A molecular biologist is preparing to clone a human gene into a bacterial plasmid. They will use restriction enzymes to cut both the isolated human gene and the plasmid vector before introducing the gene into the plasmid. What is the most critical reason for using the same restriction enzyme to digest both the gene of interest and the plasmid vector in a typical cloning experiment?
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The answer
Correct answer: A. To create compatible sticky ends on both the gene and the plasmid, facilitating their ligation.
Tested concept: Restriction enzymes and cloning
Explanation
Restriction enzymes recognize specific DNA sequences and cleave the phosphodiester backbone. When the same restriction enzyme is used to cut both the gene of interest and the plasmid vector, it generates complementary sticky ends (overhangs) on both DNA fragments. These complementary ends can then anneal through hydrogen bonding, creating a temporary association that DNA ligase can permanently seal by forming new phosphodiester bonds. This compatibility is essential for successful ligation and cloning. While orientation is important, using the same enzyme primarily ensures compatible ends, which then allows for proper orientation if the sites are asymmetric. DNA ligase forms covalent bonds, but only after the compatible ends have annealed. Restriction enzymes cut DNA; they do not activate promoter regions.
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