Question

You have two samples of covalently closed, circular DNA (ccc dsDNA)—one relaxed and one slightly negatively supercoiled—and you want to visualize the DNA using gel electrophoresis. You add a small amount of ethidium bromide to each sample. Each cccDNA contains the same number of base pairs. Interestingly, you see that the relaxed cccDNA sample migrates faster in the gel than the negatively supercoiled cccDNA sample. Which of the following is a valid explanation for the results? Select one choice from the list below (A-D) and explain your answer. A. The treatment with ethidium relaxed the negatively supercoiled cccDNA and positively supercoiled the relaxed cccDNA. B. The treatment with ethidium caused both cccDNAs to become positively supercoiled. C. The treatment with ethidium caused both cccDNAs to become relaxed. D. The treatment with ethidium decreased the linking number of the negatively supercoiled cccDNA and increased the linking number of the relaxed cccDNA.

Answer 1

The answer is

D.The treatment with ethidium decreased the linking number of the negatively supercoiled cccDNA and increased the linking number of the relaxed cccDNA.

Reason: In general Ethidium bromide intercalates in the DNA bases and induces supercoiling. It is proved by various experiments. When the circulat plasmids are treated with DNA topiisomerase which induces the positive supercoiling. and the samples is treated with ethidium bromide.When these EtBr treated plasmids are run on agarose gel electrophoresis and appeared to run at the same position equivalent to any supercoiled DNA.

However when the negatively supercoiled DNA is treated with EtBr,because of high affinity, the twist in the DNA is changes due to unwidning of the DNA. Moreover, due to removal of writhe, DNA looks more laxed.

In summary, the relaxed DNA become more supercoiled, where as supercoiled DNA become more relaxed. Therefore, relaxed DNA after EtBr treatment migrates faster.