Tools of Recombinant DNA Technology
The successful application of genetic engineering depends on the availability of specific biological tools. To create recombinant DNA (rDNA), scientists require restriction enzymes to cut the DNA, vectors to carry it, and ligases to join it. Among these, restriction enzymes act as the cornerstone of modern biotechnology.
A. Restriction Enzymes and Their Discovery
Restriction enzymes, popularly known as molecular scissors, enable scientists to selectively cut DNA at specific locations. This precise cutting is the first crucial step in isolating a gene of interest.
The journey of their discovery began in 1963. Researchers studying Escherichia coli (E. coli) bacteria noticed two specific enzymes that restricted the growth of bacteriophages (viruses that infect bacteria).
- The Protector: One enzyme added methyl groups to the bacterium’s own DNA to protect it.
- The Destroyer: The second enzyme cut the foreign viral DNA into pieces, destroying it. This DNA-cleaving enzyme was named a restriction endonuclease.
In 1968, the first true restriction endonuclease, named Hind II, was isolated. Scientists discovered that Hind II always cut DNA molecules at a particular point by recognising a specific sequence of six base pairs. This specific base sequence is known as the recognition sequence for Hind II.
B. Classification and Naming of Restriction Enzymes
Restriction enzymes belong to a broader class of enzymes called nucleases. Nucleases are broadly divided into two types based on where they cut the DNA:
- Exonucleases: These enzymes remove nucleotides from the extreme ends of the DNA molecule.
- Endonucleases: These enzymes make cuts at specific positions within the DNA molecule.
Today, scientists have identified over 900 restriction enzymes from more than 230 different strains of bacteria. Because there are so many, they are named using a strict, systematic biological convention:
- The first letter comes from the genus of the bacteria.
- The second two letters come from the species of the prokaryotic cell from which they were isolated.
- The fourth letter indicates the specific strain of the bacteria.
- The Roman numeral indicates the order in which the enzyme was discovered in that strain.
Example: Let us look at the widely used enzyme EcoRI.
- E stands for the genus Escherichia.
- co stands for the species coli.
- R stands for the strain RY13.
- I indicates it was the first enzyme isolated from this strain.
C. Recognition Sequences and Palindromic DNA
Restriction endonucleases function by inspecting the length of a DNA sequence. Once they find their specific recognition site, they bind to the DNA and cut each of the two strands of the double helix at specific points in their sugar-phosphate backbones.
A unique feature of restriction endonucleases is that they recognise palindromic nucleotide sequences in the DNA. In everyday language, a palindrome is a word that reads the same forwards and backwards. For example, the word MALAYALAM or NAMAN.
In DNA, a palindromic sequence is a sequence of base pairs that reads the exact same on the two strands when the orientation of reading is kept the same. For example, if we read from the 5′ (five prime) to 3′ (three prime) direction, the sequence is identical on both strands:
5′ — G A A T T C — 3′ 3′ — C T T A A G — 5′
This symmetrical sequence is the specific recognition site for the enzyme EcoRI.
D. Cutting the DNA: Creating "Sticky Ends"
When restriction enzymes cut the DNA, they do not always cut straight down the middle. Enzymes like EcoRI cut the DNA slightly away from the centre of the palindrome, but between the exact same two bases (G and A) on both the opposite strands.
When this off-centre cut happens, it leaves behind short, single-stranded overhangs at the ends of the DNA fragments. These overhanging stretches are called sticky ends. They are termed “sticky” because they are highly unstable and readily form hydrogen bonds with their complementary cut counterparts. This stickiness is highly advantageous for genetic engineers, as it helps distinct pieces of DNA easily find and attach to one another.
E. Use of DNA Ligase: The Molecular Glue
The generation of sticky ends is only half the battle. When foreign DNA (the gene of interest) and vector DNA (like a plasmid) are cut using the exact same restriction enzyme, they will possess perfectly compatible sticky ends.
While the sticky ends will temporarily join together through hydrogen bonds, the DNA backbone remains broken. To permanently seal the two fragments into one continuous molecule, scientists use an enzyme called DNA ligase.
DNA ligase acts as the molecular glue. It facilitates the formation of strong phosphodiester bonds between the sugar and phosphate backbones of the two DNA strands. Once the ligase completes its job, a newly formed, stable molecule of recombinant DNA is successfully created, ready to be introduced into a host cell.
