Mining Genomes

Recombinant DNA Project

This exercise casts you in the role of research geneticist. Your job is to plan a project to clone a specific gene into a plasmid vector, including the selection of the restriction enzymes and vector that you will use. You will utilize the Web sites of some of the major suppliers of biotchnology reagents in the process.

COMPREHENSION QUESTIONS^

1. List some of the effects and applications of recombinant DNA technology.

2. What common feature is seen in the sequences recognized by type II restriction enzymes?

3. What role do restriction enzymes play in bacteria? How do bacteria protect their own DNA from the action of restriction enzymes?

4. Explain how gel electrophoresis is used to separate DNA fragments of different lengths.

5. After DNA fragments are separated by gel electrophoresis, how can they be visualized?

6. What is the purpose of Southern blotting? How is it carried out?

7. What are the differences between Southern, Northern, and Western blotting?

8. Give three important characteristics of cloning vectors.

9. Briefly describe four different methods for inserting foreign DNA into plasmids, giving the strengths and weaknesses of each.

10. How are plasmids transferred into bacterial cells?

*11. Briefly explain how an antibiotic-resistance gene and the lacZ gene can be used as markers to determine which cells contain a particular plasmid.

12. How are genes inserted into bacteriophage X vectors? What advantages do X vectors have over plasmids?

*13. What is a cosmid? What are the advantages of using cosmids as gene vectors?

14. What are yeast artificial chromosomes and shuttle vectors? When are these cloning vectors used?

*15. How does a genomic library differ from a cDNA library? How is each created?

16. How are probes used to screen DNA libraries? Explain how a synthetic probe can be prepared when the protein product of a gene is known.

17. Explain how chromosome walking can be used to find a gene.

18. Discuss some of the considerations that must go into developing an appropriate cloning strategy.

*19. Briefly explain how the polymerase chain reaction is used to amplify a specific DNA sequence. What are some of the limitations of PCR?

*20. Briefly explain in situ hybridization, giving some applications of this technique.

21. What is DNA footprinting?

22. Briefly explain how site-directed mutagenesis is carried out.

*23. What are knockout mice, how are they produced, and for what are they used?

24. Describe how RFLPs can be used in gene mapping.

*25. What is DNA fingerprinting? What types of sequences are examined in DNA fingerprinting?

26. What is gene therapy?

27. As the first recombinant DNA experiments were being carried out, there was concern among some scientists about this research. What were these concerns and how were they addressed?

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