Task Chloroquine is a so-called intercalator that can bind to DNA by inserting t

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Task

Chloroquine is a so-called intercalator that can bind to DNA by inserting the molecule between base pairs. In this way, the DNA helix is twisted and opened at about 26 ° for each bound chloroquine molecule.

a. How is supercoiling of chloroquin bound to DNA affected?

b. What does this effect mean for the migration of a negative supercoiled plasmid into an agarose gel.

By electrophoresis, the mobility of plasmids is determined by the degree of supercoiling of the plasmids. However, since negative and positive supercoiled topoisomers can migrate at the same rate in the gel, this causes such topoisomeric forms to not be separated by one-dimensional electrophoresis. This problem is solved by performing two-dimensional gel electrophoresis. Electrophoresis in the first dimension takes place as normal. The second electrophoresis step is performed perpendicular to the first dimension in the presence of DNA intercalators such as EtBr or chloroquine.

c. Look at the figure and use its answers from the questions a and b to explain the gels

(N = nicked DNA circles; L = linear DNA)

Explanation / Answer

a.Chloroquine is an intercalator that unwinds DNA, relaxing the supercoiling. It makes negatively supercoiled DNA even lesser supercoiled.

b.A supercoiled version of a DNA isomer would migrate faster in agarose gel as compared to a linear or nicked form,because of its conformation. Without treatment of an intercalator like chloroquine,both isomers of DNA that is postively supercoiled and negatively supercoiled would migrate at almost the same rate. Treatment of chloroquine on negatively supercoiled DNA would relax its supercoild and it would hence migrate at a slower rate compared to positively supercoiled DNA.

c.The gel shows that "N" nicekd DNA has the slowest rate of migration, which is beacuse of the conformation rendered by a nick on one strand of the double helix. The "L" linear DNA has a higher migration rate.

While running in one phase, both positively (+) supercoiled and negatively (-) supercoiled DNA have same migration rate, but when the resolution is carried in a perpendicular direction to the first dimension of electrophoresis with chloroquine added, the positively supercoiled DNA migrates at a faster rate compared to negatively supercoiled DNA. This is because chloroquine's effect as described in part a. and b.

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