Question

A scientist is attempting to create a recombinant DNA molecule by combining a plasmid vector and a foreign DNA. A plasmid DNA and a linear DNA of the same size have a single site for the restriction enzyme EcoRI. When cut by the same RE and separated by gel electrophoresis, the plasmid shows one DNA band, and the linear DNA shows two bands.

1. What causes the difference between the number of DNA bands generated from the plasmid and from the linear DNA?
2. What is the advantage of using agarose in gel electrophoresis?
3. How does EcoRI differ from an exonuclease?

Answer 1

1. The difference in the number of DNA bands is due to the nature of the DNA. A plasmid is circular, so cutting it at a single EcoRI site produces one linear DNA fragment, resulting in one band on the gel. In contrast, linear DNA cut at a single EcoRI site produces two fragments, which appear as two bands on gel electrophoresis.
2. Agarose is chemically inert, and its large pore size allows easy and effective separation of DNA fragments based on their size.
3. EcoRI is an endonuclease that cuts DNA at a specific internal recognition site, whereas an exonuclease removes nucleotides one by one from the 5’ or 3’ end of the DNA molecule.