Recombinant DNA cloning workflow: Which enzyme is specifically used to form covalent phosphodiester bonds that join foreign DNA fragments into a vector plasmid (after compatible ends are generated)?

Introduction / Context:
In molecular cloning, foreign DNA must be inserted into a plasmid vector to create a stable recombinant molecule that can replicate inside a host cell such as Escherichia coli. The key chemical step is sealing the sugar–phosphate backbone between adjacent nucleotides, turning compatible DNA ends into a continuous duplex. The enzyme that catalyzes this covalent joining is DNA ligase, typically T4 DNA ligase in bench workflows.

Given Data / Assumptions:

• The vector and insert have compatible ends (either sticky or blunt) generated by restriction enzymes or PCR/adaptors.
• ATP is available as a cofactor for T4 DNA ligase activity.
• The goal is to obtain a closed circular recombinant plasmid capable of replication in a suitable host.

Concept / Approach:
DNA ligase creates phosphodiester bonds between a 3'-OH and a 5'-phosphate at DNA nicks or compatible overhangs. This covalent linkage is essential; without it, the insert would remain only transiently annealed to the vector via base pairing and would dissociate during transformation or replication. Ligation efficiency depends on end compatibility, DNA concentration, temperature, and buffer composition.

Step-by-Step Solution:

Verification / Alternative check:
Colony PCR, restriction digest mapping, or Sanger sequencing of candidate clones confirms that covalent joining occurred at the intended junctions, verifying ligase function in the reaction.

Why Other Options Are Wrong:

• DNA polymerase: extends DNA strands but does not seal nicks between separate duplex molecules.
• Restriction endonuclease: cuts DNA at specific sequences; it does not join DNA.
• DNA helicase: unwinds duplex DNA during replication or repair, not ligation.
• Topoisomerase: changes DNA supercoiling; specialized “TOPO cloning” vectors exist but still are not the standard ligase for routine ligations.

Common Pitfalls:
Using incompatible ends, omitting 5'-phosphorylation on PCR inserts, or incorrect vector:insert ratios can reduce ligation efficiency. Always verify buffer freshness and ATP integrity for optimal ligase activity.

Final Answer:
DNA ligase