	Recognition sequence	End after cleavage		Source
Eco RI		– G – C T T A A	A A T T C –  G –	Escherichia coli - containing drug resistant plasmid RI.
Hind III		– A – T T C G A	A G C T T – A –	Haemophilus influenzae
Bam I		– G – C C T A G	G A T C C –   G –	Bacillus amyloliquefaciens
Hae III		– G G – C C	C C – G G –	Haemophilus aegyptius

(i) Synthesizing enzymes: These enzymes are used to synthesize new strands of DNA, complementary to existing DNA or RNA template. They are of two types; reverse transcriptases and DNA polymerases.

(a) Reverse transcriptases: help in the synthesis of complementary DNA strands on RNA templates;

(b) DNA polymerases: help in the synthesis of complementary DNA strands on DNA templates.

(ii) Joining enzymes: These enzymes help in joining the DNA fragments. For example DNA ligase from Escherichia colitis used to join DNA fragments. Joining enzymes are, therefore, called molecular glues.

(iii) Alkaline phosphatases enzymes: These enzymes cut off phosphate group from the 5' end of linearised circular DNA and prevent its re-circularisation.