Restriction Enzymes

Restriction enzymes are the molecular scissors of genetic engineering. Their discovery in the 1960s made it possible to cut DNA at precise locations, which is the first step in creating recombinant DNA. Without them, we could not isolate a gene from one organism and insert it into another.

In 1963, two enzymes were isolated from Escherichia coli that together restricted the growth of a bacteriophage (a virus that infects bacteria). One enzyme added methyl groups to the bacterial DNA, protecting it from being cut. The other enzyme cut the viral DNA. This cutting enzyme was named restriction endonuclease.

Five years later, the first restriction endonuclease to be characterised was Hind II. It was special because its cutting action depended on a specific DNA sequence. Hind II always recognised a particular sequence of six base pairs and cut the DNA at that exact point. This specific sequence is called the recognition sequence for Hind II.

Today, over 900 restriction enzymes have been isolated from more than 230 strains of bacteria. Each enzyme recognises a different recognition sequence.

Naming of Restriction Enzymes

The naming convention follows a clear pattern based on the bacterial source:

• The first letter comes from the genus of the prokaryote.
• The next two letters come from the species.
• A following letter (if any) indicates the strain.
• Roman numerals (I, II, III…) show the order in which the enzymes were isolated from that strain.

EcoRI comes from Escherichia coli strain RY 13.

• E = Escherichia (genus)
• co = coli (species)
• R = RY 13 (strain)
• I = first enzyme isolated from that strain

Types of Nucleases

Restriction enzymes belong to a larger class of enzymes called nucleases, which are of two kinds:

• Exonucleases: Remove nucleotides from the ends of the DNA molecule.
• Endonucleases: Make cuts at specific positions within the DNA molecule. Restriction endonucleases are a special type of endonuclease.

How Restriction Enzymes Work

Each restriction endonuclease functions by ‘inspecting’ the length of a DNA sequence. Once it finds its specific recognition sequence, it binds to the DNA and cuts each of the two strands of the double helix at specific points in their sugar-phosphate backbones.

Palindromic Recognition Sequences

Each restriction endonuclease recognises a specific palindromic nucleotide sequence in the DNA.

A palindrome in language is a word that reads the same forwards and backwards (e.g., “MALAYALAM”). In DNA, a palindrome is a sequence of base pairs that reads the same on both strands when the direction of reading (5' → 3') is kept the same.

The recognition sequence for EcoRI is:

5' —— GAATTC —— 3'
3' —— CTTAAG —— 5'

Notice that the sequence on the top strand (5' → 3') is GAATTC, and the sequence on the bottom strand (5' → 3') is also GAATTC (since CTTAAG read backwards is GAATTC).

Sticky Ends

Restriction enzymes cut the DNA strand a little away from the centre of the palindrome site, but between the same two bases on the opposite strands. This leaves single-stranded overhanging stretches at the ends. These are called sticky ends because they can form hydrogen bonds with their complementary cut counterparts.

This stickiness is crucial — it facilitates the action of the enzyme DNA ligase, which seals the cut ends together.

Role in Recombinant DNA Technology

Restriction endonucleases are used to form recombinant molecules of DNA — molecules composed of DNA from different sources or genomes.

When the same restriction enzyme cuts both the source DNA (containing the gene of interest) and the vector DNA (the carrier molecule), both produce the same kind of sticky ends. These complementary sticky ends can then be joined together (end-to-end) using DNA ligase.

Separation and Isolation of DNA Fragments

After cutting DNA with restriction enzymes, we get fragments of different sizes. These fragments are separated by a technique called gel electrophoresis.

• DNA fragments are negatively charged molecules. …