task Circular plasmid DNA has been isolated from a bacterium and analyzed by aga
ID: 274662 • Letter: T
Question
task
Circular plasmid DNA has been isolated from a bacterium and analyzed by agarose gel electrophoresis. The result is shown in Lane 1 in the figure. Gel's poles are indicated.
a. Describe the principle of separation of DNA by agarose gel electrophoresis. In which direction does DNA migrate during gel electrophoresis and why?
b. Describe a method of visualization of DNA in an agarose gel.
Topoisomerases are enzymes that can alter the superhelicity of a plasmid. Two portions of the isolated plasmid DNA are relayed in 5 and 10 minutes with a topoisomerase, respectively, and then again analyzed by agarose gel electrophoresis. The result is shown in lane 2 and 3 respectively.
c. Compare the band patterns in lane 2 and 3 and explain the differences. What types of DNA structures represent the different bands?
d. In a new experiment, the plasmid DNA is treated with a restriction enzyme which has a single recognition sequence in the DNA. Explain what happens during the enzymatic treatment and indicate in the figure the band pattern that occurs after analysis of the treated DNA by agarose gel electrophoresis.
Explanation / Answer
DNA i.e., nucleic acids can be separated by agarose gel electrophoresis. The principle of agarose gel electrophoresis is migration of nucleic acids under the influence of electric charge through the gel. The migration distance depends upon the molecular weight of different nucleic acids. Thus nucleic acid molecules are separated according to their size. In this technique sample is loaded on the gel and the electric field with positive charge i.e., anode at farther end of sample loading wells is applied. The negatively charged molecules migrate toward anode (positive) pole. The molecules with small molecular weights migrate faster than the molecules with large molecular weight. Thus separation is observed. Visualization of the agarose gel once the migration has occurred can be done by different techniques such as staining and subsequent UV illumination. Ethidium bromide or SYBR Green are commonly used dyes, whereas UV illumination is usually done at 300nm wavelength to observe the stained nucleic acids. Lane 2 shows the presence of nucleic acids from range of high molecular weight to low molecular weight. Lane 3 shows presence of nucleic acids of high molecular weight but not the nucleic acids of low molecular weight as compared to Lane 2.
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