• Consider the two proteins A and B. Protein A has a MW = 75kDa and protein B has a MW = 15kDa. Which protein will sediment faster during rate zonal centrifugation? Which protein will migrate faster during SDS polyacrilamide gel electrophoresis? [2]

• Describe the general features of restriction enzyme sites [2]
• Explain how bacterial restriction enzymes do not cut the bacteria’s own DNA [2]
• List and describe the 2 types of DNA ends generated after cutting DNA with restriction enzymes [4]
• Describe the reaction catalyzed by DNA ligase [2]
• Describe the features of both DNA topoisomerase I and DNA topoisomerase II, including how these enzymes work. [4]
• Define the term hybridization as applied to the screening of DNA sequences [2]
• How does equilibrium sedimentation provide direct evidence for the semiconservative replication of DNA? [3]

Use the University of Florida Health Science Center e-journals or any other applicable online resource to locate the following journal article: Journal of Bacteriology, July 2000, p. 3613-3618, Vol. 182, No. 13. Use this paper to answer the following questions.

• What are the advantages of cell free extracts and fractionation to biological research? [2]
• What was the rationale for conducting early DNA replication studies on prokaryotic systems? [2]
• Studies involving phage M13 and φ X174 led to some significant discoveries relating to DNA priming and replication – list them [3]

• What is the concept behind reverse genetics and genomics? [2]

• Describe the features that distinguish plasmid, phage, BAC and YAC cloning vectors. [4]

• Does lambda DNA packaging require any enzymes or ATP hydrolysis? Explain. [2]

• Compare the differences between cDNA clones and genomic DNA clones that originate from the same region of DNA. When is it more suitable to use one preparation over the other? [2]

• What is meant y an in situ hybridization experiment? Discuss and provide an example. [2]

• Once you had obtained a highly concentrated stock of purified phage, how would you separate the recombinant phage DNA away from the phage head and tail proteins? [2]

• Define and describe an application for suicide plasmids; include in your discussion a reference for a research journal article or website that discusses this. [2]

• Briefly explain how repetitive DNA is useful for identifying individuals by the techniques of DNA fingerprinting. [2]

• Locate the following article and briefly describe how the full-length cDNA sequence for the gene KCNV1 was determined [2]

Ebihara M, Ohba H, Kikuchi M, Yoshikawa T. (2004). Gene, 325: 89-96.

• Having just read the paper above, what exactly are deletion constructs? [2]

1. What types of information can be derived from performing a Western Blot? [2]

2. In choosing an animal model for performing genetic knockout experiments, what basic characteristics would be most beneficial? [2]

3. Aseptic technique is required in a cell culture lab or dedicated area, which would be required for many of the techniques described in this module. What does this entail? [2]

4. Discuss the differences between the Maxim-Gilbert and Sanger sequencing methods. [2]

5. Briefly explain what is meant by the term “chromosome painting”.[2]

6. How might you explain an observation that the pattern of restriction fragment lengths from a region of the genome differs between homologous chromosomes and between two individuals? [2]

7. What exactly are single nucleotide polymorphisms (SNP’s) and what techniques can be used to identify them? [2]

8. Briefly discuss the pros and cons of integrating DNA microarray or chip technology into the forensic DNA laboratory [2]

9. Locate and read the following article: Roufosse CA, Direkze NC, Otto WR and Wright NA. (2004). The International Journal of Biochemistry & Cell Biology, 36, (4): 585-597.

a. What clinical implications would the identification of MPCs in adult circulating peripheral blood have? [1]
b. Exactly what was the FISH technique used to determine in these experiments? [1]

10. Locate and read the following article: Wang Z, Dooley T, Curty E, Davis R, VandeBerg J. (2004). Genomics,83(4): 588-599.

If we are able to spot thousands of human genes in a DNA microarray, what is the benefit of demonstrating cross-species application of the cDNA microarray technique described? [2]