Restriction enzymes — After EcoRI cleaves DNA at its recognition site, which ends are generated on the products?

Introduction:Understanding the chemistry of restriction endonuclease cleavage is essential for cloning. EcoRI is a classic type II enzyme that generates sticky ends. This question checks your knowledge of the terminal groups produced during the cut.

Given Data / Assumptions:

• EcoRI recognizes a palindromic 6 bp site and makes staggered cuts.
• Phosphodiester bond cleavage leaves characteristic 5' and 3' termini.
• Sticky ends facilitate ligation into vectors with compatible overhangs.

Concept / Approach:EcoRI cleaves to produce 5'-overhangs. For each break, the scissile phosphate remains attached to the 5' terminus, leaving a 5'-phosphate and a complementary 3'-hydroxyl on the opposing end. Across both strands, products present two 5'-phosphate ends and two 3'-OH ends poised for ligation by DNA ligase.

Step-by-Step Solution:

Verification / Alternative check:Ligation requires a 5'-phosphate and a 3'-OH. EcoRI-cut fragments can be directly ligated without additional phosphorylation because the necessary termini are already present.

Why Other Options Are Wrong:

• Single 3'-OH and single 5'-phosphate: undercounts ends formed across both strands.
• 3'-phosphate ends: typical type II enzymes leave 5'-phosphate, not 3'-phosphate.
• Blunt ends: EcoRI makes sticky 5' overhangs, not blunt ends.
• One 3'-OH and two 5'-phosphate: mismatched stoichiometry.

Common Pitfalls:Confusing the nature of sticky vs blunt ends and which terminus bears the phosphate after type II restriction enzyme cleavage.

Final Answer:Two 3'-OH ends and two 5'-phosphate ends.