You are trying to insert a human insulin gene into a bacterial plasmid. You cut the human DNA with the restriction enzyme EcoRI (which produces a specific sticky end) and you cut the plasmid with a different enzyme, BamHI (which produces a different sticky end). You then mix them with DNA ligase. What will be the result?

Question

You are trying to insert a human insulin gene into a bacterial plasmid. You cut the human DNA with the restriction enzyme EcoRI (which produces a specific sticky end) and you cut the plasmid with a different enzyme, BamHI (which produces a different sticky end). You then mix them with DNA ligase. What will be the result?

BlackTom AI · Accepted Answer

In this scenario, you are attempting to ligate human DNA cut with EcoRI (which generates a specific sticky end) into a plasmid cut with BamHI (which produces a different sticky end). The compatibility of the ends is crucial for successful ligation.

Option 1: 'The human gene will be inserted into the plasmid in the reverse orientation.' This would only be a possibility if the two ends were compatible and allowed directional ligation. Since EcoRI and BamHI produce non-complementary overhangs, the ends cannot base-pair, so this scenario cannot occur. The premise that orientation would be inverted relies on successful base-pairing which is not available here.

Option 2: 'The ligation will fail because the sticky ends are not complementary and cannot base-pair.' This is correct. EcoRI-generated overhangs are different from BamHI-generated overhangs. Ligation requires compatible cohesive ends or blunt ends. Because the ends do not match, the DNA fragments cannot form stable base pairs, and ligation will not proceed effectively.

Option 3: 'The plasmid will replicate, but it will effectively delete the human gene.' There is no mechanism here by which replication would selectively delete the inserted gene. If ligation fails, there is no recombinant molecule to replicate. If ligation did occur, deletion would be an unusual outcome and is not described by these restriction enzymes.

Option 4: 'A successful recombinant plasmid will be formed, but the bacteria will degrade it.' Bacterial degradation of a recombinant plasmid is not a typical outcome of incompatibility of sticky ends. If the ends do not base-pair, no recombinant plasmid forms to begin with, so degradation is not the relevant risk.

Overall, the core concept here is that incompatible sticky ends (EcoRI vs BamHI) prevent proper base-pairing, so the ligation would fail, making Option 2 the accurate choice. The other options misinterpret the consequences of using non-compatible restriction sites or assume outcomes (like orientation, deletion, or degradation) that do not align with the molecular events that occur when ends are not complementary.

Bio 93 Section CDE 2025 Final- Section E

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