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1. Genetic mutations can provide insights into the mechanisms of complex cellular or developmental processes. How might your analysis of a genetic mutation be different depending on whether a particular mutation is recessive or dominant?

2. What is a temperature-sensitive mutation? Why are temperature-sensitive mutations useful for uncovering the function of a gene?

3. Describe how complementation analysis can be used to reveal whether two mutations are in the same or in different genes. Explain why complementation analysis will not work with dominant mutations.

4. Jane has isolated a mutant strain of yeast that forms red colonies instead of the normal white when grown on a plate. To determine the mutant gene, she decides to use functional complementation with a DNA library containing a lysine selection marker. In addition to the unknown gene mutation, the yeast are lacking the gene required to synthesize the amino acids leucine and lysine. What media will Jane grow her yeast on to ensure that they have acquired the library plasmids? How will she know when a library plasmid has complemented her yeast mutation?

5. Restriction enzymes and DNA ligase play essential roles in DNA cloning. How is it that a bacterium that produces a restriction enzyme does not cut its own DNA? Describe some general features of restriction sites. What are the three types of DNA ends that can be generated after cutting DNA with restriction enzymes? What reaction is catalyzed by DNA ligase?

6. Bacterial plasmids often serve as cloning vectors. Describe the essential features of a plasmid vector. What are the advantages and applications of plasmids as cloning vectors?

7. A DNA library is a collection of clones, each containing a different fragment of DNA, inserted into a cloning vector. What is the difference between a cDNA library and a genomic DNA library? Suppose you would like to clone gene X, a gene expressed only in neurons, into a vector using a library as the source of the insert. You have the following libraries at your disposal: a genomic library from skin cells, a cDNA library from skin cells, a genomic library from neurons, and a cDNA library from neurons. Which could you use, and why?

8. In 1993, Kary Mullis won the Nobel Prize in Chemistry for his invention of the PCR process. Describe the three steps in each cycle of a PCR reaction. Why was the discovery of a thermostable DNA polymerase so important for the development of PCR?

9. Southern and Northern blotting are powerful molecular biological tools based on hybridization of nucleic acids. How are these techniques the same? How do they differ? Give some specific applications for each blotting technique.

10. A number of foreign proteins have been expressed in bacterial and mammalian cells. Describe the essential features of a recombinant plasmid that are required for expression of a foreign gene. How can the foreign protein be modified to facilitate its purification? What is the advantage of expressing a protein in mammalian cells versus bacteria?

11. Northern blotting, RT-PCR, and microarrays can be used to analyze gene expression. A lab studies yeast cells, comparing their growth in two different sugars, glucose and galactose. One student is comparing expression of the gene HMG2 under these two conditions. Which technique(s) could he use and why? Another student wants to compare expression of all the genes on chromosome 4, of which there are approximately 800. What technique(s) could she use and why?

12. In determining the identity of the protein that corresponds to a newly discovered gene, it often helps to know the pattern of tissue expression for that gene. For example, researchers have found that a gene called SERPINA6 is expressed in the liver, kidney, and pancreas but not in other tissues. What techniques might researchers use to find out which tissues express a particular gene?

13. DNA polymorphisms can be used as DNA markers. Describe the differences between SNPs and STR polymorphisms. How can these markers be used for DNA-mapping studies?

14. How can linkage-disequilibrium mapping sometimes provide a much higher resolution of gene location than classical linkage mapping?

15. Genetic linkage studies can usually locate the chromosomal position of a disease gene only roughly. How can expression analysis and DNA sequence analysis help locate a disease gene within the region identified by linkage mapping?

16. The ability to selectively modify the genome in the mouse has revolutionized mouse genetics. Outline the procedure for generating a knockout mouse at a specific genetic locus. How can the loxP-Cre system be used to conditionally knock out a gene? What is an important medical application of knockout mice?

17. Two methods for functionally inactivating a gene without altering the gene sequence involve dominant-negative alleles and RNA interference (RNAi). Describe how each method can inhibit expression of a gene.