Review The Concepts

1. Genetic mutations can provide insights into the mechanisms of complex cellular or developmental processes. What is the difference between recessive and dominant mutations? What is a temperature-sensitive mutation, and how is this type of mutation useful?

2. A number of experimental approaches can be used to analyze mutations. Describe how complementation analysis can be used to reveal whether two mutations are in the same or in different genes. What are suppressor mutations and synthetic lethal mutations?

3. 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 enzyme 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?

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

5. 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 and a genomic DNA library? How can you use hybridization or expression to screen a library for a specific gene? What oligonucleotide primers could be synthesized as probes to screen a library for the gene encoding the peptide Met-Pro-Glu-Phe-Tyr?

6. In 1993, Kerry 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 (e.g., Taq polymerase) so important for the development of PCR?

7. Southern and Northern blotting are powerful tools in molecular biology; describe the technique of each. What are the applications of these two blotting techniques?

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

9. Why is the screening for genes based on the presence of ORFs (open reading frames) more useful for bacterial genomes than for eukaryotic genomes? What are paralogous and orthologous genes? What are some of the explanations for the finding that humans are a much more complex organism than the roundworm C. elegans, yet have only less than twice the number of genes (35,000 versus 19,000)?

10. A global analysis of gene expression can be accomplished by using a DNA microarray. What is a DNA micro-array? How are DNA microarrays used for studying gene expression? How do experiments with microarrays differ from Northern botting experiments described in question 7?

11. 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?

12. Two methods for functionally inactivating a gene without altering the gene sequence are by dominant negative mutations and RNA interference (RNAi). Describe how each method can inhibit expression of a gene.

13. DNA polymorphisms can be used as DNA markers. Describe the differences among RFLP, SNP, and SSR polymorphisms. How can these markers be used for DNA mapping studies?

14. Genetic linkage studies can roughly locate the chromosomal position of a "disease" gene. Describe how expression analysis and DNA sequence analysis can be used to identify a "disease" gene.

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