Answer5 - MCB 161 Lecture 5: DNA Replication (II) MBOC...

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MCB 161 Study Problems Lecture 5: DNA Replication (II) MBOC Problems 4-84 (a-c) A. A few of the untreated plasmid molecules are relaxed because they contain one or more single-strand breaks in the DNA. DNA that is carefully handled during the isolation procedure is mostly supercoiled, as shown in Figure 4–27, lane 1. Harsh treatment during isolation will yield a DNA preparation that is almost entirely relaxed. B. The bands that run at intermediate positions are a collection of topoisomers that differ only in their linking number; that is, the number of times one strand is wrapped around the other. Adjacent bands on the gel contain topoisomers that differ by a linking number of one. The rate at which DNA molecules migrate through a gel depends on how compact they are, with more compact molecules moving faster. Relaxed circular molecules are the least compact and therefore move the slowest, whereas highly supercoiled molecules are the most compact and run the fastest. Treatment with topoisomerase removes supercoils one at a time, making the molecule progressively less compact and slower in migrating. C. The number of supercoils in the original plasmid can be estimated by counting the number of bands between the highly supercoiled and relaxed positions of the gel. About eight intermediate bands can be counted in the treated samples; thus, there must be at least nine supercoils in the original plasmid. This number is likely to be an underestimate because of the limited resolving power of such gels. Once a molecule reaches a certain degree of compactness, a gel cannot resolve molecules with further increases in supercoiling. D. Since the supercoils are removed by
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Answer5 - MCB 161 Lecture 5: DNA Replication (II) MBOC...

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