Question
What are restriction enzymes? Explain how they work, why they are called “molecular scissors,” and what are sticky ends and blunt ends.
(NEET 2022, similar pattern)
Solution — Step by Step
Restriction enzymes (restriction endonucleases) are enzymes that cut DNA at specific recognition sequences. They were discovered in bacteria, where they serve as a defence mechanism — they “restrict” the growth of bacteriophages (viruses) by cutting their DNA.
The first restriction enzyme discovered was Hind II (from Haemophilus influenzae). They are called molecular scissors because they precisely cut DNA at defined positions, just as scissors cut paper at a specific point.
Each restriction enzyme recognises a specific palindromic sequence — a sequence that reads the same on both strands in the 5’ to 3’ direction. For example, EcoRI recognises:
5’-GAATTC-3’
3’-CTTAAG-5’
EcoRI cuts between G and A on both strands, but at staggered positions. This specificity is what makes them so useful in genetic engineering — they always cut at the same sequence.
Depending on how the enzyme cuts, two types of ends are produced:
Sticky ends (cohesive ends): The enzyme cuts at staggered positions on the two strands, leaving single-stranded overhangs. Example: EcoRI produces:
5’-G…AATTC-3’ → becomes → 5’-G + AATTC-3’
3’-CTTAA…G-5’ → becomes → 3’-CTTAA + G-5’
The single-stranded overhangs (AATT) can base-pair with complementary sticky ends from another DNA fragment — making it easy to join different DNA pieces using DNA ligase.
Blunt ends: The enzyme cuts both strands at the same position, leaving no overhangs. Example: Sma I cuts at the centre of CCCGGG. Blunt ends are harder to join because they lack the complementary overhangs.
Why This Works
Restriction enzymes are the foundation of recombinant DNA technology. By cutting DNA at precise, predictable locations, scientists can:
- Cut out a specific gene from one organism
- Cut open a vector (plasmid) with the same enzyme
- Join the gene into the vector using DNA ligase (the “molecular glue”)
The sticky ends produced by the same enzyme are complementary to each other, ensuring that the gene inserts into the vector in the correct orientation. This is why the same restriction enzyme must be used to cut both the donor DNA and the vector.
Naming convention for NEET: the first letter comes from the genus, the next two from the species, and the Roman numeral indicates the order of discovery. So EcoRI = Escherichia (E) coli (co) strain R, first enzyme (I). Hind III = Haemophilus influenzae strain d, third enzyme.
Common Mistake
Students often confuse restriction endonucleases with exonucleases. Endonucleases cut DNA at internal positions within the strand. Exonucleases remove nucleotides from the ends of DNA. Restriction enzymes used in genetic engineering are always endonucleases — they cut within the DNA molecule at specific recognition sites.
Another error: thinking that the bacterium’s own DNA is cut by its restriction enzymes. Bacteria protect their own DNA by methylation — a modification enzyme (methylase) adds methyl groups to the recognition sequences in the bacterium’s own DNA, preventing the restriction enzyme from cutting it. Foreign (unmethylated) DNA is cut and destroyed. This is the restriction-modification system.