1. Look at the piano key figure on p. 25 (DNA ligase). What is the "nicked" DNA the figure refers to? What if there were two nicks instead of one? What would be the result? Would ligase still work?
Problems 2-5 refer to the second train track figure, entitled Restriction endonuclease
2. Cohen puts a mysterious phrase (Able was I ere I saw Elba) at the top of the figure. What point is he trying to make? The text speaks of "palindromes". In what way is the EcoRI site shown below the phrase a different sort of palindrome than Able was I ? How would you change the English palindrome to make it correspond more closely to the way "palindrome" is used when referring to DNA?
3. What would be the likely result if DNA ligase were added to the molecule shown at the bottom of the figure?
4. Explain this observation: When placed in the refrigerator, the molecule shown at the bottom of the figure takes the form of a circle; when placed at body temperature, the molecule is linear.
5. The restriction enzyme EcoRI is said to cleave DNA. It so happens that you can also cleave DNA by shaking it very very hard, e.g. with ultrasound. What's the advantage of using a restriction enzyme?
6. What are key differences between a plasmid and a chromosome? You might consult the figures entitled Plasmid DNA and Foreign DNA for inspiration.
Questions 7-10 refer to the figure entitled Foreign DNA
7. What is the base (G, A, T, or C) immediately to the right of AATT in the foreign DNA at the top right of the figure? (Hint: look at the legend. It says, "The plasmid is cleaved by the endonuclease ")
8. Suppose DNA of the plasmid pSC101 were red and foreign DNA were blue. Color the plasmid labeled Plasmid Chimera appropriately.
9. News flash. DNA is not red or blue. If you had microscopic eyes, is there any way you could tell the difference between pSC101 digested with EcoRI and "Foreign DNA" also digested with EcoRI?
10. Transformation, the process of getting DNA into a cell, works about one time per million cells. Therefore, for every cell that gains plasmid, as shown in the figure below the label Transformation, there are a million that don't. How can you possibly pick out the one in a million cells that has the plasmid? In answering, consider the features of pSC101 shown at the top, left of the figure.
11. Dolly the sheep is a clone of the adult sheep from which she was derived because the two are genetically identical. Consider the plasmids shown at the bottom of the figure, within the cells labeled Daughter Cells. Are they clones of the single plasmid labeled Plasmid Chimera? Suppose the "foreign DNA" at the top of the figure were taken from Dolly. In what sense would the plasmids be a clone of Dolly? (Warning! "Clone" is currently used in two very different ways. Sometimes it is used to refer to duplication of a whole organism. Other times it is used to refer to duplication and amplification of a small piece of DNA. We will refer to the first as "organismal cloning" and the second "molecular cloning")
12. DNA is isolated from a baboon and digested with EcoRI. What four bases lie at the ends of the digested DNA? Could it be ligated to DNA from broccoli also digested with EcoRI?
13. Suppose that DNA from E. coli is random (it isn't) and that the probability of any given base being a G is 1/4, of being an A is also 1/4, and so forth. How frequently would you expect to find EcoRI recognition sites in E. coli DNA? Answer in the form: "one site per X bases".