You place equal amounts of plasmid DNA in 4 different tubes and incubate the DNA
ID: 36773 • Letter: Y
Question
You place equal amounts of plasmid DNA in 4 different tubes and incubate the DNA with increasing amounts of the enzyme topoisomerase ? for 1 hour (0 enzyme units, 0.25 enzyme units, 0.5 enzyme units and 1 enzyme unit). You then analyze the plasmid DNA in each tube by agarose gel electrophoresis and obtain the figure shown below (the DNA was loaded at the top of the gel), the lane number is indicated at the bottom of the gel. 10) In the absence of any topoisomerase (see lane 1), the plasmid DNA adopts two forms that migrate at different speed even though they contain the same number of base pairs: one is the highly supercoiled plasmid form and the other is a closed circular plasmid form that arises when plasmid DNA gets nicked during the plasmid purification procedure. Which band would you expect to represent the highly supercoiled form? a) the thin, slow-migrating band at the top b) the thick, slow-migrating band at the bottom c) the thin, fast-migrating band at the top d) the thick, fast-migrating band at the bottom 11) What happened to the highly supercoiled plasmid DNA after you added a large amount of topoisomerase I (lane 4)? 12)What do the minor bands in lane 2 or 3 represent?Explanation / Answer
10. Topoisomerase is an enzyme that helps in winding of the double stranded DNA. Thus it helps in the succesful completion of transcription etc., The highle supercoiled form of plasmid DNA run faster than any other form as due to highly winding the DNA can easily penetrate through the gel pores than a linear one.
Hence the correct option is d.
11. Topoisomerase enzyme prevents the winding of DNA. Thus a plasmid DNA incubated with high amount of topoisomerase becomes linear conformation and migrates very slowly on an agarose gel.
12. Plasmid DNA takes three forms after incubation with topoisomerase. Dna may get cut at one point becoming an open circular, or it may totally unwind and become a linear form and it may not unwind and remain as supercoiled. The forms appears based on the amount of enzyme added. All the three forms appears if small amount of enzyme is added. Then the top band is the open circular form and the minor bands are the linear form bands that are cut at different base pairs and the bottom band always represent a supercoiled form.
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