Tools of Recombinant DNA Technology - Restriction Enzymes

The Breakthrough (1963): Scientists isolated two specific enzymes in Escherichia coli (E. coli) that restricted the growth of bacteriophages (viruses that infect bacteria).

• One enzyme functioned by adding methyl groups to the DNA.
• The other enzyme functioned by cutting the DNA. This DNA-cutting enzyme was called a restriction endonuclease.

The First Restriction Endonuclease: Hind II was the very first restriction endonuclease to be isolated and characterised (discovered 5 years later). It was isolated from the d-strain of Haemophilus influenzae.

Current Statistics: Today, scientists have isolated more than 900 different restriction enzymes from over 230 strains of bacteria.

Nucleases are enzymes that break nucleic acids.

Restriction enzymes follow a strict, standardised naming convention based on the bacterium they are isolated from:

• 1st Letter: Derived from the genus of the bacterium (capitalised).
• 2nd & 3rd Letters: Derived from the species of the bacterium (lowercase).
• 4th Letter (optional): Indicates the specific strain of the bacterium.
• Roman Numeral: Indicates the order of discovery or identification of the enzyme from that specific strain.

Example: EcoRI

• E = Escherichia (Genus)
• co = coli (Species)
• R = RY13 (Strain)
• I = First enzyme identified from this strain.

Inspection: Each restriction endonuclease functions by 'inspecting' the length of a DNA sequence.

Recognition: It searches for its specific recognition sequence. This sequence is always a palindromic nucleotide sequence.

• What is a palindrome? In DNA, it is a sequence of base pairs that reads exactly the same on both strands when the reading orientation is kept the same (e.g., reading 5' → 3' on both strands).
• Example (EcoRI recognition site):
  5' — GAATTC — 3'
  3' — CTTAAG — 5'
• (Note: Hind II specifically recognises a sequence of six base pairs).

Cleavage: Once it finds the palindrome, the enzyme binds to the DNA and cuts the sugar-phosphate backbones of both strands of the double helix at specific points.

1. Sticky Ends: The enzyme cuts the DNA slightly away from the centre of the palindromic site but between the same two bases on the opposite strands. This leaves single-stranded, overhanging stretches.

• These are called "sticky" because they easily form hydrogen bonds with their complementary cut counterparts. This stickiness greatly facilitates the joining action of the enzyme DNA ligase.

2. Blunt Ends: The enzyme cuts straight through, leaving no unpaired bases or overhangs. These DNA pieces cannot easily anneal (join) to each other, making them very difficult to clone.

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

To successfully create a recombinant vector molecule, both the vector DNA and the source (foreign) DNA must be cut with the exact same restriction enzyme. This ensures that both DNA fragments have complementary sticky ends that can be joined end-to-end by DNA ligases.

Diagrammatic representation of recombinant DNA technology

Once the restriction enzymes have cut the DNA into fragments, these fragments must be separated and isolated for use. This is done via gel electrophoresis.

• Step 1: The Principle: DNA fragments are negatively charged molecules. Therefore, they can be separated by applying an electric field that forces them to move towards the positive electrode (anode).
• Step 2: The Matrix: This movement takes place through a medium or matrix. The most commonly used matrix today is agarose, which is a natural polymer extracted from seaweeds.
• Step 3: The Sieving Effect: The agarose gel acts like a sieve. The DNA fragments separate based on their size. The smaller the fragment size, the farther and faster it moves down the gel.
• Step 4: Visualisation (Staining): Pure DNA fragments cannot be seen under visible light. To see them, the gel is stained with a compound called ethidium bromide.
• Step 5: UV Exposure: The stained gel is then exposed to ultraviolet (UV) radiation. The DNA bands immediately become visible as bright orange-coloured bands.
• Step 6: Elution: The desired, separated bands of DNA are physically cut out from the agarose gel and extracted from the gel piece. This extraction process is known as elution.
• Final Step: These purified DNA fragments are now ready to be joined with cloning vectors to construct recombinant DNA.

A typical agarose gel electrophoresis showing migration of undigested (lane 1) and digested set of DNA fragments (lane 2 to 4)

• Restriction enzymes, often called "molecular scissors", are specialised endonucleases that cut DNA at specific internal positions to facilitate genetic engineering.
• Over 900 restriction enzymes, including the first discovered Hind II, have been successfully isolated from more than 230 strains of bacteria.
• These enzymes are named using a standard convention based on the genus, species, strain, and discovery order of the source bacterium, such as EcoRI.
• They function by inspecting DNA to find specific palindromic nucleotide sequences, which are base pair sequences that read the same on both strands in the same orientation.
• Type II restriction enzymes are the primary ones used in genetic manipulation because they reliably recognise and cut at very specific target sequences.
• Cutting the DNA slightly away from the centre of a palindrome creates "sticky ends", which are single-stranded overhangs that easily bond with complementary DNA using the enzyme DNA ligase.
• The resulting DNA fragments are separated by size using gel electrophoresis, where smaller negatively charged fragments move faster through an agarose matrix toward an anode.
• The separated DNA is visualised as bright orange bands under UV light after ethidium bromide staining and is carefully extracted from the gel through a process called elution.