A laboratory has oat plants containing the resistance gene and a supply of plasmids. Which sequence of steps would produce bacteria containing the resistance gene in their plasmids?

• A. Cut plasmid with restriction enzyme; insert the oat gene into plasmid using DNA ligase
• B. Cut the oat gene with restriction enzyme; insert into plasmid that has not been cut
• C. Use restriction enzymes to cut both plant DNA and plasmid but join ends without ligase
• D. Cut the oat gene from plant DNA using restriction enzymes; cut the plasmid with the same enzyme; ligate ends with DNA ligase; introduce recombinant plasmid into bacterial cells and select

Answer: (D)

The concept tested is how to construct a recombinant plasmid carrying a plant gene so bacteria can acquire it. First, cut the oat resistance gene from plant DNA with a restriction enzyme to generate ends. Then cut the plasmid with the same restriction enzyme to create compatible ends. The ends from both pieces can be joined by DNA ligase to form a stable, recombinant plasmid that carries the resistance gene. Finally, introduce this recombinant plasmid into bacterial cells and use a selective condition (such as an antibiotic) to identify the cells that contain the plasmid. This sequence ensures there is a place for the gene to fit, the ends match, the backbone is sealed, and only transformed cells are recovered. The other options fail because they either don’t create compatible ends, don’t cut the plasmid or the gene properly, skip ligation, or skip the selection step needed to identify successful transformants.