Question
EcoRI is a restriction endonuclease that recognises the sequence 5’-GAATTC-3’. After cutting, what type of ends does it produce, and why are these ends called “sticky”? Also name one restriction enzyme that produces blunt ends.
(NEET 2024 pattern)
Solution — Step by Step
EcoRI is a Type II restriction enzyme isolated from Escherichia coli (that’s where the “Eco” comes from — R denotes the strain RY13, I means it was the first enzyme isolated from that strain).
It recognises the palindromic sequence 5’-GAATTC-3’ on both strands of double-stranded DNA.
In DNA, a palindrome means the sequence reads the same on both strands in the 5’→3’ direction. So if one strand is 5’-GAATTC-3’, the complementary strand is 3’-CTTAAG-5’, which is 5’-GAATTC-3’ when read in its own 5’→3’ direction.
This symmetry is why restriction enzymes can bind and cut both strands — they interact with the same sequence twice.
EcoRI cuts between G and A on both strands:
5'---G | AATTC---3'
3'---CTTAA | G---5'The cut is staggered — not straight across. This leaves a 4-nucleotide single-stranded overhang on each cut end.
After cutting, each fragment ends with a 4-base single-stranded tail:
5'---G AATTC---3'
3'---CTTAA G---5'These overhangs are 5’-AATT-3’ on one fragment and 5’-AATT-3’ on the other (complementary). Because these single-stranded tails can hydrogen-bond with each other (or with any other DNA fragment cut by the same enzyme), they “stick” together — hence sticky ends (also called cohesive ends).
This property is what makes genetic engineering possible. A human gene cut with EcoRI can be inserted into a bacterial plasmid also cut with EcoRI — the sticky ends anneal, ligase seals the backbone, and you have recombinant DNA.
HaeIII cuts straight across both strands at the same position within its recognition sequence (5’-GG↓CC-3’), leaving no overhangs. These are blunt ends — harder to ligate but useful when you don’t want sticky-end artifacts.
Why This Works
Restriction enzymes evolved in bacteria as a defence against bacteriophages — foreign DNA gets chopped up while the bacterium protects its own DNA by methylating the recognition sites. We’ve essentially borrowed this bacterial immune system as a molecular tool.
The palindromic recognition site is not accidental. A dimeric enzyme (two identical subunits) binds one side of the palindrome per subunit, and the symmetry of the sequence allows both subunits to interact identically. This makes the enzyme highly specific — EcoRI cuts at roughly one site per 4096 bp (4⁶) in random sequence DNA.
For an enzyme recognising an n-base sequence:
EcoRI (6-base cutter): ≈ once per 4 kb
Alternative Method — Identifying Sticky vs Blunt from the Cut Notation
In NEET questions, you’ll sometimes be given the cut diagram directly. The trick:
- If both cuts are at the same position relative to the recognition sequence → blunt ends
- If the cuts are offset (one strand cut before the other) → sticky ends
- Cut at 5’ side of recognition site → 5’ overhang (like EcoRI)
- Cut at 3’ side → 3’ overhang (like KpnI)
For NEET, memorise these three: EcoRI (5’ sticky, cuts G↓AATTC), BamHI (5’ sticky, cuts G↓GATCC), HaeIII (blunt, cuts GG↓CC). These three cover ~80% of restriction enzyme MCQs in PYQs.
Common Mistake
Students often say EcoRI produces “blunt ends” because they see a clean double-stranded cut in diagrams. The confusion comes from simplified textbook figures that show the two strands as a straight ladder.
The actual cut is staggered. Draw it properly:
5'-G AATTC-3'
3'-CTTAA G-5'The 4-nucleotide single-stranded overhangs (5’-AATT) are what make these sticky ends, not blunt ends. In NEET 2024, this exact distinction was tested — one option said “EcoRI produces blunt ends with 4-base overhangs,” which is self-contradictory. Any fragment with overhangs is by definition a sticky end.