Tools of Recombinant DNA Technology - Restriction Enzymes

• Restriction enzymes were first identified in bacteria, where they act as a protective mechanism by cutting foreign DNA.
• Hind II is recognised as the first restriction endonuclease for its ability to cut DNA at a specific recognition sequence.
• Their discovery became a major breakthrough in genetic engineering and recombinant DNA technology.

Nucleases are enzymes that break nucleic acids.

A restriction enzyme recognises a particular sequence of bases in DNA and cuts only at or near that site.

Palindromic sequence:

A palindromic DNA sequence reads the same on both strands when each strand is read in the 5′ to 3′ direction.

Example: EcoRI recognition site:

EcoRI recognises this sequence and cuts it in a specific staggered manner.

1. The enzyme scans the DNA molecule and finds its recognition site.
2. It binds to that specific sequence.
3. It cuts the phosphodiester bonds of the DNA backbone.
4. The DNA breaks into fragments with either sticky ends or blunt ends.
5. Compatible DNA fragments can later join through hydrogen bonding and are sealed by DNA ligase.

Steps in the formation of recombinant DNA by action of restriction endonuclease enzyme - EcoRI

Restriction enzymes are named according to the bacterium from which they are obtained.

Example: EcoRI:

One more example:

BamHI is named from Bacillus amyloliquefaciens.

Restriction enzymes are central to recombinant DNA technology.

Main uses:

• Cutting donor DNA to isolate the required gene fragment.
• Cutting vector DNA at the same recognition site.
• Producing compatible ends so that the insert can join with the vector.
• Helping in the construction of recombinant DNA molecules.

Diagrammatic representation of recombinant DNA technology

• Restriction enzymes, initially discovered as a bacterial defence mechanism, act as "molecular scissors" to cut DNA and are the fundamental tools of recombinant DNA technology.
• They belong to a class of enzymes called endonucleases, which cleave DNA at specific internal positions, unlike exonucleases that remove nucleotides from the ends.
• These enzymes recognise and bind to specific palindromic sequences, such as EcoRI's 5'-GAATTC-3', which read identically on both strands in the 5′ to 3′ direction.
• When cut at these sites, DNA produces either straight blunt ends or staggered sticky ends, which pair easily and are preferred for cloning.
• Restriction enzymes are named systematically after their bacterial source; for example, in EcoRI, 'E' is the genus, 'co' is the species, 'R' is the strain, and 'I' indicates the order of isolation.