In a control experiment, a plasmid containing a HindIII recognition sequence within a kanamycin resistance gene is cut with HindIII, re-ligated, and used to transform E. coli K12 cells. Kanamycin-resistant colonies are selected, and plasmid DNA from these colonies is subjected to electrophoresis. Most of the colonies contain plasmids that produce single bands that migrate at the same rate as the original intact plasmid. A few colonies, however, produce two bands, one of original size and one that migrates much less far down the gel. Diagram the origin of this slow band as a product of ligation.
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Understand the experimental setup: A plasmid with a HindIII site inside the kanamycin resistance gene is cut with HindIII, which creates linear DNA fragments with sticky ends.
After cutting, the plasmid is re-ligated. Since the HindIII site is within the kanamycin resistance gene, successful re-ligation restoring the original circular plasmid will regenerate the intact gene, allowing kanamycin resistance.
Most colonies show a single band matching the original plasmid size, indicating that the plasmid was re-ligated into its original circular form.
The colonies with two bands—one at the original plasmid size and one migrating more slowly—suggest the presence of a plasmid form with a different structure, such as a dimer or multimer formed by ligation of two plasmid molecules end-to-end.
Diagrammatically, the slow band corresponds to a plasmid dimer: two plasmid units joined by ligation at their HindIII sticky ends, resulting in a circular DNA molecule roughly twice the size of the original plasmid, which migrates slower during electrophoresis.
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Key Concepts
Here are the essential concepts you must grasp in order to answer the question correctly.
Restriction Enzyme Digestion and Recognition Sites
Restriction enzymes like HindIII recognize specific DNA sequences and cut at those sites, generating fragments with defined ends. In this experiment, HindIII cuts within the kanamycin resistance gene, linearizing the plasmid. Understanding how restriction sites determine cutting patterns is essential to predict the resulting DNA fragments after digestion.
DNA ligase can join compatible DNA ends, allowing linearized plasmids to re-circularize or form multimers. After HindIII digestion, re-ligation can restore the original plasmid or create dimers/multimers, which migrate differently during electrophoresis. Recognizing how ligation products affect plasmid structure helps explain the appearance of bands of varying sizes.
Agarose gel electrophoresis separates DNA molecules based on size and conformation. Larger or multimeric plasmids migrate more slowly, producing bands that appear higher on the gel. Interpreting band patterns allows identification of plasmid forms, such as monomers or dimers, crucial for understanding the experimental results.
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